"uuid","repository link","title","author","contributor","publication year","abstract","subject topic","language","publication type","publisher","isbn","issn","patent","patent status","bibliographic note","access restriction","embargo date","faculty","department","research group","programme","project","coordinates"
"uuid:15f8e22d-492b-4922-96fb-6cb7f9dac406","http://resolver.tudelft.nl/uuid:15f8e22d-492b-4922-96fb-6cb7f9dac406","An in-depth comparison of linear and non-linear joint embedding methods for bulk and single-cell multi-omics","Makrodimitris, S. (TU Delft Pattern Recognition and Bioinformatics; Erasmus MC); Pronk, I.B. (TU Delft Pattern Recognition and Bioinformatics); Abdelaal, T.R.M. (TU Delft Pattern Recognition and Bioinformatics; Leiden University Medical Center); Reinders, M.J.T. (TU Delft Pattern Recognition and Bioinformatics; Leiden University Medical Center)","","2024","Multi-omic analyses are necessary to understand the complex biological processes taking place at the tissue and cell level, but also to make reliable predictions about, for example, disease outcome. Several linear methods exist that create a joint embedding using paired information per sample, but recently there has been a rise in the popularity of neural architectures that embed paired -omics into the same non-linear manifold. This work describes a head-to-head comparison of linear and non-linear joint embedding methods using both bulk and single-cell multi-modal datasets. We found that non-linear methods have a clear advantage with respect to linear ones for missing modality imputation. Performance comparisons in the downstream tasks of survival analysis for bulk tumor data and cell type classification for single-cell data lead to the following insights: First, concatenating the principal components of each modality is a competitive baseline and hard to beat if all modalities are available at test time. However, if we only have one modality available at test time, training a predictive model on the joint space of that modality can lead to performance improvements with respect to just using the unimodal principal components. Second, -omic profiles imputed by neural joint embedding methods are realistic enough to be used by a classifier trained on real data with limited performance drops. Taken together, our comparisons give hints to which joint embedding to use for which downstream task. Overall, product-of-experts performed well in most tasks and was reasonably fast, while early integration (concatenation) of modalities did quite poorly.","dimensionality reduction; joint embedding; multi-omics; neural networks","en","review","","","","","","","","","","","Pattern Recognition and Bioinformatics","","",""
"uuid:c450abb3-38bd-4f3b-9600-909b67c29d8a","http://resolver.tudelft.nl/uuid:c450abb3-38bd-4f3b-9600-909b67c29d8a","Anionic extracellular polymeric substances extracted from seawater-adapted aerobic granular sludge","Chen, L.M. (TU Delft BT/Environmental Biotechnology); Beck, Paula (Student TU Delft); van Ede, J.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Lin, Y. (TU Delft Environmental Fluid Mechanics)","","2024","Abstract: Anionic polymers, such as heparin, have been widely applied in the chemical and medical fields, particularly for binding proteins (e.g., fibroblast growth factor 2 (FGF-2) and histones). However, the current animal-based production of heparin brings great risks, including resource shortages and product contamination. Recently, anionic compounds, nonulosonic acids (NulOs), and sulfated glycoconjugates were discovered in the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS). Given the prevalence of anionic polymers, in marine biofilms, it was hypothesized that the EPS from AGS grown under seawater condition could serve as a raw material for producing the alternatives to heparin. This study aimed to isolate and enrich the anionic fractions of EPS and evaluate their potential application in the chemical and medical fields. The AGS was grown in a lab-scale reactor fed with acetate, under the seawater condition (35 g/L sea salt). The EPS was extracted with an alkaline solution at 80 °C and fractionated by size exclusion chromatography. Its protein binding capacity was evaluated by native gel electrophoresis. It was found that the two highest molecular weight fractions (438– > 14,320 kDa) were enriched with NulO and sulfate-containing glycoconjugates. The enriched fractions can strongly bind the two histones involved in sepsis and a model protein used for purification by heparin-column. These findings demonstrated possibilities for the application of the extracted EPS and open up a novel strategy for resource recovery. Key points: • High MW EPS from seawater-adapted AGS are dominant with sulfated groups and NulOs • Fifty-eight percent of the EPS is high MW of 68–14,320 kDa • EPS and its fractions can bind histones and fibroblast growth factor 2 Graphical Abstract: [Figure not available: see fulltext.]","EPS; Granular sludge; Nonulosonic acids; Protein binding; Sulfated glycoconjugates","en","journal article","","","","","","","","2024-07-17","","","BT/Environmental Biotechnology","","",""
"uuid:5611465d-9d0c-404e-afcd-587e9631e06f","http://resolver.tudelft.nl/uuid:5611465d-9d0c-404e-afcd-587e9631e06f","Engineering Saccharomyces cerevisiae for fast vitamin-independent aerobic growth","Ehrmann, Anja K. (Technical University of Denmark); Wronska, A.K. (TU Delft BT/Industriele Microbiologie); Perli, T. (TU Delft BT/Industriele Microbiologie); de Hulster, A.F. (TU Delft BT/Industriele Microbiologie); Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Carqueija Cardoso, D.C. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2024","Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S. cerevisiae CEN.PK113-7D in the absence of pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and/or biotin were corrected by single or combined overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and Cyberlindnera fabianii BIO1, respectively. Several strategies were explored to improve growth of S. cerevisiae CEN.PK113-7D in thiamine-free medium. Overexpression of ScTHI4 and/or ScTHI5 enabled thiamine-independent growth at 83% of the maximum specific growth rate of the reference strain in vitamin-supplemented medium. Combined overexpression of seven native S. cerevisiae genes and CfBIO1 enabled a maximum specific growth rate of 0.33 ± 0.01 h−1 in vitamin-free synthetic medium. This growth rate was only 17 % lower than that of a congenic reference strain in vitamin-supplemented medium. Physiological parameters of the engineered vitamin-independent strain in aerobic glucose-limited chemostat cultures (dilution rate 0.10 h−1) grown on vitamin-free synthetic medium were similar to those of similar cultures of the parental strain grown on vitamin-supplemented medium. Transcriptome analysis revealed only few differences in gene expression between these cultures, which primarily involved genes with roles in Class-B vitamin metabolism. These results pave the way for development of fast-growing vitamin-independent industrial strains of S. cerevisiae.","","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:bf89da1d-0ef5-4ec6-b1d0-e39d4bfe3a99","http://resolver.tudelft.nl/uuid:bf89da1d-0ef5-4ec6-b1d0-e39d4bfe3a99","DeltaDTM: A global coastal digital terrain model","Pronk, M.J. (TU Delft Urban Data Science; Deltares); Hooijer, Aljosja (Deltares); Eilander, Dirk (Deltares); Haag, Arjen (Deltares); de Jong, Tjalling (Deltares); Vousdoukas, Michalis (University of the Aegean, Mytilene); Vernimmen, Ronald (Data for Sustainability, Axel); Ledoux, H. (TU Delft Urban Data Science); Eleveld, M.A. (TU Delft Mathematical Geodesy and Positioning; Deltares)","","2024","Coastal elevation data are essential for a wide variety of applications, such as coastal management, flood modelling, and adaptation planning. Low-lying coastal areas (found below 10 m +Mean Sea Level (MSL)) are at risk of future extreme water levels, subsidence and changing extreme weather patterns. However, current freely available elevation datasets are not sufficiently accurate to model these risks. We present DeltaDTM, a global coastal Digital Terrain Model (DTM) available in the public domain, with a horizontal spatial resolution of 1 arcsecond (∼30 m) and a vertical mean absolute error (MAE) of 0.45 m overall. DeltaDTM corrects CopernicusDEM with spaceborne lidar from the ICESat-2 and GEDI missions. Specifically, we correct the elevation bias in CopernicusDEM, apply filters to remove non-terrain cells, and fill the gaps using interpolation. Notably, our classification approach produces more accurate results than regression methods recently used by others to correct DEMs, that achieve an overall MAE of 0.72 m at best. We conclude that DeltaDTM will be a valuable resource for coastal flood impact modelling and other applications.","energy access; energy security; power distribution","en","journal article","","","","","","","","","","","Urban Data Science","","",""
"uuid:0eca0fc2-807b-45e8-8fb6-046e9163a8c1","http://resolver.tudelft.nl/uuid:0eca0fc2-807b-45e8-8fb6-046e9163a8c1","Demystifying polyphosphate-accumulating organisms relevant to wastewater treatment: A review of their phylogeny, metabolism, and detection","Ruiz Hadad, L. (King Abdullah University of Science and Technology); Ali, Muhammad (Trinity College Dublin); Pronk, M. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Saikaly, Pascal E. (King Abdullah University of Science and Technology)","","2024","Currently, the most cost-effective and efficient method for phosphorus (P) removal from wastewater is enhanced biological P removal (EPBR) via polyphosphate-accumulating organisms (PAOs). This study integrates a literature review with genomic analysis to uncover the phylogenetic and metabolic diversity of the relevant PAOs for wastewater treatment. The findings highlight significant differences in the metabolic capabilities of PAOs relevant to wastewater treatment. Notably, Candidatus Dechloromonas and Candidatus Accumulibacter can synthesize polyhydroxyalkanoates, possess specific enzymes for ATP production from polyphosphate, and have electrochemical transporters for acetate and C4-dicarboxylates. In contrast, Tetrasphaera, Candidatus Phosphoribacter, Knoellia, and Phycicoccus possess PolyP-glucokinase and electrochemical transporters for sugars/amino acids. Additionally, this review explores various detection methods for polyphosphate and PAOs in activated sludge wastewater treatment plants. Notably, FISH-Raman spectroscopy emerges as one of the most advanced detection techniques. Overall, this review provides critical insights into PAO research, underscoring the need for enhanced strategies in biological phosphorus removal.","Ca. accumulibacter; Ca. phosphoribacter; Enhanced biological phosphorus removal (EBPR); Knoellia; Phycicoccus; Polyphosphate-accumulating organisms (PAOs); Tetrasphaera","en","review","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:6e6c08cb-2258-4157-8179-1641372ac894","http://resolver.tudelft.nl/uuid:6e6c08cb-2258-4157-8179-1641372ac894","The water-soluble fraction of extracellular polymeric substances from a resource recovery demonstration plant: characterization and potential application as an adhesive","Chen, Le Min (Student TU Delft); Erol, Özlem (Universiteit Leiden); Choi, Young Hae (Universiteit Leiden); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Lin, Y. (TU Delft BT/Environmental Biotechnology)","","2024","Currently, there is a growing interest in transforming wastewater treatment plants (WWTPs) into resource recovery plants. Microorganisms in aerobic granular sludge produce extracellular polymeric substances (EPS), which are considered sustainable resources to be extracted and can be used in diverse applications. Exploring applications in other high-value materials, such as adhesives, will not only enhance the valorization potential of the EPS but also promote resource recovery. This study aimed to characterize a water-soluble fraction extracted from the EPS collected at the demonstration plant in the Netherlands based on its chemical composition (amino acids, sugar, and fatty acids) and propose a proof-of-concept for its use as an adhesive. This fraction comprises a mixture of biomolecules, such as proteins (26.6 ± 0.3%), sugars (21.8 ± 0.2%), and fatty acids (0.9%). The water-soluble fraction exhibited shear strength reaching 36–51 kPa across a pH range of 2–10 without additional chemical treatment, suggesting a potential application as an adhesive. The findings from this study provide insights into the concept of resource recovery and the valorization of excess sludge at WWTPs.","","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:30d19c75-2c21-4255-8591-5946428644c9","http://resolver.tudelft.nl/uuid:30d19c75-2c21-4255-8591-5946428644c9","Quantification and mitigation of byproduct formation by low-glycerol-producing Saccharomyces cerevisiae strains containing Calvin-cycle enzymes","van Aalst, A.C.A. (TU Delft BT/Industriele Microbiologie); Jansen, Mickel L.A. (DSM); Mans, R. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2023","Background: Anaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol production processes are not constant, performance of engineered strains was studied in slow-growing cultures. Results: In slow-growing anaerobic chemostat cultures (D = 0.05 h −1), an engineered PRK/RuBisCO strain produced 80-fold more acetaldehyde and 30-fold more acetate than a reference strain. This observation suggested an imbalance between in vivo activities of PRK/RuBisCO and formation of NADH in biosynthesis. Lowering the copy number of the RuBisCO-encoding cbbm expression cassette from 15 to 2 reduced acetaldehyde and acetate production by 67% and 29%, respectively. Additional C-terminal fusion of a 19-amino-acid tag to PRK reduced its protein level by 13-fold while acetaldehyde and acetate production decreased by 94% and 61%, respectively, relative to the 15 × cbbm strain. These modifications did not affect glycerol production at 0.05 h −1 but caused a 4.6 fold higher glycerol production per amount of biomass in fast-growing (0.29 h −1) anaerobic batch cultures than observed for the 15 × cbbm strain. In another strategy, the promoter of ANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2 × cbbm strain. At 0.05 h −1, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15 × cbbm strain, without affecting glycerol production. The maximum growth rate of the resulting strain equalled that of the reference strain, while its glycerol production was 72% lower. Conclusions: Acetaldehyde and acetate formation by slow-growing cultures of engineered S. cerevisiae strains carrying a PRK/RuBisCO bypass of yeast glycolysis was attributed to an in vivo overcapacity of PRK and RuBisCO. Reducing the capacity of PRK and/or RuBisCO was shown to mitigate this undesirable byproduct formation. Use of a growth rate-dependent promoter for PRK expression highlighted the potential of modulating gene expression in engineered strains to respond to growth-rate dynamics in industrial batch processes.","Chemostat; Acetaldehyde; Acetate; Phosphoribulokinase; RuBisCO; Anaerobic; Ethanol; Redox","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:7e05203c-f094-4cc1-8a86-206467b4f811","http://resolver.tudelft.nl/uuid:7e05203c-f094-4cc1-8a86-206467b4f811","Prioritization of micropollutants based on removal effort in drinking water purification treatment","Pronk, Tessa E. (KWR Water Research Institute); Fischer, A. (TU Delft Sanitary Engineering; Evides); van den Berg, Annemijne E.T. (Universiteit Utrecht); Hofman, Roberta C.H.M. (KWR Water Research Institute; Wageningen University & Research; Hogeschool Utrecht)","","2023","A main focus of water managers with regard to micropollutants is the protection of aquatic ecology. However, micropollutants also have the potential to affect the production of clean drinking water. In this paper, we propose to consider the removal effort when assessing micro-pollutants with an ‘Effort Index’ (EI). Assessments using the EI show which micropollutants need more extensive monitoring or abatement because of their difficulty to be removed using low-effort water purification treatment techniques. For water containing mixtures of micro-pollutants, the averaged EI values can indicate overall water quality. Data on the removal by different purification treatment techniques are not necessarily available for all micropollutants. Therefore, a set of data-driven indicative removal rules is derived to quantify the relation between micropollutant properties and different drinking water treatment techniques. The indicative removal rules provide a rough indication of removability. As an illustration, the water quality of the river Rhine is evaluated between 2000 and 2018. The EI value shows that the Rhine contains increasingly more difficult-to-remove micropollutants. In total, 18 of those are labeled as particularly difficult-to-remove chemicals. These micropollutants are suggested as candidates for abatement to lower the required effort in drinking water production.","micropollutants; model; prioritization; purification treatment; removal; water quality","en","journal article","","","","","","","","","","","Sanitary Engineering","","",""
"uuid:e139a76b-9fcf-4527-b49d-2a1b4fcc61ba","http://resolver.tudelft.nl/uuid:e139a76b-9fcf-4527-b49d-2a1b4fcc61ba","Optimizing the balance between heterologous acetate- and CO2-reduction pathways in anaerobic cultures of Saccharomyces cerevisiae strains engineered for low-glycerol production","van Aalst, Aafke C.A. (Student TU Delft); Geraats, Ellen H. (Student TU Delft); Martini, M.L.A. (DSM); Mans, R. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2023","In anaerobic Saccharomyces cerevisiae cultures, NADH (reduced form of nicotinamide adenine dinucleotide)-cofactor balancing by glycerol formation constrains ethanol yields. Introduction of an acetate-to-ethanol reduction pathway based on heterologous acetylating acetaldehyde dehydrogenase (A-ALD) can replace glycerol formation as 'redox-sink' and improve ethanol yields in acetate-containing media. Acetate concentrations in feedstock for first-generation bioethanol production are, however, insufficient to completely replace glycerol formation. An alternative glycerol-reduction strategy bypasses the oxidative reaction in glycolysis by introducing phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). For optimal performance in industrial settings, yeast strains should ideally first fully convert acetate and, subsequently, continue low-glycerol fermentation via the PRK-RuBisCO pathway. However, anaerobic batch cultures of a strain carrying both pathways showed inferior acetate reduction relative to a strain expressing only the A-ALD pathway. Complete A-ALD-mediated acetate reduction by a dual-pathway strain, grown anaerobically on 50 g L-1 glucose and 5 mmol L-1 acetate, was achieved upon reducing PRK abundance by a C-terminal extension of its amino acid sequence. Yields of glycerol and ethanol on glucose were 55% lower and 6% higher, respectively, than those of a nonengineered reference strain. The negative impact of the PRK-RuBisCO pathway on acetate reduction was attributed to sensitivity of the reversible A-ALD reaction to intracellular acetaldehyde concentrations.","acetate; biofuels; fermentation; NADH; redox cofactor balance","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:f51d51e1-7a30-4b5b-b905-acfe3e8740f1","http://resolver.tudelft.nl/uuid:f51d51e1-7a30-4b5b-b905-acfe3e8740f1","Impact of the anaerobic feeding mode on substrate distribution in aerobic granular sludge","Haaksman, V.A. (TU Delft BT/Environmental Biotechnology); Schouteren, M. (Student TU Delft); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV)","","2023","There is a growing interest to implement aerobic granular sludge (AGS) in existing conventional activated sludge (CAS) systems with a continuous flow-through configuration. The mode of anaerobic contact of raw sewage with the sludge is an important aspect in the adaptation of CAS systems to accommodate AGS. It remains unclear how the distribution of substrate over the sludge by a conventional anaerobic selector compares to the distribution via bottom-feeding applied in sequencing batch reactors (SBRs). This study investigated the effect of the anaerobic contact mode on the substrate (and storage) distribution by operating two lab-scale SBRs; one with the traditional bottom-feeding through a settled sludge bed similar to full-scale AGS systems, and one where the synthetic wastewater was fed as a pulse at the start of the anaerobic phase while the reactor was mixed through sparging of nitrogen gas (mimicking a plug-flow anaerobic selector in continuous flow-through systems). The distribution of the substrate over the sludge particle population was quantified via PHA analysis, combined with the obtained granule size distribution. Bottom-feeding was found to primarily direct substrate towards the large granular size classes (i.e. large volume and close to the bottom), while completely mixed pulse-feeding gives a more equal distribution of substrate over all granule sizes (i.e. surface area dependant). The anaerobic contact mode directly controls the substrate distribution over the different granule sizes, irrespective of the solids retention time of a granule as an entity. Preferential feeding of the larger granules will enhance and stabilise the granulation compared to pulse-feeding, certainly under less advantageous conditions imposed by real sewage.","aerobic granular sludge; anaerobic selector; bottom-feeding; continuous flow; PHA; pulse-feeding","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:5e049878-053c-4f59-b0db-cec1be8d7501","http://resolver.tudelft.nl/uuid:5e049878-053c-4f59-b0db-cec1be8d7501","Enhancing phosphorus removal of photogranules by incorporating polyphosphate accumulating organisms","Trebuch, Lukas M. (Netherlands Institute of Ecology; Wageningen University & Research); Sohier, Jasper (Netherlands Institute of Ecology); Altenburg, Sido (Netherlands Institute of Ecology); Oyserman, Ben O. (Netherlands Institute of Ecology; Wageningen University & Research); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); Janssen, Marcel (Wageningen University & Research); Vet, Louise E.M. (Netherlands Institute of Ecology); Wijffels, René H. (Wageningen University & Research; Nord University); Fernandes, Tânia V. (Netherlands Institute of Ecology)","","2023","Photogranules are a novel wastewater treatment technology that can utilize the sun's energy to treat water with lower energy input and have great potential for nutrient recovery applications. They have been proven to efficiently remove nitrogen and carbon but show lower conversion rates for phosphorus compared to established treatment systems, such as aerobic granular sludge. In this study, we successfully introduced polyphosphate accumulating organisms (PAOs) to an established photogranular culture. We operated photobioreactors in sequencing batch mode with six cycles per day and alternating anaerobic (dark) and aerobic (light) phases. We were able to increase phosphorus removal/recovery by 6 times from 5.4 to 30 mg/L/d while maintaining similar nitrogen and carbon removal compared to photogranules without PAOs. To maintain PAOs activity, alternating anaerobic feast and aerobic famine conditions were required. In future applications, where aerobic conditions are dependent on in-situ oxygenation via photosynthesis, the process will rely on sunlight availability. Therefore, we investigated the feasibility of the process under diurnal cycles with a 12-h anaerobic phase during nighttime and six short cycles during the 12 h daytime. The 12-h anaerobic phase had no adverse effect on the PAOs and phototrophs. Due to the extension of one anaerobic phase to 12 h the six aerobic phases were shortened by 47% and consequently decreased the light hours per day. This resulted in a decrease of phototrophs, which reduced nitrogen removal and biomass productivity up to 30%. Finally, we discuss and suggest strategies to apply PAO-enriched photogranules at large-scale.","Aerobic granular sludge; Enhanced biological phosphorus removal (EBPR); Microalgal-bacterial granules; Microbial ecology; PhotoEBPR; Wastewater","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:df930283-d6f3-45cb-8bc4-7f9905f5f9e0","http://resolver.tudelft.nl/uuid:df930283-d6f3-45cb-8bc4-7f9905f5f9e0","Aerobic granular sludge","Pronk, M. (TU Delft BT/Environmental Biotechnology); van Dijk, E.J.H. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology; University of Queensland)","Chen, Guanghao (editor); van Loosdrecht, Mark C.M. (editor); Ekama, George A. (editor); Brdjanovic, Damir (editor)","2023","","","en","book chapter","International Water Association (IWA)","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:ae94ee8d-0acb-4803-925f-547f3d263d07","http://resolver.tudelft.nl/uuid:ae94ee8d-0acb-4803-925f-547f3d263d07","Metaproteomics, metagenomics and 16S rRNA sequencing provide different perspectives on the aerobic granular sludge microbiome","Kleikamp, H.B.C. (TU Delft BT/Environmental Biotechnology); Grouzdev, Denis (SciBear OU, Tallinn); - Schaasberg, P. (TU Delft BT/Environmental Biotechnology); van Valderen, R.D. (TU Delft BT/Bioprocess Engineering); van der Zwaan, R. (TU Delft BT/Environmental Biotechnology); van de Wijgaart, R. (TU Delft BT/Environmental Biotechnology); Lin, Y. (TU Delft Environmental Fluid Mechanics); Abbas, B.A. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pabst, Martin (TU Delft BT/Environmental Biotechnology)","","2023","The tremendous progress in sequencing technologies has made DNA sequencing routine for microbiome studies. Additionally, advances in mass spectrometric techniques have extended conventional proteomics into the field of microbial ecology. However, systematic studies that provide a better understanding of the complementary nature of these 'omics' approaches, particularly for complex environments such as wastewater treatment sludge, are urgently needed. Here, we describe a comparative metaomics study on aerobic granular sludge from three different wastewater treatment plants. For this, we employed metaproteomics, whole metagenome, and 16S rRNA amplicon sequencing to study the same granule material with uniform size. We furthermore compare the taxonomic profiles using the Genome Taxonomy Database (GTDB) to enhance the comparability between the different approaches. Though the major taxonomies were consistently identified in the different aerobic granular sludge samples, the taxonomic composition obtained by the different omics techniques varied significantly at the lower taxonomic levels, which impacts the interpretation of the nutrient removal processes. Nevertheless, as demonstrated by metaproteomics, the genera that were consistently identified in all techniques cover the majority of the protein biomass. The established metaomics data and the contig classification pipeline are publicly available, which provides a valuable resource for further studies on metabolic processes in aerobic granular sludge.","16S rRNA amplicon sequencing; Aerobic granular sludge; metagenomics; Metaproteomics; Wastewater treatment","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:00e159bd-d6d2-4d56-a036-471f22ca282c","http://resolver.tudelft.nl/uuid:00e159bd-d6d2-4d56-a036-471f22ca282c","Aerobic granular sludge phosphate removal using glucose","Elahinik, A. (TU Delft BT/Environmental Biotechnology); Li, L. (TU Delft BT/Environmental Biotechnology); Pabst, Martin (TU Delft BT/Environmental Biotechnology); Abbas, B.A. (TU Delft BT/Environmental Biotechnology); Xevgenos, Dimitris (TU Delft Energie and Industrie); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV)","","2023","Enhanced biological phosphate removal and aerobic sludge granulation are commonly studied with fatty acids as substrate. Fermentative substrates such as glucose have received limited attention. In this work, glucose conversion by aerobic granular sludge and its impact on phosphate removal was studied. Long-term stable phosphate removal and successful granulation were achieved. Glucose was rapidly taken up (273 mg/gVSS/h) at the start of the anaerobic phase, while phosphate was released during the full anaerobic phase. Some lactate was produced during glucose consumption, which was anaerobically consumed once glucose was depleted. The phosphate release appeared to be directly proportional to the uptake of lactate. The ratio of phosphorus released to glucose carbon taken up over the full anaerobic phase was 0.25 Pmol/Cmol. Along with glucose and lactate uptake in the anaerobic phase, poly‑hydroxy-alkanoates and glycogen storage were observed. There was a linear correlation between glucose consumption and lactate formation. While lactate accounted for approximately 89 % of the observed products in the bulk liquid, minor quantities of formate (5 %), propionate (4 %), and acetate (3 %) were also detected (mass fraction). Formate was not consumed anaerobically. Quantitative fluorescence in-situ hybridization (qFISH) revealed that polyphosphate accumulating organisms (PAO) accounted for 61 ± 15 % of the total biovolume. Metagenome evaluation of the biomass indicated a high abundance of Micropruina and Ca. Accumulibacter in the system, which was in accordance with the microscopic observations and the protein mass fraction from metaproteome analysis. Anaerobic conversions were evaluated based on theoretical ATP balances to provide the substrate distribution amongst the dominant genera. This research shows that aerobic granular sludge technology can be applied to glucose-containing effluents and that glucose is a suitable substrate for achieving phosphate removal. The results also show that for fermentable substrates a microbial community consisting of fermentative organisms and PAO develop.","Aerobic granular sludge; Ca. Accumulibacter; Enhanced biological phosphorus removal; Fermentative GAO; Micropruina; Proteomics","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:03e6f046-787e-44f3-8355-d13617122d47","http://resolver.tudelft.nl/uuid:03e6f046-787e-44f3-8355-d13617122d47","Co-cultivation of Saccharomyces cerevisiae strains combines advantages of different metabolic engineering strategies for improved ethanol yield","van Aalst, A.C.A. (TU Delft BT/Industriele Microbiologie); van der Meulen, Igor S. (Student TU Delft); Jansen, Mickel L.A. (DSM); Mans, R. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2023","Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions. Functional expression of heterologous phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes enables yeast cells to couple a net oxidation of NADH to the conversion of glucose to ethanol. In another strategy, NADH-dependent reduction of exogenous acetate to ethanol is enabled by introduction of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD). This study explores potential advantages of co-cultivating engineered PRK-RuBisCO-based and A-ALD-based strains in anaerobic bioreactor batch cultures. Co-cultivation of these strains, which in monocultures showed reduced glycerol yields and improved ethanol yields, strongly reduced the formation of acetaldehyde and acetate, two byproducts that were formed in anaerobic monocultures of a PRK-RuBisCO-based strain. In addition, co-cultures on medium with low acetate-to-glucose ratios that mimicked those in industrial feedstocks completely removed acetate from the medium. Kinetics of co-cultivation processes and glycerol production could be optimized by tuning the relative inoculum sizes of the two strains. Co-cultivation of a PRK-RuBisCO strain with a Δgpd1 Δgpd2 A-ALD strain, which was unable to grow in the absence of acetate and evolved for faster anaerobic growth in acetate-supplemented batch cultures, further reduced glycerol formation but led to extended fermentation times. These results demonstrate the potential of using defined consortia of engineered S. cerevisiae strains for high-yield, minimal-waste ethanol production.","Acetate reduction; Bioethanol; Co-cultures; PRK-RuBisCO; Redox-engineering; Synthetic microbial consortia","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:ccba0671-1f89-490b-8e3d-05a3d0752d86","http://resolver.tudelft.nl/uuid:ccba0671-1f89-490b-8e3d-05a3d0752d86","Alterations of Glycan Composition in Aerobic Granular Sludge during the Adaptation to Seawater Conditions","Chen, L.M. (TU Delft BT/Environmental Biotechnology); Keisham, Sunanda (National Institute of Advanced Industrial Science and Technology (AIST)); Tateno, Hiroaki (National Institute of Advanced Industrial Science and Technology (AIST)); van Ede, J.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Lin, Y. (TU Delft Environmental Fluid Mechanics)","","2023","Bacteria can synthesize a diverse array of glycans, being found attached to proteins and lipids or as loosely associated polysaccharides to the cells. The major challenge in glycan analysis in environmental samples lies in developing high-throughput and comprehensive characterization methodologies to elucidate the structure and monitor the change of the glycan profile, especially in protein glycosylation. To this end, in the current research, the dynamic change of the glycan profile of a few extracellular polymeric substance (EPS) samples was investigated by high-throughput lectin microarray and mass spectrometry, as well as sialylation and sulfation analysis. Those EPS were extracted from aerobic granular sludge collected at different stages during its adaptation to the seawater condition. It was found that there were glycoproteins in all of the EPS samples. In response to the exposure to seawater, the amount of glycoproteins and their glycan diversity displayed an increase during adaptation, followed by a decrease once the granules reached a stable state of adaptation. Information generated sheds light on the approaches to identify and monitor the diversity and dynamic alteration of the glycan profile of the EPS in response to environmental stimuli.","aerobic granular sludge; extracellular polymeric substances; glycans; glycoproteins; lectin microarray","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:566d08f6-6e84-4b4f-9fe5-07282756d99d","http://resolver.tudelft.nl/uuid:566d08f6-6e84-4b4f-9fe5-07282756d99d","Performance Evaluation of a Pilot-Scale Aerobic Granular Sludge Integrated with Gravity-Driven Membrane System Treating Domestic Wastewater","Ali, Muhammad (Trinity College Dublin; King Abdullah University of Science and Technology); Singh, Yogesh (King Abdullah University of Science and Technology); Fortunato, Luca (King Abdullah University of Science and Technology); Rehman, Zahid Ur (King Abdullah University of Science and Technology); Manjunath, Sarvajith (King Abdullah University of Science and Technology); Vrouwenvelder, J.S. (King Abdullah University of Science and Technology); Pronk, M. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Saikaly, Pascal E. (King Abdullah University of Science and Technology)","","2023","This study describes a novel integration of aerobic granular sludge (AGS) with a gravity-driven membrane (GDM) system at a pilot scale with a treatment capacity of approximately 150 L per day to treat raw domestic wastewater. The treatment performance and energy consumption of the AGS-GDM system were compared to the neighboring full-scale aerobic membrane bioreactor (AeMBR), treating the same wastewater at about 4000(±500) m3 per day. The AGS-GDM system demonstrated superior nutrient (nitrogen and phosphorus) removal as compared to the AeMBR. The GDM unit was continuously supplied with AGS-treated effluent. The GDM unit started with high [ >20 L per m2 per h (LMH) ] flux, which gradually declined. The flux remained quite stable after 15 days reaching 3 LMH after 35 days without any physical or chemical cleaning. Our results suggest that AGS-GDM is a viable technology for decentralized wastewater treatment and reuse in water-scarce regions. The AGS-GDM could easily replace conventional AeMBR technology in the wastewater treatment and reclamation market.","aerobic granular sludge; decentralized wastewater treatment; gravity-driven membrane; water reuse","en","journal article","","","","","","Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.","","2023-12-20","","","BT/Environmental Biotechnology","","",""
"uuid:19d18068-24ed-4a73-9a2f-843d1b2faefe","http://resolver.tudelft.nl/uuid:19d18068-24ed-4a73-9a2f-843d1b2faefe","Sialylation and Sulfation of Anionic Glycoconjugates Are Common in the Extracellular Polymeric Substances of Both Aerobic and Anaerobic Granular Sludges","Chen, L.M. (TU Delft BT/Environmental Biotechnology); de Bruin, S. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); Sousa, Diana Z. (Wageningen University & Research); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Lin, Y. (TU Delft BT/Environmental Biotechnology)","","2023","Anaerobic and aerobic granular sludge processes are widely applied in wastewater treatment. In these systems, microorganisms grow in dense aggregates due to the production of extracellular polymeric substances (EPS). This study investigates the sialylation and sulfation of anionic glyconconjugates in anaerobic and aerobic granular sludges collected from full-scale wastewater treatment processes. Size exclusion chromatography revealed a wide molecular weight distribution (3.5 to >5500 kDa) of the alkaline-extracted EPS. The high-molecular weight fraction (>5500 kDa), comprising 16.9-27.4% of EPS, was dominant with glycoconjugates. Mass spectrometry analysis and quantification assays identified nonulosonic acids (NulOs, e.g., bacterial sialic acids) and sulfated groups contributing to the negative charge in all EPS fractions. NulOs were predominantly present in the high-molecular weight fraction (47.2-84.3% of all detected NulOs), while sulfated glycoconjugates were distributed across the molecular weight fractions. Microorganisms, closely related to genera found in the granular sludge communities, contained genes responsible for NulO and sulfate group synthesis or transfer. The similar distribution patterns of sialylation and sulfation of the anionic glycoconjugates in the EPS samples indicate that these two glycoconjugate modifications commonly occur in the EPS of aerobic and anaerobic granular sludges.","biofilm; EPS; glycoconjugates; mass spectrometry; nonulosonic acids; size exclusion chromatography","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:78d8b7b9-1d1f-434e-a22a-98f15d52eb33","http://resolver.tudelft.nl/uuid:78d8b7b9-1d1f-434e-a22a-98f15d52eb33","Respiratory reoxidation of NADH is a key contributor to high oxygen requirements of oxygen-limited cultures of Ogataea parapolymorpha","Dekker, W.J.C. (TU Delft BT/Industriele Microbiologie); Jürgens, H. (TU Delft BT/Industriele Microbiologie); Ortiz Merino, R.A. (TU Delft BT/Industriele Microbiologie); Mooiman, C. (TU Delft BT/Bioprocess Engineering); van den Berg, Remon (Student TU Delft); Kaljouw, Astrid (Student TU Delft); Mans, R. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2022","While thermotolerance is an attractive trait for yeasts used in industrial ethanol production, oxygen requirements of known thermotolerant species are incompatible with process requirements. Analysis of oxygen-sufficient and oxygen-limited chemostat cultures of the facultatively fermentative, thermotolerant species Ogataea parapolymorpha showed its minimum oxygen requirements to be an order of magnitude larger than those reported for the thermotolerant yeast Kluyveromyces marxianus. High oxygen requirements of O. parapolymorpha coincided with a near absence of glycerol, a key NADH/NAD+ redox-cofactor-balancing product in many other yeasts, in oxygen-limited cultures. Genome analysis indicated absence of orthologs of the Saccharomyces cerevisiae glycerol-3-phosphate-phosphatase genes GPP1 and GPP2. Co-feeding of acetoin, whose conversion to 2,3-butanediol enables reoxidation of cytosolic NADH, supported a 2.5-fold increase of the biomass concentration in oxygen-limited cultures. An O. parapolymorpha strain in which key genes involved in mitochondrial reoxidation of NADH were inactivated did produce glycerol, but transcriptome analysis did not reveal a clear candidate for a responsible phosphatase. Expression of S. cerevisiae GPD2, which encodes NAD+-dependent glycerol-3-phosphate dehydrogenase, and GPP1 supported increased glycerol production by oxygen-limited chemostat cultures of O. parapolymorpha. These results identify dependence on respiration for NADH reoxidation as a key contributor to unexpectedly high oxygen requirements of O. parapolymorpha.","Ogataea parapolymorpha; anaerobic growth; Custers effect; genome sequence; glycerol metabolism; thermotolerance","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:e859be6b-f1d5-40e5-a601-552f8968bce8","http://resolver.tudelft.nl/uuid:e859be6b-f1d5-40e5-a601-552f8968bce8","Metagenomic profiling and transfer dynamics of antibiotic resistance determinants in a full-scale granular sludge wastewater treatment plant","Calderon Franco, D. (TU Delft BT/Environmental Biotechnology); Sarelse, R.G. (Student TU Delft); Christou, S. (Student TU Delft); Pronk, M. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Abeel, T.E.P.M.F. (TU Delft Pattern Recognition and Bioinformatics); Weissbrodt, D.G. (TU Delft BT/Environmental Biotechnology)","","2022","In the One Health context, wastewater treatment plants (WWTPs) are central to safeguarding water resources. Nonetheless, many questions remain about their effectiveness in preventing antimicrobial resistance (AMR) dissemination. Most surveillance studies monitor the levels and removal of selected antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in intracellular DNA (iDNA) extracted from WWTP influents and effluents. The role of extracellular free DNA (exDNA) in wastewater is mostly overlooked. This study analyzed the transfer of ARGs and MGEs in a full-scale Nereda® reactor removing nutrients with aerobic granular sludge. We tracked the composition and fate of the iDNA and exDNA pools of influent, sludge, and effluent samples. Metagenomics was used to profile the microbiome, resistome, and mobilome signatures of iDNA and exDNA extracts. Selected ARGs and MGEs were analyzed by qPCR. From 2,840 ARGs identified, the genes arr-3 (2%), tetC (1.6%), sul1 (1.5%), oqxB (1.2%), and aph(3"")-Ib (1.2%) were the most abundant among all sampling points and bioaggregates. Pseudomonas, Acinetobacter, Aeromonas, Acidovorax, Rhodoferax, and Streptomyces populations were the main potential hosts of ARGs in the sludge. In the effluent, 478 resistance determinants were detected, of which 89% were from exDNA potentially released by cell lysis during aeration in the reactor. MGEs and multiple ARGs were co-localized on the same extracellular genetic contigs. Total intracellular ARGs decreased 3-42% due to wastewater treatment. However, the ermB and sul1 genes increased by 2 and 1 log gene copies mL−1, respectively, in exDNA from influent to effluent. The exDNA fractions need to be considered in AMR surveillance, risk assessment, and mitigation strategies.","Antibiotic resistance; Metagenomics; wastewater; mobile genetic elements; quantitative PCR","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:8ea7e615-0831-42f6-86a1-c244cfeea0f6","http://resolver.tudelft.nl/uuid:8ea7e615-0831-42f6-86a1-c244cfeea0f6","Reusing Timber Formwork in Building Construction: Testing, Redesign, and Socio-Economic Reflection","Pronk, Arno (Eindhoven University of Technology); Brancart, S. (TU Delft Structural Design & Mechanics); Sanders, Fred (CPONH NGO)","","2022","In 2018, the construction sector was responsible for 39% of the worldwide energy and process-related carbon dioxide emissions (Global Alliance for Buildings and Construction et al., 2019). This is partly due to the embodied carbon, which represents the carbon emissions related to building construction and material production (LETI, 2020). While zero energy buildings and zero energy renovations start to get the operational carbon down, the circular economy aims to do this by closing material loops and stimulating the reuse of discarded materials in building construction (Ellen McArthur Foundation et al., 2015). Although it is not a new phenomenon, material reuse does require a substantially different approach and is at this point not yet common in the building industry. This is especially true for load-bearing components. This article presents a pilot project for the reuse of discarded timber formwork for the construction of the façade and (load-bearing) substructure of a new house. Through this pilot case and by reflecting on a series of similar cases, it studies the remaining challenges for material reuse but also proposes and assesses redesign strategies that will allow upscaling the reuse of timber formwork. The project shows that although waste, material, and money can be saved by using reclaimed materials, it does complicate the design and construction process and, as such, does not necessarily reduce the total project budget. Moreover, for reuse to become a current practice, new design approaches and collaborations will need to be established. Finally, socio-economic factors must be considered to increase the acceptance of reclaimed materials in new building construction.","circular economy; circular housing; CO2 reduction; material reuse; resource efficiency; sustainable architecture","en","journal article","","","","","","","","","","","Structural Design & Mechanics","","",""
"uuid:b742e795-fce9-4fc2-bcaa-8e11666df89a","http://resolver.tudelft.nl/uuid:b742e795-fce9-4fc2-bcaa-8e11666df89a","Glycerol conversion by Aerobic Granular Sludge","Elahinik, A. (TU Delft BT/Environmental Biotechnology); Haarsma, M.N. (Royal HaskoningDHV); Abbas, B.A. (TU Delft BT/Environmental Biotechnology); Pabst, Martin (TU Delft BT/Environmental Biotechnology); Xevgenos, Dimitris (TU Delft BT/Biotechnology and Society); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology)","","2022","Glycerol is abundantly present in wastewater from industries such as biodiesel production facilities. Glycerol is also a potential carbon source for microbes that are involved in wastewater nutrient removal processes. The conversion of glycerol in biological phosphorus removal of aerobic granular sludge processes has not been explored to date. The current study describes glycerol utilization by aerobic granular sludge and enhanced biological phosphorus removal (EBPR). Robust granules with good phosphorus removal capabilities were formed in an aerobic granular sludge sequencing batch reactor fed with glycerol. The interaction between the fermentative conversion of glycerol and product uptake by polyphosphate accumulating organisms (PAO) was studied using stoichiometric and microbial community analysis. Metagenomic, metaproteomic and microscopic analysis identified a community dominated by Actinobacteria (Tessaracoccus and Micropruina) and a typical PAO known as Ca. Accumulibacter. Glycerol uptake facilitator (glpF) and glycerol kinase (glpK), two proteins involved in the transport of glycerol into the cellular metabolism, were only observed in the genome of the Actinobacteria. The anaerobic conversion appeared to be a combination of a substrate fermentation and product uptake-type reaction. Initially, glycerol fermentation led mainly to the production of 1,3-propanediol (1,3-PDO) which was not taken up under anaerobic conditions. Despite the aerobic conversion of 1,3-PDO stable granulation was observed. Over time, 1,3-PDO production decreased and complete anaerobic COD uptake was observed. The results demonstrate that glycerol-containing wastewater can effectively be treated by the aerobic granular sludge process and that fermentative and polyphosphate accumulating organisms can form a food chain in glycerol-based EBPR processes.","","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:8c06ffb7-ed4b-43bb-ad31-519642ec63d4","http://resolver.tudelft.nl/uuid:8c06ffb7-ed4b-43bb-ad31-519642ec63d4","Inferring the number of floors for residential buildings","Roy, E.I. (Student TU Delft); Pronk, Maarten (Deltares); Agugiaro, G. (TU Delft Urban Data Science); Ledoux, H. (TU Delft Urban Data Science)","","2022","Data on the number of floors is required for several applications, for instance, energy demand estimation, population estimation, and flood response plans. Despite this, open data on the number of floors is very rare, even when a 3D city model is available. In practice, it is most often inferred with a geometric method: elevation data is used to estimate the height of a building, which is divided by an assumed storey height and rounded. However, as we demonstrate in this paper with a large dataset of residential buildings, this method is unreliable: <70% of the buildings have a correct estimate. We demonstrate that other attributes and characteristics of buildings can help us better predict the number of floors. We propose several indicators (e.g. construction year, cadastral attributes, building geometry, and neighbourhood census data), and we present a predictive model that was trained with 172,000 buildings in the Netherlands. Our model achieves an accuracy of 94.5% for residential buildings with five floors or less, which is an improvement of about 25% over the geometric approach. Above five floors, our model has only a slight improvement on the geometric approach (5%). The main culprit is the lack of training data for tall buildings, which is uncommon in the Netherlands.","3D city modelling; floors; buildings; machine learning","en","journal article","","","","","","","","","","","Urban Data Science","","",""
"uuid:afacae91-bafa-4abc-9bec-4392797f0919","http://resolver.tudelft.nl/uuid:afacae91-bafa-4abc-9bec-4392797f0919","Displaying Decimal Numbers","Pronk, C. (TU Delft Software Engineering)","","2022","Over the years engineers have dearched for means to display decimal numbers on measuring equipment. This paper presents an overview.","numeric display technology","en","journal article","","","","","","","","","","","Software Engineering","","",""
"uuid:919245c7-3e45-4119-b5a8-48970cfad9c4","http://resolver.tudelft.nl/uuid:919245c7-3e45-4119-b5a8-48970cfad9c4","The Esiac-10 Algebraic Computer: A theoretical decomposition by the Historic Collection","Pronk, C. (TU Delft Software Engineering); Trimp, P.J. (TU Delft Electronic Instrumentation)","","2022","A description of the working pricciples of the Esiac-10 Algebraic Computer","algebraic compo=uter control engineering","en","journal article","","","","","","","","","","","Software Engineering","","",""
"uuid:a61918b0-4e20-4aca-95bb-f20229f965f7","http://resolver.tudelft.nl/uuid:a61918b0-4e20-4aca-95bb-f20229f965f7","Point clouds and Hydroinformatics","Diaz, Vitali (TU Delft Digital Technologies); Liu, H. (TU Delft GIS Technologie); van Oosterom, P.J.M. (TU Delft Digital Technologies); Meijers, B.M. (TU Delft Digital Technologies); Verbree, E. (TU Delft Digital Technologies); Baart, F. (Deltares); Pronk, M.J. (Deltares); Van Lankveld, T. (Netherlands eScience Center)","","2022","Point cloud is made up of a multitude of three-dimensional (3D) points with one or more attributes attached. Point cloud is the third data paradigm in addition to the well-established object (vector) and gridded (raster) representations, since point cloud data can be directly collected, computed, stored, and analyzed without converting to other types. Modern ways of data acquisition, including laser scanning from airborne, mobile, or static platforms, multi-beam echo-sounding, and dense image matching from photos, generate millions to trillions of 3D points with attached attributes. If the collection is carried out in different periods, one of the essential attributes is precisely time, allowing spatiotemporal analysis to be performed. Its use is widespread in some fields such as metrology and quality inspection, virtual reality, indoor/outdoor navigation, object detection, vegetation monitoring, building modeling, cultural heritage, and diverse visualization applications. There are some examples in fields related to hydroinformatics, mainly related to terrain modeling. Due to its nature of big data, over the past decades, a series of developments have been carried out in the different processing chains for the optimal use of point cloud. This research seeks to introduce the various point cloud developments from which the hydroinformatics community and research could benefit. A review of recent advances is made, mainly including the analysis and visualization of point cloud for dealing with water-related problems. Potential areas of application and development in hydroinformatics are identified. These include, for example, the topics of coastal monitoring, coastal erosion, shallow water assessment, ice sheet change analysis, sea-level rise assessment, monitoring of levels in water bodies, crop and vegetation monitoring, analysis of the effects of groundwater depletion, detail tracing of basins and channels, analysis of floods with detailed terrain models, and drought monitoring in crops and forests. The challenges to overcome and ongoing developments regarding point cloud application in hydroinformatics are also discussed.","","en","conference paper","","","","","","Abstract from EGU General Assembly 2022, Vienna, Austria, 23–27 May 2022","","","","","Digital Technologies","","",""
"uuid:719f0bdd-932b-431f-8b60-0b8504c8426a","http://resolver.tudelft.nl/uuid:719f0bdd-932b-431f-8b60-0b8504c8426a","Pathway engineering strategies for improved product yield in yeast-based industrial ethanol production","van Aalst, A.C.A. (TU Delft BT/Industriele Microbiologie); de Valk, S.C. (TU Delft BT/Industriele Microbiologie); van Gulik, W.M. (TU Delft BT/Industriele Microbiologie); Jansen, Mickel L.A. (DSM); Pronk, J.T. (TU Delft BT/Biotechnologie); Mans, R. (TU Delft BT/Industriele Microbiologie)","","2022","Product yield on carbohydrate feedstocks is a key performance indicator for industrial ethanol production with the yeast Saccharomyces cerevisiae. This paper reviews pathway engineering strategies for improving ethanol yield on glucose and/or sucrose in anaerobic cultures of this yeast by altering the ratio of ethanol production, yeast growth and glycerol formation. Particular attention is paid to strategies aimed at altering energy coupling of alcoholic fermentation and to strategies for altering redox-cofactor coupling in carbon and nitrogen metabolism that aim to reduce or eliminate the role of glycerol formation in anaerobic redox metabolism. In addition to providing an overview of scientific advances we discuss context dependency, theoretical impact and potential for industrial application of different proposed and developed strategies.","Biofuels; Energy metabolism; Metabolic engineering; Redox metabolism; Saccharomyces cerevisiae; Synthetic biology","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:e57cfcea-548b-4082-aa7b-02a6bbf3e4f7","http://resolver.tudelft.nl/uuid:e57cfcea-548b-4082-aa7b-02a6bbf3e4f7","Density measurements of aerobic granular sludge","van den Berg, L. (TU Delft Sanitary Engineering); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); de Kreuk, M.K. (TU Delft Sanitary Engineering)","","2022","Granular sludge processes are frequently used in domestic and industrial wastewater treatment. The granule buoyant density and biomass density are important parameters for the design and operation of granular sludge reactors. Different methods to measure the granule density include the pycnometer method, the Percoll density gradient method, the dextran blue method, and the settling velocity method. In this study, a comparison was made between these four methods to measure granule density for granules from a full-scale granular sludge plant treating domestic sewage. The effect of salinity on granule density was assessed as well. Three out of the four evaluated methods yielded comparable results, with granule buoyant densities between 1025.7 and 1028.1 kg/m3 and granule biomass densities between 71.1 and 71.5 g/L (based on volatile suspended solids (VSS)). The settling velocity method clearly underestimated the granule density, due to the complex relation between granule properties and settling velocity. The pycnometer method was the most precise method, but it was also quite susceptible to bias. The granule buoyant density increased proportionally with salinity, to 1049.2 kg/m3 at 36 g/L salinity. However, the granule biomass density, based on VSS, remained constant. This showed that the granule volume was not affected by salinity and that the buoyant density increase was the result of diffusion of salts into the granule pores. Overall, the granule density can be measured reliably with most methods, as long as the effect of salinity is considered. The results are discussed in light of operational aspects for full-scale granular sludge plants.","biomass density; buoyant density; density; Granular sludge; salinity","en","journal article","","","","","","","","","","","Sanitary Engineering","","",""
"uuid:e74dea6a-1124-43df-bdfb-5a2f67f0a18c","http://resolver.tudelft.nl/uuid:e74dea6a-1124-43df-bdfb-5a2f67f0a18c","On the mechanisms for aerobic granulation - model based evaluation","van Dijk, E.J.H. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); Haaksman, V.A. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV)","","2022","In this study a mathematical framework was developed to describe aerobic granulation based on 6 main mechanisms: microbial selection, selective wasting, maximizing transport of substrate into the biofilm, selective feeding, substrate type and breakage. A numerical model was developed using four main components; a 1D convection/dispersion model to describe the flow dynamics in a reactor, a reaction/diffusion model describing the essential conversions for granule growth, a setting model to track granules during settling and feeding, and a population model containing up to 100,000 clusters of granules to model the stochastic behaviour of the granulation process. With this approach the model can explain the dynamics of the granulation process observed in practice. This includes the presence of a lag phase and a granulation phase. Selective feeding was identified as an important mechanism that was not yet reported in literature. When aerobic granules are grown from activated sludge flocs, a lag phase occurs, in which not many granules are formed, followed by a granulation phase in which granules rapidly appear. The ratio of granule forming to non-granule forming substrate together with the feast/famine ratio determine if the transition from the lag phase to the granulation phase is successful. The efficiency of selective wasting and selective feeding both determine the rate of this transition. Brake-up of large granules into smaller well settling particles was shown to be an important source for new granules. The granulation process was found to be the combined result from all 6 mechanisms and if conditions for either one are not optimal, other mechanisms can, to some extent, compensate. This model provides a theoretical framework to analyse the different relevant mechanisms for aerobic granular sludge formation and can form the basis for a comprehensive model that includes detailed nutrient removal aspects.","Aerobic granular sludge; Granule formation; Key parameters; Modelling; Sensitivity analyses; Simulation","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:b1f0a296-f9e0-4f5b-9c5a-0d2d2e596c4b","http://resolver.tudelft.nl/uuid:b1f0a296-f9e0-4f5b-9c5a-0d2d2e596c4b","Hydrolysis capacity of different sized granules in a full-scale aerobic granular sludge (AGS) reactor","Toja Ortega, S. (TU Delft Sanitary Engineering); van den Berg, L. (TU Delft Sanitary Engineering); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); de Kreuk, M.K. (TU Delft Sanitary Engineering)","","2022","In aerobic granular sludge (AGS) reactors, granules of different sizes coexist in a single reactor. Their differences in settling behaviour cause stratification in the settled granule bed. In combination with substrate concentration gradients over the reactor height during the anaerobic plug-flow feeding regime, this can result in functional differences between granule sizes. In this study, we compared the hydrolytic activity in granules of 4 size ranges (between 0.5 and 4.8 mm diameter) collected from a full-scale AGS installation. Protease and amylase activities were quantified through fluorescent activity assays. To visualise where the hydrolytic active zones were located within the granules, the hydrolysis sites were visualized microscopically after incubating intact and sliced granules with fluorescent casein and starch. The microbial community was studied using fluorescent in situ hybridization (FISH) and sequencing. The results of these assays indicated that hydrolytic capacity was present throughout the granules, but the hydrolysis of bulk substrates was restricted to the outer 100 µm, approximately. Many of the microorganisms studied by FISH, such as polyphosphate and glycogen accumulating organisms (PAO and GAO), were abundant in the vicinity of the hydrolytically active sites. The biomass-specific hydrolysis rate depended mainly on the available granule surface area, suggesting that different sized granules are not differentiated in terms of hydrolytic capacity. Thus, the substrate concentration gradients that are present during the anaerobic feeding in AGS reactors do not seem to affect hydrolytic activity at the granule surfaces. In this paper, we discuss the possible reasons for this and reflect about the implications for AGS technology.","Activity staining; Aerobic granular sludge; Biomass segregation; Hydrolysis; Polymeric substrates; Wastewater treatment","en","journal article","","","","","","","","","","","Sanitary Engineering","","",""
"uuid:6f1dbcff-4dfe-45d2-b324-f4fa972d113b","http://resolver.tudelft.nl/uuid:6f1dbcff-4dfe-45d2-b324-f4fa972d113b","Full-scale aerobic granular sludge for municipal wastewater treatment - granule formation, microbial succession, and process performance","Ekholm, Jennifer (Chalmers University of Technology); Persson, Frank (Chalmers University of Technology); de Blois, Mark (H2OLAND); Modin, Oskar (Chalmers University of Technology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Suarez, Carolina (Lund University); Gustavsson, David J.I. (VA SYD; Sweden Water Research AB); Wilén, Britt Marie (Chalmers University of Technology)","","2022","Aerobic granular sludge (AGS) plants have gained growing interest and application due to their low energy demand, small footprint, and low operational costs. However, the fulfilment of strict discharge limits for nitrogen and phosphorus, vast seasonal temperature variations, and large peaks in influent flows may pose challenges to the implementation of AGS. Moreover, the knowledge about microbial community assembly and process performance under varying environmental conditions in full-scale reactors is still limited. In this study, the first implementation of the AGS process in the Nordic countries was assessed. In two full-scale AGS reactors with different seeding sludges, the start-up was associated with rapid changes in microbial community composition in both, but only successful granulation in one. As a consequence, the non-granulated reactor was eventually reseeded with biomass from the better granulated reactor. This resulted in a convergence of the microbial communities in the two reactors with the maintenance of stable sludge concentrations (6-8 g L−1) with large granules (50-80% with diameter >2 mm) and fast settling of biomass (SVI30/SVI10 of 0.9-1). Immigration from the influent wastewater was a minor factor affecting the microbial community once the granules had formed, while the seasonal variations in environmental factors were identified as important. Key guilds of AOB (Nitrosomonas), NOB (mainly Ca. Nitrotoga), PAOs (mainly Tetrasphaera), and GAOs (mainly Ca. Competibacter) varied considerably in abundance throughout the study period. After 15 months, stable organic matter, nitrogen, and phosphorus removal were attained with low effluent concentrations. During the start-up, the BOD7/N ratio, influent flow, and temperature were important factors influencing the performance of the AGS.","","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:485341ad-d201-424a-b17a-334397cb5287","http://resolver.tudelft.nl/uuid:485341ad-d201-424a-b17a-334397cb5287","Identification of fungal dihydrouracil-oxidase genes by expression in Saccharomyces cerevisiae","Bouwknegt, J. (TU Delft BT/Industriele Microbiologie); Vos, A.M. (TU Delft BT/Industriele Microbiologie); Ortiz Merino, R.A. (TU Delft BT/Industriele Microbiologie); van Cuylenburg, Daphne C. (Student TU Delft); Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2022","Analysis of predicted fungal proteomes revealed a large family of sequences that showed similarity to the Saccharomyces cerevisiae Class-I dihydroorotate dehydrogenase Ura1, which supports synthesis of pyrimidines under aerobic and anaerobic conditions. However, expression of codon-optimised representatives of this gene family, from the ascomycete Alternaria alternata and the basidiomycete Schizophyllum commune, only supported growth of an S. cerevisiae ura1Δ mutant when synthetic media were supplemented with dihydrouracil. A hypothesis that these genes encode NAD(P)+-dependent dihydrouracil dehydrogenases (EC 1.3.1.1 or 1.3.1.2) was rejected based on absence of complementation in anaerobic cultures. Uracil- and thymine-dependent oxygen consumption and hydrogen-peroxide production by cell extracts of S. cerevisiae strains expressing the A. alternata and S. commune genes showed that, instead, they encode active dihydrouracil oxidases (DHO, EC1.3.3.7). DHO catalyses the reaction dihydrouracil + O2 → uracil + H2O2 and was only reported in the yeast Rhodotorula glutinis (Owaki in J Ferment Technol 64:205–210, 1986). No structural gene for DHO was previously identified. DHO-expressing strains were highly sensitive to 5-fluorodihydrouracil (5F-dhu) and plasmids bearing expression cassettes for DHO were readily lost during growth on 5F-dhu-containing media. These results show the potential applicability of fungal DHO genes as counter-selectable marker genes for genetic modification of S. cerevisiae and other organisms that lack a native DHO. Further research should explore the physiological significance of this enigmatic and apparently widespread fungal enzyme.","5-fluorodihydrouracil; Counter-selectable marker genes; Dihydroorotate dehydrogenase; Dihydropyrimidine dehydrogenase; Dihydropyrimidine oxidase; Dihydrothymine","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:f94bed19-590e-472a-b54f-4c6f668272c5","http://resolver.tudelft.nl/uuid:f94bed19-590e-472a-b54f-4c6f668272c5","An engineered non-oxidative glycolytic bypass based on Calvin-cycle enzymes enables anaerobic co-fermentation of glucose and sorbitol by Saccharomyces cerevisiae","van Aalst, A.C.A. (TU Delft BT/Industriele Microbiologie); Mans, R. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2022","Background: Saccharomyces cerevisiae is intensively used for industrial ethanol production. Its native fermentation pathway enables a maximum product yield of 2 mol of ethanol per mole of glucose. Based on conservation laws, supply of additional electrons could support even higher ethanol yields. However, this option is disallowed by the configuration of the native yeast metabolic network. To explore metabolic engineering strategies for eliminating this constraint, we studied alcoholic fermentation of sorbitol. Sorbitol cannot be fermented anaerobically by S. cerevisiae because its oxidation to pyruvate via glycolysis yields one more NADH than conversion of glucose. To enable re-oxidation of this additional NADH by alcoholic fermentation, sorbitol metabolism was studied in S. cerevisiae strains that functionally express heterologous genes for ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK). Together with the yeast non-oxidative pentose-phosphate pathway, these Calvin-cycle enzymes enable a bypass of the oxidative reaction in yeast glycolysis. Results: Consistent with earlier reports, overproduction of the native sorbitol transporter Hxt15 and the NAD+-dependent sorbitol dehydrogenase Sor2 enabled aerobic, but not anaerobic growth of S. cerevisiae on sorbitol. In anaerobic, slow-growing chemostat cultures on glucose–sorbitol mixtures, functional expression of PRK-RuBisCO pathway genes enabled a 12-fold higher rate of sorbitol co-consumption than observed in a sorbitol-consuming reference strain. Consistent with the high Km for CO2 of the bacterial RuBisCO that was introduced in the engineered yeast strains, sorbitol consumption and increased ethanol formation depended on enrichment of the inlet gas with CO2. Prolonged chemostat cultivation on glucose–sorbitol mixtures led to loss of sorbitol co-fermentation. Whole-genome resequencing after prolonged cultivation suggested a trade-off between glucose-utilization and efficient fermentation of sorbitol via the PRK-RuBisCO pathway. Conclusions: Combination of the native sorbitol assimilation pathway of S. cerevisiae and an engineered PRK-RuBisCO pathway enabled RuBisCO-dependent, anaerobic co-fermentation of sorbitol and glucose. This study demonstrates the potential for increasing the flexibility of redox-cofactor metabolism in anaerobic S. cerevisiae cultures and, thereby, to extend substrate range and improve product yields in anaerobic yeast-based processes by enabling entry of additional electrons.","CO; Electrons; Ethanol; Fermentation; NADH; Redox engineering; Sorbitol; Yeast","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:21dd3ee8-d60c-45fb-84d1-63d566527a8a","http://resolver.tudelft.nl/uuid:21dd3ee8-d60c-45fb-84d1-63d566527a8a","Correction to: Class‑II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis (Fungal Biology and Biotechnology, (2021), 8, 1, (10), 10.1186/s40694-021-00117-4)","Bouwknegt, J. (TU Delft BT/Industriele Microbiologie); Koster, C.C. (TU Delft BT/Industriele Microbiologie); Vos, A.M. (TU Delft BT/Industriele Microbiologie; Wageningen University & Research); Ortiz Merino, R.A. (TU Delft BT/Industriele Microbiologie); Wassink, Mats (Student TU Delft); Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Hagedoorn, P.L. (TU Delft BT/Biocatalysis); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2021","Following publication of the original article [1], the authors reported errors in the text of the Results section and in Table 2. It refers to a mutation in a yeast gene as VPS1I410L and to the corresponding change in the Vps1 amino-acid sequence as I410L. The correct descriptions should read VPS1I401L and I401L, respectively. This has been corrected with this erratum.","","en","journal article","","","","","","Corrigendum DOI 10.1186/s40694-021-00117-4","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:06b5481e-d78b-430f-ae9f-782936db11c1","http://resolver.tudelft.nl/uuid:06b5481e-d78b-430f-ae9f-782936db11c1","Contrasting surface velocities between lake- and land-terminating glaciers in the Himalayan region","Pronk, J. B. (University of St Andrews); Bolch, T. (University of St Andrews); King, O. (University of St Andrews); Wouters, B. (TU Delft Physical and Space Geodesy; Universiteit Utrecht); Benn, D. I. (University of St Andrews)","","2021","Meltwater from Himalayan glaciers sustains the flow of rivers such as the Ganges and Brahmaputra on which over half a billion people depend for day-to-day needs. Upstream areas are likely to be affected substantially by climate change, and changes in the magnitude and timing of meltwater supply are expected to occur in coming decades. About 10 % of the Himalayan glacier population terminates into proglacial lakes, and such lake-terminating glaciers are known to exhibit higher-than-average total mass losses. However, relatively little is known about the mechanisms driving exacerbated ice loss from lake-terminating glaciers in the Himalaya. Here we examine a composite (2017–2019) glacier surface velocity dataset, derived from Sentinel 2 imagery, covering central and eastern Himalayan glaciers larger than 3 km2. We find that centre flow line velocities of lake-terminating glaciers (N = 70; umedian: 18.83 m yr−1; IQR – interquartile range – uncertainty estimate: 18.55–19.06 m yr−1) are on average more than double those of land-terminating glaciers (N = 249; umedian: 8.24 m yr−1; IQR uncertainty estimate: 8.17–8.35 m yr−1) and show substantially more heterogeneity than land-terminating glaciers around glacier termini. We attribute this large heterogeneity to the varying influence of lakes on glacier dynamics, resulting in differential rates of dynamic thinning, which causes about half of the lake-terminating glacier population to accelerate towards the glacier termini. Numerical ice-flow model experiments show that changes in the force balance at the glacier termini are likely to play a key role in accelerating the glacier flow at the front, with variations in basal friction only being of modest importance. The expansion of current glacial lakes and the formation of new meltwater bodies will influence the dynamics of an increasing number of Himalayan glaciers in the future, and these factors should be carefully considered in regional projections.","","en","journal article","","","","","","","","","","","Physical and Space Geodesy","","",""
"uuid:b166b69a-141f-4892-bd80-a6fb8334c840","http://resolver.tudelft.nl/uuid:b166b69a-141f-4892-bd80-a6fb8334c840","Critical parameters and procedures for anaerobic cultivation of yeasts in bioreactors and anaerobic chambers","Mooiman, C. (TU Delft BT/Bioprocess Engineering); Bouwknegt, J. (TU Delft BT/Industriele Microbiologie); Dekker, W.J.C. (TU Delft BT/Industriele Microbiologie); Wiersma, S.J. (TU Delft BT/Industriele Microbiologie); Ortiz Merino, R.A. (TU Delft BT/Industriele Microbiologie); de Hulster, A.F. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2021","All known facultatively fermentative yeasts require molecular oxygen for growth. Only in a small number of yeast species, these requirements can be circumvented by supplementation of known anaerobic growth factors such as nicotinate, sterols and unsaturated fatty acids. Biosynthetic oxygen requirements of yeasts are typically small and, unless extensive precautions are taken to minimize inadvertent entry of trace amounts of oxygen, easily go unnoticed in small-scale laboratory cultivation systems. This paper discusses critical points in the design of anaerobic yeast cultivation experiments in anaerobic chambers and laboratory bioreactors. Serial transfer or continuous cultivation to dilute growth factors present in anaerobically pre-grown inocula, systematic inclusion of control strains and minimizing the impact of oxygen diffusion through tubing are identified as key elements in experimental design. Basic protocols are presented for anaerobic-chamber and bioreactor experiments.","anaerobic; cultivation; nonconventional yeast; oxygen requirements; saccharomyces","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Bioprocess Engineering","","",""
"uuid:cecbbe89-0136-4fc7-a400-3be87ef3037c","http://resolver.tudelft.nl/uuid:cecbbe89-0136-4fc7-a400-3be87ef3037c","Identification of Oxygen-Independent Pathways for Pyridine Nucleotide and Coenzyme A Synthesis in Anaerobic Fungi by Expression of Candidate Genes in Yeast","Perli, T. (TU Delft BT/Industriele Microbiologie); Vos, A.M. (TU Delft BT/Industriele Microbiologie); Bouwknegt, J. (TU Delft BT/Industriele Microbiologie); Dekker, W.J.C. (TU Delft BT/Industriele Microbiologie); Wiersma, S.J. (TU Delft BT/Industriele Microbiologie); Mooiman, C. (TU Delft BT/Bioprocess Engineering); Ortiz Merino, R.A. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2021","Neocallimastigomycetes are unique examples of strictly anaerobic eukaryotes. This study investigates how these anaerobic fungi bypass reactions involved in synthesis of pyridine nucleotide cofactors and coenzyme A that, in canonical fungal pathways, require molecular oxygen. Analysis of Neocallimastigomycetes proteomes identified a candidate L-aspartate-decarboxylase (AdcA) and L-aspartate oxidase (NadB) and quinolinate synthase (NadA), constituting putative oxygen-independent bypasses for coenzyme A synthesis and pyridine nucleotide cofactor synthesis. The corresponding gene sequences indicated acquisition by ancient horizontal gene transfer (HGT) events involving bacterial donors. To test whether these enzymes suffice to bypass corresponding oxygen-requiring reactions, they were introduced into fms1∆ and bna2∆ Saccharomyces cerevisiae strains. Expression of nadA and nadB from Piromyces finnis and adcA from Neocallimastix californiae conferred cofactor prototrophy under aerobic and anaerobic conditions. This study simulates how HGT can drive eukaryotic adaptation to anaerobiosis and provides a basis for elimination of auxotrophic requirements in anaerobic industrial applications of yeasts and fungi. IMPORTANCE NAD (NAD +) and coenzyme A (CoA) are central metabolic cofactors whose canonical biosynthesis pathways in fungi require oxygen. Anaerobic gut fungi of the Neocallimastigomycota phylum are unique eukaryotic organisms that adapted to anoxic environments. Analysis of Neocallimastigomycota genomes revealed that these fungi might have developed oxygen-independent biosynthetic pathways for NAD + and CoA biosynthesis, likely acquired through horizontal gene transfer (HGT) from prokaryotic donors. We confirmed functionality of these putative pathways under anaerobic conditions by heterologous expression in the yeast Saccharomyces cerevisiae. This approach, combined with sequence comparison, offers experimental insight on whether HGT events were required and/or sufficient for acquiring new traits. Moreover, our results demonstrate an engineering strategy for enabling S. cerevisiae to grow anaerobically in the absence of the precursor molecules pantothenate and nicotinate, thereby contributing to alleviate oxygen requirements and to move closer to prototrophic anaerobic growth of this industrially relevant yeast.","Anaerobes; Biotechnology; Fungi; Neocallimastigomycetes; Nicotinic acid; Oxygen requirement; Pantothenate; Saccharomyces cerevisiae; Vitamin biosynthesis","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:e879b996-2f4f-40da-9c16-4c42e724ad10","http://resolver.tudelft.nl/uuid:e879b996-2f4f-40da-9c16-4c42e724ad10","Nanocellulose recovery from domestic wastewater","Pereira Espíndola, S. (TU Delft ChemE/Advanced Soft Matter); Pronk, M. (TU Delft BT/Environmental Biotechnology); Zlopasa, J. (TU Delft BT/Environmental Biotechnology); Picken, S.J. (TU Delft ChemE/Advanced Soft Matter); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology)","","2021","Wastewater solids could be an attractive source of secondary raw cellulose, mainly originating from toilet paper. Cellulose can be recovered through sieving of raw wastewater, return sludge, or excess sludge. In particular, a large fraction of cellulose (13–15%) can be found in the excess sludge of the aerobic granular sludge produced by the Nereda® wastewater technology. A cellulose extraction method was developed during this study, allowing the recovery of a pulp with over 86 wt% purity. The wastewater derived cellulose fibres could be an excellent source for production of recovered cellulose nanocrystals (rCNC). Several pre-treatment steps needed in cellulose nanocrystals (CNC) production from wood pulp are already performed in the production of toilet paper. Here, the technical feasibility of such rCNC is studied. As reference materials, microcrystalline cellulose and toilet paper were also used. The rCNC were obtained by acid hydrolysis, with yields of ∼30 wt% (pulp basis). The wastewater-based material was rod-like, with high aspect ratio (10–14), crystallinity (62–68%), and chemical structure similar to commercial CNC. The yield of rCNC per gram of cellulose recovered from the influent was 22%, while for excess sludge cellulose it was less (4%). Bio-nanocomposites of rCNC and alginate were also investigated. At 50 vol% loading of rCNC, there was a 50% relative increase in stiffness (18 GPa) compared to matrix (12 GPa). The characterization of rCNC and positive impact in composite materials confirms a suitable quality of wastewater derived CNC. Ultimately, the nanocellulose is a tangible example that recovery of high-end products from wastewater is possible, in line with a circular economy.","Cellulose; Cellulose NanoCrystals; Nanocellulose; Resource recovery; Toilet paper; Wastewater solids","en","journal article","","","","","","","","","","","ChemE/Advanced Soft Matter","","",""
"uuid:2f86b655-6641-471b-90e2-9000485b5570","http://resolver.tudelft.nl/uuid:2f86b655-6641-471b-90e2-9000485b5570","Elimination of aromatic fusel alcohols as by-products of Saccharomyces cerevisiae strains engineered for phenylpropanoid production by 2-oxo-acid decarboxylase replacement","Else-Hassing, J. (TU Delft BT/Industriele Microbiologie); Buijs, Joran (Student TU Delft); Blankerts, Nikki (Student TU Delft); Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); de Hulster, A.F. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2021","Engineered strains of the yeast Saccharomyces cerevisiae are intensively studied as production platforms for aromatic compounds such as hydroxycinnamic acids, stilbenoids and flavonoids. Heterologous pathways for production of these compounds use L-phenylalanine and/or L-tyrosine, generated by the yeast shikimate pathway, as aromatic precursors. The Ehrlich pathway converts these precursors to aromatic fusel alcohols and acids, which are undesirable by-products of yeast strains engineered for production of high-value aromatic compounds. Activity of the Ehrlich pathway requires any of four S. cerevisiae 2-oxo-acid decarboxylases (2-OADCs): Aro10 or the pyruvate-decarboxylase isoenzymes Pdc1, Pdc5, and Pdc6. Elimination of pyruvate-decarboxylase activity from S. cerevisiae is not straightforward as it plays a key role in cytosolic acetyl-CoA biosynthesis during growth on glucose. In a search for pyruvate decarboxylases that do not decarboxylate aromatic 2-oxo acids, eleven yeast and bacterial 2-OADC-encoding genes were investigated. Homologs from Kluyveromyces lactis (KlPDC1), Kluyveromyces marxianus (KmPDC1), Yarrowia lipolytica (YlPDC1), Zymomonas mobilis (Zmpdc1) and Gluconacetobacter diazotrophicus (Gdpdc1.2 and Gdpdc1.3) complemented a Pdc− strain of S. cerevisiae for growth on glucose. Enzyme-activity assays in cell extracts showed that these genes encoded active pyruvate decarboxylases with different substrate specificities. In these in vitro assays, ZmPdc1, GdPdc1.2 or GdPdc1.3 had no substrate specificity towards phenylpyruvate. Replacing Aro10 and Pdc1,5,6 by these bacterial decarboxylases completely eliminated aromatic fusel-alcohol production in glucose-grown batch cultures of an engineered coumaric acid-producing S. cerevisiae strain. These results outline a strategy to prevent formation of an important class of by-products in ‘chassis’ yeast strains for production of non-native aromatic compounds.","Ehrlich pathway; Fusel alcohols; Phenylpropanoid; Phenylpyruvate decarboxylase; Pyruvate decarboxylase; Saccharomyces cerevisiae","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:3f6ae34a-6142-48e1-b69c-039b0fd15578","http://resolver.tudelft.nl/uuid:3f6ae34a-6142-48e1-b69c-039b0fd15578","A squalene-hopene cyclase in Schizosaccharomyces japonicus represents a eukaryotic adaptation to sterol-limited anaerobic environments","Bouwknegt, J. (TU Delft BT/Industriele Microbiologie); Wiersma, S.J. (TU Delft BT/Industriele Microbiologie); Ortiz Merino, R.A. (TU Delft BT/Industriele Microbiologie); Doornenbal, E.S.R. (TU Delft Economics of Technology and Innovation); Buitenhuis, Petrik (Student TU Delft); Giera, Martin (Leiden University Medical Center); Müller, Christoph (Ludwig Maximilians University); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2021","Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requiresmolecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report [C. J. E. A. Bulder, Antonie Van Leeuwenhoek, 37, 353-358 (1971)] that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop- 17(21)-ene, hop-21(22)-ene, and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and stimulated anaerobic growth in sterol-free media, thus indicating that one or more of the hopanoids produced by SjShc1 could at least partially replace sterols. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast anaerobic growth of Sch. japonicus in sterol-free media is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.","Anaerobic; Hopanoids; Schizosaccharomyces; Sterols; Yeast","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:e292a5e5-3f05-4437-93b4-f2722a7a583a","http://resolver.tudelft.nl/uuid:e292a5e5-3f05-4437-93b4-f2722a7a583a","Anaerobic hydrolysis of complex substrates in full-scale aerobic granular sludge: enzymatic activity determined in different sludge fractions","Toja Ortega, S. (TU Delft Sanitary Engineering); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); de Kreuk, M.K. (TU Delft Sanitary Engineering)","","2021","Complex substrates, like proteins, carbohydrates, and lipids, are major components of domestic wastewater, and yet their degradation in biofilm-based wastewater treatment technologies, such as aerobic granular sludge (AGS), is not well understood. Hydrolysis is considered the rate-limiting step in the bioconversion of complex substrates, and as such, it will impact the utilization of a large wastewater COD (chemical oxygen demand) fraction by the biofilms or granules. To study the hydrolysis of complex substrates within these types of biomass, this paper investigates the anaerobic activity of major hydrolytic enzymes in the different sludge fractions of a full-scale AGS reactor. Chromogenic substrates were used under fully mixed anaerobic conditions to determine lipase, protease, α-glucosidase, and β-glucosidase activities in large granules (>1 mm in diameter), small granules (0.2–1 mm), flocculent sludge (0.045–0.2 mm), and bulk liquid. Furthermore, composition and hydrolytic activity of influent wastewater samples were determined. Our results showed an overcapacity of the sludge to hydrolyze wastewater soluble and colloidal polymeric substrates. The highest specific hydrolytic activity was associated with the flocculent sludge fraction (1.5–7.5 times that of large and smaller granules), in agreement with its large available surface area. However, the biomass in the full-scale reactor consisted of 84% large granules, making the large granules account for 55–68% of the total hydrolytic activity potential in the reactor. These observations shine a new light on the contribution of large granules to the conversion of polymeric COD and suggest that large granules can hydrolyze a significant amount of this influent fraction. The anaerobic removal of polymeric soluble and colloidal substrates could clarify the stable granule formation that is observed in full-scale installations, even when those are fed with complex wastewaters. Key points: • Large and small granules contain >70% of the hydrolysis potential in an AGS reactor. • Flocculent sludge has high hydrolytic activity but constitutes <10% VS in AGS. • AGS has an overcapacity to hydrolyze complex substrates in domestic wastewater. <!-- Query ID=""Q2"" Text="" Graphical abstract contains text below the minimum required font size of 6pts inside the artwork, and there is no sufficient space available for the text to be enlarged. Please provide replacement figure file."" -->Graphical abstract: [Figure not available: see fulltext.]","Aerobic granular sludge; Domestic wastewater; Full-scale; hydrolysis; Polymeric substrates","en","journal article","","","","","","","","","","","Sanitary Engineering","","",""
"uuid:4dbf037d-e1d2-4d96-b9c9-6a9e806e2baa","http://resolver.tudelft.nl/uuid:4dbf037d-e1d2-4d96-b9c9-6a9e806e2baa","Engineering oxygen-independent biotin biosynthesis in Saccharomyces cerevisiae","Wronska, A.K. (TU Delft BT/Industriele Microbiologie); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Perli, T. (TU Delft BT/Industriele Microbiologie); de Hulster, A.F. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2021","An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae. Implementation of this pathway required expression of seven E. coli genes involved in fatty-acid synthesis and three E. coli genes essential for the formation of a pimelate thioester, key precursor of biotin synthesis. A yeast strain expressing these genes readily grew in biotin-free medium, irrespective of the presence of oxygen. However, the engineered strain exhibited specific growth rates 25% lower in biotin-free media than in biotin-supplemented media. Following adaptive laboratory evolution in anoxic cultures, evolved cell lines that no longer showed this growth difference in controlled bioreactors, were characterized by genome sequencing and proteome analyses. The evolved isolates exhibited a whole-genome duplication accompanied with an alteration in the relative gene dosages of biosynthetic pathway genes. These alterations resulted in a reduced abundance of the enzymes catalyzing the first three steps of the E. coli biotin pathway. The evolved pathway configuration was reverse engineered in the diploid industrial S. cerevisiae strain Ethanol Red. The resulting strain grew at nearly the same rate in biotin-supplemented and biotin-free media non-controlled batches performed in an anaerobic chamber. This study established an unique genetic engineering strategy to enable biotin-independent anoxic growth of S. cerevisiae and demonstrated its portability in industrial strain backgrounds.","Anoxic growth; Biotin biosynthesis; Gene dosage; Prokaryotic pathway; Vitamin B7","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:b4cb7f7d-0c4f-40ba-944c-a4fe9e2d47cc","http://resolver.tudelft.nl/uuid:b4cb7f7d-0c4f-40ba-944c-a4fe9e2d47cc","Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth","Dekker, W.J.C. (TU Delft BT/Industriele Microbiologie); Ortiz Merino, R.A. (TU Delft BT/Industriele Microbiologie); Kaljouw, Astrid (Student TU Delft); Battjes, Julius (Student TU Delft); Wiering, Frank W. (Student TU Delft); Mooiman, C. (TU Delft BT/Bioprocess Engineering); de la Torre, P. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2021","Current large-scale, anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Responses of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for anaerobic industrial applications.","Anaerobic metabolism; Ergosterol; Ethanol production; Metabolic engineering; Tetrahymanol; Thermotolerance; Yeast biotechnology","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:51784085-07c3-4642-a4c6-3514bf9bc1ea","http://resolver.tudelft.nl/uuid:51784085-07c3-4642-a4c6-3514bf9bc1ea","Effect of an increased particulate cod load on the aerobic granular sludge process: A full scale study","Toja Ortega, S. (TU Delft Sanitary Engineering; TU Delft Water Management); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); de Kreuk, M.K. (TU Delft Sanitary Engineering; TU Delft Water Management)","","2021","High concentrations of particulate COD (pCOD) in the influent of aerobic granular sludge (AGS) systems are often associated to small granule diameter and a large fraction of flocculent sludge. At high particulate concentrations even granule stability and process performance might be compromised. However, pilot-or full-scale studies focusing on the effect of real wastewater partic-ulates on AGS are scarce. This study describes a 3-month period of increased particulate loading at a municipal AGS wastewater treatment plant. The pCOD concentration of the influent increased from 0.5 g COD/L to 1.3 g COD/L, by adding an untreated slaughterhouse wastewater source to the influent. Sludge concentration, waste sludge production and COD and nutrient removal performance were monitored. Furthermore, to investigate how the sludge acclimatises to a higher influent particulate content, lipase and protease hydrolytic activities were studied, as well as the microbial community composition of the sludge. The composition of the granule bed and nutrient removal efficiency did not change considerably by the increased pCOD. Interestingly, the biomass-specific hydrolytic activities of the sludge did not increase during the test period either. However, already during normal operation the aerobic granules and flocs exhibited a hydrolytic potential that ex-ceeded the influent concentrations of proteins and lipids. Microbial community analysis also revealed a high proportion of putative hydrolysing and fermenting organisms in the sludge, both during normal operation and during the test period. The results of this study highlight the robust-ness of the full-scale AGS process, which can bear a substantial increase in the influent pCOD concentration during an extended period.","Aerobic granular sludge; Full-scale wastewater treatment; Granule stability; Nutrient removal; Particulate COD","en","journal article","","","","","","","","","","Water Management","Sanitary Engineering","","",""
"uuid:789359ca-0f57-4ee1-9abc-524e1a8f68c6","http://resolver.tudelft.nl/uuid:789359ca-0f57-4ee1-9abc-524e1a8f68c6","Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis","Bouwknegt, J. (TU Delft BT/Industriele Microbiologie); Koster, C.C. (TU Delft BT/Industriele Microbiologie); Vos, A.M. (Wageningen University & Research); Ortiz Merino, R.A. (TU Delft BT/Industriele Microbiologie); Wassink, Mats (Student TU Delft); Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Hagedoorn, P.L. (TU Delft BT/Biocatalysis); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2021","Background: In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD. Results: Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9. Conclusions: Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure–function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.","Anaerobiosis; Neocallimastigomycota; Oxygen; Oxygen requirements; Uracil; Yeast","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:d162d3e1-ba11-4152-a544-704bbbf92436","http://resolver.tudelft.nl/uuid:d162d3e1-ba11-4152-a544-704bbbf92436","Uncoupling growth and succinic acid production in an industrial Saccharomyces cerevisiae strain","Liu, Y. (TU Delft BT/Industriele Microbiologie); Esen, Osman (Student TU Delft); Pronk, J.T. (TU Delft BT/Biotechnologie); van Gulik, W.M. (TU Delft BT/Industriele Microbiologie)","","2021","This study explores the relation between biomass-specific succinic acid (SA) production rate and specific growth rate of an engineered industrial strain of Saccharomyces cerevisiae, with the aim to investigate the extent to which growth and product formation can be uncoupled. Ammonium-limited aerobic chemostat and retentostat cultures were grown at different specific growth rates under industrially relevant conditions, that is, at a culture pH of 3 and with sparging of a 1:1 CO2–air mixture. Biomass-specific SA production rates decreased asymptotically with decreasing growth rate. At near-zero growth rates, the engineered strain maintained a stable biomass-specific SA production rate for over 500 h, with a SA yield on glucose of 0.61 mol mol−1. These results demonstrate that uncoupling of growth and SA production could indeed be achieved. A linear relation between the biomass-specific SA production rate and glucose consumption rate indicated the coupling of SA production rate and the flux through primary metabolism. The low culture pH resulted in an increased death rate, which was lowest at near-zero growth rates. Nevertheless, a significant amount of non-viable biomass accumulated in the retentostat cultures, thus underlining the importance of improving low-pH tolerance in further strain development for industrial SA production with S. cerevisiae.","high CO; low pH; near-zero growth; retentostat; succinic acid","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:aff9ca7f-4d9e-4215-91c4-009166492ce8","http://resolver.tudelft.nl/uuid:aff9ca7f-4d9e-4215-91c4-009166492ce8","Nitrous oxide emission from full-scale municipal aerobic granular sludge","van Dijk, E.J.H. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV)","","2021","The nitrous oxides emission was measured over 7 months in the full-scale aerobic granular sludge plant in Dinxperlo, the Netherlands. Nitrous oxide concentrations were measured in the bulk liquid and the off-gas of the Nereda® reactor. Combined with the batch wise operation of the reactor, this gave a high information density and a better insight into N2O emission in general. The average emission factor was 0.33% based on the total nitrogen concentration in the influent. The yearly average emission factor was estimated to be between 0.25% and 0.30%. The average emission factor is comparable to continuous activated sludge plants, using flocculent sludge, and it is low compared to other sequencing batch systems. The variability in the emission factor increased when the reactor temperature was below 14 °C, showing higher emission factors during the winter period. A change in the process control in the winter period reduced the variability, reducing the emission factors to a level comparable to the summer period. Different process control might be necessary at high and low temperatures to obtain a consistently low nitrous oxide emission. Rainy weather conditions lowered the emission factor, also in the dry weather flow batches following the rainy weather batches. This was attributed to the first flush from the sewer at the start of rainy weather conditions, resulting in a temporarily increased sludge loading.","Aerobic granular sludge; Greenhouse gas emissions; Nereda; Nitrous oxide","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:6b2bdd67-154a-4e58-b739-fd704c9d57d9","http://resolver.tudelft.nl/uuid:6b2bdd67-154a-4e58-b739-fd704c9d57d9","Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae","Perli, T. (TU Delft BT/Industriele Microbiologie); van der Vorm, Daan N.A. (Student TU Delft); Wassink, Mats (Student TU Delft); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2021","Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.","Metabolic engineering; Molybdenum cofactor; Nitrate assimilation; Nitrate reductase; Saccharomyces cerevisiae","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:c9ef9a8d-81b6-4bda-8352-69402be6a8b5","http://resolver.tudelft.nl/uuid:c9ef9a8d-81b6-4bda-8352-69402be6a8b5","Vitamin requirements and biosynthesis in Saccharomyces cerevisiae","Perli, T. (TU Delft BT/Industriele Microbiologie); Wronska, A.K. (TU Delft BT/Industriele Microbiologie); Ortiz Merino, R.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2020","Chemically defined media for yeast cultivation (CDMY) were developed to support fast growth, experimental reproducibility, and quantitative analysis of growth rates and biomass yields. In addition to mineral salts and a carbon substrate, popular CDMYs contain seven to nine B-group vitamins, which are either enzyme cofactors or precursors for their synthesis. Despite the widespread use of CDMY in fundamental and applied yeast research, the relation of their design and composition to the actual vitamin requirements of yeasts has not been subjected to critical review since their first development in the 1940s. Vitamins are formally defined as essential organic molecules that cannot be synthesized by an organism. In yeast physiology, use of the term “vitamin” is primarily based on essentiality for humans, but the genome of the Saccharomyces cerevisiae reference strain S288C harbours most of the structural genes required for synthesis of the vitamins included in popular CDMY. Here, we review the biochemistry and genetics of the biosynthesis of these compounds by S. cerevisiae and, based on a comparative genomics analysis, assess the diversity within the Saccharomyces genus with respect to vitamin prototrophy.","fermentation; growth requirements; Saccharomyces cerevisiae; synthetic media; vitamin biosynthesis","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:cd22e7d4-cca4-49d6-824c-20ae43dd6e64","http://resolver.tudelft.nl/uuid:cd22e7d4-cca4-49d6-824c-20ae43dd6e64","Squalene-Tetrahymanol Cyclase Expression Enables Sterol-Independent Growth of Saccharomyces cerevisiae","Wiersma, S.J. (TU Delft BT/Industriele Microbiologie); Mooiman, C. (TU Delft BT/Industriele Microbiologie); Giera, Martin (Leiden University Medical Center); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2020","Biosynthesis of sterols, which are considered essential components of virtually all eukaryotic membranes, requires molecular oxygen. Anaerobic growth of the yeast Saccharomyces cerevisiae therefore strictly depends on sterol supplementation of synthetic growth media. Neocallimastigomycota are a group of strictly anaerobic fungi which, instead of containing sterols, contain the pentacyclic triterpenoid ""sterol surrogate"" tetrahymanol, which is formed by cyclization of squalene. Here, we demonstrate that expression of the squalene-tetrahymanol cyclase gene TtTHC1 from the ciliate Tetrahymena thermophila enables synthesis of tetrahymanol by S. cerevisiae Moreover, expression of TtTHC1 enabled exponential growth of anaerobic S. cerevisiae cultures in sterol-free synthetic media. After deletion of the ERG1 gene from a TtTHC1-expressing S. cerevisiae strain, native sterol synthesis was abolished and sustained sterol-free growth was demonstrated under anaerobic as well as aerobic conditions. Anaerobic cultures of TtTHC1-expressing S. cerevisiae on sterol-free medium showed lower specific growth rates and biomass yields than ergosterol-supplemented cultures, while their ethanol yield was higher. This study demonstrated that acquisition of a functional squalene-tetrahymanol cyclase gene offers an immediate growth advantage to S. cerevisiae under anaerobic, sterol-limited conditions and provides the basis for a metabolic engineering strategy to eliminate the oxygen requirements associated with sterol synthesis in yeasts.IMPORTANCE The laboratory experiments described in this report simulate a proposed horizontal gene transfer event during the evolution of strictly anaerobic fungi. The demonstration that expression of a single heterologous gene sufficed to eliminate anaerobic sterol requirements in the model eukaryote Saccharomyces cerevisiae therefore contributes to our understanding of how sterol-independent eukaryotes evolved in anoxic environments. This report provides a proof of principle for a metabolic engineering strategy to eliminate sterol requirements in yeast strains that are applied in large-scale anaerobic industrial processes. The sterol-independent yeast strains described in this report provide a valuable platform for further studies on the physiological roles and impacts of sterols and sterol surrogates in eukaryotic cells.","Saccharomyces cerevisiae; anaerobic; membrane composition; oxygen requirements; sterols; tetrahymanol","en","journal article","","","","","","Accepted Author Manuscript","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:477d2241-8d3f-4e9a-8882-a41a5aa47bb0","http://resolver.tudelft.nl/uuid:477d2241-8d3f-4e9a-8882-a41a5aa47bb0","Adaptive Laboratory Evolution and Reverse Engineering of Single-Vitamin Prototrophies in Saccharomyces cerevisiae","Perli, T. (TU Delft BT/Industriele Microbiologie); Moonen, Dewi P.I. (Student TU Delft); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2020","Quantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in S. cerevisiae CEN. PK113-7D, requirements for biotin were eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA), or thiamine was omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B vitamins. In all evolution lines, specific growth rates reached at least 90% of the growth rate observed in SM supplemented with a complete B vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid, or pABA. Reaching similar results in SM lacking thiamine, pyridoxine, or pantothenate required more than 300 generations of selective growth. The genomes of evolved single-colony isolates were resequenced, and for each B vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth was selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B vitamin, the introduction of a small number of mutations sufficed to achieve a substantially increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM.","adaptive mutations; evolutionary engineering; media; nutritional requirements; prototrophy; reverse genetic analysis; Saccharomyces cerevisiae; vitamin biosynthesis","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:d0a69735-325f-48b0-aace-c6ff855e0ab7","http://resolver.tudelft.nl/uuid:d0a69735-325f-48b0-aace-c6ff855e0ab7","Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae","Wronska, A.K. (TU Delft BT/Industriele Microbiologie); Haak, Meinske P. (Student TU Delft); Geraats, Ellen (Student TU Delft); Bruins Slot, Eva (Student TU Delft); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2020","Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiaeBIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiaeIMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.","BIO1; biotin; Cyberlindnera fabianii; de novo synthesis; fungal biotin synthesis; metabolic engineering; oxygen requirement; oxygen-requiring enzyme; prototrophy; Saccharomyces cerevisiae; Saccharomycotina; vitamin B7","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:a7a92e66-6189-4717-82d6-0ea9a3ad4d6e","http://resolver.tudelft.nl/uuid:a7a92e66-6189-4717-82d6-0ea9a3ad4d6e","Improving Industrially Relevant Phenotypic Traits by Engineering Chromosome Copy Number in Saccharomyces pastorianus","Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie); Knibbe, E. (TU Delft BT/Industriele Microbiologie); van Roosmalen, Roderick (Student TU Delft); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); de la Torre, P. (TU Delft BT/Industriele Microbiologie); O’Herne, Stephanie F. (Student TU Delft); Vijverberg, Pascal A. (Student TU Delft); el Masoudi, Anissa (Student TU Delft); Brouwers, N. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2020","The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S. pastorianus remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple non-targeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same set-up yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid S. pastorianus genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement.","chromosome copy number stability; chromosome missegregation; lager beer brewing; Saccharomyces pastorianus; strain engineering","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:86c4a0d2-ba5b-4111-9827-4d6a9eb04d3e","http://resolver.tudelft.nl/uuid:86c4a0d2-ba5b-4111-9827-4d6a9eb04d3e","Stable granulation of seawater-adapted aerobic granular sludge with filamentous Thiothrix bacteria","de Graaff, D.R. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV)","","2020","Many sources of wastewater contain sulfides, which can cause excessive growth of filamentous bacteria such as Thiothrix sp. resulting in bulking sludge in conventional activated sludge systems. Granular sludge systems could potentially also suffer from the growth of filamentous bacteria. Uptake of easily degradable COD by the relatively slow growing Ca. Accumulibacter phosphatis bacteria and the absence of strong diffusion gradients due to plug flow feeding through the settled granular sludge bed are assumed to be the dominant factors for successful granulation. Sulfides will remain after this anaerobic phase and cause growth of sulfide-consuming bacteria such as Thiothrix sp. Here we observed the impact of growth of Thiothrix sp bacteria in a laboratory aerobic granular sludge reactor by feeding a mixture of acetate and thiosulfate in the influent. Thiothrix sp, proliferated when 18% of the influent COD was due to thiosulfate, forming 51.4 ± 8.3% of the total granular biomass. Despite the strong presence of these filamentous bacteria a well settling sludge was maintained (SVI10 equal to 13.3 mL/g). These results confirm that sludge morphology is not necessarily a reflection of the cell morphology of the bacteria, but is highly influence by reactor operation. It also reiterates the fact that compact biofilms are formed when the substrate consumption rate is lower than the substrate transport rate.","Aerobic granular sludge; Granulation; Seawater; Sulfide; Thiothrix","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:cf430d52-0db1-4cb5-98cd-f7686501b2cd","http://resolver.tudelft.nl/uuid:cf430d52-0db1-4cb5-98cd-f7686501b2cd","A Novel D-Galacturonate Fermentation Pathway in Lactobacillus suebicus Links Initial Reactions of the Galacturonate-Isomerase Route With the Phosphoketolase Pathway","Valk, L.C. (TU Delft BT/Industriele Microbiologie); Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); de Ram, C. (TU Delft OLD BT/Cell Systems Engineering); Pabst, Martin (TU Delft OLD BT/Cell Systems Engineering); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2020","D-galacturonate, a key constituent of pectin, is a ubiquitous monomer in plant biomass. Anaerobic, fermentative conversion of D-galacturonate is therefore relevant in natural environments as well as in microbial processes for microbial conversion of pectin-containing agricultural residues. In currently known microorganisms that anaerobically ferment D-galacturonate, its catabolism occurs via the galacturonate-isomerase pathway. Redox-cofactor balancing in this pathway strongly constrains the possible range of products generated from anaerobic D-galacturonate fermentation, resulting in acetate as the predominant organic fermentation product. To explore metabolic diversity of microbial D-galacturonate fermentation, anaerobic enrichment cultures were performed at pH 4. Anaerobic batch and chemostat cultures of a dominant Lactobacillus suebicus strain isolated from these enrichment cultures produced near-equimolar amounts of lactate and acetate from D-galacturonate. A combination of whole-genome sequence analysis, quantitative proteomics, enzyme activity assays in cell extracts, and in vitro product identification demonstrated that D-galacturonate metabolism in L. suebicus occurs via a novel pathway. In this pathway, mannonate generated by the initial reactions of the canonical isomerase pathway is converted to 6-phosphogluconate by two novel biochemical reactions, catalyzed by a mannonate kinase and a 6-phosphomannonate 2-epimerase. Further catabolism of 6-phosphogluconate then proceeds via known reactions of the phosphoketolase pathway. In contrast to the classical isomerase pathway for D-galacturonate catabolism, the novel pathway enables redox-cofactor-neutral conversion of D-galacturonate to ribulose-5-phosphate. While further research is required to identify the structural genes encoding the key enzymes for the novel pathway, its redox-cofactor coupling is highly interesting for metabolic engineering of microbial cell factories for conversion of pectin-containing feedstocks into added-value fermentation products such as ethanol or lactate. This study illustrates the potential of microbial enrichment cultivation to identify novel pathways for the conversion of environmentally and industrially relevant compounds.","anaerobic metabolism; enrichment cultivation; Entner–Doudoroff pathway; galacturonic acid; heterolactic fermentation; pectin degradation","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:64779b10-fdcc-46f5-b799-00adc1ef501f","http://resolver.tudelft.nl/uuid:64779b10-fdcc-46f5-b799-00adc1ef501f","Removal of bacterial and viral indicator organisms in full-scale aerobic granular sludge and conventional activated sludge systems","Barrios Hernandez, M.L. (TU Delft BT/Environmental Biotechnology; IHE Delft Institute for Water Education); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); Garcia, H. (IHE Delft Institute for Water Education); Boersma, Arne (Royal HaskoningDHV); Brdjanovic, Damir (TU Delft BT/Environmental Biotechnology; IHE Delft Institute for Water Education); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Hooijmans, Christine M. (IHE Delft Institute for Water Education)","","2020","The aim of this study was to evaluate the effectiveness of the novel aerobic granular sludge (AGS) wastewater treatment technology in removing faecal indicator organisms (FIOs) compared to the conventional activated sludge (CAS) treatment system. The work was carried out at two full-scale wastewater treatment plants (WWTP) in the Netherlands, Vroomshoop and Garmerwolde. Both treatment plants have a CAS and AGS system operated in parallel. The parallel treatment lines are provided with the same influent wastewater. The concentrations of the measured FIOs in the influent of the two WWTPs were comparable with reported literature values as follows: F-specific RNA bacteriophages at 106 PFU/100 mL, and Escherichia coli (E. coli), Enterococci, and Thermotolerant coliforms (TtC) at 105 to 106 CFU/100 mL. Although both systems (CAS and AGS) are different in terms of design, operation, and microbial community, both systems showed similar FIOs removal efficiency. At the Vroomshoop WWTP, Log10 removals for F-specific RNA bacteriophages of 1.4 ± 0.5 and 1.3 ± 0.6 were obtained for the AGS and CAS systems, while at the Garmerwolde WWTP, Log10 removals for F-specific RNA bacteriophages of 1.9 ± 0.7 and 2.1 ± 0.7 were found for the AGS and CAS systems. Correspondingly, E. coli, Enterococci, and TtC Log10 removals of 1.7 ± 0.7 and 1.1 ± 0.7 were achieved for the AGS and CAS systems at Vroomshoop WWTP. For Garmerwolde WWTP Log10 removals of 2.3 ± 0.8 and 1.9 ± 0.7 for the AGS and CAS systems were found, respectively. The measured difference in removal rates between the plants was not significant. Physicochemical water quality parameters, such as the concentrations of organic matter, nutrients, and total suspended solids (TSS) were also determined. Overall, it was not possible to establish a direct correlation between the physicochemical parameters and the removal of FIOs for any of the treatment systems (CAS and AGS). Only the removal of TSS could be positively correlated to the E. coli removal for the AGS technology at the evaluated WWTPs.","Activated sludge; Aerobic granular sludge; F-specific RNA bacteriophages; Faecal indicators; Pathogen removal","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:7b60aae1-f2b7-4731-a1d6-a21b0460f3ce","http://resolver.tudelft.nl/uuid:7b60aae1-f2b7-4731-a1d6-a21b0460f3ce","“Candidatus Galacturonibacter soehngenii” Shows Acetogenic Catabolism of Galacturonic Acid but Lacks a Canonical Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Complex","Valk, L.C. (TU Delft BT/Industriele Microbiologie); Diender, Martijn (Wageningen University & Research); Stouten, G.R. (TU Delft BT/Environmental Biotechnology); Petersen, Jette F. (Aalborg University); Nielsen, Per H. (Aalborg University); Dueholm, Morten S. (Aalborg University); Pronk, J.T. (TU Delft BT/Biotechnologie); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology)","","2020","Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of “Candidatus Galacturonibacter soehngenii,” from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of “Ca. G. soehngenii” was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the “Ca. G. soehngenii” MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of “Ca. G. soehngenii” enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of “Ca. G. soehngenii.”","C-labeling; acetogenesis; chemostat enrichment culture; meta-transcriptomics; Wood-Ljungdahl pathway","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:a21c570e-9f62-41df-a4ff-60266a444cc5","http://resolver.tudelft.nl/uuid:a21c570e-9f62-41df-a4ff-60266a444cc5","Biological phosphorus removal in seawater-adapted aerobic granular sludge","de Graaff, D.R. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV)","","2020","Seawater can be introduced or intrude in sewer systems and can thereby negatively influence biological wastewater treatment processes. Here we studied the impact of artificial seawater on the enhanced biological phosphate removal (EBPR) process performance by aerobic granular sludge (AGS) with synthetic wastewater. Process performance, granule stability and characteristics as well as microbial community of a seawater-adapted AGS system were observed. In seawater conditions strong and stable granules formed with an SVI5 of 20 mL/g and a lower abrasion coefficient than freshwater-adapted granules. Complete anaerobic uptake of acetate, anaerobic phosphate release of 59.5 ± 4.0 mg/L PO43--P (0.35 mg P/mg HAc), and an aerobic P-uptake rate of 3.1 ± 0.2 mg P/g VSS/h were achieved. The dominant phosphate accumulating organisms (PAO) were the same as for freshwater-based aerobic granular sludge systems with a very high enrichment of Ca. Accumulibacter phosphatis clade I, and complete absence of glycogen accumulating organisms. The effect of osmotic downshocks was tested by replacing influent seawater-based medium by demineralized water-based medium. A temporary decrease of the salinity in the reactor led to a decreased phosphate removal activity, while it also induced a rapid release of COD by the sludge, up to 45.5 ± 1.7 mg COD/g VSS. This is most likely attributed to the release of osmolytes by the cells. Recovery of activity was immediately after restoring the seawater feeding. This work shows that functioning of aerobic granular sludge in seawater conditions is as stable as in freshwater conditions, while past research has shown a negative effect on operation of AGS processes with NaCl-based wastewater at the same salinity as seawater.","Accumulibacter phosphatis; Aerobic granular sludge; Biological phosphorus removal; Salinity; Seawater","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:2d9a5e3f-c804-4a3a-b390-c5b77390562a","http://resolver.tudelft.nl/uuid:2d9a5e3f-c804-4a3a-b390-c5b77390562a","Stress-induced assays for polyphosphate quantification by uncoupling acetic acid uptake and anaerobic phosphorus release","Feng, Cuijie (Politecnico di Milano); Welles, L. (TU Delft BT/Environmental Biotechnology; IHE Delft Institute for Water Education); Zhang, X. (TU Delft Sanitary Engineering); Pronk, M. (TU Delft BT/Environmental Biotechnology); de Graaff, D.R. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology)","","2020","Phosphorus has been successfully eliminated from wastewater by biological techniques of enhanced biological phosphorus removal (EBPR) process, which relies on a specific microbiota of polyphosphate accumulating organisms (PAOs) that accumulate phosphate as polyphosphates (poly-P). Most methods for quantification of poly-P pools suffer from low accuracy and specificity. More powerful and implementable P-analysis tools are required for poly-P quantification, which will help in improved evaluation of processes in laboratory and full-scale EBPR systems. This study developed two methods to quantify poly-P pools by releasing the poly-P from the cell. During experimental optimization, it was observed that two different methods resulted in the highest phosphate release: acetate addition at a pH of 4.8 and exposure to EDTA solution with a concentration of 1% (w/v). Treatment with EDTA resulted in a higher amount of phosphate release from all sludge samples. This was characterized by P-release of 1.5–2.5 times higher than the control tests. In contrast, treatments with acetate addition at a low pH exhibited that P-release depended upon the types of the sludge samples. The highest P-release amount and rate were found in highly-enriched PAO sludge samples, but with fewer influences on the sludge collected from WWTP, which may be attributed to the lower fraction of PAOs in the sludge. Overall, the proposed approaches to quantify the poly-P concentration can be applied in simple, user-friendly, and cost-effective ways.","Enhanced biological phosphorus removal (EBPR); Polyphosphate (poly-P); Polyphosphate-accumulating organisms (PAOs); Quantification","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:5a25160c-53ec-4a1e-8fca-7d13590fd21f","http://resolver.tudelft.nl/uuid:5a25160c-53ec-4a1e-8fca-7d13590fd21f","A settling model for full-scale aerobic granular sludge","van Dijk, E.J.H. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology)","","2020","The settling behavior of aerobic granular sludge (AGS) in full-scale reactors is different from the settling of normal activated sludge. Current activated sludge models lack the features to describe the segregation of granules based on size during the settling process. This segregation plays an important role in the granulation process and therefore a better understanding of the settling is essential. The goal of this study was to model and evaluate the segregation of different granule sizes during settling and feeding in full-scale aerobic granular sludge reactors. Hereto the Patwardhan and Tien model was used. This model is an implementation of the Richardson and Zaki model, allowing for multiple classes of particles. To create the granular settling model, the most relevant parameters were identified using aerobic granular sludge from different full-scale Nereda® reactors. The settling properties of individual granules were measured as was the bulk behavior of granular sludge beds with uniform granular sludge particles. The obtained parameters were combined in a model containing multiple granule classes, which then was validated for granular sludge settling in a full-scale Nereda® reactor. In practice a hydraulic selection pressure is used to select for granular sludge. Under the same hydraulic selection pressure the model predicted that different stable granular size distributions can occur. This indicates that granular size distribution control would need a different mechanism then the hydraulic selection pressure alone. This model can be used to better understand and optimize operational parameters of AGS reactors that depend on granular sludge size, like biological nutrient removal. Furthermore insights from this model can also be used in the development of continuously fed AGS systems.","Aerobic granular sludge; Design; Full-scale; Model; Practice; Richardson and Zaki; Settling; Wastewater","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:1962bc44-c49a-4b0b-aece-062082d18540","http://resolver.tudelft.nl/uuid:1962bc44-c49a-4b0b-aece-062082d18540","Impact of aerobic availability of readily biodegradable COD on morphological stability of aerobic granular sludge","Haaksman, V.A. (TU Delft BT/Environmental Biotechnology); Mirghorayshi, M. (Razi University); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV)","","2020","Operational disturbances in aerobic granular sludge (AGS) systems can result in aerobic availability of readily biodegradable COD (rbCOD). Different from activated sludge, morphological consequences on the short and long term are not well described in literature. This study investigated the effect of incomplete anaerobic uptake of acetate on the morphological and process stability of AGS using a lab-scale reactor. A fraction of the total acetate load was dosed aerobically, which was increased stepwise while monitoring granular morphology. A good granular morphology and an SVI of 40 ml/g were obtained during initial enrichment and maintained for ≤20% aerobic acetate load dosed at 4 mg COD/g VSS/h. Biological phosphorus removal efficiency was initially unaffected, but the aerobic acetate dosage rate did decrease the aerobic phosphate uptake rate. This led to loss of phosphorus removal for >20% aerobic acetate load dosed at 8 mg COD/g VSS/h over the course of 12 days. Subsequently, significant outgrowth formed on the granular surfaces and developed over time into finger-like structures. Under these high aerobic acetate loads the SVI increased to 80 ml/g and resulted in significant biomass washout due to deteriorating settling properties of the sludge. The sludge settleability and biological phosphorus removal recovered 10 days after aerobic feeding of acetate was stopped. Aerobic presence of rbCOD can be tolerated if mostly anaerobic acetate uptake is maintained, thereby ensuring stable granular morphology and good settleability. The high enrichment of phosphate accumulating organisms in the granular sludge through bottom-feeding and selective wasting of flocs makes aerobic granular sludge resilient to morphological deterioration in aerobic presence of rbCOD.","Aerobic granular sludge; Biological phosphate removal; Bulking sludge; EBPR; Readily biodegradable COD; Sludge morphology","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:f246978b-16d5-4ce1-8b38-990e5e425dcf","http://resolver.tudelft.nl/uuid:f246978b-16d5-4ce1-8b38-990e5e425dcf","Trehalose as an osmolyte in Candidatus Accumulibacter phosphatis","de Graaff, D.R. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV)","","2020","Abstract: Candidatus Accumulibacter phosphatis is an important microorganism for enhanced biological phosphorus removal (EBPR). In a previous study, we found a remarkable flexibility regarding salinity, since this same microorganism could thrive in both freshwater- and seawater-based environments, but the mechanism for the tolerance to saline conditions remained unknown. Here, we identified and described the role of trehalose as an osmolyte in Ca. Accumulibacter phosphatis. A freshwater-adapted culture was exposed to a single batch cycle of hyperosmotic and hypo-osmotic shock, which led to the release of trehalose up to 5.34 mg trehalose/g volatile suspended solids (VSS). Long-term adaptation to 30% seawater-based medium in a sequencing batch reactor (SBR) gave a stable operation with complete anaerobic uptake of acetate and propionate along with phosphate release of 0.73 Pmol/Cmol, and complete aerobic uptake of phosphate. Microbial analysis showed Ca. Accumulibacter phosphatis clade I as the dominant organism in both the freshwater- and seawater-adapted cultures (> 90% presence). Exposure of the seawater-adapted culture to a single batch cycle of hyperosmotic incubation and hypo-osmotic shock led to an increase in trehalose release upon hypo-osmotic shock when higher salinity is used for the hyperosmotic incubation. Maximum trehalose release upon hypo-osmotic shock was achieved after hyperosmotic incubation with 3× salinity increase relative to the salinity in the SBR adaptation reactor, resulting in the release of 11.9 mg trehalose/g VSS. Genome analysis shows the possibility of Ca. Accumulibacter phosphatis to convert glycogen into trehalose by the presence of treX, treY, and treZ genes. Addition of trehalose to the reactor led to its consumption, both during anaerobic and aerobic phases. These results indicate the flexibility of the metabolism of Ca. Accumulibacter phosphatis towards variations in salinity. Key points: • Trehalose is identified as an osmolyte in Candidatus Accumulibacter phosphatis. • Ca. Accumulibacter phosphatis can convert glycogen into trehalose. • Ca. Accumulibacter phosphatis clade I is present and active in both seawater and freshwater.","Accumulibacter; EBPR; Phosphate; Salinity; Seawater; Trehalose","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:d392bceb-4b6e-4c51-9949-6b212600b233","http://resolver.tudelft.nl/uuid:d392bceb-4b6e-4c51-9949-6b212600b233","Hormones in speed-dating: The role of testosterone and cortisol in attraction","van der Meij, Leander (Eindhoven University of Technology); Demetriou, A.M. (TU Delft Multimedia Computing); Tulin, Marina (Universiteit van Amsterdam); Méndez, Ileana (Vrije Universiteit Amsterdam); Dekker, Peter (Vrije Universiteit Amsterdam); Pronk, Tila (Tilburg University)","","2019","There is evidence that testosterone and cortisol levels are related to the attraction of a romantic partner; testosterone levels relate to a wide range of sexual behaviors and cortisol is a crucial component in the response to stress. To investigate this, we conducted a speed-dating study among heterosexual singles. We measured salivary testosterone and cortisol changes in men and women (n = 79) when they participated in a romantic condition (meeting opposite-sex others, i.e., potential romantic partners), as well as a control condition (meeting same-sex others, i.e., potential friends). Over the course of the romantic speed-dating event, results showed that women's but not men's testosterone levels increased and cortisol levels decreased for both men and women. These findings indicate that men's testosterone and cortisol levels were elevated in anticipation of the event, whereas for women, this appears to only be the case for cortisol. Concerning the relationship between attraction and hormonal change, four important findings can be distinguished. First, men were more popular when they arrived at the romantic speed-dating event with elevated cortisol levels. Second, in both men and women, a larger change in cortisol levels during romantic speed-dating was related to more selectivity. Third, testosterone alone was unrelated to any romantic speed-dating outcome (selectivity or popularity). However, fourth, women who arrived at the romantic speed-dating event with higher testosterone levels were more selective when their anticipatory cortisol response was low. Overall, our findings suggest that changes in the hormone cortisol may be stronger associated with the attraction of a romantic partner than testosterone.","Attraction; Cortisol; Human mating; Popularity; Selectivity; Social relation model; Speed-dating; Testosterone","en","journal article","","","","","","","","2020-11-06","","","Multimedia Computing","","",""
"uuid:3cc590ec-fee7-4fec-9e4c-874b300b30b5","http://resolver.tudelft.nl/uuid:3cc590ec-fee7-4fec-9e4c-874b300b30b5","Quantitative Physiology of Non-Energy-Limited Retentostat Cultures of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates","Liu, Y. (TU Delft OLD BT/Cell Systems Engineering); El Masoudi, Anissa (Student TU Delft); Pronk, J.T. (TU Delft BT/Biotechnologie); van Gulik, W.M. (TU Delft OLD BT/Cell Systems Engineering)","","2019","So far, the physiology of Saccharomyces cerevisiae at near-zero growth rates has been studied in retentostat cultures with a growth-limiting supply of the carbon and energy source. Despite its relevance in nature and industry, the near-zero growth physiology of S. cerevisiae under conditions where growth is limited by the supply of non-energy substrates remains largely unexplored. This study analyzes the physiology of S. cerevisiae in aerobic chemostat and retentostat cultures grown under either ammonium or phosphate limitation. To compensate for loss of extracellular nitrogen- or phosphorus-containing compounds, establishing near-zero growth rates (μ < 0.002 h-1) in these retentostats required addition of low concentrations of ammonium or phosphate to reservoir media. In chemostats as well as in retentostats, strongly reduced cellular contents of the growth-limiting element (nitrogen or phosphorus) and high accumulation levels of storage carbohydrates were observed. Even at near-zero growth rates, culture viability in non-energy-limited retentostats remained above 80% and ATP synthesis was still sufficient to maintain an adequate energy status and keep cells in a metabolically active state. Compared to similar glucose-limited retentostat cultures, the nitrogen- and phosphate-limited cultures showed aerobic fermentation and a partial uncoupling of catabolism and anabolism. The possibility to achieve stable, near-zero growth cultures of S. cerevisiae under nitrogen or phosphorus limitation offers interesting prospects for high-yield production of bio-based chemicals.IMPORTANCE The yeast Saccharomyces cerevisiae is a commonly used microbial host for production of various biochemical compounds. From a physiological perspective, biosynthesis of these compounds competes with biomass formation in terms of carbon and/or energy equivalents. Fermentation processes functioning at extremely low or near-zero growth rates would prevent loss of feedstock to biomass production. Establishing S. cerevisiae cultures in which growth is restricted by the limited supply of a non-energy substrate therefore could have a wide range of industrial applications but remains largely unexplored. In this work we accomplished near-zero growth of S. cerevisiae through limited supply of a non-energy nutrient, namely, the nitrogen or phosphorus source, and carried out a quantitative physiological study of the cells under these conditions. The possibility to achieve near-zero-growth S. cerevisiae cultures through limited supply of a non-energy nutrient may offer interesting prospects to develop novel fermentation processes for high-yield production of bio-based chemicals.","carbon excess; near-zero growth; non-energy limitation; retentostat; yeast physiology","en","journal article","","","","","","Accepted Author Manuscript","","2020-03-15","","BT/Biotechnologie","OLD BT/Cell Systems Engineering","","",""
"uuid:ff999fe2-8ec1-44d5-960b-b71031329fb2","http://resolver.tudelft.nl/uuid:ff999fe2-8ec1-44d5-960b-b71031329fb2","Anaerobic growth of Saccharomyces cerevisiae CEN.PK113-7D does not depend on synthesis or supplementation of unsaturated fatty acids","Dekker, W.J.C. (TU Delft BT/Industriele Microbiologie); Wiersma, S.J. (TU Delft BT/Industriele Microbiologie); Bouwknegt, J. (TU Delft BT/Industriele Microbiologie); Mooiman, C. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2019","In Saccharomyces cerevisiae, acyl-coenzyme A desaturation by Ole1 requires molecular oxygen. Tween 80, a poly-ethoxylated sorbitan-oleate ester, is therefore routinely included in anaerobic growth media as a source of unsaturated fatty acids (UFAs). During optimization of protocols for anaerobic bioreactor cultivation of this yeast, we consistently observed growth of the laboratory strain S. cerevisiae CEN.PK113-7D in media that contained the anaerobic growth factor ergosterol, but lacked UFAs. To minimize oxygen contamination, additional experiments were performed in an anaerobic chamber. After anaerobic precultivation without ergosterol and Tween 80, strain CEN.PK113-7D and a congenic ole1Δ strain both grew during three consecutive batch-cultivation cycles on medium that contained ergosterol, but not Tween 80. During these three cycles, no UFAs were detected in biomass of cultures grown without Tween 80, while contents of C10 to C14 saturated fatty acids were higher than in biomass from Tween 80-supplemented cultures. In contrast to its UFA-independent anaerobic growth, aerobic growth of the ole1Δ strain strictly depended on Tween 80 supplementation. This study shows that the requirement of anaerobic cultures of S. cerevisiae for UFA supplementation is not absolute and provides a basis for further research on the effects of lipid composition on yeast viability and robustness.","S. cerevisiae; anaerobic; membrane composition; OLE1; oxygen requirement; unsaturated fatty acids","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:13c3049b-8515-4cd4-9a2c-f8f44e96971c","http://resolver.tudelft.nl/uuid:13c3049b-8515-4cd4-9a2c-f8f44e96971c","Lager-brewing yeasts in the era of modern genetics","Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2019","The yeast Saccharomyces pastorianus is responsible for the annual worldwide production of almost 200 billion liters of lager-type beer. S. pastorianus is a hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been studied for well over a century. Scientific interest in S. pastorianus intensified upon the discovery, in 2011, of its S. eubayanus ancestor. Moreover, advances in whole-genome sequencing and genome editing now enable deeper exploration of the complex hybrid and aneuploid genome architectures of S. pastorianus strains. These developments not only provide novel insights into the emergence and domestication of S. pastorianus but also generate new opportunities for its industrial application. This review paper combines historical, technical and socioeconomic perspectives to analyze the evolutionary origin and genetics of S. pastorianus. In addition, it provides an overview of available methods for industrial strain improvement and an outlook on future industrial application of lager-brewing yeasts. Particular attention is given to the ongoing debate on whether current S. pastorianus originates from a single or multiple hybridization events and to the potential role of genome editing in developing industrial brewing yeast strains.","Saccharomyces pastorianus; genome editing; hybrid heterosis; strain improvement; whole genome sequencing","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:b20bcffa-2e3f-46d7-a678-83ceb6eff228","http://resolver.tudelft.nl/uuid:b20bcffa-2e3f-46d7-a678-83ceb6eff228","Terra–ink additive earth manufacturing for emergency architecture","Venturini, T. (TU Delft Applied Mechanics); Turrin, M. (TU Delft Design Informatics); Setaki, F. (TU Delft Environmental Technology and Design); Veer, F.A. (TU Delft Structural Design & Mechanics); Pronk, Arno (Eindhoven University of Technology); Teuffel, Patrick (Eindhoven University of Technology); Moonen, Yaron (Eindhoven University of Technology); Slangen, Stefan (Eindhoven University of Technology); Vorstermans, Rens (Eindhoven University of Technology)","","2019","In recent years, natural disaster and military conflicts forced vast numbers of people to flee their home countries, contributing to the migration crisis we are facing today. According to the UNHCR, the number of forcibly displaced people worldwide reached the highest level since World War II. Post-disaster housing is by nature diverse and dynamic, having to satisfy unique socio-cultural and economical requirements. Currently, however, housing emergencies are tackled inefficiently. Post-disaster housing strategies are characterized by a high economic impact and waste production, and a low adaptability to location-based needs. As an outcome, low quality temporary shelters are provided, which often exceed by far their serving time. Focusing on temporary shelters suitable for the transitioning period between emergency accommodation and permanent housing, TERRA-ink addresses new construction methods that allow for time and cost efficiency, but also for flexibility to adapt to different contexts. TERRA-ink aims to develop a method for layering local soil, by implementing 3D printing technologies. With the aid of such a construction system, the goal is to create durable structures that can be easily de-constructed once they served their purpose. The use of locally sourced materials in combination with additive manufacturing is investigated aiming at reductions in financial investments, resources and human labor, as well as at simplified logistics, low environmental impact and adaptability to different situations and requirements. Such a building system has the potential of combining low-and high-tech technologies, in order to facilitate a fully open and universal solution for large scale 3D-printing using any type of soil.","Emergency; Extrusion; Material; Mixture; Process; Soil; Structure; Temporary","en","journal article","","","","","","Energy Innovation #5: 4TU.BOUW Lighthouse projects + PDEng ISBN 978-94-6366-246-8","","","","","Design Informatics","","",""
"uuid:0f8ed879-f0eb-4c50-8989-5de0d7bed07b","http://resolver.tudelft.nl/uuid:0f8ed879-f0eb-4c50-8989-5de0d7bed07b","Physiological responses of Saccharomyces cerevisiae to industrially relevant conditions: Slow growth, low pH, and high CO2 levels","Hakkaart, X.D.V. (TU Delft BT/Industriele Microbiologie); Liu, Y. (TU Delft OLD BT/Cell Systems Engineering); Hulst, Mandy (Student TU Delft); el Masoudi, Anissa (Student TU Delft); Peuscher, Eveline (Student TU Delft); Pronk, J.T. (TU Delft BT/Biotechnologie); van Gulik, W.M. (TU Delft OLD BT/Cell Systems Engineering); Daran-Lapujade, P.A.S. (TU Delft BT/Industriele Microbiologie)","","2019","Engineered strains of Saccharomyces cerevisiae are used for industrial production of succinic acid. Optimal process conditions for dicarboxylic-acid yield and recovery include slow growth, low pH, and high CO2. To quantify and understand how these process parameters affect yeast physiology, this study investigates individual and combined impacts of low pH (3.0) and high CO2 (50%) on slow-growing chemostat and retentostat cultures of the reference strain S. cerevisiae CEN.PK113-7D. Combined exposure to low pH and high CO2 led to increased maintenance-energy requirements and death rates in aerobic, glucose-limited cultures. Further experiments showed that these effects were predominantly caused by low pH. Growth under ammonium-limited, energy-excess conditions did not aggravate or ameliorate these adverse impacts. Despite the absence of a synergistic effect of low pH and high CO2 on physiology, high CO2 strongly affected genome-wide transcriptional responses to low pH. Interference of high CO2 with low-pH signaling is consistent with low-pH and high-CO2 signals being relayed via common (MAPK) signaling pathways, notably the cell wall integrity, high-osmolarity glycerol, and calcineurin pathways. This study highlights the need to further increase robustness of cell factories to low pH for carboxylic-acid production, even in organisms that are already applied at industrial scale.","acid stress; carbon dioxide; carboxylic acid; yeast; zero-growth","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:205dff5d-8e75-4670-bc83-5dbc6a83d910","http://resolver.tudelft.nl/uuid:205dff5d-8e75-4670-bc83-5dbc6a83d910","In vivo recombination of Saccharomyces eubayanus maltose-transporter genes yields a chimeric transporter that enables maltotriose fermentation","Brouwers, N. (TU Delft BT/Industriele Microbiologie; TU Delft OLD BT/Cell Systems Engineering); Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie; TU Delft OLD BT/Cell Systems Engineering); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie; TU Delft OLD BT/Cell Systems Engineering); Weening, S.M. (TU Delft BT/Industriele Microbiologie; TU Delft OLD BT/Cell Systems Engineering); Elink Schuurman, Tom D. (Heineken Supply Chain); Kuijpers, Niels G.A. (Heineken Supply Chain); Pronk, J.T. (TU Delft BT/Industriele Microbiologie; TU Delft OLD BT/Cell Systems Engineering); Daran, J.G. (TU Delft BT/Industriele Microbiologie; TU Delft OLD BT/Cell Systems Engineering)","","2019","Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer's wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357T, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.","","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:31774726-41c7-4b19-ab8e-8fc4229092bf","http://resolver.tudelft.nl/uuid:31774726-41c7-4b19-ab8e-8fc4229092bf","Laboratory Evolution of a Saccharomyces cerevisiae × S. eubayanus Hybrid Under Simulated Lager-Brewing Conditions","Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie); Voskamp, Maaike (Student TU Delft); van Aalst, Aafke (Student TU Delft); Kristensen, L.H. (Student TU Delft); Jansen, Liset (Student TU Delft); Salazar, A.N. (TU Delft Pattern Recognition and Bioinformatics); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Brouwers, N. (TU Delft BT/Industriele Microbiologie); Abeel, T.E.P.M.F. (TU Delft Pattern Recognition and Bioinformatics; Broad Institute of MIT and Harvard); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2019","Saccharomyces pastorianus lager-brewing yeasts are domesticated hybrids of S. cerevisiae x S. eubayanus that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid of S. cerevisiae and S. eubayanus was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploid S. pastorianus brewing strains. In contrast to the extensive aneuploidy of domesticated S. pastorianus strains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilization and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticated S. pastorianus strains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation of S. cerevisiae × S. eubayanus hybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids.","Saccharomyces pastorianus; loss of heterozygosity; ,laboratory evolution; ,domestication; maltotriose utilization; flocculation; Maltotriose utilization; Flocculation; Loss of heterozygosity; Domestication; Laboratory evolution","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:a53d14d5-3abc-4f99-9df0-d0f923eafeb7","http://resolver.tudelft.nl/uuid:a53d14d5-3abc-4f99-9df0-d0f923eafeb7","Analysis of centrifugal homogenization and its applications for emulsification & mechanical cell lysis","Singh, Kaustub (Student TU Delft); Gupta, Ankur (Princeton University); Buchner, A.J.L.L. (TU Delft Fluid Mechanics); Ibis, F. (TU Delft Intensified Reaction and Separation Systems); Pronk, J.W. (TU Delft BN/Arjen Jakobi Lab); Tam, D.S.W. (TU Delft Fluid Mechanics); Eral, H.B. (TU Delft Intensified Reaction and Separation Systems; Universiteit Utrecht)","","2019","We detail the analysis of centrifugal homogenization process by a hydrodynamic model and the model-guided design of a low-cost centrifugal homogenizer. During operation, centrifugal force pushes a multiphase solution to be homogenized through a thin nozzle, consequently homogenizing its contents. We demonstrate and assess the homogenization of coarse emulsions into relatively monodisperse emulsions, as well as the application of centrifugal homogenization in the mechanical lysis of mpkCCD mouse kidney cells. To gain insight into the homogenization mechanism, we investigate the dependence of emulsion droplet size on geometrical parameters, centrifugal acceleration, and dispersed phase viscosity. Our experimental results are in qualitative agreement with models predicting the droplet size. Furthermore, they indicate that high shear rates kept constant throughout operation produce more monodisperse droplets. We show this ideal homogenization condition can be realized through hydrodynamic model-guided design minimizing transient effects inherent to centrifugal homogenization. Moreover, we achieved power densities comparable to commercial homogenizers by model guided optimization of homogenizer design and experimental conditions. Centrifugal homogenization using the proposed homogenizer design thus offers a low-cost alternative to existing technologies as it is constructed from off-the-shelf parts (Falcon tubes, syringe, needles) and used with a centrifuge, readily available in standard laboratory environment.","Centrifugal emulsification; Centrifugation; Emulsions; Homogenizer; Lysis","en","journal article","","","","","","Green Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.","","2020-09-20","","","Fluid Mechanics","","",""
"uuid:d19bd6f5-dc13-48cc-b625-c20846fcb74f","http://resolver.tudelft.nl/uuid:d19bd6f5-dc13-48cc-b625-c20846fcb74f","Sialic acids in the extracellular polymeric substances of seawater-adapted aerobic granular sludge","de Graaff, D.R. (TU Delft BT/Environmental Biotechnology); Felz, S. (TU Delft BT/Environmental Biotechnology); Neu, Thomas R. (Helmholtz Centre for Environmental Research - UFZ); Pronk, M. (TU Delft BT/Environmental Biotechnology); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Lin, Y. (TU Delft Environmental Fluid Mechanics)","","2019","Sialic acids have been discovered in the extracellular polymeric substances (EPS) of seawater-adapted aerobic granular sludge (AGS). Sialic acids are a group of monosaccharides with a nine-carbon backbone, commonly found in mammalian cells and pathogenic bacteria, and frequently described to protect EPS molecules and cells from attack by proteases or glycosidases. In order to further understand the role of these compounds in AGS, lectin staining, genome analysis of the dominant bacterial species, and shielding tests were done. Fluorescence lectin bar-coding (FLBC) analysis showed an overlap with protein staining, indicating presence of sialoglycoproteins in the EPS matrix. Genome analysis gives a positive indication for putative production of sialic acids by the dominant bacteria Candidatus Accumulibacter. FT-IR analysis shows upon selective removal of sialic acids a decrease in carbohydrates, extension of the protein side chain, and exposure of penultimate sugars. Enzymatic removal of sialic acids results in the removal of galactose residues from the EPS upon subsequent treatment with β-galactosidase, indicating a linkage between galactose and sialic acid at the terminus of glycan chains. This work indicates the importance of sialic acids in the protection of penultimate sugar residues of glycoproteins in EPS, and provides basis for future research in the composition of EPS from biofilms and granular sludge.","Aerobic granular sludge; EPS; Glycoproteins; Neuraminic acid; Salinity; Seawater; Sialic acids","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:9bdd4b62-ac11-4614-931b-a6fc247e508f","http://resolver.tudelft.nl/uuid:9bdd4b62-ac11-4614-931b-a6fc247e508f","Phenotype-independent isolation of interspecies Saccharomyces hybrids by dual-dye fluorescent staining and fluorescence-activated cell sorting","Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie); Koster, C.C. (TU Delft BT/Industriele Microbiologie); Weening, S.M. (TU Delft BT/Industriele Microbiologie); Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); Kuijpers, N.G.A. (TU Delft BT/Biotechnologie); Geertman, Jan Maarten A. (Heineken Netherlands Supply); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2019","Interspecies hybrids of Saccharomyces species are found in a variety of industrial environments and often outperform their parental strains in industrial fermentation processes. Interspecies hybridization is therefore increasingly considered as an approach for improvement and diversification of yeast strains for industrial application. However, current hybridization methods are limited by their reliance on preexisting or introduced selectable phenotypes. This study presents a high-throughput phenotype-independent method for isolation of interspecies Saccharomyces hybrids based on dual dye-staining and subsequent mating of two strains, followed by enrichment of double-stained hybrid cells from a mating population by fluorescence-activated cell sorting (FACS). Pilot experiments on intra-species mating of heterothallic haploid S. cerevisiae strains showed that 80% of sorted double-stained cells were hybrids. The protocol was further optimized by mating an S. cerevisiae haploid with homothallic S. eubayanus spores with complementary selectable phenotypes. In crosses without selectable phenotype, using S. cerevisiae and S. eubayanus haploids derived from laboratory as well as industrial strains, 10 to 15% of double-stained cells isolated by FACS were hybrids. When applied to rare mating, sorting of double-stained cells consistently resulted in about 600-fold enrichment of hybrid cells. Mating of dual-stained cells and FACS-based selection allows efficient enrichment of interspecies Saccharomyces hybrids within a matter of days and without requiring selectable hybrid phenotypes, both for homothallic and heterothallic strains. This strategy should accelerate the isolation of laboratory-made hybrids, facilitate research into hybrid heterosis and offer new opportunities for non-GM industrial strain improvement and diversification.","FACS; Heterosis; Lager beer brewing; Marker-free mating; Non-GMO; Saccharomyces eubayanus × Saccharomyces cerevisiae hybrids","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:92e53881-f20e-4432-9106-7fe5541d8d93","http://resolver.tudelft.nl/uuid:92e53881-f20e-4432-9106-7fe5541d8d93","Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae","Else-Hassing, J. (TU Delft BT/Industriele Microbiologie); de Groot, P.A. (TU Delft BT/Industriele Microbiologie); Marquenie, Vita R. (Student TU Delft); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2019","The organic compound 2-phenylethanol (2PE) has a pleasant floral scent and is intensively used in the cosmetic and food industries. Microbial production of 2PE by phenylalanine bioconversion or de novo biosynthesis from sugar offer sustainable, reliable and natural production processes compared to chemical synthesis. Despite the ability of Saccharomyces cerevisiae to naturally synthesize 2PE, de novo synthesis in high concentration and yield remains a metabolic engineering challenge. Here, we demonstrate that improving phosphoenolpyruvate supply by expressing pyruvate kinase variants and eliminating the formation of p-hydroxy-phenylethanol without creating tyrosine auxotrophy significantly contributed to improve 2PE production in S. cerevisiae. In combination with the engineering of the aromatic amino acid biosynthesis and Ehrlich pathway, these mutations enabled better connection between glycolysis and pentose phosphate pathway optimizing carbon flux towards 2PE. However, attempts to further connect these two parts of central carbon metabolism by redirecting fructose-6P towards erythrose-4P by expressing a phosphoketolase-phosphotransacetylase pathway did not result in improved performance. The best performing strains were capable of producing 13mM of 2PE at a yield of 0.113 mol mol-1, which represents the highest yield for de novo produced 2PE in S. cerevisiae and other yeast species.","2-Phenylethanol; Aromatic amino acid pathway engineering; de novo biosynthesis; Prephenate dehydrogenase downregulation; Pyruvate kinase; Saccharomyces cerevisiae","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:e84bed8a-7b39-4b5a-9d97-34baf03454d4","http://resolver.tudelft.nl/uuid:e84bed8a-7b39-4b5a-9d97-34baf03454d4","Functional expression of a bacterial α-ketoglutarate dehydrogenase in the cytosol of Saccharomyces cerevisiae","Baldi, N. (TU Delft BT/Industriele Microbiologie); Dykstra, James C. (Student TU Delft); Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); Pabst, Martin (TU Delft OLD BT/Cell Systems Engineering); Wu, Liang (DSM); Benjamin, Kirsten R. (Amyris Inc); Vente, André (DSM); Pronk, J.T. (TU Delft BT/Biotechnologie); Mans, R. (TU Delft BT/Industriele Microbiologie)","","2019","Efficient production of fuels and chemicals by metabolically engineered micro-organisms requires availability of precursor molecules for product pathways. In eukaryotic cell factories, heterologous product pathways are usually expressed in the cytosol, which may limit availability of precursors that are generated in other cellular compartments. In Saccharomyces cerevisiae, synthesis of the precursor molecule succinyl-Coenzyme A is confined to the mitochondrial matrix. To enable cytosolic synthesis of succinyl-CoA, we expressed the structural genes for all three subunits of the Escherichia coli α-ketoglutarate dehydrogenase (αKGDH) complex in S. cerevisiae. The E. coli lipoic-acid scavenging enzyme was co-expressed to enable cytosolic lipoylation of the αKGDH complex, which is required for its enzymatic activity. Size-exclusion chromatography and mass spectrometry indicated that the heterologously expressed αKGDH complex contained all subunits and that its size was the same as in E. coli. Functional expression of the heterologous complex was evident from increased αKGDH activity in the cytosolic fraction of yeast cell homogenates. In vivo cytosolic activity of the αKGDH complex was tested by constructing a reporter strain in which the essential metabolite 5-aminolevulinic acid could only be synthetized from cytosolic, and not mitochondrial, succinyl-CoA. To this end HEM1, which encodes the succinyl-CoA-converting mitochondrial enzyme 5-aminolevulinic acid (ALA) synthase, was deleted and a bacterial ALA synthase was expressed in the cytosol. In the resulting strain, complementation of ALA auxotrophy depended on activation of the αKGDH complex by lipoic acid addition. Functional expression of a bacterial αKGDH complex in yeast represents a vital step towards efficient yeast-based production of compounds such as 1,4-butanediol and 4-aminobutyrate, whose product pathways use succinyl-CoA as a precursor.","5-Aminolevulinic acid; Compartmentalization; Lipoylation; Precursor supply; Proteomics; Succinyl-CoA","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:b6c599e8-077c-44f1-bb71-e731bcc7d81f","http://resolver.tudelft.nl/uuid:b6c599e8-077c-44f1-bb71-e731bcc7d81f","Aquaporin-2 trafficking: Studying cellular mechanisms with subcellular aspiration and cryo-electron microscopy","Pronk, J.W. (TU Delft BN/Arjen Jakobi Lab)","Engel, A.H. (promotor); Danelon, C.J.A. (copromotor); Delft University of Technology (degree granting institution)","2018","","Aquaporin-2; exocytosis; endocytosis; protein purification; membrane proteins; Fluid-FM; Atomic force microscopy; Electron microscopy; cryo-EM","en","doctoral thesis","","978-90-8593-360-1","","","","","","2020-09-05","","","BN/Arjen Jakobi Lab","","",""
"uuid:8e6d6192-2cdf-4058-9d38-3aa603e95194","http://resolver.tudelft.nl/uuid:8e6d6192-2cdf-4058-9d38-3aa603e95194","Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield","Papapetridis, I. (TU Delft BT/Industriele Microbiologie); Goudriaan, M. (TU Delft BT/Industriele Microbiologie); Vazquez Vitali, M. (TU Delft Applied Sciences); De Keijzer, Nikita A.; van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie; AlbaNova University Center); Pronk, J.T. (TU Delft BT/Industriele Microbiologie)","","2018","Background: Reduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast Saccharomyces cerevisiae. Anaerobic cultures of wild-type S. cerevisiae require formation of glycerol to maintain the intracellular NADH/NAD+ balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in S. cerevisiae was shown to enable reoxidation of NADH with CO2 as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield. Results: Multi-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the Escherichia coli GroEL/GroES chaperones and expression of PRK from the anaerobically inducible DAN1 promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting S. cerevisiae strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of GPD2, which encodes an isoenzyme of NAD+-dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO2-reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield. Conclusions: The metabolic engineering strategy presented here enables an almost complete elimination of glycerol production in anaerobic, glucose-grown batch cultures of S. cerevisiae, with an associated increase in ethanol yield, while retaining near wild-type growth rates and a capacity for glycerol formation under osmotic stress. Using current genome-editing techniques, the required genetic modifications can be introduced in one or a few transformations. Evaluation of this concept in industrial strains and conditions is therefore a realistic next step towards its implementation for improving the efficiency of first- and second-generation bioethanol production.","Anaerobic metabolism; Biofuels; CO; Fermentation; NADH; NADPH; Redox cofactor balance; Yeast","en","journal article","","","","","","","","","Applied Sciences","","BT/Industriele Microbiologie","","",""
"uuid:8acf4570-d02b-4ee4-997f-ac709ed6daa3","http://resolver.tudelft.nl/uuid:8acf4570-d02b-4ee4-997f-ac709ed6daa3","Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid","Jürgens, H. (TU Delft BT/Industriele Microbiologie); Varela, Javier A. (University College Cork); Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie); Perli, T. (TU Delft BT/Industriele Microbiologie); Gast, V.J.M. (Student TU Delft); Gyurchev, N.Y. (Student TU Delft); Rajkumar, Arun S. (University College Cork); Mans, R. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Morrissey, John P. (University College Cork); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2018","While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies ( < 1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.","CRISPR/Cas9; Hansenula polymorpha; Kluyveromyces lactis; Kluyveromyces marxianus; Ogataea polymorpha; Ribozymes","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:1a491e08-24b6-4f87-95d1-0ce8aa47aab4","http://resolver.tudelft.nl/uuid:1a491e08-24b6-4f87-95d1-0ce8aa47aab4","Structural, physiological and regulatory analysis of maltose transporter genes in Saccharomyces eubayanus CBS 12357T","Brickwedde, A. (TU Delft BT/Industriele Microbiologie); Brouwers, N. (TU Delft BT/Industriele Microbiologie); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Gallego Murillo, Joan Sebastián (TU Delft BT/Bioprocess Engineering); Fraiture, J.L. (TU Delft Medezeggenschapsondersteuning); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2018","Saccharomyces pastorianus lager brewing yeasts are domesticated hybrids of Saccharomyces cerevisiae and cold-tolerant Saccharomyces eubayanus. To understand the contribution of both parental genomes to maltose metabolism in brewing wort, this study focuses on maltose transport in the S. eubayanus type strain CBS 12357T/FM1318. To obtain complete sequences of the MAL loci of this strain, a near-complete genome assembly was generated using the Oxford Nanopore Technology MinION sequencing platform. Except for CHRXII, all sixteen chromosomes were assembled as single contigs. Four loci harboring putative maltose transporter genes (SeMALT1-4), located in subtelomeric regions of CHRII, CHRV, CHRXIII, and CHRXVI, were completely resolved. The near-identical loci on CHRV and CHRXVI strongly resembled canonical S. cerevisiae MAL loci, while those on CHRII and CHRXIII showed different structures suggestive of gene loss. Overexpression of SeMALT1-4 in a maltose-transport-deficient S. cerevisiae strain restored growth on maltose, but not on maltotriose, indicating maltose-specific transport functionality of all four transporters. Simultaneous CRISPR-Cas9-assisted deletion of only SeMALT2 and SeMALT4, which shared 99.7% sequence identity, eliminated growth of S. eubayanus CBS 12357T on maltose. Transcriptome analysis of S. eubayanus CBS 12357T established that SeMALT1 and SeMALT3, are poorly expressed in maltose-grown cultures, while SeMALT2 and SeMALT4 were expressed at much higher levels than SeMALT1 and SeMALT3, indicating that only SeMALT2/4 are responsible for maltose consumption in CBS 12357T. These results represent a first genomic and physiological characterization of maltose transport in S. eubayanus CBS 12357T and provides a valuable resource for further industrial exploitation of this yeast.","Brewing; Domestication; Gene regulation; Saccharomyces eubayanus; Transport; Wort; Yeast","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:bc4c39d6-3c8b-437a-8709-eb3b9a47170b","http://resolver.tudelft.nl/uuid:bc4c39d6-3c8b-437a-8709-eb3b9a47170b","Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae","Papapetridis, I. (TU Delft BT/Industriele Microbiologie); Verhoeven, M.D. (TU Delft BT/Industriele Microbiologie); Wiersma, S.J. (TU Delft BT/Industriele Microbiologie); Goudriaan, M.; van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2018","Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilisation of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose–xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HXK2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83G673T allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L−1 glucose and 10 g L−1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.","biofuels; yeast; industrial biotechnology; Redox engineering; fermentation; pentoses","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:4d6512c4-3a2c-4095-b941-913e73df2497","http://resolver.tudelft.nl/uuid:4d6512c4-3a2c-4095-b941-913e73df2497","Fermentation of glucose-xylose-arabinose mixtures by a synthetic consortium of single-sugar-fermenting Saccharomyces cerevisiae strains","Verhoeven, M.D. (TU Delft BT/Industriele Microbiologie); de Valk, S.C. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2018","D-Glucose, D-xylose and L-arabinose are major sugars in lignocellulosic hydrolysates. This study explores fermentation of glucose-xylose-arabinose mixtures by a consortium of three ‘specialist’ Saccharomyces cerevisiae strains. A D-glucose- and L-arabinose-tolerant xylose specialist was constructed by eliminating hexose phosphorylation in an engineered xylose-fermenting strain and subsequent laboratory evolution. A resulting strain anaerobically grew and fermented D-xylose in the presence of 20 g L-1 of D-glucose and L-arabinose. A synthetic consortium that additionally comprised a similarly obtained arabinose specialist and a pentose-non-fermenting laboratory strain, rapidly and simultaneously converted D-glucose and L-arabinose in anaerobic batch cultures on three-sugar mixtures. However, performance of the xylose specialist was strongly impaired in these mixed cultures. After prolonged cultivation of the consortium on three-sugar mixtures, the time required for complete sugar conversion approached that of a previously constructed and evolved ‘generalist’ strain. In contrast to the generalist strain, whose fermentation kinetics deteriorated during prolonged repeated-batch cultivation on a mixture of 20 g L-1 D-glucose, 10 g L-1 D-xylose and 5 g L-1 L-arabinose, the evolved consortium showed stable fermentation kinetics. Understanding the interactions between specialist strains is a key challenge in further exploring the applicability of this synthetic consortium approach for industrial fermentation of lignocellulosic hydrolysates.","bioethanol; mixed-culture fermentationt; evolutionary engineering; pentose fermentation; Yeast","en","journal article","","","","","","Accepted Author Manuscript","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:19b09f8e-efce-440d-937b-f10aa442f50d","http://resolver.tudelft.nl/uuid:19b09f8e-efce-440d-937b-f10aa442f50d","Under pressure: evolutionary engineering of yeast strains for improved performance in fuels and chemicals production","Mans, R. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie)","","2018","Evolutionary engineering, which uses laboratory evolution to select for industrially relevant traits, is a popular strategy in the development of high-performing yeast strains for industrial production of fuels and chemicals. By integrating whole-genome sequencing, bioinformatics, classical genetics and genome-editing techniques, evolutionary engineering has also become a powerful approach for identification and reverse engineering of molecular mechanisms that underlie industrially relevant traits. New techniques enable acceleration of in vivo mutation rates, both across yeast genomes and at specific loci. Recent studies indicate that phenotypic trade-offs, which are often observed after evolution under constant conditions, can be mitigated by using dynamic cultivation regimes. Advances in research on synthetic regulatory circuits offer exciting possibilities to extend the applicability of evolutionary engineering to products of yeasts whose synthesis requires a net input of cellular energy.","","en","review","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:b04fc945-12b9-4e14-983f-553127176662","http://resolver.tudelft.nl/uuid:b04fc945-12b9-4e14-983f-553127176662","Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains","Bracher, J.M. (TU Delft BT/Industriele Microbiologie); Martinez-Rodriguez, Oscar A. (Genomatica, San Diego); Dekker, W.J.C. (TU Delft BT/Industriele Microbiologie); Verhoeven, M.D. (DSM); van Maris, A.J.A. (AlbaNova University Center); Pronk, J.T. (TU Delft BT/Industriele Microbiologie)","","2018","Expression of a heterologous xylose isomerase, deletion of the GRE3 aldose-reductase gene and overexpression of genes encoding xylulokinase (XKS1) and non-oxidative pentose-phosphate-pathway enzymes (RKI1, RPE1, TAL1, TKL1) enables aerobic growth of Saccharomyces cerevisiae on d-xylose. However, literature reports differ on whether anaerobic growth on d-xylose requires additional mutations. Here, CRISPR-Cas9-assisted reconstruction and physiological analysis confirmed an early report that this basic set of genetic modifications suffices to enable anaerobic growth on d-xylose in the CEN.PK genetic background. Strains that additionally carried overexpression cassettes for the transaldolase and transketolase paralogs NQM1 and TKL2 only exhibited anaerobic growth on d-xylose after a 7-10 day lag phase. This extended lag phase was eliminated by increasing inoculum concentrations from 0.02 to 0.2 g biomass L-1. Alternatively, a long lag phase could be prevented by sparging low-inoculum-density bioreactor cultures with a CO2/N2-mixture, thus mimicking initial CO2 concentrations in high-inoculum-density, nitrogen-sparged cultures, or by using l-aspartate instead of ammonium as nitrogen source. This study resolves apparent contradictions in the literature on the genetic interventions required for anaerobic growth of CEN.PK-derived strains on d-xylose. Additionally, it indicates the potential relevance of CO2 availability and anaplerotic carboxylation reactions for anaerobic growth of engineered S. cerevisiae strains on d-xylose.","","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:94a8fbbd-d5c5-4f7c-8246-962b4d58b9d4","http://resolver.tudelft.nl/uuid:94a8fbbd-d5c5-4f7c-8246-962b4d58b9d4","Strength characterization of full-scale aerobic granular sludge","de Graaff, D.R. (TU Delft BT/Environmental Biotechnology); van Dijk, E.J.H. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Pronk, M. (TU Delft BT/Environmental Biotechnology; Royal HaskoningDHV)","","2018","For a stable operation, the aerobic granular sludge process requires mechanically strong granules in balance with the shear forces in the reactor. Despite a wide general interest in granular stability, the mechanical strength of both anaerobic and aerobic granular sludge received very little attention. In this study, a high-shear method for strength characterization has been evaluated for full-scale aerobic granular sludge (AGS). Abrasion times up to 90 min showed a stable abrasion rate coefficient (K), while prolonged periods of abrasion up to 24 h resulted in a decrease in abrasion rate. Larger granules have higher abrasion rate than smaller granules. No abrasion was observed at low shear rates, indicating a threshold shear rate for abrasion. Lab-scale AGS showed a lower abrasion rate than full-scale AGS. Incubation of full-scale granules in NaCl led to a decrease in abrasion rate at 25 g L−1 NaCl, but incubation in 50 g L−1 NaCl led to a further decrease for only half of the tested granular sludge samples.","Aerobic granular sludge; salinity; strength characterization","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:48b2d0d8-338c-404b-8e97-47109b8615c7","http://resolver.tudelft.nl/uuid:48b2d0d8-338c-404b-8e97-47109b8615c7","Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgaris Suf1 transporter","Marques, W.L. (TU Delft BT/Industriele Microbiologie; University of Campinas); van der Woude, L.N. (TU Delft Science Education and Communication); Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Nijenhuis, J.M. (TU Delft Applied Sciences); Pronk, J.T. (TU Delft BT/Biotechnologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie); Mans, R. (TU Delft BT/Industriele Microbiologie); Gombert, Andreas K. (University of Campinas)","","2018","Knowledge on the genetic factors important for the efficient expression of plant transporters in yeast is still very limited. Phaseolus vulgaris sucrose facilitator 1 (PvSuf1), a presumable uniporter, was an essential component in a previously published strategy aimed at increasing ATP yield in Saccharomyces cerevisiae. However, attempts to construct yeast strains in which sucrose metabolism was dependent on PvSUF1 led to slow sucrose uptake. Here, PvSUF1-dependent S. cerevisiae strains were evolved for faster growth. Of five independently evolved strains, two showed an approximately twofold higher anaerobic growth rate on sucrose than the parental strain (μ = 0.19 h−1 and μ = 0.08 h−1, respectively). All five mutants displayed sucrose-induced proton uptake (13–50 μmol H+ (g biomass)−1 min−1). Their ATP yield from sucrose dissimilation, as estimated from biomass yields in anaerobic chemostat cultures, was the same as that of a congenic strain expressing the native sucrose symporter Mal11p. Four out of six observed amino acid substitutions encoded by evolved PvSUF1 alleles removed or introduced a cysteine residue and may be involved in transporter folding and/or oligomerization. Expression of one of the evolved PvSUF1 alleles (PvSUF1I209F C265F G326C) in an unevolved strain enabled it to grow on sucrose at the same rate (0.19 h−1) as the corresponding evolved strain. This study shows how laboratory evolution may improve sucrose uptake in yeast via heterologous plant transporters, highlights the importance of cysteine residues for their efficient expression, and warrants reinvestigation of PvSuf1's transport mechanism.","laboratory evolution; plant sucrose facilitator; plant transporter expression; sucrose uptake; yeast physiology","en","journal article","","","","","","","","2019-09-16","Applied Sciences","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:849a4440-590a-4f01-9d1c-3a605a64d99c","http://resolver.tudelft.nl/uuid:849a4440-590a-4f01-9d1c-3a605a64d99c","Selection of Pof- saccharomyces eubayanus variants for the construction of S. cerevisiae × S. eubayanus hybrids with reduced 4-Vinyl guaiacol formation","Diderich, J.A. (TU Delft BT/Industriele Microbiologie); Weening, S.M. (TU Delft BT/Industriele Microbiologie); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2018","Saccharomyces pastorianus is an interspecies hybrid between S. cerevisiae and S. eubayanus. The identification of the parental species of S. pastorianus enabled the de novo reconstruction of hybrids that could potentially combine a wide array of phenotypic traits. Lager yeasts are characterized by their inability to decarboxylate ferulic acid present in wort, a phenotype also known as Pof-(phenolic off-flavor). However, all known S. eubayanus strains characterized so far produce clove-like aroma specific of 4-vinyl guaiacol, a decarboxylated form of ferulic acid. This study explored a non- GMO approach to construct Pof- S. eubayanus variants derived from the parental strain S. eubayanus CBS 12357. To rapidly screen a population of UV-mutagenized cells two complementary assays were developed. The first assay was based on the difference of light absorption spectra of ferulic acid and 4-vinyl guaiacol, while the second was based on the difference of sensitivity of Pof-and Pof+ strains to cinnamic acid. The S. eubayanus variant HTSE042 was selected and was confirmed not to produce 4-vinyl guaiacol. Whole genome sequencing revealed that this variant lost the subtelomeric region of the CHRXIII right arm that carried the two clustered genes SePAD1- SeFDC1 whose deletion in a naïve S. eubayanus strain (CBS 12357/FM1318) resulted in an identical phenotype. Subsequently, the Pof- variant was crossed with a Pof- S. cerevisiae partner. The resulting hybrid was not able to convert ferulic acid demonstrating the undisputable value of the mutagenized variant HTSE042 to eventually construct S. cerevisiae × S. eubayanus hybrids with phenotypic characteristics of S. pastorianus.","4-vinyl guaiacol; Brewing fermentation; Ferulic acid; Genetic; High-throughput screening (HTS); Hybridization; Wort sugar","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:3bd51393-dc45-42de-a7f6-4f664a8bdf77","http://resolver.tudelft.nl/uuid:3bd51393-dc45-42de-a7f6-4f664a8bdf77","Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics","Jürgens, H. (TU Delft BT/Industriele Microbiologie); Niemeijer, M.S. (TU Delft BT/Industriele Microbiologie; Applikon Biotechnology); Jennings-Antipov, Laura D. (Riffyn); Mans, R. (TU Delft BT/Industriele Microbiologie); Morel, Jack (Riffyn); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie; KTH Royal Institute of Technology); Pronk, J.T. (TU Delft BT/Biotechnologie); Gardner, Timothy S. (Riffyn)","","2018","Open data in science requires precise definition of experimental procedures used in data generation, but traditional practices for sharing protocols and data cannot provide the required data contextualization. Here, we explore implementation, in an academic research setting, of a novel cloud-based software system designed to address this challenge. The software supports systematic definition of experimental procedures as visual processes, acquisition and analysis of primary data, and linking of data and procedures in machine-computable form. The software was tested on a set of quantitative microbial-physiology experiments. Though time-intensive, definition of experimental procedures in the software enabled much more precise, unambiguous definitions of experiments than conventional protocols. Once defined, processes were easily reusable and composable into more complex experimental flows. Automatic coupling of process definitions to experimental data enables immediate identification of correlations between procedural details, intended and unintended experimental perturbations, and experimental outcomes. Software-based experiment descriptions could ultimately replace terse and ambiguous 'Materials and Methods' sections in scientific journals, thus promoting reproducibility and reusability of published studies.","","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:a74a63eb-f4c6-4072-b35f-406d21eae90f","http://resolver.tudelft.nl/uuid:a74a63eb-f4c6-4072-b35f-406d21eae90f","A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains","Wijsman, M. (TU Delft BT/Industriele Microbiologie); Swiat, M.A. (TU Delft BT/Industriele Microbiologie); Marques, W.L. (TU Delft BT/Industriele Microbiologie; University of Campinas; Universidade de São Paulo); Hettinga, Johanna K. (Student TU Delft); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Torre Cortés, Pilar de la (Student TU Delft); Mans, R. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie); Daran-Lapujade, P.A.S. (TU Delft BT/Industriele Microbiologie)","","2018","Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also resulted in major chromosomal rearrangements, gene loss and phenotypic changes. In the present study, CRISPR/SpCas9 was used to delete the 21 hexose transporters in an S. cerevisiae strain from the CEN.PK family in only three deletion rounds, using 11 unique guide RNAs. Even upon prolonged cultivation, the resulting strain IMX1812 (CRISPR-Hxt0) was unable to consume glucose, while its growth rate on maltose was the same as that of a strain equipped with a full set of hexose transporters. Karyotyping and whole-genome sequencing of the CRISPR-Hxt0 strain with Illumina and Oxford Nanopore technologies did not reveal chromosomal rearrangements or other unintended mutations besides a few SNPs. This study provides a new, 'genetically unaltered' hexose transporter-deficient strain and supplies a CRISPR toolkit for removing all hexose transporter genes from most S. cerevisiae laboratory strains in only three transformation rounds.","","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:113d8e73-d8f4-4825-92f0-6950a582af0b","http://resolver.tudelft.nl/uuid:113d8e73-d8f4-4825-92f0-6950a582af0b","The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae","Bracher, J.M. (TU Delft BT/Industriele Microbiologie); Verhoeven, M.D. (TU Delft BT/Industriele Microbiologie); Wisselink, H. Wouter (Isobionics); Crimi, B. (TU Delft BT/Industriele Microbiologie; UMR9002-CNRS-UM); Nijland, Jeroen G. (Rijksuniversiteit Groningen); Driessen, Arnold J.M. (Rijksuniversiteit Groningen); Klaassen, Paul (DSM); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie; AlbaNova University Center); Daran, J.G. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie)","","2018","Background: l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Results: Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (K m = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10-3 and 1.8 g L-1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L-1 l-arabinose and 20 g L-1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. Conclusion: Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.","l-Arabinose transporter; Metabolic engineering; Penicillium; Proton symport; Second-generation bioethanol; Sugar transport; Transcriptome; Yeast","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:48e11e15-caf8-48d5-b3b0-30a46fca5699","http://resolver.tudelft.nl/uuid:48e11e15-caf8-48d5-b3b0-30a46fca5699","Experimental and numerical investigation of contact heat transfer between a rotating heat pipe and a steel strip","Çelik, M. (TU Delft Energy Technology); Devendran, Kathikeyan (Student TU Delft); Paulussen, Geert (Tata Steel); Pronk, Pepijn (Tata Steel); Frinking, Ferry (Tata Steel); de Jong, W. (TU Delft Large Scale Energy Storage); Boersma, B.J. (TU Delft Process and Energy; TU Delft Energy Technology)","","2018","A new concept for energy efficient annealing of steel strip comprises of multiple rotating heat pipes. Each heat pipe extracts heat from the cooling strip which is reused to increase the temperature of the heating strip. In this context, the heat transfer between the steel strip and the rotating heat pipe is investigated. When the strip is transported over the heat pipe, gas entrains in the gap. The gas compresses into a uniform gas layer. The contact heat transfer deteriorates due to this phenomenon. A numerical model to quantify the heat transfer between the surfaces is developed. Since there is no direct way to quantify the heat transfer between two moving surfaces, the problem is divided into a gas entrainment and a heat transfer part. The model is validated with experiments executed on a rotating heat pipe test rig. The validation was made varying the strip thickness, specific tension and strip velocity. The results show a uniform gas layer forming within the first 1° of the 180° wrap angle in all cases. The heat transfer is dominated by gas conduction. Results for the uniform gas layer region yield heat transfer coefficients in the range between 4000 and 20,000 W/m2·K.","Contact heat transfer; Experimental validation; Gas entrainment; Numerical model; Rotating heat pipe","en","journal article","","","","","","","","","","Process and Energy","Energy Technology","","",""
"uuid:93a40aff-930b-4dff-9769-3410ba0e5519","http://resolver.tudelft.nl/uuid:93a40aff-930b-4dff-9769-3410ba0e5519","Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae","Marques, W.L. (TU Delft BT/Industriele Microbiologie; University of Campinas; Universidade de São Paulo); Mans, R. (TU Delft BT/Industriele Microbiologie); Henderson, Ryan K. (Rijksuniversiteit Groningen); Marella, Eko Roy; ter Horst, J. (TU Delft BT/Industriele Microbiologie); de Hulster, A.F. (TU Delft BT/Industriele Microbiologie); Poolman, Bert (Rijksuniversiteit Groningen); Daran, J.G. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Gombert, Andreas K. (University of Campinas); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie; AlbaNova University Center)","","2018","Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02 h−1 (LmSPase) and 0.06 ± 0.01 h−1 (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01 h−1 and 0.08 ± 0.00 h−1, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy.","ATP; Chemostat; Facilitated diffusion; Free-energy conservation; Phosphoglucomutase; Yeast physiology","en","journal article","","","","","","Accepted Author Manuscript","","2018-12-18","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:0501845f-fff1-4445-929b-b7e58cb0af25","http://resolver.tudelft.nl/uuid:0501845f-fff1-4445-929b-b7e58cb0af25","Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast","Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie); Couwenberg, Lucas G.F. (Student TU Delft); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); De La Torre Cortés, Pilar (External organisation); ter Horst, J. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2018","Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb and up to 1700 heterozygous nucleotides, due to replacement of sequences on the targeted chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of loss of heterozygosity were consistent with homology directed repair. The extent and frequency of loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be particularly deleterious for human gene therapy, as loss of heterozygous functional copies of antiproliferative and pro-apoptotic genes is a known path to cancer.","","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:b2753a94-bd0b-49a6-a18b-885cc4ed6650","http://resolver.tudelft.nl/uuid:b2753a94-bd0b-49a6-a18b-885cc4ed6650","A CRISPR/Cas9-based exploration into the elusive mechanism for lactate export in Saccharomyces cerevisiae","Mans, R. (TU Delft BT/Industriele Microbiologie); Else-Hassing, J. (TU Delft BT/Industriele Microbiologie); Wijsman, M. (TU Delft BT/Industriele Microbiologie); Giezekamp, Annabel (Student TU Delft); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie; KTH Royal Institute of Technology)","","2017","CRISPR/Cas9-based genome editing allows rapid, simultaneous modification of multiple genetic loci in Saccharomyces cerevisiae. Here, this technique was used in a functional analysis study aimed at identifying the hitherto unknown mechanism of lactate export in this yeast. First, an S. cerevisiae strain was constructed with deletions in 25 genes encoding transport proteins, including the complete aqua(glycero)porin family and all known carboxylic acid transporters. The 25-deletion strain was then transformed with an expression cassette for Lactobacillus casei lactate dehydrogenase (LcLDH). In anaerobic, glucose-grown batch cultures this strain exhibited a lower specific growth rate (0.15 vs. 0.25 h-1) and biomass-specific lactate production rate (0.7 vs. 2.4 mmol g biomass-1 h-1) than an LcLDH-expressing reference strain. However, a comparison of the two strains in anaerobic glucose-limited chemostat cultures (dilution rate 0.10 h-1) showed identical lactate production rates. These results indicate that, although deletion of the 25 transporter genes affected the maximum specific growth rate, it did not impact lactate export rates when analysed at a fixed specific growth rate. The 25-deletion strain provides a first step towards a 'minimal transportome' yeast platform, which can be applied for functional analysis of specific (heterologous) transport proteins as well as for evaluation of metabolic engineering strategies.","Carboxylic acid; Cas9; CRISPR; Diffusion; Genome editing; Metabolic engineering","en","journal article","","","","","","","","2018-11-14","","","BT/Industriele Microbiologie","","",""
"uuid:0a70c37e-0305-4485-b78f-f358f52b4629","http://resolver.tudelft.nl/uuid:0a70c37e-0305-4485-b78f-f358f52b4629","Success factors in the realization of large ice projects in education","Pronk, Arno (Katholieke Universiteit Leuven); Luo, Peng (Harbin Institute of Technology); Li, Q. (TU Delft Structural Design & Mechanics); Sanders, F.C. (TU Delft Environmental Technology and Design); Overtoom, M.E. (TU Delft Indoor Environment); Coar, Lancelot (University of Manitoba)","Bögle, Annette (editor); Grohmann, Manfred (editor)","2017","There has been a long tradition in making ice structures, but the development of technical improvements for making ice buildings is a new field with just a handful of researchers. Most of the projects were realized by professors in cooperation with their students as part of their education in architecture and civil engineering. The following professors have realized ice projects in this setting: Heinz Isler realized some experiments since the 1950s; Tsutomu Kokawa created in the past three decades several ice domes in the north of Japan with a span up to 25 meters; Lancelot Coar realized a number of fabric formed ice shell structures including fiberglass bars and hanging fabric as a mould for an ice shell in 2011 and in 2015 he produced an fabric-formed ice origami structure in cooperation with MIT (Caitlin Mueller) and VUB (Lars de Laet)[4]. Arno Pronk realized several ice projects such as the 2004 artificially cooled igloo, in 2014[1] and 2015[2] dome structures with an inflatable mould in Finland and in 2016 one ice dome and two ice towers in Harbin (China) as a cooperation between the Universities of Eindhoven & Leuven (Pronk) and Harbin (Wu and Luo). In this paper we will present the motivation and learning experiences of students involved in learning-by-doing by realizing one large project in ice. The 2014-2016 projects were evaluated by Sanders and Overtoom[3] using questionnaires among the participants by mixed cultural teams under extreme conditions. By comparing the results in different situations and cultures we have found common rules for the success of those kinds of educational projects. In this paper we suggest that the synergy among students participating in one main project without a clear individual goal can be very large. The paper will present the success factors for projects to be perceived as a good learning experience.","Ice composite structures; learning by doing; group dynamics; synergy in education","en","conference paper","","","","","","","","","","","Structural Design & Mechanics","","",""
"uuid:3420c6e4-5e1a-4ae8-b1a1-54481cffee6e","http://resolver.tudelft.nl/uuid:3420c6e4-5e1a-4ae8-b1a1-54481cffee6e","Form-finding and construction of ice composite shell structures","Wu, Yue (Harbin Institute of Technology); Liu, Xiuming (Harbin Institute of Technology); Li, Q. (TU Delft Structural Design & Mechanics); Chen, Boxuan (Harbin Institute of Technology); Luo, Peng (Harbin Institute of Technology); Pronk, Arno (Eindhoven University of Technology); Mergny, Elke (Université de Liège)","Bögle, Annette (editor); Grohmann, Manfred (editor)","2017","By using inflatable moulds and then spraying cellulose-water mixture, one ice dome and two ice towers were built in Harbin in December 2016. During the whole process, form-finding of the inflatable moulds as well as the construction of these ice composite shell structures are very important for the final results.
The mould for the ice dome structure was a result of the manipulation of a synclastic membrane with a rope net. The mould for the ice tower structure consisted of some anticlastic surfaces. Form-finding of the inflatable moulds was conducted by the parametric tool “EasyForm” which is a self-programed plug-in in Grasshopper based on Vector Form Intrinsic Finite Element method.
In a low-temperature work environment (-10 ℃ and below), the ice shell structures were constructed on the inflatable moulds. The cellulose-water mixture was sprayed in thin layers continuously and uniformly in order to make the surface of a shell of cellulose-reinforced ice. The construction process is introduced detailedly in this paper.","Ice composite shell; form-finding; Vector Form Intrinsic Finite Element; construction; inflatable mould; cellulose-reinforced ice","en","conference paper","","","","","","","","","","","Structural Design & Mechanics","","",""
"uuid:8cb1a309-0454-459c-a1f5-0d43b2dfd5fb","http://resolver.tudelft.nl/uuid:8cb1a309-0454-459c-a1f5-0d43b2dfd5fb","Laboratory evolution of a biotin-requiring Saccharomyces cerevisiae strain for full biotin prototrophy and identification of causal mutations","Bracher, J.M. (TU Delft BT/Industriele Microbiologie); de Hulster, A.F. (TU Delft BT/Industriele Microbiologie); Koster, C.C. (TU Delft BT/Industriele Microbiologie); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie)","","2017","Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h-1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h-1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ= 0.15 h-1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h-1. The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.","Adaptive laboratory evolution; Biotin; Prototrophy; Reverse metabolic engineering; Saccharomyces cerevisiae; Vitamin biosynthesis; Whole-genome sequencing","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:72090752-81c2-44c1-a068-d329785f24fd","http://resolver.tudelft.nl/uuid:72090752-81c2-44c1-a068-d329785f24fd","Industrial relevance of chromosomal copy number variation in Saccharomyces yeasts","Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2017","Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyces strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes.","Aneuploidy; Evolutionary adaptation; Fermentation; Genome engineering; Industrial yeast; Industrial yeast fermentation; Strain improvement","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:da0155b8-20b3-47e1-9559-f03da8d0ace8","http://resolver.tudelft.nl/uuid:da0155b8-20b3-47e1-9559-f03da8d0ace8","The acid soluble extracellular polymeric substance of aerobic granular sludge dominated by Defluviicoccus sp.","Pronk, M. (TU Delft BT/Environmental Biotechnology); Neu, Thomas R. (Helmholtz Centre for Environmental Research - UFZ); van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology); Lin, Y. (TU Delft BT/Environmental Biotechnology)","","2017","A new acid soluble extracellular polymeric substance (acid soluble EPS) was extracted from an acetate fed aerobic granular sludge reactor operated at 35 °C. Acid soluble EPS dominated granules exhibited a remarkable and distinctive tangled tubular morphology. These granules are dominated by Defluviicoccus Cluster II organisms. Acetic acid instead of the usually required alkaline extraction medium was needed to dissolve the granules and solubilise the polymeric matrix. The extracted acid soluble EPS was analysed and identified using various instrumental analysis including 1H and 13C Nuclear Magnetic Resonance, Fourier Transform Infrared Spectroscopy and Raman spectroscopy. In addition, the glycoconjugates were characterized by fluorescence lectin-binding analysis. The acid soluble EPS is α-(1 → 4) linked polysaccharide, containing both glucose and galactose as monomers. There are –OCH3 groups connected to the glucose monomer. Transmission and scanning electron microscopy (TEM, SEM) as well as confocal laser scanning microscopy (CLSM) showed that the acid soluble EPS was present as a tightly bound capsular EPS around bacterial cells ordered into a sarcinae-like growth pattern. The special granule morphology is decided by the acid soluble EPS produced by Defluviicoccus Cluster II organisms. This work shows that no single one method can be used to extract all possible extracellular polymeric substances. Results obtained here can support the elucidation of biofilm formation and structure in future research.","Aerobic granular sludge; Biofilm; Defluviicoccus; EPS extraction; Extracellular polymeric substances","en","journal article","","","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:1b0c2406-1097-4bb6-9f73-231580bb721c","http://resolver.tudelft.nl/uuid:1b0c2406-1097-4bb6-9f73-231580bb721c","Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae","Papapetridis, I. (TU Delft BT/Industriele Microbiologie); van Dijk, M. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie)","","2017","Background: Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance. Results: GPD2 encodes one of two S. cerevisiae isoenzymes of NAD+-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP+-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1Δ gpd2Δ strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L-1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Conclusions: Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP+-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.","Acetic acid; Bioethanol; NADH; NADPH; Osmotic stress; Redox engineering; Yeast","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:9e41144d-6c4a-41ee-a178-ce78a6e7e5e6","http://resolver.tudelft.nl/uuid:9e41144d-6c4a-41ee-a178-ce78a6e7e5e6","Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis","Verhoeven, M.D. (TU Delft BT/Industriele Microbiologie); Lee, Misun (Rijksuniversiteit Groningen); Kamoen, L. (Student TU Delft); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Janssen, Dick B. (Rijksuniversiteit Groningen); Daran, J.G. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie)","","2017","Combined overexpression of xylulokinase, pentose-phosphate-pathway enzymes and a heterologous xylose isomerase (XI) is required but insufficient for anaerobic growth of Saccharomyces cerevisiae on d-xylose. Single-step Cas9-assisted implementation of these modifications yielded a yeast strain expressing Piromyces XI that showed fast aerobic growth on d-xylose. However, anaerobic growth required a 12-day adaptation period. Xylose-adapted cultures carried mutations in PMR1, encoding a Golgi Ca2+/Mn2+ ATPase. Deleting PMR1 in the parental XI-expressing strain enabled instantaneous anaerobic growth on d-xylose. In pmr1 strains, intracellular Mn2+ concentrations were much higher than in the parental strain. XI activity assays in cell extracts and reconstitution experiments with purified XI apoenzyme showed superior enzyme kinetics with Mn2+ relative to other divalent metal ions. This study indicates engineering of metal homeostasis as a relevant approach for optimization of metabolic pathways involving metal-dependent enzymes. Specifically, it identifies metal interactions of heterologous XIs as an underexplored aspect of engineering xylose metabolism in yeast.","","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:cf9fb39a-8a9b-4f6f-93b2-2f6ddbd28c0b","http://resolver.tudelft.nl/uuid:cf9fb39a-8a9b-4f6f-93b2-2f6ddbd28c0b","A simulator-assisted workshop for teaching chemostat cultivation in academic classes on microbial physiology","Hakkaart, X.D.V. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie)","","2017","Understanding microbial growth and metabolism is a key learning objective of microbiology and biotechnology courses, essential for understanding microbial ecology, microbial biotechnology and medical microbiology. Chemostat cultivation, a key research tool in microbial physiology that enables quantitative analysis of growth and metabolism under tightly defined conditions, provides a powerful platform to teach key features of microbial growth and metabolism. Substrate-limited chemostat cultivation can be mathematically described by four equations. These encompass mass balances for biomass and substrate, an empirical relation that describes distribution of
consumed substrate over growth and maintenance energy requirements (Pirt equation), and a Monod-type equation that describes the relation between substrate concentration and substrate-consumption rate. The authors felt that the abstract nature of these mathematical equations and a lack of visualization contributed to a suboptimal operative understanding of quantitative microbial physiology among students who followed their Microbial Physiology B.Sc. courses. The studio-classroom workshop presented here was developed to improve student understanding of quantitative physiology by a set of question-guided simulations. Simulations are run on Chemostatus, a specially
developed MATLAB-based program, which visualizes key parameters of simulated chemostat cultures as they proceed from dynamic growth conditions to steady state.
In practice, the workshop stimulated active discussion between students and with their teachers. Moreover, its introduction coincided with increased average exam scores for questions on quantitative microbial physiology. The workshop can be easily implemented in formal microbial physiology courses or used by individuals seeking to test and improve their understanding of quantitative microbial physiology and/orchemostat cultivation.","Topology optimization; Additive manufacturing; Manufacturing process planning; Space-time optimization","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:6feb309b-c3a3-4495-99c2-1dd4468efb84","http://resolver.tudelft.nl/uuid:6feb309b-c3a3-4495-99c2-1dd4468efb84","Evolutionary engineering in chemostat cultures for improved maltotriose fermentation kinetics in saccharomyces pastorianus lager brewing yeast","Brickwedde, A. (TU Delft BT/Industriele Microbiologie); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Geertman, Jan Maarten A. (Heineken Supply Chain); Magalhães, Frederico (VTT Technical Research Center of Finland); Kuijpers, Niels G.A. (Heineken Supply Chain); Gibson, Brian (VTT Technical Research Center of Finland); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie)","","2017","The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion (""attenuation"") of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains.","Brewing; Chemostat; Evolutionary engineering; Maltose; Maltotriose consumption rate; Sacchromyces pastorianus; Transport; OA-Fund TU Delft","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:da98a321-fc15-4df8-bd83-e7d16ea1420a","http://resolver.tudelft.nl/uuid:da98a321-fc15-4df8-bd83-e7d16ea1420a","Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism","Marques, W.L. (TU Delft BT/Industriele Microbiologie; University of Campinas); Mans, R. (TU Delft BT/Industriele Microbiologie); Marella, Eko Roy (Student TU Delft); Cordeiro, Rosa Lorizolla (University of Campinas); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); Gombert, Andreas K. (University of Campinas); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie)","","2017","Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose-based processes remain to be investigated. An essential first step is to identify all native sucrose-hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated by suppressor mutations in sucrose-negative strains. A strain in which all known sucrose-transporter genes (MAL11, MAL21, MAL31, MPH2, MPH3) were deleted did not grow on sucrose after 2 months of incubation. In contrast, a strain with deletions in genes encoding sucrose-hydrolysing enzymes (SUC2, MAL12, MAL22, MAL32) still grew on sucrose. Its specific growth rate increased from 0.08 to 0.25 h-1 after sequential batch cultivation. This increase was accompanied by a 3-fold increase of in vitro sucrose-hydrolysis and isomaltase activities, as well as by a 3- to 5-fold upregulation of the isomaltase-encoding genes IMA1 and IMA5. One-step Cas9-mediated deletion of all isomaltase-encoding genes (IMA1-5) completely abolished sucrose hydrolysis. Even after 2 months of incubation, the resulting strain did not grow on sucrose. This sucrose-negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport.","Disaccharide; Isomaltase; Laboratory evolution; Multiple gene deletion; Real-time PCR; Reverse engineering","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:ccedae52-2933-4189-a121-10b1b4d8ab03","http://resolver.tudelft.nl/uuid:ccedae52-2933-4189-a121-10b1b4d8ab03","Saccharomyces cerevisiae strains tor second-generation ethanol production: from academie exploration to industrial implementation","Jansen, Mickel L.A. (DSM); Bracher, J.M. (TU Delft BT/Industriele Microbiologie); Papapetridis, I. (TU Delft BT/Industriele Microbiologie); Verhoeven, M.D. (TU Delft BT/Industriele Microbiologie); de Bruijn, J.A. (DSM); de Waal, P. (DSM); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie; AlbaNova University Center); Klaassen, P (DSM); Pronk, J.T. (TU Delft BT/Industriele Microbiologie)","","2017","The recent start-up of several full-scale ‘second generation’ ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae strains for fermentation of lignocellulosic hydrolysates of agricultural residues and energy crops. After a discussion of the challenges that these novel industrial contexts impose on yeast strains, this minireview describes key metabolic engineering strategies that have been developed to address these challenges. Additionally, it outlines how proof-of-concept studies, often developed in academic settings, can be used for the development of robust strain platforms that meet the requirements for industrial application. Fermentation performance of current engineered industrial S. cerevisiae strains is no longer a bottleneck in efforts to achieve the projected outputs of the first large-scale second-generation ethanol plants. Academic and industrial yeast research will continue to strengthen the economic value position of second-generation ethanol production by further improving fermentation kinetics, product yield and cellular robustness under process conditions.","biofuels; metabolic engineering; ndustrial fermentation; yeast biotechnology; pentose fermentation; biomass hydrolysates","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:525d41c8-8817-4b65-8e58-d231015f290b","http://resolver.tudelft.nl/uuid:525d41c8-8817-4b65-8e58-d231015f290b","A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations","Gonzalez Ramos, D. (TU Delft BT/Industriele Microbiologie); Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie); Grijseels, Sietske S. (Student TU Delft); van Berkum, M.C. (TU Delft Applied Sciences); Swinnen, Steve (Jacobs University Bremen); van den Broek, M.A. (TU Delft BT/Industriele Microbiologie); Nevoigt, Elke (Jacobs University Bremen); Daran, J.G. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie)","","2016","Background: Acetic acid, released during hydrolysis of lignocellulosic feedstocks for second generation bioethanol production, inhibits yeast growth and alcoholic fermentation. Yeast biomass generated in a propagation step that precedes ethanol production should therefore express a high and constitutive level of acetic acid tolerance before introduction into lignocellulosic hydrolysates. However, earlier laboratory evolution strategies for increasing acetic acid tolerance of Saccharomyces cerevisiae, based on prolonged cultivation in the presence of acetic acid, selected for inducible rather than constitutive tolerance to this inhibitor. Results: Preadaptation in the presence of acetic acid was shown to strongly increase the fraction of yeast cells that could initiate growth in the presence of this inhibitor. Serial microaerobic batch cultivation, with alternating transfers to fresh medium with and without acetic acid, yielded evolved S. cerevisiae cultures with constitutive acetic acid tolerance. Single-cell lines isolated from five such evolution experiments after 50-55 transfers were selected for further study. An additional constitutively acetic acid tolerant mutant was selected after UV-mutagenesis. All six mutants showed an increased fraction of growing cells upon a transfer from a non-stressed condition to a medium containing acetic acid. Whole-genome sequencing identified six genes that contained (different) mutations in multiple acetic acid-tolerant mutants. Haploid segregation studies and expression of the mutant alleles in the unevolved ancestor strain identified causal mutations for the acquired acetic acid tolerance in four genes (ASG1, ADH3, SKS1 and GIS4). Effects of the mutations in ASG1, ADH3 and SKS1 on acetic acid tolerance were additive. Conclusions: A novel laboratory evolution strategy based on alternating cultivation cycles in the presence and absence of acetic acid conferred a selective advantage to constitutively acetic acid-tolerant mutants and may be applicable for selection of constitutive tolerance to other stressors. Mutations in four genes (ASG1, ADH3, SKS1 and GIS4) were identified as causative for acetic acid tolerance. The laboratory evolution strategy as well as the identified mutations can contribute to improving acetic acid tolerance in industrial yeast strains.","Acetate; Bioethanol; Evolutionary engineering; Inhibitors; Yeast","en","journal article","","","","","","","","","Applied Sciences","","BT/Industriele Microbiologie","","",""
"uuid:3ff9d48e-7c7b-4ca2-ab20-f413b026927a","http://resolver.tudelft.nl/uuid:3ff9d48e-7c7b-4ca2-ab20-f413b026927a","Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: Pathway stoichiometry, free-energy conservation and redox-cofactor balancing","Van Rossum, H.M.; Kozak, B.U.; Pronk, J.T.; Van Maris, A.J.A.","","2016","Saccharomyces cerevisiae is an important industrial cell factory and an attractive experimental model for evaluating novel metabolic engineering strategies. Many current and potential products of this yeast require acetyl coenzyme A (acetyl-CoA) as a precursor and pathways towards these products are generally expressed in its cytosol. The native S. cerevisiae pathway for production of cytosolic acetyl-CoA consumes 2 ATP equivalents in the acetyl-CoA synthetase reaction. Catabolism of additional sugar substrate, which may be required to generate this ATP, negatively affects product yields. Here, we review alternative pathways that can be engineered into yeast to optimize supply of cytosolic acetyl-CoA as a precursor for product formation. Particular attention is paid to reaction stoichiometry, free-energy conservation and redox-cofactor balancing of alternative pathways for acetyl-CoA synthesis from glucose. A theoretical analysis of maximally attainable yields on glucose of four compounds (n-butanol, citric acid, palmitic acid and farnesene) showed a strong product dependency of the optimal pathway configuration for acetyl-CoA synthesis. Moreover, this analysis showed that combination of different acetyl-CoA production pathways may be required to achieve optimal product yields. This review underlines that an integral analysis of energy coupling and redox-cofactor balancing in precursor-supply and product-formation pathways is crucial for the design of efficient cell factories.","acetylating acetaldehyde dehydrogenase; pyruvate-formate lyase; pyruvate dehydrogenase; ATP-citrate lyase; phosphoketolase; carnitine shuttle","en","journal article","Elsevier","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:a02bdca1-8fdd-4325-9115-1322ae953034","http://resolver.tudelft.nl/uuid:a02bdca1-8fdd-4325-9115-1322ae953034","Maintenance-energy requirements and robustness of Saccharomyces cerevisiae at aerobic near-zero specific growth rates","Vos, T. (TU Delft BT/Industriele Microbiologie); Hakkaart, X.D.V. (TU Delft BT/Industriele Microbiologie); de Hulster, A.F. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); Daran-Lapujade, P.A.S. (TU Delft BT/Industriele Microbiologie)","","2016","Background: Saccharomyces cerevisiae is an established microbial platform for production of native and non-native compounds. When product pathways compete with growth for precursors and energy, uncoupling of growth and product formation could increase product yields and decrease formation of biomass as a by-product. Studying non-growing, metabolically active yeast cultures is a first step towards developing S. cerevisiae as a robust, non-growing cell factory. Microbial physiology at near-zero growth rates can be studied in retentostats, which are continuous-cultivation systems with full biomass retention. Hitherto, retentostat studies on S. cerevisiae have focused on anaerobic conditions, which bear limited relevance for aerobic industrial processes. The present study uses aerobic, glucose-limited retentostats to explore the physiology of non-dividing, respiring S. cerevisiae cultures, with a focus on industrially relevant features. Results: Retentostat feeding regimes for smooth transition from exponential growth in glucose-limited chemostat cultures to near-zero growth rates were obtained by model-aided experimental design. During 20 days of retentostats cultivation, the specific growth rate gradually decreased from 0.025 h-1 to below 0.001 h-1, while culture viability remained above 80 %. The maintenance requirement for ATP (mATP) was estimated at 0.63 ± 0.04 mmol ATP (g biomass)-1 h-1, which is ca. 35 % lower than previously estimated for anaerobic retentostats. Concomitant with decreasing growth rate in aerobic retentostats, transcriptional down-regulation of genes involved in biosynthesis and up-regulation of stress-responsive genes resembled transcriptional regulation patterns observed for anaerobic retentostats. The heat-shock tolerance in aerobic retentostats far exceeded previously reported levels in stationary-phase batch cultures. While in situ metabolic fluxes in retentostats were intentionally low due to extreme caloric restriction, off-line measurements revealed that cultures retained a high metabolic capacity. Conclusions: This study provides the most accurate estimation yet of the maintenance-energy coefficient in aerobic cultures of S. cerevisiae, which is a key parameter for modelling of industrial aerobic, glucose-limited fed-batch processes. The observed extreme heat-shock tolerance and high metabolic capacity at near-zero growth rates demonstrate the intrinsic potential of S. cerevisiae as a robust, non-dividing microbial cell factory for energy-intensive products.","Aerobic; Energetics; Heat-shock; Maintenance; Retentostat; Robustness; Yeast; Zero growth","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:5ea870b3-671e-4b02-b202-5255d5b58da2","http://resolver.tudelft.nl/uuid:5ea870b3-671e-4b02-b202-5255d5b58da2","Aerobic Granular Sludge: Effect of Substrate on Granule Formation","Pronk, M. (TU Delft BT/Environmental Biotechnology)","van Loosdrecht, Mark C.M. (promotor); Kleerebezem, R. (copromotor); Delft University of Technology (degree granting institution)","2016","Discharging untreated wastewater will contaminate the surface waters and can lead to spread of diseases and long term ecological damage. The most common method for treatment is by the activated sludge process. In this process, nutrients like nitrogen, phosphorus and COD are removed by bacteria grown in flocs. These bacterial flocs are separated from the treated water by settling. Due to the slow settling velocities of these flocs large settling tanks are needed. Settling tanks take up most of the required space for a wastewater treatment plant. Aerobic granular sludge is a compact technology designed to reduce area requirements, save energy while providing excellent effluent quality. Bacteria are grown in granules instead of flocs and have therefore a much higher settling velocity. This reduces the area requirement significantly. So much even so, that external settling tanks are completely omitted. To grow aerobic granules a few selection principles are needed. First, the influent is brought in contact with the biomass in an anaerobic environment. Here COD is converted by the bacteria into storage polymers. These storage polymers are then used for growth in the presence of oxygen hereby removing phosphorus and nitrogen from the bulk liquid. Secondly, a settling pressure is applied by which slow settling biomass is removed from the reactor, thus leading to the formation of granules. In previous research by the PhD students Janneke Beun (principle of aerobic granulation), Merle de Kreuk (basic process technology for granular sludge nutrient removal) and Mari Winkler and Joao Bassin (Microbiology and process engineering aspects of granular sludge) the basic concepts of the granular sludge technology were worked out. In this thesis the effects of several operational conditions on the conversion processes, formation and stability of aerobic granular sludge was studied. The quick implementation of the technology in practice also meant that several important subjects still needed further investigation. To ensure a well-functioning technology in domestic and industrial applications these subjects were studied in more detail (i.e. adsorption, effect of salinity, higher temperature and other substrates). Besides laboratory work also the start-up and performance of one of the first full-scale aerobic granular sludge reactors treating domestic wastewater is described. The ammonium adsorption properties of aerobic granular sludge, activated sludge and anammox granules have been investigated in Chapter 2. During operation of a pilot-scale aerobic granular sludge reactor, a positive relation between the ammonium influent concentration and the ammonium adsorbed was observed. Aerobic granular sludge exhibited much higher adsorption capacity compared to activated sludge and anammox granules. At an ammonium concentration of 30 mg N L-1, adsorption obtained with activated sludge and anammox granules was around 0.2 mg NH4+-N gVSS-1, while aerobic granular sludge from lab- and pilot scale exhibited an adsorption of 1.7 and 0.9 mg NH4+-N gVSS-1, respectively. No difference in the ammonium adsorption was observed in lab-scale reactors operated at different temperatures (20 and 30 ºC). In a lab-scale reactor fed with saline wastewater, we observed that the amount of ammonium adsorbed, decreased considerably when the salt concentration increased. The results indicate that adsorption or better: ion-exchange of ammonium should be incorporated into models for nitrification/denitrification, certainly when aerobic granular sludge is used. Salinity can adversely affect the performance of most biological processes involved in wastewater treatment (Chapter 3). The effect of salt (NaCl) on the main conversion processes in an aerobic granular sludge (AGS) process accomplishing simultaneous organic matter, nitrogen, and phosphate removal was evaluated in this chapter. Hereto an AGS sequencing batch reactor was subjected to different salt concentrations (0.2 to 20 g Cl- L-1). Granular structure was stable throughout the whole experimental period, although granule size decreased and a significant effluent turbidity was observed at the highest salinity tested. A weaker gel structure at higher salt concentrations was hypothesized to be the cause of such turbidity. Ammonium oxidation was not affected at any of the salt concentrations applied. However, nitrite oxidation was severely affected, especially at 20 g Cl- L-1, in which a complete inhibition was observed. Consequently, high nitrite accumulation occurred. Phosphate removal was also found to be inhibited at the highest salt concentration tested. Complementary experiments have shown that a cascade inhibition effect took place: first, the deterioration of nitrite oxidation resulted in high nitrite concentrations and this in turn resulted in a detrimental effect to polyphosphate-accumulating organisms (PAOs). By preventing the occurrence of the nitrification process and therefore avoiding the nitrite accumulation, the effect of salt concentrations on the bio-P removal process was shown to be negligible up to 13 g Cl- L-1. Salt concentrations equal to 20 g Cl- L-1 or higher in absence of nitrite also significantly reduced phosphate removal efficiency in the system. When aerobic granular sludge is applied for industrial wastewater treatment different soluble substrates can be present. For stable granular sludge formation on volatile fatty acids (e.g. acetate), production of storage polymers under anaerobic feeding conditions has been shown to be important. This prevents direct aerobic growth on readily available COD, which is thought to result in unstable granule formation. In Chapter 4 we investigate the impact of acetate, methanol, butanol, propanol, propionaldehyde and valeraldehyde on granular sludge formation at 35 °C. Methanogenic archaea, growing on methanol, were present in the aerobic granular sludge system. Methanol was completely converted to methane and carbon dioxide by the methanogenic archaeum Methanomethylovorans uponensis during the one-hour anaerobic feeding period, despite the relative high dissolved oxygen concentration (3.5 mg O2 L-1) during the subsequent two-hour aeration period. Propionaldehyde and valeraldehyde were fully disproportionated anaerobically into their corresponding carboxylic acids and alcohols. The organic acids produced were converted to storage polymers, while the alcohols (produced and from influent) were absorbed onto the granular sludge matrix and converted aerobically. Our observations show that easy biodegradable substrates not converted anaerobically into storage polymers could lead to unstable granular sludge formation. However, when the easy biodegradable COD is absorbed in the granules and/or when the substrate is converted by relatively slow growing bacteria in the aerobic period stable granulation can occur. The influence of sludge age on granular sludge formation and microbial population dynamics in a methanol- and acetate-fed aerobic granular sludge system operated at 35 °C is investigated in Chapter 5. During anaerobic feeding of the reactor, methanol was initially converted to methane by methylotrophic methanogens. These methanogens were able to withstand the relatively long aeration periods. Lowering the anaerobic solid retention time (SRT) from 17 to 8 days enabled selective removal of the methanogens and prevented unwanted methane formation. In absence of methanogens, methanol was converted aerobically, while granule formation remained stable. At high SRT-values (51 days) γ-Proteobacteria were responsible for acetate removal through anaerobic uptake and subsequent aerobic growth on storage polymers formed (so called metabolism of glycogen accumulating organisms). When lowering the SRT (24 days), Defluviicoccus-related organisms (cluster II) belonging to the α-Proteobacteria outcompeted acetate consuming γ-Proteobacteria at 35 ºC. DNA from the Defluviicoccus-related organisms in cluster II was not extracted by the standard DNA extraction method but with liquid nitrogen, which showed to be more effective. Remarkably, the two glycogen accumulating organisms (GAO) types of organisms grew separately in two clearly different types of granules. This work further highlights the potential of aerobic granular sludge systems to effectively influence the microbial communities through sludge age control in order to optimize the wastewater treatment processes. Recently, aerobic granular sludge technology has been scaled-up and implemented for industrial and municipal wastewater treatment under the trade name Nereda®. With full-scale references for industrial treatment application since 2006 and domestic sewage since 2009 only limited operating data have been presented in scientific literature so far. In this study performance, granulation and design considerations of an aerobic granular sludge plant on domestic wastewater at the WWTP Garmerwolde, the Netherlands were analysed (Chapter 6). After a start-up period of approximately 5 months, a robust and stable granule bed (> 8 g L-1) was formed and could be maintained thereafter, with a sludge volume index after 5 minutes settling of 45 mL g-1. The granular sludge consisted for more than 80 % of granules larger than 0.2 mm and more than 60 % larger than 1 mm. Effluent requirements (7 mg N L-1 and 1 mg P L-1) were easily met during summer and winter. Maximum volumetric conversion rates for nitrogen and phosphorus were respectively 0.17 and 0.24 kg (m3 d)-1. The energy usage was 13.9 kWh (PE150∙year)-1 that is 58 – 63 % lower than the average conventional activated sludge treatment plant in the Netherlands. Finally, this study demonstrated that aerobic granular sludge technology can effectively be implemented for the treatment of domestic wastewater.","","en","doctoral thesis","","978-94-028-0131-6","","","","","","","","","BT/Environmental Biotechnology","","",""
"uuid:e59389dc-9b03-4e98-875a-79e290907c41","http://resolver.tudelft.nl/uuid:e59389dc-9b03-4e98-875a-79e290907c41","Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6","Papapetridis, I. (TU Delft BT/Industriele Microbiologie); van Dijk, M. (TU Delft BT/Industriele Microbiologie); Dobbe, Arthur P A; Metz, B. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie)","","2016","Background: Acetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation as a mechanism for reoxidizing NADH formed in biosynthesis. An increase in the amount of acetate that can be reduced to ethanol should further decrease acetic acid concentrations and enable higher ethanol yields in industrial processes based on lignocellulosic feedstocks. The stoichiometric requirement of acetate reduction for NADH implies that increased generation of NADH in cytosolic biosynthetic reactions should enhance acetate consumption. Results: Replacement of the native NADP+-dependent 6-phosphogluconate dehydrogenase in S. cerevisiae by a prokaryotic NAD+-dependent enzyme resulted in increased cytosolic NADH formation, as demonstrated by a ca. 15 % increase in the glycerol yield on glucose in anaerobic cultures. Additional deletion of ALD6, which encodes an NADP+-dependent acetaldehyde dehydrogenase, led to a 39 % increase in the glycerol yield compared to a non-engineered strain. Subsequent replacement of glycerol formation by an acetate reduction pathway resulted in a 44 % increase of acetate consumption per amount of biomass formed, as compared to an engineered, acetate-reducing strain that expressed the native 6-phosphogluconate dehydrogenase and ALD6. Compared to a non-acetate reducing reference strain under the same conditions, this resulted in a ca. 13 % increase in the ethanol yield on glucose. Conclusions: The combination of NAD+-dependent 6-phosphogluconate dehydrogenase expression and deletion of ALD6 resulted in a marked increase in the amount of acetate that was consumed in these proof-of-principle experiments, and this concept is ready for further testing in industrial strains as well as in hydrolysates. Altering the cofactor specificity of the oxidative branch of the pentose-phosphate pathway in S. cerevisiae can also be used to increase glycerol production in wine fermentation and to improve NADH generation and/or generation of precursors derived from the pentose-phosphate pathway in other industrial applications of this yeast.","6-phosphogluconate dehydrogenase; Acetic acid; NADH; NADPH; Redox metabolism; Yeast","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:2b65286f-bbfd-4203-91a3-4723bb315d90","http://resolver.tudelft.nl/uuid:2b65286f-bbfd-4203-91a3-4723bb315d90","Excessive by-product formation: A key contributor to low isobutanol yields of engineered Saccharomyces cerevisiae strains","Milne, N.S.W.; Wahl, S.A.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.M.","","2016","It is theoretically possible to engineer Saccharomyces cerevisiae strains in which isobutanol is the predominant catabolic product and high-yielding isobutanol-producing strains are already reported by industry. Conversely, isobutanol yields of engineered S. cerevisiae strains reported in the scientific literature typically remain far below 10% of the theoretical maximum. This study explores possible reasons for these suboptimal yields by a mass-balancing approach. A cytosolically located, cofactor-balanced isobutanol pathway, consisting of a mosaic of bacterial enzymes whose in vivo functionality was confirmed by complementation of null mutations in branched-chain amino acid metabolism, was expressed in S. cerevisiae. Product formation by the engineered strain was analysed in shake flasks and bioreactors. In aerobic cultures, the pathway intermediate isobutyraldehyde was oxidized to isobutyrate rather than reduced to isobutanol. Moreover, significant concentrations of the pathway intermediates 2,3-dihydroxyisovalerate and ?-ketoisovalerate, as well as diacetyl and acetoin, accumulated extracellularly. While the engineered strain could not grow anaerobically, micro-aerobic cultivation resulted in isobutanol formation at a yield of 0.018±0.003 mol/mol glucose. Simultaneously, 2,3-butanediol was produced at a yield of 0.649±0.067 mol/mol glucose. These results identify massive accumulation of pathway intermediates, as well as overflow metabolites derived from acetolactate, as an important, previously underestimated contributor to the suboptimal yields of ‘academic’ isobutanol strains. The observed patterns of by-product formation is consistent with the notion that in vivo activity of the iron–sulphur-cluster-requiring enzyme dihydroxyacid dehydratase is a key bottleneck in the present and previously described ‘academic’ isobutanol-producing yeast strains.","saccharomyces cerevisiae; isobutanol; catabolic pathway; by-product formation; 2,3-butanediol; diacetyl","en","journal article","Elsevier","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:2ddf18b3-ec34-45b9-a64e-9e59f0026367","http://resolver.tudelft.nl/uuid:2ddf18b3-ec34-45b9-a64e-9e59f0026367","SMA-SH: Modified styrene maleic acid copolymer for functionalization of lipid nanodiscs","Lindhoud, S. (TU Delft BN/Marie-Eve Aubin-Tam Lab; Kavli institute of nanoscience Delft); Dias Ribeiro de Carvalho, V.I. (TU Delft BN/Marie-Eve Aubin-Tam Lab; Kavli institute of nanoscience Delft); Pronk, J.W. (TU Delft BN/Andreas Engel Lab; Kavli institute of nanoscience Delft); Aubin-Tam, M.E. (TU Delft BN/Marie-Eve Aubin-Tam Lab; Kavli institute of nanoscience Delft)","","2016","Challenges in purification and subsequent functionalization of membrane proteins often complicate their biochemical and biophysical characterization. Purification of membrane proteins generally involves replacing the lipids surrounding the protein with detergent molecules, which can affect protein structure and function. Recently, it was shown that styrene–maleic acid copolymers (SMA) can dissolve integral membrane proteins from biological membranes into nanosized discs. Within these nanoparticles, proteins are embedded in a patch of their native lipid bilayer that is stabilized in solution by the amphipathic polymer that wraps the disc like a bracelet. This approach for detergent-free purification of membrane proteins has the potential to greatly simplify purification but does not facilitate conjugation of functional compounds to the membrane proteins. Often, such functionalization involves laborious preparation of protein variants and optimization of labeling procedures to ensure only minimal perturbation of the protein. Here, we present a strategy that circumvents several of these complications through modifying SMA by grafting the polymer with cysteamine. The reaction results in SMA that has solvent-exposed sulfhydrils (SMA-SH) and allows tuning of the coverage with SH groups. Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy demonstrate that SMA-SH dissolves lipid bilayer membranes into lipid nanodiscs, just like SMA. In addition, we demonstrate that, just like SMA, SMA-SH solubilizes proteoliposomes into protein-loaded nanodiscs. We covalently modify SMA-SH-lipid nanodiscs using thiol-reactive derivatives of Alexa Fluor 488 and biotin. Thus, SMA-SH promises to simultaneously tackle challenges in purification and functionalization of membrane proteins.","","en","journal article","","","","","","","","","","","BN/Marie-Eve Aubin-Tam Lab","","",""
"uuid:cb4c0076-03ae-406a-acb5-8f8f7b2c255d","http://resolver.tudelft.nl/uuid:cb4c0076-03ae-406a-acb5-8f8f7b2c255d","Pichia Pastoris exhibits high viability and a low maintenance energy requirement at near-zero specific growth rates","Rebnegger, C. (TU Delft BT/Industriele Microbiologie; BOKU-University of Natural Resources and Life Sciences); Vos, T. (TU Delft BT/Industriele Microbiologie); Graf, Alexandra B. (Austrian Centre of Industrial Biotechnology (ACIB GmbH)); Valli, Minoska (Austrian Centre of Industrial Biotechnology (ACIB GmbH)); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); Daran-Lapujade, P.A.S. (TU Delft BT/Industriele Microbiologie); Mattanovicha, Diethard (Austrian Centre of Industrial Biotechnology (ACIB GmbH))","","2016","The yeast Pichia pastoris is a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented for P. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/S max) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (mu;) of 0.025 h-1 to near-zero specific growth rates (mu; -1). At these near-zero specific growth rates, viability remained at least 97%. The value of mS at near-zero growth rates was 3.10± 1 mg glucose per g biomass and h, which was 3-fold lower than themS estimated from fastergrowing chemostat cultures. This difference indicated that P. pastoris reduces its maintenance energy requirement at extremely low , a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes.","","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:79da98c8-02de-4abc-a5f7-ecfd195e202a","http://resolver.tudelft.nl/uuid:79da98c8-02de-4abc-a5f7-ecfd195e202a","Requirements for carnitine shuttle-mediated translocation of mitochondrial acetyl moieties to the yeast cytosol","Rossum, Harmen M. (TU Delft BT/Industriele Microbiologie; Zymergen); Kozak, B.U. (TU Delft BT/Industriele Microbiologie; DuPont); Niemeijer, M.S. (TU Delft BT/Industriele Microbiologie); Dykstra, James C.; Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); Daran, J.G. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie)","","2016","In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acidgrown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was L-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabled L-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle. IMPORTANCE This study demonstrates, for the first time, that Saccharomyces cerevisiae can be engineered to employ the carnitine shuttle for export of acetyl moieties from the mitochondria and, thereby, to act as the sole source of cytosolic acetyl-CoA. Further optimization of this ATP-independent mechanism for cytosolic acetyl-CoA provision can contribute to efficient, yeastbased production of industrially relevant compounds derived from this precursor. The strains constructed in this study, whose growth on glucose depends on a functional carnitine shuttle, provide valuable models for further functional analysis and engineering of this shuttle in yeast and other eukaryotes.","","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:a0717e5b-7bd5-4f1a-bf78-964a5d8f58ec","http://resolver.tudelft.nl/uuid:a0717e5b-7bd5-4f1a-bf78-964a5d8f58ec","Alternative reactions at the interface of glycolysis and citric acid cycle in Saccharomyces cerevisiae","Rossum, Harmen M. (TU Delft BT/Industriele Microbiologie); Kozak, B.U. (TU Delft BT/Industriele Microbiologie); Niemeijer, M.S. (TU Delft BT/Industriele Microbiologie); Duine, Hendrik J. (Student TU Delft); Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie); Boer, VM (DSM); Kötter, P (Goethe University); Daran, J.G. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Biotechnologie)","","2016","Pyruvate and acetyl-coenzyme A, located at the interface between glycolysis and TCA cycle, are important intermediates in yeast metabolism and key precursors for industrially relevant products. Rational engineering of their supply requires knowledge of compensatory reactions that replace predominant pathways when these are inactivated. This study investigates effects of individual and combined mutations that inactivate the mitochondrial pyruvate-dehydrogenase (PDH) complex, extramitochondrial citrate synthase (Cit2) and mitochondrial CoA-transferase (Ach1) in Saccharomyces cerevisiae. Additionally, strains with a constitutively expressed carnitine shuttle were constructed and analyzed. A predominant role of the PDH complex in linking glycolysis and TCA cycle in glucose-grown batch cultures could be functionally replaced by the combined activity of the cytosolic PDH bypass and Cit2. Strongly impaired growth and a high incidence of respiratory deficiency in pda1Δ ach1Δ strains showed that synthesis of intramitochondrial acetyl-CoA as a metabolic precursor requires activity of either the PDH complex or Ach1. Constitutive overexpression of AGP2, HNM1, YAT2, YAT1, CRC1 and CAT2 enabled the carnitine shuttle to efficiently link glycolysis and TCA cycle in l-carnitine-supplemented, glucose-grown batch cultures. Strains in which all known reactions at the glycolysis-TCA cycle interface were inactivated still grew slowly on glucose, indicating additional flexibility at this key metabolic junction.","Ach1; Cit2; PDH complex; Saccharomyces cerevisiae; carnitine shuttle; glycolysis-TCA cycle interface","en","journal article","","","","","","","","","","BT/Biotechnologie","BT/Industriele Microbiologie","","",""
"uuid:7ba8d54f-71af-4bf2-aa07-94e4f2c58e08","http://resolver.tudelft.nl/uuid:7ba8d54f-71af-4bf2-aa07-94e4f2c58e08","Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae:: Pathway stoichiometry, free-energy conservation and redox-cofactor balancing","Rossum, Harmen M. (TU Delft BT/Industriele Microbiologie); Kozak, B.U.; Pronk, J.T. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie)","","2016","","","en","review","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:23dcf3a7-2002-4078-bd2e-254758304dd7","http://resolver.tudelft.nl/uuid:23dcf3a7-2002-4078-bd2e-254758304dd7","Replacement of the initial steps of ethanol metabolism in Saccharomyces cerevisiae by ATP-independent acetylating acetaldehyde dehydrogenase","Kozak, B.U. (TU Delft BT/Industriele Microbiologie); Rossum, Harmen M. (TU Delft BT/Industriele Microbiologie); Niemeijer, M.S. (TU Delft BT/Industriele Microbiologie); van Dijk, M. (TU Delft BT/Industriele Microbiologie); Benjamin, Kirsten (Amyris Inc); Wu, Liang (DSM); Daran, J.G. (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie)","","2016","In Saccharomyces cerevisiae ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore the implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory, enables biomass yield on ethanol that is up to 40% higher. To this end, all native yeast acetaldehyde dehydrogenases (ALDs) were replaced by heterologous acetylating acetaldehyde dehydrogenase (A-ALD). Engineered Ald- strains expressing different A-ALDs did not immediately grow on ethanol, but serial transfer in ethanol-grown batch cultures yielded growth rates of up to 70% of the wild-type value. Mutations in ACS1 were identified in all independently evolved strains and deletion of ACS1 enabled slow growth of non-evolved Ald- A-ALD strains on ethanol. Acquired mutations in A-ALD genes improved affinity-Vmax/Km for acetaldehyde. One of five evolved strains showed a significant 5% increase of its biomass yield in ethanol-limited chemostat cultures. Increased production of acetaldehyde and other by-products was identified as possible cause for lower than theoretically predicted biomass yields. This study proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway with the potential to improve biomass and product yields.","Acetyl-CoA; Energetics; Evolutionary engineering; Intracellular metabolites; Precursor supply; Yeast","en","journal article","","","","","","","","","","","BT/Industriele Microbiologie","","",""
"uuid:393d1946-65ac-4e11-be2b-886916f1eaeb","http://resolver.tudelft.nl/uuid:393d1946-65ac-4e11-be2b-886916f1eaeb","Growth-rate dependency of de novo resveratrol production in chemostat cultures of an engineered Saccharomyces cerevisiae strain","Vos, T.; De la Torre Cortes, P.; Van Gulik, W.M.; Pronk, J.T.; Daran-Lapujade, P.A.S.","","2015","Introduction: Saccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures. Results: Stoichiometric analysis revealed that de novo resveratrol production from glucose requires 13 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g. 27 % at 0.03 h?1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in chemostat. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g. ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer. Conclusions: De novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways.","metabolic engineering; maintenance energy; anabolic products; qp; continuous culture; yeast; synthetic biology","en","journal article","BioMed Central","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:e45e2b46-587c-4ebd-ad7d-6e344da7b8e1","http://resolver.tudelft.nl/uuid:e45e2b46-587c-4ebd-ad7d-6e344da7b8e1","Biopolymer extraction","Lin, Y.; Al-Zuhairy, S.; Pronk, M.; Van Loosdrecht, M.C.M.","","2015","In a prior art reactor set up dense aggregates of microorganisms are formed, typically in or embedded in an extracellular matrix. Such may relate to granules, to sphere like entities having a higher viscosity than water, globules, a biofilm, etc. The dense aggregates comprise extracellular polymeric substances, or biopolymers, in particular linear polysaccharides, The present invention is in the field of extraction of a biopolymer from a granular sludge, a biopolymer obtained by said method, and a use of said method.","","en","patent","European Patent Office","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:514d0f1c-9382-4eca-afc6-41f7bd07d8fe","http://resolver.tudelft.nl/uuid:514d0f1c-9382-4eca-afc6-41f7bd07d8fe","Comparative assessment of native and heterologous 2-oxo acid decarboxylases for application in isobutanol production by Saccharomyces cerevisiae","Milne, N.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.M.","","2015","Background: Decarboxylation of ?-ketoisovalerate to isobutyraldehyde is a key reaction in metabolic engineering of Saccharomyces cerevisiae for isobutanol production with published studies relying on overexpression of either the native ARO10 gene or of the Lactococcus lactis kivD decarboxylase gene resulting in low enzymatic activities. Here, we compare relevant properties for isobutanol production of Aro10, KivD and an additional, less studied, L. lactis decarboxylase KdcA. Results: To eliminate interference by native decarboxylases, each 2-oxo acid decarboxylase was overexpressed in a ‘decarboxylase-negative’ (pdc1? pdc5? pdc6? aro10?) S. cerevisiae background. Kinetic analyses in cell extracts a superior Vmax/Km ratio of KdcA for ?-ketoisovalerate and a wide range of linear and branched-chain 2-oxo acids. However, KdcA also showed the highest activity with pyruvate which, in engineered strains, can contribute to formation of ethanol as a by-product. Removal of native decarboxylase genes eliminated growth on valine as sole nitrogen source and subsequent complementation of this growth impairment by expression of each decarboxylase indicated that based on the increased growth rate, the in vivo activity of KdcA with ?-ketoisovalerate was higher than that of KivD and Aro10. Moreover, during oxygen-limited incubation in the presence of glucose, strains expressing kdcA or kivD showed a ca. twofold higher in vivo rate of conversion of ?-ketoisovalerate into isobutanol than an ARO10-expressing strain. Finally, cell extracts from cultures grown on different nitrogen sources revealed increased activity of constitutively expressed KdcA after growth on both valine and phenylalanine, while KivD and Aro10 activity was only increased after growth on phenylalanine suggesting a difference in the regulation of these enzymes. Conclusions: This study illustrates important differences in substrate specificity, enzyme kinetics and functional expression between different decarboxylases in the context of isobutanol production and identifies KdcA as a promising alternative decarboxylase not only for isobutanol production but also for other branched-chain and linear alcohols.","saccharomyces cerevisiae; 2-oxo acid decarboxylase; lactococcus lactis; isobutanol production; fusel alcohol production; OA-Fund TU Delft","en","journal article","BioMed Central","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:63c57205-336a-492f-8a8b-52e2650fa322","http://resolver.tudelft.nl/uuid:63c57205-336a-492f-8a8b-52e2650fa322","Ureohydrolases as dominant selectable markers in yeast","Daran, J.G.; Pronk, J.T.; Romagnoli, G.","","2015","The invention relates to a nucleic acid molecule encoding a novel selection marker. Said marker is a guanidinobutyrase from Kluyveromyces lactis, which, when expressed in Saccharomyces, allows the growth of the yeast in the presence of guanidinobutyrate as the sole nitrogen source. Said marker can be used in a method for producing a microorganism having an altered genome. The invention further relates to a set of constructs, comprising a first construct comprising a recognition site for an endonuclease, a first region of homology with a target gene of a microorganism, and a first part of a nucleotide sequence encoding the selection marker, and a second construct comprising a second part of the nucleotide sequence encoding the selection marker, a second region of homology with the target gene of the microorganism, and a copy of the endonuclease recognition site. The invention further relates to methods for altering a target gene in a microorganism, to methods for producing a microorganism, and to microorganisms that are produced by the methods of the invention.","","en","patent","European Patent Office","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:0b544676-12e4-4c47-88dc-878650650541","http://resolver.tudelft.nl/uuid:0b544676-12e4-4c47-88dc-878650650541","Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains","Van den Broek, M.; Bolat, I.; Nijkamp, J.F.; Ramos, E.; Luttik, M.A.H.; Koopman, F.; Geertman, J.M.; De Ridder, D.; Pronk, J.T.; Daran, J.M.","","2015","Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Bionanoscience","","","",""
"uuid:80a6a845-4a49-4355-8e90-0dad74e291ad","http://resolver.tudelft.nl/uuid:80a6a845-4a49-4355-8e90-0dad74e291ad","Effect of sludge age on methanogenic and glycogen accumulating organisms in an aerobic granular sludge process fed with methanol and acetate","Pronk, M.; Abbas, B.; Kleerebezem, R.; Van Loosdrecht, M.C.M.","","2015","The influence of sludge age on granular sludge formation and microbial population dynamics in a methanol- and acetate-fed aerobic granular sludge system operated at 35°C was investigated. During anaerobic feeding of the reactor, methanol was initially converted to methane by methylotrophic methanogens. These methanogens were able to withstand the relatively long aeration periods. Lowering the anaerobic solid retention time (SRT) from 17 to 8 days enabled selective removal of the methanogens and prevented unwanted methane formation. In absence of methanogens, methanol was converted aerobically, while granule formation remained stable. At high SRT values (51 days), ?-Proteobacteria were responsible for acetate removal through anaerobic uptake and subsequent aerobic growth on storage polymers formed [so called metabolism of glycogen-accumulating organisms (GAO)]. When lowering the SRT (24 days), Defluviicoccus-related organisms (cluster II) belonging to the ?-Proteobacteria outcompeted acetate consuming ?-Proteobacteria at 35°C. DNA from the Defluviicoccus-related organisms in cluster II was not extracted by the standard DNA extraction method but with liquid nitrogen, which showed to be more effective. Remarkably, the two GAO types of organisms grew separately in two clearly different types of granules. This work further highlights the potential of aerobic granular sludge systems to effectively influence the microbial communities through sludge age control in order to optimize the wastewater treatment processes.","OA-Fund TU Delft","en","journal article","Wiley","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:23c43c46-0b85-496f-93d1-c3fbff15abe5","http://resolver.tudelft.nl/uuid:23c43c46-0b85-496f-93d1-c3fbff15abe5","Physiological and Transcriptional Responses of Different Industrial Microbes at Near-Zero Specific Growth Rates","Ercan, O.; Bisschops, M.M.; Overkamp, W.; Jørgensen, T.R.; Rama, A.F.; Smid, E.J.; Pronk, J.T.; Kuipers, O.P.; Daran-Lapujade, P.; Kleerebezem, M.","","2015","The current knowledge of the physiology and gene expression of industrially relevant microorganisms is largely based on laboratory studies under conditions of rapid growth and high metabolic activity. However, in natural ecosystems and industrial processes, microbes frequently encounter severe calorie restriction. As a consequence, microbial growth rates in such settings can be extremely slow and even approach zero. Furthermore, uncoupling microbial growth from product formation, while cellular integrity and activity are maintained, offers perspectives that are economically highly interesting. Retentostat cultures have been employed to investigate microbial physiology at (near-)zero growth rates. This minireview compares information from recent physiological and gene expression studies on retentostat cultures of the industrially relevant microorganisms Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger. Shared responses of these organisms to (near-)zero growth rates include increased stress tolerance and a downregulation of genes involved in protein synthesis. Other adaptations, such as changes in morphology and (secondary) metabolite production, were species specific. This comparison underlines the industrial and scientific significance of further research on microbial (near-)zero growth physiology.","","en","journal article","American Society for Microbiology","","","","","","","2015-12-06","Applied Sciences","Biotechnology","","","",""
"uuid:5563663b-39da-4476-9f9d-47914b9047d5","http://resolver.tudelft.nl/uuid:5563663b-39da-4476-9f9d-47914b9047d5","Title microbial linear polysacchrides being soluble at a low pH and precipitating at a high pH","Van Loosdrecht, M.C.M.; Lin, Y.; Pronk, M.","","2015","The present invention concerns a method of producing linear polysaccharides, bacteria capable of producing such linear polysaccharides, and uses of such linear polysaccharides, as a medicament, in agriculture, in food processing, as a coating, as a thickening agent, as a flocculating agent, and for water processing.","","en","patent","European Patent Office","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:7fc8cea1-e81d-4608-9f5f-9f20289c3cad","http://resolver.tudelft.nl/uuid:7fc8cea1-e81d-4608-9f5f-9f20289c3cad","Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae","Milne, N.; Luttik, M.A.H.; Cueto Rojas, H.F.; Wahl, A.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.G.","","2015","In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni2+-dependent enzyme and Saccharomyces cerevisiae does not express native Ni2+-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2? S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min?1 mg protein?1 and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni2+ concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni2+-dependent enzymes can be functionally expressed in S. cerevisiae.","Saccharomyces cerevisiae; ATP-independent urease; Ni-dependent enzyme; ATP conservation; nitrogen metabolism; physiology","en","journal article","Elsevier","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:fed57754-dd36-4e5a-b89f-ef77c49014b0","http://resolver.tudelft.nl/uuid:fed57754-dd36-4e5a-b89f-ef77c49014b0","A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism","Solis-Escalante, D.; Kuijpers, N.G.A.; Barrajon-Simancas, N.; Van den Broek, M.; Pronk, J.T.; Daran, J.M.; Daran-Lapujade, P.","","2015","As a result of ancestral whole-genome and small-scale duplication events, the genomes of Saccharomyces cerevisiae and many eukaryotes still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of the Saccharomyces lineage, the duplication of glycolytic genes is purported to have played an important role leading to S. cerevisiae's current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modern S. cerevisiae strains, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (the “minimal glycolysis” [MG] strain). Remarkably, a combination of a quantitative systems approach and semiquantitative analysis in a wide array of growth environments revealed the absence of a phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic of S. cerevisiae and essential for many of its industrial applications and argues against gene dosage effects as a means of fixing minor glycolytic paralogs in the yeast genome. The MG strain was carefully designed and constructed to provide a robust prototrophic platform for quantitative studies and has been made available to the scientific community.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:db68e491-0e6d-4d54-8799-68dc9a6eb670","http://resolver.tudelft.nl/uuid:db68e491-0e6d-4d54-8799-68dc9a6eb670","CRISPR/Cas9: A molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae","Mans, R.; Van Rossum, H.M.; Wijsman, M.; Backx, A.; Kuijpers, N.G.A.; van den Broek, M.; Daran-Lapujade, P.A.S.; Pronk, J.T.; Van Maris, A.J.A.; Daran, J.G.","","2015","A variety of techniques for strain engineering in Saccharomyces cerevisiae have recently been developed. However, especially when multiple genetic manipulations are required, strain construction is still a time-consuming process. This study describes new CRISPR/Cas9-based approaches for easy, fast strain construction in yeast and explores their potential for simultaneous introduction of multiple genetic modifications. An open-source tool (http://yeastriction.tnw.tudelft.nl) is presented for identification of suitable Cas9 target sites in S. cerevisiae strains. A transformation strategy, using in vivo assembly of a guideRNA plasmid and subsequent genetic modification, was successfully implemented with high accuracies. An alternative strategy, using in vitro assembled plasmids containing two gRNAs, was used to simultaneously introduce up to six genetic modifications in a single transformation step with high efficiencies. Where previous studies mainly focused on the use of CRISPR/Cas9 for gene inactivation, we demonstrate the versatility of CRISPR/Cas9-based engineering of yeast by achieving simultaneous integration of a multigene construct combined with gene deletion and the simultaneous introduction of two single-nucleotide mutations at different loci. Sets of standardized plasmids, as well as the web-based Yeastriction target-sequence identifier and primer-design tool, are made available to the yeast research community to facilitate fast, standardized and efficient application of the CRISPR/Cas9 system.","CRISPR/Cas9; S. cerevisiae, gRNA; genetic modification; webtool; plasmid","en","journal article","Oxford University Press","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:14ed6a3d-93ac-4b4c-9504-6ae1db522035","http://resolver.tudelft.nl/uuid:14ed6a3d-93ac-4b4c-9504-6ae1db522035","Effect and behaviour of different substrates in relation to the formation of aerobic granular sludge","Pronk, M.; Abbas, B.; Al-zuhairy, S.H.K.; Kraan, R.; Kleerebezem, R.; Van Loosdrecht, M.C.M.","","2015","When aerobic granular sludge is applied for industrial wastewater treatment, different soluble substrates can be present. For stable granular sludge formation on volatile fatty acids (e.g. acetate), production of storage polymers under anaerobic feeding conditions has been shown to be important. This prevents direct aerobic growth on readily available chemical oxygen demand (COD), which is thought to result in unstable granule formation. Here, we investigate the impact of acetate, methanol, butanol, propanol, propionaldehyde, and valeraldehyde on granular sludge formation at 35 °C. Methanogenic archaea, growing on methanol, were present in the aerobic granular sludge system. Methanol was completely converted to methane and carbon dioxide by the methanogenic archaeum Methanomethylovorans uponensis during the 1-h anaerobic feeding period, despite the relative high dissolved oxygen concentration (3.5 mg O2 L?1) during the subsequent 2-h aeration period. Propionaldehyde and valeraldehyde were fully disproportionated anaerobically into their corresponding carboxylic acids and alcohols. The organic acids produced were converted to storage polymers, while the alcohols (produced and from influent) were absorbed onto the granular sludge matrix and converted aerobically. Our observations show that easy biodegradable substrates not converted anaerobically into storage polymers could lead to unstable granular sludge formation. However, when the easy biodegradable COD is absorbed in the granules and/or when the substrate is converted by relatively slow growing bacteria in the aerobic period, stable granulation can occur.","aerobic granular sludge; methanol; alcohol; aldehyde; methanogens; granule formation; industrial wastewater; disproportionation; feeding strategies","en","journal article","Springer","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:c7f9a197-73d6-4ed3-a703-d064db50cf9f","http://resolver.tudelft.nl/uuid:c7f9a197-73d6-4ed3-a703-d064db50cf9f","Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of Saccharomyces cerevisiae","Kozak, B.U.; Van Rossum, H.M.; Luttik, M.A.H.; Akeroyd, M.; Benjamin, K.R.; Wu, L.; De Vries, S.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.","","2014","The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1?, E1?, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways.","","en","journal article","","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:9e21b29e-928d-4a0e-98f3-aa5c96c64baf","http://resolver.tudelft.nl/uuid:9e21b29e-928d-4a0e-98f3-aa5c96c64baf","Engineering acetyl coenzyme A supply: Functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae","Kozak, B.U.; Van Rossum, M.H.; Luttik, M.A.; Akeroyd, M.; Benjamin, K.R.; Wu, L.; De Vries, S.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.","","2014","The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1?, E1?, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker's yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker's yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker's yeast as a ""cell factory"" for sustainable production of fuels and chemicals.","","en","journal article","American Society for Microbiology: mBio","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:b4b996ab-f9a7-41e1-acaf-aae8fd99abd3","http://resolver.tudelft.nl/uuid:b4b996ab-f9a7-41e1-acaf-aae8fd99abd3","Increasing ATP conservation in maltose consuming yeast, a challenge for industrial organic acid production in non-aerated reactors","De Kok, S.; Marques, W.L.; Mans, R.; Yilmaz, D.; Suir, E.; Pronk, J.T.; Gombert, A.K.; Daran, J.M.; Van Maris, A.J.A.","","2014","","OA-Fund TU Delft","en","journal article","BioMed Central","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:b8b80a55-4fb2-4c08-8103-5eefca65a9ee","http://resolver.tudelft.nl/uuid:b8b80a55-4fb2-4c08-8103-5eefca65a9ee","Recombinant micro-organism for use in method with increased product yield","Van Maris, A.J.A.; Pronk, J.T.; Guadalupe Medina, V.G.; Wisselink, H.W.","","2014","The invention relates to a recombinant yeast cell, in particular a transgenic yeast cell, functionally expressing one or more recombinant, in particular heterologous, nucleic acid sequences encoding ribulose-1,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK). The invention further relates to the use of carbon dioxide as an electron acceptor in a recombinant chemotrophic micro-organism, in particular a eukaryotic micro-organism.","","en","patent","European Patent Office","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:7fe1abba-385d-4a30-a27c-12d405c118d3","http://resolver.tudelft.nl/uuid:7fe1abba-385d-4a30-a27c-12d405c118d3","An alternative, arginase-independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme","Romagnoli, G.; Verhoeven, M.D.; Mans, R.; Fleury Rey, Y.; Bel-Rhlid, R.; Van den Broek, M.; Maleki Seifar, R.; Ten Pierick, A.; Thompson, M.; Müller, V.; Wahl, S.A.; Pronk, J.T.; Daran, J.M.","","2014","Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in ?-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1? mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis?KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi.","","en","journal article","Wiley","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:939a8d69-1a18-461d-8385-12f65a4e6806","http://resolver.tudelft.nl/uuid:939a8d69-1a18-461d-8385-12f65a4e6806","Physiological and transcriptional responses of anaerobic chemostat cultures of Saccharomyces cerevisiae subjected to diurnal temperature cycles","Hebly, M.; De Ridder, D.; De Hulster, E.A.; De la Torre Cortes, P.; Pronk, J.T.; Daran-Lapujade, P.A.S.","","2014","Diurnal temperature cycling is an intrinsic characteristic of many exposed microbial ecosystems. However, its influence on yeast physiology and the yeast transcriptome has not been studied in detail. In this study, 24-h sinusoidal temperature cycles, oscillating between 12°C and 30°C, were imposed on anaerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. After three diurnal temperature cycles (DTC), concentrations of glucose and extracellular metabolites as well as CO2 production rates showed regular, reproducible circadian rhythms. DTC also led to waves of transcriptional activation and repression, which involved one-sixth of the yeast genome. A substantial fraction of these DTC-responsive genes appeared to respond primarily to changes in the glucose concentration. Elimination of known glucose-responsive genes revealed an overrepresentation of previously identified temperature-responsive genes as well as genes involved in the cell cycle and de novo purine biosynthesis. In-depth analysis demonstrated that DTC led to a partial synchronization of the cell cycle of the yeast populations in chemostat cultures, which was lost upon release from DTC. Comparison of DTC results with data from steady-state cultures showed that the 24-h DTC was sufficiently slow to allow S. cerevisiae chemostat cultures to acclimate their transcriptome and physiology at the DTC temperature maximum and to approach acclimation at the DTC temperature minimum. Furthermore, this comparison and literature data on growth rate-dependent cell cycle phase distribution indicated that cell cycle synchronization was most likely an effect of imposed fluctuations of the relative growth rate (?/?max) rather than a direct effect of temperature.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:68fd9d83-80b5-41de-aae9-91a046dbc3f4","http://resolver.tudelft.nl/uuid:68fd9d83-80b5-41de-aae9-91a046dbc3f4","Functional characterization of a Penicillium chrysogenum mutanase gene induced upon co-cultivation with Bacillus subtilis","Bajaj, I.; Veiga, T.; Van Dissel, D.; Pronk, J.T.; Daran, J.M.","","2014","Background Microbial gene expression is strongly influenced by environmental growth conditions. Comparison of gene expression under different conditions is frequently used for functional analysis and to unravel regulatory networks, however, gene expression responses to co-cultivation with other microorganisms, a common occurrence in nature, is rarely studied under laboratory conditions. To explore cellular responses of the antibiotic-producing fungus Penicillium chrysogenum to prokaryotes, the present study investigates its transcriptional responses during co-cultivation with Bacillus subtilis. Results Steady-state glucose-limited chemostats of P. chrysogenum grown under penillicin-non-producing conditions were inoculated with B. subtilis. Physiological and transcriptional responses of P. chrysogenum in the resulting mixed culture were monitored over 72 h. Under these conditions, B. subtilis outcompeted P. chrysogenum, as reflected by a three-fold increase of the B. subtilis population size and a two-fold reduction of the P. chrysogenum biomass concentration. Genes involved in the penicillin pathway and in synthesis of the penicillin precursors and side-chain were unresponsive to the presence of B. subtilis. Moreover, Penicillium polyketide synthase and nonribosomal peptide synthase genes were either not expressed or down-regulated. Among the highly responsive genes, two putative ?-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold, respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production. Mutanase activity was neither observed in pure cultures of P. chrysogenum or B. subtilis, nor during exposure of P. chrysogenum to B. subtilis culture supernatants or heat-inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum exposed to filter-sterilized supernatants of mixed cultures of P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that Pc12g07500 encoded an active ?-1,3 endoglucanase. Conclusion Time-course transcriptional profiling of P. chrysogenum revealed differentially expressed genes during co-cultivation with B. subtilis. Penicillin production was not induced under these conditions. However, induction of a newly characterized P. chrysogenum gene encoding ?-1,3 endoglucanase may enhance the efficacy of fungal antibiotics by degrading bacterial exopolysaccharides","time-course transcriptional analysis; mixed culture; chemostat-based transcriptomics; mutanase; heterologous expression; Penicillium chrysogenum; Bacillus subtilis; OA-Fund TU Delft","en","journal article","BioMed Central","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:aee77b3f-2ba2-41d7-9c38-1cdd1aae1e2b","http://resolver.tudelft.nl/uuid:aee77b3f-2ba2-41d7-9c38-1cdd1aae1e2b","Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis","Kozak, B.U.; Van Rossum, H.M.; Benjamin, K.R.; Wu, L.; Daran, J.G.; Pronk, J.T.; Van Maris, A.J.A.","","2013","Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1? acs2? S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h?1 were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.20 h?1) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While demonstrating that yeast ACS can be fully replaced, this study demonstrates that further modifications are needed to achieve optimal in vivo performance of the alternative reactions for supply of cytosolic acetyl-CoA as a product precursor.","yeast; acetylating acetaldehyde dehydrogenase; pyruvate-formate lyase; transcriptome; precursor supply; metabolic compartments","en","journal article","Elsevier","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:0f1c8773-f7e9-412e-b1e4-283e0cf33ee6","http://resolver.tudelft.nl/uuid:0f1c8773-f7e9-412e-b1e4-283e0cf33ee6","Stochastic Evolution Equations with Adapted Drift","Pronk, M.","Van Neerven, J.M.A.M. (promotor); Veraar, M.C. (promotor)","2013","In this thesis we study stochastic evolution equations in Banach spaces. We restrict ourselves to the two following cases. First, we consider equations in which the drift is a closed linear operator that depends on time and is random. Such equations occur as mathematical models in for instance mathematical finance and filtration theory. Second, we restrict ourselves to UMD Banach spaces with type 2. As the theory of Ito stochastic integration is insufficient for studying equations of this general type, we need to have a proper understanding of several extensions to the Ito integral. Two of such extensions that are considered rigorously in this thesis are the Skorohod integral and the forward integral. Moreover, in Chapter 5, a new solution concept is introduced. The relationship between other solution concepts is discussed. Finally, we prove existence, uniqueness and regularity of solutions to stochastic evolution equations with adapted drift.","Malliavin Calculus; Stochastic Partial Differential Equations; Stochastic Evolution Equations; Forward Integration; Truncated Skorohod Integral; Space-Time Regularity; UMD Banach space; path-wise mild solution; stochastic convolution; adapted drift; non-adapted processes","en","doctoral thesis","","","","","","","","","Electrical Engineering, Mathematics and Computer Science","Delft Institute of Applied Mathematics","","","",""
"uuid:974d45fc-0aa1-4bbf-be83-bb63b3dc5f4b","http://resolver.tudelft.nl/uuid:974d45fc-0aa1-4bbf-be83-bb63b3dc5f4b","Genome duplication and mutations in ACE2 cause multicellular, fast-sedimenting phenotypes in evolved Saccharomyces cerevisiae","Oud, B.; Guadalupe-Medina, V.; Nijkamp, J.F.; De Ridder, D.; Pronk, J.T.; Van Maris, A.J.A.; Daran, J.G.","","2013","Laboratory evolution of the yeast Saccharomyces cerevisiae in bioreactor batch cultures yielded variants that grow as multicellular, fast-sedimenting clusters. Knowledge of the molecular basis of this phenomenon may contribute to the understanding of natural evolution of multicellularity and to manipulating cell sedimentation in laboratory and industrial applications of S. cerevisiae. Multicellular, fast-sedimenting lineages obtained from a haploid S. cerevisiae strain in two independent evolution experiments were analyzed by whole genome resequencing. The two evolved cell lines showed different frameshift mutations in a stretch of eight adenosines in ACE2, which encodes a transcriptional regulator involved in cell cycle control and motherdaughter cell separation. Introduction of the two ace2 mutant alleles into the haploid parental strain led to slow-sedimenting cell clusters that consisted of just a few cells, thus representing only a partial reconstruction of the evolved phenotype. In addition to single-nucleotide mutations, a whole-genome duplication event had occurred in both evolved multicellular strains. Construction of a diploid reference strain with two mutant ace2 alleles led to complete reconstruction of the multicellular-fast sedimenting phenotype. This study shows that whole-genome duplication and a frameshift mutation in ACE2 are sufficient to generate a fast-sedimenting, multicellular phenotype in S. cerevisiae. The nature of the ace2 mutations and their occurrence in two independent evolution experiments encompassing fewer than 500 generations of selective growth suggest that switching between unicellular and multicellular phenotypes may be relevant for competitiveness of S. cerevisiae in natural environments.","whole genome sequencing; reverse engineering","en","journal article","National Academy of Sciences","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:534c0a47-83a5-4f18-bab6-f1cbd2794cc2","http://resolver.tudelft.nl/uuid:534c0a47-83a5-4f18-bab6-f1cbd2794cc2","One-step assembly and targeted integration of multigene constructs assisted by the I-SceI meganuclease in Saccharomyces cerevisiae","Kuijpers, N.G.A.; Chroumpi, S.; Vos, T.; Solis-Escalante, D.; Bosman, D.; Pronk, J.T.; Daran, J.G.; Daran-Lapujade, P.A.S.","","2013","In vivo assembly of overlapping fragments by homologous recombination in Saccharomyces cerevisiae is a powerful method to engineer large DNA constructs. Whereas most in vivo assembly methods reported to date result in circular vectors, stable integrated constructs are often preferred for metabolic engineering as they are required for large-scale industrial application. The present study explores the potential of combining in vivo assembly of large, multigene expression constructs with their targeted chromosomal integration in S. cerevisiae. Combined assembly and targeted integration of a ten-fragment 22-kb construct to a single chromosomal locus was successfully achieved in a single transformation process, but with low efficiency (5% of the analyzed transformants contained the correctly assembled construct). The meganuclease I-SceI was therefore used to introduce a double-strand break at the targeted chromosomal locus, thus to facilitate integration of the assembled construct. I-SceI-assisted integration dramatically increased the efficiency of assembly and integration of the same construct to 95%. This study paves the way for the fast, efficient, and stable integration of large DNA constructs in S. cerevisiae chromosomes.","synthetic biology; in vivo assembly; homologous recombination; pathway engineering","en","journal article","Wiley","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:e032dfe8-195e-45d7-b680-981aa0728f6e","http://resolver.tudelft.nl/uuid:e032dfe8-195e-45d7-b680-981aa0728f6e","Evolutionary engineering of a glycerol-3-phosphate dehydrogenase-negative, acetate-reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations","Guadalupe-Medina, V.; Metz, B.; Oud, B.; Van der Graaf, C.M.; Pronk, J.T.; Van Maris, A.J.A.","","2013","Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S.?cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF-expressing S.?cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1?M glucose, at a specific growth rate of 0.12?h?1. The evolved strain produced glycerol at low concentrations (0.64?±?0.33?g?l?1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.","","en","journal article","Wiley Open Access","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:41139f9b-5e86-461b-bf5f-33fc7a40b1d3","http://resolver.tudelft.nl/uuid:41139f9b-5e86-461b-bf5f-33fc7a40b1d3","Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast","Guadalupe-Medina, V.; Wisselink, H.W.; Luttik, M.A.H.; De Hulster, E.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.","","2013","Background Redox-cofactor balancing constrains product yields in anaerobic fermentation processes. This challenge is exemplified by the formation of glycerol as major by-product in yeast-based bioethanol production, which is a direct consequence of the need to reoxidize excess NADH and causes a loss of conversion efficiency. Enabling the use of CO2 as electron acceptor for NADH oxidation in heterotrophic microorganisms would increase product yields in industrial biotechnology. Results A hitherto unexplored strategy to address this redox challenge is the functional expression in yeast of enzymes from autotrophs, thereby enabling the use of CO2 as electron acceptor for NADH reoxidation. Functional expression of the Calvin cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase (Rubisco) in Saccharomyces cerevisiae led to a 90% reduction of the by-product glycerol and a 10% increase in ethanol production in sugar-limited chemostat cultures on a mixture of glucose and galactose. Co-expression of the Escherichia coli chaperones GroEL and GroES was key to successful expression of CbbM, a form-II Rubisco from the chemolithoautotrophic bacterium Thiobacillus denitrificans in yeast. Conclusions Our results demonstrate functional expression of Rubisco in a heterotrophic eukaryote and demonstrate how incorporation of CO2 as a co-substrate in metabolic engineering of heterotrophic industrial microorganisms can be used to improve product yields. Rapid advances in molecular biology should allow for rapid insertion of this 4-gene expression cassette in industrial yeast strains to improve production, not only of 1st and 2nd generation ethanol production, but also of other renewable fuels or chemicals.","metabolic engineering; synthetic biology; rubisco; ribulose-1,5-bisphosphate carboxylase; phosphoribulokinase; NADH re-oxidation; carbon dioxide fixation; Saccharomyces cerevisiae; glycerol; bioethanol","en","journal article","BioMed Central","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:701f32bb-ce64-4c9f-a195-b5541bbffeb9","http://resolver.tudelft.nl/uuid:701f32bb-ce64-4c9f-a195-b5541bbffeb9","Transcriptome-Based Characterization of Interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus in Lactose-Grown Chemostat Cocultures","Mendes, F.; Sieuwerts, S.; De Hulster, E.; Almering, M.J.; Luttik, M.A.; Pronk, J.T.; Smid, E.J.; Bron, P.A.; Daran-Lapujade, P.","","2013","Mixed populations of Saccharomyces cerevisiae yeasts and lactic acid bacteria occur in many dairy, food, and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the cocultures, five mechanisms of interaction were identified. (i) Lb. delbrueckii subsp. bulgaricus hydrolyzes lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. delbrueckii subsp. bulgaricus, is excreted and provides a carbon source for yeast. (ii) In pure cultures, Lb. delbrueckii subsp. bulgaricus grows only in the presence of increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. (iii) Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacterium. (iv) A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. delbrueckii subsp. bulgaricus. (v) Transcriptome analysis of Lb. delbrueckii subsp. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipid metabolism, suggesting either a competition of the two microorganisms for fatty acids or a response to the ethanol produced by S. cerevisiae. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigate microbial interactions in mixed populations.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:9043ecf5-5e0f-41f7-afc1-a5f4f4d8b05d","http://resolver.tudelft.nl/uuid:9043ecf5-5e0f-41f7-afc1-a5f4f4d8b05d","A versatile, efficient strategy for assembly of multi-fragment expression vectors in Saccharomyces cerevisiae using 60 bp synthetic recombination sequences","Kuijpers, N.G.; Solis-Escalante, D.; Bosman, L.; Van den Broek, M.; Pronk, J.T.; Daran, J.M.; Daran-Lapujade, P.A.S.","","2013","Background: In vivo recombination of overlapping DNA fragments for assembly of large DNA constructs in the yeast Saccharomyces cerevisiae holds great potential for pathway engineering on a small laboratory scale as well as for automated high-throughput strain construction. However, the current in vivo assembly methods are not consistent with respect to yields of correctly assembled constructs and standardization of parts required for routine laboratory implementation has not been explored. Here, we present and evaluate an optimized and robust method for in vivo assembly of plasmids from overlapping DNA fragments in S. cerevisiae. Results: To minimize occurrence of misassembled plasmids and increase the versatility of the assembly platform, two main improvements were introduced; i) the essential elements of the vector backbone (yeast episome and selection marker) were disconnected and ii) standardized 60 bp synthetic recombination sequences non-homologous with the yeast genome were introduced at each flank of the assembly fragments. These modifications led to a 100 fold decrease in false positive transformants originating from the backbone as compared to previous methods. Implementation of the 60 bp synthetic recombination sequences enabled high flexibility in the design of complex expression constructs and allowed for fast and easy construction of all assembly fragments by PCR. The functionality of the method was demonstrated by the assembly of a 21 kb plasmid out of nine overlapping fragments carrying six glycolytic genes with a correct assembly yield of 95%. The assembled plasmid was shown to be a high fidelity replica of the in silico design and all glycolytic genes carried by the plasmid were proven to be functional. Conclusion: The presented method delivers a substantial improvement for assembly of multi-fragment expression vectors in S. cerevisiae. Not only does it improve the efficiency of in vivo assembly, but it also offers a versatile platform for easy and rapid design and assembly of synthetic constructs. The presented method is therefore ideally suited for the construction of complex pathways and for high throughput strain construction programs for metabolic engineering purposes. In addition its robustness and ease of use facilitate the construction of any plasmid carrying two or more genes.","","en","journal article","BioMed Central","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:ae07be2d-7030-409a-8e9e-0b60d829ce14","http://resolver.tudelft.nl/uuid:ae07be2d-7030-409a-8e9e-0b60d829ce14","Genome-scale analyses of butanol tolerance in Saccharomyces cerevisiae reveal an essential role of protein degradation","Gonzalez-Ramos, D.; Van den Broek, M.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.M.G.","","2013","Background n-Butanol and isobutanol produced from biomass-derived sugars are promising renewable transport fuels and solvents. Saccharomyces cerevisiae has been engineered for butanol production, but its high butanol sensitivity poses an upper limit to product titers that can be reached by further pathway engineering. A better understanding of the molecular basis of butanol stress and tolerance of S. cerevisiae is important for achieving improved tolerance. Results By combining a screening of the haploid S. cerevisiae knock-out library, gene overexpression, and genome analysis of evolutionary engineered n-butanol-tolerant strains, we established that protein degradation plays an essential role in tolerance. Strains deleted in genes involved in the ubiquitin-proteasome system and in vacuolar degradation of damaged proteins showed hypersensitivity to n-butanol. Overexpression of YLR224W, encoding the subunit responsible for the recognition of damaged proteins of an ubiquitin ligase complex, resulted in a strain with a higher n-butanol tolerance. Two independently evolved n-butanol-tolerant strains carried different mutations in both RPN4 and RTG1, which encode transcription factors involved in the expression of proteasome and peroxisomal genes, respectively. Introduction of these mutated alleles in the reference strain increased butanol tolerance, confirming their relevance in the higher tolerance phenotype. The evolved strains, in addition to n-butanol, were also more tolerant to 2-butanol, isobutanol and 1-propanol, indicating a common molecular basis for sensitivity and tolerance to C3 and C4 alcohols. Conclusions This study shows that maintenance of protein integrity plays an essential role in butanol tolerance and demonstrates new promising targets to engineer S. cerevisiae for improved tolerance.","saccharomyces cerevisiae; butanol tolerance; evolutionary engineering; deletion collection screening; whole genome sequencing; proteasome; multivesicular bodies; OA-Fund TU Delft","en","journal article","BioMed Central","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:7ad7c09f-c259-4ebb-86ac-2cdce5e8a376","http://resolver.tudelft.nl/uuid:7ad7c09f-c259-4ebb-86ac-2cdce5e8a376","Crystal ball - 2013: Recombination-based DNA assembly in metabolic engineering: a goodbye to old workhorses?","Daran, J.M.; Pronk, J.T.","","2012","In this feature, leading researchers in the field of environmental microbiology speculate on the technical and conceptual developments that will drive innovative research and open new vistas over the next few years. For the better part of four decades, genetic engineering has relied on a universal toolbox containing three indispensable implements: restriction enzymes, ligases and, last but not least, Escherichia coli. Even those of us who now languish behind laptops and in meetings rather than work at the bench instantaneously recognize the smells of Luria broth and plasmid preps.","","en","journal article","Society for Applied Microbiology and Blackwell Publishing","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:21f3c07b-19ff-48f6-af54-31f7eca13e1e","http://resolver.tudelft.nl/uuid:21f3c07b-19ff-48f6-af54-31f7eca13e1e","amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae","Solis-Escalante, D.; Kuijpers, N.G.A.; Bongaerts, N.; Bolat, I.; Bosman, L.; Pronk, J.T.; Daran, J.M.; Daran-Lapujade, P.A.S.","","2012","Despite the large collection of selectable marker genes available for Saccharomyces cerevisiae, marker availability can still present a hurdle when dozens of genetic manipulations are required. Recyclable markers, counterselectable cassettes that can be removed from the targeted genome after use, are therefore valuable assets in ambitious metabolic engineering programs. In the present work, the new recyclable dominant marker cassette amdSYM, formed by the Ashbya gossypii TEF2 promoter and terminator and a codon-optimized acetamidase gene (Aspergillus nidulans amdS), is presented. The amdSYM cassette confers S. cerevisiae the ability to use acetamide as sole nitrogen source. Direct repeats flanking the amdS gene allow for its efficient recombinative excision. As previously demonstrated in filamentous fungi, loss of the amdS marker cassette from S. cerevisiae can be rapidly selected for by growth in the presence of fluoroacetamide. The amdSYM cassette can be used in different genetic backgrounds and represents the first counterselectable dominant marker gene cassette for use in S. cerevisiae. Furthermore, using astute cassette design, amdSYM excision can be performed without leaving a scar or heterologous sequences in the targeted genome. The present work therefore demonstrates that amdSYM is a useful addition to the genetic engineering toolbox for Saccharomyces laboratory, wild, and industrial strains.","counter-selectable marker; dominant marker; scarless marker removal; acetamidase; serial gene deletion; Saccharomyces cerevisiae","en","journal article","Blackwell Publishing","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:85a25098-e9d3-4262-931e-260e27a7ab82","http://resolver.tudelft.nl/uuid:85a25098-e9d3-4262-931e-260e27a7ab82","De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae","Koopman, F.W.; Beekwilder, J.; Crimi, B.; Van Houwelingen, A.; Hall, R.D.; Bosch, D.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.M.","","2012","Background Flavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide variety of antioxidant and human health-related properties. Plant production of flavonoids is limited by the low productivity and the complexity of the recovered flavonoids. Thus to overcome these limitations, metabolic engineering of specific pathway in microbial systems have been envisaged to produce high quantity of a single molecules. Result Saccharomyces cerevisiae was engineered to produce the key intermediate flavonoid, naringenin, solely from glucose. For this, specific naringenin biosynthesis genes from Arabidopsis thaliana were selected by comparative expression profiling and introduced in S. cerevisiae. The sole expression of these A. thaliana genes yielded low extracellular naringenin concentrations (<5.5 ?M). To optimize naringenin titers, a yeast chassis strain was developed. Synthesis of aromatic amino acids was deregulated by alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) and byproduct formation was reduced by eliminating phenylpyruvate decarboxylase (Aro10, Pdc5, Pdc6). Together with an increased copy number of the chalcone synthase gene and expression of a heterologous tyrosine ammonia lyase, these modifications resulted in a 40-fold increase of extracellular naringenin titers (to approximately 200 ?M) in glucose-grown shake-flask cultures. In aerated, pH controlled batch reactors, extracellular naringenin concentrations of over 400 ?M were reached. Conclusion The results reported in this study demonstrate that S. cerevisiae is capable of de novo production of naringenin by coexpressing the naringenin production genes from A. thaliana and optimization of the flux towards the naringenin pathway. The engineered yeast naringenin production host provides a metabolic chassis for production of a wide range of flavonoids and exploration of their biological functions.","saccharomyces cerevisiae; naringenin; de novo; flavonoids; metabolic engineering; OA-Fund TU Delft","en","journal article","BioMed Central","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:75361b4a-7d5e-4ba2-a9ea-a1abb639c478","http://resolver.tudelft.nl/uuid:75361b4a-7d5e-4ba2-a9ea-a1abb639c478","Similar temperature dependencies of glycolytic enzymes: An evolutionary adaptation to temperature dynamics?","Cruz, L.A.B.; Hebly, M.; Duong, G.H.; Wahl, S.A.; Pronk, J.T.; Heijnen, J.J.; Daran-Lapujade, P.; Van Gulik, W.M.","","2012","Background Temperature strongly affects microbial growth, and many microorganisms have to deal with temperature fluctuations in their natural environment. To understand regulation strategies that underlie microbial temperature responses and adaptation, we studied glycolytic pathway kinetics in Saccharomyces cerevisiae during temperature changes. Results Saccharomyces cerevisiae was grown under different temperature regimes and glucose availability conditions. These included glucose-excess batch cultures at different temperatures and glucose-limited chemostat cultures, subjected to fast linear temperature shifts and circadian sinoidal temperature cycles. An observed temperature-independent relation between intracellular levels of glycolytic metabolites and residual glucose concentration for all experimental conditions revealed that it is the substrate availability rather than temperature that determines intracellular metabolite profiles. This observation corresponded with predictions generated in silico with a kinetic model of yeast glycolysis, when the catalytic capacities of all glycolytic enzymes were set to share the same normalized temperature dependency. Conclusions From an evolutionary perspective, such similar temperature dependencies allow cells to adapt more rapidly to temperature changes, because they result in minimal perturbations of intracellular metabolite levels, thus circumventing the need for extensive modification of enzyme levels.","glycolysis; kinetic modelling; metabolomics; saccharomyces cerevisiae; temperature dynamics","en","journal article","BioMed Central","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:b4fe13a8-7cce-416f-802a-6fd618726d56","http://resolver.tudelft.nl/uuid:b4fe13a8-7cce-416f-802a-6fd618726d56","An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae","Oud, B.; Flores, C.L.; Gancedo, C.; Zhang, X.; Trueheart, J.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.","","2012","Background Pyruvate-decarboxylase negative (Pdc-) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc- strains cannot grow on high glucose concentrations and require C2-compounds (ethanol or acetate) for growth under conditions with low glucose concentrations, which hitherto has limited application in industry. Results Genetic analysis of a Pdc- strain previously evolved to overcome these deficiencies revealed a 225bp in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc- strain enabled growth on 20 g l-1 glucose and 0.3% (v/v) ethanol at a maximum specific growth rate (0.24 h-1) similar to that of the evolved Pdc- strain (0.23 h-1). Furthermore, the reverse engineered Pdc- strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h-1) than the evolved strain (0.20 h-1). The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc-S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc- strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C2-compound auxotrophy. Conclusions In this study we have discovered and characterised a mutation in MTH1 enabling Pdc- strains to grow on glucose as the sole carbon source. This successful example of reverse engineering not only increases the understanding of the glucose tolerance of evolved Pdc-S. cerevisiae, but also allows introduction of this portable genetic element into various industrial yeast strains, thereby simplifying metabolic engineering strategies.","inverse metabolic engineering; reverse metabolic engineering; whole genome sequencing; glucose tolerance; by-product reduction; MTH1 allele","en","journal article","BioMed Central","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:3f9481dd-6904-4778-af72-28c34a0e6bee","http://resolver.tudelft.nl/uuid:3f9481dd-6904-4778-af72-28c34a0e6bee","Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae","Romagnoli, G.; Luttik, M.A.H.; Kötter, P.; Pronk, J.T.; Daran, J.M.","","2012","Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxoacid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1? pdc5? pdc6? aro10? thi3? strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxobutanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:ab7adf6d-9f05-4412-8e6c-ca1f58d510e5","http://resolver.tudelft.nl/uuid:ab7adf6d-9f05-4412-8e6c-ca1f58d510e5","Galacturonic Acid Inhibits the Growth of Saccharomyces cerevisiae on Galactose, Xylose, and Arabinose","Huisjes, E.H.; De Hulster, E.; Van Dam, J.C.; Pronk, J.T.; Van Maris, A.J.A.","","2012","The efficient fermentation of mixed substrates is essential for the microbial conversion of second-generation feedstocks, including pectin-rich waste streams such as citrus peel and sugar beet pulp. Galacturonic acid is a major constituent of hydrolysates of these pectin-rich materials. The yeast Saccharomyces cerevisiae, the main producer of bioethanol, cannot use this sugar acid. The impact of galacturonic acid on alcoholic fermentation by S. cerevisiae was investigated with anaerobic batch cultures grown on mixtures of glucose and galactose at various galacturonic acid concentrations and on a mixture of glucose, xylose, and arabinose. In cultures grown at pH 5.0, which is well above the pKa value of galacturonic acid (3.51), the addition of 10 g · liter?1 galacturonic acid did not affect galactose fermentation kinetics and growth. In cultures grown at pH 3.5, the addition of 10 g · liter?1 galacturonic acid did not significantly affect glucose consumption. However, at this lower pH, galacturonic acid completely inhibited growth on galactose and reduced galactose consumption rates by 87%. Additionally, it was shown that galacturonic acid strongly inhibits the fermentation of xylose and arabinose by the engineered pentose-fermenting S. cerevisiae strain IMS0010. The data indicate that inhibition occurs when nondissociated galacturonic acid is present extracellularly and corroborate the hypothesis that a combination of a decreased substrate uptake rate due to competitive inhibition on Gal2p, an increased energy requirement to maintain cellular homeostasis, and/or an accumulation of galacturonic acid 1-phosphate contributes to the inhibition. The role of galacturonic acid as an inhibitor of sugar fermentation should be considered in the design of yeast fermentation processes based on pectin-rich feedstocks.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:106b1146-45ed-43a5-bafc-779358ecf9b6","http://resolver.tudelft.nl/uuid:106b1146-45ed-43a5-bafc-779358ecf9b6","In vivo analysis of Saccharomyces cerevisiae plasma membrane ATPase Pma1p isoforms with increased in vitro H+/ATP stoichiometry","De Kok, S.; Yilmaz, D.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.","","2012","Plasma membrane H+-ATPase isoforms with increased H+/ATP ratios represent a desirable asset in yeast metabolic engineering. In vivo proton coupling of two previously reported Pma1p isoforms (Ser800Ala, Glu803Gln) with increased in vitro H+/ATP stoichiometries was analysed by measuring biomass yields of anaerobic maltose-limited chemostat cultures expressing only the different PMA1 alleles. In vivo H+/ATP stoichiometries of wildtype Pma1p and the two isoforms did not differ significantly.","Pma1; Ser800Ala; Glu803Gln; Maltose; Yeast; Proton symport","en","journal article","Springer","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:7b0ae5aa-1553-4569-bd06-0093445b64b5","http://resolver.tudelft.nl/uuid:7b0ae5aa-1553-4569-bd06-0093445b64b5","Impact of Velvet Complex on Transcriptome and Penicillin G Production in Glucose-Limited Chemostat Cultures of a ?-Lactam High-Producing Penicillium chrysogenum Strain","Veiga, T.; Nijland, J.G.; Driessen, A.J.M.; Bovenberg, R.A.L.; Touw, H.; Van den Berg, M.A.; Pronk, J.T.; Daran, J.M.","","2012","The multicomponent global regulator Velvet complex has been identified as a key regulator of secondary metabolite production in Aspergillus and Penicillium species. Previous work indicated a massive impact of PcvelA and PclaeA deletions on penicillin production in prolonged batch cultures of P. chrysogenum, as well as substantial changes in transcriptome. The present study investigated the impact of these mutations on product formation and genome-wide transcript profiles under glucose-limited aerobic conditions, relevant for industrial production of ?-lactams. Predicted amino acid sequences of PcVelA and PcLaeA in this strain were identical to those in its ancestor Wisconsin54-1255. Controls were performed to rule out transformation-associated loss of penicillin-biosynthesis clusters. The correct PcvelA and PclaeA deletion strains revealed a small reduction of penicillin G productivity relative to the reference strain, which is a much smaller reduction than previously reported for prolonged batch cultures of similar P. chrysogenum mutants. Chemostat-based transcriptome analysis yielded only 23 genes with a consistent differential response in the PcvelA? and PclaeA? mutants when grown in the absence of the penicillin G side-chain precursor phenylacetic acid. Eleven of these genes belonged to two small gene clusters, one of which contained a gene with high homology to the aristolochene synthase. These results provide a clear caveat that the impact of the Velvet complex on secondary metabolism in filamentous fungi is strongly context dependent.","","en","journal article","Mary Ann Liebert","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:391597ac-d3da-40e6-9f27-f4b5812cef87","http://resolver.tudelft.nl/uuid:391597ac-d3da-40e6-9f27-f4b5812cef87","Metabolic engineering of ?-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis","Veiga, T.; Gombert, A.K.; Landes, N.; Verhoeven, M.D.; Kiel, J.A.K.W.; Krikken, A.; Nijland, J.G.; Touw, H.; Luttik, M.A.H.; Van der Toorn, J.C.; Driessen, A.J.M.; Bovenberg, R.A.L.; Van den Berg, M.A.; Van der Klei, I.J.; Pronk, J.T.; Daran, J.M.","","2012","Industrial production of semi-synthetic cephalosporins by Penicillium chrysogenum requires supplementation of the growth media with the side-chain precursor adipic acid. In glucose-limited chemostat cultures of P. chrysogenum, up to 88% of the consumed adipic acid was not recovered in cephalosporin-related products, but used as an additional carbon and energy source for growth. This low efficiency of side-chain precursor incorporation provides an economic incentive for studying and engineering the metabolism of adipic acid in P. chrysogenum. Chemostat-based transcriptome analysis in the presence and absence of adipic acid confirmed that adipic acid metabolism in this fungus occurs via ?-oxidation. A set of 52 adipate-responsive genes included six putative genes for acyl-CoA oxidases and dehydrogenases, enzymes responsible for the first step of ?-oxidation. Subcellular localization of the differentially expressed acyl-CoA oxidases and dehydrogenases revealed that the oxidases were exclusively targeted to peroxisomes, while the dehydrogenases were found either in peroxisomes or in mitochondria. Deletion of the genes encoding the peroxisomal acyl-CoA oxidase Pc20g01800 and the mitochondrial acyl-CoA dehydrogenase Pc20g07920 resulted in a 1.6- and 3.7-fold increase in the production of the semi-synthetic cephalosporin intermediate adipoyl-6-APA, respectively. The deletion strains also showed reduced adipate consumption compared to the reference strain, indicating that engineering of the first step of ?-oxidation successfully redirected a larger fraction of adipic acid towards cephalosporin biosynthesis.","Penicillium chrysogenum; ?-lactams; cephalosporins; ?-oxidation; adipic acid; metabolic engineering","en","journal article","Elsevier","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:69c3e926-0517-4dbc-8635-df290f0e5889","http://resolver.tudelft.nl/uuid:69c3e926-0517-4dbc-8635-df290f0e5889","De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology","Nijkamp, J.F.; Van den Broek, M.A.; Datema, E.; De Kok, S.; Bosman, L.; Luttik, M.A.H.; Daran-Lapujade, P.A.S.; Vongsangnak, W.; Nielsen, J.; Heijne, W.H.M.; Klaassen, P.; Paddon, C.J.; Platt, D.; Kötter, P.; Van Ham, R.C.; Reinders, M.J.T.; Pronk, J.T.; De Ridder, D.; Daran, J.M.","","2012","Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.","OA-Fund TU Delft","en","journal article","BioMed Central","","","","","","","","Electrical Engineering, Mathematics and Computer Science","Intelligent Systems","","","",""
"uuid:7c0d7036-46ea-4fa2-a5d9-d7f09d9be36c","http://resolver.tudelft.nl/uuid:7c0d7036-46ea-4fa2-a5d9-d7f09d9be36c","Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum","Veiga, T.; Solis-Escalante, D.; Romagnoli, G.; Ten Pierick, A.; Hanemaaijer, M.; Deshmuhk, A.; Wahl, A.; Pronk, J.T.; Daran, J.M.","","2011","The industrial production of penicillin G by Penicillium chrysogenum requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of P. chrysogenum with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-13C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis of P. chrysogenum cultures grown with phenylalanine and with (NH4)2SO4 as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free Saccharomyces cerevisiae strain CEN.PK711-7C (pdc1 pdc5 pdc6? aro10? thi3?). The introduction of Pc13g09300 restored the growth of this S. cerevisiae mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate in P. chrysogenum. These results provide a basis for the metabolic engineering of P. chrysogenum for the production of the penicillin G side chain precursor phenylacetate.","OA-Fund TU Delft","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:32dc15c7-b033-4e1a-96ad-54146e190c21","http://resolver.tudelft.nl/uuid:32dc15c7-b033-4e1a-96ad-54146e190c21","Cellular responses of Saccharomyces cerevisiae at near-zero growth rates: Transcriptome analysis of anaerobic retentostat cultures","Boender, L.G.M.; Van Maris, A.J.A.; De Hulster, E.A.F.; Almering, M.J.H.; Van der Klei, I.J.; Veenhuis, M.; De Winde, J.H.; Pronk, J.T.; Daran-Lapujade, P.A.S.","","2011","Extremely low specific growth rates (below 0.01 h?1) represent a largely unexplored area of microbial physiology. In this study, anaerobic, glucose-limited retentostats were used to analyse physiological and genome-wide transcriptional responses of Saccharomyces cerevisiae to cultivation at near-zero specific growth rates. While quiescence is typically investigated as a result of carbon starvation, cells in retentostat are fed by small, but continuous carbon and energy supply. Yeast cells cultivated near-zero specific growth rates, while metabolically active, exhibited characteristics previously associated with quiescence, including accumulation of storage polymers and an increased expression of genes involved in exit from the cell cycle into G0. Unexpectedly, analysis of transcriptome data from retentostat and chemostat cultures showed, as specific growth rate was decreased, that quiescence-related transcriptional responses were already set in at specific growth rates above 0.025 h?1. These observations stress the need for systematic dissection of physiological responses to slow growth, quiescence, ageing and starvation and indicate that controlled cultivation systems such as retentostats can contribute to this goal. Furthermore, cells in retentostat do not (or hardly) divide while remaining metabolically active, which emulates the physiological status of metazoan post-mitotic cells. We propose retentostat as a powerful cultivation tool to investigate chronological ageing-related processes.","retentostat; Saccharomyces cerevisiae; near-zero growth rates; quiescence; chronological ageing; OA-Fund TU Delft","en","journal article","Wiley","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:0157f150-926c-4d6f-9d0b-18faf360692c","http://resolver.tudelft.nl/uuid:0157f150-926c-4d6f-9d0b-18faf360692c","Effect of Elevated Salt Concentrations on the Aerobic Granular Sludge Process: Linking Microbial Activity with Microbial Community Structure","Bassin, J.P.; Pronk, M.; Muyzer, G.; Kleerebezem, R.; Dezotti, M.; Van Loosdrecht, M.C.M.","","2011","The long- and short-term effects of salt on biological nitrogen and phosphorus removal processes were studied in an aerobic granular sludge reactor. The microbial community structure was investigated by PCR-denaturing gradient gel electrophoresis (DGGE) on 16S rRNA and amoA genes. PCR products obtained from genomic DNA and from rRNA after reverse transcription were compared to determine the presence of bacteria as well as the metabolically active fraction of bacteria. Fluorescence in situ hybridization (FISH) was used to validate the PCR-based results and to quantify the dominant bacterial populations. The results demonstrated that ammonium removal efficiency was not affected by salt concentrations up to 33 g/liter NaCl. Conversely, a high accumulation of nitrite was observed above 22 g/liter NaCl, which coincided with the disappearance of Nitrospira sp. Phosphorus removal was severely affected by gradual salt increase. No P release or uptake was observed at steady-state operation at 33 g/liter NaCl, exactly when the polyphosphate-accumulating organisms (PAOs), “Candidatus Accumulibacter phosphatis” bacteria, were no longer detected by PCR-DGGE or FISH. Batch experiments confirmed that P removal still could occur at 30 g/liter NaCl, but the long exposure of the biomass to this salinity level was detrimental for PAOs, which were outcompeted by glycogen-accumulating organisms (GAOs) in the bioreactor. GAOs became the dominant microorganisms at increasing salt concentrations, especially at 33 g/liter NaCl. In the comparative analysis of the diversity (DNA-derived pattern) and the activity (cDNA-derived pattern) of the microbial population, the highly metabolically active microorganisms were observed to be those related to ammonia (Nitrosomonas sp.) and phosphate removal (“Candidatus Accumulibacter”).","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:26562c1c-97f8-46b5-94ec-bce336f2ca63","http://resolver.tudelft.nl/uuid:26562c1c-97f8-46b5-94ec-bce336f2ca63","Transcriptional responses to glucose in Saccharomyces cerevisiae strains lacking a functional protein kinase A","Livas, D.; Almering, M.J.H.; Daran, J.M.; Pronk, J.J.; Gancedo, J.M.","","2011","Background The pattern of gene transcripts in the yeast Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. In S. cerevisiae three genes, TPK1, TPK2, and TPK3, encode catalytic subunits of PKA. The lack of viability of tpk1 tpk2 tpk3 triple mutants may be suppressed by mutations such as yak1 or msn2/msn4. To investigate the requirement for PKA in glucose control of gene expression, we have compared the effects of glucose on global transcription in a wild-type strain and in two strains devoid of PKA activity, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4. Results We have identified different classes of genes that can be induced -or repressed- by glucose in the absence of PKA. Representative examples are genes required for glucose utilization and genes involved in the metabolism of other carbon sources, respectively. Among the genes responding to glucose in strains devoid of PKA some are also controlled by a redundant signalling pathway involving PKA activation, while others are not affected when PKA is activated through an increase in cAMP concentration. On the other hand, among genes that do not respond to glucose in the absence of PKA, some give a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. We show also that, for a number of genes controlled by glucose through a PKA-dependent pathway, the changes in mRNA levels are transient. We found that, in cells grown in gluconeogenic conditions, expression of a small number of genes, mainly connected with the response to stress, is reduced in the strains lacking PKA. Conclusions In S. cerevisiae, the transcriptional responses to glucose are triggered by a variety of pathways, alone or in combination, in which PKA is often involved. Redundant signalling pathways confer a greater robustness to the response to glucose, while cooperative pathways provide a greater flexibility.","","en","journal article","BioMed Central","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:d3547b05-e991-4092-9ce5-43afdbe283cc","http://resolver.tudelft.nl/uuid:d3547b05-e991-4092-9ce5-43afdbe283cc","Extreme calorie restriction and energy source starvation in Saccharomyces cerevisiae represent distinct physiological states","Boender, L.G.M.; Almering, M.J.H.; Dijk, M.; Van Maris, A.J.A.; De Winde, J.H.; Pronk, J.T.; Daran-Lapujade, P.","","2011","Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from reserve carbohydrates were 2–3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae.","retentostat; saccharomyces cerevisiae; calorie restriction; carbon starvation; reserve carbohydrates; population heterogeneity; OA-Fund TU Delft","en","journal article","Elsevier","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:abb9ca0f-9cad-4403-b811-d88fda7cfd86","http://resolver.tudelft.nl/uuid:abb9ca0f-9cad-4403-b811-d88fda7cfd86","New Oxidizer for Advanced Propellants","Chen, J.C.; Pronk, T.M.C.; Van de Wouw, A.","","2011","Document(en) uit de collectie Chemische Procestechnologie","ammonium dinitramide synthesis; spinning disc reactors; refrigeration cycle; dinitramidic acid","en","report","Delft University of Technology","","","","","","","2021-07-20","Applied Sciences","DelftChemTech","","","",""
"uuid:7ec8be66-621e-452b-a7a4-915f65465d3e","http://resolver.tudelft.nl/uuid:7ec8be66-621e-452b-a7a4-915f65465d3e","Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the β-lactam producer Penicillium chrysogenum","Daran, J.M.; Pronk, J.T.; Driessen, A.J.M.; Nijland, J.G.; Lamboo, F.; Puig-Martinez, M.; Veiga, T.; Gombert, A.K.","","2011","Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of ß-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA.","oxaloacetate hydrolase; chemostat-based transcriptomics; metabolic engineering; Penicillium chrysogenum; β-lactam; oxalate; OA-Fund TU Delft","en","journal article","Elsevier","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:be94be68-8440-4f03-a7e6-86d323a3d9b2","http://resolver.tudelft.nl/uuid:be94be68-8440-4f03-a7e6-86d323a3d9b2","Fermentative glycerol-free ethanol production","Pronk, J.T.; Van Maris, A.J.A.; Guadalupe Medina, V.G.","","2011","The present invention relates to a yeast cell, in particular a recombinant yeast cell, the cell lacking enzymatic activity needed for the NADH-dependent glycerol synthesis or the cell having a reduced enzymatic activity with respect to the NADH- dependent glycerol synthesis compared to its corresponding wild-type yeast cell, the cell comprising one or more heterologous nucleic acid sequences encoding an NAD+- dependent acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) activity. The invention further relates to the use of a cell according to the invention in the preparation of ethanol.","","en","patent","European Patent Office","","","","","","","","Applied Sciences","BT/Biotechnology","","","",""
"uuid:c1cbaa49-a299-441e-8dc1-75de27ca2324","http://resolver.tudelft.nl/uuid:c1cbaa49-a299-441e-8dc1-75de27ca2324","Rudder Model for a Manoeuvring Simulation","Pronk, Tesse","Huijsmans, R.H.M. (mentor)","2011","","hydrodynamics","","master thesis","","","","","","","","indefinite","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Ship Hydromechanics and Structures","","",""
"uuid:158df485-1a74-460b-9804-2fa9ceeeec7d","http://resolver.tudelft.nl/uuid:158df485-1a74-460b-9804-2fa9ceeeec7d","Verifying FreeRTOS; a feasibility study","Pronk, C.","","2010","This paper presents a study on modeling and verifying the kernel of Real-Time Operating Systems (RTOS). The study will show advances in formally verifying such an RTOS both by refinement and by model checking approaches. This work fits in the context of Hoare’s verification challenge. Several real-time operating systems will be discussed including some commercial ones. The focus of the latter part of the paper will be on verifying FreeRTOS. The paper investigates a number of ways to verify this operating system. A preliminary set-up of verifying FreeRTOS using model checking is presented.","","en","report","Delft University of Technology, Software Engineering Research Group","","","","","","","","Electrical Engineering, Mathematics and Computer Science","Software Technology","","","",""
"uuid:890829d0-84bd-40eb-92a8-784b353341b0","http://resolver.tudelft.nl/uuid:890829d0-84bd-40eb-92a8-784b353341b0","Anaplerotic Role for Cytosolic Malic Enzyme in Engineered Saccharomyces cerevisiae Strains","Zelle, R.M.; Harrison, J.C.; Pronk, J.T.; Van Maris, A.J.A.","","2010","Malic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO2. The Saccharomyces cerevisiae MAE1 gene encodes a mitochondrial malic enzyme whose proposed physiological roles are related to the oxidative, malate-decarboxylating reaction. Hitherto, the inability of pyruvate carboxylase-negative (Pyc–) S. cerevisiae strains to grow on glucose suggested that Mae1p cannot act as a pyruvate-carboxylating, anaplerotic enzyme. In this study, relocation of malic enzyme to the cytosol and creation of thermodynamically favorable conditions for pyruvate carboxylation by metabolic engineering, process design, and adaptive evolution, enabled malic enzyme to act as the sole anaplerotic enzyme in S. cerevisiae. The Escherichia coli NADH-dependent sfcA malic enzyme was expressed in a Pyc– S. cerevisiae background. When PDC2, a transcriptional regulator of pyruvate decarboxylase genes, was deleted to increase intracellular pyruvate levels and cells were grown under a CO2 atmosphere to favor carboxylation, adaptive evolution yielded a strain that grew on glucose (specific growth rate, 0.06 ± 0.01 h–1). Growth of the evolved strain was enabled by a single point mutation (Asp336Gly) that switched the cofactor preference of E. coli malic enzyme from NADH to NADPH. Consistently, cytosolic relocalization of the native Mae1p, which can use both NADH and NADPH, in a pyc1,2{Delta} pdc2{Delta} strain grown under a CO2 atmosphere, also enabled slow-growth on glucose. Although growth rates of these strains are still low, the higher ATP efficiency of carboxylation via malic enzyme, compared to the pyruvate carboxylase pathway, may contribute to metabolic engineering of S. cerevisiae for anaerobic, high-yield C4-dicarboxylic acid production.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:25879c96-b520-42ea-b383-02f10fabab2c","http://resolver.tudelft.nl/uuid:25879c96-b520-42ea-b383-02f10fabab2c","Towards an Accurate Stress Dependant Time & Frequency Domain VE Response Model for Bituminous Binders","Woldekidan, M.F.; Huurman, M.; Pronk, A.C.","","2010","Linear viscoelastic properties of bituminous binders for short loading times are analyzed using dynamic mechanical analysis methods. Dynamic Shear Rheometer (DSR) test with parallel plate (PP) configuration is widely used for this purpose. Due to the complex stress distribution over the cross-section of the test sample, application of this setup for nonlinear material characterization, however, has been limited. Cone-and-Plate (CP) setup on the other hand provides a relatively uniform shear stress distribution. Due to this geometrical advantage a new CP configuration were developed and used to conduct constant-stress response measurements. Application of the setup for response measurement was first verified by comparing the CP and PP results obtained at low stress levels. The effects of the non-uniform stress distribution on fatigue results were also evaluated. Using material properties obtained from dynamic tests, simulation of time-domain relaxation tests with short loading times were in good agreement with experimentally obtained relaxation test results at low stress levels. Binder response at higher stress levels exhibits a nonlinear, stress dependent, behavior.","DSR; cone and plate; fatigue; viscoelastic","en","conference paper","LCPC","","","","","","","","Civil Engineering and Geosciences","Road and Railway Engineering","","","",""
"uuid:ee84119f-c4d0-41eb-b84e-aa340a77c6eb","http://resolver.tudelft.nl/uuid:ee84119f-c4d0-41eb-b84e-aa340a77c6eb","Phosphoenolpyruvate Carboxykinase as the Sole Anaplerotic Enzyme in Saccharomyces cerevisiae","Zelle, R.M.; Trueheart, J.; Harrison, J.C.; Pronk, J.T.; Van Maris, A.J.A.","","2010","Pyruvate carboxylase is the sole anaplerotic enzyme in glucose-grown cultures of wild-type Saccharomyces cerevisiae. Pyruvate carboxylase-negative (Pyc–) S. cerevisiae strains cannot grow on glucose unless media are supplemented with C4 compounds, such as aspartic acid. In several succinate-producing prokaryotes, phosphoenolpyruvate carboxykinase (PEPCK) fulfills this anaplerotic role. However, the S. cerevisiae PEPCK encoded by PCK1 is repressed by glucose and is considered to have a purely decarboxylating and gluconeogenic function. This study investigates whether and under which conditions PEPCK can replace the anaplerotic function of pyruvate carboxylase in S. cerevisiae. Pyc– S. cerevisiae strains constitutively overexpressing the PEPCK either from S. cerevisiae or from Actinobacillus succinogenes did not grow on glucose as the sole carbon source. However, evolutionary engineering yielded mutants able to grow on glucose as the sole carbon source at a maximum specific growth rate of ca. 0.14 h–1, one-half that of the (pyruvate carboxylase-positive) reference strain grown under the same conditions. Growth was dependent on high carbon dioxide concentrations, indicating that the reaction catalyzed by PEPCK operates near thermodynamic equilibrium. Analysis and reverse engineering of two independently evolved strains showed that single point mutations in pyruvate kinase, which competes with PEPCK for phosphoenolpyruvate, were sufficient to enable the use of PEPCK as the sole anaplerotic enzyme. The PEPCK reaction produces one ATP per carboxylation event, whereas the original route through pyruvate kinase and pyruvate carboxylase is ATP neutral. This increased ATP yield may prove crucial for engineering of efficient and low-cost anaerobic production of C4 dicarboxylic acids in S. cerevisiae.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:56d5adc7-dcb3-447f-b1b2-207e01a5574d","http://resolver.tudelft.nl/uuid:56d5adc7-dcb3-447f-b1b2-207e01a5574d","Key Process Conditions for Production of C4 Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain","Zelle, R.M.; De Hulster, E.; Kloezen, W.; Pronk, J.T.; Van Maris, A.J.A.","","2010","A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter(-1) of malate at a yield of 0.42 mol (mol glucose)(-1) in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO(3), was required for efficient C(4) dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO(3) dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose)(-1), whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO(2), calcium, and O(2)). Under optimized conditions, a malate yield of 0.48 +/- 0.01 mol (mol glucose)(-1) was obtained in bioreactors, a 19% increase over yields in shake flask experiments.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:9b9b950b-d1c5-4236-80d2-8a3f9d538ed5","http://resolver.tudelft.nl/uuid:9b9b950b-d1c5-4236-80d2-8a3f9d538ed5","RAFFS: Model Checking a Robust Abstract Flash File Store","Taverne, P.; Pronk, C.","","2009","Accepted for publication in the Proceedings of the 11th International Conference on Formal Engineering Methods, ICFEM 2009. This paper presents a case study in modeling and verifying a POSIX-like file store for Flash memory. This work fits in the context of Hoare’s verification challenge and, in particular, Joshi and Holzmann’s mini-challenge to build a verifiable file store. We have designed a simple robust file store and implemented it in the form of a Promela model. A test harness is used to exercise the file store in a number of ways. Model checking technology has been extensively used to verify the correctness of our implementation. A distinguishing feature of our approach is the (bounded) exhaustive verification of power loss recovery.","","en","report","Delft University of Technology, Software Engineering Research Group","","","","","","","","Electrical Engineering, Mathematics and Computer Science","Software Computer Technology","","","",""
"uuid:ec22850b-7182-4b33-a319-277af4749448","http://resolver.tudelft.nl/uuid:ec22850b-7182-4b33-a319-277af4749448","Key Process Conditions for Production of C4 Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain","Zelle, R.M.; De Hulster, E.; Kloezen, W.; Pronk, J.T.; Van Maris, A.J.A.","","2009","A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter?1 of malate at a yield of 0.42 mol (mol glucose)?1 in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO3, was required for efficient C4 dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO3 dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose)?1, whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO2, calcium, and O2). Under optimized conditions, a malate yield of 0.48 ± 0.01 mol (mol glucose)?1 was obtained in bioreactors, a 19% increase over yields in shake flask experiments.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:8a0c3d0b-33a1-40d9-bfc6-8dba9ea4a5c8","http://resolver.tudelft.nl/uuid:8a0c3d0b-33a1-40d9-bfc6-8dba9ea4a5c8","Involvement of Vacuolar Sequestration and Active Transport in Tolerance of Saccharomyces cerevisiae to Hop Iso-?-Acids","Hazelwood, L.A.; Walsh, M.C.; Pronk, J.T.; Daran, J.M.","","2009","The hop plant, Humulus lupulus L., has an exceptionally high content of secondary metabolites, the hop -acids, which possess a range of beneficial properties, including antiseptic action. Studies performed on the mode of action of hop iso--acids have hitherto been restricted to lactic acid bacteria. The present study investigated molecular mechanisms of hop iso--acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso--acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso--acid detoxification and tolerance. This screening and further analysis of deletion mutants confirmed that yeast tolerance to hop iso--acids involves three major processes, active proton pumping into the vacuole by the vacuolar-type ATPase to enable vacuolar sequestration of iso--acids and alteration of cell wall structure and, to a lesser extent, active export of iso--acids across the plasma membrane. Furthermore, iso--acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelators.","OA-Fund TU Delft","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Department of Biotechnology","","","",""
"uuid:51f02849-3c72-4001-aa35-96fa35a83f89","http://resolver.tudelft.nl/uuid:51f02849-3c72-4001-aa35-96fa35a83f89","Elimination of Glycerol Production in Anaerobic Cultures of a Saccharomyces cerevisiae Strain Engineered To Use Acetic Acid as an Electron Acceptor","Medina, V.G.; Almering, M.J.H.; Van Maris, A.J.A.; Pronk, J.T.","","2009","In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering S. cerevisiae such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde + NAD+ + coenzyme A acetyl coenzyme A + NADH + H+), was expressed in the gpd1 gpd2 strain, anaerobic growth was restored by supplementation with 2.0 g liter–1 acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).","OA-Fund TU Delft","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","","","","",""
"uuid:3930f0ec-9634-4f83-8d1f-34baf58e3171","http://resolver.tudelft.nl/uuid:3930f0ec-9634-4f83-8d1f-34baf58e3171","Identity of the Growth-Limiting Nutrient Strongly Affects Storage Carbohydrate Accumulation in Anaerobic Chemostat Cultures of Saccharomyces cerevisiae","Hazelwood, L.A.; Walsh, M.C.; Luttik, M.A.H.; Daran-Lapujade, P.; Pronk, J.T.; Daran, J.M.","","2009","OA Fund TU Delft Accumulation of glycogen and trehalose in nutrient-limited cultures of Saccharomyces cerevisiae is negatively correlated with the specific growth rate. Additionally, glucose-excess conditions (i.e., growth limitation by nutrients other than glucose) are often implicated in high-level accumulation of these storage carbohydrates. The present study investigates how the identity of the growth-limiting nutrient affects accumulation of storage carbohydrates in cultures grown at a fixed specific growth rate. In anaerobic chemostat cultures (dilution rate, 0.10 h–1) of S. cerevisiae, the identity of the growth-limiting nutrient (glucose, ammonia, sulfate, phosphate, or zinc) strongly affected storage carbohydrate accumulation. The glycogen contents of the biomass from glucose- and ammonia-limited cultures were 10- to 14-fold higher than those of the biomass from cultures grown under the other three glucose-excess regimens. Trehalose levels were specifically higher under nitrogen-limited conditions. These results demonstrate that storage carbohydrate accumulation in nutrient-limited cultures of S. cerevisiae is not a generic response to excess glucose but instead is strongly dependent on the identity of the growth-limiting nutrient. While transcriptome analysis of wild-type and msn2 msn4 strains confirmed that transcriptional upregulation of glycogen and trehalose biosynthesis genes is mediated by Msn2p/Msn4p, transcriptional regulation could not quantitatively account for the drastic changes in storage carbohydrate accumulation. The results of assays of glycogen synthase and glycogen phosphorylase activities supported involvement of posttranscriptional regulation. Consistent with the high glycogen levels in ammonia-limited cultures, the ratio of glycogen synthase to glycogen phosphorylase in these cultures was up to eightfold higher than the ratio in the other glucose-excess cultures.","OA-Fund TU Delft","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Kluyver Centre for Genomics of Industrial Fermentation","","","",""
"uuid:c79a7900-1478-4c03-acaa-ef406d6b7b90","http://resolver.tudelft.nl/uuid:c79a7900-1478-4c03-acaa-ef406d6b7b90","Quantitative Physiology of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates","Boender, L.G.M.; De Hulster, E.A.F.; Van Maris, A.J.A.; Daran-Lapujade, P.A.S.; Pronk, J.T.","","2009","Growth at near-zero specific growth rates is a largely unexplored area of yeast physiology. To investigate the physiology of Saccharomyces cerevisiae under these conditions, the effluent removal pipe of anaerobic, glucose-limited chemostat culture (dilution rate, 0.025 h–1) was fitted with a 0.22-µm-pore-size polypropylene filter unit. This setup enabled prolonged cultivation with complete cell retention. After 22 days of cultivation, specific growth rates had decreased below 0.001 h–1 (doubling time of >700 h). Over this period, viability of the retentostat cultures decreased to ca. 80%. The viable biomass concentration in the retentostats could be accurately predicted by a maintenance coefficient of 0.50 mmol of glucose g–1 of biomass h–1 calculated from anaerobic, glucose-limited chemostat cultures grown at dilution rates of 0.025 to 0.20 h–1. This indicated that, in contrast to the situation in several prokaryotes, maintenance energy requirements in S. cerevisiae do not substantially change at near-zero specific growth rates. After 22 days of retentostat cultivation, glucose metabolism was predominantly geared toward alcoholic fermentation to meet maintenance energy requirements. The strict correlation between glycerol production and biomass formation observed at higher specific growth rates was not maintained at the near-zero growth rates reached in the retentostat cultures. In addition to glycerol, the organic acids acetate, D-lactate, and succinate were produced at low rates during prolonged retentostat cultivation. This study identifies robustness and by-product formation as key issues in attempts to uncouple growth and product formation in S. cerevisiae. Erratum: http://aem.asm.org/cgi/content/full/75/23/7578","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:9ed05621-c605-4311-b98b-27f4c2f13efd","http://resolver.tudelft.nl/uuid:9ed05621-c605-4311-b98b-27f4c2f13efd","Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: Current status and challenges","Abbott, D.A.; Zelle, R.M.; Pronk, J.T.; Van Maris, A.J.A.","","2009","To meet the demands of future generations for chemicals and energy and to reduce the environmental footprint of the chemical industry, alternatives for petrochemistry are required. Microbial conversion of renewable feedstocks has a huge potential for cleaner, sustainable industrial production of fuels and chemicals. Microbial production of organic acids is a promising approach for production of chemical building blocks that can replace their petrochemically derived equivalents. Although Saccharomyces cerevisiae does not naturally produce organic acids in large quantities, its robustness, pH tolerance, simple nutrient requirements and long history as an industrial workhorse make it an excellent candidate biocatalyst for such processes. Genetic engineering, along with evolution and selection, has been successfully used to divert carbon from ethanol, the natural endproduct of S. cerevisiae, to pyruvate. Further engineering, which included expression of heterologous enzymes and transporters, yielded strains capable of producing lactate and malate from pyruvate. Besides these metabolic engineering strategies, this review discusses the impact of transport and energetics as well as the tolerance towards these organic acids. In addition to recent progress in engineering S. cerevisiae for organic acid production, the key limitations and challenges are discussed in the context of sustainable industrial production of organic acids from renewable feedstocks.","","en","journal article","Blackwell Publishing Ltd.","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:babe3b70-7794-4a3a-bf89-963a7624eebc","http://resolver.tudelft.nl/uuid:babe3b70-7794-4a3a-bf89-963a7624eebc","Catalase Overexpression Reduces Lactic Acid-Induced Oxidative Stress in Saccharomyces cerevisiae","Abbott, D.A.; Suir, E.; Duong, G.H.; De Hulster, E.; Pronk, J.T.; Van Maris, A.J.A.","","2009","Industrial production of lactic acid with the current pyruvate decarboxylase-negative Saccharomyces cerevisiae strains requires aeration to allow for respiratory generation of ATP to facilitate growth and, even under nongrowing conditions, cellular maintenance. In the current study, we observed an inhibition of aerobic growth in the presence of lactic acid. Unexpectedly, the cyb2{Delta} reference strain, used to avoid aerobic consumption of lactic acid, had a specific growth rate of 0.25 h–1 in anaerobic batch cultures containing lactic acid but only 0.16 h–1 in identical aerobic cultures. Measurements of aerobic cultures of S. cerevisiae showed that the addition of lactic acid to the growth medium resulted in elevated levels of reactive oxygen species (ROS). To reduce the accumulation of lactic acid-induced ROS, cytosolic catalase (CTT1) was overexpressed by replacing the native promoter with the strong constitutive TPI1 promoter. Increased activity of catalase was confirmed and later correlated with decreased levels of ROS and increased specific growth rates in the presence of high lactic acid concentrations. The increased fitness of this genetically modified strain demonstrates the successful attenuation of additional stress that is derived from aerobic metabolism and may provide the basis for enhanced (micro)aerobic production of organic acids in S. cerevisiae.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:7fe3f786-1291-45e1-b1a2-812d9ec8d4bf","http://resolver.tudelft.nl/uuid:7fe3f786-1291-45e1-b1a2-812d9ec8d4bf","Exploring and dissecting genome-wide gene expression responses of Penicillium chrysogenum to phenylacetic acid consumption and penicillinG production","Harris, D.M.; Van der Krogt, Z.A.; Klaassen, P.; Raamsdonk, L.M.; Hage, S.; Van den Berg, M.A.; Bovenberg, R.A.L.; Pronk, J.T.; Daran, J.M.","","2009","OA Fund TU Delft Background Since the discovery of the antibacterial activity of penicillin by Fleming 80 years ago, improvements of penicillin titer were essentially achieved by classical strain improvement through mutagenesis and screening. The recent sequencing of Penicillium chrysogenum strain Wisconsin1255-54 and the availability of genomics tools such as DNA-microarray offer new perspective. Results In studies on ?-lactam production by P. chrysogenum, addition and omission of a side-chain precursor is commonly used to generate producing and non-producing scenarios. To dissect effects of penicillinG production and of its side-chain precursor phenylacetic acid (PAA), a derivative of a penicillinG high-producing strain without a functional penicillin-biosynthesis gene cluster was constructed. In glucose-limited chemostat cultures of the high-producing and cluster-free strains, PAA addition caused a small reduction of the biomass yield, consistent with PAA acting as a weak-organic-acid uncoupler. Microarray-based analysis on chemostat cultures of the high-producing and cluster-free strains, grown in the presence and absence of PAA, showed that: (i) Absence of a penicillin gene cluster resulted in transcriptional upregulation of a gene cluster putatively involved in production of the secondary metabolite aristolochene and its derivatives, (ii) The homogentisate pathway for PAA catabolism is strongly transcriptionally upregulated in PAA-supplemented cultures (iii) Several genes involved in nitrogen and sulfur metabolism were transcriptionally upregulated under penicillinG producing conditions only, suggesting a drain of amino-acid precursor pools. Furthermore, the number of candidate genes for penicillin transporters was strongly reduced, thus enabling a focusing of functional analysis studies. Conclusion This study demonstrates the usefulness of combinatorial transcriptome analysis in chemostat cultures to dissect effects of biological and process parameters on gene expression regulation. This study provides for the first time clear-cut target genes for metabolic engineering, beyond the three genes of the ?-lactam pathway.","","en","journal article","BioMed Central","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:df8adb48-76de-443b-b321-67fa6941a60b","http://resolver.tudelft.nl/uuid:df8adb48-76de-443b-b321-67fa6941a60b","Combinatorial effects of environmental parameters on transcriptional regulation in Saccharomyces cerevisiae: A quantitative analysis of a compendium of chemostat-based transcriptome data","Knijnenburg, T.A.; Daran, J.M.G.; Van den Broek, M.A.; Daran-Lapujade, P.A.S.; De Winde, J.H.; Pronk, J.T.; Reinders, M.J.T.; Wessels, L.F.A.","","2009","OA Fund TU delft Background: Microorganisms adapt their transcriptome by integrating multiple chemical and physical signals from their environment. Shake-flask cultivation does not allow precise manipulation of individual culture parameters and therefore precludes a quantitative analysis of the (combinatorial) influence of these parameters on transcriptional regulation. Steady-state chemostat cultures, which do enable accurate control, measurement and manipulation of individual cultivation parameters (e.g. specific growth rate, temperature, identity of the growth-limiting nutrient) appear to provide a promising experimental platform for such a combinatorial analysis. Results: A microarray compendium of 170 steady-state chemostat cultures of the yeast Saccharomyces cerevisiae is presented and analyzed. The 170 microarrays encompass 55 unique conditions, which can be characterized by the combined settings of 10 different cultivation parameters. By applying a regression model to assess the impact of (combinations of) cultivation parameters on the transcriptome, most S. cerevisiae genes were shown to be influenced by multiple cultivation parameters, and in many cases by combinatorial effects of cultivation parameters. The inclusion of these combinatorial effects in the regression model led to higher explained variance of the gene expression patterns and resulted in higher function enrichment in subsequent analysis. We further demonstrate the usefulness of the compendium and regression analysis for interpretation of shake-flask-based transcriptome studies and for guiding functional analysis of (uncharacterized) genes and pathways. Conclusion: Modeling the combinatorial effects of environmental parameters on the transcriptome is crucial for understanding transcriptional regulation. Chemostat cultivation offers a powerful tool for such an approach.","","en","journal article","BioMed Central","","","","","","","","Electrical Engineering, Mathematics and Computer Science","Mediamatics","","","",""
"uuid:b510cf81-897b-479e-ba9e-0fafe38139e8","http://resolver.tudelft.nl/uuid:b510cf81-897b-479e-ba9e-0fafe38139e8","Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: current status and challenges","Abbott, DA (TU Delft BT/Industriele Microbiologie); Zelle, RM (TU Delft BT/Industriele Microbiologie); Pronk, J.T. (TU Delft BT/Industriele Microbiologie); van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie)","","2009","","CWTS 0.75 <= JFIS < 2.00","","journal article","","","","","","","Campus only","","","","BT/Industriele Microbiologie","","",""
"uuid:8e2e31e0-c41e-4e4e-8aa2-5e7e567966ef","http://resolver.tudelft.nl/uuid:8e2e31e0-c41e-4e4e-8aa2-5e7e567966ef","Novel Evolutionary Engineering Approach for Accelerated Utilization of Glucose, Xylose, and Arabinose Mixtures by Engineered Saccharomyces cerevisiae Strains","Wisselink, H.W.; Toirkens, M.J.; Wu, Q.; Pronk, J.T.; Van Maris, A.J.A.","","2008","Lignocellulosic feedstocks are thought to have great economic and environmental significance for future biotechnological production processes. For cost-effective and efficient industrial processes, complete and fast conversion of all sugars derived from these feedstocks is required. Hence, simultaneous or fast sequential fermentation of sugars would greatly contribute to the efficiency of production processes. One of the main challenges emerging from the use of lignocellulosics for the production of ethanol by the yeast Saccharomyces cerevisiae is efficient fermentation of D-xylose and L-arabinose, as these sugars cannot be used by natural S. cerevisiae strains. In this study, we describe the first engineered S. cerevisiae strain (strain IMS0003) capable of fermenting mixtures of glucose, xylose, and arabinose with a high ethanol yield (0.43 g g–1 of total sugar) without formation of the side products xylitol and arabinitol. The kinetics of anaerobic fermentation of glucose-xylose-arabinose mixtures were greatly improved by using a novel evolutionary engineering strategy. This strategy included a regimen consisting of repeated batch cultivation with repeated cycles of consecutive growth in three media with different compositions (glucose, xylose, and arabinose; xylose and arabinose; and only arabinose) and allowed rapid selection of an evolved strain (IMS0010) exhibiting improved specific rates of consumption of xylose and arabinose. This evolution strategy resulted in a 40% reduction in the time required to completely ferment a mixture containing 30 g liter–1 glucose, 15 g liter–1 xylose, and 15 g liter–1 arabinose.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:5f89376a-67c6-4523-92ae-5828b7f040f4","http://resolver.tudelft.nl/uuid:5f89376a-67c6-4523-92ae-5828b7f040f4","Malic Acid Production by Saccharomyces cerevisiae: Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export","Zelle, R.M.; De Hulster, E.; Van Winden, W.A.; De Waard, P.; Dijkema, C.; Winkler, A.A.; Geertman, J.M.; Van Dijken, J.P.; Pronk, J.T.; Van Maris, A.J.A.","","2008","Malic acid is a potential biomass-derivable ""building block"" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:0ff0b911-56df-4e01-9327-08aad4319516","http://resolver.tudelft.nl/uuid:0ff0b911-56df-4e01-9327-08aad4319516","The Ehrlich Pathway for Fusel Alcohol Production: A Century of Research on Saccharomyces cerevisiae Metabolism","Hazelwood, L.A.; Daran, J.M.; Van Maris, A.J.A.; Pronk, J.T.; Dickinson, J.R.","","2008","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:b7c2d99c-5d50-4755-80f9-9f14ee833c00","http://resolver.tudelft.nl/uuid:b7c2d99c-5d50-4755-80f9-9f14ee833c00","Transcription factor control of growth rate dependent genes in Saccharomyces cerevisiae: A three factor design","Fazio, A.; Jewett, M.C.; Daran-Lapujade, P.; Mustacchi, R.; Usaite, R.; Pronk, J.T.; Workman, C.T.; Nielsen, J.","","2008","","","en","journal article","Biomed Central","","","","","","","","Applied Sciences","","","","",""
"uuid:bebe43d0-d57a-4810-ba3f-8b266f6cc09e","http://resolver.tudelft.nl/uuid:bebe43d0-d57a-4810-ba3f-8b266f6cc09e","New insights into the Saccharomyces cerevisiae fermentation switch: Dynamic transcriptional response to anaerobicity and glucose-excess","Van Den Brink, J.; Daran-Lapujade, P.; Pronk, J.T.; De Winde, J.H.","","2008","OA Fund","","en","journal article","Biomed Central","","","","","","","","Applied Sciences","","","","",""
"uuid:3809cf80-d848-478a-82e5-49c1e42725d4","http://resolver.tudelft.nl/uuid:3809cf80-d848-478a-82e5-49c1e42725d4","Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis","Tai, S.L.; Daran-Lapujade, P.; Walsh, M.C.; Pronk, J.T.; Daran, J.M.","","2007","Effects of suboptimal temperatures on transcriptional regulation in yeast have been extensively studied in batch cultures. To eliminate indirect effects of specific growth rates that are inherent to batch-cultivation studies, genome-wide transcriptional responses to low temperatures were analyzed in steady-state chemostats, grown at a fixed specific growth rate (0.03 h-1). Although in vivo metabolic fluxes were essentially the same in cultures grown at 12 and at 30°C, concentrations of the growth-limiting nutrients (glucose or ammonia) were higher at 12°C. This difference was reflected by transcript levels of genes that encode transporters for the growth-limiting nutrients. Several transcriptional responses to low temperature occurred under both nutrient-limitation regimes. Increased transcription of ribosome-biogenesis genes emphasized the importance of adapting protein-synthesis capacity to low temperature. In contrast to observations in cold-shock and batch-culture studies, transcript levels of environmental stress response genes were reduced at 12°C. Transcription of trehalose-biosynthesis genes and intracellular trehalose levels indicated that, in contrast to its role in cold-shock adaptation, trehalose is not involved in steady-state low-temperature adaptation. Comparison of the chemostat- based transcriptome data with literature data revealed large differences between transcriptional reprogramming during long-term low-temperature acclimation and the transcriptional responses to a rapid transition to low temperature.","","en","journal article","American Society for Cell Biology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:87ac7063-88c8-4c8e-be69-a54505eb3678","http://resolver.tudelft.nl/uuid:87ac7063-88c8-4c8e-be69-a54505eb3678","The fluxes through glycolytic enzymes in Saccharomyces cerevisiae are predominantly regulated at posttranscriptional levels","Daran, J.M.; Bakker, B.M.; Pronk, J.T.; Westerhoff, H.V.; De Winde, J.H.; Heck, A.J.R.; Slijper, M.; De Groot, M.J.L.; Luttik, M.A.H.; Van Gulik, W.M.; Rossell, S.; Daran-Lapujade, P.","","2007","Metabolic fluxes may be regulated ‘‘hierarchically,’’ e.g., by changes of gene expression that adjust enzyme capacities (Vmax) and/or ‘‘metabolically’’ by interactions of enzymes with substrates, products, or allosteric effectors. In the present study, a method is developed to dissect the hierarchical regulation into contributions by transcription, translation, protein degradation, and posttranslational modification. The method was applied to the regulation of fluxes through individual glycolytic enzymes when the yeast Saccharomyces cerevisiae was confronted with the absence of oxygen and the presence of benzoic acid depleting its ATP. Metabolic regulation largely contributed to the10-fold change in flux through the glycolytic enzymes. This contribution varied from 50 to 80%, depending on the glycolytic step and the cultivation condition tested. Within the 50–20% hierarchical regulation of fluxes, transcription played a minor role, whereas regulation of protein synthesis or degradation was the most important. These also contributed to 75–100% of the regulation of protein levels.","Open Access","en","journal article","National Academy of Sciences","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:101f94eb-a48b-471c-9570-83046cd8fdcd","http://resolver.tudelft.nl/uuid:101f94eb-a48b-471c-9570-83046cd8fdcd","Engineering of Saccharomyces cerevisiae for Efficient Anaerobic Alcoholic Fermentation of L-Arabinose","Wisselink, H.W.; Toirkens, M.J.; Del Rosario Franco Berriel, M.; Winkler, A.A.; Van Dijken, J.P.; Pronk, J.T.; Van Maris, A.J.A.","","2007","For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as L-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of L-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of L-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the L-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h–1 g [dry weight]–1) and ethanol production (0.29 g h–1 g [dry weight]–1) and a high ethanol yield (0.43 g g–1) during anaerobic growth on L-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:ea57219d-c4ef-471a-b568-4a54aecf0469","http://resolver.tudelft.nl/uuid:ea57219d-c4ef-471a-b568-4a54aecf0469","Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae.","Abbott, D.A.; Knijnenburg, T.A.; De Poorter, L.M.; Reinders, M.J.; Pronk, J.T.; Van Maris, A.J.","","2007","Transcriptional responses to four weak organic acids (benzoate, sorbate, acetate and propionate) were investigated in anaerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. To enable quantitative comparison of the responses to the acids, their concentrations were chosen such that they caused a 50% decrease of the biomass yield on glucose. The concentration of each acid required to achieve this yield was negatively correlated with membrane affinity. Microarray analysis revealed that each acid caused hundreds of transcripts to change by over twofold relative to reference cultures without added organic acids. However, only 14 genes were consistently upregulated in response to all acids. The moderately lipophilic compounds benzoate and sorbate and, to a lesser extent, the less lipophilic acids acetate and propionate showed overlapping transcriptional responses. Statistical analysis for overrepresented functional categories and upstream regulatory elements indicated that responses to the strongly lipophilic acids were focused on genes related to the cell wall, while acetate and propionate had a stronger impact on membrane-associated transport processes. The fact that S. cerevisiae exhibits a minimal generic transcriptional response to weak organic acids along with extensive specific responses is relevant for interpreting and controlling weak acid toxicity in food products and in industrial fermentation processes.","Saccharomyces cerevisiae; weak acid; acetate; benzoate; propionate; sorbate.","en","journal article","Blackwell Publishing","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:a2f6eb8e-5bba-42f0-924b-536f2eda2292","http://resolver.tudelft.nl/uuid:a2f6eb8e-5bba-42f0-924b-536f2eda2292","Development of Efficient Xylose Fermentation in Saccharomyces cerevisiae: Xylose Isomerase as a Key Component","Van Maris, A.J.A.; Winkler, A.A.; Kuyper, M.; De Laat, W.T.; Van Dijken, J.P.; Pronk, J.T.","","2007","Metabolic engineering of Saccharomyces cerevisiae for ethanol production from d-xylose, an abundant sugar in plant biomass hydrolysates, has been pursued vigorously for the past 15 years. Whereas wild-type S. cerevisiae cannot ferment d-xylose, the ketoisomer d-xylulose can be metabolised slowly. Conversion of d-xylose into d-xylulose is therefore crucial in metabolic engineering of xylose fermentation by S. cerevisiae. Expression of heterologous xylose reductase and xylitol dehydrogenase does enable d-xylose utilisation, but intrinsic redox constraints of this pathway result in undesirable byproduct formation in the absence of oxygen. In contrast, expression of xylose isomerase (XI, EC 5.3.1.5), which directly interconverts d-xylose and d-xylulose, does not have these constraints. However, several problems with the functional expression of various bacterial and Archaeal XI genes have precluded successful use of XI in yeast metabolic engineering. This changed with the discovery of a fungal XI gene in Piromyces sp. E2, expression of which led to high XI activities in S. cerevisiae. When combined with over-expression of the genes of the non-oxidative pentose phosphate pathway of S. cerevisiae, the resulting strain grew anaerobically on d-xylose with a doubling time of ca. 8 h, with the same ethanol yield as on glucose. Additional evolutionary engineering was used to improve the fermentation kinetics of mixed-substrate utilisation, resulting in efficient d-xylose utilisation in synthetic media. Although industrial pilot experiments have already demonstrated high ethanol yields from the d-xylose present in plant biomass hydrolysates, strain robustness, especially with respect to tolerance to inhibitors present in hydrolysates, can still be further improved.","","en","journal article","Springer","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:8545473c-83f1-44e6-a74e-50e5050ddc00","http://resolver.tudelft.nl/uuid:8545473c-83f1-44e6-a74e-50e5050ddc00","Control of the Glycolytic Flux in Saccharomyces cerevisiae Grown at Low Temperature: A multi-level analysis in anaerobic chemostat cultures","Tai, S.L.; Daran-Lapujade, P.; Luttik, M.A.; Walsh, M.C.; Diderich, J.A.; Krijger, G.C.; Van Gulik, W.M.; Pronk, J.T.; Daran, J.M.","","2007","","","en","journal article","American Society for Biochemistry and Molecular Biology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:1c397cff-4efa-4bca-99d8-380b7e79e4b3","http://resolver.tudelft.nl/uuid:1c397cff-4efa-4bca-99d8-380b7e79e4b3","Exploiting combinatorial cultivation conditions to infer transcriptional regulation","Knijnenburg, T.A.; De Winde, J.H.; Daran, J.M.; Daran-Lapujade, P.; Pronk, J.T.; Reinders, M.J.T.; Wessels, L.F.A.","","2007","","","en","journal article","Biomed Central","","","","","","","","Electrical Engineering, Mathematics and Computer Science","","","","",""
"uuid:921670a2-9c79-422e-b5a6-81d8ae62bbb6","http://resolver.tudelft.nl/uuid:921670a2-9c79-422e-b5a6-81d8ae62bbb6","Exploiting combinatorial cultivation conditions to infer transcriptional regulation","Knijnenburg, T.A.; De Winde, J.H.; Daran, J.M.; Daran-Lapujade, P.; Pronk, J.T.; Reinders, M.J.T.; Wessels, L.F.A.","","2007","","","en","journal article","London, Biomed","","","","","","","","Electrical Engineering, Mathematics and Computer Science","","","","",""
"uuid:d85cddf9-cf7a-4d26-8f77-d8a5b1098e24","http://resolver.tudelft.nl/uuid:d85cddf9-cf7a-4d26-8f77-d8a5b1098e24","Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: Current status","Van Maris, A.J.A.; Abbott, D.A.; Bellissimi, E.; Van den Brink, J.; Kuyper, M.; Lutik, M.A.H.; Wisselink, H.W.; Scheffers, W.A.; Van Dijken, J.P.; Pronk, J.T.","","2006","","","en","journal article","Springer","","","","","","","","Applied Sciences","","","","",""
"uuid:e2cf9ec0-fedc-4480-a8ab-ddf7a3a7afea","http://resolver.tudelft.nl/uuid:e2cf9ec0-fedc-4480-a8ab-ddf7a3a7afea","Fluidized bed heat exchangers to prevent fouling in ice slurry systems and industrial crystallizers","Pronk, P.","Witkamp, G.J. (promotor); Infante Ferreira, C.A. (promotor)","2006","Ozon layer depletion and global warming by synthetic refrigerants forces refrigeration industries to switch over to natural but hazardous refrigerants like ammonia and hydrocarbons. A promising technology to safely use the latter refrigerants is the application of indirect refrigeration systems with ice slurry as heat transfer fluid. Ice slurry, a suspension of aqueous solution and small ice crystals, has a high volumetric heat capacity, which enables to apply thermal storage and to reduce primary energy consumption. Its main disadvantage is the tendency of ice crystals to adhere to heat exchanger walls during crystallization, also known as scaling or fouling. This thesis demonstrates that liquid-solid fluidized bed heat exchangers are able to prevent this type of fouling. Experimental results show that fouling prevention is achieved when the removal rate induced by the fluidized bed exceeds the growth rate of ice crystals on the wall. The removal rate appears to be proportional with the impulse exerted by fluidized particles on the wall, while the ice growth rate depends strongly on the temperature difference and the solute type and concentration. Fluidized bed heat exchangers are economically more attractive than competitive equipment for ice slurry production, and are also able to prevent other types of fouling in industrial crystallizers.","fluidized bed; heat exchanger","en","doctoral thesis","","","","","","","","","Mechanical Maritime and Materials Engineering","","","","",""
"uuid:550a62b7-24da-4132-9d7b-2899aebe6891","http://resolver.tudelft.nl/uuid:550a62b7-24da-4132-9d7b-2899aebe6891","When transcriptome meets metabolome: Fast cellular responses of yeast to sudden relief of glucose limitation","Heijnen, J.J.; Daran, J.M.; Pronk, J.T.; Daran-Lapujade, P.; Knijnenburg, T.A.; Ras, C.; Ten Pierick, A.; Akmering, M.J.; Van Winden, W.A.; Kresnowati, M.T.","","2006","Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro-fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short-term dynamics of the transcriptome revealed a remarkably fast decrease in the average half-life of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose-induced regulation of gene expression.","Open Access Fonds","en","journal article","EMBO and NPG","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:984d3ef8-4c91-4242-acca-61d925db95d6","http://resolver.tudelft.nl/uuid:984d3ef8-4c91-4242-acca-61d925db95d6","Metabolic engineering of xylose fermenting eukaryotic cells","Winkler, A.A.; Kuyper, S.M.; De Laat, W.T.; Van Dijken, J.P.; Pronk, J.T.","","2006","The present invention relates to further genetic modifications in eukaryotic host cells that have been transformed to express a xylose isomerase that confers the host cell the ability of isomerising xylose to xylulose. The further genetic modifications are aimed at improving the efficiency of xylose metabolism and include e.g. reduction of unspecific aldose reductase activity, increased xylulose kinase activity and increased flux of the pentose phosphate pathway. The modified host cells of the invention are suitable for the production of a wide variety of fermentation products, including ethanol, in fermentation processes in which a source of xylose or a source of xylose and glucose are used as carbon source.","","en","patent","European Patent Office","","","","","","","","Applied Sciences","","","","",""
"uuid:2d02c7b5-a271-40cd-ac0c-fdf39a798960","http://resolver.tudelft.nl/uuid:2d02c7b5-a271-40cd-ac0c-fdf39a798960","Industrial Biotechnology: Drive my car","Pronk, J.T","","2006","Dies Natalis 164","164ste Dies Natalis","en","public lecture","","","","","","","","","Applied Sciences","","","","",""
"uuid:6cb4ea55-eb80-48d1-9fd7-e1832a7cc2d9","http://resolver.tudelft.nl/uuid:6cb4ea55-eb80-48d1-9fd7-e1832a7cc2d9","Carbonic anhydrase (Nce103p): An essential biosynthetic enzyme for growth of Saccharomyces cerevisiae at atmospheric carbon dioxide pressure","Aguilera, J.; Van Dijken, J.P.; De Winde, J.H.; Pronk, J.T.","","2005","","","en","journal article","The Biochemical Society","","","","","","","","","","","","",""
"uuid:fc3abb95-7ed1-44e1-8cce-1f3b0189c899","http://resolver.tudelft.nl/uuid:fc3abb95-7ed1-44e1-8cce-1f3b0189c899","Prolonged selection in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae causes a partial loss of glycolytic capacity","Jansen, M.L.A.; Diderich, J.A.; Mashego, M.; Hassane, A.; de Winde, J.H.; Daran-Lapujade, P.; Pronk, J.T.","","2005","","","en","journal article","Society for General Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:10678102-8425-47e8-b78c-d769dbcb21c8","http://resolver.tudelft.nl/uuid:10678102-8425-47e8-b78c-d769dbcb21c8","Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity 2-Oxo Acid Decarboxylase Activity of Saccharomyces cerevisiae","Vuralhan, Z.; Luttik, M.A.; Tai, S.L.; Boer, V.M.; Morais, M.A.; Schipper, D.; Almering, M.J.; Kotter, P.; Dickinson, J.R.; Daran, J.M.; Pronk, J.T.","","2005","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:09589843-7776-4efd-8a5b-ecf4d389e7ba","http://resolver.tudelft.nl/uuid:09589843-7776-4efd-8a5b-ecf4d389e7ba","Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export","van Maris, A.J.; Winkler, A.A.; Porro, D.; van Dijken, J.P.; Pronk, J.T.","","2004","","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","","","","",""
"uuid:8db72e6d-7947-41c4-a1ac-be6e154a52ba","http://resolver.tudelft.nl/uuid:8db72e6d-7947-41c4-a1ac-be6e154a52ba","Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces cerevisiae, Yielding a C2-Independent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast","van Maris, A.J.; Geertman, J.M.; Vermeulen, A.; Groothuizen, M.K.; Winkler, A.A.; Piper, M.D.; van Dijken, J.P.; Pronk, J.T.","","2004","","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","","","","",""
"uuid:fae15a7e-a1f7-4b79-91e4-d676159d259e","http://resolver.tudelft.nl/uuid:fae15a7e-a1f7-4b79-91e4-d676159d259e","Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity","Jansen, M.L.A.; Daran-Lapujade, P.; de Winde, J.H.; Piper, M.D.; Pronk, J.T.","","2004","","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:4dce9bc7-b39e-4856-b0b5-a52d1ee4c158","http://resolver.tudelft.nl/uuid:4dce9bc7-b39e-4856-b0b5-a52d1ee4c158","Overproduction of Threonine Aldolase Circumvents the Biosynthetic Role of Pyruvate Decarboxylase in Glucose-Limited Chemostat Cultures of Saccharomyces cerevisiae","van Maris, A.J.; Luttik, M.A.; Winkler, A.A.; van Dijken, J.P.; Pronk, J.T.","","2003","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:84b34402-20cf-4f3f-a5ff-19f7b83bf892","http://resolver.tudelft.nl/uuid:84b34402-20cf-4f3f-a5ff-19f7b83bf892","Identification and Characterization of Phenylpyruvate Decarboxylase Genes in Saccharomyces cerevisiae","Vuralhan, Z.; Morais, M.A.; Tai, S.L.; Piper, M.D.; Pronk, J.T.","","2003","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:41461a68-5abf-49d0-8443-529533848430","http://resolver.tudelft.nl/uuid:41461a68-5abf-49d0-8443-529533848430","Metabolic Engineering of Glycerol Production in Saccharomyces cerevisiae","Overkamp, K.M.; Bakker, B.M.; Kotter, P.; Luttik, M.A.; Van Dijken, J.P.; Pronk, J.T.","","2002","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:e3c2c829-f95f-4436-a6e7-274de92d6b8e","http://resolver.tudelft.nl/uuid:e3c2c829-f95f-4436-a6e7-274de92d6b8e","Auxotrophic Yeast Strains in Fundamental and Applied Research","Pronk, J.T.","","2002","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:9d5b92b0-f99a-4e30-91e6-a68f55ec650b","http://resolver.tudelft.nl/uuid:9d5b92b0-f99a-4e30-91e6-a68f55ec650b","Novel Pathway for Alcoholic Fermentation of delta-Gluconolactone in the Yeast Saccharomyces bulderi","van Dijken, J.P.; van Tuijl, A.; Luttik, M.A.; Middelhoven, W.J.; Pronk, J.T.","","2002","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:f148ee4e-32c7-400e-9aac-7e4a7e16fca5","http://resolver.tudelft.nl/uuid:f148ee4e-32c7-400e-9aac-7e4a7e16fca5","Hxt-Carrier-Mediated Glucose Efflux upon Exposure of Saccharomyces cerevisiae to Excess Maltose","Jansen, M.L.A.; De Winde, J.H.; Pronk, J.T.","","2002","","","en","journal article","American Society for Microbiology","","","","","","","","Applied Sciences","Biotechnology","","","",""
"uuid:cdd1c40a-c036-4923-89e5-3aa735bad3fe","http://resolver.tudelft.nl/uuid:cdd1c40a-c036-4923-89e5-3aa735bad3fe","Van kleine diertgens naar cell factories","Pronk, J.T.","","2000","","Uittreerede","nl","public lecture","","","","","","","","","","","","","",""
"uuid:fbd1ebb3-0ecf-4a41-8e3d-7abf53633701","http://resolver.tudelft.nl/uuid:fbd1ebb3-0ecf-4a41-8e3d-7abf53633701","The Mitochondrial Alcohol Dehydrogenase Adh3p Is Involved in a Redox Shuttle in Saccharomyces cerevisiae","Bakker, B.M.; Bro, C.; Kotter, P.; Luttik, M.A.; van Dijken, J.P.; Pronk, J.T.","","2000","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:f766100c-2992-42fa-a3b7-93043dc84687","http://resolver.tudelft.nl/uuid:f766100c-2992-42fa-a3b7-93043dc84687","In Vivo Analysis of the Mechanisms for Oxidation of Cytosolic NADH by Saccharomyces cerevisiae Mitochondria","Overkamp, K.M.; Bakker, B.M.; Kotter, P.; van Tuijl, A.; de Vries, S.; van Dijken, J.P.; Pronk, J.T.","","2000","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:61351ed9-ab30-4d0c-9a35-978ce7c7feb3","http://resolver.tudelft.nl/uuid:61351ed9-ab30-4d0c-9a35-978ce7c7feb3","The Saccharomyces cerevisiae ICL2 Gene Encodes a Mitochondrial 2-Methylisocitrate Lyase Involved in Propionyl-Coenzyme A Metabolism","Luttik, M.A.; Kotter, P.; Salomons, F.A.; van der Klei, I.J.; van Dijken, J.P.; Pronk, J.T.","","2000","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:52c906b2-9741-49c6-a1a8-7c7d1fedd492","http://resolver.tudelft.nl/uuid:52c906b2-9741-49c6-a1a8-7c7d1fedd492","Ontwerpen van grijpers voor stortgoed (summary)","Pronk, J.","","1999","","Design assignment","","report","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:6363cb43-1db5-4e0d-b6cf-92ee898e42df","http://resolver.tudelft.nl/uuid:6363cb43-1db5-4e0d-b6cf-92ee898e42df","Ontwerpen van grijpers voor stortgoed (summary)","Pronk, J.","","1999","","Design assignment","","report","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:be002849-f4f8-43a8-a098-addc151ade7f","http://resolver.tudelft.nl/uuid:be002849-f4f8-43a8-a098-addc151ade7f","Bagagesystemen Luchthaven Schiphol: verbeteren van de aansluiting tussen bagagesystemen en platformtransport (summary)","Pronk, J.","","1999","","","","master thesis","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:1f29dfd3-080a-4458-a8b4-7c2cbffd377f","http://resolver.tudelft.nl/uuid:1f29dfd3-080a-4458-a8b4-7c2cbffd377f","Ontwerpen van grijpers voor stortgoed","Pronk, J.","","1999","","Design assignment","","report","","","","","","","","indefinite","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:fb15620b-6b57-4ae8-9a87-6dadf36d7f24","http://resolver.tudelft.nl/uuid:fb15620b-6b57-4ae8-9a87-6dadf36d7f24","Bagagesystemen Luchthaven Schiphol: verbeteren van de aansluiting tussen bagagesystemen en platformtransport (summary)","Pronk, J.","","1999","","","","master thesis","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:184e3aac-2296-4efe-8798-6b8b5a5e5c26","http://resolver.tudelft.nl/uuid:184e3aac-2296-4efe-8798-6b8b5a5e5c26","Bagagesystemen Luchthaven Schiphol: verbeteren van de aansluiting tussen bagagesystemen en platformtransport","Pronk, J.","","1999","","","","master thesis","","","","","","","","indefinite","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:fdf05156-04ac-4b59-990e-4c21de791a14","http://resolver.tudelft.nl/uuid:fdf05156-04ac-4b59-990e-4c21de791a14","Parametrisch model van een straddle-carrier; een simulatie in Adams (summary)","Pronk, J.","","1998","","Computer assignment","","report","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:91eebc06-e995-449e-b57b-9b68a8b14169","http://resolver.tudelft.nl/uuid:91eebc06-e995-449e-b57b-9b68a8b14169","Het bedienen van hoogbouwmagazijnen","Pronk, J.","","1998","","","","report","","","","","","","","indefinite","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:b5f737a1-1ea1-4ec7-ab08-5ffcedccfabf","http://resolver.tudelft.nl/uuid:b5f737a1-1ea1-4ec7-ab08-5ffcedccfabf","Parametrisch model van een straddle-carrier; een simulatie in Adams (summary)","Pronk, J.","","1998","","Computer assignment","","report","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:cb9280fd-0518-46fd-971d-1a86aa2f7fac","http://resolver.tudelft.nl/uuid:cb9280fd-0518-46fd-971d-1a86aa2f7fac","Effect of Specific Growth Rate on Fermentative Capacity of Baker¿s Yeast","Van Hoek, P.; Van Dijken, J.P.; Pronk, J.T.","","1998","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:80f28447-634e-4299-a12c-8697875dbec4","http://resolver.tudelft.nl/uuid:80f28447-634e-4299-a12c-8697875dbec4","Parametrisch model van een straddle-carrier; een simulatie in Adams","Pronk, J.","","1998","","Computer assignment","","report","","","","","","","","indefinite","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:7c813087-b97a-4c19-b72c-3526c69e0d3e","http://resolver.tudelft.nl/uuid:7c813087-b97a-4c19-b72c-3526c69e0d3e","Het bedienen van hoogbouwmagazijnen (summary)","Pronk, J.","","1998","","","","report","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:baecc3de-b47a-42a0-88e4-23714de062ca","http://resolver.tudelft.nl/uuid:baecc3de-b47a-42a0-88e4-23714de062ca","Effects of Pyruvate Decarboxylase Overproduction on Flux Distribution at the Pyruvate Branch Point in Saccharomyces cerevisiae","van Hoek, P.; Flikweert, M.T.; van der Aart, Q.J.M.; Steensma, H.Y.; van Dijken, J.P.; Pronk, J.T.","","1998","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:09fb7126-e913-448c-a306-8e3fcccc000f","http://resolver.tudelft.nl/uuid:09fb7126-e913-448c-a306-8e3fcccc000f","Het bedienen van hoogbouwmagazijnen (summary)","Pronk, J.","","1998","","","","report","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Transport Engineering and Logistics","","",""
"uuid:5b4f46d0-76c1-4920-864d-ea09e7b6be37","http://resolver.tudelft.nl/uuid:5b4f46d0-76c1-4920-864d-ea09e7b6be37","Computer based training for robotics and payload operations in space","Pronk, Z.","","1997","This technical report contains the results of a pilot study on the application of Computer Based Training (CBT) for ERA operations training and for payload operations training onboard International Space Station.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:e8c83e1e-dd80-48fc-9f9c-c9b02fa9d014","http://resolver.tudelft.nl/uuid:e8c83e1e-dd80-48fc-9f9c-c9b02fa9d014","Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess","Flikweert, M.T.; van Dijken, J.P.; Pronk, J.T.","","1997","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:de921d0d-d78e-4927-8071-bc85144508ed","http://resolver.tudelft.nl/uuid:de921d0d-d78e-4927-8071-bc85144508ed","Development and operations in the Dutch utilisation centre","Brouwer, M.P.A.M.; Pronk, Z.; Visser, F.B.; de Haas, J.","","1996","At the early beginning (1989) of implementation of the European User Support Organisation (USO) concept, development and implementation of a Dutch Utilisation Centre (DUC) started at NLR premises. After development of several pilot DUC facilities, DUC participated in a Columbus simulation mission and in the 2nd International Micro-gravity Laboratory mission (IML-2). Different ground segment configurations were set up at different locations to meet the specific requirements for the missions. DUC development focuses on achieving a flexible support concept in teleoperations, telerobotics, and visual (video) information processing and presentation, using different communication concepts and video equipment. The configurations are prepared for the purpose of future missions onboard International Space Station. Support will be provided to both experiments and systems operations, such as operations with the European Robotic Arm (ERA). The paper will describe the technical set-up of the DUC, focusing on the communication infrastructure and the ground segment systems for receiving the various data streams.","utilisation centre; user support; DUC; crew support; IML-2; simulation; automation & robotics; International Space Station; ESA","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:6c912be4-3316-430f-a46b-1cd88f6f5baf","http://resolver.tudelft.nl/uuid:6c912be4-3316-430f-a46b-1cd88f6f5baf","EuroSim status and applications, a review","Pronk, Z.; Prins, J.J.M.","","1996","The purpose of this document is to identify applications for the European Real-time Operations Simulator, EuroSim. First, the document provides an overview of the functional capabilities of the EuroSim. An executive summary of the EuroSim tool is given dealing with what it is, its current status, and what it will become. A list of possible applications for EuroSim has been derived from the present European space programmes and other related activities. A few applications have been selected as being considered important for making EuroSim a simulation tool supporting end-to-end development of space (sub-)systems and operations. Two applications have been identified as candidate EuroSim demonstration applications.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:afac5be9-0578-4cbe-84ca-4cb13ecbd0b5","http://resolver.tudelft.nl/uuid:afac5be9-0578-4cbe-84ca-4cb13ecbd0b5","NLR robotics development and test environment real time operations simulator","Pronk, Z.; de Haas, J.","","1996","This report contains a description of work performed under contract with the ""Nederlands Instituut vor Vliegtuigontwikkeling en Ruimtevaart, NIVR"". The objective of the work was to implement and demonstrate a real-time simulation package in the development and test environment of the NLR Teleoperations and Robotics laboratory. Originally, EUROSIM was planned to be implemented, but could not be made available in time. Therefore, the NLR real-time simulation package PROSIM was used. For the demonstration, the JISFP model software was used. This software consists of dynamics model software and onboard software models. A new user interface software has been developed using the simulation control software interface package TclTk. The architecture of the software is such that a distributed simulation can be realised. The simulator, the visualisation, and the simulation control and operations control interface can run on three different workstations.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:bd42b4cd-2809-49b9-ad45-d1757f3ec6a6","http://resolver.tudelft.nl/uuid:bd42b4cd-2809-49b9-ad45-d1757f3ec6a6","High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures","Castrillo, J.I.; Kaliterna, J.; Weusthuis, R.A.; van Dijken, J.P.; Pronk, J.T.","","1996","","","en","journal article","Wiley","","","","","","","","","","","","",""
"uuid:972e4191-f337-4cdf-a623-a2dc86c91c32","http://resolver.tudelft.nl/uuid:972e4191-f337-4cdf-a623-a2dc86c91c32","The Two Acetyl-coenzyme A Synthetases of Saccharomyces cerevisiae Differ with Respect to Kinetic Properties and Transcriptional Regulation","Van den Berg, M.A.; De Jong-Gubbels, P.; Kortland, C.J.; Van Dijken, J.P.; Pronk, J.T.; Steensma, H.Y.","","1996","","","en","journal article","American Society for Biochemistry and Molecular Biology","","","","","","","","","","","","",""
"uuid:42bc0d01-93d9-4b70-8b87-ae808ce95cc5","http://resolver.tudelft.nl/uuid:42bc0d01-93d9-4b70-8b87-ae808ce95cc5","Pyruvate Decarboxylase: An Indispensable Enzyme for Growth of Saccharomyces cerevisiae on Glucose","Flikweert, M.T.; van der Zanden, L.; Janssen, W.M.Th.M.; Steensma, H.Y.; van Dijken, J.P.; Pronk, J.T.","","1996","","pyruvate decarboxylase; sugar metabolism; Succharomyces cerevisiae; metabolic compartmentation; acetyl-CoA","en","journal article","Wiley","","","","","","","","","","","","",""
"uuid:4e855902-cd08-4fb8-b185-a8c5804f79ac","http://resolver.tudelft.nl/uuid:4e855902-cd08-4fb8-b185-a8c5804f79ac","Pyruvate Metabolism in Saccharomyces cerevisiae","Pronk, J.T.; Steensma, H.Y.; van Dijken, J.P.","","1996","","Yeast; glycolysis; TCA cycle; sugar metabolism; metabolic engineering; pyruvate decarboxylase; pyruvate carboxylase; pyruvate dehydrogenase complex; alcoholic fermentation; Crabtree effect","en","journal article","Wiley","","","","","","","","","","","","",""
"uuid:0b9a4364-1aaf-41ac-8ce9-f45cebb2af20","http://resolver.tudelft.nl/uuid:0b9a4364-1aaf-41ac-8ce9-f45cebb2af20","The use of flat-floor facilities in simulation and testing","Pronk, Z.; van Woerkom, P.T.L.M.","","1995","Computer simulation and testing of zero-gravity operations in support of development of space structures becomes more and more standard. Yet, the need for hardware simulation and test facilities representing zero-gravity conditions remains necessary, because of the strong requirements for verification and validation of system responses and model performances. In the past most of the simulations and tests of zero-g operations were performed in the area of guidance and control system development, using 3-axis motion facilities, mainly to support verification of relative motion of spacecraft with respect to inertial space. Presently, space structures and their motion under zero gravity conditions are becoming more complex due to structural flexibility, sloshing of onboard liquids, and relative motion with respect to other space structures, all characterized by motion in multiple degrees of freedom. The verification and validation of these systems and their operations will mainly be performed by using computer simulation, but hardware facilities are still necessary to support model validation and real-system tests. This paper addresses an overview of facilities to be used for simulation of zero-gravity conditions and the relation between different types of facilities. The results are focused on the use of flat-floor facilities.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:cec89017-a123-4764-9198-922d21ae801c","http://resolver.tudelft.nl/uuid:cec89017-a123-4764-9198-922d21ae801c","Review of virtual reality and telepresence applications for teleoperations in space","de Brouwer, M.G.A.; Pronk, Z.","","1995","Participation of Dutch industries and institutes in future development activities of the Lunar European Demonstrator Approach has been focused on simulation and teleoperation concepts. Some studies have been initiated on the implementation of new technologies in teleoperation: Virtual Reality and Telepresence. An evaluation of state-of-the-art Virtual Reality and Telepresence technologies is given, and the factor time delays in teleoperations and the way to deal with it have been studied and discussed. Main conclusions are: •Use of Virtual Reality techniques might be used in a limited number of applications. At this moment astronaut training appears to be the only application. Use of Head Mounted Display systems here might form a part of the total training effort. •Use of Telepresence techniques for remote operations such as in Lunar telerobotics is only possible when drawbacks due to occurring time delays are reduced by using advanced technologies. Use of Head Mounted Displays here is possible, if there are enough means to display the images to the rest of the remote operations team. •Application of Virtual Reality and Telepresence techniques in The Netherlands is limited compared to the USA and some other ESA partners. Use of Virtual Reality for training seems to be technically more feasible in The Netherlands than use of Telepresence for remote operations.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:01bf7f67-b284-4b7a-8d27-292575112086","http://resolver.tudelft.nl/uuid:01bf7f67-b284-4b7a-8d27-292575112086","Remote payload operations supported by automation and robotics: Ground-based simulation with hardware-in-the-loop","de Haas, J.; Pronk, Z.; Schoonmade, M.","","1995","The specific demands and restrictions of remote payload operations in space require special attention during the development and operational phases. One of the important tools used in this process is simulation. The actual type of simulation depends on aspects like the complexity of the system, safety aspects and the development phase it is used in. NLR is developing a testbed for several aspects of payload operations in space, in which simulations play an important role. The design and use of this testbed is described. In the design of the testbed, attention was payed to communication simulation, software and hardware interfaces and easy access to configuration and state parameters. Ground-based simulation with hardware-in-the-loop (HIL) is demonstrated using communication simulation, robot operation simulation and the use of a mock-up consisting of an experiment rack and a robot system. In addition, remotely controlled video equipment is used to provide a visual monitoring capability for the ground operator.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:f4382570-fd59-43fe-952a-83e4a28c07fe","http://resolver.tudelft.nl/uuid:f4382570-fd59-43fe-952a-83e4a28c07fe","Flat-floor facilities in support of configurable space structures development","Pronk, Z.; van Woerkom, P.T.L.M.","","1995","Considering the development of various structures required in current and future space projects like the International Space Station, and lunar and planetary explorations, the development support of systems performing relative motion (rendezvous & docking), manipulations (robotics), assembly operations and inspections, becomes more and more important. Flat-floor facilities are considered to be very suitable for the purpose of development and test support of such structures by creating simulated zero-gravity conditions. A feasibility study on specification and design of a reference flat-floor facility has been performed, based on existing technologies. An extensive literature survey revealed the presence of many different types of facilities mainly built for different purposes. Based on these results the application of flat-floor facilities in support of the different phases in the development of space constructions has been investigated. This paper focuses on the results of the literature survey. In addition, a preliminary design for a reference flat-floor facility is proposed, based on the three major components for such a facility: floor construction, bearing construction, and support mechanisms. The feasibility of realising the required low friction coefficient of airbearings for heavy payloads needs further investigation.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:75f115ae-2545-4ebd-8f5c-cfe7397cf3d7","http://resolver.tudelft.nl/uuid:75f115ae-2545-4ebd-8f5c-cfe7397cf3d7","Het DAMS project (DUC in APM Mission Simulation) samenvatting","Visser, F.B.; Pronk, Z.","","1995","Als onderdeel van de ontwikkeling en evaliratie van 'crew-support tools' en mens-machine interfaces, zijn de afgelopen jaren bij ESTEC in Noordwijk enkele missie-simulaties georganiseerd. Deze simulaties vonden plaats in het 'Crew Workstation Testbed'. Een van de doelstellingen van zo'n simulatie, die gehouden zou worden in mei 1994, was het inbrengen en evalueren van Pl/gebruikersondersteuning zoals gedefinieerd in het USO-concepL In dit kader heeft een Nederlands industrieel consortium, in opdracht van ESA en het NIVR, een pilot 'Dutch Utilisation Centre' (DUC) uitgewerkt en opgezet. Dit DUC-consortium bestond uit het Nationaal Lucht- en Ruimtevaartlaboratorium als 'prime contractor' met Fokker Space & Systems, BSO Nieuwegein BV, Comprimo Consulting Services BV, Bradford Engineering en ICT Automatisering Deventer BV als 'sub contractors'. Door het consortium werden een aantal elementen voor zowel vlucht- als grondsegment ontwikkeld. Voor het vluchtsegment werd een 'payload'rek ontwikkeld met daarin twee faciliteiten ten behoeve van het uitvoeren van experimenten: de 'High Performance Capillary Electrophoresis' faciliteit en de Glovebox. Voor het grondsegment werd een DUC-operationele omgeving gerealiseerd, met daarin diverse systemen ter ondersteuning van de communicatie tussen vlucht- en grondsegment en voor het op afstand bedienen van experimenten in het vluchtsegment. Ten behoeve van de simulatie werden ook twee experimenten voorbereid. Eén experiment had als doel de bestudering van de ontwikkeling van padde-eieren en het andere de invloed van vitamine-K op het botontkalkings proces dat bij astronauten onder micro-zwaartekracht plaatsvindt. Beide experimenten hadden micro-zwaartekracht relevantie: het padde-eieren experiment is al enkele malen uitgevoerd tijdens Spacelab- en sondeerraketmissies, het vitamine-K experiment wordt voorbereid voor de EUROMIR 95 missie. Door omstandigheden werd de simulatie op ESTEC locatie afgelast. Teneinde toch de ontwikkelde systemen en het ondersteunings concept te kunnen evalueren werd een missiedemonstratie georganiseerd bij het NLR. Tijdens deze, drie dagen durende, demonstratie werden alle systemen succesvol ingezet. De demonstratie maakte de evaluatie van alle aspecten aangaande payload ontwikkeling, gebruikersondersteuning en crew-experiment operaties mogelijk, wat mede blijkt uit de reactie van ESTEC op de demonstratie (App. A). Dit rapport geeft een overzicht van de activiteiten zoals uitgevoerd door het DUC-consortium","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:4cf21695-33ed-4fca-a220-b6aa55b8cf27","http://resolver.tudelft.nl/uuid:4cf21695-33ed-4fca-a220-b6aa55b8cf27","Regulation of alcohol-oxidizing capacity in chemostat cultures of Acetobacter pasteurianus","Salgueiro Machado, S.; Luttik, M.A.H.; van Dijken, J.P.; Jongejan, J.A.; Pronk, J.T.","","1995","","","en","journal article","Springer","","","","","","","","","","","","",""
"uuid:85c51f47-a370-4c91-956b-4d92d27dd28b","http://resolver.tudelft.nl/uuid:85c51f47-a370-4c91-956b-4d92d27dd28b","Physiological and technological aspects of large-scale heterologous-protein production with yeasts","Hensing, M.C.M.; Rouwenhorst, R.J.; Heijnen, J.J.; van Dijken, J.P.; Pronk, J.T.","","1995","","fed-batch fermentation; heterologous-protein production; large-scale fermentation; yeast","en","journal article","Kluwer","","","","","","","","","","","","",""
"uuid:7f4eeacc-43e4-4392-8298-0953688dcb45","http://resolver.tudelft.nl/uuid:7f4eeacc-43e4-4392-8298-0953688dcb45","Effects of growth conditions on mitochondrial morphology in Saccharomyces cerevisiae","Visser, W.; Van Spronsen, E.A.; Nanninga, N.; Pronk, J.T.; Kuenen, J.G.; Van Dijken, J.P.","","1995","","Saccharomyces cerevisiae Mitochondria Morphology Metabolism Cell respiration Fermentation Environmental factor Ethanol Glucose Oxygen Yeast Saccharomyces cerevisiae Mitochondrie Morphologie Metabolisme Respiration cellulaire Fermentation Facteur milieu Et","en","journal article","","","","","","","","","","","","","",""
"uuid:52676974-dc71-4043-9db8-02c1a7616e1b","http://resolver.tudelft.nl/uuid:52676974-dc71-4043-9db8-02c1a7616e1b","Use of chemostat data for modelling intracellular-inulinase production by Kluyveromyces marxianus in a high-cell-density fed-batch process","Hensing, M.C.M.; Vrouwenvelder, J.S.; Hellinga, C.; van Dijken, J.P.; Pronk, J.T.","","1995","","modelling; chemostat; extracellular inulinase; Kluyveromyces marxianus; fed-batch process","en","journal article","Springer","","","","","","","","","","","","",""
"uuid:3dd5767a-ad75-4e2e-bda6-bdaad3120d20","http://resolver.tudelft.nl/uuid:3dd5767a-ad75-4e2e-bda6-bdaad3120d20","Effects of cultivation conditions on the production of heterologous alpha-galactosidase production by Kluyveromyces lactis","Hensing, M.C.M.; Bangma, K.A.; Raamsdonk, L.; de Hulster, E.; van Dijken, J.P.; Pronk, J.T.","","1995","","","en","journal article","Springer","","","","","","","","","","","","",""
"uuid:3185e5f7-baf7-483f-85c4-960d2610337d","http://resolver.tudelft.nl/uuid:3185e5f7-baf7-483f-85c4-960d2610337d","Van asielzoeker naar statushouder, huisvestingsalternatieven op lokaal niveau","Smid, I.; Kullberg, J.; Priemus, H.; Pronk, K.","","1994","","Nederland; culturele minderheden; huisvesting; vluchtelingen","nl","book","","","","","","","","","OTB Research Institute for the Built Environment","","","","",""
"uuid:626b10c7-2eb8-465f-aa62-a090d3a9b8c2","http://resolver.tudelft.nl/uuid:626b10c7-2eb8-465f-aa62-a090d3a9b8c2","Implementation of USO concept in Crew Workstation testbed activities: Executive Summary","Pronk, Z.; Visser, F.B.","","1994","As part of the evaluation of man-machine interfaces for Columbus utilisation, manned mission simulations have been performed for some years in the Crew Workstation Test-Bed at ESTEC, Noordwijk. A mission simulation was scheduled for May 1994, to include new concepts for crew and user support, and to aim at increased realism for payload operations and communications. The development of a pilot Dutch Utilisation Centre (DUC) was one of the major objectives for a Dutch consortium, studying the infrastructural elements of a DUC. Since this effort focused on Dutch candidate Space Station payload elements, the DUC was considered to be a suitable user support centre to enhance the realism of the mission simulation. The European Space Agency (ESA) and the Dutch Agency for Aero.space Programmes (NIVR) together initiated the project ""Implementation of USO concept in Crew Workstation Testbed activities (DAMS)"". A main objective of this project was to evaluate the user support concept as defined by the User Support Operations (USO) team under contract with ESA Columbus Utilisation from 1989 to 1992, by participating in a planned five-day mission simulation. To evaluate advanced man-machine interfaces and crew support, the DUC consortium prepared and implemented an end to- end manned payload operations concept. The target ""onboard"" segment was an instrumented Space Station/Columbus double-rack, the so-called Biology Facility (a class-1 facility), and two multi-user (class-2) facilities: a Glovebox based on the Shuttle Middeck Locker type, and the High Performance Capillary Electrophoresis (HPCE) instrument. In addition some experiment dedicated breadboard facilities were developed and implemented in the Biology Facility. Two realistic experiments were prepared as part of the DAMS project. One concerned the investigation of toad egg development under microgravity after in-flight fertilisation. The second experiment was to determine the effect of Vitamin K intake by astronauts on the bone demineralisation process, by analyzing astronaut's urine samples. Experiment preparation included not only payload hardware development and commissioning, but also development of timelines and crew operations software. Since the planned mission simulation at ESTEC was cancelled due to ESA budgetary problems, a three-day mission simulation was set-up at NLR, to evaluate the final DAMS payload operation concept. DUC provided (operational) support to the two experienced Principal Investigators (PI) performing the two experiments. These experiments were controlled from an ""onboard"" CrewPC and two identical ground-based systems, the GroundPCs. These systems used identical and synchronised human computer interfaces and multimedia data bases. The mission simulation was very successful in that all aspects related to payload development, user support concept, and crew operations development, implementation and execution could be demonstrated and evaluated. This report gives an overview of the activities and the results.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:82ae9023-42e4-44b7-9c03-f2fab3bcb8da","http://resolver.tudelft.nl/uuid:82ae9023-42e4-44b7-9c03-f2fab3bcb8da","Ground execution of micro-gravity experiment scenario using an end-to-end manned payload operations concept","Pronk, Z.; Roefs, H.F.A.; de Hoop, D.","","1994","","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:4987808a-9937-444b-99a3-4bb4ba9f9cc2","http://resolver.tudelft.nl/uuid:4987808a-9937-444b-99a3-4bb4ba9f9cc2","Effects of oxygen limitation on sugar metabolism in yeasts: A continuous-culture study of the Kluyver effect","Weusthuis, R.A.; Visser, W.; Pronk, J.T.; Scheffers, W.A.; Van Dijken, J.P.","","1994","","Saccharomyces cerevisiae; Candida zltilis; sugar metabolism; oxygen limitation; Kluyver effect","en","journal article","Cambridge University Press","","","","","","","","Applied Sciences","","","","",""
"uuid:d0a54df9-ebea-4606-9ab1-e1bde05201af","http://resolver.tudelft.nl/uuid:d0a54df9-ebea-4606-9ab1-e1bde05201af","Dutch participation in Columbus APM mission simulation","Pronk, Z.","","1994","Within the studies on Advanced Man-Machine Interfaces and Crew Support (AMMI & CS), being part of the Crew Workstation Testbed activities at ESTEC, a Dutch consortium is preparing the implementation of the User Support Organisation (USO) concept in a ground-based mission simulation, planned for May 1994. The objective of the implementation study is to demonstrate and validate the USO concept mainly concerning payload development operational support tasks/and organisation. The flight element is the Columbus Attached Pressurized Module (APM) mock-up at ESTEC. The ground-based support facilities are located at the Dutch Utilisation Centre (DUC/NLR, Noordoostpolder). DUC will function as the Facility Responsible Centre, which task includes specification, acceptance and operation of a class 1 Biology Facility comprising two class 2 facilities: a High Performance Capillary Electrophoresis instrument and a Glovebox. This paper discusses the scope of the project, which covers all the items needed for a real-flight situation.","Columbus, user support, mission simulation, utilisation centre, payload development, payload operations","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:62198786-80ab-493e-9d26-ec081d261c64","http://resolver.tudelft.nl/uuid:62198786-80ab-493e-9d26-ec081d261c64","Thiobacillus ferrooxidans, a versatile mineworker","Bos, P.; Meulenberg, R.; Pronk, J.T.; Hazeu, W.; Kuenen, J.G.","","1994","","review Thiobacillus mine worker","en","conference paper","","","","","","","","","","","","","",""
"uuid:241569c1-e5ac-470f-b99d-591cec1225ee","http://resolver.tudelft.nl/uuid:241569c1-e5ac-470f-b99d-591cec1225ee","Chemostat cultivation as a tool for studies on sugar transport in yeasts","Weusthuis, R.A.; Pronk, J.T.; van den Broek, P.J.; van Dijken, J.P.","","1994","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:04462b37-7058-4cb5-8f29-760829075253","http://resolver.tudelft.nl/uuid:04462b37-7058-4cb5-8f29-760829075253","Preparation and demonstration of a support technology concept for in-orbit payload operations","Pronk, C.N.A.; Visser, F.B.; Sijmonsma, R.M.M.","","1993","Within the studies on Advanced Man-Machine Interfaces and Crew Support (AMMI&CS), being part of the Crew Workstation Testbed activities at ESTEC, a Dutch consortium is preparing the implementation of the User Support Organisation (USO) concept in a ground-based mission simulation. The objective of the Implementation study is to demonstrate and validate the USO concept mainly concerning operational organisation. The flight element is Pressurized Module (APM) ground-based support facilities are located at the Dutch Utilisation Centre (DUC/NLR, Noordoostpolder). The use of similarity in user interfaces and support functionalities between ground and flight segment is emphasized. The cooperation between Crew, Principal Investigator and Facility Expert is a particular subject of the study, since all of them will be on different locations. The present concept to be implemented is based on the use of common user interfaces and functions for both ground and flight segment.","Columbus; user support; mission simulation; utilisation centre; user interfaces","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:22875b9c-6c2c-462a-a7ba-3b055eab4f4d","http://resolver.tudelft.nl/uuid:22875b9c-6c2c-462a-a7ba-3b055eab4f4d","A Review of Bioenergetics and Enzymology of Sulfur Compound Oxidation by Acidophilic Thiobacilli","Kuenen, J.G.; Pronk, J.T.; Hazeu, W.; Meulenberg, R.; Bos, P.","","1993","","oxidation","en","conference paper","MINERALS METALS & MATERIALS SOC","","","","","","","","","","","","",""
"uuid:e9618b7b-5227-4b9f-b0d8-a8b703346a71","http://resolver.tudelft.nl/uuid:e9618b7b-5227-4b9f-b0d8-a8b703346a71","Kinetics of growth and sugar consumption in yeasts","van Dijken, J.P.; Weusthuis, R.A.; Pronk, J.T.","","1993","","","en","journal article","Kluwer","","","","","","","","","","","","",""
"uuid:e8565466-4cfb-4137-8546-d7bf736fcd70","http://resolver.tudelft.nl/uuid:e8565466-4cfb-4137-8546-d7bf736fcd70","Purification and Partial Characterization of Thiosulfate Dehydrogenase from Thiobacillus-Acidophilus","Meulenberg, R.; Pronk, J.T.; Hazeu, W.; Van Dijken, J.P.; Frank, J.; Bos, P.; Kuenen, J.G.","","1993","","oxidation; sulfur-compounds thiosulfate tepidarius oxidation proteins growth","en","journal article","","","","","","","","","","","","","",""
"uuid:4413b235-a782-4b18-9429-7234bfc32a1c","http://resolver.tudelft.nl/uuid:4413b235-a782-4b18-9429-7234bfc32a1c","Experimental and Theoretical Discrepancies in Growth Yields of Acinetobacter-Calcoaceticus - a Correction of Published Data","Van Schie, B.J.; Pronk, J.T.; Van Dijken, J.P.; Kuenen, J.G.","","1993","","glucose","en","journal article","","","","","","","","","","","","","",""
"uuid:0304d657-171c-4df7-a008-e290f3e11491","http://resolver.tudelft.nl/uuid:0304d657-171c-4df7-a008-e290f3e11491","Metabolism of Tetrathionate in Thiobacillus-Acidophilus","Meulenberg, R.; Scheer, E.J.; Pronk, J.T.; Hazeu, W.; Bos, P.; Kuenen, J.G.","","1993","","thiobacillus acidophilus acidophile sulfur metabolism polythionate hydrolysis tetrathionate metabolism trithionate hydrolase reduced sulfur-compounds ferrooxidans oxidation cultures","en","journal article","","","","","","","","","","","","","",""
"uuid:aa3af2a0-c655-4d29-8734-df62130fcc02","http://resolver.tudelft.nl/uuid:aa3af2a0-c655-4d29-8734-df62130fcc02","Selection of a manipulator system for the NLR space automation and robotics laboratory","Dieleman, P.; Pronk, C.N.A.","","1992","The NLR space division is realizing a laboratory environment which supports research and development in the area of automation and robotics applied to space-based systems, including the development of automated instruments for microgravity applications. Availability of such an environment is indispensable for further participation in national and international projects in this field. An important element in such an environment is a manipulator system, which provides a flexible means for RfiJ) on automated system under development for use in space. The application of such a system may range from testing special intelligent end-effectors attached to the manipulator to the participation in training operations for manipulator systems. Prime application of the manipulator system is in the area of internal robotics, but also aspects of external robotics may be investigated. The procurement of a suitable manipulator system was the goal of the work presented in this report. This report describes the evaluation of several industrial robot systems. Procurement of the A460 Robot System from CRS Plus Inc. is proposed. CRS Plus Inc. is represented in the Netherlands by Oostendorp-Nederland B.V. The A460 system will be delivered to NLR with the present C400 Robot System Controller. Replacement by the newly developed C500 controller is suggested as soon as this controller is commercially available. The total investment needed is about KFL 100 for the robot system. Additionally the procurement of a double 19"" rack as testbench and a PC for robot system programming and commanding is proposed. A total investment of about KFL 130 is needed to realize the initial automation and robotics laboratory setup.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:e540e95b-075e-448e-bc90-20d089fe8b2c","http://resolver.tudelft.nl/uuid:e540e95b-075e-448e-bc90-20d089fe8b2c","Development concept for Dutch user support","Pronk, C.N.A.; Koopman, N.; de Hoop, D.","","1992","","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:b6733c86-59f2-4a1f-8161-8f37d9644ce4","http://resolver.tudelft.nl/uuid:b6733c86-59f2-4a1f-8161-8f37d9644ce4","HERA training facility concept","Pronk, C.N.A.; Prins, J.J.M.; Dupont, J.L.; Simkens, P.; Plas, J.J.R.; Uittenboogaard, A.","","1992","","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:8aade790-0b53-4d38-8ef6-6506b54297f8","http://resolver.tudelft.nl/uuid:8aade790-0b53-4d38-8ef6-6506b54297f8","Oxidation of reduced sulphur compounds by intact cells of Thiobacillus acidophilus","Meulenberg, R.; Pronk, J.T.; Hazeu, W.; Bos, P.; Kuenen, J.G.","","1992","","Theobacillus acidophilus; acidophilis; sulphur metabolism; sulphide; elemental sulphur; thiosulphate; tetrathionate; trithionate; sulphite","en","journal article","","","","","","","","","","","","","",""
"uuid:aca7232c-b577-4dc2-bbd0-b13e55b197fa","http://resolver.tudelft.nl/uuid:aca7232c-b577-4dc2-bbd0-b13e55b197fa","Anaerobic Growth of Thiobacillus-Ferrooxidans","Pronk, J.T.; De Bruyn, J.C.; Bos, P.; Kuenen, J.G.","","1992","","ferric iron reduction sulfur oxidation bacteria; oxidation","en","journal article","","","","","","","","","","","","","",""
"uuid:74bc7046-d5be-493b-8668-dfcace61c43b","http://resolver.tudelft.nl/uuid:74bc7046-d5be-493b-8668-dfcace61c43b","HIGH YIELD METHOD OF GROWING THIOBACILLUS FERROOXIDANS ON FORMATE","Pronk, J.T.; Van Dijken, J.P.; Bos, P.; Kuenen, J.G.","","1992","","","en","patent","European Patent Office","","","","","","","","Civil Engineering and Geosciences","","","","",""
"uuid:84e48b6f-3335-437c-8c21-d15dc3b59c08","http://resolver.tudelft.nl/uuid:84e48b6f-3335-437c-8c21-d15dc3b59c08","Anaerobic Growth of Thiobacillus ferrooxidans","Pronk, J.T.; de Bruyn, J.C.; Bos, P.; Kuenen, J.G.","","1992","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:7e7e7ff9-16c6-418d-8396-29c8140125f6","http://resolver.tudelft.nl/uuid:7e7e7ff9-16c6-418d-8396-29c8140125f6","Purification and partial characterization of a thermostable trithionate hydrolase from the acidophilic sulphur oxidizer Thiobacillus acidophilus","Meulenberg, R.; Pronk, J.T.; Frank, J.; Hazeu, W.; Bos, P.; Kuenen, J.G.","","1992","","","en","journal article","","","","","","","","","","","","","",""
"uuid:635cfdbd-05d5-446a-a7c2-2cb3184dd5e8","http://resolver.tudelft.nl/uuid:635cfdbd-05d5-446a-a7c2-2cb3184dd5e8","""Dutch Utilisation Centre stand"" op de Cospar 1990 tentoonstelling","Slippens, C.P.R.C.; Pronk, C.N.A.","","1991","Tijdens de COSPAR meeting in Den Haag van 25 juni tot en met 7 juli 1990 werd in het Congres centrum een tentoonstelling ingericht waar het Nederlandse bedrijfsleven onder stimulans van het NIVR een gezamenlijke inbreng demonstreerde met betrekking tot gebruikers-ondersteuning bij experimenten in de ruimte. De gezamenlijke presentatie van technologieën werd ondergebracht in een stand onder de naam Dutch Utilisation Centre of DUC-stand. Dit rapport geeft weer welke aktiviteiten gedemonstreerd zijn door Nederlandse bedrijven en instellingen binnen de DUC-stand of daaraan gerelateerd zijn. De demonstraties vielen zo veel mogelijk binnen de functies van de Nederlandse gebruikersondersteuningsorganisatie of DUSO. De DUSO/DUC functies worden daarom in dit verslag toegelicht. De conclusie is dat dit congres en de tentoonstelling geleid hebben tot een verdere samenwerking van bedrijven en instellingen in Nederland om te komen tot een DUC.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:37b85531-a98d-485e-8fab-7634d897fa0e","http://resolver.tudelft.nl/uuid:37b85531-a98d-485e-8fab-7634d897fa0e","Physiology of the acidophilic thiobacilli","Pronk, J.T.","Kuenen, J.G. (promotor)","1991","","","en","doctoral thesis","","","","","","","","","Applied Sciences","","","","",""
"uuid:5abb8bcb-66e7-4225-92dd-398dd2647232","http://resolver.tudelft.nl/uuid:5abb8bcb-66e7-4225-92dd-398dd2647232","The shoulder girdle, analysed and modelled kinematically","Pronk, G.M.","Stassen, H.G. (promotor); Rozendal, R.H. (promotor)","1991","","schoudergordel; biomechanische modellen","en","doctoral thesis","","","","","","","","","Mechanical Maritime and Materials Engineering","","","","",""
"uuid:06eea8b2-089e-4f13-bf52-191bea129b63","http://resolver.tudelft.nl/uuid:06eea8b2-089e-4f13-bf52-191bea129b63","Growth of Thiobacillus-Ferrooxidans on Formic-Acid","Pronk, J.T.; Meijer, W.M.; Hazeu, W.; Van Dijken, J.P.; Bos, P.; Kuenen, J.G.","","1991","","acidophilic bacteria chemostat cultures formate chemolithotroph energetics methanol sulfur; oxidation","en","journal article","","","","","","","","","","","","","",""
"uuid:a731e4a2-2da0-4098-9c5e-8023309fb0ef","http://resolver.tudelft.nl/uuid:a731e4a2-2da0-4098-9c5e-8023309fb0ef","Growth of Thiobacillus ferrooxidans on Formic Acid","Pronk, J.T.; Meijer, W.M.; Hazeu, W.; van Dijken, J.P.; Bos, P.; Kuenen, J.G.","","1991","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:11004349-707c-4081-9215-79008ed970c5","http://resolver.tudelft.nl/uuid:11004349-707c-4081-9215-79008ed970c5","Glucose metabolism and gluconic acid production by Acetobacter diazotrophicus","Attwood, M.M.; van Dijken, J.P.; Pronk, J.T.","","1991","","","en","journal article","","","","","","","","","","","","","",""
"uuid:5384ed65-66b1-4139-9cfe-d8bc3e5d2fa3","http://resolver.tudelft.nl/uuid:5384ed65-66b1-4139-9cfe-d8bc3e5d2fa3","Energy Transduction by Anaerobic Ferric Iron Respiration in Thiobacillus-Ferrooxidans","Pronk, J.T.; Liem, K.; Bos, P.; Kuenen, J.G.","","1991","","","en","journal article","","","","","","","","","","","","","",""
"uuid:f7d6dba0-3b8a-4d62-96c0-cb77d15edf6d","http://resolver.tudelft.nl/uuid:f7d6dba0-3b8a-4d62-96c0-cb77d15edf6d","Energy Transduction by Anaerobic Ferric Iron Respiration in Thiobacillus ferrooxidans","Pronk, J.T.; Liem, K.; Bos, P.; Kuenen, J.G.","","1991","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:00d4a9c3-9870-486f-a866-b655710ed96e","http://resolver.tudelft.nl/uuid:00d4a9c3-9870-486f-a866-b655710ed96e","Space automation and robotics in support of microgravity instrument applications: Research and development activities","Pronk, C.N.A.; Dieleman, P.","","1990","This document contains an overview of results from studies on Automation and Robotics (A&R) within the European space community. The scope of the overview is limited to the results related to internal vehicular activities, with emphasis on the support of microgravity experimentation. Other research areas, such as internal servicing and external servicing are shortly discussed. In order to complete the overview of present state-of-the-art space A&R, some results of non-European studies are discussed and incorporated. The overview is used to identify and classify Research and Development (R&D) needs in the field of internal automation and robotics within the European space programme, and to identify the possibilities for participation in these/' activities by industries and institutes in the Netherlands.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:ac626962-da97-491c-9e7e-ae5bb78a3752","http://resolver.tudelft.nl/uuid:ac626962-da97-491c-9e7e-ae5bb78a3752","Space automation and robotics for microgravity experiment applications: Preliminary identification of research and development topics","Dieleman, P.; Pronk, C.N.A.","","1990","A short study has been carried out by NLR to identify Research and Development (R&D) areas in the field of Automation and Robotics (A&R) for space applications which may be relevant for Dutch space industry to participate in. The report at hand gives an overview of space A&R activities and a preliminary identification of R&D topics, whereby special attention is given to items related to the use of A&R in supporting microgravity experiment facilities located inside a pressurized spacecraft. Main attention is focused on activities which take place in the ESA/ESTEC framework with emphasis on the need of A&R technology for Columbus. The report will be used as a starting point for a detailed evaluation of present requirements and identification of R&D needs. This evaluation will then be used to identify, classify and select those areas relevant for Dutch space industry and research institutes. This should result in a project proposal to NIVR by a consortium of Dutch industries and institutes. The project should aim at enhancement of the Dutch technological position within ESTEC in the field of space related A&R. An early shortlist of possible projects is included in this report.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:76d51dce-708b-47f7-a2e3-52b7f0c53998","http://resolver.tudelft.nl/uuid:76d51dce-708b-47f7-a2e3-52b7f0c53998","Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus","Pronk, J.T.; De Bruijn, P.; Van Dijken, J.P.; Bos, P.; Kuenen, J.G.","","1990","","Bacteria Mixotrophy Autotrophy Monocarbon compound Energy metabolism Enzyme Enzymatic activity Microorganism growth Bacterie Mixotrophie Autotrophie Compose monocarbone Metabolisme energetique Enzyme Activite enzymatique Multiplication microorganisme Thio","en","journal article","","","","","","","","","","","","","",""
"uuid:78f57f7f-0431-45f1-abc3-c4da9663a897","http://resolver.tudelft.nl/uuid:78f57f7f-0431-45f1-abc3-c4da9663a897","Heterotrophic Growth of Thiobacillus-Acidophilus in Batch and Chemostat Cultures","Pronk, J.T.; Meesters, P.J.W.; Van Dijken, J.P.; Bos, P.; Kuenen, J.G.","","1990","","","en","journal article","","","","","","","","","","","","","",""
"uuid:0f87db6d-9a71-473e-91b4-f26053de1de1","http://resolver.tudelft.nl/uuid:0f87db6d-9a71-473e-91b4-f26053de1de1","Mixotrophic and Autotrophic Growth of Thiobacillus acidophilus on Glucose and Thiosulfate","Pronk, J.T.; Meulenberg, R.; van den Berg, D.J.; Batenburg-van der Vegte, W.; Bos, P.; Kuenen, J.G.","","1990","","","en","journal article","American Society for Microbiology","","","","","","","","","","","","",""
"uuid:9592868a-b999-4712-a233-191b615da6c6","http://resolver.tudelft.nl/uuid:9592868a-b999-4712-a233-191b615da6c6","Oxidation of Reduced Inorganic Sulfur-Compounds by Acidophilic Thiobacilli","Pronk, J.T.; Meulenberg, R.; Hazeu, W.; Bos, P.; Kuenen, J.G.","","1990","","oxidation","en","journal article","","","","","","","","","","","","","",""
"uuid:cf51c1c7-98ec-447d-bdce-cd5b1ac8c298","http://resolver.tudelft.nl/uuid:cf51c1c7-98ec-447d-bdce-cd5b1ac8c298","Mixotrophic and Autotrophic Growth of Thiobacillus-Acidophilus on Glucose and Thiosulfate","Pronk, J.T.; Meulenberg, R.; Van den Berg, D.J.M.; Batenburg-Van de Vegte, W.H.; Bos, P.; Kuenen, J.G.","","1990","","","en","journal article","","","","","","","","","","","","","",""
"uuid:e4ea5426-fe16-4c47-b666-e3a55aa8deb3","http://resolver.tudelft.nl/uuid:e4ea5426-fe16-4c47-b666-e3a55aa8deb3","Role of NADP-dependent and quinoprotein glucose dehydrogenases in gluconic acid production by Gluconobacter oxydans","Pronk, J.T.; Levering, P.R.; Oiijve, W.; Van Dijken, J.P.","","1989","","Gluconobacter oxydans; gluconic acid; glucose metabolism; quinoproteins","en","journal article","Butterworth","","","","","","","","","","","","",""
"uuid:3bf3ed49-3279-446f-80e8-a33c09a10615","http://resolver.tudelft.nl/uuid:3bf3ed49-3279-446f-80e8-a33c09a10615","The discovery of beta-galactosidase","Rouwenhorst, R.J.; Pronk, J.T.; Van Dijken, J.P.","","1989","","","en","journal article","Elsevier","","","","","","","","","","","","",""
"uuid:134ee873-4d89-4ae2-873c-0f36d028ecb3","http://resolver.tudelft.nl/uuid:134ee873-4d89-4ae2-873c-0f36d028ecb3","Vooruitgang bit voor bit;: Liber Amicorum ac Collegarum bij het afscheid van Prof. dr. ir. W.L. van der Poel, 26 oktober 1988","Pronk, C.; Toetenel, W.J.","","1988","","informatica","nl","book","Delftse Universitaire Pers","","","","","","","","Delft University of Technology","","","","",""
"uuid:f3fb7eeb-2c9e-4b0b-ba85-d945ca7fe786","http://resolver.tudelft.nl/uuid:f3fb7eeb-2c9e-4b0b-ba85-d945ca7fe786","Preparation of D-xylulose from D-xylose: Enzyme and Microbial Technology","Pronk, J.T.; Bakker, A.W.; Van Dam, H.E.; Straathof, A.J.J.; Scheffers, W.A.; Van Dijken, J.P.","","1988","","D-xylulose; xylose isomerase; Acinetobacter calcoaceticus; immobilization","en","journal article","Butterworth","","","","","","","","Applied Sciences","","","","",""
"uuid:8d240941-86bb-4b01-9b19-5d730ac01e86","http://resolver.tudelft.nl/uuid:8d240941-86bb-4b01-9b19-5d730ac01e86","Software requirements document for the non-real time HSF software for level 3 HERA on-board control","Groothuizen, R.J.P.; Slippens, C.P.R.C.; Pronk, C.N.A.","","1988","This document contains the software requirements for the non-real-time HSF software for level 3 HERA on-board control. Software requirements for the level 3 HERA on-board control itself, having been generated in a separate study, are transformed to the non-real-time simulation facility environment and the requirements for the software supporting the incorporation of the on-board software in the simulation facility, are derived.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:c40577f1-636a-4f9d-a348-f100120bd87c","http://resolver.tudelft.nl/uuid:c40577f1-636a-4f9d-a348-f100120bd87c","Definition of the Eurosim simulation subsystem","Pronk, C.N.A.; Ersü, E.; Lippay, A.L.; Elfving, A.","","1987","Recently, the European Space Agency (ESA) initiated two studies to define a simulation facility for space operations involving space manipulators and other in-orbit servicing and assembly systems. This European Robotic Operations Simulator (EUROSIM) will play an important role during system development as well as prior and parallel to operational phases. The main task of the study EUROSIM II* was to describe the preliminary software requirements for the simulation subsystem, yielding specification of requirements for the simulation definition and control function, data structures and data flow, and mathematical models of real-world physical systems and processes. In this paper we present and discuss the results of this study. In addition a development plan will be given to arrive at a more detailed level of specification of the simulation software and to arrive at the initial operational capabilities of EUROSIM. This paper has been presented on the first European In-orbit Operations Technology Symposium, Darmstadt, 7-9 September 1987","space operations; simulation; robotics; systems analysis; software specification; dynamics","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:ab86adef-1e85-4d6d-9342-caa83c6faa4b","http://resolver.tudelft.nl/uuid:ab86adef-1e85-4d6d-9342-caa83c6faa4b","Robotics simulation and testbed, Eurosim study on simulation system definition. Vol. II. Requirements - Technical volume","Pronk, C.N.A.; de Jong, H.; Lippay, A.L.; Ersü, E.","","1987","","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:b9bf2d2a-7612-4b54-bdf6-e68c14425ef0","http://resolver.tudelft.nl/uuid:b9bf2d2a-7612-4b54-bdf6-e68c14425ef0","Robotics simulation and testbed, EUROSIM Study on simulation system definition. Volume I: Executive summary","Pronk, C.N.A.","","1987","In the framework of ESA's programma for Robotics, Tele-operation and Servicing (RTS) a powerful simulation tool has to be developed to support the various stages in the development process of systems and operations. Therefore, ESA has initiated the development of a European Robotic Operations Simulator (EUROSIM). Two parallel studies, funded and coordinated by ESA, have been performed: 1. Definition and planning of the facility (contract I, by a team with Fokker) 2. Definition and planning of the application software (contract II, by a team with NLR, CAE Electronics (Canada) and ISRA Systemtechnik (Germany)). This report contains the results of the second contract: - additional functional requirements for extended applications of EUROSIM; - preliminary software requirements for the simulation software composed of simulation definition support software, model software and data structures; - a description and evaluation of existing models and software; - an overall development plan for the simulation software. The study output consists of four volumes. Volume I is the executive summary. Volume II contains all requirements. In Volume III detailed specifications of d3mamics models, spacecraft and environment models, and manipulator models are given. Volume IV is a compilation of technical notes.","Simulators; European space programs; Robotics; Systems simulation; Signal distortion; Functional design specifications; Software tools","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:83b701ae-c91c-4f56-98b8-619ef1579663","http://resolver.tudelft.nl/uuid:83b701ae-c91c-4f56-98b8-619ef1579663","Simulation of space manipulator operations (EUROSIM)","Pronk, C.N.A.; Elfving, A.; Ersu, E.; Lippay, A.L.","","1987","Recently, the European Space Agency (ESA) initiated studies for definition of a facility for simulation of space operations, involving space manipulators and other in orbit servicing and assembly systems. This European Robotic Operations Simulator (EUROSIM) shall be used in all system and operation development phases.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:8acca6a3-39ba-453c-9ccb-73df16ec4f79","http://resolver.tudelft.nl/uuid:8acca6a3-39ba-453c-9ccb-73df16ec4f79","Functional analysis of the Agrobacterium tumefaciens octopine Ti-plasmid left and right T-region border fragments","Van Haaren, M.J.J.; Pronk, J.T.; Schilperoort, R.A.; Hooykaas, P.J.J.","","1987","","Agrobacterium tumefaciens; plant tumors; T-DNA; border repeats","en","journal article","Martinus Nijhoff","","","","","","","","","","","","",""
"uuid:d93eb39e-7cf5-43a4-9f6e-a5b73161ebaf","http://resolver.tudelft.nl/uuid:d93eb39e-7cf5-43a4-9f6e-a5b73161ebaf","Glucose-dehydrogenase-mediated solute transport and ATP synthesis in Acinetobacter calcoaceticus","Van Schie, B.J.; Pronk, J.T.; Hellingwerf, K.J.; Van Dijken, J.P.; Kuenen, J.G.","","1987","","Acinetobacter calcoaceticus Enzyme Glucose dehydrogenase Chemostat Limitation Carbon Conservation Energy Acinetobacter calcoaceticus Enzyme Glucose dehydrogenase Chemostat Limitation Carbone Conservation Energie Acinetobacter calcoaceticus Enzima Glucose","en","journal article","","","","","","","","","","","","","",""
"uuid:72dff7d6-6485-4725-a092-b61864def8b1","http://resolver.tudelft.nl/uuid:72dff7d6-6485-4725-a092-b61864def8b1","The in vivo and in vitro substrate specificity of quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus LMD 79.41","Dokter, P.; Pronk, J.T.; van Schie, B.J.; van Dijken, J.P.; Duine, J.A.","","1987","","Acinetobacter calcoaceficus; Glucose dehydrogenase; Quinoprotein; (Substrate specificity)","en","journal article","Elsevier","","","","","","","","","","","","",""
"uuid:7c8a8ca5-ba71-426d-b186-a13fd080c8da","http://resolver.tudelft.nl/uuid:7c8a8ca5-ba71-426d-b186-a13fd080c8da","Rendezvous and docking and space robotics proximity operations: part V: executive summary","Prins, J.J.M.; Pronk, C.N.A.","","1985","In the framework of ESA's Columbus programme, Europe has to master new space operations such as remotely and/or automatically controlled rendezvous and docking (RVD) and manipulation. The need for a test bed for development and verification of the critical proximity operations involved was identified. This report presents the results of a study on the realisation of such a test bed. In Part I the test requirements were derived for: - Full scale testing of final translation (last 10 meters) including docking (RVD). - Full scale testing of any manipulator operations both for free space and during mechanical contact. - Scaled testing of fly-around and translation along the docking axis (RVD). Automatic as well as human operator controlled operations need to be tested. The test bed realisation study describes in detail the test bed requirements (Part II), the conceptual design (Part III) and the development plan (Part IV). Part V is the executive summary.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:3c9b451c-1825-43c9-9a4a-175e4ebe3790","http://resolver.tudelft.nl/uuid:3c9b451c-1825-43c9-9a4a-175e4ebe3790","Rendezvous and docking and space robotics proximity operations: part III: test bed conceptual design","Prins, J.J.M.; Pronk, C.N.A.","","1985","In the framework of ESA's Columbus programme, Europe has to master new space operations such as remotely and/or automatically controlled rendezvous and docking (RVD) and manipulation. The need for a test bed for development and verifcation of the critical proximity operations involved was identified. This report presents the results of a study on the realisation of such a test bed. In Part I the test requirements were derived for: - Full scale testing of final translation (last 10 meters) including docking (RVD). - Full scale testing of any manipulator operations both for free space and during mechanical contact. Scaled testing of fly-around and translation along the docking axis (RVD). Automatic as well as human operator controlled operations need to be tested. The test bed realisation study describes in detail the test bed requirements (Part II), the conceptual design (Part III) and the development plan (Part IV). Part V is the excentive summary.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:6d482c6e-a79a-4613-9ed3-1b9fe3fb02d3","http://resolver.tudelft.nl/uuid:6d482c6e-a79a-4613-9ed3-1b9fe3fb02d3","Rendezvous and docking and space robotics proximity operations: part II: test bed requirements","Prins, J.J.M.; Pronk, C.N.A.","","1985","In the framework of ESA's Columbus programme, Europe has to master new space operations such as remotely and/or automatically controlled rendezvous and docking (RVD) and manipulation. The need for a testbed for development and verification of the critical proximity operations involved was identified. This report presents the results of a study on the realisation of such a testbed. In Part I the test requirements were derived for: - Full scale testing of final translation (last 10 meters) including docking (RVD). - Full scale testing of any manipulator operations both for free space and during mechanical contact. - Scaled testing of fly-around and translation along the docking axis (RVD). Automatic as well as human operator controlled operations need to be tested. The testbed realisation study describes in detail the testbed requirements (Part II), the conceptual design (Part III) and the development plan (Part IV). Part V is the executive summary.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:c7d1a2b1-8fdb-4546-aa7b-f6b409621d36","http://resolver.tudelft.nl/uuid:c7d1a2b1-8fdb-4546-aa7b-f6b409621d36","Rendezvous and docking and space robotics proximity operations: part I: test requirements","Prins, J.J.M.; Pronk, C.N.A.","","1985","In the framework of ESA's Columbus programme, Europe has to master new space operations such as remotely and/or automatically controlled rendezvous and docking (RVD) and manipulation. The need for a testbed for development and verification of the critical proximity operations involved was identified. This report presents the results of a study on the realisation of such a testbed. In Part I the test requirements were derived for: - Full scale testing of final translation (last 10 meters) including docking (RVD). - Full scale testing of any manipulator operations both for free space and during mechanical contact. - Scaled testing of fly-around and translation along the docking axis (RVD). Automatic as well as human operator controlled operations need to be tested. The test bed realisation study describes in detail the test bed requirements (Part II), the conceptual design (Part III) and the development plan (Part IV). Part V is the executive summary.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:be3d7ee7-0648-4f9d-a7ec-6bc22ee1ca65","http://resolver.tudelft.nl/uuid:be3d7ee7-0648-4f9d-a7ec-6bc22ee1ca65","Energization of Solute Transport by Pqq-Dependent Glucose-Dehydrogenase in Membrane-Vesicles of Acinetobacter Species","Pronk, J.T.; Van Schie, B.J.; Van Dijken, J.P.; Kuenen, J.G.","","1985","","","en","journal article","","","","","","","","","","","","","",""
"uuid:df6ca832-9385-41fc-a16c-1788d98b0a0a","http://resolver.tudelft.nl/uuid:df6ca832-9385-41fc-a16c-1788d98b0a0a","Blade spindle torque and off-design behaviour of controllable pitch propellers","Pronk, C.","Van Manen, J.D. (promotor)","1980","","","en","doctoral thesis","","","","","","","","","Mechanical, Maritime and Materials Engineering","Mechanical, Maritime and Materials Engineering","","","",""
"uuid:eb87089e-1f37-4518-90ec-bb49a921de03","http://resolver.tudelft.nl/uuid:eb87089e-1f37-4518-90ec-bb49a921de03","Blade spindle torque and off design behaviour of controllable pitch propellers","Pronk, C.","","1980","","resistance & propulsion","","doctoral thesis","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Ship Hydromechanics and Structures","","",""
"uuid:5fc1f211-2eed-49d3-8b4d-f808f85934a4","http://resolver.tudelft.nl/uuid:5fc1f211-2eed-49d3-8b4d-f808f85934a4","Aanvang voor een computersysteem voor data-aquisitie en experimentbesturing t.b.v. het lage snelheids windtunnellaboratorium","Binkhorst, H.; van Ingen, J.L.; Pronk, C.","","1978","","","nl","report","Delft University of Technology","","","","","","","","Aerospace Engineering","","","","",""
"uuid:409489a1-0e95-42f5-a582-aa0db39aa6c7","http://resolver.tudelft.nl/uuid:409489a1-0e95-42f5-a582-aa0db39aa6c7","Prediction of the dynamic stability of full span subsonic wind tunnel models","Pronk, N.","","1973","In wind tunnel tests the stability of model oscillations during the measurements is of great importance. Not only the airspeed at which oecillations become unstable is of interest, but also the damping available below this speed, because this damping determines largely the qpaiescence of the model, Special types of model supporting systems may raise problems concerning the pjresence of sufficient damping of the model oscillations. A typical example is the ""subsonic model support"" that has been designed by NLR and introduced for use in the NLR high speed wind tunnel HST (Fig, l). This system enables measurements in vdiioh different shapes of the aft fuselage are being considered. Important for the present discussion is the very slender sting on which the model is mounted through the bottom of the fuselaige. This slendernees admits relatively large deflections of the model with respect to the more conventional systems with a strut, which support the model at the aft fuselage. In view of possible troublesome oscillations of the model occurring during the measurements, the stability of the oscillations needs to be investigated already during the design of the model and the supporting system. To that purpose a computer programme has been developed at NLR which calculates damping and frequency of oscillations on a routine basis. In the following the calculation method is described briefly and some examples are shown.","","en","report","Nationaal Lucht- en Ruimtevaartlaboratorium","","","","","","Campus only","","","","","","",""
"uuid:7c33505c-c3ea-4e78-adf2-34b526ba84af","http://resolver.tudelft.nl/uuid:7c33505c-c3ea-4e78-adf2-34b526ba84af","The Controllable Pitch Propeller as a Pitch-Govenor","Pronk, C.","","1972","","resistance & propulsion","","report","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Ship Hydromechanics and Structures","","",""
"uuid:64735207-15a5-49e9-9b9a-efeb6d21bdd3","http://resolver.tudelft.nl/uuid:64735207-15a5-49e9-9b9a-efeb6d21bdd3","Selection and simulation of marine propulsion control system","Pronk, C.","","1971","","resistance & propulsion","","conference paper","","","","","","","","","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Ship Hydromechanics and Structures","","",""
"uuid:bd9c1766-b192-4220-a088-fd8e1d4cffad","http://resolver.tudelft.nl/uuid:bd9c1766-b192-4220-a088-fd8e1d4cffad","Azijnzuurbereiding uit methanol en koolmonoxyde: Bereiding van koolmonoxyde uit aardgas","Broekhuis, S.; Koppert, C.; Pronk, K.M.A.","","1970","Document(en) uit de collectie Chemische Procestechnologie","","nl","report","Delft University of Technology","","","","","","","","Applied Sciences","DelftChemTech","","","",""
"uuid:830a59ac-3198-41f8-8125-e7e222be2b25","http://resolver.tudelft.nl/uuid:830a59ac-3198-41f8-8125-e7e222be2b25","Het gedrag van een schip in achteroplopende zeegang","Pronk, C.","Gerritsma, J. (mentor)","1969","","hydrodynamics","","master thesis","","","","","","","","indefinite","Mechanical, Maritime and Materials Engineering","Marine and Transport Technology","Ship Hydromechanics and Structures","","",""