Abstract: Even though microorganisms can often grow in defined media, they are frequently cultured in rich media containing complex components like yeast extract. A drawback of using rich media is the effect of secondary substrates from complex components on the metabolism, which can change the anabolism and the formation of products, complicating the interpretation of experimental results. Rich media containing yeast extract is generally used to grow Clostridium pasteurianum. In this work, we describe the development of a minimal medium for C. pasteurianum combining rational media design, transfers in batch bottles and continuous bioreactors experiments. The media were designed based on literature, the elements needed in the metabolism, and a general chemical formula for the composition of biomass. The media were tested by cultivations in chemostat and batch bottles. Microbial growth was not sustained in an inorganic medium with glucose over batch bottles transfers. In contrast, a medium with glucose supplemented with para-aminobenzoic acid (PABA), biotin and cysteine, supported growth in chemostat (12 retention times) and in batch bottles transfers. Growth was also maintained in the same medium without cysteine during the 12 retention times of chemostat operation, but at a lower dilution rate, showing that cysteine enhanced the growth rate of C. pasteurianum despite not being essential. Microbial growth was sustained through batch bottle transfers in media with PABA only and with biotin only, apart from glucose and cysteine. Therefore, it was concluded that PABA and biotin are essential for the growth of C. pasteurianum without yeast extract, only one—any—amongst both being needed. Key points: • Clostridium pasteurianum is auxotrophic on B vitamins. • Both biotin and PABA suffice to support growth in an otherwise inorganic medium. • Cysteine was not essential but increased its growth rate.

Correction to: Scientific Reportshttps://doi.org/10.1038/s41598-023-43682-x, published online 13 October 2023 The original version of this Article contained errors in the Discussion section, where reference 34 was incorrectly cited as reference 25. Consequently, “Spirito et al.²⁵ also observed a decreased conversion rate when feeding a chain-elongating reactor microbiome with mainly ethanol. This could explain why previous studies with shorter incubation times reported no metabolic activity in the absence of acetate^25,35. The mechanism that underlies this change in rate remains elusive. Spirito et al.²⁵ proposed a thermodynamic constraint on the rate due to increased hydrogen partial pressures, but in our experiments pH₂ was low due to continuous sparging and we still observed low rates.” now reads: “Spirito et al.³⁴ also observed a decreased conversion rate when feeding a chain-elongating reactor microbiome with mainly ethanol. This could explain why previous studies with shorter incubation times reported no metabolic activity in the absence of acetate^34,35. The mechanism that underlies this change in rate remains elusive. Spirito et al.³⁴ proposed a thermodynamic constraint on the rate due to increased hydrogen partial pressures, but in our experiments pH₂ was low due to continuous sparging and we still observed low rates.” The original Article has been corrected.

Syngas fermentation has gained momentum over the last decades. The cost-efficient design of industrial-scale bioprocesses is highly dependent on quantitative microbial growth data. Kinetic and stoichiometric models for syngas-converting microbes exist, but accurate experimental validation of the derived parameters is lacking. Here, we describe a novel experimental approach for measuring substrate uptake kinetics of gas-fermenting microbes using the model microorganism Clostridium autoethanogenum. One-hour disturbances of a steady-state chemostat bioreactor with increased CO partial pressures (up to 1.2 bar) allowed for measurement of biomass-specific CO uptake- and CO₂ production rates ((Formula presented.), (Formula presented.)) using off-gas analysis. At a p_CO of 1.2 bar, a (Formula presented.) of −119 ± 1 mmol g⁻¹_X h⁻¹ was measured. This value is 1.8–3.5-fold higher than previously reported experimental and kinetic modeling results for syngas fermenters. Analysis of the catabolic flux distribution reveals a metabolic shift towards ethanol production at the expense of acetate at p_CO (Formula presented.) 0.6 atm, likely to be mediated by acetate availability and cellular redox state. We characterized this metabolic shift as acetogenic overflow metabolism. These results provide key mechanistic understanding of the factors steering the product spectrum of CO fermentation in C. autoethanogenum and emphasize the importance of dedicated experimental validation of kinetic parameters.

(Bio)fouling is the most common problem in membrane processes used in water production. It is also a reported unavoidable problem, with mitigation strategies being frequently ineffective in addressing this problem. An ultrapure water plant (UPW) in Emmen (The Netherlands), fed with secondary effluent, comprises the following subsequent treatment steps: ultrafiltration (UF), biological oxygen-dosed activated carbon (BODAC) filtration and reverse osmosis (RO). The BODAC filters were designed to prevent fouling in RO membranes, and for ten years, they have been operated without significant fouling issues. The present work aims to provide insight into the role of the full-scale UF + BODAC in fouling prevention, by conducting a mass balance (MB) analysis to assess the removal/release of common fouling precursors. Positive MB results were noticed for particulate organic compounds, iron (Fe) and manganese (Mn) meaning their constant removal in the UF + BODAC. The UF + BODAC was shown to be an effective nitrification system, effectively converting all the ammonium and nitrite to nitrate. In conclusion, the combined removal of organics, Fe, and Mn species and nitrification by UF + BODAC is most likely an important factor in downstream fouling prevention, making this system an attractive process for fouling prevention. Nevertheless, further investigations to discover the mechanisms involved are needed.

Poly(3-hydroxybutyrate) (PHB) was accumulated in full-scale municipal waste activated sludge at pilot scale. After accumulation, the fate of the PHB-rich biomass was evaluated over two weeks as a function of initial pH (5.5, 7.0 and 10), and incubation temperature (25, 37 and 55 °C), with or without aeration. PHB became consumed under aerobic conditions as expected with first order rate constants in the range of 0.19 to 0.55 d⁻¹. Under anaerobic conditions, up to 63 percent of the PHB became consumed within the first day (initial pH 7, 55 °C). Subsequently, with continued anaerobic conditions, the polymer content remained stable in the biomass. Degradation rates were lower for acidic anaerobic incubation conditions at a lower temperature (25 °C). Polymer thermal properties were measured in the dried PHB-rich biomass and for the polymer recovered by solvent extraction using dimethyl carbonate. PHB quality changes in dried biomass, indicated by differences in polymer melt enthalpy, correlated to differences in the extent of PHB extractability. Differences in the expressed PHB-in-biomass melt enthalpy that correlated to the polymer extractability suggested that yields of polymer recovery by extraction can be influenced by the state or quality of the polymer generated during downstream processing. Different post-accumulation process biomass management environments were found to influence the polymer quality and can also influence the extraction of non-polymer biomass. An acidic post-accumulation environment resulted in higher melt enthalpies in the biomass and, consequently, higher extraction efficiencies. Overall, acidic environmental conditions were found to be favourable for preserving both quantity and quality after PHB accumulation in activated sludge.

Hexanoate is a valuable chemical that can be produced by microorganisms that convert short-chain- to medium-chain carboxylic acids through a process called chain elongation. These microorganisms usually produce mixtures of butyrate and hexanoate from ethanol and acetate, but direct conversion of ethanol to hexanoate is theoretically possible. Steering microbial communities to ethanol-only elongation to hexanoate circumvents the need for acetate addition and simplifies product separation. The biological feasibility of ethanol elongation to hexanoate was validated in batch bioreactor experiments with a Clostridium kluyveri-dominated enrichment culture incubated with ethanol, acetate and butyrate in different ratios. Frequent liquid sampling combined with high-resolution off-gas measurements allowed to monitor metabolic behavior. In experiments with an initial ethanol-to-acetate ratio of 6:1, acetate depletion occurred after ± 35 h of fermentation, which triggered a metabolic shift to direct conversion of ethanol to hexanoate despite the availability of butyrate (± 40 mCmol L⁻¹). When only ethanol and no external electron acceptor was supplied, stable ethanol to hexanoate conversion could be maintained until 60–90 mCmol L⁻¹ of hexanoate was produced. After this, transient production of either acetate and butyrate or butyrate and hexanoate was observed, requiring a putative reversal of the Rnf complex. This was not observed before acetate depletion or in presence of low concentrations (40–60 mCmol L⁻¹) of butyrate, suggesting a stabilizing or regulatory role of butyrate or butyrate-related catabolic intermediates. This study sheds light on previously unknown versatility of chain elongating microbes and provides new avenues for optimizing (waste) bioconversion for hexanoate production.

Carboxydotrophic metabolism is gaining interest due to its applications in gas fermentation technology, enabling the conversion of carbon monoxide to fuels and commodities. Acetogenic carboxydotrophs play a central role in current gas fermentation processes. In contrast to other energy-rich microbial substrates, CO is highly toxic, which makes it a challenging substrate to utilize. Instantaneous scavenging of CO upon entering the cell is required to mitigate its toxicity. Experiments conducted with Clostridium autoethanogenum at different biomass-specific growth rates show that elevated ethanol production occurs at increasing growth rates. The increased allocation of electrons towards ethanol at higher growth rates strongly suggests that C. autoethanogenum employs a form of overflow metabolism to cope with high dissolved CO concentrations. We argue that this overflow branch enables acetogens to efficiently use CO at highly variable substrate influxes by increasing the conversion rate almost instantaneously when required to remove toxic substrate and promote growth. In this perspective, we will address the case study of C. autoethanogenum grown solely on CO and syngas mixtures to assess how it employs acetate reduction to ethanol as a form of overflow metabolism.

Polyhydroxyalkanoates (PHAs) can be produced with municipal waste activated sludge from biological wastewater treatment processes. Methods of selective fluorescent staining with confocal laser scanning microscopy (CLSM) were developed and optimized to evaluate the distribution of PHA storage activity in this mixed culture activated sludge microbial communities. Selective staining methods were applied to a municipal activated sludge during pilot scale PHA accumulation in replicate experiments. Visualization of stained flocs revealed that a significant but limited fraction of the biomass was engaged with PHA accumulation. Accumulated PHA granules were furthermore heterogeneously distributed within and between flocs. These observations suggested that the PHA content for the bacteria storing PHAs was significantly higher than the average PHA content measured for the biomass as a whole. Optimized staining methods provided high acuity for imaging of PHA distribution when compared to other methods reported in the literature. Selective staining methods were sufficient to resolve and distinguish between distinctly different morphotypes in the biomass, and these observations of distinctions have interpreted implications for PHA recovery methods. Visualization tools facilitate meaningful insights for advancements of activated sludge processes where systematic observations, as applied in the present work, can reveal underlying details of structure-function relationships.

The biotechnological production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) derived from organic waste streams by mixed microbial communities is well established at the pilot-level. However, there is limited research on the recovery of the biopolymer from the microbial biomass, while its impact on product quality and product costs is major. When applying solvent extraction, the choice of solvent has a profound influence on many aspects of the process design. This study provides a framework to perform a systematic solvent screening for PHBV extraction. First, a database was constructed of 35 solvents that were assessed according to six different selection criteria. Then, six solvents were chosen for further experimental analysis, including 1-butanol, 2-butanol, 2-ethyl hexanol (2-EH), dimethyl carbonate (DMC), methyl isobutyl ketone (MIBK), and acetone. The main findings are that the extractions with acetone and DMC obtained the highest yields (91-95%) with reasonably high purities (93-96%), where acetone had a key advantage of the possibility to use water as anti-solvent. Moreover, the results provided new insights in the mechanisms behind PHBV extraction by pointing out that at elevated temperatures the extraction efficiency is less determined by the solvent's solubility parameters and more determined by the solvent size. Although case-specific factors play a role in the final solvent choice, we believe that this study provides a general strategy for the solvent selection process.

Municipal activated sludge can be used for polyhydroxyalkanoate (PHA) production, when supplied with volatile fatty acids. In this work, standardized PHA accumulation assays were performed with different activated sludge to determine (1) the maximum biomass PHA content, (2) the degree of enrichment (or volume-to-volume ratio of PHA-accumulating bacteria with respect to the total biomass), and (3) the average PHA content in the PHA-storing biomass fraction. The maximum attained biomass PHA content with different activated sludge ranged from 0.18 to 0.42 gPHA/gVSS, and the degree of enrichment ranged from 0.16 to 0.51 volume/volume. The average PHA content within the PHA-accumulating biomass fraction was relatively constant and independent of activated sludge source, with an average value of 0.58 ± 0.07 gPHA/gVSS. The degree of enrichment for PHA-accumulating bacteria was identified as the key factor to maximize PHA content when municipal activated sludge is directly used for PHA accumulation. Future optimization should focus on obtaining a higher degree of enrichment of PHA-accumulating biomass, either through selection during wastewater treatment or by selective growth during PHA accumulation. A PHA content in the order of 0.6 g PHA/g VSS is a realistic target to be achieved when using municipal activated sludge for PHA production.

Abstract: Using microbial enrichment cultures for the production of waste-derived polyhydroxyalkanoates (PHAs) is a promising technology to recover secondary resources. Volatile fatty acids (VFAs) form the preferred substrate for PHA production. Isobutyrate is a VFA appearing in multiple waste valorization routes, such as anaerobic fermentation, chain elongation, and microbial electrosynthesis, but has never been assessed individually on its PHA production potential. This research investigates isobutyrate as sole carbon source for a microbial enrichment culture in comparison to its structural isomer butyrate. The results reveal that the enrichment of isobutyrate has a very distinct character regarding microbial community development, PHA productivity, and even PHA composition. Although butyrate is a superior substrate in almost every aspect, this research shows that isobutyrate-rich waste streams have a noteworthy PHA-producing potential. The main finding is that the dominant microorganism, a Comamonas sp., is linked to the production of a unique PHA family member, poly(3-hydroxyisobutyrate) (PHiB), up to 37% of the cell dry weight. This is the first scientific report identifying microbial PHiB production, demonstrating that mixed microbial communities can be a powerful tool for discovery of new metabolic pathways and new types of polymers. Key points: • PHiB production is a successful storage strategy in an isobutyrate-fed SBR • Isomers isobutyrate and butyrate reveal a very distinct PHA production behavior • Enrichments can be a tool for discovery of new metabolic pathways and polymers Graphical abstract: [Figure not available: see fulltext.].

The developments of mixed culture polyhydroxyalkanoate production has been directed to maximize the biomass PHA content with limited attention to polymer quality. Direct comparison of PHA accumulation literature is challenging, and even regularly contradicting in reported results, due to underlying differences that are not well expressed. A study was undertaken to systematically compare the commonly reported process conditions for PHA accumulation by full-scale municipal activated sludge. A biomass acclimation step combined with a pulse-wise feeding strategy resulted in maximum average PHA contents and product yields. pH control and active nitrification did not result in observable effects on the PHA productivity. Under these conditions a high molecular weight polymer (1536 ± 221 kDa) can be produced. Polymer extraction recoveries were influenced by the PHA molecular weight. A standard protocol for an activated sludge PHA accumulation test including downstream processing and standardized extraction has been developed and is available as supplementary material.

Polyhydroxyalkanoate accumulation experiments at pilot scale were performed with fullscale municipal waste activated sludge. Development of biomass PHA content was quantified by thermogravimetric analysis. Over 48 h the biomass reached up to 0.49 ± 0.03 gPHA/gVSS (n=4). Samples were processed in parallel to characterise the distribution of PHA in the biomass. Selective staining methods and image analysis were performed by Confocal Laser Scanning Microscopy. The image analysis indicated that nominally 55% of this waste activated sludge was engaged in PHA storage activity. Thus even if the biomass PHA content reached 0.49gPHA/gVSS, the accumulating fraction of the biomass was estimated to have attained about 0.64gPHA/gVSS. The combination of quantitative microscopy and polymer mass assessment enabled to distinguish the effect of level of enrichment in PHA storing bacteria and the average PHA storage capacity of the accumulating bacteria. The distribution of microbial 16S rRNA levels did not follow a measurable trend during PHA accumulation.

Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by using microbial enrichments is a promising but largely unexplored approach to obtain elastomeric biomaterials from secondary resources. In this study, several enrichment strategies were tested to select a community with a high mcl-PHA storage capacity when feeding octanoate. On the basis of analysis of the metabolic pathways, the hypothesis was formulated that mcl-PHA production is more favorable under oxygen-limited conditions than short-chain-length PHA (scl-PHA). This hypothesis was confirmed by bioreactor experiments showing that oxygen limitation during the PHA accumulation experiments resulted in a higher fraction of mcl-PHA over scl-PHA (i.e., a PHA content of 76 wt% with an mcl fraction of 0.79 with oxygen limitation, compared to a PHA content of 72 wt% with an mcl-fraction of 0.62 without oxygen limitation). Physicochemical analysis revealed that the extracted PHA could be separated efficiently into a hydroxybutyrate-rich fraction with a higher M_w and a hydroxyhexanoate/hydroxyoctanoate-rich fraction with a lower M_w. The ratio between the two fractions could be adjusted by changing the environmental conditions, such as oxygen availability and pH. Almost all enrichments were dominated by Sphaerotilus sp. This is the first scientific report that links this genus to mcl-PHA production, demonstrating that microbial enrichments can be a powerful tool to explore mcl-PHA biodiversity and to discover novel industrially relevant strains.

Activated sludge from municipal wastewater treatment processes can be used directly for the production of biodegradable polyesters from the family of polyhydroxyalkanoates (PHAs). However, municipal activated sludge typically cannot accumulate PHAs to very high levels and often low yields of polymer produced on substrate are observed. In the present work, it was found that the presence of calcium promotes selective growth and enrichment of the PHA-storing biomass fraction and significantly improved both PHA contents and yields. Calcium addition resulted in PHA contents of 0.60 ± 0.03 gPHA/gVSS and average PHA yields on substrate of 0.49 ± 0.03 gCOD_PHA/gCOD_HAc compared to 0.35 ± 0.01 gPHA/gVSS and 0.19 ± 0.01 gCOD_PHA/gCOD_HAc without calcium addition. After 48 h, three times more PHA was produced compared to control experiments without calcium addition. Higher PHA content and selective biomass production is proposed to be a consequence of calcium dependent increased levels of passive acetate uptake. Such more efficient substrate uptake could be related to a formation of calcium acetate complexes. Findings lead to bioprocess methods to stimulate a short-term selective growth of PHA-storing microorganisms and this enables improvements to the techno-economic feasibility for municipal waste activated sludge to become a generic resource for industrial scale PHA production.

Lactate production in anaerobic carbohydrate fermentations with mixed cultures of microorganisms is generally observed only in very specific conditions: the reactor should be run discontinuously and peptides and B vitamins must be present in the culture medium as lactic acid bacteria (LAB) are typically auxotrophic for amino acids. State-of-the-art anaerobic fermentation models assume that microorganisms optimise the adenosine triphosphate (ATP) yield on substrate and therefore they do not predict the less ATP efficient lactate production, which limits their application for designing lactate production in mixed-culture fermentations. In this study, a metabolic model taking into account cellular resource allocation and limitation is proposed to predict and analyse under which conditions lactate production from glucose can be beneficial for microorganisms. The model uses a flux balances analysis approach incorporating additional constraints from the resource allocation theory and simulates glucose fermentation in a continuous reactor. This approach predicts lactate production is predicted at high dilution rates, provided that amino acids are in the culture medium. In minimal medium and lower dilution rates, mostly butyrate and no lactate is predicted. Auxotrophy for amino acids of LAB is identified to provide a competitive advantage in rich media because less resources need to be allocated for anabolic machinery and higher specific growth rates can be achieved. The Matlab™ codes required for performing the simulations presented in this study are available at https://doi.org/10.5281/zenodo.4031144.

The cyclic nature of specific conversions in the nitrogen cycle imposes strict limitations to the conversions observed in nature and explains for example why anaerobic ammonium oxidation (anammox) bacteria can only use nitrite – and not nitrate – as electron acceptor in catabolism, and why nitrite is required as additional electron donor for inorganic carbon fixation in anabolism. Furthermore, the biochemistry involved in nitrite-dependent anaerobic methane oxidation excludes the feasibility of using nitrate as electron acceptor. Based on the cyclic nature of these nitrogen conversions, we propose two scenarios that may explain the ecological role of recently discovered complete ammonia-oxidizing (comammox) Nitrospira spp., some of which were initially found in a strongly oxygen limited environment: (i) comammox Nitrospira spp. may actually catalyze an anammox-like metabolism using a biochemistry similar to intra-oxic nitrite-dependent methane oxidation, or (ii) scavenge all available oxygen for ammonia activation and use nitrate as terminal electron acceptor. Both scenarios require the presence of the biochemical machinery for ammonia oxidation to nitrate, potentially explaining a specific ecological niche for the occurrence of comammox bacteria in nature.

Natural microbial communities are composed of a large diversity of interacting microorganisms, each with a specific role in the functional properties of the ecosystem. The objectives in microbial ecology research are related to identifying, understanding and exploring the role of these different microorganisms. Because of the rapidly increasing power of DNA sequencing and the rapid increase of genomic data, main attention of microbial ecology research shifted from cultivation-oriented studies towards metagenomic studies. Despite these efforts, the direct link between the molecular properties and the measurable changes in the functional performance of the ecosystem is often poorly documented. A quantitative understanding of functional properties in relation to the molecular changes requires effective integration, standardization, and parallelization of experiments. High-resolution functional characterization is a prerequisite for interpretation of changes in metagenomic properties, and will improve our understanding of microbial communities and facilitate their exploration for health and circular economy related objectives.