Plastic is completely immersed in our society, but due to its numerous negative effects on the planet and human health, bio-based alternatives are in high demand. Polyhydroxyalkanoates (PHAs) are bio polymers synthesized by microorganisms as a carbon and electron reserve under unbalanced growth. PHAs have similar properties to conventional plastics, but their biocompatibility and biodegradability give them an improved environmental footprint. In order to achieve cost-effective PHA production, sequencing batch reactor (SBR) enrichment strategies utilizing mixed microbial cultures (MMC) fed with waste streams are of particular interest. A promising strategy in MMC PHA production is the nutrient decoupled feeding strategy, with with high PHA accumulating cultures can be obtained in a single-step process. This work further characterized the metabolic behavior and PHA storage per formance of C/N decoupled enrichment cultures in five SBRs operated at varying carbon-to-nitrogen (C:N) and exchange ratios (ER). Stable and reproducible PHA accumulation of 59 wt% was achieved at a C:N of 40 molC/molN and a ER of 50%. Increasing the ER to 75% led to the highest achieved PHA content of this study, 77 wt%. The experimental performance was, however, substantially de creased compared to expectations based on theoretical and data-driven models. Investigation revealed important process parameters for further optimization. Specifically, the time of harvest, as well as the impact of harvest on the experienced C:N ratios by the culture. The most notable result of this study was the strong indication of the presence of a nitrogen storage metabolism in at least two of the five enrichment cultures. Specifically, the data pointed to the capacity for nitrogen accumulation during the famine phase and subsequent delayed growth in the feast phase. This behavoir was characterized by high nitrogen uptake rates paired with the formation of nitrogen rich, non-PHA organic solids in the famine phase. Furthermore, approximately half of the total dry cell weight (DCW) increase occurred during the nitrogen absent carbon phase. Nitrogen accumulation is predominantly thought to occur in specialized cyanobacteria and has not been described before in PHA enrichment cultures. It is therefore considered a novel metabolic insight. ... Read more

The increasing demands for chemicals and fuels combined with the fuel versus food competition over first generation feedstocks requires the development of more sustainable alternatives. Syngas fermentation offers a sustainable production of fuels and recycling of gaseous and solid waste utilizing gas
fermenting bacteria. Acetogens, such as Clostridium autoethanogenum (CA) can grow on syngas (CO, CO2 and H2) and produce acetate and ethanol via the Wood-Ljungdahl pathway (WLP). Numerous studies have been conducted to optimize the ethanol production by changing different parameters such
as the pH, the mineral medium and the ingas composition. These changes can be effectively predicted through kinetic modelling, which requires the knowledge of key kinetic parameters such as the maximum biomass specific substrate uptake rate and the maximum growth rate. Batch fermentation is not a realistic option considering the low solubility of gases such as CO in the liquid, while obtaining the kinetic parameters in chemostats by increasing the dilution rate can be time intensive. To acquire these parameters, CA was cultivated in chemostats and was grown on carbon monoxide as the sole energy and carbon source. Feeding disturbances were carried out by increasing the CO molar fraction in the
inlet gas for a short period of time to ensure constant biomass concentration levels. Continuous off-gas analysis revealed that the fermentation is mass transfer limited until the end of the pulse experiments at 85% of CO in the inlet gas. A biomass specific substrate uptake rate of 89.1 ± 0.23 mmol/gDCW /h
was calculated, which is the highest value achieved so far in literature.The calculated growth rate was 0.083 h−1. pH profile provided evidence about the metabolism of the microorganism, while the increase of CO2 to CO yield at higher CO molar fractions was closely related to acetate reduction to ethanol
so that the cells can regulate their metabolism.

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The hydrolysis of sludge solids especially for difficultly degradable sludges such as WAS is not fully understood, yet. The first-order hydrolysis rate was shown to function well for most easily degradable sludges and soluble substrates. This description for substrate hydrolysis in the context of anaerobic digestion has the benefit of being very simple and therefore applicable for many engineering applications where little data is available. On the other hand, in the last decades many studies reported that the first-order hydrolysis would need a modification to better describe the degradation of difficulty degradable solids. Guo et al. (2021) developed a cascade system for anaerobic digestion of WAS that does not seem to follow first-order hydrolysis kinetics when lowering the applied SRTs from 22 to 15 and 12 days, respectively. Based on observations by Guo et al. (2021) and a statistical analysis of the cascade system performed in the study at hand it seems that the first order hydrolysis rate constant is in fact a coefficient and that the first-order hydrolysis rate is not solely dependent on sludge characteristics and substrate concentrations. This hypothesis is tested in the thesis at hand. In Guo et al.’s study the cascade system was always compared to a reference system. To test this hypothesis and understand the kinetics of the cascade system in more detail a statistical analysis was performed for both systems from which an empirical hydrolysis model was derived. This model was implemented in ADM1 to replace the existing hydrolysis rate expression and was tested for the mentioned cascade system and the reference system. The empirical model was compared to the results of the standard ADM1 which uses a first-order hydrolysis expression. The empirical model assumed a dependency of the hydrolysis rate based on load and residence time along the cascade system to achieve a change in hydrolysis rate coefficients along the cascade system. The models were compared based on visual inspection and quantitative analysis of the simulated results. Both models showed low R² values which is likely due to the high level of detail implemented in ADM1 that does not fit to the resolution of the experimental data. However, calculated RMSE values agreed with the standard deviations of the experimental results. Therefore, the overall predictive capability for both models is given. The ADM1 managed to model the reference system with reasonable agreement to the experimental data. The performance of the empirical model for the reference was comparable. For the cascade system however the ADM1 could not fully describe the experimental at the applied low SRTs of 15 and 12 days. The empirical model in this case showed better predictive capabilities. This is an indication that a hydrolysis rate which is made dependent on system characteristics such as load and residence time might indeed have its justification and be better applicable to anaerobic digestion systems that show a concentration profile along the reactor as it is in the case with plug-flow and cascade systems. ... Read more

The first full-scale Kaumera extraction plants are in operation and increases the circularity of WWTP already. However, to reach a goal of zero waste production it is necessary to look into the waste stream of the Kaumera extraction itself. Roughly 30% of organics is extracted in the process and the remaining organics in the waste can be further recovered using anaerobic digestion (AD). In this study the continuation of the alkaline AD was used, instead of neutral digestion. Main reason for the alkaline digestion compared to the neutral digestion is the increase in CH4 content in the biogas as CO2 remains in the liquid at pH 9.6. The batch incubation uses the alkaline waste residuals stream of the Kaumera extraction plant in Epe. The digestion was done at haloalkaline conditions (pH 9.6; 0.6 M Na+). Combinations of inoculum enriched for similar substrates from a previous study and fresh soda lake sediment were used for the AD batch incubations. CH4 yields varied from 8-28% of total COD going into CH4. Compared to literature this is on lower side as these conversions are in the range of 35-50%. However, some incubations were with pre-treated substrate and already enriched incubations. Others were neutral digestion of similar substrate and the substrate used in this study. This does show the potential still for a higher conversion of methane in the alkaline digestion of the Kaumera residuals. Based on a titration, the Alkalinity need to keep the pH at 9.6 is 3 g/L of NaOH to prevent a drop from pH 9.64 to 9.34. The overall process observed takes longer than the neutral digestion due to a delay seen in acetate conversion, therefore no bottle-neck in the process could be defined and only a kinetic problem was identified. This could be tackled by transferring the process toward continuous operation, avoiding the slow growth of syntrophic acetate oxidisers once steady state is achieved. The process was modelled using two different methods, where issues surrounding the pH description arise. For the models it is essential to extend the simple buffer capacity description in order to reliably simulate the pH dynamics of the system. As of now information around the microbial community is scarce making the modelling of the alkaline ADM1 a difficult task. To improve the alkaline ADM1 work should be done to determine kinetics of the microbial community and a better description of the substrate with inoculum. The Dry matter (DM) of the process was 0.88%, which is quite low as in full-scale system usually at least 5% DM is used. The low DM used will lower the chance of bottle-necks in the process, thus with an increase of almost 6 times in DM the inhibition threshold of 420 mgNH4−N will be exceeded. Other implication that need to be solved is the highly saline and high pH waste stream after solids removal. Either this should be recycled back for subsequent digestions or added to the influent of an WWTP assuming it will be diluted enough to not have a major impact anymore. In the future continuous operation should be evaluated as an alternative strategy to prevent the syntrophs from delaying the process and operational parameters like the hydraulic retention time (HRT) and solid retention time (SRT) need to be studied for optimal digestion in such
a system. ... Read more

Sludge management has gained importance over the years due to high sludge treatment and disposal cost, stringent disposal laws, and a need to move towards sustainable energy production. Anaerobic Digestion (AD) of waste activated sludge (WAS) is a favoured sludge stabilisation technique due to its low energy footprint. Hydrolysis – the first step in AD is often the rate-limiting step and thus pre-treatment such as Thermal Hydrolysis Process (THP) are implemented before AD to improve the biodegradability of WAS and biogas production. However, THP leads to the formation of recalcitrant compounds such as melanoidins which are presumed to have a similar effect as Humic substances on AD. Also, THP leads to the release of metals incorporated in sludge flocs by the degradation of extracellular polymeric substances (EPS) structures. Therefore, this study aimed to understand the effect of melanoidins and metals on the hydrolysis step in AD.
The interaction between melanoidins and cations Fe²⁺, Ca²⁺, Mg²⁺, Cu²⁺, K⁺, and NH₄⁺ was studied using ultra-filtration and ICP-MS. The effect of these melanoidins-metals interactions on enzymatic hydrolysis of cellulose and proteins was investigated using fluorescence essay and Response Surface Methodology (RSM) modeling. The mechanism of inhibition of hydrolysis by melanoidins and metal was investigated using SEC-HPLC.
The results showed that complexation of melanoidins with metals shows the trend - Fe²⁺ > Ca²⁺ > Mg²⁺ > Cu²⁺ > K⁺ ≥ NH₄⁺. The ions with higher charge and ionic radius interacted with melanoidins more effectively. The melanoidins-metal interaction increased with an increase in melanoidins concentration most likely due to more phenolic and carboxylic functional groups available for binding. Fe³⁺ leads to an increase in molecular weight (MW) of melanoidins due to inter-molecular interaction caused by complete charge neutralisation, while no MW changes are observed with Cu²⁺ and Ca²⁺. Individually, melanoidins decreased, Fe³⁺ increased, while Cu²⁺ decreased the rate of hydrolysis of cellulose. In the presence of melanoidins, the inhibiting effect of Cu²⁺ is decreased with increasing melanoidins concentration. Similarly, the positive effect of Fe³⁺ is decreased with increasing melanoidins concentration. Protein hydrolysis was completely inhibited with 3 mM metals concentration. Melanoidins and Fe³⁺ concentrations had no major impact on protein hydrolysis while Cu²⁺ inhibits hydrolysis even at 0.15 mM concentration.
Therefore, it was hypothesised that Cu²⁺ causes enzymatic inhibition, but in the presence of melanoidins, melanoidins-Cu²⁺ complexes formation mitigates the toxic effect of Cu²⁺ on hydrolysis thus improving the rate of hydrolysis. While, Fe³⁺ improved hydrolysis by facilitating substrate-enzyme interaction but in presence of melanoidins, melanoidins-Fe³⁺ complexation leads to the non-availability of Fe³⁺ to facilitate hydrolysis process thus having no major impact on the rate of hydrolysis.
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The goal of this experiment has been to characterize storage polymers produced during anaerobic digestion under haloalkaline conditions. The project focused on three research questions: the type of storage polymers produced, which conditions selected for the production of storage polymers and which organisms are responsible for the production of the storage polymers. Based on BODIPY® staining of various batch tests with different conditions, it can be concluded that the produced storage polymer is PHA. The innate fluorescence of the methanogens was used to determine that the methanogens are not the micro-organisms producing and storing the PHA. It is unclear which organisms are
responsible for this. The characterisation and the staining of different digestions and the setup of a new batch test which focused on the influence of trace metals, acetate and toxic compounds were unable to determine the exact conditions which selected for storage polymers. Follow-up research is therefore recommended, for example new digestion series with a new inoculum from the hypersaline soda lakes in Russia instead of the mixture of the sediment from the hypersaline soda lakes and biomass from a previous digestion series. It is also recommended to perform a genomics analysis of the microbial community to find a possible PHA producer. ... Read more

Certain industrial wastewaters have posed a big challenge to biological water treatment systems because of their high toxic organic compounds concentration (e.g. phenol) and high salinity. The maximum biomass specific phenol bioconversion rate (PhCR) of a mesophilic (35 °C) anaerobic membrane bioreactor (AnMBR) under high sodium concentration [18.6 g Na+/L] condition was studied by an increase in the biomass specific phenol loading rate (PhLR) through hydraulic retention time (HRT) decrease. The maximum PhCR achieved in our research was 73 mg Ph-COD/gVSS-COD.d, with acetate as co-substrate [2g AC-COD/L]. This result was lower than that reported by the previous study of Bioxtreme (193 mg Ph-COD/gVSS-COD.d) at lower sodium concentration [8.0 g Na+/L].
On the other hand, a simplified ADM1 model was used to model the conversion of acetate to methane in batch experiments, among which, the inhibition of substrate (acetate or phenol) on microbial growth rate was described by Haldane equation. The kinetic parameters for acetate degradation were Ks,AC=300 mg COD/L, KI,AC=821mg COD/L, km,AC I =0.246 mg COD/mg COD.h without phenol addition and Ks,AC=300 mg COD/L, KI,AC=806 mg COD/L, km,AC I=0.236 mg COD/mgCOD.h with the addition of 714 mg Ph-COD/L (300 mg Ph/L) at sodium concentration of 18.6 g Na+/L, while it was Ks,AC=6.7 10-9 mg COD/L, KI,AC=5670 mg COD/L, km,AC I=0.043 mg COD/mgCOD.h at lower sodium concentration [8.0 g Na+/L] without phenol addition.
The kinetic parameters estimated for the batch experiments were applied in a mathematical model describing a dynamic experiment carried out in the AnMBR1 and validated with different HRTs. In the model, the conversion from phenol to acetate was considered and the kinetic parameters estimated for phenol degradation were Ks,Ph=20 mg COD/L, KI,Ph=300 mg COD/L, km,Ph I=0.008 mg COD/mgCOD.h. It was proved that the simplified ADM1 model could well predict the phenol and acetate concentrations in the reactor at different PhLRs.
This research has provided an experimental and modeling approach for the maximum PhCR determination and could contribute to the understanding of the inhibition effect of sodium and phenol on PhCR in the treatment process of saline phenolic wastewater.
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Certain industrial wastewaters have posed a challenge to water treatment systems because of their composition. The bitumen from the reclaimed asphalt process is heated by the BAM Infra Asfalt and produces fumes, which are usually sent to the air filters and then out through the chimney. To recover the heat lost through the fume, it is condensed. The bitumen fume condensate contains aromatic hydrocarbons of petroleum origin and this poses a threat to health and the environment. The removal of these compounds by anaerobic biodegradation was assessed with a mesophilic anaerobic membrane bioreactor (AnMBR). The bitumen condensate contained over 800 compounds, out of which some were p-cresol, o-cresol and 2-napthalenemethanol. The inhibition to the methanogenic activity and toxicity to the biomass of this wastewater on three different inocula were studied under batch-test conditions. A phenol-degrading sludge was less inhibited (IC₅₀= 870 mg COD_bitumen/L) and more resistant to the toxicity than granular sludge from a petrochemical wastewater treatment plant (IC₅₀= 187 mg COD_bitumen/L) and a municipal sludge (sludge from a municipal wastewater treatment plant, IC₅₀= 127 mg COD_bitumen/L). In continuous operation, the bitumen condensate was degraded efficiently with 89%±12% (S.D)COD removal from the influent of the AnMBR. Maximum organic conversion rate of the bitumen condensate was 26.0 mg COD/g VSS.d. This research demonstrated the efficiency of AnMBR technology to degrade bitumen condensate. Further research must be done to improve the organic conversion rate and optimise the technology for large scale implementation. ... Read more

In the context of steering product formation in anaerobic digestion systems, the present thesis work elaborates on the potential role of elevated CO2 partial pressures as an environmental driver that may influence end-product selectivity from methane towards compounds from the carboxylic platform. As an emerging field of research, organic acid production via mixed culture fermentation is currently in an exploratory phase and the understanding of basic functional principles driving each of the biochemical conversions of interest is of vital importance.The present investigation forms part of a series of studies conjunctively aimed at elucidating the effect of elevated CO2 partial pressures on glucose fermentation, which consists of a complex network of several metabolic routes. Specifically, this thesis work focuses on the effects of CO2 partial pressure on the degradation of two key metabolites that are central and/or highly relevant to the glucose conversion pathways, namely pyruvate and butyrate. ... Read more