Of all the greenhouse gases (GHGs), carbon dioxide (CO2) has been the target of most climate recovery efforts as it is the most abundantly emitted GHG by mass. In fact, in 2015 a legally binding international treaty was adopted by 196 parties in Paris, France to constrain the anthropogenic warming to 1.5-2.0˚C above the pre-industrial level. In order to meet this goal, a carbon budget was formulated as an estimate of the amount of carbon that can be emitted while limiting the anthropogenic warming to prescribed levels. However, the global CO2 emissions from industries are rapidly depleting this budget. Therefore, to mitigate the effects of climate change, CO2 emissions must be reduced by employing alternative commodities that can replace petrochemical resources. In this context, mixed culture fermentation presents an opportunity for redefining CO2 and waste streams as raw material for production of commodities traditionally derived from petrochemical resources. Previous studies by on this topic have indicated a potential association between elevated CO2 levels (pCO2) and butyrate formation from mixed culture fermentation. However, the cellular mechanism underlying this association are still poorly understood. Therefore, the principal objective of this research was to investigate the effects of initial substrate concentrations (g/L) and elevated pCO2 (bar) conditions on selectivity (moli/moltotal) of biomolecules produced from anaerobic conversion of glucose. For this purpose, a between-subject mixed factorial experimental design was developed to gauge the main and interaction effects of initial substrate concentrations (g/L) and elevated pCO2 (bar) conditions on selectivity of biomolecules. The principal findings of this research indicate that a strong positive relationship exists between the pCO2 and butyrate formation as the application of CO2 in reactor (EPBs) headspace resulted in higher butyrate selectivity compared to the control reactors (APBs). However, contrary to the conclusions reached by previous studies it was found that increasing the initial substrate concentration steered the product formation towards lactate and not butyrate. Whereas the highest recorded butyrate selectivity for EPBs was 30.41% for experimental condition with 5 g/L substrate concentration and 4 bar pCO2, the highest recorded butyrate selectivity for APBs was only 11.72% for 10 g/L substrate concentration and atmospheric pressure conditions. Conversely, the highest recorded lactate selectivity for EPBs was 15.13% for 20 g/L substrate and 3 bar pCO2 while the highest recorded lactate selectivity for APBs was 47.95% for 25 g/L substrate concentration and atmospheric pressure conditions. As a result of these investigations, theories concerning formation of butyrate and lactate were proffered in context of the role of CO2 in mixed culture fermentation. By confronting the existing understanding regarding product formation with new evidence this investigation seeks to advance theories concerning mixed culture fermentation.

Pulp and paper industries are water-intensive industries and composed of complex production processes. Untreated pulping wastewater is very toxic and lethal to aquatic life if discharged untreated. Anaerobic treatment technology has gained interest in treating these types of wastewater by reducing organic compounds. This thesis research aimed to evaluate the potential toxicity which might present in chemithermomechanical pulp (CTMP) wastewater, through the biological performance of a lab-scale expanded granular sludge bed (EGSB) reactor.
In the theoretical part CTMP process, characteristics of wastewater, toxicants, the functionality of an EGSB reactor, and any limiting factor that influences the treatment were investigated. In the experimental part, CTMP wastewaters from a mill in Sweden were analyzed using a lab-scale EGSB reactor for 182 days. Anaerobic biodegradability and toxicity test were done to measure the extent of anaerobic digestion in this wastewater. Different parameters such as chemical oxygen demand (COD) removal efficiency, volatile fatty acids (VFA), alkalinity, pH, and nutrient uptake were measured to access the biological performance of the EGSB reactor starting from unacclimated anaerobic granular biomass. Data analyzing tools including Excel and PHREEQC modeling were used in this research study. Excel tool was used to plot the graph and curve fitting whereas the PHREEQC model was done to understand the corrosivity of the biogas and calcite precipitation in the effluent discharge pipe.
The results showed that about 60% of the organic compounds in the wastewater were biodegradable, and no significant toxicity was found. Also, the performance of the EGSB was good with COD removal of roughly 50% at the stable phase. The presence of wood cellulose fiber in the wastewater had a negative impact on the performance of the reactor more specifically blockage in the recirculation and reduce methane production. However, based on the experimental results, EGSB alone would not be enough to remove most of the organic pollutants, which require additional post treatment such as aerobic system and membrane filtration to meet the discharge limit.

With increasing pressure on natural water resources, wastewater is gradually being considered as a potential source for potable water. Current WWTPs are designed for the removal of parameters like solids, nutrients, organic matter, and pathogens. For achieving a high-quality effluent, that enables reuse, it is important to also address the removal of micropollutants, particularly antibiotics, from the wastewater. Due to these antibiotics, antibiotic resistance spreads among the microorganisms and increases through various mechanisms. Antibiotics of sulfamethoxazole (SMX), trimethoprim (TMP), ciprofloxacin (CIP), and ampicillin (AMP) are known to be found abundantly in natural waters all across the globe. The abilities of an anaerobic membrane bioreactor (AnMBR) of maintaining high SRTs with low biomass losses help in treating wastewater containing antibiotics. A recently developed technique of adding limited aeration to AnMBR has the potential of removing recalcitrant antibiotics by improving the performance of the reactor. Hence, this research aims to study the removal mechanisms of the antibiotics (SMX, TMP) and the persistence of corresponding antibiotic resistance in AnMBR, followed by the effect of the antibiotics on the performance of the AnMBR. In addition, antibiotics CIP and AMP were tested via anaerobic batch tests to investigate the effect of the limited aeration on their removal.
After adding the antibiotics SMX and TMP to the reactor, no significant difference in COD and nutrients removal was observed. The biogas production was reduced slightly after the addition of SMX 150 µg/L initially, however, it increased back to the original state after few days. Total removal of SMX and TMP was 86% and 97% respectively in the reactor. Results showed that 85% of SMX and 94% of TMP were removed through biodegradation/biotransformation and 14% of SMX and only 3% of TMP were discharged through the effluent. From the adsorption batch tests conducted, it was observed that the linear adsorption isotherm fits well for TMP. With the increase in temperature, the adsorption potential of TMP was reduced with a Kd value of 1.234 L/g at 10˚C and 0.513 L/g at 37˚C. The removal of SMX was low through adsorption and high due to degradation and follows the first-order rate kinetics with a half-life of 1.71 days. After two weeks of SMX addition to the reactor, almost all the bacteria present in the effluent gained resistance either to TMP or SMX or both. Of all the ARGs measured in this study, the genes responsible for the resistance development were sul1 and sul2. The addition of antibiotics increased the presence of ARGs in the system. The correlation between the presence of sul1 and TMP resistant bacteria, and sul1 and SMX resistant bacteria was 0.91-0.93, indicating that the gene sul1 might be involved in multidrug resistance. ARGs sul1, sul2, and dfrA1 were removed respectively by 3.2 log, 3.6 log, and 7.3 log units by the membrane. In addition, the class 1 integrons and 16s rRNA were removed by 3 log and 3.2 log units respectively. Removal of CIP and AMP was found to be high with values of 82% and 84% respectively in limited aeration assisted anaerobic batch tests. The removal efficiencies of all antibiotics were more than 80% and independent of their initial concentrations in the selected range. The increase in the removal of CIP and AMP in comparison to literature points to a relation with the added limited aeration. Nevertheless, more studies need to be performed to establish this.

Medium chain length polyhydroxyalkanoate (mcl-PHA) is a form of polyhydroxyalkanoate (PHA) which has properties similar to rubber. However, most of the research on mcl-PHA has been focused on pure cultures with artificial substrates, both of which increase the cost of the production to economically unviable levels. This is especially the case since the biological and the chemical production are very cheap. To reduce the costs, a mixed culture could be used. By investigating which conditions lead to high mcl-PHA production with a mixed culture, a step can be taken towards an economically viable biodegradable rubber replacement. To develop a culture which was able to produce mcl-PHA, an enrichment was first performed in a sequential batch reactor with a feast-famine regime to select for PHA producers. Additional selection pressures like substrate selection, pH, oxygen flow rate and time between carbon source addition and nitrogen source addition were imposed on the culture. After the culture reached pseudo-steady state, an accumulation was done. The substrate was one of the most important selection pressures. Using octanoate as sole carbon and energy source, the microbial community was successfully enriched in a feast-famine regime with the capacity to produce mcl-PHA. The use of octanoate with a pH of 7 and no reduced oxygen flow resulted in mcl-PHA weight percentages of 27 wt% polyhydroxyoctanoate (PHO) and 5 wt% polyhydroxyhexanoate (PHH) during the cycle. This was raised to 30 wt% PHO and 10 wt% PHH during the accumulation. During the second enrichment, the carbon source, and the oxygen flow of the first enrichment were maintained, but the pH was increased to 8. This led to an increase in mcl-PHA production, to 33 wt% PHO and 7 wt% PHH during the cycle and 44 wt% PHO and 9 wt% PHH during the accumulation. Two other condition changes were also investigated: lowered oxygen flow and uncoupling the system. For the lowered oxygen flow, the oxygen flow rate was reduced to 5% of the original flow rate and the increased pH of 8 of the second enrichment was kept. This resulted in similar values as the enrichment with only increased pH. It is therefore unclear whether the lowered oxygen flow rate could be attributed to the high mcl-PHA production or that the culture performed well despite the lowered oxygen flow. The uncoupled system was a system where the nitrogen source was added 2 hours after addition of the carbon source. Based on the oxygen profile and preliminary results of the enrichment, the culture mainly grew after the addition of carbon and little mcl-PHA was produced. However, scl-PHA production was quite high, with a total of 51 wt% PHB. This work proves that high levels of mcl-PHA could be produced from mixed cultures. This is a good step towards producing an economically viable, biodegradable rubber replacement.

Comammox bacteria are capable of catalysing the full nitrification pathway – oxidation of ammonium to nitrate – and have been encountered in many ecosystems recently (Lawson & Lücker, 2018). What the ecological role of comammox bacteria is in hypoxic enrichment cultures remains unclear. Based on the thermodynamics and biochemistry of known nitrogen cycle conversion, we propose that comammox is oxidizing ammonium to nitrite with both oxygen and nitrate as electron acceptor in hypoxic condition. Our hypothesis suggests that when comammox cooperates with anammox, they can harvest most energy per unit of oxygen supplied. We tried to cultivate bacteria toward a community of anammox and comammox consortium by limiting the oxygen and supplying ammonium and nitrate. Although the predicted optimal state has not been achieved during this work, we did observe that the community indeed developed towards higher consumption of ammonium under limited oxygen supply.

Biofouling and scaling are ongoing challenges for reverse osmosis (RO) membranes application in wastewater reclamation. Adequate RO feed pretreatment is necessary for biofouling and scaling control. The objective of this thesis was evaluated the effectiveness of ion exchange treatment with weak acid cation (WAC) and strong base anion (SBA) resins columns, in series, after ultrafiltration (UF) treatment for the pretreatment of municipal wastewater treatment plant effluent in order to be used for RO desalination. Specifically, the performance of two SBA resins, the Amberlite SCAV4 Cl (SCAV4) and the Amberlite IRA458 Cl (IRA458) was assessed at three regeneration levels (120, 100 and 80 g NaCl/Lr). The effect of the different regeneration levels on anions removal (sulfate, phosphate and nitrate), total organic carbon (TOC) removal and the operational exchange capacity was researched. Moreover, the subsequent effect on RO biofouling and scaling potential was investigated based on the product water quality of the two SBA resins with bio-growth potential tests and software tests (WAVE design, PHREEQC 3 and Avista Ci), respectively. Phosphate and sulfate removal was above 97% for both resins at all regeneration levels. TOC removal of about 70% was achieved in all cases. It was also observed that sulfate, phosphate and TOC removal remained the same up to the point where product nitrate concentration reached its feed concentration. The removal of nitrate was influenced by the regeneration level. For both SBA resins, a lower regeneration level caused a higher nitrate baseline leakage in product water, hence to a lower removal. The macroporous SCAV4 found to have higher selectivity towards nitrate compared to the gel IRA458. Nevertheless, the anion selectivity order for both SCAV4 and IRA458 was HCO3- < NO3- < HPO42- < SO42-. A minor increase in the operational exchange capacity for both SBA resins was observed at higher regeneration levels. Overall, the influence of the different regeneration levels was found to be limited towards the product water quality and the operational exchange capacity. Also, both SBA resins resulted to similar product water quality. The operational exchange capacity of IRA458 at each regeneration level was higher than that of SCAV4, due to the former’s greater total exchange capacity. The RO feed water quality produced without ion exchange pretreatment (only UF) supported bacterial growth up to 54 ± 1.5 × 106 cells/mL. The RO feed water qualities produced with ion exchange pretreatment in the cases of SCAV4 and IRA458 supported bacterial growth up to 3.4 ± 0.3 × 106 cells/mL and to 1.25 ± 0.2 × 106 cells/mL, respectively. The resulted reduction in the bacterial growth potential was above 90% after ion exchange treatment with either one of the tested SBA resins. The growth-limiting nutrient for the RO feed water qualities produced by either SBA resin was phosphorus. The bacterial growth supported by the two RO feed water qualities produced with ion exchange pretreatment after extra phosphorus addition was 80% lower than that supported by the RO feed water quality produced without ion exchange. This difference suggests that both SBA resins removed a considerable fraction of assimilable organic carbon (AOC). It was concluded that ion exchange pretreatment with either one of the tested SBA resins resulted in nutrients removal (P and C) in the RO feed that lowers the biofouling potential compared to RO feed pretreatment with only UF. The software results suggest lower scaling potential for several scalant types in the RO feed after ion exchange treatment with either one of the tested SBA resins. Among them was calcium phosphate, which was found to have high scaling potential in RO feed without ion exchange treatment (only UF) either with or without anti-scalant dosing. However, the scaling potential of some silica and iron based minerals was high, thus anti-scalant dosing might be required.

Aniline, a toxic aromatic amine present in certain wastewaters from the petroleum-, pharmaceutical- , and textile industry is regarded recalcitrant under strict anaerobic conditions. This study assessed the feasibility of methanogenic aniline biodegradation under saline (8 gNa+/L) conditions (1) by performing biodegradability batch assays using biomass from three different origins, and (2) by treating aniline-containing synthetic wastewater in a continuous anaerobic membrane bioreactor (AnMBR), seeded with granular sludge coming from an up-flow anaerobic sludge blanket (UASB) reactor treating petrochemical wastewater. In addition, the inhibitory effect of aniline and phenol on the aceticlastic methanogenesis, as well as the toxic effect on the integrity of cell membranes of the anaerobic biomass were assessed. Methanogenic biodegradation of aniline was not observed in the AnMBR, nor in the biodegradability assays. However, the results from the AnMBR operation fed with synthetic wastewater (20-200 mg aniline/L) demonstrated a 10-20% aniline removal, which was mainly attributed to volatilisation of aniline. Results from specific methanogenic activity (SMA)-inhibition tests demonstrated a half maximal inhibitory concentration (IC50) of aniline for the aceticlastic methanogenesis of 2.5 g aniline/L. The IC50 of phenol for the aceticlastic methanogenesis was 1.0 g phenol/L. The cell membrane integrity (CMI) of the anaerobic biomass was not significantly affected after 72 hours of exposure to 4 g aniline/L or 2 g phenol/L. This research constituted the first report demonstrating the application of an AnMBR with the aim to biodegrade aniline-containing synthetic wastewater under methanogenic saline conditions. The results of this research demonstrated that, after 200 days of AnMBR operation, the methanogenic enrichment culture was not able to biodegrade aniline.

Large nitrogen emissions and depositions in countries like The Netherlands have had a negative impact on human health and natural ecosystems. Nitrogen ends up in water bodies where it causes eutrophication, which can lead to a decrease in biodiversity. Nowadays, it is removed from water at wastewater treatment plants (WWTPs) via denitrification – nitrification or the anammox process. In both of these processes the end product is N2 gas, which is again emitted into the atmosphere. In recent years, however, nitrogen in the form of ammonia (NH3) has received increased attention as a valuable resource and can be found in different types of wastewaters. To produce energy from ammonia, solid oxide fuel cells (SOFCs) are used, to which ammonia should be fed in its gaseous form. For the production of ammonia from wastewater, a low carbon-to-nitrogen ratio is desired. The overall goal of the research project is to identify nitrogen-rich wastewaters from the industry and apply a series of treatment steps for the production of ammonia. Protein-rich wastewaters are especially suitable for the production of ammonia. Effluents fitting these criteria have been identified, so far, in several industrial settings, namely the slaughterhouse, food and dairy industry. Proteins can be converted to ammonia through anaerobic digestion, while producing biogas in the form of carbon dioxide (CO2) and methane (CH4), which can also be used for energy purposes. Next to proteins, carbohydrates and volatile fatty acids (VFAs) also make part of protein-rich wastewater. However, not a lot is known about the co-digestion of proteins and these simple carbon sources. Some aspects have been investigated, but thorough research on the whole degradation is needed to fully understand the process. The master thesis research presented in this report focused on the anaerobic degradability of proteins in the presence of sugars and VFAs. Three proteins (bovine serum albumin [BSA], casein and gelatin) were selected from the identified industries and assessed based on degradation efficiency and kinetic rates, under mesophilic batch test conditions. Furthermore, the conversion of protein to ammonia was assessed and parameters to define a biological ammonia potential (BAP) are defined. An increase in the degradation coefficient after the co-digestion with sugar was observed for gelatin (1.1 to 1.6 d-1) and BSA (0.57 to 0.68 d-1). Pure proteins were degraded efficiently with 71 - 96% compared to 76 - 97% of the co-digested batches. A combination of conversion efficiencies and the newly introduced biological ammonia potential gave a good indication on the conversion of protein to NH+4 . Acidification with VFAs resulted in process instabilities with respect to the methane production rate, which was due to the applied ratio of propionate to acetate. To prevent this in future trials, amino acid analysis to predict the production of VFAs from protein was evaluated with a maximum deviation of 14% of measured to theoretical values. This study demonstrated the efficiency of co-digestion to degrade protein-rich substrates. Further research is required to unveil the impact of high ammonia concentrations and explore the microbiological aspect of protein fermentation. Finally, considerations for a test to assess the biological ammonia potential are given.

Anaerobic digestion (AD) is a promising technology to process protein-rich wastewater. However, incomplete protein degradation during the acidification of organic matter is frequently observed when carbohydrates are present. Literature reports little information to explain the mechanisms behind this phenomenon. This MSc study investigated the relationship between microbial composition and the negative effect of carbohydrates on anaerobic protein degradation in the acid-phase by restricting the carbon source to a mixed culture in a continuously stirred tank anaerobic reactor. Two continuously stirred anaerobic reactors fed with restrictive protein and/or carbohydrate substrates were operated and compared in the aspects of acidification, deamination, protease activity, and microbial composition. Results showed that the deamination degree in the protein-feeding reactor decreased from 77% to 15% and the acidification degree decreased from 75% to 34% when the restrictive carbon source shifted from proteins to mixtures of proteins and carbohydrates. A decrease in protease activity was also observed. Anaerobic protein degradation was significantly retarded by the presence of carbohydrates. Results of the microbial composition analysis showed that generalists (i.e. microorganisms that can ferment both proteins and carbohydrates) that preferentially consumed carbohydrates were the predominant populations in the microbial community when carbohydrates were present as additional substrates. Therefore, the observed negative effect of carbohydrates on protein degradation in acid-phase could be mainly attributed to the preferential substrate utilization feature of generalists. Further research on the metabolism functional analysis of the microbial community should be employed to confirm this hypothesis. Overall, this study offers a better understanding of the mechanisms behind the negative effect of carbohydrates on protein degradation, which can provide some hints of the design of the anaerobic digestion process of protein-rich wastewater.

This study aims to look at the effect of introducing controlled amounts of oxygen on biogas quantity and quality during anaerobic digestion. The anaerobic digestion process is studied, specifically, it’s biochemical processes, to understand the effect of addition of oxygen. Anaerobic sludge is aerated to mimic DAF (Dissolved Air Flotation) conditions, where tap water is pressurized (at 3 and 5 bar) and then depressurized in contact with the anaerobic sludge in a column reactor. During the course of this process, air micro-bubbles which were dissolved under high pressure are released due to contact with atmospheric conditions. To estimate and compare the methane production of the originally collected non-aerated sludge (anaerobic sludge not aerated in the column reactor, therefore, considered as 0 bar) and aerated sludge (anaerobic sludge subjected to high-pressure micro-air bubbles in the column reactor at 3 and 5 bar) a BMP test is conducted. Methane production was found to be lower in the aerated sludge with the BMP value for the 0 bar sludge being 296.17 +/- 45.15 NLCH4kg 1 and the value for 5 bar aerated sludge being 252.26 +/- 16.8 NLCH4 kg 1. Biogas composition of the aerated sludge was also examined with a Gas Chromatography (GC) machine and the percentage of methane, carbon dioxide and oxygen were measured for the 3 and 5 bar aerated sludge. For the 5 bar
aerated sludge, the overall percentages are averaged at 30%, 70%, and 1% respectively and for the 3 bar aerated sludge the average values are 20%, 80%, and 1% respectively. Furthermore, particle size distribution (PSD) analysis was done to compare variations for particle sizes between the aerated (5 bar) and non-aerated (0 bar) sludge. Very low variation was observed between these samples with the average size of the aerated samples being marginally smaller than the non-aerated sludge, indicating poor separation efficiency for the separation method adopted.