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.