Abstract: Presence of carbohydrates hampers protein degradation in anaerobic digesters. To understand this phenomenon, we used proteogenomics to identify the active protein-degraders in the presence of low and high carbohydrates concentrations. Active metabolic pathways of the identified protein-degraders were investigated using proteomics with ¹³C-protein substrates (protein stable isotope probing). Results showed that 1) Acinetobacter was the active protein-degraders under both protein-fed and protein-glucose mixture-fed conditions, 2) the relative abundance of Acinetobacter was not affected by the presence of carbohydrates, 3) the incorporation of the ¹³C-labelled protein substrate was predominantly observed in outer membrane-bound proteins and porin proteins, which are associated with proteinases or the transportation of amino acids across the cell wall. The Acinetobacter metabolic model and the incubation conditions suggested that glucose and proteins were degraded through anaerobic respiration. The negative impact of carbohydrates on protein biodegradation was attributed to Acinetobacter's preference for carbohydrates. This work highlights that efficient degradation of protein and carbohydrate mixtures in anaerobic digesters requires a staged or time-phased approach and enrichment of active protein-degraders, offering a new direction for process optimization in anaerobic digestion systems.

Bovine serum albumin (BSA) and casein (CAS) were used in batch tests to compare the protein degradation in the presence and absence of carbohydrates and volatile fatty acids (VFAs). The modified Gompertz model was applied to estimate reaction rates. The results showed that deamination was the rate-limiting step, with a rate ranging between 2.7 and 12.7 mgN·h⁻¹. Higher protein structural complexity negatively affected protein hydrolysis, deamination, and methanogenesis by a factor of 1.6–3.8; whereas a higher protein concentration improved the conversion rates. A carbohydrate:protein COD ratio of 1 improved the hydrolysis rate of BSA from 26 mg·h⁻¹ to 45 mg·h⁻¹, and that of CAS from 98 mg·h⁻¹ to 157 mg·h⁻¹; whereas the deamination rate slightly decreased from 2.7 mg N·h⁻¹ to 2.5 mg N·h⁻¹ and from 6.0 mg N·h⁻¹ to 5.6 mg N·h⁻¹. Additionally, an initial VFAs:protein COD ratio of 1 decreased the CAS deamination rate by 17 %.

Treating protein-rich wastewater using cost-effective and simple-structured single-stage reactors presents several challenges. In this study, we applied an anaerobic sequencing batch reactor (AnSBR) to treat protein-rich wastewater from a slaughterhouse. We focused on identifying the key factors influencing the removal of chemical oxygen demand (COD) and the settling performance of the sludge. The AnSBR achieved a maximum total COD removal of 90%, a protein degradation efficiency exceeding 80%, and a COD to methane conversion efficiency of over 70% at organic loading rates of up to 6.2 g COD L⁻¹ d⁻¹. We found that the variations in both the organic loading rate within the reactor and the hydraulic retention time in the buffer tank had a significant effect on COD removal. The hydraulic retention time in the buffer tank and the reactor, which determined the ammonification efficiencies and the residual carbohydrate concentrations in the reactor liquid, affected the sludge settleability. Furthermore, the genus Clostridium sensu stricto 1, known as protein- and lipids-degraders, was predominant in the reactor. Statistical analysis showed a significant correlation between the core microbiome and ammonification efficiency, highlighting the importance of protein degradation as the governing process in the treatment. Our results will provide valuable insights to optimise the design and operation of AnSBR for efficient treatment of protein-rich wastewater.

Abstract: Protein-rich agro-industrial waste streams are high in organic load and represent a major environmental problem. Anaerobic digestion is an established technology to treat these streams; however, retardation of protein degradation is frequently observed when carbohydrates are present. This study investigated the mechanism of the retardation by manipulating the carbon source fed to a complex anaerobic microbiota and linking the reactor performance to the variation of the microbial community. Two anaerobic acidification reactors were first acclimated either to casein (CAS reactor) or lactose (LAC reactor), and then fed with mixtures of casein and lactose. Results showed that when lactose was present, the microbial community acclimated to casein shifted from mainly Chloroflexi to Proteobacteria and Firmicutes, the degree of deamination in the CAS reactor decreased from 77 to 15%, and the VFA production decreased from 75 to 34% of the effluent COD. A decrease of 75% in protease activity and 90% in deamination activity of the microbiota was also observed. The microorganisms that can ferment both proteins and carbohydrates were predominant in the microbial community, and from a thermodynamical point of view, they consumed carbohydrates prior to proteins. The frequently observed negative effect of carbohydrates on protein degradation can be mainly attributed to the substrate preference of these populations. Keypoints: • The presence of lactose shifted the microbial community and retarded anaerobic protein degradation. • Facultative genera were dominant in the presence and absence of lactose. • Substrate-preference caused retardation of anaerobic protein degradation.

Total ammoniacal nitrogen (TAN) is considered to be a pollutant, but is also a versatile resource. This review presents an overview of the TAN recovery potentials from nitrogen (N)-loaded residual streams by discussing the sources, recovery technologies and potential applications. The first section of the review addresses the fate of TAN after its production. The second section describes the identification and categorisation of N-loaded (≥0.5 g L⁻¹ of reduced N) residual streams based on total suspended solids (TSS), chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), TAN, and TAN/TKN ratio. Category 1 represents streams with a low TAN/TKN ratio (<0.5) that need conversion of organic-N to TAN prior to TAN recovery, for example by anaerobic digestion (AD). Category 2 represents streams with a high TAN/TKN ratio (≥0.5) and high TSS (>1 g L⁻¹) that require a decrease of the TSS prior to TAN recovery, whereas category 3 represents streams with a high TAN/TKN ratio (≥0.5) and low TSS (≤1 g L⁻¹) that are suitable for direct TAN recovery. The third section focuses on the key processes and limitations of AD, which is identified as a suitable technology to increase the TAN/TKN ratio by converting organic-N to TAN. In the fourth section, TAN recovery technologies are evaluated in terms of the feed composition tolerance, the required inputs (energy, chemicals, etc.) and obtained outputs of TAN (chemical form, concentration, etc.). Finally, in the fifth section, the use of recovered TAN for three major potential applications (fertilizer, fuel, and resource for chemical and biochemical processes) is discussed. This review presents an overview of possible TAN recovery strategies based on the available technologies, but the choice of the recovery strategy shall ultimately depend on the product characteristics required by the application. The major challenges identified in this review are the lack of information on enhancing the conversion of organic-N into TAN by AD, the difficulties in comparing the performance and required input of the recovery technologies, and the deficiency of information on the required concentration and quality of the final TAN products for reuse.