Resource recovery through simultaneous denitrification and fermentation in engineered anaerobic systems
Authors:   , Tanvir Rahamat Ullah 
Published: November 2024

[EMBARGOED UNTIL 08/01/2025] Anaerobic digestion (AD) is widely used to process organic waste and is a promising platform for producing bioenergy and biomaterials. However, the final product of the conventional AD process (i.e., methane) has a relatively low economic value; rather, high-value products such as volatile fatty acids (VFAs) and alcohols could be harvested as the final products by selectively arresting methanogenesis. AD is often used to process nitrogen-rich wastewater and organic substrates, such as vegetable waste and crop residues. When high levels of reactive nitrogen are introduced into anaerobic systems, there emerges an intricate relationship between nitrogen transformation and fermentation processes, and such relationships must be well understood to ensure complete denitrification (including both conventional denitrification; DEN and dissimilatory nitrate reduction to ammonium; DNRA), enhance nutrient recovery, avoid greenhouse gas emissions, promote stable digestion, and attain desirable fermentation products (i.e., VFAs). Currently, DEN kinetics data reported in the literature varies significantly, along with many discrepancies. A systematic N2-based assay was developed to evaluate the DEN kinetics of 23 different carbon sources, including C1 - C8 organic acids in salt forms, alcohols, monosaccharides, and disaccharides under anaerobic conditions. Among all the carbon sources tested, acetate had the fastest specific DEN rate (9.86 [plus or minus] 1.00 mg N/g volatile suspended solids (VSS)-h). Formate exhibited slower DEN kinetics (2.82 [plus or minus] 0.18 mg N/g VSS-h) than most carbon sources tested, likely because few microorganisms could directly use this C1 substrate. The use of succinate resulted in incomplete DEN as it inhibited microbial growth. Carbohydrates had relatively slow DEN rates since they were used for both DEN and fermentation simultaneously, where possible metabolic pathways were proposed for producing VFAs such as propionate and valerate in addition to N2 production. Glucose, a monosaccharide, only had a maximum specific DEN rate of 3.78 [plus or minus] 0.09 mg N/g VSS-h. Disaccharides had even slower DEN kinetics (2.75 - 3.43 mg N/g VSS-h) than glucose, indicating that slow hydrolysis of disaccharides might adversely affect DEN. This study also investigated the competitive distribution between DEN and DNRA during simultaneous denitrification and fermentation in arrested methanogenesis. Up to 62 percent of initial NO3^- (200 mg N/L) was retained as ammonium through DNRA at a chemical oxygen demand (COD)/N ratio of 25. Significant nitrous oxide (N2O) emission occurred (1.7 - 8.0 percent of the initial NO3^-) with limited carbon supply ([less than or equal to] 1,600 mg COD/L) and sludge concentration ([less than or equal to] 3,000 mg COD/L). VFA composition shifted predominantly towards acetic acid ([greater than] 50 percent) in the presence of nitrate. For the first time, a kinetic model was developed to predict DNRA vs. DEN partitioning and NO2^- accumulation. Overall, NO3^- input, organic loading, and carbon source characteristics independently and collectively controlled competitive DNRA vs. DEN partitioning. While simultaneous fermentation and denitrification was confirmed from the above study, the short-chain fatty acids from fermentation have limited values. Hence, this study further explored high-value medium-chain fatty acids (MCFAs; C6 - C12) production during a single-stage nitrate-mediated arrested methanogenesis and microbial chain elongation (MCE) using methanol as the electron donor. A high level of nitrate input (250 mg N/L) almost completely arrested methanogenesis with no N2O production and resulted in an acetic acid-dominant (56.2 percent) mixed short-chain fatty acid (SCFA; C1 - C5) pool. When supplemented with methanol (5,000 - 20,000 mg COD/L), caproic acid production from mixed SCFAs increased with increasing methanol loading. The highest caproic acid yield (3.1 g/L) and selectivity (46.7 percent) were achieved with 15,000 mg COD/L methanol and a fixed glucose loading of 5,000 mg COD/L. However, the VFA conversion capacity sharply dropped with higher methanol loadings, e.g., 73.9 and 61.2 percent for 15,000 and 20,000 mg COD/L methanol, respectively. Interestingly, high methanol loading was greatly conducive to enhanced branched-chain caproic acid production and shifted the dominant chain elongation product from butyric acid (C4) to caproic acid (C6). Hence, controlled product tunability could be achieved through systematic methanol supplementation. The production patterns and kinetics of both branched- and straight-chain caproic acid were determined. Metabolic pathways for methanol-based chain elongation to produce caproic acid were proposed. Furthermore, MCFA production from vegetable waste and methanol was investigated during concurrent arrested methanogenesis and MCE in semi-continuous bioreactors. Up to 5.44 g/L caproic acid was produced with a selectivity of 43.8 percent using a 1:1 blend of vegetable waste and glucose as fermentable substrates and methanol as the electron donor. The maximum VFA and caproic acid production rates were 1.04 and 0.43 g COD/L-d, respectively. Internal nitrate supply from the vegetable waste alone inhibited methanogenesis by 79.9 percent. An additional one-time nitrate load (250 mg N/L) was supplied to inhibit methanogenesis completely. No N2O was produced during dissimilatory reduction of internal and externally supplied nitrate. Nonetheless, as the methanol feeding started at a rate of 2 g COD/L-d on Day 63, both fermentation and MCE were significantly inhibited by high VFA accumulation (27.47 g COD/L), high undissociated caproic acid concentration (0.25 g/L), and high residual methanol concentration (15.57 g COD/L). In summary, the overall objective of this research was to understand the implications of simultaneous denitrification and fermentation in producing high-value products such as SCFAs and MCFAs via arrested methanogenesis while systematically investigating the fate of nitrate in engineered anaerobic systems. Since high levels of nitrate are often introduced to anaerobic systems through nitrate-rich substrates, this warrants a closer look into the interaction between nitrate reduction and fermentation processes. Hence, the nitrate reduction (i.e., DEN) kinetics of different organic acids, alcohols, and carbohydrates as the carbon source were determined since they are often encountered in anaerobic systems as fermentation products, industrial waste by-products, and wastewater constituents. In doing so, a novel N2-based respirometric assay was developed to determine and compare DEN kinetics of different carbon sources (Chapter 2). Nonetheless, other competitive dissimilatory nitrate reduction processes, such as DNRA, can also occur in tandem with DEN. The DNRA pathway retains nitrate in the form of bioavailable ammonium, unlike DEN, which removes it from the ecosystem. Evidently, the DNRA vs. DEN distribution has significant implications for nutrient loss and conservation, greenhouse gas emissions, and fermentation product (i.e., VFAs) composition. Therefore, the distribution of denitrification pathways was systematically investigated under arrested methanogenesis conditions by independently varying each of the major underlying controls (e.g., NO3^- input, organic loading, and carbon sources) (Chapter 3). Since most of the VFAs produced during simultaneous denitrification and fermentation were SCFAs, they are very miscible in the fermentation broth and challenging to extract. Much higher-value MCFA could be produced by elongating the SCFAs, known as MCE. Hence, MCFA (i.e., caproic acid) production via MCE using low-cost methanol while arresting methanogenesis by NO3^- was investigated (Chapter 4). The distinct production patterns and kinetics of branched- vs. straight-chain caproic acids were determined as well. This investigation was further extended to the use of real organic waste (e.g., vegetable waste) for MCFA production with high yield and selectivity in semi-continuous bioreactors (Chapter 5). Since such waste is high in NO3^-, the effect of internal NO3^- supply on methanogenesis inhibition was also determined.