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Ammonia is a major inhibitor in anaerobic digestion of nitrogen-rich organic wastes. In this study, integrated genome-centric metagenomic and metaproteomic analyses were used to identify the key microorganisms and metabolic links causing instability by characterizing the process performance, microbial community, and metabolic responses of key microorganisms during endogenous ammonia accumulation. The identification of 89 metagenome-assembled genomes and analysis of their abundance profile in different operational phases permitted the identification of key taxa (Firmicutes and Proteobacteria) causing poor performance. Metabolic reconstruction indicated that the key taxa had the genetic potential to participate in the metabolism of C2C5 volatile fatty acids (VFAs). Further investigation suggested that during Phase I, the total ammonia nitrogen (TAN) level was maintained below 2000 mg N/L, and the reactor showed a high methane yield (478.30 ± 33.35 mL/g VS) and low VFAs concentration. When the TAN accumulated to > 2000 mg N/L, acid accumulation, mainly of acetate, began to occur, and the methane yield gradually decreased to 330.44 mL/g VS (Phase II). During this phase, the VFA degradation functions of the community were mainly mediated by Firmicutes. Approximately 61.54% of significant differentially expressed proteins (DEPs) related to acetate metabolism in Firmicutes were down-regulated, which led to an increase in acetate concentration to 4897.91 ± 1558.96 mg/L. However, the reactor performance showed spontaneous recovery without any interference (Phase III), during which Firmicutes gradually adapted to the high ammonia conditions. Approximately 75% of the significant DEPs related to acetate metabolism of Proteobacteria were also up-regulated in Phase III compared with Phase II; thus, VFA-related metabolic functions of the community were enhanced, which resulted in a decrease in the total VFA concentration to 195.39 mg/L. When the TAN increased above 4000 mg N/L, the system gradually showed acid accumulation dominated by propionate, accompanied by a second decrease in methane yield (Phase IV). During this phase, the number of up-regulated and down-regulated proteins related to acetate metabolism of Firmicutes and butyrate/valerate metabolism of Proteobacteria was comparable with that of Phase III, indicating that the metabolic functions related to acetate, butyrate, and valerate of the microbial community were not significantly affected. However, for propionate metabolism, the expression activity of fumarate hydratase from Firmicutes and Proteobacteria was severely inhibited by ammonia, as shown by down-regulation ratios of 63.64% and 85.71%, respectively. No protein with the same function that was not inhibited by ammonia could be detected, and the fumarate degradation function of the microbial community was severely damaged, leading to blocked propionate metabolism and irreversible deterioration of reactor performance. This study has provided a new perspective on the microecological mechanisms of ammonia inhibition.

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The feasibility of using food waste anaerobic digestate-derived biochar (FWDB) to mitigate ammonia toxicity in an anaerobic digester was evaluated. The optimal conditions for preparing and adding the activated FWDB were explored using response surface experiments, and the long-term effects of adding activated FWDB on digester performance under optimum conditions were verified in semi-continuous experiments. The results showed that the optimal preparation and addition conditions for activated FWDB were pyrolysis temperature of 565 °C, particle size of 0-0.30 mm, and dosage of 15.52 g·L-1. During the long-term operation of the digesters, when the total ammonia nitrogen (TAN) concentration was higher than 2000 mg·L-1, the control and experimental digesters showed deteriorated reactor performance. Volatile fatty acids in the control digester accumulated to 20,306 mg·L-1 after the TAN concentration increased to 3391 mg·L-1, the methane yield decreased to 31 mL·g VS-1, and the digester experienced process failure. In contrast, the experimental digester with added activated FWDB only suffered a slight short-term accumulation of acetate and a slight decline in methane yield. This may be attributed to the adsorption of NH4+/NH3 by activated FWDB, which reduced the TAN concentration in the anaerobic digestion (AD) system and mitigated ammonia toxicity. Microbial analysis and metagenome predictions demonstrated that the community richness, diversity, and evenness, as well as the abundance of acetogens and related key genes (ACSM1, paaF, and acdA) were higher in the experimental digester than in the control digester. This study provides a closed-loop AD enhancement strategy by pyrolysis of digestate and in-situ supplementation into the digester.

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Foaming is a key issue that threatens the efficient and stable operation of the anaerobic digestion process. This study introduced three disturbances to induce foaming and explored the responses of physicochemical parameters, microbial communities, and metabolites to reveal the foaming mechanism. Under the three disturbance conditions, extracellular polymeric substances (EPS)-related parameters are significantly positively correlated with foam height, and EPS may cause foam by lowering the surface tension. Microorganisms that are more tolerant to high acid or high ammonia stress environments were identified after foaming, and they could resist the stress environment by producing more EPS. The up-regulated expression of sphingomyelin or ceramide was discovered after foaming, involved in the signal molecular transduction process of cell apoptosis or necrosis, which might be related to EPS production. Pantothenic acid involved in pantothenate and CoA biosynthesis pathways was down-regulated expression after foaming, which might be related to the hindered degradation of EPS. The response of multi-scale parameters in the foaming process shows that EPS is the key factor in foaming events.

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Ammonia inhibition is one of the most common causes of instability during the operation of commercial biogas plants. Here, the sensitivity of different functional bacteria to ammonia stress, the ability of functional bacteria to adapt to ammonia stress, and the key phylotypes affecting anaerobic digestion (AD) performance were studied by evaluating the process performance, active microbiome, and protein expression patterns during endogenous ammonia accumulation using integrated metagenomics and metaproteomics analyses. Acetate metabolism was most sensitive to ammonia stress, and the expression activity of methyl-CoM reductase of Methanothrix was inhibited at relatively low ammonia concentrations, which resulted in the accumulation of acetate and other short-chain volatile fatty acids (VFAs) through feedback effects. As the AD process progressed, the abundance of active Methanosarcina with high ammonia tolerance increased, and the activity of their enzymes related to acetoclastic methanation was significantly up-regulated, which resulted in the complete restoration of acetate metabolism and AD performance. Thus, microbial communities can cope with acetate metabolic repression through self-optimization. In contrast, when the total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) increased to 4846.95 mg N/L and 337.46 mg N/L, respectively, propionate (and no other VFAs) accumulated in the digester, which was accompanied by a decrease in methane yield by more than 65%. At this time, the abundance of active syntrophic propionate-oxidizing bacteria (SPOB) decreased by 52%, and the expression of key enzymes related to propionate degradation was significantly down-regulated. The proportion of down-regulated differentially expressed proteins in the dominant Pelotomaculum was as high as 94%, indicating the severe suppression of the growth of these functional bacteria as well as their inability to easily acclimate to ammonia stress. Thus, SPOB appeared to be the key microbial phylotypes affecting AD performance under ammonia stress. Ammonia inhibited the methylmalonyl-CoA pathway of Pelotomaculum by inhibiting the expression of succinyl-CoA synthase, which resulted in the suppression of syntrophic propionate oxidation. The results of this study provide new insights into the microbial mechanism of ammonia inhibition and identify the key phylotypes affecting AD performance under ammonia stress. Our findings also shed light on the microbial regulatory targets of nitrogen-rich waste anaerobic digesters.

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Simple landfills lack pollution prevention measures and therefore continuously release pollutants into the surrounding environment. There are a large number of simple landfills in the mountainous and hilly areas in China, and the interflow accounts for a large proportion of runoff. However, the pollution in the interflow stemming from the simple landfill has not been extensively studied. Here, the pollution of the interflow caused by the simple landfill near the Yunxi Town Landfill in the mountainous and hilly region in Southwest China was studied. The composition and pollution release potential of aged refuse in the landfill were determined, and the water quality of interflow around the landfill was monitored for five months. Seasonal changes in water quality of the interflow were observed, and the concentration of pollutants in the interflow around the simple landfill greatly exceeded the water quality standard for the local water function zoning throughout the sampling period. Specifically, the chemical oxygen demand concentrations of the interflow were 247.90 ± 81.57 mg/L, and more than 50 types of refractory organics were detected, with as many as 10 types of polycyclic aromatic hydrocarbons and Environmental Protection Agency priority pollutants. The total nitrogen concentration of the interflow was 132.45 ± 108.68 mg/L; organic nitrogen (53.27%) was the main component, followed by nitrate nitrogen (32.28%) and nitrous nitrogen and ammonia nitrogen (14.45%). The results highlight the need for the remediation of interflow around simple landfills in mountainous and hilly areas. Generally, the basic data could be used to aid the development of remediation technology.

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Foaming is a problem that affects the efficient and stable operation of the anaerobic digestion process. Characterizing foaming mechanisms and developing early warning and foaming control methods is thus critically important. This review summarizes the correlation of process parameters, state parameters, and microbial communities with foaming in anaerobic digesters; discusses the applicability of the above-mentioned multi-scale parameters and foaming potential evaluation methods for the prediction of foaming risk; and introduces the principles and practical applications of antifoaming and defoaming methods. Multiple causes of foaming in anaerobic digestion systems have been identified, but a generalizable foaming mechanism has yet to be described. Further study of the correlation between extracellular polymeric substances and soluble microbial products and foaming may provide new insights into foaming mechanisms. Monitoring the foaming potential (including the volume expansion potential) is an effective approach for estimating the risk of foaming. An in-situ monitoring system for determining the foaming potential in anaerobic digestion sites could provide an early warning of foaming risk. Antifoaming methods based on operating parameter management and process regulation help prevent foaming from the source, and biological defoaming methods are highly targeted and efficient, which is a promising research direction. Clarifying foaming mechanisms will aid the development of active antifoaming methods and efficient biological defoaming methods.

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To further clarify the key stage and microorganisms responsible for ammonia inhibition instability, three sequential batch experiments were conducted with various ammonia concentrations and different exposure modes. Acetate metabolism was most sensitive to ammonia, however, after continuous ammonia exposure, acetate metabolism was well restored by a shift in dominant microorganisms. In contrast, the metabolism of longer-chain volatile fatty acids (LCVFAs, C3-C5) was only inhibited under a high ammonia concentration (≥6000 mg/L), however, once inhibited, continuous exposure neither restored the abundance of functional microbes nor induced new microorganisms to perform metabolic functions. Therefore, LCVFA metabolism was the key stage responsible for process instability under ammonia stress. Moreover, the deterioration of LCVFA metabolism was caused by the inhibition of syntrophic acetogenic bacteria (SAB) induced by total ammonia nitrogen, rather than the feedback inhibition from methanogenesis. That is, SAB were the key microorganisms involved in process instability.

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