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 degrees C, particle size of 0-0.30 mm, and dosage of 15.52 g center dot L-1. During the long-term operation of the digesters, when the total ammonia nitrogen (TAN) concentration was higher than 2000 mg center dot L-1, the control and experimental digesters showed deteriorated reactor performance. Volatile fatty acids in the control digester accumulated to 20,306 mg center dot L-1 after the TAN concentration increased to 3391 mg center dot L-1, the methane yield decreased to 31 mL center dot 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.

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.