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Experimental investigation and model simulation was combined to identify the effect of metal ions on mitigating ammonia inhibition during anaerobic digestion. Five metal ions (Ca, Mg, Cu, Zn, Fe) were tested in reactors with 1 g-glucose/L/d and 5 g-N/L under fed batch operation. Ca addition was considered the optimal approach with a 25% increment in methane production via balanced-strengthening dehydrogenases and reinforcing protein-binding structure. Gene-sequencing results suggested 50% and 15% increment in acetotrophic-related and hydrogenotrophic-related dehydrogenases, respectively, after Ca addition. The Anaerobic Digestion Model No.1 was modified by introducing lactate-related reactions, syntrophic acetate oxidation process, and kinetic equation of metal ions, with satisfactory predictions of methane and intermediates (R 2  > 0.80). The lowest affinity constant K I_MI value was obtained with Ca supplement, indicating the highest conversion rate of substrates to methane. The model evaluation revealed the balanced ratio on the enzyme contribution of acetotrophic to hydrogenotrophic methanogenesis., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Bioaugmentation is an attractive method to improve methane production (MP) in the anaerobic digestion (AD) process. In this study, to tackle the ammonia inhibition problem, a long-term (operating over 6 months) acclimatized consortia and a well-constructed consortia were selected as the bioaugmentation consortia for sequencing batch AD reactors fed with dairy manure and pig manure under mesophilic condition. Similar responses, in terms of the reactor performance and microorganisms structure to the different consortia, were observed with both manure kinds indicating that the effectiveness of bioaugmentation was mainly decided by the composition of the added consortia, not the feedstock. 39 - 49% increment in MP was obtained in the reactors bioaugmented with well-constructed consortia, which was higher than the acclimatized consortia (about 25% increment in MP). Both acetogenesis and methanogenesis (advantageous) steps were stimulated with well-constructed consortia bioaugmentation. According to key functional enzyme analysis, the increment of glycine hydroxymethyltransferase and phosphoglycerate mutase might be the critical point in the bioaugmented AD system. Based on the higher functional contribution rate of the well-constructed consortia bioaugmentation reactors, Methanosarcina could have expressed more comprehensive functions or performed stronger activities in different functions than Methanosaeta., (Copyright © 2022. Published by Elsevier Ltd.)

This study evaluated the methanogenic performance of typical substrates (acetate, formate, H 2 /CO 2 , and glucose) under low and high ammonia levels and the Anaerobic Digestion Model No.1 (ADM1) was extended and modified for better simulation and understanding of the process. Formate-utilizing and hydrogen-utilizing methanogenesis showed stronger ammonia resistance than acetate-utilizing methanogenesis (13-23% vs. 34% decrease in methane production (MP)). Model extension, based on foundational experiments fed with three typical precursors (R 2 > 0.92), was then validated with glucose degradation experiments, and satisfactory predictions of MP and total volatile fatty acids were obtained (R 2 > 0.91). Based on the modified ADM1, the carbon fluxes of glucose degradation were determined, and formate-utilizing methanogenesis showed its importance with a 28-34% contribution of the total methanation, becoming the dominant pathway under high ammonia level. Formate-utilizing methanogenesis also had a thermodynamic advantage among the three pathways. 16S rRNA sequencing suggested a homology between the hydrogen-utilizing and formate-utilizing methanogens. Methanobacterium and Methanobrevibacter were found to be key methanogens, and their enrichment under high ammonia level confirmed the stronger ammonia tolerance of formate-utilizing and hydrogen-utilizing methanogenesis. The microbial characterization and modified ADM1 simulations supported each other., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

The effect of bioaugmentation with different microorganisms on anaerobic digestion to mitigate the ammonia inhibition problem was investigated. Seven pure strains of microorganisms (including obligate aceticlastic methanogen, facultative aceticlastic methanogen, hydrogenotrophic methanogen, syntrophic acetate oxidizing bacteria (SAOB) were selected and thirteen bioaugmentation approaches were tested. Bioaugmentation with hydrogenotrophic methanogen Methanobrevibacter smithii (MBS) and SAOB Syntrophaceticu schinkii together was the optimal choice, methane production (MP) was 71.1% higher than that in Blank, the activity of hydrogenotrophic methanogenesis was greatly heightened according to specific methanogenic activity analysis. Bioaugmentation with facultative aceticlastic methanogen Methanosarcina barkeri (MSB) alone without SAOB addition was also proven efficient (MP was 59.7% higher than that in Blank), both aceticlastic and hydrogenotrophic methanogenesis were enhanced. Further evaluation with carbon isotope fractionations analysis indicated that balancing the activities of the aceticlastic and hydrogenotrophic methanogenic pathways is of great importance. 16s rRNA gene sequencing results showed that Methanobacterium spp. and Methanosaeta spp. were the dominant archaea in all 14 reactors. Nevertheless, bioaugmentation with Methanosaeta spp. did not result in a positive effect on MP. On the other hand, Methanobrevibacter spp. and Methanosarcina spp. were non-dominant archaea (even after bioaugmentation with MBS or MSB, the relative abundances were still poor (<2%)), but displayed pivotal roles in determining the overall microbial consortium and, in turn, improved the overall performance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Material addition is a potential method to alleviate ammonia inhibition in the anaerobic digestion process. In this study, seven materials were selected to examine their impact on methane production (MP). In the batch experiment, the reactors with "Magnetite + foam carrier" exhibited nearly a 56% increase in maximum MP rate. Different strategies of magnetite and foam carrier addition were further explored in semi-continuous experiment. Adding two materials together (simultaneous supplementation) resulted in almost 60% increase in MP, 28–41% higher than when supplemented separately. Simultaneous supplementation of the two materials was considered as the Pareto-optimal solution for MP maximization. The acetogenic and methanogenic activities analyses revealed that foam carrier addition accelerated propionate transformation (from propionate to acetate or valerate) while magnetite addition intensified the methanogenesis step. Direct interspecies electron transfer (DIET) was strengthened with over 50% enrichment in DIET-related bacteria. The Anaerobic Digestion Model No.1 was modified with the development of liquid metabolite and biofilm, result in a goodness-of-fit of R2 > 0.96.16sRNA gene sequencing demonstrated that the microbes in the biofilm had kinetic advantages over those in the liquid phase, coupled with higher k m and lower K s values, which contributed to the enhanced MP. [Display omitted] [ABSTRACT FROM AUTHOR]

The effects of ammonia on methane production, methanogenesis pathway, microbial community, and reactor performance were investigated in this study. More than 20% of methane production loss was observed in mesophilic and thermophilic reactors when the ammonia level exceeded 2 and 5 g NH 4 + -N/L, respectively. The volatile fatty acid (VFA) data indicated that acetogenesis and methanogenesis are strongly influenced by ammonia inhibition under the mesophilic condition, while methanogenesis was mainly affected under thermophilic condition. No evident methanogenesis pathway shift was found in the mesophilic reactor, whereas the increase in NH 4 + -N concentration to more than 5 g/L led to a clear shift from aceticlastic to complex and flexible pathways, thereby significantly enriching the hydrogenotrophic pathway in the thermophilic reactor. The function and response of the microbial community to ammonia inhibition were consistent despite the difference in population and diversity under mesophilic and thermophilic conditions. [ABSTRACT FROM AUTHOR]

• Bioaugmentation was confirmed stable to mitigate ammonia inhibition in long-term operation. • 49% increment was achieved in MSH + SS + MBS with both two pathways enhanced. • Bioaugmentation stains should be properly selected to achieve a synergistic effect. • Methanosarcina spp. performed different methanogenic pathway in different bottles. • Methanogenic pathway simulated by ADM1 is consistent with the microbial analysis. The effect of different bioaugmentation strategies on anaerobic digestion related to the alleviation of ammonia inhibition was investigated in a long-term operation. The long-term operation confirmed that bioaugmentation is a stable method. A 35% increase in methane production (MP) was observed in bottles bioaugmented with Methanosarcina barkeri (MSB) or Syntrophaceticu schinkii (SS) + Methanobrevibacter smithii (MBS), and a 49% increment was obtained from the bottles bioaugmented with Methanosaeta harundinacea (MSH) + SS + MBS. Results suggest that the enhancement in both aceticlastic and hydrogenotrophic methanogenic pathways should be considered, and bioaugmentation strain should be properly selected to achieve a synergistic effect. The microbial community analysis indicated Methanosarcina spp. was the dominant archaea. Combined with specific methanogenic activity and carbon isotope fractionation analysis, it was suggested that Methanosarcina spp. performed differently in methanogenic pathways in different bottles. The abundance of COG and total enzymes in the bottles with high MP (MSB and MSH + SS + MBS) was higher than that in the other bottles. The ratio of the functional enzyme tetrahydromethanopterin S-methyltransferase subunit H (EC 2.1.1.86) to formylmethanofuran dehydrogenase subunit E (EC 1.2.99.5) and the relative abundance of enolase (EC 1.2.1.2) confirmed the aceticlastic methanogenic pathway of MSB in Group 1 and the pathway enhancement balance in MSH + SS + MBS. The modified Anaerobic Digestion Model No.1 including syntrophic acetate oxidation was used for simulation with R2 > 0.96. Simulated contribution rate data indicated that the hydrogenotrophic methanogenic pathway was dominant while both two pathways got strengthened, which is consistent with the findings of the microbial analysis. [ABSTRACT FROM AUTHOR]