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High-load reactors face a significant challenge with fatty acid accumulation. Although injecting acidogenic gas (H 2 and CO 2) into methanogenic reactors improves biogas production, its mechanisms and stability enhancement remain unclear. Therefore, this study investigated the potential of using acidogenic gas to enhance methane production and system stability in a high substrate-loading co-digestion system with decreasing hydraulic retention time (HRT) and its regulatory role by considering biological pathways. Acidogenic gas injection effectively managed volatile fatty acid accumulation, with a concomitant increase in methane production from 0.042 to 0.066 L/L h as HRT decreased (16–8 days). However, the methane yield decreased from 611 to 460 mL/g volatile solids. Microbial community analysis revealed an increased abundance of hydrogenotrophic methanogens, along with several acidogenic and syntrophic fatty acid oxidizing consortia with decreasing HRT. The HRT10.67 sample exhibited the highest microbial species diversity and richness in the methanogenic reactor. Metabolomics analysis showed acidogenic gas reduced energy synthesis related to the tri-carboxylic acid cycle and oxidative phosphorylation while profoundly regulating the metabolism of amino acids, lipids, fatty acids, membrane transporters and inhibitory substances, thereby strengthening methane metabolism pathways. This study provides valuable insights into the roles of acidogenic gas in regulating energy metabolism to enhance methane production and offers potential opportunities for improving lipid and protein degradation. • H 2 content increased from 30 to 53 % as HRT decreased from 16 to 8 days. • Organic matter removal was slightly decreased by acidogenic gas injection (AGI). • AGI improved CH 4 production and yield, but the improvement decreased at lower HRT. • AGI increased abundance of hydrogenotrophic methanogens and syntrophic bacteria. • AGI triggered amino acid and lipid metabolism, reducing energy-related synthesis. [ABSTRACT FROM AUTHOR]

Optimized doses and dosing strategies for trace element application in the anaerobic digestion (AD) process still face challenges. In this study, the response surface methodology (RSM) of Box-Behnken was used to investigate the effects of Fe, Co and Ni co-supplementation dosages on the mesophilic AD process of food waste (FW). According to RSM results, the maximum experimental methane yield of 677.9 mL/g VS was obtained under the optimal condition (Fe: 113.1, Co: 0.51, and Ni: 2.44 mg/L), representing a 27.4 % increase compared to the control group without trace element addition. The noticeable decrease in Fe bioavailability in control group indicates a higher demand for Fe during AD of FW. However, while increasing the Fe dose did not enhance its easily bioavailable fraction, that of Co and Ni exhibited a negative effect on Fe bioavailability. In the optimal condition, the syntrophic methanogenesis between syntrophic genera and obligate hydrogenotrophic methanogens improved, along with the increased relative abundance of ATP-binding cassette transporter and two-component systems, which correlated with enhanced Fe bioavailability. These findings provide deeper insights into the interactions of co-supplemented trace elements during AD of FW. [Display omitted] [ABSTRACT FROM AUTHOR]

• Use of acidogenic off-gas (AOG) can improve methanogenesis in two stage AD. • Methane yield and energy yield was improved by 37.2 % and 25.6 %, respectively. • Electron transfer and syntrophic effect were enhanced after introduction of AOG. • Improved proteins degradation and lipids metabolism were confirmed. • Methanosarcina are more competitive than methanothermobacter after AOG added. Although two-stage anaerobic digestion (TSAD) technology has been investigated, the mechanisms regarding the impact of acidogenic off-gas (AOG) on successive methane production have not been well addressed. In this study, a novel TSAD system was designed. Food waste, as the main substrate, was co-digested with chicken manure and corn straw. The acidogenic gas beyond atmospheric pressure was introduced into the bottom of the methanogenesis reactor through a stainless steel diffuser. Results showed the addition of AOG increased the methane yield from 435.2 to 597.1 mL/g VS in in successive methanogenesis stage, improved by 37.2 %, and increased the energy yield from 9.0 to 11.3 kJ/g VS substrate. However, the theoretical contribution of hydrogenotrophic methanogenesis using H 2 contained in AOG was only 15.2 % of the increased methane yield. After the addition of AOG, the decreased levels of ammonia nitrogen and butyrate indicate that the stability of the AD system was improved. The electron transfer system and co-enzyme F420 activity were enhanced; however, the decrease in acetate kinase activity indicates aceticlastic methanogenesis may have been weakened. The microbial diversity and species richness were improved by the added AOG. Methanosarcina was more competitive than Methanothermobacter , enhancing the syntrophic effect. The relative abundance of protein degradation bacteria norank_f_Anaerolineaceae and lipid degradation bacteria Syntrophomonas was increased. Metabolite analysis confirmed that the addition of AOG promoted amino acid metabolism, the biosynthesis of other secondary metabolism and lipid metabolism. The improved degradation of recalcitrant organic components (lipids and proteins) in food waste was responsible for the increased methane yield. This study provides an in-depth understanding of the impact of AOG utilization on successive methane production and has practical implications for the treatment of food waste. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Bioaugmentation is applied to two-stage co-digestion system. • Obtained optimal addition mass of Thermoanaerobacterium thermosaccharolyticum of bioaugmentation was 1.12 g. • Increased cumulative production of hydrogen and methane to 68.72% and 84.45%. • Enhanced the methanogenic potential of the microbial community in anaerobic digestion. Microorganisms play a vital role in anaerobic digestion, however, bioaugmentation in a two-stage co-digestion system to directly regulate the microbial structure is limited. In this study, the different dosage of Thermoanaerobacterium thermosaccharolyticum was added in the hydrogen production stage of a two-stage anaerobic digestion system operating at a thermophilic temperature (55 °C). Results showed that Thermoanaerobacterium thermosaccharolyticum of 1.12 g had the best effect, with the cumulative hydrogen and methane yields reaching 81.54 mL/g VS and 550.98 mL/g VS respectively, which were 68.72 % and 84.45 % higher than the control group. According to the microbial analysis, the addition of Thermoanaerobacterium thermosaccharolyticum significantly changed the microbial community structure, and also its relative abundance increased in the hydrogen production stage, thereby increasing volatile fatty acids (VFAs) and hydrogen content. Although the methane production stage did not contain Thermoanaerobacterium thermosaccharolyticum , the microbial community structure changed, increasing the dominant species Anaerolineae and Limnochordia, thereby improving the efficiency of substrate decomposition and methane production. This study shows that Thermoanaerobacterium thermosaccharolyticum can promote not only hydrogen production but also the production of methane in two-stage anaerobic digestion. [ABSTRACT FROM AUTHOR]

[Display omitted] • In situ biogas upgrading via two-stage anaerobic digestion is reviewed. • H 2 addition technology seems currently one of the most promising techniques. • Crucial chemical factors of in situ biogas upgrading are pH and alkalinity. • Hydrogenotrophic methanogens are vital archaea during in situ biogas upgrading. • In situ hybrid biogas upgrading techniques via two-stage AD is proposed. High impurity concentration of biogas limits its wide commercial utilization. Therefore, the integration of two-stage anaerobic digestion process with in situ biogas upgrading technologies is reviewed, with emphasis on their principles, main influencing factors, research success, and technical challenges. The crucial factors that influence these technologies are pH, alkalinity, and hydrogenotrophic methanogenesis. Hence, pH fluctuation and low gas–liquid mass transfer of H 2 are some major technical challenges limiting the full-scale application of in situ upgrading techniques. Two-stage anaerobic digestion integration with various in situ upgrading techniques to form a hybrid system is proposed to overcome the constraints and systematically guide future research design and advance the development and commercialization of these techniques. This review intends to provide the current state of in situ biogas upgrading technologies and identify knowledge gaps that warrant further investigation to advance their development and practical implementation. [ABSTRACT FROM AUTHOR]