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This study aimed to evaluate the capacity of different inoculum sources and their bacterial diversity to generate hydrogen (H 2). The highest Simpson (0.7901) and Shannon (1.581) diversity indexes for H 2 -producing bacterial isolates were estimated for sewage inocula. The maximum cumulative H 2 production (H max) was 639.6 ± 5.49 mL/L recorded for the sewage inoculum (SS30) after 72 h. The highest H 2 -producing isolates were recovered from SS30 and identified as Clostridium saccharobutylicum MH206 and Lactobacillus brevis MH223. The H max of C. saccharobutylicum , L. brevis , and synergistic coculture was 415.00 ± 24.68, 491.67 ± 15.90, and 617.67 ± 3.93 mL/L, respectively. The optimization process showed that the H max (1571.66 ± 33.71 mL/L) with a production rate of 58.02 mL/L/h and lag phase of 19.33 h was achieved by the synergistic coculture grown on 3% molasses at 40 °C, pH 7, and an inoculum size of 25% (v/v). This study revealed the economic feasibility of the synergistic effects of coculture on waste management and biohydrogen production technology. [Display omitted] • Screening of H 2 production by different microbial communities was investigated. • Diversity of H 2 producing bacteria in sewage, rumen and wastewater was achieved. • H 2 yield from sugarcane molasses was improved using bacterial synergism. • Maximum H 2 yield of 1571.66 mL/L obtained through an optimization strategy. [ABSTRACT FROM AUTHOR]

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]

[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]