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Anaerobic digestion (AD) is a microbially-driven process enabling energy production. Microorganisms are the core of anaerobic digesters and play an important role in the succession of hydrolysis, acidogenesis, acetogenesis, and methanogenesis processes. The diversity of participating microbial communities can provide new information on digester performance for biomass valorization and biofuel production. In this study anaerobic systems were used, operating under mesophilic conditions that realized biodegradation processes of waste wheat straw pretreated with NaOH—a renewable source for hydrogen and methane production. These processes could be managed and optimized for hydrogen and methane separately but combining them in a two-stage system can lead to higher yields and a positive energy balance. The aim of the study was to depict a process of biohydrogen production from lignocellulosic waste followed by a second one leading to the production of biomethane. Archaeal and bacterial consortia in a two-stage system operating with wheat straw were identified for the first time and the role of the most important representatives was elucidated. The mixed cultures were identified by the molecular-biological methods of metagenomics. The results showed that biohydrogen generation is most probably due to the presence of Proteiniphilum saccharofermentans, which was 28.2% to 45.4% of the microbial community in the first and the second bioreactor, respectively. Archaeal representatives belonging to Methanobacterium formicicum (0.71% of the community), Methanosarcina spelaei (0.03%), Methanothrix soehngenii (0.012%), and Methanobacterium beijingense (0.01%) were proven in the methane-generating reactor. The correlation between substrate degradation and biogas accumulation was calculated, together with the profile of fatty acids as intermediates produced during the processes. The hydrogen concentration in the biogas reached 14.43%, and the Methane concentration was 69%. Calculations of the energy yield during the two-stage process showed 1195.89 kWh·t−1 compared to a 361.62 kWh·t−1 cumulative yield of energy carrier for a one-stage process.

Archaeal and Bacterial Content in a Two-Stage Anaerobic System for Efficient Energy Production from Agricultural Wastes