DNAs of two biofilms of a thermophilic two-phase leach-bed biogas reactor fed with rye silage and winter barley straw were sequenced by 454-pyrosequencing technology to assess the biofilm-based microbial community and their genetic potential for anaerobic digestion. The studied biofilms matured on the surface of the substrates in the hydrolysis reactor (HR) and on the packing in the anaerobic filter reactor (AF). The classification of metagenome reads showed Clostridium as most prevalent bacteria in the HR, indicating a predominant role for plant material digestion. Notably, insights into the genetic potential of plant-degrading bacteria were determined as well as further bacterial groups, which may assist Clostridium in carbohydrate degradation. Methanosarcina and Methanothermobacter were determined as most prevalent methanogenic archaea. In consequence, the biofilm-based methanogenesis in this system might be driven by the hydrogenotrophic pathway but also by the aceticlastic methanogenesis depending on metabolite concentrations such as the acetic acid concentration. Moreover, bacteria, which are capable of acetate oxidation in syntrophic interaction with methanogens, were also predicted. Finally, the metagenome analysis unveiled a large number of reads with unidentified microbial origin, indicating that the anaerobic degradation process may also be conducted by up to now unknown species.

Agricultural biogas plants were operated in most cases below their optimal performance. An increase in the fermentation temperature and a spatial separation of hydrolysis/acetogenesis and methanogenesis are known strategies in improving and stabilizing biogas production. In this study, the dynamic variability of the bacterial and archaeal community was monitored within a two-phase leach bed biogas reactor supplied with rye silage and straw during a stepwise temperature increase from 55 to 75 °C within the leach bed reactor (LBR), using TRFLP analyses. To identify the terminal restriction fragments that were obtained, bacterial and archaeal 16S rRNA gene libraries were constructed. Above 65 °C, the bacterial community structure changed from being Clostridiales-dominated toward being dominated by members of the Bacteroidales, Clostridiales, and Thermotogales orders. Simultaneously, several changes occurred, including a decrease in the total cell count, degradation rate, and biogas yield along with alterations in the intermediate production. A bioaugmentation with compost at 70 °C led to slight improvements in the reactor performance; these did not persist at 75 °C. However, the archaeal community within the downstream anaerobic filter reactor (AF), operated constantly at 55 °C, altered by the temperature increase in the LBR. At an LBR temperature of 55 °C, members of the Methanobacteriales order were prevalent in the AF, whereas at higher LBR temperatures Methanosarcinales prevailed. Altogether, the best performance of this two-phase reactor was achieved at an LBR temperature of below 65 °C, which indicates that this temperature range has a favorable effect on the microbial community responsible for the production of biogas.

The influence of different temperature levels on the formation of methane in a two‐phase anaerobic digestion process using rye silage and barley straw as feedstocks was investigated in this study. This process comprised a leach‐bed reactor (LBR), a reservoir for leachate from the LBR, and a separate downstream fixed‐film anaerobic filter (AF), with immobilized micro biocoenosises. The degree of degradation of the volatile solids was similar in both studies and was almost equal at 75 Ma.‐%VS. When both reactor phases were operated at thermophilic conditions, the main substrate turnover was observed in the LBR (58% of the total methane yield, with an average methane content of 41 Vol.‐%). Only the excess of organic fraction in the process‐liquid was transformed in the AF (42% of the total methane yield, with an average methane content of 74 Vol.‐%). When the parts of the reactor system operated at different temperature regimes, thermophilic hydrolysis/acidogenesis phase and mesophilic methanization, a separation of carbon dioxide and methane production, was observed. A total methane yield of 88%, with an average methane content of 85 Vol.‐%, was formed in the AF. The generated biogas with high methane concentrations is suitable for feeding the purified biomethane into the natural gas grid. Furthermore, it can be used as fuel for cars and tractors.

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