Towards molecular biomarkers for biogas production from lignocellulose-rich substrates

• Different communities of 1- and 2-phase fermenters at different temperatures.
• Distinct Clostridia were prevailing, Bacteroidetes only at mesophilic conditions.
• Many bacteria in the processes are unknown and require intensified research.
• No PCR bias: corresponding PCR/cloning and metagenome (without PCR) results.
• RT-qPCR biomarkers for hydrolytic/acidogenic bioindicator bacteria developed.

Biogas production from lignocellulose-rich agricultural residues is gaining increasingly importance in sustainable energy production. Hydrolysis/acidogenesis (H/A) of lignocellulose as the initial rate-limiting step deserves particular optimization. A mixture of straw/hay was methanized applying two-phase digester systems with an initial H/A reactor and a one-stage system at different, meso- and thermophilic temperatures. H/A was intensified with increasing pH values and increasing temperature. H/A fermenters, however, were prone to switch to methanogenic systems at these conditions. Substrate turnover was accelerated in the bi-phasic process but did not reach the methanation efficiency of the single-stage digestion. There was no indication that two different cellulolytic inocula could establish in the given process.Bacterial communities were analyzed applying conventional amplicon clone sequencing targeting the hypervariable 16S rRNA gene region V6–V8 and by metagenome analyses applying direct DNA pyrosequencing without a PCR step. Corresponding results suggested that PCR did not introduce a bias but offered better phylogenetic resolution. Certain Clostridium IV and Prevotella members were most abundant in the H/A system operated at 38 °C, certain Clostridium III and Lachnospiraceae bacteria in the 45 °C, and certain Clostridium IV and Thermohydrogenium/Thermoanaerobacterium members in the 55 °C H/A system. Clostridium III representatives, Lachnospiraceae and Thermotogae dominated in the thermophilic single-stage system, in which also a higher portion of known syntrophic acetate oxidizers was found.Specific (RT-)qPCR systems were designed and applied for the most significant and abundant populations to assess their activity in the different digestion systems. The RT-qPCR results agreed with the DNA based community profiles obtained at the different temperatures. Up to 1012 16S rRNA copies mL−1 were determined in H/A fermenters with prevalence of rRNA of a Ruminococcaceae subgroup. Besides, Thermohydrogenium/Thermoanaerobacterium rRNA prevailed at thermophilic and Prevotellaceae rRNA at mesophilic conditions. The developed (RT)-qPCR systems can be used as biomarkers to optimize biogas production from straw/hay and possibly other lignocellulosic substrates.