Determination of methanogenic Archaea abundance in a mesophilic biogas plant based on 16S rRNA gene sequence analysis

Energy production of renewable raw material is of increasing importance for sustainable energy production. As an indispensable prerequirement for further upgrading of technical equipment and operation modes of biogas plants, a deeper knowledge of the microbial community responsible for methane formation is crucial. To overcome these limitations a mesophilic biogas plant converting pig manure, maize silage, and grains of crop was sampled and subsequently analysed by construction of a methanogenic Archaea specific 16S rRNA gene clone library combined with PCR-RFLP analysis and group-specific quantitative real-time PCR (qPCR). Seventy percent of all analysed clones belonged to the order Methanomicrobiales, whereas 13% belonged to Methanosarcinales, 6% belonged to the Methanobacteriales group, and 11% of all detected clones were assigned to the CA11 and Arch1 cluster. Comparable percentages were obtained with qPCR: 84% of all detected 16S rRNA gene copy numbers were affiliated with the Methanomicrobiales, while only 14% belonged to the Methanosarcinales and 2% to the Methanobacteriales order. In conclusion, both approaches detected similar archaeal groups and revealed nearly the same abundance, pointing to a predominance of hydrogenotrophic methanogens in the biogas plant.