Enhancing biomethane production from cattle manure by integrating rumen bacteria: a microbial analyses with next-generation sequencing and quantitative PCR

Abstract This study examined the impact of mixing cow manure (CM) and rumen fluid (RF) in various proportions on methane production and microbial dynamics in batch-operated digesters at three different temperatures. The proportions of RF added to CM were 0%, 10%, 20%, 30%, 40%, 50%, and 100%. The mixed substrates in triplicate bottles were then divided into three groups and incubated at temperatures of 36 °C, 41 °C, and 43 °C each lasting 40 days. In digesters operated at 36 °C and 41 °C, the highest methane yields produced were 292 mL CH 4 /g VS added with 20% RF and 304 mL CH 4 /g VS added with 40% RF, respectively, whereas that of was 189 mL CH 4 /g VS added with 40% RF at 43 °C. The digesters achieving the highest methane yields across all temperature conditions were those inoculated with RF at ratios between 20 and 40%. These digesters demonstrated the highest methane yield increases of 129%, 95%, and 121% at 36 °C, 41 °C, and 43 °C, respectively, compared to the control digesters containing only CM. These findings underscore the substantial role of RF addition in enhancing biomethane production efficiency. Metagenomic analyses using next-generation sequencing (NGS) on the Ion PGM™ platform identified Firmicutes, Bacteroidetes, and Proteobacteria as the dominant bacterial groups. Quantitative polymerase chain reaction (qPCR) revealed that Ruminococcus albus , Fibrobacter succinogenes , and Ruminococcus flavefaciens were the main cellulose-digesting bacteria at all temperatures studied. However, the numbers of microorganisms were much lower all through the digesters operated at 43 °C, indicating that the temperature is not suitable for microbial growth. Graphical Abstract