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

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 CH₄/g VS_added with 20% RF and 304 mL CH₄/g VS_added with 40% RF, respectively, whereas that of was 189 mL CH₄/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.