Unveiling of active bacteria associated with nutrient cycling during cattle manure composting

Bacteria play a pivotal role in nutrient turnover during the composting process. However, studies relying on total DNA analysis for bacterial community may be confounded by the presence of extracellular DNA from dead cells. In this study, ethidium monoazide (EMA) was employed to extract intracellular DNA from composting samples for amplicon and metagenomic sequencing, enabling the assessment of active bacterial community dynamics during cattle manure composting. The results revealed that total DNA-based 16S rRNA sequencing could only represent 36.9 %-81.6 % of the active bacterial communities. In contrast, EMA-based 16S rRNA sequencing identified Proteobacteria as the dominant active bacterial phylum throughout the composting process, with Actinobacteria exhibiting increased activity during the maturation phase. EMA-based metagenomic sequencing further showed that carbon and nitrogen metabolism genes showed the highest activity during the initial phase. Proteobacteria were identified as key functional bacteria in nutrient turnover, with its contribution reaching 55.4 % and participating in 82.1 % (23/28) of metabolic pathways. Meanwhile, Firmicutes (bin-23, g_Capillibacterium, bin-66, c_Bacilli) were the sole active nitrogen-fixing bacteria, harboring nitrogenase genes (nifH and nifD). This study offers novel understandings regarding the contribution of active bacteria in nutrient turnover and highlights the importance of distinguishing between active and total bacterial communities for a better understanding of microbial processes in composting systems.