Validation of 16S rRNA gene sequencing and metagenomics for evaluating microbial immigration in a methanogenic bioreactor

To quantitatively evaluate the impact of microbial immigration from an upstream community on the microbial assembly of a downstream community, an ecological genomics (ecogenomics)-based mass balance (EGMB) model coupled with 16S rRNA gene sequencing was previously developed. In this study, a mock community was used to further validate the EGMB models and demonstrate the feasibility of using metagenome-based EGMB model to reveal both microbial activity and function. The mock community consisting of Aeromonas, Escherichia, and Pseudomonas was fed into a lab-scale methanogenic bioreactor together with dissolved organic substrate. Using qPCR, 16S rRNA gene, 16S rRNA gene copy number normalization (GCN), and metagenome, results showed highly comparable community profiles in the feed. In the bioreactor, Aeromonas and Pseudomonas exhibited negative growth rates throughout the experiment by all approaches. Escherichia's growth rate was negative by most biomarkers but was slightly positive by 16S rRNA gene. Still, all approaches showed a decreasing trend toward negative in the growth rate of Escherichia as reactor operation time increased. Uncultivated populations of phyla Desulfobacterota, Chloroflexi, Actinobacteriota, and Spirochaetota were observed to increase in abundance, suggesting their contribution in degrading the feed biomass. Based on metabolic reconstruction of metagenomes, these populations possessed functions of hydrolysis, fermentation, fatty acid degradation, or acetate oxidation. Overall results supported the application of both 16S rRNA gene-and metagenome-based EGMB models to measure the growth rate of microbes in the bioreactor, and the latter had advantage in providing insights into the microbial functions of uncultivated populations.