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The performance of on-site wastewater treatment systems (OWTSs) can be improved by altering digester design and by manipulating environmental variables that affect microbial community composition. Community composition can potentially be assessed using high-throughput DNA sequencing, but the two most common methods of community DNA sequencing (16S and shotgun sequencing) generally yield different taxonomic identification profiles and can perform differently according to the sampled environment. To evaluate the use of these two approaches in monitoring OWTS operation, we conducted a comparative parallel analysis using both 16S rDNA and shotgun sequencing in a controlled field study. Results indicate that when examining microorganisms above 0.1% relative abundance, 16S and shotgun sequencing produced similar results in terms of individual sample species richness and between-sample community similarity. However, shotgun sequencing provided comparatively higher taxonomic richness for the bacterial communities with lower abundance in the OWTSs. In addition, 16S sequencing resolved only 48 out of 188 bacterial communities identified by shotgun sequencing (using a 0.1% abundance cutoff). Three key bacterial genera (Desulfomicrobium, Simplicispira, and Phenylobacterium) in anaerobic digestion processes were differentially abundant for both sequencing methods. These data indicate that both sequencing methods provide similar overall profiles for bacterial communities in anaerobic digestor systems. However, shotgun sequencing provides significantly (p-value < 0.01) higher taxonomic richness overall. Thus, shotgun sequencing provides a more robust taxonomic and functional profile that can be used for the optimization of anaerobic digestor systems.

On-site wastewater treatment systems (OWTS) are primarily monitored using physiochemical factors, including chemical oxygen demand (COD) and residual total suspended solids (TSS), which are indirect measures of the microbial action during the anaerobic digestion process. Changes in anaerobic digester microbial communities can alter the digester performance, but this information cannot be directly obtained from traditional physicochemical indicators. The potential of metagenomic DNA sequencing as a tool for taxonomic and functional profiling of microbial communities was examined in both common conventional and plug flow-type anaerobic digesters (single-pass and recirculating). Compared to conventional digesters, plug flow-type digesters had higher relative levels of sulfate-reducing bacteria (Desulfovibrio spp.) and hydrogenotrophic methanogens (Methanospirillum spp.). In contrast, recirculating anaerobic digesters were enriched with denitrifier bacteria and hydrogenotrophic methanogens, and both were significantly correlated with physicochemical factors such as COD and TSS. Stratification of microbial communities was observed along the digester treatment process according to hydrolytic, acidogenic, acetogenic, and methanogenic subgroups. These results indicate that the high-throughput DNA sequencing may be useful as a monitoring tool to characterize the changes in bacterial communities and the functional profile due to differences in digester design in on-site systems.