Advanced N‐removal onsite wastewater treatment systems (OWTS) rely on nitrification and denitrification to remove N from wastewater. Despite their use to reduce N contamination, we know little about microbial communities controlling N removal in these systems. We used quantitative polymerase chain reaction and high‐throughput sequencing targeting nitrous oxide reductase (nosZ) and bacterial ammonia monooxygenase (amoA) to determine the size, structure, and composition of communities containing these genes. We analyzed water samples from three advanced N‐removal technologies in 38 systems in five towns in Rhode Island in August 2016, and in nine systems from one town in June, August, and October 2016. Abundance of nosZ ranged from 9.1 × 103 to 9 × 108 copies L−1 and differed among technologies and over time, whereas bacterial amoA abundance ranged from 0 to 1.9 × 107 copies L−1 and was not different among technologies or over time. Richness and diversity of nosZ—but not amoA—differed over time, with median Shannon diversity indices ranging from 2.61 in October to 4.53 in August. We observed weak community similarity patterns driven by geography and technology in nosZ. The most abundant nosZ‐ and amoA‐containing bacteria were associated with water distribution and municipal wastewater treatment plants, such as Nitrosomonas and Thauera species. Our results show that nosZ communities in N‐removal OWTS technologies differ slightly in terms of size and diversity as a function of time, but not geography, whereas amoA communities are similar across time, technology, and geography. Furthermore, community composition appears to be stable across technologies, geography, and time for amoA. Core Ideas We describe N‐cycling bacterial communities in advanced N‐removal septic systems. Geographic factors are weak drivers of community composition in nosZ. Technology design is a weak driver of community composition in nosZ. Community composition of amoA is similar across time, space, and among technologies Time drives differences in nosZ—but not amoA—diversity and abundance.

Biological nitrogen removal (BNR) in centralized and decentralized wastewater treatment systems is assumed to be driven by the same microbial processes and to have communities with a similar composition and structure. There is, however, little information to support these assumptions, which may impact the effectiveness of decentralized systems. We used high-throughput sequencing to compare the structure and composition of the nitrifying and denitrifying bacterial communities of nine onsite wastewater treatment systems (OWTS) and one wastewater treatment plant (WTP) by targeting the genes coding for ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ). The amoA diversity was similar between the WTP and OWTS, but nosZ diversity was generally higher for the WTP. Beta diversity analyses showed the WTP and OWTS promoted distinct amoA and nosZ communities, although there is a core group of N-transforming bacteria common across scales of BNR treatment. Our results suggest that advanced N-removal OWTS have microbial communities that are sufficiently distinct from those of WTP with BNR, which may warrant different management approaches.

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Advanced onsite wastewater treatment systems (OWTS) use biological nitrogen removal (BNR) to mitigate the threat that N-rich wastewater poses to coastal waterbodies and groundwater. These systems lower the N concentration of effluent via sequential microbial nitrification and denitrification. We used high-throughput sequencing to evaluate the structure and composition of nitrifying and denitrifying bacterial communities in advanced N-removal OWTS, targeting the genes encoding ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ) present in effluent from 44 advanced systems. We used QIIME2 and the phyloseq package in R to examine differences in taxonomy and alpha and beta diversity as a function of advanced OWTS technology, occupancy pattern (seasonal vs. year-round use), and season (June vs. September). Richness and Shannon’s diversity index for amoA were significantly influenced by season, whereas technology influenced nosZ diversity significantly. Season also had a strong influence on differences in beta diversity among amoA communities, and had less influence on nosZ communities, whereas technology had a stronger influence on nosZ communities. Nitrosospira and Nitrosomonas were the main genera of nitrifiers in advanced N-removal OWTS, and the predominant genera of denitrifiers included Zoogloea, Thauera, and Acidovorax. Differences in taxonomy for each gene generally mirrored those observed in diversity patterns, highlighting the possible importance of season and technology in shaping communities of amoA and nosZ, respectively. Knowledge gained from this study may be useful in understanding the connections between microbial communities and OWTS performance and may help manage systems in a way that maximizes N removal.