Multi-omics reveals new insights into the ammonia oxidizers in environmental samples

Abstract

Biological nitrogen removal from wastewater is a crucial step of wastewater treatment process. Ammonia oxidizers are responsible for the rate-limiting step of ammonia nitrogen removal from wastewater. However, the metabolic features, niche specifications, and microbial interactions of the recently identified ammonia oxidizers, i.e., anammox bacteria, ammonia oxidizing archaea (AOA) and complete ammonia oxidizers (comammox), are not well studied. Therefore, an integrated multi-omics approach was used in this study to discover these ammonia oxidizers from engineered and environmental samples and revealed their potential interactions with other microbial members. Using the multi-omics approach, I studied a lab-scale one-stage partial nitritation anammox (PNA) reactor to characterize the microbial community activities and microbial community interactions in anammox sludge. Genome-centric metagenomics recovered 49 metagenome-assembled genomes (MAGs), among which there were 3 MAGs of anammox bacteria (AMX) and 3 MAGs of ammonia oxidizing bacteria (AOB). The activities of anammox bacteria and AOB, NOB and denitrifiers were investigated using the generated metatranscriptomes and metaproteomes. Metabolic interactions between autotrophic ammonia oxidizers and heterotrophs were further proposed. In addition to the lab-scale system, ammonia oxidizers in a full-scale wastewater treatment plant (WWTP, Shatin WWTP, Hong Kong) were investigated. The high-throughput amplicon sequencing and metagenomic sequencing results demonstrated that marine group I.1a affiliated ammonia oxidizing archaea (AOA) outcompeted AOB and showed yearly periodical increase and decrease trends. Correlation-based net-work analysis revealed that AOA have robust intercorrelation with bacteria involved in nitrogen cycling, e.g. nitrite oxidizing bacteria and denitrifiers, and marine group bacterial lineages. To investigate the potential host-virus interaction of microorganisms in WWTPs, including ammonia oxidizers and their bacteriophages, a total of 8,478 metagenomic viral contigs ([mVCs] >5 kb) were recovered from Shatin (2,806) and Shek Wu Hui (5,672) WWTPs. Approximately 60% of the mVCs were novel viral sequences that were poorly covered by the current NCBI and IMG/VR viral database. CRISPR spacers of 85,252 bacterial and archaeal genomes were used to predict the host-virus associations. Additional temporal (~9 years Shatin WWTP AS samples) and spatial patterns of virome in wastewater treatment systems were profiled in the present study. AOA in an extreme environment (hyperthermal hot spring) was also studied. One MAG of AOA was recovered, which further extended the upper temperature for ammonia oxidation in environmental samples. A metabolic interdependencies among the AOA and other newly recovered MAGs were summarized to elucidate the biogeochemical cycles in this studied hot spring. In addition to the AOA, metabolic potentials of nitrogen fixation and methanogenesis were observed in an archaeal organism within Crenarchaeota. Finally, four MAGs of novel complete ammonia oxidizers (comammox) were recovered in drinking water distribution systems, which significantly enriched current database of comammox. More importantly, this study confirmed that the comammox could be widely identified and might outnumber the canonical ammonia oxidizers in drinking water systems. The results of this study provide new insights into ecological roles, niche specialization, microbial interactions of ammonia oxidizers and other microbial members, and contribute to the potential engineered application for different nitrogen removal systems.