Unraveling the Impact of Phosphorus, Inorganic Carbon and Different Nitrogen Sources on the Structure, Function and Activity of Mixed Nitrifying Microbial Communities

Abstract

The discovery of bacteria capable of complete ammonia oxidation (CMX) has challenged the long-held perspective of labor division between ammonia oxidizing microorganisms and nitrite oxidizing microorganisms. CMX were found to ubiquitously exist in natural and engineered environments, and co-exist with canonical ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and nitrite-oxidizing bacteria (NOB). However, the factors affecting the niche separation between CMX and canonical nitrifiers are not well understood. More CMX pure cultures and enrichments are needed to investigate the biokinetics of CMX. Many studies on CMX focused on the existence and abundance of CMX across diverse environments, but rarely investigated the activity of CMX and the interactions between CMX and canonical nitrifiers. This dissertation therefore had the following objectives: (1) To determine if phosphorus (P), inorganic carbon (IC) and nitrogen sources are factors driving the niche separation of CMX and canonical nitrifiers; (2) To understand the responses of mixed nitrifying communities under different P, IC loadings and nitrogen sources; and (3) To investigate the interactions between CMX and ammonia oxidizers, and between CMX and nitrite oxidizers.Firstly, CMX are more competitive under a P-excess condition. An increased proportion of CMX, and decreased proportion of AOB were observed upon switching from P-limited to P-excess condition. The major CMX species found in the P-limited condition was similar to that found in the P-excess condition. The mixed culture community upregulated the CMX pathway related to ammonia oxidation, nitrite oxidation, carbon dioxide (CO2) fixation, and the electron transport chain (ETC) after switching to P-excess from P-limited condition. On the contrary, the mixed culture community downregulated the AOB pathway related to ammonia oxidation CO₂ fixation, and the ETC under P-excess condition. The mixed culture community downregulated the NOB pathway related to nitrite oxidation, CO₂ fixation, and the ETC under P-excess condition. Collaboration between CMX and NOB under P-excess condition to achieve complete nitrification performance was observed showing that CMX provided nitrite to support NOB metabolism. These Findings support the possibility of incorporating CMX, as nitrite producer, into a partial nitrification process. Secondly, CMX were successfully enriched in a lab-scale reactor using urea as nitrogen source. A higher proportion of CMX was retained in the reactor fed with urea, as compared to the reactor fed with ammonia as nitrogen source. The nitrogen source is a factor driving the niche separation of different AOB and CMX species, including the observation that the major CMX and AOB species found in the urea-fed reactor were different from that in the ammonia-fed reactor. The CMX enrichment can be used to expand our understanding of the CMX biokinetic, stoichiometric and thermodynamic coefficients. Thirdly, AOB were found to outcompete CMX under low IC conditions, regardless of influent ammonia loading. In response to the low IC loading, the mixed culture community upregulated AOB pathway related to ammonia oxidation, CO₂ fixation and the ETC. On the contrary, the mixed culture community downregulated CMX pathway related to ammonia oxidation, nitrite oxidation, CO₂ fixation and the ETC in response to the low IC loading. Nitrite oxidation pathway by NOB was upregulated, while NOB pathway related to CO₂ fixation and the ETC were downregulated in the mixed culture community in response to the low IC loading. Fourthly, proliferation of CMX Nitrospira could be promoted with simultaneous co-feeding of ammonia and nitrite. In response to nitrite addition in the influent, the nitrite oxidation pathway by CMX and NOB in the mixed nitrifying community was upregulated, while the ammonia oxidation pathway by AOB was unchanged, and by CMX was downregulated. These results suggest that CMX may deteriorate the performance of energy-efficient BNR systems, such as shortcut nitrification-denitrification and partial nitritation-anammox, by contributing more to nitrite oxidation than to ammonia oxidation. In summary, this dissertation improves our understanding on the ecophysiology on the CMX and canonical nitrifiers. The results from this study can guide the enrichment of CMX to fully appreciate positive gains possible by enhancing the CMX ecophysiology. This dissertation, by applying metagenomics and metatranscriptomics, is one of the rare studies focusing on the overall system-wide responses of the mixed culture community to different conditions, rather than focusing on a single microorganism in relation to these varying conditions