Enrichment and characterisation of novel microorganisms involved in the nitrogen cycle

Abstract

Nitrification and denitrification are two critical processes in the global nitrogen cycle. Many microorganisms have been shown to contribute to these biological nitrogen transformation processes. However, our understanding of the nitrogen cycle is still evolving. In the recent years, novel microorganisms and metabolic pathways involved in the nitrogen cycle have been discovered.n Therefore, the overall aim of this thesis is to enrich and characterise novel microorganisms involved in the nitrogen cycle, in order to improve our understanding of the microbial nitrogen conversion processes and their interactions with other important nutrients cycles including carbon and metals.In terms of ammonium oxidation, ammonia-oxidising archaea (AOA) are critical ammonium oxidizers that regulate the nitrogen transformation in ubiquitous environments. AOA may have advantages when competing with ammonia-oxidising bacteria (AOB) in certain extreme environments such as acidic soils. Although 30% of the soils in the world are acidic soils, our understanding of the AOA living under low pH is insufficient. Therefore, the first research objective of this thesis is to enrich and identify a novel AOA under low pH. Using a fresh water reservoir sediments as inoculum, two novel microorganisms (an AOA and a NOB) were enriched in a bioreactor operated at pH 4.5, and proved to play a critical role in ammonium oxidation to nitrate. The AOA strain was assigned to the genus of Nitrosotalea and identified as a novel species Candidatus Nitrosotalea sp. GC1, while the NOB was clustered into a novel lineage within genus Nitrospira. According to the metagenomic analysis on Candidatus Nitrosotalea sp. GC1, the genes encoding enzymes for ammonium oxidation to nitrite were all identified and the Thaumarcheal HP/HB pathway was used for carbon fixation.As a member affiliating in the genus of Nitrosotalea, Candidatus Nitrosotalea sp. GC1 might have similar features to the other members, such as its acidophily, substrate affinity and nitrous oxide (N2O) production. As a critical step to help understand this novel AOA culture, the second research objective of this thesis is to characterise the effect of environmental conditions on Candidatus Nitrosotalea sp. GC1 and its N2O emission potential. With a series batch tests, the acidophily of strain GC was verified, with an optimal pH of 4.5. The Km of ammonium and O2 for Candidatus Nitrosotalea sp. GC1 are 39.7 p 3.2 and 35.2 p 3.0 mM, respectively, which were much higher than other AOA isolates, although still lower than most AOB. In addition, N2O emission from Candidatus Nitrosotalea sp. GC1 has a remarkable yield of 6.7%, which suggested the critical role of acidophilic AOA in greenhouse gas emission.In terms of nitrate reduction, dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are the main processes that convert nitrate to ammonium or inert dinitrogen gas respectively. Although organic carbon compounds are the main energy and electron sources for the most of known DNRA microorganisms, the mechanisms of methane driven DNRA process have not been investigated. Therefore, the third research objective of this thesis is to characterise the microorganisms and pathways involved in a culture performing methane driven DNRA process. In a bioreactor fed with methane and limited nitrate supply, continuous ammonium production from nitrate coupled with anaerobic oxidation of methane was observed, while an anaerobic methanotrophic archaea, Candidatus Methanoperedens nitroreducens (M. nitroreducens), dominated the microbial community. During batch tests, metagenomic and metatranscriptomic analyses showed that the DNRA related genes in Candidatus M. nitroreducens have significantly higher expression levels when the culture was producing ammonium, compared to their levels when the culture was fed with large quantity of nitrate to inhibit DNRA activity. Overall, the results confirmed that Candidatus M. nitroreducens can facilitate the methane-driven DNRA process.It has been shown that denitrification can couple to the oxidation of methane or metals (e.g. Fe or Mn), thus linking the nitrogen cycle to the methane and metal cycles, respectively. However, research works focusing on the interactions between nitrogen, methane and metal cycles in one system are rarely done. Therefore, the last research objective is to investigate interactions between anaerobic oxidation of methane and nitrate and metals reduction processes in one reactor. By setting up and incubating the bioreactor with methane, nitrate and ferrihydrite for more than 900 days, simultaneous methane oxidation and nitrate reduction were observed. Mass and electron balances suggested that in this system, nitrite/iron-dependent anaerobic oxidation of methane processes are coupled with nitrate-dependent iron (Fe) oxidation (NDFO) process. The community analysis suggested that Azospira sp. and Methylomirabiliaceae sp. may be responsible for these processes, while an unknown microorganism facilitated metal-dependent anaerobic oxidation of methane process.Overall, the discovery and characterisation of these novel microorganisms and metabolic pathways mediating ammonium oxidation and nitrate reduction will provide important insights into the understanding of the nitrogen cycle.n