The abundance, activity, and community composition of comammox Nitrospira and canonical ammonia oxidisers in New Zealand soils : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

Nitrification, the microbial oxidation of ammonia (NH3) via nitrite (NO2-) to nitrate (NO3-) is an important process in terrestrial ecosystems, as it contributes to the production of two environmentally significant products, nitrous oxide (N2O), and nitrate. Traditionally, nitrification was thought to be a two-step process, where ammonia is first converted to nitrite by ammonia-oxidising bacteria (AOB) before nitrite oxidising bacteria (NOB) complete the oxidation to nitrate. Later, it was discovered that a group of archaea (ammonia oxidising archaea, AOA) could also undertake ammonia oxidation, typically in oligotrophic/extreme conditions such as low ammonia availability and pH. The separation of nitrification into two steps involving different microorganisms has puzzled scientists as a single organism completing both steps of nitrification was theoretically assumed to be more efficient. The presence of complete ammonia oxidisers (comammox) was later confirmed by cultivating them from an aquaculture system and a core from a deep-sea oil well. It was found that comammox belongs to lineage II of the genus Nitrospira, a group of bacteria traditionally thought to be responsible for nitrite oxidation. Comammox Nitrospira and canonical Nitrospira can be distinguished by the presence of the ammonia monooxygenase gene (AMO). Furthermore, comammox Nitrospira can be separated into clade A and clade B based on the phylogeny of this gene. Clade A can also be further divided into sub-clades A.1, A.2.1, A2.2, and A.3. Since its initial discovery, comammox Nitrospira has been found in a variety of terrestrial ecosystems. Typically, studies have shown that clade B are more abundant in forest and paddy soils, whilst clade A.2 may prefer agricultural soils. Clade A.1 is seen to be the dominant cluster in natural and artificial aquatic ecosystems such as freshwater wells and wastewater treatment plants. However, the presence and distribution of comammox Nitrospira, relative to canonical ammonia oxidisers, in different soils and relationships with soil and environmental conditions are not fully understood. The research described in this Thesis was designed to fill this knowledge gap and improve our understanding of comammox Nitrospira and canonical ammonia oxidisers. Experiment 1 determined the abundance and community composition of comammox Nitrospira throughout New Zealand Dairy farms and quantified the abundance and community composition of comammox Nitrospira in New Zealand under various land uses. It was concluded that comammox Nitrospira are ubiquitous throughout New Zealand soils. Comammox Nitrospira amoA abundance shared a strong positive and strong negative correlation with soil moisture and pH, respectively. Interestingly, the sequencing analysis determined that the comammox Nitrospira community solely consisted of clade B. Two experiments were devised to investigate these correlations, with respect to canonical ammonia oxidisers. Experiment 2 explored the effect of soil pH, with N inputs, on comammox Nitrospira abundance and community composition in New Zealand dairy pasture soils. Comammox Nitrospira preferred the natural (6.1-6.2) soil pH with no nitrogen amendment. Comparatively, the AOB community (dominated by Nitrosospira) responded positively to soil pH and nitrogen input. This may be due to the difference in ammonia availability. Estimated ammonia availability in the synthetic urine treatments (equivalent to 700 kg N ha-1, N700) accurately predicted the AOB amoA gene abundance. Interestingly, the AOA communities (which were predominantly made up of Thaumarchaeota group I.1b clade E) seemed to prefer the natural and high pH soils over the low pH. This may be due to the lineage of AOA present. AOA did not respond to the application of nitrogen. Experiment 3 investigated the effect of soil moisture and temperature on comammox Nitrospira abundance, transcriptional activity, and community composition, relative to canonical ammonia oxidisers. AOB was the dominant nitrifier in the synthetic urine treated soil regardless of temperature or moisture. Peak AOB amoA transcript abundance was positively correlated with estimated soil ammonia availability. While the nitrification rate and changes in AOB amoA gene abundance followed a similar relationship. Ammonia oxidising archaea were strongly influenced by soil temperature. At 20 °C, AOA amoA peak transcript abundance averaged over 1 order of magnitude higher than at 8 °C. A member of the AOA community associated with the Nitrosocosmicus subclade was positively correlated with ammonium and estimated soil ammonia concentrations. The presence and relative increase of Nitrosocosmicus AOA in a high nitrogen environment poses an interesting contrast to the current scientific opinion. Contrary to Experiment 1, the abundance of comammox Nitrospira amoA was not positively correlated with soil moisture. This suggests that the association is more complex than previously thought. Further research is required to determine the drivers of comammox Nitrospira abundance in a high moisture environment. Overall, the results of this thesis indicate that in New Zealand, AOB are the dominant ammonia oxidiser in a nitrogen-rich environment, such as a dairy farm soil. While the majority of the AOA community prefer a high temperature, low nitrogen environment. However, Nitrosocosmicus-like AOA may respond positively to nitrogen amendment, which challenges our current understanding of terrestrial AOA. Comammox Nitrospira may prefer a slightly acidic, oligotrophic soil environment and do not respond to temperature change. However, they may be the main ammonia oxidiser in some high moisture environments, potentially due to biotic interactions with plants or microbes.