Effects Of Nitrogen On Methane Oxidation In Soil And Its Microbial Mechanisms

Methanotrophs, methane oxidation bacteria, play a key role in global methane emission as a bio-filter and it’s an important part of the global carbon cycle. However, the activity and growth of methanotrophs can be susceptible to many environmental factors, in which nitrogen is a key regulator. The effect of nitrogen on methane oxidation in soil had been the most investigated as a regulating factor in the pass decades. But until now, no one can illustrate exactly the results observed in natural systems. Therefore, in order to elucidate the meachnism of nitrogen impacts on methane oxidation, a series of experimental designs were setted, which included applying a series of advanced experimental methods (such as meta-transcriptome). We explored the effect of ammonium on methane oxidation and methanotriphic community in different soils, and the effect of two types of nitrogen (nitrate and ammonium) on methane oxidation. The main results are as followings:(1) The effect of ammonium on methane oxidation and methanotriphic community was compared between different soils. The results showed that methane oxidation of rice paddy soil and wetland soil were stimulated by ammonium, but methane oxidation of upland soil was inhibited by ammonium. Ammonium changed the methanotrophic community composition and population number (or both) of soil, which in return affected soil methane oxidation.(2) In order to study effects of different types of nitrogen on methane oxidation community, we established two methane oxidation enrichments from Hangzhou rice paddy soil through adding certain amount of either ammonium or nitrate and using methane as sole carbon source. Methylocystis genus was the major component of the enrichment that using ammonium, which accounted for 80% of the total community. While, the other enrichment obtained by using nitrate were dominated by Methylophilus genus (about 40%) and Methylocystis genus (about 45%). Therefore, different types of nitrogen differentially selected certain types of population to form a stable functional consortium.(3) Incubation with a series of ammonium concentrations were performed to reveal the relationship between methane oxidation and nitrogen fixation, and the effect of ammonium on methane oxidation and nitrogen fixation. Growth of enrichment was suppressed when ammonium concentration exceeded 10 mM. Nitrogen fixation were triggered to alleviate nitrogen requirement under nitrogen limited conditions. However, the rate of methane oxidation declined. Nitrogen fixation was immediatly switched off in response to additional ammonium added, meanwhile the rate of methane oxidation was elevated.(4) Meta-transcriptomics was performed to reveal the mechanisms of ammonium promoted methane oxidation. The results revealed Methylocystis genus in ammonium enrichment possesses two pMMO isozymes coding gene (pmoCAB1 and pmoCAB2). Furthermore, the pmoCABl gene was mainly used. The serine cycle, TCA cycle and Pyruvate metabolism were not affected by ammonium concentration. Based on the observation of gene expression, we proposed that Methylocystis genus was able to employ nitrogen regulating system (ntr system) to trigger nitrogen fixation under nitrogen deficiency, then major part of electron derived from the oxidation of formaldehyde and formate were distributed to nitrogen fixation. This process resulted in reduction of electron supply to the first step in methane oxidation (supply of electron to the monooxygenase), lead to the decreasing of methane oxidation rate. Nitrogen fixation was subjected to rapid "switch-off" in response to additional ammonium added. The diversion of electron flow was blocked, then methane oxidation was stimulated immediately.(5) Incubation with a series of nitrogen concentrations treatments were performed to reveal that the relationship between methane oxidation and denitrification. Our results showed that nitrogen stimulated both denitrification and methane oxidation, nitrogen stimulated methane oxidation via coupling it to denitrification.It was the first time that nitrogen fixation was revealed to have impact on methane oxidation. Our results illustrated that ammonium added stimulated methane oxidation through ammonium absolved the competition of electron between nitrogen fixation and methane oxidation. What’s more, we revealed that nitrogen stimulated methane oxidation via coupling it to denitrification. These results deepen and broaden our understanding of microbial mechanisms of nitrogen stimulated methane oxidation in soil.