Mechanisms Of Nitrogen And Oxygen Regulation On Methane Oxidation In Rice Field Soils

Rice field is one of the main methane sources, but approximately a half of methane produced in anoxic environments is consumed by methanotrophs before it is escaped to the atmosphere. The activity and growth of methanotrophs in soils are regulated by many environmental and agricultural factors including nitrogenous fertilizers and oxygen availability. In Asian rice field soils, nitrogen is a yield-limiting factor of rice production. To this aim, we conducted laboratory incubation experiments of rice soils from Hangzhou to investigate the mechanisms of nitrogen and oxygen regulation on methane oxdation and related bacteria by applying a series of chemical and molecular methods.A laboratory incubation experiment with a series of ammonium and nitrate concentrations was performed to determine the differential effects of two nitrogen forms on CH4oxidation and related bacteria. CH4oxidation was stimulated by both ammonium and nitrate, and the effects increased with increasing N addition rates for both N forms. But nitrate stimulated CH4oxidation to a greater extent than ammonium per unit N base. The community structure and abundance of total bacteira and functional populations were analyzed by T-RFLP, clone&sequencing and qPCR techniques. The methanotrophic community consisted of type I and type II methanotrophs, with the former being predominant. Nitrate promoted both types of methanotrophs, while ammonium promoted type I but suppressed type II methanotrophs. Moreover, nitrate application markedly stimulated abundance of nirS-type denitrifiers and Methylotrophic denitrifiers of the Methylophilaceae indicating the activation of denitrification. Therefore, nitrate is probably a better N source for both types of methanotrophs in rice field soil. Furthermore, in order to understand molecular bases for the nitrogen regulation on methanotrophs, we combined stable isotope probing (SIP) with metatranscriptome techniques to identify changes in gene expressions under nitrogen-limiting condition. Expression of genes involved in nitrogen assimilation, such as nitrogen transporter, increased significantly when nitrogen is limiting, suggesting that methanotrophs have more demand for nitrogen assimilation to cope with nitrogen-limiting condition. However, nitrogen limitation significantly reduced the expression of genes required for biosynthesis processes (e.g. formaldehyde assimilation, amino acids biosynthesis, protein biosynthesis and related translation) as well as the energy-generating pathways (e.g. formaldehyde oxidation via formate to CO2and electron transfer chain).Different initial concentrations of O2were tested to determine the effect of oxygen tensions on CH4oxidation and bacteria involved. CH4oxidation was more favorable to the lower O2(5%) under nitrogen-limiting condition, while it increased with O2concentration in the nitrogen treatments. But nitrate stimulated CH4oxidation more significantly than ammonium at all O2levels. We combine stable isotope probing (SIP) with T-RFLP and454pyrosequencing techniques to determine the active bacteria and methanotrophs at different phylogenetic levels. Type I methanotrophs, Methylosarcina in particular, responded fast and dominated the CH4oxidation activity with both N forms at all O2levels. In addition, nitrate could also stimulated the incorporation of13CH4into Methylobacter as well as a small proportion of nonmethanotrophic bacteria capable of denitrification at all O2levels. Moreover, Methylomonas and Methylomicrobium which could probably tolerate high ammonium concentration contributed to CH4oxidation activity mainly at0.5%O2level, and thus methanotrophs could still survive and grow even at very low oxygen tension. Furthermore, molecular bases for the oxygen regulation on methanotrophs by SIP/metatranscriptome techniques were as follows. Lower oxygen tension would significantly reduce expression of genes involved in nitrogen assimilation (e.g. nitrogen transporter and assimilatory nitrate reduction) and H4MPT-based formaldehyde oxidation. Additionally, expression of genes required for electron transfer chain and H2production was relatively increased under oxygen-limiting condition.In conclusion, by laboratory incubation experiments, our research gives a comprehensive and systematic study of the effects of nitrogen and oxygen on CH4oxidation and related bacteria, as well as the mechanisms of these effects, which could provide melecular bases for mitigation of methane oxidation from rice fields.