Effect Of Elevated Atmospheric CO₂ Concentration On The Process Of Nitrite-Dependent Anaerobic Methane Oxidation In Paddy Fields

Global warming caused by elevated cabon dioxide（CO₂）concentration and its eco-environmental effects are one of the hot issues in current research.Methane oxidation is a key biological process to reduce CH₄ emissions from paddy fields.Therefore,the response of methane oxidation to elevated CO₂ concentration has attracted much more attention.N-damo（nitrite-dependent anaerobic methane oxidation）is a new pathway of methane oxidation,which plays an important role in controlling CH₄ emissions from paddy fields.However,so far,it is not clear how elevated CO₂concentration affects the n-damo process in paddy fields.In addition,the CO₂concentration in the atmosphere increases gradually,and the effects of different ways of rising CO₂ concentration（gradual increase and abrubt increase）on the n-damo process in paddy fields may be different.This study will exlpole the effects of gradual and abrupt increase of CO₂ concentration on n-damo activity,functional microbial abundance and community structure in paddy fields,and clarify its influence mechanism.The results will help to better predict the role of n-damo in methane oxidation in paddy fields under the condition of future climate change,improve the microbial mechanism of methane oxidation in paddy soil,and provide a scientific basis for controlling CH₄ emission from paddy fields.Based on the automatic regulation platform of farmland CO₂ concentration increase,the ambient CO₂ concentration was taken as the control（CK）,with two different rinsing CO₂ treatments:gradual increase（GI）and abrubt increase（AI）of CO₂concentrations.GI is an increase of 40μL·L^-1 per year from 2016 above CK until the increase reaches 200μL·L^-1.AI is to directly increase 200μL·L^-1 above CK since 2016.The sampling period of this study is 2 years（2019 and 2020）.The 0～20 cm soil layers were collected each year in three typical growth period of rice（tillering,jointing and flowering stages）.The n-damo activity was determined through stable isotope experiments.The community composition and abundance of n-damo bacteira in soils were analyzed using high-throughput sequencing and quantitative PCR techniques.The main results are as follows:（1）The ¹³CH₄ stable isotope experiments showed the n-damo activity was0.31～8.91 nmol CO₂ g^-1（dry soil）d^-1 in 2019 and 2020.Combined with the experimental data from the two years,it is estimated that the methane oxidation capacity of n-damo is 4.36 g CH₄ m^-2 year^-1,which can reduce the CH₄ emission flux of paddy fields by about 16%.Both GI and AI promoted n-damo activity in paddy field.In 2019,GI(CK+160μL·L^-1)and AI(CK+200μL·L^-1)increased n-damo activity by5.4%and 90.9%,respectively,and AI was significantly higher than GI.In 2020,GI and AI(both CK+200μL·L^-1)significantly increased n-damo activity by 91.6%and 90.0%,respectively,but there was no significant difference between GI and AI.Therefore,the effects of different levels of rising CO₂ concentrations on n-damo activity are quite different,but the effects of different ways of rising CO₂ concentrations（GI and AI）on n-damo activity are similar.The above results indicate that the n-damo process has a positive response to elevated CO₂ concentration in the future,which is helpful to reducing CH₄ emission from paddy fields.（2）Quantitative PCR results showed that the 16S rRNA gene abundance was2.00×10⁶～8.04×10⁷ copies g^-1 dry soil in 2019 and 2020.Both GI and AI promoted the n-damo bacterial abundance in paddy fields.In 2019,GI and AI increased the n-damo bacterial abundance by 17.1%and 49.9%respectively,and AI was significantly higher than GI.In 2020,GI and AI significantly increased the n-damo bacterial abundance by67.7%and 98.6%,respectively,but there was no significant difference between GI and AI.This further suggests that different levels of rising CO₂ concentrations have different effects on the n-damo bacterial abundance,but there is no significant difference between different ways of rising CO₂ concentrations under the same CO₂level.GI and AI had no significant effect on cell-specific activity of n-damo bacteria,and there was a significantly positive correlation between n-damo bacterial abundance and n-damo activity.Therefore,elevated CO₂ concentration may enhance n-damo activity via stimulating n-damo bacterial abundance（not changing the activity of single cells）.（3）High-throughput sequencing results showed that AI treatment significantly reduced the number of OTUs and Chao1 index of n-damo bacteria,while GI had no significant effect on n-damo bacterial diversity in 2019.In addition,there was no significant difference in the composition of n-damo bacterial community among different CO₂ treatments in 2019 or 2020,indicating that GI and AI had little effect on n-damo bacterial community structure.（4）Statistical analysis showed that the changes of some physiochemical factors caused by elevated CO₂ concentration,particularly the contents of soil organic carbon,dissolved organic carbon and inorganic nitrogen,had important impacts on n-damo activity and bacterial abundance.In addition,n-damo activity and bacterial abundance in paddy soil had obvious spatial and temporal distribution characteristics.