Research On Simultaneous Reduction And Mechanisms Of Methane And Nitrous Oxide

Wetland is one of the important sources of greenhouse gas,methane?CH₄?and nitrous oxide?N2_O?.The warming potential of CH₄ and N2_O is 25 times and 310 times that of carbon dioxide?CO2?,which has an important impact on global climate change.Microorganisms play a major role in the production and reduction of CH₄ and N2_O in constructed wetlands.In the anaerobic zone of the substrate,CH₄ is produced from fermentation the organic matters,which is oxidized to form CO2 in aerobic/anaerobic area or release directly.N2_O is produced in the process of microbial nitrification and denitrification,and can be further reduced to N2.So far,the production and consumption of the three greenhouse gases?CH₄,CO2 and N2_O?has been associated by the process of CH₄ oxidation coupled to denitrification.However,the microbiological mechanism is still ambiguous.CH₄ can be used as the carbon source for denitrification.N2_O is an important product of incomplete denitrification process.There must be a correlation between the production and release of CH₄ and N2_O.However,there is little study on the relationship of CH₄ and N2_O.In the present study,the relationship between the release of the three greenhouse gases was investigated.The release of CH₄,CO2 and N2_O in constructed wetlands was discussed.By combining chemical thermodynamic analysis and pilot test,the simultaneous reduction of CH₄ and N2_O was verified.By long-term enrichment of microorganisms under laboratory conditions,the N2_O-dependent methane oxidation was verified and the influent factors as well as microbial mechanisms were finally determined.The proposed microbial mechanism provided a theoretical basis for simultaneous reduction for emission of CH₄ and N2_O in constructed wetlands.This thesis is of great significance for improving the carbon and nitrogen cycle in the constructed wetlands.The main results are as follows:?1?In constructed wetlands,the release flux of CH₄ and N2_O is in positive correlation,both of them have a negative correlation with the release of CO2.In this study,two substrate types of surface-flow constructed wetland pilots,mud substrate and soil substrate,were constructed.The release of greenhouse gases,removal of carbon and nitrogen pollutants and microbial community,as well as the relevant relationship between production and release of greenhouse gases,were determined.The results showed that,compared with soil substrate,the average release flux of N2_O and CH₄ in the mud substrate was significantly lower,and the average release flux of CO2 was significantly higher.There is a significant positive correlation between the release fluxes of CH₄ and N2_O,and they are negatively correlated with the release of CO2.There was a significant positive correlation between the release of CH₄ and N2_O and the relative abundance of Verrucomicrobia.The release of CO2 was significantly positively correlated with the relative abundance of Thiobacillus.Both mud and soil substrate constructed wetlands achieved high removal efficiency of carbon and nitrogen contaminants.The denitrification in mud substrate constructed wetlands is more active than that of soil substrate,but the removal efficiency of COD in soil substrate system is higher.The microbial abundance in the mud substrate is higher than that in soil substrate,especially the relative abundance of Thiobacillus,and the relative abundance of the Cyanobacteria in soil substrate is higher.?2?N2_O in the constructed wetland substrate can inhibit the production of CH₄ and promote the oxidation of CH₄,thus achieving the simultaneous reduction of CH₄ and N2_O.In this study,different types of wetland substrates were selected to study the production and release of CH₄ and N2_O.The stable isotope trace technology and metagenomics sequencing were used to clarify the simultaneous reduction of CH₄ and N2_O and microbial response mechanisms.The simultaneous reduction of CH₄ and N2_O was detected in the Xiaomei River and Baiyun Lake constructed wetlands substrate.The CH₄ released by the experimental group added with N2_O decreased by 58.96%and 72.90%in Xiaomei River and Baiyun Lake substrate,respectively,compared with the control group.The expression activity of the methanogen was inhibited by adding N2_O.The effects of N2_O on the oxidation of CH₄ were analyzed by further short-term culture test and stable isotope tracing.It was found that the addition of N2_O promoted the expression activity of methanotrophs by 1.1 times,the CO2 production rate was 1.45 times that of the control group,and N2_O promoted CH₄ oxidation by 3.41 mmol CO2/g dry weight sediment/d.It was also verified by Gibbs free energy calculation that the reaction process of N2_O reduction and CH₄ oxidation is spontaneous.The complete methane oxidation pathway was annotated by metagenomic sequencing.?3?The process of nitrous oxide-dependent methane oxidation was proposed and verified.The nitrous oxide-dependent methane oxidation is inter-aerobic methane oxidation process coupled to nitrous oxide reduction.In this study,an enrichment reactor was operated under anoxic conditions,and the substrate from Xiaomei River wetland was cultured by adding CH₄ and N2_O as main carbon,energy sources and nitrogen sources,respectively.The change of microbial community structure at different stages was analyzed.The process of nitrous oxide-dependent methane oxidation was verified.The results showed that,after 500 days of enrichment,the abundance ofpmoA gene,indicating methanotrophs,and nosZ gene,indicating nitrous reduction,increased by 38 and 8 times,respectively.The genus Methylocaldum under the classify of Methylococcus is the most abundant methane oxidizing bacteria,the genus Thauera the classify of Rhodocyclales became the most abundant denitrifying bacteria.Stable isotope tracing test directly confirmed that the simultaneous reduction of N2_O and CH₄ is a N2_O-dependent methane oxidation process,and nitrous oxide-dependent methane oxidation is a N2_O reduction coupled with internal aerobic CH₄ oxidation.?4?The microbial mechanism of nitrous oxide-dependent methane oxidation was coordinative operation of methanotrophs and denitrifiers,and the impact factors were clarified.In this study,two anaerobic culture bottles were used to determine CO2 production rate by setting different carbon nitrogen ratios and temperature conditions.The microbial community structure,expression activity and function were also analyzed.And the microbial mechanism of nitrous oxide-dependent oxidation of methane was identified.It was found that the enrichment conditions of high N2_O ratio?n N2_O:nCH₄=2:1?is better than loW N2_O ratio?1:1?.Low N2_O concentration is more favorable for the process of nitrous oxide-dependent methane oxidation than high N2_O concentration.In the process,the actual ratio of CH₄ and N2_O consumption is about 76:1,and the CO2 production rate is higher during the first 12 h,the highest rate was up to 1.08 ?mol/L.The mechanism ofN2_O-dependent CH₄ oxidation is the coordinative operation of methanotrophs and denitrifiers.The temperature of 20? is conducive to N2_O reduction,and 35? is beneficial to CH₄ oxidation.Aerobic methane oxidizing bacteria can utilize CH₄ as carbon source and energy source,and denitrifying bacteria use their released organic intermediates as electron donors to reduce N2_O.