Distribution Characteristics Of Anaerobic Ammonia-oxidizing Bacteria And Anaerobic Methane Oxidizing Bacteria In Winter Flooded Paddy Field In Different Seasons

The proposal of anaerobic ammonium oxidation（ANAMMOX）reaction and nitrite-dependent anaerobic methane oxidation（N-DAMO）reaction with NO₂^--N as electron acceptor not only provides new ideas for the study of global nitrogen and carbon cycles,but they also have important ecological significance for greenhouse gas emissions.In the autotrophic ANAMMOX reaction,only a small amount of N₂O is produced to the external environment.The N-DAMO reaction can oxidize CH₄ to form equimolar CO₂,and significantly reduce the greenhouse effect caused by the same amount of gas emissions.Long-term cultivation will make the soil of winter flooded paddy field rich in organic matter,which is the ideal environment for CH₄ production.Winter flooded paddy field due to abandonment in winter will make nitrogen cannot be absorbed and lost in the form of N₂O,resulting in N₂O accumulation.In addition,due to long-term flooded,the soil of winter flooded paddy field will be in an anoxic state,which provides suitable habitats for ANAMMOX bacteria and N-DAMO bacteria.Therefore,studying the distribution characteristics of ANAMMOX bacteria and N-DAMO bacteria in winter flooded paddy field in different seasons is of great significance for reducing greenhouse gas emissions in winter flooded paddy field ecosystems and developing green agricultural industries.It also provides a theoretical basis for forming a sustainable development system for winter flooded paddy field and continuously promoting the positive evolution of cultivated soil fertility.This study take different soil layers（0-20 cm,20-40 cm and 40-60 cm）of CT and RNT in different seasons（summer and winter）as the research objects,and using molecular biology and high-throughput sequencing technologies to explore the distribution characteristics of ANAMMOX bacteria and N-DAMO bacteria in winter flooded paddy field in different seasons.The main findings are as follows:1.Distribution characteristics of ANAMMOX bacteria in winter flooded paddy field in different seasons:The number of OTU,Chao1 and Ace index in SCT 20-40 cm soil layer were 57,64.80 and 66.46,respectively,which were higher than those in 0-20 cm and 40-60 cm soil layer.The number of OTU,Chao1 and Ace index in WCT 20-40 cm soil layer were 42,43.50 and 42.92,respectively,which were higher than those in 0-20 cm and40-60 cm soil layer.The number of OTU,Chao1 and Ace index in SRNT 20-40 cm soil layer were 79,83.00 and 84.08,respectively,which were higher than those in 0-20cm and 40-60 cm soil layer.In addition,the number of OTU,Chao1 and Ace index in winter flooded paddy field in summer were higher than those in winter.The results of community structure analysis showed that the OTUs sequence of ANAMMOX bacteria obtained in CT soil in different seasons belonged to Candidatus Jettenia sp.and Candidatus Brocadia sp..The OTUs sequence of ANAMMOX bacteria obtained from RNT soil in different seasons belonged to Candidatus Jettenia sp.,Candidatus Brocadia sp.and the bacteria genus without clear taxonomic status.The Student’s T test showed that the abundance of OTU93 in WRNT soil was significantly higher than that in SRNT soil（p<0.001）.And the abundance of OTU122 in SRNT soil was significantly higher than that in WRNT soil（p<0.001）.PCA analysis at OTU level showed that PC1 and PC2 explained 43.63%and 26.23%,respectively,and the cumulative explanation was 69.86%.The similarity of ANAMMOX bacteria community in SCT 40-60 cm soil layer was significantly different from that in 0-20 cm soil layer and 20-40 cm soil layer,and the similarity of ANAMMOX bacteria community in WRNT soil layer was significantly different.In addition,the similarity of ANAMMOX community in SCT 40-60 cm soil layer and WCT 40-60 cm soil layer was significantly different,and the similarity of ANAMMOX community in RNT soil was significantly different in different seasons.RDA and Heatmap analysis showed that TP,SOM,TK,p H,NO₃^--N and NH₄⁺-N were the most influential factors for the community structure of ANAMMOX bacteria in CT soil in different seasons and depths.CEC,TN,SOM,TK,p H,NO₃^--N and NH₄⁺-N were the most important factors affecting the community structure of ANAMMOX bacteria in RNT soil in different seasons and depths.2.Distribution characteristics of N-DAMO bacteria in winter flooded paddy field in different seasons:The representative OTU sequences obtained by sequencing were compared with the NCBI database,and the non-N-DAMO sequences were eliminated to ensure the accuracy of sequence analysis.The abundance of N-DAMO bacteria in winter flooded paddy field in different seasons was basically as follows:40-60 cm>20-40 cm>0-20cm.Student’s T test showed that the abundance of OTU3863 in WCT soil was significantly higher than SCT soil（p<0.05）,and the abundance of OTU2890 in SCT soil was significantly lower than WCT soil（p<0.001）.NMDS（stress=0.076）analysis at the OTU level showed that the similarity of N-DAMO community in SCT and WCT soil was significantly different,and the similarity of N-DAMO community in SRNT and WRNT soil was also significantly different.The similarity of N-DAMO community in the soil at different depths of SRNT showed great differences,and the similarity of N-DAMO community in the soil at different depths of WRNT also showed great differences.RDA and Heatmap analysis showed that TP,TN,TK,p H,NO₃^--N and NH₄⁺-N were the most important factors affecting the community structure of N-DAMO bacteria in CT soils of different seasons and depths.CEC,TN,TP,TK,p H,SOM and SWC were the major factors affecting the community structure of N-DAMO bacteria in RNT soils in different seasons and depths.In summary,the main conclusions are as follows:（1）The ANAMMOX bacteria in winter flooded paddy field in different seasons have certain spatial and temporal heterogeneity.The 20-40 cm soil layer in winter flooded paddy field is suitable for the survival of ANAMMOX bacteria.The abundance and biodiversity of ANAMMOX bacteria in winter flooded paddy field in summer are higher than those in winter.The community composition of ANAMMOX bacteria in the RNT soil in different seasons is quite different.（2）The ANAMMOX bacteria in winter flooded paddy field in different seasons are all belonging to Candidatus Jettenia sp.and Candidatus Brocadia sp..（3）SOM,TK,p H,NO₃^--N and NH₄⁺-N are the main environmental factors affecting the community structure and distribution of ANAMMOX bacteria in winter flooded paddy field in different seasons.（4）The N-DAMO bacteria in winter flooded paddy field in different seasons have certain spatial and temporal heterogeneity.The40-60 cm soil layer in winter flooded paddy field is suitable for the survival of N-DAMO bacteria.There is no significant difference in the relative abundance of N-DAMO bacteria in winter flooded paddy field in different seasons,but the community composition of N-DAMO bacteria in winter flooded paddy field varies greatly in different seasons.（5）p H,TN,TP,TK and NO₃^--N are the main environmental factors affecting the community structure and distribution of N-DAMO bacteria in winter flooded paddy field in different seasons.