The Characteristics Of Nitrate Reduction Process And The Related Function Microbes In Paddy Soils Under Different Environmental Conditions

Rice is the main food crop in the world,which occupies an important position in agricultural production in China.Due to the unique farming management practice,paddy soils are mainly flooded during rice planting,accompanying with intermittent dry.The frequent alteration of flooding and drying make the oxidation and reduction reactions in paddy soil active.At the same time,the excretive oxygen of rice root also makes the paddy soil long-term in the presence of aerobic/anaerobic state,both conducive to the occurrence of nitrification process and the nitrate reduction processes.Excess nitrogen fertilizer input in paddy soil can be reduced to N₂ via nitrate reduction pathways such as anaerobic oxidation and denitrification.Furthermore,denitrification process produce N₂O,the greenhouse gas with strong warming effect,resulting in a large loss of nitrogen and environmental problems.It is important to study the environmental impact factors and related microbial mechanisms of nitrate reduction in paddy soils,which is of great significance to improve the nitrogen use efficiency and reduce the negative impact of nitrogen on the environment.Therefore,in this study,we collected a series of 64 paddy soils representing different salinity,pH and organic carbon levels along the coastal to inland directions.We used the ¹⁵N labeling technique and combined with quantitative PCR,cloning sequencing and Illumina Miseq high throughput sequencing to investigate the reaction rate of denitrification and anaerobic ammonium oxidation in different environmental conditions and the characteristics of function microorganisms involved in the two processes.On the basis of this,the relationship between nitrate reduction processes and their functional genes in three representative soils was further explored through microcosm incubation experiment.The results are as follows:1)The results of isotopic tracer experiments on field samples showed that the rates of anaerobic ammonium oxidation（ANAMMOX）in the paddy soils varied between 0-0.479 nmol N g^-1 dry soil h^-1 and could not be detected in some samples.Anammox contributed 0-12.9% of the total N₂ production.On the other hand,the rate of denitrification process was between 0.84-31.314 nmol N g^-1 dry soil h^-1,and the contribution of the total N₂ production was 87.1%-100%,which indicated that the denitrification was the main way of N₂ loss in the paddy soils.Based on 16 S rRNA and hzsB gene cloning sequencing results showed that all of the samples had single community and only had a dominant community.For the high salinity and high pH samples TT the Candidatus Scalindua was dominant community,and Candidatus Brocadia was the advantage of paddy soil taxa.RDA results showed that soil pH,C/N,moisture content and EC significantly influenced anammox bacterial community composition and distribution.2)The nitrification and denitrification potentials of paddy soil were very high and the surface soils were higher than subsurface soils.The abundance of the archaea-nirK gene was the highest in the TT surface sediments,which was 2.42×10⁸ copies g^-1 dry soil,and decreased with the decrease of salinity gradient.The abundance of archaeanirK was about 2.73×10⁵-2.49×10⁷ copies g^-1 dry soil in the coastal saline soil,but even in inland paddy soil or even undetectable.In contrast,the distributions of bacteria-nirK gene in soil samples were more extensive,the abundance of nirK gene was 1.23×10⁷-7.36×10⁷ copies g^-1 dry soil.There was a positive correlation between the abundance of archaea-nirK gene and pH and EC,and had a negatively correlated with DEA.But for the abundance of bacteria-nirK gene was positively correlated with NH₄⁺,DOC,DON and DEA,indicating that the denitrification was only in the high pH,high EC environment archaea-nir K may play a leading role.Clustering analysis showed that the sequence of AnirK gene belonged to Thaumarchaeota,and the sequence of nirK gene from paddy soil was far away from the sequence of coastal sediments and wetland samples,while soil properties pH and EC significantly influence Thaumarchaeota-nirk gene’s community composition.Based on the nirK gene Miseq sequencing studies showed that the community diversity was higher in the paddy soil,and the surface soil richness was higher.For the high salt and high pH sites of TT,SY and FQ,the Bacteria_unclassified bacteria were the dominant bacteria,and it had 30% relative abundance in paddy soils.While the paddy soils denitrification bacteria of Rhizobium and Proteobacteria were dominant,and pH and EC significantly influence denitrifying microbial community composition.3)In order to further validate the activity of the archaea-nirK containing in denitrification and the key factors influencing its activity,we selected the tidal flat sediment（TT）representing high salt and pH and two representatives paddy soils（FQ,HZ）of medium pH and low salinity for the microcosm culture experiment.The experimental results show that: The denitrification activity of the three soils was FQ> TT> HZ,and the denitrification rate decreased with the increase of the culture time.Under the condition of saturated water content,the denitrification activity was significantly higher than that of the flooding.Correspondingly,the abundance of archaea-nirK gene was the highest in FQ samples,followed by TT,but not in HZ samples,and was significantly higher than that under flooding conditions under saturated water content.There was a significant positive correlation between the denitrification rate and the archaea-nirK gene copy number,but it was negatively correlated with the bacteria-nirK gene copy number,indicating that the archaea-nirK contributed an important contribution to the denitrification in FQ and TT samples.During the culture,the accumulation of NO+3^- and the production of N₂O in HZ soil samples were significantly lower than those in FQ and TT,and the content of NH₄⁺ was significantly higher than that of FQ and TT,which indicated that HZ soil may occur dissimilatory nitrate reduction to ammonium（DNRA）process during the culture.Thaumarchaeota-nirk genes community distribution was consistent with the field sample survey,all the AnirK sequences belonged to Thaumarchaeota,the Cluster Ⅰ and Ⅱ were mainly from paddy soil FQ,and Cluster Ⅲ and Ⅳ were from sediment TT.Meanwhile pH,EC and inorganic nitrogen significantly influence AnirK gene abundance and its community composition.