Study On Soil Anammox Process And Its Effect Factors In Paddy Soils With Paddy-upland Rotation System

At present,the anammox process as a recognized nitrogen oxidation pathway is widespread in the anoxic natural environment,but its role in the nitrogen cycle is still lack of in-depth understanding.Based on the previous research on the anammox activity in terrestrial ecosystems,anammox process in paddy field was studied by laboratory incubation and long-term fertilization experiment.15N isotope tracer technique,real time quantitative PCR,DNA-SIP,cloning library and high-throughput sequencing were used to detect the contribution of soil anammox to nitrogen production,anammox microbial community structure and its effect factors.The main findings are as follows:1)The results showed that the anammox activity was detected in both wheat and rice season,and there were significant differences between the farming seasons.The anammox rate in rice season was 0.96 nmol N2·g-1·-1,and 0.46 nmol N2·g-1·-1 in wheat season,There was no significant difference in anammo rate between different fertilization treatments.Overall,3.15%-9.62%nitrogen loss in the soil was contributed by anammox during the process of paddy and upland rotation.Furthermore,the abundance of anammox bacteria was analyzed and found that high abundance of anammox bacteria was detected in both rice and wheat season.The abundance of anammox bacteria under different fertilization treatments was as follows:PMCF>SRCF and CF>CK.The results of anammox hydrazine synthase beta subunit(hzs-?)gene sequencing showed that the anammox bacteria had higher homology with Ca.Brocadia,Ca.Scalindua and Ca.Jettenia during the rice season.In contrast,all the anammox hydrazine oxidase(hzo)gene had only high homology with Ca.Brocadia,indicating that the hzs gene was more sensitive than the hzo gene.The sequences in PMCF treatment had high homology with Ca.Jettenia and Ca.Brocadia and showed the highest diversity.2)The effects of different organic carbon sources(Non-exogenous carbon application(CK),application of urea(UR),application of straw(SR),application of pig manure(PM),application of starch(ST),application of glucose(GL))on the abundance,community structure and activity of anammox were studied by laboratory incubation experiments.The results showed that the rate of anammox and the contribution to nitrogen loss were 1.04-3.19 nmol N2·g-1·-1 and 4.22%-22.25%after incubation by adding different organic carbon sources.Although the anammox activity was detected in all treatments,the rate of anammox is different in each treatments.The highest anammox rate was in CK treatment,and the anammox rate after adding other organic carbon sources was low.The addition of organic carbon sources has a significant inhibitory effect on the anammox activity and significant effect on denitrification activity.The effect of carbon source addition on the anammox rate was as follows:GL>ST,UR>SR.The results of qPCR showed that different organic carbon sources had some inhibitory effects on the abundance of anammox bacteria,but PM treatment had the least effect on the anammox abundance.DNA-SIP analysis of the 13C anammox hydrazine synthase beta subunit(hzs-?)functional gene sequencing was carried out in PM and CK treatments which anammox rate was high.It was found that active anammox bacteria had high homology with Ca.Brocadia.3)The effects of different water conditions and dissolved oxygen concentrations(70%FC;wet-dry alternation(flooding 7 days,dry 7 days);flooding I:dissolved oxygen concentration of 5.8 mg·L-1;flooding ?:dissolved oxygen concentration of 2.6 mg·L-1)on anammox were studied by using the long-term experiment soil.The results show that the anammox rate ranged from 0.56 to 1.47 nmol N2·g-1·h-1.The lowest anammox rate was in 70%FC treatment(0.61 nmol N2·g-1·-1)and the highest(1.14 nmol N2 g-1·h-1)in flooding ? treatment,indicating that higher dissolved oxygen concentration had a certain inhibitory effect on anammox rate.Moisture treatment had a significant effect on the abundance of anammox bacteria,whether it is wet-dry alternation or flooding,the abundance of anammox bacteria was high;the abundance of anammox bacteria in CF and SRCF treatment was high in different fertilization treatments.The structure of anammox bacteria was not significantly different with different water conditions.Most of the anammox bacteria had high homology with Ca.Brocadia,while Ca.Jettenia was less.