Study On Anammox Of Farmland Soil And Its Effects On Nitrogen Cycling

The anaerobic ammonium oxidation(Anammox)process is globally an important nitrogen-cycling process mediated by specialized microbes.However,still little information is documented about the role and status of Anammox and Anammox microorganisms in agricultural soils.Based on the study of Anammox at home and abroad,Anammox process in farmland soil,especially focused on paddy soil,were studied by laboratory anaerobic incubation and long-term field fertilizer experiments.Real time quantitative PCR,PLFA-SIP,15N isotope tracer technique,DNA-SIP,cloning library and sequencing were used to study the effects of 13C-urea-PLFA on the anaerobic ammonium oxidation,the effects of tillage system and fertilization on the anaerobic ammonium oxidation process in paddy soil and the contribution and activity of different microbes in Anammox process.The main findings are as follows:(1)The results showed that the 13C was preferentially incorporated in ratios PLFAs 16:l?7c,16:lco5c and 16:0.The abundance of the Anammox bacteria(both hzs-? and hzo)were observed in vegetable soil V1 and paddy soils(R1 and R2),and also they were positively correlated with 13C-urea but were negatively correlated with NO3--N and NH4+-N concentrations.Thus,13C-PLFAs 16:1?7c,16:lco5c and 16:0 could be the biomarker as soil Anammox.The anaerobic microbial community composition of soils under different land use systems was diverse,and V1,R1 and R2 had similar microbial diversity and higher microbial biomass.The principal component analysis between soil properties and genes abundance suggested that not only pH but also soil organic matter,available P and available K were important factors of the Anammox process.This study suggested that 13C-Urea-PLFA for anaerobic incubation was a simple method to study Anammox microbial community structure through effecting the soil nutrients,and the different land use systems played important roles in determining the microbial composition of soils.(2)Anaerobic incubation experiment using 13C-Urea was conducted to study the impacts of different fertilizer treatments during wheat and rice seasons on the diversity and activity of the Anammox bacteria by using the stable-isotope probing of phospholipid fatty acids(PLFA-SIP).The results showed that the fertilization and seasonal changes can affect soil properties,microbial biomass and microbial community structure.The abundances of total 13C and Anammox microbes were higher in the pig manure compost plus 50%chemical fertilization(PMCF)and straw returning into the soil plus 100%chemical fertilization(SRCF)treatments than in the 100%chemical fertilization(CF)and no fertilization(CK)treatments in the same season,suggesting that the Anammox activities and abundances of microbes can be enhanced by employing organic-inorganic compound fertilizers.The abundances of 13C-PLFAs,especially 14:0,16:1?9c,16:1?c,16:0,18:1?9c,and 18:0,were significantly increased during the process of changing tillage from the wheat season to the rice season;therefore,these 13C-PLFAs could be used as Anammox biomarkers.The percentages of fungi were found to be approximately equal to those of bacteria and were much higher than those of actinomycetes in all soils.It was concluded that the fungi were the second microbial group that played a role in metabolizing 13C-matrix with a strong Anammox activity.(3)In this study,15N stable isotope technique combined with antibiotic inhibition was applied to explore the contribution of Anammox,co-denitrification and denitrification to nitrogen production and revealed the effects of organic fertilization on Anammox.As well,the 13C-glucose labeling was utilized in combination with DNA-SIP technology,and the role of fungi in co-denitrification and denitrification and its contribution to nitrogen cycle were deduced based on qPCR of 18S rRNA gene.We found that the contribution rates of fungi under different fertilization treatments(no fertilization(CK)treatments,the 100%chemical fertilization(CF),the pig manure compost plus 50%chemical fertilization(PMCF)treatments and straw returning into the soil plus 100%chemical fertilization(SRCF)treatments)were 4.12%,4.78%,6.73%and 8.26%,and that of anammox fungi were 10.70%,12.44%,17.51%,21.48%.Meanwhile,the correlation analysis showed that the abundance of fungi involved in 13C substrate metabolism could be regarded as the co-denitrification fungi abundance.Otherwise,principal component analysis revealed that the fertilization modes affected the co-denitrification fungal activity by influencing pH,available phosphorus and available potassium.A clone library of 13C-DNA was established to identify co-denitrification fungi,which revealed that co-denitrification fungi is very likely to be consisted ofAgaricus,Aspergillus,Phycomyces,Saitoella,Trichoderma,Our results can be used as a strong basis for further research on co-denitrification fungi.