Microbial Mechanisms Of Nitrification In Long-term Fertilized Agricultural Soils

Microorganisms play vital roles in soil energy flow and nutrient cycling,thus are important for agricultural production.Nitrogen cycling is an important part of the element cyclings in the ecosystems Nitrifiers and denitrifiers are the indicator microorganisms involved in the nitrogen cycling.Nitrous oxide(N₂O)produced by nitrification and denitrification is the largest ozone destroying substance.It has been reported that agricultural soils are sources of nitrous oxide(N₂O)due to the input of nitrogen(N)fertilizer and livestock faecal excretion.Therefore,clarifying the response of microbial community structures,N₂O microbial emission pathways and active nitrifying microbial community compositions to long-term different fertilization patterns is important for the agricultural sustainable development.In this study,the farmland soils under different fertilization treatments for 38 years were sampled.Eight different fertilization patterns were included:(1)Control treatment without any fertilizer(CK);(2)Chemical N fertilizer(N);(3)Chemical N and P fertilizer(NP);(4)Chemical N,P,and K fertilizer(NPK);(5)Manure(M);(6)Chemical N fertilizer combined with manure(MN);(7)Chemical NP fertilizer combined with manure(MNP);and(8)Chemical NPK fertilizer combined with manure(MNPK).The following experiments were carried out:(1)High throughput sequencing and Realtime Quantitative PCR(q PCR)were used to study the correlation between soil environmental factors and microbial communities,to clarify the response of microbial communities to long-term fertilization patterns;(2)The microbial pathway of N₂O emission was determined by acetylene inhibition method;(3)Stable Isotope Nucleic Acid Probe Technology(DNA-SIP)were used to explore the functional active nitrifying microorganism under long-term different fertilization patterns,and combined with Inhibitors Acetylene(C₂H₂),Simvastatin and 1-Octyne(C₈H₁₄)determined the relative contributions of ammonia oxidizing archaea(AOA),ammonia oxidizing bacteria(AOB)and complete nitrifying bacteria(Comammox)to nitrification.The main results are as follows:(1)The effect of organic fertilizer application on bacterial community was more significant than that of inorganic fertilizer application,in contrast,fungal community showed the opposite trend.The Linear Discriminant Analysis(LEf Se)showed that the number of responders of bacterial community in organic fertilizer treated soil was significantly higher than that in inorganic fertilizer treated soil,while that of fungal community showed the opposite trend.Redundancy Analysis(RDA)further proved that soil organic matter played an important role in the distribution of bacterial community rather than fungal community.(2)Organic fertilization demonstrably increased the N₂O emissions from nitrification,as well as the abundance of canonical ammonia oxidizers and complete ammonia oxidizers(clade B).Combined with the results that strong and positive relationships were detected between the abundance of bacterial amoA genes and N₂O content produced from nitrification.We proposed that organic fertilizer applications stimulated the generation of N₂O from nitrification dominated by the Nitrosospira cluster 3 lineage of AOB.In contrast,the amount of N₂O produced by denitrification were appreciably stimulated by chemical N fertilization,compared to organic fertilization.Strong and positive relationships were detected between the nirK-type denitrifying gene abundances and the N₂O emissions generated from denitrification.This suggested that single N fertilizer additions appreciably stimulated N₂O production from denitrification dominated by the nirK lineage of the Rhizobiales.These results indicated that organic and chemical N fertilizers stimulated N₂O emissions from nitrification and denitrification,respectively,which were dominated by the Nitrosospira cluster 3 lineage of AOB and the nirK lineage of Rhizobiales,respectively.(3)AOA participated in the active nitrification in CK and N treated soil,and AOB dominated the active nitrification in all fertilized soils,Comammox were not detected in the“heavy fractions”.The relative contribution rate of AOA to nitrification activity was 14.79%in the CK soil,and that of AOB to nitrification activity was higher than 85%in all soils.q PCR analysis showed that C₈H₁₄inhibited the growth and activity of AOA.Simvastain and C₈H₁₄,especially C₈H₁₄decreased the abundance of clade A and clade B.High throughput sequencing results showed that Thaumarchaeota Group I.1b AOA and Nitrosospira cluster 3a AOB were dominant,indicated that the active Thaumarchaeota Group I.1b AOA and Nitrosospira cluster 3a AOB play a leading role in the process of autotrophic nitrification,rather than Comammox.In conclusion,we conclude that organic fertilizer and inorganic fertilizer play an important role in the composition and distribution of bacterial and fungal communities respectively;organic fertilizer and chemical N fertilizer stimulated N₂O emission during nitrification and denitrification,dominated by AOB(Nitrosospira cluster 3)and nirK(Rhizobiales),respectively;traditional ammonia oxidizing microorganisms AOA(Thaumarchaeota Group I.1b)and AOB(Nitrosospira cluster 3a)play a leading role in the process of autotrophic nitrification,rather than complete nitrifying bacteria(Comammox),and AOA participated in the active nitrification in CK and N treated soil,AOB dominated the active nitrification in all fertilized soils.