Niche Differentiation And Functional Importance Of Complete And Incomplete Nitrifiers In The Soil

Nitrification in the soil is generally considered to be two consecutive steps dominated by two distinct types of microorganisms,involving both the ammonia oxidation process led by ammonia-oxidizing bacteria(AOB)and ammonia-oxidizing archaea(AOA),and the nitrite oxidation process led by nitrite-oxidizing bacteria(NOB).However,a microorganism,Complete ammonia oxidizer(Comammox),capable of both ammonia oxidation and nitrite oxidation was discovered at 2015.Correspondingly,the traditional AOA,AOB,and NOB are referred to the incomplete nitrifiers.The discovery of Comammox changed the two-step nitrification dogma that has been long held for a century,which leads to a series of scientific problems that need to be solved urgently.Although the Comammox strain has not been enriched from the soil ecosystem yet,the results of metagenomic research and quantitative PCR have found that Comammox is widespread in various soil ecosystems,indicating that it is likely to play an important role in the process of soil nitrification.There is an urgent need for more research to understand the distribution of Comammox in the soil ecosystem and its driving factors,to determine whether it will contribute to nitrification in the soil ecosystem and under what soil conditions.Comammox belongs to the Nitrospira cluster II of NOB.Comammox coexist with AOB,AOA,and NOB in the soil ecosystems,they are all autotrophic microorganisms.It is crucial to understand the niche differentiation between Comammox,AOB,AOA,and NOB,which is of great significance to reveal the microbiological mechanism of nitrification in the soil.Based on the above scientific issues,we(1)firstly selected typical forest soil samples from south to north along the latitude gradient at a large regional scale to study the spatial distribution pattern and its driving factors of Comammox and incomplete nitrifiers;(2)to find out whether different nitrogen sources can cause niche differentiation between Comammox and incomplete nitrifiers,paddy soil and forest soil were selected to study the response of nitrification and nitrifiers to different nitrogen sources;(3)in order to further prove that Comammox weather play a role in the process of soil nitrification,¹³C DNA based stable-isotope probing(DNA-SIP)combined with gene sequencing were used to study the nitrification and active nitrifying microbial community structure of forest soil and paddy soil with and without nitrogen source;(4)chlorate was used as an selective inhibitor to determine the relative contribution of Comammox to soil nitrification under different nitrogen sources,and also to investigate the responses of nitrifiers to chlorate.The main results obtained are as follows:1.Distribution patterns and driving factors of functional nitrifiers and bacterial communities in forest soils along the latitude gradient at a regional scale.The results found that the abundances of AOA and Nitrospira-NOB were generally higher than other nitrifying microorganisms.Comammox was found in all soil samples,and its abundance ranged from 1.21*10⁶ to 8.77*10⁶/g dry soil,the abundance of Clade A was higher than its counterpart Clade B in most soils.AOA,AOB and Nitrobacter had a significant correlation with soil PAO and PNO,indicating that these three nitrifying guilds may play an important role in the process of forest soil nitrification.Similar to soil nitrification activity,soil bacterial diversity showed a trend of increasing firstly and then decreasing along the latitude gradient,and the peak appeared at mid-latitude soil with a neutral p H.The proportions of the predominant microbes in each soil had significant differences.The constrained principal coordinate analysis(CPCo A)showed that the nitrifying functional groups were more affected by climate than soil bacteria.The climate type grouping could explain 50.6%and 42.7%of the total variation of nitrifying functional microbial community and bacterial community,respectively.Redundant analysis(RDA)found that soil p H,annual average precipitation(MAP),and latitude(LAT)were the main driving factors leading to the spatial distribution of nitrifying functional microbial community and bacterial community.The community of functional nitrifiers was also significantly affected by the annual average temperature(MAT),which is of great significance for the prediction of soil nitrifying microbial diversity and function under climate changing scenarios.2.The effects of different nitrogen sources on nitrification and nitrifiers in forest soil and paddy soil.The results found that in both soils,the addition of nitrogen sources significantly stimulated nitrification(P<0.05),but significantly inhibited mineralization(P<0.05).Interestingly,the nitrification of the two soils had a significant difference in response to different nitrogen sources:there was no significant difference in the net nitrification rate between the two N source treatments in the paddy soil(P>0.05);however,in the forest soil,the net nitrification rate of soil in NO₂^-treatment was significantly lower than that of NH₄⁺treatment(P<0.05).The response of bacteria to different nitrogen sources in the two soils was also different:the microbial diversity in the paddy soil was more sensitive to NH₄⁺treatment,while in acid forest soils,the microbial diversity was significantly decreased in the NO₂^-treatment(P<0.001),owing to the toxicity of a large accumulation of NO₂^-.The results of quantitative PCR found that the abundance of AOA,Comammox Clade B and Nitrospira-NOB increased significantly during the incubation in the control treatment without N.However,in the treatment with N,the abundance of AOB,Comammox Clade A and Nitrobacter-NOB increased significantly during incubation.These indicated that AOB,Comammox Clade A and Nitrobacter-NOB preferred nutrient-rich environments,while AOA,Comammox Clade B and Nitrospira-NOB preferred nutrient-poor environments,and they may contribute to soil nitrification under different nutrient conditions,respectively.Substrate availability may be an important factor driving niche differentiation of nitrifying microbial groups.Based on the above results,DNA-SIP and selective inhibitor were used to clarify whether Comammox contribute to the soil nitrification process,and to explore the relative contribution of Comammox to soil nitrification under different substrate conditions.The main results obtained were as follows:3.DNA-SIP was used to reveal the active and functional microorganisms involved in the process of soil nitrification with direct evidence.The results showed that nitrification occurred in both soil treatments with and without N addition,and the net nitrification rate of the soil with N treatment was significantly higher than that without N treatment.The addition of C₂H₂ could completely inhibit soil nitrification,indicating that nitrification occurred in all treatments were the autotrophic nitrification related to the AMO gene.Quantitative PCR results showed that the abundance of Comammox Clade B was higher than Clade A in forest soil,but its abundance in paddy soil was 14 times less than that of Clade A,indicating that the two clades of Comammox showed niche differentiation characteristics under different soil conditions.In both soils,the abundance of Comammox Clade B increased significantly without N treatment(P<0.05),and it was accompanied by an increase in nitrification activity.The distribution of Comammox amo A gene abundance along the Cs Cl gradient in ¹²CO₂ and ¹³CO₂ treatments showed that Comammox Clade B enriched in the ¹³C genome heavy fractions in forest soil and paddy soil without N addition treatment,indicating that Comammox clade B could use the trace amount of ammonium produced by mineralized organic nitrogen for nitrification.However,when exposed to high dose of inorganic ammonium,Comammox Clade B would loss its competitive advantage or be inhibited.Phylogenetic analysis showed that the amo A sequence of the labeled active Comammox Clade B belonged to two OTUs,which were closely related to the two soil uncultured sequences in the database.The labeled active AOA and AOB are mainly affiliated to Nitrososphaera and Nitrosospira Cluster 3,respectively.These results indicated that the active Comammox is the same as AOA/AOB,which are with low diversity under this culture condition.4.The relative contribution of Comammox to soil nitrification was explored by using chlorate,a selective inhibitor of NOB.The addition of chlorate significantly inhibited nitrification,and its inhibition efficiency was different in different nitrogen source treatment.In the treatment of NO₂^-as a nitrogen source,the inhibitory efficiency of chlorate on nitrification reached 98%,which was significantly higher than the other two nitrogen source treatments(?67%).The addition of chlorate did not affect the total microbial community structure and community diversity,but it had a significant impact on the abundance and community structure of nitrifying microorganisms,and the effects under different nitrogen sources were also different.The AOA abundance in the chlorate treatment did not change significantly with time(P>0.05);while the AOB abundance was significantly inhibited by chlorate(P<0.05),and the AOB abundance in the NO₂^-nitrogen source treatment was also significantly decreased(P<0.05),indicating that AOA had a certain tolerance to NO₂^-and Cl O₃^-,while AOB was sensitive to NO₂^-and Cl O₃^-.It can also be found from the changes in the AOA and AOB microbial communities,the AOA community structure was relatively stable,while the AOB community structure changed significantly after N addition.The abundance of both Comammox Clade A and Clade B were inhibited by the high concentration of NO₂^-.However,the response of Clade A and Clade B to chlorate was different,in the soil with no nitrogen and ammonium nitrogen as the nitrogen source,the addition of chlorate caused a significant decrease in the abundance of Clade A,but the abundance of Clade B did not differ significantly,indicating that compared with Clade B,Clade A was more sensitive to chlorate.The addition of chlorate significantly inhibited the growth of NOB,and significantly changed their communities.Nitrobacter-NOB and Nitrospira-NOB had different responses to chlorate and nitrite,and the responses of different subclusters of NOB to chlorate and nitrite were also different.The relative contribution of Comammox to soil nitrification in CK and NH₄⁺treatments were 87.04%and 58.73%,respectively.Since chlorate not only inhibits NOB,but also significantly inhibits the abundance of AOB and Comammox Clade A,indicating that chlorate also inhibits the ammonia oxidation process,so the relative contribution obtained here may be overestimated.Therefore,in complex soil systems,the use of chlorate as a selective inhibitor to study the relative contribution of Comammox to nitrification requires extreme caution.In summary,at a large regional scale,nitrifying microorganisms are mainly affected by soil p H,latitude and climate factors,and compared with soil bacteria,nitrifying microorganisms are more affected by temperature;different nitrifying microorganisms have different responses to nitrogen sources and chlorate inhibitors,and substrate availability is an important factor that affect the niche differentiation of Comammox and classical nitrifying microorganisms;DNA-SIP confirms that Comammox does contribute the soil nitrification process without adding nitrogen sources,but its relative contribution to nitrification remains to be studied.