Pathways And Microbes Responsible For N₂ Production In Soil

A series of human activities,e.g.industrial activities and farmland fertilization have emitted a large amount of reactive nitrogen into environment.Excessive reactive nitrogen can cause environmental problems,e.g.soil acidification,water eutrophication and greenhouse gas emission.Soil dinitrogen?N₂?emission,as the process converting reactive N back into inert N₂ gas,has important ecological and agricultural significances.The process of soil N₂ emission is not only the main process removing the excess reactive nitrogen from ecosystem,but also one of the main processes affecting nitrogen loss and greenhouse gas emission from farmland.Soil N₂ emission processes,e.g.denitrification and anammox are traditionally thought as strictly anaerobic processes.But recent studies suggest that denitrifiers,such as Pseudomonas,which are ubiquitous in soil,can produce N₂ through denitrification under aerobic conditions.As far as the literature is available,it is still unclear on the pathway and functional microbes of soil aerobic N₂ emission.In this study,the contributions of heterotrophic denitrification,codenitrification and chemical denitrification to soil aerobic N₂ emission were quantified by using the stable isotope labeling method and the double-layer He/O₂displacement measurement of directly N₂emission.In order to elucidate the microbial driving mechanism of aerobic N₂ production,the effects of aerobic-anaerobic alternation on aerobic N₂ emission rate and denitrifying microbial community structure,functional gene abundance and expression were studied by methods of molecular microbiology.The main results are as follows:1.In order to find out the change of soil aerobic N₂ emission and its contribution to soil nitrogen loss in different ecosystems,the aerobic N₂ emission rates of typical dry farming fields,forest,river bank and grassland soils were measured using the double-layer He/O₂ displacement measurement of directly N₂emission.The results showed that aerobic N₂ production rates of the four soils ranged from 2.1 to 6.2?g·g^-1 dry soil d^-1,which were 15%-31%of the rates occurring via anaerobic pathways.The highest rate of aerobic N₂emission was found in river bank soil,followed by urban turf soil and subtropical forest soil soil.The lowest rate was found in semiarid upland soil.These results indicate that the emission of N₂ plays an important role in soil nitrogen loss.The soil which experienced frequent dry-wet alternation had higher rate of aerobic N₂ emission.2.In order to find out the main pathsways of aerobic N₂ production in soil,the contributions of heterotrophic denitrification,codenitrification and chemical denitrification to aerobic N₂ emission from different soil were distinguished by using ¹⁵NO₃^-stable isotope labeling method and high temperature and pressure steam sterilization.The results showed that N₂ production from the autoclaved soil extracts was two orders of magnitude lower than that from the fresh extracts,indicating that soil aerobic N₂ emission is mainly driven by microorganisms.The addition of bacterial selective inhibitors also reduced the aerobic N₂ emission rate by two orders of magnitude,suggesting that bacteria,rather than fungi,play a dominant role in aerobic N₂ emissions.In the ¹⁵N isotope experiment,the emission rate of ²⁹N₂ accounted for 81%-89%of the total aerobic N₂emission rate.The above results indicate that bacterial codenitrification is the main pathway for soil aerobic N₂ production.3.The soil aerobic N₂O and N₂ emissions were enhanced by frequently aerobic-anaerobic alternation.The emission rate of N₂O and N₂ increased by 1-4 times after aerobic-anaerobic alternation,and the ratio of N₂O/?N₂O+N₂?increased from 11.8%to 21%under aerobic conditions.The total DNA of bacteria was collected periodically during the incubation,and the denitrifying microbial community structure was analyzed.The results showed that the abundances of Pseudomonadales,Burkholderiales,Rhodocyclaes and Rhizobiales,were significantly increased after aerobic-anaerobic alternation.The abundance of nos Z genes was determined by real-time polymerase chain reaction?real-time q PCR?.It was found that the abundance of nos Z genes in the samples was significantly increased by aerobic-anaerobic alternation.Finally,the relationship between the structure of denitrifying microbial community,the abundance and expression of functional genes and the rate of aerobic N₂ emission was analyzed.It was found that the relative abundance of Pseudomonas was significantly increased by alternating the incubation between aerobic and anaerobic conditions,and correlated positively with the aerobic N₂ production rate,while others were not.Above results show that Pseudomonas is likely to have played a key role in the aerobic N₂production in the investigated soils.In conclusion,bacterial codenitrification which performed predominantly by Pseudomonas,was the main pathway accounting for aerobic N₂production in the investigated soils.Soils experiencing frequent alternating aerobic-anaerobic conditions,high concentrations of nucleophilic N species and low concentrations of organic carbon are expected to exhibit significant aerobic N₂ production.These results are of great significance to the regulation of soil nitrogen cycling and the reduction of greenhouse gas N₂O emissions.First,the nucleophilic N substrates not only provide an N atom but also provide electrons for NO₃^-reduction in the codenitrification process.Therefore,the dependency of codenitrification on organic carbon is expected to be lower than that of traditional denitrification,which may significantly impact the nitrogen loss in soils with low levels of organic carbon.In addition,because the enzymes responsible for N₂O reduction are expected to be more sensitive to O₂ exposure than those responsible for N₂O production,the ratio of N₂O/?N₂O+N₂?in soil under aerobic condition is likely to be higher than that under anaerobic condition,which leads to more N₂O emissions.