Detection Of Nitrogen Conversion Activity And Relate Functional Genes Of Heterotrophic Ammonia Oxidizing Bacteria

Nitrification is the key link of the soil nitrogen cycle.Ammonia-oxidizing bacteria?AOB?is an important microbial group in soil nitrification and it can be classify into autotrophic ammonia-oxidizing bacteria and heterotrophic ammonia-oxidizing bacteria base on nutrient.Heterotrophic AOB has some characteristics,such as a wide variety of species and wide availability of substrates,but nitrification and its nitrogen conversion function of heterotrophic AOB are difficult to monitor in most habitats,so resulting in the nitrification contribution of this type of microorganism and its possible nitrogen conversion capacity is still unclear.In this study,three strains of heterotrophic AOB isolated from apple orchard as research objects.Strains taxonomic status preliminary identified by routine culture with 16S rRNA sequence analysis.UV spectrophotometry combine with detection of NH₄⁺-N,NO₂^--N and NO₃^--N concentration used to analyze the growth characteristic and nitrogen conversion activity of AOBs.At the same time,polymerase chain reaction?PCR?and fluorescence quantitative PCR?qPCR?are applied to detect the abundance of relate nitrogen transformation functional gene of heterotrophic AOB strains under different culture conditions.The results are as follows:?1?Three heterotrophic ammonia-oxidizing bacterium YF11 and YH9 and YH11 isolated from apple orchard soil of Shenyang Agricultural University for 5 years initially assigned as Sphingobacterium sp.,Ochrobactrum sp.and Brevundimonas sp.respectively.?2?Among the three test autotrophic media,three AOB strains all show the highest biomass and higher nitrogen conversion activity when AOB growth in the autotrophic medium I?ammonium sulfate as the nitrogen source,pH7.2?and three strains have high removal rates of ammonium in this medium when culture for 24 days.The content of nitrite nitrogen and nitrate nitrogen in culture fluid gradually increase with the decrease of ammonium,both nitrite concentration and nitrate concentration show a tendency of increase in the beginning and decrease in the end.In addition,all three heterotrophic AOB strains can also use a small amount of nitrite as the nitrogen source to growth and complete relate nitrogen conversion reaction.Compared with autotrophic culture conditions,the biomass of three AOB strains significantly increase when culture in heterotrophic medium?YMA?with yeast extract as nitrogen source and mannitol as carbon source.The changes of ammonium nitrogen and nitrite nitrogen and nitrate nitrogen can detect in three AOB culture conditions and nitrogen show an upward trend follow by a downward trend.?3?The strains cultured in different pH soil extractions.The environment with low pH?pH4?and high pH?pH9?can inhibit the growth and nitrogen transformation of strains.Strain YF11 and strain YH11 show stronger nitrogen conversion activity in the environment with pH5⁸.?4?Three AOBs culture in suitable autotrophic medium?medium I?and heterotrophic mediun?YMA?and in different soil pH extraction,respectively and hao gene,nap gene,norB gene,nirK gene and nosZ gene obtained by polymerase chain reaction?PCR?.The abundance of different nitrogen transformation gene of the strains detected by qPCR:Three AOBs involve in nitrogen transformation gene abundance show significant differences due to different culture conditions.The abundance of norB gene,nirK gene and nosZ gene are significantly increase when strains YF11 and YH11 cultured in pH6 soil extraction.The abundance of hao gene is significantly increase when strains culture in pH7soil extraction.In addition,the nap gene of strain YH11 show higher gene abundance when culture in autotrophic,heterotrophic and pH7 soil extraction while the nap gene of strain YF11 has higher abundance when strain culture in pH9 soil extraction.Strain YH9 has higher nosZ gene abundance in YMA medium and other nitrogen transform gene show higher gene abundance in medium I.?Conclusion?Three AOB strains can grow and participate in nitrogen transformation reaction in the autotrophic and heterotrophic environment.They can use ammonium,nitrite and nitrate as nitrogen source.They can also use CO₂ and organic carbon as carbon source and exhibit more extensive nutritional characteristics.The biomass of strains in heterotrophic environment?YMA medium?are significantly higher than that of autotrophic condition.Strain YF11 and YH9 and YH11 can participate in a variety of nitrogen transformation process under different culture conditions within a certain pH range,but the abundance of relevant functional gene will change according to different growth environment.