| Nitrification is important biochemical processes, including ammonia oxidation and nitrite subsequently to nitrate, the ammonia oxidation process is a limiting step. Therefore, the study of nitrification mainly focused on ammonia oxidation process. Ammonia oxidation process is a biological process, From 1890 until 2004,scientists believed that the ammonia oxidation process is completed by ammonia oxidation bacteria, the discovery of ammonia oxidizing archaea opening new doors of exciting research, it is play an important role in the global nitrogen cycle. Research the ammonia oxidation archaea and ammonia oxidation bacteria in different ecological system, still not clear whether functionally redundant or competition leads to niche differentiation, yet still not clear the oxidation of ammonia and ammonia oxidation bacteria archaea in the relative contribution in the process of ammonia oxidation. Different ecological system the ammonia oxidizing bacteria and ammonia oxidizing archaea abundance, community structure and the relative contribution of the study, have important implications for ammonia oxidation process. There are many environmental factors that considered to impact on the ammonia oxidation process, such as soil type, temperature, moisture, oxygen content, pH and ammonium nitrogen content and so on. The pH and the content of ammonium nitrogen are the two important factors.Purple Soil has special status in soil science, is mainly distributed in the subtropical areas of China, in Sichuan Basin, plays a very important role in agricultural production in China. According to the pH and carbonate content divided into acidic, neutral and calcareous of purple soil. soil pH is affecting one of the factors of soil nitrification, will not only affect the soil nitrification, but also influence nitrification microorganism population and community structure.Our experiments selected Yongchuan acidic purple soil(pH=5.3)and neutral purple soil(pH=7.2)and Sichuan Yanting calcareous purple soil(pH=8.5),using stable isotope labeling technique cultured soil sample. Each soil sample has three treatments, inclding 13CO2 labeled treatment, 12CO2 and 13CO2+C2H2 control treatment. Soil samples were cultured incubator 56 days at 28℃,each week added100 mg kg-1N and the corresponding volume of gas. After cultured experiment ends, cultured the day 0 and day 56 samples were analyzed. Analysis soil pH,ammonium nitrogen and nitrate nitrogen. Analysis the abundance of AOA and AOB amoA function gene by real-time PCR method. Clone libray method for the community structure of AOA and AOB. Miseq sequence method for the total microorganisms.The results are as follows:After the cultivation of 56 days, nitrate content in three purple soils 13CO2+C2H2 treatment compared with the initial cultured did not change, indicating that three purple soils ammonia oxidation by the autotrophic microorganisms completed; neutral purple soil 12CO2 and 13CO2 treatment nitrate contents were 536.2,472.9mg kg-1,respectively. the initial cultured nitrate contents were 12.7mgkg-1. By 12CO2 and 13CO2 treatments compared with initial cultured treatment significantly increased in neutral purple soil, nitrification significantly occurred in neutral purple soil. In acid purple soil by 12CO2 and 13CO2 treatment nitrate content were 5.2 and 4.5 mg kg-1, respectively. The initial cultured nitrate content were 6.1mg kg-1, Nitrate content in acid purple soil by 12CO2 and 13CO2 treatment not significant different compared with cultivation starting. In calcareous purple soil by 12CO2 and 13CO2 treatment nitrate content were 5.3 and 4.0mg kg-1, respectively. In initial cultured sample nitrate content was 0.6mg kg-1, compared with the neutral purple soil there a slight increase, almost no nitrification in calcareous purple soil.After 56 days of incubation, the number of ammonia-oxidizing archaea have different leves reduced in three purple soils each treatment. The number of ammonia oxidizing bacteria and ammonia-oxidizing archaea in neutral purple soil treatments are much larger than the number of copies of the acidic purple each processing soil and calcareous purple soil, indicating that ammonia-oxidizing microorganisms suitable for growing in a neutral environment.By soil NO3--N and NH4+-N content changes, we can only know the neutral purple soil nitrification occurred. The abundance of ammonia oxidizing bacteria and ammonia-oxidizing archaea changes may explain part of the ammonia oxidizing microorganisms at work. Still need further experiment to tests which ammonia oxidizing microorganisms play a role. Neutral purple soil treatments DNA were Cscl hypervelocity density gradient centrifugation, the centrifugal liquid is divided into 15 layers, centrifugal liquid purified, centrifugation was subjected to conventional PCR amplification, combined with gel electrophoresis, and each DNA was real-time fluorescence quantitative PCR. The neutral purple soil AOA and AOB while being marked, but the degree is not deep. AOA and AOB driven by ammonia oxidation process in neutral purple soil. Using the clone library method constructed phylogenetic trees. The main group of AOA play activity belong to Group1.1b in neutral purple soil of each treated sample. The main group of AOB play activity belong to Cluster3 in neutral purple soil of each treated sample.Using Miseq sequencing was analyzed microbial community structure. Shannon value represents diversity of species.Neutral purple soil shannon value> calcareous purple soil shannon value > acid purple soil shannon value, neutral purple soil microflora diversity> calcareous purple soil microflora diversity > acid purple soil microflora diversity. Rarefaction curve shows the amount of sequencing data are reasonable. Rarefaction curve were similar to flatten in three purple soils under 0.97 levels, indicating the amount of data sequencing qualified. Each processing soil samples were detected Proteobacteria, Acidobacteria, Firmicutes, chloroflexi, Actinobacteria, Bacteroidetes and Gemmatimonadetes. neutral purple soil detected Nitrospirae. Proteobacteria in three purple soils are approximately 20 percent. After 56 days of culture,adding urea in three purple soils. microbial species change and changes in different proportions, acidic soil and purple calcareous purple soil 13 C labeling process and acetylene inhibition same process variation width, while neutral purple soil 13C-labeled treatment change width greater than acetylene inhibition treatments. Ammonia oxidizing bacteria belong Nitrospira and Nitrosospira in three purple soils each treatment.This study describes the effect of pH on purple soil nitrification and ammonia oxidizing microorganisms, as well as proved main promoter of ammonia oxidation in neutral purple soil. Conclusions are as the following: Firstly, soil pH affect the abundance of ammonia oxidation microorganisms, ammonia-oxidizing microorganisms growing suitable in a neutral environment. Secondly, ammonia oxidation process is promoted by the ammonia oxidizing bacteria and ammonia-oxidizing archaea in neutral purple soil. Third, the group of ammonia-oxidizing archaea belongs to Group1.1b in neutral purple soil.the group of ammonia-oxidizing bacteria belongs to cluster 3. |
