Analysis Of Ammonia Oxidation Microbial Diversity In Typical Aquaculture Ponds In Jianghan Plain

Pond culture is the main way of freshwater aquaculture in China,but the large amount of nitrogen enrichment in ponds was caused by the extensive use of the combined feed in the process of aquaculture.The removal of nitrogen in cultured ponds was mainly dependent on microbial-led nitrification/denitrification processes,in which was the speed limiting step driven by ammonia-oxidizing bacteria and ammonia oxidizing archaea.On the basis of optimizing the amplification condition of ammonia-oxidizing microorganism(amoA)gene in this paper,the author studied the ammoxidation microorganisms in the water and sediment of 5 typical aquaculture ponds(labeled B1,B2,B3,B4,B5)in Jianghan Plain,analysised the vertical distribution characteristics of ammoxidation microorganism,and combing with the water quality and sediment properties analysised the influencing factors.The main findings of this paper were as follows:1.By using the control variable method,the optimum conditions for PCR amplification were explored under the conditions of different template concentration,annealing temperature,cycle times and whether humic acid inhibitors were added to the problems of hybridization,towing and nonspecific amplification in PCR amplification.The results showed that the amplification efficiency of amoA gene was the highest at the appropriate total DNA template concentration,and the PCR amplification was favorable when the annealing temperature was set at 58 ? and the cycle number was 32.When the template containing more humic acid was amplified,adding a certain amount of BSA could improve the activity of Taq enzyme and improve the amplification efficiency.2.In the aquaculture ponds,only the AOA amoA gene was detected in the water column,but no AOB amoA gene was detected,and AOA and AOB amoA gene were detected simultaneously in sediments,and AOA amoA gene in the water column of cultured ponds was the main ammonia-oxidizing microorga nism.In clone sequencing,the gene coverage of the number of clones was over 90%.The abundance and diversity of ammonia oxidizing microorganisms showed that the abundance of AOB amoA and AOA amoA genes was relatively high,and the diversity index was relatively low.At 97% similar levels,there were 14 OTU sequences of AOA amoA gene in the water column,18 OTU sequences of AOA amoA gene and 15 OTU sequences of AOB amoA gene in the sediments.The number of OTU in the water column is less than that in the sediments,but the OTU sequences of AOA amoA gene were both Nitrososphaera cluster?Nitrosopumilus cluster and Nitrososphaera sister group,the difference was that the OTU sequence is mainly Nitrososphaera cluster in the water column,while most OTU sequences belong to Nitrososphaera sister group in sediment.The OTU sequence of AOB amoA gene was evenly distributed in the Nitrosomonas and Nitrosospira of ?-subsets,and there was no ?-nitrosococcus species.The abundance and diversity of ammonia-oxidizing microorganisms showed that the abundance of AOB amoA gene and AOA amoA gene was higher but the diversity index was lower.By analysised the effects of water quality and sediment properties on ammonia-oxidizing microorganisms,the results showed that there was no significant correlation between water quality and the richness and diversity of AOA amoA gene in water column and AOB amoA gene in sediments,but there were significant positive correlation between pH,DO of water and Shannon-wiener index of the AOA amoA gene in sediments,while there was negatively correlated between ammonia nitrogen and the abundance of AOA amoA gene.the pH and microbiological biomass carbon(MBC)in sediment had an influence on the abundance of the AOA amoA gene in sediments,thus was pH and the Simpson index have a significant negative correlation,and the MBC has a significant positive correlation with the Shannon-Wiener index.Nitrogen concentration in sediment was negatively correlated with the abundance of the AOA amoA gene in water,and the rate of ammonia oxidation was positively correlated with the abundance of the AOA amoA gene in water.Acid phosphatase activity was positively correlated with the Simpson index of AOA amoA gene in water.3.In 8 samples of different layers sediments,enough clones of the AOA amoA gene could be selected for analysis in 7 samples,and cluster analysis showed that the AOA amoA gene of the bottom sediments were more similar,while the AOA amoA genes in the surface and middle layer were more similar.At similarity level of 97%,there were 22 OTU sequences in AOA amoA gene,the dominant species of the surface and middle layers were the same in the sediment of B4 and B5,while the dominant species at the bottom wree the same,AOA species were more abundant than non-stratified,and Nitrosotalea cluster only exists in stratified sediments.There were 5 samples could be selected enough clones to analyze,the cluster analysis showed that the AOB amoA genes of B4 and B5 sediments were significantly different in addition to the surface sediments.On the level of 97% similarity,there were 12 OTU sequences of amoA gene,the dominant species of AOB amoA gene was Otu 01,but Otu 02 was he dominant species at the bottom of B1.The species of AOB was consistent with non-stratified,but Nitrosospira was dominant in stratified sediment.The correlation analysis showed that the activity of acid phosphatase in sediments was positively correlated with the Simpson index of AOB amoA gene,and the concentration of ammonia nitrogen was negatively correlated with the abundance of AOA amoA gene,and the activity of acid phosphatase was negatively correlated with the Shannon-Wiener index and positively correlated with Simpson index of AOA amoA gene.