Diversity Analysis Of Enzyme Genes In Nitrogen Cycle And Isolation Of Heterotrophic Ammonia-oxidizing Bacteria In Shrimp Farming Water

Ammoxidation, nitrite oxidation and denitrification are key processes of nitrogen cycle.AmoA gene, nxrA gene and nirS gene are the coding genes of the key enzyme in these threeprocesses. Shrimp farming water is a complex biological nitrification, analyzing thecommunity and diversity of microorganisms involved in the key processes, ammoxidationnitrite oxidation and denitrification, of nitrogen cycle in typical aquaculture water, providingreasonable reference for directing the microbiological deterioration of NH₄⁺and NO₂^-, and thecontrol of nitrogen pollution and the valid management of nitrogen cycle in shrimp-farmingwater.In this paper, a typical water sample was decided by the analysis of Polymerase ChainReaction-Denaturing Gradient Gel Electrophoresis （PCR-DGGE） graph of8samples in thisresearch. Clone libraries of the typical sample based on the functional gene ammoniamonooxygenase gene （amoA）, nitrite oxidoreductase gene （nxrA）, and nitrite reductase gene（nirS） were constructed. These three libraries were analyzed by using Restriction FragmentLength Polymorphism （RFLP）, and phylogenetic trees were constructed basing on proteinsequeces. Heterotrophic ammonia-oxidizing bacteria were isolated from the typical shrimpfarming water. They were detected by using chemical reaction, microscopic examination andidentified by sequencing the PCR products of16SrRNA gene and amoA gene.The main results are as follows:a. The shrimp farming water collected from Gangkou Town, Zhongshan City was thetypical water among the8samples collected from different cities in Canton River Delta.b. All sequences from amoA library were clustered into-Proteobacteria, includingNitrosomonas （81%） and Nitrosospira （19%）, and Nitrosomonas was the main performer ofammoxidation.c. Clones from nxrA library were clustered into-Proteobacteria and-Proteobacteria,including Nitrobacter （92%） and Desulfobacteraceae （8%）.Nitrobacter was the mainperformer of nitrification.d. Many kinds of populations played important roles in the denitrification.Clones fromnirS library were clustered into-Proteobacteria,-Proteobacteria and Actinobacteria. β-Proteobacteria was the dominant group which consisted of Azoarcus （25%）, Brachymonas（5%）, and Thauera （20%）. In-Proteobacteria group, Sophophora （10%）, Polymorphum（25%）, Ruegeria （5%） were detected. In the Actinobacteria group, Streptomyces （10%） wasdetected.e. Aoba and AOBb were two heterotrophic ammonia-oxidizing bacteria isolatedfrom the typical shrimp faming water. they were identified as Rhodococcus sp. andAcinetobacter sp.