The Microbial Characteristics Of Nitrite Transformation And Diversity Changes Of Microbial Community In Litopenaeus Vannamei Aquaculture

The aquaculture environment is a nitrogen(N)cycle system with abundant N sources.As potentially toxic inorganic compound in this system,high concentration of nitrite has lethal effect on cultured animals,even threats human health.How nitrite accumulates in the aquaculture system and how to remove it quickly has became a focus of people's concern.Therefore,in order to understand the microbial processes participating in the accumulation and transformation of nitrite,the dynamic changes of bacterial community and its relationship with water quality parameters in different growth periods of Litopenaeus vannamei,cloning library and qPCR technique were employed to analyze diversity and abundance of seven functionnal genes(arch-amoA,bac-amoA,narG,napA,nirK,nirS and nrfA),and high-throughput sequencing tech-nique was applied to investigated the compositions of bacterial community and their dynamic changes in the water during different growth stages of Litopenaeus van-namei.1.Microbial processes participate in the accumulation and transformation of ni-trite in different aquaculture periodsFunctional gene clone library analysis showed that,in the 40th day of the aqua-culture water,the narG gene had the highest diversity and enrichness,but the nirS gene was opposite.The narG and napA gene had the highest diversity and enrichness in the 131th day of aquaculture water,and the nrfA gene was opposite.The diversity of narG gene has a little change,but for nrfA gene was opposite,and the diversity of archamoA,napA,nirK,nirS gene have different degrees of increase from D40 to D131.NCBI blast indicated that the NapA?NarG?NirK?NrfA were relatively novel.Quantification PCR analysis of functional genes indicated that that both amoA genes were at low abundance,while four denitrification genes and nrfA genes were at relative high abundance(from 3.73E+02 to 1.50E+05 copies/ng DNA)during differ-ent aquaculture period.The napA gene abundance gradually increased during aqua-culture,and ranged from 1.53E+03 to 2.05E+04 copies/ng DNA,but was about 10 times lower in D131 than in D115.The narG gene abundance gradually decreased during aquaculture,ranging from 2.25E+03 to 7.66E+04 copies/ng DNA,but was generally higher than that of napA gene.The nirK gene abundance rised in D40 and D59(from 4.06 E+03 to 6.77E+03 copies/ng DNA),then decreased in D67 to D131,ranging from 2.98E+03 to 3.73E+02 copies/ng DNA,and was lower than that of nirS gene;While the nirS gene abundance is relatively stable during the aquaculture.The abundance of nrfA gene maintained at a high level,ranged from 1.03 E+05 to 1.50E+05 copies/ng DNA.In general,ammonia-oxidizing microbes and denitrifiers may contribute to the production of nitrite,while denitrifiers futher play a role in the removal of nitrite.The difference of gene abundance for nitrate reductase genes(narG and napA)and nitrite reductase genes(nirK and nirS)may lead to accumulation of nitrite to some extent.2.The dynamic changes of bacterial community in different aquaculture peri-ods.High-throughput sequencing analysis based on 16S rRNA gene showed that the bacterial community from 19 water samples which were collected in seven different growth stage,comprised 22 phyla,37 classes,83 orders,140 families and 271 genera,which indicated a high diversity of bacterial community.There was a certain fluctua-tion of microbial composition in different aquaculture period,Proteobacteria and Bacteroidetes were widely and predominately distributed at all samples.The compo-sition and abundance of bacterial groups changed greatly at the level of class and family in different periods.The abundance of probiotics and potential pathogens is low during aquaculture.Besides,low abundance of ammonia-oxidizing bacteria,and some denitrifying bacteria were also detected,which coincide with the result of func-tional gene clone libraries.3.The relationship between bacterial community and the abundance of func-tional gene with water quality parameterThe redundancy analysis(RDA)indicated that bacterial community had the greast relevance with dissolved oxygen(DO)and temperature(T),followed by NH4+-N,NO2--N,and NO3--N.It was noteworthy that though ammonium(NH4+)concentration ranged greatly during different periods,however,its impact on bacterial community was very limited.NH4+-N,T and NO3--N had the greatest relevance with denitrifying functional gene abundance,and different water quality parameters had different relevance with different functional genes.In this study,the diversity,abundance and distribution of functional gene which participate in the accumulation and transformation of nitrite were analyzed.Besides,the diversity and dynamic changes of bacterial community in different growth stages of Litopenaeus vannamei,and its relationship with water quality parameter were also investigated.Key functional genes which participate in the accumulation and removal of nitrite were identified.This study provides some useful information for aquatic product food safety and human health.