Analysis Of The Structure And Function Of The Biofloc Bacterial In The Aquaculture Water Of Litopenaeus Vannamei

Bacteria is the builder of biofloc.Therefore,in order to better understand biofloc technology and better serve the Litopenaeus vannamei breeding industry,we want to study the bacteria in the biofloc formed in Litopenaeus vannamei culture.The related studies are divided into three parts,including the analysis of the microbial community structure of biofloc and the screening and application of high efficient functional strains in the biofloc.Part one:In order to clarify the structure and function of microbial community in the initial stage of biofloc in Litopenaeus vannamei culture,we carried out Illumina MiSeq high throughput sequencing for 16S rDNA V4 region of biofloc bacteria.The results showed that the abundance of biofloc was higher at the early stage,and the community abundance index ACE and Chao1 were 5273.65 and 2862.07,respectively,while the diversity of bacteria community was low,and the diversity index Shannon and Simpson were 2.17 and 0.22 respectively.Bacteria in Phylum level distribution mainly by Proteobacteria（58.33%）and Firmicutes（39.91%）.in Class level distribution mainly by GammaProteobacteria（46.77%）,Bacilli（39.36%）and BetaProteobacteria（8.81%）.In Genus level distribution mainly by Exiguobacterium（37.72%）,Citrobacter（21.46%）,Acinetobacter（13.13%）,Pseudomonas（8.52%）and Ramlibacter（7.6%）.The above bacteria play an important role in the construction of biofloc,as well as the assimilation of inorganic ions and water quality.In the initial stage of aquaculture,directional cultivation of these microbes will help to form biological floc.Part two:In order to understand the bacterial community structure and function of bioflocs with different sizes in Litopenaeus vannamei culture water,Illumina MiSeq high throughput sequencing of the bacterial 16SrDNA V4 region was used to compare the bacterial community characteristics of the 4 biofloc sizes,not screened（1）,>212um（2）,106-212um（3）and<106um（4）,in this study.Results showed,the abundance index ACE,Chao1 value of bacteria in each group were 3>1>2>4,diversity index Shannon value of bacteria was 3>1>2>4,Simpson value was 4>1>2>3.The biofloc size of 106-212 um had the highest abundance and diversity.However,the abundance and diversity in bioflocs of<106 um were lowest.In the 4 samples,Proteobacteria were the dominant group,Bacteroides and Firmicutes were secondary groups.The number of endemic OTU in 1-4samples reached 459,452,494 and 420.In addition,the biofloc size of<106um contained higher abundance of potential pathogenic bacteria,Vibrio（24.72%）and Arcobacter（9.17%）.But the biofloc size of 106-212 um contained pathogenic bacteria with low abundance,Vibrio（11.04%）and Arcobacter（2.91%）.There also had potential probiotics such as Oceanicola、Loktanella with high abundance in biofloc size of 106-212 um.Gene functions related carbohydrate,inorganic ions transporters and metabolics,growth and division of cells were detected in 4 samples by PICRUSt method.The abundance arrangement of whole gene functions in 4 samples was 3>1>2>4,biofloc size of 106-212 um had the highest abundance prediction function.In all,the retention of biofloc size 106-212 um in culture will be important for bacterial diversity.Part three:A nitrogen removal strain xt1 with flocculation ability was isolated from biofloc in aquaculture wastewater of Litopenaeus vannamei.The results of molecular biological,physiological,and biochemical identification showed that the strain xt1 was Bacillus pumilus.On this basis,the nitrogen removal characteristics of this strain were studied.The results show that the best carbon source of the strain xt1 was glucose.The removal rate of ammonia and nitrate was 95.56%and 57.40%with glucose as substrate,and when sucrose was used as the carbon source,the removal rates of ammonia and nitrate reached 69.95%and 49.50%,respectively.The strain could use organic nitrogen to accelerate the growth.When 0.25%,0.5%,1%and 2%peptone were added,OD₆₀₀ was increased to 0.925,1.034,1.103 and 1.314,respectively,in which the removal rate of ammonia was more than 90%,the removal rate of nitrate was over 88%.The strain could adapt to the concentration of 20—200mg/L inorganic nitrogen.The strain could use NH₄⁺-N,NO₂^--N or NO₃^--N as a sole nitrogen source,and the removal efficiency reached94.16%,47.60%and 91.17%within 84h,respectively.Among them,the nitrifying form of the strain was the conversion of ammonia into gaseous nitrogen.The form of nitrate denitrification was converted to nitrate into nitrite,and then to gaseous nitrogen.In the process of heterotrophic nitrification-aerobic denitrification,the strain xt1 showed flocculation characteristics,and the highest flocculation rate reached 82.28%,73.15%,and75.60%,respectively.Besides,the addition of strain xt1 in the wastewater could accelerate the formation of biofloc and increase the rate of nitrogen removal.Therefore,strain xt1 can be used as an alternative strain for nitrogen removal from aquaculture wastewater.