Studies On The Structure Of Bioflocs And The Abundance Of Nitrogen Metabolism Genes With Different Particle Sizes And The Screening Of Functional Microorganisms In Cultured Water Bodies Of Litopenaeus Vannamei

The accumulation of ammonia nitrogen and nitrite nitrogen in the aquaculture waters will cause disease or death of Litopenaeus vannamei to a certain extent.Therefore,farmers try to reduce the disease degree by using antibiotics or changing water in a large area.However,the abuse of antibiotics will aggravate environmental pollution,and large-scale water change will greatly increase the cost of breeding.The bioflocculation technology,which is one of the more advanced aquaculture technologies,has the natural advantages of reducing feed consumption,reducing aquaculture sewage discharge,and minimizing water exchange.The realization of various functions of bioflocs is due to the large microflora inside the bioflocs,and the different flocs size affects the composition of the microflora,so that bioflocs have different nitrogen metabolism strategies.In our study,the internal microbial structure,microbial diversity,and the abundance of the main functional genes involved in the nitrogen metabolism process of biological flocs of different particle sizes were studied,in order to have a deeper understanding of the internal composition of biological flocs and dominant bacteria species.Clarify the nitrogen utilization strategies and final products of microorganisms in biological flocs with different particle size levels,and screen and cultivate more valuable biological flocs.Meanwhile,high-efficiency heterotrophic nitrification-aerobic denitrification bacteria were isolated from the tail water of Litopenaeus vannamei culture,and their denitrification performance was studied,in order to screen more microbial species with stable denitrification ability that can efficiently treat aquaculture wastewater.Part one:In order to clarify the bacterial community structure and function of bioflocs with different size in the early and later culture of Litopenaeus vannamei,Illumina Miseq high-throughput sequencing was performed on 16S r DNA V3 region of bacteria in bioflocs with different sizes of 106μm（with this particle size only added at the later stage of culture）、48μm、25μm、5μm and 0.2μm,to compare the bacterial community composition.The results showed that in the early stage of culture,the diversity and richness of bioflocs with different particle sizes were only different in a few groups,while the overall diversity and richness of bioflocs with 0.2μm particle size in the later stage of culture were lower than those with other particle sizes.In addition,with the inflow of nitrogen,phosphorus and other nutrients,the microbial diversity in the later stage of culture was significantly higher than that in the early stage.Although Proteobacteria,Bacteroidetes,Actinobacteria,Cyanobacteria are the dominant phyla in different periods,their proportions were changed.Cyanobacteria,as obligate aerobic bacteria,had a sharp increase in particle size of 5μm and 0.2μm in the early stage of culture with nitrogen deficiency;Actinomycetes,also obligate aerobic bacteria,showed a sharp increase in particle size of 0.2μm in the later stage of culture when nitrogen was relatively abundant,replacing Proteobacteria as the dominant species with the highest abundance.All of the above showed consistent co-occurrence at the genus level.Bioflocs of different particle sizes had unique nitrogen metabolism strategies.Compared with other particle sizes in this study,the bioflocs with a particle size of 5μm、0.2μm in the oligotrophic bioflocs also had a more complete nitrogen metabolism chain.Therefore,bioflocs containing Cyanobacteria such as Cyanobium PCC-6307 and Cyanobacterium PCC-10605 or Actinobacteria such as Ilumatobacteraceae,Mycobacterium and Candidatus＿Aquiluna can be selectively screened in practical production.Therefore,it is speculated that the bioflocs with small particle size added with appropriate amount of the above bacteria have greater nitrogen metabolism potential.Part two:Fluorescence quantitative PCR（q PCR）was used to analyze the four functional denitrification genes abundance in biological flocs of different particle sizes in Litopenaeus vannamei culture water---nap A（encoding periplasmic nitrate reductase）,nar G（encoding membrane-bound nitrate reduction Enzyme）,nir S（encoding nitrite reductase）,nos Z（encoding nitrous oxide reductase）to determine the contribution of denitrification to nitrogen metabolism and the end products of nitrogen in bioflocs of different sizes.The results showed that,on the whole,the gene copies of nap A,nar G,nir S,nos Z in the later stage of culture was higher than that in the early stage of culture,and except for nir S,the bioflocs with small size（5μm、0.2μm）of nap A,nar G and nos Z had higher richness in the condition of sufficient nitrogen.The high expression of the gene（nap A,nar G）made nitrate quickly converted into nitrite,which was conducive to the progress of denitrification and promote the accumulation of nitrite.The abundance of bioflocs with smaller size of nir S decreased under nitrogen sufficient conditions,which was another important reason for the accumulation of nitrite.The high nos Z abundance and the nir S/nos Z ratio were always lower than 0.15,indicating that the bioflocs under various nutrient conditions and particle sizes had strong N₂O reduction potential,and the flocs with particle sizes of 5μm and 0.2μm under nitrogen sufficient conditions were the highest,that is,nos Z always maintains high abundance,and the nir S/nos Z ratio always remains below 0.15,indicating that the biological flocs under various nutrient conditions and particle sizes have a strong N₂O reduction potential,and the floc particle sizes of 5μm and 0.2μm under nitrogen sufficient conditions were the highest.This means that N₂ was the end product in the denitrification process.Therefore,it is speculated that the culture mode of Litopenaeus vannamei involved in this study will not cause greenhouse gas N₂O emission basically.Part three:Two heterotrophic nitrifying-aerobic denitrifying strains xc1 and xc2were isolated from the aquaculture wastewater of Litopenaeus vannamei by the combination of enrichment medium and chromogenic medium.Both strains were preliminarily identified as Pseudomonas alcaligenes by 16S r DNA.In the nitrification medium with NH₄Cl as the sole nitrogen source（initial concentration was 50 mg/L）and sodium succinate as the sole carbon source,xc1 and xc2 strains could remove the ammonia nitrogen in the medium to 0.01 mg/L and 0 mg/L,respectively,after only 3h,and the removal rate of ammonia nitrogen was both close to 100%after 24 h.The denitrification ability test was carried out in a denitrification medium with potassium nitrate as the sole nitrogen source（initial concentration was 50 mg/L）and sodium succinate as the sole carbon source.Only in 3 h,xc1 and xc2 strains could remove the nitrate nitrogen from 50 mg/L to 4.67 mg/L,4.50 mg/L,respectively,and the removal rate of nitrate nitrogen by the xc1 and xc2 strains reached 98%and 100%,respectively,after 24 h.Therefore,the experiment proved that xc1、xc2 had good performance of heterotrophic nitrification and aerobic denitrification under laboratory conditions,which laid the most important bacterial strain foundation for the development of denitrification microbial agents suitable for aerobic conditions in the future.