Bacterial Communities Diversity In Integrated Multi-trophic Ponds And Correlation With Environmental Factors

This paper in allusion to current research focus on microbial action in integratedmulti-trophic ponds, firstly introduced microbial molecular ecology PCR-DGGEtechnology in the research on microbial ecology of China’s northern prawn ponds,optimized the conditions to extract genomic DNA of pond sediment and denaturinggradient gel electrophoresis, and besides, combined plate count to study physical andchemical factors and quantity of bacteria of sediment of prawn ponds as well asbacterial community diversity with the changes of culture cycle, thus, it could bemore objective and comprehensive to acquaint and study the changes of bacterialdiversity in time and space in China’s northern culture mode to provide basis formaintaining the ecological equilibrium of breeding areas. At the same time, wecarried out clone and sequence test for the significant bands of DGGE patterns ofpond sediments to obtain the information about advantageous microbes of breedingpond, which provided basis to separate and filtrate beneficial functional microbesfrom the primary environment. Finally, the article used relevant statistical methods topreliminarily discuss the relationship of interaction between bacterial diversity andmicrobial community structure of sediment in shrimp pond of multiple nutrientlevels and provide reference to predict the possible changes of microbial mechanismof aquaculture environment with the change of physicochemical factors. The maincontent is divided into the following sections:1. Optimization of PCR-DGGE testing conditionsThe article selected DNA extraction method of CTAB to obtain full and clearmain band of high yield. The environmental total DNA was as the template, landingPCR was used to amplify the PCR action of GC-F341/R534PCR. Denaturing gradient gel electrophoresis (DGGE) was the optimal condition: the range of DGGEwas20%-60%, voltage was10v, and the time of electrophoresis was2hours, silverstaining. The DGGE of optimal resolution could be obtained in the condition afteroptimization and the belt could be clearly separated.2. Bacterial communities diversity in integrated multi-trophic pondsSediment of different ponds was carried out DGGE analysis, gel analysissoftware was used to analyze the atlas of DGGE electrophoresis，according to theresults：（1）The results of analysis showed that the bacterial species in breedingecological system were abundant; there were differences among all the samples;among total27samples in three ponds, the sample bands were very abundant and12DGGE bands in different places were founded. The number of bands of each samplewas between14and33. Among the samples in three ponds, the number of samplebands was stable, average45%was specific bands, and55%bands were generallyexisted in every sample. The content of bands had changed a lot in different stage ofbreeding and the mode of distribution of bands also had relatively great difference,which revealed that the content and structure of microbial flora in shrimp cultureecological environment had great changes.（2）The article made clustering analysis for the DGGE atlas in differentbreeding stages. UPGMA analysis chart reflected that microbial community structurehad regular changes with breeding cycle. The article calculated Shannon-Wienerbiodiversity index to reflect the law that the diversity of microbial flora of differentsamples changed with the change of time and space. The Shannon-Wiener index inthis study was at a high level, which showed that the diversity of sediment of pondsof multiple nutrition levels was high. Viewing from the space, compared with othertwo ponds, the H value of shrimp, crab and selfish polyculture was high and thedegree of change was little, which showed that the diversity of microbial flora in theway of shrimp, crab and selfish polyculture was high and the stability of communitywas high. Viewing from time, the diversity of microbial flora community of eachpond in the middle of breeding was the highest. （3）This study, through recovery and sequence test of significant bands,proved that the bacteria from the sediment of shrimp pond was mainly divided into8categories, including phylum firmicutes, proteobacteria (α-proteobacteria,β-proteobacteria, γ-proteobacteria, ε-proteobacteria and δ-proteobacteria),bacteroidetes, actinobacteria, chloroflexi, cyanobacteria, chlorobi and acidobacteria.Different samples had common bands, but also had characteristic bands. Along thetime succession gradient, new advantageous populations were reproduced and someadvantageous populations were declined.3. Aquaculture pond bacteria changes and physical and chemical factorcorrelation analysisIn the process of breeding, the scope of all physical and chemical factors werein the range of tolerance of prawn and the total number of heterotrophic bacteria andvibrio of the sediment in different breeding ponds would change with the increasingtime of breeding time. Compared with heterotrophic bacteria, the fluctuation ofnumber of vibrio was greater, which was possibly because that the number of vibrioof sediment was more sensitive to the fluctuation of physicochemical factors. Thecorrelation analysis showed that the total bacterial count of sediment in ponds waspositively related to the content of organic matter; both the content of nutrient salt ofnitrogen and temperature affected the change of bacteria quantity; there was nosignificant difference between the number of vibrio and the changes ofphysicochemical indexes. The article made a typical type analysis of the change ofmicrobial community and physicochemical factors of culture environment. Based onthe results, in this research, the detected physicochemical factors included52.6%environmental factor related to microbial community. The analysis showed that theimpact of MC on bacteria community was more significant that that of other threephysical and chemical factors.