Analysis Microbial Community Structure And Screening Antagonistic Bacteria Against Aeromonas Hydrophila In Ponds With Grass Carp As The Dominant Cultured Species

The microorganism is an important part of aquaculture system.As producers,microorganism can use sunlight or inorganic substances produce organic compounds and provide nutrients for animals;as decomposers,microorganism can remove residual feeds,excrement,dead bodies of animals and plants,and promote material recycling and energy flowing in the aquaculture environment.In addition,there are many pathogens and probiotics in aquaculture environment.Therefore,studying the microbial community structure in aquaculture environment,it can provide a theoretical basis for the monitoring of aquaculture environment,finding the probiotics and preventing fish diseases.This paper mainly focused on microbial community structure analysis,antagonistic bacteria against Aeromonas hydrophila screening and basic characteristics studying in grass carp culture environment.Firstly,the plate count method was employed to study annual variation of heterotrophic bacteria in grass carp culture water environment,the results showed that the number of heterotrophic bacteria changed seasonally,the number of heterotrophic bacteria was highest in June and August,lower in October,and lowest in April and December.Secondly,high-throughput sequencing technology was used to study changes of microbial community structure in grass carp culture water environment.The results showed that Proteobacteria,Actinobacteria,Bacteroidetes and Planctomycetes were shared by different months,Firmicutes only appeared in April and June,Chloroflexi only appeared in June,Verrucomicrobia only dispeared in April.In the level of genus,the main bacteria were Acinetobacter,Stenotrophomonas,Pseudomonas in April;Ilumatobacter,Spartobacteria in June;Acidovorax,Candidatus Pelagibacter in August;Ilumatobacter,Steroidobacter in October;and Candidatus Pelagibacter,Orientia in December.By further analysis,we found that the microbial community structure in April is great difference to other months;microbial community structure similarity is the highest between June and August,lower between June and October,and lowest between October and December.The species richness was the highest in April and the lowest in December.The Microbial diversity was highest in October and the lowest in August.Furthermore,to study probiotics from grass carp culture pond,dot-inoculating method and Oxford cup method were used to screen antagonistic bacteria against Aeromonas hydrophila in water and sediment of grass carp culture environment.Eight strains of antagonistic bacteria against Aeromonas hydrophila were screened from water and sediment.further by Oxford cup test,the diameter of inhibition zone was shown maximum of strain WM-1,minimum of strain WM-4,the diameter of inhibition zone is range from 9.1±0.58 mm from 16.2±0.76 mm.Finally,the strain WM-1 was identified as Bacillus methylotrophicus by morphological observation,physiological and biochemical activity and 16 SrDAN sequence analysis.In order to improve antibacterial activity of the strain WM-1 against Aeromonas hydrophila,investigate and obtain the best medium formula and fermentation condition,we used Aeromonas hydrophila as the index bacteria,the antibacterial activity of strain WM-1 was measured by Oxford cup test,single factor experiments and orthogonal experiments were employed to optimize the liquid medium compositions and fermentation conditions.The optimum medium included 1.0% soluble starch,1.5% beef extract,0.05% K2HPO4·3H2O and 0.07% MgSO4·7H2O.The optimal fermentation conditions were initial pH 7.5,temperature 30 ?,fermentation time 48 h,medium capacity 20%,and rotate speed 210 r/min.Under these optimum fermentation medium and culture conditions,the diameter of inhibition zone of strain WM-1 is up to 22.5±0.50 mm,which showed a great improvement compared with the initial fermentation medium and culture conditions.