A Preliminary Study On Pseudoalteromonas Sp.WCPW15015:optimization Of Medium And Antagonistic Mechanism Against Pathogenic Vibrio

Vibrio harveyi has become one of the most serious bacterial pathogens in marine aquaculture in South China.It is an important way to build an ecological farming model by making full use of the antagonistic effect between microorganisms.The optimization of the culture medium,the exploration of the antagonistic conditions and mechanisms are the pivotal prerequisites for the application of antagonistic bacteria.In this paper,we established the growth curve of the antagonistic bacterium WCPW15015(Pseudoalteromonas sp)and V.harveyi GDH1 1385(pathogenic Vibrio),and the standard curve for calculating the bacterial concentration according to the OD600 value.It was determined that the optimum pH,the temperature and the salinity was 7.6-7.8,30℃ and 30 for the antagonistic bacterium,respectively.Microscope observation resultsshowed that WCPW15015 was "long rod-shaped" and had strong moving ability,but GDH11385 was "arc-shaped" and had a moderate moving ability.Using 2216E as the basic medium,a single factor experiment was conducted by changing the nitrogen source or the carbon source.The results showed that Pseudoalteromonas WCPW15015 growed slowly when the nitrogen source was missing And the absence of carbon source had no significant effect on its growth.Through single-factor experiments,the best carbon source was brown sugar,the best nitrogen source was beef extract,and the best inorganic salt was iron phosphate for the culture of the antagonistic bacterium.On the basis of 2216E,an optimal medium was constructed by adding the above carbon source,nitrogen source or inorganic salt.The results showed that 2216E with brown sugar(lg/L)could increase the bacterial concentration from(1.183～4.213)×10~8CFU/mL to 3.375×10~9CFU/mL in the suspension,which laid an important foundation for the future establishment of the continuous fermentation culture of the antagonistic bacteria.The agar diffusion method was used to determine the antagonistic effect of suspensions,bacterial cells,extracellular products and intracellular products of Pseudoalteromonas WCPW15015.The results showed that the WCPW15015 suspension as well as the bacterial cells had significant antagonistic effect,althouth no antagonistic effect was observed in the groups of the exoproducts as well as intracellular products.Antagonist test results showed that antagonistic bacteria could produce significant antagonistic rings in the presence of Vibrio,indicating that the antagonistic bacteria could recognize Vibrio and secrete certain chemical substances to inhibit(translucent circle)or kill(full transparent circle)the pathogenic Vibrio.At the same time,the co-cultivation microscope observation results showed that the antagonistic bacteria could recognize,envelop and phagocytose(incidentally)pathogenic Vibrio,which might be another important way to antagonize pathogenic Vibrio.The agar diffusion method(pH 7.6,30℃,salinity 30)was used to determine the effect of the bacterial concentration on the antagonistic effect.The results showed that the minimum inhibitory concentrations of the Pseudoalteromonas Was 10~3 CFV/mL and the diameter of the inhibition zone was(8.40±0.16)mm when the V.harveyi concentration was 10~3 CFU/mL.When the Vibrio harveyi concentration was 104 CFU/mL and 105 CFU/mL,the minimum ihibitory bacteria concentrations of Pseudoalteromonas were 105 CFU/mL and the diameters of the zone of inhibition were(9.64±0.07)mm and(8.24±0.22)mm,respectively.These results could be matched with the multivariate quadratic equation:f(x,y)=9.09-0.3339x+0.02325y+0.00267x2-0.0002273xy+0.0002477y2.When the concentration of Vibrio harveyi was 106 CFU/mL,the minimum inhibitory concentration of Pseudoalteromonas was 107 CFU/mL and the inhibition zone diameter was(18.15±0.09)mm;when the Vibrio harveyi concentration was 107 CFU/mL and 10~8 CFU/mL,the minimum inhibitory concentrations of Pseudoalteromonas were 10~8 CFU/mL,and the diameters of the inhibition zone were(18.53±1.34)mm and(11.68±2.58)mm,respectively,which wasconsisted with the multivariate quadratic equation:f(x,y)=8.395-0.1682x+0.1161y+0.00106x2-0.00000675xy-0.0001026y2.In conclusion,we determined the optimum growth conditions for the antagonistic bacterium WCPW15015,and optimised its fermentation medium,and explored its preliminarily mechanism of antagonist in the study.All of these results were very helpful for the for the future development of probiotics and their application technologies for the prevention and control of mariculture pathogen,Vibrio harveyi.