Analysis Of EHB01 Cyanobacteria Inhibitory Active Substances And Optimization Of Culture Conditions

Cyanobacteria refer to microorganisms that directly or indirectly inhibit or even kill algae to further lyse algae cells.Most of the cyanobacteria can use algae as carbon source,nitrogen source and other energy sources to grow and participate in its metabolism and degradation process.A large number of studies have shown that the use of microbial methods for algae removal has the advantages of high efficiency,saving money,and low risk of secondary pollution.It has become the first method at home and abroad as a method of controlling eutrophication of freshwater lakes.Algae-inhibiting active substances and the mechanism in the process of algae dissolution have far-reaching practical significance.In this experiment,Microcystis aeruginosa,a representative of aquatic algae widely distributed in freshwater resources and causing the most harm,was selected as the algae control object.One strain was selected from eutrophic water by liquid infection.Efficient cyanobacteria EHB01.Using the principle of similar miscibility of organic solvents,the crude fermentation liquid of strain EHB01 was extracted by organic solvents,and the extracted substances were analyzed by GC-MS using a gas-mass spectrometer.The single factor test and response surface design method were used to optimize the cyanobacteria conditions of the strain EHB01.The main results are as follows:1.Screened a bacterium with high algae-dissolving effect in eutrophic water samples.Combined with morphological staining,scanning electron microscope observation and 16 S rRNA gene sequence analysis,this strain was combined with Ralstonia pickettii CP12(KF378754.1).The sequence similarity between them is as high as 98.33%.The strain was judged to be Ralstonia.2.The bacteria and algae were co-cultured for 72 h.By measuring the chlorophyll content of the algae cells,it can be seen that the algae lysis rate can reach 83.2%,and the bacteria EHB01 in the logarithmic and stable phases have the best effects on algae lysis,83.2% and 78.6%.The bacteria EHB01 were further cultured and centrifuged to obtain bacterial supernatant,bacterial cells and fermentation broth respectively,and they were co-cultured with algae for 60 h,and the chlorophyll content of the algae cells was measured.The results showed that the chlorophyll content of each experimental groupdecreased compared with the control group.The chlorophyll content of the bacteria group was 2.86±0.02 mg/L,and the chlorophyll content of the fermentation broth group and the sterile supernatant treatment group were 1.34±0.06 mg/L and 1.29±0.03 mg/L,indicating that the fermentation liquid of the bacteria EHB01 has a strong effect of algae.3.Organic solvents chloroform and ethyl acetate were used to repeatedly extract the crude fermentation liquid of bacteria EHB01,and the extracted cyanobacteria active substances were analyzed by GC-MS.The algae-inhibiting ability of the extract is higher than that of chloroform extract.Based on GC-MS analysis results of ethyl acetate and chloroform extracts,it was initially determined that at least 14 volatile compounds were produced in EHB01 fermentation,including 9-octadecenoic acid,(E)-octadecenoic acid,glycidyl oleate,etc.4.The environmental conditions during the algae dissolution,the concentration of the culture solution,the ambient temperature,and the light intensity were tested.The carbon-to-nitrogen ratio and the nitrogen-to-phosphorus ratio in the culture medium were further dispensed in different proportions for corresponding dissolution.Exploring algae experiments.The carbon source,pH and nitrogen source in the EHB01 fermentation broth were selected for single-factor experiment.Through the CCD experiment design method,different environmental factors were selected to achieve the best plan in the experiment.The results of the experiment show that: the increase in the rate of algae dissolution is positively correlated with the concentration of the culture solution;the temperature set for the rate of algae dissolution shows a trend of increasing first and then decreasing;the light intensity,the ratio of carbon to nitrogen in the medium,and the ratio of nitrogen to phosphorus The effect on the rate of algae lysis is gradually increased first,and then there is a clear downward trend;the internal condition medium composition of the algae lysis effect is optimized by CCD experiment,and the best carbon source,nitrogen source and medium are obtained respectively The pH value,the algae lysis rate after treatment was increased to 86.97%,which was 21.72% higher than the natural algae lysis rate before.