Isolation, Identification And Algae-lytic Characteristic Of Algaecidal Bacteria Strain A2against Microcysis Aeruginosa

With the development of society, sewage was constantly inputed to water environment,resulting in a substantial increase in the nutrient content （e.g. N and P） of the water columnthereby causing the blue-green algae and other phytoplankton blooms, causing significanteconomic losses and serious impact on the water environment and water security. Currently,cyanobacterial blooms has become a common concern all over the world. As physical,chemical and some biological methods are not satisfacted, algicidal bacteria attractedwidespread attention due to its safety and adaptability, becoming an important means for thegovernance of water pollution.The present work isolated and identificated algicidal bacteria,and further researched their impact on algal toxin secretion and finally isolated and purifiedactive compounds by separation and extraction technology from the fermentation broth of thealgicidal bacteria. Main findings are showed below:1. Co-culture method was applyed and Microcystis aeruginosa was used as indicatingalgae species, four bacteriums were islolated from eutrophication lakes and identified asAcinetobactor sp., Pseudochrobactrum saccharolyticum, Alishewanella fetalis, andLysinibacillus fusiformis based on the16S rDNA homology and physiological-biochemicalcharacteristics combained with phylogenetic analysis, and the similarity was over97%.2. The algicidal effects test showed that the algicidal rates against M. aeruginosa after7d were91.6%,70.0%,71.3%, and78.3%, respectively. Algae-lytic character studies haveshown that the four bacteria showed better algicidal activity against cyanobacteria than greenalgae. The culture of3strains A2, W10, W17still showed strong algicidal effect on M.aeruginosa after removal of the cells, high temperature treatment, and the culture filter havelittle algicidal activity, so we can see that the algicidal factor of these strains wereextracellular secretion and thermal stability substances.3. The effects of strain A2on toxin secretion of M. aeruginosa were further studied. Theresults showed that, when the bacteria was added to the algae culture （10%）, the intracellularand extracellular toxin decreased51.4%and53%, respectivly after96h exposure. Samples were collected and used for the toxin related gene expression analysis by qRT-PCR. After12hexposure, the mcyA significantly down regulated （P <0.05）, but raised after24h, theexpression was significantly decreased at96h （P <0.05）. The expression of mcyD increasedfrom12h to48h, and then declined after96h. The expression of mcyH12and24h have nosignificant differences compared woth control （P <0.05）, but significantly down-regulated at48h and96h.4. The A2strain was large scale fermentated and centrifugated. The filter was dynamicadsorpted by macroporous resin and eluented by methanol and finally extracted by n-butanolto obtain samples. Samples were separated and purified by the two-stage silica gel columnchromatography, Sephadex LH-20gel, recrystallization, etc., Finally, two pure compounds,compound I and compound II were isolated, and identified as cyclo-（Gly-L-Pro） andhydroxyacetophenone ammonia by the means of spectroscopy combined with their physicaland chemical properties. Algae activity tests showed that hydroxyacetophenone ammoniahave notable algicidal effects on on M. aeruginosa, the3d and7d EC₅₀values were22.5mg/L and10.3mg/L respectively.In summary, the Acinetobacter sp. A2strain isolated from eutrophic water has a highlytic activity and can degrade cyanobacterial toxins. The extracellular product of strain A2,hydroxyacetophenone ammonia has higher algicidal activity, showing the potential ofbecoming a new type of pollution-free algicide.