| Microcystis aeruginosa is a representative population of harmful algal blooms.If Microcystis aeruginosa is rampant in natural water,which can not only directly threaten the life safety of human and aquatic organisms by deteriorating water quality,but also indirectly damage economic development by affecting aquaculture and tourism.In this study,Pseudomonas sp.W10screened from the viscera of wild field snails in Taihu Lake Basin was selected as the research object.Its algicidal characteristics,algae-lysing kinetics,damaging effects,algal lysate products and algicidal active substances are all the key factors to reveal the algicidal mechanism and regulate the algae cell biomass.The above factors were analyzed by molecular biology identification,physiology and biochemistry,spectroscopy,GC-MS and so on.The main achievements are as follows:(1)The results of isolation,identification and algae-lysing kinetics of an algicidal bacteria Pseudomonas sp.W10 indicated that strain W10 was identified as Pseudomonas sp.that released extracellular substances to dissolve and cleavage Microcystis aeruginosa.There was no significant difference of algicidal ratio(P>0.05)between NA and starch medium,both of which were significantly better than the modified basic medium(P<0.05).However,because the composition of starch medium was simpler than that of NA medium,starch medium was selected to cultivate algicidal bacteria W10.Besides,there was no significant difference of algicidal ratio(P>0.05)between W10 and its filtrate at the same growth phase.Both of them had the best algicidal effect in the stable growth phase and the decay growth phase,followed by the logarithmic growth phase and the delay growth phase.The optimum dosage of W10 was 10%,and the highest algicidal rate was 80.05%.While the algicidal effect of W10's filtrate was positively correlated with the dosage,the highest algicidal rate of W10's filtrate was 92.15%in the 1:2 treatment.Furthermore,the growth and algicidal process of W10 followed the Logistic equation and the first-level kinetics,and the fitting effect was good.The corresponding R2 were0.9908 and 0.9882.(2)The damaging effects of Pseudomonas sp.W10 on Microcystis aeruginosa indicated that algae cells lost their normal physiological and biochemical functions,which was reflected in the significant reduction in indicators.For example,with the algae-lysing time extension,the algae cells showed a negative growth,which decreased by 156.87%.There were loopholes in the cell membrane structure and can not be regenerated and repaired,and the leakage amount of nucleoside was 2.73 times that of the blank group.Also,phycobilin concentration and net photosynthetic oxygen release rate decreased obviously.At the end of culture,phycocyanin,allophycocyanin and phycoerythrin were 1.43×10-3mg·L-1,4.36×10-3mg·L-1 and 1.07×10-3mg·L-1.The net photosynthetic oxygen release rate was only 17.54?mol O2·(mg·Chla·h)-1,and the photosynthesis was blocked.On the other hand,the index of antioxidant system of Microcystis aeruginosa increased at first and then decreased.Microcystis aeruginosa secreted more SOD and CAT to protect algal cells under aseptic stress,and MDA also increased due to the aggravation of stress.However,when more and more algae cells disintegrated and died in adversity,SOD,CAT and MDA also decreased.(3)The results of spectral analysis of algal lysate products in the algicidal process of Pseudomonas sp.W10 indicated that that the composition of algal lysate products was complex,including a variety of amino acids,proteins,carbohydrates,humic acids and aromatic compounds.The URI values of algal lysate products measured by UV-vis were 1.20,1.19 and 1.18,indicating that the degree of aromatization of algal lysate products increased gradually.The FTIR spectroscopy showed the algal lysate products contain 7 characteristic peaks during the algicidal process,and the functional groups involved were-OH,N-H,C-H,C=O,C=C,and etc.Besides,the contents of macromolecular substances such as protein,nucleic acid and cellulose decreased,while the contents of small molecular substances such as fatty acids,carbohydrates and aliphatic alkanes increased.The 3D-EMMs spectroscopy demonstated that there were 3 fluorescence peaks of algal lysate products.In the process of algae lysis,the content of amino acid components increased because of protein decomposition.However,humus components were difficult to be degraded and remained in the algal lysate products,resulting the degree of humification increased.With the results of SEM,the algicidal pathways were speculated as following:Pseudomonas sp.W10 secreted algicidal active substances,which destroyed the algae cells'structure,released the algal intracellular substances,affected the normal physiological functions and resulted in the death of algae cells finally.(4)The results of research on separation characteristics and preliminary identification of the algicidal active substances of Pseudomonas sp.W10indicated that the algicidal active substances could be well dissolved in anhydrous ethyl alcohol.However,there was still a significant difference in the algicidal effect between the ethanol-soluble phase with an algicidal rate of61.32%and the aseptic concentrate(P<0.05).so it was not ruled out that the algicidal active substances were nucleic acid,protein,polysaccharide and so on.Four kinds of CCl4,CHCl3,C2H5COOCH3 and petroleum ether extractants with little polarity had weak effect of algicidal processes in organic phase.The algicidal rates of four were all less than 52%,indicating that the active ingredient that played a major role in algicidal process was a strongly polar substance.Besides,the algicidal effects of different dialysis samples were different.The order of algicidal effects were 1.0k D>>3.5k D>7.0k D.As a result,the relative molecular weight of the algicidal active components was?1.0k D.Last,the GC-MS results indicated that the algicidal active substances might be esters,alkanes,alcohols,aromatic hydrocarbons or nitrogen-containing compounds. |
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