Isolation, Identification Of Microcystin Degrading Bacteira And Algae-lysing Bacteria And Protoplast Preparation Of T1Strain

With the rapid development of industry and agriculture, the watereutrophication has been aggravated year by year, and the cyanobacteria bloompollution has been a global issue. Some algae release microcystins, whichthreaten to human health when exposed to drinking water source. It is difficultto remove microcystin with physical and chemical methods， andmicrobiological methods show the superior to cyanobacterial bloom pollutiongradually. The algae-lysing bacteria and microcystin degrading bacteria beingjointed to remove cyanobacteria is a new research direction. There were3strains of microcystin degrading bacterium and13strains of algae-lysingbacterium isolated from the algae pulp leachate, silver carp growth in theTaihu Lake Basin and the etiolated Microcystis aeruginosa, which werenamed as R1-R3, F1-F12and TL respectively. Species identification of R3,F8and TL were done with16S rDNA sequence analysis techniques,morphologic observation and physiological-biochemistry characteristicanalysis in order to look into the action mechanism and lay a foundation forengineering bacteria construction. And the protoplast preparation andregeneration of strain T1isolated by the team, which had high efficientdegradability to microcystin-LR was researched in the study. Research resultsof the paper were as follows:(1)The microcystin-degrading strain R3isolated from the algae pulpleachate had an efficient removal to Microcystin-LR (MC-LR) and wasidentified as Lysinibacillus fusiformis. Its accession number was JQ991002inGenBank of the National Center for Biotechnology Information (NCBI) andthe number was6107in China General Microbiological Culture CollectionCenter (CGMCC). The research of strain R3for microcystin-degradingcharacteristics indicated that the suitable pH value was7.0, the best inoculumquantity was4-6%and the initial concentration of MC-LR was49.07μg/L atoptimal parameters of30℃and130r/min rotating speed. With theseoperating parameters, the removal rate of MC-LR was up to46.47%in the seventh day. In addition, added carbon source of methanol, ethanol andglucose could promote the degradation of microcystin to some extent. Andthen, the initial concentration of MC-LR with39.26μg/L, pH with7.0,inoculated with5%of bacteria inoculum, rotating speed with130r/min,temperature with30℃, and MC-LR as the sole carbon source, threeexperimental groups added different kinds of carbon sources were studied.The results showed that the strain R3had MC-LR specificity and MC-LRdecomposition rate were up to30.26%,36.90%and59.26%, respectively.And the MC-LR decomposition rate was28.66%without added carbonsource.(2)The strain F8which lysed Microcystis aeruginosa were isolated fromsilver carp growth in the Taihu Lake Basin and was identified asLysinibacillus fusiformis. Its accession number was JQ991003in theGenBank of NCBI and the number was6106in CGMCC. According to thestudy of ecological factors in its lytic character, when the temperature was28℃, the light intensity was2500lux, pH was7.0, and initial chlorophyll-a(Chla) concentration was61.2mg/m3, the optimal bacterial concentration wasnot less than2.75×10~6/mL (bacteria and algae volume ratio≥1:10), and thephotoperiodic effect was inapparent. With different treatments of the bacteriaculture, the result showed that the lytic mode of strain F8was indirectly. Theinhibition of the growth of algae could be achieved by secreting extracellularnon-protein substances and a small amount of protein substances to lysemicrocystis.aeruginosa cells.(3)Two samples of the enrichment culture of Microcystis aeruginosa inlaboratory etiolated and died after7days. Because having no other additivesin the culture, the additives had no interference on the etiolated Microcystisaeruginosa. Based on the above, infering that the etiolated Microcystisaeruginosa may be associated with bacterial infection. And one algae-lysingstrain TL which lysed Microcystis aeruginosa was isolated from the etiolatedMicrocystis aeruginosa. After analysed by morphologic observation andphysiological-biochemistry characteristic test, algae-lysing bacteria TL wasidentified as Lysinibacillus fusiformis with the16S rDNA sequence analysistechniques. Its accession number was JQ991004in the GenBank of NCBI andthe number was6108in CGMCC. When the temperature was28℃, the light intensity was2500lux, pH was7.0, and initial Chla concentration was838.58-982mg/m3, the optimal parameters were as follows: bacteria andalgae volume ratio was not less than1:10, light cycle ratio was12h:12h.Under these conditions, the microcystis aeruginosa-lysing removal rate bystrain TL was up to62.45%after96h. With different treatments of thebacteria culture, the result showed that the strain TL dissolved cyanobacteriaby the lytic mode of action was indirectly. The inhibition of the growth ofalgae could be achieved by secreting extracellular non-protein substances anda small amount of protein substances to lyse microcystis.aeruginosa cells. Themode of action of the algae-lysing bacteria had been reported was indirect ordirect contact.(4)With inactivated protoplast fusion method, the Bacillus sphaericus T1(CGMCC NO.4498) isolated by the team and algae-lysing bacteria TLseparated in the research were as parent strains to get engineering bacteriumwhich could degradate MC-LR and lyse algae at the same time. In this paper,the main research was the protoplast preparation and regeneration of strain T1to lay a foundation for subsequent work. The optimal parameters were asfollows: lysozyme concentration was0.05mg/mL, hydrolysis temperaturewas30℃, and enzymatic time was1hour. Under the condition, protoplastformation and regeneration rate was high, and the high activity of protoplastwas conducive to select fusants.Based on the systematic research on isolation and identification ofmicrocystins degrading and algae-lysing bacteria and protoplast preparationof T1, strains R3, F8and TL were all identified as Lysinibacillus fusiformis.The results laid foundation on constructing engineering bacteria withmicrocystin and algae degradation properties by protoplast technique andcontrolling cyanobacteria bloom pollution radically.