【作者】 汪辉；

【导师】 刘兆普； 刘玲；

【作者基本信息】 南京农业大学 ， 应用海洋生物学， 2011， 博士

【摘要】 由于淡水与海洋水体富营养化程度的日益加剧,有害藻类暴发日趋频繁,严重影响了水域生态系统的安全以及附近居民的生活健康,因此探索行之有效的抑制藻华暴发的途径是极为迫切的。在利用物理、化学和其它生物方法处理不甚理想的情况下,利用溶藻细菌除藻成为目前生物控藻的研究热点,具有广阔的应用前景。溶藻细菌一般从发生富营养化的水体中直接分离,具有一定的针对性,且控藻效果非常好,尤其是在藻华发生初期使用,短期内即可达到控制藻类生物量或阻止藻类大量增殖的效果。本文从江苏太湖水样和山东胶州湾海底沉积物中分别分离得到3株和2株高效溶藻细菌,在对这5株菌株进行生理生化和分子鉴定的基础上,分别研究了其对水华与赤潮常见有害藻类---铜绿微囊藻(Microcystis aeruginosa)与中肋骨条藻(Skeletonema costatum)的溶解特性和作用机理,并结合生物化学手段对细菌的溶藻活性成分进行分析,推进了溶藻细菌对藻类的抑制效应研究。1、铜绿微囊藻溶藻菌株的筛选、鉴定1)从太湖水样中共分离出24株细菌,其中3株细菌表现出较强的溶藻效果,将其编号TL1, TL2、TL3,并以这三株细菌作为试验材料进行下一步研究。2)对3株溶藻细菌的16Sr DNA进行测序,采用BLAST程序将测序结果与GenBank中的已知序列进行相似性分析,结合生理生化鉴定,结果表明：细菌TLl、TL2和TL3分别属于无色杆菌属(Achromobacter sp.)、寡养单胞菌属(Stenotrophomonas sp.)和芽孢杆菌属(Bacillus sp.)。2、铜绿微囊藻溶藻细菌的溶藻特性及溶藻方式1)试验发现,细菌TL1、TL2、TL3都能够强烈抑制铜绿微囊藻、斜生栅藻和小球藻的生长,导致其叶绿素α含量显著下降,且尤以TLl菌株的溶解效果最好。2)在细菌TL1、TL2与TL3培养液的处理下,铜绿微囊藻的叶绿素荧光动力学参数：PSⅡ的最大光能转化效率(Fv/Fm). PSⅡ的实际光能转化效率(Yield)和最大相对电子传递速率(Pm即rETRmax),随着时间的增加而持续下降。细菌TL1、TL2、TL3的溶藻机理是破坏铜绿微囊藻的光合系统,强烈地抑制铜绿微囊藻的光合活性,从而抑制铜绿微囊藻的生长。3)对细菌TL1, TL2, TL3的溶藻作用机理进行探究,发现这三株细菌的溶藻因子不是细菌菌体,而是各自的胞外分泌物。细菌TL1、TL2、TL3均是通过分泌胞外活性物质进行间接溶藻。3、铜绿微囊藻溶藻细菌发酵的优化及溶藻活性物质性质的研究1)通过单因子试验的研究,探索了溶藻细菌TL1、TL2、TL3的最佳发酵条件,结果发现：当细菌TL1的培养温度为35℃,pH为8.0,培养时间为96h时,所分泌的溶藻物质的溶藻效果最明显；当细菌TL2的培养温度为30℃,pH为7.0,培养时间为60h时,所分泌的溶藻物质的溶藻效果最明显；当细菌TL3的培养温度为30℃,pH为7.0,培养时间为72h时,所分泌的溶藻物质的溶藻效果最明显。2)蛋白酶K处理后的三株溶藻细菌的胞外分泌物都丧失溶藻活性,说明细菌TL1、TL2、TL3的溶藻因子—胞外分泌物都不是蛋白类。3)细菌TL1、TL2、TL3的胞外分泌物中也没有检出磷的存在,表明这三株溶藻细菌的胞外分泌物不是核酸。4)细菌TL1、TL2的α-萘酚反应呈阴性,细菌TL1、TL2的溶藻活性物质不属于糖类,可能是具有耐热性的其它小分子物质；TL3菌株的胞外溶藻活性成分可能是一种水溶性糖。4、中肋骨条藻溶藻细菌的筛选鉴定1)从胶州湾海底沉积物中共分离出2株对中肋骨条藻有较强溶解作用的溶藻细菌,将其编号为JZ1、JZ2、并以这两株细菌作为试验材料进行一下研究。2)鉴定结果表明,菌株JZ1及JZ2分别属于交替单胞菌属(Alteromonas sp.)和列文虎克属(Maribacter sp.)。5、中肋骨条藻溶藻细菌的溶藻特性及溶藻方式1)按照10%的体积比向中肋骨条藻藻液中加入菌株JZ1、JZ2培养液,10d后,中肋骨条藻藻细胞全部死亡。细菌JZ1、JZ2具有较强的溶藻效果。2)根据荧光显微镜的观察,经过菌株JZ1培养液后的中肋骨条藻藻细胞的膜内物质首先开始变得浑浊,接着藻细胞膜出现破裂,胞内物质外漏。因此细菌JZ1、JZ2的溶藻机理是能够破坏中肋骨条藻细胞的完整性,使藻细胞程序性死亡。3)中肋骨条藻的生长阶段对菌株JZ1、JZ2溶藻效果均有影响,不同生长阶段的中肋骨条藻对细菌JZ1溶藻效果的敏感度可认为：稳定期>对数生长期>迟缓期；而菌株JZ2在中肋骨条藻的整个生长期内均对藻细胞的生长产生抑制作用.4)细菌JZ1对供试的球等鞭金藻有较强的溶解作用,对杜氏盐藻的生长影响较小,但对周氏扁藻、鲁兹帕夫藻、微绿球藻、司西扁藻、假微型海链藻的生长却没有任何影响。5)细菌JZ2对供试的鲁兹帕夫藻和球等鞭金藻有明显的溶解作用,但对周氏扁藻、杜氏盐藻、微绿球藻、司西扁藻、假微型海链藻的生长却没有任何影响6)细菌JZ1、JZ2的溶藻因子不是细菌茵体,同细菌TL1、TL2、TL3一样,也是细菌的胞外分泌物,细菌JZ1、JZ2也是通过间接溶藻的方式进行溶藻活动。6、中肋骨条藻溶藻细菌活性物质的分离提纯1)细菌JZ1的溶藻活性物质具有一定的耐酸性,但在碱性和高温条件下失活；而细菌JZ2的溶藻活性物质具有耐酸、耐碱和耐高温性。2)经过分子筛过滤、离子交换树脂筛选、高效液相色谱分离,得到纯化的细菌JZ1的溶藻活性物质。3)根据基质辅助激光解吸质谱及高效液相色谱-电喷雾质谱联用仪分析,得知细菌JZ1的溶藻活性物质一种是分子量为1267Da的多肽。综上所述,细菌TL1、TL2、TL3分泌的胞外溶藻活性物质阻碍了铜绿微囊藻的光合作用,间接抑制了铜绿微囊藻的生长；细菌JZ1、JZ2分泌的胞外溶藻活性物质破坏了中肋骨条藻藻细胞膜的完整性,继而抑制了中肋骨条藻的生长。另外,通过对5株溶藻细菌的溶藻特性、溶藻方式的分析,以及溶藻活性物质的性质研究,我们认为将溶藻细菌所分泌的胞外溶藻活性物质做为生物除藻剂可成为控制藻华的一种可能途径。更多还原

【Abstract】 Nowdays, eutrophication and massive accumulations of planktonic microalgae as algal blooms in waterbodies have caused serious environmental problems and done harm to the health of people around. So it is important to find out an effective method for controlling the growth of the blooming-forming algae. Measures for controlling such kind of algae include physical, chemical and biological means, but the former two may damage the aquatic environment and require high-energy inputs. The alternative approach for the elimination of nuisance algae involves the application of algae-lysing bacteria. Algae-lysing bacteria as the biology of removing algae have attracted more attention and has broad application prospect.The alage-lysing bacteria are able to restrain the biomass or multiplication of the bloom-forming algae in a short time, especially in the initial stages of the water blooms. Three algae-lysing bacteria named TL1、TL2、TL3 and two algae-lysing bacteria named JZ1 and JZ2 were isolated from water collected in Tai Lake and seafloor sediment collected in Jiaozhou Bay, respectively, and these bacterial strains were identified by physiologically and biochemically. We investigated the lytic abilities and mechanisms of TL1、TL2、TL3 strains and JZ1、JZ2 on the growthes of Microcystis aeruginosa and Skeletonema costatum, respectively. Moreover, we studied the characteristics of extracellular algae-lysing components from bacterial strains.1. Isolation and identification of the bacteria against Microcystis aeruginosa1) Among 24 bacterial strains isolated from water sample of Tai Lake, three bacterial strains, named as TL1、TL2、TL3, showed strong lytic activities. Thus, they were selected to be as research materials.2) The bacterial strains were identified by the sequence analysis of 16SrDNA, and the DNA sequence similarity analysis with the obtained sequences in Genbank. According to the morphological and physiological-biochemical characteristics, strains TL1、TL2、TL3 belong to Achromobacter sp., Slenotrophomonas sp. and Bacillus sp., respectively.2. The lytic abilities and mechanisms of strain TL1、TL2、TL31) Strains TL1、TL2、TL3 were able to restrain the growth of Microcystis aeruginosa、Chlorophyta and Chlorella strongly, and contents of chlorophyll-a and phycocyanin in the algae decreased remarkable, and strain TL1 had the best algicidal activity.2) Comparing to control, the maximal photochemical efficiency of PSⅡ(Fv/Fm), the actual photochemical efficiency of PS II in the light (Yield), the maximal relative electron transport rate (Pm) reduced sharply. The photosynthesis of M. aeruginosa was greatly promoted by three bacterial strains and the growth of M. aeruginosa was inhibited, this was the algicidal mechanisms of strains TL1、TL2、TL3.3) The algae-lysing factors in strains TL1、TL2、TL3 were not the bacterium themselves, but a kind of extracellular products from them. Three bacterial strains had algicidal effects on M. aeruginosa by indirect attack.3. The optimization of fermenting conditions of bacteria and study on the characteristics of algicidal substances1) When incubation temperature was 35℃, pH condition was 8.0 and the incubation time was 96h, the algicidal substance excreted by strain TL1 had the best algicidal effect on M. aeruginosa; and when incubation temperature was 30℃, pH was 7.0, the incubation time was 60h, the algicidal substance excreted by strains TL2 had the best algaicidal effect; the algicidal substance produced by strain TL3 showed the best algicidal effects when incubation temperature was 30℃, pH was 7.0, the incubation time was 72h.2) The algicidal substances excreted by strains TL1, TL2, TL3 lost the algicidal activities under the treatment of Proteinase K, it suggested that the algicidal substances excreted by stain TL1, TL2, TL3 were not protein.3) There were not phosphorous in the algicidal substances, the algicidal substances excteted by strain TL1, TL2, TL3 were not nucleic acid.4) The molish of strain TL1 and TL3 were negative, so the algicidal substances excreted by strain TL1 and TL2 were not sugar, they may be other substances; the algicidal substance of strain TL3 was water-soluble sugar.4. Isolation and identification of bacteria against Skeletonema costatum1) Two bacterial strain which showed lytic effects on Skeletonema costatum were isolated from seafloor sediment that collected in Jiaozhou Bay. And they were named provisionally as JZ1 and JZ2.2) Strains TL1 and TL2 belong to Alteromonas sp. and Maribacter sp., respectively, on the basis of morphological and physiological-biochemical characteristics.5. The lytic abilities and mechanisms of strain JZ1、JZ21) When the volume ratio of medium to algae solution was 10%, the cells of S.costatum died absolutely after 10 days.2) After addtion of strain JZ1, cellular morphology changes with time. Condensation of the cells and chloroplasts was clearly visible as was the formation and release of membrane blebs. So the algicidal mechanisms of strain JZ1、JZ2 are because the strains could break the cells of S. costatum, and this is indicative of programmed cell death.3) The antialgal efficiencies of JZ1 and JZ2 were different to the various growth phages of algae. For strain JZ1, the removal rate of S. costatum in stable phrase was the highest, and the next was log growth phase. However, strain JZ2 showed obvious algicidal effect in all growth phases of S. costatum.4) The algae-lysing factors in strain JZ1, JZ2 were not the bacterium themselves, but a kind of extracellular products from them as stain TL1, TL2, TL3. The two bacterial strains had algicidal effects on S. costatum by indirect attack.5) Species of Isochrysis galbana, Dunaliella salina were susceptible to bacterium JZ1,while the lytic effects of this bacterium on Tetraselmis chui, Pavlova lutheri, Nannochloropsis salina, Tetraselmis suecica, Thalassiosira pseudonana were feeble.6) Strain JZ2 had effects on the growth of Pavlova lutheri and Isochrysis galbana, but didn’t show effects on Tetraselmis chui, Dunaliella salina, Nannochloropsis salina, Tetraselmis suecica, Thalassiosira pseudonana. 6. Isolation and purification of algicidal substance from strain JZ11) The algicidal substance of strain JZ1 showed a little bit effect on the growth of S. costatum with acid treatment, but that of strain JZl lost the algicidal activity under stresses with alkali or high temperature; Strain JZ2 had acid tolerance and showed obvious effect on the growth of S. costatum.2) The pure algicidal substance of strain JZ1 was got, with the isolation and purification of resins and HPLC.3) Depend on the analysis of LC-ESI-MS and MALDI-MS, the algicidal component of strain JZ2 was a peptide with 1267kDa molecular weight.As above, the excretes produced by strains TL1、TL2、TL3 inhibit the growth of Microcystis aeruginosa by hindering the photosynthesis of M. aeruginosa; the excretes procuced by strains JZ1、JZ2 inhibit the growth of Skeletonema costatum by destroying the cells of S. costatum. The biological algaecide that made from the algicidal substances produced by algae-lysing bacteria have broad application prospect, based on the analysis of algicidal characteristics and algicidal mode of 5 strains of algae-lysing bacteria and the study characteristics of algicidal substances.更多还原