【作者】 杨晓新；

【导师】 尹平河；

【作者基本信息】 暨南大学 ， 水生生物学， 2008， 博士

【摘要】 近年来赤潮灾害频繁发生,严重危害沿海地区渔业生产和经济发展,开展对有害赤潮的防治研究越来越得到各国的重视。在利用物理、化学等方法治理赤潮不甚理想的情况下,溶藻细菌作为赤潮防治的新方法和可能微生物,开始引起国内外研究人员特别关注,并逐渐成为海洋环境科学研究领域的热点。本文从珠海赤潮发生海域筛选、分离到三株溶藻细菌G、Y和W,实验表明其对球形棕囊藻均有显著的溶藻效果。分别对三株溶藻细菌的溶藻作用过程、溶藻前后的藻菌形态变化、溶藻效果与环境因子的影响、溶藻过程中荧光变化、溶藻机理、归趋及其藻际的微生态效应进行了研究,实验结果旨在为近海养殖区有害赤潮藻的微生物防治提供科学依据。主要的研究结果如下:1.从珠海赤潮发生海域中采集水样,分离出11株细菌,分别与指数生长期的棕囊藻共培养,根据溶藻效果,从中筛选出三株具有显著溶藻作用的细菌G、Y和W。实验结果表明,三株细菌均显示为革兰氏阳性,属于杆菌,形态上明显不同,G细菌多处于分裂状态,Y细菌常为族集状聚合态,W细菌以多个细胞相连的方式存在。三株细菌都对球形棕囊藻有显著的溶藻效果。2.利用原子力显微镜研究了不同藻菌作用时期的形态变化。结果表明,溶藻细菌对球形棕囊藻藻细胞有破坏和杀灭作用,可导致藻细胞破裂和胞内物质溶出而死亡;在溶藻后期,藻细胞被完全破坏为游离的碎片,藻菌共同培养液中溶藻细菌密度明显增加,并发现溶藻细菌的周围有丝状的粘质分泌物,这些分泌物可能对藻细胞产生溶蚀作用。3.实验研究表明,盐度、光照和藻密度等因素对细菌的溶藻效果影响较大。G和Y细菌均在30‰盐度时溶藻效果最好,15‰时最差,W细菌在20‰时溶藻效果最好,35‰时最差;在不同光暗比的光照条件下,细菌G、Y和W都具有溶藻能力;比较三株细菌对处于生长初期和对处于指数生长期不同藻密度的棕囊藻细胞的抑制效果,发现G、Y和W对前者溶藻效果较好,其中三株中又以细菌G抑制效果最好。4.对细菌作用前后的藻菌体进行AO染色,利用流式细胞仪进行荧光分析,结果表明,在细菌作用下,藻细胞密度明显下降的同时,伴随着藻色素的明显锐减,且在溶藻后期共同培养液里AO荧光异常增强,对照研究结果显示在体系里出现了大量增殖的溶藻细菌;利用流式细胞仪通过PI染色并结合藻的叶绿素自发荧光分析了溶藻过程藻的生理学特征,发现三株细菌的溶藻作用趋势是先破坏棕囊藻细胞膜,使其膜完整性受损,进而破坏叶绿素,造成藻体的非自然死亡,最后在细菌的持续作用下,藻菌共同培养液中约90%左右藻体细胞的核酸物质受到严重破坏,形成了最终的细胞碎片。5.利用共聚焦激光显微镜结合PI染色和PI/AO双染色,观测到在溶藻细菌作用下藻菌共培养液中棕囊藻细胞有着不同程度的膜破损和膜缺失现象,且共培养液里有大量凋亡小体和核小体的产生,以及广泛的DNA断裂、DNA片断形成和细胞空腔等现象出现。所有这些细胞的非正常凋亡都是在加入溶藻细菌后发生的,而对照组的藻细胞则为良好生长的荧光阴性表征,可见藻细胞的这种非正常凋亡跟溶藻细菌密切有关,推断正常生理生长的棕囊藻细胞是被溶藻细菌诱导凋亡,因此提出了溶藻细菌诱导藻细胞快速凋亡模型。6.通过测定β-葡萄糖苷酶来检测胞外酶活性变化,结果表明其在藻菌作用过程的活性变化和Doucette的水华种群动力学模式非常契合,从而验证并补充了溶藻细菌的动力学作用模式,表明了溶藻细菌是可以在良性循环的模式上进行赤潮治理的,其可以分离自赤潮海域,并作用于海洋水体中赤潮藻藻细胞,最后随赤潮消退而逐渐消失于海洋环境中,是天然的,且是对海域生物安全的海洋本土物种,符合目前国际上对赤潮防治剂“高效、低毒、价廉、易得”的赤潮防治要求。更多还原

【Abstract】 Red tide has frequently occurred in recent years. The blooms have seriously endangered fishery production and economic development of coastlands. Research on the mitigation of harmful algal bloom has been attracting increasing international attention. Under the circumstances that physical and chemical control methods to red tide are not effective, the use of algae-lysis bacteria as a new method of red tide control and potential microorganisms, has attracted extensive attention of national and international researchers, and gradually become an intensive area of scientific research in the field of marine environment. In this paper, three algae-lysis bacteria labeled G, Y and W, were isolated from Pearl Sea where red tide often outbreaks. The bacteria had significantly algae-lysing effect on marine alge Phaeocystis globosa Scherffel. For the purpose of exploring a new method to solve the long time problem of P. globosa outbreaks and providing a scientific basis for the control of harmful alga microorganisms in coastal zones, we studied the lysing-process of the three algae-lysis bacteria, morphological changes of the bacteria and P. globosa before and after, the relation between the effect of algae and environmental factors, the relations cycle of fluorescence changes between algae and bacteria, the molecular biology mechanisms of algae-lysing, finally, the fate of bacteria and micro-ecological effects in phycosphere. The main results were as follows:1. Eleven bacterial strains were isolated from red tide-prone areas of Pearl Sea by traditional culture, from which 3 strains with algae-lysing effects, labled G, Y, and W, were selected and confirmed as Gram-positive bacteria, bacillus, in distinctly different morphology patterns. G bacteria were more divided, Y bacteria often in cluster states, W bacteria tended to link to a number of cells. All three displayed a remarkable algae-lysing effect on P. globosa.2. Morphological studies in the pattern of algae by using atomic force microscopy showed that algae-lysis bacteria can damage and kill P. globosa cells, and cause algal cells to rupture and intracellular materials to dissolve and finally the cells die. In the late period of algae-lysing, the algal cells were seriously damaged into free debris, and a large number of algae-lysis bacteria emerged in the co-culture. Some of these bacteria were apparently surrounded by viscous materials which may be the substances secreted by algae-lysis bacteria, which chemically dissolved algal cells.3. The different effects of algae-lysing varied in different environmental conditions. G and Y bacteria at 30‰salinity were at best algae-lysing, and worst at the 15‰, W bacteria showed the best algae-lysing at salinity 20‰, and worst at 35‰. All three algae-lysis bacteria have good algae-lysing under the conditions of total dark, light cycles and full illumination. The algae-lysing ability to P. globosa in the early growth phase was better than that in the exponential growth phase. Bacteria G had the best inhibition on effects P. globosa.4. In the late stage of algae-lysing by FCM fluorescence analysis with AO staining, it was found that P. globosa cells and algal pigment were also sharply reduced, and the co-culture system contained a large number of the corresponding algae-lysis bacteria. Physiological characteristics of the algae in the algae-lysing cycle of by FCM analysis showed that the algae-lysing trends were in steps. First P. globosa cell membrane integrity was destroyed, then chlorophyll was damaged, resulted in a non-natural death of algae at last step. In the continued action of bacteria, DNA materials of algae cells were also seriously damaged and over 90% P. globosa finally formed cell debris.5. By CLSM with PI staining or PI / AO double staining analysis in the stages of algae-lysing found that after algae-lysing the membrane of P. globosa have varying degrees of damage and deficiency in co-culture system. Also a large number of apoptosis bodies, a wide range of DNA fragmentation, DNA fragment fracture formation, and cell cavity, could be found. All these phenomena in abnormal apoptosis were formed after adding the bacteria while the control group algal cells were in good growth conditions with fluorescence negative characterization. It can be concluded that algal cells with abnormal apoptosis were closely related to the additions of bacteria. Normal physiological growth of P. globosa was induced to apoptosis by algae-lysis bacteria. Therefore the model of P. globosa’s rapid apoptosis induced by algae-lysis bacteria was established.6. Experimental determination by 6-glucosidase activity could detect changes in extracellular enzyme activities. The activity changes of extracellular enzyme in the process of algae-lysisng research corroborated with the Doucette Population Dynamics Model in the water, thus it verified and complemented the dynamic of algae-lysis bacteria. The mode of action of bacteria on red tide is part of a virtuous circle. Algae-lysis bacteria, which are "drawn" from the sea, and "impact" on sea and "lose" in the sea, is natural and safe for marine native species, and also in line with treatment requirements of current international control for red tide prevention of "efficiency, low toxicity, cheap and easy".更多还原