【作者】 周文礼；

【导师】 唐学玺；

【作者基本信息】 中国海洋大学 ， 生态学， 2008， 博士

【摘要】 海水养殖过程中,由于过分注重生态系生物结构中的部分因子(养殖对象),削弱甚至排除了调节生态平衡的其它因子,简化了生态系统结构,切断了物质循环路线,造成生态平衡破坏,环境恶化.给经济和社会发展带来巨大损失。如何改善整个养殖生态环境,优化养殖模式,促进水产养殖业的稳定健康发展,已成为世界各国水产养殖业共同关注的焦点问题。因臭氧衰减而导致的UV-B(280nm-320nm)辐射增强是目前颇受关注的全球性的重大环境问题之一。研究表明,UV-B辐射增强对浮游植物和浮游细菌的生长有明显的调控作用。本文以我国常见饵料微藻-小球藻(Chlorella vulgaris)为实验对象,采用实验生态学的方法探讨了微藻与共栖异养细菌之间相互作用以及对UV-B辐射增强的响应。以期为阐明UV-B辐射增强对水生生态系统的影响机制提供科学依据,同时为海水养殖的生态修复技术提供理论支持。研究结果如下:1利用生态毒理学方法研究了小球藻和共栖异养细菌对抗生素的敏感性差异及建立小球藻无菌体系的方法。结果表明:建立小球藻无菌系的适宜抗生素为青霉素、庆大霉素、卡那霉素、新霉素。通过涂平板挑取单克隆小球藻个体去除霉菌后,将小球藻培养液进行稀释过滤处理,依次加入法加入100IU的青霉素、庆大霉素、卡那霉素,分别处理两次,获得了无菌的小球藻藻株。2小球藻除菌后,生长规律发生明显变化。除菌藻的阻滞期、对数期和稳定期都明显延长,最大细胞密度增加。UV-B辐射胁迫改变了自然带菌藻和除菌藻的生长规律。0.2 J/m~2-0.8J/m~2的UV-B辐射促进自然带菌藻和除菌小球藻的生长,相同剂量对自然带菌藻的促进作用更大;大于1.6J/m~2的UV-B辐射剂量,抑制二者的生长,相同剂量对除菌藻的抑制作用较大。3利用微生物学方法研究了小球藻不同生长时期共栖异养细菌的菌属组成和数量特征。结果如下:小球藻对异养细菌有明显的菌属选择性。小球藻的共栖异养细菌群落由芽孢杆菌属、气单胞菌属、假单胞菌属、黄杆菌属和产碱杆菌属组成。革兰氏阴性菌占据绝对优势,约为91.9%;小球藻的阻滞期、稳定期和衰退期的优势菌属为假单胞菌属,芽孢杆菌属为小球藻对数期优势菌;总菌数由大到小的顺序为:衰退期＞稳定期＞阻滞期＞对数期。4研究了小球藻不同生长时期共栖异养细菌的群落特征对UV-B辐射增强的响应变化。所有处理组3.2J/m~2和小球藻对数期1.6J/m~2的UV-B处理可以完全灭杀小球藻培养液中的气单胞菌;随UV-B辐射增强,小球藻阻滞期优势菌属由假单胞菌属逐渐被芽孢杆菌属代替。稳定期和衰退期则为假单胞菌属-黄杆菌属-芽孢杆菌属;随UV-B辐射增强,各生长期总菌数逐渐下降,以对数期下降最为明显。5运用生态毒理学方法和统计学方法研究了小球藻与共栖异养细菌之间的相互作用以及对UV-B辐射增强的响应变化。结果表明:正相互作用和负相互作用同时存在于共培养体系中。小球藻与共栖异养总菌数的相对增长率的变化总是对立的;随小球藻起始密度的增加,对细菌的抑制作用逐渐增强;低密度的细菌对小球藻的生长有促进作用,大于10×10~6cell/ml时,抑制小球藻的生长。小球藻与不同的共栖异养单株菌的相互作用机制不同,小球藻通过干扰性竞争抑制H1菌株的生长;对H2菌株的作用为资源性竞争;H1和H2菌株对小球藻的作用则分别为资源性竞争和干扰性竞争。6 UV-B辐射改变了小球藻与共栖异养细菌间的相互作用。低于0.8J/m~2的UV-B辐射剂量处理有利于小球藻的生长而对异养细菌有抑制作用,且随UV-B辐射剂量的增加,这种促进和抑制效应愈加明显。UV-B辐射处理影响小球藻与共栖异养单株菌的作用机制。小球藻滤液经UV-B辐射处理后,对H1菌株的抑制作用减轻,对H2菌株生长没有影响;H1,H2菌株滤液经UV-B辐射处理后,培养的小球藻生长规律没有发生明显改变。7生态毒理学方法和交叉培养法研究了菌株H2的密度效应响应机制以及影响密度效应的因素。结果显示:H2菌株通过分泌化学物质来实现自我行为调节和对小球藻的抑制效应,细菌的密度和分泌到培养液中的化学物质的浓度是能否启动调节机制的必要条件。8小球藻的初始接种密度、H1菌株的胁迫以及UV-B辐射增强均影响H2菌株的密度效应。3者影响H2菌株密度效应的机制不同。小球藻主要是通过资源竞争抑制H2菌株的增加;H1菌株主要是通过利用H2分泌到培养液中的化学信号分子的浓度降低H2菌株密度效应;UV-B辐射主要是抑制H2菌株的细胞分裂速度控制H2菌株的生长。更多还原

【Abstract】 In current mariculture,some cultured species were strengthened to excess,and other components were weakened and eliminated.A simple ecological structure and blocked material cycle result in a ruinate environment.Environmental improvement and optimization of cultural mode are very important to the healthy and stable development of aquaculture and has been one of the most serious problems aroused general concern in the world nowadays..Enhanced UV-B(280nm-320nm) radiation resulting from ozone depletion is one of global environmental problems.Not only marine phytoplankton,But also marine heterotrophic bacterioplankton can be affected by enhanced UV-B radiation.A common diet microalgae-Chlorella vulgaris was selected to serve as experimental materials and the relationship between C.vulgaris and associated heterotrophic bacteria was estimated under controlled laboratory conditions and change of relationship when stressed by enhanced UV-B radiation.The results could supply experimental base to answer the effect of enhanced UV-B radiation on aquatic ecosystem,while provide theoretical evidences to ecological restoration technology in mariculture.Results showed:1.The ecotoxicological method was used to determine the different effect of antibiotics on Chlorella vulgaris and associated heterotrophic bacteria,worked out a practicable method for establishing axenic Chlorella vulgaris.The results showed that: Penicillin,Gentamycin,Kanamycin and Neomycin were feasible antibiotics for removing bacteria in Chlorella vulgaris medium;after removing mould by taking monoclonal Chlorella vulgaris on the plate,Penicillin,Gentamycin,Kanamycin were added to cultural system of diluted and filtered Chlorella vulgaris in turn,which the concentrations were 100IU respectively.Axenic Chlorella vulgaris was obtained after twice treatment.2.The results about effect of enhanced UV-B radiation on Chlorella vulgaris pre and post removing bacteria indicated that there was not significantly difference between axenic and non-axenic Chlorella vulgaris by comparing their population growth.But the growth rhythm was varied obviously;the lag phase,log phase and stationary phase were prolonged significantly;and maximal cell density increased. When the dose of UV-B radiation was from 0.2 J/m2 to 0.8J/m2,axenic and non-axenic Chlorella vulgaris were promoted and the non-axenic Chlorella vulgaris had a better growth compared with the growth of axenic Chlorella vulgaris under the same dose of UV-B radiation.While two experimental groups were inhibited when the doses of UV-B radiation exceed 1.6J/m2,and axenic Chlorella vulgaris was presented more obvious inhibitory effect under the same dose of UV-B radiation.3 The microbiological and ecotoxicological method was used to study associated heterotrophic bacterial composition and quantitative character in different growth phase of Chlorella vulgaris.The results are as follows:An obvious selectivity of Chlorella vulgaris presented an obvious selectivity to heterotrophic bacteria, community composition including Bacillus,Pseudomonas,Flavobaterium and Alcaligense.Gram negative bacteria occupied absolute advantage position;the value was about 91%;Pseudomonas was the dominant bacteria in the lag phase,stationary phase and decreased phase Chlorella vulgaris and Bacillus was dominant bacteria in log phase of Chlorella vulgaris;the order of total amount of bacteria from high to low was decreased phase＞stationary phase＞lag phase＞log phase.4 Response of associated heterotrophic bacterial composition and quantitative character in different growth phase of Chlorella vulgaris to enhanced UV-B radiation was determined in lab.No change of bacteria composition under the dose of UV-B radiation from 0.2 J/m2 to 1.6J/m2;while Aeromonas was killed completely when the dose of UV-B radiation reached 3.2 J/m2.Pseudomonas was the dominant bacteria when UV-B radiation from 0.2 J/m2 to 0.8 J/m2;while Bacillus was the dominant bacteria if the dose of UV-B radiation was more than 1.6 J/m2.The total amount of bacteria was decreasing gradually with enhanced UV-B radiation.5 The ecotoxicological and statistics method was selected to examine interaction between Chlorella vulgaris and associated heterotrophic bacteria.Results indicated that the change of relative growth rates of Chlorella vulgaris and associated heterotrophic bacteria were always opposite;there were an enhanced inhibitory effect to associated heterotrophic bacteria with cell density of Chlorella vulgaris increasing; associated heterotrophic bacteria could promote the growth of Chlorella vulgaris when its density was low,while an inhibitive effect was presented to Chlorella vulgaris when cell density of associated heterotrophic bacteria exceed 10×106cell/ml. Different action mechanisms were exhibited between Chlorella vulgaris and associated heterotrophic dominant single bacteria,Chlorella vulgaris exhibited inhibitive effect to bacteria-H1(Bacillus)through interference competition and exploitation competition to bacteria-H2(Pseudomonas);while bacteria-H1 and H2 presented exploitation competition and interference competition to Chlorella vulgaris respectively.6 Interactions between Chlorella vulgaris and associated heterotrophic bacteria were changed by enhanced UV-B radiation.The lag phase of Chlorella vulgaris in all experimental groups were shortened,log phase and stationary phase were prolonged,and maximal cell density was promoted after UV-B irradiation,which the dose of UV-B radiation was under 0.8J/m2;the optimal enhancement action was presented in 0.4J/m2experimentaI group;the growth of Chlorella vulgaris were inhibited when the doses of UV-B radiation exceed 1.6J/m2.The growth of associated heterotrophic bacteria was not changed obviously when the dose of UV-B radiation was lower than 0.4 J/m2;UV-B radiation could produce inhibition effect to heterotrophic bacteria in co-cultural system when dose of UV-B radiation was higher than 0.8J/m2.Combined action of UV-B and Chlorella vulgaris enhanced inhibitory effect to associated heterotrophic bacteria,and inhibitive effect were weakened stressed by UV-B and associated heterotrophic bacteria.Inhibitive effects of Chlorella vulgaris Filtrate after treatment by UV-B radiation on bacteria-H1were lowered,and had not significant change to bacteria-H2;the growth of Chlorella vulgaris was not changed when cultured with filtrate of bacteria-H1 and H2,which stressed by UV-B radiation.7 An associated heterotrophic dominant single bacteria-H2 was selected to study its mechanism of quorum sensing and factors that affected quorum sensing by using ecotoxicological method in co-cultural system with Chlorella vulgaris.The results showed that self-regulation and inhibitive effect to Chlorella vulgaris of bacteria-H2 were conducted through excreting chemic substance.Density of bacteria-H2 and concentration of chemic substance excreted by bacteria-H2 were necessary conditions, which decided whether the regulation mechanism could be carried out or not.Initial density of Chlorella vulgaris,bacteria-Hlstress and enhanced UV-B radiation could affect quorum sensing of bacteria-H2,but there were different action mechanism among them.Chlorella vulgaris inhibited bacteria-H2 through exploitation competition;bacteria-H1 can use the chemic substance excreted to medium by bacteria-H2,by which the H1 regulated quorum sensing;UV-B radiation also affected the growth of bacteria-H2 by control the rate of cell division of bacteria-H2.更多还原