深圳大梅沙赤潮衰退期细菌群落动态及溶藻菌D84-1溶藻特性研究

发布时间：2018-09-05 12:52

【摘要】：本论文利用宏基因组测序的方法,探究了深圳大梅沙海域暴发的血红哈卡藻(Akashiwo sanguinea)赤潮衰退期海水中细菌群落结构变化情况,从中分离筛选出几株高效溶藻菌菌株,并进行溶藻特性检测、溶藻物质分离提取及溶藻菌VBNC状态转换等研究,获得如下研究结果:(1)连续四天采集2014年6月中旬的深圳大梅沙赤潮衰退期海水,并对其叶绿素含量、可培养细菌数和细菌总数变化进行了检测,发现在整个赤潮衰退期叶绿素含量持续下降,但在赤潮衰退的后期,海水中的细菌总数和可培养细菌数有一个突增的过程;通过0.22μm的水系滤膜收集水样中的细菌DNA,利用宏基因组测序,分析此次赤潮衰退过程中的细菌群落结构动态变化,结果表明赤潮衰退期开始阶段细菌群落多样性较高,优势类群为变形菌门(Proteobacteria)中的γ-变形菌纲(Gammaproteobacteria);而赤潮衰退期中间阶段厚壁菌门(Firmicutes)所占比例有明显上升,α变形菌纲和β变形菌纲和也成为优势门类;到了赤潮衰退末期主要优势门类以γ-变形菌纲为主,尤其是其中的弧菌属(Vibrio)和假交替单胞菌属(Pseudoalteromonas);赤潮衰退期海水细菌数目和群落结构的变化情况为进一步了解赤潮生消过程中的藻菌关系提供了基础;(2)从深圳大梅沙海域赤潮水样中分离获得多株溶藻菌,其中具有较强溶藻效果的菌株D84-1经16S r DNA方法鉴定其为弧菌属(Vibrio),这与弧菌属是赤潮衰退期中的优势菌属结果一致,表明有强溶藻效果的弧菌属可能在赤潮快速衰退过程中起重要作用;(3)以有毒海洋卡盾藻(Chattonella marina)为实验藻种,检测了D84-1的溶藻方式,并对其溶藻谱进行了测试,结果表明D84-1为间接溶藻,主要活性成分由细胞分泌到上清液中,对血红哈卡藻、海洋卡盾藻等在内的10种赤潮藻和1种水华蓝藻(铜绿微囊藻Microcystis aeruginosa)具有溶藻能力,具有溶藻广谱性;海洋卡盾藻加入D84-1上清后叶绿素含量降低,丙二醛(Malondialdehyde,MDA)、过氧化物酶(Peroxidase,POD)和超氧化物歧化酶(Superoxide Dismutase,SOD)含量则明显增加,说明D84-1的溶藻作用使藻细胞的膜系统收到破坏,抗氧化系统产生应激反应,活性氧代谢受到影响;在对D84-1胞外分泌物溶藻物质提取的探索过程中,发现乙酸乙酯萃取物的溶藻活性较高,而正丁醇萃取物也有一定的溶藻效果,进一步溶藻物质分离分析仍在进行中;(4)在溶藻菌D84-1的VBNC状态转换研究中,探索了低温、寡营养、陈海水等条件下溶藻菌D84-1进入活的不可培养(VBNC)状态或从这一休眠状态复苏所需的时间。结果表明在低温寡营养共同作用条件下,D84-1完全进入VBNC状态需要3 d,在陈海水诱导实验组中,温度起到重要作用,4℃下D84-1能在6 d内基本诱导进入VBNC状态,而22℃下D84-1在12 d后只有80%被诱导进入VBNC状态;在复苏实验中,赤潮衰退期水样相较于陈海水,能更快速诱导已经进入VBNC状态的D84-1复苏到正常状态,这与赤潮衰退期海水中细菌可培养数增加的结果一致。电镜观察表明两种状态下的细菌形态差异明显,正常状态下D84-1细胞长度在1~2μm,呈杆状,表面光滑,而VBNC状态细胞长度在1μm以下,中央有凹陷,细胞体积明显缩小,形态由杆状变成了椭球形。
[Abstract]:In this paper, macrogenome sequencing was used to study the changes of bacterial community structure in the seawater during the red tide recession of Akashiwo sanguinea in Dameisha waters of Shenzhen. Several algae-lysing bacteria with high efficiency were isolated and screened out. The characteristics of algae-lysing bacteria were tested, the algae-lysing substances were isolated and extracted, and the VBNC status of algae-lysing bacteria was changed. The results are as follows: (1) The chlorophyll content, the number of culturable bacteria and the total number of bacteria in the red tide recession period of Dameisha in Shenzhen in June, 2014 were detected. It was found that the chlorophyll content decreased continuously during the whole red tide recession period, but in the late period of red tide recession, the fine content of the sea water was detected. The total bacterial count and the culturable bacterial count had a sudden increase process; the bacterial DNA in water samples was collected by 0.22 micron filter membrane, and the dynamic changes of bacterial community structure during the red tide recession were analyzed by macrogenome sequencing. The results showed that the diversity of bacterial community was higher at the beginning of the red tide recession, and the dominant group was Proteus (P Gamma Proteobacteria in roteobacteria, Firmicutes in the middle stage of red tide recession, and alpha Proteobacteria and beta Proteobacteria also became the dominant class; at the end of red tide recession, gamma Proteobacteria was the dominant class, especially Vibrio. And Pseudoalteromonas; the changes of bacterial number and community structure during the red tide recession provide a basis for further understanding the relationship between algae and bacteria in the process of red tide growth and disappearance; (2) A number of algae-lysing bacteria were isolated from the red tide water samples of Dameisha sea area in Shenzhen, and the strain D84-1 with strong algae-lysing effect was obtained by 16S R. DNA method identifies Vibrio, which is consistent with the result that Vibrio is the dominant bacteria in the red tide recession, indicating that Vibrio with strong algae-lysing effect may play an important role in the rapid red tide recession; (3) The toxic marine Chattonella Marina was used as the experimental algae species to detect the algae-lysis mode of D84-1 and to dissolve it. The results showed that D84-1 was an indirect algae-dissolving agent. The main active components were secreted from cells to supernatant. 10 species of red tide algae, including Hakazakia haematochrome and Cardanella marina, and one species of cyanobacteria (Microcystis aeruginosa) had algae-dissolving ability and broad-spectrum. Chlorophyll content decreased, malondialdehyde (MDA), peroxidase (POD) and superoxide dismutase (SOD) content increased significantly, indicating that the algae cell membrane system was damaged by the algae lysis of D84-1, the antioxidant system produced stress response, and the metabolism of reactive oxygen species was affected. In the process of extracting algae-lysing substances from algae secretion, it was found that ethyl acetate extract had higher algae-lysing activity, while n-butanol extract also had certain algae-lysing effect, and further separation and analysis of algae-lysing substances were still in progress. (4) In the study of VBNC state transition of algae-lysing bacteria D84-1, algae-lysing bacteria D under the conditions of low temperature, oligotrophic and aged sea The results showed that it took 3 days for D84-1 to fully enter the state of VBNC under the condition of low temperature oligotrophic interaction. Temperature played an important role in the induction experiment group of Chen Hai Hai. D84-1 could basically induce the state of VBNC within 6 days at 4 C and D84-1 could basically enter the state of VBNC at 22 C. In the resuscitation experiment, D84-1, which had already entered the VBNC state, could be induced to recover to the normal state more quickly than that of the old sea water, which was consistent with the increase of bacterial culturable number in the sea water during the red tide recession. Obviously, the length of D84-1 cells in normal state is 1-2 micron, rod-like, and the surface is smooth. In VBNC state, the length of D84-1 cells is less than 1 micron, the center has a depression, the cell volume is obviously reduced, and the morphology changes from rod-like to ellipsoid.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X55;Q93

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