海洋生态与环境科学重点实验室知识库

  在赤潮治理方法中，微生物方法因具有特异性和生态调控能力强等特点，成为目前人们关注的热点。从赤潮暴发的藻际环境分离具有溶藻作用的藻际细菌是目前微生物防控方法研究中最为广泛、有效的途径。本论文通过在赤潮生物培养体系中添加菌类营养物质，促使体系中细菌的生长，从藻际环境中分离、筛选出4种有效溶藻菌。其中，假交替单胞菌属细菌Pseudoalteromonas shioyasakiensis SE3的溶藻作用最强。针对该株溶藻菌，本文分析了其溶藻特性、研究了其溶藻作用和作用方式。通过电镜等手段，阐释了菌株SE3的作用机制。主要研究结果如下：

  （1）高效溶藻菌的筛选：通过向多种典型赤潮藻实验室培养液中添加LB培养基，从短凯伦藻（Karenia brevis）和红色赤潮藻（Akashiwo sanguinea）藻液中筛选出4株溶藻菌，分别为Pseudoalteromonas shioyasakiensis SE3，Pseudoalteromonas tetraodonis GFC，Alteromonas abrolhosensis PEL67E，Alteromonas marina SW-47。其中菌株SE3对短凯伦藻和红色赤潮藻的溶藻率均＞95%，展现出较高的溶藻能力。在此基础上，本文以K. brevis 165和P. shioyasakiensis SE3为研究对象，进一步考察了菌株SE3在氧充足和氧限制条件下的生长特征。结果表明，氧充足时，其生长曲线符合Logistic（逻辑斯蒂）函数模型的S曲线，稳定期时细菌密度约4.30~4.50×10⁹CFU/mL；氧限制时，稳定期时细菌密度约1.45~1.50×10⁹CFU/mL；均表现出较强的生长能力。

  （2）溶藻菌SE3的溶藻特性：研究了不同温度（20~35℃）和氧气条件（氧充足及30%~90%装液量）对菌株SE3生长和溶藻效果的影响。结果表明，不同氧气条件下，菌株SE3生长量由高到低依次为：氧充足＞30%装液量（N=40.5pg/cell）＞60%装液量（N=12.3pg/cell）＞90%装液量（N=2.9pg/cell）；30℃培养条件下，作用3h时的溶藻效果为：30%装液量＞60%装液量＞90%装液量＞氧充足。氧充足条件下，菌株SE3溶藻率仅在28%~62%之间；装液量30%时，菌株SE3溶藻率最高，可达100%；装液量60%和90%时，菌株SE3的溶藻率均低于装液量30%时；说明一定氧限制条件下生长的细菌，溶藻效果更好。实验条件下，温度对细菌的生长和溶藻效果影响不大，30℃时略具优势。本文还进一步考察了菌株SE3的不同生长阶段和添加量对溶藻效果的影响，结果表明，菌株SE3的溶藻效果不受细菌生长阶段的影响，主要受细菌数量的影响。不同细菌密度处理短凯伦藻后，发现菌株SE3发挥溶藻作用需达到一定的浓度阈值，作用12h、细菌密度高于1.2×10⁷cells/mL时，溶藻效果随密度增加而提高。基于溶藻谱研究发现，菌株SE3具有特异性溶藻特点，对短凯伦藻2229、米氏凯伦藻、红色赤潮藻的溶藻率分别为98%、95%、100%，具有强效溶藻作用；对太平洋亚历山大藻有48%的溶藻率，效果较弱；对甲藻之外的几种藻类无溶藻作用。

  （3）溶藻菌SE3的溶藻作用方式及机制：将菌液分为无菌滤液、菌体重悬液分别进行溶藻实验。结果表明，菌液和以2216E液体培养基为重悬液体的菌体重悬液均能在3h后达到100%的溶藻效果，无菌滤液的作用与对照组2216E液体培养基的作用基本一致，说明溶藻菌SE3主要依靠菌体的直接作用溶藻。在光学显微镜和扫描电镜下观察溶藻过程，短凯伦藻藻细胞先形成类孢囊形态，细菌菌体大量聚集于藻细胞表面，而后藻细胞逐步裂解死亡。透射电镜观察藻细胞亚显微结构变化，结果显示，菌株SE3直接攻击藻细胞，破坏其外部膜结构，进而侵入藻细胞内部，使藻细胞内部结构紊乱、胞质外泄、空泡化，最终导致了藻细胞的死亡。

  本研究的创新点主要体现在分离、获取了对典型赤潮藻具有高效溶藻作用的溶藻菌P. shioyasakiensis SE3，发现了该株溶藻菌特异性溶藻特点和直接溶藻作用机制，为微生物方法防控赤潮技术的进一步发展和完善提供了参考。

      Among the methods to control red tides, microbial methods have become the focus of attention because of their characteristics of specificity and ecological regulation ability. It has been the most extensive and effective way to isolate algicidal bacteria from the phycosphere of red tide outbreak. In this paper, by adding bacteria culture medium to the microalgae culture system of red tides to promote the growth of bacteria in the system, four effective algicidal bacteria were isolated and screened from the phycosphere. Among them, Pseudoalteromonas shioyasakiensis SE3 had the strongest algicidal activity. In this paper, the algicidal characteristics, effects and mode of strain SE3 were analyzed. The algicidal mechanism of strain SE3 was clarified by means of electron microscopy. The main results are as follows:

      (1) Screening of efficient algicidal bacteria: By adding LB medium to the laboratory culture solution of a variety of microalgae which blong to typical red tides, 4 algicidal bacteria were screened from Karenia brevis and Akashiwo sanguinea, they were Pseudoalteromonas shioyasakiensis SE3, Pseudoalteromonas tetraodonis GFC, Alteromonas abrolhosensis PEL67E and Alteromonas marina SW-47. Among them, the algicidal rates of strain SE3 to Karenia brevis and Akashiwo sanguinea were both more than 95%, showing high algicidal ability. On this basis, K. brevis 165 and P. shioyasakiensis SE3 were selected as objects and the growth characteristics of strain SE3 under oxygen conditions of sufficiency and restriction were investigated. The results indicated that the growth curve of strain SE3 was the S-curve of Logistic function model when oxygen was sufficient, and the bacterial density was about 4.30~4.50×10⁹CFU/mL in the stable period. When oxygen was limited, the bacterial density was about 1.45~1.50×10⁹CFU/mL in the stable period. Strain SE3 showed strong growing ability under both conditions.

      (2) Algicidal characteristics of strain SE3: The effects of different temperature (20~35℃) and oxygen (sufficient oxygen and liquid loading content of 30%~90%)  conditions on the growth and algicidal activity of strain SE3 were studied. The results indicated that under different oxygen conditions, the biomass increment of strain SE3 was in the order of sufficient oxygen > liquid loading content of 30% (N=40.5pg/cell) > liquid loading content of 60% (N=12.3pg/cell) > liquid loading content of 90% (N=2.9pg/cell). After cultured under the condition of 30℃, strain SE3 was co-cultured with K. brevis 165 by 3h, then the algicidal activity of strain SE3 was in the order of liquid loading content of 30% > liquid loading content of 60% > liquid loading content of 90% > sufficient oxygen. When oxygen was sufficient, the algicidal rates of strain SE3 were about 28%~62%. When the liquid loading content was 30%, the algicidal rates of strain SE3 were the highest, up to 100%. When the liquid loading content was 60% or 90%, the algicidal rates of strain SE3 were lower than that at the liquid loading content of 30%. The results showed that the algicidal activity of strain SE3 was higher when it was cultured under certain oxygen restriction conditions. Under the experimental conditions, temperature had little effect on the growth of strain SE3 and its algicidal activity, and 30℃ had slight advantages. The effects of different growth stages and additive amount of strain SE3 on its algicidal activity were further examined, the results showed that the algicidal activity was not affected by the growth stages, but mainly by the number of bacteria. After treating K. brevis with different bacterial densities, it was found that the bacterial density of strain SE3 should reach a certain concentration threshold to make bacteria work. When the bacterial density was higher than 1.2×10⁷cells/mL for 12h, the algicidal activity increased with the increasing of bacterial density. Based on the study of algicidal range, it was found that the algicidal effect of strain SE3 was species-specific. The algicidal rates on K. brevis 2229, K. mikimotoi and A. sanguinea were 98%, 95% and 100%, respectively, which were strong. The algicidal rate on Alexandrium pacificum was 48%, which was waker. But strain SE3 has no algicidal effects on several microalgae except dinoflagellate.

      (3) Algicidal mode and mechanism of strain SE3: The bacterial culture was divided into sterile supernatant and cell suspension to carry out algicidal experiments respectively. The results indicated that both the bacterial culture and cells resuspended in 2216E liquid medium achieved 100% algicidal rates after 3h, and the effect of sterile supernatant was basically the same as that of 2216E liquid medium, indicating that strain SE3 attacked K. brevis directly through cells. The process of algicidal effect was observed under optical microscope and scanning electron microscope. Cells of K. brevis turned into cyst-like form, and cells of strain SE3 gathered on the surface of K. brevis, then cells of K. brevis gradually lysed and died. The response of submicroscopic structure of K. brevis was observed through transmission electron microscopy. The results showed that strain SE3 attacked K. brevis directly, destroyed the cytomembrane, and then invaded into the interior of K. brevis, causing the consequences of internal structure disorder, cytoplasmic leakage and void formation. And cells of K. brevis died finally.

      The innovations of this study are as follows: P. shioyasakiensis SE3, an algicidal bacterium with efficient algicidal effects on K. brevis, was isolated and obtained. It was found that the algicidal effect of this bacterium was species-specific. And the mechanism of its direct attack mode was clarified. This study provided a reference for the further development and improvement of microbial methods controlling red tides.

第1章 绪论... 1

1.1 赤潮概述... 1

1.1.1 赤潮的发生现状... 1

1.1.2 赤潮的成因... 2

1.1.3 赤潮的危害... 4

1.1.4 赤潮的治理方法... 5

1.2 藻-菌关系研究进展... 8

1.2.1 藻际环境... 8

1.2.2 藻-菌互作关系... 8

1.2.3 藻-菌关系与赤潮... 10

1.3 溶藻细菌的研究进展... 11

1.3.1 溶藻细菌的分离... 11

1.3.2 溶藻细菌的作用方式... 11

1.3.3 溶藻细菌的作用机制... 15

1.4 本文的研究目标及内容... 16

1.4.1 研究目标... 16

1.4.2 研究内容... 16

1.4.3 研究技术路线... 17

第2章 高效溶藻菌的分离、筛选与鉴定... 19

2.1 前言... 19

2.2 材料和方法... 19

2.2.1 实验藻种与材料仪器... 19

2.2.2 溶藻菌的分离... 20

2.2.3 溶藻菌的筛选... 20

2.2.4 溶藻菌的种属鉴定... 20

2.2.5 菌株SE3的形态观察... 20

2.2.6 菌株SE3的生长曲线测定方法... 20

2.2.7 数据分析... 21

2.3 结果与讨论... 21

2.3.1 溶藻菌的分离纯化... 21

2.3.2 溶藻菌的分子生物学鉴定... 23

2.3.3 溶藻菌的筛选... 25

2.3.4 菌株SE3的形态及系统发育树... 26

2.3.5 菌株SE3的生长曲线... 28

2.4 小结... 29

第3章 溶藻菌SE3的溶藻特性... 31

3.1 前言... 31

3.2 材料和方法... 31

3.2.1 细菌培养与材料仪器... 31

3.2.2 不同温度、氧气条件对SE3生长量及溶藻效果的影响... 31

3.2.3 SE3的不同生长阶段对溶藻效果的影响... 32

3.2.4 不同细菌添加量对溶藻效果的影响... 32

3.2.5 菌株SE3的溶藻谱... 33

3.2.6 数据分析... 33

3.3 结果与讨论... 33

3.3.1 不同温度、氧气条件对SE3生长量及溶藻效果的影响... 33

3.3.2 SE3的不同生长阶段对溶藻效果的影响... 37

3.3.3 不同细菌添加量对溶藻效果的影响... 38

3.3.4 菌株SE3的溶藻谱... 40

3.3.5 小结... 41

第4章 溶藻菌SE3对短凯伦藻的溶藻作用及机制... 43

4.1 前言... 43

4.2 材料和方法... 43

4.2.1 材料与仪器... 43

4.2.2 菌株SE3的溶藻作用方式... 43

4.2.3 光学显微镜与扫描电镜观察溶藻过程... 44

4.2.4 透射电镜观察短凯伦藻亚细胞结构的变化... 44

4.2.5 数据分析... 45

4.3 结果与讨论... 45

4.3.1 菌株SE3的溶藻作用方式... 45

4.3.2 菌株SE3与短凯伦藻的溶藻作用过程... 46

4.3.3 藻细胞亚显微结构对菌株SE3胁迫的响应... 49

4.4 小结... 51

第5章 总结与展望... 53

5.1 结论... 53

5.2 创新点... 54

5.3 不足与展望... 54

参考文献... 55

附录一 主要实验试剂与仪器... 71

附录二 菌株SE3的16S rDNA序列... 72

致 谢... 73

作者简历及攻读学位期间发表的学术论文与其他相关学术成果 75