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枯草芽孢杆菌对太平洋亚历山大藻的溶藻以及毒素降解作用研究
程瑞红
Subtype硕士
Thesis Advisor宋秀贤
2023-05-19
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学硕士
Degree Discipline海洋生态学
Keyword亚历山大藻 溶藻细菌 麻痹性贝类毒素 毒素降解 单过硫酸氢钾
Abstract

近年来,以产毒甲藻为原因种的有害藻华在全球近海频繁发生,对全球经济的可持续发展与人类健康构成严重威胁。因此,如何有效治理产毒甲藻及藻毒素污染问题成为当前研究热点之一。本文以典型麻痹性贝类毒素(Paralytic shellfish toxinsPSTs产毒藻太平洋亚历山大藻(Alexandrium pacificum)为研究对象,利用“以菌杀藻”的微生物除藻方法,从商用芽孢杆菌(Bacillus sp.)中筛选出了一株高效溶藻细菌,并进一步研究了溶藻细菌的溶藻特性和溶藻机理。首次阐明了产毒亚历山大藻在微生物溶藻过程中,PSTs含量和毒性的变化特征。在此基础上,利用高效氧化剂单过硫酸氢钾Potassium peroxymonosulfatePMPS对溶藻过程释放出的PSTs进行快速降解,从而降低PSTs对水体环境和海洋生物的危害,达到同时溶藻并且降解藻毒素的综合治理目标。主要研究结果如下:

1)溶藻细菌的筛选:通过比较短小芽孢杆菌(Bacillus pumilus)、侧孢短芽孢杆菌(Bacillus laterosporus)、蜡样芽孢杆菌(Bacillus cereus)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、枯草芽孢杆菌S2Bacillus subtilis S2)与S3Bacillus subtilis S3)等6商用芽孢杆菌对太平洋亚历山大藻的溶藻活性,发现解淀粉芽孢杆菌和侧孢短芽孢杆菌只能胁迫藻细胞产生暂时性孢囊,而短小、蜡样、枯草S2和枯草S3芽孢杆菌的溶藻率分别为52.00%33.33%63.16%73.12%。因此,本文选择溶藻率最高的枯草芽孢杆菌S3作为太平洋亚历山大藻的溶藻细菌进行后续研究。

2)枯草芽孢杆菌S3的溶藻特性研究:利用枯草芽孢杆菌S3对太平洋亚历山大藻进行溶藻处理,发现添加1%v/vS3菌液12 h溶藻率为80.25%,且溶藻效果随着菌液浓度和时间的增加而增强。S3无菌上清液和菌液对藻细胞表现出相同的溶藻作用,而菌体重悬液和LB培养基则无溶藻效果,由此推断S3的溶藻方式为分泌溶藻活性物质。然而,如果以LB培养基为对照组,在12 h也发生溶藻现象。本文从藻际细菌群落结构的变化对其原因进行分析,结果发现,LB培养基中的营养物质刺激了溶藻细菌快速增殖从而触发溶藻,推测该溶藻细菌为Alteromonas sp.。因此,为了排除S3菌液中残留LB培养基对溶藻结果的干扰,后续实验均在12 h内进行。

另外,研究了不同细菌发酵时长和藻细胞密度对S3溶藻效果的影响,结果表明,S3菌液发酵24-72 h组的溶藻效果高于发酵6-12 h组;当藻细胞密度为500-14000 cells/L范围时,S3菌液的溶藻效果在3 h内与藻细胞密度成正比,但在12 h24 h无明显差异。同时,选取16种常见的藻华生物,研究了S3的溶藻范围,结果表明S3对其中7种表现出溶藻效应,包括亚历山大藻属中的太平洋亚历山大藻(A. pacificum)、微小亚历山大藻(A. minutum)和链状亚历山大藻(A. catenella)。三株藻对枯草芽孢杆菌S3的溶藻敏感度从大到小为:太平洋亚历山大藻>微小亚历山大藻>链状亚历山大藻。

3)枯草芽孢杆菌S3的溶藻机理研究:利用S3无菌上清液对溶藻活性物质的性质进行初步研究发现,该溶藻活性物质是一类分子量小于500 Da,具有很好的热稳定性和酸碱稳定性,极性较强的物质,并排除蛋白质、核酸和多糖的可能性。通过显微镜观察藻细胞形态变化和生理生化实验发现,溶藻活性物质会对太平洋亚历山大藻细胞造成氧化损伤,使得抗氧化系统SODCAT活性升高,GSHMDA含量增加,导致细胞膜脂质过氧化后发生溶解,细胞内物质外流,最后裂解成细胞碎片。另外,进一步分析了S3溶藻过程中藻际细菌群落结构的变化,结果显示,S3菌液中带入的外源营养物质能够改变藻际细菌群落结构,优先刺激溶藻细菌快速增殖,这可能间接加速了藻细胞的裂解,并推测该溶藻细菌为Alteromonas sp.,与之前研究结果一致

4氧化剂对溶藻释放的PSTs降解作用研究:利用高效液相色谱法测定并揭示了S3溶藻过程中PSTs组分和含量的变化特征,结果表明,S3的溶藻作用使得胞内PSTs随着藻细胞的裂解而释放到水体中,胞内PSTs大幅度降低,而胞外PSTs12 h增加了77.48%,并在48 h内维持较高水平。溶藻过程中总PSTs组分在微生物参与下发生了一定程度的降解和转化,导致PSTs总含量和总毒性下降,但该过程发生缓慢且在48 h仍有约50% PSTs残留在体系中。因此,本研究在实现枯草芽孢杆菌S3溶藻释放藻毒素的基础上,利用氧化剂单过硫酸氢钾(Potassium peroxymonosulfatePMPS对释放出的PSTs进行快速降解。结果显示,PSTs降解率随着S3菌液浓度和PMPS添加量的增加而增加,当1%v/vS3菌液溶藻24 h后添加20 mg/L50 mg/L PMPS时,PSTs3 h内降解率分别为26.55%42.54%并且在降解过程中不会引起PSTs组分转化和毒性增加。另外,在研究S3菌液和PMPS添加方式对PSTs降解效果的影响时发现,分开添加枯草芽孢杆菌S3菌液PMPS、或同时添加二者,两种添加方式PSTs的最终降解效果相同,降解率约为75%,但同时添加可以在短时间内加大对藻细胞的损伤,加快藻细胞裂解释放毒素,从而提高对PSTs的降解效果。

综上所述,本研究首次联用溶藻微生物与氧化剂治理典型麻痹性贝类毒素产毒藻太平洋亚历山大藻。揭示了枯草芽孢杆菌S3的溶藻特性和作用机理,探明了单过硫酸氢钾对溶藻过程中释放出的PSTs的降解作用等,实现了对产毒甲藻进行同步溶藻和降解藻毒素的综合治理目标,相关研究结果可为产毒甲藻及其毒素的综合治理提供科学依据和理论基础。

Other Abstract

In recent years, the frequent occurrence of harmful algal blooms caused by toxic methanogens has caused great losses to the global economy and seriously endangered human lives. Therefore, how to effectively manage the problem of toxic algal blooms and algal toxin pollution has become one of the current research hotspots. In this paper, Alexandrium pacificum, a typical dinoflagellate producing paralytic shellfish toxins (PSTs), was studied. We screened an effective algicidal bacterial from commercial Bacillus sp. by using the microbial method of "killing algae with bacteria", and investigated its algicidal characteristics and mechanism. In addition, the changes of PSTs content and toxicity in the algicidal process of microbial were also elucidated for the first time. On the basis, the efficient oxidant potassium peroxymonosulfate (PMPS) was used to rapidly degrade the PSTs released during the algicidal process, thus reducing the harm of PSTs to the aquatic environment and marine organisms, and achieving the comprehensive treatment purpose of lysing algae and degrading toxins at the same time. The main research contents and results are as follows:

(1) Screening of algicidal bacteria. By comparing the algicidal activity of six commercial Bacillus strains, including Bacillus pumilus, Bacillus laterosporus, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus subtilis S2, and Bacillus subtilis S3, on A. pacificum, we found that B. amyloliquefaciens and B.laterosporus could only coerce the algal cells to produce temporary cysts, while the algicidal rates of B. pumilus, B.cereus, B. subtilis S2 and B. Subtilis S3 were 52.00%, 33.33%, 63.16%, and 73.12%, respectively. Therefore, B. Subtilis S3 was selected as the algicidal bacteria of A. pacificum for follow-up studies in this paper.

(2) Studies on the algicidal characteristics of B. subtilis S3. The algicidal rate of 1% (v/v) S3 cultures of A. pacificum after 12 h treatment with B. subtilis S3 was 80.25%, and the algicidal effect was enhanced with the increase of treatment concentration and time. S3 sterile supernatant and cultures showed the same algicidal effect on algal cells, while bacterial supernatant and LB medium showed no algicidal effect, which inferred that the algicidal mode of S3 was the secretion of algicidal active substances. However, if LB medium was used as the control group, algicidal effect also occurred at 12 h. In order to study the reasons, we analyzed the changes in the structure of bacterial community in phycosphere of A. pacificum, it was found that the nutrients in LB medium stimulated the rapid proliferation of the algicidal bacteria, which was presumed to be Alteromonas sp. Therefore, the subsequent experiments were conducted within 12 h to exclude the interference of the residual LB medium in S3 cultures on the results of algicidal effect.

 

 

In addition, the effects of different bacterial fermentation time and algal cell densities on the algicidal effect of S3 were investigated, and it was found that the algicidal effect of S3 cultures was higher in the 24-72 h fermentation group than in the 6-12 h fermentation group; and when the algal cell density was in the range of 500-14000 cells/L, the algicidal effect of S3 cultures was proportional to the algal cell density in 3 h, but there was no significant difference in 12 h and 24 h. Meanwhile, the algicidal range of S3 was studied using 16 common algal blooms, and it was found that S3 showed algicidal effect on seven of them, including A. pacificum, A. minutum and A. catenella in the genus Alexandrium. The sensitivity of those three algae to B. subtilis S3 in descending order were: A. pacificum>A. minutum> A. catenella.

(3) Studies on the algicidal mechanism of B. subtilis S3. Preliminary studies on the properties of the algicidal active substances using the sterile supernatant of S3, it was found that the algicidal active substances were a class of non-protein, non-nucleic acid and non-polysaccharide substances with molecular weight less than 500 Da, good thermal and acid-base stability and strong polarity. Microscopic observation of morphological changes of algal cells as well as physiological and biochemical experiments revealed that the algicidal active substances would cause oxidative damage to A. pacificum cells, resulting in increased activity of antioxidant systems SOD and CAT, increased GSH and MDA content. It resulted in cell membrane lipid peroxidation followed by lysis, efflux of intracellular material, and finally lysis into cell fragments. Furthermore, after analyzing of the changes of bacterial community structure in phycosphere of A. pacificum during the algicidal process of S3, we found that the exogenous nutrients brought in the S3 cultures could change the intercalary bacterial community structure. S3 cultures could preferentially stimulate the rapid proliferation of algicidal bacteria, which might indirectly accelerate the lysis of algal cells, and the algicidal bacteria were presumed to be Alteromonas sp., which was consistent with the results of previous studies.

(4) Studies on the degradation of PSTs released from lysed algae by oxidants. The changes in PSTs content during algicidal process of S3 were detected by high performance liquid chromatography (HPLC). It was found that the algicidal effect of S3 caused the release of intracellular PSTs into the water column with the lysis of algal cells, resulting in a significant decrease of intracellular PSTs. While the extracellular PSTs increased by 77.48% at 12 h and maintained at a higher level at 48 h. During the algicidal process, the total content and total toxicity of PSTs decreased due to the involvement of microorganisms in the degradation and transformation of PSTs components. However, this process occurred slowly and approximately 50% of the PSTs remained in the water column after 48 h. Therefore, based on the realization that B. subtilis S3 lysed algal cells and released many toxins, the efficient oxidant potassium peroxymonosulfate (PMPS) was used to degrade PSTs rapidly. The results showed that the degradation rate of PSTs increased with the increase of S3 cultures concentration and PMPS addition. When 20 mg/L and 50 mg/L PMPS were added after 24 h of algicidal effect in 1%v/vS3 cultures, the degradation rates of PSTs in 3 h were 26.55% and 42.54% , respectively. And there was no toxic transformation of PSTs components occurred during the degradation process. In addition, we found that the degradation of PSTs was the same when S3 and PMPS were added separately and simultaneously, and the degradation rate was approximately 75%, but the simultaneous addition could increase the damage to algal cells and accelerate their lysis in a short time, thus improving the degradation of PSTs.

In conclusion, this study for the first time combined algicidal microorganisms with an oxidant to control the typical paralytic shellfish toxin-producing algae A. pacificum. It was revealed that the algicidal characteristics and mechanism of B. Subtilis S3 and the degradation characteristics of PSTs released by PMPS during algicidal process, the comprehensive treatment purpose of simultaneous algal lysis and degradation of algal toxins was achieved. These results could provide a scientific basis and theoretical basis for the comprehensive treatment of toxin-producing dinoflagellate bloom and algae toxin.

Subject Area地球科学
MOST Discipline Catalogue理学
Pages108
Language中文
Table of Contents

1 绪论     1

1.1 亚历山大藻概述      1

1.1.1 亚历山大藻藻华原因种及其分类   1

1.1.2 亚历山大藻的生活史特征及孢囊   1

1.1.3 亚历山大藻的分布与危害 3

1.2 麻痹性贝类毒素概述     5

1.2.1 麻痹性贝类毒素的来源和组成       5

1.2.2 麻痹性贝类毒素的降解和转化       7

1.3 溶藻细菌的研究进展     8

1.3.1 溶藻细菌的分类   9

1.3.2 溶藻细菌的作用方式  9

1.3.3 溶藻细菌的溶藻机理  10

1.4 研究目的和内容      11

1.4.1 研究目的 11

1.4.2 研究内容 11

1.4.3 研究技术路线       12

2 溶藻细菌的筛选及其溶藻特性研究  13

2.1 引言     13

2.2 材料和方法       13

2.2.1 实验生物的培养   13

2.2.2 芽孢杆菌的纯化与鉴定     14

2.2.3 溶藻细菌的筛选   14

2.2.4 枯草芽孢杆菌S3的生长曲线测定 15

2.2.5 枯草芽孢杆菌的溶藻效果和作用方式  15

2.2.6 培养基溶藻效果和细菌群落测定   16

2.2.7 不同枯草芽孢杆菌发酵时长的溶藻效果     16

2.2.8 不同藻细胞密度的溶藻效果    16

2.2.9 枯草芽孢杆菌的溶藻范围检测       16

2.2.10 数据分析      16

2.3 结果与讨论       17

2.3.1 溶藻细菌的筛选   17

2.3.2 枯草芽孢杆菌S3的生长曲线  18

2.3.3 枯草芽孢杆菌S3的溶藻作用  19

2.3.4 藻菌条件对溶藻效果的影响    27

2.3.5 枯草芽孢杆菌S3的溶藻范围  29

2.4 小结     31

3 枯草芽孢杆菌S3的溶藻机理研究    33

3.1 前言     33

3.2 材料和方法       33

3.2.1 溶藻活性物质的性质  33

3.2.2 藻细胞形态的观察      34

3.2.3 生理生化指标测定      34

3.2.4 细菌群落测定       34

3.2.5 数据分析 35

3.3 结果与讨论       35

3.3.1 溶藻活性物质的性质初探 35

3.3.2 S3作用下藻细胞形态结构的变化   38

3.3.3 S3作用下藻细胞生理生化响应       39

3.3.4 S3作用下藻际细菌群落结构的变化      42

3.4 小结     45

4 枯草芽孢杆菌S3联合氧化剂对麻痹性贝类毒素降解作用研究     47

4.1 前言     47

4.2 材料和方法       47

4.2.1 麻痹性贝类毒素的检测方法    47

4.2.2 细菌群落测定       48

4.2.3 溶藻过程中PSTs的释放与降解     49

4.2.4 不同 PMPS添加量对PSTs的降解效果      49

4.2.5 不同 PMPS添加方式对PSTs的降解效果  49

4.2.6 PMPS含量测定     49

4.2.7 数据分析 49

4.3 结果与讨论       50

4.3.1 溶藻过程中PSTs的释放与降解     50

4.3.2 PMPS添加量对PSTs降解的影响   55

4.3.3 PMPS添加方式对PSTs降解的影响      58

4.4 小结     61

5 结论和展望       63

5.1 结论     63

5.2 创新点 64

5.3 不足与展望       64

参考文献    65

附录     75

  85

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   87

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/181247
Collection海洋生态与环境科学重点实验室
Recommended Citation
GB/T 7714
程瑞红. 枯草芽孢杆菌对太平洋亚历山大藻的溶藻以及毒素降解作用研究[D]. 中国科学院海洋研究所. 中国科学院大学,2023.
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