海洋生态与环境科学重点实验室知识库

绿潮是指海洋大型绿藻暴发性增殖并聚集形成的有害藻华现象。自2007年以来，以浒苔（Ulva prolifera）为主要原因种的绿潮在我国黄海海域连年暴发，对海洋生态环境与沿海各城市的社会经济发展构成严重的威胁。在源头区实行早期防控是目前绿潮防治的重要手段。本文针对浒苔生活史早期阶段的微观繁殖体（孢子、配子）的生物学特征，考察了多种不同类型的改性粘土对去除及抑制微观繁殖体萌发的影响，筛选出了可高效去除并有效抑制微观繁殖体萌发的改性粘土配方，从絮凝作用和生理生化等角度研究了高效改性粘土对微观繁殖体的作用机制，并模拟苏北浅滩现场环境条件考察了光照强度、温度和浊度等环境因子对高效改性粘土去除与抑制微观繁殖体萌发的影响。主要研究结果如下：

（1）高效改性粘土配方的筛选及其作用效果：通过比较聚合氯化铝改性粘土（MCI）、硫酸铝改性粘土（MCII）、十六烷基三甲基溴化铵改性粘土（MCIII）和过硫酸氢钾改性粘土（MCIV）等不同类型的改性粘土去除与抑制微观繁殖体萌发的效果，发现MCIII对微观繁殖体具有最好的去除与抑制效果。0.05g/LMCIII对孢子、配子的去除率分别为69%、99%，其作用后残留孢子和配子的相对萌发率均低于3%。因此，选择作用效果最好的MCIII作为去除微观繁殖体的改性粘土配方进行后续研究。

此外，分别考察了MCIII用量及作用时间、微观繁殖体类别和微观繁殖体密度对MCIII去除与抑制微观繁殖体萌发的影响，发现MCIII对微观繁殖体的去除与抑制效果随MCIII浓度和作用时间的增加而增强；相同浓度下MCIII对配子的去除与抑制效果均优于孢子；当微观繁殖体密度为5×10⁴-1×10⁶ cells/mL时，MCIII对各密度下的微观繁殖体均有明显的去除与抑制效果，微观繁殖体密度过高（1×10⁶ cells/mL）时，MCIII对其作用效果相对减弱，但MCIII作用后残留微观繁殖体的相对萌发率仍低至3%。

（2）MCIII对微观繁殖体的高效去除与抑制机理：通过光学显微镜观察MCIII处理后微观繁殖体的形态结构变化，发现微观繁殖体的细胞膜结构遭到破坏，微观繁殖体发生明显的细胞溶胀、细胞内物质外流等现象，最终裂解为细胞碎片。采用台盼蓝细胞染色法进一步测定了MCIII作用下微观繁殖体细胞膜的损伤情况，发现细胞膜损伤率与去除率间存在正相关关系。使用Zeta电位仪测定了MCIII颗粒的表面电性，发现去除率与MCIII表面Zeta电位间呈正相关关系，说明MCIII一方面通过破坏细胞膜结构灭杀或降低微观繁殖体活性，另一方面通过电中和作用将其絮凝沉降，实现对微观繁殖体的高效去除。

此外，分析残留微观繁殖体的生理生化参数，发现MCⅢ可对残留微观繁殖体产生氧化胁迫，使其活性氧(ROS)、总抗氧化能力（T-AOC）和丙二醛（MDA）含量升高，抑制残留微观繁殖体的生理活性。通过考察MCIII作用后残留微观繁殖体固着率与萌发率的关系，并观察萌发幼苗的形态变化，发现MCIII可使已固着微观繁殖体的增殖速度减缓，降低残留微观繁殖体的萌发率，并抑制已萌发幼苗正常的生长发育。

（3）环境条件对微观繁殖体去除与萌发的影响：模拟苏北浅滩现场环境条件考察了光照强度、温度和浊度条件对微观繁殖体去除效果的影响，发现不同光照强度、温度和浊度条件下，MCIII对微观繁殖体均有明显的去除效果，去除率在不同环境条件下有不同的变化趋势。光照强度为18-54μmol·m^-2·s^-1时，去除率无明显变化，54-144μmol·m^-2·s^-1时，去除率呈下降趋势；温度为10-30℃时，去除率无明显变化；浊度为40-140NTU时，去除率呈升高趋势。进一步考察光照强度、温度以及浊度条件对残留微观繁殖体萌发的影响，发现MCIII作用后，微观繁殖体的萌发数量均显著低于空白组，且萌发幼苗的数量随光照强度、温度和浊度的变化表现出一定的规律性。光照强度为18-54μmol·m^-2·s^-1时，萌发数量随光照强度升高而增加，高于54μmol·m^-2·s^-1后萌发数量减少。温度为10-20℃时，萌发数量随温度升高而增加，高于20℃后萌发数量减少。浊度为0-140NTU时，萌发数量随浊度升高而增加。

综上所述，本研究使用改性粘土技术治理浒苔生活史早期阶段的微观繁殖体，筛选出了能够在低浓度下高效去除并抑制微观繁殖体萌发的改性粘土配方，研究了该配方的作用效果和作用机理，并考察了不同环境条件对高效改性粘土去除与抑制微观繁殖体萌发的影响，相关研究结果为浒苔绿潮的早期防控提供了潜在的防治方法，并为改性粘土技术应用于浒苔早期防控的现场提供了科学依据与理论参考。

Green tide refers to the harmful algal bloom phenomenon formed by the explosive proliferation and aggregation of marine macroalgae. Scince 2007, the Green tide dominated by Ulva prolifera has occured in the Yellow sea every year, which has posed a serious threat to the marine ecological environment and the economic and social development of coastal cities. Early prevention and control in the source area are important strategies for green tide control. According to the biological characteristics of micropropagules (spores and gametes) in the early stage of the life history of Ulva prolifera, the effects of multiple types of modified clay on the removal and inhibition germination of micropropagules were investigated, and the modified clay formula that can effectively remove and inhibit the germination of residual micropropagules was selected. The mechanism of effective removal and inhibition of micropropagules by modified clay was studied from the view of flocculation, physiological and biochemical. In addition, by simulating the environmental conditions of Subei shoal, the effects of light intensity, temperature and turbidity conditions on the removal and inhibition efficiency on micropropagules of modified clay were investigated. The main research results are as follows:

(1) Screening of optimal modified clay and studies on its effects: The removal efficiency and inhibition effect on micropropagules were compared with different types of modified clay, namely, polyaluminum chloride modified clay (MCI), aluminum sulfate modified clay (MCII), cetyltrimethyl ammonium bromide modified clay (MCIII) and potassium persulfate modified clay (MCIV). It was found that MCIII had the best removal efficiency and inhibition effect on micropropagules. The removal efficiency of spores and gametes was 69% and 99% respectively under 0.05g/LMCIII treatment. The relative germination rate of residual spores and gametes was lower than 3% respectively. Therefore, MCIII was selected as the optimal modified clay to remove micropropagules for following research.

In addition, the influence of MCIII dosage, MCIII treatment time, micropropagule type and micropropagule density on MCIII removal and inhibition of micropropagule germination was investigated respectively. It was found that the removal efficiency and inhibition effect of MCIII on micropropagules increased with the increase of its concentration and treatment time. Under the same MCIII concentration, the removal and inhibition effect of MCIII on gametes was better than that of spores. When the density of micropropagules was 5×10⁴-1×10⁶cells/mL, MCIII had obvious removal and inhibition effect on micropropagules, but when the density of micropropagules was as high as 1×10⁶cells/mL, the effect of MCIII on micropropagules was relatively weakened, but the relative germination rate of residual micropropagules was still low to 3% after MCIII.

(2) Studies on the mechanism of MCIII in effective removing and inhibiting of micropropagules: The morphological and structural changes of micropropagules treated with MCIII were observed by optical microscopy. It was found that the cell membrane structure of the micropropagule was destroyed, the micropropagule had obvious swelling, intracellular material outflow, and finally broke into cell fragments. Trypan Blue staining method was used to further determine the damage of micropropagules cell membrane under the treatment of MCIII, and it was found that there was a positive correlation between the membrane damage rate and the removal efficiency. The surface electrical properties of MCIII particles were measured by Zeta potentiometer, and it was found that the removal efficiency was positively correlated with the surface positive charge amount of MCIII particles. These results indicated that MCIII could kill or reduce the activity of micropropagules by destroying the cell membrane structure, and flocculate and settle them by electroneutralization to achieve efficient removal of micropropagules. 

In addition, physiological and biochemical parameters of the residual micropropagules were analyzed, and it was found that MCⅢ could produce oxidative stress on the residual micropropagules, the contents of ROS, T-AOC and MDA were increased, inhibiting the physiological activity of the residual micropropagules. By investigating the relationship between the fixation rate and the germination rate of residual micropropagules, and observing the morphological changes of the germinated seedlings, it was found that MCIII could slow down the proliferation rate of fixed micropropagules, reduce the germination rate of residual micropropagules, and inhibit the normal growth and development of the germinated seedlings.

(3) Studies on the influence of environmental conditions on the removal and germination of micropropagules: The effects of light intensity, temperature and turbidity on the removal of micropropagules were investigated by simulating the environmental conditions of Subei Shoal. It was found that MCIII had obvious removal effect on micropropagules under different conditions of light intensity, temperature and turbidity, and the removal rate showed different trends under different environmental conditions.When the light intensity was 18-54μmol·m^-2·s^-1, the removal rate had no obvious change, but when the light intensity was 54-144μmol·m^-2·s^-1, the removal rate showed a downward trend. When the temperature was 10-30℃, the removal rate has no obvious change. When turbidity was 40-140NTU, the removal rate showed an increasing trend.

The effects of light intensity, temperature and turbidity conditions on the germination of residual micropropagules were further investigated. It was found that the germination number of micropropagules after MCIII was significantly lower than that in the blank group, and the germination number of seedlings showed a certain regularity with the change of light intensity, temperature and turbidity.When the light intensity was 18-54μmol·m^-2·s^-1, the germination number increased with the increase of light intensity, and when the light intensity was higher than 54μmol·m^-2·s^-1, the germination number decreased. When the temperature was 10-20℃, the germination number increased with the increase of temperature, and when the temperature was higher than 20℃, the germination number decreased. When turbidity was 0-140NTU,  the germination number increased with the increase of turbidity.

In summary, this study used modified clay to treat micropropagules which in the early stage of Ulva prolifera life history, screened out a optimal modified clay formula that can effectively remove and inhibit micropropagules germination under low concentration, investigated the mechanism of the optimal modified clay. In addition, the influence of different environmental conditions on the removal and inhibition of micropropagules of the optimal modified clay was investigated. The results provide a potential method for the early control of green tide, and provide a scientific basis and theoretical reference for the application of modified clay in the early control of green tide.

第 1 章 绪论..........................................................................................1

1.1 绿潮概述..........................................................................................1

1.1.1 绿潮的定义....................................................................................1

1.1.2 绿潮的成因...................................................................................1

1.1.3 绿潮的危害...................................................................................2

1.1.4 黄海浒苔绿潮...............................................................................3

1.2 浒苔的生物学与生态学特征...........................................................4

1.2.1 浒苔及其生活史...........................................................................4

1.2.2 浒苔微观繁殖体..........................................................................5

1.2.3 浒苔的生态学特征......................................................................7

1.3 浒苔绿潮的防治研究进展..............................................................7

1.3.1 国内外绿潮防治技术、原理与应用............................................7

1.3.2 黄海浒苔绿潮防治的现状与问题..............................................10

1.4 改性粘土在有害藻华防治中的应用..............................................10

1.4.1 改性粘土控制有害藻华的理论与方法........................................10

1.4.2 改性粘土在有害藻华治理中的应用...........................................11

1.5 研究目标与内容............................................................................12

1.5.1 研究目标.....................................................................................13

1.5.2 研究内容....................................................................................13

1.5.3 技术路线....................................................................................14

第 2 章 高效改性粘土配方的筛选及其对微观繁殖体的作用效果.........................................................................................................15

2.1 前言...............................................................................................15

2.2 材料与方法...................................................................................16

2.2.1 实验材料....................................................................................16

2.2.2 不同类型改性粘土对孢子、配子的去除与抑制萌发实验.......18

2.2.3 不同用量 MCIII 对孢子、配子的去除与抑制萌发实验...........19

2.2.4 MCIII 对不同配比的微观繁殖体的去除与抑制萌发实验.........19

2.2.5 MCIII 对不同密度配子的去除与抑制萌发实验........................19

2.2.6 数据分析....................................................................................19

2.3 结果..............................................................................................20

2.3.1 高效改性粘土配方的筛选.........................................................20

2.3.2 不同用量 MCIII 对孢子、配子的去除和抑制作用..................22

2.3.3 MCIII 对混合微观繁殖体的去除与抑制作用...........................25

2.3.4 微观繁殖体密度对 MCIII 去除与抑制作用的影响..................26

2.4 讨论.............................................................................................27

2.4.1 不同类型改性粘土对微观繁殖体去除与抑制效果的差异.......27

2.4.2 微观繁殖体类别和密度对 MCIII 作用效果的影响.................29

2.5 小结............................................................................................32

第 3 章 MCIII 对微观繁殖体的高效去除与抑制机理研究......................................................................................................33

3.1 前言............................................................................................33

3.2 材料与方法................................................................................33

3.2.1 实验材料.................................................................................33

3.2.2 MCⅢ作用下配子的细胞膜完整性测定.................................34

3.2.3 MCⅢ的 Zeta 电位测定.........................................................34

3.2.4 MCⅢ作用后残留配子的固着率测定.....................................34

3.2.5 MCⅢ作用后配子与萌发幼苗的形态观察.............................35

3.2.6 MCⅢ对残留配子作用的生理生化指标测定.........................35

3.2.7 数据分析................................................................................35

3.3 结果..........................................................................................36

3.3.1 MCⅢ对配子细胞膜损伤的作用............................................36 

3.3.2 MCⅢ表面电位对配子去除率的影响....................................36

3.3.3 MCⅢ对残留配子生理生化特性的影响................................37

3.4 讨论..........................................................................................38

3.4.1 MCⅢ对微观繁殖体的高效去除作用....................................38

3.4.2 MCⅢ对微观繁殖体的抑制萌发作用...................................40

3.5 小结..........................................................................................43

第 4 章 环境条件对微观繁殖体去除和萌发作用的影响....................................................................................................45

4.1 前言..........................................................................................45

4.2 实验材料与方法......................................................................45

4.2.1 实验材料...............................................................................45

4.2.2 光照强度对 MCIII 作用的影响实验....................................46

4.2.3 温度对 MCIII 作用的影响实验............................................46

4.2.4 浊度对 MCIII 作用的影响实验............................................47

4.2.5 数据分析..............................................................................47

4.3 结果.........................................................................................47

4.3.1 光照强度对 MCIII 去除与抑制微观繁殖体效果的影响.......47

4.3.2 温度对 MCIII 去除与抑制微观繁殖体效果的影响..............50

4.3.3 浊度对 MCIII 去除与抑制微观繁殖体效果的影响..............52

4.4 讨论.........................................................................................56

4.4.1 环境因素对 MCIII 去除效果的影响.....................................56

4.4.2 环境因素对 MCIII 抑制萌发的影响....................................57

4.4.3 微观繁殖体的抗逆性与 MCIII 抑制萌发效果分析.............57

4.5 小结........................................................................................60

第 5 章 结论和展望......................................................................61

5.1 结论.........................................................................................61

5.2 不足与展望............................................................................62

参考文献......................................................................................63

致 谢.............................................................................................75

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..................................................................................................77