实验海洋生物学重点实验室知识库

微藻具有光能利用效率高，生长快，高附加值物质含量高等特性，因此微藻资源的开发利用是获得天然蛋白、色素、药物和生物燃料的理想途径。此外，根据微藻利用营养物质固定CO₂的特性，还可以实现工业烟气中的二氧化碳及城市废水中氮磷化合物的减排。大规模持续性养殖的实现是开发利用微藻资源的前提。但是在户外，敌害生物污染是微藻大规模培养过程的限制因素之一。其中以浮游动物污染较为严重且普遍，轻者可抑制微藻生长，延长培养周期，重者会导致微藻绝收。然而，针对微藻大规模培养过程中的敌害生物污染目前尚无有效的防治方法。本论文选取敌害生物典型代表——轮虫和棘尾虫为实验对象，（1）探讨敌害生物除直接摄食微藻细胞外，通过分泌未知物质抑制微藻生长的机制，并对敌害生物分泌物的成分进行初步解析；（2）以植物源杀虫剂川楝素和苦皮藤素为例，探讨其在敌害生物污染治理中的应用效果和经济可行性，以期为微藻规模化培养中敌害生物的早期检测和防治提供理论和技术指导。

1. 研究了不同浓度轮虫分泌物对小球藻生长的影响。结果表明在新鲜培养基中添加无菌轮虫培养滤液（RCF，含轮虫分泌物）显著降低了小球藻的细胞密度，且培养基中RCF的占比（5%，10%，20% 和30%）越高小球藻的细胞密度越低。同时，不同初始细胞密度（1.4×10⁶ cells mL⁻¹、4.2×10⁶ cells mL⁻¹、7.8×10⁶ cells mL⁻¹和18.0×10⁶ cells mL⁻¹）的小球藻对10% RCF的响应也不同。增加小球藻的初始细胞密度会使10% RCF中定量的抑制性化学物质分散到更多的细胞上，从而减弱每个细胞所受的抑制作用，但随着时间增加总体抑制效果不变。说明轮虫释放的化学物质对微藻细胞生长的抑制作用与培养基中RCF浓度和小球藻暴露在其中的时间有关。同时，培养液中10%以上的RCF显著抑制了小球藻的光合作用和呼吸作用，且抑制率与RCF在培养基中的占比呈正相关。说明轮虫分泌物可以通过抑制小球藻光合作用和呼吸作用抑制其生长。同时根据已获得的结果，我们经统计计算可知，每只轮虫每小时除捕食外，释放的化学物质可以抑制45.5±3.2个小球藻细胞的生长。
2. 为了确定轮虫分泌物中抑制物质的化学本质和作用机理，本实验研究了小球藻对轮虫分泌物中不同组分的响应。结果表明煮沸RCF并不能消除其对小球藻生长的抑制作用，说明轮虫分泌物中的抑制物质不是蛋白或类蛋白。RCF中水溶性组分不会对小球藻的生长和净光合放氧速率造成抑制，但RCF中脂溶性组分显著抑制了小球藻的光系统II（PSII）活性，降低了光合电子传递速率和光合放氧速率，导致其生长速率显著下降。根据小球藻细胞的以上生理变化情况，判定RCF中的主要抑制物为脂溶性物质，非蛋白和水溶性物质。同时根据各项指标变化，我们推测，该脂溶性抑制物可能是游离脂肪酸或由不饱和脂肪酸光氧化衍生的物质。
3. 为了进一步解析轮虫脂溶性分泌物对小球藻的抑制机理，本实验对不同浓度（10% 和30%）RCF脂溶性抑制物作用下小球藻的抗氧化系统进行了研究。结果表明，低浓度抑制物（10%）作用下，小球藻超氧阴离子自由基（O₂^-）、羟自由基（·OH）和过氧化氢（H₂O₂）含量均有所升高，细胞受到过氧化伤害，同时，抗氧化系统相关酶，超氧物歧化酶（SOD）、抗坏血酸过氧化物酶（APX）和愈创木酚过氧化物酶（POD）活性均呈上升趋势，羟自由基清除能力（HFRSC）加强，还原型谷胱甘肽（GSH）水平提高，过氧化氢酶（CAT）活性不变，说明小球藻试图通过提高自身抗氧化能力来抵抗细胞内活性氧的积累。而高浓度RCF脂溶性抑制物（30%）极大地抑制了抗氧化系统相关酶APX、POD，CAT活性和HFRSC。活性氧O₂^-和·OH含量均显著上升，说明高浓度RCF脂溶性抑制物使小球藻细胞产生大量活性氧，引起氧化胁迫，细胞内发生脂质过氧化作用，细胞结构被破坏，生长受到抑制。
4. 为了寻找到有效治理微藻规模化培养中轮虫污染的方法，本实验研究了轮虫抑制微拟球藻生长的机制及植物源杀虫剂苦皮藤素（CA）：川楝素（TSN）（1:9）复配剂对轮虫的杀灭效果。结果表明，褶皱臂尾轮虫可直接吞噬微拟球藻细胞，导致藻细胞密度急剧下降。同时，轮虫污染也破坏了剩余微拟球藻PSII反应中心、光合电子受体侧和供体侧，导致光能吸收和利用的失衡，更容易引起氧化胁迫。而CA:TSN（1:9）复配剂可以阻止褶皱臂尾轮虫吞噬微拟球藻细胞，同时保护微拟球藻细胞的光合电子传递链，保证其正常生长。说明植物源杀虫剂复配剂CA:TSN（1:9）是一种很好的控制轮虫污染的选择。
5. 本实验选取微藻培养中另一种常见污染源—棘尾虫为研究对象，对比研究了川楝素和碳酸氢铵对棘尾虫的毒性作用。毒性试验表明，川楝素和碳酸氢铵对棘尾虫均为高毒性，24小时的致死中浓度（LC₅₀）分别为6.4 μg L^-1和0.8 g L^-1。当棘尾虫暴露于 ≥ 2 μg L^-1的川楝素或 ≥ 0.4 g L^-1的碳酸氢铵时，棘尾虫种群密度显著降低。此外，川楝素和碳酸氢铵对小球藻的防治效果及其安全性评价表明，14 μg L^-1的川楝素对小球藻光合作用和生长没有明显的毒害作用，与对照组相比，川楝素不仅有效地降低了棘尾虫密度，还促进了小球藻的生长。而 ≥ 0.8 g L^-1碳酸氢铵会抑制小球藻的生长和光合作用，导致微藻细胞密度下降 ≥ 5.1%。说明川楝素对棘尾虫具有高毒性，但对小球藻安全无毒，是一种很好的控制棘尾虫污染的选择。

综合以上实验结果，我们推测轮虫释放的脂溶性抑制物可能是游离脂肪酸或由不饱和脂肪酸光氧化衍生的物质。该物质通过破坏微藻细胞的光合电子传递链，导致光能吸收和利用的失衡，造成电子泄漏，产生大量活性氧，引起氧化胁迫，对质膜造成严重损伤从而使藻细胞破裂。而植物源杀虫剂川楝素和苦皮藤素在能够有效控制微藻培养过程中的轮虫和棘尾虫污染，且具有经济可行性。深入了解敌害生物对微藻细胞的毒性作用，有助于调整污染防控方案，优化藻类培养的生产力。除此之外，低成本、环境友好、使用方便的治理轮虫和棘尾虫污染的方法，可为微藻规模化培养中敌害生物防治提供理论和技术指导。

Microalgae are potential biomass feedstock for proteins, pigments, pharmaceuticals and biofuels, owing to their high efficiency of solar energy utilization, fast growth rate and ability to accumulate a high quantity of lipid. Furthermore, microalgal cultivation could be coupled with CO₂ capture from industrial flue gases and removal of nitrogen and phosphorous compounds during wastewater treatment. Sustained, large-scale, biomass production is a prerequisite for realizing these potentials of microalgae. Biological contamination is one of the limiting factors in the outdoor large-scale cultivation of microalgae. The high grazing capacity of zooplankton can inhibit the algal concentration, prolonging the culture period or resulting in aquaculture system breakdown in a few days. However, there are no effective control methods for biological contamination in large-scale cultivation of microalgae. In this paper, Brachionus plicatilis and Stylonychia mytilus were selected as the experimental objects, (1) studying the mechanism that rotifer released undefined chemicals to inhibit the microalgal growth besides predation and the components of secretion were analyzed; (2) discussing the application effects and economic feasibility of toosendanin and celangulin in the control of biological contamination, in order to provide theoretical and technical guidance for the detection and control of biological contamination in the large-scale cultivation of microalgae.

The main results are as follows:

1. The effects of different concentrations of rotifer secretion on the growth of Chlorella sp. were studied. The results showed that the bacteria-free, rotifer culture filtrate (RCF) significantly decreased Chlorella sp. cell densities during the incubation, and the microalgal cell densities decreased with increasing RCF proportions (5%, 10%, 20% and 30%) in fresh F/2 media. The responses of Chlorella sp. at different starting cell densities (1.4×10⁶ cells mL⁻¹, 4.2×10⁶ cells mL⁻¹, 7.8×10⁶ cells mL⁻¹ and 18.0×10⁶ cells mL⁻¹) to 10% RCF were different. Increasing the initial cell density of Chlorella sp. would disperse the inhibitory chemical(s) present in 10% RCF over more cells, reducing their effect on each cell, but the overall inhibitory effect was not changed with time. The results confirmed that the action of the chemical(s) released by rotifers on microalgal cell growth was dependent on both the RCF concentration and the exposure time. They also demonstrate that ≥ 10% RCF significantly inhibited photosynthesis and respiration, and inhibition rate was positively correlated with the proportion of RCF in F/2 medium. Calculations based on the data indicated that the rotifer-derived chemical(s) released hourly from each rotifer inhibits growth by 45.5±3.2 microalgal cells in addition to the rotifer predation.
2. With the aim of identifying the chemical nature and action mechanism of the inhibitor released from rotifers, the responses of Chlorella sp. to different components of rotifer secretion was studied. The results showed that the rotifer inhibition of Chlorella sp. growth could not be eliminated by boiling the RCF, indicating that the inhibitor was not likely a protein. The growth and photosynthesis of Chlorella sp. was not inhibited by the water-soluble RCF fraction, but they were significantly inhibited by the lipid-soluble RCF fraction, in a dose-dependent manner. Further, the lipid-soluble fraction decreased energy conservation and photosynthetic electron transport, which induced a severe decrease in PSII activity and a decrease in the net photosynthetic O₂ evolution rate. Based on these physiological responses of Chlorella sp. cells, the lipid-soluble fraction rather than the protein or water-soluble fractions was determined to contain the responsible inhibitor. The inhibitor, we speculated, was probably free fatty acids or substances derived from the photooxidation of unsaturated fatty acids.
3. In order to further analyze the inhibition mechanism of lipid-soluble fraction of RCF, the antioxidant system of Chlorella sp. under 10% and 30% RCF lipid-soluble inhibitors was studied. The results showed that under the lipid-soluble inhibitor of RCF (10%), the contents of superoxide anion (O₂^-), hydroxyl radical (·OH) and hydrogen peroxide (H₂O₂) were increased, which indicated that the Chlorella sp. cells treated by inhibitors were damaged by peroxidation. At the same time, the activities of superoxide dismutase (SOD), aseorbateperoxidase (APX) and peroxidase (POD) in the antioxidant system increased, the hydroxyl free radical removal capacity (HFRSC) and glutathione (GSH) increased, indicating that Chlorella sp. tried to decrease the accumulation of active oxygen by improving antioxidant capacity. However, the lipid-soluble inhibitor of RCF (30%) significantly inhibited the activities of SOD, O₂^-, ·OH and HFRSC. Although the activity of SOD increased, the content of O₂^- and ·OH also increased significantly. The high concentration lipid-soluble inhibitor of RCF made chlorella sp. produced a lot of active oxygen, causing oxidative stress, which leads to lipid peroxidation, cell structure destruction and growth inhibition.
4. In order to identify an effective technique for reducing rotifer contamination, the mechanism of Brachionus plicatilis inhibiting Nannochloropsis oculata and the efficacy of the celangulin : toosendanin (CA:TSN) (1:9) combination for rotifer extermination were investigated using chlorophyll a fluorescence transient. The results showed that B. plicatilis could directly devour N. oculata cells and sharply reduce an algal density to very low levels. B. plicatilis also inhibited the activities of PSII reaction centers, acceptor side and donor side in surviving N. oculata cells, and led to the imbalance between photosynthetic light absorption and energy utilization, even oxidative stress. However, the CA:TSN (1:9) combination could control B. plicatilis, thereby preventing B. plicatilis from devouring N. oculata cells and protecting photosynthetic electron transport chain of the surviving N. oculata cells against rotifers damage. Meanwhile, the CA:TSN (1:9) combination did not affect the growth of N. oculata. Therefore, the botanical pesticide, the binary combination of CA:TSN (1:9), is a good candidate of botanical pesticide for controlling rotifer contamination.
5. Another common biological contamination - Stylonychia mytilus was selected as the experimental object in this experiment, and the toxic effects of toosendanin and ammonium bicarbonate on Chlorella pyrenoidosa were compared. Toxicity tests showed that toosendaninand ammonium bicarbonate were highly toxic to S. mytilus, with 24 h lethal concentration 50% (LC₅₀) values of 6.4 μg L⁻¹, and 0.8 g L⁻¹, respectively. The population density of S. mytilus decreased significantly when exposed to ≥ 2 μg L⁻¹ toosendanin, or ≥ 0.4 g L⁻¹ ammonium bicarbonate. In addition, the S. mytilus control effects of toosendaninand ammonium bicarbonate and their safety in C. pyrenoidosa were evaluated. It was found that ≤ 14 μg L⁻¹ toosendanin had no obvious toxic influence on photosynthesis and growth of C. pyrenoidosa and even increased the final cell density, with the highest being 12.3% over that of untreated cultures, and effectively reduced the S. mytilus density. Ammonium bicarbonate is the most widely used optimization technique for controlling contamination, but it has limited ability to reduce S. mytilus. However, ≥ 0.8 g L⁻¹ ammonium bicarbonate inhibited photosynthesis and growth of C. pyrenoidosa, causing a 5.1% reduction in cell density or even a complete crop failure. Based on its high toxicity to S. mytilus and its relative safety to C. pyrenoidosa, toosendanin was considered to be a good potential botanical pesticide for controlling S. mytilus contamination in microalgal mass cultivation.

Based on the above results, we speculated that the lipid-soluble RCF fraction was probably free fatty acids or substances derived from the photooxidation of unsaturated fatty acids. The fraction destroyed the photosynthetic electron transport chain, causing the imbalance between photosynthetic light absorption and energy utilization, electron leakage of photosynthetic electron transport chain. Under the condition, a lot of active oxygen was produced, causing oxidative stress. Severe damage to the plasma membranes would cause cell structure destruction and growth inhibition. However, toosendanin and celangulin can effectively control B. plicatilis contamination and S. mytilus contamination in the large-scale cultivation of microalgae, with highly economic feasibility. A better understanding of the inhibition of microalgae by rotifers may help adjust contamination control schemes and optimize the productivity of algal cultures. Besides, a low-cost, environmentally friendly, easy-to-apply, pesticide treatment to control B. plicatilis and S. mytiluscontamination would provide a theoretical and technical guidance for the contamination control of harmful organisms in the large-scale cultivation of microalgae.

目 录

第1章 绪论 1

1.1 微藻规模化培养 1

1.1.1 培养模式 1

1.1.2 培养设备 2

1.1.3 生活周期与培养模式调控 5

1.1.4 培养参数优化与调控 6

1.1.5 敌害生物危害与防治 6

1.1.6 种质工程 6

1.2 微藻规模化培养中的生物污染 7

1.2.1 污染来源 7

1.2.2 主要污染物及污染机制 8

1.3 主要敌害生物——轮虫和棘尾虫 13

1.3.1 轮虫 13

1.3.2 棘尾虫 16

1.4 生物污染防控方法 18

1.4.1 污染预防 18

1.4.2过滤法 18

1.4.3化学防治 18

1.4.4改变培养条件 19

1.4.5生物控制 19

1.5 植物源杀虫剂 19

1.5.1 神经毒剂 20

1.5.2 呼吸毒剂 20

1.5.3 消化毒剂 20

1.5.4 生长抑制物质 20

1.6 研究内容及意义 21

第2章 轮虫分泌物对小球藻的化学抑制 23

2.1引言 23

2.2 材料与方法 23

2.2.1 无菌小球藻和轮虫的培养 23

2.2.2 培养基 24

2.2.3 小球藻和轮虫培养滤液的获取 25

2.2.4 小球藻在含不同浓度CCM或RCF的改良F/2培养基中的培养 25

2.2.5 不同初始密度小球藻在含定量RCF培养基中的培养 25

2.2.6 生长测量 25

2.2.7 叶绿素荧光参数测定 26

2.2.8 光合放氧能力与呼吸耗氧能力测定 26

2.2.9 数据分析 26

2.3 实验结果 26

2.3.1 CCM对小球藻细胞生长和光合的影响 26

2.3.2 RCF对小球藻细胞生长，光合和呼吸作用的影响 28

2.3.3 定量RCF对不同初始密度小球藻生长、光合和呼吸作用的影响 32

2.4 讨论 35

2.5 结论 38

第3章 轮虫分泌物中抑制性物质的化学本质及作用机理探究 39

3.1 引言 39

3.2 材料与方法 39

3.2.1 无菌小球藻，轮虫和培养基 39

3.2.2 小球藻和轮虫培养滤液获取 39

3.2.3 RCF组分分离 39

3.2.4 小球藻在含煮沸RCF的F/2培养基中的培养 39

3.2.6 生长测量 40

3.2.7 快速荧光诱导动力学曲线（OJIP）和叶绿素荧光测定 40

3.2.8 光合放氧能力与呼吸耗氧能力测定 41

3.2.9 数据分析 41

3.3 实验结果 41

3.3.1 RCF中蛋白质对小球藻的影响 41

3.3.2 RCF水溶性组分对小球藻的影响 44

3.3.3 RCF脂溶性组分对小球藻的影响 46

3.4 讨论 49

3.5 结论 51

第4章 轮虫释放的脂溶性抑制物对小球藻抗氧化系统的影响 52

4.1 引言 52

4.2 材料与方法 52

4.2.1 脂溶性抑制物的获取及小球藻培养 52

4.2.2 实验试剂及仪器设备 53

4.2.3 抗氧化系统各参数测定 53

4.3 实验结果 53

4.4 讨论 55

4.5 结论 57

第5章 植物源复配药剂对微拟球藻培养中轮虫污染的治理 58

5.1引言 58

5.2 材料与方法 58

5.2.1 微拟球藻和轮虫培养 58

5.2.2 培养基 59

5.2.3 植物源杀虫剂 59

5.2.4 微拟球藻在不同体系中的培养 60

5.2.5 生长测量 60

5.2.6 JIP测定 60

5.2.7 数据分析 60

5.3 实验结果 60

5.3.1 微拟球藻细胞密度与轮虫种群密度 60

5.3.2 快速荧光诱导动力学曲线（OJIP） 63

5.4 讨论 67

5.5 结论 69

第6章 植物源单剂川楝素对小球藻培养中棘尾虫污染的治理 70

6.1 引言 70

6.2 材料与方法 71

6.2.1 贻贝棘尾虫的获取及前处理 71

6.2.2 培养基 71

6.2.3 杀虫剂 72

6.2.4 蛋白核小球藻细胞形态鉴定 72

6.2.5 毒性试验 72

6.2.6 川楝素和碳酸氢铵在微藻养殖中的安全性评价 72

6.2.7 叶绿素荧光测定 73

6.2.8数据分析 73

6.3 实验结果 73

6.3.1 毒性试验及症状观察 73

6.3.2 棘尾虫种群数量变化 74

6.3.3 亚致死浓度川楝素和碳酸氢铵对微藻的安全性评价 75

6.4 讨论 78

6.5 结论 80

第7章 结论与展望 81

参考文献 83

致 谢 99

作者简历及攻读学位期间发表的学术论文与研究成果 101