中国科学院海洋研究所机构知识库

近年来，有害藻华的暴发频率逐年提高，规模不断扩大，已成为世界性的海洋灾害。目前有害藻华的众多治理方法中，改性粘土絮凝法被认为是最有效的方法之一，多次被应用于大规模有害藻华的现场治理，已经成为我国有害藻华应急处置的标准方法。在多次现场应用中，发现改性粘土对藻华生物的去除率达到80%时即可控制有害藻华，虽然水体残留的20%微藻密度依然较高，但未发现这部分微藻在短期内形成二次藻华。为了揭示残留微藻生长受抑制的机制，本文通过室内模拟实验，以典型有害甲藻强壮前沟藻（Amphidinium carterae Hulburt）和米氏凯伦藻（Karenia mikimotoi）为实验对象，从生理生化角度入手，考察了不同浓度改性粘土处理后，残留强壮前沟藻和米氏凯伦藻氧化应激系统和光合作用的变化；并利用实时荧光定量PCR方法，进一步研究了残留米氏凯伦藻的胁迫适应和光合作用基因表达的变化。主要成果如下：

（1）改性粘土不仅可以有效絮凝沉降强壮前沟藻，而且显著地抑制该藻的生长和影响其关键生理过程。随着改性粘土浓度的增大，对强壮前沟藻的去除率增大；当改性粘土浓度≥0.10 g/L时，残留微藻的生长受到显著抑制。经过改性粘土处理后，残留微藻的丙二醛（MDA）含量上升，说明其细胞内积累了大量活性氧（ROS）。实验组残留微藻的细胞密度与超氧化物歧化酶（SOD）活性、过氧化氢酶（CAT）活性、MDA含量均呈现出显著的负相关性，因此推测细胞内过度积累的ROS可能是抑制残留微藻生长的主要内在因素。此外，研究发现残留微藻的光合色素含量和净光合作用速率下降，表明改性粘土对残留强壮前沟藻的光合作用产生了严重伤害。叶绿素荧光动力学数据发现，单位面积上反应中心RC/CS₀显著下降，表明光合系统II（PSII）反应中心部分失去活性，引起剩余有活性的反应中心过度激发，即单位反应中心吸收的能量（ABS/RC）、捕获的能量（TR₀/RC）和用于电子传递的能量（ET₀/RC）上升。但是TR₀/ABS和ET₀/ABS下降，表明残留微藻的光能吸收和利用失去平衡。此外，0.25 g/L改性粘土处理3 h时，残留微藻W_K和V_J分别上升为对照组的1.24和1.20倍，表明其放氧复合体受到破坏，初级醌受体Q_A至次级醌受体Q_B的电子传递链受阻。改性粘土对残留微藻光合作用的以上影响，导致了叶绿体ROS的大量积累。

（2）选取我国近海第二有害藻华优势种米氏凯伦藻为实验对象，开展了不同浓度改性粘土对其生长的影响研究。在实验条件下，加入0.50 g/L改性粘土，米氏凯伦藻的去除率可达到64%，并且残留微藻的生长受到了显著抑制。各实验组残留米氏凯伦藻的过氧化氢（H₂O₂）和MDA含量显著提高，表明改性粘土导致细胞内积累较多的ROS，并引起氧化压力。细胞内H₂O₂浓度与微藻细胞密度呈现显著的负相关，进一步验证了细胞内过多ROS会抑制其生长的推论。从光合作用角度，虽然残留米氏凯伦藻的实际光化学效率未受影响，即光反应能够产生足够的NADPH和电化学势，但残留微藻的最大光化学效率F_v/F_m和光合性能指数PI_ABS下降，表明改性粘土对其光合作用产生胁迫伤害。具体来讲，残留微藻的光能吸收和利用失衡，电子传递受阻，Q_A^-过度积累，可能导致单线氧¹O₂的产生。

（3）使用实时荧光定量PCR方法，筛选了改性粘土处理条件下，米氏凯伦藻的内参基因为β-actin。定量分析了残留米氏凯伦藻的胁迫适应和光合作用相关基因表达，初步从基因水平阐释了改性粘土对残留微藻生理活动的影响。0.50 g/L改性粘土处理后，残留微藻的热休克蛋白90（HSP90）和HSP70快速参与到细胞的应激反应中，是抗逆过程的重要承担者。两者的编码基因（hsp90和dnaK）与大多数光合作用基因的表达变化呈现显著的相关性，表明两者可能参与了残留微藻光合作用的修复过程。改性粘土显著抑制了残留米氏凯伦藻光合作用的关键蛋白基因（psbC、psbB、psbA、psbD、psaB和psaA）的表达，PSII和PSI虽然受到一定程度的影响，但实际光化学效率未受影响，并经过细胞的信号传导调控，在2 d时各蛋白表达量均有不同程度的恢复。残留微藻ATP合成酶的编码基因表达量显著降低，而且保持该水平至实验后期，表明微藻对光反应提供的跨膜质子梯度利用率可能下降，合成ATP的过程受到破坏。实验组微藻核酮糖-1.5-二磷酸羧化/加氧酶（Rubisco）的大亚基编码基因rbcL表达虽然在实验前期显著下降，但在2 d时恢复至与对照无显著差别的水平，推测Rubisco蛋白含量可能不是光合作用的限制因素；根据CbbX（Rubisco活化酶）和ATP合成酶编码基因表达量的变化，推测Rubisco的活性可能降低，直接影响了CO₂固定效率，引起ROS的大量积累，从而影响其正常生长。

综上所述，本文从生理生化角度研究了改性粘土对残留微藻生长的影响，为改性粘土除藻机制提供补充。结果表明，改性粘土影响残留微藻的氧化应激系统，引起细胞内过度积累ROS，形成的氧化胁迫是抑制其生长的主要内在因素。改性粘土对残留微藻的光合作用也产生较大影响，破坏光合机构，光能吸收与利用失去平衡，电子传递受阻，可能导致叶绿体ROS的形成。此外初步研究了残留微藻光合作用相关基因的表达变化，进一步从分子水平上解释了其光合生理的变化。

In recent years, the occurrence and scale of harmful algal blooms (HABs) have increased gradually. HABs have become a worldwide marine disaster. At present, modified clay (MC) application is considered as one of the most effective strategies from varieties of methods for controlling HABs. In fact, MC application has been widely applied to large-scale treatment of HABs and has become the standard method for the emergency treatment of HABs in China. Many field applications have revealed that a removal efficiency (RE) of 80% is sufficient to control the HAB, although considerable numbers of residual algae are thought to be able to re-grow into a HAB within a short time, re-proliferation of the residual algae has rarely been observed. In the present study, to gain insight into the mechanism underlying the growth inhibition of residual microalgae, the typical harmful dinoflagellates——Amphidinium carterae Hulburt and Karenia mikimotoi were treated with different concentrations of MC, and then the oxidative stress system and photosynthesis of the residual A. carterae and K. mikimotoi were measured. Furthermore, real-time fluorescent quantitative PCR (RT-qPCR) was used to study the transcript levels of genes related to stress adaptation and photosynthesis in the residual K. mikimotoi. The main results were as follows:

(1) MC effectively removed A. carterae, and the growth of the residual algae was obviously inhibited when the MC dosage was equal to or higher than 0.10 g/L. Further investigation suggested that excessive reactive oxygen species (ROS) accumulated in the residual algae, indicated by increased malondialdehyde (MDA) contents. There were significantly negative correlations between the residual algal density and superoxide dismutase (SOD) activity, catalase (CAT) activity and MDA content, which directly indicated the accumulation of intracellular ROS. The excessive levels of ROS in residual algae might be the main internal factor inhibiting their growth. In addition, the pigment contents and net photosynthetic rate were decreased after treatment, indicating that the photosynthetic efficiency was severely decreased. Moreover, compared with the control, the active reaction centers (RCs) per excited cross section (RC/CS₀) decreased, indicating that the partial RCs became inactivated. Therefore, the residual activated RCs were over-excited, suggested by the increases in the absorption flux per photosystem II (PSII) RC (ABS/RC), the trapping flux per RC (TR₀/RC) and the electron transport flux per RC (ET₀/RC); while the TR₀/ABS and ET₀/ABS decreased, indicating MC led to an imbalance between photosynthetic light absorption and energy utilization. After adding 0.25 g/L MC at 3 h, the W_K and V_J of the residual microalgae increased to 1.24 and 1.20 times that of the control, respectively, indicating that the oxygen-evolving complex activity was impaired, the electron transport chain (ETC) from the primary quinone electron acceptor Q_A to the secondary quinone electron acceptor Q_B was blocked. These changes lead to large accumulation of ROS at PSII.

(2) K. mikimotoi, as the second dominant harmful algal bloom specise in China, was treated with different concentrations of MC and its changes of growth were studied. After treatments with 0.50 g/L MC, the RE of K. mikimotoi was 64%, and the growth of residual microalgae was significantly inhibited. Significant increases in the hydrogen peroxide (H₂O₂) content and MDA content were observed after MC treatment, indicating that MC induced significant oxidative stress in the residual K. mikimotoi. In addition, the increased ROS level was hypothesized to be the main internal factor inducing the inhibition of residual algae growth, as indicated by the significantly negative correlations between the residual algal density and H₂O₂ content. Further investigation showed that although the achieved overall photosynthetic efficiency was not significantly affected by MC, and that the light reaction of photosynthesis could provide sufficient nicotinamide adenine dinucleotide phosphate (NADPH) and transmembrane proton gradient required for the synthesis of adenosine triphosphate (ATP), MC damaged the photosynthetic apparatus, as indicated by reduction in the maximal photochemical efficiency of PSII (F_v/F_m) and the performance index (PI_ABS). The absorption energy per unit RC increased, but the capacity for energy utilization decreased, ETC was blocked, and the Q_A^- was accumulated, leading to the generation of ROS in chloroplast.

(3) The stable endogenous reference was β-actin for RT-qPCR analysis of K. mikimotoi after treatmets with MC. And to gain insight into the effects of MC on the physiological activities from the level of genes, the transcript levels of genes related to stress adaptation and photosynthesis in residual K. mikimotoi after treatment with 0.50 g/L MC were determined. The results showed that heat shock protein 90 (HSP90) and HSP70 were rapidly induced in the stress response of residual algae after MC treatment and were important regulators of the stress resistance process. The changes in their encoding genes (hsp90 and dnaK) expression were significantly correlated with the expression of the most photosynthetic genes, indicating their potential involvement in photosynthesis repair in K. mikimotoi. MC significantly inhibited the expression of six key protein genes (psbC, psbB, psbA, psbD, psaB and psaA) in the light reaction in residual K. mikimotoi, indicating that photosystem II (PSII) and PSI were damaged to a certain extent. However, this damage did not affect the actual photochemical efficiency of the light reaction. Over time, under the tight regulation of signal transduction, the expression of the 6 proteins increased to different degrees on the second day. The transcript level of genes encoding ATP synthase in residual K. mikimotoi was significantly decreased at 3 h and 1 d, and this decreased level continued to be observed until the end of the experiment, which suggested that utilization of the transmembrane proton gradient provided by the light reaction may decrease and that the process of ATP synthesis may be reduced. Although the transcript level of the ribulose-1.5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) was decreased significantly at 3 h, it recovered rapidly over time, and it is reasonable to consider that the Rubisco protein content was not the limiting factor in photosynthesis. However, changes in CbbX (a red-type Rubisco activase) and ATP synthase expression suggested that the activity of Rubisco may be reduced, which would directly affect the efficiency of CO₂ fixation, inevitably induce a large accumulation of ROS, and affect the normal growth.

In summary, the effect of MC on the growth of residual microalgae was studied from the perspective of physiological and biochemical characteristics. The results showed that MC induced the accumulation of excessive ROS, and the oxidative stress was the main inhibitor of growth. MC also exerted great influence on photosynthesis, destroyed the photosynthetic apparatus, disordered the balance in light energy absorption and utilization, and hindered ETC, which may lead to the generation of ROS in chloroplast. Furthermore, molecular biology method was also used to revel the photosynthetic activities of residual algae.