一株表面活性剂产生菌的抑藻作用过程与机理研究

Harmful algal blooms (HABs) is a serious global marine disaster. It causes a series of ecological, resource, environmental problem and major economic losses. The apparent increasing occurrence of HABs throughout the world has led to extensive researches to mitigate the blooms. As a result, some management strategies have been developed. In the factors that regulate the red tide dynamics, algal-bacterial interactions are increasingly cited as potentially regulators in the sense of both decreasing and developing algal blooms. Due to the requirements of some eco-friendly strategies in the control of harmful algal blooms, bio-surfactants produced by marine microorganisms have gradually drawn much attention. This project aims at the key problems of HABs biological control, chooses HABs causing algae as objective algae. First of all, we will try to obtain a series of marine bacterial strains with high bio-surfactant production with a high through-put screening strategy for bio-surfactant producing strains. We will optimize the culture condition of the bio-surfactant producing strain using a single-factor test、the uniform design and response surface methodology (RSM), and prepare the crude emulsifying agents from fermentation supernatant by cold centrifugation, alcohol precipitation, deproteinization and 100KD-ultrafiltration and the DEAE-Sepharose fast flow ion exchange chromatography. Then we will study the algicidal effect of bio-surfactant on the HABs causing algae. Finally we will interpret the functional process and algacidal mechanism of bio-surfactant on HABs causing algae, and provide scientific basis, bacterial resources, and biological control technology for the safe and effective control of HABs through bio-surfactant producing microbes.

有害藻华是一种严重的全球性海洋灾害，它会造成严重的生态、资源、环境问题和重大的经济损失。寻求有效的有害藻华防治方法成为当务之急。海洋细菌在藻华生消过程中有着极其重要的作用，而细菌杀藻现象的发现也为微生物防治藻华提供了新的可能途径，由微生物发酵所产的表面活性剂因其符合清洁绿色环保的要求，逐渐成为研究热点。本项目针对藻华微生物防治研究中存在的关键问题，选取典型藻华藻为研究对象，首先运用抑藻表面活性物质产生菌的高通量筛选流程，从不同生境样品中筛选高产表面活性物质的海洋细菌；其次在单因子优化的基础上，运用均匀设计和响应面法优化该菌株的发酵工艺；然后运用多级醇析、超滤法和DEAE-琼脂糖离子柱层析法分离表面活性物质，并研究该生物表面活性物质对典型藻华藻的影响，阐释表面活性物质作用下藻细胞应答机制，丰富并拓展“菌-藻关系”研究的理论，为安全有效地利用生物表面活性物质产生菌治理有害藻华提供科学依据。

生物表面活性剂是一类具备乳化、增溶、降低液面表面张力等特性的两性物质，与普通表面活性剂相比，它同时还具备了可生物降解、不易造成二次污染、环境友好等优点；研究表明，生物表面活性剂已广泛运用于污染环境的生物修复。为了探究生物表面活性剂在有害赤潮治理方面的运用潜力，本课题以抑藻生物表面活性剂BA01为对象，分析其理化特性，探究其对有害赤潮原因种--——赤潮异弯藻的抑藻机制，取得以下主要研究成果：.（1）经鉴定，生物表面活性剂粗提物BA01中总蛋白含量约为6.2%，总糖含量约为14.1%，为非离子型表面活性剂；BA01经不同温度、盐度和pH处理后，仍保持较低的表面张力和较高的杀藻活性。.（2用BA01处理赤潮异弯藻48 h后，当终浓度为2.5 μg/mL时，处理组抑藻率为24.49%，而当终浓度为15 μg/mL时，处理组抑藻率为97.76%，经计算，BA01半致死浓度为5.89 μg/mL。.（3）在半致死浓度BA01胁迫下，藻细胞整体形态略有变化，细胞膜有轻微损伤，但细胞保持相对完整，细胞器结构完整，由于半致死浓度组的藻细胞形态完整，ROS水平持续保持较高值，抗氧化物质也持续保持较高活性，以清除细胞内过多的ROS，光合作用起初受到明显抑制，随后经过细胞自身修复，光合效率逐步回升，叶绿素和类胡萝卜素等光合色素含量下降至对照组50%左右。.（4）全致死浓度组藻细胞细胞膜发生破裂，细胞器无法正常工作，细胞内容物流出，细胞裂解，抗氧化系统崩溃，无法清除细胞内的ROS，过多的ROS引起细胞膜膜脂过氧化，细胞内MDA含量显著升高，且藻细胞光合作用受到显著破坏，在高浓度表面活性剂胁迫下，藻细胞无法修复损伤，光合效率持续下降，光合色素含量仅为对照组的15%左右。