叶片山海绵共附生细菌的防污活性研究

Study on Antifouling Activity of Bacteria Associated with Marine Sponge Mycale phyllophila

从叶片山海绵(Mycale phyllophila)中分离纯化得到7株共附生细菌,经16SrDNA序列比对,分别归属于芽孢杆菌属(Bacillus,5株)、假弧菌属(Pseudovibrio,1株)和短杆菌属(Brevibacterium,1株).菌株经发酵培养后对发酵液进行提取,以翡翠贻贝(Perna viridis)足丝分泌抑制实验检测提取物的防污活性.检测结果表明,菌株NAP1和NAP7的提取物显著抑制翡翠贻贝足丝分泌,其半抑制浓度(EC50)分别为55.26和14.85μg/mL,其余5株菌株的提取物均无防污活性.结合形态观察、生理生化特征和16SrDNA序列比对,将菌株NAP7初步鉴定为脱氮假弧菌(Pseudovibrio denitrificans).另外,进一步对菌株NAP7提取物进行活性部位追踪,以不同极性的溶剂进行萃取,依次获得石油醚相、二氯甲烷相、乙酸乙酯相、正丁醇相和水相部分,进行防污活性检测,发现除水相部分无防污活性外,其余4个部分均显著抑制翡翠贻贝足丝分泌,其中正丁醇相不仅防污活性高(EC50=4.06μg/mL),对翡翠贻贝的毒性也低(半致死浓度(LC50)=158.11μg/mL),表明其中含有高效低毒的活性成分.研究结果为从海洋微生物中筛选获得新型环保海洋防污剂奠定重要基础.

Seven bacterial strains were isolated from marine sponge Mycale phyllophila.Based on comparative analysis of their 16 S rDNA sequences,five strains were affiliated to the genus Bacillus,one to the genus Pseudovibrio,and one to the genus Brevibacterium.And then,they were subjected to fermentation and the extracts of culture broth were tested for antifouling activity using the bioassay of byssus thread production with the green mussel Perna viridis.The results showed that the extracts of the bacterial strains NAP1 and NAP7significantly inhibited byssus thread production of P.viridis,with EC50 of 55.26 and 14.85μg/mL,respectively.The extracts of the other five strains had no antifouling activity.Additionally,the extract of the strain NAP7 were further investigated for its active fraction.It was separated with solvents of different polarities.Petroleum ether fraction,methylene dichloride fraction,ethyl acetate fraction,n-butanol fraction and water fraction were obtained successively and then tested for antifouling activity.Although there was no antifouling activity in the water extract,the other four fractions significantly inhibited byssus thread production of P.viridis.Especially,the n-butanol fraction was highly antifouling active（EC50=4.06μg/mL）and not very toxic（LC50=158.11μg/mL）,indicating that this fraction contained substance（s）with high antifouling activity and low toxicity.The findings of this study are important for screening environmentally friendly marine antifoulants from marine microorganisms.