滇池中溶藻细菌的分离鉴定及其溶藻效应

Identification of algicidal bacterium Sp37

摘要:

【背景】藻类水华或赤潮在世界范围内频发，带来各种危害，亟需找到有效途径控制水华或赤潮。溶藻细菌具有杀死藻类控制藻类生物量的能力，可以作为防治水华和赤潮的有效工具。【目的】分离并鉴定滇池中的铜绿微囊藻(Microcystis aeruginosa)及其溶藻细菌，对溶藻菌作用于铜绿微囊藻的溶藻效应进行研究，初步了解其溶藻特性与溶藻机制。【方法】采用LB平板稀释涂布，再经多次划线分离纯化细菌，测定16S rRNA基因序列以鉴定细菌种类；采用毛细管分离的方法分离铜绿微囊藻，并测定其cpcBA基因序列以鉴定蓝藻种类；采用热乙醇法提取叶绿素a，从而计算溶藻效率；基于过氧化氢酶(CAT)、还原型谷胱甘肽(GSH)和丙二醛(MDA)探究藻细胞在溶藻菌处理下的抗氧化系统响应。【结果】共分离获得11株微囊藻和17株针对铜绿微囊藻的高效溶藻菌。选取其中一株生长速度最快的铜绿微囊藻DCM4和一株溶藻效果最好的溶藻菌Sp37 (Bacillus siamensis)进行后续研究。Sp37对DCM4的4 d溶藻率达到92.4%±1.5%，且对微囊藻属的水华微囊藻(M. flos-aquae)和惠氏微囊藻(M. wesenbergii)均有溶藻效果，而对绿藻没有溶藻效果。Sp37的原菌液和无菌滤液对DCM4的4 d溶藻率分别为86.8%±4.3%和81.1%±2.2%，两者没有显著差异(p>0.05)。Sp37菌体对DCM4的溶藻率为25.4%±7.3%。Sp37无菌滤液经不同温度和pH处理之后的溶藻率与未经处理的无菌滤液的溶藻率无明显差异。Sp37无菌滤液处理藻细胞会使藻细胞的CAT、GSH和MDA含量发生变化。【结论】菌株Sp37对铜绿微囊藻DCM4具有高效的溶藻作用，而且对微囊藻属具有一定的溶藻特异性。Sp37是通过分泌胞外物质间接溶藻，且溶藻物质具有热稳定性和酸碱稳定性。Sp37无菌滤液处理藻细胞会触发藻细胞抗氧化系统，并且会损伤藻细胞膜。Sp37无菌滤液很可能是通过对藻细胞造成氧化胁迫，最终导致藻细胞死亡的。

Abstract:

[Background] Phytoplankton blooms are a worldwide problem that brings a serious threat to environment and human health. It is urgent to control bloom through effective methods. Algicidal bacteria have been considered as an effective strategy to control bloom because they can kill algae. [Objective] To isolate and identify Microcystis aeruginosa and its algicidal bacteria from Dianchi Lake, and further analyze the algicidal effect and inhibition mechanism of algicidal bacteria against M. aeruginosa. [Methods] Bacteria were isolated by dilution separation method and identified based on 16S rRNA genes. Algae were isolated by dilution separation and capillary separation methods, and identified based on cpcBA and 18S rRNA genes. Algicidal efficiency was calculated based on the chlorophyll-a concentration of algal culture, which was measured with heat-ethanol extraction method. Determination of CAT (catalase), GSH (glutathione) and MDA (malondialdehyde) were used to investigate the algal response of antioxidant system to the algicidal bacteria. [Results] In total 11 Microcystis and 17 algicidal bacteria strains were isolated, M. aeruginosa DCM4 and Bacillus siamensis Sp37 were selected for the further research. Sp37 showed a strong algicidal activity against M. aeruginosa DCM4 (Ae=92.4%±1.5%, t=4 d), and it also can kill M. flos-aquae and M. wesenbergii of Cyanophyta, but not Monoraphidium and Scenedesmus of Chlorophyta; there is no significant difference of algicidal ratio (P>0.05) between the culture and cell-free filtrate of Sp37 (86.8%±4.3% and 81.1%±2.2% (t=4 d), respectively), while that of washed cells were much lower (25.4%±7.3%); Sp37 cell-free filtrates that treated at different temperature and pH conditions displayed no obvious difference of algicidal effects; Cell-free filtrate of Sp37 caused great changes of CAT, GSH and MDA content of DCM4. [Conclusion] Sp37 had strong algicidal effect on M. aeruginosa DCM4, and it is likely can kill Microcystis selectively. Sp37 kills M. aeruginosa DCM4 by releasing algicidal substance(s) which has (have) good thermal and acid-base stability. Cell-free filtrate of Sp37 can activate antioxidant systems and destroy cell membrane of DCM4, and ultimately cause algal cell death.