| In recent years, eutrophication and red tides occured more and more frequently in costal area and have caused damage on fishery, tourism and aquaculture industry, even on human health. Among all the methods developed to control red tides, using microorganism as biological tools is better than using physical or chemical methods in terms of ecological security.Bacteria and algae are closely related biological groups in aquatic ecosystem. It has been found that marine algae-lysing bacteria played an important role in regulating the occurrence and termination of red tides. In this study,we used high-throughput screening method to test the algae-lysing effect of over 300 marine bacterial crude extracts isolated from Red Sea.Four crude extracts showed lysing activities on golden alga Phaeocystis globosa.Next,we isolated three algae-lysing bacterial strains from red tide seawater samples collected in Guangxi Province. We also isolated 21 strains of algae-lysing bacteria from dinoflagellate Akashiwo sanguine red tide seawater samples in Dameisha, Shenzhen. Three stains from Shenzhen showed the strongest algae-lysing effect and named as H82, H104 and H115. Some algae-lysing bacterial strains were identified based on bio-chemical characteristics and 16 S r DNA gene sequence analysis. The algae-lysing activity and VBNC state transformation of these bacterial strains were studied. The exact structure of the active substance of algae-lysing bacterium H115 is still under investigation.The main results of this research were as follows:(1) Among the 356 bacterial crude extracts provided by Hong Kong Technology University, four extracts(97, 104, 111 and 259) showed lysing effect on Phaeocystis globosa;(2) Three algae-lysing bacterial strains were isolated through liquid infection method from red tide seawater samples from Guangxi Province and showed specific inhibitous effect on A. sanguine(strain H108), Alexandrium tamarense(strain H109) and P. globosa(strain H107), respectively. During a A. sanguine red tide happened in Dameisha, Shenzhen, 21 strains of algae-lysing bacteria were isolated, among which three stains(H82, H104 and H115)showed stronger lysing effect. The 16 S r DNA gene sequence analysis suggested that H108 could be Vibrio alginolyticus strain ATCC 17749, H107 could be Cobetia marina strain NBRC 102605, H82 could be Pseudoalteromonas piscicida strain IAM 12932, H104 could be Vibrio alginolyticus strain NBRC 15630, and H115 could be Vibrio brasiliensis LMG 20546;(3) The three algae-lysing bacterial strains from Dameisha(H82, H104 and H115) could efficiently cause a motility loss of A. sanguine cells within 1 h with a volume ratio of 1:200(bacteria to algae), accompanied with quick decrease of Yield value in algal cells. After 4 h,over 97% algal cells were killed by bacterial cells. Autoclaved bacterial culture or 0.22 μm membrane filtrate still maintained the lytic effect, which indicated that the active substances were heat-resistant, non-protein bioactives secreted by bacterial cells. The increase of MDA(malondialdehyde) content and SOD(superoxide dismutase) activity in A. sanguine cells treated with algae-lysing bacterial cells suggested a stress of active oxygen metabolism and membrane lipid peroxidation;(4) Under low temperature and oligotropic conditions, cells of the strains H82, H104 and H115 could enter a viable but non-culturable(VBNC) state under which they lost the ability to form colonies on agar plates. In this study, we investigated conditions that can resuscitate the VBNC state bacterial cells, and compare their lytic activities and fatty acid profiles before and after VBNC transformation. Results showed that either the addition of growth medium or/and an uplift of temperature could resuscitate VBNC bacterial cells within24 h; compared with active state bacterial cells, cells under VBNC state showed lower lytic activity on algal cells. After entering the VBNC state, the relative level of saturated fatty acid increased while the unsaturated fatty acid level decreased; resuscitated cells showed similar level as VBNC state cells;(5) The algae-lysing substances in the algae-lysing bacterial cultures were separated and purified by polysaccharose precipitation, desalt, and concentrated with ion- exchange resin column(Amberlite GC-50-type 1), with the 5.6% ammonium hydroxide elution showed the highest algae-lysing activity. In order to test the possible toxic effect of the algae-lysing suberstance, we also performed acute toxicity tests on zebra fish(Danio rerio) and brine shrimp(Artemia salina). No acute toxic effect was found in either zebra fish or brine shrimp after addition of bacterial culture supernatant 8 times higher than the algae-lysing concentration. This indicated that the algae-lysing substances had no toxic effect on marineinvertebrates and vertebrates. |
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