Separation And Ecological Evaluation Of Algicidal Active Substances From Bucillus Tequilensis D8

Heterosigma akashiwo reproduces quickly and can take in nutrients both day and night.It is one of dominant algae species that forms harmful algal blooms and distribute in many sea areas of the world.H.akashiwo blooms have caused the death of quantity of farmed fish,seriously endangering the marine environment and the sustainable development of aquaculture.Compared with physical methods and chemical methods,which often bring high cost and secondary pollution problems,biological methods have attracted wide attention due to their high efficiency and safety.The separation and identification of algicidal bacteria and algicidal substances have become hot issues of the red tide control.In this paper,a high-efficiency algicidal strain D8 was isolated from the first wharf in Xiamen City,Fujian Province,and identified.Then,the algicidal mode,algicidal stability and spectrum of strain D8 were studied and the algicidal substances were identified by LC-MS and NMR.Finally,the ecological safety of the algicidal active substances was evaluated by constructing the Heterosigma akashiwa microcosm system.The main findings are as follows:（1）The strain D8,isolated from the first wharf of Xiamen City,had high algicidal effect against Heterosigma akashiwa.The 16S rRNA gene sequencing and SEM morphological observation showed that strain D8 belonged to Bacillus and had a 99.8%similarity to Bacillus tequilensis KCTC 13622T.（2）The algicidal spectrum of strain D8 was mainly concentrated in the Bacillariophyta,Xanthophyta and Pyrrophyta.The algicidal rates of strain D8 against Thalassiosira pseudonana and Skeletonema costatum（Bacillariophyta）reached above 90%after treatment for 72 h.The killing rates of strain D8 against Heterosigma akashiwo（Xanthophyta）and Prorocentrum donghaiense（Pyrrophyta）were 96.8%and 88.9%respectively in 72 h.But strain D8 has no obvious effect on species belong to Chlorophyta and Cyanophyta.（3）Strain D8 killed algal cells by indirect way,the algicidal ative substance existed in the supernatant of strain D8 culture.The algicidal rate reached 96.3%after 48 h treatment with 5%D8 supernatant.The algicidal activities of the D8 supernatant were insensitive to temperature,light intensity and pH value indicating that the algicidal active substance had excellent thermal stability,light stability and acid-base stability.（4）The crude algicidal substances were extracted and separated from the D8 supernatant through acid precipitation and chloroform/methanol（2:1）extraction.Different concentrations of D8 crude extract had good algicidal effect against Heterosigma akashiwo.Morphological observation and physiological parameters detection indicated that D8 crude extract primarily targetted cell membrane including plasma membrane and thylakoid membranes,which caused imperative damage to photosynthetic system and significantly reduced the content of photosynthetic pigments and electron transfer rate.Furthermore,D8 crude extract siginificantly reduced the activity of intracellular lipase,which inhibited the recycle of membrane lipid and finally resulted in the collapse of membrane system and cell lysis.5)In the microcosmic system,the crude extract of D8 still had a good effect on algae killing.After adding D8 crude extract for 2 days,the algae killing rate reached 77.7%.After the outbreak of red tide,the Alpha diversity（Sobs index,Shannon index and PD_Whole_tree index）of the community in the control,DMSO treatment and D8 crude extract treatment were significantly decreased,indicating that the outbreak of red tide would lead to the change of bacterial community diversity,which clarified the need of the control of red tide.In addition,according to Beta diversity analysis,the bacterial community composition of Control₃0 d was significantly different from that of the original seawater,while that of DMSO 30 d and D8₃0 d was closer to that of the original seawater.It was speculated that DMSO was beneficial to the restoration of the bacterial community after the outbreak of red tide,while the effect of D8 crude extract on the bacterial community composition was relatively small.At the phylum,class and family levels,the main groups in the control,DMSO treatment and D8 crude extract treatment were Proteobacteria,Alphaproteobacteria and Rhodobacteraceae,respectively.After adding D8 crude extract,the abundance of dominant taxa in the D8 crude extract treatment group was not significantly different from that in the original seawater at the phylum,class or family level.In addition,Maritimibacter changes into Marivita at the genus level.According to relevant literature,Marivita has DMSO reductase,which can reduce DMSO to DMS and release it into the air.Therefore,the change of genus level is mainly caused by DMSO.In conclusion,the addition of D8 crude extract has no significant effect on the microcosmic community structure,and it is relatively safe for red tide control.（6）The crude extract from strain D8 contained biossurfactants according to the oil drainage circle experiment.Subsequently,4 components were isolated by high performance liquid chromatography（HPLC）from D8 crude extractin which component 2,3 and 4 showed high algicidal activities.（7）After purified by ODS column chromatography and the preparative high performance liquid chromatography,component 2,3 and 4 were identified by HPLCQ-Exactive-MS/MS and NMR.The molecular weights of component 2,3 and 4 were 1008,1022 and 1036,respectively.The 1H spectrum and 13C spectrum of NMRindicated that the three components belong to homologues of surfactin,namely surfactin-C13,surfactin-C14 and surfactin-C15.