Study On Synergism Of Biological Modification On The Mitigation Of Harmful Algal Blooms By Clay Method

Harmful algal blooms,or HABs,occur when colonies of algae — simple plants that live in the sea and freshwater — grow out of control and produce toxic or harmful effects on people,fish,shellfish,marine mammals and birds.Modified clay is the most widely used method for the mitigation of harmful algal blooms,mainly through physical and chemical methods to change the surface electrical properties of clay particles,thereby improving the flocculation efficiency of clay on algal cells.It has the advantages of high efficiency,environmental friendliness,and easy operation.In recent years,with the development of research on the interaction between algae and bacteria,biological methods have provided new ideas for the mitigation of harmful algal blooms due to their environmental friendliness and long-term regulatory effects on water quality.Whether the biological method and the modified clay method can be combined to form a new mitigating method that can not only deal with harmful algal blooms in emergency,but also repair ecosystems is the content of this thesis.Therefore,in this paper,the common probiotic bacteria Effective Microorganisms(EMs)and Brevibacillus laterosporus in aquaculture were used to explore the synergism of biological modification on the mitigation of harmful algal blooms by clay method,optimize the biological modification conditions,and conduct in-depth research on the algicidal characteristics of Brevibacillus laterosporus.The algicidal substances secreted by them were isolated and identified by high-performance liquid chromatography and mass spectrometry.The main research results are as follows:1.The synergistic effect of EMs on clay.Using Prorocentrum donghaiense,a typical algal bloom organism,the synergistic effect and mechanism of EMs on modified clay were analyzed using scanning electron microscopy and flow cytometry.The results show that EMs can stably survive in algal blooms and effectively inhibit the growth of Prorocentrum donghaiense through direct contact and secretion of algicidal substances;After modifying clay,it produces a 1+1>2 algicidal effect.Therefore,EMs are a class of biomaterials suitable for composite modification with clay and improving theiralgicidal efficiency.At the same time,the modified clay has a certain enrichment effect on EMs and their secreted algicidal substances,which increases the local density of EMs and enhances their algicidal effect.In addition,suitable ripening temperature and time can promote the propagation of EMs and their adsorption on modified clay,further improving the algicidal efficiency of biologically modified clay.2.Synergistic effect of Brevibacillus laterosporus on clay.Through 16 s r DNA analysis,more than 60% of EMs are Bacillus.sp.Therefore,a strain of Brevibacillus laterosporus that has algicidal effects on algal blooms was selected as the object of further research.By analyzing the changes in biomass and algicidal efficiency of Brevibacillus laterosporus before and after clay modification,the synergistic effect of Brevibacillus laterosporus on the treatment of algal blooms by clay method was analyzed.The results showed that after modifying kaolin with Brevibacillus laterosporus for 24 hours,the biomass of Bacillus increased by about one time,and significantly improved the inhibition effect of clay on the red tide algae(P<0.01).Within 24 hours of aging,the biomass of Brevibacillus laterosporus and the removal efficiency of biologically modified clay increased with the increase of aging time;There was no significant difference in the enhancement effect of clay particle size and type on the biomass of Brevibacillus laterosporus.After 24 hours of maturation,the number of microorganisms in the microbial modified clay remained unchanged,while its algicidal efficiency increased,indicating that the algicidal effect of the microbial modified clay mainly depended on the algicidal substances secreted by Brevibacillus laterosporus.3.The algicidal effect of Brevibacillus laterosporus on the algal growth of Heterosigma akashiwo.By determining the algicidal methods of Brevibacillus laterosporus and optimizing the extraction methods of its algicidal substances,the properties of the crude extract of Brevibacillus laterosporus and its algicidal effect on Heterosigma akashiwo were studied.The research results showed that this strain mainly performs its algicidal function by secreting an algicidal substance,which is a mixture with a molecular weight of less than 3500 Da,has a large polarity,and is stable in a certain temperature(-80.0 to 70.0 ℃)and p H range(4.0 to 8.0).Within 24 hours,these algicidal substances can cause most of the cells of Heterosigma akashiwo to rupture and the content to flow out;For the remaining unbroken algae cells,the algicidal substance can activate their antioxidant system,reduce their metabolic activity,and lead to apoptosis phenomena such as shrinking of the algae cells and decreased membrane potential.4.Isolation and Identification of algicidal substances from Brevibacillus laterosporus.Using High Performance Liquid Chromatography(HPLC)and mass spectrometry,the effective algicidal components secreted by Brevibacillus laterosporus were isolated and their structures were identified.The results of HPLC and MALDI-TOF showed that there were four effective components in the algicidal crude extract of Brevibacillus laterosporus,among which components 2 and 3 had the best algicidal performance,and the components were relatively single;Through LC-Q-TOF analysis,the molecular weights of the two purified products were 1273 and 1224,respectively.After MS-MS identification,it was found that both substances were lipopeptides,and the amino acid sequences from the carbon end to the nitrogen were as follows:(1)β-Fatty acids-tyrosine-glutamic acid-aspartic acid-tyrosine-valine-asparagine-leucine/isoleucine-tyrosine and(2)β-Fatty acids-tyrosine-glutamic acid-aspartic acid-leucine/isoleucine-valine-asparagine-leucine/isoleucine-tyrosine.The innovative points of this thesis are mainly reflected in the following: the first discovery of the algicidal effect of Brevibacillus laterosporus on algal blooms in seawater;pioneering use of EMs and Bacillus to modify clay to prepare biologically modified clay and exploration of its synergistic mechanism;A new type of lipopeptide was first discovered in Brevibacillus laterosporus,which is an important synergistic mechanism for the inhibition of algae by biologically modified clays.In summary,based on the changes in bacterial biomass and algicidal efficiency of biologically modified clays,it is found that the synergistic effect of biologically modified clays is mainly reflected in the promotion of algicidal bacteria growth and the adsorption of clays on bacteria and algicidal substances.In this paper,we also studied the algicidal characteristics of Brevibacillus laterosporus,isolated and identified the algicidal lipopeptide secreted by it.The research results reveal the synergistic effect of biological modification on the treatment of harmful algal blooms by clay method and the mechanism of algicidal effect of Brevibacillus laterosporus,providing an important reference for the interaction between algae and bacteria,and having important significance for the development of a new type of biological modified clay with both ecological regulation and emergency response functions.