Photosynthetic Inhibition And Oxidative Stress In The Toxic Phaeocystis Globosa Induced By Algicidal Substance From Bacillus Sp. Strain Y4

With the rapid urbanization and industrialization, red tide caused by marine pollution has become a global problem. Phaeocystis globosa has occurred frequently in the coastal areas of China in recent years, causing devastating economic losses and health hazards. Considering high cost and secondary pollution of physical and chemical methods, biological methods that are environmental and economic are applied to to control or mitigate the harmful algal blooms,which have gotten priority of researchers. Especially, the isolation and identification of algicidal bacteria and algicidal compounds has got more attention from researchers in recent years.Bacillus sp. strain Y4 was previously isolated from surface water of the bloom of Phaeocystis globosa in zhuhai, China. In this study, the isolation and identification of algicidal compounds were studied. Physiological activity, subcellular structure and photosynthetic system in P. globosa were investigated to elucidate the mechanism involved in Hexahydropyrrolo[1,2-a]pyrazine-1,4-dione inhibition of algal growth.(1) Algicidal compound produced by Bacillus sp. strain Y4 against P. globosa was isolated and purified by silica gel column, Sephadex G-15 column and HPLC. Two algicidal compounds were obtained(named P-2 and P-3), and their algicidal activities were 60.3% and 72.9%,respectively. The one was identified as Hexahydropyrrolo[1,2-a]pyrazine-1,4-dione and the other one was amides by gas chromatography-mass spectrometry, 1H Nuclear Magnetic Resonance, Q-TOF-MS and Peak View? Software.(2) The tests showed that the level of reactive oxygen species(ROS) in P. globosa increased after exposure to Hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, and antioxidant system was activated. The level of enzymes and non-enzymatic antioxidants, such as SOD, CAT, GSH and As A, which could protect the cells from the potential damaging effects of ROS increased.These results suggested that ROS scavengers could relieve part of the damage caused by ROS,while numerous ROS exceeded the antioxidant ability. The surplus ROS increased the MDA content and lipid peroxidation of algal cells. Eventually, the cell membrane of P. globosa was broken down.(3) Flow cytometry with PI and FDA fluorescence and chlorophyll-a fluorescence was usedto investigate cell viability of algal cells. The results showed that cell membrane integrity was destroyed, then chlorophyll-a was damaged. Percentage of ruptured cells was about 90% of the control after exposure to the 20 mg L-1of Hexahydropyrrolo[1,2-a]pyrazine-1,4-dione for 96 h.TEM analysis revealed alterations of the ultrastructure of P. globosa. The results showed that the plasma membrane was detached from the cell wall. The nucleus was very heavily stained,and the chloroplasts were severely damaged. The thylakoid membrane became sparse, and the cell inclusion was released. The algal cells have been destructed by Hexahydropyrrolo[1,2-a]pyrazine-1,4-dione.(4) The results demonstrated that the contents of chlorophyll-a and carotenoid in P. globosa were decreased after exposure to Hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, and the maximum quantum yield(Fv/Fm) was also affected. The expressions of photosynthesis-related genes of psb A, psb D and rbc L were inhibited. The results showed that the transfer of photosynthetic energy was affected, and reaction center of photosystemⅡwas broken.Photosynthetic system of the algal cells was seriously destroyed.