Preparation Of Sustained-release Microspheres Of Allelochemicals From Eucalyptus Globulus Leaves And Study On Their Long-term Algal Inhibition

The outbreak of cyanobacterial blooms in eutrophic water has posed a serious threat to the aquatic ecological environment and human health.Microcystis aeruginosa(M.aeruginosa)is one of the most common harmful cyanobacteria species in the world,and its excessive reproduction can cause a series of environmental problems such as increased turbidity of water environment,decreased dissolved oxygen in water and death of aquatic organisms.The development of effective new algal suppressants is a research focus of the control of cyanobacteria blooms.In recent years,the application of sustained-release microspheres embedded with allelochemicals in long-term algal control has become a hot research topic.In order to improve the application prospect of this technology,it is very important to find environmentally friendly substances which can effectively inhibit algae as the embedding objects and explore the mechanism of its efficient inhibition.It also provides a theoretical basis for revealing the mechanism of phytoplankton growth inhibition by low dose secondary metabolites secreted by aquatic plants.Because the Eucalyptus globulus extracts of leaves(ELE)on algal cell growth has certain allelopathic inhibition,this study ELE as the embedding material to prepared ELE allelochemicals sustained-release microspheres combined with microspherification technology.Meanwhile,response surface methodology was used to optimize the preparation process of ELE allelochemicals sustained-release microspheres.The optimized microspheres were used to conduct long-term algal inhibition experiments on Microcystis aeruginosa.The inhibitory effect and mechanism of ELE allelochemicals sustained-release microspheres on Microcystis aeruginosa were studied by the characterization of cell ultrastructure and the determination of intracellular reactive oxygen species(ROS),nitric oxide(NO),cell membrane integrity,cell esterase activity,extracellular microcystins(MCs).The main research results are as follows:(1)In this study,oscillating extraction,heating extraction and ultrasonic extraction were used to extract ELE,respectively.The ELE under different extraction methods showed different inhibition degree to M.aeruginosa(FACHB 905),and the oscillating10-day extract had the best inhibition effect,with an inhibition rate of 44.32%.Using ELE of different tree ages(3,5,8 and 20 years old)to inhibit algae,the results showed that the5-year old ELE had the best inhibitory effect on algae,and the inhibitory rate was 73.94%.The results of algal inhibition experiments under different ELE exposure concentrations showed that the inhibition rate of 0.5 g/L,1 g/L,2 g/L and 3 g/L ELE on day 16 were19.52%,46.18%,76.41% and 85.16%,respectively,indicating that there was a dose-effect relationship between the ELE and the algae inhibitory rate.LC-MS was used to identify and analyze the organic compounds in the ELE,The results showed that the ELE contained catechin,gallic acid,ellagic acid and other common phenolic acid allelochemicals.(2)In this study,the effects of concentration of ELE(core material solution),concentration of sodium alginate,concentration of chitosan,concentration of calcium chloride and volume ratio of core wall solution(ELE solution volume∶sodium alginate solution volume)on loading rate and embedding rate of the microspheres during the preparation of ELE allelochemicals sustained-release microspheres were discussed.The optimal concentration of the core material was 18 g/L,and the optimal volume ratio of the core wall solution was 2∶3.Response surface optimization experiments were designed to further improve the embedding rate of ELE allelochemicals sustained-release microspheres,the optimal preparation parameters were as follows: sodium alginate concentration 2.13%(w/v),chitosan concentration 1.02%(w/v),calcium chloride concentration 0.78%(w/v).The order of the influence of the three factors on the embedding rate of ELE allelochemicals sustained-release microspheres was sodium alginate concentration>calcium chloride concentration>chitosan concentration.Under the optimal preparation conditions,the embedding rate of ELE allelochemicals sustained-release microspheres could reached 61.45%.The release performance of the optimized ELE allelochemicals sustained-release microspheres showed that the ELE allelochemicals sustained-release microspheres had good and stable sustained release performance.(3)In the long-term inhibition experiment of M.aeruginosa(FACHB 905)with different dosings(2.5 g/L,5 g/L,10 g/L)of ELE allelochemicals sustained-release microspheres,different concentrations(0.578 g/L,3 g/L)of ELE and 10 g/L blank microspheres,the 10 g/L ELE allelochemicals sustained-release microspheres showed the best algal inhibition effect,with the highest algal inhibition rate of 92.99%.The ultrastructure of M.aeruginosa cells was characterized,the results showed that the ELE allelochemicals sustained-release microspheres could destroy the internal structure of the cells seriously,leading to the disintegration and death of the cells,and the microspheres themselves had adsorption effect on the algae cells.The physiological indexes of algal cells were measured.The intracellular reactive oxygen species(ROS)levels of 10 g/L ELE allelochemicals sustained-release microspheres were 9.25,3.69 and 4.94 times higher than those of the control group at the 15 th,30th and 45 th days,respectively.The level of nitric oxide(NO)was 2.16,2.58 and 2.41 times higher than that of the control group,respectively.The proportion of cell membrane damaged cells was 21.4%,49.71%,54.26%.The results showed that the ELE allelochemicals sustained-release microspheres produced oxidative stress on M.aeruginosa,which significantly increased the levels of ROS and NO,severely damaged the integrity of cell membrane,and reduced the esterase activity of cells.Meanwhile,the ELE allelochemicals sustained-release microspheres reduced the extracellular microcystin content of algae cells,indicating that the microspheres had a certain removal effect on microcystin.In conclusion,this study simulated the algal inhibition mode of aquatic plants releasing allelopathic substances continuously at low dose,and prepared the ELE allelochemicals sustained-release microspheres with long-term inhibitory effect on the growth of M.aeruginosa(FACHB 905),which realized the effective reuse of plant waste resources.The mechanism of algal inhibition by ELE allelochemicals sustained-release microspheres was studied,which provided reference for the introduction of microspherification technology in algal control by allelochemicals.