Research On The Environmental Effect Of Protozoan Brown Flagella On The Removal Of Toxic Microcystis And The Mechanism Of Its Degradation Of Algal Toxins

Global warming and water eutrophication accelerate the outbreaks of cyanobacteria blooms,which causes great harms to the aquatic ecosystem.On one hand,cyanobacteria in lack of essential nutrients,cannot support the growth and reproduction of primary consumers well.On the other hand,the toxic cyanobacteria can produce cyanotoxins.Cladocera,rotifer and fish are directly affected by cyanotoxins,because they do not have the ability to degrade cyanotoxins.Moreover,cyanotoxins are difficult to be degraded rapidly in natural water.They will be transferred through the food chains and accumulated in the high-trophic organisms,which will cause great damages to water safety as well as human health.Biological manipulation is considered as an environment-friendly method in control of harmful algae,which is based on feeding capacity of original organisms.Protozoans are the major predators of phytoplankton in aquatic ecosystem.Many protozoan species can resist and ingest on toxic cyanobacteria,with the ability to degrade cyanotoxins,which indicates that these protozoans can effectively dredge the flow of materials and energy in the aquatic food webs dominated by toxic primary producers and reduce water risks.Therefore,relative to cladocera,copepod and other metazoans,protozoans have certain advantages in controlling toxic cyanobacteria.However,the environmental effects and the mechanism in protozoans controlling cyanobacteria and degrading cyanotoxins in still need to be systematically studied,for instance,which species are able to eliminate cyanobacterial populations and degrade cyanotoxins,how to culture these protozoans rapidly,how the environmental factors affect protozoans grazing cyanobacteria and degrading cyanotoxins,whether the protozoans can control in-situ cyanobacteria well,and how the underlying changes in genes expressions and metabolites contents in degrading microcystins.Microcystis is the most prevalent and major bloom-forming species of cyanobacteria.Thus,based on the aforementioned questions,the present research will explore the environmental effects and molecular mechanism in in-situ protozoan grazing toxic Microcystis and degrading microcystin,from the population,community and molecular levels.1.The isolation,identification and rapid cultivation of OchromonasProtozoans are major predators of phytoplankton in the aquatic ecosystem.In the present experiment,a kind of protozoan that can efficiently graze on toxic Microcystis was obtained by isolation and purification from lake water collected from Lake Taihu.By the morphological and genomic characteristics,the flagellate YZ1 strain was identified as Ochromonas gloeopara,a species of the Ochromonas genus,with mixotrophic mode.The results in growth and photosynthetic parameters showed that O.gloeopara grew at the highest rate and had the best photosynthesis capacity when grew mixotrophically.A fed-batch mode by repeated addition of glucose could result in higher biomass of Ochromonas than single addition of glucose.We concluded that the present study illuminated the nutritional characteristics and cultivation method of O.gloeopara,which provided a possible way to control Microcystis using protozoans.2.Effects of temperature and CO₂?abiotic factor?on Ochromonas grazing toxic Microcystis and degrading microcystinThis study aimed to investigate the influence of climate warming?increased temperature and CO₂ level?on the ability of Ochromonas to eliminate toxic Microcystis populations and degrade microcystin.In the temperature experiment,we exposed Ochromoans fed on toxic Microcystis at 20,25,and 30?.The results showed that rising temperature enhanced Ochromonas grazing Microcystis and degrading microcystin.In the CO₂ experiment,we exposed Ochromoans fed on toxic Microcystis at 19,22,25,29,and 33? separately under 400 and 750 ppm CO₂ level.The results showed that rising CO₂ at high temperature significantly inhibited the grazing rates of mixotrophic Ochromonas,and delayed the time to eliminate Microcystis populations.The present study suggested that there was complex combined effects of CO₂ and temperature on prey-predator interaction,and also provided some references for the prediction of protozoans controlling toxic cyanobacteria in future climate conditions.3.Effect of chlorophyte?biotic factor?on Ochromonas grazing toxic Microcystis and degrading microcystinPrevious chapter revealed that mixotrophic Ochromonas could efficiently eliminate Microcystis and degrade microcystin.However,in the field,many different algae species coexist with Microcystis and may affect protozoans eliminating Microcystis.Therefore,in this study,we assessed the impacts of chlorophytes,a type of beneficial algae for zooplankton and common competitors of cyanobacteria,on flagellate Ochromonas eliminating toxin-producing Microcystis at different temperatures.The results showed that Ochromonas still eliminated Microcystis populations and degraded the total microcystins with the addition of chlorophytes,although the time of eliminating Microcystis was prolonged and temperature-dependent.Additionally,in the grazing treatments,chlorophytes populations gradually increased with the depletion of Microcystis,whereas Microcystis dominated in the mixed algal cultures without Ochromonas.These findings indicated that although chlorophytes prolong mixotrophic Ochromonas eliminating Microcystis,the flagellate grazing Microcystis helped chlorophytes dominating in the primary producers,which is significant in improving water quality and reducing aquatic ecosystem risks.4.Ochromonas grazing causes shifts in phytoplankton community involving harmful cyanobacteriaPrevious chapters suggested the highly efficient control of Microcystis by Ochromonas in laboratory experiments.Thus,the present study investigated the effect of mixotrophic Ochromonas controlling Microcystis in the field.We separately added Ochromonas into in-situ phytoplankton community mainly dominated by small-sized Microcystis colonies and unicellular Microcystis.The results showed that mixotrophic Ochromonas eliminated the populations of small-sized Microcystis colonies and unicellular Microcystis,prompting the shift of dominated species from cyanobacteria to chlorophyte and diatom.Moreover,the phytoplankton community composition significantly changed in the present of Ochromoans with higher species diversity and species richness.The present study suggested that it is feasible and advantageous to use mixotrophic Ochromonas in control of in-situ Microcystis populations,which is of great practical significance for improving the phytoplankton community dominated by harmful cyanobacteria.5.Transcriptomic and metabolomic analyses reveal the molecular mechanisms of resisting and degrading microcystin in OchromonasBased on the previous chapters showing high resistance and degradation ability of Ochromonas to microcystin,the present study aimed to identify potential pathways involved in resisting and degrading microcystin in flagellates,through transcriptomic and metabolomic analyses.The results showed that 999 significantly differently expressed genes?DEGs?and 437 significantly differently expressed metabolites?DEMs?were identified.These DEGs and DEMs were mainly involved in translation,carbohydrate metabolism,energy metabolism and cell proliferation.Integrating transcriptomic and metabolomic analyses suggested strong resistance of mixotrophic Ochromonas to microcystin was related to enhanced antioxidant and repair activities in the cell and extracellular matrix protection strategy;microcystin degradation in Ochromonas benefited from metabolic activity in lysosome?strong oxidation of ROS?and the detoxification of GST and GSH.The present study provided a better understanding of transcriptomic and metabolomic responses of flagellates to toxic Microcystis as well as highlighted a potential mechanism of biodegrading microcystin by flagellate Ochromonas,which served as a strong theoretical support for control of toxic microalgae by protozoans.