Microbial Community Characteristics And Functional Metabolic Changes During The Progress Of Dinoflagellates Blooming Based On Metagenomic Technology

The Yangtze River is the biggest river in China.The Three Gorges Project is the backbone project for developing and harnessing of the Yangtze River;it will exert far-reaching and profound impacts on the environment after sluice,which has brought ecological and environmental concerns.After the impoundment of reservoir,flow velocity is decreased and aquatic capability is aggrandized,the aqua-environmental conditions have changed and the pollution degree was greater than that before building the dam,which can arose the rapid reproduction of phytoplankton,even leads to blooming.There are several studies for the generation of algal bloom in The Three Gorges Reservoir.However,less work has been done to investigate the mechanism of dinoflagellate bloom in The Three Gorges Reservoir.In natural state,the different stages of algal bloom,such as initiation,maintaining and extinction,involve in different physical,chemical and biological factors.As a result of specific environmental in which algal bloom occurs,these factors play different roles.Therefore,the mechanism of algal bloom has been thought as an important focus in phycology and aquatic ecology.There are some characteristics of microorganism in water,such as population diversity physiological and biochemical group diversity,ecological functional diversity,genetic diversity.Recent researches show that microbes have an extremely important role in the process of algae bloom,so the relationship between bacteria and algae have become the focus and hotspot.However,microbes in water have wide varieties and most of them belong to uncultured species,which make algae-bacteria relationship complex and unpredictable.Due to emerging of metagenome technology,we can study all of microorganisms in water bloom.Therefore,we can understand the ecological status of their habitats accurately and reveal the connection between community structure,function,and directional adjustment to guide microbial community function,as well as obtain microbial resources with special functions which make it possible of environmental protection and restoration.Ruxi River is one of the important tributaries of the Yangtze River in Three Gorges Reservoir area and lies in the upstream of Yangtze River.When Three Gorges Reservoir impounds,it impact hydrographic conditions in Ruxi River,resulting in rising water level,flowing slower,and accumulating ofnitrogen and phosphorus and other nutrients in the backwater reservoir and sediment.Analysis of the result of water eutrophication quality investigation showed that the Ruxi River water was at moderately eutrophication in spring and summer.Since spring 2013,we can continuously monitor outbreak of dinoflagellate bloom.In order to investigate the effect of bacteria on dinoflagellate blooms,this study investigated the structure composition of phytoplankton communities and microbial community during different stages of dinoflagellate bloom?early,middle and late state?of Ruxi River.In addition,different stages of microbial community were sequenced via metagenome to explore its function and metabolic pathways,analyze the relationship between dinoflagellates and surroundings microbes,as well as its role ofsecondary metabolites.To verify the dynamic changes of microbial secondary metabolites during the algal bloom,enzyme linked immunoabsorbent assay?ELISA?was applied to measure the content of microbial carbohydrate enzyme during algal bloom.Thus,the physiological and ecological relationship between dinoflagellate algal bloom and microorganisms was discussed and the microbial ecosystem factors that cause water bloom and fade was explored to provide information for the prediction of dinoflagellate bloom.These results were shown as follow:?1?During the period of monitoring time,the Ruxi river,Tujinxiang area broke out dinoflagellate blooms and the water was the color of soy sauce.The density of chlorophyll was64.76 mg·m^-33 and the biomass of dinoflagellate was up to 2.46*10⁶ cell·L^-11 in the river,suggesting a dinoflagellate bloom occurred.The water quality has come up to standard of middle-eutrophication at the middle stage of water bloom in Tujing area in terms of the quantity of some nutrient salt?nitrogen and phosphorus,etc.?.?2?With the burst of water bloom,the biomass of microbe is increasing along with the increase of phytoplankton and the biomass of microbe is decreasing when the water bloom fading away.The diversity of microbe community at different stage of water bloom is adverse to the water bloom intensity.The gene relative abundance of microbe indicated that the relationship of microbe is similar before and after the water bloom,but different in the middle stage of water bloom.The biomass of non-photosynthetic,anaerobic and inert bacteria,including Deinococcus-Thermus,Bacteroidetes,Chlorobi and Deferribacteres,was strikingly increased,but the biomass of aerobic bacteria or active bacteria was reduced dramatically at the middle stage of water bloom,for instance,Actinobacteria,Proteobacteria and Firmicutes,etc..?3?According to the analysis of the functional module using Eggnog and KEGG database,the results showed that the metagenome of aquatic microorganisms enriched a lot of genes concerning cellular processes,genetic information processing and signal transduction in the mid-term of water bloom,associated with metabolism of energy,amino acid,secondary metabolites and carbohydrate,cell membrane transport and cell motility,ect..At the earlier and later stages of water bloom,more diversity and higher relative abundance of genotypes were appeared compared with those at the mid-term.To some extent,the variation of functions of microbial community can reflect microbial community changes,indicating that the changes in species composition of phytoplankton,as observed during the water bloom,affected the function of each member in aquatic microbial community.?4?In order to further understand the potential regulation of microorganisms toward the process of water bloom,we analyzed concretely the effects of microorganisms on the aquatic nutrient cycle by KEGG,especially the cycles of C,N and P.The results showed that carbon fixation maintained active state during entire period of water bloom,while the regeneration process of CO₂released from organic compounds which degrade and generate energy was more active in the earlier and later periods;additionally,glutamate dehydrogenase?NADP+?,nitrous oxide reductase,and the genes of NADP+,rhodanase,nitrate reductase,and nitrite reductase?NADH?were metabolically active in the mid-term.Glutamine synthetase,carbonic anhydrase and glutamate synthease?NADPH/NADH?and the glutamate synthetase associated with Fe enriched greatly in the earlier and late periods of water bloom;the genes of guanosine triphosphate,phosphokinase and phosphate transporter,etc.regulated the P metabolism of microorganisms in water in the earlier and later periods;while the genes related to signal transduction mechanism and the proteins associated with P-starvation enriched more in the mid-term.The dominant microorganism species that affect metabolic pathways are different,but they have the similar tendency of succession and change.The microorganisms in the mid-term of water bloom includes Bacteroidetes,Proteobacteria,Spirochaetes,Deinococcus-Thermus,Cyanobacteria,Armatimonadetes and Firmicutes,etc.?5?The metabolism of carbohydrases in secondary metabolites of microorganisms by using CAZy database indicated that the relative abundance of the genes of glycosidase increased markedly in the mid-term of water bloom,while that of the genes of carbohydrate modules decreased greatly,which is consistent with the results of verification experiments which showed that the content of polysaccharide had a marked fall and glucoside hydrolase?including,polysaccharide hydrolase,glucosidase and lysozyme?rose significantly.These reflected the nutriment requirement of aquatic microorganisms during the water bloom.Therefore the polysaccharides in water were largely decomposed and used,and the concentration of the hydrolases decomposing saccharides increased greatly.In conclusion,different composition of phytoplankton communities facilitates the formation of various bacterial communities,which may play an important role in the organics flowing of ecosystem during water bloom.In turn,the variation of bacterial community composition provides feedback loop for the growth of phytoplankton,as well as stimulates the quality and quantity of secondary metabolites among phycomycetes to change.