| In recent years,algal blooms occur frequently all over the world due to eutrophication,and have been recognized as one of the most important ecological problems in aquatic environments.Algicidal bacteria play key roles in the termination of naturally occurring algal blooms.Hence,this thesis carried out systematic investigation on algicidal bacteria.Firstly,the microbial diversity and community structure in Xiangxi River,where algal blooms frequently occur,were analyzed,in order to reveal the spatial variation of microbial community in eutrophic rivers and its relationship with environmental factors.Then,algicidal bacteria against algae Chlorella vulgaris（C.vulgaris）were isolated,followed by identification of their algicidal substances.The impacts of the key algicidal substance on algal photosynthesis,antioxidant system,morphology,and ultrastructure,as well as algal metabolism,were analyzed.Based on the results,algicidal mechanisms were explored and proposed comprehensively.Finally,algicidal bacteria for another dominant algae species of algal blooms,Microcystis aeruginosa（M.aeruginosa）were also investigated.A bacterial strain with both algicidal and microcystin degradation abilities was isolated and identified.It could serve as a safe,efficient,and reliable agent for M.aeruginosa blooms and microcystin pollution.The structure and main results of the present study are as follows:1.The investigation of spatial variation in bacterial biomass,community composition and their relationship with environmental factors across a eutrophic river were performed in Chapter 2.According to the evaluation criteria of the Three Georges Reservoir,the nitrogen contents of the surface water samples were very high,with the value ranging from 1.67 to 2.34 mg/L,Xiangxi River was in the category of heavy eutrophication.Among the sampling sites,the midstream was with the highest value of chlorophyll a which was 16 mg/L.The similarity analysis（ANOSIM）,principal coordinate analysis（PCo A）,andβdiversity analysis were carried out to investigate the differences of microbial community structure,in both horizontal and vertical directions,respectively.The results showed that there were significant differences.Bacteria related to algal blooms and algicidal bacteria were detected with a significant higher percentage in the midstream than that of other sites,such as the genera Rhodobacter and Pirellula.This result was consistent with the functional prediction,where significant higher abundance of KEGG pathways was associated with algicidal substances in the midstream.In the vertical direction,some populations were with a significant higher percentage in the surface layer,such as the class Flavobacteriia,but some communities had significant higher abundances in the bottom layer,such as Betaproteobacteria and Acidobacteria.Overall,the formation of microbial community structure was influenced by multiple environmental factors.According to the result of the Mantel test,nutrient nitrogen（including TN,KN and NH4+）was found to be the most influential environmental factor.The alpha diversity index showed that microbial diversity increased with depth generally,and PO43-was demonstrated to be the most significant factor.2.The algicidal activities of algicidal bacteria against C.vulgaris and their algicidal substances were studied in Chapter 3.In this chapter,an algicidal bacterium named as M.paraoxydans strain M1 was isolated.This is the first time to report the algae-lysing effect of the genus Microcystis against C.vulgaris.The algicidal activities were conducted indirectly via secreting algicidal substances by the strain M1,and Atrazine-desethyl（AD）was then identified as the effective algicidal substance.It is also the first time to report the algae-lysing effect of AD.The algicidal rates of 2μg/m L AD（LAD）and 20μg/m L AD（HAD）were 62.51%and 88.16%,respectively,after co-culturing for 120 h.Furthermore,the results demonstrated that AD could cause chlorophyll a reduction,and influence photosynthetic gene expression,such as psb A and psb D.The results of scanning electron microscopy（SEM）and transmission electron microscopy（TEM）showed that AD could induce morphology alteration and ultrastructure damage in the algal cells.DNA was also lightly damaged according to the result of comet assay,after co-culturing for 120 h.To be more specific,cells shrunk,larger vacuole,lysosomes increase,membrane damage,disordered intracellular organelles,plasmolysis was observed gradually in C.vulgaris cells,along with the extended exposure duration to AD,and ultimately lead to algal cell lysis and dying.Furthermore,oxidative stress was induced by AD compared to the control.And it can be demonstrated by the excessive reactive oxygen（ROS）,changes in superoxide dismutase（SOD）and peroxidase（POD）activities,and the accumulated malonaldehyde（MDA）in the algicidal process.3.The impact of algicidal substance on algal metabolism was studied in C.vulgaris in Chapter 4,in order to reveal the mechanism of algicidal activities.Overall,metabolites and metabolic pathways were both significantly altered induced by the algicidal substance HAD in C.vulgaris compared to the control.In detail,the mean value of amino acids,amines,glycosides and urea were significantly lower than the control,after 24 h exposure to HAD（P<0.05）.Compared to the control,there were mainly five up-regulated metabolic pathways,including phenylalanine metabolism,arginine biosynthesis,arginine and proline metabolism,galactose metabolism,alanine,aspartate,and glutamate metabolism.The upregulation of most metabolism pathways was connected with metabolic resistance and defense against photosynthesis inhibition,oxidative stress,or partial cellular structure damage.It was indicated that the up-regulation of some metabolic pathways was a stress response to AD,as well as a self-protective strategy of C.vulgaris cells.On the contrary,there were seven down-regulated metabolic pathways after exposed to HAD for 24 h compared to the control,including glycine,serine,and threonine metabolism,glyoxylate and dicarboxylate metabolism,sulfur metabolism,glycolysis/gluconeogenesis,aminoacyl-t RNA biosynthesis,pyruvate metabolism,starch and sucrose metabolism.Among them,glycine,serine and threonine metabolism was demonstrated to be the most important down-regulated pathway in the present study.The down-regulated glycine,serine and threonine metabolism in algal cells was also a key step of algal-lysing process induced by AD.And the downregulation of other pathways further supported the idea,which were connected with glycine,serine and threonine metabolism.After exposed to HAD for 120 h,most metabolic pathways were down-regulated compared to the control.What’s more,glycine,serine and threonine metabolism was still the most important down-regulated pathway.It was worth noting that isoquinoline alkaloid biosynthesis were upregulated evidently compared to the control.The tyramine was accumulated in algal cells,which was detected as the synthetic precursor.It implied that tyramine may be a biomarker when C.vulgaris cells were influenced by AD.4.In the present study,the bacterial strains with algicidal effect against M.aeruginosa were isolated and identified in Chapter 5.The results showed that Pseudomonas sp.strain m10 and Bacillus sp.strain m15 were identified as two algicidal bacteria against M.aeruginosa,and the algicidal rates were 91.8%and89.24%,respectively,after co-culturing for 96 h.In addition,a bacterial strain with dual function named as Deinococcus sp.strain WL,was also isolated.The strain WL can control the growth of M.aeruginosa and degrade microcystin（MC-LR）in the meantime.Compared to the control,the degrading rate of MC-LR reached the maximum value of 56.13%after 68 h of treatment with the strain WL.In the algicidal test,the algicidal rate of strain WL was up to 96.18%,after co-culturing with M.aeruginosa for 120 h.At the same time,the extracellular microcystin secreted by M.aeruginosa was also degraded by the strain WL,with a degradation rate of 73.86%. |
