Study On The Synergistic Effects Of Abiotic Factors And Colony-Associated Bacteria On Microcystis Colony Formation And The Mechanisms

Microcystis is one of the most common dominant bloom cyanobacteria in eutrophic freshwaters.Many Microcystis strains can produce Microcystins,which together with the cyanobacterial cells seriously threaten human health and the survival of other aquatic animals.During natural cyanobacterial blooms,Microcystis mainly exists as multicellular-aggregated mucilaginous colonies,which are more conducive to resisting adverse external environmental conditions and maintaining a floating state.Therefore,the formation of colonies is considered to be a key factor to the outbreak and persistence of Microcystis blooms,suggesting that uncovering Microcystis colony-forming mechanisms is crucial for bloom control.Heterotrophic bacteria attached to Microcystis colonies（epiphytic or embedded）are called colony-associated bacteria（AB）,which have close interaction with the cyanobacteria and were found to participate in colony forming process.However,there are only a few reports about AB inducing Microcystis colony formation（referred to as"alga-aggregating"in the text）.Until now it is unclear if colony-inducing AB exist ubiquitously in different morphatypes of Microcystis.The synergestic effects of abiotic factors on the alga-aggregating of AB are also unknown.Dissolved organic matters（DOM）is an important component of the aquatic ecosystem,but there are no documents about DOM promoting Microcystis colony formation.In this study,AB capable of inducing M.aeruginosa NIES-843 to form colonies were screened from different morphatypes of Microcystis colonies,then the alga-aggregating characteristics and mechanisms of Methylobacterium sp.Z5,an AB of high and stable alga-aggregating ability,were investigated.The main research results are as follows.Prevalence of alga-aggregating AB in Microcystis colonies with different morphologies.A total of 21 strains with algal aggregation activity were obtained.AB with algal aggregation activity can be isolated from each Microcystis colony,indicating that the alga-aggregating of AB is universal.The induced colonies showed different morphologies,indicating that AB can affect the morphologies of Microcystis colonies.5 typical alga-aggregating AB strains were identified by 16S r DNA gene sequence analysis.They belong to Pseudomonas,Inhella,Kinneretia and Rhodobacter,respectively.Characterization of an AB strain Methylobacterium sp.Z5 being able to induce Microcystis NIES-843 to form colonies efficiently and stablely.Most of the isolated AB lost their alga-aggregating activity during subculture,which might result from the different nutritional conditions between natural environment in Microcystis colonies and culturing medium in lab.Z5 showed stable and high alga-aggregating activity.It could induce algal cell aggregation in 3 h.The alga aggregation efficiency of Z5increased with the increase of its inoculum sizes,and was relatively less affected by the algal cell density.The alga aggregation activities of Z5 cultured in LB medium were significantly higher than that cultured in R2A medium.The alga aggregating activities of Z5 were higher under illumination than in darkness,and red light promoted alga aggregation.The alga aggregation activities of Z5 were affected by N and P concentrations in the co-culture system.When N concentration was 16.98 mg/L and P concentration was 7.60 mg/L,the algal aggregation efficiency was the highest.Hydraulic disturbance significantly reduced the algal aggregation efficiency of Z5.When the disturbance speed reached 140 rpm,Z5 hardly aggregated algal cells to form colonies.The results of chlorophyll fluorescence parameters analysis showed that Z5could improve the photosynthetic electron transfer efficiency and photosynthesis efficiency of NIES-843 at the later stage of co-culture.The alga-aggregating mechanisms of Z5 were explored.（1）The relationship between the surface characteristics and components of algal and bacterial cells and the alga-aggregating activity.The hydrophobicity of algal cell or bacterial cell surface was not the main factor that determined Z5 aggregating NIES-843.Charge forces of algal and bacterial cell surface did not contribute to alga aggregation directly as determined by Zeta potential analysis.Z5 cells inactivated with paraformaldehyde lost algal aggregation activity,indicating that living cells are required for Z5 to aggregate Microcystis cells.Z5 living cells removed EPS displayed no algal aggregation activity at the initial stage of co-culture with NIES-843,but its algal aggregation activity recoverd with the extension of co-cultivation time.This result demonstrats that EPS of Z5 cells directly participated in algal aggregation process,and Z5 could use the organic matter of algal cells to grow and reproduce,and secreted EPS to aggregate algal cells.Fourier transform attenuated total reflection infrared spectroscopy（ATR-FTIR）analysis results showed that Z5 cells bound protein-like and polysaccharide-like components of DOM substances from water samples.The protein-like components on the surface of Z5 cells might be involved in algal aggregation process.3D fluorescence spectrum analysis also showed that the protein and aromatic components of Z5 EPS might participate in algal aggregation.However,proteinase K treatment of the Z5 cell surface did not affect its algal aggregation activity,which implys that the functional protein components might be decorated proteins,such as glycoprotein.（2）Cations were necessary for Z5 to aggregate NIES-843.Z5 showed the highes algal aggregation activity in the present of 1 m M Ca²⁺,which was close to the Ca²⁺concentration in the collected natural water samples.Both NIES-843 and Z5cells showed strong affinity to Ca²⁺.In addition to Ca²⁺,Mg²⁺and Fe³⁺in the common concentration range also significantly promoted Z5 aggregating NIES-843,while Na⁺had no obvious effect on the aggregation process.Mixed cations had a more significant promoting effect on algal aggregation activity of Z5.This result suggests that divalent or trivalent cations might accelerate algal aggregation by cation bridge or coordination.（3）DOM helped Z5 to aggregate NIES-843 to form large colonies.The coexistence of DOM and Ca²⁺significantly increased the size of the colony induced by Z5.Z5 at inoculum sizes of 5%（v/v）and 10%（v/v）showed the highest algal aggregation efficiencies when the DOM concentrations were 18.0 mg/L and 10.8 mg/L,respectively.DOM from different sources all promoted algal aggregation efficiency of Z5.When Z5 was added to water containing DOM,it aggregated to form larger flocs in 1 h,suggesting that DOM promoted the alga-aggregating activity of Z5 not only by increasing the growth ratio of Z5 but also by acting as a kind of"adhesive compound"to extend the accessable algal cells of Z5 cells.（4）Z5 affected NIES-843 gene expression.Using NIES-843 cultured alone as the control,the transcriptomic response of NIES-843 to Z5 was analyzed.The results showed that 54 genes were up-regulated and 77 genes were down-regulated.The differentially expressed genes mainly encode transporters of nutrient elements and proteins,cytochrome synthesis related enzymes and photosynthesis-related proteins,peptidoglycan synthesis-related proteins,polysaccharide synthesis-related proteins and cellular transcription factors.Taken the results of other physiological and biochemical research above,it can be speculated that Z5 had slight nutritional competition with NIES-843.Z5 induces algal cell aggregation by affecting algal cell morphology,growth and the secretion of extracellular polymeric substances.