Role Of Extracellular Polysaccharide And Associated Bacteria On Colony Formation In Microcystis

Harmful cyanobacterial blooms caused by the eutrophication of water bodies have become a global environmental and public health concern.Microcystis is one of the most commonly known bloom-forming cyanobacterial species in lakes and reservoirs worldwide.Microcystis produces extracellular polysaccharides(EPS)during growth,including the capsular polysaccharide which bound to the cell surface and the released polysaccharide which released into the surrounding environment.It has been reported that the formation of Microcystis colonies is related to the secretion of the capsular polysaccharide.It is well known that,under natural conditions,Microcystis occurs mainly as colonial aggregates constrained by mucilaginous matrix.However,during long-term cultivation under laboratory conditions,the characteristics of colonies disappear,and Microcystis exists mainly as single cells.The formation of the colony is one of the key factors for the Microcystis to gain competitive advantage and form blooms.Large amounts of bacteria have been found to be embedded in the mucilage layer of Microcystis colonies.Bacteria associated with Microcystis may play an important role in formation of Microcystis colonies and in development of cyanobacterial blooms.The aim of this study was to explore the role of extracellular polysaccharide and associated bactiria on colony formation in Microcystis through investigating the physicochemical properties of extracellular polysaccharides from the selected unicellular and colonial Microcystis,the reaggregation ability of Microcystis,and the relationship between the autoaggregation and adhesion abilities of bacteria associated with Microcystis colonies.The physicochemical properties of EPS are decisive factors affecting the formation of cell sheaths and the formation of Microcystis colonies.This paper analyzed the physicochemical properties of EPS from three common colonical Microcystis species(Microcystis wesenbergii,M.aeruginosa and M.flos-aquae).Dissolved extracellular organic matter(d EOM),dissolved EPS(d EPS)and bound EPS(b EPS)of all the Microcystis strains had negative zeta potential values at p H 7,and the absolute value of zeta potential of extracellular organic matter(EOM)could be ranked in an ascending order as follows: b EPS < d EPS < d EOM.The lower zeta potential(absolute value)of cell surface b EPS,the weaker repulsive forces between Microcystis cells,and coagulation and aggregation were more likely to occur,which is conducive to colonial formation.For each Microcystis strain,specific ultraviolet absorbance(SUVA)of b EPS was much higher than that of d EPS.Results of fluorescence excitation-emission matrix(EEM)spectrum indicated that EOM(d EOM,d EPS and b EPS)contain humic-like substances and protein-like substances.Hydrophobicity analysis using XAD resin fractionation indicated that the hydrophobicity could be ranked in an order as follows: b EPS > d EPS > d EOM for all the Microcystis strains.For each Microcystis strain,the proportion of hydrophilic(HPI)fraction in d EOM was higher than that of hydrophobic(HPO)fraction,and the proportions of HPO fraction in d EPS and b EPS were higher than those of HPI fraction.Correlation analysis showed that there was a statistically significant correlation between the amounts of carbohydrate and dissolved organic carbon(DOC)in the HPO fraction of EOM(d EOM,d EPS and b EPS),indicating that the hydrophobicity of Microcystis EOM might be related to carbohydrate.According to the data on zeta potential and hydrophobicity,b EPS would have greater hydrophobic adhesion ability than d EPS.b EPS,which bound to the cell surface,affected the hydrophobicity of cells.The stronger hydrophobicity of b EPS suggested the stronger hydrophobicity and adhesion ability of the Microcystis cells,which was more beneficial to the cells aggregation to form colonies.The coagulation removal efficiency of b EPS and d EPS of colonial Microcystis was found to be highly dependent on p H value,and it reached the highest at p H 5.For each colonial Microcystis strain,the coagulation efficiency of b EPS was higher than that of d EPS within the p H range from 3 to 10.The highest removal efficiency of b EPS and d EPS was 47.0%-80.6% and 25.7%-46.3%,respectively.This indicated that the coagulation removal efficiency of Microcystis EPS was relatively low.The lower zeta potential(absolute value)and stronger hydrophobicity of b EPS on the cell surface might be an important factor leading to the aggregation of Microcystis cells into colonies.In order to study the characteristics of cell aggregation in Microcystis colonies,this paper analyzed the reaggregation ability of colonial Microcystis.Pretreatment with sodium periodate or urea decreased the reaggregation ability of the colonial Microcystis significantly,decreased by 19.5%-92.3% and 21.3%-43.0%,respectively.When the EPS was extracted with 0.05% Na Cl at 60 °C for 30 min,the reaggregation ability of the colonial Microcystis reduced,by 21.9%-48.8%,indicating that EPS played an important role in the reaggregation.Cell hydrophobicity decreased after pretreatment with sodium periodate or urea,or removal of EPS,decreased by17.8%-55.5%,12.8%-25.4% and 48.0%-77.7%,respectively.This finding indicated that cell surface carbohydrates and proteins might affect the reaggregation ability of the colonial Microcystis by affecting cell hydrophobicity.At the 1 m M Ca2+,the reaggregation ability of the colonial Microcystis was enhanced,by 20.1%-44.1%.The results of reaggregation experiments showed that carbohydrates and proteins in EPS,and calcium cations in the water environment might affect the formation and maintenance of the Microcystis colony.It has been reported that bacteria associated with Microcystis may play an important role in formation of Microcystis colonies.Adhesion to Microcystis cells is a requirement for colonization and long-term association of bacteria with Microcystis colonies.However,up to now,there has been no report about the mechanism related to the attachment of Microcystis-associated bacteria to Microcystis colonies.This study explored the relationship between autoaggregation ability and adhesion ability of Microcystis-associated bacteria,and to evaluate the effects of different environmental factors on their autoaggregation abilities.Twelve bacterial strains were isolated from colonies of three Microcystis morphospecies.All the bacterial isolates exhibited autoaggregation abilities ranged from 2.5% to 96.6%.The adhesion abilities of 12 bacterial strains ranged from 0.2 to 4.4(OD560/OD600).A positive correlation was found between autoaggregation ability and adhesion ability.Two selected bacterial isolates,HJX5 and HJX9,exhibited strong autoaggregation abilities at p H from 3 to 11,ranged from 60.3% to 80.4%,and from 25.0% to 69.8%,respectively.Na Cl,KCl,Ca Cl2 and Mg Cl2 at 10-100 m M significantly decreased the autoaggregation ability of HJX5,by 8.4%-26.2%,4.1%-23.7%,11.8%-31.9% and11.9%-33.2%,respectively.The autoaggregation ability of HJX9 was significantly decreased by 1-100 m M Na Cl and KCl,by 4.7%-21.5% and 4.1%-19.4%,respectively;and was significantly decreased by 0.5-100 m M Ca Cl2 and Mg Cl2,by12.6%-20.7% and 12.6%-21.9%,respectively.Removal of EPS decreased autoaggregation abilities of these two bacterial strains significantly,indicating that EPS can play an important role in autoaggregation.The autoaggregation ability of Microcystis-associated bacteria may be an important trait contributing to their attachment into Microcystis colonies.