Study On The Extracellular Polymeric Substances Of Benthic Diatoms

Biofouling causes enormous economic loss in the global scope,and is a difficult problem to solve in the marine transportation,coastal engineering and other production activities.It was found in many studies that biofilms formed by the microorganisms,such as marine microalgae and bacteria,induced the adhesion of macro-organism fouling.Diatoms are the main organisms that form biofilms in visible light in the ocean.Therefore,the important strategy to prevent the adhesion of macro-organism is to stop the adhesion of diatoms Extracellular polymeric substance(EPS)of diatom is the key substance for the diatom adhesion and formation of biofilms.It could be devided into Soluble EPS(SL-EPS)and Bound-EPS(B-EPS).The relationship between EPS and the adhesion of diatom was studied by analyzing the charateristics of EPS extracted from Amphora sp.The results are as follows:1.High-speed centrifugal method.and formalin-sodium hydroxide treatment was selected to extract EPS in further study.The SL-EPS,soluble in water,was extracted by high-speed centrifugal method.For B-EPS,we compared four commonly used methods,including water bath treatment at 30? or 70?,formalin-sodium hydroxide treatment,and cationic exchange resin treatment,to extract the bound EPS of Amphora sp.The results showed that the m ain components of bound EPS varied with the extraction methods,among which the range of DNA content changed the most,the next were protein,sugar and uronic acid,the least was sulfonic acid.By comparing each substance content and the total amount in obtained B-EPS by different methods,it was found that the extraction efficiency by low temperature water bath(30?)was relatively low while the water bath treatment at 70? led to the hydrolysis and denaturation of proteins,polysaccharides and other substances,which will affect the further analysis of EPS.The cationic exchange resin treatment showed a good extraction efficiency but caused serious damages to cells.The formalin-NaOH method obtained the maximum total EPS and the highest concentrations of total sugar and glucuronic acid without causing cell rupture.Therefore,the formalin-NaOH treatment was selected to extract the bound EPS in further study.2.The concentration of phosphorus will impact the growth and substance of the diatom.Phosphorus is an important factor that restricts the growth,reproduction and biofilm formation of algae.Diatom EPS is closely related to the formation of biofilm.Therefore,the EPS composition and content for diatom Amphora sp.affected by phosphorus was investigated.In this study,we used five differen concentrations of phosphorus in modified f/2 mediums,including 1/4,1/2,1,3/2,and 2 folds of original P concentration in the standard f/2 meidium.It was found that the contents of intracellular substances,including proteins,polysaccharides,sulfate groups,and lipids,in each cell decreased,but the content of uronic acid increased,with the increase of phosphorus concentration in the culture medium.The results indicate that the content of the polysaccharide,uronic acid and sulfate groups in SL-EPS decreased with the increase of phosphorus concentration.At the same time,formalin-NaOH treatment was employed to extract the bound EPS,The content of each component in B-EPS was affected by phosphorus concentration.The contents of protein and sugar were decreased persistently while the contents of uronic acid and sulfate were increased at the biginning and then decreased with the increase of phosphorus concentration.The results indicate that the increase of phosphorus concentration slightly promoted the acculumation of soluble EPS in the culture with only a 43.4?g difference between the highest and lowest groups,while it significantly promoted the total amount of bound EPS in the culture with the difference of 1.8 folds between the highest and lowest groups.Many studies reported that SL-EPS and B-EPS can be transformed into each other,it's necessary to analyze the total amount of the EPS.We found that the total EPS production of the culture increased,although the EPS production of each cell of Amphora sp decreased,with the increase of the phosphorus concentration.3.EPS is closely related to the adhesion strength of diatoms,so the microfluidic method was employed in this study and used to test the adhesion strength of Amphora sp.cells obtained under different phosphorus concentrations.In the stability test of peristaltic pump used in present experiments,the linear equation of the relationship between the pump speed and the shear force of water flow was obtained,and the R2 of which reached 0.998,indicating that the pump had good stability.By testing the effect of the settling time of diatoms on the adhesion strength,it was found that under the same phosphorus concentration,the adhesion strength of diatom cells in the 24h group was higher than those in the 2h group.Therefore,the data measured at 24 h settlement was used in this study for the analysis of effect of phosphorus concentration on the cell adhesion.The results showed that the higher concentration of phosphorus led the stronger adhesion of Amphora sp.cell.In detail,the ?50 in the treatment of 1/4P,1P,and 2P were detected as 370.2dyn/cm2,1165.1 dyn/cm2,and 1345.0 dyn/cm2,respectively.4.As an important component of EPS,the polysaccharide plays an important role in the diatom cell adhesion.The peak time of monosaccharides in HPLC profile were determined by internal standard using 11 standard monosaccharides and the order of peak in mixed sample were Man,Glc-N,Rha,Glc-A,Gal-A,Glc-Nac,Glc,Gal,Xyl,Ara,and Fuc.The result shows that SL-EPS consists of 10 kinds of monosaccharides while B-EPS consists of 8.In SL-EPS,the dominant monosaccharides are Glc-Nac(29.01%)and Gal-A(19.90%)in 1/4P group,Glc-A(25.68%),Gal-A(20.55%)and Xyl(21.18%)in 1P group,and Glc-A(35.24%)and Xyl(26.66%)in 2P group.In the cases of B-EPS,the dominant monosaccharides are Gal-A(35.85%)and Xyl(19.91%)in 1/4-fold group,Xyl(27.30%)and Glc-N(21.65%)in 1P group,and Glc-N(27.46%)and Xyl(27.47%)in 2P group.It was found that the molar ratioes of Glc-N,Glc-A and Xyl increased in both SL-EPS and B-EPS while Rha,Gal-A and Fuc in B-EPS decreased with the increase of phosphorus concentration.The increase of the molar ratio of Glc-N,Glc-A and Xyl and the decrease of the Rha,Gal-A and Fuc in B-EPS might promote the increase of the adhesion strength of diatom cells.In summary,four kinds of B-EPS extraction methods were compared in this study,and formalin-NaOH method,which is most suitable for the extraction of B-EPS from Amphora sp was proposed.The influence of phosphorus concentration on the intracellular substances,SL-EPS and B-EPS of Amphora sp.was discussed.By comparing the EPS profiles with the adhesion strength of diatom cells,it was found that the yield of EPS per cell was inversely proportional to the adhesion strength,and the total amount of EPS in the culture was proportional to the adhesion strength.With the increase of phosphorus concentration,the adhesion strength increased.The molar ratioes of Glc-N,Glc-A and Xyl in both SL-EPS and B-EPS increased,and the content of Rha,Gal-A and Fuc in B-EPS decreased.It is proposed that the increase of total EPS in the culture and the change of molar ratios of above 6 monosaccharides might be related to the increase of adhesion strength of diatom cells.