Formation Of Extracellular Polymeric Substances Conditioning Layers And Its Regulation Mechanisms During Biofilm Formation

Biofouling is a worldwide ubiquitous phenomenon and a problem of major concern in a wide variety of industries.Taking effective measures to prevent biofouling is of great importance in order to elongate the service duration of industrial facilities,conserve energy,decrease emissions,promote medical and food safety,etc.A large amount of research has been devoted to the prevention of biofouling and corresponding antifouling strategies have been developed.However,due to the complexity of biofouling,there are still obstacles in the development of antifouling strategies with wide inhibitory spectra.Therefore,understanding the mechanisms of biofouling will provide the foundation of new antifouling methods.The adsorption of organic molecules(the formation of conditioning layers)onto material surfaces is the initial step of biofouling.Conditioning layers may significantly influence microbial adhesion and biofilm formation.This work exploited the extracellular polymeric substances(EPS)of Bacillus subtilis as the source of the condition layers.The morphologies of the pre-adsorbed EPS conditioning layers were characterized using atomic force microscopy(AFM)and the formation of conditioning layers was quantified.The results showed that EPS of B.subtilis pre-adsorbed on smooth surfaces formed porous layers or interconnected networks,depending on the concentration of the EPS solutions.The protein content of the EPS conditioning layers increased with increasing EPS concentration,but a linear correlation was not observed due to the heterogeneity of the EPS layers.In this work,the adhesion morphologies of B.subtilis and Escherichia coli were characterized using field emission scanning electron microscopy(FESEM)and biofilm growth was measured using crystal violet staining.Bacterial adhesion and cluster formation of B.subtilis were significantly promoted by the EPS conditioning layers and a positive correlation was observed between bacterial adhesion and EPS concentration.On the other hand,bacterial cluster formation of E.coli was deterred by the EPS conditioning layers,although the numbers of sessile cells were not notably decreased.This is an indication that the EPS of B.subtilis compromised the gathering and aggregating abilities of E.coli,thus rendering it more susceptible to environmental stress.To further explore the underlying mechanism of how EPS conditioning layers affected the bacterial adhesion,aggregation and biofilm formation of E.coli,the respective influence of two major constituents of B.subtilis EPS: exopolysaccharide ?-polyglutamic acid(?-PGA)and antibacterial peptide surfactin,on E.coli adhesion and biofilm formation was investigated.?-PGA conditioning layers showed no effect on the bacterial adhesion and biofilm formation of both B.subtilis and E.coli.Surfactin conditioning layers significantly promoted the extracellular matrix secretion of B.subtilis in certain areas,thereby playing an important role in facilitating the adhesion and aggregation of B.subtilis.In addition,surfactin conditioning layers diminished the size of bacterial clusters and the number of adherent cells of E.coli.Therefore,surfactin is the main reason why B.subtilis EPS promoted B.subtilis biofilm formation and inhibited E.coli aggregation.This work discussed the possible mechanisms of the influence of EPS on bacterial adhesion and provided a new route for designing biological antifouling methods.