| Microbial corrosion depends on the formation of biofilm,and the formation of biofilm starts with the adhesion of bacteria on the surface of the material,so it is very important to study the adhesion mechanism in depth.In this study,the key extracellular polysaccharides and proteins in the process of adhesion between sulfate reducing bacteria and Q235 carbon steel surface are discussed at the levels of cytology and molecular biology,and the adhesion characteristics and mechanism of the bacteria are further studied.The main content and results are as follows:(1)The SRB flora was analyzed by 16S rRNA sequencing,which mainly included five kinds of bacteria:Macellibacteroides,Desulfovibrio,Enterobacteriaceae,and Pseudomonas,Tissierella.In the planktonic state,Macellibacteroides and Enterobacteriaceae accounted for57.93%and 21.64%,respectively,and Desulfovibrio was only 0.24%.However,Desulfovibrio(accounting for as high as 35.32%at 48h)gradually dominated and played an important role in the adhesion of the carbon steel surface.Secondly,fluorescence microscopy was used to describe the adhesion law in the interaction system of SRB flora and Q235 carbon steel:After immersing for 1d,a small amount of bacteria were scattered on the surface of the carbon steel;After immersing for 2d,the bacteria grew steadily and the extracellular polymer(EPS)was produced;After immersing for 3-4d,the bacteria multiplied and more EPS was covered on the surface;By the 5th day,a complex three-dimensional biofilm structure was formed;As the environment in the biofilm was not conducive to the survival of the bacteria,the bacteria were separated from the biofilm and returned to the planktonic state..(2)By adding 0.25%trypsin and 0.05mol/L EDTA to the interaction system of SRB flora and carbon steel,the adhesion and aggregation of bacteria were affected by destroying the protein,chelating metal ions and inhibiting the production of EPS on the surface of carbon steel.These results indicated that Ca2+-dependent proteins might play an important role in the adhesion process of SRB flora.This type of protein might be a lectin present on the surface of bacteria.Secondly,mannan and dextran were used to coat the surface of carbon steel.The conditioned film provided more binding sites for bacteria to adhere to the surface of carbon steel,so more bacteria adhered to the surface of carbon steel.The results showed that mannan and glucan can adhere to the flora,and it was speculated that the lectin-mannan(dextran)system has played an important role.(3)To verify the above hypothesis,the uncoated ELISA plate was used as a blank control,and the ELISA plate was coated with 0.1 mol/L mannan,100 mg/m L dextran,and 1 mg/m L lectin ConA,respectively,to analyze the effect of single factor on the adhesion of SRB flora.The results showed that the coated ELISA plate was more favorable to the adhesion of bacteria than the blank control.Exogenously added trypsin and EDTA inhibited bacterial adhesion in a concentration-dependent manner.Exogenously addition of free ConA exhibited a complex effect in polysaccharide-flora adhesion,and the inhibitory effect decreased with increasing concentration in the range of 1×10-4-1mg/m L;Exogenously added polysaccharides also showed a more complex performance in the adhesion of ConA-flora.When the mannan was in the concentration range of 1×10-5-0.1mol/L and the dextran was in the concentration range of0.01-100mg/m L,the low concentration showed an inhibitory effect.As the concentration increased,the inhibitory effect weakened and appeared a small degree of promotion.After confirming that the above-mentioned single factor was involved in SRB flora adhesion,the EPS of the flora(polysaccharide content 56.98?g/m L,protein content 46.23?g/m L)was extracted to verify whether it can participate in flora adhesion.The results showed that the number of bacteria adhered to the EPS-coated ELISA plate increased,but the promotion degree was not high due to the low content of EPS components.The experimental group of trypsin and EDTA showed the inhibition phenomenon as mentioned above.Exogenously added mannan in the range of1×10-10-1×10-8mol/L,which was continuously inhibited as the concentration increased;In the range of 1×10-8-1×10-1mol/L,it was continuously promoted as the concentration increased.Exogenously added dextran in the range of 1×10-7-1×10-5mg/m L,which was continuously inhibited as the concentration increases;In the range of 1×10-5-100mg/m L,the adhesion of SRB flora was continuously promoted as the concentration increased.(4)The above experimental results were preliminarily verified on the surface of carbon steel to detect whether mannan and dextran coated on the surface of carbon steel can participate in the adhesion of SRB flora.The results proved that the presence of polysaccharides promoted the adhesion of flora on the surface of carbon steel.Trypsin and EDTA both inhibited the adhesion and aggregation of bacteria on the surface of carbon steel.Comprehensive analysis of the results of MTT staining and fluorescence observation further verified that the mutual combination of polysaccharides,lectins,and Ca2+played an important role in bacterial adhesion,mutual aggregation and the development of biofilms. |
PDF Full Text Request