Mechasim On Biofilm Formation And Its Application In Sequencing Batch Biofilm Reactor By Pseudomonas Stutzeri XL-2

Sequencing batch biobiofilm reactor?SBBR?is a bioreactor based on biofilm carrier degradation of pollutants,which is stable,energy saving and efficienty.However,the formation of biofilms tends to be slow,which restricts the application prospects of SBBR processes.Some studies shown that biofilm-forming bacteria play an important role in the formation of biofilms due to large amount of extracellular polymeric substances?EPS?.Thorough study of the biofilm formation mechanism can provide a reference for the rapid start-up of SBBR and maintain high-efficiency wastewater treatment capacity.The paper used a biofilm-forming ability strain called Pseudomonas stutzeri XL-2as a research object,through surface thermodynamic analysis,extended DLVO theoretical calculation,FT-IR and 3D-EEM,CLSM,XPS and other methods to investigate the role of EPS in the biofilm formation.The regulation of EPS biosynthesis-related genes and signaling molecules on EPS was discussed.Furthermore,the carrier was pretreated with strain XL-2 and then added to SBBR.The research results provided a theoretical reference for understanding the biofilm formation mechanism of biofilm-forming bacteria and its application in biofilm reactors.The conclusions reached in the paper are as follows:The composition and effect of EPS in P.stutzeri XL-2 biofilm formation were studied.The results showed that:?1?P.stutzeri XL-2 had biofilm formation ability and EPS synthesis ability.The mainly EPS component was protein and mainly composition was TB-EPS?Tightly Bound EPS?.The distribution of proteins was more uniform and tightly bound to the cell on the biofilm.?2?Protein in EPS and biofilm formation had a significant linear correlation.The biofilm formation was decreased from 2.13 to 1.57when enzymatic the protein.In the EPS composition,the TB-EPS removement reduced the biofilm-forming capacity from 60.6%to 44%,and the biofilm-forming capacity was restored to 50.8%after the addition of the extracted TB-EPS.After removing the S-EPS and LB-EPS,the biofilm-forming capacity was reduced to 52.8%and 57.5%,respectively.It revealed that the contribution rates of proteins and polysaccharides in TB-EPS to the biofilm-forming capacity were 69.2%and 29.0%after inhibiting the proteins and polysaccharides,respectively.?3?.EPS converted acid-base interactions into attractiveness,which was manifested as more hydrophobic.TB-EPS contained more hydrophobic groups and protein secondary structures that promote microbial aggregation.Proteins contain more hydrophobic amino acids.The biosynthesis-related genes of EPS and their signal regulation were studied.The results show that:?1?The genome of P.stutzeri XL-2 had 43 scaffolds and 4499ungiene were assembled and get 16009 functional annotations,there were 11 categories that may be related to EPS synthesis.There are 6 groups associated with EPS synthesis in the largest 20 annotated secondary pathway groups,and there were more secondary metabolic pathways for proteins than polysaccharides.Corresponding 83 biosynthetic genes were found in the P.stutzeri XL-2 genome.?2?P.stutzeri XL-2 produced short acyl chain AHLs,long acyl chains AHLs and AI-2 during culture.The highest concentrations of C₆-HLS,C₆-oxo-HLS and C₈-HLS produced by P.stutzeri XL-2 were determined by LC-LC/MS method to be 0.628?g/L,0.45?g/L and 0.05?g/L,respectively.The concentrations of C₆-HLS and C₆-oxo-HLS were significantly correlated with the contents of EPS protein and TB-EPS protein,but not correlated with EPS and polysaccharide.By adding exogenous sources,it was found that C₆-HLS and C₆-oxo-HLS with certain concentration thresholds can stimulate the production of TB-EPS protein by P.stutzeri XL-2.The research on the enhanced application of P.stutzeri XL-2 in SBBR showed that:?1?The biocarrier was prepared by XL-2 for two weeks,and biocarrier prepared by activated sludge was under the same conditions.The biofilm weight,biofilm thickness and EPS produced by the P.stutzeri XL-2 were more than activated sludge.?2?The P.stutzeri XL-2 modified biological carrier and the activated sludge modified biological carrier were separately added to SBBR1 and SBBR2,and it was found that the carrier of SBBR1 was basically completed on the 33 d,and SBBR2 was not completed until the42 d.The removal rate of pollutants increased with the increase of biofilm.The biofilm weight and biofilm thickness on the carrier in SBBR1 were greater than SBBR2 during the entire filming process.At the same time,the biofilm produced by SBBR1 also has higher EPS content than SBBR2.The P.stutzeri XL-2 modified biocarrier can accelerate the biofilm loading process of SBBR and enable the reactor to start more quickly.?3?It was found that the adhesion ability of P.stutzeri XL-2 bacteria on the surface of the carrier was better than that of activated sludge.So,it was found that the P.stutzeri XL-2 biofilm could adhere more microorganisms than the activated sludge biofilm.The good adhesion of P.stutzeri XL-2 may be related to the secretion of a large amount of EPS.The relative abundance of P.stutzeri XL-2 in SBBR1 was 6.7%,while SBBR2 was not detected.It was demonstrated that P.stutzeri XL-2 survived in the reactor to promote biofilm formation.In addition,the sum of the relative abundance of bacteria in SBBR1 which had ability of biofilm-forming or producing EPS was 45.1%,which was higher than SBBR2.It was indicated that the EPS produced by P.stutzeri XL-2 can also attract microbes that are favorable for biofilm formation and accelerate the formation of SBBR biofilm.