Effects Of AHLs On Pseudomonas Aeruginosa Biofilm Formation And Its Mechanism

Acyl homoserine lactones(AHLs)signal molecules are ubiquitous in bacteria in wastewater biological treatment system.They can not only act in the cell,but also regulate the physiological behavior of cells outside the cell.Pseudomonas aeruginosa is the most common microorganism in activated sludge and has the ability to degrade pollutants.It is easy to form biofilm,and it has been confirmed that its biofilm formation is regulated by a variety of signal molecules.In recent years,the formation process and structural components of Pseudomonas aeruginosa biofilm have been reported,but few scholars pay attention to the effect of signal molecules,especially the exogenous addition of AHLs signal molecules on the biofilm formation of Pseudomonas aeruginosa.In this study,four typical AHLs signaling molecules were selected from Pseudomonas aeruginosa 0.1 μM concentration addition.The effects of C4-HSL,C8oxo-HSL,C12-oxo-HSL and C12-HSL on the film-forming properties,surface properties,EPS and PS characteristics of the strains were studied.The gene changes of the strains were analyzed by prokaryotic transcriptomics,and the differential genes were analyzed,In order to reveal the influence mechanism of AHLs on Pseudomonas aeruginosa membrane forming ability,the research results can provide theoretical support for the formation of microbial aggregates in wastewater biological treatment.The main conclusions are as follows:① After adding AHLs,the film-forming ability,growth ability and self aggregation ability of the strains were significantly improved(p<0.05),and the surface characteristics such as hydrophobicity,contact angle and zeta potential were significantly increased.More EPS wrapped the strains,the biofilm structure was rougher,the electrostatic force action energy ΔGadh,the adsorption free energy and Lewis acid-base free energy are lower,and the strains are easier to agglomerate.Compared with C12-oxo-HSL and C8oxo-HSL,C12-HSL and C4-HSL had better film-forming performance,stronger surface properties,more EPS bonding and rougher biofilm structure,lower electrostatic force energy,higher adsorption free energy and Lewis acid-base free energy,and easier adhesion and aggregation.The results showed that AHLs with no substituents in the side chain had better effect on the film-forming performance and surface characteristics of the strain,and the effect of whether AHLs had carbonyl substituents in the side chain on the film-forming performance and surface characteristics of the strain was greater than that of AHLs with different side chain lengths.② After adding AHLs,the content of EPS,zeta potential and zeta potential of PS increased significantly(p<0.05).The results of Fourier transform infrared spectroscopy(FTIR)and X-ray photoelectron spectroscopy(XPS)showed that the relative proportions of O-H,C-H,C=O,hydrogen bonds and β-sheet structures in EPS and O-H,O-C=O,CH,α-glycosidic bonds and hydrogen bonds in PS had increased.The results of scanning electron microscope(SEM)showed that the structure of PS was more rough and glossy,which was conducive to the formation of biofilm by EPS and PS bridging.Compared with C12-oxo-HSL and C8-oxo-HSL,the PN content,PS content,zeta potential of EPS and PS of C12-HSL and C4-HSL were higher,and the zeta potential of EPS and PS was higher β-Folding structure α-Polysaccharides,hydrogen bonds,O-C=O and C-H ratio increased,PS structure was more porous and highly branched,PS gel characteristics were stronger,and more conducive to the formation of biofilms through PS cross-linking.The results showed that AHLs with side chains without substituents had better effects on EPS and PS characteristics,and the presence of carbonyl substituents on side chains had greater effects on EPS EPS and PS characteristics than AHLs with different side chains.③ Principal component analysis(PCA)and differential expression gene analysis showed that the distance between the control group and the AHLs group was far,which indicated that the effect of AHLs on gene expression was different.The distance between C12-HSL group,C4-HSL group and C12-oxo-HSL group,C8-oxo-HSL group was far,which indicated that the influence of carbonyl substituents in the side chain of AHLs on gene expression was different.The distance between C12-HSL and C4-HSL,C12-oxoHSL and C8-oxo-HSL were close,and the number of up-regulated/down regulated genes were small and close,which indicated that AHLs with different side chain lengths had little effect on the expression of genes.Compared with the control group,the amino sugar/nucleotide sugar metabolism,lipopolysaccharide,homologous recombination and other pathways were up-regulated after adding C12-HSL and C12-oxo-HSL,which indicated that the strain could secrete more EPS,PN and PS,and the released Edna combined with PSL to participate in the formation of biofilm and the maintenance of structural stability.The degradation of valine/leucine/isoleucine and the down-regulation of the biosynthesis pathway of non ribosomal peptide of the iron carrier group indicated that the growth,activity and hydrophobicity of the strain were enhanced,which was conducive to the enhancement of film-forming ability in response to signal molecules.Compared with C12-oxo-HSL,the metabolism of arginine and proline,pyruvate and glycolysis/gluconeogenesis were down regulated after adding C12-HSL,which indicated that the content of acetyl coenzyme a synthesized and transformed by the strain was reduced,and the energy consumption was reduced,which was conducive to the formation of biofilm.The results showed that the effects of carbonyl substituents on gene expression and metabolic pathway were different.AHLs without substituents were more conducive to biofilm formation and consumed less energy.