Characteristics Of Exopolysaccharide And Its Impacts Mechanism On Granular Aggregation And Stability

Compared with traditional activated sludge,aerobic granular sludge and anammox granular sludge have excellent biomass retention capacity,settling performance and low sludge yield,showing great potential in wastewater treatment.Their remarkable superiority is closely related to their good aggregation ability and structural stability.Under the condition of high cell density,the information exchange and cooperation between bacteria can be strengthened,and bacterial metabolism and activity will be further improved.Currently,low stability limits the popularization and application of granular sludge technology.Exopolysaccharide has been proved to play an important role in the aggregation performance and structural stability of granular sludge.However,due to its complex structure and variable properties,the action mechanism in maintaining the high aggregation performance and structural stability of granular sludge still needs further elucidation.The composition,characteristics and three-dimensional conformation of exopolysaccharide from An GS were studied in this paper.The structure and characteristics of exopolysaccharide from An GS and AGS with different aggregation ability were compared.The effects of exopolysaccharide branched chain configuration on the aggregation performance of An GS were studied.The structure-activity relationship of branched chain and branching degree of exopolysaccharide on maintaining the stability of An GS was analyzed.The mechanism of non-filamentous sludge bulking in the process of exopolysaccharide participating in aerobic granulation was explored.The main conclusions are as follows:The gel chromatography-refractive index-multi-angle laser light scattering system was used to systematically determine the molecular conformations of different exopolysaccharide fractions(PS40,PS60 and PS80)from anammox granular sludge.The results showed that the molecular weights of the three exopolysaccharide fractions were mainly concentrated in the range of 1.0×10⁵?1.0×10⁶ g/mol,accounting for more than 50%of the total polysaccharides.The PS80 had the highest number average molecular weight(M_n)and weight average molecular weight(M_w),followed by PS60.The three exopolysaccharide fractions showed typical highly branched structure,and PS80 and PS60 displayed compact uniform spherical conformation.The monosaccharide composition and ratio of exopolysaccharide fractions are obviously different.Through spectral analysis,it was found that compared with PS40 and PS60,PS80 contained a lower content of uronic acid monosaccharide residues,as well as the proportion of polar functional groups such as carboxyl,hydroxyl,and carbonyl groups.Both the monosaccharide composition and characteristic functional groups are conducive to sludge aggregation.The molecular morphology of different exopolysaccharide fractions were obtained by atomic force microscopy.PS80 exhibited a strong self-assembly ability,presenting a dense rod-like and flower-like three-dimensional structure.The research results revealed the composition characteristics and three-dimensional conformation of different molecular weight exopolysaccharides of anammox granular sludge.The aggregation difference between aerobic granular sludge and anammox granular sludge was studied through aggregation experiments.The results showed that the aggregation ability of anammox granular sludge was better than that of aerobic granular sludge,and exopolysaccharide played an important role in the process of sludge aggregation.Combining ion chromatography,gel chromatography-refractive index-multi-angle laser light scattering system and atomic force microscopy to analyze the characteristics of exopolysaccharide in depth,it is found that An GS had a higher molecular weight and molecular branched structure,and higher mechanical strength and viscosity than AGS exopolysaccharide.By Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy,it is detected that An GS exopolysaccharide has a higher proportion of hydrophobic functional groups,intermolecular hydrogen bonds and total hydrogen bonds than AGS exopolysaccharide.The results clarified the role of exopolysaccharide in granular sludge aggregation,and revealed the differences in exopolysaccharide molecular structure and characteristics between AGS and An GS.In order to elucidate the effect and mechanismof exopolysaccharide on the aggregation performance of An GS sludge,the sludge aggregation performance and characteristics were studied after branched chain glycosidic bond of exopolysaccharide were destroyed by enzyme.Through aggregation experiment,it is confirmed that the sludge aggregation performance decreased after the destruction of exopolysaccharide branched structrue.The analysis of sludge surface characteristics showed that the hydrophobicity and zeta potential decreased,which are two key indicators affecting sludge aggregation.The atomic force microscope(AFM)observation on the molecular morphology of exopolysaccharides revealed that the self-assembly performance of exopolysaccharide molecules became weak and the aggregates structure was broken and dispersed after the enzymolysis of exopolysaccharide branched chains.Throug the XDLVO theory,it was further confirm that after the destruction of exopolysaccharide branching,the energy barrier that impeded microbial aggregation was enhanced,and the compressive double layer energy(W_R)and acid-base interaction energy(W_AB)increase with the distance decrease between microorganisms,which resulted in the decrease of spontaneous aggregation ability of microorganisms.The results explained the importance of the exopolysaccharide branched chain to the aggregation performance of An GS from the perspective of the free energy of microbial aggregates,and revealed the important contribution of exopolysaccharide branched chain configuration to the anammox sludge aggregation.The mechanism of branched chain and branching degree of exopolysaccharide in maintaining the structural stability of An GS was studied.Through selective enzymatic hydrolysis,combined with turbidity tests and scanning electron microscopy technology,it was found that An GS became disintegrated after the exopolysaccharide branch chain and branch point were cut by enzyme.With the aid of rheological theory,it was detected by the rheometer that the gel properties of An GS changed significantly,and the mechanical strength and viscoelasticity of the granular sludge were reduced after the branch chain and branch point glycosidic bonds of exopolysaccharide were digested by enzyme.The aggregation morphology of exopolysaccharide molecules was measured by atomic force microscope,which confirmed that the reduction of zeta potential and hydrophobicity hindered the cross-linking and aggregation between exopolysaccharide molecular chains after hydrolysis of exopolysaccharide branch chains,resulting in deterioration of An GS gel characteristics and disintegration of granular sludge.Spectral analysis showed that the hydrolysis of the branch points and the branched glycosides of exopolysaccharide led to a higher proportion of hydrophilic and electronegative groups in the EPS matrix,revealing the internal reasons for the deterioration of sludge surface characteristics and particle stability.This investigation clarifies the structure-activity relationship between the branched chains and branching degree of exopolysaccharide and the structural stability of An GS.Aerobic granular sludge was cultivated in three typical influent modes,including direct aeration after fast feeding(R1),anaerobic stirring after fast feeding(R2)and anaerobic plug-flow slow feeding(R3).It was found that non-filamentous sludge bulking occurred in R1 and R3,while no sludge bulking occurred in R2 during start-up stage.The mechanism of non-filamentous sludge bulking in aerobic granulation process was studied by comprehensively detecting and analyzing the reactor performance,sludge characteristics,EPS composition and characteristics,content of second messenger c-di-GMP and succession of microbial community.The succession of microbial communities showed that in the non-filamentous bulking sludge the abundance of microorganisms that can secrete polysaccharides and do not use exopolysaccharide as energy sources increased.However,the abundance of communities that can decompose and utilize exopolysaccharide was significantly reduced.Exopolysaccharide was secreted and retained in large quantities due to dynamic changes of the above bacteria.The structure and characteristics of exopolysaccharide were further analyzed,and it was found that the exopolysaccharide from the non-filamentous bulking sludge had higher molecular weight,viscosity,and hydrophilicity,and the molecular chains showed a more compact and uniform spherical conformation.Through correlation analysis,it was found that organic loading rate(OLR),second messenger c-di-GMP,exopolysaccharide content and sludge volume index(SVI)were significantly correlated with each other(p<0.05)The decrease in zeta potential and hydrophobicity of sludge surface and the increase in hydrophilicity are important reasons that affect the aggregation and compression of sludge and cause the non-filamentous sludge bulking.The increase of sludge surface hydrophilicity was mainly caused by the increase of hydrophilic exopolysaccharide content.Therefore,microorganisms secrete large amounts of second messenger c-di-GMP as the driving force under high OLR selective pressure,which affected the surface characteristics of sludge by regulating the metabolism of exopolysaccharide,thus worsening the sludge aggregation,and leading to non-filamentous sludge bulking.The results can provide theoretical guidance for the smooth progress of aerobic granulation and avoiding the non-filamentous sludge bulking.