The Effect Of Metal Ions On The Extracellular Polymeric Substances Of Waste Activated Sludge And The Impact On The Anaerobic Fermentation With Lysozyme-triggered Cell Lysis

Pretreatment of waste activated sludge（WAS）for solubilization can effectively shorten the period of anaerobic fermentation and increase the yield of short chain fatty acids（SCFAs）.Lysozymes cause cell lysis and release of intracellular substances through hydrolysis of microbial cell walls.The encapsulation effect of extracellular polymeric substances（EPS）in sludge flocs hinders the transfer of lysozyme to the pellet.The hydrolysis and breakage of microbial cells are inhibited,reducing the efficiency for lysozyme WAS pretreatment and increasing the cost.Based on the research about EPS structure,this thesis 1)investigates the impact of metal ions of different valences in sludge on the composition and structure of EPS;2)reveals the mechanism of the effect of EPS structure and sludge properties on lysozyme lysis of WAS and subsequent SCFAs production;and 3)establishes a strategy to enhance WAS solubilization and anaerobic acid production based on the function and localization of metal ions in EPS structure.To clarify the functional localization of metal ions in the EPS structure,this study examined the presence of common metal elements in WAS in sewage,analyzed the effects of different metal elements on the EPS component structure,clarified the influence of metal ions on changes the structure of EPS components as well as the sludge flocs properties,and proposed the localization and function of metal ions of different valences in the EPS structure.The results showed that Na⁺,Mg²⁺,and Ca²⁺distributed in a gradient from the outer to the inner layers of the sludge flocs,while Al³⁺was mainly captured and intercepted by the outer layer LB-EPS.Na⁺did not interact with the main components of WAS EPS,contributing to balancing the osmotic pressure between the liquid and solid phases of WAS.Divalent cations such as Mg²⁺and Ca²⁺played a decisive role in EPS structure.Divalent metal ions could spontaneously（ΔG<0）bind to peptide bonds and carboxyl groups in proteins from the thermodynamic perspective,connecting large molecular proteins while causing an entropy increase in the EPS component structure.This could promote the formation of a steric network structure of EPS,and enhance the structural strength.At the same time,the protein secondary structure became more compact,the degree of polysaccharide cross-linking increased,and the hydrophilicity of the EPS structure improved.The presence of Al³⁺had a negative effect on the EPS structure,as Al³⁺thermodynamically spontaneously interacted with EPS components i with entropy increase as the main driving force.Na⁺slightly increased the hydrophobicity of sludge flocs,reduced the particle size of sludge floc,significantly increased the CST value,and deteriorated the flocculation and dewatering properties of the sludge.Ca²⁺and Mg²⁺significantly reduced the Zeta potential,CST value,and hydrophobicity of sludge,increased the sludge floc particle size,and improved the uniformity of particle size distribution.In the presence of Al³⁺,the double layers of WAS were compressed,the Zeta potential and CST value were significantly reduced.The median particle size in the sludge increased by 46%and the hydrophobicity was greatly increased.As a result,the flocculation and dewatering properties of the sludge were significantly improved.Common anions in sewage,such as NO₃^-,Cl^-,and SO₄^2-,have no significant effect on the presence and status of cations in sludge flocs and the EPS structure.Based on the function and localization of metal ions in the EPS structure,this study investigated the effects of different metal ions on the efficiency of lysozyme lysis of WAS and reveals the mechanism of how EPS structure and sludge properties influence the lysozyme lysis process in the presence of metal ions.A strategy was proposed for relieve the inhibition of multivalent cations on the lysozyme lysis.The results were as follows:The efficiency of lysozyme lysis of WAS with different metal ions varied significantly under the low dosage（5%,w/w）,ranking as Na>CG>Mg≈Ca>Al.In the presence of Na⁺,the net increase in SCOD of supernatant after 4 h of sludge lysis reached 2813.44 mg/L,while it was only 857.68 mg/L with Al³⁺.The increase of proteins in the supernatant was48.79±2.33 mg/L in the presence of Na⁺,making them the main contributors to SCOD.Multivalent metal ions such as Ca²⁺,Mg²⁺,and Al³⁺neutralized the negative charge on the sludge surface.And the specific surface area of the sludge particles reduced.Hence,it’s difficult for positively charged lysozyme molecules in the liquid phase to be adsorbed and captured by the sludge.At the end of pretreatment,29.08-40.56%of the lysozyme remained in the supernatant and could not exert a catalytic lysis effect within the sludge flocs.Under the presence of Ca²⁺and Mg²⁺,more hydrogen bonds and gel-like structures were formed in the sludge EPS,hindering the migration of lysozyme molecules to the inner layers and the release of intracellular substances to the liquid phase after pretreatment.At the same time,the activity of lysozyme was inhibited by Al³⁺,reducing the efficiency of lysozyme pretreatment.,the multivalent metal ions can be effectively solubilized with sodium citrate coupled with lysozyme.The sludge EPS structure was destroyed,the mass transfer resistance of lysozyme was relieved,and the release and solubilization of organic matter after lysis were promoted.After 4 h of coupled pretreatment,the protein and polysaccharide content in the supernatant reached 261.2%and 215.8%of that of the sole lysozyme pretreatment,and 6.6 and 4.0 times that of the sole sodium citrate pretreatment,respectively.Anaerobic acid production experiments were conducted on the WAS after lysozyme lysis in the presence of different metal ions to investigate the accumulation efficiency of SCFAs during the fermentation process of the WAS in the presence of different metal ions,and to clarify the characteristics and migration and transformation laws of organic components in the fermentation liquid.The inhibitory mechanism of multivalent metal ions on the acid production process of WAS after lysozyme lysis was revealed.The feasibility and mechanism of sodium citrate coupled with lysozyme to enhance the SCFAs production from WAS in the presence of metal ions were studied.It was found that the acid production efficiency of anaerobic fermentation of WAS after lysozyme lysis in the presence of Na⁺was excellent,with the SCFAs yield reaching 515.93 mg COD/g VSS after 8 days of fermentation,which was 130.89%of the control group.In the presence of Mg²⁺and Ca²⁺,the acid production efficiency of WAS after lysis was 232.34 mg COD/g VSS and 275.97 mg COD/g VSS,respectively.While in the presence of Al³⁺,the acid production was less than 31.7%of the Na⁺group.Acetic acid accounted for 32%to 37%of the total fatty acids.At the same time,Al³⁺had toxic effects on microorganisms,reducing the activity of key enzymes in hydrolysis and acid production pathway and affected SCFAs production.After the pretreatment with sodium citrate coupled with lysozyme（3%,w/w）,the SCFAs yield of the WAS reached 588.4 mg COD/g VSS after6 days of fermentation,which is 8.3 times that of the control group.Sodium citrate chelated multivalent cations in the WAS and releases Na⁺to disrupt the sludge EPS structure,facilitating the transfer of lysozyme molecules to the inner layers of the sludge flocs and exerting lysis efficiency.The citrate chelated with Al³⁺and heavy metal ions,maintained the activity of lysozyme and other extracellular hydrolase.As a result,anaerobic fermentation of WAS contained Al³⁺was enhanced.Moreover,the inhibition of Al³⁺on microorganisms was also blocked.leading to the increase of the richness,evenness,and diversity of microbial communities in anaerobic fermentation system,which enhanced the anaerobic fermentation and acid production of WAS containing multivalent cations.