Study On A Microbial Functional Consortium With High Cellulose Hydrolysis Ability And The High Efficient Methane Fermentation Technology Using Cassava Distillage As Raw Materials

Cassava residues composed by vast lignocellulose are the main substrates during the anaerobic digestion of cassava alcohol distillation wastewater. Because of their insolubility and complexity, they are extremely difficult to be degraded, so hydrolysis of cassava residues efficiently is the crucial step to improve the performance of anaerobic digestion and methane production rates. With the research of mechanism about cellulose degradation by synergistic relationships recently, study on complex microbial community with capacity of degrading lignocellulosic agro-industrial residues such as cassava residues and sugarcane bagasse is a promising approach which can provide efficient biomass conversion to value-added products,such as methane, hydrogen, alcohol.In this study, a microbial consortium with high effective and stable cellulosic degradation ability was constructed by successive enrichment and incubation using cassava residues and filter paper as carbon sources, where the substrates in the culture could be evidently broken down, otherwise, CMCase and xylanase activity was 38.5U and 90.2U after incubated 40 hours. This consortium could remain a stable degrading ability and high enzymatic activity after subcultured 60 generations, preserved in -80℃for half a year and treated 20min under conditions of high temperature below 90℃. what's more, PCR-DGGE technique analysis showed that the composition of this microbial complex remained stable after subcultured for several times. Moreover, some aerobic/facultative anaerobic (Beta-proteobacterium HMD444) , strictly anaerobic bacteria (Thermoanaerobacterium thermosaccharolyticum strain M18, Thermanaerovibrio acidaminovorans DSM 6589, and Clostridium sp. strains LDC-8-c12, 5-8, CO6-72, etc.) coexisted in this constructed microbial consortium. In addition, a few unidentified uncultured bacteria also stably coexisted in this consortium.The excreted extracellular enzymes of this microbial community are a group of enzyme complex community including Xylanase, CMCase,β-glucosidase, Avicelase, FPA and Pectinase. Besides, the basic enzymatic activity characteristics of CMCase and Xylanase were analysed. Both were optimally active at 60℃and pH 6.0; Both was remained 80% original activity at a temperature between 20℃and 70℃,while retained at least 70% original activity for 60min in the pH range from 5.0⁹.0; the presence of Mn²⁺ positively influenced both of activity, but the activity was greatly inhibited in the presence of Cu²⁺, Fe²⁺ and SDS, while Mg²⁺ have inhibition effect to CMCase but positively influence to xylanase; Meanwhile, tween-80, triton-100, EDTA, K+, Ca²⁺ have no difference to enzyme activity.During the degradation process of cassava residues, the key hydrolytic enzymes including CMCase, xylanase and pectinase showed a maximum enzyme activity of 34.4, 90.5 and 15.8U on the second or third day, respectively. After 10 days' fermentation, the degradation ratio of cellulose, hemicellulose and lignin of cassava residues was 79.8%, 85.9% and 19.4% respectively, meanwhile the loss ratio of cassava residues reached 61.5%. Otherwise,it was found that the dominant metabolites are acetic acid, butyric acid, caproic acid and glycerol, and the highest hydrolysis ratio was obtained on the second day by monitoring sCOD, total volatile fatty acids and total sugars. In addition, effect of culture mediums and cultural conditions on the fermentation capacity of complex microbial community bred by laboratory was investigated. The results showed that composite microbial system could directly degrade carbon source with high natural cellulose content (such as filter paper and cotton) effectively. When the addition amount of cassava residues as the sole carbon source was 20g/L, cellulase and hemicellulase showed a maxmium activity. what's more, state of hydrolysis using peptone and yeast powder as nitrogen source was higher than that of inorganic nitrogen source such as urea and ammonium nitrate. The optimum fermentation temperature, inoculum concentration, pH and rotate speed were 50⁶0℃, 5¹0%, 5.0⁹.0 and 75¹35r/min. The above results revealed that this consortium can efficitvely hydrolyze cassava residues and has great potential to be utilized for the pretreatment of cassava residues for biogas fermentation.Effect of coupled microbial consortium and anaerobic effluent circulation on hydrolysis and methanogenic of cassava distillage were studied in hydrolytic-acidogenic reactor with a valid volume 3L and methanogenic reactor with a effective volume of 13L. The results demonstrated that the maximum total volatile fatty acid concentration in the hydrohynates was accumulated from 0.89g/L to 3.57g/L after treated 24h under conditions of 1:2(the ratio of cassava residues and effluent) and 0.25vvm(The volume of aeration). The total methane production volume was improved by 17.7% compared to untreated. What's more, the biogas production rate was rose greatly. Methagenic of cassava distillations untreated and treated in ASBR, the results showed that the methanogenic reactor operated normally for OLRs lower than 12.0gCOD.L-1 during single fermentation. This behaviour was shown by the total volatile fatty acids/total alkalinity ratio, whose values were always kept lower than 0.37 and the total COD removal rate was kept around 70%. A methane yield of 0.119L CH4 g-1COD eliminated was achieved. But while operated as two phase biogas digestion, The methanogenic reactor operated with high stability for OLRs lower than 20.0 gCOD.L-1, this behaviour was shown by the volatile fatty acids/total alkalinity ratio, whose values were always kept lower than 0.12. The total COD removal rate was kept around 75%. A methane yield of 0.128L CH4 g-1COD eliminated was achieved. Once higher this OLR, the effluent acided soon and the reactor operated failure. All these results demonstrated that the integrated digestion process promoted the biotransformation of cassava residues to biogas from the high-rate hydrolytic-acidification phase to the methanogenic phase and ultimately make the methanogenic digestion process operating much more stable. More importantly, less inhibitor of alcoholic fermentation produced in this system.