The Key Technique Of High Efficient Cellulose Hydrolyzation And Its Application In Cassava-bioethanol Industry

The pollution problem with a large amount of wastewater and residues generated from cassava basedbioethanol production is the same as which existed in the production of other bulk fermentation products.At present, the end-treatment model such as “anaerobic plus aerobic” treatment and then being dischargedaccording to the criterion was generally applied in the world. However, such model, which only curing theoutside without curing the inside, is capital–intensive, hard to cure, bringing huge economic burden to theenterprises, resulting in the often occurrence of steal-discharging events. Some exploration based on theconcept of cleaner production, such as reusing wastewater directly or after filtered by membrane, still couldnot economically and effectively solve the pollution problem. Therefore, it is essential to change theexisting end-treatment model, and to explore and realize the ideal manufacture model with “zero pollutionand little energy consumption” for cassava based bioethanol production. The investigation and applicationof such kind of ideal manufacture model would promote the sustainable development of cassava bioethanolindustry and point out the direction for no-waste manufacture of other fermentation products. Suchmeasurement would increase the economic repay for enterprises and simultaneously break the pollutiondilemma, then further promote the transformation of the whole biological industry into greenermanufacture, illustrating great theoretical value and practical significance.No-waste manufacture of cassava bioethanol was utilized as the research entry point in this paper,where a circle production model of ethanol and biogas integrated fermentation was proposed according tothe theory of “ecological nutrition chain”. In the present study, the operation rule of an integrated system ofcassava bioethanol and biogas dual fermentation was investigated by the establishment of mathematicsmodels. The application potential of dilute sulfuric acid pretreatment technology was then explored on theenhancement of the methane yield from cassava residues. In addition, a microbial consortium WX-1withhigh efficient cellulolytic ability was directionally constructed and applied in the efficient biogasfermentation of cassava residues, where the key degradation factors and mechanism which realized theefficient degradation of cellulose of this consortium was further investigated. The original integratedtechnology of dual fermentation was finally improved by the process integration through coupling themicrobial consortium pretreatment technology into the integrated system of cassava bioethanol and biogasdual fermentation. The main research results were shown as follows.（1） An integrated ethanol-methane fermentation system was proposed for cassava based bioethanolproduction, where three mathematical models were established to simulate the accumulation of majorinhibitory substances, including organic compounds, total ions, volatile fatty acids （VFAs） and colorants.These inhibitory substances in the reused water reached a relative steady state after3to7batches ofrecycling fermentation, which coincided with the results of mathematical models. There were no negativeeffects of these inhibitory substances on ethanol fermentation and the final ethanol yield, fermentation time,starch utilization ratio were very close to that of the conventional process using tap water. However,approximately7.54%（w/w） of water was lost during each circulation, which was replenished in subsequentcirculations, to assure consistent fermentation broth volume. This novel process was confirmed to have astable operation over13recycles. It is concluded the stable states of the inhibitory substances in the reusedwater can assure this recycling process will run successfully.（2） The pretreatment of cassava residues by thermal-dilute sulfuric acid （TDSA） hydrolysis wasinvestigated by means of a statistically designed set of experiments. A three-factor central composite design（CCD） was employed to identify the optimum pretreatment condition of cassava residues for methaneproduction. During the pretreatment process, the individual and interactive effects of temperature, H2SO4concentration and reaction time on increase of methane yield （IMY） were evaluated by applying responsesurface methodology （RSM）. After optimization, the resulting optimum pretreatment condition was157.84℃, utilizing2.99%（w/w TS） H2SO4for20.15min, where the maximum methane yield （248mL/g VS） was56.96%higher than the control （158mL/g VS）, which was very close to the predict value56.53%.These results indicate the model obtained through RSM analysis is suit to predict the optimum pretreatmentcondition and there is great potential of using TDSA pretreatment of cassava residues to enhance methaneyield. However, the utilization of the technology of TDSA pretreatment into the double-fermentationintegrated system is not suitable.（3） A stable thermophilic microbial consortium WX-1with high cellulose-degradation ability wassuccessfully constructed. That several species of cellulolytic and non-cellulolytic microbes coexisted in thisconsortium was proved by DGGE （denaturing gradient gel electrophoresis） and sequence analysis. Thecooperation and symbiosis of these microbes in this consortium enhanced their cellulose-degradationability. The pretreatment of cassava residues mixing with distillery wastewater at the ratio of2:50（w/v）prior to anaerobic digestion was investigated by using5%（v/v） of this microbial consortium as inoculumsin batch bioreactors at55°C. The experimental results showed that the maximum methane yield （259.46mL/g-VS） of cassava residues was obtained through12hours of pretreatment by microbial consortiumWX-1, which was96.63%higher than the control （131.95mL/g-VS）.（4） The cellulose binding proteins of microbial consortium WX-1were purified by affinity digestionmethod. These purified proteins were identified consisting of8proteins by mass spectrum, it was foundonly protein CBP4is secreted by strain Paenibacillus sp., while the other proteins comprised of endo-andexo-cellulase or xylanase are all secreted by strain C. clariflavum DSM19732. According to the zymogramanalysis, it was found the proteins with higher molecular weight （66-200kDa） own both xylanase andCMCnase activities, and protein CBP6both reveal the highest xylanase and CMCnase activities, while theproteins with lower molecular weight mainly reveal CMCnase activities. In addition, all proteins （exceptCBP13） were found owning a kind of catalyze structure domain through structure domain prediction, whichbelongs to hydrolysis enzyme family8,9,10and48respectively. In addition, some proteins were foundowning carbohydrate binding domain （CBP1,2,4-6） and linking protein binding domain （CBP5-12）, andthis finding identified the efficient degradation of cellulose of microbial consortium WX-1is realized bythe formation of cellulosome.（5） The key functional microbes and proteins of microbial consortium WX-1with high efficientcellulolytic ability were elucidated by a serial dilution method. The original microbial consortium WX-1was serially diluted and incubated in fermentation medium where cassava residues and filter paper wasused as carbon sources. The critical dilution point of this consortium for effective degradation of filterpaper was10-5. Further diluting this consortium from10-5resulted in the loss of degradation ability of filterpaper accompanied with the disappearance of four bands in DGGE （denaturing gradient gel electrophoresis）profiles. The losing of C. clariflavum DSM19732（band2） and Paenibacillus （band4） during the dilutionprocess was confirmed to be the main reason for the loss of degradation ability of filter paper. These resultsindicated that the above two strains are the key functional microbes of consortium WX-1involved in thedegradation of filter paper. In addition, the serial dilution method combined with SDS-PAGE andzymogram analysis results indicated that the proteins CBP6and CBP12secreted by C. clariflavum DSM19732play key roles in the degradation of filter paper. These two proteins belong to hydrolysis enzymefamily9and48, presenting endo-and exo-cellulase activities respectively. The xylanase secreted by strainPaenibacillus sp. could promote the degradation of cellulose, but the existence of this strain with lowquantity could not solely realize the degradation of filter paper.（6） During the pretreatment of cassava distillage by microbial consortium WX-1, it would benefit theenhancement of methane yield in the subsequent anaerobic digestion when the distillery wastewatercomprised with4%（w/v） of cassava residues and anaerobic effluent was mixed at the ratio of1:2（v/v） andkept the ventilatory capacity at0.25vvm. After24h of pretreatment, the methane yield of0.5L of cassavadistillage could reach10.29L, which was17.6%higher than the control （8.75L）. In addition, it was foundthe single or two stage methane fermentation where the cassava distillage was not or pretreated bymicrobial consortium WX-1, could be stably operated when the organic loading rate was lower than12or 20g COD L^-1d^-1, and their highest methane volume yield could reach0.93or2.07L CH4L^-1d^-1respectively. The specific methane yield in the two stage system of methane fermentation could reach0.147L CH4g-1CODremoved, which was17.6%higher than that in the single system. The above results revealedthat intensification of the process of cassava distillage digestion by cellulolytic microbial consortium WX-1can obviously increase the methane yield and productivity, and simultaneously promote the anaerobicdigestion proceeding more stably.（7） In the integrated process of cassava ethanol and biogas dual fermentation where coupled with themicrobial consortium pretreatment technology,7batches of continuous recycle fermentation testsmanifested that the improved dual fermentation integrated process has no negative effect on ethanolfermentation, where the ethanol yield, starch utilization ratio and fermentation time reached about12.6%,90%and48h respectively when cassava to liquor ratio kept at1:2.7（w/w）, which was almost the samewhen fermented with tap water. During7batches of recycle process, the organic substances, volatile fattyacid, total nitrogen and ammonia nitrogen in the improved integrated system reached balanced states after2-5batches of recycle fermentation, while the total ion concentration and alkalinity showed a slow decreasetendency during the recycling process. In addition, the total methane yield and average methane producingvelocity of each recycling process could reached180-206L and42-45L CH14L-d-1respectively in theimproved technology, which was16.1-32.9%and25-40.6%higher than that of the original integratedtechnology, where its total methane yield and average methane producing velocity were only155L and32L CH4L^-1d^-1respectively.