纤维质高效水解关键技术及其在木薯燃料乙醇产业中的应用
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摘要
与其它大宗发酵产品一样，木薯燃料乙醇的生产过程中也存在着大量废水与废渣的污染问题，国际上目前普遍采用“厌氧+好氧”最后达标排放的方式进行治理，然而这种治表不治里的末端治理模式代价高昂且难以根治，给企业造成了重大的经济负担，偷排事件时有发生。一些基于清洁生产理念而提出的废水直接回用或者经膜过滤后再回用的研究探索仍然无法经济有效地解决废水的污染问题。为此，研究改变现有的废水末端治理模式，开发并实现木薯燃料乙醇的“零污染、低能耗”理想制造模式就显得尤为必要。这种理想制造模式的研究和应用将促进木薯燃料乙醇产业的可持续发展，并为其它大宗发酵产品的无废制造指明方向，在提高企业经济收益的同时有望打破产业的污染困局，进而促进整个生物工业产业向绿色制造方向的转型，具有重大的理论价值和现实意义。
本论文以木薯燃料乙醇的无废制造为研究切入点，根据“生态营养链”原理提出了将木薯燃料乙醇生产与沼气发酵相耦联的环形生产模式，通过建立数学模型的方式研究了木薯酒精沼气双发酵耦联体系的运行规律，探讨了稀硫酸预处理技术在提高木薯渣甲烷产量方面的应用潜力，定向构建了一组高效的纤维素降解复合菌系WX-1并将其应用于木薯渣的高效沼气发酵之中，研究了复合菌系WX-1实现纤维素高效降解的关键因子与降解机制，最后通过工艺集成将复合菌系预处理技术耦合于木薯酒精沼气双发酵耦联体系中，对原双发酵耦联工艺进行了改进和完善。主要研究结果如下：
(1)建立了木薯酒精沼气双发酵耦联工艺，通过构建的3个数学模型对耦联体系中主要抑制物(如有机物、总离子、挥发酸和色素等)的累积规律进行了模拟。回用水中的抑制物经过3-7批次的循环发酵后达到了一个相对稳定的平衡状态，该结论与数学模型的模拟结果基本一致。这些抑制物对酒精发酵没有明显不利影响，其酒精产量、发酵周期和淀粉利用率与传统的自来水发酵水平相当。然而，每批次的循环过程中将有7.54%(w/w)左右的水分损失，需在下批次循环时补充自来水即可达到稳定。该耦联工艺在13批次的循环过程中被证实能够稳定运行，回用水中抑制物最终所达到的平衡状态可确保该循环工艺的成功运行。
(2)通过统计学方法研究了热稀硫酸水解技术在木薯渣预处理中的应用，一组三因素的中心组合试验设计确定了木薯渣用于甲烷发酵的最佳稀硫酸预处理条件。在预处理过程中，采用响应面法评估了温度、硫酸浓度以及反应时间对甲烷产量提高率的单独和交互效应。通过优化以后，确定最佳的预处理条件为157.84℃，采用2.99%(w/w TS)的硫酸水解20.15min，此时的最大甲烷产量(248mL/g VS)比空白对照(158mL/g VS)提高了56.96%，与预测值56.53%非常接近。这些结果表明通过响应面分析得到的模型适合于预测最佳的稀硫酸预处理条件，且采用稀硫酸水解技术预处理木薯渣以增加其甲烷产量具有一定的应用潜力。但是，进一步分析发现将稀硫酸预处理技术应用于双发酵耦联体系中却并不合适。
(3)定向构建了一组稳定耐热的高效纤维素降解复合菌系WX-1，通过变性梯度凝胶电泳和序列分析证实了该复合菌系由多种纤维素降解和非降解菌所组成，这些微生物在复合菌系中的协作与共生关系提高了它们的纤维素降解能力。在厌氧消化前将木薯渣和酒精蒸馏废液按2:50(w/v)的比例混合于55℃的分批反应器中并接入5%(v/v)的复合菌系进行预处理，试验结果表明经过复合菌系WX-1预处理12h后木薯渣的甲烷产量可达到最大值(259.46mL/g-VS)，相对于空白对照(131.95mL/g-VS)提高了96.63%。
(4)采用亲和消化法提取纯化了复合菌系WX-1中的纤维结合蛋白。纯化后的纤维结合蛋白经质谱鉴定由8种蛋白所组成，其中除蛋白CBP4由Paenibacillus sp.分泌的外，其它的蛋白均由C.clariflavum DSM19732所产生的内切与外切纤维素酶或木聚糖酶所组成。由酶谱分析可见，高分子量(66-200kDa)的蛋白具有木聚糖酶和CMC酶的活性，且纤维结合蛋白CBP6的木聚糖酶和CMC酶活性均最强，而低分子量的蛋白则主要表现CMC酶活性。通过结构域预测发现除纤维结合蛋白CBP13外的所有蛋白均拥有一种催化结构域，分别隶属于第8、9、10及48家族的糖苷水解酶。此外，部分蛋白还具有碳水化合物结合域(CBP1,2,4-6)以及连接蛋白结合域(CBP5-12)，此发现证实了纤维素降解复合菌系WX~-1主要是通过形成纤维小体的模式来实现纤维素高效降解的。
(5)通过梯度稀释法阐明了纤维素高效降解复合菌系WX-1的关键功能菌和酶蛋白。原始复合菌系WX~-1经梯度稀释后置于以木薯渣和滤纸为唯一碳源的发酵培养基中进行培养，研究表明复合菌系WX~-1实现滤纸有效降解的稀释临界点为10-5，进一步提高稀释梯度将导致其丧失滤纸的降解能力同时伴随着变性梯度凝胶电泳图上4条条带的消失。在稀释的过程中菌株C. clariflavum DSM19732(条带2)和Paenibacillus(条带4)的消失被证实是该复合菌系丧失滤纸降解能力的主要原因，且这两株菌是复合菌系WX~-1中实现滤纸降解的关键功能菌。此外，由梯度稀释法结合SDS-PAGE电泳及酶谱分析结果可知，由C. clariflavum DSM19732分泌的蛋白CBP6和CBP12，在滤纸的降解中起着最为关键的作用，它们分别隶属于第9和48家族的糖苷水解酶并呈现出内切与外切的纤维素酶的活性。菌株Paenibacillus sp.所产生的木聚糖酶可以促进纤维素的降解，但少量的该菌的存在并不能单独实现滤纸的降解。
(6)在复合菌系WX-1预处理木薯酒糟的过程中将含有4%(w/v)木薯渣的酒精蒸馏废液与高温厌氧出水按1:2(v/v)的比例进行混合配比并维持0.25vvm的通气量时最有利于后续厌氧消化过程中甲烷产量的提高。经24h的预处理后，0.5L木薯酒糟的甲烷产量可以达到10.29L，相对于0h对照组的甲烷产量(8.75L)提高了17.6%。此外，研究还发现木薯酒糟不经或经过复合菌系预处理的单相或两相甲烷发酵分别能够在有机负荷小于12和20g COD L~(-1)d~(-1)的条件下稳定运行，其各自最大的甲烷体积产率分别为0.93和2.07L CH-4L1d~(-1)。两相甲烷发酵体系中厌氧消化时的比甲烷产率为0.147L CH_4g~(-1)CODremoved比单相时的0.125L CH14g-CODremoved提高了17.6%。上述结果表明通过纤维素降解复合菌系WX-1水解来强化木薯酒糟的厌氧消化过程，能够显著提高厌氧消化过程中的甲烷产量和产率，并能够使厌氧反应更加稳定地进行。
(7)在耦合复合菌系预处理技术的木薯酒精沼气双发酵耦联工艺中，连续7批次的循环发酵试验证实改进型的双发酵耦联工艺对酒精发酵没有任何不利影响，在料液比为1:2.7(w/w)的条件下酒精产量、淀粉利用率及发酵周期分别维持在12.6%、90%和48h左右，该结果与以自来水作为配料水时的酒精发酵水平相当。在7批次的循环过程中，改进型的双发酵耦联体系中的有机物、挥发酸、总氮和氨氮经过2-5批次的循环后基本达到平衡状态，而体系中的离子浓度和碱度则在循环发酵的过程中却有少许的下降趋势。此外，改进型的双发酵耦联工艺中每循环批次的厌氧消化过程中的总甲烷产量和日均产甲烷速率分别可以达到180-206L和40-45L CH4L~(-1)d~(-1)，比原耦联工艺中的总甲烷产量(155L)和日均产甲烷速率(32L CH4L~(-1)d~(-1))分别提高了16.1-32.9%和25-40.6%左右。
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~(-1)d~(-1), and their highest methane volume yield could reach0.93or2.07L CH4L~(-1)d~(-1)respectively. 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~(-1)d~(-1)respectively.
本论文以木薯燃料乙醇的无废制造为研究切入点，根据“生态营养链”原理提出了将木薯燃料乙醇生产与沼气发酵相耦联的环形生产模式，通过建立数学模型的方式研究了木薯酒精沼气双发酵耦联体系的运行规律，探讨了稀硫酸预处理技术在提高木薯渣甲烷产量方面的应用潜力，定向构建了一组高效的纤维素降解复合菌系WX-1并将其应用于木薯渣的高效沼气发酵之中，研究了复合菌系WX-1实现纤维素高效降解的关键因子与降解机制，最后通过工艺集成将复合菌系预处理技术耦合于木薯酒精沼气双发酵耦联体系中，对原双发酵耦联工艺进行了改进和完善。主要研究结果如下：
(1)建立了木薯酒精沼气双发酵耦联工艺，通过构建的3个数学模型对耦联体系中主要抑制物(如有机物、总离子、挥发酸和色素等)的累积规律进行了模拟。回用水中的抑制物经过3-7批次的循环发酵后达到了一个相对稳定的平衡状态，该结论与数学模型的模拟结果基本一致。这些抑制物对酒精发酵没有明显不利影响，其酒精产量、发酵周期和淀粉利用率与传统的自来水发酵水平相当。然而，每批次的循环过程中将有7.54%(w/w)左右的水分损失，需在下批次循环时补充自来水即可达到稳定。该耦联工艺在13批次的循环过程中被证实能够稳定运行，回用水中抑制物最终所达到的平衡状态可确保该循环工艺的成功运行。
(2)通过统计学方法研究了热稀硫酸水解技术在木薯渣预处理中的应用，一组三因素的中心组合试验设计确定了木薯渣用于甲烷发酵的最佳稀硫酸预处理条件。在预处理过程中，采用响应面法评估了温度、硫酸浓度以及反应时间对甲烷产量提高率的单独和交互效应。通过优化以后，确定最佳的预处理条件为157.84℃，采用2.99%(w/w TS)的硫酸水解20.15min，此时的最大甲烷产量(248mL/g VS)比空白对照(158mL/g VS)提高了56.96%，与预测值56.53%非常接近。这些结果表明通过响应面分析得到的模型适合于预测最佳的稀硫酸预处理条件，且采用稀硫酸水解技术预处理木薯渣以增加其甲烷产量具有一定的应用潜力。但是，进一步分析发现将稀硫酸预处理技术应用于双发酵耦联体系中却并不合适。
(3)定向构建了一组稳定耐热的高效纤维素降解复合菌系WX-1，通过变性梯度凝胶电泳和序列分析证实了该复合菌系由多种纤维素降解和非降解菌所组成，这些微生物在复合菌系中的协作与共生关系提高了它们的纤维素降解能力。在厌氧消化前将木薯渣和酒精蒸馏废液按2:50(w/v)的比例混合于55℃的分批反应器中并接入5%(v/v)的复合菌系进行预处理，试验结果表明经过复合菌系WX-1预处理12h后木薯渣的甲烷产量可达到最大值(259.46mL/g-VS)，相对于空白对照(131.95mL/g-VS)提高了96.63%。
(4)采用亲和消化法提取纯化了复合菌系WX-1中的纤维结合蛋白。纯化后的纤维结合蛋白经质谱鉴定由8种蛋白所组成，其中除蛋白CBP4由Paenibacillus sp.分泌的外，其它的蛋白均由C.clariflavum DSM19732所产生的内切与外切纤维素酶或木聚糖酶所组成。由酶谱分析可见，高分子量(66-200kDa)的蛋白具有木聚糖酶和CMC酶的活性，且纤维结合蛋白CBP6的木聚糖酶和CMC酶活性均最强，而低分子量的蛋白则主要表现CMC酶活性。通过结构域预测发现除纤维结合蛋白CBP13外的所有蛋白均拥有一种催化结构域，分别隶属于第8、9、10及48家族的糖苷水解酶。此外，部分蛋白还具有碳水化合物结合域(CBP1,2,4-6)以及连接蛋白结合域(CBP5-12)，此发现证实了纤维素降解复合菌系WX~-1主要是通过形成纤维小体的模式来实现纤维素高效降解的。
(5)通过梯度稀释法阐明了纤维素高效降解复合菌系WX-1的关键功能菌和酶蛋白。原始复合菌系WX~-1经梯度稀释后置于以木薯渣和滤纸为唯一碳源的发酵培养基中进行培养，研究表明复合菌系WX~-1实现滤纸有效降解的稀释临界点为10-5，进一步提高稀释梯度将导致其丧失滤纸的降解能力同时伴随着变性梯度凝胶电泳图上4条条带的消失。在稀释的过程中菌株C. clariflavum DSM19732(条带2)和Paenibacillus(条带4)的消失被证实是该复合菌系丧失滤纸降解能力的主要原因，且这两株菌是复合菌系WX~-1中实现滤纸降解的关键功能菌。此外，由梯度稀释法结合SDS-PAGE电泳及酶谱分析结果可知，由C. clariflavum DSM19732分泌的蛋白CBP6和CBP12，在滤纸的降解中起着最为关键的作用，它们分别隶属于第9和48家族的糖苷水解酶并呈现出内切与外切的纤维素酶的活性。菌株Paenibacillus sp.所产生的木聚糖酶可以促进纤维素的降解，但少量的该菌的存在并不能单独实现滤纸的降解。
(6)在复合菌系WX-1预处理木薯酒糟的过程中将含有4%(w/v)木薯渣的酒精蒸馏废液与高温厌氧出水按1:2(v/v)的比例进行混合配比并维持0.25vvm的通气量时最有利于后续厌氧消化过程中甲烷产量的提高。经24h的预处理后，0.5L木薯酒糟的甲烷产量可以达到10.29L，相对于0h对照组的甲烷产量(8.75L)提高了17.6%。此外，研究还发现木薯酒糟不经或经过复合菌系预处理的单相或两相甲烷发酵分别能够在有机负荷小于12和20g COD L~(-1)d~(-1)的条件下稳定运行，其各自最大的甲烷体积产率分别为0.93和2.07L CH-4L1d~(-1)。两相甲烷发酵体系中厌氧消化时的比甲烷产率为0.147L CH_4g~(-1)CODremoved比单相时的0.125L CH14g-CODremoved提高了17.6%。上述结果表明通过纤维素降解复合菌系WX-1水解来强化木薯酒糟的厌氧消化过程，能够显著提高厌氧消化过程中的甲烷产量和产率，并能够使厌氧反应更加稳定地进行。
(7)在耦合复合菌系预处理技术的木薯酒精沼气双发酵耦联工艺中，连续7批次的循环发酵试验证实改进型的双发酵耦联工艺对酒精发酵没有任何不利影响，在料液比为1:2.7(w/w)的条件下酒精产量、淀粉利用率及发酵周期分别维持在12.6%、90%和48h左右，该结果与以自来水作为配料水时的酒精发酵水平相当。在7批次的循环过程中，改进型的双发酵耦联体系中的有机物、挥发酸、总氮和氨氮经过2-5批次的循环后基本达到平衡状态，而体系中的离子浓度和碱度则在循环发酵的过程中却有少许的下降趋势。此外，改进型的双发酵耦联工艺中每循环批次的厌氧消化过程中的总甲烷产量和日均产甲烷速率分别可以达到180-206L和40-45L CH4L~(-1)d~(-1)，比原耦联工艺中的总甲烷产量(155L)和日均产甲烷速率(32L CH4L~(-1)d~(-1))分别提高了16.1-32.9%和25-40.6%左右。
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~(-1)d~(-1), and their highest methane volume yield could reach0.93or2.07L CH4L~(-1)d~(-1)respectively. 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~(-1)d~(-1)respectively.
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