常压甘油自催化预处理麦草浓醪发酵纤维素乙醇
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摘要
目前纤维素乙醇成本偏高的根本原因在于没有达到淀粉质乙醇发酵水平的"三高"(高浓度、高转化率和高效率)指标,提高水解糖液浓度和避免发酵抑制物来实现浓醪发酵,是解决问题的关键。文中以常压甘油自催化预处理麦草为底物,尝试采用不同发酵策略,探讨其浓醪发酵产纤维素乙醇的可行性。在优化培养条件(15%底物浓度,加酶量30 FPU/g干底物,温度37℃,接种量10%)下同步糖化发酵72 h,纤维素乙醇产量为31.2 g/L,转化率为73%,发酵效率0.43 g/(L·h);采用半同步(预酶解24 h)糖化发酵72 h,纤维素乙醇浓度达到33.7 g/L,转化率为79%,发酵效率为0.47 g/(L·h),其中(半)同步糖化发酵中90%以上纤维素已被糖化水解用于发酵;采用分批补料式半同步糖化发酵,补料到基质浓度相当于30%,发酵72 h时纤维素乙醇产量达到51.2 g/L,转化率为62%,发酵效率为0.71 g/(L·h)。在所有浓醪发酵中乙酸不足3 g/L,无糠醛和羟甲基糠醛等发酵抑制物。以上结果表明,常压甘油自催化预处理木质纤维素基质适用于纤维素乙醇发酵;分批补料式半同步糖化发酵策略可用来进行浓醪纤维素乙醇发酵;未来工作中提高基质纯度和强化酶解产糖是浓醪纤维素乙醇达到"三高"指标的关键。
The expensive production of bioethanol is because it has not yet reached the ‘THREE-HIGH'(High-titer, high-conversion and high-productivity) technical levels of starchy ethanol production. To cope with it, it is necessary to implement a high-gravity mash bioethanol production(HMBP), in which sugar hydrolysates are thick and fermentation-inhibitive compounds are negligible. In this work, HMBP from an atmospheric glycerol autocatalytic organosolv pretreated wheat straw was carried out with different fermentation strategies. Under an optimized condition(15% substrate concentration, 10 g/L(NH4)2SO4, 30 FPU/g dry matter, 10%(V/V) inoculum ratio), HMBP was at 31.2 g/L with a shaking simultaneous saccharification and fermentation(SSF) at 37 °C for 72 h, and achieved with a conversion of 73% and a productivity of 0.43 g/(L·h). Further by a semi-SFF with pre-hydrolysis time of 24 h, HMBP reached 33.7 g/L, the conversion and productivity of which was 79% and 0.47 g/(L·h), respectively. During the SSF and semi-SSF, more than 90% of the cellulose in both substrates were hydrolyzed into fermentable sugars. Finally, a fed-batch semi-SFF was developed with an initial substrate concentration of 15%, in which dried substrate(= the weight of the initial substrate) was divided into three portions and added into the conical flask once each 8 h during the first 24 h. HMBP achieved at 51.2 g/L for 72 h with a high productivity of 0.71 g/(L·h) while a low cellulose conversion of 62%. Interestingly, the fermentation inhibitive compound was mainly acetic acid, less than 3.0 g/L, and there were no other inhibitors detected, commonly furfural and hydroxymethyl furfural existing in the slurry. The data indicate that the lignocellulosic substrate subjected to the atmospheric glycerol autocatalytic organosolv pretreatment is very applicable for HMBP. The fed-batch semi-SFF is effective and desirable to realize an HMBP.
The expensive production of bioethanol is because it has not yet reached the ‘THREE-HIGH'(High-titer, high-conversion and high-productivity) technical levels of starchy ethanol production. To cope with it, it is necessary to implement a high-gravity mash bioethanol production(HMBP), in which sugar hydrolysates are thick and fermentation-inhibitive compounds are negligible. In this work, HMBP from an atmospheric glycerol autocatalytic organosolv pretreated wheat straw was carried out with different fermentation strategies. Under an optimized condition(15% substrate concentration, 10 g/L(NH4)2SO4, 30 FPU/g dry matter, 10%(V/V) inoculum ratio), HMBP was at 31.2 g/L with a shaking simultaneous saccharification and fermentation(SSF) at 37 °C for 72 h, and achieved with a conversion of 73% and a productivity of 0.43 g/(L·h). Further by a semi-SFF with pre-hydrolysis time of 24 h, HMBP reached 33.7 g/L, the conversion and productivity of which was 79% and 0.47 g/(L·h), respectively. During the SSF and semi-SSF, more than 90% of the cellulose in both substrates were hydrolyzed into fermentable sugars. Finally, a fed-batch semi-SFF was developed with an initial substrate concentration of 15%, in which dried substrate(= the weight of the initial substrate) was divided into three portions and added into the conical flask once each 8 h during the first 24 h. HMBP achieved at 51.2 g/L for 72 h with a high productivity of 0.71 g/(L·h) while a low cellulose conversion of 62%. Interestingly, the fermentation inhibitive compound was mainly acetic acid, less than 3.0 g/L, and there were no other inhibitors detected, commonly furfural and hydroxymethyl furfural existing in the slurry. The data indicate that the lignocellulosic substrate subjected to the atmospheric glycerol autocatalytic organosolv pretreatment is very applicable for HMBP. The fed-batch semi-SFF is effective and desirable to realize an HMBP.
引文
[1]Ragauskas AJ,Williams CK,Davison BH,et al.The path forward for biofuels and biomaterials.Science,2006,311(5760):484–489.
[2]Yue GJ,Wu GQ,Lin X.Insights into engineering of cellulosic ethanol.Chin J Biotech,2014,30(6):816–827(in Chinese).岳国君,武国庆,林鑫.纤维素乙醇工程化探讨.生物工程学报,2014,30(6):816–827.
[3]Galbe M,Sassner P,Wingren A,et al.Process engineering economics of bioethanol production.Biofuels,2007,108(6):303–327.
[4]Gray KA,Zhao L,Emptage M.Bioethanol.Curr Opin Chem Biol,2006,10(2):141–146.
[5]Himmel ME,Ding SY,Johnson DK,et al.Biomass recalcitrance:engineering plants and enzymes for biofuels production.Science,2007,315(5813):804–807.
[6]Hodge DB,Karim MN,Schell DJ,et al.Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose.Biores Technol,2008,99(18):8940–8948.
[7]Hodge DB,Karim MN,Schell DJ,et al.Model-based fed-batch for high-solids enzymatic cellulose hydrolysis.Appl Biochem Biotechnol,2009,152(1):88–107.
[8]Cazetta ML,Celligoi M,Buzato JB,et al.Fermentation of molasses by Zymomonas mobilis:effects of temperature and sugar concentration on ethanol production.Biores Technol,2007,98(15):2824–2828.
[9]Song AD,Ren TB,Zhang LL,et al.Optimization of corn stover hydrolysis by fed-batch process.Chin J Biotech,2011,27(3):393–397(in Chinese).宋安东,任天宝,张玲玲,等.玉米秸秆分批补料获得高还原糖浓度酶解液的条件优化.生物工程学报,2011,27(3):393–397.
[10]Shen Y,Ge XM,Bai FW.Impact of fermentation system initial status on oscillations in very high gravity ethanol continuous fermentation process and analysis of fermentation efficiency improvement.Chin J Biotech,2010,26(5):604–609(in Chinese).申渝,葛旭萌,白凤武.初始条件对高浓度乙醇连续发酵过程的影响及振荡行为提高发酵效率的机理分析.生物工程学报,2010,26(5):604–609.
[11]Xu HX,Duan G.Dry solid staging fermentation.Chin J Biotech,2009,25(2):200–206(in Chinese).许宏贤,段钢.固态间歇补料乙醇生料发酵新工艺.生物工程学报,2009,25(2):200–206.
[12]Fan C,Qi K,Xia XX,et al.Efficient ethanol production from corncob residues by repeated fermentation of an adapted yeast.Biores Technol,2013,136(5):309–315.
[13]Zhang M,Wang F,Su R,et al.Ethanol production from high dry matter corncob using fed-batch simultaneous saccharification and fermentation after combined pretreatment.Biores Technol,2010,101(13):4959–4964.
[14]Zhang J,Chu D,Huang J,et al.Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor.Biotechnol Bioeng,2010,105(4):718–728.
[15]J?rgensen H,Vibe-Pedersen J,Larsen J,et al.Liquefaction of lignocellulose at high-solids concentrations.Biotechnol Bioeng,2007,96(5):862–870.
[16]Cannella D,J?rgensen H.Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol production?Biotechnol Bioeng,2014,111(1):59–68.
[17]Sun F,Chen H.Organosolv pretreatment by crude glycerol from oleochemicals industry for enzymatic hydrolysis of wheat straw.Biores Technol,2008,99(13):5474–5479.
[18]Sun FB,Chen HZ.Enhanced enzymatic hydrolysis of wheat straw by aqueous glycerol pretreatment.Biores Technol,2008,99(14):6156–6161.
[19]Romani A,Ruiz HA,Pereira FB,et al.Fractionation of eucalyptus globulus wood by glycerol-water pretreatment:optimization and modeling.Ind Eng Chem Res,2013,52(40):14342–14352.
[20]Sluiter A,Hames B,Ruiz R,et al.NREL laboratory analytical procedure“determination of structural carbohydratesand lignin in biomass”.[EB/OL].[2012-09-28].http://www.nrel.gov/biomass/analyti cal_procedures.html.42618.
[21]Ghose T.Measurement of cellulase activities.Pure Appl Chem,1987,59(2):257–268.
[22]Lau MW,Dale BE.Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST).Proc Natl Acad Sci USA,2009,106(5):1368–1373.
[23]Wingren A,Galbe M,Zacchi G.Techno-economic evaluation of producing ethanol from softwood:comparison of SSF and SHF and identification of bottlenecks.Biotechnol Progr,2003,19(4):1109–1117.
[24]Modenbach AA,Nokes SE.Enzymatic hydrolysis of biomass at high-solids loadings–areview.Biomass Bioenergy,2013,56(9):526–544.
[25]Tian S,Li Y,Wang Z,et al.Evaluation of simultaneous saccharification and ethanol fermentation of undetoxified steam-exploded corn stover by Saccharomyces cerevisiae Y5.Bioenergy Res,2013,6(4):1142–1146.
[26]Liu ZH,Qin L,Zhu JQ,et al.Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature.Biotechnol Biofuels,2014,7(1):167–182.
[27]Zhang J,Chu DQ,Yu ZC,et al.Process strategy for ethanol production from lignocellulose feedstock under extremely low water usage and high solids loading conditions.Chin J Biotech,2010,26(7):950–959(in Chinese).张建,楚德强,于占春,等.低水用量约束条件下的高固体含量纤维乙醇生物加工技术策略.生物工程学报,2010,26(7):950–959.
[28]Wyman CE.Ethanol from lignocellulosic biomass-technology,economics,and opportunities.Biores Technol,1994,50(1):3–15.
[29]Xin CB,Zi LH,Liu CG,et al.Response surface methodology application for optimizing corncobs pretreatment conditions during simultaneous saccharification and ethanol production.J Agric Sci Technol,2013,15(5):173–180(in Chinese).辛崇博,孜力汗,刘晨光,等.响应面法优化玉米芯同步糖化发酵预处理条件.中国农业科技导报,2013,15(5):173–180.
[30]Liu HC,Qian TT,Wang YH,et al.Primary study on simultaneous saccharification and fernmentation of high temperature tolerance yeast and straw powder.Liquor-making Sci Technol,2008,7(11):25–28(in Chinese).刘海臣,钱甜甜,王玉慧,等.耐高温酵母利用稻草粉同步糖化发酵的初步研究.酿酒科技,2008,7(11):25–28.
[31]Shen J,Agblevor FA.Modeling semi-simultaneous saccharification and fermentation of ethanol production from cellulose.Biomass Bioenergy,2010,34(8):1098–1107.
[32]Bayrock D,Ingledew WM.Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology.J Ind Microbiol Biotechnol,2001,27(2):87–93.
[33]Park JM,Oh BR,Seo JW,et al.Efficient production of ethanol from empty palm fruit bunch fibers by fed-batch simultaneous saccharification and fermentation using Saccharomyces cerevisiae.Appl Biochem Biotechnol,2013,170(8):1807–1814.
[34]Chang C,Xu GZ,Ma XJ.Optimization of process parameters for ethanol production from alkaline-pretreated wheat straw by fed-batch simultaneous saccharification and fermentation.Adv Mater Res,2011,236(5):922–925.
[35]Kim Y,Ximenes E,Mosier NS,et al.Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass.Enzyme Microb Technol,2011,48(4):408–415.
[36]Palmqvist E,Hahn-H?gerdal B.Fermentation of lignocellulosic hydrolysates.II:inhibitors and mechanisms of inhibition.Biores Technol,2000,74(1):25–33.
[37]Silverstein RA,Chen Y,Sharma-Shivappa RR,et al.A comparison of chemical pretreatment methods for improving saccharification of cotton stalks.Biores Technol,2007,98(16):3000–3011.
[38]J?rgensen H,Kristensen JB,Felby C.Enzymatic conversion of lignocellulose into fermentable sugars:challenges and opportunities.Biofuels,Bioprod Biorefin,2007,1(2):119–134.
[39]Wang G,Pan X,Zhu J,et al.Sulfite pretreatment to overcome recalcitrance of lignocellulose(SPORL)for robust enzymatic saccharification of hardwoods.Biotechnol Prog,2009,25(4):1086–1093.
[40]Olofsson K,Bertilsson M,Lidén G.A short review on SSF-an interesting process option for ethanol production from lignocellulosic feedstocks.Biotechnol Biofuels,2008,1(7):1–14.
[41]Hu J,Arantes V,Pribowo A,et al.The synergistic action of accessory enzymes enhances the hydrolytic potential of a“cellulase mixture”but is highly substrate specific.Biotechnol Biofuels,2013,6(8):112–123.
[42]Yang B,Wyman CE.BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates.Biotechnol Bioeng,2006,94(4):611–617.
[43]Chen N,Fan JB,Xiang J,et al.Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles.BBA-Proteins Proteom,2006,1764(6):1029–1035.
[44]Njoku SI,Uellendahl H,Ahring BK.Comparing oxidative and dilute acid wet explosion pretreatment of cocksfoot grass at high dry matter concentration for cellulosic ethanol production.Energy Sci Eng,2013,1(2):89–98.
[2]Yue GJ,Wu GQ,Lin X.Insights into engineering of cellulosic ethanol.Chin J Biotech,2014,30(6):816–827(in Chinese).岳国君,武国庆,林鑫.纤维素乙醇工程化探讨.生物工程学报,2014,30(6):816–827.
[3]Galbe M,Sassner P,Wingren A,et al.Process engineering economics of bioethanol production.Biofuels,2007,108(6):303–327.
[4]Gray KA,Zhao L,Emptage M.Bioethanol.Curr Opin Chem Biol,2006,10(2):141–146.
[5]Himmel ME,Ding SY,Johnson DK,et al.Biomass recalcitrance:engineering plants and enzymes for biofuels production.Science,2007,315(5813):804–807.
[6]Hodge DB,Karim MN,Schell DJ,et al.Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose.Biores Technol,2008,99(18):8940–8948.
[7]Hodge DB,Karim MN,Schell DJ,et al.Model-based fed-batch for high-solids enzymatic cellulose hydrolysis.Appl Biochem Biotechnol,2009,152(1):88–107.
[8]Cazetta ML,Celligoi M,Buzato JB,et al.Fermentation of molasses by Zymomonas mobilis:effects of temperature and sugar concentration on ethanol production.Biores Technol,2007,98(15):2824–2828.
[9]Song AD,Ren TB,Zhang LL,et al.Optimization of corn stover hydrolysis by fed-batch process.Chin J Biotech,2011,27(3):393–397(in Chinese).宋安东,任天宝,张玲玲,等.玉米秸秆分批补料获得高还原糖浓度酶解液的条件优化.生物工程学报,2011,27(3):393–397.
[10]Shen Y,Ge XM,Bai FW.Impact of fermentation system initial status on oscillations in very high gravity ethanol continuous fermentation process and analysis of fermentation efficiency improvement.Chin J Biotech,2010,26(5):604–609(in Chinese).申渝,葛旭萌,白凤武.初始条件对高浓度乙醇连续发酵过程的影响及振荡行为提高发酵效率的机理分析.生物工程学报,2010,26(5):604–609.
[11]Xu HX,Duan G.Dry solid staging fermentation.Chin J Biotech,2009,25(2):200–206(in Chinese).许宏贤,段钢.固态间歇补料乙醇生料发酵新工艺.生物工程学报,2009,25(2):200–206.
[12]Fan C,Qi K,Xia XX,et al.Efficient ethanol production from corncob residues by repeated fermentation of an adapted yeast.Biores Technol,2013,136(5):309–315.
[13]Zhang M,Wang F,Su R,et al.Ethanol production from high dry matter corncob using fed-batch simultaneous saccharification and fermentation after combined pretreatment.Biores Technol,2010,101(13):4959–4964.
[14]Zhang J,Chu D,Huang J,et al.Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor.Biotechnol Bioeng,2010,105(4):718–728.
[15]J?rgensen H,Vibe-Pedersen J,Larsen J,et al.Liquefaction of lignocellulose at high-solids concentrations.Biotechnol Bioeng,2007,96(5):862–870.
[16]Cannella D,J?rgensen H.Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol production?Biotechnol Bioeng,2014,111(1):59–68.
[17]Sun F,Chen H.Organosolv pretreatment by crude glycerol from oleochemicals industry for enzymatic hydrolysis of wheat straw.Biores Technol,2008,99(13):5474–5479.
[18]Sun FB,Chen HZ.Enhanced enzymatic hydrolysis of wheat straw by aqueous glycerol pretreatment.Biores Technol,2008,99(14):6156–6161.
[19]Romani A,Ruiz HA,Pereira FB,et al.Fractionation of eucalyptus globulus wood by glycerol-water pretreatment:optimization and modeling.Ind Eng Chem Res,2013,52(40):14342–14352.
[20]Sluiter A,Hames B,Ruiz R,et al.NREL laboratory analytical procedure“determination of structural carbohydratesand lignin in biomass”.[EB/OL].[2012-09-28].http://www.nrel.gov/biomass/analyti cal_procedures.html.42618.
[21]Ghose T.Measurement of cellulase activities.Pure Appl Chem,1987,59(2):257–268.
[22]Lau MW,Dale BE.Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST).Proc Natl Acad Sci USA,2009,106(5):1368–1373.
[23]Wingren A,Galbe M,Zacchi G.Techno-economic evaluation of producing ethanol from softwood:comparison of SSF and SHF and identification of bottlenecks.Biotechnol Progr,2003,19(4):1109–1117.
[24]Modenbach AA,Nokes SE.Enzymatic hydrolysis of biomass at high-solids loadings–areview.Biomass Bioenergy,2013,56(9):526–544.
[25]Tian S,Li Y,Wang Z,et al.Evaluation of simultaneous saccharification and ethanol fermentation of undetoxified steam-exploded corn stover by Saccharomyces cerevisiae Y5.Bioenergy Res,2013,6(4):1142–1146.
[26]Liu ZH,Qin L,Zhu JQ,et al.Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature.Biotechnol Biofuels,2014,7(1):167–182.
[27]Zhang J,Chu DQ,Yu ZC,et al.Process strategy for ethanol production from lignocellulose feedstock under extremely low water usage and high solids loading conditions.Chin J Biotech,2010,26(7):950–959(in Chinese).张建,楚德强,于占春,等.低水用量约束条件下的高固体含量纤维乙醇生物加工技术策略.生物工程学报,2010,26(7):950–959.
[28]Wyman CE.Ethanol from lignocellulosic biomass-technology,economics,and opportunities.Biores Technol,1994,50(1):3–15.
[29]Xin CB,Zi LH,Liu CG,et al.Response surface methodology application for optimizing corncobs pretreatment conditions during simultaneous saccharification and ethanol production.J Agric Sci Technol,2013,15(5):173–180(in Chinese).辛崇博,孜力汗,刘晨光,等.响应面法优化玉米芯同步糖化发酵预处理条件.中国农业科技导报,2013,15(5):173–180.
[30]Liu HC,Qian TT,Wang YH,et al.Primary study on simultaneous saccharification and fernmentation of high temperature tolerance yeast and straw powder.Liquor-making Sci Technol,2008,7(11):25–28(in Chinese).刘海臣,钱甜甜,王玉慧,等.耐高温酵母利用稻草粉同步糖化发酵的初步研究.酿酒科技,2008,7(11):25–28.
[31]Shen J,Agblevor FA.Modeling semi-simultaneous saccharification and fermentation of ethanol production from cellulose.Biomass Bioenergy,2010,34(8):1098–1107.
[32]Bayrock D,Ingledew WM.Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology.J Ind Microbiol Biotechnol,2001,27(2):87–93.
[33]Park JM,Oh BR,Seo JW,et al.Efficient production of ethanol from empty palm fruit bunch fibers by fed-batch simultaneous saccharification and fermentation using Saccharomyces cerevisiae.Appl Biochem Biotechnol,2013,170(8):1807–1814.
[34]Chang C,Xu GZ,Ma XJ.Optimization of process parameters for ethanol production from alkaline-pretreated wheat straw by fed-batch simultaneous saccharification and fermentation.Adv Mater Res,2011,236(5):922–925.
[35]Kim Y,Ximenes E,Mosier NS,et al.Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass.Enzyme Microb Technol,2011,48(4):408–415.
[36]Palmqvist E,Hahn-H?gerdal B.Fermentation of lignocellulosic hydrolysates.II:inhibitors and mechanisms of inhibition.Biores Technol,2000,74(1):25–33.
[37]Silverstein RA,Chen Y,Sharma-Shivappa RR,et al.A comparison of chemical pretreatment methods for improving saccharification of cotton stalks.Biores Technol,2007,98(16):3000–3011.
[38]J?rgensen H,Kristensen JB,Felby C.Enzymatic conversion of lignocellulose into fermentable sugars:challenges and opportunities.Biofuels,Bioprod Biorefin,2007,1(2):119–134.
[39]Wang G,Pan X,Zhu J,et al.Sulfite pretreatment to overcome recalcitrance of lignocellulose(SPORL)for robust enzymatic saccharification of hardwoods.Biotechnol Prog,2009,25(4):1086–1093.
[40]Olofsson K,Bertilsson M,Lidén G.A short review on SSF-an interesting process option for ethanol production from lignocellulosic feedstocks.Biotechnol Biofuels,2008,1(7):1–14.
[41]Hu J,Arantes V,Pribowo A,et al.The synergistic action of accessory enzymes enhances the hydrolytic potential of a“cellulase mixture”but is highly substrate specific.Biotechnol Biofuels,2013,6(8):112–123.
[42]Yang B,Wyman CE.BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates.Biotechnol Bioeng,2006,94(4):611–617.
[43]Chen N,Fan JB,Xiang J,et al.Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles.BBA-Proteins Proteom,2006,1764(6):1029–1035.
[44]Njoku SI,Uellendahl H,Ahring BK.Comparing oxidative and dilute acid wet explosion pretreatment of cocksfoot grass at high dry matter concentration for cellulosic ethanol production.Energy Sci Eng,2013,1(2):89–98.