基于高底物浓度的纤维素酶三段水解桑木粉得率与形貌分析
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摘要
为改善高底物浓度酶水解过程中产物抑制问题,采用三段酶水解方法,通过在水解过程中及时移除反应产物纤维二糖和葡萄糖,降低产物抑制作用,增加酶反应速率,从而提高酶水解得率、缩短酶反应时间。与原料和经NaOH预处理的桑木比较,NaOH-Fenton预处理后的桑木中木聚糖含量明显降低,纤维素含量相对增加,木质素含量变化较小。无论是一段水解还是三段水解,纤维素酶水解得率均随底物质量浓度的升高而下降。在0.30 g/m L(m/V)底物质量浓度下,当酶用量增加为40 U/g(以纤维素质量计)时,三段(10+10+10)h酶水解得率74.16%,比一段水解72 h得率45.61%增长了62.60%,并且水解时间缩短了42 h。该研究结果对提高纤维素酶水解得率、降低纤维资源制取燃料乙醇成本具有指导意义。
Since the large-scale bioethanol industrialization is not fully mature,as one of the main technical steps,the enzymatic hydrolysis process needs to be further studied. In this paper,the three-stage enzymatic hydrolysis method was used to solve the problem of high glucose concentration inhibition during the high substrate concentration hydrolysis process. The enzymatic hydrolysis rate was improved by the three-stage enzymatic hydrolysis method,which could increase the enzymatic hydrolysis rate and shorten the enzymatic reaction time by removing the reaction products,i.e.,cellobiose and glucose,in the hydrolysis process,reducing the product inhibition and increasing the enzyme reaction rate. The hydroxyl radical produced by the fenton reaction caused the loose structure and changed the threedimensional structure of the cellulose in the biomass,which was propitious to improve the hydrolysis efficiency of the subsequent enzyme by NaOH-Fenton pretreatment method. The results showed that 2% NaOH-Fenton pretreated sample had higher cellulose content and lower hemicellulose content compared with the materials with 2% NaOH pretreated sample. The enzymatic hydrolysis yield decreased with the increase of substrate concentration,either in onestage or three-stages enzymatic hydrolysis. The NaOH-Fenton pretreated mulberry at 30% substrate concentration were performed the three-stage hydrolysis,using the enzyme dosage 40 U/g cellulose,and the three-stage hydrolysis could reach much higher hydrolysis yields of 74. 16% in 30 h. Compared with the 72 h yield(45. 61%) of the one-stage hydrolysis,a 62. 60% increase in hydrolysis yield was achieved and 42 h hydrolysis time was reduced. The results have practical significance for improving the yield of cellulase hydrolysis and reducing the cost of making fuel ethanol from biomass resources.
Since the large-scale bioethanol industrialization is not fully mature,as one of the main technical steps,the enzymatic hydrolysis process needs to be further studied. In this paper,the three-stage enzymatic hydrolysis method was used to solve the problem of high glucose concentration inhibition during the high substrate concentration hydrolysis process. The enzymatic hydrolysis rate was improved by the three-stage enzymatic hydrolysis method,which could increase the enzymatic hydrolysis rate and shorten the enzymatic reaction time by removing the reaction products,i.e.,cellobiose and glucose,in the hydrolysis process,reducing the product inhibition and increasing the enzyme reaction rate. The hydroxyl radical produced by the fenton reaction caused the loose structure and changed the threedimensional structure of the cellulose in the biomass,which was propitious to improve the hydrolysis efficiency of the subsequent enzyme by NaOH-Fenton pretreatment method. The results showed that 2% NaOH-Fenton pretreated sample had higher cellulose content and lower hemicellulose content compared with the materials with 2% NaOH pretreated sample. The enzymatic hydrolysis yield decreased with the increase of substrate concentration,either in onestage or three-stages enzymatic hydrolysis. The NaOH-Fenton pretreated mulberry at 30% substrate concentration were performed the three-stage hydrolysis,using the enzyme dosage 40 U/g cellulose,and the three-stage hydrolysis could reach much higher hydrolysis yields of 74. 16% in 30 h. Compared with the 72 h yield(45. 61%) of the one-stage hydrolysis,a 62. 60% increase in hydrolysis yield was achieved and 42 h hydrolysis time was reduced. The results have practical significance for improving the yield of cellulase hydrolysis and reducing the cost of making fuel ethanol from biomass resources.
引文
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[19]SLUITER A D,HAMES B,RUIZ R O,et al.Determination of structural carbohydrates and lignin in biomass:laboratory analytical procedure[R].Golden:National Renewable Energy Laboratory,2008.
[20]黄爱玲,周美华.玉米秸秆酶水解影响因素的研究[J].中国资源综合利用,2004(8):25-27.HUANG A L,ZHOU M H.Study on affecting factor of enzymolysis corn stalk[J].China Resources Comprehensive Utilization,2004(8):25-27.
[2]WENG J,LI X,BONAWITZ N,et al.Emerging strategies of lignin engineering and degradation for cellulosic biofuel production[J].Current Opinion in Biotechnology,2008,19(2):166-172.
[3]CHEN M,XIA L M,XUE P.Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate[J].International Biodeterioration&Biodegradation,2007,59(2):85-89.
[4]HODGE D B,KARIM M N,SCHELL D J,et al.Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose[J].Bioresource Technology,2008,99(18):8940-8948.
[5]ZACCHI G,AXELSSON A.Economic evaluation of preconcentration in production of ethanol from dilute sugar solutions[J].Biotechnology and Bioengineering,1989,34(2):223-233.
[6]STENBERG K,BOLLOK M,RECZEY K,et al.Effect of substrate and cellulase concentration on simultaneous saccharification and fermentation of steam-pretreated softwood for ethanol production[J].Biotechnology and Bioengineering,2000,68(2):204-210.
[7]WINGREN A,GALBE M,ZACCHI G.Techno-economic evaluation of producing ethanol from softwood:comparison of SSF and SHF and identification of bottlenecks[J].Biotechnology Progress,2003,19(4):1109-1117.
[8]KNUTSEN J S,DAVIS R H.Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis[J].Applied Biochemistry and Biotechnology,2004,114(1/3):585-599.
[9]YANG S,DING W Y,CHEN H Z.Enzymatic hydrolysis of rice straw in a tubular reactor coupled with UF membrane[J].Process Biochemistry,2006,41(3):721-725.
[10]瞿丽莉.β-葡萄糖苷酶的分离纯化及在纤维素水解上的应用[D].南京:南京林业大学,2008.QU L L.Separation and purification ofβ-glucosidase and its application in cellulose hydrolysis[D].Nanjing:Nanjing Forestry U-niversity,2008.
[11]MICHALSKA K,MIAZEK K,KRZYSTEK L,et al.Influence of pretreatment with Fenton's reagent on biogas production and methane yield from lignocellulosic biomass[J].Bioresource Technology,2012,119:72-78.
[12]DE HEREDIA J B,TORREGROSA J,DOMINGUEZ J R,et al.Kinetic modelfor phenolic compound oxidation by Fenton's reagent[J].Chemosphere,2001,45:85-90.
[13]ERDEN G,FILIBELI A.Improving anaerobic biodegradability of biological sludges by Fenton pre-treatment:effects on single stage and two-stage anaerobic digestion[J].Desalination,2010,251:58-63.
[14]王燕云,杨静.稀碱-芬顿试剂预处理对云南苦竹酶水解得率的影响[J].生物质化学工程,2015,49(5):17-22.WANG Y Y,YANG J.The influence of alkaline-fenton pretreatment on the enzymatic hydrolysis yield of Yunnan bamboo[J].Biomass Chemical Engineering,2015,49(5):17-22.
[15]BALLESTEROS I,OLIVA J M,NEGRO M J,et al.Enzymic hydrolysis of steam exploded herbaceous agricultural waste(Brassica carinata)at different particule sizes[J].Process Biochemistry,2002,38(2):187-192.
[16]CHEN M,ZHAO J,XIA L M.Enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars[J].Carbohydrate Polymers,2008,71(3):411-415.
[17]杨静.木质纤维原料分段酶水解技术的研究[D].南京:南京林业大学,2010.YANG J.Multi-stage enzymatic saccharification of lignocellulosic materials[D].Nanjing:Nanjing Forestry University,2010.
[18]朱圆圆,顾夕梅,朱均均,等.两步碱法预处理对玉米秸秆组分及结构的影响[J].中国科技论文,2015,10(12):1376-1381.ZHU Y Y,GU X M,ZHU J J,et al.Effects of two-step alkaline pretreatment on components and structure of corn stover[J].China Science Paper,2015,10(12):1376-1381.
[19]SLUITER A D,HAMES B,RUIZ R O,et al.Determination of structural carbohydrates and lignin in biomass:laboratory analytical procedure[R].Golden:National Renewable Energy Laboratory,2008.
[20]黄爱玲,周美华.玉米秸秆酶水解影响因素的研究[J].中国资源综合利用,2004(8):25-27.HUANG A L,ZHOU M H.Study on affecting factor of enzymolysis corn stalk[J].China Resources Comprehensive Utilization,2004(8):25-27.
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