三倍体毛白杨原料预处理提高纤维素生物转化效率的研究
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摘要
本文对比研究了不同预处理技术对纤维素生物转化效率的影响,重点研究了木质纤维素原料经过预处理后物料化学成份、结构以及酶水解效率的变化。为了实现木质纤维素原料的综合利用,本文也对半纤维素成份的高值化利用进行了探索。得到的主要结论如下：
采用0.5%H2SO4对三倍体毛白杨处理2h,在100-200℃的温度范围评估了反应温度对处理效率的影响。并以1.5%NaOH-70%乙醇水溶液在回流条件下对稀酸处理后的物料抽提3h,回收残渣中的木素组分并对其进行结构表征。结果表明：(1)半纤维素的明显降解发生在130℃处理温度下,半纤维素脱除率为79.9%；升高处理温度也使水解液中的可溶性糖进一步降解生成甲酸、乙酸、乙酰丙酸、糠醛和5-羟甲基糠醛。(2)随着稀酸处理温度的升高,半纤维素和部分纤维素发生降解,物料回收率由92.4%下降至41.2%,而残渣中木素含量由20.8%上升至50.4%。(3)综合考虑物料回收率和纤维素水解效率发现,120℃下0.5%H2SO4处理具有可观的物料回收率(84.4%)和纤维素酶水解效率(60.8%,为原料纤维素酶水解效率的1.6倍),经稀碱抽提后,纤维素水解效率进一步提高至69.8%。(4)稀碱回收木素组分的结构表征发现,稀酸处理使木素β-O-4键断裂,并在木素单元之间生成了新的碳-碳键联接。回收木素的质量占原料克拉森木素的17%-30%。
采用白腐菌Trametes velutina D10149对三倍体毛白杨处理后以1.0%NaOH-70%乙醇溶液在回流条件下对物料抽提3h。通过对处理样品的成份及水解效率分析发现,白腐菌对S型木素单元的降解比G型木素更为明显。稀碱抽提能够有效地提高白腐菌处理的效率,将处理时间缩短至8周。处理后样品的比表面积由1.7m2/g提高至10.6m2/g。随着样品可及度的增加,纤维素水解效率由4.6%(原料)升高至63.0%。
分别以离子液体AmimCl、BmimCl和EmimAc将三倍体毛白杨木粉全溶后以5%NaOH溶液为溶剂再生回收碳水化合物,纤维素回收率分别为70.8%、71.1%和73.4%。三种离子液体处理使纤维素分子量由317000g/mo1分别降低至214000g/mol,258000g/mol和229000g/mol.此外,AmimCl和BmimCl处理使纤维素发生润胀,经EmimAc处理后纤维素Ⅰ转变成为纤维素Ⅱ。离子液体处理的样品纤维素酶水解效率(约87%)提高了30%(5%NaOH处理纤维素酶水解率为67.2%)。
对比不同半纤维素含量的纤维素样品的酶水解效率发现：(1)样品中木聚糖含量由24.7%下降至4.1%时,纤维素水解效率由64.2%提高至85.8%。而当木聚糖完全脱除以后,纤维素水解效率反而下降至78.4%。(2)5IU/g木聚糖的木聚糖的添加量能使纤维素转化效率由50.0%提高至59.5%,但木聚糖酶用量的增加,木聚糖酶对纤维素水解效率的影响作用减小。
以三倍体毛白杨木聚糖为底物,采用毕赤酶母木聚糖酶水解能够在14h内将36.8%的木聚糖水解转化成为低聚木糖。水解产物中聚合度2-4的糖占总低聚木糖的98%。当木聚糖溶于1%NaOH溶液中以超声波处理30min能将木聚糖转化率提高到43.8%,低聚木糖产物中含有98%聚合度2-4的糖。
Lignocellulosic materials have been considered as a potential sustainable source of sugars for biofuels, biochemicals and other biomaterials. However, the intricate structure of plant cell wall, the high ordered cellulose structure, and the seals of hemicellulose and lignin contribute to the recalcitrance of lignocellulosic materials and respond for the high cost of lignocellulose conversion. In this paper, the effect of dilute acid pretreatment, white-rot fungal pretreatment, ionic liquid pretreatment on the structural changes and the enzymatic digestibility of triploid poplar cellulose were comparatively studied. The role of hemicelluloses on the cellulose digestibility was also investigated. In addition, xylan preparation of triploid poplar was also converted to xylooligosaccharides (XOS) by xylanase for the integrational ulitization of lignocellulosic materials. The results of this study were summarized as follows:
1. Pretreatment of triploid poplar with0.5wt%H2SO4were conducted at various temperatures (100-200℃), and the effect of temperature on cellulose structure and enzymatic digestibility was evaluated. The results indicated that79.9%of the hemicellulosic polymers were degraded at130℃. With the increasing of pretreatment temperature, the mono-sugars in the hydrolyzete were further degraded to formic, acetic and levulic acids, furfural and5-hydroxymethyl furfural (5-HMF). The content of residual lignin increased from20.8%to50.4%. Taking both of feedstock recovery and cellulose digestibility into considerateon, the optimum pretreatment temperature was found to be120℃. In this case,84.4%lignocellulose was recovered and achieved60.8%of cellulose conversion via enzymatic hydrolysis. Further fractation of the residue from dilute acid pretreatment with1.5%NaOH-70%aqueous ethanol solution increased the bioconversion of cellulose to69.8%. Alkaline fractionation also recovered17%-30%Klason lignin from the dilute acid pretreated residues. However, dilute acid pretreatment led to the degradation of β-O-4bonds and condensation of lignin units.
2. Pretreatment of triploid poplar with white-rot fungus Trametes velutina D10149(T. velutina D10149) and1.0%(w/v) NaOH-70%(v/v) ethanol aqueous were proposed to reduce the recalcitrance of lignocelluloses. The results suggeseted that syringly type lignin substrate was more susceptible to white-rot fungus for degradation than guaiacyl type. The effect of biologic pretreatment was enhanced by the subsequent alkaline fractionation. Incultivation with Trametes velutina D10149for8weeks and subsequent alkaline fractionation could effectively facilitated the carbohydrate availability and achieved the maximum cellulose enzymatic digestibility as63.0%. After the stepwise pretreatment, the BET surface area of lignocellulose increased from1.7m2/g to10.7m2/g.
3. AmimCl, BmimCl and EmimAc were used to dissolve ball-milled triploid poplar, and the cellulose-rich preparations were subsequently recovered via incubation in5%NaOH aqueous solution and filtration. After pretreatment, the recoveries of cellulose were70.8%,71.1%and73.4%, respectively. Dissolution with these three ionic liquids also decreased the molecular weight of cellulose from317000g/mol to214000g/mol,258000g/mol and229000g/mol, respectively. After reconstitution, the digestibility of the three cellulosic preparations was all improved, yielding87%cellulose conversion, which were1.3-fold times as that without IL pretreatment (67.2%for5%NaOH pretreated sample).
4. The enzymatic digestibilities of poplar cellulosic preparations with different contents of hemicelluloses were investigated. The results indicated that cellulose digestibility increased with the decreasing of hemicelluloses. The cellulose conversion increased from64.2%to85.8%as the xylan content decreased from24.7%to4.1%. Moreover, supplementation of xylanase can hydrolysis xylan and improve the cellulose conversion in return. The result indicated that5IU/g xylans additional xylanase supplementation was effective for enhancing cellulose digestibility, increasing the cellulose conversion from50.0%to59.5%.
5. The production of xylooligosaccharides (XOS) from lignocellulosic material was performed by combined chemical-enzymatic methods. Xylans were prepared from triploid Populas tomentosa, and were conversed into XOS by crude xylanase solution obtained from Pichia stipitis. Under the optimal condition of the xylanase (25IU/g substrate and pH5.4at50℃),36.8%of the xylans preparation were converted to XOS after14h. Xylobiose, xylotriose and xylotetrose were analyzed to be the main products of the enzyme hydrolysis, which accounted98.0%of the released oligosaccharides. In addition, pretreatment of the xylans preparation with sonication in1%NaOH for30min increased the xylans conversion to43.8%. XOS with DP2-4were also the main products of the enzymatic hydrolysis, accounting98.0%of the total XOS.
采用0.5%H2SO4对三倍体毛白杨处理2h,在100-200℃的温度范围评估了反应温度对处理效率的影响。并以1.5%NaOH-70%乙醇水溶液在回流条件下对稀酸处理后的物料抽提3h,回收残渣中的木素组分并对其进行结构表征。结果表明：(1)半纤维素的明显降解发生在130℃处理温度下,半纤维素脱除率为79.9%；升高处理温度也使水解液中的可溶性糖进一步降解生成甲酸、乙酸、乙酰丙酸、糠醛和5-羟甲基糠醛。(2)随着稀酸处理温度的升高,半纤维素和部分纤维素发生降解,物料回收率由92.4%下降至41.2%,而残渣中木素含量由20.8%上升至50.4%。(3)综合考虑物料回收率和纤维素水解效率发现,120℃下0.5%H2SO4处理具有可观的物料回收率(84.4%)和纤维素酶水解效率(60.8%,为原料纤维素酶水解效率的1.6倍),经稀碱抽提后,纤维素水解效率进一步提高至69.8%。(4)稀碱回收木素组分的结构表征发现,稀酸处理使木素β-O-4键断裂,并在木素单元之间生成了新的碳-碳键联接。回收木素的质量占原料克拉森木素的17%-30%。
采用白腐菌Trametes velutina D10149对三倍体毛白杨处理后以1.0%NaOH-70%乙醇溶液在回流条件下对物料抽提3h。通过对处理样品的成份及水解效率分析发现,白腐菌对S型木素单元的降解比G型木素更为明显。稀碱抽提能够有效地提高白腐菌处理的效率,将处理时间缩短至8周。处理后样品的比表面积由1.7m2/g提高至10.6m2/g。随着样品可及度的增加,纤维素水解效率由4.6%(原料)升高至63.0%。
分别以离子液体AmimCl、BmimCl和EmimAc将三倍体毛白杨木粉全溶后以5%NaOH溶液为溶剂再生回收碳水化合物,纤维素回收率分别为70.8%、71.1%和73.4%。三种离子液体处理使纤维素分子量由317000g/mo1分别降低至214000g/mol,258000g/mol和229000g/mol.此外,AmimCl和BmimCl处理使纤维素发生润胀,经EmimAc处理后纤维素Ⅰ转变成为纤维素Ⅱ。离子液体处理的样品纤维素酶水解效率(约87%)提高了30%(5%NaOH处理纤维素酶水解率为67.2%)。
对比不同半纤维素含量的纤维素样品的酶水解效率发现：(1)样品中木聚糖含量由24.7%下降至4.1%时,纤维素水解效率由64.2%提高至85.8%。而当木聚糖完全脱除以后,纤维素水解效率反而下降至78.4%。(2)5IU/g木聚糖的木聚糖的添加量能使纤维素转化效率由50.0%提高至59.5%,但木聚糖酶用量的增加,木聚糖酶对纤维素水解效率的影响作用减小。
以三倍体毛白杨木聚糖为底物,采用毕赤酶母木聚糖酶水解能够在14h内将36.8%的木聚糖水解转化成为低聚木糖。水解产物中聚合度2-4的糖占总低聚木糖的98%。当木聚糖溶于1%NaOH溶液中以超声波处理30min能将木聚糖转化率提高到43.8%,低聚木糖产物中含有98%聚合度2-4的糖。
Lignocellulosic materials have been considered as a potential sustainable source of sugars for biofuels, biochemicals and other biomaterials. However, the intricate structure of plant cell wall, the high ordered cellulose structure, and the seals of hemicellulose and lignin contribute to the recalcitrance of lignocellulosic materials and respond for the high cost of lignocellulose conversion. In this paper, the effect of dilute acid pretreatment, white-rot fungal pretreatment, ionic liquid pretreatment on the structural changes and the enzymatic digestibility of triploid poplar cellulose were comparatively studied. The role of hemicelluloses on the cellulose digestibility was also investigated. In addition, xylan preparation of triploid poplar was also converted to xylooligosaccharides (XOS) by xylanase for the integrational ulitization of lignocellulosic materials. The results of this study were summarized as follows:
1. Pretreatment of triploid poplar with0.5wt%H2SO4were conducted at various temperatures (100-200℃), and the effect of temperature on cellulose structure and enzymatic digestibility was evaluated. The results indicated that79.9%of the hemicellulosic polymers were degraded at130℃. With the increasing of pretreatment temperature, the mono-sugars in the hydrolyzete were further degraded to formic, acetic and levulic acids, furfural and5-hydroxymethyl furfural (5-HMF). The content of residual lignin increased from20.8%to50.4%. Taking both of feedstock recovery and cellulose digestibility into considerateon, the optimum pretreatment temperature was found to be120℃. In this case,84.4%lignocellulose was recovered and achieved60.8%of cellulose conversion via enzymatic hydrolysis. Further fractation of the residue from dilute acid pretreatment with1.5%NaOH-70%aqueous ethanol solution increased the bioconversion of cellulose to69.8%. Alkaline fractionation also recovered17%-30%Klason lignin from the dilute acid pretreated residues. However, dilute acid pretreatment led to the degradation of β-O-4bonds and condensation of lignin units.
2. Pretreatment of triploid poplar with white-rot fungus Trametes velutina D10149(T. velutina D10149) and1.0%(w/v) NaOH-70%(v/v) ethanol aqueous were proposed to reduce the recalcitrance of lignocelluloses. The results suggeseted that syringly type lignin substrate was more susceptible to white-rot fungus for degradation than guaiacyl type. The effect of biologic pretreatment was enhanced by the subsequent alkaline fractionation. Incultivation with Trametes velutina D10149for8weeks and subsequent alkaline fractionation could effectively facilitated the carbohydrate availability and achieved the maximum cellulose enzymatic digestibility as63.0%. After the stepwise pretreatment, the BET surface area of lignocellulose increased from1.7m2/g to10.7m2/g.
3. AmimCl, BmimCl and EmimAc were used to dissolve ball-milled triploid poplar, and the cellulose-rich preparations were subsequently recovered via incubation in5%NaOH aqueous solution and filtration. After pretreatment, the recoveries of cellulose were70.8%,71.1%and73.4%, respectively. Dissolution with these three ionic liquids also decreased the molecular weight of cellulose from317000g/mol to214000g/mol,258000g/mol and229000g/mol, respectively. After reconstitution, the digestibility of the three cellulosic preparations was all improved, yielding87%cellulose conversion, which were1.3-fold times as that without IL pretreatment (67.2%for5%NaOH pretreated sample).
4. The enzymatic digestibilities of poplar cellulosic preparations with different contents of hemicelluloses were investigated. The results indicated that cellulose digestibility increased with the decreasing of hemicelluloses. The cellulose conversion increased from64.2%to85.8%as the xylan content decreased from24.7%to4.1%. Moreover, supplementation of xylanase can hydrolysis xylan and improve the cellulose conversion in return. The result indicated that5IU/g xylans additional xylanase supplementation was effective for enhancing cellulose digestibility, increasing the cellulose conversion from50.0%to59.5%.
5. The production of xylooligosaccharides (XOS) from lignocellulosic material was performed by combined chemical-enzymatic methods. Xylans were prepared from triploid Populas tomentosa, and were conversed into XOS by crude xylanase solution obtained from Pichia stipitis. Under the optimal condition of the xylanase (25IU/g substrate and pH5.4at50℃),36.8%of the xylans preparation were converted to XOS after14h. Xylobiose, xylotriose and xylotetrose were analyzed to be the main products of the enzyme hydrolysis, which accounted98.0%of the released oligosaccharides. In addition, pretreatment of the xylans preparation with sonication in1%NaOH for30min increased the xylans conversion to43.8%. XOS with DP2-4were also the main products of the enzymatic hydrolysis, accounting98.0%of the total XOS.
引文
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