预处理方法对甜高粱茎秆汁液及残渣乙醇发酵的影响
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摘要
甜高粱作为一种非粮作物，越来越成为各国学者的研究热点。研究以甜高粱为原料制备燃料乙醇对于缓解目前日益短缺的能源危机具有重要的现实意义。
对于基于甜高粱的燃料乙醇而言，目前甜高粱原料供应无法满足生物乙醇周年生产的需求，发酵效率相对较低严重限制了甜高粱乙醇的产业化。本文以甜高粱为研究对象，研究了添加甲酸和壳聚糖对甜高粱茎秆汁液进行储藏，并验证发酵效果，延长了甜高粱茎秆汁液的储藏周期；对甜高粱茎秆汁液进行澄清处理，以及添加不同的微量元素研究，有效提高了甜高粱茎秆汁液的乙醇发酵效率；对甜高粱籽粒部分进行了糖化条件的优化及糖化醪与甜高粱茎秆汁液混合发酵，从而有效的提高了茎秆汁液与甜高粱籽粒的高效利用；采用五种预处理方法对甜高粱茎秆残渣进行预处理，并结合酶水解制取乙醇，比较得到较好的预处理方法及酶水解效果，预处理后的残渣是甜高粱乙醇的重要补充原料。
添加甲酸和壳聚糖储存甜高粱茎秆汁液的研究结果表明，甲酸与壳聚糖都有利于甜高粱茎秆汁液中的糖分保存及储藏后汁液的乙醇发酵。甜高粱茎秆汁液中添加体积分数为0.1%的甲酸是文中提到的几种处理中效果最好的处理方法，在该储藏条件下储藏40天后甜高粱茎秆汁液中总可溶性糖的损失率为15.9%，接种固定化酵母粒子发酵30h后，乙醇浓度和乙醇产率分别为39.94g/L和75.49%。
对甜高粱茎秆汁液采用壳聚糖溶液进行澄清处理，通过响应面方法优化得到最佳的澄清处理效果为壳聚糖用量为0.42g/L，汁液pH值5.4，温度29.6℃。在最优条件下进行验证试验，预处理后汁液澄清度为90.62±0.12%，总可溶性糖含量为130.32±0.22g/L，优化结果真实可信。研究了澄清处理与对照汁液的发酵动力学参数，动力学方程的拟合结果表明，实验数据拟合度较好，拟合得到的动力学相关参数进一步表明，澄清处理提高了酵母细胞的最大比生长速率，增加酵母细胞的数量，加快了甜高粱茎秆汁液中糖分的消耗速度，从而提高乙醇产量。
采用Plackett-Burman设计方法对供试的8种微量元素进行筛选得到对以甜高粱茎秆汁液为原料发酵制取乙醇具有积极影响的元素，分别为Biotin、CoCl_2·6H_2O和MnCl_2·4H_2O，并采用单因素法确定得到这3中微量元素的添加量的范围以及采用Box-Behken方法进行优化，得到最佳的微量元素的添加量为MnCl_2·4H_2O7.70mg/L, CoCl_2·6H_2O15.74mg/L，Biotin11.97mg/L，在最佳条件下进行发酵验证，相比较不添加微量元素的汁液而言，乙醇产率提高了5.63%，这为未来甜高粱茎秆汁液的中试生产中微量元素的添加提供参考。
对甜高粱籽粒进行液化和糖化，采用单因素法分别确定了液化和糖化过程参数的水平范围，通过CCD响应面优化得到合适的液化和糖化工艺条件，分别为液化过程：底物浓度20%，液化酶酶活5.0u/g底物，液化pH值为5.5，液化温度为65℃，液化时间30min，糖化酶活浓度为594u/g底物，pH值为4.1，糖化温度为51.4℃，糖化时间为80h。将糖化得到的糖化醪液的澄清液与甜高粱茎秆汁液混合并接种酵母发酵制备乙醇得到的研究结果表明，添加澄清糖化液到甜高粱茎秆汁液中可以增加发酵醪中还原糖含量及自由氨基氮含量并提高发酵速率。混合汁液中澄清糖化液的体积分数越大，发酵速率越快。含有80%澄清糖化液的混合汁液具有最高的乙醇浓度、乙醇生产力和乙醇产率，分别为65.64±0.57gL~(1)、5.47±0.04gL~(1)h~(1)和89.29±0.77%，这比对照纯甜高粱茎秆汁液分别提高了6.8%、33.5%和13.7%。
对甜高粱茎秆残渣进行了原料分析和预处理，研究结果表明文中所用的五种预处理方法均有利于提高甜高粱茎秆残渣的纤维素水解效率，其中先2%氢氧化钠溶液浸泡并高温高压处理再使用5%双氧水处理甜高粱茎秆残渣是这五种方法中最为合适的预处理方法，这种预处理方法预处理后的残渣具有最高的纤维素水解率、总糖得率和乙醇浓度，分别达到了74.29%、90.94g糖/100g干物质和6.12g/L，这分别是对照的5.88倍、9.54倍和19.13倍。在扫描电镜分析中可以看出预处理后的甜高粱茎秆残渣的显著结构变化，傅立叶红外光谱分析可以推断预处理造成了一些化学键的断裂和变化，甜高粱茎秆残渣的纤维素含量和酶糖化率有关。该预处理方法将为甜高粱茎秆残渣制乙醇提高提供参考。
综上所述，甲酸储藏可以至少延长甜高粱茎秆汁液的储藏周期至40天，壳聚糖的澄清以及添加Biotin、MnCl_2·4H_2O、CoCl_2·6H_2O可以提高乙醇产率。汁液和糖化液的混合发酵能够提高发酵液中乙醇浓度及发酵速率，稀碱液高温高压处理辅以双氧水浸泡预处理后提高了甜高粱茎秆残渣的水解率，拓宽了甜高粱乙醇的原料来源，为乙醇的周年生产提供充足的原料。
As a kind of non-food crop, sweet sorghum is becoming a worldwideresearch hotspot recently. There are realistic significances to resolve theenergy shortage problems by developing bioethanol production from sweetsorghum. As far as bioethanol production from sweet sorghum concernednowadays, the seasonally harvested raw material can’t meet the requirementof continuous bioethanol production of the whole year. Also, the lowfermentation efficiency severely restricts its industrialization progress.
The objective in this thesis is to utilize the whole plant of sweetsorghum for bioethanol production. Firstly, storage study applying formicacid and chitosan addition to juice from the stalk was carried out to prolongthe storage period of sweet sorghum juice. And the verification tests to determine the fermentation effect have been done. Then different traceelements were added into the juice which has been clarified. Results showedthat some trace elements could significantly improve the ethanolfermentation efficiency. After that, optimization studies of saccharificationconditions using the grain have been performed. And co-fermentation testsusing the saccharification mash and sweet sorghum juice were conducted inorder to realize the high efficiency utilization of the whole plant forbioenergy production. Finally for the residue of sweet sorghum stalk, fivedifferent pretreatment methods were applied to make this kind oflignocellulosic raw material much easier to be saccharified. The enzymatichydrolysis and ethanol fermentation results showed that the applied range ofbioethanol production from sweet sorghum was greatly broadened.
Results from the storage tests applying formic acid addition andchitosan to preserve sweet sorghum juice showed that both of themcontributes to sugar conservation in juice as well as bioethanol fermentationafter storage. Among the treatments mentioned in the thesis, adding0.1%(v/v) formic acid into the juice was the best method. Under this conditionthe soluble sugar loss rate in sweet sorghum juice after storage for40d is15.90%. And the ethanol content and ethanol yield after fermentation for30h using immobilized yeast was39.94g/L and75.49%, respectively.
Clarification treatment was applied to sweet sorghum juice usingchitosan solution. The optimal treatment condition was chitosanconcentration0.42g/L, pH value5.4and temperature29.6℃, respectively,which were determined by Respond Surface Method. The verificationexperiments showed that the optimization results bore quite a high degree ofconfidence level. The clarity of juice with optimized conditions was90.62±0.12%and the total soluble sugar content was130.32±0.22g/L,which showed that the optimization results were genuine and believable.Furthermore, fermentation kinetics studies were carried out both ofclarification juice and the control. The results showed that experimental datafitting degree is reasonable. And the kinetic related parameters indicated thatclarification not only increased the maximum specific growth rate andpopulation of yeast cell, but also accelerated the consumption rate of sugarcontent thereby improved the bioethanol yield.
Plackett-Burman design was carried out to screen3kinds of traceelements (Biotin, CoCl_2·6H_2O and MnCl_2·4H_2O) which would exertpositively impact to bioethanol production from sweet sorghum juice from8trace elements. The addition range was determined by single factorexperiments. After that Box-Behken method was conducted to get theoptimized addition amount of the3trace elements, which were7.70mg/L, 5.74mg/L and11.97mg/L for MnCl_2·4H_2O, CoCl_2·6H_2O and Biotin,respectively. Verification tests were performed under the optimizedconditions. Results showed that the ethanol yield efficiency was5.63%higher than the control, which would offer a reference to the future pilotscale ethanol production with trace elements addition.
Single factor experiments were brought to determine the extent ofliquefaction and saccharification effects for sweet sorghum grain. CCDmethod was applied to get the applicable technological conditions forliquefaction and saccharification. The optimal conditions for liquefactionwere as follows:20%of substrate concentration,5.0u/g substrate ofenzymatic activity of liquifying enzyme,5.50of pH value,65℃oftemperature, and30min of liquefaction time. And the optimalsaccharification conditions for saccharification were enzymatic activity ofsaccharifying enzyme:594u/g substrate, pH value:4.1, temperature:51.4℃;time:80h. Then the grain saccharification mash after clarification wasco-fermented with sweet sorghum juice to produce ethanol. And the resultsdemonstrated that co-fermentation could increase the reducing sugar contentand free amino nitrogen (FAN) content in the fermentation broth as well asthe fermentation rate. Moreover, the higher the volumetric percent ofclarified saccharification mash was added, the higher the fermentation rate was obtained. The highest ethanol concentration, productivity and yieldwere obtained at the80%volumetric percentage of clarified saccharificationmash in the mixed fermentation liquor, which were65.64±0.57gL~(1)、5.47±0.04gL~(1)h~(1)and89.29±0.77%, respectively. These results were6.8%、33.5%and13.7%higher than the control which using pure sweet sorghumjuice.
The residue of sweet sorghum stalk was analyzed to determine its maincharacteristic and then was pretreated. The results manifested that the fivedifferent methods mentioned in this thesis all benefited the cellulosehydrolysis efficiency of stalk residue. Preatment with2%NaOH plus hightemperature and pressure and then immersed by5%H_2O2was the mostappropriate pretreatment method, after which the highest glucose and xylosecontent in hydrolyzate were obtained. Besides, the highest cellulosehydrolysis rate (74.29%), enzyme saccharification rate (90.94g sugar/100gdry material) and ethanol concentrate (6.12g/L) was obtained with thismethod, which were5.88,9.54and19.13times higher than the control,respectively. Furthermore, it could be observed by the SEM analysis thatthere were significant structural changes of stalk residue after thepretreatments. Also some certain breakage and changes of the chemicalbonds caused by pretreatment were deduced though FITR analysis. Characteristic analysis after pretreatments dedicated that cellulose contentand total sugar yield were related to each other, so were glucoseconcentration, cellulose hydrolysis yield and ethanol concentration. Thiskind of pretreatment method would offer a reference for the bioethanolproduction from lignocellulosic stalk residue in the future.
In sum, the storage period of sweet sorghum juice will be extended atleast by40days by adding certain mount of formic acid. And clarification ofthe juice using chitosan as well as adding trace elements including Biotin,MnCl_2·4H_2O and CoCl_2·6H_2O will improve the bioethanol yield effectively.Co-fermentation by applying sweet sorghum juice and saccharification mashfrom grain will increase the ethanol concentration in the fermentation broth.And pretreatment at high temperature and pressure with diluted alkaline plushydrogen peroxide soaking pretreatment will contribute to the hydrolysisrate of residue of sweet sorghum stalk. All of these methods will broadenthe range of raw material source as well as prolonged the industrializedproduction period of bioethanol.
对于基于甜高粱的燃料乙醇而言，目前甜高粱原料供应无法满足生物乙醇周年生产的需求，发酵效率相对较低严重限制了甜高粱乙醇的产业化。本文以甜高粱为研究对象，研究了添加甲酸和壳聚糖对甜高粱茎秆汁液进行储藏，并验证发酵效果，延长了甜高粱茎秆汁液的储藏周期；对甜高粱茎秆汁液进行澄清处理，以及添加不同的微量元素研究，有效提高了甜高粱茎秆汁液的乙醇发酵效率；对甜高粱籽粒部分进行了糖化条件的优化及糖化醪与甜高粱茎秆汁液混合发酵，从而有效的提高了茎秆汁液与甜高粱籽粒的高效利用；采用五种预处理方法对甜高粱茎秆残渣进行预处理，并结合酶水解制取乙醇，比较得到较好的预处理方法及酶水解效果，预处理后的残渣是甜高粱乙醇的重要补充原料。
添加甲酸和壳聚糖储存甜高粱茎秆汁液的研究结果表明，甲酸与壳聚糖都有利于甜高粱茎秆汁液中的糖分保存及储藏后汁液的乙醇发酵。甜高粱茎秆汁液中添加体积分数为0.1%的甲酸是文中提到的几种处理中效果最好的处理方法，在该储藏条件下储藏40天后甜高粱茎秆汁液中总可溶性糖的损失率为15.9%，接种固定化酵母粒子发酵30h后，乙醇浓度和乙醇产率分别为39.94g/L和75.49%。
对甜高粱茎秆汁液采用壳聚糖溶液进行澄清处理，通过响应面方法优化得到最佳的澄清处理效果为壳聚糖用量为0.42g/L，汁液pH值5.4，温度29.6℃。在最优条件下进行验证试验，预处理后汁液澄清度为90.62±0.12%，总可溶性糖含量为130.32±0.22g/L，优化结果真实可信。研究了澄清处理与对照汁液的发酵动力学参数，动力学方程的拟合结果表明，实验数据拟合度较好，拟合得到的动力学相关参数进一步表明，澄清处理提高了酵母细胞的最大比生长速率，增加酵母细胞的数量，加快了甜高粱茎秆汁液中糖分的消耗速度，从而提高乙醇产量。
采用Plackett-Burman设计方法对供试的8种微量元素进行筛选得到对以甜高粱茎秆汁液为原料发酵制取乙醇具有积极影响的元素，分别为Biotin、CoCl_2·6H_2O和MnCl_2·4H_2O，并采用单因素法确定得到这3中微量元素的添加量的范围以及采用Box-Behken方法进行优化，得到最佳的微量元素的添加量为MnCl_2·4H_2O7.70mg/L, CoCl_2·6H_2O15.74mg/L，Biotin11.97mg/L，在最佳条件下进行发酵验证，相比较不添加微量元素的汁液而言，乙醇产率提高了5.63%，这为未来甜高粱茎秆汁液的中试生产中微量元素的添加提供参考。
对甜高粱籽粒进行液化和糖化，采用单因素法分别确定了液化和糖化过程参数的水平范围，通过CCD响应面优化得到合适的液化和糖化工艺条件，分别为液化过程：底物浓度20%，液化酶酶活5.0u/g底物，液化pH值为5.5，液化温度为65℃，液化时间30min，糖化酶活浓度为594u/g底物，pH值为4.1，糖化温度为51.4℃，糖化时间为80h。将糖化得到的糖化醪液的澄清液与甜高粱茎秆汁液混合并接种酵母发酵制备乙醇得到的研究结果表明，添加澄清糖化液到甜高粱茎秆汁液中可以增加发酵醪中还原糖含量及自由氨基氮含量并提高发酵速率。混合汁液中澄清糖化液的体积分数越大，发酵速率越快。含有80%澄清糖化液的混合汁液具有最高的乙醇浓度、乙醇生产力和乙醇产率，分别为65.64±0.57gL~(1)、5.47±0.04gL~(1)h~(1)和89.29±0.77%，这比对照纯甜高粱茎秆汁液分别提高了6.8%、33.5%和13.7%。
对甜高粱茎秆残渣进行了原料分析和预处理，研究结果表明文中所用的五种预处理方法均有利于提高甜高粱茎秆残渣的纤维素水解效率，其中先2%氢氧化钠溶液浸泡并高温高压处理再使用5%双氧水处理甜高粱茎秆残渣是这五种方法中最为合适的预处理方法，这种预处理方法预处理后的残渣具有最高的纤维素水解率、总糖得率和乙醇浓度，分别达到了74.29%、90.94g糖/100g干物质和6.12g/L，这分别是对照的5.88倍、9.54倍和19.13倍。在扫描电镜分析中可以看出预处理后的甜高粱茎秆残渣的显著结构变化，傅立叶红外光谱分析可以推断预处理造成了一些化学键的断裂和变化，甜高粱茎秆残渣的纤维素含量和酶糖化率有关。该预处理方法将为甜高粱茎秆残渣制乙醇提高提供参考。
综上所述，甲酸储藏可以至少延长甜高粱茎秆汁液的储藏周期至40天，壳聚糖的澄清以及添加Biotin、MnCl_2·4H_2O、CoCl_2·6H_2O可以提高乙醇产率。汁液和糖化液的混合发酵能够提高发酵液中乙醇浓度及发酵速率，稀碱液高温高压处理辅以双氧水浸泡预处理后提高了甜高粱茎秆残渣的水解率，拓宽了甜高粱乙醇的原料来源，为乙醇的周年生产提供充足的原料。
As a kind of non-food crop, sweet sorghum is becoming a worldwideresearch hotspot recently. There are realistic significances to resolve theenergy shortage problems by developing bioethanol production from sweetsorghum. As far as bioethanol production from sweet sorghum concernednowadays, the seasonally harvested raw material can’t meet the requirementof continuous bioethanol production of the whole year. Also, the lowfermentation efficiency severely restricts its industrialization progress.
The objective in this thesis is to utilize the whole plant of sweetsorghum for bioethanol production. Firstly, storage study applying formicacid and chitosan addition to juice from the stalk was carried out to prolongthe storage period of sweet sorghum juice. And the verification tests to determine the fermentation effect have been done. Then different traceelements were added into the juice which has been clarified. Results showedthat some trace elements could significantly improve the ethanolfermentation efficiency. After that, optimization studies of saccharificationconditions using the grain have been performed. And co-fermentation testsusing the saccharification mash and sweet sorghum juice were conducted inorder to realize the high efficiency utilization of the whole plant forbioenergy production. Finally for the residue of sweet sorghum stalk, fivedifferent pretreatment methods were applied to make this kind oflignocellulosic raw material much easier to be saccharified. The enzymatichydrolysis and ethanol fermentation results showed that the applied range ofbioethanol production from sweet sorghum was greatly broadened.
Results from the storage tests applying formic acid addition andchitosan to preserve sweet sorghum juice showed that both of themcontributes to sugar conservation in juice as well as bioethanol fermentationafter storage. Among the treatments mentioned in the thesis, adding0.1%(v/v) formic acid into the juice was the best method. Under this conditionthe soluble sugar loss rate in sweet sorghum juice after storage for40d is15.90%. And the ethanol content and ethanol yield after fermentation for30h using immobilized yeast was39.94g/L and75.49%, respectively.
Clarification treatment was applied to sweet sorghum juice usingchitosan solution. The optimal treatment condition was chitosanconcentration0.42g/L, pH value5.4and temperature29.6℃, respectively,which were determined by Respond Surface Method. The verificationexperiments showed that the optimization results bore quite a high degree ofconfidence level. The clarity of juice with optimized conditions was90.62±0.12%and the total soluble sugar content was130.32±0.22g/L,which showed that the optimization results were genuine and believable.Furthermore, fermentation kinetics studies were carried out both ofclarification juice and the control. The results showed that experimental datafitting degree is reasonable. And the kinetic related parameters indicated thatclarification not only increased the maximum specific growth rate andpopulation of yeast cell, but also accelerated the consumption rate of sugarcontent thereby improved the bioethanol yield.
Plackett-Burman design was carried out to screen3kinds of traceelements (Biotin, CoCl_2·6H_2O and MnCl_2·4H_2O) which would exertpositively impact to bioethanol production from sweet sorghum juice from8trace elements. The addition range was determined by single factorexperiments. After that Box-Behken method was conducted to get theoptimized addition amount of the3trace elements, which were7.70mg/L, 5.74mg/L and11.97mg/L for MnCl_2·4H_2O, CoCl_2·6H_2O and Biotin,respectively. Verification tests were performed under the optimizedconditions. Results showed that the ethanol yield efficiency was5.63%higher than the control, which would offer a reference to the future pilotscale ethanol production with trace elements addition.
Single factor experiments were brought to determine the extent ofliquefaction and saccharification effects for sweet sorghum grain. CCDmethod was applied to get the applicable technological conditions forliquefaction and saccharification. The optimal conditions for liquefactionwere as follows:20%of substrate concentration,5.0u/g substrate ofenzymatic activity of liquifying enzyme,5.50of pH value,65℃oftemperature, and30min of liquefaction time. And the optimalsaccharification conditions for saccharification were enzymatic activity ofsaccharifying enzyme:594u/g substrate, pH value:4.1, temperature:51.4℃;time:80h. Then the grain saccharification mash after clarification wasco-fermented with sweet sorghum juice to produce ethanol. And the resultsdemonstrated that co-fermentation could increase the reducing sugar contentand free amino nitrogen (FAN) content in the fermentation broth as well asthe fermentation rate. Moreover, the higher the volumetric percent ofclarified saccharification mash was added, the higher the fermentation rate was obtained. The highest ethanol concentration, productivity and yieldwere obtained at the80%volumetric percentage of clarified saccharificationmash in the mixed fermentation liquor, which were65.64±0.57gL~(1)、5.47±0.04gL~(1)h~(1)and89.29±0.77%, respectively. These results were6.8%、33.5%and13.7%higher than the control which using pure sweet sorghumjuice.
The residue of sweet sorghum stalk was analyzed to determine its maincharacteristic and then was pretreated. The results manifested that the fivedifferent methods mentioned in this thesis all benefited the cellulosehydrolysis efficiency of stalk residue. Preatment with2%NaOH plus hightemperature and pressure and then immersed by5%H_2O2was the mostappropriate pretreatment method, after which the highest glucose and xylosecontent in hydrolyzate were obtained. Besides, the highest cellulosehydrolysis rate (74.29%), enzyme saccharification rate (90.94g sugar/100gdry material) and ethanol concentrate (6.12g/L) was obtained with thismethod, which were5.88,9.54and19.13times higher than the control,respectively. Furthermore, it could be observed by the SEM analysis thatthere were significant structural changes of stalk residue after thepretreatments. Also some certain breakage and changes of the chemicalbonds caused by pretreatment were deduced though FITR analysis. Characteristic analysis after pretreatments dedicated that cellulose contentand total sugar yield were related to each other, so were glucoseconcentration, cellulose hydrolysis yield and ethanol concentration. Thiskind of pretreatment method would offer a reference for the bioethanolproduction from lignocellulosic stalk residue in the future.
In sum, the storage period of sweet sorghum juice will be extended atleast by40days by adding certain mount of formic acid. And clarification ofthe juice using chitosan as well as adding trace elements including Biotin,MnCl_2·4H_2O and CoCl_2·6H_2O will improve the bioethanol yield effectively.Co-fermentation by applying sweet sorghum juice and saccharification mashfrom grain will increase the ethanol concentration in the fermentation broth.And pretreatment at high temperature and pressure with diluted alkaline plushydrogen peroxide soaking pretreatment will contribute to the hydrolysisrate of residue of sweet sorghum stalk. All of these methods will broadenthe range of raw material source as well as prolonged the industrializedproduction period of bioethanol.
引文
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