纸浆污泥纤维素酶水解糖化与增效工艺及机理的研究
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摘要
纸浆污泥是制浆造纸工业的主要固体废弃物,含有约40%～60%的纤维素资源,可以作为生产纤维素乙醇的原料。相对木质纤维素原料,纸浆污泥具有价格低廉、纤维结构疏松等优点。纸浆污泥通过纤维素酶水解发酵乙醇,将会拓宽生物质乙醇的原料来源,同时,减小纸浆污泥排放对环境造成的压力,是工业废弃物一种极具潜力的能源转化方式。本论文以纸浆纤维为底物,探讨了纤维素酶在纤维上的吸附以及酶水解过程中一系列物理和化学的变化,建立了纸浆污泥酶水解的动力学模型,为纸浆污泥的酶水解提供理论依据。另外,提出了一种使用阳离子聚合物,提高纤维素酶水解效率的新方法,可以大大降低酶解糖化的生产成本。
纤维素酶在纤维上的吸附是酶水解反应的第一步。纤维素酶在纤维上吸附60 min时基本达到吸附平衡;纤维素酶吸附的过程满足二级吸附动力学模型,可表示为(?)的形式;纤维素酶在纤维上的吸附平衡满足Langmuir等温吸附;纤维素酶在短纤维上有最大的吸附量,而在长纤维上的吸附平衡常数最大,最易达到吸附平衡。纤维素酶吸附的热力学研究表明,纤维素酶吸附过程的吉布斯自由能△G°小于0,是自发过程,吸附同时存在物理吸附和化学吸附;吸附的反应焓变△H?为负值,表明吸附为放热过程;吸附的反应熵变△S°大于0,表明纤维素酶的吸附是不可逆的。纤维素酶在48目纤维上有最大的吸附焓变△H°,在28目纤维上有最大的吸附熵变△S°。反应温度、pH值和溶液中的离子强度是影响纤维素酶活力的主要因素。工业用纤维素酶可使用的温度为40～60℃,pH值为3～7,离子强度为20～100 mmol/L(以柠檬酸浓度表示)。响应面法优化漂白木浆酶水解的结果表明,经条件优化后,可溶糖转化率最大可达81.5%;葡萄糖转化率最大可达54.3%。木浆纤维在酶水解过程中,纤维形态是不断变化的,纤维长度和扭结角随酶水解时间的增加而减小,但漂白针叶木浆纤维和漂白阔叶木浆纤维形态的变化有明显的不同,而且两种纤维葡萄糖和可溶性糖的溶出速率也不同。纸浆的游离度和回用次数也是影响纤维酶水解的因素,游离度越低,水解效率越高;回用次数越多,水解效率越低。纤维素酶降解纤维是吸附和催化两个过程的综合作用,在实验温度范围内,纤维素酶的吸附与温度呈负相关关系,而纤维素酶的催化活性与温度呈正相关关系,两者的综合作用造成了不同长度的纤维酶水解与温度的依赖关系不同。细小纤维的酶水解更容易受到温度的影响,而长纤维的酶水解受温度的影响很小。酶水解活化能的计算证明了不同长度的纤维底物受温度影响的差异。因此,酶水解工业中应避免盲目升温到50℃进行酶水解而带来不必要的能量消耗。
阳离子聚丙烯酰胺以电荷补丁或电荷桥接的机理,增加纤维素酶分子在纸浆纤维上的吸附,进而提高酶水解效率。中等电荷量(40%)、低分子量(约3.4～4.5 MDA)的阳离子聚丙烯酰胺,用量为250 mg/L对增加纸浆纤维酶水解效率的效果最明显。适宜的搅拌强度可以提高阳离子聚丙烯酰胺存在时,纤维素酶水解的效率,实验表明,反应器中流体的雷诺数Re为298时最佳。当纤维被切断到一定程度后即延迟添加阳离子聚丙烯酰胺,也可提高聚合物的作用效果。阳离子聚丙烯酰胺对酶水解的促进作用具有广泛性,同样适用于淀粉酶水解系统。
纸浆污泥经过纤维素酶处理后,长纤维被切断成短纤维,提高了纤维粒子间结合的紧密程度,减少脱水后滤饼的孔隙率,增加其固含量,提高率最高可达6%。阳离子聚丙烯酰胺的加入可以减小脱水的阻力。纤维素酶提高纸浆污泥脱水固含量的方法,其投入和产出在经济上可达到平衡,随着纤维素酶性价比的提高以及污泥处理费用的持续增高,该方法有望得到广泛应用。
纸浆污泥酶水解系统中加入少量的氯胺T,可抑制反应过程中细菌的增长,并保持纤维素酶的活性。调节系统初始的pH值很重要,可使酶水解过程的pH值保持在对纤维素酶适宜的范围内。根据纸浆污泥酶水解的机理,建立酶水解过程的动力学模型:(?)。利用该模型,可根据初始纤维素酶用量和酶水解时间,准确预测酶水解的转化率。纸浆污泥酶水解在工厂的中试表明,500 mg/L用量的阳离子聚丙烯酰胺可以使葡萄糖产量和可溶性糖产量分别提高32%和24%,将为企业带来良好的经济效益。
Paper sludge is a major solid waste from pulp and paper industry. Because it contains 40% ~ 60% cellulose in the dry components, paper sludge could be used as a raw material for cellulose ethanol industry. Comparing with the traditional lignocellulosic materials, such as wood, crop residues and straws, paper sludge has its own advantages: low price and loose structure. Paper sludge as a material for ethanol product through enzymatic hydrolysis and fermentation, will expand the material resource of ethanol. In the meantime, it also decreases the total sludge discharge, lessens the cost for sludge disposal, reduces the pressure for environment. It would be a highly potential method for energy conversion of industry waste. In this thesis, using pulp fiber as substrate, the adsorption of cellulase on cellulose fiber and the physical and chemical changes during the enzymatic hydrolysis process were discussed. These would provide the theoretical support for the enzymatic hydrolysis of paper sludge. Additionally, a new method for improving the efficiency of enzymatic hydrolysis by cationic polymer was also proposed, which could significantly reduce the cost of enzymatic saccharification.
The adsorption of cellulase on cellulose fiber is the first step of the whole enzymatic hydrolysis process. It was found that the adsorption equilibrium was reached at the time of about 60 min. The kinetics of the adsorption process could be described by second order adsorption model, as the expression of(?). The adsorption equilibrium fits the Langmuir isotherm. The maximum adsorption amount was found in adsorption on short fiber, and the maximum Langmuir adsorption equilibrium constant in adsorption on long fiber, which indicated that cellulase shows the highest adsorption affinity on long fiber. Thermodynamics parameters were also calculated. The value of Gibbs energy change ?G ? was less than 0, value of enthalpy change ?H ? less than 0, value of entropy change ? S? higher than 0 , indicating that adsorption of cellulase on cellulose fiber is a spontaneous, exothermic and irreversible process. This adsorption process contains both physical adsorption and chemical adsorption. The highest enthalpy change ?H ? and entropy change ? S? were found in adsorption on 48 mesh fiber and 28 mesh fiber, respectively. Temperature, pH value and ionic strength in solution are the main factors affecting the activity of cellulase. For the industrial cellulase, it was found that temperature of 40～60℃, pH value of 3～7 and ionic strength of 20～100 mmol/L (expressed by concentration of citric acid) were the workable conditions. Response Surface Methodology was employed, to study the effects of temperature, cellulase dosage and pH on the efficiency of enzymatic hydrolysis of bleached pulp. At optimal conditions, the soluble sugar conversion could reach to 81.5%, and the glucose conversion to 54.3%. During the enzymatic hydrolysis process, the fiber configuration and quality change with reaction time. The changes were different for bleached softwood fiber and bleached hardwood fiber. In addition, the relative rate for glucose generation and soluble sugar generation in the hydrolysis was also not alike. The freeness and recycle times of pulp fiber also affect the enzymatic hydrolysis. With lower freeness, the efficiency was higher; with higher recycle times, the lower efficiency.
The hydrolysis process of cellulose by cellulase is a combination of two process units, adsorption and catalytic step. The adsorption of the enzyme to cellulosic fiber is inversely dependent on temperature, while catalytic step is directly temperature dependent at the experimental conditions. The integration of these two effects causes the difference of temperature dependence of enzymatic hydrolysis for fibers with different length. The fines were high temperature dependent and easy to be affected by temperature, but the longer fiber shows the opposite performance. The activation energy for enzymatic hydrolysis was calculated through first order rate equation, which could confirm the proposed theory. The avoidance cost (capital and operational) of heating this material to 50oC for the enzymatic hydrolysis is significant.
Cationic polyacrylamides(CPAM) could increase the adsorption of cellulase on cellulose fiber and then enchace the efficiency of enzymatic hydrolysis, by the mechanisms of charged patches or charged bridging. Cationic polyacrylamides with medium cationicity (40%), relatively low molecular weight (3.4～4.5 MDA) and dosage of 250 mg/L were found to have best efficiency-improving for enzymatic hydrolysis. Proper agitation could also enhance the efficiency, with the best Reynolds number of 298. It was found it had advantage to delay the addition of CPAM until the fiber was first shortened by the enzyme to a certain extent. The effect of CPAM in enzymatic hydrolysis system is universal, which could also be well used in the system of enzymatic hydrolysis of starch.
After cellulase treatment, the fiber in paper sludge was cleanly cut into smaller units, cake consolidation would be improved because it would be easier to pack short fibers into a cake than longer ones. In other words, a lower void volume and a higher cake density would obtain. The highest improvement for cake solids could reach 6%. Cationic polyacrylamides could reduce the resistance in the dewatering process. The application of this method in industry is break even now, but will become more attactive as the cost:performace of cellulase increases and the cost of sludge disposal is trending higher.
In the system of enzymatic hydrolysis of paper sludge, addition of small amount of Chloramine-T would restrain the growing of bacteria, and keep the cellulase active. Adjusting the initial pH value of the hydrolysis system is vital for controlling the whole process. The change of pH value during the hydrolysis would stay in the range suitable for cellulase. According to the mechanism of enzymatic hydrolysis, a kinetic model was proposed: (?). With varied intial enzyme loading and reaction time, precise conversion of hysrolysis could be expected. In the pilot scale of enzymatic hydrolysis of paper sludge, CPAM with dosage of 500 mg/L increased 32% of glucose production and 24% of soluble sugar production, respectively. It will highly benefit the industry.
纤维素酶在纤维上的吸附是酶水解反应的第一步。纤维素酶在纤维上吸附60 min时基本达到吸附平衡;纤维素酶吸附的过程满足二级吸附动力学模型,可表示为(?)的形式;纤维素酶在纤维上的吸附平衡满足Langmuir等温吸附;纤维素酶在短纤维上有最大的吸附量,而在长纤维上的吸附平衡常数最大,最易达到吸附平衡。纤维素酶吸附的热力学研究表明,纤维素酶吸附过程的吉布斯自由能△G°小于0,是自发过程,吸附同时存在物理吸附和化学吸附;吸附的反应焓变△H?为负值,表明吸附为放热过程;吸附的反应熵变△S°大于0,表明纤维素酶的吸附是不可逆的。纤维素酶在48目纤维上有最大的吸附焓变△H°,在28目纤维上有最大的吸附熵变△S°。反应温度、pH值和溶液中的离子强度是影响纤维素酶活力的主要因素。工业用纤维素酶可使用的温度为40～60℃,pH值为3～7,离子强度为20～100 mmol/L(以柠檬酸浓度表示)。响应面法优化漂白木浆酶水解的结果表明,经条件优化后,可溶糖转化率最大可达81.5%;葡萄糖转化率最大可达54.3%。木浆纤维在酶水解过程中,纤维形态是不断变化的,纤维长度和扭结角随酶水解时间的增加而减小,但漂白针叶木浆纤维和漂白阔叶木浆纤维形态的变化有明显的不同,而且两种纤维葡萄糖和可溶性糖的溶出速率也不同。纸浆的游离度和回用次数也是影响纤维酶水解的因素,游离度越低,水解效率越高;回用次数越多,水解效率越低。纤维素酶降解纤维是吸附和催化两个过程的综合作用,在实验温度范围内,纤维素酶的吸附与温度呈负相关关系,而纤维素酶的催化活性与温度呈正相关关系,两者的综合作用造成了不同长度的纤维酶水解与温度的依赖关系不同。细小纤维的酶水解更容易受到温度的影响,而长纤维的酶水解受温度的影响很小。酶水解活化能的计算证明了不同长度的纤维底物受温度影响的差异。因此,酶水解工业中应避免盲目升温到50℃进行酶水解而带来不必要的能量消耗。
阳离子聚丙烯酰胺以电荷补丁或电荷桥接的机理,增加纤维素酶分子在纸浆纤维上的吸附,进而提高酶水解效率。中等电荷量(40%)、低分子量(约3.4～4.5 MDA)的阳离子聚丙烯酰胺,用量为250 mg/L对增加纸浆纤维酶水解效率的效果最明显。适宜的搅拌强度可以提高阳离子聚丙烯酰胺存在时,纤维素酶水解的效率,实验表明,反应器中流体的雷诺数Re为298时最佳。当纤维被切断到一定程度后即延迟添加阳离子聚丙烯酰胺,也可提高聚合物的作用效果。阳离子聚丙烯酰胺对酶水解的促进作用具有广泛性,同样适用于淀粉酶水解系统。
纸浆污泥经过纤维素酶处理后,长纤维被切断成短纤维,提高了纤维粒子间结合的紧密程度,减少脱水后滤饼的孔隙率,增加其固含量,提高率最高可达6%。阳离子聚丙烯酰胺的加入可以减小脱水的阻力。纤维素酶提高纸浆污泥脱水固含量的方法,其投入和产出在经济上可达到平衡,随着纤维素酶性价比的提高以及污泥处理费用的持续增高,该方法有望得到广泛应用。
纸浆污泥酶水解系统中加入少量的氯胺T,可抑制反应过程中细菌的增长,并保持纤维素酶的活性。调节系统初始的pH值很重要,可使酶水解过程的pH值保持在对纤维素酶适宜的范围内。根据纸浆污泥酶水解的机理,建立酶水解过程的动力学模型:(?)。利用该模型,可根据初始纤维素酶用量和酶水解时间,准确预测酶水解的转化率。纸浆污泥酶水解在工厂的中试表明,500 mg/L用量的阳离子聚丙烯酰胺可以使葡萄糖产量和可溶性糖产量分别提高32%和24%,将为企业带来良好的经济效益。
Paper sludge is a major solid waste from pulp and paper industry. Because it contains 40% ~ 60% cellulose in the dry components, paper sludge could be used as a raw material for cellulose ethanol industry. Comparing with the traditional lignocellulosic materials, such as wood, crop residues and straws, paper sludge has its own advantages: low price and loose structure. Paper sludge as a material for ethanol product through enzymatic hydrolysis and fermentation, will expand the material resource of ethanol. In the meantime, it also decreases the total sludge discharge, lessens the cost for sludge disposal, reduces the pressure for environment. It would be a highly potential method for energy conversion of industry waste. In this thesis, using pulp fiber as substrate, the adsorption of cellulase on cellulose fiber and the physical and chemical changes during the enzymatic hydrolysis process were discussed. These would provide the theoretical support for the enzymatic hydrolysis of paper sludge. Additionally, a new method for improving the efficiency of enzymatic hydrolysis by cationic polymer was also proposed, which could significantly reduce the cost of enzymatic saccharification.
The adsorption of cellulase on cellulose fiber is the first step of the whole enzymatic hydrolysis process. It was found that the adsorption equilibrium was reached at the time of about 60 min. The kinetics of the adsorption process could be described by second order adsorption model, as the expression of(?). The adsorption equilibrium fits the Langmuir isotherm. The maximum adsorption amount was found in adsorption on short fiber, and the maximum Langmuir adsorption equilibrium constant in adsorption on long fiber, which indicated that cellulase shows the highest adsorption affinity on long fiber. Thermodynamics parameters were also calculated. The value of Gibbs energy change ?G ? was less than 0, value of enthalpy change ?H ? less than 0, value of entropy change ? S? higher than 0 , indicating that adsorption of cellulase on cellulose fiber is a spontaneous, exothermic and irreversible process. This adsorption process contains both physical adsorption and chemical adsorption. The highest enthalpy change ?H ? and entropy change ? S? were found in adsorption on 48 mesh fiber and 28 mesh fiber, respectively. Temperature, pH value and ionic strength in solution are the main factors affecting the activity of cellulase. For the industrial cellulase, it was found that temperature of 40～60℃, pH value of 3～7 and ionic strength of 20～100 mmol/L (expressed by concentration of citric acid) were the workable conditions. Response Surface Methodology was employed, to study the effects of temperature, cellulase dosage and pH on the efficiency of enzymatic hydrolysis of bleached pulp. At optimal conditions, the soluble sugar conversion could reach to 81.5%, and the glucose conversion to 54.3%. During the enzymatic hydrolysis process, the fiber configuration and quality change with reaction time. The changes were different for bleached softwood fiber and bleached hardwood fiber. In addition, the relative rate for glucose generation and soluble sugar generation in the hydrolysis was also not alike. The freeness and recycle times of pulp fiber also affect the enzymatic hydrolysis. With lower freeness, the efficiency was higher; with higher recycle times, the lower efficiency.
The hydrolysis process of cellulose by cellulase is a combination of two process units, adsorption and catalytic step. The adsorption of the enzyme to cellulosic fiber is inversely dependent on temperature, while catalytic step is directly temperature dependent at the experimental conditions. The integration of these two effects causes the difference of temperature dependence of enzymatic hydrolysis for fibers with different length. The fines were high temperature dependent and easy to be affected by temperature, but the longer fiber shows the opposite performance. The activation energy for enzymatic hydrolysis was calculated through first order rate equation, which could confirm the proposed theory. The avoidance cost (capital and operational) of heating this material to 50oC for the enzymatic hydrolysis is significant.
Cationic polyacrylamides(CPAM) could increase the adsorption of cellulase on cellulose fiber and then enchace the efficiency of enzymatic hydrolysis, by the mechanisms of charged patches or charged bridging. Cationic polyacrylamides with medium cationicity (40%), relatively low molecular weight (3.4～4.5 MDA) and dosage of 250 mg/L were found to have best efficiency-improving for enzymatic hydrolysis. Proper agitation could also enhance the efficiency, with the best Reynolds number of 298. It was found it had advantage to delay the addition of CPAM until the fiber was first shortened by the enzyme to a certain extent. The effect of CPAM in enzymatic hydrolysis system is universal, which could also be well used in the system of enzymatic hydrolysis of starch.
After cellulase treatment, the fiber in paper sludge was cleanly cut into smaller units, cake consolidation would be improved because it would be easier to pack short fibers into a cake than longer ones. In other words, a lower void volume and a higher cake density would obtain. The highest improvement for cake solids could reach 6%. Cationic polyacrylamides could reduce the resistance in the dewatering process. The application of this method in industry is break even now, but will become more attactive as the cost:performace of cellulase increases and the cost of sludge disposal is trending higher.
In the system of enzymatic hydrolysis of paper sludge, addition of small amount of Chloramine-T would restrain the growing of bacteria, and keep the cellulase active. Adjusting the initial pH value of the hydrolysis system is vital for controlling the whole process. The change of pH value during the hydrolysis would stay in the range suitable for cellulase. According to the mechanism of enzymatic hydrolysis, a kinetic model was proposed: (?). With varied intial enzyme loading and reaction time, precise conversion of hysrolysis could be expected. In the pilot scale of enzymatic hydrolysis of paper sludge, CPAM with dosage of 500 mg/L increased 32% of glucose production and 24% of soluble sugar production, respectively. It will highly benefit the industry.
引文
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