利用玉米芯半纤维素水解液发酵生产木糖醇的研究
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摘要
玉米芯是廉价的可再生资源,利用玉米芯半纤维素水解液发酵生产木糖醇,与传统的加氢催化工艺相比,具有原料成本低、反应条件温和、能耗小、产品质量好等优势。本文对半纤维素水解液的制备工艺以及水解液的脱毒处理、菌种的驯化改良、木糖醇发酵过程的调控与优化以及产品的提取结晶等关键技术进行了深入研究,主要结果如下:
(1)对玉米芯半纤维素水解过程中的主要影响因素进行了研究,确立了水解工艺条件为:1.00%硫酸,液固比8:1,108℃,水解3h。
(2)对玉米芯半纤维素水解液的成分进行了检测分析,发现乙酸、糠醛是主要的发酵抑制物。当乙酸浓度高于1.20g/L、糠醛浓度高于0.50g/L时,木糖醇发酵被明显抑制。比较了石灰中和、活性炭吸附、汽提挥发和离子交换树脂吸附等方法对玉米芯半纤维素水解液脱毒效果的影响。结果表明,采用活性炭吸附或离子交换树脂脱毒,虽然能够除去水解液中大部分乙酸和糠醛,但成本相对较高;石灰中和对水解液中乙酸的去除率为5.20%,糠醛的去除率为6.66%,同时结合浓缩汽提挥发脱毒,乙酸的去除率可达55.20%,糠醛的去除率为73.30%。该方法经济简便,具有良好的实际应用价值。
(3)采用逐步提高培养基中发酵抑制物浓度的方法,对木糖醇发酵菌种(Candida sp.)进行驯化改良。驯化后的菌株耐毒性能增强,对乙酸浓度的耐受力从1.20g/L提高到5.41g/L;利用驯化后的菌株可以直接发酵只经简单脱毒处理的半纤维素水解液生产木糖醇,并且木糖醇产率比出发菌株提高了4.18倍。
(4)对木糖醇发酵的主要工艺参数进行了优化,确立了适宜的发酵条件为:初始木糖浓度100g/L,玉米浆2.00%,初始pH6.00,温度32℃,初始细胞浓度0.60g/L。对半纤维素水解液发酵生产木糖醇的时间进程进行了研究,结果表明:发酵前期为菌体的营养生长阶段,需要较高的溶氧和营养用作细胞增殖;菌体生长进入平衡阶段后,木糖醇开始大量累积。适当延长平衡阶段,有利于提高木糖醇产率。
(5)研究了通气条件对木糖醇发酵的影响,发现前期采用较大的通气速率,有利于发酵抑制物的挥发和细胞的营养生长;发酵后期采用相对低的通气速率,可以提高假丝酵母细胞酶系中NADH/NAD~+比值,抑制依赖NAD~+的木糖醇脱氢酶活力,有利于木糖醇的积累。在3.7L发酵罐中采用分阶段改变通气速率(0～24h,3.75L/min,24～96h,0.75L/min)发酵生产木糖醇,木糖醇产率和体积生
Corncob is a kind of cheap renewable lignocellulosic resource. The bioconversion of hemicellulosic hydrolysate to xylitol by microorganisms could be a cheaper alternative to the traditional chemical process, since it is a simple process, with great specificity and low energy requirements. Key factors in xylitol production, such as detoxification of hemicellulosic hydrolysate, strain adaptation, fermentation parameters optimization and xylitol recovery were investigated in this work. The main results were as follows:The optimal kinetic parameters for hydrolysis were concentration of H_2SO_4 of 1.00%, hydrolysis reaction temperature of 108 ℃, hydrolysis reaction time of 3 h and the ratio of liquid/solid of 8.It was found that acetic acid and furfural were the primary fermentation inhibitor in corncob hemicellulosic hydrolysate. When the concentrations of acetic acid and furfural exceeded 1.20 g/L and 0.50 g/L respectively, xylitol fermentation process ceased. Detoxification methods, such as lime neutralization, evaporation, absorption with activated charcoal and treatment with anion-exchange resins could remove a given amount of inhibitor. The results indicated that lime neutralization followed by evaporation could remove 55.20% of acetic acid and 73.30% of furfural in the hemicellulosic hydrolysate.Strain adaptation was carried out by increasing the inhibitor concentration in hemicellulosic hydrolysate medium gradually. A well-adapted strain of Candida sp. ZU04 was isolated in the 19th batch by this continuous adaptation process. Candida sp. ZU04 adaptability of acetic acid was increased from 1.20 g/L to 5.41 g/L compared with initial strain. With the strain, xylitol was produced from hemicellulosic hydrolysate untreated with activated carbon or ion-exchange resins, and the xylitol yield increased 4.18 folds compared with initial strain. This process can effectively reduce the detoxification treatment costs, showing broad prospects of industrial applications.The optimum fermentation conditions by Candida sp.ZU04 were as follows: xylose concentration, 100 g/L;corn steep liquor concentration, 2.00%;temperature,32 ℃;initial pH value, 6.00;initial cell concentration, 0.60 g/L. Time course of xylitol production from corn cob hemicellulosic hydrolysate by Candida sp.ZU04 was investigated. Time course showed that biomass increased at the expense of oxygen uptake and xylose consumption at the initial stage of fermentation, then xylitol began
to accumulate when the cells grew well. If this fermentation stage was prolonged, xylitol yield would be improved to some extent.According to the xylitol fermentation mechanism, high aeration rate was applied in the initial stage of fermentation process, and partial inhibitors were decomposed and glucose was consumed by Candida sp.ZU-04 for biomass growth. In the latter fermentation phase aeration rate was reduced, the high ratio of NADH/NAD+ reduce activities of xylitol dehydrogenase(XDH), so xylitol yield could be improved. The maximum xylitol yield (76.00%) and volumetric productivity (0.76 g-L^-h"1) were obtained with the two-phase aeration (0~24 h, 3.75 L/min, 24~96 h, 0.75 L/min).Xylitol production from hemicellulosic hydrolysate by recycling cells of Candida sp.ZU04 was proved to be an effective method. In 10 repeated fermentation batches using recycling cells, the average xylitol yield, xylitol concentration and fermentation time were 77.72%, 98.20 g/L and 55.10 h respectively.Xylitol recovery from fermentation broth is a difficult step due to the low xylitol concentration and complex components in the fermentation broth. In order to optimize the decoloration of xylitol broth, macroreticular resins and activated carbons were applied in xylitol fermentation broth decoloration. Results showed that activated carbon AC3 performed best in the absorption of coloring matters. The absorption ratio of coloring matters reached 96.00% and the loss of xylitol was 3.90% when treated with 3% activated carbon AC3 at pH 6.0, 80°C for 40 min. The clarified xylitol fermentation broth was treated with a strong cation-exchange resin D001 and a weak anion-exchange resin D301 to desalt the salt and absorb the impurity after which xylitol crystallization was attempted. Results showed that most of salt was removed from the xylitol broth by the combined D001 and D301 resins. After treating with activated carbon and combined resins, xylitol purity degree was increased from 61.19% to 91.00%.Xylitol crystallization kinetics was investigated in the xylitol broth and xylitol solution. As far as xylitol solution is concerned, xylitol concentration does not drop in the initial crystallization phase, so xylitol solution has longer crystallization time than xylitol broth. Temperature and initial xylitol concentration are two key factors in xylitol crystallization process. Xylitol crystallization rate increased with the increased xylitol concentration. However, xylitol crystallization rate decreased when the crystallization temperature was enhanced. By adding l%0 xylitol crystal seeds, crystallization time was shortened, and improved crystallization rate was obtained. The best result in term of xylitol crystal yield (58.00%) was obtained with
concentrated xylitol broth (750 g/L of xylitol) at temperature (—5°C) by adding l%o xylitol crystal seeds.The xylitol crystallization kinetic model was as follows:(C-C*) 05- (Co-C*) °-5=-0.5KtThe mathematic model was set up for this complex xylitol crystallization process. Comparing with the experiment data, this model could excellently simulate to the experiment data.The xylitol crystal obtained from the xylitol broth has regular tetrahedral shape, and its purity reached 99.9% (analysed by HPLC). After identifying by the IR . MS and tfMF, the crystal is ascertained as xylitol, and xylitol crystal matches the trade grade level.The research work developed an environmental-friendly and economical xylitol production process by fermentation on corncob hemicellulosic hydrolysate using the adapted strain of Candida sp.Z\J04, showing broad prospects of industrial applications.
(1)对玉米芯半纤维素水解过程中的主要影响因素进行了研究,确立了水解工艺条件为:1.00%硫酸,液固比8:1,108℃,水解3h。
(2)对玉米芯半纤维素水解液的成分进行了检测分析,发现乙酸、糠醛是主要的发酵抑制物。当乙酸浓度高于1.20g/L、糠醛浓度高于0.50g/L时,木糖醇发酵被明显抑制。比较了石灰中和、活性炭吸附、汽提挥发和离子交换树脂吸附等方法对玉米芯半纤维素水解液脱毒效果的影响。结果表明,采用活性炭吸附或离子交换树脂脱毒,虽然能够除去水解液中大部分乙酸和糠醛,但成本相对较高;石灰中和对水解液中乙酸的去除率为5.20%,糠醛的去除率为6.66%,同时结合浓缩汽提挥发脱毒,乙酸的去除率可达55.20%,糠醛的去除率为73.30%。该方法经济简便,具有良好的实际应用价值。
(3)采用逐步提高培养基中发酵抑制物浓度的方法,对木糖醇发酵菌种(Candida sp.)进行驯化改良。驯化后的菌株耐毒性能增强,对乙酸浓度的耐受力从1.20g/L提高到5.41g/L;利用驯化后的菌株可以直接发酵只经简单脱毒处理的半纤维素水解液生产木糖醇,并且木糖醇产率比出发菌株提高了4.18倍。
(4)对木糖醇发酵的主要工艺参数进行了优化,确立了适宜的发酵条件为:初始木糖浓度100g/L,玉米浆2.00%,初始pH6.00,温度32℃,初始细胞浓度0.60g/L。对半纤维素水解液发酵生产木糖醇的时间进程进行了研究,结果表明:发酵前期为菌体的营养生长阶段,需要较高的溶氧和营养用作细胞增殖;菌体生长进入平衡阶段后,木糖醇开始大量累积。适当延长平衡阶段,有利于提高木糖醇产率。
(5)研究了通气条件对木糖醇发酵的影响,发现前期采用较大的通气速率,有利于发酵抑制物的挥发和细胞的营养生长;发酵后期采用相对低的通气速率,可以提高假丝酵母细胞酶系中NADH/NAD~+比值,抑制依赖NAD~+的木糖醇脱氢酶活力,有利于木糖醇的积累。在3.7L发酵罐中采用分阶段改变通气速率(0～24h,3.75L/min,24～96h,0.75L/min)发酵生产木糖醇,木糖醇产率和体积生
Corncob is a kind of cheap renewable lignocellulosic resource. The bioconversion of hemicellulosic hydrolysate to xylitol by microorganisms could be a cheaper alternative to the traditional chemical process, since it is a simple process, with great specificity and low energy requirements. Key factors in xylitol production, such as detoxification of hemicellulosic hydrolysate, strain adaptation, fermentation parameters optimization and xylitol recovery were investigated in this work. The main results were as follows:The optimal kinetic parameters for hydrolysis were concentration of H_2SO_4 of 1.00%, hydrolysis reaction temperature of 108 ℃, hydrolysis reaction time of 3 h and the ratio of liquid/solid of 8.It was found that acetic acid and furfural were the primary fermentation inhibitor in corncob hemicellulosic hydrolysate. When the concentrations of acetic acid and furfural exceeded 1.20 g/L and 0.50 g/L respectively, xylitol fermentation process ceased. Detoxification methods, such as lime neutralization, evaporation, absorption with activated charcoal and treatment with anion-exchange resins could remove a given amount of inhibitor. The results indicated that lime neutralization followed by evaporation could remove 55.20% of acetic acid and 73.30% of furfural in the hemicellulosic hydrolysate.Strain adaptation was carried out by increasing the inhibitor concentration in hemicellulosic hydrolysate medium gradually. A well-adapted strain of Candida sp. ZU04 was isolated in the 19th batch by this continuous adaptation process. Candida sp. ZU04 adaptability of acetic acid was increased from 1.20 g/L to 5.41 g/L compared with initial strain. With the strain, xylitol was produced from hemicellulosic hydrolysate untreated with activated carbon or ion-exchange resins, and the xylitol yield increased 4.18 folds compared with initial strain. This process can effectively reduce the detoxification treatment costs, showing broad prospects of industrial applications.The optimum fermentation conditions by Candida sp.ZU04 were as follows: xylose concentration, 100 g/L;corn steep liquor concentration, 2.00%;temperature,32 ℃;initial pH value, 6.00;initial cell concentration, 0.60 g/L. Time course of xylitol production from corn cob hemicellulosic hydrolysate by Candida sp.ZU04 was investigated. Time course showed that biomass increased at the expense of oxygen uptake and xylose consumption at the initial stage of fermentation, then xylitol began
to accumulate when the cells grew well. If this fermentation stage was prolonged, xylitol yield would be improved to some extent.According to the xylitol fermentation mechanism, high aeration rate was applied in the initial stage of fermentation process, and partial inhibitors were decomposed and glucose was consumed by Candida sp.ZU-04 for biomass growth. In the latter fermentation phase aeration rate was reduced, the high ratio of NADH/NAD+ reduce activities of xylitol dehydrogenase(XDH), so xylitol yield could be improved. The maximum xylitol yield (76.00%) and volumetric productivity (0.76 g-L^-h"1) were obtained with the two-phase aeration (0~24 h, 3.75 L/min, 24~96 h, 0.75 L/min).Xylitol production from hemicellulosic hydrolysate by recycling cells of Candida sp.ZU04 was proved to be an effective method. In 10 repeated fermentation batches using recycling cells, the average xylitol yield, xylitol concentration and fermentation time were 77.72%, 98.20 g/L and 55.10 h respectively.Xylitol recovery from fermentation broth is a difficult step due to the low xylitol concentration and complex components in the fermentation broth. In order to optimize the decoloration of xylitol broth, macroreticular resins and activated carbons were applied in xylitol fermentation broth decoloration. Results showed that activated carbon AC3 performed best in the absorption of coloring matters. The absorption ratio of coloring matters reached 96.00% and the loss of xylitol was 3.90% when treated with 3% activated carbon AC3 at pH 6.0, 80°C for 40 min. The clarified xylitol fermentation broth was treated with a strong cation-exchange resin D001 and a weak anion-exchange resin D301 to desalt the salt and absorb the impurity after which xylitol crystallization was attempted. Results showed that most of salt was removed from the xylitol broth by the combined D001 and D301 resins. After treating with activated carbon and combined resins, xylitol purity degree was increased from 61.19% to 91.00%.Xylitol crystallization kinetics was investigated in the xylitol broth and xylitol solution. As far as xylitol solution is concerned, xylitol concentration does not drop in the initial crystallization phase, so xylitol solution has longer crystallization time than xylitol broth. Temperature and initial xylitol concentration are two key factors in xylitol crystallization process. Xylitol crystallization rate increased with the increased xylitol concentration. However, xylitol crystallization rate decreased when the crystallization temperature was enhanced. By adding l%0 xylitol crystal seeds, crystallization time was shortened, and improved crystallization rate was obtained. The best result in term of xylitol crystal yield (58.00%) was obtained with
concentrated xylitol broth (750 g/L of xylitol) at temperature (—5°C) by adding l%o xylitol crystal seeds.The xylitol crystallization kinetic model was as follows:(C-C*) 05- (Co-C*) °-5=-0.5KtThe mathematic model was set up for this complex xylitol crystallization process. Comparing with the experiment data, this model could excellently simulate to the experiment data.The xylitol crystal obtained from the xylitol broth has regular tetrahedral shape, and its purity reached 99.9% (analysed by HPLC). After identifying by the IR . MS and tfMF, the crystal is ascertained as xylitol, and xylitol crystal matches the trade grade level.The research work developed an environmental-friendly and economical xylitol production process by fermentation on corncob hemicellulosic hydrolysate using the adapted strain of Candida sp.Z\J04, showing broad prospects of industrial applications.
引文
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66. Sene L. Effects of Environmental Confitions on Xylose Reductase and Xylitol Dehydrogenase Production by Candida guilliermondii [J], Appl Biochem Biotechnol.,2000, 84-86: 371-380
67. Kastner JR, Roberts RS, Jones W J, Effects of pH on cell viability and product yields in D-xylose fermentations by Candida shehatae [J], Appl. Microbiol. BIotechnol.,1996, 45: 224-228
68. Maria GA, et al. Fermentation of sugar cane bagasse hemicellulosic hydrolysate for xylitol production: effect of pH [J]. Bioresour. Bioenerg.,1997, 11 : 11-14
69. Olsson L, Hahn-Hagerdal B. Fermentation of lignocellulosic hydrolysates for enthanol production[J].Enzyme Microb Technol,1986, 18:312-331
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72. Girio FM, Roseiro JC, S P, Duarte-Reis AR, Amaral-Collaco MT. Effect of oxygen transfer rate on levels of keyenzymes of xylose metabolism in Debaryomyces hansenii[J]. Enzyme Microb. Technol.,1994, 16:1074-1078
73. Girio FM, Amaro C, Azinheira H, Pelica F, Amaral-Collaco MT. Polyols production during single and mixed substrate fermentation in Debaryomyces hansenii[J]. Bioresour Technol., 2000, 71:245-251
74. Granstrom TB, Aristidou AA, Jokela J, Leisola M. Growth characteristics and metabolic flux analysis of Candida milleri[J]. Biotechnol Bioeng.,2000, 70:197-207
75. Dominguez JM, Gong CS, Tsao GT. Pretreatment of sugar cane bagasse hemicellulose hydrolysate for xylitol production by yeasts[J]. Appl Biochem Biotechnol, 1996, 57-58: 49-56.
76. Felipe MGA, Vitolo M, Mancilha IM, Silva SS. Environmental parameters affecting xylitol production from sugar cane bagasse hemicellulosic hydrolyzate by Candida guilliermondii[J]. J Ind Microbiol Biotechno1,1997, 18: 251-254
77. Denise C, Rodrigues C, et al. Xylose Reductase Activity of Candida guilliermondii During Xyltiol Production by Fed-Batch Fermentation[J]. Appl. Biochem. Biotechnol., 2002, 98-100: 875-883
78. Tavares JM, et al. The influence of hexoses addition on the fermentation of D-xylose in Debaryomyces hansenii under continuous cultivation [J], Enzyme Microb. Technol., 2000, 26: 743—747
79. Mayerhoff ZD, Roberto I C, Silva S S, Production of Xylitol by Candida mogii from Rice Straw Hydrolysate [J], Appl. Biochem. Biotechnol.,1998, 70-72:150-159
80. Dahiya JS. Xylitol production by Petromyces albertensis grown on medium containing D-xylose [J]. Can. J. Microbiol, 1991, 37:14-18
81. Barbosa MFS, Medeiros MB, Mancilha IM, Schneider H, Lee H. Screening of yeasts for production of xylitol from D-xylose and some factors which affect xylitol yield in Candida guilliermondii[J]. J Ind Microbiol., 1988, 3:241-251
82. Paul A. Belter EL, Cussler, Wei-shou Hu, Bioseparations: downstream processing for biotechnology[M], A Wiley-Interscience Publication JOHN WILEY SONS,1988:273-303
83.E.B.哈姆斯基.《化学工业中的结晶》[M]化学工业出版社.1984:36—40
84. Hartel, RW, Shastry AV. (1991). Sugar crystallization in food products [J]. Critical Reviews in Food Science and Nutrition, 1(1) : 49-112
85. Pyne A., Surana R., Suryanarayanan R. Crystallization of mannitol below Tg during freeze-drying in binary and ternary aqueous systems[J]. Pharm. Res., 2002, 19:901-908
86. Yu L, Milton N, Groleau EG. Mishra, D. S., Vansickle, R. E. Existence of a mannitol hydrate during freeze-drying and practical implications[J]. J. Pharm. Sci., 1999, 88:196-198
87. Murase N, Franks F. Salt precipitation during the freeze-concentration of phosphate buffer solutions[J]. Biophys. Chem., 1989, 34:293-300
88. Telang C, Yu L, Suryanarayanan R. Effective inhibition of mannitol crystallization in frozen solutions by sodium chloride[J]. Pharm. Res.,2003, 20:660-667
89. Torrado S, Torrado S. Characterization of physical state of mannitol after freeze-drying: effect of acetylsalicylic acid as a second crystalline cosolute[J]. Chem. Pharm. Bull., 2002, 50: 567-570
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