玉米芯半纤维素水解液发酵生产木糖醇的关键技术研究
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摘要
木糖醇是一种甜度与蔗糖相当、具有防龋齿、胰岛素不依赖性代谢等多种优良性质的五碳糖醇,在食品、医药、轻工业等领域具有广泛用途。传统的木糖醇生产方法是采用化学还原法,在高温高压下催化氢化纯木糖制得,该法生产成本较高,且镍催化剂会污染环境。本论文以假丝酵母(Candida sp.)为生产菌株,采用微生物发酵技术转化生产木糖醇,高效、节能、无污染,具有重要的社会及经济价值。
通常发酵法生产木糖醇多利用纯木糖为底物,成本过高,不利于大规模推广。本论文针对自然界中丰富的植物纤维原料玉米芯,通过稀酸水解法将其中的半纤维素转化为可发酵的木糖,再以水解液为原料发酵生产木糖醇,廉价而易得。在优化工艺条件下制得的玉米芯半纤维素水解液中,总糖含量为39.4 g/L(洗液重复利用、水解液未浓缩时),其中木糖占81.9%,乙酸、糠醛等发酵抑制物浓度低,发酵性能良好,无需经脱色及复杂脱毒处理便可由驯化的假丝酵母细胞直接转化为木糖醇,经济而高效,为工业化应用奠定了良好的基础。
对木糖醇发酵过程中的主要工艺参数进行了优化,并在3.7 L发酵罐中进行了放大试验。间歇发酵时,假丝酵母细胞转化纯木糖的生产速率为2.46 g/(L·h),木糖醇得率为0.83 g木糖醇/g木糖；转化半纤维素水解液的生产速率为0.66 g/(L·h),木糖醇得率为0.75 g木糖醇/g木糖。进一步采取补料分批发酵方式,对半纤维素水解液而言,可减轻底物、产物及乙酸、糠醛等发酵抑制物的抑制作用,有利于提高发酵效率。若采用连续发酵方式,当控制半纤维素水解液的稀释率为0.015 h-1时,木糖醇生产速率和得率分别比间歇发酵时提高了63.23%和5.33%,酵母细胞的发酵潜能得到较大发挥。
由于假丝酵母在木糖醇发酵前期需要较高的氧气供应,以促进菌体快速生长,而后期则需要微氧环境,以利于木糖醇的大量积累；因而本论文采用固定化酵母细胞发酵,可省去菌体的营养生长阶段,直接进入产物合成阶段,有利于缩短发酵周期,便于实现连续生产及自动化控制。在2.0 L鼓泡式生物反应器中,海藻酸钙凝胶包埋固定的假丝酵母细胞发酵玉米芯半纤维素水解液生产木糖醇,持续稳定,经济高效,可以重复利用,发酵效果良好,具有明显的特色。
对木糖醇发酵液的脱色、纯化与结晶工艺条件进行了初步有效的探索,证实了从纯化后的发酵液中获得高纯度木糖醇晶体的可行性,显示出良好的工业应用前景。本文采用驯化后的假丝酵母细胞,发酵经过简单脱毒处理的玉米芯半纤维素水解液生产木糖醇,工艺简单、对环境友好、能耗低、发酵效率高、产品质量好；相关研究结果对于促进作为重要可再生资源的玉米芯的转化利用及木糖醇工业化生产的可持续发展具有重要意义。
Xylitol, a five-carbon sugar alcohol, has many applications in the food, pharmaceutical, and odontological industries, owing to its sweetening power equal to sucrose, anticariogenic properties, and insulin-independent metabolism. Xylitol is currently manufactured by catalytic hydrogenation of xylose, an expensive process mainly because it involves a large number of purification steps. In addition, the nickel catalyst used can pollute the environment. This research investigated the bioconversion of xylose to xylitol by Candida sp., a biological xylitol production process, which holds great economic and social value compared to the chemical process. This microbial production of xylitol requires neither xylose purification nor high temperatures and pressures. It bears many advantages, including high specificity and low energy requirements, and more importantly, no environmental pollution.
Generally, synthetic xylose solution was used as the fermentation medium for producing xylitol. However, the high overall processing costs prevent it from a large scale application. Corn cob, consisting of mainly lignocellulosic residue, has many advantages: renewable, widespread, inexpensive, and xylose-abundant. Firstly, the degradation of corn cob hemicellulose into fermentable xylose, which can be performed by dilute sulphuric acid hydrolysis. Subsequently, the corn cob hemicellulosic hydrolysates could be efficiently converted to xylitol by the adapted Candida sp. cells. Totally,39.4 g/L reducing sugar exists in the corn cob hemicellulosic hydrolysates, which were prepared under the optimal process parameters (Reuse of wash water and unconcentrated hemicellulosic hydrolysates). Xylose constitutes 81.9% of the total reducing sugar, and concentrations of acetic acid, furfuraldehyde and other fermentation inhibitors were lower than the maximum allowable concentration of each inhibitor. In this thesis, the corn cob hemicellulosic hydrolysates could be directly and efficiently converted to xylitol without decolorization or ion-exchanged by the adapted Candida sp. cells. This process can effectively reduce the pretreatment costs, and its fermentation results are desirable, laying favourable foundation for industrial applications.
In this study, we optimized the major processing parameters for xylitol fermentation by an adapted Candida sp. Moreover, a series of scale-up xylitol fermentation experiments were performed in a 3.7-L fermentator. Meanwhile, batch xylitol fermentations were carried out under the optimum culture conditions by Candida sp. Fermentation on synthetic xylose solutions produces xylitol at an average rate of 2.46 g/(L·h) and yield of 0.83 g xylitol/g xylose, whereas fermentation on hemicellulosic hydrolysates generates the average xylitol productivity (0.66 g/(L·h)) and xylitol yield (0.75 g xylitol/g xylose). In addition, fed-batch cultures of hemicellulosic hydrolysates were employed, which could alleviate the inhibition of xylose, xylitol, acetic acid, furfuraldehyde, and other inhibitors, and therefore might increase the fermentation efficiency. As for the continuous xylitol fermentation on hemicellulosic hydrolysates, the best results were obtained with a dilution rate of 0.015 h-1, compared to batch fermentation, the xylitol productivity and xylitol yield increased by 63.23% and 5.33%, respectively.
In the early stage of the xylitol fermentation by Candida sp., relatively high oxygen supply favoured higher cell growth, but in the later stage, relatively low oxygen supply, led to substantial accumulation of xylitol. Therefore, in this research, immobilized Candida sp. cells were used for xylitol fermentations on corn cob hemicellulosic hydrolysates, which can omit the cell growth phase, direct toward xylitol biosynthesis immediately, shorten the fermentation period, readily employ continuous production, and easily automate the process. In a 2.0 L bubble column bioreactor, xylitol fermentations on corn cob hemicellulosic hydrolysates were carried out under the optimized conditions by Candida sp. cells immobilized in calcium alginate, the fermentation results were good and continuously stable, the immobilized cells could be successively reused, rendering the process efficient and economical, and other beneficial characteristics.
The process parameters of decolorization, purification and crystallization of the xylitol fermented broths had been investigated primarily, and the results confirmed the feasibility of xylitol crystallization from purified fermentation broths, showing broad prospects of industrial applications. This work developed an environmental-friendly, simple, efficient, and economical xylitol production process by fermentation on corn cob hemicellulosic hydrolysates using the adapted Candida sp. The results should not only favor higher biconversion of corn cob, an important renewable resource, but also help the persistent development of xylitol industrial production.
通常发酵法生产木糖醇多利用纯木糖为底物,成本过高,不利于大规模推广。本论文针对自然界中丰富的植物纤维原料玉米芯,通过稀酸水解法将其中的半纤维素转化为可发酵的木糖,再以水解液为原料发酵生产木糖醇,廉价而易得。在优化工艺条件下制得的玉米芯半纤维素水解液中,总糖含量为39.4 g/L(洗液重复利用、水解液未浓缩时),其中木糖占81.9%,乙酸、糠醛等发酵抑制物浓度低,发酵性能良好,无需经脱色及复杂脱毒处理便可由驯化的假丝酵母细胞直接转化为木糖醇,经济而高效,为工业化应用奠定了良好的基础。
对木糖醇发酵过程中的主要工艺参数进行了优化,并在3.7 L发酵罐中进行了放大试验。间歇发酵时,假丝酵母细胞转化纯木糖的生产速率为2.46 g/(L·h),木糖醇得率为0.83 g木糖醇/g木糖；转化半纤维素水解液的生产速率为0.66 g/(L·h),木糖醇得率为0.75 g木糖醇/g木糖。进一步采取补料分批发酵方式,对半纤维素水解液而言,可减轻底物、产物及乙酸、糠醛等发酵抑制物的抑制作用,有利于提高发酵效率。若采用连续发酵方式,当控制半纤维素水解液的稀释率为0.015 h-1时,木糖醇生产速率和得率分别比间歇发酵时提高了63.23%和5.33%,酵母细胞的发酵潜能得到较大发挥。
由于假丝酵母在木糖醇发酵前期需要较高的氧气供应,以促进菌体快速生长,而后期则需要微氧环境,以利于木糖醇的大量积累；因而本论文采用固定化酵母细胞发酵,可省去菌体的营养生长阶段,直接进入产物合成阶段,有利于缩短发酵周期,便于实现连续生产及自动化控制。在2.0 L鼓泡式生物反应器中,海藻酸钙凝胶包埋固定的假丝酵母细胞发酵玉米芯半纤维素水解液生产木糖醇,持续稳定,经济高效,可以重复利用,发酵效果良好,具有明显的特色。
对木糖醇发酵液的脱色、纯化与结晶工艺条件进行了初步有效的探索,证实了从纯化后的发酵液中获得高纯度木糖醇晶体的可行性,显示出良好的工业应用前景。本文采用驯化后的假丝酵母细胞,发酵经过简单脱毒处理的玉米芯半纤维素水解液生产木糖醇,工艺简单、对环境友好、能耗低、发酵效率高、产品质量好；相关研究结果对于促进作为重要可再生资源的玉米芯的转化利用及木糖醇工业化生产的可持续发展具有重要意义。
Xylitol, a five-carbon sugar alcohol, has many applications in the food, pharmaceutical, and odontological industries, owing to its sweetening power equal to sucrose, anticariogenic properties, and insulin-independent metabolism. Xylitol is currently manufactured by catalytic hydrogenation of xylose, an expensive process mainly because it involves a large number of purification steps. In addition, the nickel catalyst used can pollute the environment. This research investigated the bioconversion of xylose to xylitol by Candida sp., a biological xylitol production process, which holds great economic and social value compared to the chemical process. This microbial production of xylitol requires neither xylose purification nor high temperatures and pressures. It bears many advantages, including high specificity and low energy requirements, and more importantly, no environmental pollution.
Generally, synthetic xylose solution was used as the fermentation medium for producing xylitol. However, the high overall processing costs prevent it from a large scale application. Corn cob, consisting of mainly lignocellulosic residue, has many advantages: renewable, widespread, inexpensive, and xylose-abundant. Firstly, the degradation of corn cob hemicellulose into fermentable xylose, which can be performed by dilute sulphuric acid hydrolysis. Subsequently, the corn cob hemicellulosic hydrolysates could be efficiently converted to xylitol by the adapted Candida sp. cells. Totally,39.4 g/L reducing sugar exists in the corn cob hemicellulosic hydrolysates, which were prepared under the optimal process parameters (Reuse of wash water and unconcentrated hemicellulosic hydrolysates). Xylose constitutes 81.9% of the total reducing sugar, and concentrations of acetic acid, furfuraldehyde and other fermentation inhibitors were lower than the maximum allowable concentration of each inhibitor. In this thesis, the corn cob hemicellulosic hydrolysates could be directly and efficiently converted to xylitol without decolorization or ion-exchanged by the adapted Candida sp. cells. This process can effectively reduce the pretreatment costs, and its fermentation results are desirable, laying favourable foundation for industrial applications.
In this study, we optimized the major processing parameters for xylitol fermentation by an adapted Candida sp. Moreover, a series of scale-up xylitol fermentation experiments were performed in a 3.7-L fermentator. Meanwhile, batch xylitol fermentations were carried out under the optimum culture conditions by Candida sp. Fermentation on synthetic xylose solutions produces xylitol at an average rate of 2.46 g/(L·h) and yield of 0.83 g xylitol/g xylose, whereas fermentation on hemicellulosic hydrolysates generates the average xylitol productivity (0.66 g/(L·h)) and xylitol yield (0.75 g xylitol/g xylose). In addition, fed-batch cultures of hemicellulosic hydrolysates were employed, which could alleviate the inhibition of xylose, xylitol, acetic acid, furfuraldehyde, and other inhibitors, and therefore might increase the fermentation efficiency. As for the continuous xylitol fermentation on hemicellulosic hydrolysates, the best results were obtained with a dilution rate of 0.015 h-1, compared to batch fermentation, the xylitol productivity and xylitol yield increased by 63.23% and 5.33%, respectively.
In the early stage of the xylitol fermentation by Candida sp., relatively high oxygen supply favoured higher cell growth, but in the later stage, relatively low oxygen supply, led to substantial accumulation of xylitol. Therefore, in this research, immobilized Candida sp. cells were used for xylitol fermentations on corn cob hemicellulosic hydrolysates, which can omit the cell growth phase, direct toward xylitol biosynthesis immediately, shorten the fermentation period, readily employ continuous production, and easily automate the process. In a 2.0 L bubble column bioreactor, xylitol fermentations on corn cob hemicellulosic hydrolysates were carried out under the optimized conditions by Candida sp. cells immobilized in calcium alginate, the fermentation results were good and continuously stable, the immobilized cells could be successively reused, rendering the process efficient and economical, and other beneficial characteristics.
The process parameters of decolorization, purification and crystallization of the xylitol fermented broths had been investigated primarily, and the results confirmed the feasibility of xylitol crystallization from purified fermentation broths, showing broad prospects of industrial applications. This work developed an environmental-friendly, simple, efficient, and economical xylitol production process by fermentation on corn cob hemicellulosic hydrolysates using the adapted Candida sp. The results should not only favor higher biconversion of corn cob, an important renewable resource, but also help the persistent development of xylitol industrial production.
引文
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