从农副产品生产乙醇的新技术

木材和农作物残余物,主要由纤维素和半纤维素组成,在发酵法生产乙醇过程中,纤维素水解成D-萄葡糖,半纤维素被水解成五碳糖D-木糖,普通的酵母使葡萄糖转变成乙醇,但不能使木糖发酵。因此在通常的发酵过程中原料中大量的半纤维素都变成了废物。最近美国北部地区农业研究中心的科学家们正在开发一项新的有希望的方法,利用法国科学家1957年在鞣革废物中发现的酵母菌Pachysolen tannophilus的特殊胁变,可使D-木糖直接发酵转变成乙醇。在他们早期的间歇法试验中,每克木糖产生0.34克乙醇,这种酵母的生长需要有氧条件。在32℃温度     

遗传工程提高乙醇产量

美国太阳能研究所 (SERI) 的研究人员正通过遗传工程技术来降低以纤维质的生物量原料如麦秸、木屑等生产燃料乙醇的成本。技术关键是木糖异构酶的生产,这是一种能挺高将五碳糖小糖发酵成乙醇功效的细     

与膜耦合的细胞固定化串联发酵制乙醇的研究

利用植物纤维作为廉价的糖源生产燃料乙醇是解决世界能源危机的最有效途径.今研究采用海藻酸钙固定普通酿酒酵母细胞和嗜鞣管囊酵母细胞于两个串联的发酵罐内,连续发酵葡萄糖和木糖组成的糖液并与膜耦合来制取酒精.通过硅橡胶膜(PDMS)的渗透蒸发过程,将产品乙醇从发酵液中移出,减少了产物乙醇对发酵的抑制作用.实验结果表明,这套采用海藻酸钙固定酵母细胞进行连续发酵并与膜耦合的生物反应器系统,在稀释率为0.321 h-1下稳定运行,剩余葡萄糖和木糖浓度分别为0.134、4.921 g·L-1,乙醇得率为O.457 g(乙醇)·g-1(糖),是理论得率的92.64%.生产能力达到10.996 g·L-1·h-1.与其它发酵方式相比较,用海藻酸钙来固定细胞并与膜耦合的发酵过程可增大酵母细胞浓度,明显降低乙醇对酵母的抑制作用,并提高糖的转化率.     

四株酵母乙醇发酵的初步研究

比较了休哈塔假丝酵母NLP21、树干毕赤酵母NLP22、NLP23和NLP31,在30 g/L的木糖和混合糖(葡萄糖15 g/L+木糖15 g/L)发酵培养基上以及在培养基中氮源浓度降低到原来1/2和1/10时的发酵性能。结果表明,在30 g/L木糖发酵培养基上,NLP23和NLP31产乙醇质量浓度最高,分别为(11.14±0.13)和(11.15±0.08) g/L。在15 g/L葡萄糖+15 g/L木糖混合糖发酵培养基上,NLP31产乙醇质量浓度最高,为(10.91±0.12) g/L。当发酵培养基中氮源浓度降低到原来的1/2时,NLP23和NLP31产乙醇能力相当,但后者产木糖醇的量增大;当氮源质量浓度降低到原来的1/10时,NLP23和NLP31产乙醇能力随着发酵轮数的增加,逐渐下降,氮源浓度低,降低了乙醇的产量。     

木糖发酵酒精代谢工程的研究进展

木糖发酵是生物转化木质纤维素产生酒精及其他化工产品最为重要的一环,但自然界中缺少能将上述生物质有效转化为乙醇的微生物菌种. 近年来,根据代谢工程原理,利用基因工程技术对酵母和细菌进行遗传改造,或将木糖代谢途径引入传统的酒精发酵菌酿酒酵母及高酒精产生菌运动发酵单胞菌中,从而拓展其底物利用范围;或使原本可以利用多种糖底物的细菌获得选择性产生酒精的能力,构建了各种不同类型的木糖发酵重组菌株. 虽然这些重组菌株在木糖转化酒精方面均显示出良好的应用前景,但仍存在诸多问题. 有必要在对木糖代谢调控机制深入系统研究的基础上,进一步改造现有菌株,并结合生化工程技术对重组菌株发酵条件进行优化,以实现高效生物转化木质纤维素原料制取乙醇. 本工作介绍了近年来代谢工程改造微生物菌种发酵木糖生产酒精的研究进展.     

Pro-sigmaK inhibitor-BofA基因敲除对嗜热厌氧杆菌SCUT27葡萄糖和木糖利用的影响

嗜热厌氧杆菌SCUT27(Thermoanaerobacterium aotearoense SCUT27,SCUT27)能同时利用木糖和葡萄糖,是发酵廉价木质纤维素获得乙醇及乳酸的优势菌株。为了提高该菌株对木糖和葡萄糖的利用效率,对其在葡萄糖、木糖和混合糖下不同时间点转录组数据分析的基础上,敲除pro-sigmaK抑制因子BofA(pro-sigmak processing inhibitor BofA,BofA)基因,获得基因工程菌ΔbofA。摇瓶发酵结果显示,与出发菌相比,当以木糖为唯一碳源时,突变株ΔbofA对木糖的代谢速率提高51.43%,乳酸产率提高23.53%;以混合糖(葡萄糖:木糖为=1:1)为碳源时,ΔbofA对木糖的代谢速率提高44.44%,乳酸和乙醇的产率分别提高43.75%和20.00%。由此可见,pro-sigmaK抑制因子BofA是木糖利用的负调控因子,其功能有待进一步研究。     

LSM蛋白增强木糖发酵重组酵母抗逆性能的研究

木质纤维素原料预处理过程中产生的弱酸、呋喃醛类和酚类化合物等对酿酒酵母的乙醇发酵有抑制作用,提高基因重组酵母对抑制物的耐受性,是利用植物秸秆水解液生产燃料乙醇的关键技术之一。研究从前期构建的戊糖、己糖共发酵重组酿酒酵母Saccharomyces cerevisiae ZU-E8基因组DNA中克隆出RNA结合蛋白LSM6,将其连入含有PADH启动子的质粒构成表达载体pR-LSM,进而转入ZU-E8宿主细胞中。通过高浓度醋酸根平板筛选,得到高抗逆性木糖发酵重组酵母ZU-910。在醋酸浓度为2 g·L-1的木糖培养基中发酵96 h后,ZU-910的木糖利用率和乙醇浓度为90.2%和26.9 g·L-1,分别是出发菌株ZU-E8的8.5和10倍,并且ZU-910对糠醛和硫酸根的耐受能力也较ZU-E8大大增强。在玉米秸秆酶解液发酵中,ZU-910的木糖利用率和乙醇产量在ZU-E8基础上增加了10.5%和7.7%.证明LSM6蛋白确实能够增强木糖发酵重组酵母的抗逆能力,提高其发酵性能。该研究成果在木质纤维素替代粮食生产乙醇的产业化进程中具有良好的应用前景。     

可使用多种不同原料的乙醇生产技术

一种用纤维素废物生产燃料级乙醇的技术将在明年下半年开始商业化应用于意大利南部萨莱诺的一家工厂。初期,这家年产37850L乙醇的工厂使用的原料是小麦(包括谷壳和麦杆)。晚些时候将增加废物原料(包括生物质和多余的废面团)。这家萨莱诺工厂现在用甜菜生产乙醇。瑞典的Chematur工程公司将为美国生物开发公司对这家工厂进行技术改造。原料将用美国天鹅生物质公司开发的方法加工。用酸性水解配合酶将纤维素和半纤维素分解成C6糖(葡萄糖和C5糖,主要是木糖)。通常的酵母不能使木糖发酵,此法使用一种遗传修饰的酵母可使C6和C5糖发酵。此法使用…     

纤维素乙醇生产重组酿酒酵母菌株的构建与优化研究进展

寻找化石能源的替代品以及开发和利用生物能源已引起国内外研究者的广泛关注。提高酿酒酵母利用来源广泛、贮存丰富的农林废弃物等木质纤维素原料生产燃料乙醇的效率是生物能源的重要研究内容,但是,重组酿酒酵母木糖发酵性能低是限制纤维素乙醇经济性的关键问题。本文总结了酿酒酵母中木糖代谢途径的构建和优化以及木糖转运对木糖利用的影响,分析了重组酵母利用纤维素水解液进行乙醇发酵的研究现状,并对进一步提高重组酿酒酵母纤维素乙醇生产效率的研究趋势进行了展望。目前国内外已经构建了可有效利用木糖产乙醇的重组酵母,但对其木糖代谢机制的研究还尚未深入,限制了重组菌株的定向改造。此外,目前缺少在纤维素生物质水解液发酵实际应用过程中对重组菌株的评价。因此,加强重组酵母菌株对木糖利用相关代谢调控机理的分析,注重多种抑制物对菌株发酵性能的影响,结合真实底物纤维素乙醇发酵过程进行重组菌株的构建和优化,从而进一步提高纤维素乙醇生产的经济性,是未来菌株构建的重要研究方向。     

木糖利用融合子D2的制备与乙醇发酵特性

以树干毕赤酵母（Pichia stipitis）1960（Ps1960）与酿酒酵母（Saccharomyces cerevisiae）AADY（ScAADY）为亲本菌株,采用双亲灭活原生质体技术制备木糖利用融合子,并对其制备条件进行了优化。优化后的原生质体制备条件为Ps1960采用2%蜗牛酶和1%纤维素酶在28℃酶解45 min,20W紫外灯距离10 cm照射3 min灭活;ScAADY采用1.5%蜗牛酶和1%纤维素酶28℃酶解50 min,55℃水浴50 min灭活;均采用0.6 mol/L山梨醇为渗透压稳定剂。在该条件下,共得到22株融合子。通过测定各融合子在不同培养基条件下的生物量来评价其木糖代谢和乙醇耐受能力,最终获得能利用木糖高效发酵产乙醇、遗传性状稳定的融合子D2,并进行乙醇发酵条件优化。结果表明,在混合糖质量分数8%、木糖和葡萄糖质量比6：1、5%接种量、30℃、160 r/min、培养72 h条件下,融合子D2发酵产乙醇的产量为40.58 g/L。     

A FEASIBILITY ANALYSIS OF A NOVEL APPROACH FOR THE CONVERSION OF XYLOSE TO ETHANOL

Economic production of ethanol from plant biomass could be significantly increased if the feedstock for the fermentation is more completely utilized. Currently, simple sugars (mostly D-glucose and D-xylose) can be recovered from lignocellulose by enzymatic or acid hydrolysis. However, while glucose can be readily converted to ethanol by yeasts, the xylose is not fermentable by many of the same species of yeasts that are able to convert glucose into ethanol. Nevertheless, xylose can be converted to its ketose isomer, xylulose, by the enzyme xylose isomerase and this isomer can be converted to ethanol. A major obstacle, however, in converting the xylose to xylulose and then simultaneously converting the xylulose to ethanol is that the pH at which xylose isomerase displays its optimal activity (pH of 7.0-8.0) is much different from the pH at which the fermentation of the xylulose and glucose is best carried out (pH of 4.0-5.0).

Herein we propose a novel scheme to provide a means by which the isomerization and the fermentation can both take place simultaneously. The xylose isomerase is immobilized in a porous polymer pellet and this pellet is then coated with an additional layer in which the enzyme urease is immobilized. These bilayered pellets are dispersed in a fermentation broth which contains a prespecified concentration of urea in addition to all the other necessary ingredients. The xylose isomerase, immobilized in the core region of the pellet, catalyses the conversion of xylose to xylulose, and this latter substrate at the same time also becomes available for the fermentation reaction immediately. It is also possible to sustain the necessary spatial pH gradient between the bulk liquid phase and the core region of the pellet, because as hydrogen ions diffuse into the pellet they are neutralized by the ammonia produced in the outer layer of the pellet by the hydrolysis of urea by urease. A mathematical model is developed to demonstrate that the required pH gradient can be achieved. The model evaluates the effect of pellet design parameters (enzyme loading and thickness of the outer layer) and reactor conditions (bulk concentration of urea, and the effect of weak acids) on the ability to generate the required pH gradient across the outer layer of the pellet.     

Optimization of fermentation condition for co-production of ethanol and 2,3-butanediol (2,3-BD) from hemicellolosic hydrolysates by Klebsiella oxytoca XF7

Microbial production of ethanol and 2,3-butanediol (2,3-BD) from agro-residues has been attracting interest because of their applications in various industries, including generation of biofuel molecules. In the present investigation, the hemicellulosic fraction of corncob was hydrolyzed by indigenous holocellulase from novel psychrotolerant Aspergillus niger SH3 resulting in high xylose release (34.61?g?L^?1), followed by the bioconversion of xylose to ethanol and 2,3-BD. Taguchi design was adopted to optimize the process which resulted in 5.25- and 3.31-fold increase in 2,3-BD (12.18?±?0.53?g?L^?1) and ethanol (4.08?±?0.03?g?L^?1), as compared with un-optimized condition. For the first time, co-production of ethanol and 2,3-BD from the corncob hemicellulosic hydrolysate was performed using a newly isolated Klebsiella oxytoca XF7 strain, under the optimized fermentation conditions. These results suggest that K. oxytoca XF7 is a promising candidate for co-production of ethanol and 2,3-BD, with high xylose conversion efficiency (96.65%), facilitating the economical production of biofuel molecules.     

Mathematical model for ethanol production from mixed sugars by Pichia stipitis

A mathematical model for estimating the dynamic behavior of ethanol production from mixed sugars such as glucose and xylose is presented. This model was constructed by introducing the term for ethanol production into the diauxic growth model proposed previously by the authors. It was assumed that the first substrate, glucose, is metabolized by a constitutive enzyme and the second substrate, xylose, is utilized by an inducible enzyme. The synthesis of the inducible enzyme is controlled by the catabolite repression caused by glucose and with the induction caused by xylose as an inducer, and the xylose is then converted into cell mass and ethanol by the inducible enzyme. The parameters of the model were estimated from the experimental data in the medium containing glucose and xylose, singly or in combination in a batch culture. The application of the model was examined for experiments in both batch and continuous culture, with glucose and xylose as carbon sources. The calculated values, according to the model, corresponded satisfactorily with experimental data, such as cell growth, substrate consumption, and ethanol production, especially in the estimation of the lag times between the first log phase and the second log phase in cell growth and ethanol production. © 2001 Society of Chemical Industry     

嗜单宁管囊酵母木糖酒精发酵的研究

综合利用丰富、廉价且可再生的农作物秸秆资源生产酒精的研究已经渐渐成为全球范围的热点之一。文章研究了嗜单宁管囊酵母木糖酒精发酵的发酵性能,分离、筛选、驯化培养出一株高效木糖酒精发酵菌株 g-13,在最适条件下其木糖酒精发酵的转化率为 0.446 g/g(酒精/消耗的糖),为发酵秸秆纤维水解液中的戊糖生产燃料酒精提供菌种支持。     

THE GROWTH OF CLOSTRIDIUM THERMO - HYDROSULFURICUM ON MULTIPLE SUBSTRATES

Clostridium thermohydrosulfuricum has a number of characteristics which make it an attractive organism to use in a process to convert pretreated, lignocellulosic materials into alcohol. One such characteristic is its ability to convert the major products of cellulose hydrolysis, glucose, cellobiose, xylose and xylobiose into ethanol at relatively high yields. Experiments were carried out to investigate the kinetics of multiple sugar utilization. Results showed that glucose repressed the utilization of cellobiose, xylose and xylobiose all of which could otherwise be catabolized simultaneously in the absence of glucose. Further experiments with the glucose/cellobiose system showed that both transient and permanent repression occurred. A mathematical model was developed which successfully described the growth of Cl. thermohydrosulfuricum on mixtures of glucose and cellobiose in both batch and continuous culture.     

不同非氧化磷酸戊糖途径基因的过表达对酿酒酵母木糖发酵性能的影响

以双拷贝过表达木糖代谢上游途径关键酶(木糖还原酶XR、木糖醇脱氢酶XDH和木酮糖激酶XKS)的酿酒酵母菌株为背景,在过表达非氧化磷酸戊糖(PP)途径中转醛酶基因TAL1的基础上,对途径中其他基因TKL1(转酮酶)、RPE1(核酮糖-5-磷酸差向异构酶)和RKI1(核酮糖-5-磷酸异构酶)进行了不同程度的过表达,以研究PP途径基因过表达对酿酒酵母木糖代谢的影响。在不同培养基条件下对重组菌株木糖代谢进行研究,结果显示,在过表达TAL1的基础上不同组合过表达PP途径其他基因不同程度改善了酿酒酵母木糖发酵性能,重组菌株能在36~48 h耗完质量分数(下同)为5%的木糖。其中,过表达PP途径全部基因比其他过表达基因组合表现出明显的优势,在8%木糖发酵条件下其乙醇产量达到了每1 g木糖0.337 g,较对照菌株提高了7.86%。这说明同步过表达PP途径基因更有利于酿酒酵母木糖发酵。     

Kinetic studies of xylan hydrolysis of corn stover in a dilute acid cycle spray flow-through reactor

Xylan of corn stover was pretreated with 1%, 2% and 3% (w/w) sulfuric acid at relatively low temperatures (90°C, 95°C and 100°C) in a dilute acid cycle spray flow-through reactor (DCF). The hydrolysis of xylan to its monomeric xylose was modeled by a series of first-order reactions. Both biphasic and Saeman hydrolysis models were applied to fit the experimental data. The results confirmed that the kinetic data of xylan hydrolysis fitted a first-order irreversible reaction model and the experimental data. The reaction rates of xylose monomer formation and degradation were sensitive to catalyst concentration and temperature. Higher catalyst concentration and lower reaction temperature result in high xylose yield. The activation energy for xylose formation and degradation were determined to be 112.9 and 101.0 kJ·mol^-1, respectively. Over 90% theoretical xylose obtained from corn stover can be used to produce ethanol, xylitol and fumaric acid by fermentation.