酵母对抑制剂耐受的系统分析

纤维素生产乙醇的关键问题之一是水解产生的抑制性物质对乙醇发酵具有明显的抑制效应,因而引起了国内外研究者的广泛关注.研究发现,在抑制剂存在下,酵母在基因表达水平,蛋白水平和代谢物水平都有相应的耐受响应,且这些响应错综复杂.从系统角度运用组学的方法研究这一体系将有助于全面深入了解酵母的耐受机制.本文综述了系统研究的思路和方法在酵母对抑制剂耐受方面的研究状况;对主要研究手段和成果进行了回顾;并对酵母发酵乙醇系统分析的前景进行了展望.     

酵母对抑制剂耐受的系统分析

纤维素生产乙醇的关键问题之一是水解产生的抑制性物质对乙醇发酵具有明显的抑制效应,因而引起了国内外研究者的广泛关注.研究发现,在抑制剂存在下,酵母在基因表达水平,蛋白水平和代谢物水平都有相应的耐受响应,且这些响应错综复杂.从系统角度运用组学的方法研究这一体系将有助于全面深入了解酵母的耐受机制.本文综述了系统研究的思路和方法在酵母对抑制剂耐受方面的研究状况;对主要研究手段和成果进行了回顾;并对酵母发酵乙醇系统分析的前景进行了展望.     

生物预处理对甘蔗渣转化的影响

为了研究不同种类产纤维素酶的菌株降解甘蔗渣的效果,结合已得出的最佳化学预处理方法,利用枯草芽孢杆菌、绿色木霉和烟曲霉三种高产纤维素酶的菌株对甘蔗渣进行生物预处理,比较降解效果;并用腺嘌呤缺陷型和非缺陷型酵母进行发酵,比较乙醇产量。结果表明:分解10 g甘蔗渣,枯草芽孢杆菌组还原糖产量为427.56 mg,绿色木霉组还原糖产量为887.36 mg,烟曲霉组还原糖产量为982.84mg。相同还原糖含量的烟曲霉组和绿色木霉组滤液用腺嘌呤缺陷型酵母发酵时,在27℃发酵25.5 h,乙醇浓度达到最高值,绿色木霉组为5.4%,烟曲霉组为5.5%。在三种产纤维素酶菌株中,烟曲霉降解甘蔗渣的效果最好。     

基于纤维小体展示技术的酿酒酵母纤维素乙醇研究进展

利用联合生物加工工艺生产第二代燃料乙醇(纤维素乙醇)是国内外的研究热点.前期的研究结果表明,酿酒酵母分泌或展示非复合型纤维素酶体系的应用效果并不理想,而复合型纤维素酶体系(即纤维小体)因对纤维素的降解能力比非复合型纤维素酶体系更强,所以其在酿酒酵母细胞表面的组装研究受到越来越多的关注.目前,单支架和双支架纤维小体在酵母细胞表面的完全自组装以及多细胞协同参与的非完全自组装均已实现.纤维小体展示型酿酒酵母已能直接利用结晶型纤维素发酵生产乙醇,但由于降解模块的结构缺陷,纤维素乙醇的产量仍然偏低.本文对纤维小体的酵母展示技术及其在纤维素乙醇发酵中的应用研究进行了论述,并对该领域的发展方向进行了展望.     

高效液相色谱法测定克鲁维酵母菊芋发酵液中的乙醇,糖和有机酸类代谢成分

建立了高效液相色谱法同时分析菊芋发酵液中的乙醇和有机酸的方法.采用HPLC有机酸分析柱, 流动相为0.01 mol/L H2SO4,流速为0.5 mL/min, 以紫外和示差折光检测器作为双通道检测手段,同时对克鲁维酵母菊芋发酵液中的柠檬酸、α-酮戊二酸、葡萄糖、丙酮酸、果糖、琥珀酸、乳酸、甘油、乙酸、乙醇进行了定量分析,本方法的回收率为95.8%～109.6%;RSD为0.33%～4.0%,结果表明,本方法简单、快速、准确,适用于监控克鲁维酵母发酵产物并指导整个发酵过程条件的优化.     

拉曼光谱分析有机氮源促进乙醇发酵的机制

应用拉曼光谱和单细胞分析技术监测有机氮源尿素和酵母粉、无机氮源硝酸铵和硫酸铵对酿酒酵母乙醇发酵的影响及发酵过程胞内主要生物大分子的变化动态,以期从光谱学角度获知有机氮源促进乙醇发酵的机制。结果表明,利用酵母粉和尿素的乙醇发酵速度最快,14~18 h即可达到乙醇浓度的最大值;在有机氮源下,酵母细胞的RNA合成无明显的迟滞期,发酵前期,782 cm!1峰平均强度比无机氮源的高,其最大峰强是初始强度的1.9~2.1倍,而无机氮源仅为1.2~1.4倍;以酵母粉为氮源的不同发酵阶段,部分细胞的蛋白质二级结构以β折叠为主,而其它氮源的细胞则是以α螺旋为绝对主导。因此,尿素、酵母粉等有机氮源促进乙醇发酵的可能原因是缩短酵母的迟滞期,促进胞内RNA的快速合成,促进相关基因的快速转录和表达。     

钙离子在电针镇痛、电针耐受和吗啡耐受发展中的作用

本文研究了Ca~(2+)在电针镇痛、电针耐受和吗啡耐受发展中的作用,同时了解电针耐受发展是否能被蛋白质合成抑制剂抑制。实验结果表明,和吗啡耐受一样,电针耐受动物的脑Ca~(2+)和cAMP水平均明显增高。用蛋白质合成抑制剂茴香霉素(Anisomycin)、放线菌素(Actinomycin)或环己亚胺(Cycloheximide)处理后,电针镇痛耐受发展被抑制,同时使脑Ca~(2+)和cAMP水平降低.从脑Ca~(2+)和cAMP水平的变化看,电针、吗啡和镧系元素引起的镇痛,以及电针和吗啡的耐受发展不仅相似,很可能还有共同的离子基础和作用机理,同时提示蛋白质合成抑制剂阻断电针和吗啡的耐受发展似与新的多肽或RNA合成相关。     

酵母AHAS酶与磺酰脲类抑制剂作用模型的分子对接研究

基于酵母乙酰羟酸合成酶(AHAS)与磺酰脲类抑制剂复合物的晶体结构, 用分子对接方法对AHAS与5个磺酰脲类抑制剂相互作用的方式进行了系统的分子对接研究. 晶体复合物对接和假复合物对接两种模式对接的结果基本相同, 并与实验结果吻合. 在进一步的对接中逐级考虑了辅酶FAD和TPP的影响, 结果表明, 辅酶FAD和TPP的加入, 对AHAS酶与磺酰脲类抑制剂的结合顺序基本没有影响. 其中FAD的加入使AHAS与抑制剂的结合更加稳定, 这主要是由于抑制剂的R2取代基与FAD中的平面基团Flavin环间存在的范德华相互作用所致; 抑制剂与TPP间存在的静电相互作用可能是加速TPP降解的原因.     

提高纤维素酶水解效率和降低水解成本

在我国可大量转化乙醇的是纤维质材料.纤维质材料转化乙醇的关键问题是纤维质转化为糖的过程,提高纤维素酶转化效率的方法有:(1)对纤维质材料进行预处理;(2)研究纤维素酶的最适作用条件;(3)纤维素酶的重复利用;(4)合理的发酵工艺等.本文分析了纤维素的结构以及纤维素酶的作用方式,总结了目前研究较多的几种纤维质材料预处理方法,及其对纤维素酶水解率的影响,并对研究纤维素酶的最适作用条件、纤维素酶的重复利用以及合理的发酵工艺进行了综述和分析.     

气相色谱法测定亚硫酸盐废液发酵过程中的醋酸、糠醛和乙醇

发酵亚硫酸盐废液中的单糖制酒精的过程中,抑制剂醋酸和糠醛[1]及产物乙醇的测定是发酵研究的重要方面。文献中用气相色谱法测定水溶液中的脂肪酸[2,3]、亚硫酸废液中的糠醛[4]及同时测定发酵液中的脂肪酸和乙醇[5—7]的报道虽然很多,但有的是用溶剂萃取法[2,4],有的是用程序升温法,     

Enhanced cofermentation of glucose and xylose by recombinantSaccharomyces yeast strains in batch and continuous operating modes

Agricultural residues, such as grain by-products, are rich in the hydrolyzable carbohydrate polymers hemicellulose and cellulose; hence, they represent a readily available source of the fermentable sugars xylose and glucose. The biomass-to-ethanol technology is now a step closer to commercialization because a stable recombinant yeast strain has been developed that can efficiently ferment glucose and xylose simultaneously (coferment) to ethanol. This strain, LNH-ST, is a derivative ofSaccharomyces yeast strain 1400 that carries the xylose-catabolism encoding genes ofPichia stipitis in its chromosome. Continuous pure sugar cofermentation studies with this organism resulted in promising steady-state ethanol yields (70.4% of theoretical based on available sugars) at a residence time of 48 h. Further studies with corn biomass pretreated at the pilot scale confirmed the performance characteristics of the organism in a simultaneous saccharification and cofermentation (SSCF) process: LNH-ST converted 78.4% of the available glucose and 56.1% of the available xylose within 4 d, despite the presence of high levels of metabolic inhibitors. These SSCF data were reproducible at the bench scale and verified in a 9000-L pilot scale bioreactor.     

Study the oxidative injury of yeast cells by NADH autofluorescence

Autofluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex system such as biological cells and tissues. NADH is an important fluorescent substance in living cells. The time courses of intracellular NADH autofluorescence in the process of yeast cells exposed to H(2)O(2) and ONOO(-) have been recorded in detail in this work. In the presence of different amounts of H(2)O(2) and ONOO(-), necrosis, apoptosis and reversible injury are initiated in yeast cells, which are confirmed by acridine orange/ethidum bromide and Annexin V/propidium iodide staining. It is found that intracellular NADH content increases momently in the beginning of the apoptotic process and then decreases continually till the cell dies. The most remarkable difference between the apoptotic and the necrotic process is that the NADH content in the latter case changes much more sharply. Further in the case of reversible injury, the time course of intracellular NADH content is completely different from the above two pathways of cell death. It just decreases to some degree firstly and then resumes to the original level. Based on the role of NADH in mitochondrial respiratory chain, the time course of intracellular NADH content is believed to have reflected the response of mitochondrial redox state to oxidative stress. Thus, it is found that the mitochondrial redox state changes differently in different pathways of oxidative injury in yeast cells.     

Cellulosic fuel ethanol

Iogen (Canada) is a major manufacturer of industrial cellulase and hemicellulase enzymes for the textile, pulp and paper, and poultry feed industries. Iogen has recently constructed a 40 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. The integration of enzyme and ethanol plants results in significant reduction in production costs and offers an alternative use for the sugars generated during biomass conversion. Iogen has partnered with the University of Toronto to test the fermentation performance characteristics of metabolically engineered Zymomonas mobilis created at the National Renewable Energy Laboratory. This study focused on strain AX101, a xylose- and arabinose-fermenting stable genomic integrant that lacks the selection marker gene for antibiotic resistance. The “Iogen Process” for biomass depolymerization consists of a dilute-sulpfuric acid-catalyzed steam explosion, followed by enzymatic hydrolysis. This work examined two process design options for fermentation, first, continuous cofermentation of C₅ and C₆ sugars by Zm AX101, and second, separate continuous fermentations of prehydrolysate by Zm AX101 and cellulose hydrolysate by either wildtype Z. mobilis ZM4 or an industrial yeast commonly used in the production of fuel ethanol from corn. Iogen uses a proprietary process for conditioning the prehydrolysate to reduce the level of inhibitory acetic acid to at least 2.5 g/L. The pH was controlled at 5.5 and 5.0 for Zymomonas and yeast fermentations, respectively. Neither 2.5 g/L of acetic acid nor the presence of pentose sugars (C₆:C₅ = 2:1) appreciably affected the high-performance glucose fermentation of wild-type Z. mobilis ZM4. By contrast, 2.5 g/L of acetic acid significantly reduced the rate of pentose fermentation by strain AX101. For single-stage continuous fermentation of pure sugar synthetic cellulose hydrolysate (60 g/L of glucose), wild-type Zymomonas exhibited a four-fold higher volumetric productivity compared with industrial yeast. Low levels of acetic acid stimulated yeast ethanol productivity. The glucose-to-ethanol conversion efficiency for Zm and yeast was 96 and 84%, respectively.     

Ethanol fermentation of various pretreated and hydrolyzed substrates at low initial pH

Lignocellulosic materials represent an abundant feedstock for bioethanol production. Because of their complex structure pretreatment is necessary to make it accessible for enzymatic attack. Steam pretreatment with or without acid catalysts seems to be one of the most promising techniques, which has already been applied for large variety of lignocellulosics in order to improve enzymatic digestibility. During this process a range of toxic compounds (lignin and sugar degradation products) are formed which inhibit ethanol fermentation. In this study, the toxicity of hemicellulose hydrolysates obtained in the steam pretreatment of spruce, willow, and corn stover were investigated in ethanol fermentation tests using a yeast strain, which has been previously reported to have a resistance to inhibitory compounds generated during steam pretreatment. To overcome bacterial contamination, fermentations were carried out at low initial pH. The fermentability of hemicellulose hydrolysates of pretreated lignocellulosic substrates at low pH gave promising results with the economically profitable final 5 vol% ethanol concentration corresponding to 85% of theoretical. Adaptation experiments have shown that inhibitor tolerance of yeast strain can be improved by subsequent transfer of the yeast to inhibitory medium.     

溶液电导率法对碳酸钙结晶动力学的研究

溶液电导率法对碳酸钙结晶动力学的研究;结垢;电导率     

Electrochemical insights into the ethanol tolerance of Saccharomyces cerevisiae

It is expected that intracellular redox activity may closely related to catabolic states of living cells, based on which a mediated electrochemical method has been proposed to measure the ethanol tolerance of the yeast Saccharomyces cerevisiae AS 3800. The couple menadione/ferricyanide was employed as a carrier mediator system, sensing intracellular redox activity. Microelectrode voltammetric method was introduced to assay the ferrocyanide accumulations arising from menadione mediated reduction of ferricyanide by the yeast. The mediated electrochemical study show that the maximal ethanol tolerance limit of S. cerevisiae is about 25% (v/v) ethanol, which is consistent with the result obtained by the conventional fermentative ability measurement. Moreover, the electrochemical method for the first time confirmed that the specific activities of the glycolytic and alcohologenic enzymes within intact living cells remained high by the presence of sublethal ethanol, which was only predicted by in vitro enzymatic assay and cannot be measured by conventional method. The new method can be used as an easy and rapid method to determine the maximal ethanol tolerance of yeast cells.     

Improvements of Tolerance to Stress Conditions by Genetic Engineering in Saccharomyces Cerevisiae during Ethanol Production

Saccharomyces cerevisiae, industrial yeast isolate, has been of great interest in recent years for fuel ethanol production. The ethanol yield and productivity depend on many inhibitory factors during the fermentation process such as temperature, ethanol, compounds released as the result of pretreatment procedures, and osmotic stress. An ideal strain should be able to grow under different stress conditions occurred at different fermentation steps. Development of tolerant yeast strains can be achieved by reprogramming pathways supporting the ethanol metabolism by regulating the energy balance and detoxicification processes. Complex gene interactions should be solved for an in-depth comprehension of the yeast stress tolerance mechanism. Genetic engineering as a powerful biotechnological tool is required to design new strategies for increasing the ethanol fermentation performance. Upregulation of stress tolerance genes by recombinant DNA technology can be a useful approach to overcome inhibitory situations. This review presents the application of several genetic engineering strategies to increase ethanol yield under different stress conditions including inhibitor tolerance, ethanol tolerance, thermotolerance, and osmotolerance.     

A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis

In this paper we compare the effects of three representative PCR inhibitors using quantitative PCR (qPCR) and multiplex STR amplification in order to determine the effect of inhibitor concentration on allele dropout and to develop better ways to interpret forensic DNA data. We have used humic acid, collagen and calcium phosphate at different concentrations to evaluate the profiles of alleles inhibited in these amplifications. These data were correlated with previously obtained results from quantitative PCR including melt curve effects, efficiency changes and cycle threshold (Ct) values. Overall, the data show that there are two competing processes that result from PCR inhibition. The first process is a general loss of larger alleles. This appears to occur with all inhibitors. The second process is more sequence specific and occurs when the inhibitor binds DNA, altering the cycle threshold and the melt curve. This sequence-specific inhibition results in patterns of allele loss that occur in addition to the overall loss of larger alleles. The data demonstrate the applicability of utilizing real-time PCR results to predict the presence of certain types of PCR inhibition in STR analysis.     

Vendor test studies supporting the design of a biomass-to-ethanol pilot plant

Applied Biochemistry and Biotechnology - In support of the effort to develop the biomass-to-ethanol process, the National Renewable Energy Laboratory (NREL) is building a pilot plant based on the...     

Production of oxychemicals from precipitated hardwood lignin

Lignin is a major byproduct in the biomass-to-ethanol process. The lignin produced from acid treatment of biomass has characteristics suitable for further conversion to organic chemicals. It is free of contaminants and has a relatively low molecular weight. In this study, catalytic oxidative conversion of the acid-soluble lignin precipitated from acid hydrolysates of hardwood was investigated. The process is based on aqueous alkaline oxidation of lignin with dissolved O₂ in the presence of Fe³⁺ and Cu²⁺ catalysts at moderate reaction temperatures (160–180°C). Aromatic aldehydes, ketones, and organic acids are found to be the primary products identifiable on extraction with ether. The combined weight yield of the total ether extractable products is about 20–25% of the initial lignin. The yield of the aldehydes (vanillin + syringaldehyde) is in the vicinity of 15% with an additional 3 to 4% of aromatic ketones. The yields of aldehydes plus ketones observed in this work far exceeded those obtainable from the conventional alkaline air oxidation of spent sulfite liquors. This article also provides comprehensive batch reaction data on conversion and product distribution.