紫外诱变结合驯化提高酿酒酵母对抑制物耐受性

[目的]提高工业酿酒酵母Sc4126对木质纤维素预处理过程中产生的发酵抑制物的耐受性。[方法]采用紫外诱变结合驯化对工业酿酒酵母Sc4126进行选育,对筛选出的三株菌株Sc4126-1、Sc4126-2、Sc4126-3在复合抑制剂和单一抑制剂存在下对比考查其发酵性能。[结果]相比原始菌株,3株菌株对抑制剂的耐受性均不同程度地提高。在复合抑制剂存在下,优势菌株Sc4126-1发酵时间为48 h,乙醇平均产率0.19 g/L·h,而原始菌株Sc4126发酵时间138 h,乙醇平均产率0.06 g/L·h,相比原始菌株,优势菌株发酵时间缩短65.22%,乙醇平均产率提高2.17倍。对于单一抑制剂,改造后的菌株对糠醛、香草醛的耐受性明显提高,而对乙酸的耐受性提高较少。[结论]通过紫外诱变结合驯化的方法,有效提高了工业酿酒酵母Sc4126对抑制剂的耐受性。     

过表达长链鞘氨醇激酶基因LCB4提高酿酒酵母抑制物耐受性

木质纤维素预处理过程中产生的有毒副产物严重影响了纤维素乙醇发酵,提高酿酒酵母抑制物耐受性是提高纤维素乙醇发酵效率的有效方法。文中通过过表达LCB4基因,研究了重组菌株S288C-LCB4在乙酸、糠醛和香草醛胁迫下的细胞生长和乙醇发酵性能。结果表明,LCB4过表达菌株在分别含有10 g/L乙酸、1.5 g/L糠醛和1 g/L香草醛的平板中生长均优于对照菌株;在分别含有10 g/L乙酸、3 g/L糠醛和2 g/L香草醛的液体乙醇发酵过程中,重组菌株S288C-LCB4乙醇发酵产率分别为0.85 g/(L·h)、0.76 g/(L·h)和1.12 g/(L·h),比对照菌株提高了34.9%、85.4%和330.8%;且糠醛和香草醛胁迫下发酵时间分别缩短了30 h和44 h。根据发酵终点发酵液代谢物分析发现重组菌株比对照菌株产生了更多甘油、海藻糖和琥珀酸,这些物质有利于增强菌株的抑制物耐受性。综上所述,LCB4基因过表达可显著提高酿酒酵母S288C在乙酸、糠醛和香草醛胁迫下的乙醇发酵性能。     

常压甘油自催化预处理麦草浓醪发酵纤维素乙醇

目前纤维素乙醇成本偏高的根本原因在于没有达到淀粉质乙醇发酵水平的"三高"(高浓度、高转化率和高效率)指标,提高水解糖液浓度和避免发酵抑制物来实现浓醪发酵,是解决问题的关键。文中以常压甘油自催化预处理麦草为底物,尝试采用不同发酵策略,探讨其浓醪发酵产纤维素乙醇的可行性。在优化培养条件(15%底物浓度,加酶量30 FPU/g干底物,温度37℃,接种量10%)下同步糖化发酵72 h,纤维素乙醇产量为31.2 g/L,转化率为73%,发酵效率0.43 g/(L·h);采用半同步(预酶解24 h)糖化发酵72 h,纤维素乙醇浓度达到33.7 g/L,转化率为79%,发酵效率为0.47 g/(L·h),其中(半)同步糖化发酵中90%以上纤维素已被糖化水解用于发酵;采用分批补料式半同步糖化发酵,补料到基质浓度相当于30%,发酵72 h时纤维素乙醇产量达到51.2 g/L,转化率为62%,发酵效率为0.71 g/(L·h)。在所有浓醪发酵中乙酸不足3 g/L,无糠醛和羟甲基糠醛等发酵抑制物。以上结果表明,常压甘油自催化预处理木质纤维素基质适用于纤维素乙醇发酵;分批补料式半同步糖化发酵策略可用来进行浓醪纤维素乙醇发酵;未来工作中提高基质纯度和强化酶解产糖是浓醪纤维素乙醇达到"三高"指标的关键。     

混合糖发酵重组酿酒酵母的菌株构建和菊芋秸秆同步糖化发酵研究

【目的】构建可用于纤维素乙醇高效生产的混合糖发酵重组酿酒酵母菌株,并利用菊芋秸秆为原料进行乙醇发酵。【方法】筛选在木糖中生长较好的酿酒酵母YB-2625作为宿主菌,构建木糖共代谢菌株YB-2625 CCX。进一步通过r DNA位点多拷贝整合的方式,以YB-2625 CCX为出发菌株构建木糖脱氢酶过表达菌株,并筛选得到优势菌株YB-73。采用同步糖化发酵策略研究YB-73的菊芋秸秆发酵性能。【结果】YB-73菌株以90 g/L葡萄糖和30 g/L木糖为碳源进行混合糖发酵,乙醇产量比出发菌株YB-2625 CCX提高了13.9%,副产物木糖醇产率由0.89 g/g降低至0.31 g/g,下降了64.6%。利用重组菌YB-73对菊芋秸秆进行同步糖化发酵,48 h最高乙醇浓度达到6.10%(体积比)。【结论】通过转入木糖代谢途径以及r DNA位点多拷贝整合过表达木糖脱氢酶基因可有效提高菌株木糖发酵性能,并用于菊芋秸秆的纤维素乙醇生产。这是首次报道利用重组酿酒酵母进行菊芋秸秆原料的纤维素乙醇发酵。     

酿酒酵母在纤维素乙醇生产中对毒性化合物的耐受机理研究进展

利用木质纤维素生产燃料乙醇的过程中,前期预处理所产生的抑制剂会影响酵母的正常生长和后续的发酵过程。为减小抑制剂的影响所采取的一些脱毒策略往往造成糖的损失和生产成本的增加,这在实际生产与经济上是不可行的。因此,具有强的抑制剂耐受性的酿酒酵母菌株对于提高纤维素乙醇产率是十分重要的。近十年来,对于酿酒酵母胁迫耐受机制的研究取得了一些重要的进展,着重介绍目前酿酒酵母对抑制剂耐受机制的研究现状,包括一些关键性基因的表达及代谢通路过程分析等。同时也介绍一些应对抑制剂提高酵母发酵能力的措施。     

分别利用产甘油假丝酵母抗逆转录因子CgSTB5和CgSEF1对酿酒酵母菌株糠醛耐受改造

【背景】纤维素是生物转化解决能源问题的主要原料之一,其水解物中存在严重影响抑制菌株生长的糠醛,需脱毒才可应用于发酵,提高菌株耐受性是解决纤维素水解液实际生产应用的关键。【目的】酿酒酵母(Saccharomyces cerevisiae)是主要的纤维素水解液发酵工业菌株,但糠醛耐受性较低,通过分子改造获得具有高糠醛耐受性的菌株。【方法】利用新获得的产甘油假丝酵母(Candidaglycerinogenes)的相关抗逆转录因子CgSTB5、CgSEF1和CgCAS5,通过分子技术进行S.cerevisiae改造,考察其对酿酒酵母糠醛耐受性的影响,并尝试应用于未脱毒纤维素乙醇发酵。【结果】单个表达CgSTB5和CgSEF1的酿酒酵母,通过菌株点板实验表明菌株的糠醛耐受性提高25%以上,并且摇瓶发酵结果显示糠醛降解性能明显提高,生长延滞期明显缩短,S.cerevisiae W303/p414-CgSTB5的未脱毒纤维素乙醇发酵生产效率提高12.5%左右。【结论】转录因子CgSTB5和CgSEF1均能对提高酿酒酵母糠醛耐受性起到重要作用,并且有助于提高酿酒酵母菌株未脱毒纤维素乙醇发酵性能。     

过表达MRP8提高酿酒酵母乙酸耐性及乙醇发酵效率

乙酸是木质纤维素水解液中含量较多的抑制物,因此提高酿酒酵母菌株对乙酸的耐受性有助于提高纤维素乙醇生产效率。本文中,笔者利用基于CRISPR/Cas9系统的基因组编辑技术过表达了酿酒酵母(Saccharomyces cerevisiae)S288c线粒体核糖体蛋白编码基因MRP8,并比较了过表达MRP8的菌株与对照菌株的生长和发酵特性。平板耐性检测发现,MRP8过表达明显提高了菌株的乙酸胁迫耐受性;乙醇发酵结果表明,在4.8 g/L乙酸胁迫条件下,过表达菌株MRP8-3在51 h消耗全部的葡萄糖,发酵时间缩短了25 h,显著优于相同时间的对照菌株。本研究结果为构建高效纤维素乙醇发酵的酿酒酵母菌株提供了新思路。     

酿酒酵母发酵高浓度乳清粉生产燃料乙醇

利用酿酒酵母NL22对乳清粉进行分步糖化发酵(SHF)和同步糖化发酵(SSF),对其生产燃料乙醇的条件进行比较,同时考察pH、温度和底物浓度对SHF和SSF过程的影响。结果表明:SHF工艺和SSF工艺都可以实现酿酒酵母NL22对高浓度乳清粉的发酵,但SSF工艺可明显缩短生产周期,提高生产效率。在pH 6和30℃的条件下进行补料同步糖化发酵,最终乙醇质量浓度为118.52 g/L,产率为1.74 g/(L·h)。     

纤维素乙醇发酵液中复杂组分对乙醇渗透汽化传质过程的影响

考察了木质纤维素乙醇发酵液中各组分对乙醇透过聚二甲基硅氧烷(PDMS)-silicalite-1渗透汽化膜传质性能的影响。结果表明:酵母细胞、玉米秸秆残渣和发酵用无机盐可增加乙醇通过膜的通量和选择性;而葡萄糖和甘油的存在会对乙醇的透膜传质产生负面影响;木质纤维素水解后的产物如糠醛和羟基丙酮,表现出对膜分离乙醇轻微的抑制作用。本文建立了渗透汽化优先透醇与纤维素乙醇发酵集成过程,批次发酵20 h后乙醇产率从最初的12.95下降到10.22 g/(L·h),60 h后乙醇产率下降为0,葡萄糖消耗速率与乙醇消耗产率呈同样趋势;连续发酵过程中,乙醇产率较稳定地维持在13.30 g/(L·h)。实验证明,集成过程可及时地将产物乙醇分离出去,能够有效地消除产物抑制,提高乙醇生产速率和葡萄糖转化率。     

木质纤维素水解液副产物对东方伊萨酵母乙醇发酵的影响

为了客观评判耐高温东方伊萨酵母HN-1利用木质纤维素水解液生产燃料乙醇的潜力,本文采用单因素试验和响应面中心组合试验研究了木质纤维素水解液有毒副产物甲酸钠(1.0-5.0 g/L)、乙酸钠(2.5-8.0 g/L)、糠醛(0.2-2.0 g/L)、5-羟甲基糠醛(0.1-1.0 g/L)和香草醛(0.5-2.0 g/L)对其乙醇发酵的影响。结果表明,木质纤维素水解液有毒副产物对东方伊萨酵母HN-1乙醇发酵的影响较小,除添加2 g/L香草醛或添加1 g/L 5-羟甲基糠醛可使乙醇产量分别降低20.38%和11.2%外,其他抑制物的添加对乙醇的生成未有显著影响。但是,当副产物浓度较高时,可以显著抑制菌体生长,添加1-5 g/L甲酸钠、2.5-8.0 g/L乙酸钠、0.4-2 g/L糠醛或0.5-2 g/L香草醛,发酵36 h时菌体细胞干重分别较对照下降了25.04%-37.02%、28.83%-43.82%、20.06%-37.60%和26.39%-52.64%。中心组合试验结果表明各抑制物交互作用对乙醇的生成影响不显著。该研究表明木质纤维素水解液副产物对东方伊萨酵母HN-1乙醇发酵的影响较小,适合用于纤维乙醇发酵。     

Comparison of glucose/xylose cofermentation of poplar hydrolysates processed by different pretreatment technologies

The inhibitory effects of furfural and acetic acid on the fermentation of xylose and glucose to ethanol in YEPDX medium by a recombinant Saccharomyces cerevisiae strain (LNH‐ST 424A) were investigated. Initial furfural concentrations below 5 g/L caused negligible inhibition to glucose and xylose consumption rates in batch fermentations with high inoculum (4.5–6.0 g/L). At higher initial furfural concentrations (10–15 g/L) the inhibition became significant with xylose consumption rates especially affected. Interactive inhibition between acetic acid and pH were observed and quantified, and the results suggested the importance of conditioning the pH of hydrolysates for optimal fermentation performance. Poplar biomass pretreated by various CAFI processes (dilute acid, AFEX, ARP, SO₂‐catalyzed steam explosion, and controlled‐pH) under respective optimal conditions was enzymatically hydrolyzed, and the mixed sugar streams in the hydrolysates were fermented. The 5‐hydroxymethyl furfural (HMF) and furfural concentrations were low in all hydrolysates and did not pose negative effects on fermentation. Maximum ethanol productivity showed that 0–6.2 g/L initial acetic acid does not substantially affect the ethanol fermentation with proper pH adjustment, confirming the results from rich media fermentations with reagent grade sugars. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Expression of aldehyde dehydrogenase 6 reduces inhibitory effect of furan derivatives on cell growth and ethanol production in Saccharomyces cerevisiae

Yeast dehydrogenases and reductases were overexpressed in Saccharomyces cerevisiae D452-2 to detoxify 2-furaldehyde (furfural) and 5-hydroxymethyl furaldehyde (HMF), two potent toxic chemicals present in acid-hydrolyzed cellulosic biomass, and hence improve cell growth and ethanol production. Among those enzymes, aldehyde dehydrogenase 6 (ALD6) played the dual roles of direct oxidation of furan derivatives and supply of NADPH cofactor to their reduction reactions. Batch fermentation of S. cerevisiae D452-2/pH-ALD6 in the presence of 2 g/L furfural and 0.5 g/L HMF resulted in 20-30% increases in specific growth rate, ethanol concentration and ethanol productivity, compared with those of the wild type strain. It was proposed that overexpression of ALD6 could recover the yeast cell metabolism and hence increase ethanol production from lignocellulosic biomass containing furan-derived inhibitors.     

Ethanol production from glucose and dilute-acid hydrolyzates by encapsulated S. cerevisiae

The performance of encapsulated Saccharomyces cerevisiae CBS 8066 in anaerobic cultivation of glucose, in the presence and absence of furfural as well as in dilute-acid hydrolyzates, was investigated. The cultivation of encapsulated cells in 10 sequential batches in synthetic media resulted in linear increase of biomass up to 106 g/L of capsule volume, while the ethanol productivity remained constant at 5.15 (+/-0.17) g/L x h (for batches 6-10). The cells had average ethanol and glycerol yields of 0.464 and 0.056 g/g in these 10 batches. Addition of 5 g/L furfural decreased the ethanol productivity to a value of 1.31 (+/-0.10) g/L x h with the encapsulated cells, but it was stable in this range for five consecutive batches. On the other hand, the furfural decreased the ethanol yield to 0.41-0.42 g/g and increased the yield of acetic acid drastically up to 0.068 g/g. No significant lag phase was observed in any of these experiments. The encapsulated cells were also used to cultivate two different types of dilute-acid hydrolyzates. While the free cells were not able to ferment the hydrolyzates within at least 24 h, the encapsulated yeast successfully converted glucose and mannose in both of the hydrolyzates in less than 10 h with no significant lag phase. However, since the hydrolyzates were too toxic, the encapsulated cells lost their activity gradually in sequential batches.     

Effects of furfural on ethanol fermentation bySaccharomyces cerevisiae: Mathematical models

Different inocula with high yeast concentration were investigated as a means of overcoming the inhibitory effect of furfural in ethanol fermentation. In order to verify the toxicity of the furfural, a series of fermentation runs were made with 0.25, 5.50, and 9.00 g/L (dry weight) ofSaccharomyces cerevisiae inoculum and 1, 3, and 5 g/L of furfural. The extent of cell death occurring in the early phase of fermentation was dependent on the initial cell concentration. With high initial yeast concentration, the effect of furfural is canceled, because it is depleted at an early stage of fermentation. The ethanol weight yield averaged 0.45 on the basis of sugar consumed. The ethanol productivity and specific growth rate decreased with the increase of furfural concentration, and the inhibitory effect almost disappeared with high cell concentration (9 g/L). Mathematical models were developed that relate productivity and growth rate with furfural and cell concentration.     

Conversion of furfural into furfuryl alcohol by saccharomyces cervisiae 354

The influence of aeration, stirring conditions, and the addition of furfural on the yield and productivity of furfural – furfuryl alcohol bioconversion by the yeast strain Saccharomyces cerevisiae 354 was investigated. The formation of furfuryl alcohol increases up to 32 hours of incubation corresponding to the addition of furfural, while the cell growth essentially ceased at 20 hours. The conversion of furfural into furfuryl alcohol under anaerobic and low aeration conditions was 70% and the productivity 0.5g · 1^?1 · h^?1, when the final concentrationof of furfural amounted to 35 g · 1^?1.     

Selection and adaptation of Saccharomyces cerevisae to increased ethanol tolerance and production

A total of 24 yeast strains were tested for their capacity to produce ethanol, and of these, 8 were characterized by the best ethanol yields (73.11-8 1.78%). The most active mutant Saccharomyce s cerevisiae ER-A, resistant to ethanol stress, was characterized by high resistance to acidic (pH 1.0 and 2.0), oxidative (1 and 2% of H2O2), and high temperature (45 and 52 degrees C) stresses. During cultivation under all stress conditions, the mutants showed a considerably increased viability ranging widely from about 1.04 to 3.94-fold in comparison with the parent strain S. cerevisiae ER. At an initial sucrose concentration of 150 g/l in basal medium A containing yeast extract and mineral salts, at 300C and within 72 h, the most active strain, S. cerevisiae ER-A, reached an ethanol concentration of 80 g/1, ethanol productivity of 1.1 g/Il/h, and an ethanol yield (% of theoretical) of 99.13. Those values were significantly higher in comparison with parent strain (ethanol concentration 71 g/1 and productivity of 0,99 g/l/h). The present study seems to confirm the high effectiveness of selection of ethanol-resistant yeast strains by adaptation to high ethanol concentrations, for increased ethanol production.     

Effect of air supplement on the performance of continuous ethanol fermentation system

For the purpose of improving ethanol productivity, the effect of air supplement on the performance of continuous ethanol fermentation system was studied. The effect of oxygen supplement on yeast concentration, cell yield, cell viability, extracellular ethanol concentration, ethanol yield, maintenance coefficient, specific rates of glucose assimilation, ethanol production, and ethanol productivity have been evaluated, using a high alcohol tolerant Saccharomyces cerevisiae STV89 strain and employing a continuous fermentor equipped with an accurate air metering system in the flow rate range 0-11 mL air/L/h. It was found that, when a small amount of oxygen up to about 80mu mol oxygen/L/h was supplied, the ethanol productivity was significantly enhanced as compared to the productivity of the culture without any air supplement. It was also found that the oxygen supplement improved cell viability considerably as well as the ethanol tolerance level of yeast. As the air supply rate was increased, from 0 to 11 mL air/L/h while maintaining a constant dilution rate at about 0.06 h(-1), the cell concentration increased from 2.3 to 8.2 g/L and the ethanol productivity increased from 1.7 to 4.1 g ethanol/L/h, although the specific ethanol production rate decreased slightly from 0.75 to 0.5 g ethanol/g cell/h. The ethanol yield was slightly improved also with an increase in air supply rate, from about 0.37 to 0.45 ethanol/g glucose. The maintenance coefficient increased by only a small amount with the air supplement. This kind of air supplement technique may very well prove to be of practical importance to a development of a highly productive ethanol fermentation process system especially as a combined system with a high density cell culture technique.