乙酸、糠醛和5-羟甲基糠醛对产酸克雷伯氏菌发酵生产2,3-丁二醇的影响

为了解产酸克雷伯氏菌对木质纤维素水解液中主要抑制物的耐受和代谢,考察了产酸克雷伯氏菌发酵生产2,3-丁二醇(2,3-butanediol,2,3-BDO)过程中对3种发酵抑制物乙酸、糠醛和5-羟甲基糠醛(5-hydroxymethylfurfural HMF)的耐受以及抑制物浓度的变化,检测了糠醛和HMF的代谢产物.结果表明:产酸克雷伯氏菌对乙酸、糠醛和HMF的耐受浓度分别为30 g/L、4 g/L和5 g/L.并且部分乙酸可作为生产2,3-丁二醇的底物,在0～30 g/L浓度范围内可提高2,3-丁二醇的产量.发酵过程中产酸克雷伯氏菌可将HMF和糠醛全部转化,其中约70％HMF被转化为2,5-呋喃二甲醇,30％HMF和全部糠醛被菌体代谢.研究表明在木质纤维素水解液生产2,3-丁二醇的脱毒过程中可优先考虑脱除糠醛,一定浓度的乙酸可以不用脱除.     

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。     

玉米秸秆酸解副产物对重组酿酒酵母6508-127发酵的影响

将木质纤维素类生物质如玉米秸秆等用稀酸水解预处理,在半纤维素水解为单糖的同时,水解液中还会产生一些可能对后续发酵有影响的副产物。本实验分别考查了在玉米秸秆稀酸水解液中检测出的乙酸、甲酸、香草醛、糠醛和羟甲基糠醛对重组木糖发酵菌株S. cerevisiae 6508-127生长和发酵的影响。结果表明,甲酸和乙酸对菌体生长的抑制强于乙醇生成,且甲酸的抑制程度远大于乙酸;2g/L香草醛可使菌体生长延滞期明显延长,而在较低浓度（≤1.2g/L）此现象不明显。糠醛在0.5-1.5g/L范围内对菌体生长有抑制作用,但使乙醇得率提高;羟甲基糠醛在0.2g/L浓度存在就使乙醇得率有明显降低,但使生物量得率提高;研究中还发现,糠醛、羟甲基糠醛和香草醛可被S. cerevisiae 6508-127代谢。     

木质纤维素酸解副产物对D-乳酸生产菌Sporolactobacillus sp.Y2-8生长及发酵的影响

选择乙酸根、糠醛、5-羟甲基糠醛、苯酚、香草酸和丁香醛等6种典型木质纤维素酸解副产物,考察它们对D-乳酸生产菌Sporolactobacillus sp.Y2-8生长及发酵的影响。实验结果表明:酚类物质抑制作用最强烈,0.25 g/L丁香醛已经完全抑制了菌体的生长和D-乳酸的发酵;苯酚和香草酸在低浓度(≤1.0 g/L)时抑制作用较小,但质量浓度达到3 g/L时对D-乳酸产量的抑制率分别为99%和70%;3 g/L糠醛和5-羟甲基糠醛对产物的抑制率分别为60%与20%,抑制作用小于酚类;乙酸根的影响最小,10 g/L的乙酸钠对菌体的生长和发酵几乎无抑制作用;当抑制物混合时,存在着相互促进作用,抑制作用更强烈。     

纤维素降解产物对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响

研究纤维素酸水解产生的4种副产物乙酸、甲酸、糠醛、5-羟甲基糠醛及发酵产物乙醇对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响。结果表明:5.0 g/L乙酸和1.0 g/L甲酸对葡萄糖和木糖共发酵具有明显的抑制作用;1.0 g/L糠醛和5-羟甲基糠醛基本不影响K.marxianus 1727发酵葡萄糖,且能够被K.marxianus1727转化为毒性相对较低的物质。由于5-羟甲基糠醛的转化速率慢,对K.marxianus 1727发酵木糖的抑制程度大于糠醛。乙醇对K.marxianus 1727发酵木糖具有抑制作用,当乙醇质量浓度大于20 g/L时,生物量及木糖利用率约是对照的44%和70%。     

抗氧化基因过表达提高运动发酵单胞菌糠醛耐受性

糠醛是木质纤维素水解液的主要抑制物,能诱发活性氧自由基(Reactive oxygen species,ROS)产生。探索抗氧化基因对于糠醛耐受性的影响有助于改善菌株表型。过表达运动发酵单胞菌Zymomonas mobilis自身的NADH氧化酶(ZMO1885)、NADP+还原酶(ZMO1753)和谷胱甘肽还原酶(ZMO1211)基因,检测胞内ROS、NADH/NAD+以及其耐受性能。结果表明,在2g/L糠醛条件下,相比对照菌株,ZM4/ZMO1885的胞内ROS降低了52.1%,生物量提高了38.9%,且未引起胞内辅因子扰动。过表达ZMO1753和ZMO1211没有显著提高菌株耐受性。在模拟木质纤维素水解液中,ZM4/ZMO1885的生物量较对照菌株提高了46.9%,糖耗速率提高了110.3%,乙醇生产力提高了195.2%。综上,过表达抗氧化酶基因能够影响糠醛胁迫耐受性,其中过表达ZMO1885不仅不影响细胞正常生长,还能降低ROS水平,提高糠醛去除率,改善了细胞的糠醛耐受性。     

糠醛对粘红酵母生长与油脂积累的影响

为了探究纤维素水解液中常见的发酵抑制物糠醛对粘红酵母Rhodotorula glutinis生长与油脂积累的影响,对比了不同的糠醛浓度(0.1、0.4、0.6、1.5 g/L)下粘红酵母的生物量和油脂积累情况,并探究了1.0 g/L的糠醛对粘红酵母不同碳源(葡萄糖和木糖)利用的影响。研究表明,当糠醛浓度达1.5 g/L时,粘红酵母的延迟期延长至96 h,残糖高达17.7 g/L,生物量最高6.6 g/L,仅为正常积累量的47%,油脂含量也减少了约50%;以木糖为碳源时,糠醛对粘红酵母的抑制程度小于葡萄糖为碳源时的情况;在糠醛存在的逆境中,粘红酵母倾向于生成更多的18碳脂肪酸或18碳不饱和脂肪酸。     

玉米芯半纤维素水解液中抑制物对丁醇发酵的影响北大核心CSCD

半纤维素水解液抑制物对微生物细胞的毒性限制了其在丁醇发酵中的应用,旨在探讨其对产丁醇共生体系TSH06的抑制作用并为其应用于丁醇发酵奠定基础。通过稀酸水解半纤维素制得水解液,采用P2培养基稀释的半纤维素水解液为底物,分别利用NaOH和氨水调节培养基pH值,结合实时荧光定量PCR方法来研究水解液对产丁醇共生体系TSH06的抑制作用。以NaOH调节pH抑制菌体的生长,氨水调节pH菌体可生长发酵。产丁醇菌株TSH06可以在50%稀释度以下的水解液中发酵生长,并且能够耐受并降解抑制物糠醛与5-羟甲基糠醛,最终丁醇产量达到4.16-5.16 g/L,低于P2培养基中的丁醇产量(8.83 g/L)。稀释水解液中,48 h之后乙酸浓度在3.18-4.16 g/L,远大于P2培养基中的乙酸浓度(低于2 g/L)。相对于P2培养基,在50%水解液中培养的TSH06有机酸生成途径关键基因的基因转录水平明显提高,而有机酸返耗途径以及丁醇生成途径的关键基因的基因转录水平则明显下降。水解液中过多的乙酸抑制了产酸期到产溶剂期的转化,而酸的累积使得菌体在底物被完全消耗之前就趋于衰退死亡,从而造成丁醇产量的降低与底物的不完全利用。     

玉米芯半纤维素水解液中抑制物对丁醇发酵的影响

半纤维素水解液抑制物对微生物细胞的毒性限制了其在丁醇发酵中的应用,旨在探讨其对产丁醇共生体系TSH06的抑制作用并为其应用于丁醇发酵奠定基础。通过稀酸水解半纤维素制得水解液,采用P2培养基稀释的半纤维素水解液为底物,分别利用NaOH和氨水调节培养基pH值,结合实时荧光定量PCR方法来研究水解液对产丁醇共生体系TSH06的抑制作用。以NaOH调节pH抑制菌体的生长,氨水调节pH菌体可生长发酵。产丁醇菌株TSH06可以在50%稀释度以下的水解液中发酵生长,并且能够耐受并降解抑制物糠醛与5-羟甲基糠醛,最终丁醇产量达到4.16-5.16 g/L,低于P2培养基中的丁醇产量(8.83 g/L)。稀释水解液中,48 h之后乙酸浓度在3.18-4.16 g/L,远大于P2培养基中的乙酸浓度(低于2 g/L)。相对于P2培养基,在50%水解液中培养的TSH06有机酸生成途径关键基因的基因转录水平明显提高,而有机酸返耗途径以及丁醇生成途径的关键基因的基因转录水平则明显下降。水解液中过多的乙酸抑制了产酸期到产溶剂期的转化,而酸的累积使得菌体在底物被完全消耗之前就趋于衰退死亡,从而造成丁醇产量的降低与底物的不完全利用。     

紫外诱变驯化提高酿酒酵母木糖发酵的抑制物耐受性

利用驯化和紫外处理结合驯化的手段对一株木糖发酵工业菌株的抑制物耐受性进行提升。在反复批次培养过程中不断提高含9种抑制物的混合抑制物浓度,使细胞的生长和发酵逐渐适应高浓度抑制物环境,分离突变菌株并进行评价。紫外处理结合驯化比直接驯化能更有效的提升细胞对高浓度抑制物的耐受能力;通过突变菌株分离和筛选,获得3株抑制物耐受能力高于出发菌株的突变菌株,它们在抑制物浓度100%MI(甲酸1 g/L,乙酸3.5 g/L,乙酰丙酸1.5 g/L,糠醛1.5 g/L,5-羟甲基糠醛1.5g/L,丁香醛0.1 g/L,香草醛0.1 g/L,松柏醛0.025 g/L,肉桂酸0.025 g/L)条件下的木糖消耗率比出发菌株高出11.3%-23.2%。紫外诱变处理结合驯化过程可以有效提高酿酒酵母对混合抑制物的耐受性。     

Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid

This paper explores the use of the hydrolysate from the dilute sulfuric acid pretreatment of wheat straw for microbial oil production. The resulting hydrolysate was composed of pentoses (24.3 g/L) and hexoses (4.9 g/L), along with some other degradation products, such as acetic acid, furfural, and hydroxymethylfurfural (HMF). Five oleaginous yeast strains, Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium toruloides, Lipomyces starkeyi, and Yarrowia lipolytica, were evaluated by using this hydrolysate as substrates. The results showed that all of these strains could use the detoxified hydrolysate to produce lipids while except R. toruloides non-detoxified hydrolysate could also be used for the growth of all of the selective yeast strains. C. curvatus showed the highest lipid concentrations in medium on both the detoxified (4.2 g/L) and non-detoxified (5.8 g/L) hydrolysates. And the inhibitory effect studies on C. curvatus indicated HMF had insignificant impacts at a concentration of up to 3 g/L while furfural inhibited cell growth and lipid content by 72.0% and 62.0% at 1 g/L, respectively. Our work demonstrates that lipid production is a promising alternative to utilize hemicellulosic sugars obtained during pretreatment of lignocellulosic materials.     

发酵产丁二酸过程中废弃细胞的循环利用

对厌氧发酵产丁二酸后的废弃细胞进行破壁处理,考察了以细胞水解液作为有机氮源重新用于丁二酸发酵的可行性。比较了超声破碎、盐溶、酶解3种方法破碎细胞获得的水解液作为氮源发酵产丁二酸的效果,结果表明酶解制得的细胞水解液效果最佳。以总氮含量为1.11g/L的酶解液(相当于10g/L酵母膏)作为氮源发酵,丁二酸产量可达42.0g/L,继续增大酶解液用量对耗糖、产酸能力没有显著提高。将细胞酶解液与5g/L酵母膏联用发酵36h后,丁二酸产量达75.5g/L,且丁二酸生产强度为2.10g/(L·h),比使用10g/L酵母膏时提高了66.7%。因此,厌氧发酵产丁二酸结束后的废弃细胞酶解液可以替代原培养基中50%的酵母膏用于发酵。     

Butanol production from agricultural residues: Impact of degradation products on Clostridium beijerinckii growth and butanol fermentation

During pretreatment and hydrolysis of fiber-rich agricultural biomass, compounds such as salts, furfural, hydroxymethyl furfural (HMF), acetic, ferulic, glucuronic, rho-coumaric acids, and phenolic compounds are produced. Clostridium beijerinckii BA101 can utilize the individual sugars present in lignocellulosic [e.g., corn fiber, distillers dry grain solubles (DDGS), etc] hydrolysates such as cellobiose, glucose, mannose, arabinose, and xylose. In these studies we investigated the effect of some of the lignocellulosic hydrolysate inhibitors associated with C. beijerinckii BA101 growth and acetone-butanol-ethanol (ABE) production. When 0.3 g/L rho-coumaric and ferulic acids were introduced into the fermentation medium, growth and ABE production by C. beijerinckii BA101 decreased significantly. Furfural and HMF are not inhibitory to C. beijerinckii BA101; rather they have stimulatory effect on the growth of the microorganism and ABE production.     

采用玉米秸秆水解糖和玉米浆发酵生产丁二酸

研究了以玉米秸秆水解糖为碳源,不同氮源条件下琥珀酸放线杆菌Actinobacillus succinogenesSF-9的丁二酸发酵产酸能力。结果表明玉米浆可以替代酵母膏作为丁二酸发酵的廉价氮源。厌氧摇瓶丁二酸发酵单因素试验,得到在初糖浓度50 g/L时,玉米浆的较佳用量为20 g/L。在5 L搅拌罐上,考察了不同初始玉米秸秆水解糖浓度对A.succinogenes SF-9发酵生产丁二酸的影响,结果显示高初始秸秆糖浓度对琥珀酸放线杆菌的生长有抑制作用。采用补料分批发酵,发酵60 h丁二酸的产量达到42.7g/L,丁二酸产率82.7%,生产强度0.81 g/(L·h)。丁二酸的产量和生产强度较分批发酵有明显提高。     

Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.     

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.     

Ultrasonic pretreatment and acid hydrolysis of sugarcane bagasse for succinic acid production using Actinobacillus succinogenes

Immense interest has been devoted to the production of bulk chemicals from lignocellulose biomass. Diluted sulfuric acid treatment is currently one of the main pretreatment methods. However, the low total sugar concentration obtained via such pretreatment limits industrial fermentation systems that use lignocellulosic hydrolysate. Sugarcane bagasse hemicellulose hydrolysate is used as the carbon and nitrogen sources to achieve a green and economical production of succinic acid in this study. Sugarcane bagasse was ultrasonically pretreated for 40 min, with 43.9 g/L total sugar obtained after dilute acid hydrolysis. The total sugar concentration increased by 29.5 %. In a 3-L fermentor, using 30 g/L non-detoxified total sugar as the carbon source, succinic acid production increased to 23.7 g/L with a succinic acid yield of 79.0 % and a productivity of 0.99 g/L/h, and 60 % yeast extract in the medium could be reduced. Compared with the detoxified sugar preparation method, succinic acid production and yield were improved by 20.9 and 20.2 %, respectively.     

Isolation and characterization of Cupriavidus basilensis HMF14 for biological removal of inhibitors from lignocellulosic hydrolysate

The formation of toxic fermentation inhibitors such as furfural and 5-hydroxy-2-methylfurfural (HMF) during acid (pre-)treatment of lignocellulose, calls for the efficient removal of these compounds. Lignocellulosic hydrolysates can be efficiently detoxified biologically with microorganisms that specifically metabolize the fermentation inhibitors while preserving the sugars for subsequent use by the fermentation host. The bacterium Cupriavidus basilensis HMF14 was isolated from enrichment cultures with HMF as the sole carbon source and was found to metabolize many of the toxic constituents of lignocellulosic hydrolysate including furfural, HMF, acetate, formate and a host of aromatic compounds. Remarkably, this microorganism does not grow on the most abundant sugars in lignocellulosic hydrolysates: glucose, xylose and arabinose. In addition, C. basilensis HMF14 can produce polyhydroxyalkanoates. Cultivation of C. basilensis HMF14 on wheat straw hydrolysate resulted in the complete removal of furfural, HMF, acetate and formate, leaving the sugar fraction intact. This unique substrate profile makes C. basilensis HMF14 extremely well suited for biological removal of inhibitors from lignocellulosic hydrolysates prior to their use as fermentation feedstock.