文章信息
- 郭雪娇, 查健, 姚坤, 王昕, 李炳志, 元英进.
- GUO Xue-jiao, ZHA Jian, YAO Kun, WANG Xin, LI Bing-zhi, YUAN Ying-jin.
- 选育耐受复合抑制剂酿酒酵母提高乙醇产量
- Accelerated Ethanol Production by a Tolerant Saccharomyces cerevisiae to Inhibitor Mixture of Furfural, Acetic Acid and Phenol
- 中国生物工程杂志, 2016, 36(5): 97-105
- China Biotechnology, 2016, 36(5): 97-105
- http://dx.doi.org/DOI:10.13523/j.cb.20160514
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文章历史
- 收稿日期: 2015-11-06
- 修回日期: 2016-12-06
2. 天津化学化工协同创新中心合成生物学研究平台 天津 300072
2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering(Tianjin, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
近年来生物乙醇作为化石燃料的代替物受到广泛关注,普遍认为其在一定程度上可以缓解全球性或区域性的危机,如过度依赖原油进口、温室气体排放及石油储量日益枯竭等问题[1-3]。与淀粉或甘蔗产乙醇相比,纤维素乙醇将是最有潜力、最稳定的液体能源[4-5]。纤维素向乙醇的转化步骤复杂,包括预处理、酶解及微生物发酵等[6]。预处理主要有稀酸法、蒸汽爆破法及水热法等高温、高压策略,然而这些方法会产生多种抑制剂等副产品[7-9]。预处理过程产生的主要抑制剂分为三类:呋喃类、弱酸类及酚类。呋喃类包括糠醛和5-羟甲基糠醛(HMF)等;弱酸类包括乙酸、甲酸及乙酰丙酸等;酚类包括香草醛、丁香醛、松柏醛等[10],其中低分子质量的酚类毒性更大[11]。这些抑制剂对微生物的生长及乙醇的生产有着严重的抑制作用[10, 12-13]。
因此,发酵前脱毒或选育高耐受性菌株对乙醇生产至关重要。然而外加脱毒过程,如离子交换或灰碱处理过度,既大大地增加了工业投入和成本,也造成了可利用糖分的损失[14-15]。因而选育在纤维素水解液中具有高耐受性和高生产能力的乙醇生产菌株更方便、经济[6, 16-18]。酿酒酵母由于乙醇生产效率高,并对乙醇及一些有毒物质有较高的耐受能力,因此被广泛应用于乙醇生产工业中。利用酵母这些特性更有望选育出耐受多种抑制剂物质的乙醇生产菌株[19-21]。
在增强菌体对纤维素水解液中单一抑制剂的耐受性及抑制剂作用机制方面已有了较多的研究[22-24]。然而相对于单一抑制剂,复合抑制剂不仅仅是累加作用,协同作用使其更加复杂[25-26],其作用机制尚未清晰。选育复合抑制剂耐受性菌株更有益于从生物质水解液生产乙醇。
本研究通过紫外诱变和驯化工程手段筛选到一株高耐受性工业酿酒酵母。鉴于抑制剂的复杂性,选择糠醛、乙酸和苯酚三种不同类型抑制剂做为代表形成复合抑制剂,其pH低于4.0或高于5.5。另外,在水热法处理所得玉米秸秆水解液中进行了耐受菌株和原始菌株的发酵对照研究。
1 材料与方法 1.1 菌 株本实验所用酿酒酵母菌株为安琪耐高温酿酒高活性干酵母(Saccharomyces cerevisiae,以下称该菌株为“S”),购自中国湖北安琪酵母有限公司[27]。耐受酿酒酵母菌株YYJ003为本实验室通过逐步驯化得到,保藏在中国普通微生物菌种保藏管理中心,保存号为CGMCC 2757。
1.2 复合抑制剂耐受酵母的选育将细胞悬浮液按照每平板1~2×105细胞密度涂板,将铺好的平板距离15W紫外线30cm下照射210s,使存活率约为1%。处理后的细胞避光培养3天,存活的细胞转移至含有复合抑制剂(1.3g/L糠醛、1.0g/L苯酚、5.3g/L乙酸)的YPD培养基中培养。每24h收集细胞并转移至新鲜含有复合抑制剂YPD培养基(pH 4.0)中培养。经过30天筛选和富集选育出一株上述复合抑制剂耐受菌株-YYJ003。
1.3 种子培养分别从固体YPD培养基上的单菌落上挑一株原始菌株S及复合抑制剂耐受菌株YYJ003,分别接种到含有100ml YPD培养基的250ml锥形瓶中,其中YYJ003的培养基中加入复合抑制剂,180r/min、30℃培养12h至对数中期。4 000r/min离心5min收集沉淀细胞备用。
1.4 模拟水解液培养基制备本研究所用模拟水解液培养基为含有葡萄糖20g/L、蛋白胨20g/L、酵母粉10g/L的YPD培养基,121℃灭菌20min,在接种前加入除菌的抑制剂糠醛、苯酚和乙酸,使糠醛的含量为1.3g/L、乙酸的含量为5.3g/L、苯酚的含量为0.5g/L。
1.5 玉米秸秆水解液预处理玉米秸秆由天津农村收集得到,经过风干后打碎成约4mm的碎片。预处理过程在10L反应器中进行,生物质-水的比例为6%(m/V)。540g干玉米秸秆加入到反应器中并升温至100℃,利用250℃水蒸气将反应体系温度维持在220℃,30min后冷却至40℃,利用过滤器将固液分离。
过滤所得液体组分含有4.24g/L乙酸、0.97g/L HMF及0.55g/L糠醛。加入等体积乙酸乙酯,涡旋0.5h,去除体系中抑制剂成分,后利用氮吹法去除乙酸乙酯。脱毒后水解液含有3.15g/L乙酸、0.49g/L HMF及0.03g/L糠醛,在水解液中补加葡萄糖至终浓度100g/L,即为本研究使用的水解液。
1.6 发 酵本研究中所有分批发酵均在5L发酵罐(BXBIO,中国上海)中完成。原始菌株S和耐受菌株YYJ003以初始OD600=0.1接种至含有复合抑制剂(1.3g/L糠醛、5.3g/L乙酸及1.0g/L苯酚)YPD培养基中,在30℃和300r/min下发酵。发酵过程中使用2mol/L NaOH维持pH4.0~5.5。
玉米秸秆水解液发酵在含有100ml水解液的250ml摇瓶中进行,摇瓶瓶口使用插有针头的橡胶塞封住。
发酵过程中不同间隔时间检测菌体密度,并取样进行后期代谢物质分析。每组发酵进行两组平行。
1.7 样品分析菌体生长曲线用紫外可见分光光度计(吸收波长为600nm,OD600)测定。发酵液中葡萄糖、乙醇、甘油及乙酸浓度用HPLC检测,流速为0.6ml/min。葡萄糖和乙醇的检测使用伯乐Aminex HPX-87H糖分析柱对发酵液中的物质进行分离,柱温为65℃,5mmol/L硫酸溶液作为流动相用示差检测器检测。糠醛的测定用Kromasil C18柱毛细管色谱柱以40%的甲醇溶液作为流动相(浓盐酸调节pH至3.0);糠醛的检测利用紫外检测器于室温下检测,吸收波长为284nm。
2 结果与讨论本研究测试了YYJ003菌株与S菌株在含有糠醛、乙酸及苯酚复合抑制剂的培养基中的发酵情况。YPD中乙酸及糠醛的浓度分别按照稀酸法水解液中乙酸和所有呋喃类物质浓度折算[28],苯酚终浓度为1.0g/L。研究中利用含复合抑制剂的培养基来对比研究YYJ003菌株的耐受性。研究中也进行了两个菌株在玉米秸秆水解液中的发酵表征。
2.1 复合抑制剂下原始菌株和耐受菌株在高pH培养基中的生长和发酵性能比较原始菌株S和耐受菌株YYJ003在pH为5.5的含复合抑制剂的培养基中发酵表征,结果如图 1所示。YYJ003菌株的延滞期约为12h,比S菌株的延滞期降低了至少6h;YYJ003在16h内已经耗尽葡萄糖,乙醇产量为0.48g/g(乙醇/葡萄糖,下同),而S菌株需近22h达到相同乙醇产量。如表 1所示,两个菌株的最终细胞密度相近,OD600为2.10~2.20。YYJ003菌株的乙醇产率比S菌株高61%,甘油产量低15%。
发酵过程中两菌株的糠醛降解能力相似,糠醛在发酵初期就开始降解,约12h降解完全。但当培养基中糠醛量降至12.5%时S菌株才开始生长,而YYJ003菌株在糠醛剩余量为60%就开始生长。培养基中乙酸浓度基本保持不变,普遍认为无氧发酵时苯酚浓度也保持不变[26]。因此我们可以看出YYJ003菌株对糠醛的高耐受性提高了其乙醇产量。
已有的研究表明,当细胞受到糠醛、乙酸、邻苯二酚或对羟基苯甲酸的协同抑制时,糠醛是抑制细胞生长的主要因素[26]。酿酒酵母在延滞期内可将糠醛和5-HM降解为糠醇,而这种抑制剂转化效率与其自身的耐受性有关[29]。研究证明,在糠醛转化期间只有耐受菌株可生长,而非耐受菌株只有当糠醛耗尽时才开始生长[30-31]。Martin等研究表明,通过复合抑制剂驯化而选育的菌株虽对糠醛的转化能力增强,但当糠醛存在时仍不能生长[32]。本研究中S和YYJ003两菌株均具有较高的糠醛耐受能力,可能由于二者为工业菌株。
Parameters | pH 5.5 | pH 4.0 | ||
YYJ 003 | S strain | YYJ 003 | S strain | |
Initial cell density (OD600) | 0.1 | 0.1 | 0.1 | 0.1 |
Ethanol yield on sunstrate(g/g glucose) | 0.48 | 0.48 | 0.46 | 0.40 |
Theoretical yielda(%) | 94% | 94% | 90.2% | 78.4% |
Glycerol concentrationb (g/L) | 0.39 | 0.46 | 0.23 | 0.78 |
Furfural conversion ratec [g/(h·L)] | 9.2×10-2 | 9.2×10-2 | 2.2×10-1 | 3.3×10-2 |
Ethanol productivity [g/(h·L)] | 0.624 | 0.386 | 0.476 | 6.10×10-2 |
Note:a:Experimental produced ethanol concentration divided by theoretical ethanol concentration; b:Final concentration at the end of fermentation: c:Conversion time was calculated from the beginning of the fermentation |
2.2 复合抑制剂下原始菌株和耐受菌株在低pH培养基中的生长和发酵性能比较
研究证明,乙酸的形成影响培养基的pH,继而影响菌株的性能[33]。因此本研究中将pH降低至4.0以对比研究YYJ00菌株3与S菌株的发酵性能,结果如图 2所示。由于细菌在pH高于5.0时生长速率大于酿酒酵母[34],因此降低pH可防止菌体污染。另外,低pH发酵减少了碱性物质对水解物的中和作用,因此发酵速率和产量并未明显降低。
低pH发酵条件下,S菌株的延滞期约为90h,远远超过耐受菌株YYJ003,后者约为12h。延滞期后S菌株快速生长,于144h耗尽葡萄糖,细胞终密度OD600为1.98,而YYJ003于24h内生长到稳定期,最终细胞密度OD600为1.5,相较于S菌株降低了约20%。
原始菌株S的乙醇产率在72h内较慢,96h后积累速率增加,其葡萄糖消耗趋势与乙醇的生成相一致。YYJ003菌株乙醇的产率相对较快。YYJ003菌株和S菌株的最终乙醇产量为9.05g/L和7.8g/L,分别是理论产量的90.2%和78.4%。YYJ003菌株的甘油产量比S菌株低70.5%,说明相对较低的生物量可能是YYJ003菌株乙醇产量较高的原因。
YYJ003菌株对糠醛的转化相对较快,18h内完全转化为糠醇,平均转化速率为0.072g/(L·h)。原始菌株S在延滞期内糠醛转化速率较低,但后期转化速率有较大提高,其转化速率是YYJ003菌株的1/6。这两株菌降解糠醛时均伴有细胞生长。YYJ003菌株的培养基中乙酸浓度保持不变,但S菌株的培养基中乙酸终浓度较初始浓度有15%的提高。
2.3 乙酸及pH对原始菌株和耐受菌株的综合作用为研究pH及复合抑制剂对菌株发酵性能的影响,对细胞量、乙醇产量及未解离酸进行整理比较,结果如表 2所示。以前的研究结果表明,加入1.3g/L糠醛或0.5g/L苯酚对原始菌株S几乎没有影响。不同pH主要影响未解离的乙酸浓度:当pH为5.5时,约84.7%的乙酸解离为乙酸盐,然而当pH为4.0时只有12.3%的乙酸进行了解离。苯酚和糠醛在发酵过程中基本保持不变,因此pH主要影响复合抑制剂中乙酸的状态。
Parameters | pH= 5.5 | pH 4.0 | pH 5.5 | pH 4.0 |
YYJ003 | S | |||
Undissociated acetic acid(g/L) | 0.81 | 4.65 | 0.81 | 4.65 |
Fermentation time(h) | 16 | 24 | 22 | 144 |
Biomass (OD600) | 2.2 | 1.5 | 2.1 | 1.98 |
Ethanol yield(g/g sugar) | 0.48 | 0.46 | 0.46* | 0.40 |
Glycerol yield(g/g sugar) | 0.020 | 0.012 | 0.023 | 0.039 |
* Value is significantly different (P<0.05) from that in pH 4.0 |
原始菌株S的发酵周期延长至7天,表明低pH条件对细胞的影响较大;原始菌株需更长时间以适应外界恶劣环境,这与4天的延滞期相吻合。而较低的pH对YYJ003菌株并没有显著影响。相较于pH 5.5的发酵条件下,YYJ003菌株的发酵周期延长了8h,这说明YYJ003菌株能够快速从抑制剂造成的损伤中恢复活力。
在低pH条件下YYJ003菌株的生物量有所降低,然而S菌株的生物量变化不大,这有可能是由于二者的耐受能力不同。原始菌株S需要更多的细胞来降解糠醛并耐受其他抑制剂物质,但YYJ003菌株的耐受性更好,即其单位细胞降解糠醛能力更强。因此不同的生物量展现不同的抑制剂耐受能力和应答模式。
pH的变化对乙醇和甘油的产量有一定影响。早期研究也观察到YYJ003菌株的甘油产量随pH的降低而降低[35],然而原始菌株的甘油产量随pH的降低而升高,这说明二者具有不同的代谢模式,如不同的脱氢酶活性等。不同pH条件下,YYJ003菌株的乙醇产量基本相同,而S菌株在pH4.0时产量较低,这与之前的研究结果中降低pH可提高乙醇产量相悖[33]。以前的研究证明乙醇产量主要受三种抑制剂协同作用的影响,而不仅仅是乙酸。虽然本研究中的主要变量是乙酸,但其对乙醇产量和生物量的影响复杂[26]。复合抑制剂对两个菌株乙醇产量的这些特殊影响反映了不同的应答机制和对复合抑制剂的耐受性。
2.4 水热法预处理玉米秸秆水解液中发酵表征上述结果表明,在含有复合抑制剂的培养基中YYJ003菌株能有效代谢葡萄糖,因此我们对其是否能在生物质水解液中发酵产生乙醇及工业应用价值进行了研究。本研究对原始菌株S和耐受菌株YYJ003分别在经过脱毒和未经过脱毒的水解液中进行发酵表征。本研究针对的是水热法预处理的水解液,因为水热法是一种较为经济的预处理方法[7, 36]。
在含有100g/L葡萄糖的未脱毒预处理水解液中,耐受菌株YYJ003能快速利用葡萄糖,延滞期较短,22h内到达稳定期(图 3),乙醇产量为50.2g/L,产率为0.502g/g,结果如表 3所示。然而原始菌株S在22h内未生长,且未消耗葡萄糖。YYJ003菌株在发酵期间葡萄糖消耗速率最高达到10.63g/(L·h),平均乙醇产率为2.28g/(L·h)。
Parameters | Non-detoxified hydrolysate | Detoxified hydrolysate | ||
YYJ 003 | Parent strain | YYJ 003 | Parent strain | |
Initial cell density (OD600) | 1.0 | 1.0 | 1.0 | 1.0 |
Ethanol yield on substrate (g/g glucose) | 0.50 | - | 0.50 | 0.50 |
% theoretical yield(%) | 98 | - | 98 | 98 |
Maximal sugar consumption rate (g/L) | 10.63 | - | 16.04 | 15.00 |
Ethanol productivity [g/(L·h)] | 2.28 | - | 4.16 | 4.57 |
水解液经过乙酸乙酯脱毒前,乙酸浓度为4.24g/L,是复合抑制剂浓度的80%,HMF和糠醛浓度分别为0.99g/L和0.55g/L,总苯酚浓度未检测。预处理后水解液pH为3.6,即其含有4.0g/L未解离的乙酸,这些乙酸分子经扩散进入细胞中而造成损伤[12, 37]。另外,乙酸会与HMF及糠醛协同抑制细胞生长[38]。发酵过程中并未观测到明显的延滞期,这说明虽然水解液中呋喃类物质总浓度为1.52g/L,比模拟水解液培养基的浓度稍高,但模拟培养基中的糠醛浓度足够完成耐受菌株的驯化。水解液中含有HMF及其他不存在于模拟培养基中的抑制剂物质,但YYJ003仍具有较短的延滞期,说明虽然其是通过模拟培养基驯化而得,但在发酵初期已快速提高对这些物质的耐受能力。这也证明了本研究所用的复合抑制剂适用于耐受菌株的选育及驯化。
原始菌株S和耐受菌株YYJ003在乙酸乙酯脱毒后的水解液发酵情况如图 3所示。脱毒后水解液中乙酸、HMF和糠醛分别减少了25%、49.4%和94.5%,毒性也随之大幅度降低。S菌株和YYJ003菌株均在发酵初期开始快速利用葡萄糖,发酵周期分别为10h和12h,乙醇产量分别为48.6g/L及48.7g/L,产率均约为0.50g/g。YYJ003的乙醇平均产率为4.16g/(L·h),是未脱毒水解液中的1.82倍。有研究认为耐受性的提高与新酶或新辅因子有关[10],重排的代谢通路也有助于提高菌株耐受性[39]。这些变化可能与YYJ003菌株在弱毒性水解液中糖耗速率降低有关。
水解液脱毒后,部分乙酸、呋喃及苯酚等亲油性抑制剂会被去除,这些亲油性抑制剂会跨膜扩散至细胞内,进而降低细胞内pH,造成阴离子富集、抑制糖酵解酶等关键酶的活性等一系列毒性作用[10, 40]。在未脱毒水解液中,原始菌株S完全失去代谢能力,而YYJ003仍具有相对较高的乙醇产量和产率,约为脱毒发酵的54.5%。研究证明增加接种量可缩短发酵时间并降低成本[19],因此利用高耐受性菌株代替复杂昂贵的脱毒步骤有助于提高生物乙醇产业的经济性和可行性[41]。将本研究所获得的复合抑制剂耐受菌株YYJ003与已有研究报道的比较领先的具有抑制剂耐受能力的菌株进行比较,如表 4所示。表 4中所列菌株乙醇产量均低于0.46g/g(乙醇/葡萄糖),不同接种量、发酵方法或抑制剂组成,菌株的乙醇产率为0.05~2.41g/(L·h)。本研究分别针对复合抑制剂培养基和水热法预处理的生物质水解液,使用远低于其他研究的初始细胞浓度,为0.055g DCW/L(干细胞重/L)和0.55g DCW/L,同时为防止染菌将培养基pH控制在4.0。在上述条件下YYJ003菌株的乙醇产量达到0.46g/g,并具有相对较高的乙醇产率。借助代谢工程技术改造YYJ003菌株,有望利用木糖或阿拉伯糖等戊糖生产乙醇,这将更进一步优化纤维素乙醇工业的生产效率和成本[42-43]。
Strain | Inhibitors/Concentration(g/L) | YEtOH (%) | QEtOH [g/(L·h)] | Reference |
Y1528 | SW SSL(furfural 0.05a+HMFb0.16+HACc 10) | 0.37 | 1.90 | [19] |
Y1528 | HW SSL(furfural 0.18+HMF 0.16+HAC 10) | 0.32 | 0.42 | [19] |
Tembec T1 | SW SSL(furfural 0.05 + HMF 0.16+HAC 10) | 0.34 | 1.87 | [19] |
Tembec T1 | HW SSL(furfural 0.18 +HMF 0.16 +HAC 10) | 0.34 | 0.43 | [19] |
Bakers’ yeast | Furfural 1.0+HAC 5.0 + POHd 1.0 | 0.41 | 2.41 | [25] |
Adapted BCRC 21777 | Sulfate 15+HAC17 + furfural 1.0 | 0.44 | 0.24 | [44] |
Adapted TMB3001 | Hydrolysate SA100 (Furaldehyde 4.5 + Aliphatic acids 10.1+Phenols 2.8) Hydrolysate SA75 (75 % dilution) Hydrolysate SA50 (50% dilution) | 0.09 0.11 0.38 | 0.05 0.09 0.51 | [45] |
PichiastipitisPS101 | HW SSL HAC 10+furfural 1.8+HMF 1.1 | 0.192 | 0.038 | [46] |
Adapted Y5 | HAC 5.12+Levulincid acid 0.31+HMF2.51+ furfural1.99+formic acid 3.59 | 0.44 | 0.31 | [47] |
YYJ003 | HAC 5.3 + furfural 1.3+ phenol 1.0 | 0.48 | 0.43(low inoculums) | This study |
YYJ003 | LHW pre-treated corn stover hydrolysate | 0.50 | 2.28 | This study |
Note:a: The symbol means the inhibitor and corresponding concentration in hydrolysates; b: HMF means 5-hydroxymethyl-furfural; c: HAC represents acetic acid; d: POH represents p-hydroxybenzoic acid |
[1] | Li Z, Ji X, Kan S, et al. Past, present, and future industrial biotechnology in China. Adv Biochem Eng Biotechnol,2010, 122 : 1 –42. |
[2] | Buraimoh O M, Ilori M O, Amund O O, et al. Assessment of bacterial degradation of lignocellulosic residues (sawdust) in a tropical estuarine microcosm using improvised floating raft equipment. International Biodeterioration & Biodegradation,2015, 104 : 186 –193. |
[3] | Zhong C, Cao Y X, Li B Z, et al. Biofuels in China: past, present and future. Biofuels, Bioproducts and Biorefining,2010, 4 (3) : 326 –342. |
[4] | Farrell A E, Plevin R J, Turner B T, et al. Ethanol can contribute to energy and environmental goals. Science,2006, 311 (5760) : 506 –508. |
[5] | Solomon B D, Barnes J R, and Halvorsen K E. Grain and cellulosic ethanol: history, economics, and energy policy. Biomass and Bioenergy,2007, 31 (6) : 416 –425. |
[6] | Parawira W, Tekere M. Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review. Crit Rev Biotechnol,2011, 31 (1) : 20 –31. |
[7] | Ingram T, Wormeyer K, Lima J C, et al. Comparison of different pretreatment methods for lignocellulosic materials. Part I: conversion of rye straw to valuable products. Bioresour Technol,2011, 102 (8) : 5221 –5228. |
[8] | Marker T L, Felix L G, Linck M B, et al. Integrated hydropyrolysis and hydroconversion (IH2) for the direct production of gasoline and diesel fuels or blending components from biomass, part 1: Proof of principle testing. Environmental Progress & Sustainable Energy,2012, 31 (2) : 191 –199. |
[9] | Silveira M H L, Morais A R C, da Costa Lopes A M, et al. Current pretreatment technologies for the development of cellulosic ethanol and biorefineries. ChemSusChem,2015, 8 (20) : 3366 –3390. |
[10] | Palmqvist E, Hahn-Hgerdal B. Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification.. Bioresource Technology,2000, 74 (1) : 17 –24. |
[11] | Field S, Ryden P, Wilson D, et al. Identification of furfural resistant strains of Saccharomyces cerevisiae and Saccharomyces paradoxus from a collection of environmental and industrial isolates. Biotechnology for Biofuels,2015, 8 (1) : 1 –8. |
[12] | Narendranath N V, Thomas K C, Ingledew W M. Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium. J Ind Microbiol Biotechnol,2001, 26 (3) : 171 –177. |
[13] | Li H, Zhang X, Shen Y, et al. Inhibitors and their effects on Saccharomyces cerevisiae and relevant countermeasures in bioprocess of ethanol production from lignocellulose. Chin J Biotech,2009, 25 (9) : 1321 –1328. |
[14] | Jonsson L J, Alriksson B, Nilvebrant N O. Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnol Biofuels,2013, 6 (1) : 16 . |
[15] | Guo X, Cavka A, Jonsson L J, et al. Comparison of methods for detoxification of spruce hydrolysate for bacterial cellulose production. Microb Cell Fact,2013, 12 : 93 . |
[16] | Nieves L M, Panyon L A, Wang X. Engineering sugar utilization and microbial tolerance toward lignocellulose conversion. Front Bioeng Biotechnol,2015, 3 : 17 –27. |
[17] | Zhang M M, Zhao X Q, Cheng C, et al. Improved growth and ethanol fermentation of Saccharomyces cerevisiae in the presence of acetic acid by overexpression of SET5 and PPR1. Biotechnology Journal,2015, 10 (12) : 1903 –1911. |
[18] | Wallace-Salinas V, Gorwa-Grauslund M F. Adaptive evolution of an industrial strain of Saccharomyces cerevisiae for combined tolerance to inhibitors and temperature. Biotechnol Biofuels,2013, 6 (1) : 151 . |
[19] | Keating J D, Panganiban C, Mansfield S D. Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds. Biotechnol Bioeng,2006, 93 (6) : 1196 –1206. |
[20] | Zha J, Li B Z, Shen M H, et al. Optimization of CDT-1 and XYL1 expression for balanced Co-production of ethanol and xylitol from cellobiose and xylose by engineered Saccharomyces cerevisiae. PLoS One,2013, 8 (7) : e68317 . |
[21] | Wang X, Bai X, Chen D F, et al. Increasing proline and myo-inositol improves tolerance of Saccharomyces cerevisiae to the mixture of multiple lignocellulose-derived inhibitors. Biotechnol Biofuels,2015, 8 : 142 –150. |
[22] | Li B Z, Yuan Y J. Transcriptome shifts in response to furfural and acetic acid in Saccharomyces cerevisiae. Appl Microbiol Biotechnol,2010, 86 (6) : 1915 –1924. |
[23] | Strijbis K, Distel B. Intracellular acetyl unit transport in fungal carbon metabolism. Eukaryot Cell,2010, 9 (12) : 1809 –1815. |
[24] | Oshoma C E, Greetham D, Louis E J, et al. Screening of non-Saccharomyces cerevisiae strains for tolerance to formic acid in bioethanol fermentation. PLoS One,2015, 10 (8) : e0135626 . |
[25] | Palmqvist E, Grage H, Meinander N Q, et al. Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts. Biotechnol Bioeng,1999, 63 (1) : 46 –55. |
[26] | Oliva J M, Negro M J, Sáez F, et al. Effects of acetic acid, furfural and catechol combinations on ethanol fermentation of Kluyveromyces marxianus. Process Biochemistry,2006, 41 (5) : 1223 –1228. |
[27] | Li B Z, Cheng J S, Qiao B, et al. Genome-wide transcriptional analysis of Saccharomyces cerevisiae during industrial bioethanol fermentation. J Ind Microbiol Biotechnol,2010, 37 (1) : 43 –55. |
[28] | Martinez A, Rodriguez M E, Wells M L, et al. Detoxification of dilute acid hydrolysates of lignocellulose with lime. Biotechnol Prog,2001, 17 (2) : 287 –293. |
[29] | Liu Z L, Slininger P J, Gorsich S W. Enhanced biotransformation of furfural and hydroxymethylfurfural by newly developed ethanologenic yeast strains. Appl Biochem Biotechnol,2005, 121 (2) : 451 –460. |
[30] | Wallace-Salinas V, Brink D P, Ahren D, et al. Cell periphery-related proteins as major genomic targets behind the adaptive evolution of an industrial Saccharomyces cerevisiae strain to combined heat and hydrolysate stress. BMC Genomics,2015, 16 (1) : 514 . |
[31] | Heer D, Sauer U. Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain. Microb Biotechnol,2008, 1 (6) : 497 –506. |
[32] | Smith J, van Rensburg E, Gorgens J F. Simultaneously improving xylose fermentation and tolerance to lignocellulosic inhibitors through evolutionary engineering of recombinant Saccharomyces cerevisiae harbouring xylose isomerase. BMC Biotechnol,2014, 14 : 41 –58. |
[33] | Casey E, Sedlak M, Ho N W, et al. Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cerevisiae. FEMS Yeast Res,2010, 10 (4) : 385 –393. |
[34] | Sanchezi Nogue V, Narayanan V, Gorwa-Grauslund M F. Short-term adaptation improves the fermentation performance of Saccharomyces cerevisiae in the presence of acetic acid at low pH. Appl Microbiol Biotechnol,2013, 97 (16) : 7517 –7525. |
[35] | Bellissimi E, van Dijken J P, Pronk J T, et al. Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces cerevisiae strain. FEMS Yeast Res,2009, 9 (3) : 358 –364. |
[36] | Yu Q, Liu J, Zhuang X, et al. Liquid hot water pretreatment of energy grasses and its influence of physico-chemical changes on enzymatic digestibility. Bioresource Technology,2016, 199 : 265 –270. |
[37] | Svanstrom A, Boveri S, Bostrom E, et al. The lactic acid bacteria metabolite phenyllactic acid inhibits both radial growth and sporulation of filamentous fungi. BMC Res Notes,2013, 6 : 464 –473. |
[38] | Liu Z L, Slininger P J, Dien B S, et al. Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran. J Ind Microbiol Biotechnol,2004, 31 (8) : 345 –352. |
[39] | Yi X, Gu H, Gao Q, et al. Transcriptome analysis of Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic aldehyde inhibitors from lignocellulose pretreatment. Biotechnol Biofuels,2015, 8 : 153 –168. |
[40] | Shui Z X, Qin H, Wu B, et al. Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors. Applied Microbiology and Biotechnology,2015, 99 (13) : 5739 –5748. |
[41] | Izmirlioglu G, Demirci A. Enhanced bio-ethanol production from industrial potato waste by statistical medium optimization. International Journal of Molecular Sciences,2015, 16 (10) : 24490 . |
[42] | Qi X, Zha J, Liu G G, et al. Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae. Front Microbiol,2015, 6 : 1165 . |
[43] | Shen M H, Song H, Li B Z, et al. Deletion of D-ribulose-5-phosphate 3-epimerase (RPE1) induces simultaneous utilization of xylose and glucose in xylose-utilizing Saccharomyces cerevisiae. Biotechnol Lett,2015, 37 (5) : 1031 –1036. |
[44] | Huang C F, Lin T H, Guo G L, et al. Enhanced ethanol production by fermentation of rice straw hydrolysate without detoxification using a newly adapted strain of Pichia stipitis. Bioresour Technol,2009, 100 (17) : 3914 –3920. |
[45] | Martin C, Marcet M, Almazan O, et al. Adaptation of a recombinant xylose-utilizing Saccharomyces cerevisiae strain to a sugarcane bagasse hydrolysate with high content of fermentation inhibitors. Bioresour Technol,2007, 98 (9) : 1767 –1773. |
[46] | Bajwa P K, Shireen T, D'Aoust F, et al. Mutants of the pentose-fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor. Biotechnol Bioeng,2009, 104 (5) : 892 –900. |
[47] | Tian S, Zhu J, Yang X. Evaluation of an adapted inhibitor-tolerant yeast strain for ethanol production from combined hydrolysate of softwood. Applied Energy,2011, 88 (5) : 1792 –1796. |