Comparative metabolic profiling of parental and inhibitors-tolerant yeasts during lignocellulosic ethanol fermentation

The metabolic responses of parental and inhibitors-tolerant yeasts in presence of the combination of three inhibitors (furfural, phenol and acetic acid) during ethanol fermentation were investigated by comparative metabolic profiling. Samples of parental and tolerant yeasts with/without three inhibitors in fermentation medium represented significantly different metabolic states. Further investigation on the specific responses of two strains revealed that the levels of most amino acids, inositol, and phenethylamine were dramatically increased in presence of inhibitors in parental yeast, while they kept relatively stable in tolerant yeast. It suggested that the protein degradation was increased and oxygen stress was induced by combined inhibitors in parental yeast. In addition, carbon metabolism (glycolysis and TCA) and pyrimidine ribonucleotides pathway (uracil and cytosine) were reduced in both strains in presence of combined inhibitors, which was considered as the general stress response. Higher levels of pyridimines in tolerant yeast suggested that they were responsible for counteracting the stress of combined inhibitors. These findings provided new insights into underlying mechanisms of yeast in resistance to the synergistic effects of inhibitors in lignocellulose hydrolysates.     

Effects of some inhibitors on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides and preparation of biodiesel by enzymatic transesterification of the lipid

Microbial lipid produced using yeast fermentation with inexpensive carbon sources such as lignocellulosic hydrolyzate can be an alternative feedstock for biodiesel production. Several inhibitors that can be generated during acid hydrolysis of lignocellulose were added solely or together into the culture medium to study their individual inhibitory actions and their synergistic effects on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides. When the inhibitors were present in isolation in the medium, to obtain a high cell biomass accumulation, the concentrations of formic acid, acetic acid, furfural and vanillin should be lower than 2, 5, 0.5 and 1.5 g/L, respectively. However, the synergistic effects of these compounds could dramatically decrease the minimum critical inhibitory concentrations leading to significant growth and lipid production inhibitions. Unlike the above-cited inhibitors, sodium lignosulphonate had no negative influence on biomass accumulation when its concentration was in the range of 0.5-2.0 g/L; in effect, it was found to facilitate cell growth and sugar-to-lipid conversion. The fatty acid compositional profile of the yeast lipid was in the compositional range of various plant oils and animal tallow. Finally, the crude yeast lipid from bagasse hydrolyzate could be well converted into fatty acid methyl ester (FAME, biodiesel) by enzymatic transesterification in a tert-butanol system with biodiesel yield of 67.2% and lipid-to-biodiesel conversion of 88.4%.     

Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds

Synthetic mixtures of predominant lignocellulosic hexose sugars were supplemented with separate aliquots of three inhibitory compounds (furfural, hydroxymethylfurfural (HMF), and acetic acid) in a series of concentrations and fermented by the spent sulfite liquor (SSL)-adapted yeast strain Tembec T1 and the natural isolate Saccharomyces cerevisiae (S. cerevisiae) Y-1528 to compare tolerance and assess fermentative efficacy. The performance of Y-1528 exceeded that of Tembec T1 by a significant margin, with faster hexose sugar consumption, higher ethanol productivity, and in the case of furfural and HMF, faster inhibitor consumption. Nevertheless, furfural had a dose-proportionate effect on sugar consumption rate and ethanol productivity in both strains, but did not substantially affect ethanol yield. HMF had a similar effect on sugar consumption rate and ethanol productivity, and also lowered ethanol yield. Surprisingly, acetic acid had the least impact on sugar consumption rate and ethanol productivity, and stimulated ethanol yield at moderate concentrations. Sequential iterations of softwood (SW) and hardwood (HW) SSL were subsequently inoculated with the two yeast strains in order to compare adaptation to, and performance in lignocellulosic substrates in a cell recycle batch fermentation (CRBF) regime. Both strains were severely affected by the HW SSL, which was attributed to specific syringyl lignin-derived degradation products and synergistic interactions between inhibitors. Though ethanologenic capacity was preserved, a net loss of performance was evident from both strains, indicating the absence of adaptation to the substrates, regardless of the sequence in which the SSL types were employed.     

Inhibition analysis of inhibitors derived from lignocellulose pretreatment on the metabolic activity of Zymomonas mobilis biofilm and planktonic cells and the proteomic responses

Lignocellulose pretreatment produces various toxic inhibitors that affect microbial growth, metabolism, and fermentation. Zymomonas mobilis is an ethanologenic microbe that has been demonstrated to have potential to be used in lignocellulose biorefineries for bioethanol production. Z. mobilis biofilm has previously exhibited high potential to enhance ethanol production by presenting a higher viable cell number and higher metabolic activity than planktonic cells or free cells when exposed to lignocellulosic hydrolysate containing toxic inhibitors. However, there has not yet been a systematic study on the tolerance level of Z. mobilis biofilm compared to planktonic cells against model toxic inhibitors derived from lignocellulosic material. We took the first insight into the concentration of toxic compound (formic acid, acetic acid, furfural, and 5‐HMF) required to reduce the metabolic activity of Z. mobilis biofilm and planktonic cells by 25% (IC₂₅), 50% (IC₅₀), 75% (IC₇₅), and 100% (IC₁₀₀). Z. mobilis strains ZM4 and TISTR 551 biofilm were two‐ to three fold more resistant to model toxic inhibitors than planktonic cells. Synergetic effects were found in the presence of formic acid, acetic acid, furfural, and 5‐HMF. The IC₂₅ of Z. mobilis ZM4 biofilm and TISTR 551 biofilm were 57 mm formic acid, 155 mm acetic acid, 37.5 mm furfural and 6.4 mm 5‐HMF, and 225 mm formic acid, 291 mm acetic acid, 51 mm furfural and 41 mm 5‐HMF, respectively. There was no significant difference found between proteomic analysis of the stress response to toxic inhibitors of Z. mobilis biofilm and planktonic cells on ZM4. However, TISTR 551 biofilms exhibited two proteins (molecular chaperone DnaK and 50S ribosomal protein L2) that were up‐regulated in the presence of toxic inhibitors. TISTR 551 planktonic cells possessed two types of protein in the group of 30S ribosomal proteins and motility proteins that were up‐regulated.     

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。     

Proteomic research reveals the stress response and detoxification of yeast to combined inhibitors

The tolerant mechanism of yeast to the combination of three inhibitors (furfural, phenol and acetic acid) was investigated using 2-DE combined with MALDI-TOF/TOF-MS. The stress response and detoxification related proteins (e.g., Ahp1p, Hsp26p) were expressed higher in the tolerant yeast than in the parental yeast. The expressions of most nitrogen metabolism related proteins (e.g. Gdh1p, Met1p) were higher in the parental yeast, indicating that the tolerant yeast decreases its nitrogen metabolism rate to reserve energy, and possesses high resistance to the stress of combined inhibitors. Furthermore, upon exposure to the inhibitors, the proteins related to protein folding, degradation and translation (e.g., Ssc1p, Ubp14p, Efb1p) were all significantly affected, and the oxidative stress related proteins (e.g., Ahp1p, Grx1p) were increased. Knockdown of genes related to the oxidative stress and unfolded protein response (Grx1, Gre2, Asc1) significantly decreased the tolerance of yeast to inhibitors, which further suggested that yeast responded to the inhibitors mainly by inducing unfolded protein response. This study reveals that increasing the detoxification and tolerating oxidative stress, and/or decreasing the nitrogen metabolism would be promising strategies in developing more tolerant strains to the multiple inhibitors in lignocellulose hydrolysates.     

选育耐受复合抑制剂酿酒酵母提高乙醇产量

能够耐受纤维素预处理中抑制剂的酿酒酵母对高效、经济生产纤维素乙醇至关重要。利用诱变结合驯化工程选育了一株可耐受复合抑制剂(1.3g/L糠醛、5.3g/L乙酸及1.0g/L苯酚)的工业酿酒酵母YYJ003。在pH 4.0的含有抑制剂的培养基中,耐受菌株乙醇产率是原始菌株的7.8倍,糠醛转化速率提高了5倍。在pH 5.5的复合抑制剂条件下,YYJ003发酵时间(16h)比野生菌株发酵时间(22h)缩短6h。在pH 4.0的未脱毒的玉米秸秆水热法预处理水解液中YYJ003的乙醇产率达到0.50g/g(乙醇/葡萄糖),乙醇产速达到4.16g/(L·h),而对照菌株无乙醇产出。     

Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 II: production of xylitol and ethanol in the presence of inhibitors

A systematic study was conducted characterizing the effect of furfural, 5-hydroxymethylfurfural (5-HMF), and acetic acid concentration on the production of xylitol and ethanol by a novel endophytic yeast, Rhodotorula mucilaginosa strain PTD3. The influence of different inhibitor concentrations on the growth and fermentation abilities of PTD3 cultivated in synthetic nutrient media containing 30?g/l xylose or glucose were measured during liquid batch cultures. Concentrations of up to 5?g/l of furfural stimulated production of xylitol to 77?% of theoretical yield (10?% higher compared to the control) by PTD3. Xylitol yields produced by this yeast were not affected in the presence of 5-HMF at concentrations of up to 3?g/l. At higher concentrations of furfural and 5-HMF, xylitol and ethanol yields were negatively affected. The higher the concentration of acetic acid present in a media, the higher the ethanol yield approaching 99?% of theoretical yield (15?% higher compared to the control) was produced by the yeast. At all concentrations of acetic acid tested, xylitol yield was lowered. PTD3 was capable of metabolizing concentrations of 5, 15, and 5?g/l of furfural, 5-HMF, and acetic acid, respectively. This yeast would be a potent candidate for the bioconversion of lignocellulosic sugars to biochemicals given that in the presence of low concentrations of inhibitors, its xylitol and ethanol yields are stimulated, and it is capable of metabolizing pretreatment degradation products.     

Cofactor dependence in furan reduction by Saccharomyces cerevisiae in fermentation of acid-hydrolyzed lignocellulose

A decreased fermentation rate due to inhibition is a significant problem for economic conversion of acid-pretreated lignocellulose hydrolysates to ethanol, since the inhibition gives rise to a requirement for separate detoxification steps. Together with acetic acid, the sugar degradation products furfural and 5-hydroxymethyl furfural are the inhibiting compounds found at the highest concentrations in hydrolysates. These aldehydes have been shown to affect both the specific growth rate and the rate of fermentation by yeast. Two strains of Saccharomyces cerevisiae with different abilities to ferment inhibiting hydrolysates were evaluated in fermentations of a dilute acid hydrolysate from spruce, and the reducing activities for furfural and 5-hydroxymethyl furfural were determined. Crude cell extracts of a hydrolysate-tolerant strain (TMB3000) converted both furfural and 5-hydroxymethyl furfural to the corresponding alcohol at a rate that was severalfold higher than the rate observed for cell extracts of a less tolerant strain (CBS 8066), thereby confirming that there is a correlation between the fermentation rate in a lignocellulosic hydrolysate and the bioconversion capacity of a strain. The in vitro NADH-dependent furfural reduction capacity of TMB3000 was three times higher than that of CBS 8066 (1,200 mU/mg protein and 370 mU/mg protein, respectively) in fed-batch experiments. Furthermore, the inhibitor-tolerant strain TMB3000 displayed a previously unknown NADH-dependent reducing activity for 5-hydroxymethyl furfural (400 mU/mg protein during fed-batch fermentation of hydrolysates). No corresponding activity was found in strain CBS 8066 (<2 mU/mg). The ability to reduce 5-hydroxymethyl furfural is an important characteristic for the development of yeast strains with increased tolerance to lignocellulosic hydrolysates.     

Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts

The influence of the factors acetic acid, furfural, and p-hydroxybenzoic acid on the ethanol yield (YEtOH) of Saccharomyces cerevisiae, bakers' yeast, S. cerevisiae ATCC 96581, and Candida shehatae NJ 23 was investigated using a 2(3)-full factorial design with 3 centrepoints. The results indicated that acetic acid inhibited the fermentation by C. shehatae NJ 23 markedly more than by bakers' yeast, whereas no significant difference in tolerance towards the compounds was detected between the S. cerevisiae strains. Furfural (2 g L-1) and the lignin derived compound p-hydroxybenzoic acid (2 g L-1) did not affect any of the yeasts at the cell mass concentration used. The results indicated that the linear model was not adequate to describe the experimental data (the p-values of curvatures were 0.048 for NJ 23 and 0.091 for bakers' yeast). Based on the results from the 2(3)-full factorial experiment, an extended experiment was designed based on a central composite design to investigate the influence of the factors on the specific growth rate (mu), biomass yield (Yx), volumetric ethanol productivity (QEtOH), and YEtOH. Bakers' yeast was chosen in the extended experiment due to its better tolerance towards acetic acid, which makes it a more interesting organism for use in industrial fermentations of lignocellulosic hydrolysates. The inoculum size was reduced in the extended experiment to reduce any increase in inhibitor tolerance that might be due to a large cell inoculum. By dividing the experiment in blocks containing fermentations performed with the same inoculum preparation on the same day, much of the anticipated systematic variation between the experiments was separated from the experimental error. The results of the fitted model can be summarised as follows: mu was decreased by furfural (0-3 g L-1). Furfural and acetic acid (0-10 g L-1) also interacted negatively on mu. Furfural concentrations up to 2 g L-1 stimulated Yx in the absence of acetic acid whereas higher concentrations decreased Yx. The two compounds interacted negatively on Yx and YEtOH. Acetic acid concentrations up to 9 g L-1 stimulated QEtOH, whereas furfural (0-3 g L-1) decreased QEtOH. Acetic acid in concentrations up to 10 g L-1 stimulated YEtOH in the absence of furfural, and furfural (0-2 g L-1) slightly increased YEtOH in the absence of acetic acid whereas higher concentrations caused inhibition. Acetic acid and furfural interacted negatively on YEtOH.     

Cofactor Dependence in Furan Reduction by Saccharomyces cerevisiae in Fermentation of Acid-Hydrolyzed Lignocellulose

A decreased fermentation rate due to inhibition is a significant problem for economic conversion of acid-pretreated lignocellulose hydrolysates to ethanol, since the inhibition gives rise to a requirement for separate detoxification steps. Together with acetic acid, the sugar degradation products furfural and 5-hydroxymethyl furfural are the inhibiting compounds found at the highest concentrations in hydrolysates. These aldehydes have been shown to affect both the specific growth rate and the rate of fermentation by yeast. Two strains of Saccharomyces cerevisiae with different abilities to ferment inhibiting hydrolysates were evaluated in fermentations of a dilute acid hydrolysate from spruce, and the reducing activities for furfural and 5-hydroxymethyl furfural were determined. Crude cell extracts of a hydrolysate-tolerant strain (TMB3000) converted both furfural and 5-hydroxymethyl furfural to the corresponding alcohol at a rate that was severalfold higher than the rate observed for cell extracts of a less tolerant strain (CBS 8066), thereby confirming that there is a correlation between the fermentation rate in a lignocellulosic hydrolysate and the bioconversion capacity of a strain. The in vitro NADH-dependent furfural reduction capacity of TMB3000 was three times higher than that of CBS 8066 (1,200 mU/mg protein and 370 mU/mg protein, respectively) in fed-batch experiments. Furthermore, the inhibitor-tolerant strain TMB3000 displayed a previously unknown NADH-dependent reducing activity for 5-hydroxymethyl furfural (400 mU/mg protein during fed-batch fermentation of hydrolysates). No corresponding activity was found in strain CBS 8066 (<2 mU/mg). The ability to reduce 5-hydroxymethyl furfural is an important characteristic for the development of yeast strains with increased tolerance to lignocellulosic hydrolysates.     

Biomass Conversion Inhibitors Furfural and 5-Hydroxymethylfurfural Induce Formation of Messenger RNP Granules and Attenuate Translation Activity in Saccharomyces cerevisiae

Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.     

Effect of fermentation inhibitors in the presence and absence of activated charcoal on the growth of Saccharomyces cerevisiae

The acidic hydrolysis of biomass generates numerous inhibitors of fermentation, which adversely affect cell growth and metabolism. The goal of the present study was to determine the effects of fermentation inhibitors on growth and glucose consumption by Saccharomyces cerevisiae. We also conducted in situ adsorption during cell cultivation in synthetic broth containing fermentation inhibitors. In order to evaluate the effect of in situ adsorption on cell growth, five inhibitors, namely 5-hydroxymethylfurfural, levulinic acid, furfural, formic acid, and acetic acid, were introduced into synthetic broth. The existence of fermentation inhibitors during cell culture adversely affects cell growth and sugar consumption. Furfural, formic acid, and acetic acid were the most potent inhibitors in our culture system. The in situ adsorption of inhibitors by the addition of activated charcoal to the synthetic broth increased cell growth and sugar consumption. Our results indicate that detoxification of fermentation media by in situ adsorption may be useful for enhancing biofuel production.     

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。     

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.     

Development of a phenotypic assay for characterisation of ethanologenic yeast strain sensitivity to inhibitors released from lignocellulosic feedstocks

Inhibitors released by the breakdown of plant cell walls prevent efficient conversion of sugar into ethanol. The aim of this study was to develop a fast and reliable inhibitor sensitivity assay for ethanologenic yeast strains. The assay comprised bespoke 96-well plates containing inhibitors in isolation or combination in a format that was compatible with the Phenotypic Microarray Omnilog reader (Biolog, hayward, CA, USA). A redox reporter within the assay permits analysis of inhibitor sensitivity in aerobic and/or anaerobic conditions. Results from the assay were verified using growth on spot plates and tolerance assays in which maintenance of viability was assessed. The assay allows for individual and synergistic effects of inhibitors to be determined. It was observed that the presence of both acetic and formic acid significantly inhibited the yeast strains assessed, although this impact could be partially mitigated by buffering to neutral pH. Scheffersomyces stipitis, Candida spp., and Pichia guilliermondii demonstrated increased sensitivity to short chain weak acids at concentrations typically present in lignocellulosic hydrolysates. S. cerevisiae exhibited robustness to short chain weak acids at these concentrations. However, S. stipitis, Candida spp., and P. guilliermondii displayed increased tolerance to HMF when compared to that observed for S. cerevisiae. The results demonstrate that the phenotypic microarray assay developed in the current study is a valuable tool that can be used to identify yeast strains with desirable resistance to inhibitory compounds found in lignocellulosic hydrolysates.