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High-titer-ethanol production from cellulosic hydrolysate by an engineered strain of Saccharomyces cerevisiae during an in situ removal process reducing the inhibition of ethanol on xylose metabolism
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Short communication
High-titer-ethanol production from cellulosic hydrolysate by an engineered strain of Saccharomyces cerevisiae during an in situ removal process reducing the inhibition of ethanol on xylose metabolism Bo Zhang a,1 , Hongbing Sun a,b , Jing Li c , Yinhua Wan c , Yin Li a , Yanping Zhang a,∗ a
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China c Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China b
a r t i c l e
i n f o
Article history: Received 17 July 2015 Received in revised form 12 April 2016 Accepted 20 April 2016 Available online xxx Keywords: Cellulosic ethanol Ethanol inhibition Fermentation–pervaporation Saccharomyces cerevisiae Xylose catabolism
a b s t r a c t Efficient xylose utilization is critical for the production of fuels from biomass hydrolysates. It is known that xylose catabolism is inhibited by glucose. In this study, we showed that ethanol also inhibits xylose catabolism. By introducing a xylose metabolic pathway into Saccharomyces cerevisiae and using evolutionary engineering, an engineered S. cerevisiae strain, W32N55, was obtained that can anaerobically ferment xylose to ethanol. The effect of ethanol on xylose utilization was investigated. The results showed that xylose catabolism was inhibited upon the addition of ethanol, and it resumed once ethanol was removed. Based on these results, a fermentation–pervaporation coupling process was developed. After the in situ removal of ethanol, 150 g/L glucose and 31 g/L xylose were consumed in 72 h, providing a total of 76 g/L ethanol and an overall total sugar yield of 0.42 g/g. We believe that this strain will be valuable to the bio-ethanol industry. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulose, one of the world’s most abundant renewable resources, is the most favorable alternative feedstock for the production of fuels and chemicals [6]. Lignocellulosic biomass is generally composed of cellulose, hemicellulose, and lignin. Upon pretreatment via alkaline, acidic, and/or enzymatic hydrolyses, lignocellulose is decomposed to glucose, xylose, and a small amount of arabinose, and such treatments also result in the production of some microbial growth inhibitors [23]. To use lignocellulosic resources for biotechnological purposes, a desirable strain is expected to co-utilize glucose and xylose, and tolerate inhibitors. However, natural strains of Saccharomyces cerevisiae cannot utilize xylose for growth or ethanol production. Because S. cerevisiae possesses a metabolic pathway to convert xylulose, a metabolic product of xylose, into ethanol, many efforts have been made to introduce metabolic pathways, including the xylose reductase–xylitol dehydrogenase (XR-XDH) and xylose isomerase (XI) pathways, which convert xylose to xylulose, into
∗ Corresponding author. E-mail address: [email protected] (Y. Zhang). 1 Present address: College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
S. cerevisiae [7,5,24]. However, the rates of xylose consumption and ethanol production in such recombinant strains are unsatisfactory. In addition, most of the previously engineered strains can only ferment xylose under aerobic or microaerobic conditions [24,15], which is not favorable for the development of the bio-ethanol industry. Therefore, evolutionary engineering is the key to the development of xylose-fermenting S. cerevisiae strains, and many such examples have been demonstrated [22,11]. However, few strains can co-utilize glucose and xylose in high-sugar concentration hydrolysates to produce a high ethanol yield. In an attempt to obtain an engineered S. cerevisiae strain that is capable of efficiently co-utilizing glucose and xylose, a xylose metabolic pathway was introduced into a S. cerevisiae strain, and it was allowed to evolve under oxygen-limiting conditions. The resulting strain, W32N55, fermented xylose to ethanol under anaerobic conditions. Using this strain, we found that the consumption rate of xylose decreased significantly during the late fermentation period, when a high concentration of ethanol was obtained. These results led to the hypothesis that in addition to glucose, ethanol might inhibit xylose catabolism. To verify this, we investigated the effect of ethanol on xylose catabolism. This was followed by the development of a fermentation–pervaporation coupling process, from which the efficient production of ethanol from corn stover hydrolysates was achieved.
http://dx.doi.org/10.1016/j.procbio.2016.04.019 1359-5113/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: B. Zhang, et al., High-titer-ethanol production from cellulosic hydrolysate by an engineered strain of Saccharomyces cerevisiae during an in situ removal process reducing the inhibition of ethanol on xylose metabolism, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.04.019
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2 Table 1 Plasmids and strains used in this study. Strain/plasmid
Relevant genotype
Source
Plasmids plasmid-2 pXYL1-2 (LEU2) pXYL2-2 (LEU2) Xks1-2 (LEU2) pXYL12-2u(LEU2) pXYL12-XK-2u (LEU2)
plasmid-2 , leu, E. coli ori, Apr plasmid-2 -xyl1, leu, E. coli ori, Apr plasmid-2 -xyl2, leu, E. coli ori, Apr plasmid-2 -xks1, leu, E. coli ori, Apr plasmid-2 - xyl1-xyl2, leu, E. coli ori, Apr plasmid-2 - xyl1-xyl2-xks1, leu, E. coli ori, Apr
Our lab stored This study This study This study This study This study
MATa/MAT␣ ura3-52/ura3-52; trp1 2/trp12; leu2-3,112/leu2-3,112;his3-11/his3-11; ade2-1/ade2-1; can1-100/can1-100 F-,80dlacZM15, (lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44, -, thi-1, gyrA96, relA1
EUROSCARF
Strains S. cerevisiae W303-1B (2n) E. coli DH5␣
2. Materials and methods 2.1. Strain construction Plasmids and yeast strains used in this study are listed in Table 1. The plasmid construction steps were as follows. The XR-encoding gene xyl1 and the XDH-encoding gene xyl2 were polymerase chain reaction (PCR)-amplified from the genomic DNA of Pichia stipites, and inserted into the BamHI site of plasmid-2 (LEU2), resulting in plasmids pXYL1-2 (LEU2) and pXYL2-2 (LEU2), respectively, in which the expression of the target genes was controlled by the GPD promoter. Similarly, the xylulokinase (XK)-encoding xk gene from S. cerevisiae was inserted into plasmid-2 (LEU2) under the control of GPD promoter, resulting in plasmid pXK-2 (LEU2). Next, a xyl2 cassette containing a promoter, the xyl2 gene, and a terminator was PCR-amplified from pXYL2-2 (LEU2) and introduced into pXYL1-2 (LEU2), resulting in plasmid pXYL12-2 (LEU2). An xk cassette containing a promoter, the xk gene, and a terminator was PCR-amplified from pXK-2 (LEU2) and ligated into pXYL122 (LEU2), resulting in the final expression vector pXYL12-XK-2 (LEU2) (Fig. S1). S. cerevisiae strain W303-1B (2N), a diploid strain that is easy to genetically manipulate, was used as the host strain. By transforming pXYL12-XK-2 (LEU2) into strain W303-1B (2N), the engineered strain W32N01 was obtained and identified by PCR and enzymatic activity assays. Minimal medium (yeast nitrogen base without amino acids, 0.67%) supplemented with 20 g/L xylose was used for strain evolution at 30 ◦ C. First, strain W32N01 was grown under aerobic conditions on a rotary shaker at 220 rpm. The cells were cultivated until they reached the mid-exponential phase (optical density at 600 nm (OD600 ) = 4), and then they were transferred to fresh medium. The process was repeated until the strain growth rate improved significantly. Then, the dissolved oxygen availability was reduced by gradually lowering the shaker speed from the original 220 rpm to 100 rpm. After 55 transfers (a 2% inoculum was used for each transfer), a single clone, named W3N55, was obtained from the last shake flask. W32N55 was used for ethanol fermentation in this study.
2.2. Enzyme activity assays XR, XDH, and XK activities were measured according to the following protocol. Target strains (W32N01, W32N55, and a control strain) were cultivated in yeast-peptone-dextrose (YPD) medium (10 g/L yeast extract, 20 g/L peptone, and 50 g/L glucose) until the OD600 reached 7–8 under aerobic conditions. Five milliliters of broth was centrifuged at 5000g. The resulting cell pellets were washed with sterile water, and resuspended in 300 l of buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8), and
Takara
1 mM Na2 EDTA). The cells were transferred to a 1.5-ml Eppendorf tube containing 0.2 g of glass beads (0.45–0.5 mm), and the cells were disrupted by vortexing in an ice bath. Cell debris was removed by centrifugation (10,000g for 20 min at 4 ◦ C), which resulted in a cell-free extract that was subsequently used for enzymatic activity assays. XR activity was measured using a multimode reader by monitoring the oxidation of NADPH at 340 nm in a reaction mixture with the following composition: 0.1 M sodium phosphate buffer (pH 7), 0.2 M xylose, and 0.15 mM NADPH. XDH activity was measured by monitoring the reduction of NAD+ at 340 nm in a reaction mixture with the following composition: 0.1 M Tris-HCl (pH 7), 1 mM MgCl2 , 50 mM xylitol, and 5 mM NAD+ [26]. XK activity was measured by monitoring the oxidation of NADH at 340 nm [20]. The specific enzyme activities were expressed as micromoles of converted substrate per milligram of protein per minute (U mg/ml). 2.3. Medium and culture conditions YPD medium was used for cell growth. The yeast strains used for fermentation were first grown in 50 ml of YPD medium in 250-ml Erlenmeyer flasks with shaking at 220 rpm. After a 16–18-h cultivation period, cells were collected by centrifugation, washed twice with sterile water, and transferred to fresh fermentation medium for ethanol production. The initial cell density in the fermentation medium was maintained at 3 g/L dry cell weight (DCW). Anaerobic ethanol fermentation was performed at 30 ◦ C in a closed bottle equipped with a bubbling CO2 outlet. Yeast extractpeptone-xylose (YPX) medium (10 g/L yeast extract, 20 g/L peptone, and 50 g/L xylose, pH 5.5) was used for xylose fermentation. To investigate the effect of ethanol on the xylose metabolism of S. cerevisiae strain W32N55, various concentrations of ethanol were added to the YPX medium. A corn stover hydrolysate containing 107 g/L glucose, 35 g/L xylose, and the microbial inhibitors acetic acid (7.2 g/L) and furaldehyde (3.8 mg/L) was used to produce ethanol. The hydrolysate was kindly provided by Prof. Jianmin Xing (Institute of Process Engineering, Chinese Academy of Sciences). The biomass pretreatment and enzymatic hydrolysis protocol was similar to that described by Yang et al. [28], except that the number of washes was reduced to three. To determine whether xylose metabolism could resume after removing the ethanol, the ethanol was separated from the culture broth by evaporation using rotary evaporators when glucose was depleted, and the metabolic profile of strain W32N55 was investigated. 2.4. Ethanol fermentation–pervaporation coupling process Cellulosic ethanol fermentation was performed at 30 ◦ C in a 2-L fermenter using the evolved strain W32N55 and a corn
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stover hydrolysate containing 150 g/L glucose and 50 g/L xylose, which was concentrated from the aforementioned corn stover hydrolysate (107 g/L glucose and 35 g/L xylose) by distillation. To facilitate the fermentation by lowering the ethanol concentration below inhibitory levels, membrane separation, coupled with the fermenter, was used to recover the ethanol as reported previously [30]. Cells obtained from an aerobic culture in YPD medium were harvested and transferred to the cellulosic corn stover hydrolysate using an inoculum of 3 g/L DCW. After 17 h of fermentation, ethanol was collected by separation at 12-h intervals, and samples of the fermentation product were collected at various intervals for analysis. During the pervaporation, some water was removed with the ethanol. Therefore, to maintain the fermentation volume, some water was added to the fermenter during the fermentation. Changes in the ethanol concentration and the volume of water added are shown in Tables S2 and S3. 2.5. Cell growth, and sugar and fermentation product assays Cell growth was monitored by measuring the OD600 with a spectrophotometer. The concentrations of sugars and fermentation products containing glucose, xylose, xylitol, glycerol, and ethanol were measured using a high-performance liquid chromatograph (Shimadzu, Kyoto, Japan) equipped with a refractive index detector and an HPX-87H ion-exclusion column (Bio-Rad, Hercules, CA, USA). The system was operated at a flow rate of 0.6 ml/min and a column temperature of 55 ◦ C. It took 25 min to test each sample. 3. Results and discussion 3.1. Introduction of a xylose metabolic pathway into S. cerevisiae, and characterization of its xylose metabolism As described in Section 2, the expression vector pXYL12-XK2 (LEU2) bearing the xyl1, xyl2, and xk genes was introduced into the leucine-deficient strain W303-1B (2N), resulting in the recombinant S. cerevisiae strain W32N01. After a 24-h cultivation, strain W32N01 and its parent strain W303-1B (2N) were processed to test their enzyme activities. XR and XDH activities were not detected in the control strain W303-1B (2N), while the specific XR and XDH activities in strain W32N01 reached 0.528 ± 0.11 and 0.32 ± 0.09 U/mg, respectively. Meanwhile, the XK specific activity increased from 0.105 ± 0.08 to 0.621 ± 0.06 U/mg (Table S1). The three genes involved in xylose metabolism were well expressed in the engineered strain W32N01 (data not shown). When cultivated in YPX medium, the strain grew well under aerobic conditions (Fig. S2), but not under anaerobic conditions (data not shown). 3.2. Adaptive evolution of strain W32N01 increases its xylose metabolism To increase its xylose fermentation ability under anaerobic conditions, the engineered strain W32N01 was subjected to a continuous evolution process in which the dissolved oxygen concentration was gradually decreased. An adaptive strain named W32N55 was obtained after 55 transfers and 210 d (Fig. S3). The specific XR, XDH, and XK activities of this strain reached 0.758 ± 0.03, 0.601 ± 0.10, and 0.876 ± 0.07 U/mg, respectively (Table S1), 40–90% higher than those in the originally engineered strain W32N01. As shown in Fig. 1B, the evolved strain W32N55 fermented xylose to ethanol under anaerobic conditions when xylose was the only carbon source in the fermentation medium. Xylose (50 g/L) was consumed by strain W32N55 in 56 h, and 16 g/L ethanol was produced. In comparison, the parental strain W32N01 produced 12.4 g/L ethanol from 50 g/L xylose in 144 h
3
(Fig. 1A). The ethanol yield (0.32 g/g xylose) and ethanol productivity (0.285 g/L/h) of strain W32N55 were increased by 29% and 231%, respectively, compared with those of strain W32N01, thereby demonstrating the efficiency of the strain evolution. These results are consistent with previous reports that showed that evolutionary engineering improved the xylose fermentation abilities of other S. cerevisiae strains [3,11,21,27]. Notably, the xylose consumption rates of strains W32N01 and W32N55 decreased rapidly during the late fermentation period, especially when the ethanol concentration reached 10 g/L or higher. This observation prompted us to hypothesize that the production of a high ethanol titer might inhibit the utilization of xylose by S. cerevisiae. 3.3. High concentrations of ethanol inhibit xylose utilization To investigate the inhibitory effects of ethanol on the metabolism of strain W32N55, varying amounts of ethanol were added to the YPD and YPX media. As shown in Fig. 2A, when no additional ethanol was added, 50 g/L xylose was consumed in 56 h, with a xylose consumption rate of 0.9 g/L/h. However, the xylose consumption rates decreased sharply, from 0.9 to 0.2 g/L/h, when 1–10% ethanol was added (Fig. 2A), suggesting that xylose catabolism was retarded in the presence of ethanol. The ethanol yields (0.32 g/g xylose) were not changed obviously by the addition of ethanol. In comparison, low concentrations of ethanol did not affect the metabolism of glucose, as the glucose consumption rate was approximately 2.1 g/L/h. Only ethanol concentrations greater than 5% obviously inhibited glucose metabolism, as the glucose yield decreased from 1.35 to 0.32 g/L/h when 5–10% ethanol was added (Fig. 2B), which is consistent with previous studies [2,17]. Additionally, the effect of the same ethanol concentration on xylose catabolism was greater than its effect on glucose catabolism. As described by Zhang et al. [29], we suggest that the aforementioned results be explained as follows. First, many studies reported that ATPase activity in the cell membrane is reduced by ethanol [1,18]. In the xylose XR-XDH pathway, the mutant strain maintained a NAD+ /NADH imbalance during the anaerobic fermentation of xylose. The byproducts glycerol and xylitol may, to some extent, ameliorate the excess production of NADH, thereby leading to an overall consumption of ATP. Thus, ATPases are necessary for xylose fermentation. Second, acetic acid, another end product of xylose alcoholic fermentation, also enhances the toxicity of ethanol, affecting the growth, fermentation and viability of bacterial cells [1]. Acetic acid can directly affect the sugar transport proteins or other related pH dependence of enzymatic activity. That is why xylose consumption is related with the pH value [31]. The exact mechanism of the ethanol-mediated inhibition of xylose catabolism requires further study. We suggest that the cofactor imbalance, which leads to the incomplete transfer of carbohydrates to ethanol, resulted in an ethanol yield (0.32 g/g xylose) that was lower than the theoretical value (0.51 g/g). In the future, we will try to use the XI pathway to replace the redox pathway, which can largely reduce the production of the byproduct xylitol. 3.4. Removal of ethanol from the fermentation broth leads to the resumption of xylose catabolism To further investigate the hypothesis, an ethanol removal experiment was performed. As shown in Fig. 3, when strain W32N55 was used to ferment the corn stover hydrolysate containing 107 g/L glucose and 35 g/L xylose, glucose was depleted within 40 h, but only 5 g/L xylose was consumed after 40 h (Fig. 3). Interestingly, even after the glucose was depleted, the strain could not utilize xylose (data not shown). Pervaporation was performed to remove
Please cite this article in press as: B. Zhang, et al., High-titer-ethanol production from cellulosic hydrolysate by an engineered strain of Saccharomyces cerevisiae during an in situ removal process reducing the inhibition of ethanol on xylose metabolism, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.04.019
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Fig. 1. Anaerobic fermentation of the engineered strain W32N01 (A) and adaptive evolution of the engineered strain W32N55 (B) in YPX medium containing 50 g/L xylose as the sole carbon source. Data points shown are the average of three independent experiments. Error bars represent standard deviations (n = 3).
Fig. 2. Anaerobic fermentation of sugars using strain W32N55 in the presence of different concentrations of added ethanol. (A) xylose fermentation and (B) glucose fermentation. Data points shown are the average of three independent experiments. Error bars represent standard deviations (n = 3).
catabolism, and they revealed that xylose catabolism could resume once the ethanol was separated from the fermentation system.
3.5. The fermentation–pervaporation coupling process produces a high ethanol titer from a cellulosic hydrolysate
Fig. 3. Fermentation of a cellulosic hydrolysate using strain W32N55. Ethanol was separated by pervaporation after 40 h of fermentation when glucose was depleted. Data points shown are the average of three independent experiments. Error bars represent standard deviations (n = 3).
the ethanol from the hydrolysate fermentation broth after the glucose was depleted at 40 h. Under these conditions, the consumption of xylose resumed, with 30 g/L xylose consumed and 10 g/L ethanol produced in 80 h (Fig. 3), with an ethanol yield of 0.33 g/g xylose. These results confirmed that ethanol is an inhibitor of xylose
By coupling the fermentation process with the use of a pervaporation system, we developed a coupling process from which the ethanol produced in the bioreactor could be continuously separated in situ, which allowed the ethanol concentration in the broth to be maintained at approximately 30 g/L. Using this process, the corn stover hydrolysate containing 150 g/L glucose and 50 g/L xylose was fermented by the evolved strain W32N55. As shown in Fig. 4, strain W32N55 consumed 150 g/L glucose and 31 g/L xylose within 72 h, and it produced 76 g/L ethanol. These results indicate that a lower ethanol concentration is a prerequisite for the initiation of xylose catabolism by strain W32N55. In addition, we showed that a pervaporation system can be coupled with a fermentation system containing a corn stover hydrolysate. In this coupling process, the initial cell density of strain W32N55 in the fermentation medium was maintained at 3 g/L DCW. This is lower than that of the engineered S. cerevisiae strain 424A(LNH-ST) (9 g/L DCW), which can anaerobically ferment hydrolysates [19]. In this study, by separating the ethanol in situ, a 76 g/L ethanol yield was obtained from hydrolyzed sugars in 72 h, with an ethanol yield of 0.42 g/g total sugar, compared with a 47 g/L ethanol yield that was achieved in 140 h, with a yield of 0.432 g/g total sugar, in a previous study by Sedlak and Ho [19]. Although there have been many
Please cite this article in press as: B. Zhang, et al., High-titer-ethanol production from cellulosic hydrolysate by an engineered strain of Saccharomyces cerevisiae during an in situ removal process reducing the inhibition of ethanol on xylose metabolism, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.04.019
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Fig. 4. Fermentation of a lignocellulose hydrolysate using the adaptive evolution-engineered strain W32N55 and an ethanol-separation membrane. Data points shown are the average of three independent experiments. Error bars represent standard deviations (n = 3).
other studies of the co-fermentation of glucose and xylose, most of these studies that obtained high ethanol yields (greater than 0.4 g/g total sugar) used a complex medium (e.g., YPDX), not hydrolyzed sugars [11,13]. Ultimately, difference in microorganisms (pentose vs. non-pentose-fermenting strains) and fermentation conditions makes it difficult to directly compare such studies. As shown in Fig. 4, although xylose consumption continued following the removal of ethanol by the pervaporation system, ethanol production was very slow, especially from 42 to 72 h. This might be due to the relatively high ethanol concentration (about 30 g/L) that was maintained in the fermenter. Because the above results were obtained by removing the ethanol at 12-h intervals, continuous pervaporation was applied to decrease the ethanol concentration in the fermenter, and the results of this procedure lowered the ethanol concentration in the fermentation broth by approximately 1%, at which point ethanol production and xylose utilization could not be further increased. The mechanism underlying the lower ethanol productivity during the latter fermentation remains unclear. Although it has such some associated problems, as mentioned above, and requires extra energy, a fermentation–pervaporation coupling system is still a promising technology for removing ethanol from fermentation broth in situ, and overcoming the disadvantage of ethanol inhibition, especially during high-sugar concentration fermentations. However, the recovery of a concentrated ethanol stream requires the low-temperature condensation of permeate vapors, which uses additional energy and increases costs. In addition, there are still technical and economic problems, such as the actual membrane lifespan under industrial fermentation conditions, as well as operational considerations for continuous fermentation systems, which can only be solved by further research [16]. Considering its overall operational cost, intermittent pervaporation is a promising choice. If a better process that uses less energy were developed, it would have greater potential in the fermentation industry.
4. Conclusions Xylose utilization is critical for the cellulosic ethanol fermentation industry. The engineered strain S. cerevisiae W32N55 fermented xylose anaerobically to ethanol. However, the xylose utilization was inhibited by high concentrations of ethanol, although the removal of ethanol allowed the resumption of xylose metabolism. Using a fermentation–pervaporation coupling process, the efficient utilization of xylose in a steam-exploded corn stover hydrolysate was achieved by strain W32N55. After 72 h of fermentation, 150 g/L glucose and 31 g/L xylose were consumed,
producing 76 g/L ethanol, with an overall yield of 0.42 g/g total sugar. Acknowledgments This work was supported by the National High Technology Research and Development Program of China (grant nos. 2012AA022106 and 2012AA101807) and the Knowledge Innovation Project of the Chinese Academy of Sciences (grant nos. KSCX2-EW-Q-14 and KSCX1-YW-11C3). We thank Haifeng Zhang, Taicheng Zhu, and Zhen Cai (Institute of Microbiology, Chinese Academy of Sciences) for helpful discussions regarding strain construction, Peng He (Institute of Microbiology, Chinese Academy of Sciences) for providing the host strain W303-1B(2N), and Jianmin Xing (Institute of Process Engineering, Chinese Academy of Sciences) for providing the corn stover hydrolysate. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.procbio.2016.04. 019. References [1] H. Alexandre, C. Charpentier, Biochemical aspects of stuck and sluggish fermentation in grape must, J. Ind. Microbiol. Biotechnol. 20 (1) (1998) 20–27. [2] H. Alexandre, V. Ansanay-Galeote, S. Dequin, B. Blondin, Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae, FEBS Lett. 498 (1) (2001) 98–103. [3] Z. Cai, B. Zhang, Y. Li, Engineering Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: reflections and perspectives, Biotechnol. J. 7 (1) (2012) 34–46. [5] A. Eliasson, C. Christensson, C.F. Wahlbom, B. Hahn-Hägerdal, Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures, Appl. Environ. Microbiol. 66 (8) (2000) 3381–3386. [6] B. Hahn-Hägerdal, C.F. Wahlbom, M. Gárdonyi, W.H. van Zyl, R.R.C. Otero, L.J. Jönsson, Metabolic engineering of Saccharomyces cerevisiae for xylose utilization, Metab. Eng. 73 (2001) 53–84. [7] N.W. Ho, Z. Chen, A.P. Brainard, Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose, Appl. Environ. Microbiol. 64 (5) (1998) 1852–1859. [11] M. Kuyper, M.J. Toirkens, J.A. Diderich, A.A. Winkler, J.P. Dijken, J.T. Pronk, Evolutionary engineering of mixed sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain, FEMS Yeast Res. 5 (10) (2005) 925–934. [13] A. Matsushika, H. Inoue, K. Murakami, O. Takimura, S. Sawayama, Bioethanol production performance of five recombinant strains of laboratory and industrial xylose-fermenting Saccharomyces cerevisiae, Bioresour. Technol. 100 (8) (2009) 2392–2398. [15] H. Ni, J.M. Laplaza, T.W. Jeffries, Transposon mutagenesis to improve the growth of recombinant Saccharomyces cerevisiae on d-xylose, Appl. Environ. Microbiol. 73 (7) (2007) 2061–2066.
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Short communication
High-titer-ethanol production from cellulosic hydrolysate by an engineered strain of Saccharomyces cerevisiae during an in situ removal process reducing the inhibition of ethanol on xylose metabolism Bo Zhang a,1 , Hongbing Sun a,b , Jing Li c , Yinhua Wan c , Yin Li a , Yanping Zhang a,∗ a
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China c Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China b
a r t i c l e
i n f o
Article history: Received 17 July 2015 Received in revised form 12 April 2016 Accepted 20 April 2016 Available online xxx Keywords: Cellulosic ethanol Ethanol inhibition Fermentation–pervaporation Saccharomyces cerevisiae Xylose catabolism
a b s t r a c t Efficient xylose utilization is critical for the production of fuels from biomass hydrolysates. It is known that xylose catabolism is inhibited by glucose. In this study, we showed that ethanol also inhibits xylose catabolism. By introducing a xylose metabolic pathway into Saccharomyces cerevisiae and using evolutionary engineering, an engineered S. cerevisiae strain, W32N55, was obtained that can anaerobically ferment xylose to ethanol. The effect of ethanol on xylose utilization was investigated. The results showed that xylose catabolism was inhibited upon the addition of ethanol, and it resumed once ethanol was removed. Based on these results, a fermentation–pervaporation coupling process was developed. After the in situ removal of ethanol, 150 g/L glucose and 31 g/L xylose were consumed in 72 h, providing a total of 76 g/L ethanol and an overall total sugar yield of 0.42 g/g. We believe that this strain will be valuable to the bio-ethanol industry. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulose, one of the world’s most abundant renewable resources, is the most favorable alternative feedstock for the production of fuels and chemicals [6]. Lignocellulosic biomass is generally composed of cellulose, hemicellulose, and lignin. Upon pretreatment via alkaline, acidic, and/or enzymatic hydrolyses, lignocellulose is decomposed to glucose, xylose, and a small amount of arabinose, and such treatments also result in the production of some microbial growth inhibitors [23]. To use lignocellulosic resources for biotechnological purposes, a desirable strain is expected to co-utilize glucose and xylose, and tolerate inhibitors. However, natural strains of Saccharomyces cerevisiae cannot utilize xylose for growth or ethanol production. Because S. cerevisiae possesses a metabolic pathway to convert xylulose, a metabolic product of xylose, into ethanol, many efforts have been made to introduce metabolic pathways, including the xylose reductase–xylitol dehydrogenase (XR-XDH) and xylose isomerase (XI) pathways, which convert xylose to xylulose, into
∗ Corresponding author. E-mail address: [email protected] (Y. Zhang). 1 Present address: College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
S. cerevisiae [7,5,24]. However, the rates of xylose consumption and ethanol production in such recombinant strains are unsatisfactory. In addition, most of the previously engineered strains can only ferment xylose under aerobic or microaerobic conditions [24,15], which is not favorable for the development of the bio-ethanol industry. Therefore, evolutionary engineering is the key to the development of xylose-fermenting S. cerevisiae strains, and many such examples have been demonstrated [22,11]. However, few strains can co-utilize glucose and xylose in high-sugar concentration hydrolysates to produce a high ethanol yield. In an attempt to obtain an engineered S. cerevisiae strain that is capable of efficiently co-utilizing glucose and xylose, a xylose metabolic pathway was introduced into a S. cerevisiae strain, and it was allowed to evolve under oxygen-limiting conditions. The resulting strain, W32N55, fermented xylose to ethanol under anaerobic conditions. Using this strain, we found that the consumption rate of xylose decreased significantly during the late fermentation period, when a high concentration of ethanol was obtained. These results led to the hypothesis that in addition to glucose, ethanol might inhibit xylose catabolism. To verify this, we investigated the effect of ethanol on xylose catabolism. This was followed by the development of a fermentation–pervaporation coupling process, from which the efficient production of ethanol from corn stover hydrolysates was achieved.
http://dx.doi.org/10.1016/j.procbio.2016.04.019 1359-5113/© 2016 Elsevier Ltd. All rights reserved.
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2 Table 1 Plasmids and strains used in this study. Strain/plasmid
Relevant genotype
Source
Plasmids plasmid-2 pXYL1-2 (LEU2) pXYL2-2 (LEU2) Xks1-2 (LEU2) pXYL12-2u(LEU2) pXYL12-XK-2u (LEU2)
plasmid-2 , leu, E. coli ori, Apr plasmid-2 -xyl1, leu, E. coli ori, Apr plasmid-2 -xyl2, leu, E. coli ori, Apr plasmid-2 -xks1, leu, E. coli ori, Apr plasmid-2 - xyl1-xyl2, leu, E. coli ori, Apr plasmid-2 - xyl1-xyl2-xks1, leu, E. coli ori, Apr
Our lab stored This study This study This study This study This study
MATa/MAT␣ ura3-52/ura3-52; trp1 2/trp12; leu2-3,112/leu2-3,112;his3-11/his3-11; ade2-1/ade2-1; can1-100/can1-100 F-,80dlacZM15, (lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44, -, thi-1, gyrA96, relA1
EUROSCARF
Strains S. cerevisiae W303-1B (2n) E. coli DH5␣
2. Materials and methods 2.1. Strain construction Plasmids and yeast strains used in this study are listed in Table 1. The plasmid construction steps were as follows. The XR-encoding gene xyl1 and the XDH-encoding gene xyl2 were polymerase chain reaction (PCR)-amplified from the genomic DNA of Pichia stipites, and inserted into the BamHI site of plasmid-2 (LEU2), resulting in plasmids pXYL1-2 (LEU2) and pXYL2-2 (LEU2), respectively, in which the expression of the target genes was controlled by the GPD promoter. Similarly, the xylulokinase (XK)-encoding xk gene from S. cerevisiae was inserted into plasmid-2 (LEU2) under the control of GPD promoter, resulting in plasmid pXK-2 (LEU2). Next, a xyl2 cassette containing a promoter, the xyl2 gene, and a terminator was PCR-amplified from pXYL2-2 (LEU2) and introduced into pXYL1-2 (LEU2), resulting in plasmid pXYL12-2 (LEU2). An xk cassette containing a promoter, the xk gene, and a terminator was PCR-amplified from pXK-2 (LEU2) and ligated into pXYL122 (LEU2), resulting in the final expression vector pXYL12-XK-2 (LEU2) (Fig. S1). S. cerevisiae strain W303-1B (2N), a diploid strain that is easy to genetically manipulate, was used as the host strain. By transforming pXYL12-XK-2 (LEU2) into strain W303-1B (2N), the engineered strain W32N01 was obtained and identified by PCR and enzymatic activity assays. Minimal medium (yeast nitrogen base without amino acids, 0.67%) supplemented with 20 g/L xylose was used for strain evolution at 30 ◦ C. First, strain W32N01 was grown under aerobic conditions on a rotary shaker at 220 rpm. The cells were cultivated until they reached the mid-exponential phase (optical density at 600 nm (OD600 ) = 4), and then they were transferred to fresh medium. The process was repeated until the strain growth rate improved significantly. Then, the dissolved oxygen availability was reduced by gradually lowering the shaker speed from the original 220 rpm to 100 rpm. After 55 transfers (a 2% inoculum was used for each transfer), a single clone, named W3N55, was obtained from the last shake flask. W32N55 was used for ethanol fermentation in this study.
2.2. Enzyme activity assays XR, XDH, and XK activities were measured according to the following protocol. Target strains (W32N01, W32N55, and a control strain) were cultivated in yeast-peptone-dextrose (YPD) medium (10 g/L yeast extract, 20 g/L peptone, and 50 g/L glucose) until the OD600 reached 7–8 under aerobic conditions. Five milliliters of broth was centrifuged at 5000g. The resulting cell pellets were washed with sterile water, and resuspended in 300 l of buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8), and
Takara
1 mM Na2 EDTA). The cells were transferred to a 1.5-ml Eppendorf tube containing 0.2 g of glass beads (0.45–0.5 mm), and the cells were disrupted by vortexing in an ice bath. Cell debris was removed by centrifugation (10,000g for 20 min at 4 ◦ C), which resulted in a cell-free extract that was subsequently used for enzymatic activity assays. XR activity was measured using a multimode reader by monitoring the oxidation of NADPH at 340 nm in a reaction mixture with the following composition: 0.1 M sodium phosphate buffer (pH 7), 0.2 M xylose, and 0.15 mM NADPH. XDH activity was measured by monitoring the reduction of NAD+ at 340 nm in a reaction mixture with the following composition: 0.1 M Tris-HCl (pH 7), 1 mM MgCl2 , 50 mM xylitol, and 5 mM NAD+ [26]. XK activity was measured by monitoring the oxidation of NADH at 340 nm [20]. The specific enzyme activities were expressed as micromoles of converted substrate per milligram of protein per minute (U mg/ml). 2.3. Medium and culture conditions YPD medium was used for cell growth. The yeast strains used for fermentation were first grown in 50 ml of YPD medium in 250-ml Erlenmeyer flasks with shaking at 220 rpm. After a 16–18-h cultivation period, cells were collected by centrifugation, washed twice with sterile water, and transferred to fresh fermentation medium for ethanol production. The initial cell density in the fermentation medium was maintained at 3 g/L dry cell weight (DCW). Anaerobic ethanol fermentation was performed at 30 ◦ C in a closed bottle equipped with a bubbling CO2 outlet. Yeast extractpeptone-xylose (YPX) medium (10 g/L yeast extract, 20 g/L peptone, and 50 g/L xylose, pH 5.5) was used for xylose fermentation. To investigate the effect of ethanol on the xylose metabolism of S. cerevisiae strain W32N55, various concentrations of ethanol were added to the YPX medium. A corn stover hydrolysate containing 107 g/L glucose, 35 g/L xylose, and the microbial inhibitors acetic acid (7.2 g/L) and furaldehyde (3.8 mg/L) was used to produce ethanol. The hydrolysate was kindly provided by Prof. Jianmin Xing (Institute of Process Engineering, Chinese Academy of Sciences). The biomass pretreatment and enzymatic hydrolysis protocol was similar to that described by Yang et al. [28], except that the number of washes was reduced to three. To determine whether xylose metabolism could resume after removing the ethanol, the ethanol was separated from the culture broth by evaporation using rotary evaporators when glucose was depleted, and the metabolic profile of strain W32N55 was investigated. 2.4. Ethanol fermentation–pervaporation coupling process Cellulosic ethanol fermentation was performed at 30 ◦ C in a 2-L fermenter using the evolved strain W32N55 and a corn
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stover hydrolysate containing 150 g/L glucose and 50 g/L xylose, which was concentrated from the aforementioned corn stover hydrolysate (107 g/L glucose and 35 g/L xylose) by distillation. To facilitate the fermentation by lowering the ethanol concentration below inhibitory levels, membrane separation, coupled with the fermenter, was used to recover the ethanol as reported previously [30]. Cells obtained from an aerobic culture in YPD medium were harvested and transferred to the cellulosic corn stover hydrolysate using an inoculum of 3 g/L DCW. After 17 h of fermentation, ethanol was collected by separation at 12-h intervals, and samples of the fermentation product were collected at various intervals for analysis. During the pervaporation, some water was removed with the ethanol. Therefore, to maintain the fermentation volume, some water was added to the fermenter during the fermentation. Changes in the ethanol concentration and the volume of water added are shown in Tables S2 and S3. 2.5. Cell growth, and sugar and fermentation product assays Cell growth was monitored by measuring the OD600 with a spectrophotometer. The concentrations of sugars and fermentation products containing glucose, xylose, xylitol, glycerol, and ethanol were measured using a high-performance liquid chromatograph (Shimadzu, Kyoto, Japan) equipped with a refractive index detector and an HPX-87H ion-exclusion column (Bio-Rad, Hercules, CA, USA). The system was operated at a flow rate of 0.6 ml/min and a column temperature of 55 ◦ C. It took 25 min to test each sample. 3. Results and discussion 3.1. Introduction of a xylose metabolic pathway into S. cerevisiae, and characterization of its xylose metabolism As described in Section 2, the expression vector pXYL12-XK2 (LEU2) bearing the xyl1, xyl2, and xk genes was introduced into the leucine-deficient strain W303-1B (2N), resulting in the recombinant S. cerevisiae strain W32N01. After a 24-h cultivation, strain W32N01 and its parent strain W303-1B (2N) were processed to test their enzyme activities. XR and XDH activities were not detected in the control strain W303-1B (2N), while the specific XR and XDH activities in strain W32N01 reached 0.528 ± 0.11 and 0.32 ± 0.09 U/mg, respectively. Meanwhile, the XK specific activity increased from 0.105 ± 0.08 to 0.621 ± 0.06 U/mg (Table S1). The three genes involved in xylose metabolism were well expressed in the engineered strain W32N01 (data not shown). When cultivated in YPX medium, the strain grew well under aerobic conditions (Fig. S2), but not under anaerobic conditions (data not shown). 3.2. Adaptive evolution of strain W32N01 increases its xylose metabolism To increase its xylose fermentation ability under anaerobic conditions, the engineered strain W32N01 was subjected to a continuous evolution process in which the dissolved oxygen concentration was gradually decreased. An adaptive strain named W32N55 was obtained after 55 transfers and 210 d (Fig. S3). The specific XR, XDH, and XK activities of this strain reached 0.758 ± 0.03, 0.601 ± 0.10, and 0.876 ± 0.07 U/mg, respectively (Table S1), 40–90% higher than those in the originally engineered strain W32N01. As shown in Fig. 1B, the evolved strain W32N55 fermented xylose to ethanol under anaerobic conditions when xylose was the only carbon source in the fermentation medium. Xylose (50 g/L) was consumed by strain W32N55 in 56 h, and 16 g/L ethanol was produced. In comparison, the parental strain W32N01 produced 12.4 g/L ethanol from 50 g/L xylose in 144 h
3
(Fig. 1A). The ethanol yield (0.32 g/g xylose) and ethanol productivity (0.285 g/L/h) of strain W32N55 were increased by 29% and 231%, respectively, compared with those of strain W32N01, thereby demonstrating the efficiency of the strain evolution. These results are consistent with previous reports that showed that evolutionary engineering improved the xylose fermentation abilities of other S. cerevisiae strains [3,11,21,27]. Notably, the xylose consumption rates of strains W32N01 and W32N55 decreased rapidly during the late fermentation period, especially when the ethanol concentration reached 10 g/L or higher. This observation prompted us to hypothesize that the production of a high ethanol titer might inhibit the utilization of xylose by S. cerevisiae. 3.3. High concentrations of ethanol inhibit xylose utilization To investigate the inhibitory effects of ethanol on the metabolism of strain W32N55, varying amounts of ethanol were added to the YPD and YPX media. As shown in Fig. 2A, when no additional ethanol was added, 50 g/L xylose was consumed in 56 h, with a xylose consumption rate of 0.9 g/L/h. However, the xylose consumption rates decreased sharply, from 0.9 to 0.2 g/L/h, when 1–10% ethanol was added (Fig. 2A), suggesting that xylose catabolism was retarded in the presence of ethanol. The ethanol yields (0.32 g/g xylose) were not changed obviously by the addition of ethanol. In comparison, low concentrations of ethanol did not affect the metabolism of glucose, as the glucose consumption rate was approximately 2.1 g/L/h. Only ethanol concentrations greater than 5% obviously inhibited glucose metabolism, as the glucose yield decreased from 1.35 to 0.32 g/L/h when 5–10% ethanol was added (Fig. 2B), which is consistent with previous studies [2,17]. Additionally, the effect of the same ethanol concentration on xylose catabolism was greater than its effect on glucose catabolism. As described by Zhang et al. [29], we suggest that the aforementioned results be explained as follows. First, many studies reported that ATPase activity in the cell membrane is reduced by ethanol [1,18]. In the xylose XR-XDH pathway, the mutant strain maintained a NAD+ /NADH imbalance during the anaerobic fermentation of xylose. The byproducts glycerol and xylitol may, to some extent, ameliorate the excess production of NADH, thereby leading to an overall consumption of ATP. Thus, ATPases are necessary for xylose fermentation. Second, acetic acid, another end product of xylose alcoholic fermentation, also enhances the toxicity of ethanol, affecting the growth, fermentation and viability of bacterial cells [1]. Acetic acid can directly affect the sugar transport proteins or other related pH dependence of enzymatic activity. That is why xylose consumption is related with the pH value [31]. The exact mechanism of the ethanol-mediated inhibition of xylose catabolism requires further study. We suggest that the cofactor imbalance, which leads to the incomplete transfer of carbohydrates to ethanol, resulted in an ethanol yield (0.32 g/g xylose) that was lower than the theoretical value (0.51 g/g). In the future, we will try to use the XI pathway to replace the redox pathway, which can largely reduce the production of the byproduct xylitol. 3.4. Removal of ethanol from the fermentation broth leads to the resumption of xylose catabolism To further investigate the hypothesis, an ethanol removal experiment was performed. As shown in Fig. 3, when strain W32N55 was used to ferment the corn stover hydrolysate containing 107 g/L glucose and 35 g/L xylose, glucose was depleted within 40 h, but only 5 g/L xylose was consumed after 40 h (Fig. 3). Interestingly, even after the glucose was depleted, the strain could not utilize xylose (data not shown). Pervaporation was performed to remove
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Fig. 1. Anaerobic fermentation of the engineered strain W32N01 (A) and adaptive evolution of the engineered strain W32N55 (B) in YPX medium containing 50 g/L xylose as the sole carbon source. Data points shown are the average of three independent experiments. Error bars represent standard deviations (n = 3).
Fig. 2. Anaerobic fermentation of sugars using strain W32N55 in the presence of different concentrations of added ethanol. (A) xylose fermentation and (B) glucose fermentation. Data points shown are the average of three independent experiments. Error bars represent standard deviations (n = 3).
catabolism, and they revealed that xylose catabolism could resume once the ethanol was separated from the fermentation system.
3.5. The fermentation–pervaporation coupling process produces a high ethanol titer from a cellulosic hydrolysate
Fig. 3. Fermentation of a cellulosic hydrolysate using strain W32N55. Ethanol was separated by pervaporation after 40 h of fermentation when glucose was depleted. Data points shown are the average of three independent experiments. Error bars represent standard deviations (n = 3).
the ethanol from the hydrolysate fermentation broth after the glucose was depleted at 40 h. Under these conditions, the consumption of xylose resumed, with 30 g/L xylose consumed and 10 g/L ethanol produced in 80 h (Fig. 3), with an ethanol yield of 0.33 g/g xylose. These results confirmed that ethanol is an inhibitor of xylose
By coupling the fermentation process with the use of a pervaporation system, we developed a coupling process from which the ethanol produced in the bioreactor could be continuously separated in situ, which allowed the ethanol concentration in the broth to be maintained at approximately 30 g/L. Using this process, the corn stover hydrolysate containing 150 g/L glucose and 50 g/L xylose was fermented by the evolved strain W32N55. As shown in Fig. 4, strain W32N55 consumed 150 g/L glucose and 31 g/L xylose within 72 h, and it produced 76 g/L ethanol. These results indicate that a lower ethanol concentration is a prerequisite for the initiation of xylose catabolism by strain W32N55. In addition, we showed that a pervaporation system can be coupled with a fermentation system containing a corn stover hydrolysate. In this coupling process, the initial cell density of strain W32N55 in the fermentation medium was maintained at 3 g/L DCW. This is lower than that of the engineered S. cerevisiae strain 424A(LNH-ST) (9 g/L DCW), which can anaerobically ferment hydrolysates [19]. In this study, by separating the ethanol in situ, a 76 g/L ethanol yield was obtained from hydrolyzed sugars in 72 h, with an ethanol yield of 0.42 g/g total sugar, compared with a 47 g/L ethanol yield that was achieved in 140 h, with a yield of 0.432 g/g total sugar, in a previous study by Sedlak and Ho [19]. Although there have been many
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Fig. 4. Fermentation of a lignocellulose hydrolysate using the adaptive evolution-engineered strain W32N55 and an ethanol-separation membrane. Data points shown are the average of three independent experiments. Error bars represent standard deviations (n = 3).
other studies of the co-fermentation of glucose and xylose, most of these studies that obtained high ethanol yields (greater than 0.4 g/g total sugar) used a complex medium (e.g., YPDX), not hydrolyzed sugars [11,13]. Ultimately, difference in microorganisms (pentose vs. non-pentose-fermenting strains) and fermentation conditions makes it difficult to directly compare such studies. As shown in Fig. 4, although xylose consumption continued following the removal of ethanol by the pervaporation system, ethanol production was very slow, especially from 42 to 72 h. This might be due to the relatively high ethanol concentration (about 30 g/L) that was maintained in the fermenter. Because the above results were obtained by removing the ethanol at 12-h intervals, continuous pervaporation was applied to decrease the ethanol concentration in the fermenter, and the results of this procedure lowered the ethanol concentration in the fermentation broth by approximately 1%, at which point ethanol production and xylose utilization could not be further increased. The mechanism underlying the lower ethanol productivity during the latter fermentation remains unclear. Although it has such some associated problems, as mentioned above, and requires extra energy, a fermentation–pervaporation coupling system is still a promising technology for removing ethanol from fermentation broth in situ, and overcoming the disadvantage of ethanol inhibition, especially during high-sugar concentration fermentations. However, the recovery of a concentrated ethanol stream requires the low-temperature condensation of permeate vapors, which uses additional energy and increases costs. In addition, there are still technical and economic problems, such as the actual membrane lifespan under industrial fermentation conditions, as well as operational considerations for continuous fermentation systems, which can only be solved by further research [16]. Considering its overall operational cost, intermittent pervaporation is a promising choice. If a better process that uses less energy were developed, it would have greater potential in the fermentation industry.
4. Conclusions Xylose utilization is critical for the cellulosic ethanol fermentation industry. The engineered strain S. cerevisiae W32N55 fermented xylose anaerobically to ethanol. However, the xylose utilization was inhibited by high concentrations of ethanol, although the removal of ethanol allowed the resumption of xylose metabolism. Using a fermentation–pervaporation coupling process, the efficient utilization of xylose in a steam-exploded corn stover hydrolysate was achieved by strain W32N55. After 72 h of fermentation, 150 g/L glucose and 31 g/L xylose were consumed,
producing 76 g/L ethanol, with an overall yield of 0.42 g/g total sugar. Acknowledgments This work was supported by the National High Technology Research and Development Program of China (grant nos. 2012AA022106 and 2012AA101807) and the Knowledge Innovation Project of the Chinese Academy of Sciences (grant nos. KSCX2-EW-Q-14 and KSCX1-YW-11C3). We thank Haifeng Zhang, Taicheng Zhu, and Zhen Cai (Institute of Microbiology, Chinese Academy of Sciences) for helpful discussions regarding strain construction, Peng He (Institute of Microbiology, Chinese Academy of Sciences) for providing the host strain W303-1B(2N), and Jianmin Xing (Institute of Process Engineering, Chinese Academy of Sciences) for providing the corn stover hydrolysate. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.procbio.2016.04. 019. References [1] H. Alexandre, C. Charpentier, Biochemical aspects of stuck and sluggish fermentation in grape must, J. Ind. Microbiol. Biotechnol. 20 (1) (1998) 20–27. [2] H. Alexandre, V. Ansanay-Galeote, S. Dequin, B. Blondin, Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae, FEBS Lett. 498 (1) (2001) 98–103. [3] Z. Cai, B. Zhang, Y. Li, Engineering Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: reflections and perspectives, Biotechnol. J. 7 (1) (2012) 34–46. [5] A. Eliasson, C. Christensson, C.F. Wahlbom, B. Hahn-Hägerdal, Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures, Appl. Environ. Microbiol. 66 (8) (2000) 3381–3386. [6] B. Hahn-Hägerdal, C.F. Wahlbom, M. Gárdonyi, W.H. van Zyl, R.R.C. Otero, L.J. Jönsson, Metabolic engineering of Saccharomyces cerevisiae for xylose utilization, Metab. Eng. 73 (2001) 53–84. [7] N.W. Ho, Z. Chen, A.P. Brainard, Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose, Appl. Environ. Microbiol. 64 (5) (1998) 1852–1859. [11] M. Kuyper, M.J. Toirkens, J.A. Diderich, A.A. Winkler, J.P. Dijken, J.T. Pronk, Evolutionary engineering of mixed sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain, FEMS Yeast Res. 5 (10) (2005) 925–934. [13] A. Matsushika, H. Inoue, K. Murakami, O. Takimura, S. Sawayama, Bioethanol production performance of five recombinant strains of laboratory and industrial xylose-fermenting Saccharomyces cerevisiae, Bioresour. Technol. 100 (8) (2009) 2392–2398. [15] H. Ni, J.M. Laplaza, T.W. Jeffries, Transposon mutagenesis to improve the growth of recombinant Saccharomyces cerevisiae on d-xylose, Appl. Environ. Microbiol. 73 (7) (2007) 2061–2066.
Please cite this article in press as: B. Zhang, et al., High-titer-ethanol production from cellulosic hydrolysate by an engineered strain of Saccharomyces cerevisiae during an in situ removal process reducing the inhibition of ethanol on xylose metabolism, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.04.019
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Please cite this article in press as: B. Zhang, et al., High-titer-ethanol production from cellulosic hydrolysate by an engineered strain of Saccharomyces cerevisiae during an in situ removal process reducing the inhibition of ethanol on xylose metabolism, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.04.019