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Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides
Bioresource Technology 100 (2009) 4843–4847
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Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides Cuimin Hu a,c, Xin Zhao a,c, Jin Zhao a, Siguo Wu a,c, Zongbao K. Zhao a,b,* a
Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian 116023, PR China Dalian National Laboratory of Clean Energy, Dalian 116023, PR China c Graduate School of the Chinese Academy of Sciences, Beijing 100049, PR China b
a r t i c l e
i n f o
Article history: Received 28 January 2009 Received in revised form 13 April 2009 Accepted 22 April 2009 Available online 3 June 2009 Keywords: Rhodosporidium toruloides Y4 Microbial lipid Lignocellulose hydrolysate Biomass Inhibitor
a b s t r a c t Lignocellulosic biomass hydrolysis inevitably coproduces byproducts that may have various affects on downstream biotransformation. It is imperative to document the inhibitor tolerance ability of microbial strain in order to utilize biomass hydrolysate more effectively. To achieve better lipid production by Rhodosporidium toruloides Y4, we performed fermentation experiments in the presence of some representative inhibitors. We found that acetate, 5-hydroxymethylfurfural and syringaldehyde had slightly inhibitory effects; p-hydroxybenzaldehyde and vanillin were toxic at a concentration over 10 mM; and furfural and its derivatives furfuryl alcohol and furoic acid inhibited cell growth by 45% at around 1 mM. We further demonstrated that inhibition is generally additive, although strong synergistic inhibitions were also observed. Finally, lipid production afforded good results in the presence of six inhibitors at their respective concentrations usually found in biomass hydrolysates. Fatty acid compositional profile of lipid samples indicated that those inhibitors had little effects on lipid biosynthesis. Our work will be useful for optimization of biomass hydrolysis processes and lipid production using lignocellulosic materials. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulosic biomass, mainly composed of cellulose, hemicellulose and lignin, is the most abundant and renewable organic compound in the biosphere. To utilize biomass with a biochemical route more effectively, complete hydrolysis of lignocellulose is a prerequisite, because most microorganisms have much better bioconversion rate with monomeric carbohydrates. Unfortunately, the release of monosaccharides during hydrolysis is routinely accompanied by the generation of non-carbohydrate compounds, such as furfural and 5-hydroxymethylfurfural (HMF) from the dehydration of pentoses and hexoses, acetic acid from the acetyl group in hemicellulose, and phenolic compounds including syringaldehyde, p-hydroxybenzaldehyde, vanillin, etc. derived from lignin (Almeida et al., 2007; Palmqvist and Hahn-Hagerdal, 2000a,b). The distribution of these inhibitors in a given hydrolysate sample is depending on both the raw material and the operational conditions employed for hydrolysis (Marzialetti et al., 2008; Schirmer-Michel et al., 2008). These byproducts had various affects on microbial cell growth, metabolism, as well as on product titer, presenting a major challenge in biological conversion of biomass (Almeida et al., * Corresponding author. Address: Dalian Institute of Chemical Physics, 457 Zhongshan Road, Dalian 116023, PR China. Tel./fax: +86 411 84379211. E-mail address: [email protected] (Z.K. Zhao). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.04.041
2007). Previous studies investigated the toxic effects of aldehyde, alcohol and acid components from hemicellulose hydrolysate for ethanologenic Escherichia coli, and demonstrated that the toxicity of hydrolysate resulted from the aggregate effects of different compounds rather than a single agent (Zaldivar and Ingram, 1999; Zaldivar et al., 1999). Studies on yeasts with different inhibitors also reached similar conclusions (Palmqvist et al., 1999a,b). To deal with these problems, one possibility is to develop detoxification methods, such as further processing with active charcoal, overliming, ion-exchange resins, or inhibitor-degrading microorganisms (Nichols et al., 2005). On the other hand, engineering superior strains with global stress resistance using either traditional approach or rational design is also extensively pursued recently (Almeida et al., 2007; Larsson et al., 2001). Yet, it is equally important to identify strains or processes with excellent inhibitor tolerance. Microorganisms that can accumulate lipids to more than 20% of their biomass are defined as oleaginous species (Ratledge and Wynn, 2002). Some yeast strains, such as Cryptococcus sp., Lipomyces sp., Rhodosporidium sp. and Rhodotorula sp. can accumulate intracellular lipids as high as 60% of its cell dry weight when using glucose as the carbon source (Li et al., 2007). Constitutive fatty acids of those lipids are mainly long chain ones that are quite similar to those of conventional vegetable oil. Therefore, oleaginous microorganisms have recently been suggested as alternative lipid
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producer to fuel a more sustainable biodiesel industry (Zhao et al., 2005). Making lipids through microorganisms is potentially a new technology of arable land-independent, continuous and controllable. However, the carbon sources for oleaginous microbes need extend to lignocellulosic biomass and related raw materials so that large volume of microbial lipids can be secured. Our previous work demonstrated that oleaginous yeast Rhodosporidium toruloides Y4 was a powerful lipid producer capable of accumulating lipid over 70% and with a titer over 100 g/L (Li et al., 2007). In this study we took some representative byproducts found in biomass hydrolysate to check their effects on the lipid production. We demonstrated that R. toruloides Y4 was a robust strain for lipid production using biomass hydrolysates. 2. Methods
All experiments were carried out in duplicates, and data were averaged. 2.3. Analytical methods Cell biomass, expressed as cell dry weight (CDW), was obtained from cell pellet in 30 ml of culture broth, and dried in an oven at 105 °C to a constant weight. Residual glucose was monitored with a SBA-50B glucose analyzer (Shandong Academy of Sciences, Jinan, China). Total lipid was extracted by the known procedure using a mixture of chloroform and methanol (Li et al., 2007). Lipid content was expressed as gram lipid per gram dry biomass. The fatty acid compositional profiles of lipid samples were determined using a 7890F gas chromatography (GC) instrument according to the published procedure (Li et al., 2007).
2.1. Yeast strain, media and chemicals Oleaginous yeast R. toruloides Y4 was a domesticated strain of R. toruloides AS 2.1389 obtained from the China General Microbiological Culture Collection Center. Inoculum was grown in YPD liquid medium contained (g/L): glucose 20, yeast extract 10, and peptone 10, pH 6.0). YPD agar slants were made with YPD liquid medium supplemented with 20 g/L agar. The fermentation media contained (g/L): glucose 54, (NH4)2SO4 0.215, yeast extract 1.0, KH2PO4 0.4, and MgSO47H2O 1.5, pH 6.0. This medium had a carbon-to-nitrogen (C/N, mol/mol) ratio around 80, and was formulated for higher lipid production (Li et al., 2006). Yeast extract (containing 1.4% ammonium-N and 8.7% total nitrogen) and peptone (containing 0.73% ammonium-N and 15.3% total nitrogen) were obtained from Aoboxing Biotech. Co. Ltd. (Beijing, China). Compounds 5-hydroxymethylfurfural (HMF), furoic acid and syringaldehyde were from Aldrich. Acetic acid, furfural, furfuryl alcohol, p-hydroxybenzaldehyde (PHB), vanillin and other chemicals were obtained from local supplier, and used as received. 2.2. Lipid production in the presence of inhibitors Precultures were inoculated from YPD agar slants (one loopful) and grown at 30 °C for 24–28 h in 50 ml of YPD medium in 250-ml shake flasks, and then 5 ml of cell culture was transferred into 45 ml of fermentation medium. Fermentation experiments were carried out in 250 ml flasks at a rotary rate of 200 rpm at 30 °C. In our experiments, inhibitor and their concentrations were acetic acid (0–120 mM), HMF (0–14.7 mM), furfural (0–10 mM), PHB (0–10 mM), vanillin (0–12 mM), syringaldehyde (0–12 mM), furoic acid (0–4 mM) and furfuryl alcohol (0–2 mM). For experiments with acetate, sodium acetate was introduced prior to sterilization. To introduce other single inhibitors, an appropriate amount of the corresponding stock solution was directly added to the inoculated media by pipetting. The control samples were prepared with no inhibitors. For binary combination experiments, two individual inhibitors at various concentrations were added to the media. Besides a control with no inhibitors, we conducted additional control experiments with each single inhibitor at the selected concentrations. Experiments with mixed inhibitors were carried out in the presence of acetate 52.2 mM, furfural 10.2 mM, HMF 15.0 mM, vanillin 1.0 10 3 mM, PHB 0.44 10 3 mM, and syringaldehyde 0.117 10 3 mM, as found elsewhere (Persson et al., 2002). Cells were cultured at 30 °C, 200 rpm until the residue sugar in the control experiments reached below 2 g/L, then collected by centrifugation, washed with distilled water, and subjected to biomass and lipid analysis. The supernatants were collected for glucose analysis.
3. Results and discussion To explore lipid production using inexpensive carbon sources, we conducted flask culture experiments by R. toruloides Y4 in the presence of some representative inhibitors found in biomass acid hydrolysate. 3.1. Effects of individual inhibitor on R. toruloides Y4 When R. toruloides Y4 cells were cultured at 30 °C for 4–5 days in the absence of an inhibitor (the control experiment), biomass, residue sugar and lipid were 15 g/L, 2 g/L and 62 wt.%, respectively. However, when an inhibitory compound was included, the fermentation performance changed. Fig. 1A–C shows R. toruloides Y4 cultural changes relative to the control sample in substrate consumption, biomass and lipid content, respectively, in the presence of single inhibitor at various concentrations. Acetate is a major inhibitory compound found in biomass hydrolysate. For example, 49.8 mM and 34.3 mM acetate were found in soybean hull and spruce acid hydrolysate (Nilsson et al., 2005; Schirmer-Michel et al., 2008). Acetic acid at 83 mM showed strong inhibitory effect on ethanol production by Pichia stipitis (Delgenes et al., 1996). However, our results showed that acetate had little effect on lipid production up to 70 mM (4.2 g/L). Moreover, excess acetate improved cell performance as lipid content reached 68% in the presence of 120 mM acetate (Fig. 1C). This was likely due that R. toruloides Y4 used acetate as building block for lipid biosynthesis. On the other hand, high concentration of acetate also led to an increase in C/N ratio such that lipid accumulation process was greatly augmented. It was demonstrated that acetic acid in its dissociated form was difficult to cross the plasma membrane and exhibit cellular toxicity (Palmqvist and Hahn-Hagerdal, 2000a,b). Three lignin derived inhibitors had different effects on lipid production. Syringaldehyde was a weaker inhibitor, as in the presence of 12 mM syringaldehyde, glucose consumption, biomass and lipid content were 39 g/L, 12.4 g/L and 58.6%, which were about 87.6%, 84.3% and 94.8%, respectively, those of the control sample. Vanillin and PHB had similar inhibition profile, although PHB showed slightly stronger effect. When vanillin and PHB were added to 9 mM, glucose consumption was suppressed by 8.4% and 40.2%, respectively. However, almost complete inhibition occurred in the presence of 12 mM of vanillin or 10 mM of PHB (Fig. 1B). Thus, the order of inhibition ability by these aldehydes was PHB > vanillin > syringaldehyde, which was in good agreement with their hydrophobicities indicated by the octanol/water partition coefficients. The more hydrophobilic the aldehyde is, the stronger the inhibitory effect. A same conclusion has been previously reported
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Concentration of acetic acid (mM)
Relative substrate consumption (%)
0
20
40
60
80
100
A 100
100
80
Furfural Syringaldehyde PHB Vanillin HMF Acetic acid
60
40
20
Furoic acid Furfuryl alcohol Furfural
120
Relative substrate consumption (%)
A
80
60
40
20
0 0
2
0
4
8
12
16
6
8
12
B 100 Furoic acid Furfuryl alcohol Furfural
Concentration of acetic acid (mM) 0
20
40
60
80
100
80
120
Relative biomass (% )
B 100
80
60
Furfural Syringaldehyde PHB Vanillin HMF Acetic acid
40
20
0
0
4
12
8
16
60
40
20
0 0
2
4
6
8
20
Concentration of acetic acid (mM) 0
20
40
60
80
100
12
C 100 Relative lipid content (%)
C
10
Concentration of inhibitors (mM)
Furoic acid Furfuryl alcohol Furfural
Concentration of other inhibitors (mM)
120
100
Relative lipid content (%)
10
20
Concentration of other inhibitors (mM)
Relative biomass (%)
4
Concentration of inhibitors (mM)
0
80
80
60
40
20
60
Furfural Syringaldehyde PHB Vanillin HMF Acetic acid
40
20
0
4
8
12
16
2
4
6
8
10
12
Concentration of inhibitors (mM) Fig. 2. The effects of furan derivatives on lipid production by R. toruloides Y4.
0 0
0
20
Concentration of other inhibitors (mM) Fig. 1. The effects of selected inhibitors on lipid production by R. toruloides Y4. (A) Glucose consumption, (B) biomass concentration and (C) lipid content.
for Escherichia coli (Zaldivar et al., 1999). Because PHB, syringaldehyde and vanillin were normally found at very low concentrations of far below 0.1 mM (Chen et al., 2006), these compounds would
likely had little impacts on lipid production by R. toruloides Y4 when actual biomass hydrolysate was fed. One typical hexoses decomposed byproduct during biomass hydrolysis is HMF. In a representative corn stover hydrolysate, HMF concentration was 10.2 mM (Agbogbo and Wenger, 2007). Fortunately, HMF had little inhibitory effects on lipid production up to 14.7 mM. Furfural is a dehydrated product of pentoses and hemicellulose, and its concentrations in hydrolysate of spruce and corn stover
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were determined at 2.9 mM and 9.8 mM, respectively (Agbogbo and Wenger, 2007; Nilsson et al., 2005), which was a major byproduct in the acid-promoted biomass hydrolysis (Schell et al., 2003). This compound showed strong inhibitory effects on cell growth and ethanol fermentation by P. stipitis and Candida shehatae (Delgenes et al., 1996). Several mechanisms may explain the inhibitory effects of ethanol fermentation by furfural. For Saccharomyces cerevisiae, furfural was reduced to furfuryl alcohol, the less toxic form of furfural, at the expense of NADH during anaerobic growth (Palmqvist et al., 1999a,b). This transformation consumed cellular redox equivalent, resulting in major inhibition on dehydrogenases, and thus cell growth (Modig et al., 2002; Nilsson et al., 2005). Yeasts were found very sensitive to furfural, as xylitol and ethanol production were significantly inhibited by furfural at concentrations of 7.3 mM (0.7 g/L) and 19.8 mM (1.9 g/L), respectively (Converti et al., 2000; Gutierrez-Padilla and Karim, 2005). It was also pointed out that furfural could lead to membrane leakage for E. coli (Almeida et al., 2007; Zaldivar et al., 1999). Our data also indicated that furfural had drastic inhibitory effect on R. toruloides Y4. When 1 mM of furfural was present, glucose consumption, biomass and lipid content dropped by 41.1%, 45.5% and 26.5% of those of the control sample to 26.5 g/L, 8.2 g/L and 45.9%, respectively. Furthermore, the effects of furfuryl alcohol and furoic acid, two potential derivatives of furfural, were tested. The results were shown in Fig. 2. A similar trend as furfural was observed for these two derivatives, though furoic acid appeared slightly less toxic. The biomass decreased about 45% at 1 mM level for both furfural and furfuryl alcohol while 31.7% for furoic acid at the same level. It also revealed that biomass production was in general more sensitive to those inhibitors than lipid content did, suggesting that these compounds likely repressed cell growth more severely than lipid biosynthesis. Taken together, furfural and its derivatives imposed major inhibition on cell growth and lipid production by R. toruloides Y4. Thus, future work is needed either to separate the furfural containing fluent during biomass hydrolysis or to develop a costeffective detoxification method for furfural removal. 3.2. Effects of inhibitor combinations on R. toruloides Y4 We further designed experiments using binary combinations of inhibitors to look into the interactions among these inhibitors. In these trials, whenever two inhibitors were mixed, the calculated relative growth data were obtained by adding data found in experiments with single inhibitor. If the experimental data was equal or close to the calculated one, the inhibition by the two inhibitors was
100
calculated
Table 1 Results of lipid production by R. toruloides Y4 in the presence of all six inhibitorsa. Samples
Biomass (g/L)
Lipid content (%)
Lipid (g/L)
Control Experiment
15.8 ± 0.33 13.2 ± 0.12
65.2 ± 0.13 65.9 ± 0.13
10.3 ± 0.24 8.7 ± 0.09
a Acetate 52.2 mM, furfural 10.2 mM, HMF 15.0 mM, vanillin 1.0 10 0.44 10 3 mM, and syringaldehyde 0.117 10 3 mM.
3
mM, PHB
defined as additive. If the experimental data exceeded the calculated one, the inhibition was defined as synergistic. Each two inhibitors were combined at various levels. In the whole concentration range tested, most of the binary combinations were roughly additive, as the relative growth data from the experimental and the calculated were comparable (Fig. 3). However, a few exceptions were obvious. Combination sets including H/ V = 8/9, V/S = 9/6, S/P = 6/9 and V/P = 4.5/6 gave almost complete inhibition, suggesting that strong synergistic inhibition occurred. One particular combination, F/P = 0.8/9, was interesting in that cell growth was even 1.6-fold faster than the calculated value, indicating that there was cancellation of inhibitory effects between these two compounds. These data suggested that combinations of different hydrolysate by-products would give a much complex effects on fermentation processes. Fortunately, for most inhibitors the concentrations would be much lower than these concentrations tested herein. Finally we did lipid fermentation experiments in the presence of six inhibitors at their respective concentrations usually found in biomass hydrolysates. The concentration for individual inhibitor was arbitrarily chosen as found in spruce hydrolysate (Persson et al., 2002). Obviously, when all inhibitors existed in the fermentation broth, there was noticeable inhibition on biomass production. However, cellular lipid content was near identical to the control sample (Table 1). This result further suggested that inhibition was mainly due to a slowdown in cell growth, while lipid accumulation process remained nearly intact. These data indicated that R. toruloides Y4 could produce lipid in the presence of various inhibitors. Future works thus can focus on lipid production from lignocellulosic biomass hydrolysate. 3.3. Analysis of lipid compositions The fatty acid compositions of lipid were analyzed by GC, and the results were shown in Table 2. The distribution of major fatty acids, namely, C16:0 (palmitic acid), C18:0 (stearic acid) and
actual
Relative growth (%)
90 80 70 60 50 40 30 20 10
/6 V/ S= 3/ 3 V /S =9 /6 F/ H =0 .6/ 8 F/ H =0 .8/ F/ 4 H =0 .6 /1 F/ 1 V =0 .6 /4 .5 F/ V =0 .6 /9 F/ P= 0. 6/ 6 F/ P= 0. 8/ 9 H /P =8 /6 H /P =4 /9 S/ P= 3/ 3 S/ P= 6/ 9 V /P =3 /3 V /P =4 .5 /6
H
/S
=8
=8 /V
H
H /V
=8
/4 .
5
/9
0
Binary concentrations of inhibitors (mM) Fig. 3. The combined effects of selected inhibitors on cell growth of R. toruloides Y4. Relative growth was expressed as a percentage of the control. A combination indicated as ‘‘A/B = X/Y” means the experiment was done in the presence of X mM inhibitor A plus Y mM inhibitor B. H: HMF; V: vanillin; S: syringaldehyde; P: PHB; F: furfural.
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C. Hu et al. / Bioresource Technology 100 (2009) 4843–4847 Table 2 Fatty acid compositional data of lipid samples. Inhibitor
Control Acetic acid (120 mM) HMF (15 mM) Syringaldehyde (12 mM) PHB (9 mM) Vanillin (4.5 mM) Furfural (0.6 mM) Mixture of six inhibitors
Relative fatty acid content (%) C14:0
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
Others
1.8 1.5 1.4 1.8 2.3 1.8 1.4 2.4
33.8 29.8 27.7 30.3 39.9 33.0 25.1 37.3
0.5 0.4 0.6 0.6 0.4 0.6 1.1 nd
13.4 16.0 11.2 9.6 15.1 13.4 10.9 11.6
48.3 50.4 53.3 50.6 40.2 47.0 52.2 43.9
1.1 0.9 4.5 3.1 nd 1.3 6.9 0.9
nd nd 0.8 0.7 0.6 nd 1.8 nd
1.0 1.0 0.5 3.3 1.5 2.9 0.5 3.9
nd: not detectable.
C18:1 (oleic acid), were not significantly changed although cells were cultured in the presence of different inhibitors. These data implied that inhibitors did not affect fatty acid biosynthetic pathway. Oleic acid was the most abundant one, made up about 50% of the total and was followed by palmitic acid and stearic acid. Such a fatty acid compositional profile was quite similar to that of plant oil, indicating that microbial lipid produced from biomass hydrolysate is of great potential as biodiesel feedstock (Liu and Zhao, 2007). 4. Conclusion Release of monomeric carbohydrates from lignocellulose biomass routinely accompanied by the formation of various non-sugar compounds. Tolerance of these compounds is one of the most imperative characteristics for a successful biochemical process in order to utilize biomass. We demonstrated that R. toruloides Y4 had considerable ability to accumulate lipid in the presence of those representative inhibitors. We are now working on evolutionary engineering to further improve the stress resistance profile of oleaginous microorganisms and on developing cost-effective methods to attain biomass hydrolysate with lower inhibitor content. Acknowledgements Financial supports provided by the National High Technology Research and Development Program of China (2007AA05Z403) and the Knowledge Innovation Program of CAS (KGCX2-YW-336) are greatly acknowledged. References Agbogbo, F.K., Wenger, K.S., 2007. Production of ethanol from corn stover hemicellulose hydrolyzate using Pichia stipitis. J. Ind. Microbiol. Biotechnol. 34, 723–727. Almeida, J.R., Modig, T., Petersson, A., Hahn-Hagerdal, B., Liden, G., GorwaGrauslund, M.F., 2007. Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J. Chem. Technol. Biotechnol. 82, 340–349. Chen, S.F., Mowery, R.A., Castleberry, V.A., van Walsum, G.P., Chambliss, C.K., 2006. High-performance liquid chromatography method for simultaneous determination of aliphatic acid, aromatic acid and neutral degradation products in biomass pretreatment hydrolysates. J. Chromatogr. A 1104, 54–61. Converti, A., Perego, P., Torre, P., da Silva, S.S., 2000. Mixed inhibitions by methanol, furfural and acetic acid on xylitol production by Candida guilliermondii. Biotechnol. Lett. 22, 1861–1865. Delgenes, J.P., Moletta, R., Navarro, J.M., 1996. Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces
cerevisiae, Zymomonas mobilis, Pichia stipitis, and Candida shehatae. Enzyme Microb. Technol. 19, 220–225. Gutierrez-Padilla, M.G.D., Karim, M.N., 2005. Influence of furfural on the recombinant Zymomonas mobilis strain CP4 (pZB5) for ethanol production. J. Am. Sci. 1, 24–27. Larsson, S., Cassland, P., Jonsson, L.J., 2001. Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase. Appl. Environ. Microbiol. 67, 1163–1170. Li, Y.H., Zhao, Z.B., Bai, F.W., 2006. Optimization of culture conditions for lipid production by Rhodosporidium toruloides. Chin. J. Biotechnol. 22, 650–656. Li, Y.H., Zhao, Z.B., Bai, F.W., 2007. High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzyme Microb. Technol. 41, 312–317. Liu, B., Zhao, Z.B., 2007. Biodiesel production by direct methanolysis of oleaginous microbial biomass. J. Chem. Technol. Biotechnol. 82, 775–780. Marzialetti, T., Olarte, M.B.V., Sievers, C., Hoskins, T.J.C., Agrawal, P.K., Jones, C.W., 2008. Dilute acid hydrolysis of Loblolly pine: a comprehensive approach. Ind. Eng. Chem. Res. 47, 7131–7140. Modig, T., Liden, G., Taherzadeh, M.J., 2002. Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochem. J. 363, 769–776. Nichols, N.N., Dien, B.S., Guisado, G.M., Lopez, M.J., 2005. Bioabatement to remove inhibitors from biomass-derived sugar hydrolysates. Appl. Biochem. Biotechnol. 121, 379–390. Nilsson, A., Gorwa-Grauslund, M.F., Hahn-Hagerdal, B., Liden, G., 2005. Cofactor dependence in furan reduction by Saccharomyces cerevisiae in fermentation of acid-hydrolyzed lignocellulose. Appl. Environ. Microbiol. 71, 7866–7871. Palmqvist, E., Hahn-Hagerdal, B., 2000a. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 74, 25–33. Palmqvist, E., Hahn-Hagerdal, B., 2000b. Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification. Bioresour. Technol. 74, 17–24. Palmqvist, E., Almeida, J.S., Hahn-Hagerdal, B., 1999a. Influence of furfural on anaerobic glycolytic kinetics of Saccharomyces cerevisiae in batch culture. Biotechnol. Bioeng. 62, 447–454. Palmqvist, E., Grage, H., Meinander, N.Q., Hahn-Hagerdal, B., 1999b. Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts. Biotechnol. Bioeng. 63, 46–55. Persson, P., Andersson, J., Gorton, L., Larsson, S., Nilvebrant, N.O., Jonsson, L.J., 2002. Effect of different forms of alkali treatment on specific fermentation inhibitors and on the fermentability of lignocellulose hydrolysates for production of fuel ethanol. J. Agric. Food Chem. 50, 5318–5325. Ratledge, C., Wynn, J.P., 2002. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv. Appl. Microbiol. 51, 1–51. Schell, D.J., Farmer, J., Newman, M., McMillan, J.D., 2003. Dilute-sulfuric acid pretreatment of corn stover in pilot-scale reactor – investigation of yields, kinetics, and enzymatic digestibilities of solids. Appl. Biochem. Biotechnol. 105, 69–85. Schirmer-Michel, A.C., Flores, S.H., Hertz, P.F., Matos, G.S., Ayub, M.A., 2008. Production of ethanol from soybean hull hydrolysate by osmotolerant Candida guilliermondii NRRL Y-2075. Bioresour. Technol. 99, 2898–2904. Zaldivar, J., Ingram, L.O., 1999. Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01. Biotechnol. Bioeng. 66, 203–210. Zaldivar, J., Martinez, A., Ingram, L.O., 1999. Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli. Biotechnol. Bioeng. 65, 24–33. Zhao, Z.B., Hua, Y.Y., Liu, B., 2005. Strategies to secure feedstock supply for Chinese biodiesel industry. China Biotechnol. 25, 1–6.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides Cuimin Hu a,c, Xin Zhao a,c, Jin Zhao a, Siguo Wu a,c, Zongbao K. Zhao a,b,* a
Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian 116023, PR China Dalian National Laboratory of Clean Energy, Dalian 116023, PR China c Graduate School of the Chinese Academy of Sciences, Beijing 100049, PR China b
a r t i c l e
i n f o
Article history: Received 28 January 2009 Received in revised form 13 April 2009 Accepted 22 April 2009 Available online 3 June 2009 Keywords: Rhodosporidium toruloides Y4 Microbial lipid Lignocellulose hydrolysate Biomass Inhibitor
a b s t r a c t Lignocellulosic biomass hydrolysis inevitably coproduces byproducts that may have various affects on downstream biotransformation. It is imperative to document the inhibitor tolerance ability of microbial strain in order to utilize biomass hydrolysate more effectively. To achieve better lipid production by Rhodosporidium toruloides Y4, we performed fermentation experiments in the presence of some representative inhibitors. We found that acetate, 5-hydroxymethylfurfural and syringaldehyde had slightly inhibitory effects; p-hydroxybenzaldehyde and vanillin were toxic at a concentration over 10 mM; and furfural and its derivatives furfuryl alcohol and furoic acid inhibited cell growth by 45% at around 1 mM. We further demonstrated that inhibition is generally additive, although strong synergistic inhibitions were also observed. Finally, lipid production afforded good results in the presence of six inhibitors at their respective concentrations usually found in biomass hydrolysates. Fatty acid compositional profile of lipid samples indicated that those inhibitors had little effects on lipid biosynthesis. Our work will be useful for optimization of biomass hydrolysis processes and lipid production using lignocellulosic materials. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulosic biomass, mainly composed of cellulose, hemicellulose and lignin, is the most abundant and renewable organic compound in the biosphere. To utilize biomass with a biochemical route more effectively, complete hydrolysis of lignocellulose is a prerequisite, because most microorganisms have much better bioconversion rate with monomeric carbohydrates. Unfortunately, the release of monosaccharides during hydrolysis is routinely accompanied by the generation of non-carbohydrate compounds, such as furfural and 5-hydroxymethylfurfural (HMF) from the dehydration of pentoses and hexoses, acetic acid from the acetyl group in hemicellulose, and phenolic compounds including syringaldehyde, p-hydroxybenzaldehyde, vanillin, etc. derived from lignin (Almeida et al., 2007; Palmqvist and Hahn-Hagerdal, 2000a,b). The distribution of these inhibitors in a given hydrolysate sample is depending on both the raw material and the operational conditions employed for hydrolysis (Marzialetti et al., 2008; Schirmer-Michel et al., 2008). These byproducts had various affects on microbial cell growth, metabolism, as well as on product titer, presenting a major challenge in biological conversion of biomass (Almeida et al., * Corresponding author. Address: Dalian Institute of Chemical Physics, 457 Zhongshan Road, Dalian 116023, PR China. Tel./fax: +86 411 84379211. E-mail address: [email protected] (Z.K. Zhao). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.04.041
2007). Previous studies investigated the toxic effects of aldehyde, alcohol and acid components from hemicellulose hydrolysate for ethanologenic Escherichia coli, and demonstrated that the toxicity of hydrolysate resulted from the aggregate effects of different compounds rather than a single agent (Zaldivar and Ingram, 1999; Zaldivar et al., 1999). Studies on yeasts with different inhibitors also reached similar conclusions (Palmqvist et al., 1999a,b). To deal with these problems, one possibility is to develop detoxification methods, such as further processing with active charcoal, overliming, ion-exchange resins, or inhibitor-degrading microorganisms (Nichols et al., 2005). On the other hand, engineering superior strains with global stress resistance using either traditional approach or rational design is also extensively pursued recently (Almeida et al., 2007; Larsson et al., 2001). Yet, it is equally important to identify strains or processes with excellent inhibitor tolerance. Microorganisms that can accumulate lipids to more than 20% of their biomass are defined as oleaginous species (Ratledge and Wynn, 2002). Some yeast strains, such as Cryptococcus sp., Lipomyces sp., Rhodosporidium sp. and Rhodotorula sp. can accumulate intracellular lipids as high as 60% of its cell dry weight when using glucose as the carbon source (Li et al., 2007). Constitutive fatty acids of those lipids are mainly long chain ones that are quite similar to those of conventional vegetable oil. Therefore, oleaginous microorganisms have recently been suggested as alternative lipid
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producer to fuel a more sustainable biodiesel industry (Zhao et al., 2005). Making lipids through microorganisms is potentially a new technology of arable land-independent, continuous and controllable. However, the carbon sources for oleaginous microbes need extend to lignocellulosic biomass and related raw materials so that large volume of microbial lipids can be secured. Our previous work demonstrated that oleaginous yeast Rhodosporidium toruloides Y4 was a powerful lipid producer capable of accumulating lipid over 70% and with a titer over 100 g/L (Li et al., 2007). In this study we took some representative byproducts found in biomass hydrolysate to check their effects on the lipid production. We demonstrated that R. toruloides Y4 was a robust strain for lipid production using biomass hydrolysates. 2. Methods
All experiments were carried out in duplicates, and data were averaged. 2.3. Analytical methods Cell biomass, expressed as cell dry weight (CDW), was obtained from cell pellet in 30 ml of culture broth, and dried in an oven at 105 °C to a constant weight. Residual glucose was monitored with a SBA-50B glucose analyzer (Shandong Academy of Sciences, Jinan, China). Total lipid was extracted by the known procedure using a mixture of chloroform and methanol (Li et al., 2007). Lipid content was expressed as gram lipid per gram dry biomass. The fatty acid compositional profiles of lipid samples were determined using a 7890F gas chromatography (GC) instrument according to the published procedure (Li et al., 2007).
2.1. Yeast strain, media and chemicals Oleaginous yeast R. toruloides Y4 was a domesticated strain of R. toruloides AS 2.1389 obtained from the China General Microbiological Culture Collection Center. Inoculum was grown in YPD liquid medium contained (g/L): glucose 20, yeast extract 10, and peptone 10, pH 6.0). YPD agar slants were made with YPD liquid medium supplemented with 20 g/L agar. The fermentation media contained (g/L): glucose 54, (NH4)2SO4 0.215, yeast extract 1.0, KH2PO4 0.4, and MgSO47H2O 1.5, pH 6.0. This medium had a carbon-to-nitrogen (C/N, mol/mol) ratio around 80, and was formulated for higher lipid production (Li et al., 2006). Yeast extract (containing 1.4% ammonium-N and 8.7% total nitrogen) and peptone (containing 0.73% ammonium-N and 15.3% total nitrogen) were obtained from Aoboxing Biotech. Co. Ltd. (Beijing, China). Compounds 5-hydroxymethylfurfural (HMF), furoic acid and syringaldehyde were from Aldrich. Acetic acid, furfural, furfuryl alcohol, p-hydroxybenzaldehyde (PHB), vanillin and other chemicals were obtained from local supplier, and used as received. 2.2. Lipid production in the presence of inhibitors Precultures were inoculated from YPD agar slants (one loopful) and grown at 30 °C for 24–28 h in 50 ml of YPD medium in 250-ml shake flasks, and then 5 ml of cell culture was transferred into 45 ml of fermentation medium. Fermentation experiments were carried out in 250 ml flasks at a rotary rate of 200 rpm at 30 °C. In our experiments, inhibitor and their concentrations were acetic acid (0–120 mM), HMF (0–14.7 mM), furfural (0–10 mM), PHB (0–10 mM), vanillin (0–12 mM), syringaldehyde (0–12 mM), furoic acid (0–4 mM) and furfuryl alcohol (0–2 mM). For experiments with acetate, sodium acetate was introduced prior to sterilization. To introduce other single inhibitors, an appropriate amount of the corresponding stock solution was directly added to the inoculated media by pipetting. The control samples were prepared with no inhibitors. For binary combination experiments, two individual inhibitors at various concentrations were added to the media. Besides a control with no inhibitors, we conducted additional control experiments with each single inhibitor at the selected concentrations. Experiments with mixed inhibitors were carried out in the presence of acetate 52.2 mM, furfural 10.2 mM, HMF 15.0 mM, vanillin 1.0 10 3 mM, PHB 0.44 10 3 mM, and syringaldehyde 0.117 10 3 mM, as found elsewhere (Persson et al., 2002). Cells were cultured at 30 °C, 200 rpm until the residue sugar in the control experiments reached below 2 g/L, then collected by centrifugation, washed with distilled water, and subjected to biomass and lipid analysis. The supernatants were collected for glucose analysis.
3. Results and discussion To explore lipid production using inexpensive carbon sources, we conducted flask culture experiments by R. toruloides Y4 in the presence of some representative inhibitors found in biomass acid hydrolysate. 3.1. Effects of individual inhibitor on R. toruloides Y4 When R. toruloides Y4 cells were cultured at 30 °C for 4–5 days in the absence of an inhibitor (the control experiment), biomass, residue sugar and lipid were 15 g/L, 2 g/L and 62 wt.%, respectively. However, when an inhibitory compound was included, the fermentation performance changed. Fig. 1A–C shows R. toruloides Y4 cultural changes relative to the control sample in substrate consumption, biomass and lipid content, respectively, in the presence of single inhibitor at various concentrations. Acetate is a major inhibitory compound found in biomass hydrolysate. For example, 49.8 mM and 34.3 mM acetate were found in soybean hull and spruce acid hydrolysate (Nilsson et al., 2005; Schirmer-Michel et al., 2008). Acetic acid at 83 mM showed strong inhibitory effect on ethanol production by Pichia stipitis (Delgenes et al., 1996). However, our results showed that acetate had little effect on lipid production up to 70 mM (4.2 g/L). Moreover, excess acetate improved cell performance as lipid content reached 68% in the presence of 120 mM acetate (Fig. 1C). This was likely due that R. toruloides Y4 used acetate as building block for lipid biosynthesis. On the other hand, high concentration of acetate also led to an increase in C/N ratio such that lipid accumulation process was greatly augmented. It was demonstrated that acetic acid in its dissociated form was difficult to cross the plasma membrane and exhibit cellular toxicity (Palmqvist and Hahn-Hagerdal, 2000a,b). Three lignin derived inhibitors had different effects on lipid production. Syringaldehyde was a weaker inhibitor, as in the presence of 12 mM syringaldehyde, glucose consumption, biomass and lipid content were 39 g/L, 12.4 g/L and 58.6%, which were about 87.6%, 84.3% and 94.8%, respectively, those of the control sample. Vanillin and PHB had similar inhibition profile, although PHB showed slightly stronger effect. When vanillin and PHB were added to 9 mM, glucose consumption was suppressed by 8.4% and 40.2%, respectively. However, almost complete inhibition occurred in the presence of 12 mM of vanillin or 10 mM of PHB (Fig. 1B). Thus, the order of inhibition ability by these aldehydes was PHB > vanillin > syringaldehyde, which was in good agreement with their hydrophobicities indicated by the octanol/water partition coefficients. The more hydrophobilic the aldehyde is, the stronger the inhibitory effect. A same conclusion has been previously reported
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Concentration of acetic acid (mM)
Relative substrate consumption (%)
0
20
40
60
80
100
A 100
100
80
Furfural Syringaldehyde PHB Vanillin HMF Acetic acid
60
40
20
Furoic acid Furfuryl alcohol Furfural
120
Relative substrate consumption (%)
A
80
60
40
20
0 0
2
0
4
8
12
16
6
8
12
B 100 Furoic acid Furfuryl alcohol Furfural
Concentration of acetic acid (mM) 0
20
40
60
80
100
80
120
Relative biomass (% )
B 100
80
60
Furfural Syringaldehyde PHB Vanillin HMF Acetic acid
40
20
0
0
4
12
8
16
60
40
20
0 0
2
4
6
8
20
Concentration of acetic acid (mM) 0
20
40
60
80
100
12
C 100 Relative lipid content (%)
C
10
Concentration of inhibitors (mM)
Furoic acid Furfuryl alcohol Furfural
Concentration of other inhibitors (mM)
120
100
Relative lipid content (%)
10
20
Concentration of other inhibitors (mM)
Relative biomass (%)
4
Concentration of inhibitors (mM)
0
80
80
60
40
20
60
Furfural Syringaldehyde PHB Vanillin HMF Acetic acid
40
20
0
4
8
12
16
2
4
6
8
10
12
Concentration of inhibitors (mM) Fig. 2. The effects of furan derivatives on lipid production by R. toruloides Y4.
0 0
0
20
Concentration of other inhibitors (mM) Fig. 1. The effects of selected inhibitors on lipid production by R. toruloides Y4. (A) Glucose consumption, (B) biomass concentration and (C) lipid content.
for Escherichia coli (Zaldivar et al., 1999). Because PHB, syringaldehyde and vanillin were normally found at very low concentrations of far below 0.1 mM (Chen et al., 2006), these compounds would
likely had little impacts on lipid production by R. toruloides Y4 when actual biomass hydrolysate was fed. One typical hexoses decomposed byproduct during biomass hydrolysis is HMF. In a representative corn stover hydrolysate, HMF concentration was 10.2 mM (Agbogbo and Wenger, 2007). Fortunately, HMF had little inhibitory effects on lipid production up to 14.7 mM. Furfural is a dehydrated product of pentoses and hemicellulose, and its concentrations in hydrolysate of spruce and corn stover
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were determined at 2.9 mM and 9.8 mM, respectively (Agbogbo and Wenger, 2007; Nilsson et al., 2005), which was a major byproduct in the acid-promoted biomass hydrolysis (Schell et al., 2003). This compound showed strong inhibitory effects on cell growth and ethanol fermentation by P. stipitis and Candida shehatae (Delgenes et al., 1996). Several mechanisms may explain the inhibitory effects of ethanol fermentation by furfural. For Saccharomyces cerevisiae, furfural was reduced to furfuryl alcohol, the less toxic form of furfural, at the expense of NADH during anaerobic growth (Palmqvist et al., 1999a,b). This transformation consumed cellular redox equivalent, resulting in major inhibition on dehydrogenases, and thus cell growth (Modig et al., 2002; Nilsson et al., 2005). Yeasts were found very sensitive to furfural, as xylitol and ethanol production were significantly inhibited by furfural at concentrations of 7.3 mM (0.7 g/L) and 19.8 mM (1.9 g/L), respectively (Converti et al., 2000; Gutierrez-Padilla and Karim, 2005). It was also pointed out that furfural could lead to membrane leakage for E. coli (Almeida et al., 2007; Zaldivar et al., 1999). Our data also indicated that furfural had drastic inhibitory effect on R. toruloides Y4. When 1 mM of furfural was present, glucose consumption, biomass and lipid content dropped by 41.1%, 45.5% and 26.5% of those of the control sample to 26.5 g/L, 8.2 g/L and 45.9%, respectively. Furthermore, the effects of furfuryl alcohol and furoic acid, two potential derivatives of furfural, were tested. The results were shown in Fig. 2. A similar trend as furfural was observed for these two derivatives, though furoic acid appeared slightly less toxic. The biomass decreased about 45% at 1 mM level for both furfural and furfuryl alcohol while 31.7% for furoic acid at the same level. It also revealed that biomass production was in general more sensitive to those inhibitors than lipid content did, suggesting that these compounds likely repressed cell growth more severely than lipid biosynthesis. Taken together, furfural and its derivatives imposed major inhibition on cell growth and lipid production by R. toruloides Y4. Thus, future work is needed either to separate the furfural containing fluent during biomass hydrolysis or to develop a costeffective detoxification method for furfural removal. 3.2. Effects of inhibitor combinations on R. toruloides Y4 We further designed experiments using binary combinations of inhibitors to look into the interactions among these inhibitors. In these trials, whenever two inhibitors were mixed, the calculated relative growth data were obtained by adding data found in experiments with single inhibitor. If the experimental data was equal or close to the calculated one, the inhibition by the two inhibitors was
100
calculated
Table 1 Results of lipid production by R. toruloides Y4 in the presence of all six inhibitorsa. Samples
Biomass (g/L)
Lipid content (%)
Lipid (g/L)
Control Experiment
15.8 ± 0.33 13.2 ± 0.12
65.2 ± 0.13 65.9 ± 0.13
10.3 ± 0.24 8.7 ± 0.09
a Acetate 52.2 mM, furfural 10.2 mM, HMF 15.0 mM, vanillin 1.0 10 0.44 10 3 mM, and syringaldehyde 0.117 10 3 mM.
3
mM, PHB
defined as additive. If the experimental data exceeded the calculated one, the inhibition was defined as synergistic. Each two inhibitors were combined at various levels. In the whole concentration range tested, most of the binary combinations were roughly additive, as the relative growth data from the experimental and the calculated were comparable (Fig. 3). However, a few exceptions were obvious. Combination sets including H/ V = 8/9, V/S = 9/6, S/P = 6/9 and V/P = 4.5/6 gave almost complete inhibition, suggesting that strong synergistic inhibition occurred. One particular combination, F/P = 0.8/9, was interesting in that cell growth was even 1.6-fold faster than the calculated value, indicating that there was cancellation of inhibitory effects between these two compounds. These data suggested that combinations of different hydrolysate by-products would give a much complex effects on fermentation processes. Fortunately, for most inhibitors the concentrations would be much lower than these concentrations tested herein. Finally we did lipid fermentation experiments in the presence of six inhibitors at their respective concentrations usually found in biomass hydrolysates. The concentration for individual inhibitor was arbitrarily chosen as found in spruce hydrolysate (Persson et al., 2002). Obviously, when all inhibitors existed in the fermentation broth, there was noticeable inhibition on biomass production. However, cellular lipid content was near identical to the control sample (Table 1). This result further suggested that inhibition was mainly due to a slowdown in cell growth, while lipid accumulation process remained nearly intact. These data indicated that R. toruloides Y4 could produce lipid in the presence of various inhibitors. Future works thus can focus on lipid production from lignocellulosic biomass hydrolysate. 3.3. Analysis of lipid compositions The fatty acid compositions of lipid were analyzed by GC, and the results were shown in Table 2. The distribution of major fatty acids, namely, C16:0 (palmitic acid), C18:0 (stearic acid) and
actual
Relative growth (%)
90 80 70 60 50 40 30 20 10
/6 V/ S= 3/ 3 V /S =9 /6 F/ H =0 .6/ 8 F/ H =0 .8/ F/ 4 H =0 .6 /1 F/ 1 V =0 .6 /4 .5 F/ V =0 .6 /9 F/ P= 0. 6/ 6 F/ P= 0. 8/ 9 H /P =8 /6 H /P =4 /9 S/ P= 3/ 3 S/ P= 6/ 9 V /P =3 /3 V /P =4 .5 /6
H
/S
=8
=8 /V
H
H /V
=8
/4 .
5
/9
0
Binary concentrations of inhibitors (mM) Fig. 3. The combined effects of selected inhibitors on cell growth of R. toruloides Y4. Relative growth was expressed as a percentage of the control. A combination indicated as ‘‘A/B = X/Y” means the experiment was done in the presence of X mM inhibitor A plus Y mM inhibitor B. H: HMF; V: vanillin; S: syringaldehyde; P: PHB; F: furfural.
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C. Hu et al. / Bioresource Technology 100 (2009) 4843–4847 Table 2 Fatty acid compositional data of lipid samples. Inhibitor
Control Acetic acid (120 mM) HMF (15 mM) Syringaldehyde (12 mM) PHB (9 mM) Vanillin (4.5 mM) Furfural (0.6 mM) Mixture of six inhibitors
Relative fatty acid content (%) C14:0
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
Others
1.8 1.5 1.4 1.8 2.3 1.8 1.4 2.4
33.8 29.8 27.7 30.3 39.9 33.0 25.1 37.3
0.5 0.4 0.6 0.6 0.4 0.6 1.1 nd
13.4 16.0 11.2 9.6 15.1 13.4 10.9 11.6
48.3 50.4 53.3 50.6 40.2 47.0 52.2 43.9
1.1 0.9 4.5 3.1 nd 1.3 6.9 0.9
nd nd 0.8 0.7 0.6 nd 1.8 nd
1.0 1.0 0.5 3.3 1.5 2.9 0.5 3.9
nd: not detectable.
C18:1 (oleic acid), were not significantly changed although cells were cultured in the presence of different inhibitors. These data implied that inhibitors did not affect fatty acid biosynthetic pathway. Oleic acid was the most abundant one, made up about 50% of the total and was followed by palmitic acid and stearic acid. Such a fatty acid compositional profile was quite similar to that of plant oil, indicating that microbial lipid produced from biomass hydrolysate is of great potential as biodiesel feedstock (Liu and Zhao, 2007). 4. Conclusion Release of monomeric carbohydrates from lignocellulose biomass routinely accompanied by the formation of various non-sugar compounds. Tolerance of these compounds is one of the most imperative characteristics for a successful biochemical process in order to utilize biomass. We demonstrated that R. toruloides Y4 had considerable ability to accumulate lipid in the presence of those representative inhibitors. We are now working on evolutionary engineering to further improve the stress resistance profile of oleaginous microorganisms and on developing cost-effective methods to attain biomass hydrolysate with lower inhibitor content. Acknowledgements Financial supports provided by the National High Technology Research and Development Program of China (2007AA05Z403) and the Knowledge Innovation Program of CAS (KGCX2-YW-336) are greatly acknowledged. References Agbogbo, F.K., Wenger, K.S., 2007. Production of ethanol from corn stover hemicellulose hydrolyzate using Pichia stipitis. J. Ind. Microbiol. Biotechnol. 34, 723–727. Almeida, J.R., Modig, T., Petersson, A., Hahn-Hagerdal, B., Liden, G., GorwaGrauslund, M.F., 2007. Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J. Chem. Technol. Biotechnol. 82, 340–349. Chen, S.F., Mowery, R.A., Castleberry, V.A., van Walsum, G.P., Chambliss, C.K., 2006. High-performance liquid chromatography method for simultaneous determination of aliphatic acid, aromatic acid and neutral degradation products in biomass pretreatment hydrolysates. J. Chromatogr. A 1104, 54–61. Converti, A., Perego, P., Torre, P., da Silva, S.S., 2000. Mixed inhibitions by methanol, furfural and acetic acid on xylitol production by Candida guilliermondii. Biotechnol. Lett. 22, 1861–1865. Delgenes, J.P., Moletta, R., Navarro, J.M., 1996. Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces
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