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Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid
Xiaochen Yu, Yubin Zheng, Kathleen M. Dorgan, Shulin Chen ⇑
Department of Biological Systems Engineering, Washington State University, Pullman, WA 99164-6120, USA
a r t i c l e
i n f o
a b s t r a c t
This paper explores the use of the hydrolysate from the dilute sulfuric acid pretreatment of wheat straw for microbial oil production. The resulting hydrolysate was composed of pentoses (24.3 g/L) and hexoses (4.9 g/L), along with some other degradation products, such as acetic acid, furfural, and hydroxymethylfurfural (HMF). Five oleaginous yeast strains, Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium toruloides, Lipomyces starkeyi, and Yarrowia lipolytica, were evaluated by using this hydrolysate as substrates. The results showed that all of these strains could use the detoxiﬁed hydrolysate to produce lipids while except R. toruloides non-detoxiﬁed hydrolysate could also be used for the growth of all of the selective yeast strains. C. curvatus showed the highest lipid concentrations in medium on both the detoxiﬁed (4.2 g/L) and non-detoxiﬁed (5.8 g/L) hydrolysates. And the inhibitory effect studies on C. curvatus indicated HMF had insigniﬁcant impacts at a concentration of up to 3 g/L while furfural inhibited cell growth and lipid content by 72.0% and 62.0% at 1 g/L, respectively. Our work demonstrates that lipid production is a promising alternative to utilize hemicellulosic sugars obtained during pretreatment of lignocellulosic materials. Ó 2011 Elsevier Ltd. All rights reserved.
Article history: Received 17 December 2010 Received in revised form 18 February 2011 Accepted 18 February 2011 Available online 3 April 2011 Keywords: Microbial oil Lignocellulosic hydrolysate Detoxiﬁcation Oleaginous yeast Cryptococcus curvatus
1. Introduction Biofuels and their potentials as renewable alternatives to conventional fossil fuels have been the topic of increasing focus in recent years. However, biofuel commercialization depends on the availability of feedstock in both large quantities and at low cost. Lignocellulosic materials, due to their abundance and sustainability, have attracted much attention from the biofuel production industry. Wheat straw, for example, is a lignocellulosic material that is an abundant byproduct in many wheat production regions. In 2008 the worldwide wheat production was estimated to be over 650 tonnes, thus about 850 tonnes of wheat straw were produced annually based on the straw/crop ratio of 1.3 (Talebnia et al., 2010). Wheat straw consists of 35–45% cellulose, 20–30% hemicellulose, and 8–15% lignin (Saha et al., 2005). Cellulose consists of glucose while hemicellulose contains predominant amounts of pentoses and a few hexoses. Although these two carbohydrate components in the biomass can be converted to fermentable sugar monomers for biofuel production, the direct enzymatic hydrolysis is impeded due to the physico-chemical and structural cell wall composition of the biomass. Thus, biomass pretreatment prior to enzymatic hydrolysis is essential to enhance the accessibility of
⇑ Corresponding author. Tel.: +1 509 335 3743; fax: +1 509 335 2722.
E-mail address: email@example.com (S. Chen). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.02.081
cellulase to cellulose. Among various chemical pretreatment methods, dilute sulfuric acid is the most commonly applied catalyst (Taherzadeh and Karimi, 2008). At moderate temperatures, most of the hemicellulose is effectively removed and recovered by dilute sulfuric acid as dissolved sugars and acetic acid in the hydrolysate (Wyman et al., 2005). Since hemicellulose is the second most abundant polysaccharide in lignocellulosic biomass, the full utilization of these sugars in the hydrolysate is required for economical biofuel production. Pentose utilization for biofuel production, mainly xylose from hemicellulose, has been one of the focuses of much of the research in recent decades. For ethanol production, pentose can be utilized by naturally occurring or genetically engineered microbial strains. However, some of them have poor ethanol yields or low ethanol tolerance, or are extremely sensitive to metabolic inhibitors (Smolke, 2009). For example, Saccharomyces cerevisiae is the most widely used ethanol-fermenting strain in industry, which can produce ethanol from hexose with high yields and productivities, and is capable of a high ethanol tolerance (Olsson and Hahn-Haegerdal, 1996). Nevertheless, in order to achieve pentose sugar fermentation, engineering of S. cerevisiae is required (Bettiga et al., 2009). For xylose-fermenting recombinant strains of S. cerevisiae, the yield is presently limited by: (1) the cofactor imbalance in the xylose reductase and xylitol dehydrogenase steps, (2) the absence of speciﬁc xylose transporters, (3) the limitations of the pentose phosphate pathway, (4) poor tolerance to fermentation inhibitors, and (5) instability of metabolically engineered strains with
furfural and chitosan has also been explored with certain success (Gravitis et al. Huang et al. Fig. many oleaginous yeasts can accumulate lipids on different substrates. And the market for lipids is huge because they can be easily converted into transportation fuels either via transesteriﬁcation or hydrotreatment. furfural from pentose. Idaho. followed by ﬁltration using a 0. The processed straw was then sealed in plastic bags and stored at room temperature for further use. there are several studies on converting hemicellulose hydrolysate into lipids by oleaginous yeast strains (Chen et al. Both the non-detoxiﬁed liquid hydrolysate (NDLH) and detoxiﬁed liquid hydrolysate (DLH) were used as substrates in yeast fermentation. the pentose utilization and high energy density of the lipids makes this an attractive alternative for energy production.. and these compounds strongly inhibit microorganisms during ethanol fermentation. 2010.2.. the straw was washed. 2010). C. and milled to pass through a 2 mm sieve. Thus. 2009). and Y. Rhodosporidium toruloides. Wheat straw was pretreated with dilute sulfuric acid and the liquid fraction obtained was separated via vacuum ﬁltration and divided into two portions. L. Huang et al. Organization of experimental work Fig. the main goal of this paper is to screen strains capable of growing in the non-detoxiﬁed hydrolysate for viable microbial lipid production. So a detoxiﬁcation treatment was required prior to the fermentation. Preparation of the dilute acid pretreated wheat straw hydrolysate The diluted acid pretreatment condition used was similar to those described elsewhere (Chen et al. Moreover. The ﬁltrate was then 2. which mainly include acetic acid from acetyl groups in hemicellulose. curvatus.. 2007. whey permeate (Akhtar et al. 2002). 2001. 2005). And the novelty of our work is (1) to select wheat straw as the lignocellulosic feedstock due to its largest annual production than other agricultural residues in Washington State (Frear et al. and thereafter the mixture was maintained at 50 °C and stirred for 30 min using the heater stir plate. in dilute sulfuric acid pretreated wheat straw hydrolysate various degradation products are present. After cooling.. 2009). 2009. sugar cane molasses (Alvarez et al. which is critical for local biofuel economics. And ﬁnally the lipids produced were quantiﬁed.1. Five oleaginous yeast strains. Methods 2. 2009).. and rice straw hydrolysate (Huang et al. which can produce lipids in amounts up to 70% of the total dry cell weight (Chen et al.. R. some oleaginous microorganisms. calcium hydroxide was then added to increase the pH to 10.. and (2) to make a comparatively comprehensive screening from the oleaginous strains with reported high lipid contents by using the lignocellulosic hydrolysate from dilute acid pretreatment (Meng et al. 2. In order to investigate the capability of oleaginous yeast to produce lipids by using dilute sulfuric acid pretreated wheat straw hydrolysate as a growth substrate.2 g/g sugar consumed) were lower than those of ethanol (0. Flow chart of the process to convert lignocellulosic biomass into lipids. At this time. The effects of hydrolysate detoxiﬁcation and the tolerance degrees to the inhibitors furfural and HMF were studied. Tai et al. monosodium glutamate wastewater (Xue et al. 2. starkeyi. The mixture was then treated in an autoclave at 121 °C for 60 min. oleaginous yeast has been suggested as a favorable feedstock for a sustainable biodiesel industry (Zhao et al. Rhodotorula glutinis. these strains were unable to efﬁciently produce lipids in the presence of the inhibitors in the hydrolysate.. These strains were Cryptococcus curvatus. Hu et al. 1. wheat straw was suspended and stirred at room temperature in 2% (v/v) dilute sulfuric acid solution at a solid loading of 10% (w/v). One promising approach is the production of lipids from the carbon sources present in the hydrolysate generated during acid hydrolysis.. 1 shows the process to convert the hydrolysate resulting from dilute sulfuric acid pretreatment of wheat straw to lipids. 1992). In addition. toruloides. and 5hydroxymethylfurfural (HMF) from hexose.. Preparation of the detoxiﬁed liquid hydrolysate The original hydrolysate was ﬁrst heated to 42 °C while stirring using a stir bar. 2009. However.1–0. Although.3. the development of other high value products such as xylitol. have been shown to utilize xylose efﬁciently.. ﬁve strains were chosen based on their physiological oil-producing characteristics during pentose fermentation. the market for these products is not large enough to absorb the volumes of hemicellulose derived sugars that could be produced during the pretreatment of lignocellulosic materials. . such as industrial glycerol (Papanikolaou and Aggelis. Because the fatty acid proﬁle of the microbial oils is quite similar to that of conventional vegetable oils.4–0. lipolytica were used as microbial lipid producers.0 in a process called overliming. 2.. (2006) and the other one not. Preparation of wheat straw Wheat straw was obtained from Moscow.. the autoclaved liquid hydrolysate was separated by centrifugation and vacuum ﬁltration and then stored at 4 °C prior to use.5 g/g sugar consumed) on average. Although yields of lipids (0. 2008). 2009). Yu et al. Brieﬂy. Huang et al. One fraction was detoxiﬁed as described by Mohagheghi et al. which will lead to the cost increase of the process. 2009). and the ﬁltrate was allowed to cool to 30 °C... / Bioresource Technology 102 (2011) 6134–6140 6135 subsequent mutagenesis (Hahn-Hagerdal et al. 1998)..22 lm membrane (Millipore. R. After collection.. Lipomyces starkeyi. The temperature of the hydrolysate increased to 50–52 °C by addition of calcium hydroxide. 2010). glutinis.4. air-dried. and Yarrowia lipolytica. On the other hand. MA). Additionally.X. 2001). 2008). sewage sludge (Angerbauer et al..
3 g/L malt extract. 0. The strains were incubated in the medium containing 10 g/L yeast extract.0001 g/L CuSO4Á5H2O. Yu et al. 0. The total reducing sugars were determined by the dinitrosalicylic acid method as described by Miller (1959). Statistical analysis Experimental data were statistically analyzed using the GLM procedure. glutinis (ATCC 204091). The cell pellet was then washed twice with distilled water. Analytical procedures 2.0 12.5 ± 0. This detoxiﬁed liquid hydrolysate was then ready for use as a fermentation substrate.05. Santa Clara.8. and galactose and glucose (hexoses) were Table 1 Chemical composition of wheat straw on dry solid basis. The NDLH obtained should be toxic to the oleaginous yeasts according to the results reported by Huang et al. while HMF at 0–2 g/L had no signiﬁcant inhibition on all the strains. lipolytica (ATCC 20460) were grown in the medium containing 3 g/L yeast extract. yeast cells were harvested and freeze-dried overnight. 0. a ﬂame ionization detector.5 ± 0. 2. 0. Analytes were detected and quantiﬁed against standard curves by electrochemical detection in a pulsed amperiometric detector. 10 g/L peptone.1 1. 3. and 5-hydroxymethyl-furfural (HMF) analysis Acetic acid. CA) and a refractive index detector as described by Sluiter et al.1. which performed best among the tested strains. 2. (2009).20 lm capillary column (Supelco) installed on a Hewlett Packard 5890 gas chromatograph equipped with a Hewlett Packard 3396 Series II integrator and 7673 controller. v/v) were then added to the culture medium. a method of least squares designed to ﬁt general linear models (SAS Institute.6% (v/v). followed by 0.25 mm Â 0. Arabinose and xylose (pentoses).4. and 1.3% xylan. The wheat straw contained 36. glucose.8. The medium was ﬁrst incubated at 30 °C and 150 rpm for 24 h as a preculturing step.5. starkeyi (ATCC 12659). 2. L. / Bioresource Technology 102 (2011) 6134–6140 re-acidiﬁed to pH 5.005 M sulfuric acid at a ﬂow rate of 0.5 ± 0.22 lm ﬁltration to remove any precipitate formed.6. Dry cell weight determination and fatty acids analysis To determine the amount of biomass.8. R. w/w) 36. Samples were ﬁltered through a 0.7.3 Lignin (%) = acid soluble lignin (%) + acid insoluble lignin (%). which came from cellulose and hemicellulose. NJ) and HMF (Sigma–Aldrich. And then the mixture was ﬁltered using a 0.7. The peaks obtained in the chromatographs were assigned to acetic acid. and the ﬁnal mass expressed as dry cell weight (DCW). 2. 1996 (2007)). toruloides (ATCC 10788). as well as 0..22 lm membrane (Millipore.22 lm membrane before injection and were subsequently eluted isocratically with 0. Chemical compositions of untreated wheat straw. CA). The calibration curves were linear in the range of concentrations studied. The mobile phase was 0. Seed inoculums (10%. 2. Strain cultivation in NDLH and DLH C.4 g/L MgSO4Á7H2O. NDLH and DLH Table 1 shows the chemical compositions of the untreated wheat straw. After the dilute sulfuric acid pretreatment.7 ± 0.5 with sulfuric acid.6 mL/min. respectively. 2. Seed inoculums (10%.5.5.0 ± 0. 2.003 g/L MnSO4ÁH2O.8. and Y. Thus. Results and discussion 3. Composition Glucan Xylan Arabinan Galactan Lignina Extractive a Dry solids (%. The wheat straw was prepared and the glucan. CA). Preparation of the non-detoxiﬁed liquid hydrolysate Calcium hydroxide was added to the original liquid hydrolysate at room temperature until the pH was 5. (2006).6 9.5 g/L yeast extract. in our experiment.7 ± 0. 100 m Â 0. v/v) were then transferred to the culture medium (as described above using 20 g/L glucose instead of NDLH or DLH). 1. and xylose in the NDLH and DLH were analyzed using a Dionex ICS-3000 ion chromatography system equipped with a CarboPac TM PA 20 (4 Â 50 mm) analytical column. and furfural (Acros Organics. . All experiments were performed in duplicate. (2009) that the oleaginous yeast Trichosporon cutaneum ceased to grow at the concentration of furfural greater than 2 g/L. 0. dried in a pre-weighed aluminum dish at 105 °C for 3 h.5. and split injection (Agilent Technologies Inc. or 3 g/L.3. sugar monomers from most of hemicellulose dissolved in the hydrolysate with a total liquid yield of 81.01 M NaOH at a ﬂow rate of 0. Cultures were maintained at 28 °C and 200 rpm in 250 mL ﬂasks unless mentioned otherwise.. curvatus (ATCC 20509). MO) were introduced at a concentrations of 0. Acetic acid.3 3.3 ± 0. R. And then the liquor was evaporated under vacuum and the extractive content was determined gravimetrically.1. Wheat straw compositional analysis Two grams of wheat straw were extracted with the ethanol–toluene solution for about 8 h in the Soxhlet extraction apparatus as described (ASTM.3. For fatty acids analysis. and CarboPac TM PA 20 (3 Â 30 mm) guard column (Dionex Corporation. acid soluble lignin and acid insoluble lignin were then analyzed according to NREL Procedure LAP-002 (Sluiter et al. Lipid production in the presence of furfural and HMF It was reported by Chen et al. a 5 mL cell suspension sample was centrifuged at 2500 rpm for 5 min. 2. furfural and HMF with the same residence times as the standards. NC). galactose. 5 g/L peptone. curvatus. and 10 g/L xylose.8.5 mL/min. Cultures were maintained at 28 °C and 200 rpm in 250 mL ﬂasks unless mentioned otherwise. 2 g/L KH2PO4. The fatty acid composition of the fatty acid methyl esters (FAME) was determined according to O’Fallon et al. and Table 2 presents the chemical compositions of the non-detoxiﬁed liquid hydrolysate (NDLH) and detoxiﬁed liquid hydrolysate (DLH).6136 X.6 ± 0. xylan. The signiﬁcance level of a was established at p < 0. 2008). where its hydrolysate applied had the similar inhibitor (furfural and HMF) levels with ours. the inhibition effects of furfural and HMF were tested in the range of 0–3 g/L on C.5% glucan and 21. (2007) by capillary gas chromatography on a SP-2560.1. furfural. This ﬁltrate was prepared as the non-detoxiﬁed liquid hydrolysate and ready for use as a fermentation substrate. which included 50 mL each of either non-detoxiﬁed liquid hydrolysate (NDLH) or detoxiﬁed liquid hydrolysate (DLH).5 21. MA). Sugar analysis Arabinose.8. and 20 g/L glucose (YPD medium) at 30 °C and 150 rpm for 24 h as a preculturing step. furfural and HMF in the NDLH and DLH were determined via High-performance liquid chromatography (HPLC) with a Biorad Aminex HPX-87H column (Bio-Rad Laboratories.2.
1 25.0 5.1 14. starkeyi was unaffected by the various concentrations of acetic acid. 3 shows the overall mass balance of the strategy tested. To explore the inhibition of these compounds on lipid production.7 ± 0. 3. lipolytica C. and HMF tested. 16. Fig.2 ± 0. As indicated in Table 3.5 Lipid concentration in medium (g/L) NDLH 0. glutinis.2. curvatus. and then the resulting hydrolysate was subject to the fermentation using the yeast C.2 0. which is in agreement with the loss of 7–34% xylose reported by Mohagheghi et al. Interestingly.8 g/L and 4. 3.2 ± 0.6 N/D 31.0 ± 0. Strain DCW(g/L) NDLH Y. Inhibitory effects were Table 3 Effect of NDLH and DLH on yeast strain cell growth and lipid accumulation.. However.2 ± 0.5 ± 0. / Bioresource Technology 102 (2011) 6134–6140 Table 2 Monosaccharide concentrations in the non-detoxiﬁed and detoxiﬁed hydrolysates. . curvatus showed the highest lipid concentrations at 5. there were large differences in lipid concentrations among the strains tested.2 ± 0. C.8 ± 0. the 100 g dry wheat straw generated 3. are shown in Fig. and 33.0 g glucose.4 ± 0. R.9 g ethanol).1 ± 0. oleic (C18:1). After detoxiﬁcation.3 9.1% in the NDLH and DLH. and lipid concentrations in the medium of the ﬁve yeast strains are shown in Table 3. Overall.2 Lipid content(%.0 DLH 0.9 24.3 27. which resulted from the degradation of pentoses and hexoses.00 DLH 3.2 15.0 N/A 4. curvatus was tested in the presence of furfural and HMF since this strain showed the highest lipid production originally. the lipid yield was 140 mg lipid/g substrate (total reducing sugar + acetic acid) consumed and the speciﬁc growth rate is 0.7 ± 0. curvatus to produce 4. In addition to sugar monomers. toruloides L. N/A: not available. Y. respectively.1 ± 0.3 g acetic acid in the hydrolysate after the dilute sulfuric acid pretreatment.0 Experiments were conducted at 28 °C and 200 rpm for 6 days.2 3. toruloides were slightly higher in the NDLH than in the DLH.5 ± 0. which is due to the removal of most of the hemicellulose during dilute sulfuric acid pretreatment.4 ± 0.8 g arabinose. were detected in low concentrations.6% xylose.2 19. 1. and can be seen in Tables 4. These results show that there are varying tolerances to inhibitors exhibited in different strains of yeast. and most of the total reducing sugars were depleted within the ﬁrst 2 days. (2009) due to the use of accumulated lipids in the biomass for cells’ growth by C.7 ± 0.9 g/L and 37. Lipid concentrations in all yeast strains except R. 4 shows the effect of furfural and HMF concentration on biomass and lipid content of C. and linoleic (C18:2).6 ± 0.8 ± 0.3% galactose.3% arabinose. lipolytica. however.5 4. which is in agreement with a previous report by Taherzadeh et al.6 ± 0. and (2) the strains could tolerant the inhibitors at the concentrations in the NDLH. (2006). lipid contents. curvatus in the NDLH.5% glucose. N/D: not detected.7 ± 0.4 ± 0. C.X. Based on the data obtained.44 ± 0. curvatus was more sensitive to furfural than HMF.3 DLH 4. achieved by C. 3.0 g/L in both NDLH and DLH. (2009) reported that detoxiﬁcation was necessary to improve the fermentability of the rice straw hydroly- sate by Trichosporon fermentans.7 29.2 ± 1.0 4. C. respectively.74 dayÀ1.1 3. curvatus. 28. curvatus were 10. while the biomass concentrations kept increasing until 19.6 ± 1.8 g/L. Huang et al. monosaccharides and acetic acid were retained in the DLH.2 g/L in NDLH and 15. Acetic acid and furfural were almost completely consumed within the ﬁrst day of fermentation.8 ± 0.6 ± 0.0 ± 1. 2.2%.01 0. which led to higher biomass and lipid concentrations in NDLH. Four of the ﬁve yeast strains were able to grow in both the NDLH and the DLH.0 g galactose and 3.5% and 27.1 0. The lipid concentrations were constant at about 5. the highest biomasses obtained were 17. stearic (C18:0). and L.3 ± 0. The concentration of the pentoses was approximately ﬁve times that of the hexoses. 21.2 ± 0.0 ± 0.6 20.7 g/L from day 2 through day 7. which either attributed to the strains’ inherent capabilities or large inoculum size (Huang et al. Effects of various yeast strain fermentation on biomass and lipid production with NDLH and DLH The biomass. curvatus.7 ± 0.02 ± 0. indicating that the non-detoxiﬁed hydrolysate did not have a negative impact on lipid accumulation.1 4.00 6137 the main monosaccharides in both the NDLH and the DLH. glutinis R. curvatus R. respectively. Inhibitory effect of furfural and HMF on C. and HMF was present in extremely low concentrations in both the NDLH and the DLH.6 ± 0.7 ± 0.1 1.5 DLH 7. curvatus.2 ± 0.1 3. with the lipid contents of 33.3. starkeyi 7. R. the biomass and lipid content in C. Yu et al.0 g glucose. HMF showed insigniﬁcant inhibition on biomass and lipid production at concentrations of up to 3 g/L. The compositions of the fatty acid proﬁles were different among the yeast strains.7 17.9 ± 0. the next most abundant compound in the hydrolysates was acetic acid formed via the de-acetylation of hemicellulose.4 13. (2000).0 11. w/w) NDLH 4. furfural. Lipid production in Y.4 ± 0.03 ± 0.2 2. which were less than 1.7 ± 0. The predominant fatty acids found in all of the yeast strains were palmitic (C16:0). respectively.0 33.8 ± 0. Fig.0 12. curvatus biomass and lipid production Furfural and HMF are reported to be inhibitory in many yeast strains during ethanol production. In the absence of furfural and HMF (the control experiment).2 g/L in the NDLH and DLH. toruloides was not able to grow in the NDLH. Consumptions of the total reducing sugars and inhibitors. Monosaccharide Concentration (g/L) NDLH Glucose Xylose Arabinose Galactose Acetic acid Furfural HMF 3. In our experiments. 2009).6 g/L in DLH.1 0.8 ± 0. Furfural and HMF. These similar phenomena were also observed by Huang et al. while furfural was essentially completely removed.3 N/D 14. as well as the lipid concentrations in medium during fermentation using C.00 0.0 4. The overliming process resulted in the loss of 13.05 ± 0. lipolytica showed the lowest lipid concentrations in medium. The possible reasons were (1) the sugar concentrations in NDLH were higher than those in DLH. and no significant changes were observed in the same yeast strain when grown in the NDLH versus DLH.7 g lipids (equivalent to 7. and C.1 2.
Hu et al.3 37.2 ± 0. the growth of C.0 ± 0. 2007). / Bioresource Technology 102 (2011) 6134–6140 Table 4 Fatty acid proﬁles of lipids accumulated utilizing NDLH and DLH. Our results showed that.1% (w/v).1 15. cerevisiae during ethanol fermentation (Palmqvist and Hahn-Hagerdal. Time (days) Fig. 2009).85 g/L (14. furfural inhibited biomass and lipid production by 72.0 2.5 ± 0.9 ± 3.0 0.0 42.1 ± 0.3 ± 1. lipolytica C. chromatin. Additionally. (2009) mentioned that furfural and HMF did not affect the fatty acid biosynthetic pathway of R. cerevisiae growth rate.0 Biomass/lipid concentration (g/L) A 40 35 30 25 20 15 10 Lipid concentration Biomass Total reducing sugar 50 40 30 20 10 5 0 0 1 2 3 4 5 6 7 Time (days) 0 B Inhibitor concentration (g/L) 3.567).0 ± 3. 3. the mechanism for furfural inhibition in the oleaginous yeast is most likely different than that of HMF. R. 2009).1 5. and 2 g/L in T. The overall mass balance of converting lignocellulosic biomass into lipids.5 1.2%. and furfural causes reactive oxygen species to accumulate.0 ± 0. cutaneum. curvatus has a higher tolerance to furfural and HMF than reported in other strains.3 C18:1 NDLH 56.3 ± 0.5 ± 0.9 ± 0. curvatus may be the reason that furfural was absent after the ﬁrst day of fermentation as shown in Fig. 2009).1 ± 0. 2. Furfural is always reduced to the less inhibitory furfuryl alcohol by S.5 0.7 ± 0. 1. curvatus R. mitochondrial membrane. glutinis. distinct from HMF.0 ± 0. as well as vacuole membrane. and in our experiments it was also concluded that the distributions of major fatty acids (Table 5) were not changed signiﬁcantly by HMF (p = 0..4 ± 0. (A) Biomass. 2000). toruloides (Hu et al.3 ± 0.6138 X. In comparison.0% and 62. inhibitors consumed in the NDLH.4 ± 0.0 0 1 2 3 4 5 6 7 Acetic acid Furfural HMF Total reducing sugar (g/L) Fig.4 DLH 55.0 ± 0.3 N/A 46.3 15.8 ± 0. and aldehyde dehydrogenase (ALDH).7 g/L (7.9 ± 0. When HMF was present at 3 g/L.0 23..1 DLH 5. In the presence of 0. curvatus.0 ± 0.0 1.3 ± 0.6 13.5 2.1 15.3 mM) furfural. and this potential conversion by C. toruloides (Hu et al.1 45.3 7.3 53. Furfural and HMF directly inhibit alcohol dehydrogenase (ADH).7 ± 0. Yu et al. pyruvate dehydrogenase (PDH). biomass production decreased by 44.4 ± 0. This same inhibition was achieved with 0. These results indicate that C.9 ± 0. in comparison with ethanol-producing microorganisms is the tolerance to acetic acid inhibition. 2.0 45. as indicated in Fig. toruloides L. and (B) the inhibitors’ concentration.5 ± 0.0 C18:2 NDLH 19.0%.. 2000).0 22.05–0. toruloides (Chen et al.7 ± 1. biomass and lipid concentrations in the medium during fermentation by C. and actin damage (Almeida et al.42 ± 0.5 3. the biomass and lipid contents greatly decreased with the increased concentrations of furfural. toruloides. 4. acetic acid reduced S.7 mM) in R. which was also seen during ethanol production (Palmqvist and Hahn-Hagerdal. starkeyi (Chen et al. lipid and total reducing sugar concentration.3 ± 0.2 9..2 9..2 DLH 20. reported at 1 g/L in L.0 ± 0. and R.2 N/A 4.516) or furfural (p = 0. At concentrations as low as 0.0 17.4 ± 0.3 27.8 ± 0. biomass and lipid content dropped only by 5.5 47. glutinis R.5 6.9%.3 19. starkeyi 6. and rates of glucose consumption and ethanol production decreased as the concentration of acetic . thus this was the reason why there was no change in the fatty acid proﬁles for the strains when growing in the NDLH or DLH.0 DLH 0. 2009). Another advantage of oleaginous yeast.2 ± 0.8 N/A 36. curvatus was more sensitive to furfural than was lipid accumulation. However.5 ± 0.1 g/L furfural in R.3 N/A 3.4 ± 0. 2000). At 1 g/L.1 4.9 ± 0.4 5. Strain Relative fatty acid content (%) C16:0 NDLH Y.3 43. Total reducing sugars. Several mechanisms had been reported to explain the inhibition of furans on ethanol fermentation.8 25. however.7% and 7. respectively. compared to the control samples. respectively.2 C18:0 NDLH 2. although the two chemicals have similar inhibition mechanisms in other yeasts for ethanol fermentation (Palmqvist and Hahn-Hagerdal. furfural had obvious negative impact on biomass and lipid production.
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