Ethanol production from enzymatic hydrolysates of sugarcane bagasse

Enzyme and Microbial Technology 31 (2002) 274–282

Ethanol production from enzymatic hydrolysates of sugarcane bagasseusing recombinant xylose-utilisingSaccharomyces cerevisiae

Carlos Mart́ına,b, Mats Galbec, C. Fredrik Wahlboma,Bärbel Hahn-Hägerdala, Leif J. Jönssona,∗

a Applied Microbiology, Lund University, P.O. Box 124, SE-221 00 Lund, Swedenb Department of Chemistry and Chemical Engineering, University of Matanzas, 44 740 Matanzas, Cuba

c Chemical Engineering I, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden

Received 23 March 2001; accepted 19 March 2002

Abstract

Sugarcane bagasse was pre-treated by steam explosion at 205 and 215◦C and hydrolysed with cellulolytic enzymes. The hydrolysateswere subjected to enzymatic detoxification by treatment with the phenoloxidase laccase and to chemical detoxification by overliming.Approximately 80% of the phenolic compounds were specifically removed by the laccase treatment. Overliming partially removed thephenolic compounds, but also other fermentation inhibitors such as acetic acid, furfural and 5-hydroxy-methyl-furfural. The hydrolysateswere fermented with the recombinant xylose-utilisingSaccharomyces cerevisiae laboratory strain TMB 3001, a CEN.PK derivative withover-expressed xylulokinase activity and expressing the xylose reductase and xylitol dehydrogenase ofPichia stipitis, and theS. cerevisiaestrain ATCC 96581, isolated from a spent sulphite liquor fermentation plant. The fermentative performance of the lab strain in undetoxifiedhydrolysate was better than the performance of the industrial strain. An almost two-fold increase of the specific productivity of the strainTMB 3001 in the detoxified hydrolysates compared to the undetoxified hydrolysates was observed. The ethanol yield in the fermentationof the hydrolysate detoxified by overliming was 0.18 g/g dry bagasse, whereas it reached only 0.13 g/g dry bagasse in the undetoxifiedhydrolysate. Partial xylose utilisation with low xylitol formation was observed.© 2002 Elsevier Science Inc. All rights reserved.

Keywords: Ethanol; Lignocellulose; Bagasse; Fermentation; Inhibitors; Detoxification; Xylose

1. Introduction

To reduce the net contribution of greenhouse gases tothe atmosphere, ethanol has been recognised as a potentialalternative to petroleum-derived transportation fuels. Lig-nocellulosic materials are considered the most abundantrenewable resource available for the production of ethanol.The cellulose and hemicellulose fractions can be hydrolysedto sugars, which can then be fermented to ethanol. Hydroly-sis can be catalysed by acids or cellulolytic enzymes. Sinceenzymatic hydrolysis has several advantages over acid hy-drolysis, it is a very promising method for saccharificationof lignocellulose polysaccharides[1]. However, when en-zymatic hydrolysis is the method of choice, lignocellulosicmaterials need to be pre-treated for making the cellulosemacromolecules accessible for the enzymes[2].

∗ Corresponding author. Present address: Biochemistry, Division forChemistry, Karlstad University, SE-651 88 Karlstad, Sweden.Tel.: +46-54-7001801; fax:+46-54-7001457.

E-mail address: [email protected] (L.J. Jönsson).

The sugarcane plant (Saccharum officinarum) can pro-vide food, energy and feedstock for industry. Since sugarprizes on the world market have been very low for a longtime, the diversification of the sugar industry is an urgentrequirement in sugar-exporting countries[3]. Bagasse, thesolid residue after extraction of the sugarcane juice, ismostly utilised for producing steam and electricity requiredfor the cane processing plant. Bagasse, as a process waste,has traditionally been burnt in low efficiency boilers toproduce modest amounts of energy and to limit the dis-posal problem. Because of its high carbohydrate content,relatively low lignin content and its availability as an in-dustrial waste product, bagasse is a particularly appropriatesubstrate for bioconversion to ethanol. Xylan is, after glu-can, the most abundant carbohydrate in bagasse. Xylosecan account for almost one-third of the total sugar contentin bagasse hydrolysates[4]. Therefore, micro-organismsable to ferment both glucose and xylose are required for anefficient conversion of bagasse to ethanol.

Most previous studies of using sugarcane bagasse asraw material for fuel ethanol production deal with acid

0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.PII: S0141-0229(02)00112-6

C. Martı́n et al. / Enzyme and Microbial Technology 31 (2002) 274–282 275

hydrolysis, but there are also some reports about usingsteam pre-treatment[5–7]. Different pre-treatment condi-tions for preparing bagasse enzymatic hydrolysates havebeen investigated with focus on obtaining high sugaryields. Aspects deserving further attention include theuse of recombinant micro-organisms for utilisation of xy-lose and the identification and removal of fermentationinhibitors.

During the saccharification of lignocellulose polysaccha-rides, aliphatic acids, e.g. acetic, formic and levulinic acid,furan derivatives, e.g. furfural and 5-hydroxy-methyl-furfural(HMF), and phenolic compounds are formed in additionto the released sugars. These compounds might seriouslyinhibit the subsequent fermentation[8–10]. Therefore, hy-drolysates have to be detoxified in order to improve thefermentability. Several detoxification methods can be usedfor removing the inhibitory compounds from wood hy-drolysates[9,11]. A novel approach to detoxify lignocellu-lose hydrolysates is enzymatic detoxification, for examplewith the phenoloxidase laccase[9,12]. Ion exchange resins[10,13,14], active charcoal[14,15], and neutralisation andpH adjustments with different forms of alkali[10,13–15]have been employed for detoxification of bagasse acidhydrolysates.

Saccharomyces cerevisiae (baker’s yeast) has been usedfor producing food and beverages for several millennia. Ithas several advantages for ethanol production from ligno-cellulose: it is an efficient ethanol producer from glucose,does not require oxygenation, has low pH optimum and arelatively high tolerance to ethanol and inhibitors. The onlydrawback ofS. cerevisiae is that it cannot metabolise xy-lose[11]. Engineering organisms that would be able to fer-ment all the sugars in lignocellulose hydrolysates to ethanolis a challenge for molecular biologists and engineers. Oneapproach is the transformation of a natural xylose-utilisingorganism with genes from an efficient ethanologenic organ-ism [16]. Another approach is the introduction intoSaccha-romyces of a pathway for xylose metabolism from a naturalxylose-utilising organism[17–19].

SinceS. cerevisiae is able to ferment xylulose to ethanol,it can potentially be metabolically engineered to fermentxylose to ethanol by the introduction of genes encoding theenzymes xylose reductase (XR) and xylitol dehydrogenase(XDH), which are present in the natural xylose-utilisingyeastPichia stipitis [20]. However, only low amounts ofethanol have been obtained from xylose withS. cerevisiaedue to redox cofactor imbalances and to low expressionlevels of xylulokinase (XK). Over-expression of XK hasbeen shown to enhance ethanol production from xylose[18]and from xylulose[21]. Whereas recombinant bacteria con-taining the genes encoding theZymomonas mobilis ethanolpathway have been used for fermentation of bagasse hy-drolysates[22,23], no previous attempts to perform ethanolicfermentation of bagasse hydrolysates using xylose-utilisingrecombinant strains ofS. cerevisiae were found inthe literature.

In this work, the effect of two different detoxifica-tion methods, laccase treatment and overliming, on thecomposition and fermentability of enzymatic hydrolysatesof sugarcane bagasse was compared, the performance oftwo differentS. cerevisiae strains was investigated and thesubstrate utilisation and product formation of one of thestrains, a xylose-utilising genetically engineeredS. cere-visiae, was analysed in detail.

2. Materials and methods

2.1. Preparation of the hydrolysates

Fresh bagasse was obtained from “Jaime López” sugarfactory, Matanzas, Cuba. The bagasse was fractionated to aparticle size between 2.2 and 10 mm and subjected to steampre-treatment as previously described[7]. The bagasse waspre-treated at either 205 or 215◦C during 10 min.

Pre-treated bagasse was hydrolysed by the cellulase mix-ture Celluclast 2L (75 FPU/g and 12 Cellobiase IU/g) and the�-glycosidase preparation Novozym 188 (392 CellobiaseIU/g)(Novo Industri A/S, Bagsværd, Denmark) as pre-viously described[7]. The hydrolysates obtained afterpre-treatments at 205 and 215◦C are hereafter referred toas H205 and H215, respectively. The composition of thehydrolysates is shown inTable 1.

2.2. Micro-organisms

Two strains were used,S. cerevisiae ATCC 96581, isolatedfrom a spent sulphite liquor fermentation plant[24], and therecombinantS. cerevisiae strain TMB 3001. The TMB 3001strain is a CEN.PK derivative which expresses XR and XDHfrom the chromosomally integratedP. stipitis genesXYL1andXYL2, respectively, and over-expresses the homologousXKS1 gene encoding XK[19]. The strains were maintainedon YPD-agar plates containing 10 g/l yeast extract (Merck,Darmstadt, Germany), 20 g/l peptone (Difco, Detroit, MI,USA), 20 g/l glucose (BDH Laboratory Supplies, Poole,UK), and 20 g/l agar-agar (Merck, Darmstadt, Germany).

Table 1Content of the utilised hydrolysates

Component (g/l) H205 H215

Glucose 23.5 21.0Xylose 9.0 4.5Arabinose 1.8 1.1Mannose 0.5 0.4Acetic acid 4.0 4.5Formic acid 0.8 1.4Furfural 1.1 1.6HMF 0.2 0.5Phenols 4.1 4.5

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2.3. Detoxification

Enzymatic detoxification with the phenoloxidase laccasefrom Coriolus (Trametes) versicolor (E 014, Jülich En-zyme Products, Germany) was performed at pH 5.3 (afterpH adjustment with 10% (w/v) NaOH). The laccase solu-tion (90.7 U/ml, syringaldazine assay) was dialysed againstdeionised water overnight giving a two-fold increase of thevolume. The dialysed solution was added to the hydrolysateat a concentration of 10% (v/v). A control experiment(hereafter referred to as the laccase control), in which thesame amount of water instead of the enzyme solution wasadded to the hydrolysate, was also included. The sampleswere incubated at 30◦C for 12 h in a rotary shaker (90 rpm)following a previously described procedure[12].

Chemical detoxification by overliming was performed byadding 20% (w/w) Ca(OH)2 until pH 10 was reached. After1 h at pH 10, the hydrolysate was filtered. The filtrate wasthen neutralised to pH 5.5 with H2SO4, and filtered again[9].

2.4. Fermentation

Two separate fermentation experiments were carried out.In the first experiment, the ability of the two different yeaststrains to ferment the sugars in an undetoxified enzymatichydrolysate of bagasse was compared. In the second ex-periment, the effect of detoxification and the fermentationperformance of the recombinant xylose-fermentingS. cere-visiae strain TMB 3001 in bagasse hydrolysates were anal-ysed in detail.

Hydrolysates were supplemented with nutrients to finalconcentrations of 1 g/l yeast extract, 0.5 g/l (NH4)2HPO4,0.025 g/l MgSO4·7H20 and 1.38 g/l NaH2PO4 as previouslydescribed[9]. All chemicals used were purchased fromMerck (Darmstadt, Germany) if not otherwise stated. ThepH was adjusted to 5.5 with 2 M NaOH.

Pre-cultures, inoculated from agar plates, were grown in100 ml mineral medium[25] at 30◦C overnight in 250 mlbaffled Erlenmeyer flasks with agitation (150 rpm) in anorbital incubator (Gallenkamp, Leicester, UK). The cellswere harvested in a refrigerated centrifuge (J-25I, Beckman,Fullerton, CA, USA) at 5000 rpm for 5 min, resuspended in0.9% (w/v) NaCl and used for inoculation of the fermen-tors. The initial cell concentration was 4 g/l dry weight inall fermentations. Fermentations were run for 24 h undernon-sterile conditions at 30◦C. Fermentations of solutionscontaining glucose (BDH Laboratory Supplies, Poole, UK)and xylose (Acros Organics, NJ, USA) in the same con-centration as in the hydrolysate and supplemented with thesame nutrients were performed as references.

In the first experiment, the fermentations were conductedin 21 ml flasks containing 20 ml hydrolysate H215, sealedwith rubber stoppers and equipped with syringes for sam-pling and cannulas for CO2 venting. The flasks were inocu-lated and incubated at 30◦C with magnetic stirring. Samples

of 100�l were withdrawn at the start and after 4, 8, 12, and24 h. Two fermentations with ATCC 96581 and TMB 3001in the hydrolysate and two fermentations with each strain inthe reference solution were performed and this was repeatedtwo times. The produced ethanol (g/l), the total ethanol yield(g/g) (calculated as maximum produced ethanol divided bytotal initial sugar content), the maximum volumetric produc-tivity of ethanol (g/(lh)) and maximum specific productiv-ity (g/(gh)) were used as criteria of the ability of the yeaststrains to ferment the bagasse hydrolysate.

In the second experiment, the fermentations were carriedout in computer-controlled 1 l glass fermentors (BelachBioteknik AB, Stockholm, Sweden) with a working volumeof 600 ml. The fermentations were conducted at 30◦C. ThepH was kept at 5.5 by automatic titration with 3 M NaOH.The agitation was set at 200 rpm. For ensuring anaerobicconditions, the fermentors were flushed with 0.3 l/min ni-trogen. The gas outlet condenser was cooled to 2◦C bymeans of water circulation from a cooling bath. Dow Corn-ing Antifoam (BDH Laboratory Supplies, Poole, UK) wasmanually added when required. Two fermentations withhydrolysate detoxified by overliming and two with hy-drolysate detoxified by laccase treatment were conductedfor hydrolysates H205 and H215, respectively. Fermen-tations of undetoxified hydrolysates and of the referencewere also performed.S. cerevisiae TMB 3001 was usedin all these fermentations. Samples were withdrawn at thestart and after 2, 4, 6, 8, 10, 12, and 24 h for dry weightdetermination and HPLC analysis.

2.5. Analyses

All samples were analysed by HPLC (Gilson, Middle-town, WI, USA) with an RI-detector (Shimadzu RID-6A,Kyoto, Japan). The samples were filtered through a 0.20�mfilter and diluted prior to analysis. In the hydrolysissamples, cellobiose, glucose, xylose, galactose and man-nose/arabinose were separated on an HPX-87P column(Bio-Rad, Hercules, CA, USA) operating at 80◦C with de-gassed ultrapure water as the mobile phase at a flow rate of0.6 ml/min. Since mannose and arabinose cannot be sepa-rated by the HPLC method used[2], another method wasemployed to separate these sugars, namely anion exchangechromatography with pulsed amperometric detection (PAD,Dionex, Sunnyvale, CA, USA) using a CarboPac PA 10column (Dionex, Sunnyvale, CA, USA). A GP40 gradientpump (Dionex, Sunnyvale, CA, USA) was used for propor-tioning NaOH and deionised water as the mobile phase at aflow rate of 1 ml/min. Acetic, formic and levulinic acid, aswell as furfural, HMF and total contents of phenols werealso analysed. In fermentation samples, glucose, ethanol,glycerol, xylitol, lactic acid, acetic acid, furfural and HMFwere analysed on an Aminex HPX-87H column (Bio-Rad,Hercules, CA, USA) operating at 45◦C with 5 mM H2SO4as the mobile phase at a flow rate of 0.6 ml/min. Furfuraland HMF were detected with a UV-spectrophotometric


detector (Shimadzu SPD-6A, Kyoto, Japan). The totalcontent of phenolic compounds was determined colorimet-rically by the Folin-Ciocalteu method[26] with vanillinas calibration standard, using a U-1100 spectrophotometer(Hitachi Ltd., Tokyo, Japan).

Cell mass content was analysed gravimetrically. Fivemillilitre samples were vacuum filtered through 0.45�mpre-weighed filters (Gelman Sciences, Ann Arbor, MI,USA). The filters were dried in a microwave oven(Whirlpool, MI, USA) during 10 min at the power of 3.5and weighed. Biomass concentration was also determinedby optical density measurements at 620 nm. This analysisgave only approximate results due to the interference of theantifoam addition.

3. Results

3.1. Fermentation of non-detoxified bagasse hydrolysates

With the purpose of choosing a micro-organism able toutilise most sugars and to withstand the inhibitory sub-stances contained in bagasse hydrolysates, the undetoxifiedhydrolysate H215 was fermented with two different yeaststrains: ATCC 96581 isolated from a spent sulphite liquorfermentation plant[24] and the recombinant xylose-utilisingstrain TMB 3001[19]. Hydrolysate H215 was selected forthis experiment because it contained the highest amountsof inhibitors (Table 1). The hydrolysate was enriched withnutrients to exclude any nutritional deficiency. A high in-oculum was utilised to avoid bacterial contamination. Theinitial cellmass concentrations (dry weight) were 4.1 g/lfor ATCC 96581 and 3.9 g/l for TMB 3001. The cell massincreased slightly for both strains during the course of thefermentation.

Fermentations in hydrolysate were inhibited comparedto the reference fermentations (Table 2). The total ethanolyield was less affected by the inhibition than the volumetricand specific productivities. The ethanol yield was 16–18%lower in the hydrolysate than in the reference fermenta-tions. The volumetric productivity was 48–69% lower in thehydrolysate. For the calculation of the productivities, theethanol produced within the first 8 h was considered, sincethe maximum productivity in the reference fermentationswas achieved after 8 h.

ATCC 96581 was more affected by the inhibition thanTMB 3001. The recombinant strain consumed all the glucose

Table 2Fermentative performance of theS. cerevisiae strains

Yeast strain Medium Total sugar (g/l) Ethanol (g/l) Total yield (g/g) Maximum volumetricproductivity (g/(lh))

Maximum specificproductivity (g/(gh))

ATCC 96581 H215 26.0 7.4 0.28 0.37 0.09ATCC 96581 Reference 26.5 9.1 0.34 1.19 0.31TMB 3001 H215 26.2 8.2 0.31 0.65 0.15TMB 3001 Reference 26.6 9.9 0.37 1.26 0.32

Fig. 1. Effect of laccase treatment on the phenol content of the hy-drolysates.

present in the hydrolysate and a part of the xylose, whereasATCC 96581 left some glucose and, as expected, did notutilise the xylose (data not shown). ATCC 96581 gave a vol-umetric productivity of 0.37 g/(lh) in the hydrolysate, whichis less than one-third of the productivity in the reference fer-mentation. On the other hand, the volumetric productivity0.65 g/(lh) for the recombinant strain TMB 3001in the fer-mentation of the hydrolysate was more than a half of thatof the reference.

TMB 3001 exhibited a higher specific productivity, with0.15 g/(gh), which corresponds to 47% of the productivity ofthe reference. In contrast, the specific productivity obtainedwith ATCC 96581 was only 0.09 g/(gh), which correspondsto 29% of the productivity of the reference.

3.2. Detoxification

In order to improve the ethanolic fermentation, the hy-drolysates were detoxified. Two separate detoxificationmethods were used: enzymatic treatment with the phe-noloxidase laccase and chemical treatment with Ca(OH)2,so called overliming (Figs. 1 and 2).

The enzymatic treatment had a marked effect upon theremoval of phenolic compounds: approximately 80% of thephenols were removed from both hydrolysate H205 andH215 (Fig. 1). The concentrations of the other inhibitors in-vestigated were not affected (data not shown). The decreasein the concentration of phenolic compounds in the laccasecontrol only shows the dilution effect of adding the enzymesolution.

Overliming affected the concentration of acetic acid, fur-fural, HMF and phenolic compounds. Around 18% of theacetic acid was removed in hydrolysate H205 and the con-centration of furan derivatives was reduced by more than


Fig. 2. Effect of overliming on the content of acetic acid, furans andphenols in the hydrolysates. (A) H205, (B) H215. Same symbols as inFig. 1.

25%. Roughly 17% of the phenols were removed. Sugarswere only marginally affected by the detoxification treat-ments and the changes could be attributed to dilution bythe detoxifying agents (data not shown). Comparable resultswere obtained with both hydrolysates. However, the removalof inhibitors in H215 was slightly lower than in H205.

3.3. Fermentation of detoxified hydrolysates

For obtaining more information on the performance of therecombinant strain in bagasse hydrolysates a new fermenta-tion experiment was conducted. The effect of detoxificationon the fermentability of the hydrolysates was evaluated.Sugar consumption, ethanol and by-product formation wereanalysed during fermentation. The analysis of the bagassehydrolysates by anion exchange chromatography (Table 1)gave an arabinose:mannose ratio of 4:1. Therefore, thecombined concentration of these sugars is given as arabi-nose inFig. 3. As the results of sugar consumption for bothdetoxification methods used were very close, only thosefor the fermentation of overlimed hydrolysates are shown.Since sugar utilisation in both hydrolysates followed thesame trend, only the results for H205 are shown (Fig. 3).

Fig. 3. Sugar consumption during fermentation of bagasse hydrolysates withS. cerevisiae TMB 3001. (A) Hydrolysate H205 detoxified by overliming,(B) undetoxified hydrolysate H205. Glucose (�); xylose (�); arabinose (�).

Fig. 4. Effect of detoxification treatment on ethanol production duringfermentation withS. cerevisiae TMB 3001. Hydrolysate detoxified by lac-case treatment (�); hydrolysate detoxified by overliming (�); undetoxi-fied hydrolysate (�); reference (�).

In the detoxified hydrolysates, glucose was readily fer-mented, whereas in the undetoxified ones some glucosestill remained after 12 h. Xylose consumption during fer-mentation was slow in the hydrolysates, but in the detox-ified hydrolysates more xylose was consumed than in theundetoxified ones. After 24 h, 33% of the initial xylosehad been consumed in the overlimed hydrolysate and 31%in the laccase-treated hydrolysate, whereas only 19% hadbeen consumed in the undetoxified hydrolysate. The arabi-nose fraction was not utilised in either treated or untreatedhydrolysates.

Ethanol was produced readily from both detoxified sam-ples, whereas a 2 h lag was observed in the fermentation ofthe undetoxified hydrolysate (Fig. 4). The rate of ethanolformation was slightly faster for the overlimed hydrolysatesthan for the laccase-treated ones. The ethanol yield increasedfrom 0.38 g/g glucose in the fermentation of the undetoxi-fied hydrolysate H205 to 0.47 and 0.52 g/g glucose in thehydrolysates treated with laccase and overliming, respec-tively (Table 3). The ethanol yield from dry bagasse in-creased from 0.13 to 0.16 and 0.18 g/g dry bagasse whenthe hydrolysate was detoxified by laccase treatment andoverliming, respectively. The specific productivity in thedetoxified hydrolysates was almost twice of that in the un-treated hydrolysate. Both detoxification methods increasedthe biomass yield from less than 50% to roughly 84–90%of the yield achieved in the reference fermentation. The fer-mentability of H205 was better than H215. All analysed pa-rameters had higher or equal values for H205 compared toH215, with the exception of the biomass yield on total sugar.

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Fig. 5. Variation of the composition of inhibiting substances and by-products during fermentation of hydrolysate H205 withS. cerevisiae TMB 3001.(A) Detoxified by overliming, (B) undetoxified, (C) reference sugar solution. Acetic acid (�); glycerol (�); furfural (�); HMF (×); xylitol (�).

Furfural in the detoxified hydrolysates was converted bythe yeast cells within the first 2–4 h of fermentation whereasin the undetoxified hydrolysates it took 6–8 h (Fig. 5). HMFwas converted at a lower rate than furfural. The acetic acidconcentration remained roughly constant during the courseof fermentation. Glycerol formation in the hydrolysates waslower than in the reference solution and it started only aftercomplete consumption of furfural.

Xylitol production was relatively low and it was formedonly at the end of the fermentation. The average yield ofxylitol was 0.16 g/g consumed xylose in the hydrolysatesand 0.26 g/g consumed xylose in the fermentation of thereference solution. However, if only the xylose consumedduring the last 12 h (when xylitol was formed) is taken inaccount, then the average xylitol yield increased to 0.58 g/gconsumed xylose in the hydrolysates and 0.97 g/g consumedxylose in the reference fermentation. These differences werestatistically significant at the 95% confidence level of theFisher’s least significant differences (LSD) procedure usingthe software STATGRAPHICS Plus 2.1 for Windows.

4. Discussion

The results showing that the strain TMB 3001, a recom-binant laboratoryS. cerevisiae strain, performed better thanATCC 96581, an industrial isolate ofS. cerevisiae, in thefermentation of the bagasse hydrolysates were unexpected,since the latter strain was isolated from a spent sulphiteliquor fermentation plant[24] and therefore has a high tol-erance to inhibitors. The most appropriate measurement forcomparing the fermentation capacity of different microor-ganisms is the specific productivity[11]. Since S. cere-visiae TMB 3001 exhibited a higher specific productivitythan didS. cerevisiae ATCC 96581, the former appears to

be more suitable than the latter for fermentation of bagassehydrolysates.

Both laccase treatment and overliming proved to be effi-cient for detoxification of the bagasse hydrolysates. The en-zymatic treatment selectively removed the phenolics, whileoverliming lowered the concentration of different typesof inhibitors. The slightly lower effectiveness of detoxi-fication of hydrolysate H215 was probably due to higherbackground levels of inhibitors compared to hydrolysateH205. Although the level of fermentation-inhibiting com-pounds in bagasse enzymatic hydrolysates was lower thanin bagasse acid hydrolysates[10], it was high enoughfor causing a clear inhibitory effect compared to refer-ence solutions having similar sugar composition. Since theconcentration of furan derivatives was relatively low, theinhibition could probably mainly be attributed to phenoliccompounds and acetic acid, which is a well-known in-hibitor of micro-organisms, especially when combined withacidic pH[27,28]. The positive effect of laccase treatment,which specifically removed the phenolic inhibitors, directlyshowed the inhibitory effect of the phenolics. Similar ef-fects have previously been shown in a hardwood (willow)[12] and a softwood (spruce) hydrolysate[9].

The results of the fermentation of detoxified hydrolysatesindicate that both detoxification methods were capable ofimproving the performance of the yeast in the hydrolysates.However, there was still some inhibition in the detoxified hy-drolysates due to the fact that the toxic compounds were notcompletely removed by the detoxification treatments. Lac-case treatment removed most of the phenolics, but not theacetic acid or the furaldehydes. Overliming removed someof the inhibitors, but a considerable fraction of the inhibitors,acetic acid, furaldehydes and phenolics, were still left. Thelag in ethanol formation in the non-detoxified hydrolysateand the slightly lower fermentation rate of the laccase-treated


hydrolysate (Fig. 4) compared to the overlimed hydrolysate,where the concentrations of furfural and HMF were lowermay possibly be connected with inhibition by furfural. Fur-fural has previously been found to inhibit the ethanol pro-duction rate in the early phase of batch fermentation beforeit is reduced by the yeast cells to non-inhibitory furfuryl al-cohol [29–31]. The longer depletion time for furfural andHMF in hydrolysate H215 compared to hydrolysate H205may be a consequence of the higher initial concentrationsof those compounds in H215.

The ethanol yield from total sugar achieved in the fer-mentation of detoxified hydrolysates with the recombinantS. cerevisiae strain TMB 3001 (0.32–0.35 g/g total sugar)was lower than what was achieved in the fermentation ofbagasse hemicellulose hydrolysates with the genetically en-gineeredEscherichia coli strain KO11 (0.44–0.51 g/g drybagasse)[23]. This does not demerit the yeast strain, sincethe hydrolysates used for the bacterial strain were generouslysupplemented with either 10 g/l tryptone and 5 g/l yeast ex-tract or nutrients such as corn steep liquor (up to 5% byvolume) and crude yeast autolysate (up to 2% by volume),whereas the hydrolysates for fermentation with TMB 3001were supplemented only with 1 g/l yeast extract and mini-mal amounts of salts. However,E. coli KO11 metabolisedall the sugars in the hydrolysate, whereas TMB 3001 did notmetabolise arabinose and only partially xylose.

The higher than theoretical ethanol yield from glucoseobserved in the reference sugar solution and in the over-limed hydrolysate H205 may be due to the utilisation ofother sugars in addition to glucose. The results showed that:(i) utilisation of arabinose could not be detected, (ii) thecontent of mannose was negligible, and (iii) the formationof xylitol was very low. Taken together, these findings sug-gest that the additional ethanol was produced from xylose.Previously, recombinantS. cerevisiae strains harbouringthe XYL1 and XYL2 genes yielded low ethanol from xy-lose and xylitol was formed in considerable amounts[32].Over-expression of theTAL gene encoding the pentosephosphate pathway enzyme transaldolase gave a higherbiomass yield but the ethanol formation was not improved[33]. In the present study, the over-expression of the ho-mologousXKS gene encoding XK improved the ethanolformation from xylose and considerably reduced the xylitolyield compared to the results obtained with the strain withover-expressed transaldolase activity[33].

The slow xylose consumption during fermentation maybe a consequence of the competitive inhibition of xylosetransport by glucose. It is known, that inS. cerevisiae glucoseand xylose share the same transport system, which has amuch higher affinity for glucose than for xylose[34].

The absence of xylitol formation during the first 12 h indi-cates that in the presence of glucose this metabolite was ef-ficiently metabolised further through the pentose phosphatepathway and xylose was mostly converted into ethanol andbiomass. The stimulation of xylose metabolism by glucosehas been attributed to the generation of key intermediates

for the xylose metabolism through glucose metabolism[35].Nevertheless, the more efficient xylose conversion to xylitolafter glucose depletion may also be associated with the re-lease of a possible glucose inhibition of xylitol transport outfrom the cell. However, evidence of such an inhibition can-not be found unless intracellular accumulation of xylitol isdemonstrated. Xylitol formation could also be decreased byother components in the hydrolysate, as indicated by higherxylitol formation during the fermentation of the referencesugar solution.

The retardation observed in glycerol formation at the be-ginning of the fermentation of the hydrolysates, as well asthe lower amounts of this by-product compared to the fer-mentation of the reference solution may be linked with trans-formations occurring during furfural assimilation by yeast.Glycerol is synthesised by yeast for equilibrating the intra-cellular redox balance by converting the surplus of NADHgenerated during biomass formation to NAD+ [36]. The lackof glycerol formation during the reduction of furfural maybe attributed to the reoxidation of NADH to NAD+ by thereduction of furfural by a NADH-dependent furfuryl alcoholdehydrogenase instead of by the reduction of dihydroxyace-tone phosphate by glycerol-3-phosphate dehydrogenase.

The lower amounts of produced glycerol in the hy-drolysates may also be a consequence of the lower biomassproduction compared to fermentation of the referencesugar solution. Furthermore, low glycerol formation maybe linked with the high concentration of acetic acid in thehydrolysates, as has previously been indicated[37].

In this work, the potential of a genetically engineeredxylose-utilising S. cerevisiae strain for fermenting sugar-cane bagasse enzymatic hydrolysates was demonstrated.Both laccase treatment and overliming effectively detox-ified the bagasse hydrolysates. Since detoxification pro-cesses are costly and time consuming, the adaptation ofthe xylose-utilising yeast strain used in this work to un-detoxified bagasse hydrolysate would be a desirable futureachievement.

Acknowledgment

The Swedish Institute, the University of Matanzas and theSwedish National Energy Administration are gratefully ac-knowledged for their financial support. Prof. Marcelo Marcetis thanked for critical reading of the manuscript.

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Ethanol production from enzymatic hydrolysates of sugarcane bagasse

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