Simultaneous saccharification and fermentation of steam-pretreated barley straw at low enzyme loadings and low yeast concentration

Enzyme and Microbial Technology 40 (2007) 1100–1107

Simultaneous saccharification and fermentation of steam-pretreated barley straw at low enzyme loadings and low yeast concentration M. Linde ∗ , M. Galbe, G. Zacchi Department of Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Received 29 May 2006; received in revised form 11 August 2006; accepted 18 August 2006

Abstract The maximum concentration of water-insoluble solids (WIS) in simultaneous saccharification and fermentation (SSF) is restricted due to inhibition of the enzymes and the yeast, as well as mass transport problems caused by the viscosity of the pretreated material. However, the higher the concentration of WIS during SSF the less energy is needed in the subsequent distillation and evaporation steps. In this study, SSF was performed on barley straw sprayed with H2 SO4 and steam pretreated at conditions yielding a highly digestible material, aiming to increase the WIS concentration and decrease the enzyme loading and the yeast concentration in SSF, in order to reduce the production cost. Three concentrations of WIS (5, 7.5 and 10%), and three enzyme loadings (5, 10 and 20 FPU/g cellulose) of Celluclast 1.5 L complemented with Novozym 188 were investigated in terms of ethanol yield. Ordinary cultivated Baker’s yeast and Baker’s yeast cultivated on barley straw hydrolyzate were also evaluated in terms of ethanol yield. The highest ethanol yield, 82% of the theoretical based on the glucose content in barley straw, was obtained after SSF with 5% WIS at an enzyme loading of 20 FPU/g cellulose together with 5 g/L ordinary cultivated yeast. Increased WIS concentration and decreased enzyme loading decreased the ethanol yield. However, by cultivating the yeast in hydrolyzate from pretreated barley straw the WIS concentration in SSF could be increased from 5% to 7.5% and the yeast concentration could be reduced from 5 to 2 g/L, while still attaining a yield of approximately 80%. © 2006 Elsevier Inc. All rights reserved. Keywords: Ethanol; Steam pretreatment; Simultaneous saccharification and fermentation; SSF; Barley straw

1. Introduction Producing bioethanol from cellulose-rich organic materials is considered a promising technology for meeting one of the greatest challenges to today’s society: namely replacing fossil fuels, especially in the transport sector, with renewable fuels with a zero net contribution of carbon dioxide to the atmosphere when combusted [1]. Bioethanol is an interesting alternative fuel as it could partially replace gasoline and at the same time mitigate greenhouse gas emission. The choice of raw material depends on the location, and softwood and corn stover have been thoroughly studied regarding ethanol production in Sweden and the USA, respectively. In the southern part of Europe one source of cellulose-rich organic material is the agricultural residue barley straw [2]. Although they have approximately the same composition, the ethanol yield from different celluloserich organic materials differs when applying the same conditions

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0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.08.014

in the cellulose-to-ethanol process. It is thus important to study each raw material to determine the appropriate production conditions. The process for producing ethanol from lignocellulosic material includes three main steps: steam pretreatment, enzymatic hydrolysis and fermentation. Steam pretreatment, which is performed at a high temperature, facilitates enzymatic hydrolysis of the cellulose and hemicellulose. The subsequent enzymatic hydrolysis of the cellulose and hemicellulose can be performed simultaneously with fermentation of the produced monomeric glucose and is then denoted simultaneous saccharification and fermentation (SSF). However, when lignocellulosic material is steam pretreated a hydrolyzate rich in substances inhibitory to the yeast and the enzymes is formed. These inhibitors are degradation products produced from the three main constituents of lignocellulosic biomass, i.e. cellulose, hemicellulose and lignin [3–5], and reduce the ethanol productivity of the yeast and the final ethanol yield. As a result of the inhibition of the enzymes and the yeast, as well as mass transport problems caused by the viscosity of the pretreated material, the maximum concentration of water-

M. Linde et al. / Enzyme and Microbial Technology 40 (2007) 1100–1107

insoluble solids (WIS) in SSF is restricted. However, the higher the concentration of WIS during SSF, the less energy is needed in the subsequent distillation and evaporation steps [6–8]. Thus, both high yields and as high a concentration of ethanol as possible are required to decrease the production cost in a full-scale process [6,9]. It is thus important to solve the problems associated with a high concentration of WIS. The production cost can also be reduced further by decreasing the enzyme loading [10] and by decreasing the yeast concentration in SSF. An additional problem during SSF is that there is often a lag phase in fermentation due to the change from cultivation to fermenting conditions [11]. The lag phase increases the total time required for SSF and thus increases the production cost. In a study on SSF of steam-pretreated spruce the lag phase has been reported to be reduced or disappear when growing the yeast on hydrolyzate similar to the one to be fermented [12]. However, different raw materials produce different hydrolyzates. It is therefore important to determine whether the lag phase can be avoided in SSF. In this study SSF was performed on steam-pretreated barley straw at three different concentrations of WIS and at three different enzyme loadings to study how the ethanol yield and the ethanol concentration respond to an increase in WIS concentration and a decrease in enzyme loading. Pretreatment conditions yielding good digestibility of cellulose were chosen from a previous study where the pretreatment conditions were investigated using enzymatic hydrolysis [13]. To study whether the lag phase can be avoided in SSF of pretreated barley straw, yeast was cultivated on hydrolyzate. The results were compared with yeast cultivated on an ordinary synthetic medium composed of glucose and nutrients. 2. Methods 2.1. Raw material Barley straw was kindly provided by Abengoa Bioenergy, Spain. The straw was delivered as pieces of approximately 50–100 mm in length and had a drymatter content of 93%. The straw was ground in a mill (Retsch GmbH, Haan, Germany), sieved to obtain pieces of 2–10 mm, and stored at room temperature.

2.2. Steam pretreatment Prior to steam pretreatment the raw material was impregnated with dilute sulphuric acid. The acid was sprayed over the barley straw through a nozzle creating a mist, until the desired concentration of acid in the liquid of the straw was reached. The straw was agitated in a cement mixer lined with stainless steel while being sprayed to ensure the even distribution of the acid. The drymatter content of the straw after impregnation was 60% (w/w). Spraying took approximately 5 min to complete. The acid-sprayed straw was stored for 1 h in a sealed bucket to prevent evaporation, and was then steam pretreated. The impregnated barley straw was pretreated in a steam pretreatment unit comprising a 10-L reactor, which has been described elsewhere [14], and the amount of straw was 500 g dry matter. The temperature was maintained using saturated steam. Pretreatment conditions producing high yields of glucose and xylose were chosen from a previous study [13]: (I) 1.0% H2 SO4 (based on the liquid content of the impregnated straw), 210 ◦ C for 5 min, and (II) 2.0% H2 SO4 , 190 ◦ C for 5 min. When the desired pretreatment time had been reached, the pressure was released and the material collected in a tank. Several batches of impregnated barley straw were steam pretreated and thoroughly mixed to form one large batch before subsequent analysis and use in SSF.

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2.3. Cell cultivation The inoculum culture was prepared on an agar plate from pure Baker’s yeast (Saccharomyces cerevisiae) produced by J¨astbolaget AB, Rotebro, Sweden. The cells were added to a 300-mL Erlenmeyer-flask with 70 mL water solution containing 23.8 g/L glucose, 10.8 g/L (NH4 )2 SO4 , 5.0 g/L KH2 PO4 and 1.1 g/L MgSO4 ·7H2 O. The water solution also contained 14.4 mL/L trace metal solution and 1.4 mL/L vitamin solution, prepared according to Taherzadeh et al. [15]. The pH was adjusted to pH 5 with 0.25 M NaOH. The Erlenmeyer flask was sealed with a cotton plug and incubated at 30 ◦ C for 24 h on a shaking table. Batch cultivation was then performed in a 2-L fermentor (Infors AG, Bottmingen, Switzerland) with a working volume of 500 mL, according to the procedure described by Rudolf et al. [16]. Cultivation was started by adding 60 mL inoculum to a medium containing 40.0 g/L glucose, 22.5 g/L (NH4 )2 SO4 , 10.5 g/L KH2 PO4 , 2.2 g/L MgSO4 ·7H2 O, 60.0 mL/L trace metal solution and 6.0 mL/L vitamin solution. The pH was continuously adjusted to pH 5 with 2.5 M NaOH. The stirrer speed was 700 rpm and the aeration rate was 0.5 L/min, corresponding to an average of 1 vvm. The dissolved oxygen concentration was continuously measured throughout batch cultivation with an oxygen sensor. Batch cultivation was changed to fed-batch cultivation when the ethanol produced during batch cultivation had been depleted, thus creating a rapid increase in oxygen concentration. 2.3.1. Fed-batch cultivation on glucose solution Fed-batch cultivation was performed by feeding 1 L solution containing 80 g/L glucose, 11.3 g/L (NH4 )2 SO4 , 5.3 g/L KH2 PO4 and 1.1 g/L MgSO4 ·7H2 O to the fermentor at a constant flow rate for 14 h. The pH was maintained at pH 5 with 2.5 M NaOH. The stirrer speed was 1000 rpm and the aeration rate was 2.3 L/min, corresponding to 1.5 vvm at the end of fed-batch cultivation. 2.3.2. Fed-batch cultivation on hydrolyzate Fed-batch cultivation was performed with hydrolyzate by the continuous addition of 904 mL hydrolyzate supplemented with glucose and salt solution to a total volume of 1 L. The glucose concentration in the pretreatment liquid solution was adjusted to 80 g/L. Salts were added to the solution to a concentration of 11.3 g/L (NH4 )2 SO4 , 5.3 g/L KH2 PO4 g/L and 1.1 g/L MgSO4 ·7 H2 O. The final concentration of diluted hydrolyzate was equivalent to that obtained when the slurry from pretreatment had been diluted to 7.5% WIS. The hydrolyzate was added to the fermentor at constant flow rate for 14 h. The pH was continuously adjusted to 5 with 2.5 M NaOH. The stirrer speed was 1000 rpm and the aeration rate was 2.3 L/min, corresponding to 1.5 vvm at the end of fed-batch cultivation.

2.4. Simultaneous saccharification and fermentation The SSF experiments presented in Table 1 were performed with unwashed slurry of pretreated barley straw in 2-L fermentors (Infors AG, Bottmingen, Switzerland) with a total working weight of 1.5 kg. The slurry was diluted with deionized water to WIS concentrations of 5, 7.5 and 10% (w/w). The diluted slurry was sterilized together with the fermentor in an autoclave at 121 ◦ C for 20 min. Nutrients were mixed together, sterilized and added to final concentrations of 0.5, 0.025 and 1.0 g/L of (NH4 )2 HPO4 , MgSO4 ·7H2 O and yeast extracts, respectively. A commercial cellulase mixture, supplied by Novozymes A/S (Bagsværd, Denmark), consisting of Celluclast 1.5 L (65 FPU/g and 17 ␤glucosidase IU/g), supplemented with the ␤-glucosidase preparation Novozym 188 (376 ␤-glucosidase IU/g) was employed in SSF. Three loadings of enzymes were investigated: 5, 10 and 20 FPU of Celluclast 1.5 L/g cellulose, with 6, 13 and 25 IU of Novozym 188 per gram cellulose, respectively. The yeast cell suspension was added to the fermentor to a concentration of either 2 or 5 g dry yeast cells/L. The SSF experiments were performed at pH 5 and 35 ◦ C, and the pH was set using 5% NH3 OH before sterilization. 2.4.1. Prehydrolysis Prehydrolysis prior to SSF was carried out to investigate whether the mass transport of enzymes and substrate could be increased, thus increasing the productivity of enzymatic hydrolysis and fermentation in SSF. Prehydrolysis was performed at 35 ◦ C or 45 ◦ C for 24 h. Prehydrolysis was preformed with 5 and

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Table 1 Concentration of WIS, enzymes and yeast in SSF experiments Experiment number

WIS (%)

Celluclast 1.5 L (FPU/g)

Novozym 188 (IU/g)

Yeast (g/L)

I II 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A B

5 5 5 5 5 7.5 7.5 7.5 10 10 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5

20 20 5 10 20 5 10 20 10 20 5 10 20 5 10 20 5 10

25 25 6 13 25 6 13 25 13 25 6 13 25 6 13 25 6 13

5 5 5 5 5 5 5 5 5 5 5 5 5 2 2 2 2 2

A and B were preceded by prehydrolysis. Experiment nos. I and II: compressed Baker’s yeast; experiment nos. 1–8: Baker’s yeast cultivated on glucose solution; experiment nos. 9–14, A and B: Baker’s yeast cultivated on hydrolyzate supplemented with glucose.

10 FPU of Celluclast 1.5 L per gram cellulose and 6 and 13 IU of Novozym 188 per gram cellulose, respectively. The WIS concentration was 7.5% in both cases. The subsequent SSF was performed with 2 g/L yeast cultivated on hydrolyzate with no additional enzymes. All nutrients were added before prehydrolysis (Table 1, SSF A and B).

2.5. Analysis Dry-matter contents were determined by drying samples in an oven at 105 ◦ C until constant weight was obtained. The starch content in the barley straw was analyzed by hydrolyzing the starch to monomeric sugars using Termamyl 120 L and AMG 300 L, kindly provided by Novozymes A/S. The straw-to-water ratio was 1:10 (w/w), and the starch removal procedure was divided into two steps. In the first, the starch was liquefied using thermostable ␣-amylases (24 mL termamyl 120 L/kg dry straw) for 4 h at 80 ◦ C and pH 6, which randomly hydrolysis ␣-amylases ␣-1,4 linkages in amylose and amylopectin to dextrin and ␣-limited dextrin, respectively. In the second step, saccharification of the remaining ␣-1,4 linkages and ␣-1,6 linkages was performed with amyloglucosidase (72 ml AMG 300 L/kg straw) at 55 ◦ C and pH 5 for 48 h [17]. The composition of the straw and the starch-free straw as well as the WIS after pretreatment, was determined according to the National Renewable Energy Laboratory (NREL) procedure for determination of structural carbohydrates and lignin in biomass [18] and that for the determination of extractives [19]. The Kjeldahl nitrogen content was estimated with a Kjeltec System 1026 distilling unit (Tecator, Sweden) and a nitrogen-to-protein factor of 6.2 was used for estimation of the protein concentration. Before SSF the composition of the pretreated barley straw was analyzed by separating a small part of it into a solid fraction and a hydrolyzate fraction by filtration. The solid fraction was washed with deionized water to remove water-soluble solids, WS, from the WIS and then analyzed regarding its composition. The hydrolyzate was analyzed regarding its content of oligosaccharides, using the NREL dilute-acid procedure for the determination of sugars, byproducts, and degradation products in liquid fraction process samples [20]. The oligosaccharide concentration was determined as the difference in monomer sugar concentration before and after acid hydrolysis. The liquids from the starch hydrolysis, the liquids from determination of carbohydrates in biomass and the liquids after acid hydrolysis for oligosaccharide determination as well as the filtrate from the pretreated material and the

samples from SSF were analyzed regarding their content of monomeric sugars using high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) equipped with a refractive index detector (Shimadzu). The column used for the separation of glucose, xylose, galactose, arabinose, and xylitol was an Aminex HPX-87P (Bio-Rad, Hercules, CA, USA) at 85 ◦ C with water as eluent, at a flow rate of 0.5 mL/min. The hydrolyzate and the samples from SSF were also analyzed regarding their content of ethanol, lactic acid, acetic acid, HMF, and furfural, using an Aminex HPX-87H column (Bio-Rad) at 65 ◦ C, with 5 mM H2 SO4 as eluent, at a flow rate of 0.5 mL/min. All samples were filtered through a 0.2-␮m filter before analysis to remove particles.

3. Results and discussion All ethanol yields in SSF are expressed as percentage of the theoretical, based on the glucose content of the pretreated material, and are calculated after 120 h of SSF, unless otherwise stated. The overall yield, including both pretreatment and SSF, was based on the glucose content of the raw material. The recovery of a specific sugar after pretreatment is defined as the sum of monomeric, oligomeric, and polymeric sugars in the hydrolyzate and in the WIS as % of the theoretical, based on the content in the raw material. When the enzymatic loading is stated, only the FPU of Celluclast 1.5 L/g cellulose will be given. However, for every 5, 10 or 20 FPU of Celluclast 1.5 L/g cellulose added, 6, 13 and 25 IU of Novozym 188 per gram cellulose, respectively, was also added. 3.1. Steam pretreatment The composition of the barley straw is presented in Table 2. The content of cellulose, hemicellulose, lignin and remaining components in the straw was within the range found in the literature [13,21–23]. The total composition adds up to 102.5 g/100 g, which is within the margin of error, as some uncertainties exist in the analyses, especially for proteins and extractives. In a previous study on the optimization of pretreatment of barley straw, two conditions resulted in high yields of glucose: Table 2 Composition of barley straw and of WIS after pretreatment with 1% H2 SO4 at 210 ◦ C for 5 min Barley straw

WIS in pretreated straw

Carbohydrate Glucan Xylan Galactan Arabinan Starch as glucan

36.8 17.2 2.2 5.3 0.4

Acetate Protein (6.2 × N) Extractives

2.4 ± 0.1 3.9 ± 0.1 11.1 ± 1.4

Ash Acid-insoluble Acid-soluble

2.6 ± 0.1 6.2 ± 0.8

4.2 ± 0.1 1.4 ± 0.8

Lignin Acid-insoluble Acid-soluble

12.1 ± 0.1 2.2 ± 0.3

26.0 ± 0.1 0.8 ± 0.0

102.4 ± 3.9

95.0 ± 3.9

Total

± ± ± ± ±

0.3 0.7 0.0 0.0 0.0

56.9 ± 4.5 ± 0.3 ± 0.9 ± –

2.6 0.2 0.0 0.1

– – –

M. Linde et al. / Enzyme and Microbial Technology 40 (2007) 1100–1107

(I) 210 ◦ C for 5 min with 1% H2 SO4 -impregnated barley straw, and (II) 190 ◦ C for 5 min with 2% H2 SO4 -impregnated barley straw [13]. SSF was performed on barley straw pretreated at both conditions to determine which pretreatment conditions that gave the highest yield in SSF (Table 1, SSF I and II). The ethanol yield from SSF after 72 h was 55% and 49% in SSF (I) and SSF (II), respectively. However, in both cases lactic acid production was observed after 12–24 h. This was due to the Baker’s yeast, which contains small amounts of lactic acid bacteria. Lactic acid is produced from the released sugars and competes with the ethanol production. However, within the first 12 h no lactic acid was formed and barley straw pretreated under the condition (I) resulted in a higher ethanol yield than straw pretreated under condition (II); 45% and 41%, respectively. Furthermore, the concentration of sulphuric acid in (I) was half that in (II), and thus more attractive for full-scale production as less neutralizing chemicals are required. It was therefore decided to continue the study with barley straw impregnated with 1% H2 SO4 , pretreated at 210 ◦ C for 5 min. In this study, the composition of the pretreated barley straw was defined in two fractions, WIS and hydrolyzate. The pretreated material contained 12% WIS and 7% water-soluble solids (WS) which gives 19% total solids. The composition of the WIS from the pretreated straw is also given in Table 2. The total concentrations of monomeric and oligomeric glucose and xylose in the hydrolyzate were 10.7 and 26.8 g/L, respectively. The concentrations of degradation products such as HMF, furfural and acetic acid, which are inhibitory to the yeast, were 0.2, 1.1 and 4.4 g/L, respectively. The concentration of acetic acid increased slightly during SSF indicating that the hemicellulose was not totally hydrolyzed during pretreatment. The concentration of acetic acid did not increase above 2, 3 and 5 g/L at a WIS concentrations of 5, 7.5 and 10%, respectively, which is below the concentration at which acetic acid alone starts to inhibit the yeast [5,24]. The recovery of glucose and xylose after steam pretreatment of the impregnated barley straw are presented in Table 3. Although the barley straw was exposed to pretreatment conditions chosen from a previous study [13] the recovery of glucose and xylose in this study differ from that in the previous study. The hemicellulose was significantly more resistant to degradation and as a result more xylose was recovered in both the WIS

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and the hydrolyzate. In contrast, more cellulose was hydrolyzed during the pretreatment, increasing the recovery of glucose in the hydrolyzate while maintaining a high total recovery. The difference between the two studies can be due to natural variations in the straw, as both the pretreatment conditions and equipment were the same as in the previous study. 3.2. Simultaneous saccharification and fermentation 3.2.1. WIS and enzyme concentrations SSF with three different WIS concentrations (5, 7.5 and 10%), at three enzyme activities (5, 10 and 20 FPU/g cellulose), was evaluated with regard to ethanol yield (Table 1, SSF 1-8). Fig. 1 shows the concentration profiles of glucose and ethanol in SSF

Table 3 Recovery of glucose and xylose after pretreatment of barley straw with 1% H2 SO4 at 210 ◦ C for 5 min, as % of the theoretical WIS Glucose Xylose

84.2 14.3

Hydrolyzate Glucosea Xyloseb

11.4 65.2

Total recovery Glucose Xylose

95.6 79.5

a b

0.2% monomers and 11.2% oligomers. 4.3% monomers and 60.9% oligomers.

Fig. 1. Ethanol and glucose concentrations in SSF. (A) 5% WIS. (B) 7.5% WIS. (C) 10% WIS. () Glucose 5 FPU/g. () Ethanol 5 FPU/g. () Glucose 10 FPU/g. () Ethanol 10 FPU/g. () Glucose 20 FPU/g. (䊉) Ethanol 20 FPU/g. The line drawn between the symbols is only to guide the eye.

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under these conditions. As seen in the figure, a long lag phase in ethanol production was observed at WIS concentrations of 7.5 and 10% and resulted in a high increase in glucose concentration at the beginning of SSF. The duration of the lag phase increased with increasing WIS and at 10% WIS with 20 FPU/g cellulose the glucose concentration reached 34 g/L before fermentation finally started after 96 h. For this SSF experiment the concentrations for the first 10 h are very uncertain as it was difficult to take representative liquid samples due to the high amount of solids. With 5% WIS there was no apparent lag phase in ethanol production at any enzyme loading. However, at the highest enzyme loading, 20 FPU/g cellulose, an increase in glucose concentration was seen during the first 4 h, indicating that the fermentation rate was lower than the hydrolysis rate. A lag phase was also observed by Stenberg et al. during SSF on softwood [25] for WIS concentrations of 7.5 and 10%. At 10% fermentation did not start within the total time of SSF they investigated (96 h), which is similar to some of the results in this study. Stenberg et al. also found that the risk of contamination in SSF of softwood was high despite the fact that pure cultivated Baker’s yeast was used. This was not the case in this study on SSF of barley straw, which indicates a concentration of substances in barley straw hydrolyzate that suppresses the growth of lactic acid bacteria [26]. Yeast often exhibits a lag phase in ethanol production when a change in medium has occurred. During this lag phase the yeast adapts to the new medium and no growth takes place [11]. Substances that are inhibiting to the yeast are present in the hydrolyzate. HMF and furfural are two inhibitors formed when hexose sugars and pentose sugars, respectively, are degraded [27] in the pretreatment step. The concentrations of HMF and furfural are often used as indicators of the severity of inhibition of the yeast in the new environment, i.e. the hydrolyzate [28]. Furfural is metabolized by the yeast to furfuryl alcohol, reducing the ethanol productivity until all the furfural has been consumed [29] thus also creating a lag in fermentation. In this study, fermentation started several hours after the furfural and HMF had been depleted thus indicating the presence of other inhibiting substances. Fig. 2 shows the ethanol yield after SSF for 120 h. The highest ethanol yield in SSF, 82%, was obtained with 5% WIS and 20 FPU/g cellulose. This corresponds to an overall ethanol yield of 79% and an ethanol concentration of 15.5 g/L. Previous results from enzymatic hydrolysis of barley straw pretreated under the same conditions showed that the digestibility in enzymatic hydrolysis was 94%, with 2% WIS [13]. If the digestibility is assumed to be the same in this study, the fermentation yield would have been 87% of the theoretical. However, it is likely that that the fermentation yield is higher as the cellulose conversion would be lower at 5% WIS. Increased WIS concentration, as well as decreased enzyme loading, lowered the ethanol yield in SSF. The highest ethanol concentration, 21.9 g/L, was obtained using 10% WIS and 20 FPU/g cellulose. This concentration resulted in an ethanol yield of only 50% of the theoretical after 120 h SSF. However, all the released glucose had not been fermented at 120 h. Increas-

Fig. 2. Ethanol yield in SSF with cultivated Baker’s yeast, as % of theoretical, after 120 h. The ethanol concentration (in g/L) after 120 h is given above the bars.

ing the time may have increased the ethanol concentration and ethanol yield further. To our knowledge there is no reported study on SSF of slurry from steam-pretreated barley straw. However, in a study on wheat straw by Ballesteros et al. [30] an overall ethanol yield of 62.5% (equivalent to 18.1 g/L) was reached after 82 h using WIS only, at a concentration of 10%. The enzyme loading was 15 FPU/g WIS (equivalent to 27 FPU/g cellulose), which could partially explain the high ethanol yield compared to SSF on barley straw. Additionally, as only the WIS was used in SSF of wheat straw, inhibitors normally present in the hydrolyzate were avoided, which could have influenced the yield. 3.2.2. Yeast cultivated on hydrolyzate The yeast was cultivated on hydrolyzate to establish whether this would reduce the duration of the lag phase and increase the ethanol productivity and yield. As previously, SSF was performed with 5 g/L yeast and enzyme activities of 5, 10 and 20 FPU/g cellulose, but only with 7.5% WIS. Fig. 3 shows the concentrations of glucose and ethanol throughout SSF for the different enzyme activities. When yeast cultivated on hydrolyzate was used in SSF there was no visible lag phase, and ethanol production started already within the first hour. Fig. 4 shows the ethanol yield after 120 h of SSF with Baker’s yeast cultivated on hydrolyzate. The yields were higher than those obtained with ordinary cultivated yeast and the same WIS, even when the glucose concentration fell to 0 before 120 h of SSF with ordinary cultivated yeast (see Fig. 2). This could be because the yeast had access to excess glucose, which it used to produce by-products instead of ethanol, or the yeast cultivated on hydrolyzate detoxified the slurry more efficiently, which decreased the enzyme deactivation caused by inhibitors. The highest ethanol yield in SSF with 5 g/L yeast cultivated on hydrolyzate was 80%, and was obtained with an enzyme loading of 20 FPU/g cellulose. This corresponds to an overall

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Fig. 3. Ethanol and glucose concentration in SSF with 7.5% WIS and 5 g/L of yeast cultivated on hydrolyzate. () Glucose 5 FPU/g. () Ethanol 5 FPU/g. () Glucose 10 FPU/g. () Ethanol 10 FPU/g. () Glucose 20 FPU/g. (䊉) Ethanol 20 FPU/g. The line drawn between the symbols is only to guide the eye.

ethanol yield of 76% and an ethanol concentration of 22.4 g/L. The yields were almost the same as that in SSF with only 5% WIS and yeast cultivated on pure sugar solution (Fig. 2). The increase in WIS concentration from 5 to 7.5%, when using yeast cultivated on hydrolyzate at a concentration of 5 g/L, increased the ethanol concentration from 15.5 to 22.4 g/L and from 12.4 to 18.5 g/L for enzyme activities of 20 and 10 FPU/g cellulose, respectively. SSF was also performed with a WIS concentration of 7.5% with only 2 g/L yeast cultivated on hydrolyzate as the presence of WIS in SSF makes recirculation of the yeast difficult. A lower yeast concentration requires less sugar for cell growth and thus reduces the total cost of ethanol production. Fig. 4 shows the ethanol yield after 120 h of SSF. Using 2 g/L yeast and 20 FPU/g cellulose slightly decreased the ethanol yield from 80 to 76%. In SSF with 10 FPU/g cellulose and a yeast concentration of 2 g/L the ethanol yield increased from 65 to 67%, and in SSF

Fig. 5. Ethanol and glucose concentration during SSF with 7.5% WIS and 2 g/L yeast cultivated on hydrolyzate. (A) 20 FPU/g cellulose. (B) 10 FPU/g cellulose. (C) 5 FPU/g cellulose. () Glucose. (䊉) Ethanol. () Glucose (45 ◦ C prehydrolysis). () Ethanol (45 ◦ C prehydrolysis). () Glucose (35 ◦ C prehydrolysis). () Ethanol (35 ◦ C prehydrolysis). The line drawn between the symbols is only to guide the eye.

Fig. 4. Ethanol yield in SSF, as % of theoretical, after 120 h with 7.5% WIS and 2 or 5 g/L yeast cultivated on hydrolyzate. The ethanol concentration (in g/L) after 120 h is given above the bars.

with 5 FPU/g cellulose the yield was the same for both yeast concentrations. Thus, lowering the yeast concentration from 5 to 2 g/L did not have any significant influence on the ethanol yield. Fig. 5 shows the concentration profiles of ethanol and glucose for enzyme activities of 5, 10 and 20 FPU/g cellulose in SSF with 2 g/L yeast cultivated on hydrolyzate. A peak in glucose concentration could be detected and the lower the enzyme loading, the later the peak in glucose concentration appeared. Thus, the concentration of yeast should not be decreased further as the rate of fermentation was, for a short time, lower than the rate of hydrolysis.

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SSF has been performed on the whole pretreated slurry in ¨ studies with other raw materials. Ohgren et al. [31] performed SSF on corn stover at 10% WIS and an enzyme loading of 15 FPU/g WIS (equivalent to 25 FPU/g cellulose) with both 2 g/L ordinary compressed Baker’s yeast and yeast cultivated on hydrolyzate, and obtained the same overall ethanol yield, 73% and 74%, respectively, after 72 h. A lag phase was observed with compressed Baker’s yeast but not with yeast cultivated on hydrolyzate, which resulted in increased productivity. In a study on H2 SO4 -impregnated and steam-pretreated Salix performed by Sassner et al. [32] an ethanol yield of 76% was obtained after 72 h of SSF with 9% WIS, 2 g/L yeast cultivated on hydrolyzate and 15 FPU/g WIS (equivalent to 20 FPU/g cellulose). However, when ordinary Baker’s yeast was used the overall ethanol yield was approximately 20%, due to the low tolerance of the compressed Baker’s yeast to inhibitors present in the Salix hydrolyzate. Although the ethanol yield obtained with Salix and Baker’s yeast was low, it was higher than that obtained with barley straw at 10% WIS and ordinary cultivated yeast after 72 h of SSF. This shows that the inhibition of the yeast is raw-material dependent and cultivating the yeast on hydrolyzate significantly increases the ethanol yield for both Salix and barley straw, while ordinary Baker’s yeast performed as well as cultivated yeast for corn stover. Prehydrolysis was performed at 45 ◦ C, which is more suitable for the enzymes, to decrease the total time of saccharification and fermentation of the pretreated barley straw. Prehydrolysis was also performed at 35 ◦ C, thus exposing the enzymes to the same temperature as without prehydrolysis. Fig. 5(B and C) shows the concentration profiles of ethanol and glucose in SSF with prehydrolysis compared with SSF only, with 2 g/L yeast cultivated on hydrolyzate. During the first 24 h of prehydrolysis at 45 ◦ C with 10 and 5 FPU/g cellulose, 29% and 21% of the total polymeric and oligomeric glucose, respectively, was hydrolyzed to monomeric glucose. After adding the yeast the glucose concentration decreased below 1 g/L within 8 and 4 h with 10 and 5 FPU/g cellulose, respectively. The ethanol yield was approximately the same with and without prehydrolysis at 45 ◦ C until 56 and 104 h SSF with 10 and 5 FPU/g cellulose, respectively. However, after 120 h the ethanol yield was only 54% and 41% with 10 and 5 FPU/g cellulose, respectively, which was lower than without prehydrolysis. During the first 24 h of prehydrolysis at 35 ◦ C with 10 and 5 FPU/g cellulose, 21% and 14% of the total polymeric and oligomeric glucose, respectively, was hydrolyzed to monomeric glucose. As expected, the conversion was lower at 35 ◦ C prehydrolysis than at 45 ◦ C. Theoretically, the conversion should be the same as with no prehydrolysis at 35 ◦ C, as no cellobiose was detected during the prehydrolysis. However, when fermentation was initiated after 24 h the ethanol concentration never increased to the level that was reached when SSF was performed with no prehydrolysis. This could be due to the fact that the yeast had access to excess glucose when added to the prehydrolysis slurry at the start of SSF, and it is likely that the yeast used some glucose for by-product formation, thus lowering the final ethanol yield. The ethanol yield after 120 h of SSF with prehydrolysis at 35 ◦ C was 57% and 36% with 10 and 5 FPU/g cellulose, respec-

tively. Thus, 10 FPU/g cellulose at 35 ◦ C gave a higher yield than at 45 ◦ C, while 5 FPU/g cellulose gave a lower yield. However, at 168 h of SSF the ethanol yield with 5 FPU/g cellulose was approximately the same at 35 and 45 ◦ C prehydrolysis. The low ethanol yield after SSF with prehydrolysis could also be due to enzyme deactivation caused by the increased temperature during prehydrolysis. It has previously been shown that the optimal temperature during hydrolysis is time dependent, due to enzyme deactivation. Enzymatic hydrolysis at 40 ◦ C with Celluclast 2 L may give a higher degree of cellulose conversion after 48 h than at 45 ◦ C [33]. Tengborg et al. [34] also showed that the degree of cellulose conversion decreased with increasing temperature in enzymatic hydrolysis with Celluclast 1.5 L for 144 h. Thus, enzyme deactivation and the fact that the yeast most likely consumed excess glucose for by-product formation when fermentation was initiated decreased the final ethanol yield in SSF of steam-pretreated barley straw when prehydrolysis was preformed. 4. Conclusions In a full-scale process the yeast will most likely be produced at the ethanol plant. Using hydrolyzate as the cultivation medium is thus a realistic alternative, and in this study cultivation of the yeast on the barley straw hydrolyzate increased the final ethanol yield. The highest ethanol yield after 120 h of SSF, 82%, equivalent to an ethanol concentration of 15.5 g/L, was obtained with 5% WIS, 20 FPU/g cellulose and 5 g/L ordinary cultivated Baker’s yeast. However, by cultivating the yeast on hydrolyzate the WIS concentration could be increased to 7.5%, increasing the ethanol concentration to 22.4 g/L, while maintaining a high ethanol yield of 80%. As it is difficult to recycle the yeast when performing saccharification and fermentation simultaneously instead of separately, decreasing the yeast concentration in SSF is essential as the yeast consumes glucose during cultivation and thus decreases the final ethanol yield. In this study a decrease from 5 to 2 g/L yeast cultivated on hydrolyzate produced the same final ethanol yield after 120 h of SSF, which increases the overall ethanol yield and decreases the cost of a full-scale ethanol plant. Twenty-four hours of prehydrolysis prior to SSF was investigated whether prehydrolysis at a more suitable temperature for the enzymes would decrease the total time of saccharification and fermentation. The results obtained showed that SSF without prehydrolysis of pretreated barley straw resulted in a higher final ethanol yield. However, prehydrolysis reduced the viscosity of the slurry and facilitated stirring in SSF. Acknowledgement This study was financed by the European Commission Framework V, contract no. NNE5-2001-00685. References [1] Galbe M, Zacchi G. A review of the production of ethanol from softwood. Appl Microbiol Biotechnol 2002;59:618–28.

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