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Controlled feeding of cellulases improves conversion of xylose in simultaneous saccharification and co-fermentation for bioethanol production
Journal of Biotechnology 145 (2010) 168–175
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Controlled feeding of cellulases improves conversion of xylose in simultaneous saccharification and co-fermentation for bioethanol production Kim Olofsson ∗ , Magnus Wiman, Gunnar Lidén Department of Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden
a r t i c l e
i n f o
Article history: Received 6 April 2009 Received in revised form 28 September 2009 Accepted 1 November 2009 Keywords: SSF SSCF Co-fermentation Modeling Enzymatic hydrolysis Saccharomyces cerevisiae
a b s t r a c t Simultaneous saccharification and fermentation (SSF) is an interesting option for ethanol production from lignocellulosic materials. To meet desired overall yields during ethanol production from lignocellulosic materials, it is important to use both hexoses and pentoses. This can be achieved by co-fermentation of sugars in SSF, so called SSCF (simultaneous saccharification and co-fermentation), using genetically modified yeast strains. However, high concentration of glucose in the pretreated material makes xylose utilization challenging due to competitive inhibition of sugar transport. The present work demonstrates a new approach for controlling the glucose release rate from the enzymatic hydrolysis by controlling the addition of enzymes in SSCF using spruce as the raw material. Enzyme kinetics and yeast sugar uptake rates for a recombinant xylose utilizing strain of Saccharomyces cerevisiae, TMB3400, were determined in a real hydrolyzate medium. A simplified model for glucose release and uptake was created, to be used as a tool for control of the glucose concentration in a SSCF process. With help of this model, an SSCF process with efficient co-utilization of glucose and xylose was successfully designed. The results showed that the total xylose uptake could be increased from 40% to as much as 80% by controlling the enzyme feed. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Simultaneous saccharification and fermentation (SSF) (Takagi et al., 1977) has been demonstrated to be an advantageous option for ethanol production from lignocellulosic materials (Wingren et al., 2003). Residues from, e.g. forest industry and agriculture are today potential interesting feedstocks. However, a high final ethanol concentration and a high overall ethanol yield are crucial factors for the process economy (von Sivers and Zacchi, 1996). To meet desired overall yields during ethanol production from lignocellulosic materials, it is important to use both hexoses and pentoses. Saccharomyces cerevisiae, the most commonly used organism for industrial ethanol production, does not naturally ferment pentoses, but genetically modified S. cerevisiae strains with xylose fermenting abilities are now available (Jeffries and Jin, 2004; Hahn-Hägerdal et al., 2007). Strains have been designed principally by introduction of heterologous genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from fungi (Kötter et al., 1990; Eliasson et al., 2000) or genes encoding xylose isomerase (XI) from bacteria and fungi (Karhumaa et al., 2005; Kuyper et al., 2005). Xylose flux is further enhanced by over-expression of xylulose kinase and selected other genes associated with the PPP.
∗ Corresponding author. Tel.: +46 46 2220863; fax: +46 46 149156. E-mail address: [email protected] (K. Olofsson). 0168-1656/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2009.11.001
The glucose concentration must be low in order to obtain efficient xylose uptake with S. cerevisiae, since xylose transport into the cell is inhibited by glucose. The main reasons for this are that xylose and glucose compete for the same transport systems (Kilian and Uden, 1988; Meinander and Hahn-Hägerdal, 1997) and the affinity for xylose is approximately 200-fold lower than for glucose (Kötter and Ciriacy, 1993). However, glucose has also shown to enhance xylose utilization at low but non-zero concentrations (Meinander et al., 1999; Pitkänen et al., 2003), which can be attributed to induction of transporter systems (Pitkänen et al., 2003; Bertilsson et al., 2008), induction of glycolytic enzymes (Boles et al., 1996) and improved co-factor generation (Pitkänen et al., 2003). The positive effects of glucose (in low concentrations) on xylose uptake make SSF an interesting process concept for co-fermentation, since the glucose released to the medium will be simultaneously fermented. Additionally, after pretreatment of the lignocellulosic material, the hemicellulosic sugars in spruce, mainly mannose, glucose, and xylose, are present as monomers, while a large fraction of the glucose content is present as glucan in the fibers. In spruce, a potential feedstock that grows in the far northern hemisphere, the proportion of xylose is relatively low. Still, complete conversion of xylose would increase the ethanol yield by as much as 8% (Sassner et al., 2007), which is significant for an industrial process. The xylose utilizing strain of S. cerevisiae used in this work, TMB3400 (Wahlbom et al., 2003), has previously been used for SSF of different xylose rich lignocellulosic materials from the agriculture with satisfactory results (Öhgren et al., 2006; Olofsson
K. Olofsson et al. / Journal of Biotechnology 145 (2010) 168–175 Table 1 Composition of the pretreated spruce material. Content in solid fraction (% of WIS)
Glucan Mannan Galactan Xylan Lignin
a
Material 1
Material 2
54.6 1.1 ∼0 ∼0 39.3
50.4 1.3 ∼0 ∼0 41.1
Content in liquid fraction (g L−1 )
Glucosea Mannosea Galactosea Xylosea Furfural HMF Acetic acid
Material 1
Material 2
19.8 33.9 5.9 12.4 1.1 3.5 5.9
28 26.2 4.6 9.1 1.8 2.3 6.4
Both monomeric and oligomeric form is included.
et al., 2008; Rudolf et al., 2008). The highest xylose utilization previously achieved was about 40%, and a total ethanol yield, based on all available sugars, was approximately 80% (Olofsson et al., 2008). However, in spruce the low ratio between xylose and glucose makes xylose conversion particularly challenging. Furthermore, due to the relatively severe pretreatment conditions, the fermentability is expected to be relatively low in the spruce hydrolyzate (Palmqvist and Hahn-Hägerdal, 2000). This is due to the fact that during acid pretreatment, some of the sugars are degraded to furaldehydes, organic acids, and other compounds which are acting as inhibitors for the yeast (Almeida et al., 2007). A suitable process design for simultaneous saccharification and co-fermentation (SSCF) is therefore needed. Recently, a first step toward increasing the xylose uptake was taken by lowering the initial glucose concentration in the SSCF using a scheme termed prefermentation (Bertilsson et al., 2009). However, although the initial glucose concentration was indeed decreased, resulting in improved xylose utilization, the glucose was not maintained at sufficiently low levels throughout the fermentation. In the present work a new approach for controlling the glucose release rate from the enzymatic hydrolysis is made by using a model-based addition of enzyme throughout the SSCF. Initially, studies of enzyme kinetics and yeast sugar uptake rates were carried out on pretreated spruce material in order to design and calibrate a simple and robust model for sugar release and sugar uptake in the SSCF process. Subsequently, enzyme feed profiles for the SSCF process were designed and experimentally tested using spruce hydrolyzate. 2. Material and methods 2.1. Raw material and pretreatment Two different spruce materials were used and pretreated separately, but with the same pretreatment conditions. The wood chips (with 50% and 60% w/w moisture respectively) were impregnated in closed plastic bags for 20 min with SO2 (2.5% w/w moisture). Subsequently, the impregnated chips were steam-pretreated batch-wise at 210 ◦ C for 5 min in a 10L reactor. Further description of the equipment is given by Palmqvist et al. (1996). The pretreated material was stored at 4 ◦ C. The composition of the two pretreatment slurries is shown in Table 1. The water insoluble and liquid fractions were analyzed using NREL (National Renewable Energy Laboratories) standard procedures (Ruiz and Ehrman, 1996; Sluiter et al., 2004). The WIS content of the pretreated slurry was 15.1% for material 1 and 12.4% for material 2. 2.2. Cell cultivation The recombinant xylose fermenting strain S. cerevisiae TMB3400 (Wahlbom et al., 2003) was used in all SSCF experiments. Yeast cells to be used in SSCF was produced by aerobic batch cultiva-
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tion on glucose, followed by an aerobic fed-batch cultivation on spruce hydrolyzate liquid, in order to improve inhibitor tolerance by adaptation as previously shown by Alkasrawi et al. (2006). The yeast was inoculated in 300 ml flasks containing 100 ml media supplemented with 16.5 g L−1 glucose, 7.5 g L−1 (NH4 )2 SO4 , 3.5 g L−1 KH2 PO4 , 0.74 g L−1 MgSO2 ·7H2 O, trace metals and vitamins. The cells were grown for 24 h at 30 ◦ C and pH 5 in a rotary shaker at 180 rpm. Aerobic batch cultivations were performed in a 2.5 L bioreactor (Biostat A, B. Braun Biotech International, Melsungen, Germany) at 30 ◦ C. The working volume was 0.7 L and the medium contained 20.0 g L−1 glucose, 20.0 g L−1 (NH4 )2 SO4 , 10.0 g L−1 KH2 PO4 , 2.0 g L−1 MgSO4 , 27.0 mL L−1 trace metal solution and 2.7 mL L−1 vitamin solution. The pH was maintained at 5.0 throughout the cultivation, by automatic addition of 3 M NaOH. The trace metal and vitamin solutions were prepared according to Taherzadeh et al. (1996). The cultivation was initiated by adding 20.0 mL of the inoculum to the bioreactor. Aeration was maintained at 1.0 L min−1 and the stirrer speed was kept at 800 rpm. Upon depletion of the ethanol produced in the batch phase, the feeding of pretreatment liquid from spruce was initiated. 1.0 L of pretreatment liquid was added with an initial feed rate of 0.04 L h−1 that was increased linearly to 0.10 L h−1 during 16 h of cultivation. The aeration during the fed-batch phase was maintained at 1.4 L min−1 and the stirrer speed was kept at 800 rpm. After cultivation, the cells were harvested by centrifugation in 700 mL flasks for 8 min at 3000 rpm using a HERMLE Z 513K centrifuge (HERMLE Labortechnik, Wehingen, Germany). The pellets were resuspended in 0.9% NaCl solution in order to obtain a cell suspension with a cell mass concentration of 75 g dry weight L−1 . The time between cell harvest and initiation of the SSCF was no longer than 3 h. 2.3. Enzymatic hydrolysis Enzymatic hydrolysis was carried out in 600 mL flasks equipped with stirrers, which were placed in a temperature controlled water bath at 34 ◦ C. The flasks were filled with 300 mL slurry of pretreated spruce at a WIS content of 8%. To obtain the desired WIS content, the pretreatment slurry was diluted with sterile deionized water. Some of the experiments were performed without the hydrolyzate liquid, i.e. using only washed fibers in water. All hydrolysis experiments were performed in duplicates, showing good reproducibility. The enzyme preparation used, Celluclast, was provided by Novozymes A/S, Bagsvaerd, Denmark, and had a cellulase activity of 42 FPU g−1 and a -glucosidase activity of 20 IU g−1 . This was used together with Novozyme 188 (Novozymes A/S, Bagsvaerd, Denmark) with a -glucosidase activity of 340 IU g−1 . 2.4. Simultaneous saccharification and co-fermentation (SSCF) All SSCF experiments were carried out in 2.5 L bioreactors (Biostat A, B. Braun Biotech International, Melsungen, Germany; Biostat A plus, Sartorius, Melsungen, Germany and BIOFLO III, New Brunswick Scientific, Edison, NJ, USA) with a final working weight of 1.4 kg. The batch experiments were carried out with a WIS content of 8%. To obtain the desired WIS content, the pretreatment slurry was diluted with sterile deionized water. All SSCF experiments were carried out at 34 ◦ C for 96 h, beginning with a short prefermentation phase at 30 ◦ C (Bertilsson et al., 2009). The pH was maintained at 5.0 throughout the fermentation by automatic addition of 3 M NaOH and the stirring speed was kept at 400 rpm. The SSCF medium was supplemented with 0.5 g L−1 NH4 H2 PO4 , 0.025 g L−1 MgSO4 × 7H2 O and 1.0 g L−1 yeast extract. A yeast concentration of 3 g dry weight L−1 was used. The enzyme preparations used were same as in the hydrolysis experiments. The total amount
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of enzyme added was same for all the SSCF experiments and corresponded to a cellulase activity of 30 FPU (g glucan)−1 , and a total -glucosidase activity of 60 IU g−1 glucan. Peristaltic pumps connected to a computer with control software (LabView® , National Instruments) were used for the enzyme addition. The enzyme solutions were stored in a refrigerator (4 ◦ C) until just before the pump feed was started. The enzyme solution was then fed from a sealed bottle through a sterile rubber hose into the reactor. Samples for HPLC-analysis were taken repeatedly throughout the SSCF. 2.5. Sugar uptake rate experiments In order to be able to measure sugar uptake rates in real hydrolyzates, an experiment was designed which mimicked an SSCF experiment, but without enzymes added. Instead a concentrated glucose solution (500 g L−1 ) was used to simulate glucose release. The experiments were carried out on both whole slurry and hydrolyzate liquid. The conditions and concentrations were identical to the SSCF experiments. 2.6. Analysis Cell mass concentrations were measured in duplicates from 10 mL samples. The samples were centrifuged (1000 × g) for 5 min at 3000 rpm (Z200 A, HERMLE Labortechnik, Wehingen, Germany). The supernatants were discarded, and the pellets were washed with 0.9% NaCl solution and centrifuged a second time. The pellets were dried at 105 ◦ C overnight and subsequently weighed. HPLC was used for analysis of the metabolites and substrates. Samples of the fermentation liquid were centrifuged (16,000 × g) in 2 mL eppendorf tubes at 14,000 rpm for 5 min (Z 160 M, HERMLE Labortechnik, Wehingen, Germany). The supernatant was filtered using 0.2 m filters, and the filtered samples were stored at −20 ◦ C. The sugar concentrations were determined using a polymer column (Aminex HPX-87P, Bio-Rad Laboratories, München, Germany) at 85 ◦ C. MilliQ-water was used as eluent, with a flow rate of 0.6 mL min−1 . Ethanol, glycerol, acetate, HMF and furfural were analyzed using an Aminex HPX-87H column (Bio-Rad Laboratories, München, Germany) at 60 ◦ C. The eluent was 5 mM H2 SO4 with a flow rate of 0.6 mL min−1 . The compounds of interest were detected with a refractive index detector (Waters 2410, Waters, Milford, MA, USA) or with a UV detector at a wavelength of 210 nm (Waters 2487, Waters, Milford, MA, USA). 2.7. Yield calculations The ethanol yield, YSE , was calculated based on the total amount of fermentable sugars added to the SSCF, i.e. the sum of available glucose, mannose, galactose and xylose present in the pretreatment slurry, including monomers, oligomers and polymers (glucan fibers). The theoretical mass of glucose released during the hydrolysis was (due to the addition of water) 1.11 times the mass of glucan. Based on the maximum ethanol yield of 0.51 g g−1 (yield on hexoses), the conversion efficiency, Y*SE, was calculated as (YSE /0.51) × 100. 2.8. Theory – modeling the SSCF Due to the complexity of an SSCF process, it is difficult to apply models based on pure enzyme kinetics. Hence, a simplified empirical hydrolysis model was created. In this model, the rate of hydrolysis, vhydrolysis , was assumed to be a function of enzyme concentration (E), glucose concentration (Cglu ), inhibitors present in the hydrolyzate (), and the fractional degradation of the glucan
(˛), i.e.:
vhydrolysis = f (E, Cglu , , ˛)
(1)
The influence of these parameters was investigated by performing hydrolysis experiments using spruce substrate. First, the impact of different enzyme loads on the glucose release rate was studied. This was done by testing different enzyme additions (10, 20 and 40 FPU g−1 ) in hydrolysis experiments with washed fibers. Subsequently, the product inhibition by glucose was investigated by measuring the initial rate of glucose release from washed fibers at several different initial glucose concentrations: 0, 1, 3, 7 and 14 g L−1 , respectively. Two different enzyme loads were used in these tests; 20 and 40 FPU g−1 glucan, respectively. To compensate for inhibitory compounds that may affect the enzymes, an inhibition factor () correcting for difference in sugar release rate between whole slurry and washed fibers with the same glucose concentration in the liquid, was included in the model. Finally, the time dependence of the glucose release rate was considered by including the factor ˛, representing the fractional degradation. This correlation was found from hydrolysis experiment, where the initial release rate was determined for freshly washed fibers at different time points, i.e. at different stages of degradation. The different factors considered in the model were assumed to be independent and multiplicative. The sugar uptake rate of the yeast must be obtained under process relevant conditions. For this reason, model SSCF experiments were carried out (as described above) from which the uptake rate of glucose in the spruce hydrolyzate could be calculated. Finally, by combining the information of the glucose uptake rate and the glucose release rate, the glucose concentration in the SSCF could be calculated for different enzyme feed profiles and a suitable profile could be selected. Calculations and data fitting were made in MATLAB® R2008b (The MathWorks, Inc.). 3. Results 3.1. Obtaining data for modeling the SSCF process The aim of the experimental work was to improve the xylose uptake in an SSCF process through controlling the glucose concentration by adjusting the rate at which cellulase is added to the fermentation. The overall strategy was to maintain the glucose concentration at a low but none-zero concentration by controlling the addition of enzymes. For this purpose, a sufficiently robust model describing the glucose release rate as a function of known process variables was needed. The basis for this model was measurements of yeast glucose uptake rates and kinetics of glucose release rate in the hydrolysis of spruce fibers. 3.1.1. Sugar uptake Initially, the impact of continuous glucose addition per se on xylose uptake was investigated. In one bioreactor a glucose feed (500 g L−1 ) was employed in pretreated hydrolyzate liquid without fibers to simulate enzymatic sugar release. In another, identical reactor, no glucose was fed. The results shown in Fig. 1 indeed demonstrate that the presence of glucose (at low concentrations) enhances xylose utilization in pretreated hydrolyzate of spruce. To obtain appropriate data for the glucose uptake rate of the yeast, experiments were made using pretreated spruce hydrolyzate including the fibers. The sugar release was obtained by feeding glucose (using a solution of 500 g L−1 ) to the bioreactor instead of adding enzymes in these experiments. No cell growth takes place and the same cell density, 3 g L−1 , as in the subsequent SSCF experiments was used. By varying the glucose feed rate and observing the glucose concentration in the reactor, the maximum sugar uptake
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Fig. 3. Normalized rate of glucose release as a function of the glucose concentration. Polynomial function: y = 3 × 10−5 x4 − 0.0013x3 + 0.0257x2 − 0.2189x + 0.9999. Polynomial fit: r2 = 0.9956.
to increase with increasing glucose concentration and, as could be expected, the sugar release rate decreased rapidly with increasing glucose concentration up to around 7 g L−1 (Fig. 3). The experiments also showed that the spruce hydrolyzate did not affect the enzymes to any greater extent, i.e. the inhibition factor could be disregarded in the model leaving only three variables to be considered:
vhydrolysis = f (E, Cglu , ˛)
Fig. 1. Mimicked SSCF in spruce hydrolyzate liquid (no fibers) showing glucose (䊉) and xylose (). (A) No glucose is fed, resulting in very little xylose uptake. (B) A continuous glucose feed is employed, resulting in complete consumption of the xylose.
rate for the yeast in spruce hydrolyzate containing 8% WIS could be determined to 2.5 g glucose L−1 h−1 (results not shown). 3.1.2. Hydrolysis The sugar release rate was found to be proportional to the enzyme concentration in the interval 10–40 FPU (g glucan)−1 , in the experiments made (Fig. 2). The end-product inhibition was found
Fig. 2. Measured rate of glucose release as a function of enzyme load.
(2)
In the hydrolysis experiments, the relation between the glucose release rate and the fractional degradation (˛) was also studied (Fig. 4). As expected, the rate of hydrolysis at high ˛ values is clearly lower than for lower values. However, one should note that the glucose release rate in fact has a maximum around ˛ = 0.25, i.e. is it not highest in the very beginning. 3.2. SSCF experiments 3.2.1. Batch SSCF Standard batch SSCF with 8% WIS were carried out as a reference experiment. The experiments were carried out in duplicates (material 1) and triplicates (material 2) respectively, and the results are shown in Fig. 6A and Table 2.
Fig. 4. Measured rate of glucose release as a function of the fractional degradation (˛). The data was obtained from hydrolysis experiments of pretreated spruce using an enzyme load of 20 FPU (g glucan)−1 . In an SSCF a constant rate, based on the yeast’s uptake rate, is desired. Hence, the curve needs to be flattened (represented by the dotted line) in order to obtain an efficient SSCF.
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Table 2 Results from SSCF of spruce with 8% WIS at 34 ◦ C after 96 h. Raw material
Mode of operation
Glucose release rate (g L−1 h−1 )
Xylose consumptiona (%)
Xylose conc. (g L−1 )
Xylitol formationb (%)
Glycerol conc. (g L−1 )
Ethanol conc. (g L−1 )
Ethanol yield (g g−1 )
Ethanol yieldc (%)
Spruce 1 Spruce 1 Spruce 1 Spruce 1 Spruce 1 Spruce 1
Ref. batch Ref. batch Enz. feed Enz. feed Enz. feed Enz. feed
No enz. feed No enz. feed 1.4 2.0 2.5 2.0d
44 40 47 69 51 82
3.2 3.3 3.2 1.7 2.7 1.1
16 10 14 10 18 22
2.6 2.4 2.9 3.7 2.9 3.7
28.5 30.4 30.8 32.2 30.8 32.9
0.34 0.36 0.38 0.38 0.37 0.39
66 70 74 75 72 77
Spruce 2 Spruce 2 Spruce 2 Spruce 2 Spruce 2 Spruce 2
Ref. batch Ref. batch Ref. batch Enz. feed Enz. feed Enz. feed
No enz. feed No enz. feed No enz. feed 2.0d 2.0d 2.0d
35 61 59 81 83 85
3.5 2.1 2.3 1.0 0.9 0.8
26 23 24 18 19 20
2.8 3.6 3.6 3.5 3.3 3.6
35.5 35.5 35.3 33.4 34.9 35.2
0.40 0.41 0.40 0.38 0.40 0.40
79 79 79 75 78 78
In all experiments an enzyme load of 30 FPU (g glucan)−1 and a yeast load of 3 g L−1 were used. a Related to total amount of available xylose. b Related to consumed xylose. c Corresponding to the maximum theoretical yield on total available sugars. d Slightly modified feed profile (see text).
3.2.2. SSCF with controlled enzyme feed A polynomial mathematical model for the rate of hydrolysis during SSCF was created based on the glucose release rate from the hydrolysis experiments (Eq. (3)).
vhydrolysis = E × (−2.19˛2 + 1.18˛ + 0.28) × (3 × 10−5 Cglu 4 − 0.0013Cglu 3 + 0.026Cglu 2 − 0.22Cglu + 1.00)
(3)
where ˛=
vSSCF × t × V glucantot × 1.11
(4)
By inserting values for the desired glucose concentration in the SSCF (Cglu ) and the desired glucose release rate in the SSCF (vSSCF ) in Eqs. (3) and (4), the glucose release rate as a function of time could be calculated. Subsequently, the desired amount of enzyme at any time (and thereby indirectly the addition profile) could be calculated from: menz ∝
E
vhydrolysis
× vSSCF × glucantot
(5)
A constant release rate of glucose, slightly less than the maximum uptake rate for the yeast, was expected to be preferable in the SSCF in order to improve the xylose uptake. A hypothetically desired hydrolysis rate is given by the dotted line in Fig. 4. When the rate of hydrolysis slows down due to the higher degree of degradation (a higher value of ˛), more enzymes are needed. By controlling the amount of enzymes added it should in principle be possible to maintain the desired glucose concentration. The actual feed profile was kept as close as possible to the profile given by the model, however, with a slight simplification (cf. Fig. 5). Since removal of enzyme from the reactor is generally not feasible, an amount that is slightly too low was added initially (t0 ). From time t1 it was possible to follow the curve again, and a constant enzyme feed was initiated. Finally, the feed rate was increased (t2 ), and kept until the total amount of enzyme specified had been added (t3 ). The SSCF experiments were started with a prefermentation phase (Bertilsson et al., 2009), i.e. no enzymes were added initially in order to decrease the glucose concentration. After the glucose concentration had decreased sufficiently (time I, Figs. 6 and 7), enzymes were added according to the model. At time II the enzyme feed was started, and at time III the feed rate was increased (Figs. 6 and 7). Fig. 6 shows measured concentration profiles during a standard batch SSCF as well as three SSCF experiments conducted with spruce material 1 according to the procedure described above.
Obtained yields and xylose conversion are shown in Table 2. A glucose release rate of 2.0 g L−1 h−1 resulted in the highest total xylose uptake; 69% (Fig. 6C). A feed profile corresponding to a desired glucose release rate of 1.4 g L−1 h−1 (Fig. 6B) resulted in a xylose consumption of 47%, whereas at a glucose release rate of 2.5 g L−1 h−1 (Fig. 6D) the total xylose consumption was 51% of the theoretical. These findings indicate that a glucose release rate around 2.0 g L−1 h−1 , i.e. somewhat lower than the maximum glucose uptake rate determined in the spruce hydrolyzate, is optimal with respect to the xylose conversion for this process. In order to increase the xylose uptake further, a slight modification of the best case, i.e. a release rate of 2.0 g L−1 h−1 was carried out (Table 2). By changing the addition times II and III marginally, an even flatter glucose concentration profile could be achieved, resulting in a xylose consumption as high as 82%. The final ethanol concentrations and the corresponding ethanol yields are shown in Table 2. A release rate of 2.0 g L−1 h−1 proved to give the highest ethanol yield; 0.39 g g−1 of total sugars (i.e. glucose, xylose, mannose, galactose, glucan and mannan) for material 1. This corresponds to 77% of the maximum theoretical yield. This glucose release rate also resulted in the highest final ethanol concentration.
Fig. 5. Qualitative figure of the theoretically desired (solid line) and actual (dotted line) amount of enzyme in the SSCF. The shape of the curve will change with different glucose uptake rates. However, the total amount of enzymes added was the same for all the different SSCF experiments, i.e. 30 FPU (g glucan)−1 . The actual feed profile (dotted line) was always kept as close as possible to the profile given by the model.
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Fig. 6. Measured concentrations during SSCF of spruce (material 1) with 8% WIS showing glucose (䊉), xylose () and ethanol () for batch (A) and enzyme feed using a model for a glucose release rate of 1.4 g L−1 h−1 (B), 2.0 g L−1 h−1 (C) and 2.5 g L−1 h−1 (D). I, first enzyme addition; II, start of enzyme feed; III, increased enzyme feed rate.
Fig. 7. Measured concentrations during triplicate SSCF of spruce (material 2) with 8% WIS showing glucose (䊉), xylose () and ethanol () when using a model for a glucose release rate of 2.0 g L−1 h−1 with a slightly modified feed profile (see text). I, first enzyme addition; II, start of enzyme feed; III, increased enzyme feed rate.
In order to verify the reproducibility of the SSCF experiments a second spruce material was prepared, using the same pretreatment conditions (material 2, Table 1). Triplicate reference batch SSCF experiments, as well as triplicate SSCF experiments using the best enzyme feed profile, were performed (Table 2). As the results show (Fig. 7), the reproducibility was very good, which confirms that the feeding protocol is robust. Also with the second material, the enzyme feed improved xylose utilization significantly, although the ethanol yields showed no difference between the batch SSCF and the SSCF with enzyme feeding. All SSCF experiments carried out with enzyme feed resulted in a higher xylose consumption, compared to the respective reference batch SSCF (Table 2). 4. Discussion Given the high ratio of glucose to xylose in the spruce material, co-fermentation of xylose and glucose is a challenging task. SSCF
is an interesting process option, since it allows a slow constant release of glucose throughout the process which is beneficial for xylose uptake by xylose fermenting strains of S. cerevisiae (Öhgren et al., 2006; Olofsson et al., 2008). However, the glucose concentration should be kept low, i.e. the release rate during the process should be tuned to the consumption rate in order to increase the xylose uptake. In the current study, a simple and robust kinetic model for simultaneous saccharification and co-fermentation of spruce was developed in order to allow an open-loop control of sugar concentration, by making a programmed addition of enzyme feed during the process. The results showed a substantial increase in xylose consumption as a consequence of the controlled glucose release rate as afforded by enzyme feeding. The increased xylose consumption can mainly be explained by lower competitive glucose inhibition of the xylose uptake, made possible by the control of glucose at low concentration throughout the process. By employing prefermentation, i.e. by first fermenting the glucose present in the medium before adding enzymes (Bertilsson et al., 2009), the competitive glucose inhibition could be reduced at an early stage. Furthermore, the glucose concentration was maintained at a relatively low level throughout the experiment. The highest xylose consumption was achieved with an enzyme feed corresponding to a glucose release rate of 2.0 g L−1 h−1 . This glucose release rate was slightly lower than the maximum glucose uptake rate determined for the yeast strain used in the spruce hydrolyzate. This very nicely agrees with the competitive uptake system and the beneficial effects of a concomitant glucose and xylose uptake. The glucose must not accumulate in the system, and there should also be some spare transport capacity for xylose uptake, i.e. one should expect the optimal release rate to be somewhat below the maximum value. The simple kinetic model developed in this work proved to be a useful tool, capable of predicting the glucose release and uptake rate for the system and the particular raw material used at a sufficient accuracy for the intended purpose, i.e. calculation of the enzyme feed. However, the model needs to be recalibrated for use in a different SSCF process or a different raw material. Pri-
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marily, new sugar uptake and/or release rates will have to be measured. As the results show, the feed profile is dependent on a correct uptake rate of glucose since the shape of the feed profile (Fig. 5) will change with the desired glucose release rate. Hence, it is important to use a glucose uptake rate in the model that is somewhat below the maximum uptake rate of the yeast. The results obtained in SSCF (Figs. 6 and 7) correlated in principle well with what could be expected from the model, even though the feed profile could not be completely mimicked. However, as expected from the model, the best results were obtained using a glucose release rate of 2.0 g L−1 h−1 (Figs. 6C and 7). When a lower glucose uptake rate than the optimal was used in the model, the model showed a low feed rate in the beginning, and forecasted a too fast addition rate closer to the end. This was observed during the experiment with a desired glucose release rate of 1.4 g L−1 h−1 . Evidently, there was a somewhat too small enzyme addition in the beginning and a too large enzyme addition towards the end of feeding, resulting in an increased glucose concentration (Fig. 6B). Consequently, when a higher glucose uptake rate was used as an input value in the hydrolysis model, a large enzyme addition in the beginning and a slow enzyme feed towards the end was predicted. This was seen in the experiment with a desired glucose release rate of 2.5 g L−1 h−1 . Here the glucose was probably released too fast in the beginning – even if the glucose concentration was kept low – and hence, only little glucose could be released from around 20 h and on (Fig. 6D). This resulted in lower xylose uptake from this time and on, since a sufficient glucose uptake is needed for xylose uptake. It should also be noticed that the first ∼40 h of SSCF are most important when it comes to increased xylose uptake. After that, the concentration profiles looks very similar to the reference batch SSCF. Clearly, this is the time period during which the feed should be carefully controlled during the SSCF. There is ample evidence that xylose fermentation in SSCF becomes more challenging with increasing WIS content (Öhgren et al., 2006; Olofsson et al., 2008; Rudolf et al., 2008) due to higher levels of glucose and inhibitors, as well as due to mixing problems. Both previous (Bertilsson et al., 2009) and present results show that control of the glucose concentration is a key for good xylose utilization. Also fed-batch addition of substrate, which previously has been used primarily due to other benefits, gives a positive effect on the xylose uptake due to a lower glucose concentration in the reactor (Öhgren et al., 2006; Olofsson et al., 2008). Previous studies for SSCF of spruce conducted in our laboratory (Bertilsson et al., 2009) resulted in a total ethanol yield of 86% and 80% on total sugars for 7% and 10% WIS respectively, using prefermentation. Despite generally higher xylose consumption in the present study, the total ethanol yield only reached 77% (material 1) and 79% (material 2) with 8% WIS. This is most likely due to differences in the pretreated material. Although good reproducibility has been shown for the SSCF experiments, the variation in pretreatment is somewhat larger. Furthermore, a comparison of experiments with different WIS contents is often difficult. A study conducted on pretreated spruce material at 8% WIS, resulted in a ethanol yield of 86% of the theoretical (Alkasrawi et al., 2006). However, this number was based only on hexoses, and another yeast strain (commercial Baker’s yeast) at a much higher loading (5 g L−1 ) was used. Due to the relatively low xylose content in spruce, the increased xylose consumption resulted in no, or only a slight, increase of the ethanol yield. The insignificant difference in ethanol yields, as well as the overall higher ethanol yield for the second material in comparison to the first material, could be explained by a slightly different outcome of the pretreatment, resulting in more sugars in the liquid as well as a higher degree of more accessible fibers for the cellulases. The two batches of pretreated material are thus slightly different.
Most likely, the impact of xylose consumption on the ethanol yield would be higher with a more xylose rich substrate. Furthermore, spruce is considered to be a material which is not very easy to ferment by yeast. In xylose rich materials, e.g. wheat straw or sugar cane bagasse, there is a more favorable ratio of xylose and glucose, which should facilitate xylose consumption. On the other hand, there are indications that the residual xylose after fermentation is higher for these feedstocks (Öhgren et al., 2006; Olofsson et al., 2008). Consequently, there is a need for improved process design also in this case. There may also be possible advantage of using an enzyme feed also for cases in which only hexose fermentation takes place. By keeping the glucose concentration low throughout the SSF, product inhibition of the enzymatic hydrolysis can possibly be reduced and therefore a faster hydrolysis may occur. In conclusion, our study has shown that xylose conversion in SSCF can be significantly improved by employing an enzyme feed during the process. A simple model for glucose release and uptake, calibrated at process conditions, proved to be useful for calculating an enzyme feed profile that provides a constant glucose release. By employing this feed profile, the total xylose consumption could be as much as doubled in comparison to a batch SSCF. This approach will most likely be useful also for more xylose rich materials, for which improvements of the overall ethanol yields will result. Abbreviations ˛ fractional degradation of the fibers (0 ≤ ˛ ≥ 1, 1 = complete degradation) Cglu glucose concentration in liquid (g L−1 ) E enzyme concentration (FPU (g glucan)−1 ) menz amount of enzyme (g) FPU filter paper units inhibition factor (in this work = 1) t time (h) glucantot initial total amount glucan in fibers (g) vhydrolysis glucose release rate due to hydrolysis (g L−1 h−1 ) vSSCF desired glucose release rate in SSCF (g L−1 h−1 ) V volume (L) WIS water insoluble solids Acknowledgements This work was financed by the European Commission through the EU Project “New Improvements for Lignocellulosic Ethanol” (NILE, FP6, EU contract No. 019882) and the Swedish Energy Agency. References Alkasrawi, M., Rudolf, A., Lidén, G., 2006. Influence of strain and cultivation procedure on the performance of simultaneous saccharification and fermentation of steam pretreated spruce. Enzyme Microb. Technol. 38, 279–287. Almeida, J.R.M., Modig, T., Petersson, A., Hahn-Hagerdal, B., Lidén, G., GorwaGrauslund, M.F., 2007. Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J. Chem. Technol. Biotechnol. 82, 340–349. Bertilsson, M., Andersson, J., Lidén, G., 2008. Modeling simultaneous glucose and xylose uptake in Saccharomyces cerevisiae from kinetics and gene expression of sugar transporters. Bioprocess Biosyst. Eng. 31, 369–377. Bertilsson, M., Olofsson, K., Lidén, G., 2009. Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce. Biotechnol. Biofuels 2, 1–10. Boles, E., Müller, S., Zimmermann, F.K., 1996. A multi-layered sensory system controls yeast glycolytic gene expression. Mol. Microbiol. 19, 641–642. Eliasson, A., Christensson, C., Wahlbom, C.F., Hahn-Hägerdal, B., 2000. Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures. Appl. Environ. Microbiol. 66, 3381–3386. Hahn-Hägerdal, B., Karhumaa, K., Fonseca, C., Spencer-Martins, I., Gorwa-Grauslund, M., 2007. Towards industrial pentose-fermenting yeast strains. Appl. Microbiol. Biotechnol. 74, 937–953.
K. Olofsson et al. / Journal of Biotechnology 145 (2010) 168–175 Jeffries, T.W., Jin, Y.S., 2004. Metabolic engineering for improved fermentation of pentoses by yeasts. Appl. Microbiol. Biotechnol. 63, 495–509. Karhumaa, K., Hahn-Hägerdal, B., Gorwa-Grauslund, M.F., 2005. Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering. Yeast 22, 359–368. Kilian, S.G., Uden, N., 1988. Transport of xylose and glucose in the xylose-fermenting yeast Pichia stipitis. Appl. Microbiol. Biotechnol. 27, 545–548. Kuyper, M., Hartog, M.M.P., Toirkens, M.J., Almering, M.J.H., Winkler, A.A., van Dijken, J.P., Pronk, J.T., 2005. Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Res. 5, 399–409. Kötter, P., Amore, R., Hollenberg, C.P., Ciriacy, M., 1990. Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant. Curr. Genet. 18, 493–500. Kötter, P., Ciriacy, M., 1993. Xylose fermentation by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 38, 776–783. Meinander, N.Q., Boels, I., Hahn-Hägerdal, B., 1999. Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without overexpression of TAL1. Bioresour. Technol. 68, 79–87. Meinander, N.Q., Hahn-Hägerdal, B., 1997. Influence of cosubstrate concentration on xylose conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: a comparison of different sugars and ethanol as cosubstrates. Appl. Environ. Microbiol. 63, 1959–1964. Olofsson, K., Rudolf, A., Lidén, G., 2008. Designing simultaneous saccharification and fermentation for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae. J. Biotechnol. 134, 112–120. Öhgren, K., Bengtsson, O., Gorwa-Grauslund, M.F., Galbe, M., Hahn-Hägerdal, B., Zacchi, G., 2006. Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400. J. Biotechnol. 126, 488–498. Palmqvist, E., Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 74, 25–33.
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Palmqvist, E., Hahn-Hägerdal, B., Galbe, M., Larsson, M., Stenberg, K., Szengyel, Z., Tengborg, C., Zacchi, G., 1996. Design and operation of a bench-scale process development unit for the production of ethanol from lignocellulosics. Bioresour. Technol. 58, 171–179. Pitkänen, J.-P., Aristidou, A., Salusjärvi, L., Ruohonen, L., Penttilä, M., 2003. Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture. Metab. Eng. 5, 16–31. Rudolf, A., Baudel, H., Zacchi, G., Hahn-Hägerdal, B., Lidén, G., 2008. Simultaneous saccharification and fermentation of steam-pretreated bagasse using Saccharomyces cerevisiae TMB3400 and Pichia stipitis CBS6054. Biotechnol. Bioeng. 99, 783–790. Ruiz, R., Ehrman, T., 1996. Dilute Acid Hydrolysis Procedure for Determination of Total Sugars in the Liquid Fraction of Process Samples. NREL, Golden, CO. Sassner, P., Galbe, M., Zacchi, G., 2007. Techno-economic evaluation of bioethanol production from three different lignocellulosic materials. Biomass Bioenerg. 32, 422–430. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Tempelton, D., 2004. Determination of Structural Carbohydrates and Lignin in Biomass. NREL, Golden, CO. Taherzadeh, M.J., Lidén, G., Gustafsson, L., Niklasson, C., 1996. The effects of pantothenate deficiency and acetate addition on anaerobic batch fermentation of glucose by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 46, 176– 182. Takagi, M., Abe, S., Suzuki, S., Emert, G.H., Yata, N., 1977. A method for production of alcohol directly from cellulose using cellulase and yeast. In: Bioconversion Symposium, New Delhi, India, pp. 551–571. von Sivers, M., Zacchi, G., 1996. Ethanol from lignocellulosics: a review of the economy. Bioresour. Technol. 56, 131–140. Wahlbom, C.F., van Zyl, W.H., Jonsson, L.J., Hahn-Hägerdal, B., Otero, R.R.C., 2003. Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054. FEMS Yeast Res. 3, 319–326. Wingren, A., Galbe, M., Zacchi, G., 2003. Techno-economic evaluation of producing ethanol from softwood: comparison of SSF and SHF and identification of bottlenecks. Biotechnol. Progr. 19, 1109–1117.
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Controlled feeding of cellulases improves conversion of xylose in simultaneous saccharification and co-fermentation for bioethanol production Kim Olofsson ∗ , Magnus Wiman, Gunnar Lidén Department of Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden
a r t i c l e
i n f o
Article history: Received 6 April 2009 Received in revised form 28 September 2009 Accepted 1 November 2009 Keywords: SSF SSCF Co-fermentation Modeling Enzymatic hydrolysis Saccharomyces cerevisiae
a b s t r a c t Simultaneous saccharification and fermentation (SSF) is an interesting option for ethanol production from lignocellulosic materials. To meet desired overall yields during ethanol production from lignocellulosic materials, it is important to use both hexoses and pentoses. This can be achieved by co-fermentation of sugars in SSF, so called SSCF (simultaneous saccharification and co-fermentation), using genetically modified yeast strains. However, high concentration of glucose in the pretreated material makes xylose utilization challenging due to competitive inhibition of sugar transport. The present work demonstrates a new approach for controlling the glucose release rate from the enzymatic hydrolysis by controlling the addition of enzymes in SSCF using spruce as the raw material. Enzyme kinetics and yeast sugar uptake rates for a recombinant xylose utilizing strain of Saccharomyces cerevisiae, TMB3400, were determined in a real hydrolyzate medium. A simplified model for glucose release and uptake was created, to be used as a tool for control of the glucose concentration in a SSCF process. With help of this model, an SSCF process with efficient co-utilization of glucose and xylose was successfully designed. The results showed that the total xylose uptake could be increased from 40% to as much as 80% by controlling the enzyme feed. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Simultaneous saccharification and fermentation (SSF) (Takagi et al., 1977) has been demonstrated to be an advantageous option for ethanol production from lignocellulosic materials (Wingren et al., 2003). Residues from, e.g. forest industry and agriculture are today potential interesting feedstocks. However, a high final ethanol concentration and a high overall ethanol yield are crucial factors for the process economy (von Sivers and Zacchi, 1996). To meet desired overall yields during ethanol production from lignocellulosic materials, it is important to use both hexoses and pentoses. Saccharomyces cerevisiae, the most commonly used organism for industrial ethanol production, does not naturally ferment pentoses, but genetically modified S. cerevisiae strains with xylose fermenting abilities are now available (Jeffries and Jin, 2004; Hahn-Hägerdal et al., 2007). Strains have been designed principally by introduction of heterologous genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from fungi (Kötter et al., 1990; Eliasson et al., 2000) or genes encoding xylose isomerase (XI) from bacteria and fungi (Karhumaa et al., 2005; Kuyper et al., 2005). Xylose flux is further enhanced by over-expression of xylulose kinase and selected other genes associated with the PPP.
∗ Corresponding author. Tel.: +46 46 2220863; fax: +46 46 149156. E-mail address: [email protected] (K. Olofsson). 0168-1656/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2009.11.001
The glucose concentration must be low in order to obtain efficient xylose uptake with S. cerevisiae, since xylose transport into the cell is inhibited by glucose. The main reasons for this are that xylose and glucose compete for the same transport systems (Kilian and Uden, 1988; Meinander and Hahn-Hägerdal, 1997) and the affinity for xylose is approximately 200-fold lower than for glucose (Kötter and Ciriacy, 1993). However, glucose has also shown to enhance xylose utilization at low but non-zero concentrations (Meinander et al., 1999; Pitkänen et al., 2003), which can be attributed to induction of transporter systems (Pitkänen et al., 2003; Bertilsson et al., 2008), induction of glycolytic enzymes (Boles et al., 1996) and improved co-factor generation (Pitkänen et al., 2003). The positive effects of glucose (in low concentrations) on xylose uptake make SSF an interesting process concept for co-fermentation, since the glucose released to the medium will be simultaneously fermented. Additionally, after pretreatment of the lignocellulosic material, the hemicellulosic sugars in spruce, mainly mannose, glucose, and xylose, are present as monomers, while a large fraction of the glucose content is present as glucan in the fibers. In spruce, a potential feedstock that grows in the far northern hemisphere, the proportion of xylose is relatively low. Still, complete conversion of xylose would increase the ethanol yield by as much as 8% (Sassner et al., 2007), which is significant for an industrial process. The xylose utilizing strain of S. cerevisiae used in this work, TMB3400 (Wahlbom et al., 2003), has previously been used for SSF of different xylose rich lignocellulosic materials from the agriculture with satisfactory results (Öhgren et al., 2006; Olofsson
K. Olofsson et al. / Journal of Biotechnology 145 (2010) 168–175 Table 1 Composition of the pretreated spruce material. Content in solid fraction (% of WIS)
Glucan Mannan Galactan Xylan Lignin
a
Material 1
Material 2
54.6 1.1 ∼0 ∼0 39.3
50.4 1.3 ∼0 ∼0 41.1
Content in liquid fraction (g L−1 )
Glucosea Mannosea Galactosea Xylosea Furfural HMF Acetic acid
Material 1
Material 2
19.8 33.9 5.9 12.4 1.1 3.5 5.9
28 26.2 4.6 9.1 1.8 2.3 6.4
Both monomeric and oligomeric form is included.
et al., 2008; Rudolf et al., 2008). The highest xylose utilization previously achieved was about 40%, and a total ethanol yield, based on all available sugars, was approximately 80% (Olofsson et al., 2008). However, in spruce the low ratio between xylose and glucose makes xylose conversion particularly challenging. Furthermore, due to the relatively severe pretreatment conditions, the fermentability is expected to be relatively low in the spruce hydrolyzate (Palmqvist and Hahn-Hägerdal, 2000). This is due to the fact that during acid pretreatment, some of the sugars are degraded to furaldehydes, organic acids, and other compounds which are acting as inhibitors for the yeast (Almeida et al., 2007). A suitable process design for simultaneous saccharification and co-fermentation (SSCF) is therefore needed. Recently, a first step toward increasing the xylose uptake was taken by lowering the initial glucose concentration in the SSCF using a scheme termed prefermentation (Bertilsson et al., 2009). However, although the initial glucose concentration was indeed decreased, resulting in improved xylose utilization, the glucose was not maintained at sufficiently low levels throughout the fermentation. In the present work a new approach for controlling the glucose release rate from the enzymatic hydrolysis is made by using a model-based addition of enzyme throughout the SSCF. Initially, studies of enzyme kinetics and yeast sugar uptake rates were carried out on pretreated spruce material in order to design and calibrate a simple and robust model for sugar release and sugar uptake in the SSCF process. Subsequently, enzyme feed profiles for the SSCF process were designed and experimentally tested using spruce hydrolyzate. 2. Material and methods 2.1. Raw material and pretreatment Two different spruce materials were used and pretreated separately, but with the same pretreatment conditions. The wood chips (with 50% and 60% w/w moisture respectively) were impregnated in closed plastic bags for 20 min with SO2 (2.5% w/w moisture). Subsequently, the impregnated chips were steam-pretreated batch-wise at 210 ◦ C for 5 min in a 10L reactor. Further description of the equipment is given by Palmqvist et al. (1996). The pretreated material was stored at 4 ◦ C. The composition of the two pretreatment slurries is shown in Table 1. The water insoluble and liquid fractions were analyzed using NREL (National Renewable Energy Laboratories) standard procedures (Ruiz and Ehrman, 1996; Sluiter et al., 2004). The WIS content of the pretreated slurry was 15.1% for material 1 and 12.4% for material 2. 2.2. Cell cultivation The recombinant xylose fermenting strain S. cerevisiae TMB3400 (Wahlbom et al., 2003) was used in all SSCF experiments. Yeast cells to be used in SSCF was produced by aerobic batch cultiva-
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tion on glucose, followed by an aerobic fed-batch cultivation on spruce hydrolyzate liquid, in order to improve inhibitor tolerance by adaptation as previously shown by Alkasrawi et al. (2006). The yeast was inoculated in 300 ml flasks containing 100 ml media supplemented with 16.5 g L−1 glucose, 7.5 g L−1 (NH4 )2 SO4 , 3.5 g L−1 KH2 PO4 , 0.74 g L−1 MgSO2 ·7H2 O, trace metals and vitamins. The cells were grown for 24 h at 30 ◦ C and pH 5 in a rotary shaker at 180 rpm. Aerobic batch cultivations were performed in a 2.5 L bioreactor (Biostat A, B. Braun Biotech International, Melsungen, Germany) at 30 ◦ C. The working volume was 0.7 L and the medium contained 20.0 g L−1 glucose, 20.0 g L−1 (NH4 )2 SO4 , 10.0 g L−1 KH2 PO4 , 2.0 g L−1 MgSO4 , 27.0 mL L−1 trace metal solution and 2.7 mL L−1 vitamin solution. The pH was maintained at 5.0 throughout the cultivation, by automatic addition of 3 M NaOH. The trace metal and vitamin solutions were prepared according to Taherzadeh et al. (1996). The cultivation was initiated by adding 20.0 mL of the inoculum to the bioreactor. Aeration was maintained at 1.0 L min−1 and the stirrer speed was kept at 800 rpm. Upon depletion of the ethanol produced in the batch phase, the feeding of pretreatment liquid from spruce was initiated. 1.0 L of pretreatment liquid was added with an initial feed rate of 0.04 L h−1 that was increased linearly to 0.10 L h−1 during 16 h of cultivation. The aeration during the fed-batch phase was maintained at 1.4 L min−1 and the stirrer speed was kept at 800 rpm. After cultivation, the cells were harvested by centrifugation in 700 mL flasks for 8 min at 3000 rpm using a HERMLE Z 513K centrifuge (HERMLE Labortechnik, Wehingen, Germany). The pellets were resuspended in 0.9% NaCl solution in order to obtain a cell suspension with a cell mass concentration of 75 g dry weight L−1 . The time between cell harvest and initiation of the SSCF was no longer than 3 h. 2.3. Enzymatic hydrolysis Enzymatic hydrolysis was carried out in 600 mL flasks equipped with stirrers, which were placed in a temperature controlled water bath at 34 ◦ C. The flasks were filled with 300 mL slurry of pretreated spruce at a WIS content of 8%. To obtain the desired WIS content, the pretreatment slurry was diluted with sterile deionized water. Some of the experiments were performed without the hydrolyzate liquid, i.e. using only washed fibers in water. All hydrolysis experiments were performed in duplicates, showing good reproducibility. The enzyme preparation used, Celluclast, was provided by Novozymes A/S, Bagsvaerd, Denmark, and had a cellulase activity of 42 FPU g−1 and a -glucosidase activity of 20 IU g−1 . This was used together with Novozyme 188 (Novozymes A/S, Bagsvaerd, Denmark) with a -glucosidase activity of 340 IU g−1 . 2.4. Simultaneous saccharification and co-fermentation (SSCF) All SSCF experiments were carried out in 2.5 L bioreactors (Biostat A, B. Braun Biotech International, Melsungen, Germany; Biostat A plus, Sartorius, Melsungen, Germany and BIOFLO III, New Brunswick Scientific, Edison, NJ, USA) with a final working weight of 1.4 kg. The batch experiments were carried out with a WIS content of 8%. To obtain the desired WIS content, the pretreatment slurry was diluted with sterile deionized water. All SSCF experiments were carried out at 34 ◦ C for 96 h, beginning with a short prefermentation phase at 30 ◦ C (Bertilsson et al., 2009). The pH was maintained at 5.0 throughout the fermentation by automatic addition of 3 M NaOH and the stirring speed was kept at 400 rpm. The SSCF medium was supplemented with 0.5 g L−1 NH4 H2 PO4 , 0.025 g L−1 MgSO4 × 7H2 O and 1.0 g L−1 yeast extract. A yeast concentration of 3 g dry weight L−1 was used. The enzyme preparations used were same as in the hydrolysis experiments. The total amount
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of enzyme added was same for all the SSCF experiments and corresponded to a cellulase activity of 30 FPU (g glucan)−1 , and a total -glucosidase activity of 60 IU g−1 glucan. Peristaltic pumps connected to a computer with control software (LabView® , National Instruments) were used for the enzyme addition. The enzyme solutions were stored in a refrigerator (4 ◦ C) until just before the pump feed was started. The enzyme solution was then fed from a sealed bottle through a sterile rubber hose into the reactor. Samples for HPLC-analysis were taken repeatedly throughout the SSCF. 2.5. Sugar uptake rate experiments In order to be able to measure sugar uptake rates in real hydrolyzates, an experiment was designed which mimicked an SSCF experiment, but without enzymes added. Instead a concentrated glucose solution (500 g L−1 ) was used to simulate glucose release. The experiments were carried out on both whole slurry and hydrolyzate liquid. The conditions and concentrations were identical to the SSCF experiments. 2.6. Analysis Cell mass concentrations were measured in duplicates from 10 mL samples. The samples were centrifuged (1000 × g) for 5 min at 3000 rpm (Z200 A, HERMLE Labortechnik, Wehingen, Germany). The supernatants were discarded, and the pellets were washed with 0.9% NaCl solution and centrifuged a second time. The pellets were dried at 105 ◦ C overnight and subsequently weighed. HPLC was used for analysis of the metabolites and substrates. Samples of the fermentation liquid were centrifuged (16,000 × g) in 2 mL eppendorf tubes at 14,000 rpm for 5 min (Z 160 M, HERMLE Labortechnik, Wehingen, Germany). The supernatant was filtered using 0.2 m filters, and the filtered samples were stored at −20 ◦ C. The sugar concentrations were determined using a polymer column (Aminex HPX-87P, Bio-Rad Laboratories, München, Germany) at 85 ◦ C. MilliQ-water was used as eluent, with a flow rate of 0.6 mL min−1 . Ethanol, glycerol, acetate, HMF and furfural were analyzed using an Aminex HPX-87H column (Bio-Rad Laboratories, München, Germany) at 60 ◦ C. The eluent was 5 mM H2 SO4 with a flow rate of 0.6 mL min−1 . The compounds of interest were detected with a refractive index detector (Waters 2410, Waters, Milford, MA, USA) or with a UV detector at a wavelength of 210 nm (Waters 2487, Waters, Milford, MA, USA). 2.7. Yield calculations The ethanol yield, YSE , was calculated based on the total amount of fermentable sugars added to the SSCF, i.e. the sum of available glucose, mannose, galactose and xylose present in the pretreatment slurry, including monomers, oligomers and polymers (glucan fibers). The theoretical mass of glucose released during the hydrolysis was (due to the addition of water) 1.11 times the mass of glucan. Based on the maximum ethanol yield of 0.51 g g−1 (yield on hexoses), the conversion efficiency, Y*SE, was calculated as (YSE /0.51) × 100. 2.8. Theory – modeling the SSCF Due to the complexity of an SSCF process, it is difficult to apply models based on pure enzyme kinetics. Hence, a simplified empirical hydrolysis model was created. In this model, the rate of hydrolysis, vhydrolysis , was assumed to be a function of enzyme concentration (E), glucose concentration (Cglu ), inhibitors present in the hydrolyzate (), and the fractional degradation of the glucan
(˛), i.e.:
vhydrolysis = f (E, Cglu , , ˛)
(1)
The influence of these parameters was investigated by performing hydrolysis experiments using spruce substrate. First, the impact of different enzyme loads on the glucose release rate was studied. This was done by testing different enzyme additions (10, 20 and 40 FPU g−1 ) in hydrolysis experiments with washed fibers. Subsequently, the product inhibition by glucose was investigated by measuring the initial rate of glucose release from washed fibers at several different initial glucose concentrations: 0, 1, 3, 7 and 14 g L−1 , respectively. Two different enzyme loads were used in these tests; 20 and 40 FPU g−1 glucan, respectively. To compensate for inhibitory compounds that may affect the enzymes, an inhibition factor () correcting for difference in sugar release rate between whole slurry and washed fibers with the same glucose concentration in the liquid, was included in the model. Finally, the time dependence of the glucose release rate was considered by including the factor ˛, representing the fractional degradation. This correlation was found from hydrolysis experiment, where the initial release rate was determined for freshly washed fibers at different time points, i.e. at different stages of degradation. The different factors considered in the model were assumed to be independent and multiplicative. The sugar uptake rate of the yeast must be obtained under process relevant conditions. For this reason, model SSCF experiments were carried out (as described above) from which the uptake rate of glucose in the spruce hydrolyzate could be calculated. Finally, by combining the information of the glucose uptake rate and the glucose release rate, the glucose concentration in the SSCF could be calculated for different enzyme feed profiles and a suitable profile could be selected. Calculations and data fitting were made in MATLAB® R2008b (The MathWorks, Inc.). 3. Results 3.1. Obtaining data for modeling the SSCF process The aim of the experimental work was to improve the xylose uptake in an SSCF process through controlling the glucose concentration by adjusting the rate at which cellulase is added to the fermentation. The overall strategy was to maintain the glucose concentration at a low but none-zero concentration by controlling the addition of enzymes. For this purpose, a sufficiently robust model describing the glucose release rate as a function of known process variables was needed. The basis for this model was measurements of yeast glucose uptake rates and kinetics of glucose release rate in the hydrolysis of spruce fibers. 3.1.1. Sugar uptake Initially, the impact of continuous glucose addition per se on xylose uptake was investigated. In one bioreactor a glucose feed (500 g L−1 ) was employed in pretreated hydrolyzate liquid without fibers to simulate enzymatic sugar release. In another, identical reactor, no glucose was fed. The results shown in Fig. 1 indeed demonstrate that the presence of glucose (at low concentrations) enhances xylose utilization in pretreated hydrolyzate of spruce. To obtain appropriate data for the glucose uptake rate of the yeast, experiments were made using pretreated spruce hydrolyzate including the fibers. The sugar release was obtained by feeding glucose (using a solution of 500 g L−1 ) to the bioreactor instead of adding enzymes in these experiments. No cell growth takes place and the same cell density, 3 g L−1 , as in the subsequent SSCF experiments was used. By varying the glucose feed rate and observing the glucose concentration in the reactor, the maximum sugar uptake
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Fig. 3. Normalized rate of glucose release as a function of the glucose concentration. Polynomial function: y = 3 × 10−5 x4 − 0.0013x3 + 0.0257x2 − 0.2189x + 0.9999. Polynomial fit: r2 = 0.9956.
to increase with increasing glucose concentration and, as could be expected, the sugar release rate decreased rapidly with increasing glucose concentration up to around 7 g L−1 (Fig. 3). The experiments also showed that the spruce hydrolyzate did not affect the enzymes to any greater extent, i.e. the inhibition factor could be disregarded in the model leaving only three variables to be considered:
vhydrolysis = f (E, Cglu , ˛)
Fig. 1. Mimicked SSCF in spruce hydrolyzate liquid (no fibers) showing glucose (䊉) and xylose (). (A) No glucose is fed, resulting in very little xylose uptake. (B) A continuous glucose feed is employed, resulting in complete consumption of the xylose.
rate for the yeast in spruce hydrolyzate containing 8% WIS could be determined to 2.5 g glucose L−1 h−1 (results not shown). 3.1.2. Hydrolysis The sugar release rate was found to be proportional to the enzyme concentration in the interval 10–40 FPU (g glucan)−1 , in the experiments made (Fig. 2). The end-product inhibition was found
Fig. 2. Measured rate of glucose release as a function of enzyme load.
(2)
In the hydrolysis experiments, the relation between the glucose release rate and the fractional degradation (˛) was also studied (Fig. 4). As expected, the rate of hydrolysis at high ˛ values is clearly lower than for lower values. However, one should note that the glucose release rate in fact has a maximum around ˛ = 0.25, i.e. is it not highest in the very beginning. 3.2. SSCF experiments 3.2.1. Batch SSCF Standard batch SSCF with 8% WIS were carried out as a reference experiment. The experiments were carried out in duplicates (material 1) and triplicates (material 2) respectively, and the results are shown in Fig. 6A and Table 2.
Fig. 4. Measured rate of glucose release as a function of the fractional degradation (˛). The data was obtained from hydrolysis experiments of pretreated spruce using an enzyme load of 20 FPU (g glucan)−1 . In an SSCF a constant rate, based on the yeast’s uptake rate, is desired. Hence, the curve needs to be flattened (represented by the dotted line) in order to obtain an efficient SSCF.
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Table 2 Results from SSCF of spruce with 8% WIS at 34 ◦ C after 96 h. Raw material
Mode of operation
Glucose release rate (g L−1 h−1 )
Xylose consumptiona (%)
Xylose conc. (g L−1 )
Xylitol formationb (%)
Glycerol conc. (g L−1 )
Ethanol conc. (g L−1 )
Ethanol yield (g g−1 )
Ethanol yieldc (%)
Spruce 1 Spruce 1 Spruce 1 Spruce 1 Spruce 1 Spruce 1
Ref. batch Ref. batch Enz. feed Enz. feed Enz. feed Enz. feed
No enz. feed No enz. feed 1.4 2.0 2.5 2.0d
44 40 47 69 51 82
3.2 3.3 3.2 1.7 2.7 1.1
16 10 14 10 18 22
2.6 2.4 2.9 3.7 2.9 3.7
28.5 30.4 30.8 32.2 30.8 32.9
0.34 0.36 0.38 0.38 0.37 0.39
66 70 74 75 72 77
Spruce 2 Spruce 2 Spruce 2 Spruce 2 Spruce 2 Spruce 2
Ref. batch Ref. batch Ref. batch Enz. feed Enz. feed Enz. feed
No enz. feed No enz. feed No enz. feed 2.0d 2.0d 2.0d
35 61 59 81 83 85
3.5 2.1 2.3 1.0 0.9 0.8
26 23 24 18 19 20
2.8 3.6 3.6 3.5 3.3 3.6
35.5 35.5 35.3 33.4 34.9 35.2
0.40 0.41 0.40 0.38 0.40 0.40
79 79 79 75 78 78
In all experiments an enzyme load of 30 FPU (g glucan)−1 and a yeast load of 3 g L−1 were used. a Related to total amount of available xylose. b Related to consumed xylose. c Corresponding to the maximum theoretical yield on total available sugars. d Slightly modified feed profile (see text).
3.2.2. SSCF with controlled enzyme feed A polynomial mathematical model for the rate of hydrolysis during SSCF was created based on the glucose release rate from the hydrolysis experiments (Eq. (3)).
vhydrolysis = E × (−2.19˛2 + 1.18˛ + 0.28) × (3 × 10−5 Cglu 4 − 0.0013Cglu 3 + 0.026Cglu 2 − 0.22Cglu + 1.00)
(3)
where ˛=
vSSCF × t × V glucantot × 1.11
(4)
By inserting values for the desired glucose concentration in the SSCF (Cglu ) and the desired glucose release rate in the SSCF (vSSCF ) in Eqs. (3) and (4), the glucose release rate as a function of time could be calculated. Subsequently, the desired amount of enzyme at any time (and thereby indirectly the addition profile) could be calculated from: menz ∝
E
vhydrolysis
× vSSCF × glucantot
(5)
A constant release rate of glucose, slightly less than the maximum uptake rate for the yeast, was expected to be preferable in the SSCF in order to improve the xylose uptake. A hypothetically desired hydrolysis rate is given by the dotted line in Fig. 4. When the rate of hydrolysis slows down due to the higher degree of degradation (a higher value of ˛), more enzymes are needed. By controlling the amount of enzymes added it should in principle be possible to maintain the desired glucose concentration. The actual feed profile was kept as close as possible to the profile given by the model, however, with a slight simplification (cf. Fig. 5). Since removal of enzyme from the reactor is generally not feasible, an amount that is slightly too low was added initially (t0 ). From time t1 it was possible to follow the curve again, and a constant enzyme feed was initiated. Finally, the feed rate was increased (t2 ), and kept until the total amount of enzyme specified had been added (t3 ). The SSCF experiments were started with a prefermentation phase (Bertilsson et al., 2009), i.e. no enzymes were added initially in order to decrease the glucose concentration. After the glucose concentration had decreased sufficiently (time I, Figs. 6 and 7), enzymes were added according to the model. At time II the enzyme feed was started, and at time III the feed rate was increased (Figs. 6 and 7). Fig. 6 shows measured concentration profiles during a standard batch SSCF as well as three SSCF experiments conducted with spruce material 1 according to the procedure described above.
Obtained yields and xylose conversion are shown in Table 2. A glucose release rate of 2.0 g L−1 h−1 resulted in the highest total xylose uptake; 69% (Fig. 6C). A feed profile corresponding to a desired glucose release rate of 1.4 g L−1 h−1 (Fig. 6B) resulted in a xylose consumption of 47%, whereas at a glucose release rate of 2.5 g L−1 h−1 (Fig. 6D) the total xylose consumption was 51% of the theoretical. These findings indicate that a glucose release rate around 2.0 g L−1 h−1 , i.e. somewhat lower than the maximum glucose uptake rate determined in the spruce hydrolyzate, is optimal with respect to the xylose conversion for this process. In order to increase the xylose uptake further, a slight modification of the best case, i.e. a release rate of 2.0 g L−1 h−1 was carried out (Table 2). By changing the addition times II and III marginally, an even flatter glucose concentration profile could be achieved, resulting in a xylose consumption as high as 82%. The final ethanol concentrations and the corresponding ethanol yields are shown in Table 2. A release rate of 2.0 g L−1 h−1 proved to give the highest ethanol yield; 0.39 g g−1 of total sugars (i.e. glucose, xylose, mannose, galactose, glucan and mannan) for material 1. This corresponds to 77% of the maximum theoretical yield. This glucose release rate also resulted in the highest final ethanol concentration.
Fig. 5. Qualitative figure of the theoretically desired (solid line) and actual (dotted line) amount of enzyme in the SSCF. The shape of the curve will change with different glucose uptake rates. However, the total amount of enzymes added was the same for all the different SSCF experiments, i.e. 30 FPU (g glucan)−1 . The actual feed profile (dotted line) was always kept as close as possible to the profile given by the model.
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Fig. 6. Measured concentrations during SSCF of spruce (material 1) with 8% WIS showing glucose (䊉), xylose () and ethanol () for batch (A) and enzyme feed using a model for a glucose release rate of 1.4 g L−1 h−1 (B), 2.0 g L−1 h−1 (C) and 2.5 g L−1 h−1 (D). I, first enzyme addition; II, start of enzyme feed; III, increased enzyme feed rate.
Fig. 7. Measured concentrations during triplicate SSCF of spruce (material 2) with 8% WIS showing glucose (䊉), xylose () and ethanol () when using a model for a glucose release rate of 2.0 g L−1 h−1 with a slightly modified feed profile (see text). I, first enzyme addition; II, start of enzyme feed; III, increased enzyme feed rate.
In order to verify the reproducibility of the SSCF experiments a second spruce material was prepared, using the same pretreatment conditions (material 2, Table 1). Triplicate reference batch SSCF experiments, as well as triplicate SSCF experiments using the best enzyme feed profile, were performed (Table 2). As the results show (Fig. 7), the reproducibility was very good, which confirms that the feeding protocol is robust. Also with the second material, the enzyme feed improved xylose utilization significantly, although the ethanol yields showed no difference between the batch SSCF and the SSCF with enzyme feeding. All SSCF experiments carried out with enzyme feed resulted in a higher xylose consumption, compared to the respective reference batch SSCF (Table 2). 4. Discussion Given the high ratio of glucose to xylose in the spruce material, co-fermentation of xylose and glucose is a challenging task. SSCF
is an interesting process option, since it allows a slow constant release of glucose throughout the process which is beneficial for xylose uptake by xylose fermenting strains of S. cerevisiae (Öhgren et al., 2006; Olofsson et al., 2008). However, the glucose concentration should be kept low, i.e. the release rate during the process should be tuned to the consumption rate in order to increase the xylose uptake. In the current study, a simple and robust kinetic model for simultaneous saccharification and co-fermentation of spruce was developed in order to allow an open-loop control of sugar concentration, by making a programmed addition of enzyme feed during the process. The results showed a substantial increase in xylose consumption as a consequence of the controlled glucose release rate as afforded by enzyme feeding. The increased xylose consumption can mainly be explained by lower competitive glucose inhibition of the xylose uptake, made possible by the control of glucose at low concentration throughout the process. By employing prefermentation, i.e. by first fermenting the glucose present in the medium before adding enzymes (Bertilsson et al., 2009), the competitive glucose inhibition could be reduced at an early stage. Furthermore, the glucose concentration was maintained at a relatively low level throughout the experiment. The highest xylose consumption was achieved with an enzyme feed corresponding to a glucose release rate of 2.0 g L−1 h−1 . This glucose release rate was slightly lower than the maximum glucose uptake rate determined for the yeast strain used in the spruce hydrolyzate. This very nicely agrees with the competitive uptake system and the beneficial effects of a concomitant glucose and xylose uptake. The glucose must not accumulate in the system, and there should also be some spare transport capacity for xylose uptake, i.e. one should expect the optimal release rate to be somewhat below the maximum value. The simple kinetic model developed in this work proved to be a useful tool, capable of predicting the glucose release and uptake rate for the system and the particular raw material used at a sufficient accuracy for the intended purpose, i.e. calculation of the enzyme feed. However, the model needs to be recalibrated for use in a different SSCF process or a different raw material. Pri-
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marily, new sugar uptake and/or release rates will have to be measured. As the results show, the feed profile is dependent on a correct uptake rate of glucose since the shape of the feed profile (Fig. 5) will change with the desired glucose release rate. Hence, it is important to use a glucose uptake rate in the model that is somewhat below the maximum uptake rate of the yeast. The results obtained in SSCF (Figs. 6 and 7) correlated in principle well with what could be expected from the model, even though the feed profile could not be completely mimicked. However, as expected from the model, the best results were obtained using a glucose release rate of 2.0 g L−1 h−1 (Figs. 6C and 7). When a lower glucose uptake rate than the optimal was used in the model, the model showed a low feed rate in the beginning, and forecasted a too fast addition rate closer to the end. This was observed during the experiment with a desired glucose release rate of 1.4 g L−1 h−1 . Evidently, there was a somewhat too small enzyme addition in the beginning and a too large enzyme addition towards the end of feeding, resulting in an increased glucose concentration (Fig. 6B). Consequently, when a higher glucose uptake rate was used as an input value in the hydrolysis model, a large enzyme addition in the beginning and a slow enzyme feed towards the end was predicted. This was seen in the experiment with a desired glucose release rate of 2.5 g L−1 h−1 . Here the glucose was probably released too fast in the beginning – even if the glucose concentration was kept low – and hence, only little glucose could be released from around 20 h and on (Fig. 6D). This resulted in lower xylose uptake from this time and on, since a sufficient glucose uptake is needed for xylose uptake. It should also be noticed that the first ∼40 h of SSCF are most important when it comes to increased xylose uptake. After that, the concentration profiles looks very similar to the reference batch SSCF. Clearly, this is the time period during which the feed should be carefully controlled during the SSCF. There is ample evidence that xylose fermentation in SSCF becomes more challenging with increasing WIS content (Öhgren et al., 2006; Olofsson et al., 2008; Rudolf et al., 2008) due to higher levels of glucose and inhibitors, as well as due to mixing problems. Both previous (Bertilsson et al., 2009) and present results show that control of the glucose concentration is a key for good xylose utilization. Also fed-batch addition of substrate, which previously has been used primarily due to other benefits, gives a positive effect on the xylose uptake due to a lower glucose concentration in the reactor (Öhgren et al., 2006; Olofsson et al., 2008). Previous studies for SSCF of spruce conducted in our laboratory (Bertilsson et al., 2009) resulted in a total ethanol yield of 86% and 80% on total sugars for 7% and 10% WIS respectively, using prefermentation. Despite generally higher xylose consumption in the present study, the total ethanol yield only reached 77% (material 1) and 79% (material 2) with 8% WIS. This is most likely due to differences in the pretreated material. Although good reproducibility has been shown for the SSCF experiments, the variation in pretreatment is somewhat larger. Furthermore, a comparison of experiments with different WIS contents is often difficult. A study conducted on pretreated spruce material at 8% WIS, resulted in a ethanol yield of 86% of the theoretical (Alkasrawi et al., 2006). However, this number was based only on hexoses, and another yeast strain (commercial Baker’s yeast) at a much higher loading (5 g L−1 ) was used. Due to the relatively low xylose content in spruce, the increased xylose consumption resulted in no, or only a slight, increase of the ethanol yield. The insignificant difference in ethanol yields, as well as the overall higher ethanol yield for the second material in comparison to the first material, could be explained by a slightly different outcome of the pretreatment, resulting in more sugars in the liquid as well as a higher degree of more accessible fibers for the cellulases. The two batches of pretreated material are thus slightly different.
Most likely, the impact of xylose consumption on the ethanol yield would be higher with a more xylose rich substrate. Furthermore, spruce is considered to be a material which is not very easy to ferment by yeast. In xylose rich materials, e.g. wheat straw or sugar cane bagasse, there is a more favorable ratio of xylose and glucose, which should facilitate xylose consumption. On the other hand, there are indications that the residual xylose after fermentation is higher for these feedstocks (Öhgren et al., 2006; Olofsson et al., 2008). Consequently, there is a need for improved process design also in this case. There may also be possible advantage of using an enzyme feed also for cases in which only hexose fermentation takes place. By keeping the glucose concentration low throughout the SSF, product inhibition of the enzymatic hydrolysis can possibly be reduced and therefore a faster hydrolysis may occur. In conclusion, our study has shown that xylose conversion in SSCF can be significantly improved by employing an enzyme feed during the process. A simple model for glucose release and uptake, calibrated at process conditions, proved to be useful for calculating an enzyme feed profile that provides a constant glucose release. By employing this feed profile, the total xylose consumption could be as much as doubled in comparison to a batch SSCF. This approach will most likely be useful also for more xylose rich materials, for which improvements of the overall ethanol yields will result. Abbreviations ˛ fractional degradation of the fibers (0 ≤ ˛ ≥ 1, 1 = complete degradation) Cglu glucose concentration in liquid (g L−1 ) E enzyme concentration (FPU (g glucan)−1 ) menz amount of enzyme (g) FPU filter paper units inhibition factor (in this work = 1) t time (h) glucantot initial total amount glucan in fibers (g) vhydrolysis glucose release rate due to hydrolysis (g L−1 h−1 ) vSSCF desired glucose release rate in SSCF (g L−1 h−1 ) V volume (L) WIS water insoluble solids Acknowledgements This work was financed by the European Commission through the EU Project “New Improvements for Lignocellulosic Ethanol” (NILE, FP6, EU contract No. 019882) and the Swedish Energy Agency. References Alkasrawi, M., Rudolf, A., Lidén, G., 2006. Influence of strain and cultivation procedure on the performance of simultaneous saccharification and fermentation of steam pretreated spruce. Enzyme Microb. Technol. 38, 279–287. Almeida, J.R.M., Modig, T., Petersson, A., Hahn-Hagerdal, B., Lidén, G., GorwaGrauslund, M.F., 2007. Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J. Chem. Technol. Biotechnol. 82, 340–349. Bertilsson, M., Andersson, J., Lidén, G., 2008. Modeling simultaneous glucose and xylose uptake in Saccharomyces cerevisiae from kinetics and gene expression of sugar transporters. Bioprocess Biosyst. Eng. 31, 369–377. Bertilsson, M., Olofsson, K., Lidén, G., 2009. Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce. Biotechnol. Biofuels 2, 1–10. Boles, E., Müller, S., Zimmermann, F.K., 1996. A multi-layered sensory system controls yeast glycolytic gene expression. Mol. Microbiol. 19, 641–642. Eliasson, A., Christensson, C., Wahlbom, C.F., Hahn-Hägerdal, B., 2000. Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures. Appl. Environ. Microbiol. 66, 3381–3386. Hahn-Hägerdal, B., Karhumaa, K., Fonseca, C., Spencer-Martins, I., Gorwa-Grauslund, M., 2007. Towards industrial pentose-fermenting yeast strains. Appl. Microbiol. Biotechnol. 74, 937–953.
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