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Fermentation of biomass sugars to ethanol using native industrial yeast strains
Bioresource Technology 102 (2011) 3246–3253
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Fermentation of biomass sugars to ethanol using native industrial yeast strains Dawei Yuan a,1, Kripa Rao b,1, Patricia Relue a, Sasidhar Varanasi b,⇑ a b
Department of Bioengineering, University of Toledo, 1610 N. Westwood Ave. MS 303, Toledo, OH 43606, United States Department of Chemical and Environmental Engineering, University of Toledo, 1610 N. Westwood Ave. MS 305, Toledo, OH 43606, United States
a r t i c l e
i n f o
Article history: Received 1 July 2010 Received in revised form 7 November 2010 Accepted 9 November 2010 Available online 13 November 2010 Keywords: Cellulosic ethanol Xylose isomerase Simultaneous isomerization and fermentation (SIF) with native yeast Co-immobilized enzymes Mixed sugar fermentation (MSF)
a b s t r a c t In this paper, the feasibility of a technology for fermenting sugar mixtures representative of cellulosic biomass hydrolyzates with native industrial yeast strains is demonstrated. This paper explores the isomerization of xylose to xylulose using a bi-layered enzyme pellet system capable of sustaining a micro-environmental pH gradient. This ability allows for considerable flexibility in conducting the isomerization and fermentation steps. With this method, the isomerization and fermentation could be conducted sequentially, in fed-batch, or simultaneously to maximize utilization of both C5 and C6 sugars and ethanol yield. This system takes advantage of a pH-dependent complexation of xylulose with a supplemented additive to achieve up to 86% isomerization of xylose at fermentation conditions. Commercially-proven Saccharomyces cerevisiae strains from the corn–ethanol industry were used and shown to be very effective in implementation of the technology for ethanol production. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction One of the crucial issues in utilizing lignocellulosic biomass as a feedstock is the ability to ferment to ethanol both the C6 and C5 sugars resulting from biomass saccharification. Research efforts have focused on the utilization of xylose and native strains capable of fermenting xylose have been identified among bacteria, yeast and filamentous fungi (Skoog and Hahn-Hägerdal, 1988). However, these strains do not display many industrially desired characteristics, such as resistance to inhibitors commonly found in hydrolyzates (Hahn-Hägerdal et al., 2007a; Olofsson et al., 2008). As a result none of these strains are likely to find industrial use. Significant progress has been made recently in the development and testing of genetically modified yeast and bacterial strains capable of metabolizing both sugars (Bera et al., 2010; Dutta et al., 2010; Govindaswamy and Vane, 2007; Hahn-Hägerdal et al., 2007b; Kuyper et al., 2005; Lau and Dale, 2010; Zhang et al., 1995). However, the long-term viability of these engineered-strains in the demanding operating environment associated with ‘‘cellulosic ethanol’’ production is yet to be established (Lau et al., 2008). Therefore, approaches that are able to employ native strains as opposed to genetically modified organisms (GMOs) are still desirable. Saccharomyces cerevisiae is well-adapted to industrial use due to its near theoretical ethanol yields and tolerance to a wide spectrum of inhibitors and elevated osmotic pressure (Hahn-Hägerdal et al., ⇑ Corresponding author. Tel.: +1 419 530 8093; fax: +1 419 530 8086. 1
E-mail address: [email protected] (S. Varanasi). These authors contributed equally to the research.
0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.034
2007a). S. cerevisiae contains all necessary enzymes for the conversion of xylose to ethanol, except xylose isomerase (XI) (Gong et al., 1981). Hence, addition of exogenous xylose isomerase for isomerization of xylose to xylulose and fermentation of xylulose to ethanol using native yeast strains was proposed (Gong et al., 1981). Until now, due to the unfavorable xylose:xylulose (6:1) equilibrium, xylulose formation has been the bottleneck in commercially implementing this strategy. Two different approaches have been used to partially overcome this unfavorable isomerization equilibrium. In one approach, reagents that selectively complex to xylulose, such as borax, are employed to shift the isomerization equilibrium in favor of more xylulose formation (Gong et al., 1981). Indeed, borax, when added to an isomerization medium at pH 7.5 and 34 °C at a molar ratio to xylose of 1:8, is able to improve the xylulose yield at equilibrium from 14% to about 50% (Rao et al., 2008). In a second approach, simultaneous isomerization and fermentation (SIF) has been attempted to improve xylose utilization (Hahn-Hägerdal et al., 1986). Unfortunately, the pH optima for the isomerization (7.5) and the fermentation (4.5) steps are vastly different. In SIF operating under suboptimal pH conditions, the utilization of xylose is unsatisfactory due to the limited concentrations of xylulose available to the yeast (Chiang et al., 1981; Gong et al., 1981). In a previous paper, a novel technique was introduced to sustain two pH microenvironments in a single vessel. The technique involves coating immobilized XI particles with urease and dispersing the particles in a low pH fermentation broth which contains urea (Rao et al., 2008; Varanasi et al., 2009). As hydrogen ions diffuse
D. Yuan et al. / Bioresource Technology 102 (2011) 3246–3253
from the broth into the pellets, they are neutralized by the ammonia produced in the hydrolysis of urea by urease. Thus, a significant pH gradient is established between the bulk liquid and the core region of the pellet. The XI in the pellet interior catalyzes the isomerization of the xylose to xylulose; xylulose diffuses into the fermentation broth where it is available for fermentation. Interestingly, this bi-layered pellet configuration has an inherent feature that enables very high conversions of xylose to xylulose when the fermentation broth is supplemented with a xylulose ‘‘complexing agent’’ such as borax (Rao et al., 2008). Under conditions that establish a pH gradient between the pellet interior and the bulk solution, borax sets up a ‘‘facilitated transport’’ shuttle for xylulose in this bi-layered enzyme pellet design (Rao et al., 2008). This shuttle leads to xylulose yields that are higher than those achievable through simple equilibrium shift via the complexing agent. In a recently published study, it was shown that by adding borax at a molar ratio of 1:8 to sugar in the low pH bulk solution, up to 86% conversion of xylose to xylulose was achieved in the bi-layered pellet system (Rao et al., 2008). This conversion is well-above that possible through the use of borax when it is used merely to shift the equilibrium at pH 7 (Rao et al., 2008). While the feasibility of generating high xylulose concentrations with the bilayer pellets suspended in low pH media was demonstrated (Rao et al., 2008), the implementation of this technology using native yeasts to produce ethanol was not reported. The bi-layered pellet technology as described above allows high conversions of xylose to xylulose via the two pH microenvironment even without having to concurrently ferment xylulose to ethanol. The main features that distinguish this technology from the traditional isomerization-followed-by-fermentation (sequential) approach known in the literature are the ability (1) to initiate fermentation without having to adjust temperature or pH; and (2) to perform isomerization at temperatures of fermentation as opposed to 50–60 °C (which significantly extends the half-life of XI from days to months (Lim and Saville, 2007)). These two features can significantly impact the overall process economics, especially when coupled with recovery and reuse of the bi-layer pellets (Varanasi et al., 2009). As such, flexibility is afforded to the overall process: isomerization and fermentation can be conducted simultaneously (SIF), sequentially, or yeast can be added in a fed-batch mode at different stages of the isomerization. Results are presented in this paper for the implementation of this technology using baker’s yeast as well as robust ethanol-tolerant adapted strains used in the corn–ethanol industry. 2. Methods 2.1. Enzymes GensweetÒ IGI-HF was a gift from Genencor International Inc. (Palo Alto, CA). The GensweetÒ pellets were stored at 4 °C. Soluble jack bean urease used for generating the bi-layered enzyme pellets was purchased from Worthington Biochemical Corporation (Lakewood, NJ); urease was stored at 20 °C. The bi-layered enzyme pellets were prepared by physical adsorption of urease on the GensweetÒ pellets as described elsewhere (Rao et al., 2008). 2.2. Organisms and chemicals Baker’s yeast (BY) (S. cerevisiae, Type II) was purchased in dried pellet form from Sigma–Aldrich (St. Louis, MO). The commerciallyproven yeasts from the corn–ethanol industry, namely ethanol red (ER) (Fermentis, Lesaffre, Marcq-en-Baroeul, France) and bio-ferm XR (BXR) (North American Bio Products, Atlanta, GA) were provided by the companies for research use. Yeasts were stored at 4 °C and were added to the fermentation media as dry yeast. All
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other reagents and chemicals were purchased from Sigma–Aldrich (St. Louis, MO). 2.3. Fermentations Fermentation media were prepared with yeast extract (0.3%), peptone (0.6%), and diammonium phosphate (0.17%) in 0.05 M sodium citrate buffer (pH 4.5). Fermentations were performed at 34 °C in shake flasks (100 or 250 ml) filled to 20–25% capacity (to allow for rapid CO2 generation and resultant foaming) and agitated at 130 rpm on an orbital shaker. Shake flasks were capped with rubber stoppers, and a 21 gauge needle was inserted to vent CO2 generated during fermentation. In SIF experiments, bi-layered pellets (18 g/l) were added to the media with the yeast (200 g/l). In experiments in which xylose was pre-isomerized, the xylose (30 or 60 g/ l) media containing 0.05 M borax and 0.01–0.1 M urea was isomerized for 24 h using the bi-layered enzyme pellets, addition of yeast and/or glucose (for mixed sugar fermentations) followed. Addition of azide as a respiratory inhibitor has been shown to aid fermentation of glucose by S. cerevisiae, significantly enhancing ethanol production (Hahn-Hägerdal and Mattiasson, 1982). Accordingly, some of the experiments were conducted with 4.6 mM azide in the broth. The yeast strains (BY, ER, and XBR) were first tested for ethanol productivity and by-product formation on glucose in the presence of the additives borax, urea, and azide. Two different yeast loadings, either 50 or 100 g/l, were used at glucose concentrations of 90 and 150 g/l (see Section 3 for justification of yeast loadings used). The overall yields of ethanol from BY and ER strains were very similar at the two glucose and yeast loadings. No significant difference in glycerol and acetic acid by-product formation (0.06–0.07 g/g glucose with 150 g/l glucose; 0.075–0.095 g/g glucose with 90 g/l glucose) was observed between the three strains. Overall, the benefit of azide reported with S. cerevisiae on by-product formation (Hahn-Hägerdal and Mattiasson, 1982) is paralleled by the two adapted strains (ER and BXR) as well. BXR fermentations, however, were significantly slower than the other two yeasts; hence, BXR was not considered for additional use in our experiments. 2.4. Analytical techniques and data analysis For analysis of metabolites from fermentation, a 200–300 ll sample was collected at each time point and centrifuged to separate yeast. The supernatant was collected and diluted 1:3 with deionized water and filtered through a 0.2 lm filter. Calibration standards for glucose, xylose, xylulose, glycerol, acetic acid, xylitol, urea and ethanol were prepared in a similar manner. All standards and samples were analyzed by HPLC using a 30 ll injection volume with a 100 ll injection loop. The HPLC system used was a Shimadzu Series 10A HPLC unit equipped with a SIL-10Ai autosampler and a refractive index detector (RID 10A). For sugar, glycerol, acetic acid and ethanol detection and analysis, Bio-Rad Aminex HPX-87 H (300 7.8 mm) and Phenomenex Rezex RFQ fast acid (150 7.8 mm) ion exchange columns were used in series. The column temperature was maintained at 50 °C with a mobile phase of 0.4 ml/min of 5 mM H2SO4. At these operating conditions, when xylulose concentrations are low relative to xylose, the peaks are not well-resolved and the calculated xylulose concentrations may be subject to higher uncertainty. For xylitol and urea analysis, a Bio-Rad Aminex HPX-87 C (300 7.8 mm) column was used at 80 °C with a 0.6 ml/min mobile phase of ultra-pure water. The stoichiometric fermentations of glucose and xylulose to ethanol are given by:
C6 H12 O6 ! 2C2 H5 OH þ 2CO2 and 3C5 H10 O5 ! 5C2 H5 OH þ 5CO2
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Table 1 Carbon mass balance, carbon recovery, and ethanol yields for xylose fermentations with BY at 72 h. For Experiments 1 and 2, 60 g/l xylose was pre-isomerized to achieve initial xylulose concentrations of 33 and 52.2 g/l, respectively. For Experiment 3, 30 g/l xylose was pre-isomerized to achieve an initial xylulose concentration of 25.8 g/l. All fermentation media contained 4.6 mM azide. Experiment 1 was initiated with 100 g/l yeast, while Experiments 2 and 3 were initiated with 100 g/l yeast with fed-batch addition of 50 g/l yeast twice to a total of 200 g/l BY. Compound
MW (g/mol)
Xylose 150 Xylulose 150 Ethanol 46 CO2 44 Glycerol 92 Acetic acid 60 Xylitolc 152 Total carbon out Total carbon in Carbon recovery (%) g Ethanol/g total sugar
Mass of carbon (g C/mol compound)
60 60 24 12 36 24 60
Carbon mass fractiona (g C/g compound)
0.400 0.400 0.522 0.273 0.391 0.400 0.395
(1) Fig. 1 100 g/l yeast
(2) Fig. 2a, curve B 100 + 50 + 50 g/l yeast
(3) Fig. 2a, curve A 100 + 50 + 50 g/l yeast
Conc. from HPLC (g/l)
Carbon (g/1)
Conc. from HPLC (g/l)
Carbon (g/1)
Conc. from HPLC (g/l)
Carbon (g/1)
15.8 6.2 18.2 17.4b 3.9 2.4 N/A
6.3 2.5 9.5 4.7 1.5 1.0 – 25.5 24.0d
3.7 3.3 25.0 23.0b 3.4 2.0 N/A
1.5 1.3 13.0 6.2 1.3 0.8 – 24.2 24.0d
1.1 3.2 15.6 15.0b 2.2 1.0 N/A
0.4 1.3 8.1 4.1 0.9 0.4 – 15.2 12.0d
106 0.30
100 0.40
127 0.51
a The mass fraction of carbon is computed based on the molecular weight and molecular formula of the compound. For example, xylose/xylulose has a molecular weight of 150 g/mol and 5 C/molecule, so the sugar contains 60 g C/mol or 0.40 g C/g sugar. b CO2 production was not measured, but was estimated based on a 1:1 stoichiometry with ethanol production. c In this experiment, the column configuration used resulted in xylitol co-eluting with borate. However, based on the reproducible area of the borate peak, xylitol formation was likely negligible. d For Experiments 1 and 2, which have an initial xylose concentration of 60 g/l prior to isomerization, the g C coming in is (0.40 g C/g sugar) (60 g sugar/l) = 24 g C/l. In Experiment 3, which has 30 g/l xylose coming in, the carbon in is 12 g C/l.
The yield of ethanol on a mass basis was calculated on the basis of the total initial sugar concentration (g/l). Per the stoichiometry of the above equations, the theoretical yield of ethanol is 0.51 g/g sugar for both glucose and xylulose. Specific details on the carbon balance calculations for the fermentations are provided in the footnotes to Table 1. 3. Results and discussion As shown previously (Chiang et al., 1981), high yeast densities can promote better utilization of C5 sugars. Since the ultimate goal is fermentation of mixtures of C6 and C5 sugars, high cell densities were used in the experiments although these are not required for the fermentation of glucose alone. While such high densities may pose operational issues in traditional stirred tank fermentors, novel fermentor configurations that permit use of high yeast densities for multiple fermentation cycles are being developed (Varanasi et al., 2009). All of the data presented is based on the results of duplicate experiments. Data represent average values with standard deviation typically less than ±8%.
with the two strains is shown in Fig. 1. The initial fermentation rates are higher for BY but the overall ethanol yields are similar for both strains, as was the case for glucose. Both strains were able to convert the xylulose to near theoretical yield for ethanol. Total acetic acid and glycerol amounts of 0.06–0.08 g/g total sugar were observed in both cases, which are similar to literature reports for BY (Hahn-Hägerdal et al., 1986). Xylitol has been found to be a measurable by-product in xylose/ xylulose fermentations (Chiang et al., 1981) when a significant quantity of xylose is present in the medium. Xylitol production tends to increase as the temperature decreases (less than 35 °C) or the pH increases (pH > 5) (Chiang et al., 1981). For these experiments at 34 °C and pH 4.5 with azide, xylitol formation is not likely. A significant reduction in not only xylitol but also glycerol and acetic acid formation was shown previously in SIF with soluble XI and S. cerevisiae in the presence of 4.6 mM azide at pH 6 and 30 °C (Hahn-Hägerdal et al., 1986).
3.1. Xylulose fermentations with BY and ER As noted in Section 2, the additives used for isomerization of xylose had no adverse effect on the glucose fermentation using either BY and ER. The experiments in this section were conducted to assess the relative effectiveness of BY and ER in fermenting xylulose to ethanol. Xylose (60 g/l) was partially isomerized and fermented using BY or ER. The xylose was pre-isomerized to xylulose with 0.05 M borax and 0.01 M urea for a 55% conversion (33 g xylulose/l). By allowing a significant proportion of xylose to remain in the media, the effectiveness of azide in suppressing xylitol formation on the two yeast strains can be assessed. In addition, the 33 g/l xylulose concentration is typical of pentose yields in biomass hydrolyzates following CAFI protocols (Wyman et al., 2005). Fermentation of the 33/27 g/l of xylulose/xylose mixtures was initiated by adding 100 g/l yeast to the media. Ethanol production
Fig. 1. Fermentation of 33 g/l xylulose with both ER and BY. Xylulose was produced by the partial isomerization of 60 g/l xylose with the bi-layered enzyme pellets. Both strains fermented xylulose to ethanol with high yield, but with significantly slower kinetics than for glucose. Both experiments contained 4.6 mM azide to block respiration.
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Fig. 2. Effect of BY addition and initial pentose concentration on xylulose fermentation. Xylose in all experiments (30 or 60 g/l) was pre-isomerized to near 86% conversion to xylulose. Initially, 100 g/l BY was added to the fermentation and in (a) and (b), an additional 50 g/l BY was added at 24 and 40 h. Fed-batch addition of BY significantly improved the ethanol yield at the higher sugar loading. All fermentations contained 4.6 mM azide.
3.2. Effect of initial xylulose concentration and mode of BY addition on xylulose utilization The complete saccharification of 20% (w/v) woody biomass typically yields around 30 g/l of soluble xylose; however, saccharification of other kinds of biomass could lead to higher xylose concentrations. Accordingly, fermentation of 30 and 60 g/l xylose were compared. In the previous experiment on strain evaluation, a significant quantity of xylose was allowed to remain in the media. However, from a practical standpoint, high ethanol yield requires complete xylose isomerization. Accordingly, both 30 and 60 g/l xylose samples were pre-isomerized with 0.1 M urea for 24 h to achieve 86% xylulose, or about 25.8 and 52.2 g/l xylulose, respectively. For BY fermentations at each substrate loading, the change in ethanol, xylose, and xylulose with time are shown in Fig 2. For yeast loadings of 100 g/l (data not shown) or fed-batch addition to 200 g/l, the 25.8 g/l xylulose was fermented completely to ethanol (see Fig. 2a, curve A). When the initial concentration of xylulose was doubled with 100 g/l yeast loading, only 40 g/l xylulose was fermented (Fig. 2a, curve C), yielding 20.6 g/l ethanol (0.4 g/g) in 70 h. To determine if fed-batch addition of yeast could lead to more effective sugar utilization at the higher xylulose concentration, 100 g/l yeast was added initially, followed by addition of 50 g/l yeast at 24 and 40 h (Fig. 2, curve B). The addition of dry yeast cells at 24 h resulted in an increase in the kinetics of the fermentation (compare Fig. 2a, curves B and C after 24 h). The final ethanol yield increased from 0.4 to 0.51 g EtOH/g xylulose. The total formation of by-products (acetic acid and glycerol) was similar at both cell densities (5.2–5.3 g/l). The fact that doubling the yeast density through fed-batch addition did not result in a significant increase in by-product formation might suggest that the by-products derive primarily from the minor quantity of xylose in the initial sugar mixture (Fig. 2b). The higher initial xylulose concentrations may lead to rapid accumulation of ethanol intracellularly, approaching levels that could be inhibitory for pentose fermentations (Chiang et al., 1981). Fermentation by the fresh yeast is not biased by intracellular accumulation of ethanol, and hence the yeast added in fedbatch mode is able to more efficiently utilize the available xylulose. Just as ER is adapted to ethanol tolerance for glucose fermentation, yeasts could perhaps be adapted to ethanol tolerance for xylulose fermentation as well.
Thus, a properly-strategized fed-batch addition of yeast provides an opportunity to not only achieve high utilization of xylulose but also to significantly shorten the time required for fermentation. 3.3. Carbon balance for xylose fermentations In addition to ethanol, other compounds such as glycerol, acetic acid, xylitol, and CO2 are produced during fermentation. The carbon balances for the fermentations of xylose/xylulose mixtures corresponding to the cases discussed in Figs. 1 and 2 are presented in Table 1. Details on the carbon balance procedure are provided in the footnotes to Table 1. Cell mass of the cultures was not measured as yeast growth was expected to be minimal under anaerobic fermentation on xylulose with azide. The major by-products detected in the fermentations were glycerol and acetic acid. The carbon balances for Experiments 1 and 2 with 60 g/l initial sugar were satisfactory; the mass balance for Experiment 3 shows an accumulation of carbon, possibly due to reduced accuracy in HPLC measurements at the lower concentrations measured. A similar over-estimation of carbon (10%) was reported in the literature for xylulose fermentations using S. cerevisiae ATCC 24860 (Yu et al., 1995). 3.4. Fermentation of mixed sugars The ultimate goal of the bi-layered enzyme technology is its implementation with biomass hydrolyzates. Hydrolyzates may contain various fermentation inhibitors depending on the method of biomass pretreatment employed. Accordingly, detoxification/ conditioning may be necessary prior to the isomerization/fermentation. Removal of fermentation inhibitors prior to isomerization will also eliminate the risk of inhibition of the XI (Lindén and Hahn-Hägerdal, 1989) and urease activities. A recently developed ionic liquid pretreatment method (Varanasi et al., 2008) was shown to produce hydrolyzate free of the fermentation inhibitors traditionally found with dilute acid pretreatment. Thus, as new pretreatment methods are developed, the need for hydrolyzate preconditioning may be eliminated. Even in the absence of fermentation inhibitors, when S. cerevisiae are presented with mixtures of glucose and xylulose, the overall ethanol yields can be much different than that achieved with either sugar separately. Accordingly, the experiments in this section focus on simulated glucose/xylulose mixtures.
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Fig. 3. Mixed sugar fermentation of 90 g/l glucose and 30 g/l xylose (pre-isomerized to xylulose) without azide (a–c) and with 4.6 mM azide (d–f). Three yeast loadings were tested: 50 g/l (a,d); 100 g/l (b,e); and 200 g/l (c,f).
3.4.1. Effect of yeast loading on fermentation of mixed sugars Mixed sugar fermentations were conducted with 90 g/l glucose and 30 g/l xylose both with and without the addition of azide. To ensure immediate fermentable sugar availability, the xylose was first pre-isomerized for 24 h to obtain 86% xylulose. Fermentations were performed with three yeast loadings (50, 100, and 200 g/l). Results of these fermentations in the absence of azide are shown in Fig. 3a–c. In all cases, glucose was consumed within the first 5 h. As yeast loading increases from panels a to c, the overall ethanol yield increases and the by-product formation decreases by about 20%. With 50 g/l yeast, more than 6 g/l xylulose remain after 28 h of fermentation. At this low yeast loading, uptake of xylulose may limit its utilization. At the higher yeast loadings shown in Fig 3b and c, xylulose utilization is much improved as is the overall ethanol production, reflecting better pentose utilization kinetics and co-utilization of the sugars. To assess the impact of azide in mixed sugar fermentations, the fermentations shown in Fig. 3a–c were repeated with 4.6 mM azide (see Fig. 3d–f). By-product formation was about half of that seen without azide while ethanol yields are generally increased. However, xylulose uptake is significantly slower in the presence of azide. This indicates that the observed increase in ethanol yield in the presence of azide results from the glucose portion but not necessarily from xylulose utilization. In pure xylulose fermentations, close to theoretical yields of ethanol were achieved under similar conditions. The incomplete utilization of xylulose in mixed sugar fermentations suggests that ethanol produced from glucose fermentation may inhibit xylulose uptake and utilization. If this were the case, fed-batch addition of yeast or use of an ethanol-tolerant yeast strain (i.e. ER) could prove beneficial; these strategies are tested next. 3.4.2. Effect of mode of addition and strain of yeast on mixed sugar fermentation The fermentation of two mixed sugar compositions, (1) 90 g/l glucose and 30 g/l xylose and (2) 130 g/l glucose and 50 g/l xylose,
were conducted with fed-batch addition of 50 g/l yeast at time 0, 4, 7 and 24 h to a final concentration of 200 g/l. The fermentations were conducted with either BY or ER in the presence of 4.6 mM azide. Xylose was pre-isomerized to 86% conversion prior to fermentation for all cases. In an attempt to more-closely produce anaerobic conditions, nitrogen was bubbled into the shake flasks for 60 s to purge oxygen after each yeast addition. Ethanol production for 120 g/l total sugars fermented with 200 g/l yeast added either initially or in fed-batch mode are shown in Fig. 4a. The overall ethanol yield increased from 0.38 to 0.42 g/g total sugar when yeast was switched from initial to fed-batch addition. Under these conditions, ER proved to be a marginally better fermentor of mixed sugars (ethanol yield of 0.46 g/g total sugar) than BY with fed-batch addition. At very high biomass loadings (30%) during saccharification, the hydrolyzates generated could have up to 180 g/l of mixed sugars. Complete conversion of these sugars to ethanol would yield 9% (w/v) ethanol in the fermentation broth – a concentration level that is known to inhibit BY. Since ER is known to tolerate up to 15% (w/v) ethanol in corn-to-ethanol fermentations, and it performed well with 120 g/l mixed sugars, its performance at these very high sugar concentrations was tested next. Ethanol yields from the fermentation of 130 g/l glucose and 50 g/l xylose with fed-batch yeast addition are shown in Fig. 4b. Ethanol yields at the end of 72 h were 0.35 and 0.41 g ethanol/g total sugar from BY and ER, respectively. For BY, some reassimilation of ethanol occurred after the first 24 h. The carbon balances for these experiments closed very well (see Table 2). The closure of the carbon balances is likely due to the high yeast loadings in combination with the anaerobic nitrogen blanket in the fermenting flask. Fed-batch yeast addition resulted in better sugar utilization and about 25% higher total product formation, which is reflected as higher ethanol as well as by-product production (see Table 2). ER clearly shows less ethanol inhibition at these high sugar loadings than BY. This result is consistent with the hypothesis that an ethanol-tolerant yeast strain may be less
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Fig. 4. Mixed sugar fermentations with BY and ER in fed-batch yeast addition mode. Xylose was pre-isomerized to 86% conversion for all experiments prior to fermentation; 4.6 mM sodium azide was used in all experiments. The fed-batch yeast addition was implemented by adding 50 g/l of yeast at 0, 4, 7 and 24 h to a total of 200 g/l. Nitrogen was purged into the shake flask after each yeast addition and sample collection to maintain anaerobic conditions. Ethanol production is shown for (a) for 120 g/l sugar (90 g/l glucose and 30 g/l xylose) fermentation and (b) 180 g/l total sugar (130 g/l glucose and 50 g/l xylose) fermentation.
Table 2 Summary of mixed sugar fermentation results from Fig. 4 with carbon balance results. Values shown are at 72 h for fed-batch (fb) yeast addition and at 64 h for initial (i) yeast addition. All experiments contain a total of 200 g/l yeast. Xylitol was not measured as these experiments contain low starting xylose concentrations and are nitrogen-blanketed; carbon balances close well for all experiments. Total sugar (g/l)
Yeast – addition mode
Initial carbon (g/l)
Ethanol (g/l)
Ethanol yields (g/g total sugar)
Sugar remaining (g/l)
Glycerol (g/l)
Acetic acid (g/l)
Carbon accounted (g/l)
Carbon recovery (%)
120
BY – i BY – f b ER – f b BY – f b ER – f b
48 48 48 72 72
45.26 50.51 55.62 62.71 73.46
0.377 0.421 0.463 0.348 0.408
11.51 9.54 7.71 32.65 24.23
5.71 7.57 7.63 8.66 9.10
1.79 1.41 2.17 1.56 2.32
42.98 46.9 50.5 66.2 71.7
89.53 97.7 105.1 91.9 99.5
180
3.4.3. Xylulose uptake in mixed sugar fermentations for BY Transient xylulose concentrations for three different fermentations with 100 g/l BY loading and the same initial xylulose concentration are compared in Fig. 5. Curve C shows that the most rapid uptake of xylulose occurred in the mixed sugar fermentation without azide (see also Fig. 3b). In contrast, when azide is added, xylulose uptake rate is reduced (Fig. 5, curve B, see also Fig. 3e). Indeed, the xylulose uptake in curve B is even slower than observed with pure xylulose alone (curve A) where ethanol inhibition resulting from glucose fermentation is not possible. It may be that xylulose utilization kinetics are hampered by the combined effects of azide and accumulated intracellular ethanol. Based on the experiments summarized in Figs. 3–5, it was concluded that the most efficient implementation of SIF for mixed sugar fermentations with BY is high yeast loadings without addition of azide to the media. Under such conditions, xylulose uptake is benefited by the availability of sufficient transporters (Hamacher et al., 2002; Hector et al., 2008; Lee et al., 2002) as well as reduced ethanol inhibition. Accordingly, these conditions were selected for a comparison of SIF to partial SIF. Fig. 5. Xylulose uptake in pure and mixed sugar fermentations. All experiments were conducted with an initial yeast loading of 100 g/l and with 30 g/l xylose preisomerized to yield approximately 25.8 g/l xylulose; Experiments A and B contained 4.6 mM azide. Mixed sugar fermentations contained 90 g/l glucose which was consumed within the first 4 h (not shown).
susceptible to the synergistic inhibition effects of azide and ethanol. This inhibitory effect may be more clearly seen in Fig. 5 where xylulose uptake rates are compared for BY with and without azide on pure pentose and mixed sugars.
3.4.4. Simultaneous-isomerization-and-fermentation of mixed sugars Isomerizations and fermentations were conducted simultaneously on mixed sugars containing 60 g/l glucose and 30 g/l xylose in media without azide. Three separate experiments were conducted to assess the effect of the extent of xylose isomerization to xylulose on ethanol yield. In these experiments, the bi-layered pellets were (a) not added to the fermentation medium (control run); (b) added with borax and yeast; or (c) added first, followed
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xylose) was 67%; about 20% of the xylose was consumed. Byproducts formed included acetic acid, glycerol and xylitol. When the bi-layer pellets were added with 0.05 M borax (Fig. 6b), the overall ethanol yield increased from 67% to 74% (±1%), with about one third of the initial xylose remaining at 10 h. The facilitated transport shuttle set up by borax allows appreciable conversion of xylose to xylulose, presenting yeast with xylulose to ferment. However, as the SIF proceeds, the rate of xylose isomerization decreases due to xylitol accumulation within the fermentation broth. Xylitol formation began after glucose was depleted and rose gradually to 2.5 g/l. These experiments, while proving the viability of SIF, point to the need to address issues associated with the slow rate of isomerization in this configuration. The activity of XI can be adversely affected by the xylitol formed from xylose during fermentation in SIF. Xylitol formation from xylose fermentation is known to be an issue under aerobic conditions; strict anaerobic conditions were difficult to achieve. Recently, XI that is much less prone to xylitol inhibition was successfully produced as a recombinant protein (Brat et al., 2009). Using this new XI enzyme in combination with strict anaerobic conditions during SIF can lead to higher xylulose concentrations and higher ethanol yield. Since xylitol-inhibitionresistant XIs are not yet commercially available, in the subsequent experiment, the bi-layered pellets were used to achieve high xylulose concentrations at pH conditions suitable for fermentation prior to adding yeast to the media. In Fig 6c, the xylose was pre-isomerized to 70% xylulose and then yeast were added. These high initial xylulose concentrations led to a further improvement over Fig. 6b with ethanol yield approaching 81%. Indeed, the partial isomerization to xylulose prior to fermentation leads to lower xylitol production contributing to the increased ethanol productivity. The fact that XI isomerizes both glucose and xylose does not adversely affect either the xylulose yield or the C6 fermentation. The affinity of XI for xylose is 160 times greater than its affinity for glucose (Bhosale et al., 1996). Even in proportions found in biomass hydrolyzates (concentration of glucose 3–6 times higher than xylose), isomerization of xylose to xylulose will dominate. Moreover, since glucose is rapidly fermented by yeast, fructose formation is likely to be very low (fructose was not detected in the experiments described in Fig. 6b and c). Even if small quantities of fructose are formed, S. cerevisiae ferments fructose as well as glucose. 4. Conclusions
Fig. 6. Simultaneous isomerization and fermentation (SIF) of 60 g/l glucose and 30 g/l xylose. Fermentation with (a) no isomerization of xylose; (b) SIF; and (c) preisomerization of xylose followed by SIF. Data shown are the average values of duplicate experiments; glucose values have standard deviation within ±8%; all other values have standard deviation within ±3%. For all experiments shown in Fig. 3, carbon balances closed to within 3% (data not shown). Ethanol produced in (c) is slightly lower than (b) due to lower initial sugar concentrations. Yield of ethanol per total sugar for the experiments are (a) 67%, (b) 74%, and (c) 81%.
Recently, a method for isomerizing xylose to xylulose at high yield was demonstrated wherein the xylulose complexing agent, pH, and temperature are compatible with fermentation. Thus, isomerization and fermentation could be conducted sequentially, in fed-batch, or simultaneously to maximize utilization of both C5 and C6 sugars and ethanol yield. For the first time, it was demonstrated that industrially proven yeast strains such as ethanol red can convert high concentrations of mixed sugars to ethanol in times of about 30 h with yields comparable to engineered strains. This exogenous isomerization method avoids several issues with which GMO yeasts incorporating XI have to contend. Acknowledgements
by yeast addition after partial isomerization of the sugars with borax. As shown in Fig. 6, glucose was completely consumed within 1–2 h for all three cases. In the absence of added bi-layer pellets (Fig. 6a), the ethanol yield based on total sugar (glucose and
The authors acknowledge the support of this research through grants from United States Department of Energy Office of the Biomass Program (GO18163) and Ohio Third Frontier Advanced Energy Program (08-021). Mention of trade names or commercial products does not constitute an endorsement or recommendation for use.
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References Bera, A., Sedlak, M., Khan, A., Ho, N., 2010. Establishment of L-arabinose fermentation in glucose/xylose co-fermenting recombinant Saccharomyces cerevisiae 424A(LNH-ST) by genetic engineering. Appl. Microbiol. Biotechnol. 87 (5), 1803–1811. Bhosale, S.H., Rao, M.B., Deshpande, V.V., 1996. Molecular and industrial aspects of glucose isomerase. Microbiol. Rev. 60 (2), 280–300. Brat, D., Boles, E., Wiedemann, B., 2009. Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 75 (8), 2304– 2311. Chiang, L.C., Gong, C.S., Chen, L.F., Tsao, G.T., 1981. D-Xylulose fermentation to ethanol by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 42 (2), 284–289. Dutta, A., Dowe, N., Ibsen, K.N., Schell, D.J., Aden, A., 2010. An economic comparison of different fermentation configurations to convert corn stover to ethanol using Z. mobilis and Saccharomyces. Biotechnol. Prog. 26 (1), 64–72. Gong, C.S., Chen, L.F., Flickinger, M.C., Chiang, L.C., Tsao, G.T., 1981. Production of ethanol from D-xylose by using D-xylose isomerase and yeasts. Appl. Environ. Microbiol. 41 (2), 430–436. Govindaswamy, S., Vane, L.M., 2007. Kinetics of growth and ethanol production on different carbon substrates using genetically engineered xylose-fermenting yeast. Bioresour. Technol. 98, 677–685. Hahn-Hägerdal, B., Karhumaa, K., Fonseca, C., Spencer-Martins, I., Gorwa-Grauslund, M.F., 2007a. Towards industrial pentose-fermenting yeast strains. Appl. Microbiol. Biotechnol. 74 (5), 937–953. Hahn-Hägerdal, B., Karhumaa, K., Jeppsson, M., Gorwa-Grauslund, M.F., 2007b. Metabolic engineering for pentose utilization in Saccharomyces cerevisiae. Adv. Biochem. Eng./Biotechnol. 108, 147–177. Hahn-Hägerdal, B., Mattiasson, B., 1982. Azide sterilization of fermentation media – ethanol production from glucose using immobilized Saccharomyces cerevisiae. Eur. J. Appl. Microbiol. Biotechnol. 14 (3), 140–143. Hahn-Hägerdal, B., Skoog, K., Berner, S., 1986. Improved ethanol production from xylose with glucose isomerase and Saccharomyces cerevisiae using the respiratory inhibitor azide. Appl. Microbiol. Biotechnol. 24 (4), 287–293. Hamacher, T., Becker, J., Gardonyi, M., Hahn-Hägerdal, B., Boles, E., 2002. Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology 148 (9), 2783–2788. Hector, R., Qureshi, N., Hughes, S., Cotta, M., 2008. Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption. Appl. Microbiol. Biotechnol. 80 (4), 675–684.
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Kuyper, M., Hartog, M.M., Toirkens, M.J., Almering, M.J., 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 (4–5), 399–409. Lau, M.W., Dale, B.E., 2010. Effect of primary degradation-reaction products from Ammonia Fiber Expansion (AFEX)-treated corn stover on the growth and fermentation of Escherichia coli KO11. Bioresour. Technol. 101 (20), 7849– 7855. Lau, M.W., Dale, B.E., Balan, V., 2008. Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation. Biotechnol. Bioeng. 99 (3), 529–539. Lee, W.J., Bisson, L.F., Seo, J.H., Kim, M.D., Ryu, Y.W., 2002. Kinetic studies on glucose and xylose transport in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 60 (1/2), 186–191. Lim, L., Saville, B., 2007. Thermoinactivation mechanism of glucose isomerase. Appl. Biochem. Biotechnol. 137–140 (1), 115–130. Lindén, T., Hahn-Hägerdal, B., 1989. Fermentation of lignocellulose hydrolysates with yeasts and xylose isomerase. Enzyme Microb. Technol. 11 (9), 583–589. Olofsson, K., Bertilsson, M., Liden, G., 2008. A short review on SSF – an interesting process option for ethanol production from lignocellulosic feedstocks. Biotechnol. Biofuels 1 (1), 7. Rao, K., Chelikani, S., Relue, P., Varanasi, S., 2008. A novel technique that enables efficient conduct of simultaneous isomerization and fermentation (SIF) of xylose. Appl. Biochem. Biotechnol. 146 (1–3), 101–117. Skoog, K., Hahn-Hägerdal, B., 1988. Xylose fermentation. Enzyme Microb. Technol. 10, 66–80. Varanasi, S., Rao, K., Relue, P., Yuan, D., 2009. Methods for fermentation of xylose and hexose sugars. WO 2009/089103. Jul. 16, 2009. Varanasi, S., Schall, C.A., Dadi, A.P., Anderson, J., Rao, K., 2008. Biomass pretreatment. US Patent 2008/0227162. Sep. 18, 2008. Wyman, C.E., Dale, B.E., Elander, R.T., Holtzapple, M., Ladisch, M.R., Lee, Y.Y., 2005. Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover. Bioresour. Technol. 96 (18), 2026– 2032. Yu, S., Hahn-Hägerdal, B., Jeppsson, H., 1995. Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains. Appl. Microbiol. Biotechnol. 44 (3/4), 314–320. Zhang, M., Eddy, C., Deanda, K., Finkelstein, M., Picataggio, S., 1995. Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. Science 267 (5195), 240–243.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Fermentation of biomass sugars to ethanol using native industrial yeast strains Dawei Yuan a,1, Kripa Rao b,1, Patricia Relue a, Sasidhar Varanasi b,⇑ a b
Department of Bioengineering, University of Toledo, 1610 N. Westwood Ave. MS 303, Toledo, OH 43606, United States Department of Chemical and Environmental Engineering, University of Toledo, 1610 N. Westwood Ave. MS 305, Toledo, OH 43606, United States
a r t i c l e
i n f o
Article history: Received 1 July 2010 Received in revised form 7 November 2010 Accepted 9 November 2010 Available online 13 November 2010 Keywords: Cellulosic ethanol Xylose isomerase Simultaneous isomerization and fermentation (SIF) with native yeast Co-immobilized enzymes Mixed sugar fermentation (MSF)
a b s t r a c t In this paper, the feasibility of a technology for fermenting sugar mixtures representative of cellulosic biomass hydrolyzates with native industrial yeast strains is demonstrated. This paper explores the isomerization of xylose to xylulose using a bi-layered enzyme pellet system capable of sustaining a micro-environmental pH gradient. This ability allows for considerable flexibility in conducting the isomerization and fermentation steps. With this method, the isomerization and fermentation could be conducted sequentially, in fed-batch, or simultaneously to maximize utilization of both C5 and C6 sugars and ethanol yield. This system takes advantage of a pH-dependent complexation of xylulose with a supplemented additive to achieve up to 86% isomerization of xylose at fermentation conditions. Commercially-proven Saccharomyces cerevisiae strains from the corn–ethanol industry were used and shown to be very effective in implementation of the technology for ethanol production. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction One of the crucial issues in utilizing lignocellulosic biomass as a feedstock is the ability to ferment to ethanol both the C6 and C5 sugars resulting from biomass saccharification. Research efforts have focused on the utilization of xylose and native strains capable of fermenting xylose have been identified among bacteria, yeast and filamentous fungi (Skoog and Hahn-Hägerdal, 1988). However, these strains do not display many industrially desired characteristics, such as resistance to inhibitors commonly found in hydrolyzates (Hahn-Hägerdal et al., 2007a; Olofsson et al., 2008). As a result none of these strains are likely to find industrial use. Significant progress has been made recently in the development and testing of genetically modified yeast and bacterial strains capable of metabolizing both sugars (Bera et al., 2010; Dutta et al., 2010; Govindaswamy and Vane, 2007; Hahn-Hägerdal et al., 2007b; Kuyper et al., 2005; Lau and Dale, 2010; Zhang et al., 1995). However, the long-term viability of these engineered-strains in the demanding operating environment associated with ‘‘cellulosic ethanol’’ production is yet to be established (Lau et al., 2008). Therefore, approaches that are able to employ native strains as opposed to genetically modified organisms (GMOs) are still desirable. Saccharomyces cerevisiae is well-adapted to industrial use due to its near theoretical ethanol yields and tolerance to a wide spectrum of inhibitors and elevated osmotic pressure (Hahn-Hägerdal et al., ⇑ Corresponding author. Tel.: +1 419 530 8093; fax: +1 419 530 8086. 1
E-mail address: [email protected] (S. Varanasi). These authors contributed equally to the research.
0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.034
2007a). S. cerevisiae contains all necessary enzymes for the conversion of xylose to ethanol, except xylose isomerase (XI) (Gong et al., 1981). Hence, addition of exogenous xylose isomerase for isomerization of xylose to xylulose and fermentation of xylulose to ethanol using native yeast strains was proposed (Gong et al., 1981). Until now, due to the unfavorable xylose:xylulose (6:1) equilibrium, xylulose formation has been the bottleneck in commercially implementing this strategy. Two different approaches have been used to partially overcome this unfavorable isomerization equilibrium. In one approach, reagents that selectively complex to xylulose, such as borax, are employed to shift the isomerization equilibrium in favor of more xylulose formation (Gong et al., 1981). Indeed, borax, when added to an isomerization medium at pH 7.5 and 34 °C at a molar ratio to xylose of 1:8, is able to improve the xylulose yield at equilibrium from 14% to about 50% (Rao et al., 2008). In a second approach, simultaneous isomerization and fermentation (SIF) has been attempted to improve xylose utilization (Hahn-Hägerdal et al., 1986). Unfortunately, the pH optima for the isomerization (7.5) and the fermentation (4.5) steps are vastly different. In SIF operating under suboptimal pH conditions, the utilization of xylose is unsatisfactory due to the limited concentrations of xylulose available to the yeast (Chiang et al., 1981; Gong et al., 1981). In a previous paper, a novel technique was introduced to sustain two pH microenvironments in a single vessel. The technique involves coating immobilized XI particles with urease and dispersing the particles in a low pH fermentation broth which contains urea (Rao et al., 2008; Varanasi et al., 2009). As hydrogen ions diffuse
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from the broth into the pellets, they are neutralized by the ammonia produced in the hydrolysis of urea by urease. Thus, a significant pH gradient is established between the bulk liquid and the core region of the pellet. The XI in the pellet interior catalyzes the isomerization of the xylose to xylulose; xylulose diffuses into the fermentation broth where it is available for fermentation. Interestingly, this bi-layered pellet configuration has an inherent feature that enables very high conversions of xylose to xylulose when the fermentation broth is supplemented with a xylulose ‘‘complexing agent’’ such as borax (Rao et al., 2008). Under conditions that establish a pH gradient between the pellet interior and the bulk solution, borax sets up a ‘‘facilitated transport’’ shuttle for xylulose in this bi-layered enzyme pellet design (Rao et al., 2008). This shuttle leads to xylulose yields that are higher than those achievable through simple equilibrium shift via the complexing agent. In a recently published study, it was shown that by adding borax at a molar ratio of 1:8 to sugar in the low pH bulk solution, up to 86% conversion of xylose to xylulose was achieved in the bi-layered pellet system (Rao et al., 2008). This conversion is well-above that possible through the use of borax when it is used merely to shift the equilibrium at pH 7 (Rao et al., 2008). While the feasibility of generating high xylulose concentrations with the bilayer pellets suspended in low pH media was demonstrated (Rao et al., 2008), the implementation of this technology using native yeasts to produce ethanol was not reported. The bi-layered pellet technology as described above allows high conversions of xylose to xylulose via the two pH microenvironment even without having to concurrently ferment xylulose to ethanol. The main features that distinguish this technology from the traditional isomerization-followed-by-fermentation (sequential) approach known in the literature are the ability (1) to initiate fermentation without having to adjust temperature or pH; and (2) to perform isomerization at temperatures of fermentation as opposed to 50–60 °C (which significantly extends the half-life of XI from days to months (Lim and Saville, 2007)). These two features can significantly impact the overall process economics, especially when coupled with recovery and reuse of the bi-layer pellets (Varanasi et al., 2009). As such, flexibility is afforded to the overall process: isomerization and fermentation can be conducted simultaneously (SIF), sequentially, or yeast can be added in a fed-batch mode at different stages of the isomerization. Results are presented in this paper for the implementation of this technology using baker’s yeast as well as robust ethanol-tolerant adapted strains used in the corn–ethanol industry. 2. Methods 2.1. Enzymes GensweetÒ IGI-HF was a gift from Genencor International Inc. (Palo Alto, CA). The GensweetÒ pellets were stored at 4 °C. Soluble jack bean urease used for generating the bi-layered enzyme pellets was purchased from Worthington Biochemical Corporation (Lakewood, NJ); urease was stored at 20 °C. The bi-layered enzyme pellets were prepared by physical adsorption of urease on the GensweetÒ pellets as described elsewhere (Rao et al., 2008). 2.2. Organisms and chemicals Baker’s yeast (BY) (S. cerevisiae, Type II) was purchased in dried pellet form from Sigma–Aldrich (St. Louis, MO). The commerciallyproven yeasts from the corn–ethanol industry, namely ethanol red (ER) (Fermentis, Lesaffre, Marcq-en-Baroeul, France) and bio-ferm XR (BXR) (North American Bio Products, Atlanta, GA) were provided by the companies for research use. Yeasts were stored at 4 °C and were added to the fermentation media as dry yeast. All
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other reagents and chemicals were purchased from Sigma–Aldrich (St. Louis, MO). 2.3. Fermentations Fermentation media were prepared with yeast extract (0.3%), peptone (0.6%), and diammonium phosphate (0.17%) in 0.05 M sodium citrate buffer (pH 4.5). Fermentations were performed at 34 °C in shake flasks (100 or 250 ml) filled to 20–25% capacity (to allow for rapid CO2 generation and resultant foaming) and agitated at 130 rpm on an orbital shaker. Shake flasks were capped with rubber stoppers, and a 21 gauge needle was inserted to vent CO2 generated during fermentation. In SIF experiments, bi-layered pellets (18 g/l) were added to the media with the yeast (200 g/l). In experiments in which xylose was pre-isomerized, the xylose (30 or 60 g/ l) media containing 0.05 M borax and 0.01–0.1 M urea was isomerized for 24 h using the bi-layered enzyme pellets, addition of yeast and/or glucose (for mixed sugar fermentations) followed. Addition of azide as a respiratory inhibitor has been shown to aid fermentation of glucose by S. cerevisiae, significantly enhancing ethanol production (Hahn-Hägerdal and Mattiasson, 1982). Accordingly, some of the experiments were conducted with 4.6 mM azide in the broth. The yeast strains (BY, ER, and XBR) were first tested for ethanol productivity and by-product formation on glucose in the presence of the additives borax, urea, and azide. Two different yeast loadings, either 50 or 100 g/l, were used at glucose concentrations of 90 and 150 g/l (see Section 3 for justification of yeast loadings used). The overall yields of ethanol from BY and ER strains were very similar at the two glucose and yeast loadings. No significant difference in glycerol and acetic acid by-product formation (0.06–0.07 g/g glucose with 150 g/l glucose; 0.075–0.095 g/g glucose with 90 g/l glucose) was observed between the three strains. Overall, the benefit of azide reported with S. cerevisiae on by-product formation (Hahn-Hägerdal and Mattiasson, 1982) is paralleled by the two adapted strains (ER and BXR) as well. BXR fermentations, however, were significantly slower than the other two yeasts; hence, BXR was not considered for additional use in our experiments. 2.4. Analytical techniques and data analysis For analysis of metabolites from fermentation, a 200–300 ll sample was collected at each time point and centrifuged to separate yeast. The supernatant was collected and diluted 1:3 with deionized water and filtered through a 0.2 lm filter. Calibration standards for glucose, xylose, xylulose, glycerol, acetic acid, xylitol, urea and ethanol were prepared in a similar manner. All standards and samples were analyzed by HPLC using a 30 ll injection volume with a 100 ll injection loop. The HPLC system used was a Shimadzu Series 10A HPLC unit equipped with a SIL-10Ai autosampler and a refractive index detector (RID 10A). For sugar, glycerol, acetic acid and ethanol detection and analysis, Bio-Rad Aminex HPX-87 H (300 7.8 mm) and Phenomenex Rezex RFQ fast acid (150 7.8 mm) ion exchange columns were used in series. The column temperature was maintained at 50 °C with a mobile phase of 0.4 ml/min of 5 mM H2SO4. At these operating conditions, when xylulose concentrations are low relative to xylose, the peaks are not well-resolved and the calculated xylulose concentrations may be subject to higher uncertainty. For xylitol and urea analysis, a Bio-Rad Aminex HPX-87 C (300 7.8 mm) column was used at 80 °C with a 0.6 ml/min mobile phase of ultra-pure water. The stoichiometric fermentations of glucose and xylulose to ethanol are given by:
C6 H12 O6 ! 2C2 H5 OH þ 2CO2 and 3C5 H10 O5 ! 5C2 H5 OH þ 5CO2
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Table 1 Carbon mass balance, carbon recovery, and ethanol yields for xylose fermentations with BY at 72 h. For Experiments 1 and 2, 60 g/l xylose was pre-isomerized to achieve initial xylulose concentrations of 33 and 52.2 g/l, respectively. For Experiment 3, 30 g/l xylose was pre-isomerized to achieve an initial xylulose concentration of 25.8 g/l. All fermentation media contained 4.6 mM azide. Experiment 1 was initiated with 100 g/l yeast, while Experiments 2 and 3 were initiated with 100 g/l yeast with fed-batch addition of 50 g/l yeast twice to a total of 200 g/l BY. Compound
MW (g/mol)
Xylose 150 Xylulose 150 Ethanol 46 CO2 44 Glycerol 92 Acetic acid 60 Xylitolc 152 Total carbon out Total carbon in Carbon recovery (%) g Ethanol/g total sugar
Mass of carbon (g C/mol compound)
60 60 24 12 36 24 60
Carbon mass fractiona (g C/g compound)
0.400 0.400 0.522 0.273 0.391 0.400 0.395
(1) Fig. 1 100 g/l yeast
(2) Fig. 2a, curve B 100 + 50 + 50 g/l yeast
(3) Fig. 2a, curve A 100 + 50 + 50 g/l yeast
Conc. from HPLC (g/l)
Carbon (g/1)
Conc. from HPLC (g/l)
Carbon (g/1)
Conc. from HPLC (g/l)
Carbon (g/1)
15.8 6.2 18.2 17.4b 3.9 2.4 N/A
6.3 2.5 9.5 4.7 1.5 1.0 – 25.5 24.0d
3.7 3.3 25.0 23.0b 3.4 2.0 N/A
1.5 1.3 13.0 6.2 1.3 0.8 – 24.2 24.0d
1.1 3.2 15.6 15.0b 2.2 1.0 N/A
0.4 1.3 8.1 4.1 0.9 0.4 – 15.2 12.0d
106 0.30
100 0.40
127 0.51
a The mass fraction of carbon is computed based on the molecular weight and molecular formula of the compound. For example, xylose/xylulose has a molecular weight of 150 g/mol and 5 C/molecule, so the sugar contains 60 g C/mol or 0.40 g C/g sugar. b CO2 production was not measured, but was estimated based on a 1:1 stoichiometry with ethanol production. c In this experiment, the column configuration used resulted in xylitol co-eluting with borate. However, based on the reproducible area of the borate peak, xylitol formation was likely negligible. d For Experiments 1 and 2, which have an initial xylose concentration of 60 g/l prior to isomerization, the g C coming in is (0.40 g C/g sugar) (60 g sugar/l) = 24 g C/l. In Experiment 3, which has 30 g/l xylose coming in, the carbon in is 12 g C/l.
The yield of ethanol on a mass basis was calculated on the basis of the total initial sugar concentration (g/l). Per the stoichiometry of the above equations, the theoretical yield of ethanol is 0.51 g/g sugar for both glucose and xylulose. Specific details on the carbon balance calculations for the fermentations are provided in the footnotes to Table 1. 3. Results and discussion As shown previously (Chiang et al., 1981), high yeast densities can promote better utilization of C5 sugars. Since the ultimate goal is fermentation of mixtures of C6 and C5 sugars, high cell densities were used in the experiments although these are not required for the fermentation of glucose alone. While such high densities may pose operational issues in traditional stirred tank fermentors, novel fermentor configurations that permit use of high yeast densities for multiple fermentation cycles are being developed (Varanasi et al., 2009). All of the data presented is based on the results of duplicate experiments. Data represent average values with standard deviation typically less than ±8%.
with the two strains is shown in Fig. 1. The initial fermentation rates are higher for BY but the overall ethanol yields are similar for both strains, as was the case for glucose. Both strains were able to convert the xylulose to near theoretical yield for ethanol. Total acetic acid and glycerol amounts of 0.06–0.08 g/g total sugar were observed in both cases, which are similar to literature reports for BY (Hahn-Hägerdal et al., 1986). Xylitol has been found to be a measurable by-product in xylose/ xylulose fermentations (Chiang et al., 1981) when a significant quantity of xylose is present in the medium. Xylitol production tends to increase as the temperature decreases (less than 35 °C) or the pH increases (pH > 5) (Chiang et al., 1981). For these experiments at 34 °C and pH 4.5 with azide, xylitol formation is not likely. A significant reduction in not only xylitol but also glycerol and acetic acid formation was shown previously in SIF with soluble XI and S. cerevisiae in the presence of 4.6 mM azide at pH 6 and 30 °C (Hahn-Hägerdal et al., 1986).
3.1. Xylulose fermentations with BY and ER As noted in Section 2, the additives used for isomerization of xylose had no adverse effect on the glucose fermentation using either BY and ER. The experiments in this section were conducted to assess the relative effectiveness of BY and ER in fermenting xylulose to ethanol. Xylose (60 g/l) was partially isomerized and fermented using BY or ER. The xylose was pre-isomerized to xylulose with 0.05 M borax and 0.01 M urea for a 55% conversion (33 g xylulose/l). By allowing a significant proportion of xylose to remain in the media, the effectiveness of azide in suppressing xylitol formation on the two yeast strains can be assessed. In addition, the 33 g/l xylulose concentration is typical of pentose yields in biomass hydrolyzates following CAFI protocols (Wyman et al., 2005). Fermentation of the 33/27 g/l of xylulose/xylose mixtures was initiated by adding 100 g/l yeast to the media. Ethanol production
Fig. 1. Fermentation of 33 g/l xylulose with both ER and BY. Xylulose was produced by the partial isomerization of 60 g/l xylose with the bi-layered enzyme pellets. Both strains fermented xylulose to ethanol with high yield, but with significantly slower kinetics than for glucose. Both experiments contained 4.6 mM azide to block respiration.
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Fig. 2. Effect of BY addition and initial pentose concentration on xylulose fermentation. Xylose in all experiments (30 or 60 g/l) was pre-isomerized to near 86% conversion to xylulose. Initially, 100 g/l BY was added to the fermentation and in (a) and (b), an additional 50 g/l BY was added at 24 and 40 h. Fed-batch addition of BY significantly improved the ethanol yield at the higher sugar loading. All fermentations contained 4.6 mM azide.
3.2. Effect of initial xylulose concentration and mode of BY addition on xylulose utilization The complete saccharification of 20% (w/v) woody biomass typically yields around 30 g/l of soluble xylose; however, saccharification of other kinds of biomass could lead to higher xylose concentrations. Accordingly, fermentation of 30 and 60 g/l xylose were compared. In the previous experiment on strain evaluation, a significant quantity of xylose was allowed to remain in the media. However, from a practical standpoint, high ethanol yield requires complete xylose isomerization. Accordingly, both 30 and 60 g/l xylose samples were pre-isomerized with 0.1 M urea for 24 h to achieve 86% xylulose, or about 25.8 and 52.2 g/l xylulose, respectively. For BY fermentations at each substrate loading, the change in ethanol, xylose, and xylulose with time are shown in Fig 2. For yeast loadings of 100 g/l (data not shown) or fed-batch addition to 200 g/l, the 25.8 g/l xylulose was fermented completely to ethanol (see Fig. 2a, curve A). When the initial concentration of xylulose was doubled with 100 g/l yeast loading, only 40 g/l xylulose was fermented (Fig. 2a, curve C), yielding 20.6 g/l ethanol (0.4 g/g) in 70 h. To determine if fed-batch addition of yeast could lead to more effective sugar utilization at the higher xylulose concentration, 100 g/l yeast was added initially, followed by addition of 50 g/l yeast at 24 and 40 h (Fig. 2, curve B). The addition of dry yeast cells at 24 h resulted in an increase in the kinetics of the fermentation (compare Fig. 2a, curves B and C after 24 h). The final ethanol yield increased from 0.4 to 0.51 g EtOH/g xylulose. The total formation of by-products (acetic acid and glycerol) was similar at both cell densities (5.2–5.3 g/l). The fact that doubling the yeast density through fed-batch addition did not result in a significant increase in by-product formation might suggest that the by-products derive primarily from the minor quantity of xylose in the initial sugar mixture (Fig. 2b). The higher initial xylulose concentrations may lead to rapid accumulation of ethanol intracellularly, approaching levels that could be inhibitory for pentose fermentations (Chiang et al., 1981). Fermentation by the fresh yeast is not biased by intracellular accumulation of ethanol, and hence the yeast added in fedbatch mode is able to more efficiently utilize the available xylulose. Just as ER is adapted to ethanol tolerance for glucose fermentation, yeasts could perhaps be adapted to ethanol tolerance for xylulose fermentation as well.
Thus, a properly-strategized fed-batch addition of yeast provides an opportunity to not only achieve high utilization of xylulose but also to significantly shorten the time required for fermentation. 3.3. Carbon balance for xylose fermentations In addition to ethanol, other compounds such as glycerol, acetic acid, xylitol, and CO2 are produced during fermentation. The carbon balances for the fermentations of xylose/xylulose mixtures corresponding to the cases discussed in Figs. 1 and 2 are presented in Table 1. Details on the carbon balance procedure are provided in the footnotes to Table 1. Cell mass of the cultures was not measured as yeast growth was expected to be minimal under anaerobic fermentation on xylulose with azide. The major by-products detected in the fermentations were glycerol and acetic acid. The carbon balances for Experiments 1 and 2 with 60 g/l initial sugar were satisfactory; the mass balance for Experiment 3 shows an accumulation of carbon, possibly due to reduced accuracy in HPLC measurements at the lower concentrations measured. A similar over-estimation of carbon (10%) was reported in the literature for xylulose fermentations using S. cerevisiae ATCC 24860 (Yu et al., 1995). 3.4. Fermentation of mixed sugars The ultimate goal of the bi-layered enzyme technology is its implementation with biomass hydrolyzates. Hydrolyzates may contain various fermentation inhibitors depending on the method of biomass pretreatment employed. Accordingly, detoxification/ conditioning may be necessary prior to the isomerization/fermentation. Removal of fermentation inhibitors prior to isomerization will also eliminate the risk of inhibition of the XI (Lindén and Hahn-Hägerdal, 1989) and urease activities. A recently developed ionic liquid pretreatment method (Varanasi et al., 2008) was shown to produce hydrolyzate free of the fermentation inhibitors traditionally found with dilute acid pretreatment. Thus, as new pretreatment methods are developed, the need for hydrolyzate preconditioning may be eliminated. Even in the absence of fermentation inhibitors, when S. cerevisiae are presented with mixtures of glucose and xylulose, the overall ethanol yields can be much different than that achieved with either sugar separately. Accordingly, the experiments in this section focus on simulated glucose/xylulose mixtures.
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Fig. 3. Mixed sugar fermentation of 90 g/l glucose and 30 g/l xylose (pre-isomerized to xylulose) without azide (a–c) and with 4.6 mM azide (d–f). Three yeast loadings were tested: 50 g/l (a,d); 100 g/l (b,e); and 200 g/l (c,f).
3.4.1. Effect of yeast loading on fermentation of mixed sugars Mixed sugar fermentations were conducted with 90 g/l glucose and 30 g/l xylose both with and without the addition of azide. To ensure immediate fermentable sugar availability, the xylose was first pre-isomerized for 24 h to obtain 86% xylulose. Fermentations were performed with three yeast loadings (50, 100, and 200 g/l). Results of these fermentations in the absence of azide are shown in Fig. 3a–c. In all cases, glucose was consumed within the first 5 h. As yeast loading increases from panels a to c, the overall ethanol yield increases and the by-product formation decreases by about 20%. With 50 g/l yeast, more than 6 g/l xylulose remain after 28 h of fermentation. At this low yeast loading, uptake of xylulose may limit its utilization. At the higher yeast loadings shown in Fig 3b and c, xylulose utilization is much improved as is the overall ethanol production, reflecting better pentose utilization kinetics and co-utilization of the sugars. To assess the impact of azide in mixed sugar fermentations, the fermentations shown in Fig. 3a–c were repeated with 4.6 mM azide (see Fig. 3d–f). By-product formation was about half of that seen without azide while ethanol yields are generally increased. However, xylulose uptake is significantly slower in the presence of azide. This indicates that the observed increase in ethanol yield in the presence of azide results from the glucose portion but not necessarily from xylulose utilization. In pure xylulose fermentations, close to theoretical yields of ethanol were achieved under similar conditions. The incomplete utilization of xylulose in mixed sugar fermentations suggests that ethanol produced from glucose fermentation may inhibit xylulose uptake and utilization. If this were the case, fed-batch addition of yeast or use of an ethanol-tolerant yeast strain (i.e. ER) could prove beneficial; these strategies are tested next. 3.4.2. Effect of mode of addition and strain of yeast on mixed sugar fermentation The fermentation of two mixed sugar compositions, (1) 90 g/l glucose and 30 g/l xylose and (2) 130 g/l glucose and 50 g/l xylose,
were conducted with fed-batch addition of 50 g/l yeast at time 0, 4, 7 and 24 h to a final concentration of 200 g/l. The fermentations were conducted with either BY or ER in the presence of 4.6 mM azide. Xylose was pre-isomerized to 86% conversion prior to fermentation for all cases. In an attempt to more-closely produce anaerobic conditions, nitrogen was bubbled into the shake flasks for 60 s to purge oxygen after each yeast addition. Ethanol production for 120 g/l total sugars fermented with 200 g/l yeast added either initially or in fed-batch mode are shown in Fig. 4a. The overall ethanol yield increased from 0.38 to 0.42 g/g total sugar when yeast was switched from initial to fed-batch addition. Under these conditions, ER proved to be a marginally better fermentor of mixed sugars (ethanol yield of 0.46 g/g total sugar) than BY with fed-batch addition. At very high biomass loadings (30%) during saccharification, the hydrolyzates generated could have up to 180 g/l of mixed sugars. Complete conversion of these sugars to ethanol would yield 9% (w/v) ethanol in the fermentation broth – a concentration level that is known to inhibit BY. Since ER is known to tolerate up to 15% (w/v) ethanol in corn-to-ethanol fermentations, and it performed well with 120 g/l mixed sugars, its performance at these very high sugar concentrations was tested next. Ethanol yields from the fermentation of 130 g/l glucose and 50 g/l xylose with fed-batch yeast addition are shown in Fig. 4b. Ethanol yields at the end of 72 h were 0.35 and 0.41 g ethanol/g total sugar from BY and ER, respectively. For BY, some reassimilation of ethanol occurred after the first 24 h. The carbon balances for these experiments closed very well (see Table 2). The closure of the carbon balances is likely due to the high yeast loadings in combination with the anaerobic nitrogen blanket in the fermenting flask. Fed-batch yeast addition resulted in better sugar utilization and about 25% higher total product formation, which is reflected as higher ethanol as well as by-product production (see Table 2). ER clearly shows less ethanol inhibition at these high sugar loadings than BY. This result is consistent with the hypothesis that an ethanol-tolerant yeast strain may be less
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Fig. 4. Mixed sugar fermentations with BY and ER in fed-batch yeast addition mode. Xylose was pre-isomerized to 86% conversion for all experiments prior to fermentation; 4.6 mM sodium azide was used in all experiments. The fed-batch yeast addition was implemented by adding 50 g/l of yeast at 0, 4, 7 and 24 h to a total of 200 g/l. Nitrogen was purged into the shake flask after each yeast addition and sample collection to maintain anaerobic conditions. Ethanol production is shown for (a) for 120 g/l sugar (90 g/l glucose and 30 g/l xylose) fermentation and (b) 180 g/l total sugar (130 g/l glucose and 50 g/l xylose) fermentation.
Table 2 Summary of mixed sugar fermentation results from Fig. 4 with carbon balance results. Values shown are at 72 h for fed-batch (fb) yeast addition and at 64 h for initial (i) yeast addition. All experiments contain a total of 200 g/l yeast. Xylitol was not measured as these experiments contain low starting xylose concentrations and are nitrogen-blanketed; carbon balances close well for all experiments. Total sugar (g/l)
Yeast – addition mode
Initial carbon (g/l)
Ethanol (g/l)
Ethanol yields (g/g total sugar)
Sugar remaining (g/l)
Glycerol (g/l)
Acetic acid (g/l)
Carbon accounted (g/l)
Carbon recovery (%)
120
BY – i BY – f b ER – f b BY – f b ER – f b
48 48 48 72 72
45.26 50.51 55.62 62.71 73.46
0.377 0.421 0.463 0.348 0.408
11.51 9.54 7.71 32.65 24.23
5.71 7.57 7.63 8.66 9.10
1.79 1.41 2.17 1.56 2.32
42.98 46.9 50.5 66.2 71.7
89.53 97.7 105.1 91.9 99.5
180
3.4.3. Xylulose uptake in mixed sugar fermentations for BY Transient xylulose concentrations for three different fermentations with 100 g/l BY loading and the same initial xylulose concentration are compared in Fig. 5. Curve C shows that the most rapid uptake of xylulose occurred in the mixed sugar fermentation without azide (see also Fig. 3b). In contrast, when azide is added, xylulose uptake rate is reduced (Fig. 5, curve B, see also Fig. 3e). Indeed, the xylulose uptake in curve B is even slower than observed with pure xylulose alone (curve A) where ethanol inhibition resulting from glucose fermentation is not possible. It may be that xylulose utilization kinetics are hampered by the combined effects of azide and accumulated intracellular ethanol. Based on the experiments summarized in Figs. 3–5, it was concluded that the most efficient implementation of SIF for mixed sugar fermentations with BY is high yeast loadings without addition of azide to the media. Under such conditions, xylulose uptake is benefited by the availability of sufficient transporters (Hamacher et al., 2002; Hector et al., 2008; Lee et al., 2002) as well as reduced ethanol inhibition. Accordingly, these conditions were selected for a comparison of SIF to partial SIF. Fig. 5. Xylulose uptake in pure and mixed sugar fermentations. All experiments were conducted with an initial yeast loading of 100 g/l and with 30 g/l xylose preisomerized to yield approximately 25.8 g/l xylulose; Experiments A and B contained 4.6 mM azide. Mixed sugar fermentations contained 90 g/l glucose which was consumed within the first 4 h (not shown).
susceptible to the synergistic inhibition effects of azide and ethanol. This inhibitory effect may be more clearly seen in Fig. 5 where xylulose uptake rates are compared for BY with and without azide on pure pentose and mixed sugars.
3.4.4. Simultaneous-isomerization-and-fermentation of mixed sugars Isomerizations and fermentations were conducted simultaneously on mixed sugars containing 60 g/l glucose and 30 g/l xylose in media without azide. Three separate experiments were conducted to assess the effect of the extent of xylose isomerization to xylulose on ethanol yield. In these experiments, the bi-layered pellets were (a) not added to the fermentation medium (control run); (b) added with borax and yeast; or (c) added first, followed
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xylose) was 67%; about 20% of the xylose was consumed. Byproducts formed included acetic acid, glycerol and xylitol. When the bi-layer pellets were added with 0.05 M borax (Fig. 6b), the overall ethanol yield increased from 67% to 74% (±1%), with about one third of the initial xylose remaining at 10 h. The facilitated transport shuttle set up by borax allows appreciable conversion of xylose to xylulose, presenting yeast with xylulose to ferment. However, as the SIF proceeds, the rate of xylose isomerization decreases due to xylitol accumulation within the fermentation broth. Xylitol formation began after glucose was depleted and rose gradually to 2.5 g/l. These experiments, while proving the viability of SIF, point to the need to address issues associated with the slow rate of isomerization in this configuration. The activity of XI can be adversely affected by the xylitol formed from xylose during fermentation in SIF. Xylitol formation from xylose fermentation is known to be an issue under aerobic conditions; strict anaerobic conditions were difficult to achieve. Recently, XI that is much less prone to xylitol inhibition was successfully produced as a recombinant protein (Brat et al., 2009). Using this new XI enzyme in combination with strict anaerobic conditions during SIF can lead to higher xylulose concentrations and higher ethanol yield. Since xylitol-inhibitionresistant XIs are not yet commercially available, in the subsequent experiment, the bi-layered pellets were used to achieve high xylulose concentrations at pH conditions suitable for fermentation prior to adding yeast to the media. In Fig 6c, the xylose was pre-isomerized to 70% xylulose and then yeast were added. These high initial xylulose concentrations led to a further improvement over Fig. 6b with ethanol yield approaching 81%. Indeed, the partial isomerization to xylulose prior to fermentation leads to lower xylitol production contributing to the increased ethanol productivity. The fact that XI isomerizes both glucose and xylose does not adversely affect either the xylulose yield or the C6 fermentation. The affinity of XI for xylose is 160 times greater than its affinity for glucose (Bhosale et al., 1996). Even in proportions found in biomass hydrolyzates (concentration of glucose 3–6 times higher than xylose), isomerization of xylose to xylulose will dominate. Moreover, since glucose is rapidly fermented by yeast, fructose formation is likely to be very low (fructose was not detected in the experiments described in Fig. 6b and c). Even if small quantities of fructose are formed, S. cerevisiae ferments fructose as well as glucose. 4. Conclusions
Fig. 6. Simultaneous isomerization and fermentation (SIF) of 60 g/l glucose and 30 g/l xylose. Fermentation with (a) no isomerization of xylose; (b) SIF; and (c) preisomerization of xylose followed by SIF. Data shown are the average values of duplicate experiments; glucose values have standard deviation within ±8%; all other values have standard deviation within ±3%. For all experiments shown in Fig. 3, carbon balances closed to within 3% (data not shown). Ethanol produced in (c) is slightly lower than (b) due to lower initial sugar concentrations. Yield of ethanol per total sugar for the experiments are (a) 67%, (b) 74%, and (c) 81%.
Recently, a method for isomerizing xylose to xylulose at high yield was demonstrated wherein the xylulose complexing agent, pH, and temperature are compatible with fermentation. Thus, isomerization and fermentation could be conducted sequentially, in fed-batch, or simultaneously to maximize utilization of both C5 and C6 sugars and ethanol yield. For the first time, it was demonstrated that industrially proven yeast strains such as ethanol red can convert high concentrations of mixed sugars to ethanol in times of about 30 h with yields comparable to engineered strains. This exogenous isomerization method avoids several issues with which GMO yeasts incorporating XI have to contend. Acknowledgements
by yeast addition after partial isomerization of the sugars with borax. As shown in Fig. 6, glucose was completely consumed within 1–2 h for all three cases. In the absence of added bi-layer pellets (Fig. 6a), the ethanol yield based on total sugar (glucose and
The authors acknowledge the support of this research through grants from United States Department of Energy Office of the Biomass Program (GO18163) and Ohio Third Frontier Advanced Energy Program (08-021). Mention of trade names or commercial products does not constitute an endorsement or recommendation for use.
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