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The influence of presaccharification, fermentation temperature and yeast strain on ethanol production from sugarcane bagasse
Bioresource Technology 109 (2012) 63–69
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The influence of presaccharification, fermentation temperature and yeast strain on ethanol production from sugarcane bagasse Carlos J.A. de Souza a, Daniela A. Costa a,b, Marina Q.R.B. Rodrigues a, Ancély F. dos Santos a, Mariana R. Lopes a, Aline B.P. Abrantes a, Patrícia dos Santos Costa a, Wendel Batista Silveira b, Flávia M.L. Passos b, Luciano G. Fietto a,⇑ a b
Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Av. PH Rolfs s/n, Campus Universitário, Viçosa 36571-000, MG, Brazil Departamento de Microbiologia, Universidade Federal de Viçosa, Av. PH Rolfs s/n, Campus Universitário, Viçosa 36571-000, MG, Brazil
a r t i c l e
i n f o
Article history: Received 12 September 2011 Received in revised form 20 December 2011 Accepted 7 January 2012 Available online 15 January 2012 Keywords: Simultaneous saccharification and fermentation Ethanol Sugarcane bagasse Thermotolerant yeast
a b s t r a c t Ethanol can be produced from cellulosic biomass in a process known as simultaneous saccharification and fermentation (SSF). The presence of yeast together with the cellulolytic enzyme complex reduces the accumulation of sugars within the reactor, increasing the ethanol yield and saccharification rate. This paper reports the isolation of Saccharomyces cerevisiae LBM-1, a strain capable of growth at 42 °C. In addition, S. cerevisiae LBM-1 and Kluyveromyces marxianus UFV-3 were able to ferment sugar cane bagasse in SSF processes at 37 and 42 °C. Higher ethanol yields were observed when fermentation was initiated after presaccharification at 50 °C than at 37 or 42 °C. Furthermore, the volumetric productivity of fermentation increased with presaccharification time, from 0.43 g/L/h at 0 h to 1.79 g/L/h after 72 h of presaccharification. The results suggest that the use of thermotolerant yeasts and a presaccharification stage are key to increasing yields in this process. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction During the past decades, the increasing demand for energy has encouraged the search for renewable alternative energy sources. Among these, ethanol fuel has received much attention. Recently, the international ethanol market has been stimulated by government incentive offers for using renewable fuels (Demain, 2009; Hahn-Hagerdal et al., 2006; Van Maris et al., 2006). Lignocellulosic biomass is one of the salient alternatives that is being considered for large-scale ethanol production and will likely be the major feedstock for ethanol production in the near future (Mussatto et al., 2010). Lignocellulosic wastes, such as sugarcane bagasse, are alternative, low-cost feedstocks that can be enzymatically hydrolyzed to fermentable sugars for subsequent biofuel production. Brazil has a large supply of this feedstock, which is an abundant product of the sugar and alcohol industries; it is estimated that about 186 million tons of bagasse are generated in Brazil per year (Cardona et al., 2010; Soccol et al., 2010). According to Pandey et al. (2000), half of the sugarcane bagasse generated is used in power plants for energy production, while the other half has no set destination. Thus, it is necessary to develop new technologies for the utilization of sugarcane bagasse generated in Brazil. In this
⇑ Corresponding author. Tel.: +55 31 3899 3046; fax: +55 31 3899 2373. E-mail address: lgfi[email protected] (L.G. Fietto). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.01.024
sense, this feedstock emerges as a promising alternative for use in ethanol production (Cardona et al., 2010). The first step in the conversion of sugarcane bagasse into ethanol is pretreatment, which alters the lignocellulosic structure to allow enzyme access in the hydrolysis step (Alvira et al., 2010; Hendriks and Zeeman, 2009). After pretreatment, there are several ways to conduct subsequent steps in the process. One promising method is to simultaneously carry out the hydrolysis and fermentation steps in a process called simultaneous saccharification and fermentation (SSF) (Cardona et al., 2010; Olofsson et al., 2008; Takagi et al., 1977). SSF was designed to decrease the inhibition of cellulases by hydrolysis products, mainly glucose, which is readily consumed by yeast cells. Therefore, the SSF process costs are decreased due to the reduced load of enzymes required for the enzymatic hydrolysis of biomass (Olofsson et al., 2008; Sun and Cheng, 2002). In addition, the use of a single fermenter for the entire process may reduce the investment costs (Ballesteros et al., 2004). The major problem associated with the SSF process is the different optimum temperatures for saccharification and fermentation. The optimum temperature for cellulase enzymes is higher than can be tolerated by most yeast strains used for industrial ethanol production. This drawback can be overcome by using thermotolerant yeast strains (Hari-Krishna et al., 2001; Olofsson et al., 2008). In fact much effort has been put forth to identify thermotolerant yeasts that have potential for use in the ethanol industry, mainly in the SSF process for second generation ethanol
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production, with the most promising results being obtained with Kluyveromyces marxianus strains. The present study evaluated and compared the fermentation capacity of sugarcane bagasse in SSF processes at 37 and 42 °C using two thermotolerant strains, Saccharomyces cerevisiae LBM1 isolated from fermentation vats in Brazil and UFV-3, a K. marxianus strain identified by Silveira et al. (2005) that showed good fermentation results when converting lactose in cheese whey to ethanol. Furthermore, the influence of presaccharification, fermentation temperature and yeast strain on ethanol production were evaluated. 2. Methods 2.1. Isolation of yeast strain and growth conditions The S. cerevisiae strain used in this study was isolated from fermentation vats in the state Minas Gerais, Brazil using the modified method of Oliveira et al. (2008). Samples were collected from fermentation vats in sterile 150-mL flasks. Samples were diluted 1:100,000 in sterile water in triplicate and were inoculated on YNB medium supplemented with ampicillin at 30 °C for 3 days. The isolates were first classified on the basis of morphological characteristics and then tested for their ability to grow at different temperatures (37 and 42 °C) in the presence of 10% (w/v) ethanol. Selected colonies were grown on YP medium containing 2% (w/v) glucose supplemented with 10% (w/v) ethanol at 37 °C. The selected isolates underwent molecular identification of the D1/D2 region of large subunit rRNA gene (Rosa and Lachance, 1998). PCR was performed from total DNA samples using the primers NL1 and NL4 as previously described (Rosa and Lachance, 1998). The amplified DNA was concentrated, cleaned using the PEG method (Sambrook and Russell, 2001) and sequenced in a Mega-BACE™ 1000 automated sequencing system (Amersham Biosciences) in the Institute of Biological Sciences of the Federal University of Minas Gerais, Brazil. DNA sequences were analyzed using the program blastn (Basic Local Alignment Search Tool 2.2.24-BLAST version 2.0) available at National Center for Biotechnology Information (NCBI) website (Altschul et al., 1997). The sequences were compared with sequences already deposited in GenBank. Sequence alignment was performed using the program CLC Main Workbench 5.7.1. Active cultures of K. marxianus UFV-3 and S. cerevisiae LBM-1 for inoculation were prepared by growing the organisms using a rotary shaker at 180 rpm for 16 h at 28 °C in YPD medium (1% yeast extract, 2% peptone and 2% glucose).Yeast growth at different temperatures was analyzed by plating 5 ll of culture at dilutions of 1:0, 1:10 and 1:100 on agar plates containing YPD medium (1% yeast extract, 2% peptone, 4% glucose and 2% agar). The plates were incubated at 28, 37, 42 or 45 °C for 72 h. The dilutions were prepared in sterilized saline solution (0.85% NaCl) from active cultures with an optical density (OD)600nm of 0.5. 2.2. Raw material and pretreatment 2.2.1. Acid pretreatment The crushed sugarcane bagasse, Saccharum sp., at a concentration of 10% (w/v) in 0.5% (v/v) H2SO4 was pretreated by autoclaving at 121 °C for 30 min. The solid and liquid fractions were then separated by vacuum filtration through filter paper (Whatman No. 5). The solid residue was washed with 500 mL of deionized water and dried at 50 °C for 24 h. 2.2.2. Acid and alkaline pretreatment The solid residue obtained upon acid pretreatment of sugarcane bagasse was treated with alkali to increase cellulose accessibility
by partially removing lignin. This pretreatment was conducted by mixing 10 g of solid with a 4% (w/v) NaOH solution, which was then subjected to thermal treatment at 121 °C for 30 min. The solid residue was washed with 500 mL of deionized water and dried at 50 °C for 24 h. 2.3. Hydrolysis tests Hydrolysis was carried out in duplicate under mild agitation at 180 rpm in 125-mL flasks. Hydrolysis was performed using the solid fraction obtained after pretreatment of the substrate at 8% (w/v) concentration suspended in 30 mL of the fermentation medium (yeast extract, 2.5 g/L; peptone, 2.5 g/L; NH4Cl, 2 g/L; KH2PO4, 1 g/L and MgSO47H2O, 0.3 g/L) in citrate buffer (50 mM, pH 4.8), supplemented with 15 Filter Paper Units (FPU) per gram of substrate. The commercial cellulase preparation used in this work was Celluclast 1.5 L (Sigma–Aldrich, Brazil). Samples were collected every 12 h and centrifuged for 15 min at 10,000g, and the supernatants were used to quantify carbohydrates by high performance liquid chromatography (HPLC). 2.4. Saccharification and fermentation tests SSF (simultaneous saccharification and fermentation) and SHF (separate hydrolysis and fermentation) experiments were carried out according to the pretreatment and presaccharification conditions described above. In the SSF and SHF assays, flasks were inoculated with yeast cultures using the following presaccharification conditions: 0, 24 or 72 h at 37, 42 or 50 °C. The fermentation was initiated by adding yeast culture to an OD600nm of 2. SSF assays were conducted under sterile conditions at 37 or 42 °C for 24 h. Samples were collected every 2 h and centrifuged for 15 min at 10,000g. The supernatants were used to quantify carbohydrates and ethanol by HPLC. In the SHF assays the supernatants of hydrolysis were separated from biomass by centrifuging prior the yeast inoculation. 2.5. Determination of hydrolysis and fermentation parameters The presaccharification yield (YG/B) was calculated by dividing the difference between the initial (Glui) and final (Gluf) glucose mass (g) by the initial mass of pretreated biomass (g) (Biomass):
Y G=B ¼
Gluf Glui Biomass
ð1Þ
The ethanol yield (YE/B) was calculated at the end (8 h) of fermentation by dividing the difference between the final (EtOHf) and initial (EtOHi) ethanol mass (g) by the initial pretreated biomass mass (g):
Y E=B ¼
EtOHf EtOHi Biomass
ð2Þ
The volumetric productivity (Qp) was calculated by dividing the maximum concentration of ethanol (g/L) achieved by the time of fermentation in hours:
QP ¼
EtOHf t
ð3Þ
2.6. Analytical methods 2.6.1. Glucose and ethanol concentrations Quantitative determinations of glucose and ethanol concentrations were carried out by HPLC using an ion exclusion column Aminex HPX-87H (Bio-Rad) kept at 60 °C. The eluent for separation
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was 5 mM H2SO4 applied at an elution rate of 0.7 mL/min. The column was coupled to the HP 1047 A refractive index detector. 2.7. Statistical analysis Data for comparison of the pretreatment, presaccharification and fermentation processes were obtained by experiments repeated in triplicate and analyzed using ANOVA followed by Duncan’s test for means. The statistical analyses were performed with SAEG demo version using 95% confidence (a = 0.05). The influence of presaccharification time, fermentation temperature and yeast strain on the ethanol yield was assessed using a factorial design repeated twice (23 2 = 16 assays) (Table 1). This experiment was conducted at 50 °C during the presaccharification step. The data from this experiment were analyzed by ANOVA followed by a t-test for the effects of each factor and interactions between them. The analysis and graphing were performed with MINITAB15 using a cutoff of 95% confidence (a = 0.05). 3. Results and discussion 3.1. Growth pattern of the yeast strains S. cerevisiae LBM-1 and K. marxianus UFV-3 at different temperatures Of the 50 strains of S. cerevisiae isolated from Brazilian alcohol manufacturing plants that were able to grow at 37 °C in YNB medium containing 4% glucose (w/v) and 10% ethanol (w/v), S. cerevisiae LBM-1 was chosen for this study because it had the fasted growth rates in assays conducted at different temperatures. The strain was identified based on sequence analysis of the D1/D2 region of large subunit rRNA gene. The sequence was 100% identical to the S. cerevisiae sequence in GenBank. The K. marxianus UFV-3 strain used in this work was identified by Silveira et al. (2005). This strain was chosen for this work because it is able to convert lactose to ethanol with a yield close to the theoretical value. Furthermore, this strain is also able to convert glucose to ethanol efficiently. Fig. 1 shows that all tested dilutions of the two strains grew at temperatures of 30, 37 and 42 °C, though only K. marxianus UFV-3 was able to grow at 45 °C. The thermotolerance of the species K. marxianus is well documented, while it is rare in S. cerevisiae with few strains described (Sree et al., 2000). Thermotolerant K. marxianus strains isolated by Banat et al. (1992) were tested for the ability to convert biomass into ethanol in the SSF process. Faga et al. (2010) showed that the K. marxianus strain IMB3 was able to ferment switchgrass in the SSF process at 45 °C. The same study found that the ethanol yield produced by IMB3 was similar to that of S. cerevisiae D5A at 37 °C. However, processes using S. cerevisiae as a fermenting organism are performed at 37 °C because these yeasts generally are not able to grow at temperatures above 40 °C (Santos et al., 2010; Rudolf et al., 2007; Suryawati et al., 2008). Because K. marxianus UFV-3 and S. cerevisiae LBM-1 grew at both 37 and 42 °C, it was decided to evaluate the steps of hydroly-
sis and fermentation to produce cellulosic ethanol at these temperatures. Yields from each step were calculated to determine whether a cooling step at the transition from hydrolysis to fermentation could be avoided, which would reduce processing costs. 3.2. Pretreatment analysis Mill-dried in natura sugarcane bagasse was pretreated with diluted sulfuric acid and resulted in a 55% yield in relation to the initial bagasse mass. In the acid–alkali pretreatment, there was a 30% yield. Sugarcane bagasse contains around 20–24% lignin, 27–32% hemicelluloses, 32–44% cellulose and 4.5–9.0% ashes (Soccol et al., 2010). In the presented experiments, the acid treatment removed around 45% of the initial mass, which is equivalent to the hemicellulose fraction and the portion of cellulose fraction that may undergo partial hydrolysis during treatment with diluted acid. In addition to the hemicellulose fraction, the acid–alkali treatment removed the lignin fraction, representing approximately 30% of the biomass. Therefore, the final yield of 30% is compatible with a 70% loss in mass and is equivalent to the cellulose fraction of the bagasse. To verify which pretreatment was the most effective in terms of enzyme accessibility to the substrate, hydrolysis was conducted using bagasse with and without alkali pretreatment. After 24 h of hydrolysis at 42 °C using only acid-pretreated bagasse, 8.9 g/L of glucose were released while 18.36 g/L of glucose were released when the acid–alkali pretreatment was used. The average glucose yields for the two treatments differed significantly according to ANOVA (p-value = 0.005). Twofold more glucose was released after hydrolysis with the acid–alkali pretreatment than the acid pretreatment. With the removal of lignin and hemicellulose by the acid–alkali pretreatment, cellulose became more accessible to the enzymes in the hydrolysis step. According to Santos et al. (2010), the presence of lignin limits the process of enzyme diffusion in the substrate and consequently the release of glucose. Vásquez et al. (2007) noted the same effect when analyzing the hydrolysis of sugarcane bagasse subjected to sulfuric acid pretreatment or to acid–alkali pretreatment (sulfuric acid and sodium hydroxide). In that study, the authors observed that after 24 h of hydrolysis at 50 °C using 7% (w/v) bagasse and 20 FPU of cellulases per gram of substrate, approximately 10 and 20 g/L of glucose were released with the acid pretreatment and the acid–alkali pretreatment, respectively. In this work similar results were obtained with the acid–alkali pretreatment using 15 FPU of enzyme and 8% (w/v) of pretreated bagasse. The acid–alkali pretreatment was selected for use in subsequent experiments because it showed the best hydrolysis yield. 3.3. Hydrolysis Enzymatic hydrolysis and fermentation processes were conducted in the same batch and were assumed to be a SSF process. Initially, the alkali-delignified biomass underwent a presaccharifi-
Table 1 b E=B Þ in factorial design (23) for time of presaccharification, temperature of fermentation and yeast strain. Means obtained ðY E=B Þ and fitted ð Y Time of presaccharification (h)
Temperature of fermentation (°C)
Yeast strain
Y E=B
^E=B y
24 24 24 24 72 72 72 72
37 37 42 42 37 37 42 42
S. cerevisiae LBM-1 K. marxianus UFV-3 S. cerevisiae LBM-1 K. marxianus UFV-3 S. cerevisiae LBM-1 K. marxianus UFV-3 S. cerevisiae LBM-1 K. marxianus UFV-3
0.0927 0.0907 0.8625 0.0980 0.1655 0.1535 0.1630 0.1795
0.1104 0.1051 0.1100 0.1158 0.1653 0.1538 0.1630 0.1798
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Fig. 1. Growth of the S. cerevisiae LBM-1 and K. marxianus UFV-3 strains on YPD medium (4% glucose) over a 24-h period at 30, 37, 42 or 45 °C. The numbers above the figures represent the dilutions of a culture with an OD(600nm) of 0.5.
cation step to provide a source of fermentable carbon for the yeast. An assay was conducted to evaluate the timing of hydrolysis for better yield, i.e., the best time to inoculate the yeast to start the fermentation process. By comparing the SSF process with and without the presaccharification period, Santos et al. (2010) noted that presaccharification led to faster conversion of cellulose into glucose and more conversion of cellulose into ethanol. Moreover, presaccharification led to higher solubilization of the substrate in the mixture. This effect was also noted by Martín et al. (2008) when assessing the effect of presaccharification in fermentation yield of clover–ryegrass. In the present work, the hydrolysis process was performed at 50 °C for 72 h, as shown in Fig. 2A. After 48 h, the rate of glucose release decreased. It should be noted that in the 0–24 h period of hydrolysis, there was an increase of 20 g/L, while in the 48–72 h period, the increase was only 2.5 g/L of glucose. Santos et al. (2010) obtained similar results when studying delignified bagasse hydrolysis, and it is likely that in this period, cellulases start to be inhibited by the hydrolysis products, including glucose and cellobiose. In this study, approximately 2% glucose was detected after 24 h of hydrolysis at 50 °C. At this point, the cellulases were not yet inhibited by the hydrolysis products and the amount of glucose released was sufficient to start fermentation. Furthermore, the same hydrolysis assay was performed at 37 and 42 °C. After 24 h of hydrolysis, 12.72 g/L glucose was obtained at 37 °C and 16.19 g/L at 42 °C (Fig. 2B). The glucose yields for the three temperatures are significantly different according to ANOVA (p-value = 0.000) followed by Duncan means test (comparison done at a = 0.05). Thus, 24 h of presaccharification at 37, 42 or 50 °C were chosen for this stage of the process. 3.4. Simultaneous saccharification and fermentation Saccharification and fermentation were performed simultaneously at 37 or 42 °C after 24 h of presaccharification at 37, 42 or 50 °C. For all temperatures tested, results indicated that after 8 h of fermentation, the glucose concentration was close to zero and no additional increases in ethanol concentration were seen after that time (Fig. 3), indicating the end of the fermentation process. When presaccharification and fermentation processes were performed at 37 °C, the final ethanol concentration was 6.12 g/L for S.
Fig. 2. Time course of enzymatic hydrolysis of pretreated bagasse at laboratory scale at 50 °C (A) and enzymatic hydrolysis after 24 h at 37, 42 and 50 °C (B) with 8% initial solids content (w/v) and an enzyme dosage of 15 FPU per gram dry matter.
cerevisiae LBM-1 and 6.14 g/L for K. marxianus UFV-3 (Fig. 3A). At 42 °C, the two strains exhibited the same fermentation behavior,
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and similar ethanol concentrations were obtained at the end of the process (Fig. 3B). However, with presaccharification at 50 °C followed by fermentations at 37 or 42 °C, higher ethanol concentrations were attained for both species (Fig. 3C and D). Higher ethanol yields were observed when fermentation was initiated after presaccharification at 50 °C than at 37 or 42 °C (Table 2), indicating that for the two strains, the presaccharification temperature is more important to ethanol yield than the fermentation temperature. When analyzing the data showed in Fig. 2 and in Table 2, it can be seen that ethanol yield depends on the initial glucose concentration, as higher yields were achieved when fermentation was initiated with a higher glucose concentration. To confirm this result, another SSF experiment was carried out in which fermentation was started after 72 h of presaccharification at 50 °C with an initial yeast concentration equal to an OD600nm of 2. Under these conditions, the final ethanol concentration was approximately 15 g/L (Fig. 3E and F). Thus, it was clear that the ethanol yield was higher when fermentation was started after 72 h of
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presaccharification compared to 24 h (Table 2) due to the higher glucose concentration at the start of fermentation. Positive influences on ethanol production can be seen when the ethanol yields of strains UFV-3 and LBM-1 are compared with those obtained by Santos et al. (2010), Tomás-Pejó et al. (2009) and Ballesteros et al. (2004). Santos et al. (2010) used an S. cerevisiae strain that ferments at 328 °C, which is not useful in the SSF process and resulted in a lower ethanol yield compared to that of strains UFV-3 and LBM-1. Tomás-Pejó et al. (2009) obtained higher ethanol concentrations than those achieved in this study by using a K. marxianus strain that ferments at 42 °C. However, these authors evaluated crystalline cellulose as a substrate, which is known to be more accessible to enzymes in the hydrolysis stage than bagasse. Ballesteros et al. (2004) obtained ethanol concentrations close to those attained in this study using thermotolerant yeasts, emphasizing the importance of the use of these strains in the fermentation process to increase ethanol yield and avoid contamination, especially when envisioning a larger-scale process.
Fig. 3. Glucose consumption and ethanol formation over 10 h by thermotolerant K. marxianus UFV-3 and S. cerevisiae LBM-1, with (A) SSF at 37 °C and (B) SSF at 42 °C after 24 h of presaccharification at 37 and 42 °C, respectively; (C) SSF at 37 °C and (D) SSF at 42 °C after 24 h of presaccharification at 50 °C; (E) SSF at 37 °C and (F) SSF at 42 °C after 72 h of presaccharification at 50 °C with (N)UFV-3/glucose, (4) UFV-3/ethanol, (j) LBM-1/glucose, (h) LBM-1/ethanol.
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Table 2 Ethanol yields (YE/B) from different combinations of presaccharification and fermentation temperatures for LBM-1 and UFV-3. Temperature of presaccharification (°C)
Temperature of fermentation (°C)
YE/B S. cerevisiae LBM-1
YE/B K. marxianus 2
37 42 50 50
37 42 37 42
0.0734 0.0643 0.1124 0.1082
0.0737 0.0821 0.1079 0.1139
A B C C
A A C C
Means followed by the same letter are not significantly different by Duncan’s test (comparisons done at a = 0.05).
To evaluate the influence of the presaccharification time and fermentation temperature (quantitative factors) and of the yeast strain (qualitative factor) on ethanol yield, a factorial design (23) was performed (Table 1). The mean ethanol yields obtained from two repetitions and the adjusted means according the analyses are presented in Table 1, and the comparison shows that the experiment was precise. The results were analyzed by Analysis of Variance (ANOVA) followed by a t-test for the main and interaction effects, both using a confidence level of 95% (a = 0.05). According to this analysis, the hydrolysis time (p-value = 0.000) and fermentation temperature (p-value = 0.009) factors had significant and positive effects on the yields, while the yeast strain factor had no significant effect (p-value > 0.05) (Fig. 4A). These results suggest that the timing of the presaccharification step is the most important factor when considering the three studied factors, which influences ethanol yield when the presaccharification is performed at 50 °C, is in agreement with the principle that the glucose concentration is a decisive factor for fermentative metabolism in yeast cells. Fig. 4B shows a significant (p-value = 0.004) interaction between the fermentation temperature and yeast strain factors. The intersection of the straight lines indicates a complex interaction between these factors, which means that the best temperature for fermentation depends on the yeast strain being used. In this way, the best temperature for the fermentation of S. cerevisiae was 37 °C, while it was 42 °C for K. marxianus. In addition, it should be noted that the highest ethanol yield was obtained by using K. marxianus for fermentation at 42 °C. Therefore, this study showed that the thermotolerant yeast strains S. cerevisiae LBM-1 and K. marxianus UFV-3 have potential for use in the batch undergoing an enzymatic hydrolysis process because they grow and ferment at high temperatures, producing yields close to those already described in the literature for other strains (Santos et al., 2010; Suryawati et al., 2008). S. cerevisiae LBM-1 was capable of fermenting at 42 °C with yields close to those observed with K. marxianus UFV-3 at the same temperature. Moreover, these results show that presaccharification at the enzyme’s optimal temperature and during its maximum action time are the parameters that most affect ethanol yield when thermotolerant yeasts are used. Santos et al. (2010) showed that presaccharification led to a reduction in the total time of the process when compared to separate saccharification and fermentation processes. Higher ethanol yields were also obtained when comparing the SSF process with presaccharification to the SSF process without presaccharification. However, the ethanol concentration obtained using the SSF process was not higher than 18 g/L, probably as a consequence of low conversion of cellulose into glucose. These results reaffirm the need to develop and use enzymatic preparations with ideal proportions of the different enzymes in the cellulose complex (Castro, 2010), as well as pretreatment technologies for the sugarcane bagasse, as these components are key to obtain fermentable sugars in the hydrolysis stage (Mosier et al., 2005). Rudolf et al. (2007) achieved ethanol yields that were approximately twofold of those obtained in the present study; however, their higher yields resulted from the use of a higher enzyme load in the hydrolysis, especially of b-glycosidase, as a way of reducing cellulase inhibition by the concentration of cellobiose.
Fig. 4. (A) Column graphs of the adjusted means showing the influences of presaccharification time (24 and 72 h), fermentation temperature (37 and 42 °C) and yeast strain (LBM-1 and UFV-3) on ethanol yield (YE/B) from the SSF process. Symbol () indicates the significance (p-value <0.05) of the tested variable. (B) The effect of the interactions between the yeast strains (LBM-1 and UFV-3) and fermentation temperatures 37 °C (h) and 42 °C (j) on the ethanol yield (YE/B) after SSF.
To further support our interpretation that the presaccharification has a positive effect on ethanol production, we performed SHF and SSF with and without presaccharification. Comparisons of volumetric productivity (Qp) and ethanol yields of the variable conditions are depicted in Fig. 5. The results show that the volumetric productivity of fermentation and ethanol yields increase with presaccharification time, from 0.43 g/L/h at 0 h to 1.79 g/L/h after 72 h of presaccharification. The Qp of SSF with 72 h of presaccharification was superior to the Qp of the SHF process with 72 h of saccharification (Fig. 5). The volumetric productivity was calculated using the minimum time required to reach the maximum concentration of ethanol in the process. This occurred after 24 h for SSF without presaccharification and after 8 h for the SHF and SSF experiments with presaccharification. However, the total time
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Fig. 5. Volumetric productivity (Qp) and yields (YE/B) of ethanol by K. marxianus UFV-3 at 42 °C (gray bars and white bars, respectively). SHF: 72 h of presaccharification and 8 h of fermentation in separate vessels. SSF-0: 0 h of presaccharification and 24 h of simultaneous saccharification and fermentation. SSF-24: 24 h of presaccharification at 50 °C and 8 h of simultaneous saccharification and fermentation. SSF-72: 72 h of presaccharification at 50 °C and 8 h of simultaneous saccharification and fermentation. The symbols () and () indicate the means of yields and volumetric productivity with significant differences (p-value <0.05).
of the process was lower for SSF without presaccharification; 24 h were required to reach the maximum concentration of ethanol (10.31 g/L), whereas SSF after 72 h of presaccharification and 8 h after fermentation yielded 14.36 g/L of ethanol. This article provides incremental knowledge on alternative ways for improving conversion technologies of cellulose into ethanol. In particular, the utilization of thermotolerant yeast strains during SSF allows the process to occur at higher temperatures that are closer to the optimum temperature of cellulose hydrolysis using commercial cellulases. The results obtained show that presaccharification prior to fermentation, conducted in the same batch allows for increased ethanol yields. 4. Conclusion This investigation reported the isolation of the S. cerevisiae LBM-1 strain, which is capable of growth at 42 °C. In addition, the capacity of S. cerevisiae LBM-1 and K. marxianus UFV-3 to ferment sugarcane bagasse in SSF processes at 37 and 42 °C were demonstrated. The results show that these two yeast strains have a potential for the production of ethanol through the SSF process and suggest that the use of thermotolerant yeasts and a presaccharification stage are key to increasing yields in this process. Acknowledgements This research was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico CNPq and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG). C.J.A.S. is supported by fellowship from CAPES. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25 (17), 3389–3402. Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101 (13), 4851–4861. Banat, I.M., Nigam, P., Marchant, R., 1992. Isolation of thermotolerant, fermentative yeasts growing at 52 °C and producing ethanol at 45 °C and 50 °C. World J. Microbiol. Biotechnol. 8 (3), 259–263. Ballesteros, M., Oliva, J.M., Negro, M.J., Manzanares, P., Ballesteros, I., 2004. Ethanol from lignocellulosic materials by a simultaneous saccharification and
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fermentation process (SSF) with Kluyveromyces marxianus CECT 10875. Process Biochem. 39 (12), 1843–1848. Cardona, C.A., Quintero, J.A., Paz, I.C., 2010. Production of bioethanol from sugarcane bagasse: status and perspectives. Bioresour. Technol. 101 (13), 4754–4766. Castro, A.M., 2010. Produção, propriedades e aplicação de celulases na hidrólise de resíduos agroindustriais. Quim. Nova 33 (1), 181–188. Demain, A.L., 2009. Biosolutions to the energy problem. J. Ind. Microbiol. 36 (3), 319–332. Faga, B.A., Wilkins, M.R., Banat, I.M., 2010. Ethanol production through simultaneous saccharification and fermentation of switchgrass using Saccharomyces cerevisiae D5A and thermotolerant Kluyveromyces marxianus IMB strains. Bioresour. Technol. 101 (7), 2273–2279. Hahn-Hagerdal, B., Galbe, M., Gorwa-Grauslund, M.F., Lidén, G., Zacchi, G., 2006. Bio-ethanol – the fuel of tomorrow from the residues of today. Trends Biotechnol. 24 (12), 549–556. Hari-Krishna, S., Reddy, T.J., Chowdary, G.V., 2001. Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast. Bioresour. Technol. 77 (2), 193–196. Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100 (1), 10–18. Martín, C., Thomsen, M.H., Hauggaard-Nielsen, H., Thomsen, A.B., 2008. Wet oxidation pretreatment, enzymatic hydrolysis and simultaneous saccharification and fermentation of clover–ryegrass mixtures. Bioresour. Technol. 99 (18), 8777–8782. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M., 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96 (6), 673–686. Mussatto, S.I., Dragone, G., Guimarães, P.M., Silva, J.P., Carneiro, L.M., Roberto, I.C., Vicente, A., Domingues, L., Teixeira, J.A., 2010. Technological trends, global market, and challenges of bio-ethanol production. Biotechnol. Adv. 28 (6), 817– 830. Oliveira, V.A., Vicente, M.A., Fietto, L.G., Castro, I.M., Coutrim, M.X., Schuller, D., Alves, H., Casal, M., Santos, J.O., Araujo, L.D., Silva, P.H., Brandao, R.L., 2008. Biochemical and molecular characterization of Saccharomyces cerevisiae strains obtained from sugarcane juice fermentations and their impact in cachaca production. Appl. Environ. Microbiol. 74 (3), 693–701. Olofsson, K., Bertilsson, M., Lidén, G., 2008. A short review on SSF – an interesting process option for ethanol production from lignocellulosic feedstocks. Biotechnol. Biofuels 1 (1), 1–14. Pandey, A., Soccol, C.R., Nigam, P., Soccol, V.T., 2000. Biotechnological potential of agro-industrial residues. Part I. Sugarcane bagasse. Bioresour. Technol. 74 (1), 69–80. Rosa, C.A., Lachance, M.A., 1998. The yeast genus Starmerella gen. nov. and Starmerellabombicola sp. nov., the teleomorph of Candida bombicola (Spencer, Gorin&Tullock) Meyer & Yarrow. Int. J. Syst. Bacteriol. 48 (4), 1413–1417. Rudolf, R., Baudel, H., Zacchi, G., Hahn-HaGerdal, B., Liden, G., 2007. Simultaneous saccharification and fermentation of steam-pretreated bagasse using Saccharomyces cerevisiae TMB3400 and Pichia stipitis CBS6054. Biotechnol. Bioeng. 89 (4), 783–790. Sambrook, J., Russell, D.W., 2001 Molecular Cloning: A Laboratory Manual, third ed. CSHL Press. Santos, J.R.A., Souto-Maior, A.M., Gouveia, E.R., 2010. Comparação entre processos em shf e em ssf de bagaço de cana-de-açúcar para a produção de etanol por Saccharomyces cerevisiae. Quim. Nova 33 (4), 904–908. Silveira, W.B., Passos, F.J.V., Mantovani, H.C., Passos, F.M.L., 2005. Ethanol production from cheese whey permeate by Kluyveromyces marxianus UFV-3: a flux analysis of oxido-reductive metabolism as a function of lactose concentration and oxygen levels. Enzyme Microb. Technol. 36 (7), 930–936. Soccol, C.R., Vandenberghe, L.P.S., Medeiros, A.B.P., Karp, S.G., Buckeridge, M., Ramos, L.P., Pitarelo, A.P., Ferreira-Leitão, V., Gottschalk, L.M.F., Ferrara, M.A., Bom, E.P.S., Moraes, L.M.P., Araújo, J.A., Torres, F.A.G., 2010. Bioethanol from lignocelluloses: status and perspectives in Brazil. Bioresour. Technol. 101 (13), 4820–4825. Sree, N.K., Sridhar, M., Suresh, K., Banat, I.M., Rao, L.V., 2000. Isolation of thermotolerant, osmotolerant, flocculating Saccharomyces cerevisiae for ethanol production. Bioresour. Technol. 72 (1), 43–46. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83 (1), 1–111. Suryawati, L., Wilkins, M.R., Bellmer, D.D., Huhnke, R.L., Maness, N.O., Banat, I.M., 2008. Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluyveromycesmarxianus IMB4. Biotechnol. Bioeng. 101 (5), 894–902. Takagi, M., Abe, S., Suzuki, S., Emert, G., Yata, N., 1977. A method for production of alcohol directly from cellulose using cellulase and yeast. In: Ghose, T. (Ed.), Proceedings of Bioconversion of Cellulosic Substances into Energy, Chemicals, and Microbial Protein. IIT, New Delhi, pp. 551–571. Tomás-Pejó, E., Garcia-Aparicio, M., Negro, M.J., Oliva, J.M., Ballesteros, M., 2009. Effect of different cellulase dosages on cell viability and ethanol production by Kluyveromyces marxianus in SSF processes. Bioresour. Technol. 100 (2), 890–895. Van Maris, A.J.A., Abbott, D.A., Bellissimi, E., Van Den Brink, J., Kuyper, M., Luttik, M.A.H., Wisselink, H.W., Scheffers, W.A., Van Dijken, J.P., Pronk, J.T., 2006. Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie van Leeuwenhoek 90 (4), 391–418. Vásquez, M.P., Silva, J.N.C., Souza, M.B., Pereira Jr, N., 2007. Enzymatic hydrolysis optimization to ethanol production by simultaneous saccharification and fermentation. Appl. Biochem. Biotechnol. 136–140 (1–12), 104–114.
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The influence of presaccharification, fermentation temperature and yeast strain on ethanol production from sugarcane bagasse Carlos J.A. de Souza a, Daniela A. Costa a,b, Marina Q.R.B. Rodrigues a, Ancély F. dos Santos a, Mariana R. Lopes a, Aline B.P. Abrantes a, Patrícia dos Santos Costa a, Wendel Batista Silveira b, Flávia M.L. Passos b, Luciano G. Fietto a,⇑ a b
Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Av. PH Rolfs s/n, Campus Universitário, Viçosa 36571-000, MG, Brazil Departamento de Microbiologia, Universidade Federal de Viçosa, Av. PH Rolfs s/n, Campus Universitário, Viçosa 36571-000, MG, Brazil
a r t i c l e
i n f o
Article history: Received 12 September 2011 Received in revised form 20 December 2011 Accepted 7 January 2012 Available online 15 January 2012 Keywords: Simultaneous saccharification and fermentation Ethanol Sugarcane bagasse Thermotolerant yeast
a b s t r a c t Ethanol can be produced from cellulosic biomass in a process known as simultaneous saccharification and fermentation (SSF). The presence of yeast together with the cellulolytic enzyme complex reduces the accumulation of sugars within the reactor, increasing the ethanol yield and saccharification rate. This paper reports the isolation of Saccharomyces cerevisiae LBM-1, a strain capable of growth at 42 °C. In addition, S. cerevisiae LBM-1 and Kluyveromyces marxianus UFV-3 were able to ferment sugar cane bagasse in SSF processes at 37 and 42 °C. Higher ethanol yields were observed when fermentation was initiated after presaccharification at 50 °C than at 37 or 42 °C. Furthermore, the volumetric productivity of fermentation increased with presaccharification time, from 0.43 g/L/h at 0 h to 1.79 g/L/h after 72 h of presaccharification. The results suggest that the use of thermotolerant yeasts and a presaccharification stage are key to increasing yields in this process. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction During the past decades, the increasing demand for energy has encouraged the search for renewable alternative energy sources. Among these, ethanol fuel has received much attention. Recently, the international ethanol market has been stimulated by government incentive offers for using renewable fuels (Demain, 2009; Hahn-Hagerdal et al., 2006; Van Maris et al., 2006). Lignocellulosic biomass is one of the salient alternatives that is being considered for large-scale ethanol production and will likely be the major feedstock for ethanol production in the near future (Mussatto et al., 2010). Lignocellulosic wastes, such as sugarcane bagasse, are alternative, low-cost feedstocks that can be enzymatically hydrolyzed to fermentable sugars for subsequent biofuel production. Brazil has a large supply of this feedstock, which is an abundant product of the sugar and alcohol industries; it is estimated that about 186 million tons of bagasse are generated in Brazil per year (Cardona et al., 2010; Soccol et al., 2010). According to Pandey et al. (2000), half of the sugarcane bagasse generated is used in power plants for energy production, while the other half has no set destination. Thus, it is necessary to develop new technologies for the utilization of sugarcane bagasse generated in Brazil. In this
⇑ Corresponding author. Tel.: +55 31 3899 3046; fax: +55 31 3899 2373. E-mail address: lgfi[email protected] (L.G. Fietto). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.01.024
sense, this feedstock emerges as a promising alternative for use in ethanol production (Cardona et al., 2010). The first step in the conversion of sugarcane bagasse into ethanol is pretreatment, which alters the lignocellulosic structure to allow enzyme access in the hydrolysis step (Alvira et al., 2010; Hendriks and Zeeman, 2009). After pretreatment, there are several ways to conduct subsequent steps in the process. One promising method is to simultaneously carry out the hydrolysis and fermentation steps in a process called simultaneous saccharification and fermentation (SSF) (Cardona et al., 2010; Olofsson et al., 2008; Takagi et al., 1977). SSF was designed to decrease the inhibition of cellulases by hydrolysis products, mainly glucose, which is readily consumed by yeast cells. Therefore, the SSF process costs are decreased due to the reduced load of enzymes required for the enzymatic hydrolysis of biomass (Olofsson et al., 2008; Sun and Cheng, 2002). In addition, the use of a single fermenter for the entire process may reduce the investment costs (Ballesteros et al., 2004). The major problem associated with the SSF process is the different optimum temperatures for saccharification and fermentation. The optimum temperature for cellulase enzymes is higher than can be tolerated by most yeast strains used for industrial ethanol production. This drawback can be overcome by using thermotolerant yeast strains (Hari-Krishna et al., 2001; Olofsson et al., 2008). In fact much effort has been put forth to identify thermotolerant yeasts that have potential for use in the ethanol industry, mainly in the SSF process for second generation ethanol
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production, with the most promising results being obtained with Kluyveromyces marxianus strains. The present study evaluated and compared the fermentation capacity of sugarcane bagasse in SSF processes at 37 and 42 °C using two thermotolerant strains, Saccharomyces cerevisiae LBM1 isolated from fermentation vats in Brazil and UFV-3, a K. marxianus strain identified by Silveira et al. (2005) that showed good fermentation results when converting lactose in cheese whey to ethanol. Furthermore, the influence of presaccharification, fermentation temperature and yeast strain on ethanol production were evaluated. 2. Methods 2.1. Isolation of yeast strain and growth conditions The S. cerevisiae strain used in this study was isolated from fermentation vats in the state Minas Gerais, Brazil using the modified method of Oliveira et al. (2008). Samples were collected from fermentation vats in sterile 150-mL flasks. Samples were diluted 1:100,000 in sterile water in triplicate and were inoculated on YNB medium supplemented with ampicillin at 30 °C for 3 days. The isolates were first classified on the basis of morphological characteristics and then tested for their ability to grow at different temperatures (37 and 42 °C) in the presence of 10% (w/v) ethanol. Selected colonies were grown on YP medium containing 2% (w/v) glucose supplemented with 10% (w/v) ethanol at 37 °C. The selected isolates underwent molecular identification of the D1/D2 region of large subunit rRNA gene (Rosa and Lachance, 1998). PCR was performed from total DNA samples using the primers NL1 and NL4 as previously described (Rosa and Lachance, 1998). The amplified DNA was concentrated, cleaned using the PEG method (Sambrook and Russell, 2001) and sequenced in a Mega-BACE™ 1000 automated sequencing system (Amersham Biosciences) in the Institute of Biological Sciences of the Federal University of Minas Gerais, Brazil. DNA sequences were analyzed using the program blastn (Basic Local Alignment Search Tool 2.2.24-BLAST version 2.0) available at National Center for Biotechnology Information (NCBI) website (Altschul et al., 1997). The sequences were compared with sequences already deposited in GenBank. Sequence alignment was performed using the program CLC Main Workbench 5.7.1. Active cultures of K. marxianus UFV-3 and S. cerevisiae LBM-1 for inoculation were prepared by growing the organisms using a rotary shaker at 180 rpm for 16 h at 28 °C in YPD medium (1% yeast extract, 2% peptone and 2% glucose).Yeast growth at different temperatures was analyzed by plating 5 ll of culture at dilutions of 1:0, 1:10 and 1:100 on agar plates containing YPD medium (1% yeast extract, 2% peptone, 4% glucose and 2% agar). The plates were incubated at 28, 37, 42 or 45 °C for 72 h. The dilutions were prepared in sterilized saline solution (0.85% NaCl) from active cultures with an optical density (OD)600nm of 0.5. 2.2. Raw material and pretreatment 2.2.1. Acid pretreatment The crushed sugarcane bagasse, Saccharum sp., at a concentration of 10% (w/v) in 0.5% (v/v) H2SO4 was pretreated by autoclaving at 121 °C for 30 min. The solid and liquid fractions were then separated by vacuum filtration through filter paper (Whatman No. 5). The solid residue was washed with 500 mL of deionized water and dried at 50 °C for 24 h. 2.2.2. Acid and alkaline pretreatment The solid residue obtained upon acid pretreatment of sugarcane bagasse was treated with alkali to increase cellulose accessibility
by partially removing lignin. This pretreatment was conducted by mixing 10 g of solid with a 4% (w/v) NaOH solution, which was then subjected to thermal treatment at 121 °C for 30 min. The solid residue was washed with 500 mL of deionized water and dried at 50 °C for 24 h. 2.3. Hydrolysis tests Hydrolysis was carried out in duplicate under mild agitation at 180 rpm in 125-mL flasks. Hydrolysis was performed using the solid fraction obtained after pretreatment of the substrate at 8% (w/v) concentration suspended in 30 mL of the fermentation medium (yeast extract, 2.5 g/L; peptone, 2.5 g/L; NH4Cl, 2 g/L; KH2PO4, 1 g/L and MgSO47H2O, 0.3 g/L) in citrate buffer (50 mM, pH 4.8), supplemented with 15 Filter Paper Units (FPU) per gram of substrate. The commercial cellulase preparation used in this work was Celluclast 1.5 L (Sigma–Aldrich, Brazil). Samples were collected every 12 h and centrifuged for 15 min at 10,000g, and the supernatants were used to quantify carbohydrates by high performance liquid chromatography (HPLC). 2.4. Saccharification and fermentation tests SSF (simultaneous saccharification and fermentation) and SHF (separate hydrolysis and fermentation) experiments were carried out according to the pretreatment and presaccharification conditions described above. In the SSF and SHF assays, flasks were inoculated with yeast cultures using the following presaccharification conditions: 0, 24 or 72 h at 37, 42 or 50 °C. The fermentation was initiated by adding yeast culture to an OD600nm of 2. SSF assays were conducted under sterile conditions at 37 or 42 °C for 24 h. Samples were collected every 2 h and centrifuged for 15 min at 10,000g. The supernatants were used to quantify carbohydrates and ethanol by HPLC. In the SHF assays the supernatants of hydrolysis were separated from biomass by centrifuging prior the yeast inoculation. 2.5. Determination of hydrolysis and fermentation parameters The presaccharification yield (YG/B) was calculated by dividing the difference between the initial (Glui) and final (Gluf) glucose mass (g) by the initial mass of pretreated biomass (g) (Biomass):
Y G=B ¼
Gluf Glui Biomass
ð1Þ
The ethanol yield (YE/B) was calculated at the end (8 h) of fermentation by dividing the difference between the final (EtOHf) and initial (EtOHi) ethanol mass (g) by the initial pretreated biomass mass (g):
Y E=B ¼
EtOHf EtOHi Biomass
ð2Þ
The volumetric productivity (Qp) was calculated by dividing the maximum concentration of ethanol (g/L) achieved by the time of fermentation in hours:
QP ¼
EtOHf t
ð3Þ
2.6. Analytical methods 2.6.1. Glucose and ethanol concentrations Quantitative determinations of glucose and ethanol concentrations were carried out by HPLC using an ion exclusion column Aminex HPX-87H (Bio-Rad) kept at 60 °C. The eluent for separation
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was 5 mM H2SO4 applied at an elution rate of 0.7 mL/min. The column was coupled to the HP 1047 A refractive index detector. 2.7. Statistical analysis Data for comparison of the pretreatment, presaccharification and fermentation processes were obtained by experiments repeated in triplicate and analyzed using ANOVA followed by Duncan’s test for means. The statistical analyses were performed with SAEG demo version using 95% confidence (a = 0.05). The influence of presaccharification time, fermentation temperature and yeast strain on the ethanol yield was assessed using a factorial design repeated twice (23 2 = 16 assays) (Table 1). This experiment was conducted at 50 °C during the presaccharification step. The data from this experiment were analyzed by ANOVA followed by a t-test for the effects of each factor and interactions between them. The analysis and graphing were performed with MINITAB15 using a cutoff of 95% confidence (a = 0.05). 3. Results and discussion 3.1. Growth pattern of the yeast strains S. cerevisiae LBM-1 and K. marxianus UFV-3 at different temperatures Of the 50 strains of S. cerevisiae isolated from Brazilian alcohol manufacturing plants that were able to grow at 37 °C in YNB medium containing 4% glucose (w/v) and 10% ethanol (w/v), S. cerevisiae LBM-1 was chosen for this study because it had the fasted growth rates in assays conducted at different temperatures. The strain was identified based on sequence analysis of the D1/D2 region of large subunit rRNA gene. The sequence was 100% identical to the S. cerevisiae sequence in GenBank. The K. marxianus UFV-3 strain used in this work was identified by Silveira et al. (2005). This strain was chosen for this work because it is able to convert lactose to ethanol with a yield close to the theoretical value. Furthermore, this strain is also able to convert glucose to ethanol efficiently. Fig. 1 shows that all tested dilutions of the two strains grew at temperatures of 30, 37 and 42 °C, though only K. marxianus UFV-3 was able to grow at 45 °C. The thermotolerance of the species K. marxianus is well documented, while it is rare in S. cerevisiae with few strains described (Sree et al., 2000). Thermotolerant K. marxianus strains isolated by Banat et al. (1992) were tested for the ability to convert biomass into ethanol in the SSF process. Faga et al. (2010) showed that the K. marxianus strain IMB3 was able to ferment switchgrass in the SSF process at 45 °C. The same study found that the ethanol yield produced by IMB3 was similar to that of S. cerevisiae D5A at 37 °C. However, processes using S. cerevisiae as a fermenting organism are performed at 37 °C because these yeasts generally are not able to grow at temperatures above 40 °C (Santos et al., 2010; Rudolf et al., 2007; Suryawati et al., 2008). Because K. marxianus UFV-3 and S. cerevisiae LBM-1 grew at both 37 and 42 °C, it was decided to evaluate the steps of hydroly-
sis and fermentation to produce cellulosic ethanol at these temperatures. Yields from each step were calculated to determine whether a cooling step at the transition from hydrolysis to fermentation could be avoided, which would reduce processing costs. 3.2. Pretreatment analysis Mill-dried in natura sugarcane bagasse was pretreated with diluted sulfuric acid and resulted in a 55% yield in relation to the initial bagasse mass. In the acid–alkali pretreatment, there was a 30% yield. Sugarcane bagasse contains around 20–24% lignin, 27–32% hemicelluloses, 32–44% cellulose and 4.5–9.0% ashes (Soccol et al., 2010). In the presented experiments, the acid treatment removed around 45% of the initial mass, which is equivalent to the hemicellulose fraction and the portion of cellulose fraction that may undergo partial hydrolysis during treatment with diluted acid. In addition to the hemicellulose fraction, the acid–alkali treatment removed the lignin fraction, representing approximately 30% of the biomass. Therefore, the final yield of 30% is compatible with a 70% loss in mass and is equivalent to the cellulose fraction of the bagasse. To verify which pretreatment was the most effective in terms of enzyme accessibility to the substrate, hydrolysis was conducted using bagasse with and without alkali pretreatment. After 24 h of hydrolysis at 42 °C using only acid-pretreated bagasse, 8.9 g/L of glucose were released while 18.36 g/L of glucose were released when the acid–alkali pretreatment was used. The average glucose yields for the two treatments differed significantly according to ANOVA (p-value = 0.005). Twofold more glucose was released after hydrolysis with the acid–alkali pretreatment than the acid pretreatment. With the removal of lignin and hemicellulose by the acid–alkali pretreatment, cellulose became more accessible to the enzymes in the hydrolysis step. According to Santos et al. (2010), the presence of lignin limits the process of enzyme diffusion in the substrate and consequently the release of glucose. Vásquez et al. (2007) noted the same effect when analyzing the hydrolysis of sugarcane bagasse subjected to sulfuric acid pretreatment or to acid–alkali pretreatment (sulfuric acid and sodium hydroxide). In that study, the authors observed that after 24 h of hydrolysis at 50 °C using 7% (w/v) bagasse and 20 FPU of cellulases per gram of substrate, approximately 10 and 20 g/L of glucose were released with the acid pretreatment and the acid–alkali pretreatment, respectively. In this work similar results were obtained with the acid–alkali pretreatment using 15 FPU of enzyme and 8% (w/v) of pretreated bagasse. The acid–alkali pretreatment was selected for use in subsequent experiments because it showed the best hydrolysis yield. 3.3. Hydrolysis Enzymatic hydrolysis and fermentation processes were conducted in the same batch and were assumed to be a SSF process. Initially, the alkali-delignified biomass underwent a presaccharifi-
Table 1 b E=B Þ in factorial design (23) for time of presaccharification, temperature of fermentation and yeast strain. Means obtained ðY E=B Þ and fitted ð Y Time of presaccharification (h)
Temperature of fermentation (°C)
Yeast strain
Y E=B
^E=B y
24 24 24 24 72 72 72 72
37 37 42 42 37 37 42 42
S. cerevisiae LBM-1 K. marxianus UFV-3 S. cerevisiae LBM-1 K. marxianus UFV-3 S. cerevisiae LBM-1 K. marxianus UFV-3 S. cerevisiae LBM-1 K. marxianus UFV-3
0.0927 0.0907 0.8625 0.0980 0.1655 0.1535 0.1630 0.1795
0.1104 0.1051 0.1100 0.1158 0.1653 0.1538 0.1630 0.1798
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Fig. 1. Growth of the S. cerevisiae LBM-1 and K. marxianus UFV-3 strains on YPD medium (4% glucose) over a 24-h period at 30, 37, 42 or 45 °C. The numbers above the figures represent the dilutions of a culture with an OD(600nm) of 0.5.
cation step to provide a source of fermentable carbon for the yeast. An assay was conducted to evaluate the timing of hydrolysis for better yield, i.e., the best time to inoculate the yeast to start the fermentation process. By comparing the SSF process with and without the presaccharification period, Santos et al. (2010) noted that presaccharification led to faster conversion of cellulose into glucose and more conversion of cellulose into ethanol. Moreover, presaccharification led to higher solubilization of the substrate in the mixture. This effect was also noted by Martín et al. (2008) when assessing the effect of presaccharification in fermentation yield of clover–ryegrass. In the present work, the hydrolysis process was performed at 50 °C for 72 h, as shown in Fig. 2A. After 48 h, the rate of glucose release decreased. It should be noted that in the 0–24 h period of hydrolysis, there was an increase of 20 g/L, while in the 48–72 h period, the increase was only 2.5 g/L of glucose. Santos et al. (2010) obtained similar results when studying delignified bagasse hydrolysis, and it is likely that in this period, cellulases start to be inhibited by the hydrolysis products, including glucose and cellobiose. In this study, approximately 2% glucose was detected after 24 h of hydrolysis at 50 °C. At this point, the cellulases were not yet inhibited by the hydrolysis products and the amount of glucose released was sufficient to start fermentation. Furthermore, the same hydrolysis assay was performed at 37 and 42 °C. After 24 h of hydrolysis, 12.72 g/L glucose was obtained at 37 °C and 16.19 g/L at 42 °C (Fig. 2B). The glucose yields for the three temperatures are significantly different according to ANOVA (p-value = 0.000) followed by Duncan means test (comparison done at a = 0.05). Thus, 24 h of presaccharification at 37, 42 or 50 °C were chosen for this stage of the process. 3.4. Simultaneous saccharification and fermentation Saccharification and fermentation were performed simultaneously at 37 or 42 °C after 24 h of presaccharification at 37, 42 or 50 °C. For all temperatures tested, results indicated that after 8 h of fermentation, the glucose concentration was close to zero and no additional increases in ethanol concentration were seen after that time (Fig. 3), indicating the end of the fermentation process. When presaccharification and fermentation processes were performed at 37 °C, the final ethanol concentration was 6.12 g/L for S.
Fig. 2. Time course of enzymatic hydrolysis of pretreated bagasse at laboratory scale at 50 °C (A) and enzymatic hydrolysis after 24 h at 37, 42 and 50 °C (B) with 8% initial solids content (w/v) and an enzyme dosage of 15 FPU per gram dry matter.
cerevisiae LBM-1 and 6.14 g/L for K. marxianus UFV-3 (Fig. 3A). At 42 °C, the two strains exhibited the same fermentation behavior,
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and similar ethanol concentrations were obtained at the end of the process (Fig. 3B). However, with presaccharification at 50 °C followed by fermentations at 37 or 42 °C, higher ethanol concentrations were attained for both species (Fig. 3C and D). Higher ethanol yields were observed when fermentation was initiated after presaccharification at 50 °C than at 37 or 42 °C (Table 2), indicating that for the two strains, the presaccharification temperature is more important to ethanol yield than the fermentation temperature. When analyzing the data showed in Fig. 2 and in Table 2, it can be seen that ethanol yield depends on the initial glucose concentration, as higher yields were achieved when fermentation was initiated with a higher glucose concentration. To confirm this result, another SSF experiment was carried out in which fermentation was started after 72 h of presaccharification at 50 °C with an initial yeast concentration equal to an OD600nm of 2. Under these conditions, the final ethanol concentration was approximately 15 g/L (Fig. 3E and F). Thus, it was clear that the ethanol yield was higher when fermentation was started after 72 h of
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presaccharification compared to 24 h (Table 2) due to the higher glucose concentration at the start of fermentation. Positive influences on ethanol production can be seen when the ethanol yields of strains UFV-3 and LBM-1 are compared with those obtained by Santos et al. (2010), Tomás-Pejó et al. (2009) and Ballesteros et al. (2004). Santos et al. (2010) used an S. cerevisiae strain that ferments at 328 °C, which is not useful in the SSF process and resulted in a lower ethanol yield compared to that of strains UFV-3 and LBM-1. Tomás-Pejó et al. (2009) obtained higher ethanol concentrations than those achieved in this study by using a K. marxianus strain that ferments at 42 °C. However, these authors evaluated crystalline cellulose as a substrate, which is known to be more accessible to enzymes in the hydrolysis stage than bagasse. Ballesteros et al. (2004) obtained ethanol concentrations close to those attained in this study using thermotolerant yeasts, emphasizing the importance of the use of these strains in the fermentation process to increase ethanol yield and avoid contamination, especially when envisioning a larger-scale process.
Fig. 3. Glucose consumption and ethanol formation over 10 h by thermotolerant K. marxianus UFV-3 and S. cerevisiae LBM-1, with (A) SSF at 37 °C and (B) SSF at 42 °C after 24 h of presaccharification at 37 and 42 °C, respectively; (C) SSF at 37 °C and (D) SSF at 42 °C after 24 h of presaccharification at 50 °C; (E) SSF at 37 °C and (F) SSF at 42 °C after 72 h of presaccharification at 50 °C with (N)UFV-3/glucose, (4) UFV-3/ethanol, (j) LBM-1/glucose, (h) LBM-1/ethanol.
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Table 2 Ethanol yields (YE/B) from different combinations of presaccharification and fermentation temperatures for LBM-1 and UFV-3. Temperature of presaccharification (°C)
Temperature of fermentation (°C)
YE/B S. cerevisiae LBM-1
YE/B K. marxianus 2
37 42 50 50
37 42 37 42
0.0734 0.0643 0.1124 0.1082
0.0737 0.0821 0.1079 0.1139
A B C C
A A C C
Means followed by the same letter are not significantly different by Duncan’s test (comparisons done at a = 0.05).
To evaluate the influence of the presaccharification time and fermentation temperature (quantitative factors) and of the yeast strain (qualitative factor) on ethanol yield, a factorial design (23) was performed (Table 1). The mean ethanol yields obtained from two repetitions and the adjusted means according the analyses are presented in Table 1, and the comparison shows that the experiment was precise. The results were analyzed by Analysis of Variance (ANOVA) followed by a t-test for the main and interaction effects, both using a confidence level of 95% (a = 0.05). According to this analysis, the hydrolysis time (p-value = 0.000) and fermentation temperature (p-value = 0.009) factors had significant and positive effects on the yields, while the yeast strain factor had no significant effect (p-value > 0.05) (Fig. 4A). These results suggest that the timing of the presaccharification step is the most important factor when considering the three studied factors, which influences ethanol yield when the presaccharification is performed at 50 °C, is in agreement with the principle that the glucose concentration is a decisive factor for fermentative metabolism in yeast cells. Fig. 4B shows a significant (p-value = 0.004) interaction between the fermentation temperature and yeast strain factors. The intersection of the straight lines indicates a complex interaction between these factors, which means that the best temperature for fermentation depends on the yeast strain being used. In this way, the best temperature for the fermentation of S. cerevisiae was 37 °C, while it was 42 °C for K. marxianus. In addition, it should be noted that the highest ethanol yield was obtained by using K. marxianus for fermentation at 42 °C. Therefore, this study showed that the thermotolerant yeast strains S. cerevisiae LBM-1 and K. marxianus UFV-3 have potential for use in the batch undergoing an enzymatic hydrolysis process because they grow and ferment at high temperatures, producing yields close to those already described in the literature for other strains (Santos et al., 2010; Suryawati et al., 2008). S. cerevisiae LBM-1 was capable of fermenting at 42 °C with yields close to those observed with K. marxianus UFV-3 at the same temperature. Moreover, these results show that presaccharification at the enzyme’s optimal temperature and during its maximum action time are the parameters that most affect ethanol yield when thermotolerant yeasts are used. Santos et al. (2010) showed that presaccharification led to a reduction in the total time of the process when compared to separate saccharification and fermentation processes. Higher ethanol yields were also obtained when comparing the SSF process with presaccharification to the SSF process without presaccharification. However, the ethanol concentration obtained using the SSF process was not higher than 18 g/L, probably as a consequence of low conversion of cellulose into glucose. These results reaffirm the need to develop and use enzymatic preparations with ideal proportions of the different enzymes in the cellulose complex (Castro, 2010), as well as pretreatment technologies for the sugarcane bagasse, as these components are key to obtain fermentable sugars in the hydrolysis stage (Mosier et al., 2005). Rudolf et al. (2007) achieved ethanol yields that were approximately twofold of those obtained in the present study; however, their higher yields resulted from the use of a higher enzyme load in the hydrolysis, especially of b-glycosidase, as a way of reducing cellulase inhibition by the concentration of cellobiose.
Fig. 4. (A) Column graphs of the adjusted means showing the influences of presaccharification time (24 and 72 h), fermentation temperature (37 and 42 °C) and yeast strain (LBM-1 and UFV-3) on ethanol yield (YE/B) from the SSF process. Symbol () indicates the significance (p-value <0.05) of the tested variable. (B) The effect of the interactions between the yeast strains (LBM-1 and UFV-3) and fermentation temperatures 37 °C (h) and 42 °C (j) on the ethanol yield (YE/B) after SSF.
To further support our interpretation that the presaccharification has a positive effect on ethanol production, we performed SHF and SSF with and without presaccharification. Comparisons of volumetric productivity (Qp) and ethanol yields of the variable conditions are depicted in Fig. 5. The results show that the volumetric productivity of fermentation and ethanol yields increase with presaccharification time, from 0.43 g/L/h at 0 h to 1.79 g/L/h after 72 h of presaccharification. The Qp of SSF with 72 h of presaccharification was superior to the Qp of the SHF process with 72 h of saccharification (Fig. 5). The volumetric productivity was calculated using the minimum time required to reach the maximum concentration of ethanol in the process. This occurred after 24 h for SSF without presaccharification and after 8 h for the SHF and SSF experiments with presaccharification. However, the total time
C.J.A. de Souza et al. / Bioresource Technology 109 (2012) 63–69
Fig. 5. Volumetric productivity (Qp) and yields (YE/B) of ethanol by K. marxianus UFV-3 at 42 °C (gray bars and white bars, respectively). SHF: 72 h of presaccharification and 8 h of fermentation in separate vessels. SSF-0: 0 h of presaccharification and 24 h of simultaneous saccharification and fermentation. SSF-24: 24 h of presaccharification at 50 °C and 8 h of simultaneous saccharification and fermentation. SSF-72: 72 h of presaccharification at 50 °C and 8 h of simultaneous saccharification and fermentation. The symbols () and () indicate the means of yields and volumetric productivity with significant differences (p-value <0.05).
of the process was lower for SSF without presaccharification; 24 h were required to reach the maximum concentration of ethanol (10.31 g/L), whereas SSF after 72 h of presaccharification and 8 h after fermentation yielded 14.36 g/L of ethanol. This article provides incremental knowledge on alternative ways for improving conversion technologies of cellulose into ethanol. In particular, the utilization of thermotolerant yeast strains during SSF allows the process to occur at higher temperatures that are closer to the optimum temperature of cellulose hydrolysis using commercial cellulases. The results obtained show that presaccharification prior to fermentation, conducted in the same batch allows for increased ethanol yields. 4. Conclusion This investigation reported the isolation of the S. cerevisiae LBM-1 strain, which is capable of growth at 42 °C. In addition, the capacity of S. cerevisiae LBM-1 and K. marxianus UFV-3 to ferment sugarcane bagasse in SSF processes at 37 and 42 °C were demonstrated. The results show that these two yeast strains have a potential for the production of ethanol through the SSF process and suggest that the use of thermotolerant yeasts and a presaccharification stage are key to increasing yields in this process. Acknowledgements This research was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico CNPq and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG). C.J.A.S. is supported by fellowship from CAPES. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25 (17), 3389–3402. Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101 (13), 4851–4861. Banat, I.M., Nigam, P., Marchant, R., 1992. Isolation of thermotolerant, fermentative yeasts growing at 52 °C and producing ethanol at 45 °C and 50 °C. World J. Microbiol. Biotechnol. 8 (3), 259–263. Ballesteros, M., Oliva, J.M., Negro, M.J., Manzanares, P., Ballesteros, I., 2004. Ethanol from lignocellulosic materials by a simultaneous saccharification and
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