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Organosolv pretreatment of rice straw for efficient acetone, butanol, and ethanol production
Bioresource Technology 152 (2014) 450–456
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Organosolv pretreatment of rice straw for efficient acetone, butanol, and ethanol production Hamid Amiri a, Keikhosro Karimi a,b,⇑, Hamid Zilouei a a b
Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran Industrial Biotechnology Group, Institute of Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Production of ABE was significantly
improved by organosolv pretreatment. The pretreatment resulted in 60% lignin removal. After pretreatment, more than 311 g sugar was produced from each kg of rice straw. More than 80 g butanol and 21 g acetone were produced from each kg of rice straw.
a r t i c l e
i n f o
Article history: Received 14 September 2013 Received in revised form 13 November 2013 Accepted 16 November 2013 Available online 23 November 2013 Keywords: ABE fermentation Organosolv Lignocellulose Pretreatment Rice straw
a b s t r a c t Acetone–butanol–ethanol (ABE) was produced from rice straw using a process containing ethanol organosolv pretreatment, enzymatic hydrolysis, and fermentation by Clostridium acetobutylicum bacterium. Pretreatment of the straw with 75% (v/v) aqueous ethanol containing 1% w/w sulfuric acid at 150 °C for 60 min resulted in the highest total sugar concentration of 31 g/L in the enzymatic hydrolysis. However, the highest ABE concentration and productivity (10.5 g/L and 0.20 g/L h, respectively) were obtained from the straw pretreated at 180 °C for 30 min. Enzymatic hydrolysis of the straw pretreated at 180 °C for 30 min with 5% solid loading resulted in glucose yield of 46.2%, which was then fermented to 80.3 g butanol, 21.1 g acetone, and 22.5 g ethanol, the highest overall yield of ABE production. Thus, the organosolv pretreatment can be applied for efficient production of the solvents from rice straw. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Acetone–butanol–ethanol (ABE) fermentation is one of the first large-scale industrial fermentation processes to be developed (Jones and Woods, 1986). Up to the 1950s, about two thirds of butanol and 10% of acetone in the world were produced by this process from corn starch and molasses (Bahl and Dürre, 2001). However, during the 1960s, production of butanol and acetone
⇑ Corresponding author at: Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran. Tel.: +98 3113915623; fax: +98 3113912677. E-mail address: [email protected] (K. Karimi). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.11.038
shifted from biological to petrochemical based process due to increasing the price of corn and molasses as well as availability of cheaper petrochemical derived butanol and acetone (García et al., 2011). In recent years, by increasing concerns about unstable crude oil supplies and dramatic environmental impact of fossil fuels, there is a high interest in production of green butanol from biomass (Kumar and Gayen, 2011). Even though economically production of acetone, butanol, and ethanol as bulk chemicals from renewable sources is considered as a breakthrough toward reduction of dependency on fossil resources, it is the unique characteristics of butanol as a biofuel that renewed the interest in ABE fermentation (Kumar and Gayen, 2011). In comparison to ethanol, butanol has a higher energy
H. Amiri et al. / Bioresource Technology 152 (2014) 450–456
content, lower solubility in water, lower vapor pressure, and a similar air-to-fuel ratio to gasoline. More importantly, butanol is compatible with the current automobile engines and the transportation pipelines. These facts make the biobutanol as an ideal candidate to replace gasoline (Fortman et al., 2008; Kumar and Gayen, 2011). The bioconversion of lignocellulosic materials to monomeric sugars and its consequent fermentation has been suggested for economical production of ABE (Jurgens et al., 2012). Since 1898, when the first cellulosic ethanol production was industrialized in Germany, different technologies have been suggested and developed for economical conversion of lignocelluloses to fermentable sugars (Mosier et al., 2005). However, most of these technologies have been developed for ethanol production, while they do not match well with requisites of ABE fermentation (Fischer et al., 2008). In addition, the negative effects of inhibitors are more serious problems in ABE fermentation compared with ethanolic fermentation (Jang et al., 2012). Therefore, in the production of fermentable sugars, not only the yield of conversion but also the concentration of sugars and inhibitors in the hydrolysate are among the factors affecting the possibility and economy of ABE production from lignocelluloses. Enzymatic hydrolysis is a widely used process for conversion of lignocellulosic materials to monomeric sugars. However, the recalcitrant structure of the lignocelluloses makes the hydrolysis inefficient, and it is essential to open up the structure through a pretreatment (Karimi et al., 2013). The role of pretreatment is even more important in ABE production from lignocelluloses, since the process is more sensitive to inhibitors. Diluted sulfuric acid pretreatment has been commonly used for ABE production from lignocellulosic resources (Gottumukkala et al., 2013; Qureshi et al., 2007, 2010a). However, different inhibitors which are generated in this process severely affect the growth and fermentation ABE producing microorganism; thus, an additional detoxification process is necessary to obtain a high yield which is accompanied with some sugar loss and additional capital investment (Qureshi et al., 2010b). Recently, concentrated alkaline and phosphoric acid pretreatments were evaluated for production of ABE from rice straw (Moradi et al., 2013). However, the recovery of the chemical agents in these processes accompanied with high energy consumption and production of environmental pollutant wastewaters. Thus, the organosolv pretreatment can be applied for efficient production of the solvents from rice straw. Among the pretreatment technologies, ethanol organosolv process has been considered as one of the most promising methods for biorefining of lignocelluloses (Alvira et al., 2010). Ethanol organosolv pretreatment involves the use of ethanol and water to partially hydrolyze lignin and lignin–carbohydrate bonds to remove lignin from lignocelluloses (Hu et al., 2011), the major barrier to the enzymatic hydrolysis (McMillan, 1994). Lignin hinders hydrolysis by blocking access of cellulases to cellulose due to irreversibly binding to the hydrolytic enzymes. Therefore, removal of lignin can considerably improve the hydrolysis rate (McMillan, 1994). The organosolv pretreatment is usually carried out using a strong inorganic acid catalyst, e.g., sulfuric acid, which takes a role to hydrolyze the lignin bonds in biomass (Zhao et al., 2009). Compared to other chemical pretreatments, one of the main and unique advantages of organosolv process is recovery of lignin as a valuable by-product, which is relatively pure, primarily unaltered, and less condensed than Kraft lignins (Mesa et al., 2011; Zhao et al., 2009). Furthermore, the recovery of solvent is typically performed with minimal energy consumption. From the environmental point of view, separation of lignin as a product could reduce the problem with wastewater treatment. Considering the special requirements of ABE fermentation, organosolv pretreatment may offer several advantages over the other pretreatments. In addition, lignin and its derivatives were
451
found to be the main inhibitors affecting the ABE fermentation (Wang and Chen, 2011). Moreover, enrichment of cellulose content by partial removal of lignin is a possible approach to increase the maximum obtainable glucose concentration. As a result, removal of lignin as a purified product through the organosolv pretreatment can improve the economy of ABE process by improving the ABE production and addition of ethanol organosolv lignin as a value-added product. To our knowledge, there is no publication on evaluation of organosolv pretreatment for improvement of ABE production. The main drawbacks and limitations of the previous studies on ABE production from lignocelluloses are production of hydrolysates containing different inhibitors, necessity of detoxification which is accompanied with some sugar loss (Qureshi et al., 2013), and environmental problems pertaining to wastewater containing different degraded materials produced during the pretreatment. In this study, ethanol organosolv pretreatment was evaluated for production of ABE from rice straw. This pretreatment does not have the mentioned drawbacks and separate a lignin with high purity, which is a value added product. The straw is one of the most favorable substrates for biological products in terms of quantity and world wide availability (Kim and Dale, 2004). The pretreated rice straw was hydrolyzed by two commercial hydrolytic enzymes and fermented by Clostridium acetobutylicum. One of the goals was obtaining higher yield of ABE from the straw compared to the results obtained by other pretreatment methods. 2. Methods 2.1. Raw materials and enzymes Rice straw, used as a substrate in all experiments, was obtained from Lenjan field with a cultivar named ‘‘Sazandegi’’ (Isfahan, Iran). It was partly ball-milled and screened to achieve a size of between 833 lm (20 mesh) and 177 lm (80 mesh). Dry weight content of the sample was measured by drying at 105 °C in a convection oven. The materials were stored at room temperature in resealable plastic bags. Two commercial enzymes of cellulase (Celluclast 1.5L, Novozyme, Denmark) and b-glucosidase (Novozyme 188, Novozyme, Denmark) were used for the hydrolysis. The cellulase activity was 65 FPU/ml according to the method presented by Adney and Baker (1996), and the protein content of the enzyme was 42 mg/ml as measured by Bradford assay. The b-glucosidase activity was 210 IU/ml, measured by the method presented by Ximenes et al. (1996). 2.2. Pretreatment methods Fifty grams (dry weight) of the straw was mixed with 400 g (solid-to-liquid ratio of 1:8) of 75% (v/v) aqueous ethanol containing 1% w/w (based on the straw dried mass) sulfuric acid as a catalyst. Treatments were carried out in a high-pressure stainless steel vessel as a batch reactor with a working volume of 500 ml (Amiri et al., 2010). The reaction mixture was heated at a rate of 3 °C/min. After reaching the desired temperature, i.e., 150 or 180 °C, the mixture was maintained at the temperature for 30 or 60 min. Afterwards, it was cooled in an ice bath. The pretreated materials were then washed with 60 °C aqueous ethanol (75% (v/ v), 3 100 ml) and air dried overnight. Finally, the pretreated materials were stored in resealable plastic bags at 5 °C until use. 2.3. Enzymatic hydrolysis The untreated and pretreated materials were subjected to enzymatic hydrolysis by cellulase and b-glucosidase in 50 mM sodium
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citrate buffer (pH 4.8). The hydrolysis was conducted at 45 °C and 140 rpm for 72 h using 25 FPU cellulase and 40 IU b-glucosidase per gram of either treated or untreated biomass at two solid loadings of 5% and 8%. The cellulase loadings were 33 mg protein per gram of cellulose for untreated rice straw, whereas it was varied from 26 to 30 mg protein per gram of cellulose for the pretreated straw. The hydrolysates were separated from the remaining solid fraction by centrifugation (5 min, 5000 rpm) and then fermented.
0.6 ml/min eluent of deionized water. Concentrations of sugars, acetone, butanol, and ethanol were determined by RI detector, while acetic acid and butyric acid were quantified on UV chromatograms at 210 nm. All experiments were performed in duplicates and the averages of the results are presented.
2.4. Microorganism and inoculum preparation 3. Results and discussion C. acetobutylicum NRRL B-591, obtained from Persian type culture collection (PTCC) (Tehran, Iran), was used in all fermentations. In order to prepare the preculture, 1.5 g cooked meat medium (Sigma–Aldrich) was added to 25 ml distilled water. After addition of 0.27 g glucose, the mixture was autoclaved (at 121 °C for 20 min) and cooled to 75 °C. Spore suspension (0.5 ml) was heat shocked at 75 °C for 2 min after addition to the medium (Moradi et al., 2013). Subsequently, the medium containing spores was cooled in ice-cold water for 1 min. The heat shocked spores were then incubated at 37 °C for 16–18 h (Moradi et al., 2013). For biomass preparation, a 100 ml medium containing (g/L): 30 glucose, 5 yeast extract, 2 (NH4)2SO4, 1 NaCl, 0.75 KH2PO4, 0.75 K2HPO4, 0.2 MgSO4, 0.01 MnSO47H2O, and 0.01 FeSO47H2O was prepared. Then, the medium was autoclaved (at 121 °C for 20 min) and cooled to 35 °C (Moradi et al., 2013). Afterwards, 5 ml of the preculture was added to the medium, and the growth was conducted at 37 °C for 16–18 h. 2.5. ABE fermentation After addition of 0.05 g yeast extract to 50 ml of the hydrolysates in 118 ml serum bottles, pH of the solutions was adjusted to 6.5 using 5 M NaOH. The solution was autoclaved (at 120 °C for 10 min) and cooled to room temperature. Subsequently, 0.5 ml of a buffer (50 g/L KH2PO4, 50 g/L K2HPO4, and 220 g/L CH3COONH4), a vitamin (0.1 g/L para-aminobenzoic acid, 0.1 g/L thiamin, and 0.001 g/L biotin), and a mineral (20 g/L MgSO47H2O, 1 g/L MnSO4H2O, 1 g/L FeSO47H2O, 1 g/L NaCl) solutions, was filter sterilized (Millipore filter; 0.22 lm) and added to each bottle (Moradi et al., 2013). The bottle was then purged with pure nitrogen passed over a heated reduced copper column to remove trace oxygen and sealed using a butyl rubber stopper fastened with an aluminum crimp. After inoculation with 6 ml of actively growing culture (optical density of 1.2–1.6 at 610 nm), fermentation was conducted at 37 °C for 72 h. During fermentation, liquid samples were periodically withdrawn using a sterile syringe through the rubber stopper. The samples were centrifuged at 9000 rpm for 25 min and stored at 18 °C before sugar and ABE content analyses. 2.6. Analytical methods Moisture and total solids (Sluiter et al., 2008a), structural carbohydrates, lignin, and ash contents (Sluiter et al., 2008b) of the untreated and pretreated straw were determined. The cell concentration was estimated from optical density (OD) using a predetermined correlation between OD at 610 nm and the dry cell weight (at 105 °C until constant weight) (Moradi et al., 2013). Concentration of the fermentation products as well as sugars were analyzed by high-performance liquid chromatography (HPLC), equipped with UV/VIS and RI detectors (Jasco International Co., Tokyo, Japan). Concentration of acetone, butanol, ethanol, acetic acid, and butyric acid were determined by an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) at 60 °C with 0.6 ml/min eluent of 0.005 M sulfuric acid. Sugars were analyzed on an Aminex HPX-87P column (Bio-Rad, Richmond, CA, USA) at 80 °C with
3.1. Pretreatment Rice straw was subjected to ethanol organosolv pretreatment prior to hydrolysis in order to improve the production yield and concentration of ABE in the subsequent fermentation. The pretreatment temperature and time were selected by considering the previous studies results (Obama et al., 2012) and preliminary investigations. The solid recovery and chemical composition of the pretreated materials are summarized in Table 1. The untreated straw consisted of 49.2% glucan, 29.2% xylan, 3.5% arabinan, and 16.3% Klason lignin. Different parts of the straw were differently affected by the pretreatment. The organosolv pretreated material was considered as cellulosic fibers, which depending on the applied conditions, contained various amounts of hemicellulose and residual lignin (Zhao et al., 2009). The organosolv pretreatment at 180 °C for 60 min resulted in the highest lignin removal (60%), obtaining a pretreated material with 9.8% Klason lignin. In addition, the pretreatment at 180 °C for 30 min resulted in 56% lignin removal. Furthermore, the pretreatment at the lower temperature, i.e., 150 °C, resulted in 41% and 45% lignin removal after 30 and 60 min treatment, respectively. In other words, increasing the temperature from 150 to 180 °C resulted in more than 33% higher lignin removal. It was in agreement with previous studies that showed that the lignin removal is highly affected by the process temperature and increased by increasing temperature (Shatalov and Pereira, 2005). Through the organosolv pretreatment, in addition to lignin, the hemicellulosic sugars are partially dissolved in the organic liquor. After pretreatment, the organic liquor was evaporated to recover the solvent and diluted with water to precipitate the lignin, as one of the valuable products (Zhao et al., 2009). The remaining aqueous solution is rich in hemicellulosic sugars, mainly xylose in the case of rice straw, and is considered as a product which can be utilized by pentose fermenting microorganisms or converted to furfural, xylitol, and some other chemicals (Zhao et al., 2009). In this study, the remaining liquor, after evaporation of ethanol, was diluted with water and subjected to ABE fermentation. However, using different dilution ratios (1:1.5, 1:2, and 1:5 the initial to final liquor), no detectable butanol was produced through the fermentation (data not shown). Through the pretreatment, the xylan content decreased by 40% and 49% after 30 and 60 min pretreatment at 180 °C, while it was 42% and 45% after 30 and 60 min pretreatment at 150 °C, respectively. Increasing the temperature and retention time of the organosolv pretreatment, generally, increases the lignin and xylan removal (Koo et al., 2012). In the case of rice straw, the lignin removal was considerably increased by temperature, whereas the conditions had a minor effect on xylan removal. The organosolv pretreatment of rice straw generally resulted in enrichment of cellulose content as a result of partial solubilisation of both hemicellulose and lignin. Cellulose content of the untreated rice straw was 49.2% which was increased up to 62.3% by the pretreatment at 180 °C for 60 min, while the pretreatment at 180 °C for 30 min resulted in the material with 58.9% cellulose.
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H. Amiri et al. / Bioresource Technology 152 (2014) 450–456 Table 1 Chemical composition and solid recovery of untreated and organosolv pretreated rice straw. Pretreatment conditions
Composition
T (°C)
Time (min)
Glucan (%)
Xylan (%)
Arabinnan (%)
Acid soluble lignin (%)
Acid insoluble (Klason) lignin (%)
Ash (%)
Solid recovery (%)
30 60 30 60
49.2 ± 1.2 55.6 ± 0.8 54.8 ± 0.7 58.9 ± 1.2 62.3 ± 1.2
29.2 ± 1.4 21.7 ± 0.1 20.9 ± 0.6 24.4 ± 0.1 22.4 ± 0.6
3.5 ± 0.2 2.4 ± 0.1 2.7 ± 0.1 2.3 ± 0.4 1.9 ± 0.4
0.7 ± 0.1 1.7 ± 0.1 1.7 ± 0.2 1.5 ± 0.1 1.3 ± 0.1
16.3 ± 1.3 12.4 ± 0.2 11.7 ± 0.2 9.9 ± 0.2 9.8 ± 0.2
6.7 ± 0.2 7.9 ± 0.3 8.4 ± 0.2 6.6 ± 0.1 7.5 ± 0.2
– 78.3 ± 0.9 76.8 ± 1.5 72.1 ± 1.3 66.9 ± 1.0
Untreated 150 150 180 180
3.2. Enzymatic hydrolysis The pretreated and untreated straws were subjected to enzymatic hydrolysis at 45 °C for 72 h using 25 FPU cellulase and 40 IU b-glucosidase per gram of substrate with 5% and 8% solid loadings. Concentration of sugars and the yield of glucose are shown in Fig. 1. Total concentration of sugars was increased after the pretreatment in all applied conditions. After 72 h enzymatic hydrolysis with 5% solid loading, 15.1 g/L glucose, 5.6 g/L xylose, and 0.9 g/L arabinose were obtained from the straw pretreated at 180 °C for 30 min; thus, the obtained total sugar concentration was 76% higher than that obtained from the untreated straw. In other words, glucose yield of 46.2% was obtained. At 8% solid loading, glucose yield of 32.9% was obtained from the straw pretreated at 180 °C for 30 min while it was 44.2% from the straw pretreated at 150 °C for 60 min. Despite 18% higher lignin content, the straw pretreated at 150 °C for 60 min was converted to glucose with 34% higher yield. This observation may be related to the fact that the lignin content is not the only parameter affecting the hydrolysis. Change in the structure of lignin and its interaction with other lignocellulosic parts are also among the important factors. Furthermore, the hemicellulose removal (mainly xylan in the case of rice straw) can improve the yield of hydrolysis (Karimi et al., 2013). Therefore, without considering the other affecting parameters, the yield of hydrolysis could not be correlated with lignin content. Enzymatic hydrolysis (with 2% solid loading) of alkaline (using 12% w/v NaOH at 0 °C for 180 min) and concentrated phosphoric acid (85% at 50 °C for 30 min) pretreated rice straw had been previously evaluated for ABE production in which total sugar concentration of less than 10.8 g/L was obtained (Moradi et al., 2013).
Increasing the solid loading in the hydrolysis process form 5% to 8% increased the total sugar concentration. The highest total sugar concentration of 31.0 g/L, containing 21.5 g/L glucose, 7.9 g/L xylose, and 1.5 g/L arabinose, was obtained after 72 h hydrolysis with 8% solid loading from the rice straw pretreated at 150 °C for 60 min. In this process, the glucose yield of 44.2% was obtained. 3.3. ABE fermentation Rice straw which was pretreated at different conditions (at 150 and 180 °C for 30 and 60 min) was hydrolyzed using 5% and 8% solid loadings, and the hydrolysates were subsequently subjected to ABE fermentation by C. acetobutylicum at 37 °C for 72 h. Depending on the pretreatment conditions and the solid loading in the hydrolysis step, different amounts of butanol, acetone, ethanol, acetic acid, and butyric acid were detected through the fermentation. Concentrations and yields of the fermentation products are shown in Fig. 2 and Table 2, respectively. In addition, the results of ABE production from rice straw using different pretreatment technologies from the current and previous studies are summarized in Table 3. The organosolv pretreatment of the straw increased the concentration of butanol and acetone. Pretreatment at 180 °C for 30 min and the subsequent hydrolysis using 5% solid loading resulted in production of 29.2 g/L sugars, and fermentation of the sugars resulted in production of 5.1 g/L butanol, 1.34 g/L acetone, 1.43 g/L ethanol, 1.18 g/L acetic acid, and 1.23 g/L butyric acid. In comparison with acetone and ethanol, butanol concentration in the fermentation product was more affected by the pretreatment conditions. Even though prolonging the pretreatment at 150 °C enhanced the concentration of butanol from rice straw, the
40
50 45
35
40
Sugar (g/l)
35 25
30
20
25 20
15
15
Glucose yield (%)
30
10 10 5
5
0
0 Untreated
30, 150
60, 150
30, 180
Pretreatment conditions (Time (min), Temperature (°C))
60, 180
Untreated
30, 150
60, 150
30, 180
60, 180
Pretreatment conditions (Time (min), Temperature (°C))
Fig. 1. Glucose (black), xylose (dark gray), and arabinose (light gray) concentrations, and theoretical glucose yield (s) after 72 h enzymatic hydrolysis of organosolv pretreated rice straw at 5% (a) and 8% (b) solid loading. Theoretical glucose yield (%) = produced glucose (g/L) 100/(1.111 substrate concentration (g/L) biomass glucan fraction).
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10
12 10
8
ABE (g/l)
8 6 6 4 4 2
2 0
Acetic acid (g/l); Butyric acid (g/l)
b
a
0 Untreated
30, 150
60, 150
30, 180
60, 180
Untreated
Pretreatment conditions (Time (min), Temperature ( C))
30, 150
60, 150
30, 180
60, 180
Pretreatment conditions (Time (min), Temperature ( C))
Fig. 2. Concentration of butanol (white), acetone (dark gray), ethanol (light gray), acetic acid (), and butyric acid () after 72 h fermentation of hydrolysate of organosolv pretreated rice straw by hydrolysis at solid loading of 5% (a) and 8% (b).
Table 2 Overall yield of butanol, acetone, ethanol, acetic acid, and butyric acid production from rice straw using different pretreatments.* Pretreatment conditions Temperature (°C) Untreated 150 150 180 180 Untreated 150 150 180 180 *
Solid loading (%) Time (min) 30 60 30 60 30 60 30 60
5 5 5 5 5 8 8 8 8 8
Overall yields (g/kg rice straw) Butanol
Acetone
Ethanol
Acetic acid
Butyric acid
43.6 57.0 77.8 80.3 61.2 7.6 27.7 58.8 69.8 60.3
22.7 20.5 18.2 21.1 21.9 17.4 19.4 24.6 18.7 20.3
23.1 18.3 23.1 22.5 24.1 15.7 33.5 27.1 14.7 14.0
12.8 15.4 121.4 18.6 11.8 <1 37.4 48.4 62.2 18.1
35.6 23.6 37.8 19.3 14.7 <1 8.2 4.1 3.0 1.9
The volume changes through addition of P2 solutions and the culture were considered in the final yields.
pretreatment at 180 °C for 30 min was more effective than that for 60 min. Increasing the solid loading in the hydrolysis from 5% to 8% enhanced the total concentration of acetone, butanol, and ethanol. Using 8% solid loading, enzymatic hydrolysis of rice straw which was pretreated at 180 °C for 30 min and the subsequent fermentation resulted in production of 7.1 g/L butanol, 1.9 g/L acetone, 1.5 g/L ethanol, 6.3 g/L acetic acid, and 0.3 g/L butyric acid. Concentration of the acids in the final product was less than 64% of ABE concentration except in the case of using the straw pretreated at 180 °C for 30 min with 5% solid loading in which acids concentration was 34% higher than ABE concentration. It should be noted that the acids mainly form in the exponential (acidogenesis) phase of growth while the solvents produce at the stationary (solventogenesis) phase. Considering the solvents as the desired products, the fermentation was continued to reach the solventogenesis phase, and the reported data are belonging to the samples analyzed at the end of fermentation (72 h). However, the yield of acids is high at acidogenesis phase, and the fermentation should be interrupted at this phase if the acids are the desired products. ABE fermentation of hydrolysates by C. acetobutylicum using pretreatments by steam explosion (121 °C; 15 lb; 30 min), diluteacid hydrolysis (1% H2SO4; 60 °C; 24 h and 121 °C; 15 min), and enzyme assisted hydrolysis (enzyme and 1% HCl; 60 °C; 24 h and 121 °C; 15 min) resulted in production of 2.07, 2.12, and 2.99 g/L ABE, respectively (Ranjan and Moholkar, 2011). In addition, through a hydrolysis and fermentation of alkaline (12% w/v NaOH;
at 0 °C; for 180 min) and phosphoric acid (85%; at 50 °C; for 30 min) pretreated rice straw by C. acetobutylicum, ABE concentration of 2–2.85 g/L had been obtained (Moradi et al., 2013). Concentration profiles during ABE fermentation of hydrolysates obtained from pretreated (at 180 °C for 30 min in which the highest butanol concentration was obtained) and untreated rice straw are depicted in Fig. 3. During the first 8 h of fermentation, less than 0.4 g/L ABE was produced (Fig. 3). More than 40% of butanol obtained from the pretreated rice straw was produced during 12 to 24 h of fermentation. Fermentation of the hydrolysate obtained from the rice straw pretreated at 180 °C for 30 min resulted in ABE productivity of 0.12 and 0.20 g/L/h at solid loadings of 5% and 8% in enzymatic hydrolysis, respectively. In addition to the concentration, the overall production yield of acetone, butanol, and ethanol as well as acetic acid and butyric acid based on the initial rice straw utilized were evaluated (Table 2). The overall yield of ABE production from rice straw was improved by the organosolv pretreatment. After organosolv pretreatment at 180 °C for 30 min and enzymatic hydrolysis at 5% solid loading, the ABE fermentation resulted in 80.3 g butanol, 21.1 g acetone, 22.5 g ethanol, 18.6 g acetic acid, and 19.3 g butyric acid from each kg of initial rice straw. The maximum overall ABE yield (i.e., 123.9 g/kg rice straw) obtained in this work was about twice as high as the overall yield that previously obtained through the alkaline pretreatment (i.e., 64.1 g/kg rice straw) and the phosphoric acid pretreatment (i.e., 63.0 g/kg rice straw) (Moradi et al., 2013)
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H. Amiri et al. / Bioresource Technology 152 (2014) 450–456 Table 3 ABE production form rice straw using different pretreatment technologies. Microorganisms
Pretreatment
Hydrolysis
Treatment to remove inhibitors
Highest butanol (ABE) (g/L)
Highest productivity (g/L/h)
Butanol (ABE) yield (g/kg raw rice straw)
Reference
C. acetobutylicum NRRL B-591 C. acetobutylicum NRRL B-591 C. acetobutylicum NRRL B-591 C. acetobutylicum MTCC 481
Ethanol organosolv (75% EtOH; 1% H2SO4 180 °C; 30 min) Alkaline (12% NaOH; 0 °C; 180 min)
Enzymatic hydrolysis Enzymatic hydrolysis Enzymatic hydrolysis –
–
7.10 (10.5)
0.200
80.3 (123.9)
This study
–
1.40 (2.00)
0.030
45.2 (64.1)
–
2.00 (2.85)
0.030
44.2 (63.0)
–
1.72 (2.07)
0.017
34.4 (41.4)*
C. acetobutylicum MTCC 481
Dilute acid (1% H2SO4; 60 °C; 24 h and 121 °C; 15 min)
–
–
1.60 (2.12)
0.017
32.0 (42.4)*
C. acetobutylicum MTCC 481
Enzyme assisted hydrolysis (enzyme and 1% HCl; 60 °C; 24 h and 121 °C; 15 min) Acid (4% H2SO4; 121 °C; 60 min)
–
–
2.10 (2.99)
0.025
42 .0 (59.8)*
Enzymatic hydrolysis Enzymatic hydrolysis Enzymatic hydrolysis
Anionic resin
3.43 (5.32)
0.050
34.3 (53.2)*
–
3.00 (4.5)
0.060
–
XAD-4 resin
6.5 (9.3)
0.100
77.4 (110.7)*
Moradi et al. (2013) Moradi et al. (2013) Ranjan and Moholkar (2011) Ranjan and Moholkar (2011) Ranjan and Moholkar (2011) Gottumukkala et al. (2013) Soni et al. (1982) Qureshi et al. (2008)
Clostridium sporogenes BE01 Cl. saccharoper butylacetonicum C. beijerinckii BA101**
Alkaline (2.25% NaOH; 120 °C; 60 min) Dilute acid (1% H2SO4; 121 °C; 60 min)
The yields were calculated based on butanol (or ABE) and substrate concentrations, and the solid recovery of pretreatments assumed to be 100%. In this study corn fiber was used for ABE production.
12
b
ABE (g/l)
a 10
10
8
8
6
6
4
4
2
2
16
12
8
4
0 12
0
0
20
12
cc
ABE (g/l)
20
12
Sugar (g/l)
**
Steam explosion (121 °C; 15 lb; 30 min)
d
10
10
8
8
6
6
4
4
2
2
16
12
8
Sugar (g/l)
*
Phosphoric acid (85%; 50 °C; 30 min)
4
0
0
0
0
12
24
36
48
60
Fermentation time (h)
72 0
12
24
36
48
60
72
Fermentation time (h)
Fig. 3. Concentration profile of ABE (s), butanol (}), acetone (h), ethanol (4), glucose (d), xylose (j), and arabinose (N) by fermentation of hydrolysates obtained after 72 h enzymatic hydrolysis of untreated (a and b) and organosolv pretreated rice straw at 180 °C for 30 min (c and d) by hydrolysis with solid loading of 5% (a and c) and 8% (b and d).
(Table 3). Even though the hydrolysis of the pretreated rice straw at 8% solid loading resulted in higher ABE concentration, the overall yield of ABE was higher at 5% solid loading. Increasing substrate
concentration led to a highly concentrated sugar solution, which in turn fermented to higher ABE concentration. However, high solid loading was accompanied with difficulties in mixing and
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end-product enzyme inhibition which was negatively affected the yield of hydrolysis (Karimi et al., 2013). In addition, butanol has been shown to have an inhibitory effect on ABE fermentation (García et al., 2011); thus, the lower yield of ABE production may be related to increasing the solid loading as a result of butanol inhibition at higher concentrations. Therefore, utilizing pretreated straw at higher solid loading resulted in production of ABE with higher concentration but lower overall yield. Therefore, the maximum ABE concentration of 10.5 g/L was obtained through the organosolv pretreatment at 180 °C for 30 min, enzymatic hydrolysis at 8% solid loading, and the ABE fermentation. 4. Conclusion The organosolv pretreatment can be applied for efficient production of the solvents from rice straw. The yield of ABE production from rice straw as well as the concentration of ABE was improved by organosolv pretreatment without necessity of detoxifications. A relatively high yield of 123.9 g ABE was obtained from each kg of rice straw using the pretreatment followed by enzymatic hydrolysis and fermentation. In addition, the concentration of ABE was increased up to 10.5 g/L through the fermentation of the organosolv pretreated rice straw hydrolysates. References Adney, B., Baker, J., 1996. Measurement of cellulase activities. In: LAP-006 NREL Analitical Procedure. National Renewable Energy Laboratory, Golden, CO. Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101, 4851–4861. Amiri, H., Karimi, K., Roodpeyma, S., 2010. Production of furans from rice straw by single-phase and biphasic systems. Carbohydr. Res. 345, 2133–2138. Bahl, H., Dürre, P., 2001. Clostridia: Biotechnology and Medical Applications. WileyVCH Verlag GmbH, Weinheim, pp. 57–63. Fischer, C.R., Klein-Marcuschamer, D., Stephanopoulos, G., 2008. Selection and optimization of microbial hosts for biofuels production. Metab. Eng. 10, 295– 304. Fortman, J.L., Chhabra, S., Mukhopadhyay, A., Chou, H., Lee, T.S., Steen, E., Keasling, J.D., 2008. Biofuel alternatives to ethanol: pumping the microbial well. Trends Biotechol. 26, 375–381. García, V., Päkkilä, J., Ojamo, H., Muurinen, E., Keiski, R.L., 2011. Challenges in biobutanol production: how to improve the efficiency? Renew. Sust. Energ. Rev. 15, 964–980. Gottumukkala, L.D., Parameswaran, B., Valappil, S.K., Mathiyazhakan, K., Pandey, A., Sukumaran, R.K., 2013. Biobutanol production from rice straw by a non acetone producing Clostridium sporogenes BE01. Bioresour. Technol. 140, 182–187. Hu, G., Cateto, C., Pu, Y., Samuel, R., Ragauskas, A.J., 2011. Structural characterization of switchgrass lignin after ethanol organosolv pretreatment. Energy Fuels 26, 740–745. Jang, Y.S., Malaviya, A., Cho, C., Lee, J., Lee, S.Y., 2012. Butanol production from renewable biomass by clostridia. Bioresour. Technol. 123, 653–663. Jones, D.T., Woods, D.R., 1986. Acetone–butanol fermentation revisited. Microbiol. Rev. 50, 484–524. Jurgens, G., Survase, S., Berezina, O., Sklavounos, E., Linnekoski, J., Kurkijärvi, A., Väkevä, M., van Heiningen, A., Granström, T., 2012. Butanol production from lignocellulosics. Biotechnol. Lett. 34, 1415–1434.
Karimi, K., Shafiei, M., Kumar, R., 2013. Progress on physical and chemical pretreatment of lignocellulosic biomass. In: Gupta, V.K., Tuohy, M.G. (Eds.), Biofuels and Biorefineries: Recent Developments. Springer Science Publishers, Berlin, pp. 53–96. Kim, S., Dale, B.E., 2004. Global potential bioethanol production from wasted crops and crop residues. Biomass Bioenergy 26, 361–375. Koo, B.-W., Min, B.-C., Gwak, K.-S., Lee, S.-M., Choi, J.-W., Yeo, H., Choi, I.-G., 2012. Structural changes in lignin during organosolv pretreatment of Liriodendron tulipifera and the effect on enzymatic hydrolysis. Biomass Bioenergy 42, 24–32. Kumar, M., Gayen, K., 2011. Developments in biobutanol production: new insights. Appl. Energy 88, 1999–2012. McMillan, J.D., 1994. Pretreatment of lignocellulosic biomass. In: Himmel, M.E., Baker, J.O., Overend, R.P. (Eds.), Enzymatic Conversion of Biomass for Fuels Production. American Chemical Society, Washington, DC, pp. 292–324. Mesa, L., González, E., Cara, C., González, M., Castro, E., Mussatto, S.I., 2011. The effect of organosolv pretreatment variables on enzymatic hydrolysis of sugarcane bagasse. Chem. Eng. J. 168, 1157–1162. Moradi, F., Amiri, H., Soleimanianzad, S., Ehsani, M.R., Karimi, K., 2013. Improvement of acetone, butanol and ethanol production from rice straw by acid and alkaline pretreatments. Fuel 112, 8–13. 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, 673–686. Obama, P., Ricochon, G., Muniglia, L., Brosse, N., 2012. Combination of enzymatic hydrolysis and ethanol organosolv pretreatments: effect on lignin structures, delignification yields and cellulose-to-glucose conversion. Bioresour. Technol. 112, 156–163. Qureshi, N., Saha, B.C., Cotta, M.A., 2007. Butanol production from wheat straw hydrolysate using Clostridium beijerinckii. Bioprocess Biosyst. Eng. 30, 419–427. Qureshi, N., Ezeji, T.C., Ebener, J., Dien, B.S., Cotta, M.A., Blaschek, H.P., 2008. Butanol production by Clostridium beijerinckii. Part I: Use of acid and enzyme hydrolyzed corn fiber. Bioresour. Technol. 99, 5915–5922. Qureshi, N., Saha, B.C., Dien, B., Hector, R.E., Cotta, M.A., 2010a. Production of butanol (a biofuel) from agricultural residues: Part I – use of barley straw hydrolysate. Biomass Bioenergy 34, 559–565. Qureshi, N., Saha, B.C., Hector, R.E., Dien, B., Hughes, S., Liu, S., Iten, L., Bowman, M.J., Sarath, G., Cotta, M.A., 2010b. Production of butanol (a biofuel) from agricultural residues: Part II - use of corn stover and switchgrass hydrolysates. Biomass Bioenergy 34, 566–571. Qureshi, N., Liu, S., Ezeji, T., 2013. Cellulosic butanol production from agricultural biomass and residues: recent advances in technology. In: Lee, J.W. (Ed.), Advanced Biofuels and Bioproducts. Springer, New York, pp. 247–265. Ranjan, A., Moholkar, V., 2011. Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels. Fuel 112, 567–571. Shatalov, A.A., Pereira, H., 2005. Kinetics of organosolv delignification of fibre crop Arundo donax L. Ind. Crop. Prod. 21, 203–210. Sluiter, A., Hames, B., Hyman, D., Payne, C., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Wolfe, J., 2008a. Determination of total solids in biomass and total dissolved solids in liquid process samples. In: NREL/TP-510-42621 Analitical Procedure. National Renewable Energy Laboratory, Golden, CO. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Wolfe, J., 2008b. Determination of structural carbohydrates and lignin in biomass. In: NREL/TP510-42618 Analitical Procedure. National Renewable Energy Laboratory, Golden, CO. Soni, B., Das, K., Ghose, T., 1982. Bioconversion of agro-wastes into acetone butanol. Biotechnol. Lett. 4, 19–22. Wang, L., Chen, H., 2011. Increased fermentability of enzymatically hydrolyzed steam-exploded corn stover for butanol production by removal of fermentation inhibitors. Process Biochem. 46, 604–607. Ximenes, E.A., Felix, C.R., Ulhoa, C.J., 1996. Production of cellulases by Aspergillus fumigatus and characterization of one b-glucosidase. Curr. Microbiol. 32, 119– 123. Zhao, X., Cheng, K., Liu, D., 2009. Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis. Appl. Microbiol. Biotechnol. 82, 815–827.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Organosolv pretreatment of rice straw for efficient acetone, butanol, and ethanol production Hamid Amiri a, Keikhosro Karimi a,b,⇑, Hamid Zilouei a a b
Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran Industrial Biotechnology Group, Institute of Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Production of ABE was significantly
improved by organosolv pretreatment. The pretreatment resulted in 60% lignin removal. After pretreatment, more than 311 g sugar was produced from each kg of rice straw. More than 80 g butanol and 21 g acetone were produced from each kg of rice straw.
a r t i c l e
i n f o
Article history: Received 14 September 2013 Received in revised form 13 November 2013 Accepted 16 November 2013 Available online 23 November 2013 Keywords: ABE fermentation Organosolv Lignocellulose Pretreatment Rice straw
a b s t r a c t Acetone–butanol–ethanol (ABE) was produced from rice straw using a process containing ethanol organosolv pretreatment, enzymatic hydrolysis, and fermentation by Clostridium acetobutylicum bacterium. Pretreatment of the straw with 75% (v/v) aqueous ethanol containing 1% w/w sulfuric acid at 150 °C for 60 min resulted in the highest total sugar concentration of 31 g/L in the enzymatic hydrolysis. However, the highest ABE concentration and productivity (10.5 g/L and 0.20 g/L h, respectively) were obtained from the straw pretreated at 180 °C for 30 min. Enzymatic hydrolysis of the straw pretreated at 180 °C for 30 min with 5% solid loading resulted in glucose yield of 46.2%, which was then fermented to 80.3 g butanol, 21.1 g acetone, and 22.5 g ethanol, the highest overall yield of ABE production. Thus, the organosolv pretreatment can be applied for efficient production of the solvents from rice straw. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Acetone–butanol–ethanol (ABE) fermentation is one of the first large-scale industrial fermentation processes to be developed (Jones and Woods, 1986). Up to the 1950s, about two thirds of butanol and 10% of acetone in the world were produced by this process from corn starch and molasses (Bahl and Dürre, 2001). However, during the 1960s, production of butanol and acetone
⇑ Corresponding author at: Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran. Tel.: +98 3113915623; fax: +98 3113912677. E-mail address: [email protected] (K. Karimi). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.11.038
shifted from biological to petrochemical based process due to increasing the price of corn and molasses as well as availability of cheaper petrochemical derived butanol and acetone (García et al., 2011). In recent years, by increasing concerns about unstable crude oil supplies and dramatic environmental impact of fossil fuels, there is a high interest in production of green butanol from biomass (Kumar and Gayen, 2011). Even though economically production of acetone, butanol, and ethanol as bulk chemicals from renewable sources is considered as a breakthrough toward reduction of dependency on fossil resources, it is the unique characteristics of butanol as a biofuel that renewed the interest in ABE fermentation (Kumar and Gayen, 2011). In comparison to ethanol, butanol has a higher energy
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content, lower solubility in water, lower vapor pressure, and a similar air-to-fuel ratio to gasoline. More importantly, butanol is compatible with the current automobile engines and the transportation pipelines. These facts make the biobutanol as an ideal candidate to replace gasoline (Fortman et al., 2008; Kumar and Gayen, 2011). The bioconversion of lignocellulosic materials to monomeric sugars and its consequent fermentation has been suggested for economical production of ABE (Jurgens et al., 2012). Since 1898, when the first cellulosic ethanol production was industrialized in Germany, different technologies have been suggested and developed for economical conversion of lignocelluloses to fermentable sugars (Mosier et al., 2005). However, most of these technologies have been developed for ethanol production, while they do not match well with requisites of ABE fermentation (Fischer et al., 2008). In addition, the negative effects of inhibitors are more serious problems in ABE fermentation compared with ethanolic fermentation (Jang et al., 2012). Therefore, in the production of fermentable sugars, not only the yield of conversion but also the concentration of sugars and inhibitors in the hydrolysate are among the factors affecting the possibility and economy of ABE production from lignocelluloses. Enzymatic hydrolysis is a widely used process for conversion of lignocellulosic materials to monomeric sugars. However, the recalcitrant structure of the lignocelluloses makes the hydrolysis inefficient, and it is essential to open up the structure through a pretreatment (Karimi et al., 2013). The role of pretreatment is even more important in ABE production from lignocelluloses, since the process is more sensitive to inhibitors. Diluted sulfuric acid pretreatment has been commonly used for ABE production from lignocellulosic resources (Gottumukkala et al., 2013; Qureshi et al., 2007, 2010a). However, different inhibitors which are generated in this process severely affect the growth and fermentation ABE producing microorganism; thus, an additional detoxification process is necessary to obtain a high yield which is accompanied with some sugar loss and additional capital investment (Qureshi et al., 2010b). Recently, concentrated alkaline and phosphoric acid pretreatments were evaluated for production of ABE from rice straw (Moradi et al., 2013). However, the recovery of the chemical agents in these processes accompanied with high energy consumption and production of environmental pollutant wastewaters. Thus, the organosolv pretreatment can be applied for efficient production of the solvents from rice straw. Among the pretreatment technologies, ethanol organosolv process has been considered as one of the most promising methods for biorefining of lignocelluloses (Alvira et al., 2010). Ethanol organosolv pretreatment involves the use of ethanol and water to partially hydrolyze lignin and lignin–carbohydrate bonds to remove lignin from lignocelluloses (Hu et al., 2011), the major barrier to the enzymatic hydrolysis (McMillan, 1994). Lignin hinders hydrolysis by blocking access of cellulases to cellulose due to irreversibly binding to the hydrolytic enzymes. Therefore, removal of lignin can considerably improve the hydrolysis rate (McMillan, 1994). The organosolv pretreatment is usually carried out using a strong inorganic acid catalyst, e.g., sulfuric acid, which takes a role to hydrolyze the lignin bonds in biomass (Zhao et al., 2009). Compared to other chemical pretreatments, one of the main and unique advantages of organosolv process is recovery of lignin as a valuable by-product, which is relatively pure, primarily unaltered, and less condensed than Kraft lignins (Mesa et al., 2011; Zhao et al., 2009). Furthermore, the recovery of solvent is typically performed with minimal energy consumption. From the environmental point of view, separation of lignin as a product could reduce the problem with wastewater treatment. Considering the special requirements of ABE fermentation, organosolv pretreatment may offer several advantages over the other pretreatments. In addition, lignin and its derivatives were
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found to be the main inhibitors affecting the ABE fermentation (Wang and Chen, 2011). Moreover, enrichment of cellulose content by partial removal of lignin is a possible approach to increase the maximum obtainable glucose concentration. As a result, removal of lignin as a purified product through the organosolv pretreatment can improve the economy of ABE process by improving the ABE production and addition of ethanol organosolv lignin as a value-added product. To our knowledge, there is no publication on evaluation of organosolv pretreatment for improvement of ABE production. The main drawbacks and limitations of the previous studies on ABE production from lignocelluloses are production of hydrolysates containing different inhibitors, necessity of detoxification which is accompanied with some sugar loss (Qureshi et al., 2013), and environmental problems pertaining to wastewater containing different degraded materials produced during the pretreatment. In this study, ethanol organosolv pretreatment was evaluated for production of ABE from rice straw. This pretreatment does not have the mentioned drawbacks and separate a lignin with high purity, which is a value added product. The straw is one of the most favorable substrates for biological products in terms of quantity and world wide availability (Kim and Dale, 2004). The pretreated rice straw was hydrolyzed by two commercial hydrolytic enzymes and fermented by Clostridium acetobutylicum. One of the goals was obtaining higher yield of ABE from the straw compared to the results obtained by other pretreatment methods. 2. Methods 2.1. Raw materials and enzymes Rice straw, used as a substrate in all experiments, was obtained from Lenjan field with a cultivar named ‘‘Sazandegi’’ (Isfahan, Iran). It was partly ball-milled and screened to achieve a size of between 833 lm (20 mesh) and 177 lm (80 mesh). Dry weight content of the sample was measured by drying at 105 °C in a convection oven. The materials were stored at room temperature in resealable plastic bags. Two commercial enzymes of cellulase (Celluclast 1.5L, Novozyme, Denmark) and b-glucosidase (Novozyme 188, Novozyme, Denmark) were used for the hydrolysis. The cellulase activity was 65 FPU/ml according to the method presented by Adney and Baker (1996), and the protein content of the enzyme was 42 mg/ml as measured by Bradford assay. The b-glucosidase activity was 210 IU/ml, measured by the method presented by Ximenes et al. (1996). 2.2. Pretreatment methods Fifty grams (dry weight) of the straw was mixed with 400 g (solid-to-liquid ratio of 1:8) of 75% (v/v) aqueous ethanol containing 1% w/w (based on the straw dried mass) sulfuric acid as a catalyst. Treatments were carried out in a high-pressure stainless steel vessel as a batch reactor with a working volume of 500 ml (Amiri et al., 2010). The reaction mixture was heated at a rate of 3 °C/min. After reaching the desired temperature, i.e., 150 or 180 °C, the mixture was maintained at the temperature for 30 or 60 min. Afterwards, it was cooled in an ice bath. The pretreated materials were then washed with 60 °C aqueous ethanol (75% (v/ v), 3 100 ml) and air dried overnight. Finally, the pretreated materials were stored in resealable plastic bags at 5 °C until use. 2.3. Enzymatic hydrolysis The untreated and pretreated materials were subjected to enzymatic hydrolysis by cellulase and b-glucosidase in 50 mM sodium
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citrate buffer (pH 4.8). The hydrolysis was conducted at 45 °C and 140 rpm for 72 h using 25 FPU cellulase and 40 IU b-glucosidase per gram of either treated or untreated biomass at two solid loadings of 5% and 8%. The cellulase loadings were 33 mg protein per gram of cellulose for untreated rice straw, whereas it was varied from 26 to 30 mg protein per gram of cellulose for the pretreated straw. The hydrolysates were separated from the remaining solid fraction by centrifugation (5 min, 5000 rpm) and then fermented.
0.6 ml/min eluent of deionized water. Concentrations of sugars, acetone, butanol, and ethanol were determined by RI detector, while acetic acid and butyric acid were quantified on UV chromatograms at 210 nm. All experiments were performed in duplicates and the averages of the results are presented.
2.4. Microorganism and inoculum preparation 3. Results and discussion C. acetobutylicum NRRL B-591, obtained from Persian type culture collection (PTCC) (Tehran, Iran), was used in all fermentations. In order to prepare the preculture, 1.5 g cooked meat medium (Sigma–Aldrich) was added to 25 ml distilled water. After addition of 0.27 g glucose, the mixture was autoclaved (at 121 °C for 20 min) and cooled to 75 °C. Spore suspension (0.5 ml) was heat shocked at 75 °C for 2 min after addition to the medium (Moradi et al., 2013). Subsequently, the medium containing spores was cooled in ice-cold water for 1 min. The heat shocked spores were then incubated at 37 °C for 16–18 h (Moradi et al., 2013). For biomass preparation, a 100 ml medium containing (g/L): 30 glucose, 5 yeast extract, 2 (NH4)2SO4, 1 NaCl, 0.75 KH2PO4, 0.75 K2HPO4, 0.2 MgSO4, 0.01 MnSO47H2O, and 0.01 FeSO47H2O was prepared. Then, the medium was autoclaved (at 121 °C for 20 min) and cooled to 35 °C (Moradi et al., 2013). Afterwards, 5 ml of the preculture was added to the medium, and the growth was conducted at 37 °C for 16–18 h. 2.5. ABE fermentation After addition of 0.05 g yeast extract to 50 ml of the hydrolysates in 118 ml serum bottles, pH of the solutions was adjusted to 6.5 using 5 M NaOH. The solution was autoclaved (at 120 °C for 10 min) and cooled to room temperature. Subsequently, 0.5 ml of a buffer (50 g/L KH2PO4, 50 g/L K2HPO4, and 220 g/L CH3COONH4), a vitamin (0.1 g/L para-aminobenzoic acid, 0.1 g/L thiamin, and 0.001 g/L biotin), and a mineral (20 g/L MgSO47H2O, 1 g/L MnSO4H2O, 1 g/L FeSO47H2O, 1 g/L NaCl) solutions, was filter sterilized (Millipore filter; 0.22 lm) and added to each bottle (Moradi et al., 2013). The bottle was then purged with pure nitrogen passed over a heated reduced copper column to remove trace oxygen and sealed using a butyl rubber stopper fastened with an aluminum crimp. After inoculation with 6 ml of actively growing culture (optical density of 1.2–1.6 at 610 nm), fermentation was conducted at 37 °C for 72 h. During fermentation, liquid samples were periodically withdrawn using a sterile syringe through the rubber stopper. The samples were centrifuged at 9000 rpm for 25 min and stored at 18 °C before sugar and ABE content analyses. 2.6. Analytical methods Moisture and total solids (Sluiter et al., 2008a), structural carbohydrates, lignin, and ash contents (Sluiter et al., 2008b) of the untreated and pretreated straw were determined. The cell concentration was estimated from optical density (OD) using a predetermined correlation between OD at 610 nm and the dry cell weight (at 105 °C until constant weight) (Moradi et al., 2013). Concentration of the fermentation products as well as sugars were analyzed by high-performance liquid chromatography (HPLC), equipped with UV/VIS and RI detectors (Jasco International Co., Tokyo, Japan). Concentration of acetone, butanol, ethanol, acetic acid, and butyric acid were determined by an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) at 60 °C with 0.6 ml/min eluent of 0.005 M sulfuric acid. Sugars were analyzed on an Aminex HPX-87P column (Bio-Rad, Richmond, CA, USA) at 80 °C with
3.1. Pretreatment Rice straw was subjected to ethanol organosolv pretreatment prior to hydrolysis in order to improve the production yield and concentration of ABE in the subsequent fermentation. The pretreatment temperature and time were selected by considering the previous studies results (Obama et al., 2012) and preliminary investigations. The solid recovery and chemical composition of the pretreated materials are summarized in Table 1. The untreated straw consisted of 49.2% glucan, 29.2% xylan, 3.5% arabinan, and 16.3% Klason lignin. Different parts of the straw were differently affected by the pretreatment. The organosolv pretreated material was considered as cellulosic fibers, which depending on the applied conditions, contained various amounts of hemicellulose and residual lignin (Zhao et al., 2009). The organosolv pretreatment at 180 °C for 60 min resulted in the highest lignin removal (60%), obtaining a pretreated material with 9.8% Klason lignin. In addition, the pretreatment at 180 °C for 30 min resulted in 56% lignin removal. Furthermore, the pretreatment at the lower temperature, i.e., 150 °C, resulted in 41% and 45% lignin removal after 30 and 60 min treatment, respectively. In other words, increasing the temperature from 150 to 180 °C resulted in more than 33% higher lignin removal. It was in agreement with previous studies that showed that the lignin removal is highly affected by the process temperature and increased by increasing temperature (Shatalov and Pereira, 2005). Through the organosolv pretreatment, in addition to lignin, the hemicellulosic sugars are partially dissolved in the organic liquor. After pretreatment, the organic liquor was evaporated to recover the solvent and diluted with water to precipitate the lignin, as one of the valuable products (Zhao et al., 2009). The remaining aqueous solution is rich in hemicellulosic sugars, mainly xylose in the case of rice straw, and is considered as a product which can be utilized by pentose fermenting microorganisms or converted to furfural, xylitol, and some other chemicals (Zhao et al., 2009). In this study, the remaining liquor, after evaporation of ethanol, was diluted with water and subjected to ABE fermentation. However, using different dilution ratios (1:1.5, 1:2, and 1:5 the initial to final liquor), no detectable butanol was produced through the fermentation (data not shown). Through the pretreatment, the xylan content decreased by 40% and 49% after 30 and 60 min pretreatment at 180 °C, while it was 42% and 45% after 30 and 60 min pretreatment at 150 °C, respectively. Increasing the temperature and retention time of the organosolv pretreatment, generally, increases the lignin and xylan removal (Koo et al., 2012). In the case of rice straw, the lignin removal was considerably increased by temperature, whereas the conditions had a minor effect on xylan removal. The organosolv pretreatment of rice straw generally resulted in enrichment of cellulose content as a result of partial solubilisation of both hemicellulose and lignin. Cellulose content of the untreated rice straw was 49.2% which was increased up to 62.3% by the pretreatment at 180 °C for 60 min, while the pretreatment at 180 °C for 30 min resulted in the material with 58.9% cellulose.
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H. Amiri et al. / Bioresource Technology 152 (2014) 450–456 Table 1 Chemical composition and solid recovery of untreated and organosolv pretreated rice straw. Pretreatment conditions
Composition
T (°C)
Time (min)
Glucan (%)
Xylan (%)
Arabinnan (%)
Acid soluble lignin (%)
Acid insoluble (Klason) lignin (%)
Ash (%)
Solid recovery (%)
30 60 30 60
49.2 ± 1.2 55.6 ± 0.8 54.8 ± 0.7 58.9 ± 1.2 62.3 ± 1.2
29.2 ± 1.4 21.7 ± 0.1 20.9 ± 0.6 24.4 ± 0.1 22.4 ± 0.6
3.5 ± 0.2 2.4 ± 0.1 2.7 ± 0.1 2.3 ± 0.4 1.9 ± 0.4
0.7 ± 0.1 1.7 ± 0.1 1.7 ± 0.2 1.5 ± 0.1 1.3 ± 0.1
16.3 ± 1.3 12.4 ± 0.2 11.7 ± 0.2 9.9 ± 0.2 9.8 ± 0.2
6.7 ± 0.2 7.9 ± 0.3 8.4 ± 0.2 6.6 ± 0.1 7.5 ± 0.2
– 78.3 ± 0.9 76.8 ± 1.5 72.1 ± 1.3 66.9 ± 1.0
Untreated 150 150 180 180
3.2. Enzymatic hydrolysis The pretreated and untreated straws were subjected to enzymatic hydrolysis at 45 °C for 72 h using 25 FPU cellulase and 40 IU b-glucosidase per gram of substrate with 5% and 8% solid loadings. Concentration of sugars and the yield of glucose are shown in Fig. 1. Total concentration of sugars was increased after the pretreatment in all applied conditions. After 72 h enzymatic hydrolysis with 5% solid loading, 15.1 g/L glucose, 5.6 g/L xylose, and 0.9 g/L arabinose were obtained from the straw pretreated at 180 °C for 30 min; thus, the obtained total sugar concentration was 76% higher than that obtained from the untreated straw. In other words, glucose yield of 46.2% was obtained. At 8% solid loading, glucose yield of 32.9% was obtained from the straw pretreated at 180 °C for 30 min while it was 44.2% from the straw pretreated at 150 °C for 60 min. Despite 18% higher lignin content, the straw pretreated at 150 °C for 60 min was converted to glucose with 34% higher yield. This observation may be related to the fact that the lignin content is not the only parameter affecting the hydrolysis. Change in the structure of lignin and its interaction with other lignocellulosic parts are also among the important factors. Furthermore, the hemicellulose removal (mainly xylan in the case of rice straw) can improve the yield of hydrolysis (Karimi et al., 2013). Therefore, without considering the other affecting parameters, the yield of hydrolysis could not be correlated with lignin content. Enzymatic hydrolysis (with 2% solid loading) of alkaline (using 12% w/v NaOH at 0 °C for 180 min) and concentrated phosphoric acid (85% at 50 °C for 30 min) pretreated rice straw had been previously evaluated for ABE production in which total sugar concentration of less than 10.8 g/L was obtained (Moradi et al., 2013).
Increasing the solid loading in the hydrolysis process form 5% to 8% increased the total sugar concentration. The highest total sugar concentration of 31.0 g/L, containing 21.5 g/L glucose, 7.9 g/L xylose, and 1.5 g/L arabinose, was obtained after 72 h hydrolysis with 8% solid loading from the rice straw pretreated at 150 °C for 60 min. In this process, the glucose yield of 44.2% was obtained. 3.3. ABE fermentation Rice straw which was pretreated at different conditions (at 150 and 180 °C for 30 and 60 min) was hydrolyzed using 5% and 8% solid loadings, and the hydrolysates were subsequently subjected to ABE fermentation by C. acetobutylicum at 37 °C for 72 h. Depending on the pretreatment conditions and the solid loading in the hydrolysis step, different amounts of butanol, acetone, ethanol, acetic acid, and butyric acid were detected through the fermentation. Concentrations and yields of the fermentation products are shown in Fig. 2 and Table 2, respectively. In addition, the results of ABE production from rice straw using different pretreatment technologies from the current and previous studies are summarized in Table 3. The organosolv pretreatment of the straw increased the concentration of butanol and acetone. Pretreatment at 180 °C for 30 min and the subsequent hydrolysis using 5% solid loading resulted in production of 29.2 g/L sugars, and fermentation of the sugars resulted in production of 5.1 g/L butanol, 1.34 g/L acetone, 1.43 g/L ethanol, 1.18 g/L acetic acid, and 1.23 g/L butyric acid. In comparison with acetone and ethanol, butanol concentration in the fermentation product was more affected by the pretreatment conditions. Even though prolonging the pretreatment at 150 °C enhanced the concentration of butanol from rice straw, the
40
50 45
35
40
Sugar (g/l)
35 25
30
20
25 20
15
15
Glucose yield (%)
30
10 10 5
5
0
0 Untreated
30, 150
60, 150
30, 180
Pretreatment conditions (Time (min), Temperature (°C))
60, 180
Untreated
30, 150
60, 150
30, 180
60, 180
Pretreatment conditions (Time (min), Temperature (°C))
Fig. 1. Glucose (black), xylose (dark gray), and arabinose (light gray) concentrations, and theoretical glucose yield (s) after 72 h enzymatic hydrolysis of organosolv pretreated rice straw at 5% (a) and 8% (b) solid loading. Theoretical glucose yield (%) = produced glucose (g/L) 100/(1.111 substrate concentration (g/L) biomass glucan fraction).
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10
12 10
8
ABE (g/l)
8 6 6 4 4 2
2 0
Acetic acid (g/l); Butyric acid (g/l)
b
a
0 Untreated
30, 150
60, 150
30, 180
60, 180
Untreated
Pretreatment conditions (Time (min), Temperature ( C))
30, 150
60, 150
30, 180
60, 180
Pretreatment conditions (Time (min), Temperature ( C))
Fig. 2. Concentration of butanol (white), acetone (dark gray), ethanol (light gray), acetic acid (), and butyric acid () after 72 h fermentation of hydrolysate of organosolv pretreated rice straw by hydrolysis at solid loading of 5% (a) and 8% (b).
Table 2 Overall yield of butanol, acetone, ethanol, acetic acid, and butyric acid production from rice straw using different pretreatments.* Pretreatment conditions Temperature (°C) Untreated 150 150 180 180 Untreated 150 150 180 180 *
Solid loading (%) Time (min) 30 60 30 60 30 60 30 60
5 5 5 5 5 8 8 8 8 8
Overall yields (g/kg rice straw) Butanol
Acetone
Ethanol
Acetic acid
Butyric acid
43.6 57.0 77.8 80.3 61.2 7.6 27.7 58.8 69.8 60.3
22.7 20.5 18.2 21.1 21.9 17.4 19.4 24.6 18.7 20.3
23.1 18.3 23.1 22.5 24.1 15.7 33.5 27.1 14.7 14.0
12.8 15.4 121.4 18.6 11.8 <1 37.4 48.4 62.2 18.1
35.6 23.6 37.8 19.3 14.7 <1 8.2 4.1 3.0 1.9
The volume changes through addition of P2 solutions and the culture were considered in the final yields.
pretreatment at 180 °C for 30 min was more effective than that for 60 min. Increasing the solid loading in the hydrolysis from 5% to 8% enhanced the total concentration of acetone, butanol, and ethanol. Using 8% solid loading, enzymatic hydrolysis of rice straw which was pretreated at 180 °C for 30 min and the subsequent fermentation resulted in production of 7.1 g/L butanol, 1.9 g/L acetone, 1.5 g/L ethanol, 6.3 g/L acetic acid, and 0.3 g/L butyric acid. Concentration of the acids in the final product was less than 64% of ABE concentration except in the case of using the straw pretreated at 180 °C for 30 min with 5% solid loading in which acids concentration was 34% higher than ABE concentration. It should be noted that the acids mainly form in the exponential (acidogenesis) phase of growth while the solvents produce at the stationary (solventogenesis) phase. Considering the solvents as the desired products, the fermentation was continued to reach the solventogenesis phase, and the reported data are belonging to the samples analyzed at the end of fermentation (72 h). However, the yield of acids is high at acidogenesis phase, and the fermentation should be interrupted at this phase if the acids are the desired products. ABE fermentation of hydrolysates by C. acetobutylicum using pretreatments by steam explosion (121 °C; 15 lb; 30 min), diluteacid hydrolysis (1% H2SO4; 60 °C; 24 h and 121 °C; 15 min), and enzyme assisted hydrolysis (enzyme and 1% HCl; 60 °C; 24 h and 121 °C; 15 min) resulted in production of 2.07, 2.12, and 2.99 g/L ABE, respectively (Ranjan and Moholkar, 2011). In addition, through a hydrolysis and fermentation of alkaline (12% w/v NaOH;
at 0 °C; for 180 min) and phosphoric acid (85%; at 50 °C; for 30 min) pretreated rice straw by C. acetobutylicum, ABE concentration of 2–2.85 g/L had been obtained (Moradi et al., 2013). Concentration profiles during ABE fermentation of hydrolysates obtained from pretreated (at 180 °C for 30 min in which the highest butanol concentration was obtained) and untreated rice straw are depicted in Fig. 3. During the first 8 h of fermentation, less than 0.4 g/L ABE was produced (Fig. 3). More than 40% of butanol obtained from the pretreated rice straw was produced during 12 to 24 h of fermentation. Fermentation of the hydrolysate obtained from the rice straw pretreated at 180 °C for 30 min resulted in ABE productivity of 0.12 and 0.20 g/L/h at solid loadings of 5% and 8% in enzymatic hydrolysis, respectively. In addition to the concentration, the overall production yield of acetone, butanol, and ethanol as well as acetic acid and butyric acid based on the initial rice straw utilized were evaluated (Table 2). The overall yield of ABE production from rice straw was improved by the organosolv pretreatment. After organosolv pretreatment at 180 °C for 30 min and enzymatic hydrolysis at 5% solid loading, the ABE fermentation resulted in 80.3 g butanol, 21.1 g acetone, 22.5 g ethanol, 18.6 g acetic acid, and 19.3 g butyric acid from each kg of initial rice straw. The maximum overall ABE yield (i.e., 123.9 g/kg rice straw) obtained in this work was about twice as high as the overall yield that previously obtained through the alkaline pretreatment (i.e., 64.1 g/kg rice straw) and the phosphoric acid pretreatment (i.e., 63.0 g/kg rice straw) (Moradi et al., 2013)
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H. Amiri et al. / Bioresource Technology 152 (2014) 450–456 Table 3 ABE production form rice straw using different pretreatment technologies. Microorganisms
Pretreatment
Hydrolysis
Treatment to remove inhibitors
Highest butanol (ABE) (g/L)
Highest productivity (g/L/h)
Butanol (ABE) yield (g/kg raw rice straw)
Reference
C. acetobutylicum NRRL B-591 C. acetobutylicum NRRL B-591 C. acetobutylicum NRRL B-591 C. acetobutylicum MTCC 481
Ethanol organosolv (75% EtOH; 1% H2SO4 180 °C; 30 min) Alkaline (12% NaOH; 0 °C; 180 min)
Enzymatic hydrolysis Enzymatic hydrolysis Enzymatic hydrolysis –
–
7.10 (10.5)
0.200
80.3 (123.9)
This study
–
1.40 (2.00)
0.030
45.2 (64.1)
–
2.00 (2.85)
0.030
44.2 (63.0)
–
1.72 (2.07)
0.017
34.4 (41.4)*
C. acetobutylicum MTCC 481
Dilute acid (1% H2SO4; 60 °C; 24 h and 121 °C; 15 min)
–
–
1.60 (2.12)
0.017
32.0 (42.4)*
C. acetobutylicum MTCC 481
Enzyme assisted hydrolysis (enzyme and 1% HCl; 60 °C; 24 h and 121 °C; 15 min) Acid (4% H2SO4; 121 °C; 60 min)
–
–
2.10 (2.99)
0.025
42 .0 (59.8)*
Enzymatic hydrolysis Enzymatic hydrolysis Enzymatic hydrolysis
Anionic resin
3.43 (5.32)
0.050
34.3 (53.2)*
–
3.00 (4.5)
0.060
–
XAD-4 resin
6.5 (9.3)
0.100
77.4 (110.7)*
Moradi et al. (2013) Moradi et al. (2013) Ranjan and Moholkar (2011) Ranjan and Moholkar (2011) Ranjan and Moholkar (2011) Gottumukkala et al. (2013) Soni et al. (1982) Qureshi et al. (2008)
Clostridium sporogenes BE01 Cl. saccharoper butylacetonicum C. beijerinckii BA101**
Alkaline (2.25% NaOH; 120 °C; 60 min) Dilute acid (1% H2SO4; 121 °C; 60 min)
The yields were calculated based on butanol (or ABE) and substrate concentrations, and the solid recovery of pretreatments assumed to be 100%. In this study corn fiber was used for ABE production.
12
b
ABE (g/l)
a 10
10
8
8
6
6
4
4
2
2
16
12
8
4
0 12
0
0
20
12
cc
ABE (g/l)
20
12
Sugar (g/l)
**
Steam explosion (121 °C; 15 lb; 30 min)
d
10
10
8
8
6
6
4
4
2
2
16
12
8
Sugar (g/l)
*
Phosphoric acid (85%; 50 °C; 30 min)
4
0
0
0
0
12
24
36
48
60
Fermentation time (h)
72 0
12
24
36
48
60
72
Fermentation time (h)
Fig. 3. Concentration profile of ABE (s), butanol (}), acetone (h), ethanol (4), glucose (d), xylose (j), and arabinose (N) by fermentation of hydrolysates obtained after 72 h enzymatic hydrolysis of untreated (a and b) and organosolv pretreated rice straw at 180 °C for 30 min (c and d) by hydrolysis with solid loading of 5% (a and c) and 8% (b and d).
(Table 3). Even though the hydrolysis of the pretreated rice straw at 8% solid loading resulted in higher ABE concentration, the overall yield of ABE was higher at 5% solid loading. Increasing substrate
concentration led to a highly concentrated sugar solution, which in turn fermented to higher ABE concentration. However, high solid loading was accompanied with difficulties in mixing and
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end-product enzyme inhibition which was negatively affected the yield of hydrolysis (Karimi et al., 2013). In addition, butanol has been shown to have an inhibitory effect on ABE fermentation (García et al., 2011); thus, the lower yield of ABE production may be related to increasing the solid loading as a result of butanol inhibition at higher concentrations. Therefore, utilizing pretreated straw at higher solid loading resulted in production of ABE with higher concentration but lower overall yield. Therefore, the maximum ABE concentration of 10.5 g/L was obtained through the organosolv pretreatment at 180 °C for 30 min, enzymatic hydrolysis at 8% solid loading, and the ABE fermentation. 4. Conclusion The organosolv pretreatment can be applied for efficient production of the solvents from rice straw. The yield of ABE production from rice straw as well as the concentration of ABE was improved by organosolv pretreatment without necessity of detoxifications. A relatively high yield of 123.9 g ABE was obtained from each kg of rice straw using the pretreatment followed by enzymatic hydrolysis and fermentation. In addition, the concentration of ABE was increased up to 10.5 g/L through the fermentation of the organosolv pretreated rice straw hydrolysates. References Adney, B., Baker, J., 1996. Measurement of cellulase activities. In: LAP-006 NREL Analitical Procedure. National Renewable Energy Laboratory, Golden, CO. Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101, 4851–4861. Amiri, H., Karimi, K., Roodpeyma, S., 2010. Production of furans from rice straw by single-phase and biphasic systems. Carbohydr. Res. 345, 2133–2138. Bahl, H., Dürre, P., 2001. Clostridia: Biotechnology and Medical Applications. WileyVCH Verlag GmbH, Weinheim, pp. 57–63. Fischer, C.R., Klein-Marcuschamer, D., Stephanopoulos, G., 2008. Selection and optimization of microbial hosts for biofuels production. Metab. Eng. 10, 295– 304. Fortman, J.L., Chhabra, S., Mukhopadhyay, A., Chou, H., Lee, T.S., Steen, E., Keasling, J.D., 2008. Biofuel alternatives to ethanol: pumping the microbial well. Trends Biotechol. 26, 375–381. García, V., Päkkilä, J., Ojamo, H., Muurinen, E., Keiski, R.L., 2011. Challenges in biobutanol production: how to improve the efficiency? Renew. Sust. Energ. Rev. 15, 964–980. Gottumukkala, L.D., Parameswaran, B., Valappil, S.K., Mathiyazhakan, K., Pandey, A., Sukumaran, R.K., 2013. Biobutanol production from rice straw by a non acetone producing Clostridium sporogenes BE01. Bioresour. Technol. 140, 182–187. Hu, G., Cateto, C., Pu, Y., Samuel, R., Ragauskas, A.J., 2011. Structural characterization of switchgrass lignin after ethanol organosolv pretreatment. Energy Fuels 26, 740–745. Jang, Y.S., Malaviya, A., Cho, C., Lee, J., Lee, S.Y., 2012. Butanol production from renewable biomass by clostridia. Bioresour. Technol. 123, 653–663. Jones, D.T., Woods, D.R., 1986. Acetone–butanol fermentation revisited. Microbiol. Rev. 50, 484–524. Jurgens, G., Survase, S., Berezina, O., Sklavounos, E., Linnekoski, J., Kurkijärvi, A., Väkevä, M., van Heiningen, A., Granström, T., 2012. Butanol production from lignocellulosics. Biotechnol. Lett. 34, 1415–1434.
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