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Improvement of acetone, butanol and ethanol production from rice straw by acid and alkaline pretreatments
Fuel 112 (2013) 8–13
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Improvement of acetone, butanol and ethanol production from rice straw by acid and alkaline pretreatments Farzad Moradi a, Hamid Amiri a, Sabihe Soleimanian-Zad b, Mohammad Reza Ehsani a, Keikhosro Karimi a,c,⇑ a
Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran c Industrial Biotechnology Group, Institute of Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran b
h i g h l i g h t s ABE production from rice straw was significantly improved by alkali and acid pretreatments. After pretreatments, over 67% of glucan and 17% of xylan were recovered. After pretreatment, more than 163 g glucose was produced from each kg of rice straw. More than 44 g butanol and 17 g acetone were produced from each kg of rice straw.
a r t i c l e
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Article history: Received 29 May 2012 Received in revised form 11 March 2013 Accepted 1 May 2013 Available online 18 May 2013 Keywords: ABE fermentation Alkaline Phosphoric acid Pretreatment Rice straw
a b s t r a c t Rice straw was hydrolyzed and fermented to acetone, butanol, and ethanol by Clostridium acetobutylicum bacterium. Concentrated phosphoric acid and alkaline treatment with NaOH were used for pretreatment of the straw prior to enzymatic hydrolysis using commercial cellulase and b-glucosidase. The enzymatic hydrolysates were then anaerobically fermented by C. acetobutylicum. Hydrolysis of the alkaline pretreated straw resulted in production of 163.5 g glucose from each kg of untreated rice straw which was then fermented to 45.2 g butanol, 17.7 g acetone, and 1.2 g ethanol. Additionally, concentrated phosphoric acid pretreatment and subsequent hydrolysis resulted in production of 192.3 g glucose from each kg straw from which 44.2 g butanol, 18.2 g acetone, and 0.6 g ethanol were produced after 72 h fermentation. Increasing the produced ABE from less than 10 g to higher than 62 g from each kg straw by the treatments suggested the alkaline and phosphoric acid pretreatments as promising processes for efficient production of ABE from rice straw. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Butanol, main product of acetone butanol ethanol (ABE) fermentation, is a solvent, a chemical intermediate, an extractant, and more importantly a potential biofuel [1]. By 1950, ABE fermentation by solventogenic Clostridium species using corn starch and molasses as substrates was one of the main fermentative products in industrial scale [2]. However, many ABE plants were closed during the 1960s as a result of increased price of corn and molasses and availability of cheaper petrochemical derived butanol [2]. Growing concerns regarding volatility of oil supply and global warming result in recent expansion in research relating to ABE fer-
⇑ 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). 0016-2361/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fuel.2013.05.011
mentation for production of butanol which has unique characteristics as a potential biofuel [1]. Even though biological production of butanol through ABE fermentation is challenging due to its commercial obstacles, interesting attributes of biobutanol as a biofuel represents its potential new market [3]. Not only biobutanol is less volatile, explosive, corrosive, and hygroscopic than ethanol, but also it has more energy content and could easily mix with gasoline in any portion [3]. Considering the important effects of substrate cost on economical feasibility of biobutanol production, efficient utilization of lignocellulosic wastes instead of costly food-based substrates, e.g., corn and molasses, was suggested to make the butanol production economically viable [4,5]. A number of lignocellulosic residues such as wheat straw [6], barely straw [7], corn stover [8], and switchgrass [8] have previously been used for biobutanol production. Among agricultural wastes, rice straw is one of the low cost and mainly useless lignocellulosic materials that may properly be used for biological
F. Moradi et al. / Fuel 112 (2013) 8–13
butanol production. Additionally, rice straw is potentially one of the most favorable feedstocks in terms of quantity as a substrate for biological products [9]. Solventogenic Clostridia used in ABE fermentation are able to ferment a wide variety of carbohydrates including lactose, sucrose, glucose, fructose, mannose, dextrin, starch, xylose, arabinose, and inulin [4]. Therefore, the prerequisite in the utilization of lignocelluloses for butanol production is to prepare a hydrolysate rich in fermentable sugars. Application of enzymes for the hydrolysis of lignocellulose offers several advantages of higher yields, minimal byproduct formation, low energy requirements, mild operating conditions, and environmentally friendly processing over the other chemical conversion routes. However, the recalcitrant structure of the native straw, similar to other lignocelluloses, makes its enzymatic hydrolysis inefficient [10]. Dilute sulfuric acid pretreatment, which is among the best pretreatment methods for improvement of cellulosic ethanol production, has recently been used for improvement of ABE fermentation from lignocellulosic resources [6,7]. However, generation of different byproducts in this process has inhibited the ABE producing microorganism growth and fermentation [8]. Overliming of the dilute acid pretreated hydrolysates was also applied to reduce the negative effects of the inhibitors; however, lime treatment of the hydrolysates resulted in reduction of the produced sugars levels and also inefficient fermentation [8]. Furthermore, steam explosion without addition of any acid was also used for pretreatment of ABE production from lignocelluloses. However, the explosion process should be conducted at high pressures and temperatures [11]. Different pretreatment processes have been developed for improvement of ethanol production from lignocelluloses [10]. Alkaline pretreatment is one of the most promising technologies for improvement of agricultural residues hydrolysis. The process resulted in increasing the swelling capacity and internal surface area, decreasing cellulose crystallinity and degree of polymerization, and also disrupting the compact lignin-carbohydrate structure [12]. On the other hand, the concentrated phosphoric acid pretreatment at modest reaction conditions has been recently demonstrated to be an effective method for improvement of hydrolysis of lignocelluloses [13]. These pretreatment technologies may also be applied prior to ABE fermentation. To our knowledge, there is no publication on utilization of these pretreatments prior to ABE fermentation. In the current study, alkaline and concentrated phosphoric acid pretreatments were evaluated for production of acetone, butanol, and ethanol from rice straw. The pretreated straw was hydrolyzed by two commercial hydrolytic enzymes and fermented by C. acetobutylicum.
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2.2. Pretreatment methods Prior to enzymatic hydrolysis, alkaline and concentrated phosphoric acid pretreatments were used. Alkaline pretreatment was performed using 12% w/v NaOH with 5% w/w solids loading at 0 °C for 3 h. After the pretreatment, the mixture was washed with distilled water until pH 7 was reached. The solid was then filtered and dried at 50 °C [17]. Concentrated phosphoric acid pretreatment was performed by mixing one gram straw with 8 ml of H3PO4 (85%) at 50 °C for 30 min. Pre-cold acetone was consequently added for quenching the mixture. After centrifugation for 10 min, the supernatant was collected. The solid pellet was suspended in 40 ml acetone and centrifuged three times. The precipitated solids were consequently washed three times with excess distilled water and centrifuged [13]. The treated solid was then dried at 50 °C. 2.3. Enzymatic hydrolysis Pretreated and untreated rice straws were enzymatically hydrolyzed in 50 mM sodium citrate buffer (pH 4.8) using cellulase and b-glucosidase. The straw was soaked in the buffer for 4 h prior to enzymatic hydrolysis. Hydrolysis was performed at 140 rpm and 45 °C with 2% solid loading for 72 h. The enzyme loadings were 25 FPU cellulase and 50 IU b-glucosidase per gram of biomass. 2.4. Microorganism and inoculum preparation 2.4.1. Clostridium acetobutylicum NRRL B-591 was obtained from Persian type culture collection (PTCC) (Iranian Research Organization for Science and Technology, Tehran, Iran). The culture was stored in sterile distilled water at 2– 4 °C. In order to prepare the inoculum, 2.5 g cooked meat medium (Sigma–Aldrich) was soaked in 15–18 ml distilled water. After addition of 0.2 g glucose, the prepared medium was autoclaved at 121 °C for 20 min and cooled to 75 °C. One half ml of spore suspension was added to the medium and heat shocked at 75 °C for 2 min, and the mixture was then cooled in ice-cold water for 1 min. The heat shocked spores were then incubated in an anaerobic jar at 35 °C for 24 h. For cultivation of the strain, a 100 ml medium containing 30 g/L glucose, 5 g/L yeast extract, 2 g/L ammonium acetate, 1 g/L sodium chloride, 0.75 g/L KH2PO4, 0.75 g/L K2HPO4, 0.2 g/L MgSO4, 0.01 g/L MnSO47H2O, and 0.01 g/L FeSO47H2O was prepared and autoclaved at 121 °C for 20 min. After cooling to 35 °C, 0.5 g/L cysteine HClH2O was filtered (0.45 lm Millipore filter, GEMA Medical SL) and added to the medium. About 5 ml of the prepared bacterial culture was then added to the medium and the growth was conducted at 37 °C for 24 h [18].
2. Materials and methods
2.5. ABE fermentation
2.1. Rice straw and enzymes
Fermentation of 50 ml of the enzymatic hydrolysate was performed in 118 ml serum bottles (717561, Pajuhesh Setayesh Sepahan, Isfahan, Iran) sealed with butyl rubber and aluminum crimp cap. After addition of 0.05 g yeast extract to each bottle, pH of the solutions was adjusted to 6.5 using 5 M NaOH. These solutions were sterilized at 121 °C for 20 min followed by cooling to room temperature. Prior to inoculation, 0.5 ml of P2 stock solution was filter sterilized (Millipore filter; 0.22 lm) and added to each bottle. P2 stock solutions contained a buffer (50 g/L KH2PO4, 50 g/L K2HPO4, and 220 g/L CH3COONH4), vitamin (0.1 g/L para-aminobenzoic acid, 0.1 g/L thiamin, and 0.001 g/L biotin), and mineral (20 g/L MgSO47H2O, 1 g/L MnSO4H2O, 1 g/L FeSO47H2O, 1 g/L NaCl) solutions [19]. The bottles were then inoculated with 6 ml of actively growing culture (optical density 1.2–1.6 at 610 nm). These bottles were sparged with pure nitrogen in order to provide
Rice straw used in all the experiments was obtained from Lenjan field (Isfahan province, Iran). It was dried at 60 ± 5 °C for 1 day. The straw with original length between 20 and 50 mm was partially ball-milled and screened to achieve a size of less than 833 lm (20 mesh) and larger than 299 lm (48 mesh) prior to the enzymatic hydrolysis. Two commercial enzymes, cellulase (Celluclast 1.5L, Novozyme, Denmark) and b-glucosidase (Novozyme 188, Novozyme, Denmark), were used for hydrolysis. The cellulase activity was 80 FPU/ml, measured by the method presented by Adney and Baker [14] and it contained 42 mg/ml protein, as measured by Bradford assay [15]. The b-glucosidase activity was 240 IU/ml according to method presented by Ximenes et al. [16].
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the anaerobic conditions. Fermentation was conducted at 37 °C for 72 h. During fermentation, liquid samples for analysis of sugars and ABE contents were periodically withdrawn with a sterile syringe through the rubber stopper. The samples were centrifuged at 9000 rpm for 25 min and stored at 18 °C before the analysis. 2.6. Analytical procedures Moisture and total solid contents of the biomass were measured by drying at 105 °C to a constant weight [20]. Ash content of the biomass was also analyzed following the procedure described by Sluiter et al. [21]. Structural carbohydrates and lignin in biomass were determined according to a standard method presented by Sluiter et al. [22]. The cell concentration was estimated by optical density (OD) and the dry cell weight (DCW) was calculated using a predetermined correlation between OD at 610 nm and DCW. Concentration of fermentation products (ABE, acetic acid, and butyric acid) as well as sugars were analyzed by high-performance liquid chromatography (HPLC) (Jasco International Co., Tokyo, Japan), equipped with UV/VIS and RI detectors (Jasco International Co., Tokyo, Japan). Fermentation products (ABE, acetic acid, and butyric acid) were analyzed on 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. Concentrations of sugars were determined using an Aminex HPX-87P column (Bio-Rad, Richmond, CA, USA) at 80 °C. Deionized water was used as an eluent at a flow rate of 0.6 ml/ min. Concentrations of glucose, acetone, butanol, and ethanol were determined by RI detector, while acetic 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.
Table 3 Glucose yield after enzymatic hydrolysis of pretreated and untreated rice straw. Yield of enzymatic hydrolysis
Alkaline pretreated Phosphoric acid pretreated Untreated rice straw
Theoretical glucose yield (%)a
(g Glucose/kg rice straw)
(g Glucose/kg pretreated rice straw)
60.4
163.5
407.9
69.8
192.3
354.1
31.1
101.8
–
a Theoretical glucose yield (%) = produced glucose (g/L) 100/(1.111 substrate concentration (g/L) biomass glucan fraction).
As can be seen in Table 1, the recovery of the solid was higher for the phosphoric acid pretreatment than that in the alkaline treatment. Considering composition of pretreated materials (Table 2), different parts of rice straw were differently affected by the pretreatments. More than 66% of glucan was recovered after the pretreatments, while only 17% of xylan after the treatments was recovered. On the other hand, alkaline pretreatment reduced 76% of lignin, while phosphoric acid pretreatment reduced only 27% of lignin content. Higher acid insoluble lignin removal by the alkaline pretreatment compared with the phosphoric acid treatment could be the reason for lower solid recovery by the alkaline pretreatment. Considering the recovery of the solid, rice straw lost 63% and 30% of its ash content through the alkaline and phosphoric acid pretreatments, respectively. However, weight percentage of ash in the solid increased after pretreatments due to further reduction of other parts.
3. Results 3.2. Enzymatic hydrolysis
Rice straw was pretreated with alkaline and concentrated phosphoric acid prior to hydrolysis in order to improve the yield of ABE production in the consequent fermentation. The solid recovery and chemical composition of the pretreated materials are summarized in Tables 1 and 2, respectively.
Table 1 Solid recovery and ash content of pretreated and untreated rice straw. Pretreatment
Solid recovery (w/w%)
Ash (wt.%)
Alkaline pretreatment Phosphoric acid pretreatment Untreated rice straw
40.1 54.3 –
14.2 19.9 15.4
Table 2 Chemical composition of pretreated and untreated rice straw. Components
Untreated rice straw
Alkaline pretreated rice straw
Phosphoric acid pretreated rice straw
Glucan (wt.%) Xylan (wt.%) Arabinan (wt.%) Acid insoluble residue (wt.%) Acid insoluble ash (wt.%) Acid insoluble lignin (wt.%) Acid soluble lignin (wt.%)
36.5 13.9 2.7 20.3
60.8 5.8 2.1 16.1
45.7 4.3 0.0 33.5
7.6
7.8
14.2
12.7
8.3
19.3
Pretreated materials were subsequently subjected to enzymatic hydrolysis using cellulase and extra b-glucosidase enzymes with 2% solid loading. The results of hydrolysis are shown in Fig. 1 and Table 3. As can be seen in Fig. 1, both of the pretreatments improved the yield of glucose production. After 72 h of enzymatic hydrolysis, glucose was produced with a yield of 35.4 and 40.8 g per 100 g of alkali and phosphoric acid pretreated straw, respectively, while the yield from untreated rice straw was only 10.2 g per 100 g of untreated straw.
g glucose/ 100 g pretreated rice straw
3.1. Pretreatment
45 40 35 30 25 20 15 10 5 0 0
10
20
30
40
50
60
70
Time (h) 2.4
0.6
0.9 Fig. 1. Production of glucose in the enzymatic hydrolysis of untreated (s), alkaline pretreated (h), and H3PO4 pretreated (D) rice straw.
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3
12
B
A 2.5
Products (g/ L)
Glucose (g/L)
10 8 6 4
1.5 1 0.5
2 0
2
0 0
10
20
30
40
50
60
70
0
10
20
30
Time (h)
40
50
60
70
Time (h)
Fig. 2. Glucose (A) and solvents (B) concentrations in fermentation of reference medium. The symbols represents glucose (j), acetone (e), butanol (), ethanol (D), acetic acid (s), butyric acid (h), and total ABE () concentrations.
Enzymatic hydrolysis of phosphoric acid pretreated rice straw resulted in the highest theoretical glucose yield of 69.8%. Considering the pretreatments solid recovery, alkaline and phosphoric acid pretreatments resulted in production of 163.5 g and 192.3 g glucose per kg of the initial straw, while hydrolysis of untreated rice straw resulted in only 101.8 g glucose per kg of the straw.
concentration of 0.69 g/L, 75% of its final concentration. At the same time, acetic and butyric acid concentrations reached 1.55 and 0.42 g/L, respectively, which were slightly decreased during the fermentation. As shown in Fig. 3, production of ABE was significantly affected by the pretreatments. During the first 13 h of fermentation, glucose content of alkaline pretreated hydrolysate was sharply consumed to its half concentration through the growth phase of fermentation, while no detectable ABE was produced. Concentration of glucose remained constant during the next 23 h. The residual glucose was consumed between 36 and 48 h of fermentation and over 60% of total ABE was produced throughout the stationary phase of fermentation. Remaining 40% of total ABE was gradually produced during the next 34 h. Fermentation of alkaline pretreated rice straw resulted in 2.85 g/L ABE which contained 2 g/L butanol, 0.8 g/L acetone, and 0.05 g/L ethanol (Fig. 3a and d). In addition, 1.22 g/L acetic acid and 1.47 g/L butyric acid were produced through the fermentation of alkaline pretreated rice straw. Fermentation of phosphoric acid pretreated straw hydrolysate was also resulted in consumption of sugars and production of
3.3. Fermentation Hydrolysates obtained from the enzymatic hydrolysis of pretreated and untreated rice straw, and P2 medium with 10 g/L glucose as a reference, were anaerobically fermented by C. acetobutylicum. The fermentation results of the reference and hydrolysates are shown in Figs. 2 and 3, respectively. Although the fermentation of the reference was conducted for 72 h, the culture consumed entire glucose content of the medium within 33 h and produced 1.36 g/L butanol. After 33 h, concentration of products were slightly changed except for ethanol which was decreased from 0.23 g/L at 57 h to lower than 0.04 g/L at 70 h. After 12 h fermentation, acetone was produced with
8
A
Consumed sugars (g/l)
7
B
C
E
F
6 5 4 3 2 1 0 3
D Products (g/l)
2.5 2 1.5 1 0.5 0 0
10
20
30
40
Time (h)
50
60
70
80
0
10
20
30
40
50
Time (h)
60
70
80
0
10
20
30
40
50
60
70
80
Time (h)
Fig. 3. Consumption of sugars and production of ABE from hydrolysates of NaOH pretreated (A and D), H3PO4 pretreated (B and E), and untreated (C and F) rice straw. The symbols represents glucose (j), xylose (d), arabionse (N), acetone (e), butanol (), ethanol (D), acetic acid (s), butyric acid (h), and total ABE () concentrations.
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Table 4 Yields of acetone, butanol, and ethanol productions from rice straw.a Butanol
Acetone
Ethanol
Acetic acid
Butyric acid
1.2 0.6 <0.1
27.6 45.6 83.2
33.4 39.2 74.7
(g/kg rice straw) Alkaline pretreatment Phosphoric acid pretreatment Untreated rice straw
45.2 44.2 9.0
17.7 18.2 <0.1
Butanol
Acetone
Ethanol
Acetic acid
Butyric acid
68.9 83.9 –
83.2 72.3 –
(g/kg pretreated rice straw) 112.7 81.4 –
44.2 33.6 –
3.0 1.1 –
The volume changes through addition of P2 solutions and the culture were considered in the final yields. a Yield (g/kg rice straw) = % solid recovery yield (g/kg pretreated rice straw).
ABE. Fermentation started with gradual consumption of glucose and xylose without ABE production. Glucose consumption was intensified after 20 h, and it was entirely consumed at 60 h of fermentation. Similar to fermentation of hydrolysate of the alkaline pretreated straw, 59% of total ABE were produced between 36 and 48 h in fermentation of the acid pretreated hydrolysate. Fermentation of the acid pretreated hydrolysate resulted in 1.4 g/L butanol, 0.6 g/L acetone, less than 0.1 g/L ethanol, 1.48 g/L acetic acid, and 1.28 g/L butyric acid (Fig. 3b and e). Fermentation of hydrolysate of untreated rice straw resulted in 0.16 g/L butanol which showed over 12-fold increase with alkaline pretreatment and 9-fold increase with phosphoric acid pretreatment (Fig. 3c and f). As presented in Table 4, using alkaline pretreatment and phosphoric acid pretreatments, 64.1 and 63.0 g ABE were produced from each kg of rice straw, respectively, while without pretreatment only 9 g ABE were produced from the same amount of rice straw. Butanol comprised 70% of ABE which were produced from both pretreated and untreated rice straws. Butyric acid, which is a product of acidogenesis phase of fermentation, was gradually produced up to 1.5 and 1.3 g/L after 82 h of fermentation of the alkaline and phosphoric acid pretreated hydrolysates, respectively. At the beginning of the fermentation, acetic acid was presented in alkaline and phosphoric pretreated hydrolysates with concentration of 1.3 and 1.2 g/L, respectively. The concentration of acetic acid was changed during fermentation of each hydrolysate, and its concentration in the fermented hydrolysates of the alkaline and phosphoric pretreated rice straw finally reached to 1.2 and 1.5 g/L, respectively.
4. Discussion Butanol, a potential substitute for fossil fuels, is the most desired product of ABE fermentation. In this fermentation process, species of Clostridium acetobutylicum bacteria metabolize different sugars to butanol and other solvents [1]. Even though ABE fermentation is one of the oldest fermentation technologies, its large-scale production remained challenging. High substrate costs and low volumetric productivity were considered as the main bottlenecks that preclude large-scale application of ABE fermentation [4]. Butanol is also a value-added byproduct of bioconversion of glycerol with Clostridum pasteurianum [23]. Available in large quantities, lignocellulosic sources were suggested as a unique feedstocks to revive the biobutanol industries [4,5]. In the current study, C. acetobutylicum was used to produce ABE from hydrolysates of pretreated rice straw. The prerequisite in the utilization of lignocellulose for butanol production is to efficiently produce a hydrolysate rich in fermentable sugars. Enzymatic hydrolysis is considered as a proper strategy with advantages over other chemical conversion routes [24]. However, like other lignocelluloses, native rice straw are recalcitrant to enzymatic hydrolysis, and a pretreatment is required prior to the enzymatic hydrolysis to render the cellulose to enzymatic attack [10]. In view of cellulosic ethanol production, a number of
pretreatment approaches have been investigated on a wide variety of feedstocks [10]. Dilute sulfuric acid pretreatment is among the best pretreatment methods; however, generation of some byproducts inhibited cell growth and ABE fermentation [6–8]. Thus, alkaline and phosphoric acid pretreatments were evaluated for improvement of ABE production in this study. Alkaline pretreatment, one of the most effective pretreatment technologies, is basically a delignification process, in which a significant amount of hemicellulose is solubilized as well [25]. In this study, the alkaline pretreatment and subsequent enzymatic hydrolysis of the treated residues resulted in production of 407.9 g glucose per kg of pretreated rice straw which was corresponded to 163.5 g glucose per kg of initial rice straw. Concentrated phosphoric acid pretreatment could be used to open up the structure of lignocelluloses and decrease the crystallinity of cellulose at a relatively low temperature [13]. Concentrated phosphoric acid could dissolve crystalline cellulose and hemicellulose portion which consequently precipitate to amorphous forms by addition of an anti-solvent such as acetone. Having large difference in volatility, phosphoric acid and acetone could easily recovered avoiding acid dilution with water which facilitates acid re-concentration [13]. In this study, the phosphoric acid pretreatment was used for improvement of butanol production by ABE fermentation, and the treated rice straw was recovered after the pretreatment which contained 68%, 17%, and 83% of the initial glucan, xylan, and lignin, respectively. The alkaline and phosphoric acid pretreatments not only increase the glucan, but also they decrease the xylan, and lignin parts [26]. In comparison with alkaline pretreatment, phosphoric acid pretreatment resulted in recovery of almost the same amounts of glucan and xylan but higher amount of lignin. Highest theoretical glucose yield of 69.8% was obtained through enzymatic hydrolysis of phosphoric acid pretreated rice straw. On the other hand, the alkaline pretreated straw had a higher glucan fraction which resulted in higher glucose concentration in its hydrolysate. However, phosphoric acid pretreatment, as a result of its higher solid recovery, resulted in higher glucose production from the same amounts of straw after enzymatic hydrolysis. Phosphoric acid pretreatment and subsequent enzymatic hydrolysis of the solid residues resulted in production of 354.1 g glucose per kg of the pretreated straw which corresponded to 192.3 g glucose per kg of initial rice straw. Butanol may be produced from hydrolysates of lignocellulosic biomass containing sugars. Hydrolysates of dilute acid pretreated wheat straw [6], barley straw [7], maize stover [8], and switchgrass [8] have been used for ABE fermentation in which a detoxification process such as overliming is necessary prior to the fermentation especially for barley straw and maize stover. In this study, hydrolysates of alkaline and phosphoric acid pretreated rice straw were used for ABE fermentation. Using the hydrolysate of alkaline treated rice straw, 45.2 g butanol, 17.7 g acetone, 1.2 g ethanol, 27.6 g acetic acid, and 33.4 g butyric acid were produced from each kg of initial rice straw. On the other hand, 44.2 g butanol, 18.2 g acetone, 0.6 g ethanol, 45.6 g acetic acid, and 39.2 g butyric acid per kg rice straw were produced through the phosphoric acid pretreatment, enzymatic hydrolysis and ABE fermentation. Comparing the results
F. Moradi et al. / Fuel 112 (2013) 8–13
of ABE production from the treated and untreated rice straws, both alkaline and phosphoric acid pretreatments resulted in a nearly 5fold increase in the butanol yield. Furthermore, no detoxification treatment is required for ABE fermentation of the hydrolysates. 5. Conclusions Rice straw can be a suitable substrate for production of butanol, acetone, and ethanol by fermentation. Alkaline and phosphoric acid pretreatments were two promising processes for improvement of the ABE from rice straw without necessity of detoxifications. More than 44 g butanol and 17 g acetone were produced from each kg of rice straw using both of the pretreatments followed by enzymatic hydrolysis and fermentation. References [1] García V, Päkkilä J, Ojamo H, Muurinen E, Keiski RL. Challenges in biobutanol production: how to improve the efficiency? Renew Sust Energy Rev 2011;15:964–80. [2] Jones DT, Woods DR. Acetone–butanol fermentation revisited. Microbiol Rev 1986;50:484–524. [3] Cascone R. Biobutanol – a replacement for bioethanol? Chem Eng Prog 2008;104:s4–9. [4] Qureshi N. Agricultural residues and energy crops as potentially economical and novel substrates for microbial production of butanol (a biofuel). CAB Rev 2010;5:1–8. [5] Zverlov V, Berezina O, Velikodvorskaya G, Schwarz W. Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery. Appl Microbiol Biotechnol 2006;71:587–97. [6] Qureshi N, Saha BC, Cotta MA. Butanol production from wheat straw hydrolysate using Clostridium beijerinckii. Bioproc Biosyst Eng 2007;30:419–27. [7] Qureshi N, Saha BC, Dien B, Hector RE, Cotta MA. Production of butanol (a biofuel) from agricultural residues: Part I – Use of barley straw hydrolysate. Biomass Bioenergy 2010;34:559–65. [8] Qureshi N, Saha BC, Hector RE, Dien B, Hughes S, Liu S, et al. Production of butanol (a biofuel) from agricultural residues: Part II – Use of corn stover and switchgrass hydrolysates. Biomass Bioenergy 2010;34:566–71. [9] Kim S, Dale BE. Global potential bioethanol production from wasted crops and crop residues. Biomass Bioenergy 2004;26:361–75.
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[10] Taherzadeh MJ, Karimi K. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int J Mol Sci 2008;9:1621–51. [11] Ranjan A, Moholkar VS. Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels. Fuel 2013;112:567–71. [12] Fan LT, Gharpuray MM, Lee YH. Cellulose Hydrolysis. Biotechnology Monographs, vol. 3. Springer-Verlag; 1987. [13] Zhang YHP, Ding SY, Mielenz JR, Cui JB, Elander RT, Laser M, et al. Fractionating recalcitrant lignocellulose at modest reaction conditions. Biotechnol Bioeng 2007;97:214–23. [14] Adney B, Baker J. Measurement of cellulase activities. NREL LAP-006 National Renewable Energy Laboratory, Golden, Colorado; 1996. [15] Noori MS. Effect of Lignin and Hemicellulose Separation on Enzymatic Bioadsorption of Lignocellulosic Compounds. M.Sc Thesis. Department of Chemical Engineering, Isfahan University of Technology; 2012. [16] Ximenes EA, Felix CR, Ulhoa CJ. Production of cellulases by Aspergillus fumigatus and characterization of one b-glucosidase. Curr Microbiol 1996;32:119–23. [17] Jafari V, Labafzadeh SR, Jeihanipour A, Karimi K, Taherzadeh MJ. Construction and demolition lignocellulosic wastes to bioethanol. Renew Energy 2011;36:2771–5. [18] Qureshi N, Meagher M, Huang J, Hutkins R. Acetone butanol ethanol (ABE) recovery by pervaporation using silicalite–silicone composite membrane from fed-batch reactor of Clostridium acetobutylicum. J Membrane Sci 2001;187:93–102. [19] Qureshi N, Blaschek HP. Butanol recovery from model solution/fermentation broth by pervaporation: evaluation of membrane performance. Biomass Bioenergy 1999;17:175–84. [20] Sluiter A, Hames B, Hyman D, Payne C, Ruiz R, Scarlata C, et al. Determination of total solids in biomass and total dissolved solids in liquid process samples. NREL/TP-510–42621. National Renewable Energy Laboratory, Golden, Colorado; 2008. [21] Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D. Determination of ash in biomass. Technical Report TP-510-42622. National Renewable Research Laboratory, Golden, Colorado; 2008. [22] Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, et al. Determination of structural carbohydrates and lignin in biomass. NREL/TP510-42618. National Renewable Research Laboratory, Golden, Colorado; 2004. [23] Khanna S, Goyal A, Moholkar VS. Production of n-butanol from biodiesel derived crude glycerol using Clostridium pasteurianum immobilized on Amberlite. Fuel 2013;112:557–61. [24] Taherzadeh MJ, Karimi K. Enzymatic-based hydrolysis processes for ethanol from lignocellulosic materials: a review. Bioresource 2007;2:707–38. [25] Zheng Y, Pan Z, Zhang R. Overview of biomass pretreatment for cellulosic ethanol production. Int J Agric Biol Eng 2009;2:51–68. [26] Karimi K, Shafiei M, Kumar R. Progress on physical and chemical pretreatment of lignocellulosic biomass. In: Gupta VK, editor. Biofuels and Biorefineries: Recent Developments. Germany: Springer Science Publishers; 2012. p. 53–96.
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Improvement of acetone, butanol and ethanol production from rice straw by acid and alkaline pretreatments Farzad Moradi a, Hamid Amiri a, Sabihe Soleimanian-Zad b, Mohammad Reza Ehsani a, Keikhosro Karimi a,c,⇑ a
Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran c Industrial Biotechnology Group, Institute of Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran b
h i g h l i g h t s ABE production from rice straw was significantly improved by alkali and acid pretreatments. After pretreatments, over 67% of glucan and 17% of xylan were recovered. After pretreatment, more than 163 g glucose was produced from each kg of rice straw. More than 44 g butanol and 17 g acetone were produced from each kg of rice straw.
a r t i c l e
i n f o
Article history: Received 29 May 2012 Received in revised form 11 March 2013 Accepted 1 May 2013 Available online 18 May 2013 Keywords: ABE fermentation Alkaline Phosphoric acid Pretreatment Rice straw
a b s t r a c t Rice straw was hydrolyzed and fermented to acetone, butanol, and ethanol by Clostridium acetobutylicum bacterium. Concentrated phosphoric acid and alkaline treatment with NaOH were used for pretreatment of the straw prior to enzymatic hydrolysis using commercial cellulase and b-glucosidase. The enzymatic hydrolysates were then anaerobically fermented by C. acetobutylicum. Hydrolysis of the alkaline pretreated straw resulted in production of 163.5 g glucose from each kg of untreated rice straw which was then fermented to 45.2 g butanol, 17.7 g acetone, and 1.2 g ethanol. Additionally, concentrated phosphoric acid pretreatment and subsequent hydrolysis resulted in production of 192.3 g glucose from each kg straw from which 44.2 g butanol, 18.2 g acetone, and 0.6 g ethanol were produced after 72 h fermentation. Increasing the produced ABE from less than 10 g to higher than 62 g from each kg straw by the treatments suggested the alkaline and phosphoric acid pretreatments as promising processes for efficient production of ABE from rice straw. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Butanol, main product of acetone butanol ethanol (ABE) fermentation, is a solvent, a chemical intermediate, an extractant, and more importantly a potential biofuel [1]. By 1950, ABE fermentation by solventogenic Clostridium species using corn starch and molasses as substrates was one of the main fermentative products in industrial scale [2]. However, many ABE plants were closed during the 1960s as a result of increased price of corn and molasses and availability of cheaper petrochemical derived butanol [2]. Growing concerns regarding volatility of oil supply and global warming result in recent expansion in research relating to ABE fer-
⇑ 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). 0016-2361/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fuel.2013.05.011
mentation for production of butanol which has unique characteristics as a potential biofuel [1]. Even though biological production of butanol through ABE fermentation is challenging due to its commercial obstacles, interesting attributes of biobutanol as a biofuel represents its potential new market [3]. Not only biobutanol is less volatile, explosive, corrosive, and hygroscopic than ethanol, but also it has more energy content and could easily mix with gasoline in any portion [3]. Considering the important effects of substrate cost on economical feasibility of biobutanol production, efficient utilization of lignocellulosic wastes instead of costly food-based substrates, e.g., corn and molasses, was suggested to make the butanol production economically viable [4,5]. A number of lignocellulosic residues such as wheat straw [6], barely straw [7], corn stover [8], and switchgrass [8] have previously been used for biobutanol production. Among agricultural wastes, rice straw is one of the low cost and mainly useless lignocellulosic materials that may properly be used for biological
F. Moradi et al. / Fuel 112 (2013) 8–13
butanol production. Additionally, rice straw is potentially one of the most favorable feedstocks in terms of quantity as a substrate for biological products [9]. Solventogenic Clostridia used in ABE fermentation are able to ferment a wide variety of carbohydrates including lactose, sucrose, glucose, fructose, mannose, dextrin, starch, xylose, arabinose, and inulin [4]. Therefore, the prerequisite in the utilization of lignocelluloses for butanol production is to prepare a hydrolysate rich in fermentable sugars. Application of enzymes for the hydrolysis of lignocellulose offers several advantages of higher yields, minimal byproduct formation, low energy requirements, mild operating conditions, and environmentally friendly processing over the other chemical conversion routes. However, the recalcitrant structure of the native straw, similar to other lignocelluloses, makes its enzymatic hydrolysis inefficient [10]. Dilute sulfuric acid pretreatment, which is among the best pretreatment methods for improvement of cellulosic ethanol production, has recently been used for improvement of ABE fermentation from lignocellulosic resources [6,7]. However, generation of different byproducts in this process has inhibited the ABE producing microorganism growth and fermentation [8]. Overliming of the dilute acid pretreated hydrolysates was also applied to reduce the negative effects of the inhibitors; however, lime treatment of the hydrolysates resulted in reduction of the produced sugars levels and also inefficient fermentation [8]. Furthermore, steam explosion without addition of any acid was also used for pretreatment of ABE production from lignocelluloses. However, the explosion process should be conducted at high pressures and temperatures [11]. Different pretreatment processes have been developed for improvement of ethanol production from lignocelluloses [10]. Alkaline pretreatment is one of the most promising technologies for improvement of agricultural residues hydrolysis. The process resulted in increasing the swelling capacity and internal surface area, decreasing cellulose crystallinity and degree of polymerization, and also disrupting the compact lignin-carbohydrate structure [12]. On the other hand, the concentrated phosphoric acid pretreatment at modest reaction conditions has been recently demonstrated to be an effective method for improvement of hydrolysis of lignocelluloses [13]. These pretreatment technologies may also be applied prior to ABE fermentation. To our knowledge, there is no publication on utilization of these pretreatments prior to ABE fermentation. In the current study, alkaline and concentrated phosphoric acid pretreatments were evaluated for production of acetone, butanol, and ethanol from rice straw. The pretreated straw was hydrolyzed by two commercial hydrolytic enzymes and fermented by C. acetobutylicum.
9
2.2. Pretreatment methods Prior to enzymatic hydrolysis, alkaline and concentrated phosphoric acid pretreatments were used. Alkaline pretreatment was performed using 12% w/v NaOH with 5% w/w solids loading at 0 °C for 3 h. After the pretreatment, the mixture was washed with distilled water until pH 7 was reached. The solid was then filtered and dried at 50 °C [17]. Concentrated phosphoric acid pretreatment was performed by mixing one gram straw with 8 ml of H3PO4 (85%) at 50 °C for 30 min. Pre-cold acetone was consequently added for quenching the mixture. After centrifugation for 10 min, the supernatant was collected. The solid pellet was suspended in 40 ml acetone and centrifuged three times. The precipitated solids were consequently washed three times with excess distilled water and centrifuged [13]. The treated solid was then dried at 50 °C. 2.3. Enzymatic hydrolysis Pretreated and untreated rice straws were enzymatically hydrolyzed in 50 mM sodium citrate buffer (pH 4.8) using cellulase and b-glucosidase. The straw was soaked in the buffer for 4 h prior to enzymatic hydrolysis. Hydrolysis was performed at 140 rpm and 45 °C with 2% solid loading for 72 h. The enzyme loadings were 25 FPU cellulase and 50 IU b-glucosidase per gram of biomass. 2.4. Microorganism and inoculum preparation 2.4.1. Clostridium acetobutylicum NRRL B-591 was obtained from Persian type culture collection (PTCC) (Iranian Research Organization for Science and Technology, Tehran, Iran). The culture was stored in sterile distilled water at 2– 4 °C. In order to prepare the inoculum, 2.5 g cooked meat medium (Sigma–Aldrich) was soaked in 15–18 ml distilled water. After addition of 0.2 g glucose, the prepared medium was autoclaved at 121 °C for 20 min and cooled to 75 °C. One half ml of spore suspension was added to the medium and heat shocked at 75 °C for 2 min, and the mixture was then cooled in ice-cold water for 1 min. The heat shocked spores were then incubated in an anaerobic jar at 35 °C for 24 h. For cultivation of the strain, a 100 ml medium containing 30 g/L glucose, 5 g/L yeast extract, 2 g/L ammonium acetate, 1 g/L sodium chloride, 0.75 g/L KH2PO4, 0.75 g/L K2HPO4, 0.2 g/L MgSO4, 0.01 g/L MnSO47H2O, and 0.01 g/L FeSO47H2O was prepared and autoclaved at 121 °C for 20 min. After cooling to 35 °C, 0.5 g/L cysteine HClH2O was filtered (0.45 lm Millipore filter, GEMA Medical SL) and added to the medium. About 5 ml of the prepared bacterial culture was then added to the medium and the growth was conducted at 37 °C for 24 h [18].
2. Materials and methods
2.5. ABE fermentation
2.1. Rice straw and enzymes
Fermentation of 50 ml of the enzymatic hydrolysate was performed in 118 ml serum bottles (717561, Pajuhesh Setayesh Sepahan, Isfahan, Iran) sealed with butyl rubber and aluminum crimp cap. After addition of 0.05 g yeast extract to each bottle, pH of the solutions was adjusted to 6.5 using 5 M NaOH. These solutions were sterilized at 121 °C for 20 min followed by cooling to room temperature. Prior to inoculation, 0.5 ml of P2 stock solution was filter sterilized (Millipore filter; 0.22 lm) and added to each bottle. P2 stock solutions contained a buffer (50 g/L KH2PO4, 50 g/L K2HPO4, and 220 g/L CH3COONH4), vitamin (0.1 g/L para-aminobenzoic acid, 0.1 g/L thiamin, and 0.001 g/L biotin), and mineral (20 g/L MgSO47H2O, 1 g/L MnSO4H2O, 1 g/L FeSO47H2O, 1 g/L NaCl) solutions [19]. The bottles were then inoculated with 6 ml of actively growing culture (optical density 1.2–1.6 at 610 nm). These bottles were sparged with pure nitrogen in order to provide
Rice straw used in all the experiments was obtained from Lenjan field (Isfahan province, Iran). It was dried at 60 ± 5 °C for 1 day. The straw with original length between 20 and 50 mm was partially ball-milled and screened to achieve a size of less than 833 lm (20 mesh) and larger than 299 lm (48 mesh) prior to the enzymatic hydrolysis. Two commercial enzymes, cellulase (Celluclast 1.5L, Novozyme, Denmark) and b-glucosidase (Novozyme 188, Novozyme, Denmark), were used for hydrolysis. The cellulase activity was 80 FPU/ml, measured by the method presented by Adney and Baker [14] and it contained 42 mg/ml protein, as measured by Bradford assay [15]. The b-glucosidase activity was 240 IU/ml according to method presented by Ximenes et al. [16].
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the anaerobic conditions. Fermentation was conducted at 37 °C for 72 h. During fermentation, liquid samples for analysis of sugars and ABE contents were periodically withdrawn with a sterile syringe through the rubber stopper. The samples were centrifuged at 9000 rpm for 25 min and stored at 18 °C before the analysis. 2.6. Analytical procedures Moisture and total solid contents of the biomass were measured by drying at 105 °C to a constant weight [20]. Ash content of the biomass was also analyzed following the procedure described by Sluiter et al. [21]. Structural carbohydrates and lignin in biomass were determined according to a standard method presented by Sluiter et al. [22]. The cell concentration was estimated by optical density (OD) and the dry cell weight (DCW) was calculated using a predetermined correlation between OD at 610 nm and DCW. Concentration of fermentation products (ABE, acetic acid, and butyric acid) as well as sugars were analyzed by high-performance liquid chromatography (HPLC) (Jasco International Co., Tokyo, Japan), equipped with UV/VIS and RI detectors (Jasco International Co., Tokyo, Japan). Fermentation products (ABE, acetic acid, and butyric acid) were analyzed on 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. Concentrations of sugars were determined using an Aminex HPX-87P column (Bio-Rad, Richmond, CA, USA) at 80 °C. Deionized water was used as an eluent at a flow rate of 0.6 ml/ min. Concentrations of glucose, acetone, butanol, and ethanol were determined by RI detector, while acetic 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.
Table 3 Glucose yield after enzymatic hydrolysis of pretreated and untreated rice straw. Yield of enzymatic hydrolysis
Alkaline pretreated Phosphoric acid pretreated Untreated rice straw
Theoretical glucose yield (%)a
(g Glucose/kg rice straw)
(g Glucose/kg pretreated rice straw)
60.4
163.5
407.9
69.8
192.3
354.1
31.1
101.8
–
a Theoretical glucose yield (%) = produced glucose (g/L) 100/(1.111 substrate concentration (g/L) biomass glucan fraction).
As can be seen in Table 1, the recovery of the solid was higher for the phosphoric acid pretreatment than that in the alkaline treatment. Considering composition of pretreated materials (Table 2), different parts of rice straw were differently affected by the pretreatments. More than 66% of glucan was recovered after the pretreatments, while only 17% of xylan after the treatments was recovered. On the other hand, alkaline pretreatment reduced 76% of lignin, while phosphoric acid pretreatment reduced only 27% of lignin content. Higher acid insoluble lignin removal by the alkaline pretreatment compared with the phosphoric acid treatment could be the reason for lower solid recovery by the alkaline pretreatment. Considering the recovery of the solid, rice straw lost 63% and 30% of its ash content through the alkaline and phosphoric acid pretreatments, respectively. However, weight percentage of ash in the solid increased after pretreatments due to further reduction of other parts.
3. Results 3.2. Enzymatic hydrolysis
Rice straw was pretreated with alkaline and concentrated phosphoric acid prior to hydrolysis in order to improve the yield of ABE production in the consequent fermentation. The solid recovery and chemical composition of the pretreated materials are summarized in Tables 1 and 2, respectively.
Table 1 Solid recovery and ash content of pretreated and untreated rice straw. Pretreatment
Solid recovery (w/w%)
Ash (wt.%)
Alkaline pretreatment Phosphoric acid pretreatment Untreated rice straw
40.1 54.3 –
14.2 19.9 15.4
Table 2 Chemical composition of pretreated and untreated rice straw. Components
Untreated rice straw
Alkaline pretreated rice straw
Phosphoric acid pretreated rice straw
Glucan (wt.%) Xylan (wt.%) Arabinan (wt.%) Acid insoluble residue (wt.%) Acid insoluble ash (wt.%) Acid insoluble lignin (wt.%) Acid soluble lignin (wt.%)
36.5 13.9 2.7 20.3
60.8 5.8 2.1 16.1
45.7 4.3 0.0 33.5
7.6
7.8
14.2
12.7
8.3
19.3
Pretreated materials were subsequently subjected to enzymatic hydrolysis using cellulase and extra b-glucosidase enzymes with 2% solid loading. The results of hydrolysis are shown in Fig. 1 and Table 3. As can be seen in Fig. 1, both of the pretreatments improved the yield of glucose production. After 72 h of enzymatic hydrolysis, glucose was produced with a yield of 35.4 and 40.8 g per 100 g of alkali and phosphoric acid pretreated straw, respectively, while the yield from untreated rice straw was only 10.2 g per 100 g of untreated straw.
g glucose/ 100 g pretreated rice straw
3.1. Pretreatment
45 40 35 30 25 20 15 10 5 0 0
10
20
30
40
50
60
70
Time (h) 2.4
0.6
0.9 Fig. 1. Production of glucose in the enzymatic hydrolysis of untreated (s), alkaline pretreated (h), and H3PO4 pretreated (D) rice straw.
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F. Moradi et al. / Fuel 112 (2013) 8–13
3
12
B
A 2.5
Products (g/ L)
Glucose (g/L)
10 8 6 4
1.5 1 0.5
2 0
2
0 0
10
20
30
40
50
60
70
0
10
20
30
Time (h)
40
50
60
70
Time (h)
Fig. 2. Glucose (A) and solvents (B) concentrations in fermentation of reference medium. The symbols represents glucose (j), acetone (e), butanol (), ethanol (D), acetic acid (s), butyric acid (h), and total ABE () concentrations.
Enzymatic hydrolysis of phosphoric acid pretreated rice straw resulted in the highest theoretical glucose yield of 69.8%. Considering the pretreatments solid recovery, alkaline and phosphoric acid pretreatments resulted in production of 163.5 g and 192.3 g glucose per kg of the initial straw, while hydrolysis of untreated rice straw resulted in only 101.8 g glucose per kg of the straw.
concentration of 0.69 g/L, 75% of its final concentration. At the same time, acetic and butyric acid concentrations reached 1.55 and 0.42 g/L, respectively, which were slightly decreased during the fermentation. As shown in Fig. 3, production of ABE was significantly affected by the pretreatments. During the first 13 h of fermentation, glucose content of alkaline pretreated hydrolysate was sharply consumed to its half concentration through the growth phase of fermentation, while no detectable ABE was produced. Concentration of glucose remained constant during the next 23 h. The residual glucose was consumed between 36 and 48 h of fermentation and over 60% of total ABE was produced throughout the stationary phase of fermentation. Remaining 40% of total ABE was gradually produced during the next 34 h. Fermentation of alkaline pretreated rice straw resulted in 2.85 g/L ABE which contained 2 g/L butanol, 0.8 g/L acetone, and 0.05 g/L ethanol (Fig. 3a and d). In addition, 1.22 g/L acetic acid and 1.47 g/L butyric acid were produced through the fermentation of alkaline pretreated rice straw. Fermentation of phosphoric acid pretreated straw hydrolysate was also resulted in consumption of sugars and production of
3.3. Fermentation Hydrolysates obtained from the enzymatic hydrolysis of pretreated and untreated rice straw, and P2 medium with 10 g/L glucose as a reference, were anaerobically fermented by C. acetobutylicum. The fermentation results of the reference and hydrolysates are shown in Figs. 2 and 3, respectively. Although the fermentation of the reference was conducted for 72 h, the culture consumed entire glucose content of the medium within 33 h and produced 1.36 g/L butanol. After 33 h, concentration of products were slightly changed except for ethanol which was decreased from 0.23 g/L at 57 h to lower than 0.04 g/L at 70 h. After 12 h fermentation, acetone was produced with
8
A
Consumed sugars (g/l)
7
B
C
E
F
6 5 4 3 2 1 0 3
D Products (g/l)
2.5 2 1.5 1 0.5 0 0
10
20
30
40
Time (h)
50
60
70
80
0
10
20
30
40
50
Time (h)
60
70
80
0
10
20
30
40
50
60
70
80
Time (h)
Fig. 3. Consumption of sugars and production of ABE from hydrolysates of NaOH pretreated (A and D), H3PO4 pretreated (B and E), and untreated (C and F) rice straw. The symbols represents glucose (j), xylose (d), arabionse (N), acetone (e), butanol (), ethanol (D), acetic acid (s), butyric acid (h), and total ABE () concentrations.
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Table 4 Yields of acetone, butanol, and ethanol productions from rice straw.a Butanol
Acetone
Ethanol
Acetic acid
Butyric acid
1.2 0.6 <0.1
27.6 45.6 83.2
33.4 39.2 74.7
(g/kg rice straw) Alkaline pretreatment Phosphoric acid pretreatment Untreated rice straw
45.2 44.2 9.0
17.7 18.2 <0.1
Butanol
Acetone
Ethanol
Acetic acid
Butyric acid
68.9 83.9 –
83.2 72.3 –
(g/kg pretreated rice straw) 112.7 81.4 –
44.2 33.6 –
3.0 1.1 –
The volume changes through addition of P2 solutions and the culture were considered in the final yields. a Yield (g/kg rice straw) = % solid recovery yield (g/kg pretreated rice straw).
ABE. Fermentation started with gradual consumption of glucose and xylose without ABE production. Glucose consumption was intensified after 20 h, and it was entirely consumed at 60 h of fermentation. Similar to fermentation of hydrolysate of the alkaline pretreated straw, 59% of total ABE were produced between 36 and 48 h in fermentation of the acid pretreated hydrolysate. Fermentation of the acid pretreated hydrolysate resulted in 1.4 g/L butanol, 0.6 g/L acetone, less than 0.1 g/L ethanol, 1.48 g/L acetic acid, and 1.28 g/L butyric acid (Fig. 3b and e). Fermentation of hydrolysate of untreated rice straw resulted in 0.16 g/L butanol which showed over 12-fold increase with alkaline pretreatment and 9-fold increase with phosphoric acid pretreatment (Fig. 3c and f). As presented in Table 4, using alkaline pretreatment and phosphoric acid pretreatments, 64.1 and 63.0 g ABE were produced from each kg of rice straw, respectively, while without pretreatment only 9 g ABE were produced from the same amount of rice straw. Butanol comprised 70% of ABE which were produced from both pretreated and untreated rice straws. Butyric acid, which is a product of acidogenesis phase of fermentation, was gradually produced up to 1.5 and 1.3 g/L after 82 h of fermentation of the alkaline and phosphoric acid pretreated hydrolysates, respectively. At the beginning of the fermentation, acetic acid was presented in alkaline and phosphoric pretreated hydrolysates with concentration of 1.3 and 1.2 g/L, respectively. The concentration of acetic acid was changed during fermentation of each hydrolysate, and its concentration in the fermented hydrolysates of the alkaline and phosphoric pretreated rice straw finally reached to 1.2 and 1.5 g/L, respectively.
4. Discussion Butanol, a potential substitute for fossil fuels, is the most desired product of ABE fermentation. In this fermentation process, species of Clostridium acetobutylicum bacteria metabolize different sugars to butanol and other solvents [1]. Even though ABE fermentation is one of the oldest fermentation technologies, its large-scale production remained challenging. High substrate costs and low volumetric productivity were considered as the main bottlenecks that preclude large-scale application of ABE fermentation [4]. Butanol is also a value-added byproduct of bioconversion of glycerol with Clostridum pasteurianum [23]. Available in large quantities, lignocellulosic sources were suggested as a unique feedstocks to revive the biobutanol industries [4,5]. In the current study, C. acetobutylicum was used to produce ABE from hydrolysates of pretreated rice straw. The prerequisite in the utilization of lignocellulose for butanol production is to efficiently produce a hydrolysate rich in fermentable sugars. Enzymatic hydrolysis is considered as a proper strategy with advantages over other chemical conversion routes [24]. However, like other lignocelluloses, native rice straw are recalcitrant to enzymatic hydrolysis, and a pretreatment is required prior to the enzymatic hydrolysis to render the cellulose to enzymatic attack [10]. In view of cellulosic ethanol production, a number of
pretreatment approaches have been investigated on a wide variety of feedstocks [10]. Dilute sulfuric acid pretreatment is among the best pretreatment methods; however, generation of some byproducts inhibited cell growth and ABE fermentation [6–8]. Thus, alkaline and phosphoric acid pretreatments were evaluated for improvement of ABE production in this study. Alkaline pretreatment, one of the most effective pretreatment technologies, is basically a delignification process, in which a significant amount of hemicellulose is solubilized as well [25]. In this study, the alkaline pretreatment and subsequent enzymatic hydrolysis of the treated residues resulted in production of 407.9 g glucose per kg of pretreated rice straw which was corresponded to 163.5 g glucose per kg of initial rice straw. Concentrated phosphoric acid pretreatment could be used to open up the structure of lignocelluloses and decrease the crystallinity of cellulose at a relatively low temperature [13]. Concentrated phosphoric acid could dissolve crystalline cellulose and hemicellulose portion which consequently precipitate to amorphous forms by addition of an anti-solvent such as acetone. Having large difference in volatility, phosphoric acid and acetone could easily recovered avoiding acid dilution with water which facilitates acid re-concentration [13]. In this study, the phosphoric acid pretreatment was used for improvement of butanol production by ABE fermentation, and the treated rice straw was recovered after the pretreatment which contained 68%, 17%, and 83% of the initial glucan, xylan, and lignin, respectively. The alkaline and phosphoric acid pretreatments not only increase the glucan, but also they decrease the xylan, and lignin parts [26]. In comparison with alkaline pretreatment, phosphoric acid pretreatment resulted in recovery of almost the same amounts of glucan and xylan but higher amount of lignin. Highest theoretical glucose yield of 69.8% was obtained through enzymatic hydrolysis of phosphoric acid pretreated rice straw. On the other hand, the alkaline pretreated straw had a higher glucan fraction which resulted in higher glucose concentration in its hydrolysate. However, phosphoric acid pretreatment, as a result of its higher solid recovery, resulted in higher glucose production from the same amounts of straw after enzymatic hydrolysis. Phosphoric acid pretreatment and subsequent enzymatic hydrolysis of the solid residues resulted in production of 354.1 g glucose per kg of the pretreated straw which corresponded to 192.3 g glucose per kg of initial rice straw. Butanol may be produced from hydrolysates of lignocellulosic biomass containing sugars. Hydrolysates of dilute acid pretreated wheat straw [6], barley straw [7], maize stover [8], and switchgrass [8] have been used for ABE fermentation in which a detoxification process such as overliming is necessary prior to the fermentation especially for barley straw and maize stover. In this study, hydrolysates of alkaline and phosphoric acid pretreated rice straw were used for ABE fermentation. Using the hydrolysate of alkaline treated rice straw, 45.2 g butanol, 17.7 g acetone, 1.2 g ethanol, 27.6 g acetic acid, and 33.4 g butyric acid were produced from each kg of initial rice straw. On the other hand, 44.2 g butanol, 18.2 g acetone, 0.6 g ethanol, 45.6 g acetic acid, and 39.2 g butyric acid per kg rice straw were produced through the phosphoric acid pretreatment, enzymatic hydrolysis and ABE fermentation. Comparing the results
F. Moradi et al. / Fuel 112 (2013) 8–13
of ABE production from the treated and untreated rice straws, both alkaline and phosphoric acid pretreatments resulted in a nearly 5fold increase in the butanol yield. Furthermore, no detoxification treatment is required for ABE fermentation of the hydrolysates. 5. Conclusions Rice straw can be a suitable substrate for production of butanol, acetone, and ethanol by fermentation. Alkaline and phosphoric acid pretreatments were two promising processes for improvement of the ABE from rice straw without necessity of detoxifications. More than 44 g butanol and 17 g acetone were produced from each kg of rice straw using both of the pretreatments followed by enzymatic hydrolysis and fermentation. References [1] García V, Päkkilä J, Ojamo H, Muurinen E, Keiski RL. Challenges in biobutanol production: how to improve the efficiency? Renew Sust Energy Rev 2011;15:964–80. [2] Jones DT, Woods DR. Acetone–butanol fermentation revisited. Microbiol Rev 1986;50:484–524. [3] Cascone R. Biobutanol – a replacement for bioethanol? Chem Eng Prog 2008;104:s4–9. [4] Qureshi N. Agricultural residues and energy crops as potentially economical and novel substrates for microbial production of butanol (a biofuel). CAB Rev 2010;5:1–8. [5] Zverlov V, Berezina O, Velikodvorskaya G, Schwarz W. Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery. Appl Microbiol Biotechnol 2006;71:587–97. [6] Qureshi N, Saha BC, Cotta MA. Butanol production from wheat straw hydrolysate using Clostridium beijerinckii. Bioproc Biosyst Eng 2007;30:419–27. [7] Qureshi N, Saha BC, Dien B, Hector RE, Cotta MA. Production of butanol (a biofuel) from agricultural residues: Part I – Use of barley straw hydrolysate. Biomass Bioenergy 2010;34:559–65. [8] Qureshi N, Saha BC, Hector RE, Dien B, Hughes S, Liu S, et al. Production of butanol (a biofuel) from agricultural residues: Part II – Use of corn stover and switchgrass hydrolysates. Biomass Bioenergy 2010;34:566–71. [9] Kim S, Dale BE. Global potential bioethanol production from wasted crops and crop residues. Biomass Bioenergy 2004;26:361–75.
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