Bioresource Technology 203 (2016) 252–258
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Pilot-scale steam explosion for xylose production from oil palm empty fruit bunches and the use of xylose for ethanol production Sairudee Duangwang a,⇑, Taweesak Ruengpeerakul b, Benjamas Cheirsilp c, Ram Yamsaengsung a, Chayanoot Sangwichien a a b c
Department of Chemical Engineering, Faculty of Engineering, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Computer Engineering, Faculty of Engineering, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
h i g h l i g h t s Superheated steam (SHS) explosion can recover xylose from palm empty fruit bunches. Acid pretreatment prior to SHS explosion increased xylose recovery. Alkali pretreatment prior to SHS explosion increased enzymatic accessibility. Xylose recovered in this way can directly be used for conversion to ethanol.
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Article history: Received 26 September 2015 Received in revised form 20 December 2015 Accepted 21 December 2015 Available online 24 December 2015 Keywords: Enzymatic accessibility Ethanol Oil palm empty fruit bunch Pilot-scale steam explosion Xylose
a b s t r a c t Pilot-scale steam explosion equipments were designed and constructed, to experimentally solubilize xylose from oil palm empty fruit bunches (OPEFB) and also to enhance an enzyme accessibility of the residual cellulose pulp. The OPEFB was chemically pretreated prior to steam explosion at saturated steam (SS) and superheated steam (SHS) conditions. The acid pretreated OPEFB gave the highest xylose recovery of 87.58 ± 0.21 g/kg dried OPEFB in the liquid fraction after explosion at SHS condition. These conditions also gave the residual cellulose pulp with high enzymatic accessibility of 73.54 ± 0.41%, which is approximately threefold that of untreated OPEFB. This study has shown that the acid pretreatment prior to SHS explosion is an effective method to enhance both xylose extraction and enzyme accessibility of the exploded OPEFB. Moreover, the xylose solution obtained in this manner could directly be fermented by Candida shehatae TISTR 5843 giving high ethanol yield of 0.30 ± 0.08 g/g xylose. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction It is nowadays common to consider fossil fuels, petroleum, and natural gas as finite and unsustainable resources, so alternative renewable energy sources are actively sought. Not only does gasohol present a cleaner burning alternative fuel, but it can be derived from highly abundant biomass including waste lignocellulose materials such as oil palm empty fruit bunches (OPEFB). The amounts of OPEFB produced in Indonesia, Malaysia and Thailand, when converted to ethanol, could make up almost 2% of the gasoline consumption in these countries. However, while many Southeast Asian countries have relatively large ethanol production from other sources that are generally used as food supply, little ethanol is produced from this OPEFB source (Yano et al., 2009). ⇑ Corresponding author. E-mail address: [email protected] (S. Duangwang). http://dx.doi.org/10.1016/j.biortech.2015.12.065 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Xylose and glucose from lignocellulose has been used extensively to produce a wide variety of fuels or chemical compounds by chemical or biotechnological processes. To maximize total sugars, mainly xylose and glucose, biomass has to be pretreated. As a preliminary step, OPEFB is normally pretreated to remove physical and chemical impediments that might hamper the subsequent steps, which include enzymatic hydrolysis and fermentation. These pretreatments are intended to increase the content of sugars and to avoid degradation (loss of carbohydrates) and the formation of inhibitory products. However, energy intensive and expensive steps are problematic and not practically feasible for the production of ethanol from biomass (Sharma et al., 2015). The steam explosion process exposes the lignocellulosic plant materials to high-pressure steam, followed by a rapid decompression, which forces the fibrous material to ‘‘explode” into separated fibers and fiber bundles. During this process, the high temperature steam causes the release of acids from the acetylated wood compo-
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nents, which catalyzes hydrolytic reactions in the wood polymers. These auto hydrolysis reactions result in the loss of hemicellulose, which is dissolved in hot water and recovered for further conversion into value-added products, such as ethanol. Steam explosion is commonly used because most of hemicellulose can be extracted by hot water from the exploded materials (Martín-Sampedro et al., 2012). Moreover, this method might also enhance the enzymatic accessibility of the exploded materials. The hydrolysis of cellulose in lignocellulosic materials is an important step in a process that produces ethanol. The cellulose can be hydrolyzed either by high acid concentration or by enzymes. Enzymatic hydrolysis is more attractive than acid hydrolysis because it produces less inhibitors (Rocha et al., 2012). However, the high cost of enzymes is problematic for ethanol production. Therefore there is a need to enhance the enzymatic accessibility of these raw materials. It is expected that steam explosion might not only specifically solubilize xylose from the materials, but could also enhance the enzymatic accessibility of the residual solid fraction (cellulose pulp). This study aimed to design and construct pilot-scale steam explosion equipment and to determine suitable conditions that maximize xylose extraction from OPEFB. The enzymatic accessibility of the exploded OPEFB was also evaluated. In addition, the potential use of the xylose, extracted by steam explosion, in ethanol production was assessed and compared with using xylose extracted by acid hydrolysis.
2. Methods 2.1. Materials The OPEFB samples were obtained from Trang Palm Oil Industry Co., Ltd. (Trang, Thailand). They were washed, sun-dried at ambi-
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ent temperature for a day and oven-dried at 105 °C for 4 h until the moisture content was approximately 6–7% dry basis. The dried OPEFB were then shredded into the particle size of 10–20 mm by a knife mill and stored in sealed plastic bags at 30 °C. A culture of Candida shehatae TISTR 5843 was purchased from the Culture Collection of the Thailand Institute of Scientific and Technological Research, Bangkok, Thailand. It was maintained on YPD agar slants containing (g/L): glucose 20.0, yeast extract 10.0, peptone 20.0, agar 15.0, and pH 6. Stock cultures were stored at 4 °C and re-suspended in sterile distilled water.
2.2. Design and construction of pilot scale equipment A schematic diagram of the steam explosion equipment is shown in Fig. 1 and the process design is shown in Fig. 2. The specifications of the pilot scale equipment are: 118 L stainless steel vessel (SS316) with an internal diameter of 450 mm, height of 600 mm, wall thickness of the vessel of 8 mm, and cap thickness of 8 mm. This design is able to handle 5 kg of dried OPEFB per experiment. The vessel is equipped with a pressure gauge, temperature indicator, relief valve, blow-off valve, and a panel for thermocouple insertions. A pipe cluster was constructed to serve as the boiler that can produce temperatures up to 250 °C at a maximum pressure of 1 MPa. A set of gas heaters was used to heat the pipe boiler, and another gas heater above the piping section is set up to heat the reactor during operation in order to maintain the desired temperature and pressure. Relief valves are installed in the pipe section before the steam enters the reactor and at the cap of the reactor. The relief valves are set to release at 1 MPa, as a safety measure. The reactor and piping are insulated with 50.8 mm thick insulating wool. Steam is depressurized abruptly to a collection tank at the end of each treatment to give the explosion effect.
Fig. 1. Sizing of steam explosion vessel.
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Fig. 2. Schematic flow diagram of the steam explosion process (feed unit is not included in the scope of this project).
2.3. Experimental set up for steam explosion Fig. 3 shows the flow chart of xylose production from OPEFB using the pilot scale steam explosion process and its usage for ethanol production. A 100 g sample of dry OPEFB was exploded in the reactor under saturated steam (SS) and superheated steam (SHS) conditions at a temperature of 160–200 °C, pressure of 0.6–1 MPa, and a retention time of 5 min for explosion reactions. The explosive discharge was actuated by suddenly opening a valve to storage tank collection. The severity factor of each treatment was defined by S₀ = log (e((T 100)/14.75)t) following MartínSampedro et al. (2012), where T is the temperature (°C) and t the duration of the treatment (min). To investigate the effects of chemical pretreatment, the OPEFB was soaked in either acid or alkali before steam explosion. In the acid pretreatment, 100 g samples were soaked in 2 L of 0.02 M H2SO4 for 4 h, then held at 121 °C and 103.42 kPa for 20 min. After that, the OPEFB was filtered, oven dried at 105 °C for 4 h, and steam exploded after holding at the selected optimum temperature and pressure for 5 min. In the alkali pretreatment, 100 g of the sample was soaked in 2 L of 0.5 M NaOH (instead of in 0.02 M H2SO4) and was steam exploded. The pretreated OPEFB was collected and the xylan was extracted with 2 L of hot water (80 °C) for 30 min. The extracted xylan was hydrolyzed in an autoclave at 121 °C with 0.1 M H2SO4 for 30 min according to Pumiput et al. (2008). Then,
OPEFB
Steam explosion
the solid fraction was dried at 105 °C temperature for 4 h. The post-hydrolyzed liquid was analyzed for sugar and inhibitors, while the solid fraction of cellulose pulp was evaluated for chemical composition and enzymatic accessibility. 2.4. Enzymatic hydrolysis of OPEFB Enzymatic hydrolysis of the raw and exploded OPEFB was performed according to NREL standard procedure No. 009. The cellulase enzyme used in this study was a commercial product from Trichoderma reesei (Sigma–Aldrich, Co. LLC.) with a calculated filter paper activity of 447.8 FPU/kg of enzyme at pH 4.8 and 50 °C according to the method used by Pan et al. (2005). The cellobiase enzyme was a commercial product from Aspergillus niger (Sigma– Aldrich, Co. LLC.) with a calculated cellubiose assay of 55.9 U/mL for the enzyme solution (1 mL of enzyme/10 mL buffer) at pH 4.8 and at 50 °C according to the method used by Merino and Cherry (2007). The enzymatic hydrolysis experiments were conducted in test tubes with screw caps with a total working volume of 10 mL at a substrate concentration of 1% (w/v). The pre-warmed test tube contained 50 mmol/L sodium citrate buffer (pH = 4.8). Enzymatic accessibility (defined as the glucose produced divided by the potential glucose) was determined by hydrolyzing the cellulose in a pretreated OPEFB sample with an enzyme loading of 20 FPU g/(g of dried OPEFB) for cellulase and 4 U/(g of dried OPEFB)
Liquid fraction of xylan
Xylan hydrolysis
Solid fraction of cellulose pulp
Enzymatic hydrolysis
Xylose
Ethanol fermentation
Ethanol
Hot-water extraction of xylan Enzymatic accessibility
Fig. 3. Flow chart of xylose production from OPEFB in the pilot-scale steam explosion, and of its use in ethanol production.
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for cellobiase and digesting it for 48 h as suggested by Hamzah et al. (2011). The triplicate reaction test tubes were incubated at 50 °C with 160 rpm shaking speed. The enzymatic accessibility of pretreated OPEFB expressed as percentage is calculated as follows (Sun and Cheng, 2005):
Glucose ðgÞ Enzymatic accessibility ¼ 100 Glucan in solid fraction ðgÞ
ð1Þ
2.5. Ethanol production from xylose The xylose obtained after steam explosion was concentrated to the desired level by vacuum evaporation at temperatures below 60 °C and pH was adjusted to 5.5 before use as a medium for ethanol production. The xylose obtained by acid hydrolysis of OPEFB was also used for ethanol production. The acid hydrolysis of OPEFB was done using 0.75 M H2SO4 at 119 °C for 60 min (Rahman et al., 2007). The xylose was autoclaved at 110 °C for 15 min before supplemented with 1.5 g/L yeast extract, 2 g/L KH2PO4, 0.5 g/L NH4Cl, 0.1 g/L CaCl22H2O, 0.1 g/L FeCl32H2O and 0.5 g/L MgSO47H2O. The fermentation runs were carried out in 15 mL test tubes containing 10 mL of fermentation media, at 30 °C on a shaker set at 140 rpm for 72 h. 2.6. Analytical methods Xylose and glucose from the liquid fraction of steam explosion and enzymatic hydrolysis were analyzed by High Performance Liquid Chromatography (HPLC, 1100, Hewlett Packard, Germany) using Luna NH2 column of size 250 4.6 mm and an RI detector. Aqueous acetonitrile (75%) was used as the mobile phase with a flow rate of 0.5 mL/min, and the oven temperature was maintained at 35 °C. Furfural and hydroxyl-methylfurfural (HMF) in the liquid fraction of steam explosion were analyzed using Aminex HP-87H column, operating at 65 °C with 0.005 N H2SO4 as the mobile phase with a flow rate of 0.6 mL/min, together with a UV detector (280 nm wavelength). Acetic acid was analyzed by GC using the column HP-Innowax (30 m 0.32 mm id, 0.5 lm) and FID detector at 275 °C. The solid fractions after steam explosion were analyzed for xylan and glucan with HPLC according to NREL standard procedure No. 002. Scanning electron microscopy (SEM) used SEM model JEOL 5200. Ethanol was estimated by gas chromatography (GC) with an HP-FFAP polyethylene glycol column (30 m 0.25 mm) at 120 °C, and flame ionization detectors (FID) at 250 °C with injector set at 150 °C. The carrier gas was helium with flow rate set at 2 mL/min.
3. Results and discussion 3.1. Steam explosion of OPEFB This study aimed to maximize xylose production from OPEFB by steam explosion. After steam explosion, the xylan was recovered from exploded OPEFB with hot water. The xylan in the liquid fraction was further hydrolyzed to xylose by dilute acid (Fig. 3). The saturated steam (SS) explosion was studied at three different temperatures and pressures, namely 160 °C/0.6 MPa, 170 °C/0.8 MPa and 180 °C/1.0 MPa. The severity factor increases with temperature (Table 1). However, the increase in SS temperature and pressure decreased the xylose recovery in the liquid fraction from 19.71 ± 0.33 to 14.15 ± 0.10 g/kg dried OPEFB. Overend and Chornet (1987) reported that hemicellulose could be extracted with SS explosion, but at a low severity factor. With increasing severity factor, the total mass of hemicellulose-derived material might diminish and the soluble material is progressively converted to furfural and/or incorporated into pseudolignin by condensation reactions. In this study, since increasing pressure to produce SS at higher temperatures did not enhance the solubility of xylan from OPEFB, superheated steam (SHS) was then used to increase the severity factor at the same pressure with SS (0.6 MPa). The SHS explosion at 180 °C/0.6 MPa gave a slightly improved xylose recovery of 20.79 ± 0.11 g/kg dried OPEFB. Table 1 also shows that at the same severity factor of 3.05, xylose was more solubilized by SHS explosion (180 °C/0.6 MPa) than by SS explosion (160 °C/0.6 MPa). Since SHS stores more heat and energy than does SS at the same pressure, the movement of its molecules is more rapid. When the sudden release of pressure occurred, the structure of material after SHS explosion was more disrupted than that after SS explosion. However, the SHS explosion at a higher temperature (200 °C/0.6 MPa) or higher pressure (200 °C/1.0 MPa) yielded very low xylose recoveries. This was likely because the structure of OPEFB had been burnt before the explosion, as suggested by the color of the treated OPEFB. To increase the xylose recovery, alkali and acid pretreatments were performed prior to SS and SHS explosion. It should be noted that the liquid after acid and alkali pretreatments contained certain amounts of xylose, namely 4.28 g/L and 1.13 g/L respectively, but very low amounts of glucose (0.01–0.06 g/L), furfural (0.02–0.4 g/L), and acetic acid (0.5–0.98 g/L), and no 5-hydroxymethylfurfural (HMF). The pretreated OPEFB was than subjected to SS or SHS explosion. Among the cases tested, only the SHS explosion of acid pretreated OPEFB gave a high xylose
Table 1 Sugar recovery in liquid fraction and byproduct concentration after steam explosion. Material code
Severity factor
Xylose (g/kg)
Glucose (g/kg)
Saturated steam (SS) 160 °C/0.6 MPa 170 °C/0.8 MPa 180 °C/1.0 MPa
Acetic acid (g/L)
2.47 2.76 3.05
19.71 ± 0.33b 18.64 ± 0.22c 14.15 ± 0.10d
1.37 ± 0.10fg 1.38 ± 0.01fg 4.17 ± 0.05c
0.07 ± 0.01e 0.09 ± 0.01d 0.13 ± 0.02c
0.02 ± 0.01de 0.02 ± 0.00de 0.03 ± 0.00cd
Superheated steam (SHS) 180 °C/0.6 MPa 200 °C/0.6 MPa 200 °C/1.0 MPa
3.05 3.64 3.64
20.79 ± 0.11b 1.61 ± 0.11h 3.50 ± 0.07g
5.01 ± 0.11b 0.62 ± 0.06h 1.58 ± 0.05f
0.15 ± 0.01b 0.27 ± 0.00a 0.26 ± 0.01a
0.05 ± 0.00cd 0.14 ± 0.02a 0.12 ± 0.02a
Acid pretreatment prior to steam explosion 160 °C/0.6 MPa (SS) 2.47 180 °C/0.6 MPa (SHS) 3.05
18.43 ± 0.15c 87.58 ± 0.21a
2.03 ± 0.02d 10.97 ± 0.18a
0.08 ± 0.00de 0.16 ± 0.00b
0.02 ± 0.00ab 0.07 ± 0.01b
Alkali pretreatment prior to steam explosion 160 °C/0.6 MPa (SS) 2.47 180 °C/0.6 MPa (SHS) 3.05
12.33 ± 0.12e 13.34 ± 0.12f
1.63 ± 0.15f 1.79 ± 0.08e
0.04 ± 0.01f 0.06 ± 0.01f
0.00 ± 0.00f 0.01 ± 0.02e
Note: different superscripts in the same column mean that the values are significantly different at 95% confidence level (p 6 0.05).
Furfural (g/L)
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recovery of 87.58 ± 0.21 g/kg dried OPEFB. It should be noted that using acid pretreatment prior to SHS explosion also reduced the dark coloration of the exploded OPEFB. It has been reported that the byproducts, such as acetic acid and furfural, released during hemicellulose solubilization may strongly inhibit microorganism activity during fermentation (Felipe et al., 1995; Scholl et al., 2015). In this study, the concentrations of acetic acid and furfural in the liquid fraction were very low in the ranges of 0.04–0.27 g/L and 0–0.14 g/L, respectively (Table 1). Therefore, it is a reasonable hypothesis that the liquid fraction from steam explosion in this study could be directly used as fermentable sugar solution.
3.2. Physical properties of OPEFB The color of the exploded OPEFB with SS changed from light brown to dark brown, and subjectively the smell was similar to burnt sugar. The color samples exploded with SHS were changed from light brown to darker brown and the smell was subjectively stronger than those exploded with SS (Fig. 1S). The coloration effects are attributed to photochemical reactions of carbohydrate and lignin at high steam temperature (Bahrin et al., 2012). This also caused the reduction of carbohydrate compounds possibly due to elimination of the waxy layer on the outer surface of the OPEFB, as described by Bahrin et al. (2012). Moreover, the samples treated at temperatures exceeding 200 °C became brittle and were easily broken by milling. This indicates that the use of steam at too high temperatures could destroy the mechanical strength of OPEFB. In plant structures the lignin acts like glue filling the spaces between cellulose and hemicellulose. The raw OPEFB fibers clearly contained impurities, wax, fatty substances and globular protrusions called ‘‘tyloses’’ (Rout et al., 2000). The acid pretreatment changed the morphological structure of the OPEFB fibers: the fiber surfaces became cleaner and roughness was reduced (Fig. 2S). This might be because almost all impurities were removed (Izani et al., 2013). Chemically-treated OPEFB appeared more porous than the exploded OPEFB without chemical pretreatment, consistent with the findings of Asadieraghi and Wan Daud (2014), who described this as evidence of leaching the fibers after steam explosion pretreatment. Not only did some changes occur in the biomass stomata and epidermis due to dissolving of some hemicellulose and probably cellulose, but also there was partial dissolution by acid of mineral constituents and amorphous hemicellulose, which increased the surface area and pore volume. Therefore, the chemically pretreated lignocellulose has an increased pore volume
accessible to enzymatic attack. These results confirmed that hemicellulose was removed during the pretreatment process (Ariffin et al., 2008).
3.3. Chemical composition and enzymatic accessibility of OPEFB Table 2 shows the chemical composition and enzymatic accessibility of untreated and exploded OPEFB. Obviously the xylan content in the solid fraction decreased after steam explosion, as also previously observed for other types of biomass such as elephant grass (Scholl et al., 2015), cane bagasse (Scholl et al., 2015) and Eucalyptus grandis (Scholl et al., 2015). After SS explosion the xylan and glucan in solid fraction of exploded OPEFB slightly changed, while they were clearly altered by SHS explosion. The SHS explosion at 0.6 MPa increased the glucan content of the solid fraction from 36.56 ± 0.18% up to 64.54 ± 0.12%, and decreased the xylan content from 33.34 ± 0.15% to 13.58 ± 0.13%. It can be concluded that during the steam explosion the most severely affected biomass component was hemicellulose (Scholl et al., 2015; Sharma et al., 2015). The solid fraction of cellulose pulp after steam explosion was enzymatic hydrolyzed to evaluate its enzymatic accessibility. The enzymatic accessibility was tested for raw OPEFB and exploded OPEFB. The raw OPEFB had low enzymatic accessibility of 24.61 ± 0.28%. This could be because of high lignin and hemicellulose contents, as well as high crystallinity of the cellulose. The enzymatic accessibility of OPEFB increased when OPEFB was exploded by SS (37.01–46.69%). Scholl et al. (2015) also reported that the steam exploded elephant grass could be easily hydrolyzed and yielded higher sugar recovery than that without treatment. However, the explosion with SHS decreased the enzymatic accessibility of the exploded OPEFB. This could be due to the decomposition of carbohydrates and lignin at high steam temperatures (Bahrin et al., 2012). In addition, pore collapse might occur in the micro-structure of biomass, leading to the decreased enzymatic release of glucose from the cellulose. However, it has been reported that chemical pretreatment of lignocellulose could increase the pore fraction accessible for enzymatic attack (Ariffin et al., 2008). When OPEFB was chemically pretreated by acid or alkaline prior to SHS explosion, the enzymatic accessibility of the exploded OPEFB was increased up to 73.54 ± 0.41% and 91.71 ± 0.39%, respectively. These levels are approximately 3- and 3.8-fold higher than that of untreated OPEFB. It should be noted that the enzymatic accessibilities of the acid and alkaline pretreated OPEFB were 2.89 ± 0.19% and 51.37 ± 0.21%, respectively. Since the OPEFB
Table 2 Chemical compositions and enzymatic accessibility of solid fraction after steam explosion. Material code
Severity factor
Xylan (%)
Glucan (%)
Enzymatic accessibility (%)
Saturated steam (SS) 160 °C/0.6 MPa 170 °C/0.8 MPa 180 °C/1.0 MPa
2.47 2.76 3.05
19.06 ± 0.15a 15.83 ± 0.18b 14.80 ± 0.09c
32.36 ± 0.27h 32.92 ± 0.09h 41.85 ± 0.11f
42.98 ± 0.35e 46.69 ± 0.44d 37.01 ± 0.23f
Superheated steam (SHS) 180 °C/0.6 MPa 200 °C/0.6 MPa 200 °C/1.0 MPa
3.05 3.64 3.64
13.58 ± 0.13d 12.16 ± 0.20e 9.76 ± 0.11g
64.54 ± 0.12a 42.69 ± 0.11e 64.05 ± 0.17a
12.92 ± 0.47g 4.83 ± 0.37h 3.07 ± 0.33h
Acid pretreatment prior to steam explosion 160 °C/0.6 MPa (SS) 2.47 180 °C/0.6 MPa (SHS) 3.05
10.88 ± 0.20f 2.04 ± 0.08h
34.80 ± 0.13g 43.08 ± 0.17d
16.18 ± 0.39e 73.54 ± 0.41c
Alkali pretreatment prior to steam explosion 160 °C/0.6 MPa (SS) 2.47 180 °C/0.6 MPa (SHS) 3.05
12.22 ± 0.17e 10.85 ± 0.13f
50.50 ± 0.33b 48.75 ± 0.12c
99.52 ± 0.32a 91.71 ± 0.39b
Note: different superscripts in the same column mean that the values are significantly different at 95% confidence level (p 6 0.05).
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Ethanol (g/L)
(a)
25
Xylose (g/L)
20
Concentration (g/L)
Concentration (g/L)
25
15 10 5
Ethanol (g/L)
(b)
Xylose (g/L)
20 15 10 5 0
0 0
20
40 Time (h)
60
80
0
20
40 Time (h)
60
80
Fig. 4. Ethanol productions from xylose obtained by acid hydrolysis (a), and from xylose in the liquid fraction after steam explosion (b).
samples were treated in various ways, the extent of irreversible pore collapse for each sample might also vary during the subsequent drying of treated OPEFB, prior to enzymatic hydrolysis. Therefore, the extent of enzymatic accessibility was also affected by drying step. 3.4. Fermentation of xylose for ethanol production The objective of this section was to evaluate the potential use of xylose extracted by steam explosion as fermentable sugar for ethanol production. The liquid fraction from SHS explosion of acid pretreated OPEFB, with high xylose concentration, was used in this section. The fermentation of xylose by the specific yeast C. shehatae for ethanol production was tested (Fig. 4). For comparison, the xylose obtained by direct acid hydrolysis of OPEFB was also used for ethanol production (Fig. 4a). This direct acid hydrolysate contained 19.22 g/L xylose and 2.34 g/L glucose and also furfural and acetic acid at high concentrations of 0.82 g/L and 2.51 g/L, respectively. Fig. 4a shows that the yeast could not ferment xylose in this direct acid hydrolysate. This could be due to the high concentrations of inhibitors present in the hydrolysate. Chandel et al. (2007) have reported that the acid hydrolysate needed to be detoxified and each detoxification method gave different ethanol yields i.e., overliming (0.302 g ethanol/g xylose), laccase (0.374 g ethanol/ g xylose), activated charcoal (0.425 g ethanol/g xylose) and ion exchange (0.482 g ethanol/g xylose). While the liquid fraction from steam explosion in this study contained initial xylose, glucose, furfural and acetic acid at 13.87 g/L, 0.23 g/L, 0.03 g/L and 1.19 g/L, respectively. The low furfural and acetic acid concentrations in this liquid fraction suggest the high potential use for fermentation. It was obvious that the yeast could assimilate xylose in the liquid fraction from steam explosion and produced ethanol at a high yield of 0.30 g ethanol/g xylose (Fig. 4b). 4. Conclusions This study has shown that the acid pretreatment prior to superheated steam explosion was the most suitable among the tested conditions for xylose production from OPEFB. The exploded OPEFB by this manner also had high accessibility for enzymatic hydrolysis due to its high porosity after steam explosion. However, the alkali pretreatment prior to superheated steam explosion was more suitable for obtaining the exploded OPEFB with the highest enzymatic accessibility. The xylose recovered in the liquid fraction from superheated steam explosion could directly be used as fermentable sugar for ethanol production, as this was facilitated by its low inhibitor concentrations.
Acknowledgements This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, and PTT Global Chemical Public Company Limited. The first author was granted additional support from the Prince of Songkla University (PSU) Graduate School Research Support Funding, and also received a scholarship from PSU. The use of facilities and equipment at the PSU Department of Chemical Engineering, Faculty of Engineering, is gratefully acknowledged. Also thanks to the PSU research and development office (RDO) and Assoc. Prof. Seppo Karrila, Ph.D. (Chem Eng). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.12. 065. References Ariffin, H., Hassan, M.A., Umi Kalsom, M.S., Abdullah, N., Shirai, Y., 2008. Effect of physical, chemical and thermal pretreatments on the enzymatic hydrolysis of oil palm empty fruit bunch (OPEFB). J. Trop. Agric. Fd. Sc. 36, 259–268. Asadieraghi, M., Wan Daud, W.M.A., 2014. Characterization of lignocellulosic biomass thermal degradation and physiochemical structure: effects of demineralization by diverse acid solutions. Energy Convers. Manage. 82, 71–82. Bahrin, E.K., Baharuddin, A.S., Ibrahim, M.F., Razak, M.N.A., Sulaiman, A., Aziz, S.A., Nishida, H., 2012. Physicochemical property changes and enzymatic hydrolysis enhancement of oil palm empty fruit bunches treated with superheated steam. BioResources 7, 1784–1801. Chandel, A.K., Kapoor, R.K., Singh, A., Kuhad, R.C., 2007. Detoxification of sugarcane bagasse hydrolysate improves ethanol production by Candida shehatae NCIM 3501. Bioresour. Technol. 98, 1947–1950. Felipe, M.G.A., Veira, M.V., Vitolo, M., Mancilha, I.M., Roberto, I.C., Silva, S.S., 1995. Effect of acetic acid on xylose fermentation to xylitol by Candida guilliermondii. J. Basic Microbiol. 35, 171–177. Hamzah, F., Idris, A., Shuan, T.K., 2011. Preliminary study on enzymatic hydrolysis of treated oil palm (Elaeis) empty fruit bunches fibre (EFB) by using combination of cellulase and b 1–4 glucosidase. Biomass Bioenergy 35, 1055–1059. Izani, N., Paridah, M.T., Anwar, U.M.K., MohdNor, M.Y., H’ng, P.S., 2013. Effects of fiber treatment on morphology, tensile and thermo gravimetric analysis of oil palm empty fruit bunches fibers. Compos. Part B 45, 1251–1257. Martín-Sampedro, R., Eugenio, M.E., García, J.C., Lopez, F., Villar, J.C., Diaz, M.J., 2012. Steam explosion and enzymatic pre-treatments as an approach to improve the enzymatic hydrolysis of Eucalyptus globulus. Biomass Bioenergy 42, 97–106. Merino, S.T., Cherry, J., 2007. Progress and challenges in enzyme development for biomass utilization. Adv. Biochem. Eng. Biotechnol. 108, 95–120. Overend, R.P., Chornet, E., 1987. Fractionation of lignocellulosics by steam-aqueous pretreatment. Philos. Trans. R. Soc. Lond. 321, 523–536. Pan, X., Arato, C., Gilkes, N., Gregg, D., Mabee, W., Pye, K., Saddler, J., 2005. Biorefining of softwoods using ethanol organosolv pulping: preliminary evaluation of process streams for manufacture of fuel-grade ethanol and coproducts. Biotechnol. Bioeng. 90, 473–481.
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Pumiput, S., Chuantranuluck, S., Kitpreechavanich, V., Punsuvon, V., Vaithanomsat, P., 2008. Production process of hydrolysate from steam explosion of oil palm trunk for xylitol fermentation. Kasetsart J. Nat. Sci. 42, 73–78. Rahman, S.H.A., Choudhury, J.P., Ahmad, A.L., Kamaruddin, A.H., 2007. Optimization studies on acid hydrolysis of oil palm empty fruit bunch fiber for production of xylose. Bioresour. Technol. 98, 554–559. Rocha, G.J.M., Gonçalves, A.R., Oliveira, B.R., Olivares, E.G., Rossell, C.E.V., 2012. Steam explosion pretreatment reproduction and alkaline delignification reactions performed on a pilot scale with sugarcane bagasse for bioethanol production. Ind. Crop. Prod. 35, 274–279. Rout, J., Tripathy, S.S., Nayak, S.K., Misra, M., Mohanty, A.K., 2000. Scanning electron microscopystudy of chemically modified coir fibers. J. Appl. Polym. Sci. 79, 1169–1177.
Scholl, A.L., Menegol, D., Pitarelo, A.P., Fontana, R.C., Filho, A.Z., Ramos, L.P., Dillon, A. J.P., Camassola, M., 2015. Elephant grass (Pennisetum purpureum Schum.) pretreated via steam explosion as a carbon source for cellulases and xylanases in submerged cultivation. Ind. Crop. Prod. 70, 280–291. Sharma, S., Kumar, R., Gaur, R., Agrawal, R., Gupta, R.P., Tuli, D.K., Das, B., 2015. Pilot scale study on steam explosion and mass balance for higher sugar recovery from rice straw. Bioresour. Technol. 175, 350–357. Sun, Y., Cheng, J.J., 2005. Dilute acid pretreatment of rye straw and bermudagrass for ethanol production. Bioresour. Technol. 96, 1599–1606. Yano, S., Murakami, K., Sawayama, S., Imou, K., Yokoyama, S., 2009. Ethanol production potential from oil palm empty fruit bunches in Southeast Asian countries considering xylose utilization. J. Jpn. Inst. Energy 88, 923–926.
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Pilot-scale steam explosion for xylose production from oil palm empty fruit bunches and the use of xylose for ethanol production Sairudee Duangwang a,⇑, Taweesak Ruengpeerakul b, Benjamas Cheirsilp c, Ram Yamsaengsung a, Chayanoot Sangwichien a a b c
Department of Chemical Engineering, Faculty of Engineering, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Computer Engineering, Faculty of Engineering, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
h i g h l i g h t s Superheated steam (SHS) explosion can recover xylose from palm empty fruit bunches. Acid pretreatment prior to SHS explosion increased xylose recovery. Alkali pretreatment prior to SHS explosion increased enzymatic accessibility. Xylose recovered in this way can directly be used for conversion to ethanol.
a r t i c l e
i n f o
Article history: Received 26 September 2015 Received in revised form 20 December 2015 Accepted 21 December 2015 Available online 24 December 2015 Keywords: Enzymatic accessibility Ethanol Oil palm empty fruit bunch Pilot-scale steam explosion Xylose
a b s t r a c t Pilot-scale steam explosion equipments were designed and constructed, to experimentally solubilize xylose from oil palm empty fruit bunches (OPEFB) and also to enhance an enzyme accessibility of the residual cellulose pulp. The OPEFB was chemically pretreated prior to steam explosion at saturated steam (SS) and superheated steam (SHS) conditions. The acid pretreated OPEFB gave the highest xylose recovery of 87.58 ± 0.21 g/kg dried OPEFB in the liquid fraction after explosion at SHS condition. These conditions also gave the residual cellulose pulp with high enzymatic accessibility of 73.54 ± 0.41%, which is approximately threefold that of untreated OPEFB. This study has shown that the acid pretreatment prior to SHS explosion is an effective method to enhance both xylose extraction and enzyme accessibility of the exploded OPEFB. Moreover, the xylose solution obtained in this manner could directly be fermented by Candida shehatae TISTR 5843 giving high ethanol yield of 0.30 ± 0.08 g/g xylose. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction It is nowadays common to consider fossil fuels, petroleum, and natural gas as finite and unsustainable resources, so alternative renewable energy sources are actively sought. Not only does gasohol present a cleaner burning alternative fuel, but it can be derived from highly abundant biomass including waste lignocellulose materials such as oil palm empty fruit bunches (OPEFB). The amounts of OPEFB produced in Indonesia, Malaysia and Thailand, when converted to ethanol, could make up almost 2% of the gasoline consumption in these countries. However, while many Southeast Asian countries have relatively large ethanol production from other sources that are generally used as food supply, little ethanol is produced from this OPEFB source (Yano et al., 2009). ⇑ Corresponding author. E-mail address: [email protected] (S. Duangwang). http://dx.doi.org/10.1016/j.biortech.2015.12.065 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Xylose and glucose from lignocellulose has been used extensively to produce a wide variety of fuels or chemical compounds by chemical or biotechnological processes. To maximize total sugars, mainly xylose and glucose, biomass has to be pretreated. As a preliminary step, OPEFB is normally pretreated to remove physical and chemical impediments that might hamper the subsequent steps, which include enzymatic hydrolysis and fermentation. These pretreatments are intended to increase the content of sugars and to avoid degradation (loss of carbohydrates) and the formation of inhibitory products. However, energy intensive and expensive steps are problematic and not practically feasible for the production of ethanol from biomass (Sharma et al., 2015). The steam explosion process exposes the lignocellulosic plant materials to high-pressure steam, followed by a rapid decompression, which forces the fibrous material to ‘‘explode” into separated fibers and fiber bundles. During this process, the high temperature steam causes the release of acids from the acetylated wood compo-
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nents, which catalyzes hydrolytic reactions in the wood polymers. These auto hydrolysis reactions result in the loss of hemicellulose, which is dissolved in hot water and recovered for further conversion into value-added products, such as ethanol. Steam explosion is commonly used because most of hemicellulose can be extracted by hot water from the exploded materials (Martín-Sampedro et al., 2012). Moreover, this method might also enhance the enzymatic accessibility of the exploded materials. The hydrolysis of cellulose in lignocellulosic materials is an important step in a process that produces ethanol. The cellulose can be hydrolyzed either by high acid concentration or by enzymes. Enzymatic hydrolysis is more attractive than acid hydrolysis because it produces less inhibitors (Rocha et al., 2012). However, the high cost of enzymes is problematic for ethanol production. Therefore there is a need to enhance the enzymatic accessibility of these raw materials. It is expected that steam explosion might not only specifically solubilize xylose from the materials, but could also enhance the enzymatic accessibility of the residual solid fraction (cellulose pulp). This study aimed to design and construct pilot-scale steam explosion equipment and to determine suitable conditions that maximize xylose extraction from OPEFB. The enzymatic accessibility of the exploded OPEFB was also evaluated. In addition, the potential use of the xylose, extracted by steam explosion, in ethanol production was assessed and compared with using xylose extracted by acid hydrolysis.
2. Methods 2.1. Materials The OPEFB samples were obtained from Trang Palm Oil Industry Co., Ltd. (Trang, Thailand). They were washed, sun-dried at ambi-
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ent temperature for a day and oven-dried at 105 °C for 4 h until the moisture content was approximately 6–7% dry basis. The dried OPEFB were then shredded into the particle size of 10–20 mm by a knife mill and stored in sealed plastic bags at 30 °C. A culture of Candida shehatae TISTR 5843 was purchased from the Culture Collection of the Thailand Institute of Scientific and Technological Research, Bangkok, Thailand. It was maintained on YPD agar slants containing (g/L): glucose 20.0, yeast extract 10.0, peptone 20.0, agar 15.0, and pH 6. Stock cultures were stored at 4 °C and re-suspended in sterile distilled water.
2.2. Design and construction of pilot scale equipment A schematic diagram of the steam explosion equipment is shown in Fig. 1 and the process design is shown in Fig. 2. The specifications of the pilot scale equipment are: 118 L stainless steel vessel (SS316) with an internal diameter of 450 mm, height of 600 mm, wall thickness of the vessel of 8 mm, and cap thickness of 8 mm. This design is able to handle 5 kg of dried OPEFB per experiment. The vessel is equipped with a pressure gauge, temperature indicator, relief valve, blow-off valve, and a panel for thermocouple insertions. A pipe cluster was constructed to serve as the boiler that can produce temperatures up to 250 °C at a maximum pressure of 1 MPa. A set of gas heaters was used to heat the pipe boiler, and another gas heater above the piping section is set up to heat the reactor during operation in order to maintain the desired temperature and pressure. Relief valves are installed in the pipe section before the steam enters the reactor and at the cap of the reactor. The relief valves are set to release at 1 MPa, as a safety measure. The reactor and piping are insulated with 50.8 mm thick insulating wool. Steam is depressurized abruptly to a collection tank at the end of each treatment to give the explosion effect.
Fig. 1. Sizing of steam explosion vessel.
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Fig. 2. Schematic flow diagram of the steam explosion process (feed unit is not included in the scope of this project).
2.3. Experimental set up for steam explosion Fig. 3 shows the flow chart of xylose production from OPEFB using the pilot scale steam explosion process and its usage for ethanol production. A 100 g sample of dry OPEFB was exploded in the reactor under saturated steam (SS) and superheated steam (SHS) conditions at a temperature of 160–200 °C, pressure of 0.6–1 MPa, and a retention time of 5 min for explosion reactions. The explosive discharge was actuated by suddenly opening a valve to storage tank collection. The severity factor of each treatment was defined by S₀ = log (e((T 100)/14.75)t) following MartínSampedro et al. (2012), where T is the temperature (°C) and t the duration of the treatment (min). To investigate the effects of chemical pretreatment, the OPEFB was soaked in either acid or alkali before steam explosion. In the acid pretreatment, 100 g samples were soaked in 2 L of 0.02 M H2SO4 for 4 h, then held at 121 °C and 103.42 kPa for 20 min. After that, the OPEFB was filtered, oven dried at 105 °C for 4 h, and steam exploded after holding at the selected optimum temperature and pressure for 5 min. In the alkali pretreatment, 100 g of the sample was soaked in 2 L of 0.5 M NaOH (instead of in 0.02 M H2SO4) and was steam exploded. The pretreated OPEFB was collected and the xylan was extracted with 2 L of hot water (80 °C) for 30 min. The extracted xylan was hydrolyzed in an autoclave at 121 °C with 0.1 M H2SO4 for 30 min according to Pumiput et al. (2008). Then,
OPEFB
Steam explosion
the solid fraction was dried at 105 °C temperature for 4 h. The post-hydrolyzed liquid was analyzed for sugar and inhibitors, while the solid fraction of cellulose pulp was evaluated for chemical composition and enzymatic accessibility. 2.4. Enzymatic hydrolysis of OPEFB Enzymatic hydrolysis of the raw and exploded OPEFB was performed according to NREL standard procedure No. 009. The cellulase enzyme used in this study was a commercial product from Trichoderma reesei (Sigma–Aldrich, Co. LLC.) with a calculated filter paper activity of 447.8 FPU/kg of enzyme at pH 4.8 and 50 °C according to the method used by Pan et al. (2005). The cellobiase enzyme was a commercial product from Aspergillus niger (Sigma– Aldrich, Co. LLC.) with a calculated cellubiose assay of 55.9 U/mL for the enzyme solution (1 mL of enzyme/10 mL buffer) at pH 4.8 and at 50 °C according to the method used by Merino and Cherry (2007). The enzymatic hydrolysis experiments were conducted in test tubes with screw caps with a total working volume of 10 mL at a substrate concentration of 1% (w/v). The pre-warmed test tube contained 50 mmol/L sodium citrate buffer (pH = 4.8). Enzymatic accessibility (defined as the glucose produced divided by the potential glucose) was determined by hydrolyzing the cellulose in a pretreated OPEFB sample with an enzyme loading of 20 FPU g/(g of dried OPEFB) for cellulase and 4 U/(g of dried OPEFB)
Liquid fraction of xylan
Xylan hydrolysis
Solid fraction of cellulose pulp
Enzymatic hydrolysis
Xylose
Ethanol fermentation
Ethanol
Hot-water extraction of xylan Enzymatic accessibility
Fig. 3. Flow chart of xylose production from OPEFB in the pilot-scale steam explosion, and of its use in ethanol production.
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for cellobiase and digesting it for 48 h as suggested by Hamzah et al. (2011). The triplicate reaction test tubes were incubated at 50 °C with 160 rpm shaking speed. The enzymatic accessibility of pretreated OPEFB expressed as percentage is calculated as follows (Sun and Cheng, 2005):
Glucose ðgÞ Enzymatic accessibility ¼ 100 Glucan in solid fraction ðgÞ
ð1Þ
2.5. Ethanol production from xylose The xylose obtained after steam explosion was concentrated to the desired level by vacuum evaporation at temperatures below 60 °C and pH was adjusted to 5.5 before use as a medium for ethanol production. The xylose obtained by acid hydrolysis of OPEFB was also used for ethanol production. The acid hydrolysis of OPEFB was done using 0.75 M H2SO4 at 119 °C for 60 min (Rahman et al., 2007). The xylose was autoclaved at 110 °C for 15 min before supplemented with 1.5 g/L yeast extract, 2 g/L KH2PO4, 0.5 g/L NH4Cl, 0.1 g/L CaCl22H2O, 0.1 g/L FeCl32H2O and 0.5 g/L MgSO47H2O. The fermentation runs were carried out in 15 mL test tubes containing 10 mL of fermentation media, at 30 °C on a shaker set at 140 rpm for 72 h. 2.6. Analytical methods Xylose and glucose from the liquid fraction of steam explosion and enzymatic hydrolysis were analyzed by High Performance Liquid Chromatography (HPLC, 1100, Hewlett Packard, Germany) using Luna NH2 column of size 250 4.6 mm and an RI detector. Aqueous acetonitrile (75%) was used as the mobile phase with a flow rate of 0.5 mL/min, and the oven temperature was maintained at 35 °C. Furfural and hydroxyl-methylfurfural (HMF) in the liquid fraction of steam explosion were analyzed using Aminex HP-87H column, operating at 65 °C with 0.005 N H2SO4 as the mobile phase with a flow rate of 0.6 mL/min, together with a UV detector (280 nm wavelength). Acetic acid was analyzed by GC using the column HP-Innowax (30 m 0.32 mm id, 0.5 lm) and FID detector at 275 °C. The solid fractions after steam explosion were analyzed for xylan and glucan with HPLC according to NREL standard procedure No. 002. Scanning electron microscopy (SEM) used SEM model JEOL 5200. Ethanol was estimated by gas chromatography (GC) with an HP-FFAP polyethylene glycol column (30 m 0.25 mm) at 120 °C, and flame ionization detectors (FID) at 250 °C with injector set at 150 °C. The carrier gas was helium with flow rate set at 2 mL/min.
3. Results and discussion 3.1. Steam explosion of OPEFB This study aimed to maximize xylose production from OPEFB by steam explosion. After steam explosion, the xylan was recovered from exploded OPEFB with hot water. The xylan in the liquid fraction was further hydrolyzed to xylose by dilute acid (Fig. 3). The saturated steam (SS) explosion was studied at three different temperatures and pressures, namely 160 °C/0.6 MPa, 170 °C/0.8 MPa and 180 °C/1.0 MPa. The severity factor increases with temperature (Table 1). However, the increase in SS temperature and pressure decreased the xylose recovery in the liquid fraction from 19.71 ± 0.33 to 14.15 ± 0.10 g/kg dried OPEFB. Overend and Chornet (1987) reported that hemicellulose could be extracted with SS explosion, but at a low severity factor. With increasing severity factor, the total mass of hemicellulose-derived material might diminish and the soluble material is progressively converted to furfural and/or incorporated into pseudolignin by condensation reactions. In this study, since increasing pressure to produce SS at higher temperatures did not enhance the solubility of xylan from OPEFB, superheated steam (SHS) was then used to increase the severity factor at the same pressure with SS (0.6 MPa). The SHS explosion at 180 °C/0.6 MPa gave a slightly improved xylose recovery of 20.79 ± 0.11 g/kg dried OPEFB. Table 1 also shows that at the same severity factor of 3.05, xylose was more solubilized by SHS explosion (180 °C/0.6 MPa) than by SS explosion (160 °C/0.6 MPa). Since SHS stores more heat and energy than does SS at the same pressure, the movement of its molecules is more rapid. When the sudden release of pressure occurred, the structure of material after SHS explosion was more disrupted than that after SS explosion. However, the SHS explosion at a higher temperature (200 °C/0.6 MPa) or higher pressure (200 °C/1.0 MPa) yielded very low xylose recoveries. This was likely because the structure of OPEFB had been burnt before the explosion, as suggested by the color of the treated OPEFB. To increase the xylose recovery, alkali and acid pretreatments were performed prior to SS and SHS explosion. It should be noted that the liquid after acid and alkali pretreatments contained certain amounts of xylose, namely 4.28 g/L and 1.13 g/L respectively, but very low amounts of glucose (0.01–0.06 g/L), furfural (0.02–0.4 g/L), and acetic acid (0.5–0.98 g/L), and no 5-hydroxymethylfurfural (HMF). The pretreated OPEFB was than subjected to SS or SHS explosion. Among the cases tested, only the SHS explosion of acid pretreated OPEFB gave a high xylose
Table 1 Sugar recovery in liquid fraction and byproduct concentration after steam explosion. Material code
Severity factor
Xylose (g/kg)
Glucose (g/kg)
Saturated steam (SS) 160 °C/0.6 MPa 170 °C/0.8 MPa 180 °C/1.0 MPa
Acetic acid (g/L)
2.47 2.76 3.05
19.71 ± 0.33b 18.64 ± 0.22c 14.15 ± 0.10d
1.37 ± 0.10fg 1.38 ± 0.01fg 4.17 ± 0.05c
0.07 ± 0.01e 0.09 ± 0.01d 0.13 ± 0.02c
0.02 ± 0.01de 0.02 ± 0.00de 0.03 ± 0.00cd
Superheated steam (SHS) 180 °C/0.6 MPa 200 °C/0.6 MPa 200 °C/1.0 MPa
3.05 3.64 3.64
20.79 ± 0.11b 1.61 ± 0.11h 3.50 ± 0.07g
5.01 ± 0.11b 0.62 ± 0.06h 1.58 ± 0.05f
0.15 ± 0.01b 0.27 ± 0.00a 0.26 ± 0.01a
0.05 ± 0.00cd 0.14 ± 0.02a 0.12 ± 0.02a
Acid pretreatment prior to steam explosion 160 °C/0.6 MPa (SS) 2.47 180 °C/0.6 MPa (SHS) 3.05
18.43 ± 0.15c 87.58 ± 0.21a
2.03 ± 0.02d 10.97 ± 0.18a
0.08 ± 0.00de 0.16 ± 0.00b
0.02 ± 0.00ab 0.07 ± 0.01b
Alkali pretreatment prior to steam explosion 160 °C/0.6 MPa (SS) 2.47 180 °C/0.6 MPa (SHS) 3.05
12.33 ± 0.12e 13.34 ± 0.12f
1.63 ± 0.15f 1.79 ± 0.08e
0.04 ± 0.01f 0.06 ± 0.01f
0.00 ± 0.00f 0.01 ± 0.02e
Note: different superscripts in the same column mean that the values are significantly different at 95% confidence level (p 6 0.05).
Furfural (g/L)
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recovery of 87.58 ± 0.21 g/kg dried OPEFB. It should be noted that using acid pretreatment prior to SHS explosion also reduced the dark coloration of the exploded OPEFB. It has been reported that the byproducts, such as acetic acid and furfural, released during hemicellulose solubilization may strongly inhibit microorganism activity during fermentation (Felipe et al., 1995; Scholl et al., 2015). In this study, the concentrations of acetic acid and furfural in the liquid fraction were very low in the ranges of 0.04–0.27 g/L and 0–0.14 g/L, respectively (Table 1). Therefore, it is a reasonable hypothesis that the liquid fraction from steam explosion in this study could be directly used as fermentable sugar solution.
3.2. Physical properties of OPEFB The color of the exploded OPEFB with SS changed from light brown to dark brown, and subjectively the smell was similar to burnt sugar. The color samples exploded with SHS were changed from light brown to darker brown and the smell was subjectively stronger than those exploded with SS (Fig. 1S). The coloration effects are attributed to photochemical reactions of carbohydrate and lignin at high steam temperature (Bahrin et al., 2012). This also caused the reduction of carbohydrate compounds possibly due to elimination of the waxy layer on the outer surface of the OPEFB, as described by Bahrin et al. (2012). Moreover, the samples treated at temperatures exceeding 200 °C became brittle and were easily broken by milling. This indicates that the use of steam at too high temperatures could destroy the mechanical strength of OPEFB. In plant structures the lignin acts like glue filling the spaces between cellulose and hemicellulose. The raw OPEFB fibers clearly contained impurities, wax, fatty substances and globular protrusions called ‘‘tyloses’’ (Rout et al., 2000). The acid pretreatment changed the morphological structure of the OPEFB fibers: the fiber surfaces became cleaner and roughness was reduced (Fig. 2S). This might be because almost all impurities were removed (Izani et al., 2013). Chemically-treated OPEFB appeared more porous than the exploded OPEFB without chemical pretreatment, consistent with the findings of Asadieraghi and Wan Daud (2014), who described this as evidence of leaching the fibers after steam explosion pretreatment. Not only did some changes occur in the biomass stomata and epidermis due to dissolving of some hemicellulose and probably cellulose, but also there was partial dissolution by acid of mineral constituents and amorphous hemicellulose, which increased the surface area and pore volume. Therefore, the chemically pretreated lignocellulose has an increased pore volume
accessible to enzymatic attack. These results confirmed that hemicellulose was removed during the pretreatment process (Ariffin et al., 2008).
3.3. Chemical composition and enzymatic accessibility of OPEFB Table 2 shows the chemical composition and enzymatic accessibility of untreated and exploded OPEFB. Obviously the xylan content in the solid fraction decreased after steam explosion, as also previously observed for other types of biomass such as elephant grass (Scholl et al., 2015), cane bagasse (Scholl et al., 2015) and Eucalyptus grandis (Scholl et al., 2015). After SS explosion the xylan and glucan in solid fraction of exploded OPEFB slightly changed, while they were clearly altered by SHS explosion. The SHS explosion at 0.6 MPa increased the glucan content of the solid fraction from 36.56 ± 0.18% up to 64.54 ± 0.12%, and decreased the xylan content from 33.34 ± 0.15% to 13.58 ± 0.13%. It can be concluded that during the steam explosion the most severely affected biomass component was hemicellulose (Scholl et al., 2015; Sharma et al., 2015). The solid fraction of cellulose pulp after steam explosion was enzymatic hydrolyzed to evaluate its enzymatic accessibility. The enzymatic accessibility was tested for raw OPEFB and exploded OPEFB. The raw OPEFB had low enzymatic accessibility of 24.61 ± 0.28%. This could be because of high lignin and hemicellulose contents, as well as high crystallinity of the cellulose. The enzymatic accessibility of OPEFB increased when OPEFB was exploded by SS (37.01–46.69%). Scholl et al. (2015) also reported that the steam exploded elephant grass could be easily hydrolyzed and yielded higher sugar recovery than that without treatment. However, the explosion with SHS decreased the enzymatic accessibility of the exploded OPEFB. This could be due to the decomposition of carbohydrates and lignin at high steam temperatures (Bahrin et al., 2012). In addition, pore collapse might occur in the micro-structure of biomass, leading to the decreased enzymatic release of glucose from the cellulose. However, it has been reported that chemical pretreatment of lignocellulose could increase the pore fraction accessible for enzymatic attack (Ariffin et al., 2008). When OPEFB was chemically pretreated by acid or alkaline prior to SHS explosion, the enzymatic accessibility of the exploded OPEFB was increased up to 73.54 ± 0.41% and 91.71 ± 0.39%, respectively. These levels are approximately 3- and 3.8-fold higher than that of untreated OPEFB. It should be noted that the enzymatic accessibilities of the acid and alkaline pretreated OPEFB were 2.89 ± 0.19% and 51.37 ± 0.21%, respectively. Since the OPEFB
Table 2 Chemical compositions and enzymatic accessibility of solid fraction after steam explosion. Material code
Severity factor
Xylan (%)
Glucan (%)
Enzymatic accessibility (%)
Saturated steam (SS) 160 °C/0.6 MPa 170 °C/0.8 MPa 180 °C/1.0 MPa
2.47 2.76 3.05
19.06 ± 0.15a 15.83 ± 0.18b 14.80 ± 0.09c
32.36 ± 0.27h 32.92 ± 0.09h 41.85 ± 0.11f
42.98 ± 0.35e 46.69 ± 0.44d 37.01 ± 0.23f
Superheated steam (SHS) 180 °C/0.6 MPa 200 °C/0.6 MPa 200 °C/1.0 MPa
3.05 3.64 3.64
13.58 ± 0.13d 12.16 ± 0.20e 9.76 ± 0.11g
64.54 ± 0.12a 42.69 ± 0.11e 64.05 ± 0.17a
12.92 ± 0.47g 4.83 ± 0.37h 3.07 ± 0.33h
Acid pretreatment prior to steam explosion 160 °C/0.6 MPa (SS) 2.47 180 °C/0.6 MPa (SHS) 3.05
10.88 ± 0.20f 2.04 ± 0.08h
34.80 ± 0.13g 43.08 ± 0.17d
16.18 ± 0.39e 73.54 ± 0.41c
Alkali pretreatment prior to steam explosion 160 °C/0.6 MPa (SS) 2.47 180 °C/0.6 MPa (SHS) 3.05
12.22 ± 0.17e 10.85 ± 0.13f
50.50 ± 0.33b 48.75 ± 0.12c
99.52 ± 0.32a 91.71 ± 0.39b
Note: different superscripts in the same column mean that the values are significantly different at 95% confidence level (p 6 0.05).
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Ethanol (g/L)
(a)
25
Xylose (g/L)
20
Concentration (g/L)
Concentration (g/L)
25
15 10 5
Ethanol (g/L)
(b)
Xylose (g/L)
20 15 10 5 0
0 0
20
40 Time (h)
60
80
0
20
40 Time (h)
60
80
Fig. 4. Ethanol productions from xylose obtained by acid hydrolysis (a), and from xylose in the liquid fraction after steam explosion (b).
samples were treated in various ways, the extent of irreversible pore collapse for each sample might also vary during the subsequent drying of treated OPEFB, prior to enzymatic hydrolysis. Therefore, the extent of enzymatic accessibility was also affected by drying step. 3.4. Fermentation of xylose for ethanol production The objective of this section was to evaluate the potential use of xylose extracted by steam explosion as fermentable sugar for ethanol production. The liquid fraction from SHS explosion of acid pretreated OPEFB, with high xylose concentration, was used in this section. The fermentation of xylose by the specific yeast C. shehatae for ethanol production was tested (Fig. 4). For comparison, the xylose obtained by direct acid hydrolysis of OPEFB was also used for ethanol production (Fig. 4a). This direct acid hydrolysate contained 19.22 g/L xylose and 2.34 g/L glucose and also furfural and acetic acid at high concentrations of 0.82 g/L and 2.51 g/L, respectively. Fig. 4a shows that the yeast could not ferment xylose in this direct acid hydrolysate. This could be due to the high concentrations of inhibitors present in the hydrolysate. Chandel et al. (2007) have reported that the acid hydrolysate needed to be detoxified and each detoxification method gave different ethanol yields i.e., overliming (0.302 g ethanol/g xylose), laccase (0.374 g ethanol/ g xylose), activated charcoal (0.425 g ethanol/g xylose) and ion exchange (0.482 g ethanol/g xylose). While the liquid fraction from steam explosion in this study contained initial xylose, glucose, furfural and acetic acid at 13.87 g/L, 0.23 g/L, 0.03 g/L and 1.19 g/L, respectively. The low furfural and acetic acid concentrations in this liquid fraction suggest the high potential use for fermentation. It was obvious that the yeast could assimilate xylose in the liquid fraction from steam explosion and produced ethanol at a high yield of 0.30 g ethanol/g xylose (Fig. 4b). 4. Conclusions This study has shown that the acid pretreatment prior to superheated steam explosion was the most suitable among the tested conditions for xylose production from OPEFB. The exploded OPEFB by this manner also had high accessibility for enzymatic hydrolysis due to its high porosity after steam explosion. However, the alkali pretreatment prior to superheated steam explosion was more suitable for obtaining the exploded OPEFB with the highest enzymatic accessibility. The xylose recovered in the liquid fraction from superheated steam explosion could directly be used as fermentable sugar for ethanol production, as this was facilitated by its low inhibitor concentrations.
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