High temperature dilute phosphoric acid pretreatment of corn stover for furfural and ethanol production

Industrial Crops and Products 50 (2013) 478–484

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High temperature dilute phosphoric acid pretreatment of corn stover for furfural and ethanol production Ayse Avci a,b,∗ , Badal C. Saha a , Gregory J. Kennedy a , Michael A. Cotta a a Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture1 , 1815 N. University Street, Peoria, IL 61604, USA b Department of Food Engineering, Faculty of Engineering, Sakarya University, Sakarya 54187, Turkey

a r t i c l e

i n f o

Article history: Received 30 May 2013 Received in revised form 16 July 2013 Accepted 21 July 2013 Keywords: Corn stover Furfural production Dilute phosphoric acid pretreatment Enzymatic hydrolysis Ethanol fermentation Saccharomyces cerevisiae

a b s t r a c t Furfural was produced from corn stover by one stage pretreatment process using dilute H3 PO4 and solid residues following furfural production were used for ethanol production by Saccharomyces cerevisiae NRRL-Y2034. A series of experiments were conducted at varied temperatures (140–200 ◦ C) and acid doses (0.0–2.0%, v/v) in order to determine optimal conditions. The effects of time (5–25 min) and substrate concentration (5–15%, w/w) on furfural production were determined at optimal temperature (200 ◦ C) and acid dose (0.75%, v/v). Maximum furfural yield (10.8 ± 0.3 g/100 g stover) was achieved at 20–25 min duration with 5% (w/w) corn stover which corresponds to 61.6% of the potential yield. About 74% of the glucan content of corn stover was converted to glucose after enzymatic digestion of solid residues. S. cerevisiae NRRL-Y2034 fermented glucose from the solid residues efficiently to ethanol. It produced 0.47–0.50 g ethanol per g glucose which corresponds to 92–99% of the theoretical yield. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Lignocellulosic biomass is the most abundant sustainable feedstock in the world and is considered as a promising source for production of fuels and chemicals to replace fossil fuels (Saha, 2003; Mamman et al., 2008). It consists of mainly cellulose (38–50%), hemicellulose (23–32%) and lignin (15–25%) (Saha, 2004). Due to its heterogeneous structure and recalcitrance, complex and expensive processes are required to convert lignocellulosic biomass to fuels and chemicals (Lange et al., 2012). For the last two decades, many efforts have focused on the hydrolysis of lignocellulosic feedstock into simple sugars for effective utilization of pentose and hexose sugars present in hemicellulose and cellulose, respectively (Dutta et al., 2012). Dilute acid hydrolysis of lignocellulosic biomass at high temperature helps to remove lignin from the structure, and solubilizes and converts hemicellulose into simple sugars (xylose, arabinose, galactose, mannose). The cellulose fraction remains unaltered during acid hydrolysis, however, accessibility of cellulase enzymes increases (Saha et al., 2005; Vazquez et al., 2007).

∗ Corresponding author at: Department of Food Engineering, Faculty of Engineering, Sakarya University, Sakarya 54187, Turkey. Tel.: +90 264 295 5464; fax: +90 264 295 5608. E-mail address: [email protected] (A. Avci). 1 Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.07.055

The fractionation of hemicellulose and cellulose allows processing of each fraction separately into fuels and chemicals (Saha, 2003; Dutta et al., 2012). Acid-catalyzed hydrolysis of the hemicellulosic pentosan fraction of biomass yields xylose (most predominant pentose in most feedstocks) and arabinose which are further converted to furfural (2-furaldehyde, C5 H4 O2 ) by means cyclodehydration (Rong et al., 2012; Telleria et al., 2011). Antal et al. (1991) showed that dehydration of xylose to furfural is primarly catalyzed by Brønsted acids (donors of H+ ), which catalyze formation of a 1,2-enodiol, and then it dehydrates to furfural. During acid treatment, both depolymerization and dehydration occur simultaneously. Furfural is a useful chemical solvent which has been used for separating saturated and unsaturated compounds in petroleum refining, gas, oil and diesel fuel (Mamman et al., 2008). The unsaturated bonds and aldehyde group in the structure make it highly versatile that can be involved in the production of a broad range of industrial chemicals such as plastics, pharmaceuticals and agrochemicals (Rong et al., 2012; Zhang et al., 2012). There is no synthetic route available for production of furfural; hence, it is exclusively produced from hemicellulose (Zhang et al., 2013). Any kind of lignocellulosic biomass containing hemicellulose such as corncobs, cotton seed hulls, oat hulls, bran, saw dust, bagasse, and rice hulls can be used for this purpose (Uppal et al., 2008). Two types of process technology are mainly used for furfural production. In single stage process, pentosans present in lignocellulose are hydrolyzed by acid into sugars (xylose and arabinose) and then

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dehydrated to furfural simultaneously. In two-stage process, the acid hydrolysis of pentosans to pentose sugars occurs under mild conditions, followed by dehydration of the sugars to furfural (Sanchez et al., 2013; Mansilla et al., 1998). This process has the advantage of preventing degradation of lignocellulosic residues. In industrial production, one stage process is preferred because of lower process cost. The remaining solid residue is burned for steam production (Mansilla et al., 1998; Sanchez et al., 2013). Sulfuric acid is used as catalyst in the current commercial production of furfural (Telleria et al., 2011; Molina et al., 2012). Reaction temperature is maintained by steam injection to the system. Furfural formed during the process is continuously stripped out (Mao et al., 2012). However, this process is not economical due to degradation of furfural to organic acids, aldehydes and condensation products (Gairola and Smirnova, 2012). About 40–50% of cellulosic fraction is also lost by degradation of glucose to HMF, formic acid and levulinic acid. Hydrochloric acid, nitric acid (Yemis and Mazza, 2011), phosphoric (Vazquez et al., 2007) and formic acid (Yang et al., 2012) can also be used for the production of furfural. Phosphoric acid is environmentally friendly and the solid residues can be used as fertilizers. Moreover furfural is less degraded when phosphoric acid is used (Avci et al., 2013). Several other techniques have been proposed for production of furfural including addition of salts (NaCl, KCl, CaCl2 , MgCl2 , FeCl2 , ZnCl2 ) to enhance conversion of xylose to furfural (Marcotullio and de Jong, 2011; Rong et al., 2012; Yoo et al., 2012; Molina et al., 2012) or for effective stripping of furfural with N2 or supercritical CO2 (Tellaria et al., 2011; Gairola and Smirnova, 2012). Some methods are based on prevention of furfural degradation during processing by using solvents such as toluene, n-butanol, methyl isobutyl ketone, dichloromethane and cyclopentylmethyl ether in biphasic system (Amiri et al., 2010; Rong et al., 2012; Lange et al., 2012). Although there has been much efforts to increase furfural yield from lignocellulosic materials, research on the utilization of residues containing cellulose is limited (Mao et al., 2012). Production of ethanol from lignocellulosic biomass requires effective utilization of pentose and hexose sugars by microorganisms. Native ethanol producers (Saccharomyces cerevisiae and Zymomonas mobilis) are not able to utilize pentose sugars. Various strains of Escherichia coli and S. cerevisiae have been modified genetically to produce ethanol and metabolize pentoses to ethanol, respectively (Saha and Cotta, 2012), but challenges remain (Lange et al., 2012). In this research paper, we report the production of furfural from corn stover in one stage process using dilute H3 PO4 . Our aim was to convert pentosans into furfural at the same time preventing the degradation of cellulose in order to use it for ethanol production. The effects of temperature, acid dose, time, solid concentration on the furfural production from corn stover were determined. To our knowledge it is the first report demonstrating that H3 PO4 is a better catalyst than H2 SO4 for furfural production. Further studies have been conducted to investigate fermentability of residual solids to ethanol by using conventional yeast strain (S. cerevisiae NRRL-Y2034).

2. Materials and methods 2.1. Materials Corn stover (92.6 ± 0.2% dry matter) was harvested in the fall of 2011 (Peoria, IL). It was ground in a hammer mill so as to pass through a 1.27 mm screen and stored at ambient temperature in a tightly closed plastic bag. Composition of the corn stover (37.10 ± 0.03% glucan, 21.00 ± 0.10% xylan, 2.10 ± 0.01% galactan, 3.10 ± 0.4% arabinan, 1.80 ± 0.02% acid soluble lignin, 17.10 ± 0.02%

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acid insoluble lignin and 5.04 ± 0.02% ash on dry basis) was determined by using National Renewable Energy Laboratory Procedures (NREL), Golden, CO (Sluiter et al., 2008a,b). Celluclast 1.5 L (cellulase) and Novozym 188 (␤-glucosidase) were purchased from Brentag Great Lakes (Milwaukee, WI, USA). Fiberzyme (hemicellulase) was provided by Dyadic Corp., Jupiter, FL. Glucose, xylose, arabinose, galactose, H2 SO4 , HNO3 , 2furfuralaldehyde (furfural, 99%), 5-hydroxymethylfurfural (HMF, 99%) and KOH were purchased from Sigma-Aldrich, St. Louis, MO, USA. H3 PO4 and Ca(OH)2 were purchased from Fisher Scientific, Pennington, NJ, USA; yeast extract, peptone and agar were from Difco Laboratories, Detroit, MI, USA. The filter sterilization unit (0.2 mm) was purchased from Nalgene Company, Rochester, NY, USA. Aminex HPX 87P column (300 × 78 mm), Aminex HPX 87H column, De-ashing cartridge (30 × 4.6 mm), Carbo-P microguard cartridge (30 × 4.6 mm) and Cation H micro-guard cartridge (30 × 4.6 mm) were purchased from Bio-Rad Laboratories Inc., Hercules, CA, USA. 2.2. Dilute acid pretreatment of corn stover Milled corn stover (5–15%, w/w) was slurried in dilute H3 PO4 (0.0–2.0%, v/v) and pretreated in a rotating stainless steel reactor with infrared heating (Labomat BFA-12, Mathis USA, Inc., Concord, NC, USA) at 140–200 ◦ C for 5–25 min. Twelve reactors (each 200 mL capacity) were used at one time. The heating and cooling times of the reactors were not considered part of the reported pretreatment time, even though the heat-up ramp took about 49 min (3.6 ◦ C/min) to reach the final temperature at 200 ◦ C. The reactors were water-cooled (6 ◦ C/min) using tap water following pretreatment. The cooling time after pretreatment at 200 ◦ C was about 28 min. The reactors were routinely rotated at 50 rpm with 60s clockwise followed by 60-s counter clockwise rotations during pretreatment for proper mixing. Effects of temperature (140, 160, 180 and 200 ◦ C) and H3 PO4 dose (0.00, 0.25, 0.5, 0.75, 1.0 and 2.0%; v/v) on the pretreatment of corn stover (10%, w/w) for 10 min duration were investigated. Pretreatment of corn stover (10%, w/w) was also performed at 200 ◦ C for 10 min using H2 SO4 at 200 ◦ C with 0.25, 0.50, 0.75 and 1.0% (v/v) concentrations for comparison. Effects of corn stover solid loading (5–15%, w/w) and pretreatment time (5–25 min) on furfural production were investigated at 200 ◦ C and 0.75% (v/v) H3 PO4 dose which were determined as the optimum temperature and optimum acid dose, respectively. The pH was adjusted to 5.0 using solid Ca(OH)2 just after each pretreatment. Samples were taken, centrifuged at 25,000 × g and analyzed for furfural, HMF, and released sugars. The yield of furfural was calculated according to the following equation: Furfural (%) =

Furfural produced (g) × 100 Xylose + arabinose in dry corn stover (g)

2.3. Enzymatic hydrolysis Enzymatic digestibility of dilute acid pretreated corn stover was performed at pH 5.0 and 45 ◦ C for 72 h by gentle shaking at 135 rpm using a filter sterilized cocktail of 3 commercial enzyme (cellulase, ␤-glucosidase and hemicellulase) preparations. The enzyme cocktail contained 15 FPU cellulase (Celluclast 1.5 L) and 9 U ␤-glucosidase (Novozym 188) per g glucan content and 1578 U xylanase (Fiberzyme) per g hemicellulose content, unless otherwise specified. The solid residues were separated from the liquid portion by centrifugation at 25,000 × g for 10 min before using the liquid portion as enzymatically saccharified corn stover hydrolyzate. The commercial enzyme preparations contained small

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quantities of glucose. For simplification purpose, the quantity of glucose present in the enzyme cocktail was subtracted from the measured glucose in each case. A modified procedure of the above procedure was applied for determination of enzymatic digestibility of solid residues obtained after pretreatment. After adjustment of pH to 5.0, liquid part of pretreated material was removed by centrifugation at 25,000 × g for 10 min. The solid residues were then washed thoroughly with water (1:10) twice and suspended in 25 mL deionized water for enzymatic hydrolysis which was carried out at 45 ◦ C and pH 5.0 for 72 h using 30 FPU cellulase and 18 U ␤-glucosidase per g glucan. Glucose recovery was calculated as follows: Glucose recovery(%) Total amount ofglucose after anzymatic hydrolysis (g) = Theoretical amount of glucoe in dry untreated corn stover (g) × 100

2.4. Fermentation of enzymatically saccharified solid residues The pH (5.0) of enzymatically saccharified corn stover hydrolyzate was adjusted to 5.5 by using 2 N KOH and it was supplemented with peptone (10 g/L) and yeast extract (5 g/L). The medium was sterilized using a filter sterilization unit. Fermentations were performed in pre-sterilized 50 mL Erlenmeyer flasks containing 20 mL medium. The flasks were covered with rubber stoppers with a syringe needle mounted on for gas exhaust. The S. cerevisiae NRRLY2034 was grown in YMP medium (glucose, 10 g/L; yeast extract, 3 g/L; peptone, 5 g/L; malt extract, 3 g/L) for 24 h prior to its use as seed culture. A 5% (v/v) of active culture (with 2.0 OD600nm ) was inoculated. Fermentations were carried out at 30 ◦ C for 72 h. Samples were taken periodically to analyze for ethanol production and sugar consumption. The yield of ethanol was calculated as follows: Ethanol yield =

Ethanol produced (g) × 100 Initial sugar load (g)

The theoretical maximum yield of ethanol from glucose is 0.511 g/g glucose. 2.5. Analytical procedures The cellulase activity in terms of filter paper activity was assayed and expressed as filter paper unit (FPU) by the procedure described by Ghose (1987). The ␤-glucosidase and xylanase activities were determined by the procedures described previously (Saha and Cotta, 2008). All enzyme assays were performed at pH 5.0 and 45 ◦ C and the activities were expressed in terms of international units (IU, ␮mol product formed per min). Moisture content was analyzed using a moisture analyzer (Mark 2, Sartorious Mechatronics Corp., Bohemia, NY, USA). Growth was determined by measuring the optical density at 600 nm. Sugars, furfural, HMF and ethanol were analyzed using high performance liquid chromatography (HPLC) (Saha and Bothast, 1999). The separation system consisted of a solvent delivery system (P2000 Pump, Spectra-Physics, San Jose, CA) equipped with an autosampler (717, Waters Chromatography Division, Millipore Corp., Milford, MA), a refractive index detector (410 Differential Refractometer, Waters), a dual ␭ absorbance detector (2487, Waters), and a computer software based integration system (Chromquest 4.0, Spectra-Physics). Sugars and ethanol were separated using an Aminex HPX-87P column (300 × 78 mm) and detected using the refractive index detector. Two guard columns [De-ashing (30 × 4.6 mm) and Carbo-P micro guard (30 × 4.6 mm)

cartridges] were used with Aminex HPX-87P column. Column temperature was maintained at 85 ◦ C and elution was made at a flow rate of 0.6 mL/min using Milli-Q (Millipore Corp., Bedford, MA, USA) filtered water. Furfural and HMF were separated in Aminex HPX87H column and detected by using the dual ␭ absorbance detector at 277 nm. The column temperature was 65 ◦ C and 10 mM HNO3 in Milli-Q filtered water was used for elution at a flow rate of 0.6 mL/min.

3. Results and discussion 3.1. Effects of H3 PO4 dose and temperature on furfural production Composition of the liquid portion after dilute acid pretreatment of lignocellulosic biomass depends on pretreatment process conditions such as feedstock concentration, temperature, acid type, acid dose and duration (Gamez et al., 2004; Vazquez et al., 2007). In order to determine the optimum temperature and acid dose for furfural production, corn stover (10%, w/w) was pretreated at 140–200 ◦ C using 0 to 2.0% (v/v) H3 PO4 for 10 min duration. The ranges of temperature and acid dose were chosen based on our previous study (Avci et al., 2013). There was no furfural generated at 140 ◦ C and it was very low (0.0–0.7 g/100 g stover) at 160 ◦ C at all acid doses used (Fig. 1A). However, remarkable increases in furfural production were observed at 180 and 200 ◦ C. Effect of acid dose was also prominent at 180 and 200 ◦ C. At 180 ◦ C, furfural production increased gradually from 0.55 ± 0.1 at 0.25% (v/v) acid dose to 5.5 ± 0.2 g/100 g (10 fold increase) at 2.0% (v/v) acid dose. There was significant increase in furfural production at 200 ◦ C even at lower acid doses. It was 1.3 ± 0.0, 6.5 ± 0.3, 8.7 ± 0.5, 9.1 ± 0.2, 9.0 ± 0.2 and 8.7 ± 0.4 g/100 g stover at 0.0, 0.25, 0.50, 0.75, 1.0 and 2.0% (v/v) acid dose, respectively. It seems that degradation of furfural started over 1.0% (v/v) of H3 PO4 at 200 ◦ C as the furfural content decreased at 2.0% (v/v) acid dose (from 9.1 ± 0.2 g/L to 8.7 ± 0.2 g/L, Fig. 1A). The optimal temperature and acid dose for production of maximum furfural were at 200 ◦ C and 0.75% (v/v), respectively, at which 9.1 ± 0.2 g furfural was obtained per 100 g stover (34.3% yield based on theoretical xylose and arabinose content). There was little or no HMF production at 140 and 160 ◦ C at all acid doses investigated (Fig. 1B). However, the HMF production increased with the increase of acid dose at 180 ◦ C and 200 ◦ C, but at 200 ◦ C, the effect of acid dose on HMF production was at least 5 fold higher than at 160 ◦ C. The glucose, xylose and total sugars (glucose, xylose, arabinose and galactose) generated from dilute acid pretreated corn stover at 140–200 ◦ C for 10 min duration using 0 to 2.0% (v/v) H3 PO4 are shown in Fig. 2(A–C). Glucose release was positively influenced by the increase of both temperature and acid dose of pretreatment. Cellulose is less susceptible to acids and high temperatures than hemicellulose due to its crystalline structure (Saha, 2004; Mamman et al., 2008). The glucose yield in the pretreated corn stover did not exceed 6.8 ± 0.2 g/100 g stover which was the highest yield obtained at 200 ◦ C with 2.0% (v/v) H3 PO4 (Fig. 2A). On the other hand, hemicellulose conversion to component sugars (mainly pentose sugars) and their further degradation to furans was found to be highly dependent on acid dose, temperature and duration of pretreatment (Avci et al., 2013). The xylose yield was maximum (16.6 ± 0.3 g/100 g stover) at 160 ◦ C with 2.0% (v/v) H3 PO4 and decreased to 1.3 ± 0.1 g/100 g stover at 200 ◦ C with the same acid dose due to the formation of furfural (Fig. 2B, Fig. 1A). The total sugar yield in the pretreated corn stover was maximum (27.6 ± 0.7 g/100 g stover) at 160 ◦ C and 2.0 (%, v/v) acid dose. It decreased to 10.3 ± 0.5 g/100 g stover at 200 ◦ C with the same acid dose (Fig. 2C).

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Fig. 1. Effects of temperature (140–200 ◦ C) and H3 PO4 dose (0.0–2.0%, v/v) of pretreatment of corn stover (10%, w/w) for 10 min on furfural (A) and HMF (B) production. The data presented are averages of 2 separate experiments.

The enzymatic hydrolysis of pretreated corn stover was performed using a cocktail of 3 commercial enzyme (cellulose, ␤-glucosidase and hemicellulase) preparations. The data on the yields of glucose, xylose and total sugars are presented in Fig. 3(A–C). Cellulose hydrolysis increased with the increase of temperature and acid dose. The glucose yield was lower at 140 ◦ C and 160 ◦ C (Fig. 3A). The glucose yield was 32.3 ± 0.1 g/100 g at 180 ◦ C with 2.0% (v/v) H3 PO4 and 32.4 ± 0.5 g per 100 g stover at 200 ◦ C with 0.5% (v/v) H3 PO4. These values correspond to 79.1 and 79.4% yield based on theoretical glucose yield from corn stover. On the other hand, xylose yield (18.8 ± 0.3 g/100 g) was maximum at 160 ◦ C with 0.75% (v/v) H3 PO4 which is equivalent to 81.4% yield based on theoretical xylose content of corn stover (Fig. 3B). Maximum total sugars (49.0 ± 0.0) was obtained at 180 ◦ C with 0.75% (v/v) acid dose which is equivalent to 70.3% yield of sugars based on total carbohydrate content of corn stover (Fig. 3C). For comparison, dilute H2 SO4 pretreatment of corn stover was performed at 200 ◦ C under the same conditions used for H3 PO4 (Data not shown). Maximum amount of furfural (6.5 ± 0.2 g/100 g

Fig. 2. Effects of temperature (140–200 ◦ C) and H3 PO4 dose (0.0–2.0%, v/v) of pretreatment of corn stover (10%, w/w) for 10 min on glucose (A) and xylose (B) and total sugars (C) yield prior to enzymatic hydrolysis. The data presented are averages of 2 separate experiments.

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stover) was obtained with 0.5% (v/v) H2 SO4 at 200 ◦ C and glucose yield was 30.6 ± 0.8 g/100 g stover which is also the maximum. The HMF production reached a maximum of 1.2 ± 0.2 g/100 g stover at 200 ◦ C with 0.75% (v/v) H2 SO4. As H2 SO4 is a stronger acid than H3 PO4 , this pretreatment process appears to have resulted in furfural degradation and as well as cellulose loss. These results indicate that H3 PO4 is more efficient than H2 SO4 for production of furfural at elevated temperatures, although H2 SO4 is the most commonly used acid in industry for this purpose. The glucose yield from the glucan fraction can be increased if dilute H3 PO4 is used for furfural production. 3.2. Effect of duration and corn stover loading on furfural production by dilute H3 PO4 pretreatment

Fig. 3. Effects of temperature (140–200 ◦ C) and H3 PO4 dose (0.0–2.0%, v/v) of pretreatment of corn stover (10%, w/w) for 10 min on glucose (A) and xylose (B) and total sugars (C) yield after enzymatic hydrolysis using a cocktail of 3 commercial enzyme (cellulase, ␤-glucosidase and hemicellulose) preparations. The data presented are averages of 2 separate experiments.

The effect of duration (5, 10, 15, 20 and 25 min) of pretreatment of corn stover (5, 10, 15%; w/w) on the production of furfural was investigated at 200 ◦ C using 0.75% (v/v) H3 PO4 . Furfural production increased gradually up to 20 min at all substrate concentrations (Table 1). The furfural yields after 5 min duration were 6.1 ± 0.5, 6.0 ± 0.2 and 4.8 ± 0.1 g/100 g stover at 5, 10 and 15% (w/w) corn stover, respectively. The xylose yield under these conditions were 6.8 ± 1.4, 4.6 ± 0.8 and 4.8 ± 0.1 g/100 g stover at 5, 10 and 15% (w/w) stover, respectively. After 20 min duration, furfural yields were 10.8 ± 0.3, 9.1 ± 0.3 and 7.6 ± 0.3 g/100 g stover at 5, 10 and 15% (w/w) stover, respectively. Also, 2.4 ± 0.0, 0.5 ± 0.0 and 0.9 ± 0.0 g xylose per 100 g stover were detected in the pretreated stover hydrolyzates at 5, 10 and 15% (w/w) corn stover, respectively. The furfural yield was found to decrease at 25 min duration for 10 and 15% corn stover presumably due to decomposition of furfural to humin and other products (Mamman et al., 2008). This suggests that for furfural production at 0.75% (v/v) acid dose at 200 ◦ C using 10 and 15% corn stover, the duration of pretreatment cannot be increased over 20 min with this one stage process under the conditions used. Maximum yields of furfural were achieved at 20 min pretreatment time. The detailed product (furfural, HMF, xylose, arabinose and glucose) yields obtained at different concentrations of corn stover (5–15%, w/w) pretreated at 200 ◦ C using 0.75% (v/v) H3 PO4 for 5–25 min duration are presented in Table 1. Lowest furfural yields were obtained at 5 min duration at all substrate concentrations with the highest yields of both pentose sugars. Maximum furfural yield (10.8 ± 0.3 g/100 g) was achieved at 20–25 min duration with 5% (w/w) corn stover (Table 1). This value corresponds to 40.75% conversion of pentose sugars to furfural based on theoretical yield of pentose sugars (26.5 g/100 g stover) from corn stover. Theoretically, 72.72% of pentose sugars can be converted to furfural (Yoo et al., 2012). Based on this number, 44.8% of pentosans of corn stover was converted to furfural which is actually 61.6% of potential furfural yield from corn stover. Practically, the yield of furfural is generally below 60% of the theoretical using the one stage process when lignocellulosic biomass is used as substrate (Mao et al., 2012). The furfural loss can be attributed to low hydrolysis of lignocellulosic substrates as well as furfural loss caused by the side reactions (Bamufleh et al., 2013; Mao et al., 2012). Sanchez et al. (2013) showed that furfural production was strongly influenced by acid concentration, duration and temperature of microwave-assisted pretreatment using corn cob autohydrolyzed liquor as substrate. They obtained a maximum of 37.06% furfural with respect to hemicellulose content of corn cobs at 180 ◦ C, 5 min pretreatment and 2% (v/v) HCl using microwave heating. In this research, a similar yield (37.5%) of furfural was obtained from corn stover hemicellulose using dilute H3 PO4 pretreatment. Suxia et al. (2012) obtained a maximum of 8.9% furfural from rice husk using H2 SO4 and a solid acid catalyst through a two-stage process under optimized conditions. Yoo et al. (2012) obtained 10.2 g furfural per 100 g corn stover using a hybrid two

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Table 1 Effect of pretreatment duration on product (furfural, HMF, xylose, arabinose and glucose) yield after dilute H3 PO4 (0.75%, v/v) pretreatment of corn stover (5–15%, w/w) at 200 ◦ C. Corn stover (%, w/w)

Pretreatment time (min) (g/100 g)

5 10 15 20 25

6.1 ± 8.9 ± 15 10.8 ± 10.8 ±

0.5 0.4

5 10 15 20 25

6.0 ± 7.3 ± 15 9.1 ± 8.9 ±

0.2 0.0

5 10 15 20 25

4.8 ± 6.0 ± 15 7.6 ± 7.4 ±

0.1 0.1

0.3 0.2

0.3 0.3

0.3 0.3

Furfural (g/100 g)

HMF (g/100 g)

Xylose (g/100 g)

Arabinose (g/100 g)

Glucose (g/100 g)

0.6 1.2 10.1 2.3 2.5

± ± ± ± ±

0.1 0.1 0.4 0.2 0.0

6.8 3.6 1.4 2.4 1.9

± ± ± ± ±

1.4 0.2 0.0 0.0 0.2

1.8 1.0 2.5 0.8 0.8

± ± ± ± ±

0.5 0.8 0.3 0.3 0.1

4.5 6.0 1.1 7.6 7.8

± ± ± ± ±

0.4 0.4 0.1 0.3 0.2

6.9 ± 0.3

0.6 1.0 8.9 2.0 2.1

± ± ± ± ±

0.0 0.0 0.1 0.0 0.1

4.6 2.5 1.4 1.4 0.7

± ± ± ± ±

0.8 0.3 0.0 0.1 0.2

1.3 1.0 1.6 0.5 0.5

± ± ± ± ±

0.0 0.2 0.1 0.0 0.1

2.6 3.1 1.0 4.0 3.7

± ± ± ± ±

0.1 0.1 0.1 0.1 0.3

3.5 ± 0.2

0.5 0.8 7.5 1.7 1.8

± ± ± ± ±

0.0 0.0 0.2 0.1 0.1

4.8 2.0 1.2 0.9 0.4

± ± ± ± ±

0.1 0.2 0.1 0.0 0.0

1.3 0.7 1.1 0.4 0.2

± ± ± ± ±

0.0 0.0 0.1 0.0 0.0

1.9 2.2 0.6 2.8 2.4

± ± ± ± ±

0.1 0.0 0.2 0.2 0.2

2.3 ± 0.1

The data presented are averages of two separate experiments.

stage pretreatment process (150 ◦ C, 10 min; 170 ◦ C, 10 min with 5 min for temperature shifting) using ZnCl2 as catalyst (0.937 g/g stover) and then converting the liquid fraction to furfural by heating at 150 ◦ C for 120 min. Only a few reports are available on furfural production from lignocelluloses biomass using dilute H3 PO4 as catalyst. Vazquez et al. (2007) studied the hydrolysis of sorghum straw using 2–6% (v/v) H3 PO4 at 134 ◦ C. They obtained the highest furfural production (13.36 g/100 g) with 6% acid in 300 min in a one stage process. Cellulose digestibility was not reported. In our study, using 8 fold dilute H3 PO4 , we have shown that good yields (61.5% of theoretical yield based on pentosan content of corn stover) of furfural can be achieved in a short duration (20 min) of pretreatment at 200 ◦ C. Recently, Yemis and Mazza (2011) obtained 27.6% furfural from xylose with H3 PO4 at 180 ◦ C by microwave assisted pretreatment. The process conditions were a temperature of 180 ◦ C, a solid liquid ratio of 1:100, a residence time of 20 min, and a pH 0f 1.12. The yield was lower than the yield (31.9%) they obtained with H2 SO4 using the same conditions. They reported furfural yield of 25.6% from birchwood xylan which is also lower than 29.0% obtained using H2 SO4 under the same conditions. This is the first report, to our knowledge, on the superior performance of dilute H3 PO4 over H2 SO4 as a catalyst for furfural production using corn stover as feedstock. 3.3. Enzymatic saccharification of solid residues Liquid fraction containing furfural was separated by centrifugation and solid residues were washed with distilled water in order to remove residual furfural and other soluble products. The washed solid residues were subjected to enzymatic digestion using a cocktail of commercial cellulase and ␤-glucosidase preparations.

Glucose was the sole sugar detected in all enzymatic hydrolyzates. Table 2 shows the results as glucose yield in g per 100 g of corn stover after enzymatic hydrolysis of solid residues obtained from dilute H3 PO4 (0.75%, v/v) pretreatment at 200 ◦ C for 5–25 min duration. Highest glucose yields [30.2 ± 0.3, 30.1 ± 1.0, 27.5 ± 0.6 g/100 g at 5, 10, 15% (w/w) corn stover, respectively] were obtained at 5 min duration and the yield decreased with the increase of duration of pretreatment. Increased production of HMF was also observed with the increase of duration of pretreatment (Table 1). Glucose yield (maximum yield, 74% based on glucan content of corn stover) is satisfactory when compared with the previously published results which were made either stripping furfural during process or in two stage process. This corresponds to 85% glucose yield taking into account of the quantity of glucose present in the liquid fraction after pretreatment. 3.4. Ethanol production using S. cerevisiae S. cerevisiae NRRL Y-2034, a non-recombinant laboratory strain was used for ethanol fermentation of the enzymatic hydrolyzates of washed solid residues obtained after dilute H3 PO4 pretreatment (0.75%, v/v acid; 200 ◦ C, 5–25 min duration) of corn stover (5–15%, w/w) containing glucose only as fermentable sugar. The fermentation results of ethanol production at pH 5.5 and 30 ◦ C after 72 h are presented in Table 3. Glucose was fully utilized by the yeast at the end of 72 h and almost quantative yields of ethanol (0.47–0.50 g/g glucose; 92–99%) were obtained. Maximum amounts of ethanol was 31.6 g/L with 10% (w/w) corn stover and 42 g/L with 15% (w/w) stover under the experimental conditions used. Ethanol yield (0.13 g ethanol per g of corn stover) was very consistent in all cases. These findings are quite satisfactory, because only the cellulosic fraction of corn stover was involved in ethanol fermentation. Yoo

Table 2 Effect of pretreatment duration (5–25 min) on glucose yield in g/100 g corn stover after enzymatic hydrolysis of washed solid residues obtained from dilute H3 PO4 pretreated (0.75%, v/v; 200 ◦ C) corn stover (5–15%, w/w). Corn stover (%, w/w)

Pretreatment time (min) 5

5 10 15

30.2 (4.5 30.1 (2.6 27.5 (2.0

10 ± ± ± ± ± ±

0.3 0.3) 1.0 0.1) 0.6 0.1)

28.8 (6.0 28.3 (3.1 25.7 (2.2

15 ± ± ± ± ± ±

0.4 0.4) 0.6 0.1) 1.5 0.0)

26.7 (6.9 26.9 (3.6 26.2 (2.3

20 ± ± ± ± ± ±

0.1 0.2) 0.5 0.2) 0.4 0.0)

25.7 (7.6 28.1 (4.1 25.6 (2.8

25 ± ± ± ± ± ±

0.0 0.0) 0.3 0.1) 0.1 0.1)

25.2 (7.9 26.4 (3.7 26.6 (2.4

± ± ± ± ± ±

0.8 0.2) 0.2 0.1) 0.4 0.1)

The data presented are averages of 2 separate experiments. The data in parenthesis is the quantity of glucose present in the liquid fraction after dilute H3 PO4 pretreatment.

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A. Avci et al. / Industrial Crops and Products 50 (2013) 478–484

Table 3 Summary of fermentation of glucose generated after enzymatic hydrolysis of solid residues obtained from dilute H3 PO4 pretreated (0.75%, v/v; 200 ◦ C; 5–25 min duration) corn stover (5–15%, w/w) by S. cerevisiae NRRL Y-2034 at pH 5.5 and 30 ◦ C for 72 h. Corn stover (%, w/w) 5

10

15

Pretreatment time (min) 5 10 15 20 25 5 10 15 20 25 5 10 15 20 25

Solid residue (g/L) 104.0 113.0 107.0 130.0 118.0 146.0 142.0 146.0 154.0 160.0 167.0 187.0 187.0 204.0 210.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.2 0.5 0.0 0.5 0.6 0.5 0.1 0.0 0.2 0.1 1.7 0.2 0.3 0.2

Glucose (g/L) 36.3 38.7 37.5 36.9 35.8 60.4 59.0 60.7 64.6 63.5 70.3 71.6 76.4 80.0 83.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.6 0.5 0.1 1.3 1.1 0.2 0. 0.1 0.2 1.4 2.3 1.2 0.9 0.1

Ethanol (g/L) 17.2 17.1 18.0 17.7 17.9 30.0 29.1 28.5 31.2 31.6 31.8 34.4 35.9 40.0 42.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0 0.6 0.5 0.1 0.3 0.5 0.3 0.4 0.3 0.1 0.7 0.1 0.1 0.4 0.1

Ethanol (g/g glu)

Ethanol (g/g stover)

0.47 0.44 0.48 0.48 0.50 0.50 0.49 0.47 0.49 0.50 0.45 0.48 0.47 0.50 0.50

0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13

The data presented are averages of two separate experiments. glu, glucose.

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