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Dilute alkali pretreatment of softwood pine: A biorefinery approach
Accepted Manuscript Dilute alkali pretreatment of softwood pine: A biorefinery approach Ali Safari, Keikhosro Karimi, Marzieh Shafiei PII: DOI: Reference:
S0960-8524(17)30300-0 http://dx.doi.org/10.1016/j.biortech.2017.03.030 BITE 17734
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
27 January 2017 3 March 2017 4 March 2017
Please cite this article as: Safari, A., Karimi, K., Shafiei, M., Dilute alkali pretreatment of softwood pine: A biorefinery approach, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.03.030
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Dilute alkali pretreatment of softwood pine: A biorefinery approach Ali Safaria, Keikhosro Karimia,b, Marzieh Shafieic,*
a
Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran
b
Industrial Biotechnology Group, Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran
c
Department of Chemical Engineering, Faculty of Engineering, University of Isfahan, Isfahan 8174673441, Iran *
Corresponding Author
Tel: +983137934558, Fax: +983137934031, E-mail address: [email protected]
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Abstract
Dilute alkali pretreatment was performed on softwood pine to maximize ethanol and biogas production via a biorefinery approach. Alkali pretreatments were performed with 0-2% w/v NaOH at 100-180 °C for 1–5 h. The liquid fraction of the pretreated substrates was subjected to anaerobic digestion. The solid fraction of the pretreatment was used for separate enzymatic hydrolysis and fermentation. High ethanol yields of 76.9‒78.0% were achieved by pretreatment with 2% (w/v) NaOH at 180 °C. The highest biogas yield of 244 mL/g volatile solid (at 25 ºC, 1 bar) was achieved by the pretreatment with 1% (w/v) NaOH at 180 °C. The highest gasoline equivalent (sum of ethanol and methane) of 197 L per ton of pinewood and the lowest ethanol manufacturing cost of 0.75 €/L was obtained after pretreatment with 1% NaOH at 180 °C for 5 h. The manufacturing cost of ethanol from untreated wood was 4.12 €/L.
Keywords: Biorefinery; Ethanol; Biogas; Dilute Alkali Pretreatment; Softwood Pine.
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1. Introduction Ethanol and biomethane are two renewable fuels that can be used in the transportation sector. Currently, ethanol is industrially produced from sugar- or starch-based raw materials. However, due to limited resources of corn and wheat, this has led to a food vs. fuel dilemma (Noori and Karimi, 2016a; Kumar et al., 2016). Lignocellulosic materials such as agricultural and forest residues are available to a large extent at low prices. These materials have been considered non-food based feedstocks for biofuel production. However, they resist enzymatic or microbial attacks, and a pretreatment step is necessary prior to their enzymatic or biological conversion (Shafiei et al., 2015; Karimi and Taherzadeh, 2016a, 2016b). Alkaline pretreatment by sodium hydroxide is one of the most effective chemical pretreatments for ethanol production (Kim et al., 2016; Karimi et al., 2013). This pretreatment modifies cellulose structure and partially solubilizes hemicellulose and lignin, and thus increases the accessibility of lignocelluloses. Hemicellulose and lignin removal were shown to enhance the hydrolysis of lignocelluloses (Karimi et al., 2013; Hendriks et al., 2009; Menegol et al., 2016). Pretreatment by sodium hydroxide can be performed at severe (0.5–4% NaOH at high temperature) or moderate conditions (at least 6–8% NaOH and low temperature) (Karimi et al., 2013). Considering the high cost of sodium hydroxide, using lower concentrations is economically more favorable. Several NaOH pretreatments of softwoods have been studied by Mirahmadi et al. (2010), Noori and Karimi (2016a), and Salehian and Karimi (2013) with 7–8% NaOH. These studies have revealed that pretreatment with higher concentrations of NaOH was most efficient for agricultural residues and hardwood rather than for softwood of which maximum hydrolysis yields were less than 55% (Mirahmadi et al., 2010; Salehian and Karimi, 2013). Zhao et al. (2008) have investigated presoaking spruce with 12% urea and 3% NaOH. Pretreatment of the presoaked wood with 3% NaOH for 24 h at −15 °C yielded 60% glucose. To our knowledge, no previous investigation has been reported on pretreatment of softwood with dilute NaOH at high temperatures. Additionally, previous studies which were preformed at higher alkaline 3
concentrations or lower temperatures did not result in high saccharification yields. Thus, the first aim of this study was to investigate dilute alkali pretreatment of softwood pine at elevated temperatures. After completion of the pretreatment by NaOH, a slurry containing solid and liquid fractions is produced. The solid fraction is filtrated from the liquid fraction (called pre-hydrolysate) and enzymatically hydrolyzed for ethanol fermentation. However, a portion of carbohydrates which are recovered in the pre-hydrolysate are not fermentable by ordinary ethanolic microorganisms, e.g., Saccharomyces cerevisiae (Shafiei et al., 2015; FazeliNejad et al., 2016). In order to have an economically viable process, the pre-hydrolysate portion should be converted to byproducts or products, such as ethanol, methane, or biomass (FazeliNejad et al., 2016; McMillan et al., 1999; Dien et al., 2003). Pre-hydrolysate contains solubilized monomer or short chain carbohydrates. The carbohydrates are mainly solubilized hemicelluloses which have been released during the pretreatment (Karimi et al., 2013). For the conversion of both pentoses and hexoses to ethanol, separate hydrolysis and co-fermentation (SHCF) or simultaneous saccharification and co-fermentation (SSCF) was suggested (McMillan et al., 1999; Dien et al., 2003). Xylose assimilation in the former process suffers from glucose and ethanol inhibition (Taherzadeh and Karimi, 2011; Jin et al., 2012). SSCF process can be performed by two different or one recombinant microorganism (Kumar et al., 2016; Talebnia, 2015). In SSCF, glucose inhibition is reduced; however, ethanol yield in SSCF from both glucose and xylose is considerably lower than that formed from glucose by ordinary yeasts (e.g., Saccharomyces cerevisiae) (McMillan et al., 1999; Koppram et al., 2013). Furthermore, differences in the optimal fermentation condition of the two strains, e.g., pH and temperature, and the challenges of using genetically modified organisms, i.e., safety issues and gene instabilities, are practical bottlenecks (Ho et al., 2001; Dien et al., 2000). Therefore, simultaneous conversion of both hexoses and pentoses to ethanol on an industrial scale is still challenging (Kumar et al., 2016; Dien et al., 2003). In this paper, biogas production was suggested as an alternative method to maximize biofuel production since 4
methane contributes to 50–70% of the biogas. A biorefinery for combination of ethanol production from solid fraction and biogas production from pre-hydrolysate was used to maximize the conversion of the available sugars in softwood pine to biofuels. Although many researches were performed on ethanol and biogas production from the solid fraction of pretreated materials; however, to our knowledge, no previous research on biogas production from the pre-hydrolysate obtained in dilute alkali pretreatment has been reported in the literature. This study was aimed at optimizing dilute sodium hydroxide pretreatment for maximized biofuel production in a biorefinery based on softwood pine. To find the optimum pretreatment conditions, total amounts of energy produced in terms of gasoline equivalent were determined and compared. Furthermore, the effects of alkali pretreatment on the wood structure were investigated. Finally, a preliminary economic assessment for the biorefinery was carried out for determination of the most economically viable pretreatment condition.
2. Material and Methods 2.1. Raw Materials Pinewood (Pinus eldarica) was obtained from the city of Delijan (Delijan, Iran, 33°59' N, 50°41' E). The wood was debarked, ground, and milled to powder. Afterwards, the milled wood was screened and sieved through 20- and 80-mesh screen to obtain particles less than 1 mm. The dry weight of pinewood powder was measured by oven drying at 105 °C (Sluiter et al., 2008). Two commercial enzymes, Cellic® CTec2 (VCNI0013) (as the main source of cellulase and βglucosidase), and Cellic® HTec2 (VHN00002) (as the source of hemicellulase and cellulase) were kindly provided by Novozymes A/S (Bagsvaerd, Denmark). The cellulase activities of the enzymes were measured to 122 and 34 filter paper units (FPUs), respectively, based on the filter paper method (Adney and Baker, 1996). A mixture of 90% Cellic® CTec2 and 10% Cellic® HTec2 was prepared and used in 5
enzymatic hydrolysis. A block flow diagram (BFD) of the procedure that presents the experiments is depicted in Fig. 1. The procedure includes pretreatment, enzymatic hydrolysis, fermentation and anaerobic digestion, which are discussed in the next sections.
2.2. Pretreatment A Pretreatment with dilute sodium hydroxide was performed in a 500 mL high pressure stainless steel batch reactor (Kayhan Steel Sanat Co., Isfahan, Iran) equipped with pressure indicator and thermometer (Amiri et al., 2010). Sodium hydroxide solutions of 0, 1, and 2% w/v were prepared and used for the pretreatment of pinewood powder at the concentration of 10 wt% wood in the final slurry. The pretreatment experiments were performed for 1, 2, and 5 h at 100, 140, and 180 °C. Afterwards, the reactor was cooled to about 70 °C in an ice chamber. The pretreated solids were separated from the liquid phase by filtration and washed with distilled water until they reached pH 7. Finally, the solid fractions were dried at room temperature for one day and stored in plastic bags. 2.3. Neutralization For biogas-producing bacteria to survive, it is desirable to decrease the alkaline pH to about neutral. Removing the Na+ ions from the slurry was also necessary, since these ions at high concentrations have an inhibitory effect on the bacterial consortia even at neutral pH. For neutralization, H3PO4 was used (3 NaOH + H3PO4 Na3PO4 + 3 H2O) because of the low solubility of Na3PO4 salt in water (5.4 g/100 mL). Furthermore, the temperature was decreased to 0 °C to further increase salt precipitation. Neutralization of pre-hydrolysates was performed by the addition of 2 M H3PO4 to obtain pH 7. Ice chamber was used to decrease the temperature of the mixture. The precipitated lignin and salts of Na3PO4 were removed by centrifugation and the liquid fractions were kept frozen until use.
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2.4. Biogas Production Anaerobic digestions of the neutralized pre-hydrolysates, were performed in batch reactors (118 mL dark glass serum bottles) at mesophilic conditions (37 °C) (Hansen et al., 2004). The inoculum was obtained from an anaerobic digester (7500 m3) operating at 37 °C in Isfahan Municipal Wastewater Treatment Plant (Isfahan, Iran). The inoculum was sieved twice to remove the particles larger than 2 mm. The percentage of total solid (TS) and volatile solid (VS) of the sieved inoculum were 5.10% ± 0.01 and of 2.52% ± 0.01, respectively. Each biogas bottle contained 20 mL of inoculum and 5 mL of prehydrolysate. Furthermore, a blank test was prepared with 20 mL of inoculum and 5 mL of distilled water in order to determine the amount of biogas produced from the inoculum alone. The bottles were closed and sealed with butyl rubber and aluminum caps. A gas mixture containing 80% nitrogen and 20% carbon dioxide was purged into the bottles for 2 min to obtain anaerobic conditions in each bottle. Pure methane and carbon dioxide gas were used for calibration. All experiments in anaerobic digestion were performed in duplicate. 2.5. Separate Enzymatic Hydrolysis and Fermentation (SHF) Enzymatic hydrolysis of the pretreated and untreated pinewood was performed in 118 mL serum glass bottles. An amount of 1.5 g of the pretreated or untreated wood was suspended in 30 ml of 50 mM sodium citrate buffer at pH 4.8. Then, the suspensions were autoclaved for 20 min at 121 °C. Next, 20 filter paper unit (FPU) of enzyme mixture (90% Cellic® CTec2 and 10% Cellic® HTec2) per gram of dry wood was added to each bottle (Satari et al., 2015). Hydrolysis was performed at 45 °C in a shaking incubator at 120 rpm for 72 h. Liquid samples were taken periodically and analyzed for sugar content by HPLC. Fermentation was performed in 118 mL bottles using a flocculating strain of Saccharomyces cerevisiae (CCUG 53310, Culture Collection of University of Gothenburg, Sweden) under anaerobic conditions. The strain maintenance on solid medium and inoculum preparation in the liquid medium was performed as 7
described by Shafiei et al. (2010). A volume of 20 mL of pre-hydrolysate was supplemented with 5 mL of required nutrients (5 g/L yeast extract, 7.5 g/L (NH4)2SO4, 3.5 g/L K2HPO4, 0.75 g/L MgSO4.7H2O, and 1.0 g/L CaCl2.2H2O). Then, pH was adjusted to 5 and the mixture was autoclaved at 121 °C for 20 min. Finally, 1 g/L S. cerevisiae was added to the cooled mixture and inoculated at 32 °C for 24 h (Shafiei et al., 2010). All experiments of hydrolysis and fermentation were performed in duplicate and the average values of the data are reported. The theoretical yield of glucose and ethanol production were calculated using the Eqs. (1–3) (Bateni et al., 2014; Ostovareh et al., 2015). % = . ×
/ ×
(1)
!"# $$%#%!$ !$ &''! /×#$ %
()ℎ+, % = . × .- ×
%&#$ / × !"# $$%#%!$ !$ &''! /×#$ %
Average Ethanol productivity (g/L.h) =
%&#$ / ./ &
(2)
(3)
In the above equations, 1.111 is the hydration factor for the conversion of glucan to glucose, and 0.51 is theoretical yield for ethanol production from glucose. Average ethanol productivity was calculated as the average ethanol production rate after 24 h of fermentation. 2.6. Analytical Methods Methods presented by Sluiter et al. (2008a, 2008b) were applied for measuring TS, VS, carbohydrate content, lignin, and ash of the untreated and pretreated pinewood. Fourier transform infrared (FTIR) spectrometer (TENSOR 27 FT-IR, Bruker, Leipzig, Germany) was employed for investigating the effects of pretreatment on the crystallinity and chemical structure of pinewood. The spectra were collected with an average of 60 scans at a range of 600–4000 cm−1. Additionally, scanning electron microscopy (SEM) was performed to quantify the crystallinity changes of the wood after pretreatment. Samples were freeze dried and coated with gold (SBC-12 ion sputter coater, KYKY, Shanghai, China) and analyzed by SEM (KYKY-EM3200, Shanghai, China) at 15 kV. 8
A high-performance liquid chromatograph (HPLC) equipped with RI detector (Jasco International Co. Tokyo, Japan) was used to measure the concentrations of the dissolved materials. Ethanol concentration was measured using an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, USA) at 60 °C using 5 mM sulfuric acid as a mobile phase at a flow rate of 0.6 mL/min. To determine the concentration of glucose, mannose, xylose, galactose, and arabinose, an ion exchange column (Aminex HPX-87P Bio-Rad Laboratories, Hercules, USA) was used at 85 °C using 0.6 mL/min deionized water as a mobile phase. Gas samples of biogas were taken with a 250 µl pressure-lock syringe (SGE Analytical Science, Waterford, Ireland). The samples were analyzed for methane and carbon dioxide content using a gas chromatograph (GC) (SP-3420A, thermal conductivity detector, Beijing Beifen Ruili Analytical Instrument Co., Beijing, China). The GC was equipped with a packed column (Porapack Q column, Chrompack, Engstingen, Germany) and a thermal detector operating at an injection temperature of 150 °C. Nitrogen at 50 °C was used as a carrier gas with 20 mL/min flow rate (Mirmohamadsadeghi et al., 2014).
2.7. Calculation of Total Biofuel Production In order to determine the optimum pretreatment conditions, the total amount of produced biofuel should be considered. Thus, the sum of gasoline equivalent volumes of methane and ethanol produced from one ton of pinewood were calculated for each process. For this calculation, lower heating values of the fuels at 25 ºC and 1 bar, which are 21.2 MJ/L for ethanol, 32.7 MJ/m3 for methane, and 32.0 MJ/L for gasoline, were considered (Shafiei et al., 2014). 2.8. Process Mass Balance and Economic Analysis Before building a commercial scale plant with a new technology, several steps including experiments in lab scale, pilot scale, and demonstration plants are performed. After completion of each step, an economic evaluation is necessary for detection of the process bottle necks. If the process is economically attractive, the design process continuous to the next step. Otherwise the process is modified and bottlenecks are 9
resolved before proceeding to the next step. In the present study, the economic assessments were conducted based on lab experiments and shows a preliminary evaluation of the economic potential. A process for production of ethanol and biogas from pinewood by dilute alkali pretreatment was proposed. The process includes units for feed handling and pretreatment, hydrolysis and fermentation, distillation and dehydration, neutralization and electrodialysis, and anaerobic and aerobic digestion (Fig. 2). The required materials and utilities such as steam, nutrients, enzyme, and NaOH for pH control were estimated based on the data presented by Khoshnevisan et al. (2016) and Shafiei et al. (2014). Preliminary calculations have shown that the process was not economically viable if NaOH was not recycled. Therefore a unit for recovery of NaOH and also acid by electrodialysis was assumed to reduce the costs of the alkali and acid. Electrodialysis is a process for exchange of salt ions through ion-exchange membranes. The ions are transferred from one solution to other solutions under influence of direct electric current between cathode and anode. Among several types of process, application of bipolar membrane electrodialysis has been widely used for recovery of sodium hydroxide, and sulfuric, hydrochloric, and nitric acid (Koter and Warszawski, 2000). The required energy for such a system was estimated to 2 kWh per kg of NaOH while the efficiency was 80%. In order to evaluate the economic viability of the pretreatments, economic potential as well as manufacturing cost of ethanol was calculated based on the following equations: Economic Potential (EP) $/ton pinewood = Revenue of products – Costs for Raw materials, chemicals and utilities (1) Ethanol Manufacturing Cost = Costs for Raw materials, chemicals and main utilities+ Other Direct Costs + Fixed charges + General Expenses
(2)
2.9. Statistical Analysis Statistical validation of results was performed by the analysis of variance (ANOVA) method and general linear model (GLM) using Minitab 17.1.0 software (Minitab Inc., State College, USA). For comparison and estimation of significant differences among the means, Tukey method with 95% 10
confidence was used. The means within the same lettered group did not have significant differences at a 5% probability level (P < 0.05).
3. Results and Discussion Pinewood was treated with dilute sodium hydroxide (1 and 2% w/v) at 100–180 °C for 1–5 h. Pretreatment by sodium hydroxide can be performed at severe (0.5–4% NaOH at high temperature) or moderate conditions (at least 6–8% NaOH and low temperature) (Karimi et al., 2013). Considering the high cost of sodium hydroxide, application of lower concentrations is economically more favorable. The untreated and treated solids were subjected to enzymatic hydrolysis for 72 h. Subsequently, fermentation under anaerobic conditions for 24 h was performed by S. cerevisiae to produce ethanol. In order to obtain the maximal conversion of carbohydrates to biofuels, anaerobic digestion of prehydrolysate from pretreatment was performed. The total amount of biofuel produced from each pretreatment was calculated as the sum of gasoline equivalent volumes of ethanol and methane. Total volume was used for comparison of the pretreatments.
3.1. Biogas Production The results of 50 days’ anaerobic digestion of the pre-hydrolysates are summarized in Fig. 3. The maximum yield of methane was 244 mL/g VS, obtained by the pretreatment at 180 °C for 1 h with 1% NaOH. Although pretreatment at higher NaOH concentration could solubilize greater amounts of carbohydrates, the results of pretreatment at 100 and 140 °C indicated that increasing the concentration of NaOH negatively affects methane production. Degradation of glucose and xylose during alkali pretreatment was reported to produce several aliphatic, cyclic enols and phenolic compounds (Forsskahl et al., 1976), which might inhibit biogas-producing bacteria. Furthermore, release 11
of acetyl groups during pretreatment as well as the remainder of the sodium ions inside the prehydrolysate could inhibit the bacteria. The presence of Na+ ions in the pre-hydrolysate solution increases the osmotic pressure of the cell wall of microorganisms, and thus kills or deactivates the cells (Deublein and Steinhauser, 2011). Increasing the alkali concentration to 2% w/v NaOH decreased the biogas production and had negative effects on the methane production. This can be related to the solubilization of more sugars at temperature of 180 °C (compared with lower temperature) and less toxic effect of inhibitor compounds at 1% NaOH (compared with 2%). Furthermore, prolongation of the pretreatment time from 1 to 5 h (at 180 °C and 1% NaOH) reduced the methane production yield. This result can be related to the decomposition of sugars at the latter phase of the pretreatment.
3.2. Enzymatic Hydrolysis Enzymatic hydrolysis was carried out for the solid fractions of the pretreated and untreated wood at 100, 140, and 180 ºC (Fig. 4). The highest glucose concentrations were obtained for the pretreatments at 180 ºC. The results show that glucose concentrations did not significantly change by increasing the pretreatment time at constant temperature and alkali concentration. However, glucose concentrations were augmented by increasing the NaOH loading from 1% to 2% w/v. Furthermore, pretreatment at higher temperature resulted in higher glucose concentration. Maximum glucose concentrations of 12.4, 21.5, and 31.0 g/L were achieved, respectively, by pretreatment at 100 °C, 140 °C, and 180 °C with 2% w/v NaOH for 5 h (Fig. 4). The optimum pretreatment conditions are based on results of hydrolysis or anaerobic digestions from treatment at 180 ºC with 2% or 1% NaOH, respectively. The glucose yields corresponding to 72 h hydrolysis of untreated wood as well as wood pretreated at 180 °C are presented in Table 1. While the glucose yield of untreated wood was only 8.6%, this yield was increased to 58.3‒70.7% and 80.7‒83.5% 12
by pretreatment with 1% and 2% w/v NaOH, respectively. The maximum glucose yield was 9.7-fold higher than the yield from the untreated pinewood.
Noori and Karimi (2016a, 2016b) investigated pretreatment of softwood pine and hardwood elm with 8% NaOH for 2 h at 0‒80 °C. They revealed that pretreatment at 0 °C resulted in maximum glucose yields of 39.2% and 79.8%, respectively. These hydrolysis yields had been enhanced by addition of Tween-20 (Sigma-Aldrich). In another study, maximum glucose yield of 55% was achieved by pretreatment of pine bark at 100 °C with 8% NaOH concentration (Salehian and Karimi, 2013). Mirahmadi et al. (2010) investigated pretreatment of softwood spruce with 7.0% NaOH solution at −10‒100 °C. They showed that maximum glucose yield of 35.7% was achieved by pretreatment at −5 °C for 2 h. In the current study, significantly higher glucose yields were achieved compared with the previous studies.
3.3. Effect of Pretreatment on Pinewood Composition The compositions of the untreated and pretreated pinewood were analyzed according to the method presented by Sluiter et al. (2008b). The composition results corresponding to the best conditions for hydrolysis or anaerobic digestion, i.e., pretreatment at 180 °C with 2% and 1% NaOH, respectively, are shown in Table 1. The untreated wood contained 48.4% glucan, 21.3% hemicellulose, and 24.7% lignin. The results showed that all hemicelluloses were removed, while acid insoluble lignin content of the samples was not significantly decreased. The glucan content rose from 65.7% to 70.2% when the pretreatment duration was increased from 1 to 5 h at constant temperature (180 °C) and 2% NaOH loading (Table 1). This increase in the glucan content compared to untreated pinewood can be explained by hemicellulose solubilization. Similar trends for the amount of glucan and hemicellulose were observed in previous studies (Mirmohamadsadeghi et al., 2014; Salehi et al., 2012).
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Lignin removal as well as increasing the swelling capacity of the lignocellulosic materials is among the reasons for enhancement of hydrolysis yield by alkali pretreatments (Taherzadeh and Karimi, K., 2008). Lignin and hemicellulose polymers protect native cellulose chains, and removal of these barriers improves the hydrolysis of cellulose (Taherzadeh and Karimi, K., 2008). Based on the results of this study, all of the xylan, galactan, mannan, and arabinan content of the wood were removed by the pretreatment. However, lignin content of the pretreated wood was only decreased by pretreatment with 2% NaOH concertation. This indicates that lignin removal occurs only at higher NaOH concentrations, e.g., (2% w/v), and hemicellulosic sugars were the main constituents removed in the current study. Nevertheless, elimination of hemicelluloses is also advantageous for improvement of enzymatic hydrolysis (Karimi et al., 2013).
3.4. Ethanol Production After enzymatic hydrolysis, the hydrolysates of the untreated and pretreated pinewood (the best pretreatments based on results of hydrolysis or anaerobic digestions) were subjected to ethanol production by S. cerevisiae under anaerobic conditions for 24 h. Results of ethanol production are presented in Table 2. All pretreatments improved the ethanol yield. The highest ethanol yields of 76.9‒78.0% were obtained by the pretreatment at 180 °C with 2% NaOH, while the yield for the untreated wood was only 5.8%. The results indicated that the pretreatment at high temperature and NaOH concentration increased the ethanol yield to a higher extent. Mirahmadi et al. (2010) showed that pretreatment with 7.0% NaOH solution for 2 h at 5 °C improved the ethanol yield of softwood spruce to 26.1%. Maximum ethanol yield of 42% was achieved by pretreatment of pine bark at 100 °C with 8.0% NaOH concentration Salehian and Karimi (2013). Simultaneous saccharication and fermentation of pinewood and elmwood which were pretreated with 8.0% (w/v) NaOH at 0 °C for 2 h resulted in ethanol yields of 32.7% and 57.3%, respectively (Noori and 14
Karimi, 2016a, 2016b). The ethanol yield achieved in this study was significantly higher than those of previous studies in which higher concentrations of NaOH and lower temperatures were used for the pretreatment. The results of this study revealed that pretreatment using dilute sodium hydroxide at temperatures over 100 °C resulted in significant improvements in ethanol and glucose yields.
3.5. Effects of Pretreatment on the Wood Structure FTIR spectroscopy was used to study the effects of the dilute NaOH pretreatment on the structure of pinewood. Sample spectra of untreated as well as pretreated wood at 180 °C with 1% w/v NaOH for 1 h and 2% w/v NaOH for 5 h. Crystallinity index (CI) and total crystallinity index (TCI) were calculated as the absorbance ratio of A1098/A898 and A1377/A2922, respectively (Table 3) (Shafiei et al., 2010). The results indicated that the treatments at high temperatures (180 °C) increased CI from 1.54 for the untreated pinewood to 1.63 and 1.74 for pretreatment with 1% w/v NaOH for 1 h and 2% w/v NaOH for 5 h, respectively. However, TCI of 1.72, corresponding to the untreated pinewood, decreased to 1.57 and 1.60 after the pretreatment with 1% w/v NaOH for 1 h and 2% w/v NaOH for 5 h, respectively. The absorption band at 1098 was assigned to cellulose I and the band at 898 cm-1 was assigned to cellulose II and amorphous cellulose. Thus, the increase in CI could be explained by the removal of amorphous celluloses (Karimi et al., 2013). Similar result of increasing crystallinity from 43% to 60% was reported by Kim and Holtzapple (2006) after alkaline pretreatment of corn stover. Decrease in TCI of pretreated pinewood by NaOH pretreatment at lower temperatures was reported by Salehian and Karimi (2013). The main reactions which occur in alkali pretreatments are: dissolution of polysaccharides; peeling-off reactions which result in cleavage of the sugar monomers and formation of alkali-stable end-groups; hydrolysis of polysaccharides via cleavage of glycosidic bonds and acetyl groups; and decomposition reactions of the dissolved polysaccharides. These reactions were well described by Karimi et al. (2013).
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The main consequence of these reactions is endwise peeling of the cellulose and hemicellulose chains, which results in release of monomeric sugars and lowers the cellulose and hemicellulose degree of polymerization (DP). Reduction in cellulose DP by alkali pretreatment is another mechanism for enhancement of enzymatic saccharification (Karimi et al., 2013; Taherzadeh and Karimi, 2008). However, direct measurement of cellulose DP is not yet possible (Hallac and Ragauskas, 2011). Hydrolytic reactions are more efficient at temperatures above 150 °C. These reactions are faster in hemicelluloses than in cellulose chains, and they may stop for cellulose chains. This stop could be due to formation of a non-reactive metasaccharinic end group or to reaching the crystalline regions of the cellulose chain (Glaus and Van Loon, 2008). Therefore, after NaOH pretreatment, hemicelluloses and amorphous celluloses are solubilized to monomeric sugars and short chain oligosaccharides, while the crystalline regions of cellulose remain intact. The results of this study are in line with the proposed mechanisms.
3.6. Gasoline Equivalent The volumes of ethanol, biogas, and gasoline equivalent based on one ton of pinewood are summarized in Table 4. From each ton of untreated pinewood, only 24.0 L of ethanol equivalent to 16 L of gasoline can be produced. The application of dilute alkali pretreatment with 1% w/v NaOH at 180 °C for 5 h resulted in the maximum amount of biofuel from pinewood, presented as gasoline equivalent volume. The amount of ethanol, biogas, and total gasoline equivalent at this condition were 268 L, 18.5 m3 (at normal condition: 25 ºC, 1 bar), and 197 L, respectively (Table 4). These values are in line with the previous works for ethanol and biogas production from pinewood reported by Bahmani et al. (2016) and Safari et al. (2016). In the study by Bahmani et al. (2016), anaerobic digestion was investigated as a pretreatment for ethanol production. They reported production of 123 L of ethanol as well as 47 m3 of methane from 1 ton of pinewood. The sum of energy which can be produced from these biofuels is 16
equivalent to energy from combustion of 130 L of gasoline. Safari et al. (2016) demonstrated that pretreatment of pinewood with 0.5% H2SO4 at 140 °C for 5 min was the optimum condition for production of ethanol from pretreated solid and biogas from pre-hydrolysate. They reported production of 101 L of ethanol and 162 m3 of methane (total, equivalent to 233 L of gasoline or 6.29 GJ) per ton of pinewood.
3.7. Process mass balance and economic analysis Mass balance for the processes were calculated based on the results of experiments as well as data provided by Shafiei et al. (2014) and Khoshnevisan et al. (2016). Main expenses including raw materials, chemicals, and utilities for the best pretreatment results were calculated and presented in Table 5. The calculations were performed based on 1 ton of pinewood. The amount of chemicals and utilities which were required in each process step were estimated based on the amount of input material to that unit operation. For instance, if the solid recovery after pretreatment was 74%, the required amount of enzyme for hydrolysis was calculated for 0.74 ton of pretreated wood which entered the hydrolysis reactor. Ethanol manufacturing cost was estimated based on the method by Peters and Timmerhaus (2003). In the equation 2, direct manufacturing costs includes the costs for operating labor, direct supervisory, maintenance and repairs as well as operating charges. The estimated values for other direct costs, fixed charges and general expenses were 0.046, 0.088 and 0.300 €/L of ethanol. The values were estimated based on the results of a techno-economic analysis for manufacturing of ethanol and biogas from pinewood by dilute acid pretreatment. The most significant expenses for the processes were expenditures for raw materials, enzyme, steam boiler, NaOH for pretreatment and acid for neutralization (Table 5). The results indicated that production of second generation of ethanol requires very cheap raw materials which can effectively enhance the manufacturing costs. Furthermore, considering the costs of chemicals, application of dilute solutions of NaOH is economically more favorable. Additionally, usage of a cheap acid such as sulfuric acid for 17
neutralization is unavoidable. It is worth to mention that the costs for acid and base would be 5 times more than the current value if electrodialysis was not applied for recovery of the chemicals. The manufacturing costs of ethanol as well as economic potentials of the proposed processes are presented in Table 6. Based on the values for economic potentials, the most promising pretreatment conditions were pretreatment with 1% NaOH for 5 and 2 h. The economic potentials for production of ethanol and biogas were 161 and 136 $/kg of pinewood, respectively; while manufacturing costs of ethanol were 0.75 and 0.79 $/kg of pinewood. In the current calculations the cost differences between pretreatment equipment were not considered, e.g., the capital cost for pretreatment reactor which operated for 2 h should have been lower than reactor cost which operates for 5 h. Therefore, the differences in the economic potentials as well as manufacturing costs of the above two processes may decrease, if the capital costs were estimated more precisely, e.g., by Aspen Economic Analyzer. The results indicate that increase in the biogas yield is essential for enhancement of the process economy. Biogas yield may increase if the inhibition effects are reduced in the anaerobic digestion.
4. Conclusions Yield and economy of ethanol production from pinewood was enhanced significantly after pretreatment with 1–2% w/v NaOH at 100–180 ºC for 1–5 h. Pretreatment enhanced the ethanol yield from the solid fraction to maximum 78%, much higher than that from the untreated wood (6%). Additionally biogas was successfully produced from the pre-hydrolysate of NaOH pretreatment, i.e., the solubilized hemicellulosic part. Preliminary economic evaluations indicated pretreatment with 1% NaOH at 180 °C for 2 and 5 h had the lowest manufacturing costs of 0.79 and 0.75 €/L. This value for the untreated wood was 4.12 €/L.
Electronic Annex
18
FTIR spectra of untreated as well as the wood pretreated at 180 °C with 1% w/v NaOH for 1 h and 2% w/v NaOH for 5 h are presented as supplementary data Fig. S1. SEM images were captured to observe the structural modifications of pinewood by pretreatment, and selected images are provided as supplementary data (Fig. S2). Untreated pinewood had a highly compact structure, while the pretreated substrate had more porous surface area for the cellulolytic enzymes. Compared with Fig. S2 a, more sheet-like in Fig. S2 b and the appearance of more wood pits holes in Fig. S2 c is clearly observable. Acknowledgements This work was financially supported by the Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology.
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Figure captions Fig. 1. BFD of the experiments conducted for ethanol and biogas production from pinewood after dilute NaOH pretreatment. Fig. 2. BFD of a process for ethanol and biogas production from pinewood by dilute NaOH pretreatment. LP and MP steam represent low pressure and medium pressure steam, respectively. Fig. 3. Accumulated methane (mL/g VS at 25 ºC, 1 bar) produced from solution pretreated at (A) 100 °C, (B) 140 °C, (C) 180 °C, after 3 (), 15 (), and 50 () days of anaerobic digestion. The letter on the top of the bars represent the results of statistical analysis. Fig. 4. Glucose concentration from enzymatic hydrolysis of pretreated at (A)100 °C, (B) 140 °C and (C) 180 °C with 0-2% w/v NaOH for 4 h (), 24 h (), and 72 h () of enzymatic hydrolysis.
Table Captions Table 1. Composition of untreated and pretreated pinewood. Table 2. Ethanol yield from untreated and pretreated pinewood. Table 3. Crystallinity and total crystallinity index of untreated and pretreated pinewood. Table 4. The volume of ethanol, biogas, and gasoline equivalents obtained at the best pretreatment conditions. Table 5. Process main expenses for biofuel production from 1 ton of pinewood. Table 6. Process main incomes and manufacturing cost for ethanol production from 1 ton of pinewood.
24
Fig. 1. NaOH
Pinewood
Enzyme
Heat
Pretreatment
Solid fraction
Hydrolysate
Neutralization
Anaerobic Digestion
Lignin, Salt
25
CO2
Fermentation
pH control
Liquid fraction H3PO4
Hydrolysis
Nutrients
Biogas
Ethanol
Fig. 2. NaOH Pinewood
Feed handling and Pretreatment
MP Steam Liquid fraction
Acid
Enzyme Nutrients
LP Steam Solid fraction
Hydrolysis and Fermentation
Recovered NaOH
Neutralization
Lignin
CO2 Vent vapor
Beer
pH control
MP Steam
NaOH and acid recovery (Electrodialysis)
Recovered Acid
Electricity
26
Distillation and Dehydration
Anaerobic Digestion
Ethanol Solid residue Stillage
Aerobic Digestion Water Biogas
Waste water
Fig. 3.
Methane Production (mL/gr.Vs)
300
200
A
A
A
100
B 0 300
Methane Production (mL/gr.Vs)
(A)
200
B
B
B
(B) AB
A AB
100
ABC
BC C
0 300
(C) Methane Production (mL/gr.Vs)
B
B
AB
A
C
C
C
AB AB
200
BCD
BC
100
CD D
0
Pretreatment Conditions
27
CD
Fig. 4.
Glucose (g/L)
30
20
C
10
D
D
BC
B
A B
BC
D
D
0
Glucose (g/L)
30
A 20
B C
10
D
D
C
B
C
D
D
0
Glucose (g/L)
30
C
BC
ABC
AB
AB
A
20
10
D
D
D D
0
Pretreatment Conditions
28
Table 1.
Pretreatment conditions
a
Glucan
Xylan
Mannan
Galactan
Arabinan
AINSLb
Ash
(%)
(%)
(%)
(%)
(%)
(%)
(%)
180 °C
1%
1h
61.7±0.4
N
N
N
N
25.0
0.20
180 °C
1%
2h
63.7±0.5
N
N
N
N
25.6
0.37
180 °C
1%
5h
67.2±0.4
N
N
N
N
28.2
0.40
180 °C
2%
1h
65.7±0.5
N
N
N
N
21.8
0.63
180 °C
2%
2h
66.1±0.6
N
N
N
N
22.8
0.83
180 °C
2%
5h
70.2±0.5
N
N
N
N
23.9
0.45
48.4±0.4
6.20±0.1
12.10±0.2
1.75
1.25
24.7
0.36
Untreated pinewood a
Pretreatment conditions are temperature, NaOH concentration, and time. Acid insoluble lignin. N = Negligible. b
29
Table 2.
Pretreatment conditions
Glucose yield (%) a
Ethanol yield (%) a
Ethanol productivity (g/L.h) a
Temperature
NaOH wt%
time
180 °C
1%
1h
58.3 ± 4.1
54.2 ± 1.5
0.376 ± 0.010
180 °C
1%
2h
67.2 ± 2.5
62.5 ± 0.6
0.448 ± 0.004
180 °C
1%
5h
70.7 ± 2.8
64.4 ± 1.5
0.487 ± 0.012
180 °C
2%
1h
81.7 ± 1.7
78.0 ± 1.1
0.576 ± 0.008
180 °C
2%
2h
80.7 ± 1.0
76.9 ± 1.9
0.572 ± 0.014
180 °C
2%
5h
83.5 ± 0.3
77.1 ± 1.9
0.609 ± 0.015
8.6 ± 0.3
5.8 ± 0.2
0.032 ± 0.005
Untreated pinewood a
The data indicate average values ± standard deviations.
30
Table 3.
Pretreatment conditions a
CI (A1098/A898)
TCI (A1377/A2922)
180 °C
1%
1h
1.63
1.57
180 °C
2%
5h
1.74
1.60
1.54
1.72
Untreated pinewood a
Pretreatment conditions are temperature, NaOH concentration, and time.
31
Table 4.
Pretreatment conditions a
Solid recovery %
Prehydrolysate VS%
180 °C, 1%, 1 h
74
180 °C, 1%, 2 h
Produced Biofuels
Energy of Biofuels Total gasoline equivalent (L)
Ethanol (L)
Methan 3 b e (m )
Ethanol (MJ)
Methane (MJ)
Total (MJ)
0.93
211
15.2
4465
497
4962
155
70
1.31
238
18.5
5037
606
5643
176
180 °C, 1%, 5 h
72
1.31
268
18.5
5683
606
6289
197
180 °C, 2%, 1 h
55
1.88
240
1.4
5097
45
5143
161
180 °C, 2%, 2 h
51
1.26
223
1.6
4719
52
4772
149
180 °C, 2%, 5 h
54
1.73
249
7.2
5279
235
5514
172
Untreated wood
100
0.00
24
0.0
509
0
509
16
a b
Pretreatment conditions are temperature, NaOH concentration, and time. at 25 ºC and 1 bar.
32
Table 5.
Costs (€/ton)
Pretreatment a
Hydrolysis and Fermentation b
Distillation and Dehydration
Pretreatment Condition
Pinewood 0.06 €/kg
NaOH (50% Soln.) 0.24 €/kg
Nutrients 0.6 €/kg
Enzyme 1.23 €/kg
MP Steam 0.008 €/kg
180 °C, 1%, 1 h
60.0
9.1
4.4
13.6
180 °C, 1%, 2 h
60.0
9.1
4.2
180 °C, 1%, 5 h
60.0
9.1
180 °C, 2%, 1 h
60.0
180 °C, 2%, 2 h
Neutralization Acid type c
ElectroDialysis
Total Expenses c $/ton
H3PO4 0.7 €/kg
H2SO4 0.2 €/kg
Electricity 0.03 $/kWh
9.43
34.4
8.4
5.7
115.1
89.1
12.9
8.93
30.6
8.4
5.7
106.9
84.6
4.3
13.4
9.27
30.6
8.4
5.7
107.6
85.4
18.2
3.3
10.2
7.03
61.2
16.7
11.4
165.2
120.8
60.0
18.2
3.1
9.5
6.56
61.2
16.7
11.4
164.0
119.6
180 °C, 2%, 5 h
60.0
18.2
3.2
10.0
6.89
61.2
16.7
11.4
159.1
114.7
Untreated
60.0
0.0
6.0
18.5
12.81
0.0
0.0
0.0
88.5
88.5
a
H3PO4
H2SO4
The costs for LP steam, MP steam and wood milling in the pretreatment and feed handling unit were 0.001, 0.01, and 0.6 €/ton of pinewood, respectively. The respective unit price were 0.004 €/kg LP steam, 0.008 €/kg MP steam and 0.03 €/kWh electricity. b The cost for NaOH solution in the hydrolysis and fermentation for controlling pH was 0.2-0.5 €/ton of pinewood. c The expenses were calculated considering neutralization process either with H3PO4 or H2SO4.
33
Table 6.
a
Economic Potential $/ton pinewooda H3PO4 H2SO4
Manufacturing cost of ethanol €/La H3PO4 H2SO4
Pretreatment Condition
Ethanol 0.85 €/L
Biogas 1 €/m3 of CH4
Solid residue 0.04 €/kg
Total income $/ton
180 °C, 1%, 1 h
179.0
15.2
7.4
194
79
105
1.08
0.86
180 °C, 1%, 2 h
202.0
18.5
7.0
220
114
136
0.96
0.79
180 °C, 1%, 5 h
227.9
18.5
7.2
246
139
161
0.91
0.75
180 °C, 2%, 1 h
204.4
1.4
5.5
206
41
85
1.24
0.94
180 °C, 2%, 2 h
189.2
1.6
5.1
191
27
71
1.30
0.97
180 °C, 2%, 5 h
211.7
7.2
5.4
219
60
104
1.19
0.90
Untreated
20.4
0.0
10.0
20
-68
-68
4.77
4.12
The expenses were calculated considering neutralization process either with H3PO4 or H2SO4.
34
Graphical Abstract SHF
Solid fraction (Cellulose) 1 ton Pinewood
Enzymatic Hydrolysis
Fermentation by S.cerevisiae
268 L Ethanol 0.75 €/L
Dilute-alkali Pretreatmen Anaerobic Digestion
Liquid fraction (Pentoses)
35
18.5 m3 Biogas
197 L Gasoline Equivalent (6.29 GJ)
Highlights: •
Dilute alkali pretreatment enhanced the yield and economy of biofuel production.
•
Biogas was produced from hemicelluloses that were not fermentable by S. cerevisiae.
•
The maximum ethanol yield from the solid fraction of pretreated pinewood was 78%.
•
The maximum total energy of 6.29 GJ/ton can be generated from pretreated pinewood.
•
Biogas yield and the economic viability were reduced at higher NaOH concentration.
36
S0960-8524(17)30300-0 http://dx.doi.org/10.1016/j.biortech.2017.03.030 BITE 17734
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
27 January 2017 3 March 2017 4 March 2017
Please cite this article as: Safari, A., Karimi, K., Shafiei, M., Dilute alkali pretreatment of softwood pine: A biorefinery approach, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.03.030
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Dilute alkali pretreatment of softwood pine: A biorefinery approach Ali Safaria, Keikhosro Karimia,b, Marzieh Shafieic,*
a
Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran
b
Industrial Biotechnology Group, Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran
c
Department of Chemical Engineering, Faculty of Engineering, University of Isfahan, Isfahan 8174673441, Iran *
Corresponding Author
Tel: +983137934558, Fax: +983137934031, E-mail address: [email protected]
1
Abstract
Dilute alkali pretreatment was performed on softwood pine to maximize ethanol and biogas production via a biorefinery approach. Alkali pretreatments were performed with 0-2% w/v NaOH at 100-180 °C for 1–5 h. The liquid fraction of the pretreated substrates was subjected to anaerobic digestion. The solid fraction of the pretreatment was used for separate enzymatic hydrolysis and fermentation. High ethanol yields of 76.9‒78.0% were achieved by pretreatment with 2% (w/v) NaOH at 180 °C. The highest biogas yield of 244 mL/g volatile solid (at 25 ºC, 1 bar) was achieved by the pretreatment with 1% (w/v) NaOH at 180 °C. The highest gasoline equivalent (sum of ethanol and methane) of 197 L per ton of pinewood and the lowest ethanol manufacturing cost of 0.75 €/L was obtained after pretreatment with 1% NaOH at 180 °C for 5 h. The manufacturing cost of ethanol from untreated wood was 4.12 €/L.
Keywords: Biorefinery; Ethanol; Biogas; Dilute Alkali Pretreatment; Softwood Pine.
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1. Introduction Ethanol and biomethane are two renewable fuels that can be used in the transportation sector. Currently, ethanol is industrially produced from sugar- or starch-based raw materials. However, due to limited resources of corn and wheat, this has led to a food vs. fuel dilemma (Noori and Karimi, 2016a; Kumar et al., 2016). Lignocellulosic materials such as agricultural and forest residues are available to a large extent at low prices. These materials have been considered non-food based feedstocks for biofuel production. However, they resist enzymatic or microbial attacks, and a pretreatment step is necessary prior to their enzymatic or biological conversion (Shafiei et al., 2015; Karimi and Taherzadeh, 2016a, 2016b). Alkaline pretreatment by sodium hydroxide is one of the most effective chemical pretreatments for ethanol production (Kim et al., 2016; Karimi et al., 2013). This pretreatment modifies cellulose structure and partially solubilizes hemicellulose and lignin, and thus increases the accessibility of lignocelluloses. Hemicellulose and lignin removal were shown to enhance the hydrolysis of lignocelluloses (Karimi et al., 2013; Hendriks et al., 2009; Menegol et al., 2016). Pretreatment by sodium hydroxide can be performed at severe (0.5–4% NaOH at high temperature) or moderate conditions (at least 6–8% NaOH and low temperature) (Karimi et al., 2013). Considering the high cost of sodium hydroxide, using lower concentrations is economically more favorable. Several NaOH pretreatments of softwoods have been studied by Mirahmadi et al. (2010), Noori and Karimi (2016a), and Salehian and Karimi (2013) with 7–8% NaOH. These studies have revealed that pretreatment with higher concentrations of NaOH was most efficient for agricultural residues and hardwood rather than for softwood of which maximum hydrolysis yields were less than 55% (Mirahmadi et al., 2010; Salehian and Karimi, 2013). Zhao et al. (2008) have investigated presoaking spruce with 12% urea and 3% NaOH. Pretreatment of the presoaked wood with 3% NaOH for 24 h at −15 °C yielded 60% glucose. To our knowledge, no previous investigation has been reported on pretreatment of softwood with dilute NaOH at high temperatures. Additionally, previous studies which were preformed at higher alkaline 3
concentrations or lower temperatures did not result in high saccharification yields. Thus, the first aim of this study was to investigate dilute alkali pretreatment of softwood pine at elevated temperatures. After completion of the pretreatment by NaOH, a slurry containing solid and liquid fractions is produced. The solid fraction is filtrated from the liquid fraction (called pre-hydrolysate) and enzymatically hydrolyzed for ethanol fermentation. However, a portion of carbohydrates which are recovered in the pre-hydrolysate are not fermentable by ordinary ethanolic microorganisms, e.g., Saccharomyces cerevisiae (Shafiei et al., 2015; FazeliNejad et al., 2016). In order to have an economically viable process, the pre-hydrolysate portion should be converted to byproducts or products, such as ethanol, methane, or biomass (FazeliNejad et al., 2016; McMillan et al., 1999; Dien et al., 2003). Pre-hydrolysate contains solubilized monomer or short chain carbohydrates. The carbohydrates are mainly solubilized hemicelluloses which have been released during the pretreatment (Karimi et al., 2013). For the conversion of both pentoses and hexoses to ethanol, separate hydrolysis and co-fermentation (SHCF) or simultaneous saccharification and co-fermentation (SSCF) was suggested (McMillan et al., 1999; Dien et al., 2003). Xylose assimilation in the former process suffers from glucose and ethanol inhibition (Taherzadeh and Karimi, 2011; Jin et al., 2012). SSCF process can be performed by two different or one recombinant microorganism (Kumar et al., 2016; Talebnia, 2015). In SSCF, glucose inhibition is reduced; however, ethanol yield in SSCF from both glucose and xylose is considerably lower than that formed from glucose by ordinary yeasts (e.g., Saccharomyces cerevisiae) (McMillan et al., 1999; Koppram et al., 2013). Furthermore, differences in the optimal fermentation condition of the two strains, e.g., pH and temperature, and the challenges of using genetically modified organisms, i.e., safety issues and gene instabilities, are practical bottlenecks (Ho et al., 2001; Dien et al., 2000). Therefore, simultaneous conversion of both hexoses and pentoses to ethanol on an industrial scale is still challenging (Kumar et al., 2016; Dien et al., 2003). In this paper, biogas production was suggested as an alternative method to maximize biofuel production since 4
methane contributes to 50–70% of the biogas. A biorefinery for combination of ethanol production from solid fraction and biogas production from pre-hydrolysate was used to maximize the conversion of the available sugars in softwood pine to biofuels. Although many researches were performed on ethanol and biogas production from the solid fraction of pretreated materials; however, to our knowledge, no previous research on biogas production from the pre-hydrolysate obtained in dilute alkali pretreatment has been reported in the literature. This study was aimed at optimizing dilute sodium hydroxide pretreatment for maximized biofuel production in a biorefinery based on softwood pine. To find the optimum pretreatment conditions, total amounts of energy produced in terms of gasoline equivalent were determined and compared. Furthermore, the effects of alkali pretreatment on the wood structure were investigated. Finally, a preliminary economic assessment for the biorefinery was carried out for determination of the most economically viable pretreatment condition.
2. Material and Methods 2.1. Raw Materials Pinewood (Pinus eldarica) was obtained from the city of Delijan (Delijan, Iran, 33°59' N, 50°41' E). The wood was debarked, ground, and milled to powder. Afterwards, the milled wood was screened and sieved through 20- and 80-mesh screen to obtain particles less than 1 mm. The dry weight of pinewood powder was measured by oven drying at 105 °C (Sluiter et al., 2008). Two commercial enzymes, Cellic® CTec2 (VCNI0013) (as the main source of cellulase and βglucosidase), and Cellic® HTec2 (VHN00002) (as the source of hemicellulase and cellulase) were kindly provided by Novozymes A/S (Bagsvaerd, Denmark). The cellulase activities of the enzymes were measured to 122 and 34 filter paper units (FPUs), respectively, based on the filter paper method (Adney and Baker, 1996). A mixture of 90% Cellic® CTec2 and 10% Cellic® HTec2 was prepared and used in 5
enzymatic hydrolysis. A block flow diagram (BFD) of the procedure that presents the experiments is depicted in Fig. 1. The procedure includes pretreatment, enzymatic hydrolysis, fermentation and anaerobic digestion, which are discussed in the next sections.
2.2. Pretreatment A Pretreatment with dilute sodium hydroxide was performed in a 500 mL high pressure stainless steel batch reactor (Kayhan Steel Sanat Co., Isfahan, Iran) equipped with pressure indicator and thermometer (Amiri et al., 2010). Sodium hydroxide solutions of 0, 1, and 2% w/v were prepared and used for the pretreatment of pinewood powder at the concentration of 10 wt% wood in the final slurry. The pretreatment experiments were performed for 1, 2, and 5 h at 100, 140, and 180 °C. Afterwards, the reactor was cooled to about 70 °C in an ice chamber. The pretreated solids were separated from the liquid phase by filtration and washed with distilled water until they reached pH 7. Finally, the solid fractions were dried at room temperature for one day and stored in plastic bags. 2.3. Neutralization For biogas-producing bacteria to survive, it is desirable to decrease the alkaline pH to about neutral. Removing the Na+ ions from the slurry was also necessary, since these ions at high concentrations have an inhibitory effect on the bacterial consortia even at neutral pH. For neutralization, H3PO4 was used (3 NaOH + H3PO4 Na3PO4 + 3 H2O) because of the low solubility of Na3PO4 salt in water (5.4 g/100 mL). Furthermore, the temperature was decreased to 0 °C to further increase salt precipitation. Neutralization of pre-hydrolysates was performed by the addition of 2 M H3PO4 to obtain pH 7. Ice chamber was used to decrease the temperature of the mixture. The precipitated lignin and salts of Na3PO4 were removed by centrifugation and the liquid fractions were kept frozen until use.
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2.4. Biogas Production Anaerobic digestions of the neutralized pre-hydrolysates, were performed in batch reactors (118 mL dark glass serum bottles) at mesophilic conditions (37 °C) (Hansen et al., 2004). The inoculum was obtained from an anaerobic digester (7500 m3) operating at 37 °C in Isfahan Municipal Wastewater Treatment Plant (Isfahan, Iran). The inoculum was sieved twice to remove the particles larger than 2 mm. The percentage of total solid (TS) and volatile solid (VS) of the sieved inoculum were 5.10% ± 0.01 and of 2.52% ± 0.01, respectively. Each biogas bottle contained 20 mL of inoculum and 5 mL of prehydrolysate. Furthermore, a blank test was prepared with 20 mL of inoculum and 5 mL of distilled water in order to determine the amount of biogas produced from the inoculum alone. The bottles were closed and sealed with butyl rubber and aluminum caps. A gas mixture containing 80% nitrogen and 20% carbon dioxide was purged into the bottles for 2 min to obtain anaerobic conditions in each bottle. Pure methane and carbon dioxide gas were used for calibration. All experiments in anaerobic digestion were performed in duplicate. 2.5. Separate Enzymatic Hydrolysis and Fermentation (SHF) Enzymatic hydrolysis of the pretreated and untreated pinewood was performed in 118 mL serum glass bottles. An amount of 1.5 g of the pretreated or untreated wood was suspended in 30 ml of 50 mM sodium citrate buffer at pH 4.8. Then, the suspensions were autoclaved for 20 min at 121 °C. Next, 20 filter paper unit (FPU) of enzyme mixture (90% Cellic® CTec2 and 10% Cellic® HTec2) per gram of dry wood was added to each bottle (Satari et al., 2015). Hydrolysis was performed at 45 °C in a shaking incubator at 120 rpm for 72 h. Liquid samples were taken periodically and analyzed for sugar content by HPLC. Fermentation was performed in 118 mL bottles using a flocculating strain of Saccharomyces cerevisiae (CCUG 53310, Culture Collection of University of Gothenburg, Sweden) under anaerobic conditions. The strain maintenance on solid medium and inoculum preparation in the liquid medium was performed as 7
described by Shafiei et al. (2010). A volume of 20 mL of pre-hydrolysate was supplemented with 5 mL of required nutrients (5 g/L yeast extract, 7.5 g/L (NH4)2SO4, 3.5 g/L K2HPO4, 0.75 g/L MgSO4.7H2O, and 1.0 g/L CaCl2.2H2O). Then, pH was adjusted to 5 and the mixture was autoclaved at 121 °C for 20 min. Finally, 1 g/L S. cerevisiae was added to the cooled mixture and inoculated at 32 °C for 24 h (Shafiei et al., 2010). All experiments of hydrolysis and fermentation were performed in duplicate and the average values of the data are reported. The theoretical yield of glucose and ethanol production were calculated using the Eqs. (1–3) (Bateni et al., 2014; Ostovareh et al., 2015). % = . ×
/ ×
(1)
!"# $$%#%!$ !$ &''! /×#$ %
()ℎ+, % = . × .- ×
%&#$ / × !"# $$%#%!$ !$ &''! /×#$ %
Average Ethanol productivity (g/L.h) =
%&#$ / ./ &
(2)
(3)
In the above equations, 1.111 is the hydration factor for the conversion of glucan to glucose, and 0.51 is theoretical yield for ethanol production from glucose. Average ethanol productivity was calculated as the average ethanol production rate after 24 h of fermentation. 2.6. Analytical Methods Methods presented by Sluiter et al. (2008a, 2008b) were applied for measuring TS, VS, carbohydrate content, lignin, and ash of the untreated and pretreated pinewood. Fourier transform infrared (FTIR) spectrometer (TENSOR 27 FT-IR, Bruker, Leipzig, Germany) was employed for investigating the effects of pretreatment on the crystallinity and chemical structure of pinewood. The spectra were collected with an average of 60 scans at a range of 600–4000 cm−1. Additionally, scanning electron microscopy (SEM) was performed to quantify the crystallinity changes of the wood after pretreatment. Samples were freeze dried and coated with gold (SBC-12 ion sputter coater, KYKY, Shanghai, China) and analyzed by SEM (KYKY-EM3200, Shanghai, China) at 15 kV. 8
A high-performance liquid chromatograph (HPLC) equipped with RI detector (Jasco International Co. Tokyo, Japan) was used to measure the concentrations of the dissolved materials. Ethanol concentration was measured using an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, USA) at 60 °C using 5 mM sulfuric acid as a mobile phase at a flow rate of 0.6 mL/min. To determine the concentration of glucose, mannose, xylose, galactose, and arabinose, an ion exchange column (Aminex HPX-87P Bio-Rad Laboratories, Hercules, USA) was used at 85 °C using 0.6 mL/min deionized water as a mobile phase. Gas samples of biogas were taken with a 250 µl pressure-lock syringe (SGE Analytical Science, Waterford, Ireland). The samples were analyzed for methane and carbon dioxide content using a gas chromatograph (GC) (SP-3420A, thermal conductivity detector, Beijing Beifen Ruili Analytical Instrument Co., Beijing, China). The GC was equipped with a packed column (Porapack Q column, Chrompack, Engstingen, Germany) and a thermal detector operating at an injection temperature of 150 °C. Nitrogen at 50 °C was used as a carrier gas with 20 mL/min flow rate (Mirmohamadsadeghi et al., 2014).
2.7. Calculation of Total Biofuel Production In order to determine the optimum pretreatment conditions, the total amount of produced biofuel should be considered. Thus, the sum of gasoline equivalent volumes of methane and ethanol produced from one ton of pinewood were calculated for each process. For this calculation, lower heating values of the fuels at 25 ºC and 1 bar, which are 21.2 MJ/L for ethanol, 32.7 MJ/m3 for methane, and 32.0 MJ/L for gasoline, were considered (Shafiei et al., 2014). 2.8. Process Mass Balance and Economic Analysis Before building a commercial scale plant with a new technology, several steps including experiments in lab scale, pilot scale, and demonstration plants are performed. After completion of each step, an economic evaluation is necessary for detection of the process bottle necks. If the process is economically attractive, the design process continuous to the next step. Otherwise the process is modified and bottlenecks are 9
resolved before proceeding to the next step. In the present study, the economic assessments were conducted based on lab experiments and shows a preliminary evaluation of the economic potential. A process for production of ethanol and biogas from pinewood by dilute alkali pretreatment was proposed. The process includes units for feed handling and pretreatment, hydrolysis and fermentation, distillation and dehydration, neutralization and electrodialysis, and anaerobic and aerobic digestion (Fig. 2). The required materials and utilities such as steam, nutrients, enzyme, and NaOH for pH control were estimated based on the data presented by Khoshnevisan et al. (2016) and Shafiei et al. (2014). Preliminary calculations have shown that the process was not economically viable if NaOH was not recycled. Therefore a unit for recovery of NaOH and also acid by electrodialysis was assumed to reduce the costs of the alkali and acid. Electrodialysis is a process for exchange of salt ions through ion-exchange membranes. The ions are transferred from one solution to other solutions under influence of direct electric current between cathode and anode. Among several types of process, application of bipolar membrane electrodialysis has been widely used for recovery of sodium hydroxide, and sulfuric, hydrochloric, and nitric acid (Koter and Warszawski, 2000). The required energy for such a system was estimated to 2 kWh per kg of NaOH while the efficiency was 80%. In order to evaluate the economic viability of the pretreatments, economic potential as well as manufacturing cost of ethanol was calculated based on the following equations: Economic Potential (EP) $/ton pinewood = Revenue of products – Costs for Raw materials, chemicals and utilities (1) Ethanol Manufacturing Cost = Costs for Raw materials, chemicals and main utilities+ Other Direct Costs + Fixed charges + General Expenses
(2)
2.9. Statistical Analysis Statistical validation of results was performed by the analysis of variance (ANOVA) method and general linear model (GLM) using Minitab 17.1.0 software (Minitab Inc., State College, USA). For comparison and estimation of significant differences among the means, Tukey method with 95% 10
confidence was used. The means within the same lettered group did not have significant differences at a 5% probability level (P < 0.05).
3. Results and Discussion Pinewood was treated with dilute sodium hydroxide (1 and 2% w/v) at 100–180 °C for 1–5 h. Pretreatment by sodium hydroxide can be performed at severe (0.5–4% NaOH at high temperature) or moderate conditions (at least 6–8% NaOH and low temperature) (Karimi et al., 2013). Considering the high cost of sodium hydroxide, application of lower concentrations is economically more favorable. The untreated and treated solids were subjected to enzymatic hydrolysis for 72 h. Subsequently, fermentation under anaerobic conditions for 24 h was performed by S. cerevisiae to produce ethanol. In order to obtain the maximal conversion of carbohydrates to biofuels, anaerobic digestion of prehydrolysate from pretreatment was performed. The total amount of biofuel produced from each pretreatment was calculated as the sum of gasoline equivalent volumes of ethanol and methane. Total volume was used for comparison of the pretreatments.
3.1. Biogas Production The results of 50 days’ anaerobic digestion of the pre-hydrolysates are summarized in Fig. 3. The maximum yield of methane was 244 mL/g VS, obtained by the pretreatment at 180 °C for 1 h with 1% NaOH. Although pretreatment at higher NaOH concentration could solubilize greater amounts of carbohydrates, the results of pretreatment at 100 and 140 °C indicated that increasing the concentration of NaOH negatively affects methane production. Degradation of glucose and xylose during alkali pretreatment was reported to produce several aliphatic, cyclic enols and phenolic compounds (Forsskahl et al., 1976), which might inhibit biogas-producing bacteria. Furthermore, release 11
of acetyl groups during pretreatment as well as the remainder of the sodium ions inside the prehydrolysate could inhibit the bacteria. The presence of Na+ ions in the pre-hydrolysate solution increases the osmotic pressure of the cell wall of microorganisms, and thus kills or deactivates the cells (Deublein and Steinhauser, 2011). Increasing the alkali concentration to 2% w/v NaOH decreased the biogas production and had negative effects on the methane production. This can be related to the solubilization of more sugars at temperature of 180 °C (compared with lower temperature) and less toxic effect of inhibitor compounds at 1% NaOH (compared with 2%). Furthermore, prolongation of the pretreatment time from 1 to 5 h (at 180 °C and 1% NaOH) reduced the methane production yield. This result can be related to the decomposition of sugars at the latter phase of the pretreatment.
3.2. Enzymatic Hydrolysis Enzymatic hydrolysis was carried out for the solid fractions of the pretreated and untreated wood at 100, 140, and 180 ºC (Fig. 4). The highest glucose concentrations were obtained for the pretreatments at 180 ºC. The results show that glucose concentrations did not significantly change by increasing the pretreatment time at constant temperature and alkali concentration. However, glucose concentrations were augmented by increasing the NaOH loading from 1% to 2% w/v. Furthermore, pretreatment at higher temperature resulted in higher glucose concentration. Maximum glucose concentrations of 12.4, 21.5, and 31.0 g/L were achieved, respectively, by pretreatment at 100 °C, 140 °C, and 180 °C with 2% w/v NaOH for 5 h (Fig. 4). The optimum pretreatment conditions are based on results of hydrolysis or anaerobic digestions from treatment at 180 ºC with 2% or 1% NaOH, respectively. The glucose yields corresponding to 72 h hydrolysis of untreated wood as well as wood pretreated at 180 °C are presented in Table 1. While the glucose yield of untreated wood was only 8.6%, this yield was increased to 58.3‒70.7% and 80.7‒83.5% 12
by pretreatment with 1% and 2% w/v NaOH, respectively. The maximum glucose yield was 9.7-fold higher than the yield from the untreated pinewood.
Noori and Karimi (2016a, 2016b) investigated pretreatment of softwood pine and hardwood elm with 8% NaOH for 2 h at 0‒80 °C. They revealed that pretreatment at 0 °C resulted in maximum glucose yields of 39.2% and 79.8%, respectively. These hydrolysis yields had been enhanced by addition of Tween-20 (Sigma-Aldrich). In another study, maximum glucose yield of 55% was achieved by pretreatment of pine bark at 100 °C with 8% NaOH concentration (Salehian and Karimi, 2013). Mirahmadi et al. (2010) investigated pretreatment of softwood spruce with 7.0% NaOH solution at −10‒100 °C. They showed that maximum glucose yield of 35.7% was achieved by pretreatment at −5 °C for 2 h. In the current study, significantly higher glucose yields were achieved compared with the previous studies.
3.3. Effect of Pretreatment on Pinewood Composition The compositions of the untreated and pretreated pinewood were analyzed according to the method presented by Sluiter et al. (2008b). The composition results corresponding to the best conditions for hydrolysis or anaerobic digestion, i.e., pretreatment at 180 °C with 2% and 1% NaOH, respectively, are shown in Table 1. The untreated wood contained 48.4% glucan, 21.3% hemicellulose, and 24.7% lignin. The results showed that all hemicelluloses were removed, while acid insoluble lignin content of the samples was not significantly decreased. The glucan content rose from 65.7% to 70.2% when the pretreatment duration was increased from 1 to 5 h at constant temperature (180 °C) and 2% NaOH loading (Table 1). This increase in the glucan content compared to untreated pinewood can be explained by hemicellulose solubilization. Similar trends for the amount of glucan and hemicellulose were observed in previous studies (Mirmohamadsadeghi et al., 2014; Salehi et al., 2012).
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Lignin removal as well as increasing the swelling capacity of the lignocellulosic materials is among the reasons for enhancement of hydrolysis yield by alkali pretreatments (Taherzadeh and Karimi, K., 2008). Lignin and hemicellulose polymers protect native cellulose chains, and removal of these barriers improves the hydrolysis of cellulose (Taherzadeh and Karimi, K., 2008). Based on the results of this study, all of the xylan, galactan, mannan, and arabinan content of the wood were removed by the pretreatment. However, lignin content of the pretreated wood was only decreased by pretreatment with 2% NaOH concertation. This indicates that lignin removal occurs only at higher NaOH concentrations, e.g., (2% w/v), and hemicellulosic sugars were the main constituents removed in the current study. Nevertheless, elimination of hemicelluloses is also advantageous for improvement of enzymatic hydrolysis (Karimi et al., 2013).
3.4. Ethanol Production After enzymatic hydrolysis, the hydrolysates of the untreated and pretreated pinewood (the best pretreatments based on results of hydrolysis or anaerobic digestions) were subjected to ethanol production by S. cerevisiae under anaerobic conditions for 24 h. Results of ethanol production are presented in Table 2. All pretreatments improved the ethanol yield. The highest ethanol yields of 76.9‒78.0% were obtained by the pretreatment at 180 °C with 2% NaOH, while the yield for the untreated wood was only 5.8%. The results indicated that the pretreatment at high temperature and NaOH concentration increased the ethanol yield to a higher extent. Mirahmadi et al. (2010) showed that pretreatment with 7.0% NaOH solution for 2 h at 5 °C improved the ethanol yield of softwood spruce to 26.1%. Maximum ethanol yield of 42% was achieved by pretreatment of pine bark at 100 °C with 8.0% NaOH concentration Salehian and Karimi (2013). Simultaneous saccharication and fermentation of pinewood and elmwood which were pretreated with 8.0% (w/v) NaOH at 0 °C for 2 h resulted in ethanol yields of 32.7% and 57.3%, respectively (Noori and 14
Karimi, 2016a, 2016b). The ethanol yield achieved in this study was significantly higher than those of previous studies in which higher concentrations of NaOH and lower temperatures were used for the pretreatment. The results of this study revealed that pretreatment using dilute sodium hydroxide at temperatures over 100 °C resulted in significant improvements in ethanol and glucose yields.
3.5. Effects of Pretreatment on the Wood Structure FTIR spectroscopy was used to study the effects of the dilute NaOH pretreatment on the structure of pinewood. Sample spectra of untreated as well as pretreated wood at 180 °C with 1% w/v NaOH for 1 h and 2% w/v NaOH for 5 h. Crystallinity index (CI) and total crystallinity index (TCI) were calculated as the absorbance ratio of A1098/A898 and A1377/A2922, respectively (Table 3) (Shafiei et al., 2010). The results indicated that the treatments at high temperatures (180 °C) increased CI from 1.54 for the untreated pinewood to 1.63 and 1.74 for pretreatment with 1% w/v NaOH for 1 h and 2% w/v NaOH for 5 h, respectively. However, TCI of 1.72, corresponding to the untreated pinewood, decreased to 1.57 and 1.60 after the pretreatment with 1% w/v NaOH for 1 h and 2% w/v NaOH for 5 h, respectively. The absorption band at 1098 was assigned to cellulose I and the band at 898 cm-1 was assigned to cellulose II and amorphous cellulose. Thus, the increase in CI could be explained by the removal of amorphous celluloses (Karimi et al., 2013). Similar result of increasing crystallinity from 43% to 60% was reported by Kim and Holtzapple (2006) after alkaline pretreatment of corn stover. Decrease in TCI of pretreated pinewood by NaOH pretreatment at lower temperatures was reported by Salehian and Karimi (2013). The main reactions which occur in alkali pretreatments are: dissolution of polysaccharides; peeling-off reactions which result in cleavage of the sugar monomers and formation of alkali-stable end-groups; hydrolysis of polysaccharides via cleavage of glycosidic bonds and acetyl groups; and decomposition reactions of the dissolved polysaccharides. These reactions were well described by Karimi et al. (2013).
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The main consequence of these reactions is endwise peeling of the cellulose and hemicellulose chains, which results in release of monomeric sugars and lowers the cellulose and hemicellulose degree of polymerization (DP). Reduction in cellulose DP by alkali pretreatment is another mechanism for enhancement of enzymatic saccharification (Karimi et al., 2013; Taherzadeh and Karimi, 2008). However, direct measurement of cellulose DP is not yet possible (Hallac and Ragauskas, 2011). Hydrolytic reactions are more efficient at temperatures above 150 °C. These reactions are faster in hemicelluloses than in cellulose chains, and they may stop for cellulose chains. This stop could be due to formation of a non-reactive metasaccharinic end group or to reaching the crystalline regions of the cellulose chain (Glaus and Van Loon, 2008). Therefore, after NaOH pretreatment, hemicelluloses and amorphous celluloses are solubilized to monomeric sugars and short chain oligosaccharides, while the crystalline regions of cellulose remain intact. The results of this study are in line with the proposed mechanisms.
3.6. Gasoline Equivalent The volumes of ethanol, biogas, and gasoline equivalent based on one ton of pinewood are summarized in Table 4. From each ton of untreated pinewood, only 24.0 L of ethanol equivalent to 16 L of gasoline can be produced. The application of dilute alkali pretreatment with 1% w/v NaOH at 180 °C for 5 h resulted in the maximum amount of biofuel from pinewood, presented as gasoline equivalent volume. The amount of ethanol, biogas, and total gasoline equivalent at this condition were 268 L, 18.5 m3 (at normal condition: 25 ºC, 1 bar), and 197 L, respectively (Table 4). These values are in line with the previous works for ethanol and biogas production from pinewood reported by Bahmani et al. (2016) and Safari et al. (2016). In the study by Bahmani et al. (2016), anaerobic digestion was investigated as a pretreatment for ethanol production. They reported production of 123 L of ethanol as well as 47 m3 of methane from 1 ton of pinewood. The sum of energy which can be produced from these biofuels is 16
equivalent to energy from combustion of 130 L of gasoline. Safari et al. (2016) demonstrated that pretreatment of pinewood with 0.5% H2SO4 at 140 °C for 5 min was the optimum condition for production of ethanol from pretreated solid and biogas from pre-hydrolysate. They reported production of 101 L of ethanol and 162 m3 of methane (total, equivalent to 233 L of gasoline or 6.29 GJ) per ton of pinewood.
3.7. Process mass balance and economic analysis Mass balance for the processes were calculated based on the results of experiments as well as data provided by Shafiei et al. (2014) and Khoshnevisan et al. (2016). Main expenses including raw materials, chemicals, and utilities for the best pretreatment results were calculated and presented in Table 5. The calculations were performed based on 1 ton of pinewood. The amount of chemicals and utilities which were required in each process step were estimated based on the amount of input material to that unit operation. For instance, if the solid recovery after pretreatment was 74%, the required amount of enzyme for hydrolysis was calculated for 0.74 ton of pretreated wood which entered the hydrolysis reactor. Ethanol manufacturing cost was estimated based on the method by Peters and Timmerhaus (2003). In the equation 2, direct manufacturing costs includes the costs for operating labor, direct supervisory, maintenance and repairs as well as operating charges. The estimated values for other direct costs, fixed charges and general expenses were 0.046, 0.088 and 0.300 €/L of ethanol. The values were estimated based on the results of a techno-economic analysis for manufacturing of ethanol and biogas from pinewood by dilute acid pretreatment. The most significant expenses for the processes were expenditures for raw materials, enzyme, steam boiler, NaOH for pretreatment and acid for neutralization (Table 5). The results indicated that production of second generation of ethanol requires very cheap raw materials which can effectively enhance the manufacturing costs. Furthermore, considering the costs of chemicals, application of dilute solutions of NaOH is economically more favorable. Additionally, usage of a cheap acid such as sulfuric acid for 17
neutralization is unavoidable. It is worth to mention that the costs for acid and base would be 5 times more than the current value if electrodialysis was not applied for recovery of the chemicals. The manufacturing costs of ethanol as well as economic potentials of the proposed processes are presented in Table 6. Based on the values for economic potentials, the most promising pretreatment conditions were pretreatment with 1% NaOH for 5 and 2 h. The economic potentials for production of ethanol and biogas were 161 and 136 $/kg of pinewood, respectively; while manufacturing costs of ethanol were 0.75 and 0.79 $/kg of pinewood. In the current calculations the cost differences between pretreatment equipment were not considered, e.g., the capital cost for pretreatment reactor which operated for 2 h should have been lower than reactor cost which operates for 5 h. Therefore, the differences in the economic potentials as well as manufacturing costs of the above two processes may decrease, if the capital costs were estimated more precisely, e.g., by Aspen Economic Analyzer. The results indicate that increase in the biogas yield is essential for enhancement of the process economy. Biogas yield may increase if the inhibition effects are reduced in the anaerobic digestion.
4. Conclusions Yield and economy of ethanol production from pinewood was enhanced significantly after pretreatment with 1–2% w/v NaOH at 100–180 ºC for 1–5 h. Pretreatment enhanced the ethanol yield from the solid fraction to maximum 78%, much higher than that from the untreated wood (6%). Additionally biogas was successfully produced from the pre-hydrolysate of NaOH pretreatment, i.e., the solubilized hemicellulosic part. Preliminary economic evaluations indicated pretreatment with 1% NaOH at 180 °C for 2 and 5 h had the lowest manufacturing costs of 0.79 and 0.75 €/L. This value for the untreated wood was 4.12 €/L.
Electronic Annex
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FTIR spectra of untreated as well as the wood pretreated at 180 °C with 1% w/v NaOH for 1 h and 2% w/v NaOH for 5 h are presented as supplementary data Fig. S1. SEM images were captured to observe the structural modifications of pinewood by pretreatment, and selected images are provided as supplementary data (Fig. S2). Untreated pinewood had a highly compact structure, while the pretreated substrate had more porous surface area for the cellulolytic enzymes. Compared with Fig. S2 a, more sheet-like in Fig. S2 b and the appearance of more wood pits holes in Fig. S2 c is clearly observable. Acknowledgements This work was financially supported by the Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology.
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Figure captions Fig. 1. BFD of the experiments conducted for ethanol and biogas production from pinewood after dilute NaOH pretreatment. Fig. 2. BFD of a process for ethanol and biogas production from pinewood by dilute NaOH pretreatment. LP and MP steam represent low pressure and medium pressure steam, respectively. Fig. 3. Accumulated methane (mL/g VS at 25 ºC, 1 bar) produced from solution pretreated at (A) 100 °C, (B) 140 °C, (C) 180 °C, after 3 (), 15 (), and 50 () days of anaerobic digestion. The letter on the top of the bars represent the results of statistical analysis. Fig. 4. Glucose concentration from enzymatic hydrolysis of pretreated at (A)100 °C, (B) 140 °C and (C) 180 °C with 0-2% w/v NaOH for 4 h (), 24 h (), and 72 h () of enzymatic hydrolysis.
Table Captions Table 1. Composition of untreated and pretreated pinewood. Table 2. Ethanol yield from untreated and pretreated pinewood. Table 3. Crystallinity and total crystallinity index of untreated and pretreated pinewood. Table 4. The volume of ethanol, biogas, and gasoline equivalents obtained at the best pretreatment conditions. Table 5. Process main expenses for biofuel production from 1 ton of pinewood. Table 6. Process main incomes and manufacturing cost for ethanol production from 1 ton of pinewood.
24
Fig. 1. NaOH
Pinewood
Enzyme
Heat
Pretreatment
Solid fraction
Hydrolysate
Neutralization
Anaerobic Digestion
Lignin, Salt
25
CO2
Fermentation
pH control
Liquid fraction H3PO4
Hydrolysis
Nutrients
Biogas
Ethanol
Fig. 2. NaOH Pinewood
Feed handling and Pretreatment
MP Steam Liquid fraction
Acid
Enzyme Nutrients
LP Steam Solid fraction
Hydrolysis and Fermentation
Recovered NaOH
Neutralization
Lignin
CO2 Vent vapor
Beer
pH control
MP Steam
NaOH and acid recovery (Electrodialysis)
Recovered Acid
Electricity
26
Distillation and Dehydration
Anaerobic Digestion
Ethanol Solid residue Stillage
Aerobic Digestion Water Biogas
Waste water
Fig. 3.
Methane Production (mL/gr.Vs)
300
200
A
A
A
100
B 0 300
Methane Production (mL/gr.Vs)
(A)
200
B
B
B
(B) AB
A AB
100
ABC
BC C
0 300
(C) Methane Production (mL/gr.Vs)
B
B
AB
A
C
C
C
AB AB
200
BCD
BC
100
CD D
0
Pretreatment Conditions
27
CD
Fig. 4.
Glucose (g/L)
30
20
C
10
D
D
BC
B
A B
BC
D
D
0
Glucose (g/L)
30
A 20
B C
10
D
D
C
B
C
D
D
0
Glucose (g/L)
30
C
BC
ABC
AB
AB
A
20
10
D
D
D D
0
Pretreatment Conditions
28
Table 1.
Pretreatment conditions
a
Glucan
Xylan
Mannan
Galactan
Arabinan
AINSLb
Ash
(%)
(%)
(%)
(%)
(%)
(%)
(%)
180 °C
1%
1h
61.7±0.4
N
N
N
N
25.0
0.20
180 °C
1%
2h
63.7±0.5
N
N
N
N
25.6
0.37
180 °C
1%
5h
67.2±0.4
N
N
N
N
28.2
0.40
180 °C
2%
1h
65.7±0.5
N
N
N
N
21.8
0.63
180 °C
2%
2h
66.1±0.6
N
N
N
N
22.8
0.83
180 °C
2%
5h
70.2±0.5
N
N
N
N
23.9
0.45
48.4±0.4
6.20±0.1
12.10±0.2
1.75
1.25
24.7
0.36
Untreated pinewood a
Pretreatment conditions are temperature, NaOH concentration, and time. Acid insoluble lignin. N = Negligible. b
29
Table 2.
Pretreatment conditions
Glucose yield (%) a
Ethanol yield (%) a
Ethanol productivity (g/L.h) a
Temperature
NaOH wt%
time
180 °C
1%
1h
58.3 ± 4.1
54.2 ± 1.5
0.376 ± 0.010
180 °C
1%
2h
67.2 ± 2.5
62.5 ± 0.6
0.448 ± 0.004
180 °C
1%
5h
70.7 ± 2.8
64.4 ± 1.5
0.487 ± 0.012
180 °C
2%
1h
81.7 ± 1.7
78.0 ± 1.1
0.576 ± 0.008
180 °C
2%
2h
80.7 ± 1.0
76.9 ± 1.9
0.572 ± 0.014
180 °C
2%
5h
83.5 ± 0.3
77.1 ± 1.9
0.609 ± 0.015
8.6 ± 0.3
5.8 ± 0.2
0.032 ± 0.005
Untreated pinewood a
The data indicate average values ± standard deviations.
30
Table 3.
Pretreatment conditions a
CI (A1098/A898)
TCI (A1377/A2922)
180 °C
1%
1h
1.63
1.57
180 °C
2%
5h
1.74
1.60
1.54
1.72
Untreated pinewood a
Pretreatment conditions are temperature, NaOH concentration, and time.
31
Table 4.
Pretreatment conditions a
Solid recovery %
Prehydrolysate VS%
180 °C, 1%, 1 h
74
180 °C, 1%, 2 h
Produced Biofuels
Energy of Biofuels Total gasoline equivalent (L)
Ethanol (L)
Methan 3 b e (m )
Ethanol (MJ)
Methane (MJ)
Total (MJ)
0.93
211
15.2
4465
497
4962
155
70
1.31
238
18.5
5037
606
5643
176
180 °C, 1%, 5 h
72
1.31
268
18.5
5683
606
6289
197
180 °C, 2%, 1 h
55
1.88
240
1.4
5097
45
5143
161
180 °C, 2%, 2 h
51
1.26
223
1.6
4719
52
4772
149
180 °C, 2%, 5 h
54
1.73
249
7.2
5279
235
5514
172
Untreated wood
100
0.00
24
0.0
509
0
509
16
a b
Pretreatment conditions are temperature, NaOH concentration, and time. at 25 ºC and 1 bar.
32
Table 5.
Costs (€/ton)
Pretreatment a
Hydrolysis and Fermentation b
Distillation and Dehydration
Pretreatment Condition
Pinewood 0.06 €/kg
NaOH (50% Soln.) 0.24 €/kg
Nutrients 0.6 €/kg
Enzyme 1.23 €/kg
MP Steam 0.008 €/kg
180 °C, 1%, 1 h
60.0
9.1
4.4
13.6
180 °C, 1%, 2 h
60.0
9.1
4.2
180 °C, 1%, 5 h
60.0
9.1
180 °C, 2%, 1 h
60.0
180 °C, 2%, 2 h
Neutralization Acid type c
ElectroDialysis
Total Expenses c $/ton
H3PO4 0.7 €/kg
H2SO4 0.2 €/kg
Electricity 0.03 $/kWh
9.43
34.4
8.4
5.7
115.1
89.1
12.9
8.93
30.6
8.4
5.7
106.9
84.6
4.3
13.4
9.27
30.6
8.4
5.7
107.6
85.4
18.2
3.3
10.2
7.03
61.2
16.7
11.4
165.2
120.8
60.0
18.2
3.1
9.5
6.56
61.2
16.7
11.4
164.0
119.6
180 °C, 2%, 5 h
60.0
18.2
3.2
10.0
6.89
61.2
16.7
11.4
159.1
114.7
Untreated
60.0
0.0
6.0
18.5
12.81
0.0
0.0
0.0
88.5
88.5
a
H3PO4
H2SO4
The costs for LP steam, MP steam and wood milling in the pretreatment and feed handling unit were 0.001, 0.01, and 0.6 €/ton of pinewood, respectively. The respective unit price were 0.004 €/kg LP steam, 0.008 €/kg MP steam and 0.03 €/kWh electricity. b The cost for NaOH solution in the hydrolysis and fermentation for controlling pH was 0.2-0.5 €/ton of pinewood. c The expenses were calculated considering neutralization process either with H3PO4 or H2SO4.
33
Table 6.
a
Economic Potential $/ton pinewooda H3PO4 H2SO4
Manufacturing cost of ethanol €/La H3PO4 H2SO4
Pretreatment Condition
Ethanol 0.85 €/L
Biogas 1 €/m3 of CH4
Solid residue 0.04 €/kg
Total income $/ton
180 °C, 1%, 1 h
179.0
15.2
7.4
194
79
105
1.08
0.86
180 °C, 1%, 2 h
202.0
18.5
7.0
220
114
136
0.96
0.79
180 °C, 1%, 5 h
227.9
18.5
7.2
246
139
161
0.91
0.75
180 °C, 2%, 1 h
204.4
1.4
5.5
206
41
85
1.24
0.94
180 °C, 2%, 2 h
189.2
1.6
5.1
191
27
71
1.30
0.97
180 °C, 2%, 5 h
211.7
7.2
5.4
219
60
104
1.19
0.90
Untreated
20.4
0.0
10.0
20
-68
-68
4.77
4.12
The expenses were calculated considering neutralization process either with H3PO4 or H2SO4.
34
Graphical Abstract SHF
Solid fraction (Cellulose) 1 ton Pinewood
Enzymatic Hydrolysis
Fermentation by S.cerevisiae
268 L Ethanol 0.75 €/L
Dilute-alkali Pretreatmen Anaerobic Digestion
Liquid fraction (Pentoses)
35
18.5 m3 Biogas
197 L Gasoline Equivalent (6.29 GJ)
Highlights: •
Dilute alkali pretreatment enhanced the yield and economy of biofuel production.
•
Biogas was produced from hemicelluloses that were not fermentable by S. cerevisiae.
•
The maximum ethanol yield from the solid fraction of pretreated pinewood was 78%.
•
The maximum total energy of 6.29 GJ/ton can be generated from pretreated pinewood.
•
Biogas yield and the economic viability were reduced at higher NaOH concentration.
36