Integrated ethanol and biogas production from pinewood

Abstract

Ethanol and biogas production from softwood pine was maximized by dilute acid pretreatment. Pretreatment was performed with 0.25 and 0.5% w/w H₂SO₄ at 100, 140 and 180 °C for 5–30 min. Untreated wood and the solid fraction of the pretreatments, mainly containing cellulose, were used for ethanol production. The liquid fraction of the pretreated materials, mainly containing hemicellulosic sugars, was detoxified and subjected to anaerobic digestion for biogas production. The highest methane production yield was obtained by pretreatment with 0.5% H₂SO₄ at 140 °C for 5 min, resulting in methane yield of 162 m³ per ton of pinewood, whereas the highest ethanol yield of 53% was achieved by treatment with 0.5% H₂SO₄ at 180 °C for 30 min. Pretreatment at high temperature, i.e., 180 °C, increased the production of inhibitory compounds. These compounds had more significant inhibitory effects on biogas production than ethanol production. The highest gasoline equivalent, 61.5 gallons per ton, was obtained after wood pretreatment with 0.5% acid at 140 °C for 5 min, whereas the gasoline equivalent was only 4.9 gallons per ton for untreated wood. The reason for the improvements was followed by compositional and structural changes made by the pretreatment.

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1 Introduction

While fossil fuels are finite resources of energy, their consumption is continuously increasing. Emission of carbon dioxide from the combustion of these fuels has led to undesired climate changes.¹ Replacement of fossil fuels by renewable biofuels, e.g., ethanol and biogas, is the most applicable solution to this problem.^2,3 Currently, ethanol is produced from sugar- or starch-based raw materials. Due to limited resources of these food resources and competition for the production of fuels, food prices have increased.^4,5 Meanwhile, the availability of lignocellulosic materials to a large extent has resulted in increasing attention to these materials for ethanol and biogas production.⁶

Lignocellulosic materials are mainly composed of cellulose, hemicellulose and lignin. Highly crystalline cellulose chains are protected by lignin and hemicellulose. The main drawback of lignocellulosic materials is their resistance to enzymatic or microbial attacks. Hence, a pretreatment step is necessary prior to any enzymatic or biological conversion.⁷ Dilute-acid hydrolysis is the most practiced pretreatment for ethanol production. This pretreatment improves the conversion of cellulose by hemicellulose and lignin removal, which increases the accessible surface area of cellulose.^7–9 However, production of a single product, i.e., ethanol or biogas, is not economically feasible.^10,11 The most practical approach to have an industrial ethanol production process from lignocelluloses is conversion of all constituents of the biomass to biofuels. After pretreatment, a slurry is produced consisting of solid and liquid fractions.⁴ The pretreatment hydrolysate, i.e., the liquid fraction, contains mono- and short-chain oligosaccharides. These sugars mainly originate from hemicelluloses and contain solubilized pentoses.⁷ To date, no industrial strain of alcoholic fermentation yeast, fungi or bacteria is able to ferment these two types of sugars simultaneously.^3,11

In order to convert both pentoses and hexoses to ethanol, separate hydrolysis and co-fermentation (SHCF) was suggested.¹² This process should be conducted in two separate stages, since the presence of glucose inhibits xylose assimilation.¹³ To overcome this problem, simultaneous saccharification and co fermentation (SSCF) of hexoses and pentoses by one recombinant or two types of microorganisms was suggested.^3,10 The basic practical bottleneck of this method is that hexose fermentation is performed anaerobically while pentose fermentation requires aerobic conditions.^13,14 Aerobic cultivation on pentose is not successful since ethanol is assimilated at the same time of xylose fermentation and biomass is the main product of cultivation, instead of ethanol. Afterwards, micro-aerobic SSCF process has been proposed to reduce the assimilation of ethanol; however, several challenges is accompanied at the industrial scales of this process.^11,15 On one hand, there are several operational problems besides the challenges of using genetically modified organisms, i.e., safety issues and gene instabilities. On the other hand, in the case of using two different types of microorganisms, the compatibility of the strains, e.g., differences in optimum temperature and pH as well as tolerance to high ethanol concentration, are practical bottlenecks.^3,10 Therefore, simultaneous conversion of both hexoses and pentoses to ethanol is still challenging.³

Production of biogas from the pretreatment hydrolysate, which contains both hexoses and pentoses, is a possible method for maximizing the conversion of all available sugars to a biofuel. In contrast to pentose fermentation for ethanol production, which has not been proved in commercial scale yet,³ biogas production is a well-developed technology.¹⁶ To our knowledge, there is no previous report on the biogas production from the hydrolysate which was produced in the improvement of ethanol production from lignocelluloses by dilute acid pretreatment.

The main scope of this study was optimization of the pretreatment conditions for maximized biofuel production from pinewood, which is among the hardest to convert of the lignocelluloses. The wood was pretreated with dilute sulfuric acid. The solid fraction was subjected to enzymatic hydrolysis and fermentation, whereas anaerobic digestion was used for the production of methane from the detoxified pretreatment hydrolysate. Total amounts of energy produced, in terms of gasoline equivalent, were calculated and compared to find the best pretreatment conditions. Finally, the effects of acid pretreatment on wood structural changes were investigated.

2 Material and methods

2.1 Raw materials

Native species of pinewood were obtained from Delijan city (Delijan, Markazi province, Iran 33°59′ N, 50°41′ E). The wood was debarked, ground, and milled. Afterwards, the powder was sieved through 20- and 80-mesh panels to obtain particle sizes of 0.841–0.177 mm. Total solid content of the materials was measured by oven drying at 105 °C.¹⁷

2.2 Pretreatment

The wood powder was pretreated by dilute sulfuric acid in a 500 mL high pressure stainless steel batch reactor equipped with pressure indicator and thermometer.¹⁸ The pretreatment was performed with 0, 0.25 and 0.5% w/w H₂SO₄ at a solid-to-liquid ratio of 1 : 10 (based on dry mass). The experiments were accomplished within 5, 10 and 30 min at 100, 140, and 180 °C. After pretreatment, the reactor was cooled for 5 min to about 70 °C. The pretreated solids were separated from the liquid fraction and washed with distilled water until the pH of the wash liquid reached 7. In the end, the solids were dried at room temperature for 24 h and stored in plastic bags. The liquid fraction was kept frozen to be used for biogas production.

2.3 Detoxification

At elevated temperatures, a portion of the solubilized sugars and lignin are degraded to inhibitory compounds. These inhibitors can reduce or even prevent the subsequent microbial growth and activity.¹⁹ After dilute-acid pretreatment, overliming was used to reduce the amount of toxic compounds in the pretreatment hydrolysate. In this method, calcium hydroxide (lime) is added to the pretreatment hydrolysate to increase pH to 10–12. The materials are kept at this pH for a period of time, and then the pH is decreased by acid.^20,21 Suspensions of 100 g L⁻¹ Ca(OH)₂ were added to the pretreatment hydrolysates to adjust the pH to 11. The materials were kept for 20 h and then neutralized by 2 M H₂SO₄ to obtain pH 7. Afterwards, the precipitated materials were removed by centrifugation. The detoxified pretreatment hydrolysates were kept frozen until use.^22,23

2.4 Separate enzymatic hydrolysis and fermentation (SHF)

Enzymatic hydrolysis of the untreated and pretreated pinewood was performed in 118 mL glass bottles. Thus, 5% w/w of the solid materials were suspended in 50 mM sodium citrate buffer at pH 4.8 (ref. 24) and autoclaved for 20 min at 121 °C. Hydrolysis was performed at 45 °C for 72 h in a shaker incubator (120 rpm). The enzyme loadings were 20 FPU cellulase (Cellic® CTec2, VCNI0013, Novozyme, Bagsvaerd, Denmark) and 50 IU β-glucosidase (Cellic® HTec2, VHN00002, Novozyme, Bagsvaerd, Denmark) per gram of dry wood.²⁵ Samples were taken at 4, 24, 48 and 72 h of hydrolysis and analyzed for sugar content by high performance liquid chromatography (HPLC). All experiments were run in duplicate.

Fermentation was performed in 118 mL bottles at pH 5 and 32 °C under anaerobic conditions using Saccharomyces cerevisiae (CCUG 53310, Culture Collection, University of Gothenburg, Sweden). An amount of 20 mL of hydrolysate was supplemented with required nutrients, which were 5 g L⁻¹ yeast extract, 7.5 g L⁻¹ (NH₄)₂SO₄, 3.5 g L⁻¹ K₂HPO₄, 0.75 g L⁻¹ MgSO₄·7H₂O and 1.0 g L⁻¹ CaCl₂·2H₂O. pH was adjusted to 5 and then the hydrolysate was autoclaved at 121 °C for 20 min.²⁶ Next 1 g L⁻¹ S. cerevisiae was added to the mixture and incubated at 32 °C for 24 hours.²⁷ All experiments were run in duplicate and the average values of the data were reported. The theoretical yield of ethanol production was calculated according to the following equation:²⁸

2.5 Biogas production

Anaerobic digestions of the pinewood hydrolysates (the liquid fraction produced during the dilute acid pretreatment) were performed according to the method presented by Hansen et al.²⁹ Biogas was produced at mesophilic conditions (37 °C) in 118 mL serum glass bottles. The inoculum was obtained from a 7000 m³ anaerobic digester (Isfahan Municipal Wastewater Treatment Plant, Isfahan, Iran) operating at 37 °C. The inoculum was sieved to remove large particles. For biogas production from the hydrolysates, 20 mL of inoculum was added to a certain amount of hydrolysate. The amount of hydrolysate was precalculated to maintain a volatile solid ratio of 3 : 1 (inoculum : substrate). A blank test consisting of inoculum and distilled water was used to determine the biogas production from the inoculum itself. Butyl rubber septum and aluminum caps were used to seal the bottles. Each bottle was purged with 80% N₂ and 20% CO₂ gas mixture for 2 min to obtain anaerobic conditions.

Samples of 100 μL from the headspace of each bottle 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 gas chromatography. Methane yield was calculated as:

In the above formula, VS stands for volatile solids and 0.75 is the liquid recovery (cf. Section 2.8). The numerical value of mL g⁻¹ is equal to m³ per ton. Pure methane and carbon dioxide gas were used as calibration standards. All anaerobic digestion experiments were performed in duplicate.

2.6 Analytical methods

A gas chromatograph (SP-3420A, TCD detector, Beijing Beifen Ruili Analytical Instrument Co., Beijing, China) was used to analyze the composition of methane and carbon dioxide produced in the anaerobic digestion. The system was equipped with a packed column (Porapack Q column, Chrompack, Engstingen, Germany) and a thermal detector with 150 °C injection temperature. Nitrogen was used as carrier gas with a flow rate of 20 mL min⁻¹ at 50 °C.³⁰

Total solids, volatile solids, ash, lignin, and carbohydrate contents of the untreated and pretreated wood were measured according to the method presented by National Renewable Energy Laboratory (USA).³¹

Ethanol and sugar contents of separate hydrolysis and fermentation (SHF) and carbohydrate analysis were determined using high-performance liquid chromatography (HPLC) equipped with UV and RI detectors (Jasco International Co. Tokyo, Japan). Concentration of glucose, mannose, xylose, galactose and arabinose were determined by an ion exchange column (Aminex HPX-87P Bio-Rad Laboratories, Hercules, USA) at 85 °C with an eluent of deionized water at a flow rate of 0.6 mL min⁻¹. Concentrations of ethanol, acetic acid, furfural, and 5-hydroxymethylfurfural (HMF) were determined by Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, USA) at 60 °C using 0.6 mL min⁻¹ 5 mM sulfuric acid as eluent.

The effects of pretreatment on the chemical structure and crystallinity of the treated and untreated pinewood were investigated using a Fourier transform infrared (FTIR) spectrometer (TENSOR 27 FT-IR, Bruker, Leipzig, Germany) equipped with a universal ATR (attenuated total reflection) accessory and a DTGS (deuterated triglycine sulfate) detector. The spectra were obtained with an average of 60 scans at a range of 600–4000 cm⁻¹.

Structural change in the treated and untreated pinewood was examined by SEM (scanning electron microscopy). The dried samples were coated with gold (SBC-12 ion sputter coater, KYKY, Shanghai, China) and analyzed by SEM (KYKY-EM3200, Shanghai, China) at a voltage of 15 kV.

2.7 Statistical analysis

Statistical validation of results was performed by ANOVA test and general linear model (GLM) using Minitab 17.1.0 software (Minitab Inc., State College, USA). Tukey method with 95% confidence was used for comparison and estimation of significant differences among the means. There are no significant differences among the means with the same lettered group at a 5% probability level (P < 0.05).

2.8 Sensitivity analysis

A sensitivity analysis was performed to determine the most efficient pretreatment in the parallel process of ethanol and biogas production. For better comparison of the pretreatments, the gasoline equivalent of the whole process was calculated using the lower heating values of the fuels (at 25 °C and 1 atm), which are 21.2 MJ L⁻¹ for ethanol, 32.7 MJ Nm⁻³ for methane and 32.0 MJ L⁻¹ for gasoline.³² The gasoline equivalents were calculated based on one ton of pinewood. Other assumptions were 10% solid loading in the pretreatment (based on experimental data of this study) and complete solid recovery. The liquid recovery after filtration was measured to be 75%. This was due to the sorption of 25% of the hydrolysate to the solid. Thus this 25% liquid was practically lost since the solid fraction was washed with pure water in the next step.

3 Results and discussion

Pinewood was treated with dilute sulfuric acid at different temperatures and treatment durations. The untreated and treated solids were enzymatically hydrolyzed for 72 h. Afterwards, fermentation was performed by S. cerevisiae for 24 h. Moreover, the liquid fractions from dilute acid pretreatment (pretreatment hydrolysates) were anaerobically digested for biogas production. Gasoline equivalent was used to find the total amounts of energy produced in the form of ethanol and biogas and to compare the results.

3.1 Effect of pretreatment on pinewood composition

The carbohydrate and lignin contents of the untreated and pretreated pinewood were analysed according to the method presented by Sluiter et al.³¹ Results corresponding to the best conditions (based on the hydrolysis and anaerobic digestion results) are presented in Table 1. The untreated wood contained 24.7% lignin, 48.4% glucan, and 21.3% of other carbohydrates (Table 1). The results indicated that the glucan content was increased from 56.8% to 61.1% by increasing the pretreatment duration from 5 min to 30 min at 180 °C and 0.5% acid loading. Meanwhile, under these conditions, acid insoluble lignin was decreased from 40.0% to 37.8% while all hemicelluloses were removed. In the wood cell wall, the main molecular force for the arrangement of cellulose chains in crystalline structure is hydrogen bonding. During dilute acid pretreatment, acid penetrates inside the cell wall and breaks some of the hydrogen bonds. Furthermore, dilute acid pretreatment efficiently hydrolyses hemicelluloses by breaking glycosidic bonds, while cellulose is slightly hydrolyzed. Besides, a part of lignin, mainly acid-soluble lignin, is released during the pretreatment condition.⁷ Thus, in this study, in spite of hydrolysis of a minor fraction of glucan, its percentage was increased after pretreatment. This increase was mainly due to the reduction of hemicellulose and lignin content by the pretreatment. Specifically, using the acid pretreatment, all of the xylan, galactan, mannan, and arabinan content (Table 1), contributed to 21% of wood dry weight, were dissolved to the pretreatment hydrolysate. Increasing the pretreatment temperature at constant acid concentration resulted in more hemicellulose removal. Similar trends for the amount of glucan and other sugar polymers were observed in previous studies.^30,33

Table 1 Composition of untreated and treated pinewood at different pretreatments^c

Pretreatment conditions^a			Glucan (%)	Xylan (%)	Mannan (%)	Galactan (%)	Arabinan (%)	AINSL^b (%)	Ash (%)
a Pretreatment conditions are temperature, acid concentration and time.b AINSL: acid insoluble lignin. The amount of acid soluble lignin was measured to less than 1%.c ND = not determined.
100 °C	0%	5 min	51.6 ± 0.2	6.01 ± 0.4	11.80 ± 0.1	1.61	1.11	20.0	0.83
140 °C	0%	5 min	51.8 ± 0.3	5.92 ± 0.5	11.60 ± 0.2	1.55	1.03	22.2	0.65
180 °C	0%	5 min	53.8 ± 0.5	4.14 ± 0.3	9.21 ± 0.2	1.31	0.88	26.3	0.45
140 °C	0.5%	5 min	54.2 ± 0.3	ND	ND	ND	ND	31.9	0.27
180 °C	0.5%	5 min	56.8 ± 0.2	ND	ND	ND	ND	40.0	0.75
180 °C	0.5%	10 min	57.4 ± 0.3	ND	ND	ND	ND	42.7	0.67
180 °C	0.5%	30 min	61.1 ± 0.3	ND	ND	ND	ND	37.8	0.58
Untreated pinewood			48.4 ± 0.4	6.20 ± 0.1	12.10 ± 0.2	1.75	1.25	24.7	0.36

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3.2 Effects of pretreatment on the wood structure

Effects of dilute acid pretreatment on the structure of pinewood were followed by FTIR spectroscopy. Sample spectra for untreated and treated pinewood are presented in Fig. 1. The spectra represent data for the wood pretreated with 0.5% w/w acid for 5 min at 140 °C and for 30 min at 180 °C.

Fig. 1 FTIR spectra of (a) untreated pinewood, and of wood pretreated at (b) 0.5% w/w sulfuric acid for 5 min at 140 °C and (c) 0.5% w/w sulfuric acid for 30 min at 180 °C.

Crystallinity index (CI) was calculated as the absorbance ratio of A1098/A898. The absorption bands at 1098 and 898 cm⁻¹ denote cellulose I and cellulose II, respectively.^34–35 The results indicated that treatments at high temperatures increased the CI. This value was 1.54 for the untreated pinewood, which increased to 1.70 when treatment was performed at high temperatures. The increase in the crystallinity could be explained by the removal of amorphous celluloses.³⁶ Similar results of increasing crystallinity were reported after dilute acid pretreatment.³⁷

Furthermore, the changes in the pinewood by pretreatment were observed using SEM, and some of the images are presented in Fig. 2. The untreated pinewood had a highly compact and crystalline structure. Through pretreatment, this structure was altered to have a more accessible surface area for the cellulolytic enzymes (Fig. 2).

Fig. 2 Scanning electron micrographs (SEM) of (a) untreated pinewood and wood, pretreated at (b) 0.5% w/w sulfuric acid for 5 min at 140 °C and at (c) 0.5% w/w sulfuric acid for 30 min at 180 °C. Magnification: ×500.

3.3 Effect of detoxification on the inhibitors

The possible inhibitors produced or released during the acid treatments are classified into organic acids (acetic acid, formic acid and levulinic acid), furans (5-hydroxymethylfurfural (HMF) and furfural), and phenolic compounds.^38,39 Biogas production from the non-detoxified hydrolysates failed to produce methane. Thus, overliming, treatment with Ca(OH)₂, was used to partially remove furfural, HMF and phenolic compounds.

The concentration of all sugars as well as furfural, HMF and acetic acid for the selected pretreatments hydrolysates is summarized in Table 2. The results show a decrease in the concentration of sugars and inhibitors through overliming. For samples pretreated with 0% acid, overliming reduced the concentration of these compounds to negligible amounts. The concentrations of inhibitors in the pretreatment hydrolysates were increased by elevation of the treatment time and temperature. However, under these conditions, the concentration of total sugars was also decreased. After overliming at high pH, degradation of a part of sugars occurred, resulting in the lower sugar concentration.³⁹ Similar reductions in sugar concentration were observed after overliming in previous studies.^22,40

Table 2 Concentration of total sugars and inhibitors in the pretreatment hydrolysate before and after detoxification^c

Pretreatment conditions^a			Pretreatment hydrolysate				Pretreatment hydrolysate after detoxification
			Total sugars^b	Acetic acid	Furfural	HMF	Total sugars^b	Acetic acid	Furfural	HMF
a Pretreatment conditions are temperature, acid concentration, and time.b Concentration of glucose, mannose, xylose, arabinose, and galactose, measured by HPLC using Aminex HPX-87P column.c ND: non detectable.
100 °C	0%	5 min	3.28	ND	ND	ND	ND	ND	ND	ND
140 °C	0%	5 min	3.47	ND	0.034	ND	ND	ND	ND	ND
140 °C	0.5%	5 min	30.5	2.58	0.085	0.05	12.5	1.87	0.01	0.01
180 °C	0.5%	5 min	22.4	0.24	2.47	1.72	18.7	0.11	1.08	0.72
180 °C	0.5%	10 min	12.2	0.17	2.83	1.85	9.39	0.49	0.57	0.52
180 °C	0.5%	30 min	10.1	1.28	4.43	1.95	9.30	0.42	1.29	0.76

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3.4 Biogas production

The results of anaerobic digestion of detoxified pretreatment hydrolysates and the total amount of methane produced after 50 days are shown in Fig. 3–5. The data in Fig. 3–5 indicate that methane production is more enhanced by increasing the pretreatment temperature at a constant time and acid concentration. Without the addition of acid (0% acid), the total amount of methane was increased by increasing pretreatment temperature. But with acid pretreatment, the maximum value of methane production was obtained by pretreatment at 140 °C, and increasing the pretreatment temperature to 180 °C resulted in less methane production.

Fig. 3 Total methane (m³ per ton of initial dry pinewood) produced from the detoxified hydrolysates obtained in pretreatment at 100 °C. The symbols represent biogas yield after 3 (), 15 () and 50 () days of anaerobic digestion.

Fig. 4 Total methane (m³ per ton of initial dry pinewood) produced from the detoxified hydrolysates obtained in pretreatment at 140 °C. The symbols represent biogas yield after 3 (), 15 () and 50 () days of anaerobic digestion.

Fig. 5 Total methane (m³ per ton of initial dry pinewood) produced from the detoxified hydrolysates obtained in pretreatment at 180 °C. The symbols represent the biogas yield after 3 (), 15 () and 50 () days of anaerobic digestion.

This result can be related to decomposition of sugars and formation of inhibitors by increasing temperature. Increasing the acid concentration at a constant time and temperatures of 100–140 °C increased biogas production. In addition, by treatment at 180 °C, the application of higher acid concentrations had negative impact on methane production. The maximum production volume of methane was 162 m³ per ton of untreated pinewood, obtained by pretreatment at 140 °C for 5 min and with 0.5% acid. Pretreatment at more severe conditions, i.e., higher temperature, time, and acid concentration, resulted in the production of more inhibitor compounds. In spite of detoxification, these compounds significantly reduced biogas production. Application of more efficient detoxification methods is suggested for further improvements in biogas production; however, it is accompanied with higher sugar loss and production of more pollutants.

3.5 Enzymatic hydrolysis

The results of enzymatic hydrolysis of the untreated and pretreated wood (the solid fraction of the dilute acid pretreatment) by cellulase and β-glucosidase are shown in Fig. 6. The results indicated that glucose concentration was increased by increasing the temperature. These enhancements were not considerable from 100 to 140 °C, while the improvements were significant at 180 °C. Furthermore, the prolongation of pretreatment time did not affect the glucose yield at temperatures of 100 and 140 °C. Nevertheless, increasing time in pretreatment at 180 °C enhanced the efficiency of hydrolysis. The glucose concentration from the untreated wood was 3.4 g L⁻¹ after 72 h hydrolysis. However, pretreatment with 0.5% acid at 180 °C resulted in the maximum glucose concentration of 11.7 g L⁻¹. This value is 3.4 folds higher than the glucose concentration from untreated wood. Dilute acid pretreatment followed by an enzymatic hydrolysis of olive tree biomass were performed by Cara et al.⁴¹ The maximum glucose yield of 42% was achieved after pretreatment with 0.6% sulfuric acid at 210 °C and 72 h hydrolysis.

Fig. 6 Glucose concentrations from enzymatic hydrolysis of pretreated and untreated pinewood after 4 h (), 24 h (), 72 h () of hydrolysis. The pretreatments were performed at (a) 100 °C, (b) 140 °C and (c) 180 °C.

3.6 Ethanol production

Results of ethanol production (theoretical ethanol yield) from optimum treated and untreated pinewood are presented in Table 3. The theoretical ethanol yield from untreated pinewood was only 6.4%, and pretreatment improved the yields to 53.1%. The results indicated that pretreatment at more severe conditions can more efficiently enhance ethanol yield. In a similar study by Nguyen et al.,⁴² dilute acid pretreatment of a mixture of different softwoods was performed, and ethanol yield of 61% was detected after the pretreatment with 0.4% H₂SO₄ at 200 °C followed by simultaneous saccharification and fermentation (SSF). The lower yield obtained in this study might be due to the type of substrate, as pinewood is among the most recalcitrant woods among the softwoods. Another reason could be difference between the pretreatment conditions used, i.e., higher temperature, as well as fermentation methods used.

Table 3 Ethanol yield from untreated and pretreated pinewood

Pretreatment conditions^a			Ethanol yield (%)
a Pretreatment conditions were temperature, acid concentration and time.
100 °C	0%	5 min	10.96 ± 0.27
140 °C	0%	5 min	15.15 ± 0.34
180 °C	0%	5 min	26.84 ± 0.20
140 °C	0.5%	5 min	30.34 ± 0.26
180 °C	0.5%	5 min	40.73 ± 0.06
180 °C	0.5%	10 min	48.05 ± 0.25
180 °C	0.5%	30 min	53.07 ± 0.35
Untreated pinewood			6.44 ± 0.07

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3.7 Gasoline equivalent

The results of sensitivity analysis and volume of gasoline equivalent are reported in Table 4. Each ton of untreated pinewood produced only 4.9 gallons of gasoline. However, the application of dilute acid pretreatment increased the volume of gasoline equivalent to 61.5 gallons.

Table 4 Comparison of volume of ethanol, biogas and gasoline equivalents from best pretreatment conditions^a

Pretreatment conditions^b			Ethanol (gal)	methane (m^3)	Gasoline equivalents (gal)
a All data were calculated based on one tone of initial dry pinewood.b Pretreatment conditions were temperature, acid concentration and time.
100 °C	0%	5 min	12.4	12.6	11.6
140 °C	0%	5 min	16.5	20.9	16.6
180 °C	0%	5 min	25.4	89.2	40.9
140 °C	0.5%	5 min	26.8	162	61.5
180 °C	0.5%	5 min	31.1	72.9	40.3
180 °C	0.5%	10 min	37.8	59.8	41.2
180 °C	0.5%	30 min	42.5	51.6	42.1
Untreated pinewood			7.39	0.00	4. 9

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The best ethanol yield was achieved after pretreatment at higher temperature and the maximum gasoline equivalent value was 28.2 gallons. However, pretreatment with 0.5% w/w acid at 140 °C for 5 min was the optimum condition in terms of gasoline equivalent production. Laurens et al.⁴³ used acid-catalyzed pretreatment for ethanol production from algal biomass. The pretreatment improved the ethanol and gasoline equivalents to 59 gallons and 39 gallons per ton of algal biomass, respectively.

4 Conclusions

Pinewood has a highly recalcitrant structure and its conversion to ethanol was inefficient. Dilute acid pretreatment at high temperature was practiced to maximize the production of biofuels from this wood. After pretreatment, most of the solubilized hemicelluloses were not fermentable by S. cerevisiae. Thus, the production of biogas from the pretreatment hydrolysate was performed as an efficient method for the conversion of these compounds to a biofuel. Biogas was successfully produced from the pretreatment hydrolysates with the maximum amount of 162 m³ per ton of untreated pinewood. Furthermore, the acid pretreatment enhanced the yield of ethanol production from the solid fraction to 53%, while it was only 6% for the untreated wood. Gasoline equivalent amounts of ethanol and biogas produced from the wood were calculated and sum of these values were compared. The gasoline equivalent was improved from 4.9 gallons per ton for the untreated wood to 61.5 gallon per ton of pretreated wood. While pretreatment at higher temperature increases ethanol yield, the production of higher amounts of inhibitors reduces biogas yield. Thus, the best pretreatment condition to obtain the maximum gasoline equivalent value was treatment by 0.5% acid at 140 °C and 5 min.

Acknowledgements

The authors are grateful to Novozymes (Denmark) for donation of Cellic® CTec enzymes for this work, and to Dr Akram Zamani, University of Borås, for her valuable assistance with the experiments.

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Footnote

† Electronic supplementary information (ESI) available: Results of ANOVA test. See DOI: 10.1039/c5ra27901a

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