Chemical and structural analysis of alkali pretreated pinewood for efficient ethanol production

Abstract

Improvement of enzymatic hydrolysis and ethanol production from softwood pine was conducted by pretreatment with 8% (w/v) NaOH at different temperatures of 0, 25, and 80 °C for 2 h. Compared with the untreated wood, the lowest temperature resulted in the highest improvement of 271.1 and 218.2% in glucose and ethanol production yields, respectively. In addition to the pretreatment, Tween-20 as a non-ionic surfactant was used to enhance the enzymatic hydrolysis and fermentation yields by decreasing the ineffective adsorption of cellulases to the substrate. Addition of Tween-20 modified the glucose and ethanol yields from 10.9 and 10.4% for untreated wood to 39.2 and 47.8%, respectively. Structural analyses, including SEM and FTIR along with swelling and buffering capacity measurements, indicated that alkaline pretreatment modified the structure of pinewood by decreasing cellulose crystallinity and lignin content as well as increasing water absorbance and resistance to pH.

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1. Introduction

The increasing world population together with industrial developments has led to an increase in energy demand which is still supplied from conventional fossil fuels.¹ Concerns about the future of fossil fuels along with environmental problems associated with their consumption have increased interest in alternative fuels.² Ethanol is one of the most important renewable fuels that has attracted much attention over the last decades. It is currently produced from edible and easily degradable sugar- and starch-based materials, which are limited and result in food–fuel conflict.^3,4 Accordingly, lignocellulosic substrates, e.g., agricultural and forest residues, are considered an abundant and inexpensive alternative feedstock for ethanol production.^5,6

One of the major lignocellulosic resources is softwoods which are available in many geographical areas.⁷ Among the softwood trees, pine trees are evergreen, resistant and fast-growing, resinous, and grown widely in most temperate areas.⁸ Pine's availability has made it one of the most affordable softwoods on the market with a variety of applications which can result in a huge amount of leftover wood waste. This leftover is often selected to burn for heating purposes, while it has a high potential for biofuel production.⁴ However, the recalcitrant structure of lignocellulosic materials, particularly softwoods, is the major obstacle for further biological processes. Hence, a pretreatment process to destruct the lignin–carbohydrate complex is suggested to improve ethanol production from these materials.⁹ It can lead to reduce cellulose crystallinity, increase accessible surface area, and remove lignin and hemicelluloses.^10,11 Among several pretreatment methods, alkaline pretreatment using high sodium hydroxide (NaOH) concentration (6–20%) at low temperature and ambient pressure is one of the economically and environmentally preferable pretreatment methods since NaOH solution can be reused and recycled.^9,12

Substrate properties are crucial elements for the optimization of pretreatment conditions, i.e., temperature, NaOH concentration, and the retention time, which consequently influence the bioconversion yield.⁸ It has been reported that alkaline pretreatment of waste parts of pine tree (e.g. needle leaves, branches, cones, and bark) using 8% NaOH solution could significantly improve biogas production yields at both high (100 °C) and low (0 °C) temperatures; however, higher temperature for short duration was more successful.⁸ Additionally, the effect of pretreatment temperature with 7% NaOH solution for 2 h on the yield of further microbial processes, including enzymatic hydrolysis, fermentation (SHF), and anaerobic digestion, were investigated on softwood spruce and hardwood birch. The results showed that high temperature (100 °C) alkaline pretreatment could drastically improve the enzymatic hydrolysis yield of hardwood, whereas the pretreatment at lower temperature (e.g., 5 °C) showed relatively better results in the case of the softwood.¹² Another study showed similar results for alkaline pretreated softwood spruce at low temperature (0 °C), in which enzymatic hydrolysis yield was increased by 40% with respect to the untreated substrate.¹³ Similarly, the effect of pretreatment temperature using 8% NaOH solution for 2 h was investigated on the chemical composition and fermentable sugar production from hardwood elm. The results illustrated that decreasing the pretreatment temperature from 80 to 0 °C improved the enzymatic hydrolysis and ethanol production yields.⁴

After pretreatment, the hydrolysis process convert accessible carbohydrates to fermentable sugars.¹¹ Non-biospecific and irreversible adsorption of the enzyme on lignin^14,15 and enzyme inactivation due to the hydrophobic interactions with lignin¹⁶ are two dominant obstacles for the enzymatic saccharification of lignocellulosic materials. Although higher enzyme loading can decrease these problems, it significantly increases the process cost.¹⁷ Utilization of surfactants, especially Tween series, can eliminate the need for high enzyme loading.¹¹ Surfactants can weaken the hydrophobic interaction,¹⁶ prevent the surface deactivation of enzyme,¹⁸ improve the desorption of cellulase from the insoluble substrate.¹⁹ Furthermore, they can displace with adsorbed enzyme on lignin and consequently improve the saccharification process²⁰ and the ethanol production yield.²¹

Although many studies have been carried out on ethanol production from alkali pretreated softwoods, to the best of our knowledge, there is no report in the literature on enhancing ethanol production from NaOH pretreated pinewood.

The main objective of this study was using NaOH pretreatment at different temperatures to enhance ethanol production from pinewood via simultaneous saccharification and fermentation (SSF). Fig. 1 illustrates the schematic of ethanol production from pinewood. Furthermore, effects of the pretreatment on different characteristics of the wood were studied by different analyses, including swelling capacity and buffering capacity measurements. Moreover, the improvement of enzymatic hydrolysis and fermentation by addition of non-ionic surfactant, Tween-20, was studied, and its effect on the lignocellulose structural changes and cellulase adsorption and desorption have been investigated.

Fig. 1 Simple schematic of ethanol production from pinewood using alkali pretreatment.

2. Materials and methods

2.1. Materials and microorganism

Pinewood was collected from Isfahan University of Technology Forest (Isfahan, Iran). It was debarked, milled, screened to achieve particle sizes between 0.18 mm (mesh 80) and 0.85 mm (mesh 20), and used in all experiments. The wood dry weight was measured by drying in an oven at 105 °C until constant weight.⁴

Enzymatic hydrolysis was performed using two commercial enzymes, cellulase (Celluclast 1.5 L, Novozymes, Denmark) and β-glucosidase (Novozyme 188, Novozymes, Denmark). The activity of cellulase and β-glucosidase was 52.5 FPU mL⁻¹ and 240 IU mL⁻¹, measured by Adney and Baker²² and Ximenes et al.²³ methods, respectively. The protein content of the cellulase enzyme was 42 mg mL⁻¹, measured by Bradford assay.²⁴ The fermentation experiments were carried out using a flocculating strain of Saccharomyces cerevisiae (CCUG 53310, Culture Collection, University of Gothenburg, Sweden). The yeast biomass preparation were performed according to the Karimi et al.²⁵ method.

2.2. Pretreatment

The alkaline pretreatment process was performed using 8% (w/v) NaOH solution. An amount of 95 g NaOH solution was added to 5 g pinewood (based on dry weight) and thoroughly mixed for 10 min at room temperature. Then, the suspension was placed in a laboratory water bath (WNE 14, Memmert, Germany) at appropriate temperature (0, 25, or 80 °C) for 2 h and manually mixed every 10 min. After that, the slurry was centrifuged at 4000 rpm and room temperature for 6 min. The sediments were washed using distilled water until pH 7 and then freeze-dried (alpha 1–2/LD plus, Christ, Germany) for 72 h. The dried substrates were kept in resealable bags at room temperature until use.⁴

2.3. Enzymatic hydrolysis

The untreated and pretreated pinewoods were used to prepare 30 mL of 50 g L⁻¹ suspension in 50 mM citrate buffer at pH 4.8 in 118 mL glass bottles. The suspension was autoclaved at 121 °C for 20 min and placed under biosafety cabinet to cool down to room temperature, and then 30 FPU cellulase and 60 IU β-glucosidase per grams of dry substrates were added. Furthermore, 0.5 g L⁻¹ sodium azide was added to the mixture in order to prevent any microbial growth during hydrolysis. The untreated and pretreated substrates were enzymatically hydrolyzed at 45 °C and 130 rpm for 72 h, and liquid samples were periodically taken to measure the released glucose. The glucose formation yield was calculated using eqn (1), in which 1.111 is glucan to glucose hydration factor:³

Moreover, in separate experiments, 2.5 g L⁻¹ Tween-20 was added to the hydrolysis mixture to investigate the effect of this surfactant on the enzymatic hydrolysis.

2.4. Simultaneous saccharification and fermentation (SSF)

An amount of 30 mL of suspension containing 5 g L⁻¹ yeast extract, 7.5 g L⁻¹ ammonium sulfate, 3.5 g L⁻¹ dipotassium phosphate, 0.75 g L⁻¹ magnesium sulfate heptahydrate, 1 g L⁻¹ calcium chloride dehydrate, and 50 g L⁻¹ of the untreated or pretreated wood in 50 mM citrate buffer at pH 5 in 118 mL glass bottle was used as a fermentation medium. The mixture was autoclaved at 121 °C for 20 min and placed in biosafety cabinet to cool to ambient temperature, and then 1 g L⁻¹ S. cerevisiae (based on the dry weight), 15 FPU cellulase, and 30 IU β-glucosidase per gram of substrate were added to perform the SSF at 36 °C and 130 rpm under anaerobic conditions for 72 h. In contrast to enzymatic hydrolysis, less enzyme loading is needed for SSF process due to the consumption of glucose upon its production which can prevent the inhibition effect of glucose.^26,27 In a separate set of samples, 2.5 g L⁻¹ of Tween-20 was added to each bottle to investigate the effect of the surfactant on ethanol yield. Liquid samples were periodically taken and stored in a freezer prior to ethanol content analysis. Eqn (2) was applied to determine the ethanol yield:²where 0.51 is the theoretical yield of glucose to ethanol and 1.111 is glucan hydration factor.

2.5. Swelling capacity

Swelling capacity was used to indicate the accessibility of interior surface area of the pinewood to enzymatic hydrolysis.²⁸ In order to measure the swelling capacity of untreated or pretreated pinewood, 0.1 g (based on dry weight) of wood in a bag of non-woven materials was immersed in distilled water for 1 h. Then, based on the weight of dry material (W₁) and swollen material (W₂), the water absorbency was calculated using eqn (3):^4,29

2.6. Buffering capacity

The buffering capacity measurement, which shows the resistance against pH variations, was done using 1 g of the untreated or pretreated wood (based on dry weight) that gently swirled in 80 mL distilled water for 2 h. Then, the initial pH was measured and when it was lower than 7, 0.1 N NaOH was gradually added to raise the initial pH to 7. The volume of the required base (mL) was stand for titratable alkalinity. After that, hydrochloric acid (0.1 N) was used as a titrant to change the pH of all mixtures from 7 to 4, and the volume of the applied hydrochloric acid (mL) was defined as titratable acidity.

The base and acid buffering capacities was calculated using eqn (4) and (5), respectively:^4,30,31

2.7. Enzyme adsorption and desorption

Two individual set of samples were prepared in order to evaluate the kinetics of cellulase adsorption–desorption according to the method presented by Kumar and Wyman.³² For both evaluations, 5 mL of 50 mM citrate buffer (pH 4.8) was poured into 15 mL centrifuged tube. Then, 0.05 g of the untreated or pretreated substrate and 478 μL cellulase (400 mg g⁻¹ solid) were added and mixed. All tubes were placed in a shaking water bath (Labcon, South Africa), which turns the tubes end-over-end at 4 °C for 2 h at 100 rpm. Then, the slurries of adsorption set were centrifuged at 4000 rpm and 4 °C for 15 min, and the protein content of unabsorbed enzyme in the supernatants were measured in accordance with Bradford assay.²⁴ For enzyme desorption measurements, a set of the prepared samples was diluted with 5 mL citrate buffer at 4 °C and kept in the shaking bath for two more hours at 4 °C, and then centrifuged at 4000 rpm and 4 °C for 15 min and the protein content was similarly determined. Eqn (6) was used to determine the average percentages of cellulase desorption:³²where C and D are the average amounts of cellulase adsorbed on the pretreated solids (mg g⁻¹) over 2 h and the protein left on the pretreated solids (mg g⁻¹) after desorption over 2 h, respectively.

2.8. Analytical methods

The NREL/TP-510-42619 ³³ and TP-510-42618 ³⁴ standard methods were used to determine extractives (ethanol and water extractive) and chemical composition (carbohydrates and lignin content) of the substrates, respectively.

Fourier transform infrared (FTIR) spectrometer equipped with a universal Attenuated Total Reflection (ATR) accessory and a deuterated triglycine sulfate (DTGS) detector (Bruker Tensor 27 FT-IR, Billerica, USA) was used to examine the influence of pretreatments on the pinewood structure with an average of 60 scans between 600 and 4000 cm⁻¹. All absorption values were normalized to adjust the values between 0 and 1.

Scanning electron microscopy (SEM) was used to study the morphological changes of the pinewood before and after pretreatment. All the substrates were coated with gold (BAL-TEC SCD 005, BalTec Maschinenbau AG, Pfäffikon, Switzerland) and analyzed by SEM (Zeiss, Germany) at 15 kV.

Ethanol and sugars concentrations were analyzed using a high-performance liquid chromatography (HPLC) equipped with a refractive index (RI) detector (Jasco International Co., Tokyo, Japan). An Aminex HPX-87H column (Bio-Rad, USA) at 60 °C and an ion-exchange Aminex HPX-87P column (Bio-Rad, USA) at 85 °C with 0.6 mL min⁻¹ respective eluents of 5 mN sulfuric acid and deionized water were employed for analyses of ethanol and sugars, respectively.

3. Results and discussion

Pinewood has a variety of applications; therefore, a large amount of its processing residuals is produced annually. These residues have a high potential for ethanol production. However, similar to other softwoods, its compact structure increases resistance towards hydrolysis, and consequently decreases its efficient conversion to ethanol.³⁵ In this work, concentrated alkali pretreatment as a relatively inexpensive techniques³⁶ was used to improve the hydrolysis and ethanol production of pinewood.

3.1. Effects of pretreatment on the composition and structure of pinewood

Fig. 2 shows the composition of pinewood before and after the pretreatment. Water and ethanol extractive contents of untreated pinewood were 5.2 and 5.1%, respectively. Glucan was the dominant parts of pinewood by 34.9%, and the alkaline pretreatments increased its content by 16.9–25.5%. The polysaccharide part of hemicellulose is typically consisted of xylan, mannan, galactan, glucosan, rhamnan, and arabinan;³⁷ however, the amount of last three sugars was negligible in the pinewood samples. The xylan, galactan, and mannan contents in the native pinewood were 7.5, 6.5, and 12.4%, respectively, which were decreased by increasing the alkali pretreatment temperature, where their minimum concentrations (5.6, 3.7 and 10.3%, respectively) were detected after the treatment at 80 °C. According to Fig. 2, pretreatment with cold NaOH removed more lignin from the wood, while kept more glucan and hemicellulosic sugars.

Fig. 2 Composition of untreated and pretreated pinewood. The pretreatment conducted at different temperatures with 8% (w/v) NaOH for 2 h. In hemicellulose figure, black, dark gray, and light gray colors represent xylan, mannan, and galactan contents, respectively.

FTIR analysis was applied to investigate the effects of the pretreatment on the wood structure (Table 1 and Fig. 3). Cellulose I is denoted to the absorption band at 1430 cm⁻¹ that is very resistance to hydrolysis, whereas cellulose II is more amenable to hydrolysis and assigned to the band at 896 cm⁻¹.^35,37 As a result of pretreatments, crystallinity index, which is defined as the absorbance ratio of A1430/A896,³⁵ was reduced by the pretreatment (Table 2). As indicated in Table 1, the absorbance of hydroxyl groups (3175 and 1335 cm⁻¹) and acetyl groups (1730 cm⁻¹) were increased and decreased after the pretreatment, respectively. Acetyl groups connected hemicellulose and lignin together which can prevent the hydrolysis of hemicellulosic polysaccharides.⁴

Table 1 Characteristics and variations of bands in the FTIR spectra of treated and untreated pinewood

Wavenumber (cm^−1)	Functional group	Band assignment	Untreated pinewood	NaOH pretreatment at
				0 °C	25 °C	80 °C
3175	–OH stretching intramolecular hydrogen bonds	Cellulose II	0.123	0.176	0.188	0.147
2918	C–H stretching	Cellulose	0.099	0.034	0.175	0.069
1730	C=O stretching of acetyl or carboxylic acid	Hemicellulose & lignin	0.106	0.072	0.055	0.050
1627	C=C stretching of the aromatic ring	Lignin	0.211	0.189	0.159	0.129
1598	C=C	Lignin	0.244	0.211	0.189	0.151
1510	C=C stretching of the aromatic ring	Lignin	0.318	0.285	0.218	0.224
1465	Asymmetric bending in C–H3	Lignin	0.256	0.210	0.208	0.181
1430	C–H2 bending	Cellulose	0.229	0.265	0.255	0.235
1423	C–H2 symmetric bending	Cellulose	0.216	0.292	0.273	0.244
1375	C–H bending	Cellulose	0.142	0.254	0.253	0.222
1335	–OH (in plane bending)	Cellulose	0.140	0.259	0.261	0.234
1315	C–H2 wagging	Cellulose	0.154	0.272	0.280	0.255
1158	C–O–C asymmetric stretching	Cellulose	0.358	0.348	0.368	0.347
896	Asym., out of phase ring stretching (cellulose)	Cellulose	0.304	0.405	0.375	0.351

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Fig. 3 The FTIR spectra of untreated pinewood (1) and the wood pretreated by 8% NaOH for 2 h at 25 °C (2), 80 °C (3), and 0 °C (4).

Table 2 Crystallinity index and swelling and buffering capacities of untreated and alkali pretreated pinewood

Pretreatment conditions	Crystallinity index (CrI)	Swelling capacity (g g^−1)	Base buffering capacity (mL)	Acid buffering capacity (mL)
Untreated pinewood	0.75 ± 0.02	1.41 ± 0.08	0.05	0.38
NaOH pretreatment at 0 °C	0.65 ± 0.03	1.99 ± 0.05	0	0.62
NaOH pretreatment at 25 °C	0.68 ± 0.01	1.61 ± 0.06	0	0.68
NaOH pretreatment at 80 °C	0.67 ± 0.01	1.75 ± 0.13	0	0.75

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To investigate the effects of pretreatment on morphological modification and surface structure of the pinewood, SEM images were captured (Fig. 4). It could be inferred that the pretreatment opened up the structure of pinewood, and therefore more accessible area was available for enzyme penetration in the pretreated woods. High temperature pretreatment (80 °C) showed demolishing effects on the pinewood, while low temperature pretreatment (0 °C) showed swelled shape. The sample pretreated at 25 °C had both characteristics of low and high temperature pretreatment, a swollen structure with some small pores that shows degrading effect.

Fig. 4 SEM image (1000× magnification) of (a) untreated pinewood and the wood pretreated with 8% NaOH for 2 h at (b) 0 °C, (c) 25 °C, and (d) 80 °C.

The results of FTIR and SEM analyses showed that the alkali pretreatment at high and low temperatures have different effects on the pinewood. Pretreatment at low temperature resulted in swelled structure and lower cellulose crystallinity, while pretreatment at high temperature demolished the wood with creating deep pores. Generally, structural deformation and deeply etched surface of pretreated samples by alkali pretreatment created the high accessible surface for the cellulase. According to the crystallinity index analyses, the ability of cold NaOH (0 °C) treatment to reduce the cellulose crystallinity was more than that of hot NaOH (80 °C) pretreatment. In the other words, due to stronger Na⁺ and OH⁻ binding to water at lower temperature, cold NaOH solution can break the hydrogen binds of cellulosic structure and transform cellulose I to cellulose II that is more desirable for enzymatic hydrolysis.^35,37 Furthermore, cellulase with ellipsoid core is small enough (length of 18.0–21.5 nm and core diameter of 4.0–6.5 nm)^4,38 to penetrate readily into the pores of three different pretreated pinewoods.

Swelling capacities of the samples are reported in Table 2. Water adsorption capacity increased for all the pretreated woods, compared to that of untreated one. The maximum increase was detected for the wood pretreated at 0 °C by 41.1%. During the NaOH pretreatment, cross-linking ester bonds (1730 cm⁻¹) between the three main segments of lignocellulosic materials, i.e., hemicellulose, cellulose, and lignin, are breakdown with Na⁺ ions penetration in small pores of wood.^35,37 Along with this phenomena, hydroxyl ions participation in lignin and hemicellulose fragmentation increases biomass porosity.³⁷ Thereupon, cellulose swelling is a result of alkali solution entrance in the amorphous regions of cellulosic materials, which improved the mobility of cellulose chains, reduced the strength of hydrogen bonds within the cellulosic structure, and reduced size of crystalline parts.^35,39 Higher swelling capacity of the pretreated pinewood compared to the untreated one (Table 2) can confirm more accessible area of the pretreated substrates, leading to increase enzymatic hydrolysis yield and ethanol production.^37,40 However, the pretreatment at high temperature led to disintegrate the polysaccharides and decreased the sugars content (Fig. 2), which has a negative effect on ethanol production.³⁷

Buffering capacity analysis measures the resistance of wood to change in pH, and its results are presented in Table 2. The untreated pinewood was the only substrate that had the base buffering capacity by 0.05 mL. In addition, the alkaline pretreatment increased the acid buffering capacity of pinewood, and the maximum capacity was observed for the pretreatment at 80 °C. Furthermore, NaOH pretreatment increases the free hydroxyl groups on cellulose chains which could improve the hydrophilicity.^4,41 Increase in acid buffering capacity of the pretreated samples compared to the untreated pinewood, confirmed the increase in hydrophilicity of the pretreated substrates which could also improve the resistance to pH change. Higher buffering capacity helps to stabilize the pH at its optimum value, which is very important in enzymatic hydrolysis.⁴²

Adsorption and desorption of cellulase on the pinewood with and without Tween-20 are shown in Table 3. The important step in controlling enzymatic hydrolysis rate is the adsorption of enzyme on the cellulose chains.^32,43 Substrate porosity, its accessible surface area, and lignin concentration are the main parameters affecting the adsorption rate of cellulase. During enzymatic hydrolysis, lignin can adsorb and inactivate the cellulase enzyme by irreversible adsorption.³⁷ In the absence of Tween-20, the maximum adsorption of cellulase was unexpectedly observed for the untreated pinewood, while the presence of Tween-20 led to the maximum adsorption for the wood pretreated at 80 °C. This may be due to the reduction of the irreversible enzyme binding on the cellulose surface in the presence of the softwood extractives. Naturally, the major fraction of pine extractives are located in resin canals and in the parenchyma cells which are mainly cyclic terpenes and terpenoids to protect the plant from microbic attack (e.g., resin acids, fatty acids, fatty acid esters, waxes, triglycerides, fatty alcohols, and sterols).⁴⁴ Deposition of pinewood extractives, e.g., abietic acid, the primary component of resin acid, and pinene, on the cellulose surface can inhibit the cellulase activity by adsorbing the cellulase on their surface⁴⁵ and increasing the cellulase adsorption. On the other hand, alkali pretreatment with 8% NaOH solution can dissolve and remove the majority of extractive compounds of the pinewood,^44,46 reducing the cellulase adsorption on pretreated samples in the absence of Tween-20. However, more detailed analysis of the extractives and investigation are necessary to find the main reason. In this work, by increasing accessible surface area and reducing the lignin content, the alkali pretreatment enhanced cellulase desorption, which resulted in improving the enzymatic hydrolysis and ethanol fermentation yield (Table 3). Moreover, the pretreatment at lower temperature resulted in higher desorbed quantity either with or without addition of the surfactant. Besides, the presence of the surfactant reduced both adsorption and desorption of cellulase.

Table 3 Average amount of cellulase adsorption and desorption for untreated and pretreated pinewood in the absence and presence of Tween-20

Pretreatment conditions	Adsorption^a (mg g^−1)		Desorption^b (mg g^−1)		Desorption (%)
	Without Tween-20	With Tween-20	Without Tween-20	With Tween-20	Without Tween-20	With Tween-20
a The average amounts of cellulase adsorbed on the pretreated solids (mg g^−1) over 2 h.b The average amounts of cellulase desorbed from the pretreated solids (mg g^−1) after desorption over 2 h.
Untreated pinewood	89.6 ± 4.7	5.2 ± 2.6	33.8 ± 1.6	1.6 ± 0.7	37.7	31.5
NaOH pretreatment at 0 °C	48.3 ± 2.8	37.2 ± 3.1	43.9 ± 2.2	25.0 ± 2.3	90.1	67.3
NaOH pretreatment at 25 °C	75.4 ± 3.3	13.1 ± 1.5	58.8 ± 3.4	5.7 ± 1.1	78.0	44.0
NaOH pretreatment at 80 °C	81.0 ± 2.1	47.6 ± 4.7	47.8 ± 2.5	15.2 ± 1.7	59.0	31.9

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3.2. Enzymatic hydrolysis of untreated and treated pinewood

Enzymatic hydrolysis of the untreated and pretreated pinewood was performed using cellulase and β-glucosidase. Table 4 shows the hydrolysis results in the absence and presence of Tween-20. Generally, the alkaline pretreatment increased the enzymatic hydrolysis yields of pinewood and pretreatment at the lowest temperature showed the highest hydrolysis yield either in the presence or in absence of Tween-20.

Table 4 Glucose yield of untreated and alkali treated pinewood in the absence and presence of Tween-20

Pretreatment conditions	Initial glucose formation yield^a (%)		Glucose yield^b (%)
	Without Tween-20	With Tween-20	Without Tween-20	With Tween-20
a The initial glucose formation yield over first 4 h.b Glucose yield after 72 h, 30 FPU cellulase and 60 IU β-glucosidase per grams of dry substrates were loaded.
Untreated pinewood	6.3 ± 0.4	6.4 ± 0.5	10.1 ± 0.1	10.93 ± 0.7
NaOH pretreatment at 0 °C	9.2 ± 0.7	11.0 ± 0.4	37.37 ± 0.1	39.2 ± 0.1
NaOH pretreatment at 25 °C	8.8 ± 0.5	9.0 ± 0.6	33.4 ± 0.5	33.8 ± 0.1
NaOH pretreatment at 80 °C	8.6 ± 0.7	9.7 ± 0.8	32.5 ± 0.5	33.3 ± 0.6

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3.3. Simultaneous saccharification and fermentation (SSF)

Fig. 5 shows the effects of the alkaline pretreatment on ethanol production from the pinewood through SSF process. The alkaline pretreatment showed positive impacts on ethanol production yield. The maximum improvement of ethanol yield was achieved after pretreatment at 0 °C, resulting in increasing the yield for untreated wood from 10.3% to 32.7%. Addition of Tween-20 in SSF process resulted in significant improvement on the ethanol yield from the wood pretreated at 0 °C, which was around five times higher than that from the untreated wood.

Fig. 5 Ethanol yield obtained after 72 h SSF of untreated and alkali pretreated pinewood in the (a) absence and (b) presence of Tween-20.

The non-ionic surfactant, Tween-20, significantly decreased irreversible adsorption of the enzyme by preventing the formation of undesired bond between cellulase and lignin²¹ which enhanced ethanol formation. Tween-20 price is considerably lower than cellulase; therefore, the presence of this non-ionic surfactant not only increases ethanol yield but also enhances the process economics.²¹ Results of the current work showed that alkali pretreatment at 0 °C in the presence of Tween-20 improved ethanol yield from 20 g kg⁻¹ of untreated pinewood to 119 g kg⁻¹ from the pretreated one.

4. Conclusions

Enzymatic hydrolysis and ethanol production from pinewood was considerably enhanced by alkaline pretreatment with 8% (w/v) NaOH at different temperatures. The pretreatment at 0 °C for 2 h showed the highest effect on opening up the wood structure and crystallinity reduction, resulted in higher ethanol yield. The pretreatment enhanced desorption of cellulase from cellulose, resistance to pH changes, and swelling capacity. Addition of non-ionic surfactant Tween-20 reduced the irreversible adsorption of cellulase on untreated and pretreated samples, positively improved the ethanol yield.

Acknowledgements

This work was funded by the Research Center of Biotechnology and Bioengineering, Isfahan University of Technology, Iran.

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