Comparison of steam explosion, dilute acid, and alkali pretreatments on enzymatic saccharification and fermentation of hardwood sawdust

Abstract

Effects were compared for three low-cost pretreatment methods (dilute acid, alkali, and steam explosion) relative to the effectiveness of environmentally friendly enzymatic hydrolysis and ethanol fermentation of aspen, birch, and oak chips. The highest monomeric sugar yield was achieved with the alkali pretreatment of the aspen chips (22 g/L of glucose and 6 g/L of xylose). Additionally, the concentration of lignocellulose degradation products formed during this pretreatment was relatively low, and so the hydrolysis and fermentation efficiencies were 80% and 85%, respectively. The application of dilute acid pretreatment led to lower yield of enzymatic hydrolysis in comparison with alkali pretreatment, resulting in 41% to 62% of theoretical yield for aspen and birch chips, respectively. Increasing the NaOH concentration led to an increase in the monomeric sugar yield, and consequently increased the hydrolysis and fermentation yields. By contrast, increasing the acid concentration resulted in a higher sugar yield, and the fermentation efficiency decreased. The applied steam explosion conditions resulted in the formation of 6.8 to 15.4 g glucose/L, with hydrolysis yield in the range 34 to 42% of theoretical. The most susceptible for pretreatment and enzymatic hydrolysis was found to be aspen biomass.

Full Article

Comparison of Steam Explosion, Dilute Acid, and Alkali Pretreatments on Enzymatic Saccharification and Fermentation of Hardwood Sawdust

Urszula Dziekońska-Kubczak,* Joanna Berłowska, Piotr Dziugan, Piotr Patelski, Maria Balcerek, Katarzyna Pielech-Przybylska, Agata Czyżowska, and Jarosław Domański

Effects were compared for three low-cost pretreatment methods (dilute acid, alkali, and steam explosion) relative to the effectiveness of environmentally friendly enzymatic hydrolysis and ethanol fermentation of aspen, birch, and oak chips. The highest monomeric sugar yield was achieved with the alkali pretreatment of the aspen chips (22 g/L of glucose and 6 g/L of xylose). Additionally, the concentration of lignocellulose degradation products formed during this pretreatment was relatively low, and so the hydrolysis and fermentation efficiencies were 80% and 85%, respectively. The application of dilute acid pretreatment led to lower yield of enzymatic hydrolysis in comparison with alkali pretreatment, resulting in 41% to 62% of theoretical yield for aspen and birch chips, respectively. Increasing the NaOH concentration led to an increase in the monomeric sugar yield, and consequently increased the hydrolysis and fermentation yields. By contrast, increasing the acid concentration resulted in a higher sugar yield, and the fermentation efficiency decreased. The applied steam explosion conditions resulted in the formation of 6.8 to 15.4 g glucose/L, with hydrolysis yield in the range 34 to 42% of theoretical. The most susceptible for pretreatment and enzymatic hydrolysis was found to be aspen biomass.

Keywords: Sawdust; Pretreatment; Enzymatic hydrolysis; Fermentation; Bioethanol

Contact information: Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Wolczanska 171/173, 90-924 Lodz, Poland;

* Corresponding author: urszula.dziekonska-kubczak@p.lodz.pl

INTRODUCTION

Currently, production of second-generation biofuels is of major interest to many researchers. Biofuels can be produced from lignocellulosic materials that are not applicable in food production. The availability of lignocellulosic biomass, its composition, renewability, and non-food characteristics are the main reasons for using it for the production of bioethanol. Lignocellulosic biomass includes agricultural residues, wood residues, energy crops, municipal solid waste, and various industrial wastes. Lignocellulose is the main structural component of plant cell walls and consists of cellulose, hemicellulose, and lignin. The chemical composition of agricultural wastes or woody biomass depends on the species, age, growing conditions, fractionation degree, and pretreatment (Sjöström 1993). Typically, most hardwoods are comprised of approximately 18% to 25% lignin, 13% to 40% hemicellulose, and 40% to 55% cellulose (Kumar et al. 2009; Vena et al. 2013; Chen et al. 2016). Cellulose is the most abundant biopolymer on Earth and is an important structural component of plant cell walls. Because of its strong linkage to hemicelluloses and lignin, the isolation of cellulose requires multi-step processing, which includes pretreatment, enzymatic hydrolysis, and fermentation (Xiao et al. 2012). Pretreatment of lignocellulosic materials is used to make cellulose more accessible to hydrolyzing enzymes by inducing changes in the cell wall chemical composition. The goal is to remove lignin and hemicellulose, reduce the cellulose crystallinity, and increase the porosity of the material (Kumar et al. 2009). Among all of the currently available and widely used pretreatment methods, acid hydrolysis (Karapatsia et al. 2017), alkaline hydrolysis (Karp et al. 2015), and steam explosion (SE) (Li et al. 2015) are the most widely used for economic reasons.

Pretreatment with the SE method is the most widely studied and used method for the physico-chemical treatment of biomass, especially in the case of raw materials with low lignin contents (e.g., hardwood). With this method, the raw material is treated with saturated steam (160 °C to 260 °C) under high pressure (0.7 MPa to 4.8 MPa) (Kumar et al. 2009, 2017; Sun and Cheng 2002). These conditions are maintained for a period of time that can range from several seconds to several minutes (in that time the hydrolysis of hemicellulose occurs), and then the pressure is suddenly reduced. During this process, hemicelluloses are degraded and lignin is transformed, and thereby the accessibility of the cellulose to hydrolytic enzymes is increased (Hendriks and Zeeman 2009). Hemicellulose is degraded by acids generated during autohydrolysis (mostly acetic acid), and lignin is only removed to a certain extent, but it is distributed on the surface of fibers. The rapid exposure of biomass to atmospheric pressure also results in fragmentation of the raw material, which increases the available surface area (Kumar et al. 2009). However, this method does not result in substantial delignification (Sannigrahi et al. 2011). Other problems related to SE are relatively low yield of hemicelluloses, formation of inhibitory compounds and incomplete destruction of lignin-carbohydrate matrix (Singh et al. 2015)

The use of dilute acid causes the removal and allows recovery of most of the hemicelluloses, so higher productivity of glucose can be achieved. This is due to the removal of hemicelluloses that facilitates access to the cellulose (Mosier et al. 2005). Pretreatment of lignocellulosic biomass with diluted acids (0.5 wt.% to 1.0 wt.%) at high temperatures (160 °C to 190 °C) for 30 min to 60 min has been performed within the industry (Gupta and Demirbas 2010; Gírio et al. 2017). Sulfuric acid (H₂SO₄) is mainly used, though hydrochloric, phosphoric, and nitric acids have also been employed. This method, however, has some drawbacks. The most important one concerns the corrosion of the equipment (which occurs even at low acid concentrations), and thus the need for expensive materials used for construction (Mussatto 2016). Acid treatment demands high energy consumption associated with the use of high temperatures and biomass comminution (Mosier et al. 2005; Mussatto 2016). Other very important issues are related to the presence of toxic inhibitory compounds in the hydrolysate, which can inhibit microbial activity and the need of neutralization of hydrolysate or washing of biomass. The neutralization step results in formation of insoluble salts which need to be separated and removed (Singh et al. 2015).

Alkali pretreatment uses alkaline reagents, such as calcium hydroxide (lime), ammonia, and sodium hydroxide (NaOH). Most of these chemicals specifically target hemicellulose acetyl groups and lignin–carbohydrate linkages, which results in high lignin removal and solubilization of the lignocellulosic complex (da Costa Sousa et al. 2009). Alkali hydrolysis requires milder conditions and a lower severity compared with other pretreatment technologies, which leads to a lower sugar degradation (Mosier et al. 2005). Alkali treatment is particularly useful for the processing of hardwoods and agricultural residues, and its effectiveness is highly dependent upon the initial lignin content in the raw material (Alvira et al. 2010). Alkaline treatment has one major issue, which is conversion of alkali into irrecoverable salts and their incorporation into the biomass (Mosier et al. 2005). Also, another limitation is a problem with disposal and purification of hydrolysate, also related with the presence of salts, and formation of inhibitors under harsh conditions (Bensah and Mensah 2013).

The efficiency of downstream processes (i.e., hydrolysis and fermentation) is highly dependent upon both the raw material and pretreatment method employed. Therefore, the aim of this study was to evaluate the impact of three different pretreatment methods (dilute acid, alkali, and SE) on three hardwood biomasses (aspen, birch, and oak) with efficient conversion of the biomass to ethanol.

EXPERIMENTAL

Materials

Aspen (Populus tremula L.), birch (Betula sp. Ehrh.), and oak (Quercus robur L.) wood chips were obtained from a private sawmill located in the northeastern region of Poland. The materials were dried at room temperature, chopped into fragments approximately 1.0 mm to 2.0 mm thick, 3.0 cm to 5.0 cm long, and 0.5 cm to 1.0 cm wide, and stored at room temperature. Table 1 shows the chemical composition of the raw materials.

Table 1. Chemical Composition of the Raw Materials

Enzymatic hydrolysis was performed using commercial enzyme preparations consisting of cellulase from Trichoderma longibrachiatum (C9748, Sigma Aldrich) and β-glucosidase (Novozyme 188, Novozymes, Denmark). For the fermentation experiments, the commercial dry distillers yeast Thermosacc Dry (Lallemand Ethanol Technology, Montreal, Canada) was used, which is a Saccharomyces cerevisiae strain dedicated to the production of biofuels and alcoholic beverages.

Methods

Pretreatment methods

For the dilute acid pretreatment (ACP), wood chips were treated with 0.5% and 2.0% (w/v) H₂SO₄ at 121 °C and a substrate solid loading of 5% (w/v) (dry weight basis) for 1 h. The pretreated biomass was then neutralized to a pH of 5.0 with a 1-M NaOH solution. When the alkali pretreatment (ALP) was used, the wood chips were treated with 0.5% and 2.0% (w/v) NaOH solutions at 121 °C and a substrate solid loading of 5% (w/v, dry weight basis) for 1 h. After pretreatment, the biomass was washed with deionized water until the filtrate was neutral. The SE pretreatment was performed in a batch pilot unit equipped with a 5-L reaction vessel. The wood chips were introduced into the reaction vessel and exposed to saturated steam at 1.5 MPa (198 °C) for 15 min. After saturated steam exposure, a ball valve at the bottom of the reactor was opened suddenly to rapidly bring the reactor to atmospheric pressure. The pretreated fractions were stored at 4 °C before using them for hydrolysis.

Enzymatic Hydrolysis

The pretreated samples were enzymatically hydrolyzed with 5% (w/v, dry weight basis) 50 mM sodium citrate buffer at a pH of 4.8. Penicillin (50 U/mL) and streptomycin (50 μg/mL) (Sigma Aldrich) were added to prevent bacterial growth during the reaction period. Cellulase and β-glucosidase were added at doses of 20 filter paper units/g of cellulose and 15 cellobiose units/g of glucose, respectively. The hydrolysis experiments were performed at 50 °C for 72 h. Untreated samples were hydrolyzed simultaneously as reference samples. After hydrolysis was completed, the samples were collected to determine the concentrations of monomeric sugars and various lignocellulose degradation products with high-performance liquid chromatography (HPLC).

Fermentation

Fermentation was conducted in 500-mL flasks that contained 200 mL of each hydrolysate and supplemented with (NH₄)₂HPO₄ (0.3 g/L). After inoculation with yeast (0.5 g/L), the hydrolysates were incubated at 30 °C for 96 h under anaerobic conditions. When fermentation was completed, the residual sugars and ethanol concentrations of the samples were analyzed with HPLC and gas chromatography (GC), respectively.

Analytical Methods

The total solids, ash, and acid insoluble lignin contents in the raw materials were analyzed according to National Renewable Energy Laboratory protocols (Sluiter et al. 2008a; Sluiter et al.2008b; Sluiter et al. 2012). The cellulose content was analyzed according to the Kurschner–Hoffer method (Kurschner and Hoffer 1993), in which milled raw material is treated three times with a mixture of ethanol and nitric acid (4:1) at 100 °C for 1 h, the cellulose in then washed and dried to constant weight. The hemicelluloses content was analyzed according to Ermakov method (Arasimovich and Ermakov 1987), where hemicelluloses were hydrolyzed with dilute sulfuric acid, and resulting monosaccharides were assayed by the Miller method (Miller 1959). The hydrolysis yield of the substrate was defined as the ratio of reducing sugars in the hydrolysates to the polysaccharides content in the raw material. To quantify the monomeric sugars and lignocellulose degradation products after enzymatic hydrolysis and fermentation, HPLC analysis (Agilent 1260 Infinity, Agilent Technologies, Santa Clara, CA, USA) was performed according to Balcerek et al. (2016a). The ethanol concentration was analyzed using GC analysis (Agilent 7890A, Agilent Technologies, Santa Clara, CA, USA) with a mass spectrometer (Agilent MSD 5975C, ), as was described by Balcerek et al. (2016b). The ethanol yield was calculated based on available glucose present in the hydrolysate, according to the stoichiometric Gay-Lussac equation and expressed as a percentage of the theoretical yield.

Statistical Analysis

All of the samples were prepared and analyzed in triplicate. The statistical analysis was performed using Statistica 10 software (StatSoft, Tulsa, OK, USA). The obtained results were expressed as the mean with the standard deviation and analyzed by a one-way or twoway analysis of variance (ANOVA), followed by Tukey’s post-hoc test at a significance level (p) of 0.05.

RESULTS AND DISCUSSION

Enzymatic Hydrolysis of the Pretreated Biomass

To increase the accessibility of the lignocellulosic materials to enzymatic hydrolysis, various pretreatment methods were used. Of the pretreatment methods commonly used for lignocellulosic biomass, the dilute acid, alkali, and SE methods were compared in this study. The applied conditions were selected based on the presupposition that milder process conditions result in fewer inhibitors obtained in the prehydrolysate. Typically, the pretreatment of hardwoods with SE is carried out at temperatures between 200 °C and 260 °C for 2 to 10 min. However, under more severe conditions hemicellulose is degraded and furan compounds are generated; thus lower temperature and longer residence time were found to be more advantageous (Martin-Sampedro et al. 2011; Singh et al. 2015). Dilute acid pretreatment is mostly carried out at a temperature range from 120 °C and 210 °C for several minutes to an hour, and similar to SE, the lower severity of pretreatment results in less sugar degradation and formation of inhibitors (Xu and Huang 2014). For that reason, the application of milder process conditions of dilute acid and SE treatments, were chosen for evaluation. On the other hand, alkaline treatment can be held at low, or even ambient, temperature and pressure. However, a process carried under such conditions may require long reaction time ranging from an hour to days. In order to accelerate the pretreatment, higher temperature (up to 121 °C) is often applied (de Carvalho et al. 2016). From the hydrolysates obtained, the monomeric sugars concentrations were evaluated. Subsequently the glucose content was determined, which was later metabolized by yeast during fermentation. The results are presented in Table 2. Additionally, the lignocellulose degradation products concentration was also analyzed.

In the samples obtained after acid pretreatment and enzymatic hydrolysis of the aspen chips, the main content of the reducing sugars was glucose (13.3 g/L and 9.7 g/L for 0.5% and 2.0% H₂SO₄ (w/v), respectively). After enzymatic hydrolysis of untreated aspen sawdust, the concentration of xylose was 1.5 g/L. The application of dilute acid pretreatment resulted in a significantly (p < 0.05) higher xylose concentration (7 g/L), which may have been because of the decomposition of hemicellulose contained in the raw material. By contrast, in the hydrolysates obtained from the birch chips, the glucose content was low and did not exceed 4 g/L. The xylose content in the samples that were not pretreated was 0.25 g/L, and applying the dilute acid pretreatment increased the concentration to almost 8 g/L (p < 0.05). The hydrolysates from the oak chips contained almost twice as much arabinose as those obtained from the aspen or birch chips, possibly because of a different chemical composition of the hemicellulose fraction in the raw materials. The glucose and xylose contents in the oak hydrolysates, as was the case for the birch chips, did not exceed 3.5 g/L and 8 g/L, respectively (p > 0.05). The low sugar contents in all of the hydrolysates may have been caused by the high resistance of the lignocellulosic raw materials to acid hydrolysis because of the presence of a high lignin content in the biomass. Cara et al. (2008) studied the effect of different sulfuric acid concentration (0.2, 0.6, 1.0 and 1.4% w/w) and temperatures (range from 170 to 210 °C) applied during dilute acid pretreatment on subsequent saccharification of olive tree biomass. The authors obtained the best hemicellulose recovery rate (83%) in the prehydrolysate after pretreatment at 170 °C with 1% w/w sulfuric acid. The maximum cellulose hydrolysis yield (76.5%) was achieved from solids pretreated at 210 °C with 1.4% acid concentration.

Table 2. Concentration of Monomeric Sugars in Liquid Fraction after 72 h of Enzymatic Hydrolysis of Biomass Obtained after Various Pretreatment Methods

After the alkali pretreatment, the derived hydrolysates from the aspen chips contained mainly glucose (approximately 70% to 80% of the released reducing sugars). The glucose content reached 23 g/L, while the xylose concentration was only 5 g/L to 6 g/L. The arabinose concentration was low (0.02 g/L) in the hydrolysates from the aspen and birch chips. The glucose and xylose concentrations in the hydrolysates obtained from the alkali-treated oak and birch wood were significantly lower (p < 0.05) than those from the aspen wood, which reached 7 g/L to 10 g/L and 4 g/L to 5 g/L, respectively. The hydrolysates from the oak chips contained slightly more arabinose than those obtained from the aspen and birch chips (p < 0.05). Mirahmadi et al. (2010), determined the effect of alkaline pretreatment of birch and spruce wood, at a temperature between -15 and 100 °C, for 2 h, with 7.0 w/w NaOH solution. The authors have found that as a result of pretreatment the significant reduction of hemicellulose and the crystallinity of cellulose occurred, resulting in improvement of enzymatic hydrolysis of birch from 6.9% to 82.3% (at 100 °C), and for spruce from 14.1% to 35.7% (at 5 °C).

Steam explosion is one of the most frequently used pretreatment methods for lignocellulosic raw materials. In this work, the application of SE did not considerably affect the reducing sugar contents released during enzymatic hydrolysis of the raw materials (Table 2). This may be the result of mild conditions used in experiments. As reported by Ibrahim and Glasser (1999), and Josefsson et al. (2002) the glucose content in solid fraction increased with increasing severity of steam pretreatment, from 68.4 to 76.7% and from 88.8 to 96.9%, respectively for oak and aspen wood. In the present study, the impact of the pretreatment method was strongly dependent upon the biomass (p < 0.05). The best pretreatment resulted in 15 g/L glucose and 2 g/L xylose for the aspen chips, 10 g/L glucose and 2 g/L xylose for the birch chips, and 6.8 g/L glucose and 1.5 g/L xylose for the oak chips. For all of the tested materials, 90% of the sugars released after pretreatment were glucose. The arabinose content in all of the samples was at a similar level (approximately 0.022 g/L (p > 0.05)). Vivekanand et al. (2013) conducted an autohydrolysis study of birch chips at various temperature (170 °C to 230 °C) and time conditions (5 min to 15 min). They found that at severe pretreatment conditions, a higher degree of xylan degradation and pseudo-lignin formation occurred. They obtained the highest glucose concentration (corresponding to a 97% yield) after autohydrolysis at 220 °C for 10 min.

Based on the released monomeric sugar content, the hydrolysis yield was calculated (Fig. 1). The raw material most susceptible to hydrolysis was the aspen chips, as even the untreated aspen chips furnished a 27% yield, which was significantly (p < 0.05) higher than that from the other two raw materials. The untreated birch and oak chips resulted in similar hydrolysis yields (p > 0.05) of 5.6% and 8.6%, respectively. The H₂SO₄ pretreatment of the aspen chips at both the 0.5% and 2% concentrations (w/v) gave similar hydrolysis yield results (p > 0.05), at approximately 60%.

The alkali pretreatment using the 0.5% (w/v) NaOH concentration resulted in a similar yield as that with acid at higher concentration (p > 0.05). However, increasing the NaOH concentration to 2% (w/v) resulted in a significant (p < 0.05) increase in the hydrolysis efficiency to almost 80% of the theoretical yield. The hydrolysis efficiency of the birch and oak biomass for all of the pretreatment methods was similar (p > 0.05); with 2% H₂SO₄, higher yields were obtained from hydrolysis of the oak chips compared with that from the birch chips (p < 0.05) (51% and 58% efficiency, respectively).

Fig. 1. Enzymatic hydrolysis yield of the aspen, birch, and oak chips treated with H₂SO₄, NaOH (at concentrations of 0.5% and 2.0% (w/v)), and SE; Error bars show the standard deviation; Different lower-case letters indicate significant differences (p < 0.05) between the mean values of the hydrolysis yield (two-way ANOVA and Tukey’s post-hoc test)

Effect of the Pretreatments on the Formation of Lignocellulose Degradation Products and Ethanol Production Efficiency

After hydrolysis was completed, all of the samples were subjected to fermentation using dry distillers yeast. The susceptibility of the hydrolysates to fermentation was reliant on the concentrations of specific chemical compounds present in the starting material, formed during pretreatment, or formed during the fermentation processes. The concentrations of these compounds during fermentation depends largely on the configuration of the downstream process. All of the hydrolysates were also analyzed for the concentrations of sugar degradation products, such as aliphatic acids (lactic, formic, and acetic acids) and furan compounds (furfural and 5-Hydroxymethylfurfural (5-HMF)) (Table 3).

Among the aliphatic acids, the highest concentration was observed for acetic acid, which may have been related to the chemical structure of the hardwood hemicellulose, in which the xylan chain is highly substituted with acetyl groups (Larsson et al. 1999). The concentration of acetic acid in the untreated samples was approximately 0.09 g/L to 0.18 g/L. The alkali pretreatment led to a reduction in the acetic acid concentration to 0.03 g/L to 0.05 g/L, regardless of the raw material used (p > 0.05). However, the dilute acid pretreatment caused a significant increase in the acetic acid concentration to 0.78 g/L to 1.63 g/L, 1.61 g/L to 1.71 g/L, and 1.77 g/L to 1.85 g/L for the oak, aspen, and birch chips, respectively (p < 0.05). The lactic and formic acids concentrations were generally lower than the acetic acid concentration, and strongly depended on both the raw material and pretreatment method (p < 0.05). The hydrolysates obtained from the hardwood biomass and agricultural wastes contained noticeably higher concentrations of these compounds than those obtained from softwood (Larsson et al. 1999).

Table 3. Concentration of Select Lignocellulose Degradation Products in the Liquid Fraction after 72 h of Enzymatic Hydrolysis of Biomass Obtained after Various Pretreatment Methods

Taking into consideration furan derivatives, the furfural concentration was significantly higher than the 5-HMF and generally increased in the following order for the different pretreatments: SE < ALP < ACP. In the case of the dilute acid pretreatment, increasing the acid concentration from 0.5% to 2% (w/v) caused 3- and 4-fold higher furfural concentrations for the birch and oak chips, respectively. For the aspen chips, both 0.5% and 2% (w/v) H₂SO₄ resulted in high furfural concentrations of approximately 10 g/L. The 5-HMF concentration was also dependent upon the raw material and pretreatment method (p < 0.05). The highest amount was detected in the case of the aspen chips pretreated with dilute acid. Both furfural and 5-HMF are the dominant furan compounds present in lignocellulosic hydrolysates from the dehydration of pentoses and hexoses, respectively. The presence of furfural impairs ethanol productivity by yeast, similar to 5-HMF, but the latter is remarkably more toxic at the same doses (Larsson et al. 1999). Wikandari et al. (2010) showed that the addition of furfural at doses above 0.5 g/L results in a 73% decrease in the ethanol yield. Fermentation of hydrolysates produced from this study generated a relatively high ethanol yield based on glucose available in hydrolysates (Fig. 2).

Fig. 2. Ethanol yield from the fermentation of the aspen, birch, and oak chips treated with H₂SO₄, NaOH (at concentrations of 0.5% and 2.0%), and SE; Error bars show the standard deviation; Different lower-case letters indicate significant differences (p < 0.05) between the mean values of the hydrolysis yield (two-way ANOVA and Tukey’s post-hoc test)

For the untreated chips, the ethanol yield was approximately 90% of the theoretical yield (p > 0.05), and the obtained efficiency was similar when the biomass was pretreated with 0.5% NaOH (p > 0.05). The concentration of toxic compounds in hydrolysates from untreated biomass was negligible; thus the fermentation activity of yeast had not been inhibited. However, it is worth mention, that the total amount of glucose in untreated samples was lower (p < 0.05) in comparison with samples after pretreatment. Fermentation of the hydrolysates obtained after the SE pretreatment resulted in 83% and 86% efficiencies for the oak and aspen chips, respectively. However, in the case of the birch chips, the obtained ethanol yield was significantly lower (p < 0.05). The lowest fermentation yield from the hydrolysates was obtained after the dilute acid pretreatment, wherein increasing the H₂SO₄ concentration from 0.5% to 2% resulted in a significant decrease in the ethanol yield. Jung et al. (2014) obtained over 70% of the theoretical ethanol yield after fermentation of the hydrolysate from the acid-base pretreatment at 190 °C for 2 min.

In order to better compare the total ethanol yield obtained after fermentation of biomass pretreated by different methods, the obtained ethanol concentration was calculated and expressed in liters of pure ethanol per tonne of raw wood (Fig.3).

Fig. 3. Ethanol yield expressed in L of ethanol per tonne of raw wood biomass, obtained after the fermentation of the aspen, birch, and oak chips treated with H₂SO₄, NaOH (at concentrations of 0.5% and 2.0%), and SE; Error bars show the standard deviation; Different lower-case letters indicate significant differences (p < 0.05) between the mean values of the hydrolysis yield (two-way ANOVA and Tukey’s post-hoc test)

The highest ethanol yield (252 L/tonne) was achieved after fermentation of alkali-pretreated aspen sawdust. In general, the biomass of aspen was found to be most susceptible to hydrolysis and fermentation processes, as even untreated chips gave relatively high ethanol yield (112 L/tonne). The biomass of birch and oak was more resistant to applied pretreatment conditions. The highest achieved ethanol yield was only 105 L/tonne (for both birch and oak) when alkali pretreatment was used. The obtained results are lower than those reported by Stephen et al. (2012), who gave the theoretical yield (based on C6 sugars only) as 318 and 439 L/bone dry ton for poplar stem and Douglas fir heartwood, respectively.

CONCLUSIONS

1. The highest hydrolysis yield (79.6% of the theoretical yield) was obtained for the aspen chips with 2% NaOH, while the most efficient pretreatment method for the oak chips was the dilute acid pretreatment with 2% H₂SO₄.
2. When considering both the enzymatic hydrolysis and ethanol fermentation yields, the best results were obtained with the aspen chips pretreated with 2% NaOH.
3. The oak biomass was the most resistant raw material, regardless of the pretreatment applied.

ACKNOWLEDGMENTS

This research was funded by the Polish National Center for Research and Development under the project BIOSTRATEG2/296369/5/NCBR/2016.

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Article submitted: May 9, 2018; Peer review completed: July 7, 2018; Revised version received and accepted: July 27, 2018; Published: July 31, 2018.

DOI: 10.15376/biores.13.3.6970-6984