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Bioorganosolv pretreatments of P. radiata by a brown rot fungus (Gloephyllum trabeum) and ethanolysis
Enzyme and Microbial Technology 47 (2010) 11–16
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Bioorganosolv pretreatments of P. radiata by a brown rot fungus (Gloephyllum trabeum) and ethanolysis ˜ a,b , Victoria Melin a,b , Jaime Baeza a,b , Mariel Monrroy a , Jeniffer Ibanez a,c Regis Teixeira Mendonc¸a , David Contreras a,b , Juanita Freer a,b,∗ a b c
Renewable Resources Laboratory, Biotechnology Center, Universidad de Concepción, Casilla 160-C, Concepción, Chile Faculty of Chemical Sciences, Universidad de Concepción, Casilla 160-C, Concepción, Chile Faculty of Forest Sciences, Universidad de Concepción, Casilla 160-C, Concepción, Chile
a r t i c l e
i n f o
Article history: Received 29 November 2009 Received in revised form 28 January 2010 Accepted 29 January 2010 Keywords: Brown rot fungi Organosolv Bioethanol
a b s t r a c t Pinus radiata wood chips were pretreated with the brown rot fungus Gloephyllum trabeum for 3 weeks followed by an organosolv delignification with ethanol:water mixture at pH 2. The organosolv process was assessed using biotreated material that showed a high viscosity loss and a mass loss ranging from 6% to 8%. The experiment was designed to optimize the organosolv conditions, ethanol:water ratio and H factor (factor that combines temperature and time in one variable) to obtain the highest ethanol yield by simultaneous enzymatic saccharification and fermentation (SSF) for 96 h at 10% consistency. The optimized conditions for organosolv process for biotreated material were: ethanol:water mixture (60/40, v/v) and 1156 H factor (185 ◦ C, 18 min); the optimized conditions for the control (chips without biotreatment) were: ethanol:water mixture (60/40, v/v) and 6000 H factor (200 ◦ C, 32 min). The experimental ethanol yields obtained at these conditions were 63.8% and 64.3% (wood basis) for biotreated material and control, respectively. The maximum amount of ethanol that could be produced from P. radiata is 252 g ethanol/kg wood, assuming total glucose conversion into ethanol. The results indicate that the obtained ethanol was 161 g ethanol/kg wood from both materials. The biotreatment of the wood before the organosolv process improved solvent accessibility. To obtain the same ethanol yield, lower process severity was required in the biotreated samples in comparison with the control. © 2010 Published by Elsevier Inc.
1. Introduction Oil depletion and the greenhouse effect have increased the need for alternative non-fossil transportation fuels [1,2]. Research in bioethanol production from lignocellulosic biomass (LCB) has grown significantly over the last few decades. Lignocellulose is the most abundant biomass that can be converted into liquid fuels by enzymatic hydrolysis and microbial fermentation. According to U.S. Department of Energy, it can be estimated that terrestrial plants produce 13 × 1010 metric tons of biomass per year or about 6 times of the world’s energy requirement [3]. LCB includes materials such as agricultural and forestry residues, municipal solid waste, and industrial wastes. LCB can be used as an inexpensive feedstock for production of renewable fuels and chemicals [4].
∗ Corresponding author at: Centro de Biotecnología, Universidad de Concepción, Barrio Universitario s/n, Concepción, Chile. Tel.: +56 41 2204601; fax: +56 41 2204074. E-mail address: [email protected] (J. Freer). 0141-0229/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.enzmictec.2010.01.009
However, due to their structural features, LCB has limited accessibility to enzymes or microorganisms. LCB is made up of cellulose, hemicellulose, and lignin. Since the carbohydrate polymers are tightly bound to the lignin, mainly by hydrogen bonds but also by some covalent bonds [5], pretreatment is required to make biomass more accessible to the enzymes that convert the carbohydrate polymers into fermentable sugars. In short, pretreatment alters the physical features and chemical composition, specifically improving enzyme access and effectiveness by breaking the lignin seal, removing hemicellulose, disrupting the crystalline structure of cellulose, and expanding the structure to increase pore volume [1,6–8]. A large number of pretreatments have been tested by many researchers, which can be broadly classified into physical, chemical, physicochemical, biological and combined pretreatments [5,6,9–11]. Pretreatments using organic solvents have been reported as efficient processes. These pretreatments break the internal lignin and hemicellulose bonds and separate the lignin and hemicellulose fractions that can be potentially converted to useful products. Methanol, ethanol, butanol, n-butylamine, acetone, ethylene glycol, and other compounds have been used in the organosolv process [5,12]. These treatments have sometimes been combined with biological treatments [11,13].
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In biological pretreatments, natural wood attacking microorganisms that can degrade lignin and holocellulose are allowed to grow on the biomass, producing lignin–holocellulose complex degradation. The main biological pretreatments include fungi and their enzymes. The principal organism used for biological pretreatment of lignocellulose is white rot fungi [5,14]. These fungi have been shown to effectively disrupt the lignin–cellulose complex. Other types of wood rot fungi, such as the brown rot fungi, are responsible for naturally breaking down the highly ordered cellulose crystalline structure. These fungi preferentially degrade the wood polysaccharides, but also partially oxidize lignin [15]. They degrade holocellulose in an unusual manner, causing a rapid decrease in degree of polymerization with a low mass loss. It has been proposed that initial depolymerization is caused by producing small, diffusible, extracellular oxidants (free radicals), operating at a distance from the hyphae [15,16]. As a result of the initial attack by brown rot fungi and the depolymerization of the cellulose, wood strength rapidly decays. The aim of this work was to use a fungal pretreatment prior to the organosolv process (ethanolysis) in order to improve the solvent accessibility, decreasing the H factor. The ethanolysis process was optimized for conversion of Pinus radiata into ethanol by SSF. 2. Experimental 2.1. Raw material and preparation P. radiata D. Don samples were chipped and screened to approximately 2.0 cm × 2.5 cm × 0.5 cm. The wood chips were air-dried until reaching 10% (w/w) moisture, and then stored in plastic bags until its use. Prior to the biodegradation experiments, wood chips were immersed in water for 36 h and the excess of water was drained. Moist wood chips were sterilized (121 ◦ C/30 min) and brought to room temperature. 2.2. Fungus, inoculum preparation and wood biodegradation Gloephyllum trabeum (ATCC 11539) was used for the wood chips treatment. The strain was maintained on 2% (w/v) malt extract, 0.5% (w/v) soybean peptone, 1.5% (w/v) agar culture plates at 24 ◦ C for 1 week. Then, fungal mycelium was grown in 2 L Erlenmeyer flasks with 200 mL liquid culture medium (sterilized at 121 ◦ C, 30 min) containing 2% (w/v) malt extract and 0.5% (w/v) soybean peptone. Each flask was inoculated with 20 discs (8 mm in diameter) of the strain pre-cultured in solid medium and maintained unshaken for 2 weeks at 25 ◦ C. The grown mycelium was filtered and washed with 300 mL sterilized water. Washed mycelium obtained from several cultures was blended with 50 mL of sterilized water in two cycles of 10 s. The mycelium suspension was used to inoculate the sterilized wood chips in bioreactors. Each bioreactor was loaded with 300 g wood chips and inoculated with a suspension volume corresponding to 500 mg of fungal mycelium per kg of dry wood. The inoculated wood chips were incubated in an acclimatized room at 25 ◦ C and 55% relative humidity (RH) for 3 weeks. After the biodegrading treatment, the superficial mycelium was removed from the wood chips by brushing and the decayed wood chips were dried at 40 ◦ C for 48 h. The moisture of each sample was determined and the calculated initial and final dry weights were used to determine mass loss due to fungal biotreatment. The samples obtained in this process were named “biotreated material”. Control wood chip samples were prepared under the same conditions but without fungus addition. All the experiments were carried out in sextuplicate.
2.3. Organosolv pretreatment (ethanolysis) Organosolv pretreatment was carried out in a 1-L Parr reactor (Moline, IL, USA) loaded with 40 g of wood chips (dry weight base) of the biopretreated material or control and 240 mL of an ethanol (95%):water mixture (60/40, v/v) containing sulfuric acid as catalyst (0.13%, w/v pH approximately 2). The solvent:wood ratio inside the reactor was 6:1 (w/w). The H factor was 1156 (185 ◦ C) and 6000 (200 ◦ C) for biotreated and control materials, respectively. The cooking severity in the organosolv process was described by the H factor. This factor relates the time and temperature in a single variable. These experimental conditions were previously determined by a factorial analysis, where these values were optimized for a maximal ethanol yield. The effect of the ethanol:water mixture (50/60–70/30, v/v) and H factor (800–6000) were evaluated (Table 1). The pressures obtained were 1.8 MPa (at 185 ◦ C) and 2.5 MPa (at 200 ◦ C) for biotreated material and control, respectively. Once the process ended, the reactor was cooled in a water–ice bath. The supernatant was removed by decantation and the solid pressed out on 130-nylon mesh. The solid was washed with water, disintegrated in a blender for 5 min, and then passed by a Fiber Classifier (Regmed, Brazil) to segregate fibers from the rejects. The pulp was centrifuged. The samples were then stored in plastic bags at 4 ◦ C. 2.4. Chemical characterization of wood and organolsolv pulp samples Milled wood samples were extracted with acetone for 16 h according to standard TAPPI procedure T280 pm99. Wood and pulp samples were characterized for their carbohydrate composition using the methodology described by Ferraz et al. [17]. In a test tube, 300 mg of extractive-free milled wood was weighed and 3 mL of 72% (w/w) H2 SO4 was added. The hydrolysis was carried out in a water bath at 30 ◦ C for 1 h with stirring every 10 min. Later, the acid was diluted to 4% (w/w) with 79 mL of distilled water and the mixture transferred to a 250-mL Erlenmeyer flask and autoclaved for 1 h at 121 ◦ C. The residual material was cooled and filtered through a sintered glass filter number 4. The solid fraction, insoluble lignin, was dried and weighed. The concentration of monomeric sugars in the soluble fraction was determined by high-performance liquid chromatography (HPLC) in a Hewlett Packard 1050 using a Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA), at 45 ◦ C, eluted at 0.6 mL/min with 5 mM H2 SO4 and with a refractive index detector. Glucose, mannose and arabinose were used as external calibration standards (since the retention time and response for mannose, galactose and xylose are similar, these three sugars were expressed as mannose). The factors used to convert sugar monomers to anhydromonomers were 0.90 for glucose to glucan and for mannose to mannan, and 0.88 for arabinose to arabinan. All samples were analyzed in triplicate. The holocellulose and viscosity were determined according to standard TAPPI T 9m54 and T230 om04. 2.5. Inoculum preparation The yeast strain used in this work was a thermal acclimatized (40 ◦ C) Saccharomyces cerevisiae IR2T9 [9]. The inoculum was grown in 100 mL of liquid culture consisting in glucose, 50 g/L; yeast extract, 5 g/L; peptone, 5 g/L; KH2 PO4 , 1.0 g/L; MgSO4 ·7H2 O, 0.50 g/L; NH4 Cl, 2 g/L in a 500-mL Erlenmeyer flask. The culture was incubated for 48 h at 40 ◦ C in an orbital shaker at 150 rpm. 2.6. Simultaneous saccharification and fermentation (SSF) The simultaneous saccharification and fermentation were performed at 10% substrate consistency. In a 125-mL Erlenmeyer flask, 3 g dry weight of pretreated material (65% humidity) was suspended in a total reaction volume of 30 mL 0.05 M citrate buffer solution (pH 4.8). Nutrients, consisting in KH2 PO4 , 1.0 g/L; MgSO4 ·7H2 O, 0.50 g/L; peptone, 5.0 g/L; yeast extract, 5.0 g/L, were added. The enzymatic hydrolysis of the samples was performed using a commercial preparation of Trichoderma reesei cellulases Celluclast (70 FPU/mL; Novozymes, NC, USA), supplemented with a -glucosidase Novozym 188 (230 IU/mL; Novozymes, NC, USA),
Table 1 Experimental design for organosolv pretreatment of P. radiata. Experimental number
1 2 3 4 5 6 7 8 9 10 11
Control
Biotreated
H factor
Organic solvent (%)
H factor
Organic solvent (%)
5000 (−1) 7000 (1) 5000 (−1) 7000 (1) 4586 (−1.41) 7423 (1.41) 6000 (0) 6000 (0) 6000 (0) 6000 (0) 6000 (0)
50 (−1) 50 (−1) 70 (1) 70 (1) 60 (0) 60 (0) 46 (−1.41) 74 (1.41) 60 (0) 60 (0) 60 (0)
900 (−1) 1300 (1) 900 (−1) 1300 (1) 817 (−1.41) 1383 (1.41) 1100 (0) 1100 (0) 1100 (0) 1100 (0) 1100 (0)
50 (−1) 50 (−1) 70 (1) 70 (1) 60 (0) 60 (0) 46 (−1.41) 74 (1.41) 60 (0) 60 (0) 60 (0)
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where the amounts of enzyme were 20 FPU and 40 IU/g of pretreated material. After enzyme addition, the yeast inoculum was added with a cell density 6.0 g/L (3.5 × 108 to 3.3 × 109 yeast cell/mL). SSF was performed at 40 ◦ C and 150 rpm for 96 h. Samples were taken at 24, 48, 72 and 96 h and analyzed for ethanol content by gas chromatography (GC) on a Perkin-Elmer autosystem XL Headspace using a FID detector and a column HPS MS30m. The GC program was: 50 ◦ C × 3 min; 10 ◦ C/min, 100 ◦ C × 1 min; 25 ◦ C/min, 125 ◦ C × 1 min. The temperatures of the injector and detector were 200 and 300 ◦ C, respectively. Ethanol yield were calculated as a percentage of the theoretical yield. The theoretical yield was calculated by dividing the amount of ethanol obtained (g) by the amount of glucose in the pretreated material (g), assuming that all the potential glucose in the pretreated material is available for fermentation, with a fermentation yield of 0.51 g ethanol/g glucose multiplied by 100. All the determinations were realized in duplicate. Fig. 1. Holocellulose viscosity of P. radiata wood chips under brown degradation by G. trabeum.
2.7. Response surface methodology In order to determine the conditions to obtain maximal ethanol yield from the organosolv pulps, the variables organic solvent concentration and H factor were studied. The influence of each variable was determined using response surface methodology (RSM) [18]. This model is based on a central composite circumscribed design made of a factorial design and star points. The variable values were coded and normalized in unitary values: −1 was defined as the lowest value of a variable and +1 was defined as the highest value. From the extreme variable values, the central point (coded 0) was set and assayed in triplicate. Four star points distributed at a distance of 1.41 from the central point were included. The complete experimental design for the reaction conditions of the two methods optimized for ethanol yield is presented in Table 1. A second-order function that best describes the system’s behavior was determined by a multiple lineal regression method (MLR). The statistical validation was performed by a one-way ANOVA test with 95% confidence level. The optimal condition values were determined based on the response surface calculated using the SIMPLEX method [18]. All the calculations were performed with the software Modde 7.0.0.1 (Umetrics, USA).
3.2. Organosolv pulps characterization
3. Results and discussion
Table 3 summarizes the chemical composition and pulp yield for the organosolv pulps produced from biotreated material and control. The pulp yields for the biotreated material were 30% lower for H factors between 4000 and 7000 (data not shown), probably due to the process’s high severity. Therefore, H factors employed in the factorial design for the biotreated material were lower than for the control. The organosolv pulps yields obtained were 27–43% and 23–41% for the biotreated material and control, respectively. In general, there were few rejects (<5) in the pulping process. The glucan content was slightly higher for control pulps (81–85%) than biotreated pulps (75–80%). Almost all hemicelluloses were solubilized or degraded. The hemicelluloses remaining in the pulps were below 3%. The lignin content varied between 10% and 20% in the pulps, and was slightly higher for biotreated pulp.
3.1. Biodegradation of P. radiata
3.3. Simultaneous saccharification and fermentation (SSF)
Wood chips of P. radiata were decayed by G. trabeum for different periods (data not shown). During the first month of biodegradation, the mass loss ranged between 6% and 8%. Weight loss was calculated by the difference between the weights of the wood sample before and after the fungus treatment and the value obtained was 6%. We consider that this value is underestimated since during the fungus treatment, the cellulose chains were fragmented, and a water molecule is incorporated for each broken bond. The weight loss calculated based on the main component content was 8.2%. However, this value is overestimated; for example, the glucan content is calculated by multiplying the glucose content in the hydrolysate by 0.9 (162/180) to reflect the weight gain of glucose by the addition of water to the anhydroglucan during hydrolysis. It is not considered that there are many chains, for that reason more water is subtracted. The hemicellulose (mannan + galactan + xylan) and glucans decreased approximately 5% and 3%, respectively. Holocellulose viscosity decreased linearly over time until the third week (Fig. 1), but without a large carbohydrate mineralization. The material biotreated for 3 weeks was selected to assay the organosolv process. Table 2 summarizes the chemical composition of the biotreated and control P. radiata wood chips.
The hydrolysis and fermentation of the cellulose were performed by SSF process. Previous studies have shown that SSF processes have several advantages over a separate hydrolysis and fermentation (SHF) process: lower enzyme inhibition by hydrolysis products, less overall processing times, and higher ethanol yields. SSF is also considered a less capital intensive process [11,19]. In order to assure greater conversion in all experiments, the SSF was evaluated for 96 h. The ethanol yields obtained from the biotreated pulps varied between 73% and 91% (pulp basis) and 51% and 64% (wood basis). For the control sample, ethanol yields varied between 82% and 87% (pulp basis) and 43% and 65% (wood basis). The results indicated that the amounts of ethanol obtained were 129–161 g ethanol/kg wood and 108–164 g ethanol/kg wood for the biotreated and control pulps, respectively. The ethanol yields from biotreated and control pulps were very similar, but the organosolv process conditions were less severe for the biotreated samples. A probable explanation for this can be physical structural features of biotreated material, such as distribution of lignin in the biomass matrix, crystallinity, pore volume and biomass particle size, where the material is more accessible in the organosolv process. These features have been considered relevant for conversion of lignocellulosic biomass to liquid fuels [1,7,20].
Table 2 Chemical composition of wood chips from control and biotreated P. radiata.
a
Wood samples
Glucan (%)
Mannan + galactan + xylan (%)
Arabinan (%)
Lignin (%)
Extractives (%)
Viscosity (dL/g)
Control Biotreated Biotreated (cwl)a
44.5 ± 0.2 44.1 ± 0.1 41.5 ± 0.1
19.0 ± 0.4 15.4 ± 0.1 14.4 ± 0.4
1.2 ± 0.03 0.83 ± 0.02 0.8 ± 0.02
27.5 ± 0.6 29.4 ± 0.3 27.6 ± 0.3
1.5 ± 0.04 1.2 ± 0.02 1.1 ± 0.02
5.8 ± 0.1 2.5 ± 0.2 2.5 ± 0.2
Values corrected for weight loss due to fungal biodegradation.
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Table 3 Chemical composition of organosolv pulps from control and biotreated P. radiata. Experimental number
Wood samples
H factor
Organic solvent (%)
Pulp yield (%)
Rejects (%)
Glucan (%)
Mannan + galactan + xylan (%)
Lignin (%)
1 2 3 4 5 6 7 8 9 10 11
Control Control Control Control Control Control Control Control Control Control Control
5060 7000 5077 7008 4825 7423 6103 6049 6048 6051 6032
50 50 70 70 60 60 46 74 60 60 60
38 27 34 23 38 33 37 38 40 41 40
2 0 2 1 5 1 5 5 4 2 2
84 81 84.4 82.6 87 84.8 81 83 85 84 83
± ± ± ± ± ± ± ± ± ± ±
1 1 1 0.5 1 0.4 2 1 1 1 1
1.6 1.3 1.6 0.5 1.3 1.2 0.6 0.8 0.6 0.9 0.6
± ± ± ± ± ± ± ± ± ± ±
0.4 0.3 0.02 0.01 0.6 0.3 0.02 0.02 0.04 0.1 0.1
14.1 16.5 14.7 15.4 10.3 11.4 11.4 14.4 15.1 13.5 13.0
± ± ± ± ± ± ± ± ± ± ±
0.4 0.1 0.2 0.5 0.1 0.1 0.1 0.7 0.1 0.3 0.3
1 2 3 4 5 6 7 8 9 10 11
Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated
900 1312 901 1312 811 1368 805 1088 1092 1085 1073
50 50 70 70 60 60 46 74 60 60 60
34 37 38 39 40 41 27 39 41 43 39
7 6 2 4 4 4 18 3 3 4 4
78.4 78.2 78 74.3 76.2 78 75.4 78.9 78.4 77.7 80
± ± ± ± ± ± ± ± ± ± ±
0.5 0.6 0.2 0.2 0.2 1 0.4 0.5 0.6 0.1 1
1.2 1.2 2.7 2.2 2.5 1.9 1.2 1.2 1.6 2.3 2.2
± ± ± ± ± ± ± ± ± ± ±
0.01 0.05 0.1 0.1 0.1 0.1 0.02 0.2 0.1 0.2 0.2
16.5 17.3 17.7 16.3 16.7 16.5 20.2 16.5 16.3 16.8 15.4
± ± ± ± ± ± ± ± ± ± ±
0.6 0.2 0.2 0.2 0.2 0.4 0.3 0.5 0.1 0.1 0.4
3.4. Response surface methodology for the organosolv pretreatment The main purpose of this study was to compare biotreated and control material in terms of their response to organosolv pretreatments in order to optimize these pretreatments to achieve maximum ethanol yield. From the experimental design data (Table 1) and its corresponding ethanol yield (Table 4), a quadratic polynomial was determined (Eqs. (1) and (2)) and validated by the ANOVA test for the biotreated and control pulps. The variables in quadratic polynomial were scaled and centered. Biotreated material: Y = 64.27 ± 1.34 + 1.86 ± 0.84Z1 + 0.98 ± 0.98Z2 − 2.67 ± 1.02Z1 2 − 8.30 ± 1.04Z2 2
(1)
Control: Y = (122e18 ± 410e17 − 214e17 ± 267e17 Z1 − 5.41e16 ± 253e17 Z2 − 3.97e17 ± 335e17 Z1 2 − 424 e17 ± 2.97e17 Z2 2 )
Z=
Xi − X0 Xmax − X0
1/10
(2)
(3)
where Z denotes the scaled and centered value from original variables (X) (Eq. (3)), Z1 correspond to H factor, Z2 to the organic solvent concentration (ethanol, %, v/v), X0 is midrange and Y is the ethanol yield (%, wood basis). The error values corresponded to a 95% confidence level. The response surfaces for the polynomials (plot inside the dominium limited for the experimental planning) are shown in Fig. 2. For the biotreated pulp, the linear terms for H factor and the organic solvent concentration have positive coefficients, meaning that the ethanol yield incremented with the increase in these variables until a maximum value, as shown by the negative quadratic terms for all variables. According to the polynomial, the H factor is the main determining factor for maximum ethanol yield. For the control pulp, the linear terms for the H factor and the organic solvent concentration have negative coefficients, meaning that the ethanol yield increases with the decrease in these variables until a maximum value. Considering the confidence intervals, the inter-
Fig. 2. Surface response for the ethanol yield. (A) Control and (B) biotreated samples.
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Table 4 Experimental ethanol yield and response polynomial calculated values. Experimental number
1 2 3 4 5 6 7 8 9 10 11
Control (ethanol yield (%))
Biotreated (ethanol yield (%))
Experimental
Calculated
Experimental
Calculated
62.5 43.0 53.1 36.8 63.0 56.1 57.3 60.0 63.5 63.7 65.6
60.6 54.7 59.5 51.5 62.6 50.7 58.2 55.9 64.4 64.4 64.4
51.6 54.9 52.8 55.3 56.6 61.8 36.5 49.5 63.8 63.1 64.6
50.4 54.2 52.4 56.1 56.3 61.6 37.7 49.0 64.3 64.3 64.3
action between variables did not have a significant effect on ethanol yield. Based on the experimental conditions used in the experimental design, the polynomial was used to predict the ethanol yield. The responses were close to the experimental values (Table 4) with a correlation coefficient (r2 ) = 0.99 and (r2 ) = 0.79 for the biotreated and control pulps, respectively. These values together with the ANOVA test statistically validated the model. The optimum values of the variables for maximal ethanol yield were determined by the SIMPLEX method using the maximum values of the response surface [18]. The predicted values for biotreated pulps were: 1156 H factor and 60% (v/v) organic solvent (at 185 ◦ C for 17 min). According to the polynomial, the maximum ethanol yield was 64.6 ± 1.2% (at 95% confidence level), while the experimental ethanol yield for this pulp obtained at these conditions was 63.8%, agreeing with the predicted value (Fig. 2). The predicted values for control pulps were: 6000 H factor and 60% (v/v) organic solvent (at 200 ◦ C for 32 min). According to the polynomial, the ethanol yield was 64.4 ± 2.2%, while the experimental ethanol yield for this pulp obtained at these conditions was 64.3%, agreeing with the predicted value (Fig. 3). Ethanol production was 40 g/L for both materials (Fig. 2). The maximal ethanol amount that could be produced from P. radiata is 252 g ethanol/kg wood considering total conversion of glucose into ethanol. The results indicate that the amount of ethanol obtained is 161 g ethanol/kg wood for both materials. The ethanol yield obtained in this study is similar to the amount ˜ et al. [11] from beech and P. obtained by Itoh et al. [13] and Munoz radiata, respectively, with white rot fungi although the organosolv conditions used in these studies were significantly higher. Itoh et al. [13] employed 200 ◦ C at 120 min and 180 ◦ C at 60 min for the ˜ control and biotreated materials, respectively. Munoz et al. [11]
Fig. 3. Ethanol production from SSF of P. radiata organosolv pulps. Ethanol yield () biotreated material and () control. Ethanol amount () biotreated material and (䊉) control.
employed 200 ◦ C at 60 min for both materials. The present study used organosolv at lower severity for the control (200 ◦ C, 32 min) and for the biotreated material (185 ◦ C, 18 min), obtaining the same ethanol yield. 4. Conclusions The biotreatment of the wood prior to the organosolv process improved solvent accessibility. A lower process severity was required in the biotreated samples with brown rot fungus in comparison with the control to obtain the same ethanol yield. Acknowledgement Financial support from FONDECYT (Grant 1080303) is gratefully acknowledged. References [1] Kristensen J, Thygesen L, Felby C, Jorgensen H, Elder T. Cell-wall structural changes in wheat straw pretreated for bioethanol production. Biotechnol Biofuels 2008;1:5. [2] Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY. Coordinated development of leading biomass pretreatment technologies. Bioresour Technol 2005;96:1959–66. [3] U.S. Department of Energy, Oak Ridge National Laboratory, Bioenergy Information Network, Bioenergy Feedstock Development Program. Bioenergy Frequently Asked Questions. 2001. Available on-line at http://bioenergy.ornl.gov/faqs/index.html. [4] Ramakrishnan A, Mala R. Bioethanol from lignocellulosic biomass: part III hydrolysis and fermentation. In: Pandey A, editor. Handbook of plant-based biofuels. Boca Raton: Taylor & Francis Group; 2009. p. 159–76. [5] Laxman RS, Lachke AH. Bioethanol from lignocellulosic biomass. In: Pandey A, editor. Handbook of plant-based biofuels. Boca Raton: Taylor & Francis Group; 2009. p. 121–38. [6] Um BH, Karim MN, Henk LL. Effect of sulfuric and phosphoric acid pretreatments on enzymatic hydrolysis of corn stover. Appl Biochem Biotechnol 2003;105:115–25. [7] Zhu L, O’Dwyer JP, Chang VS, Granda CB, Holtzapple MT. Structural features affecting biomass enzymatic digestibility. Bioresour Technol 2008;99:3817–28. [8] Mosier NS, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, et al. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 2005;96:673–86. [9] Araque E, Parra C, Freer J, Contreras D, Rodríguez J, Mendonc¸a R, et al. Evaluation of organosolv pretreatment for the conversion of Pinus radiata D. Don to ethanol. Enzyme Microb Technol 2008;43:214–9. [10] Ballesteros I, Negro MJ, Oliva JM, Cabanas A, Manzanares P, Ballesteros M. Ethanol production from steam-explosion pretreated wheat straw. Appl Biochem Biotechnol 2006, 129–132:496–508. ˜ C, Mendonc¸a R, Baeza J, Berlin A, Saddler J, Freer J. Bioethanol production [11] Munoz from bio-organosolv pulps of Pinus radiata and Acacia dealbata. J Chem Technol Biotechnol 2007;82:767–74. [12] Zhao X, Cheng K, Liu D. Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis. Appl Microbiol Biotechnol 2009;82:815–27. [13] Itoh H, Wada M, Honda Y, Kuwahara M, Watanabe T. Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi. J Biotechnol 2003;103:273–80. [14] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 2002;83:1–11.
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[15] Jensen Jr KA, Houtman CJ, Ryan ZC, Hammel KE. Pathways for extracellular fenton chemistry in the brown rot basidiomycete Gloeophyllum trabeum. Appl Environ Microbiol 2001;67:2705–11. [16] Flournoy DS, Kent Kirk T, Highley TL. Wood decay by brown-rot fungi: changes in pore structure and cell wall volume. Holzforschung 1991;45:383–8. [17] Ferraz A, Baeza J, Rodriguez J, Freer J. Estimating the chemical composition of biodegraded pine and eucalyptus wood by DRIFT spectroscopy and multivariate analysis. Bioresour Technol 2000;74:201–12.
[18] Bruns RE, Scarminio IS, Barros NB. Statistical design—chemometrics. In: Data handling in science and technology. 1st ed. Elsevier Science; 2001. [19] Söderström J, Galbe M, Zacchi G. Separate versus simultaneous saccharification and fermentation of two-step steam pretreated softwood for ethanol production. J Wood Chem Technol 2005;25:187–202. [20] Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, et al. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 2005;96:673–86.
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Bioorganosolv pretreatments of P. radiata by a brown rot fungus (Gloephyllum trabeum) and ethanolysis ˜ a,b , Victoria Melin a,b , Jaime Baeza a,b , Mariel Monrroy a , Jeniffer Ibanez a,c Regis Teixeira Mendonc¸a , David Contreras a,b , Juanita Freer a,b,∗ a b c
Renewable Resources Laboratory, Biotechnology Center, Universidad de Concepción, Casilla 160-C, Concepción, Chile Faculty of Chemical Sciences, Universidad de Concepción, Casilla 160-C, Concepción, Chile Faculty of Forest Sciences, Universidad de Concepción, Casilla 160-C, Concepción, Chile
a r t i c l e
i n f o
Article history: Received 29 November 2009 Received in revised form 28 January 2010 Accepted 29 January 2010 Keywords: Brown rot fungi Organosolv Bioethanol
a b s t r a c t Pinus radiata wood chips were pretreated with the brown rot fungus Gloephyllum trabeum for 3 weeks followed by an organosolv delignification with ethanol:water mixture at pH 2. The organosolv process was assessed using biotreated material that showed a high viscosity loss and a mass loss ranging from 6% to 8%. The experiment was designed to optimize the organosolv conditions, ethanol:water ratio and H factor (factor that combines temperature and time in one variable) to obtain the highest ethanol yield by simultaneous enzymatic saccharification and fermentation (SSF) for 96 h at 10% consistency. The optimized conditions for organosolv process for biotreated material were: ethanol:water mixture (60/40, v/v) and 1156 H factor (185 ◦ C, 18 min); the optimized conditions for the control (chips without biotreatment) were: ethanol:water mixture (60/40, v/v) and 6000 H factor (200 ◦ C, 32 min). The experimental ethanol yields obtained at these conditions were 63.8% and 64.3% (wood basis) for biotreated material and control, respectively. The maximum amount of ethanol that could be produced from P. radiata is 252 g ethanol/kg wood, assuming total glucose conversion into ethanol. The results indicate that the obtained ethanol was 161 g ethanol/kg wood from both materials. The biotreatment of the wood before the organosolv process improved solvent accessibility. To obtain the same ethanol yield, lower process severity was required in the biotreated samples in comparison with the control. © 2010 Published by Elsevier Inc.
1. Introduction Oil depletion and the greenhouse effect have increased the need for alternative non-fossil transportation fuels [1,2]. Research in bioethanol production from lignocellulosic biomass (LCB) has grown significantly over the last few decades. Lignocellulose is the most abundant biomass that can be converted into liquid fuels by enzymatic hydrolysis and microbial fermentation. According to U.S. Department of Energy, it can be estimated that terrestrial plants produce 13 × 1010 metric tons of biomass per year or about 6 times of the world’s energy requirement [3]. LCB includes materials such as agricultural and forestry residues, municipal solid waste, and industrial wastes. LCB can be used as an inexpensive feedstock for production of renewable fuels and chemicals [4].
∗ Corresponding author at: Centro de Biotecnología, Universidad de Concepción, Barrio Universitario s/n, Concepción, Chile. Tel.: +56 41 2204601; fax: +56 41 2204074. E-mail address: [email protected] (J. Freer). 0141-0229/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.enzmictec.2010.01.009
However, due to their structural features, LCB has limited accessibility to enzymes or microorganisms. LCB is made up of cellulose, hemicellulose, and lignin. Since the carbohydrate polymers are tightly bound to the lignin, mainly by hydrogen bonds but also by some covalent bonds [5], pretreatment is required to make biomass more accessible to the enzymes that convert the carbohydrate polymers into fermentable sugars. In short, pretreatment alters the physical features and chemical composition, specifically improving enzyme access and effectiveness by breaking the lignin seal, removing hemicellulose, disrupting the crystalline structure of cellulose, and expanding the structure to increase pore volume [1,6–8]. A large number of pretreatments have been tested by many researchers, which can be broadly classified into physical, chemical, physicochemical, biological and combined pretreatments [5,6,9–11]. Pretreatments using organic solvents have been reported as efficient processes. These pretreatments break the internal lignin and hemicellulose bonds and separate the lignin and hemicellulose fractions that can be potentially converted to useful products. Methanol, ethanol, butanol, n-butylamine, acetone, ethylene glycol, and other compounds have been used in the organosolv process [5,12]. These treatments have sometimes been combined with biological treatments [11,13].
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In biological pretreatments, natural wood attacking microorganisms that can degrade lignin and holocellulose are allowed to grow on the biomass, producing lignin–holocellulose complex degradation. The main biological pretreatments include fungi and their enzymes. The principal organism used for biological pretreatment of lignocellulose is white rot fungi [5,14]. These fungi have been shown to effectively disrupt the lignin–cellulose complex. Other types of wood rot fungi, such as the brown rot fungi, are responsible for naturally breaking down the highly ordered cellulose crystalline structure. These fungi preferentially degrade the wood polysaccharides, but also partially oxidize lignin [15]. They degrade holocellulose in an unusual manner, causing a rapid decrease in degree of polymerization with a low mass loss. It has been proposed that initial depolymerization is caused by producing small, diffusible, extracellular oxidants (free radicals), operating at a distance from the hyphae [15,16]. As a result of the initial attack by brown rot fungi and the depolymerization of the cellulose, wood strength rapidly decays. The aim of this work was to use a fungal pretreatment prior to the organosolv process (ethanolysis) in order to improve the solvent accessibility, decreasing the H factor. The ethanolysis process was optimized for conversion of Pinus radiata into ethanol by SSF. 2. Experimental 2.1. Raw material and preparation P. radiata D. Don samples were chipped and screened to approximately 2.0 cm × 2.5 cm × 0.5 cm. The wood chips were air-dried until reaching 10% (w/w) moisture, and then stored in plastic bags until its use. Prior to the biodegradation experiments, wood chips were immersed in water for 36 h and the excess of water was drained. Moist wood chips were sterilized (121 ◦ C/30 min) and brought to room temperature. 2.2. Fungus, inoculum preparation and wood biodegradation Gloephyllum trabeum (ATCC 11539) was used for the wood chips treatment. The strain was maintained on 2% (w/v) malt extract, 0.5% (w/v) soybean peptone, 1.5% (w/v) agar culture plates at 24 ◦ C for 1 week. Then, fungal mycelium was grown in 2 L Erlenmeyer flasks with 200 mL liquid culture medium (sterilized at 121 ◦ C, 30 min) containing 2% (w/v) malt extract and 0.5% (w/v) soybean peptone. Each flask was inoculated with 20 discs (8 mm in diameter) of the strain pre-cultured in solid medium and maintained unshaken for 2 weeks at 25 ◦ C. The grown mycelium was filtered and washed with 300 mL sterilized water. Washed mycelium obtained from several cultures was blended with 50 mL of sterilized water in two cycles of 10 s. The mycelium suspension was used to inoculate the sterilized wood chips in bioreactors. Each bioreactor was loaded with 300 g wood chips and inoculated with a suspension volume corresponding to 500 mg of fungal mycelium per kg of dry wood. The inoculated wood chips were incubated in an acclimatized room at 25 ◦ C and 55% relative humidity (RH) for 3 weeks. After the biodegrading treatment, the superficial mycelium was removed from the wood chips by brushing and the decayed wood chips were dried at 40 ◦ C for 48 h. The moisture of each sample was determined and the calculated initial and final dry weights were used to determine mass loss due to fungal biotreatment. The samples obtained in this process were named “biotreated material”. Control wood chip samples were prepared under the same conditions but without fungus addition. All the experiments were carried out in sextuplicate.
2.3. Organosolv pretreatment (ethanolysis) Organosolv pretreatment was carried out in a 1-L Parr reactor (Moline, IL, USA) loaded with 40 g of wood chips (dry weight base) of the biopretreated material or control and 240 mL of an ethanol (95%):water mixture (60/40, v/v) containing sulfuric acid as catalyst (0.13%, w/v pH approximately 2). The solvent:wood ratio inside the reactor was 6:1 (w/w). The H factor was 1156 (185 ◦ C) and 6000 (200 ◦ C) for biotreated and control materials, respectively. The cooking severity in the organosolv process was described by the H factor. This factor relates the time and temperature in a single variable. These experimental conditions were previously determined by a factorial analysis, where these values were optimized for a maximal ethanol yield. The effect of the ethanol:water mixture (50/60–70/30, v/v) and H factor (800–6000) were evaluated (Table 1). The pressures obtained were 1.8 MPa (at 185 ◦ C) and 2.5 MPa (at 200 ◦ C) for biotreated material and control, respectively. Once the process ended, the reactor was cooled in a water–ice bath. The supernatant was removed by decantation and the solid pressed out on 130-nylon mesh. The solid was washed with water, disintegrated in a blender for 5 min, and then passed by a Fiber Classifier (Regmed, Brazil) to segregate fibers from the rejects. The pulp was centrifuged. The samples were then stored in plastic bags at 4 ◦ C. 2.4. Chemical characterization of wood and organolsolv pulp samples Milled wood samples were extracted with acetone for 16 h according to standard TAPPI procedure T280 pm99. Wood and pulp samples were characterized for their carbohydrate composition using the methodology described by Ferraz et al. [17]. In a test tube, 300 mg of extractive-free milled wood was weighed and 3 mL of 72% (w/w) H2 SO4 was added. The hydrolysis was carried out in a water bath at 30 ◦ C for 1 h with stirring every 10 min. Later, the acid was diluted to 4% (w/w) with 79 mL of distilled water and the mixture transferred to a 250-mL Erlenmeyer flask and autoclaved for 1 h at 121 ◦ C. The residual material was cooled and filtered through a sintered glass filter number 4. The solid fraction, insoluble lignin, was dried and weighed. The concentration of monomeric sugars in the soluble fraction was determined by high-performance liquid chromatography (HPLC) in a Hewlett Packard 1050 using a Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA), at 45 ◦ C, eluted at 0.6 mL/min with 5 mM H2 SO4 and with a refractive index detector. Glucose, mannose and arabinose were used as external calibration standards (since the retention time and response for mannose, galactose and xylose are similar, these three sugars were expressed as mannose). The factors used to convert sugar monomers to anhydromonomers were 0.90 for glucose to glucan and for mannose to mannan, and 0.88 for arabinose to arabinan. All samples were analyzed in triplicate. The holocellulose and viscosity were determined according to standard TAPPI T 9m54 and T230 om04. 2.5. Inoculum preparation The yeast strain used in this work was a thermal acclimatized (40 ◦ C) Saccharomyces cerevisiae IR2T9 [9]. The inoculum was grown in 100 mL of liquid culture consisting in glucose, 50 g/L; yeast extract, 5 g/L; peptone, 5 g/L; KH2 PO4 , 1.0 g/L; MgSO4 ·7H2 O, 0.50 g/L; NH4 Cl, 2 g/L in a 500-mL Erlenmeyer flask. The culture was incubated for 48 h at 40 ◦ C in an orbital shaker at 150 rpm. 2.6. Simultaneous saccharification and fermentation (SSF) The simultaneous saccharification and fermentation were performed at 10% substrate consistency. In a 125-mL Erlenmeyer flask, 3 g dry weight of pretreated material (65% humidity) was suspended in a total reaction volume of 30 mL 0.05 M citrate buffer solution (pH 4.8). Nutrients, consisting in KH2 PO4 , 1.0 g/L; MgSO4 ·7H2 O, 0.50 g/L; peptone, 5.0 g/L; yeast extract, 5.0 g/L, were added. The enzymatic hydrolysis of the samples was performed using a commercial preparation of Trichoderma reesei cellulases Celluclast (70 FPU/mL; Novozymes, NC, USA), supplemented with a -glucosidase Novozym 188 (230 IU/mL; Novozymes, NC, USA),
Table 1 Experimental design for organosolv pretreatment of P. radiata. Experimental number
1 2 3 4 5 6 7 8 9 10 11
Control
Biotreated
H factor
Organic solvent (%)
H factor
Organic solvent (%)
5000 (−1) 7000 (1) 5000 (−1) 7000 (1) 4586 (−1.41) 7423 (1.41) 6000 (0) 6000 (0) 6000 (0) 6000 (0) 6000 (0)
50 (−1) 50 (−1) 70 (1) 70 (1) 60 (0) 60 (0) 46 (−1.41) 74 (1.41) 60 (0) 60 (0) 60 (0)
900 (−1) 1300 (1) 900 (−1) 1300 (1) 817 (−1.41) 1383 (1.41) 1100 (0) 1100 (0) 1100 (0) 1100 (0) 1100 (0)
50 (−1) 50 (−1) 70 (1) 70 (1) 60 (0) 60 (0) 46 (−1.41) 74 (1.41) 60 (0) 60 (0) 60 (0)
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where the amounts of enzyme were 20 FPU and 40 IU/g of pretreated material. After enzyme addition, the yeast inoculum was added with a cell density 6.0 g/L (3.5 × 108 to 3.3 × 109 yeast cell/mL). SSF was performed at 40 ◦ C and 150 rpm for 96 h. Samples were taken at 24, 48, 72 and 96 h and analyzed for ethanol content by gas chromatography (GC) on a Perkin-Elmer autosystem XL Headspace using a FID detector and a column HPS MS30m. The GC program was: 50 ◦ C × 3 min; 10 ◦ C/min, 100 ◦ C × 1 min; 25 ◦ C/min, 125 ◦ C × 1 min. The temperatures of the injector and detector were 200 and 300 ◦ C, respectively. Ethanol yield were calculated as a percentage of the theoretical yield. The theoretical yield was calculated by dividing the amount of ethanol obtained (g) by the amount of glucose in the pretreated material (g), assuming that all the potential glucose in the pretreated material is available for fermentation, with a fermentation yield of 0.51 g ethanol/g glucose multiplied by 100. All the determinations were realized in duplicate. Fig. 1. Holocellulose viscosity of P. radiata wood chips under brown degradation by G. trabeum.
2.7. Response surface methodology In order to determine the conditions to obtain maximal ethanol yield from the organosolv pulps, the variables organic solvent concentration and H factor were studied. The influence of each variable was determined using response surface methodology (RSM) [18]. This model is based on a central composite circumscribed design made of a factorial design and star points. The variable values were coded and normalized in unitary values: −1 was defined as the lowest value of a variable and +1 was defined as the highest value. From the extreme variable values, the central point (coded 0) was set and assayed in triplicate. Four star points distributed at a distance of 1.41 from the central point were included. The complete experimental design for the reaction conditions of the two methods optimized for ethanol yield is presented in Table 1. A second-order function that best describes the system’s behavior was determined by a multiple lineal regression method (MLR). The statistical validation was performed by a one-way ANOVA test with 95% confidence level. The optimal condition values were determined based on the response surface calculated using the SIMPLEX method [18]. All the calculations were performed with the software Modde 7.0.0.1 (Umetrics, USA).
3.2. Organosolv pulps characterization
3. Results and discussion
Table 3 summarizes the chemical composition and pulp yield for the organosolv pulps produced from biotreated material and control. The pulp yields for the biotreated material were 30% lower for H factors between 4000 and 7000 (data not shown), probably due to the process’s high severity. Therefore, H factors employed in the factorial design for the biotreated material were lower than for the control. The organosolv pulps yields obtained were 27–43% and 23–41% for the biotreated material and control, respectively. In general, there were few rejects (<5) in the pulping process. The glucan content was slightly higher for control pulps (81–85%) than biotreated pulps (75–80%). Almost all hemicelluloses were solubilized or degraded. The hemicelluloses remaining in the pulps were below 3%. The lignin content varied between 10% and 20% in the pulps, and was slightly higher for biotreated pulp.
3.1. Biodegradation of P. radiata
3.3. Simultaneous saccharification and fermentation (SSF)
Wood chips of P. radiata were decayed by G. trabeum for different periods (data not shown). During the first month of biodegradation, the mass loss ranged between 6% and 8%. Weight loss was calculated by the difference between the weights of the wood sample before and after the fungus treatment and the value obtained was 6%. We consider that this value is underestimated since during the fungus treatment, the cellulose chains were fragmented, and a water molecule is incorporated for each broken bond. The weight loss calculated based on the main component content was 8.2%. However, this value is overestimated; for example, the glucan content is calculated by multiplying the glucose content in the hydrolysate by 0.9 (162/180) to reflect the weight gain of glucose by the addition of water to the anhydroglucan during hydrolysis. It is not considered that there are many chains, for that reason more water is subtracted. The hemicellulose (mannan + galactan + xylan) and glucans decreased approximately 5% and 3%, respectively. Holocellulose viscosity decreased linearly over time until the third week (Fig. 1), but without a large carbohydrate mineralization. The material biotreated for 3 weeks was selected to assay the organosolv process. Table 2 summarizes the chemical composition of the biotreated and control P. radiata wood chips.
The hydrolysis and fermentation of the cellulose were performed by SSF process. Previous studies have shown that SSF processes have several advantages over a separate hydrolysis and fermentation (SHF) process: lower enzyme inhibition by hydrolysis products, less overall processing times, and higher ethanol yields. SSF is also considered a less capital intensive process [11,19]. In order to assure greater conversion in all experiments, the SSF was evaluated for 96 h. The ethanol yields obtained from the biotreated pulps varied between 73% and 91% (pulp basis) and 51% and 64% (wood basis). For the control sample, ethanol yields varied between 82% and 87% (pulp basis) and 43% and 65% (wood basis). The results indicated that the amounts of ethanol obtained were 129–161 g ethanol/kg wood and 108–164 g ethanol/kg wood for the biotreated and control pulps, respectively. The ethanol yields from biotreated and control pulps were very similar, but the organosolv process conditions were less severe for the biotreated samples. A probable explanation for this can be physical structural features of biotreated material, such as distribution of lignin in the biomass matrix, crystallinity, pore volume and biomass particle size, where the material is more accessible in the organosolv process. These features have been considered relevant for conversion of lignocellulosic biomass to liquid fuels [1,7,20].
Table 2 Chemical composition of wood chips from control and biotreated P. radiata.
a
Wood samples
Glucan (%)
Mannan + galactan + xylan (%)
Arabinan (%)
Lignin (%)
Extractives (%)
Viscosity (dL/g)
Control Biotreated Biotreated (cwl)a
44.5 ± 0.2 44.1 ± 0.1 41.5 ± 0.1
19.0 ± 0.4 15.4 ± 0.1 14.4 ± 0.4
1.2 ± 0.03 0.83 ± 0.02 0.8 ± 0.02
27.5 ± 0.6 29.4 ± 0.3 27.6 ± 0.3
1.5 ± 0.04 1.2 ± 0.02 1.1 ± 0.02
5.8 ± 0.1 2.5 ± 0.2 2.5 ± 0.2
Values corrected for weight loss due to fungal biodegradation.
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Table 3 Chemical composition of organosolv pulps from control and biotreated P. radiata. Experimental number
Wood samples
H factor
Organic solvent (%)
Pulp yield (%)
Rejects (%)
Glucan (%)
Mannan + galactan + xylan (%)
Lignin (%)
1 2 3 4 5 6 7 8 9 10 11
Control Control Control Control Control Control Control Control Control Control Control
5060 7000 5077 7008 4825 7423 6103 6049 6048 6051 6032
50 50 70 70 60 60 46 74 60 60 60
38 27 34 23 38 33 37 38 40 41 40
2 0 2 1 5 1 5 5 4 2 2
84 81 84.4 82.6 87 84.8 81 83 85 84 83
± ± ± ± ± ± ± ± ± ± ±
1 1 1 0.5 1 0.4 2 1 1 1 1
1.6 1.3 1.6 0.5 1.3 1.2 0.6 0.8 0.6 0.9 0.6
± ± ± ± ± ± ± ± ± ± ±
0.4 0.3 0.02 0.01 0.6 0.3 0.02 0.02 0.04 0.1 0.1
14.1 16.5 14.7 15.4 10.3 11.4 11.4 14.4 15.1 13.5 13.0
± ± ± ± ± ± ± ± ± ± ±
0.4 0.1 0.2 0.5 0.1 0.1 0.1 0.7 0.1 0.3 0.3
1 2 3 4 5 6 7 8 9 10 11
Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated Biotreated
900 1312 901 1312 811 1368 805 1088 1092 1085 1073
50 50 70 70 60 60 46 74 60 60 60
34 37 38 39 40 41 27 39 41 43 39
7 6 2 4 4 4 18 3 3 4 4
78.4 78.2 78 74.3 76.2 78 75.4 78.9 78.4 77.7 80
± ± ± ± ± ± ± ± ± ± ±
0.5 0.6 0.2 0.2 0.2 1 0.4 0.5 0.6 0.1 1
1.2 1.2 2.7 2.2 2.5 1.9 1.2 1.2 1.6 2.3 2.2
± ± ± ± ± ± ± ± ± ± ±
0.01 0.05 0.1 0.1 0.1 0.1 0.02 0.2 0.1 0.2 0.2
16.5 17.3 17.7 16.3 16.7 16.5 20.2 16.5 16.3 16.8 15.4
± ± ± ± ± ± ± ± ± ± ±
0.6 0.2 0.2 0.2 0.2 0.4 0.3 0.5 0.1 0.1 0.4
3.4. Response surface methodology for the organosolv pretreatment The main purpose of this study was to compare biotreated and control material in terms of their response to organosolv pretreatments in order to optimize these pretreatments to achieve maximum ethanol yield. From the experimental design data (Table 1) and its corresponding ethanol yield (Table 4), a quadratic polynomial was determined (Eqs. (1) and (2)) and validated by the ANOVA test for the biotreated and control pulps. The variables in quadratic polynomial were scaled and centered. Biotreated material: Y = 64.27 ± 1.34 + 1.86 ± 0.84Z1 + 0.98 ± 0.98Z2 − 2.67 ± 1.02Z1 2 − 8.30 ± 1.04Z2 2
(1)
Control: Y = (122e18 ± 410e17 − 214e17 ± 267e17 Z1 − 5.41e16 ± 253e17 Z2 − 3.97e17 ± 335e17 Z1 2 − 424 e17 ± 2.97e17 Z2 2 )
Z=
Xi − X0 Xmax − X0
1/10
(2)
(3)
where Z denotes the scaled and centered value from original variables (X) (Eq. (3)), Z1 correspond to H factor, Z2 to the organic solvent concentration (ethanol, %, v/v), X0 is midrange and Y is the ethanol yield (%, wood basis). The error values corresponded to a 95% confidence level. The response surfaces for the polynomials (plot inside the dominium limited for the experimental planning) are shown in Fig. 2. For the biotreated pulp, the linear terms for H factor and the organic solvent concentration have positive coefficients, meaning that the ethanol yield incremented with the increase in these variables until a maximum value, as shown by the negative quadratic terms for all variables. According to the polynomial, the H factor is the main determining factor for maximum ethanol yield. For the control pulp, the linear terms for the H factor and the organic solvent concentration have negative coefficients, meaning that the ethanol yield increases with the decrease in these variables until a maximum value. Considering the confidence intervals, the inter-
Fig. 2. Surface response for the ethanol yield. (A) Control and (B) biotreated samples.
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Table 4 Experimental ethanol yield and response polynomial calculated values. Experimental number
1 2 3 4 5 6 7 8 9 10 11
Control (ethanol yield (%))
Biotreated (ethanol yield (%))
Experimental
Calculated
Experimental
Calculated
62.5 43.0 53.1 36.8 63.0 56.1 57.3 60.0 63.5 63.7 65.6
60.6 54.7 59.5 51.5 62.6 50.7 58.2 55.9 64.4 64.4 64.4
51.6 54.9 52.8 55.3 56.6 61.8 36.5 49.5 63.8 63.1 64.6
50.4 54.2 52.4 56.1 56.3 61.6 37.7 49.0 64.3 64.3 64.3
action between variables did not have a significant effect on ethanol yield. Based on the experimental conditions used in the experimental design, the polynomial was used to predict the ethanol yield. The responses were close to the experimental values (Table 4) with a correlation coefficient (r2 ) = 0.99 and (r2 ) = 0.79 for the biotreated and control pulps, respectively. These values together with the ANOVA test statistically validated the model. The optimum values of the variables for maximal ethanol yield were determined by the SIMPLEX method using the maximum values of the response surface [18]. The predicted values for biotreated pulps were: 1156 H factor and 60% (v/v) organic solvent (at 185 ◦ C for 17 min). According to the polynomial, the maximum ethanol yield was 64.6 ± 1.2% (at 95% confidence level), while the experimental ethanol yield for this pulp obtained at these conditions was 63.8%, agreeing with the predicted value (Fig. 2). The predicted values for control pulps were: 6000 H factor and 60% (v/v) organic solvent (at 200 ◦ C for 32 min). According to the polynomial, the ethanol yield was 64.4 ± 2.2%, while the experimental ethanol yield for this pulp obtained at these conditions was 64.3%, agreeing with the predicted value (Fig. 3). Ethanol production was 40 g/L for both materials (Fig. 2). The maximal ethanol amount that could be produced from P. radiata is 252 g ethanol/kg wood considering total conversion of glucose into ethanol. The results indicate that the amount of ethanol obtained is 161 g ethanol/kg wood for both materials. The ethanol yield obtained in this study is similar to the amount ˜ et al. [11] from beech and P. obtained by Itoh et al. [13] and Munoz radiata, respectively, with white rot fungi although the organosolv conditions used in these studies were significantly higher. Itoh et al. [13] employed 200 ◦ C at 120 min and 180 ◦ C at 60 min for the ˜ control and biotreated materials, respectively. Munoz et al. [11]
Fig. 3. Ethanol production from SSF of P. radiata organosolv pulps. Ethanol yield () biotreated material and () control. Ethanol amount () biotreated material and (䊉) control.
employed 200 ◦ C at 60 min for both materials. The present study used organosolv at lower severity for the control (200 ◦ C, 32 min) and for the biotreated material (185 ◦ C, 18 min), obtaining the same ethanol yield. 4. Conclusions The biotreatment of the wood prior to the organosolv process improved solvent accessibility. A lower process severity was required in the biotreated samples with brown rot fungus in comparison with the control to obtain the same ethanol yield. Acknowledgement Financial support from FONDECYT (Grant 1080303) is gratefully acknowledged. References [1] Kristensen J, Thygesen L, Felby C, Jorgensen H, Elder T. Cell-wall structural changes in wheat straw pretreated for bioethanol production. Biotechnol Biofuels 2008;1:5. [2] Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY. Coordinated development of leading biomass pretreatment technologies. Bioresour Technol 2005;96:1959–66. [3] U.S. Department of Energy, Oak Ridge National Laboratory, Bioenergy Information Network, Bioenergy Feedstock Development Program. Bioenergy Frequently Asked Questions. 2001. Available on-line at http://bioenergy.ornl.gov/faqs/index.html. [4] Ramakrishnan A, Mala R. Bioethanol from lignocellulosic biomass: part III hydrolysis and fermentation. In: Pandey A, editor. Handbook of plant-based biofuels. Boca Raton: Taylor & Francis Group; 2009. p. 159–76. [5] Laxman RS, Lachke AH. Bioethanol from lignocellulosic biomass. In: Pandey A, editor. Handbook of plant-based biofuels. Boca Raton: Taylor & Francis Group; 2009. p. 121–38. [6] Um BH, Karim MN, Henk LL. Effect of sulfuric and phosphoric acid pretreatments on enzymatic hydrolysis of corn stover. Appl Biochem Biotechnol 2003;105:115–25. [7] Zhu L, O’Dwyer JP, Chang VS, Granda CB, Holtzapple MT. Structural features affecting biomass enzymatic digestibility. Bioresour Technol 2008;99:3817–28. [8] Mosier NS, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, et al. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 2005;96:673–86. [9] Araque E, Parra C, Freer J, Contreras D, Rodríguez J, Mendonc¸a R, et al. Evaluation of organosolv pretreatment for the conversion of Pinus radiata D. Don to ethanol. Enzyme Microb Technol 2008;43:214–9. [10] Ballesteros I, Negro MJ, Oliva JM, Cabanas A, Manzanares P, Ballesteros M. Ethanol production from steam-explosion pretreated wheat straw. Appl Biochem Biotechnol 2006, 129–132:496–508. ˜ C, Mendonc¸a R, Baeza J, Berlin A, Saddler J, Freer J. Bioethanol production [11] Munoz from bio-organosolv pulps of Pinus radiata and Acacia dealbata. J Chem Technol Biotechnol 2007;82:767–74. [12] Zhao X, Cheng K, Liu D. Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis. Appl Microbiol Biotechnol 2009;82:815–27. [13] Itoh H, Wada M, Honda Y, Kuwahara M, Watanabe T. Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi. J Biotechnol 2003;103:273–80. [14] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 2002;83:1–11.
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