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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.50 No.4 pp.35-44
DOI : https://doi.org/10.14397/jals.2016.50.4.35

Effect of Steam Explosion Pretreatment on Bioethanol Production from Populus euramericana as Alternative Feedstock

Jong-Soo Jo1, Ji Young Jung2, Jae-Kyung Yang2*
1Department of Interior Materials Engineering, Gyeongnam National University of Science and Technology, Jinju, 52725, Korea
2Division of Environmental Forest Science and Institute of Agriculture & Life Science, Gyeongsang National University, Jinju, 52828, Korea
Corresponding author: Jae-Kyung Yang +82-55-772-1862+82-55-772-1869jkyang@gnu.ac.kr
May 14, 2016 June 7, 2016 June 23, 2016

Abstract

Populus euramericana is emerging as a viable feedstock for producing bioethanol from renewable resources. Steam explosion pretreatment of P. euramericana can solubilize a significant portion of the hemicellulosic component and enhance the glucose conversion of the remaining cellulose for fermentation into ethanol. In this study, steam explosion condition of P. euramericana is performed in a steam explosion reactor at severity log Ro 4.02 and severity log Ro 4.37. Glucose conversion varied from 72.3% to 80.1% of steam exploded P. euramericana at severity log Ro 4.02 and severity log Ro 4.37. Ethanol yields(%) based on sugar content after enzymatic hydrolysis after 48 h fermentation ranged from 87.0% to 88.4%. As a result, from 100 g of raw material, 14.0 g of ethanol are recovered of 47.3 g available cellulose content. This research of steam explosion pretreatment was a promising method to improve glucose conversion and ethanol yield for bioethanol production.


초록


    Korea Forest Service
    No. S211316L010140 Gyeongnam National University of Science and Technology

    Introduction

    Lignocellulosic biomass abundantly available in the nature can be used as a feedstock for the thermochemical and biochemical processes to derive energy(Klass, 1988). Lignocellulosic biomass to be considered for ethanol production include wood, crops from annual plants, agricultural residue and waste paper(Landucci et al., 1996; Ladisch 2002; Kim & Dale 2004; Karimi et al., 2006; Linoj et al., 2006; Tabka et al., 2006).

    Among the various lignocellulosic biomass for producing ethanol, the choice of Populus euramericana are considered to have great potential as a biorefinery feedstock in the worldwide, due to their widespread availability and good productivity(Bose et al., 2009). Poplars are representing an important source as short rotation energy crops for use as alternative energy resources(Duff & Murray 1996; Smits et al., 1998). However, challenges for lignocellulose-to-biofuel conversion still exist because cellulose, being different in chemistry from starch.

    Cellulose is a polysaccharide that closely is associated with both hemicelluloses and lignin in the plant cell wall complex. Also its hydrolysis to glucose requires significant investment of energy compared to starch-to-glucose conversion(Swana et al., 2011). Enzymatic utilization of the cellulose in lignocellulosic biomass requires effective pretreatment to make the recalcitrant cellulose more accessible to cellulaseenzymes (Chang & Holtzapple, 2000).

    Pretreatment is an important tool for practical cellulose conversion processes(Mosier et al., 2005). Pretreatment also has great potential for improvement of efficiency and lowering of cost through research and development(Lynd et al., 1996). Pretreatment technologies can be classified into biological(Patel et al., 2007), emical(Silverstein et al., 2007) and physicochemical pretreatment(Alizadeh et al., 2005; Zhang et al., 2007). However, recent research trends show that physicochemical pretreatment could be most favorable for biofuels production. Hydrothermal, steam explosion, dilute acid, ammonia fiber explosion(AFEX) and lime treatment are the leading technologies for biomass pretreatment(Wyman et al., 2005).

    Steam explosion pretreatment is one of the most attractive pretreatment processes because of its low use of chemicals and low energy consumption. Steam explosion pretreatment consists to expose the biomass up to high pressure(1500 - 5000 kPa) and temperature(180 - 250°C) in presence of steam during a determined time, up to 90 min, followed by a rapid reduction in pressure, in order to breakdown the lignocellulosic structure. The treatment leads to a partial self - hydrolysis of hemicelluloses, depolymerization of lignin and a destruction of cellulose, largely dependent on treatment temperature(Sassner et al., 2008). Steam explosion pretreatment(uncatalyzed) of poplar was reported by Ando et al.(1986), Grouset al.(1986), Toussaint et al.(1991), Oliva et al.(2001) and Cantarellaet al.(2004). However, the ethanol yield based on dry weight of untreated original materials was not provided; thus, it is difficult to compare with other results.

    In this study, we have evaluated the potential of P. euramericana as a bioresource for ethanol production. Also, the effect of different severity log Ro conditions on glucose conversion and ethanol yield assessed by enzymatic hydrolysis and fermentation from steam exploded P. euramericana.

    Materials and Methods

    1.Raw material

    The raw material(12 years old Populus euramericana) was prepared from the Korea Forest Research Institute(Suwon, Korea). The raw material debarked, chipped to an average size 1 × 1 × 2 cm3 for steam explosion pretreatment. The chipped raw material was milled into 1-2 mm and then sieved through a -20 mesh/+80 mesh screen and collected for chemical composition analysis as described below.

    2.Chemical composition of raw material

    Chemical composition of raw material was determined according to the analytical methods of National Renewable Energy Laboratory(NREL) procedures(Sluiter et al., 2004a, Sluiter et al., 2004b). The analysis included the determination of carbohydrates, lignin(acid-insoluble and acid soluble), extractives and ash. The carbohydrate and lignin content of the raw material was determined based on monomer content after a two-step acid hydrolysis procedure. The first hydrolysis step was carried out by treating wood with 72%(w/w) H2SO4 at 30°C for 60 min. The resultant hydrolysate was diluted to 4%(w/w) H2SO4 and autoclaved at 121°C for 1 h in a second hydrolysis step. The hydrolysate from the second hydrolysis step was then analyzed for sugar determinations. The remaining acid-insoluble residue was considered as acid-insoluble lignin.

    3.Steam explosion pretreatment

    Steam explosion pretreatment, which was used as a physicochemical pretreatment, was carried out in a batch pilot unit equipped with a 1L reaction vessel. The “severity parameter” (Ro) was used to map the destruction, desegregation, and depolymerization of P. euramericanaRo was calculated using the following equation(Fernandez et al., 1999).

    where T is the temperature(°C) and tthe time (min). A steam temperature of 213 °C and 225 °C, pretreatment time of 5 min were applied to realize a severity parameter value of log Ro 4.02 and log Ro 4.37. After the saturated steam exposure, a ball valve at the bottom of the reactor was opened suddenly to bring the reactor rapidly to atmospheric pressure. The exploded material was recovered in a cyclone and after cooling to about 40 °C. The recovered solid was extracted with distilled water for 2 h at 60 °C, and filtered. The water insoluble fraction was used for enzymatic hydrolysis.

    4.Enzymatic hydrolysis

    The raw material and steam exploded material were hydrolyzed with Celluclast 1.5® L (Novo Co., Denmark), a mixture of cellulases obtained from Trichoderma reeseiand Viscozyme® L(Novo Co., Denmark) a β-glucosidase. The substrate(1 g) was suspended in 50 mL of 0.1 M citrate buffer(pH 4.8) contained in a 250 mL Erlenmeyer flask. Afterwards appropriate aliquots of cellulase(20 FPU/g) and β -glucosidase(24 CBU/g) were introduced into flask and placed on a shaking incubator(IS-97IR Jeio-Tech Co., South Korea) for 72 h at 50 °C. Aliquots of samples from the culture flasks were withdrawn under aseptic conditions after 0, 12, 24, 48 and 72 h to monitor the progress of the enzyme reaction. The sugar from the hydrolysate was measured as sugar monomer from supernatants obtained after centrifugation for 15 min at 3,000 rpm(Hanilmicro-12, Hanil Science Industrial Co., Korea) of heat treated(100 °C for 10 min) samples by HPLC (Shimadzu, Kyoto, Japan) with a refractive-index detector. The HPLC column used for the separation of the sugars; glucose, xylose, galactose, arabinose and mannose was an Aminex HPX-87P(Bio-Rad, Hercules, CA, USA) maintained at 85 °C, with water as an eluent at a flow rate of 0.6 mL/min. All samples were filtered through a 0.2 μm filter before analysis to remove any coarse particles. All analytical determinations were performed in duplicate and average results are shown.

    5.Fermentation

    The fermentation of the hydrolysate was carried out with Saccharomyces cerevisiae KCTC 7296. The inoculum of required for the initiation of fermentation was prepared by culturing S. cerevisiae on GPY(40 g L-1 glucose, 5 g L-1 peptone and 5 g L-1 yeast extract) medium by incubating at 35°C on an orbital shaker for 24 h. For fermentation studies 5%(v/v) inoculum with a cell density of 1.5 × 108 mL-1 was incorporated in to culture medium. Fermentation was performed in 100 mL glass flasks with a working volume of 20 mL, consisting of 18.5 mL hydrolysate, 0.5 mL nutrients(0.5 g L-1 (NH4)2HPO4, 0.025 g L-1 MgSO4·7H2O, 0.1 M NaH2PO4 and 1 g L-1 yeast extract) (Taherzadeh et al., 1996) and 1 mL inoculum. The flasks were sealed with rubber stoppers through which hypodermic needles had been inserted for removal of the CO2 produced, as well as for withdrawal of samples. The fermentation flasks were incubated at 35°C for 48 h and monitored for ethanol and sugars from samples withdrawn after 0, 8, 12, 24 and 48 h. The ethanol from samples withdrawn during fermentation was analyzed using Aminex HPX-87H column(Bio-Rad) maintained at 6 5°C, with 5 mM H2SO4 as an eluent with a flow rate of 0.6 mL/min. All analytical determinations were performed in duplicate and average results are shown.

    Results and Discussion

    1.Chemical composition

    The chemical composition of P. euramericana, the raw material used in this study, was presented in Table 1. Sugars ware expressed as monomers. Glucose, which was derived from both the P. euramericana fiber and plant cell wall, was major component(47.3 %). Xylose, as the major hemicellulose constituent, constituted up to 15.1%. The lignin content of P. euramericana was higher than agriculture residue (Samayam & Schall 2010; McIntosh & Vancov 2010; Jorgensen et al., 2007). Additionally, P. euramericana contained extractives(6.2%), ash(1.7%) and other unknown component. Glucose and xylose can be converted to ethanol using organism capable for fermenting pentose and hexose) (Han et al., 2011). The carbohydrate(Cellulose and hemicellulose) accounted for more than 66.8% of the whole plant, similarly to that of other major sources of lignocellulosic biomass, such as wheat straw(54.6%) (Petersen et al., 2009), barley hull(64.1%) (Kim et al., 2008), rice straw(58.3%) (Zhu et al., 2005) and corn stover (62.8%) (Qing et al., 2011), indicating that P. euramericana was a potentially useful feedstock resource for bioethanol production.

    2.Steam explosion pretreatment

    Lignocellulosic biomass requires pretreatment, mainly because the lignin in plant cell walls forms a barrier against enzyme attack. An ideal pretreatment reduces the lignin content and crystallinity of the cellulose and increases surface area(Balat et al., 2008).

    The raw material was subjected to steam explosion pretreatment during 5 min and temperatures of 213(severity log Ro 4.02) and 225°C(severity log Ro 4.37). Though the steam explosion processes occur in temperature ranges from 200°C to 280°C with retention time varying from 2 to 10 min but under these conditions, thermal degradation of cellulose into sugars takes place Steam explosion conditions(log Ro 4.02 and log Ro 4.37) were optimized to obtain maximum cellulose recovery(data not shown). Table 2 shows the solid recovery and main component of water insoluble fraction resulting from steam explosion pretreatment at different severity log Ro value. The solid recovery ranged between 64.0 and 87.0%. As expected, a decrease of solid recovery was detected as the severity log Ro increased. This is mainly attributed to the decrease of cellulose and hemicellulosic fraction. Similarly, a more severe pretreatment condition resulted in higher cellulose and hemicellulose degradation(Ballesteros et al., 2000). The maximum cellulose content(expressed as glucose, g) in water insoluble fraction was obtained at severity log Ro 4.02. The proportion of sugars that are decreased in the solids after pretreatment was dependent on the type of hemicellulosic sugar. Galactose and mannose were completely removed at the lowest tested severity log Ro 4.02 while the increase in the severity log Ro valueto 4.37 resulted, in general, in increased solubilization of xylose and arabinose. The contents in lignin of the solid pretreated residue, referred to raw material, showed a slight decrease.

    In the biomass feedstock, cellulose is the main reservoir of glucose, the desired fermentation substrate for bioenergy source(Jeoh, 1998). In optimizing steam explosion pretreatment conditions for enzymatic hydrolysis and fermentation process, cellulose losses is an important considerable factor.

    Table 3 shows the sugar weight of the filtrate after pretreatment. Results are referred to 100 g raw material, taking into account both concentrations and filtrate volumes obtained. There was increased hemicellulose degradation at higher severity log Ro value(log Ro 4.02 < log Ro 4.37). The pH values of filtrates, ranging from 4.5 to 4.3, were also shown on Table 3. At higher severity log Ro value lower final pH values were obtained. The low pH include acetic acid, formic acid, caprylic acid and levulinicacid(Hongqiang & Hongzhang, 2008). During steam explosion pretreatment, some degradation products are formed that may be potential inhibitors during fermentation of the sugar fraction. Acetic acid from hydrolysis of hemicellulose and furfural from degradation of xylose were obtained as a consequence of the high xylan content in hardwood. Vanillin, which is formed by degradation of guaiacylpropane units of lignin and syringaldehyde, which are formed in turn by the degradation of syringyl propane units, has been reported in hydrolysates from other hardwoods such as poplar(Luo et al., 2002) and red oak(Tran & Chambers, 1985).

    3.Enzymatic hydrolysis

    Enzymatic hydrolysis was performed on the raw material and the steam exploded material(water insoluble fraction). The water insoluble fraction was composed of mostly cellulose, lignin and residual hemicellulose. we used the degree of hydrolysis of water insoluble fraction as a measure of the effectiveness of the pretreatment method. Glucose conversion and glucose release from enzyme hydrolysis of steam exploded P. euramericana were shown in Fig. 1. Conversion was expressed as a percent of the released glucose compared to the glucose equivalent of the cellulose content of the substrate. The rate and extent of glucose conversion from steam exploded material was much higher than the control(raw material). The maximum glucose conversion(80.1%) and glucose release(31.2 g / 100 g raw material) were obtained severity log Ro 4.37 after 72 h of enzymatic hydrolysis. Similarly, Ruiz et al.(2008) reported a linear increase in cellulose conversion with increased severity for steam exploded sunflower stalk. The hydrolysate from severity log Ro 4.37 contained high glucose, which was more suitable for subsequent fermentation process. The severity log Ro 4.37 were easily hydrolysable than severity log Ro 4.02. Their high susceptibility to enzyme may arise from the low content of inert components(lignin and the like) (Margeot et al., 2009) (Table 2).

    4.Fermentation

    We have also investigated the feasibility of steam explosion pretretment at different severity log Ro value, using fermentation process. The effect of steam explosion on ethanol yields from fermentation of P. euramericana was analyzed as a percentage of the theoretical ethanol yield. The theoretical ethanol yield was calculated from stoichiometry. Thus, 51 g of ethanol per 100 g of sugar can be produced under ideal fermentation conditions. Ethanol concentration reached its highest peak at 24 h. No difference in ethanol concentrations was observed at 48 h. Ethanol yield increased with increased severity log Ro value(Fig. 2). The highest yield(88.4% and 14.0 g / 100 g raw material) was achieved at severity log Ro 4.37. The high theoretical ethanol yields indicate that most of the sugar in the biomass was utilized by the microorganisms(date not shown). The highest fermentation efficiency lies between 90 and 93% (Ingledew, 1999) because some glucose has to be used for production of cell mass, reactions of cell maintenance, and production of minor end metabolism products. Similar to the trend observed in ethanol yield(87-88.4%), fermentation efficiency for steam exploded material were greater than those for raw material.

    5.Mass balance

    Using dry matter recoveries and several composition analyses after each step we developed an overall mass balance for our operation including pretreatment step, enzymatic hydrolysis step and fermentation step. Steam explosion pretreatment condition(severity log Ro 4.37) were optimized to obtain maximum recovery of glucose(31.2 g / 100 g raw material) and ethanol (14.0 g / 100 g raw material) in P. euramericana. (Fig. 3)

    The results obtained in this study suggested that the steam explosion pretreatment of P. euramericana was a promising pretreatment. Steam explosion condition at severity log Ro 4.37 improves glucose conversion by Three times compared to that from raw material(from 24.1% to 80.1%) and rendered a relatively concentrated glucose broth suitable for fermentation. The ethanol yield increased from 51.4% to 88.4% comparing raw material and severity log Ro 4.37 steam exploded material respectively. This study represents that steam explosion pretreatment of P. euramericana can be advantageous for ethanol production with cost and environmental benefits.

    Acknowledgement

    This work was supported by Gyeongnam National University of Science and Technology Grant 2014 and also partially supported of “Forest science & Technology projects(project No. S211316L010140)” Provide by Korea Forest service.

    Figure

    JALS-50-4-35_F1.gif

    Enzymatic hydrolysis of steam exploded P. euramericana at different severity log Ro. Glucose conversion: closed symbols(■, ●, ◆), glucose released: open symbols(□, ○, ◇).

    JALS-50-4-35_F2.gif

    Fermentation of steam exploded P. euramericana at different severity log Ro. Ethanol yield(%): closed symbols(■, ●, ◆), ethanol yield(g / 100g raw material): open symbols(□, ○, ◇).

    JALS-50-4-35_F3.gif

    Mass balance diagram of steam explosion pretreatment followed by fermentation.

    Table

    Chemical composition of raw material1)

    1)Data in the table are based on oven dry samples.
    2)Values are mean ± S.D of three separate experiments.

    Solid recovery and main component of solid resulting from steam explosion pretreatment at different severity log Ro in P. euramericana

    1)Solids remaining after pre-treatment divided by original oven-dried weight.
    2)Acid insoluble lignin.
    3)Data are expressed in parentheses as a percentage based on dry weight of raw material.
    4)Not detected.

    Sugar weight(g / 100 g raw material) and pH of the filtrate resulting from steam explosion pretreatment at different severity log Ro in P. euramericana

    1)Not detected.

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