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Producing bioethanol from pretreated-wood dust by simultaneous saccharification and co-fermentation process
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Producing bioethanol from pretreated-wood dust by simultaneous saccharification and co-fermentation process Wei-Chuan Chen a, Yin-Chen Lin a, Ya-Lian Ciou a, I-Ming Chu b, Shen-Long Tsai c, John Chi-Wei Lan d, Yu-Kaung Chang e, Yu-Hong Wei a,∗ a
Graduate school of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan 320, Taiwan Department of Chemical Engineering, National Tsing Hua University, Hsin Chu 300, Taiwan Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 100, Taiwan d Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan 320, Taiwan e Department & Graduate Institute of Chemical Engineering & Graduate Institute of Biochemical Engineering, Ming Chi University of Technology, Taishan New Taipei City 243, Taiwan b c
a r t i c l e
i n f o
Article history: Received 21 December 2016 Revised 19 April 2017 Accepted 19 April 2017 Available online xxx Keywords: Bioethanol production Pretreated-wood dust medium Supercritical fluid extraction Steam explosion Saccharification enzyme activity
a b s t r a c t The aim of this study was to utilize a new and highly effective bioreactor system, i.e., simultaneous saccharification and fermentation (SSCF), for bioethanol production by the cocultivation of Trichoderma reesei, Aspergillus niger, and Zymomonas mobilis by using a direct conversion process of pretreated-wood dust medium. Wood dust has been effectively used to obtain reducing sugars (glucose, xylose, and other byproducts) through the use of either a supercritical fluid extraction (SFE) or steam explosion (SE) pretreatment step. In addition, experimental results showed that polyurethane as a porous carrier could enhance total saccharification enzyme activity at an inoculum proportion of 1/1 of T. reesei, and A. niger and at a total inoculum concentration of 6.5 × 106 spores/ml. Furthermore, the concentration of alginate beads (3%) and immobilized proportion of Z. mobilis to alginate beads (1:4) were also examined. In accordance with previous reports, bioethanol production was carried out in a SSCF bioreactor by the cocultivation of T. reesei and A. niger in the polyurethane carrier and Z. mobilis immobilized in alginate beads using pretreated-wood dust medium. Experimental results revealed that, after 24 h of cultivation, the yield of bioethanol produced using pretreated-wood dust medium (1%) were 0.069 g/g and 0.049 g/g, for SFE and SE, respectively. Meanwhile, the sugar conversion rate reached 20.72% and 24.39% for SFE and SE, respectively. Thus, the results of this study show that pretreated-wood dust medium has significant potential for use in bioethanol production. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Continued industrialization and population growth have increased annual energy consumption [1]. Increasing fossil fuel prices and greenhouse gases have motivated the research and development of renewable resources in many countries. The principal substitute for petrol in road vehicles is bioethanol. One of the advantages of using bioethanol is that doing so reduces greenhouse gas emissions. Also, blending bioethanol with petrol, as in E85, helps to extend diminishing oil supplies, increase fuel security, and eliminate heavy reliance on oil producing nations. Bioethanol is seen as the most promising prospective renewable energy source, which can be produced from microbial fermentation by converting sugars from cellulosic materials such as wood dust [2]. ∗
Corresponding author. E-mail address: [email protected] (Y.-H. Wei).
Lignocellulose is an abundant natural carbohydrate that can be transformed into a substitute renewable energy resource by microbial conversion [3]. The advantages of using cellulosic materials for bioethanol production are their low cost, ready availability, lack of conflict with use for food, and the potential production of fuel from lignin [4]. A pretreatment step is necessary for modifying structural characteristics of lignocellulose and increasing the availability of glucan and xylan for enzymatic saccharification [5]. The pretreatment step can destroy the lignin structure to allow for easier break down of the cellulose by the saccharification enzyme [4]. Traditional pretreatments of biomass for producing bioethanol include physical methods and chemical methods, involving liquid hot water and alkali (or acid), for example [6]. However, several disadvantages for all the different pretreatment options exist, and it is necessary to adapt suitable pretreatments based on the properties of the raw material. Physical and chemical pretreatments inhibit the
http://dx.doi.org/10.1016/j.jtice.2017.04.025 1876-1070/© 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: W.-C. Chen et al., Producing bioethanol from pretreated-wood dust by simultaneous saccharification and cofermentation process, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.025
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hydrolysis of cellulose and hemicellulose and lignin fraction owing to the close among the components of lignocellulosic biomass. Therefore, in this study, steam explosion (SE) and supercritical fluid extraction (SFE) were used in this work to pretreat wood dust to avoid the aforementioned problems. Steam explosion (SE) technology has become one of the most common and widely employed physicochemical pretreatments for lignocellulosic biomass. The process of SE, developed by William H. Mason in 1925, is used for the pretreatment of agricultural wastes, i.e., wood dust in this study [5]. SE technology is a hydrothermal pretreatment that consists of three main steps: (1) the treatment step, (2) the explosion step, and (3) the impact step [5]. The first step is a process wherein lignocellulosic biomass is treated with pressurized steam for a certain period. The SFE rapid release of pressure causes the explosion of lignocellulosic biomass. Finally, the impact of the lignocellulosic biomass mixture is done to form a raw material used for enzyme hydrolysis. Supercritical fluid extraction is also one of the pretreatment methods for disrupting the crystalline structure of lignocellulose under certain conditions such as temperature and pressure above its critical point [7]. SFE is becoming necessary to reduce the environmental hazards of common chemicals and solvents used in traditional methods. SFE shows excellent potential as a method for lignocellulosic biomass pretreatment [8]. Reports have shown that SFE has been successfully used for the pretreatment of commercial cellulosic materials, recycled paper mix, and sugarcane bagasse [7,8]. In addition, the use of SFE, as a green solvent for biomass pretreatment in a biorefinery concept, is increasing and it is expected to continue growing in the future. Saccharification enzymes can be produced by fungi such as Trichoderma reesei and Aspergillus niger, and agricultural waste can be converted into bioethanol by Zymomonas mobilis. Conversion of agricultural waste reduces its environmental impacts by efficiently using it to produce secondary energy. The conversion of cellulose into monosaccharides by microorganisms has been proved and does not cause secondary environmental pollution. A modified bioreactor that supports simultaneous saccharification and cofermentation (SSCF) was used for the effective conversion of agricultural waste into bioethanol. Previous studies have reported the conversion of cellulose into monosaccharides using microorganisms [9,10]. The SSCF has recently been used to produce bioethanol according to its properties, i.e., reduced inhibition of cellulase by fermented hydrolyzed sugars, higher product yield, lower cellulase requirements, lower requirements for sterile conditions, shorter process time, and reduced reactor volume. Additionally, SSCF has many advantages over bioethanol production such as a higher yield, shorter processing time, and lower reactor volume. This work has two parts. First, wood dust is pretreated by SFE and SE to form substrates on which Z. mobilis can produce bioethanol. The respective amounts of bioethanol produced are compared. Second, the activities of saccharification enzymes were evaluated by cocultivating T. reesei and A. niger. Pre-treated wood dust is applied as a medium in the SSCF bioreactor to evaluate its potential for bioethanol production. The whole experimental procedures were shown into the Fig. 1A. 2. Materials and methods 2.1. Microorganisms and maintenance media T. reesei BCRC 31,863 and A. niger BCRC 31,130 were obtained from the Bioresource Collection and Research Centre (BCRC) of Taiwan. The stock culture was maintained aseptically on potatodextrose-agar (PDA) Petri plates (BD, New Jersey, USA). The PDA plates were incubated at 30 °C for 7 days until good sporulation was reached and then stored at 4 °C. Z. mobilis BCRC 10,809 was
obtained from the BCRC of Taiwan. Plate stock culture medium consisted of yeast extract (5.0 g/L, BD), glucose 20 (g/l, Sigmaaldrich, Maryland, USA), Bacto peptone (5 g/l, BD), MgSO4 ·7H2 O (1.5 g/l, Sigma), KH2 PO4 (2 g/l, Sigma), and (NH4 )2 HPO4 (1.5 g/l, Sigma) at pH 6.8, with cultivation at 30 °C for 2 days. BushnellHaas selection D medium (BHSD) was used for cultivation of T. reesei and A. niger spores, and Z. mobilis, and consisted of carboxymethylcellulose (CMC, 10 g/l, Sigma), MgSO4 7H2 O (0.4 g/l, Sigma), KH2 PO4 (1 g/l, Sigma), (NH4 )2 HPO4 (1 g/l, Sigma), CaCl2 (0.02 g/l, Sigma), and FeCl3 (0.04 g/l, Sigma). For ethanol production Bushnell–Haas selection W medium (BHSW), consisting of wood dust (dried weight, 10 g/l, Far Eastern New Century, Taoyuan City, Taiwan), MgSO4 7H2 O (0.4 g/l, Sigma), KH2 PO4 (1 g/l, Sigma), (NH4 )2 HPO4 (1 g/l, Sigma), KNO3 (1 g/l, Sigma), CaCl2 (0.02 g/l, Sigma), and FeCl3 (0.04 g/l, Sigma). The pH was adjusted to 5.5–6 with NaOH or H2 SO4 . 2.2. Pretreatment by steam explosion Wood dusts (Chang Chun Plastics Co., LTD, Taipei City, Taiwan) was immersed in 4% sulfuric acid solution for 2 h After removing out the residual water, 10 kg mass samples of the water-riched wood dusts were treated with high-pressure steam in a 250-l steam-explosion reactor. The final temperature in the reactor was 120 °C for 5 min. Then the process temperature varied to 180 °C for steam treatment around 3 min. After cooling, the solids from the steam-explosion process were filtrated with water. The resulting aqueous hydrolyzate solutions were rich in hemicellulose-derived sugars and acetic acid. The hydrolyzates were first subjected to an over-liming process with Ca(OH)2 to remove the lignin-derived phenolics and final pH adjusted to near 7.0. Next, 12–15 (FPU)/g of acid-treated residue was further processed with glucan CTec 2 enzyme (Sigma) at 50 °C for 72 h After filtration, the glucose was removed to harvest 1.8 kg of lingocellulose. 2.3. Pretreatment by supercritical fluid extraction The experiments were performed using ten kilograms of wood dusts (moisture content of 80%) that was placed inside a 50-ml stainless steel reactor vessel (Thar - Pittsburgh, USA). Pressurized CO2 was fed into the reactor until 20.6 MPa was reached. The temperature was raised to 120 °C, 150 °C, or 180 °C for 1 h to create the supercritical state. At the end of each experiment, the supercritical CO2 was suddenly released into the atmosphere via an expansion valve. The residues were collected and washed extensively with deionized water. After supercritical CO2 pretreatment, the samples were stored in sealed bags at 4 °C to prevent contamination until needed for composition analysis after enzymatic hydrolysis. 2.4. Evaluation of enzyme activities of endo-1,4-β -glucanase, exo-1,4-β -glucanase, and 1,4-β -glucosidase The activities of endo-1,4-β -glucanase (EC 3.2.1.4, Sigma), exoβ −1,4-glucanase (EC 3.2.1.155, Sigma), and 1,4-β -glucosidase (EC 3.2.1.21, Sigma) were determined using 1.0 mL of the following substrate preparations in 50 mol/l acetate buffer pH 4.8, respectively [11]: 1 wt% of Avicel type 50, 2 wt% of CMC, and 15 mmol/l of cellobiose. These substrates were incubated with 1.0 ml of the enzyme preparation for 60 min (Avicel type 60) or 30 min (CMC and cellobiose), depending on the assay. For total cellulase activity, the reaction was stopped by the addition of 3.0 ml of 3,5dinitrosalicylic acid (DNS) solution, including 3,4-dinitrosalicyclic acid (10 g/l), potassium sodium tartrate tetrahydrate (403 g/l) and NaOH (anhydrous, 16 g/l). One unit (U) of each enzyme activity of endo-1,4-β -glucanase, exo-β −1,4-glucanase and 1,4-β -glucosidase
Please cite this article as: W.-C. Chen et al., Producing bioethanol from pretreated-wood dust by simultaneous saccharification and cofermentation process, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.025
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Fig. 1. The whole experimental procedures of this study (A); the scheme of the modified bioreactor used in this study (B).
are defined as the amount of enzyme, which releases amounts of glucose (0.5 mg for endo-1,4-β -glucanase, exo-β −1,4-glucanase and 1 mg for 1,4-β -glucosidase) per minute in the reaction mixture under these assay conditions [11].
carried out for another 24 h at pH 5.5–6 and 30 °C. The supernatant was harvested from the collected broth that was centrifuged at 12,0 0 0 rpm for 10 min per 2 h 2.7. HPLC analysis of reducing sugars and bioethanol
2.5. Cell immobilization Concentrated Z. mobilis (3.25 × 106 cells/ml) was mixed with a series of sodium alginate solutions from 0.5 to 4% [w/v] to yield cell-embedded beads. The alginate beads were prepared by syringing the well-mixed sodium alginate (Sigma) solution by means of a 16-gauge needle into a 0.1 M CaCl2 (Sigma) solution. Finally, the alginate beads with bacteria were harvested by washing with phosphate buffer. In addition, the amount ratio of sodium alginate solution to Z. mobilis was varied from 1/1 to 1/4 to determine the most effective immobilization ratio.
The concentration of reducing sugars and bioethanol was determined using a HITACHI HPLC system equipped with a ICESep COREGEL 87H3 Column (Transgenomic Inc., USA). The analyses were carried out at 30 °C using 8 mM sulfuric acid as the mobile phase. The column was eluted at a flow rate of 0.6 ml/min, and the eluate was analyzed using a refractive index detector. 3. Results and discussions 3.1. Identification of the composition of wood dust and the byproduct effects
2.6. Bioethanol production in the modified bioreactor This modified bioreactor (Fig. 1B) was operated in accordance with the protocol used in our previous study. Briefly, this procedure involved cocultivation of the three species (T. reesei, A. niger, and Z. mobilis). First, T. reesei and A. niger (6 × 106 spores/ml) were cultivated on a polyurethane (PU) carrier with fresh BHSD medium to significantly increase microbial proliferation (the precultivation step) for 3 days at 30 °C, and Z. mobilis was entrapped onto alginate beads in the meantime. Second, the BHSD medium was removed, and the microbe-laden alginate beads were placed into the lower anaerobic section of the bioreactor after the precultivation step. The fresh medium for bioethanol production was fed into the bioreactor to produce bioethanol via SSCF. Total experiments were
Three major parameters, i.e., cellulose, hemicellulose, and lignin were observed when analyzing the composition of wood dust by SFE and SE. The remainder of the composition of wood dust by SFE and SE was made up of acetate, water and ash. According to the experimental results presented in Fig. 2, the percentages of cellulose, hemicellulose, lignin, and the others of wood dust were 39.6%, 11.7%, 39.40%, and 9.3%, respectively. The experimental results revealed that the combined content of cellulose and hemicellulose was around 80% in wood dust. To further determine the reducing sugar (i.e., glucose and xylose) content of wood dust, the commercial enzyme (i.e., CTec 2) was used to degrade the cellulose, hemicellulose, and lignin for 3 days. As shown in Table 1, the concentrations of glucose and xy-
Please cite this article as: W.-C. Chen et al., Producing bioethanol from pretreated-wood dust by simultaneous saccharification and cofermentation process, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.025
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W.-C. Chen et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2017) 1–6 Table 1. Comparison of procedures and types of byproduct from pretreated-wood dust substrate under enzymatic hydrolysis.
Pre-treatment supercritical fluid extraction steam explosion
byproduct concentration (g/L) Glucose Xylose Acetic acid
HMF
Furfural
LDP
2.57 ± 0.21
1.59 ± 0.19
1.51 ± 0.23
0
0
0.21 ± 0.07
2.12 ± 0.14
2.58 ± 0.32
2.69 ± 0.27
0.25 ± 0.05
0.33 ± 0.08
0.72 ± 0.02
Fig. 2. The composition of wood dust after the pretreatment process used in this study.
Fig. 3. Effect of producing bioethanol from Zymomonas mobilis using BHSD medium.
lose were 2.57 g/l and 1.59 g/l by SFE and 2.12 g/l and 2.58 g/l by SE. The experimental results revealed that wood dust is rich in xylose. However, xylose as a nutrient source could not be taken up by the bioethanol-producing bacteria, Z. mobilis in this study. In addition, as shown in Table 1, hydroxymethylfurfural (HMF) and furfural were not observed as byproducts of wood dust by SFE, which is contrary to the results obtained by SE. These results indicated that using wood dust as a nutrient source should facilitate to microorganism proliferation and bioethanol production. To understand the effects of the byproduct, BHSD medium was used as a nutrient source for Z. mobilis. The CTec 2 enzyme was used to treat the pretreated wood dust solution for use with Z. mobilis. The experimental results presented in Fig. 3 showed that the byproducts of the pretreatment steps might affect bioethanol production significantly. Bioethanol production from Z. mobilis using SFE reached a plateau (around 1.4 g/l) after 9-h of cultivation. Ad-
Fig. 4. Saccharification enzyme activities of single cultivation of A. niger or T. reesei and cocultivation of A. niger and T. reesei.
ditionally, when glucose was used as a nutrient source, its uptake was zero g/L from the beginning of cultivation to the 9th hour. However, little to no bioethanol production from Z. mobilis was observed using SE from the beginning to the end of the cultivation period. Reports indicated that the byproducts of HMF, acetic acid, and furfural might play an inhibitory role, reducing bioethanol production [12–14]. Miller et al. proved that byproducts, such as furfural, of the pretreatment process affect microbial growth and fermentation by inhibiting the assimilation of sulfate [12]. Palmqvist et al. examined the effect of the acetic acid, furfural, and p-hydroxybenzoic acid on ethanol yield by varying their concentrations [14]. Their results proved that higher concentrations of acetic acid and furfural were associated with lower ethanol yield and interacted with each other. Moreover, the species of byproducts of SFE pretreatment and their concentrations lower than those of SE pretreatment showed the great potential of bioethanol production owing to the preservation of most of the cellulose in a greater accessible surface area and pore volume and finally, significant disruption and redistribution of lignin. 3.2. Optimization of enzyme activities, the concentration of alginate beads, and immobilized proportion of Z. mobilis and alginate beads To enhance bioethanol production, SSCF was carried out in the modified bioreactor. Meanwhile, pretreated wood dust medium was used as a nutrient source for Z. mobilis when using SFE and SE for bioethanol production. Several parameters were evaluated to optimize the fermentation process, including enzyme activities, concentration of alginate beads, and immobilized proportion of Z. mobilis and alginate beads. Reports have shown that A. niger and T. reesei can produce a saccharification enzyme to break down wood dust to produce glucose and xylose [15,16]. To prove high enzyme activities can be achieved, A. niger and T. reesei were cocultivated with BHSD medium as a nutrient source. As shown by the results in Fig. 4, the
Please cite this article as: W.-C. Chen et al., Producing bioethanol from pretreated-wood dust by simultaneous saccharification and cofermentation process, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.025
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Yabannavar et al. also confirmed the substantial buildup of inhibitory product in large beads in an analysis of the effects of substrate and product transport. Therefore, the 3% alginate bead was selected for further experimentation [18]. Subsequently, the 3% alginate bead was fixed for evaluating the various immobilized proportions of Z. mobilis to alginate beads. Four proportion conditions were chosen for evaluating the effects of bioethanol production. As shown in Fig. 5B, the proportion of 1:3 of Z. mobilis to alginate beads showed the highest bioethanol production. However, the highest bioethanol production yield (around 2.5 g/l) occurred at the proportion of 1:4 of Z. mobilis to alginate beads. Based on these results, the proportion of 1:4 of Z. mobilis to alginate beads was chosen as the most optimal. Based on the aforementioned experimental results, the optimized conditions for the SSCF process were 3% alginate beads and a 1:4 ratio of alginate beads to Z. mobilis. Therefore, these conditions were used in a subsequent experiment in which bioethanol was produced using a modified bioreactor. 3.3. Bioethanol production by a modified bioreactor
Fig. 5. Bioethanol production and productivity of Z. mobilis using various concentration of alginate (A) and inoculum proportions of Z. mobilis to alginate (B).
enzyme activities of endo-glucanase (11.83 U/ml), exo-glucanase (12.27 U/ml, and β -glucosidase (13.49 U/ml) by the cocultivation of A. niger and T. reesei were higher than that by single bacterial cultivation. Yang et al. developed a cocultivation of T. reesei RUT-C30 and P. chrysosporium Burdsall for biodegradation of lignocellulosic pumpkin residues producing soluble saccharide. Their results demonstrated that cocultivation of the two fungi with Phanerochaete chrysosporium Burdsall inoculation delayed for 1.5 days produced remarkably higher saccharide yield compared with that from monoculture of P. chrysosporium Burdsall or T. reesei RUTC30, respectively [17]. Therefore, we believe that the enzyme synergistic effect of the cocultivation of A. niger and T. reesei would raise the enzyme activities for bioethanol production. To evaluate the effect of alginate concentration and immobilized proportion of Z. mobilis to alginate beads, BHSD medium was applied as a basic nutrient for bioethanol production. Five alginate concentrations, 0.5%, 1%, 2%, 3%, and 4% were examined to determine the optimal concentration for bioethanol production by SSCF. The immobilized proportion of Z. mobilis and alginate beads was fixed at 1:4 first and 0.1 M CaCl2 was used to form the beads. Fig. 5 A shows that the concentration of highest bioethanol production (around 6.5 g/l) increased along with an increase of alginate bead concentration. The highest bioethanol production was achieved at 3% alginate beads. Our previous study showed that the limitation of mass transfer in gel beads would affect bioethanol production [16]. Moreover, mathematical simulations performed by
A modified bioreactor with an upper aerobic reactor and a lower aerobic reactor was used to enhance bioethanol production (Fig. 1B). T. reesei and A. niger (inoculum ratio and concentration were 1:1 and 6 × 106 spores/ml, respectively) were evenly distributed on PU carriers, the total medium volume of modified bioreactor was immediately reduced to one third, and the exposed volume of PU carriers in the air was increased to enhance growth of aerobic fungi at the single stage. After 72 h preculture, Z. mobilis alginate beads were put into the lower reactor with fresh BHSW medium to initiate ethanol production. The experimental results presented in Fig. 6A show that the bioethanol production and conversion rate by Z. mobilis alginate beads using the wood dust pretreated by SFE were 0.069 g/g (g (ethanol) / g (wood dust), equal to 0.69 g/l) and 20.72%, respectively, after 2 h of cultivation. After 4 h of cultivation, the glucose had been totally consumed. Similar results were also observed for the SE group, where the bioethanol production and conversion rate were 0.049 g/g and 24.39%, respectively. Actually, the bioethanol production in the SSCF bioreactor (0.69 g/l) was far less than the flask (1.44 g/l), around 2 folds when the BHSW medium was applied. Interestingly, the xylose from SFE pretreatment, like the glucose, was consumed after 2 h of cultivation, but the xylose from SE pretreatment seemed to be consumed less from the beginning to the end of the experiment. A report showed that A. niger might take up xylose as its nutrient source to promote secretion of the saccharification enzyme, which would then facilitate bioethanol production [19]. Besides, computational and experimental approaches were also used to identify and characterize xylose-transporting proteins from the industrial cell factories A. niger and T. reesei and to reveal the presence of various xylose transporters [19]. Additionally, bioethanol production using pretreated wood dust via SFE pretreatment was higher than that via SE pretreatment. The reason for the low bioethanol production may be explained by the byproduct effect. According to the results presented in Table 1, the byproducts of HMF and furfural appeared when the SE pretreatment was applied. Reports indicate that the byproducts of HMF and furfural might play an inhibitory role, acting to reduce bioethanol production [12–14]. In addition, because of the nutritional competition of T. reesei, A. niger, and Z. mobilis, when the reducing sugars of the 1% pretreated wood dust solution were used as a nutrient source, they were almost completely consumed, leading to a low bioethanol production. Since 1% pretreated wood dust solution as a nutrient source may not be effective for producing bioethanol in the SSCF bioreactor, adding sufficient solution of wood dust that has been
Please cite this article as: W.-C. Chen et al., Producing bioethanol from pretreated-wood dust by simultaneous saccharification and cofermentation process, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.025
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SFE pretreatment and 0.049 g/g for SE pretreatment) by cultivating A. niger and T. reesei in a PU carrier and immobilizing Z. mobilis onto alginate beads using 5% of pretreated-wood dust as its nutrient source was successful. Collectively, the findings of this study highlight the enormous potential of applying the pretreatment method of SFE to process wood dust for bioethanol production. Future studies should attempt to increase bioethanol production by optimizing the medium composition. Conflict of interest The authors declare no competing interests. Acknowledgements The authors gratefully acknowledge the financial support by the Ministry of Science and Technology of the Republic of China under grant number MOST 104-2622-8-0 07-0 01- and MOST 105-26228-0 07-0 09- and MOST 105-2221-E-155-070-. Valuable discussions and suggestions from Dr. Yu-Kuo Liu are gratefully acknowledged. References
Fig. 6. Bioethanol production and reducing sugar concentration by SSCF process using 1% of pretreat-wood dust with BHSW medium (A). The comparison of using 1%, 5% and 10% of pretreated-wood dust with BHSW medium for bioethanol production (B).
pretreated by SFE to the BHSW medium may improve bioethanol production. However, the reflux column of the SSCF bioreactor may become blocked if the solution that is added to the BHSW medium constitutes more than 10% by volume). Therefore, three concentrations (1%, 5%, and 10%) were used and their effects on bioethanol production in the SSCF bioreactor were evaluated. As presented in Fig. 6B, the production of bioethanol and rate of conversion thereof by Z. mobilis alginate beads were 0.138 g/g (around 2 folds) and 11.71% when 5% pretreated wood dust solution was applied. However, no bioethanol was produced when 10% pretreated wood dust solution was applied. 4. Conclusions Our experimental results suggest that the pretreatment methods used in this study are feasible by using either SFE or SE. In addition, the advantage of using SFE pretreatment is that it can effectively reduce byproduct production because there is no acid treatment step in the whole process. This study also showed the synergistic effect of the cocultivation of A. niger and T. reesei on saccharification enzyme activity; the results also suggest the optimal alginate conditions used for immobilizing Z. mobilis, including the alginate concentration and inoculum proportion. Furthermore, using a new SSCF bioreactor to produce bioethanol (0.069 g/g for
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Producing bioethanol from pretreated-wood dust by simultaneous saccharification and co-fermentation process Wei-Chuan Chen a, Yin-Chen Lin a, Ya-Lian Ciou a, I-Ming Chu b, Shen-Long Tsai c, John Chi-Wei Lan d, Yu-Kaung Chang e, Yu-Hong Wei a,∗ a
Graduate school of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan 320, Taiwan Department of Chemical Engineering, National Tsing Hua University, Hsin Chu 300, Taiwan Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 100, Taiwan d Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan 320, Taiwan e Department & Graduate Institute of Chemical Engineering & Graduate Institute of Biochemical Engineering, Ming Chi University of Technology, Taishan New Taipei City 243, Taiwan b c
a r t i c l e
i n f o
Article history: Received 21 December 2016 Revised 19 April 2017 Accepted 19 April 2017 Available online xxx Keywords: Bioethanol production Pretreated-wood dust medium Supercritical fluid extraction Steam explosion Saccharification enzyme activity
a b s t r a c t The aim of this study was to utilize a new and highly effective bioreactor system, i.e., simultaneous saccharification and fermentation (SSCF), for bioethanol production by the cocultivation of Trichoderma reesei, Aspergillus niger, and Zymomonas mobilis by using a direct conversion process of pretreated-wood dust medium. Wood dust has been effectively used to obtain reducing sugars (glucose, xylose, and other byproducts) through the use of either a supercritical fluid extraction (SFE) or steam explosion (SE) pretreatment step. In addition, experimental results showed that polyurethane as a porous carrier could enhance total saccharification enzyme activity at an inoculum proportion of 1/1 of T. reesei, and A. niger and at a total inoculum concentration of 6.5 × 106 spores/ml. Furthermore, the concentration of alginate beads (3%) and immobilized proportion of Z. mobilis to alginate beads (1:4) were also examined. In accordance with previous reports, bioethanol production was carried out in a SSCF bioreactor by the cocultivation of T. reesei and A. niger in the polyurethane carrier and Z. mobilis immobilized in alginate beads using pretreated-wood dust medium. Experimental results revealed that, after 24 h of cultivation, the yield of bioethanol produced using pretreated-wood dust medium (1%) were 0.069 g/g and 0.049 g/g, for SFE and SE, respectively. Meanwhile, the sugar conversion rate reached 20.72% and 24.39% for SFE and SE, respectively. Thus, the results of this study show that pretreated-wood dust medium has significant potential for use in bioethanol production. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Continued industrialization and population growth have increased annual energy consumption [1]. Increasing fossil fuel prices and greenhouse gases have motivated the research and development of renewable resources in many countries. The principal substitute for petrol in road vehicles is bioethanol. One of the advantages of using bioethanol is that doing so reduces greenhouse gas emissions. Also, blending bioethanol with petrol, as in E85, helps to extend diminishing oil supplies, increase fuel security, and eliminate heavy reliance on oil producing nations. Bioethanol is seen as the most promising prospective renewable energy source, which can be produced from microbial fermentation by converting sugars from cellulosic materials such as wood dust [2]. ∗
Corresponding author. E-mail address: [email protected] (Y.-H. Wei).
Lignocellulose is an abundant natural carbohydrate that can be transformed into a substitute renewable energy resource by microbial conversion [3]. The advantages of using cellulosic materials for bioethanol production are their low cost, ready availability, lack of conflict with use for food, and the potential production of fuel from lignin [4]. A pretreatment step is necessary for modifying structural characteristics of lignocellulose and increasing the availability of glucan and xylan for enzymatic saccharification [5]. The pretreatment step can destroy the lignin structure to allow for easier break down of the cellulose by the saccharification enzyme [4]. Traditional pretreatments of biomass for producing bioethanol include physical methods and chemical methods, involving liquid hot water and alkali (or acid), for example [6]. However, several disadvantages for all the different pretreatment options exist, and it is necessary to adapt suitable pretreatments based on the properties of the raw material. Physical and chemical pretreatments inhibit the
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hydrolysis of cellulose and hemicellulose and lignin fraction owing to the close among the components of lignocellulosic biomass. Therefore, in this study, steam explosion (SE) and supercritical fluid extraction (SFE) were used in this work to pretreat wood dust to avoid the aforementioned problems. Steam explosion (SE) technology has become one of the most common and widely employed physicochemical pretreatments for lignocellulosic biomass. The process of SE, developed by William H. Mason in 1925, is used for the pretreatment of agricultural wastes, i.e., wood dust in this study [5]. SE technology is a hydrothermal pretreatment that consists of three main steps: (1) the treatment step, (2) the explosion step, and (3) the impact step [5]. The first step is a process wherein lignocellulosic biomass is treated with pressurized steam for a certain period. The SFE rapid release of pressure causes the explosion of lignocellulosic biomass. Finally, the impact of the lignocellulosic biomass mixture is done to form a raw material used for enzyme hydrolysis. Supercritical fluid extraction is also one of the pretreatment methods for disrupting the crystalline structure of lignocellulose under certain conditions such as temperature and pressure above its critical point [7]. SFE is becoming necessary to reduce the environmental hazards of common chemicals and solvents used in traditional methods. SFE shows excellent potential as a method for lignocellulosic biomass pretreatment [8]. Reports have shown that SFE has been successfully used for the pretreatment of commercial cellulosic materials, recycled paper mix, and sugarcane bagasse [7,8]. In addition, the use of SFE, as a green solvent for biomass pretreatment in a biorefinery concept, is increasing and it is expected to continue growing in the future. Saccharification enzymes can be produced by fungi such as Trichoderma reesei and Aspergillus niger, and agricultural waste can be converted into bioethanol by Zymomonas mobilis. Conversion of agricultural waste reduces its environmental impacts by efficiently using it to produce secondary energy. The conversion of cellulose into monosaccharides by microorganisms has been proved and does not cause secondary environmental pollution. A modified bioreactor that supports simultaneous saccharification and cofermentation (SSCF) was used for the effective conversion of agricultural waste into bioethanol. Previous studies have reported the conversion of cellulose into monosaccharides using microorganisms [9,10]. The SSCF has recently been used to produce bioethanol according to its properties, i.e., reduced inhibition of cellulase by fermented hydrolyzed sugars, higher product yield, lower cellulase requirements, lower requirements for sterile conditions, shorter process time, and reduced reactor volume. Additionally, SSCF has many advantages over bioethanol production such as a higher yield, shorter processing time, and lower reactor volume. This work has two parts. First, wood dust is pretreated by SFE and SE to form substrates on which Z. mobilis can produce bioethanol. The respective amounts of bioethanol produced are compared. Second, the activities of saccharification enzymes were evaluated by cocultivating T. reesei and A. niger. Pre-treated wood dust is applied as a medium in the SSCF bioreactor to evaluate its potential for bioethanol production. The whole experimental procedures were shown into the Fig. 1A. 2. Materials and methods 2.1. Microorganisms and maintenance media T. reesei BCRC 31,863 and A. niger BCRC 31,130 were obtained from the Bioresource Collection and Research Centre (BCRC) of Taiwan. The stock culture was maintained aseptically on potatodextrose-agar (PDA) Petri plates (BD, New Jersey, USA). The PDA plates were incubated at 30 °C for 7 days until good sporulation was reached and then stored at 4 °C. Z. mobilis BCRC 10,809 was
obtained from the BCRC of Taiwan. Plate stock culture medium consisted of yeast extract (5.0 g/L, BD), glucose 20 (g/l, Sigmaaldrich, Maryland, USA), Bacto peptone (5 g/l, BD), MgSO4 ·7H2 O (1.5 g/l, Sigma), KH2 PO4 (2 g/l, Sigma), and (NH4 )2 HPO4 (1.5 g/l, Sigma) at pH 6.8, with cultivation at 30 °C for 2 days. BushnellHaas selection D medium (BHSD) was used for cultivation of T. reesei and A. niger spores, and Z. mobilis, and consisted of carboxymethylcellulose (CMC, 10 g/l, Sigma), MgSO4 7H2 O (0.4 g/l, Sigma), KH2 PO4 (1 g/l, Sigma), (NH4 )2 HPO4 (1 g/l, Sigma), CaCl2 (0.02 g/l, Sigma), and FeCl3 (0.04 g/l, Sigma). For ethanol production Bushnell–Haas selection W medium (BHSW), consisting of wood dust (dried weight, 10 g/l, Far Eastern New Century, Taoyuan City, Taiwan), MgSO4 7H2 O (0.4 g/l, Sigma), KH2 PO4 (1 g/l, Sigma), (NH4 )2 HPO4 (1 g/l, Sigma), KNO3 (1 g/l, Sigma), CaCl2 (0.02 g/l, Sigma), and FeCl3 (0.04 g/l, Sigma). The pH was adjusted to 5.5–6 with NaOH or H2 SO4 . 2.2. Pretreatment by steam explosion Wood dusts (Chang Chun Plastics Co., LTD, Taipei City, Taiwan) was immersed in 4% sulfuric acid solution for 2 h After removing out the residual water, 10 kg mass samples of the water-riched wood dusts were treated with high-pressure steam in a 250-l steam-explosion reactor. The final temperature in the reactor was 120 °C for 5 min. Then the process temperature varied to 180 °C for steam treatment around 3 min. After cooling, the solids from the steam-explosion process were filtrated with water. The resulting aqueous hydrolyzate solutions were rich in hemicellulose-derived sugars and acetic acid. The hydrolyzates were first subjected to an over-liming process with Ca(OH)2 to remove the lignin-derived phenolics and final pH adjusted to near 7.0. Next, 12–15 (FPU)/g of acid-treated residue was further processed with glucan CTec 2 enzyme (Sigma) at 50 °C for 72 h After filtration, the glucose was removed to harvest 1.8 kg of lingocellulose. 2.3. Pretreatment by supercritical fluid extraction The experiments were performed using ten kilograms of wood dusts (moisture content of 80%) that was placed inside a 50-ml stainless steel reactor vessel (Thar - Pittsburgh, USA). Pressurized CO2 was fed into the reactor until 20.6 MPa was reached. The temperature was raised to 120 °C, 150 °C, or 180 °C for 1 h to create the supercritical state. At the end of each experiment, the supercritical CO2 was suddenly released into the atmosphere via an expansion valve. The residues were collected and washed extensively with deionized water. After supercritical CO2 pretreatment, the samples were stored in sealed bags at 4 °C to prevent contamination until needed for composition analysis after enzymatic hydrolysis. 2.4. Evaluation of enzyme activities of endo-1,4-β -glucanase, exo-1,4-β -glucanase, and 1,4-β -glucosidase The activities of endo-1,4-β -glucanase (EC 3.2.1.4, Sigma), exoβ −1,4-glucanase (EC 3.2.1.155, Sigma), and 1,4-β -glucosidase (EC 3.2.1.21, Sigma) were determined using 1.0 mL of the following substrate preparations in 50 mol/l acetate buffer pH 4.8, respectively [11]: 1 wt% of Avicel type 50, 2 wt% of CMC, and 15 mmol/l of cellobiose. These substrates were incubated with 1.0 ml of the enzyme preparation for 60 min (Avicel type 60) or 30 min (CMC and cellobiose), depending on the assay. For total cellulase activity, the reaction was stopped by the addition of 3.0 ml of 3,5dinitrosalicylic acid (DNS) solution, including 3,4-dinitrosalicyclic acid (10 g/l), potassium sodium tartrate tetrahydrate (403 g/l) and NaOH (anhydrous, 16 g/l). One unit (U) of each enzyme activity of endo-1,4-β -glucanase, exo-β −1,4-glucanase and 1,4-β -glucosidase
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Fig. 1. The whole experimental procedures of this study (A); the scheme of the modified bioreactor used in this study (B).
are defined as the amount of enzyme, which releases amounts of glucose (0.5 mg for endo-1,4-β -glucanase, exo-β −1,4-glucanase and 1 mg for 1,4-β -glucosidase) per minute in the reaction mixture under these assay conditions [11].
carried out for another 24 h at pH 5.5–6 and 30 °C. The supernatant was harvested from the collected broth that was centrifuged at 12,0 0 0 rpm for 10 min per 2 h 2.7. HPLC analysis of reducing sugars and bioethanol
2.5. Cell immobilization Concentrated Z. mobilis (3.25 × 106 cells/ml) was mixed with a series of sodium alginate solutions from 0.5 to 4% [w/v] to yield cell-embedded beads. The alginate beads were prepared by syringing the well-mixed sodium alginate (Sigma) solution by means of a 16-gauge needle into a 0.1 M CaCl2 (Sigma) solution. Finally, the alginate beads with bacteria were harvested by washing with phosphate buffer. In addition, the amount ratio of sodium alginate solution to Z. mobilis was varied from 1/1 to 1/4 to determine the most effective immobilization ratio.
The concentration of reducing sugars and bioethanol was determined using a HITACHI HPLC system equipped with a ICESep COREGEL 87H3 Column (Transgenomic Inc., USA). The analyses were carried out at 30 °C using 8 mM sulfuric acid as the mobile phase. The column was eluted at a flow rate of 0.6 ml/min, and the eluate was analyzed using a refractive index detector. 3. Results and discussions 3.1. Identification of the composition of wood dust and the byproduct effects
2.6. Bioethanol production in the modified bioreactor This modified bioreactor (Fig. 1B) was operated in accordance with the protocol used in our previous study. Briefly, this procedure involved cocultivation of the three species (T. reesei, A. niger, and Z. mobilis). First, T. reesei and A. niger (6 × 106 spores/ml) were cultivated on a polyurethane (PU) carrier with fresh BHSD medium to significantly increase microbial proliferation (the precultivation step) for 3 days at 30 °C, and Z. mobilis was entrapped onto alginate beads in the meantime. Second, the BHSD medium was removed, and the microbe-laden alginate beads were placed into the lower anaerobic section of the bioreactor after the precultivation step. The fresh medium for bioethanol production was fed into the bioreactor to produce bioethanol via SSCF. Total experiments were
Three major parameters, i.e., cellulose, hemicellulose, and lignin were observed when analyzing the composition of wood dust by SFE and SE. The remainder of the composition of wood dust by SFE and SE was made up of acetate, water and ash. According to the experimental results presented in Fig. 2, the percentages of cellulose, hemicellulose, lignin, and the others of wood dust were 39.6%, 11.7%, 39.40%, and 9.3%, respectively. The experimental results revealed that the combined content of cellulose and hemicellulose was around 80% in wood dust. To further determine the reducing sugar (i.e., glucose and xylose) content of wood dust, the commercial enzyme (i.e., CTec 2) was used to degrade the cellulose, hemicellulose, and lignin for 3 days. As shown in Table 1, the concentrations of glucose and xy-
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W.-C. Chen et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2017) 1–6 Table 1. Comparison of procedures and types of byproduct from pretreated-wood dust substrate under enzymatic hydrolysis.
Pre-treatment supercritical fluid extraction steam explosion
byproduct concentration (g/L) Glucose Xylose Acetic acid
HMF
Furfural
LDP
2.57 ± 0.21
1.59 ± 0.19
1.51 ± 0.23
0
0
0.21 ± 0.07
2.12 ± 0.14
2.58 ± 0.32
2.69 ± 0.27
0.25 ± 0.05
0.33 ± 0.08
0.72 ± 0.02
Fig. 2. The composition of wood dust after the pretreatment process used in this study.
Fig. 3. Effect of producing bioethanol from Zymomonas mobilis using BHSD medium.
lose were 2.57 g/l and 1.59 g/l by SFE and 2.12 g/l and 2.58 g/l by SE. The experimental results revealed that wood dust is rich in xylose. However, xylose as a nutrient source could not be taken up by the bioethanol-producing bacteria, Z. mobilis in this study. In addition, as shown in Table 1, hydroxymethylfurfural (HMF) and furfural were not observed as byproducts of wood dust by SFE, which is contrary to the results obtained by SE. These results indicated that using wood dust as a nutrient source should facilitate to microorganism proliferation and bioethanol production. To understand the effects of the byproduct, BHSD medium was used as a nutrient source for Z. mobilis. The CTec 2 enzyme was used to treat the pretreated wood dust solution for use with Z. mobilis. The experimental results presented in Fig. 3 showed that the byproducts of the pretreatment steps might affect bioethanol production significantly. Bioethanol production from Z. mobilis using SFE reached a plateau (around 1.4 g/l) after 9-h of cultivation. Ad-
Fig. 4. Saccharification enzyme activities of single cultivation of A. niger or T. reesei and cocultivation of A. niger and T. reesei.
ditionally, when glucose was used as a nutrient source, its uptake was zero g/L from the beginning of cultivation to the 9th hour. However, little to no bioethanol production from Z. mobilis was observed using SE from the beginning to the end of the cultivation period. Reports indicated that the byproducts of HMF, acetic acid, and furfural might play an inhibitory role, reducing bioethanol production [12–14]. Miller et al. proved that byproducts, such as furfural, of the pretreatment process affect microbial growth and fermentation by inhibiting the assimilation of sulfate [12]. Palmqvist et al. examined the effect of the acetic acid, furfural, and p-hydroxybenzoic acid on ethanol yield by varying their concentrations [14]. Their results proved that higher concentrations of acetic acid and furfural were associated with lower ethanol yield and interacted with each other. Moreover, the species of byproducts of SFE pretreatment and their concentrations lower than those of SE pretreatment showed the great potential of bioethanol production owing to the preservation of most of the cellulose in a greater accessible surface area and pore volume and finally, significant disruption and redistribution of lignin. 3.2. Optimization of enzyme activities, the concentration of alginate beads, and immobilized proportion of Z. mobilis and alginate beads To enhance bioethanol production, SSCF was carried out in the modified bioreactor. Meanwhile, pretreated wood dust medium was used as a nutrient source for Z. mobilis when using SFE and SE for bioethanol production. Several parameters were evaluated to optimize the fermentation process, including enzyme activities, concentration of alginate beads, and immobilized proportion of Z. mobilis and alginate beads. Reports have shown that A. niger and T. reesei can produce a saccharification enzyme to break down wood dust to produce glucose and xylose [15,16]. To prove high enzyme activities can be achieved, A. niger and T. reesei were cocultivated with BHSD medium as a nutrient source. As shown by the results in Fig. 4, the
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Yabannavar et al. also confirmed the substantial buildup of inhibitory product in large beads in an analysis of the effects of substrate and product transport. Therefore, the 3% alginate bead was selected for further experimentation [18]. Subsequently, the 3% alginate bead was fixed for evaluating the various immobilized proportions of Z. mobilis to alginate beads. Four proportion conditions were chosen for evaluating the effects of bioethanol production. As shown in Fig. 5B, the proportion of 1:3 of Z. mobilis to alginate beads showed the highest bioethanol production. However, the highest bioethanol production yield (around 2.5 g/l) occurred at the proportion of 1:4 of Z. mobilis to alginate beads. Based on these results, the proportion of 1:4 of Z. mobilis to alginate beads was chosen as the most optimal. Based on the aforementioned experimental results, the optimized conditions for the SSCF process were 3% alginate beads and a 1:4 ratio of alginate beads to Z. mobilis. Therefore, these conditions were used in a subsequent experiment in which bioethanol was produced using a modified bioreactor. 3.3. Bioethanol production by a modified bioreactor
Fig. 5. Bioethanol production and productivity of Z. mobilis using various concentration of alginate (A) and inoculum proportions of Z. mobilis to alginate (B).
enzyme activities of endo-glucanase (11.83 U/ml), exo-glucanase (12.27 U/ml, and β -glucosidase (13.49 U/ml) by the cocultivation of A. niger and T. reesei were higher than that by single bacterial cultivation. Yang et al. developed a cocultivation of T. reesei RUT-C30 and P. chrysosporium Burdsall for biodegradation of lignocellulosic pumpkin residues producing soluble saccharide. Their results demonstrated that cocultivation of the two fungi with Phanerochaete chrysosporium Burdsall inoculation delayed for 1.5 days produced remarkably higher saccharide yield compared with that from monoculture of P. chrysosporium Burdsall or T. reesei RUTC30, respectively [17]. Therefore, we believe that the enzyme synergistic effect of the cocultivation of A. niger and T. reesei would raise the enzyme activities for bioethanol production. To evaluate the effect of alginate concentration and immobilized proportion of Z. mobilis to alginate beads, BHSD medium was applied as a basic nutrient for bioethanol production. Five alginate concentrations, 0.5%, 1%, 2%, 3%, and 4% were examined to determine the optimal concentration for bioethanol production by SSCF. The immobilized proportion of Z. mobilis and alginate beads was fixed at 1:4 first and 0.1 M CaCl2 was used to form the beads. Fig. 5 A shows that the concentration of highest bioethanol production (around 6.5 g/l) increased along with an increase of alginate bead concentration. The highest bioethanol production was achieved at 3% alginate beads. Our previous study showed that the limitation of mass transfer in gel beads would affect bioethanol production [16]. Moreover, mathematical simulations performed by
A modified bioreactor with an upper aerobic reactor and a lower aerobic reactor was used to enhance bioethanol production (Fig. 1B). T. reesei and A. niger (inoculum ratio and concentration were 1:1 and 6 × 106 spores/ml, respectively) were evenly distributed on PU carriers, the total medium volume of modified bioreactor was immediately reduced to one third, and the exposed volume of PU carriers in the air was increased to enhance growth of aerobic fungi at the single stage. After 72 h preculture, Z. mobilis alginate beads were put into the lower reactor with fresh BHSW medium to initiate ethanol production. The experimental results presented in Fig. 6A show that the bioethanol production and conversion rate by Z. mobilis alginate beads using the wood dust pretreated by SFE were 0.069 g/g (g (ethanol) / g (wood dust), equal to 0.69 g/l) and 20.72%, respectively, after 2 h of cultivation. After 4 h of cultivation, the glucose had been totally consumed. Similar results were also observed for the SE group, where the bioethanol production and conversion rate were 0.049 g/g and 24.39%, respectively. Actually, the bioethanol production in the SSCF bioreactor (0.69 g/l) was far less than the flask (1.44 g/l), around 2 folds when the BHSW medium was applied. Interestingly, the xylose from SFE pretreatment, like the glucose, was consumed after 2 h of cultivation, but the xylose from SE pretreatment seemed to be consumed less from the beginning to the end of the experiment. A report showed that A. niger might take up xylose as its nutrient source to promote secretion of the saccharification enzyme, which would then facilitate bioethanol production [19]. Besides, computational and experimental approaches were also used to identify and characterize xylose-transporting proteins from the industrial cell factories A. niger and T. reesei and to reveal the presence of various xylose transporters [19]. Additionally, bioethanol production using pretreated wood dust via SFE pretreatment was higher than that via SE pretreatment. The reason for the low bioethanol production may be explained by the byproduct effect. According to the results presented in Table 1, the byproducts of HMF and furfural appeared when the SE pretreatment was applied. Reports indicate that the byproducts of HMF and furfural might play an inhibitory role, acting to reduce bioethanol production [12–14]. In addition, because of the nutritional competition of T. reesei, A. niger, and Z. mobilis, when the reducing sugars of the 1% pretreated wood dust solution were used as a nutrient source, they were almost completely consumed, leading to a low bioethanol production. Since 1% pretreated wood dust solution as a nutrient source may not be effective for producing bioethanol in the SSCF bioreactor, adding sufficient solution of wood dust that has been
Please cite this article as: W.-C. Chen et al., Producing bioethanol from pretreated-wood dust by simultaneous saccharification and cofermentation process, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.025
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SFE pretreatment and 0.049 g/g for SE pretreatment) by cultivating A. niger and T. reesei in a PU carrier and immobilizing Z. mobilis onto alginate beads using 5% of pretreated-wood dust as its nutrient source was successful. Collectively, the findings of this study highlight the enormous potential of applying the pretreatment method of SFE to process wood dust for bioethanol production. Future studies should attempt to increase bioethanol production by optimizing the medium composition. Conflict of interest The authors declare no competing interests. Acknowledgements The authors gratefully acknowledge the financial support by the Ministry of Science and Technology of the Republic of China under grant number MOST 104-2622-8-0 07-0 01- and MOST 105-26228-0 07-0 09- and MOST 105-2221-E-155-070-. Valuable discussions and suggestions from Dr. Yu-Kuo Liu are gratefully acknowledged. References
Fig. 6. Bioethanol production and reducing sugar concentration by SSCF process using 1% of pretreat-wood dust with BHSW medium (A). The comparison of using 1%, 5% and 10% of pretreated-wood dust with BHSW medium for bioethanol production (B).
pretreated by SFE to the BHSW medium may improve bioethanol production. However, the reflux column of the SSCF bioreactor may become blocked if the solution that is added to the BHSW medium constitutes more than 10% by volume). Therefore, three concentrations (1%, 5%, and 10%) were used and their effects on bioethanol production in the SSCF bioreactor were evaluated. As presented in Fig. 6B, the production of bioethanol and rate of conversion thereof by Z. mobilis alginate beads were 0.138 g/g (around 2 folds) and 11.71% when 5% pretreated wood dust solution was applied. However, no bioethanol was produced when 10% pretreated wood dust solution was applied. 4. Conclusions Our experimental results suggest that the pretreatment methods used in this study are feasible by using either SFE or SE. In addition, the advantage of using SFE pretreatment is that it can effectively reduce byproduct production because there is no acid treatment step in the whole process. This study also showed the synergistic effect of the cocultivation of A. niger and T. reesei on saccharification enzyme activity; the results also suggest the optimal alginate conditions used for immobilizing Z. mobilis, including the alginate concentration and inoculum proportion. Furthermore, using a new SSCF bioreactor to produce bioethanol (0.069 g/g for
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Please cite this article as: W.-C. Chen et al., Producing bioethanol from pretreated-wood dust by simultaneous saccharification and cofermentation process, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.025