Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1209-2
ORIGINAL PAPER
An integrative process of bioconversion of oil palm empty fruit bunch fiber to ethanol with on-site cellulase production Youshuang Zhu • Fengxue Xin • Ying Zhao Yunkang Chang
•
Received: 16 November 2013 / Accepted: 28 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract The aim of this study was to efficiently convert oil palm empty fruit bunch fiber (OPEFB), one of the most commonly generated lingo-wastes in Southeast Asia, into both cellulase and bioethanol. The unprocessed cellulase crude (37.29 %) produced under solid-state fermentation using OPEFB as substrate showed a better reducing sugar yield using filter paper than the commercial enzyme blend (34.61 %). Organosolv pretreatment method could efficiently reduce hemicellulose (24.3–18.6 %) and lignin (35.2–22.1 %) content and increase cellulose content (40.5–59.3 %) from OPEFB. Enzymatic hydrolysis of pretreated OPEFB using the crude cellulase with 20 % solid content, enzyme loading of 15 FPU/g OPEFB at 50 °C, and pH 5.5 resulted in a OPEFB hydrolysate containing 36.01 g/L glucose after 72 h. Fermentation of the hydrolysate medium produced 17.64 g/L ethanol with 0.49 g/g yield from glucose and 0.088 g/g yield from OPEFB at 8 h using Saccharomyces cerevisiae. Keywords Cellulase Organosolv pretreatment OPEFB Solid state fermentation Ethanol
Y. Zhu and F. Xin contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s00449-014-1209-2) contains supplementary material, which is available to authorized users. Y. Zhu Y. Zhao Y. Chang Department of Biological Science, Jining Medical University, Jining 272067, China F. Xin (&) Department of Civil and Environmental Engineering, National University of Singapore, T-Lab 07-03 N, 5A Engineering Drive 1, Singapore 117576, Singapore e-mail: [email protected]
Introduction Bioconversion of lignocellulosic waste materials to chemicals and fuels is receiving great attention as they are low cost, renewable, and widespread in nature. According to recent techno-economic evaluations, the main contributors to the overall costs of producing ethanol from biomass are the raw material (30–40 %) and the capital investment (30–45 %), followed by the cellulase enzymes (10–20 %) [1]. Southeast Asia is well known for its potential in renewable resources such as oil palm waste, sugar cane bagasse, and rice straw. In the process of extraction of palm oil from oil palm fruit, a lignocellulosic material oil palm empty fruit bunch (OPEFB), which consists of 41.3–46.5 % cellulose, 25.3–33.8 % hemicellulose, and 27.6–32.5 % lignin is generated as a waste product [2–5]. The palm oil industries in Indonesia and Malaysia generate approximately 55.73 million tons of lignocellulosic agricultural waste and by-products per year [6]. Lignocellulosic OPEFB is potentially one of the lower-cost materials, as well as an alternative renewable bioresource, in comparison to using food sources, such as corn, sugarcane, and other food stocks, for the production of bioethanol [4–6]. However, OPEFB contains a relatively high percentage of hemicellulose and lignin per gram biomass as compared to other food and feedstocks: biomass (% cellulose: % hemicelluloses: % lignin); birch (40:23:21); wheat straw (38.2:21.2:23.4); corn stover (37.5:22.4:17.6); switch grass (31.0:20.4:17.6); Alfalfa (33:18:8) [7]. Thus, pretreatment is necessary to reduce the hemicellulose and lignin contents for effective bioethanol fermentation. The pretreatment process would ideally also enhance the proportion of cellulose in the OPEFB [2–5]. Moreover, removal of hemicellulose increases the pore size of the biomass and, therefore, increases the accessibility and the digestibility of
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Bioprocess Biosyst Eng Oil palm empty fruit bunch fiber Solid state fermentation Fungi: Trichoderma reesei RUT-C30 Substrate: OPEFB Medium: Mendel's medium
Pretreatment with ethanol at 70°C for 6 hours , 1st wash using ethanol for 1h Post-treatment with 4% H2O2 at 45°C for 16 hours, 2nd wash using water for 1h
Extration
50°C, pH 5.5, 72 h Crude enzymes (15FPU/ g OPEFB)
Treated OPEFB
Enzymatic hydrolysate Saccahromyces cerevisiae yeast extract and peptone
Bioethanol
Fig. 1 Experiment layout for saccharification and fermentation of pretreated OPEFB into fuel ethanol
cellulase toward the pretreated biomass [7]. Acid or alkali pretreatments, combined with high temperature or high pressure, have been applied in conventional chemical treatment processes [8, 9], while being achieved at the expense of increased thermal energy cost. A modified organosolv pretreatment has been developed in our prior work to remove lignin and hemicellulose of softwood under non-corrosive and mild conditions and increase accessibility of the cellulose [10]. So in this study, our first objective was to pretreat OPEFB using this organosolv modified method to efficiently remove hemicelluloses and lignin. The production economics of bio-ethanol is also largely dependent on the cost of cellulases [1, 7]. Efforts towards cost reduction have been directed at increasing enzyme production by finding hyperactive microbial strains, efficient fermentation techniques, and recovery systems [7]. The production of cellulases has been widely studied in submerged culture processes, but solid-state fermentation (SSF) has gained a fresh attention in the recent years, mainly due to its advantages over submerged fermentation such as low capital investment, solid waste management, reduced energy requirements, improved product recovery, etc. [11, 12]. SSF is a process whereby an insoluble substrate is fermented with sufficient moisture but without free water [12]. The substrates generally used are low-cost agro residues such as wheat straw, wheat bran, baggase, soya hulls, horticultural waste, etc. [13–18]. The use of SSF using cheap biomass resources as substrate can further improve production economics [11, 12]. So the second objective of the present study was to utilize the inexpensive and abundantly available lignocellulosic biomass, OPEFB
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for crude cellulase production under SSF by Trichoderma reesei RUT-C30. The organosolv pretreated OPEFB was then hydrolyzed using the above crude cellulase mixture for bioethanol production (Fig. 1).
Materials and methods Materials OPEFB was collected from Masai Palm Oil Mill, Johore, Malaysia. The collected OPEFB was first dried and mechanically milled with a lab mill (Ultra Centrifugal Mill ZM 200, Retsch GmbH, Germany) and sieved through standard mesh sieves (200–500 lm) using an electronic sieve shaker model RP09 (Barcelona, Spain) to obtain the powder of 200–500 lm particle sizes. All chemicals were of analytical grade and obtained from Sigma–Aldrich (St. Louis, MO, USA). A commercial Trichoderma reesei cellulase (Celluclast 1.5 L) and a b-glucosidase preparation (Novozyme 188) were both kindly donated by Novozymes Malaysia sdn bhd. Microorganisms and culture conditions The cellulase hyper-producing fungus Trichoderma reesei RUT-C30 ATCC 56765 and Saccharomyces cerevisiae ATCC 96581 were obtained from ATCC (American Type Culture Collection). T. reesei RUT-C30 was routinely maintained and sporulated on potato dextrose agar plate for 2 weeks till spore crop was developed. Four milliliters of
Bioprocess Biosyst Eng
sterile 0.05 % Tween 80 solution was added to the plate and swirled to gently release the spores. Approximately, 1 mL of the spore suspension consisting of 106–107 spores was used as the inoculum. YPD medium containing (g/L) glucose, 20.0; yeast extract, 10.0; and peptone 10.0 was used as the seed culture medium for S. cerevisiae. The OPEFB enzymatic hydrolysate containing 36.01 g/L of glucose and 2.55 g/L xylose was supplemented to a final concentration of (g/L) KH2PO4, 2.0; MgSO47H2O, 1.0; yeast extracts, 10.0, and urea, 6.4. The pH was adjusted to 5.5 ± 0.2 unless otherwise mentioned using 2 M NaOH. The synthetic medium containing the same amount of sugar (25.06 g/L of glucose and 1.64 g/L xylose) with the same composition and initial pH was used as the control medium. Enzyme production and enzymatic assays SSF was carried out according to our previous work. The culture temperature was kept at 30 ± 1 °C for 7 days, and the initial pH was 5.0. [18]. Filter paper (FPase) and endoglucanase (CMCase) activities were measured according to IUPAC recommendations [19, 20]. FPase and CMCase activities were determined by measuring the reducing sugars produced from Whatman no.1 filter paper (grade 3, catalogue number 1003 150, Whatman international, UK) and 1 % (w/v) carboxymethyl cellulose, respectively. Both reactions were carried out in 0.05 M citrate buffer at pH 5.5. The reaction mixtures were incubated at 50 °C for 1 h and for 30 min for the FPase and CMCase activity assays, respectively. Xylanase activity was measured according to Ghose [20]. The assay was carried out in the total reaction mixture of 1.5 mL containing 0.5 mL of suitably diluted enzyme and 1.0 mL of 1 % (w/v) xylan solution in phosphate buffer (0.05 M, pH 6.5). This mixture was incubated at 40 °C for 10 min. The amount of reducing sugar released from FPase, CMCase, and xylanase activity assays was measured using the 3,5-dinitrosalicylic acid (DNS) method [21]. b-Glucosidase and b-xylosidase activities were determined using the method described by Bailey et al. [22]. In this method, the p-nitrophenol released from 1 mM p-nitrophenyl-b-D-glucopyranoside (Sigma N2132) and p-nitrophenyl-b-D-xylopyranoside (Sigma N-7006) was measured using a spectrophotometer (UV-1601 PC, Shimadzu, Japan). One unit (IU) of enzyme activity was defined as the amount of enzyme required to liberate 1 lmol of product from their respective substrate per minute of crude filtrate under the assay conditions. The enzymatic hydrolysis was conducted in 50-mL Falcon tubes in 20 mL of 50 mM sodium citrate buffer. The experiments were performed in duplicate at 50 ± 1 °C, pH 5.5 in a shaking water bath (Memmert GmbH ? Co. KG, ScOPEFBabach, Germany) with maximum strokes with an
activity of 15 FPU/g biomass. Effect of solid content (5–25 %, based on biomass dry weight) was studied to obtain the final highest possible sugar concentration for ethanol fermentation. The glucose yields were calculated as follows: reducing sugar (g) 9 0.9 9 100/initial cellulose (g) in biomass. Hydrolysis of polysaccharides involves water. For each mole of reducing sugar released, 1 mol of H2O is required. A correction factor of 0.9 was, therefore, included in the calculation of the amount of polysaccharides hydrolyzed. Experiments were carried out in duplicates. Pretreatment and chemical characterization of OPEFB Mild-condition organosolv pretreatment was carried out according to our previous work (Fig. 1) [10]. Briefly, 5.0 g of OPEFB (dry weight base) was treated at 70 °C for 6 h with 100 mL of 70:30 (v/v) ethanol:water mixture. The solid to liquid ratio was maintained at 1:20 (w/v) throughout all the experiments unless otherwise stated. The pretreated OPEFB was washed with first washing solution using 95 % ethanol at 60 °C for 4 h and then using 70 % ethanol at 30 °C for 1 h. The residue was collected and subjected to a post-treatment with 4 % (v/v) hydrogen peroxide solution at 45 °C for 16 h. The final pretreated OPEFB biomass was washed with water at 40 °C for 4 h. The residue was subsequently collected for further analysis and enzymatic hydrolysis. The amount of hemicellulose, cellulose, and lignin in the lignocellulosic biomass was determined according to Yang et al. [23] with modification. To determine the amount of hemicellulose, 0.5 M NaOH solution was added to the dried biomass and the temperature was held at 80 °C for 3.5 h. After that, the sample was washed until no more Na? was detected (indicated by the pH value of the solution approaching 7), and it was then dried to a constant weight. The difference between the sample weight before and after this treatment is the hemicellulose content. To determine the acid-soluble lignin content, 98 % H2SO4 was added to the hemicellulose-free biomass, followed by 2-h incubation at 30 °C. The mixture was then diluted to 4 % H2SO4 with de-ionized water. The diluted solution was autoclaved at 121 °C for 1 h. The mixture was filtered. Aliquots of the filtrate were measured at the absorbance of 205 nm using 4 % H2SO4 as the control. Dilutions were done to obtain optical density readings of 0.2–0.8. Acidsoluble lignin content was calculated based on the sample absorbance at 205 nm. The rest of the biomass residue was washed until the sulfate ion in the filtrate was undetectable (via titration of a 10 % barium chloride solution); it was then dried to a constant weight. The weight of the residue is recorded as the lignin content. Finally, the content of cellulose is calculated by the difference, assuming that
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hemicellulose, lignin, and cellulose are the only components of the entire biomass. Ethanol fermentation S. cerevisiae was grown for 24 h at 150 rpm on a rotary shaker at 30 °C. 2 mL of such seed culture was inoculated to each 250 mL Erlenmeyer flask (Sartorius, USA) containing 100 mL of the culture medium to make an initial biomass concentration of around 1.5 g/L. The OPEFB enzymatic hydrolysate and the control medium were fermented using S. cerevisiae in 250 mL Erlenmeyer flasks sealed with screwed caps for 32 h. The shaking speed was kept at 150 rpm and temperature at 30 °C. Samples were withdrawn periodically to determine the concentration of sugar, ethanol, and cell biomass in the fermentation broth. Fermentation experiments were conducted in duplicate. Analytical methods Cell biomass was monitored spectrophotometrically by measuring absorbance at 600 nm using a spectrophotometer (UV-1601 PC, Shimadzu, Japan). The measurement was made such that the optical density (OD600) of the samples was smaller than 0.70, as obtained by sample dilution. This is to ensure that the Beer–Lambert law applies. Forty-milliliter samples of whole culture were filtered through 0.45 lm pre-dried, preweighed glass fiber membrane filters and dried at 60 °C till constant weight was obtained. Biomass dry weight was calculated as the difference between the membrane dry weight before and after the culture broth filtration. A calibration curve was prepared between OD600 and biomass dry weight. The OD value was then converted to biomass dry weight using such calibration curve. Biomass concentration (grams per liter) was found to follow the regression equation X (g/ L) = 0.314 9 (OD600). Samples were filtered through 0.45 lm filters and stored at 20 °C until analyzed by a 1200 Series HPLC system (Agilent Technologies Inc.) equipped with a refractive index detector. Glucose and ethanol were analyzed on an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) at 75 °C with 0.6 mL/min eluent of 5 mM sulfuric acid.
Results and discussion Crude cellulase production by solid state fermentation (SSF) Moisture content is a critical factor on SSF processes because this variable has influences on both cell growth and the biosynthesis and secretion of enzymes [11]. Lower
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moisture content causes the reduction in the solubility of substrate nutrients, low degree of swelling, and high water tension [11, 12]. On the other hand, higher moisture levels can cause a reduction in the enzyme yield due to its steric hindrance to cell growth of the enzyme producing strain that resulted from the reduction in the solid matrix porosity (inter particle spaces). Less porosity interferes with oxygen transfer and in turn influences the cell growth [11, 12]. As oxygen transfer affects both the growth and the metabolism of fungi, the solid substrate should contain suitable amount of water to enhance mass transfer. The effect of initial moisture content (50–90 %) on enzyme activities by T. reesei grown under SSF was tested. A positive relationship between the activities of FPase and CMCase and the moisture content was observed when it was lower than 80 %. However, further increase in moisture content influenced the activities of these two enzymes negatively. On the other hand, higher moisture contents (85–90 %) were necessary for maximal xylanase, b-glucosidase, and b-xylosidase production. Based on high FPase and CMCase, 80 % of moisture content was chosen for further enzyme production. When T. reesei was grown on OPEFB as solid support in SSF at 30 ± 1 °C, initial pH value of 5.0, and 80 % moisture content, the time course profiles of enzyme production was shown in Fig. 2. The maximum FPase and xylanase activities were 20.2 IU g-1 in 4 days, 70.2 IU g-1 in 5 days, respectively. The highest CMCase activity of 132.5 IU g-1, b-glucosidase activity of 101.1 IU g-1 and b-xylosidase activity of 16.1 IU g-1 were obtained after 5–6 days of fermentation. Since comparisons of the results obtained in this study with those obtained by other researchers are difficult, the yields of each enzyme were presented in Table 1. T. reesei was able to produce a higher activity of cellulases and hemicellulases in SSF using OPEFB than those reported by other researchers; however, the CMCase activity was lower in some cases. In order to investigate the hydrolytic potential of the crude enzyme complex produced by T. reesei RUT-C30 under SSF using OPEFB, a comparison of the effectiveness of crude cellulase bend, which has FPase and b-glucosidase activities of 15 and 90 U/ml, respectively, and a commercial preparation of cellulase in the saccharification of Whatman filter paper is shown in Fig. 3. This commercial enzyme was diluted to approximately the same FPase activity as cellulase produced by T. reesei RUT-C30, and it had FPase and b-glucosidase activities of 15 and 21 U/ml, respectively. The rate of reducing sugar production was very high during the initial stages of saccharification and became constant after about 72 h. Although the rate of saccharification of filter paper by cellulases of T. reesei RUT-C30 was slightly lower than the rate obtained by commercial cellulase, the maximum amount of reducing
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25 FPase Xylosidase CMCase Xylanase Xylosidase
FPase and Xylosidase (U/g substrate)
20
140 120 100
15
80 10
60 40
5 20 0
CMCase, Xylanase and Glucosidase (U/g substate)
Fig. 2 Time course of cellulase and hemicellulase production from untreated OPEFB under SSF
0 0
1
2
3
4
5
6
7
8
Time (days)
Table 1 Enzyme yields by solid-state fermentation from other strains grown on lignocellulosic biomass
Organism
Aspergillus ustus Penicillum capsulatum
Substrate
Enzyme activity (IU g-1)
References
FP
CMC
BG
XYL
BX
Rice straw
5.8
12.6
15.8
740
–
Wheat bran
3.8
11.8
60.0
615
–
Beet pulp
[13]
2.7
632
14.8
98
1.2
7.7
704
4.6
386
7.7
Wheat bran/beet pulp (1:1)
18
265
33
350
2.9
[15]
Wheat straw
4.3
956
46.1
2,973
40.7
[16]
Aspergillus niger KK2
Rice straw
19
130
94
5,070
176
[17]
Trichoderma reesei RUTC30
Horticultural waste
15.0
90.5
61.6
52.1
10.4
[18]
Trichoderma reesei RUTC30
OPEFB
20.2
132.5
101.1
70.2
16.1
This study
Trichoderma reesei MCG77 Talaromyces emersonii
[14]
Trichoderma reesei MCG77 Talaromyces emersonii
FP FPase, CMC CMCase, BG b-glucosidase, XYL xylanase, BX b-xylosidase, OPEFB oil palm empty fruit bunch
sugars obtained was slightly higher. The maximum concentration of reducing sugar obtained at the end of the saccharification process was about 46.62 g/L, which corresponded to about 0.37 g reducing sugar/g filter paper. Hydrolysis of organosolv pretreated OPEFB using crude cellulase Production of bioethanol from lignocellulosic biomass requires four main steps: physical and chemical pretreatment of the lignocellulosic biomass, enzymatic hydrolysis of the lignocellulosic biomass, fermentation of the resulting sugars, and distillation of the ethanol [6, 9]. Of these, pretreatment of the biomass is the most important step in increasing the saccharification efficiency of the enzymatic hydrolysis, ultimately determining the yield of bioethanol
production [6, 7, 9]. The selection of the pretreatment procedure depends on the proportion of cellulose, hemicellulose, and lignin in a biomass and can remove interfering materials that would impact the overall bioprocess [6, 7, 9]. The cellulose, hemicellulose, and lignin contents were 40.5, 24.3, and 35.2 %, respectively, per 100 g of dry OPEFB according to the modified method (Table 2). A relatively high amount of cellulose (40.5 %) was observed per 100 g of dry OPEFB versus other biomasses: straw (36.6 %), poplar (33.1 %), and switchgrass (29.7 %) [6]. Hemicellulose (24.3 %) with around half the percentage of cellulose (40.5 %) was present in the biomass. Additionally, a high content of lignin (35.2 %) was observed in the biomass. To increase cellulosic biomass for the production of bioethanol, something was needed to reduce the contents of hemicellulose and lignin in OPEFB. A modified
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Reducing Sugar Concentration (g/L)
Conversion Yield (g/g)
50 Conversion yield (crude cellulase) Conversion yield (commercial cellulase)
40
30
20
10
0 0
20
40
60
50 5% (w/v) 10% (w/v) 15%( w/v) 20% (w/v) 25% (w/v)
40
30
20
10
0 0
80
10
20
Fig. 3 Comparison of the hydrolysis potentials of Whatman filter paper using the crude enzyme sample and the commercial enzyme mixture
Table 2 Oil palm empty fruit bunch fiber composition
40
50
60
70
80
Fig. 4 Effect of the solid content (w/v) on the reducing sugar concentration (g/L) in the organosolv treated OPEFB hydrolysate obtained after hydrolysis at 50 °C, pH 5.5, 15 FPU/g OPEFB at 72 h
Cellulose (%)
Hemicellulose (%)
Lignin (%)
Sugar yield (%)
Untreated
40.5 ± 0.37
24.3 ± 0.14
35.2 ± 0.11
9.7 ± 0.32
Organolsolv treated
59.3 ± 0.29
18.6 ± 0.34
22.1 ± 0.43
56.4 ± 0.25
organosolv pretreatment method which could efficiently remove hemicellulose and improve cellulose content of horticultural waste has been developed under non-corrosive and mild conditions and it is potentially more cost effective and energy efficient. So, to remove the hemicellulose and lignin, this organosolv pretreatment method was assessed using OPEFB (Fig. 1). From Table 2, it can be seen that organosolv pretreatment could efficiently remove lignin (35.2–22.1 %) and hemicellulose (24.3–18.6 %) and increase accessibility of the cellulose, which resulted in a higher sugar yield (59.3 %) and cellulose content (56.4 %). Furthermore, the operational costs can be reduced by recycled usage of the solvent ethanol from the system using appropriate extraction and separation techniques, e.g. evaporation and condensation in industrial scale. Meanwhile, the solid part (residue) can be further hydrolyzed to glucose by cellulase and fermented to biofuel in the later part, while the hemicellulosic hydrolysate which contains the main component of xylose from hemicellulose and furfural, vanillin, etc., from lignin can be further detoxified and also used as carbon source for biofuel production. Substrate concentration is another main factor that affects the yield and the initial rate of enzymatic hydrolysis of cellulose. At low substrate levels, an increase of substrate concentration normally results in an increase of the sugar yield and reaction rate of the hydrolysis. However, high substrate concentration can cause substrate inhibition,
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30
Time (hours)
Time (hours)
which substantially lowers the rate of the hydrolysis, and the extent of substrate inhibition depends on the ratio of total substrate to total enzymes [24]. During industrial operations, higher biomass loadings of 15 % (w/v) or higher can result in higher sugar concentrations and greater ethanol titers, which in turn require less energy and in some areas, smaller equipment for a given throughput [10]. Therefore, experiments were conducted to determine the impact of varying the solid content (w/v) from 5 to 25 % using crude cellulase mixture on reducing sugar concentration (Fig. 4). The final sugar concentrations with varying solid contents after enzymatic hydrolysis of organosolvtreated OPEFB are given in Fig. 4. The cellulase dosage was 15 FPU/g OPEFB. Experiments were conducted at 50 °C, pH 5.5 for 72 h. Reducing sugar concentration increased with the increase of OPEFB loading and it reached a plateau when the solid content was 20 % (w/v). The maximum sugar concentration obtained was 38.56 g/L. Further increase in the solid content did not result in any increase in sugar concentration. This might be due to the saturation of the enzyme active sites or be attributed to the insufficient mass transfer caused by the high biomass loading. Furthermore, higher substrate concentration may cause by end-products (glucose and cellobiose) or other inhibitors which become noticeable at a high-solids loading. This substantially lowers the rate of the hydrolysis. The extent of substrate inhibition depends on the ratio of total substrate
concentration to total enzyme loading. The results listed here suggest that the highest sugar concentration can be obtained from the pretreated OPEFB using the optimized condition in this study was 38.56 g/L although higher sugar concentration was reported from other lignocellulosic biomass. This might be due to the much lower density of OPEFB compared with other lignocellulosic biomass. Ethanol fermentation After saccharification of organosolv-treated OPEFB using crude cellulase complex, 36.01 g/L glucose and 2.55 g/L xylose released were further used for ethanol production by S. cerevisiae, which is the favored microorganism for converting glucose into ethanol. Generally, the hydrolysate made from lignocellulosic biomass contains a variety of inhibitors, such as furfural, 5-hydroxymethyl furfural (HMF), hydroxybenzaldehyde (HBA), syringaldehyde (SGA), and vanillin, which have a negative influence on the efficiency and fermentation rate during the ethanol production process [25, 26]. In order to compare whether the enzymatic hydrolase of OPEFB has the negative effect on the fermentation performance of S. cerevisiae, the fermentation results were compared with the control media containing the same concentration glucose, 36.01 g/L and xylose, 2.55 g/L without any inhibitor. Figure 5 shows the time course of fermentation profile using the hydrolysate and the synthetic medium. The fermentation was performed at 30 °C (pH 5.5), 150 rpm for 32 h. All of the glucose was consumed in the two media, while due to the inability of utilizing xylose by S. cerevisiae, xylose was almost unconsumed although a slight decrease was observed initially for both cases (data not shown). This was consistent to what we observed before [10]. The maximum cell and ethanol concentration was found to be 3.96 and 17.64 g/L, respectively, after 8 h of fermentation in the hydrolysate (Table 3). The ethanol yield (Yp/s) and productivity (Qp) were estimated to be 0.49 g per gram of glucose (91.98 % of theoretical yield) and 0.088 g per gram of OPEFB and 2.21 g/L/h, respectively. The same in synthetic media (supplemented with synthetic sugar glucose) ethanol yield and productivity were 0.47 g/g and 2.12 g/L/h, respectively, with specific ethanol productivity of 0.47 g/h/g biomass (Table 3). The similar ethanol yield and productivity obtained from OPEFB hydrolysate and the control medium (Table 2) indicate that OPEFB hydrolysate is quite fermentable and no prior detoxification is required. The highest yield obtained by Gupta et al. [24] was given as 0.49 g/g using commercial enzyme for hydrolysis and fermentation by S. cerevisiae which is similar to the present study. In addition, OPEFB hydrolysate seemed to have a slightly positive effect in ethanol production evidenced by the higher specific ethanol productivity. This is
Glucose, Biomass and Ethanol (g/L)
Bioprocess Biosyst Eng 40 Glucose in synthetic media Glucose in hydrolysate Biomass in synthetic media Biomass in hydrolysate Ethanol in synthetic media Ethanol in hydrolysate
30
20
10
0 0
5
10
15
20
25
30
35
Time (h) Fig. 5 Fermentation profile of enzymatic hydrolysate of organosolv treated OPEFB and the control medium by S. cerevisiae
Table 3 Fermentation parameters for bioethanol production in enzymatically hydrolyzed (crude enzyme) sugars and synthetic sugars media by S. cerevisiae Kinetic parameters
Hydrolysate media
Synthetic media
Initial glucose, S0 (g/L)
36.01 ± 0.37
36.01 ± 0.14
Maximum ethanol concentration (g/L)
17.64 ± 0.11
16.92 ± 0.35
Maximum cell concentration (g/L)
3.96 ± 0.04
4.52 ± 0.09
Maximum time (hours)
8
8
Glucose consumed (%) Ethanol yield coefficient, Yp/s (g/g)
100 0.49 ± 0.001
100 0.47 ± 0.004
Biomass yield coefficient, Yx/s (g/g)
0.11 ± 0.04
0.13 ± 0.02
Ethanol productivity, Qp (g/L/h)
2.21 ± 0.07
2.12 ± 0.02
Specific ethanol productivity (g/L/h/g biomass)
0.56 ± 0.03
0.47 ± 0.18
Max. sugar consumption rate, Qs (g/L/h)
4.50 ± 0.02
4.50 ± 0.11
probably owing to the presence the hydrolysis by-product, such as acetic acid, which is known to enhance the fermentation rate at low concentrations [24, 27]. The fermentation results presented here show that the organosolv pretreated OPEFB using the crude cellulase enzymes produced from OPEFB under SSF is a promising process for bioconversion of OPEFB to value-added products, such as fuels and chemicals. By achieving the integration of crude cellulase production using OPEFB under SSF and efficient pretreatment of OPEFB using organosolv, the costs of ethanol production could be reduced 10–30 % using OPEFB [1]. Further improvement of ethanol production from organosolv pretreated OPEFB can be achieved by fed-batch fermentation.
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Conclusions In Southeast Asia, oil palm empty fruit bunch fiber (OPEFB) is the most commonly generated lignobiomass waste, which is a potential feedstock for both fuel ethanol and cellulase production. In the present study, organosolvtreated OPEFB was first saccharified using crude enzymes produced from untreated OPEFB under solid-state fermentation instead of commercial enzymes and then fermented to fuel ethanol. 8.8 grams of ethanol could be produced from 0.1 kg of OPEFB without any usage of commercial cellulase. Such process that is using one substrate (OPEFB) for both cellulase and fuel production, will not only exert pressure on municipal landfilling or incineration but also create its added value.
References 1. Gnansounou E, Dauriat A (2010) Techno-economic analysis of lignocellulosic ethanol: a review. Bioresour Technol 101:4980–4991 2. Ariffin H, Hassan MA, Shah UK, Abdullah N, Ghazali FM, Shirai Y (2008) Production of bacterial endoglucanase from pretreated oil palm empty fruit bunch by Bacillus pumilus EB3. J Biosci Bioeng 106:231–236 3. Yunus R, Salleh SF, Abdullah N, Biak DR (2010) Effect of ultrasonic pretreatment on low temperature acid hydrolysis of oil palm empty fruit bunch. Bioresour Technol 101:9792–9796 4. Hamzah F, Idris A, Shuan TK (2011) Preliminary study on enzymatic hydrolysis of treated oil palm (Elaeis) empty fruit bunches fibre by using combination of cellulose and b1-4 glucosidase. Biomass Bioenerg 35:1055–1059 5. Piarpuza´n D, Quintero JA, Cardona CA (2011) Empty fruit bunches from oil palm as a potential raw material for fuel ethanol production. Biomass Bioenerg 35:1130–1137 6. Abdullah MA, Nazir MS, Wahjoedi BA (2011) Development of value-added biomaterials from oil palm agro-wastes. IPCBEE 7:32–35 7. Chandel AK, Singh OV (2011) Weedy lignocellulosic feedstock and microbial metabolic engineering: advancing the generation of ‘biofuel’. Appl Microbiol Biotechnol 89:1289–1303 8. da Costa Sousa L, Chundawat SP, Balan V, Dale BE (2009) ‘Cradle-to-grave’ assessment of existing lignocellulose pretreatment technologies. Curr Opin Biotechnol 20:339–347 9. FitzPatrick M, Champagne P, Cunningham MF, Whitney RA (2010) A biorefinery processing perspective: treatment of lignocellulosic materials for the production of value-added products. Bioresour Technol 101:8915–8922 10. Geng AL, Xin FX, Ip JY (2012) Ethanol production from horticultural waste treated by a modified organosolv method. Bioresour Technol 104:715–721
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11. Singhania RR, Patel AK, Soccol CR, Pandey A (2009) Recent advances in solid state fermentation. Biochemical Eng J 44:13–18 12. Kim J, Hosobuchi M, Kishimoto M, Seki T, Yoshida T, Taguchi H (1985) Cellulase production by a solid state culture system. Biotechnol Bioeng 27:1445–1450 13. Shamala TR, Sreekantiah KR (1986) Production of cellulases and D-xylanase by some selected fungal isolates. Enzyme Microb Technol 8:178–182 14. Considine PJ, O’Rorke A, Hackett TJ, Coughlan MP (1988) Hydrolysis of beet pulp polysaccharides by extracts if solid state cultures of Penicillium capsulatum. Biotechnol Bioeng 31:433–438 15. Tuohy MG, Coughlan TL, Coughlan MP (1990) Solid state versus liquid cultivation of Talaromyces emersonii on straws and pulps: enzyme productivity. In: Coughlan M (ed), Advances in biological treatments of lignocellulosic materials. Elsevier Applied Science, pp 153–175 16. Kalogeris E, Fountoukides G, Kekos D, Macris BJ (1999) Design of solid state bioreactor for thermophilic microorganisms. Bioresour Technol 67:313–315 17. Kang SW, Park YS, Lee JS, Hong SI, Kim SW (2004) Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresour Technol 91:153–156 18. Xin FX, Geng AL (2010) Horticultural waste as the substrate for cellulase and hemicellulase production by Trichoderma reesei under solid-state fermentation. Appl Biochem Biotechnol 162:295–306 19. Ghose TK (1987) Measurement of cellulase activity. Pure Appl Chem 59:257–268 20. Ghose TK, Bisaria VS (1987) Measurement of hemicellulase activity. Pure Appl Chem 59:1739–1752 21. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 31:426–428 22. Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23:257–270 23. Yang HP, Yan R, Chen HP, Zheng CG, Lee DH, Liang DT (2006) An in-depth investigation of biomass pyrolysis based on three major components: xylan, cellulose and lignin. Energy Fuels 20:388–393 24. Gupta R, Sharma KK, Kuhad RC (2009) Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, a woody substrate, for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498. Bioresour Technol 100:1214–1220 25. Mosier N, Wyman CE, Dale BE, Elander RT, Lee YY, Holtzapple M, Ladisch MR (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 96:673–686 26. Araque E, Parra C, Freer J, Contreras D, Rodriguez J, Mendonca R, Baeza J (2008) Evaluation of organosolv pretreatment for the conversion of Pinus radiate D. Don to ethanol. Enzyme Microb Technol 3:214–219 27. Cheung SW, Anderson BC (1997) Laboratory investigation of ethanol production from municipal primary wastewater solids. Bioresour Technol 59:81–96
ORIGINAL PAPER
An integrative process of bioconversion of oil palm empty fruit bunch fiber to ethanol with on-site cellulase production Youshuang Zhu • Fengxue Xin • Ying Zhao Yunkang Chang
•
Received: 16 November 2013 / Accepted: 28 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract The aim of this study was to efficiently convert oil palm empty fruit bunch fiber (OPEFB), one of the most commonly generated lingo-wastes in Southeast Asia, into both cellulase and bioethanol. The unprocessed cellulase crude (37.29 %) produced under solid-state fermentation using OPEFB as substrate showed a better reducing sugar yield using filter paper than the commercial enzyme blend (34.61 %). Organosolv pretreatment method could efficiently reduce hemicellulose (24.3–18.6 %) and lignin (35.2–22.1 %) content and increase cellulose content (40.5–59.3 %) from OPEFB. Enzymatic hydrolysis of pretreated OPEFB using the crude cellulase with 20 % solid content, enzyme loading of 15 FPU/g OPEFB at 50 °C, and pH 5.5 resulted in a OPEFB hydrolysate containing 36.01 g/L glucose after 72 h. Fermentation of the hydrolysate medium produced 17.64 g/L ethanol with 0.49 g/g yield from glucose and 0.088 g/g yield from OPEFB at 8 h using Saccharomyces cerevisiae. Keywords Cellulase Organosolv pretreatment OPEFB Solid state fermentation Ethanol
Y. Zhu and F. Xin contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s00449-014-1209-2) contains supplementary material, which is available to authorized users. Y. Zhu Y. Zhao Y. Chang Department of Biological Science, Jining Medical University, Jining 272067, China F. Xin (&) Department of Civil and Environmental Engineering, National University of Singapore, T-Lab 07-03 N, 5A Engineering Drive 1, Singapore 117576, Singapore e-mail: [email protected]
Introduction Bioconversion of lignocellulosic waste materials to chemicals and fuels is receiving great attention as they are low cost, renewable, and widespread in nature. According to recent techno-economic evaluations, the main contributors to the overall costs of producing ethanol from biomass are the raw material (30–40 %) and the capital investment (30–45 %), followed by the cellulase enzymes (10–20 %) [1]. Southeast Asia is well known for its potential in renewable resources such as oil palm waste, sugar cane bagasse, and rice straw. In the process of extraction of palm oil from oil palm fruit, a lignocellulosic material oil palm empty fruit bunch (OPEFB), which consists of 41.3–46.5 % cellulose, 25.3–33.8 % hemicellulose, and 27.6–32.5 % lignin is generated as a waste product [2–5]. The palm oil industries in Indonesia and Malaysia generate approximately 55.73 million tons of lignocellulosic agricultural waste and by-products per year [6]. Lignocellulosic OPEFB is potentially one of the lower-cost materials, as well as an alternative renewable bioresource, in comparison to using food sources, such as corn, sugarcane, and other food stocks, for the production of bioethanol [4–6]. However, OPEFB contains a relatively high percentage of hemicellulose and lignin per gram biomass as compared to other food and feedstocks: biomass (% cellulose: % hemicelluloses: % lignin); birch (40:23:21); wheat straw (38.2:21.2:23.4); corn stover (37.5:22.4:17.6); switch grass (31.0:20.4:17.6); Alfalfa (33:18:8) [7]. Thus, pretreatment is necessary to reduce the hemicellulose and lignin contents for effective bioethanol fermentation. The pretreatment process would ideally also enhance the proportion of cellulose in the OPEFB [2–5]. Moreover, removal of hemicellulose increases the pore size of the biomass and, therefore, increases the accessibility and the digestibility of
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Bioprocess Biosyst Eng Oil palm empty fruit bunch fiber Solid state fermentation Fungi: Trichoderma reesei RUT-C30 Substrate: OPEFB Medium: Mendel's medium
Pretreatment with ethanol at 70°C for 6 hours , 1st wash using ethanol for 1h Post-treatment with 4% H2O2 at 45°C for 16 hours, 2nd wash using water for 1h
Extration
50°C, pH 5.5, 72 h Crude enzymes (15FPU/ g OPEFB)
Treated OPEFB
Enzymatic hydrolysate Saccahromyces cerevisiae yeast extract and peptone
Bioethanol
Fig. 1 Experiment layout for saccharification and fermentation of pretreated OPEFB into fuel ethanol
cellulase toward the pretreated biomass [7]. Acid or alkali pretreatments, combined with high temperature or high pressure, have been applied in conventional chemical treatment processes [8, 9], while being achieved at the expense of increased thermal energy cost. A modified organosolv pretreatment has been developed in our prior work to remove lignin and hemicellulose of softwood under non-corrosive and mild conditions and increase accessibility of the cellulose [10]. So in this study, our first objective was to pretreat OPEFB using this organosolv modified method to efficiently remove hemicelluloses and lignin. The production economics of bio-ethanol is also largely dependent on the cost of cellulases [1, 7]. Efforts towards cost reduction have been directed at increasing enzyme production by finding hyperactive microbial strains, efficient fermentation techniques, and recovery systems [7]. The production of cellulases has been widely studied in submerged culture processes, but solid-state fermentation (SSF) has gained a fresh attention in the recent years, mainly due to its advantages over submerged fermentation such as low capital investment, solid waste management, reduced energy requirements, improved product recovery, etc. [11, 12]. SSF is a process whereby an insoluble substrate is fermented with sufficient moisture but without free water [12]. The substrates generally used are low-cost agro residues such as wheat straw, wheat bran, baggase, soya hulls, horticultural waste, etc. [13–18]. The use of SSF using cheap biomass resources as substrate can further improve production economics [11, 12]. So the second objective of the present study was to utilize the inexpensive and abundantly available lignocellulosic biomass, OPEFB
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for crude cellulase production under SSF by Trichoderma reesei RUT-C30. The organosolv pretreated OPEFB was then hydrolyzed using the above crude cellulase mixture for bioethanol production (Fig. 1).
Materials and methods Materials OPEFB was collected from Masai Palm Oil Mill, Johore, Malaysia. The collected OPEFB was first dried and mechanically milled with a lab mill (Ultra Centrifugal Mill ZM 200, Retsch GmbH, Germany) and sieved through standard mesh sieves (200–500 lm) using an electronic sieve shaker model RP09 (Barcelona, Spain) to obtain the powder of 200–500 lm particle sizes. All chemicals were of analytical grade and obtained from Sigma–Aldrich (St. Louis, MO, USA). A commercial Trichoderma reesei cellulase (Celluclast 1.5 L) and a b-glucosidase preparation (Novozyme 188) were both kindly donated by Novozymes Malaysia sdn bhd. Microorganisms and culture conditions The cellulase hyper-producing fungus Trichoderma reesei RUT-C30 ATCC 56765 and Saccharomyces cerevisiae ATCC 96581 were obtained from ATCC (American Type Culture Collection). T. reesei RUT-C30 was routinely maintained and sporulated on potato dextrose agar plate for 2 weeks till spore crop was developed. Four milliliters of
Bioprocess Biosyst Eng
sterile 0.05 % Tween 80 solution was added to the plate and swirled to gently release the spores. Approximately, 1 mL of the spore suspension consisting of 106–107 spores was used as the inoculum. YPD medium containing (g/L) glucose, 20.0; yeast extract, 10.0; and peptone 10.0 was used as the seed culture medium for S. cerevisiae. The OPEFB enzymatic hydrolysate containing 36.01 g/L of glucose and 2.55 g/L xylose was supplemented to a final concentration of (g/L) KH2PO4, 2.0; MgSO47H2O, 1.0; yeast extracts, 10.0, and urea, 6.4. The pH was adjusted to 5.5 ± 0.2 unless otherwise mentioned using 2 M NaOH. The synthetic medium containing the same amount of sugar (25.06 g/L of glucose and 1.64 g/L xylose) with the same composition and initial pH was used as the control medium. Enzyme production and enzymatic assays SSF was carried out according to our previous work. The culture temperature was kept at 30 ± 1 °C for 7 days, and the initial pH was 5.0. [18]. Filter paper (FPase) and endoglucanase (CMCase) activities were measured according to IUPAC recommendations [19, 20]. FPase and CMCase activities were determined by measuring the reducing sugars produced from Whatman no.1 filter paper (grade 3, catalogue number 1003 150, Whatman international, UK) and 1 % (w/v) carboxymethyl cellulose, respectively. Both reactions were carried out in 0.05 M citrate buffer at pH 5.5. The reaction mixtures were incubated at 50 °C for 1 h and for 30 min for the FPase and CMCase activity assays, respectively. Xylanase activity was measured according to Ghose [20]. The assay was carried out in the total reaction mixture of 1.5 mL containing 0.5 mL of suitably diluted enzyme and 1.0 mL of 1 % (w/v) xylan solution in phosphate buffer (0.05 M, pH 6.5). This mixture was incubated at 40 °C for 10 min. The amount of reducing sugar released from FPase, CMCase, and xylanase activity assays was measured using the 3,5-dinitrosalicylic acid (DNS) method [21]. b-Glucosidase and b-xylosidase activities were determined using the method described by Bailey et al. [22]. In this method, the p-nitrophenol released from 1 mM p-nitrophenyl-b-D-glucopyranoside (Sigma N2132) and p-nitrophenyl-b-D-xylopyranoside (Sigma N-7006) was measured using a spectrophotometer (UV-1601 PC, Shimadzu, Japan). One unit (IU) of enzyme activity was defined as the amount of enzyme required to liberate 1 lmol of product from their respective substrate per minute of crude filtrate under the assay conditions. The enzymatic hydrolysis was conducted in 50-mL Falcon tubes in 20 mL of 50 mM sodium citrate buffer. The experiments were performed in duplicate at 50 ± 1 °C, pH 5.5 in a shaking water bath (Memmert GmbH ? Co. KG, ScOPEFBabach, Germany) with maximum strokes with an
activity of 15 FPU/g biomass. Effect of solid content (5–25 %, based on biomass dry weight) was studied to obtain the final highest possible sugar concentration for ethanol fermentation. The glucose yields were calculated as follows: reducing sugar (g) 9 0.9 9 100/initial cellulose (g) in biomass. Hydrolysis of polysaccharides involves water. For each mole of reducing sugar released, 1 mol of H2O is required. A correction factor of 0.9 was, therefore, included in the calculation of the amount of polysaccharides hydrolyzed. Experiments were carried out in duplicates. Pretreatment and chemical characterization of OPEFB Mild-condition organosolv pretreatment was carried out according to our previous work (Fig. 1) [10]. Briefly, 5.0 g of OPEFB (dry weight base) was treated at 70 °C for 6 h with 100 mL of 70:30 (v/v) ethanol:water mixture. The solid to liquid ratio was maintained at 1:20 (w/v) throughout all the experiments unless otherwise stated. The pretreated OPEFB was washed with first washing solution using 95 % ethanol at 60 °C for 4 h and then using 70 % ethanol at 30 °C for 1 h. The residue was collected and subjected to a post-treatment with 4 % (v/v) hydrogen peroxide solution at 45 °C for 16 h. The final pretreated OPEFB biomass was washed with water at 40 °C for 4 h. The residue was subsequently collected for further analysis and enzymatic hydrolysis. The amount of hemicellulose, cellulose, and lignin in the lignocellulosic biomass was determined according to Yang et al. [23] with modification. To determine the amount of hemicellulose, 0.5 M NaOH solution was added to the dried biomass and the temperature was held at 80 °C for 3.5 h. After that, the sample was washed until no more Na? was detected (indicated by the pH value of the solution approaching 7), and it was then dried to a constant weight. The difference between the sample weight before and after this treatment is the hemicellulose content. To determine the acid-soluble lignin content, 98 % H2SO4 was added to the hemicellulose-free biomass, followed by 2-h incubation at 30 °C. The mixture was then diluted to 4 % H2SO4 with de-ionized water. The diluted solution was autoclaved at 121 °C for 1 h. The mixture was filtered. Aliquots of the filtrate were measured at the absorbance of 205 nm using 4 % H2SO4 as the control. Dilutions were done to obtain optical density readings of 0.2–0.8. Acidsoluble lignin content was calculated based on the sample absorbance at 205 nm. The rest of the biomass residue was washed until the sulfate ion in the filtrate was undetectable (via titration of a 10 % barium chloride solution); it was then dried to a constant weight. The weight of the residue is recorded as the lignin content. Finally, the content of cellulose is calculated by the difference, assuming that
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hemicellulose, lignin, and cellulose are the only components of the entire biomass. Ethanol fermentation S. cerevisiae was grown for 24 h at 150 rpm on a rotary shaker at 30 °C. 2 mL of such seed culture was inoculated to each 250 mL Erlenmeyer flask (Sartorius, USA) containing 100 mL of the culture medium to make an initial biomass concentration of around 1.5 g/L. The OPEFB enzymatic hydrolysate and the control medium were fermented using S. cerevisiae in 250 mL Erlenmeyer flasks sealed with screwed caps for 32 h. The shaking speed was kept at 150 rpm and temperature at 30 °C. Samples were withdrawn periodically to determine the concentration of sugar, ethanol, and cell biomass in the fermentation broth. Fermentation experiments were conducted in duplicate. Analytical methods Cell biomass was monitored spectrophotometrically by measuring absorbance at 600 nm using a spectrophotometer (UV-1601 PC, Shimadzu, Japan). The measurement was made such that the optical density (OD600) of the samples was smaller than 0.70, as obtained by sample dilution. This is to ensure that the Beer–Lambert law applies. Forty-milliliter samples of whole culture were filtered through 0.45 lm pre-dried, preweighed glass fiber membrane filters and dried at 60 °C till constant weight was obtained. Biomass dry weight was calculated as the difference between the membrane dry weight before and after the culture broth filtration. A calibration curve was prepared between OD600 and biomass dry weight. The OD value was then converted to biomass dry weight using such calibration curve. Biomass concentration (grams per liter) was found to follow the regression equation X (g/ L) = 0.314 9 (OD600). Samples were filtered through 0.45 lm filters and stored at 20 °C until analyzed by a 1200 Series HPLC system (Agilent Technologies Inc.) equipped with a refractive index detector. Glucose and ethanol were analyzed on an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) at 75 °C with 0.6 mL/min eluent of 5 mM sulfuric acid.
Results and discussion Crude cellulase production by solid state fermentation (SSF) Moisture content is a critical factor on SSF processes because this variable has influences on both cell growth and the biosynthesis and secretion of enzymes [11]. Lower
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moisture content causes the reduction in the solubility of substrate nutrients, low degree of swelling, and high water tension [11, 12]. On the other hand, higher moisture levels can cause a reduction in the enzyme yield due to its steric hindrance to cell growth of the enzyme producing strain that resulted from the reduction in the solid matrix porosity (inter particle spaces). Less porosity interferes with oxygen transfer and in turn influences the cell growth [11, 12]. As oxygen transfer affects both the growth and the metabolism of fungi, the solid substrate should contain suitable amount of water to enhance mass transfer. The effect of initial moisture content (50–90 %) on enzyme activities by T. reesei grown under SSF was tested. A positive relationship between the activities of FPase and CMCase and the moisture content was observed when it was lower than 80 %. However, further increase in moisture content influenced the activities of these two enzymes negatively. On the other hand, higher moisture contents (85–90 %) were necessary for maximal xylanase, b-glucosidase, and b-xylosidase production. Based on high FPase and CMCase, 80 % of moisture content was chosen for further enzyme production. When T. reesei was grown on OPEFB as solid support in SSF at 30 ± 1 °C, initial pH value of 5.0, and 80 % moisture content, the time course profiles of enzyme production was shown in Fig. 2. The maximum FPase and xylanase activities were 20.2 IU g-1 in 4 days, 70.2 IU g-1 in 5 days, respectively. The highest CMCase activity of 132.5 IU g-1, b-glucosidase activity of 101.1 IU g-1 and b-xylosidase activity of 16.1 IU g-1 were obtained after 5–6 days of fermentation. Since comparisons of the results obtained in this study with those obtained by other researchers are difficult, the yields of each enzyme were presented in Table 1. T. reesei was able to produce a higher activity of cellulases and hemicellulases in SSF using OPEFB than those reported by other researchers; however, the CMCase activity was lower in some cases. In order to investigate the hydrolytic potential of the crude enzyme complex produced by T. reesei RUT-C30 under SSF using OPEFB, a comparison of the effectiveness of crude cellulase bend, which has FPase and b-glucosidase activities of 15 and 90 U/ml, respectively, and a commercial preparation of cellulase in the saccharification of Whatman filter paper is shown in Fig. 3. This commercial enzyme was diluted to approximately the same FPase activity as cellulase produced by T. reesei RUT-C30, and it had FPase and b-glucosidase activities of 15 and 21 U/ml, respectively. The rate of reducing sugar production was very high during the initial stages of saccharification and became constant after about 72 h. Although the rate of saccharification of filter paper by cellulases of T. reesei RUT-C30 was slightly lower than the rate obtained by commercial cellulase, the maximum amount of reducing
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25 FPase Xylosidase CMCase Xylanase Xylosidase
FPase and Xylosidase (U/g substrate)
20
140 120 100
15
80 10
60 40
5 20 0
CMCase, Xylanase and Glucosidase (U/g substate)
Fig. 2 Time course of cellulase and hemicellulase production from untreated OPEFB under SSF
0 0
1
2
3
4
5
6
7
8
Time (days)
Table 1 Enzyme yields by solid-state fermentation from other strains grown on lignocellulosic biomass
Organism
Aspergillus ustus Penicillum capsulatum
Substrate
Enzyme activity (IU g-1)
References
FP
CMC
BG
XYL
BX
Rice straw
5.8
12.6
15.8
740
–
Wheat bran
3.8
11.8
60.0
615
–
Beet pulp
[13]
2.7
632
14.8
98
1.2
7.7
704
4.6
386
7.7
Wheat bran/beet pulp (1:1)
18
265
33
350
2.9
[15]
Wheat straw
4.3
956
46.1
2,973
40.7
[16]
Aspergillus niger KK2
Rice straw
19
130
94
5,070
176
[17]
Trichoderma reesei RUTC30
Horticultural waste
15.0
90.5
61.6
52.1
10.4
[18]
Trichoderma reesei RUTC30
OPEFB
20.2
132.5
101.1
70.2
16.1
This study
Trichoderma reesei MCG77 Talaromyces emersonii
[14]
Trichoderma reesei MCG77 Talaromyces emersonii
FP FPase, CMC CMCase, BG b-glucosidase, XYL xylanase, BX b-xylosidase, OPEFB oil palm empty fruit bunch
sugars obtained was slightly higher. The maximum concentration of reducing sugar obtained at the end of the saccharification process was about 46.62 g/L, which corresponded to about 0.37 g reducing sugar/g filter paper. Hydrolysis of organosolv pretreated OPEFB using crude cellulase Production of bioethanol from lignocellulosic biomass requires four main steps: physical and chemical pretreatment of the lignocellulosic biomass, enzymatic hydrolysis of the lignocellulosic biomass, fermentation of the resulting sugars, and distillation of the ethanol [6, 9]. Of these, pretreatment of the biomass is the most important step in increasing the saccharification efficiency of the enzymatic hydrolysis, ultimately determining the yield of bioethanol
production [6, 7, 9]. The selection of the pretreatment procedure depends on the proportion of cellulose, hemicellulose, and lignin in a biomass and can remove interfering materials that would impact the overall bioprocess [6, 7, 9]. The cellulose, hemicellulose, and lignin contents were 40.5, 24.3, and 35.2 %, respectively, per 100 g of dry OPEFB according to the modified method (Table 2). A relatively high amount of cellulose (40.5 %) was observed per 100 g of dry OPEFB versus other biomasses: straw (36.6 %), poplar (33.1 %), and switchgrass (29.7 %) [6]. Hemicellulose (24.3 %) with around half the percentage of cellulose (40.5 %) was present in the biomass. Additionally, a high content of lignin (35.2 %) was observed in the biomass. To increase cellulosic biomass for the production of bioethanol, something was needed to reduce the contents of hemicellulose and lignin in OPEFB. A modified
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Reducing Sugar Concentration (g/L)
Conversion Yield (g/g)
50 Conversion yield (crude cellulase) Conversion yield (commercial cellulase)
40
30
20
10
0 0
20
40
60
50 5% (w/v) 10% (w/v) 15%( w/v) 20% (w/v) 25% (w/v)
40
30
20
10
0 0
80
10
20
Fig. 3 Comparison of the hydrolysis potentials of Whatman filter paper using the crude enzyme sample and the commercial enzyme mixture
Table 2 Oil palm empty fruit bunch fiber composition
40
50
60
70
80
Fig. 4 Effect of the solid content (w/v) on the reducing sugar concentration (g/L) in the organosolv treated OPEFB hydrolysate obtained after hydrolysis at 50 °C, pH 5.5, 15 FPU/g OPEFB at 72 h
Cellulose (%)
Hemicellulose (%)
Lignin (%)
Sugar yield (%)
Untreated
40.5 ± 0.37
24.3 ± 0.14
35.2 ± 0.11
9.7 ± 0.32
Organolsolv treated
59.3 ± 0.29
18.6 ± 0.34
22.1 ± 0.43
56.4 ± 0.25
organosolv pretreatment method which could efficiently remove hemicellulose and improve cellulose content of horticultural waste has been developed under non-corrosive and mild conditions and it is potentially more cost effective and energy efficient. So, to remove the hemicellulose and lignin, this organosolv pretreatment method was assessed using OPEFB (Fig. 1). From Table 2, it can be seen that organosolv pretreatment could efficiently remove lignin (35.2–22.1 %) and hemicellulose (24.3–18.6 %) and increase accessibility of the cellulose, which resulted in a higher sugar yield (59.3 %) and cellulose content (56.4 %). Furthermore, the operational costs can be reduced by recycled usage of the solvent ethanol from the system using appropriate extraction and separation techniques, e.g. evaporation and condensation in industrial scale. Meanwhile, the solid part (residue) can be further hydrolyzed to glucose by cellulase and fermented to biofuel in the later part, while the hemicellulosic hydrolysate which contains the main component of xylose from hemicellulose and furfural, vanillin, etc., from lignin can be further detoxified and also used as carbon source for biofuel production. Substrate concentration is another main factor that affects the yield and the initial rate of enzymatic hydrolysis of cellulose. At low substrate levels, an increase of substrate concentration normally results in an increase of the sugar yield and reaction rate of the hydrolysis. However, high substrate concentration can cause substrate inhibition,
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30
Time (hours)
Time (hours)
which substantially lowers the rate of the hydrolysis, and the extent of substrate inhibition depends on the ratio of total substrate to total enzymes [24]. During industrial operations, higher biomass loadings of 15 % (w/v) or higher can result in higher sugar concentrations and greater ethanol titers, which in turn require less energy and in some areas, smaller equipment for a given throughput [10]. Therefore, experiments were conducted to determine the impact of varying the solid content (w/v) from 5 to 25 % using crude cellulase mixture on reducing sugar concentration (Fig. 4). The final sugar concentrations with varying solid contents after enzymatic hydrolysis of organosolvtreated OPEFB are given in Fig. 4. The cellulase dosage was 15 FPU/g OPEFB. Experiments were conducted at 50 °C, pH 5.5 for 72 h. Reducing sugar concentration increased with the increase of OPEFB loading and it reached a plateau when the solid content was 20 % (w/v). The maximum sugar concentration obtained was 38.56 g/L. Further increase in the solid content did not result in any increase in sugar concentration. This might be due to the saturation of the enzyme active sites or be attributed to the insufficient mass transfer caused by the high biomass loading. Furthermore, higher substrate concentration may cause by end-products (glucose and cellobiose) or other inhibitors which become noticeable at a high-solids loading. This substantially lowers the rate of the hydrolysis. The extent of substrate inhibition depends on the ratio of total substrate
concentration to total enzyme loading. The results listed here suggest that the highest sugar concentration can be obtained from the pretreated OPEFB using the optimized condition in this study was 38.56 g/L although higher sugar concentration was reported from other lignocellulosic biomass. This might be due to the much lower density of OPEFB compared with other lignocellulosic biomass. Ethanol fermentation After saccharification of organosolv-treated OPEFB using crude cellulase complex, 36.01 g/L glucose and 2.55 g/L xylose released were further used for ethanol production by S. cerevisiae, which is the favored microorganism for converting glucose into ethanol. Generally, the hydrolysate made from lignocellulosic biomass contains a variety of inhibitors, such as furfural, 5-hydroxymethyl furfural (HMF), hydroxybenzaldehyde (HBA), syringaldehyde (SGA), and vanillin, which have a negative influence on the efficiency and fermentation rate during the ethanol production process [25, 26]. In order to compare whether the enzymatic hydrolase of OPEFB has the negative effect on the fermentation performance of S. cerevisiae, the fermentation results were compared with the control media containing the same concentration glucose, 36.01 g/L and xylose, 2.55 g/L without any inhibitor. Figure 5 shows the time course of fermentation profile using the hydrolysate and the synthetic medium. The fermentation was performed at 30 °C (pH 5.5), 150 rpm for 32 h. All of the glucose was consumed in the two media, while due to the inability of utilizing xylose by S. cerevisiae, xylose was almost unconsumed although a slight decrease was observed initially for both cases (data not shown). This was consistent to what we observed before [10]. The maximum cell and ethanol concentration was found to be 3.96 and 17.64 g/L, respectively, after 8 h of fermentation in the hydrolysate (Table 3). The ethanol yield (Yp/s) and productivity (Qp) were estimated to be 0.49 g per gram of glucose (91.98 % of theoretical yield) and 0.088 g per gram of OPEFB and 2.21 g/L/h, respectively. The same in synthetic media (supplemented with synthetic sugar glucose) ethanol yield and productivity were 0.47 g/g and 2.12 g/L/h, respectively, with specific ethanol productivity of 0.47 g/h/g biomass (Table 3). The similar ethanol yield and productivity obtained from OPEFB hydrolysate and the control medium (Table 2) indicate that OPEFB hydrolysate is quite fermentable and no prior detoxification is required. The highest yield obtained by Gupta et al. [24] was given as 0.49 g/g using commercial enzyme for hydrolysis and fermentation by S. cerevisiae which is similar to the present study. In addition, OPEFB hydrolysate seemed to have a slightly positive effect in ethanol production evidenced by the higher specific ethanol productivity. This is
Glucose, Biomass and Ethanol (g/L)
Bioprocess Biosyst Eng 40 Glucose in synthetic media Glucose in hydrolysate Biomass in synthetic media Biomass in hydrolysate Ethanol in synthetic media Ethanol in hydrolysate
30
20
10
0 0
5
10
15
20
25
30
35
Time (h) Fig. 5 Fermentation profile of enzymatic hydrolysate of organosolv treated OPEFB and the control medium by S. cerevisiae
Table 3 Fermentation parameters for bioethanol production in enzymatically hydrolyzed (crude enzyme) sugars and synthetic sugars media by S. cerevisiae Kinetic parameters
Hydrolysate media
Synthetic media
Initial glucose, S0 (g/L)
36.01 ± 0.37
36.01 ± 0.14
Maximum ethanol concentration (g/L)
17.64 ± 0.11
16.92 ± 0.35
Maximum cell concentration (g/L)
3.96 ± 0.04
4.52 ± 0.09
Maximum time (hours)
8
8
Glucose consumed (%) Ethanol yield coefficient, Yp/s (g/g)
100 0.49 ± 0.001
100 0.47 ± 0.004
Biomass yield coefficient, Yx/s (g/g)
0.11 ± 0.04
0.13 ± 0.02
Ethanol productivity, Qp (g/L/h)
2.21 ± 0.07
2.12 ± 0.02
Specific ethanol productivity (g/L/h/g biomass)
0.56 ± 0.03
0.47 ± 0.18
Max. sugar consumption rate, Qs (g/L/h)
4.50 ± 0.02
4.50 ± 0.11
probably owing to the presence the hydrolysis by-product, such as acetic acid, which is known to enhance the fermentation rate at low concentrations [24, 27]. The fermentation results presented here show that the organosolv pretreated OPEFB using the crude cellulase enzymes produced from OPEFB under SSF is a promising process for bioconversion of OPEFB to value-added products, such as fuels and chemicals. By achieving the integration of crude cellulase production using OPEFB under SSF and efficient pretreatment of OPEFB using organosolv, the costs of ethanol production could be reduced 10–30 % using OPEFB [1]. Further improvement of ethanol production from organosolv pretreated OPEFB can be achieved by fed-batch fermentation.
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Bioprocess Biosyst Eng
Conclusions In Southeast Asia, oil palm empty fruit bunch fiber (OPEFB) is the most commonly generated lignobiomass waste, which is a potential feedstock for both fuel ethanol and cellulase production. In the present study, organosolvtreated OPEFB was first saccharified using crude enzymes produced from untreated OPEFB under solid-state fermentation instead of commercial enzymes and then fermented to fuel ethanol. 8.8 grams of ethanol could be produced from 0.1 kg of OPEFB without any usage of commercial cellulase. Such process that is using one substrate (OPEFB) for both cellulase and fuel production, will not only exert pressure on municipal landfilling or incineration but also create its added value.
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