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Synergistic effect of pretreatment and hydrolysis enzymes on the production of fermentable sugars from date palm lignocellulosic waste
Journal of Industrial and Engineering Chemistry 19 (2013) 413–415
Contents lists available at SciVerse ScienceDirect
Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Short communication
Synergistic effect of pretreatment and hydrolysis enzymes on the production of fermentable sugars from date palm lignocellulosic waste Sulaiman Al-Zuhair *, Khalda Ahmed, Ashir Abdulrazak, Muftah H. El-Naas Chemical and Petroleum Engineering Department, UAE University, 17555 Al-Ain, United Arab Emirates
A R T I C L E I N F O
Article history: Received 20 July 2012 Accepted 17 September 2012 Available online 13 October 2012 Keywords: Enzymatic pretreatment Lignocelluloses Synergic effect Reducing sugars
A B S T R A C T
The sequential addition of the enzymes, laccase for lignin degrading, followed by xylanase for hemicelluloses hydrolysis, then cellulase for cellulose hydrolysis, was compared to the synergistic action of using the three enzymes together. It was shown that the reducing sugars yield increased from 5.6% using cellulase only to 45.6% by pretreatment with laccase and xylanase, prior to the enzymatic hydrolysis. A higher conversion of 60% was achieved by using the three enzymes together for the same incubation period. The proposed synergistic enzymes approach is a simpler and less energy intensive alternative compared to the conventional lignocelluloses pretreatment techniques. ß 2012 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
1. Introduction Lignocellulosic materials consist of a complex form of cellulose, hemicelluloses and lignin [1]. The main disadvantage of using lignocelluloses for bioethanol production is the requirement of more process steps than those required when conventional bioethanol feedstock, such as starch, is used. Particularly the lignocelluloses have to be pretreated in order to make the cellulose accessible to hydrolysis. In a successful pretreatment process lignin structure is broken down and the crystalline structure of cellulose is disrupted, which enhance the rate and extent of the hydrolysis by chemical or biochemical reagents [2]. Conventional pretreatment techniques consist of mechanical processing, acid hydrolysis, alkali swelling, ammonia, steam and other explosive techniques, and exposure to supercritical fluids [3–5]. These conventional techniques are usually carried out at high temperature and pressure that render this step the most expensive in biomass-to-fuels conversion. In addition, conventional pretreatment processes usually result in the formation of several degradation products that are inhibitory to downstream biological processes and utilize large amounts of water that have a negative effect on the overall process. Enzymes, on the other hand, provide a cost effective alternative for the pretreatment of lignocellulosic materials, in which lignin polymers are degraded to a degree that allows access to cellulose and hemicelluloses. The most important lignin degrading enzyme is laccase, which has been successfully used for lignin degradation
* Corresponding author. Tel.: +971 37133636; fax: +971 37624262. E-mail address: [email protected] (S. Al-Zuhair).
of lignocelluloses [6–8]. Laccase has also been used for the degradation of nonphenolic lignin [9]. The pretreatment can further be improved by the use of hemicellulases, such as xylanases, which have been effectively used for the hydrolysis of hemicelluloses to pentoses [10,11]. Lignin degrading enzymes and hemicellulases have been successfully applied in the pulp and paper industry [12]. However, the application of these enzymes in bioethanol production for the pretreatment of lignocelluloses prior to the hydrolysis step has seen much less attention. During the hydrolysis step, simpler sugars are produced from the pretreated cellulose. Pretreated cellulose can be chemically hydrolyzed using dilute or concentrated acids. However, these processes are corrosive and produce toxic by-products that require a detoxification step before fermentation. The utility cost of enzymatic hydrolysis is much lower because it is carried out at mild conditions and does not require any treatment step prior to fermentation. United Arab Emirates has more than 40 million palm trees and these are important sources of cellulose. Each palm tree produces about 15 kg of waste fronds per year. Palm fronds contain 58% of cellulose and 22% of hemicelluloses. That means under ideal conditions 80% of palm fronds can be converted to bioethanol, which is a renewable energy and present a good alternative to conventional, non-renewable, fossil fuels. In this work it has been proposed to replace the conventional pretreatment and hydrolysis steps with biological processes, using enzymes. Using biocatalysts makes the process less energy intensive and less harmful by-products are expected to be produced. The successful use of the approach proposed in this work provides a promising simplification to the overall bioethanol production from lignocellulosic waste available in abundance in the UAE.
1226-086X/$ – see front matter ß 2012 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jiec.2012.09.022
S. Al-Zuhair et al. / Journal of Industrial and Engineering Chemistry 19 (2013) 413–415
2. Materials and methods 2.1. Chemical and enzymes Laccase from Trametes versicolor (10 unit mg 1), xylanase from Thermomyces anuginosus (2.5 unit mg 1), cellulase from Aspergillus niger (0.3 unit mg 1), 1-hydroxybenzotriazole hydrate (HOBT) and D-glucose were obtained from Sigma–Aldrich, USA. A unit of laccase activity is defined as the amount of enzyme needed to oxidize 1 mmol of 2,6-dimethoxyphenol per min at pH 4.5 and 30 8C [13], a unit activity of xylanase is defined as the amount of enzyme needed to release 1 mmol of xylose equivalent sugar from xylan per minute at pH 4.5 at 30 8C [14] and a unit of cellulase is defined as the amount of enzyme needed to liberate 1.0 mmole of glucose from cellulose in 1 h at pH 5.0 at 37 8C [15]. The materials used to prepare the DNS reagent include 3,5-dinitrosalicylic acid, potassium sodium tartate (Rochelle salt), phenol, sodium hydroxide and sodium sulfite anhydrous were all obtained from Sigma– Aldrich. Acetate buffer with pH 4.6 was prepared from acetic acid and ammonium acetate obtained from Sigma–Aldrich, USA. 2.2. Experimental procedure The substrate was prepared by grinding samples of palm tree fronds and stirring them vigorously in distilled water. The solids were then vacuum-filtered and dried in an oven. The weight of the washed, dried solids was determined and the washing process was repeated several times until the weight remained constant. The reactions took place in four 100 ml Erlenmeyer flasks, each containing 0.5 g of washed, dried samples, which were mixed in 50 ml distilled water of 1 mM acetate buffer (pH 4.7) and 1 mM HOBT. The addition of HOBT was essential because laccase by itself is incapable of delignifying lignocelluloses due to its large size that does not allow it to penetrate the wood cell walls and thus cannot get in to direct contact with the substrate. By the addition of small amounts of low-molecular weight aromatic compounds such as HOBT, the lignin degradation is done indirectly by activating the mediators that can diffuse into wood cell walls and perform oxidations of the lignin. The enzymes can then interact directly with lignin in a later phase of the degradation process, when pores have been opened in the plant cell wall [9]. All flasks had the same composition, except for the amounts of enzymes in each one. The first flask (F1) was left as a blank, the second flask (F2) contained initially 20 units laccase per gram solid sample, the third flask (F3) contained 0.6 units cellulase per gram solid sample, and the fourth flask (F4) contained a combination of three enzymes (20 units laccase, 5 units xylanase and 0.6 units cellulase per gram solid sample). The flasks were covered tightly to prevent evaporation and kept in an incubator shaker (Innova 40 benchtop) at 45 8C. The blank flask, F1, was left for 72 h, then the sample was filtered, and the sugar content in the filtrate was determined by the 3,5-dinitrosalicylic acid (DNS) method using Dglucose as the standard [16]. Flasks 3 and 4 were incubated for 24 h, and then the samples were filtered, and sugar contents in their respective filtrates were determined. The sample is flask F2 was initially incubated for 2 h with laccase, then the sample was filtered and the sugar content in the filtrate was determined. The solids were oven dried and placed back in the flask (F2*), which was filled with 50 ml of the blank solution as in F1 and contained 5 units xylanase per gram sample. After 48 h, the sample in F2* was filtered, and the sugar content in the filtrate was determined. The solids were oven dried and placed back in the flask (F2**), which was filled with 50 ml of the blank solution as in F1 and contained 0.6 units cellulase per gram sample. The pretreated dried sample was incubated in cellulase for 24 h, and then the sample was filtered and the sugar content in the filtrates were determined. The
incubation periods by each enzyme was determined from a preliminary experiments showing that beyond the time selected for each enzyme, negligible changes were observed. 3. Results and discussions The yield percentages of reducing sugars produced from the four flasks are shown in Fig. 1. The reducing sugar yield was determined by dividing the amount of reducing sugars produced per weight of dried solid sample. As show in Fig. 1, in absence of any enzyme, a negligible reducing sugars yield of almost 0.4% was achieved in the blank solution (F1) after 72 h of incubation. Incubating with 0.6 units of cellulase per gram solids alone without pretreatment in sample (F3) resulted in a small yield of only 5.6% after 24 h of conversion. The addition of the pretreatment enzymes in a sequential manner resulted in small conversion of 2.7% after treatment with laccase for 2 h (F2) and the yield increased to 4.1% after further treatment with xylanase for 48 h (F2*). The pretreatment with laccase and xylanase resulted in a aromatic enhancement in the activity of cellulase and an appreciable yield of 45.6% was achieved after incubating the pretreated samples (F2**) with the same activity concentration of cellulase and for the same incubation time as in (F3). The success of a pretreatment process is evaluated by the improving action of hydrolysis enzyme to produce sugars, while avoiding loss of potential sugars and formation of inhibitory by-product. The results clearly prove the successful pretreatment of the lignocelluloses using the two enzymes, which resulted in a much higher effectiveness of cellulase. To assess the synergic action of the three enzymes, sample (F4) had the three enzymes combined together in the same activity concentrations as in the sequential addition sample (F2). In this case, a higher conversion yield of 60.0% was achieved after incubating for 24 h. The incubation periods by each enzyme in the sequential addition text, in sample (F2), were determined from a preliminary experiments showing that beyond the times used for each enzyme, negligible changes were observed. The reactions using each pretreatment enzyme separately may have been inhibited by the treated product. This drawback has been overcome using the combined enzymes, as in this case the treated products have been reacted immediately by the other enzymes, and hence a higher yield was achieved. The higher yield using the combined enzymes can also be due to the gradual production of accessible substrate, which reduces the effect of substrate inhibition that cellulase is known to encounter. In addition to the higher yield, using the three enzymes together provides a promising simplification to the overall bioethanol production. The pretreatment and hydrolysis can be done in one simple bioreactor. 70 Yield of reducing sugar produced
414
F4
60 50
F2 **
40 30
20 10 0
F1
F2
F2 *
F3
Fig. 1. Reducing sugars yield after 72 h in the blank sample (F1), 2 h treatment with laccase (F2) followed by 48 h treatment with xylanase (F2*) followed by 24 h treatment with cellulase (F2**), 24 h of cellulase alone (F3) and 24 h of the combined catalysts (laccase, xylanase and cellulase) (F4).
S. Al-Zuhair et al. / Journal of Industrial and Engineering Chemistry 19 (2013) 413–415
However, each enzyme has its own optimum conditions, which are not necessarily be the same as of the other enzymes. Using the enzymes in a sequential addition allows using each enzyme at its optimum condition, which is not possible in when the combined enzymes are used. 4. Conclusion Reducing sugars have been enzymatically produced from lignocellulosic agricultural waste from date palm trees. The effectiveness of the sequential pretreatment by laccase, followed by xylanase, prior to hydrolysis by cellulase has been investigated and compared to the synergistic action of combined use of the enzymes. A total reducing sugars yield of 45.6% was achieved by the sequential pretreatment, compared to only 5.6% for enzymatic hydrolysis without pretreatment. The use of the three enzymes together resulted in a higher yield of 60%. The successful pretreatment and hydrolysis in one step found in this work provides a significant simplification to the overall bioethanol production. In addition, the need for high energy, as in the conventional pretreatment techniques, is avoided.
415
References [1] D. Klemm, B. Heublein, H. Fink, A. Bohn, Angewandte Chemie 44 (2005) 3358–3393. [2] A.T.W.M. Hendriks, G. Zeeman, Bioresource Technology 100 (2009) 10–18. [3] Y. Sun, J. Cheng, Bioresource Technology 83 (2002) 1–11. [4] R. Bura, S.D. Mansfield, J.N. Saddler, R.J. Bothast, Applied Biochemistry and Biotechnology 98 (2002) 59–72. [5] J.D. McMillan, in: M.E. Himmel, J.O. Baker, R.P. Overend (Eds.), Enzymatic Conversion of Biomass for Fuels Production, American Chemical Society, Washington, DC, 1994, pp. 292–324. [6] D.S. Arora, M. Chander, P.K. Gill, International Biodeterioration and Biodegradation 50 (2002) 115–120. [7] U. Moilanen, M. Kellock, S. Galkin, L. Viikari, Enzyme and Microbial Technology 49 (2011) 492–498. [8] V. Ibrahim, L. Mendoza, G. Mamo, R. Hatti-Kaul, Process Biochemistry 46 (2011) 379–384. [9] E. Srebotnik, K.E. Hammel, Biotechnology 81 (2000) 179–188. [10] V. Menon, G. Prakash, A. Prabhune, M. Rao, Bioresource Technology 101 (2010) 5366–5373. [11] F. Shen, L. Kumar, J. Hu, J.N. Saddler, Bioresource Technology 102 (2011) 8945–8951. [12] P. Bajpai, Biotechnology Progress 15 (1999) 147–157. [13] A.M. Barbosa, R.F.H. Dekker, I. Kurtbo¨ke, G. Hardy, Letters in Applied Microbiology 23 (1996) 93–96. [14] T.K. Ghose, V.S. Bisaria, Pure and Applied Chemistry 59 (1987) 1739–1752. [15] L. Hankin, S.L. Anagnostakis, Mycologia 67 (1975) 67–73. [16] G.L. Miller, Analytical Chemistry 31 (1959) 426–428.
Contents lists available at SciVerse ScienceDirect
Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Short communication
Synergistic effect of pretreatment and hydrolysis enzymes on the production of fermentable sugars from date palm lignocellulosic waste Sulaiman Al-Zuhair *, Khalda Ahmed, Ashir Abdulrazak, Muftah H. El-Naas Chemical and Petroleum Engineering Department, UAE University, 17555 Al-Ain, United Arab Emirates
A R T I C L E I N F O
Article history: Received 20 July 2012 Accepted 17 September 2012 Available online 13 October 2012 Keywords: Enzymatic pretreatment Lignocelluloses Synergic effect Reducing sugars
A B S T R A C T
The sequential addition of the enzymes, laccase for lignin degrading, followed by xylanase for hemicelluloses hydrolysis, then cellulase for cellulose hydrolysis, was compared to the synergistic action of using the three enzymes together. It was shown that the reducing sugars yield increased from 5.6% using cellulase only to 45.6% by pretreatment with laccase and xylanase, prior to the enzymatic hydrolysis. A higher conversion of 60% was achieved by using the three enzymes together for the same incubation period. The proposed synergistic enzymes approach is a simpler and less energy intensive alternative compared to the conventional lignocelluloses pretreatment techniques. ß 2012 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
1. Introduction Lignocellulosic materials consist of a complex form of cellulose, hemicelluloses and lignin [1]. The main disadvantage of using lignocelluloses for bioethanol production is the requirement of more process steps than those required when conventional bioethanol feedstock, such as starch, is used. Particularly the lignocelluloses have to be pretreated in order to make the cellulose accessible to hydrolysis. In a successful pretreatment process lignin structure is broken down and the crystalline structure of cellulose is disrupted, which enhance the rate and extent of the hydrolysis by chemical or biochemical reagents [2]. Conventional pretreatment techniques consist of mechanical processing, acid hydrolysis, alkali swelling, ammonia, steam and other explosive techniques, and exposure to supercritical fluids [3–5]. These conventional techniques are usually carried out at high temperature and pressure that render this step the most expensive in biomass-to-fuels conversion. In addition, conventional pretreatment processes usually result in the formation of several degradation products that are inhibitory to downstream biological processes and utilize large amounts of water that have a negative effect on the overall process. Enzymes, on the other hand, provide a cost effective alternative for the pretreatment of lignocellulosic materials, in which lignin polymers are degraded to a degree that allows access to cellulose and hemicelluloses. The most important lignin degrading enzyme is laccase, which has been successfully used for lignin degradation
* Corresponding author. Tel.: +971 37133636; fax: +971 37624262. E-mail address: [email protected] (S. Al-Zuhair).
of lignocelluloses [6–8]. Laccase has also been used for the degradation of nonphenolic lignin [9]. The pretreatment can further be improved by the use of hemicellulases, such as xylanases, which have been effectively used for the hydrolysis of hemicelluloses to pentoses [10,11]. Lignin degrading enzymes and hemicellulases have been successfully applied in the pulp and paper industry [12]. However, the application of these enzymes in bioethanol production for the pretreatment of lignocelluloses prior to the hydrolysis step has seen much less attention. During the hydrolysis step, simpler sugars are produced from the pretreated cellulose. Pretreated cellulose can be chemically hydrolyzed using dilute or concentrated acids. However, these processes are corrosive and produce toxic by-products that require a detoxification step before fermentation. The utility cost of enzymatic hydrolysis is much lower because it is carried out at mild conditions and does not require any treatment step prior to fermentation. United Arab Emirates has more than 40 million palm trees and these are important sources of cellulose. Each palm tree produces about 15 kg of waste fronds per year. Palm fronds contain 58% of cellulose and 22% of hemicelluloses. That means under ideal conditions 80% of palm fronds can be converted to bioethanol, which is a renewable energy and present a good alternative to conventional, non-renewable, fossil fuels. In this work it has been proposed to replace the conventional pretreatment and hydrolysis steps with biological processes, using enzymes. Using biocatalysts makes the process less energy intensive and less harmful by-products are expected to be produced. The successful use of the approach proposed in this work provides a promising simplification to the overall bioethanol production from lignocellulosic waste available in abundance in the UAE.
1226-086X/$ – see front matter ß 2012 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jiec.2012.09.022
S. Al-Zuhair et al. / Journal of Industrial and Engineering Chemistry 19 (2013) 413–415
2. Materials and methods 2.1. Chemical and enzymes Laccase from Trametes versicolor (10 unit mg 1), xylanase from Thermomyces anuginosus (2.5 unit mg 1), cellulase from Aspergillus niger (0.3 unit mg 1), 1-hydroxybenzotriazole hydrate (HOBT) and D-glucose were obtained from Sigma–Aldrich, USA. A unit of laccase activity is defined as the amount of enzyme needed to oxidize 1 mmol of 2,6-dimethoxyphenol per min at pH 4.5 and 30 8C [13], a unit activity of xylanase is defined as the amount of enzyme needed to release 1 mmol of xylose equivalent sugar from xylan per minute at pH 4.5 at 30 8C [14] and a unit of cellulase is defined as the amount of enzyme needed to liberate 1.0 mmole of glucose from cellulose in 1 h at pH 5.0 at 37 8C [15]. The materials used to prepare the DNS reagent include 3,5-dinitrosalicylic acid, potassium sodium tartate (Rochelle salt), phenol, sodium hydroxide and sodium sulfite anhydrous were all obtained from Sigma– Aldrich. Acetate buffer with pH 4.6 was prepared from acetic acid and ammonium acetate obtained from Sigma–Aldrich, USA. 2.2. Experimental procedure The substrate was prepared by grinding samples of palm tree fronds and stirring them vigorously in distilled water. The solids were then vacuum-filtered and dried in an oven. The weight of the washed, dried solids was determined and the washing process was repeated several times until the weight remained constant. The reactions took place in four 100 ml Erlenmeyer flasks, each containing 0.5 g of washed, dried samples, which were mixed in 50 ml distilled water of 1 mM acetate buffer (pH 4.7) and 1 mM HOBT. The addition of HOBT was essential because laccase by itself is incapable of delignifying lignocelluloses due to its large size that does not allow it to penetrate the wood cell walls and thus cannot get in to direct contact with the substrate. By the addition of small amounts of low-molecular weight aromatic compounds such as HOBT, the lignin degradation is done indirectly by activating the mediators that can diffuse into wood cell walls and perform oxidations of the lignin. The enzymes can then interact directly with lignin in a later phase of the degradation process, when pores have been opened in the plant cell wall [9]. All flasks had the same composition, except for the amounts of enzymes in each one. The first flask (F1) was left as a blank, the second flask (F2) contained initially 20 units laccase per gram solid sample, the third flask (F3) contained 0.6 units cellulase per gram solid sample, and the fourth flask (F4) contained a combination of three enzymes (20 units laccase, 5 units xylanase and 0.6 units cellulase per gram solid sample). The flasks were covered tightly to prevent evaporation and kept in an incubator shaker (Innova 40 benchtop) at 45 8C. The blank flask, F1, was left for 72 h, then the sample was filtered, and the sugar content in the filtrate was determined by the 3,5-dinitrosalicylic acid (DNS) method using Dglucose as the standard [16]. Flasks 3 and 4 were incubated for 24 h, and then the samples were filtered, and sugar contents in their respective filtrates were determined. The sample is flask F2 was initially incubated for 2 h with laccase, then the sample was filtered and the sugar content in the filtrate was determined. The solids were oven dried and placed back in the flask (F2*), which was filled with 50 ml of the blank solution as in F1 and contained 5 units xylanase per gram sample. After 48 h, the sample in F2* was filtered, and the sugar content in the filtrate was determined. The solids were oven dried and placed back in the flask (F2**), which was filled with 50 ml of the blank solution as in F1 and contained 0.6 units cellulase per gram sample. The pretreated dried sample was incubated in cellulase for 24 h, and then the sample was filtered and the sugar content in the filtrates were determined. The
incubation periods by each enzyme was determined from a preliminary experiments showing that beyond the time selected for each enzyme, negligible changes were observed. 3. Results and discussions The yield percentages of reducing sugars produced from the four flasks are shown in Fig. 1. The reducing sugar yield was determined by dividing the amount of reducing sugars produced per weight of dried solid sample. As show in Fig. 1, in absence of any enzyme, a negligible reducing sugars yield of almost 0.4% was achieved in the blank solution (F1) after 72 h of incubation. Incubating with 0.6 units of cellulase per gram solids alone without pretreatment in sample (F3) resulted in a small yield of only 5.6% after 24 h of conversion. The addition of the pretreatment enzymes in a sequential manner resulted in small conversion of 2.7% after treatment with laccase for 2 h (F2) and the yield increased to 4.1% after further treatment with xylanase for 48 h (F2*). The pretreatment with laccase and xylanase resulted in a aromatic enhancement in the activity of cellulase and an appreciable yield of 45.6% was achieved after incubating the pretreated samples (F2**) with the same activity concentration of cellulase and for the same incubation time as in (F3). The success of a pretreatment process is evaluated by the improving action of hydrolysis enzyme to produce sugars, while avoiding loss of potential sugars and formation of inhibitory by-product. The results clearly prove the successful pretreatment of the lignocelluloses using the two enzymes, which resulted in a much higher effectiveness of cellulase. To assess the synergic action of the three enzymes, sample (F4) had the three enzymes combined together in the same activity concentrations as in the sequential addition sample (F2). In this case, a higher conversion yield of 60.0% was achieved after incubating for 24 h. The incubation periods by each enzyme in the sequential addition text, in sample (F2), were determined from a preliminary experiments showing that beyond the times used for each enzyme, negligible changes were observed. The reactions using each pretreatment enzyme separately may have been inhibited by the treated product. This drawback has been overcome using the combined enzymes, as in this case the treated products have been reacted immediately by the other enzymes, and hence a higher yield was achieved. The higher yield using the combined enzymes can also be due to the gradual production of accessible substrate, which reduces the effect of substrate inhibition that cellulase is known to encounter. In addition to the higher yield, using the three enzymes together provides a promising simplification to the overall bioethanol production. The pretreatment and hydrolysis can be done in one simple bioreactor. 70 Yield of reducing sugar produced
414
F4
60 50
F2 **
40 30
20 10 0
F1
F2
F2 *
F3
Fig. 1. Reducing sugars yield after 72 h in the blank sample (F1), 2 h treatment with laccase (F2) followed by 48 h treatment with xylanase (F2*) followed by 24 h treatment with cellulase (F2**), 24 h of cellulase alone (F3) and 24 h of the combined catalysts (laccase, xylanase and cellulase) (F4).
S. Al-Zuhair et al. / Journal of Industrial and Engineering Chemistry 19 (2013) 413–415
However, each enzyme has its own optimum conditions, which are not necessarily be the same as of the other enzymes. Using the enzymes in a sequential addition allows using each enzyme at its optimum condition, which is not possible in when the combined enzymes are used. 4. Conclusion Reducing sugars have been enzymatically produced from lignocellulosic agricultural waste from date palm trees. The effectiveness of the sequential pretreatment by laccase, followed by xylanase, prior to hydrolysis by cellulase has been investigated and compared to the synergistic action of combined use of the enzymes. A total reducing sugars yield of 45.6% was achieved by the sequential pretreatment, compared to only 5.6% for enzymatic hydrolysis without pretreatment. The use of the three enzymes together resulted in a higher yield of 60%. The successful pretreatment and hydrolysis in one step found in this work provides a significant simplification to the overall bioethanol production. In addition, the need for high energy, as in the conventional pretreatment techniques, is avoided.
415
References [1] D. Klemm, B. Heublein, H. Fink, A. Bohn, Angewandte Chemie 44 (2005) 3358–3393. [2] A.T.W.M. Hendriks, G. Zeeman, Bioresource Technology 100 (2009) 10–18. [3] Y. Sun, J. Cheng, Bioresource Technology 83 (2002) 1–11. [4] R. Bura, S.D. Mansfield, J.N. Saddler, R.J. Bothast, Applied Biochemistry and Biotechnology 98 (2002) 59–72. [5] J.D. McMillan, in: M.E. Himmel, J.O. Baker, R.P. Overend (Eds.), Enzymatic Conversion of Biomass for Fuels Production, American Chemical Society, Washington, DC, 1994, pp. 292–324. [6] D.S. Arora, M. Chander, P.K. Gill, International Biodeterioration and Biodegradation 50 (2002) 115–120. [7] U. Moilanen, M. Kellock, S. Galkin, L. Viikari, Enzyme and Microbial Technology 49 (2011) 492–498. [8] V. Ibrahim, L. Mendoza, G. Mamo, R. Hatti-Kaul, Process Biochemistry 46 (2011) 379–384. [9] E. Srebotnik, K.E. Hammel, Biotechnology 81 (2000) 179–188. [10] V. Menon, G. Prakash, A. Prabhune, M. Rao, Bioresource Technology 101 (2010) 5366–5373. [11] F. Shen, L. Kumar, J. Hu, J.N. Saddler, Bioresource Technology 102 (2011) 8945–8951. [12] P. Bajpai, Biotechnology Progress 15 (1999) 147–157. [13] A.M. Barbosa, R.F.H. Dekker, I. Kurtbo¨ke, G. Hardy, Letters in Applied Microbiology 23 (1996) 93–96. [14] T.K. Ghose, V.S. Bisaria, Pure and Applied Chemistry 59 (1987) 1739–1752. [15] L. Hankin, S.L. Anagnostakis, Mycologia 67 (1975) 67–73. [16] G.L. Miller, Analytical Chemistry 31 (1959) 426–428.