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IRJET- Hydrothermal Pretreatment Enhanced Enzymatic Hydrolysis and Glucose Production from Lignocellulose Biomass
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IRJET- Hydrothermal Pretreatment Enhanced Enzymatic Hydrolysis and Glucose Production from Lignocellulose Biomass
1.
International Research Journal
of Engineering and Technology (IRJET) e-ISSN: 2395-0056 Volume: 06 Issue: 08 | Aug 2019 www.irjet.net p-ISSN: 2395-0072 © 2019, IRJET | Impact Factor value: 7.34 | ISO 9001:2008 Certified Journal | Page 1309 Hydrothermal Pretreatment Enhanced Enzymatic Hydrolysis and Glucose Production from Lignocellulose Biomass May Zon Kyawt Oo1, Tint Tint Kywe2 1Professor, Department of Chemical Engineering, Mandalay Technological University, Mandalay, Myanmar 2Professor, Department of Chemical Engineering, Yangon Technological University, Yangon, Myanmar ----------------------------------------------------------------------***--------------------------------------------------------------------- Abstract - The present works investigate hydrothermal pretreatment of Lignocellulose biomass, such as wood and agricultural residues, on chemical composition of sawdust samples from hardwood and softwood and subsequent bioethanol production applying pre-enzymatic hydrolysis and fermentation. Sawdust samples from hardwood and softwood were treated by preheating to boil 80±5˚C followed by adding sodium hydroxide solution (5% wt of sawdust) for 60min to 240min in the hydrothermal treatment. At reaction time (180min), the best condition for hydrothermal treatment gave the higher cellulose content from 52.5% to 84.9% and lesser lignin content from 26% to 3% than the other pretreatment conditions. Liberation of cellulose was confirmed by X-ray Diffraction (XRD).Thepretreatedsawdustwashydrolysedwith inoculum containing crude enzymes from Trichoderma and Aspergillus niger. After enzymatic hydrolysis, the maximum glucose yield was 7.4% and 6% by inoculum containing crude enzymes from Trichoderma and Aspergillus niger by absorbance measurement using the UV spectrophotometer. The total solid conversion were 30% for sawdust sample from softwood hydrolysed with inoculum containing crude enzyme from Trichoderma of 52 FPU/ml and 34% for sawdust sample from softwoodhydrolyzed with crude enzymefromAspergillus niger of 45 FPU/ml. Key Words: Hydrothermal Pretreatment, Enzymatic Hydrolysis, Lignocellulosic Biomass, Sawdust, Glucose Production 1. INTRODUCTION Recent economicdevelopmentsinmanycountriesall around the globe have heightened the need for alternative energy resources due to the well-documented drawbacks of fossil fuels: (1) their finite supply (2) greenhouse gasses emission and global warming and (3) increasingpriceandunexpected fluctuation. All these weaknesses have strengthened the interest in alternatives, renewable, sustainable, and economically viable fuel such as bioethanol [1]. In the first generation bioethanol production, expensive starch and sugar derived from sugar cane and maize are employed as feedstock but in the second generation process, lignocellulosic materials, which are cheap, abundant and renewable, are used [2]. Myanmar forests consists of many species of wood which, after harvesting and processing, leave behind wood wastes in the forests and wood residues in the wood processing factories. Lignocellulose biomass, such as wood and agricultural residues, is attractive materials for the ethanol production since it is the most abundant reproducible resources on earth. Ethanol production from lignocelluloses biomass depends on the hydrolysis of cellulose and hemicellulose into simple reducing sugars that can be fermented into ethanol by microorganisms [3]. However, the natural recalcitrance of lignocellulosic biomass has hindered its potential applications such as biochemical andbiofuel productionif no pretreatment is performed. Pretreatment is necessary to remove barriers such as hemicellulose and lignin that limit the penetration of enzyme to cellulose. Hydrothermal pretreatmenthasadvantagesoverotherpretreatmentssince the system only use water and the hydronium ion from water ionization act as catalyst in the reaction medium [4]. Under hydrothermal pretreatment process, several soluble inhibitors were produced, which hampered the efficiency of enzymatic hydrolysis and fermentation [5]. The technology for bioethanol production from lignocelluloses biomass is well defined; however, production from other feed stocks such as biomass still requires extensive research to develop a feasible production method. So, this work focused at improving its yield by using source of lignocelluloses namely: sawdust from hardwood and softwood [6]. The aim of the current study was to investigate the chemical changes in the process of hydrothermal pretreatment and the effect of the sawdust on the yield of inoculum containing crude enzymes from Trichoderma and Aspergillus niger. Factors affecting maximal conversion of cellulose to glucose was highlighted and discussed. 2. MATERIALS AND METHODS 2.1 Raw Materials The substrates used for this work are sawdust samples from hardwood and softwood;theyarecheapandreadilyavailable sources of lignocelluloses from one of the twig in Phyu Township, Bago Division, in Myanmar for the pretreatment process. The substrates were individually screen analyzedin the British Standard, BSS 410 test sieve shaker and each sample was made to pass through 100 mesh number. 2.2 Hydrothermal Pretreatment The 50g of sawdust samples were added to400mlwater.The slurry was controlled to 80±5˚C by thermostat. Sodium hydroxidesolution(5%wtofsawdust)wasaddedtosawdust slurry. The mixture was heated to boil for various
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International Research Journal
of Engineering and Technology (IRJET) e-ISSN: 2395-0056 Volume: 06 Issue: 08 | Aug 2019 www.irjet.net p-ISSN: 2395-0072 © 2019, IRJET | Impact Factor value: 7.34 | ISO 9001:2008 Certified Journal | Page 1310 predetermined reaction time (60-240min) at 80±5˚C. After completing the boiling,solidandliquidportionofthemixture was separated by filtering. Then, the solid sample was washed several times with water to achieve neutral condition. The solid sample was dried at room temperature for 2days. At reaction time (180min), the best condition for hydrothermal treatment gave the higher cellulose content and lesser lignin content than the other pretreatment conditions. For optimum condition (180min), the treated sawdust samples were designated as HS-5 for hardwoodand SS-5 for softwood. 2.3 Inoculum Preparation for Enzymatic Hydrolysis The pure culture of Aspergillus niger and Trichoderma were provided by the Department of Biotechnology, Mandalay Technological University. The organismsweremaintainedas direct stockculturefromwhichinoculawereprepared.100ml of medium (Sabouraud broth) of sample with Aspergillus niger and 100ml of medium (Sabouraud broth) of sample with Trichoderma were used inoculum prepared in 250ml.The inoculum was shaken continuously on an environment-controlled at 25˚C before it was used for enzymatic hydrolysis and fermentation process. 2.4 Enzymatic Hydrolysis The inoculumcontainingcrudeenzymeswereusedcellulases from Aspergillus niger and Trichoderma worked in Department of Biotechnology. The pretreated sawdust samples from hardwood and softwood were hydrolysed by cellulases from Aspergillus niger and Trichoderma at 50˚C, 85rpm in a water bath shaker with cellulose 5% (w/v).The cellulose powder was dissolved in1mlof0.05Mcitratebuffer (pH 4.8).At each reaction time of 60min, 0.5ml of samplewas taken and diluted forthe glucose andthetotalreducingsugar analysis. In the enzymatic hydrolysis, filter paper, pretreated hardwood (HS-5) and softwood (SS-5) were used as the substrates. At the end of the hydrolysis period, DNS reagent was added to stop the reaction. Then the process for colour development was continued. The undigested pulps were settled, separated and absorbance of liquid portion was measured to find the amount of glucose produced. The untreated pulp was washed with water, dried at 100 °C and weighed for determination of solid conversion. 2.6 Analytical Methods The cellulose content ,hemicellulose content and lignin content of pretreatment sawdust were analyzed by heat-of- dilution dichromate method, extraction of alkali method,72%(v/v)sulphuricacidmethodrespectively[3].The degree of crystallinity and the crystal structure of sawdust were characterized by X-ray diffractometer (XRD). Total reducing sugars were determined by the DNS method using glucose as the standard [5]. Cellulase activity was assayed as filter paper units [7].The presence of glucose can be detected by absorbance measurement using the UV spectrophotometer. 3. RESULTS AND DISCUSSION 3.1 Chemical Compositions of sawdust samples from Hydrothermal Pretreatment According to results from hydrothermal treatment, the percentage of cellulose content is as shown in Fig. 1. The Figure shows time versus cellulose percent of the hardwood by using hydrothermal treatment process. At reaction time (180min), Sample No. (HS-5) gave the higher percentage of cellulose for optimum condition.Themorereactiontime, the higher percentage of cellulose. So, hardwood sample (HS-5) is the best conditions. Then,sawdustsfromsoftwoodtreated by preheating to boil 80±5˚C followed by adding sodium hydroxide solution (5% by wt of sawdust) for 60min to 240min.So, sawdusts from softwood (SS-5) treated by preheating to boil 80±5˚C followed by adding sodium hydroxide solution (5% wt of sawdust) for180min wasbest condition compared to that other conditions . Fig -1: Percentage of cellulose from hardwood According to results from hydrothermal treatment, these experimental data can be seen in Table 1. Table -1: Compositions of Hardwood and Softwood from Hydrothermal Pretreatment Sample No. Cellulose (%) Hemicellulose (%) Lignin (%) HS-0a HS-5b 43.5 83.5 23.5 8 24 4 SS-0a SS-5b 52.5 84.9 9 6.3 26 3 aHS-0 andSS-0= untreated sawdust samples from hardwood and softwood bHS-5 and SS-5= pretreatedsawdustsamplesfromhardwood and softwood
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International Research Journal
of Engineering and Technology (IRJET) e-ISSN: 2395-0056 Volume: 06 Issue: 08 | Aug 2019 www.irjet.net p-ISSN: 2395-0072 © 2019, IRJET | Impact Factor value: 7.34 | ISO 9001:2008 Certified Journal | Page 1311 The compositions of untreated and pretreated sawdusts from hardwood and softwood are compared. According to Table 1, the percentage of cellulose content of untreated sawdust sample from softwood (SS-0) was morethanthat of hardwood (HS-0).But hardwood had lower lignin content than softwood. The hemicellulose could be produced significantly in hardwood. As softwood have higher lignin content which makes the hydrolysis step more difficult,they have generally produced less hemicellulose. For hydrothermal treatment, sample HS-5and SS-5 of the lignin contents were decreased more than that of sample HS-0and SS-0. 3.2 XRD Patterns of Untreated and Pretreated Sawdust Samples The XRD patterns of untreated and pretreated sawdust samples from hardwood and softwood for hydrothermal treatment are shown in Fig. 2 and Fig. 3. According to Fig. 2, the strongest peak, 2θ≈23˚, originates from the cellulose crystalline plane. It could be seen that the longer reaction time heated more linkage with increase in the percentage of intensity reduction. Increasing the percentage of intensity reduction shows decreasing the degree of crystalline. Fig -2: XRD patterns of untreated and pretreated sawdust samples from hardwood for hydrothermal treatment After the heating time 180min, this crystalline peak noticeably disappeared. So, sawdustsamplefrompretreated hardwood (HS-5) was better condition compared to that of (HS-0) pretreatment condition. Sample (HS-5) could reduce the linkages between lignin, hemicellulose, and cellulose in sawdust. According to untreated and pretreated sawdust samples from hardwoodfor hydrothermal treatmentresults, hydrothermal treatment on sawdust from hardwood was effective. According to Fig. 3, the strongest peak, 2θ≈23˚, originates from the cellulose crystalline plane. It could be seen that the longer reaction time heated more linkage with increase in the percentage of intensity reduction. Increasing the percentage of intensity reduction shows decreasing the degree of crystallinity. After the heating time 180min, this crystalline peak noticeably disappeared.So,sawdustsample from pretreated hardwood (SS-5) was better condition compared to that of (SS-0) pretreatment condition. Fig -3: XRD patterns of untreated and pretreated sawdust samples from softwood for hydrothermal treatment Sample (SS-5) could reduce the linkages between lignin, hemicellulose, and cellulose in sawdust. According to untreated and pretreated sawdust samples from softwood for hydrothermal treatment results,hydrothermal treatment was best condition observed in this study.It is surethatHS-5 and SS-5 were continued to treat for enzymatic hydrolysis and fermentation process. 3.3 Glucose Concentration by using Enzymatic Hydrolysis Inoculum containing crude enzymes concentrations (v/v) against glucose liberated from filter paper, pretreated sawdust samples from softwood (SS-5) and hardwood (HS- 5) were plotted and shown in Fig. 4 and Fig. 5. Fig -4: Amount of glucose liberated against Inoculum containing crude enzyme concentration from Trichoderma
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International Research Journal
of Engineering and Technology (IRJET) e-ISSN: 2395-0056 Volume: 06 Issue: 08 | Aug 2019 www.irjet.net p-ISSN: 2395-0072 © 2019, IRJET | Impact Factor value: 7.34 | ISO 9001:2008 Certified Journal | Page 1312 Fig -5: Amount of glucose liberated against Inoculum containing crude enzymes concentration from Aspergillus niger According to Fig. 4, concentration of inoculum containing crude enzymes from Trichoderma that released 2 mg of glucose was 0.0068 for filter paper, 0.007 for pretreated softwood (SS-5) and 0.008 for pretreated hardwood (HS-5). According to Fig. 5, concentration of inoculum containing crude enzymes from Aspergillus niger that released 2 mg of glucose was 0.007 for filter paper, 0.0082 for pretreated softwood (SS-5)and0.0084forpretreatedhardwood(HS-5). Comparing to Fig. 4 and Fig. 5, the cellulase activity of inoculum containing crude enzyme from Trichoderma was 54FPU/ml for filter paper, 52FPU/ml and 46FPU/ml for pretreated softwood (SS-5) and hardwood (HS-5). The cellulase activity of inoculum containing Crude enzyme from Aspergillus niger was 52FPU/ml for filter paper, 45FPU/ml and 46FPU/ml for pretreated softwood (SS-5) and hardwood (HS-5). Inoculum containing crude enzyme dilution from Trichoderma was less concentrated and more enzymatic cellulase activity. 4. CONCLUSION Hydrothermal pretreatment and enzymatic hydrolysis of Lignocellulosic biomass demonstrated high conversion of cellulose to glucose. Pretreated sawdusts from hardwood and softwood were then hydrolysed with two types of inoculum containing crude enzymes from Trichoderma and Aspergillus niger to produce glucose. The maximum glucose yield was 7.4% and 6% by crude enzymes from Trichoderma and Aspergillus niger. ACKNOWLEDGEMENT The author would like to express her deepest gratitude to her beloved parents and teachers, for their kindness, patience, encouragement and support to reach her destination without any trouble. REFERENCES [1] Hahn HagerdalB, GalbeM, Gorwa-GrauslundMF, LidenG,ZacchiG. Bio-ethanol the fuel of tomorrow from the residues of today. Trends in Biotechnology2006; 24(549):56.M. Young,TheTechnical Writer’sHandbook. Mill Valley, CA: University Science, 1989. [2] KootstraAMJ, MosierNS, ScottEL, BeeftinkHH, SandersJPM. Differential effects of mineral and organic acids on the kinetics of arabinose degradation under lignocellulose pretreatment conditions. Biochemical Engineering Journal 2009; 43:92–7. [3] Mie Mie Kyaw, Mechanical andchemical pretreatmentof woody biomass for bioethanol production, thesis submitted for the degree of philosophy, Mandalay techanological University, Myanmar, (2007).. [4] Möller, M., Nilges, P., Harnisch, F., Schröder, U., 2011. Subcritical water as reaction environment: fundamentals of hydrothermal biomasstransformation. ChemSustChem. 4, 566–579. [5] Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal.Chem. 31, 426- 428, 1959. [6] Sun Y, Cheng J, Hydrolysisoflignocellulosicmaterialsfor ethanol production: a review, Biores Technol 83: 1-11. 2002. [7] Ghose, Measurement of cellulose activities. Pure Appl. Chem.59, 257-268, 1987 BIOGRAPHIES The author has got her doctoral degree in Chemical Engineering from Mandalay Technological University, Myanmar in 2009. During her academic studies, she carried out postgraduate researches on PolymerTechnology and Renewable Energy. At First, she studied to the Environmental Protection Technology and Management for Air Pollution at Korea and Japan, 2010. After her graduation, she worked for Chemical EngineeringDepartment,
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International Research Journal
of Engineering and Technology (IRJET) e-ISSN: 2395-0056 Volume: 06 Issue: 08 | Aug 2019 www.irjet.net p-ISSN: 2395-0072 © 2019, IRJET | Impact Factor value: 7.34 | ISO 9001:2008 Certified Journal | Page 1313 Mandalay Technological University, Myanmar between 2012 and 2013.After that, she studied to the Renewable Energy and Efficiency at India,2014. Since March, 2014, she is currently working for Department of Chemical Engineering, Mandalay Technological University, teaching some undergraduate courses and guiding some masterstudentswho are including the analysis and protection of environmental pollution. The author has got her doctoral degree in Chemical Engineering from Mandalay Technological University, Myanmar in 2007. As a chemical engineer, she studied to enhance the ability to develop the Renewable Energy by the various lecturers and field visit. After her graduation, she worked Biodiesel Project for Chemical Engineering Department, Mandalay Technological University, Myanmar between 2009and 2015. Now, she is currently working for Department of Chemical Engineering,Yangon Technological University, teaching some undergraduate courses and guiding some master students. At present, she wants to serve her ministry and her country to protect her environmental fresh.
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