The using of enzymes for degradation of cellulose substrate for the ...

Slovak Society of Chemical EngineeringInstitute of Chemical and Environmental EngineeringSlovak University of Technology in BratislavaPROCEEDINGS37 th International Conference of Slovak Society of Chemical EngineeringHotel HutníkTatranské Matliare, SlovakiaMay 24 – 28, 2010Editor: J. MarkošISBN 978-80-227-3290-1Mackul’ak, T., Prousek, J., Olejníková, P., Bodík, I.: The using of enzymes for degradation of cellulose substratefor the production of biogas, Editor: Markoš, J., In Proceedings of the 37th International Conferenceof Slovak Society of Chemical Engineering, Tatranské Matliare, Slovakia, 1407–1412, 2010.

37th International Conference of SSCHEMay 24–28, 2010, Tatranské Matliare, SlovakiaPo-Th-6, 116p.pdfThe Using Of Enzymes For Degradation Of Cellulose Substrate For TheProduction Of BiogasTomáš Mackuľak, Josef Prousek, Petra Olejníková a , Igor Bodík,Department of Environmental Engineering, Institute of Chemical and Environmental Engineering,Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskeho 9, 812 37Bratislava, Slovak RepublicaDepartment of Biochemistry and Microbiology, Institute of Biochemistry, Nutrition and HealthProtection, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskeho9, Bratislava 812 37, Slovak Republic,e-mail: mackulakt@azet.skKey wordsenzymatic hydrolysis, cellulose substrate, glucoseAbstractThe main purpose of this article is to describe different kinds of cellulose degradation processes such asenzymatic hydrolysis. As the cellulose substrates the sawdust, leaves and cartons were used. Anaerobicdegradation processes utilized for the biogas production can be accelerated by this way.1. IntroductionAs substrates for biogas production waste materials which contain polysaccharides, lipids and proteinscan be used. Plant origin substrates contain mainly cellulose (Fig. 1), hemicellulose and lignin [1]. Thestructure of cellulose consists of parallel glucan chains and is stabilized by hydrogen bonds.Fig. 1. Cellulose structureThe main structural unit of cellulose is D – glucose [3]. Cellulose is resistant to different types ofdegradation [2]. The cellulose substrate contains various representation of lignin, which influences theresistance of substrate and also increases the cost of pretreatment. Usage of hay, straw and recycledpaper as a substrate containing lignocelluloses for increasing biogas production is currently beingtested and interesting topic.A mechanical pretreatment is the first step in the pretreatment of lignocelluloses substrate. In next stepsis possible to degrade cellulose by chemical or enzymatic hydrolysis [5, 8]. The chemical hydrolysis byH 2 SO 4 and HCl leads to breaking cellulose into cellotetrose and cellobiose [2, 4, 8]. By Fenton reactionis possible to degrade cellotetrose and cellobiose to glucose, a simple aldehydes and ketones. Directway to degrade cellulose into glucose is enzymatic hydrolysis.Enzymatic hydrolysis of cellulose takes place with a mixture of enzymes – cellulases [1, 5-9], at 36 –37 °C. Cellulases break down cellulose in three ways. Endoglucanases convert chains of cellulose from1407

37th International Conference of SSCHEMay 24–28, 2010, Tatranské Matliare, SlovakiaPo-Th-6, 116p.pdfthe inside, creating new ends of shorter chains [1]. Exoglucanases separated from non-reducting endsof new chains cellobiotic units. In consequent steps cellotetrose and cellobiose are decomposed toglucose in liquid phase by the enzyme β-glucosidase [1]. Cellulases are produced by different types ofbacteria and filamentous fungi [5,7]. Research of different tribes of filamentous fungi isolated fromwaste waters from paper production, which are able to produce cellulose in large scale seems to beinteresting nowadays [5,7]. Lignocellullase substrate pre-treated in this way can be used to increase thequality and quantity of produced biogas.2. Experimental partCellulose substrateCellulose substrate was inactivated with lime (Fig. 2). Total solids were 43% and volatile solids were44%.Fig. 2. Cellulose substratePrincipes of glucose determinationOxidation of glucose with oxygen was preformed with enzymatic catalysis by glucose oxidase forhydrogen peroxide and gluconate. Formed hydrogen peroxide was determinated by oxidationcopulation with substitued phenol and 4- aminoantipyrine and cotalised with peroxidase.Determination of glucoseOperating solution was mixed with serum or sample, standard glucose solution and distilled water(control solution) in the ratio 100:1 or 150:1 in three tubes. Incubation was preformed for 30 min. at15- 25 °C or for 15 min. at 37°C. The incubation mixture must be protected from direct light. Whitin40 min. after incubation the absorbance of sample (A 1 ) and absorbance of sample (A 2 ) was measuredand compared with control solution.CalculationGlucose (mmol.l -1 ) = a. A 1 /A 2 ,where a is glucose contentration in standard solution [mmol.l -1 ].Enzymatic hydrolysisIn various experiments with enzymes different amounts of enzyme mixture for cellulose substratedegradation were used (Fig. 3). The effectiveness of individual experiments on the measurement ofCOD value (mg.l -1 ) and glucose concentration was determined (g.l -1 ). Procedures and results indegradation of the substrate are given in experiments No. 1-3, and Fig. 4-5.1408

37th International Conference of SSCHEMay 24–28, 2010, Tatranské Matliare, SlovakiaPo-Th-6, 116p.pdfFig. 3. Enzymatic hydrolysisExperiment No. 1 (E1)In the first series of experiments with compound of enzymes 5mg of enzymes were added into 200 mlof deionized water. The solution was stirred until dissolved enzymes. In the next step 3 g of cellulosesubstrate were added. The temperature was 37°C and mixture wasn’t stirred. Degradation of substrateto glucose was determined by glucose- oxidation test and by measurement of COD values.Experiment No. 2 (E2)In another series of experiments 50 mg of compound of enzymes were dissolved in 200 ml of deionizedwater. The solution was stirred until dissolved enzymes. In the next step 3 g of cellulose substrate wereadded. The temperature was 37°C and mixture wasn’t stirred. Degradation of substrate to glucose wasdetermined by glucose- oxidation test and by measurement of COD values.Experiment No. 3 (E3)In final series of experiments with compound of enzymes 450 mg of enzymes were dissolved in 200 mlof deionized water. The solution was stirred until dissolved enzymes. In the next step 3 g of cellulosesubstrate were added. The temperature was 37°C and mixture wasn’t stirred. Degradation of substrateto glucose was determined by glucose- oxidation test and measurement of COD values.3. Results and discussionEnzymatic hydrolysis decomposes cellulose to cellotetrose and cellobiose in first step. Subsequentlycellotetrose and cellobiose are degradated to glucose, lower aldehydes and ketones (Fig. 4). Products ofenzymatic hydrolysis depends on the type and composition of the substrate, the reaction time, the typeand quantity of the enzymes and the reaction temperature. Glucose itself can be enzymatic degraded toaldehydes and ketones.Compound of enzymes was used for the degradation of cellulosic substrate. Time dependence ofglucose production resulting from substrate degradation is shown in figure 5. From each experiment isappeared that the production of glucose is affected by the amount of enzymes used, quantity ofsubstrate, reaction temperature and time. The highest glucose yield was achieved in the experiments no.3. In experiment No. 2 and 3. a decrease in glucose concentration after 24 hrs. was observed, whichwas caused by the amount of used enzymes (Tab. 1, Fig. 4).1409

37th International Conference of SSCHEMay 24–28, 2010, Tatranské Matliare, SlovakiaPo-Th-6, 116p.pdfFig. 4. Expected mechanism of cellulose degradation by enzymatic hydrolysis0,350,300,25glucose g.l -10,200,150,10E 1E 2E 30,050,000 10 20 30 40 50 60 70 80t(h)Fig. 5. Time dependance of glucose production by enzymatic degradation of cellulose substrate.Tab. 1. Time dependance of glucose production by enzymatic degradation of cellulose substrate inexperiments no. 1-3.Time (hours) Glucose (g.l -1 )E 1 E 2 E 36 0.1 0.2 0.2224 0.2 0.26 0.3248 0.22 0.3 0.2872 0.26 0.26 0.241410

37th International Conference of SSCHEMay 24–28, 2010, Tatranské Matliare, SlovakiaPo-Th-6, 116p.pdfTab. 2. Time dependence of COD value in experiments no. 1- 3 of glucose production by enzymaticdegradation of cellulose substrate.Time (hours) COD (mg.l -1 )E 1 E 2 E 36 200 250 34024 360 540 59048 430 580 62072 600 650 6807 0 06 0 05 0 0CHSK (mg.l -1 )4 0 03 0 02 0 0E 1E 2E 31 0 000 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0t ( h )Fig. 6. Comparation of experiments no. 1-3 by COD value.4. ConclusionFrom results it can be concluded that lignocellulosic substrates can be succesfully modified byenzymatic hydrolysis. We dealt with the use of enzymatic hydrolysis in experiments No. 1-3. It isobtained possible to decompose cellulose substrate to glucose by enzymes. Yield of glucose is dependson quantity of substrate used, amount of used enzymes used, reaction temperature and time (Fig. 5).Substrate is decomposed to glucose and lower aldehydes for prolonged exposure to enzymes.Efficiency and progress of enzymatic hydrolysis is affected by content of lignin in substrate. Celluloseis covered by lignin and thereby increase the resistance of cellulose to enzymatic degradation. It istherefore possible to combine the procedures for remove lignin (e.g., chemical hydrolysis) andsubsequently enzymatic hydrolysis or microbial degradation can be used. The effectiveness ofenzymatic hydrolysis can be regulated or completely inhibited by temperature increasing. Enzymatichydrolysis is one of the processes by which we can pretreated cellulose substrates such as sawdust,leaves, hay, straw, recycled paper and thus accelerated the processes of degradation under anaerobicconditions. Products of enzymatic degradation of substrates are glucose and lower aldehydes, which are1411

37th International Conference of SSCHEMay 24–28, 2010, Tatranské Matliare, SlovakiaPo-Th-6, 116p.pdfhighly degraded in anaerobic processes. Substrates treated in this way are easier and fasterbiodegradabiled and can be used to increase biogas yield and quantity.5. References1. Straka F. a kol. (2006).: Bioplyn. Praha: GAS s.r.o.,706 s.2. Görner F., Valík Ľ. (2004).: Aplikovaná mikrobiológia požívatín. Malé Centrum, Bratislava.3. Šulák M., Šmogrovičová D. (2008).: Bioetanol : Súčasné trendy vo výskume a v praxi : Chem.Listy 102, 108−115.4. Sun Y., Cheng J. (2002).: Hydrolysis of lignocellulosic materials for ethanol production.Bioresource Technology., 83, 1-11.5. Tanaka H., Koike K., Itakura S., Enoki A. (2009).: Degradation of wood and enzyme productionby Ceriporiopsis subvermispora. Enzyme Microb. Technol. doi:10.1016/j.enzmictec.2009.06.003.6. Romsaiyud A, Songka S.W., Nopharatana A., Chaiprasert P. (2009).: Combination effect of pHand acetate on enzymatic cellulose hydrolysis. J. Environ. Sci. 21, 965–970.7. Solange I.M., Marcela F., Adriane M.F.M, Inˆes C.R. (2008).: Effect of hemicellulose and ligninon enzymatic hydrolysis of cellulose from brewer’s spent grain. Enzyme Microb. Technol. 43,124–129.8. Voet D., Voetová J.G. (1995).: Biochemie. Victoria Publishing a.s.,Praha, 268-283.9. Mackuľak T., Prousek J., Bodík I. (2009).: Predúprava a využitie celulózového substrátu z výrobypapiera na produkciu bioplynu. Zborník konferencie: Výstavba a provoz bioplynových stanic.Třeboň, október 15.-16, str. 83-89. ISBN-978-80-254-5455-8.AcknowledgementsThe presented contributions was created as a part of project SK00023 financed by Norwegian FinancialMechanism, Financial Mechanism of EEA and the State Budget of the Slovak Republic(www.eeagrants.com). This work was supported by the Slovak Research and Development Agencyunder the contract No. LPP-0019-09.1412