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, 8 (5): 520-524, 2010 ISSN 1818-6769
c IDOSI Publications, 201 0
Investigation of Olive Stones as Lignocellulose Material for Bioethanol Production
'Hemmat Ahmadi, lVahid Akbarpour and 2Abdolali Shojaeian
'Department of Horticulture, Sari University of Agricultural Sciences and Natural Resources, Sari, Iran 2Department of Horticulture, Tarbiat Modares University, Iran
Abstract: In Iran, the area under olive cultivation is growing. With increasing the production of olive a lot of waste cake is produced. Olive stone as a lignocelluloses biomass is a raw material suitable for enzymatic hydrolysis and bioethanol production that now only starchy and sugar plants use. In this study, the olive stones were delignified with sodium hydroxide at 50°C and simultaneous velocity of 250 rpm. It has undergone enzymatic hydrolysis. Various quantities of cellolase in different concentrations of achieved cellulose materials have been used. The findings of the study indicated that, although using more enzymes (80 cc) yields more sugar, the amount of sugar produced will be half of the quantity (40 cc) used, with regard to high cost of enzymes. The velocity of reactions dropped dramatically after 8 h. With an 8-h double cycle of hydrolysis and enzymatic recovery, sugar concentration of about 20 gil is achieved using 200 g of primary cellulose compounds (100 g per cycle) and 40 cc of enzymes. Thus, with 0.55 cc of alcohol efficiency to 1 g of sugar, 11 cc of alcohol is produced in fermentation.
Key words: olive stone . Enzymatic hydrolysis . Bioethanol . Lignocellulosic compounds
Olive tree (Olea europae L.) is one that has been used in producing oil and table olives for many years. Over 95% of the olive trees are in the Mediterranean region . Outside this region i.e., in Iran, its cultivation has grown in recent years and has reached 50,000 ha based on the latest statistics (17). As a result, large quantities of fruit will be produced and extracted soon. Based on extraction technology, press, biphase, or triphase decanter, in return for every ton of olive fruit, 200 kg of oil, 110, 400, and 1000 liters of vegetable water, and 400 kg of olive pumace are made, respectively. The olive pumace contains 10-11 % skin, 21-23% flesh, 36-54% stone, 5-8% oil, and 25% water, which changes to 22-20% and 30-55% in continuous systems [2, 3]. This cake contains 94.23% organic matter, 33.5% lignin, 20.5% cellulose, and 40.6% hemicellulose [4,5]. Usage of these organic matters hasn't developed in Iran yet. It is used more to increase olive-garden soil fertility and lower quantities are used to feed livestock . However, olive cake has various uses in different countries. Energy production by burning it alone [4, 7, 8], or with other organic and some mineral substances [2, 9, 10], feeding livestock [6, 11], compost making , improving soil
conditions [3, 12], producing active carbon , biogas production [14-17], and hemicelluloses extraction  are some of the cases. Yet this lignocellulosic compound has never been used to produce bioethanol . Lignocelluloses compounds refer to substances that contain cellulose andhemicelluloses of polymers made up of sugar monomers that are converted into ethanol after primary treatments, hydrolysis, and microbial fermentation [19,20]. Many countries including America, Brazil, China, Taiwan, India, and Thailand have decided to substitute part of their fossil fuels for ethanol in the coming years. Some of the reasons for this are less environmental pollution, energy supply security, higher octane, and the production of fewer green house gases [19, 21]. Up to 10% bioethanol can be added to gasoline to boost its combustion; however, there are some modes of transportation that are able to consume up to 85% ethanol . Today's technologies use grains (ccirn and wheat) and sugar plants (sugar cane and beetroot) to produce bioethanol. High demand of bioethanol resulted in the study of other sources of biomass [18, 19].
Some of these compounds are produced in large quantities as by-products in agricultural-based industries and are quite cheap . In a study, Peterson, et al.  used rye chaff, colza, and faba beans as lignocelluloses
Corresponding Author: Vahid Akbarpour, Department of Horticulture, Faculty of Crop Sciences, Sari University of Agricultural Sciences & Natural Resources, Khazar Bvd., Km 20, Sari, Mazandaran Province, Iran.
Cell: +989177319907, E-mail: firstname.lastname@example.org.
Am-Euras. J. Agric. & Environ. Sci., 8 (5): 520-524, 2010
resources to produce bioethanol . By pre-treating an aquatic oxidation, they could produce 66%, 70%, and 52% alcohol out of rye, colza, and faba beans, respectively. Adrados et al.  could also produce ethanol by using wheat bran that comprises 19% of the grain weight (1). Gnansonou et al.  suggested that sorghum bagass is more suitable for producing bioethanol than burning it (14]). In an experiment, Silverstein, et al.  compared different methods of pre-treatment to enhance scarification of cotton stalks (27). In the near future, bioethanol will be produced by using lignocelluloses compourtds such as hard woods, soft woods, and herbaceous materials. Movagharnejad et al.  have suggested models for enzymatic hydrolysis in heterogeneous liquid-soiled systems (21, 22). This study aims to analyzing enzymatic hydrolysis of cellulose stock in olive stone and bioethanol production. Such study has had no place in Iran because of the abundance of oil and gas. However, this will be appreciated soon because of the elimination of petrol subsidies. Appling mathematical models for shrinking particles based on the data gathered in this study can be used to make an accurate prediction regarding the production of sugar during the hydrolysis and fermentation processes.
MATERIALS AND METHODS
Applied Materials: Olive waste cake provided by the Ganje factory in Roudbar (north of Iran) was used to perform this experiment. Olive waste cake usually contains non-cellulose parts that should be separated. After separating the skin and flesh, the stone of the olive was ground and kept in a mortar at 90°C for 1 day. The soiled materials (compounds) were dried and prepared for the experiment . The enzyme used was celloclast (22.85- 4) and was supplied by Danish Novo. The enzymatic activity of celloclast was determined to be 42 fpulml .
Experimentation: A total of 100 g of oven-dried compound was added into 1 liter of sodium hydroxide solution (5 gil). The resulting compound was boiled at 100°C for 1 h. Large quantities of lignin were obtained from the produced substances and prepared for enzymatic hydrolysis. Through this process, the mixture was stirred well to avoid settlement of the solid particles. Then, the compound was cooled for 1 h and the modified solid particles were separated. The solid particles were then leached to eliminate any alkaline substances and turned into neutral pH by distilled water. After that, the modified solid particles (deligninized) were redried in the oven at 90°C for 1 day.
Enzymatic Hydrolysis: In this experiment, the considered quantities of the achieved cellulose compounds were added into the reaction container and 500cc of buffer acetate (pH=5) was added. Care should be taken when the solid particles are added to the reaction container to stop them from sticking to the walls of the container. Then the container of cellulose compounds was kept in water bath at 50°C. The temperature of the reaction vessel is kept at 50°C during the hydrolysis process. After fixing the vessel temperature, the mechanical mixer is put in the reaction vessel and the velocity is set at 250 rpm to keep the cellulose particles suspended (22). Following this, the necessary amount of enzymes is added to the compound. The addition of enzymes is the starting point of the process. At intervals of 1 h, 5-ml samples are taken out of the reaction container and added into the test tube and it is immediately put in the boiling vessel for 15 min to prevent any remaining enzymatic activity in the samples. After being cooled, the solid particles are separated by filter paper and the remaining solution is kept until the concentration of the produced sugar is measured. The experiments were over up to the time that the process velocity became too slow. The sugar level of the experiment samples were measured after dilution with dinitrosalicylic acid using the standard method [28, 29]. The primary experiments indicate that when the concentration of the primary substances exceeds 8-10% w/w (80-100 gIL), it may lead to encrusted mass transfer resistances and may block the enzymes from reaching the reactive spot on the outer surface of the cellular particles. Because of this, cellulose compounds at 3 levels of 60,80, and 100 gr/L concentration were used in the experiments. The enzyme was also used at 4 levels of 10, 20, 40, and 80 mlz L,
Compound Recovery Test: This experiment is in fact the continuation of the previous hydrolysis analysis and is done to absorb part of the free remaining e=ymes in the solution container. After an 8-h period, using hydrolysis based on the applied methods in the test, the hydrolysis mixture contents were separated using a filter and the solid part was discarded. The necessary quantities of cellulose compounds were added into the remaining solution and the enzymatic hydrolysis process was continued for another 8-h. This action results in significant progress in the process of cellulose enzymatic hydrolysis. This study has been done as a factorial experiment in a completely randomized design with three replications and the comparison of means was conducted by Duncan test.
RESUL TS AND DISCUSSION
Am-Euras. J. Agric. & Environ. Sci., 8 (5): 520-524, 2010
The produced sugar was different in vanous concentrations of cellulose compounds and the highest sugar level was in lOO gil. There was also a significant difference between used enzymes concentration and the highest produced sugar observed in 80 cc (Fig. 3). By passing time, the produced sugar concentration has increased, however, after 8 11, the speed of the reaction slowed and the trials inevitably stopped (Fig. 3). Although, in laboratory scales, it is possible to continue any experiment up to the desired time but time is important in practical processes and it is undesirable to continue the reaction at a low speed. Usually achievement to the highest level of final product is the goal of chemical processes. In such processes, the cost of catalyst versus the cost of primary materials, production expenses, or the cost of the final product are dispensable. However, in hydrolysis trials, the main costs are related to the cost of enzymes and the process is more economical if fewer enzymes are used. On the other hand, ethyl alcohol purification is one of the major costs of the project, so it is necessary to produce more sugar in the solution. This issue has been ignored in most of the conducted research and process efficiency has been defined as the produced glucose over the utilized primary materials. According to the results obtained by these researchers, optimum process efficiency is achieved when higher quantities of enzymes are added to the primary materials. However, due to the high costs of enzymes utilized, it is not economical. Therefore, the interaction effects of the applied treatments in the trials must be noted.
The trial results have shown that althciugh the highest level of sugar concentration has been obtained in 100 gil of cellulose compound concentration with the presence of 80 cc enzyme, there has been no significant
difference with 80 g concentration and 40 cc enzyme (Fig. 2). Even good results were achieved by using 20 cc of enzymes in a 60 gil concentration of cellulose com pounds (Fig. 1). In all utilized concentrations of the cellulose compounds, the obtained sugar reached its peak after 8 h and the speed of reaction slowed down despite of achieving the maximum sugar concentration in the presence of lOO gil cellulose compounds. However, it seems that the resulting sugars are desirable 6 or 7 h after the trial as well, and is more than the concentration of 80 g in 8 h. Provided that the time factor is considered important in such processes, it is possible to mark this result as practical.
The interactions of the utilized treatment have indicated that the maximum level of sugar is obtained after 8 h with 100 gil concentration of cellulose compounds and with the presence of 80 cc of enzymes, but the sugar produced after 7 h (even 6 h) is also acceptable. At the same concentration of cellulose compounds, when the used enzyme is halved, the acceptable sugar is produced after 8 h. At lower concentrations of cellulose compounds, using more enzymes does not result in more sugar.
To achieve a higher concentration of sugar solution and to economize using enzymes, it is necessary to utilize a higher concentration of the primary materials and to reuse the enzymes.
Several cycles of enzyme recovery were tested. The results showed that the best practical method to recover enzymes is to apply fresh primary materials. The study results have demonstrated that using a 100 gil concentration of cellulose compounds with 40 cc of enzymes (42 fpu) and conducting one recovery operation in two cycles of 8-h with the same amount of enzymes provides the best results. In other words, two 8-h cycles of hydrolysis and enzyme recovery with 200 g of
__ 20 _'_40 ____ 80
Fig. 1: The produced sugar for 60 gil cellulose compounds and the different amounts of enzyme
Am-Euras. J. Agric. & Environ. Sci., 8 (5): 520-524, 2010
Fig. 2: The produced sugar for 80 gil cellulose compounds and different amounts of enzyme 25,---------------------------------------------------,
~ 15 ~
__._ 10(ccA) ---.- 20
9 1011 12 13 14 15
Fig. 3: The produced sugar for 100 gil cellulose compounds and different amounts of enzyme
primary materials prepared (100 g per cycle) and 40 cc of enzymes provides 20 gil of sugar concentration (2%). Since it is possible to produce about 55% alcohol volume out of 1 % sugar in fermentation, it is concluded that it is possible to produce approximately 11 cc of alcohol out of every 200 g primary materials.
The authors wish to thank Mr. Zenozi, general director of Khorramshahar Oil factory for his comments and supports.
1. Movagharnejad, K., M. Sohrabi, T. Kaghazchi and F. Vahabzadeh, 2000. A model for the rate of enzymatic hydrolysis of cellulose in heterogeneous solid-liquid systems. Biochemical Engineering J., 4197-206.
2. Atimtay, A.T. andH. Topal, 2004. Co-Combustion of olive cake with lignite coal in a circulating fluidized bed. Fuel, 83: 859-867.
3. Lopez-Villatta,L.C., 1998. The olive tree, the oil, the olive. International Olive Oil Council Publications, Madrid.
4. Caputo, AC., F. Scacchia and P.M. Pelagagge, 2002.
Disposal of by-products in olive oil industry: Wasteto-Energy Solutions. Applied Thermal Engineering, 23: 197-214.
5. Mnios T., 2004. The composting potential of different organic solid waste: Experience from the island Crete. Envir International, 29: 1079-1089.
6. Teimoury, A, H. Sadeghi and Z. Anasarri, 2007.
Ruminal dry matter and nutrient degradability of different olive cake by-products after incubation in the rumen using nylon bag technique, Int; J. Agriculture and Biol., 9: 439-442.
7. Al-Widyan, M., G. Tashtoush and A.M. Ham ash, 2006. Combustion and emissions of pulverized olive cake in tube furnace. Energy Conversion and Management, 47: 1588-1596.
8. Derrich, R. andK.S. Berrahmoune, 2006. Valorization of olive oil cake by extraction of hemicelluloses. J. Food Engineering, 4: 1149-1154.
Am-Euras. J. Agric. & Environ. Sci., 8 (5): 520-524, 2010
9. Al kharnis, T.M and MM. Kablan, 1996.
A process for producing carbonaceous matter from tar, sand oil shale and olive cake. Energy, 24: 937-881.
10. Armesto, L., A Bahillo and A Cabanillas, 2003. Cocombustion of coal and olive oil industry residues in Fluidized bed. Fuel, pp: 82: 819-828.
11. Chiofalo, B., L. Liotta, A Zumbo and V. Chiofalo, 2004. Administration of olive cake for ewe feeding: effect on milk yield and composition. Small Ruminant Res., pp: 55.
12. Melloui, H.I., R. Hatman, D. Gabriels and W.M. Cornelis, 1998. The use of olive mill effluents (magrines) as soil conditioner mulch to reduce evaporation losses. Soil and Tillage Res., 49: 85-91.
13. Bacicaui, A, A Yaacoubi, A Dahbi, C. Bennouna, T.L. Phan, F.l Maldonado, 1 Rivera and C. Moreno, 2001. Optimization of conditions for the preparation of activated carbons from olive-waste cake. Carbon, 39: 425-432.
14. Al-Masri, M.R., 2001. Changes in biogas production due to different ratios of some animal and agricultural wastes. Bioresource Technol., 77: 1125-1130.
15. Abdi, N., F. Hamdache, D. Belhocine, H. Grib, D.L. Pivon and N. Mameri, 2000. Enzymatic saccharin fiction of solid residue of olive mill in batch reactor. Biochemistry Engineering, 6: 177-183.
16. Dem irbas, A and 1. Dernirbas, 2007. Importance of rural bioenergy for developing countries. Energy Conversion and Management, 48: 2386-2298.
17. Vitolos, v, L. Petarcaand B. Bresci, 1999. Treatment of olive oil industry wastes, Bioresource Technol., 67: 124-137.
18. Kabel, M.A, G. Bos, 1 Zeevalking, A Vorgen and H.A Schols, 2007. Effects of pretreatment severity on xylan solubility and enzymatic breakdown of remaining cellulose from wheat straw. Bioresource Technol., 98: 2034-2042.
19. Demirbas, A, 2007. Progress and recent trends in biofuels.Progress in Energy and Combustion Sci., 33: 1-18.
20. Gnansounou, E., A Dauriat and C.E. Wyman, 2005.
Refining sweet sorghum to ethanol and sugar: economic trade offs in the context of North China. Bioresource Technol., 96: 958-1002.
21. Gan, Q., S.l Allen and G. Taylor, 2002. Kinetic dynamics in heterogeneous enzymatic hydrolysis of cellulose: an overview and experimental study and mathematical modeling process Biochemistry, 90: 1-16.
22. Peterson, A, M.H. Thomsen, H.H. Nielsen and AB. Thomsen, 2007. Potential bioethanol and biogas production using lignocellulosic biomass from winter rye, oilseed rape and faba bean, Biomass and Bioenergy, In press.
23. Adrados, B.P., P. Choteborska, M. Galbe and G. Zacchi, 2005. Ethanol production from non-starch carbohydrates of wheat bran, Bioresouce Technol., 96: 843-850.
24. Silverstein, K.A, Y. Chen, R.R. Shivappa, MD.
Boyette and 1 Osborne, 2007. A comparison of chemical pretreatment methods for improving saccharification of cotton stalks, Bioresource Technol., 98: 3000-3011.
25. Movagharnejad, M., 2005. Modified shrinking particle model for the rate of enzymatic hydrolysis of impure cellulose waste materials enzyme reuse by the substrate replacement, Biochemical Engineering L, 24: 217-223.
26. Ingesson, H., G. Zacchi, B. Yang, A Esteghlalian and IN. Saddler, 2001. The effect of shaking regime on the rate and extent of enzymatic hydrolysis of cellulose. 1 Biotechnol., 88: 177-182.
27. Wood, T.M, 1998. Methods in Enzymology, Academic press, 160-165.
28. Miler, L.G., 1959. Use of denitrosalicylic acid reagent for determination of reducing sugar, Anal. Chern., 31: 426-428.
29. Mandels, M. andR. Andreotti, 1976. Measurement of saccharifying cellulose, Biotechnol Bioengineering System, 6: 21-33.