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De Prados, M., Segu, L. Fito, P.

Instituto Universitario de Ingeniera de Alimentos para el Desarrollo (IUIAD), Universidad Politcnica de Valencia, camino de Vera s/n, C.P. 46822, Valencia, Spain. email:

INTRODUCTION At present, the transportation sector is almost entirely dependent on petroleum-based fuel, it being responsible for around 60% of the world oil consumption. Biofuels represent an alternative to petroleum-based fuel; in particular, bioethanol is the most widely used biofuel for transportation (Balat, 2010). Bioethanol can be produced from different raw materials which are commonly classified into three categories: sucrose-containing feedstocks (sugar cane, sugar beet, sweet sorghum), starch materials (corn, potatoes, wheat) and lignocellulosic materials (wood, grasses). One major problem with bioethanol is the availability of raw materials for the production, besides, the price of these raw materials is highly unstable and has a big impact on the production costs of bioethanol. Nowadays, research is mainly focused on lignocellulosic biomass, this being considered the most promising feedstock due to availability and low cost; however, an successfully effective conversion of lignocellulosic materials into bioethanol has not still been achieved, and so large-scale production of bioethanol from this source is not feasible at the moment. Nowadays, industrial bioethanol production is mainly focused on corn, wheat and sugarcane, as well as on highly abundant agricultural wastes. The use of residual biomass for bioethanol productions has the added advantage of transforming a waste material into a valorized product. Pineapple waste is a material rich in sugars and lignocellulosic components. In the present work the feasibility of obtaining ethanol from pineapple waste have been assayed, with the purpose of obtaining a valuable product from the residues of the juice and canning industries.

MATERIALS AND METHODS Golden Sweet pineapples (Annanas comosus cv. MD2) were used in the experiments. Pineapple waste (core and peel) was separated, blended and characterized in terms of total

soluble solids (TSS) by refractometry (Brix), fermentable sugars by ionic chromatography (sucrose, glucose and fructose), pH and water content (xw). The pineapple waste (batches of 9 fruits each) was pressed in pilot plant scale pneumatic equipment, which yielded a liquid phase (liquor) and a press cake. Both streams were also characterized in terms of TSS, pH and sugar content (liquor) and moisture content (cake). Three different processes were used for obtaining bioethanol form pineapple waste: direct fermentation (DF) of the extracted liquor, consecutive saccharification and fermentation (CSF) of the blended waste, and simultaneous saccharification and fermentation (SSF) of the blended waste. Three different industrial yeasts were tested (CECT: Saccharomyces bayanus 1926, Saccharomyces cerevisiae 11020, Saccharomyces cerevisiae 1319), and cellulase and hemicellulase (Sigma Aldrich, Spain) were used for the hydrolysis of the cellulosic materials (1g/kg 1.2 U/g hemicellulase and 6 g/kg 0.87 U/g cellulose).

RESULTS AND DISCUSSION Characterization of pineeaple waste is shown in Table 1, for three batches of 9 pineapples each. The mass of waste material over the total mass of the pineapples, in percentage, is shown in the lower row, from which it can be deduced that peel and core represent almost half of the fruit. Other authors (Bardiya et al., 1996) report lower values, but it is known that the amount of waste produced depends on the industrial process and the product/s obtained. Total soluble solids (10.1 1.0) are in the lower limit of the published by other authors which report values among 10.8 and 17.5 (Hulme 1971, Ban-koffi et al., 1990); however, results were considered consistent taking into account that they correspond only to the peel and core, parts that contain less soluble solids than the whole fruit with pulp, to which the other published values correspond. Table 2 shows the sugar content of the liquid phase extracted after blending and pressing the waste material. Results were in the range of the published by other authors (Nigam, 1999). It was considered that the amount of simple sugars present in the waste was sufficient for undergoing fermentation. With regard to fermentation experiments, for non-hydrolysed material best results were obtained when sterilizing the waste material, pH was adjusted to 5 and after 72 hours of fermentation; which yielded a 5% ethanol medium as an average. The evolution of the soluble solids content and alcoholic degree for thermally treated and non-thermally treated liquor is presented in figure 1. Once adopted this conditions as optimal for fermentation, statistically significant differences among the different yeasts assayed were not observed.

Saccharification with cellulose and hemicellulase was tested at 28 C (approximate optimum temperature for yeasts) and 40 C (approximate optimum temperature for the used enzymes), during 26 hours, and at pH 4, 5 and 6, in order to optimize the process with regard to the amount of fermentable sugars yielded. As expected, the higher temperature (40 C) yielded more fermentable sugars, the amount of sugars yielded during the enzymatic hydrolysis being completely stabilized after 24 hours of treatments. A 24 hours saccharification, at pH6 and 40 C was settled for the consecutive saccharification and fermentation (CSF) process. For the simultaneous saccharification and fermentation (SSF) process, the optimum temperature for yeasts (28 C) was preferred against the optimum temperature of the enzymes (40 C). CSF did not improve ethanol yield comparing to fermentation of non-hydrolysed material; however, SSF improved the yield by 12-15%. For illustration, table 3 compares some of the results obtained. First row indicates the initial amount of fermentable sugars present in the waste material; next, the sugar content after the different processes applied is indicated; and finally, the ethanol yield (gethanol/ginitial glucose) is shown.

CONCLUSIONS It was concluded that pineapple waste might be used as a low-cost material for bioethanol production, this representing the partial valorisation of pineapple industrial residues. Among the methodologies analyzed, SSF showed a significant improvement of the ethanol yield; however, in order to select one or other process, it should be considered whether this improvement is sufficient to afford the cost of the enzymes.

REFERENCES Balat, M. (2010). Production of bioethanol from lignocellulosic materials via the biochemical pathway: A review. Energy Conversion and Management, in press. doi:10.1016/j.enconman. 2010.08.013. Ban-koffi, L. and Han, Y.W. 1990. Alcohol production from pineapple waste. World Journal of Microbiology and Biotechnology, 6, 281-284. Bardiya, N., Somayaji, D. And Khanna, S. (1996). Biomethanation of banana peel and pineapple waste. Bioresource Technology, 58 (1), 73-76. Hulme, A.C. 1971. The Pineapple. In: The biochemistry of fruits and their products. Eds. London, New York, Academia Press, pp. 303-331. Nigam J.N. 1999. Continuous etanol production from pineapple cannery waste. Journal of Biotechnology. 72, 197- 202.

Table 1. Characterization of the pineapple waste (core and peel). TSS: total soluble solids.
TSS (Brix) pH Moisture (xw) Waste (%) Batch 1 10,8 1,0b 3,8 0,1a 0,84 0,01a 49,0 2,3a Batch 2 9,4 0,3 a 3,40 0,04b 0,87 0,01b 48,0 2,8a Batch 3 10,2 1,2 ab 3,58 0,03c 0,84 0,04a 48,8 4,6b Average 10,1 1,0 3,6 0,2 0,85 0,03 48,6 3,3

Table 2. Simple sugars content in the pineapple waste liquor.

Fermentable sugars (g/L) Glucose Fructose Sucrose 21,72,1 14,61,2 66,814,5

Table 3. Comparison among direct fermentation (DF), consecutive saccharification and fermentation (CSF) and simultaneous saccharification and fermentation (SSF) processes. Fermentable sugars (yielded, consumed) and ethanol yield.
S. bayanus 1926 FERMENTABLE SUGARS (g/L) ETHANOL YIELD (gethanol/gglucose) INITIAL DF 24 h sacch. CSF 72 h ferm. SFS DF CSF SSF 00 5.1 0.7 4.9 0.3 0.36 0.02 0.36 0.03 0.418 0.008 S. cerevisiae 11020 103 15 0.4 0.4 80 12 5.9 1.1 5.3 0.2 0.38 0.03 0.380 0.005 0.430 0.017 S. cerevisiae 13190 0.09 0.10 5.9 0.3 5.5 0.5 0.384 0.003 0.39 0.04 0.428 0.015

Figure 1. Evolution of the total soluble solid content (TSS) and alcoholic degree during fermentation of sterilized and non-sterilized pineapple waste liquor.