International Journal of Scientific Research in Knowledge (IJSRK), 1(9), pp. 343-348, 2013 Available online at http://www.ijsrpub.

com/ijsrk ISSN: 2322-4541; 2013 IJSRPUB http://dx.doi.org/10.12983/ijsrk-2013-p343-348

Full Length Research Paper Effect of Starch Granule Morphology on Production of Bioethanol from Cassava and Sweet Potato
Oluremi Isola Adeniran*, Onoja Peter Onoja
Department of Chemistry, Faculty of Science, University of Abuja, Nigeria *Corresponding Author: benadole@yahoo.com; Tel: 08027263867
Received 3 July 2013; Accepted 15 August 2013

Abstract. The need for renewable biofuels alerted by increasing global energy demand which failed to be met with fossil fuels has led to a search for suitable alternatives like bioethanol to meet this great challenge. In this study, starch was extracted separately from both cassava (Manihot esculenta Crantz) and sweet potato (Ipomea batatas Lam), hydrolysed with -amylase followed by fermentation of the hydrolysates at room temperature with bakers yeast at varying fermentation durations (between 24 and 120 h) and pH values (between 4.5 and 6.5). From the experimental results, the optimum fermentation duration is 48 h for sweet potato and 120 h for cassava, while the optimum pH is 4.5 and 6.5 for sweet potato and cassava respectively. These results show that although both duration and pH of fermentation affect the production of ethanol; their effects are not independent of the presence of readily fermentable sugars in the fermenting mash which is predicated on the granule morphology of the starch in the feedstock. Key words: Bioethanol, granule morphology, cassava, sweet potato

1. INTRODUCTION The continuous growth of world population and attempt by more countries, including Nigeria, to reach developmental targets through industrialization with a consequent skyrocketing global energy demand and rise in prices of crude oil and its fractions is one of the greatest challenges to be met by modern society. Invariably this has necessitated the development of efficient, renewable and sustainable alternative energy sources since the quantities of conventional energy sources such as fossil fuels are limited and cannot meet the increasing energy demand. Biofuels especially bioethanol is one of the promising future energy substitutes contributing to the reduction of negative environmental impacts generated by the use of fossil fuels (McMillan, 1997). It has proven to be a viable alternative to fossil fuels being renewable, efficient, degradable, cost-effective and environmentally friendly with a relatively lower conservatory gas emission rate. Goldemberg (2008) estimated that more than 70% of CO emissions and 19% of global CO2 emissions are accounted for by motor vehicles which problem could be alleviated by using biofuels. In addition, report by Kim and Dale (2005), revealed that greenhouse gas emissions can be reduce by 4161% per kilometer driven using ethanol fuel E85 (gasoline with 85% of bioethanol) in a moderate passenger vehicle compared to gasolinefueled vehicles. Likewise, a reduction in particulate and nitrogen oxides emissions from combustion has

been reported because ethanol is an oxygenated fuel containing 35% oxygen (Balat and Balat, 2009). Presently sugar cane is the most widely used crop for bioethanol in the Tropics because it provides the simple sugars, as sucrose, glucose and fructose that can be readily fermented by yeasts (Amorim et al., 2009). However, sugar cane requires a lot of water thereby making sites suitable for its cultivation very limited (and on most of such sites sugar cane plantations have already been established). Nigeria being one of the leading oil-producing countries in the world is presently facing a challenge of fast depletion of her fossil fuels. With increasing industrial demand for fuel, there is need to explore new and alternative sources of energy. Bioethanol could offer this opportunity as its production shows economic and environmental advantages over the traditional extraction of fossil fuels. Nigeria has great propensities to harness her abundantly produced root and tuber crops for bioethanol production among these are cassava and sweet potato. Her cassava production is by far the largest in the world responsible for 18% of world cassava production, about 35% more than the production in Brazil and almost double the production in Indonesia and Thailand (Tewe et al., 2003). Sweet potato on the other hand has a high agronomic yield potential realizable within a relatively short growing season as well as a wide range of ecological adaptability and shows no marked seasonality under suitable climatic conditions. Thus, the aim of this study is to compare the yield of bioethanol obtained

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from cassava and sweet potato under the same range of fermentation conditions (duration and pH) from the perspective of granule morphology; it also aims at finding suitable feedstock that can combine great potential for ethanol production with achievement of cost-effective bioethanol technologies in Nigeria. 2. MATERIALS AND METHODS 2.1. Collection of materials Freshly harvested cassava and sweet potato tubers as well as Vahine dry Bakers yeast (Saccharomyces cerevisiae) were obtained from Minna central market, Niger state, Nigeria. lpha ()-amylase enzyme obtained from Benue Breweries Limited, Markudi, Benue state, Nigeria, was used for the hydrolysis of the cassava and sweet potato starch. 2.2. Production of starch Fresh cassava and sweet potato tubers were separately washed, peeled and rinsed with water. The peeled tubers were milled and 2 kg of each weighed into a container. Distilled water was added to each milled sample, allowed to soak for 30 minutes and then washed manually in the same water so that the starch could run into solution. The starch containing water was then separated into a different container using a filter cloth. The process of washing and filtration was repeated two more times to ensure complete extraction of the starch. The water-starch suspension was allowed to stand for six hours after which the supernatant was carefully decanted to obtain white compact mass of starch at the bottom of the container. 2.3. Starch hydrolysis Enzyme hydrolysis of each of cassava and sweet potato starch produced was accomplished by gelatinizing separately with 1500 ml of distilled water and application of heat. Alpha-amylase enzyme (4 g) was dispensed to the gelatinized starch and within a few minutes the gelatinized starch was completely liquefied. The hydrolyzed starch was heated up to 100oC to stop further enzyme action and thereafter allowed to cool. 2.4. Effect of fermentation duration on ethanol yield This experiment was carried out to determine optimum fermentation duration for cassava and sweet

potato separately. The filtered hydrolysate (1500 ml) was diluted with another 1500 ml of distilled water and dispensed into five different 1000 ml Erlenmeyer flasks labeled A, B, C, D and E, each holding 600ml of the sample. To each of the flasks was added 6 g of dry bakers yeast (1% w/v) and allowed to ferment at room temperature (302 oC); the samples were then subjected to varying fermentation durations 24, 48, 72, 96 and 120 h respectively. Each set of five samples was distilled at 24 h intervals to obtain the ethanol produced in each case. The time at which maximum ethanol yield was obtained was noted for both cassava and sweet potato. The optimum duration in each case was used in subsequent experiment on pH. 2.5. Effect of pH on ethanol yield To evaluate the effect of pH on ethanol yield, 1500 ml of each hydrolysate was filtered and diluted with 1500 ml of water after which it was dispensed into five different Erlenmeyer flasks (labeled P, Q, R, S and T), each holding 600 ml of the sample. The pH of each set of five samples was adjusted by adding dilute H2SO4 and NaOH solutions in drops until the desired pH values of 4.5, 5.0, 5.5, 6.0 and 6.5 for samples P, Q, R, S and T respectively were obtained. To each of the flasks was introduced 6 g of dry bakers yeast and fermented for a duration that resulted in maximum ethanol production (48 and 120 h for potato and cassava respectively). During fermentation, the pH of each set-up was monitored at intervals for stabilization with appropriate buffer when necessary. At the expiration of the respective fermentation duration, the pH that gave highest yield of ethanol was noted. 3. RESULTS The results of fermentation duration for cassava and sweet potato are as presented in Tables 1 and 2. These results obtained on the effect of fermentation duration on the rate of ethanol yield showed that ethanol yield increased progressively throughout for cassava while for sweet potato it reduced steadily after 48 h with increase in fermentation duration. Optimum ethanol yield obtained was at 48 h for sweet potato and 120 h for cassava. Similarly, the maximum ethanol concentration achieved in the effect of pH experiments on the yield of ethanol was 2.09 % v/v at pH 4.5 and 2.18 % v/v at pH 6.5 for sweet potato and cassava respectively (Tables 3 and 4).

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Table 1: Results of fermentation duration on cassava

Duration (hours) 24 48 72 96 120 W3-W1 (g) 48.85 48.72 48.70 48.59 48.58 Sg= 0.9849 0.9823 0.9819 0.9796 0.9794 Ethanol % w/w 9.11 10.93 11.22 12.91 13.06 Ethanol % v/v 11.34 13.58 13.94 16.00 16.18 Ethanol volume (ml) 11.34 13.58 13.94 16.00 16.18 Actual yield (% v/v) 1.89 2.26 2.32 2.67 2.70

Table 2: Results of fermentation duration on sweet potato

Duration (hours) 24 48 72 96 120 W3-W1 (g) 49.01 48.94 48.96 48.97 48.99 Sg= 0.9881 0.9867 0.9871 0.9873 0.9877 Ethanol % w/w 6.95 7.88 7.61 7.48 7.21 Ethanol % v/v 8.68 9.84 9.51 9.34 9.01 Ethanol volume (ml) 8.68 9.84 9.51 9.34 9.01 Actual yield (% v/v) 1.45 1.64 1.59 1.56 1.50

Table 3: Results of fermentation pH on Cassava

pH 4.5 5.0 5.5 6.0 6.5 W3-W1 (g) 49.10 49.05 48.98 48.94 48.75 Sg= 0.9899 0.9889 0.9875 0.9867 0.9829 Ethanol % w/w 5.78 6.42 7.35 7.88 10.50 Ethanol % v/v 7.23 8.03 9.17 9.84 13.06 Ethanol volume (ml) 7.23 8.03 9.17 9.84 13.06 Actual yield (% v/v) 1.21 1.34 1.53 1.64 2.18

Table 4: Results of fermentation pH on sweet potatoes

pH 4.5 5.0 5.5 6.0 6.5 W3-W1 (g) 48.78 48.88 48.92 48.93 49.03 Sg= 0.9835 0.9855 0.9863 0.9865 0.9885 Ethanol % w/w 10.08 8.69 8.15 8.02 6.68 Ethanol % v/v 12.53 10.84 10.17 10.00 8.35 Ethanol volume (ml) 12.53 10.84 10.17 10.00 8.35 Actual yield (% v/v) 2.09 1.81 1.70 1.67 1.39

Fig. 1: Graph of effect of fermentation duration on the yield of bioethanol for cassava and sweet potato

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Adeniran and Onoja Effect of Starch Granule Morphology on Production of Bioethanol from Cassava and Sweet Potato

Fig. 2: Graph of effect of pH on the yield of bioethanol for cassava and sweet potato

4. DISCUSSION The yield of bioethanol increases progressively from 24 h up to 120 h for cassava (Table 1). However, for sweet potato, an initial increase (from 24 up to 48 h) and a subsequent decline (from 48 up to 120 h) in yield were observed (Table 2). The initial increase in alcohol yield might be due to more microbial activity owing to multiplication with consequent increase in metabolism of the sugar in the fermenting mash and concomitant production of more alcohol. After 48 hours, however, a decline in yield was observed; it might be that the nutrient present in the mash is almost depleted by the microbes thus resulting in dwindling microbial activity, dormancy or death. In addition, the observed period of fermentation could be ascribed to the presence of readily fermentable sugars and the nutrients (wort composition). According to Briggs et al. (1981) wort composition was reported to have some great influences on the speed of fermentation and the extent of fermentation. Still from the results (Tables 1 and 2), it is obvious that the yield of ethanol from cassava is higher than the yield from sweet potato and the optimum duration of fermentation differs in each case (Fig. 1). It could then be affirmed from the foregoing observations that the wort composition of cassava starch hydrolysate is higher than that of sweet potato. A possible explanation for this is the enzyme susceptibility of the starch granules to hydrolysis. Research has shown that among non-cereal starches, cassava starch has comparatively higher enzyme susceptibility than other starches (Rickard et al., 1991). Also reports by Delpeuch and Favier (1980); Hizukuri et al. (1988) revealed that sweet potato starch is less susceptible than cassava starch in that it showed stronger resistance to -amylase and glucoamylase attack. This

affirmation is further supported by the effect of starch granule size on enzymatic susceptibility; large size of starch granule has been responsible for less susceptibility of starch to -amylase hydrolysis (Franco and Ciacco, 1992). Most morphological studies of starches from root and tuber crops have reported average granule size range of 2 to 72 m for sweet potato starch granules while cassava starch granules exhibit size ranging from 4 to 43m (Wickramasinghe et al., 2009; Nwokocha et al., 2009; Aboubakar et al., 2008). In addition, the higher yield of bioethanol from cassava compared to sweet potato suggests a higher extent of hydrolysis of cassava starch than sweet potato starch. Studies have shown that hydrolysis of starches occurs in two stages; first the rapid destruction of the amorphous region characterized by a higher hydrolysis rate and secondly the slow degradation of the crystalline phase typified by a lower rate of hydrolysis (Hoover, 2000). From this it can be inferred that the amorphous regions of sweet potato starch are more compactly packed than those of cassava starch. Although the exact mechanism of pH effect on fermentation is not very clear, the difference in optimum fermentation pH (4.5 and 6.5 for sweet potato and cassava respectively) as recorded in Tables 3 and 4 could be attributed to significant differences in starch molecular structure and morphology. Researchers including Ring et al. (1988) have established that starch susceptibility to enzyme attack is influenced by several factors, such as amylose and amylopectin content, crystalline structure, particle size and the presence of enzyme inhibitors. Since different starches show varied enzyme-inhibiting actions when the reaction products of hydrolysis are not removed (Franco et al., 1988); these results indicated that the hydrolysis products obtained from sweet potato starch

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were more efficient in inhibiting enzyme action. Hence sweet potato hydrolysate required more acidic pH for optimum fermentation conditions than cassava hydrolysate (Fig. 2). 5. CONCLUSION From the analysis of the results, it was found that cassava produced a higher concentration of glucose than sweet potato. It is very clear that while the duration and pH of fermentation affect the production of ethanol in different ways, the effects are all subject to the concentration of the reducing sugars in the fermenting medium which is a function of the starch granule morphology. Ethanol from cassava starch holds great potential due to the widespread availability, abundance, and feasibility of emergence of relatively cost-effective technologies. This finding is one too important to be overlooked and information hitherto unavailable. REFERENCES Aboubakar NYN, Scher J, Mbofung CM (2008). Physicochemical, thermal properties and microstructure of six variety of taro (Colocasia esculenta L. Schott) flours and starches. Journal of Food Engineering, 86: 294-305. Amorim HV, Basso LC, Lopes ML (2009). Sugar cane juice and molasses, beet molasses and sweet sorghum: composition and usage, p 3946. In The Alcohol Text Book (5th edition), Ingledew,WM, Kelsall, DR, Austin, GD and Kluhspies, C (ed.), Nottingham University Press, Nottingham, 541p. Balat M, Balat H (2009). Recent trends in global production and utilization of bio-ethanol fuel. Appl. Energy, 86: 22732282 . Briggs DE, Hough JS, Stevens R, Young TW (1981). Malting and Brewing Science. 2nd Ed. Chapman and Hall Ltd. Vol 1. Delpeuch F, Favier JC (1980). Characteristics of starches from tropical food plants; malpha amylase hydrolysis swelling and solubility patterns. Ann. Technol. Agri., 29: 53-67.

Franco CML, Ciacco CF (1992). Factors that Affect the Enzymatic Degradation of Natural Starch Granules. Effect of the Size of the Granules. Starch/Strke, 44: 422426. Franco CML, Ciacco CF, Tavares DQ (1988). Studies on the Susceptibility of Granular Cassava and Corn Starches to Enzymatic Attack. Part 2. Study of the Granular Structure. Starch/ Strke, 40: 2932. Goldemberg, J (2008). In: Proceedings of the conference on the ecological dimensions of biofuels, Washington DC p.12 Hizukuri S, Takeda Y, Shitaozono T, Abe J, Ohtakara A, Takeda C, Suzuki A (1988).Structure and properties of water chestnut starch. Starch/Strke, 40: 165-171. Hoover R (2000). Acid-treated starches. Food Reviews International, 16: 369-392 . Kim S, Dale BE (2005). Biomass Bioenerg., 28: 475489 McMillan, JD (1997). Bioethanol production: Status and prospects. Renew Energ., 10 (2), 295-302. Nwokocha LM, Aviara, NA, Senan C, Williams PA (2009). A comparative study of some properties of cassava (Manihot esculenta, Crantz) and cocoyam (Colocasia esculenta, Linn) starches. Carbohydrate Polymers, 76 (3): 362-367. Rickard JE, Asaoka M, Blanshard JMV (1991). Review of the physicochemical properties of cassava starch. Tropical Science, 31: 189-207. Ring SG, Gee MJ, Whittam M, Orford P, Johnson IT (1988). Resistant starch: its chemical form in food stuffs and effect on digestibility in vitro.Food Chemistry. 28: 97-109. Tewe OO, Ojeniyi FE, Abu OA (2003). Sweet Potato Production, Utilization And Marketing In Nigeria. The International Potato Center (CIP) & University Of Ibadan. Wickramasinghe HAM, Takigawa S, Matsuura-Endo C, Yamauchi H, Noda T (2009). Comparative analysis of starch properties of different root and tuber crops of Sri Lanka. Food Chemistry, 112: 98-103.

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Mr Adeniran Oluremi Isola is a Lecturer in Organic Chemistry (Natural Products/Medicinal Unit), Department of Chemistry, Faculty of Science, University of Abuja, Nigeria. Mr Adeniran holds an M. Sc. Degree in Organic Chemistry from University of Ibadan in 2003. To date, he has published some scientific articles related to Natural product and Medicinal Chemistry.

Onoja Peter Onoja graduated from University of Abuja, Nigeria with a B. Sc. Degree in Industrial Chemistry. He is currently preparing for his postgraduate programme to enhance his research capacity.

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