International Journal of Science, Technology & Management Volume No.04, Issue No. 01, January 2015

www.ijstm.com ISSN (online): 2394-1537

OPTIMIZATION FOR SACCHARIFICATION OF SWEET POTATO (IPOMOEA BATATAS L) FLOUR FOR ENHANCED ETHANOL PRODUCTION Suman Jagatee1, Chinmay Pradhan2, Preeti Krishna Dash3, Santilata Sahoo4, Rama Chandra Mohanty5 1, 2,3,4,5

Division of Microbiology, Post Graduate Department of Botany, Vani Vihar, Utkal University, Bhubaneswar, Odisha, (India)

ABSTRACT Sweet potato (Ipomoea batatas L) premises an important and alternative biomass resource for fuel ethanol production by fermentation using microorganisms because of its high density of starch (17 – 60 %) in comparison to the other forms of biomass. Saccharification followed by dextrinisation, is the most important process to convert the starch present in sweet potato to simpler sugar (glucose) which further produces ethanol. Keeping this in view the present research work focuses on the optimization of process parameters (Incubation time, pH, temperature, enzyme concentration) for enzymatic hydrolysis of sweet potato using one variable at a time (OVAT) methodology. Two amylase enzymes Palkolase ® HT (an α-amylase) and Palkodex ® HT (a glucoamylase) were employed for dextrinisation and saccharification of sweet potato starch respectively. The results concluded that the optimised parameters for dextrinisation and saccharification are incubation time of 45 mins and 24 h, pH of 5.5 and 4.5, temperature of 90 ◦C and 65◦C, enzyme concentration of 20µl and 224µl respectively. The maximum sugar of 423 mg/g and 876 mg/g are released after dextrinisation and saccharification which is 26.71 % and 64.61 % more than the initial concentration respectively.

Keywords: Dextrinisation, Enzymatic Hydrolysis, OVAT Method, Saccharification, Sweet Potato I. INTRODUCTION There is a considerable interest in developing biorenewable alternatives to substitute fossil fuels such as bioethanol as transportation fuel. Bioethanol contributes to diminish petroleum dependency, generates new development opportunities in the agricultural and agro-industrial sectors, more farm work and environmental benefits. Main feedstocks for bioethanol production are sugarcane (Brazil) and corn grain (USA). Because of the increasing demand for ethanol, alternative and non-conventional raw materials are under research [1]. Sweet potato (Ipomoea batatas L) has been considered a promising substrate for alcohol fermentation since it has a higher starch yield per unit land cultivated than grains [2,3,4,5]. Industrial sweet potatoes are not intended for use as a food crop. They are bred to increase its starch content, significantly reducing its attractiveness as a food crop when compared to other conventional food cultivars (visual aspect, color, taste). Therefore, they offer potentially greater fermentable sugar yields from a sweet potato crop for industrial conversion processes and the opportunity to increase planted acreage (even on marginal lands) beyond what is in place for food. It has been reported that some industrial sweet potatoes breeding lines developed could produce ethanol yields of 4500– 6500 L/ha compared to 2800–3800 L/ha for corn [2,5]. Sweet potato has several agronomic characteristics that

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International Journal of Science, Technology & Management Volume No.04, Issue No. 01, January 2015

www.ijstm.com ISSN (online): 2394-1537

determine its wide adaptation to marginal lands such as drought resistant, high multiplication rate and low degeneration of the propagation material, short grow cycle, low illness incidence and plagues, cover rapidly the soil and therefore protect it from the erosive rains and controlling the weed problem [2]. Previous transformation of the raw material into chips or flour (powder) can be done in order to facilitate its transport and/or plant conservation. Fresh sweet potato contains high water content. The drying process of this material is an aspect to be studied to optimize its transport, storing and processing. The use of flour of sweet potato would allow working with higher sugar concentration during the fermentation than fresh sweet potato without the addition of water. In this case, it should be assessed the energy saving of manipulating lesser amount of material, the handling of high viscous material, the extra cost of drying and the effect of drying on the performance of the process (conversion of starch to fermentable sugars) [6]. The conventional process for bioethanol production from starch-based materials includes the conversion of starch into fermentable sugars which generally takes place in two enzymatic steps: liquefaction using thermostable alpha-amylase and saccharification by addition of amyloglucosidase (AMG). In dextrinisation αamylase randomly hydrolyses internal α-1, 4-glucosidic bonds in starch, liberating, soluble dextrin and oligosaccharides. During saccharification, glucoamylase hydrolyses 1, 4 and 1, 6-α linkages in liquefied starch and thereby converting the dextrinized starch to sugar [7]. Most studies of starch hydrolysis use enzymes, temperature conditions and reaction times which have been done for grains, such as corn. The starch of sweet potatoes is considered more complex than cereal starches, making it more challenging to hydrolyse into fermentable sugars. Besides, the digestibility of starch by enzymes varies among different cultivars [2, 6]. Yet there is still a need to establish a more defined biologically based approach to sweet potato starch conversion and evaluate the enzymes and processing conditions suitable for effective fermentable sugar production [2]. Keeping the above in view, the present study aims at optimizing the process parameters for hydrolysis of sweet potato root flour (SPRF) by using commercial grade enzymes, in the processes following initial dextrinisation followed by saccharification.

II. MATERIALS AND METHODS 2.1 Substrate Collection Freshly harvested sweet potato (Ipomoea batatas L) were collected from the experimental farm of Odisha University of Agriculture and Technology, Bhubaneswar, Odisha and used as substrate for our experiment during the month of December, 2013. The fresh roots were choffed manually, shade dried and finally grinded to flour by dry milling. Further the slurry was prepared by taking dried flour and water in ratio (w/v) of 1:10 at room temperature.

2.2 Reagents and Enzymes Palkolase ® - HT (a heat stable high performance α-amylase) and Palkodex ® - HT (glucoamylase i.e. high performance starch saccharification enzyme) were supplied by M/s Maps Enzymes Ltd, India. Palkolase ® - HT is a bacterial α-amylase preparation produced from the selected strain of Bacillus sp. The enzyme randomly hydrolyses 1,4-α-D-glucosidic linkages in gelatinized starch into soluble dextrins and oligosaccharides. Palkodex ® was isolated from a selected strain of Aspergillus sp. The enzyme randomly hydrolyses 1,4- and 1,6- α linkages in liquefied starch into glucose units in a step wise manner from the non reducing end of the

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International Journal of Science, Technology & Management Volume No.04, Issue No. 01, January 2015

www.ijstm.com ISSN (online): 2394-1537

molecule(Technical bulletin by M/s Maps enzymes Ltd, India). All other chemicals used for the quantification of starch, total and reducing sugar were of analytical grade.

2.3 Analytical Methods The reducing sugars, total sugars and starch were assayed by Dinitrosalicylic acid and Anthrone reagent.

2.4 Standardization of the Optimum Conditions for Dextrinisation of Sweet Potato Different optimum conditions for dextrinisation of sweet potato i.e. pH, temperature, incubation time, enzyme concentrations studied by the process OVAT methodology by analyzing the total sugar content of the substrate. 2.4.1 Incubation time- The slurry with enzyme and pH of 5.5 was incubated at temperature 90 0C with different incubation period (15 - 90mins). 2.4.2 pH- The slurry (w/v) of 1:10 with different pH (5-7.5) inoculated with enzyme was incubated at 90 0C for 45 mins. 2.4.3 Temperature- The slurry with enzyme and pH of 5.5 was incubated at different temperatures (50- 110 0C) for 45 mins. 2.4.4 Enzyme concentration- The slurry with pH 5.5 and temperature 90 0C was incubated for 45mins with different enzyme concentrations (15- 60 µl)

2.5 Standardization of the Optimum Conditions for Saccharification of Sweet Potato Further using optimum parameters of dextrinisation different optimum conditions for dextrinisation of sweet potato i.e. pH, temperature, incubation time, enzyme concentrations studied by the process OVAT methodology by analyzing the total sugar content of the substrate. 2.5.1 Incubation time- The dextrinised sweet potato slurry with the saccharifying enzyme and pH of 4.5 was incubated at temperature 650C with different incubation period (6 hr - 96hr). 2.5.2 pH- The slurry with different pH (4-5.5) inoculated with saccharifying enzyme was incubated at 65 0C for 24hr. 2.5.3 Temperature- The slurry with enzyme and pH of 4.5 was incubated at different temperatures (50- 70 οC) for 24 hr. 2.5.4 Enzyme concentration- The slurry with pH 4.5 and temperature 65 0C was incubated for 24 hr with different enzyme concentrations (100- 275 µl)

2.6 Statistical Analysis Three replicates (duplicate analysis/replicate) were maintained for each experiment and the standard deviation (S.D.) value were computed as described by Panse and Sukhatme, [8].

III. RESULTS AND DISCUSSIONS Sweet potato is a starchy crop which underway to produce higher starch content with larger roots for production of bioethanol [9]. Bioethanol production from sweet potato requires conversion of complex carbohydrates into simpler form which will be fermented by yeast strain [10]. For effective ethanol fermentation an optimization process of dextrinisation and saccharification is required to SPRF which are discussed as below.

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International Journal of Science, Technology & Management Volume No.04, Issue No. 01, January 2015

www.ijstm.com ISSN (online): 2394-1537

3.1 Optimizing Enzyme Action for Dextrinising Sweet Potato Slurry The result from the optimization of the parameters for dextrinising activity by palkolase-HT is presented in Fig 1-4. The results showed that an initial thinning of SPRF by Palkolase ®-HT facilitate hydrolysis at complex carbohydrate in sweet potato. The optimum condition for Palkolase®-HT liquefaction of SPRF were pH 5.5, temperature 90ᵒC, enzyme concentration 20 µl and incubation time 45 mins). Approximately 42 % conversion of sweet potato total sugar could be achieved at this stage. Earlier studies on the starch hydrolysis of sweet potato exhibited

that liquezyme X (Novo Industries, Denmark), a thermostable α-amylase require a higher

operating pH~7.0 and temperature of 90ºC with incubation time of 1h [11]. Further study on the enzymatic liquefication of sweet potato starch showed that Stargen TM 001 could liquefy the starch at pH 6.5, temperature of 90 ºC and 1h incubation period [12]. Shanavas et al., [13] almost obtained the same reducing sugar group (15.3 %) , when used the Spezyme Xtra enzyme concentration 20.0 mg for 10% cassava starch slurry at pH 5.5, temperature 90 ºC for 30 min as incubation period. Nikolic et al. [14] found that higher liquefaction was obtained from the corn meal at incubation temperature 85 ºC and pH ~ 6.0 for 1h of incubation period by adding 0.02 % (v/w starch) enzyme Termamyl SC.

3.2 Optimizing Enzyme Action for Saccharification for Sweet Potato Slurry Saccharification involves the conversion of maltodextrins into reducing sugars by using moderately thermostable glucoamylse. The optimum condition for the saccharifying enzyme were pH 4.5, temperature 65°C, enzyme concentration 224 µl and incubation time 24 hr). The effects of varying levels of Palkodex® on the release of total sugar formed from sweet potato are given in Figure 5-9. It was found that as high as 87 % of SPRF could be hydrolyzed by the activity of Palkodex® (224 µl) on 10 % (w v-1) SPRF slurry respectively. Similar trend of saccharification results were observed by several researches from starches and flour of different tuber crops [13, 15, 16].

Fig 1: Effect of Incubation Period on Dextrinisation of Sweet Potato for Release of Maximum Sugar

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International Journal of Science, Technology & Management Volume No.04, Issue No. 01, January 2015

www.ijstm.com ISSN (online): 2394-1537

Total sugar (mg/g of the substrate)

500

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0 5

5.5

6

6.5

7

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pH Total sugar (mg/g) Fig 2: Effect of Ph on Dextrinisation of Sweet Potato for Release of Maximum Sugar

Total sugar (mg/g of substrate)

500

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0 50

60

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Temperature (°C) Total sugar (mg/g) Fig 3: Effect of Temperature on Dextrinisation of Sweet Potato for Release of Maximum Sugar

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International Journal of Science, Technology & Management Volume No.04, Issue No. 01, January 2015

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Total sugar (mg/g of substrate)

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0 15

16

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Enzyme concentration (µl) Total sugar (mg/g)

Fig 4: Effect of Enzyme Concentration on Dextrinisation of Sweet Potato for Release of Maximum Sugar

Total sugar (mg/g of substrate)

600

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100 6

12

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Incubation period (h) Total sugar (mg/g)

Fig 5: Effect of Incubation Period on Saccharification of Sweet Potato for Release of Maximum Sugar

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Fig 6: Effect of Ph on Saccharification of Sweet Potato for Release of Maximum Sugar

Fig 7: Effect of Temperature on Saccharification of Sweet Potato for Release of Maximum Sugar

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Fig 8: Effect of Enzyme Concentration on Saccharification of Sweet Potato for Release of Maximum Sugar

Fig 9: Optimization of Enzyme Concentration on Saccharification of Sweet Potato for Release of Maximum Sugar

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International Journal of Science, Technology & Management Volume No.04, Issue No. 01, January 2015

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IV. CONCLUSIONS The present study concluded that the enzymatic hydrolysis can be a sustainable improved technology for bioethanol production. The amount of total reducing sugars in the enzyme hydrolysate sweet potato increased significantly which finally impacts a higher bioethanol concentration by fermenting microorganisms. In search of an economic way for producing ethanol from starchy substrates, the enzymatic hydrolysis of SPRF shown in the present study has been demonstrated to be an excellent alternative to commercial process for the further scale-up for production of bioethanol.

V. ACKNOWLEDGEMENT The University Grants Commission, New Delhi is gratefully acknowledged for the financial support of research to carry out this research work at P.G. Department of Botany, Utkal University, Vani Vihar [F. No. 41-478/2012 (SR)]. The authors are thankful to the M/s Maps Enzymes Ltd, Ahmedabad, India for providing the enzymes. We thank the Head, P.G. Department of Botany, Utkal University, Vani Vihar for providing laboratory facilities for this research work.

REFERENCE [1]

S. Mussatto, G. Dragone, P. Guimarães, J. Silva, and L. Carneiro, “Technological trends, global market, and

[2]

challenges of bio-ethanol production.” Biotechnology Advertisement, Vol. 28, 2010, pp.817-830.

W. H. Duvernay, M. S. Chinn, and G. C. Yencho, “Hydrolysis and fermentation of sweet potatoes for production of fermentable sugars and ethanol.” Industrial crops and products, Vol. 42, 2013, pp.527– 537.

[3]

W.S. Lee, I.C. Chen, C.H. Chang, and S.S. Yang, “Bioethanol production from sweet potato by coimmobilization of saccharolytic molds and Saccharomyces cerevisiae.” Reneweble Energy, Vol. 39, 2012, pp.216-222.

[4]

S. M.Srichuwong, X. Fujiwara, T. Wang, S. Seyama, “Simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash for the production of ethanol.” Biomass and Bioenerergy, Vol, 33, 2009,

[5]

pp. 890-898.

L. H. Ziska, G. B. Runionb, M. Tomeceka, S. A. Priorb, H. A. Torbetb, and R. Sichera,“An evaluation of cassava, sweet potato and field corn as potential carbohydrate sources for bioethanol production in Alabama and Maryland.” Biomass and Bioenergy, Vol, 33, 2009, pp.1503-1508.

[6]

S. N. Moorthy, “Physicochemical and functional properties of tropical tuber starches: A review.” Starch/Starke, Vol, 54, No, 12, 2002, pp. 559–592.

[7]

M. J. E. C.van der Maarel, B.van der Veen, and J. C. M.Uitdehaag, “Properties and applications of starchconverting enzymes of the α-amylase family.” Journal of Biotechnology, Vol, 94, No, 2, 2002, pp.137– 155

[8]

V. G. Panse, and P. V. Sukhatme, “Statistical methods for agricultural workers. New Delhi, India: Indian Council of Agricultural Research.” Record Number 19561604178. 1967.

[9]

M. Santa-Maria, K.V. Pecota, C.G. Yencho, Allen, and G. B. Sosinski, “Rapid shot regeneration in industrial „high starch‟ sweet potato (Ipomoea batatas L.) genotypes.” Plant Cell Tissue Organ Culture, Vol, 97, 2009, pp.109–17.

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International Journal of Science, Technology & Management Volume No.04, Issue No. 01, January 2015 [10]

www.ijstm.com ISSN (online): 2394-1537

S. Nikolic, L. Mojovic, M. Rakin, and D. Pejin, “Bioethanol production from corn meal by simultaneous enzymatic saccharification and fermentation with immobilized cells of Saccharomyces cerevisiae var. ellipsoideus.” Fuel, Vol, 200988, No, 9,2009, pp.1602–1607.

[11]

S. Regy Johnson, N. Moorthy, and G. Padmaja, “Optimized parameters for the enzyme catalysed liquefaction and saccharification of sweet potato starch.” Journal of Root Crops Vol 31, pp: 7-13.

[12]

S. Regy Johnson, N. Moorthy and G. Padmaja, “Enzyme kinetics in the liquefaction and saccharification of cassava starch for High Fructose Syrup (HFS) production.” In: National seminar on root and tuber crops NSRTC 1, 29-31 October, 2004 Bhubaneswar, Orissa, 2004, pp.

[13]

S. Nikolic, L. Mojovic, M. Rakin, D. Pejin, and J. Pejin, “Ultrasoundassisted production of bioethanol by simultaneous saccharification and fermentation of corn meal.” Food Chemistry, Vol, 122, 2010, pp.21-22.

[14]

S. Shanavas, G. Padmaja, S.N. Moorthy, M.S. Sajeev, and J.T. Sheriff. “Process optimization for bioethanol production from cassava starch using novel eco-friendly enzymes.” Biomass Bioenergy, Vol, 35, 2011, pp. 901-909.

[15]

S. Srichuwong, M. Arakane, M. Fujiwara, Z. Zhang, H. Takahashi, and K.Tokuyasu, “Alkali-aided enzymatic viscosity reduction of sugar beet mash for novel bioethanol production process.” Biomass Bioenergy, Vol, 34, 2010, pp.1336-1341.

[16]

G. Duan and J.K. Shetty, “ Novel enzyme for tapioca fermentation to make alcohol without high temperature cooking.” Bangkok: Pacific Ethanol and Biofuel; 2004.

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