Optimization of Very High Gravity (VHG) Finger Millet (ragi) Medium for Ethanolic Fermentation by Yeast

116 Chiang Mai J. Sci. 2010; 37(1) Chiang Mai J. Sci. 2010; 37(1) : 116-123 www.science.cmu.ac.th/journal-science/josci.html Contributed Paper Optimization of Very High Gravity (VHG) Finger Millet (ragi) Medium for Ethanolic Fermentation by Yeast Puligundla Pradeep [a], Gangaraju K. Goud [b], and Obulam V. S. Reddy*[a] [a] Department of Biochemistry, Sri Venkateswara University, Tirupati - 517 502, India. [b] M/s. Sagar Sugars & Allied Products Ltd., Nelavoy - 517 167, Chittoor District (A.P), India. *Author for correspondence; e-mail: ovsreddy@yahoo.com Received: 29 April 2009 Accepted: 16 July 2009 ABSTRACT Very high gravity (VHG) media with finger millet (ragi) flour were prepared and fermented with ethanol tolerant yeast, Saccharomyces bayanus. Maximum ethanol concentration of 15.6% (v/v) was obtained in the nutrients supplemented medium. About 86.6% fermentation efficiency was obtained. The supplementation of peptone and yeast extract led to increase in the ethanol productivity and yeast viable cell concentration. Separate hydrolysis of the finger millet mash followed by fermentation was found to be better for ethanol production than simultaneous saccharification and fermentation. Keywords: ethanol, very high gravity medium, finger millet flour, Saccharomyces bayanus. 1. INTRODUCTION Ethanol ranks top among the renewable alternatives to the depleting crude oil reserves in today s world. Global fuel ethanol production has doubled in the past 5 years, and will continue to grow at 20% plus annually through 2010 [1]. Bioethanol can be produced from a variety of raw materials, sugar or grain (starch) at present to lignocellulosics in future. Feedstocks would crucially determine the profitability of fuel ethanol production. About 60% of the production cost is from raw material consumption like sugarcane molasses and starchy substrates, which drives the R&D of cheaper lignocellulosic biomass for ethanol production [2]. During the past few years, although significant progress has been made in lignocellulosic conversion, it is still economically problematic to replace sugar and starch materials in the near-future, even long-term [3]. Whereas, energy consumption cost is the second largest, this is about 30% of the total production cost. The very high gravity (VHG) fermentation technology emerging as promising one at present to cut down ever increasing energy costs. It allows significant increase in the final ethanol concentration, from 7-10 to 15-18 + % (v/v) or more [4]. In addition, considerable reduction in the capital costs, less process water requirement reduces distillation costs, and there by a significant decrease in overall energy costs per liter ethanol production. About 40% of world total ethanol is being produced from starchy materials [5]. Numerous starchy substrates have been

Chiang Mai J. Sci. 2010; 37(1) 117 experimented for very high gravity ethanol fermentation. Majority work has been carried out using wheat, corn, barley and oats, the predominant sources of fuel ethanol in North America and Europe. However, negligible or no attempts have been made with using starches/grains available in tropical regions. It has been shown earlier that finger millet malt [6], horse gram flour [7] and cowpea malt [8] could be used for successful VHG fermentation of the glucose medium to ethanol. This study was undertaken to evaluate the suitability of whole finger millet flour for VHG media preparation and fermentation to ethanol. 2. MATERIALS AND METHODS 2.1 Finger Millet (ragi) Finger millet was procured from local agricultural market. It was powdered in an Apex mill. Moisture content of starch flours was analysed using AOAC Method no. 925.10 (Air Oven Method) (AOAC, 2000), and was found to be 12 0.38% (dry basis). The starch content of the flour was 67.4 2.89% (dry basis). 2.2 Yeast and Enzymes Yeast, Saccharomyces bayanus, was kindly provided by Dr. Roberto Ambrosoli, university of Turin, Italy. The strain was maintained and preserved in refrigerator on conventional slants containing Wickerham (MPYD) agar [9] by periodic transfer method. It was utilized in all the experiments conducted. For liquefaction, the enzyme Biotempase L (Biocon India) was applied. It is a heat-stable -amylase preparation with an activity of 1,00,000 BAA units/g, specific gravity of 1.18 g/ml, and optimal activity in the ph range of 5.5-6.5 Amylo 300L, a mixture of glucoamylase (260 GAU/g) and pullulanase (390 ASPU/g) having specific gravity of 1.10 g/ml was used for saccharification of liquefied starch. 2.3 Preparation of Finger millet mash Finger millet (ragi) flour and water were taken at 1:2 ratio, mixed well, ph was adjusted to 6.0. Then, α-amylase in the dose range of 0.1-0.5% (v/w) was tested for its optimal concentration for liquefaction, and also the use of 1 mm CaCl 2 in starch conversion as cofactor of α-amylase. It was autoclaved for 30 min at 105-110 o C and cooled to room temperature. The % dissolved solids were noted in order to determine the optimal dose concentration of amylase enzyme. After liquefaction, the temperature was lowered to 60 o C and ph was adjusted to 4.4 using diluted HCl. Glucoamylase was added at different dosages and kept in a hot air oven at 60 o C for 24 h duration, allowed for complete sachharification of liquefied starch. After saccharification, slurry was filtered through a double layered cheese cloth to remove undissolved solids. The filtrate was used for batch fermentation studies. 2.4 Inoculum Preparation Finger millet hydrolysate containing 10% reducing sugars, 1% urea and 0.2% yeast extract were taken in different flasks and autoclaved (121 o C for 15-20 min). After cooling to room temperature, one loopful of yeast colony from YPD plates was transferred to each flask. The precultures were grown at 30 o C on a rotary shaker (130 rpm) for 24 h. 2.5 Fermentation Conditions An amount of preculture yielding a concentration of 1.5 107 yeast cells ml-1 was added to the pre-saccharified fermenting medium. The ph of fermenting medium was adjusted to 5.0 and the temperature was maintained at 30 o C. Nutrients such as yeast extract, peptone, ammonium sulphate and magnesium sulphate were added [10] into the medium in order to accelerate the growth associated ethanol yield. Fermentation was

118 Chiang Mai J. Sci. 2010; 37(1) allowed to take place for a period of 72 h. 2.6 Simultaneous Saccharification and Fermentation (SSF) All the steps in SSF process are virtually similar to that of separate hydrolysis and fermentation, except both saccharification and fermentation processes occur simultaneously in single reactor in the former process. Flour slurry was prepared by adding (1:2 ratio) hot tap water (60 o C) containing 1mM CaCl 2. ph was adjusted to 6.0 using diluted HCl and then 0.4% (v/w) thermo-stable α-amylase was added. Both gelatinization and liquefaction processes were allowed to take place in a single step by using autoclaving temperature (105-110 o C) for 20 min. After liquefaction, un-dissolved solids were removed by filtration. The mash temperature was cooled to 60 o C, ph was adjusted to 4.5. Glucoamylase at a dose of 0.5% (v/w), starch and nutrients such as peptone, yeast extract and magnesium sulphate were added. Then, allowed the enzyme to react on liquefied starch for 1 h at 60 o C. After that, the medium was cooled to 30 o C, ph adjusted to 5.0 and inoculated with Saccharomyces bayanus yeast. Saccharification along with fermentation process was allowed to occur simultaneously for 60 h. 2.7 Sampling and Analyses Sampling was done at regular intervals for different fermentation parameters. Samples were analysed for total reducing sugars (TRS) by Fehling s method [11] after enzymatic degradation of starch to simple sugars. The dissolved solids (Brix) content of the mash was measured by brix hydrometer. Specific gravity of the mashes was determined using density hydrometer. The yeast cells viability was determined by the methylene blue staining technique [12]. Ethanol in the fermented wash, and fermentation by-products such as aldehydes, methanol, total esters, total fusel oils and acetone contents, were estimated by using gas chromatography (GC) according to Anthony [13]. All analyses were conducted in triplicates and results are presented as the mean of triplicate values. Statistical analysis of data was done by using GraphPad InStat software. 3. RESULTS AND DISCUSSION 3.1 Optimal Dose of α-amylase for Liquefaction Total dissolved solids concentration was found to increase with higher enzyme dosage. Table 1 presents the formation of increased dissolved solids concentration media with increased enzyme concentration up to 0.3% (v/w), thereafter no improvement was found using still higher concentrations. One mm CaCl 2 addition was found ineffective in increasing the dissolved solids content, final brix of hydrolysate did not exceed 26.0 o Bx when 0.2% (v/w) enzyme dosage supplemented with calcium ions. The duration of autoclaving for 1 h was found to be effective for liquefaction. 3.2 Saccharification As given in the Table 1, the reducing sugar concentration increased with increasing concentration of the added glucoamylase. After 24 h saccharification period, a dose of 0.4% glucoamylase liquid enzyme yielded maximal reducing sugars, and it was used as the optimal dose for further experimentation with finger millet flour. 3.3 Batch Fermentations In this study, saccharified finger millet VHG mash having 27-28% (w/v) reducing sugars was used for batch ethanolic fermentations. After 72 h fermentation duration, significant (P<0.05) increase in the final ethanol concentration was observed in

Chiang Mai J. Sci. 2010; 37(1) 119 Table 1. Effect of different dosages of liquefying enzyme (α-amylase) on dissolved solids concentration and saccharifying enzyme (glucoamylase) on reducing sugars in finger millet (ragi) hydrolysates. Liquefaction α-amylase Brix Gluco- Reducing conditions dosage % ( o Bx) Saccharification amylase sugars % (w/v) (v/w) dosage (v/w) after 24 h ph of the 0.1 25.2 After liquefaction, 27 o Bx 0.1% 21.8 slurry was 6.0 0.2 26.0 media allowed for 24h 0.2% 23.6 and autoclaved 0.3 27.4 saccharification at 60 o C 0.3% 25.8 at 105-110 o C 0.3 + 26.8 using glucoamylase enzyme 0.4% 28.0 for 30 minutes. CaCl 2 26.4 0.5% 27.8 0.4 27.0 0.5 the nutrients supplemented media compared to the control (Table 2). Ethanol concentration as high as 15.6% (v/v) was obtained upon supplementation with increased concentrations of nutrients. The addition of glycine has shown positive effect on final ethanol yield, which is in good agreement with previously observed values [14]. About 86.6% fermentation efficiency was obtained in nutrients supplemented medium. Figure 1 shows the time dependent decrease in the residual sugars and ethanol production by yeasts. Supplementation with yeast nutrients was shown significant effect on increased final yield of ethanol and productivity. Ethanol productivity was Figure 1. Time dependent changes in the residual sugars and ethanol production profile during fermentation of high gravity finger millet mashes.

120 Chiang Mai J. Sci. 2010; 37(1) increased to 2.56 g/l/h in the nutrients supplemented medium. Nutrients, especially the addition of yeast assimilable nitrogen (Free Amino Nitrogen) sources were found to have profound influence on yeast viability and fermenting ability, thereby increasing the productivity and final ethanol yield. In media containing 120 g saccharides/l, adequate usable nitrogen must be in the 140 150 mg FAN/l range [15]. In the media of higher gravity, however, the recommended free nitrogen concentrations increase considerably [16]. So, the lower ethanol concentration in the unsupplemented medium could be due to insufficient FAN content. In addition, these nutrients provide sufficient lipids, minerals and vitamins to tolerate gravity induced stress on the yeast cells [17]. VHG media having initial specific gravities of 1.14-1.142 (30-33 o Bx) were fermented rapidly upon supplementation (Table 2). During 0 to 72 h of fermenting period, the fall in the specific gravities could be correlated with the reduction in the reducing sugars of all the fermentation media. Table 2. Influence of nutrients on final ethanol concentrations in finger millet mash fermentations (n=3). Reducing Specific gravity Residual Ethanol % Fermentation sugar % Supplements added After sugar % (v/v) efficiency % Initial (w/v) 72 h 27.5 Un-supplemented 1.140 1.078 6.4 0.6 9.1 0.1 51.1 28.0 Yeast extract-0.1%, 1.142 1.062 2.1 0.3 11.0 0.1* 60.6 Urea-0.1% and Glycine-0.1% 27.8 Peptone-0.5%, Yeast 1.140 1.044 0.7 0.1 15.6 0.1* 86.6 extract-0.3%, (NH 4 ) 2 SO 4-0.3% and MgSO 4.7H 2 O - 0.1% Data expressed as mean SD; *p < 0.05. 3.4 Yeast Growth and Viability The yeast cell count was found to increase to 4.2 10 8 cells/ml in nutrients supplemented medium from initial 1.5 10 7 cells/ml over a period of 48h. In unsupplemented medium, a cell count of 3.2 10 8 cells/ml was observed. At 48 h of fermentation time, as high as 70% cell viability was observed in the nutrients supplemented medium, whereas in unsupplemented medium not more than 39% viability was observed. This clearly indicates that the yeast cells are provided with certain nutrients in order to overcome the osmotic stress at initial and ethanol induced oxidative stress conditions at the end of fermentation. 3.5 Simultaneous Saccharification and Fermentation (SSF) Final ethanol concentration was comparatively low in SSF process (Table 3) of production than that of separate hydrolysis and fermentation (SHF), even upon supplementation. Decreased yield was due to removal of solids after liquefaction, these

Chiang Mai J. Sci. 2010; 37(1) 121 Table 3. Influence of yeast nutrients on final ethanol concentrations in SSF of finger millet (ragi) mash (n=3). Brix Nutrients added % (w/v) Residual sugar % Final Brix Ethanol % (v/v) 27-28 o Bx Urea-0.1, Yeast extract-0.1 and 1.4 0.3 12 o Bx 12.01 0.46 Magnesium sulphate-0.05 28 o Bx Peptone-0.4, Yeast extract-0.6, Mg 2+ -20mM & Glycine-40mM 0.8 0.2 10 o Bx 13.1 0.16 Data expressed as mean SD. solids would contain a fraction of starch which could be hydrolysed and converted to fermentable sugars during a long saccharification step (24 h). It may also be due to removal of osmoprotectants/nutrients such as polyphenols in the residue [18], which is evident by high final brix and low viability of yeasts at the end of fermentation. 3.6 Congeners Total fusel oils concentration as high as 470 mg/100 ml @100% alcohol formed during the fermentation of glycine supplemented medium (Table 4). Among these fusels, iso-amyl alcohol (higher alcohol) was formed in high concentration. Higher alcohols are synthesized as by-products of amino acid assimilation (the catabolic route) by yeast [19]. High concentration of amino acids favors the catabolic pathway. 4. CONCLUSIONS From the above results, it can be concluded that the very high gravity (VHG) Table 4. Congeners profile in the glycine supplemented and nutrients without glycine supplemented media. Concentration (mg/100 ml @100% alc.). Contents Glycine Nutrients without supplementation glycine Acetaldehyde 45.86 2.57 54.37 3.12 Methanol 48.34 4.32 36.96 2.65 Total esters: Ethyl acetate 9.57 1.09 2.98 0.56 Total fusel oils: n-propanol 28.32 1.45 27.77 0.98 Butanol 0.00 0.00 iso-butanol 99.81 2.23 37.55 2.22 act-amyl Alcohol 32.85 1.12 19.43 2.11 iso-amyl Alcohol 308.95 7.30 99.21 3.32 Data expressed as mean SD.

122 Chiang Mai J. Sci. 2010; 37(1) fermentations could be successfully applied for batch ethanolic fermentations using finger millet as the sole substrate. Final ethanol concentrations >15 + % (v/v) in the fermentors would have greater economic advantage over the existing batch system. From the industry point of view, further refinement of the SSF process is needed to achieve the yield above 15% (v/v) ethanol. ACKNOWLEDGEMENTS We thank University Grants Commission (UGC), New Delhi for financial assistance and thanks to Dr. S.C. Basappa, Former Deputy Director and Scientist, Central Food Technological Research Institute (CFTRI), Mysore, for his encouragement and critical comments on the manuscript. We also thank Dr. T.N. Bhavanishankar, Plant Manager, Bacardi-Martine India Limited (Nanjangud, Karnataka State) for his support in the GLC analysis. REFERENCES [1] David Zhuang, Potential and development of cellulosic ethanol. Novozymes, 2007. [2] Bai F.W., Process oscillations in continuous ethanol fermentation with Saccharomyces cerevisiae. PhD Thesis, University of Waterloo, Waterloo, Ontario, Canada, 2007. [3] Bungay H.R., Confessions of a bioenergy advocate. Trends Biotechnol., 2004; 22: 67-71. [4] Ingledew W.M., Improvements in alcohol technology through advancements in fermentation technology. Getreidetechnologie., 2005; 59(5): 308-311. [5] Sergio Trindade, Assessing the Biofuels option, IEA, Paris, June 2005: 20-21. [6] Reddy L.V.A. and Reddy O.V.S., Rapid and enhanced production of ethanol in very high gravity (VHG) sugar fermentation by Saccharomyces cerevisiae: Role of finger millet (Eleusine coracana L.) flour. Process Biochem., 2006; 41: 726-729. [7] Reddy L.V.A. and Reddy O.V.S., Improvement of ethanol production in very high gravity (VHG) fermentation by horse gram (Dolichos biflorus) flour supplementation. Lett. Appl. Microbiol., 2005; 41: 440-444. [8] Pradeep P. and Reddy O.V.S., Effect of supplementation of malted cowpea (Vigna unguiculata L.) flour in the enhancement of yeast cell viability and ethanol production in VHG fermentation. Asian Jr. of Microbiol. Biotech. Env. Sc., 2008; 10(4): 767-772. [9] Wickerham, In Technical Bulletin No, 1029, US Dept of Agriculture, Washington DC, 1951. [10] Wang F.Q., Gao C.J., Yang C.Y., and Xu P., Optimization of an ethanol production medium in very high gravity fermentation. Biotechnol. Lett., 2007; 29: 233 236. [11] Lane J.H. and Eynon L., Determination of reducing sugars by means of Fehling s solution with methylene blue as internal indicator. J. Soc. Chem. Ind. Trans., 1923: 32-36. [12] Postgate J.P., Viable counts and viability. In: Norris, J.R., Ribbons, D.W. (Eds.), Methods in Microbiology. New York Academic press, 1967: 611-28. [13] Anthony J.C., Malt beverages and malt brewing materials: Gas chromatographic determination of ethanol in beer. J. Assoc. Annal. Chem., 1984; 67: 192-193. [14] Thomas K.C. and Ingledew W.M., Fuel alcohol production: Effects of free amino nitrogen on fermentation of very high gravity wheat mashes. Appl. Environ. Microbiol., 1990: 2046-2050. [15] O Connor-Cox E.S.C., Paik J. and Ingledew W.M., Improved ethanol yields

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