KKEK 3171 Laboratory communication 2 P1E3: Fermentation

Identification and Optimization of the Important Parameters in Simultaneous Saccharification-Fermentation using Ragi Tapai
Heng Joe Shen, Aqilah Syafiqa binti Yaacob, Tan Li Xiu and Muhammad Safwan
Department of Chemical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia

Abstract Ragi Tapai is a traditional starter culture for production of alcoholic food and drinks. The main objectives of this study were to identify and optimize the important parameters in enzymatic saccharification process of cassava starch for glucose production and the growth of Ragi Tapai. The experiments were carried out by using the Box-Behnken response surface methodology (RSM) with the aid of Design Expert 6.0 in total of 17 different experiments. The experiments were carried out to determine the effect of cassava starch concentration, yeast concentration and saccharification time on the glucose yield and biomass weight. The growth medium for Ragi inoculum contained 0.05g of peptones, 0.05g of yeast extract and 0.025g of sodium chloride, and incubated at 37C, 150rpm for 30 minutes. The inoculum was then used to ferment the microwaved pre-treated substrate, starch. DNS reagents and absorbance at 560nm were used to detect the glucose concentration while Whatman grade No.1 filter paper was used to filter the sample. The samples were dried in oven at 60C for 1 day before measurement. From the fermentation, 3.870g of biomass and 5.148mg/mL of glucose concentration were obtained at the optimized conditions at 8.99 hours of saccharification, 5.17% of ragi and 4.99% of starch content. Glucose concentration was found to be relatively independent of the parameters while the biomass (fermented product in terms of cell weight) largely depended on the saccharification time and the initial Ragi content. Keywords: Ragi Tapai, starch, saccharification time, microwave pre-treatment, response surface methodology. 1. INTRODUCTION 1.1 Introduction Tapai (or tape) is a traditional fermented food found in Asia region. It is sweet, fragrant, round-shaped delicacy and quite popular especially during the Ramadan season. Tapai tastes sweet because of high sugar content while its fragrant smell comes from ethanol. The main process in the preparation of Tapai is by fermenting the flour with Ragi powder (Ko, 1972). Ragi actually comes from a type of plant known as finger millet and normally found in Africa and Asia. Scientifically, Ragi is known as Eleusine coracana (Saifuddin & Refal, 2011) and it is found to contain a lot of different types of microorganisms. Among the microbes, moulds are primary responsible for the saccharification process that produces sugar in the Tapai through its strong amylolytic activity while yeasts are capable of fermenting the glucose into ethanol (Banerjee, Debnath, & Majumdar, 1988). This process of simultaneous saccharification and fermentation has been of vast research recently. This is because enzymatic hydrolysis of the starch into glucose is an important parameter for the later bioethanol production. In Europe, ethanol is sold up to $2.4/gallon. The uniqueness of Ragi Tapai made it possible for multiple productions besides ethanol within a single fermentor. Starch is produced from grain or root crops. It is mainly used as food, but is capable of being converted into many other useful products through chemical, physical and biological process. At present, starch is used to produce such diverse products as food, paper, textiles, adhesives, beverages, confectionery, pharmaceuticals, and building materials. Cassava starch especially, has many remarkable characteristics, including high paste viscosity, high paste clarity, and high freezethaw stability, which are advantageous to many industries. Cassava starch comes from cassava / tapioca plant, which is also known as Kamoting

Kahoy. It is a small woody plant that is grown in Africa, Asia, and South America and widely available throughout Malaysia as well. Scientifically, it is known as Manihot esculenta Crantz (James, 1983). In Malaysia, Johor has the largest tapioca growing area, which is about 875 hectare and produced 19506 metric tonnes per year. There are 2 general methods to hydrolyze the starch into glucose before it can be fermented into ethanol: Acid hydrolysis (Agrawal, Pradeep, Chandraraj & Gummadi, 2005) or enzymatic hydrolysis (Kraiyot, Yaowaluk & Aran, 2007). Although the rate of production by enzymatic hydrolysis is lower, it is much more economical compared to acid hydrolysis because it occurs at milder condition, more environmental-friendly and the microbes could grow and be reused after every batch of fermentation. The current trend in this enzymatic saccharification and fermentation is the investigation for multiple productions including ethanol using microbes within a single fermentor. Cassava starch is a choice for bio-ethanol production due to its high starch content. Therefore, bio-ethanol production from cassava starch would open the market of ethanol manufacturing in Malaysia. Problem statement: To convert starch into simple sugar for fermentation requires different culture respectively in saccharification and fermentation. Important growth parameters have to be identified and eventually optimized for microbial growth and production of the desired product. 1.2 Research objectives The objectives of this research were: 1.2.1 To study the feasibility of saccharification of cassava starch using Ragi Tapai 1.2.2 To identify the important parameters in saccharification process 1.2.3 To study the effect of saccharification time, initial ragi content and amount of starch present on the saccharification process and the microbial growth 1.2.4 To optimize the important parameters for microbial growth 1.3 Research hypothesis The first hypothesis: The higher amount of starch present would lead to higher glucose production as well as the microbial growth. The second hypothesis: The microbes would continue to grow and duplicate for as long as there is sufficient nutrients for them.

2. LITERATURE REVIEW In the past, vast researches have been done intensively to break down the starch into simple monomers and subsequently utilize the monomers to produce desired fermentation products. 2.1 Substrate Starch, being the main source of energy in the human diet and animal feed, is the most abundant and universally distributed forms of storage polysaccharide in plants, and occurs as granules in the chloroplast of green leaves and amyloplast of seeds, pulses and tubers (Tester, Karkalas & Qi, 2004). Being a polysaccharide, it is mainly comprised of long chains of glucose as well as other simple monomers. In one of the study, cassava flour is reported to contain high amount of starch as shown in Table 2.1. This makes it an ideal substrate for conversion into ethanol. Table 2.1: Composition (Worawikunya, 2007) of cassava starch

2.2 Microbes Among millions of microbes, only a portion of those are capable of converting the monomers, specifically glucose into ethanol. Saccharomyces cerevisia strain is one of them that being reported to be very efficient with yield of 85.71% (Hector, etc. 2011). However, yeast itself is not capable of hydrolysing the starch provided it is amylolytic or otherwise, tedious treatment of the starch has to be done before Saccharomyces cerevisia ferment the glucose into ethanol and biomass. Coincidentally, Saccharomyces cerevisia strain has been reported in a type of plant, known as Ragi tapai (Kraiyot, 2007). The microbes detected in Ragi are moulds (Rhizopus oryzae, Amylomyces rouxii, Mucor sp. and Candida utilis) and yeasts (Saccharomyces cerevisiae, Saccharomycopsis fibuliger, Endomycopsis) (Gandjar, 2003). In another study, Ragi tapai is reported to carry out simultaneous saccharification and fermentation

(SSF) process (Azlin, 2011). Her research has yielded as high as 65.05% of bio-ethanol. These studies simplified the procedure into a single bioreactor to avoid contamination. 2.3 Glucose assays Dinitrosalicylic (DNS) Acid Reagent is reported to be capable of detecting the presence of reducing sugar in a mixture. This reagent is composed of dinitrosalicylic acid, Rochelle salt, phenol, sodium bisulfate and sodium hydroxide. Glucose and DNS undergo a reduction-oxidation reaction during DNS detection as shown in Figure 2.3. In the absence of Rochelle salt, the colour obtained after reaction is unstable. An optimum of 40% Rochelle salt solution is added into the solution after reaction. The solution obtained should stop at reddish-brown after Rochelle salt is added and absorbed strongly at 540nm (Miller, 1958).

((C12H10O10)20)5 5 (C12H10O10)20 ... (2.1) Starch Dextrin (C12H10O10)20 + H2O + O2 20 C6H12O12 ... (2.2) Dextrin Glucose The above reactions showed that -amylase hydrolyzes the straight chain bonds in large starch molecules by attacking them randomly and breaks into stable dextrin. This enables further reaction by -amylase to break it down into glucose. 2.5 Fermentation Fermentation is a form of anaerobic digestion that generates ATP by the oxidation of certain organic compounds, such as carbohydrates. Fermentation uses an endogenous, organic electron acceptor (Lansing, 2005). In yeast, fermentation is carried out by metabolizing the glucose to pyruvic acid via glycolysis. The pyruvic acid is converted to acetaldehyde and then to ethyl alcohol. Two molecules of ATP are normally produced as the result. In the process, electrons and hydrogen ions are removed from NADH. The effect is to free the NAD so it can participate in future reactions of glycolysis. The net gain to the yeast cell of two ATP molecules permits it to remain alive for some time. 2.6 Microbial growth It is generally accepted that microbial growth can be measured in mass of dry or wet weight per sample. Dry weight is the pure weight of the sample after the water is removed and it provides a more consistent result than the wet weight. In several studies, ethanol production was reported to increase with cell dry weight (Najafpour, 2004 and Nand Lal, 2009). Thus, it is an indirect indication of amount of product obtained. 2.7 Operating condition Generally, factors such as the temperature, shaking speed, initial pH value and inoculums size were found to be rather insignificant on the production on ethanol. The temperature is usually set at 37C, which is warm enough for the microbes to grow but not too hot to inhibit its enzyme activity and neither it is too cold to slow down the rate of reaction. Shaking speed is set at 150rpm to achieve a homogeneous mixture. In this study, several significant parameters had been selected. They are inoculation time, starch content and initial concentration of Ragi tapai. These parameters have great economic impact because they determine the feasibility of a pilot plant for mass production.

Figure 2.3: Reaction in glucose assay 2.4 Saccharification Saccharification is the process of breaking a complex carbohydrate (as starch or cellulose) into its monosaccharide components such as glucose, fructose or galactose (Merriam-Webster Online dictionary). In enzymatic Saccharification of starch, it utilizes the diastatic enzymes (amylases) to hydrolyze the straight chain bonds between the individual glucose molecules. As early as 1980, enzymatic hydrolysis had proved to achieve higher yields, and this led to research in the area of enzyme-based processing (Hall et al., 1956). The general enzymatic saccharification and fermentation process is given in the Figure 2.4.1 and equation (2.1) and (2.2):
Starch (lignocelluloses) Liquefaction by microwave Amorphous gel Enzymatic saccharification by mould Glucose Fermentation by yeast Ethanol and various products

Figure 2.4.1: Enzymatic saccharification of starch and the fermentation process

3. EXPERIMENTAL METHODS 3.1 Pre-fermentation 3.1.1 Preparation of nutrients medium Materials: Deionized water (DI), 1 %w/v peptone, 1 %w/v yeast extract, 0.5 %w/v NaCl & 70 %v/v Ethanol solution Apparatus: 12 x 150mL Erlenmeyer flasks, Spatulas, 3 x weighing boats, Tissue, 12 x cotton bundles (cotton and gauze), Aluminium foil, Wash bottle (DI), 1 x 10mL measuring cylinder & Bunsen burner 12 filter papers were placed into the oven. 0.05g of peptone was weighed and introduced carefully into each of the 12 Erlenmeyer flask. The sticky peptone residue was washed with 2.5mL DI water and the diluted content was then poured gently into the filled Erlenmeyer flask. The same procedure was repeated for 0.05g of yeast extract. 0.025g of NaCl was weighed and introduced carefully into the Erlenmeyer flask filled with 10mL DI water. The aluminium foil was wiped with ethanol solution. Cotton bundles were inserted and the flasks (including the control flask) were sealed with aluminium foil. *The sealed flasks were autoclaved at 121 C for 20 minutes and the content was then allowed to cool to room temperature. 3.1.2 Preparation of starch suspension for microwave pre-treatment Materials: Cotton bundles, Tapioca flour, 70 %w/v ethanol solution, DI water Apparatus: 12 x 150 mL beaker, 8 x magnetic stirrer, Glass rod, Bunsen burner, Weighing boat, Spatula, Tissue, 24 Centrifuge tubes 12 x 150 mL beakers were prepared and filled with 50 mL DI water. One magnetic stirrer was inserted into each beaker and openings of the beakers were sealed with aluminium foil. *The sealed flasks were autoclaved at 121 C for 20 minutes and the content was then allowed to cool to room temperature. Mass of tapioca flour was weighed respectively (0.5g for 1% w/v) and introduced into respective filled beakers (50 mL). After that, the flour was suspended in the water with a glass rod that had been flamed with Bunsen burner and the flask was sealed with aluminium foil immediately to reduce contamination.

The starch suspensions prepared were then subjected to microwave treatment at 600W with stirring until the temperature of the suspension reach 80C. A thermometer was used to measure the temperature. After microwave treatment, the beakers were sealed immediately with aluminium foil and allowed to cool down to room temperature. 3.2 Simultaneous Saccharification Fermentation process (SSF) 3.2.1 Incubation Materials: Aluminium foil and ethanol solution. Apparatus: Bunsen burner and incubator shaker. and

The incubator was switched on with the parameters set accordingly half an hour before the process. Squared-shaped aluminium foils and spatula were sterilized with 70 %v/v ethanol solution. Mass of ragi tapai was weighed respectively (1g for 1 %w/v) on the sterilized aluminium foil. Cotton bundles were removed and opening of the nutrient-filled Erlenmeyer flasks were flamed with a Bunsen burner. Ragi was transferred immediately into the flasks with a flamed spatula followed by closure of the opening with the cotton bundle. This step was repeated for the remaining 12 flasks. The Erlenmeyer flasks were then placed in an incubator shaker at 37C, 150 rpm for 30 minutes. 3.2.2 Inoculation The 12 inoculated flasks and control flask were placed in the incubator shaker at 37C and 150rpm. The filter papers that had been placed in the oven were weighed. Entire content of every flask were filtered at specific hour of analysis. The filtrate was agitated before collecting in centrifuge tubes. The filtrate was then centrifuged at 4 C and 3500 rpm for 20 minutes. 10ml of the supernatant was withdrawn. The remaining supernatant and solids were poured into the filter paper. The filter papers (with retentate) were dried in oven for 1 day while 10mL of the supernatant were stored at -20 C. 3.3 Analytical methods 3.3.1 Determination of glucose concentration Materials: Ultrapure water (DI), standard glucose powder, Reverse osmosis water (RO), sodium bisulfite, aluminium foil, , Rochelle salt, 70 %v/v Ethanol solution, pH paper, 1ml pipette tips, KimWipe

paper, tissue and DNS reagent (1% dinitrosalicyclic acid, 0.2% phenol, 0.05% sodium sulphide, 1% NaOH). Apparatus: 24 glass test tubes, 1ml pipette with box of tips, 5ml pipette with tip, 1000ml beaker, 500ml beaker filled with ultrapure water, a bottle of ultrapure water, cuvette and samples. The water was boiled in water bath machine. 1.5ml from each sample was pipette from the centrifuge tube into the glass test tube. 5 standard solutions of glucose were prepared respectively as shown in Table 3.4.1. Table 3.4.1: Standard glucose concentrations Final glucose Stock DI water concentration (mg/mL) (mL) (mL) 1 0.1 1.4 2 0.2 1.3 3 0.3 1.2 4 0.4 1.1 5 0.5 1 The samples were neutralized with NaOH before analysis to maintain a pH around 6-8. 2 beakers were prepared: First beaker (volume = sample x 3ml) was filled with 0.05% of sodium bisulfite and fully covered with aluminium foil while the second beaker (V1 = sample x 1ml) was filled with 40% of Rochelle salt, filled with (V1) of ultrapure water and sealed the mouth with tape. All the fluorescent light bulbs in the lab were switched off and the DNS reagent was brought out from the fridge. DNS reagent was poured (V2 = sample x 3ml) into the first beaker and was stirred with pipette. 3ml of the DNS reagent was added into each sample. The samples were boiled immediately for 5 minutes in the water bath machine. The samples were removed from the boiling water bath and Rochelle salt was added to stabilize the colour. The samples were allowed to cool to room temperature. The computer and UV-Vis spectrophotometer was turned on. VisionLite programme, method: test, mode: wavelength was entered and the wavelength was set to 540nm. The spectrophotometer was auto-zero with ultrapure water. The reading without the cuvette was maintained around -0.035A. 2.5ml of ultrapure water and 0.2ml of the sample was added into the cuvette. It was stirred with pipettes tip and tested with spectrophotometer. The absorbance was recorded for each sample.

The cuvette was cleaned with ultrapure water and wiped with KimWipe paper. Steps 4-7 were repeated until all absorbance were taken. A linear calibration graph was obtained when absorbance was plotted against standard glucose concentration. The glucose concentrations of the samples were then obtained using the formula: Glucose concentration = (Absorbance graph constant) / (gradient of the graph) 3.3.2 Determination of biomass weight Apparatus: Electronic mass balance and spatula. Each filter paper was weighed before used. The filter papers were placed in the oven at 60C. After filtered, the cell cake/paste was dried in oven until a constant weight was achieved. The dry sample was weighed again and the difference in weight was calculated. The biomass weight was determined by deducting the mass of filter paper, starch and nutrients. 3.3.3 pH measurement Apparatus: Electronic pH meter, KimWipe paper and a bottle of DI water. The pH meter was re-calibrated with the specific pH solution of 4, 7 and 9. pH meter was rinsed with DI water and dried with KimWipe paper. The sample in the centrifuge tube was shaken vigorously to ensure a homogeneous mixture. The pH of each sample was recorded. After measuring each sample, the pH meter was rinsed thoroughly with DI water to avoid contamination. 3.3.4 Optimization of glucose and concentration Apparatus: Laptop with Design Expert 6.0 software. . cell

Box-Behnken response surface methodology was applied using the software Design Expert 6.0. Range of saccharification time, starch content and ragi content was keyed into the table. A table that consists of 17 different experiments was carried out. All the data obtained from the glucose and cell concentration from the experiment was also keyed into the software.

4. RESULTS AND DISCUSSION Glucose Absorbance concentration (mg/ml) 0.00 0.0005 1.00 0.0495 2.00 0.0930 3.00 0.1430 4.00 0.1805 5.00 0.2050 Table 4.1: Absorbance of Standard glucose solutions

Absorbance against glucose concentration

0.2500

Absorbance

0.2000 0.1500 0.1000 0.0500 0.0000 0.00 1.00

y = 0.0419x + 0.0072 R = 0.9897

2.00

3.00

4.00

5.00

6.00

Glucose concentration (mg/mL) Figure 4.1: Graph of absorbance against glucose concentration

A linear graph with R-squared value of 0.9897 was obtained when absorbance at 560nm against standard glucose concentration is plotted. Glucose was observed to turn reddish brown upon boiling with DNS reagent. Rochelle salts did stop further reaction between the glucose and DNS reagent. As the concentration increased, the colour of the glucose after boiling tended to be darker. Thus, a higher absorbance was expected because lower intensity of light would be able to penetrate through the darker glucose solution. With the aid of Microsoft Excel software, a calibration graph was obtained: Absorbance = 0.0419*(Glucose concentrations) + 0.0072 Equation (4.1) Analysis of the glucose concentration in each sample can then be obtained using their absorbance: Glucose concentration = (Absorbance of sample 0.0072) / 0.0419 Equation (4.2) Table 4.2: Weight of samples obtained and glucose concentrations at various fermentation conditions. Time Ragi Starch Weight of Weight of filter Weight Absorbance Glucose (hours) (%) (%) filter paper + biomass concentration paper (g) biomass (g) (g) (mg/ml) 1 1.00 5.00 3.00 0.8311 1.8383 1.0072 0.1300 2.93 2 1.00 10.00 1.00 0.8278 1.5219 0.6941 0.1645 3.75 3 1.00 15.00 3.00 0.8221 2.5697 1.7476 0.2860 6.65 4 1.00 10.00 5.00 0.8140 2.9740 2.1600 0.2150 4.96 5 5.00 10.00 3.00 0.8398 2.1807 1.3409 0.3705 8.67 6 5.00 10.00 3.00 0.8200 1.7999 0.9799 0.3050 7.11 7 5.00 15.00 5.00 0.8240 4.1284 3.3044 0.3225 7.53 8 5.00 10.00 3.00 0.8340 3.8693 3.0353 0.2795 6.50 9 5.00 5.00 1.00 0.8097 1.2865 0.4768 0.1325 2.99 10 5.00 10.00 3.00 0.8329 2.4333 1.6004 0.1870 4.29 11 5.00 10.00 3.00 0.8217 3.4592 2.6375 0.1740 3.98 12 5.00 5.00 5.00 0.8147 3.9380 3.1233 0.1065 2.37 13 5.00 15.00 1.00 0.8258 1.6966 0.8708 0.1570 3.58 14 9.00 15.00 3.00 0.8197 2.3447 1.5250 0.3730 8.73 15 9.00 10.00 1.00 0.8175 1.6265 0.8090 0.0580 1.21 16 9.00 10.00 5.00 0.8123 4.3516 3.5393 0.3365 7.86 17 9.00 5.00 3.00 0.8292 2.8654 2.0362 0.1925 4.42 *0.2500g of ragi is required for every 5% and 0.5000g of starch is required for every 1%.

Table 4.3 gives the ANOVA results and it was found that the glucose concentration was relatively independent of the manipulated variables. This was because upon saccharification of starch by ragi, it would be eventually converted into ethanol, an important component in biofuels. In order to verify this, a simple experiment was carried out twice using 1% starch and 1% ragi for 48 hours of saccharification as shown in Figure 4.2. Optimum glucose concentration was found to be between 5 and 8 hours of saccharification. On the other hand, weight of dry biomass was found to be dependent on the saccharification time and initial amount of ragi content. This was because the microbes duplicated and grew as it fed on the starch and nutrients in the flask. The longer the period, the heavier the sample measured given similar initial amount of ragi and starch content. It was observed that from 1 to 9 hours, at 5% ragi and 3% starch, the sample increased from 1.0072g to 2.0362 which are about 2-fold. Table 4.3: ANOVA results
Glucose concentrations = +5.14882 Biomass weight = +0.10435A 0.020232A2 0.011545AB where A = Saccharification time & B = Ragi content
8.000 7.000 6.000 5.000 4.000 3.000 2.000 1.000 0.000 0 10 20 30 40

Glucose concentration (mg/ml)

Saccharification time (hours) Figure 4.2: Glucose concentration against saccharification time of ragi tapai As shown in Figure 4.3, the growth of ragi was optimum at 5% ragi content after 9 hours of saccharification. This may due to the limited amount of nutrients available in the flask. Intra-species competition was therefore higher when there was a higher amount of ragi content. At 15% ragi content, the cell concentration initially increased before decreased. The possible explanation could be that all the nutrients available in the flask were consumed. Without any nutrients available, the population of microbes eventually decreased. Microbes present in the fermentation process would produce acids as a by-product. The solution eventually turned acidic and no longer suitable for the growth of microbes. This was confirmed upon pH measurement of 1% starch and ragi after 48 hours of saccharification as shown in Figure 4.4.

Biomass weight

Figure 4.3: 3D graph of ragi content, Saccharification time and cell concentration at 3% starch content.

7 6.5 6 5.5 5 4.5 4 3.5 3 2.5 2 0 10 20 30 40 50

pH value

Saccharification time (hours) Figure 4.4: pH value against saccharification time of ragi tapai When 10 cycles per optimization and default level of duplicate solution filter were used to achieve maximum glucose and cell concentrations, different conditions were obtained for desirability of 0.863. From Table 4.4, it was found that the glucose concentration and weight of biomass was optimize at the highest amount of starch content. So, although the highest amount of starch used in the experiment is 5%, it was expected that the growth of ragi would be much higher if higher amount of starch content is used. However, this would require a longer period of saccharification time and a bigger flask was required. The viscosity of the solution would tend to increase at higher amount of starch. With lower amount of ragi, a longer period of saccharification was needed to yield a heavier amount of biomass. Thus, the optimal weight of biomass was found to be 3.870g at 8.99 hours, 5.17% of ragi and 4.99% of starch content. Desirability of 0.863 is achieved with 5.148mg/mL of glucose is produced. Despite longer period of fermentation, 8.99 hours is found to be sufficient to have optimum cell growth. Table 4.4: Optimization results from Box-behnken RSM
Number 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Saccharification Ragi time content 8.99 5.17 6.21 14.98 8.37 14.87 8.98 14.76 5.65 14.95 8.94 13.84 4.95 15.00 Starch content 4.99 4.98 4.92 5.00 5.00 5.00 5.00 Glucose concentrations 5.148 5.148 5.148 5.148 5.148 5.148 5.148 Weigh of biomass 3.870 3.640 3.768 3.860 3.561 3.561 3.448 Desirability 0.863 0.863 0.863 0.863 0.863 0.863 0.787

5. CONCLUSIONS The production of glucose from saccharification of cassava starch was possible using Ragi Tapai in a medium that contained only yeast extract, peptones, sodium chloride and starch. The important parameters identified in the saccharification process are saccharification time, initial ragi content and amount of starch present. The glucose production in the saccharification process is independent of the parameters but dry biomass weight is dependent on the saccharification time and initial ragi content. Highest glucose yield was determined to be around 5-8 hours of saccharification depending on the fermentation period, initial ragi content and amount of starch present. Using Box-Behnken response surface methodology, optimum weigh of biomass was found to be 3.870g at 5.148mg/mL, 8.99 hours, 5.17% of ragi and 4.99% of starch content. It is expected that a higher glucose production and dry biomass weight would be obtained if a higher amount of starch is used. Although this experiment was done in a small-scale, it showed possible exploration to enlarge the scale into mass production of bio-ethanol since Ragi Tapai grew well in the medium.

6. REFERENCE
Agrawal, M., Pradeep, S. Chandraraj, K. and Gummadi, S.N. 2005. Hydrolysis of starch by amylase from Bacillus sp. KCA102: a statistical approach. Process Biochemistry, 40: 2499-2507 Altaf, M.D., Naveeena, B.J., Venkateshwar, M., Kumar, E.V. & Gopal, R. (2005). Single step fermentation of starch to L(+) lactic acid by lactobacillus amylophilus GV6 in SSF using inexpensive nitrogen sources to replace peptone and yeast extract: Optimization by RSM. Department of microbiology, Osmania University, Hyderabad, Andhra Prdesh 500007, India. Azlin, S.A., Ngoh, G.C. & Maizirwan, M. (2011). Prediction of significant factors in the production of ethanol by ragi tapai co-culture using Taguchi methodology. Department of Chemical engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia. Banerjee, M., Debnath, S. & Majumdar, S.K. 1988. Production of alcohol from starch by direct fermentation. Biotechnol. Bioeng. 32 : 831-834. Gandjar, I. 2003. Tapai from cassava and cereals. Proceedings of the 1st International Symposium and Workshop on insight into the World of Indigenous Fermented Foods for Technology Development and Food Safety: 1-10, August 13-17, Kasetsart University. Hector, A.R., Daniel, P.S, Denise, S.R., Luis, F.L., Antonio, A.V. & Jose, A.T. (2011). Bioethanol production from hydrothermal pretreated wheat straw by a flocculating Saccharomyces cerevisiae strain: Effect of process conditions. Institute for Biotechnology and bioengineering, Centre of biological engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal. James A. D. 1983. Handbook of Energy Crops. Unpublished. Jianan, Z., Dehua, L., Dongming, X., Yueyun, W. & Yan Sun. (2001). Production of glycerol by fermentation using osmophilic yeast Candida krusei with different starchy substrates. Department of Chemical Engineering, Tsinghua University, Beijing 100084, China. Ko, S.D. 1972. Tape Fermentation. Applied Microbiology, Vol. 23 No. 5: 976-978. Agricultural University, Department of Food Science, Wageningen, Netherlands. Kraiyot, S., Yaowaluk, D. & Aran, H. 2007. Saccharification of cassava starch by Saccharomycopsis fibuligera YCY1 isolated from Loog-Pang (rice cake starter). Songklanakarin J. Sci. Technol, 30 (Suppl. 1), 65-71. Department of Industrial biotechnology, Faculty of agro-industry. Li, P.H., Jin, B., Paul, L. & Jiti, Z. (2004). Simultaneous saccharification and fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rihzopus arrhizus. Department of Chemical engineering, University of Queensland, St. Lucia, Qld. 4072, Australia. Linko, Y. & Javanainen, P. (1996). Simultaneous liquefaction, saccharification and lactic acid fermentation on barley starch. Laboratory of Biotechnology and Food Engineering, Department of Chemical engineering, Helsinki University of Technology, Espoo, Finland. Najafpour, G., Younesi, H. & Ismail, K.S.K. 2004. Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae. Bioresource Technology 92, 251-260. School of chemical engineering, Engineering campus, Universiti Sains Malaysia, Malaysia. Nand Lal, S., Srivastava, P. & Mishra, P.K. 2009. Studies on ethanol production using immobilized cells of Kluyveromyces thermotolerans in a packed bed reactor. Journal of Scientific & Industrial Research 68, 617-623. Department of Chemical Engineering and Technology, Institute of Technology, Banaras Hindu University, India. Saifuddin, N. & Refal, H. 2011. Microwave assisted bioethanol production from Sago starch by co-culturing of Ragi Tapai and Saccharomyces Cerevisiae. Journal of Mathematics and Statistics 7(3): 198-206. 2011 Science Publications. Shanavas, S., Padmaja, G., Moorthy, S.N., Sajeev, M.S. & Sheriff, J.T. (2010). Process optimization for bioethanol production form cassava starch using novel eco-friendly enzymes. Division of crop utilization, Central tuber Crops research institute, Thiruvananthapuram, 695 017 Kerala, India. Yuefang, D., Shuang Li, Qing Xu, Min Gao & He Huang (2011). Production of fumaric acid by simultaneous saccharification and fermentation of starchy materials with 2-deoxyglucose-resistant mutant strains of Rhizopus oryzae. State key laboratory of Material-oriented, Chemical engineering, College of Biotechnology and Pharmaceutical engineering, Nanjing University of Technology, No. 5, Xinmofan road, Nanjing 210009, PR China.

APPENDIX JOB SAFETY ANALYSIS

Job step Prepare the ethanol solution Sterilize the tip of Erlenmeyer flasks with Bunser burner Autoclave the nutrients Microwave the starch Prepare DNS reagent Boil the sample Dry the filter paper Possible hazard Pour out ethanol on the floor Burn our clothes or paper nearby The lock is not shut properly and the heated the entire room Burn the aluminium foil or plastic cover Inhale or in-contact with the toxic DNS Burn our skin upon contact with hot water Damage our eye by the heat in the high temperature microwave oven Recommendation Always wear glove and cautious when preparing Ensure that the workplace is always neat and tidy Ensure that the machine is shut properly Ensure that only materials that can be microwave to be put inside Wear glove and mask. Always be cautious not to inhale the phenol Wear thick cotton glove when handling Ensure a minimum distance from the microwave and use a handler to place the filter paper into the oven

Figure 1: Finger millet (ragi)

Figure 2: Ragi in powder form

Figure 3: Tapioca plant (roots)

Figure 4: Tapioca starch in powder form

Figure 5: Incubator shaker at biochemical laboratory

Figure 6: Preparation of Erlenmeyer flasks

Figure 7 & 8: The biomass product after dried in the oven for 1 day

Figure 9: Glucose concentration, cell density and production of ethanol in batch fermentation with initial 50 g/l glucose versus time. (Najafpour, 2004)

Figure 10: ANOVA results using Design Expert 6.0

Figure 11: 3D graph of biomass weight, ragi content and saccharification time at 3% starch content.