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 37ºC, 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 60ºC 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 freeze-

thaw 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 of cassava starch

(Worawikunya, 2007)

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).

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

((C12H10O10)20)5 → 5 (C12H10O10)20 ... (2.1)

Starch Dextrin

(C12H10O10)20 + H2O + ½ O2 → 20 C6H12O12 ... (2.2)

Dextrin Glucose

The above reactions showed that α-amylase

hydrolyzes the straight chain bonds in large starch

molecules by attacking them randomly and breaks

into stable dextrin. This enables further reaction by

β-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 37ᴼC,

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.

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 80°C. 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 and

Fermentation process (SSF)

3.2.1 Incubation

Materials:

Aluminium foil and ethanol solution.

Apparatus:

Bunsen burner and incubator shaker.

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 37°C, 150 rpm for 30 minutes.

3.2.2 Inoculation

The 12 inoculated flasks and control flask were

placed in the incubator shaker at 37°C 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

concentration (mg/mL)

Stock

(mL)

DI water

(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 pipette’s 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 60°C. 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 cell

concentration

Apparatus:

Laptop with Design Expert 6.0 software. .

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.

y = 0.0419x + 0.0072 R² = 0.9897

0.0000

0.0500

0.1000

0.1500

0.2000

0.2500

0.00 1.00 2.00 3.00 4.00 5.00 6.00 A

bso

rban

ce

Glucose concentration (mg/mL)

Absorbance against glucose concentration

4. RESULTS AND DISCUSSION

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

(hours)

Ragi

(%)

Starch

(%)

Weight of

filter

paper (g)

Weight of filter

paper +

biomass (g)

Weight

biomass

(g)

Absorbance

Glucose

concentration

(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%.

Glucose

concentration

(mg/ml)

Absorbance

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

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

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.

Figure 4.3: 3D graph of ragi content,

Saccharification time and cell concentration

at 3% starch content.

0.000

1.000

2.000

3.000

4.000

5.000

6.000

7.000

8.000

0 10 20 30 40

Glu

cose

conce

ntr

atio

n

(mg/m

l)

Saccharification time (hours)

Bio

mas

s w

eigh

t

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 Saccharification

time Ragi

content Starch

content Glucose

concentrations Weigh of

biomass Desirability

1.00 8.99 5.17 4.99 5.148 3.870 0.863

2.00 6.21 14.98 4.98 5.148 3.640 0.863

3.00 8.37 14.87 4.92 5.148 3.768 0.863

4.00 8.98 14.76 5.00 5.148 3.860 0.863

5.00 5.65 14.95 5.00 5.148 3.561 0.863

6.00 8.94 13.84 5.00 5.148 3.561 0.863

7.00 4.95 15.00 5.00 5.148 3.448 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.

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

0 10 20 30 40 50

pH

val

ue

Saccharification time (hours)

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

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APPENDIX

JOB SAFETY ANALYSIS Job step Possible hazard Recommendation

Prepare the ethanol solution Pour out ethanol on the floor Always wear glove and cautious when

preparing

Sterilize the tip of Erlenmeyer

flasks with Bunser burner

Burn our clothes or paper nearby Ensure that the workplace is always neat

and tidy

Autoclave the nutrients The lock is not shut properly and the

heated the entire room

Ensure that the machine is shut properly

Microwave the starch Burn the aluminium foil or plastic

cover

Ensure that only materials that can be

microwave to be put inside

Prepare DNS reagent Inhale or in-contact with the toxic

DNS

Wear glove and mask. Always be

cautious not to inhale the phenol

Boil the sample Burn our skin upon contact with hot

water

Wear thick cotton glove when handling

Dry the filter paper Damage our eye by the heat in the

high temperature microwave oven

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.