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DIRECT FERMENTATION OF SUGAR CANE SYRUP TO ETHANOL Lee Shee Yen QP 60 1 lA77 20 13 Bachelor of Science with Honours (Resource Biotechnology) 2013
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DIRECT FERMENTATION OF SUGAR CANE SYRUP TO … Fermentation of Sugar Cane Syrup... · Direct Fermentation of Sugar Cane Syrup to Ethanol Lee Shee Yen Resource Biotechnology Programme,

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Page 1: DIRECT FERMENTATION OF SUGAR CANE SYRUP TO … Fermentation of Sugar Cane Syrup... · Direct Fermentation of Sugar Cane Syrup to Ethanol Lee Shee Yen Resource Biotechnology Programme,

DIRECT FERMENTATION OF SUGAR CANE SYRUP TO ETHANOL

Lee Shee Yen

QP 601 lA77 2013

Bachelor of Science with Honours (Resource Biotechnology)

2013

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DECLARATION

I hereby declare that no portion of the work referred in this project has been submitted in

support of an application for another degree qualification of this or any other university or

institution of higher learning.

________________

(Lee Shee Yen)

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

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ACKNOWLEDGEMENTS

I would like to thank my supervisor Professor Dr. Kopli bin Bujang for his dedicated

internal review of this paper, patience and helpful suggestions throughout this project.

Also, I am gratefully thanks to my co-supervisor Assoc.Prof. Dr. Cirilo for his guidance

and provided the yeast strain.

Next, millions of thanks to postgraduate students of the Biochemistry Laboratory, Faculty

of Resource Science and Technology especially to Miss Rubena Malfia Kamal, Miss Nur

Jannah and also Miss Seha Zul for their assistance in this project.

I would also like to thank my family for their encouragement, motivation and support

during the development of this project. Last but not least, my greatest appreciation to my

course mates and friends.

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TABLE OF CONTENT

DECLARATION I

ACKNOWLEDGEMENTS II

TABLE OF CONTENT III

LIST OF ABBREVIATIONS VII

ABSTRACT VIII

1. INTRODUCTION 1

2. LITERATURE REVIEW 2

2.1. Sugar Cane 2

2.1.1. Production of Sugar Cane in Malaysia 2

2.1.2. Sugar Cane for Ethanol Production 3

2.2. Bioethanol 4

2.3. Saccharomyces cerevisiae 4

2.4. Batch fermentation 5

3. MATERIALS AND METHODS 6

3.1. Materials 6

3.1.1. Fresh Sugar Cane 6

3.1.2. Commercial glucose 6

3.1.3. Microorganism 7

3.2. Methods 7

3.2.1. Preparation and extraction of sugar cane syrup 7

3.2.2 Acid hydrolysis of SCS 10

3.2.3. Fermentation medium 10

3.2.3.1 Utilization of Baker’s yeast 10

3.2.3.2 Utilization of CSI-1 11

3.2.4 Batch fermentation 11

3.2.5. Sampling 12

3.2.6. Analytical techniques 13

3.2.6.1. Moisture Content 13

3.2.6.3 Dry cell weight determination 14

3.2.6. 4 Ethanol Concentration Determination 15

3.2.6.5 Fermentation efficiency 16

4. RESULT AND DISCUSSION 17

4.1 Characterization of fresh sugar cane 17

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4.2 Batch ethanol fermentation from sugar cane syrup (SCS) 21

4.2.1 Utilization of Baker’s yeast 21

4.2.1.1 CG fermentation 21

4.2.1.2 SCS fermentation 22

4.2.2 Utilization of CSI-1 26

4.2.2.1 CG fermentation 26

4.2.2.2 SCS fermentation I 27

4.2.2.3 SCS fermentation II 30

4.2.2.4 SCS fermentation III 31

4.3 Overall results of analyses 36

4.3.1 Dry cell weight analysis 36

4.3.1.1 Lag phase 37

4.3.1.1.1 Diauxic growth phase 37

4.3.1.2 Exponential phase 38

4.3.1.3 Deceleration growth phase 39

4.3.1.4 Stationary phase 39

4.3.1.5 Death phase 39

4.3.1.6Maintenance and survival 40

4.3.2 Batch ethanol fermentation by Baker’s yeast and CSI-1 40

5. CONCLUSION 45

6. REFERENCES 46

Appendix A 51

Appendix B 52

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LIST OF FIGURES

Figure 2.1 Sugar cane plantation in Malaysia. 2

Figure 3.1 Fresh sugar cane of different species and maturity 6

Figure 3.2 Mauri-pan dry instant yeast (Saccharomyces cerevisiae) is used as

microorganism for fermentation process. 7

Figure 3.3 Preparation and extraction of SCS. 8

Figure 3.4 Dark colour of SCS after autoclaved. 9

Figure 3.5 De-skinned sugar cane for moisture content analysis and products of sugar cane

extraction by household blender. 9

Figure 3.6 2 L labscale benchtop fermentor with 1L working volume. 13

Figure 3.7 Chopped sugar cane pith for moisture content analysis. 13

Figure 3.8 DNS method for reducing sugar analysis of SCS. 14

Figure 3.9 Cell pellet of yeasts obtained after centrifugation of samples to be used for dry

cell weight determination. 15

Figure 3.10 Purification of SCS using vacuum pump for reducing sugar and ethanol

analyse. 17

Figure 4.1 Ethanol fermentation of CG. 22

Figure 4.2 Ethanol fermentation of SCS. 23

Figure 4.3 Ethanol yield comparison of CG and SCS. 24

Figure 4.4 Ethanol fermentation of CG. 27

Figure 4.5 Ethanol fermentation of SCS. 29

Figure 4.6Ethanol fermentation of SCS II (after 1 week storage period). 31

Figure 4.7 Ethanol fermentation of SCS III. 33

Figure 4.8 Ethanol yield (%) for CG, SCS II and SCS III. 33

Figure A1 Standard curve of sucrose. 51

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LIST OF TABLES

Table 4.1 Analysis sample of CG fermentation. 21

Table 4.2 Analysis sample of SCS fermentation. 23

Table 4.3 Reducing sugar consumption of CG and SCS fermentation. 25

Table 4.4 Ethanol concentration of CG and SCS fermentation. 25

Table 4.5 Analysis of sample of CG. 26

Table 4.6 Analysis of sample of SCS. 28

Table 4.7 Analysis of sample of SCS II (SCS kept for 1 week). 30

Table 4.8 Analysis sample of SCS fermentation III. 32

Table 4.9 Reducing sugar consumption of CG and SCS fermentation. 34

Table 4.10 Ethanol production over time for CG and SCS. 35

Table 4.11 DCW analysis in exponential phase. 38

Table 4.12 Comparison of Baker's yeast fermentation with previous study. 41

Table 4.13 Comparison of CSI-1 fermentation with previous study. 42

Table A1 OD reading on sucrose 51

Table A2 Reducing sugar determination of SCS sample 1 52

Table A3 Reducing sugar determination of SCS sample 2 53

Table A4 Reducing sugar determination of GSC sample 3 53

Table A5 Reducing sugar determination of SCS III sample 54

Table A6 Juice recovery of GSC from raw material using juice extractor 54

Table A7 Juice recovery of GSC from raw material using household blender 55

Table A8 Green SCS moisture content 55

Table A9 Red SCS moisture content 55

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VII

LIST OF ABBREVIATIONS

oC Degree Celsius

DCW Dry cell weight

DNS Dinitrosalicylic acid

FE Fermentation Efficiency

H2O Water

KI Potassium Iodide

OD Optical density

g Gram

g/L Gram per litre

cm centimeter

kg Kilogram

mL Milliliters

L Liters

rpm Revolution per minute

µl Microlite

w/v weight per volume

SCS Sugarcane syrup

CG Commercial glucose

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Direct Fermentation of Sugar Cane Syrup to Ethanol

Lee Shee Yen

Resource Biotechnology Programme, Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Ethanol production from pure sugar cane syrup (SCS) were carried out to compare the efficiency of

batch fermentation with commercial glucose using Saccharomyces cerevisiae baker’s yeast and

Saccharomyces cerevisiae CSI-1 (JCM 15097). Fermentation of Baker’s yeast and CSI-1 in SCS

medium supplemented with 0.5% (w/v) of yeast extract at 37oC, initial pH adjusted to 5.5, agitation

200 rpm, produced high concentration of ethanol in 1.5L lab scale fermentor. Ethanol production

by dry Bakers’s reached 138g/L and yield 156.9% of theoretical yield. For CSI-1, a maximal

concentration of 73.3g/L and a yield of 88.9% of theoretical yield were obtained from fresh SCS.

The result from this research demonstrate that in Malaysia, sugar cane syrup could be employed as

an alternative renewable carbon source for ethanol production using batch fermentation with

Saccaharomyces cerivisiae.

Key words: Ethanol, fermentation, Saccharomyces cerevisiae, sugar cane syrup.

ABSTRAK

Penelitian telah dijalankan untuk berbanding effisensi fermentasi etanol antara glukosa(CG) dan

sirap air tebu (SCS) dengan menggunakan Saccharomyces cerevisiae Baker’s yeast dan

Saccharomyces cerevisiae CSI-1 (JCM 15097). Fermentasi Baker’s Yeast dan CSI-1 di dalam

media yang disuplimen dengan 0.5% (w/v) yis extrak, suhu 37oC, pH asal ditentukan pada

5.5,pegadukan 200rpm berjaya menghasilkan kadar etanol yang maksima di dalam 1.5L fermentor

skala lab.Kadar maksima etanol daripada fermentasi SCS oleh Baker’s yeast mencapai 138g/L

dengan fermentasi efisensi 156.9% manakala CSI-1 mencapai kadar maksima etanol 73.3g/L

dengan fermentasi efisensi 88.9%. Penelitian ini membahas SCS sesuai digunakan sebagai satu

sumber substrak untuk fermentasi etanol dengan menggunakan Saccaharomyces cerivisiae.

Kata Kunci: etnanol, fermentasi, Saccharomyces cerevisiae , sirap gula tebu.

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1. INTRODUCTION

Several renewable substrates are under investigation as feedstock for bioconversion to

fuel ethanol. In temperate climates, grain starch is the basic raw material for

fermentation ethanol due to its avaibility and low cost. In tropical country such as

Malaysia, sugar cane juice can be the sources of sugar for fermentation into ethanol.

Sugar cane is a highly productive land plant that uses the C4 pathway of

photosynthesis, which confers higher potential light, water and nitrogen use

efficiencies, than the alternative C3 pathway (De Souza & Buckeridge, 2010). In

general, the ethanol fermentation of these saccharine materials is much simpler than the

fermentation of grain starch (Hodge and Hildebrand, 1954; Maiorella, 1985). Unlike

grain starch, in which starch has to be broken down into sugars with expensive

enzymes before it can be fermented, the entire sugar cane stalk is already contain high

sugar and it starts to ferment almost as soon as it’s cut . Currently in Brazil, sugar cane

worts of 16%w/v to 20% w/v are routinely fermented to produce 7.5 to 10.0 % (w/v)

ethanol (Laluce, 1991).

This project studied the use of sugar cane syrup for ethanol production by Mauri-

pan dried instant yeast and Saccharomyces cerevisiae CSI-1 (JCM 15097) using a non-

aerated bench-top fermentor. Comparative studies were performed between 2 strains of

Saccharomyces cerevisiae grown on 15% to 19% (w/v) of either commercial glucose

(CG) or sugar cane syrup (SCS) with the initial pH set at 5.5 and uncontrolled

throughout the experiment. The objectives of this study were to quantify the production

of ethanol from sugar cane syrup under batch fermentation using Baker’s yeast and

CSI-1 and to determine the feasibility of planting sugar cane locally to reduce import of

the raw material.

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2. LITERATURE REVIEW

2.1. Sugar Cane

2.1.1. Production of Sugar Cane in Malaysia

The cultivation of sugar cane (Saccharum officinarum) in Malaysia is small. Figures 2.1

and 2.2 illustrate the sugar cane plantation in Chuping, Perlis. According to the study done

by Food and Agriculture Organization of the United Nations (1997), production is

concentrated in the Northwest extremity of peninsular Malaysia in the states of Perlis and

Kedah because these areas have a distinct dry season needed for cost-efficient sugar cane

production. The study also revealed that plantings in the states of Perak and Negeri

Sembilan were unsuccessful due to high unit costs as producing conditions were less

suitable. Areas for potential expansion have been identified in the state of Johor and in

Sarawak, but no projects have yet been undertaken. Most of the cane areas are under the

management of three sugar cane plantations, two in the State of Perlis and one in the state

of Kedah, with smallholders contributing only about 15 percent of the total. The lack of

growth in cane areas largely reflects the higher remuneration received by farmers for other

crops, especially oil palm.

Figure 2.1 Sugar cane plantations in Malaysia. (A) Sugar cane plantation. (B) Sugar Cane Plantation in

Chuping, Perlis.

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2.1.2. Sugar Cane for Ethanol Production

Sugar cane is so far the world’s largest source and most efficient raw material for

bioethanol production because the consumption of fossil energy during sugar cane

processing is much smaller compared to others common raw materials (Macedo et al.,

2008). Another advantage of sugar cane is that the by-product of sugar cane production or

bagasse can also be utilized for producing of steam and electricity required for the cane

processing plant as well as an ideal substrate for bioconversion to ethanol (Martin et al.,

2002). Furthermore, optimization of bioethanol production process from sugar cane is still

possible, and significant reduction of energy consumption can be achieved. Sugar cane is a

high biomass tropical crop (Limtong et al., 2007) .It contains 12 to 17% total sugars, of

which 90% is sucrose and 10% is glucose and/or fructose (Wheals et al., 1999). The cane

plant takes up moisture and nutrients from the soil; the chlorophyll in the plant uses

sunlight to manufacture sucrose, which is stored in the stalk. Therefore, a load of cane

stalks transported to the factory contains not only sucrose but also all the other materials

that make up this complex natural material. Study done by Limtong et al. (2007) revealed

that sugar cane juice normally has sufficient organic nutrients and minerals that make it

suitable for ethanol production by fermentation with yeast (Saccharomyces cerevisiae).

Sugar cane yields 600 to 800 gallons of ethanol an acre, and it starts to ferment almost as

soon as it’s cut (McKibben, 2007).Brazil is the world largest fuel ethanol producer using

sugar cane juice (Moreira, 2000) and/or molasses as substrates (Moreira,2000; Wheals et

al., 1999). Goldemberg (2007) also reported that Brazil has been using sugar cane as raw

material for large scale bioethanol production for more than 30 years. Besides Brazil,

Thailand is another tropical country where sugar cane is cultivated in large scale for the

sugar production (Limtong et al., 2007).

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2.2. Bioethanol

Bioethanol is an energy saving fuel which is readily available for combustion engines

made from biomass. Production of bioethanol lowers both consumption of crude oil and

greenhouse gas emissions from transport and traffic because it only releases the same

amount of carbon dioxide as plants bound while growing. Because of its high octane

number, low octane number and high heat of vaporization impede self-ignition in the diesel

engine; it is suitable for the mixed fuel in the gasoline engine (Balat et al., 2008).

Therefore, ignition improver, glow-plug, surface ignition, and pilot injection are applied to

promote self-ignition by using diesel-bioethanol-blended fuel. Disadvantages of bioethanol

include its lower energy density than gasoline, its corrosiveness, low flame luminosity,

lower vapor pressure (making cold starts difficult), miscibility with water, and toxicity to

ecosystems (Balat et al., 2008).

Bioethanol is produced by a biological fermentation process in which organic

material is converted by microorganisms to simpler compounds, such as sugars. These

fermentable compounds are then fermented by microorganisms to produce ethanol and

CO2. Besides, enzyme also produced by the microorganisms to catalyze chemical reactions

that hydrolyze the complicate substrates into simpler compounds for carbon source.

Several strains of microorganisms, bacteria, yeasts, and fungi have been reportedly used

for the production of ethanol (Lin &Tanaka, 2006).

2.3. Saccharomyces cerevisiae

Historically, the most preferred used microbe for most ethanol fermentation is the

Saccharomyces cerevisiae, which is capable of very rapid rates of glycolysis and ethanol

production under optimal condition to give concentration as high as 18% of the

fermentation broth (Lin &Tanaka, 2006). It produces over 50mmol of ethanol per g of cell

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protein (Dombek & Ingram, 1987). This yeast can grow both on simple sugars, such as

glucose, and on the disaccharide sucrose .It is also ideal for producing alcoholic beverages

and for leavening bread since it is recognized safe as a food additive for human

consumption (Lin & Tanaka, 2006).It has the ability to cause the flocculants to adhere

carbon dioxide and rise at the top of the fermentation vessel. . CSI-1 (JCM 15097) was

able to produce ethanol at high concentration and high rate.

2.4. Batch fermentation

Batch fermentation is considered a closed system. In batch fermentation, substrate and

yeast culture are charged into the bioreactor together with nutrients (Hassan, 2008). At the

time of zero, the sterilized nutrient solution in the fermentor is inoculated with

microorganism and incubation is allowed to proceed under optimal physiological

conditions. During the entire fermentation process, nothing is added except oxygen (in a

form of air), an antifoam, and acid or base to control the pH. Most of the ethanol produced

today is done by the batch fermentation as the investment costs are low, do not require

much control and can be accomplished with unskilled labour (Belkis & Sukan, 1998).

Complete sterilization and management of feedstock are easier than in the other processes.

The other advantage of batch operation is the greater flexibility that can be achieved by

using a bioreactor for various product specifications. The disadvantages are

microorganisms are either slow growing or strongly affected by product inhibition

(Najafpour et al., 2004).

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3. MATERIALS AND METHODS

3.1. Materials

3.1.1. Fresh Sugar Cane

The fresh sugar cane (Figure 3.1) was obtained from different locations and different

vendors for each SCS fermentation.

Figure 1.1Fresh sugar cane of different species and maturity. (A) Variety species of sugar cane. (B) Fibre of

sugar cane. (C) Variety species and maturity.

3.1.2. Commercial glucose

The commercial glucose that used as control was obtained from laboratory of Faculty of

Resource Science and Technology.

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3.1.3. Microorganism

Baker’s yeast (Saccharomyces cerevisiae) (Figure 3.2) was obtained from local market

whereas CSI-1 was obtained from laboratory of Faculty of Resource Science and

Technology.

Figure 3.2 Mauri-pan dry instant yeast (Saccharomyces cerevisiae) is the microorganism used for

fermentation process.

3.2. Methods

For all experiments and analysis, an average of three trials was used as analytical data. The

ethanol production from fermentation process of the yeast using CG will also be conducted

as the control treatment.

3.2.1. Preparation and extraction of sugar cane syrup

Approximate 3 to 5kg weight of fresh sugar cane was used for this research. Sugar cane

was washed, peeled and extracted using juice extractor as shown in Figures 3.3. The SCS

was filtered using double fold cheesecloth. The SCS obtained was secondary filtered using

filter paper to remove the fibre. Next, the SCS was hydrolysed by concentrated

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hydrochloric acid for the analysis of reducing sugar concentration using DNS. Percentage

of dry matter will also be calculated from the sugar cane bagasse collected from after SCS

extraction.

Figure 3.3 Preparation and extraction of SCS. (A) Extraction of SCS by juice extractor.(B) Filtration of SCS

to remove fibre.(C) Filtration of SCS by cheesecloth.(D) Sugar cane bagasse after SCS extraction.

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Figure 3.4 shows the darken in colour of SCS after autoclaved.

Figure 3.4 Dark colour of SCS after autoclaved.

Figure 3.5(A) shows the de-skinned sugar cane for moisture content analysis and Figure

3.5(B) & (C) show the products of extraction of SCS in laboratory using household food

processor.

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Figure 3.5 (A) De-skinned sugar cane showing the white and greenish pith. (B) Filtration of SCS upon

blending. (C) Fibre of sugar cane after blending of the pith.

3.2.2 Acid hydrolysis of SCS

A separate set of the samples containing SCS was chemically digested in triplicate by

adding 3µl concentrated hydrochloric acid (HCl) to 450µl of each sample. These samples

were placed in a 90oC hot water bath for 15 minutes, removed, and left to sit at room

temperature for another 15 minutes. Approximately 40µl of sodium bicarbonate was added

in 10µl portions to each sample in order to neutralize the acid. The pH was checked to

ensure a pH of 7. Water was then added to bring each sample to the designated volume,

900µL. Small quantities of sample was taken for reducing sugar analysis using DNS

method. (Miloski et al., 2008).

3.2.3. Fermentation medium

3.2.3.1 Utilization of Baker’s yeast

The working medium in the 1L non-aerated bench-top fermentor consisted of 1 L pure

SCS, 5g/L of yeast extract and 10g/L of Mauri-pan dry instant yeast (Saccharomyces

cerevisiae) . The initial pH was adjusted to 5.5 with 1M NaOH before autoclaving.

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3.2.3.2 Utilization of CSI-1

CSI-1 was first propagated at 30oC for 24 h without agitation by transferring 1ml of stock

culture to 10ml of liquid medium contained 5g/L of yeast extract and 20g/L of glucose.

The starter culture was then transferred to 200mL of seed culture medium and was grown

in incubator shaker under non-aerated conditions at 30oC for 8 h. Cultures were centrifuged

at 3000rpm for 5min at 27o

C and harvested as inoculum for fermentation process. The

working medium in the 1L non-aerated bench-top fermentor consisted of 1 L of pure SCS,

5g/L of yeast extract and 10% w/v of CSI-1 inoculum. The initial pH was adjusted to 5.5

with 1M NaOH before autoclaving.

3.2.4 Batch fermentation

The medium was autoclaved at 121oC at 15 psi for 15 minutes. Upon cooling, yeast was

added in batch fermentation study. Pure SCS was fermented by yeast to produce ethanol

with basic parameters of temperature 37oC and agitation rate of 200 rpm in order to

maintain homogenous culture. The initial pH was set at pH 5.5 but not controlled

throughout the fermentation process. The reducing sugar, ethanol and DCW measurement

was determined until the depletion of reducing sugar in medium. Figures 3.6 shows the

setting up of bench top fermentor for fermentation process.

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Figure 3.6 (A) The 2 L labscale benchtop fermentor with 1L working volume. (B) The 2 L labscale benchtop

fermentor with 1L working volume. (C) Bubbles indicated CO2 produced during fermentation.

3.2.5. Sampling

An aliquot of 20ml of the sample was obtained at every 6 hours for at least 36 hours

manually and stored at 4oC for subsequent analysis. Sampling was performed with strict

aseptic technique to avoid risk of contamination.

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3.2.6. Analytical techniques

3.2.6.1. Moisture Content

Thin slices of fresh sugar cane was weighed and dried at 60oC for 24 hours (Figures 3.7),

then cooled in dessicator and weighed again. The process of drying, cooling and weighing

is repeated every 24 hours until there is no weight different of the sample. The moisture

content was calculated using formula as the following.

Moisture content

w1-w2 X 100%

w1-w

w = empty crucible

w1 = weight of sample + crucible

w2 = weight of sample + crucible after drying

Figure 3.7 Chopped sugar cane piths for moisture content analysis. 2 Freshly chopped sugar cane pith. (B)

Dried sugar cane pith upon drying in oven for 24 hours at 60oC.

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3.2.6.2. Reducing Sugar Analysis

3ml of DNS reagent was added to 3ml of the test solution and heated in the boiling water

for 15 minutes. Then, 1 ml of 40% Rochelle salts was added to stabilize the colour formed.

The test tubes were cooled under running water before measuring it in a spectrophotometer

at 575nm (Figure 3.8). A standard curve was plotted using sucrose as standards to read off

sucrose equivalent values. (Miller et al., 1960 & Miloski et al., 2008)

3.2.6.3 Dry cell weight determination

a) 10 ml of sample were centrifuged at 7000rpm, 4oc for 10 minutes. Supernatant was

discarded. Cell pellet was suspended with distilled water and centrifuged again.

Cell pellet kept in the oven for dry cell weight determination (Figures 3.9).

Using the formula ,

DCW (g/L) = (Wt. Of dried tube + cells)g-(Wt. Of dried tube)g X 103

Sample volume (mL)

Figure 3.8 Reducing sugar was heating with 3,5 dinitrosalyclic acid which produce a red brown product.

Concentration of coloured complex was determined with spectrophotometer at absorbance 575nm.

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Figure 3.9 Cell pellets obtained after centrifugation of samples to be used for dry cell weight determination.

(A) Cell pellet of yeasts before drying in oven. (B)Dried yeast cells.

3.2.6. 4 Ethanol Concentration Determination

Dilution was carried out to dilute the samples to 10 times by adding 9ml of distilled water

to 1 ml of ethanol. Then 30µl aliquot of samples was injected into Biosensor and the

reading was taken. Samples were filtered using vacumm pump to remove the yeast cell

(Figure 3.10).

Figure 3.10 Purification of SCS using vacuum pump for reducing sugar and ethanol analyse.