Report Biorec Lab 1

UNIVERSITI TEKNOLOGI MARA FAKULTI KEJURUTERAAN KIMIA BIOPROCESS ENGINEERING LABORATORY (CBE 661) NAME AND MATRIC NO

: MUHAMMAD ARSHAD BIN ABDUL RASHID (2014683386)

GROUP

: 5

EXPERIMENT

: LAB 1 (GROWTH KINETICS STUDY OF MICROORGANISM)

DATE PERFORMED

: 15 SEPTEMBER 2015

SEMESTER

: 5

PROGRAMME CODE

: EH242 5E

SUBMIT TO

: PN SUHAILA BT MOHD SAUID

No. 1. 2. 3. 4. 5. 6 7. 8. 9. 10. 11. 12. 13.

Title Abstract/Summary Introduction Aims Theory Apparatus Methodology/Procedure Results Calculations Discussion Conclusion Recommendations Reference Appendices TOTAL

Allocated Marks 5 5 5 5 5 10 10 10 20 10 5 5 5 100

Marks

Remarks: Checked by:

Rechecked by:

…………………………….........

……………………………..

Date:

Date: 1

Table of Contents

NO.

TITLE

PAGES

1

Abstract

3

2

Introduction

4

3

Objectives

5

4

Theory

5

5

Apparatus

8

6

Methodology/Procedure

8

7

Results

11

8

Calculations

17

9

Discussion

17

10

Conclusion

20

11

Recommendations

21

12

Reference

21

13

Appendices

22

2

Abstract In this experiment the study is for the growth kinetics of microorganisms in shake flask. E.coli is grown in a LB and TB broth mediums and being fermented for 24 hours at 350 rpm and 370C. Throughout the fermentation, the cell culture is taken out every 1 hours from 0 hour to 4th hour and continued with every 2 hour until 20th hour and the absorbance analysis, glucose analysis and cell dry weight are being performed. As for the optical density analysis, the absorbance reading from the spectrophotometer is taken while for the glucose test, the reading of glucose level is taken from the YSI 2700 Select Biochemical Analyzer. These substrate concentration values will then being plotted on graph with growth rate. The cell dry weight, in the other hand, is taken after the mass concentration is being dried overnight in the oven. The weight of the tube which contains the biomass before and after the drying process is recorded to get the cell dry weight. For the optical density of the cell, the absorbance value showed an increment which indicating that the cell was growing and number of cell is increased in the shake flask. The growth rate, however, cannot be determined as the absorbance values were increased and decreased unevenly and calculation of rate of microbial growth cannot be made as the data for cell dry weight concentration, X are not consistent giving some of the results negative value thus some of the rate of growth, μ and substrate constant, Ks cannot be calculated. Supposedly, as the number of cell increased inside the shake flask, the cell dry weight also should be increased. Therefore no conclusion can be made about the cell growth rate in the shake flask. Another reason because the electronic weight balance shows different reading when weighted twice.

3

Introduction There are many ways to carried out Fermentation process such as batch, continuous and fedbatch processes. In this experiment, we decided to use the shake flask fermentation. The shake flask fermentation is an example of batch fermentation. In shake flask, the culture flask usually Erlenmeyer flask is being used to place and growing the microorganisms. It is a small scale equipment which equivalent to stirred tank bioreactor and it is also the cheapest and easiest way to culture microorganism aerobically, in small volumes of nutrient broth. To ensure the prevention of any contamination to the culture, shake flask must be plugged. Different plug can be made of cotton-wool, glass wool, polyurethane foam, gauze or synthetic fibrous material. In this experiment, we used cotton plugged. The plug function to prevent airborne microorganism from getting into the medium while at the same time allowing free flow of air enter the flask. The cultures are incubated at certain temperature 37o C and shaking frequency 350 rpm for 4 hours in an incubator shaker to achieve a required growth rate. The shaking agitates the medium and the culture to keep the mixture relatively homogeneous and also to ensure aeration, creating an aerobic condition. In batch culture, it is closed environment that means there is neither input supplied nor output generated throughout the fermentation. The medium culture is initially inoculated with the microorganism. The growth keeps increasing until at certain extent, the growth is inhibited because of the decreasing substrate concentration and the presence of toxic metabolites. The microorganism that we used to study in this experiment is E.coli. There are many specific media for growth of E.coli but in this experiment we used Luria Bertani (LB) a Terrific Broth (TB) media because it is the most commonly used medium in molecular biology for E. coli cell culture. The relationship between the specific growth rate (μ) of a microbial population and the substrate concentration (s), is an indispensable tool in all fields of microbiology, be it physiology, genetics, ecology, or biotechnology, and therefore it is an important part of the basic teaching of microbiology (Kovárová-Kovar. K, et. al, 1998)

4

Objectives –

To study the growth kinetics of microorganism in shake flask experiment

–

To construct a growth curve including lag, log, stationary and death phases

–

To determine the Monod parameters

Theory Shake flask fermentation is one of the examples of batch fermentation. Batch culture is an example of a closed culture system which contains an initial, limited amount of nutrient. The inoculated culture will pass through a number of phases. After an inoculation there is a period during which no growth appears to take place. This period is referred as the lag phase and may be considered as a time of adaptation. In a commercial process, the length of the lag phase should be reduced as much as possible. Following a period during which the cell gradually increases, the cell grows at constant, maximum rate and this period is known as the exponential phase. The exponential phase may be described by the equation below:

𝒅𝒙 𝒅𝒕

= µx -------------------1

where x is the concentration of microbial biomass t is the time, in hours µ is the specific growth rate, in hour -1 on integration, equation (1) gives 𝒙𝒕 = 𝒙𝒐 𝒆µ𝒕 ------------------2 where 𝒙𝒐 is the original biomass concentration 𝒙𝒕 is the biomass concentration after time interval, t hours During the exponential phase, the organism is growing at its maximum specific growth rate, µ𝒎𝒂𝒙 for the prevailing conditions.

5

Equation 2 predicts that growth will continue indefinitely. However, growth results in the consumption of nutrients and the excretion of microbial products. Thus after a certain time the cell growth rate will decrease until growth ceases. The cessation of growth may be due to the depletion of some essential nutrient in the medium when there is limitation in substrate. The decrease in growth rate and the cessation of growth due to the depletion of substrate may be described by the relationship between µ and the residual growth-limiting substrate as follows:

µ=

µ𝒎𝒂𝒙 𝒔 𝑲𝒔 + 𝒔

where µ𝒎𝒂𝒙

= maximum growth rate

s

= residual substrate concentration

𝑲𝒔

= substrate utilization constant

Figure 4.1: The graph showing the relationship between the parameter of the Monod equation.

The stationary phase in batch culture is the point where the growth rate has declined to zero. In the other word the growth rate is equivalent to the death rate. The cell death is might due to the nutrient limitations due to their incorporation into cells during log-phase growth or a build-up of toxins due to their release of fermentation products also during log-phase growth. 6

The death phase is the result of the inability of the bacteria to carry out further reproduction as condition in the medium become less and less supportive of cell division. The nutrient is extremely insufficient for the growth of the microorganism. Eventually, the number of viable bacterial cells begins to decline at an exponential rate. Industrial fermentation is usually interrupted at the end of the exponential growth phase or before the death phase begins.

Figure 4.2: Growth curve of microorganism based on cell number analysis

7

Apparatus 

Microbe: Escherichia Coli



Shake flask (250mL flasks and 1000 mL flasks)



Eppendorf tubes / falcon tube (1.5 mL)



Cuvettes (spectrophotometer)



Incubator Shaker



Refrigerated Centrifuge



Media (for specific microbe)



Ethanol (70% ethanol for swabbing for sterility)



Spectrophotometer



Bunsen burner for sterility



Graduated Flask for measuring media (1000mL, 100mL, 10mL)



Laminar Flow hood for sterility



Biochemical Analyzer



HPLC for product measurement like ethanol



Cotton plugged



pH meter

Procedure

(i) Preparation of media

a)

Terrific Broth (TB) preparation 1. The recipe as stated at the bottle is followed. 2. The media is autoclaved at 121 0C for 20 minutes 3. Glycerol and media has been autoclaved together. 4. pH reading should be near 7 as the media is a readied phosphate buffer solution

b)

Luria Bertani (LB) preparation 1. The recipe as stated at the bottle is followed for LB media. 2. A certain amount of phosphate buffer is added to give a specific pH which is pH 7. Refer to table below. 8

3. Glucose solution is prepared and it should be autoclaved separately before cooled down in a different bottle. 4. Distilled water is autoclaved which will be used for filling up volume to the needed volume prepared. 5. When both solutions are cooled, then only both solutions are added.

Table 6.1: Buffer recipe according to 50 mM Buffer Strength No

Phosphate buffer

Concentration

components 1

K2HPO4

8 g/l

2

KH2PO4

3 g/l

(ii) Preparation of cell culture

a)

Seed culture preparation (inoculum) 1. 5 loops of grown E coli is taken on agar plates and added to the sterilized media of 150mL in 1000mL shake flask. (you may need 2 of 1000mL shake flask to ensure enough inoculum needed) 2. Sterility must be sustained during transfer. 3. The media is grown at 350 rpm for 4 hours and it is assumed as exponential growth of E coli. 4. At this stage, the seed cultures are assumed to be at its most active condition. 5. OD reading is taken for seed culture using spectrophotometer during this time.

b)

Main experiment 1. 10% of inoculum to the main experiment media is transferred using aseptic technique. (For instance, if the working volume is 150ml, therefore, 10% of inoculum would be 15mL of seed culture needed).

2. The shake flask is then capped (cotton plugged) and swabbed with 70% ethanol before incubation in a thermostated rotary shaker at required rotational speed and temperature for 24 hours.

9

(iii)

Sampling 1. Required amount of sample is transferred into the sampling tube with interval time for every hour or every 2 or 3 hours. 2. 5 mL of sample is withdrawn every time sampling is done during fermentation for measuring optical density (OD), glucose analysis and total cell number (biomass concentration: g/L). 3. Table below is referred for planned usage of sample volume: Table 6.2: Sampling

No.

Sample Name

1

OD

1000

Optical measurement using spectrophotometer

2

CDW

1000

For Cell Dry Weight measurement

3

S

1500

Glucose Analysis

iv)

Volume (uL)

Use for

Absorbance Analysis (Optical Density) (OD) 1. 1 mL of sample is transferred into a cuvette and the optical density measurement is made using a spectrophotometer with the wavelength set at 600nm. 2. The spectrophotometer is calibrated to zero by blank consisting 1 mL chosen media. 3. This method is used to measure cell growth; higher number of cells means more absorbance, which is caused by low transmittance and vice versa.

v)

Cell Dry Weight. (Biomass Concentration) (X) (g/L) 1. Dried centrifuge tubes is weighted and noted as initial mass.(empty container) 2. 1 mL sample is added to weighted centrifuge tube. 3. The sample is centrifuged at 10,000 rpm and at T of 4 oC. for 20 minutes 4. The supernatant is taken out and washing with distilled water and centrifuging may be repeated 5. The centrifuge tube is dried(left with biomass only) in oven at 80 oC for overnight 6. The dried centrifuged tubes is left in desiccator for half an hour. 7. The centrifuge tube weighted and note this as final mass (with biomass = Cell Dry Weight) Cell Dry Weight = Final mass – Initial mass

10

vi)

Glucose Analysis. (Substrate Concentration) (S) (g/L) 1. Sample of 1.5 mL is transferred into the micro centrifuge tube and refrigerated centrifuged for 10 minutes at 10,000 rpm. 2. Then, the supernatant is taken out into cuvette and put onto turntable of YSI 2700 Select Biochemical Analyzer for direct analysis of glucose (dextrose) concentration. 3. The glucose analysis is based on Glucose Oxidase that has been immobilized in the YSI Dextrose Membrane (YSI 2365).

Results Luria Bertani Results

Seed Culture Preparation (Inoculum) Initial OD : 1.278 (12.40pm) Final OD : 2.532 (4.40pm)

Table 7.1: Shows The Data For Luria Bertani (LB) No

Time (h)

OD (nm)

M1 (g)

M2 (g)

X (g/L)

ln X (g/L) (M2-M1)

S (g/L)

μ (1/h)

Ln (X/X0)

1

0

0.188

1.084

1.031

-26.5

-

8.71

-

0.000

2

1

0.189

1.076

1.085

4.5

1.504

8.82

-

-

3

2

0.307

1.076

1.243

83.5

4.401

8.70

2.897

-

4

3

0.806

1.097

1.091

-3

-

7.92

-

-2.179

5

4

0.853

1.103

1.095

-4

1.386

8.09

-

-1.891

6

6

0.655

1.098

1.098

-

-

5.79

-

-

7

8

0.757

1.097

1.078

-9.5

-

5.72

-

-1.026

8

10

0.783

1.093

1.093

-

-

4.83

-

-

9

12

0.802

1.098

1.109

5.5

1.705

4.21

-0.2695

-

10

14

0.282

1.104

1.155

25.5

3.239

3.89

0.767

-

11

16

0.406

1.089

1.091

1

0

3.47

-1.609

-

12

18

0.409

1.098

1.104

3

1.099

3.30

0.549

-

13

20

0.498

1.113

1.095

-9

-

2.90

-

-1.080 11

OD – Absorbance Optical Density M1 – Empty Centrifuge M2 – Dried Centrifuge Tube + Sample X – Cell Dry Weight Concentration S – Substrate

Graph of Absorbance (nm) against Time 1 0.8

Absorbance (nm)

0.6 0.4 0.2 0 0

5

10

15

20

25

Time(h) Figure 7.1: The growth curve of E.coli plotted using absorbance optical density.

ln X against Time 5 4.5 4 3.5 3

ln X

2.5

2 1.5 1 0.5 0 0

5

10

15

20

Time (h)

Figure 7.2: Graph of ln X against time which is plotted to construct the growth curve of the cell by using cell dry weight

12

By referring Figure 7.2, it show that the exponential phase may be between at 1th and 14th hour. This is because at 1st to 2nd hour at 12th and 14th hour shows an increasing data. Thus, the data between one of this period will be used to find the value µmax. I take period of 1st to 2nd hour.

ln X/Xo against Time 0 0

5

10

15

20

25

-0.5

y = 0.0554x - 2.0291 R² = 0.5576

-1

ln X/X0 -1.5 -2

-2.5

Time (h)

Figure 7.3: Graph of ln X/X0 against time is plotted to find the value of µmax

From Figure 7.3, the value of µmax which is equal to the slope of the graph is 0.0554. Thus, the doubling time is 12.51 h.

Graph Of Growth Rate Against Substrate Concentration 3.5 3 2.5 2

μ

1.5 1 0.5 0 -0.5 0

1

2

3

4

5

6

7

8

9

10

-1 -1.5 -2

S

Figure 7.4: Graph of growth rate, μ against substrate concentration, S

13

From Figure 7.4 we cannot find the Ks we lack the value of μ thus make the graph uncomplete. The value of Ks can be determined if the graph complete by using ½ μmax. By using Monod Equation Ks value can be determined. Terrific Broth Results Seed culture preparation (inoculum) Initial OD : 0.168 Final OD : 1.203 Table 7.2: Absorbance value at 600 nm obtained using Terrific Broth as medium (fermentation) No.

Time (h)

Absorbance Optical Density (nm)

1

0

0.191

2

1

0.442

3

2

0.763

4

3

1.541

5

4

0.888

6

6

0.991

7

8

0.994

8

10

1.110

9

12

1.103

10

14

1.129

11

16

1.526

12

18

1.178

13

20

1.068

14

Graph of Absorbance (nm) against Time

Absorbance (nm)

1.4 1.2 1 0.8 0.6 0.4 0.2 0 0

5

10

15

20

25

Time(h) Figure 7.5: The growth curve of E.coli plotted using absorbance optical density. Table 7.3: Results of Cell Dry Weight (CDW) obtained using Terrific Broth as medium No.

Time

Empty

Empty tube +

Cell dry

Cell mass

(h)

tube +

sticker + cell

weight (g)

concentration,

sticker (g),

(g), m2

(m2 – m1)

X (g/L)

ln x

ln X/X0

m1 1

0

1.10991

1.10

0.0009

0.45

-0.7985

0.000

2

1

1.1030

1.11

0.0070

3.50

1.2528

2.0513

3

2

1.0946

1.09

-0.0046

2.30

0.8329

1.6314

4

3

1.0861

1.09

0.0039

1.95

0.6678

1.4663

5

4

1.1064

1.11

0.0036

1.80

0.5878

1.3863

6

6

1.1021

1.11

0.0079

3.95

1.3737

2.1722

7

8

1.0983

1.11

0.0117

5.85

1.7664

2.5649

8

10

1.1032

1.11

0.0068

3.40

1.2238

2.0223

9

12

1.1106

1.13

0.0194

9.70

2.2721

3.0707

10

14

1.1005

1.11

0.0095

4.75

1.5581

2.3567

11

16

1.0920

1.10

0.0080

4.00

1.3863

2.1848

12

18

1.1029

1.11

0.0071

3.55

1.2669

2.0655

13

20

1.1025

1.11

0.0075

3.75

1.3218

2.1203

15

ln X against Time 2.5 2 1.5

ln X 1

0.5 0 0

5

10

15

20

25

Time (h)

Figure 7.6: Graph of ln X against time which is plotted to construct the growth curve of the cell by using cell dry weight By referring Figure 7.6, it show that the exponential phase may at between at 4th and 12th hour. Thus, the data between that particular times will be used to find the value µmax.

ln X/Xo against Time 3.5 y = 0.1997x + 0.8006 R² = 0.9213

3 2.5 2

ln X/X0

Y-Values

1.5

Linear (Y-Values)

1 0.5 0 0

5

10

15

Time (h)

Figure 7.7: Graph of ln X/X0 against time is plotted to find the value of µmax From Figure 7.7, the value of µmax which is equal to the slope of the graph is 0.1997. Thus, the doubling time is 3.4709.

16

Calculations Cell dry weight, X final weight – initial weight Volume of Sample

X=

=

1.085 g – 1.076 g

=

0.009 g

Growth Rate, μ μ =

ln x1 – ln x0 Time1 – Time0

=

ln 83.5 – ln 4.5 2–1

=

2.897 1/h 𝑥

Yield of substrate, (Y𝑠 ) 𝒙

Y𝒔 = -

∆𝑿 ∆𝑺

(g cells/ g substrate)

- 25.5 – 5.5 3.89 – 4.21

= 62.5 g cells/ g substrate

Saturation constant (Ks) μg = μmax . S Ks +S No example due to results error Discussion Microorganism population growth studies require inoculation of viable cells into a sterile broth medium and incubation of the culture under optimum temperature, pH, and gaseous conditions. This experiment is carried out to study the kinetic growth of microorganism. E.coli is selected as the cell and being cultivated inside a shake flask. We used the growth of microorganism in shake flask which is a simple method of fermentation. The nutrients for the microorganism are being supplied by the media which contain the carbon sources. In this experiment we used Luria Bertani (LB) and Terrific Broth (TB) as media. The flask is shaken during the cultivation to ensure constant mixing of the cell and the media therefore increase the homogeneity between these two and also to provide aeration for the cells. The culture is gone through the fermentation process for 20 hours. 17

Within that period, the biomass/cell sample is taken out for every 1 hours from 0 hour to 4 hour and continued every 2 hour from 4 hour to 20 hour. This is done to analyze the concentration of the cell (g/L), the cell dry weight and the glucose concentration.

In order to analyze the concentration of the cell inside the flask, absorbance reading for the optical density is taken from the spectrophotometer. The higher the absorbance reading means higher number of cell presence inside the flask at a particular time. As for this experiment, the absorbance reading for LB is increase from the beginning of the experiment until the 6th hour and decrease slightly until the 8th hour then increase back before decrease until 20th hour. It can be explained that the number of cell increase and decrease inconsistently throughout the cultivation. In the other hand, the decrease in cell number in

6th

and 14th hour indicating that the cell growth

has been interrupted maybe because there are contamination occur to the sample. The absorbance reading for TB shows increasing until 10th hour then it decrease a bit before increase until 18th hour. For TB media, the results shows better reading maybe there are no contamination occur during conducting the experiment. When there is other microorganism in the culture, the growth will be interrupted thus the growth of E. coli started to slow down. When there are different microorganism during the growth they will compete each other to keep living. Only the strong microorganism can withstand starvation. There is much turnover of protein for the culture to cope with this period of low substrate availability. In cell growth, the cell will go through several phases like lag, exponential, deceleration, stationary and death phase. We can say the absorbance experiment is fail due to inconsistence result.

In cell cultivation, the cells themselves need food or carbon sources like glucose for growth. In batch fermentation for example in this experiment, the glucose can be the limiting factor for the cell growth or we called it as substrate limiting growth. For this condition, the Monod equation can be used to predict the growth rate and the cell concentration inside the shake flask. In addition, the glucose concentration can be known by testing the cell sample into the glucose analyzer and the direct glucose concentration can be obtained. The LB media experiment, there are glucose concentration in there and the reading was obtained by using glucose analyzer. From the results of substrate concentration, we manage to get what we suppose to get which is the reading will decrease against time. Although the starting reading is lower than the second reading, the third until the last reading shows decreasing.

18

For the graph ln X against time, we also fail to get the estimated result. This graph is plotted to determine the gradient of exponential phase which is the μmax. The estimated exponential phase for LB media is from 1st hour until 14th hour. The graph shows decreasing at 2nd hour might be because of contamination. The reason why I estimate the exponential growth until 14th hour because the growth occur until that hour. The µmax for this period is 0.0554. For TB media, the estimated exponential phase may at between at 4th and 12th hour thus, the data between that particular times will be used to find the value µmax which is 0.1997. The graph below shows the result that we should achieved but we fail due to so many error.

Figure 9.1: Graph the number of cell against time Another analysis that can be performed to analyze the cell sample is by taking the dry weight of the cell. In this method, the cell is being taken out from cultivation flask and transferred into centrifuged tube. Before that, the empty centrifuged tube is weighted. Then the tube is the being centrifuged to separate the supernatant with the cell. For LB media, the supernatant is separate and will be used for glucose analysis but for TB media, the supernatant is separated and removed. The remained cell is washed with distilled water and centrifuged again before being dried inside an oven for 24 hours. The dry cell weight with tube is finally taken and the result will be subtract with the weight of initial empty tube to know the weight of the cell that present at particular time during the cultivation. In this experiment, the cell dry weight should increased from 0th hour until the 20th hour. Unfortunately, we did not manage to get like that because the electronic weight balance is not accurate. We weight each tube for two times but still get different reading. The worst part comes when the final reading of cell dry weight with tube is smaller than the initial reading of empty tube. This will give negative reading thus it is impossible for us to produce a good graph. When we get negative reading for cell dry weight, we cannot calculate the growth rate and this happen for LB media. 19

Conclusion

After conducting the experiment we can ensure that, E.coli is suitable to be fermented inside a shake flask and it is a simple method to investigate the growth kinetics of the microorganism. Knowledge of microbial growth kinetics is essential to determine when to harvest the culture for different purposes. Although we fail to achieve the objectives due to some errors during conducting the experiment, we try to learn our mistake for next experiment. Microorganism will go through several phases in their growth, several analyses on the cell need to be done to know the growth kinetics of the cell and the duration for each phase. This includes the cell concentration, glucose concentration and also the cell dry weight analyses. This method can be done in the laboratory before the fermentation or the cultivation of microbes in large scale is performed. Growth kinetics deals with the rate of cell growth and how it is affected by various chemical and physical conditions. During the course of growth, the cells is continuously changing and adapting itself in the media environment, which is also continuously changing in physical and chemical conditions. In conclusion, the microbial culture in batch culture system that is shake flask system goes through a lag phase, exponential growth phase, decelerating growth phase, stationary phase and sometimes the death phase depends on the end product desired. The substrate concentration in the culture medium and growth parameters, such as glucose concentration changes correspondingly throughout the growth phases. Thus, the physiology of the microorganisms is always in a transient stage, subjected to a continually changing culture conditions. Consequently, product formation is confined to a certain period of cultivation, for example antibiotics would only be produced in the decelerating and stationary growth phases. The batch culture system is still widely used in certain industrial processes for example brewery industry because of its easy management of feed stocks. These advantages allow the use of unskilled labor and low risk of financial loss. Low level of microbial contamination in fermented products is at time tolerable, as long as the microbial contaminants are not pathogenic and do not alter the desired properties of the product, such as taste, color and texture.

20

Recommendation –

Aseptic technique must be practised when handling biomass concentration to avoid any contamination.

–

Cuvette must be wiped cleanly to prevent any scratch that would affect the spectrophotometer reading during protein test.

–

Disinfect the work area with 70% alcohol before handling the culture.

–

Dispose of all contaminated materials in appropriate containers.

–

The cap of the viral must be opened to fasten the drying process of the biomass in the oven.

–

The supernatant of cell concentration should be taken out carefully without any taking out of the biomass.

–

This experiment must be carried out under the laminar flow to prevent any contamination to the culture.

–

Wash hand after handling the culture.

References

Ardestani, F., (2011). Investigation of the Nutrient Uptake and Cell Growth Kinetics with Monod and Moser Models for Penicillium brevicompactum ATCC 16024 in Batch Bioreactor. culture, Iranica Journal of Energy and Environment (IJEE), 2(2): 117-121

Crueger W and Crueger A., (1990). Biotechnology: A Textbook of Industrial Microbiology. Sinauer Associates, Sunderland Massachusetts.

Kovárová-Kovar, K., & Egli, T. (1998). Growth Kinetics of Suspended Microbial Cells: From Single-Substrate-Controlled Growth to Mixed-Substrate Kinetics. Retrieved on October 4, 2015 from http://mmbr.asm.org/content/62/3/646.full

PF Stanbury and A Whitaker.,1984. Principles Of Fermentation Technology. Pergamon Press

21

Appendix

Figure 13.1: Result of the experiment after being approved

Figure 13.2: After being heated, the sample is being cooled inside a desiccator.

Figure 13.3: Glucose analysis machine model YSI 2700

Figure 13.4: Incubator Shaker

22