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BTEN 3184

BIOTECHNOLOGY ENGINEERING LAB IV


ASSOC. PROF. DR. MAIZIRWAN MEL
ASST. PROF. DR. NURUL SAKINAH BINTI ENGLIMAN

EXPERIMENT 1
GROWTH KINETICS OF E.COLI IN A SHAKE FLASK
20/2/2019

AMIRUL IZWAN BIN RAMLAN


1614807

Content Mark

Introduction 5
Objectives 3
Procedures 4
Result and Discussion 10
Conclusion 5
References 3

Total 30 pts
INTRODUCTION
In this experiment, the shake flask fermentation is being used. Shake flask
fermentation is the example of batch fermentation. In shake flask, the culture flask usually
Erlenmeyer flask is being used to place and growing the microorganisms. It is the cheapest
and easiest way to culture microorganism aerobically, in small volumes of nutrient broth. It is
a small-scale equipment which equivalent to stirred tank bioreactor. The bacterial growth
curve is a fundamental part of introductory microbiology (Monod, 1949). Escherichia coli are
often used both as a model organism to understand fundamental biological process and as a
tool to produce biomolecules, including plasmids and proteins. The growth and physiology of
Escherichia coli cells are studied in a batch culture.

In order to prevent 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. The plug has to prevent airborne microorganism from getting into
the medium while at the same time allowing free flow of air into the flask.

The cultures are incubated at certain temperature and shaking frequency 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 to ensure aeration, creating an
aerobic condition. In batch culture, 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.

OBJECTIVES
1. To study the growth kinetics of E. coli in shake flask under batch process.
2. To evaluate the protein concentration produce by the microbes during fermentation.
3. To analyse the glucose uptake by the microbes during fermentation process.
\

MATERIALS & APPARATUS


Materials
1) Media (for specific microbe)
2) Ethanol (70% ethanol for swabbing for sterility)
3) Microbe: Escherichia Coli
4) Distilled water
Apparatus
1) Shake flask

2) Eppendorf tubes

3) Cuvettes (spectrophotometer)

4) Thermostat rotary shaker/incubator shaker

5) Refrigerated centrifuge

6) Spectrophotometer

7) Bunsen burner

8) Graduated Flask

9) Laminar Flow hood

10) Spectrophotometer

11) HPLC for product measurement like ethanol

12) Cotton plugged

PROCEDURE
Part 1: Preparation of inoculated fermentation medium

The media was prepared by the lab asistant

Part 2: Sampling for cell dry weight

1.5 ml of biomass concentration is taken out.

The 1.5 ml biomass concentration is transferred into micro centrifuge tube. An empty micro
centrifuge tube must be weighted first.

The sample is then centrifuged for 30 minutes at 10000 rpm.

After that, the supernatant of the sample is taken out carefully without taking out any
biomass.

The biomass is then left dried inside an oven at 80C for overnight.

The dried biomass is then being placed inside a desiccator to let it cool before rapidly
weighing on an analytical balance.
Part 3: Glucose analysis

1.5 ml of biomass concentration is taken out.

The 1.5 ml biomass concentration is transferred into micro centrifuge tube.

The supernatant was extracted and put in the fridge

1ml l of DNS reagent is added into 1ml of the supernatant sample inside a capped test tube

The mixture is heated at 90℃ for 10 minutes to develop the red-brown colour.

The heated mixture is then cooled to the room temperature for 2-3 minutes in iced water.

The absorbance is checked with a spectrophotometer at 575 nm.

Part 4: Protein analysis

1.5 ml of biomass concentration is taken out.

The 1.5 ml biomass concentration is transferred into micro centrifuge tube.

The pellet was obtained from the centrifuged sample.

1 ml of distilled water was added.

Sonification was done on the sample for cell lysis.

200 µl of the sample was added together with 800 µl of Bradford solution and 1ml of distilled
water.

The sample was then measured with spectrophotometer at 595nm.

Part 5: Sampling for optical density

1ml of biomass concentration is taken out and being transferred into micro centrifuge tube
every 30 minute.

The spectrophotometer is calibrated to zero by blank consisting of 1ml LB Broth.

The biomass concentration is then being transferred into a cuvette and optical density
measurement is taken with wavelength set at 600nm.

More absorbance means higher number of cell.


RESULT

Protein (mg/ml) Optical Density (OD)

Blank 0.001

125 0.630

250 1.073

500 1.317

750 1.387

1000 1.504

Table 1: Standard Curve Protein

Time (min) OD Protein Concentration (g/L)

30 0.311 0.359

60 0.614

90 0.529 0.636

120 1.133

150 1.254 0.691

180 1.379

210 1.476 0.625

240 1.599

270 1.628

300 1.686

390 1.717

Table 2: Analysis of growth Kinetics value of E.coli


Time (min) Aluminium boat Aluminium boat + Cell Dry Weight Cell Density, Ln X
weight, m1 (g) Dried sample, m2 (g) X (g/L)
(g)
30 1.1646 1.1757 0.0111 7.40 2.00

60 1.1637 1.1872 0.0235 16.67 2.81

90 1.1665 1.1826 0.0161 10.73 2.37

120 1.1583 1.1750 0.0167 11.13 2.41

150 1.1657 1.1713 0.0056 3.73 1.32

180 1.1616 1.1733 0.0117 7.80 2.05

210 1.1830 1.2261 0.0431 28.73 3.36

240 1.1816 1.2502 0.0686 45.73 3.82

270 1.2013 1.2430 0.0417 27.8 3.33

300 1.1955 1.1999 0.0044 2.93 1.08

390 1.1859 1.2048 0.0189 12.6 2.53

Table 3: Cell dry weight (Concentration of the Biomass)

Optical Density vs Time


2
1.8
1.6
1.4
Optical Density

1.2
1
0.8
0.6
0.4
0.2
0
0 50 100 150 200 250 300 350 400 450
Time (min)

Figure 1 - The Growth Curved Of E. Coli


Cell Density, X vs Time (min)
50
45
40
35
30
25
20
15
10
5
0
0 50 100 150 200 250 300 350 400 450

Figure 2 - Graph of Cell Density, X versus Time

1200

1000

800

600

400

200

0
0.63 1.073 1.317 1.387 1.504

Figure 3 – Standard Protein Curve

Protein vs time
0.8
0.7
0.6
0.5
Protein

0.4
0.3
0.2
0.1
0 TIme (min)
60 120 180 240

Figure 4 – Protein vs time


CALCULATION

Sample of Calculation for Determination of Dry Cell Weight (g),


Cell Dry Weight = Final Mass (m2) – Initial Mass (m1)
For 0th hour,
Cell Dry Weight = 1.1757g - 1.1646g
Cell Dry Weight = 0.0111 g
Sample of Calculation for Concentration of Cell Mass, X
Concentration of Cell (g/L)= Cell Dry Weight (g)/Volume of sample (L)
For 0th hour,
Concentration of Cell (g/L)= 0.001g/0.001L
Concentration of Cell (g/L)= 1g/L
Sample Calculation for ln X/Xo
For 0th hour, ln(𝑋𝑋0)= ln(1g/L1g/L)=0
Sample Calculation for Net Growth Rate, μ
μ= (ln𝑋2−ln𝑋1) / (t2−t1)
= (2.81-2.00)/(60-30)
= 0.027 /min or 1.62 /L
Doubling time, d
d=ln2/ μ
d= ln2/0.027=25.67 min
DISCUSSION
This experiment is done to study the growth kinetics of microorganism in shake flask
experiment. In this experiment, the microorganism that are desired to be growth is known as
E.coli. This experiment is done by using batch culture process where there is not inlet of
substrates and outlet of product throughout the fermentation in the given time. The Terrific
broth medium is inoculated with the E.coli which then the growth of E.coli kept increasing
until it reached one point where the growth decelerates due to limited substrates concentration
and the presence of toxic metabolites (Growth Kinetics Study of Microorganism in Shake
Flask, 2017).
The growth curve plotted in Figure 1 shows that there is no lag phase occur, this is
probably an error, or the lag phase occur below 30 minutes. The lag phase occurs immediately
after inoculation of E. coli into the medium. It is known as the period of the adaptation of cells
into new environment where the cells started to embower their habitat. They started to
reorganize their molecular constituent every time they are transferred or introduced to a new
environment. Also, the exponential phase can be seen through the growth curve starting from
time 0 to 300. At this phase, cell started to grow rapidly along with their maximum growth rate.
In this phase, the cell mass and density increase exponentially with time. The nutrient
concentration contain in the medium is high in this phase resulting in substantive progress
which elevate the growth rate of cell. The exponential growth of this cell is assumed to be first
order.
Next, the deceleration growth phase should follows the exponential phase. This phase
growth of E.coli decelerates a little bit due to depletion of one or more essential nutrients or
the accumulation of toxic by-products of growth. It occurs for a very short period of time.
However, from the graph in Figure 1, it can be seen that there is no deceleration phase. This is
maybe due to the cell do not detect the depletion of nutrients thus it straight forward carried on
to the stationary phase. It is assumed that the cell were readily restructured themselves to
prepare for cellular survival in the stationary phase due to limited sources of nutrient as their
food to grow.
The cell density obtained from the experiment is not in order. This error may happen
because of error when weighing the cell or during taking sample of the cell dry weight. 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
analyser and the direct glucose concentration can be obtained. In other way, the glucose
concentration is also being obtained by mixing the sample with DNS reagent. The DNS reagent
will be reduced to 3-amino,5-nitrosalicylic acid in the presence of free carboxyl group
(glucose) and absorbance reading can be taken through the spectrophotometer. The sugar
concentration and sugar concentration were not recorded because there was an error during the
preparation of the DNS reagent.
CONCLUSION
The growth kinetics of E.coli is studied using the shake flask experiment by plotting the
graph of real absorbance optical density versus time. The lag phase were not present in the
graph. The growth rate, μ obtained is 0.027 /min and the doubling time, d is 25.67 min. The
deceleration phase cannot be determined. Unfortunately, due to limited sources of data, the
yield coefficient (YX/S) and saturation constant (Ks) cannot be determined. The first and
second object were achieved but the last objective was not achieved due to error, the
experiment is not completely successful.

REFERENCE
1) Fogler, H. S. (2006). Element of Chemical Reaction Engineering. Michigan: Prentice Hall.
2) Growth Kinetics Study of Microorganism in Shake Flask. (February 2017). Lab Manual
Faculty of Chemical Engineering UiTM Shah Alam. Shah Alam, Selangor: UiTM Shah Alam.
3) Jitendra, P. (1 October 2017). Bacterial Growth Monod Equation. Retrieved from Slide
Share: https://www.slideshare.net/chondu100/bacterial-growth-curve-monods-equation
4) Microbial Growth. (2016). Retrieved from Bio CS Montana:
https://www.cs.montana.edu/webworks/projects/stevesbook/contents/chapters/chapter002/sec
tion002/black/page001.html
5) Monod Equation. (n.d.). Retrieved from Wikipedia:
https://en.wikipedia.org/wiki/Monod_equation
6) Panikov, N. (1995). Microbial Growth Kinetics. Springer Science & Business Media.
7) Shuler, M. L., & Kargi, F. (2002). How Cells Grow. In M. L. Shuler, & F. Kargi, Bioprocess
Engineering Basic Concepts Second Edition (pp. 155-200). Prentice Hall PTR