CHE506 - Lab Report On Growth Study of E. Coli in Shake Flask (2018) PDF

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UNIVERSITI TEKNOLOGI MARA FAKULTI KEJURUTERAAN KIMIA REACTION ENGINEERING LABORATORY (CHE506) NAME: STUDENT NO : KHAIRUL AMIRIN BIN KHAIRUL ANUAR 2017632082 PUTERA NAJMEEN FARITH BIN ABDUL RAZAK 2017632096 NURUL AMIRAH BINTI MUSDAFA KAMAL 2017632124 NURUL AIDA BINTI MOHAMMAD 2017632132 NURUL KAMILAH...

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UNIVERSITI TEKNOLOGI MARA FAKULTI KEJURUTERAAN KIMIA REACTION ENGINEERING LABORATORY (CHE506) NAME: STUDENT NO : KHAIRUL AMIRIN BIN KHAIRUL ANUAR 2017632082 PUTERA NAJMEEN FARITH BIN ABDUL RAZAK 2017632096 NURUL AMIRAH BINTI MUSDAFA KAMAL 2017632124 NURUL AIDA BINTI MOHAMMAD 2017632132 NURUL KAMILAH BINTI KHAIROL ANUAR 2017632192 NURLINA SYAHIIRAH BINTI MD TAHIR 2017632214 GROUP : EH2205I EXPERIMENT : GROWTH STUDY OF E.COLI IN SHAKE FLASK DATE PERFORMED : 18th OCTOBER 2018 SEMESTER :5 PROGRAMME / CODE : CHEMICAL ENGINEERING / EH220 SUBMIT TO : MADAM SYAZANA MOHAMAD PAUDZI 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 Appendix TOTAL MARKS

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TABLE OF CONTENT

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ABSTRACT ................................................................................................................... 2

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INTRODUCTION......................................................................................................... 3

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OBJECTIVES ............................................................................................................... 3

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THEORY ....................................................................................................................... 7

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MATERIALS & APPARATUS ................................................................................... 9

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METHODOLOGY ..................................................................................................... 10

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RESULTS .................................................................................................................... 12

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CALCULATIONS ...................................................................................................... 16

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DISCUSSION .............................................................................................................. 16

10.0 CONCLUSION ........................................................................................................... 21 11.0 RECOMMENDATIONS............................................................................................ 22 12.0 REFERENCES ............................................................................................................ 23 13.0 APPENDICES ............................................................................................................. 24

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ABSTRACT

The bacterial growth curve is a fundamental part of introductory microbiology. The growth kinetics of E.coli is studied using the shake flask fermentation experiment by plotting the graph of real absorbance optical density versus time. The Terrific Broth is prepared by autoclaving the media at 121⁰C for 20 minutes. The E.coli inoculum is grow for 5 hours at 300 rpm. 10% of the inoculum which 15 mL is added into sterilized media of 150mL then incubated in thermostat rotary shaker at required rotational speed and temperature for 16 hours. The required amount of sample was transferred into the sampling tube with interval time for every hour or every 2 hours. The reading for the absorbance optical density is measured using the spectrophotometer. The lag phase occur from t = 0h until t = 1h as the E.coli familiarize themselves with new environment in the media. There two exponential phase from t = 1 h until t = 6 h and from t = 8h until t = 14h. The maximum growth rate of the E.coli is believed to be found from the exponential curve which yield to µmax = 0.016 h-1. The presence of the second exponential phase is due to the cryptic growth and the possibility of the presence of second microorganism consuming the E.coli as the food supplies. The deceleration phase cannot be determined. The death phase occur at t = 14h until t = 16h where the second microorganism is believed to drastically die due to the abrupt loss of food supplies and their inability in surviving with their own metabolism. The µnet for the respective phase are µnet, lag phase = 0.6931 h-1, µnet, exponential phase =

µmax = 0.016 h-1, µnet,

stationary phase

= 0.0000 h-1, µnet,

death phase

= 0.0000 h-1.

Unfortunately, due to limited sources of data, the yield coefficient (YX/S) and saturation constant (Ks) cannot be determined.

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INTRODUCTION

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 cultures. When batch cultures are used, E. coli cells from an overnight culture are usually inoculated into Erlenmeyer flasks containing a complex or a defined medium. Like any other living system, microorganisms also require a source of energy, carbon, nitrogen, oxygen, iron and other minerals, micronutrients, and water for growth, and multiplication. All these nutrients that are essential for the growth and multiplication of microbial organisms are supplied in the form of nutrient media. For commercial purposes, there commended media should be cheap and available year round. The following are the minimum components required in a microbial medium for cultivation of microbes in a laboratory: 1) Carbon source A simple carbon source, which is simple to use and easily available, can be used. Sugars such as glucose, lactose, sucrose, and complex polysaccharides such as starch, glycogen cellulose, a mixture of various carbohydrates, and other compounds such as cereal grain powders, cane molasses, etc., are usually used as carbon sources in microbial culture media. The main purpose of the carbon source is to provide energy and carbon skeleton for the synthesis of various other biological compounds. 2) Nitrogen sources The major types of nitrogen sources used in culture media are ammonium salts, urea, animal tissue extracts, amino acid mixtures, and plant-tissue extracts. 3) Micro elements or trace elements Elements required in small amounts or in traces are to be added into the medium as salts in required amounts. The elements such as copper, cobalt, iron, zinc, manganese, magnesium, etc., are the microelements. Typically, to understand and define the growth of a particular microbial isolate, cells are placed in a liquid medium in which the nutrients and environmental conditions are controlled. If the medium supplies all nutrients required for growth and environmental parameters are optimal, the increase in numbers or bacterial mass can be measured as a function of time to

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obtain a growth curve. Based on Figure 1, several distinct growth phases can be observed within a growth curve. These include: 1) Lag Phase 2) The Exponential Or Log Phase 3) The Stationary Phase 4) The Death Phase. Each of these phases represents a distinct period of growth that is associated with typical physiological changes in the cell culture. As will be seen in the following sections, the rates of growth associated with each phase are quite different.

Figure 1 - A typical growth curve for bacterial population. Lag phase represents immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity. The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium. The second phase is exponential phase. The cells begin to proliferate with their maximum growth rate. The doubling time of E.coli is 20 minutes. Exponential phase is important for determining the maximum growth rate, µ and doubling time, d since the growth

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at this time is the most constant and ideal. The third phase of growth is the stationary phase. The stationary phase in a batch culture can be defined as a state of no net growth. Although there is no net growth in stationary phase, cells still grow and divide. Growth is simply balanced by an equal number of cells dying. The final phase of the growth curve is the death phase, which is characterized by a net loss of culturable cells. Even in the death phase there may be individual cells that are metabolizing and dividing, but more viable cells are lost than are gained so there is a net loss of viable cells. The death phase is often exponential, although the rate of cell death is usually slower than the rate of growth during the exponential phase. LB Media (Luria-Bertani) is common bacterial growth media for Escherichia Coli. Although already described in the fifties in the early days of phage genetics these media are still widely used in molecular biology. The two main components of LB media are Tryptone and Yeast Extract. Tryptone is used in a concentration of 10 gram / litre and Yeast Extract in a concentration of 5 gram / litre. Many variations of LB medium only differ in the concentration of NaCl. All LB-Media are listed in order of increasing concentration of NaCl. Tryptone broth is a moderately rich medium for growth and cultivation of Escherichia Coli. Terrific Broth is a rich medium compared to LB and Tryptone Media. The medium is developed for higher density growth of Escherichia Coli cells and higher yield of plasmid DNA compared to LB and Tryptone broth. Super Broth is an even richer medium developed for obtaining high yields of lambda bacteriophage in liquid lysates, Botstein, D. et al.

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OBJECTIVES

The objectives of the experiment are: 1) To study and observe the growth kinetics of microorganism in shake flask experiment. 2) To construct a growth curve including lag, log, stationary and death phases. 3) To determine the monod parameters of maximum growth rate (µmax), yield of substrate (YX/S), mass doubling time (td), saturation constant (Ks) and specific growth rate (µnet).

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4.0

THEORY

Shake flask fermentation is one of the examples of batch fermentation. Batch culture is an example of closed culture system which contains an initial, limited amount of nutrient. The inoculated 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 a lag phase and may be considered as a time adaptation. In a commercial process, the length of lag phase should be reduced as much as possible. Following a period during which cell gradually increases, the cell grows at constant, maximum rate and this period is known as the log phase or exponential phase. During lag phase dX/dt ans dS/dt are essentially zero. However, as exponential growth phase begins it is possible to measure dX/dt and dS/dt values which are very useful for defining important microbial kinetic parameters. Using corresponding observations of dS/dt and dX/dt obtained just after the onset of exponential growth phase, we can compute the specific growth rate, μ and yield coefficients, YXS as: Rate of microbial growth (μnet) is characterized by specific growth rate:

μ net 

1 dX X dt

Yield coefficients (YX/S) are defined based on the amount of consumption of another material:

YX/S  

ΔX ΔS

Mass doubling time (τd) is calculated based on cell numbers and the net specific rate of replication: τd 

ln2 μ net

The monod equation is a mathematical model for the growth of microorganisms. It is named for Jacques Monod who proposed using an equation of this form to relate microbial growth rate in an aqueous environment to the concentration of limiting nutrients.

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For substrate limited growth Monod equation is applicable in cellular system. Monod equation is as the following: μg 

μ mS KS  S

Where, μm = maximum specific growth rate when S >> KS μg = μnet when endogeneous metabolism is unimportant KS = saturation constant or half-velocity constant KS = S when μg = 1/2μm S >> KS, μg = μm S...