LAB 1 Edited 4 Mac 2015

CBE 661 Lab 1 Lab Manual Edited 4 Mar 2015

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Laboratory 1: Growth Kinetics Study of Microorganism in Shake Flask

1. Introduction

In the shake flask fermentation, the culture flasks (usually Erlenmeyer) of 250 or 500 mL or larger are used for growing microorganisms. Shake flask fermentation is the cheapest and simplest technique to grow bacteria or fungi, aerobically, in small volumes of nutrient broth. The broth is poured into Erlenmeyer Flasks equipped with cotton-wool stoppers, and autoclaved. After cooling, some microbes are "seeded" into the flask, and it is placed on a Shaker machine. The shaking agitates the content and so ensures aeration, so that the microbes could breathe. These flasks are shaken, generally, by an incubator shaker at a suitable agitation speed, which is usually in r.p.m. Shaken cultures are usually applied to aerobic processes. In general, filamentous microorganisms are grown for the production of secondary metabolites, which begins 1 to 3 days after inoculation and continues 3 to 4 days thereafter, for instance. In all such cases, the shaken cultures are used for strain improvement as well as for determination of the optimum conditions for the fermentation process. In many industrial processes, it is also used for the initial stages of inoculum development. Shaken cultures are a convenient method of growing microorganisms in submerged cultures under aerobic conditions created by shaking; it is a small scale equivalent of stirred tank bioreactor. Both the devices are extensively used with filamentous microorganisms and, often, with other types of microorganisms as well.

Usually, complex media are used for shake flask cultures. However, to enhance the

growing the synthetic medium is being devised for the fermentation process. Studies on inoculum size, temperature, agitation, nutrition are initially done using these cultures to monitor their influences on growth and product formation.

Objectives :

To study/observe 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 of maximum growth rate (max),

yield of substrate (Yx/s), mass doubling time (td), saturation constant

(Ks), specific growth rate (net).


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Figure 1 Phases of a typical growth curve of E.coli in a batch culture In a 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. Lag phase is the time between inoculation and reaching the maximum growth rate. There are two sub phases in the lag phase. In the first phase, there is no growth identified whereas in the second sub phase which is also known as acceleration phase, there is a constant growth begins. 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, and doubling time, d since the growth at this time is the most constant and ideal. Retardation phase is the period between exponential and stationary phase, or in other words, the phase before the growth becomes stationary. Among the factors that inhibit the growth are reduced dissolved oxygen tension (DOT), substrate concentration, pH changes and presence of inhibiting metabolites. After retardation phase, the growth phase enters stationary phase where the growth becomes constant for a period of time before it declines. Finally, the growth declines from its stationary phase due to the cells lysation. This is indicated by the decrease of the viable cell number. There are many specific media for certain microorganisms like Luria Bertani (Lennox) and Terrific Broth media. Bacterial E.coli growth media: LB Miller broth/LB Lennox broth is the most commonly used medium in molecular biology for E.coli cell culture. LB broth contains the enzymatic digestion product of casein commonly known as peptone (some vendors term it Tryptone), yeast extract, and sodium chloride. Peptone is rich in amino acids and peptides. Its amino acid and peptide compositions reflect those of casein. In addition to amino acids and peptides, yeast extract also contains nucleic acids, lipids and other nutrients which are needed for bacterial growth. (LB Miller, Lennox)

Other media is Bacterial E. coli growth medium TB or Terrific Broth TB or Terrific broth is a phosphate buffered rich medium. In addition to 20% more peptone and 380% more yeast extract than LB broth, TB also has an added 0.4% glycerol as an extra carbon source. All these nutrients in TB can support E. coli growth to OD600 5 to 8 under normal


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shaking incubation conditions. TB is commonly used for protein expression and plasmid production in a laboratory scale. (TB broth)

2. Theories

Rate of microbial growth net is characterized by specific growth rate:

net dt

dX

X

1 [1/h]

Yield Coefficients ( sXY / ) are defined based on the amount of consumption of another material

S

XY sX

/ [g cells/g substrate]

Mass doubling time ( d ) is calculated based on cell numbers and the net specific rate of

replication

net

d

2ln

[h]

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

SK

S

s

m

g

[1/h]

m = maximum specific growth rate when S >> sK

g = net when endogeneous metabolism is unimportant

sK = saturation constant or half-velocity constant

sK = S when g = m

S>> sK , g = m

S


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3. Apparatus and Reagent

Microbe: Escherichia coli

Shake flask (250mL flasks and 1000 mL flasks)

Eppendorf tubes/falcon tube (1.5mL)

Cuvettes (spectrophotometer)

Thermostated rotary shaker/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

4. Experimental Procedures

The experimental parameters will be as the following

Group Temperature

(oC)

Shaking frequency

(rpm)

Shake flask size

Filling/Working Volume

(mL)

Media Type

Inoculum percentage

pH Carbon source

1 37 350 500 150

TB 10 7

Glycerol

LB Glucose

2 37 350 500 150 TB

10 7

Glycerol

LB Glucose

3 37 350 500 150 TB

10 7 Glycerol

LB Glucose

4 37 350 500 150 TB

10 7 Glycerol

LB Glucose

5 37 350 500 150 TB

10 7 Glycerol

LB Glucose

6 37 350 500 150 TB

10 7 Glycerol

LB Glucose

Table 1: Experimental Parameters


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(i) Preparation of media Media must be prepared according to the needs of microorganism used. Microorganism used is Escherichia coli. There are many kinds of media for E coli for instance Luria Bertani broth or Terrific Broth. Terrific Broth is a readied phosphate buffer media. Prepare the chosen broth according to the recommended formula or recipe stated at the chemical bottle. The example readied recipe for the broths are as the following: Please further read the instruction of bottle

No Name of Broth Brand Recipe (g/L

1 Terrific Broth SRL Chem or Merck

47.60 g to be filled up to 1L volume Glycerol as carbon source (4mL/1L)

2 Luria Bertani (Lennox)

SRL Chem 20 .0 g to be filled up to 1L volume Glucose as carbon source (10g/L)

Table 2: Broth

a) Terrific Broth preparation Follow the recipe as stated at the bottle. Autoclave the media at 121 oC for 20 minutes Glycerol and media can be autoclaved together. pH reading should be near 7 as the media is a readied phosphate buffer solution

b) Luria Bertani (Lennox) preparation Follow the recipe as stated at the bottle for LB media. A certain amount of phosphate buffer is added to give a specific pH which is pH 7. Refer to table below. Prepare glucose solution and it should be autoclaved separately and cooled down in a different bottle. Autoclave distilled water which will be used for filling up volume to the needed volume prepared. When both solutions are cooled, then only both solutions are added.

No Phosphate buffer

components Concentration

1 K2HPO4 8 g/L

2 KH2PO4 3 g/L

Table 3: Buffer recipe according to 50 mM Buffer Strength (Buffer calculator)

(ii) Preparation of cell culture

Cell culture used must be maintained on an agar plate and liquid broth for the inoculum preparation. A suitable media is needed in order to ensure that the


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microorganism is growing. Inoculum preparation refers to seed culture which will be the feed for the main experiment. a) Seed culture preparation (inoculum)

Take 5 loops of grown E coli 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) Sterility must be sustained during transfer. Grow the media at 350 rpm for 4 hours assuming exponential growth of E coli. At this stage, the seed cultures are assumed to be at its most active condition. Take note the OD for seed culture using spectrophotometer

Temperature (oC)

Shaking frequency

(rpm)

Shake flask size

Filling/ Working Volume

(mL)

Media Type

Inoculum percentage

pH

Carbon source

Fermentation time (h)

Initial and final

OD of seed

culture **

37 350 1000 150 TB

5 loops from agar

plate 7

Glycerol

4

OD initial

OD final

Table 4 Take note: (please prepare enough seeds for all main experiments) ** Please take note the initial OD (after 5 loops inoculation) and final OD (after 4 hours of fermentation)

b) Main experiment

Using aseptic technique, transfer 10% of inoculum to the main experiment media. For instance, if the working volume is 150ml, therefore, 10% of inoculum would be 15mL of seed culture needed 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. The main experiment is stated in Table 1. .

(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. Refer to Table below for planned usage of sample volume: No Sample

Name Volume (uL)

Use for

1 OD 1000 Optical measurement using spectrophotometer

2 CDW 1000 For Cell Dry Weight measurement


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3 S 1500 Glucose Analysis

4 P remaining Product analysis i.e ethanol

Table 5: Sampling

v) 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. Suggested method: Certain tenth-time dilution is proposed for the OD measurement by using spectrophotometer. For instance, with 1 mL sample, take only 100 uL sampl e being added to 900 uL of Distilled Water for OD measurement in 1000 uL Cuvette.

vi) Cell Dry Weight. (Biomass Concentration) (X) (g/L)

1. Weigh dried centrifuge tubes and note this 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. Take out the supernatant and you may repeat washing with distilled water and centrifuging 5. Dry the centrifuge tube (left with biomass only) in oven at 80 oC for overnight 6. Leave the dried centrifuged tubes in dessicator. 7. Weigh the centrifuge tube and note this as final mass (with biomass = Cell Dry Weight)

Cell Dry Weight = Final mass Initial mass Alternative method

1. Aluminum weight of boat are dried in an oven at 80C for 6-8 hours and placed in a dessicator containing a drying agent for cooling before weighing (for 30min).

2. The cell pellet (after sample is centrifuged at 10,000 rpm) is suspended in 10 mL centrifuge tube with distilled water. 3. The cell then transferred to aluminum foil boat. The tube was rinsed with water

and placed in an oven at 80C for overnight. 4. The sample is then removed from the oven with tongs and placed in a dessicator to cool and weighed rapidly on an analytical balance. The weight of the cell pellet is recorded.

vii) 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).


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Suggested method: This method is to analyze the concentration of S: substrate which is glucose as the main source of carbon. The glucose being consumed is monitored and denoted as S in the unit of g/L.

Take note: only for media having glucose only.

viii) Product analysis (optional)

1. The remaining sample is transferred into the centrifuge tube and centrifuged for 10 minutes at 10,000 rpm. 2. Then, the supernatant is taken out to analyze the product desired by following the suitable methods.


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ix) Proposed Table Data Collection

Seed/Inoculum No Time

(h) OD (10 times dilution)

Real OD

1 0

Main experiment

No Time (h)

Absorbance

Optical Density

OD

(10 times dilution)

OD read

Absorbance

Real OD

(ODread times 10)

Empty Centrifuge

m1

Dried Centrifuge tube + sample

m2

Cell Dry Weight X

(g/L) (m2-m1)

Substrate S

(g/L)

Product P

(g/L)

1 0

2 3

3 6

4 9

5 12

6 15

7 18

8 21

9 24


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5. Report: (100M) Plagiarism is highly prohibited.

1 Abstract/Summary (5M)

2 Introduction (5M)

3 Aims/Objective (5M)

4 Theory (10M)

5 Apparatus (5M)

6 Methodology/Procedure (10M)

7 Results (10M)

8 Calculations (10M)

9 Discussion (20M)

10 Conclusion (10M)

11 Recommendation (5M)

12 Reference/Appendix (5M)

6. References Buffer calculator: http://home.fuse.net/clymer/buffers/phos2.html Shuler, Michael L., and Fikret Kargi. Bioprocess engineering. New York: Prentice Hall, 2002.

Garvie, Ellen I. "The growth of Escherichia coli in buffer substrate and distilled water." Journal of bacteriology 69.4 (1955): 393.

Documents

LAB 1 Edited 4 Mac 2015