EXPERIMENT NO. 2

BATCH FERMENTATION OF RECOMBINANT E.COLI IN BIOREACTOR

USING DIFFERENT CONTROL CONDITION

OBJECTIVES

To study the growth kinetics of E coli in benchtop bioreactor using different

control condition

BACKGROUND

Escherichia coli Bacteria

E. coli is classified as nonphotosynthetic and mesophiles bacteria. There are

hundreds of different types of E. coli recognized by the combination of sugars and

proteins displayed on the bacterial surface. E. coli bacilli have long rods without

separation when grown under limiting conditions. It is enterotoxigenic – secretes toxins

that specifically affecting cells of intestinal mucosa, causing vomiting and diarrhea. The

toxins are; Cholera-like, heat-labile toxin (LT) - adenylate cyclase activator and heat-

stable toxin (ST) - intestinal membrane-bound guanylate cyclase activator. The toxicity

factor must be taken into consideration to ensure safety of handling and product.

Growth kinetic of E. coli (doubling time) has been recognized at 40°C is 0.35

hours. Meanwhile, the minimum, optimum and maximum growth temperature of E. coli

are 10°C, 37°C, and 45°C respectively. As a neutrophile, pH optimum of E. coli has been

identified between 6 and 7 with the lower limit 4.4 and upper limit 9.0.

There are several advantages of using E. coli. It is one of the most-studied

organisms for recombinant protein synthesis and the best-studied microorganism where

its genetics and physiology are far better understood than any other living organism,

which greatly facilitates genetic manipulations. E. coli also do not require any growth

factors: they can synthesize all essential purines, pyrimidines, amino acids and vitamins,

starting with their carbon source, as part of their own intermediary metabolism. Peptone

used in this project (in the media preparation) contains carbon source for that purpose. In

addition, a wide range of mutations with specific characteristics and the well-defined

vectors and promoters are available, which has greatly speeded up the development of an

appropriate biological catalyst. Furthermore, it is easy to attain a very high volumetric

productivity because E. coli has a relatively high growth rate and it can reach a very high

cell concentration (>50 g dry wt/l), as well as high expression levels by selecting a

combination of specific vector and promoter (about 25% to 50% more of total protein).

Finally, the production cost of protein is low since E. coli can grow on simple and

inexpensive media, and the operational cost of the fermentation process is also relatively

low.

However there are also disadvantages of using E. coli. One major problem is it

does not normally secrete protein, therefore the active protein present intracellularly is

limited due to the either proteolytic degradation or the formation of inclusion body and it

makes the product separation and purification difficult. This problem can be solved by

protein secretion. And also, production of recombinant proteins in E. coli typically, is

severely curtailed under slow-growth conditions, such as during the production stage of

fed-batch cultivation, due to the intense competition between normal cell function

maintenance and recombinant protein production for the limited available metabolic

machinery. So, careful medium selection and controlled growth rate is important.

Growth Kinetics

Growth phase of microorganism consists of 4 phases; first, the lag phase – in this

phase cell synthesizes new components and ready to divide. Second, the exponential/log

phase – microorganism grows and divides at maximum rate possible. Third, the

stationary phase – population growth ceases and growth curve becomes horizontal.

Usually attained at a population level of around 109 cells per ml. Reasons are nutrient

limitation, limited oxygen availability and accumulation of toxic waste products. And the

final phase is, death phase – decline in number of viable cells, caused by depletion of

nutrients and buildup of toxic wastes.

There is also mathematical consideration of growth. During the exponential phase

each microorganism is dividing at constant intervals. Thus, the population will double in

number during a specific length of time called the generation time or doubling time.

Because the population is doubling every generation the increase in population is always

2n, n is number of generations. The resulting population increases is exponential or

logarithmic.

Bioreactor Experimental Design

The bench top bioreactor, 2-litre B-Braun fermenter, is set to operate at the

optimum growth condition for E. coli. The design of experiment is shown in Figure 1 and

Bioreactor’s parameters are set according to Table 1.

1: Low Level 2: High Level F: Factor

Table 1: Design of Experiment (DOE) by Taguchi Method

Table 1: Reference for designed experiment for bioreactor condition optimization

Parameters F1

Airflow (vvm)

F2

Agitation

F3

Temp (oC)

Run 1 0.5 200 35

Run 2 0.5 300 37

Run 3 1 200 37

Run 4 1 300 35 F: Factor

Prior to the inoculation, the DCU Tower control unit is switched on, and the culture

medium parameter such as pH, partial oxygen pressure (pO2), agitation, aeration, and

temperature are set according to the predetermined optimum values. pH is calibrated by

using standard solution of pH 4.0, 7.0 and 10. pO2 is calibrated by using nitrogen gas

(N2).

Media for fermentation is prepared without autoclave. Top plate of the bioreactor

vessel is unscrewed and lifted-off. The media then is poured into the bioreactor vessel,

and closed by screwing back the top plate. The bioreactor vessel then equipped with air

sparger, 1M sodium hydroxide (NaOH) solution, 1M hydrochloric acid (HCl) solution,

antifoam solution, pH probe, pO2 probe, and temperature probe. The bioreactor vessel is

then autoclaved.

Preparation of E. coli cell culture

E. coli source is taken from stock culture stored at -80C freezers. The tube is thawed, and a wire loop was used to transfer E. coli into a bijou bottle containing 10ml

media. The culture is incubated for overnight at 37 °C.

Preparation of inoculum

The first requirement for a batch scale process is sufficient inoculum culture to

start full-scale process. A one tenth volume (10%) inoculum was used to achieve rapid

growth without a lag phase.

Using aseptic technique, 1 ml of E. coli liquid culture is pipetted into a bijou

bottle containing 9 ml of media, and incubated for 5 hours at 37 °C. The 10ml starter

culture in the bijou bottle is then transferred into a 250 ml flask containing 90 ml media

resulting 100 ml culture, and then allowed to incubate in a thermostatted rotary shaker set

at 37 °C and 300 rpm for 6 hours (for inoculation in bioreactor). The 100 ml inoculum is

transferred aseptically into the inoculum flask.

Cell Cultivation in Stirred Tank Bioreactor

The inoculum flask is taken to the sterilized bioreactor vessel. By using aseptic

technique, the connection tube of the inoculum flask is connected the inoculum pipeline

of the bioreactor vessel. The connection tube was clamped-off, and by gravity action, the

inoculum inside the inoculum flask will flow through the connection tube into the culture

vessel. The inoculum vessel needs to be lifted quite high to ensure stable flow of the

inoculum. Finally, after all the inoculum has been transferred into the sterilized bioreactor

vessel, the connection tube was disconnected aseptically.

Do fermentation with conditions as shown in Table 1. The agitation, temperature,

pO2 level, and pH are monitored during the fermentation process.

Sampling

Sampling will be carried out by closing the airflow supply first. Then the plunger

of the syringe is pushed down, which opened the outlet tubing. Hose clamp is removed

from the connected tube to the culture vessel, and outlet tubing is clamped-off. The

plunger of the syringe is slowly pulled to suck the sample through the sampling tube into

the sampling reservoir tube. Required amount of sample is transferred into the sampling

reservoir tube, and then the plunger is pushed back a little to empty the sampling tube

from the culture. This action avoids residues of previous samples in the sampling tube.

The hose clamp is removed from the outlet tubing and the transfer tubing from the culture

vessel is clamped-off. The outlet tubing is led into a collector (bijou bottle or yellow-

capped test tube). Sample is transferred into the collector by slowly pushing the plunger

of the syringe. Sample is driven through the rising tubing of the sample and the outlet

tubing into the collector. Flame and ethanol 70% is used during sample collecting for

aseptic purpose.

Sample of 10 ml will be withdrawn every 60 minutes a bijou bottle during

fermentation for measuring the optical density/absorbance (OD, A600), TCN ad CDW,

substrate (glucose), and protein concentration. For protein and glucose concentration

assay, 5ml samples is withdrawn into a yellow-capped test tube, and centrifuged at 5000

rpm before undergo analysis.

Optical density (OD), Total Cell Number and Cell Dry Weight Analysis

Please refer to Experiment 1.

Substrate analysis (Glucose)

Innova YSI 2700 Select biochemistry analyzer with YSI 2710 Turntable is used

for direct analysis of glucose (dextrose) concentration. The enzyme Glucose Oxidase is

immobilized in the YSI Dextrose Membrane (YSI 2365):

Glucose (Dextrose) + O2 H2O2 + D-Glucono-£-lactone

Glucose-oxidase

1ml of sample is transferred into an Eppendorf tube, and centrifuged for 5 minutes

at 10,000 rpm. Then, the sample was transferred into a cuvette, put onto the turntable, and

analyzed.

Protein concentration assay

The supernatant obtained from the cell lysis is used to determine the protein

content. Seven hundred and eighty micro liter phosphate buffer (100mM) and 20μl

supernatant is pipetted into Eppendorf tubes. Then, 200μl Biorad Protein Assay reagent is

added carefully, as it is highly viscous and toxic. The mixtures are shaken and left for a

minimum time of 5 minutes and maximum 1 hour, to allow the reaction. The content of

the Eppendorf tubes then transferred into cuvettes. The spectrophotometer is set to a

wavelength of 595nm and calibrated using a blank, which consist of 800μl phosphate

buffer and 200μl Biorad Protein Assay. Standard curve was prepared earlier by using

Bovine Serum Albumin (BSA), which replaces the supernatant. The standard curve then

was used to determine the concentration of the protein from the OD value measured.

Growth kinetic study

μmax, maximum specific growth rates are determined after the specific growth rate

became constant. The OD600 values of the cultures are determined every one hour until 8

hour. The natural logarithms of these OD600 values were plotted against time and the

slope of the resulting linear regression line was taken as µmax. The values obtained

for

µmax were highly reproducible. Using Michelis-Menten kinetics; for batch growth culture,

specific growth rate μ, is equal to maximum specific growth rate μmax

Ks is known as the saturation constant or half-velocity constant and is equal to the

concentration of the rate limiting substrate. In other words, the Monod model implies that

Ks can be calculated from measured µmax and residual glucose concentrations when

µmax/2, Ks been determined.

True yield such as YX/S, and YP/S are often difficult to evaluate. Although true

yields are essentially stoichiometric coefficients, the stoichiometry of biomass production

and product formation is only known for relatively simple fermentations. However,

theoretical yields can be related to observed yields such as Y’X/S and Y’P/S, which more

easily to determine by plotting graph. The true yields can only determined by expression

from theoretical yields.

ANALYSIS

Parameters for Run 1-4

Time

(min)

RPM pO2 pH OD Glucose Protein Enzyme

60

120

180

240

300

360

420

480

Question:

1) Plot the graph and compare for other Runs

2) Which condition to get the highest μ, td and yield coefficient (YX/S and YP/S)

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