3.3

Preparation of

Microorganism

1. Inoculate the E. coli cells on agar media by inoculation loop in

aseptic conditions, firstly. Culture the cells at 37 ^C.

2. After colonies grown on petri, take a single colony from petri

and transfer into 5 mL of broth media. Scale up until 40 mL of

broth media in 250 mL of Erlenmeyer with following the

culture grown (see Notes 9 and 10).

3. Use the 40 mL of culture to inoculate bioreactor.

3.4

Operation of the

STR

1. After the sterilization process, transfer the sterile growth

medium into bioreactor in aseptic conditions.

2. Inoculate 2% (v/v) of the cells corresponding to 40 mL into

the bioreactor in aseptic conditions.

3. Connect

the

air

pump

to

head

of

sparger

port

with

silicone pipe.

4. Operate the STR under suitable conditions at 150 rpm and

1 vvm (37 ^C) with following from the control unit of bioreac-

tor (see Notes 11 and 12).

3.5

Measurement of

the Oxygen Transfer

Coefficient: kLa

1. Check continuously the optical density of cells for following

the growth.

2. After the cells reach to logarithmic growth phase, be ready for

determination of kLa through dynamic method in bioreactor

(see Note 13).

3. For de-oxygenation of the broth at time, connect the nitrogen

tank (see Note 14).

4. Sparge nitrogen gas into the bioreactor for displacing with

oxygen.

5. Use DO probe and monitor the changes in DO concentration

(see Notes 15 and 16).

6. In this period, C drops (Fig. 1).

7. After observing the value of DO as 0% saturation (or near %0),

cut the nitrogen supply (see Notes 17 and 18).

8. After C drops, aerate the fermentation broth through pumping

at specific operating conditions like a known constant air flow

rate (see Note 18).

9. As a function of time, C increases. Monitor this change in DO

concentration with following the air inflow start (Fig. 1).

10. Measure C values in different times in the bioreactor and

record these several values with respect to time using chronom-

eter for the plot (see Note 19).

11. Assume that the re-oxygenation of the fermentation broth is

fast compared to cell growth, so that reaching of level of DO to

a steady-state value will be shortly. C^* which is the steady-state

value shows an equilibrium between supply and consumption

of the oxygen (see Note 20).

Volumetric Mass Transfer Coefficient Measurement

21