AIM:
* To autoclave the 5L – Fermenter.
* To inoculate the required volume of the shaker flask culture to the fermenter.
* To maintain the conditions required for the growth of Pichia pastoris in the fermenter.
* To determine the KLa of the process by dynamic gassing out method.

COMPONENTS OF 5L – FERMENTER:
v Vessel made of Borosilicate Glass
v Control Panel
v DO probe
v 0.2 air filters (air-in, exhaust-out, addition bottles)
v pH probe
v Temperature probe
v Transmission lines ( Acid, Alkali & Antifoam)
v 2 Rushton turbine type impllers on a shaft mounted on a motor
v Baffle cage
v Condensor
v Sparger
v Rotameter
v Sampling line
v Chiller
v Peristaltic pumps – 3 for acid, alkali, antifoam dosing
v Inoculum transfer bottle + needle inoculation assembly + inoculum port
v Dosing bottles for above
v Silicon tubings for attachments

WORKING CONDITIONS FOR Pichia pastoris:
Working volume : 3 Litres
pH : 5.0
Temperature : 30.0oC
RPM : 200
Media: Tryptone Soya Broth [3Litres]

PROCEDURE :

Ø PREPARING THE FERMENTER FOR AUTOCLAVING
Prepared TSB media was added.
The lid of thefermenter was closed properly.
The pH probe was calibrated and inserted into the fermenter.
The jacket was filled with water.
KCl elctrolyte was added into the DO probe and it was inserted into the fermenter.
The condenser, acid, base and dummy ports were connected and the tubings were clamped properly.
Tubings to the sampling line and the harvest line were connected. The ends were covered with cotton dipped in alcohol and foil.
The motor shaft (dry mechanical seal portion protruding out of the vessel top plate), and DO probes were covered with cotton and foil.
After making sure that the whole assembly had been connected properly, a pressure hold test was conducted. For this, air was started in to the fermentor at a very low rate, with one end of the condensor connected to the fermenter, and the other end to a beaker containing water. As bubbles were formed, it was ensured that the connections were proper and there is no leakage.
To the inoculation bottle used for this fermenter, tubings and filter were connected.
The fermenter and all its accessories were kept in the autoclave at 121oC for 30 minutes.
After autoclaving the fermentor was removed from the autoclave and immediately filtered air was connected to the sparger and the air was started to break the vaccume build-up during the cooling and to cool the contents. Also, all the probes were connected, especially the DO probe which being a polarographic probe, needs at least six hours of polarization for proper results.
Ø INOCULATION OF THE 5L FERMENTER
1. An aeration rate of 2 LPM was maintained in the fermenter.
2. XXXXXX(DO probe was calibrated. )XXXXXX
3. Agitator speed was set at 200 RPM, temperature at 30oC.
4. After aeration and agitation at set temperature was maintained for at least 30-60 minutes, DO probe was calibrated to 100% DO saturation.
5. Shaker flask culture was transferred to the inoculation bottle used for the fermenter under sterile conditions.
It was optimized that 600 units had to be transferred to the 5L fermenter. OD of shake flask was 7.4, so by formula:
Vol of seed transferred= 600 Units/ OD =600/7.4 = 81ml
6. Inoculation bottle was connected through the peristaltic pump to the fermenter. Connect the inoculation needle was connected onto the fermenter under sterile conditions and inoculum was tranferred.
7. The fermenter was run for 24 hours so that the culture attained the required growth before transferring to the next fermenter.
8. Care was taken to ensure that all the set conditions in the fermenter remained same throughout the run. If there was any change in the pH of the culture, acid/base was added as the case may be.
Ø DETERMINATION OF KLa

1. Aeration was stopped to the fermentor when the DO level reached 70%.

2. The DO concentration (CL) was allowed to come to a level just above the critical oxygen concentration level. Aeration was started again at this level.

3. Timer was started and the increase in DO level was noted over a specific interval of time till it reached maximum DO level, i.e. saturation oxygen concentration level.

4. Saturation oxygen concentration (C*) was noted with this medium.

5. ln ( C* – CL ) was calculated.

6. A graph was plotted between ln (C* – CL) versus time and the slope of the graph was calculated.