Experiment 1 Bioprocess Tech
Answers to Questions:

Q1.
1. A mammalian cell bioreactor has four rotameters while a microbial bioreactor only has one.
2. A mammalian cell bioreactor does not have baffles, unlike the microbial bioreactor. This is to reduce the shear forces in the bioreactor as mammalian cells are very fragile.
3. A mammalian cell bioreactor uses marine impellers instead of the flat-blade impellers used by microbial bioreactors as it gives a more gentle agitation.

Q2.

1st stage:
- Get familiar with the different parts of the fermentor, their function and operation procedures. A brief idea about what is the fermentation process is given.
- E.g. motor is used to turn the impeller. If lots of foaming is formed during fermentation process, antifoam is added to reduce the foaming.

2nd stage:
- The media (Luria-Bertani Medium) for seed-culture was prepared. It contains Bacto-tryptone (10g), yeast extract (5g), NaCl (10g), d H2O (1000ml). The final pH of medium was adjusted to pH 7.5.
-Preparation of bioreactor was done by calibrate the pH electrode; install the pH probe, pO2 probe, foam and level probe, connect addition lines for acid, base and antifoam.
- Preparation of seed culture is done – streak plate of E.coli was performed on LB/Amp/Ara plate. The cells growth after incubation is used for scaling up fermentation.

3rd stage:
- One loop of colonies (E.coli cells) was transferred from the agar plate to the culture medium (ampicilin is added to the culture, final concentration is 0.2%)
- The control parameters of the fermentor are set.
E.g. pH= 7.5, stirred speed: min 10%, max 90% (about 500rpm), temperature=32°C or below, airflow: min 25%, max 100%.
- Culture (cells) is inoculated into the fermentor, and 10ml of sample is taken every hour after the culture is inoculated. 11 samples are taken (including the blank).
- 10ml of sample was taken from harvested culture.

4th stage:
- Isolation if green fluorescent protein (GFP) is done. Supernatant was taken out.
- Isoenzyme was used to resuspend the supernatant, in order to digest the bacteria cell wall.
- The tube (contains cells) was placed in liquid nitrogen and then warm time for 3 times to completely rupture the bacteria cell wall. Cell disruption is completed by sonication using ultrasonic waves.
- The protein mixture (contains product) is purified by gel filtration. This is done by separating mixture of molecules by size.


Done by Miss LoanShark ;)