Day 1 - Monday - 29/10/2007

Bioprocess Technology Lab

Contents
Overall objective:
To grow and extract green florescent protein

Individual objectives:

Experiment 1

To be familiarized with the parts and components of microbial and mammalian bioreactors

To introduce the basic operation procedure of a bioreactor

Experiment 2

To describe the steps to prepare a bioreactor

To prepare the media for seed culture and scale up fermentation

To prepare seed culture for scale-up fermentation

Experiment 3

To carry out scale up fermentation process to increase the yield of desired protein product

To monitor cell growth and product formation through manual sampling and computer data logging

Part of Bioreactor	Purpose
Motor	To power impeller
Impeller	To spin culture and to mix media. This ensures that there is no settling of cells
Sparger	Introduce air into fermenter so that cells can take up dissolved oxygen
Baffles	Introduce turbulence and for uniform mixing
Inlet Air Filter	To remove all contaminants found in the air
Exhaust Air Filter	To prevent contaminants from being released into the surrounding
Rotameter	To measure air flow
Pressure Gauge	To measure air pressure of fermenter
Temperature Probe	To monitor temperature changes and to maintain optimal temperature for cell growth
Cooling Jacket	To cool down reactor and control the temperature of reactor
pH Probe	To monitor pH level changes and to maintain optimal pH for cell growth
Dissolved Oxygen Probe	To measure the amount of dissolved oxygen so that cells get adequate oxygen for growth
Level Probe	To maintain constant amount of media
Foam Probe	To measure the level of foam produced so that it does not choke up the fermenter system
Acid	Added when pH level is too high to provide optimal pH for cell growth
Base	Added when pH level is too low to provide optimal pH for cell growth
Antifoam	To reduce the foams produced
Sampling tube	To monitor growth of cells
Control Panel	To specify all parameters

Procedure for preparation of equipment, media and culture:

Media for Seed-Culture and Scale-up Fermentation

Today was the first day of our bioprocess technology (BPT) lab. It was quite fun coz we managed to do quite a number of hands on stuff. The first thing that we had to make after the briefing was preparing the media. We were asked to make about 2.0 litres of Luria-Bertani (LB) media. We had to make that first coz the LB media was needed for both the seed-culture and for the fermentation media.

The commercially made LB broth powder, which made our life easier.

Making the media was quite easy as we only had to measure and add the ingredients together. The only challenge was how accurate our measurements were. The recipe was given to us, which was 10g of Bacto-tryptone, 5g of yeast extract, 10g of NaCl, 1000ml of distilled water and maintaining the pH at 7.5. Since the lecturers were nice enough to make our life easier, we only needed to add the pre-mixed water with 1000ml of distilled water. The other ingredients were already mixed for us. Even the overall pH! How lucky was that.

Immanuel and Filza measuring the LB broth powder.

Look at how precise it was. We were suppose to measure 50g of the LB powder.

Anwar and Filza adding the smelly powder into the distilled water.

Anwar making sure that the LB broth was exactly 2 litres.

Anyway, we transferred about 100ml of the media into a 500ml shaker flask and the remaining 1.900ml of the media into the 2-litre bioreactor. Both the shaker flask and the bioreactor was autoclave at 121^oC for 20 minutes. We were lucky (yet again) that the lab technician helped us to do this part of the procedure and even assists us in adding the ampicillin to a final concentration of 100ug/ml (for both flask and fermentation media), after the broth was cooled to about 50^oC.
The media preparation is complete so the media was kept at 4^oC till inoculation.

Wahyuna excitedly poured the LB broth into the 2 litre jar.

Our LB media all done! (looks like urine though~)

Next we had to prepare the bioreactor. This was a little more complicated. However, we had the assistance of the lab technician to help us in majority of the preparation work. He explained to us what had to be done. So, first thing first, we had to calibrate the pH electrode using the standard buffer solution. After calibrating it, all the probes were installed like the probes for pH, pO₂, foam and level. The foam and level probe was also adjusted. When the probes were placed correctly, the addition agent lines for acid, base and antifoam were connected. The levels in the storage bottles were also checked and it was brought to our attention that hydrochloric acid is so not suitable as a corrective agent for pH.

The next step was installing all the other necessary accessories such as the exhaust condensers, air inlet and exhaust filters and manual sampler unit were.
After installing, we checked the water jacket to see if it is filled with water.

When all the installing was completed, we prepared the bioreactor for sterilization. To do this, we had to disconnect all cables except the temperature probe, which is also autoclavable. We also had to clamp all the silicone tubings’ except for the exhaust filter and female STT coupling of sampling unit, cover all the filters and sockets with aluminum foil to protect from condensing moisture. Finally when we finished all the preparation, the bioreactor was autoclaved with steam at 121^oC for 20 minutes. The pO₂ probe was polarized for 6 hours and calibrated by aerating it with nitrogen.

Finally, the final two steps in preparing the bioreactor. First, was having to connect the addition lines to the peristaltic pumps. Of course we made sure that the pumping direction was correct. Last but not the least, the switch was changed to "Auto" or "manual" where needed.

NOW’s THE GROWING OF OUR BABIES part!
Transformed E.coli with the GFP protein had to be grown up in a media plate with Amp and Ara. WHY?! The reason is partly to remove other types of bacterial strains growing in the media before streaking, or removing those asses that were transformed with GFP. That will be Ampicillin, Amp’s job! After streaking, it’s time to let them grow and they can be transferred to a larger flask the next day.

Just FYI:

1. On media preparation:
a. Bacto tryptone is a protein source for the growing bacteria like E. coli.

b. Yeast extract is used to provide the nutrients for the bacteria growing.

c. DH₂O is used to dilute the solution and to dissolve all the components.

d. Ampicillin is an antibiotic used to prevent other bacteria besides the targeted bacteria from forming.

e. Ampicillin is only added after autoclaving as the heat from the autoclaving can deactivate the antibiotic.

2. On equipment preparation:
a. Calibration of the pH probe meant that the probe reading had to be adjusted for optimal and accurate reading of pH during the run.

b. Hydrochloric acid is not suitable as a correction agent for pH because it is a strong acid and by adding a small amount of acid, it can create a big change in the solution.

c. A voltage is applied across the electrodes of the Dissolved Oxygen probe. Silver ions are oxidized at the anode producing silver chloride and electrons. Oxygen is reduced at the cathode accepting electrons and producing hydroxide ions. The current generated by the chemical reactions is conducted through the filling solution. This current is proportional to the oxygen concentration and is converted by the meter to a dissolved oxygen reading. Polarization is the term used to describe when the probe has reached equilibrium, and is able to produce stable, reliable measurements. For the probe to be polarized, voltage must continually be applied to the probe, allowing the oxygen reaction to proceed. When voltage stops, the reaction no longer continues and the probe is no longer considered polarized.

d. A peristaltic pump is a type of positive displacement pump used for pumping a variety of fluids. The fluid is contained within a flexible tube fitted inside a circular pump casing (though linear peristaltic pumps have been made). A rotor with a number of 'rollers', 'shoes' or 'wipers' attached to the external circumference compresses the flexible tube. As the rotor turns, the part of tube under compression closes (or 'occludes') thus forcing the fluid to be pumped to move through the tube. Additionally, as the tube opens to its natural state after the passing of the cam ('restitution') fluid flow is induced to the pump.

3a. The arabinose serves a GFP inducer. It is an inducer that will induce the cell to produce more GFP when the cell comes into contact with the arabinose in the agar plate. The cells in this study uses arabinose as a carbon source, and if the amount of arabinose available to act as an inducer was low, the production of GFP would be lowered as well.

3b. The environment and materials in the flow hood had to be swabbed with ethanol; the hands, with gloves had to be cleaned with ethanol;

3c. Step-wise scaling up would not result in the cell getting a “culture-shock”. In this way, it is also possible to monitor the optimum growing conditions rather than in a large scale medium. If in a large scale medium, it is very difficult to measure the culture parameters. Also, there are considerations to be decided before upscale such as Stir rate, Temp of media, Factors affecting yield of prod.

Nic & Fil