CSTRworkshop: 2nd day (24th November 2011)

It is our 2^nd day!!

After finished our class on that morning, we went straight to our lovely lab at 2^nd floor and and quickly put on our lab coats. When entering the lab, we saw Mr. Shaman had waited us for a long time. Firstly, we had a mission which was to prepare the base as a buffer for our media. We climb the stairs of Industrial Technology School to go the lab at 3^rd floor and went to the laminar hood. The base of sodium hydroxide has to be prepared in the sterilization condition and was then filled inside the reagent bottle. The base was then installed in the holder and the peristaltic pump head on the bioreactor body with the labeling of base.

Adding of NaOH under laminar flow

After that, we got ready and sat on our own place to listen Mr. Shaman’s lecture, with full attention of course ! He explained about the functions of the probes involved in the fermentation. The probes include peristaltic pump, pH electrode, pO₂ and others. The peristaltic pump is used to stabilize pH in the fermentor. While the tip of pH electrode is immersed in an electrolyte to measure the pH of inoculums and Mr.Shaman also mentioned that there are some precaution steps to take into account. We shall not leave probes aside after fermentation because media will seek into it. We should wipe the pH electrodes using paper towel CO₂ that contain fiber. pO₂ electrode is needed to mountain in polarized and sense dissolved O₂ found in media. If there is too much O₂ pumped in the probe would not show any reading. The rotameter is used adjusting the volume of air that sparged into fermentor in the unit of L/min. It is adjusted by the large knob at the bottom of the housing and turned clockwise in order to open a needle valve. This will allow more gas to pass through, thus further the steel ball, the graduated glass tube is rises up. He also taught us how to calibrate and set the probes.

Temperature calibration

Anti-foam calibration

The rotameter used for controlling flow rate.

Installing the probes on top plate

Back view of the fermentor. It is connected to gas supply and water from tape for colling.

After autoclave those probes, they were calibrated. All of the filters have to be wrapped with aluminum foils to prevent from leakage. Aeration rate is measured in unit of VVM (volume vessels per minute) refer to one min equal to one working vessel volume. There is a formula to obtain flow rate:

1VVM x working volume fermentor = flow rate

The bottom of the ball was indicated as flow rate and was set for 1.5VVM based on the working volume. The speed of stirrer was adjusted to 300rpm. The faster the stirrer stirred,the higher O₂diffusion in the fermentor. The pH control is turned on and calibrated to 5.8, the temperature was adjusted to 30^oC, pO₂ was calibrated to the point of 40% and control turned off. Meanwhile, antifoam was used to reduce the foam forming and it was turned on. The acid, base and feed controls were turned automatically and also peristaltic pump was set to be aut 000.

Before adding inoculums, the rotameter has to be reduced to 0.5L/min while vessels O₂ content increase to 150rpm.

Setting up of fermentor done : )

Aseptic technique used for inoculation

Inoculum was added !

The reading of some parameters involved in the bioreactor by using IRIS software helps us to observe the activity of microorganism and its development. Mr. Shaman had installed IRIS V5 at each group’s computer respectively. We can select the parameters that we want to observe that are pO₂, stirrer, temperature, pH, antifoam, base pump and antifoam pump. The sampling of the fermentation had to be taken from time to time. So it was started at 2p.m., then followed for the next 2 hours, 4p.m., 6p.m., 8p.m. and 10p.m. For the next day, it was continued at 8a.m. and finally at 10a.m.