Continuing Our LABathon

A fresh morning to start our second day of fermentation! Complete the first day task which filled the reagent bottle with sodium hydroxide instead of distilled water. We transfer it in a laminar flow cabinet to maintain the sterile condition. First, we wrapped the table with 70% ethanol. We carried out the procedures near the flame to maintain sterile condition. Distilled water was poured into a beaker, and then we transfer the base which is sodium hydroxide into reagent bottle as shown in the photo below.

After that, we connect all the connections such as pH probes, base pump and air inlet. We took off clamps and aluminium foil which is along the connections. 

Mr. Shaman shared us the about ‘Points & Pitfalls in Fermentation’ and ‘Handling Pumps and Probes’ which are a guideline for us to handle the preparation, calibration and avoid some faults in fermentation.  He taught us how to use the control panel to set up the various parameters such as ̊C, rpm, pH, pO₂ and pumps. It is quite difficult to learn the way of setting, look like very complicated. May be is first time for me to access and manipulate the real bioreactor. 

Besides that, Mr. Shaman also taught us how to distinguish the aeration rate and flow rate….as well as how to convert the VVM (Vessel Volumes per Minute) to ℓ/min. The VVM is set up based on working volume.  All of us were concentrated on his lectures. 

Before inoculation, Mr. Shaman demonstrated how to set the parameter of the minifors. The parameter depends on the types on inoculums in the fermentor. As mentioned by Mr. Shaman, the settings for parameter of yeast Saccharomyces cerevisiae as follow,

Aeration rate: 1L/min

Speed: 500rpm

Temperature: 30°C

pH: 5

pO₂: 100

Antifoam: no foam

Base: Auto

For 100% calibration, we need to turn on all the services available, such as the tap and air compressor. Thus, the bioreactor must be at the operating temperature. Mr. Shaman demonstrated how to set up of Iris. Iris is software which has been installed in our computer in lab. It provides the data, graph and control which is able to link between all parameter in the minifors.

Followed by we was learnt how to pour the inoculum into vessel taught by Mr. Shaman. To avoid contamination all the steps must be carried out carefully. Prior to inoculation into bioreactor, we must shake the shake flask which contain the seed culture. This action is to avoid any cells precipitated in the bottom of shake flask. Then, we need to spray the ethanol on the spare port, mouth of shake flask and carefully pour the inoculums of flask into the vessel. After that, we quickly place the port closure to cover the hole and screw it down. This step was not easily to carried out. For perfectly do this step, we must always practice. It is because we are inexperienced to perform aseptic technique during inoculation. 

After the inoculum has been successfully transferred into the bioreactor, it was already 2pm!! Mr. Shaman showed us how to take the sample aseptically from the bioreactor so that it will reduce the risk of contamination. We take 3ml of the sample and measure the growth of cell by using spectrophotometer to determine the optical density at 600nm.

At 4pm, we took second sample and adjust the sterile speed from 500rpm to 300rpm. At 6pm, we set the cascade of speed, which is 600rpm and 150rpm for the maximum and minimum speed respectively. We take the samples every two hours in order to observe the growth rate of the cells until 10pm of the day and it will continue to observe on the next day.