THIRD DAY: 24 Hours Lab

THE ONE DAY FERMENTATION

Date : 6 October 2010

Time : 9.00 a.m

Venue : Lab 148

Let's continue the story....

Before fermentation started, we need to do some set up for our mini fermenter. First, what we need to do is to prepare vessel...

These are the checklist :

• Medium already contain all nutrient needed for batch operation. (filled up before sterilization)
• Calibrate pH electrode, with buffer pH 7 for high and buffer pH 4 for low.
• Calibrate pO2. ( we used nitrogen zero O2 calibration and air compressor for 100% O2 calibration.

Now, the bioreactor is ready to be set up.. This is the suitable condition for the little yeast :

• Agitator speed : 300 rpm
• Temperature : 30 C
• pH value : pH 5.0
• pO2 (dissolved O2) : 30 %
• Antifoam control

 !!!!! make sure all the controls were turned ON.

After finishing all set up, we inoculated the seed culture of Saccharomyces cerevisiae  which had been fermented overnight in shake flask at 30°C and 200 rpm.

Now, it is the time for our little yeast to be transferred into their new home:

Mr. Fermenter -MINIFORS.

Scale up : Approximately 10% inoculums added into 1.5 ml medium

We took T₀ sample right after the inoculation.

Here, how we draw out the sample from the vessel.

First, we need to unclamp the tube which connect the vessel and the sampling bottle.

Secondly, pull out some sample by using syringe. Do not too much, as long as enough for OD and glucose reading.

Thirdly, we need to clamp again the tube from the vessel to the sample bottle and unclamp the sample bottle to the outlet. as maintain aseptic technique, sprayed alcohol to the end of the tube.

Now, push the entire sample out from the bottle.

Finish sampling.. Then, take the OD reading using spectrophotometer at 600nm.

While glucose content can be determined by using diabetic blood glucose meter available along with the test strips.

The OD value represent the cell viability inside the bioreactor, while by taking the glucose value we could know how much carbon source they (our yeast) have eaten.

Thus, as fermentation go on, the OD reading should increase where as glucose level decrease because we just feed them once.

Every one hour, we took a sample out from vessel to check the OD value and glucose level. We did it for 24 hours. So that, we could know how our little yeast have been doing…

See the cloudiness of medium??

This picture is taken around 3.00AM after 15 hours fermentation. The cell now is mostly saturated and they has limited medium left.

Here Comes The Problems....

While we are handling our first bioreactor, of course there will be some problem occur. Although we learn theoretically perfectly, but it doesn’t mean that we are mastering how to handle the bioreactor. 

After approximately 7 hour fermentation, foam drastically started to increase. So, we increase the addition of antifoam manually. When we are adding antifoam, the air supply must be stopped because if not, the pressure inside will force the medium to enter to the antifoam tube.

Thankfully we have seen how Mr. Sharman handles the foam formation while calibrating the pO_2. So that, we know what button we should press.

Foaming can cause a big trouble to a fermentor. The foam might reach to the top plate of vessel and enter to the each open line there.

One more problem is pressure. When we handle a bioreactor we should know that pressure inside is higher than outside. So that when draw a sample, ensure which clamps should open or off. If not, oversampling might be happen.