Day 3 - Let's Have Some Action... Inoculation, Fermentation and Monitoring I

Objective

• To carry out scale-up fermentation to process to increase the yield of desired protein product (Green fluorescent Protein)

Procedure - Scale up fermentation

The ampicillin and arabinose stock solutions are already prepared and filter-sterilized.

1. When the medium broth was cooled to below 50° C, ampicillin was added to a final concentration of 100ug/ml and arabinose to a final concentration of 0.2%.
   
2. The control parameters was set as shown:
   
3. The fermentor was inoculated with 100ml of seed culture. The fermentation was continued for 24 hours at the above conditions.
   
4. 10ml of blank sample was taken before the inoculation and 10ml of the sample was taken for each subsequent hour.
   
5. The fermentation broth was harvested after 24 hours of fermentation. 10ml of culture was then aliquot into a sterile, disposable test tube.

Record of culture absorbance and fermentor parameters

Between the first and initial hour, there is no noticeable increase in the E. coli bacteria level as they are at the lag phase. During this period of time, the bacteria is adapting to the new environment and preparing the necessary enzymes to metabolise the nutrients in the medium. Thus there is no increase in the bacteria level.

From hour’s 1 to 4, the rate of bacteria growth increases almost exponentially. This is the log phase and bacteria are able to multiply quickly as there is an abundance of nutrients available for their metabolism.

From hour’s 4 to 6, the rate of growth slows down. This is due to the continuously falling concentrations of nutrients and the continuously increasing of toxic substances. This phase, where the increase of the rate of growth is checked, is the deceleration phase or late growth phase

From hour’s 6 to 11, the rate of growth remains constant and the culture enters a stationary phase or a steady state. At this state, the growth rate of the E. coli is equivalent to the death rate. The medium has minimal amount of nutrients left and a lot of waste material is accumulated. The green fluorescent protein was harvested during the stationary phase.




Here's our precious Bioreactor... (Look! It's cloudy.)


Drawing sample.....


Sample in the tube...


Demostrating to collect our culture of cells.


Collection of culture of cells... Easy as ABC..


Paying full attention to the demostration...


These are all the parameters to control our biorectors..


1, 2, 3... off we go.... to collect our "baby" culture!



live demostration



mastering the art of drawing samples..