Experiment 1 (Day 1) : Further Questions

The questions found in our lab manual are adressed here.

1) State the differences you observe between a microbial bioreactor and a mammalian cell bioreactor.

Mammalian cells are more prone to damage by shear forces and hence require more gentle aeration and agitation as compared to microbial cells. Hence, the bioreactors used for mammalian cells are usually stirred tank with modified impellers (e.g. marine propeller type) at lower speeds of approximately 10-100 rpm and modified sparger for gentler aeration (e.g. bubbleless aeration).

Airlift fermenters or bubble columns maybe also used for mammalian cells to provide an environment even lower shear forces. Perfusion bioreactors used with immobilisation techniques are also used to provide a low shear force environment. (E.g. using micro-carriers or hollow fibres).

Since they are also more sensitive to deviations in culture conditions such as pH and dissolved oxygen, such parameters are more tightly regulated.

Microbial cells being more sturdy and are able to withstand higher shear forces and more extreme culture conditions are instead grown in standard stirred tank reactors with growth parameters not as tightly regulated.


2) Study the work flow on page 1 of your laboratory manual. Describe the typical activities that are performed for each stage in the fermentation process.

Experiment 1: familiarization with the bioreactor and its function.

We got to know about the location of different parts of the bioreactor, their names and function. We also took turns to learn how to do sampling.

Experiment 2: Equipment, media and seed culture preparation.

MEDIA PREPARATION:
We used powdered LB medium to make 2 litres of the media which to be used for culturing the transformed E. coli in the seed flask and in the fermenter. After autoclaving, ampicillin and arabinose were added to the medium in the fermenter.

SEED CULTURE PREPARATION:
Streaking of pGLO transformed Escherichia coli from a thawed cryovial on LB/Amp/Ara plate was first done. After incubation, several colonies of pGLO transformed E.coli from a fresh LB/Amp/Ara plate was transferred to a shake flask containing LB medium with ampicillin and incubated.

EQUIPMENT PREPARATION:
The Fermenter was sterilised and the various probes were calibrated and installed in the fermenter and connected to the computer. Additional accessories (exhaust condensers, air filters and manual sampler unit) are also installed. Reagent bottles (containing antifoam, acid and base) are hooked up to the fermenter via the reagent lines which will run through the peristaltic pumps. Finally, parameters are set via the control panel and the fermenter is ready for use.

Note: Equipment preparation was done by our TSO, Mr Wee Yong Hao. :)

Experiment 3: Inoculation, fermentation and monitoring

100ml of the seed culture from the shake flask was inoculated into the culture medium in the fermenter using a pump. Control parameters are set, and the fermentation was allowed to take place for 24hours. The readings of the control parameters were plotted on a graph by the computer over the course of the fermentation. This graph was analysed later.

For the first 10 hours of fermentation, samples of the culture broth in the fermenter were taken every hour. The absorbance of these samples were taken later and analysed.

Experiment 4: isolation and purification of product

ISOLATION:
Green fluorescent product is an intracellular product; hence the bacteria cells need to be lysed first to release the protein. 3 methods of cell disruption (using enzymes, freezing and thawing, and sonication) were performed on the bacteria cells. After cell disruption, the extract is obtained from the supernatant after centrifuging.

PURIFICATION:
The extract that was obtained from isolation was purified using size exclusion chromatography. After purification, the absorbance reading of 8 fractions and blank was taken using the spectrophotometer set at 476 nm, recorded and analysed.