DAY THREE!
Inoculation, Fermentation and Monitoring
For scale-up fermentation, we let the medium broth cool to below 50degreeC before adding Ampicillin to a final concentration of 100ug/ml. We then added arabinose to a final concentration of 0.2%. With the control parameters set as shown below, we inoculated the fermentor with 100ml of seed culture [5% of fermentation medium volume]. Fermentation was allowed to continue for 24hrs at the conditions listed below.
The cloudy culture!
Monitoring the conditions of the fermentor
We took a 10ml blank sample before inoculation and subsequently a sample of 10ml was taken every hour. This is to monitor the fermentation process. After 24hrs of fermentation, the broth was harvested. 10ml of culture was then transferred into a sterile, disposable test tube.
Here are our results!
below is our Cell Growth Plot...
what does it tell us?
From the graph, we can see that cell concentration is steadily increasing over time for the first 5 hours. The trend shown in the graph should be in the exponential phase as the cell have already adapt to the media and fermenter conditions with its enzymes needed for cell metabolism have already been synthesizes and activated through cofactors and thus begin to proliferate quickly in the medium. Around 6 hours later, we observed that the batch culture is entering the stationary phase as we can see the graph forming a straight line. The cells are not growing very quickly at this stage. The number of cells growing is almost equivalent to the number of cells dying.
our glowing pellet!
Familiarization with the Bioreactor and its Operation
Q1)State the differences you observe between a microbial bioreactor and a mammalian cell bioreactor.
In a microbial bioreactor, both impeller and bubble column device can be installed. Whereas in a mammalian cell bioreactor, only bubble column can be installed. This is so as mammalian cell are very fragile cells, and would be damaged upon the strong propelling force of the impeller.
Q2)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.
Familiarization with the Bioreactor and its Operation (Exp 1)
In this stage, parts and functions of the bioreactor is being introduced and elaborated on. This is to ensure that the operators are clear about operations to carry upon with a bioreactor, such as sampling, throughout the fermentation process.
Equipment, Media and Seed Culture Preparation (Exp 2)
This is the stage where by the input of the fermentation process is being prepared. Equipment has to be ensured sterility and set in the correct operations, media prepared with exact proportion, and that the culture to be seeded is genetically modified to produce the desired product. All in all ensuring that these 3 key factors : Equipment, Media and seed culture, to commence the fermention process to be carried out to be a success generate the desired product.
Inoculation, Fermentation and Monitoring (Exp 3)
During this stage, accurate analyze of the entire process is crucial. This is by inoculating sample consistently (hourly), thus monitoring the progress inside the bioreactor tank. By doing so, graphs can be plot and hence operators are able to deduce the process of the fermentation taking place in the bioreactor. If any point of time the fermentation goes wrong, the operator would be able to make necessary changes to factors such as pH and temperature, so as to divert the fermentation back on track.
Isolation and Purification of Product (Exp 4)
Final stage of a fermentation process is be isolate the desired products out of the bioreactor tank. This can be done through dilution, column chromatography and sonification, and filtering, so as to purify the desired product and thus obtained it from the fermentation process.
Equipment, media and seed preparation
Q1) Media Preparation
a. Explain the purpose of each ingredient found in the LB media.
Bacto-tryptone – Provides essential amino acids needed for E.coli growth
Yeast extract – Provides vitamins and certain trace elements
NaCl – Provides sodium ions for transport and osmotic balance
Water – To dissolve the LB powder and provide an aqueous environment for
E.coli to thrive
b. What is the purpose of ampicillin?
The purpose of adding ampicillin is to screen out any contaminating bacteria as well as to select for only E.coli which have been transformed presumably successfully with the gfp gene which was encoded together with the same plasmid containing the gene for ampicillin resistance(bla gene).
c. Why is ampicillin added only after autoclaving?
Ampicillin is added only after autoclaving as the high heat(121oC for 20min) involved would disrupt the structure of the functional group(beta lactam ring) involved in its antimicrobial activity, rendering the antibiotic useless.
Q2) Equipment Preparation
a. What is meant by calibration of the pH probe?
Calibration of the pH probe involves immersing it into at least 2 buffers, 1 with a pH of 7 and another to match the desired range of pH being measured. The meter is adjusted until it measures the correct pH in the 2 solutions. This makes the probe accurate in measuring pH within that range.
b. Why is Hydrochloric acid not suitable as a correction agent for pH?
HCl is a strong acid and so does not make a good buffer. Usually, a weak acid and a conjugate base is used.
c. What is meant by polarisation of the pO2 probe?
Probe polarization is essential for stable measurements with the same recurring degree of accuracy. With the probe properly polarized, oxygen is continually used up by passing through the sensitive diaphragm and dissolving in the electrolyte solution contained within the probe. If this operation is interrupted, the electrolyte solution continues to be enriched with oxygen until it reaches an equilibrium with the surrounding solution.
Whenever measurements are taken with a non-polarized probe, the oxygen level revealed is both that of the tested solution as well as that present in the electrolyte solution, which therefore results in an incorrect reading being taken.
d. What is a peristaltic pump?
A peristaltic pump is a positive displacement pump that is used for pumping fluids. It works in a peristaltic motion much like the muscle walls of the gastrointestinal tract and is often used to maintain the sterile conditions of the fluid in the bioprocess context.
Q3) Seed Preparation
a. What is the purpose of arabinose?
Arabinose provides an alternative carbon source to glucose for E.coli to metabolize.
b. Describe the sterile techniques used in seed preparation.
Hands were sprayed with ethanol prior to work done which is all within the fume hood.
c. Why do we perform step-wise scale-up instead of tranferring directly to the fermentor?
Conditions within the fermenter is much different than that of a lab scale culture. As such, factors such as aeration, distribution of nutrients must be taken into consideration. Step-wise scale up allows for us to monitor how the organisms adapt to the changing environment better and to adjust the parameters accordingly to cater to the organisms growth.
Inoculation, Fermentation and Monitoring
Q1) Explain the control philosophy for pH, temperature and dissolved oxygen as was used in the fermentation process.
The pH probe measures pH of the culture broth in real time. This information is fed into the main computer control system which provides a negative
feedback mechanism such that any fluctuation in pH is corrected in order to stabilize the pH at the value set. This correction is done by the addition of
appropriate amounts of acid/alkali/buffer through the use of an external containment unit connected to pumps and interfaced with the main computer control system.
The maintenance of a constant temperature and dissolved oxygen content works in a similar way, with the temperature probe and dissolved oxygen probe measuring
their respective parameters and the main computer control providing the negative feedback calculations to allow for the amount of oxygen to be sparged into the
culture broth or the increase/decrease in the rate of flow of cooling water within the cooling jacket.
