Today (i.e. DAY 3 of BPT!!) has got to be the longest day for us! o.O
I mean, seriously!
In a nutshell, Day 3 of BPT involves 'Inoculation, Fermentation and Monitoring'.
What it basically means was that we were to perform a scale-up fermentation process to increase the yield of desired protein product (which was the Green Fluorescent Protein a.k.a. GFP).
Day 3 also involved monitoring the cell growth and product formation via manual sampling and computer data logging.
First, we had to be in the BPT lab by 8 in the morning. After a short briefing by our esteemed lecturer-slash-practical-tutor Miss Ang, we then divided ourselves into groups of pairs for rotating shifts for the collection of samples. This had to be done as we were required to collect one sample subsequently for each hour starting from 8am and all the way to 6pm.
Would you like to see what awaited us that we had to be in school as early as 8 in the morning?
The FIRST thing we saw was our fermentor with the media we previously concocted in it already added in! (The yellow solution was rather clear and that was the media we had prepared a few days back!)
Once the medium broth was cooled to below 50°C, YongHao (our expert laboratory technician) aided us in adding ampicillin to a final concentration of 100µg/ml and arabinose to a final concentration of 0.2%.
While YongHao was helping us to add the amphicilin and arabinose into the fermentor, Nurul then busied herself by labelling the Sample Tubes in her neat-and-pretty-much-tiny handwriting. HAHAHA! ^.^
Note: The diverse timings labelled and indicated on the tubes with a black permanent marker were the various timings required to draw the samples. (Recall that we had to collect samples every hour!)
You can see how we neatly arranged all the sample tubes in order of time ranging from 9am to 6pm!
We are so efficient, I know right! :D
Anyways, right after all those preparations were accomplished, the ever cool and skillful YongHao then aided us to set the parameters control appropriately. The various parameters utilized would be for measuring of pH, temperature, stirring speed and dissolved oxygen. The measurement would be auto recorded to the machine screen, where it is directly connected to the parameters used.
This is the screen where all the diverse parameters' readings could be observed and also recorded.
Advances in techonology in this twenti-first century amazes me, honestly! ^.^
Above, it shows a fermentor with the sparger activated and running and err whirring? (Note: Observe the cute bubbles) LOL.
Not too long afterwards, Geraldine took the very first sample required which was the BLANK. (:
You can see here how carefully she is drawing out the sample from the fermentor via a syringe.
Meticulous girl, she is....
Here, she is seen transferring the sample collected into a tube previously labelled as BLANK.
Afterwards, the seed culture was FINALLY added into the fermentor. It was observed that after addition of the seed culture, the fermentor turned slightly chalky.
A little of a close up of the chalky yellow solution..
Below, you can see the full set up of our uber cute fermentor! (:
(Yes, by now you might have noticed I keep saying how various things are cute.. Well, that's cause they are okay!!)
Sampling time!
First and foremost, the first clump must be removed. This was to allow media from the fermentor to flow through into the anticipating sampling tube.
Cool, huh?
Secondly, the stringe was then pulled upwards to collect a sample into the 'Mini-Collector' or perhaps to be more specific the sampling tube? Hmmmm...
Thirdly, the syringe was pulled downwards and thereafter upwards to clear any remaining media found in the rubber tube connected to the media in the fermentor.
Subsequently, the clump must then be screwed back as quickly as possible. This is to prevent any other media from the fermentor to flow through.
Then, the syringe was temporarily detached from the filter to pull it downward (this is to put in air into the filter so as to 'push' the collected sample into the labelled sample tube), after which the syringe was attached back to the filter again.
Afterwards, the second clump was removed.
Consequently, the sample was dispensed into a tube previously labelled as Xo. This indicates the sample taken at time = 0, just after inoculation of the seed culture!
After which, the syringe was pushed upward to release the sample found in the 'Mini-Collector' (or Sampling Tube) into the tube labelled Xo.
Last but not least, the clump was then finally screwed back on. And yay! All was DONE for the first two successful samples!
Budding scientists, UNITE! ^^V
Thus, this post basically demonstrates how exactly the first two tubes (the blank and the Xo) of samples were collected!
Other samples was also collected the exact same way, only they were taken during different timings of course! :D
After 9 hours of sample collection...
And in the picture above, it depicts the record of Culture Absorbance and Fermenter Parameters!
It basically shows the results of the samples' absorbance values starting from when time = 0 hours (which is also the Control a.k.a Xo), which was just after inoculation of seed culture, until time = 10 hours.
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Discussion of graph of
log (x/x0) against time.
As you can see, we have 10 readings which represents the 10 samples taken hourly throughout the day during the fermentation process. The absorbance value was then taken for these 10 readings. As we can see that the OD600 increased throughout the day, we conclude that the cell density also increased throughout the fermentation.
From this graph, we can roughly see how the cells within the fermentors are growing throughout the day. During the first hour, very little cell growth was being observed. This might be due to the cells trying to adapt to their new environment and not being able to grow that fast. After the first hour till the fourth hour, we can see a steady growth of the cells. This phase is also called the exponential growth. As the cells are now more settled to the environment of the fermentor, it can now grow at its full 'speed' with the presence of rich nutrients. At the fourth hour till the sixth hour, we can see a drop in gradient after hitting a maximum growth at the fourth hour. This can be due to the fermentor reaching its highest capacity to hold and there might not be enough nutrients in the fermentor to support the huge amount of cells, thus, some of cells die. It can also be the accumulation of toxic waste that cause some cells to die, thus, the drop in cell density during that period. However, in some cases, the cells did not die but instead that disintegrate as the cytoplasmic components of the lysed cells let out into the nutrient broth and may serve as nutrients for other cell and thus promote secondary growth which can be seen from the sixth to ninth hour. After the death phase, we can see that there is another growth within the 10 hours fermentation period. This is due to the dead cells providing the 'extra' nutrients need for more cells to grow, thus , another exponential phase appears where there is a steady and high growth of cells. From the ninth to tenth hour, we can see a drop in gradient for the second time. This time, the fermentor maybe too overcrowded and again not enough nutrients to support thus the drop in cell density during this period as some cells might have died.
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