Day 3 with Minifors-4

On the third day, we arrived at the lab at 10am also. This should be the last time to take the sample from our lovely bioreactor. What we can be summarized as:

•          Continue collecting result
•          Measure for glucose content of the medium inside the bioreactor manually using diabetic blood glucose meter. Glucose content is in mg/dL
•          Result is noted.

Result

Table 1: OD and glucose reading of Saccharomyces cerevisiae within fermentation time.

Time (hours)	OD	Glucose (mg/dL)
0	0.049	1828.0
2	0.172	1756.0
4	0.228	1656.0
6	0.579	1183.9
8	1.448	952.2
18	1.975	438.0
20	3.328	276.0
23	2.940	61.2
25	2.620	Low

Fig 1: Graph of absorbance versus time 

Fig 2 : Graph of glucose concentration versus time

Figure 1 has shows the increasing in OD reading. This indicates that cells are continuing growing until 20 hours. After this point, cells are starting to die and available living cell start to use up all the available content in the cell debris. So, OD measurement is decreasing. Overall, the growth curve obtain still a sigmoid (follow the theory). This is because there are no adding medium or nutrient such as glucose into the fermentor.  By referring to figure 2, glucose is decreasing by hour. Cells continue to use the available glucose in the medium. After 23 hours, there is no more glucose that cell can get in order to continue living. So, they are starting to die because of glucose depletion.  We can conclude that stirred tank bioreactor that we used is very efficient in controlling the environment inside vessel. It can control every parameter affect growth of cells which are agitation, aeration, foam formation, pH and temperature in order to get perfect cell growth pattern.

Closedown and cleaning

After the results were obtained, Mr Shaman announced that our bioreactor need to be closed down and cleaned. He also demonstrated the ways to clean a bioreactor. In order to maintain the conditions of the bioreactor, there are several techniques that must be followed. The technique are as below:-

•      Any feeding or reagent was stopped added.
•      The stirring speed was reduced to 300rpm and active aeration was stopped.
•    The remote control from Iris is OFF.
•   The temperature was set to 60⁰C and it was allowed to hold there for about 30 minutes.
•   The reagent bottle lines were emptied and the reagents were carefully discarded.
•    The reagent lines were washed with DDW, the bottle was rinsed and reassemble.
•    The pH probe and pO₂ electrodes were removed, cleaned and kept to the boxes. The vessel was allowed to cool then the top plate was removed.
•    Lastly, empty the vessel and it was rinsed thoroughly and then reassembled.

However, our group, group 4 is a little it different from the other groups because....... We are the pioneers! and our yeast cells are pioneers too! The cells in our bioreactor seemed to be growing quite happily, so Mr Shaman encouraged us to wait for some time until the afternoon before discarding the fermentation process. From the last sample taken at 10am, we've taken another 2 more samples at 1pm and 3pm respectively. 

Here, we had some pictures to share with all of you:

IRIS software showing the Oxygen level is decreasing as the cells consume it.

The calculation of the glucose level in the bioreactor went like this:

1. We need to note that the the glucose meter has a detection limit of 20 - 600mg/dL and our sample must be diluted before readings can be taken so that they fall between the detection range.
2. The initial glucose concentration that we used for fermentation was 2%. This is equivalent to 2g/ 100mL of medium. Converting them into the same units as what glucose meter could read, it would be 2000mg/dL.
3. Therefore, in order to ensure that the glucose level is within the glucose meter detection range, we made a 4 times dilution for the sample.
4. The calculation for glucose level for sample 1 at t=0 was done like this..

Glucose meter to measure the glucose level

The conversion table for blood glucose monitoring.

We use it to calculate the glucose level of sample.

 

Glucose meter gave a reading of 25.4mmol/L.

Referring to the conversion table for blood glucose monitoring, 25.4mmol/L falls within 25 and 26.4mmol/L.

Interpolation of the data has to be calculated. The same calculation was done for other samples.

Besides the 5 Minifors that we had in our lab, we recently also bought a 15L bioreactor, and it looked huge as compared with our Minifors. Mr Shaman introduced to us the Techfors-S and how to operate the bioreactor in a correct way.

15L CSTR

Sophisticated isn't it?

Also, we've taken some photos during the fermentation of yeast cells:

Bioreactor filled with fermentation medium and yeast cells..

while the experiment is running, do you see the cloudiness of the medium..?

With all the cleaning process being carried out, our 3-days Bioreactor Operation workshop had come to an end! It's time to wave goodbye to Mr. Shaman and also put a fullstop to our romantic story with Hawrylenko Technik.

Finally, a group photo with Mr. Shaman

Oh ya!

Did you notice the title of our first post?

'I.N.teresting Fermentation Of a Romantic Story with Hawrylenko Technik'...

Have you ever wonder why it was so long? No...? Well, think again!

The answer behind it: INFORS HT.