Members of group 4

Fauzan

"It was one of the coolest experiments I have ever done!" 

Estevan

"Learnt a lot. First time handling such an expensive piece of equipment all by myself. But it won't be my last."

 Azie

"A marathon laboratory experiment isn't so bad after all. I really enjoy working as a team."

Result Interpretation

This growth curve that we obtained from the experiment is not a common growth curve of the Saccharomyces cerevisiae. 

As we can see from the curve, the cell culture achieved 2 stages of log phase with a stationary phase in between.


Interpretation for the growth curve is as below:

Lag phase
Inoculation was done at 0th hour, and the cells went into lag phase for 2 hours. During this stage, the cells which were originated from a 500 ml shake flask were trying to adjust themselves to the new environment. Rate of growth of cells was low at this stage. Lag phase lasted for approximately 2 hours.

Log phase 1

At around 2nd hour, the cell biomass started to exhibit rapid growth. At this stage, the glucose substrate in the fermenter was fully utilized by the cells for growth and cell division. We observed a gradual increment of optical density from 1.600 to 8.270 within 4 hours.

Stationary phase

During the 6th hour to 12th hour, we observed the cells were experiencing fluctuation of growth. The Optical density of the cell biomass was maintained at 7.360, 8.900 and 8.630 for 8th, 10th and 12th hour respectively. The initial assumption we made was that, the cell biomass had reached its maximum due to depletion of the glucose substrate.

Log phase 2 

At 12th hour, we increased the air flow rate of the fermenter from 1.5 vvm to 3.0 vvm to test for test on our initial assumption. We found out that, increasing the air flow rate had also promoted the cells to enter another log phase. It appeared that our initial assumption on the depletion of glucose that had limited the growth of the cells were inaccurate. The actual limiting factor that had limited the growth of Saccharomyces cerevisiae from 8th to 12th hour was the oxygen concentration in the fermenter.  A rapid increment of cell biomass was observed from 12th to 14th hours, followed by a slower increment of cell biomass from 14th to 16th hour.

Decline phase

Starting from 16th to 18th hour, optical density of the cell biomass started to show decreasing trend. At this stage, we had observed another uncommon phenomenon of cell growth where the microorganism had skipped the stationary phase and directly entered the declining phase. A possible rationale is that, the glucose substrate in the culture had really depleted this time. An increase in air flow rate afterwards didn't increase the optical density of the cell biomass. Another reasoning our group came up with was that, the build up of toxic in the culture has inhibited the growth of the cell biomass. However, future study and experimental testing would have to be done to prove that assumption.

Result

The following are the results we obtained from the analysis:

Table 1: The result of the Optical Density of Cell biomass Saccharomyces Cerevisiae in 18 hours.

Graph 1 represent the grow curve of the Saccharomyces cerevisiae

Graph 2: Glucose Concentration over Time. 

Note: The glucose concentration was only measurable until the 4th hours, later than that, the concentration of glucose was too low to be detected by the glucose level device. 

Graph 2 is obtain from the Minifors Iris program that records and monitors the changes along the fermentation process

For result interpretation, please refer to the post on "Result interpretation".

Day 3 (Final day)

The fermentation was completed on the 3rd day. We got our last reading at 7.30am. All the analysis was completed. Mr. Sharman showed us how we could retrieve informations from the Iris software as well as to interpret the graph and data we obtained.

Soon after that, we switched off the bioreactor and disassemble the parts to be cleaned and dried.

We had also thanked and said goodbye to Mr. Sharman for being with us for the past few days. It was indeed a great experience working with Mr. Sharman. Hopefully, in the near future, we would still have the chance to meet up and share experiences again.

Sitting: Dr. Rosma, MR. Sharman, Pn. Wan Nadia (left to right)

with 3rd year students of Bioprocess Technology Industry 2012.

PO2 PROBE CALIBRATION

This process was shown by Mr.Shamam,

He took the pO2 electrode and removed the green plastic end cap.

Then, the bottom metal section was unscrewed to check whether there was liquid electrolyte in the membrane cartridge inside.

There is a bottle provided from the company to top up the liquid electrolyte if the level of the liquid is low.

After that, he replaced the end section, and fit it into the vessel vertically and secured it.

Inoculation

MEDIA PREPARATION FOR INNOCULATION OF SACCHAROMYCES CEREVISIAE

After Mr. Shaman gave us some explanations about the components of the bioreactor and their functions, we had move to second part of our lab which was the preparation of media for the innoculation of Saccharomyces Cerevisiae, which is the yeast that we will use in this experiment.

The media that we used in this experiment was YEPG medium which contain yeast extract (1%), Peptone (2%) and glucose (2%). The total medium that needed for the innoculation was 200ml, so the weight for yeast extract, peptone and glucose needed were :

Yeast Extract (1%) = 2g

Peptone (2%) = 4g

Glucose (2%) = 4g

and after that we dissolved them with distilled water.

Image 1: medium of innoculation

After the medium was done, the media was then autoclaved before the innoculation of the yeast step was taken.

After 3o minutes we prepared seed culture, we using sterile toothpicks to scrape up a single colony aseptically. The work will be completed by transferring the colony into the shake flask which contained medium. We left it overnight (18-24 hours) in the incubator shaker.

How to prepare a bioreactor for autoclaving

     Before undergo the fermentation process bioreactor and all its compartment must be sterile first in autoclave to prevent contamination. Below shows all steps preparation a bioreactor for autoclaving.

1. First, the buffer pH 4.01 and buffer pH 7.01 was prepared.

 2. Then the pH probe was took and the cap was removed  on the bottom and platinum was scraped to make sure it is neutral.

3. The probe was put in the pH buffer to set the point of the calibration curve, C1 is pH 7.01 and C2 is pH 4.01.

4. When calibrated step, the pH probe  need to insert together with the temperature probe to allow comparison.

5. The medium was poured  into the vessel with the adding of 15ml distilled water to make sure that no evaporation lost during autoclaving the medium.

6. The pO2 electrode was took and the green plastic end cap ws removed. Then, the bottom metal section was unscrewed to check whether there was liquid electrolyte in the membrane cartridge inside. There is a bottle provided from the company to top up the liquid electrolyte if the level of the liquid is low. After that, the end section was replaced, and fit it into the vessel vertically and secured it.

7. The reagent which is antifoam liquid was prepared by adding around 10ml in the 250ml bottle. A silicone dip tube was connected to one metal pipe on the head plate was checked. The tubing between the bottle and the pump head is clamped off.

8. Then the other parts of the fermentor was set up, exit gas cooler with filter, inlet filter connected to sparger, free port for inoculation and sample device. Need to double check all these accessories already completely set up. Then need to use aluminium foil to close all the parts that will direct exposed to the air before autoclaved, so that the fermentor will remain sterile after taking out from the autoclave.

9.Bioreactor was ready to autoclave.