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.

Parts of a bioreactor

       In the first day meet Mr.Shamam from HT Company at room 148,  he was introduced all compartments of bioreactor. All this part is very important to maintain and standardize the fermentation process for small and large volume production. Below shows a list of compartments of the bioreactor and its functions that was introduced by Mr.Shamam.

 Image 1: compartments inside the vessel of bioreactor

         The function of the vessel is where the fermentation take place and inside it has many of compartments to complete the fermentation process.

1. Head plate:
to cover the top of vessel of bioreactor

2. Antifoam probe:
to prevent contamination in fermentation process

3. Stirrer shaft seal:
To hold the stirrer and head plate

4. Acid/base probe:
A flow to add acid and bases during fermentation process

5. Shaft:
Not allow the medium to escape or microorganism enter

6. 2.5 glass jar bioreactor:
Material of the vessel of bioreactor

7. Baffles:
To prevent a whirl pool effect that could impede proper mixing
per mixing
8. Sampling port:
A valve to get the sample of the fermentation

9. 6- bladed disk impeller:  
a. To diminish the size of air bubbles to give a bigger interfacial area for O₂ transfer and to decrease diffusion path.

b. To maintain uniform environment throughout the vessel content.

10. Air sparger:
To ensure better dispersal of air.

                           Image 2:  All fittings on head plate of the bioreactor

                                                      Image 3: rotameter

 The rotameter is the means of manually controlling the amount of air entering the vessel and for controlling the gas flow rate.

Image 4: holder of reagent bottles

        The function of this holder is where the reagent bottles are usually placed during  

         the fermentation.

                                                 Image 5: reagent bottles

             The reagent bottles that usually needed during the fermentation take place are, reagent bottle for base, antifoam and acid, but in this experiment we do not need acid because we dealing with microorganism, which is yeast (Saccharomyces Cerevisiae) which usually will produce acid trough out the process.

                                                   Image 6: pO2 electrode

            The pO2 electrode should already be connected to the bioreactor base unit which has been powered for at least 2 hours to ensure the electrode is polarized.

 

                                             Image 7: Air compressor

    The function of air compressor  is used to provide air during fermentation process.

 Thats all for the introducing session with bioreactor compartments. Then, we are ready to go the experiment operation.

The sweet treat at KFC

Besides working, we were also invited to have a little enjoyment together at a nearby restaurant. 

It was really enjoyable that all the third year students, Pn Wan Nadia, Dr. Rosma and the postgraduate students who helped in the laboratory were able to gather for a lunch treat by Mr. Sharman. 

The chicken tasted especially delicious that day. We wonder whether it was because the chickens were provided free for us or it was due to the fact that we were having it together that makes it so. Maybe it was both.  =)

Day 2 (Part II)

Diagram 1: Fauzan measuring the Optical density of the sample

After the inoculation, the rest of the day was about sampling. The sampling was done at 2 hours interval. About 10 ml of culture was collected each time into a test tube. The cell biomass of the culture was monitor by measuring the optical density of the culture using a UV-Vis spectrophotometer. To monitor the glucose consumption by the cells, glucose tests were done using digital glucose testing device. Readings were recorded for both the optical density measurements as well as the glucose concentration in the samples.

The sampling was carried out in a total of 18 hours, from 10.30 pm on 20/11/2012 (Tuesday) to 6.30 pm on 21/11/2012 (Wednesday). A total of 10 samples were collected and analysed.

For the first time, we had our overnight experience in the lab. Through this experiment, we could see that teamwork is really important. We took turn, sacrificed our sleep time to make sure that the fermentation process was going smoothly throughout the night and sampling and analysis were done at the specific time. It was great experience!

Diagram 2: Samples collected to be analysed 

Day 2 (Part I)

The day started with briefing on the inoculation process. It is crucial to carry out the aseptic techniques during the inoculation process to keep the contamination risk to the minimal.

With Mr. Sharman's supervision, we inoculated the yeast culture into the bioreactor around 10.30am.

Oxygen told by Mr. Sharman is one of the main factors that control the growth rate of the culture cells in the fermenter. The higher the oxygen transfer rate, the higher the cell growth rate.

The Oxygen transfer rate which is as the KLa is controlled by a few parameters; the rotatory speed of the impeller, the air flow rate, concentration of the oxygen supplied and the pressure in the vessel.

While using Minifors for the fermentation, we can easily manipulate 3 of the above parameters, which are the rotatory speed (rpm), the air flow rate (vvm) and the concentration of oxygen supplied (either we obtain oxygen from the air or from the oxygen tank).

Minifors is built in such a way that makes controlling the fermentation process easier and more efficient. The cascade mode is making use of an intelligent technology that constantly monitor the oxygen uptake rate by the cells in the fermenter. When the oxygen update rate by the cells increases, the concentration of the oxygen in the fermenter decreases, this change is detected by the oxygen regulatory system of the fermenter. With the cascade mode technology, rotatory speed will be increased spontaneously. Thus, the oxygen diffusing rate to the media will increase as well. 


Further reading on:
1. Inoculation process, please refer to the post on "Inoculation"
2. Calibration of the PO2 probe, please refer to the post on "PO2 probe Calibration"

Day 1

Introduction of bioreactor by Mr. Sharman, the Asian South Pacific Regional Manager from INFORS HT Company.

The first day of the workshop, we were greeted with Mr. Sharman, a very humorous Indian man who was the speaker and mentor for our bioreactor workshop. 

The day started with a little introduction of Mr. Sharman himself follow by general background of the INFORS HT company.

Next, we were given a tour on the anatomy of Minifors bioreactor. Functions of every single part of the bioreactor were explained in great details. 

We were given a brief lessons on the basics of scaling up process of a shake flask to a lab scale/ seed fermentor. 

"The parameters of fermentation such as, the temperature, pH, aeration, agitation and antifoam are the important things that we must take note throughout the fermentation process."  - Mr. Sharman

After the presentation, we were shown the demo of how to disassemble as well as to prep the bioreactor for steam sterilization in the autoclave. 

For the details of:

1. Functions of each part of the bioreactor please refer to the post on "Bioreactor parts".

2. Sterilization please refer to the post on "How to prep a bioreactor for autoclaving"

 

  

Minifors Bioreactor

Minifors Bioreactor

Hi ! Welcome and thank you for visiting our blog ! 

In this blog, you will read about our 3 days Bioreactor Workshop in the Bioprcessing Technology lab, operating on the very stylish, high-tech., table-top Continuous Stir Tank Bioreactor.    

We must say that the experience is both wonderful and beneficial. We all enjoyed working with the "little smart glass jar". 

We would also like to take this chance to express our gratitude to Pn. Wan Nadiah, the lecturer of IBG 302 Bioreactor Operation for her work on arranging such an excellent workshop. To, Mr. Sharman, we couldn't thank you enough for travelling all the way from Kuala Lumpur, spending your time and sharing your precious knowledge with us. 

Do enjoy spending your time to look around.