1st Day (23rd November 2011)

Woo wooo~our first day in the three days workshop started on 23rd November 2011. For the first time in our life, we met with the fermentor which is one of the main tools used in Bioprocess Technology. 

Mr Shaman Gaspar who is the Regional Manager from INFORS HG explained to us about the details of this vessel. Also, he introduced to us the background of INFORS ,the company which had supplied their most best selling fermentors to many places. 

Mr.Shaman talked about the importance of the lovely tank and the electrodes. According to him, there are 3 types of fermentor used:


 *Bacterial Fermentor
*Plant Cell Fermentor
 *Animal Cell Fermentor

During the preparation of setting up the fermentor, we shall understand its importance. Fermentor is used to multiply the cells in large quantity homogenously in sterile condition. Lighter fermentor builds up yields at higher rate as it carry  fewer burdens.

Different configuration of fermentor has its specific functions on different microorganism and solutions. There are many types of cells, for example:

1.    Animal

2.    Plant

3.    Algae (need light source around vessels)

4.    Bacteria

5.    Fungi

6.    Yeast

7.    Pathogenic microbes

However, there are a few problems while handling with microbes such as fungi that will attach on the glass surface, hence create difficulties when harvesting. Ways are needed to overcome this problem and to upgrade the fermentor to yield better quality products.

In order for us to understand the fermentation process, Mr Shaman told the story about bacterial growth.  Bacteria growth has four stages which are: lag phase, log phase, stationary phase and death phase. 

Lag phase is the phase where microbes are adapting to the medium and nutrients given in the vessel. At this phase, abundant of nutrients is available and oxygen transfer rate is much larger. The microbes which cannot adapt to this phase will die while those that can adapt will survive and continue to the next phase which is the log phase. 

Log phase is a point of successful transfer. At this stage, the cells start to consume the nutrients in large quantity and uses lots of oxygen to grow. Carbon source like glucose, ethanol or glycerol is served for the growth of the cells. At early stage of log phase, the cells are young like babies, but at the end of this phase, the cells grown old to reach adulthood.  During this stage, heat will be produced by some microbes, hence the vessel has to be cooled down by connecting it to the water tab. Antifoam is needed since protein molecule will produce foam in the fermentation process.  

The cells entering another new phase when they approach to senescene. Stationary phase is a stage whereby most toxins present due to the cell stress. Metabolic by-products that inhibit cell growth might be produced and the medium in the process start to turn acidic. Therefore, pH must be managed well.  The carbon source also finished since they are used by the cells during log phase. For the fed-batch fermentation, nutrient will be added to maintain at log phase but in batch fermentation, the nutrient is only added once which is during the preparation stage of medium. 

Cells enter death phase when the cells are dying due to lack of  nutrients available. 

These four stages form the cycle of cells or bacterial growth that happens in fermentor vessel.

Fermentor poses better advantages than shake flask because fermentor enable us to check on the pH during entire fermentation process and most importantly to optimize the media.

We then get to know more about the vessel. 
Mr Shaman introduced to us the configurations of the vessel.

The vessel is equipped with:

(a) Oxygen supplement: when there's not enough oxygen, the air can be supplied immediately to the vessel. Drive motor at the top plate uses to turn the stirrer.

(b) Drive motor: Turn stirrer

(c) Ruston impeller: for bacterial fermentation

(d) Flat Bottom vessel

(e) Baffles: better mixing

(f) Ring sparger: provide bigger surface area or better contact of air with culture

(g) Seal: mechanical seal (lubricate with glycerin). Mechanical seal can prevent contamination.

(h) Steam: during autoclave, steam out pipe should not be clamped  for steam going out during autoclave since the medium is boiling.

(i) Sampling point: rinse with water not with acid, base or solvent to avoid cell aggregates.

Head plate of the bioreactor

The medium is then prepared by adding:
  *Peptone 2%
  * Glucose 2%
  *Yeast extract 1%

with 1.5 liter of distilled water. The tank is 1.7 liter. Hence the head space is left approximately 20%.

The medium then stirred with magnetic bar to dilute the material. After that,the medium is poured into the vessel with addition of 70 ml of distilled water because the medium will evaporate during autoclave process.

Preparation of the medium

The mouth of the vessel and mechanical seal are lubricated with the glycerin. The foam detector was also adjusted to the correct level so that when foam occurs, it can break 85% of foam.

Before putting the vessel along with the medium into the autoclave machine, the silicon tubes must be clamped. Filters of sparger must be clamped to prevent media come up to the filter. Cable tie is used to tie the tube at the connector with the probes of vessel.

Mr.Shaman explaining. 

This is not a very nice take i guess! XP

So what about this ?

Mr.Shaman demo-ing with our group's fermentor.

The medium was autoclaved for about 1 hour before it was taken out. Meanwhile, the starter or inoculum is prepared.

Bioreactor together with the medium gone autoclavation

Our group members had prepared the starter for two times. Supposingly we can leave early, how sad we were ! T.T

The first starter prepared had some problem of vessel leakage therefore we prepared the second starter. Yet, it was time consuming since we needed to wait other groups to autoclave together.

Our members prepared 10% of starter out of the medium 1.5 ml and autoclave it. Then, after cooling the medium, 1 colony of yeast was selected and mixed with starter medium. The whole process was done in aseptic condition.Lysol and flames were used in order to avoid any contamination occur. Then, the starter medium was left  in the shake flask for 18 hours for the yeast to grow.