Practical 3 : Introduction to Minifors & Growth Culture of S. cerevisiae

INTRODUCTION

On 19^th of November, we had experienced a good times with the Minifors and Mr Shaman. The experiment involving a brief explaination on the types of fermentors, the processes, the basic parameter in incubator shaker, cascade control and strategy, and the whole steps on working the Minifors.

Our group have been assisted to conduct Bioreactor 3, which unluckily, we encounter some minor problem at the early stage, but with the teamworks, help and guidance from Mr Shaman, and not forgotten the Guidance Assisstants, we been enlighted with the results.

In the morning, we have been briefed with some important informations on fermentation. Mr Shaman did explained about the lag, exponential, and stationary phase, and he reflected all of it onto the aspect of a successful fermentation. The best time to inoculate, or to transfer from the shaker flask (small volume) onto a fermentor (larger volume), is at the middle of log phase. This is because all the cells at this phase has become viable cells. They intend to be actively dividing and growing.

  

However, in stationary phase, the oxygen supply is becoming a limited supply. But then, there are some processes that need a continuous supply of oxygen gas to obtain the secondary products. In a fed-batch fermentation, we can harvest a meaningful amount of biomass or products without the need to undergoes a repetitive runs. 

Also, in some part, our generous Mr Shaman did touch a lil bit on the special requirements of fermentation. Among them are anaerobic type fermentation, a halophiles (metal bleaching), a genetically manipulated microbes, viscous culture, thermophilic bacteria, and extremophiles ( too high or too low pH). Thus, as a student, or perhaps, as a scientis that work in this area, one must be clear enough what type fermentation he/she experimented. He/she must pick a right type of fermentor, processes to suit with the requirements, just like making cake, but more hideous.

Also, we learnt something new, which as new as a baby to us, CASCADE CONTROL. What is it exactly? It a process where we manipulate few variables to keep a certain variable constant. Hmm, sounds hard is it? But eventually, cascade control did help the fermentation went well.

In addition to this, we had the chances to learn more on the aspect of foaming. No no, not anyone on the Earth who conduct the fermentation favors the foaming. Foaming is caused by the cells that produce proteins. Protein then exit the cells, and due to the presence of air, it being trapped and form the bubbles. The antifoam is added either, manually or automated to reduce the foaming.

The air sparger must be fixed under the impeller, so that if the speed of agitation is increase, air bubbles, which are useful to transfer the dissolved oxygen, are then being broken down to smaller sizes, and increase the K_La (and this will be conducted on Practical 5 later).

Here there are, a lot of informations to be digested on a single day. A very tiring day, where in the same time of operating the Minifors for the fermenatation purpose ( The growth culture of Saccromyces cerevisiae ).

Methodology

Preparation of media and reagents

1. Yeast extract, peptone and glucose is weighed in the ratio of 3:1:1 and is added into a flask.

2. 300ml of distilled water is added.

3. The solution is stirred until homogeneous solution is obtained.

4. The opening of flask is closed by using cotton wool wrapped with aluminium foil.

5. The media is autoclaved at 121^oC with 15 minutes retention time. 

6. The flask is stored in an incubator shaker under the conditions of 200rpm and 30^oC overnight.

Setting up bioreactor

1. The bioreactor is cleaned with distilled water to remove any residue from previous usage.

2. Upon cleaning, the orifice of the sparger is inspected for any blockage by running water through the air sparger tubes.

3. The agitator and agitator driver is lubricated using glysol solution until the lubricant flooded at the top of the drive.

4. The bioreactor is assembled. Glass vessel is connected to the base unit, lifting with the handles of the support frame and the stud is placed at the back of the vessel into the metal fork on the support frame.

5. The drive arm is lowered into the horizontal position.

6. The side shield is replaced to keep it out together with the base unit.

7. The exit gas cooler is fit into a free port and the water connecting tubing is checked for length and connections are made ready for the next day.

8. The pO₂ electrode is located and the green plastic end cap is removed. The bottom metal section is unscrewed and the membrane cartridge inside is checked whether has liquid electrolyte in it. If not, up to half is topped from the bottle provided.

9. The pO₂ electrode is fit into the vessel loosely. The top plastic cap is removed from the electrode.

10. The connection point between the vessel and head plate is smeared with high vacuum grease.

11. All internal part of the bioreactor (vessel, sparger, impeller, agitator and head plate) is sprayed with alcohol solution. The head plate and vessel is then connected.

12. Two reagent bottles are prepared (anti-foam and base). Anti-foam is poured into one of the reagent bottles while the one for the base is filled with distilled water prior to autoclave.

13. Yeast extract: Peptone: Glucose (YEPG) medium is prepared with the same ratio of 3:1:1 and distilled water is added until the volume reaches 900ml. The medium is then added into the vessel.

14. All openings, filters and pump are wrapped with aluminium foil and all tubings are clamped before autoclave.

15. The bioreactor is autoclaved for 15 minutes at temperature of 121^oC.

Inoculation into the media and fermentation.

1. After autoclaving, all aluminium foil is unwrapped and all clamps are removed.

2. Base is filled into the reagent bottles and both the pumps of the reagent bottles are fitted to the correct motors and the tubing connected to the multi-way inlet and clamped closely.

3. Temperature, pH and pO₂ electrode are fitted into the vessel and are calibrated.

4. Inoculum is poured into the vessel under aseptic condition.

5. Speed is adjusted to 300rpm and cascade, pH and pO₂ are all on. Fermentation is run and the sample is collected every 2 hours.

RESULT

Table 1 shows the results of the cell density during the 18 hours of fermentation and Figure 1 shows the graph of cell density versus the time along the fermentation. 

Time	OD (abs)
(hours)	Reading 1	Reading 2	Reading 3	Average reading
0	0.643	0.643	0.643	0.643
2	1.840	1.840	1.850	1.840
4	4.440	4.440	4.440	4.440
6	7.350	7.350	7.350	7.350
8	7.960	7.960	7.960	7.960
10	7.670	7.670	7.670	7.670
12	8.600	8.600	8.600	8.600
14	8.300	8.300	8.300	8.300
16	1.470	1.470	1.470	1.470
18	1.750	1.750	1.750	1.750

Figure 1: Graph of cell density, OD (abs) vs. time (hours)

From Figure 1, from the time 0 to 6 hours, the OD is increasing sharply, because the yeast is at the log phase. When the yeast is inoculates into the CSTR, it skips the log phase. This is because the yeast has undergoes the lag phase when we culture it in the shake flask for about 24 hours. When the yeast reach log phase, we inoculate it into the CSTR. These can also shorten the time for yeast to grow faster.

During this phase, the yeast is very active and grows very fast.  It utilizes the nutrients and the oxygen present in the medium very fast. The pH will drop because carbon dioxide is released during the cell respiration. When the pH drops to low, the base will be added automatically to increase the pH to normal range. 

 

From the time 6 to 14 hours, the yeast reaches the stationary phase. The OD reading is almost the same. During this phase, most of the nutrients are used up. Thus, the yeast grows slower. The nutrients become the limiting factor. Besides that, some inhibitors also were produced by the yeast. These substances also inhibit the growth of the yeast. 

Mostly of secondary metabolite is being synthesized during this phase. Thus, if we need to synthesis this metabolites, we need to maximize this phase to get optimize products.

After the 14 hours, the yeast starts to die. The OD starts to decrease. This is because the nutrients in the medium finish.  The cell die because lack of food. If new medium or glucose is adding in, the cell will use up the nutrients and grow very fast.

From 16 to 18 hours, we can see the OD is slightly increases. Maybe the yeast uses the dead cell as nutrients for growth. But, the increasing of cell density is not very much.

Table 2 shows the glucose level that present in the medium and Figure 2 shows the glucose level versus time during the fermentation.

Table 2: the glucose level in the medium during fermentation

 

Time (hours)		Glucose level
Blank		236
0		252
2		209
4		75

 

               Figure 2: Graph of the glucose level vs. time during the fermentation         

 

From Table 2, the glucose level at 0 hour is higher than the glucose level in the blank. This is because when we add in the inoculums, we also add in extra sugar that presence in the inoculums. From time 0 to 4 hour, the glucose level is decreasing, because the yeast uses up the glucose during fermentation. After 6 hour, the glucose that presence in the medium is very little that cannot be detected, because the yeast almost uses up the glucose in the medium. At this time, the yeast reach stationary phase (in Figure 1).

DISCUSSION:

1.  Using of cascade mode

What is cascade? Cascade is one of the applications that provided in the inforst bioreactor to maintain the high yield of the fermentation product. This cascade is most like the auto pilot in the airplane system. This cascade is function to regulate the condition in the bioreactor to become the suitable for the microbial growth in it.

How cascade works? Cascade operates with the help of some equipment like oxygen probe, pH probe, antifoam probe, temperature probe, agitator and aeration. This equipment will perform in one system that makes it operate to get the maximum production of biomass. By referring to the graph that shows in the monitor, we can know all the condition and the action that occur by the equipment in the bioreactor.

For example the rotation per minutes (rpm) of the agitator, the percentage of the oxygen in the medium, the value of pH in the medium, the temperature of the medium, the usage of the antifoam solution and the usage of acid and base in the bioreactor. All this things will be shown in the continuous graph that represents each line that will be referring to the each function by using specific colour.

How to make cascade functions? First of all, we will set up all the equipment according to their place correctly. And then, we will calibrate the entire probe according to their function. First of all, we will calibrate the pH probe. To calibrate it, we will select the pH mode in the bioreactor monitor and we will select the function to calibrate it. Then, we will start calibrate by using the pH 4.01 buffer. During the calibration occur, be makes sure that the temperature probe also included because of the pH will also affected by the temperature. After we done calibrating the first buffer, press enter and do the second pH calibration by using the pH 7.00 buffer and follow the instruction exactly same like the calibration of the first pH 4.01 buffer. After we done both the calibration of pH, we will put the both probe into their place. Then, we will go to the second probe calibration that is pO₂ calibration. This probe will be calibrated by using the different method like the first one. First of all, we will calibrate the maximum percentage of the oxygen in the medium by the increasing the rpm of an agitator to the maximum that is 900 rpm. And then, we also increase the aeration of the vessel up to 1.5 vvm. Then, after the pO₂ reading become constant, we will adjust the value up to the 100 if the value is lower that that and vice versa. After that, we will press the enter button to save the modification. Then, we will go straight to the third calibration that is rpm. In this calibration is regulated by adding the minimum value of the rpm that is between ranges of 200 to 300 rpm while the maximum range of the rpm is between 800 to 900 rpm. The rpm of the bioreactor required is depending on the morphology of the microbial used in the medium. After we already set all the parameter calibration, we can perform the cascade by press the button on at the auto cascade. Then, the cascade mode will be operated.

EXPERIENCES:

During operation of this experiment, our group facing a lot of experiences that teaches us a lot of lesson.

Firs of all, during the set up apparition period, we didn’t aware about the miss connect of the pO₂ probe that make that probe cannot work properly. Because of our group mistake, we cannot know the exact value of O₂ concentration on the medium during the experiment progressing and this problem also affecting to our cascade mode because it cannot depending on the pO₂ reading for making the maximum suitable environment for microbial to growth. Then, we monitor the growth of the microbial in the vessel by referring to the pH value of the medium. The value of the microbial is usually slightly acidic. By referring to the pH we can know that the microbial are happily growth in the medium. In addition, we also referring to the foam production during this experiment progressing. It is because as we know that, the production of foam because of the production of the protein that makes the bubble cannot break easily due to protein bond.

            We also cannot show all of our result in this blog because of the problem occur in our as well. During the set up of all apparatus and the pc from the bioreactor to the computer, we realize that the connector port from bioreactor to the pc was already rusted. But the rusting occur was little bit worst. And due to that problem, we had problem to connect the bioreactor to the pc. But finally, we can connect it after repeating it several times and change the pc with the laptop. Unfortunately, some of the problems also occur when our group was trying to save the data to the laptop and there someone was accidently touch the connector wire and then, all of our group data were missing due to disconnected to the bioreactor. That’s why our group results only depending to the analysis data from the spectrometer reading.

Conclusion

By using bioreactor, we can produce our desired products in large quantity. Besides that, all of the process can be controlled automatically, thus it can save our time. However, all of the process must do in sterile condition. A strict aseptic technique must be used to prevent contaminations that can cause a lot of loses.