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

She was there every time we did our lab.
Just like she was staring at us.
We did not dare to approach her.
She was so inapproachable...

To us, 

She looked so precious.
She sounded so high tech.
She seemed so fragile.

........ Finally, the day had arrived.

We were told that we can have a date with her for three days.

We were so excited!

 

Can u see the excited face? 

We couldn't wait for the day to come!

The details of our first date:

Place: Lab 148
Time: 10am
Date: 23 Dec 2011 

We were in Lab 148 before 10am on that day. We needed time to prepare ourselves physically and mentally. 'Tik Tok Tik Tok..' Clock is ticking away. Suddenly, a special guest stepped into the lab. "Hello and Good Morning" he said to all of us. Automatically, we replied "good morning". After our lecturer Puan Wan Nadiah introduced him to us, we knew that our special guest named Mr Shaman, and he was invited to provide us a "Three Days Fermentation Training" programme. Without wasting much of time, he began his presentation about some general knowledge on fermentation.

Mr Shaman introduced the history background of INFORS HT, the company which manufactured our bench-scale bioreactor called MINIFORS. That's how we started to get to know her and a three days precious date with her- our lovely bioreactor!

Bioreactor is known as a fermentor too, and it can be used for bacterial/microbial fermentation, Plant cells and animal cells culture. Bioreactor enables the cells to multiply with high efficiency and high density.

In his presentation, Mr. Shaman also explained the growth curve to us stage by stage, and what we should take note in each phase.

• Lag Phase: When cells are transferred from shake flask into a bioreactor, they will experience 'culture shock', and need time to adapt to the new environment. (Mr. Shaman used a very interesting comparison, Shake flask resembles a 1 room flat to the cells while Bioreactor resembles Equatorial hotel with buffet breakfast, swimming pool and etc.)

• Log Phase: Cells start to grow rapidly and causes heavy nutrient consumption, this is where the exponential part of the graph begins. Carbon dioxide stripping occurs, and also foam will be formed. The speed of the agitation can be increased depending on the types of cells, but for our fermentation, yeast cells can go up to 900rpm.

• Stationary Phase: Cell growth is close to cell death. This situation is due to the toxins produced by the cells and nutrients depletion. Oxygen level in the reactor will start to increase again as the cells cannot combine carbon sources with oxygen to from carbon dioxide.

That's Mr. Shaman explaining the growth curve

After some very useful knowledge being pumped into our head, it's time for Hands-On-Experience!!! We'll be handling the bioreactor ourselves, yay! Whole class which consisted of 24 students was divided into 5 groups as we had 5 MINIFORS in our lab, and we, the group 4, had Chua Kee Yu nominated as our group leader! Cheers :)  With this, we introduce the super agents who had taken good care of MINIFORS 4 throughout the three days workshop:

The most important step before beginning with our experiment was to get familiar with our Minifors friend here. The photos below showed the labelled parts of the bioreactor and their respective functions:

 

1. Filter : Removal of microorganism , dust , etc from process air or gas. 
2. Condenser : As a heat exchanger, through which cool water is passed. Volatile materials and water is passed. Volatile materials and water vapor condense on the inner surface which allow to minimized water evaporation and loss of volatiles. 
3. Sparger : Breaks incoming air into small bubbles. The smaller bubbles will stay up longer without joining with others again. Hence, it increase the chances to be take up by cells. (      oxygen transfer rate)    
4. Antifoam : A chemical additives to reduce and hinder the formation of foam. 
5. Base : pH buffer. To maintain the pH in the medium. 
6. Sterilized  syringe : To return culture in the tubing into the vessel by pushing down on the syringe plunger. 
7. Syringe : To draw the culture into the sample. 
8. Drive motor : To drive the agitated shaft.
9.  pH probe : Sensors for pH which connected to the fixed cables. 
10. pO2 probe : Sensors for pO2 which are connected to the fixed cables.
11. Head plate : Consists a series of ports , items attached to the head plate gives the gives the bioreactor its functionality. 
12. Water jacket :  Function as watercooling system. 
13. Rotameter : The means of manually controlling the amount of air entering the vessel. 
14. Glass vessel :  The body of the fermentor which is Round bottomed.
15. Control panel : The 2 LED displays show the actual values and set points etc for the various parameters which are measured and controlled. 
16. Impeller : To diminish the size of air bubbles to give a bigger interfacial area for O2 transfer and to decrease diffusion path. To maintain uniform environment throughout the vessel content. 
17. Baffle:  To break the liquid flow ( increase turbulence and moxing efficiency).

Also, we have to get used to the operation of the control panel on the bioreactor itself.

• Alternative Function Key: Select the one parameter value to be permanently displayed in the left hand LED display. 
• Second Display: Parameter values shown cycle every few seconds. 
• Function Key: To select a chosen parameter or use the options choices. 
• '+'  key: Increase a set point or calibration value in the LED display. 
• '-'  key: Decrease a value in the LED display. 
• ON/OFF Key: For switching parameters On and Off and starting procedures such as calibration. 
• Options Key: To cycle through choices available for each parameter.
• Icons:

(a) Relating to right hand display- parameter values displayed, foam warming & pump settings.
(b) Relating to left hand display plus power on, external control and other functions.

• Left Hand Display: Show one parameter value permanently when in operation and provides information about functions such as calibration when used with right hand display. 

  

Introducing the parts of the top plate too!

Top plate of our Minifors vessel

Before we can actually work on our own, Mr. Shaman demonstrated to us how to set up the Minifors for fermentation. First of all, he switched on the Minifors. After that, Mr. Shaman poured buffers pH 7 and pH 4 from the packet into two labeled bottles respectively (these buffers are reusable, so we should keep them properly). We were told that the pH electrode had to be stored by dipping the tip in 3 mol of potassium chloride (KCl) when we are not using the electrode. To use the electrode, the cap filled with KCl had to be removed and then the pH electrode was rinsed with distilled water. He opened the top of the pH electrode and made a connection from the Minifors to the pH electrode by pulling down the red top collar and screwed them tightly into place.

 

Putting the electrode aside, the calibration can now be carried out for pH electrode:

1. Press F(forward) button till the light on the panel shows 'pH'.
2. Press the Menu button twice so that the “cAL” (calibration) is showed on the control panel. 
3. [2 points calibration is done, as we have 2 buffers] Press the “Increase” button till a number “2” appeared on the control panel.
4. Press 'Enter'.
5. Dip the cleaned pH electrode into the bottle filled with buffer 7.01. Along with this, temperature probe is also immersed into the buffer together with the electrode. (This is done to compensate the pH and temperature. Reason: pH will change with the shift in temperature. For instance, if we do the calibration at 25 ⁰C and the temperature of the culture is 30 ⁰C, then we will have a degree of error on pH).
6. Wait for the reading on the control panel to be stable, and adjust to 7.01 if it doesn't show the correct reading.
7. The same procedure was applied for calibration on buffer 4.01, after rinsing both the electrode and temperature probe.
8. At the end of calibration, remove the pH probe from the Minifors store it in KCl.
9. Switch off Minifors.

Followed next, was the preparation of the pO2 probe. It is very important to take note, that every time when you're done using the pO2 probe, you have to store it in its box carefully in a horizontal position because there is very thin membrane at the tip of the probe which could be very fragile.

1. Open the cap of the probe, and check whether it is clean.
2. Suck up some electrolyte, which is provided along with the probe and fill the cap till it is half full.
3. Reinstall the cap back onto the probe. (it's normal if some electrolyte overfill) and set the probe aside.

Now, it's time for the preparation of fermentation medium.

1. We added 15 g of yeast extract, 30 g of peptone and 30 g glucose into a beaker.
2. Fill the volume up to 1.5 L with distilled water.
3. Put the beaker on a hot plate till all the ingredients are dissolved. 
   

The components added into the fermentation medium.

Nurul carefully weighing the components.

Then, we set up all the parts of the vessel.

1. Remove the 3 black screws which lock the headplate to the glass vessel.
2. Pour the fermentation medium (~ 1.5L) which was prepared previously into the glass vessel.
3. Add in about 60ml distilled water (to account for the loss of medium due to evaporation during the autoclave).
4. Lubricate the top plate with silicone grease/glycerin. (Put on gloves)
5. Make sure the sparger is fixed at the right position whereby it is located at the split on the baffle cage.
6. Fix back the black screws.
7. Adjust the height of the antifoam probe to a suitable level with a spanar.
8. By using a syringe, put some glycerin into the mechanical seal on top of the agitator shaft.
9. Fix pO2 probe into its position on the top plate and make sure there is an O ring.
10. Fix pH probe into its position also. Use a HEX spanar to tighten the probe.
11. Fix exit gas cooler.
12. Fix the super-safe sampler.
13. Only base is used in this fermentation as a buffer system, hence the other 2 reagents inlet are blocked with silicone tubing connecting both of them.
14. Acid/base are never autoclave. So seal the base inlet on the top plate with a blanking port with one end tied.
15. Two filters with 0.2-0.45 microns size each, are fixed individually at gas inlet and exit gas outlet.
16. Fit filters on the reagent bottles. For the antifoam bottle, fill with diluted antifoam. For base bottle, just fill with a little bit of distilled water.
17. Label the direction of flow on the peristaltic pump (Left out, right in).
18. Cover all the openings with non-absorbent cotton and a layer of aluminium foil on the outside. All the tubes have to be clamped before and after the filter to prevent the flow of medium to the filter and also the steam from going in to the filter.
19. All inlets are covered except gas outlet tube.
20. Take the assembled vessels to the autoclave and allow it to be sterilised.
21. After sterilization, cool the vessel and fix it on the bioreactor structure. Then connect the pO2 probe to allow polarisation for overnight.

Putting the assembled vessel into the autoclave machine

The last step that we performed on the first day was the preparation of seed culture.

Yeast cells that were inoculated on agar medium was transferred aseptically into pre-sterilised medium in a shake flask. About 1 or 2 loops of yeast cells were taken. Then the shake flask culture was incubated overnight at room temperature.

Aseptically transferring of yeast cells into shake flask culture.

Mr. Shaman told us quite an interesting joke: Do you know how people used to shake their shake flask culture during incubation in the old times?
Answer: They actually hold it with their two hands and shake it physically!

Hence, our very funny leader...

Trying to imitate how people in the olden days 'SHAKE' their flasks..

The look of medium in the vessel after autoclaving and before inoculation. It's crystal clear!

That marked the end of our first day with MINIFORS-4! 

Day 2 with Her- Minifors!

Early morning on the second day, we were having class before we go and see our lovely Minifors. We rushed to the lab after the class. Mr. Shaman was there waiting for us.

Preparing the base

The first mission we told to complete was fill the reagent bottle with base i.e. 0.5 M sodium hydroxide (NaOH) that used to adjust the pH value of the medium in the Minifors. This step was carried out in a sterile condition by using a laminar hood. The bottle was labeled with its content before filling. After that, the reagent bottle was put on the holder and the base was connected to the peristaltic pump head and then the head plate.  

Preparing base in the laminar hood.

pO₂ calibration 

Why we need to calibrate pO2 probe?

Mr shaman explained that the pO2 varied in different vessel, resulting in each probe to have different characteristics. Hence, there is necessity to calibrate the pO2 probe.

1.Air and water services available and turned on.

2. Bioreactor is set at 30°C. ( put temp probe into pt-100 port)

3. The speed is adjusted to 500 rpm.

4. Anti foam is added manually. ( Before using, antifoam is shaken. The antifoam is drawn from the reagent bottle to the antifoam probe after removing the clamp. 3 to 4 drops of antifoam is sufficient.)

5. The aeration is adjusted to 2.25 L/min. ( reading from the center of the ball in rotameter)

Unfortunately, the filter of our bioreactor is clogged due to the use of water-absorbance cotton.

Hence, the aeration rate cannot rise further as the filter is partially or completely blocked! Mr. Shaman said that we should used non-water-absorbance cotton to avoid this problem. However, he managed to solve problem by replacing a new filter for the bioreactor. ( THANKS TO MR SHAMAN !) 

We were told to hold the filter high before Mr. Shaman fixed it.

Mr. Shaman came to rescue! Yay!

6. The last part is connect to the base pump. ( wear gloves to do it)

7. Alcohol is sprayed on the tube and the tube is filled with alcohol inside.

8. After open the clamp, the tube is quickly fitted into the reagent probe.

9. Use a cable tie to tie the probe in order to avoid the base from leaking out.

10. Remove all the clamps ( except the clamp on the sample probe) and aluminum foil.

11. The base is drawn until it reach the top of the headplate.

12. The pH control is turned on and a set point of 1 is given.

13. pO2 set point to 40% and control turn on.

Differences between aeration rate and flow rate.

Mr shaman explained that aeration rate is measured by volume vessel per minutes (vvm), the universal unit. The amount / volume of air which is sparged into the vessel in 1 minute is equal to the working volume of the vessel. For example, the working volume of out bioreactor is 1.5 L . So, 1 vvm refer to the amount of air sparged to the vessel.   

So, in total is 1.5 L in 1 minute.

For flow rate, it is measured by Liter/ mins. Rotameter is used to adjust the flow rate of the volume of the air pump into the vessel .  Using conversion method, we calculate that the aeration rate for the bioreactor is 2.25 L/min in 1.5 vvm. 

Inoculation part

-          We have to inoculate in the “ old traditional way” since the shake flask for our seed culture did not have the side arm.

1. Glass funnel and beaker is spray with alcohol.
2. Put in on flame. (Mr. Shaman demonstrated Magic by using a FLAME gun!) We were stunt!
3. Use hexagonal spanar to open the inoculum port. Spray alcohol at the inoculum port. 
4. The plug is opened and a bit of alcohol is sprayed around the port.
5. The plug is taken out. The glass funnel is put on.
6. The mouth of shake flask is burned with flame.
7. The culture is poured into the vessel.

Mr. Shaman demonstrating the 'traditional way' of inoculating seed culture into bioreactor.

Once the inoculum had been added, observation was carried out using IRIS SOFTWARE . If viable cell are inoculated, we can observe the oxygen level is decreasing.

• For 5% inoculum = the oxygen level will decrease slowly.
• For 10 % inoculum = the oxygen level will decrease faster.

(Time for the inoculum to put into the inoculum port was noted: 1.38pm)  

 

 If seed culture is carried  out in the shake flask with side arm, the alcohol is sprayed at the pipe opening.

The inoculum port is sprayed with the alcohol  and then the shake flask tube is put in. After that, the clamp is opened and the culture is allowed to flow in. The inoculum port is quickly sprayed with the alcohol and closed it.  

Iris

Iris, the software which help us to analysis the various parameters was installed in the computer before the experiment started. Minifors was already be connected to the computer running Iris. The parameters that we going to analyse were dissolved oxygen concentration (pO₂), stirrer speed, temperature of the medium, pH value, antifoam and its pump and base pump. Iris is a convenient tool because we can easily observe the parameters by using it.

Observing what IRIS software reads in our bioreactor.

After everything was in place, the clock showed 2pm, it's time for lunch!!! We were extremely excited today because Mr. Shaman had decided to treat all of us KFC meal! Hooray :D

It was awesome! All of us enjoyed the delicious food. After that, we headed back to our lab to continue with sampling. 

Sampling

The sample from the fermentation had to be taken from time to time. Taking a sample without letting contaminants get into the vessel is made simple with the use of a dedicated device, SUPER SAFE SAMPLER. Thanks to INFORS-HT for this convenient innovation! To draw sample with super safe sampler, a syringe is fitted to the air filter connected to a short pipe on the head plate of the sample device. The syringe plunger was pulled back to draw the culture till the syringe was 1/3 full. Then, the pipe connected to the vessel was clamped and the syringe plunger which was connected to the air filter was pulled back too to suck out all the remaining culture in the pipe. After that, the syringe that filled with culture was disconnected from the pipe and then its plunger was pushed down to fill the culture into a sample bottle. The optical density and the glucose content of the sample were measured.  

Drawing samples with Super-Safe Sample!

The sampling was carried out at the following time:

24th Dec 2011

t= 0 (2pm)

t= 2 (4pm)

t= 4 (6pm)

t= 6 (8pm)

t= 8 (10pm)

25th Dec 2011

t= 18 (8am)

t= 20 (10am)