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)