Thank You !!!

The three days practical class ended ,yet the path leading us to knowledge is still far long to go.
Output had been given by Mr.Shaman, our lovely trainer from Infors while we, Bioprocess Technology students gained a lots of input!


We had learnt the correct ways and safety precautions when operating the bioreactors used in laboratory, understanding each small parts of the bioreactors and about the parameters of bioreactor (agitation speed, oxygen content, flow rate and pH,etc) that are suitable for the cell growth at different stages. Also,we get to know some available solutions to solve the practical problems we might face in an experiment. It was really a great opportunity for us to gain experiences on handling the bioreactor on our own.


Here, we would like to thank our course IBG 302 lecturer, Pn.Wan Nadiah, lab assistants (Pn.Najmah and En.Asmaizan) as well as our graduate assistants for giving us guides and helps throughout the workshop. We appreciate your patience and guidance.
Once again, a special thanks to Mr.Shaman for spending precious time, sharing all sorts of information regarding this bioreactors for us. We are lucky to attend this workshop : )

Result

Result of our one day fermentation is obtained through the measurement of absorbance and the glucose uptake.

Samples were taken at different time, starting on 24th November 2pm, then 4pm, 6pm, 8pm, 10pm and the next day (25th November) at 8am and 10am.

First, we shall look at the absorbance reading using spectrophotometer at 600nm.

Table 1: Absorbance reading at different time.

Figure 1: Graph of Absorbance vs Time

 From the first diagram, we can see that the cells start growing since 0 hr (2pm), it was at the log phase until approximately 4 hours later, it entered lag phase. Cells grew at higher rate and it reach maximum yield at time 6 hr.

After that, the cells slowly decreased. We suspect that the condition in the bioreactor inhibit the cells growth. The cells growth continuously dropped.

Other than the optical density of cells, we also estimate the cells growth by using glucose level measurement. The glucose content of the samples can be tested with the blood glucose meter.

The reading shown in the meter is in mmol/L. So, we have to convert it to mg/dL using the conversion table as shown in figure 2.

Figure 2: Conversion table for blood glucose monitoring.

Simple conversion can be calculated as the example below:

The glucose content read from biosensor at time 0 hr is 25.3 mmol/L.

Simple calculation needed for conversion:

 Table 2: Glucose level at different time. 

To calculate glucose consumed throughout the fermentation, we have to substract the glucose content used from the initial glucose content inside the bioreactor which was 2000 mg/dL.
Glucose used = 2000 mg/dL - 1821.6 mg/dL
                     = 178.4 mg/dL


Total volume of the medium was 1.5 L  (15 dL)
Therefore, 
initial glucose content in the medium 
= 2000mg/dL x 1.5 L x 10dL/1L x 1g/1000mg
= 30 g
At time=0, glucose content consumed 
= 178.4 mg/dL x 1.5 x 10dL/1L x 1g/1000mg
= 2.676 g

Table 3: Total glucose consumed at different time.

Figure 3: Total glucose consumed vs Time

From the graph, we can observe that the glucose consumption was gradually increasing until time = 4.7 hr, it had a sudden drop. However, after some times, the cells consumed glucose again at a higher rate. The cells kept utilizing the glucose supply after that.

We had not much idea with the sudden drop of glucose, it might be some error done when testing the glucose using the biosensor.

However, if we were to compare the relationship between absorbance and glucose uptake, we faced another question. Absorbance graph showed us that the cells stopped growing after sometimes. However, the continuous utilization of glucose indicate that 'some things' were using the glucose supply. Therefore, we highly doubted that there might be some other contaminants which were utilizing the glucose content.

Figure 4: The graph shown by IRIS at time=4 hr

3rd day (25th November 2011)

3^rd day – cleaning up our mess!!

Well, here we are back to the lab 148 again.

:) Luckily today is the last day as we are going to clean up our bioreactor!!  After obtaining the last 2 results (8am and 10am), here comes the star, Mr. Shaman!!

First and foremost, he checked through all the bioreactors to make sure they were still in good conditions. However, some of the bioreactor seemed not that 'well', such as our group. ><  

Anyway, Mr. Shaman claimed that that should be okay if we were to increase the air flow rate. Therefore we increased it from 1vvm to 1.5vvm.

Later, here comes Mr. Shaman again to give us another brief presentation again regarding the large scale bioreactor models. In the presentation (a video actually), it showed how the people in the Ipoh Veterinary Center handle a 30 liter bioreactor. Due to the quality of the video was not that good; we couldn't really see the bioreactor clearly. However, with Mr. Shaman’s explanation, we were able to catch up with the content of the video.

After finishing the video, finally was the time for us to end everything. The yeasts were going breath for their last time. (In fact the yeasts in our group already dead as the graph indicates that there were no more glucose in the medium and no oxygen uptake.) Well, just rest in peace my dear yeasts.

After we switched off the main switch, everything stops. The impellers stop moving, the sparger stop functioning and everything else stops. We carefully extract the last sample required and take it for analysis. We did the procedure of cleaning as below:

1.       Switched off all the functions on the control panel.

2.       Switched off the main switch.

3.       Stopped the pump.

4.       Removed the tube and pipe connected to the headplate.

5.       Removed the antifoam bottle and the alkaline bottle.

6.       Removed the pH probe as well as the O₂ probe.

7.       Unscrewed the headplate and poured away the medium. (goodbye Saccharomyces cerevisiae~~~)

8.       Washed the vessel and the holding part of it as well.

9.       Kept the vessel and the headplate on the table and let it dry.

10.   Incubated the filters and the outlet gas pipes at 70°C.

Sebastian removing the sparger 

Rozi carefully move the vessel containing medium

Pouring the medium 

Hoho...washing done !

 Here we let them dry : )

 After the cleaning part, a big surprise for us!! A 15 liter bioreactor was right in front of us. It was quite bulky if compared to the nice and cute little mini forz that we were using. 

Here comes our ''star of the day'' !

Dr. Shaman explained to us patiently each part of the bioreactor and the usage of it.

Everyone paid full attention

 A 15 liter bioreactor basically has all the features similar to a mini bioreactor, other than they have a trolley of its own. Some of us did rise up a few questions after the briefing of the bioreactor. It was a fruitful interaction session!

Well, that’s basically the end of our final day. We really appreciate that Mr. Shaman who spent the past 3 days with us teaching us the new knowledges and allowed us to have some hands on experience on the bioreactor.

(FYI: pity group 4 as they still need to keep their bioreactor on to see how far that their Saccharomyces cerevisiaecan go on. XD)

Short clip (adding of inoculum)

Mr.Shaman demonstrating how to add inoculum into bioreactor under sterile condition.

Beware!

He hold a GUN !

a flame gun.

haha

2nd day (24th November 2011)

It is our 2^nd day!!

After finished our class on that morning, we went straight to our lovely lab at 2^nd floor and and quickly put on our lab coats. When entering the lab, we saw Mr. Shaman had waited us for a long time. Firstly, we had a mission which was to prepare the base as a buffer for our media. We climb the stairs of Industrial Technology School to go the lab at 3^rd floor and went to the laminar hood. The base of sodium hydroxide has to be prepared in the sterilization condition and was then filled inside the reagent bottle. The base was then installed in the holder and the peristaltic pump head on the bioreactor body with the labeling of base.

Adding of NaOH under laminar flow 

After that, we got ready and sat on our own place to listen Mr. Shaman’s lecture, with full attention of course ! He explained about the functions of the probes involved in the fermentation. The probes include peristaltic pump, pH electrode, pO₂ and others. The peristaltic pump is used to stabilize pH in the fermentor. While the tip of pH electrode is immersed in an electrolyte to measure the pH of inoculums and Mr.Shaman also mentioned that there are some precaution steps to take into account. We shall not leave probes aside after fermentation because media will seek into it. We should wipe the pH electrodes using paper towel CO₂ that contain fiber. pO₂ electrode is needed to mountain in polarized and sense dissolved O₂ found in media. If there is too much O₂ pumped in the probe would not show any reading. The rotameter is used adjusting the volume of air that sparged into fermentor in the unit of  L/min. It is adjusted by the large knob at the bottom of the housing and turned clockwise in order to open a needle valve. This will allow more gas to pass through, thus further the steel ball, the graduated glass tube is rises up. He also taught us how to calibrate and set the probes.

Temperature calibration

Anti-foam calibration

The rotameter used for controlling flow rate.

Installing the probes on top plate

Back view of the fermentor. It is connected to gas supply and water from tape for colling.

After autoclave those probes, they were calibrated. All of the filters have to be wrapped with aluminum foils to prevent from leakage. Aeration rate is measured in unit of VVM (volume vessels per minute) refer to one min equal to one working vessel volume. There is a formula to obtain flow rate:

 1VVM x working volume fermentor = flow rate

The bottom of the ball was indicated as flow rate and was set for 1.5VVM based on the working volume. The speed of stirrer was adjusted to 300rpm. The faster the stirrer stirred,the higher O₂ diffusion in the fermentor. The pH control is turned on and calibrated to 5.8, the temperature was adjusted to 30^oC, pO₂ was calibrated to the point of 40% and control turned off. Meanwhile, antifoam was used to reduce the foam forming and it was turned on. The acid, base and feed controls were turned automatically and also peristaltic pump was set to be aut 000.

Before adding inoculums, the rotameter has to be reduced to 0.5L/min while vessels O₂ content increase to 150rpm. 

 Setting up of fermentor done : )

Aseptic technique used for inoculation 

 Inoculum was added !

The reading of some parameters involved in the bioreactor by using IRIS software helps us to observe the activity of microorganism and its development. Mr. Shaman had installed IRIS V5 at each group’s computer respectively. We can select the parameters that we want to observe that are pO₂, stirrer, temperature, pH, antifoam, base pump and antifoam pump. The sampling of the fermentation had to be taken from time to time. So it was started at 2p.m., then followed for the next 2 hours, 4p.m., 6p.m., 8p.m. and 10p.m. For the next day, it was continued at 8a.m. and finally at 10a.m. 

 Fermentor was connected to IRIS software.
Our group member, Rozi checking on it.

Oxygen level dropped. Meaning that microbial activity was present in our baby fermentor.

 Reading OD of samples using spectrophotometer

Then that’s the end of our story with Mr. Bioreactor for 2^nd day. We will meet again next day!!

bye2… ^^

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.

CSTR

CSTR !?!?


In case you don't know what's CSTR mean,
here it stands for Continuous Stirred Tank Reactor ! 
It is a common ideal reactor used in fermentation process.
CSTR run at a steady state with continuous flow of reactants and products.
The feed assumes a uniform composition throughout the reactor while exit stream has the same composition as in the tank.
CSTR give some advantages, for example: continuous operation, good control of temperature and it is kinda easy to operate or construct.


So what had we done during the workshop? 


Day 1
Introduction of Infors
Concise guide into Fermentation
Preparation of the fermentor (Demo)
Preparation of seed culture


Day 2
Calibration of the O2 probes
Steps for inoculation
Starting of IRIS software
Maintainence procedures and "Do's and Don'ts"
Software lectures
Sampling and parameter optimization


Day 3
Video of fermentors in industry
Viewing of 15 liter fermentor
Cleaning and emptying the fermentation vessel




The fermentation carried out by us is basically the same as the diagram shown below:

and the fermentor we used is as belows:

Minifors 

Some basic understanding of Minifors:

Available in both bacterial and cell culture versions, Minifors is supplied as a package with everything necessary to take the next step in capacity and control from shake flasks or spinners. For example, the basic bacterial Minifors gives you pH control, feeding, sampling and software for data-logging straight out of the box. 

The control system provides all the necessary parameters for a wide range of applications and the operating is based on the instrumentation for our incubator shakers, so making it easy to use. 

Introduction

Hi all welcome to our website, this blog is a summary of a 3-days workshop/training done by Bioprocess Technology 3rd year students (2011) from School of Industrial Technology, University Sains Malaysia.
We are group 1 and we are going to write out the knowledge and experiences gained within these three days with our workshop trainer, Mr.Shaman Gaspar who is the Regional Manager of INFORS HG, Southeast Asia.
Stay tuned !