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last updated: october/18/10
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HELLO TECHNOLOGISTS
welcome to our kawaii blog. feel free to read around our activities.
this blog was designed for our course IBG 302. this is for our lovely lecturer ;Puan Wan Nadiah
COPYCATS, rippers & spammers are not welcome here!
strictly NO RIPPING
US
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 Saturday, October 23, 2010  The 1st day @ 11:06 PM Today, 04/10/2010 is the first day of our IBG 302 practical class. Special thanks to our School of Industrial Technology, Dr. Rosma, Puan Wan Nadiah, Lab assistants, graduate assistants for guiding us during this practical class and giving us this golden opportunity to have an in-depth look and understanding about the installation, utilization, and some precaution steps when dealing with the bioreactors. Not to miss out, we appreciate Mr. Shaman from Infors-HT for presenting the information about the bioreactor we are going to use in this practical class, from the very beginning to the end.
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| Mr.Shaman |
This is Mr. Shaman!! In the beginning of the practical class, he gave a little bit presentation about the history of the company, the organization, company’s philosophy and also the location.
First of all, allow me to introduce to you our Bioreactor, it is a 2.5L minifors bioreactor. And the title for our practical class is the fermentation process using yeast, S.cerevisiae. There are normally 3 parts of a bioreactor, instrumentation, control cabinet and the vessel. To ensure optimum fermentation process, the maximum volume of medium we can add into our fermentor is 1.8L.
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Parts of the Minifors (bioreactor) we used for this lab
The reason of using bioreactor instead of shake flask is due to some reasons. First, if we use shake flask, medium can be used up and this leads to death of cells. However, if bioreactor is being used, sterilized medium can be fed in and bioreactor can be used in large scale, up to few thousand liters. Bioreactor is also suitable for the production of vaccine, penicillin and some culture drinks like vitagen and yakult.
For fermentation process involved bacteria or yeast, acid is normally produced itself. Therefore, we do not need to add in acid buffer. Base buffer is used in this case.
After explaining and showing us how to install a bioreactor, we continue with the media preparation. The medium we are going to use consists of 1% yeast extract, 2% peptone and 2% glucose. Then, we added in 1.5L of distilled water into the medium and stirred it. Then, we put the medium and the inoculation flask into autoclave.
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Monday, October 18, 2010 the 4th day @ 10:08 PM On the 4th day of our fermentation experiment, we continued taking hourly samples and ran the process until 11 am. After 11 am, we started to close down the bioreactors and finish the experiment.
On that day, Mr. Shaman lectured us about finishing procedures, disposal techniques, preparing the fermenter and equipment for the next experiment and analyzing the data and results with computer and computer program; Iris. He also showed us how to shut down the fermenter, step by step.
The samples This is how we did it:
Closedown and cleaning.
- Stop all the feeding or reagent addition, aeration, and stirring.
- Stop all the other functions from the control panel.
- Disconnect all tubings and connections between the vessel and the base unit.
- Unscrew the cover plate of the vessel.
- Lift the vessel from the base unit.
- Discharge the vessel to the drain (since the microorganism used was not phatogenic).
- Take off all tubings and equipment.
- Unscrew the top plate and lift it up.
- Wash the vessel and all the metal parts separately with some mild detergent.
- Let dry or wipe dry with tissue papers.
- Maintain and prepare all the parts and equipments for future usage. Store properly.
Discharging the vessel
After the closedown, we talked about the data and how to analyze it. We were shown how to use Iris in order to collect and handle the data and the results gained. Multiple parameters can be chosen and various styles used when we were handling the data. Always use the ones that fit the best on your purposes.
Observation:
OD 600 value and glucose reading.
L - Low value of glucose.
Graph Absorbance at OD600 vs time
From the data, we can conclude that as the time inccrease, the turbidity which is the number of cells is increasing. It is because the yeast is growing as their nutrient and condition still enough. We can see that from T1 until T21. But at T22, the OD value starts to decrease. This is due to the limited nutrient or substrate in the media. The yeast begin to starve and will undergo the stationary phase. For the glucose value, as time increase the glucose reading decreased. The yeast consumed glucose to grow. We cannot determine the value after T4 because we only use the diabetic test kit.
Graph:
Based on the graph, our group has longer lag phase than the other group. According to Mr Shaman, it is maybe because of our culture has less viable cell or maybe because we cultured the older cell. It is because by the time we inoculate the loop of cell from the colony on the Petri dish, we cannot see and determine which one is viable and new cell. So the cell might be different.
As we can see, the red line and blue line indicate the pO2 value and speed of stirrer respectively are complementing each other. pO2 is the parameter that determines the dissolve oxygen in the media. When pO2 start o decrease, the stirrer will increase their speed in other to overcome the depletion amount of oxygen dissolve.
According to Mr Shaman again, the pH of the culture in the vessel will increase after it is going down, it is because the cells are producing toxin or maybe it has been contaminated with other foreign microbe. That is why we need to do sampling and check it under microscope. The green line and yellow line indicate the antifoam and pH value respectively. This two parameters are complementary each other. As we can see when the antifoam was added to overcome the foaming problem, the pH starts to decrease. The antifoam contains 30% aqueous emulsion of silicon polymer. Lastly as the time of fermentation increase the temperature also increase. This is because the cells themselves produce heat.
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Sunday, October 17, 2010 the 3rd day @ 2:10 PM Innoculation time…
After waiting for 2 days for the fermenter to be fully set up, today Mr Shaman shows us how to do inoculation and take sample aseptically. Now is time to inoculate our lovely wild type yeast, Saccharomyces cerevisiae into the fermenter and let them grow.
But before inoculate the culture we need to set the parameter required by the yeast at the fermenter.
- Aeration rate: 1L /minute
- Speed: 700 rpm
- Calibrate pH
· 1st calibration for 4.01
· 2nd calibration for 7.01
- Temperature
- Calibrate the oxygen
· Sparge the Nitrogen gas into the vessel – to remove the out O2 in the media. Then stop adding N2 when the PO2 data is stable and indicate as 0%.
· Start aeration to increase the O2 level in the media and let the data of PO2 stable. Then indicate it is as 100 %.
- Antifoam
- Base line
Figure 1: Inoculate the yeast into fermenter
Here are several important steps that we need to follow:
1. Cut off the aeration while inoculate the sample
2. Spray the inoculation inlet with 70% ethanol
3. Open inlet probe and then spray again
4. Spray the end of tube at conical flask containing our culture with 70% ethanol.
5. Quickly place the tube to the inlet probe.
6. Start aeration again after finish inoculation. The reason is to increase the pressure in the vessel so that we can reduce the risk of contamination.
Sampling time...
Let me explain how to do sampling without taking out the sample bottle. It is because we want to reduce the risk of contamination.
1. Put syringe at the filter.
2. Unclamp the sample line A
3. Put back on syringe then the sample will flow out into the sampling bottle.
4. Reclamp the sample line.
5. Unclamp the sample line B
6. Spray the end tube of sample line B with 70% ethanol.
7. Push the syringe and the sample will flow into the test tube.
8. The sample now ready to be observed
9. Don’t forget to reclamp the sample line B
10. Spray again the end tube and wrap it with cotton and aluminum foil.
We need to do sampling from 12.30 for first reading at time 0. After that we need to sample out to take OD600 value and glucose reading every hour from 2pm until 11 am on the next day.
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Saturday, October 16, 2010 the 2nd day @ 12:01 PM Second day, October 5th, 2010.
On the second day, Mr. Shaman once again came to demonstrate the preparation of fermenters for autoclaving. Before he came to the laboratory, we prior inoculate one colony of S. Cerevisiae (Saccharomyces cerevisiae) into the inoculation medium. Then the medium containing inoculate is incubated in shaker at 30°C and 200 rpm for 24 hours.
Figure 1: The area of the workplace is sterilized with Lysol
Figure 2: Scraping up a single colony
Figure 3: The lid of the agar plate is lifted and a single colony is scrapped up using sterilized inoculating loop
Figure 4: The medium is inoculated with the colony on the inoculating loop
Figure 5: The flask is incubated in the incubator shaker
After that, we prepare the fermentation medium using the same formula as the day before, 1% of yeast extract, 2% peptone and 2% galactose. The amount of fermentation medium made is 1.5 l/ fermenter. The fermenter is then loaded with the fermentation medium that has been prepared before hand. After that, the fermenter together with the fermentation medium is autoclaved.
Figure 6: 2% of glucose
Figure 7: 1% of peptone
Figure 8: 1% of yeast extract
Figure 9: 1.5 L of water
Figure 10: The YEPG mixture is dissolved in 1.5 L of distilled water
Figure 11: The medium is mixed using stirrer until all the mixture is properly mixed
Figure 12: The fermentation medium is loaded into the fermenter vessel
Figure 13
Before the pH electrode can be used in the fermentation, it needs to be calibrated first. The silicone that covered the tip of the electrode is removed. The calibration option in the bioreactor instrumentation is set with high calibration point (pH 7). Then, the probe tip is immersed in buffer solution pH 7 until it get a stable reading. After stable reading is achieved, the value of the buffer is entered in Minifors. The electrode is washed and ready for the second calibration. The steps for buffer 4 are similar as the buffer 7 and then confirm the calibration by pressing Enter. Then the electrode is washed and fitted into vessel. When placing the electrode into the vessel, the stirrers need to be switch of to avoid breaking the electrode. The pO2 electrode is fitted the same way as the pH electrode.
Figure 14: pH probe is calibrated before use during fermentation
Figure 15: buffer solution at pH 4.01 is used for the calibration of the pH electrode
Figure 16: Buffer solution at pH 7.01 that is used in calibrating the pH electrode
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6ix of us
Jack - the class leader! :DD
Tiina - the stunning Finnish girl :))
Minx - blog creator. ;)
Rat - the craziest. :P
Salwa - as sweet as her name. ;;)
Dilla - the brainiac. =B |
Saturday, October 23, 2010 The 1st day @ 11:06 PM Today, 04/10/2010 is the first day of our IBG 302 practical class. Special thanks to our School of Industrial Technology, Dr. Rosma, Puan Wan Nadiah, Lab assistants, graduate assistants for guiding us during this practical class and giving us this golden opportunity to have an in-depth look and understanding about the installation, utilization, and some precaution steps when dealing with the bioreactors. Not to miss out, we appreciate Mr. Shaman from Infors-HT for presenting the information about the bioreactor we are going to use in this practical class, from the very beginning to the end.
|
| Mr.Shaman |
This is Mr. Shaman!! In the beginning of the practical class, he gave a little bit presentation about the history of the company, the organization, company’s philosophy and also the location.
First of all, allow me to introduce to you our Bioreactor, it is a 2.5L minifors bioreactor. And the title for our practical class is the fermentation process using yeast, S.cerevisiae. There are normally 3 parts of a bioreactor, instrumentation, control cabinet and the vessel. To ensure optimum fermentation process, the maximum volume of medium we can add into our fermentor is 1.8L.
|
Parts of the Minifors (bioreactor) we used for this lab
The reason of using bioreactor instead of shake flask is due to some reasons. First, if we use shake flask, medium can be used up and this leads to death of cells. However, if bioreactor is being used, sterilized medium can be fed in and bioreactor can be used in large scale, up to few thousand liters. Bioreactor is also suitable for the production of vaccine, penicillin and some culture drinks like vitagen and yakult.
For fermentation process involved bacteria or yeast, acid is normally produced itself. Therefore, we do not need to add in acid buffer. Base buffer is used in this case.
After explaining and showing us how to install a bioreactor, we continue with the media preparation. The medium we are going to use consists of 1% yeast extract, 2% peptone and 2% glucose. Then, we added in 1.5L of distilled water into the medium and stirred it. Then, we put the medium and the inoculation flask into autoclave.
|
|
Monday, October 18, 2010 the 4th day @ 10:08 PM On the 4th day of our fermentation experiment, we continued taking hourly samples and ran the process until 11 am. After 11 am, we started to close down the bioreactors and finish the experiment.
On that day, Mr. Shaman lectured us about finishing procedures, disposal techniques, preparing the fermenter and equipment for the next experiment and analyzing the data and results with computer and computer program; Iris. He also showed us how to shut down the fermenter, step by step.
The samples This is how we did it:
Closedown and cleaning.
- Stop all the feeding or reagent addition, aeration, and stirring.
- Stop all the other functions from the control panel.
- Disconnect all tubings and connections between the vessel and the base unit.
- Unscrew the cover plate of the vessel.
- Lift the vessel from the base unit.
- Discharge the vessel to the drain (since the microorganism used was not phatogenic).
- Take off all tubings and equipment.
- Unscrew the top plate and lift it up.
- Wash the vessel and all the metal parts separately with some mild detergent.
- Let dry or wipe dry with tissue papers.
- Maintain and prepare all the parts and equipments for future usage. Store properly.
Discharging the vessel
After the closedown, we talked about the data and how to analyze it. We were shown how to use Iris in order to collect and handle the data and the results gained. Multiple parameters can be chosen and various styles used when we were handling the data. Always use the ones that fit the best on your purposes.
Observation:
OD 600 value and glucose reading.
L - Low value of glucose.
Graph Absorbance at OD600 vs time
From the data, we can conclude that as the time inccrease, the turbidity which is the number of cells is increasing. It is because the yeast is growing as their nutrient and condition still enough. We can see that from T1 until T21. But at T22, the OD value starts to decrease. This is due to the limited nutrient or substrate in the media. The yeast begin to starve and will undergo the stationary phase. For the glucose value, as time increase the glucose reading decreased. The yeast consumed glucose to grow. We cannot determine the value after T4 because we only use the diabetic test kit.
Graph:
Based on the graph, our group has longer lag phase than the other group. According to Mr Shaman, it is maybe because of our culture has less viable cell or maybe because we cultured the older cell. It is because by the time we inoculate the loop of cell from the colony on the Petri dish, we cannot see and determine which one is viable and new cell. So the cell might be different.
As we can see, the red line and blue line indicate the pO2 value and speed of stirrer respectively are complementing each other. pO2 is the parameter that determines the dissolve oxygen in the media. When pO2 start o decrease, the stirrer will increase their speed in other to overcome the depletion amount of oxygen dissolve.
According to Mr Shaman again, the pH of the culture in the vessel will increase after it is going down, it is because the cells are producing toxin or maybe it has been contaminated with other foreign microbe. That is why we need to do sampling and check it under microscope. The green line and yellow line indicate the antifoam and pH value respectively. This two parameters are complementary each other. As we can see when the antifoam was added to overcome the foaming problem, the pH starts to decrease. The antifoam contains 30% aqueous emulsion of silicon polymer. Lastly as the time of fermentation increase the temperature also increase. This is because the cells themselves produce heat.
|
Sunday, October 17, 2010 the 3rd day @ 2:10 PM Innoculation time…
After waiting for 2 days for the fermenter to be fully set up, today Mr Shaman shows us how to do inoculation and take sample aseptically. Now is time to inoculate our lovely wild type yeast, Saccharomyces cerevisiae into the fermenter and let them grow.
But before inoculate the culture we need to set the parameter required by the yeast at the fermenter.
- Aeration rate: 1L /minute
- Speed: 700 rpm
- Calibrate pH
· 1st calibration for 4.01
· 2nd calibration for 7.01
- Temperature
- Calibrate the oxygen
· Sparge the Nitrogen gas into the vessel – to remove the out O2 in the media. Then stop adding N2 when the PO2 data is stable and indicate as 0%.
· Start aeration to increase the O2 level in the media and let the data of PO2 stable. Then indicate it is as 100 %.
- Antifoam
- Base line
Figure 1: Inoculate the yeast into fermenter
Here are several important steps that we need to follow:
1. Cut off the aeration while inoculate the sample
2. Spray the inoculation inlet with 70% ethanol
3. Open inlet probe and then spray again
4. Spray the end of tube at conical flask containing our culture with 70% ethanol.
5. Quickly place the tube to the inlet probe.
6. Start aeration again after finish inoculation. The reason is to increase the pressure in the vessel so that we can reduce the risk of contamination.
Sampling time...
Let me explain how to do sampling without taking out the sample bottle. It is because we want to reduce the risk of contamination.
1. Put syringe at the filter.
2. Unclamp the sample line A
3. Put back on syringe then the sample will flow out into the sampling bottle.
4. Reclamp the sample line.
5. Unclamp the sample line B
6. Spray the end tube of sample line B with 70% ethanol.
7. Push the syringe and the sample will flow into the test tube.
8. The sample now ready to be observed
9. Don’t forget to reclamp the sample line B
10. Spray again the end tube and wrap it with cotton and aluminum foil.
We need to do sampling from 12.30 for first reading at time 0. After that we need to sample out to take OD600 value and glucose reading every hour from 2pm until 11 am on the next day.
|
Saturday, October 16, 2010 the 2nd day @ 12:01 PM Second day, October 5th, 2010.
On the second day, Mr. Shaman once again came to demonstrate the preparation of fermenters for autoclaving. Before he came to the laboratory, we prior inoculate one colony of S. Cerevisiae (Saccharomyces cerevisiae) into the inoculation medium. Then the medium containing inoculate is incubated in shaker at 30°C and 200 rpm for 24 hours.
Figure 1: The area of the workplace is sterilized with Lysol
Figure 2: Scraping up a single colony
Figure 3: The lid of the agar plate is lifted and a single colony is scrapped up using sterilized inoculating loop
Figure 4: The medium is inoculated with the colony on the inoculating loop
Figure 5: The flask is incubated in the incubator shaker
After that, we prepare the fermentation medium using the same formula as the day before, 1% of yeast extract, 2% peptone and 2% galactose. The amount of fermentation medium made is 1.5 l/ fermenter. The fermenter is then loaded with the fermentation medium that has been prepared before hand. After that, the fermenter together with the fermentation medium is autoclaved.
Figure 6: 2% of glucose
Figure 7: 1% of peptone
Figure 8: 1% of yeast extract
Figure 9: 1.5 L of water
Figure 10: The YEPG mixture is dissolved in 1.5 L of distilled water
Figure 11: The medium is mixed using stirrer until all the mixture is properly mixed
Figure 12: The fermentation medium is loaded into the fermenter vessel
Figure 13
Before the pH electrode can be used in the fermentation, it needs to be calibrated first. The silicone that covered the tip of the electrode is removed. The calibration option in the bioreactor instrumentation is set with high calibration point (pH 7). Then, the probe tip is immersed in buffer solution pH 7 until it get a stable reading. After stable reading is achieved, the value of the buffer is entered in Minifors. The electrode is washed and ready for the second calibration. The steps for buffer 4 are similar as the buffer 7 and then confirm the calibration by pressing Enter. Then the electrode is washed and fitted into vessel. When placing the electrode into the vessel, the stirrers need to be switch of to avoid breaking the electrode. The pO2 electrode is fitted the same way as the pH electrode.
Figure 14: pH probe is calibrated before use during fermentation
Figure 15: buffer solution at pH 4.01 is used for the calibration of the pH electrode
Figure 16: Buffer solution at pH 7.01 that is used in calibrating the pH electrode
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affiliates
Those Close ones
 friend @ MINX
friend @ RAT
friend @ TIINA
friend @ SALWA
friend @ DILLA
friend @ JACK
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The 1st day
