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This research includes measuring the amount of oxygen needed by a plant known as P.pastoris. The procedure is carried out in a laboratory under carefully monitored artificial conditions. P.pastoris requires oxygen for aerobic reactions that result in fermentation. Silva et al. (2012) Plants that respire in the presence of oxygen undergo a related reaction. The specimen under inspection is put in a fermenter, and the oxygen is pumped in by a reactor equipped with a meter that measures the amount of oxygen used. The term kL.a stands for oxygen mass transfer coefficient. Oxygen mass transfer coefficient is a common term where the transfer of oxygen in stirred Fermenters is involved. It is therefore used fundamentally in facilitating the fermentation of the contents of the fermenter which more often than not contain aerobic organisms (Scargiali, Busciglio, Grisafi and Brucato, 2014). Given the importance of the kL.a, its measurement should be done, and the outcome critically analyzed which is the aim of this experiment.
The setup of the experiment is a reactor containing oxygen and a fermenter with P.pastoris. The two apparatus are connected with pipes which are definitely small in diameter to increase the pressure of the gas. The air is then passed over the contents of the fermenter at given revolutions per minute using different impellers. An impeller is a blade that causes motion in a fluid. The rate of oxygen uptake entirely depends on the oxygen requirements of the plant. (Karimi, et al., 2013). Some plants grow massively as compared to others and as a result, require more oxygen for metabolic purposes. This is because they definitely show incredibly high rates of respiration.
The change in the rate of oxygen transfer can be varied using different impellers. One reason as to why that is done is because different organisms require different percentages of oxygen. Since the experiment is being carried out in the lab and on a small scale, there has to be a control experiment. A control experiment is usually set usually the counter of the primary test. As the name suggests, the research controls the main test by comparing the results of the primary experiment with those of the main experiment.
In the event of measurement of dissolved oxygen, oxygen electrodes do not measure the absolute amounts of dissolved oxygen instead they the partial pressure of the dissolved gas. The electrodes need to be calibrated in zero and oxygen saturated solutions to produce a scale from 0-100% dissolved oxygen tension. The experiment is executed all the conditions kept constant, and the results analyzed and interpreted accordingly. The results are calculated using a formula that will be eloquently explained under the results sections (Klein, Schneider, & Heinzle, 2013).
The above process is repeated this time using nitrogen gas in the place oxygen gas all the other conditions kept constant. The oxygen electrode is connected to an oxygen meter which still measures the rate of nitrogen being aerated. The dissolved oxygen tension is measured using a stopwatch. Working with your fermenter, you should aim to perform 3-6 runs under different conditions. Each run will involve purging the vessel before the run with nitrogen, establishing your experimental conditions, then aerating and recording the dissolved oxygen tension trace. This data will be used to estimate the kLa for that particular run.
For this to work, it is vital that you do a few things before beginning.
Become familiar with the actual controls at your disposal _x0096_ stirrer speed and aeration rate.
"Zero" the system by purging with nitrogen until no further decrease is observed in the signal on the meter and on the chart recorder. If necessary, adjust the zero on the meter.
Now aerate the fermenter by connecting the air line. You should see the DO value on the meter begin to increase. Depending on the conditions you set, this could take 3- 10 minutes. Once there is no further increase, ensure the meter reading is 100% (adjust if necessary by adjusting the "span").
In smaller groups, students should aim to move around the apparatus, so you get at least 3-4 measurements from at least three different fermenters. One will be a bubble column fermenter (you can only adjust flow rate of air), and the others will be stirred tanks (you can adjust stirrer speed and/or air flow rate)
For each run;
Zero the fermenter by purging it with nitrogen
Set-your experimental conditions. Record these clearly in your lab book
Switch air on and start your stopwatch
Record data until at least 80% of air saturation is reached. You will want to record DO data every 5 to 20s
One of your group adds this to the spreadsheet being collected on the lab computer. (Stanbury, Whitaker, & Hall, (2013).
A bioreactor will be operating in the lab with a P.pastoris culture. These will be operated under controlled temperature and with oxygen measured. You will be required to estimate the specific oxygen uptake (QO2 in mg O2 gX-1h-1) of the yeast using the following protocol
Record the temperature, dissolved oxygen, stirrer speed and air flow rate
Sample as shown, dilute and estimate the OD600 of the sample. Use your data from lab 1 to convert this into g DCW per liter
With stopwatch ready, shut off the air supply and briefly sparge the headspace with nitrogen if possible. Record the DO levels every 10s
Convert the %DO readings into concentration (mg/l) values using the chart below.
5. To estimate the QO2, you need to do the following:
Estimate the bioreactor OCR (oxygen consumption rate) (mg O2 l-1h-1) (average data when the DO appears to be declining linearly.
Convert the OD600 reading into gX.l-1 using the data from your other lab (as a guide, 1 OD600 should be equal to approx. 0.25-0.30 gX.l-1 for P.pastoris (Gullo, Verzelloni, and Canonico, (2014).
QO2 = OCR/X
Repeat the calculation for other data collected by other groups.
The rate of mass oxygen transfer is given by the formula;
(1) OTR=dCo2,l/ dt= kLa(Co2,l*-C02,l) integrating gives the following relationship.
Where; t = time period of measurement (h)
CO2,l* = saturated [O2] in the liquid (mM or mg l-1)
CO2,l = actual [O2] in the liquid (mM or mg l-1)
kLa = O2 mass transfer coefficient (h-1)
The measure of dissolved oxygen tension is given by the formula;
DOT=100*C02,l/C02,l* , therefore
The results that were obtained after the experiment were fed into an excel sheet. Using the formula given above the rate of oxygen transfer and dissolved oxygen transfer can be calculated by merely substituting the results into the equation. In this case, the following results were used to calculate the rate of oxygen uptake by the P.pastoris.
JL Table 1: Oxygen Uptake; Best Group in the World
Time 3.00 P.M 0 78.3 OD 14.1 10 78.2 WCW 22.1 20 76.6 pH 5.8 30 73.2 Temperature 28.2 40 68.4 Airflow Rate (L/min) 2 50 62.4 Stirrer Speed (rpm) 900 60 55.8 70 48.8 80 41.5 90 33.7 100 25.5
Substituting the corresponding values in the formula elaborately estimates the rate of oxygen transfer. As for the dissolved oxygen tension, the following results carried the day.
Table 2: Dissolved Oxygen Tension (DOT); Best Group in the World
Temperature 23.8 23.8 23.8 Airflow Rate (L/min 3 3 3 Stirrer Speed (rpm) 1000 703 1150 Time (s) dO2 (%) dO2 (%) dO2 (%) 0 0 0 0 5 0 0 0 10 - 0 0.2 15 8.7 4.1 10.6 20 25.1 11.8 29.2 25 42 22.9 47.8 30 57.4 34.4 60.2 35 69.6 45.3 73.5 40 77.1 52.9 83.1 45 84.1 63.2 50 69.2 55 75.8 60 81.1
Using the formula, DOT=100*C02,l/C02,l* the estimated value of the dissolved oxygen tension can be estimated.
Oxygen supports life in that the gas is involved in many important processes that is crucial for life in this case fermentation. Fermentation takes place through aerobic respiration in the presence of oxygen. On a few occasions agents like yeast are used to speed up the process as catalysts (Stanbury, Whitaker, & Hall, 2013). In the world today is applicable in many manufacturing and food processing industries such as those that brew wine and bake bread. The fact that fermentation is crucial for living gives rise to need to study it and find out how the process takes place hence such experiments as the one discussed above.
Silva, J. P. A., Mussatto, S. I., Roberto, I. C., & Teixeira, J. A. (2012). Fermentation medium and oxygen transfer conditionsl by Pichia stipitis. Renewable Energy, 37(1), 259-265.
Gabelle, J. C., Jourdier, E., Licht, R. B., Chaabane, F. B., Henaut, I., Morchain, J., & Augier, F. (2012). Impact of rheology on the mass transfer coefficient during the growth phase of Trichoderma reesei in stirred bioreactors. Chemical engineering science, 75, 408-417.
Kirk, T. V., & Szita, N. (2013). Oxygen transfer characteristics of miniaturized bioreactor systems. Biotechnology and bioengineering, 110(4), 1005-1019.
Stanbury, P. F., Whitaker, A., & Hall, S. J. (2013). Principles of fermentation technology. Elsevier.
Scargiali, F., Busciglio, A., Grisafi, F., & Brucato, A. (2014). Mass transfer and hydrodynamic characteristics of unbaffled stirred bio-reactors: influence of impeller design. Biochemical Engineering Journal, 82, 41-47.
Klein, T., Schneider, K., & Heinzle, E. (2013). A system of miniaturized stirred bioreactors for parallel continuous cultivation of yeast with online measurement of dissolved oxygen and off‐gas. Biotechnology and bioengineering, 110(2), 535-542.
Gullo, M., Verzelloni, E., & Canonico, M. (2014). Aerobic submerged fermentation by acetic acid bacteria for vinegar production: process and biotechnological aspects. Process Biochemistry, 49(10), 1571-1579.
Karimi, A., Golbabaei, F., Mehrnia, M. R., Neghab, M., Mohammad, K., Nikpey, A., & Pourmand, M. R. (2013). Oxygen mass transfer in a stirred tank bioreactor using different impeller configurations for environmental purposes. Iranian journal of environmental health science & engineering, 10(1), 6.
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