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Bioprocess Engineering I
Practical Course Manual Static KLa estimation

compiled by Dr. Sonja Diercks-Horn Fall 2011

Introduction
The aim of this practical is to determine the oxygen mass transfer coefficient, KLa, by static gassing out method. This method is carried out in the absence of respiring organisms, thus there is no oxygen consumption.

Simple mass balance considerations for the concentration of dissolved oxygen give:

= KLa (C*AL CAL) qO2x

(1)

where C C* AL t KLa qO2 x

= = = = = = =

oxygen concentration O2 concentration at saturation from air, in liquid time mass transfer coefficient specific oxygen uptake rate biomass concentration

Since the static KLa estimation is carried out in the absence of organisms equation (1) is reduce to the following expression:

= KLa (C*AL CAL)

(2)

Equation (2) shows only the variation of CAL over time, the integration between the limits t = 0 and CAL = 0 gives the following equation:

-KLa t= ln

(3)

The value C*AL for oxygen at room temperature is 10 mg L-1 (0.3125 mM) or in our case 100 %, so equation (3) is a slope of ln(C*AL - CAL) vs t, with a slope value = - KLa. The experimental data to evaluate KLa must derive from different CAL vs t values. This can be performed in a batch reactor, in which the dissolved oxygen has been eliminated prior to any measurements by bubbling nitrogen until CAL = 0.

3 For example: Raw data withdrawn from Erazo et al 2001

Variation of oxygen at a spirrer speed of 500 RPM

0.35 0.3 CAL (mMO2/L) 0.25 0.2 0.15 0.1 0.05 0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 time (sec)

Fig. 1. Variation of oxygen measure in mM O2 per Litre at 500 RPM stirrer speed

The ln(C*AL - CAL) was calculated and plotted against the time for the exponential increase of the measured values. The linear regression was added as well as the formula. The KLa for this data is 41.76 h-1 calculated from the slope.

0.000 -0.500 -1.000 ln(C*AL- CAL) -1.500 -2.000 -2.500 -3.000 -3.500 -4.000 500 rpm time (sec) Linear (500 rpm) y = -0.0116x - 1.1372 R = 0.9971 0 50 100 150 200 250

Materials and Methods

Materials Fermenter with 6 L volume (20C) Carboxymethyl cellulose (Merck, 217274), 2110 mPas Stopwatch

Done by course instructor: Fill the vessel with 6 L water through one of the ports on the vessel lid Calibrate the PO2

Experiment 1 different oxygen rates

1. 2. 3. 4. 5. Set the spirrer speed to 500 rpm Sparge vessel contents with N2, displacing O2 Monitor variation in dissolved oxygen concentration using pO2 electrode Allow the dissolve oxygen to fall to 0% saturation, then turn off N2 flow Sparge vessel contents with different air ratios either at 0.2 VVM, 1 VVM or 1.5 VVM. Stopwatches must be started at the same time as when air flow starts. 6. Monitor and record variation of dissolved oxygen concentration with respect to time every 10 seconds until the same value is recorded for a span of 60 seconds. 7. Stop the fermenter run and finally remove the water from the fermenter using the sample valve

5 Table 1. different oxygen rates: 0.2 VVM

seconds 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

PO2

seconds 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

PO2

seconds 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500

PO2

Table 2. different oxygen rates: 1 VVM

seconds 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

PO2

seconds 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

PO2

seconds 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500

PO2

6 Table 3. different oxygen rates: 1.5 VVM

seconds 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

PO2

seconds 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

PO2

seconds 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500

PO2

Experiment 2 different stirrer speeds

1. 2. 3. 4. 5. Set the spirrer speed either to (1) 300 rpm, (2) 500 rpm (done before) or (3) 800 rpm Sparge vessel contents with N2, displacing O2 Monitor variation in dissolved oxygen concentration using pO2 electrode Allow the dissolve oxygen to fall to 0% saturation, then turn off N2 flow Sparge vessel contents with air at 1 VVM (liters of O2 per liter of medium per minute). Stopwatches must be started at the same time as when air flow starts. 6. Monitor and record variation of dissolved oxygen concentration with respect to time every 10 seconds until the same value is recorded for a span of 60 seconds.

7 Table 4. different spirrer speeds: 300 RPM

seconds 0 10 20 30 40 50 60 70 80 90 100 110 120 130

PO2

seconds 140 150 160 170 180 190 200 210 220 230 240 250 260 270

PO2

seconds 280 290 300 310 320 330 340 350 360 370 380 390 400 410

PO2

Table 5. different spirrer speeds: 800 RPM

seconds 0 10 20 30 40 50 60 70 80 90 100 110 120 130

PO2

seconds 140 150 160 170 180 190 200 210 220 230 240 250 260 270

PO2

seconds 280 290 300 310 320 330 340 350 360 370 380 390 400 410

PO2

Experiment 3 different viscosity of media using sodium carboxymethyl cellulose (CMC)

1. 2. 3. 4. Fill the fermenter with 6 L of medium 1 (0.25 % CMC) using a port on the vessel lid and a funnel Set the spirrer speed to 500 rpm Sparge vessel contents with N2, displacing O2 Monitor variation in dissolved oxygen concentration using pO2 electrode

8 5. Allow the dissolve oxygen to fall to 0% saturation, then turn off N2 flow 6. Sparge vessel contents with air at 1 VVM. Stopwatches must be started at the same time as when air flow starts. 7. Monitor and record variation of dissolved oxygen concentration with respect to time every 10 seconds until the same value is recorded for a span of 60 seconds. Table 6. different oxygen rates: 0.25% CMC

seconds 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270
8. 9. 10. 11. 12.

PO2

seconds 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550

PO2

seconds 560 570 580 590 600 610 620 630 640 650 660 670 680 690 700 710 720 730 740 750 760 770 780 790 800 810 820 830

PO2

Stop the fermenter run and remove medium 1 from the fermenter using the sample valve Fill the fermenter with 6 L of medium 2 (0.5 % CMC) using a port on the vessel lid and a funnel Set the spirrer speed to 500 rpm Sparge vessel contents with N2, displacing O2 Monitor variation in dissolved oxygen concentration using pO2 electrode

9 13. Allow the dissolve oxygen to fall to 0% saturation, then turn off N2 flow 14. Sparge vessel contents with air at 1 VVM. Stopwatches must be started at the same time as when air flow starts. 15. Monitor and record variation of dissolved oxygen concentration with respect to time every 10 seconds until the same value is recorded for a span of 60 seconds. Table 7. different oxygen rates: 0.5% CMC

seconds 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

PO2

seconds 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550

PO2

seconds 560 570 580 590 600 610 620 630 640 650 660 670 680 690 700 710 720 730 740 750 760 770 780 790 800 810 820 830

PO2

16. Stop the fermenter run and remove medium 1 from the fermenter using the sample valve and clean the fermenter with water.

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References
Erazo, R. E.; Crdenas J. L. R. (2001): Determinacin experimental del coeficiente de transferencia de oxgeno (KLa) en un bioreactor batch. Rev. Per. Qum. Ing. Qum., Vol. 4 (2), 22-27

Lab Report Writing Guidelines

Every student has to submit one report for the experiments. The main purpose of lab report writing is to communicate the results to others and to enable others to duplicate the work in a straightforward manner. A report should be as short as possible but contain all essential information. The lab report should be organized like a scientific publication which contains the following subjects:

1. Title: This page includes a short descriptive title, the name of the person(s) submitting the report, date of the lab course and the name of the students in the group, the date the report is submitted, the name of the instructor. This page is not numbered. 2. Table of Content: The Table of Contents provides page locations of major sections. 3. Introduction: Explains its objectives, significance, and provides the background necessary to understand the experiment. When appropriate, the background should indicate theoretical prediction. This section is not intended to be a simple reproduction of some texts (do not forget to cite your references in the text, see how to cite below); instead it must reflect your understanding about information pertaining to the experiment, and must emphasize the importance and applications of the experimental subject. Direct reproductions from earlier reports, books, and/or internet will be considered as cheating and be subjected to a penalty. 4. Materials and methods: if the procedure is the same as in the manual, cite the manual. Write only the changes you made into the lab report. 5. Results: present all data obtained and calculated, graphs and description of data, e.g. SDS gels. When presenting graphs, make sure that your specific measurement data points are indicated on the graphs. Do not just show a line or a curve. Sufficiently detailed explanations should precede each table and/or graph in regard to the experimental conditions, range of parameters, so as to allow the reader to follow and understand the meaning of the information presented. Each table or graph has to have a caption (Figures have legends while Tables have headings!), proper labels and subheadings, names of parameters (with units) used in axes of graphs or in the table column headings. The symbols used must be the same as those used in the text. The results section further includes any unexpected observations that you made during the experiment (they could help to explain unexpected outcomes in the discussion). If you made a mistake do not try to cover it up but describe it. Do not yet interpret or discuss your data, this belongs to the discussion! 6. Discussion: This section places specific results into the context of the experiment as a whole. Analyze the results and discuss their implications, compare experimental results and

11 expected, acknowledge possible sources of error. What would you make better if you have to do the experiment again. 7. Reference: Good scientific practice includes that all information taken from other sources (textbooks, articles, Lab Manuals, Internet) needs to be indicated as such. In addition, the different sources of information have to be cited in the correct way. The correct way means the way citations are placed in scientific articles. Every listed reference must be cited in the text of the report by author and year (for example, Bird et al., 1960). In the last part of the lab report, you place the list of citations in alphabetical order based on first author and you write out all authors, year of publishing, title, Journal or Publisher. The internet sources are given at the end.

Examples for a reference list: Abramowitz, M., Stegun, I.A., (1965): Handbook of Mathematical Functions. Dover, New York Winslow, F.H., and Matreyek, W., (1951): Pyrolysis of Crosslinked Styrene Polymers. Journal of Polymer Science, 22, pp.315-324.

Tasks for the lab report

Plot the measurement of the three different experiments (RPM, VVM, viscosity) in three different plots with the PO2 values on the y-axis and the time on the x-axis Calculate the ln(C*AL - CAL) and plot the three different experiments (RPM, VVM, viscosity) in three different plots with ln(C*AL - CAL) on the y-axis and the time on the x-axis Calculate the KLa from the slope and present the values in a table. Discuss your results

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MATERIAL SAFETY DATA SHEET

Name (chemical and trivial) Chemical formula Structural formula Sodium carboxymethyl cellulose

Molar mass/Molecular weight Density General classification Functional groups

GHS Pictograms H statements P statements Signal word Gloves Safety goggles Hood

Role in the experiment

Additional notes

_________________________________ Printed Name and Signature