Fermentation Lab Report

Fermentation Lab Module II

Minor Lab Report Presented to Dr. Claire Komives San José State University

ChE - 194

by Priyanka Tiwari February 15, 2012

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1

Objective The purpose of this experiment is to grown a strain Bl21(DE3) pET-GFPuv E. coli under aerobic fermentation by fed batch process. The experiment determines key parameters such as optical density, fluorescence, glucose uptake rate and cell yield. Optical density is an important parameter to find cell concentration. Glucose uptake is important to find cell yield. 2

Results The experiment starts with preparation of LB media(5g/l), glucose solution (250g/l) and set up of inoculum flask. Autoclave of bioreactor (filled with LB media), glucose solution and inoculum flask is then carried out. The fermenter is inoculated after autoclave. The bioreactor is setup and its pH and temperature are adjusted to 7.0 and 35 ˚C respectively. Once the fermentation is initiated (inoculation started), samples are collected which are analyzed for its optical density and fluorescence. Table1. Sample absorbance data from spectrophotometer

Sample

Time

time (mins)

1 2 3 4 5 6 7 8 9 10 11

9:55am 12:00pm 1:25pm 4:30pm 5:43pm 5:59pm 6:30pm 7:05pm 7:32pm 8:00pm 8:33pm

0 125 210 401 468 484 515 550 581 605 638

Time (hrs) 0 2.08 3.50 6.68 7.80 8.07 8.58 9.17 9.68 10.08 10.63

cell Total cell Dilution Actual Absorbance concentration weight factor Absorbance (g/l) (g) 0.33 n/a 0.33 0.1089 0.163 0.209 10 2.09 0.6897 1.035 0.258 25 6.45 2.1285 3.193 0.157 50 7.85 2.5905 3.886 0.132 50 6.6 2.178 3.267 0.143 50 7.15 2.3595 3.539 0.19 50 9.5 3.135 4.703 0.137 50 6.85 2.2605 3.391 0.121 50 6.05 1.9965 2.995 0.133 50 6.65 2.1945 3.292 0.147 50 7.35 2.4255 3.638

2

10

3.5 3

8 7

2.5

6

2

5 4

1.5

3

1

cell density g/l

optical density AU 600nm

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optical density Vs time cell concentration Vs time

2 0.5

1 0

0 0

2

4

6 time, hours

8

10

12

Figure1. The figure shows the variation of optical density (AU) and cell density (g/l) vs. time. The values of optical density show an increase as the bacteria multiply. Similarly a graph of cell concentration with time also shows an increase in cell density with time. This behavior is expected and follows microbial growth curve. The graph is analyzed in discussion section. Table 2. Sample Fluorescence obtained from Fluorimeter. The values are normalized with optical density to find the amount of Green fluorescent protein expressed per cell. Sample

Time

time (mins)

1 2 3 4 5 6 7 8 9 10 11

9:55am 12:00pm 1:25pm 4:30pm 5:43pm 5:59pm 6:30pm 7:05pm 7:32pm 8:00pm 8:33pm

0 125 210 401 468 484 515 550 581 605 638

Time (hrs)

UV1

UV2

UV3

UV average

0 2.08 3.50 6.68 7.80 8.07 8.58 9.17 9.68 10.08 10.63

255.8 271.2 310.8 345.8 313.1 281.9 309.2 301.5 360.1 423.4 515.5

258.1 271 315 341.7 315.1 279.9 306.3 302.2 361.5 421.4 517.6

256.5 268.8 316.2 339.7 313.6 277.6 304.8 301.1 358.9 423.9 513.1

256.8 270.3 314.0 342.4 313.9 279.8 306.8 301.6 360.2 422.9 515.4

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UV normalized (FU) 778.1818182 1293.460925 1217.054264 2180.89172 2378.282828 1956.643357 1614.561404 2201.459854 2976.584022 3179.699248 3506.122449

Fluorescence, FU

Normalised fluorescence Vs. Time 4000 3500 3000 2500 2000 1500 1000 500 0

Fluorescence Vs. Time

0

2

4

6 8 Time, hours

10

12

Figure 2. Shows the variation of Fluorescence with time. Table 3. Condensed report from Fermworks. Shows averaged out values for 1 hour interval. Temp Time 8:45-9:45 AM 9:45 -10:45 AM 10:45 -11:45 AM 11:45- 12:45PM 12:45-1:45PM 1:45-2:45PM 2:45-3:45PM 3:45 - 4:45PM 4:45 - 5:45 PM 5:45-6:45PM 6:45-7:45 PM 7:45-8:45 PM 8:45–9:20 PM

pH 6.964 6.863 6.665 6.726 6.768 7.036 7.104 6.907 6.742 6.726 6.762 6.763 6.817

˚C 35.720 35.480 34.949 36.009 36.435 36.172 36.010 36.015 35.819 35.670 36.470 36.142 36.442

% DO 113.926 103.193 86.900 81.632 82.992 81.403 78.074 75.593 71.989 75.120 79.738 86.713 80.898

%CO2 2.389 4.454 3.304 2.618 2.107 1.729 1.426 1.186 0.996 0.834 1.147 2.224 2.065

%O2 6.475 13.835 15.295 16.940 18.167 19.117 19.824 20.347 20.739 21.061 21.732 21.729 22.023

Flowrate 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Stir Speed (rpm) 639.063 638.842 628.509 757.197 914.005 913.555 914.098 913.814 913.718 913.856 913.869 913.869 712.417

Flow Sensor 0.001 0.002 0.001 0.000 0.000 0.001 0.000 0.000 0.001 0.000 0.378 0.754 0.623

Weight 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Table 3. Data shows zero readings for flow rate and weight as we did not measure them. The above table shows averaged values (obtained from Fermworks for an interval of one hour). The values of pH and temperature are controlled at 7 and 35 ˚C. Figures 3, 4, 5 are plotted from the log file generated by Fermworks. These plots show variation of DO, pH and Temperature with time. 4

Sparger (VVM) 2.007 1.116 0.992 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.013

Saturation Dissolved Oxygen, %

DO vs. time 140 120 100 80 60

DO vs. time

40 20 0 0

2

4

6 8 Time, hours

10

12

14

Figure 3. Depicts saturation dissolved oxygen (DO) percentage in fermentation broth with time.

pH

pH vs. time 7.2 7.1 7 6.9 6.8 6.7 6.6 6.5 6.4 6.3

pH vs. time

0

2

4

6 8 time, hours

10

12

14

Figure 4. Figure presents pH vs. time. We can see that spike in pH is obtained after 4.5 hours from inoculation i.e., something around 2:30pm. The spike shows that the amount of substrate, i.e., glucose is consumed. After this the cells start eating protein from the media. This results in the production of NH4OH and the pH rises. The reactor is fed with glucose at 2:30p.m after the spike is observed. The values go back to normal after this.

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Temperature vs. time 40

Temperature, ˚C

35 30 25 20 Temperature vs. time

15 10 5 0 0

2

4

6

8 time, hours

10

12

14

Figure 5. The figure shows variation of Temperature vs. Time. The value is maintained at 35 ˚C Table 4. Shows the data obtained for glucose feed. The feed was shut down at 6:15p.m. Time(min) 0 1 2 3

Glucose Feed (g) 2:30 PM 5:00PM 2246 2078 2242 2069 2239 2058 2237 2048

9:55AM 2246 2246 2246 2246

6:15PM 2048 2048 2048 2048

Table 5. Shows the calculated values for total glucose consumed and Yield (YX/S). The calulations are shown in the discussion section. time glucose feed (hours)

Rate (g/hour)

0 2.08 3.5 4.58 7.08 8.33 8.58 9.68 10.63

125 125 125 0 65.12 24 0 0 0

Total glucose consumed (g) 125 125 125 125 237.8 273 273 273 273

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Yield (X/S) g/g 0 0.0083 0.0255 0.0311 0.0134 0.0130 0.0172 0.0124 0.0110

glucose feed rate vs. time 140 120 rate, g/l

100 80 60 glucose feed rate vs. time

40 20 0 0

2.08

3.5

4.58

7.08

8.33

8.58

9.68 10.63

time, hours

Figure 6.The figure presents the dependence of glucose feed rate vs. time

Glucose consumed glucose consumption, g

300 250 200 150 Glucose consumed

100 50 0 0

2

4

6 time, hours

8

10

12

Figure 7. The figure represents variation of total glucose consumed with time during fermentation.

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Yield

Yield 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

Yield

0

2

4

6

8

10

12

time, hours

Figure 8. The figure presents yield vs. time plot. ‘X’ denotes cell density (g/l) and ‘S’ shows amount of substrate, g/l (glucose)

2.1

Interpretation of data

2.1.1 Figure 1 and 2. Optical density and Fluorescence with time. With an initial rise, the graph shows a decline in optical density after 8hours of fermentation. Ideally the optical density should be a rising curve. This is because of the bacteria density increases in the fermentation broth as they multiply. However during the experiment, bioreactor was fed with glucose at 5:00pm, which caused the dilution of the broth resulting in a dip in optical density measured in the sample collected after 5:00pm i.e., 5:43p.m.The feed was shut down at 6:15pm. The level of fluorescence also shows an increase after induction. The fluorescence is an indicator of the expression level of GFP (green fluorescent protein) that helps in identifying the levels of recombinant protein expression in bacteria. The rising graph shows that fluorescence is increasing as the cells multiply. The dip is observed after the feed was started at 5:00pm that resulted in dilution of the fermentation solution.

2.2

Figure 3. Dissolved Oxygen vs. Time The inoculation was done at 9:55am. After inoculation, the values of dissolved oxygen are erratic. This is because the culture adapts to the new environment of nutrients and glucose. DO readings stabilize eventually and remain constant. It was observed during the off-gas set up analysis that there was a missing clamp in the fermentation set up. This was the cause of air leakage from the headplate port of fermenter. The 8

issue was fixed at 7:05p.m. A DO spike is observed when the issue was fixed. The values were steady after this. 2.3

Figure 4. pH vs. time From figure 4, we can see that a spike in pH is obtained after 4.5 hours from inoculation i.e., something around 2:30pm. The spike shows that the amount of substrate, i.e., glucose is low and cells consume protein from the media. This results in the production of NH4OH and the pH rises. The reactor is fed with glucose at 2:30p.m after the spike is observed. The values go back to normal after this. 2.4

Figure 5. Feed rate vs. time The glucose feed is assumed to be completely consumed. Therefore initial rate is 125g/l. Calculations are attached in the appendix A. We have assumed that bugs have consumed all the glucose. Therefore the feed rate goes to zero. The batch reactor was fed at 2:30p.m.The feeding is represented by a spike in plot. 2.5

Figure 6. Glucose consumed vs. Time We have assumed that the initial amount of glucose (125g/l) is completely consumed by the culture. The feed was started at 2:30 and thus there is a rise in the graph of total glucose consumed. The feed rate was resumed at 5:00pm and the feed was shut down at 6:15pm. Therefore feed rate goes to zero in the end. 2.6

Figure 7. The figure shows the total glucose consumed over time. The initial amount of glucose present in the bioreactor was 125g. With the growth of the bacteria in their exponential phase, all glucose was consumed. This was confirmed from the pH spike observed. The bioreactor was fed with glucose at 2:30 p.m. The feed was shut down and then restarted at 5:00pm. The graph becomes constant once the glucose feed was shut down at 6:15p.m. 2.7

Figure 8. Yield vs. Time The figure clearly shows that the yield is increased as the bacteria enter the exponential growth phase. The values of yield coefficient are however very low in our case. This is may be a result of dilution of solution from glucose. 2.8

Comparison of measured values with literature results In order to check the goodness of our experimental data, we have tried to fit the experimental data in the net specific growth rate equation.

is growth rate expressed as h-1.

Where, X is cell density expressed as g/l and Linearizing the above equation we get,

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Values of lnX is plotted with time ‘t’. Table 6, shows values of lnX and time ‘t’. Table 6. Data presenting variation of lnX vs. time. Time (hours) 0 2.08 3.50 6.68 7.80 8.07 8.58 9.17 9.68 10.08 10.63

X (g/l) 0.1089 0.6897 2.1285 2.5905 2.178 2.3595 3.135 2.2605 1.9965 2.1945 2.4255

ln X -2.217325 -0.371499 0.7554175 0.9518509 0.778407 0.8584497 1.1426292 0.815586 0.6913956 0.7859542 0.8860377

From the data that we have collected, DO, fluorescence, pH(spike) etc., we can say that the cells were in their exponential phase before 4:30p.m. Therefore plotting the results for the data obtained from first four samples (exponential phase), we get Figure 9. lnX vs.time 2 y = 0.4626x - 1.6389 R² = 0.7982

1.5 1 lnX, g/l

0.5 0 -0.5 0

2

4

6

8

lnX vs.time Linear (lnX vs.time)

-1 -1.5 -2 -2.5

Time, hours

Figure 9. The figure shows the exponential growth phase of the recombinant cells. From figure 9, it is found that the value of R2 is 0.798 ≈ 0.8. The value of growth rate is 0.4626 ≈0.5 h-1. The values of R2 shows a good fit of data in the growth rate equation. Thus we can say that the experimental data is good.

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2.9

Sources of Error There can be following sources of error.    



 

pH probe, DO probe, spectrophotometer, fluorimeter are not calibrated properly. Sterilization is not performed with caution before fermentation. Dilutions are not performed correctly. Concentration of dissolved oxygen is maintained above saturation. However if extra air is blown, pocket of air gets created which eventually leads to notable drop in power supply to the mixer. Overfeeding/Underfeeding of substrate is another source of error. Overfeeding results in cells producing organic acid which causes the pH to drop. Underfeeding on the other hand causes production of bases, resulting in a pH rise. Off-gas valves and knobs are not inspected properly for flow of off-gas. Leakage of air due to a missing clamp from any head port outlet. The gas should flow only from the off-gas port on headplate. The readings are not accurate if leakage is there.

2.10 Mistakes made during the experiment  The culture was overfed for 1 hour. This led to an extreme dilute fermentation solution. We got off results for optical density and fluorescence from the samples collected after this.  By mistake a wrong off-gas knob was open. We could not perform off - gas analysis since the readings of % O2out and % CO2out were false. The values of important parameters such as OUR, CER, kla, respiratory quotient can’t be done due to this.  A clamp was missing in the set-up that affected the saturated dissolved oxygen percent in the bioreactor. As the issue was fixed, the DO level got stabilized.  Off-gas measurements were also affected due to missing clamp in the setup.  The final weight of glucose left in the bottle was not taken due to which we had to assume a constant rate of feeding. 3

Conclusions The glucose uptake rate is = 43.661 g/hr The value of cell yield for the fermentation process is obtained = 0.0216 g/g The value of growth rate is 0.5h-1. The value of R2 obtained is 0.798 ≈ 0.8

Therefore from the results of cell growth and cell density, it can be interpreted that microbial growth started soon after inoculation. This is shown by a rising cell concentration curve with time. The value of growth rate 0.5h-1 also shows that bacteria were growing fast. R2 = 0.8, also shows that the fit was good.

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