Fermentation Lab Report

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Fermentation Lab Module II

Minor Lab Report Presented to Dr. Claire Komives

San José State University

ChE - 194

by Priyanka Tiwari February 15, 2012

2 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) Time (hrs) Absorbance Dilution factor Actual Absorbance cell concentration (g/l) Total cell weight (g) 1 9:55am 0 0 0.33 n/a 0.33 0.1089 0.163 2 12:00pm 125 2.08 0.209 10 2.09 0.6897 1.035 3 1:25pm 210 3.50 0.258 25 6.45 2.1285 3.193 4 4:30pm 401 6.68 0.157 50 7.85 2.5905 3.886 5 5:43pm 468 7.80 0.132 50 6.6 2.178 3.267 6 5:59pm 484 8.07 0.143 50 7.15 2.3595 3.539 7 6:30pm 515 8.58 0.19 50 9.5 3.135 4.703 8 7:05pm 550 9.17 0.137 50 6.85 2.2605 3.391 9 7:32pm 581 9.68 0.121 50 6.05 1.9965 2.995 10 8:00pm 605 10.08 0.133 50 6.65 2.1945 3.292 11 8:33pm 638 10.63 0.147 50 7.35 2.4255 3.638

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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)

Time

(hrs) UV1 UV2 UV3 UV average

UV normalized (FU) 1 9:55am 0 0 255.8 258.1 256.5 256.8 778.1818182 2 12:00pm 125 2.08 271.2 271 268.8 270.3 1293.460925 3 1:25pm 210 3.50 310.8 315 316.2 314.0 1217.054264 4 4:30pm 401 6.68 345.8 341.7 339.7 342.4 2180.89172 5 5:43pm 468 7.80 313.1 315.1 313.6 313.9 2378.282828 6 5:59pm 484 8.07 281.9 279.9 277.6 279.8 1956.643357 7 6:30pm 515 8.58 309.2 306.3 304.8 306.8 1614.561404 8 7:05pm 550 9.17 301.5 302.2 301.1 301.6 2201.459854 9 7:32pm 581 9.68 360.1 361.5 358.9 360.2 2976.584022 10 8:00pm 605 10.08 423.4 421.4 423.9 422.9 3179.699248 11 8:33pm 638 10.63 515.5 517.6 513.1 515.4 3506.122449 0 0.5 1 1.5 2 2.5 3 3.5 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 10 12 ce ll dens it y g /l o ptic a l dens it y AU 6 0 0 nm time, hours

optical density Vs time cell concentration Vs time

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Figure 2. Shows the variation of Fluorescence with time.

Table 3. Condensed report from Fermworks. Shows averaged out values for 1 hour interval.

Time pH

Temp

˚C % DO %CO2 %O2 Flowrate

Stir Speed (rpm) Flow Sensor Weight Sparger (VVM) 8:45-9:45 AM 6.964 35.720 113.926 2.389 6.475 0.000 639.063 0.001 0.000 2.007 9:45 -10:45 AM 6.863 35.480 103.193 4.454 13.835 0.000 638.842 0.002 0.000 1.116 10:45 -11:45 AM 6.665 34.949 86.900 3.304 15.295 0.000 628.509 0.001 0.000 0.992 11:45- 12:45PM 6.726 36.009 81.632 2.618 16.940 0.000 757.197 0.000 0.000 1.000 12:45-1:45PM 6.768 36.435 82.992 2.107 18.167 0.000 914.005 0.000 0.000 1.000 1:45-2:45PM 7.036 36.172 81.403 1.729 19.117 0.000 913.555 0.001 0.000 1.000 2:45-3:45PM 7.104 36.010 78.074 1.426 19.824 0.000 914.098 0.000 0.000 1.000 3:45 - 4:45PM 6.907 36.015 75.593 1.186 20.347 0.000 913.814 0.000 0.000 1.000 4:45 - 5:45 PM 6.742 35.819 71.989 0.996 20.739 0.000 913.718 0.001 0.000 1.000 5:45-6:45PM 6.726 35.670 75.120 0.834 21.061 0.000 913.856 0.000 0.000 1.000 6:45-7:45 PM 6.762 36.470 79.738 1.147 21.732 0.000 913.869 0.378 0.000 1.000 7:45-8:45 PM 6.763 36.142 86.713 2.224 21.729 0.000 913.869 0.754 0.000 1.000 8:45–9:20 PM 6.817 36.442 80.898 2.065 22.023 0.000 712.417 0.623 0.000 1.013 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. 0 500 1000 1500 2000 2500 3000 3500 4000 0 2 4 6 8 10 12 F luo re sce nce, F U Time, hours

Normalised fluorescence Vs. Time

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

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. 0 20 40 60 80 100 120 140 0 2 4 6 8 10 12 14 Sa tura tio n Dis so lv ed O x y g en, % Time, hours DO vs. time DO vs. time 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7 7.1 7.2 0 2 4 6 8 10 12 14 pH time, hours

pH vs. time

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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) Glucose Feed (g) 9:55AM 2:30 PM 5:00PM 6:15PM 0 2246 2246 2078 2048 1 2246 2242 2069 2048 2 2246 2239 2058 2048 3 2246 2237 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) Total glucose

consumed (g) Yield (X/S) g/g 0 125 125 0 2.08 125 125 0.0083 3.5 125 125 0.0255 4.58 0 125 0.0311 7.08 65.12 237.8 0.0134 8.33 24 273 0.0130 8.58 0 273 0.0172 9.68 0 273 0.0124 10.63 0 273 0.0110 0 5 10 15 20 25 30 35 40 0 2 4 6 8 10 12 14 T em pera ture, ˚C time, hours

Temperature vs. time

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

Figure 7. The figure represents variation of total glucose consumed with time during fermentation. 0 20 40 60 80 100 120 140 0 2.08 3.5 4.58 7.08 8.33 8.58 9.68 10.63 ra te, g /l time, hours

glucose feed rate vs. time

glucose feed rate vs. time

0 50 100 150 200 250 300 0 2 4 6 8 10 12 g luco se co ns um ptio n, g time, hours

Glucose consumed

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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 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0 2 4 6 8 10 12 Yield time, hours

Yield

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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.

Where, X is cell density expressed as g/l and is growth rate expressed as h-1.

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) X (g/l) ln X 0 0.1089 -2.217325 2.08 0.6897 -0.371499 3.50 2.1285 0.7554175 6.68 2.5905 0.9518509 7.80 2.178 0.778407 8.07 2.3595 0.8584497 8.58 3.135 1.1426292 9.17 2.2605 0.815586 9.68 1.9965 0.6913956 10.08 2.1945 0.7859542 10.63 2.4255 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.

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.

y = 0.4626x - 1.6389 R² = 0.7982 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 0 2 4 6 8 lnX, g /l Time, hours lnX vs.time lnX vs.time Linear (lnX vs.time)

11 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.