Difference between revisions of "Team:Aachen/Lab/Bioreactor/Characterization of Bioreactor"

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{{Team:Aachen/Header}}
 
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= Evaluation of the Bioreactor Design =
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As our bioreactor is built following the do-it-yourself principle, it is most crucial that the obtained results are reproducible.
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We, therefore, assessed the reliablitity of the reactor system design in a series of experiments.
  
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=Experiments=
 
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We ran several experiments throughout the development of the complete bioreactor. After the first few times, we realized that the pumps needed individual calibrations due to a slight error seen after a few days of running it. It was also observed that aeration pump was not fast enough and so, the same stepper motor was worked on and made faster, to aerate at 10 mL per minute. Meanwhile the spring structure of the pump was modified time and again to make a sturdy and stable structure.
 
  
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=== Preliminery Runs ===
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Preliminary experiments were conducted to optimize and validate the complete bioreactor setup and its individual components. The configurations of the aeration pump (the 3D spring structure) was enhanced to pump air at a rate of 10 mL/min, to achieve an aeration rate of 1 vvm in the reactor.
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The first experiment was carried out using only the pumps and the stirrer, with continuous samples being manually taken to observe the OD using the laboratory spectrophotometer and monitor the growth. We used <i> Escherechia Coli </i> DH5alpha strain in LB medium and ran the experiments for 110 hours and obtained the following results:
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{{Team:Aachen/Figure|Aachen_Reactor0ODvsD.png|title=Plot of Optical Density versus Dilution rate|size=large}}
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== Preliminary Runs ==
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Before the development of our continuous OD device, an initial experiment was carried out in the mini-bioreactor and growth was manually monitored using the laboratory spectrophotometer. We used ''E. coli'' DH5α strain in LB medium and the experiment was run for 110 hours.
  
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=== Final Experiment ===
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{{Team:Aachen/Figure|Aachen_Reactor0ODvsD.png|title=Plot of optical density and dilution rate with respect to time, in hours|subtitle=''E. Coli'' DH5α was grown in LB medium. We were able to observe stable OD for different dilution rates for 5 reactor volumes|size=large}}
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== Final Experiment ==
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The experiment for characterization of the chemostat bioreactor consisted of a batch mode followed by the continuous mode with 3 different dilution rates. We used BL21 Gold (DE3) pSB1KRDP strain in minimal carbon medium M9.
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The experiment was designed to keep the dilution rates constant for 3 reactor volumes, so as to obtain constant optical density, an indication of the biomass, within human sampling error, to achieve proof of concept.
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{{Team:Aachen/Figure|Aachen_Reactor1ODvsD.png|size=large|title = Plot of optical density and dilution versus time, in hours|subtitle =Chemostat cultivation of  in medium M9. We observed stable OD for 3 reactor volumes, with slight variations due to change in the conditions.|size=large}}
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The batch mode ran for 5 hours until we observed OD above the threshold of 1.0 in the reactor.
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The continuous mode of operation was initiated with a dilution rate of 0.1 h{{sup|-1}} at hour 5. Constant OD was obtained for 9 hours. However, the feed pump was observed to be pumping more medium than the harvest removed by the harvest pump. Hence at our 30, the Master Control Program was modified to change the speed of the harvest pump accordingly. The harvest pump rate was changed to pump 30 % faster than the feed pump. The change made is reflected in reduction of OD from an average value of 0.78 to 0.76.
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At hour 48, the first step change in dilution rate was made to 0.3 h{{sup|-1}}. The OD was observed to be stable with few variations within pipetting error.
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A final step increase in dilution rate was carried out at hour 63, where the dilution rate was increased to 0.4 h{{sup|-1}}. This resulted in OD values for 3 reactor volumes, which lasted for 7.5 hours.
  
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The experiment for characterisation of the chemostat Bioreactor consisted of a Batch mode followed by the Continuous mode with 3 different dilution rates.
 
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The experiment was designed to keep the dilution rates constant for 3 reactor volumes, so as to obtain constant OD within human sampling error, to achieve Proof of Concept.
 
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{{Team:Aachen/Figure|Aachen_Reactor1ODvsD.png|size=large}}
 
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The Batch phase ran for 5 hours until we observed OD above the threshold of 1.0 in the reactor to start increasing the dilution rate.
 
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The continuous mode was started with dilution rate of 0.1 /h at hour 5. Constant OD was obtained for 9 hours but soon it was observed that the feed pump supplies more medium than the harvest removed by the harvest pump. At hour 30, we decided to modify the Master Control Program to change the speed of the harvest pump. Therefore, the harvest pump rate was changed to pump 30% faster than the feed pump. This can be observed in the reduction in the OD from an average of 0.78 to 0.76. 
 
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At hour 48, the dilution rate was changed for step 2 of the experiment, to 0.3 /h. The OD was observed to be stable with few variations within pipetting error. 
 
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The step 3 was carried out at hour 63, with dilution rate at 0.4 /h. This resulted in OD values for 3 reactor volumes, which lasted for 7.5 hours.
 
  
 
{{Team:Aachen/Footer|color=green}}
 
{{Team:Aachen/Footer|color=green}}

Latest revision as of 03:50, 19 September 2015

Evaluation of the Bioreactor Design

As our bioreactor is built following the do-it-yourself principle, it is most crucial that the obtained results are reproducible. We, therefore, assessed the reliablitity of the reactor system design in a series of experiments.


Preliminary experiments were conducted to optimize and validate the complete bioreactor setup and its individual components. The configurations of the aeration pump (the 3D spring structure) was enhanced to pump air at a rate of 10 mL/min, to achieve an aeration rate of 1 vvm in the reactor.

Preliminary Runs

Before the development of our continuous OD device, an initial experiment was carried out in the mini-bioreactor and growth was manually monitored using the laboratory spectrophotometer. We used E. coli DH5α strain in LB medium and the experiment was run for 110 hours.

Aachen Reactor0ODvsD.png
Plot of optical density and dilution rate with respect to time, in hours
E. Coli DH5α was grown in LB medium. We were able to observe stable OD for different dilution rates for 5 reactor volumes

Final Experiment

The experiment for characterization of the chemostat bioreactor consisted of a batch mode followed by the continuous mode with 3 different dilution rates. We used BL21 Gold (DE3) pSB1KRDP strain in minimal carbon medium M9. The experiment was designed to keep the dilution rates constant for 3 reactor volumes, so as to obtain constant optical density, an indication of the biomass, within human sampling error, to achieve proof of concept.

Aachen Reactor1ODvsD.png
Plot of optical density and dilution versus time, in hours
Chemostat cultivation of in medium M9. We observed stable OD for 3 reactor volumes, with slight variations due to change in the conditions.


The batch mode ran for 5 hours until we observed OD above the threshold of 1.0 in the reactor.


The continuous mode of operation was initiated with a dilution rate of 0.1 h-1 at hour 5. Constant OD was obtained for 9 hours. However, the feed pump was observed to be pumping more medium than the harvest removed by the harvest pump. Hence at our 30, the Master Control Program was modified to change the speed of the harvest pump accordingly. The harvest pump rate was changed to pump 30 % faster than the feed pump. The change made is reflected in reduction of OD from an average value of 0.78 to 0.76.


At hour 48, the first step change in dilution rate was made to 0.3 h-1. The OD was observed to be stable with few variations within pipetting error.


A final step increase in dilution rate was carried out at hour 63, where the dilution rate was increased to 0.4 h-1. This resulted in OD values for 3 reactor volumes, which lasted for 7.5 hours.


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