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.
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.
|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
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.
|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.