Continuous production

Because of the time limit, our propane producing E.coli strain wasn't competely constructed. We decided to try chemostat production with E.coli BL21 (DE3 ΔyjgB ΔyqhD, pET-TPC4 + pCDF-cAD + pACYC-Fdx-Fpr) made by Kallio et al. which differs from our own strain with two enzymes at the beginning of the reaction pathway. Thus, experiments with Kallio's strain largely reveals how our own strain would have behaved. More information available at the Project page.

The experiment was successful. With 1,0 l/h aeration, the propane content of reactor's gas phase was determined to be 4,5-22,7 ug/l with GC/MS. The result were calculated when 0,1-0,5 ug of propane was gathered into a 22ml gas chromatography vial. However, these values were below GC-standard line so the accuracy may have decreased. The GC-samples were also gathered after the second steady state was over. Nevertheless, the result proves that propane can be formed industrially with continuous production.

The batch phase took six hours, and soon after starting the process dissolved oxygen amount decreased into zero. Therefore, oxygen became the limiting factor of growth. Continuous phase was started and the first steady state was reached 39 h after starting to feed fresh media into the reactor. Overall, the steady state lasted for 15 hours. The density of cell population was calculated to be 9,85 g/L at this point, which can be assumed relatively high. Interestingly, bacteria acted as facultative anaerobe when changing its oxygen uptake depending on the growth phase.

IPTG induction started with 2mM concentration and the population growth started to react to changed process conditions. New steady state was achieved 40 h later and the population density became 9,28 g/L. Thus, there was a 0,57 g/L difference of the biomass between the normal growth and the growth with propane production. Reactor's glucose consentration decreased from original 20 g/l to zero during the exponential grow phase as the cells used it as a carbon source. However, it was hard to recognize which was the limiting factor of growth and production, glucose or oxygen? During the first steady state, glucose concentration increased when the need for the catabolism of biomass was lower. Before starting to feed fresh media, The end of batch phase was determined from decreased pH-level when the cells produced acids as a response for nutrient deficiency. TB-media used for cultivation contained phosphates which had buffer capacity but it wasn't enough. pH-control was assembled in order to maintain pH-values above 5,0.

More information about the experiment available at the Process data sheet and the Lab book. Details about standards, peaks etc. for GS/MS can be seen in this file.