Sustainability

Total complete efficiency converting reactant to product is incredibly difficult, and in some cases, waste or side product formation is inevitable. The PACE papers describe a variety of different cultivation conditions that unavoidably result in the consumption of chemicals and generation of waste product [1, 2]. A goal of our iGEM project was to reduce the consumption of these chemicals and waste production by optimizing the cultivation process. The team reached out to the department of Bioprocess Engineering where they got caught on the idea of the project and two students decided to join the team. The department for Bioprocess Engineering made it possible to use their laboratories and resources for the cultivation experiment. Furthermore they supported us with their experience in continuous E. coli cultivation.

With the goal of increasing the sustainability the cultivation a balance sheet was drawn up for the process. This increased the survey of the process and helped to identify 3 parts of the process which were giving us the possibility to improve the sustainability.

Minimize waste volume

The previous PACE experiments used a CSR with dilutions between 1 - 2.5 h^-1 to console the cell density in the bioreactor. To achieve the desired flow rate in the lagoon, most of the flow from the CSR was forwarded into the waste. This waste had to be minimized in order for efficiency to increase. Therefore the flowrates and the volumes were adapted so that no waste from the chemostat was produced. The cell density is controlled over the supplement concentration in the medium.

The idea was to calculate the CSR setup from the end to the beginning. This means from the phage output to the fresh medium input. With regard to the paper of Dickinson et al. [1] a dilution rate of 1 h^-1 was chosen (1 lagoon volume change per hour respectively). In order to avoid medium waste production the phage output flow had to be equal to the fresh medium input. Furthermore a minimal volume of medium was to be used in the stirred tank reactor (250 mL for a 1 L Applikon bioreactor).

CSR Parameter approximation:

The cultivation period was set to 72 hours and maximum medium reservoir volume of 5 L was chosen. With the following equation a maximum flow rate of 69.4 mL h^-1 was calculated.

F_L,0 =

V_R / t_c

Where F_L,0 is the medium flow through the system, V_R is the reservoir volume and t_c is the total cultivation time. Previous cultivations with E. coli done at the Institute of Bioprocess Engineering had shown a dilution rate above 0.15 h^-1 was suitable for cultivation. The following equation was used to approximate the dilution rate in the CSR at a medium flow of 69.4 mL h^-1.

D₁ =

F_L,0 / V_R

The calculated dilution rate (D₁) corresponded to a value of roughly 0.28 h^-1, which is sufficiently larger than the minimal dilution rate of 0.15 h^-1. To ensure enough medium for the a 72 h cultivation, a dilution rate of 0.25 h^-1 was chosen for the experiments. This result in a lower flow rate of 62.5 mL h^-1 which will allow a reserve volume of 500 mL. Respectively this flow rate will result in a lagoon volume of roughly 63 mL (according to a set dilution rate of 1 h^-1 inside the lagoon).

Minimize medium volume

The growth of the E. coli is controlled by the glucose and nitrogen concentration, which are calculated by the yield of the bacteria’s growth. This system makes it possible to control the growth and reduce the used supplements to a minimum. As a buffer system and phosphate source a phosphate buffer is used.

To limit cell growth inside the CSR a carbon limitation has to be used. As carbon source a glucose solution was used. To reach a final cell density of 5 ∙ 10¹¹ cells per liter corresponding to 0.25 g bio dry mass (BDM) per liter the limiting glucose concentration was calculated. With a yield of 0.5 gram BDM per gram glucose, a glucose concentration of 0.5 g L^-1 had to be used.

Medium nutrient	g L^-1
KH₂PO₄	2.7
Na₂HPO₄ · 12 H₂O	7.2
NH₄Cl	0.5
Na₂SO₄	1.1
MgCl₂	0.02
FeCl₃ · 6 H₂O	0.005
MnCl · 4 H₂O	0.0004
NaCl	5
Glucose	0.25

Chloramphenicol	25 · 10^-6 μg mL^-1
Trace elements solution	0.02 ml L^-1

Trace elements solution: 25 g FeSO₄ · 7 H₂O, 25 g ZnSO₄ · 7 H₂O, 5 g CuSO₄ · 5 H₂O, 5 g MnSO₄ · 4 H₂O, 1 g CoSO · 7 H₂O, 1 g H₃BO₃, 0.5 g Na₂MoO₄ · 2 H₂O, 0.5 g NiSO₄ · 6 H₂O, and 0.5 g KI.

Reduce antibiotics

During the growth of the E. coli it is possible for the organism to lose its plasmid. The most common way to prevent the loss of the plasmid is the addition of an antibiotic. In these experiments chloramphenicol was used. However bacteria containing a resistance are able to reduce the concentration making the growth for all bacteria possible. Therefore the stability of the plasmid has to be analyzed by using plates containing antibiotics and no antibiotics. If the plasmid is stable than it can be possible to run the CSR without antibiotics.

During the cultivations it was desired to leave out the antibiotic with the goal of reducing its waste. With the goal of identifying the plasmid stability, during different times of the cultivation, samples were taken and plated on LB-plates without antibiotics and than transferred on plates with antibiotics. To get an idea of the plasmid stability the ration of the grown cell is calculated. If the plasmids seem to be stable without the antibiotics they can be left away.

References

Dickinson, B. C., Packer, M. S., Badran, A. H., Liu, D. R. (2014). A system for the continuous directed evolution of proteases rapidly reveals drug-resistance mutations. Nature Communications, 5, Article number: 5352.
Esvelt, K. M., Carlson, J. C., Liu, D.R. (2011). A system for the continuous directed evolution of biomolecules. Nature, 472(7344), 499-503.