Substrate-source flexibility of an exponential-fed perfusion process to produce plasmid DNA for use as leishmaniasis vaccine

Figures & data

Figure 1. Modes of operation of bioreactors for pDNA production and medium colour comparison.

Figure 2. Kinetics of batch fermentation. Biomass growth and substrate-consumption kinetics using glucose (a) and glycerol (b). Experimental mean with standard deviation (±SD) of cell [○] and substrate [■] concentrations. Plasmid formation kinetics using glucose (c) and glycerol (d). Experimental mean with SD of VY [♦], SY [▲]. [—] Adjusted model.

Table 1. Operating and kinetic parameters of batch and EFP cultures.

Symbol	Glucose	Glycerol	Units	Description
Batch parameters
X0	1.00	1.00	OD600	Initial biomass concentration
Xm	16.36	12.50	OD600	Maximum biomass concentration
µm	0.55	0.50	1/h	Maximum specific growth rate
YX/S	1.16	0.86	OD600 L/g	Yield coefficient (biomass/glucose)
YP/X	1.28	2.78	mg/(L OD600)	Yield coefficient (plasmid/biomass)
EFP parameters
X0	1.00	1.00	OD600	Initial biomass concentration
Xm	86.00	66.50	OD600	Maximum biomass concentration
Ks	0.10	0.10	g/L	Saturation constant
µp	0.25	0.25	1/h	Maximum specific growth rate
VD	3.00	3.00	L	Total volume fed
YX/Sp	1.16	0.86	OD600/(g/L)	Yield coefficient for the perfusion phase (biomass/glucose)
YP/Xb	1.44	2.85	mg/(L OD600)	Yield coefficient for the batch phase (plasmid/biomass)
YP/Xp	1.90	3.65	mg/(L OD600)	Yield coefficient for the perfusion phase

Figure 3. Agarose gel electrophoresis of cell lysate of the batch fermentation. Glucose culture (a) lanes 1–5 correspond to time 0, 2, 4, 5, and 5.5 h respectively. Glycerol culture, (b) lanes 6–9 correspond to 0, 2, 4 and 5 h respectively. (M) Molecular weight marker.

Figure 4. Kinetics of the EFP fermentation. Exponential feed with μ_p = 0.25 h⁻¹. Biomass and substrate consumption kinetics using glucose (a) and glycerol (b). Experimental cell concentration [○] and substrate concentration [■]. Plasmid formation kinetics using glucose (c) and glycerol (d). Experimental VY [♦] and SY [▲]. [—] Fitted model.

Figure 5. Agarose gel electrophoresis of cell lysate of the EFP fermentation. Glucose culture (a) lanes 1–8 correspond to samples at time 0, 4, 5, 5.5, 6, 8, 10, and 12 h respectively. Glycerol culture (b) lanes 9–16 correspond to time 0, 4, 5, 6, 8, 10, 11, and 12 h respectively. (M) Molecular weight marker.

Figure 6. Dissolved oxygen control. Batch (a) and EFP (b) experiments using glucose and batch (c) and EFP (d) experiments using glycerol.