During the initial batch period, a specific growth rate (μ) of 0.65 h-1 was achieved, whereas a lower specific growth rate
of 0.07 h-1 was maintained during the fed batch period. It has been reported that low growth rates lead to a higher specific
plasmid DNA yield than if cell growth is not inhibited. A reduced growth rate is critical for high quality, high yield fermentations
for plasmid production because it allows for plasmid amplification and greater stability.14
The cellular yield achieved from the fermentation was 0.42 g dry cell/g of glucose, which is similar to what has been reported
in the literature (0.5 g dry cell/g of glucose) for E. coli, based on the theory described by Carnes.14 On the other hand, the total protein concentration levels were high (around 7 g/L) after feeding started, because of the
yeast extract, which contained high quantities of protein.
The highest cell mass (29±1.7 g dry cell/L), plasmid yield (154±2.8 mg plasmid DNA/L), and specific plasmid DNA yield (0.44±0.02
mg plasmid DNA/g dry cell weight) were obtained after 24 h of culture (Figure 1), so we suggest that culture be stopped after
Taking into account that several studies have shown that the supercoiling linking number is an important factor to consider
when processing pDNA for therapeutic use,8 we analyzed the percentage of supercoils at this time point in the culture (24 h) and found that the level was approximately
80% of the total plasmid DNA (Figure 2). Several articles have reported similar results because supercoiled pDNA is known
to be more resistant to certain growth conditions than other isoforms.8
Figure 2. Plasmid DNA yield kinetics of recombinant E. coli DH10B transformed with pIDKE2 in the designed medium in a 5-L fed batch fermentation process
The behavior of E. coli (DH10B) in the fed batch fermentation was normal because the state of the system changed from one with a low initial cellular
concentration (0.1 g dry cell weight/L) to a state with very high biomass and product concentrations.
Comparing Glucose and Glycerol as Carbon Sources
Glucose is a conventional carbon source because it is inexpensive and very efficiently metabolized. However, high glucose
levels are known to cause undesirable acetate production as a result of metabolic overflow. The application of glycerol avoids
the repression of intermediate metabolites and accumulations of inhibitive organic acids to some extent. Therefore, the effect
of glycerol additions in culture and feed medium on plasmid DNA production in E. coli was also examined. The effects of glycerol and glucose as a carbon source in culture medium on plasmid production are shown
in Figure 3.
Figure 3. Cell growth kinetics of recombinant E. coli DH10B transformed with pIDKE2 in a 5-L fed batch fermentation process. Effects of carbon source on plasmid production and
The results showed that the highest cell mass (27.71±1.5 g dry cell weight/L) and specific plasmid yield (0.45±0.15 mg plasmid
DNA/g dry cell weight) were obtained after 24 h of culture when glucose was chosen as the carbon source for the medium, whereas
both cell mass and plasmid productivity were reduced when glycerol was used. Thus, compared to glycerol, glucose was the optimal
carbon source when the initial concentration was 5 g glucose/L, and 265 g/L was added to the feed medium.
The feeding of nutrients, usually glucose, has been extensively researched and incorporates a range of approaches that span
from simple to very elaborate, each presenting its own advantages and disadvantages.2