Team:Lethbridge/Acetic Acid Production

Results

Acetic Acid Production

Acetic Acid Production and Export by E. coli

Overview

To test the effectiveness of overexpressing and co-localizing PTA and ACK with the transporter Aata, we first needed to determine intrinsic acid production by Escherichia coli BL21(DE3). Glucose was used as an energy source for E. coli, since this will be produced by Synechococcus elongatus in our optimized system.

Experimental Setup

Overnight cultures of E. coli were used to inoculate fresh LB media that was supplemented with various concentrations of glucose (0-16 g/L). Growth of the cultures was monitored by measuring the optical density at 600 nm every hour for 6 hours. In addition, 4 mL of each culture were collected and centrifuged to obtain a clear supernatant. Titrations were performed with standardized 0.05 M NaOH in order to determine the concentration of acid in the media.

Figure 1. Growth curves of E. coli BL21(DE3) in different starting concentrations of glucose.

All cultures grew at a similar rate and to a similar final density (Fig. 1). This indicates that glucose concentration does not significantly affect the growth of E. coli. The amount of acid produced by the E. coli was also not significantly influenced by glucose concentration (Fig. 2). In the culture grown without the addition of glucose, an average amount of 13 mM acid was released into the media. This concentration remained stable over the six hour time window. However, a slight upward trend can be seen for most of the cultures supplemented with glucose, with the media for those cultures reaching a final acid concentration close to 30 mM, or approximately 2-fold when compared to the control culture. Further replication of this data will be necessary to determine acid production rates by E. coli.

Figure 2. Concentration of acid in the culture media of growing E. coli. Data points represent an average concentration from three separate trials ± standard deviation.

The acid produced by these cultures was due entirely to intrinsic acid production by E. coli, and we will be able to use these values to compare acid production by E. coli after overexpression of the acetogenesis enzymes PTA and ACK, as well as after using a scaffold to co-localize those enzymes with the acetic acid transporter Aata. This will indicate the level of expression needed for optimal levels of acetic acid production for CAB extraction. With the overexpression of Aata, we will also expect increased resistance to low pH, which should be reflected in the growth curve of E. coli growing in higher concentrations of acetic acid.