Pure-culture growth of fermentative bacteria, facilitated by H2 removal: bioenergetics and H2 production

Abstract

We used an H2-purging culture vessel to replace an H2-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (DeltaG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H2 production changed such that DeltaG remained nearly constant for each organism (-11.1 +/- 1.4 kJ mol butyrate(-1) for S. lipocalidus and -58.2 +/- 1.0 kJ mol alanine(-1) for A. colombiense). The cellular maintenance energy, determined from the DeltaG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 x 10(-13) kJ cell(-1) day(-1) for S. lipocalidus at 55 degrees C and 6.2 x 10(-13) kJ cell(-1) day(-1) for A. colombiense at 37 degrees C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.

FIG. 1.

Catabolism and bioenergetics of S. lipocalidus and A. colombiense. (A) Consumption of butyrate (open circles) and production of acetate (filled circles) in S. lipocalidus. (B) Consumption of alanine (open triangles) and production of acetate (filled triangles) in A. colombiense. (C and D) Time course changes of hydrogen partial pressure. (E and F) ΔG values calculated for measured concentrations of products and reactants.

FIG. 2.

Effect of purging gas flow rate on S. lipocalidus. (A) P_H2 over the course of the experiment. Vertical dashed lines indicate the times the purging rate was changed, with the rates given in cm³ min⁻¹. Horizontal dotted lines represent the P_H2 expected if the H₂ production rate remained constant at its initial value while the purging rate was changed from 20 cm³ min⁻¹. (B) H₂ production rate after stabilization at each purging rate. (C) Response of ΔG, calculated for steady-state P_H2 at each purging rate. The dashed line indicates the ΔG values expected if the H₂ production rate had remained constant at its initial value at the 20 cm³ min⁻¹ purging rate.

FIG. 3.

Effect of acetate concentration on S. lipocalidus. (A) Response of P_H2 to acetate. Dashed lines indicate times of acetate additions, and measured concentrations (mM) are noted. (B) Response of ΔG, calculated for the steady-state P_H2, at each acetate concentration. The dashed line indicates the ΔG values that would be expected if P_H2 had remained constant while acetate was added.

FIG. 4.

Effect of temperature on P_H2 of S. lipocalidus (A) and A. colombiense (C). Dashed lines indicate times of temperature changes, and the temperatures (°C) are noted. (B and D) Responses of ΔG, calculated for the steady-state P_H2 at each temperature, for S. lipocalidus (B) and A. colombiense (D). Dashed lines indicate the ΔG values that would be expected if the concentrations of products and reactants had remained at their initial values while the temperature changed.