TABLE 2

Analysis of metabolites in spent culture medium of D. alaskensis G20 wild type and CycA mutant I2a

Medium and strainConcn of substrate or accumulated metabolite remainingbProtein yield (mg/liter)Recovery (%)Initial electron donor/acceptor concn (mM)i
PyruvateMalateSuccinateLactateFumarateFormateAcetateCOH2eH2SfCarbongReductanth
Lactate-sulfate
    G20NDc0.20.727.4NDND24.1NQd0.613.7102.110211251.9/30.0
    I2NDNDND32.0ND1.620.3ND1.08.895.310110152.0/30.0
Pyruvate-sulfate
    G2034.50.30.4NDND1.916.4NQ0.4485.59510354.5/15.0
    I247.6NDNDNDND0.65.8ND0.9ND12.01009953.4/15.0
Pyruvate
    G2041.0ND0.2ND0.13.27.3ND0.7ND31.310210248.7
    I240.4NDNDNDND0.77.6ND0.9ND32.4999548.0
  • a Duplicate 200-ml lactate-sulfate and pyruvate-sulfate cultures were harvested when their growth reached an OD600 of ∼0.5, with the exception of I2 on pyruvate-sulfate, which was harvested at an OD600 of 0.17. Cells fermenting pyruvate were harvested at an OD600 of ∼0.2.

  • b The soluble metabolite concentrations in the filtrates of harvested cultures. Typically, from the inoculum, there was measurable carryover of acetate to the experimental cultures. This carryover amount was subtracted from the concentration reported in the table. Data are in millimolar unless indicated otherwise and are averages of measurements from duplicate cultures.

  • c ND, not detected.

  • d NQ, detected but not quantified. H2 interfered with the precise measurement of CO with the gas chromatograph.

  • e The H2 was collected, and the amounts (mmol) produced in a 200-ml culture were calculated by use of the ideal gas law.

  • f No H2S was detected in the collected gaseous end products, and dissolved H2S was quantified (34). The 0.5 mM sulfide added at the time of inoculation, to reduce the medium and to provide a sulfur source for fermenting cultures, was subtracted from the sulfide concentration measured at harvest. The lower limit of detection in this experiment was 0.5 mM.

  • g The carbon recovered in culture filtrates was the ratio of the concentration of carbon present in measured end products and the remaining unused substrate to the initial concentration of carbon provided as the electron donor. For the purpose of this calculation, it was assumed that as a primary end product of organic acid oxidation, the CO2 concentration was essentially equal to that of the acetate generated during growth minus the concentration of any other C1 or C4 end products, such as formate, fumarate, and succinate, which were identified and quantified by HPLC. The amount of carbon present in the biomass produced was not taken into account in these calculations.

  • h Reductant recovery was the ratio of the number of mole equivalents of electrons in the measured end products as well as the electrons remaining in unused substrate to the total number of moles of electrons available for consumption in the substrate added.

  • i Measured concentrations of electrons and the carbon donor determined by HPLC, corrected for inoculum carryover, and calculated concentrations of sulfate in the growth media for G20 and I2 at the initiation of growth.