The representative Desulfobulbaceae
species, D. propionicus
(DSMZ 2032), was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures; Braunschweig, Germany) and was grown in a slightly modified version of NB Basal medium (14
), which contained the following (per liter): 0.42 g of KH2
, 0.22 g of K2
, 0.2 g of NH4
Cl, 0.38 g of KCl, 0.36 g of NaCl, 0.75 g of CaCl2
O, 0.10 g of MgCl2
, 1.8 g of NaHCO3
, and 0.5 g of Na2
, as well as 1 μM Na2
and trace minerals and vitamins. This medium differed from that typically used to culture D. propionicus
(DSMZ medium 194) in that it contained selenium and fivefold more calcium. Sulfate other than that present in the trace metal solution (300 μM) was omitted to prevent extensive sulfide production with subsequent abiotic reduction of Fe(III). Strict anaerobic techniques were used throughout, and cultures were incubated at 30°C in the dark. Organic acids (21
), cell numbers (19
), Fe(II), and total iron (19
) were monitored as previously described. Growth with various electron donors and acceptors was considered positive only after six consecutive transfers.
When D. propionicus was grown on pyruvate alone, 6.67 ± 0.33 mM (n = 3) pyruvate was fermented to 4.22 ± 0.51 mM acetate and 2.23 ± 0.35 mM propionate according to the following reaction: 3CH3COCOO− + 3H2O→2CH3COO− + CH3CH2COO− + 2HCO3− + 2H+.
These results are similar to those of previous studies that have shown that acetate and propionate are formed in a 2:1 ratio when Desulfobulbus propionicus
is grown on pyruvate in the absence of an electron acceptor (26
Pyruvate consumption by D. propionicus differed significantly from fermentation when a soluble form of iron was provided as an electron acceptor. For example, when pyruvate (mean ± standard deviation, 19.20 ± 0.68 mM; n = 3) was provided as the electron donor with Fe(III)-citrate (50 mM) as the electron acceptor, Fe(III) was reduced and 18.65 ± 0.36 mM acetate was formed, accompanied by cell growth (Fig. ). The stoichiometry of pyruvate consumption and Fe(III) reduction was consistent with the following reaction: CH3COCOO− + 2Fe3+ + 2H2O→CH3COO− + HCO3− + 2Fe2+ + 3H+.
FIG. 1. (A) Growth of D. propionicus with pyruvate (19.2 mM) as the electron donor and Fe(III)-citrate (50 mM) as the electron acceptor. (B) Growth with Fe(III)-oxide (100 mmol/liter) as the electron acceptor and hydrogen (101 kPa) as electron donor. (C) Growth (more ...)
D. propionicus was able to grow with several other soluble electron acceptors, including Fe(III)-NTA (5 mM), Fe(III)-pyrophosphate (10 mM), and anthraquinone-2,6-disulfonate (AQDS; 5 mM), with pyruvate as the electron donor. Propionate, lactate, and hydrogen also served as electron donors for growth on all forms of soluble Fe(III) evaluated as well as AQDS. The mechanism(s) for Fe(III) reduction appeared to be independent of the mechanism(s) for sulfate reduction, because D. propionicus continued to reduce Fe(III)-citrate in the presence of 1 to 10 mM molybdate, an inhibitor of sulfate reduction.
There was a mixture of pyruvate fermentation and Fe(III) reduction when poorly crystalline Fe(III)-oxide (100 mM) (17
) was provided as the electron acceptor with pyruvate (7.15 mM) as the electron donor. In the presence of Fe(III)-oxide approximately twice as much acetate was formed from pyruvate oxidation than would be expected from pyruvate fermentation alone; 6.87 ± 0.75 mM (n
= 3) of Fe(III)-oxide was reduced, and 5.13 ± 0.36 mM acetate and 1.84 ± 0.25 mM propionate accumulated (Fig. ). In addition, Fe(III)-oxide reduction during pyruvate metabolism appeared to yield energy to support cell growth, as the final cell numbers in cultures grown in the presence of Fe(III)-oxide, 1.0 × 108
± 1.53 × 107
cells/ml (starting cell number was 1.20 × 106
± 2.32 × 105
= 3), were significantly higher than those in cultures grown with pyruvate alone (3.72 × 107
± 2.5 × 106
cells/ml of culture; starting cell number was 2.43 × 106
± 6.35 × 105
= 3). The stoichiometry of this mixed reaction was consistent with the following reaction: 3.4CH3
and 3.75CH3COCOO− + 3.75H2
+ 2.5HCO3− + 2.5H+ for a combined reaction of 7.15CH3
+ 10.55H2O→5.9CH3COO− + 5.9HCO3− + 6.8Fe2+ +1.25CH3
D. propionicus could also be continually cultured with hydrogen as the electron donor and poorly crystalline Fe(III)-oxide as the electron acceptor when acetate (0.1 mM) was provided as a carbon source (Fig. ).