Evaluation of <i>Thiobacillus denitrificans</i>’ sustainability in nitrate-reducing Fe(II) oxidation and the potential significance of Fe(II) as a growth-supporting reductant

The betaproteobacterium Thiobacillus denitrificans (ATCC 25259) oxidizes Fe(II) while reducing nitrate, yet its capacity for autotrophic growth as a nitrate-reducing Fe(II)-oxidizer remains uncertain. This study explored this capacity through cultivation experiments across multiple transfers, using growth medium with Fe(II) and nitrate as sole electron donor and acceptor, respectively. This setup necessitated nitrate reduction coupled to Fe(II) oxidation as the primary energy-yielding mechanism and Fe(II) as the exclusive electron donor for CO2 fixation and biomass production. Thiosulfate/nitrate pregrown T. denitrificans oxidized 42% of 10 mM Fe(II), reduced 54% of 3.5 mM nitrate, and accumulated 1.6 mM nitrite, but showed no cell growth. Subsequent transfers from this Fe(II)/nitrate culture to fresh medium with Fe(II) and nitrate showed no nitrate-reducing Fe(II) oxidation or population growth. While bacterial activity [Fe(II) oxidation, nitrate reduction] occurred in the first transfer from thiosulfate/nitrate to Fe(II)/nitrate, nitrite was produced, further reacting with Fe(II) abiotically (chemodenitrification). A kinetic model assessed enzymatic versus abiotic Fe(II) oxidation, revealing enzymatic oxidation accounted for twice as much (ca. 70%) as abiotic denitrification (ca. 30%) within 22 days. These findings suggest T. denitrificans performs the first step of denitrification with Fe(II) as an electron donor but does not achieve autotrophic growth under these conditions.