Polysulfides as intermediates in the oxidation of sulfide to sulfate by Beggiatoa spp

Abstract

Zero-valent sulfur is a key intermediate in the microbial oxidation of sulfide to sulfate. Many sulfide-oxidizing bacteria produce and store large amounts of sulfur intra- or extracellularly. It is still not understood how the stored sulfur is metabolized, as the most stable form of S(0) under standard biological conditions, orthorhombic α-sulfur, is most likely inaccessible to bacterial enzymes. Here we analyzed the speciation of sulfur in single cells of living sulfide-oxidizing bacteria via Raman spectroscopy. Our results showed that under various ecological and physiological conditions, all three investigated Beggiatoa strains stored sulfur as a combination of cyclooctasulfur (S8) and inorganic polysulfides (Sn(2-)). Linear sulfur chains were detected during both the oxidation and reduction of stored sulfur, suggesting that Sn(2-) species represent a universal pool of bioavailable sulfur. Formation of polysulfides due to the cleavage of sulfur rings could occur biologically by thiol-containing enzymes or chemically by the strong nucleophile HS(-) as Beggiatoa migrates vertically between oxic and sulfidic zones in the environment. Most Beggiatoa spp. thus far studied can oxidize sulfur further to sulfate. Our results suggest that the ratio of produced sulfur and sulfate varies depending on the sulfide flux. Almost all of the sulfide was oxidized directly to sulfate under low-sulfide-flux conditions, whereas only 50% was oxidized to sulfate under high-sulfide-flux conditions leading to S(0) deposition. With Raman spectroscopy we could show that sulfate accumulated in Beggiatoa filaments, reaching intracellular concentrations of 0.72 to 1.73 M.

FIG 1

Example of H₂S (○), total sulfide (●), and SO₄²⁻ (□) profiles in a Beggiatoa sp. 35Flor culture after 16 days of incubation with a low sulfide flux. The Beggiatoa mat was located at a depth of 0.5 to 0.7 cm.

FIG 2

(A) Optical micrograph of a Beggiatoa sp. 35Flor filament with a low sulfide flux. (B) Point spectra of sulfur inclusions measured 5, 8, and 16 days after inoculation using constant 180-μW laser power and 1-s exposure time, compared to sulfur standards (Std). A spectrum of cytochrome c (cyt c)identified in the Beggiatoa membrane region is also shown. (C) Raman map of a filament, showing the 473-cm⁻¹ sulfur peak in yellow and autofluorescence of the cell in green.

FIG 3

(A) Optical micrograph of Beggiatoa sp. 35Flor from the upper mat of a culture grown with a high sulfide flux. (B) Corresponding Raman map showing the occurrence of the 473-cm⁻¹ sulfur peak in yellow/red and autofluorescence of the cell in green. (C) Representative point spectra of sulfur inclusions from upper and lower mat filaments together with polysulfide and cyclooctasulfur standards (Std).

FIG 4

(A) From top to bottom: (i) a background-corrected spectrum of live Beggiatoa sp. 35Flor grown on sulfate-free medium exhibiting a sulfate peak at 997 cm⁻¹ and (ii) sulfate detected in a dried filament in comparison to (iii) an Na₂SO₄ standard. (B) Raman map of a dried Beggiatoa sp. 35Flor filament, with the 473-cm⁻¹ S—S peak shown in yellow/red, the 997-cm⁻¹ S=O peak of sulfate in blue, and autofluorescence of the cell in green.

FIG 5

Porewater sulfate concentrations in the upper 2 cm of agar medium from cultures grown with different sulfide concentrations in the bottom agar: uninoculated control (■), 4 mM (□), 12 mM (▲), and 16 mM (○).

FIG 6

(A) From top to bottom: (i) representative point spectra of sulfur inclusions from B. alba B18LD, (ii) extracellular sulfur from B. alba B15LD, (iii) a polysulfide standard, (iv) cytochrome c, and (v) PHB. (B and C) Optical micrographs of strain B18LD (B) and strain B15LD (C) grown with 8 mM sulfide in the bottom agar. (D and E) Raman map of a B15LD filament, with the 2,900-cm⁻¹ C—H peak in pink and the 757-cm⁻¹ cytochrome c resonance peak in green (D), and the corresponding optical image (E). The red square outlines the Raman map.