Investigating Abiotic and Biotic Mechanisms of Pyrite Reduction

Abstract

Pyrite (FeS₂) has a very low solubility and therefore has historically been considered a sink for iron (Fe) and sulfur (S) and unavailable to biology in the absence of oxygen and oxidative weathering. Anaerobic methanogens were recently shown to reduce FeS₂ and assimilate Fe and S reduction products to meet nutrient demands. However, the mechanism of FeS₂ mineral reduction and the forms of Fe and S assimilated by methanogens remained unclear. Thermodynamic calculations described herein indicate that H₂ at aqueous concentrations as low as 10^-10 M favors the reduction of FeS₂, with sulfide (HS^-) and pyrrhotite (Fe_{1- x} S) as products; abiotic laboratory experiments confirmed the reduction of FeS₂ with dissolved H₂ concentrations greater than 1.98 × 10^-4 M H₂. Growth studies of Methanosarcina barkeri provided with FeS₂ as the sole source of Fe and S resulted in H₂ production but at concentrations too low to drive abiotic FeS₂ reduction, based on abiotic laboratory experimental data. A strain of M. barkeri with deletions in all [NiFe]-hydrogenases maintained the ability to reduce FeS₂ during growth, providing further evidence that extracellular electron transport (EET) to FeS₂ does not involve H₂ or [NiFe]-hydrogenases. Physical contact between cells and FeS₂ was required for mineral reduction but was not required to obtain Fe and S from dissolution products. The addition of a synthetic electron shuttle, anthraquinone-2,6-disulfonate, allowed for biological reduction of FeS₂ when physical contact between cells and FeS₂ was prohibited, indicating that exogenous electron shuttles can mediate FeS₂ reduction. Transcriptomics experiments revealed upregulation of several cytoplasmic oxidoreductases during growth of M. barkeri on FeS₂, which may indicate involvement in provisioning low potential electrons for EET to FeS₂. Collectively, the data presented herein indicate that reduction of insoluble FeS₂ by M. barkeri occurred via electron transfer from the cell surface to the mineral surface resulting in the generation of soluble HS^- and mineral-associated Fe_{1- x} S. Solubilized Fe(II), but not HS^-, from mineral-associated Fe_{1- x} S reacts with aqueous HS^- yielding aqueous iron sulfur clusters (FeS _aq ) that likely serve as the Fe and S source for methanogen growth and activity. FeS _aq nucleation and subsequent precipitation on the surface of cells may result in accelerated EET to FeS₂, resulting in positive feedback between cell activity and FeS₂ reduction.

Keywords: dissolution; extracellular electron transfer; hydrogen; methanogens; pyrite (FeS2); pyrrhotite (Fe1–xS).

FIGURE 1

Gibb’s free energy calculated for abiotic pyrite (FeS₂) reduction to pyrrhotite (Fe_1–xS) and HS^– by H₂ at 38°C, an ionic strength of 0.05 M, and at a pH of 7.0. The vertical dashed line depicts the minimum H₂ concentration that abiotic FeS₂ reduction was detected experimentally (see Figure 2).

FIGURE 2

Production of total sulfide (aqueous plus gas phase) in reactors containing 2 mM synthetic FeS₂ nanoparticles when incubated at 38°C in the presence of H₂ ranging from 0 to 1.98 × 10^{– 3} M aqueous H₂ (equivalent to 0 to 2.5 bar). All abiotic reactors contained 35 mL of base salts medium and 35 mL of headspace (balance of headspace as N₂ gas).

FIGURE 3

Dissolved ferrous iron [Fe(II)] concentration in abiotic dialysis experiment reactors following 7 days of incubation at 38°C. One hundred sixty-five milliliters serum bottles containing base salts medium were provided with no Fe (No added Fe), 0.1 g of specimen pyrrhotite sequestered in 50 kDa dialysis tubing (Sequestered Fe_1–xS), or 20 μM FeCl₂. Reactors were either provided with no sulfur source (0 μM HS^–) or 500 μM Na₂S (500 μM HS^–). Following incubation, subsamples were collected under anoxic conditions and were leached with 1N HCl at 4°C for 16 h before quantifying Fe(II).

FIGURE 4

Production of (A) total CH₄, (B) dissolved H₂, (C) total sulfide (aqueous plus gas phase), and (D) total biomass (DNA) by Methanosarcina barkeri strain Fusaro during growth with 2 mM synthetic pyrite (FeS₂) nanoparticles, 20 μM ferrous iron [Fe(II)] and 2 mM HS^–, or no added Fe or S source (No Fe/S). The H₂ concentration is reported for the dissolved phase. Averages and standard deviations for triplicates are shown. Protein data as an additional proxy for growth is presented in Supplementary Figure 3. Data depicting growth kinetics and activities of M. barkeri strain MS with FeS₂ are presented in Supplementary Figure 4.

FIGURE 5

Production of (A) methane (CH₄), (B) total sulfide (aqueous plus gas phase), and (C) total biomass (DNA) in cultures of Methanosarcina barkeri Fusaro with deletions in each of the four operons encoding all five [NiFe]-hydrogenases in its genome. The M. barkeri Fusaro hydrogenase mutant was grown either with no provided iron (Fe) or sulfur (S) source (No Fe/S); with 20 μM ferrous iron [Fe(II)] and 0.4 mM sulfide (HS^–) [Fe(II)/HS^–]; or with 2 mM synthetic pyrite (FeS₂) nanoparticles as the sole sources of Fe and S. Hydrogen (H₂) was not detected (detection limit 0.1 μM) in the headspace of any of the culture conditions tested (data not shown). Averages and standard deviations for triplicates are shown.

FIGURE 6

Production of (A) total methane (CH₄), (B) total sulfide (aqueous plus gas phase), (C) total biomass (DNA), and (D) reduced anthrahydroquinone-2,6-disulfonate (AH₂QDS) from anthraquinone-2,6-disulfonate (AQDS) by Methanosarcina barkeri strain Fusaro when grown with pyrite (FeS₂) free in solution (green) or sequestered in 100 kDa dialysis tubing (orange) to prevent physical association. Negative control cultures contained no added Fe or S source (red), while positive control cultures were provided with 20 μM Fe(II) and 2 mM HS^– (red; same data as presented in Figure 2). AQDS was provided at a final concentration of 20 mM. In (A–C), culture conditions with added AQDS are represented by dashed lines and culture conditions that do not contain AQDS are shown by solid lines. Only conditions provided AQDS are shown panel d, and absorbance at 325 nm (AQDS) is depicted by solid lines while absorbance at 450 nm (AH₂QDS) depicted by long-dashed lines. Averages and standard deviations for triplicates are shown. Protein data as an additional proxy for growth is presented in Supplementary Figure 3.

FIGURE 7

Production of (A) total CH₄ and (B) total biomass (DNA) of M. barkeri Fusaro wild-type grown with no added Fe (No added Fe), with 0.1 g specimen pyrrhotite (Fe_1–xS; 63–125 μm size fraction) sequestered in 100 kDa dialysis tubing (Sequestered Fe_1–xS), or with 20 μM FeCl₂. For each of these conditions, no sulfide (0 μM HS^–) or 500 μM sulfide (500 μM HS^–) was added as Na₂S. The data represent the difference for each analyte between the initial measurement (day 0) and the final measurement (day 10). Averages and standard deviations for triplicates are shown. Sampling was infrequent to minimize potential damage to dialysis membranes from sharp, freshly fractured, Fe_1–xS grains and their edges.

FIGURE 8

(A) Methanosarcina barkeri MS gene transcripts that were significantly upregulated in cells provided with synthetic pyrite (FeS₂) nanoparticles compared to those provided with ferrous iron [Fe(II)]/cysteine (Cys) and those provided with Fe(II)/sulfide (HS^–) as the sole iron and sulfur source. The log fold change (LFC) of transcript abundance in FeS₂- versus Fe(II)/HS^–-grown cells (y-axis) is plotted as a function of the LFC of transcript abundance in FeS₂- versus Fe(II)/Cys-grown cells (x-axis). Each point represents a single gene and the size of the point represents the relative mean normalized transcript abundance detected across FeS₂ replicate cultures. (B) A cassette of genes whose expression (transcripts) were upregulated when Methanosarcina barkeri MS cells were provided with synthetic FeS₂ nanoparticles when compared to cells provided with 20 μM ferrous iron [Fe(II)] and 2 mM Cys or 20 μM ferrous iron [Fe(II)] and 2 mM HS^– as sole sources of Fe and S. Truncated locus tags and annotations of gene functions as assessed by UniProt are provided (“MSBRM_” has been removed due to space limitations). Gene arrows colored blue represent significant differential expression in M. barkeri MS cells grown on FeS₂ compared to cells grown with Fe(II)/Cys or Fe(II)/HS^– (p < 0.05, LFC > 0.5). The green gene arrows were significantly upregulated (p < 0.05) in the FeS₂ growth condition relative to both the Fe(II)/Cys or Fe(II)/HS^– growth conditions but the LFC was below 0.5. The gene depicted in yellow was upregulated on the FeS₂ growth condition relative to both the Fe(II)/Cys or Fe(II)/HS^– growth conditions but not significantly (p > 0.05). The gene depicted in white was not differentially regulated among growth conditions. Importantly, an analysis of proteomes of Methanococcus voltae A3 cells provided with 2 mM FeS₂ or 26 μM Fe(II) and 2 mM HS^– independently identified homologs of many of the same proteins as being up-expressed under the FeS₂ condition (Payne et al., 2021a), and these are indicated with an asterisk (*). Gene abbreviations: Fld, flavodoxin; AKR, aldo/keto reductase; Fdx, ferrodoxin; mABH, membrane-associated alpha/beta hydrolase; FeoAB, ferrous iron transporter subunits (A,B).

FIGURE 9

A model for Methanosarcina barkeri growth on pyrite (FeS₂). See main text for description.