Sulfate Reduction Drives Elevated Methylmercury Formation in the

Overview

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Key numbers

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accounted for over 50% of the hgcA gene transcription, despite representing less than 10% of the hgcA-carrying microbial
in low oxygen (<0.5 mg/L) and anoxic (oxygen below more highly conserved and is therefore most often used as a
than actively regulated.26 However, hgcA expression is the range of redox conditions found in the hypolimnion with
formation and accumulation was specifically investigated incubations, enriched inorganic 198Hg (198Hg(II); 92.8%
interdisciplinary approach, including field experiments and methyl-204Hg (Me204Hg; 98.1% isotope abundance), to track
descriptions of the sites and sample collection can be found in epilimnetic sulfate (∼30.5 mg/L).28,29 Samples were gently
were collected from the deepest basin (∼24 m) in Lake withdrawn from the bags and preserved to 1% HCl in a 125
Teflon tubing. Sulfide/sulfate samples were preserved in 1% at the USGS MRL and passed required quality assurance and
1% nitric acid. Filter-passing metal samples were filtered using tion�Section S1.3. Ambient total Hg (THg) in filter-passing
The filtrate was preserved to 1% hydrochloric acid (HCl) and analysis was conducted by bromine monochloride oxidation,
Hg(II) = THg MeHg relative abundance = × 100%
rather than a true rate since the 198Hg(II) in the pre- strategies53−57 and subsequently dereplicated using a 96%
In eq 3, Kmet and nonSRBKmet represent the mean Kmet and to the internal standard, ISlength is the length in base pairs of the
MS on an Agilent 8900 (Agilent, Santa Clara, CA). Dissolved transcript concentration = × NFmRNA
period.24 Additionally, this period immediately precedes lake (Mn) in the oxycline (maximum at oxycline = 0.101 mg/L,
turnover, which is a common driver of MeHg uptake into the mean overall = 0.019 ± 0.005 mg/L) and filter-passing Mn just
elevated primary production and subsequent high biological interface = 0.383 mg/L, mean overall = 0.178 ± 0.023 mg/L)
oxygen depletion started in July (Figure S2).24,63−67 Complete mean values are presented as “mean ± standard error of the
shown little to no detectable nitrate or nitrite in the due to the elevated sulfide levels leading to FeS precipitation.69
metalimnion and hypolimnion in Lake Mendota after August Sulfate levels were over 20 mg/L in the epilimnion. During fall,
error bars represent the standard error of the mean (SEM; n = 4) of Kmet (A−C). Horizontal error bars represent the standard deviation of the hgcA
minimum measured sulfate concentration of 4.7 mg/L (Figure been conducted in mesotrophic or oligotrophic systems10,76 or
maximum THg in 2021 = 1.41 ng/L; Table S1). Filter-passing and/or used glass serum bottles, which could scavenge or sorb
L), which was also reflected in the percent MeHg data (2020 lake, with up to 50−60% of the 198Hg(II) being methylated
MeHg did not solely account for the increase in THg with reached ∼1.25 mg/L, suggesting possible abiotic demethyla-
in 2021, accounting for a maximum of 2.1% of the THg. This conditions (Kdem = ∼0 day−1). The higher Kmet values in 2020
incubations are presented in Table S2.60 Incubation validation tion. MeHg can be photochemically demethylated by UVA,
both date and depth of the incubation. Kmet ranged from 0.001 demethylation. While we identified several homologues of
for water column methylation assays in freshwater (range = amino acid residues83 and were not contained within a mer
(Table S7).7−10,23,70−80 This rapid methylation could be due Methanogens and methanotrophs have also been shown to
documented to reduce up to 5% of the MeHg pool after across the different incubations, and propose that the decrease
MeHg in 2020 than 2021 (Figure 1). Kdem values were higher (Tables S4−S6). Relative hgcA gene abundance ranged from
in 2021, which could also contribute to reduced overall MeHg 0.3 to 16.3% of the total microbial community (mean = 7.5 ±
in the water column. Two lines of evidence suggest that 1.6%). This range is consistent with previously reported values
has been reached, and assuming the absence of significant transcript abundance ranged from 1.0 to 7.9 million transcripts
external sources or sinks, we can thus assume that Kmet/Kdem = per liter (mean = 3.1 ± 0.7 million transcripts per liter); to our
of sulfide (∼2.4 mg/L in 2020, ∼3.8 mg/L in 2021), Kmet sulfide threshold leading to the canonical unimodal Goldilocks
represent the standard error of the mean (SEM; n = 4) of Kmet (A), the standard deviation of the hgcA abundance when duplicate metagenomes
Hg(II) bioavailability, regardless of the underlying biogeo- redox conditions. SRBKmet ranged from 0.003 to 0.129 day−1
chemical constraints. They also highlight the complexity of the (mean = 0.054 ± 0.018 day−1). When Kmet was above 0.002
influence of sulfur cycling and DOM chemistry on MeHg day−1 (at 8 locations), SRBKmet accounted for 57.2 to 84.0% of
production and the need for future investigations of these the total Kmet (mean = 70.3 ± 3.9%). Both SRBKmet and

Methods (brief)

hgcA-carrying (hgcA+) SRB in environments where sulfate collected into N2-flushed serum bottles, then transferred to
regulator homologous to arsR, a gene involved in arsenic Samples for Hg methylation incubations were collected into
production rates to microbial biogeochemical cycles and gene from each depth was collected in a bag to prepare the enriched
Water and Nucleic Acid Sample Collection. Detailed
descriptions of the sites and sample collection can be found in epilimnetic sulfate (∼30.5 mg/L).28,29 Samples were gently
the Supporting Information�Section S1.1. Briefly, samples mixed and then an initial sample (t0) was immediately
were collected from the deepest basin (∼24 m) in Lake withdrawn from the bags and preserved to 1% HCl in a 125
Mendota in Madison, Wisconsin, USA (Figure S1). Sampling mL PETG bottle. An unfiltered sample for sulfide analysis was
and turbidity profiles were measured using a multiparameter Sample collection was repeated after approximately 24 h (t1)
sonde (YSI, Yellow Spring, OH). Samples were collected with and either 80 or 48 h in 2020 and 2021, respectively (t2).
Teflon tubing. Sulfide/sulfate samples were preserved in 1% at the USGS MRL and passed required quality assurance and
zinc acetate. Iron and manganese samples were preserved in control metrics; complete details are in Supporting Informa-
1% nitric acid. Filter-passing metal samples were filtered using tion�Section S1.3. Ambient total Hg (THg) in filter-passing
a 0.45 μm Acrodisc filter. Water for Hg analysis was collected and particulate samples was analyzed by U.S. Environmental
technique, then filtered onto an ashed quartz fiber filter vapor atomic fluorescence spectrometry (CVAFS) using a
the particulate samples were frozen until analysis. Dissolved tin reduction, dual-stage gold amalgamation, and quantification
gaseous mercury (DGM) was collected onto gold-coated bead by ICP-MS using a Merx-T coupled to an iCAP-RQ ICP-MS
traps in the field by purging 1L of sample water with high (Thermo-Fisher, Waltham, MA). DGM samples were analyzed

Implications

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Update history

The five most recent substantive edits to this page, classified major (evidence or structure moved), correction (a published value or statement was wrong and has been fixed), or minor (narrative rewritten without changing the underlying evidence). Each description is derived from what the edit did to this page; the linked commit is the authoritative record, routine regeneration passes are excluded, and the full version history lives in git. When DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.

Commit	Date	Change	Description
b01ec52c	2026-08-04	major	2 sections added
d49e450f	2026-08-03	major	5 sections added; narrative text revised