I am excited to share our manuscript, titled Sulfate Reduction Drives Elevated Methylmercury Formation in the Water Column of a Eutrophic Freshwater Lake, investigating the microbial formation of toxic methylmercury in the water column of a eutrophic freshwater lake. Link here: https://pubs.acs.org/doi/10.1021/acs.est.4c12759. I encourage you to check it out if you are interested in mercury contamination, microbial-contaminant interactions, or overambitious experimental methods, but I’ll provide a quick overview here. If you’re just here for the overambitious experimental methods, you can skip to the end for field work photos. This project has been a long time coming for me… I began developing the incubation methods in my second year of grad school, conducted most of the experiments during my first postdoc, did most of the writing during my second postdoc, and finalized and submitted the manuscript as an assistant professor. Even after all that time, I’m excited about the project and what it tells us about the microbial constraints on methylmercury formation in the environment, particularly with respect to sulfide.

Lake Mendota is a well-studied and eutrophic lake with an anoxic hypolimnion for most of the stratified year. Methylmercury concentrations can reach up to 80% (!!!) of total mercury in the hypolimnion, and sulfate reduction appears to be a key pathway driving microbial metabolism. The first chapter of my PhD work investigated the microbes carrying the mercury methylation gene cluster hgcAB in the hypolimnion of Mendota (Peterson et al., 2020), which showed that there were few sulfate-reducing bacteria (SRB) with hgcA throughout the lake. Combined with other studies from my circle of collaborators (Peterson et al., 2023, Peterson et al., 2023) and others (Jones et al, 2019, Capo et al., 2022, among many others) showing that SRB with hgcA are relatively low in abundance at many different sites, we decided to dig further into how this functionally affected our understanding of environmental methylmercury formation.

To do this, we designed a method for conducting enriched stable isotope mercury transformations assays to pair with our microbial measurements. We were adamant about mimicking in situ conditions as much as possible… probably why the first trial runs were conducted in 2017 but the first data used for the study was collected in 2020. We collected samples at night into custom-designed trace-metal clean bags with no headspace, then resuspended them in the lake for the duration of the incubation. We injected enriched isotope mercury to the lowest concentration that we could while obtaining reasonable measures (approximately equal to the ambient concentration) and pre-equilibrated the enriched isotope spike with filter water taken from the same depth. While this was often a huge pain (ask any of the many people who helped with sampling), it resulted in some very clean data by mercury work standards. In parallel to these incubations, we filtered water for microbial sequencing efforts, collected water samples for water chemistry measurements (filtered and unfiltered metals, sulfate/sulfide, dissolved organic matter, mercury speciation), and measured bacterial production.

Overall, we showed that methylation rate potentials were high. Consistent with our previous study (and other similar studies), the gene abundance of hgcA increased with sulfide, with a maximum of ~16% of the total microbial community carrying hgcA. However, hgcA expression peaked along with the rate potential, at moderate sulfide concentrations. We used an internal standard to calculate absolute concentrations of hgcA transcripts in the water column, which peaked at 7.9 million transcripts per milliliter. This data, paired with the demethylation measurements and mercury speciation measurements, strongly suggested that in situ methylation was the primary source of methylmercury in the water column. This is consistent with recent literature but in contrast to early consensus in the field that most methylmercury in freshwater lakes was derived from the sediment.

Given the prominent role of sulfate reduction in Lake Mendota and the historical emphasis on sulfate-reducing, we specifically investigated the role of sulfate-reducing bacteria in methylmercury production. To do this, we amended molybdate, a (putatively) SRB-specific inhibitor, to a subset of the methylation assays. Molybdate amendment resulted in a drastic decrease (up to 79%) in MeHg production across all redox conditions, which was a surprise given the sequencing results from our previous study. When looking at the sequencing data, sulfate-reducing bacteria (SRB) still only accounted for <10% of the hgcA gene abundance, but represented the majority (>50%) of the hgcA gene transcription. In digging in why this was, we found that overall transcriptional activity was also higher in SRB. However, we also identified an arsR-like transcriptional regulator preceding hgcA, as has been previously reported (McDaniel et al., 2020, Gionfriddo et al., 2023). What has not been described is that the arsR-like element was associated with lower hgcA transcription, but the relative transcription of hgcA did not change over the redox gradient. Overall, this adds some more mystery to the intrigue surrounding transcriptional regulation of hgcA and ultimately the physiological role that it plays for hgcA-carrying microbes.

Check it out, and let us know what you think! This represented a major effort from a large team, and I’m proud to see this work out in the world. For me, this work highlights the value in pairing detailed characterization of the microbial community with rate measurements of the transformation of interest. My experiences with this project have had a major impact on how I approach experimental design, and I look forward to continuing to explore how to improve this approach. I hope you enjoy it and learn something from it.

Check out some more pictures: