Carbon Amendments Alter Microbial Community Structure and Net Mercury Methylation Potential in Sediments

Abstract

Neurotoxic methylmercury (MeHg) is produced by anaerobic Bacteria and Archaea possessing the genes hgcAB, but it is unknown how organic substrate and electron acceptor availability impacts the distribution and abundance of these organisms. We evaluated the impact of organic substrate amendments on mercury (Hg) methylation rates, microbial community structure, and the distribution of hgcAB⁺ microbes with sediments. Sediment slurries were amended with short-chain fatty acids, alcohols, or a polysaccharide. Minimal increases in MeHg were observed following lactate, ethanol, and methanol amendments, while a significant decrease (∼70%) was observed with cellobiose incubations. Postincubation, microbial diversity was assessed via 16S rRNA amplicon sequencing. The presence of hgcAB⁺ organisms was assessed with a broad-range degenerate PCR primer set for both genes, while the presence of microbes in each of the three dominant clades of methylators (Deltaproteobacteria, Firmicutes, and methanogenic Archaea) was measured with clade-specific degenerate hgcA quantitative PCR (qPCR) primer sets. The predominant microorganisms in unamended sediments consisted of Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria Clade-specific qPCR identified hgcA⁺Deltaproteobacteria and Archaea in all sites but failed to detect hgcA⁺Firmicutes Cellobiose shifted the communities in all samples to ∼90% non-hgcAB-containing Firmicutes (mainly Bacillus spp. and Clostridium spp.). These results suggest that either expression of hgcAB is downregulated or, more likely given the lack of 16S rRNA gene presence after cellobiose incubation, Hg-methylating organisms are largely outcompeted by cellobiose degraders or degradation products of cellobiose. These results represent a step toward understanding and exploring simple methodologies for controlling MeHg production in the environment.IMPORTANCE Methylmercury (MeHg) is a neurotoxin produced by microorganisms that bioacummulates in the food web and poses a serious health risk to humans. Currently, the impact that organic substrate or electron acceptor availability has on the mercury (Hg)-methylating microorganisms is unclear. To study this, we set up microcosm experiments exposed to different organic substrates and electron acceptors and assayed for Hg methylation rates, for microbial community structure, and for distribution of Hg methylators. The sediment and groundwater was collected from East Fork Poplar Creek in Oak Ridge, TN. Amendment with cellobiose (a lignocellulosic degradation by-product) led to a drastic decrease in the Hg methylation rate compared to that in an unamended control, with an associated shift in the microbial community to mostly nonmethylating Firmicutes This, along with previous Hg-methylating microorganism identification methods, will be important for identifying strategies to control MeHg production and inform future remediation strategies.

Keywords: 16S; hgcA; hgcAB; mercury; methylmercury; qPCR.

FIG 1

Methylmercury production potential (MPP) in microcosm experiments from each of three sites receiving one of seven carbon amendment treatments. MPP is the amount of MeHg produced per day (micrograms of MeHg per kilogram of THg spiked per day) or the fraction methylated (MeHg produced per THg added per day). Bars and error bars represent the mean ± standard deviation from triplicate microcosms. HC, Hinds creek (background site); NOAA, upstream site in the contaminated EFPC; NH, downstream site in EFPC.

FIG 2

Relative abundances of bacterial phyla (A) or families (B) and of archaeal phyla (C) or families (D) detected for three sites (HC, NOAA, and NH) amended separately with cellobiose, lactate, or ethanol.

FIG 3

NMDS plots with ADONIS analysis for Bacteria (A) and Archaea (B) to determine the effect of carbon amendment on the β-diversity of the three sites used in this study. Black, Hinds Creek (HC) (the background site); red, NOAA (NO) (upstream); blue, New Horizon (NH) (downstream). Amendments: ●, unamended; ⬥, lactate; ▲, ethanol; ■, cellobiose.

FIG 4

qPCR for hgcA specific to Deltaproteobacteria (top), Firmicutes (middle), and Archaea (bottom) at the three sites (HC, NOAA, and NH) for each of the treatments. hgcA quantification is shown. Detection limits (genome copies per 1 ng of gDNA template): Deltaproteobacteria, 2,000; Firmicutes, 200; and Archaea, 2,000 (empirically determined from a representative strain from each clade).