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The contamination of groundwater with mercury (Hg) is an increasing problem worldwide. Yet, little is known about the interactions of Hg with microorganisms and their processes in subsurface environments. We tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge Integrated Field Research Challenge site, where nitrate is a major contaminant and where bioremediation efforts are in progress. We observed an inverse relationship between Hg concentrations and onset and rates of denitrification in nitrate enrichment cultures containing between 53 and 1.1 μM of inorganic Hg; higher Hg concentrations increasingly extended the time to onset of denitrification and inhibited denitrification rates. Microbial community complexity, as indicated by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA genes, declined with increasing Hg concentrations; at the 312 nM Hg treatment, a single tRFLP peak was detected representing a culture of Bradyrhizobium sp. that possessed the merA gene indicating a potential for Hg reduction. A culture identified as Bradyrhizobium sp. strain FRC01 with an identical 16S rRNA sequence to that of the enriched peak in the tRFLP patterns, reduced Hg(II) to Hg(0) and carried merA whose amino acid sequence has 97 % identity to merA from the Proteobacteria and Firmicutes. This study demonstrates that in subsurface sediment incubations, Hg may inhibit denitrification and that inhibition may be alleviated when Hg resistant denitrifying Bradyrhizobium spp. detoxify Hg by its reduction to the volatile elemental form.

Aim: Factors affecting faecal indicator bacteria (FIB) and pathogen survival/persistence in sand remain largely unstudied. This work elucidates how biological and physical factors affect die-off in beach sand following sewage spills. Methods and Results: Solar disinfection with mechanical mixing was pilot-tested as a disinfection procedure after a large sewage spill in Los Angeles. Effects of solar exposure, mechanical mixing, predation and/or competition, season, and moisture were tested at bench scale. First-order decay constants for Escherichia coli ranged between −0·23 and −1·02 per day, and for enterococci between −0·5 and −1·0 per day. Desiccation was a dominant factor for E. coli but not enterococci inactivation. Effects of season were investigated through a comparison of experimental results from winter, spring, and fall. Conclusions: Moisture was the dominant factor controlling E. coli inactivation kinetics. Initial microbial community and sand temperature were also important factors. Mechanical mixing, common in beach grooming, did not consistently reduce bacterial levels. Significance and Impact of the Study: Inactivation rates are mainly dependent on moisture and high sand temperature. Chlorination was an effective disinfection treatment in sand microcosms inoculated with raw influent.

Recent studies have shown that rice consumption is another critical route of human exposure to methylmercury (MeHg), the most toxic and accumulative form of mercury (Hg) in the food web. Yet, the mechanisms that underlie the production and accumulation of MeHg in the paddy ecosystem are still poorly understood. In 2013 and 2014, we conducted field campaigns and laboratory experiments over a rice growing season to examine Hg and MeHg cycling, as well as associated biogeochemistry in a suite of paddies close to a municipal solid waste incinerator and a coal-fired power plant station in Taiwan. Concentrations of total Hg and MeHg in paddy soil and rice grain at both sites were low and found not to exceed the control standards for farmland soil and edible rice in Taiwan. However, seasonal variations of MeHg concentrations observed in pore water samples indicate that the in situ bioavailability of inorganic Hg and activity of Hg-methylating microbes in the rhizosphere increased from the early-season and peaked at the mid-season, presumably due to the anoxia created under flooded conditions and root exudation of organic compounds. The presence of Hg-methylators was also confirmed by the hgcA gene detected in all root soil samples. Subsequent methylation tests performed by incubating the root soil with inorganic Hg and an inhibitor or stimulant specific for certain microbes further revealed that sulfate-reducers might have been the principal Hg-methylting guild at the study sites. Interestingly, results of hydroponic experiments conducted by cultivating rice in a defined nutrient solution amended with fixed MeHg and varying levels of MeHg-binding ligands suggested that chemical speciation in soil pore water may play a key role in controlling MeHg accumulation in rice, and both passive and active transport pathways seem to take place in the uptake of MeHg in rice roots.

Biofilms can methylate mercury (Hg) at higher rates than unattached bacteria and are increasingly recognized as important Hg methylation sites in the environment. Our previous study showed that methylation rates in biofilm cultures were up to 1 order of magnitude greater than those in planktonic cultures of a sulfate-reducing bacterium. To probe whether the differential Hg methylation rates resulted from metabolic differences between these two cultures, Hg methylation assays following molybdate or chloroform inhibition (a specific inhibitor of the acetyl-CoA pathway) were conducted on biofilm and planktonic cultures of Desulfovibrio desulfuricans strains M8 and ND132. Molybdate was as effective in inhibiting Hg methylation as well as growth in both planktonic and biofilm cultures. The addition of chloroform only impacted Hg methylation in biofilm cultures, suggesting that different pathways are used for methylation in biofilm compared to planktonic cultures. To investigate this further, expression of the cooS gene, which encodes for carbon monoxide dehydrogenase, a key enzyme in the acetyl-CoA pathway, was compared in biofilm and planktonic cultures of ND132. Biofilm cultures showed up to 4 times higher expression of cooS than planktonic cultures. On the basis of these results, the acetyl-CoA pathway appears to play an important role in methylation in biofilm cultures of this organism, possibly by supplying the methyl group to Hg methylating enzymes; methylation in planktonic cultures appears to be independent of this pathway. This observation has important implications, particularly in developing reliable models to predict Hg methylation rates in different environments and perhaps eventually in being able to control this undesirable chemical transformation.

The speciation of mercury (Hg) is a major determinant of its methylation rate by sulfate-reducing bacteria (SRB), considered the primary methylators. Under anoxic conditions, sulfur (S) cycling may have a significant influence on Hg complexation and methylation, by influencing both SRB activity and the pool of available reduced S ligands, as the presence of zero-valent sulfur (S(0)) in sulfidic water results in the formation of polysulfides. While SRB frequently coexist with S-oxidizing bacteria in natural environments, the effect that these organisms may have on methylation by SRB is not understood. In this study, we investigate the role of S(0) in methylation by SRB monocultures and cocultures with phototrophic green or purple S-oxidizing bacteria. In the coculture experiments, the presence of S-oxidizers was found to increase Hg methylation rates, apparently by maintaining favorable chemical speciation in the environment. The measured Hg methylation rates were in accord with predictions based on geochemical modeling of speciation. In SRB monoculture experiments conducted in the presence and absence of S(0), the data showed that at limited total Hg, the presence of polysulfides resulted in decreased Hg methylation, presumably by causing a decrease in the most bioavailable Hg–sulfide complexes. These results indicate that models of Hg speciation and methylation in the environment should include a detailed investigation of S redox speciation.

A phenanthrene-degrading bacterium, Sphingomonas paucimobilis EPA505 was used to construct two fluorescence-based reporter strains. Strain D harboring gfp gene was constructed to generate green fluorescence when the strain started to biodegrade phenanthrene. Strain S possessing gef gene was designed to die once phenanthrene biodegradation was initiated and thus to lose green fluorescence when visualized by a live/dead cell staining. Confocal laser scanning microscopic observation followed by image analysis demonstrates that the fluorescence intensity generated by strain D increased and the intensity by strain S decreased linearly at the phenanthrene concentration of up to 200 mg/L. Such quantitative increase and decrease of fluorescence intensity in strain D (i.e., from 1 to 11.90 ± 0.72) and strain S (from 1 to 0.40 ± 0.07) were also evident in the presence of Ottawa sand spiked with the phenanthrene up to 1000 mg/kg. The potential use of the reporter strains in quantitatively determining biodegradable or toxic phenanthrene was discussed.

While biofilms are now known to be the predominant form of microbial growth in nature, very little is yet known about their role in environmental mercury (Hg) methylation. Findings of Hg methylation in periphyton communities have indicated the importance of investigating how environmental biofilms affect Hg methylation, as periphyton can be the base of the food webs in aquatic ecosystems. Chemical speciation influences the microbial uptake and methylation of inorganic Hg by planktonic cultures of sulfate-reducing bacteria; however, the effect of speciation on Hg methylation by biofilm cultures of these organisms has previously not been studied. In the present study, Hg methylation rates in biofilm and planktonic cultures of two isolates of Desulfovibrio desulfuricans from a coastal wetland were compared. Notably, the specific Hg methylation rate found was approximately an order of magnitude higher (0.0018 vs. 0.0002 attomol cell(-1) day(-1)) in biofilm cells than in planktonic cells, suggesting an important role for environmental biofilms in Hg methylation. To investigate the role of chemical speciation of Hg, experiments were conducted at two levels of sulfide. Both biofilm and planktonic cultures produced methylmercury at roughly twice the rate at low sulfide, when HgS(0)(aq), rather than HgHS2-, was the dominant Hg species. This indicates that the presence of a biofilm does not alter the relative availability of the dominant Hg species in sulfidic medium, in accordance with our previous studies of Hg uptake by Escherichia coli along a chloride gradient.

Monitoring the water quality of recreational beaches is only one step toward understanding microbial contamination -- the primary cause of beach closings. The surf zone sediment reservoir is typically overlooked and may also be important. This study involved monitoring the fecal indicator bacteria (FIB) levels in water and sediment at three ocean beaches (two exposed and one enclosed) during a storm event, conducting laboratory microcosm experiments with sediment from these beaches, and surveying sediment FIB levels at 13 beaches (some exposed and some enclosed). Peaks in Escherichia coli and enterococci concentrations in water and sediment coincided with storm activity at the two exposed beaches, while levels of both FIB were consistently high and irregular at the enclosed beach. Results from microcosm experiments showing similar, dramatic growth of FIB in both overlying water and sediment from all beaches, as well as results from the beach survey, support the hypothesis that the quiescent environment rather than sediment characteristics can explain the elevated sediment FIB levels observed at enclosed beaches. This work has implications for the predictive value of FIB measurements, and points to the importance of the sediment reservoir.

While it is known that microbial uptake of mercury (Hg) by planktonic cultures is influenced by the extracellular speciation of mercury in aquatic systems, Hg uptake in biofilm cultures is understudied. We compared the importance of Hg(II) speciation in toxicity to both planktonic and biofilm cultures of the Gram-negative bacterium Escherichia coli 055. Variable chloride chemistry experiments were carried out to modify mercury speciation. Biofilms were observed to be more resistant to Hg than planktonic cells. In both planktonic and biofilm cultures, the toxicity of Hg increased and then decreased along the chloride gradient. The percent reduction in cell viability was linearly related to the concentration of HgCl2(0) when Hg-chloro complexes dominated the speciation, consistent with a passive diffusion model. However, toxicity to both planktonic cells and biofilms at low salinities could not be explained by passive diffusion alone, which suggests that microbial uptake of Hg in both planktonic cells and biofilms may occur by both passive diffusion of neutral species and facilitated uptake. The relationship between toxicity and chloride concentration was similar in the presence and absence of a biofilm, indicating that the presence of the biofilm does not drastically change the relative availability of the dominant mercury species.

In southern California, USA, wildfires may be an important source of mercury (Hg) to local watersheds. Hg levels and Hg accumulation rates were investigated in dated sediment cores from two southern California lakes, Big Bear Lake and Crystal Lake, located approximately 40-km apart. Between 1895 and 2006, fires were routinely minimized or suppressed around Big Bear Lake, while fires regularly subsumed the forest surrounding Crystal Lake. Mean Hg concentrations and mean Hg accumulation rates were significantly higher in Crystal Lake sediments compared to Big Bear Lake sediments (Hg levels: Crystal Lake 220 ± 93 ng g−1, Big Bear Lake 92 ± 26 ng g−1; Hg accumulation: Crystal Lake 790 ± 1,200 μg m−2 year−1, Big Bear 240 ± 54 μg m−2 year−1). In Crystal Lake, the ratio between post-1965 and pre-1865 Hg concentrations was 1.1, and several spikes in Hg levels occurred between 1910 and 1985. Given the remote location of the lake, the proximity of fires, and the lack of point sources within the region, these results suggested wildfires (rather than industrial sources) were a continuous source of Hg to Crystal Lake over the last 150 years.