1. Reduction of Hg(II) to Hg(0) by dissimilatory metal reducing bacteria Heather Wiatrowski and Tamar Barkay Department of Biochemistry and Microbiology, Rutgers University, Cook College wiatrows@rci.rutgers.edu Abstract The reduction of mercuric mercury (Hg[II]) to elemental (Hg[0]) affects the bioavailability and mobility of mercury in the environment. In surface waters, mercury may be reduced by sunlight or by mercury resistant bacteria that possess a mer system. Because mer-mediated microbial reduction of Hg(II) occurs at nM to µM concentrations of Hg(II), these processes are irrelevant in ground water aquifers that are contaminated with lower concentrations of mercury. Yet, presence Hg(0) in groundwater has been documented and the mechanisms leading to its formation are presently unknown. We therefore examined if dissimilatory metal reducing bacteria (DMRB), which are often found in subsurface sediments and are known for their ability to reduce toxic metals and radionuclides, also reduce Hg(II). We show that Shewanella oneidensis MR-1 reduced 67.73.69% of an initial concentration of 150 nM HgCl 2 in 24 hours, when grown anaerobically with fumarate as a terminal electron acceptor. Under the same conditions, uninoculated media removed 6.433.57% of the added HgCl 2. Reduction of Hg(II) showed a dependence on the presence of an electron donor and an electron acceptor, as incubation of cells in media which lacked either resulted in activity not significantly different from that of autoclaved cells. Unlike mer-mediated Hg(II) reduction, this activity is not inducible, as exposed cells and unexposed cells had a specific activity for reduction of Hg(II) of 3.14  0.25 and 3.07  0.35 nmol min -1 mg protein - 1 respectively. Reduction by MR-1 was enhanced five fold in iron reducing conditions relative to fumarate reducing conditions. Two other DMRB, Geobacter sulfurreducens PCA and Geobacter metallireducens GS- 15, reduced Hg(II) at comparable rates to MR-1. However, Hg(II) reduction is not universal among DMRB or anaerobic bacteria, as the iron reducing bacterium Anaeromyxobacter dehalogenans 2CP-C and the denitrifier Pseudomonas stutzeri OX1 did not reduce Hg(II). Our observations of constitutive reduction of Hg(II) by DMRB suggest a mechanism that may affect mercury speciation, and thus bioavailability and environmental mobility, in environments contaminated with a low level of mercury that do not receive day light. Materials and Methods Unless otherwise noted, Hg(II) was added as HgCl 2 at a concentration of 300 nM. Mercury was analyzed using Cold Vapor Atomic Absorbance Spectroscopy on a Leeman Labs Hydra AA Mercury analyzer or by Liquid Scintillation Counting using 203 Hg (provided by Delon Barfus) as a tracer. N2N2 Hg(0) cells, Hg(II) KMnO 4, H 2 SO 4 Figure 2: Reduction of Hg(II) to Hg(0) by dissimilatory metal reducing bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens PCA S. oneidensis MR-1G. sulfurreducens PCA Cells and mercury were added to growth media and bubbled with nitrogen for the indicated time, allowing Hg to be collected in a trap of acidified permanganate. Mercury was analyzed by CVAAS both in the growth media and in the trapping solution before and after bubbling. For strain MR-1, fumarate was used as an electron acceptor and ferric oxyhydroxide was used as an electron acceptor for strain PCA. Hg (nmol) Time (h)03 live 050305 S. oneidensis MR-1, G. sulfurreducens PCA, and G. metallireducens GS-15 are able to reduce Hg(II) (Figs. 1-5). This reduction occurs at low concentrations of mercury (Table 1) and does not require induction. By contrast, the mer system is inducible and is effective at high concentrations of Hg(II). Endogenous reduction of Hg(II) by MR-1 requires presence of an electron donor and acceptor, occurs in aerobic conditions, fumarate reducing conditions, and is enhanced in iron reducing conditions (Figs. 1, 3, 4). Reduction of Hg(II) by Geobacter spp. requires the presence of an electron acceptor (Fig. 5). Conclusions To facilitate comparison, a mer system was introduced into MR-1 A spontaneous rifampicin resistant mutant was selected for. This strain was called MR-1H This strain was mated to Pseudomonas aeruginosa PAO1 containing a mer system on the plasmid R388::Tn501, generating a transconjugate called MR-1H/R388::Tn501 MR-1 is not resistant to mercury MR-1 is inhibited by 0.5 M Hg(II) MR-1H/R388::Tn501 can grow in the presence of 25 M Hg(II) A comparison of endogenous reduction of Hg(II) by MR-1 to reduction of Hg(II) by the mer system - - + ++ + + + Figure 5: Reduction of Hg(II) by Geobacter spp. in the presence of ferric oxyhydroxide TEA preincubation autoclaved FeOOH none inoculated +---- -+ -+- -++ specific activity (nmol /min/mg protein) G. sulfurreducens PCA G. metallireducens GS-15 i ii i heat-killed M-207

2. Geobacter metallireducens GS-15 was grown in ATCC medium 1768 and Geobacter sulfurreducens PCA was grown in ATTC medium 1957 with ferric citrate (20 mM) as an electron acceptor. In all experiments for which electron donating or electron accepting conditions varied from the culture conditions, cells were washed two times in media containing no electron donor and/or electron acceptor. Care was taken to ensure that anoxic conditions were maintained throughout the washes. Cell concentrations were normalized to extractable protein, and a typical inoculum was ~ 0.4 g protein/ml, which corresponds to approximately 10 5 cells/ml Data represents means of three replicates, and all errors are standard deviations. Means that are not significantly (p >.05) are indicated with the same roman numeral. ? Microbial transformations of mercury Hg(II) CH 3 Hg Hg(0) methyl mercury ionic mercury elemental mercury merB sulfate reducing bacteria catalasemerA, Fe(II) dependent reduction by Acidithiobacilli aerobic microbes anaerobic microbes ionic mercury Results MR-1 reduces Hg(II) at ~5 X higher rate in iron versus fumarate reducing conditions A 24h preincubation period in FeOOH was required for Hg(II) reduction activity. specific activity (nmol Hg/min/ / mg protein) + - TEA preincubation autoclaved pregrown in fumarate pregrown in ferric citrate fumarateFeOOHnonefumarate - --- - - ------++ ++ none FeOOH i ii i iii oxygenfumarate Hg(II) (nM) Time (h)0240 0 0 sterile media sterile media MR-1 Fig 1: Shewanella oneidensis MR-1 causes loss of Hg(II) from culture media Mercury was analyzed using 203 Hg as a tracer. Reduction of Hg(II) to Hg(0) increases its mobility, because Hg(II) sorbs to sediments and Hg(0) is a volatile gas that is poorly soluble in water. Reduction of Hg(II) to Hg(0) may make it less available for methylation by anaerobic bacteria. Many sites are contaminated with low levels of Hg(II). Because reduction by MR-1 is constitutive and functions at low Hg(II) concentrations, it may be more applicable to subsurface environments. A strategy in development to remediate sites contaminated by metals and radionuclides is to stimulate endogenous dissimilatory metal reducing bacteria to reduce and immobilize these contaminants. Hg(0) has been discovered in groundwater in New Jersey, and it is not likely due to point- source contamination, and this process is a potential cause. Acknowledgements Future directions We would like to thank D. Lovley for strains, Delon Barfus for providing the 203 Hg, and Costantino Vetriani and Jeffra Schaefer for advice. This research was funded by the Natural and Accelerated Bioremediation Research (NABIR) program, Biological and Environmental Research (BER), U.S. Department of Energy (Grant No. DE-FG02-99ER62864). Can anaerobic microorganisms reduce Hg(II)? Table 1: Endogenous reduction of Hg(II) is effective at low concentrations of Hg(II), but not at high concentrations Figure 3: Electron donors and electron acceptors are required for Hg(II) reduction by MR-1 lactate (10 mM) fumarate (10 mM) hydrogen (ND) autoclaved +++ ++ + + + + + -- - - --- - -- specific activity (nmol Hg/min/mg protein) i ii Figure 4: A higher reduction rate of Hg(II) occurs with iron as compared to fumarate as an electron acceptor for MR-1 Environmental Relevance We hypothesize that these organisms reduce Hg(II) through their electron transport chains. mer-independent reduction is not an inducible process MR-1 cells pregrown in 300 nM Hg(II) and unexposed cells had specific activities of 3.14  0.25 and 3.07  0.35 nmol Hg(II)/min/mg protein respectively. Investigate mer-independent reduction of Hg(II) in intact sediment samples Determine what terminal-electron accepting conditions maximize mer-independent Hg(II) reduction. Microbial community analysis on Hg(II) reducing anoxic sediments