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Interactions of Chemosynthetic Bacteria with Mercury at
Deep-sea Hydrothermal Vents
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Melitza Crespo-Medina1, Nicolas Bloom2, Aspassia Chatziefthimiou1,
John Reinfelder1, Costantino Vetriani1 and Tamar Barkay1
1Cook
College, Rutgers University, New Brunswick, NJ, 2Studio GeoChimica, Seattle, WA
ABSTRACT
Microorganisms in geothermal environments, such as deep-sea
hydrothermal vents, have likely evolved in presence of elevated
concentrations of mercury. Because this mercury is likely
complexed with sulfur, chemolithoautotrophic microbes, that use
reduced sulfur and iron as a energy source are likely exposed to
the toxicity of mercury. To test this hypothesis we collected
water for chemical and microbiological analyses at different
distances from diffuse flow vents at 9 ºN on the East Pacific
Rise (EPR), during oceanographic expeditions in 2004 and
2005. Total mercury (THg) concentrations in the 2004 samples
ranged from 2.8 to 88.7 ng/L (methylmercury concentration <
0.3 ng/L). Growth medium, designed to enrich for
chemolithoautotrophic thiosulfate oxidizing microorganisms, was
used to enumerate total and mercury resistant (10 µM) microbes
by the MPN method. Mercury speciation in this medium,
determined with MINEQL+ (Version 4.5), showed the exclusive
presence of negatively charges -2 (61.3%) and -4 (38.7%)
complexes of mercury thiosulfate. The bioavailability of these
complexes to microorganisms was determined using mer-lux
bio-reporters, which emit light as mercury enters the cell.
Results suggested an enhanced bioavailability of mercurythiosulfate relative to mercuric-nitrate complexes. The
abundance of mercury resistant microbes was 6% and 13% of
total thiosulfate oxidizing chemolithoautotrophs in vent waters
with THg concentrations of 6.5 and 88.7 ng/L, respectively.
Thus, the number of resistant microbes was directly related to
the concentration of THg in the water suggesting acclimation of
these microbes to life in the presence of toxic mercury.
Isolations from vent water samples led to the identification of
mercury resistant bacteria belonging to several genera,
including, Thiomicrospira, Thioclava and Halomonas whose
mechanisms of mercury resistance is currently being
investigated. These initial results begin to reveal the processes
that facilitate microbial life in metal rich geothermal environment
and will lead to an understanding of the role of these microbes
in mercury biogeochemistry in these ecosystems.
RESULTS
Table 1. Selected chemical measurements of diffuse
flow waters from EPR 9ºN (Apr. 2004 expedition)
1 MeHg
concentration was <0.3 ng/L in all samples
HgT concentrations varied and were not always
related to temperature and sulfide
Bioavailability of Hg(II) in medium 142-A
MPN counts of chemosynthetic microbes in
samples collected on the EPR 9oN
Table 3. Summary of MPN Results
The percentage of resistant MPN counts was between
0.1 and 13.9 %
The highest resistant MPN counts was found in the
sample with the highest mercury concentration
Figure 5. 16S rRNA gene phylogeny of isolates from
diffuse flow vents
I. MINEQL+ modeling of 142-A + 10 µM HgCl2
Figure 2. Hg-thiosulfate speciation
OBJECTIVE
Study the interactions between mercury and
chemosynthetic microorganisms isolated from
deep-sea hydrothermal vents
METHODS
All Hg(II) in 142-A with 10 µM HgCl2 was present as
thiosulfate complexes, 61% as Hg(S2O3)2-2 and 39% as
Hg(S2O3)3-4
Samples were collected on East the Pacific Rise 9oN
(Fig. 1), during DSV Alvin dives in April 2004 and 2005.
Samples were preserved on board of R/V Atlantis
immediately upon retrieval
II. Hg speciation in the mer-lux biosensor assay
media
Samples from the 2004 cruise were analyzed for
chemical compositions at Frontier GeoScience Inc.
(Seattle, WA)
Table 2. The effect of increasing thiosulfate
concentrations on the speciation of Hg(II), added as 10
nM Hg(NO3)2 to mer-lux biosensor assay medium
MINEQL+ Chemical Equilibrium Modeling System
(Schecher et al., 1994), was used to determine mercury
speciation in the growth (142-A) and the biosensor assay
media
mer-lux biosensor assays were done according to Barkay
et al., (1998)
The Most Probable Number (MPN) technique and growth
medium 142-A were used to determine the number of
chemosynthetic and chemosynthetic-mercury (10 µM
HgCl2) resistant bacteria. Total number of cells was
determined by Acridine Orange Direct Counts.
16S rDNA gene phylogeny was obtained using Clustal X
and NJ analysis
* mercury resistant strains
A merA PCR fragment from EPR84 was sequenced and
found most closely related to the sequence of merA of
Tn501 and Tn21
At 2 mM S2O3-2 Hg(II) speciation in mer-lux assay
medium simulates that of 10 µM Hg(II) in 142-A growth
medium
III. Bioavailability of varied Hg-S2O3 species
Figure 1. Study sites on the East Pacific Rise 9ºN
50´N/104° 17´W
Figure 3. Induction of mer-lux
increasing concentrations of S2O3-2
decreased
with
CONCLUSIONS
Hydrothermal fluids from diffuse flow vents are
enriched in mercury with concentrations ranging from
2.8 to 88.7 ng/mL (Table 1)
Thiosulfate is the favored ligand of Hg in medium 142A,
a
growth
medium
formulated
for
chemolithoautotrophic marine bacteria (Fig. 2)
 The use of the biosensor assay in conjunction with
MINEQL+ speciation is a useful approach to determine
bioavailability of Hg(II) under specific conditions (Table 2
and Fig. 3)
Thiosulfate reduces Hg(II) bioavailability, yet it does
not abolish it
The bioavailability of Hg(II) decreases with the
increased negative charge of the Hg-thiosulfate complex
(Fig. 3)
In diffuse flow samples with higher mercury
concentrations, the microbial community is likely to be
adapted to the presence of mercury (Table 3)
Chemosynthetic
bacteria
from
diffuse
flow
environments are distributed among  and Proteobacteria; four of the isolates were resistant to 10
µM HgCl2 (Fig. 4)
LITERATURE CITED
Barkay et al. 1998. Lux-facilitated detection of mercury in natural waters. in:
Bioluminescent Protocols, R. LaRossa (ed). Humana Press, Inc. Totowa,
NJ.pp. 231-246.
Induction of mer-lux decreased with S2O3-2 (A)
At 2 mM S2O3-2 bioavailability decreased about 55%
relative to assay media devoid of thiosulfate (B)
Bioavailability decreases in inverse proportion to an
increase in the ratio of Hg(S2O3)3-4 :Hg(S2O3)-2 (B)
Schecher et al. MINEQL+: A Chemical Equilibrium Program for Personal
Computers; Environmental Research Software: Hallowell, ME, 1994.
Acknowledgements
This work was supported by a National Science Foundation Graduate
Research Fellowship to MCM and by an NSF Grant OCE 03-27353 to C.
Vetriani