Download Sulfate-reducing bacterium grows with Cr(VI), U(VI), Mn(IV)

FEMS Microbiology Letters 162 (1998) 193^198
Sulfate-reducing bacterium grows with Cr(VI), U(VI), Mn(IV),
and Fe(III) as electron acceptors
Bradley M. Tebo *, Anna Ya. Obraztsova
Marine Biology Research Division and Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,
University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0202, USA
Received 8 December 1997; revised 12 March 1998; accepted 13 March 1998
Abstract
A spore-forming sulfate-reducing bacterium Desulfotomaculum reducens sp. nov. strain MI-1 has been isolated from heavy
metal contaminated sediments. Strain MI-1 grows with Cr(VI), Mn(IV), Fe(III), and U(VI), in addition to various sulfur
compounds, as electron acceptors. This organism shares physiological properties with both the sulfate-reducing and metalreducing groups of bacteria and is the first sulfate-reducing bacterium described that can grow with metals or U(VI) as sole
electron acceptors. z 1998 Published by Elsevier Science B.V. All rights reserved.
Keywords : Spore-forming sulfate-reducing bacterium; Iron; Manganese; Uranium; Chromium ; Metal reduction
1. Introduction
Sulfate-reducing bacteria are of economic, environmental and biotechnological importance. They
fall into two distinct groups, the spore-forming
Gram-positive species and the others which all are
placed in the N-subdivision of the proteobacteria [1].
Sulfate-reducing bacteria couple the oxidation of organic compounds or molecular H2 with the reduction of sulfate as an external electron acceptor under
anaerobic conditions [2], a process known as dissimilatory sulfate reduction. The end product of this
reaction, hydrogen sul¢de, can react with heavy-metal ions to form insoluble metal sul¢des or reduce
soluble toxic metals, often to less toxic or less soluble
* Corresponding author: Tel.: +1 (619) 534-5470;
Fax: +1 (619) 534-7313; E-mail: [email protected]
forms [3]. Furthermore, some sulfate-reducing bacteria can reduce nitrate, oxygen [2], or arsenate [4],
disproportionate thiosulfate or elemental sulfur to
sulfate and sul¢de [5], or oxidize thiosulfate and elemental sulfur to sulfate with Mn(IV) as an electron
acceptor [6]. It has recently been shown that some
sulfate-reducing bacteria can enzymatically reduce
Fe(III), U(IV) and Cr(VI), but are unable to grow
with these metals as terminal electron acceptors [7,8].
A second major group of anaerobes, the dissimilatory metal(loid)-reducing bacteria, has been studied
extensively in recent years [9^11]. These bacteria,
which are phylogenetically diverse, occupy similar
ecological niches as sulfate-reducing bacteria and
are also involved in the decomposition of organic
matter. In addition to metal(loid)s, some sulfur compounds, but not sulfate, can serve as electron acceptors for growth for these bacteria. Sulfate-reducing
0378-1097 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved.
PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 1 2 2 - 0
FEMSLE 8132 27-4-98
194
B.M. Tebo, A.Ya. Obraztsova / FEMS Microbiology Letters 162 (1998) 193^198
and metal-reducing bacteria are widespread in nature
and in£uence the geochemical cycling of carbon, sulfur and metals in marine and aquatic sediments and
terrestrial subsurface environments. We report here
the discovery of a new spore-forming sulfate-reducing bacterium, Desulfotomaculum reducens sp. nov.
strain MI-1, that grows with Cr(VI), Mn(IV),
Fe(III), and U(VI), in addition to various sulfur
compounds, as electron acceptors. This organism
shares physiological properties with both the sulfate-reducing and metal-reducing groups of bacteria.
2. Materials and methods
2.1. Source of organism
Sediments from Mare Island Naval Shipyard located in the San Francisco Bay estuary, California
are contaminated with high concentrations of Cr(VI)
and other heavy metals as a result of ship building
and maintenance operations for over 50 years. Samples of surface sediment cores taken with 60 ml syringes served as inocula for enrichment cultures of
sulfate-reducing bacteria.
2.2. Medium and cultivation conditions
Brackish medium for sulfate-reducing bacteria recommended by Widdel and Bak [1] supplemented
with 20 mM lactate or other carbon sources and
28 mM sulfate was used for enrichment and pure
cultures. The same medium with 1.5% Difco agar
was used for single colony isolation. All procedures
during preparation of medium and manipulations
with bacteria were performed using standard anaerobic techniques [12]. Cells were grown at 37³C in 25ml Balch tubes (Bellco Glass, Inc.) ¢lled with 10 ml
medium (pH 7.2^7.4) and sealed with black butyl
rubber stoppers and aluminum crimp seals under
an N2 atmosphere. For growth on alternative e3
acceptors 10 mM Fe(III) (as soluble ferric citrate),
200 WM Mn(IV) (as NMnO2 ), 500 WM U(VI) (as
uranyl acetate) or 60 WM Cr(VI) (as 30 WM
Na2 Cr2 O7 ) replaced the sulfate. No other known
sulfate source (e.g., in the trace minerals) is present
in the basal medium and no reductant was added in
order to avoid chemical reduction of the metals. All
experiments reported were started with inocula obtained after three sequential transfers and growth on
each e3 acceptor alone. Samples for cell number and
concentrations of HS3 , Fe(II), Mn(IV) or U(VI)
were taken by syringe and needle and assayed immediately.
2.3. Analytical methods
Cell numbers were determined by direct counting
by phase-contrast microscopy using a Petro¡-Hauser
counting chamber. Sul¢de concentration was measured by the CuSO4 spectrophotometric assay [13].
Fe(III) reduction was monitored by measuring
Fe(II) production by the ferrozine technique [14].
Mn(IV) concentration was analyzed using the leucoberbelin blue colorimetric method [15]. U(VI) was
measured spectrophotometrically at 380 nm with
benzohydroxamic acid in 1-hexanol [16]. Cr(VI)
was determined colorimetrically using diphenylcarbazide [8]. Carbon monoxide di¡erence spectra
were used to assay cytochromes in cytoplasmic membrane fractions using a Beckman DU 640 spectrophotometer. Acetate was determined on a Beckman
HPLC system with an Aminex HPX-87U column
(7.8 mmU300 mm) and UV detection at 210 nm.
vG³P values were calculated from the G³f [17].
2.4. Phylogenetic analysis
DNA was isolated and puri¢ed using a QIAamp
tissue kit (Qiagen Corp.). PCR was conducted with
10 ng bacterial DNA in a ¢nal volume of 0.1 ml
using 2.5 U Taq polymerase, 200 WM of each
dNTP, bu¡er (Boehringer Mannheim), and 100 nM
each of eubacterial primer 27F and universal primer
1492R [18] with a temperature cycle of 94³C (1 min),
50³C (1 min), 72³C (1 min), 30 cycles, and ¢nal
elongation step at 72³C for 7 min. The PCR product
was sequenced using standard small subunit rRNA
primers [18] with £uorescently labeled dideoxynucleotide terminators in a standard cycle sequencing
protocol (Applied Biosystems, Inc., Prism Ready Reaction Kit FS) and run on an Applied Biosystems
373A automated sequencer. A total of 1516 base
pairs were sequenced corresponding to position 32
to position 1400 of the Heliobacterium chlorum sequence. The sequence was aligned to other sequences
FEMSLE 8132 27-4-98
B.M. Tebo, A.Ya. Obraztsova / FEMS Microbiology Letters 162 (1998) 193^198
from GenBank (organism, accession number: D.
auripigmentum; U85624; D. thermoacidovans,
Z26315; B. subtilis, M10606; D. orientis, M34417;
D. ruminis, Y11572; D. geothermicum, X80789; D.
thermobenzoicum, LI5628; D. australicum, M96665;
D. nigri¢cans, X62176; Desul¢tovibrio dehalogenans,
L28946). Portions that could not be con¢dently
aligned or were missing from some of the sequences
were excluded from the analysis leaving a matrix of
960 positions. The phylogenetic tree was constructed
by maximum parsimony using the branch and bound
search algorithm in PAUP [19]. Relationships were
tested by bootstrapping the dataset 100 times. Signi¢cantly supported nodes ( s 80%) are labeled with
the percent of bootstrap replications that support
that node.
3. Results and discussion
Active enrichment cultures obtained with sulfate
and butyrate or lactate as carbon sources contained
numerous spore-forming bacteria. Enrichment cul-
195
tures were transferred to a de¢ned medium imitating
natural brackish sea water and a pure culture, strain
MI-1, was isolated from colonies in agar. Cells were
motile and slightly curved rods (0.8^1.0U5^10 Wm).
Optimum growth occurred at 37³C, pH 7.0^7.2, and
0^2% NaCl concentrations. Strain MI-1 used a wide
range of organic compounds including short chain
fatty acids (C3 ^C5 ; propionate, butyrate, and valerate); alcohols (C1 ^C4 ; methanol, ethanol, n-propanol, and n-butanol); and lactate, pyruvate, and glucose. Acetate, fumarate, and H2 /CO2 (80:20) were
not utilized. These substrates are common for
many other spore-forming sulfate-reducing bacteria
[20]. Besides 28 mM sulfate, 10 mM thiosulfate, 10
mM dithionite and elemental S³ (V1%) served as
electron acceptors.
The unique feature of strain MI-1 is that it uses
metals as electron acceptors for growth in the absence of sulfate. With butyrate as the carbon source,
strain MI-1 could grow with Mn(IV), Fe (III),
U(VI), or Cr(VI) in the absence of sulfate (Figs. 1
and 2). Other electron donors such as lactate or valerate also supported growth with these electron ac-
Fig. 1. Growth and reduction of (A) SO23
4 , (B) Fe(III), (C) Mn(IV) and (D) U(VI) by strain MI-1 on butyrate. Symbols : F, cell number,
no e3 acceptor ; b, cell number, +e3 acceptor ; R, HS3 , Fe(II), Mn(IV) or U(VI) concentrations without cells; 8, HS3 , Fe(II), Mn(IV)
and U(VI) concentrations with cells.
FEMSLE 8132 27-4-98
196
B.M. Tebo, A.Ya. Obraztsova / FEMS Microbiology Letters 162 (1998) 193^198
Fig. 2. Growth of strain MI-1 on butyrate with Cr(VI) as an
electron acceptor. Symbols : F, cell number, no Cr(VI); b, cell
number, +Cr(VI); R, Cr(VI) concentrations without cells; 8,
Cr(VI) concentrations with cells. Cr(VI) was rapidly reduced by
2 days; at days 2 and 4, amendments of additional 50 WM
Cr(VI) (arrows) stimulated a concomitant increase in number of
cells.
ceptors. In all cases the carbon sources were oxidized
incompletely to acetate; in the absence of e3 acceptors, no growth occurred. Thermodynamic calculations supporting these modes of growth are shown
in Table 1. Strain MI-1 grew with approximately the
same doubling time (about 20 h) with these metals as
with sulfate. In contrast to Fe(III), Mn(IV) and
U(VI), Cr(VI) is highly toxic for many microorganisms. Thus, to demonstrate growth with Cr(VI) as
the electron acceptor we added it incrementally in
relatively small amounts (Fig. 2). After each addition
of Cr(VI) to the culture, Cr(VI) was depleted and
there was an incremental increase in cell number,
demonstrating that growth of strain MI-1 depended
on Cr(VI). Cr(VI) was presumably reduced to Cr(III)
as a ¢ne gray precipitate characteristic of Cr(OH)3
formed in the bottom of tubes. Concentrations of
Cr(VI) greater than 200 WM inhibited growth of
this isolate (data not shown).
The mechanism by which strain MI-1 reduces metals such as Cr(VI) and Mn(IV) is unknown. It is
important to note that it is possible that Fe(II),
which is present at V27 WM in the medium from
the trace element solution, acts as the reductant
due to recycling of iron in the culture. Thus,
Cr(VI) and Mn(IV) which are well known to be reduced quite rapidly by Fe(II) [21,22] may be reduced
indirectly via the Fe(III)-reducing [Fe(II)-producing]
activities of strain MI-1. However, in control experiments without cells, the initial rates of abiotic reduction are signi¢cantly slower than when cells are
present (Figs. 1 and 2). These results strongly suggest
that Cr(VI), U(VI) and Mn(IV) reduction, like
Fe(III) reduction, are directly mediated by strain
MI-1.
The question arises how strain MI-1 might catalyze the reduction of these metals. Carbon monoxide
di¡erence spectra of cytoplasmic fractions exhibited
various absorption peaks between 400 to 595 nm.
Absorption maxima at 520, 483, and 419 nm suggest
that cytochromes b and c are present [23]. Thus, the
mechanism of dissimilatory metal reduction may involve cytochromes in a manner similar to that observed for Cr(VI) and U(VI) reduction by other sulfate-reducing bacteria [7,8]. It is unclear, however,
whether strain MI-1 can compete with other metalreducing organisms for electron acceptors or whether
utilization of these metals as electron acceptors represents an adaptation for survival under unfavorable
conditions. Further characterization of the mechanism(s) involved in metal reduction are needed to
understand how heavy metals are reduced by the
cell, whether there is a hierarchy of electron acceptor
preferences, and how the cells tolerate toxic metals
such as Cr(VI).
Phylogenetic 16S rDNA analysis shows that strain
MI-1 belongs to the genus Desulfotomaculum [20];
Table 1
Reactions and thermodynamics of dissimilatory metal and sulfate reduction by strain MI-1
vG³P (kJ mol31 )
Reaction
3
3‡
3
2‡
‡
CH3 CH2 CH2 COO +4Fe +2H2 OC2CH3 COO +4Fe +5H
4
1 ‡
3 4
CH3 CH2 CH2 COO3 +23 Cr2 O23
7 ‡ 3H2 O+3H C2CH3 COO +3Cr(OH)3
CH3 CH2 CH2 COO3 +2MnO2 +3H‡ C2CH3 COO3 +2Mn2‡ +2H2 O
CH3 CH2 CH2 COO3 +2UO2 2‡ +2H2 OC2CH3 COO3 +2UO2 +5H‡
3 1
3 1 ‡
CH3 CH2 CH2 COO3 +14 SO23
4 C2CH3 COO +2HS +2H
vG³P (pH 7) were calculated from the G³ values.
FEMSLE 8132 27-4-98
3409
3333
3291
3130
328
B.M. Tebo, A.Ya. Obraztsova / FEMS Microbiology Letters 162 (1998) 193^198
197
Fig. 3. Phylogenetic relationships of Desulfotomaculum reducens sp. nov. strain MI-1 and relatives based on analysis of small subunit
(16S) rRNA genes. The sequence for strain MI-1 has been deposited in GenBank under accession number U95951. The arrow indicates
the outgroup used in the analysis, Bacillus subtilis.
the di¡erence between D. reducens and the nearest
known relatives, D. nigri¢cans and D. ruminis, in the
1516 bp that were sequenced is V8%, counting the
large insertion in D. reducens as one di¡erence and
discounting ambiguities (Fig. 3). Strain MI-1 is more
metabolically diverse than these species; it is able to
utilize a greater number of electron donors and acceptors. These observations suggest that strain MI-1
is a new species within the genus of spore-forming
sulfate-reducing bacteria, Desulfotomaculum. We
propose to name the isolate Desulfotomaculum reducens sp. nov. D. reducens is also di¡erent from most
other Desulfotomaculum species [20] in its ability to
reduce elemental sulfur. Recently a new sulfate-reducing bacterium most closely related to D. orientis,
Desulfotomaculum auripigmentum sp. nov., was discovered that can grow by respiring As(V), but not
Fe(III) or Mn(IV) [4]. These limited data suggest
that dissimilatory reduction of some metals or metalloids by sulfate-reducing bacteria may be speci¢c to
spore-forming sulfate-reducing bacteria found in the
Gram-positive Bacteria rather than other non-sporeforming sulfate-reducing bacteria belonging to the
delta Proteobacteria.
Although it is well established that sulfate-reducing bacteria can reduce di¡erent metals enzymatically or through hydrogen sul¢de production, and
that low concentrations of certain heavy metals,
such as Cr(VI), Hg(II), and Zn(II), enhance the
growth and sulfate-reducing activity of sulfate-reduc-
ing bacteria [24,25], to our knowledge the results
presented here are the ¢rst that demonstrate that
sulfate-reducing bacteria can grow by coupling the
oxidation of organic compounds with the reduction
of Cr(VI) to Cr(III), Mn(IV) to Mn(II), Fe(III) to
Fe(II) or U(VI) to U(IV), and the ¢rst to demonstrate that Cr(VI) reduction supports anaerobic
growth in any organism. Although other microorganisms may reduce Cr(VI) when provided as the
sole electron acceptor, in no case has Cr(VI) reduction de¢nitively been shown to be coupled to energygenerating metabolism to support anaerobic growth
[9].
Organisms like D. reducens may play an important
role in the biogeochemical cycling of metals and actinides in anoxic sediments. For example in marine
sediments, reactions carried out by D. reducens may
lead to siderite formation, U(IV) accumulation, or
the release of phosphate and other trace metals
from Fe and Mn oxides. D. reducens may also
have useful applications for the removal of heavy
metals or radionuclides from waters or for the bioremediation of metal contaminated environments because of its ability to reductively precipitate Cr(VI)
and U(VI) and possibly other elements. In any case,
the activities of D. reducens probably lead to the
natural attenuation of Cr(VI) toxicity in contaminated marine sediments, such as those found at the
Mare Island Naval Shipyard. We are presently evaluating this possibility.
FEMSLE 8132 27-4-98
198
B.M. Tebo, A.Ya. Obraztsova / FEMS Microbiology Letters 162 (1998) 193^198
Acknowledgments
We thank Linda Park and Margo Haygood for
help with the 16S sequencing and phylogenetic analysis. This work was funded by the University of California Toxic Substances Research and Teaching
Program, by a grant from the National Sea Grant
College Program, National Oceanic and Atmospheric Administration, US Department of Commerce,
under Grant Number NA36RG0537, Project Number 39-C-N through the California Sea Grant College System, and by the California State Resources
Agency.
References
[1] Widdel, F. and Bak, F. (1992) Gram-negative mesophilic sulfate-reducing bacteria. In: The Prokaryotes, Vol. 4 (Ballows,
A., Truper, H.G., Dworkin, M., Harder, W. and Shleifer,
K.-H., Eds.), pp. 3352^3378. Springer, Berlin.
[2] Barton, L.L. and Tomei, F.A. (1995) Characteristics and activities of sulfate-reducing bacteria. In : Sulfate-reducing Bacteria (Barton, L.L., Ed.), pp. 1^32. Plenum Press, New York.
[3] Tebo, B.M. (1995) Metal precipitation by marine bacteria:
potential for biotechnological applications. In: Genetic Engineering ^ Principles and Methods, Vol. 17 (Setlow, J.K., Ed.),
pp. 231^263. Plenum Press, New York.
[4] Newman, D.K., Kennedy, E.K., Coates, J.D., Ahmann, D.,
Ellis, D.J., Lovley, D.R. and Morel, F.M.M. (1997) Dissimilatory arsenate and sulfate reduction in Desulfotomaculum
auripigmentum sp. nov. Arch. Microbiol. 168, 380^388.
[5] Janssen, P.H., Schuhmann, A., Bak, F. and Liesack, W.
(1996) Disproportionation of inorganic sulfur compounds by
the sulfate-reducing bacterium Desulfocapsa thiozymogenes
gen. nov., sp. nov. Arch. Microbiol. 166, 184^192.
[6] Lovley, D.R. and Phillips, E.J.P. (1994) Novel processes for
anaerobic sulfate production from elemental sulfur by sulfatereducing bacteria. Appl. Environ. Microbiol. 60, 2394^2399.
[7] Lovley, D.R., Roden, E.E., Phillips, E.J.P. and Woodward,
J.C. (1993) Enzymatic iron and uranium reduction by sulfatereducing bacteria. Mar. Geol. 113, 41^53.
[8] Lovley, D.R. and Phillips, E.J.P. (1994) Reduction of chromate by Desulfovibrio vulgaris and its C3 cytochrome. Appl.
Environ. Microbiol. 60, 726^728.
[9] Lovley, D.R. (1993) Dissimilatory metal reduction. Annu.
Rev. Microbiol. 47, 263^290.
[10] Laverman, A.M., Blum, J.S., Schaefer, J.K., Phillips, E.J.P.,
Lovley, D.R. and Oremland, R.S. (1995) Growth of strain
SES-3 with arsenate and other diverse electron acceptors.
Appl. Environ. Microbiol. 61, 3556^3561.
[11] Macy, J.M. (1996) Chrysiogenes arsenatis gen. nov., sp. nov., a
new arsenate-respiring bacterium isolated from gold mine
wastewater. Int. J. Syst. Bacteriol. 46, 1153^1157.
[12] Balch, W.E., Fox, G.E., Margrum, L.J. and Woese, R.S.
(1979) Methanogens : reevaluation of a unique biological
group. Microbiol. Rev. 43, 260^296.
[13] Cord-Ruwish, R. (1985) A quick method for the determination of dissolved and precipitated sul¢des in cultures of sulfate-reducing bacteria. J. Microbiol. Methods 4, 33^36.
[14] Stookey, L.L. (1970) Ferrozine ^ a new spectrophotometric
reagent for iron. Anal. Chem. 42, 779^781.
[15] Krumbein, W.E. and Altmann, H.J. (1973) A new method for
the detection and enumeration of manganese oxidizing and
reducing microorganisms. Helgoland Wiss. Meeresunters. 25,
347^356.
[16] Meloan, C., Holkeboer, P. and Brandt, W.W. (1960) Spectrophotometric determination of uranium with benzohydroxamic
acid in 1-hexanol. Anal. Chem. 32, 791^793.
[17] Stumm, W. and Morgan, J.J. (1996) Aquatic Chemistry:
Chemical Equilibria and Rates in Natural Waters. John Wiley
and Sons, New York.
[18] Lane, D.S. (1990) 16S and 23S rRNA sequencing. In: Nucleic
Acid Techniques in Bacterial Systematics (Stackebrandt, E.
and Goodfellow, M., Eds.), pp. 115^148. John Wiley, New
York.
[19] Swo¡ord, D.L. (1990) PAUP: Phylogenetic Analysis Using
Parsimony.
[20] Widdel, F. (1992) The genus Desulfotomaculum. In: The Prokaryotes, Vol. 4 (Ballows, A., Truper, H.G., Dworkin, M.,
Harder, W. and Shleifer, K.-H., Eds.), pp. 1792^1799. Springer, Berlin.
[21] Fendorf, S.E. and Li, G. (1996) Kinetics of chromate reduction by ferrous iron. Environ. Sci. Technol. 30, 1614^1617.
[22] Postma, D. (1985) Concentration of Mn and separation from
Fe in sediments. I. Kinetics and stoichiometry of the reaction
between birnessite and dissolved Fe(II) at 10³C. Geochim.
Cosmochim. Acta 49, 1023^1033.
[23] Jones, H.E. (1972) Cytochromes and other pigments of dissimilatory sulphate-reducing bacteria. Arch. Mikrobiol. 84,
207^224.
[24] Bharathi, P.A.L., Sathe, V. and Chandramohan, D. (1990)
E¡ect of lead, mercury and cadmium on a sulphate-reducing
bacterium. Environ. Pollut. 67, 361^374.
[25] Karnachuk, O.V. (1995) In£uence of hexavalent chromium on
hydrogen sul¢de formation by sulfate-reducing bacteria. Microbiology 64, 262^265.
FEMSLE 8132 27-4-98