Mn(IV), and Fe(III) as electron acceptors - Wiley Online Library

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

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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

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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-

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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.

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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.

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3409 3333 3291 3130 328


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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.

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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.

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