Extracellular metal-binding activity of the sulphate-reducing bacterium Desulfococcus multivorans

Toni A. M. Bridge1, Chris White1 and Geoffrey M. Gadd1

Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK1

Author for correspondence: Geoffrey M. Gadd. Tel:+44 1382 344266. Fax:+44 1382 344275. e-mail: g.m.gadd{at}dundee.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Polarography was used to measure the copper-binding ability of culture filtrates from a range of sulphate-reducing bacteria (SRB), including pure cultures and environmental isolates. Of those tested, Desulfococcus multivorans was shown to have the greatest copper-binding capacity and this organism was used for further experiments. Extracellular copper- and zinc-binding activities of Dc. multivorans culture filtrates from batch cultures increased over time and reached a maximum after 10 d growth. The culture filtrate was shown to bind copper reversibly and zinc irreversibly. Twelve-day-old Dc. multivorans culture filtrates were shown to have a copper-binding capacity of 3·64±0·33 µmol ml-1 with a stability constant, log10K, of 5·68±0·64 (n=4). The metal-binding compound was partially purified from culture growth media by dichloromethane extraction followed by HPLC using an acetonitrile gradient.

Keywords: sulphate-reducing bacteria, copper binding, zinc binding, metal binding, Desulfococcus multivorans

Abbreviations: SRB, sulphate-reducing bacteria


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Sulphate-reducing bacteria (SRB) are a group of bacteria which grow in fresh or salt water anoxic environments and are defined by their ability to reduce sulphate to sulphide, using it as a terminal electron acceptor for the heterotrophic oxidation of organic compounds or hydrogen (Hansen, 1993 ; Ensley & Suflita, 1995 ; Hao et al., 1996 ). SRB are strictly anaerobic and so are found in environments with a low redox potential, such as marshes, mud, marine and freshwater sediments and sludge reactors (Widdel & Hansen, 1991 ). Active laboratory cultures of SRB can produce millimolar concentrations of sulphide, and in environments such as anaerobic sediments, SRB can also generate significant amounts of sulphide. The high environmental sulphide concentrations produced by SRB lead to the precipitation of any metal ions present as metal sulphides. Metal sulphides, other than those of the alkali metals, have very low solubility products [e.g. copper(II) sulphide 8·5x10-45; zinc sulphide 1·2x10-23], meaning that metal sulphides are essentially insoluble under neutral, anaerobic conditions (Lide, 1995 ). As a result of the combination of neutral pH, low Eh and high sulphide conditions, soluble metal ion concentrations in the microenvironment of SRB are therefore extremely low. This allows SRB to grow in environments containing high levels of toxic metals, and SRB have been utilized in processes for the bioremediation of metal-contaminated land and water by the precipitation of contaminating metal species as metal sulphides (Barnes et al., 1992 ; Gadd & White, 1993 ;White & Gadd, 1996 , 1998 ; White et al., 1998 ). However, such precipitation of metal sulphides must also decrease the availability of essential metals to SRB. Metals such as iron, copper and zinc are necessary for most micro-organisms including SRB; copper and iron, being redox-active under physiological conditions, are important as cofactors in electron transport and in redox-active metalloenzymes, while zinc plays a structural role in many proteins as well as being a component of some enzymes (Hughes & Poole, 1989 ; Beveridge et al., 1997 ). In fact, SRB are known to require high levels of iron in culture media in order to compensate for that precipitated by sulphide (Postgate, 1984 ).

Micro-organisms release a diverse range of specific and non-specific metal-binding compounds into the environment, which can act to ameliorate the presence of toxic metals and/or mediate uptake. Non-specific metal-binding microbial metabolites, e.g. organic acids, can complex metals and affect mobility and thus toxicity (Burgstaller & Schinner, 1993 ; Sayer & Gadd, 1997 ; Gadd, 1999 ). Undefined, microbially produced macromolecules can also bind significant amounts of potentially toxic metals. These include humic and fulvic acids arising from lignocellulose degradation and extracellular polymeric substances (EPS), a mixture of polysaccharides, mucopolysaccharides and proteins produced by bacteria, algae and fungi (Schreiber et al., 1990 ; Beech & Cheung, 1995 ; Spark et al., 1997 ). The composition of EPS produced by SRB has been shown to be modified by the presence of carbon steel, which may increase its metal-binding capacity (Zinkevich et al., 1996 ). In contrast, specific metal-binding compounds are produced directly in response to high levels of toxic metals. Intracellular, low-molecular-mass, metal-binding proteins produced by mammals (metallothioneins) and also plants and algae (phytochelatins) have been well studied (Birch & Bachofen, 1990 ; Rauser, 1995 ; Howe et al., 1997 ). However, some micro-organisms are known to produce extracellular, specific metal-binding compounds. Examples include Sarcina urea, which has been shown to produce metal-binding proteins in response to Pd2+, Sr2+, In2+ and Ce4+ (Beveridge, 1986 ), a Pseudomonas sp. which has been shown to produce cadmium-binding proteins in response to cadmium (Rayner & Sadler, 1989 ) and Vibrio alginolyticus, which produces copper-binding proteins in response to high levels of copper (Harwood-Sears & Gordon, 1990 ). Specific extracellular metal-binding compounds can also be produced by micro-organisms in response to low levels of metals, in order to facilitate the uptake of essential metals. The most studied system is the production of siderophores in response to low environmental iron concentrations. Siderophores are low-molecular-mass Fe(III) coordination compounds (500–1000 Da) produced by many micro-organisms and act by complexing and solubilizing insoluble Fe(III) in a form which can be transported into the cell using specific transport mechanisms (Neilands, 1981 ). Although siderophores are iron(III)-binding compounds, they are also able to bind other metals such as magnesium, manganese, chromium(III), gallium(III) and radionuclides such as plutonium(IV) (Bulman, 1978 ; Birch & Bachofen, 1990 ). However, siderophores are not produced under the anaerobic conditions in which SRB grow and where iron is predominately in the Fe(II) form (Guerinot, 1994 ).

It can be theorized that, since metals are effectively immobilized by the sulphide produced by SRB, these organisms must require a specific metal-binding and/or uptake mechanism in order to obtain essential metals such as copper and zinc. Such a metal acquisition system should also have an affinity greater than or comparable to that of sulphide in order to compete successfully for any available metal cations with the sulphide present. This study was therefore undertaken to determine whether SRB produce extracellular, soluble metal-binding compounds in order to provide further understanding of metal metabolism in these organisms.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
SRB strains and culture media.
Desulfovibrio vulgaris (DSM 644), Desulfovibrio desulfuricans (DSM 1926) and Desulfococcus multivorans (DSM 2059) were kindly provided by Dr K. Purdy (University of Essex, UK). Environmental isolates from Alaska and Indonesia were kindly provided by Dr I. B. Beech (University of Portsmouth, UK; see Zinkevich et al., 1996 ). All the SRB used in this work used lactate as a carbon and energy source and were routinely cultured in SL10-lactate media at pH 7·0, in either 10 ml volumes in 12 ml Hach tubes (screw-top test tubes with an airtight seal) or 50 ml volumes in 60 ml Weaten vials. SL10 defined liquid media (Widdel & Pfennig, 1981 ) contained the salts (g l-1) Na2SO4 (4·0), KH2PO4 (0·2), NH4Cl (0·25), NaCl (1·0), MgCl2 . 6H2O (0·4), KCl (0·5) and CaCl2 (0·15) with trace elements and vitamins (µg l-1) MnCl2 . 4H2O (100), CoCl2 . 6H2O (190), ZnSO4 . 7H2O (144), H3BO3 (6), NiCl2 . 6H2O (24), CuCl2 . 2H2O (2), NaMoO4 . 2H2O (36), FeSO4 . 7H2O (2·1), Na2SeO3 . 5H2O (6), Na2WO4 . 2H2O (8); (µg l-1) 4-aminobenzoic acid (0·4), D(+)-biotin (0·1), nicotinic acid (1·0), Ca-D(+)-pantothenate (0·5), pyridoxine . 2HCl (1·5) and thiamin . 2HCl (1·0). The media also contained 1·5 mM sodium sulphide and 20 mM sodium lactate, in order to lower the redox potential of the medium and act as a carbon source. Cultures were incubated at 30 °C in the dark and samples of spent media for analysis were filtered through 0·45 µm pore size cellulose nitrate membranes (Whatman), which effectively removed cells (control experiments showed that protein concentrations in filtered spent medium were negligible), snap-frozen in liquid nitrogen and stored at -20 °C.

Polarographic measurement of metal binding by SRB spent medium.
Differential pulse polarography was carried out using a Metrohm 663 VA stand incorporating a multi-wavelength electrode set to the static mercury drop electrode facility. The analysis was carried out using Autolab General Purpose Electrochemical System (GPES) for Windows, version 4.3 (Ecochemie, The Netherlands). Measurements of free metal ion concentrations were carried out in 25 ml electrolyte containing 1 M KCl, buffered with either 10 mM PIPES (pH 6·0) or 50 mM MES (pH 5·5) buffer, adjusted to the appropriate pH value using tetramethylammonium hydroxide. These buffers were chosen for their negligible metal-binding activities (Good et al., 1986 ). In most experiments, free metal cation concentrations, from added metal chloride solutions, were measured immediately after the reaction mixture was purged with N2. The electrolyte was purged for 180 s and the current measured over three scans between potentials of 0 and -0·4 V for copper or -0·8 and -1·2 V for zinc with a step potential of 0·0036 V. Each metal has a characteristic half wave potential, -0·18 V for copper and -0·97 V for zinc, with the measured current being proportional to the amount of metal ion present. Free metal concentrations were calculated with respect to a standard curve over the range 10–100 µM. Two approaches were used to measure the metal-binding activity present in SRB culture filtrates. The first involved measuring the decrease in free metal concentration on the addition of a known volume of sample and calculating the concentration of metal-binding compound as the amount of metal bound (ml culture filtrate)-1 (Shuman, 1994 ). This method was used to measure the increase in metal-binding activity over time in Dc. multivorans batch cultures and to identify which HPLC fractions contained metal-binding activity. The second approach (the reverse of the method described above) involved the addition of aliquots of metal ion solution to buffered culture filtrate. These titration data could then be transformed using the method of Ruzic (1982) and van den Berg (1982) to obtain stability constants for the metal-binding ligands.

Purification of a metal-binding compound from SRB spent media.
The metal-binding compound was extracted and purified using a method suggested by Professor R. O. Hallberg, University of Stockholm (personal communication). Each sample of spent medium was extracted by shaking for 1 h with approximately 10% (v/v) dichloromethane (CH2Cl2), the organic phase being collected using a separating funnel, and the extraction repeated. Both extracts were pooled and evaporated under nitrogen to a volume of less than 1 ml; acetonitrile (CH3CN) was added and the residual CH2Cl2 was removed under nitrogen. All the apparatus used for the purification was washed with dichloromethane and rinsed with Milli-Q water.

HPLC was carried out using a 5 µM adsorbosphere, C18 reverse-phase column (Alltech) linked to a Waters 600 system controller, a Waters 486 programmable multiwavelength detector, a Waters 600 pump and a Waters 717plus autosampler controlled by Millipore (Waters) Millennium chromatography software. The mobile phase consisted of an initial gradient from 10 to 100% (v/v) CH3CN over 10 min followed by isocratic 100% CH3CN for 15 min. The detector monitored the A318 of the eluate (R. O. Hallberg, personal communication). Timed fractions of eluate were collected at 30 s intervals using a Pharmacia FRAC-100 fraction collector. Fractions from three runs were collected in the same tubes and most of the acetonitrile was removed by evaporation under nitrogen (final volume {approx}0·1 ml) in order to prevent interference with the detection of the copper-binding compound. Each fraction was tested for copper-binding activity as the decrease in free ion concentration.

Analytical techniques.
The Bradford method was used to estimate protein (Bradford, 1976 ). The reagent consisted of a filtered solution of 100 mg Coomassie BBG-250 l-1 dissolved in 5% (v/v) ethanol acidified with 10% (v/v) phosphoric acid. One hundred microlitres of an appropriate dilution of the unknown sample was mixed with 1 ml reagent and incubated in the dark for 2 min before measuring the A595 in a Unicam SP600 series 2 spectrophotometer. Bovine serum albumin (BSA) was used to construct a standard curve over the range 1–10 µg (100 µl)-1. Sulphide was measured using polarography. Twenty millilitres of 0·1 M NaOH electrolyte was purged with N2 for 300 s, the unknown sample was added and the current was measured over three scans between potentials of -0·6 and -1 V, using a step potential of 0·0036 V. The electrolyte was purged for 10 s after the sample had been added to mix the solution but was not purged between scans in order to minimize sulphide loss during the analysis. Sodium sulphide (2·08 mM in 0·1 M NaOH) was used to construct a standard curve over the range 0–50 µM.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Metal binding by SRB culture filtrates
A comparison of sulphide content and copper-binding capacity of spent media from five strains of 12-d-old SRB cultures is shown in Table 1. Metal-binding concentrations were measured by titration of buffered spent media with aliquots of copper chloride solution. Initially, as free metal cations were added to the culture filtrate, all the metal was bound and free metal ions were not detected. However, subsequent additions of metal ions resulted in a point at which all the binding sites on the ligand present were complexed and further metal ion additions were measured as an increase in free metal ion concentration. A plot of free metal ion concentration against added metal ion concentration gave a straight line with an intercept on the x-axis equivalent to the metal-binding capacity, i.e. the concentration of ligand multiplied by the number of binding sites per ligand (see Fig. 3a for a typical titration plot). Dc. multivorans culture filtrate had the highest copper-binding capacity of those tested (Table 1). There was no correlation between the amount of sulphide present in each culture filtrate and the ability of that culture filtrate to bind copper, indicating that the metal binding measured in some SRB culture filtrates was due to a compound other than sulphide. In each case the amount of copper binding measured was less than would be expected with a theoretical 1:1 binding of any sulphide present with copper, which also suggests that measurements do not reflect copper sulphide precipitation. A low level of copper-binding capacity was detected in control samples of uninoculated SL10 culture medium, probably due to medium components, including the vitamins. Since Dc. multivorans is capable of the complete oxidation of lactate, in contrast to several other SRB which incompletely oxidize lactate, acetate was not a contributor to copper binding by control culture filtrate.


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Table 1. Copper-binding capacity and sulphide content of culture filtrates from a range of SRB

 


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Fig. 3. Calculation of metal-binding capacities and stability constants of four replicate 12-d-old Dc. multivorans culture filtrates. Buffered 12-d-old culture filtrate (0·5 ml) (1 M KCl, 50 mM MES, pH 5·5; total volume, 25 ml) was titrated with 20 µM additions of Cu2+. (a) Titration plots of free copper against added copper giving a copper-binding ligand concentration (CL) of 2·90±0·62 mM. (b) Ruzic/van den Berg plots of free metal ion concentration/bound metal ion concentration, [M]/(MT-[M]), against free metal ion concentration, [M]. This graph has a gradient of 1/CL and a y intercept of 1/KCL, giving a copper-binding ligand concentration (CL) of 3·64±0·33 mM (n=4) and a stability constant (log10K) of 5·68±0·64 (n=4).

 
Time course of Dc. multivorans metal-binding capacity
Dc. multivorans culture filtrates showed higher metal-binding capacities than the other SRB tested and so this organism was used in further experiments. To determine metal-binding activity at different stages of Dc. multivorans growth, 10 ml anaerobic batch cultures were grown in SL10 medium and four replicate cultures were destructively sampled at 0, 1, 2, 5, 10 and 20 d. The metal-binding capacities and sulphide concentrations obtained are shown in Fig. 1. Metal-binding capacities were measured as the decrease in free metal ion concentration on the addition of culture filtrate to 100 µM metal ion solution buffered with 1 M KCl and 10 mM PIPES (pH 6·0). This method assumes that the concentration of metal removed from solution was proportional to the amount of culture filtrate added and was validated in preliminary work using the range of metal concentrations examined (data not shown). This method was also used by Shuman (1994) for analysis of aluminium–ligand equilibria. Fig. 1 shows that the metal-binding capacity of the culture filtrate increased with time to a maximum after 10 d incubation. Spent medium sampled after 10 d contained [(ml culture filtrate)-1] 4·50±0·11 µmol sulphide and bound 4·88±0·35 µmol copper and 6·27±1·54 µmol zinc (n=4). Although such a sulphide concentration could suggest that metal ion disappearance from solution was due to metal sulphide formation, subsequent experiments have shown that this was not the sole explanation.



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Fig. 1. Time course of copper- and zinc-binding capacities and sulphide concentrations in batch cultures of Dc. multivorans. Each point is the mean of four measurements with error bars being standard deviations.

 
Polarographic analysis of Dc. multivorans culture filtrate
Polarographic traces of the addition of aliquots of Dc. multivorans culture filtrate to buffered metal solutions are shown in Fig. 2, along with relevant controls for comparison. The traces can be used to calculate the copper- and zinc-binding capacities of the culture filtrate and to determine the type of metal binding taking place. Reversible metal binding, such as chelation, usually causes a shift in the half wave potential of the metal and irreversible binding causes a decrease in the peak height (Nurnberg, 1984 ; Lund, 1986 ). Fig. 2(a) illustrates the decrease in free metal concentration observed on addition of Dc. multivorans culture filtrate to electrolyte containing copper. The decrease in the height of the copper peak was concurrent with a peak shift, suggesting reversible binding, and an increase in a peak with a lower half wave potential (-0·30 V). This peak at -0·30 V was not due to a constituent of the culture filtrate as it was not observed when electrolyte without copper was titrated with culture filtrate (Fig. 2b). Control titrations also showed that the copper binding was due to a component of the culture filtrate other than sulphide, as the addition of abiotic sulphide, equal to that present in titrations using Dc. multivorans culture filtrate, to electrolyte containing copper only resulted in a small shift in the half wave potential of the copper peak (Fig. 2c) and the addition of abiotic sulphide to electrolyte without copper only resulted in a small peak at -0·25 V (Fig. 2d). The addition of aliquots of culture filtrate to buffered zinc solution caused a decrease in the height of the zinc peak (Fig. 2e) without a shift in the half wave potential, suggesting that irreversible binding was taking place. No peaks were detected in controls without metal, and zinc binding was not observed when equal concentrations of abiotic sodium sulphide were added to buffered zinc solution (Fig. 2f–h). In both copper- and zinc-binding experiments the polarographic electrolyte remained at a constant pH 5·5 throughout, ruling out the possibility that the observed effects were due to pH changes. Due to the very low solubility product constants of both copper and zinc sulphides, the stoichiometry between sulphide added and metal precipitated as sulphide would be expected to be approximately 1:1. However, the reduction in peak current indicated that approximately 0·36 µmol copper and 0·054 µmol zinc were removed from solution on addition of 1·04 µmol sulphide, giving stoichiometries of 0·35:1 and 0·052:1, respectively (Fig. 2c, g). This was probably due to combined effects of pH and the high ionic strength of the polarograph buffer where pH, especially, can prevent zinc sulphide formation if the H+ concentration is high enough to significantly reduce dissociation of H2S. CuS precipitation is also pH-dependent as it forms a colloidal dispersion under mildly acidic conditions which could result in the copper remaining polarographically active (Svehla, 1996 ).



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Fig. 2. Typical polarographic traces of copper and zinc binding by 15-d-old Dc. multivorans culture filtrate in electrolyte containing 1 M KCl and 50 mM MES at pH 5·5. (a) CuCl2 (100 µM) in electrolyte with the addition of five aliquots of Dc. multivorans culture filtrate with a sulphide equivalent of 100 µl 2·08 mM sodium sulphide; (b) electrolyte with the addition of five aliquots of Dc. multivorans culture filtrate with a sulphide equivalent of 100 µl 2·08 mM sodium sulphide; (c) 100 µM CuCl2 in electrolyte with the addition of 5x100 µl aliquots of 2·08 mM sodium sulphide; (d) electrolyte with the addition of 5x100 µl aliquots of 2·08 mM sodium sulphide. Graphs (e)–(h) show the same titrations as graphs (a)–(d) with 100 µM ZnCl2 in place of CuCl2.

 
Polarographic measurement of copper-binding ligand concentrations and related stability constants for Dc. multivorans culture filtrate
Titration data, such as that of 12-d-old Dc. multivorans culture filtrate titrated with copper (shown in Fig. 3a), can be linearized using the method of Ruzic (1982) and van den Berg (1982) in order to determine both the ligand concentration and stability constant. The abbreviations used are: CM, total metal; CL, total ligand; [M], free metal; [L], free ligand; ML, complex; and K, stability constant of ML. For a simple model of 1:1 complex formation, in the absence of an insoluble form of the metal ion, the following equations apply:


The titration curve produced on the addition of trace metal ({Delta}M) can be described using the equation

This can be rearranged to give

and if MT equals CM+{Delta}M, then this last equation can be rearranged to give

Plotting free metal ion concentration/bound metal ion concentration, [M]/(MT-[M]), against free metal ion concentration, [M], produces a straight line with a gradient of 1/CL and a y intercept of 1/KCL. The titration data plotted in Fig. 3(a) were thereby transformed and the graph of free copper/bound copper against free copper (Fig. 3b) was used to calculate ligand concentrations and binding constants for Dc. multivorans culture filtrates, assuming 1:1 metal–ligand binding. A copper-binding ligand concentration of 3·64±0·33 µmol ml-1 was obtained which compared to that of 2·90±0·62 µmol ml-1 calculated by titration (n=4). Stability constants (K=[ML]/[M][L]) are traditionally compared as their log values and the log10K for the copper ligand complex was calculated as 5·68±0·64 (n=4). This value is similar to that obtained for the binding of copper by oxalic acid (log10K=6·23; Smith & Martell, 1976 ) but several orders of magnitude lower than that of copper or iron(III) complexed by organic ligands in sea water [copper log10K{approx}9–12; iron(III) log10K{approx}21; van den Berg & Donat, 1992 ; van den Berg, 1995 ] and either that of copper bound by sulphide (log10K=36·1; Smith & Martell, 1976 ) or typical siderophores. For example, catecholate-type siderophores isolated from the marine bacterium Alteromonas luteoviolacea and marine cyanobacterium Synechococcus were found to bind iron(III) with a log10K of 37·6–43·6 (Lewis et al., 1995 ) and hydroxamate siderophores have been shown to form iron(III) complexes with stability constants in the range 29–32·5 (Crumbliss, 1991 ). The relatively low stability constant obtained for the copper-binding compound from Dc. multivorans culture filtrates as compared to metal-sulphide binding suggests that unless the stability constant has been underestimated due to other ligands in the culture filtrate, the copper-binding compound identified is rather inefficient and therefore unlikely to have a role in the acquisition of essential metals for metabolism. It can be postulated that there is an alternative, more competitive specific metal-binding and uptake system, possibly similar to one of the range of cell surface (as opposed to extracellular) metal transport systems that have been characterized in microbes for both the import and export of ions. Metal ions can be transported as either free ions (e.g. the transport of Cu+ by Saccharomyces cerevisiae) or metal–ligand complexes [e.g. siderophore-mediated Fe(III) transport]. S. cerevisiae has surface metal reductase enzymes that convert insoluble extracellular Fe3+ to the more soluble Fe2+ and Cu2+ into Cu+, for uptake into the cell. Each metal is taken up using either a high-affinity system which is expressed at low metal levels or a low-affinity system which is expressed under high levels of metal (Eide, 1998 ). Cell membrane metal-ion transport systems tend to involve one or more transmembrane proteins and often other substrate or ATP-binding proteins. Transport requires energy, either in the form of ATP (e.g. the cadmium and copper ATPases found in Gram-positive bacteria) or by the chemiosmotic co-transport of ions such as H+ (e.g. the arsenite efflux system in Gram-negative bacteria; Silver & Phung, 1996 ).

Both the ligand concentrations and stability constants obtained for Dc. multivorans culture filtrates were for complexes formed over a few minutes, i.e. the culture filtrate was added to metal ion solution (or vice versa) and the free metal ion concentration was measured almost immediately. However, work carried out by van den Berg (1995) on complexation of copper in sea water allowed the reaction mixtures to equilibrate over 5 h before polarographic analysis. In order to find out if long-term incubation influenced the copper–ligand complexes, copper-binding capacities of Dc. multivorans culture filtrate were measured after 0, 2, 5 and 72 h (data not shown). It was found that the ligand concentrations and stability constants obtained were similar to those described above and there was no apparent change with time.

Purification of the metal-binding compound from Dc. multivorans culture filtrate
The metal-binding compound from Dc. multivorans culture filtrate was partly purified by organic solvent extraction. The copper-binding capacity and sulphide concentration of the original culture filtrate and the dichloromethane extract transferred into acetonitrile are given in Table 2. Data presented in Table 2 show that extraction of the Dc. multivorans culture filtrate using dichloromethane resulted in the separation of non-dichloromethane-soluble medium constituents such as sulphide from the copper-binding activity. The extraction process resulted in an approximate 1/33 concentration in volume with approximately 6% of the total activity being extracted. The absence of sulphide in the extract confirms that the metal-binding capacity measured in Dc. multivorans culture filtrates was not due to the presence of sulphide.


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Table 2. Separation of copper-binding activity and sulphide from a Dc. multivorans culture filtrate

 
A section of a typical HPLC trace showing the separation of a Dc. multivorans culture filtrate extract is shown in Fig. 4. The HPLC trace shows that the extract contained several compounds that absorbed at 318 nm. Analysis of copper-binding abilities of the concentrated eluate fractions showed that the fraction collected over 18·5–19·0 min contained copper-binding activity as measured by a decrease in free metal concentration (Fig. 5a). This figure shows a slight peak shift in the half wave potential suggestive of reversible binding; however, in contrast to Fig. 2(a), no peak was detected at -0·30 V. No copper-binding activity was present in controls containing a similar amount of acetonitrile (Fig. 5b). The peak containing the copper-binding compound had a retention time of 18·7 min using this HPLC method.



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Fig. 4. A section of a typical chromatogram of HPLC separation of a dichloromethane extract of Dc. multivorans culture filtrate (transferred into acetonitrile) using a 10–100% acetonitrile gradient. The peak marked with an asterisk (retention time 18·7 min) was shown to contain copper-binding activity.

 


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Fig. 5. Copper binding by purified metal-binding compound from Dc. multivorans visualized as the decrease in free metal ion. (a) The 18·5–19·0 min fraction from the HPLC run and (b) acetonitrile control, added to 100 µM CuCl2 in 25 ml electrolyte containing 1 M KCl and 50 mM MES at pH 5·5.

 
Conclusions
This work has shown that certain SRB have an extracellular metal-binding capacity that is unrelated to the production of sulphide. The ability to bind copper was strain-dependent with Dc. multivorans culture filtrate, the highest of those tested, having a copper-binding capacity in the µmol ml-1 range. Although this work substantiates the production of an extracellular metal-binding compound by SRB, the unpurified copper-binding compound from Dc. multivorans was found to have a stability constant of 5·68±0·64 (n=4). This is significantly lower than that of metal sulphides (log10K=36·1; Smith & Martell, 1976 ) and so the compound studied is unlikely to be the sole method of metal binding or acquisition by Dc. multivorans. A potential metal-uptake system for SRB could theoretically involve a cell surface enzyme or transporter protein able to bind metal sulphide directly, thus enabling uptake of essential metal ions into the cell without the need for a soluble metal-binding intermediate.


   ACKNOWLEDGEMENTS
 
G.M.G. gratefully acknowledges research support from the BBSRC (94/SPC05211) for this work. We are also grateful to Dr Kevin Purdy (University of Essex, UK) and Dr Iwona Beech (University of Portsmouth, UK) for supplying the bacteria used in this work and to Professor Rolf Hallberg (University of Stockholm) for advice on the separation of the metal-binding compound.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Barnes, L. J., Janssen, F. J., Scheeren, P. J. H., Versteegh, J. H. & Koch, R. O. (1992). Simultaneous microbial removal of sulfate and heavy-metals from waste-water. Trans Inst Min Metall Sect C 101, C183-189.

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Received 14 January 1999; revised 13 May 1999; accepted 7 June 1999.



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