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FEMS Microbiology Letters 183 (2000) 313^318

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Copper accumulation by sulfate-reducing bacterial biolms


C. White, G.M. Gadd *
Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, Scotland, UK
Received 26 November 1999; accepted 27 December 1999

Abstract
Sulfate-reducing bacterial biofilms were grown in continuous culture. When exposed to medium containing 20 or 200 WM Cu, biofilms
accumulated Cu. Energy-dispersive X-ray analysis (EDXA) showed that accumulation of Cu occurred in the form of sulfides while EDXA
mapping of Cu and S in biofilm sections indicated that they were not uniformly distributed but located in the surface of the biofilm. While
the polymer content of biofilm exposed to 20 WM Cu did not appear to increase relative to control Cu-free biofilms, biofilms exposed to 200
WM Cu accumulated carbohydrate and smaller amounts of protein throughout the incubation period. The mechanism of uptake, therefore,
appeared to be precipitation of Cu sulfides at the biofilm surface or in the liquid phase followed by entrapment of precipitated Cu sulfide by
the exopolymer-enhanced biofilm. 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All
rights reserved.
Keywords : Sulfate-reducing bacterium ; Copper accumulation ; Metal^biolm interaction; Bioprecipitation

1. Introduction
The ability of sulfate-reducing bacteria (SRB) to remove
toxic metals from waters and waste waters by precipitating
highly insoluble suldes [1] has bioremediation potential
and is already used in the large-scale biological treatment
for toxic metals [2,3]. However, current bioreactors, often
based on those used in water-treatment technology, are
large, in contrast to biolm reactors which represent a
means of increasing process intensity thus reducing residence time, working volume and cost. Free-living and biolm SRB may interact with metals by precipitation of
metal suldes [1,4,5] which can be enhanced by metabolic
processes leading to raised pH [1]. Metals may also undergo biosorption by cell surfaces [3,6,7] and extracellular
polymeric substances (EPS) [8]. EPS comprises a mixture
of polysaccharides, mucopolysaccharides and proteins
which varies in composition between species and culture
conditions [9] and can take up soluble metals [10] or ne
particulates [11,12]. In a previous study, SRB biolms exposed to Cd accumulated solid CdS and both the polysaccharide and protein content of the biolms increased simultaneously [5]. The distribution of CdS was such that
bound Cd occurred mainly at or near the biolm surface

* Corresponding author. Tel. : +44 (1382) 344266;


Fax: +44 (1382) 344275; E-mail : g.m.gadd@dundee.ac.uk

which indicated that precipitated Cd adhered to, but did


not penetrate, the biolm [5]. Although Cu readily forms
suldes, its interactions with microorganisms can dier in
several important respects from those of Cd [13] with its
sulde chemistry diering from that of Cd in that it forms
mixed Cu(I) and Cu(II) suldes which are less stable than
CdS, especially at low pH [14^16]. The present study was
therefore undertaken in order to explore whether these
aspects of biology and chemistry aect the interactions
between Cu and SRB biolms.
2. Materials and methods
2.1. Organisms and maintenance
The mixed sulfate-reducing culture was initially selected
from natural sediment samples by chemostat culture [1].
No fermentative activity was evident when lactate was
used as a substrate and methanogenesis was not detected
on any substrate [1,17]. The culture was selected for biolm growth and biolms were maintained in anaerobic
batch culture in 10 ml volumes of SL10 medium [18] in
screw-topped glass tubes each containing a sterile 5U20
mm glass coupon and incubated at 20C for 14 days.
Biolm growth was then preferentially subcultured by
aseptically transferring the coupon to a further tube of
SL10 medium containing a similar coupon [5].

0378-1097 / 00 / $20.00 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 0 0 2 - 1

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2.2. Experimental culture


Experimental cultures were grown on 10 mm wide `Plastikard' polystyrene coupons (Slaters Ltd, Matlock, UK)
suspended in a 1 l `Quickt' vessel equipped for continuous culture [5]. Mixed SRB biolm culture inoculum was
developed in continuous culture in 10 ml syringe barrels
and inoculated into the 1 l culture vessels as described
previously [5]. After 48 h batch growth, continuous medium ow was started and the biolm was allowed to
develop for 7 days in SL10 medium at 20C and a dilution
rate of 0.2 h31 to produce a mature biolm prior to the
start of the experimental run. Experimental runs were carried out under identical conditions except that an appropriate volume of 100 mM CuSO4 was added to the SL10
medium to yield the required nal Cu concentration and
Na2 S was omitted from both control and metal-containing
cultures to avoid premature precipitation of the added Cu.
Neither redox poising agents nor metal chelating agents
were present in this medium. CuSO4 stocks were made up
in Milli-Q ultrapure water (Millipore UK Ltd, Watford,
UK) with the addition of 0.01% (v/vaq ) analar H2 SO4
(Merck, Lutterworth, UK) and stored in acid-rinsed
(0.01% (v/vaq ) H2 SO4 ) polypropylene bottles.

then cooled to 4C to solidify the gelatin. The coupon


was removed and the excess gelatin trimmed o leaving
the complete biolm embedded in gelatin. Approximately
5 mm cubes of gelatin containing the embedded biolm
were xed in 2.5% v/vaq glutaraldehyde in 100 mM sodium
phosphate buer, pH 7.0, at 4C overnight and dehydrated through a 10% incremental ethanol series. The gelatin had a wax-like consistency following this treatment
and a thinner section (approximately 2 mm thick) was cut
using a hand razor and air-dried prior to sputter coating
with carbon. Scanning EM and EDXA analysis were carried out using a Jeol JSM-35 scanning electron microscope
and EDXA spectra were analyzed using an Apple computer equipped with a Link Interface P1445 and software
(Link Systems Ltd, High Wycombe, UK).
3. Results
3.1. Copper accumulation by SRB biolm
Biolms accumulated Cu when it was present, the

2.3. Sampling and analysis


Samples were taken by removing entire coupons in a
predetermined random sequence. Four 10 mm long subsamples were removed from the remainder of the coupon
for electron microscopy and chemical assays of biolm
Cu, protein and carbohydrate. This procedure ensured
that the samples were taken from areas that were submerged under a minimum of 10 mm of medium during
growth. Cu was assayed by atomic absorption spectrophotometry (AAS) following digestion in 6 M HNO3 [1,19].
Protein was extracted by vortexing the coupon in 1.0 or
2.0 ml of 0.5 M NaOH with the addition of approximately
0.5 cm3 of 0.5 mm diameter glass beads at 200 rpm for
5 min followed by extraction of the resuspended biolm
material for 30 min and assaying 100 or 200 Wl of extract
by the Bradford method [1]. Carbohydrate was assayed
using the anthrone method [20] after vortexing in distilled
water with glass beads in the same way and removing a
suitable volume of suspended biolm. Previous experiments had indicated that no further protein or carbohydrate was extracted with more prolonged treatment (data
not shown). Dissolved sulde was assayed polarographically in whole culture [1].
2.4. Electron microscopy and EDXA analysis
For electron microscopy and EDXA analysis, segments
of coupon 5U10 mm in size, with attached biolms, were
embedded in 1.5 ml of melted 5% pig-skin gelatin at 50C.
They were immersed for 2.5 h to allow inltration and

Fig. 1. Accumulation of (a) Cu, (b) carbohydrate and (c) protein in continuous mixed culture SRB biolms grown with no added Cu (control)
(a), 20 WM Cu (b) and 200 WM Cu (E) over a period of 14 days following 7 days cultivation with no Cu present in all cases. Each point is
the mean of at least two separate samples and the bars indicate the
standard error of the mean (S.E.M.).

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amount being about ten-fold greater at 200 WM than at 20


WM (0.047 and 0.489 Wmol cm32 day31 at 20 and 200 WM
Cu respectively) (Fig. 1, Table 1). Trace metals were
present in the medium in only 0.1^2.0 WM concentrations
and were undetectable in the biolm with the exception of
Fe (73 WM in the medium). Fe accumulated in the biolm
in both control and Cu-containing cultures. The amount
of Fe accumulated was the same in both the presence and
absence of Cu (0.28 0.07 and 0.26 0.08 Wmol cm32 respectively over 21 days) which indicated that there was
little or no interference between Fe and Cu uptake.
3.2. Protein and carbohydrate content of SRB biolms
Neither the protein nor the carbohydrate content of
control biolms (no added Cu) increased signicantly during the experiment (Fig. 1) and both protein and polysaccharide increased only slightly in the biolms exposed to
20 WM Cu but this was not statistically signicant (Table
1). On the other hand, at 200 WM Cu the biolms accumulated signicantly larger amounts of EPS, with approximately twice the protein and almost ten times the carbohydrate content of the controls (Table 1). This indicated a
signicant role for carbohydrate in the uptake or retention
of copper. There was a lower increase in protein content,
both in absolute terms and as a proportion of control
levels (Fig. 1, Table 1) so its role appears to be of lesser
importance than polysaccharide in relation to Cu uptake.
3.3. Electron microscopy and EDXA analysis
The gelatin sections containing the biolm showed no
appreciable distortion and remained at following drying
and carbon coating. The biolm was visible under S.E.M.
as an area of etched appearance to one side of the section
which was separated from the smooth surface of the gelatin by an irregular boundary following the original surface of the biolm. This section showed the biolm to be
spatially uneven, containing water spaces similar to those
observed in other bacterial biolms [21]. The at base of
the biolm (which was in contact with the support during
growth) corresponded to one side of the gelatin section

315

(Fig. 2a). EDXA spectra of biolms exposed to 200 WM


Cu showed the presence of large Cu peaks which were
considerably smaller or absent in the controls where only
traces of Cu were present. The Cu-exposed specimens also
showed strong peaks for S in addition to Cu, although
these were also present (but smaller) in the control specimens (Fig. 3). These data are consistent with the deposition of Cu suldes in the biolm. The EDXA spectra were
also used to provide X-ray energy windows for mapping
both Cu and S. In the EDXA map, each dot results from
one X-ray count within the energy window for the element
as the electron beam scans the object and a higher density
of dots in an area of the image indicates a greater concentration of the target element, showing its distribution [22].
Both Cu and S were found to be present in the greatest
concentrations in the surface layers of the biolm (Fig.
2b,c) which indicated that Cu suldes were either precipitated in the bulk phase or at the interface with the biolm
and were accumulated by entrapment in the biolm.
4. Discussion
Cu appeared to be mainly accumulated by SRB biolms
as solid copper suldes. Suldes were the dominant copper
species because of the presence of sulde produced by the
SRB biolm (in concentrations 1.5^4.5 mM) and very low
solubilities of CuS and Cu2 S (solubility products being
1.27U10336 and 2.26U10348 respectively) [16] resulting
in precipitation of the suldes. This also occurred when
Cu2 was fed to a chemostat containing the free-living
mixed SRB culture [1]. In addition, the biolm also entrapped precipitated solid Cu suldes and responded to
their presence by accumulation of additional EPS. It was
not possible to distinguish whether this resulted from additional production or reduced sloughing. Accumulation of
EPS also accompanied Cd uptake by SRB biolms [5] and
particulate accumulation has been observed to have a similar eect on other bacterial biolms [12]. The higher accumulation of carbohydrate rather than protein with both
Cu and Cd suggests that the secretion of extracellular
polysaccharide may be a response to the accumulation

Table 1
Copper, EPS and protein accumulation by mixed sulfate-reducing biolm cultures exposed to varying copper concentrations
Run no.

Medium Cu concentration
(WM)

Biolm Cu
(Wmol cm32 )

Biolm protein
(mg BSA eq. cm32 )

Biolm carbohydrate
(Wmol glucose eq. cm32 )

1
2
3
LSD

0
20
200

0.03
0.95
7.82
0.73

0.66
0.71
1.36
0.121

0.38
0.74
3.70
0.107

The amounts shown were measured after 14 days incubation at the Cu concentration indicated.
Each value is the mean of eight determinations with the least signicant dierence (LSD) being calculated by variance analysis and comprising the residual standard error of the whole data set for the three runs multiplied by an appropriate `t' value for the required signicance level at 21 degrees of freedom [23].
Mean values diering by more than the LSD shown are distinct at a P 6 0.01 signicance level.

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Fig. 2. Scanning electron micrographs and EDXA maps of SRB biolms showing sections of biolms grown with (a) no added Cu and (b) 200 WM Cu
embedded in 5% pig-skin gelatin. The biolm is visible as a pale area at the lower part of each frame and its surface is visible as an irregular boundary
with the embedding gelatin which appears darker. The EDXA maps show the distribution of (c) Cu (815^825 keV, 44 722 counts) and (d) S (210^220
keV, 664 281 counts) in the biolm section shown in (b). Both Cu and S can be seen to be concentrated in the surface of the biolm although S is also
visible throughout due to the presence of suldes of metals other than Cu. A control map of the same eld with the energy window covering no elemental peaks for relevant elements (800^810 keV, 19 473 counts) (e) shows the absence of topographical emissions. The scale bar shows 100 Wm.

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Acknowledgements
G.M.G. gratefully acknowledges nancial support from
the BBSRC (LINK Programme: Biological treatment of
soil and water, Ref: 94/BSW 05375). This work was carried out as part of the overall project `Metal-biolm interactions in sulfate-reducing bacterial systems' in collaboration with British Nuclear Fuels plc, Preston, Lancashire
and Westlakes Research Institute, Whitehaven, Cumbria.
The authors also wish to thank Martin Kierans of this
Department for assistance with electron microscopy and
EDXA.

References

Fig. 3. EDXA spectra obtained from mixed culture SRB biolms following 21 days continuous culture. The spectra were obtained from
(a) control cultures grown in the absence of Cu and (b, c) cultures
grown in 20 and 200 WM Cu respectively for 14 days following 7 days
development in the absence of Cu. The horizontal axis represents X-ray
energy (0^1023 keV) and the vertical axis shows emission counts (total
counts for each spectrum = 3.1^3.5U105 ).

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Cu and Cd accumulation in SRB biolms were fundamentally similar, being dominated by sulde precipitation and
entrapment of the resulting metal sulde particles by the
biolm EPS. As discussed above, sulde precipitation is an
ecient means of separating toxic metals from solution,
while entrapment in a biolm is also a further means of
reducing the mobility of the metals, which could be of
value in bioremediation.

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