Geobiology (2007), 5, 211–218
DOI: 10.1111/j.1472-4669.2007.00111.x
The impact of metabolic state on Cd adsorption onto
bacterial cells
Metabolism
O
RIGINA
Leffects
A R Ton
I CCd
L E adsorption
S
by bacteria
Blackwell
Publishing
Ltd
K . J . J OH N SON , 1, * D . A . A M S , 1 A . N . W ED EL , 2 J. E. S. SZY M ANOW SKI, 1 D. L . W EBER, 2
M. A. SC H N E E GU RT 2 A N D J . B . F E I N 1
1
Department of Civil Engineering and Geological Sciences, University of Notre Dame, Notre Dame, Indiana, USA
Department of Biological Sciences, Wichita State University, Wichita, Kansas, USA
2
ABSTRACT
This study examines the effect of bacterial metabolism on the adsorption of Cd onto Gram-positive and
Gram-negative bacterial cells. Metabolically active Gram-positive cells adsorbed significantly less Cd than nonmetabolizing cells. Gram-negative cells, however, showed no systematic difference in Cd adsorption between
metabolizing and non-metabolizing cells. The effect of metabolism on Cd adsorption to Gram-positive cells was
likely due to an influx of protons in and around the cell wall from the metabolic proton motive force, promoting
competition between Cd and protons for adsorption sites on the cell wall. The relative lack of a metabolic effect
on Cd adsorption onto Gram-negative compared to Gram-positive cells suggests that Cd binding in Gramnegative cells is focused in a region of the cell wall that is not reached, or is unaffected by this proton flux. Thermodynamic modeling was used to estimate that proton pumping causes the pH in the cell wall of metabolizing
Gram-positive bacteria to decrease from the bulk solution value of 7.0 to approximately 5.7.
Received 3 July 2006; accepted 13 April 2007
Corresponding author: K. J. Johnson. Tel.: 650 329 4380; fax: 650 329 4545; e-mail: [email protected].
INTRODUCTION
Bacterial cell walls can adsorb a wide range of aqueous metal
cations, potentially altering the mobility of the metals in geologic
systems (e.g., Beveridge & Murray, 1976, 1980; Beveridge &
Koval, 1981; Crist et al., 1981; Harvey & Leckie, 1985;
Goncalves et al., 1987). Most previous studies of bacterial
surface adsorption have involved bacterial cells that were not
metabolically active during the period of adsorption, focusing
on the passive binding that occurs between bacterial surface
functional groups and aqueous metal cations (e.g., Mullen
et al., 1989; Fein et al., 1997; Haas et al., 2001; Ngwenya
et al., 2003). Metal cations appear to bind predominantly to
deprotonated sites within the bacterial cell wall. The extent of
metal adsorption onto bacterial surface functional groups
decreases markedly with decreasing pH due to protonation,
and hence neutralization, of negatively charged surface functional
groups at lower pHs. The decrease in adsorption can be viewed
as competitive adsorption of H+ and aqueous metal cations on
available surface sites.
*Present address: US Geological Survey, Water Resources Division,
Menlo Park, CA, USA.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Previous experimental studies of bacterial metal adsorption
mimic adsorption conditions in nutrient-poor oligotrophic
environments. However, metabolically active bacteria may
exhibit significantly different adsorption behaviors than inactive cells, thereby changing the mobility, speciation, and bioavailability of metals. Bacterial metabolic activity can create an
electric potential across the plasma membrane, called a proton
motive force (PMF). During aerobic metabolism, protons are
pumped across the plasma membrane toward the outside of the
cell, and electrons or negatively charged species such as OH–
concentrate inside the cell (Ehrlich, 1996). The PMF is an
essential component of bacterial metabolism, for the movement
of protons back into the cell, down the concentration gradient
through plasma membrane ATPases, enables electrical potential
energy to be captured as chemical potential energy in ATP. If
protons are pumped out of the cytoplasm faster than protons
can diffuse back through and move away from the plasma
membrane, then the cell wall region influenced by the proton
accumulation by metabolizing bacterial cells should possess H+
activities that are elevated relative to that in the bulk solution.
It has been postulated that the lower pH associated with the
cell wall environment of metabolizing cells could diminish the
extent of metal cation adsorption onto cell wall functional groups
211
212 K. J. JOHNSON et al.
(Urrutia Mera et al., 1992). Clearly, in order to model bacterial adsorption in systems where a significant fraction of the
bacteria are undergoing active metabolism, the effect of
metabolism on metal cation adsorption must be determined.
Urrutia Mera et al. (1992) observed less adsorbed uranyl
and scandium onto active Gram-positive cells compared to that
observed onto cells that were inactivated through exposure
to sodium azide (NaN3), carbonyl cyanide m-chlorophenylhydrazone (CCCP), or gamma radiation. Urrutia Mera
et al. (1992) concluded that the PMF of the active cells caused
the lower extent of adsorption due to enhanced H+ competition at the adsorption sites. These results are consistent with
similar experiments by Kemper et al. (1993), who also observed
enhanced cation adsorption by inactivated Bacillus subtilis
cell walls. Although the work of Urrutia Mera et al. (1992)
suggests that the PMF exerts a significant effect on adsorption,
a number of their experimental procedures may have affected
the results. The metal concentrations used by Urrutia Mera et al.
(1992) were relatively high and mineral precipitation may account
for some of the observed metal loss from solution. For
example, the solubility of schoepite ((UO2)8O2(OH)12·(H2O))
under the experimental conditions of a solution open to the
atmosphere at pH 6.4 is less than 1 µM. Even considering
aqueous uranyl–acetate complexation, the 1 mM experimental
solutions were significantly oversaturated with respect to
schoepite, and precipitation may have occurred in some of
the experiments. Additionally, the ‘active’ bacterial controls
consisted of cell suspensions in distilled water and the level of
metabolic activity may have been quite low.
Claessens et al. (2006) studied the effects of bacterial
metabolism on cell wall site protonation and surface charge of
a Gram-negative (Shewanella putrefaciens) species, and they
examined the role of cell wall structure by comparing the
protonation behavior of metabolically active S. putrefaciens to
that of metabolically active B. subtilis. Live S. putrefaciens cells
exhibited rapid initial consumption of acid under all pH conditions studied, likely reflecting the initial protonation behavior
of cell wall functional groups. At pH 4, proton uptake by
suspensions of live cells stopped after 50 min, likely due to
loss of viability. However, at pH 8 and 10, Claessens et al. (2006)
observed that deprotonation continued at a slower rate for
the entire 5-h duration of the experiment, likely due to active
respiration and PMF generation by the cells. Inactivation of
S. putrefaciens cells caused no effect on initial acid or base consumption by the cells; however, the inactivation caused longterm protonation/deprotonation to cease or proceed at a very
slow rate. Active B. subtilis cells exhibited a greater extent of
initial proton consumption than did active S. putrefaciens cells,
indicating that the B. subtilis cells have more functional groups
present on the cell wall. In contrast, the S. putrefaciens cells
exhibited much higher extents of long-term deprotonation at
pH 8 and 10 than did the B. subtilis cells, suggesting that the
Gram-negative species had a larger PMF effect on protonation
of the cell wall functional groups.
The objective of our study is to further investigate the effect
of metabolic activity on the ability of bacteria to adsorb aqueous metal cations. These experiments are similar to those conducted by Urrutia Mera et al. (1992) in that we compare metal
uptake onto inactivated cells to that observed for metabolically
active control suspensions. However, in this study the adsorption of a range of concentrations of aqueous Cd2+ is studied
under clearly undersaturated conditions, metal-cell suspensions
are allowed to equilibrate for 2.5 h, and all experiments are
conducted in a nutrient growth medium to insure substantial
metabolic activity in our untreated control cultures. Varying
the Cd2+ concentration allows us to observe the effects of
metabolism on Cd adsorption at different metal–bacteria ratios
and to demonstrate that effects on Cd binding are not due to
metal toxicity, but rather to changes in the metabolic state of
the cells. Furthermore, we examine a range of both Grampositive (B. subtilis and Bacillus cereus) and Gram-negative
(Pseudomonas fluorescens and Shewanella oneidensis) bacterial
species in order to determine if cell wall structure influences
the metabolic effects on adsorption.
METHODS
Bacterial strains and culture conditions
Cultures of P. fluorescens str. ATCC 11764, B. cereus str. ATCC
6462, S. oneidensis str. MR1, and B. subtilis str. ATCC 6051
were maintained as liquid 100-ml shake-flask cultures (150
rpm; 1-in stroke dia) at room temperature or at 32 °C in LB
broth (Sambrook et al., 1989). Fresh liquid cultures were
inoculated every 2 weeks from clonal colonies on LB agar
plates. Actively growing log-phase subcultures were used for
growth and binding experiments.
Metabolism treatment and cadmium binding
Although Urrutia Mera et al. (1992) and Kemper et al. (1993)
used a range of metabolic inhibitors, the current study used
formalin (a 37% by weight formaldehyde solution in water)
treatments of cell suspensions to inhibit the metabolism of
both Gram-positive and Gram-negative bacterial cells. Our
measurements (see below) demonstrate that formalin is faster
and more effective than sodium azide at inhibiting cellular
metabolism, and is unlikely to have the potentially damaging
effects that high doses of radiation may have on cell wall
structures. However, in order to facilitate direct comparison
of these results to those of Urrutia Mera et al. (1992) and
Kemper et al. (1993), separate experiments were conducted
using only B. subtilis in which formalin was replaced with
sodium azide as the metabolic inhibitor.
Two 100-mL aliquots of bacterial inoculum from a 500-ml
stock culture were distributed into 250-mL polypropylene
bottles. One aliquot was treated with 2.5% (w/v) formalin,
and one aliquot was left as an untreated control. Both the
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Metabolism effects on Cd adsorption by bacteria
formalin-treated culture and the untreated control culture
were incubated under identical conditions (at 25° C) for 30
min. The B. subtilis- sodium azide experiments were conducted in the same manner; however, one aliquot was treated
with 0.5% (w/v) sodium azide and the experimental cultures
were incubated for 6 h. Preliminary respirometry experiments
demonstrated that bacteria require a longer exposure period
to sodium azide to inactivate cells. Note that in the untreated
control experiments, cell reproduction occurred and culture
density increased during the incubation period, while this did
not occur to a significant extent in the treated suspensions. To
account for this unavoidable difference in cell concentration,
Cd adsorption concentration results were normalized using
cell mass present at the end of the incubation. Since the
formalin treatment requires a shorter incubation time to be
effective, there was less of a difference between the cell concentrations in the treated and untreated systems, and this was
another reason why the formalin approach was favored over
the sodium azide treatment to inactivate the bacterial cells.
After the incubation period, the bacterial cultures were harvested by centrifugation for 10 min at 6000 g. Bacterial cell
pellets were then resuspended in 100 mL of assay medium [in
g L−1: KNO3, 0.55; NaNO3, 0.47; HEPES, 2.37; with glycerol used as the carbon and energy source at 0.5% (w/v), and
with an ionic strength of 0.005 M], and supplemented with
the appropriate Cd concentration (3, 10, or 20 ppm from a
1000-ppm Cd nitrate reference solution). The solution pH
was adjusted to ~7.0 with HCl or NaOH and maintained by
the HEPES buffer. Cd concentrations were well below saturation values. The assay medium was designed to limit competing ions such as phosphate and chloride, although the ionic
strength was similar to typical basal salts media for bacteria. All
adsorption experiments were performed with three or more
replicates. All metal incubations were performed while rotating the reaction vessel end over end on a carousel for 2.5 h.
Preliminary experiments showed that this duration was more
than sufficient to ensure binding equilibrium.
After the 2.5 h reaction period, the pH of each assay culture
was measured and cell density determined by measuring
absorbance at 600 nm (Cary 300 spectrophotometer, Varian
Inc., Palo Alto, CA, USA). Experiments were performed with
each bacterial species to determine a conversion factor between
absorbance and wet weight (see Borrok et al., 2004a for a discussion of the relative merits of wet vs. dry weights), with 1 OD
unit at 600 nm corresponding to 2.613, 1.558, 1.743, and
2.920 g L−1 wet weight for B. subtilis, B. cereus, P. fluorescens,
and S. oneidensis, respectively. These values were used to normalize the Cd adsorption data, and adsorption results are
reported in terms of microgram of Cd bound per milligram
wet weight of bacteria. After cell densities were determined
for each experiment, the supernatant from each assay was collected by centrifugation at 10 000 g for 3 min and filtered with
a 0.45 µm filter (Osmonics Cameo 30N, Minnetonka, MN,
USA). The filtered solutions were then acidified with a small
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
213
aliquot of concentrated HNO3 and stored at 4° C for no
longer than 1 week. The final dissolved Cd concentration in
each of the assay supernatants was determined by inductively
coupled plasma–optical emission spectroscopy (ICP-OES),
with matrix-matched standards for calibration. The amount of
Cd that was adsorbed onto the bacteria during each assay was
determined as the difference between the amount of Cd in
solution at the end of the assay and the initial Cd concentration. Control experiments without bacteria were performed
simultaneously with the cell binding assays to determine the
amount of nonspecific Cd adsorption onto the experimental
apparatus. The average adsorption of three controls was 0.2-ppm
Cd loss from an initial 10-ppm Cd solution, and this value was
subtracted from each experimental value to account for loss of
Cd. Data are plotted in the figures as a mean ± one standard
deviation. Determination of the statistical difference between
experiments conducted with active and inactive cells was calculated using the two-tailed t-test for paired samples, with resulting
P values < 0.05 indicating statistical difference (within a 95%
confidence interval) in the extent of adsorption.
Metabolic activity measurements
The effectiveness of the inactivation treatments was determined
by viability staining, iodonitrophenyl tetrazolium chloride
(INT) staining, and O2 respiration measurements. Tetrazolium
chloride is a redox indicator that can indicate cellular respiration.
Sodium azide affects cells in such a way that for some time
after treatment, the cells appear alive with viability staining and
INT staining. Therefore, respiration was also measured in
treated cultures using a Clark-type oxygen electrode (YSI 5000)
calibrated with air-saturated water. INT staining was performed
following the method of Bovill et al. (1994) where cells
treated with a tetrazolium dye were harvested, washed,
resuspended in buffer, and examined microscopically for
refractile spots diagnostic of metabolic activity. Viability staining
used as a measure of membrane integrity was performed
following the manufacturer’s instructions (BacLight Kit,
Molecular Probes, Eugene, OR, USA).
RESULTS
A variety of techniques were used to measure the effectiveness
of the formalin and sodium azide treatments in inhibiting cell
metabolism, and to confirm that metabolic activity occurred in
the untreated systems. Figure 1 shows respirometer measurements
for suspensions of both treated and untreated B. subtilis cells,
with and without Cd (10 ppm) present in the system. These
results are representative of all of the bacterial species used in
the current study, but for clarity only the B. subtilis results are
shown. The respirometer measurements show that untreated
cells, with or without Cd present, remove 90–95% of the
oxygen in the system within the first minute of the assay. Cells
treated with either formalin or sodium azide remove a
214 K. J. JOHNSON et al.
Fig. 1 Representative oxygen consumption by treated and non-treated
bacterial cells.
maximum of 10–15% of the oxygen in the system over the
entire length of the experiment. Thus, the respirometry
experiments provide evidence that untreated bacteria maintain
active metabolism under the conditions of the binding assays,
even in the presence of Cd, and that cells treated with either
formalin or sodium azide metabolize at a greatly diminished
rate. It should be noted that respiration tests reflect the
metabolic state of the cells at the beginning of the experiment,
not oxygen content over the duration of the overall formalin
and sodium azide treatment experiments. A scanning electron
microscope (SEM) image (Fig. 2) depicts formalin-treated B.
subtilis cells, indicating that the bacteria retain their shape and
are visually unaffected after the formalin treatment. Similar
results were seen for the sodium azide-treated B. subtilis and
formalin-treated B. cereus, P. fluorescens, and S. oneidensis cells.
These images suggest that the formalin and sodium azide
treatments had a negligible effect on the macro-structure of
the cell wall, and suggest that any changes in metal uptake that
we observed associated with these treatments are due to the
effect of the treatments on cell metabolism. The results of INT
staining and viability staining of formalin-treated cells were
consistent with the respiration results, demonstrating that the
cells were no longer metabolically active. For the azide-treated
cells, the plasma membrane apparently remained intact and
Fig. 2 Scanning electron microscope (SEM) image, taken under ultrahigh vacuum at 20 kv, of formalin-treated B. subtilis cells. The cell suspension solution was
diluted by a 1:10 ratio prior for imaging, due to the salt content of the assay medium. Once B. subtilis samples were mounted on the SEM stubs, they were rinsed
briefly with deionized water to remove residual salt.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Metabolism effects on Cd adsorption by bacteria
215
Fig. 3 Comparison of the amount of Cd adsorbed onto metabolizing and non-metabolizing bacterial cells for experiments containing 3, 10, and 20 ppm Cd. F =
formalin treatment and SA = sodium azide treatment (for B. subtilis only), where ‘Active’ denotes untreated cells incubated for identical times to ‘Inactive’ cells treated
with formalin or sodium azide, respectively: (a) B. subtilis; (b) B. cereus; (c) P. fluorescens; and (d) S. oneidensis. The error bars represent the standard error of the
mean, and an asterisk (*) indicates the t-test showed the data from the active and inactive cell experiments to be significantly different.
INT staining is not an appropriate measure of electron transport
activity, as azide blocks the system beyond the point detected
by the INT reaction.
The design of these experiments allows for comparison of
the amount of Cd bound to metabolically active cells relative
to that bound to inactivated cells. Preliminary experiments
showed a measurable increase in bacterial culture density over
the course of the experiments with untreated cells, another
indication that bacterial metabolism was active in the assay
medium. Conversely, there was no difference between the initial and final bacterial culture densities in experiments with
treated cells. Since the final bacterial culture densities differed
in each experiment due to normal variations in growth, the
concentrations of adsorbed Cd given in Fig. 3(a–d) were
normalized to the final bacterial culture density. Error bars
in Fig. 3(a–d) represent the standard error of all replicate
experiments. The final pH for all adsorption experiments was
between 6.7 and 7.2. The uncertainties for these experiments
are potentially higher than those typically seen for metal
adsorption experiments due to the treatment of the bacterial
cells prior to the metal adsorption experiments. Also, the cells
were not acid washed or rinsed with an electrolyte to remove
cations that may have adsorbed onto the cells from the growth
medium.
In all of the assays with Gram-positive cells, higher Cd
adsorption was observed with inactivated cells than in the systems that contained metabolically active cells. Depending on
the experimental Cd concentration, Cd adsorption onto metabolically active B. subtilis was between 24 and 48% less than
that exhibited by the inactive cells that were treated with
formalin; and between 22 and 32% lower than cells that were
treated with sodium azide (Fig. 3a). Cd adsorption onto
untreated metabolizing B. cereus cells was between 30 and 53%
lower than formalin-treated cells (Fig. 3b). Although the inactivation treatment had a consistent effect on the extent of Cd
adsorption onto the two Gram-positive species, there was no
distinguishable trend in the effect as a function of Cd concen© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Table 1 Two-tailed t-test results
Bacteria
Cd(ppm)
Treatment
N
P
B. subtilis
3
10
20
3
10
20
3
10
20
3
10
20
3
10
20
F
F
F
SA
SA
SA
F
F
F
F
F
F
F
F
F
7
3
6
7
3
6
11
9
9
9
9
9
12
9
9
0.037
0.005
0.140
7.24E-06
0.030
0.081
0.004
2.09E-18
1.35E-16
0.001
1.38E-05
1.33E-08
0.311
0.058
3.16E-04
B. cereus
P. fluorescens
S. oneidensis
Treatment: F, formalin; SA, sodium azide; N, number of replicates.
tration for either species. The results of the B. subtilis experiments suggest that sodium azide had a consistently smaller
impact on cell metabolism, as cells inhibited with formalin
exhibited higher metal adsorption than those treated with
sodium azide (Fig. 3a). Table 1 shows the number of replicates (N) for each experimental condition and the statistical
difference in Cd adsorption by active and inactive cells at each
Cd concentration. For the 3- and 10-ppm B. subtilis experiments, the two-tailed paired t-test indicates that the inactive
cells exhibited significantly enhanced Cd adsorption relative
to the active cells. Although the t-test result for the 20-ppm
experiments suggests no statistical difference between the
active and inactive cells, the overall trend from the B. subtilis
experiments indicates enhanced Cd adsorption by the inactive
cells relative to the active cells. The t-tests for the B. cereus cells
indicate that there is a statistical difference in the extent of
Cd adsorption by active and inactive cells at each of the Cd
concentrations tested.
216 K. J. JOHNSON et al.
The results for B. subtilis adsorption are in agreement with
those of Urrutia Mera et al. (1992) in that an increase in cation adsorption was observed in the experiments that involved
inactivated cells. However, the effect observed by Urrutia
Mera et al. (1992) was larger than that reported here, possibly
due to differences in wash procedures between the two studies
or differences in the metals used in each study. Urrutia Mera
et al. (1992) also observed a greater change in bulk solution
pH during their experiments. The initial pH of 7.0 in the
Urrutia Mera et al. (1992) experiments decreased to 6.5 for
their azide-treated cells and to 6.4 for their control cells,
whereas the pH in the current experiments decreased from 7.0
to 6.8.
In contrast to what was observed in the experiments with
Gram-positive bacterial cells, the results from experiments
with Gram-negative bacterial cells show no systematic effect of
metabolism on the extent of Cd adsorption onto the cells. The
inactivated cells exhibited approximately the same extent of
Cd adsorption as the actively metabolizing cells. For P. fluorescens, paired two-tailed t-tests comparing the active and inactive
cells suggest that there is a statistical difference between the
active and inactive cell experiments (Table 1). However, the
difference between the observed extent of adsorption by active
and inactive cells for each Cd concentration studied was less
than 12% (Fig. 3c), and there is no consistent trend as a function of Cd concentration. Similar behavior for S. oneidensis to
that seen for P. fluorescens is depicted in Fig. 3(d): no consistent difference was observed in the extent of Cd adsorption
onto active cells relative to the inactive cells, and this is true for
all Cd concentrations studied. Results of t-tests for the active
and inactive S. oneidensis experiments suggest that there is not
a statistical difference in the Cd adsorption at 3- and 10-ppm
Cd concentrations. The 10-ppm experiments with P. fluorescens and S. oneidensis exhibited a slight increase in the extent
of adsorption onto the treated cells, but the opposite was
observed in the 20-ppm experiments. The observation of a
larger metabolic effect in Gram-positive species relative to that
observed for Gram-negative species is inconsistent with the
potentiometric titrations conducted by Claessens et al. (2006),
who documented greater long-term proton release in suspensions of metabolically active Gram-negative cells compared
with Gram-positive cells.
DISCUSSION
The results of this study suggest that adsorption of Cd onto
Gram-positive bacteria is significantly diminished by the
presence of a PMF, while the effect on Gram-negative bacteria
is small to negligible. As suggested by Urrutia Mera et al.
(1992), who conducted similar experiments to these using
only the Gram-positive species B. subtilis, the decrease in
adsorption associated with active metabolism observed for
Gram-positive bacteria is likely due to increased competition
between Cd2+ and the H+ ions effluxed from the cell
membrane, adjacent to negatively charged functional groups
on the cell walls of metabolizing bacteria. Urrutia Mera et al.
(1992) hypothesized that the PMF exerts a similar effect on
both Gram-positive and Gram-negative bacterial species.
However, the differences in adsorption behavior between
Gram-positive and Gram-negative species observed here suggest
that metal binding to Gram-negative and Gram-positive bacteria
is affected by the PMF in substantially different ways.
Gram-positive bacterial cell walls consist of a thick layer of
peptidoglycan overlying the plasma membrane (Beveridge &
Murray, 1980). For Gram-positive bacteria, the PMF is generated by protons effluxed from the plasma membrane and the
peptidoglycan layers, thus, acidifying to some extent the cell
wall region. Gram-negative bacterial cell walls consist of a plasma
membrane overlain with a relatively thin peptidoglycan layer,
which in turn is overlain by a lipopolysaccharide outer membrane.
The PMF in Gram-negative species, like that in Gram-positive
species, should at least partially neutralize the electronegativity
of the peptidoglycan layer. The observation that metabolizing
and non-metabolizing Gram-negative bacteria exhibit similar
extents of Cd adsorption can be explained by the following
hypotheses: either (1) the extent of the influx of protons into
the cell wall is smaller for Gram-negative bacterial species than
it is for Gram-positive species; or (2) the primary sites of binding
of Cd in Gram-negative cells under these experimental conditions is exterior to the peptidoglycan layer, and the PMF does
not extend out to these primary sites of metal binding within the
Gram-negative cell wall structure. It is possible that the flux of
protons during Gram-negative metabolism neutralizes the electronegativity within the peptidoglycan layer, but does not affect
the more external surface of the outer membrane and associated structures. This interpretation suggests that a significant
component of the metal adsorbed onto Gram-negative cells is
bound to the phosphoryl groups of the outer membrane and
associated structures, likely involving the functional groups
within the outer membrane phospholipids and lipopolysaccharides, and does not involve the peptidoglycan layer as much as
is suggested for Gram-positive species (Beveridge, 1989).
In this study, B. subtilis cells treated with formalin adsorbed
more Cd than those treated with sodium azide (Fig. 3a); this
suggests that formalin is more effective at retarding metabolism in these bacteria. The lower oxygen consumption exhibited
by the formalin-treated cells relative to those treated with sodium
azide (Fig. 1) also supports this premise, however it should be
noted that the two are likely within experimental error. In an
analysis of the effectiveness of various treatments in retarding
microbial activity in sediment trap material, Lee et al. (1992)
found that formalin and chloroform treatments reduced microbial activity to undetectable levels, but that the activity of azidetreated samples were reduced only to 10–20% of their initial
value. The difference in Cd adsorption between the formalin- and
the sodium azide-treated cells (Fig. 3a) is likely due to the manner
in which the treatments impact the bacterial cell. Sodium
azide is a proton-conducting uncoupler; it inhibits oxidative
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Metabolism effects on Cd adsorption by bacteria
phosphorylation by binding to the terminal cytochromes of most
respiring bacteria, preventing electron transfer (Harold, 1972).
Cytochromes are generally membrane-bound proteins that carry
out electron transport or catalyze reductive/oxidative reactions.
Conversely, formalin inactivates proteins by forming covalent
crosslinks with several functional groups; this dehydrates the
cells and replaces the normal fluid with a gel-like rigid complex.
Surface complexation modeling can be used to quantify
the proton flux responsible for the observed decrease in Cd
adsorption capacity in metabolically active Gram-positive cells.
As discussed above, the decreased adsorption associated with
the metabolizing Gram-positive cells is likely due to a lower
local pH in the near-surface region, inhibiting adsorption onto
these cells. Since surface complexation modeling can be used
to account for the pH dependence of adsorption (e.g., Plette
et al., 1995, 1996; Fein et al., 1997; Cox et al., 1999), it is
possible that this modeling approach could be used to model
the effects of metabolism on adsorption if the extent of the
surface pH change was known. Conversely, a thermodynamic
modeling approach can be used to estimate the pH of the cell
wall region of metabolizing Gram-positive bacteria. Based on
the decrease in adsorption that accompanies bacterial metabolism, surface complexation modeling was used to determine
the effective pH of the region where Cd binding occurs on the
B. subtilis cell wall. This calculation can only be conducted
for the B. subtilis experiments, because acidity constants of
the cell wall functional group sites and associated Cd binding
constants have not been determined for the other bacterial
species used in this study. The surface complexation model
accounts for the protonation behavior of the cell wall using the
deprotonation constants and surface site concentrations of
discrete cell wall functional groups in the four-site model
developed by Fein et al. (2005). The deprotonation reactions
are represented by the following equation:
R − Ln H 0 ⇔ R − Ln− + H +
(1)
where R is the bacterium to which each functional group type
Ln is attached. The mass action equation for the above
deprotonation reaction is
Ka =
[R − Ln− ]aH +
[R − Ln H 0]
(2)
where Ka represents the acidity constant, a is the activity of the
subscripted species, and brackets denote the concentration of
surface sites in mol L−1 of solution. Although Fein et al. (2005)
model the bacterial cell wall acidity using four sites, under near
neutral pH conditions, Sites 2 and 3, with log Ka values of 4.8
and 6.8, respectively, are the metal adsorption sites of interest.
The two-site model of Borrok et al. (2004b) was used to
account for Cd adsorption onto B. subtilis:
Cd 2+ + R − L n(−1) ⇔ R − Ln − Cd +
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
(3)
217
The corresponding mass action equation relates the Cd
binding constant, Kads , to components of reaction (3):
K ads =
[R − Ln − Cd + ]
aCd 2+ [R − Ln− ]
(4)
The log Kads values for Cd adsorption onto cell wall Sites 2
and 3 are 3.4 and 4.6, respectively. The above mass action
equations (equations 2 and 4), along with mass action equations
for aqueous Cd hydrolysis, and mass balance constraints on total
Cd and bacterial site concentrations were used to determine
pH conditions at the binding sites of the actively metabolizing
bacteria. The initial bulk solution pH of both metabolizing and
non-metabolizing experiments was 7.0, and it was assumed
that the observed decrease in adsorption was due exclusively
to a decrease in pH at the binding sites. The 3.0-ppm Cd
experiments involving formalin-treated B. subtilis cells exhibited
a change in adsorbed Cd from 1.3 ppm to 0.7 ppm associated
with metabolic activity. Calculations show that this decrease
in adsorption would be caused by a decrease in pH at the
binding sites from 7.0 to 5.7. Similarly, the calculations for
the 10- and 20-ppm experiments suggest a pH decrease that
is associated with bacterial metabolism from 7.0 to 6.3 and
from 7.0 to 6.2, respectively, yielding an average calculated
effective pH at the binding sites of actively metabolizing cells
of 6.1 ± 0.3. The experiments involving sodium azide-treated
B. subtilis cells yield pH changes (from the bulk solution pH
of 7.0) associated with metabolism to values of 6.1, 6.4, and
6.0 for the 3-, 10-, and 20-ppm Cd experiments, respectively,
with an average calculated effective pH at the Cd binding sites
on the actively metabolizing cells of 6.2 ± 0.2.
CONCLUSIONS
This study demonstrates that the metabolic activity of Grampositive bacteria has a significant impact on Cd adsorption
onto cell wall functional groups. Metabolizing Gram-positive
cells adsorbed significantly less Cd than did non-metabolizing
Gram-positive cells. Conversely, metabolizing and nonmetabolizing Gram-negative cells exhibited roughly similar
extents of Cd adsorption. The effect of bacterial metabolism
on Cd adsorption onto Gram-positive cells is likely due to a
local decrease in pH in the cell wall region where Cd is bound.
The lack of an observable effect of metabolism on Cd adsorption
onto Gram-negative cells suggests that Cd binding occurs at a
greater distance from the inner plasma membrane than occurs
within the cell wall of Gram-positive cells. A surface complexation
modeling approach was used to estimate that the effect of the
metabolic proton motive force on the Gram-positive cell wall
was to decrease the pH of this region by approximately one
pH unit. This study demonstrates that bacterial metabolic state
can influence the extent of passive metal adsorption onto cell
wall functional groups, at least for bacterial species similar to
the Gram-positive species studied here. Adsorption onto actively
218 K. J. JOHNSON et al.
metabolizing bacterial cells has been modeled using a surface
complexation approach and the results suggest that the decrease
in pH in Gram-positive cell walls due to metabolism was approximately one pH unit. The results also suggest that biosorption
remediation strategies that involve Gram-positive bacterial
species may be more efficient at metals removal from solution
if bacterial metabolism is inhibited during the sorption process.
ACKNOWLEDGEMENTS
This research was supported by NSF grant EAR02-07169,
and by a NSF Environmental Molecular Science Institute grant
(EAR02-21966). Three anonymous journal reviews greatly
improved the manuscript.
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