Marine Biology Research Division and Center
for Marine Biotechnology and Biomedicine, Scripps Institution of
Oceanography, University of California at San Diego, La Jolla,
California 92093-0202
 |
INTRODUCTION |
Divalent manganese [Mn(II)]
oxidation and subsequent formation of Mn (hydr)oxides are
major controls on the speciation of Mn in sediments, soils, and natural
waters. Mn (hydr)oxides are very reactive components of
natural environments and affect the fate, transport, and
bioavailability of a variety of heavy metals and organic compounds
(6). Abiotic Mn(II) oxidation is a thermodynamically favorable but kinetically slow process under most natural conditions. In the environment, however, Mn(II) oxidation is believed to be largely
due to the activities of microorganisms that catalyze the reaction
(22) and increase the rate of Mn(II) oxidation by up to 5 orders of magnitude compared to abiotic Mn(II) oxidation (22,
37). For example, the Mn(II) oxidation rate near the sediment
surface in a eutrophic lake calculated from a 4-year record of sediment
trap data showed a distinct seasonal pattern, with maxima of up to 2.8 mmol m
2 day1 during the summer
(46). The average half-life of Mn(II) during stagnation in
the summer was 1.4 days. This oxidation rate cannot be explained with
the available knowledge concerning abiotic surface catalysis but is
within the range typical of microbiological oxidation rates.
A wide variety of bacteria have been reported to oxidize Mn(II) in
fresh and marine waters, soils, and sediments (10). Marine Bacillus sp. strain SG-1, which was isolated from a
near-shore sediment enrichment culture, is one bacterium that has been
extensively studied in terms of Mn(II) and Co(II) oxidation
(40). In this bacterium, it is the spores that catalyze
Mn(II) and Co(II) oxidation (19, 29) under environmentally
relevant pH, temperature, and metal concentration conditions. The
spores also bind a variety of other heavy metals, such as Cd and Zn
(38). Mn(II)-oxidizing activity is located on the spore
coats and apparently is catalyzed by a protein (7, 42, 45).
Recent evidence suggests that a spore surface protein related to the
multicopper oxidase family of proteins is involved in Mn(II) oxidation
(45). Consistent with this idea, low amounts of Cu(II)
stimulate Mn(II) oxidation (45).
Although Mn(II) oxidation appears to be protein catalyzed, the
mechanism of oxidation and precipitation of Mn(II) and Co(II) is not
understood well. The process is envisaged as having two steps, binding
and oxidation-precipitation, based on the observation that the
precipitated Mn is generally found outside the cell (39). Soluble Mn(II) ions first bind to the negative sites on the spore surface; the bound ions are then enzymatically oxidized to Mn(III, IV),
which precipitates as Mn (hydr)oxide solid phases. This two-step process is believed to be closely related to the surface charge properties of the spores.
In order to better understand heavy metal binding and the possible role
of copper in Mn(II) oxidation by SG-1 spores, information about the
characteristics of the surface charge and metal adsorption behavior of
SG-1 spores is needed. In this study, we initiated investigations to
examine the interactions of Cu(II) with SG-1 spores. The surface of
SG-1 spores was first characterized in terms of surface area, surface
site density, and surface charge. Cu(II) adsorption by SG-1 spores was
investigated to quantify adsorption kinetics, adsorption capacity, and
the effect of pH.
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MATERIALS AND METHODS |
Unless indicated otherwise, all reagents used in this study were
analytical grade or better. All vessels and storage containers were
polyethylene or polycarbonate and were acid washed prior to use.
Solutions were made with water purified by passage through a Milli-Q
water system.
Preparation, purification, and pretreatment of SG-1 spores.
Bacillus sp. strain SG-1 was grown and maintained in K
medium. K medium contains (per liter of 75% seawater) 2 g of
peptone and 0.5 g of yeast extract. After autoclaving, 20 ml of 1 M filter-sterilized N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid
(HEPES) buffer (pH 7.4 to 7.8) and 0.1 ml of 1 M filter-sterilized
MnCl2 were added per liter. For agar plates, 15 g of
agar per liter was added before autoclaving.
SG-1 cultures were grown at room temperature in 1 liter of K medium in
2-liter flasks placed on a rotary shaker at 150 rpm. Usually more than
95% of the bacteria produced endospores within 5 days. Fully
sporulated cultures consisted of spore clumps and few individual
spores, as observed by phase-contrast microscopy. Spores were harvested
by centrifugation at 16,000 × g and 4°C for 10 min
and were purified to remove precipitated Mn oxides and adsorbed trace
metals from the spore surface as follows. Spore pellets were washed
with water by using a Teflon tissue homogenizer and recollected by
centrifugation. Spores were then homogenized and treated with 0.01 M
ascorbic acid with shaking for 10 min, washed three times with 1 M
NaNO3-0.01 M EDTA, and finally washed five times with
Milli-Q water. Unless indicated otherwise, prior to the ascorbic acid
treatment spores were treated with 2% glutaraldehyde for 2 h at
37°C (29, 30) to prevent spore germination that would have
resulted in changes in resistance and could have caused trace metal
release. The glutaraldehyde-treated spores still possessed the ability
to oxidize Mn(II), as observed in previous experiments (29).
After homogenization, spores were stored in Milli-Q water at 4°C;
spores in the suspensions usually formed aggregates. The number of
spores in a suspension was determined by direct counting with a
Petroff-Hausser counting chamber (Arthur H. Thomas Co., Philadelphia,
Pa.) after passage through a French pressure cell at 8,000 lb/in2 to disperse the clumps. A total of 44 small squares
were counted. The average number of spores in each small square was
23.4, and the standard deviation was 6.8.
Surface area. (i) BET method.
The purified spore suspension
was freeze-dried, and the dried spores were stored in a desiccator
until they were used. The specific spore surface area was measured with
a model NOVA-1000 BET surface area analyzer. The dried spores were
first degassed by evacuation for 2 h at 80°C, and the surface
area was then determined by using five datum points.
(ii) Dimension measurement.
Spore surface area was
calculated from spore dimensions obtained by scanning electron
microscopy. The lengths and widths of 30 individual spores were
measured, and the surface area (Sa) was
calculated with the following equation by assuming that each spore is a
perfect prolate ellipsoid of revolution (5):
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(1)
|
where a and b are the semimajor and
semiminor axes, respectively. The volume of a spore (V) was
obtained by using the following equation:
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(2)
|
(iii) Methylene blue adsorption method.
The methylene blue
adsorption technique has been used to successfully measure the surface
areas of kaolinite, illite, and montmorillonite (25).
Methylene blue has the molecular structure shown in Fig.
1. This molecule can be regarded as an
approximately rectangular box with dimensions of 1.7 by 0.76 by 0.325 nm (25), and the projected areas of the molecule surfaces
are 1.3, 0.55, and 0.25 nm2. The methylene blue used in
this study (Aldrich Chemical Co., Inc., Milwaukee, Wis.) has a
molecular weight of 373.9, which corresponds to the molecular weight of
the methylene blue hydrochloride with three H2O molecules.
The methylene blue adsorption experiments were conducted by using
concentrations of <7 µM since methylene blue molecules form dimers
when the methylene blue concentration exceeds 7 µM (4).
A spore suspension containing 107 spores per ml was
prepared and adjusted to pH 8.5. Aliquots (40 ml) were placed into
50-ml centrifuge tubes. A stock solution of methylene blue was added to
give total methylene blue concentrations ranging from 0.5 to 7 µM.
The volumes of methylene blue stock solution added ranged from 0.01 to
0.28 ml. Each suspension was then shaken continuously on a rotary
shaker (100 rpm). The adsorption kinetics of methylene blue indicated
that the adsorption reached equilibrium in 4 h. Each spore
suspension was centrifuged for 10 min at 16,000 × g, and the supernatant was analyzed to determine the amount of methylene blue remaining. From the amount of methylene blue retained in solution,
the quantity adsorbed was determined. To obtain a calibration curve, a
blank experiment without spores was also conducted at the same time by
using the same concentrations of methylene blue that were used for the
spore suspensions. The concentration of methylene blue after adsorption
by spores was measured spectrophotometrically at a wavelength of 661 nm. Solutions containing 0.5 to 7 µM methylene blue gave
A661 values of 0.05 to 0.7.
During adsorption of methylene blue it is assumed that complete
monolayer coverage has occurred when the isotherm reaches a plateau.
The monolayer adsorption value can be obtained by fitting the data to
the Langmuir equation:
|
(3)
|
where 
is the amount of adsorbed methylene blue,
Ceq is the equilibrium concentration of
methylene blue, K is a coefficient related to adsorption
energy (affinity), and m is the monolayer
adsorption capacity. The amount of methylene blue adsorbed as a
monolayer can then be related to the spore surface area (S) (in square micrometers) by the following equation:
|
(4)
|
where m is the monolayer amount of dye
adsorbed (in moles per spore), NA is Avogadro's
number (6.02 × 1023 molecules mol1),
and 
is the area of the methylene blue molecule, which is 0.55 × 1018 m2 when maximum monolayer adsorption
occurs (25).
Surface site density.
Proton exchange was used to measure
the surface site density of SG-1 spores (33). Duplicate
titrations were performed as follows. A 100-ml spore suspension in 0.01 M NaNO3 was placed into a 120-ml glass bottle, and nitrogen
gas was bubbled through the preparation for 30 min to remove
CO2 prior to titration. The suspension was acidified to pH
3.5 with 0.1 M HNO3 (standardized; Aldrich), and the
suspension was then titrated to pH 8.5 with 0.1 M NaOH (standardized;
Aldrich). A preliminary experiment indicated that the spores did not
break down at the pH extremes used in the titrations. After 30 min of
equilibration with N2 purged at pH 8.5, spores were removed
by filtration by using 0.2-µm-pore-size prewashed 47-mm-diameter
Nuclepore polycarbonate membrane filters. The pH of the filtrate did
not change after filtration. The filtrate was titrated back to pH 3.5 with 0.1 M HNO3. The difference between the number of moles
of NaOH (mNaOH) necessary to raise the pH to 8.5 and the number of moles of HNO3
(mHNO3) necessary to restore the pH of the
filtrate to pH 3.5 is an estimate of the number of moles of surface
sites (H+sur):
|
(5)
|
This technique may underestimate the absolute concentration of
surface sites due to charging effects (6).
Surface charge.
The surface charge was measured as a
function of pH by acid-base titration (47). A 100-ml spore
suspension in 0.01 M NaNO3 (108 spores
ml1) was used, and nitrogen gas (>99.95% pure) was
bubbled through the preparation for 30 min to remove CO2
prior to titrations. Standardized 0.1 M NaOH was added to the spore
suspension to bring the pH up to ~8.5, and the suspension was
equilibrated for an additional 20 min. The suspension was then titrated
with standardized 0.01, 0.1, and 1.0 M HNO3 solutions. The
points in the titration curve were determined after the pH stabilized
(usually after 2 to 4 min). Titrations were duplicated. In this
one-sample titration, subtraction of the amount of base added
(nB) (in moles) from the amount of acid added
(nA) (in moles) yielded the net amount
(
n) of base or acid added (in moles):
|
(6)
|
When n is negative, it is the net amount of base
added. When n is zero, the amount of base added equals
the amount of acid added and there is no net addition of acid or base.
When n is positive, it is the net amount of acid added.
An acid-base titration curve was obtained by plotting pH versus net
addition (n) of acid or base (in moles).
The surface charge density (0) (in moles per square
meter) can be calculated from the titration curve by the following
expression:
|
(7)
|
where S is the total spore surface area (in square
meters) and nH and nOH
are the numbers of moles of H+ and OH in the
suspension at a measured pH, respectively. A detailed derivation of
equation 7 has been given by Schulthess and Sparks (31). The
values of nH and nOH are
given by the following equations:
|
(8)
|
|
(9)
|
where V is the volume of the suspension and 
is
the activity coefficient that may be calculated from the Davies
equation:
|
(10)
|
where Z is ionic valence and I is ionic
strength given by:
|
(11)
|
where Ci is the concentration of the
ith ion.
Cu(II) adsorption kinetics.
A 100-ml spore suspension in
0.01 M NaNO3 was placed into a 125-ml flask. The suspension
was stirred with a Teflon-coated magnetic stirrer. A Cu(II) stock
solution [Cu(NO3)2] was added to the
suspension to start the experiment. Aliquots (2 ml) were removed at
various times by using 5-ml syringes with needles. Each sample was
filtered through a 0.45-µm-pore-size Gelman type GHP acrodisc filter,
and the filtrate was collected in 3-ml Wheaton polypropylene vials,
stored in a 4°C cold room, and analyzed within 2 days. A control
(blank) experiment indicated that filtration did not affect the Cu
concentration in the filtrate.
Cu(II) adsorption isotherm.
A plot of the concentration (in
moles per liter) of adsorbate in the supernatant solution (or filtrate)
versus the amount adsorbed (in moles per gram or moles per square
meter) by a solid at a fixed temperature and applied pressure is an
adsorption isotherm. If the adsorption of a metal follows the Langmuir
equation (equation 3), the adsorption capacity and affinity can be
calculated. The Cu(II) adsorption isotherm for SG-1 spores was obtained
by the following procedures. A spore suspension in 0.01 M
NaNO3 was adjusted to the desired pH, and 20-ml aliquots
were placed in 50-ml centrifuge tubes. A copper stock solution was then
added to the suspensions so that the Cu(II) concentrations in the
suspensions varied over 2 orders of magnitude (0.3 to 30 µM). Each
suspension was shaken to reach equilibrium; a shaking time of 2 h
was determined to be appropriate from the adsorption kinetics described
above. The suspension was centrifuged and filtered through
0.45-µm-pore-size acrodisc filters.
Distribution coefficients can be used to describe the efficiency of
removal of trace elements from solutions by solids. The distribution
coefficient (Kd) for Cu(II) sorbed by SG-1
spores may be calculated by the following equation:
|
(12)
|
where is the amount of Cu(II) sorbed (in millimoles per
gram) and Ceq is the equilibrium concentration
of Cu(II) in solution (in moles per liter). Thus,
Kd has units of milliliters per gram. At low
concentrations, Kd is related to K (a
measure of affinity) and may be expressed as follows:
|
(13)
|
where m and K can be
obtained from equation 3.
Cu(II) adsorption as a function of pH.
Copper(II) adsorption
experiments were carried out in batch systems to determine adsorption
edges [percentages of Cu(II) adsorbed as a function of solution pH]
(13, 14). Portions (20-ml) of a spore suspension in 0.01 M
NaNO3 were placed in 50-ml centrifuge tubes, and an equal
amount of Cu(II) stock solution was added to each tube. After the
Cu(II) solution was added, the suspensions were adjusted to the desired
pH values by using 0.1 M HNO3 or 0.1 M NaOH (the total
volume was changed by less than 2%). The changes in ionic strength due
to acid or base additions were less than 0.003 M. The treated
suspensions were shaken for 2 h at 150 rpm. The pH of each
suspension was determined with a pH meter and a Ross combination
electrode. The samples were centrifuged, the supernatants were
filtered, and the filtrates were analyzed for Cu as described below.
The exact volume of solution in each tube was calculated from the
volume of the suspension, the volume of Cu(II) stock solution added,
the volume of acid or base added, and the volume of spores in
suspension.
Dissolved Cu analysis.
Dissolved Cu concentrations were
measured by graphite furnace atomic absorption spectrophotometry
performed with a Perkin-Elmer model Zeeman 5000 spectrophotometer
equipped with a model HGA 400 programmable graphite furnace and a model
AS 40 autosampler. Standard metal solutions were prepared from SPEX
plasma standards and contained 0.01 M NaNO3. The external
standard calibration method was used in the measurements. Calibration
curves were obtained by using three standards that covered the
concentrations of the samples. Three absorbance values were averaged to
obtain values for standards. The standard calibration was repeated
every 10 samples. The Zeeman effect background correction was used to
significantly improve the signal-to-noise ratio. Samples were analyzed
with pyrolytically coated graphite tubes equipped with L'vov
platforms. The graphite furnace system was set up with a sample volume
of 20 µl and six steps consisting of time-temperature sequences. All
of the samples (in polypropylene autosampler cups) were analyzed in
duplicate.
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RESULTS |
Spore surface area.
The specific surface area of freeze-dried
SG-1 spores determined with a BET surface area analyzer was 6.86 m2 g1, which is similar to the surface area
(7 m2 g1) obtained for Bacillus
subtilis spores (23) and slightly higher than the value
(5.04 m2 g) reported by Berlin et al.
(5), who used the same technique.
The individual spore sizes, as determined by scanning electron
microscopy, were 1.26 ± 0.13 by 0.60 ± 0.09 µm. The spore
surface area calculated from the spore dimensions was 2.02 µm2 per spore, and the spore volume was 0.237 µm3 per spore. The ratio of surface area to spore volume
was 8.5. The number of spores per gram of dried spores was calculated
by using a previously published value for average spore density of 1.36 g cm3 (5). The value obtained was
3.103 × 1012 spores g1. From the values
presented above, the specific surface area of SG-1 spores was
calculated to be 6.27 m2 g1, which is
comparable to the value determined with the BET surface area analyzer.
A similar value (6 m2 g1) was also calculated
for Bacillus spores by Neihof et al. (23). Since
the specific surface area was small, we postulated that there is no
external porosity and the spore surface is smooth (5).
Figure 2 shows a plot of the amounts of
methylene blue adsorbed by SG-1 spores versus the equilibrium
concentrations of methylene blue. The surface area of spores was
calculated from the monolayer adsorption value that was obtained by
fitting the data to the Langmuir equation (equation 3). The methylene
blue monolayer adsorption capacity was estimated to be 7.27 nmol per
108 spores (Fig. 2). As described previously, when the
maximum adsorption of methylene blue by clay minerals is reached, the
surface area of a methylene blue molecule is 0.55 nm2
(25). If it is assumed that this value is applicable to the spore-water system, the spore surface area is 24.1 µm2
per spore, which corresponds to a specific surface area of 74.7 m2 per g of dry weight and a ratio of spore surface area to
spore volume of ~100. Because we believe that the wet spore surface area measured by methylene blue adsorption is the best estimate of the
true spore surface area (see below), this value was used for all
calculations of site density, charge density, and adsorption capacity
presented in this paper.
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FIG. 2.
Methylene blue adsorption isotherm obtained with SG-1
spores (107 spores ml1, pH 8.5). The curve
was obtained by fitting the data to the Langmuir equation. MB,
methylene blue.
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Spore surface charge.
The surface exchange capacity of SG-1
spores as determined by the proton exchange technique was 30.6 µmol
m2 (2.29 mmol per g of dry spores), which is equivalent
to 18.3 sites nm2. The acid-base titration data for the
spore suspension in 0.01 M NaNO3 are presented in Fig.
3. The surface charge density
(0) as calculated by equation 5 is shown in Fig.
4. The zero point of charge (ZPC) is pH
4.5, which is identical to the ZPC of the cell wall of a gram-positive
bacterium (28). Nearly all Fe and Al (hydr)oxides have ZPC
values of pH >8, while the ZPC of birnessite (
-MnO2) is
approximately pH 2.2 and the ZPC of kaolinite is pH 4.6 (47). Powder materials with low ZPCs should exhibit high capacities for binding metals at low pH values.
At pH >4.5, negatively charged sites predominate (0 is
negative), and the maximum 0 is 27.3 µmol
m2 (2.04 mmol g1) at pH 8.59, which is
comparable to the surface exchange capacity (30.6 µmol
m2 at pH 8.50) determined by the proton exchange method.
At pH <4.5, the net surface charge is positive, but the positive
charge density at pH 2.5, 5 µmol m2 (0.37 mmol
g1), is much lower than the negative charge density,
indicating that fewer positively charged sites exist on the spore
surface. Plette et al. (28) reported that the
0 for a soil bacterium changed from +0.25 to 
0.75 mmol
per g of cell walls in the pH range from 3 to 10.
Mineral ions may leach from spores during acid-base titration
processes, particularly at low pH. Mineral leaching should not have
interfered with the results of surface site density determinations since we used the back-titration method, which takes into account all
ions, such as leached metals that consume H+ ions
(31). On the other hand, since back-titration was not used
for surface charge determinations, mineral leaching may affect the
surface charge results via metal leaching at low pH or through precipitation and hydrolysis of the leached metals at high pH. However,
because the concentrations of metal leached over the pH range used in
the experiments were low, the released mineral ions should not have
significantly influenced the surface charge results reported here.
Cu(II) adsorption by spores.
The Cu(II) adsorption reaction
with spores was rapid, and more than 60% of the Cu(II) was sequestered
from the solution at pH 7.2 within the first 1 min (Fig.
5). In many biosorption systems, most of
the metal sorption occurs within 5 to 15 min after solid-liquid contact
(44). The Cu(II) adsorption curve for glutaraldehyde-treated spores leveled off within 10 min, whereas Cu(II) adsorption by the
untreated spores reached its highest value in minutes and then
decreased gradually with time. The difference in Cu(II) adsorption kinetics between the treated and untreated spores may be attributed to
spore germination because it was found that the concentrations of
dissolved Mn and Zn increased with time in the untreated spore suspensions but not the glutaraldehyde-fixed spore suspensions (data
not shown). Although release of Mn and Zn is not the best indicator of
germination, there was greater release of these metals and lower
adsorption of Cu(II) by the spores at room temperature than by the
spores at 4°C (Fig. 5). Based on these results, glutaraldehyde-fixed spores and a 2-h equilibrium time were used for the subsequent experiments in this study.
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FIG. 5.
Kinetics of adsorption of Cu(II) by SG-1 spores that
were treated with 2% glutaraldehyde or not treated at 4 and 25°C
[108 spores ml1, 2 mM Cu(II), 0.01 M
NaNO3, pH 7.2].
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The isotherm of Cu(II) adsorption by spores was characterized by a
large increase in the amount of adsorbed Cu(II) with increasing Cu(II)
concentration at low equilibrium concentrations and leveling off or
saturation at higher concentrations (Fig.
6). This L-type curve indicates that the
spores had a high affinity for Cu(II). The Cu(II) adsorption affinity
coefficient (K) and adsorption capacity
(m) of the spores were obtained by fitting the experimental data to the Langmuir equation (equation 3). The K value was found to be 2.08 × 106 liters
mol1. This affinity value is 2 to 4 orders of magnitude
higher than the affinities of Cu(II) determined for a variety of
biomasses, such as fungi, bacteria, and marine algae (20),
or for an alginate gel (17). The Cu(II) adsorption capacity
at pH 7.0 was 10.77 µmol m2, equivalent to 0.829 mmol
per g of dried spores, which is the same order of magnitude as the
Cu(II) adsorption capacities of various biomasses, such as bacteria,
algae, fungi (20). Pirszel et al. (26) reported
that the ion-exchange capacities were 0.825 mmol g of dry
weight1 for cells for Anacystis nidulans (a
cyanobacterium), 0.205 mmol g for Synechocystis
aquatilis (a cyanobacterium), 0.260 mmol g1 for
Stichococcus bacillaris (a green alga), and 0.041 mmol
g1 for Vaucheria sp. (a macroalga). The
capacity at pH 7.0 is one-half of the charge density of SG-1 spores
(Fig. 5). This might indicate that Cu(II) is adsorbed on the spore
surface by the bidentate mechanism; i.e., one Cu(II) ion shares two
surface sites on SG-1 spores. It is also possible, however, that Cu(II)
binds to the spore surface in nonbidentate modes because Cu(II)-binding
sites might differ from sites to which H+ ions bind, as
described for Pb(II) sorption onto Al and Fe oxides by Bargar et al.
(2, 3).
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FIG. 6.
Cu(II) adsorption isotherm obtained with SG-1 spores
(108 spores ml1, 0.01 M NaNO3, pH
7.0). The curve was obtained by fitting the data to the Langmuir
equation.
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The percentage of Cu(II) adsorption was lower at lower pH values and
increased as the pH increased (Fig. 7).
Copper adsorption was zero around pH 3 and rapidly increased to
~100% at pH 6. This adsorption behavior is similar to metal
adsorption by microbial biomasses (44) and metal oxides
(35).
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FIG. 7.
Cu(II) adsorption by SG-1 spores as a function of pH
[108 spores ml1, 0.01 M NaNO3, 2 µM Cu(II)]. The adsorption value for 100% adsorption is 1.5 µmol
m2.
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Using the Cu(II) adsorption isotherm data obtained at pH 7 (Fig. 6) and
equation 12, we obtained Kd values ranging from
1.4 × 105 to 1.3 × 106 ml
g1 (average, 7.3 × 105 ml
g1). These values are 2 to 4 orders of magnitude higher
than those obtained for most inorganic materials. It is important to
note that Kd values vary with pH. From the
Cu(II) sorption data obtained at different pH values (Fig. 7) we
calculated that the Kd values were between
3.1 × 104 ml g1 at pH 4.16 and 1.0 × 107 ml g1 at pH 8.38 and increased with
solution pH. The Kd value calculated by equation
13 by using the m and K values
obtained at pH 7 (Fig. 6) was 1.6 × 106 ml
g1, which was in reasonable agreement with the values
calculated by equation 12.
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DISCUSSION |
Spore surface area.
The surface area of a solid is more
important than the mass for understanding and interpreting the ion
sorption properties of the solid. There are two categories of methods:
(i) physical determination of the size and morphology of solid
particles and (ii) measurement of the adsorption of gas or solute
molecules having known dimensions and interpretation of the resulting
data with a particular adsorption model. Both of these types of methods were used to measure the specific surface area of SG-1 spores in this
study.
It is relatively easy to obtain the geometric surface area of spores
because the number of spores in a suspension can be counted easily and
spores have nearly uniform size and shape. It should be noted that
geometric surface area is calculated with the assumption that spores
are nonporous. N2 gas adsorption (the BET method) is the
most popular technique for measuring the specific surface areas of
powder samples. The BET method has some serious limitations, however.
For example, the structure and surface characteristics of solids,
particularly biological materials, can change significantly in the
drying step required in the BET method. For SG-1 spores, the surface
area determined by the BET method was low and very similar to the
geometric surface area. This similarity indicates that either the
spores were smooth (nonporous) or the drying step altered the surface
structure of the spores.
To test this hypothesis, a dye adsorption method, in which the drying
step was avoided, was used to measure the surface area of SG-1 spores.
Dyes with well-characterized dimensions have been used by many
investigators to measure the specific surface areas of porous oxide
materials (43), clay minerals (25), activated carbon (24), and sludge (1, 34). The spore
surface area determined by the methylene blue adsorption method was
11-fold higher than spore surface areas determined by the BET technique and the spore dimension measurement technique. A similar specific surface area (74 m2 g1) for spores was
reported by Neihof et al. (23), who used a solvent
replacement procedure. These authors washed spores six times with
absolute ethanol and then four times with dry pentane and measured the
surface area of the treated spores with the BET technique. Neihof et
al. (23) indicated that although the water in spores was
removed by the solvent replacement procedure, considerable porosity was
retained. This was verified by the fact that the surface area decreased
after the solvent-replaced spores were rewetted and dried.
In this study, the dye methylene blue was used to measure the specific
surface area of SG-1 spores. The dimensions of methylene blue have been
quantified very well, and the orientation of adsorbed methylene blue
molecules has been described well (25). The large surface
area determined by the methylene blue adsorption method suggests that
in the water-wet state, a spore is swollen and there is a water-filled
porous structure. When the spore is dried in air or even freeze-dried,
this porous structure collapses, resulting in a much smaller surface
area. Even molecules as small as nitrogen are unable to get in. We
believe that measurements obtained by using methylene blue better
reflect the real surface area of the spore material because drying is
not required.
Dyes other than methylene blue have also been used to measure the
surface areas of powder materials. Andreadakis (1) found that the specific surface area of an activated sludge determined by
adsorption of lissamine scarlet 4R was 1 or 2 orders of magnitude greater than the geometric surface area of the sludge. Depending on the
assumptions concerning the amount of absorbed dye and the orientation
of the molecules used, the specific surface area varied between 60 and
189 m2 g1, suggesting that the activated
sludge was porous (1). Sorensen and Wakeman (34)
believed that the surface area of activated sludge measured by
rhodamine B adsorption was a realistic measure of the solid-liquid
interfacial area.
Spore surface charge.
Surface charge and site density may be
experimentally measured by acid-base titration (H+
exchange), tritium (3H2O) exchange, or other
ion (e.g., Ca, Mg, F, and PO43)
adsorption techniques. Site density estimates obtained with different
methods typically differ by factors of 2 to 3 and sometimes more
(9). Acid-base titration is widely used for measuring the
surface charge of powder materials and bacterial cells (28, 31) and is the simplest technique since only the pH must be measured. The surface site density measured by acid-base titration depends to some extent on ionic strength. A low ionic strength results
in underestimates of surface site density because the surface sites are
not totally ionized. In this study, we used an ionic strength of 0.01 M, which is between the ionic strengths of freshwater and seawater.
The surface site density of SG-1 spores (18.3 sites nm2)
is on the same order of magnitude as the surface site densities of most
mineral colloids, such as Fe, Mn, and Al (hydr)oxides (2 to 20 sites
nm2) (6, 36). Theoretically, the maximum
surface site density for oxides is 39 µmol m2 (23.3 sites nm2) if the radius of H+ is 0.145 nm
and surface groups are located on two layers (36). The site
density of SG-1 spores (2.29 mmol g of spores1) is
similar to the previously reported values for cell walls of other
bacteria and algae, which range between ~1 and 2.5 mmol g of cell
wall1 (28).
While spores clearly differ from vegetative cells in structure and heat
and chemical resistance, our results suggest that similar surface
functional groups (i.e., carboxylate, phosphate, and amino groups) are
present. The carboxylate and phosphate groups carry negative charges
that allow the cells to be potent scavengers of cations. In
gram-positive bacteria, the carboxylate and phosphate groups are
primarily associated with the peptidoglycan; the peptidoglycan backbone
is rich in carboxylate groups, and the associated teichoic acids are
rich in phosphate groups (32). In gram-negative bacteria, the carboxylate and phosphate groups are primarily in the outer membrane lipopolysaccharide (32). In both types of bacteria, amino groups are associated with the peptidoglycan or other proteins on
the cell surface.
In SG-1 spores, the surface charge is primarily associated with the
outermost layer of the spore, the exosporium, which in Bacillus
cereus is composed mainly of protein, polysaccharide, and lipids
(21). The surface charge density (absolute value) (strictly
speaking, it should be called the net surface charge density) increases
linearly from pH 4.5 to 7.5 and seems to level off at pH values greater
than 8 (Fig. 5). This leveling off at higher pH values suggests that
the spore surface carries phosphate groups which have pK2
(the second dissociation constant of phosphoric acid) values of 7 to 8 (16). The negative charge between pH 4.5 and 6 could develop
from carboxylate groups that have pK values of 4 to 5 (16,
27). The net positive charge at pH values of less than 4.5 is
probably due to amino groups on the SG-1 spore surface. Our results are
consistent with previous reports. The electrophoretic mobility of
spores of three Bacillus species suggested that in these
spores carboxylate groups are the principal surface charge groups and
there are some phosphate and amino functional groups (8). It
should be noted that macroscopic analyses based on titration curves are
indirect methods for examining surface functional groups. Direct
spectroscopic techniques, such as Fourier transformed infrared
spectroscopy and X-ray adsorption fine-structure spectroscopy, should
be employed to identify the surface functional groups on SG-1 spores.
Results obtained by using Fourier transformed infrared spectroscopy
suggest that carboxylate is the most important surface functional group
for a variety of biomasses, including algae, diatoms, fungi, seaweed,
and terrestrial plants (11, 12, 18). Other functional
groups, such as amino and sulfonate groups, have also been identified.
The mechanism of surface charge development for SG-1 spores and other
biomasses (i.e., protonation or deprotonation) is similar to the
mechanism of surface charge development for metal (hydr)oxides, but the
surface groups are different. Metal (hydr)oxides have amphoteric
surfaces that may be charged negatively or positively at the same site,
depending on the pH, whereas SG-1 spores are zwitterionic surfaces,
like latexes, which carry carboxylate, sulfonate, and amino surface
groups (15). The ZPC of SG-1 spores determined in this study
is pH 4.5. This means that at pH 4.5 the net surface charge is zero and
the positive charge of the spore surface equals the negative charge.
When the pH is greater than the ZPC, there is a net negative charge,
which largely results from deprotonation of the carboxylate and
phosphate groups. When the pH is less than the ZPC, protonation of all
three major groups (carboxylate, phosphate, and amino groups) results
in a net positive charge.
Cu(II) adsorption by spores.
The adsorption of Cu(II) by SG-1
spores was extremely rapid. Because of the fast kinetics of the Cu(II)
sorption reaction, it is believed that the overall reaction rate is
controlled by mass transfer of Cu(II) ions to the reactive sites of
SG-1 spores. The Cu(II) adsorption kinetics are different in spores
treated with glutaraldehyde and spores not treated with glutaraldehyde. We believe that this difference is due to spore activation and germination in experiments performed with untreated spores because, consistent with germination, the untreated spores released Mn and Zn,
whereas the fixed spores did not. Although glutaraldehyde treatment
might alter the surface properties of SG-1 spores by reducing the
numbers of charged groups on the surface, this alteration seems small
since the initial (
4-h) Cu(II) adsorption capacity was nearly the
same in the treated and untreated spores (Fig. 5).
The L-curve isotherm, into which Cu(II) adsorption by SG-1 spores
falls, is characterized by an initial slope that does not increase with
the concentration of adsorbate in solution. This type of isotherm is
the result of a high relative affinity of the solid particles for the
adsorbate at low surface coverage coupled with a decreasing amount of
adsorbing surface remaining as the surface excess of adsorbate
increases. By fitting experimental data to the Langmuir equation we
obtained the values for two parameters, K and
m. The parameter K is related to
adsorption energy and determines the magnitude of the initial slope of
the isotherm. The magnitude of the slope indicates the affinity of an
adsorbate for an adsorbent. The Cu(II) adsorption by SG-1 spores in
this study (Fig. 7) showed a relatively high affinity since at low Cu(II) concentrations Cu(II) was almost totally adsorbed by SG-1 spores, as demonstrated by a high K value (2.08 × 106 liters mol1).
The K value obtained from the Langmuir equation is the
inverse of the Km value determined from the
Lineweaver-Burk or Eadie-Hofstee plot that was described by Lee and
Tebo (19) for Co(II) oxidation by SG-1 spores. The
Km values for Co(II) oxidation obtained from the
Eadie-Hofstee plot range from 3.3 × 108 to 5.2 × 106 mol liter1, corresponding to a range
of K values between 1.92 × 105 and
3.03 × 107 liters mol1. Therefore, the
K value obtained in this study for Cu(II) adsorption by SG-1
spores falls in the range of the K values obtained for Co(II) oxidation (19), indicating that SG-1 spores have
similarly high affinities for both Co(II) and Cu(II).
SG-1 spores have proved to be an excellent model system for studying
oxidative precipitation reactions, such as Mn(II) and Co(II) oxidation
(19, 29, 41). Although the biochemical mechanism of metal
oxidation has not been fully elucidated, copper has been shown to be
important, both from genetic investigations that have implicated a
multicopper oxidase-like protein in Mn(II) oxidation and because Cu(II)
stimulates Mn(II) oxidation (45). While the results
presented here are not conclusive proof, the high affinity and binding
capacity for Cu(II) may be related to the use of Cu(II) as a cofactor
for Mn(II) oxidation.
SG-1 spores are an attractive system for possible use in metal removal
and recovery applications (38). They actively oxidize Mn(II)
and Co(II) over a wide range of environmental conditions and are
naturally resistant to a variety of physical and chemical stresses
which may be encountered in a waste stream or polluted environment, and
their growth does not have to be sustained. SG-1 is capable of binding
or oxidizing metals both directly on the spore surface. This study
shows that SG-1 spores have both a high affinity and a high capacity
for Cu(II). Thus, SG-1 spores have a metal removal capability that
exploits both active metal precipitation and passive adsorption
processes, and the spores may be well-suited for solving problems where
mixed metals occur.
This research was funded in part by grant MCB94-07776 from The
National Science Foundation, by grant NA36RG0537 from the National Sea
Grant College Program (National Oceanic and Atmospheric Administration, U.S. Department of Commerce; project R/CZ-123 of the California Sea
Grant College), and by a grant from the California State Resources Agency. L.M.H. received partial support as a postdoctoral trainee from
the University of California Toxic Substances Research and Teaching
Program.
We appreciate two elaborate anonymous reviews and most useful
criticisms. We thank John Bargar at Stanford University and Margo
Haygood and Karen Casciotti at the Scripps Institution of Oceanography
for critically reviewing an early draft of the manuscript. Laboratory
assistance rendered by Deeanne Edwards, Karen Casciotti, Ron Caspi,
Chris Francis, Larry Knight, and Donna Givens is greatly appreciated.
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Abstract
Introduction
