Applied and Environmental Microbiology, November 1998, p. 4610-4613, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Metal Toxicity Reduction in Naphthalene
Biodegradation by Use of Metal-Chelating Adsorbents
Pomthong
Malakul,
Keeran R.
Srinivasan, and
Henry Y.
Wang*
Department of Chemical Engineering,
University of Michigan, Ann Arbor, Michigan 48109-2136
Received 30 April 1998/Accepted 19 August 1998
 |
ABSTRACT |
A model system comprising microbial degradation of naphthalene in
the presence of cadmium has been developed to evaluate metal toxicity
associated with polyaromatic hydrocarbon biodegradation and its
reduction by the use of unmodified and surfactant-modified clays in
comparison with a commercially available chelating resin (Chelex 100;
Bio-Rad). The toxicity of cadmium associated with naphthalene
biodegradation was shown to be reduced significantly by using the
modified-clay complex and Chelex resin, while unmodified clay has no
significant impact on this reduction. The degree of metal toxicity
reduction can be quantitatively related to the metal adsorption
characteristics of these adsorbents, such as adsorption capacity and selectivity.
| |
TEXT |
In recent years, the concern about
the presence, persistence, and disposition of polyaromatic hydrocarbons
(PAHs) in the environment (air, soil, and water systems) has increased
since this important class of chemicals has been shown to be
carcinogenic in experimental animals and thus pose a potential human
health risk (4, 5). Microbial degradation has been proposed
as an inexpensive and efficient method to remove PAHs from the
environment. However, heavy metals often occur as cocontaminants and
reportedly have adverse effects on biodegradation. These effects
include extended acclimation periods, reduced biodegradation rates, and
failure of target compound biodegradation (16, 22). A number
of research efforts have been directed toward overcoming this metal
toxicity problem. Some studies used metal-resistant strains which can
tolerate high metal concentrations (12-14, 24). Others
attempted to reduce metal toxicity by using a natural clay, such as
montmorillonite or kaolinite (2, 3, 15). However, either
metal toxicity was reduced insignificantly or large amounts of
adsorbents were needed due to a poor selectivity of the clay toward
target metal ions. We have been preparing several surfactant-modified
clay complexes through a simple surface modification method of grafting metal-chelating ligands in order to impart a higher metal capturing capacity and selectivity to the base clays (19). The
resulting clay complexes have been shown to have high metal adsorption
capacities and high affinities for heavy metals, such as cadmium and
copper. In this study, we used a model system comprising microbial
degradation of naphthalene in the presence of cadmium to evaluate metal
toxicity associated with PAH biodegradation and its reduction by the
use of natural and modified-clay complexes in comparison with Chelex 100 resin.
Bacterial strain and culture media.
Pseudomonas putida
ppo200 carrying a naphthalene-degrading plasmid (NAH) was kindly
provided by Ronald Olsen (University of Michigan Medical School). This
strain is capable of growing on naphthalene as the sole source of
carbon and energy. The strain was grown and maintained on tryptone
nutrient agar (7, 23). It was then stored at 4°C until
required. All liquid cultures were carried out with mineral medium
(MMO) (23), with a minor modification; the medium was
buffered with 50 mM Tris-HCl (Trizma; Sigma), pH 7, instead of a
phosphate buffer to avoid precipitation of insoluble metal phosphates.
Phosphorus was provided in the form of sodium 
-glycerophosphate (3 mM) (9, 12). Naphthalene was added to the medium, using 1 ml
of a concentrated stock solution in the solvent
N,N-dimethylformamide (DMF) to achieve the desired concentration. It was reported that this solvent, at the concentration used, had no effect on substrate oxidation (10).
Naphthalene biodegradation studies.
All biodegradation
experiments were carried out in batch mode, using 50 ml of MMO medium
per 250-ml Erlenmeyer flask. A stock culture stored at 4°C was
regrown on a tryptone nutrient agar plate and maintained at 30°C for
24 h. The strain was then transferred to an MMO agar plate with
naphthalene supplied in vapor form from crystals in the lid of the
plate. The plate was incubated at 30°C for 48 h. A microbial
inoculum was prepared by the transfer of one full loop of growth from
the MMO agar plate to MMO liquid medium containing 1 g of
naphthalene per liter. Cells were allowed to grow overnight to an
optical density at 600 nm of 1. A 1-ml portion of the culture was then
used as an inoculum for each of the degradation study flasks. The
control culture was prepared by adding 1 ml of the solvent DMF alone
(without naphthalene). The cell suspensions were incubated on a rotary
shaker (250 rpm) at 30°C for 24 h. Growth of the bacteria was
monitored turbidimetrically at 600 nm.
Figure 1 shows the effect of various
naphthalene concentrations (0.1, 0.2, 0.5, 1.0, and 1.5 g/liter) on the
growth of P. putida. No growth was observed in the control
culture (with DMF alone), suggesting that the solvent DMF cannot serve
as a carbon or energy source for P. putida ppo200(NAH). It
can be seen that the growth of P. putida increases with
increasing naphthalene concentration, indicating that naphthalene is
the only carbon and energy source in the mineral medium and that the
bacterial growth is purely a result of naphthalene degradation. In
addition, the growth varies linearly with the naphthalene concentration in the observed range (0.1 to 1.5 g/liter). These results suggest that
microbial growth can be used as an indicator of naphthalene biodegradation as well as a means of quantifying the heavy metal (cadmium) toxicity associated with the biodegradation.
Effect of cadmium on microbial growth of P. putida
ppo200(NAH).
To assess quantitatively the effect of a heavy metal
on a P. putida culture, similar experiments were carried out
in MMO medium containing 1 g of naphthalene per liter and with
various concentrations of cadmium (Cd), ranging from 5 to 200 ppm. The
cell suspensions were incubated as described above. A control culture
was grown under the same conditions but in the absence of cadmium. Test cultures and the control culture were prepared in triplicate. Cadmium
was added to the medium from concentrated stock solutions of
CdCl2 which were prepared with Milli-Q water (Millipore,
Bedford, Mass.). The stock solutions were sterilized by passing them
through 0.2-µm-pore-size membrane filters (Gelman, Ann Arbor, Mich.). Cadmium concentrations were analyzed by using an atomic absorption spectrometer (model 3100; Perkin-Elmer).
Since we observed bacterial growth over a wide range of Cd
concentrations, a relatively high naphthalene concentration (1 g/liter)
was used in this study to obtain an appreciable growth level which
could be measured accurately even when high Cd concentrations were
used. Figure 2 shows the growth of
P. putida in liquid MMO medium containing various cadmium
concentrations. Growth was expressed as a percentage of the final cell
growth in the control culture (without cadmium). It can be seen that a
Cd concentration of less than 10 ppm (0.09 mM) has no effect on the
growth of P. putida. Inhibition of growth was first detected
at 10 ppm, and a significant decrease in growth was observed at a Cd
concentration of 80 ppm (0.71 mM). A reduction in final growth by 50%
was observed at a Cd concentration of 100 ppm (0.89 mM). Complete
inhibition of bacterial growth was observed at a Cd concentration of
170 ppm (1.51 mM). Thus, cadmium toxicity associated with naphthalene degradation can easily be divided into three distinct regions (Fig. 2)
in terms of the cadmium concentration: (a) no inhibition (line a; less
than 10 ppm), (b) partial inhibition of growth (line b; 10 to <170
ppm), and (c) complete inhibition (
170 ppm).
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FIG. 2.
Effect of cadmium on growth of P. putida
ppo200(NAH). (a) No inhibition; (b) partial inhibition; (c) complete
inhibition.
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|
In previous studies of toxicity of cadmium using various organisms,
growth inhibition was first noted at Cd concentrations between 0.1 to
10 ppm while complete inhibition was observed at Cd concentrations
between 100 to 500 ppm (1, 8, 15, 17, 18). Higham et al.
(12, 13), when studying cadmium transport in P. putida, reported that uptake of Cd during the lag phase was
critical for cell survival after inoculation. They found that Cd was
taken up in two different phases. An initial, rapid, linear influx
during the first 2 to 3 min was followed by a slower, second phase. The
initial phase exhibited Michaelis-Menten kinetics, suggesting uptake
via saturable Cd-binding sites, presumably on the cell membrane. In the
second phase, little uptake of Cd occurred at low Cd concentrations,
but upon reaching a threshold level of 0.75 mM (approximately 84 ppm),
uptake increased with increasing Cd concentration. These findings are
in good agreement with our results in the present study (Fig. 2). A
significant reduction in growth was observed at Cd concentrations
higher than 80 ppm, which was the threshold level reported by Higham et
al. The complete-inhibition concentration (170 ppm) observed here
corresponded to their finding that the cadmium uptake rate in the first
phase reached its maximum.
Metal toxicity reduction via metal-chelating adsorbents.
In
our laboratory, various modified-clay adsorbents have been designed and
constructed to remove and concentrate heavy metals in various liquid
and solid media. The preparation and metal adsorption characteristics
of several modified-clay complexes have been previously described
(19). The modified-clay complex was prepared by a simple
two-step method involving adsorption of a cationic surfactant, such as
cetyl benzyl dimethyl ammonium (CBDA), and then anchoring of various
metal-complexing ligands, such as palmitic acid (PA), through
hydrophobic interactions to form a stable mixed bilayer of CBDA and PA
on the clay surface. In this study, the modified-clay complex
montmorillonite-CBDA-PA was used with the hope of reducing the toxicity
of cadmium to P. putida ppo200(NAH). Unmodified clay (cleaned montmorillonite) and a commercial chelating resin, Chelex 100 (50/100 mesh, sodium form; Bio-Rad, Hercules, Calif.), were used for
comparison purposes. Chelex 100 is a chelating ion-exchange resin which
has been used to selectively adsorb divalent metal ions such as
Pb2+ and Cd2+ (6, 21). These metal
adsorbents (0.5 g) were added to the flasks containing MMO medium which
had previously been amended with different cadmium concentrations (5 to
500 ppm). Following the addition of the adsorbents, the flasks were
inoculated and incubated as described above.
The effects of equal amounts of these metal adsorbents on the toxicity
of cadmium to P. putida ppo200(NAH) are shown in Fig. 3. Adsorbent was not added to the control
(Fig. 2). In the presence of the modified-clay complex (line c) or
Chelex resin (line d), the toxicity of cadmium was reduced
significantly, as indicated by growth being normal even at high cadmium
concentrations. The complete-inhibition concentration observed in the
control culture (170 ppm, or 1.51 mM) had no effect on microbial growth
when Chelex was added to the medium; in the presence of the modified
clay, approximately 90% of control growth was observed. Complete
inhibition of bacterial growth was observed at cadmium concentrations
of 400 ppm (3.6 mM) and 490 ppm (4.4 mM) in the presence of the
modified clay and Chelex, respectively. Therefore, addition of these
adsorbents makes it possible to conduct naphthalene biodegradation at a
much higher cadmium concentration in the same medium. It can also be seen from Fig. 3 that the control curve (line a) and unmodified-clay curve (line b) are indistinguishable, indicating that unmodified clay
has no effect on the reduction of cadmium toxicity.
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FIG. 3.
Effect of cadmium on growth of P. putida
ppo200(NAH) in the absence (a) and presence (b to d) of various
adsorbents: unmodified clay (b), modified clay (c), and Chelex 100 (d).
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|
The adsorption characteristics of the adsorbent, such as adsorption
capacity and selectivity, play an important role in the metal toxicity
reduction, as quantitatively shown in the following analysis. The
dose-response curve shown previously (Fig. 2 and 3) can adequately be
described by an exponential function proposed by Duxbury
(9):
|
(1)
|
where Y is the level of growth of the bacterium in
medium containing a heavy metal at millimolar concentration C and
a is the level of growth in the control (in the absence of
metal). The parameter b is a measure of metal toxicity
(inverse millimolar concentration); this value indicates the degrees of
toxicity of different metal species to the microorganisms and can be
used to quantify the effect of the metal-chelating adsorbent on metal toxicity reduction. By taking the natural logarithm (ln) of equation 1, one should get
|
(2)
|
In the presence of a metal adsorbent, equation 2 can be rewritten
as
![<UP>ln </UP>(Y/a)=<UP>−</UP>b[C−(QM/V)]](/content/vol64/issue11/fulltext/4610/img003.gif)
|
(3)
|
where Q and M are the adsorption capacity
(in milligrams per gram) and the amount of metal adsorbent (in grams),
respectively, and V is the volume of the medium (in liters).
By rearranging equation 3, we get
![<UP>ln </UP>(Y/a)=<UP>−</UP>bC[1−(QM/VC)]](/content/vol64/issue11/fulltext/4610/img004.gif)
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(4)
|
or
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(5)
|
where
|
(6)
|
Equation 6 shows that is actually a ratio of the amount of
metal adsorbed by the adsorbent to the total amount of metal ions in
the medium. It can be seen that the term 1 
in equation 5 represents a reduction term for the toxicity value b
attributable to the effect of the metal adsorbent. Therefore, in the
presence of the metal adsorbent, the b value will be
modified to b(1 ), with ranging from 0 to 1. In the absence of the metal-chelating adsorbent (M = 0), equals 0, resulting in b(1 ) being
equal to the original value b. On the other hand, if total
adsorption occurs (QM = VC; thus, = 1),
b(1 ) will be 0, which leads to maximum bacterial
growth (Y = a). This analysis demonstrates that the
reduction in metal toxicity depends strongly on the adsorption capacity
(Q) of the adsorbent, which can be determined from the adsorption isotherm. For high metal concentrations or complex media,
the metal adsorption can be described by the Freundlich isotherm,
|
(7)
|
where Kf and n are constants
which can be related to adsorption capacity and adsorption intensity,
respectively. The constants Kf and n
of various metal adsorbents used in this study and the toxicity values
b, which were obtained from fitting the experimental data in Fig. 3 to equation 1, are shown in Table
1.
In the absence of the metal adsorbent (i.e., in the control), the
toxicity parameter is the original value (b). Unmodified clay has no effect on metal toxicity reduction, as indicated by the
unchanged b value, due to the small adsorption capacity
(Kf) and intensity (n) of the clay.
This is attributed to the adsorption mechanism of the unmodified clay,
which is purely ion exchange or nonselective in nature. The toxicity of
cadmium was greatly reduced when the modified clay or Chelex 100 was
added to the medium. The b value was reduced from its
original value of 0.45 mM
1 to 0.18 and 0.08 mM1 for the modified clay and Chelex, respectively.
Cadmium ions in the medium were selectively adsorbed onto these
metal-chelating adsorbents and thus become unavailable to inhibit the
bacterial growth. This is attributed to the metal complexation
mechanism of these adsorbents (11, 19) which results in a
high adsorption capacity as indicated by the high
Kf and n values. These results clearly show the effective reduction of metal toxicity and its relation
to the adsorption characteristics of these metal-chelating adsorbents.
We have further investigated the sorption of naphthalene onto the
metal-chelating adsorbents (modified clay and Chelex) and its effect on
the bioavailability of naphthalene. Our preliminary data have shown
that while using the more-hydrophobic Chelex resin significantly
reduces the bioavailability of substrate (naphthalene) to the bacteria,
there is less of a bioavailability problem when the modified-clay
complex is used (20). This phenomenon must be taken into
consideration when we design or select the metal adsorbents for
mixed-waste treatment. We are currently investigating this further by
both analysis and experimentation.
In summary, we have quantified the inhibitory effect of cadmium on the
growth of P. putida ppo200(NAH) solely for naphthalene biodegradation. We have demonstrated that the toxicity of cadmium to
P. putida can be greatly reduced by the addition of a
modified-clay complex or a commercial chelating resin (Chelex).
Unmodified natural clay, as a control, has no effect on this metal
toxicity reduction. We have also shown that the reduction of metal
toxicity can be quantitatively related to the adsorption
characteristics of these adsorbents. This study shows that the
surfactant-modified clay adsorbent is a promising and economical
candidate with potential applications in mixed-waste biotreatment, in
which toxic organics and heavy metals coexist.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Chemical Engineering, University of Michigan, Ann Arbor, MI 48109-2136. Phone: (734) 763-5659. Fax: (734) 763-0459. E-mail:
hywang{at}engin.umich.edu.
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Applied and Environmental Microbiology, November 1998, p. 4610-4613, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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