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Applied and Environmental Microbiology, July 2000, p. 3083-3087, Vol. 66, No. 7
Department of Applied Science, Brookhaven
National Laboratory, Upton, New York 11973,1
and Biological Science Department, Florida State
University, Tallahassee, Florida 323062
Received 27 January 2000/Accepted 25 April 2000
Heavy metal resistance by bacteria is a topic of much importance to
the bioremediation of contaminated soils and sediments. We report here
the isolation of a highly cadmium-resistant Klebsiella planticola strain, Cd-1, from reducing salt marsh sediments. The strain grows in up to 15 mM CdCl2 under a wide range
of NaCl concentrations and at acidic or neutral pH. In
growth medium amended with thiosulfate, it precipitated significant
amounts of cadmium sulfide (CdS), as confirmed by x-absorption
spectroscopy. In comparison with various other strains tested, Cd-1 is
superior for precipitating CdS in cultures containing thiosulfate.
Thus, our results suggest that Cd-1 is a good candidate for the
accelerated bioremediation of systems contaminated by high levels of cadmium.
Cadmium, a highly toxic metal and a
group B1 human carcinogen, is commonly ranked among the top 10 priority pollutants by U.S. regulatory agencies. Significant amounts of
this metal often infiltrate the groundwater at hazardous waste sites. A
practical approach to minimizing Cd(II) levels in the subsurface
systems is precipitating it with hydrogen sulfide as the highly
insoluble cadmium sulfide (CdS). In reducing environments,
sulfate-reducing bacteria (SRB) generate hydrogen sulfide through
dissimilatory sulfate reduction. However, little is known about the
heavy metal resistance and growth of these bacteria in contaminated
systems. Recent studies by Poulson et al. (14) and White and
Gadd (22) indicate that even low levels of free Cd(II),
Zn(II), or Ni(II) ions, i.e., 20 to 200 µM, are toxic to SRB, such as
Desulfovibrio and Desulfotomaculum. Our
unpublished studies (P. K. Sharma and M. A. Vairavamurthy)
also indicate that at least the SRB from coastal salt marshes from
Shelter Island, New York, are sensitive to cadmium. However, a new
Klebsiella planticola strain, Cd-1, from the same coastal
environment grew to saturation in 15 mM Cd(II) ions in a minimal
anaerobic medium, and after thiosulfate was added to the growth medium,
it transformed significant amounts of the dissolved cadmium to CdS, up
to 50 times higher than that reported for highly purified and
continuously fed sulfate-reducing biofilms (22) or batch
cultures of a genetically engineered Escherichia coli
with overexpressed thiosulfate reductase (S. W. Bang, D. S. Clark, and J. D. Keasling, Abstr. 99th Gen. Meet. Am. Soc.
Microbiol., abstr. Q-302, 1999). In general, facultative anaerobes,
such as Cd-1, are attractive candidates for accelerated bioremediation
applications, as large amounts of them can be grown rapidly with
various nonhazardous substrates in aerobic reactors and can then be
injected into anaerobic subsurface environments. In contrast, SRB often
are slow growers and also may require expensive substrates, nutrients,
or cofactors for growth.
In this report, we describe the isolation and basic characteristics of
strain Cd-1. We show that it can grow in high levels of Cd(II) ions
under a wide range of environmental conditions, including high salinity
and acidic pH. Importantly, our studies demonstrate that Cd-1 can
transform high levels of cadmium to CdS in the presence of thiosulfate.
We compared the growth and the ability of Cd-1 to produce CdS in
media containing high levels of cadmium with those of several other
metal-resistant, metal-transforming, or metal-reducing ubiquitous
facultative anaerobes. These results suggest that Cd-1 is a
superior candidate for the anaerobic growth and transformation of
cadmium, particularly in the presence of thiosulfate.
Isolation of Cd-1.
Strain Cd-1 was isolated from near-surface
sediments (up to 6 in. deep) from a coastal salt marsh on Shelter
Island, New York. The isolate was enriched by adding sediment (ca.
13 g per liter) to a medium containing 0.3 M NaCl, 10 mM
Na2SO4, and 0, 50, 200, or 500 µM
CdCl2; 2 g of powdered Spartina
alterniflora tissue per liter was added as the source of carbon
and nutrients. S. alterniflora is the dominant source of
organic matter sustaining the microbial populations in the salt marshes
in Shelter Island. The pH of the microcosms before incubation was
5.5. They were incubated in an anaerobic chamber (Coy Laboratory
Products, Inc., Grass Lake, Mich.) at 24.5°C. The headspace of
the sealed microcosms contained the chamber atmosphere, i.e.,
H2-CO2-N2 at a ratio of 5:5:90.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A New Klebsiella planticola Strain
(Cd-1) Grows Anaerobically at High Cadmium Concentrations and
Precipitates Cadmium Sulfide
ABSTRACT
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-D-glucose, 100 mM NaCl, 10 mg of yeast extract per liter, and 5 mM Ches [2-(cyclohexylamino)ethanesulfonic
acid], along with a 4:1 mineral solution-to-metal solution ratio
(solutions described later). Following a further 10-day
incubation, a portion was transferred into fresh media; the resulting
second-generation enrichment was incubated for 1 day and then was
serially diluted. The serial dilutions were streaked onto nutrient agar
plates containing 0.5 mM CdCl2, which were then incubated
both inside and outside the anaerobic chamber. Only one morphotype
appeared within 24 to 72 h under both redox conditions.
Identification and characterization of Cd-1. (i) Basic
characteristics.
Strain Cd-1 is a gram-negative, nonmotile,
facultative anaerobe. It can grow in >6% (wt/vol) NaCl in nutrient
broth both aerobically and anaerobically; thus, it is a halotolerant
bacterium. It is nutritionally diverse, with the ability to utilize all
but 25 of the 95 substrates on Biolog GN MicroPlate. The compounds it was unable to use are cyclodextrin, erythritol, lactulose, xylitol, D-galactonic acid lactone, D-glucosaminic acid,
-, 
-, or 
-hydroxy butyric acid, itaconic acid,
-keto-butyric acid, glutaric acid, valeric acid, propionic acid,
sebacic acid, L-leucine, L-ornithine, L-pyroglutamic acid, L-threonine,
D- or L-carnitine, -amino butyric acid,
urocanic acid, phenyl ethylamine, 2-amino ethanol, and 2,3-butanediol.
(ii) 16S rRNA gene analysis. A segment of approximately 1,500 bases of Cd-1's 16S rRNA gene (nearly the entire gene) was amplified and sequenced with an Applied Biosystems 373A automated DNA sequencer (Perkin-Elmer-Applied Biosystems, Foster City, Calif.), using the Taq DyeDeoxy terminator cycle-sequencing method. The PCR amplification primers were fD1 and rP1 (21), while the DNA sequencing primers were A, C, G, H, H-complement, P, and P-complement (1). The assembled sequence, corresponding to positions 20 to 1350 of the Escherichia coli 16S rDNA sequence, was aligned with sequences for selected reference strains, i.e., those with the most similar 16S rDNA sequences, obtained from RDB and the GenBank and EMBL databases. The phylogenetic position of Cd-1 was analyzed with standard methods: distance matrix, maximum likelihood, and parsimony. Distances were calculated by the methods described by Jukes and Cantor (12). The PHYLIP version 3.5c programs (8) were used for distance matrix analyses, after which phylogenies were estimated using the algorithms of DeSoete (7) and/or Fitch and Margoliash (10). The DNAML component of the PHYLIP package was used for maximum likelihood analyses, while the PAUP program (17) was employed to construct the most parsimonious phylogenetic tree.
The 16S rDNA sequences of Cd-1 and an official strain of K. planticola (GenBank accession no. X93215) were 100% similar over the 1,327 bases that we compared, indicating that Cd-1 is a strain of K. planticola. These observations were confirmed with phylogenetic analysis; all three methods, i.e., distance matrix, maximum likelihood, and parsimony, showed a close relationship between Cd-1 and K. planticola.(iii) Metabolic and biochemical analyses. The Biolog System identified Cd-1 as K. planticola. The similarity index (SI) value for K. planticola and two different Cd-1 cultures was 0.514 and 0.651; it was below 0.14 for any other gram-negative strain.
The MIDI/Hewlett-Packard Microbial Identification System also placed Cd-1 under Enterobacteriaceae.Growth of Cd-1 in Cd(II). (i) Composition of growth medium.
The anaerobic medium for strain Cd-1 contained the following per liter:
5 mM pyruvate, 1 mM -D-glucose, 5 ml of mineral
solution, 1.25 ml of metal solution, and 5 mM Trizma buffer (Sigma
Chemical Co., St. Louis, Mo.). The mineral solution had the
following composition per liter: 40 g of NaCl, 5 g of
NH4SO4, 5 g of KCl, 5 g of
-glycerophosphate (C3H7O6PNa2), 5 g
of MgCl2 · 6H2O, and 2 g of
CaCl2 · 2H2O. A liter of the metal
solution contained the following: 2 g of
C10H14O8N2Na2 · 2H2O (pH was adjusted to 6.0 with freshly prepared KOH
before adding the other compounds), 1 g of MnSO4
· H2O, 600 mg of FeCl2 · 4H2O, 200 mg of CoCl2 · 6H2O, 200 mg of (CH3COO)2Zn
· 2H2O, 20 mg of CuCl2 · 2H2O, 20 mg of NiCl2 · 6H2O,
and 20 mg of NaMoO4 · 2H2O. Any changes
made in the medium's composition are listed elsewhere. The pH of the
growth medium was around 7.1, except in experiments assessing the
effect of different initial pH values on the growth in Cd(II). A 2%,
i.e., 1:50, (vol/vol) inoculum was used unless indicated otherwise.
(ii) Role of NaCl.
The presence of 40 to 100 mM NaCl shortened
the lag phase or accelerated the growth rate in 1 mM Cd(II) (Fig.
1). However, in the absence of Cd(II),
the growth rate was maximal when no NaCl was added (the growth medium
contained around 3.4 mM NaCl from mineral solution; see also above).
Studies with equimolar LiCl showed similar trends. These observations
suggest that either an enhanced expression of Na+ extrusion
systems (13) or an osmotic shock (9) protected the cells against Cd(II) toxicity. Also, less-toxic Cd---Cl
coordination complexes may have been formed at a high NaCl
concentration (6). Often, 100 mM NaCl was added to the
growth medium, except in experiments to determine the effect of the
varying NaCl concentration on Cd-1 in Cd(II).
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(iii) Growth kinetics. Under minimal fermentative conditions, Cd-1 reached the stationary phase within 1 to 2 days in 0.2 to 2.0 mM Cd(II), within 3 days in 5 mM Cd(II), and within 7 to 10 days in 7 to 15 mM Cd(II). Although the lag phase was longer with Cd(II) present, the apparent doubling time was nearly identical with or without 1 mM Cd(II), i.e., ~3.5 h. However, in 5 mM Cd(II), the apparent doubling time was ~10 h (doubling time values are averages from replicate cultures, and the ranges were negligible).
With or without Cd(II), Cd-1 did not grow to an optical density (OD) of >0.12 when supplied with 5 mM pyruvate and 1 mM glucose (initial OD, ~0.008). For example, the average OD of five different cultures was 0.0801 after 28 h [0.2 to 5.0 mM Cd(II)] (Table 1). Since this maximum OD value was small, we monitored the concentrations of the viable cells to determine whether significant growth occurred. As Table 1 shows, there was an increase to about 61-fold in CFU within 30 h in Cd(II), ranging from 0.2 to 2.0 mM, and a ~30-fold increase in 5 mM Cd(II) cultures (2× inoculum; with a 1× inoculum, a ~10-fold increase occurred after 50 h, a time frame consistent with that observed in other experiments). Without Cd(II), a 50-fold increase in CFU was observed, a value comparable to that for cultures grown in 0.2 to 2.0 mM Cd(II) (Table 1).
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(iv) Growth in Cd(II) at high salinity and low pH. Since Cd-1 was isolated from coastal salt marsh sediments, we tested whether it could grow in high Cd(II) levels (0.5 to 5.0 mM) with concentrations of NaCl typical of estuarine and coastal systems (300 to 500 mM). Indeed, it grew well (final OD, ~0.1), though at rates lower than those at the 100 mM NaCl level. We noticed that growth rates in 300 or 500 mM NaCl were lower than those in 100 mM NaCl, even in the absence of Cd(II).
Cd-1 grew well (OD, ~0.1) in 2 mM Cd(II) at an initial external pH ranging from 4.16 to 7.18. However, its growth was faster under acidic pH, i.e., 4.16 to 5.78, in which cadmium exists as divalent cations (6). Without Cd(II), optimal growth occurred at near-neutral pH.Transformation of cadmium to cadmium sulfide. (i) Synchrotron X-ray absorption spectroscopy. Cd-1 cultures containing cadmium turned yellow during the stationary phase of growth, indicating that the isolate had transformed the added Cd(II) into CdS. We confirmed the identity of CdS with X-ray absorption spectroscopy using washed and freeze-dried late stationary-phase cells (harvested by centrifugation at 5,000 × g, 20 min, 4°C). Analyses included X-ray absorption near-edge structure (XANES) spectroscopy to characterize sulfur species and extended X-ray absorption fine structure (EXAFS) spectroscopy to examine the coordination environment around cadmium. We used the X-19A and X-18B beamlines at the National Synchrotron Light Source, Brookhaven National Laboratory, Upton, New York, for XANES and EXAFS studies, respectively.
(i) XANES data.
Sulfur K-edge XANES spectroscopy is a valuable
technique for determining sulfur speciation because the absorption fine
structure of the variety of sulfur forms are richly endowed with
characteristic features, including edge energy, allowing the
identification among various oxidation states and structures (18,
19). Consequently, an XANES spectrum of a sample can be
deconvoluted to derive information of the different sulfur constituents
in the sample. Figure 2 shows the XANES
spectra of reference CdS and Cd-1 cells grown with or without 5 mM
Cd(II) under fermentative conditions; the sum of the spectral fit is
also shown. Deconvolution was performed according to published
procedures (18). The spectra of Cd-1 show that cells grown
in Cd(II) contained more reduced sulfur; the fit indicates that this
difference may be attributed to CdS production. The deconvolution of
the spectra indicate that CdS contributed to about 60% of reduced
sulfur in cells grown with Cd(II).
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(ii) EXAFS data.
We used EXAFS to gain information on the
local coordination environment of cadmium to verify its chemical
identity in the Cd-1 samples. EXAFS data were analyzed by UWXAFS
software (15). Figure 3a gives
EXAFS data [k2-weighted 
(k),
where k is the photoelectron wave vector] extracted from
the averaged absorption coefficient after removing a smooth background
function using the program AUTOBK. It is well established that when
(k) is Fourier transformed over a finite k
range, the result is a radial structure function exhibiting a
series of peaks whose positions and amplitudes are related to the
interatomic distances and the number of atoms in different
coordination shells, respectively. The Fourier transform magnitudes (in
the k range, from 2 to 12 Å
1) are shown in
Fig. 3b. Essentially, Cd---O and Cd---S coordination numbers and
distances and their disorder, together with their uncertainties, were
obtained by a nonlinear least-squares fit of the theoretical EXAFS
equation (16) to the data of strain Cd-1. The scattering
amplitudes and phases used in the fits were extracted from the
experimental standards CdS and Cd(ClO4)2
· H2O, respectively. The Cd---O and Cd---S distances in
Cd(ClO4)2 and reference CdS are very different:
2.28 Å (4) and 2.52 Å (23), respectively. This
difference in the first nearest-neighbor distance is manifested as the
shift of the first peak in the Fourier transform magnitude of reference
CdS toward higher distances. The strain Cd-1 sample is shifted to
higher distances relative to Cd(ClO4)2, an
independent indication that sulfur is present in the nearest environment around cadmium. Figure 3b also shows the fit to the strain
Cd-1 data. The coordination numbers for the Cd---O and Cd---S bonds
were 1.5 ± 1.0 (mean ± standard deviation) and 3.8 ± 1.0 Å, respectively.
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(iii) Sulfide measurement.
Sulfide concentration reflecting
the production of CdS by strain Cd-1 in the presence of
S2O32 was determined by the
methylene blue assay (Hach Company, Loveland, Ohio). The reagent
produces a stable blue color within a few minutes in the presence of
H2S. The estimated detection limit in deionized water
matrix was ~0.05 (± 0.02) nM, as suggested by the manufacturer. The
reagent reacts positively to
S2O32 as the latter dissociates
to H2S at acidic pH. Therefore, we ensured the removal of
S2O32 from the late-stationary
phase cells before the methylene blue assay: centrifugation
(5,000 × g, 20 min, 4°C) to harvest the cell pellet,
followed by a rinse in deionized water. Sodium sulfide (Na2S · 9H2O) served as the source of
sulfide standards; its concentration ranged from 100 to 500 µM.
Absorption was measured at 665 nm with a Shimadzu UV-2101PC UV-visible
light Scanning Spectrophotometer equipped with UVPC Personal
Spectroscopy Software Version 3.9 (Shimadzu Scientific Instruments,
Inc., Columbia, Md.). Our data indicate that strain Cd-1 produced
around 1 mM CdS when grown in 5 mM Cd(II) in the presence of 5 or 10 mM
S2O32 (average CdS value for 10 mM S2O32 duplicate cultures was
0.978 mM, with a standard deviation of ±0.052).
Growth of Cd-1 in other toxic metals. Cd-1 grew within 2 to 10 days in the presence of 1 mM Cr(VI), As(V), Se(IV), Co(II), or Zn(II) and also in 0.5 mM Pb(II). Significant amounts of precipitates were formed in some cultures. For example, pink precipitates were formed in cultures with Se(IV) present indicating that the metal was reduced to elemental selenium. Precipitates also were formed in cultures with Co(II), Zn(II), or Pb(II) present. The color of the Cr(VI) media changed, but it is unclear whether significant precipitation occurred. As expected, no precipitates were formed in cultures with As(V) present.
Comparison of Cd-1 with closely related Klebsiella
strains.
Comparisons of Cd-1 were made with strains of K. planticola ATCC 33531 and Klebsiella orinithinolytica
ATCC 31898. Both strains grew in 5 mM Cd(II) in Cd-1's minimal medium
described earlier or in Cd-1's medium amended with 5 mM
NO3 or
S2O32. However, the lag phase of
the ATCC strains was three to four times longer than that of Cd-1 (data
not shown). The growth rate of K. planticola ATCC 33531 also
was about five times slower. The lag phases and growth rates of the
ATCC strains were comparable to those of Cd-1 in Cd(II)-free medium.
The ATCC strains produced noticeable amounts of CdS in the presence of
S2O32; K. ornithinolytica produced around 1 mM CdS, but the amount was not
determined for K. planticola. Here also, CdS production occurred primarily during the stationary phase of growth.
Comparison with other genera. We also compared Cd-1 with many nonrelated strains, including several pseudomonads, two Shewanella strains (Shewanella oneidensis MR-1 and Shewanella putrefaciens CN32), Ralstonia eutropha ATCC 43123, Enterobacter agglomerans ATCC 27993, and Bacillus subtilis ATCC 35946. The pseudomonads included Comamonas testosteroni ATCC 1996, Pseudomonas putida ATCC 17484, and Pseudomonas aeruginosa CW-96-1 and three new isolates (Cd-2, Cd-4, and Cd-6).
Strains Cd-2 and Cd-4 were isolated from surficial sediments (top 4 in.) from the Mashomack Preserve, near a creek within 2 to 3 miles of the salt marsh from which strain Cd-1 was isolated. Biolog analysis suggested a close match of Cd-2 and Cd-4 with Pseudomonas fluorescens type C (SI, 0.575) and Pseudomonas resinovorans (SI, 0.257), respectively. Strain Cd-6 was isolated from a tetrachloroethene- to -cis-1,2-dichloroethene -dehalogenating anaerobic culture derived from tetrachloroethene-contaminated aquifer material obtained from Victoria, Tex. (V. Warikoo and P. K. Sharma, unpublished data). Cd-6 showed a close match with P. aeruginosa (Biolog SI, 0.556). All strains grew well on aerobic nutrient agar containing 1 mM CdCl2. Past studies on metal resistance by pseudomonads mainly emphasized aerobic growth. For example, P. aeruginosa CW-96-1 was shown to grow well aerobically in up to 5 mM cadmium in a medium containing citrate and S2O32
(20). In contrast, our experiments were performed
anaerobically with 5 mM CdCl2. We used Cd-1's minimal
growth medium with or without 5 mM NO3 or
S2O32. Our studies showed no
growth of the pseudomonads (including P. aeruginosa CW-96-1)
in the presence of 5 mM cadmium under fermentative, nitrate-respiring,
or thiosulfate-respiring conditions. Although S. oneidensis
MR-1 grew well (OD of 0.06 within 82 h) and generated sulfide
under thiosulfate-respiring conditions, it failed to grow when Cd(II)
was present. Of the different strains tested, R. eutropha CH34 (3) was the only one that grew in 5 mM Cd(II) (final OD of 0.076 after 360 h). However, CH34 did not grow under
thiosulfate-respiring conditions, even in the absence of cadmium. Thus,
this bacterium lacks the potential to stabilize cadmium as CdS.
Cd(II) resistance in Klebsiella: a widespread phenomenon? We found that at least three Klebsiella strains grew in high levels of Cd(II). A previous study (5) described a marine Klebsiella pneumoniae culture that grew in up to 20 mM Cd(II) under nutrient-rich aerobic conditions. However, it is not clear whether the ability to resist and grow in high Cd(II) levels is a widespread phenomenon in Klebsiella. Bhattacharyya et al. (2) screened 50 enterics, including 16 Klebsiella aerogenes (K. pneumoniae) strains, and showed that only one strain each of K. aerogenes, E. coli, and Serratia marcenes resisted 50 µM CdCl2 aerobically. Holmes et al. (11) found that the composition of the medium affects Cd(II) resistance in K. pneumoniae; the organism becomes highly sensitive to Cd(II) when some specific buffers are added to a defined growth medium [e.g., tolerance level decreases from 2 mM Cd(II) in a phosphate- or Tricine-buffered medium to 10 to 150 µM Cd(II) in a medium buffered with Tris or other buffers]. However, our strain, Cd-1, grew in mM Cd(II) levels in a Tris-buffered minimal medium.
Conclusions. Our results clearly show the superiority of the new isolate, Cd-1, in anaerobically transforming Cd(II) to CdS, particularly when the metal is present as complexes of thiosulfate. It grew in up to 15 mM Cd(II) ions in a minimal anaerobic medium and thrived both aerobically and anaerobically under various environments, even with high salinities (>6% [wt/vol] NaCl) and pH range of 4 to 8. In fact, we are not aware of a previous study showing resistance by any eubacterium or archaebacterium to levels as high as 15 mM Cd(II) ions in a minimal anaerobic medium. Cd-1 also grew in high levels (0.5 to 1.0 mM) of various toxic metals and cocontaminants, such as Cr(VI), As(V), Se(IV), Co(II), Pb(II), and Zn(II). Thus, we believe strain Cd-1 to be a useful organism for accelerated bioremediation processes under diverse geochemical conditions.
American Type Culture Collection (ATCC) accession number. Strain Cd-1 has been deposited in ATCC under accession no. 7008340.
Nucleotide sequence accession number. The 16S rDNA sequence (bases 1 to 1339) of strain Cd-1 has been deposited in GenBank under accession no. AF175281.
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ACKNOWLEDGMENTS |
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This study was supported by the Office of Biological and Environmental Research of the U.S. Department of Energy under Prime Contract no. DE-AC02-98CH10886 with the Brookhaven National Laboratory (BNL).
We thank Mike McInerney of University of Oklahoma for helpful suggestions.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Applied Science, Brookhaven National Laboratory, Bldg. 815, Upton, NY 11973. Phone: (631) 344-5337. Fax: (631) 344-5526. E-mail: vmurthy{at}bnl.gov.
Present address: Millipore Corporation, Bioprocess Division,
Bedford, MA 01730.
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Applied and Environmental Microbiology, July 2000, p. 3083-3087, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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