Journal of Environmental Quality 32:1228-1233 (2003)
© 2003 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
TECHNICAL REPORTS
Bioremediation and Biodegradation
Chromate Reduction by Chromium-Resistant Bacteria Isolated from Soils Contaminated with Dichromate
F. A. O. Camargo,
F. M. Bento,
B. C. Okeke and
W. T. Frankenberger*
Department of Environmental Science, Univ. of California, Riverside, CA 92521
* Corresponding author (william.frankenberger{at}ucr.edu)
Received for publication May 2, 2002.
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ABSTRACT
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Extensive use of hexavalent chromium [Cr(VI)] in various industrial applications has caused substantial environmental contamination. Chromium-resistant bacteria isolated from soils can be used to remove toxic Cr(VI) from contaminated environments. This study was conducted to isolate chromium-resistant bacteria from soils contaminated with dichromate and describes the effects of some environmental factors such as pH, temperature, and time on Cr(VI) reduction and resistance. We found that chromium-resistant bacteria can tolerate 2500 mg L-1 Cr(VI), but most of the isolates tolerated and reduced Cr(VI) at concentrations lower than 1500 mg L-1. Chromate reduction activity of whole cells was detected in five isolates. Most of these isolates belong to the genus Bacillus as identified by the 16S rRNA gene sequencing. Maximal Cr(VI) reduction was observed at the optimum pH (7.0–9.0) and temperature (30°C) of growth. One bacterial isolate (Bacillus sp. ES 29) was able to aerobically reduce 90% of Cr(VI) in six hours. The Cr(VI) reduction activity of the whole cells of five isolates had a KM of 0.271 (2.61 mM) to 1.51 mg L-1 (14.50 mM) and a Vmax of 88.4 (14.17 nmol min-1) to 489 mg L-1 h-1 (78.36 nmol min-1). Our consortia and monocultures of these isolates can be useful for Cr(VI) detoxification at low and high concentrations in Cr(VI)-contaminated environments and under a wide range of environmental conditions.
Abbreviations: LB, Luria–Bertani
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INTRODUCTION
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HEXAVALENT CHROMIUM [Cr(VI)] has many industrial applications and often causes environmental contamination in marine and freshwater sediments from urban and industrial discharges (Losi et al., 1994a). Chromate 
is a strong oxidizing agent that is reduced intracellularly to Cr5+ and reacts with nucleic acids and other cell components to produce mutagenic and carcinogenic effects on biological systems (McLean and Beveridge, 2001; Clark, 1994). Although reduction to Cr5+ is responsible for chromate toxicity, further reduction to trivalent chromium leads to the formation of stable, less soluble, and less toxic Cr(III). Reduction of Cr(VI) to Cr(III) is therefore a potentially useful process for remediation of Cr(VI)-affected environments (Michel et al., 2001).
Conventional methods for removing toxic CrO2-4 include chemical reduction followed by precipitation, ion exchange, and adsorption on activated coal, alum, kaolinite, and ash. However, most of these methods require high energy or large quantities of chemical reagents (Komori et al., 1990). Microbial reduction of toxic hexavalent chromium has practical importance, because biological strategies provide green technology that is cost-effective (Ganguli and Tripathi, 2002).
Bioreduction of Cr(VI) can occur directly as a result of microbial metabolism (enzymatic) or indirectly, mediated by a bacterial metabolite (such as H2S) (Losi et al., 1994a). A number of chromium-resistant microorganisms have been reported, including Pseudomonas spp. (Mondaca et al., 1998; Alvarez et al., 1999; Oh and Choi, 1997), Microbacterium (Pattanapipitpaisal et al., 2001), Desulfovibrio (Michel et al., 2001), Enterobacter spp. (Wang et al., 1990; Clark, 1994), Escherichia coli (Shen and Wang, 1993), Shewanella alga (Guha et al., 2001), Bacillus spp. (Campos et al., 1995), and several other bacterial isolates (Basu et al., 1997; Losi and Frankenberger, 1994; Holman et al., 1999). However, most of them have been isolated from tannery sludge, industrial sewage, evaporation ponds, or discharge water, or were purchased from culture collections.
In this paper, we describe the isolation of Cr(VI)-resistant bacteria from chromium contaminated soils in the USA and Brazil and the characterization of factors involved in hexavalent chromium reduction by the monoculture isolates.
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MATERIALS AND METHODS
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Chromate-Resistant Bacteria
Chromate-resistant bacteria were isolated from potassium dichromate–contaminated soil samples obtained from a landfarming site in southern Brazil and waste disposal site in California, USA (Table 1)
. For the isolation and enumeration of bacteria, samples were serially diluted and plated on Luria–Bertani (LB) agar (tryptone, 10 g L-1; yeast extract, 5 g L-1; NaCl, 10 g L-1; glucose, 0.1 g L-1) at three pH values (5.0, 7.0, and 9.0). Adjustment of pH was made by adding aliquots of either HCl or NaOH. The molten medium was supplemented with Cr(VI) as K2Cr2O7 to final concentrations ranging from 500 to 5000 mg L-1 with sterile Cr(VI) stock solutions. The Cr(VI) stock solutions were filter sterilized with a 0.22-µm membrane filter (Millipore Corp., Bedford, MA). Plates were incubated at 30°C in the dark and were read after one week. Bacterial colonies were purified on LB agar containing 500 mg L-1 of Cr(VI) and incubated for 4 d at 30°C.
Identification of Chromium-Resistant Bacteria
The bacterial isolates were identified by 16S rRNA sequencing as follows. Bacterial isolates were grown on Luria–Bertani agar containing 500 mg of Cr(VI) L-1 for 2 d at 30°C. Bacterial colonies were then suspended in nuclease-free water. DNA was extracted from the suspension according to the method described by Asubel et al. (1997). Colonies of the isolates, suspended in a mixture of TE buffer, SDS (10%), and proteinase K, were incubated for 1 h at 37°C. The NaCl (5 M) and CTAB–NaCl solution (4.1 g NaCl and 10 g CTAB [N-cetyl-N,N,N-trimethylammoniumbromide] in 100 mL of prewarmed, distilled water) were added and incubated for 10 min at 65°C. The solution was extracted with 780 mL of chloroform–isoamyl alcohol (24:1) and centrifuged for 5 min, and the aqueous phase was further extracted with an equal volume of phenol–chloroform–isoamyl alcohol (25:24:1). After centrifugation for 5 min, the DNA present in the aqueous phase was precipitated with 0.6 vol isopropanol and the precipitate was washed with 70% ethanol. The DNA pellet was dried with a lyophilizer and resuspended in nuclease-free water. Universal bacterial primers corresponding to E. coli positions 27F and 519R were used for polymerase chain reaction (PCR) amplification of the 16S rRNA gene. A PCR master mix (Catalog no. M7502; Promega, Madison, WI) was used according the manufacturer's instructions. Genomic DNA template (1 mL) was amplified with a 35-cycle PCR (initial denaturation, 95°C for 3 min; subsequent denaturation, 95°C for 1 min; annealing temperature, 55°C for 1 min; extension temperature, 72°C for 1 min; and final extension, 72°C for 5 min). The PCR product was analyzed on 2% agarose gel and purified with a QIAEX II gel extraction kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Briefly, the gel slice was suspended in buffer QXI and the QIAEXII suspension was added and mixed by vortexing. The suspension was incubated at 50°C for 10 min and centrifuged for 30 s, and the pellet was washed twice with buffer QXI and once with buffer PE. After air-drying for 15 min, the DNA was eluted with nuclease-free water. The DNA cycle sequencing was performed with a BigDye terminator kit and an ABI 3100 genetic analyzer (both from Applied Biosystems, Foster City, CA). MEGABLAST (Altschul et al., 1997) was used for homology searching.
Characterization of Chromium Reduction by Isolates
Chromate-resistant bacterial isolates were inoculated into LB broth (pH 9.0) containing 500 µg L-1 of Cr(VI) and incubated for 24 h at 30°C with orbital shaking (200 rpm). Bacterial cell density of the liquid cultures was determined by measuring optical density at 600 nm with a Spectronic 1001 spectrophotometer (Milton Roy Co., Rochester, NY). Cells were collected by centrifugation (5000 rpm, 15 min) and the supernatant was analyzed for residual chromium. The Cr(VI) reduction by whole cells was analyzed as follows. Cells were washed twice with 100 mM phosphate buffer (pH 7.0) and resuspended in 1 mL of the same buffer. For chromate reduction activity, 0.5 mL of resuspended cells were incubated with 500 µg L-1 of Cr(VI) in phosphate buffer (0.1 M) at 30°C for 30 min and residual Cr(VI) was analyzed. Hexavalent chromium was determined colorimetrically (l540) with a spectrophotometer (Spectronic 1001) with the s-diphenylcarbazide method (Bartlett and James, 1996) with a detection limit of 5 µg L-1. Five isolates that reduced chromate were selected for further characterization.
Effect of pH and Temperature
The influence of pH and temperature on bacterial growth and chromate reduction were assessed with the LB medium and culture conditions described for Cr(VI) reduction by the isolates. For the effect of pH, autoclaved culture medium was adjusted to pH 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with predetermined amounts of filter-sterilized (0.22 m) 1 M HCl or 1 M NaOH and incubated at 30°C. Incubation temperature was varied at 25, 30, 35, and 40°C. The inoculum used was 5% of the total volume and was the logarithmic-phase cultures of bacterial isolates (l600 = 0.288, 0.075, 0.182, 0.210, and 0.146 for isolates ES 04, ES 23, ES 29, ES 32, and ES 39, respectively) prepared in LB broth plus 0.5 mg L-1 of Cr(VI). For cell density determination, cultures were diluted 10-fold with water before measuring the absorbance.
Effect of Chromate Concentration
The effect of varying concentrations (0, 250, 500, 750, 1000, 1250, and 1500 mg L-1) of Cr(VI) as K2Cr2O7 on the tolerance of bacteria was examined in triplicate with the LB medium. Culture, inoculum, growth, and Cr(VI) reduction conditions were the same as described for Cr(VI) reduction by the isolates. The time course of Cr(VI) reduction was evaluated with Cr(VI) as K2Cr2O7 at five concentrations (0, 0.5, 1.0, 1.5, and 2.0 mg L-1) and incubated for 24 h at 30°C. The initial pH was adjusted to 7.0 for isolates ES 23 and ES 32, 8.0 for isolate ES 39, and 9.0 for isolates ES 04 and 29. The Cr(VI) reduction and growth of the isolates were evaluated at 0, 3, 6, 9, 12, and 24 h.
Determination of Kinetic Parameters
Kinetic parameters (KM and Vmax) were determined with the hyperbolic equation described for Michaelis–Menten (vo = Vmax[S]/[S]1/2 + [S]; where [S]1/2 = KM) (Farrell and Ranallo, 2000). Time course data at different chromium concentrations were subjected to an exponential decay equation [y = a exp(-bx)] to estimate the rate constant (b) of Cr reduction. Parameters were obtained after fitting the results to the equations with the software Sigma Plot (Jandel Scientific, 2002).
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RESULTS
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Chromate-Resistant Bacterial Isolates
The tolerance of Cr(VI)-resistant bacteria at different pH and Cr(VI) concentration is presented in Fig. 1
. The highest number (CFU g-1 soil, where CFU is colony-forming units) of Cr(VI)-resistant bacteria and the highest resistance were obtained at pH 9.0. At pH 7.0, Cr resistance was low at concentrations greater than 500 mg L-1 of Cr(VI). This concentration (500 mg L-1) was limiting for growth of Cr(VI)-resistant bacteria at pH 5.0 (Fig. 1).
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Fig. 1. Tolerance of Cr(VI)-resistant bacteria cultured on Luria–Bertani agar supplemented with Cr(VI) at different pH values. Error bars represent standard deviation (n - 1). CFU, colony-forming units.
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In the first isolation step, 29 Cr(VI)-resistant bacteria were isolated from the Brazilian soil and 14 from the USA soil. In the second step, Cr(VI) resistant bacterial isolates were further purified on LB agar containing 500 mg L-1 of Cr(VI). Nine isolates from the Brazilian soil and six isolates from the USA soil sample grew luxuriantly at 500 mg L-1 and were selected for further studies.
Growth and chromate reduction by the 15 bacterial isolates from both soils are presented in Table 2
. After 24 h, the percentage of Cr(VI) reduced was different for each isolate. Some isolates reduced only 8% of Cr(VI) added, while others reduced more than 80%. One isolate (ES 29) completely removed Cr(VI) (500 µg L-1) from the medium within 24 h. Isolate ES 29 is a Gram-positive rod forming long and branched chains. The isolate formed heat-stable spores (80°C for 15 min) and was catalase positive. Strain ES 29 hydrolyzed casein and gelatin and utilized carbohydrates under aerobic and facultatively anaerobic conditions.
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Table 2. Growth and Cr(VI) reduction activity of isolates from Brazil and USA soil samples exposed to 500 µg L-1 of Cr(VI).
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The isolates purified on LB agar containing 500 mg L-1 of Cr(VI) were identified by the 16S rRNA sequence technique (Table 3)
. We identified two genus and three species with more than 96% similarity. The isolates from the Brazilian soil sample (ES 04–ES 29) belong to the genus Bacillus and this genus was also detected as a USA soil isolate (ES 39). Isolate ES 32 was identified as an Arthrobacter of the Micrococcaceae family of the Actinomycetales order. According to the scientific literature, these species identified with the 16S rRNA gene sequence have never been reported as chromium-resistant bacteria.
Chromate reduction activity of whole cells [µmol Cr(VI) reduced h-1] was detected in five bacterial isolates and these isolates were selected for further studies. The influence of pH, temperature, and concentration of Cr on growth and Cr(VI) reduction was evaluated to determine the potential use of these isolates to reduce Cr(VI) at different environmental conditions.
Factors Affecting Hexavalent Chromium Reduction
The five Cr(VI)-resistant bacterial isolates were exposed to seven different Cr(VI) concentrations, and bacterial density (OD600) was measured after 24 h (Fig. 2)
. Bacterial isolates ES 04 and ES 29 showed the highest Cr(VI) resistance. Growth of the other isolates were inhibited by 250 mg L-1 of Cr(VI) and decreased cell density by more than 50%. A decrease in growth was observed for isolate ES 29 only in presence of a high concentration of Cr(VI) at 1500 mg L-1 (Fig. 2). However, at this concentration, ES 29 effectively reduced Cr(VI).
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Fig. 2. Growth of Cr(VI)-resistant bacterial isolates in a Luria–Bertani medium supplemented with different Cr(VI) concentrations. Error bars represent standard deviation (n - 1).
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Initial culture pH of the medium was considered as a factor for growth and Cr(VI) reduction by the isolates (Fig. 3)
. Isolates ES 23 and ES 32 grew better at pH 7.0, while isolate ES 39 grew better at pH 8.0 (Fig. 3a). The isolates that were more resistant to Cr(VI) grew better at pH 9.0. Values for pH of 5.0 and 10.0 restricted bacterial growth and Cr(VI) reduction. Optimal Cr(VI) reduction was directly related to the optimum pH for growth of the isolates.
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Fig. 3. Effect of pH on the (a) growth and (b) Cr(VI) reduction of Cr-resistant bacterial isolates. Error bars represent standard deviation (n - 1).
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Reduction of Cr(VI) by the isolates was dependent on temperature. The highest Cr(VI) reduction (82%) was observed at 30°C. The optimum temperature of the five isolates was between 25 and 30°C. Isolate ES 29, which displayed high resistance to Cr(VI) and greater Cr(VI) reduction, was severely affected by temperatures above 30°C. High temperatures (35–40°C) severely decreased bacterial growth and chromate reduction except for isolate ES 23, which grew and reduced chromate at 40°C. On the average, 65% of the Cr(VI) was reduced by the isolates at 25°C, but at 40°C reduction was only 48% (Fig. 4b)
.
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Fig. 4. Effect of temperature on the (a) growth and (b) Cr(VI) reduction of Cr-resistant bacterial isolates. Error bars represent standard deviation (n - 1).
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Time Course of Hexavalent Chromium Reduction
The analysis of the average of all isolates at different Cr(VI) concentrations showed a reduction of 7% of the initial Cr(VI) concentration after three hours of incubation. On the next three hours, 62% of the added Cr(VI) was reduced, and after 24 h, less than 5% of Cr(VI) was remaining (data not shown).
The time course of Cr(VI) reduction by the isolates (at 2.0 mg of Cr L-1) and isolate ES 29 at different Cr(VI) concentrations is shown in Fig. 5
. Among the isolates, ES 29 showed the highest reduction rate (0.192 h-1), reducing 82% of Cr(VI) added (2.0 mg L-1) to the medium in less than 6 h (Fig. 5a). Considering the average of all Cr(VI) added at different concentrations, isolate ES 29 was responsible for more than 90% of Cr(VI) reduction after six hours (data not shown). The rate of Cr(VI) reduction by the isolate ES 29 was highest at the lower Cr(VI) concentration (0.5 mg L-1, 0.230 h-1) and the lowest at the highest Cr(VI) concentration (2.0 mg L-1, 0.172 h-1) (Fig. 5b).
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Fig. 5. Time course of Cr(VI) reduction (a) at 2.0 mg Cr(VI) L-1 by the Cr-resistant bacterial isolates and (b) at different Cr concentration by the isolate ES 29. Error bars represent standard deviation (n - 1).
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Kinetic Parameters
The nonspecific half-saturation or Michaelis–Menten constant, KM, was estimated for each isolate (Table 4)
. The highest KM (14.5 mM) was observed with isolate ES 29 and the lowest KM (2.61 mM) was noted with isolate ES 32. The maximum nonspecific reduction rate or maximum velocity, Vmax, is presented in Table 2. Isolate ES 29 displayed the highest Vmax at 78.36 nmol min-1, while the lowest Vmax (14.17 nmol min-1) was observed with isolate ES 32. Among the isolates, the lower affinity for the substrate Cr(VI) was observed with isolate ES 29. The highest affinity (KM = 2.61 mM) for Cr(VI) was with the isolate ES 32.
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Table 4. Kinetic parameters of Cr(VI) reduction in whole cells of Cr-resistant bacterial isolates obtained by fitting the data to a hyperbolic equation described by Michaelis–Menten (vo = Vmax[S]/[S]1/2 + [S]; where [S]1/2 = KM).
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DISCUSSION
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Microorganisms with the ability to tolerate and reduce Cr(VI) can be used for detoxification of environments contaminated with Cr(VI). In this study, we describe the isolation and screening of 43 Cr(VI)-resistant bacterial isolates and the characterization of Cr(VI) reduction by selected isolates. Most of the isolates were able to reduce Cr(VI), but at different rates. Five isolates that displayed substantial reduction of Cr(VI) were further studied.
The bacterial isolates tolerated Cr(VI) over a wide concentration range (500–2500 mg L-1). Chromium(VI) bacterial resistance above 2500 mg L-1 has only been reported by Shakoori et al. (2000). They isolated a gram-positive bacterial strain (probably a Bacillus species) from a tannery effluent that grew in media containing potassium dichromate up to 80 mg mL-1. Most Cr(VI)-resistant microorganisms are tolerant up to 10 to 1500 mg L-1 of Cr(VI) (McLean and Beveridge, 2001; Basu et al., 1997; Losi and Frankenberger, 1994; Bopp et al., 1983). In our study, isolate ES 29 rapidly reduced 0.5 mg L-1 of Cr(VI) to a nondetectable level within 24 h. Four other isolates also showed substantial Cr(VI) reduction activity.
Both growth of the isolates and Cr(VI) reduction were dependent on pH, temperature, and Cr concentration. The optimum pH for growth of the isolates was 7.0 to 9.0. Extreme pH (5.0 and 10.0) restricted bacterial growth and Cr(VI) reduction. Variation of the culture initial pH highly affected Cr(VI) reduction. For most of the isolates, the optimal pH for growth correlated with the highest rate of Cr(VI) reduction. The relationship between pH and Cr(VI) reduction was not surprising because chromate is the dominant Cr(VI) species in an aqueous environment at pH 6.5 to 9.0 (McLean and Beveridge, 2001). Optimal pH for growth of Cr(VI)-resistant bacteria was reported at 7.0 to 7.8 (Losi et al., 1994a), but Cr(VI) forms are soluble over a wide pH range and generally mobile in soil–water systems (Losi et al., 1994a). Wang et al. (1990) reported that Cr(VI) reduction by Enterobacter cloacae occurred at pH 6.5 to 8.5 and was strongly inhibited at pH 5.0 and 9.0. Losi et al. (1994b) did not observe any significant effect of pH on the rate of chromate reduction in soil at different manure amendments. However, since Cr(VI) reduction is enzyme-mediated, changes in pH will affect the degree of ionization of the enzyme, changing the protein's conformation and affecting the enzyme activity (Farrell and Ranallo, 2000).
Both growth and Cr(VI) reduction by the five isolates were influenced by temperature. We report an optimum temperature of 30°C for Cr(VI) reduction among our bacterial isolates. Losi et al. (1994a) reported an optimal temperature of 30 to 37°C for Cr(VI) reduction. Wang et al. (1990) reported that no chromate reduction was observed at 4 and 60°C. Temperature is an important selection factor for bacterial growth and will affect enzymatic reactions necessary for Cr(VI) reduction.
The Cr(VI) concentration affected the growth of the isolates and Cr(VI) reduction. One isolate (ES 29) from a soil sample collected in Brazil showed the highest rate of Cr(VI) resistance and reduction. Isolate ES 29 can grown at high concentrations of Cr(VI) (>1000 mg L-1) and growth decreased to 50% only in the presence of 1500 mg L-1 of Cr(VI). The toxic and mutagenic effects of chromium on microorganisms have been reported to occur at concentrations between 10 and 12 mg Cr(VI) L-1, which are inhibitory to most soil bacteria in liquid media. These toxic effects are attributed to alteration of genetic material and altered metabolic and physiological reactions (Losi et al., 1994a).
The effects of different concentrations of Cr showed that Cr(VI) reduction activity by the isolates was possibly performed via enzymatic reaction, since the curve fit of the data (data not shown) and the hyperbolic shape of the data showed a clear dependence on Cr(VI). Among the isolates, the highest Vmax and invariably the lowest KM were found with isolate ES 29, indicating weak binding of the catalytic system of Cr(VI) reduction. The KM values of the five bacterial isolates in our study compare with the KM of 13 to 1730 mM reported by Oh and Choi (1997), indicating that our isolates have a high affinity for Cr(VI).
The factors that affected Cr(VI) reduction studied here (pH, temperature, Cr concentration, and kinetic parameters) are important environmental factors regulating remediation strategies for ecosystems polluted with natural or anthropogenic Cr(VI). The isolates described in this work show promise for Cr(VI) reduction and their characteristics will be useful in Cr(VI) bioremediation, which is potentially more cost-effective than traditional physical or chemical methods in the treatment of environments contaminated with Cr(VI).
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ACKNOWLEDGMENTS
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F.A.O. Camargo and F.M. Bento are grateful to the Brazilian National Research Council (CNPq) for a scholarship concession, especially to SEBIE staff member Jose Airton de Souza and to the Department of Environmental Science (UCR) for the opportunity to participate as post-doctoral scientists.
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ABSTRACT
INTRODUCTION
