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Journal of Environmental Quality 31:1831-1841 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORTS
Bioremediation and Biodegradation

Low-Temperature Chromium(VI) Biotransformation in Soil with Varying Electron Acceptors

Julia K. Tseng and Angela R. Bielefeldt*

Department Civil, Environmental, and Architectural Engineering, Univ. of Colorado, 428 UCB, Boulder, CO 80309-0428

* Corresponding author (angela.bielefeldt{at}colorado.edu)

Received for publication November 13, 2001.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Effective and low-cost strategies for remediating chromium (Cr)-contaminated soil are needed. Chromium(VI) leaching from contaminated soil into ground water and surface water threatens water supplies and the environment. This study tested indigenous Cr(VI) microbial transformation in batch systems at 10°C in the presence of various electron acceptors. The effects of carbon addition, spiked Cr(VI), and mixing highly contaminated soil with less contaminated soil were investigated. The results indicated that Cr(VI) can be biotransformed in the presence of different electron acceptors including oxygen, nitrate, sulfate, and iron. Sugar addition had the greatest effect on enhancing Cr(VI) removal. Less dissolved organic carbon (DOC) was consumed per amount of Cr(VI) transformed under anaerobic conditions [0.8–93 mg DOC/mg Cr(VI)] compared with aerobic conditions [1.4–265 mg DOC/mg Cr(VI)]. Toxicity of high concentrations (<160 mg/L) of spiked Cr(VI) were not evident. At Cr(VI) concentrations > 40 mg/L, aerobic conditions promoted faster Cr(VI) reduction than anaerobic conditions with nitrate or sulfate present. Biotransformation of Cr(VI) in highly contaminated soil (22 000 mg Cr/kg) was facilitated by mixing with less-contaminated soil. The study results provide a framework for evaluating indigenous Cr(VI) microbial transformation and enhance the ability to develop strategies for soil treatment.

Abbreviations: BSM, basic salt medium • DOC, dissolved organic carbon • HI, high chromium contamination • LO, low chromium contamination • MD, medium chromium contamination


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
EXCESSIVE CHROMIUM (Cr) is present in the natural environment due to chrome plating and polishing operations, inorganic chemical production, cooling tower and steel mill effluents, wood-preserving facilities, and petroleum refineries (USEPA, 1990; Allen et al., 1998). According to the USEPA (1978, 1990), industries in the USA use more than 50 000 Mg of Cr every year and release 4500 kg/d into the environment. Without adequate treatment before disposal, these Cr wastes create a serious threat to water quality and the environment.

Chromium(VI) is toxic to biological systems due to its strong oxidizing potential that can damage cells (Kotas and Stasicka, 2000). Inside cells, Cr(III) formed from Cr(VI) can complex with organic compounds, interfering with metallo–enzyme systems at high concentrations (Kotas and Stasicka, 2000). Chromium(VI) has been shown to have carcinogenic and allergenic effects in humans and animals, while Cr(III) is considered a trace element essential for living systems (Costa, 1997; Nies, 1999). Due to toxicity concerns, in the USA concentrations of total Cr are regulated at 0.1 mg/L in drinking water, 5 mg/L leached from solids in the Toxicity Characteristic Leaching Procedure (TCLP) under the Resource Conservation and Recovery Act (RCRA), and as low as 200 mg/kg in biosolids (USEPA, 1990, 1994). In 1999, California set a public health goal (PHG) for chromium in drinking water at 2.5 µg/L (Montgomery Watson, 2000); however, this PHG was withdrawn in 2001 and is currently under review. Total chromium in natural soils ranges from 1 to 3016 mg/kg, with a typical concentration around 53 mg/kg (Richard and Bourg, 1991; Angeloni and Bini, 1992).

The fate of chromium entering the environment depends on the pH and redox potential of the site. Chromium(III) and Cr(VI) are stable in the pH–Eh range of most natural environments. In the normal pH range of 4 to 9, chromium exists as insoluble Cr(III) in more reducing environments, versus more soluble Cr(VI) forms (including chromate and dichromate) in oxidizing environments (Kotas and Stasicka, 2000). Various inorganics can affect the fate of chromium in the environment. Chromium(VI) has been shown to be reduced by Mn(II) to Cr(III), while colloidal MnO2 oxidized Cr(III) to Cr(VI) (Perez-Benito and Arias, 2001).

One of the treatment strategies for chromium-contaminated soil and ground water is to reduce soluble Cr(VI) to insoluble Cr(III) either ex situ or in situ. Taking advantage of microbial activity to reduce Cr(VI) could be cost effective and environmentally friendly. Natural microbes of many genera, including Bacillus, Enterobacter, Escherichia, and Pseudomonas, have been reported to enzymatically reduce Cr(VI) to Cr(III) (Bopp and Ehrlich, 1988; Chen and Hao, 1998; Garbisu et al., 1998; Ishibashi et al., 1990; Philip et al., 1998; Shen and Wang, 1994; Wang et al., 1989). In liquid treatment systems, biotransformation Cr(VI) by pure cultures has been studied under a range of redox (aerobic and anaerobic), temperature (10–45°C), and pH (6.5–9.5) conditions. Escherichia coli ATCC 33456 transformed Cr(VI) faster at 10 to 45°C under anaerobic than at 10 to 35°C under aerobic conditions, with temperature effects following Arrenhuis equations (Shen and Wang, 1994). Bhide et al. (1996) found that Pseudomonas mendocina aerobically removed Cr(VI) when molasses was added, with optimum activity at 25 to 30°C based on studies at 20 to 45°C. Philip et al. (1998) found that Bacillus coagulans, isolated from chromium-contaminated soil, reduced Cr(VI) with soluble enzymes and that cell respiration was not necessary for Cr(VI) reduction. In two studies, Cr(VI) (as dichromate and chromate) was used as an electron acceptor under anaerobic conditions by pure cultures of bacteria (Wang et al., 1989; Romanenko and Koren'kov, 1977). Based on studies at 10 to 40°C, optimum chromate reduction by Enterobacter cloacae strain H01 occurred at 30°C (Wang et al., 1989).

Indirect "biotransformations" or biologically mediated reduction of Cr(VI) to Cr(III) has been reported under sulfate- and ferric iron–reducing conditions (Apel et al., 1990; Gerlach et al., 1999; Caccavo, 2001; Petersen, 2001). At pH 7.5 and 37°C, a consortium of sulfate-reducing bacteria (SRB) showed a first-order Cr(VI) removal constant of 0.15/h for an initial Cr(VI) concentration of 1000 mg/L due to H2S production and subsequent precipitation of metal sulfide (Fude et al., 1994). Reactive permeable subsurface barriers containing SRB and iron-reducing microbes have been used to treat dissolved reducible metals including Cr(VI) in contaminated ground water (Gerlach et al., 1999; Gerlach, 2001). The Fe(II) that is produced by the iron-reducing bacteria chemically reduces Cr(VI) to Cr(III) (Gerlach et al., 1999).

Few studies provide quantitative information about Cr(VI) reduction by indigenous microbes in soil. Organic amended soil in pots reduced Cr(VI) in ground water from 1 mg/L to <50 µg/L (Losi et al., 1994b). In a study by Cifuentes et al. (1996), at room temperature indigenous microbes aerobically reduced 100 mg/L of Cr(VI) to Cr(III) in a shaker flask after 15 d with supplemental yeast extract. Turick et al. (1998) reported that under anaerobic conditions indigenous microbes reduced 65% of Cr(VI) from contaminated soil (200 mg/kg) in 128 d at 20 to 22°C with supplemental glucose. Highly contaminated soil [12 400 mg Cr(VI)/kg] reduced aqueous Cr(VI) from 1840 to 1240 mg/L in 21 d under aerobic conditions in LB (Luria–Bertani) broth in shaker flasks at 27°C (Bader et al., 1999). Indigenous microbes from uncontaminated soil reduced spiked Cr(VI) (1000 mg/L) by 23 to 24% in 15 d under the same experimental conditions (Bader et al., 1999). The mechanisms of Cr(VI) removal were not characterized in any of the aforementioned soil studies but were probably a combination of nonspecific reductase reactions and/or respiration of Cr(VI). None of the aforementioned studies with soils were conducted at lower temperatures that may occur during winter months in many ground waters.

The potential toxic inhibition of Cr(VI) on bacteria is important to biological treatment strategies. Chen and Hao (1998) summarized a number of studies on chromium resistance in bacteria. Concentrations of Cr(VI) below 65 mg/L were generally not toxic while concentrations above 100 mg/L often exhibited some level of inhibition. Therefore, microbial remediation of high concentrations of Cr(VI) in the environment may be limited by toxicity.

To design an in situ treatment strategy for contaminated soil, the abilities of indigenous microbes to biotransform Cr(VI) under various conditions must be quantified. In this work, indigenous microbial activity was evaluated at 10°C. Chromium(VI) removal was studied in the presence of various electron acceptors including oxygen, sulfate, nitrate, or Fe(III). Sugar at varying levels was tested to determine carbon requirements. Finally, the potential toxicity of high Cr(VI) concentrations was evaluated by spiking Cr(VI) into soil and mixing highly contaminated soil with less contaminated soil. This information will improve the understanding of optimal conditions to enhance microbial reduction of Cr(VI).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Soil Samples
Chromium-contaminated soil was collected from various locations at an active chrome plating facility located southeast of Denver, Colorado. Topsoil, less than 15 cm deep, was collected by spatula into clean plastic bags and stored at 4°C prior to use. Total chromium concentrations in soils with high (HI), medium (MD), and low (LO) amounts of contamination were about 22 000, 4000, and 1000 mg/kg, respectively (based on air-dry soil weights; see the Analytic Methods section for the measurement procedure). The sandy soil averaged 37, 54, 8, and 1% gravel (2–5 mm), sand, silt, and clay by weight, respectively (per ASTM [American Society for Testing and Materials] size classifications). The specific gravity of solids and soil bulk density were 2.42 and 1.35 g/cm3, respectively (Klute, 1986). The soils had an average organic matter content (Method 1-4) of 7% and pH of 8.04 (Method 12-2) (Page, 1982).

Batch Test Method
All experiments were conducted in 40-mL brown glass vials with screw caps lined with teflon septa. Each test vial contained 10 g of contaminated soil and 20 mL of basic salt medium (BSM) with various supplements and headspace gas. The BSM contained 700 mg/L KH2PO4, 1000 mg/L K2HPO4, 20 mg/L NH4Cl, 50 mg/L CaCl2, and 1 mL/L Pfennig trace nutrient mixture (Pfennig, 1974). The measured pH of the BSM was 6.8. Brown sugar (food grade) was used as a carbon supplement. Nitrogen (500 mg/L NH4Cl) was added in some of the aerobic tests to determine if nitrogen availability limited chromium removal. The ammonia concentration added is higher than would normally be present in ground water, but given the high organic content of the soil, this would ensure that the C to N ratio in the vial was no more than 135:1 (by mass, neglecting the unmeasured N content of the soil). To serve as potential alternate electron acceptors under anaerobic conditions, NaNO3, Na2SO4, and FeCl3 at 500 mg/L were added. In tests where extra Cr(VI) was added, appropriate volumes of a 1000 mg/L standard solution (K2Cr2O7) were added. The vial headspace contained air for aerobic conditions. For anaerobic conditions, vials were sparged with >99% purity N2 at the beginning of the experiment and once every week for the first month, followed by resparging after each sampling event. All test conditions were replicated with autoclaved soils as sterilized controls to account for abiotic processes. Soil microbes were killed by autoclaving in a Sterilmatic sterilizer (Market Forge Industries, Everett, MA) at 121°C for 30 min on two consecutive days. All tests and controls were set up as duplicates and incubated statically in the dark at 10°C. It is important to note that due to static incubation, microzones of different redox may have been present in the soil compared with the overlying liquid that was sampled. Experimental conditions are summarized in Table 1.


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  		Table 1. Batch experimental conditions for Cr(VI) biotransformation studies.

 
Aqueous samples of 0.2 to 1 mL were collected from the vials at selected times via syringe through the septa. Under aerobic conditions, the liquid volume that was removed from the vial during sampling was replaced with air. Gas samples were periodically tested to ensure that oxygen remained in the headspace of the aerobic vials and was absent from the anaerobic vials. A 50- to 100-µL gas sample from the headspace was injected into a GOWMAC (Lehigh, PA) Series 350 gas chromatograph with a thermal conductivity detector. Oxygen was measured on a Supelco (Bellefonte, PA) 5A 80/100 mesh molecular sieve with an 8-ft x 1/8-in (2.4-m x 3.2-mm) stainless steel packed column. The column temperature was 60°C, the detector temperature was 120°C, and the bridge current was 200 mA. At the end of the test period, the total chromium concentration in the soil was measured.

Analytical Methods
Aqueous concentrations of Cr(VI) were measured with a colorimetric method (American Public Health Association, 1992). Samples were centrifuged in 1.5-mL eppendorf tubes for 10 min at 10 000 rpm to remove turbidity. Supernatant was then diluted with MilliQ water (Millipore, Bedford, MA) to 5 mL, and 50 µL of nitric acid (1:4 HNO3 [12 M] and H2O) was added followed by 100 µL of 1,5-diphenylcarbazide. The solution was read at 540 nm on a Shimadzu (Kyoto, Japan) UV160 UV-visible recording spectrophotometer. The linear Cr(VI) detection range was from 0.05 to 2.5 mg/L.

Two methods were used to measure total Cr from soil samples. USEPA Method 3050B (performed by the Laboratory of Environmental & Geological Studies at the University of Colorado) is an acid digestion procedure using 1 g of air-dry soil followed by inductively coupled plasma analysis (USEPA, 1986). In an effort to improve total Cr recovery, a second method (American Public Health Association, 1992) used 1 to 10 g of soil, nitric acid (12 M) digestion, and potassium permanganate oxidation to transform total Cr into Cr(VI). The Cr(VI) was then measured by the colorimetric assay. Both methods cannot distinguish Cr(III) from Cr(VI), as the strong acid and oxidant solutions required to extract the Cr from the soil also results in Cr(III) oxidation to Cr(VI).

Ammonia in aqueous samples was measured by the Nessler method (American Public Health Association, 1992). The detection range was from 0.1 to 10 mg/L with a spectrophotometer at the 425-nm wavelength. Nitrate in aqueous samples was measured by spectrophotometric absorbance at 220 and 275 nm (American Public Health Association, 1992). The detection range for nitrate was from 0.1 to 10 mg/L, with samples diluted as needed with MilliQ water prior to analysis. Standard curves containing varying nitrate and Cr(VI) concentrations were used to correct for Cr(VI) interference on the nitrate analysis. Initial redox in some of the vials was measured with an Orion (Beverly, MA) Model 720A benchtop meter with a Cole Parmer (Vernon Hills, IL) combination ORP electrode. The electrode was inserted into the open vials, with the anaerobic systems under active nitrogen gas sparging.

To measure dissolved organic carbon (DOC), aqueous samples were diluted as needed with Milli-Q water to 6 mL, filtered with 0.45-µm glass microfiber filters (Whatman, Maidstone, UK), and analyzed on Shimadzu 5000 TOC analyzer. Based on known sugar standards, the measured relationship to DOC was: DOC in mg/L = 0.4223 (brown sugar, mg/L) - 0.1441. The quantification range of DOC was from 1 to 20 mg/L, with a detection limit of around 0.2 mg/L.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Desorption of Chromium(VI) from the Site Soil
In tests without chromium spiked into the liquid, no Cr(VI) would be initially present in solution. As Cr(VI) partitioned from the soil into the aqueous phase, it was detected with the analytic method used. The use of the phosphate-buffered basal salt medium in the experiments probably led to significant dissolution of so-called "exchangeable" Cr(VI) from the soil. Because of competition for adsorption sites, KH2PO4 has been found to be a good extraction agent for Cr(VI) from soil (Bartlett and Kimble, 1976; James and Bartlett, 1983).

In sterilized controls, the aqueous Cr(VI) concentrations increased over time for up to 65 d. Chromium desorption was influenced by the different electron acceptors, carbon addition, and initial Cr concentration in the soil (selected results shown in Fig. 1) . Carbon addition decreased aqueous Cr(VI) concentrations by 0 to 25%. In tests with aerobic sterilized soil, Losi et al. (1994a) found approximately 40% lower extractable Cr(VI) in manure-amended soil compared with unamended soil and attributed this difference to organics serving as an electron donor for the reduction of Cr(VI) to Cr(III). This same abiotic reduction of Cr(VI) may account for the lower aqueous Cr(VI) concentrations measured in the sugar-amended controls in this study.



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  		Fig. 1. Aqueous Cr(VI) concentrations measured over time in sterilized controls with (A) low-contamination soil (LO) and (B) high-contamination soil (HI), with and without carbon addition and selected electron acceptor conditions shown.

 
In sterilized controls with LO soil and HI soil without carbon addition, the most Cr(VI) desorbed from the following conditions: sulfate > nitrate {cong} aerobic > anaerobic > iron. The largest differences occurred early in the experiment with 16 to 63% versus 2 to 36% more aqueous Cr(VI) in the sulfate-amended vials compared with the other electron acceptor conditions on Days 3 and 63, respectively. In the sterilized controls with HI soil and carbon addition, the conditions that resulted in the highest aqueous Cr(VI) concentrations were: nitrate > sulfate > anaerobic > iron > aerobic. Sulfate has been found to reduce Cr(VI) adsorption to soil, presumably by competitive ion effects (James and Bartlett, 1983). Competitive anion effects toward Cr(VI) sorption to spent grain have also been reported for sulfate > nitrate > phosphate (Low et al., 2001). Baron et al. (1996) reported that ferric-chromate precipitates were found in soils at a chromium-plating facility. A similar interaction of Fe(III) with Cr(VI) possibly occurred in these experiments to account for the lowest aqueous Cr(VI) concentrations measured in the Fe(III)-amended sterilized controls.

Redox measurements in the batch systems were complicated by the "open" system required to allow insertion of the probe. The initial redox in vials with HI soil under aerobic and aerobic + NH4 conditions, either with or without carbon added, averaged 196 ± 10 mV. All of the anaerobic conditions with nitrogen headspace had a similar measured initial redox of 115 ± 26 mV. While it is logical that the anaerobic conditions had a lower redox than the aerobic conditions, the exact values in the anaerobic systems are somewhat unreliable due to the "open" vial during measurement versus closed caps during final nitrogen sparging. Redox in the soil and its pore water may also have been different than the overlying solution that was measured. In addition, the redox probably declined over time in the nonsterile systems due to bioactivity.

Because "background" aqueous Cr(VI) concentrations changed over time, microbial Cr(VI) removal was computed by comparing aqueous concentrations in the live vials to their respective sterilized controls. For example, if the aqueous Cr(VI) concentrations measured on Day 4 were 3 and 2 mg/L Cr(VI) in sterilized and live vials, respectively, and on Day 9 were 3.5 and 1 mg/L Cr(VI), the Cr(VI) removal by bioactivity was 1 mg/L on Day 3 and 2.5 mg/L on Day 9, with an average rate of Cr(VI) removal of 0.3 mg/(L d). Alternatively, Cr(VI) reductions due to bioactivity are expressed as the aqueous Cr(VI) concentration in live soil - aqueous Cr(VI) concentration in sterilized soil.

Electron Acceptor Effects on Chromium(VI) Reduction in Low-Chromium Soil
In tests with the LO soil, aqueous Cr(VI) concentrations in the sterilized controls increased from 2.5 to 4 mg/L over 3 to 63 d. As shown in Fig. 2A , between 3 to 63 d there was 25 to 60% less Cr(VI) in the aqueous phase of live versus sterilized controls. There were no definite trends in these removal percentages over time, indicating that most of the bioactivity occurred within the first 3 d. The aerobic with additional NH4 condition had the most aqueous Cr(VI) removal relative to the sterilized control, indicating that ammonia addition may have provided a nutrient source allowing enhanced biogrowth and therefore chromium removal. Headspace gas analyses did not detect oxygen in the anaerobic vials, while oxygen was depleted by 19 to 32% in the aerobic vials. These results indicate that bioactivity reduced aqueous Cr(VI) concentrations somewhat, but not sufficiently for remediation.



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  		Fig. 2. Effect of electron acceptors on the aqueous Cr(VI) concentration in live vials over time shown as a percentage of the concentration measured in appropriate sterilized controls. (A) No carbon; (B) 500 mg/L sugar added.

 
Effect of Carbon Addition on Chromium(VI) Reduction in Low-Chromium Soil
The addition of 500 mg/L of sugar to the LO soil significantly increased the microbial removal of Cr(VI), as shown in Fig. 2B. At 7 d and beyond, Cr(VI) was not detected in the aqueous phase of the live nitrate- and sulfate-amended anaerobic vials. At 14 d and beyond aqueous Cr(VI) was not detected in aerobic + NH4, anaerobic, and iron-amended vials. Aqueous Cr(VI) remained at 20% of the sterilized control in the aerobic without additional NH4 vials from 40 to 63 d. This indicates that nitrogen addition was beneficial for microbial Cr(VI) removal under aerobic conditions. By comparing Fig. 2A and 2B it is clear that significantly better Cr(VI) biotransformation occurred under all electron acceptor conditions when carbon was added. Similar benefits of carbon addition on microbial Cr(VI) removal have been previously reported under aerobic, anoxic, and anaerobic conditions (Cifuentes et al., 1996; Losi et al., 1994b; Fujie et al., 1996; Turick et al., 1998).

Of the 136 mg/L NH4–N added to the aerobic + N vials, 113 mg/L NH4–N was measured in the aqueous phase at Day 63 in the sterilized vial compared with 83 mg/L NH4–N in the live vials. In a 1-d test with sterilized soil, 30 mg/L of NH4–N was "lost" from the aqueous phase due to soil interaction. This result confirms that ammonia N was microbially consumed and was probably limiting in the aerobic vial to which only 5 mg/L NH4–N was added. Aqueous concentrations of nitrate N at the end of 63 d averaged 74 mg/L in the sterilized vials and 0.5 mg/L in the live vials, compared with the 82 mg/L nitrate N dosed. In a second sterilized control test, 72 mg/L aqueous nitrate N was measured after 1 d, indicating that the soil influenced nitrate recovery. This indicates that microbial nitrate reduction occurred under anaerobic conditions in the nitrate-amended vials.

Total chromium was measured at the end of the carbon-amended experiment, with results summarized in Table 2. Total Cr should be the same in the pre-treated, sterile, and bioactive conditions since chromium cannot be created nor destroyed in the experiments. Soil heterogeneity probably led to somewhat different initial Cr concentrations in the replicate vials, as evidenced by the standard deviation in the measurements. As expected, there was no significant difference (at 90% confidence using Student's t test) between the total Cr recovered from the live conditions (except sulfate amended), sterilized soil, and pre-treated soil.


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  		Table 2. Total chromium in low-contamination soil before treatment and at the end of 63 d of incubation with added carbon under different electron acceptor conditions.

 
The significantly lower total Cr recovered from the live sulfate-amended soils may be due to the fact that USEPA Method 3050 does not break down all mineral components from soil samples (USEPA, 1986). Pintail Systems (1998) reported that anaerobic bioreactors produced complex chromium minerals such as chromite and tarapacaite from chromic acid wastewater. The "losses" of chromium due to abiotic reactions were explored further. In systems containing BSM (no soil) and Cr(III) with and without 300 mg/L Na2S, similar total Cr was measured by Method 3050B. In vials with BSM (no soil) and Cr(VI), total Cr concentrations were reduced from 49.4 to 16.7 mg/L when 300 mg/L Na2S was added. This indicates that sulfide and elements in the BSM reacted with Cr(VI) to form precipitates and complexes from which the Cr was not extracted to allow detection. It is therefore speculated that in the live anaerobic vials with added sulfate that microbial sulfide formation occurred, which then reacted with the Cr(VI) and rendered it undetectable by Method 3050B. Because the chromium was not extracted from the soil under a vigorous acid-digestion procedure, it indicates that the sulfide reaction with chromium may be the most stable way to sequester toxic Cr from the environment.

The DOC concentrations in the aerobic + N and carbon vials were measured at the end of the experiment. The live vial contained 29% less carbon than the sterilized control. This indicates that some carbon was consumed during biological Cr(VI) removal.

Chromium(VI) Reduction in the High-Chromium Soil
No significant aqueous Cr(VI) removal in the live vials compared with sterile controls was noted over the first 30 d with the HI soil under any electron acceptor condition. At Day 63 an average of 36 mg/L (19%) less aqueous Cr(VI) was present in the nonsterile vials (data not shown). The performance was not significantly better when 500 mg/L sugar was added, with an average of 41 mg/L (21%) Cr(VI) removal under all six electron acceptor conditions. The aqueous Cr(VI) concentrations of 100 to 200 mg/L are in the range where Cr(VI) toxicity to bacteria has been reported (Chen and Hao, 1998). Although the percent removal is significantly lower in the HI soil compared with the LO soil, the concentration of Cr(VI) removed was greater than the 4 mg/L in the LO soil with carbon. The approximate 30-d lag in Cr(VI) removal may be due to low bacterial populations in the HI soil requiring time for adaptation and growth before evidence of activity, particularly in light of potential Cr(VI) toxicity. The DOC analysis on the aqueous phase of the aerobic vials measured an average of 6% less sugar in the live plus carbon vials compared with the sterilized controls, evidence of some level of bioactivity. The significantly lower carbon removal compared with the LO soil indicates that the carbon consumption in the LO vials was probably not fully due to microbial Cr(VI) transformation but may have been due to more general bioactivity in the soils associated with aerobic heterotrophs and nitrate-reducing heterotrophs.

Chromium(VI) Reduction in Mixed High- and Medium-Chromium Soil
Due to poor removal efficiency of Cr(VI) in the HI soil and suspected toxicity, tests were conducted with mixtures of HI and MD soil to investigate different Cr concentrations. Carbon (500 mg/L sugar) was added to enhance Cr(VI) removal. Aqueous concentrations of Cr(VI) were measured after 9 and 16.5 wk of incubation under aerobic + N and anaerobic nitrate- and sulfate-amended conditions. Aqueous Cr(VI) concentrations with 100% HI or MD soil in sterile controls ranged from 160 to 300 and 2 to 4 mg/L, respectively. Results of Cr(VI) removal compared with sterilized controls are summarized in Fig. 3 . After 9 wk, aqueous Cr(VI) concentrations were below the detection limit for up to 50 and 30% HI soil mixtures under aerobic + N and sulfate-amended conditions, respectively [representing 100% aqueous Cr(VI) removal]. With 50% HI soil under sulfate-amended conditions, 69 and 93% Cr(VI) removal were measured at 9 and 16.5 wk, respectively. At 70% HI soil, about 50% removal was noted at 9 wk under both aerobic and sulfate amended conditions, increasing to around 60% removal at 16.5 wk. With 100% HI soil, there was 18 to 30% Cr(VI) removal after 16.5 wk.



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  		Fig. 3. Percent aqueous Cr(VI) removal measured in mixtures of high- (HI) and medium-contamination (MD) soil compared with sterilized controls. Close hatch bars represent removal observed after 9 wk, and the bars with wider hatch represent removal between 9 and 16.5 wk.

 
Nitrate-reducing conditions exhibited the least Cr(VI) removal, with a lag greater than 9 wk until activity occurred with >=50% HI soil. This may be due to inhibition of Cr(VI) reduction by nitrate, as reported by Petersen (2001) for Shewanella oneidensis MR1 at 28 mg/L nitrate N. In contrast, Philip et al. (1998) found that Cr(VI) reduction in aerobic aqueous solution by a pure culture of Bacillus coagulans was not changed by sulfate and nitrate up to 1000 mg/L. Chromium(VI) transformation under anaerobic conditions by Escherichia coli strain ATCC 33456 was not affected by the presence of up to 8000 mg/L nitrate or 4000 mg/L sulfate (Shen and Wang, 1994). The mechanism of nitrate inhibition of chromium may be due to serving as a competitive electron acceptor against Cr(VI), causing a higher redox, which inhibits microbial Cr(VI) reduction, or that neither the MD or HI site soils contained appropriate bacteria (such as the B. coagulans or E. coli) to function under nitrate-amended anaerobic conditions.

The average Cr(VI) removal rate was calculated by dividing the concentration of Cr(VI) removed from the live versus sterilized conditions at 9 and 16.5 wk with the elapsed time, as summarized in Fig. 4 . Removal rates are plotted against the Cr(VI) concentration that best represents the range encountered by the bacteria under the electron acceptor conditions of interest over the 9- to 16.5-wk period. For example, if the sterile 100% HI soil condition had an aqueous Cr(VI) concentration of 250 mg/L at Week 9 while the nonsterile 100% HI soil vial contained 0 mg/L, the rate was (250 mg/L - 0 mg/L)/9 wk = 4.0 mg/(L d) at an average Cr(VI) concentration of 125 mg/L. It appears that the removal rate increases with increasing amounts of HI soil (increasing Cr(VI) concentration) up to 50% and then levels off and/or decreases. However, it is likely that the time until 100% Cr(VI) removal occurred in <=30% HI soil mixtures was prior to the measurement at 9 wk, causing the calculated Cr(VI) removal rate to be lower than the actual rate. Toxicity could result in the slower rates at >50% HI soil, corresponding to aqueous concentrations of Cr(VI) of >100 mg/L for aerobic soil and >150 mg/L for sulfate-reducing soil. Alternatively, the observed trends could be due to lower populations or different species of bacteria in the HI soil compared with the MD soil. The trends may be following traditional Monod kinetics, with biotransformation of high Cr(VI) concentrations in the zero order regime combined with lower biomass concentrations in the HI soil giving a net result of lower Cr(VI) removal rates.



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  		Fig. 4. The Cr(VI) biotransformation rate in the presence of different electron acceptors as calculated by comparison with sterile controls.

 
Final DOC concentrations in the vials were measured in the aerobic and sulfate-reducing vials (data not shown). In the live aerobic vials, measured DOC concentrations were 31 and 63% of the DOC measured in the sterilized controls for the 100% and 0% HI soil mixtures, respectively. In the live sulfate-reducing vials, measured DOC concentrations were 47 and 97% of the DOC measured in the sterilized controls for the 100% and 0% HI soil mixtures, respectively. This indicates a general trend to more sugar consumed with higher Cr present. It also shows that less carbon was consumed under sulfate-amended versus aerobic conditions.

The amount of DOC consumed per amount of Cr(VI) removal from the aqueous phase was calculated. For the 100% MD soil under aerobic and sulfate conditions there was 20 and 14 kg DOC/kg Cr(VI), respectively. Under aerobic conditions with 30 to 100% HI soil the average was 1.4 ± 0.4 kg DOC/kg Cr(VI). Under sulfate-amended conditions with 30 to 70% HI soil the average was 0.8 ± 0.1 kg DOC/kg Cr(VI), versus 1.7 to 1.8 kg DOC/kg Cr(VI) with 10 and 100% HI soil. The reasons for these differences are not known. It is speculated that heterotrophic activity not directly related to Cr(VI) removal [and subjected to minimal potential for Cr(VI) toxicity] occurred at the 10% HI soil while at 100% HI soil Cr(VI) toxicity resulted in less efficient carbon use. The DOC concentrations in the nitrate-reducing vials were not measured.

Chromium(VI) Reduction When Spiked into Low-Chromium Soil
Because chromium toxicity cannot be conclusively determined from the mixed soil study due to suspected differences in the indigenous bacteria present in the MD versus HI soils, a spike test was conducted by adding 500 mg/L sugar and varying amounts of Cr(VI) to the LO soil. All six electron acceptor conditions were tested with a 30 mg/L Cr(VI) spike and analyzed from Day 3 to 63, while only three conditions (aerobic + N, nitrate, and sulfate) were tested with variable Cr(VI) spikes from 12.5 to 200 mg/L and measured from Day 2 to 28. In the sterilized controls, measured aqueous Cr(VI) concentrations decreased from Day 2 to 5 in the spiked vials and then remained fairly constant (data not shown). Of the initial Cr(VI) spike concentration of 12.5 to 200 mg/L, 7 to 24% sorbed to the soil in the sterilized controls [giving final steady state aqueous Cr(VI) concentrations ranging from 11.6 to 156 mg/L, respectively].

With a 30 mg/L spike (Fig. 5) , the aerobic + N condition removed the most Cr(VI), with 100% Cr(VI) removed at 63 d. The average rate of Cr(VI) removal over the first 21 d was 1.55 mg Cr(VI)/(L d). This is faster than the Cr(VI) removal rate in the unspiked soil of about 0.30 mg/(L d). The Cr(VI) removal was similar under all of the other electron acceptor conditions, with increasing Cr(VI) removal over the first 30 d to around 40%, after which Cr(VI) was fairly constant. Because the Cr(VI) biotransformation did not continue to completion, it is likely that carbon or nutrients became limiting and/or that inhibition stopped bioactivity.



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  		Fig. 5. Aqueous Cr(VI) reduction over time in low-contamination (LO) soil spiked with 30 mg/L of Cr(VI) and carbon under six electron acceptor conditions.

 
Another example of Cr(VI) removal over time is shown for the 12.5 mg/L spike in Fig. 6 . There is a "lag" for 6 to 8 d (probably due to bacterial dormancy caused by approximately 3 mo of soil storage at 4°C), after which Cr(VI) is removed at an average of 0.41, 0.15, and 0.12 mg Cr(VI)/(L d) under aerobic, sulfate, and nitrate conditions, respectively. The average Cr(VI) removal rates for all six spiked concentrations were computed over the first 4 wk, and results are shown as a function of the average Cr(VI) concentrations measured in the sterilized controls in Fig. 7 . The aerobic microbes with added ammonia generally performed better than nitrate- and sulfate-reducing microbes at most of the Cr(VI) spike levels. Ignoring the somewhat unusual point at 45 mg/L Cr(VI) for the nitrate and sulfate conditions, there is no evidence that increasing aqueous Cr(VI) concentrations up to 160 mg/L [due to 200 mg/L Cr(VI) spike less soil sorption] are toxic. Under aerobic conditions increasing Cr(VI) concentrations up to 160 mg/L increases the rate of Cr(VI) removal, with an almost first order relationship evident. Aerobic conditions seem increasingly favorable for Cr(VI) removal at higher aqueous Cr(VI) concentrations compared with nitrate and sulfate available under anaerobic conditions.



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  		Fig. 6. Aqueous Cr(VI) reduction over time in low-contamination (LO) soil spiked with carbon and 12.5 mg/L Cr(VI).

 


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  		Fig. 7. The Cr(VI) biotransformation rate versus aqueous Cr(VI) concentration in Cr(VI) spiked soil with carbon under three electron acceptor conditions.

 
The spike test results indicate that the decreasing chromium transformation rates at high concentrations observed in the mixed soil study were probably due to differences in microbial species and/or populations, rather than inherent toxicity of Cr(VI). Comparing the Cr(VI) removal rates in Fig. 7 and 4, significantly higher Cr(VI) removal occurred under sulfate-amended anaerobic conditions in the mixed soil, which may indicate that higher populations of appropriate microbes were present in the MD soil than the LO soil. It is also likely that the HI soil contained lower populations of appropriate microbe species than the MD soil. Therefore, increasing Cr(VI) transformation rates at higher Cr(VI) concentrations were overshadowed by decreasing microbes available for Cr transformation in the highly contaminated soil. In addition, the total Cr concentrations in the HI soil were much higher than in the LO soil, indicating potential bacterial toxicity of sorbed Cr(VI) and/or Cr(III).

Effect of Carbon Concentration on Chromium(VI) Reduction
Earlier studies have shown that carbon addition enhanced chromium biotransformation. However, the levels of carbon required were not determined nor was carbon consumption directly measured. To optimize biotreatment of Cr(VI), the dose of carbon required should be determined. Fig. 8 shows the change of aqueous Cr(VI) over 15 d in vials containing MD soil amended with 0.16 to 500 mg/L of sugar under aerobic + N and anaerobic (without supplemental electron acceptor) conditions. The average Cr(VI) concentration in the sterilized controls due to desorption of Cr(VI) is also shown. Under aerobic conditions with 500 or 100 mg/L sugar, aqueous Cr(VI) concentrations were below detection limit within 6 d. Lower amounts of Cr(VI) removal correlated with lower amounts of added carbon, indicating that carbon limited Cr(VI) removal. Under anaerobic conditions, similar results were achieved.



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  		Fig. 8. Aqueous Cr(VI) concentrations over time as a function of variable spiked concentrations of brown sugar in mg/L to live and sterilized medium-contamination (MD) soil. (A) aerobic + N; (B) anaerobic.

 
Dissolved organic carbon concentrations were measured for the high carbon-amended conditions, with results shown in Fig. 9 . The average of the replicate vials is shown by the data point (the range of the replicates was generally within the size of the symbol, so error bars are not shown). The "initial" Day 0 DOC concentration is the calculated DOC equivalent of the added sugar. With 100 mg/L sugar added (42 mg/L DOC, Fig. 8A), DOC removal was similar in the live vials under aerobic and anaerobic conditions. The Day 15 DOC concentrations in the sterilized controls were similar to the carbon represented by the 100 mg/L sugar; the reason for the low DOC value measured in the sterilized controls on Day 6 is not known. When 500 mg/L sugar was added (Fig. 9B), aerobically the DOC consumption was much higher than anaerobically. The increasing DOC concentration in the anaerobic sterilized control may be due to natural organic matter desorbing from the soil, although this was not observed in the other sterilized controls. Carbon consumption was not directly dependent on chromium removal, since significant sugar uptake occurred in comparison with sterilized controls while aqueous Cr(VI) concentrations remained zero (Days 6 to 15). However, this could be due to bacteria continuing to transform Cr(VI) as it desorbed from the soil into solution, thereby maintaining near-zero aqueous concentrations. This seems likely since aqueous Cr(VI) concentrations continued to increase in the sterilized controls during this time period.



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  		Fig. 9. Average dissolved organic carbon (DOC) concentration measured over time in live and sterilized vials under aerobic and anaerobic conditions. (A) 42 mg/L DOC spike; (B) 211 mg/L DOC spike.

 
The amount of DOC consumed per amount of Cr(VI) removed was calculated by difference between the live and sterile conditions, with results summarized in Table 3. Over the first 6 d a similar amount of DOC was consumed per mass of Cr(VI) removed under aerobic and anaerobic conditions, but with significantly more carbon consumed at the higher dose. The Day 6 to 15 quantities are similar to the Day 0 to 6 values under anaerobic conditions, but were significantly higher under aerobic conditions due to the very low amounts of additional Cr(VI) removed. Under aerobic conditions carbon consumption may have been largely dominated by non-Cr(VI) reducing aerobic heterotrophs. Under anaerobic conditions, the amount of carbon consumed is also much larger than should be required if Cr(VI) were the only electron acceptor linked to sugar utilization as an electron donor. The stoichiometry for "sugar" (represented by glucose) mineralization is:



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  		Table 3. Carbon consumption during Cr(VI) biotransformation at doses of 500 or 100 mg/L brown sugar.

 
Only 0.17 mg C/mg Cr(VI) would be needed if Cr(VI) were a primary electron acceptor under anaerobic conditions. Therefore, a significant amount of sugar was probably consumed by bacteria not related to Cr(VI) removal or by bacteria that removed Cr(VI) via nonspecific reductases.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
This study provides information relevant to designing in situ microbial treatment of Cr(VI)-contaminated soil beyond what currently exists in the literature. All the studies were conducted at 10°C, which is below the temperature that has been tested in previous soil work. In contrast to studies emphasizing a single "form" of microbial activity, direct comparison of multiple electron acceptor environments with the same site soil indicates how engineered interventions to add electron acceptors would work in the field. Results indicate that Cr(VI) biotransformation occurs in the presence and absence of a wide range of electron acceptors including oxygen, nitrate, sulfate, and ferric iron. The optimal conditions are anaerobic at lower Cr(VI) concentrations and aerobic at higher Cr(VI) concentrations. In situ, natural soil will contain microzones of varying redox even if the bulk conditions are predominantly a single redox (such as aerobic in vadose zone soil and anaerobic in deeper saturated soils with varying alternate electron acceptors depending on ground water conditions). Therefore, it does not appear that strict control of redox or electron acceptors is necessary to ensure Cr(VI) biotransformation.

The most critical factor limiting chromium biotransformation appears to be carbon availability. More carbon will need to be added under aerobic conditions due to "nonproductive" carbon consumption that is not coupled with Cr(VI) biotransformation. Depending on the levels of Cr(VI) present in the soil, high concentrations of carbon addition may be needed. A simple sugar served as an adequate carbon source, and other substrates such as molasses could be equally as effective. Turick et al. (1998) found that basic salt medium with glucose achieved the same anaerobic Cr(VI) reduction as tryptic soy broth medium, while mineral salts without a carbon source did not. The result (Table 3) from various concentrations of carbon suggest that the relationship between organic carbon dosing and its efficiency of reducing Cr(VI) can be estimated to make the cost of supplements more economical for soil treatment projects.

Additional nitrogen appeared favorable to enhancing aerobic transformation of Cr(VI). All test conditions had 5 mg/L NH4–N added, and under aerobic conditions 136 mg/L NH4–N achieved better Cr(VI) transformation than the 5 mg/L NH4–N. The effect of varying levels of supplemental nitrogen on aiding anaerobic biotransformation was not studied.

The rates of Cr(VI) removal from aqueous solutions in contact with contaminated soil varied depending on the availability of sugar and electron acceptors. The maximum rates under aerobic conditions with added carbon were 1.6 mg Cr(VI)/(L d), and 1.4 mg Cr(VI)/(L d) under nitrate- and sulfate-reducing conditions. The anaerobic rates are in the range of results from Turick et al. (1998) under anaerobic conditions at 20°C of 1.16 to 1.3 mg/(L d). The aerobic rates measured in this study are lower than the literature-reported aerobic rates that ranged from 8.7 to 67 mg/(L d) at 20 to 27°C under well-mixed conditions (Cifuentes et al., 1996; Bader et al., 1999). Significantly higher carbon concentrations (such as 2000–5000 mg/L glucose, yeast extract, etc.) were present in all of the previously referenced studies. Note that since biomass concentrations in soil are not known it is not possible to compare the reported biotransformation rates from the soil studies with reported rates from suspended culture studies.

With 160 mg/L aqueous Cr(VI) due to spiking into the LO soil, the rate of microbial Cr(VI) removal under aerobic conditions was three to five times the rate under anaerobic conditions (Fig. 8). Results from the mixed soil study indicated that sulfate-reducing microbes had greater Cr(VI) biotransformation rates than aerobes with HI soil. Mixed soil had much more total Cr (due to adsorbed Cr) than was present in the spiked study, such that direct comparisons and assumptions about biomass concentrations cannot be made. Cifuentes et al. (1996) found that 104 mg/L of Cr(VI) was reduced in 12 d in soil at 22°C. In the study by Bader et al. (1999), the highly contaminated soil aerobically reduced aqueous Cr(VI) from 1840 to 1240 mg/L in 21 d, and the addition of more nutrients further reduced 30% Cr(VI) over the next 16 d. All these results show that indigenous soil microbes appear well suited for Cr(VI) transformation in highly contaminated soils.

The total Cr analysis (Table 2) indicated that under sulfate-amended conditions, about 60% of the total chromium was not recovered by a strong acid digestion and oxidation procedure. This was attributed to various mineral forms of chromium and sulfide. Therefore, it appears that very stable final chromium forms can be achieved as a result of microbial activity, with minimal risk of rerelease of Cr(VI).

Our results echo previous studies indicating that indigenous microbes are suitable for in situ Cr(VI) treatment, regardless of redox or electron acceptor availability, provided that sufficient carbon is available. Microbial activity can probably be maintained year-round at many sites, since favorable results were achieved at low temperatures (10°C). Pilot scale studies with soil columns are needed to explore issues such as dosing frequency, kinetics, nutrient delivery, bioclogging, potential for rerelease of Cr(VI), and other issues pertaining to field application.


   ACKNOWLEDGMENTS
 
The authors thank Mike Hansen from Power Engineering for help with soil collection, Sung-kil Park for soil characterization, and Myoungsuk Song for DOC analysis.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 





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