Waste Management 22 (2002) 195–200 www.elsevier.com/locate/wasman

Effect of microbial activity on the mobility of chromium in soils V. Desjardina,*, R. Bayarda, N. Hucka, A. Manceaub, R. Gourdona a LAEPSI, INSA de Lyon Baˆt. 404, 69621 Villeurbanne Cedex, France LGIT-IRIGM Observatoire de Grenoble, Universite´ J. Fourier, BP 53 38041 Grenoble Cedex 09, France

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Abstract The effect of microbial activity on the chemical state of chromium, in a contaminated soil located in the Rhoˆne-Alpes region (France), has been investigated. This soil contained 4700 mg kg 1 Cr, with about 40% present in the soluble hexavalent form. Indigenous microbial activity was found to significantly reduce Cr(VI) to the less mobile form (III) when the soil was incubated at 30  C in an aqueous medium containing glucose and nutrients. A Cr(VI)-reducing strain of Streptomyces thermocarboxydus was isolated from the contaminated soil. The strain was found to metabolize Cr(VI) in a similar manner as an exogenous inoculum of Pseudomonas fluorescens LB300, and to precipitate chromium as a Cr oxyhydroxide with a gCrOOH-like local structure. The Cr(VI)-reducing activity of S. thermocarboxydus was induced, or significantly accelerated, by the aggregation of bacterial cells or their adhesion to suspended solid particles, and was stimulated in pure culture by glycerol and chromate. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cr(VI) reduction; Bio-reduction; Streptomyces; Mobility

1. Introduction Chromium has a large number of industrial applications, such as the manufacture of anticorrosive agents, metal plating, leather tanning, etc. Chromate (CrO24 ) and dichromate (Cr2O27 ) are discharged frequently in the environment [1]. The migration of chromium in soils and surface- or ground-waters, from its source of pollution, and its toxicity mainly depend on its speciation [2,3]. In its oxidized Cr(VI) form, chromium is highly soluble in water and therefore mobile, whereas the reduced Cr(III) form is almost insoluble in water, and thus immobile, at pH levels higher than 5. In contrast to chromic ions, chromates are also highly toxic. Human exposure to Cr(VI) can result in ulceration of the skin, eyes and mucus membranes. Finding methods to reduce Cr(VI) to its less toxic and mobile Cr(III) form is important in order to decrease the chemical risk and to prevent Cr migration to underground water. In natural systems, Cr(VI) can be reduced to Cr(III) by abiotic chemical reactions involving the oxidation of organic substances or mineral ions, such as sulfides or Fe2+ [4]. Microorganisms, such as Pseudomonas fluorescens LB300, also have the ability to reduce chromium [5]. * Corresponding author. Tel.: +33-4-7243-8753; fax: +33-4-72438717. E-mail address: [email protected] (V. Desjardin).

Under aerobic or anaerobic conditions, microbial processes can drastically accelerate chromate reduction. The present study investigates the bioreduction of Cr(VI) by a new Cr(VI)-reducing bacterium, isolated from a contaminated soil: Streptomyces thermocarboxydus, named NH50.

2. Materials 2.1. Bacterial strains and growth conditions Two mineral salts media, modified Vogel-Bonner (VB) (K2HPO4 60 mM, Na2HPO4.10H2O 20 mM, NH4Cl 15 mM, MgSO4.7H2O 1 mM, and citric acid 2 g l 1) [3] and M63 (KH2PO4 0.1 M, (NH4)2SO4 15 mM, FeSO4 3.2 mM, MgSO4 1.6 mM, thiamine 0.0001%; adjusted to pH 6.8 with KOH 6M) supplemented with a carbon source (glucose 7.5 or 10 g l 1 in VB or M63, respectively, or glycerol 3 g l 1) were used for growing Streptomyces thermocarboxydus NH50 and Pseudomonas fluorescens LB300. Luria broth (LB) (bactotryptone 10 g l 1, yeast extract 5 g l 1 and NaCl 5 g l 1 adjusted to pH 7 with NaOH) was used to obtain exponential cultures of P. fluorescens LB300. Spore suspensions of S. thermocarboxydus were prepared, as described by Hopwood et al. [6], and spore concentration was measured by spreading 0.1 ml of a 10 6 and a 10 8 dilution

0956-053X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0956-053X(01)00069-1

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on LB Petri plates containing 1.5% agar (w/v). Media were autoclaved at 120  C for 20 min. Glucose was autoclaved separately. Liquid media were inoculated with 1–2109 spores l 1 of Streptomyces and cells were grown at 30  C in an orbital incubator at 105 rpm. 2.2. Experiments with contaminated soil A soil contaminated by leaking drums of metal finishing effluents was selected for this study. The soil contained 4700 mg Cr per kg of dry matter, with about 40% present as Cr(VI), and it was also contaminated with other metals, including lead (4600 mg kg 1) and nickel (5100 mg kg 1). The bioreduction of Cr(VI) was studied in the laboratory by incubating, in 500 ml shaker flasks at 30  C and under aerobic conditions, 5 g of soil in 50, 100 or 200 ml of modified VB medium (L/S ratio of 10, 20 or 40, respectively). In some assays, sterile soil suspensions, obtained by autoclaving the soil at 120  C for 20 min, were inoculated with an exponential exogenic culture of P. fluorescens LB300, already reported as being capable of reducing Cr(VI) aerobically [5,7]. Other assays were performed without autoclaving the soil and in the presence or absence of added exogenic inoculum. Finally, blanks were prepared under sterile conditions without inoculation. The concentration of Cr(VI) in solution was followed as a function of time in the blanks and assays. At least three replicates of each blank and assay were performed. 2.3. Preparation of samples for XANES and EXAFS spectroscopy The speciation of metabolized chromium was studied by non-destructive XANES (X-ray Absorption Near Edge Structure) and EXAFS (Extended X-ray Absorption Fine Structure) spectroscopy. Cultures of P. fluorescens and S. thermocarboxydus were grown separately at 30  C until sufficient biomass was obtained, in VB glucose medium (with no soil added), spiked with 1 mM  10% Cr(VI) added as K2CrO4. Microbial biomass was then recovered by centrifugation and a portion of the pellet mounted in a cell with kapton windows for spectroscopic measurements. XANES and EXAFS spectra were collected at the synchrotron radiation source (SRS) in Daresbury (UK). Details on data collection and reduction have been described elsewhere [8].

were inoculated with 109 spores l 1, and 1 mM of Cr(VI), added as K2CrO4. To locate the reducing activity, S. thermocarboxydus was grown in M63 glycerol medium, with or without 1 mM of Cr(VI), for 1 week in 5 ml test tubes. Cultures were centrifuged at 3000 g and supernatants were recovered and filtered on a 0.2 mm Millipore filter to remove the bacterial cells. The Cr(VI) concentration of the supernatants was measured and adjusted to 1 mM to follow chromate reduction in both supernatant S [from a Cr(VI)-free culture] and supernatant SCr (from a Cr(VI) culture) under the same conditions. 2.5. Determination of Cr(VI) concentration Cr(VI) was titrated using the diphenylcarbazide (DPCZ) color reaction. One hundred ml of DPCZ solution (0.3 g of chemically pure DPCZ, diluted to a final volume of 100 ml with 95% ethanol and 400 ml of sulphuric acid at 176 g l 1) was added to 1 ml of sample diluted appropriately (generally 1/200). DPCZ reacts with chromate, forming a purple complex with a maximum absorbance at 540 nm. If necessary, an aliquot was centrifuged before dilution to remove any cells or soil particles.

3. Results and discussion 3.1. Cr(VI)-reducing activity in soil Results obtained from the batch assays on soil in VB glucose medium, at a liquid to solid ratio of 40, are shown in Fig. 1. Similar results (data not shown) were obtained at lower L/S ratios, although a longer time was needed to reach a final Cr(VI) concentration close to zero suggesting a possible inhibitory effect in Cr(VI) and/or other metals on the bacterial activity. Only a slight decrease of Cr(VI) concentration was observed in

2.4. Cr(VI) reduction by S. thermocarboxydus NH50 The effect of the carbon source was studied with cultures of S. thermocarboxydus either in water or in M63 minimal medium, supplemented with a carbon source (glucose 10 g l 1 or glycerol 3 g l 1). Experiments with the soil were carried out using 2.5 g of soil in 100 ml of liquid medium, and cultures of S. thermocarboxydus

Fig. 1. Reduction of Cr(VI) in a bath at 30  C in VB medium, L/ S=40, agitation 140 rpm. ^, Sterile soil; &, autoclaved soil inoculated with Pseudomonas fluorescens LB300; ~, fresh soil (no autoclaving) inoculated with P. fluorescens LB300; , non sterile soil.

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the abiotic assays, indicating that the soil or the solution did not contain significant amounts of any substance capable of spontaneously reducing Cr(VI) under the experimental conditions used. In assays inoculated with P. fluorescens LB300, the Cr(VI) concentration was lowered in 2 days from an initial value of ca. 30 mg l 1 (0.6 mM of Cr(VI)) to a final concentration close to zero, whether the soil was initially autoclaved or not. In an assay with the indigenous microflora only (no inoculum), a similar decrease in Cr(VI) to that found in the inoculated assays was obtained after a lag time of about 1 day, indicating the presence of active indigenous micro-organisms. 3.2. Isolation of Cr(VI)-reducing strains and characterization of Cr(VI)-reducing activity Samples from a 15-day-old culture of contaminated soil were plated onto LB agar medium containing 1 mM of chromate, as K2CrO4, in order to obtain bacterial isolates [9]. These plates were incubated for 5 days at 30  C. Three isolates, representing the dominant Cr(VI)resistant population, were transferred into a 250-ml flask containing 50 ml of Vogel-Bonner medium spiked with Cr(VI) at an initial concentration of 50 mg l 1 (1 mM).

Fig. 2. Reduction of Cr(VI) by Streptomyces thermocarboxydus NH50 in VB medium in the absence of suspended particles (T=30  C, agitation 140 rpm).

Only one of the three isolates showed an ability to reduce Cr(VI) concentration. This isolate was characterized as S. thermocarboxydus by identifying the DNA sequence encoding for the 16S rRNA, which has been recently described in the literature for other metabolic characteristics [10]. The isolated Cr(VI)-reducing strain was named NH50 and the 16S rRNA gene sequence of this strain has been deposited in the EMBL Nucleotide Sequence Database under the accession number AJ249627. 3.3. Cr(VI)-reducing activity stimulated by a solid surface The capacity of S. thermocarboxydus NH50 isolate to reduce Cr(VI) in solution was found to be induced and significantly accelerated by the presence of solid particles, such as sand or soil suspended in the medium, as illustrated in Table 1. In the absence of any suspended particles in the medium, no significant decrease in Cr(VI) concentration was observed during the 3 days of incubation, whereas the disappearance from solution was almost quantitative when sand or soil particles were added. This result suggested that the adhesion of the cells onto solid surfaces induced or stimulated the Cr(VI) disappearance process. In the absence of suspended particles, the decrease in Cr(VI) concentration was observed in VB medium only after about 5 days of incubation (Fig. 2). The decrease in Cr(VI) concentration started when the formation of aggregates (pellets) became visible in the liquid medium. The aggregation of the cells therefore acted in a similar manner as their adhesion to inert solid surfaces, possibly through a mechanism (genetic or enzymatic) of induction not yet elucidated, or by reducing the availability of O2 to the cells in favor of the utilization of Cr(VI) ions. The observed decrease in Cr(VI) concentration in solution, in the experiments where active cells of P. fluorescens LB300 or S. thermocarboxydus NH50 were present (Figs. 1 and 2 and Table 1), may be related to a metabolic

Table 1 Effect of the presence of suspended particles on the reduction of Cr(VI) by Streptomyces thermocarboxydus in VB medium (30  C, 3 days of agitation at 140 rpm) Nature of sterile particles added

Liquid to solid ratio

Inoculation by S. thermocarboxydus

% reduction of Cr(VI) after 3 days of incubation

20 10 10 20

Yes Yes No Yes

98 83 0 100

Coarse sand (< 1 mm)

10 10 20

Yes No Yes

97 0 100

Agricultural soil (