World J Microbiol Biotechnol DOI 10.1007/s11274-009-0047-x ORIGINAL PAPER Removal of toxic chromate using free and immobilized Cr(VI)-reducing bacterial cells of Intrasporangium sp. Q5-1 Jinxia Yang Æ Minyan He Æ Gejiao Wang Received: 11 February 2009 / Accepted: 8 April 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Chromate-reducing microorganisms with the Introduction ability of reducing toxic chromate [Cr(VI)] into insoluble trivalent chromium [Cr(III)] are very useful in treatment of The wide use of chromium (Cr) in industries such as leather Cr(VI)-contaminated water. In this study, a novel chro- tanning, metallurgy, electroplating, textile, and pigment mate-reducing bacterium was isolated from Mn/Cr-con- manufacturing has resulted in large quantities of chromium- taminated soil. Based on morphological, physiological/ containing industrial effluent in China and in the World biochemical characteristics and 16S rRNA gene sequence (Wang and Xiao 1995; Pattanapipitpaisal et al. 2001; Sultan analyses, this strain was identified as Intrasporangium sp. and Hasnain 2007). Haxavalent chromium [chromate, strain Q5-1. This bacterium has high Cr(VI) resistance with Cr(VI)] and trivalent chromium Cr(III) are the most com- a MIC of 17 mmol l-1 and is able to reduce Cr(VI) aero- mon oxidation states (Megharaj et al. 2003). Cr(VI) is highly bically. The best condition of Cr(VI) reduction for Q5-1 is toxic, mobile and soluble, which generally exists as an 2- 2- pH 8.0 at 37°C. Strain Q5-1 is also able to reduce Cr(VI) in oxyanion (CrO4 ) in aqueous systems. CrO4 is a strong resting (non-growth) conditions using a variety of carbon oxidizing agent, which reacts with nucleic acids and other sources as well as in the absence of a carbon source. cell components and results in toxicity, mutants and carci- Acetate (1 mmol l-1) is the most efficient carbon source nogenesis (Clark 1994; Codd et al. 2001; Ackerley et al. for stimulating Cr(VI) reduction. In order to apply strain 2006). Cr(VI) has been placed as a priority pollutant, and Q5-1 to remove Cr(VI) from wastewater, the bacterial cells classified as a class A human carcinogen by the US Envi- were immobilized with different matrices. Q5-1 cells ronmental Protection Agency (USEPA; Cieslak-Golonka embedded with compounding beads containing 4% PVA, 1995; Costa and Klein 2006). Cr(III), on the other hand, is 3% sodium alginate, 1.5% active carbon and 3% diatomite insoluble and less toxic (Mclean and Beveridge 2000; Upreti showed a similar Cr(VI) reduction rates to that of free cells. et al. 2004). Therefore reduction of Cr(VI) to Cr(III) is an In addition, the immobilized Q5-1 cells have the advanta- effective way for remediation of Cr(VI)-polluted water. ges over free cells in being more stable, easier to re-use and Conventional technologies for Cr(VI)-contaminated minimal clogging in continuous systems. This study pro- wastewater treatments include chemical reduction followed vides potential applications of a novel immobilized chro- by precipitation, ion exchange, and adsorption on alum or mate-reducing bacterium for Cr(VI) bioremediation. kaolinite etc. However, most of these technologies need high energy or large input of chemical reagents that may Keywords Bioremediation Á Bacterial immobilization Á cause secondary environmental pollutions (Komori et al. Cr Á Chromate-reducing bacterium Á Intrasporangium 1990). Alternatively, an increasing attention has been paid to use bioremediation approaches through selective microorganisms that capable of reducing Cr(VI) to the less J. Yang Á M. He Á G. Wang (&) toxic and insoluble Cr(III). State Key Laboratory of Agricultural Microbiology, College of Reduction of Cr(VI) has been reported in several bac- Life Science and Technology, Huazhong Agricultural University, 430070 Wuhan, People’s Republic of China teria under aerobic or anaerobic conditions, including e-mail: [email protected]; [email protected] Bacillus (Campos et al. 1995; Camargo et al. 2003b; 123 World J Microbiol Biotechnol Elangovan et al. 2006; Liu et al. 2006; Desai et al. 2008), county, Hunan province, P. R. China. This region is highly Pseudomonas (Salunkhe et al. 1998;Parketal.2000; heavy metal contaminated with up to 3.27 g kg-1 Mn in Mclean and Beveridge 2000; Ganguli and Tripathi 2002), the soil (Guo et al. 2006). For enrichment, 100 g soil Escherichia coli (Bae et al. 2004), Microbacterium (Pat- sample was amended with K2CrO4 at a final concentration tanapipitpaisal et al. 2001) and Shewanella (Myers et al. of 5 mM and kept in dark at 37°C for 1 week. Cr(VI) 2000; Vaimajala et al. 2002) etc. In the presence of oxygen, resistant bacteria were isolated from the soil sample by microbial reduction of Cr(VI) is catalyzed by soluble adding 10 g soil to 100 ml of 0.85% NaCl solution and enzymes (Cervantes and Campos 2007). Under anaerobic shaking at room temperature for 10 min. The extraction conditions, both soluble and membrane associated enzymes solution was serially diluted and plated onto Luria Broth of the electron transfer system were reported to associate (LB) plates (10 g tryptone, 10 g NaCl, 5 g yeast extract in Cr(VI) reduction that coupled to the oxidation of an elec- 1 l distilled water) containing 2 mM K2CrO4. The plates tron donor substrate. In this process, Cr(VI) serves as the were incubated at 37°C for 1 week. Single colonies were terminal electron acceptor of an electron transfer chain that re-streaked several times to obtain pure isolates. Bacterial frequently involves cytochrome b/c (Cervantes and Cam- chromate resistant level was checked by inoculating 1% pos 2007). The Cr(III) species forms an insoluble precipi- original culture of the pure isolate (OD600 = 1.0) into LB tate, such as Cr(OH)3, which can be removed from medium that amended with different concentrations of wastewater (Jeyasingh and Philip 2005). K2CrO4. The growth of each treatment was observed after Chromate-reducing bacteria have been used in labora- incubation at 37°C for 3 days. The MIC, defined as the tory study for bacterial chromate removal from water lowest K2CrO4 concentration that completely inhibits the system (Camargo et al. 2003a; Desai et al. 2008). However, growth of strain Q5-1, was determined (Sarangi and for industrial purposes, using free bacterial cells is disad- Krishnan 2007). vantageous due to the difficulty of biomass/effluent sepa- Quantitative characterization of bacterial Cr(VI) reduc- ration (White et al. 1995) etc. These problems may be tion abilities was carried out under an aerobic condition in overcome by the use of immobilized bacterial cells with 250 ml culture flasks containing 50 ml LB medium. The the advantages of stability, regeneration, solid–liquid sep- flasks with the LB medium was supplemented with 1 mM aration and minimal clogging in continuous systems (Po- K2CrO4 and inoculated with 1% bacterial culture -1 opal and Laxman 2008). Cell immobilization has been (OD600 = 1.0) and incubated at 37°C with 160 rev min accomplished using a variety of supporting materials such shaking for up to 2 days. Controls without bacterial inoc- as natural (agar, alginate, active carbon, and diatomite etc.) ulation were also incubated in the same condition to and synthetic matrices (polyacrylamide, polyethylene gly- monitor the abiotic Cr(VI) reduction. The samples were col, and polyvinyl alcohol etc.; Seung et al. 2005). The aseptically taken at about every 4 h, centrifuged at choice of immobilization materials is different with various 6,000 rev min-1 for 10 min. Cr(VI) concentration in the microorganisms. Combination parameter of supporting supernatant was measured using 1,5-diphenyl carbazide matrices is an also key factor of an immobilized biocatalyst (DPC) reagent at absorbance value of 540 nm using a UV (Poopal and Laxman 2008). spectrophotometer (DU800, Beckman, CA, USA; APHA The objectives of this research were to: (1) isolate a 1995). A standard curve was generated using 0.2, 0.8, 1.0, novel chromate-reducing bacterium and study its chromate 1.5, and 2 mM K2CrO4. The growth of the bacterium was removal efficiency. Bacterial identification was performed determined at the Optimal Density of 600 nm (OD600). using morphological, biochemical/biophysical and 16S rRNA gene analyses; (2) evaluate the Cr(VI) reducing Morphological and biochemical/biophysical analyses abilities using growing, resting, and immobilized chro- of the Cr(VI)-reducing bacterium mate-reducing bacterial cells. The immobilized bacterium may provide a superior potential in bioremediation of Colony morphologies of the bacterium were observed on chromate pollution in various environments. LB plates after incubating at 37°C for 3 days. Cell mor- phologies were examined under a JSM-6390/LV scanning electron microscope (SEM; JSM-6390, JEOL, Japan) with Materials and methods 20,000 V accelerating voltage and 10,000 times enlarge- ment. Gram staining was performed using bacterial colo- Sample collection and isolation of Cr(VI)-reducing nies on LB plates as described by Bailey and Scott (1966). bacteria Biochemical and biophysical analyses were performed according to the Bergey’s manual (Holt et al. 1994). Soil sample was collected from the surface horizon Characteristics of catalase and oxidase activities, hydroly- (0–15 cm) in a manganese/chromium mine in Huayuan sis of starch, gelatin liquefaction, Voges-Proskauer (V.P.) 123 World J Microbiol Biotechnol and Methyl Red (M.R.) reactions, indole production, H2S In a repeated-adding of Cr(VI) aliquot experiment, production and the utilization of sole carbon/nitrogen 250 ml LB (pH 8.0) was amended with 1 mM K2CrO4 and sources were tested. incubated as described above. Cr(VI) reduction was mon- itored at about 8 h time interval.