Elsevier

Bioresource Technology

Volume 99, Issue 14, September 2008, Pages 6059-6069
Bioresource Technology

Evaluation of In vitro Cr(VI) reduction potential in cytosolic extracts of three indigenous Bacillus sp. isolated from Cr(VI) polluted industrial landfill

https://doi.org/10.1016/j.biortech.2007.12.046Get rights and content

Abstract

Three efficient Cr(VI) reducing bacterial strains were isolated from Cr(VI) polluted landfill and characterized for in vitro Cr(VI) reduction. Phylogenetic analysis using 16S rRNA gene sequencing revealed that the newly isolated strains G1DM20, G1DM22 and G1DM64 were closely related to Bacillus cereus, Bacillus fusiformis and Bacillus sphaericus, respectively. The suspended cultures of all Bacillus sp. exhibited more than 85% reduction of 1000 μM Cr(VI) within 30 h. The suspended culture of Bacillus sp. G1DM22 exhibited an ability for continuous reduction of 100 μM Cr(VI) up to seven consecutive inputs. Assays with the permeabilized cells and cell-free extracts from each of Bacillus sp. demonstrated that the hexavalent chromate reductase activity was mainly associated with the soluble fraction of cells and expressed constitutively. The Cr(VI) reduction by the cell-free extracts of Bacillus sp. G1DM20 and G1DM22 was maximum at 30 °C and pH 7 whereas, Bacillus sp. G1DM64 exhibited maximum Cr(VI) reduction at pH 6. Addition of 1 mM NADH enhanced the Cr(VI) reductase activity in the cell-free extracts of all three isolates. Amongst all three isolates tested, crude cell-free extracts of Bacillus sp. G1DM22 exhibited the fastest Cr(VI) reduction rate with complete reduction of 100 μM Cr(VI) within 100 min. The apparent Km and Vmax of the chromate reductase activity in Bacillus sp. G1DM22 were determined to be 200 μM Cr(VI) and 5.5 μmol/min/mg protein, respectively. The Cr(VI) reductase activity in cell-free extracts of all the isolates was stable in presence of different metal ions tested except Hg2+ and Ag+.

Introduction

Hexavalent chromium Cr(VI), an extensively used anthropogenic pollutant is released into the environment due to its applications in metallurgical and refractory industries including corrosion inhibition, electroplating, paints, pigment manufacturing, leather tanning and wood preservatives (Viti et al., 2003, Sultan and Hasnain, 2007). Chromium can exist in oxidation states ranging from 2− to 6+ (Avudainayagam et al., 2003). Hexavalent chromium Cr(VI) and trivalent chromium Cr(III) are the most dominant oxidation states of chromium that exist in the environment (Megharaj et al., 2003). Cr(VI) is highly mobile and water soluble as compared to Cr(III), whereas Cr(III) is relatively inert, chemically more stable than Cr(VI) and less bioavailable due to its negligible permeability to biomembranes (Myers et al., 2000, Megharaj et al., 2003, Pal et al., 2005).

In aqueous systems Cr(VI) exists as oxyanions (CrO42-) which are structurally analogous to sulfate and phosphate ions, hence can be easily taken up by anionic transport systems of both bacterial and eukaryotic cells (Mattagajasingh et al., 1996, Cervantes et al., 2001, Asatiani et al., 2004, Cheung and Gu, 2007). The intracellular Cr(VI) is reduced to Cr(V), Cr(IV) and Cr(III) valence states by intracellular components with generation of reactive oxygen species (ROS) which in turn results in chromate induced toxicity by formation of ternary adducts of intracellular Cr(III) with DNA and proteins, whereas the oxidative DNA damage is caused by Cr(V) and ROS (Mattagajasingh et al., 1996, Bose et al., 1999, Quievryn et al., 2003, Ackerley et al., 2006). Due to its strong oxidizing properties intracelluar Cr(VI) exerts mutagenic and teratogenic effects. In view of the seriousness of Cr(VI) pollution and its alarming effects on human health, it has been listed as a priority pollutant and classified as class A human carcinogen by the US Environmental Protection Agency (USEPA) (Cieslak-Golonka, 1995, Costa and Klein, 2006).

Metal pollutants are non-degradable and can only be transformed to less toxic oxidation states or removed either by adsorption/accumulation or by physicochemical treatments. Amongst the various approaches currently employed for environmental clean-up of Cr(VI), biological Cr(VI) detoxification is an ecofriendly and a cost-effective alternative to the conventional physicochemical methods (Camargo et al., 2003a, Megharaj et al., 2003). Several microorganisms have the exceptional ability to adapt and colonize the noxious metal polluted environments by developing mechanisms to evade metal toxicity like metal efflux channels, metal resistance plasmids, adsorption uptake, DNA methylation and metal biotransformation either directly by specific enzymes or indirectly by cellular metabolites.

Biotransformation of Cr(VI) to Cr(III) using bacteria is the most pragmatic approach with a well-established feasibility in bioremediation. Reduction of Cr(VI) has been demonstrated in various bacterial species including Bacillus sp. (Campos et al., 1995, Camargo et al., 2004, Elangovan et al., 2006, Liu et al., 2006), Pseudomonas sp. (Bopp and Ehrlich, 1988, Ishibashi et al., 1990, Suzuki et al., 1992, Salunkhe et al., 1998, Park et al., 2000, Mclean and Beveridge, 2001, Ganguli and Tripathi, 2002), Escherichia coli (Bae et al., 2005), Desulfovibrio sp. (Mabbett and Macaskie, 2001), Microbacterium sp. (Pattanapipitpaisal et al., 2001), Shewanella sp. (Myers et al., 2000, Vaimajala et al., 2002) and Arthrobacter sp. (Megharaj et al., 2003, Asatiani et al., 2004). It is imperative to gauge the enzymatic mechanisms involved in hexavalent chromium reduction in order to develop a rational Cr(VI) bioreduction process and such mechanisms have been reported in Bacillus sp. AND 303 (Pal et al., 2005), Bacillus sp. ES 29 (Camargo et al., 2004), Bacillus sp. QC-2 (Campos et al., 1995), Bacillus sp. (Elangovan et al., 2006), Pseudomonas ambigua G-1 (Suzuki et al., 1992), Pseudomonas putida (Ishibashi et al., 1990, Park et al., 2000), Escherichia coli ATCC 33456 (Bae et al., 2005). Bacterial enzymes with their biological function as hydrogenases (Chardin et al., 2003), nitroreductases (Kwak et al., 2003) and quinone reductases (Gonzalez et al., 2005) have been reported to exhibit chromate reductase activity.

The present study evaluates the chromate reductase activity associated with the cell-free extracts of three indigenous Bacillus sp. isolated from a highly polluted industrial landfill site. The optimal conditions for Cr(VI) reduction as well as, the stability of the Cr(VI) reduction under different in vitro conditions for each of the Bacillus sp. have been elucidated in this study.

Section snippets

Isolation and cultivation of bacteria

Cr(VI) resistant and reducing bacterial strains were isolated from long term chromium polluted site of Gorwa Industrial Estate, Vadodara, Gujarat, India. Sediment sample of this landfill was contaminated with chromium level of 10,703 mg/kg of soil as estimated by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using Perkin–Elmer ICP Optima-3300RL (Perkin Elmer, Norwalk, Conn). The bacterial strains were screened from the serial dilutions of the sediment suspensions plated onto

Screening and identification of bacterial isolates

Heavy metal contamination is known to cause shifts in microbial communities with emergence of bacterial species with elevated metal tolerance (Smit et al., 1997, Stepanauskas et al., 2005). Chromium contamination exerts a selective pressure on microbial flora of tannery soils (Viti et al., 2003). Industrial landfill site receiving long-term augmentations of hexavalent chromium Cr(VI) was chosen in this study with an aim to screen for Cr(VI) resistant and Cr(VI) reducing bacteria.

Conclusion

The present study demonstrates the expediency of Cr(VI) reduction and evaluates the Cr(VI) reductase activity under different in vitro conditions in the cell-free extracts of three indigenous Bacillus sp. isolates of a Cr(VI) polluted landfill. Resting cells of all the three Bacillus sp. effectively reduced 100 μM Cr(VI) and cell-permeabilization further increased the Cr(VI) reduction potential. The suspended cells of Bacillus sp. G1DM22 exhibited an ability for continuous reduction of 100 μM

Acknowledgements

This work was financially supported by Department of Biotechnology, Ministry of Science and Technology, New Delhi, India and Puri Foundation, Nottingham, UK. Authors are also grateful to the Sophisticated Instrumentation Center for Applied Research and Testing (SICART), Vallabh Vidyanagar, Gujarat, India for providing facilities for ICP-AES and electron microscopy analysis.

References (46)

  • F.M. Ausubel et al.

    Current Protocols in Molecular Biology, Unit 24

    (1997)
  • S. Avudainayagam et al.

    Chemistry of chromium in soils with emphasis, on tannery waste sites

    Rev. Environ. Contam. Toxicol.

    (2003)
  • W.C. Bae et al.

    Purification and characterization of NADPH-dependent Cr(VI) reductase from Escherichia coli ATCC 33456

    J. Microbiol.

    (2005)
  • L.H. Bopp et al.

    Chromate resistance and reduction in Pseudomonas fluorescence strain LB300

    Arch. Microbiol.

    (1988)
  • R.N. Bose et al.

    Oxidative damage of DNA by chromium(V) complexes: relative importance of base versus sugar oxidation

    Nucleic Acids Res.

    (1999)
  • M.M. Bradford

    A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding

    Anal. Biochem.

    (1976)
  • F.A.O. Camargo et al.

    Chromate reduction by chromium resistant bacteria isolated from soils contaminated with dichromate

    J. Environ. Qual.

    (2003)
  • F.A.O. Camargo et al.

    In vitro reduction of hexavalent chromium by a cell-free extract of Bacillus sp. ES 29 stimulated by Cu+2

    Appl. Microbiol. Biotechnol.

    (2003)
  • F.A.O. Camargo et al.

    Hexavalent chromium reduction by immobilized cells and the cell-free extract of Bacillus sp. ES 29

    J. Bioremed.

    (2004)
  • J. Campos et al.

    Hexavalent-chromium reduction by a chromate-resistant Bacillus sp. strain

    Anton. Leeuw.

    (1995)
  • B. Chardin et al.

    Hydrogenases in sulfate-reducing bacteria function as chromium reductase

    Appl. Microbiol. Biotechnol.

    (2003)
  • M. Cieslak-Golonka

    Toxic and mutagenic effects of Cr(VI) – a review

    Polyhedron

    (1995)
  • M. Costa et al.

    Toxicity and carcinogenicity of chromium compounds in humans

    Crit. Rev. Toxicol.

    (2006)
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