Bioreduction of Cr(VI) by Viable Cells and Cell-Free Extract of a Moderate Halophile, Halomonas Smyrnensis KS802 and Evaluation of Their in Vitro Efficacy in Tannery Effluent

The present study was aimed to characterize the chromate reducing ability of cells and cell-free extract (CFE) of Halomonas smyrnensis KS802 (GenBank Accession No. KU982965) and evaluate their effectiveness in tannery effluents. Viable cells of the strain reduced 200 µM Cr(VI) in basal medium for halophiles (MH) in 10 h and was inversely proportional to Cr(VI) concentrations. The rapid reduction by cells (10⁹ cells/mL) was achieved with 7.5% NaCl, at pH 7 and 37°C which increased with increasing cell density (10¹° cells/mL). While acetate, Cu³⁺, Fe³⁺, SO₄²⁻, and CO₃²⁻ were stimulating the reduction, the inhibitors retarded the process significantly. The NADH-dependent chromate reduction of the CFE was found to be constitutive with Km and Vmax values of 56.58 µM and 3.37 µM/min/mg protein respectively. The optimal reductase activity of the CFE was evident at 200 µM Cr(VI), 10% NaCl, pH 8.0 and at 45°C. A higher concentration of CFE and electron donors increased the enzyme activity but was impacted negatively by toxic metals and anions. Both the cells and CFE were capable of reducing Cr(VI) remarkably from tannery effluent. FTIR and XRD spectra of chromate reducing cells confirmed possible complexation of reduced Cr-species with functional groups on cell surface.

NfoR: Chromate Reductase or Flavin Mononucleotide Reductase?

ABSTRACT Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 μM. The enzyme then binds NADPH with a dissociation constant of 140 μM and reduces the flavin at a rate of 1,350 s−1. Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s−1. The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes. IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed.

Methods of biological treatment intensification aimed at removal of heavy metals

Introduction. The application of microorganisms, capable of using chemical elements with variable valency as terminal electron acceptors, in the wastewater treatment technology can improve the ecological and energy efficiency of biological treatment plants. Materials and methods. The co-authors employed the analytical generalization of findings, including the overview of literary sources, laboratory researches involving standard and advanced methodologies and analytical equipment. Results. Cr(6+) Мn(4+), highly concentrated in wastewaters of versatile production facilities, deserve attention as terminal electron acceptors. The ability of Асіnеtobacter, a non-adapted aerobic bacteria, to reduce Мn(4+), Сr(6+) in the culture medium under a layer of vasseline oil, was studied in the laboratory environment. In the course of the laboratory research, different aspects of Сr(6+)- и Мn(4+) reduction were studied, namely, the bacteria’s chromate reduction ability, as Сr(6+) concentration change curves were obtained for the process of reduction; the influence of Сr(6+) and Мn(2+) reductions on processes of chromate and manganese reduction was identified; water treatment efficiency boosted by Сr(6+) was identified; changes in the reduction sequence in case of their co-presence in the culture medium were checked; the sequence of Сr(6+) and Мn(4+) reduction in case of their co-cultivation together with the bacteria was identified, as well. A succession of experiments with Pseudomonas bacteria was held to validate the results. Conclusions. The experiments have proven that aerobic bacteria, including Асіnеtobacter, P. aeruginosa P-1, P. flurescens var. Pseudo-iodinum P-11, P. Mendocina P-13, P. stutzeri P-19, can develop anaerobic respiration ability under certain conditions. The co-authors have identified that Сr(6+)- and Мn(4+) reduction is applicable by microorganisms, belonging to varied taxonomic groups, in anaerobic cultivation environments, if Сr(6+)- and Мn(4+) act as terminal acceptors of electrons; the co-authors have also reconstructed the reduction sequence: denitrifying bacteria use МnО2 much more efficiently than Сr(6+). Microbial chromate reduction precedes Мn(4+) reduction, while products of their metabolism are less toxic. Acknowledgements. The co-authors would like to express gratitude to the executives of the St. Petersburg University of Architecture and Civil Engineering for the research grant and to all reviewers and the editorial team for the publication of this material.

Microbial Chromate Reductases: Novel and Potent Mediators in Chromium Bioremediation-A Review

Heavy metal pollution from the growing industrialization are a significant cause of environmental concern.Chromium (Cr) is commonly used in the production of stainless steel, textile dyeing, electroplating, as nuclear coolants and largely in chrome tanning of hides and skins. About 90% of leather is produced by chrome tanning and the leather industry contributes to an overload of Cr toxicity in tannery effluents. Accumulation of Cr6+ is carcinogenic, genotoxic and teratogenic to organisms. Biological methods are ‘green’ approaches for chromium bioremediation and microorganisms are the desired candidates for pollution abatement. Microbial chromate reduction is mediated by chromate reductases (ChrRs) which may be expressed constitutively or inducibly. ChrRs have been produced by a number of bacteria, fungi and yeasts and may be extracellular or localized in the membrane or cytosol. ChrRs are dependent on electron donors such as reduced Nicotinamide adenine dinucleotide (NADH) or reduced Nicotinamide adenine dinucleotide phosphate (NADPH) or reduced Glutathione (GSH) as cofactors. In chromate reduction by ChrRs, Cr6+ undergo one electron transfer to producean unstable Cr5+ radical that is converted to stable and less toxic Cr3+. Putative ChrR genomic sequences have been studied with 99% sequence similarity in Gram negative bacteria. ChrRs are valuable resources in different environments for chromate reduction. This review is to discuss the expression and characteristics of ChrRs and their mechanisms in reduction of Cr6+ toxicity in order to provide a comprehensive understanding of this novel class of enzymes for promisingapplications in Cr bioremediation.