Detoxification, Active Uptake, and Intracellular Accumulation of Chromium Species by a Methane-Oxidizing Bacterium

Abstract

Despite the wide-ranging proscription of hexavalent chromium, chromium(VI) remains among the major polluting heavy metals worldwide. Aerobic methane-oxidizing bacteria are widespread environmental microorganisms that can perform diverse reactions using methane as the feedstock. The methanotroph Methylococcus capsulatus Bath, like many other microorganisms, detoxifies chromium(VI) by reduction to chromium(III). Here, the interaction of chromium species with M. capsulatus Bath was examined in detail by using a range of techniques. Cell fractionation and high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) indicated that externally provided chromium(VI) underwent reduction and was then taken up into the cytoplasmic and membranous fractions of the cells. This was confirmed by X-ray photoelectron spectroscopy (XPS) of intact cultures that indicated negligible chromium on the surfaces of or outside the cells. Distribution of chromium and other elements within intact and sectioned cells, as observed via transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS), was consistent with the cytoplasm/membrane location of the chromium(III), possibly as chromium phosphate. The cells could also take up chromium(III) directly from the medium in a metabolism-dependent fashion and accumulate it. These results indicate a novel pattern of interaction with chromium species distinct from that observed previously with other microorganisms. They also suggest that M. capsulatus and similar methanotrophs may contribute directly to chromium(VI) reduction and accumulation in mixed communities of microorganisms that are able to perform methane-driven remediation of chromium(VI).IMPORTANCEM. capsulatus Bath is a well-characterized aerobic methane-oxidizing bacterium that has become a model system for biotechnological development of methanotrophs to perform useful reactions for environmental cleanup and for making valuable chemicals and biological products using methane gas. Interest in such technology has increased recently owing to increasing availability of low-cost methane from fossil and biological sources. Here, it is demonstrated that this versatile methanotroph can reduce the toxic contaminating heavy metal chromium(VI) to the less toxic form chromium(III) while accumulating the chromium(III) within the cells. This is expected to diminish the bioavailability of the chromium and make it less likely to be reoxidized to chromium(VI). Thus, M. capsulatus has the capacity to perform methane-driven remediation of chromium-contaminated water and other materials and to accumulate the chromium in the low-toxicity chromium(III) form within the cells.

Keywords: Methylococcus; bioavailability; bioremediation; heavy metals; methanotrophs.

FIG 1

Effect of M. capsulatus Bath cultures on Cr(VI) at various concentrations. Experiments were biological triplicates. Results are plotted as the ratio of supernatant chromium(VI) concentration at each time point (C) to initial concentration (C₀), and data are means and standard deviations (SD). Parallel triplicate controls without M. capsulatus Bath cells were performed at each initial chromium(VI) concentration, which were constant to within 4% of the initial chromium(VI) concentration.

FIG 2

Reduction and accumulation of chromium species by M. capsulatus Bath after addition of Cr(VI) to 20 mg liter⁻¹. Values are the means from biological triplicates and SD. Concentrations in each of the fractions were normalized to the volume of the original culture.

FIG 3

Speciation and distribution of chromium species analyzed after fractionation of cells into separate cell wall, cytoplasm, and membrane fractions. The initial Cr(VI) concentration was 20 mg liter⁻¹. Error bars show the standard deviations for three biological replicates.

FIG 4

Effect of adding 20 mg liter⁻¹ of Cr(III) to M. capsulatus Bath cultures with and without methane. The abiotic controls were culture medium plus methane (A) and culture medium without methane (B).

FIG 5

EEL spectra of M. capsulatus Bath cells compared with chromium standards. Insets show the areas of the samples (circled) that were analyzed by EELS. Initial Cr(VI) concentration was 20 mg liter⁻¹.

FIG 6

HAADF-STEM and EDX of sectioned cells showing the distribution of chromium. (A) HAADF images of cells without exposure to chromium; (B) HAADF images of cells exposed to 20 mg liter⁻¹ chromium(VI) for 144 h; (C) spatial distribution of chromium in the EDX map of the sample shown in B. Green and yellow boxes on the micrographs in panels A and B show the areas of the sample analyzed in the EDX spectra of the samples without (D) and with (E) exposure to chromium(VI).