ENVIRONMENTAL MICROBIOLOGY

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Detoxification, Active Uptake, and Intracellular Accumulation

of Chromium Species by a Methane-Oxidizing Bacterium
Salaheldeen Enbaia,a Abdurrahman Eswayah,a,b Nicole Hondow,c Philip H. E. Gardiner,a Thomas J. Smitha

a Biomolecular Sciences Research Centre, Sheffield Hallam University, Sheffield, United Kingdom
b Biotechnology Research Centre, Tripoli, Libya
c
School of Chemical and Process Engineering, University of Leeds, Leeds, United Kingdom

ABSTRACT Despite the wide-ranging proscription of hexavalent chromium, chromi-

um(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 chromi-
um(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 frac-
tionation 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 indi-
cate a novel pattern of interaction with chromium species distinct from that ob-
served 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).
IMPORTANCE M. capsulatus Bath is a well-characterized aerobic methane-oxidizing
Citation Enbaia S, Eswayah A, Hondow N,
bacterium that has become a model system for biotechnological development of Gardiner PHE, Smith TJ. 2021. Detoxification,
methanotrophs to perform useful reactions for environmental cleanup and for mak- active uptake, and intracellular accumulation of
chromium species by a methane-oxidizing
ing valuable chemicals and biological products using methane gas. Interest in such bacterium. Appl Environ Microbiol 87:e00947-
technology has increased recently owing to increasing availability of low-cost meth- 20. https://doi.org/10.1128/AEM.00947-20.
ane from fossil and biological sources. Here, it is demonstrated that this versatile Editor Ning-Yi Zhou, Shanghai Jiao Tong
University
methanotroph can reduce the toxic contaminating heavy metal chromium(VI) to the
Copyright © 2021 Enbaia et al. This is an open-
less toxic form chromium(III) while accumulating the chromium(III) within the cells. access article distributed under the terms of
This is expected to diminish the bioavailability of the chromium and make it less the Creative Commons Attribution 4.0
likely to be reoxidized to chromium(VI). Thus, M. capsulatus has the capacity to per- International license.
Address correspondence to Thomas J. Smith,
form methane-driven remediation of chromium-contaminated water and other mate-
[email protected].
rials and to accumulate the chromium in the low-toxicity chromium(III) form within Received 23 April 2020
the cells. Accepted 20 October 2020
Accepted manuscript posted online 30
October 2020
KEYWORDS Methylococcus, bioavailability, bioremediation, heavy metals, Published 4 January 2021
methanotrophs

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Enbaia et al. Applied and Environmental Microbiology

D espite worldwide regulation of the use of hexavalent chromium in industry, this

highly toxic, and bioavailable form of chromium continues to be a substantial
environmental problem. Hence, environmental microorganisms that can detoxify and
sequester chromium species are of biotechnological interest. Despite a fall in commer-
cial use of hexavalent chromium per se, various forms of chromium are heavily used in
industry, especially in the production of chromium-iron alloys such as stainless steel
and in leather manufacture, in which chromium(III) salts are used. Mining of chromite,
the major chromium-containing ore, also causes substantial environmental release of
chromium species. Such anthropogenic contamination contains significant hexavalent
chromium, which may leach into the aqueous environment (1, 2). Chromium(III), from
natural or human sources, may be solubilized by organic acids produced by living
systems and reoxidized in the soil under oxic conditions, particularly in the presence of
manganese compounds that can mediate O2-driven oxidation of chromium(III) to
chromium(VI) (3–5). Siderophores, which naturally act as microbial iron-scavenging
molecules, can also bind and solubilize chromium(III) (6). In most jurisdictions, the
maximum contaminant level (MCL) for chromium(VI) in drinking water has not been
fixed, though 10 ␮g liter⫺1, which was in force in California between 2014 and 2017,
has been used as a benchmark of the safe level of chromium(VI) (7). Substantial
concentrations of Cr(VI) are found in groundwater, for example, up to 1 mg liter⫺1 in
certain areas of California (7) and up to 34 ␮g liter⫺1 in drinking water wells in the
Piedmont area of North Carolina (8). The particulate fraction of the exhaust from
biodiesel combustion may contain up to 2 mg/g of chromium (9). Worldwide, waste
materials and contaminated sites requiring remediation have reported chromium(VI)
concentrations up to tens of parts per million (10, 11).
Many bacteria can bioremediate chromium(VI) via reduction to the less harmful
trivalent form (12). The metabolic diversity of prokaryotes provides a wide range of
natural and artificial electron donors for chromium(VI) reduction (12, 13). Among these,
methane is particularly attractive because it is available in large quantities from fossil
sources and biogas. It has aroused greater interest in recent years due to falling
methane prices (14). Aerobic methanotrophs, a diverse group of environmental bac-
teria that are able to use methane as their carbon and energy source, are significant as
a global methane sink. Methanotrophs and their enzymes have been explored for a
range of biotechnologically valuable methane-driven processes, including bioremedia-
tion and production of single-cell protein, and as catalysts for oxygenation of unfunc-
tionalized carbon atoms in organic molecules (15–19).
The gammaproteobacterial methanotroph Methylococcus capsulatus Bath is able to
reduce chromium(VI) to chromium(III) over a wide range of concentrations (tested
across 1.4 to 1,000 mg liter⫺1) (20). Methane-driven chromium(VI) reduction has also
been achieved in a methane-fed polymicrobial biofilm reactor system (21), where some
of the reduction of Cr(VI) is attributed to nonmethanotrophs scavenging nutrients
(multicarbon compounds and more generally accessible C1 substrates such as metha-
nol) produced by the methanotrophs.
Methanotrophs are known to bind and transform a range of toxic metals and
metalloids in addition to chromium. The alphaproteobacterial methanotrophs, such as
Methylosinus trichosporium OB3b, produce a range of structurally related copper-
scavenging molecules termed methanobactins (22). Methanobactin-bound copper is in
the ⫹1 oxidation state; binding of Cu(II) to methanobactin results in its reduction to
Cu(I) (23), possibly by electrons derived from water (22). Methanobactin is able to bind
a wide range of cations; a subset of these, including Hg(II) and Au(III), undergo
reduction upon binding, in a similar manner to copper, to give metallic mercury and
metallic gold nanoparticles, respectively (24–27). Methanobactin binds methylmercury
(MeHg⫹); in M. trichosporium, methanobactin is necessary for detoxification of MeHg⫹
as well as promoting in vivo methylation of Hg(II) (24, 27, 28). The gammaproteobac-
terial methanotroph M. capsulatus Bath reduces Hg2⫹ ions to metallic mercury (29) and
takes up but does not detoxify MeHg⫹. Methanotrophs respond to lanthanide ele-
ments that are required for activity of a key metabolic methanol dehydrogenase (25, 30,

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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) concen-
tration, which were constant to within 4% of the initial chromium(VI) concentration.

31). They also convert selenite (SeO32-) to selenium-containing nanoparticles and

volatile methylated selenium species (32, 33).
Previous work on remediation of Cr(VI) by M. capsulatus Bath showed that chromi-
um(III) accumulated in the particulate fraction of the culture and (based on extended
X-ray absorption spectroscopy fine structure [EXAFS] results) was likely coordinated by
oxygen and phosphorus (20). Previously, electron microscopy techniques coupled with
spectroscopic analysis have been used with other systems to characterize the distribu-
tion and speciation of chromium associated with bacteria at the cellular or subcellular
level (21, 34–37). Here, we used a range of cell fractionation, analytical, electron-
microscopic, and spectroscopic techniques to obtain spatially resolved information
about the interaction of chromium species with M. capsulatus, to determine how its
specific properties might be exploited for bioremediation and to gain insights into the
role such methanotrophs may play in the environmental chromium cycle.

RESULTS
Effect of concentration on chromium(VI) removal. High-performance liquid chro-
matography (HPLC)–inductively coupled plasma mass spectrometry (HPLC–ICP-MS) is a
well-established technique for quantifying and determining the speciation of heavy
metals and other elements that has been previously used to characterize reduction of
chromium(VI) at a bulk culture level (for example, see reference 38). In order to
characterize the range of chromium(VI) concentrations over which M. capsulatus could
remediate all or most of the added chromium(VI), various concentrations of chromi-
um(VI) were added to cultures of M. capsulatus Bath (optical density at 600 nm [OD600]
of 0.7 to 0.9), and then the cultures were incubated at 45°C in the presence of methane
and air. Chromium species in the culture supernatant were quantified by using HPLC–
ICP-MS (Fig. 1). M. capsulatus Bath achieved complete removal of detectable chromi-
um(VI) at initial concentrations up to 5 mg liter⫺1 and was able to substantially
decrease the chromium(VI) of the culture supernatant from initial concentrations of
ⱕ40 mg liter⫺1. No other detectable chromium species appeared in the culture super-
natant. A concentration of chromium(VI) of 20 mg liter⫺1 was chosen for further
experiments, since this concentration resulted in the largest amount of chromium(VI)
removed within 144 h.
Reduction and accumulation of chromium species within cellular fractions. In
order to gain more information about the possible location of chromium species within
the culture, cultures were incubated with an initial concentration of chromium(VI) of
20 mg liter⫺1, and samples were taken over a period of 144 h. Cells within each sample
were then broken and separated into cell walls and a combined fraction of membranes
and cytoplasm, as detailed in Materials and Methods, before quantification of the
chromium species via HPLC–ICP-MS (Fig. 2).
Over a period of 144 h, the concentration of chromium(VI) in the culture supernatant
declined, while there was a corresponding increase in the concentration of chromium-

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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.

(III) in the cytoplasm-membrane fraction. No other chromium species were detected at

significant concentrations in any of the samples. The constant total chromium (the sum
of the chromium detected in all fractions) (Fig. 2) indicates that the appearance of
chromium(III) in the cytoplasm-membrane fraction accounted, within experimental
error, for the decrease in chromium(VI) in the culture supernatant. Hence, cells of M.
capsulatus Bath not only were able to reduce chromium(VI) to chromium(III) but also
appeared to be able to accumulate all the chromium(III) within the biomass.
To gain additional information about the location of chromium species within the
cells, cultures were exposed to chromium(VI) (20 mg liter⫺1), and cells were fraction-
ated to produce separate membrane and cytoplasm fractions. The results showed that
the distribution of chromium(III) between the two fractions was approximately two-
thirds in the cell membrane fraction and one-third in the cytoplasm fraction (Fig. 3).
Uptake of Cr(III). The fact that all of the cell-associated chromium was in the ⫹3
oxidation state, even though the cells had been exposed to chromium in the hexava-
lent form, raised the question of whether reduction and uptake of chromium were
necessarily linked or whether cells could take up trivalent chromium directly. When
exposed in exactly the same way to 20 mg liter⫺1 of chromium(III) in the presence of
methane and air, the M. capsulatus cells took up the chromium(III) completely into the
cytoplasm-membrane fraction within 1 h (Fig. 4A), much more quickly than the ⬎144
h taken for reduction and accumulation of the same amount of chromium(VI). Previous
work has shown that the reduction of chromium(VI) to chromium(III) by M. capsulatus
Bath is an active process requiring the presence of the carbon and energy source
methane (20). In order to investigate whether the uptake of chromium(III) was also an
active process, cultures were exposed to 20 mg liter⫺1 of chromium(III) aerobically but
in the absence of methane. When the cells were grown to an OD600 of 0.7 to 0.9 in the

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.

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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).

presence of methane and then methane was removed and chromium(III) added
immediately, all the chromium(III) was taken up by the cells within 1 h (Fig. 4B). If,
however, the cells were starved of methane overnight (16 h) before addition of the
chromium(III), only 25% of the chromium(III) was taken up (Fig. 4C). Addition of the
metabolic inhibitor sodium azide to 0.05% (wt/vol) (Fig. 4D) abolished approximately
half of the uptake of chromium(III) within a 1-h period. When the cells were starved of
methane overnight and sodium azide was added at the same time as the chromium(III),
uptake of chromium(III) was completely abolished (Fig. 4E). Heat killing (autoclaving) of
the cells also completely abolished chromium(III) uptake (Fig. 4F). These results indicate
that uptake of chromium(III) is an active process but also that when methane is

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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.

removed from a growing culture, it has sufficient reserves of energy to take up a

substantial amount of chromium(III).
Spatially resolved spectroscopic characterization of cells. Electron energy loss
spectroscopy (EELS) coupled with TEM of whole M. capsulatus Bath cells exposed to
20 mg liter⫺1 of chromium(VI) for 96 h or 144 h confirmed, via comparison with spectra
of chromium standards, that the cell-associated chromium was in the ⫹3 oxidation
state (Fig. 5). Cells exposed to 20 mg liter⫺1 of chromium(VI) for 144 h were also
prepared as thin sections to see how chromium and other elements were distributed
within the cells. High-angle annular dark-field (HAADF) scanning transmission electron
microscopy (STEM)– energy-dispersive X-ray spectroscopy (EDX) showed the presence

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of chromium in the chromium-treated cells and its absence from the chromium-
untreated control (Fig. 6). The spatial distribution of chromium (Fig. 6C) indicated that
the chromium was largely cell-associated and distributed throughout the cell. This is
consistent with the approximate 40:60 distribution of the chromium between the
cytoplasm and membrane fractions, when it is borne in mind that under the particulate
methane monooxygenase (pMMO)-expressing conditions of these experiments, M.
capsulatus Bath is expected to have intracellular as well as peripheral membranes (19).
The spatial distribution of chromium and a number of other elements (carbon,
phosphorus, and oxygen) was also determined within whole M. capsulatus cells via EDX
(Fig. S1 to S4). These results indicated that there was inhomogeneity in the distribution
of all four elements, which appeared to correlate with structural features of the cells
visible in the electron micrographs. Since these features showed elevated concentra-
tions of chromium, phosphorus, and oxygen and decreased concentrations of carbon,
they are consistent with the deposition of chromium phosphate (containing Cr, O, and
P) associated with the cells. This is also consistent with the phosphorus and oxygen
ligation of M. capsulatus cell-associated chromium(III) inferred from EXAFS data (20).
The EDX analysis also indicated an increase in calcium associated with the cells that had
been exposed to chromium (Fig. S1 to S4).
Surface analysis of samples of M. capsulatus cells treated with 20 mg of chromi-
um(VI) liter⫺1 was performed, in comparison with chromium-untreated samples, by
using X-ray photoelectron spectroscopy (XPS) (Fig. S5 and S6; Tables S1 and S2). While
the depth of penetration of XPS into the sample is small (several nanometers), the areas
of data acquisition were 400 ␮m in diameter. This is large compared to the cells
(approximate diameter, 1 ␮m), and so these data show the properties of the surface of
the whole sample rather than individual cells and subcellular structures seen by other
techniques.
The carbon and oxygen X-ray photoelectron spectra of chromium(VI)-treated and
chromium(VI)-untreated cells show features that are consistent with the presence of
peptides, carbohydrates, lipids, and phosphate groups that are likely to be associated
with the surfaces of the cells (Tables S1 and S2). The signals at binding energies of
400.1 eV [chromium(VI)-treated sample] and 399.8 eV [chromium(VI)-untreated sample]
were attributed to N 1s, which is widely found in amino acids, and amino sugars (amino
sugars are found in cell wall peptidoglycan, and amino acids are constituents of both
peptidoglycan and proteins) (Fig. S5 and S6). No substantial differences in the spectra
of carbon, oxygen, and nitrogen were observed between the chromium(VI)-treated and
-untreated cells (Fig. S5 and S6; Tables S1 and S2).
Most notably, the small peak at a binding energy of 577.8 eV in the chromium(VI)-
treated sample (Fig. S5A and E), which may be due to emission of photoelectrons from
the 2p orbitals of chromium, was not substantially above the noise in the baseline of
the spectrum. This indicates the presence of very little chromium on the surfaces of the
cells, which is consistent with the results from analysis of the cell fractions that suggest
that chromium is within the cells, associated with the membranes and cytoplasm.

DISCUSSION
The results reported here indicate the capacity of M. capsulatus Bath to reduce
chromium(VI) at concentrations up to several milligrams per liter or milligrams per
gram, which are relevant to current contaminated groundwater and solid waste
problems (7, 9–11). M. capsulatus Bath can, for example, take the concentration of
hexavalent chromium from 3 mg liter⫺1 to below the level of detection of HPLC–ICP-MS
(ⱕ0.005 mg liter⫺1 in these experiments) within 48 h (Fig. 1). The nearly straight plots
of C/C0 [ratio of supernatant chromium(VI) concentration at each time point to initial
concentration] versus time (Fig. 1) at an initial chromium concentration of ⱕ10 mg
liter⫺1 suggest near-zero-order kinetics at these low chromium concentrations. The rate
of chromium(VI) removal during the first 48 h of incubation (Fig. S7) shows a depen-
dence of chromium(VI) removal on the initial concentration, with a maximum at 20 mg
liter⫺1 and a clear decline as the system becomes inhibited at higher concentrations of

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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).

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chromium(VI). In these experiments, total removal of detectable chromium(VI) was not

achieved within 144 h from an initial concentration of 10 mg liter⫺1 or above. Although
no significant removal of chromium(VI) was observed from an initial chromium con-
centration of 50 mg liter⫺1, reduction of a small proportion of added chromium(VI) by
M. capsulatus must be possible at higher concentrations, since the cells from a culture
exposed to 1,000 mg liter⫺1 of chromium(VI) contained chromium solely in the ⫹3
oxidation state, although the amount of chromium(VI) reduced within the culture was
not measured (20).
Previously, BLAST sequence similarity searches of the M. capsulatus Bath genome
with representatives of three classes of chromium(VI) reductases and a chromate efflux
pump were performed to identify proteins possibly involved in reduction of chromi-
um(VI) (20). Since the genome of M. trichosporium OB3b (39), which does not reduce
chromium(VI) (20), is now available, this analysis was repeated with both genomes in
parallel (Table S3). The numbers of potential chromate reductases identified in the
genomes of the two organisms are the same. Also, neither has a homologue of the
chromate efflux pump. The chromate reductase ChrA of Pseudomonas putida (40) has
a homologue in M. trichosporium OB3b but not in M. capsulatus Bath. The chromate
reductase Fre of Escherichia coli (41) has only two homologues in M. trichosporium OB3b
but three in M. capsulatus Bath. Among these, M. capsulatus alone has the gene
encoding a putative F subunit of a Na⫹-translocating NADH-quinone reductase, which
is part of a six-gene cluster encoding a possible transmembrane complex that transfers
electrons between NADH and quinones (42) and that is missing from M. trichosporium
OB3b. One possibility is that chromium(VI) reduction in M. capsulatus Bath is an
adventitious activity of this complex, although it is also possible that other factors [such
as the access of chromium(VI) to the reductase due to permeability properties] are the
reason that M. trichosporium OB3b cells do not reduce chromium. The complexity of the
reaction kinetics is consistent with a system in which the rate is influenced by multiple
factors, such as enzyme activities, permeability, and toxicity.
Here, as in other studies, HAADF-STEM-EDX was used to study the distribution of
metals in bacterial cells (33, 43, 44). The formation of cell-associated structures com-
posed of precipitated metal ions has been observed previously, such as chromium-
containing particles on the surface of a chromium(VI)-reducing Pseudomonas synxantha
strain (45) and extracellular fibers and stalks of Fe(III) oxides formed by Gallionella (46).
Chromium(VI) exposure of Shewanella oneidensis (35) and Desulfovibrio vulgaris (36) also
results in deposition of chromium(III) in the form of precipitate on the surfaces of the
cells, although intracellular chromium(II) has been observed under strictly anaerobic
conditions in S. oneidensis and may be an intermediate in the conversion of chromi-
um(VI) under conditions where the extracellular precipitated chromium(III) is produced
by this organism (43). The cellular breakage and fractionation technique used here with
M. capsulatus Bath showed the presence of all detectable chromium, after cell break-
age, as chromium(III) within soluble cell-associated material and in association with
membranes (or other small cell-derived particulate material with similar sedimentation
properties). While the possibility of redistribution of chromium(III) after breakage of the
cells cannot be excluded, the absence of extracellular precipitated chromium-
containing material from TEM images, together with the shielding of chromium from
XPS, give strong independent evidence of the intracellular location of the chromium(III)
product. This intracellular chromium(III), which may be in the form of chromium(III)
phosphate or chromium associated with organic phosphate groups, is visible to EDX
and EELS in whole cells, as these are transmission techniques (results generated
throughout the thickness of the sample).
M. capsulatus Bath was originally isolated from the Roman baths in Bath, United
Kingdom, which are fed by water from a geothermal spring that is consistently low in
toxic heavy metals, including chromium (⬍0.5 ␮g liter⫺1) (47). A strain of Methylomonas
koyamae capable of removing chromium(VI) has also been isolated from river sediment
with substantial heavy metal pollution (including chromium at up to 25 mg kg⫺1) (48).

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Hence, chromium(VI)-removing methanotrophs can be found in environments with

greatly differing heavy metal contamination.
A number of studies have reported methane-driven chromium(VI) reduction by
mixed communities of microorganisms (21, 49–51). These studies have attributed
chromium(VI) reduction in such communities to the activities of nonmethanotrophs.
The fact that a pure methanotroph strain is able to reduce chromium(VI) (20) indicates
that methanotrophs may contribute to methane-driven chromium(VI) reduction in
polymicrobial communities, including those which occur naturally in the environment.
Studies of methane-driven reduction of chromium(VI) by mixed communities of micro-
organisms have shown that the bulk of the chromium in such polymicrobial systems is
in the form of extracellular precipitate that is visible to XPS (21), in contrast to the
intracellular location of the chromium(III) in pure cultures of M. capsulatus Bath.
Intracellular sequestration of chromium(III) by M. capsulatus Bath may help to reduce
its bioavailability to other organisms and, since immobilization of chromium(III) has
been linked to preventing its environmental reoxidation (3), likely make it less suscep-
tible to reoxidation to chromium(VI). It is also possible that, under environmental
conditions on a sufficiently long timescale, the cell-associated chromium(III) may be
released into the environment.
While it is generally accepted that chromium(III) has lower toxicity than chromium(VI),
previous studies have indicated detectable genotoxicity of chromium(III) to Escherichia coli
(52), toxicity through generation of reactive oxygen species in Gram-positive and Gram-
negative bacteria (53), and harmful morphological changes in Shewanella oneidensis (54).
Hence, it is likely that the cells are substantially damaged by their uptake of chromium(III).
They evidently maintain sufficient metabolism to allow the observed reduction and accu-
mulation of chromium species observed, though it may be accumulation of the chromi-
um(III) within the cells that causes decline in chromium(VI) removal as the externally applied
chromium(VI) concentration is increased.
Previously, removal of chromium(III) by microorganisms has been largely attributed to
adsorption to biomass; for example, biofilms of Bacillus subtilis were found to be more
effective in immobilizing chromium(III) produced from reduction of chromium(VI) than
planktonic cells, possibly owing to adsorption to extrapolymeric substances within the
biofilm (55). The results with M. capsulatus suggest a rather different pattern, where uptake
of external chromium(III) into the interior of the cell (cytoplasm and membranes) is an
active process. Since most studies of chromium(VI) bioremediation have not investigated
what happens if chromium(III) is added, it may be that active uptake of chromium(III) is
found in other microorganisms also. The presence of chromium(III) to a substantial extent
in soluble form in the cytoplasm fraction of M. capsulatus is consistent with observations
that organic acids, amino acids, and other small biomolecules can maintain chromium(III)
in soluble form in the presence of macromolecular biomass (56).
In order to establish the pathway of electrons between methane and chromium(VI),
it will be necessary to identify the enzymes involved. Additionally, the effects of the
availability of copper and lanthanides (which control the expression of different forms
of methane monooxygenase, respectively) (30, 57–59), may offer a means of studying
the pathway of electrons into chromium(VI) reduction.
Work in recent years has highlighted the key environmental role played in the
environmental cycling of metals by aerobic methanotrophs (30), which show a much
greater diversity than was previously realized (16). This study has demonstrated that
one member of this important group of organisms shows a novel pattern of interaction
with chromium species that may make it suitable for applications in bioremediation of
chromium species and open the possibility for a role for methanotrophs in transfor-
mation and bioavailability of chromium species in the environment.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The methanotrophic bacterium M. capsulatus Bath was
grown aerobically at 45°C in sterile nitrate mineral salts (NMS) medium with shaking (180 rpm) or NMS
agar plates inside airtight jars, as described previously (60). Methanotrophs such as M. capsulatus Bath
have a “copper switch” that controls the expression of the membrane-associated copper-dependent

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particulate methane monooxygenase (pMMO) versus the cytoplasmic iron-dependent soluble methane
monooxygenase (sMMO) on the basis of the copper-to-biomass ratio of the culture (57, 61). In our hands,
flask cultures grown at a copper concentration of 0.4 ␮M in the NMS used here do not attain the cell
density needed to express sMMO, and so the experiments described were performed under pMMO-
expressing conditions. Chromium(VI) bioremediation experiments were performed in 50-ml liquid cul-
tures in 250-ml conical Quickfit flasks capped with Suba-Seals (Sigma-Aldrich). Plates were incubated in
a 1:4 (vol/vol) methane-air mixture. To add methane to flasks, 50 ml of headspace gas was removed, after
which 60 ml of methane was added (60). Liquid cultures (50 ml) were grown to late logarithmic phase
(OD600 of 0.6 to 0.9), which typically took 40 to 48 h. Chromium species were added from filter-sterilized
stock solutions containing 1,000 mg liter⫺1 of Cr [K2CrO4 for Cr(VI), and Cr(NO3)3 for Cr(III)] to the
concentrations stated for each experiment.
Quantitation and characterization of chromium species. Separation and quantification of aque-
ous chromium(VI) and chromium(III) were achieved via ion exchange HPLC coupled to ICP-MS, as follows.
An aliquot of the sample (20 ␮l) was injected by using a PerkinElmer LC Flexar autosampler into a
PerkinElmer Flexar HPLC pump attached to a Hamilton PRP-X100 column, 4.6 by 250 mm, and coupled
to a PerkinElmer ICP-MS NexION 350X instrument. The column flow rate was 1.2 ml min⫺1; the mobile
phase was 0.5 mmol liter⫺1 EDTA disodium salt (Na2-EDTA) containing nitric acid (70% [wt/wt], 0.875 ml
per liter of solution); aqueous ammonia was added to adjust the pH to 7. The limits of detection were
0.01 mg liter⫺1 for chromium(III) and 0.005 mg liter⫺1 for chromium(VI).
Fractionation of cultures and cells. Aliquots (5 ml) of cultures exposed to chromium(VI) or chro-
mium(III) species as detailed above were collected at intervals and centrifuged (11,000 ⫻ g; 10 min; room
temperature) to remove cells and other debris. The remaining culture supernatant was analyzed via
HPLC–ICP-MS as detailed above.
Cells were fractionated via a modification of a published method (62), as follows. The whole process
was performed at 0 to 4°C, to minimize sample degradation. Harvested cell pellets from 5-ml samples of
culture were washed with 5 ml of 25 mM MOPS (morpholinepropanesulfonic acid, pH 7), centrifuged
(11,000 ⫻ g; 10 min), and resuspended in 5 ml of the same buffer. The suspension was passed twice
through a French pressure cell (8.2 MPa) in order to break the cell walls. The suspension of broken cells
was centrifuged (3,000 ⫻ g, twice for 2 min each) to remove unbroken cells before being centrifuged
(27,000 ⫻ g, 20 min) to sediment cell wall fragments and any other large broken cell fragments. The
pellet was washed twice by resuspension in 25 mM MOPS (pH 7) and then resuspended in the same
buffer; the resulting fraction was used as the cell wall fraction. The supernatant from the first centrifu-
gation after cell breakage, which contained the cytoplasm and membrane fragments, was centrifuged
again (27,000 ⫻ g, 20 min) to remove remaining large particulate material. The resulting supernatant was
used as the cytoplasm-membrane fraction. When cytoplasm and membranes were analyzed separately,
this fraction was further separated via ultracentrifugation at 105,000 ⫻ g for 60 min. The pellet was
washed in 25 mM MOPS (pH 7), ultracentrifuged again under the same conditions, and resuspended in
the same buffer to give the cell membrane fraction. The supernatant from the first ultracentrifugation
was centrifuged again under the same conditions to remove remaining membranous material, to give
the cytoplasm fraction.
Imaging and surface analysis. For electron microscopy, cells from samples (5 ml) of chromium(VI)-
exposed cultures and control cultures without chromium were pelleted by centrifugation (11,000 ⫻ g;
10 min; room temperature) and washed under the same conditions with 0.1 M sodium phosphate buffer
(pH 7.4). The specimens were then fixed in 3% glutaraldehyde in the same buffer overnight at room
temperature and washed again under the same conditions in the same buffer. Secondary fixation was
carried out in 1% (wt/vol) aqueous osmium tetroxide for 1 h at room temperature followed by the same
washing procedure. Fixed cells were dehydrated through a graded series of ethanol dehydration steps
(75, 95 and 100% [vol/vol]) and then placed in a 50/50 (vol/vol) mixture of ethanol and hexamethyld-
isilazane followed by 100% hexamethyldisilazane. The specimens were then allowed to air dry overnight.
A small portion of the fixed sample was crushed and dispersed in methanol, with a drop placed on a
holey carbon-coated copper grid (Agar Scientific). Transmission electron microscopy (TEM) was con-
ducted on an FEI Titan3 Themis G2 instrument operating at 300 kV and fitted with 4 EDX silicon drift
detectors, a Gatan OneView charge-coupled device (CCD) camera, and a Gatan GIF quantum ER 965
imaging filter for electron energy loss spectroscopy (EELS). Energy-dispersive X-ray (EDX) spectroscopy
and mapping were undertaken using Bruker Esprit v1.9 software and a high-angle annular dark-field
(HAADF) scanning TEM (STEM) detector.
For thin-section analysis, after the ethanol dehydration steps, the cells were embedded in EMbed 812
epoxy resin and cut into thin sections (90 nm) using a diamond knife on a Reichert Ultracut S
ultramicrotome. The sections were supported on copper grids and coated with carbon. The samples were
examined in an FEI Tecnai F20 field emission gun (FEG) transmission electron microscope operating at
200 kV and fitted with a Gatan Orius SC600A CCD camera, an Oxford Instruments XMax SDD energy-
dispersive X-ray (EDX) detector, and a HAADF-STEM detector.
XPS measurements were made on a Kratos Supra photoelectron spectrometer at 10 kV and 20 mA
using a monochromatic Al K (alpha) X-ray source (1,486.6 eV). The takeoff angle was fixed at 90°. For each
sample, the data were collected from three randomly selected locations, and the area corresponding to
each acquisition was 400 ␮m in diameter. Each analysis consisted of a wide survey scan (pass energy,
160 eV; 1.0-eV step size), and high-resolution scan (pass energy, 20 eV; 0.1-eV step size) for identification
of component species. The binding energies of the peaks were determined using the C 1s peak at
284.5 eV. The software Casa XPS 2.3.17 was used to fit the XPS spectrum peaks. No constraint was applied

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Enbaia et al. Applied and Environmental Microbiology

to the initial binding energy values, and the full width at half maximum (FWHM) was kept constant for
the carbon contributions in a particular spectrum.
Bioinformatics. BLAST searches of the proteins encoded by the M. capsulatus Bath and M. tricho-
sporium OB3b genomes were conducted via the IMG platform (63).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 1.8 MB.

ACKNOWLEDGMENTS
S.E. gratefully acknowledges financial support from the Libyan Government in the
form of a Ph.D. scholarship.
We thank Michael Cox (Sheffield Hallam University) for assistance with HPLC-ICP-MS
measurements. We thank Chris Hill (University of Sheffield) for sectioning samples
for TEM.
We declare no competing financial interests.

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