Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Hexavalent chromium reduction by bacterial consortia and

pure strains from an alkaline industrial effluent
H.A. Piñón-Castillo1, E.M.S. Brito2, M. Goñi-Urriza3, R. Guyoneaud3, R. Duran3,
G.V. Nevarez-Moorillon4, J.F. Gutiérrez-Corona1, C.A. Caretta5 and G.E. Reyna-López1
1 Departamento de Biologı́a, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, Gto., Mexico
2 Departamento de Ingenierı́a Civil, División de Ingenierı́as, Universidad de Guanajuato, Guanajuato, Gto., Mexico
3 Equipe Environnement et Microbiologie, IPREM UMR CNRS5254, Bâtiment IBEAS, Université de Pau et des Pays de l’Adour, Pau Cedex, France
4 Facultad de Ciencias Quı́micas, Universidad Autónoma de Chihuahua. Chihuahua, Chih., Mexico
5 Departamento de Astronomı́a, División de Ciencias Naturales y Exactas, Universidad de Guanajuato. Guanajuato, Gto., Mexico

Keywords Abstract
adsorption, bacterial communities, chromium
(VI), reduction, t-RFLP. Aims: To characterize the bacterial consortia and isolates selected for their role
in hexavalent chromium removal by adsorption and reduction.
Correspondence Methods and Results: Bacterial consortia from industrial wastes revealed signi-
Georgina E. Reyna-López, Departamento de ficant Cr(VI) removal after 15 days when incubated in medium M9 at pH 6Æ5
Biologia, División de Ciencias Naturales y
and 8Æ0. The results suggested chromium reduction. The bacterial consortia
Exactas, Universidad de Guanajuato. Cjon. de
jalisco s ⁄ n, Col. valenciana, 36000
diversity (T-RFLP based on 16S rRNA gene) indicated a highest number of
Guanajuato, Gto., Mexico. operational taxonomic units in an alkaline carbonate medium mimicking
E-mail: [email protected] in situ conditions. However, incubations under such conditions revealed low
Cr(VI) removal. Genomic libraries were obtained for the consortia exhibiting
2010 ⁄ 0663: received 22 April 2010, revised 5 optimal Cr(VI) removal (M9 medium at pH 6Æ5 and 8Æ0). They revealed the
August 2010 and accepted 23 August 2010 dominance of 16S rRNA gene sequences related to the genera Pseudomonas ⁄
Stenotrophomonas or Enterobacter ⁄ Halomonas, respectively. Isolates related to
doi:10.1111/j.1365-2672.2010.04849.x
Pseudomonas fluorescens and Enterobacter aerogenes were efficient in Cr(VI)
reduction and adsorption to the biomass.
Conclusions: Cr(VI) reduction was better at neutral pH rather than under in
situ conditions (alkaline pH with carbonate). Isolated strains exhibited signifi-
cant capacity for Cr(VI) reduction and adsorption.
Significance and Impact of Study: Bacterial communities from chromium-
contaminated industrial wastes as well as isolates were able to remove Cr(VI).
The results suggest a good potential for bioremediation of industrial wastes
when optimal conditions are applied.

most dangerous substances by the US EPA (1998). Cr(VI)

Introduction
compounds are mutagenic, carcinogenic (Losi et al. 1994)
Chromium is a metal existing under several oxidation and inhibit enzymes and nucleic acid synthesis (Gunarat-
states, ranging from )2 to +6. Among them, Cr(VI) and nam and Grant 2008). By contrast, Cr(III) is less toxic
Cr(III) are of major environmental significance because and much less mobile. It forms stable complexes with
of their persistence and stability. Cr(VI) is a strong oxi- organic ligands (Zayed and Terry 2003) and precipitates
dizing agent, commonly present as hydrochromate at physiological pH as hydroxide [Cr(OH)3] or hydrated
(HCrO4)), chromate (CrO4)) or dichromate (Cr2O7) oxide (Cr2O.H2O) (Ehrlich 2002).
oxyanions, depending on pH (United States, Environ- In contaminated soils and industrial wastes, chromium
mental Protection Agency, US EPA 1998). Because of its availability is influenced by many processes such as organic
high solubility in water (Losi et al. 1994; Barceloux 1999), and inorganic complexes formation, oxidation ⁄ reduction,
Cr(VI) is highly toxic and has been listed as one of the precipitation ⁄ dissolution or adsorption ⁄ desorption. These

Journal of Applied Microbiology 109, 2173–2182 ª 2010 The Society for Applied Microbiology
No claim to Mexican Government works 2173
Bioreduction of hexavalent chromium H.A. Piñón-Castillo et al.

processes can be influenced by microbial activities (Gadd Table 1 Physicochemical characteristics of the samples from industrial
2005), but chronic chromium stress can decrease microbial wastes lixiviates
diversity, biomass and activity. Some micro-organisms can Metal content (lg g)1)
resist to this stress and are more likely to survive and thus
influence chromium speciation (Francisco et al. 2002; Sample Ca Total Cr Cu Fe Zn Salinity (%) pH

Branco et al. 2005). Therefore, the success of microbial- Dry season 43780 7040 10 19590 80 100 12Æ0
based chromium remediation technologies requires a Wet season 3660 5100 <DL 2695 30 100 12Æ0
better understanding of the microbial community diversity
<DL, under detection limit.
and response to these stress conditions. Some indigenous
bacterial species from chromium-contaminated environ-
ments are highly resistant to Cr(VI) (Agrawal et al. 2006), minimal medium contained the following per litre of dis-
and the mechanisms of interaction with chromium have tilled water: K2HPO4, 3 g; NaH2PO4, 6 g; and NH4SO4,
been considered of importance for the development of 10 g. This basal medium was autoclaved, and after cool-
new cleaning technologies (Viamajala et al. 2007). The ing the following solutions were added: 2 ml of 1 mol l)1
most studied mechanisms of chromium removal are MgSO4 and 100 ll of 1 mol l)1 CaCl2. The medium with
related to the Cr(VI) reduction to Cr(III) and chromium high CO3 concentration, employed to mimic environ-
biosorption (Cervantes et al. 2001; Gadd 2005). mental conditions (addition of CaCO3 to precipitate
The industrial area of Guanajuato (Mexico) is charac- chromium in the in situ tailings), contained per litre of
terized by both many tannery manufactures and industrial distilled water: CaCl2 2H2O 0Æ05 g, NH4Cl 0Æ5 g, KH2PO4
chromium production from mineral chromite [(Fe, 0Æ2 g, NaCl 20 g, KCl 0Æ2 g, MgSO4.7H2O 1 g, MgCl.
Mg)Cr2O4]. For more than 50 years, this industry has 6H2O 2 g, Na2CO3 40 g and yeast extract 0Æ5 g. Both
produced and accumulated large amounts of wastes. The media were adjusted to pH 6Æ5 with 1 mol l)1 HCl and
aim of this study was to characterize bacterial consortia to pH 8Æ0 or 10Æ0 with 1 mol l)1 NaOH. After pH adjust-
build-up to diminish chromium (VI) concentrations. ment, 10 g of sediment derived from the industrial wastes
The consortia were obtained from an extremely alkaline was suspended in 30 ml of medium, stirred during 5 min
and chromium-contaminated site. The bacterial consortia and centrifuged at 4600 g for 10 min (IEC HN-SII; Inter-
and their efficiency in diminishing Cr(VI) were evaluated national Equipment Company, Needham, MA, USA). The
with two culture media and at three pH values. Microbial pellet was then dissolved in the same volume of medium,
diversity of these consortia was assessed using t-RFLP and the overall process continued until the supernatant
fingerprints based on 16S rRNA gene amplification. The was colourless. The pellet obtained at the last stage was
consortia exhibiting the best efficiency in terms of chro- dissolved in 10 ml of medium and served as inoculum
mium remediation were further characterized by clone (1 ⁄ 10, v ⁄ v) for the enrichments under different incuba-
libraries analyses. Finally, three bacterial isolates obtained tion conditions (media and pH). All incubations were
from these consortia were evaluated for their capacity to processed in the presence of glucose as carbon and elec-
reduce or adsorb Cr(VI). The overall results are discussed tron sources (2%) and 50 mg l)1 of Cr(VI) from a stock
in the view of possible application of biological technolo- solution of K2CrO7. They were incubated at 28C at
gies for chromium remediation in industrial wastes. 200 rev min)1 under oxic conditions for 30 days (Shaking
incubator; LabTech Co. Ltd, Seoul, South Korea). All
experiments were carried out in triplicate, and the
Materials and methods
standard deviation for chromium measurements was
Samples were lixiviates originating from the waste depos- obtained. The consortium exhibiting the best efficiency in
its of a chromite-processing industry located in León, Gto diminishing Cr(VI) concentration was used to analyse
(Guanajuato state), in central Mexico (latitude N Cr(VI) reduction, adjusting the initial O.D. at 620 nm to
2102¢32¢¢, longitude W 10147¢29¢¢). Chemical analyses 0Æ8 (Labsystem Multiskan MS, Finland).
of the samples were carried out by flame atomic absorp-
tion (FLAA) using standard methods (EPA, 1996). Some
Molecular characterization of the consortia
of the characteristics of the samples are given in Table 1.
Community diversity was determined for all consortia by
t-RFLP in samples obtained after 15 and 30 days of incu-
Consortia enrichments
bation. Clone libraries were obtained from the 15- day
Consortia enrichments were carried out in modified M9 incubation for consortia grown in modified M9 media at
minimal medium and on a mineral minimal medium pH 6Æ5 and 8Æ0. The samples (1Æ5 ml) were centrifuged
with high concentration of carbonates. The modified M9 and the pellet immediately frozen at )70C.

Journal of Applied Microbiology 109, 2173–2182 ª 2010 The Society for Applied Microbiology
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H.A. Piñón-Castillo et al. Bioreduction of hexavalent chromium

DNA extraction and amplification conditions of step, electrophoresis was carried out for 30 min, applying
16s rRNA gene a voltage of 15 kV. T-RFLP profiles were analysed using
The samples were mixed with 400 ll Tris–glucose–EDTA Gene Scan Software (Applied Biosystem). Only the
(25 mmol l)1 pH 8Æ0, 50 mmol l)1, 10 mmol l)1 pH 8Æ0, T-RFLP peaks with intensity above 1% were considered
respectively) solution containing lysozyme (final concen- (Bordenave et al. 2004). Comparison analysis of T-RFLP
tration 1 mg ml)1) and achromopeptidase (final concen- data (abundance of OTUs, Operational Taxonomic Units)
tration 20 mg ml)1 or 1500 U ml)1) and incubated at in relation to growth conditions was performed by prin-
37C for 10 min. Two microlitres of proteinase K cipal component analysis (PCA) using mvsp software
(20 mg ml)1) and 20 ll sodium dodecyl sulfate (SDS (Multivariate statistical package 3.12d, Kovach Computing
10%) were then added, and the solution was incubated for Service, 1985–2001, Anglesey, UK). Linear regression anal-
30 min at 37C. Twenty microlitres of SDS 10% was ysis was performed to avoid over interpretation of the
added afterwards, and the solution incubated for 10 min correlation and synthesis errors.
at 60C. These lysis steps were followed by a classical
organic extraction: phenol, phenol–chloroform and chlo- Genomic libraries
roform–isoamyl alcohol (Ogram et al. 1987). Finally, To complement the community diversity analysis by
nucleic acids were precipitated with 10% cold sodium ace- T-RFLP, genomic libraries of the 16S rRNA gene were
tate (3 mol l)1; pH 5Æ2) and 95% cold ethanol (overnight constructed, using PCR products generated by unlabelled
at )20C) and centrifuged (15 200 g) for 30 min. DNA primers 8F and 907R. These fragments were inserted on
was dissolved in sterile deionized water and incubated standard cloning vector (pCR2.1 Topo TA cloning kit;
with 1 ll RNase (1 mg ml)1) at 37C for 30 min. DNA Invitrogen, Inc., Carlsbad, CA) and transformed into Esc-
quality was verified by electrophoresis on 1% agarose gel herichia coli TOP10 cells. Clones containing inserts were
in Tris–acetate–EDTA buffer (TAE). DNA solutions were digested with restriction enzymes HaeIII and HinP1I, and
preserved at )20C. Bacterial 16S rRNA gene was ampli- the restriction profiles were analysed on 3% agarose gel
fied by PCR in a PTC 200 thermocycler (Corbett Research, electrophoresis (small-fragment resolution agarose, QA
Mortlake, NSW, Australia) using forward primer 8F agarose; QBiogene Inc., Illkirch, France) (Sklarz et al.
(5¢-AGAGTTTGATCCTGGCTCAG-3¢) and reverse primer 2009). Two clones were selected for every restriction pro-
907R (5¢-CCGTCAATTCCTTTRAGTTT-3¢), both specific file and sequenced using the Big Dyes Terminator ver. 3.1
for the domain Bacteria (Lane et al. 1985; Felske et al. cycles sequencing kit. The sequences obtained were com-
1997). The reactions were cycled with an initial denatur- pared with the GenBank nucleotide database library, by
ation step (95C for 5 min) followed by 35 cycles of dena- BLAST online searches. Multiple sequences alignment and
turation (95C for 45 s), annealing (52C for 45 s) and phylogenetic analysis were performed using mega 4.1
elongation (72C for 10 min) and then a final elongation software and the neighbour-joining algorithm model. The
step (72C for 15 min). PCR products were purified with significance of branching order was determined using
the GFX PCR DNA purification kit (Amersham, Orsay, bootstrap analysis with 500 replacing data set (Tamura
France) according to the manufacturer. The samples were et al. 2007).
analysed by triplicate to avoid false positive amplifications
and the introduction of artefacts. Negative controls with
Isolation and phylogenetic analyses of bacterial strains
no DNA template or PCRs performed only with forward
or reverse primers were usually included. Isolation procedures
The consortia exhibiting the highest chromium reduction
T-RFLP analysis potential (M9 medium, pH 8) were selected for bacterial
T-RFLP analysis was performed with the primers isolations. Bacterial density was estimated by the Most
described previously labelled with carboxifluorescein Probable Number (MPN) method on the same medium
(FAM). Purified PCR products (100 ng) were digested supplemented with glucose, lactate-Na or pyruvate-Na
with 10 U of HaeIII or HinP1I (New England Biolabs, (10 mmol l)1 each) as carbon sources. The samples were
Ipswich, MA). The length of terminal fluorescently diluted in the same medium to obtain a theoretical bacte-
labelled fragments, from the digested PCR products, was rial abundance of one cell per 100 ll. Thousand aliquots
determined by capillary electrophoresis on an ABI prism of 100 ll were then distributed in 384 microtiter plates
310 (Applied Biosystems, Foster City, CA, USA). Approxi- supplemented with 50 and 100 mg l)1 of Cr(VI) and
mately 50 ng of digested DNA was mixed with 15Æ5 ll of incubated for 48 to 96 h at 30C. Among these 1000 sub-
deionized formamide, and 0Æ5 ll of TAMRA size standard cultures, those exhibiting chromium reduction were
was added (Applied Biosystems) and then denatured at transferred to solid medium [10 mmol l)1 glucose and
94C for 5 min and chilled on ice. After a 10 s injection 50 mg l)1 of Cr(VI)] to check for purity. Three strains

Journal of Applied Microbiology 109, 2173–2182 ª 2010 The Society for Applied Microbiology
No claim to Mexican Government works 2175
Bioreduction of hexavalent chromium H.A. Piñón-Castillo et al.

exhibiting the best growth and Cr(VI) reduction potential ditions (6Æ5, 8Æ0 and 10Æ0). A decrease in dissolved
were chosen to test for the influence of pH on Cr(VI) Cr(VI) concentration was evidenced whatever the incu-
reduction and adsorption. bations, nevertheless the highest decreases (100% in
15 days) were obtained for bacterial consortia incubated
Phylogenetic analyses of isolated strains in modified M9 medium at pH 8Æ0 and 6Æ5 (Fig. 1).
The 16S rRNA encoding genes were amplified by PCR Incubations with carbonate medium, tested to mimic
using primers 8F (5¢-AGAGTTTGATCCTGGCTCAG-3¢) in situ alkaline and carbonated conditions, never reached
and 1489R (5¢-TACCTTGTTACGACTTCA-3¢ (Weisburg 100% removal of dissolved Cr(VI) in the culture, even
et al. 1991). Amplified 16S rRNA-encoding genes were after 30 days, and best removal was evidenced for lower
partially sequenced (from nt 8 to nt 1489 according to pH (Fig. 1).
E. coli numbering) using the BigDye sequencing kit The bacterial diversity in the consortia was evaluated
(Applied Biosystem). The sequences obtained were com- by T-RFLP, in samples taken after 15 and 30 days of
pared with the GenBank nucleotide database library and incubation (see Fig. S1). The highest diversity in terms of
analysed, as described for Genomic libraries. OTU was found in the consortia incubated in the carbon-
ate medium (from 10 to 12 OTU, six of them being com-
mon for all pH conditions) when compared with M9
Evaluation of chromium interactions for consortia and
medium enrichments (3 to 8 OTU, three of them being
bacterial strains
common for all pH conditions).
During the enrichment procedure of the bacterial consor- PCA (Fig. 2) clearly discriminate the bacterial commu-
tia, aliquots were taken at different sampling times and nities as a function of the incubation medium according
centrifuged at 15 200 g for 5 min. In the supernatant, to the second axis explaining 23Æ99% of the dissimilarities
hexavalent chromium was quantified by the colorimetric between the samples. PCA also showed no differences
method employing diphenylcarbazide (DPC) (Greenberg between the communities analysed after 15 and 30 days
et al. 1981). Total chromium in culture medium was for both pH 8Æ0 and 6Æ5 incubated in modified M9
measured directly from culture medium filtrates, using culture medium. Growth was probably already finished
0Æ45 lm membranes (Millipore, Billerica, MA, USA). To after 15 days as revealed by Cr(VI) removal (see Fig. 1).
determine chromium in the biomass, 50 ml cultures was Higher differences were found between both incubation
prepared, and the biomass was recovered by centrifuga- times for all other samples. The overall data also showed
tion, frozen and lyophilized (Labconco, Kansas City, MO, less dissimilarity between all communities grown on car-
USA). Total chromium content was measured on acid bonate medium, which could be linked to less growth
digests by FLAA (EPA, 1996). (Fig. 2).
To evaluate the capability to diminish Cr(VI) and con- The enrichment consortia exhibiting the more efficient
vert Cr(VI) to Cr(III), the isolated strains were incubated chromium removal (modified M9 medium, pH 6Æ5 and
with modified M9 medium at pH 6Æ5 and 8Æ0. From these 8Æ0) were selected for further studies on short time scaled
cultures, those with the highest observed efficiency for
decreasing chromium were transferred to fresh liquid
medium and incubated overnight at 200 rev min)1 and 100
28C under oxic conditions. These cultures were used
Cr(VI) remaining

80
to inoculate fresh medium containing 50 mg l)1 of
Percentage of

Cr(VI), using an initial O.D. (620 nm) of 0Æ8. Subsamples 60

were taken every 48 h to measure growth (biomass by 40
dry weight) as well as Cr(VI) in the culture medium
20
<DL
<DL

<DL
<DL

(Greenberg et al. 1981) and total chromium on biomass

determined by standard methods (EPA, 1996). 0
15 30
Time (day)
Results Figure 1 Percentage of soluble Cr(VI) in the culture medium for all
bacterial consortia. The initial concentration was 50 mg l)1 correspond-
Bacterial consortia: Cr(VI) removal and diversity ing to 100% Cr(VI) at initial time (solid line). The bars for 15 and
analyses by T-RFLP 30 days correspond to bacterial consortia incubated in modified M9
medium at pH 6Æ5, 8Æ0 (both under detection limit) and 10Æ0 (light
Bacterial consortia incubations were conducted with grey) or in the carbonate medium at pH 6Æ5 (black), 8Æ0 (white) and
modified M9 medium and with the mineral medium 10Æ0 (dark grey). Standard deviations were calculated from triplicate
supplemented with carbonate, both under three pH con- experiments. <DL, under detection limit (0Æ1 mg l)1 per 0Æ05%).

Journal of Applied Microbiology 109, 2173–2182 ª 2010 The Society for Applied Microbiology
2176 No claim to Mexican Government works
H.A. Piñón-Castillo et al. Bioreduction of hexavalent chromium

PCA case scores

3·4
6·5 M9 30
2·7
6·5 M9 15
2·1
10 M9 15

1·4

Axis 2: 23·99%
8·0 M9 15
Figure 2 Principal component analysis from 0·7
T-RFLP profiles obtained for bacterial consortia 10 M9 30
8·0 M9 30
incubated under different incubation
conditions. t-RFLP’s profiles were obtained –2·1 –1·4 –0·7 0·7 1·4 2·1 2·7 3·4
from 16SrRNA gene amplicons digested with 10 CO3 15 –0·7
10 CO3 30
two restriction enzymes (see text). The dark 6·5 CO3 30 8·0 CO3 30
triangles represent samples incubated with –1·4
6·5 CO3 15
the carbonate medium and the open circles 8·0 CO3 15
the modified M9 medium. Results shown –2·1
are the average of triplicates. Axis 1: 26·86%

experiments under the same incubation conditions. 6Æ5 were related to Pseudomonas putida and Stenotropho-
Growth was significantly faster and biomass production monas malthophilia (Fig. 4).
higher when grown at pH 6Æ5 when compared with pH
8Æ0 (Fig. 3a). Nevertheless, the decrease in soluble Cr(VI)
Bacterial strains: isolation, identification and chromium
was only slightly more efficient at pH 6Æ5 and in both
removal
cases reached 100% after 144 h of incubation when cells
were present (Fig. 3b). This decrease in soluble Cr(VI) One hundred strains were obtained from the bacterial
could be partially related to biomass adsorption because consortia incubated in M9 medium at pH 6Æ5 and 8Æ0.
total soluble chromium decreased in the first 48 h and From these, the strains BCGc1, BCG42 and BCG70 were
was stable afterwards (Fig. 3c) with values ranging from selected for their high tolerance to chromate (tested up to
73Æ03 (pH 6Æ5) to 85Æ15% (pH 8Æ0). Chromium adsorp- 600 mg l)1) in solid medium. The 16S rRNA gene
tion to the biomass could be estimated from 413 to sequence from strains BCGc1 and BCG42 showed high
665 lg Cr mg)1 biomass at pH 8Æ0 and 85 to relatedness to Pseudomonas fluorescens, whereas strain
282 lg Cr mg)1 biomass at pH 6Æ5 (data not shown). BCG70 was related (98% similarity) to Ent. aerogenes
These overall results indicate that most of the Cr(VI) was (Fig. 4). These strains were analysed for their capacity of
probably reduced to Cr(III) in the culture medium. Cr(VI) removal and chromium adsorption potentials at
pH 6Æ5 and 8Æ0. Strains BCGc1 and BCG42, closely related
phylogenetically, exhibited good Cr(VI) removal at both
Bacterial consortia: identification of bacterial populations
pH values, while strain BCG70 only had this capacity at
by clone libraries
pH 8Æ0 (Fig. 5). Strains BCG42 and BCG70 were selected
The consortia incubated in modified M9 medium at for further investigations at pH 8Æ0. Their growth was not
both pH 6Æ5 and 8Æ0 were selected for precise analysis of significantly different in the presence or absence of Cr(VI)
the bacterial community composition by genomic library (Fig. 6a) and, as already obtained for bacterial consortia,
analyses. Hundred and forty clones were obtained, the percentage of Cr(VI) remaining in the culture med-
grouped according to their restriction patterns (AR- ium was very low with values around 2% after 96 h of
DRA), and 17 phylotypes were sequenced. BLAST analy- incubation (Fig. 6b). On the other hand, the amount of
ses and dendrogram construction showed completely soluble total chromium decreased in the first 48 h and
different community composition for the two enrich- reached minimum values ranging from 77Æ5 (strain
ment conditions (Fig. 4). The 16S rRNA gene sequences BCG42) to 72Æ7% (strain BCG70). In contrast to bacterial
obtained for the consortia incubated at pH 8Æ0 showed consortia, chromium adsorption to the biomass was
two main clusters related to Enterobacter sp., Enterobact- much lower for strain BCG42 as it was estimated from 44
er aerogenes and Halomonas chromatireducens (similari- to 67 lg Cr mg)1 biomass. It was higher for strain
ties from 93 to 97%, depending on the clone tested). BCG70 (92 to 529 lg Cr mg)1 biomass) but decreasing
The data obtained for the consortium incubated at pH all over the incubation period (data not shown).

Journal of Applied Microbiology 109, 2173–2182 ª 2010 The Society for Applied Microbiology
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Bioreduction of hexavalent chromium H.A. Piñón-Castillo et al.

(a) 2·50 induces chromate–carbonate interactions and probably

reduces chromate spills from wastes even if chromate
Growth (Ln mg ml–1)

2·00
concentrations in lixiviates are extremely high, ranging
1·50 from 5000 to 7000 ppm. Therefore, chemical treatment
of the tailings is not enough to avoid chromate con-
1·00 tamination, and bioprocesses could represent a good
0·50 alternative.
The enrichments made with the carbonate medium,
0·00 realized to mimic in situ conditions of alkalinity and
0 48 96 144
Time (h) salinity, revealed more diversity when compared with the
(b) 100
consortia obtained with modified M9 medium. Never-
theless, under such conditions chromate removal was less
Cr(VI) remaining
Percentage of

80 efficient. This could be attributed to a decrease in

60 Cr(VI) toxicity, as a result of carbonate addition, which
could influence chromate availability to micro-organisms
40
(Shukla et al. 2009). As already demonstrated for estua-
20 rine sediments (Grahamand and Bouwer 2010), the effi-
0 ciency of Cr(VI) removal was better when pH was
0 48 96 144 decreased when compared to the in situ conditions.
Time (h)
Cultures at pH 8Æ0 conserved only 2Æ99 ± 0Æ33 mg l)1 of
(c) 100 Cr(VI) after 144 h of incubation, a value close to the
total chromium

legally allowed environmental limits for this contami-

Percentage of

80
nant. This is especially interesting because these results
60
were obtained under conditions resembling those usually
40 found in tannery industrial wastes (Lefebvre et al. 2006;
20 Sivaprakasam et al. 2008), characterized by high alkalin-
0 ity. Therefore, it could be possible to use bioprocesses
0 48 96 144 directly to lixiviate by the addition of organic carbon
Time (h)
and electron sources for growth of the bacterial commu-
Figure 3 Growth, soluble Cr(VI) and total chromium in culture nity. Nevertheless, the enriched consortia obtained from
medium for selected bacterial consortia. Growth (a) was measured by industrial wastes lixiviates were much more efficient to
dry weigh in bacterial consortia grown in modified M9 medium at pH perform chromium removal in the modified M9 med-
6Æ5 with (black squares) and without Cr(VI) (open squares) and at pH ium at pH 6Æ5 and 8Æ0 even if diversity analysis revealed
8Æ0 with (black circles) and without Cr(VI) (open circles). Percentage of
a limited number of OTUs. Therefore, another possibil-
soluble Cr(VI) (b) and total chromium (c) in the culture medium was
evaluated at pH 6Æ5 (grey bars) and pH 8Æ0 (black bars). In both cases,
ity of bioprocessing these lixiviates would be a dilution
black lines (pH 8Æ0) and grey lines (pH 6Æ5) correspond to controls (for salinity) and acidification of the wastes before treat-
incubated without cells. Standard deviations were calculated from ment because the organisms able to remove the Cr(VI)
triplicate experiments. are present and efficient. The microbial diversity analysis
of Cr(VI) reducing consortia showed that on M9
medium at pH 8Æ0, two main groups related to gamma
proteobacteria were predominant: Ent. aerogenes and
Discussion
H. chromatireducens. In the same medium at pH 6Æ5
The sample site consisted of lixiviate from a chromite Ps. putida and S. malthophilia were found as the major
waste tailings. According to site main characteristics, it groups.
can be considered an extreme environment because of Some of these micro-organisms have already been
high pH, salinity and also metal contain (Table 1). Such described with regard to their chromium reducing capaci-
conditions of pH and salinity are close to those found ties. Pseudomonas putida, for example, has been observed
in soda lakes (Foti et al. 2008; Joshi et al. 2008). More- to reduce Cr(VI), and the enzymatic activity responsible
over, such high content of heavy metals, among them for this process has been characterized (Park et al. 2000).
chromium, is a condition rare in nature. The high Regarding the genus Halomonas, only H. chromatiredu-
amounts of calcium and the high pH found in lixiviates cens, isolated from a hot spring, has been described as a
are the result of the addition of CaCO3 to the wastes Cr(VI) reducer under high salinity (Shapovalova et al.
to avoid acid bioleaching. Such carbonate addition 2009). Some reports have indicated the biotechnological

Journal of Applied Microbiology 109, 2173–2182 ª 2010 The Society for Applied Microbiology
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H.A. Piñón-Castillo et al. Bioreduction of hexavalent chromium

58 C5 pH 6·5
60 C27 pH 6·5
84 C11 pH 6·5
96 C19 pH 6·5
6 C22 pH 6·5
95
Pseudomonas putida (AF094741)
98 Pseudomonas stutzieri (EU883663)
Pseudomonas fluorescens (AM181176.4)
63 Strain BCGc1
99
88
Strain BCG42
96 C39 pH 6·5
100 Stenotrophomonas malthophilia (AB294557)
C7 pH 8
52 Halomonas chromatireducens (EU447163)
Figure 4 Phylogenetic tree based on 16S 96
52 C3 pH 8
rDNA gene sequences of the clones (indicated 27 55 C8 pH 8
as Cx pHz) and the isolated strains (bold C5 pH 8
C4 pH 8
typed). The relatedness of the newly obtained
14
C2 pH 8
sequences to closest reference organism is C10 pH 8
shown. GenBank accession numbers of the C1 pH 8
12
reference sequences are given in parenthesis. C11 pH 8
The bootstrap neighbour-joining tree was 18 C9 pH 8
C6 pH 8
constructed with mega 4.1 software.
Strain BCG70
Numbers at nodes show occurrences in Enterobacter aerogenes (AB244472)
bootstrap samples and provide an estimate of Enterobacter sp. (CP000653.1)
confidence of the analysis. Bar represents 0Æ2
substitutions per site. 0·2

100 the other hand, Ent. aerogenes has already been character-
Cr(VI) remaining

ized for its resistance to antibiotics by pump efflux mech-

Percentage of

80
anisms (Chevalier et al. 2008; Moreira et al. 2009),
60
although resistance mechanisms to Cr(VI) have not been
40 reported.
20 The results for Cr(VI) removal obtained with the pure
0 strains were quite similar as those obtained for the
BCGc1 BCG42 BCG70
Isolates bacterial consortia, i.e., total removal of Cr(VI) with
only partial chromium adsorbed to the biomass. Never-
Figure 5 Effect of pH on Cr(VI) removal in culture medium by theless, the adsorption of chromium to the biomass was
isolated strains. Levels of Cr(VI) remaining in culture of strains BCGc1, quite low in our experiments (maximum of about
BCG42 and BCG70 at pH 6Æ5 (grey) and 8 (black). The initial concen- 600 lg Cr g)1) when compared to data obtained with
tration of Cr(VI) was 50 mg l)1. Data shown represent the average of
cyanobacteria exhibiting up to 18000 lg Cr g)1(Colica
three separate experiments, and deviation bars are indicated.
et al. 2010). Nevertheless, these data are comparable to
some obtained elsewhere with Pseudomonas strains
potential of bacterial strains belonging to the genus (Pérez Silva et al. 2009). Reduction to Cr(III) and
Halomonas (Khijniak et al. 2003; Llamas et al. 2006).The adsorption to biomass are the two major ways for chro-
efforts aimed at isolating the culturable strains from the mium removal. The decrease in Cr(VI) concentrations
enrichment consortia led to the recovery of few strains: in the spent medium and the constant levels of total
two similar to Ps. fluorescens and only one similar to chromium over incubation time, combined with the low
Ent. aerogenes. This poor diversity was probably attributed levels of chromium incorporated into the biomass, were
to selective pressure by chromium presence and culture indicative of biologically mediated chemical reduction of
conditions. Pseudomonas fluorescens has been described Cr(VI) to Cr(III), as was also the case with the enriched
with regard to heavy metal solubilization and phyto- consortia. Cr(VI) detoxification by its conversion to
extraction (Braud et al. 2006) and has also been studied Cr(III) has been proposed as a mechanism of bacterial
for its response to heavy metals in planktonic cells and resistance to the oxyanion (Ramı́rez-Dı́az et al. 2007). As
biofilms (Workentine et al. 2008). Pseudomonas putida discussed before, Cr(VI) reduction is a well-known pro-
has been described with regard to their pump efflux cess in Pseudomonas and Halomonas strains, and the
mechanisms acting as protection against Cr(VI) damage same process probably occurs in our Enterobacter strain
(Jiménez-Mejı́a et al. 2006; Diaz-Perez et al. 2007). On BCG70.

Journal of Applied Microbiology 109, 2173–2182 ª 2010 The Society for Applied Microbiology
No claim to Mexican Government works 2179
Bioreduction of hexavalent chromium H.A. Piñón-Castillo et al.

(a) 2·3 nological processes for chromium transformation and

detoxification in industrial wastes or for bioremediation
1·8 of chromium-contaminated areas. The strategies for treat-
Ln mg ml–1

ment could involve the amendment with cultured isolated

1·3
micro-organisms and ⁄ or enriched consortia and the use
0·8 of cheap organic nutrients. More work is needed to
improve chromium removal under conditions similar to
0·3
those found in situ, considering the use of consortia or
–0·2 pure culturable isolates adapted to saline conditions, such
0 48 96 144
Time (h) as the Halomonas representatives found in this study. An
additional aspect of interest is the search for a biotechno-
(b) 100 logical process for the recovery and recycling of Cr(III)
produced by reduction of Cr(VI).
Cr(VI) remaining
Percentage of

80
60
Acknowledgements
40
This work was supported by Grants from ECOS-
20
NORD–SEP-CONACyT-ANUIES (M07A01), FONCICyT
0 (Ref. 95887) and Universidad de Guanajuato (129 ⁄ 09),
0 48 96 144
Time (h) México. H.A. Piñón-Castillo received a fellowship from
CONACyT, Mexico. Thanks are given to Cruz E. Marti-
(c) 100 nez-Palacios for technical assistance.
total chromium
Percentage of

80
Author contributions
60
40 Conceived and designed the experiments: H.A.P.C.,
G.E.R.L., E.M.S.B., R.G., M.G.U., F.G.C., R.D. Performed
20
the experiments: H.A.P.C., E.M.S.B., V.N.M. Analysed the
0 data: H.A.P.C., G.E.R.L., C.C., E.M.S.B., V.N.M., R.G.,
0 48 96 144
Time (h) M.G.U., R.D., F.G.C., C.C. Wrote the paper: H.A.P.C.,
G.E.R.L., R.G., R.D., F.G.C., C.C.
Figure 6 Growth, soluble Cr(VI) and total chromium in culture
medium for selected bacterial strains. Growth (a) was measured by
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2182 No claim to Mexican Government works