Ecotoxicology and Environmental Safety 147 (2018) 102109

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Hexavalent chromium reduction potential of Cellulosimicrobium sp. isolated MARK

from common euent treatment plant of tannery industries

Ram Naresh Bharagava , Sandhya Mishra
Laboratory for Bioremediation and Metagenomics Research (LBMR), Department of Environmental Microbiology, Babasaheb Bhimrao Ambedkar University (A Central
University), Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India

A R T I C L E I N F O A B S T R A C T

Keywords: Present study deals with the isolation and characterization of a bacterium capable for the eective reduction of
Tannery euent Cr(VI) from tannery wastewater. Based on the 16S rRNA gene sequence analysis, this bacterium was identied as
Chromium reduction Cellulosimicrobium sp. (KX710177). During the Cr(VI) reduction experiment performed at 50, 100, 200,and
Cellulosimicrobium sp. 300 mg/L of Cr(VI) concentrations, the bacterium showed 99.33% and 96.98% reduction at 50 and 100 mg/L at
16S rRNA gene
24 and 96 h, respectively. However, at 200 and 300 mg/L concentration of Cr(VI), only 84.62% and 62.28%
SEM analysis
reduction was achieved after 96 h, respectively. The SEM analysis revealed that bacterial cells exposed to Cr(VI)
EDX analysis
showed increased cell size in comparison to unexposed cells, which might be due to either the precipitation or
adsorption of reduced Cr(III) on bacterial cells. Further, the Energy Dispersive X-ray (EDX) analysis showed some
chromium peaks for cells exposed to Cr(VI), which might be either due to the presence of precipitated reduced Cr
(III) on cells or complexation of Cr(III) with cell surface molecules. The bacterium also showed resistance and
sensitivity against the tested antibiotics with a wide range of MIC values ranging from 250 to 800 mg/L for
dierent heavy metals. Thus, this multi-drug and multi-metal resistant bacterium can be used as a potential
agent for the eective bioremediation of metal contaminated sites.

1. Introduction Andhra Pradesh, Bihar, Gujarat, and Maharashtra, generating total ~

The contamination of environments (soil and water) with various
toxic metals is a serious threat for ecosystem and human health, and
requires the implementation of appropriate remedial measures. Heavy
metals, such as chromium, cadmium, mercury, arsenic, lead etc. are
considered as major environmental pollutants due to their toxic eects
on environment as well as on human health (Ray and Ray, 2009). In
developing countries, dierent types of industrial wastes (solid and li-
quid) containing a number of toxic metals in high concentration are
directly or indirectly discharged into the environment without adequate
treatment (Dixit et al., 2015; Chandra et al., 2009). Industries such as
metallurgical, chemical, refractory brick, leather, wood preservation,
pigments and dyes are the major sources of toxic metals contamination
in environment (USEPA, 1998; Ryan et al., 2002).
However, tannery industries are the major source of chromium
contamination into the environment. Tannery industries consume a
huge volume of water in tanning of hides and skin, as it is wholly a wet
process and generate ~ 3035 L of wastewater per kg skin/hides pro-
cessed (Nandy et al., 1999). There are ~ 3000 tanneries in India,
mainly located in the states of Tamil Nadu, West Bengal, Uttar Pradesh,
1,75,000 m3 wastewater per day (Kaul et al., 2005). In Uttar Pradesh, ~
444 tanneries are in operation mainly in Kanpur and Unnao region
generating 22.1 MLD of wastewater per day (CPCB, 2013) and this
wastewater is reported to contain 0.014.24 mg/L of Cr(VI) (MOWR,
2013). However, most of the tanneries (nearly 80%) are engaged in
chrome tanning process that releases ~ 20003200 t of Cr into the
environment annually (Belay, 2010). The Cr concentration in tannery
wastewater ranges between 2000 and 5000 mg/L, which is much
higher than the permissible limit of 2 mg/L for wastewater discharge
(Belay, 2010).
Like organic pollutants, metals are not degraded and tend to accu-
mulate into the environment, may enter the food chain and cause toxic,
genotoxic, mutagenic and carcinogenic eects (Chandra et al., 2011).
Chromium compounds are well known to have toxic, genotoxic, mu-
tagenic, and carcinogenic eects on humans, animals, plants, and as
well as in microbes (Cheung and Gu, 2007; Mishra and Bharagava,
2016). In nature, chromium exists in several oxidation states ranging
from 2 to + 6, but only trivalent (III) and hexavalent (VI) forms of
chromium is most prevalent and stable. Out of these two forms, hex-
avalent chromium [Cr(VI)] is highly toxic, mutagenic, teratogenic,

Corresponding author.
E-mail addresses: bharagavarnbbau11@gmail.com, bharagavabiotech77@gmail.com (R.N. Bharagava).

http://dx.doi.org/10.1016/j.ecoenv.2017.08.040
Received 20 May 2017; Received in revised form 1 August 2017; Accepted 17 August 2017
0147-6513/ 2017 Elsevier Inc. All rights reserved.
R.N. Bharagava, S. Mishra
carcinogenic to human and animals and has been designated as priority
pollutant by US Environmental Protection Agency (USEPA) (1998). If
Cr(VI) concentration into the environment exceeds > 0.05 mg/L, then
it may aect the human physiology and if enter the food chain, it may
cause severe health hazards such as skin irritation, nasal irritation, ul-
ceration, eardrum perforation, and lung carcinoma etc. (WHO, 2011;
Srinath et al., 2002).
Cr(VI) also acts as a strong oxidizing agent and exists only in oxy-
genated forms as hydro-chromate (HCrO4-), chromate (CrO4-) and di-
chromate (Cr2O72) ionic species in aqueous systems. Cr(VI) com-
pounds are comparatively more toxic than Cr(III) compounds due to
their higher solubility in water, rapid permeability through biological
membranes and subsequent interaction with intracellular proteins and
nucleic acids (Thacker et al., 2006; Cheung and Gu, 2007). Although a
number of conventional/traditional methods are reported either for
removal or detoxication of Cr(VI) from industrial wastes such as
chemical precipitation, reverse osmosis, ion-exchange, ltration,
membrane technologies, evaporation recovery, absorption on coal, ac-
tivated carbon, alum, kaolinite, and y ash etc. (Saxena et al., 2016;
Ahluwalia and Goyal, 2007). These methods are very costly, less ef-
fective and also generate a metal rich sludge as secondary pollutants.
Therefore, it becomes very essential to develop an eco-friendly, cost-
competitive and eective method for removal/detoxication of Cr(VI)
for the safety of environment and human health protection.
However, microbial reduction of toxic Cr(VI) to non-toxic Cr(III) by
chromium resistant bacteria (CRB) is the most pragmatic approach that
oers an economical as well as eco-friendly option for chromate de-
toxication and bioremediation. Microbes have diverse resistance me-
chanisms to cope with chromate toxicity that enable them to survive in
such harsh environmental conditions (Cervantes and Campos-Gracia,
2007). These detoxication strategies include biosorption, bioaccumu-
lation and biotransformation by enzymatic reduction, diminished in-
tracellular accumulation through either direct obstruction of ion uptake
system or active chromate eux, precipitation, and reduction of Cr(VI)
to less toxic and less mobile Cr(III) (Cheung and Gu, 2003; Ramirez-
Diaz et al., 2008). Hence, the objectives of this study were to isolate and
characterize chromium resistant bacteria, which should be capable to
reduce/detoxify the toxic Cr(VI) into less toxic and less mobile Cr(III)
for environmental cleanup and human health safety.
2. Materials and methods
2.1. Collection of tannery wastewater
The tannery wastewater was collected from Common Euent
Treatment Plant (CETP) of Jajmau Unit, Kanpur (2626'59.7228"N and
8019'54.7335"E), Uttar Pradesh, India in a pre-sterilized conical ask
(Cap. 2 L), brought to laboratory, maintained at 4 C and used in ana-
lysis of physico-chemical parameters as well as for the isolation of
bacterial strains capable for the reduction of hexavalent chromium.
2.2. Physico-chemical analysis of tannery wastewater
The physico-chemical analysis of tannery wastewater was made in
triplicate as per the standard methods for the examination of water and
wastewaters (APHA, 2012). The collected tannery wastewater was
analyzed for pH, conductivity, BOD (5 days method), COD (open reux
method), total solids (TS), total dissolve solids (TDS) and total sus-
pended solids (TSS) (drying method), Total nitrogen (TN) (Kjeldhal
method) and Chloride (AgNO3 titration method). Phosphate and sul-
phate was measured (Vanadomolybdo-phosphoric acid) colourimetric
and (BaCl2 precipitation) methods, respectively (APHA, 2012).
2.2.1. Analysis of heavy metals in tannery wastewater
The concentration of heavy metals (Cr, Zn, Mn Ni, Cd, and Fe) in
collected tannery wastewater was determined by the acid digestion
103
Ecotoxicology and Environmental Safety 147 (2018) 102109
method and Atomic Absorption Spectrophotometer (AAS) (VARIAN
AS240FS, Australia), following the standard methods for the examina-
tion of water and wastewater (APHA, 2012). The digestion of waste-
water sample was performed by taking 100 mL lter sterilized waste-
water sample in a conical ask containing 6 mL of conc. Nitric acid and
1 mL of perchloric acid (6:1). This mixture was swirled gently covered
with watch glass and heated on hot plate at room temperature. The
sample was digested on hot plate until yellow fumes were released and
the solution become clear. After cooling, the acid solution was ltered
by Whattman's lter paper No. 44 and the volume of samples was make
up 10 mL by using deionized water and used for metal analysis with
their respective standard metal solutions.
2.3. Isolation of chromium resistant bacterial strain and growth conditions
To isolate chromium resistant bacteria, the collected tannery was-
tewater was serially diluted and spreaded on Luria-Bertani (LB) agar
plates amended with potassium dichromate (100 mg/L) and incubated
at 37 C for 2448 h (Farag and Zaki, 2010). The morphologically dis-
tinct colonies appeared on potassium dichromate amended LB agar
plates were screened for maximum chromium tolerance potential by
subsequent transferring/sub-culturing on LB agar plates amended with
increasing concentration (2001000 mg/L) of potassium dichromate.
Out of ten bacterial isolates, only one bacterium (SCRB10) was found
capable to tolerate 800 mg/L concentration of Cr(VI) and selected for
biochemical characterization, identication by 16S rRNA gene se-
quencing analysis and other studies.
2.4. Characterization and identication of bacterial isolate
2.4.1. Morphological and biochemical characterization
The isolated bacterium was characterized morphologically and
biochemically following the standard protocols of Cowan and Steel's
manual for the identication of medical bacteria (Barrow and Feltham,
1993) and identied based on 16S rRNA gene sequencing analysis.
2.4.2. 16S rRNA gene sequencing analysis and gene-bank accession number
The genomic DNA was prepared from overnight grown bacterial
culture following the alkaline lysis method described by Kapley et al.
(2001). About 5 L DNA was used to amplify 16S rDNA gene using
universal eubacterial primers (27F) 50-AGAGTTTGATCMTGGCTCAG-
30 and (1492R) 50-TACGGYTACCTTGTTACGACTT-30 (Narde et al.,
2004) and a 1500 bp product was amplied. The reaction mixture
contained 5 L template, 1X PCR buer, 200 M of each dNTP, 3.0 mM
MgCl2, 25 pmol of primer, and 2.5 units of Amplitaq DNA polymerase
(Perkin Elmer) in a nal reaction volume of 50 L (Bharagava et al.,
2009).
The thermocycling reactions were carried out by using Veriti 96-
well Thermal Cycler (Applied Biosystems, USA). The 16S rDNA frag-
ment was amplied by 35-cycles, PCR initial denaturation at 95C for
5min, subsequent denaturation at 94C for 30s, annealing temperature
at 50C for 30s, extension temperature 72C for 1.30min and nal ex-
tension at 72C for 7min). The PCR product was analyzed on 1%
agarose gel and puried by using gel extraction kit (Merk Biosciences,
Bangalore). The gel puried PCR products were made sequenced by
Chromous Biotech, Pvt Ltd. (Bangalore, India) on ABI 3500 Genetic
Analyzer, using Big Dye Terminator Version 3.1. The partial sequences
obtained were subjected to BLAST analysis using the online option
available at www.ncbi.nlm.nih.gov/BLAST (Altschul et al., 1997) sug-
gesting the identity of isolated bacterium. The phylogenetic tree was
constructed by neighbour-joining method using NCBI database online
phylogenetic tree builder (http://www.ncbi.nlm.nih.gov). Further, the
sequences were also deposited to Gene-Bank under the accession no.
KX710177.
R.N. Bharagava, S. Mishra
2.5. Evaluation of growth and Cr (VI) reduction potential of isolated
bacterium
The growth and Cr(VI) reduction potential of bacterial isolate was
evaluated by growing the bacterium in Erlenmeyer asks (250 mL)
containing 100 mL of autoclaved LB broth supplemented with dierent
concentrations (50, 100, 200 & 300 mg/L) of Cr(VI) followed by in-
cubation at 35 C under shaking condition at 120 rpm (Innova 4230,
USA). The broth containing Cr(VI), but without bacterial culture was
taken as control. The bacterial growth was monitored spectro-
photometrically (Evolution 201, Australia) in terms of increase in ab-
sorbance at 600 nm and Cr(VI) reduction potential was evaluated by a
colorimetric method in terms of decrease in Cr(VI) concentration by
using a Cr(VI) specic colorimetric S-diphenylcarbazide (DPC) method
at 540 nm (APHA, 2012; Thacker et al., 2007). The Cr(VI) reduction
was calculated by using the following formula:
Ci Cf
Cr(VI)% reduction = 100
Ci
[where, Ci = initial Cr(VI) conc. (mg/L) and Cf = nal Cr(VI) conc.
(mg/L)].
2.6. Scanning Electron Microscopic analysis (SEM)
The SEM analysis was performed to observe the eects of Cr(VI) on
cell morphology of isolated bacterium. The bacterium was grown in LB
broth containing 100 mg/L of Cr(VI) at 35 C under shaking ask
conditions (120 rpm) for 24 h whereas bacterial cells grown in LB broth
without Cr(VI) was taken as control. After 24 h, the bacterial cells were
harvested by centrifugation at 8000 rpm for 10 min at 4 C and the
pellets obtained were washed thrice with phosphate buer saline (PBS)
and pre-xed with 2.5% glutaraldehyde for 46 h at 4 C. These pre-
xed cells were washed twice with PBS (7.2 pH) and post-xed with 1%
osmium tetraoxide for 1 h followed by washing with PBS and thrice
dehydration with 30%, 50%, 70%, 90%, 95% and 100% (v/v) of
acetone. The xed cells were dried with critical point dryer (CPD) and
platinum-coated ion sputter coater (JEOL, Japan JFC 1600 Auto Fine
Coater), and examined under SEM (JEOL JSM-6490LV) to observe the
changes in cell morphology.
2.7. Fourier transform infrared spectrophotometric (FT-IR) analysis
The FTIR analysis was carried out to identify the functional groups
involved in the adsorption of chromium ions on bacterial cell surface.
The active culture of isolated bacterium was inoculated into LB broth
containing Cr(VI) (100 mg/L) and control without Cr(VI). After 24 h of
incubation, the bacterial cells were pelleted by centrifugation at
8000 rpm for 10 min at 4 C. The bacterial pellets were washed thrice
with 0.85% sodium chloride solution followed by DDI water and dried
in air at 50 C (Kamnev et al., 1997). About one milligram of nely
crushed air-dried bacterial biomass was mixed with 400 mg of po-
tassium bromide. The mixture was ground to ne powder and trans-
lucent sample disks were obtained by using a manual hydraulic press at
a pressure of 100 kg cm2 for 10 min. The disks were xed in a FTIR
Spectrophotometer (Nicolet 6700, Thermo Scientic, USA) for ana-
lysis. The FTIR spectrum was recorded at 4004000 cm1 (Franois
et al., 2012).
2.8. Evaluation of antibiotic resistance property of isolated bacterium
The antibiotic resistance property of isolated bacterium was studied
by disk diusion method using Muller-Hinton agar medium against the
following antibiotics: Vancomycin (30 mcg), Tetracycline (30 mcg),
Chloramphenicol (30 mcg), Kanamycin (30 mcg), Erythromycin
(15 mcg), Azithromycin (15 mcg), Streptomycin (10 mcg), Noroxacin
(10 mcg), Ampicillin (10 mcg), Gentamycin (10 mcg), Penicillin G (10
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Ecotoxicology and Environmental Safety 147 (2018) 102109
units), Lomeoxacin (10 mcg), Gatioxacin (5 mcg), Levooxacin
(5 mcg) (Himedia, India). The plates were swabbed with a faintly
opalescent culture, and then antibiotic disks were applied followed by
incubation at 30 C for 24 h (Yadav et al., 2016; Bharagava et al.,
2014). The inhibition zones were measured after 24 h of growth period
and the bacterium was classied as resistant, intermediate, or suscep-
tible based on the zone size following the standard antibiotic disc
sensitivity testing method (DIFCO, 1984).
2.9. Evaluation of metal resistant property for chromium and other toxic
metals
The metal resistant property of isolated bacterium for Cu, Cr, Cd,
Co, Zn, Fe, Ni, Pb, Mo and As was evaluated in terms of minimum in-
hibitory concentration (MIC). The stock solutions of the analytical
grade salts of CuSO4, K2Cr2O7, CdCl2, CoCl2, ZnSO4, FeCl3, NiCl2,
PbNO3, Na2MoO4 and NaAsO2 forCu2+,Cr6+, Cd2+, Co2+, Zn2+, Fe3+,
Ni2+, Pb3+, Mo6+ and As3+ ions, respectively were prepared in
Millipore water and autoclaved. The nutrient agar plates amended with
the increasing concentration (501000 mg/L) of selected metals were
prepared, streaked with the log phase of isolated bacterium followed by
incubation at 35 C for 48 h. The MIC of dierent heavy metals for
bacterial isolates was designated as the minimum concentration of
metal ions at which no visible growth of test organisms occurred. The
nutrient agar plates without metal solutions were taken as control
(Yadav et al., 2016; Bharagava et al., 2014; Sagar et al., 2012).
2.10. Statistical analysis
All observations were carried out in triplicates (n = 3) to improve
the analytical precision of the experiment and the data was recorded.
To conrm the variability of data obtained and validity of results, the
data were subjected for the statistical analysis using one way Analysis
of Variance (ANOVA) and the means were compared by (Post Hoc)
Tukey's test (p < 0.05) using SPSS software (IBM SPSS Statistics version
20).
3. Results and discussion
3.1. Physico-chemical characteristics of tannery wastewater
Thephysico-chemical analysis of tannery wastewater reveals that it
was alkaline in nature (pH 8.49 0.2), light yellowish in color and
decient in dissolved oxygen. In addition, the BOD, COD, total solids,
TDS, TSS, phenol, sulphate, and total chromium content was
160 15.8, 322 28.6, 11,028 805.2, 3491.3 239.4,
194 23.5, 12.7 1.2, 1445 67.9 and 5.7 0.2 mg/L, respectively
as shown in Table 1. The alkaline pH and high EC of collected tannery
wastewater could aect the biological properties of receiving water
bodies (Vijayanand and Hemapriya, 2014). The high EC
(4.16 0.07 mS/cm) and TDS (3491.3 239.4 mg/L) values indicate
the presence of high salt content and inorganic substances in tannery
wastewater (Sultan and Hasnain, 2007). The EC depends on the che-
lating properties of water bodies and reported to create an imbalance in
free metal availability for aquatic fauna and ora (Akan et al., 2007),
whereas high TDS value causes osmotic stress and disrupts the osmor-
egulatory functions (Thacker et al., 2006). On the other hand, the set-
tled particles may cause damage to soil fauna and ora, various detri-
mental changes in soil properties and reduction in water holding
capacity (Chowdhury et al., 2013).
3.2. Isolated chromium resistant bacterium and its characteristics
Initially, ten morphologically dierent bacterial strains
(SCRB1SCRB10) were isolated from the collected tannery wastewater
samples and out of these ten bacterial strains, only one bacterium
R.N. Bharagava, S. Mishra Ecotoxicology and Environmental Safety 147 (2018) 102109

Table 1
Physicochemical characteristics of collected tannery wastewater sample.

S. no. Parameters Collected euent Permissible limit (CPCB,

2013)

1. Color Light coloured Colorless

2. Odour Pungent No odour
3. pH 8.49 0.2 5.59.0
4. Temperature (C) 22 C 40 C
5. Conductivity 4.16 0.07 NM
(mS/cm)
6. Alkalinity (mg/L) 680 500
7. BOD (mg/L) 160 15.8 30
8. COD (mg/L) 322 28.6 250
9. TS (mg/L) 11,028 805.2
10. TDS (mg/L) 3491.3 239.4 2100
11. TSS (mg/L) 194 23.5 100
12. Chloride (mg/L) 44.32 4.3 1000
13. Sulphate (mg/L) 1445 67.9 1000
14. Phosphate (mg/L) 3.7 0.2 5.0
15. Total nitrogen 10.64 0.8 100
(mg/L)
16. Phenol (mg/L) 12.7 1.2 0.1
17. PCP (mg/L) 14.6 1.3 0.1
18. Nitrate (mg/L) 9.8 0.4 10

Heavy metal concentration

i. Cr (mg/L) 5.7 0.2 2.0
ii. Zn (mg/L) 0.039 0.003 1.0
iii. Mn (mg/L) 0.025 0.003
iv. Fe (mg/L) 3.93 0.02 3.0
v. Ni (mg/L) 1.10 0.02 3.0
vi. Cd (mg/L) ND 2.0
Fig. 2. a: Growth curve pattern of Cellulosimicrobium sp. (SCRB10) at dierent Cr(VI)
concentrations b: Chromium reduction by Cellulosimicrobium sp. (SCRB10) at 50300 mg/
(SCRB10) was found capable to tolerate 800 mg/L of Cr(VI) con- L of Cr(VI) concentration. The dierences between treatments are statistically dierent
and signicant determined by using one-way ANOVA. Dierent letters (i.e. af) above the
centration in screening tests. Whereas, bacterial strains SCRB 1, SCRB 2,
bars represent the signicant dierences according to ANOVA followed by the (Post-hoc)
SCRB 3, SCRB 4, SCRB 5, SCRB 6, SCRB 7, SCRB 8, and SCRB 9 were
Tukey's test (p < 0.05).
found capable to tolerate 200, 300, 250, 500, 300, 150, 500, 250, and
550 mg/L of Cr(VI), respectively. This SCRB10 bacterium appeared as
golden white colonies on LB agar plates and was gram positive, rod 3.3. Growth pattern and chromium reduction by isolated bacterium
shaped, non-spore forming, and motile bacterium. This bacterium also SCRB10
showed positive reactions for catalase, oxidase, gelatinase, amylase,
cellulase and nitrate reductase activities, but negative reactions for In present study, it was observed that bacterial growth was stimu-
starch hydrolysis, indole test, lipase, urease, citrate utilization and lated upto 100 mg/L of Cr(VI) concentration and further increase in Cr
voges-proskauer tests. Further, the 16S rRNA gene sequence analysis (VI) concentration had inhibitory eects on bacterial growth (Fig. 2a).
showed that isolated bacterium has closest relatedness (99%) with that The bacterium also exhibited concentration dependent reduction pat-
of genus Cellulosimicrobium (Fig. 1) and thus, based on the sequence tern of Cr(VI) that is the reduction potential of bacterium decreases as
similarity and blast analysis, the isolated bacterium (SCRB10) was the concentration of Cr(VI) increases (Fig. 2b). Fig. 2a shows bacterial
identied as Cellulosimicrobium sp. with accession number KX710177. growth and Fig. 2b shows reduction potential at 50, 100, 200 and
300 mg/L of Cr(VI) concentration. The maximum reduction of Cr(VI)

Fig. 1. Phylogenetic tree showing the relationship of isolated

bacterium with their neighbouring sp. The numbers in bracket
represent gene accession numbers.

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R.N. Bharagava, S. Mishra Ecotoxicology and Environmental Safety 147 (2018) 102109

Fig. 3. Proposed mechanism of Cr(VI) uptake and reduction in a

bacterial cell (Modied from Vincent, 1994; Cheung and Gu,
2007).

Fig. 4. SEM analysis of untreated (a) and chromium treated (b) cells of Cellulosimicrobium sp. (SCRB10).

Table 2
Elemental content in bacterial isolate SCRB10 grown on LB agar amended with 100 mg/L
Cr(VI).

Element Weight % Atomic %

CK 71.62 81.95
OK 12.20 10.48
Na K 4.65 2.78
Mg K 0.66 0.37
PK 6.48 2.87
Cl K 1.96 0.76
KK 0.91 0.32
Ca K 0.82 0.28
Cr K 0.71 0.19

Fig. 5. EDX analysis of Cellulosimicrobium sp. (SCRB10) grown on LB agar amended with
100 mg/L of Cr(VI)for elemental analysis.
i.e. 99.33% and 96.98% by isolated bacterium was achieved at 50 and
100 mg/L of Cr(VI) concentration at 24 and 96 h, respectively, whereas
only 84.62% and 62.28% reduction of Cr(VI) was achieved at 200 and
300 mg/L concentration of Cr(VI) after 96 h, respectively. The dier-
ence in percentage reduction between dierent treatments are statisti-
cally dierent and signicant (p < 0.05) as determine by the one-way
Analysis of Variance (ANOVA) followed by (Post Hoc) Tukey's Test.
The bacterial growth varies inversely with increasing Cr(VI)
Total 100.00
concentration that might be attributed to the toxicity of Cr(VI). The
bacterial growth curve after a certain period of time appears saturated
suggesting the adaptive mechanisms allowing isolate to develop re-
sistance for toxic Cr(VI) and grow in its presence (Cervantes and
Campos-Garcia, 2007). The decrease in chromium reduction potential
of bacterium at higher concentrations might be associated to the mu-
tagenic and toxic eects of Cr(VI) on bacterial cell metabolism (Thacker
et al., 2006). Various researchers have reported that only few Cellulo-
simicrobium sp. were found capable to reduce Cr(VI) at higher

106
R.N. Bharagava, S. Mishra Ecotoxicology and Environmental Safety 147 (2018) 102109

Fig. 6. FTIR analysis of untreated (a) and chromium treated (b) cells of Cellulosimicrobium sp. (SCRB10).

Table 3 Table 4
Antibiotic resistance prole of isolated bacterium Cellulosimicrobium sp. (SCRB10). Minimum inhibitory concentration (MIC) of dierent metal ions for the isolated bac-
terium (SCRB10).
S. no. Antibiotic disc (conc.) Observation
Metal ions used Minimum inhibitory concentration (MIC) (g mL1) of
1. Azithromycin (15 mcg) R in study metal ions for the isolated bacterium (SCRB10)
2. Chloramphenicol (30 mcg) R
3. Erythromycin (15 mcg) R Cu 250
4. Gatioxacin (5 mcg) S Cr 500
5. Kanamycin (30 mcg) R Cd 160
6. Gentamycin (10 mcg) S Co 190
7. Lomeoxacin (10 mcg) R Zn 600
8. Tetracycline (30 mcg) R Fe 700
9. Noroxacin (10 mcg) R Ni 500
10. Ampicillin (10 mcg) R Pb 380
11. Levooxacin (5 mcg) S Mo 600
12. Penicillin G (10 units) R As 350
13. Streptomycin (10 mcg) R

R = Resistant; S = Sensitive. Cellulosimicrobium cellulans CrK16 and Exiguobacterium CrK19 at 200

and 400 g/mL of Cr(VI) concentration and found that Cellulosimicro-
concentrations (Naeem et al., 2013; Chatterjee et al., 2011). Rehman bium cellulans CrK16 was more eective resulting 34% and 27% re-
and Faisal (2015), studied Cr(VI) reduction potential of duction as compared to Exiguobacterium CrK19 resulting only 34% and

107
R.N. Bharagava, S. Mishra
18% reduction of Cr(VI) within 24 h at 37 C. In addition, Zahoor and
Rehman (2009) also checked the chromium reduction potential of Ba-
cillus sp. and S. capitis at 100 g/mL of Cr(VI) and found that Bacillus sp.
JDM-2-1 could reduce 40%, 66%, 77%, and 85%, whereas S. capitis
reduced 29%, 53%, 65%, and 81% Cr(VI) from medium after 24, 48,
72, and 96 h.
The uptake and reduction of Cr(VI) by bacteria is thought to be
mediated by acid adsorption mechanism in which liquid should have
enough protons to cause anion exchange. After accumulation, the Cr
(VI) may act as terminal electron acceptor getting reduced into Cr(III)
and binds to cell wall (Cheung and Gu, 2007; Cabrera et al., 2007;
Srinath et al., 2002). The reduction of chromium occurs mainly by two
processes i.e. bioaccumulation of metal inside cells or biosorption on
microbial cell surface. Bioaccumulation involves two phases: an initial
rapid phase of physical adsorption or ion exchange at cell surface fol-
lowed by slow phase of active metabolism-dependent transport of Cr
(VI) into the bacterial cell (Chandra et al., 2011; Cabrera et al., 2007).
However, the biosorption of heavy metals is a metabolism dependent
process that depends on dierent mechanism of ion exchange, co-or-
dination, adsorption, chelation, complexation (Srinath et al., 2002).
It has been reported that chromate ions actively cross the biological
membranes through sulphate pathway, which reects the chemical
analogy between two oxyanions (Cervantes and Campos-Gracia, 2007).
Inside the cell, Cr(VI) is readily reduced into Cr(III) by enzymatic or
non-enzymatic activities, resulting in diverse toxic eects in the cyto-
plasm (Cervantes et al., 2001). The reduction of Cr(VI) into Cr(III) by
bacteria may occur under aerobic and anaerobic conditions through
electron transport system containing cytochromes. Under aerobic con-
ditions, the reduction of Cr(VI) into the stable end product Cr(III) oc-
curs in two to three steps with generation of Cr(V) and Cr(IV) short
lived intermediates (Fig. 3) (Vincent, 1994; Cheung and Gu, 2007).
However, the reduction of Cr(V) to Cr(IV) and Cr(IV) to Cr(III) is not
known to be either spontaneous or enzyme mediated but, NADH,
NADPH and electron from the endogenous reserve are supposed to act
electron donor in Cr(VI) reduction process (Cheung and Gu, 2007).
3.4. SEM analysis
The SEM analysis revealed, that the bacterial cells exposed to Cr(VI)
become rough along with surface depression and showed increase in
cell size in comparison to unexposed cells having smooth cell surface
(Fig. 4a and b). This might be due to either the precipitation or ad-
sorption of reduced Cr(III) on bacterial cell surface (Liu et al., 2006;
Dhal et al., 2010; Kumari et al., 2016). Further, the Energy Dispersive
X-ray (EDX) analysis showed some chromium peaks (with 0.19 wt%
analysis) for cells exposed to Cr(VI) as shown in (Fig. 5), which might
be associated either with the presence of precipitated reduced Cr(III)
species on cells or complexation of Cr(III) species with cell surface
molecules. However, carbon, oxygen, phosphorus, sodium, magnesium,
and potassium as shown in Table 2 are well reported to participate in
surface localization of chromium on bacterial cells (Niftrik et al., 2008).
3.5. FTIR analysis
The FTIR analysis of bacterial biomass exposed to chromium has
shown a number of absorption peaks indicating the presence of dif-
ferent functional groups such as alkene (C=C), carbonyl (C=O), nitro
(-NO2), carboxyl (-COOH), amines (-NH2) and sulphonic (-SO3) at dif-
ferent wavelengths (Fig. 6a and b). The peak observed at 3425 and
3245 cm1 resulted from -NH2 asymmetric stretching mode of amines
indicated the overlapping of amines and hydroxyl stretching on bac-
terial cell surface (Mungasavalli et al., 2007). However, the absorption
peak appeared at 2967 cm1 remains unchanged in case of both Cr(VI)
exposed and unexposed cells indicating the asymmetric stretching of
lipids, proteins, polysaccharides and nucleic acid in cell wall (Das and
Guha, 2007).
108
Ecotoxicology and Environmental Safety 147 (2018) 102109
In addition, the spectrum also showed presence of some prominent
absorption peaks at 1406, 1240, 1548 and 1648 cm1 indicating the
presence of carboxyl and amide groups on bacterial cell surface.
However, a slight shifting in absorption peak from lower (1647 cm1) to
higher (1648 cm1) was also observed indicating the presence of -C=O
stretching mainly conjugated with a NH deformation mode (Doshi
et al., 2007). The chromium exposed bacterial biomass also showed two
prominent peaks at 974 and 879 cm1 (not observed in unexposed
bacterial samples) representing -CH=CH of trans-di-substituted alkenes
and -CH out of plane deformation showing the strong interaction of
chromium with bacterial cell wall. The peak appeared at 1063 cm1
(showing -C-O stretching) becomes more prominent and signicant in
Cr(VI) exposed cell biomass indicating the shifting of frequency
1066 cm1 and suggesting the involvement of -SO3H due to the sym
stretching of sulphonic moiety in interaction with chromium.
3.6. Antibiotic and metal resistant property of isolated bacterium
In this study, results revealed that the isolated bacterium was re-
sistant for azithromycin, chloramphenicol, erythromycin, kanamycin,
lomeoxacin, tetracycline, noroxacin, ampicillin, penicillin G, and
streptomycin whereas sensitive for gatioxacin, gentamycin and levo-
oxacin as shown in Table 3. The antibiotic and metal resistance
property in microbes is a potential health hazard because these prop-
erties are generally associated with transmissible plasmids. These
properties might be also appearing because of exposure to antibiotics/
metal contaminated environments, which may cause a coincidental co-
selection for resistance factors for antibiotics and metal ions (Verma
et al., 2009; Jain et al., 2009). The bacterial heavy metals and antibiotic
resistance is generally conned to plasmid DNA, but sometime it can
also be coupled to the chromosomal DNA.
Further, the isolated bacterium also showed a wide range of MIC
values for tested metals ranging from 250, 500, 160, 190, 600, 700,
500, 380, 600 and 350 g mL1 for Cu, Cr, Cd, Co, Zn, Fe, Ni, Pb,Mo
and As, respectively (Table 4). The resistance for toxic metals in bac-
teria probably reects the degree of environmental contamination with
toxic metals and might be directly related to the exposure of bacterial
cells with toxic metals (Bharagava et al., 2014). However, the un-
polluted environments may also harbour metal resistant organisms or
organisms that readily adapted to high concentrations of toxic metals.
Malik and Jaiswal (2000) have suggested that the incidence of a high
metal resistant population resulted from increasing environmental
pollution. However, the bacterial resistance to heavy metals is an im-
portant consideration when bacteria are to be introduced into soils for
enhancing the bioremediation of metal contaminated sites. Although
some heavy metals are required in low concentrations for normal me-
tabolic activities, but at elevated levels, these may act as carcinogenic,
mutagenic or teratogenic agents (Feuerpfeil et al., 1999).
4. Conclusion
Metals such as chromium, cadmium, mercury, arsenic, lead etc. are
considered as a major environmental pollutant due to their toxic eects
on environment as well as on human health. Based on the results of this
study, it can be concluded that the isolated bacterium can be a good
agent for the reduction/detoxication of hexavalent chromium from
contaminated environments. During the chromium reduction experi-
ment, the bacterium was found capable to reduce 99.33% and 96.98%
Cr(VI) at 50 and 100 mg/L at 24 and 96 h, respectively and 84.62% and
62.28% at 200 and 300 mg/L concentration of Cr(VI) after 96 h, re-
spectively. It indicates that the isolated bacterium is capable for the
eective reduction/detoxication of hexavalent chromium from con-
taminated environments and hence, can be used as a potential agent for
the eective bioremediation of metal contaminated sites for environ-
mental safety and human/animal health protection.
R.N. Bharagava, S. Mishra
Acknowledgements
Authors are highly grateful to the University Grant Commission
(UGC), Government of India (GOI), New Delhi for UGC Fellowship.
Authors also acknowledge the support from USIC B. B. Ambedkar
University for SEM and FTIR analysis.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.ecoenv.2017.08.040.
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