Journal of Environmental Protection, 2011, 2, 76-82
doi:10.4236/jep.2011.21008 Published Online March 2011 (http://www.SciRP.org/journal/jep)
Studies on Chromate Removal by
Chromium-Resistant Bacillus sp. Isolated
from Tannery Effluent
Manoj Kumar Chaturvedi
Nano Mission, Department of Science and Technology, Government of India, Technology Bhawan, India.
Received September 23rd, 2010; revised November 10th, 2010; accepted December 26th, 2010.
ABSTRACT
A chromate-removing strain was isolated from spent chrome effluent and identified as Bacillus circulans strain MN1.
The isolated strain was studied for resistance to Cr (VI) and its ability to remove Cr (VI). The strain was found to to ler-
ate Cr (VI) concentration as high as 4500 mg/L, but the cells growth was heavily influenced when initial Cr (VI) con-
centration was increased between 1110 mg/L and 4500 mg/L while Cr(VI) at 500 mg/L to 1110 mg/L did not s uppressed
the cells growth. The experiments also demonstrated that the cells removed toxic Cr (VI) more efficiently at 30˚C com-
pared with that at 25˚C and 35˚C. The optimum initial pH for Cr (VI) removal was 5.6 and final pH values of 5.1-5.6
were observed for initial pH 5.2-5.7.
Keywords: Bacillus sp., Bioremediation, Cr (VI) Removal, Tannery Effluent
1. Introduction
Hexavalent chromium is recognized as one of the most
dangerous environmental pollutant due to its ability to
cause mutations and cancer in humans. Chromium is a
heavy metal with large industrial application, such as in
textile dyeing, chemicals and pigments production, wood
preservation, tanning activity and electroplating for sur-
face treatment [1]. The extensive application of chro-
mium in a variety of industries and disposal of the
chrome laded wastewaters without appropriate treatment
pose a great threat to the environmental and human
health. Chromium generally exists in two stable oxida-
tion states, trivalent chromium and hexavalent chromium.
The trivalent chromium is less toxic and mobile, while
hexavalent chromium is easily soluble and 100-fold more
toxic than trivalent chromium. The hexavalent form of
chromium, usually present in form of chromate (CrO4
–)
and dichromate (Cr2O7
–) possesses significant higher
levels of toxicity than other valence states [2]. Chromate
(CrO4
–) is a strong oxidizing agent that is reduced in-
tracellularly to Cr5+ and reacts with nucleic acids and
other cell components to produce mutagenic and car-
cinogenic effect on biological systems [3]. Accordingly,
the decontamination of hexavalent chromium is of great
importance.
The conventional methods for heavy metals removal
from industrial effluents are precipitation, coagulation,
ion exchange, cementation, elctro-dialysis, elctro-winning,
elctro-coagulation, reverse osmosis [4]; evaporation, sol-
vent extraction and membrane separation [1]. These pro-
cesses are expensive and present some technological
problems, mainly when applied to diluted metal solution.
Biosorption is a process in which certain types of bio-
masses, viable or dead, may bind and concentrate heavy
metals from aqueous solutions [5]. Microorganisms have
a high surface area-to-volume ratio because of their small
size and therefore, they can provide a large contact inter-
face, which would interact with metals from the sur-
rounding environment [6]. The structural polymers in the
bacteria cell provide acidic functional groups like car-
boxyl, phosphoryl and amino groups that are directly
responsible for reactivity of the bacterial cells [7]. All the
surfaces of the bacteria are intrinsically reactive towards
dissolved metals, despite the different surface formats
between different types of bacteria. It has been proved
that, in some cases, growing cells are able to remove
metals continuously through internal detoxification mech-
anisms [8].
Microbial removal of toxic hexavalent chromium has
practical importance, because biological strategies pro-

Studies on Chromate Removal by Chromium-Resistant Bacillus sp. Isolated from Tannery Effluent77
vide green technology that is cost-effective [9]. Isolation
and identification of chromium (VI)-resistant and chro-
mium (VI)-reducing strain are fundamentally significant.
Cr (VI) reduction by different microorganisms has been
well documented in different studies [10]. In previous
researches, many species of microorganisms, including
strain of Pseudomonas [11-13]; Escherichia [14,15]; En-
terobacter [16-18]; Bacillus [19-21]; Shewanella [22,23];
have been found to be able to reduce Cr (VI). It is re-
ported that a chrome-resistant P. ambigua strain GI re-
duced chromate anaerobically [24]. The mechanisms by
which these microorganisms reduce Cr (VI) are variable
and are species dependent. Some species use Cr (VI) as
the final electron acceptor in the respiratory chain [25,26]
whiles in some other strains certain soluble enzymes are
responsible for reduction of Cr (VI) to Cr (III) [20,14,27,
28]. Reduced trivalent chromium is less toxic than hex-
avalent chromium and it readily precipitates, forming
less soluble chromium hydroxide at normal pH. There-
fore these bacterial ability to reduce chromate would be
useful not only for detoxification but for removal of total
chromium from wastewaters. Microbial chromate reduc-
tion becomes a bit complicated as a result of the effect of
environmental conditions under which microbial Cr (VI)
reduction proceeds, thus, determining the optimum con-
ditions is also quite important for the maximum conver-
sion of chromium (VI). The ability of these microbial
strains for detoxification and removal of total chromium
from wastewaters can be exploited the best under opti-
mum condition of controlling parameters.
The aim of present study is to isolate and identify the
chromate-resistant and chromate removing bacteria from
the spent chrome effluent where the hexavalent chro-
mium level is quite high, research the bacterial chromate
removal, and determine the preferable conditions for
bacterial chromate removal. This paper describes the
effect of temperature, initial chromate concentration on
Cr (VI) removal and resistance and growth of the isolated
strain. The paper also study about the variation in pH.
2. Material and Methods
2.1. Bacterial Strain and Cultivation Conditions
Chromate-resistant bacterium was isolated from spent
chrome effluent containing high level of chromium. The
spent chrome-effluent was obtained from a local tannery
in Kanpur, U.P. India. For isolation and enumeration,
sample of spent chrome effluent was serially diluted to
obtain the serial 9-fold dilution sample suspension at a
dilution of 10-7 . One milliliter aliquots were withdrawn
from 10-7 sample suspension dilution and dropped re-
spectively to the sterilized culture plates, followed by
pouring nutrient agar media. The media did not contain
Cr (IV) as the spent chrome-effluent already contained
high concentration of chromate and allowed the growth
of chromate-resistant microbial strains, only. The plates
were incubated at 30˚C for 24 hours. Colonies were than
streaked on separate nutrient agar plate, incubated at
30˚C for 24 hours. Finally the bacterium was inoculated
from the plate onto agar slant and stored at 4˚C until
needed for further experiments. The agar medium con-
sisted of beef extract (3.0 g), peptone (5.0 g), glucose
(1.0 g) NaCl (2.5 g), agar (20.0 g) in 1 liter distilled wa-
ter. The pH value of the medium was adjusted to 7.0 by
adding aliquot of either 10% (w/v) HCL or 10% (w/v)
NaOH.
The operations of gram-staining and morphological
studies followed by biochemical tests were first per-
formed for preliminary characterization of the isolate
before the isolate was identified by Bergeys’ Methods of
Determinative Bacteriology [29]. The present bacterial
strain has been preserved with Microbial Type Culture
Collection and Gene Bank (MTCC) at the Institute of
Microbial Technology (C.S.I.R.), Sector 39-A, Chandi-
garh-160 036, India, under accession number MTCC
3918.
2.2. Growth Media and Culture Conditions
The isolated strain was grown under microaerophilic
conditions at 30˚C for 24 hours in sterilized nutrient
broth containing in gram per liter distilled water glucose,
1.0 g; peptone, 5.0 g; and beef extract, 3.0 g. Adjustment
of pH to 6.8 ± 0.1 was made by adding aliquots of either
HCl or NaOH. Suspension for inoculums was obtained
by growing the isolated strain MN1, in 5 mL sterilized
nutrient broth, incubated at 30˚C for 24 hours under mi-
croaerophilic condition. Higher volumes of inoculums
were obtained by inoculating pre-sterilized nutrient broth
with inoculums having one-tenth volume of required
final volume of inoculums and incubated at 30˚C for 24
hours.
2.3. Cr (VI) Removal Experiments
The isolated strain was enriched by transferring one loop
of cells from the agar slant to 100 mL of previously ster-
ilized liquid nutrient medium in 250 mL flasks and incu-
bated at 30˚C for 24 hours. The liquid medium contained
the same components described as above in agar medium
except agar and the pH value was adjusted to 7.0 in the
same way as mentioned above. The media were auto-
claved at 120˚C for 20 minutes before used in Cr (VI)
removal experiments. The 50 mL flasks containing vary-
ing concentrations (500 mg/L to 4500 mg/L) of Cr (VI)
as K2Cr2O7 were inoculated with 20 mL of enriched cells
suspension and incubated under the same conditions de-
scribed above. The liquid media was supplemented with

Studies on Chromate Removal by Chromium-Resistant Bacillus sp. Isolated from Tannery Effluent 78
1000 mg/L glucose as the electron donors. All of the
stock solutions were autoclaved as described above be-
fore used in Cr (VI) removal experiments. The experi-
ments were performed in triplicate. Cells suspension
volume, growth phase and Cr (VI) reduction conditions
were the same in all the sets. Incubation temperature was
varied at 25˚C, 30˚C and 35˚C to study the effect of
temperature on Cr (VI) removal. The cells suspension
used was 20 mL and was the logarithmic phase culture of
isolated strain (MLVSS, 3000 mg/L) prepared in nutrient
broth. A layer of paraffin was used to maintain mi-
croaerophilic conditions.
2.4. Analytical Methods
Samples were drawn and filtered using 0.45 μm filter
paper (47 mm, Cat. No. HAWP 04700 Millipore India
Pvt. Ltd.). The chromate concentration, growth of the
bacterial strain and pH was evaluated at 0 h and 24 h. Cr
(VI) concentration in supernatant was determined col-
orimeterically using diphynelcarbazide reagent in acid
solution with a spectrophotometer (SpectronicR Geneys
TM 2) following standard methods [30]. The Cr (IV)
determination analysis involved dilution of the initial Cr
(IV) concentrations to the level sensitive enough to be
determined by employing colorimetric method. Final Cr
(IV) values were obtained by incorporating dilution fac-
tor into the calculations. Bacterial cell density of the liq-
uid culture was determined as MLVSS following Stan-
dard methods [30]. The growth of the isolated strain in
experimental sets containing varying concentration of the
chromate (500 mg/L to 4500 mg/L) as potassium di-
chromate indicated the chromate resistance of the iso-
lated strain. The pH was determined using a pH meter
(EUTECH Cyber Scan ISO 9001 Certified) with an ac-
curacy of ± 0.01. The pH meter was calibrated with
standard pH meter. All the chemicals used in the present
study were of analytic grade when available.
3. Results and Discussion
3.1. Identification of the Strain
The tests of gram-reaction showed that the strain is
gram-positive. In the following operations the strain was
identified by Bergeys’ Methods of Determinative Bacte-
riology [29]. The strain was found to belong to genus
Bacillus. This may imply that Bacillus sp. probably have
become dominant strains in the high level of Cr (VI)-
containing spent chrome effluent and other bacteria wh-
ich cannot tolerate the toxicity of the Cr (VI) are ex-
cluded from the spent chrome effluent because of the
selective pressure. The biochemical characteristics of the
isolated strain are shown in Table 1. The strain is desig-
nated as Bacillus circulans MN1. The bacterium of strain
Bacillus circulans MN1 was eventually used in the fol-
lowing Cr (VI) reduction experiments. A variety of mi-
croorganisms with Cr (VI)-resistant and Cr (VI)-reducing
ability have been isolated from chrome-contaminated en-
vironment [12,20,31-33].
3.2. Effect of Temperature and Cr (VI) on the
Cells Growth
The effect of Cr (VI) on the growth of Cr (VI)-resistant
strain Bacillus circulans MN1 was evaluated at 25˚C,
30˚C, and 35˚C. Figure 1 shows the relationship between
growth of the cells and initial Cr (VI) concentration at
the three temperatures. The cells were grown in media
supplemented with varying Cr (VI) concentrations. The
biomass concentration (mg/L, dry wt.) was tested after
incubation of 24 h Initial biomass concentration was con-
stant in all the experimental sets. It was obvious that the
growth of the cells was heavily influenced when Cr (VI)
Table 1. Characteristics of the isolated strain.
Biochemical Tests Results Acid production
from carbohydratesResults
Growth on MacConkey agar(-) ve Adonitol (-) ve
Indole test (-) ve Arabinose (+) ve
Methyle Red test (+) ve Cellobiose (+) ve
Voges Proskaure test (-) ve Dextrose (+) ve
Citrate Utilization (-) ve Dulcitol (+) ve
Casein hydrolysis (+) ve Fructose (-) ve
Strach hydrolysis (+) ve Galactose (+) ve
Urea hydrolysis (-) ve Inositol (-) ve
ONPG hydrolysis (-) ve Inulin (-) ve
Nitrate reduction (+) ve Lactose (-) ve
Nitrite reduction (+) ve Maltose (+) ve
H2S production (-) ve Mannitol (+) ve
Cytochrome Oxidase test (+) ve Mannose (-) ve
Catalase test (+) ve Melibiose (-) ve
Oxidation/fermentation F Raffinose (-) ve
Gelatine liquefaction (+) ve Rhamnose (+) ve
Arginine dihydrolase (+) ve Salicin (+) ve
Lysine decarboxylase (+) ve Sorbitol (-) ve
Ornithine decaroxylase (-) ve Sucrose (+) ve
Trehalose (+) ve
Xylose (+) ve
The (-) ve (negative) and (+) ve (positive) results indicated in the Table 1
implies that desired reaction has not taken place/has taken place, respec-
ively. t

Studies on Chromate Removal by Chromium-Resistant Bacillus sp. Isolated from Tannery Effluent 79
Figure 1. Effect of initial Cr (VI) concentration on growth of Bacillus circulans MN1 at 25˚C, 30˚C and 35˚C respectively.
concentration was added up to 4500 mg/L, at all the three
temperatures. The highest growth of Cr (VI)-resistant
cells (MLVSS, 1780 mg/L) was observed at 30˚C at ini-
tial Cr (VI) concentration of 1110 mg/L. The result indi-
cated that the isolated strain Bacillus circulans MN1
could tolerate Cr (IV) concentration as high as 1110
mg/L. It was observed that growth of cells was heavily
influenced when initial Cr (VI) concentration was in-
creased beyond 1110 mg/L and 4500 mg/L while Cr (VI)
at 500 mg/L to 1110 mg/L did not suppressed the cells
growth. This indicated the greater toxicity of Cr (VI) to
the cells at higher Cr (VI) concentrations. It is also re-
ported that chromate at 52 mg/L significantly affected
cells growth of Bacillus subtilis and the cells failed to
grow and reduce chromate at 104 mg/L chromate [19].
The tolerance of Cr (VI)-resistant cells decreased at
25˚C for all initial Cr (VI) concentration studied. The
cells exhibited minimum tolerance toward Cr (VI) at
35˚C for all initial Cr (VI) concentration studied.
3.3. Effect of Temperature and Cr (VI) on
Cr (VI) Removal
Temperature is an important factor that has effect on mi-
crobial Cr (VI) removal. Cr (VI) removal by the strain
Bacillus circulans MN1, was evaluated under three dif-
ferent temperature: 25˚C, 30˚C, and 35˚C. The results are
presented in Figure 2. The final Cr (VI) concentration
was tested after incubation of 24 h. Initial biomass con-
centration was constant in all the experimental sets. Cr
(VI) was removed effectively (71.4%) at 30˚C for initial
Cr (VI) concentration of 1100 mg/L, after 24 hours. The
Cr (IV) concentration determined in experimental set,
after completion of incubation period was subtracted
from the initial chromate concentration in the experi-
mental set and divided by 100, to arrive at the percent
chromate removal by the strain Bacillus circulans MN1.
The Cr (VI) removal by the cells was severely affected at
35˚C and 25˚C temperature. This indicates that strain
removed Cr (VI) better at 30˚C compared with that at
35˚C and 25˚C. The initial Cr (VI) concentration above
1100 mg/L affected the Cr (VI) removal ability of the
strain Bacillus circulans MN1, at all temperatures i.e.
25˚C, 30˚C and 35˚C. Chromium (VI) bacterial resis-
tance up to 2500 mg/L has been reported by Camargo
[31]. Chromium (VI) bacterial resistance above 2500
mg/L has only been reported by Shakoori [32].
3.4. Variation of pH
Initial culture pH of the medium was considered as a
factor for growth and Cr (VI) removal by strain Bacillus
circulans MN1. This study tested the variation of pH in
every experimental set and data are listed in Table 2. In
general the pH value has the trend of being decreased.
This variation may be caused by the metabolites secreted
by cells.
The strain Bacillus circulans MN1 exhibited maxi-
mum Cr (VI) resistant at initial pH 5.6 at 25˚C (MLVSS,
550 mg/L); 30˚C (MLVSS, 1781 mg/L) and 35˚C
(MLVSS, 410 mg/L) for initial Cr (VI) concentration of
1110 mg/L. Optimum Cr (VI) reduction at varying tem-
perature was directly related to the optimum pH for
growth of the strain Bacillus circulans MN1. Value for
pH of 5.4 and 5.5 restricted bacterial growth and Cr (VI)
removal at the temperature studied (data not shown).

Studies on Chromate Removal by Chromium-Resistant Bacillus sp. Isolated from Tannery Effluent 80
Figure 2. Effect of initial Cr (VI) concentration on Cr (VI) removal by Bacillus circulans MN1 at 25˚C, 30˚C and 35˚C respec-
tively.
Table 2. Variation of pH in the medium used for Cr (VI)
removal by Bacillus circulans MN1.
pH value after 24h at varying
temperature Initial Cr (VI)
Con., mg/L
Initial pH
valuea
25˚C 30˚C 35˚C
550 5.7 5.6 5.6 5.6
1100 5.6 5.5 5.5 5.5
1550 5.5 5.4 5.4 5.4
2000 5.4 5.3 5.3 5.3
3000 5.2 5.2 5.2 5.3
4500 5.2 5.1 5.1 5.1
aInitial pH values of the replicates were adjusted to be identical.
Many other researchers reported the optimum pH value
for bacterial Cr (VI) reduction but not the optimum initial
pH value. It is reported that the optimum pH was 9 for Cr
(VI) reduction by gram-negative bacterium [32] but it
was found that the optimum pH was 7 in case of Pseu-
domonas aeruginosa and Bacillus coagulans [13,20,21].
The difference in optimum pH value suggests that pH
modification is important for different cultures to achieve
the maximum Cr (VI) reduction in the bioremediation of
chromate. The pH value is an important index reflecting
the microbial activity. Evaluation of pH variation in the
course of bacterial Cr (VI) reduction is helpful for under-
standing the mechanisms of bacterial Cr (VI) reduction.
4. Conclusions
The bacterium isolated from spent chrome effluent was
capable of Cr (VI) removal. The isolated strain was iden-
tified as species Bacillus circulans MN1 and it was used
further in Cr (VI) removal experiments, under mi-
croaerophilic conditions.
The cells removed toxic Cr (VI) more efficiently at
30˚C when compared with that at 25˚C and 35˚C. The
optimum initial pH was 5.6. The maximum chromate
removal (71.4%) at initial chromate concentration of
1110 mg/L at 30˚C was achieved during 24 hours of in-
cubation period. However, the growth of the bacterium
strain Bacillus circulan s MN1, was significantly affected
at higher chromate concentration varying from 2000
mg/L to 4500 mg/L at 25˚C, 30˚C and 35˚C. The strain
Bacillus circulans MN1, tolerated Cr (VI) over a wide
concentration range (500-4500 mg/l).
This result suggests that controlling temperature would
be critical for maintaining the bacterial processes for
chromate removal. High initial concentrations of the
chromate were toxic to the cells. Hence it is imperative
that bacterial ability to remove chromate can be achieved
by increasing their resistance to chromate. Further re-
searches will be conducted on the mechanisms by which
the bacteria remove Cr (VI).
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