Biosorption of Heavy Metals from Aqueous Solution using Bacillus Licheniformis

Int. J. Pure Appl. Sci. Technol., 10(2) (2012), pp. 12-19
International Journal of Pure and Applied Sciences and Technology
ISSN 2229 - 6107
Available online at www.ijopaasat.in
Research Paper
Biosorption of Heavy Metals from Aqueous Solution using
Bacillus Licheniformis
Deepti Pranay Samarth 1 , Chandrashekhar Jangluji Chandekar 2
Bhadekar 3,*
and Rama Kaustubh
1, 3
Department of Microbial Biotechnology, Rajiv Gandhi Institute of IT and Biotechnology, Bharati
Vidhyapeeth University, Pune- 411046, India.
2
Department of Microbiology and Biotechnology, Shivaji Science College, Congress nagar, Nagpur-
440012, India.
* Corresponding author, e-mail: (neetabhadekar@gmail.com)
(Received: 24-4-12; Accepted: 4-6-12)
Abstract: Current problems of heavy metal pollution in urban and rural ground
water resources as well as sea water are reducing the potability of drinking water.
Besides, there are several ill effects on human/animal health due to toxicity of
heavy metals in soil and water. Conventionally physiochemical methods are used
for the removal of metals. However, considering their limitations and certain
disadvantages, biological methods mainly biosorption are preferred. In this work
we have used living biomass of Bacillus licheniformis, which is already employed
in sewage treatment. Aerobic batch biosorption experiments were carried out for
removal of Cr (VI), Fe (III), Cu (II) ions from aqueous metal solutions. % removal
efficiency for iron, chromium and copper ions was 95, 52 and 32 (w/v) after 48
hrs at 120 rpm. Optimum pH was found to be 3.5 for Fe (III) and Cr (VI) whereas
for Cu (II) it was 2.5. Optimum incubation temperature was found to be 28 ºC.
Metal tolerance studies were also carried out using aqueous metal solutions of
these metals individually and in combination. The maximum tolerable
concentration was 1200 mg L -1 for Cr (VI) while it was above 1500 mg L -1 for Fe
(III) and Cu (II). MTC for the set having combination of all metals in equal
amount was 1000 mg L -1. Interestingly the organism was found to tolerate upto
1000 mg L -1 concentration of heavy metals when a mixture of them (each at equal
concentration) was used. Our study is an attempt to provide a multipurpose
alternative for waste water treatment.
Keywords: Bacteria, heavy metals, living biomass, metal removal, metal
tolerance.

Int. J. Pure Appl. Sci. Technol., 10(2) (2012), 12-19. 13
Introduction:
Certain species of micro organisms have found to adsorb surprisingly large quantities of heavy metals.
Many of these metals include those causing toxicity to humans and metals of commercial and
economic importance. Removal of metals and their recovery is one of the major concerns in sewage
and industrial effluent treatment, which is both in municipal and industrial interest. The heavy metals
of widespread concern to human health are mercury, cadmium, lead, arsenic, chromium, copper, and
zinc (Lazrova et al., 2005). Nriagu (1988) estimated that over one billion human beings are currently
exposed to elevated concentrations of toxic metals and metalloids in the environment and several
million people may be suffering from subclinical metal poisoning. Adverse effect of heavy metals
include i) suppression of the immune system ii) carcinogenicity (Peakall, 1992), iii) neurotoxicity,
mainly in children (Cohen, 2005) iv) inhibition of the activity of some critical enzymes related to
synthesis of vital bio-molecules v) accumulation in the body of different organisms causing biomagnification
(Paknikar et al., 2003).
Physico-chemical methods, such as chemical precipitation, oxidation or reduction, electrochemical
treatment, evaporative recovery, filtration, ion exchange and membrane technologies are widely used
to remove heavy metal ions from industrial wastewater. However, application of these treatment
processes has been found to be sometimes restricted, because of investment, operational costs and the
potential generation of secondary pollution. These processes may be ineffective or expensive,
especially when the heavy metal ion concentrations in solutions are 1-100 mg L -1 (Volesky, 1990a, b).
It was only in the 1990s that a new scientific area developed that could help to recover heavy metals
using biological means i.e. biosorption. The first reports described how abundant biological materials
could be used to remove; at very low cost, even small amounts of toxic heavy metals from industrial
effluents (Vieira and Volesky 2000). The technique of biosorption utilizes the characteristics of
microoganisms to adsorb metals in a commercial manner. Microorganisms uptake metal, either
actively (bioaccumulation) and/or passively (biosorption) (Fourest and Roux, 1992). This is due to
affinity of bacterial surfaces for heavy metals leading to their adsorption and precipitation. The
biosorption is passive non-metabolic process of binding various chemicals on biomass (Volesky,
1990a). Most studies of biosorption for metal removal deal with the use of either laboratory-grown
microorganisms or biomass generated by the pharmacology and food processing industries or
wastewater treatment units (Tsezos and Volesky, 1981; Hussein et al. 2004).
Bacillus licheniformis (B.licheniformis) is frequently used in waste water treatment in combination
with Bacillus subtilis (Hiatt 2000, Kalia et al. 1994) due to the production of extracellular enzymes,
mainly protease and lipase. The extraordinary characters of organism which support its application in
sewage treatment are its diverse habitat, ease of cultivation, non pathogenic nature to humans, spore
forming ability and its tolerance to environmental stress, starvation etc. There are many reports
describing metal binding sites of B. licheniformis. Other than peptidoglycan (basic component of cell
wall), B. licheniformis cell wall has high proportion of teichoic and teichouronic acid, both of which
are responsible for about 60% of metal binding (Beveridge, 1982).
These characteristics of B.licheniformis prompted us to study efficacy of its living biomass in heavy
metal removal. This work mainly deals with biosorption of hexavalent chromium ion, cupric ion and
ferric ion by whole cell broth of B.licheniformis (NCIM 2471). Effects of change in pH and
incubation temperature on metal removal were also studied. Our studies also aimed at determination
of heavy metal tolerance of the organism using the metal ions individually and in combination.
Materials and Methods:
All the chemicals were procured from Merck, India and were of A.R grade whereas all media
components were from Hi Media, India. B. licheniformis (NCIM 2471) was obtained from National
Collection of Industrial Microorganisms, National Chemical Laboratory, Pune, India. The strain was
maintained by subculturing on nutrient agar. The culture was stored at 4° C between transfers and
subcultured before experimental use.

Int. J. Pure Appl. Sci. Technol., 10(2) (2012), 12-19. 14
Metal Solutions:
Experimental metals used in the study were chromium (VI), copper (II) and iron (III) in the form of
their respective metal solutions. A synthetic multi-element standard solution of liquid media (1%
tryptone, 0.5% yeast extract, 0.5% NaCl) containing 150 mg L -1 of each Cr, Cu and Fe ions was
prepared from their respective stock solutions (1000 mg L -1 ). Chemicals used for preparing these
stock solutions where potassium dichromate, copper sulphate and ferric chloride. Stock solutions of
these metals were prepared using deionized water and autoclaved separately. Prior to addition of stock
solution of metals the liquid media were autoclaved at 121°C for 20 min. All the additions were
performed aseptically.
Effect of pH on Biosorption:
The experiments were carried out in the batch mode for the measurement of biosorption capabilities.
Medium containing synthetic multi-element solution (15 mg % of each metal) in 500 ml Erlenmeyer
flask was used. Before the addition of the biosorbant inoculum to the solution the samples were
adjusted to different pH values viz. pH- 2.5, 3, 3.5, 4, 4.5. The pH of solution was adjusted with 1 M
HCl and 1 M NaOH solutions. Samples were inoculated with 10% overnight grown culture of
B.licheniformis and then incubated at 120 rpm for 48 hrs. The inoculated samples were then incubated
at 28°C for 48 hrs. After the incubation period the cells were harvested by centrifugation for 30 min at
5000 rpm. Total metal concentration biosorbed by B. licheniformis was analysed using Inductive
Coupled Plasma-Atomic Emission Spectrophotometer (ICP-AES) (JY-24, Jobin yvon, France).
Effect of Incubation Temperature on Biosorption:
A set of three samples was prepared as described above and pH was adjusted to 3.5 for all three
samples. After inoculation, samples were incubated at temperatures i.e. 20°C, 28°C, 37°C respectively
and incubated at 120 rpm for 48 hrs. Total metal concentration biosorbed by B. licheniformis was
analyzed using ICP-AES as described above.
Determination of Metal Tolerance:
Colorimetric method was used to determine maximum tolerable concentration (MTC) of B.
licheniformis for hexavalent chromium ion, cupric ion and ferric ion. Four sets of experiments were
prepared using liquid media as mentioned above. First three sets had varying concentrations (200 mg
L -1 to 1500 mg L -1 ) each of chromium, cupric and ferric ion respectively. In the fourth set, the samples
contained all metal ions in the range of 200 mg L -1 to 1500 mg L -1 . Thus total metal concentration
varied from 600 mg L -1 to 4500 mg L -1 in the samples in fourth set of experiments. The pH was
maintained at 3.5 for all four sets. 10 % overnight grown culture of B. licheniformis was inoculated in
these samples and incubated at 28°C, 120 rpm for 48 hrs.
Growth was determined by measuring absorbance at 540 nm. Samples showing zero absorbance were
further confirmed for growth by examining total viable count (TVC). Maximum tolerable
concentration (MTC) of heavy metal was designated as the highest concentration of heavy metal that
allowed growth after 2 days (Schmidt and Schlegal, 1994). All the experiments were carried out in
triplicates and the results indicate the mean values.
Results and Discussion:
Biosorption Studies:
The living biomass of B. licheniformis (NCIM 2471) was used for biosorption studies of Cr (VI), Cu
(II) and Fe (III) ions. Effect of pH and incubation temperature was studied on removal efficiency of
the organism.

Int. J. Pure Appl. Sci. Technol., 10(2) (2012), 12-19. 15
Effect of pH:
The pH of the solution is the most critical parameter for metal sorption as it influences both the
bacterial surface chemistry as well as solution chemistry of soluble metal ion. Different metals show
different pH optima for their biosorption (Donmez et al. 1999; Saeed and Iqbal 2003).
Fig.1 shows % removal of heavy metal ions at pH- 2.5, 3, 3.5, 4, 4.5. It was found to be in the range
of 14 – 95 %. Maximum biosorption was observed at pH 3.5 for iron and chromium ion with removal
efficiency of 95% and 52% respectively. pH 2.5 was found to be optimum for Cu ion giving the
maximum removal efficiency of 32%. The results suggested that acidic pH (2.5 to 3.5) was optimum
for biosorption of these heavy metals using B.licheniformis. Among the three metal ions removal
efficency for iron (63-95 %) was found to be maximum.
Zhou et al, (2007) have also reported acidic pH optimum (pH 2.5) for Cr (VI) ion using dead biomass
of B. licheniformis isolated from polluted soil sample with 85 % metal removal efficency. B.
licheniformis CCO1 reported by Clausen (2000) exhibited removal efficiency of 93% and 8% for
copper and chromium respectively. However our results indicated 60% removal efficiency when a
mixture of Cr (VI), Cu (II) and Fe (III) ions was used. This showed higher metal removal efficiency
of living biomass of B. licheniformis (NCIM 2471), thereby suggesting its possible application in
multiple metal removal in effluent treatment.
Effect of Incubation Temperature:
Fig.2 shows effect of incubation temperature on metal biosorption. Maximum % removal of metal ion
was observed at 28°C. Removal efficiencies were 95%, 52% and 38% for Fe (III), Cr (VI) and Cu (II)
respectively at 28°C. Such high binding efficiency of iron ion by B.licheniformis was also reported by
Beveridge et al. (1982). Teichoic acid, teichouronic acid and peptidoglycan were shown to be
important biomolecules involved in metal binding as mentioned earlier. Iron binding was found to be
reduced by 38% when teichoic acid was removed and by 60 % when both teichoic acid and
teichouronic acid were removed. McLean et al. (1990) observed similar results in uptake of iron and
suggested participation of gamma-glutamyl capsular polymer of the organism, other than these
biomolecules. To our knowledge this is the first report showing 95% biosorption of iron using living
cells of B.licheniformis.
Determination of MTC:
Using colorimetric method, MTC of B.licheniformis for three metal ions viz. hexavalent chromium
ion, cupric ion, ferric ion and for mixture of these three metal ions in a same sample were determined
(Fig 3). As indicated in fig.3, MTC for hexavalent chromium ion was 1200 mg L -1. In case of cupric
ion and ferric ion, the MTC was above 1500 mg L -1 , whereas the MTC for set having mixture of all
the metal in a sample was 1000 mg L -1 . These results suggested that the organism has high metal
tolerance for copper and iron as compared to chromium. In light of our observations, living biomass
of B.licheniformis could be used efficiently and effectively in removal of heavy metals from effluents.
Conclusions
Our results demonstrated that the use of live cells of B.licheniformis in waste water treatment would
prove an eco-friendly, multipurpose and cost efficient alternative to conventional methods. This work
will be helpful in i) developing technologies useable for in situ bioremediation using B.licheniformis
ii) construction of large scale bioreactor iii) promoting its detail study of binding mechanism and
other technological parameters to improve the feasibility in the process application.

Int. J. Pure Appl. Sci. Technol., 10(2) (2012), 12-19. 16
Acknowledgement
The authors are indebted to Analytical instrumentation division, National Environmental Engineering
Research Institute, Nagpur for providing us the analytical facility.
Figures
100
90
80
70
% Rem o val o f m etal io n
60
50
40
30
Ferrous ions
Chromium (VI) ion
Cupric ion
20
10
0
2.5 3 3.5 4 4.5
pH
Fig.1

Int. J. Pure Appl. Sci. Technol., 10(2) (2012), 12-19. 17
100
90
80
Ferrous ions
% Rem oval of m etal ion
70
60
50
40
30
Chromium (VI) ion
Cupric ion
20
10
0
20 28 37
Temperature ( o C)
Fig.2

Int. J. Pure Appl. Sci. Technol., 10(2) (2012), 12-19. 18
2
1.8
1.6
Absorbance (O.D. at 540 n m)
1.4
1.2
1
0.8
0.6
0.4
Ferrous ions
Chromium (VI) ion
Cupric ion
Mixure of all three metal
ions
0.2
0
200 500 700 1000 1200 1500
Concentration of metal ion (mg L -1 )
Fig .3
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