Removal of Cr(VI) from aqueous solution
using Bacillus subtilis, Pseudomonas
aeruginosa and Enterobacter cloacae
P. Sethuraman1, N. Balasubramanian2*
1
Department of Chemical Engineering, A.C. Tech Campus, Anna University Chennai, Chennai-600 025, India 2
Department of Chemical Engineering, A.C. Tech Campus, Anna University Chennai, Chennai-600 025, India (E-mail: N. Balasubramanian* nbsbala@annauniv.edu)
Abstract
The objective of this study is to investigate the removal efficiency of Cr(VI) by Bacillus subtilis, Pseudomonas aeruginosa and Enterobacter cloacae from aqueous solution under different process conditions. Batch mode experiments were carried out as a function of solution pH, biosorbent dosage, Cr(VI) concentration and contact time. The FT-IR spectra and SEM analysis of the biosorbent were recorded to analyse the number and position of the functional groups available for the binding of Cr(VI) ions and to study the morphology of biosorbent. The batch isothermal equilibrium data were analyzed with Freundlich and Langmuir isotherm models. The kinetic models were examined with pseudo first order and pseudo second order kinetics. The results revealed that the Cr(VI) is considerably adsorbed on bacterial biomass and it could be an economical method for the removal of Cr(VI) from aqueous solution.
Keywords: Biosorption, Cr(VI) removal, Bacillus subtilis, Pseudomonas aeruginosa, Enterobacter cloacae, Waste water treatment.
1. Introduction
Heavy metal pollution holds a threat for human health and as such life in general. The disposal of heavy metals is a consequence of industrial activities like chemical manufacturing, painting and coating, mining, extractive metallurgy, nuclear and other industries. These metals exert a deleterious effect on the flora and fauna that grow in lakes and streams [1]. Chromium, a highly reactive element with an oxidation state of 6 exhibits stability as Cr(III) and Cr(VI). But Cr(VI) is more toxic to living organisms than the Cr(III) [2]. Furthermore, Cr(III) has limited hydroxide solubility making it relatively immobile and less available for biological uptake. Cr(VI) being powerfully carcinogenic, modifies DNA transcription process thereby causing important chromosomic aberration as quotedby The International Agency for Research on Cancer [3]. The Cr(VI) has also been classified as a group A carcinogen by USEPA based on its chronic effects [4]. Strong exposure of Cr(VI) causes cancer in the digestive tract and lungs [5] and may cause epigastric pain, nausea, vomiting, severe diarrhoea and hemorrhage [6]. Chromium has adverse impacts on aquatic species. It accumulates in fish tissues and at higher concentration causes reduction in fish production [7].
In live bacterial cells besides surface accumulation, metal ions have the possibility to enter into the cytoplasm through the specific carrier system and thus transport processes in bacteria can been studied [16,17]. Bacteria are able to synthesis macromolecules such as polysaccharides, protein, nucleic acid, humic substances and uronic acid usually called extracellular polymeric substances (EPS). EPS are metabolic products of bacteria and they originate from their lysis or hydrolysis [18] and they restrain the respective functional compounds like carboxyl, phosphoric, amine and hydroxyl groups. Extracellular polymeric substances react with the biological matter in the effluent that is to be treated [19]. EPS involved in cell aggregation [20], produce a protective fence for cells to keep at bay harmful substances and accept the accumulation of inorganic ions from the environment [21]. In existing researches, several microorganisms can reduce Cr(VI) concentration, including genus of Pseudomonas, Bacillus, Enterobacter,
Escherichia coli, Shewanella, and several other bacterial isolates were also reported.
This work aims to investigate the biosorption behavior of B.subtilis, P.aeruginosa and E.cloacae. These biomass growths were taken to treat Cr(VI) ion present in aqueous solutions. The effect of solution pH, contact time & biosorbent dosage on the removal of Cr(VI) ion were studied. The experimental data were analysed by fitting it into Langmuir and Freundlich adsorption isotherm models and pseudo first order and pseudo second order kinetic models. The Cr(VI) samples were characterized by Atomic absorption spectroscopy (AAS). The presence of functional compounds in the biomass that may have a role in the biosorption process was confirmed by Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopic (SEM) investigations.
2. Materials and Methods
2.1. Microorganism growth and preparation for biosorption
Bacterial cultures of B.subtilis (MTCC-121), P.aeruginosa (MTCC-424) and E.cloacae (MTCC-509) were received from Microbial Type Culture Collection (MTCC) Chandigarh, India. The nutrient broth was prepared using the prescribed growth medium containing beef extract 1.0g, yeast extract 0.1g, peptone 5.0g, sodium chloride 5.0g and distilled water 1.0 litre. The bacterial culture was sterilized in an autoclave maintained at 15 lbs for 15 minutes and maintained as per the guidelines of MTCC.
2.2. Preparation of samples
Synthetic Cr(VI) solution was prepared using potassium dichromate salt of Cr(VI). All chemicals used in the study are of analytical grade and were obtained from Ranbaxy Fine Chemicals Ltd., India. The potassium dichromate (K2Cr2O7) solutions were prepared using double distilled water. Cr(VI) solution of varying concentrations were acquired by diluting the stock solution. 1N Sodium hydroxide (NaOH) and 1N hydrochloric acid (HCl) solutions were used to adjust the solution pH. Characterization of the biosorbent was carried out using Scanning electron microscope (SEM) and FT-IR studies. Scanning electron microscopic (SEM; Model 6360-JSM; JEOL, Japan) study was also conducted to observe the surface texture and porosity of biosorbent. Fourier transform infrared spectroscopy (FTIR; Model Tensor 27, Bruker Optic GmbH, Germany) spectrometer was used to determine the type of functional groups in bacteria responsible for the Cr(VI) metal bioadsorption. Atomic absorption spectrophotometer (AAnalyst 800; PerkinElmer, USA) was used for the determination of Cr(VI) content in standard and treated solutions respectively. The pH of the solution was measured using a pH Meter (L1 120; Elico - India) containing standard buffer solutions. Centrifuge (R-24, Research Centrifuge. REMI- India) was used for the biosorbent centrifugation. Incubated shaker (Scingenic Biotech/ORBITEK- with temperature mode) at a constant speed of 150 rpm was used for different biosorbents.
2.3. Batch biosorption of Cr(VI)
100
)
(
(%)
0
0
C
C
C
R
e (1)where C0 and Ce represent initial and final Cr(VI) concentration. The biosorption capacity can be estimated as
V
M
C
C
q
0
e
e (2)
where qe is the amount of adsorbed metal ion onto the biomass at equilibrium (mg/l), M represents the amount of biomass in the suspension (L), and V is the volume of the suspension (L).
3. Result and Discussion
This work, studied the effect of Cr(VI) ions on the growth rate of B.subtilis, P.aeruginosa and E.cloacae cells and the biosorption properties of Cr(VI).
3.1. Bacterial Growth curve
The growth rate of B.subtilis, P.aeruginosa and E.cloacae in Nutrient medium under aseptic conditions is studied. It can be noticed from Fig. 1 the growth rate of bacteria has four distinct phases, such i) lag phase, ii) log phase, iii) stationary phase and iv) death phase. In lag phase, bacteria adapt to the environment & begin to grow. In this phase, the bacteria are not yet able to divide. The bacterial growth cycle, synthesis of RNA, enzymes and protein molecules may occur. Exponential phase or log phase is characterized by doubling of cell. During stationary phase, the growth rate slows down as a result of nutrient depletion and adsorption of toxic products. During death phase, due to lack of nutrients, nonavailability of space and Oxygen lead to the decay of bacteria. The optical density recorded at 600nm taken for B.subtilis, P.aeruginosa and E.cloacae shows good exponential phase within 4 hrs. Maximum biomass growth occurred in 20 hrs for B.subtilis, while it took 8 hrs to 16 hrs for P.aeruginosa and E.cloacae to show good exponential growth. The biomass for biosorption process for Cr(VI) removal was taken at the highest exponential phases for all the bacteria.
Fig. 1. Growth curve of B.subtilis, P.aeruginosa and E.cloacae on nutrient medium. pH = 6.6, dosage = l g, agitation rate = 150 rpm, temperature
3.2. Effect of initial pH
In these batch biosorption experiments, the influence of pH on Cr(VI) biosorption was studied using synthetic solutions. In synthetic solution, Cr(VI) biosorption strongly depends on initial solution pH Fig. 2. The effect of pH in batch system was studied by varying the pH from 2 to 9. Due to the activity of hydrogen ions, at low pH values the Cr(VI) uptake was observed to be on the higher side in B.subtilis and E.cloacae. As the pH increases to 6 the amount of Cr(VI) uptake also increases in P.aeruginosa. At lower pH values, the functional groups transfer H+ ions which indicates that majority of the binding sites were occupied. When the pH increases, the concentration of H+ ions decrease and negatively charged biomass surface can interact with the positively charged metal ions. At different pH condition the plenty of carboxyl groups of alginate, sulfonate groups of fucoidan and hydroxyl groups in other polysaccharides are found to play an important role in metal binding [22]. Fig. 2, very clearly shows that
E.cloacae has a higher percentage removal of Cr(VI) (94.9%) compared to B.subtilis (37.5%) and P.aeruginosa
(67.9%).
Fig. 2. Effect of initial pH on the biosorption of Cr(VI) ion (conditions: in B.subtilis;biosorbent dosage = 0.2 g, contact time = 6 h, temperature =
27 ºC, concentration = 100 mg/l, agitation rate = 150 rpm, the same parameter condition were kept as a constant for P.aeruginosa and E.cloacae.
3.3. Effect on biomass dosage
Fig. 3. Effect of Biomass dosage on biosorption of Cr(VI) ion (conditions: in B.subtilis and E.cloacae; pH = 2, time = 6 h, concentration =
100mg/l, temperature = 27 ºC, agitation rate = 150 rpm. P.aeruginosa; (pH-6 and other parameter constants are same as the B.subtilis).
3.4. Effect of initial metal concentration
To investigate the effect of initial metal concentration, the experimental results for the biosorption of Cr(VI) ions onto B.subtilis, P.aeruginosa and E.cloacae. The initial Cr(VI) ion concentrations were changed between 25-200 mg/l values in each experiment set shown in Fig. 4. In the given time duration of 6 hrs the maximum Cr(VI) removal percentage in B.subtilis was (63 % at 100 mg/l) while in as P.aeruginosa it was (77.9 % at 25 mg/l) and in E.cloacae
it was (86.2 % at 25 mg/l). In 200 mg/l B.subtilis gave the best removal rate compared to other bacteria, but initially in the lag phase growth removal was less in B.subtilis. The maximal removal percentage noted in 25 mg/l was
P.aeruginosa (78%), E.cloacae (86%) and B.subtilis (33.5%) respectively. This figure clearly shows that as concentration increases there is a gradual decrease in the percentage removal in all bacteria in their biosorption capacities. In the metal ion adsorption due to increasing electrostatic interactions, present on the site affinity for metal ions gradually moves downward [25].
Fig. 4. Effect of Concentration on biosorption of Cr(VI) ion (conditions: B.subtilis; biosorbent dosage = 1g, pH = 2. P.aeruginosa; biosorbent
dosage = 1 g, pH = 6. E.cloacae; biosorbent dosage = 0.2 g, pH = 2 for all bacteria other parameter conditions are same such as agitation rate =
3.5. Biosorption isotherms
In biosorption, the metal ions adsorbed at the surface of bacterium cell wall & can well be represented by conventional isotherms. An attempt is made to test for the Langmuir and Freundlich isotherms models on the experimental data. Biosorption process availing batch technique needs an understanding of the interaction between metal ions and biosorbent. The Langmuir and Freundlich isotherm models are first applied to these interactions. In this study, the Scatchard plot has been used in order to obtain more compact information about the interaction between metal ions and the biosorbent [26].
3.5.1. Freundlich isotherm
The Freundlich isotherm is an empirical model that relates the adsorption intensity of the sorbent to the biosorbent. The isotherm is adopted to describe reversible adsorption and is not restricted to monolayer formation. The mathematical expression of the Freundlich isotherm model can be given as
Inq
e
InK
F
b
FInC
e (3)A plot of Inqe versus InCe gives a straight line with slope KF and intercept bF. The values of KF and bF along with the linear regression co-efficient (R2) for the present experimental conditions have been obtained and are given in Table 1. Fig. 5, the best correlation coefficient (R2) was observed from Freundlich isotherm model on P.aeruginosa
(0.998) that matches satisfactorily with the experimental observation.
Table 1. Langmuir and Freundlich biosorption isotherm for Cr(VI) on B.subtilis, P.aeruginosa and E.cloacae.
Fig. 5. Freundlich isotherm models for B.subtilis, P.aeruginosa and E.cloacae on Cr(VI) ion.
Bacteria
Langmuir constant Freundlich constant
KL b R2 KF bF R2
B.subtilis 10.526 0.0874 0.997 2.7179 1.9503 0.972
P.aeruginosa 3.496 0.2977 0.988 15.299
9.5499 0.998
3.5.2. Langmuir isotherm
The Langmuir isotherm assumes monolayer deposition of adsorbents on homogenous biosorbent can be given as
e e L e
bC
bC
K
q
1
(4)The bindingconstant (KL,) and the sorbent capacity (b) are estimated by plotting Ce/qe against Ce. The model simulations along with experimental observations for Cr(VI) with the experimental values of KL and b along with the linear regression co-efficient (R2) are given in Table 1 and Fig. 6 respectively. The B.subtilis (0.997), E.cloacae
(0.999) showed a best correlation coefficient (R2) respectively.
Fig. 6. Langmuir isotherm models for B.subtilis, P.aeruginosa and E.cloacae on Cr(VI) ion.
3.5.3. Kinetics
The prediction of batch kinetics is required for the design of industrial scale reactors. Kinetic models were employed to analyze the adsorption rates of chromium. The experimental batch biosorption kinetics data was modeled using pseudo first order and pseudo second order kinetics. The linear form of pseudo first and second order kinetic equations are given as,
303
.
2
)
log(
)
log(
1 e t et
k
q
q
q
(5)and e 2 e 2
1
q
t
q
k
q
t
(6)Table 2. Biosorption kinetics for Cr(VI) on Bacillus subtilis.
Table 3. Biosorption kinetics for Cr(VI) on P.aeruginosa.
Table 4. Biosorption kinetics for Cr(VI) on E.cloacae.
B.subtilis
Concentration (mg/l)
Pseudo first order Pseudo second order
qe K1 R2 qe K2 R2 25 1.986 0.350 0.987 2.146 0.386 0.907 50 2.747 0.439 0.991 3.058 0.416 0.952 100 9.204 0.868 0.964 7.813 0.193 0.965 150 6.223 0.812 0.958 8.621 0.561 0.995 200 5.742 0.854 0.975 8.772 0.618 0.996
P.aeruginosa
Concentration (mg/l)
Pseudo first order Pseudo second order
qe K1 R2 qe K2 R2 25 2.372 0.688 0.992 2.353 0.349 0.901 50 2.958 0.739 0.972 2.849 0.543 0.958 100 4.246 0.905 0.974 4.046 0.565 0.982 150 3.845 0.787 0.927 3.584 0.568 0.972 200 2.249 0.796 0.971 3.076 1.487 0.994
E.cloacae
Concentration (mg/l)
Pseudo first order Pseudo second order
Fig. 7. Pseudo first order plot of time versus t/qe/qt for the Cr(VI) on E.cloacae at various metal concentration
Fig. 8. Pseudo second order plot of time versus t/qt for the Cr(VI) on E.cloacae at various metal concentration
3.6. Characterization of the biosorbent and biosorption mechanism
3.6.1. FT-IR spectral analysis
2915 and 2841 cm−1 [27] corresponding to stretching of the C-H bonds in the methyl and methylene groups present in the cell wall structure. The IR spectrum with loaded and unloaded biomass indicates that amine groups are present, and these are likely to be responsible for Cr(VI) binding.
Table 5. Assignments of Infrared absorption bands
Fig. 9. FTIR Spectra of B.subtilis in Cr(VI), (a) with and (b) with out metal loaded
Wave numbers
(cm
-1)
Intensity shape
Assignment
3600-3750 Sharp O-H
stretching
3400-3550 Sharp O-H
stretching
3100-3500 Strong-broad N-H
stretching
2500-3400 Weak-broad O-H
stretching
2700-2950 Variable C-H
stretching
1400-1660 Variable
N-H
bending
1280-1430 Variable
C-H
bending
1160-1420 Variable
O-H
bending
900-1350 Variable C-N
stretching
900-1380 Variable C-O
stretching
Fig. 10. FTIR Spectra of P.aeruginosa in Cr(VI), (a) with and (b) with out metal loaded
3.6.2. Scanning electron microscopy (SEM) analysis
The biosorption of three different bacteria B.subtilis, P.aeruginosa and E.cloacae were analyzed by scanning electron microscopy to understand its surface morphological characterization of bacteria with the magnification of 10,000 x. An SEM micrograph of unloaded and loaded biomass is shown for B.subtilis in Fig. 12, P.aeruginosa in Fig. 13 and E.cloacae in Fig. 14. Over the biosorption period, the morphology of the bacteria had undergone remarkable physical disintegration. Fig. 12, represents the integrated and cluster arrangement that occurs before and after biosorption which clearly shows that metal particles adhere on the surface of the B.subtilis cell wall. Fig. 13, shows that flakes like morphology with inscribed cluster formation occurred, after usage of metal ion in biosorbent thus increasing the active surface area. Fig. 14, both loaded and unloaded metal ion on biosorbent, fly ash particles are covered by precipitated and complex formed by the heavy metal ions.
(a)
(b)
(a)
(b)
(a)
(b)
Fig. 14. SEM micrograph of (a) before and (b) after Cr(VI) loaded on E.cloacae
4. Conclusion
The Biosorption experiments were carried out to remove Cr(VI) using B.subtilis, P.aeruginosa and E.cloacae in batch systems. The following conclusions can be made based on the analysis:
1. The investigation shows that B.subtilis, P.aeruginosa and E.cloacae are abundantly available and are cost effective. They can be used as biosorbent for the removal of Cr(VI) metal ion from aqueous solutions.
3. The kinetic and equilibrium data fitted well with the pseudo second order kinetic model and the Langmuir isotherm model, respectively. The biosorption of Cr(VI) onto B.subtilis, P.aeruginosa and E.cloacae was found as biosorption on the homogeneous surface via chemisorption process.
4. Of these three bacteria, the maximum biosorption capacity with good rate constant and correlation coefficient was obtained in E.cloacae.
5. The proposed biosorption system was successfully applied to real effluent with Cr(VI) metal ions.
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