Ph.D Paper Publication dated 12.08.2016

Int J Pharm Bio Sci 2016 July ; 7(3): (B) 1121 - 1134
This article can be downloaded from www.ijpbs.net
B - 1121
Original Research Article Biotechnology
International Journal of Pharma and Bio Sciences ISSN
0975-6299
PROFICIENT DECOLORIZATION OF METHYLENE BLUE BY RETURNABLE
BACILLUS COAGULANS IMMOBILIZED BEADS: KINETICS, ISOTHERMS
AND THERMODYNAMIC STUDIES
PALRAJ RANGANATHAN 1
, V. KASIVISWANATHAN2
, AND R. SAYEE KANNAN2*
1
Institute of Organic and Polymeric Materials, National Taipei University of Technology, No. 1, Section 3,
Chung-Hsiao East Road, Taipei 106, Taiwan, ROC
2*
Department of Chemistry, Thiagarajar College, Madurai, India - 625 009
ABSTRACT
The decolorization equilibria and kinetics of a cationic dye (methylene blue) were examined in this study
using sodium alginate (polymer), agriculture waste stuff such as saw dust (individual and consortium)
immobilized Bacillus coagulans beads as the adsorbent. Factors affecting the variety of adsorption
processes: concentrations of dye (50-90 ppm), time, pH (7.0), inoculums size, temperature were
investigated. The effective dye decolorization was attained within 25 hrs and MB dye removal was 98%.
Captivatingly, MB dye decolorization performance of bacteria immobilized sodium alginate and saw dust
beads was same that of free bacteria. We have also tested the reusability of bacteria immobilized sodium
alginate (SA) and saw dust (SD) beads. The decolorization process followed by the pseudo-first-order
kinetic and the Langmuir isotherm models. The determination of the thermodynamic parameters (∆G, ∆H
and ∆S) indicates the spontaneous and exothermic nature of the adsorption process. Overall, batch study
indicated that this environmentally friendly adsorbent may be an alternative for the removal of methylene
blue dye from contaminated media.
KEYWORDS: Methylene blue, bacillus coagulans, Freundlich, Langmuir, adsorption and decolorization
R. SAYEE KANNAN
Department of Chemistry, Thiagarajar College, Madurai, India - 625 009

B - 1122
INTRODUCTION
Methylene blue (MB), a basic and cationic dye, has
been widely used as a colorant, an indicator, and an
antiseptic agent in clinical therapy.
1,2
However, removal
of MB-containing water can cause plain harm to the
environment. They not only do grave harm to aquatic
species by affecting photosynthetic activity, but also
cause cancer and mutation in humans.
3,4
Numerous
human diseases have been conveyed to be closely
associated to MB, such as hemolytic anemia,
hyperbilirubinemia and acute renal failure.
5
It has been
reported that even micro molar levels of MB can
persuade cytotoxicity in SK-N-MC human
neuroblastoma and U-373 MG human astrocytoma
cells.
6
Hence, the removal of MB is a very significant
chore in the defense of our environment and health.
Thus, a number of biological and physicochemical
methods such as photo catalytic degradation,
ultrafiltration and physical adsorption on activated
carbon have been developed for the efficient removal of
MB from gorgeous wastewater.
7-10
These methods are
attractive for their high effectiveness, but are difficult and
exclusive. Biochemical methods including biosorption
with algae, plant powders and baker's yeast are
substitute ways to desire MB-containing wastewater. In
recent years, a number of studies have fixated on some
microorganisms capable of degrading and absorbing
dyes from wastewater.
11,12
A wide diversity of
microorganisms is reported to be proficient of
decolorization of dyes. It has been described that
microorganisms are capable of degrading dyes and
could be used in sewage treatment plants for removal of
these dyes.
13,14
Microbial decolorization has been
projected as a less luxurious and less environmentally
intrusive alternative. Immobilization improves stability
and allows reuse of the enzymes including laccase.
15,16
Selection of immobilization conditions and
immobilization matrix is essential to design a system
appropriate to each particular purpose.
17
On the other
hand, when adsorption technologies are used, the
adsorption efficiency of the reported adsorbent and
flocculants is quite low, and neither the organic matter
nor treatment agents can be easily recycled.
18,19
The
objective of this work is to investigate the decolorization
equilibria and kinetics of a cationic dye (methylene blue)
by using Bacillus coagulans free cells. Challenge has
also been made for whole cells immobilization using
sodium alginate entrapment, due to the moderate
gelation procedure associated to that of chemical
polymerization procedures. SA beads are porous
materials that form spontaneously when sodium alginate
solution reacts with calcium chloride. Analogous to that
of free cells, studies were also carried out consuming
immobilized beads with SD.
MATERIALS AND METHODS
Assemblage of bacteria
Bacillus coagulans was isolated from forest (kodaikanal)
soil by using the nutrient agar medium. Pure cultures of
Bacillus coagulans are inoculated in nutrient broth
contained in sterile Erlenmeyer flasks and incubated in
an incubator with shaker at a temperature of 30 ºC. The
bacteria are developed to early stationary phase (20
hrs). Subsequently the culture was transferred into the
50 ml falcon tubes and centrifuged at 8000 rpm for 10
min, the supernatant was surplus; and the pellet was
washed in pure water.
Enrichment and isolation of dye degrading microbes
Isolated bacterial sample was used as the close relative
foundation of inoculums in this study. For enrichment of
total inhabitants of dye mortifying isolates in the
samples, all colonies were aseptically transferred to 100
ml of enrichment medium, containing 1% (w/v) glucose
as carbon source. The flasks were incubated in shaker
condition at 150 rpm at 28 ºC for 6 days. Behind
incubation, the culture is plated in nutrient agar medium
then diverse colony morphology were preferred and
maintained on nutrient agar slants at 4 ºC.
20
Selection of efficient isolates for dye decolorization
Plate analyzation was performed for detection of MB
dye decolorization activity by the isolated Bacillus
coagulans. Prepared nutrient agar medium is
supplemented with different dyes (50 ppm/100 ml)
individually. Then it was autoclaved at 121 ºC for 30
min. The isolated culture was inoculated in centre of the
medium. All the plates are incubated at 37 ºC for 2 days.
Clear zone was formed around the colony, which
indicated that those bacteria are proficient to degrade
the dye.
21
Biosorption of dyes by the isolated bacteria
Prepared 100 ml of nutrient broth medium containing
various concentration of dye (50-90 ppm) in 250 ml
conical flask, inoculated with 5 ml of Bacillus coagulans
culture for bacterial decolorization study. These
solutions were incubated in an incubator with shaker
(200 rpm) at 30 ºC. Samples of 5 ml of each of the
mixtures were collected at every 5 hrs. The samples
were then centrifuged at 3000 rpm for 10 min, apparent
supernatant was measured in UV-Vis
spectrophotometer (HITACHI U-2000). Percentage of
decolorization was determined by monitoring the
decrease in absorbance at the maximum wavelength of
the dye (i.e., 663 nm for methylene blue). Biosorption
activity was calculated as follows:
Diagnostic methods
Decolorization of dye was analyzed
spectrophotometrically, monitoring reduce in absorption
spectrum at λ=663 nm (λmax for MB K2RL) using the UV-
Vis spectrophotometer. 5 ml samples were inhibited at
regular time intervals and centrifuged at 3000 rpm for 10
min. Supernatant was collected and scanned (200-700
nm). Remaining dye was resolute at maximum
absorption of dye spectrophotometrically and used was
to resolve the percentage of dye decolorized.
Immobilization of bacteria
Dissolve 3.6% of sodium alginate to 0.1% NaCl solution
slowly with continuous stirring. Leave it for 6 to 8 hrs for
air bubbles to escape. Add 5 ml/100 ml bacteria cell to
the alginate slurry and mix well. Take the slurry in a

B - 1123
syringe and extrude drop wise into the 4% CaCl2
solution. The drops formed into spherical beads of 2 to 3
mm size keep the beads solution for gelation. Wash the
beads in sterile distilled water repeatedly till there is no
leakage. Activate the beads in rich medium for 1 to 2
hrs.
Immobilization of agricultural waste with bacteria
5 gm of saw dust waste was prepared as fine powder
mixed with 5 ml of 24 hrs culture to make a wide history
and to make small beads. The beads were dipped in the
sodium alginate solution for the immobilization. The 10
immobilized beads were added with 100 ml solution of
dissimilar concentration of MB dye. All the procedures
were carried out aseptically. The obvious supernatant
was collected by centrifugation at 3000 rpm for 10 min.
The intensity of the color was calculated at maximum
absorbance wavelength of dye. The percent of
decolorization dye was calculated from the above
mentioned formula.
Reusability experiments for bacteria immobilized SA
and SD beads
MB decolorization studies were implemented 5 times to
measure the reusability of the bacteria immobilized SA
and SD beads for an initial concentration of 50 ppm (5
mg/100 ml). Before each cycle, SA and SD beads were
washed three times with sterile PBS buffer and
incubated overnight in PBS to remove any uncommitted
bacteria. MB decolorization experiments were
performed at 100 rpm and 30 ºC for 25 hrs after each
washing step, for a total of 5 cycles. Dye concentrations
were measured at 0 and 25 hrs. Each cycle was
terminated after 25 hrs of total incubation and washing
steps were repeated for both beads samples before the
initiation of the next cycle.
Statistical Analysis
Mean and standard deviation values were calculated
from set of experiments. All statistical analysis were
performed using Microsoft Excel 2007, Version office Xp.
RESULTS AND DISCUSSION
Segregation, selection and classification of dye
degrading bacteria
A secluded bacterium was identified by microscopic and
biochemical tests. Also it was established as Bacillus
coagulans by 16S RNA gene sequencing and it was
deposited in NCBI gene bank (Accessionno. eu732701).
Pseudomonas sp. and Shewanella strains were
secluded from dye effluent due to its dye removal
capacity and has been studied expansively for azo dye
decolorization.
22,23
The Schematic Illustration of
adsorption using an alginate beads with Bacillus
coagulans and Alginate beads, Bacillus coagulans with
saw dust is represented in scheme 1.
Scheme 1
Schematic Illustration of Adsorption using an Alginate beads with
Bacillus coagulans and Alginate beads + Bacillus coagulans
with Saw dust Evaluation of dye decolorization

B - 1124
Figure 1
UV-VIS Spectra of (a) Initial concentration of inoculated medium
(b) Decolorized medium (after 30 hrs incubation)
Figure 2
(a) culture medium (b) Bacillus coagulans bacteria (Microscopic view) (c) Decolorized medium (d) MB solution
(e& f) immobilized bacterial SA beads using decolorization before adsorption and after adsorption (g & i, and
h & j) immobilized bacterial SD beads before adsorption and after adsorption.
Analyzing the product of MB dye reduction process,
using spectrophotometer and photographic evidence
was used to monitor the color change taking place
during treatment. We got main peaks (663 nm) for
standard dye Fig 1. After 30 hrs incubation of dye with
bacterial culture, the degradation products of dye were
analyzed in the visible regions and the peak gets
completely disappeared. The microorganisms can
degrade MB Fig 2 to less harmful product and can
biosorb the compound through fixation and secretion of
secondary metabolites (enzymes and organic acid) by
the functional groups present on the cell’s surface. The
nitrogen source peptone used in this study helps the
growth of microbes more ultimately.

B - 1125
Effect of various concentration of MB dye, deviation of inoculums size and pH
Figure 3
a) Percentage of removal of MB on Bacillus coagulans at various concentration and same inoculum size (5
ml) b) Percentage of removal of MB on Bacillus coagulans at same concentration and various inoculum size
(1-5 ml) c) Percentage of removal of MB on Bacillus coagulans at various pH and same inoculum size (5 ml).
The consequence of primary concentration and
inoculum size of MB on its removal by microbial
sorbents was investigated. The pH of the solution is very
significant parameter in adsorption experiment, as it
affects the electrostatic state of both the sorbate and the
sorbent. The growth of the bacteria and the matching
adsorption process were fundamentally controlled by the
pH of the medium. The percentage biosorption of the
dyes, using free bacteria at various initial maximum
decolorization for all the dyes considered in this work
was observed at a pH range of 4.0-8.0. The percent
decolorization decreases with increased primary
concentration of dye. This can be recognized to the
information that, at lower dye concentration, the ratio of
adsorbent sites to the dye concentration was superior,
whereas, at superior dye concentration, the adsorbent
sites were finally saturated.
24,25
Similar studies have
been reported former.
26,27 A,B
Percentage decolorization
of MB was established to vary with initial concentrations
(50-90 ppm) and same inoculums size (5 ml) when
studied up to 30 hrs. Ponraj et al
13
reported that
Pseudomonas sp, have exceedingly decolorized 50%
with 10% inoculums at 144 hrs. Used a mixed culture
for decolorization of reactive azo dye and reported 98%
at 10% inoculums size.
28
Unlike these studies
conducted by, the optimum pH for growth of
Burkholderia sp. was found to be 8.0 and, optimum pH
was 6.8.
29-31
Fig 3a and 3b showed that the maximum
biosorption was found on 50 ppm (98.64% at 25 hrs)
concentration and the inoculum size of 5 ml. The same
size of inoculum was used for all supplementary studies.
The optimum pH range for the decolorization of the MB
dye was found to be pH 7 (87.04%) in 25 hrs and the
least amount decolorization of the dye MB was found to
be pH 4 (70.02%) Fig 3c. Experimental results on the
evaluation of best possible concentration of the
inoculums are needed to achieve maximum
decolorization.

B - 1126
Effect of SA beads, immobilized SD beads (empty and consortium beads),
Various temperature and reusability result
Figure 4
a) Percentage of removal of MB at SA beads (empty-10 beads) b) Percentage of removal of MB on Bacillus
coagulans at immobilized Bacterial SA beads (10 beads) c) Percentage of removal of MB at SD beads (empty-
10 beads).

B - 1127
Figure 5
a) Percentage of removal of MB on Bacillus coagulans at immobilized SD beads (bacterial beads-10
beads) b) Percentage of removal of MB on Bacillus coagulans at various Temperature c) Reusability
test of the 5 cycles of MB dye decolorization experiment at the initial concentration of 50 ppm.
The decolorization process was extremely partial by the
physicochemical nature of reactive immobilized SA
beads. SA is one of the most widely studied gel matrices
for cell. Entrapment with SA gel beads offering high
biomass loading and good substrate diffusion within the
matrix. Immobilized cells have some advantages over
free cells, such as higher cell density per volume of
reactor, easier separation from the reaction medium.
The major restriction in the commercialization of
industrial bioremediation is their high processing cost.
The use of readily available cheap agro-industrial
residues as the carbon sources may reduce the high
cost. In our study, cheap substrate like SD and SA was
utilized for MB decolorization and percentage removal of
MB dye at various temperature and reusability test was
investigated.By offering many advantages over other
methods, immobilized microbial cell technology has
been applied widely in the field of wastewater treatment.
For decolorization of azo dyes in wastewater, natural
gels such as alginate; carrageenan; synthetic gels such
as polyvinyl alcohol.
32
Immobilizing bacteria will increase
the density of bacteria contained by the bioreactor,
which in terms will increase the rate of degradation
inside the bioreactor.
21
Studies steered to assess the
effect of temperature on dye decolorization prophesied
that higher temperature ranges stimulate higher dye
decolorization. With increase in temperature from 30 to
50
◦
C, time taken for dye removal decreased. Similarly,
Chen et al reported increase in decolorization of
Remazol Black B by K. Marxianus from 25 to 45
◦
C.
33
Dye decolorization (%) increased from 62% at 25
◦
C to
98% at 45
◦
C at an initial dye concentration of 50 mg/L
beyond which it tends to decrease (10% decolorization
at 50
◦
C). In difference to this, Meehan et al reported 30
◦
C as the best temperature for growth of Candida
tropicalis and also for the removal of disperse dye.
34
Lower dye removal was reported elsewhere 35
◦
C.
Thermal inactivation of the enzyme and low biomass
production could have resulted in the decreased color
removal. For useful applications, the flat of reusability is
an important problem for dye decolorization of
wastewater in textile industry.
35
The Fig 4a shows the
effect of decolorization of MB dye by SA (empty) beads.
Maximum percentage of decolorization was experiential
in (56.09%) MB (50 ppm), at 25 hrs the SA
polysaccharides can adsorb the dye compounds. The
minimum percentage of decolorization was observed in
(17.01%) MB (90 ppm) at 25 hrs. Fig 4b show the
maximum percentage of decolorization was observed
(98%) in MB at 25 hrs (immobilized bacterial SA beads).
In the present study SD immobilized with SA beads
(empty beads) were utilized for MB decolorization and
the maximum of 18% of decolorization achieved in 25
hrs Fig 4c. In additional, study of SA immobilized SD
beads with bacteria Fig 5a were utilized for MB
decolorization and the maximum of 98.66% of
decolorization achieved in 25 hrs. Subsequently 25 hrs
of incubation, 81.42% dye decolorization was obtained

B - 1128
at an incubation temperature of 30
◦
C, respectively Fig
5b. But at 50
◦
C, dye decolorization increased to 97.14%
respectively. This can be recognized to the increased
rate of bacteria growth at higher temperature ranges.
The reusability results Fig 5c showed that, after the four
cycles of renewal, favorable percentage removal of MB
dye was detected as 51% and 54% for Bacillus
coagulans immobilized SA and SD beads, respectively.
For the 5th renewal cycle, the MB decolorization
dropped to 40% and 43% for Bacillus coagulans
immobilized SA and SD beads, respectively. The
declorization capacity observed in the current study was
higher than that of the above mentioned references.
Kinetic isotherms
Adsorption isotherms are essential for the explanation of
how adsorbates will cooperate with an adsorbent and
are serious in optimizing the use of adsorbent.
36
Two
legendary isotherm equations, the Langmuir, Freundlich
isotherm, were employed for additional elucidation of the
obtained adsorption data. In the current analysis,
isothermal study of MB was conducted at different
concentrations by keeping the inoculums size present at
5 ml.
Langmuir isotherms
The hypothetical Langmuir isotherm is applicable for
adsorption of absolute from a liquid solution as
monolayer adsorption on a surface containing a fixed
number of identical sites. The Langmuir equation is
generally expressed as follows:
Where ‘ eq ’ is the amount of MB dye adsorbed at equilibrium (mg/gm), ‘ maxq ’ is the monolayer sorption capacity (mg/gm), ‘ aK ’ is
Langmuir constant ‘ eC ’ is concentration of MB dye in solution at equilibrium.
The Freundlich isotherm
Figure 6
Freundlich isotherm model for the adsorption of MB dye onto Bacillus coagulansat
concentration a) 50 ppm b) 60 ppm c)70 ppm d) 80 ppm and e) 90 ppm.

B - 1129
Figure 7
Langmuir isotherm model for the adsorption of MB dye onto Bacillus coagulans
concentration at a) 50 ppm b) 60 ppm c) 70 ppm d) 80 ppm and e) 90 ppm.
Table 1
Langmuir and Freundlich isotherm parameters for the
adsorption of MB dye onto Bacillus coagulans bacteria at various concentrations
Freundlich isotherm model is the most primitive known
equation describing the adsorption process. It is an
experiential equation and can be used for non-ultimate
sorption that involves heterogeneous adsorption.
37,38
The Freundlich isotherm can be consequent assuming a
logarithmic diminish in the enthalpy of adsorption with
the increase in the division of engaged sites and is
generally given by the following non linear equation: The
Freundlich isotherm is commonly presented as,
Isotherms Conc (µg) R2
Freundlich isotherms
100
200
300
400
500
0.9981
0.985
0.8661
0.9315
0.8507
Langmuir isotherms
100
200
300
400
500
0.9996
0.9821
0.954
0.999
0.9992

B - 1130
Where KF is a constant investigative of the adsorption
capacity of and n is an observed constant correlated to
the magnitude of the adsorption driving force.In the
present investigation, the isotherm study for
decolorization of MB by Bacillus coagulans was
conducted at different concentrations (50-90 ppm). The
Freundlich and Langmuir isotherm models were used to
describe the equilibrium adsorption data Fig 6 and 7.
The parameters obtained from the Langmuir (Ce/qe
versus Ce) and Freundlich (log qe versus log Ce)
isotherm plots are listed in Table 1. To quantitatively
compare the accuracy of the models, the correlation
coefficients (R
2
) were also calculated and are also listed
in Table 1. Analysis of the R
2
values suggests that the
Langmuir isotherm model provides best fit to the
equilibrium adsorption data than the Freundlich isotherm
models at all studied concentration implying monolayer
coverage of MB molecules onto the adsorbent surface.
Kinetics of adsorption and Intra particles - diffusion
Figure 8
Pseudo kinetic models for the adsorption of MB dye onto bacillus coagulans
bacteria at various cells mass a) first-order b) second-order and c) Intra
particle diffusion kinetic plots for MB onto Bacillus coagulans
Table 2
Pseudo-first-order and Pseudo-second-order kinetic models for the adsorption
of MB dye onto Bacillus coagulans bacteria at various cells mass
Inoculums
size (ml)
qe Cal (mg/gm) k1 R2
First-order-kinetic
5
10
15
20
25
1071.5
549.54
416.86
338.8
213.79
0.0612
0.121
0.168
0.124
0.169
0.9952
0.9664
0.9138
0.9666
0.9938
Second-order-kinetic
5
10
15
20
25
1666.66
500
486.3
454.54
277.77
2.48x10-5
5.33x10-4
2.48 x10-4
3.58 x10-4
7.85 x10-4
0.9468
0.9832
0.9996
0.9956
0.9983
Table 3
Intra particle diffusion and thermodynamic parameters
for the adsorption of MB dye
Intra particle Diffusion
T (K) Ki C R2
303K 43.146 27.038 0.9941
313K 46.966 -9.93 0.9925
323K 60.641 -93.83 0.9723
Van’t Hoff
T (K) ∆G (J mol-1
) ∆H (J mol-1
) ∆S(J mol-1
k-1
)
303K -25005.22
313K -23062.17 -83879 -194.306
323K -21119.102
Eyring plot
T(K) ∆G#
(J mol-1
) ∆H#
(J mol-1
) ∆S#
(Jmol-1
k-1
)
303K -0.77899
313K -0.77859 -0.79149 0.0000415
323K -0.7780

B - 1131
The adsorption kinetics is important for adsorption
studies because it can predict the rate of adsorption of
dye on bacterial surface and provide valuable data for
understanding the mechanism of adsorption reactions.
Kinetic models were experienced to obtain the rate
constant and equilibrium adsorption capability at
different inoculums.The pseudo-first-order and pseudo-
second-order reaction rate equations are the two most
commonly applied models to investigate the adsorption
mechanism and description based on experimental
data. Linear form of pseudo-first-order kinetic equation
is expressed as,
where qe (pg/cell) is the amount of methylene blue
adsorbed at the point of equilibrium on the surface of
bacterial cell, qt is the amount of methylene blue
adsorbed at time t and k1 (min
−1
) is the rate constant
which can be calculated from the straight line plot of log
(qe−qt) versus time (h).Linear form of pseudo-second-
order kinetic equation is expressed as,
The second order rate constant k2 and qe values were
determined from the slopes and intercepts of the plots.
The applicability of two models (pseudo-first-order
model, pseudo-second-order model) was tested by
linear fitting of log (qe− qt) versus t, (t/qt) versus t,
respectively. The results showed that the correlation
coefficients (R
2
) obtained at different inoculums size for
the pseudo-first-order and pseudo-second-order kinetic
model Fig.8a and 8b The pseudo-first-order rate
constant k2, the calculated qe values and corresponding
linear regression correlation coefficients values R
2
are
given in Table 2. As seen in Table 2, the calculated qe
values agree with experimental qe values well, and also,
the correlation coefficients for the pseudo-first-order
kinetic plots at all the studied inoculums sizes are
significantly higher (R
2
>0.99). In addition, the theoretical
and experimental equilibrium adsorption capacities, qe
obtained from the pseudo second order kinetic model
varied widely at all inoculums size. These findings
suggest that adsorption of MB on Bacillus coagulans
cannot be described by the pseudo-second-order kinetic
model. Conversely, the kinetic data exhibited an
excellent compliance with pseudo-first-order kinetic
equation. Any adsorption process consists of different
steps, the surface diffusion followed by intra-particle
diffusion. In general, the adsorption was governed by
the liquid phase mass transport or by intra particle mass
transport. The mass transfer rate can be expressed as a
function of the square root of time (t). The intra-particle-
diffusion model was expressed by,
Where qt is the amount of dye adsorbed onto the
adsorbent at time t (mg/gm), C is the intercept, and Kdif
is the intra-particle diffusion rate constant (mg g
-1
min
-1
).
The plot of qt vs t
1/2
for intra-particle diffusion in the
adsorption of methylene blue onto Bacillus coagulans at
various temperatures was used to obtain the diffusion
rate parameters. Though intra particle diffusion curves
give good agreement to the linear fitting (R
2
= 0.97 to
0.97) they do not pass through the origin Fig 8c which
implies that intra particle diffusion is not the rate-
controlling step of the adsorption process. Through the
simultaneous calculation of the linear relationship, the
results were shown in Table 3.

B - 1132
Thermodynamic parameters, Activation parameters and Eyring plot
Figure 9
a) Van’t Hoff equation isotherm
b) Arrhenius plot and c) Eyring plot
Thermodynamic parameters such as free energy
change ( Δ G), enthalpy change ( Δ H) and entropy
change ( Δ S) were calculated to evaluate the
thermodynamic feasibility and the spontaneous nature
of the process. Therefore, the thermodynamic
parameters were calculated from the change of
distribution coefficient, with the respect to temperature.
Therefore, the thermodynamic parameters can be obtained from the following equations,
ΔH and ΔG can be obtained from the slope and intercept of Van't Hoff plot of ln K versus 1/T.39,40
The data are presented in Fig 9a and Table 3. The
negative ∆G values confirm the spontaneous nature and
feasibility of the adsorption process. The negative
values of ΔH further confirm the exothermic nature of
the adsorption process. The negative Δ S value
corresponds to a decrease in randomness at the
solid/liquid interface during the biosorption of dye on
adsorbent while low value of S indicates that no
remarkable change on entropy occurs.
41
Activation
energy gives information about the adsorption
mechanism. A low activation energy (<42 kJ mol
−1
)
value indicates physical adsorption, while a high
activation energy indicates chemical adsorption. The
activation energy, Ea is calculated by using Arrhenius
equation.
42,43
Where A is the Arrhenius pre-exponential factor, R is the
universal gas constant, and T is the temperature in
Kelvin. Ea can be obtained by plotting (Arrhenius plot) ln
k against the reciprocal of the absolute temperature
T.The activation energy for the adsorption of MB onto
Bacillus coagulans from the slope of the plot of ln k vs
1/T Fig 9b which is found to be 111.89 kJ/mol. It clearly
indicates that the process is governed by chemical

B - 1133
adsorption.The standard enthalpy (∆H
#
), entropy of
activation (∆S
#
) and free energy (∆G
#
) of activation for
dye adsorption can also be calculated using the Eyring
equation as follows:
Where k is the rate constant, KB is the Boltzmann
constant (1.3807x 10
-23
), h is the Plank constant
(6.6261x10
-34
), R is the ideal gas constant (8.314 kJmol
-
1
K
-1
), and T is temperature (K). The values of ∆H
#
and
∆S
#
can be determined from the slope and intercept of a
plot of ln k/T versus 1/T Fig 9c a negative value of
enthalpy of activation implies that the adsorption
process is exothermic. A positive value of entropy
suggests that the adsorption process involves a
dissociative mechanism.
CONCLUSION
Sodium alginate and Saw dust beads containing a
consortium of Bacillus coagulans can be an effective
and alternative adsorbent material used for MB dye
removal in wastewater treatment processes.
ACKNOWLEDGEMENTS
The authors wish to acknowledge the management and
Department of Chemistry, Thiagarajar college, Madurai-
9, for providing lab facilities.
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