Langmuir 2006, 22, 7265-7272 7265

Adsorption Behavior of Rhodamine B on Rhizopus oryzae Biomass

Sujoy K. Das,† Jayati Bhowal,† Akhil R. Das,‡ and Arun K. Guha*,†
Department of Biological Chemistry, Indian Association for the CultiVation of Science, JadaVpur,
Kolkata 700 032, India, and Polymer Science Unit, Indian Association for the CultiVation of Science,
JadaVpur, Kolkata 700 032, India

ReceiVed September 28, 2005. In Final Form: March 31, 2006

The removal of a carcinogenic dye rhodamine B (C. I. 45170) from wastewater by biomass of different moulds
and yeasts is described. Among all of the fungal species tested, the biomass of Rhizopus oryzae MTCC 262 is found
to be the most effective. Dye adsorption reaches maximum with the biomass harvested from the early stationary phase
of growth. The optimum temperature and pH for adsorption are observed to be 40 °C and 7.0, respectively. The
adsorption rate is very fast initially and attains equilibrium after 5 h. The adsorption isotherm follows the Langmuir
isotherm model satisfactorily within the studied dye concentration range. Of the different metabolic inhibitors tested,
2,4-ditrophenol (DNP) and N,N′-dicyclohexylcarbodiimide (DCCD) decrease dye adsorption by ∼30% suggesting
the role of energy metabolism in the process. Spectrophotometric study indicates that the removal of rhodamine B
by R. oryzae biomass involves an adsorption process. Scanning (SEM) and transmission (TEM) electron microscopic
investigations have been carried out to understand the probable mechanism of the dye-biomass interaction.

Introduction biomass2-3,6,11-14 to adsorb or degrade dyes present in waste-

A huge quantity of dyes, approximately 5-10% of the annual
global production (ca. 107 kg), is discharged as effluent mainly
by paint and textile industries.1,2 The majority of the dyes are
toxic and even carcinogenic and cause damage not only to aquatic
life but also to humans.2-4 Photosynthesis5 is also reduced due
to inhibition of sunlight penetration. Because of environmental
legislations,6 industrial concerns are forced to treat dye bearing
effluents before discharging into water streams. Most of the
commercial dyes are of synthetic origin having complex aromatic
structures which make them stable against photodegradation5
and oxidation.7 As a result, removal of color from wastewaters
becomes difficult by conventional techniques, such as aerobic
digestion.2 Current research is now focused on the removal of
dye from effluent using the adsorption technique, which does
not generate a huge amount of sludge or harmful substances.
Activated carbon is the most efficient and popular choice of
adsorbent but the high cost and huge requirement6,8 restrict its
use in many countries including India. Thus, there is much interest
in the development of new adsorbents8-10 for the treatment of
biological and industrial wastes. Due to the low adsorption
capacity of these materials, a huge amount is required; hence,
highly effective and economic adsorbents are needed. In recent
years many studies have been conducted with microbial
* Corresponding author. E-mail: bcakg@mahendra.iacs.res.in. Fax: +91
33 2473 2805. Phone: +91 33 2473 4971/5904 Ext. 502.
† Department of Biological Chemistry.
‡ Polymer Science Unit.
(1) Wong, Y.; Yu, J. Water Res. 1999, 33, 3512-3520.
(2) Banate, I. M.; Nigam, P.; Singh, D.; Marchant, R. Bioresour. Technol.
1996, 58, 217-227.
(3) Fu, Y.; Viraraghavan, T. Bioresour. Technol. 2001, 79, 251-262.
(4) Hartman, C. P.; Fulk, G. E.; Andrews, A. W. Mutat. Res. 1978, 58, 125-
132.
(5) Ramakrishna, K. R.; Viraraghavan, T. Waste Manage. 1997, 17, 483-488.
(6) Robinson, T.; McMullan, G.; Marchant, R.; Poonam, N. Bioresour. Technol.
2001, 77, 247-255.
(7) Poots, V. J. P.; McKay, G.; Healy, J. J. Water Res. 1976, 10, 1061-1066.
(8) Figueiredo, S. A.; Boaventura, R. A.; Loureiro, J. M. Sep. Purif. Technol.
2000, 20, 129-141.
(9) Namasivayam, C.; Kumar, M. D.; Selvi, K.; Begum, R. A.; Vanathi, T.;
Yamuna, R. T. Biomass Bioeng. 2001, 21, 477-483.
(10) Chatterjee, S.; Chatterjee, S.; Chatterjee, B. P.; Das, A. R.; Guha, A. K.
J. Colloid Interface Sci. 2005, 288, 30-35.
water. However, the removal of rhodamine B (C. I. 45170), a
xanthine dye, used in the textile, printing, and paint industries15
from wastewater using microbial biomass remains unexplored.
In this paper, we describe for the first time the efficacy of using
different fungal biomass to adsorb rhodamine B from its aqueous
solution and the effect of different physicochemical parameters
involved in the process.
Experimental Section
Materials. Rhodamine B (C. I. 45170) used in this study was
purchased from BDH, England. Microbiological media and ingre-
dients were procured from Himedia, India. Sodium azide (NaN3),
2,4-dinitrophenol (DNP), and N,N′-dicyclohexylcarbodiimide (DCCD)
were obtained from Sigma, USA. All other chemicals and bio-
chemicals were purchased from Merck, Germany. The chemical
structure and characteristics of the dye are described in the Supporting
Information (Figure S1 and Table S1).
The fungi Rhizopus oryzae (MTCC 262), Mucor rouxii (MTCC
386), Penicillium ochrochloron (MTCC 517), Aspergillus Viridie
(MTCC 1782), and Pleurotus sajor-caju (MTCC 141) were obtained
from the Institute of Microbial Technology, Chandigarh, India.
Termitomyces clypeatus, Saccharomyces cereVisiae MATa, Sac-
charomyces cereVisiae MATa,R and Candida utilis were kindly
supplied by Dr. A. K. Ghosh, Indian Institute of Chemical Biology,
Kolkata, India.
Methods. Mould and yeast strains were maintained on potato-
dextrose (20% potato extract and 2% dextrose) and yeast extract-
peptone-dextrose (0.3% yeast extract, 1% peptone and 2% dextrose)
slants, respectively. Organisms were subcultured at regular intervals
of 30 days to maintain viability.
Preparation of Biosorbents. Potato-dextrose broth, yeast extract-
peptone-dextrose broth, and deproteinised whey medium supple-
mented with 0.8% diammonium hydrogen phosphate were used for
the cultivation of mould, yeast, and R. oryzae, respectively. The
(11) Zheng, Z.; Levin, R. E.; Pinkham, J. L.; Shetty, K. Process Biochem.
1999, 34, 31-37.
(12) Bell, J. P.; Tsezos, M. Water Sci. Technol. 1987, 19, 409-16.
(13) Juhasz, A. L.; Naidu, R. J. Micrbiol. Methods 2000, 39, 149-158.
(14) Juhasz, A. L.; Smith, E.; Smith, J.; Naidu, R. J. Indus Microbiol. Biotechnol.
2002, 29, 162-169.
(15) Namasivayam, C.; Radhika, R.; Suba, S. Waste Manage. 2001, 21, 381-
387.

10.1021/la0526378 CCC: $33.50 © 2006 American Chemical Society

Published on Web 07/25/2006
7266 Langmuir, Vol. 22, No. 17, 2006
media were dispensed in aliquots of 75 mL in 250-mL Erlenmeyer
flasks and sterilized by autoclaving at 121 °C for 15 min. The flasks
containing the medium were inoculated with 3.5 × 107/mL mould
spores or 1 mL yeast culture (5 × 109 cells/mL). Inoculated media
were incubated under submerged condition (130 rpm) at 30 °C for
96 and 48 h for mould and yeast strains, respectively. At the end
of incubation, yeast cells and fungal mycelia were harvested by
centrifugation (Sorval RC-5B refrigerated centrifuge) and washed
with deionized water. Dead biomass was prepared by autoclaving
the culture before harvesting at 121 °C for 15 min. Starved biomass
was obtained by suspending live biomass in isotonic saline for 24
h at 4 °C. Starved biomass was resuscitated by incubating in peptone
water (0.5% w/v) for 2 h. All of the biomasses were dried by
lyophilization.
Dye Solution and Estimation. A stock solution (1000 mg/L) of
rhodamine B was prepared in 10 mM Tris-HCl buffer (pH 7.0) and
diluted with the same buffer to get the desired concentrations of the
dye. A calibration curve of rhodamine B was prepared by measuring
the absorbance of different concentrations of the dye at the optimum
λmax (554 nm) using UV-vis spectrophotometer (Varian model
CARY 50 Bio). The dye concentrations in the experimental samples
were calculated from the calibration curve.
Batch Biosorption Experiments. Screening of Biosorbent. To
25 mL of rhodamine B (100 mg/L, pH 7.0) solution taken in different
100-mL Erlenmeyer flasks was added 0.25 g (dry weight) of biomass
of mould or yeast. The flasks were incubated at 30 °C for 24 h with
shaking (130 rpm). At the end of incubation, the biomass was
separated by centrifugation, and the concentration of dye in the
supernatant was estimated spectrophotometrically. The biomass
which showed maximum adsorption of rhodamine B was selected
for further studies.
Metabolism of Rhodamine B. Deprotienised whey medium (75
mL) containing 5 mg/L rhodamine B was taken in different 250-mL
Erlenmeyer flasks. Control flasks contain only deproteinised whey
medium. Flasks were inoculated with a spore suspension of R. oryzae
as described earlier and incubated at 30 °C for 72 h under submerged
condition (130 rpm). At the end of incubation, mycelia and broth
were separated by centrifugation at 10 000 rpm for 15 min.
Supernatants were scanned in the range of 200-800 nm using a
UV-vis spectrophotometer to monitor any change in the spectral
shift due to degradation of the dye.
Growth Phase of R. oryzae and Dye Adsorption. Mycelia of R.
oryzae were harvested at different growth phases of the organism,
washed, dried, and used for dye adsorption as described in the batch
biosorption experiment.
pH and Temperature on Dye Adsorption. The effect of pH on
rhodamine B adsorption was studied over a pH range of 3.0-10.0.
Prior to this, the biomass was conditioned in a buffer solution of
required pH under experimental conditions for 2 h with shaking.
Other experimental parameters were the same as described under
the screening of biosorbent. This experiment was repeated at optimum
pH 7.0 by varying the incubation temperature from 20 to 60 °C.
Equilibrium Adsorption Isotherm. This experiment was done as
described in the screening of biosorbent using varied dye concentra-
tions from 10 to 1000 mg/L. The pH and temperature of incubation
were 7.0 and 40 °C, respectively.
Metabolic Inhibitors and Nutrients on Dye Adsorption. ROB (0.25
g) was incubated in 50 mL of Tris buffer (pH 7.0) containing
individually DNP (1 mM), DCCD (400 µM), NaN3 (2 mM), sucrose
(0.5%, w/v), and peptone (0.5%, w/v) for 60 min at 30 °C. ROB
incubated in Tris buffer only served as a control. Conditioned ROB
was collected after centrifugation, washed, and used for adsorption
study. The concentration of rhodamine B used in this experiment
was 1000 mg/L and the procedure described in the equilibrium
adsorption isotherm was followed.
Kinetics Study. The rate of rhodamine B adsorption was studied
at regular intervals of time up to 500 min using different dye
concentrations (100, 250, 400, and 1000 mg/L) at optimum pH 7.0
and temperature 40 °C. The other experimental conditions were
same as described earlier.
Das et al.
Figure 1. Rhodamine B adsorption on biomass of different
organisms. Data represent an average of four independent experiments
( SD shown by error bar.
Zeta Potential. The zeta potential of the R. oryzae biomass (ROB)
at different pH was measured by Zetasizer (Malvern Zetasizer)
following the procedure of Li et al.16
Scanning and Transmission Electron Microscopy. The samples
for scanning (SEM) and transmission (TEM) electron microscopy
before and after dye adsorption were prepared as described by Sastry
et al.17 Electron micrographs for SEM and TEM were recorded on
FESEM (JEOL JSM-6700F) and HRTEM (JEOL JEM 2010),
respectively.
Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectrum
of ROB after pelleting with spectroscopic grade KBr was recorded
with a NICOLET Magma 750 FTIR spectrometer in the range of
4000-400 cm-1.
Results and Discussion
Screening of Microorganism. The biomass of different
nonpathogenic moulds and yeasts has been initially screened to
study their capacity to adsorb rhodamine B from its solution.
Among all of the biosorbents tested, live biomass of Rhizopus
oryzae is found to be the most potent in this respect (Figure 1).
It is observed that the adsorption capacity of ROB is more than
4-fold larger in comparison to S. cereVisiae MATa. It is reported
that the type of biomass has a significant effect on the adsorption
process, especially in the case of organic pollutants.18-20 The
observed difference in adsorption capacity is probably due to the
variation in the cell size, morphology, as well as the number of
active binding sites and their distribution on the cell surface.18
Further, it is observed that the dead biomass adsorbs 20-35%
less rhodamine B than the corresponding live biomass under
identical conditions. Reduction in dye adsorption by the dead
biomass may be attributed to the loss of some binding sites due
to the high temperature required for inactivation of the biomass.
Because of better adsorption capacity toward rhodamine B, only
live biomass of R. oryzae was used to carry out further studies
in this respect.
Biodegradability of Rhodamine B. To understand the process
of dye removal from its solution, R. oryzae was allowed to grow
in deproteinised whey medium in the presence of rhodamine B.
(16) Li, N.; Bai, R. Sep. Purif. Technol. 2005, 42, 237-247.
(17) Sastry, M.; Ahmad, A.; Khan, M. I.; Kumar, R. Curr. Sci. 2003, 85,
162-170
(18) Aksu, Z. Process Biochem. 2005, 40, 997-1026.
(19) Tsezoz, M.; Bell, J. P. Water Res. 1988, 22, 391-394.
(20) Tsezoz, M.; Bell, J. P. Water Res. 1989, 23, 561-568.
Adsorption of Rhodamine B on R. oryzae Biomass Langmuir, Vol. 22, No. 17, 2006 7267

Figure 2. UV-vis spectrum of rhodamine B before and after growth

of R. oryzae.

The UV-vis spectra of the fermented broth along with the control
were recorded (Figure 2). No significant spectral shift or
development of a new peak was noted. This indicates that
rhodamine B is not transformed or degraded by R. oryzae and
its removal occurs by adsorption11 only.
Growth Kinetics of ROB and Effects of Different Factors
on the Adsorption Process. Since biochemical composition of
mycelia depends on the growth phase of the organism,21 studies
on the adsorption of rhodamine B in relation to the age of biomass
are highly interesting. Figure 3a shows that the dye adsorption
capacity of ROB depends on the time of harvest of the mycelia
and increases during the entire exponential growth phase of the
organism. Maximum adsorption occurs with the biomass
harvested from the early stationary phase of growth (70 h); the
biomass harvested thereafter has reduced adsorption capacity.
Adsorption of dye depends on the binding sites as well as
metabolic activities, which are related to the age of the mycelia.
It may be noted that the chemical composition of cells in the
exponential phase is different from those in the stationary phase.22
Increased dye adsorption exhibited by mycelia harvested at the
exponential growth phase indicates that the binding of rhodamine
B to ROB is probably facilitated by membrane bound functional
groups such as carboxyl, amino, sulfate, phosphate, amide,
hydroxyl, imidazole, etc. as well as metabolic activities which
undergo changes with the progress of growth process.23,24
pH is an important factor in dye adsorption. It has been reported
that adsorption of dye may be dependent15,25 or independent26,27
of solution pH. Annadurai et al.25 reported that maximum
adsorption of rhodamine B on orange peel occurs at pH 7.0,
whereas Namasivayam et al. reported that rhodamine B adsorption
on orange peel26 and waste banana pith27 was not significantly Figure 3. Dye adsorption by ROB under different conditions. Effect
altered within pH 3.0-11.0. It appears from Figure 3b that the of (a) harvesting period, (b) pH, and (c) temperature. Symbols: (b)
pH value of the solution does not appreciably affect the adsorption dye adsorbed, (O) biomass production, and (0) zeta potential. Data
capacity. Zeta potentials have been measured to analyze the represent an average of four independent experiments ( SD shown
surface charge properties of ROB at different pH values to by error bar.

(21) Gottlieb, D.; Etten, J. L. V. J. Bacteriol. 1964, 88, 114-121.
(22) Freeman, B. A. Burrows Textbook of Microbiology, 22nd ed.; W. B.
Saunders Co: Philadelphia, 1985; p 52.
(23) Tobin, J. M.; Cooper, D. G.; Neufeld, R. J. Appl. EnViron. Microbiol.
1984, 47, 821-824.
(24) Fu, Y.; Viraraghavan, T. AdV. EnViron. Res. 2002, 7, 239-247.
(25) Annadurai, G.; Juang, R.-S.; Lee, D.-J. J. Hazard. Mater. 2002, 92, 263-
274.
(26) Namasivayam, C.; Muniasamy, N.; Gayatri, K.; Rani, M.; Ranganathan,
K. Bioresour. Technol. 1996, 57, 37-43.
(27) Namasivayam, C.; Kanchana, N.; Yamuna, R. T. Waste Manage. 1993,
13, 89-95.
understand the role of electrostatic forces on the adsorption
process.13 Figure 3b shows that the zeta potentials of the adsorbent
(ROB) suspensions decrease from +5.4 to -39.3 mV corre-
sponding to a change in pH from 3.0 to 10.0, but dye adsorption
does not increase to a great extent. Because rhodamine B is basic
dye, it is expected that adsorption will increase appreciably with
an increase in net negative surface charge of the adsorbent.
Therefore, it is most likely that, in addition to electrostatic force
of attraction, other factors such as intracellular accumulation,
7268 Langmuir, Vol. 22, No. 17, 2006 Das et al.

chemical interaction between the dye molecules and different

functional groups present on the fungal cell walls, or physical
force of attraction also play a role in the present adsorption process.
The adsorption process by microbial biomass has been reported
to be dependent10,28-29 or independent30 with incubation tem-
perature. Again, the biochemical constituents of microbial cells
may be changed at high-temperature resulting in low adsorption.31
In view of this, we have decided to study the effect of temperature
on adsorption of rhodamine B by ROB. The results presented
in Figure 3c show that the adsorption capacity is dependent on
the temperature of incubation with an optimum at 40 °C. Higher
adsorption noted at increased temperature is probably due to
higher affinity of the sites for rhodamine B. At a higher
temperature, the energy of the system is likely to facilitate the
attachment of rhodamine B on the cell surface. Adsorption
decreases with a further increase in temperature probably due
to the decreased surface activity as noted by Aksu et al.29 in the
case of biosorption of Remazol Black B reactive dye by R.
arrhizus.
Equilibrium Adsorption Isotherm. Studies on the adsorption
isotherm are a prerequisite to understand the adsorbate-adsorbent
interaction and to optimize the use of the adsorbent. In the present
study, the equilibrium adsorption of rhodamine B by ROB (Figure
4a) shows that the adsorption capacity increases with an increase
in equilibrium concentration and ultimately attains a saturated
value. The experimental data have been analyzed by the Langmuir
and Freundlich adsorption isotherms.32,33 Langmuir’s model is
based on a few postulates, e.g., (a) the monolayer coverage of
the adsorbate at specific homogeneous sites of the outer surface
of adsorbent and (b) all adsorption sites are identical and
energetically equivalent. Thus, from a theoretical standpoint, an
adsorbent can adsorb a definite amount of an adsorbate. The
linearized form of Langmuir (eq 1) isotherm can be expressed
as

Ce 1 aL
) + Ce (1)
qe KL KL

where qe and Ce are the concentrations at equilibrium in the solid

phase (mg/g) and aqueous phase (mg/L), respectively, and aL
(L/mg) and KL (L/g) are the Langmuir isotherm constants. The
theoretical monolayer saturation capacity Qmax can be calculated
from the straight line plot of Ce/qe against Ce (eq 1). Plotting of
the experimental data of Figure 4a as presented in Figure 4b
shows that the adsorption of rhodamine B on ROB follows the
Langmuir model reasonably well. From the slope of the curve,
the theoretical monolayer saturation capacity (Qmax) of the
adsorbate on the adsorbent has been calculated to be 39.21 mg/g
against 39.08 mg/g obtained experimentally. The maximum
saturation capacity Qmax obtained from the plot is higher than
other types of biosorbents.25-27 The general shape of the curve
and the sharp curvature close to saturation indicate the char-
Figure 4. Equilibrium adsorption isotherm of rhodamine B
acteristics of Langmuir equilibrium and a high degree of adsorption (a); adsorption isotherm following Langmuir model (b);
irreversibility34 with a correlation coefficient of 0.999. The adsorption isotherm following Freundlich model (c). Data represent
Langmuir constant has been calculated (Supporting Information, an average of four independent experiments ( SD shown by error
Table S2). The essential features of the Langmuir isotherm can bar.

(28) Ju, Y.-H.; Chen, T.-C.; Liu, J. C. Colloids Surf. B 1997, 9, 187-196. be expressed in terms of a dimensionless constant separation
(29) Aksu, Z.; Tezer, S. Process Biochem. 2000, 36, 431-439.
(30) Mogollon, L.; Rodriguez, R.; Larrota, W.; Ramirez, N.; Torres, R. Appl. factor or equilibrium parameter, RL which is defined as34
Biochem. Biotechnol. 1998, 70-72. 593-601.
(31) Kuyucak, N.; Volesky, B. Biotechnol. Bioeng. 1989, 33, 823-831. 1
(32) Langmuir, I. J. Am. Chem. Soc. 1916, 38, 2221-2295. RL )
(33) Glastone, S. Textbook of physical chemistry, 2nd ed.; MacMillan Publishing 1 + aLC0
Co: New York, 1962; p 1196.
(34) McKay, G.; Blair, H. S.; Gardner, J. R. J. Appl. Polym. Sci. 1982, 27,
3043-3057. where aL is the Langmuir constant as described above and C0
Adsorption of Rhodamine B on R. oryzae Biomass
Figure 5. Adsorption kinetics corresponding to different initial dye
concentrations. Data represent an average of four independent
experiments ( SD shown by error bar.
is the initial dye concentration (mg/L). The RL values within the
range 0 < RL < 1 indicate favorable adsorption. The calculated
RL value in the present adsorption process is found to be 0.03
for an initial dye concentration of 1000 mg/L. This indicates that
the process is favorable for rhodamine B removal. On the other
hand, the Freundlich equation describes the adsorption on a
heterogeneous surface with uniform energy. The linearized form
of Freundlich isotherm (eq 2) can be expressed as
1
log qe ) log KF + log Ce (2)
n under identical conditions. This is due to less intracellular
where KF (L/g) is the Freundlich constant and 1/n is the
heterogeneity factor. Experimental data have also been analyzed
according to Freundlich isotherm model by plotting log qe against
log Ce and presented in Figure 4c. It appears from the figure that
the present adsorption process does not ideally follow Freundlich
isotherm model and exhibits deviation from linearity over the
entire concentration range. However, if the total concentration
range is divided into three regions, good fits to the experimental
data can be noted specially in the lower concentration range,
e.g., region 1 and 2. Thus, the Freundlich equation cannot describe
the adsorption process at higher concentration ranges (region 3).
In the present system, the correlation coefficient comes out to
be 0.999 and 0.952 for Langmuir and Freundlich models,
respectively. The Langmuir isotherm model is thus found to
provide the better prediction for the sorption of rhodamine B for
total concentration range.35
Kinetics Study. The rate of rhodamine B adsorption by live
ROB collected from the early stationary phase of growth has
been investigated under optimum conditions at pH (7.0) and
temperature (40 °C) using four different dye concentrations. The
kinetic results are summarized in Figure 5, which show that the
rate of adsorption is initially rapid in all of the cases, and this
rapid rate continues for about 60 min and then gradually slows
down to reach equilibrium after 5 h. About 70% adsorption takes
place during this initial rapid rate period. This has significant
practical importance requiring smaller reactor volumes ensuring
high efficiency and economy.
Accumulation of the Dye in the Presence of Metabolic
Inhibitors and Nutrients. Dye adsorption by fungal biomass in
the presence of metabolic inhibitors and nutrients is an unexplored
(35) Wong, Y. C.; Szeto, Y. S.; Cheung, W. H.; McKay, G. Langmuir 2003,
19, 7888-7894.
Langmuir, Vol. 22, No. 17, 2006 7269
area although such reports are available with respect to metals.36,37
Adsorption of the dye by ROB that had been previously incubated
in the presence of different metabolic inhibitors or nutrients was
followed for 5 h in order to understand the possible energy
requirement in the process. Both DNP (uncoupler) and DCCD
(ATP synthetase inhibitor) inhibit dye adsorption to the extent
of ∼30%; on the other hand, sucrose (carbon nutrient) and peptone
(both carbon and nitrogen nutrients) increase the adsorption by
almost the same proportion. NaN3 (terminal oxidase inhibitor)
has practically no effect on the process. Uncouplers of oxidative
phophorylation prevent ATP synthesis38 in mitochondria by
dissipating the energized membrane state while substrate oxidation
and oxygen consumption proceed normally. Thus, it is expected
that the active transport process requiring energy would be
inhibited where primary source of ATP generation is oxidative
phophorylation. Inhibition of dye adsorption by ∼33% by DNP
(uncoupler) indicates that ATP derived by oxidative phospho-
rylation is required in this process. This view gets further support
from the observation that DCCD reduces the dye adsorption
almost to the same extent (∼30%) as DNP. DCCD, a true inhibitor
of oxidative phosphorylation, inactivates the ATP synthetase
function38 by inhibiting proton translocation through the F0 subunit
of the enzyme. Berger39 has also shown the involvement of
phosphate bond energy (ATP) in the accumulation of glutamine
and proline in Escherichia coli by studying the reduction of
uptake in the presence of DNP and carbonyl cyanide-p-
trifluromethoxyphenyl-hydrazone. Incubation of ROB in the
presence of sucrose or peptone increases dye adsorption by 1.3-
fold. These compounds are easily metabolized generating more
ATP and thus enhance dye accumulation. Further, it is observed
that starved ROB adsorbs less rhodamine B as against live biomass
accumulation of the dye in starved cells compared to live cells
as revealed from transmission electron micrographs (Figure 7,
panels B and C). Energy rich compound (ATP) required for this
purpose is likely to be present at low concentrations in starved
cells. However, the starved biomass recovers the loss in adsorption
capacity after incubation for 2 h in peptone water (Supporting
Information, Table S4) indicating that the energy rich compound
(ATP) is synthesized to enhance the dye adsorption. The dead
biomass which contains no ATP adsorbs a lesser amount of dye
supporting the above view of phosphate-bond energy involvement
in the intracellular accumulation of the dye.
Scanning Electron Microscopy (SEM). Scanning electron
microscopy has been used extensively as a tool for biosorbent
characterization30 because it can indicate the accumulation of
dye on the surfaces. Figure 6, panels C and CH, shows the surface
morphology of the biosorbent, which appears to be rough and
irregular with a large area for dye-surface interaction. Significant
change in the surface morphology of ROB is noted, which became
compact after rhodamine B adsorption (Figure 6D). SEM image
of dye adsorbed ROB at a higher magnification (Figure 6DH)
exhibits the adsorbed dye molecules in organized form. The
exact reason for this is not known at this moment. Optical
microscopic images (Figure 6, panels A and B) also support the
adsorption of rhodamine B on ROB.
Transmission Electron Microscopy (TEM). Figure 7 rep-
resents the micrographs of the thin section of the fungal cells
(36) Sar, P.; Kazy, S. K.; Asthana, R. K.; Sing, S. P. Curr. Microbiol. 1998,
37, 306-311.
(37) Kazy, S. K.; Sar, P.; Asthana, R. K.; Sing, S. P. World J. Microbiol.
Biotechnol. 1999, 15, 599-605.
(38) Smith, E. L.; Hill, R. L.; Lehman, I. R.; Lefkowiz, R,; Handler, P,; White,
A. Principles of biochemistry: General Aspects, 7th ed.; McGraw-Hill Book Co:
Singapore, 1985; p 352.
(39) Berger, E. A. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 1514-1518.
7270 Langmuir, Vol. 22, No. 17, 2006
Figure 6. Optical microscopic image (40×) of ROB before (A) and
after rhodamine B adsorption (B). SEM micrographs of ROB before
dye adsorption (C, low magnification; CH, high magnification) and
after dye adsorption (D, low magnification; DH, high magnification).
Figure 7. TEM micrographs of ROB before dye adsorption (A).
TEM micrographs of dye adsorbed on live biomass (B, low
magnification; BH, high magnification), and dye adsorbed on starved
biomass (C). Arrows indicate the location of the dye molecules.
before and after dye adsorption. The cells exposed to dye
molecules exhibit electron dense molecules mainly in the region
of cell surfaces, whereas in control cells (Figure 7A), these are
absent. In living cells (Figure 7B,BH), the dye molecules are
observed to penetrate the cell membrane and accumulate in the
cytoplasm as granules. Figure 7C shows that in starved cells dye
molecules mainly bind on the cell surface and a very small amount
is transported to the cytoplasm compared to the living cells.
Modeling. The adsorption of rhodamine B on ROB has been
studied under continuous agitating condition and hence the process
can be considered as a combination of the following steps: (a)
transport of the dye from bulk solution to the surface of the solid
Das et al.
Figure 8. Diffusion controlled kinetic model.
particulates including intraparticle diffusion and (b) binding of
the dye molecules on the active sites of the adsorbent. Initially
the surface of the biomass is free of dye molecules and when
the dye molecules reach the surface it may attach instantly to the
binding sites. Hence, the adsorption rate may be dominated by
the number of dye molecules diffused from the bulk solution to
the surfaces of adsorbent. The adsorption process following
diffusion controlled dynamics40 with time can be presented as
qt ) 2C0SxDt/π ) kdt0.5 (3)
where qt (mg/g) and C0 (mg/L) represent the amount of rhodamine
B adsorbed per unit weight of ROB at time “t” and initial
rhodamine B concentration in the bulk solution, respectively. D
is the diffusion coefficient, and S is the specific surface area of
ROB. Therefore, the plot of qt versus t0.5 would be a straight line
under a diffusion controlled transport mechanism. The results
presented in Figure 8 show a multilinearity of three steps at
higher concentration (>400 mg/L). The initial sharp portion can
be attributed to the instantaneous adsorption stage at the external
surface of the adsorbent where maximum adsorption takes place.
In the latter stage, dye adsorption data do not obey the model
in eq 3, indicating that this gradual adsorption stage is controlled
by intraparticle diffusion41 because most of the binding sites are
occupied. The third portion is the final equilibrium stage where
the rate is further slowed with scarcity of the available binding
sites and would probably transit for diffusion control to attachment
control process.
Mechanism. The complexity of the microbial structure makes
the biosorption process much more complicated. Biosorption42
may be classified as cell surface adsorption/precipitation and
intracellular accumulation depending on the location of the
adsorbate. Further, on the basis of cellular activity, this can be
divided into metabolism dependent and metabolism independent
adsorption.42 Surface adsorption is generally assisted through
ionic, chemical, and physical interaction. Scanning and transmis-
sion electron microscopic studies provide important information
regarding the possible mechanism in dye adsorption. SEM (Figure
6) micrographs of ROB before and after dye adsorption indicate
that the dye molecules adsorb on the cell surfaces. Cell walls of
(40) McKay, G.; Poots, V. J. P. J. Chem. Technol. Biotechnol. 1980, 30, 279-
292.
(41) McKay, G.; Otterburn, M. S., Sweeney, A. G. Water Res. 1980, 14,
15-20.
(42) Gadd, G. M. In Biotechnology; Rehm, H. J., Reed, G., Eds.; VCH:
Weinheim, Germany, 1988; Vol. 6b, p 401.
Adsorption of Rhodamine B on R. oryzae Biomass Langmuir, Vol. 22, No. 17, 2006 7271

Figure 9. (A) Schematic presentation of rhodamine B adsorption on the cell surface; (a) electrostatic interactions between the different
functional groups of cell surface and the dye molecule, (b) H-bonding between the hydroxyl groups of the polysaccharides and the aromatic
rings in dye molecule, and (c) hydrogen bonding between the hydroxyl groups of polysaccharides and electronegative groups in the dye.
“-S-” denotes the sugar moieties present in the envelope structure, and cell surface proteins are shown by cylindrical structures. (B)
Schematic presentation of proposed biosorprtion mechanism; (a) initial binding of dye molecule on the cell surface and (b) subsequent
transportation of dye into the cytoplasm through plasma membrane.
fungi including that of R. arrhizus contain chitin, chitosan, β-1,3- phosphate, and amino) results in a net positive charge. The FTIR
D-glucans, β-1,6-D-glucans, and mannoproteins which are spectrum of the R. oryzae biomass is also recorded to explain
abundant sources of different functional groups such as carboxyl, the binding of rhodamine B with different active groups present
amine, hydroxyl, phosphate, and sulfonate.18,23,43-44 Cell wall in the cell surface as the functional groups have a unique energy
functional groups can be identified on the basis of the cell surface absorption band.44,48 The FTIR spectrum of ROB (Supporting
charge density45,46 at different pH values and FTIR spectroscopic Information, Figure S2) shows the presence of amino, carboxyl,
analysis.44 It is observed from Figure 3b that the net negative hydroxyl, phosphate, and sulfonate groups on the biomass. The
charge density increases linearly from pH 3.5 to 7.5 and levels strong bands at the region 3300-3500 cm-1 are characteristic
off at higher pH values. This observation suggests that the cell of N-H and O-H stretching vibrations. The characteristic
surface carries phosphate groups which have pK2 (the second absorption bands are observed at 3450.2 and 3327.0 cm-1 for
dissociation constant of phosphoric acid) values within 7-8.47 amine and hydroxyl groups and at 2925.8-2852.5 cm-1 for alkyl
The negative charge between pH 3.5 and 6 could develop from chains, respectively. A distinct peak at 1745.3 cm-1 can be
carboxylate groups that have pK values within 3.5-5.0.47 The attributed to the CdO stretching band from the protonated
net positive charge at low pH (<3.5) might be due to amino carboxylic groups or ester groups, 1708.1 cm-1 for CdO of the
groups on the cell surface. The zero point charge (ZPC) is found carboxylic groups of amino acids. The amide I band is primarily
to be at pH 3.5 in this study. This means that at pH 3.5 the net a CdO stretching mode and is centered at 1643.7 cm-1; the
surface charge is zero. When the pH is higher than ZPC, there amide II band is a combination of N-H bending and C-N
is a net negative charge, which results from deprotonation of stretching and is centered near 1550 cm-1. The more complex
carboxylate and phosphate groups. When the pH is less than the amide III band is located near 1330 cm-1. Peak positions at
ZPC, protonation of all three major functional groups (carboxylic, 1541.0 and 1411.8 cm-1 can be attributed to COO- of the
carboxylate group present in the biomass. The strong band around
(43) Ruiz-Herrera, J. Fungal Cell Wall: Structure, Synthesis and Assembly; 1100-1000 cm-1 is due to the C-O bond, which is the
CRC Press: Boca Raton, FL, 1992.
(44) Naja, G.; Mustin, C.; Berthelin, J.; Volesky, B. J. Colloid Interface Sci. characteristic peak for polysaccharides. The absorption peak in
2005, 292, 537-543. the region of 1153.4-1033.8 and 1075.0-970.0 cm-1 for P-OH
(45) Douglas, H. W. J. Appl. Bacteriol. 1957, 20, 390-403.
(46) He, L. M.; Tebo, B. M. Appl. EnViron. Microbiol. 1998, 64, 1123-1129. (48) Aranvindhan, R.; Madhan, B.; Rao, J. R.; Nair, B. U.; Ramasami, T.
(47) James, R. O.; Parks, G. A. Surf. Colloid Sci. 1982, 12, 119-216. EnViron. Sci. Technol. 2004, 38, 300-306
7272 Langmuir, Vol. 22, No. 17, 2006
and P-O-C stretching, respectively, and 800-850 cm-1 for the
SdO bond indicate the presence of phosphate and sulfonate
groups in the biomass. All of these groups may interact with the
dye. Thus, the initial attachment of the dye molecules with the
cell surfaces follows a complicated pattern. The adsorption of
rhodamine B on R. oryzae biomass may be attributed to (1)
chemical interaction between dye molecules and fungal cell wall
components, (2) electrostatic interaction between the dye
molecules and the electron rich sites on the cell surface, and (3)
weak physical forces such as hydrogen bonding and van der
Waals interactions between the hydrophobic parts of the dye
molecules (e.g., the aromatic rings) and the polysaccharides of
the biomass as proposed by Blackburn.49 A schematic diagram
of dye-biomass interactions on the cell surface is presented in
Figure 9A.
After surface adsorption, dye molecules are transported into
the cytoplasm due to the scarcity of the available binding sites
on the cell surface. TEM micrographs of ROB before and after
dye adsorption indicate that dye molecules not only adsorb on
the cell surface but also accumulate in the cytoplasm. Dye
adsorption in the presence of different metabolic inhibitors
(Supporting Information, Table S3) as well as with the starved
(Supporting Information, Table S4) and dead biomass (Figure
1) indicates that energy rich compounds are required for
intracellular accumulation. Thus, transportation of the dye into
cytoplasm is associated with the active transport system of the
cell. A scheme of the possible biosorption mechanism involved
in rhodamine B adsorption by R. oryzae biomass is shown in
Figure 9B.
(49) Blackburn, R. S. EnViron. Sci. Technol. 2004, 38, 4905-4909.
Das et al.
Conclusions
Rhizopus oryzae biomass adsorbs about 90% of rhodamine B
from its aqueous solution (100 mg/L). DNP and DCCD strongly
inhibit the dye adsorption. The adsorption process depends both
on the pH and temperature of the solution. The optimum pH and
temperature of the adsorption process are found to be 7.0 and
40 °C, respectively. The adsorption isotherm follows Langmuirian
behavior within the studied dye concentration range. The
adsorption process is very fast initially and then slows down to
reach equilibrium after 5 h. In the initial stage, the adsorption
process obeys transport controlled adsorption kinetics but in the
latter stage may transit to attachment controlled adsorption
kinetics. Chemical interaction, ionic interaction, and other physical
forces may be responsible for the binding of rhodamine B with
the cell wall components of the fungal biomass. Electron
microscopic analyses indicate that the dye molecules initially
bind with the cell surfaces and then accumulate in the cytoplasm
by the active transport process of the cell.
Acknowledgment. We thank Mr. S. Dey (IICB, Kolkata) for
his cooperation during the TEM experiments.
Supporting Information Available: Calculation of the amount
of dye adsorbed and additional figures and tables concerning dye
adsorption. This material is available free of charge via the Internet at
http://pubs.acs.org.
LA0526378