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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 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:
[email protected]. 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, 125132. (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.
biomass2-3,6,11-14 to adsorb or degrade dyes present in wastewater. 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 ingredients 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 biochemicals 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, Saccharomyces 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 potatodextrose (20% potato extract and 2% dextrose) and yeast extractpeptone-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 extractpeptone-dextrose broth, and deproteinised whey medium supplemented 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, 381387.
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 concentrations 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.
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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.
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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 altered within pH 3.0-11.0. It appears from Figure 3b that the pH value of the solution does not appreciably affect the adsorption capacity. Zeta potentials have been measured to analyze the surface charge properties of ROB at different pH values to
Figure 3. Dye adsorption by ROB under different conditions. Effect of (a) harvesting period, (b) pH, and (c) temperature. Symbols: (b) dye adsorbed, (O) biomass production, and (0) zeta potential. Data represent an average of four independent experiments ( SD shown 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, 263274. (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 corresponding 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,
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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 temperature. 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
aL Ce 1 ) + Ce qe KL KL
(1)
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 characteristics of Langmuir equilibrium and a high degree of irreversibility34 with a correlation coefficient of 0.999. The Langmuir constant has been calculated (Supporting Information, Table S2). The essential features of the Langmuir isotherm can (28) Ju, Y.-H.; Chen, T.-C.; Liu, J. C. Colloids Surf. B 1997, 9, 187-196. (29) Aksu, Z.; Tezer, S. Process Biochem. 2000, 36, 431-439. (30) Mogollon, L.; Rodriguez, R.; Larrota, W.; Ramirez, N.; Torres, R. Appl. Biochem. Biotechnol. 1998, 70-72. 593-601. (31) Kuyucak, N.; Volesky, B. Biotechnol. Bioeng. 1989, 33, 823-831. (32) Langmuir, I. J. Am. Chem. Soc. 1916, 38, 2221-2295. (33) Glastone, S. Textbook of physical chemistry, 2nd ed.; MacMillan Publishing Co: New York, 1962; p 1196. (34) McKay, G.; Blair, H. S.; Gardner, J. R. J. Appl. Polym. Sci. 1982, 27, 3043-3057.
Figure 4. Equilibrium adsorption isotherm of rhodamine B adsorption (a); adsorption isotherm following Langmuir model (b); adsorption isotherm following Freundlich model (c). Data represent an average of four independent experiments ( SD shown by error bar.
be expressed in terms of a dimensionless constant separation factor or equilibrium parameter, RL which is defined as34
RL )
1 1 + aLC0
where aL is the Langmuir constant as described above and C0
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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 n
(2)
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.
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 phosphorylation 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-ptrifluromethoxyphenyl-hydrazone. Incubation of ROB in the presence of sucrose or peptone increases dye adsorption by 1.3fold. 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 under identical conditions. This is due to less intracellular 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 represents 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.
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Figure 8. Diffusion controlled kinetic model.
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
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 transmission 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, 279292. (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.
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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-1,3D-glucans, β-1,6-D-glucans, and mannoproteins which are abundant sources of different functional groups such as carboxyl, amine, hydroxyl, phosphate, and sulfonate.18,23,43-44 Cell wall functional groups can be identified on the basis of the cell surface charge density45,46 at different pH values and FTIR spectroscopic analysis.44 It is observed from Figure 3b that the net negative charge density increases linearly from pH 3.5 to 7.5 and levels off at higher pH values. This observation suggests that the cell surface carries phosphate groups which have pK2 (the second dissociation constant of phosphoric acid) values within 7-8.47 The negative charge between pH 3.5 and 6 could develop from carboxylate groups that have pK values within 3.5-5.0.47 The net positive charge at low pH (
