Environ. Sci. Technol. 2007, 41, 8281–8287
A Study on the Adsorption Mechanism of Mercury on Aspergillus versicolor Biomass SUJOY K. DAS,† AKHIL R. DAS,‡ AND A R U N K . G U H A * ,† 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 April 06, 2007. Revised manuscript received September 12, 2007. Accepted September 25, 2007.
The adsorption behavior of mercury on Aspergillus versicolor biomass (AVB) has been investigated in aqueous solution to understand the physicochemical process involved and to explore the potentiality of AVB in pollution control management. This biomass has been successfully used for reducing the mercury concentration level in the effluent of chloralkali and battery industries to a permissible limit. The results establish that 75.6 mg of mercury is adsorbed per gram of biomass. The adsorption process is found to be a function of pH of the solution, with the optimum range being pH 5.0–6.0. The process obeys the Langmuir-Freundlich isotherm model. Scanning electron microscopic analysis demonstrates a conspicuous surface morphology change of the mercury-adsorbed biomass. A nearly uniform distribution of metal ions on the mycelial surface excepting a few aggregation points is revealed by X-ray elemental mapping profiles. The results of zeta potential measurement, Fourier transform infrared (FTIR) spectroscopy, and blocking of the functional groups by chemical modification reflect the binding of mercury on the biomass occurs through electrostatic and complexation reactions. The accumulation of mercury on the cell wall associated with negligible diffusion and or transportation into cytoplasm finds support from the results of adsorption kinetics and transmission electron micrographs. Mercury adsorption on biomass also leads to elongation of cells and cytoplasmic aggregation of spheroplast/protoplasts, indicating that the cell wall acts as a permeation barrier against this toxic metal.
Introduction Mercury is considered as one of the most hazardous of the different heavy metal elements and is included on the list of priority pollutants (1) prepared by the U.S. Environmental Protection Agency. Well-documented cases of severe mercury poisoning have been reported from Japan (Minamata Bay, 1956) and Iraq (1971). The important wastewater sources of this metal include chloralkali plants and battery industries, etc. (2). The removal of mercury employing conventional methodologies such as ion exchange, chemical precipitation, or * 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. 10.1021/es070814g CCC: $37.00
Published on Web 11/08/2007
2007 American Chemical Society
reverse osmosis (3, 4) suffer from limitations like high operating cost, incomplete precipitation, and sludge generation, etc. Biosorption, (5, 6) an emerging technology, is receiving increasing attention for the removal and recovery of heavy metals from contaminated effluents. The process is based on the ability of certain biological materials to adsorb organic or inorganic substances from their solution. Different types of adsorbents (7–11) such as bark, carbon aerogel, chitin, lignin, peat, and seaweeds, etc., have been used for the removal of this toxic metal. However, lack of affinity and specificity of these materials toward mercury result in low removal, requiring a long equilibrium time. Lately, efforts have been directed toward the development of specific biosorbents for mercury. Since the performance of any biosorbent depends on the characteristics of biomass and the microenvironment of the target metal-solution, search for new biosorbents is always a continuous process. In this respect research focus has now converged to microbial biomass, (12–15) because cell walls of these types of biosorbents contain polysaccharides and proteins with different functional groups such as amine, carboxyl, hydroxyl, sulfates, and phosphates responsible for interaction with metal ions. The uptake of heavy metals by microbial biomass is sometimes classified into three different categories: (16, 17) (1) cell surface binding, (2) intracellular accumulation, and (3) extracellular accumulation. Because of metabolism independence, the cell surface binding can occur in either living or inactivated organisms, whereas the intracellular and extracellular accumulation occurs only in a living organism. The initial surface binding is attributed to physical adsorption, ion-exchange, complexation, precipitation, and crystallization within the multilaminate, microfibrillar cell wall structure. To enhance the applicability of biosorption in wastewater treatment, it is important to understand the mechanism involved in the process. This article deals with the use of fungal biomass of Aspergillus versicilor for the removal of mercury from aqueous solution. The role of different physicochemical parameters related to the adsorption of mercury from its aqueous solution by A. versicolor through batch method is described. The mechanism and binding sites involved in the adsorption of mercury on A. versicolor biomass (AVB) have also been reported at molecular level by Fourier transform infrared spectroscopy (FTIR), and scanning and transmission electron microscopy equipped with energy dispersive X-ray analysis.
Materials and Methods Chemicals. Dehydrated microbiological media and ingredients were procured from Himedia, India. Cellulase (EC 3.2.1.4, 1.92 units/mg solid) and Chitinase (EC 3.2.1.14, 78 units/g solid) were obtained from Sigma, USA. All the other reagents were of analytical reagent grade and procured from Merck, Germany. Metal Solution and Analysis. Stock solution of mercury (500 mg/L) was prepared by dissolving mercuric chloride (HgCl2) in double distilled water and diluted to get the desired concentration. The concentration of mercury was measured by a cold vapor atomic absorption spectrometer (Varian Spectra AA 55). Adsorbent Preparation. Aspergillus versicolor used in this study was maintained and cultivated in potato dextrose (20% potato extract and 2% dextrose) slant and broth, respectively. Preparation of A. versicolor biomass is described in the Supporting Information. Preparation of Fungal Spheroplasts. Spheroplasts of A. versicolor at different degrees were prepared following the VOL. 41, NO. 24, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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protocol of White et al. (18) as described in the Supporting Information. Batch Experiment. To study the effects of pH, adsorption kinetics, and equilibrium adsorption isotherm on the mercury adsorption process, lyophilized A. versicolor biomass (4 g/L) was added to 25 mL of mercury solution taken in different 100-mL Erlenmeyer flasks and incubated with shaking (130 rpm) at 30 °C (room temperature) for 24 h unless stated otherwise. The biomass was separated by centrifugation (10,000 rpm for 10 min) at the end of incubation, and the concentration of mercury in the supernatant was measured spectrometrically. The optimum pH for mercury adsorption was determined by suspending A. versicolor biomass (AVB) in 25 mL of citrate-phosphate buffer (50 mM, pH 2.0–8.0) containing 20 and 50 mg/L mercury taken in 100-mL Erlenmeyer flasks. The adsorption kinetics of mercury by AVB at pH 6.0 was followed at regular intervals of time up to 24 h. Each data point was obtained from an individual flask and, therefore, no correction was necessary due to withdrawal of sampling volume. Similarly, the adsorption experiments with spheroplasts of AVB were carried out at pH 6.0. The equilibrium adsorption isotherm experiment was conducted by varying the concentrations of mercury from 5 to 500 mg/L. For recyclability testing, mercury was eluted from the adsorbed biomass with 0.1 M HCl, washed with deionized water, and used again for adsorption study. The amount of mercury adsorbed by the biomass was calculated using the mass balance equation (see Supporting Information). Chemical Modification. A detailed description for the modification of functional groups of AVB is provided in the Supporting Information. Surface Area Measurement. The specific surface area of the lyophilized AVB was determined by a BET surface area analyzer (Quantachrome Instruments, Autosorb-1-C). The dried biomass was first degassed by evacuation for 2 h at 80 °C, and the surface area was then determined by N2 sorption method. Adsorbent Characterization. The experimental details of the zeta potential measurements, FTIR analysis, electron micrographs, and energy dispersive X-ray analysis are described in the Supporting Information. Adsorption of Mercury from Industrial Effluents. The removal of mercury by A. versolor biomass was also tested in the case of an actual system. The effluents of chloralkali and battery industries were collected from the Northern region of Kolkata, India and the concentration of mercury along with other ions present in the effluents is summarized in Table S1 in the Supporting Information. The concentration of different ions was determined following standard methods. The removal of mercury was carried out as described earlier with 25 mL of effluent after adjusting its pH to 6.0.
Result and Discussion Mercury Adsorption Experiment. The removal of mercury from aqueous solution was tested by adding AVB (4 g/L) to 25 mL of mercury (20 mg/L) solution (pH 6.0) and it was found that more than 95% of mercury was eliminated from aqueous solution. The reduction of mercuric ions (Hg2+) to its volatile state (Hg0) has been well studied in bacterial systems, (19) but to our knowledge no such report is available in fungal systems. We have, therefore, attempted to estimate the mercury volatilized by AVB. However, we could not detect the presence of any volatilized mercury thereby ruling out the removal of mercury through volatilization process. Effect of pH. The interaction between the metal ions and the functional groups of the biomass depends on the nature of the adsorbent as well as on the solution chemistry of the adsorbate, which in turn depends on the pH of the solution (20) considerably influencing metal speciation, sequestration, 8282
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and/ or mobility. (21–23) The surface charge of the AVB determined by measuring its zeta potential at different pH values shows that zeta potential decreased from +15.1 to -35.5 mV corresponding to increase in pH from 2.0 to 8.0, with the zero point charge being noted at pH 3.5. This clearly indicates that at low pH values, cell surface being positively charged would not be favorable for the attachment of positively charged mercuric ions due to Coloumbic repulsion. With increasing pH values, the cell surface becomes more negatively charged, favorable for adsorption. It is observed that the adsorption of mercury increases with increasing values of solution pH, reaching an optimum value at pH 5.0–6.0 (Figure 1A). The observation is similar to that reported (24) for other hydrolyzable metal cations, whereby adsorption increases to its maximum value over a certain pH range, giving rise to a sharp adsorption edge. This high adsorption is believed to be associated with the formation of positively charged metal-hydroxy species, having strong affinity for the surface functional groups. It is well established from speciation studies that mercury exists as Hg2+ in a solution of pH value 6.0, the adsorption of mercury decreases as Hg(OH)2 becomes the dominant species in solution. Vieira et al. (9) and Bae et al. (20) also reported the maximum adsorption of mercury at pH 6.0 and decreases thereafter due to the formation of Hg(OH)2. Desorption of mercury with 0.1 M HCl shows that ∼92% can be eluted from the loaded biomass, and the regenerated biomass can be used for the next cycle of adsorption experiment. The adsorption-desorption experiments were carried out for up to five repetitive cycles when the regeneration and recovery capacity of the biomass exhibited considerable decrease beyond the fourth cycle probably due to lysis of the cells (data not shown). The regeneration and reusability efficiency make the biomass valuable for practical purposes. Kinetics Study. Kinetics study provides an insight into the rate as well as mechanism of the adsorption process. The batch experiments have been carried out to determine the mercury adsorption rate by AVB. It is observed from the Figure 1B that contact time significantly affects the mercury adsorption. The rate of adsorption is very fast initially; about 75% of total mercury is removed within a few minutes and tapers off thereafter as equilibrium is approached. The initial high rate is due to the abundance of free binding sites, which with time become saturated resulting in decreased adsorption rate. The relatively rapid uptake indicates that the process occurs mainly through the phenomenon of surface binding.
FIGURE 1. Adsorption of mercury on A. versicolor biomass. (A) Effect of pH on mercury adsorption at 30 °C with metal ion concentrations 20 and 50 mg/L. (B) Adsorption kinetics of mercury at 30 °C with metal ion concentrations of 20 and 50 mg/L. (C) Adsorption isotherm of mercury on the biomass with different metal ion concentrations, (inset) best fitting Langmuir-Freundlich isotherm model. Data represent an average of four independent experiments ( SD shown by error bar: -O- 20 mg/L mercury, -•- 50 mg/L mercury, -*- zeta potential. This has significant practical importance requiring smaller reactor volumes ensuring high efficiency and economy. Adsorption Isotherm. Efficiency of an adsorbent is ascertained by its capacity to adsorb a particular adsorbate. The adsorption capacity of AVB toward mercury was determined by plotting the amount of mercury adsorbed by the AVB (qe) against equilibrium concentration of mercury in solution (Ce), as shown in Figure 1C. The adsorption capacity of AVB toward mercury (75.6 mg/g) is found to be
much higher in comparison with most other adsorbents such as 35 mg/g by carbon aerogel within the pH range 5.0–6.0, (8) 25.3 mg/g by chitosan at pH 6.0, (9) 9.32 mg/g by rice husk ash at pH 6.0, (10) 22 mg/g by Lactuca sativa and 8.6 mg/g by Zae mays at pH 5.0, (11) and 61 mg/g by P. chrysosporium at pH 7.0. (27) The adsorption of metal ions from its aqueous solution is generally governed by surface chemistry and also on surface area of the adsorbent. BET surface area of AVB was found to be 5.1 m2/g as against VOL. 41, NO. 24, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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200–3000 m2/g for most other adsorbents. (8, 28, 29) It is interesting to note that one gram of carbon aerogel (8) having 700 m2 BET surface area can adsorb 35 mg mercury. Therefore, we may surmise that besides BET surface area, other properties such as functional groups present on the biomass interacting with mercuric ions may play an important role in the adsorption process. Moreover, mercury and biomass are soft acid and soft base, respectively, (30) hence they will prefer to coordinate as explained by the Pearson rule. (31) To understand the adsorption process, it is necessary to correlate the equilibrium adsorption data to different isotherm models such as Langmuir, (32) Freundlich, (33) and the Langmuir-Freundlich dual model. (34) The experimental results also show that the overall shape of the isotherm displays the characteristics of the LangmuirFreundlich isotherm model. At low metal ion concentration isotherm correlation is closest to a Langmuir type; however, at higher concentrations it is certainly more typical of Freundlich isotherm. The isotherm model graphs presented in Figure 1C show that mercury isotherm data exhibit the best fit to the Langmuir-Freundlich (inset Figure 1C) model, because of higher linear regression coefficient, r 2 (0.999), and the lowest Chi-square, χ2 (4.16) values with 10 degrees of freedom (φ) (Table S2 in the Supporting Information) correspond to the significance level between 90% and 95% according to the Fischer and Yates chart. (35). X-ray elemental mapping on mercury adsorption (Figure S1A Supporting Information) depicts the distribution pattern of mercury over the surface of AVB, on which mercury is nearly uniformly distributed over the entire surface area except some points (marked area in Figure S1A Supporting Information) where increased accumulation is also noticed. The distribution pattern of the images of carbon, nitrogen, and oxygen on the adsorbent surface is found to be relatively more homogeneous compared with that of mercury. Adsorption Mechanism. The description of metal uptake by microorganism in addition to adsorption isotherm (13, 36) should also include processes such as precipitation, diffusion, and/or transportation into the cytoplasm. (37, 38) To investigate the characteristics of the adsorption process including the mechanism involved, we studied the scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDXA) profile. The post-adsorbed biomass (Figure S1C Supporting Information) is conspicuously different from that of the pristine one (Figure S1B Supporting Information). Figure S1D in the Supporting Information demonstrates the high-resolution image of the mercury nanostructures formed on the biomass surface. EDXA spectra taken in spot profile mode record the signals of carbon, nitrogen, and oxygen (Figure S1F Supporting Information) likely to be present in polysaccharides and proteins on the cell wall of the biomass. An additional signal of mercury observed in EDXA profile (Figure S1E Supporting Information) indicates its presence due to adsorption on the AVB biomass. The adsorption of heavy metals on microorganisms is considered to be a two-phase process: (16) (i) initial rapid phase of metabolism-independent binding on the cell wall followed by (ii) relatively slower energy-dependent active uptake or intracellular accumulation. Transmission electron microscopic (TEM) studies provide information regarding the metal binding location with reference to the individual cell and thereby help obtain a better understanding of the adsorption phenomenon. The micrographs of the pristine biomass along with the mercury-adsorbed whole cell were taken to identify the location of mercury in the cell. TEM images revealed the electron-dense region/layer throughout the cell wall only, with no intracellular accumulation (Figure 2, B and C). Figure 2A depicts the control cells free of metal accumulation. EDXA spectra demonstrate the peaks due to mercury in the metal adsorbed cells (Figure 2H) compared 8284
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FIGURE 2. Transmission electron micrographs of thin section of A. versicolor: (A) control cell; (B) and (C) indicate accumulation of mercury on the cell surface using mercury concentration 50 and 500 mg/L, respectively. (D) phase contrast micrograph of A. versicolor protoplast. Thin section of (E) spheroplast and (F) protoplast after adsorption of mercury from a solution containing 50 mg/L mercury. Spheroplast and protoplast were generated as described in the text. EDXA spectrum of (G) pristine biomass, (H) mercury-adsorbed biomass and, (I) mercury-adsorbed protoplast and spheroplast. Arrows indicate the location of mercury. with the control ones (Figure 2G), however, no such peak of mercury was detected when the spectra were recorded on cytosolic region of the metal-adsorbed cell, thereby confirming the accumulation of mercury species only on the cell wall. The peaks of carbon, nitrogen, and oxygen originated from the biomass, while that of mercury was due to adsorption on the cell surface. Microscopic copper grid used to support the specimen was responsible for the enhanced copper peak in the spectra. It is well-known that A. versicolor is a non-sulfide-producing strain; (39) therefore, precipitation of mercury as mercuric sulfide is ruled out. The absence of S peak in the EDXA spectrum on adsorption (Figure S1E Supporting Information and Figure 2, H and I) also supports our proposition. Thus, we may conclude that the cell wall components of the biomass have the major binding sites of mercury, while diffusion and/or transportation into cytoplasm as well as surface precipitation is not significant. This indicates that the phenomenon occurs only through the surface binding process. Extracellular association of mercury on the cell surface (Figure 2, B and C) of AVB as revealed from the micrographs (high contrast area) is probably due to chemical interactions involving electrostatic/or ion exchange, and complexation. It is found from the micrograph of the whole cell (Figure 2C), that mercury is adsorbed homogeneously on the cell surface with a few sites of multilayer formation. Aspergillus versicolor biomass was incubated with cellulase and chitinase (cell wall digestive enzymes) for 1 and 3 h to obtain spheroplasts containing different amounts of residual cell wall. It was noted that spheroplasts obtained after 1 and 3 h incubation with enzymes adsorbed 50% and 30% mercury, respectively, compared to that of whole cells. TEM images show electron dense layer throughout the cell wall of the whole cell (Figure 2, B-C) and in the residual wall portion of spheroplast (Figure 2, E and F) following adsorption of mercury. TEM and EDXA studies (Figure 2, H and I) also corroborate the low mercury adsorption by spheroplast compared to that of the whole cell. These indicate that the cell wall of the fungus is mainly responsible for binding of mercuric ions since the cell wall component in spheroplast is less in comparison with that of the whole cell. However, it is interesting to note that small amounts of intracellular
mercury accumulation in the spheroplast (enzyme digested AVB) also occurred (Figure 2, E and F) due to the removal of cell wall. When the cell wall barrier is removed, as in spheroplast, a small amount of mercury enters into the cytoplasm through passive diffusion as noted. The micrographs also exhibit that the adsorption of mercury on AVB induces significant structural alterations of the cell, with elongation of cells (Figure 2C) and cytoplasmic aggregation (Figure 2, E and F) of spheroplast with respect to the control cell. These observations indicate that mercury is localized on the cell wall acting as a permeation barrier for this toxic metal. Thus we may suggest that the cell wall of Aspergillus versicolor acts as a defense against mercury toxicity. The morphological changes of bacterial cell under stress condition has been reported earlier, (40, 41) but to date we are not aware of any report regarding the fungal cell elongation by mercury. Functional groups present on the cell surface can be identified on the basis of the surface charge density (42, 43) at different pH values and Fourier transform infrared spectroscopic analysis. It is observed from Figure 1A that the net negative charge density increases linearly from pH 3.5 to 8.0. The negative charge between pH 3.5 and 5.0 could develop due to the carboxylate group with pK values in the range of 3.5-5.0, whereas phosphate and hydroxyl groups are responsible for negative charges at higher pH values (5.0–8.0). The net positive charge at low pH (
