Journal of Biotechnology 63 (1998) 121 – 136
Recovery of gold from solutions using Cladosporium cladosporioides biomass beads A.V. Pethkar, K.M. Paknikar * Di6ision of Microbial Sciences, Agharkar Research Institute, G.G. Agarkar Road, Pune 411 004, India Received 4 September 1997; received in revised form 27 May 1998; accepted 29 May 1998
Abstract A fungal isolate, Cladosporium cladosporioides was used for biosorption of gold from solutions. The fungal biomass was granulated by mixing it with a matrix derived from keratinous material of natural origin. The resulting biosorbent beads adsorbed 100 mg gold per gram from a solution of gold. Maximum biosorption of gold (80%) occurred under acidic pH conditions (pH 1–5). The contact time required for 80% biosorption of gold could be reduced to 20 min by pre-soaking the beads in deionized distilled water. Gold uptake by the beads was found to increase linearly as a function of metal concentration. The data could be fitted into Freundlich model of adsorption isotherms. A column packed with 3 g biosorbent beads was used for continuous adsorption of gold. The gold loading capacity obtained in the system was to the tune of 110 mg g − 1. Gold was removed from an electroplating unit effluent with 55% efficiency in batch experiment and the loading capacity was 36 mg g − 1. It was found that gold could be removed from solutions in the presence of carbonate and complexing agents like citrate, sulfite and thiosulfate albeit with less efficiency. The beads were found to biodegrade in soil in about 140 days. The process, thus, has the prospect of becoming an efficient and environmental friendly method to recover gold from aqueous solutions. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Biosorption; Cladosporium cladosporioides; Gold
1. Introduction The importance of gold is not restricted to its role as a value standard or a jeweler’s metal. As a * Corresponding author. Tel.: + 91 212 354357/+91 212 353680; fax: +91 212 351542; e-mail:
[email protected]
noble metal, it finds many industrial applications such as soldering components in high-tech electronics, electrical contacts, plating materials, wear-resistant contacts in rockets, submarines, computers, signaling devices, manufacture of printed circuit boards, etc. Gold electroplating is one of the oldest industries encompassing many walks of human life ranging from household,
0168-1656/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0168-1656(98)00078-9
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decorative and hi-fashion materials to electronics, communication systems and space applications. The commercial use of gold electroplating of various materials started in the 1840s; but real advances in the field came in the 1940s with the emergence of the electronics industry. The technical field makes use of the excellent electrical, chemical and optical properties of gold. A variety of electroplating baths has been formulated for specific applications (Goldie, 1974). The alkaline cyanide bath is commonly used in jewelry and the electronics industry. High purity alkaline gold electrolytes typically contain KAu(CN)2, 4 – 500 g l − 1; KCN, 7.5–60 g l − 1; conducting salts such as (NH4)2HPO4, K2HPO4 and sodium potassium tartarate in the range of 15 – 125 g l − 1; K2CO3, 0 – 30 g l − 1; KOH, 0 – 40 g l − 1 in addition to wetting agents. The final pH is 10.5 – 13. Other metals such as silver, nickel, zinc, cadmium, etc. are added to give color deposits. The industrial gold electrolytes contain KAu(CN)2, 4 – 20 g l − 1; KAg(CN)2, 0.1–0.4 g l − 1; KCN, 80 – 100 g l − 1; KOH, 80 g l − 1; K2CO3, 20 g l − 1; Na2S2O3, 5 – 35 g l − 1 with brightners, wetting agents, etc. The pH is less than 12. Neutral electrolytes used mainly for decorative purposes contain sodium phosphate, 28 g l − 1; copper cyanide, 7 g l − 1; ferrocyanide, 3 g l − 1; gold cyanide, 7 g l − 1; EDTA, 16 g l − 1 and copper EDTA, 8 g l − 1 (final pH 6.8 – 8.5). Industrial neutral electrolytes contain citrates to the extent of 50 – 125 g l − 1 along with the above constituents. The acid gold electrolytes (pH 3–4.5) typically contain a variety of complexing agents such as citrate, 40 – 120 g l − 1; EDTA, 1 g l − 1, tetraethylene pentamine, 20 g l − 1, etc. The chelating agents not only serve as buffers, but also help in controlled co-deposition of metals such as nickel, cobalt, indium, etc. and reduce the activity of metallic impurities. The acid electrolytes are used mainly in electronic applications. To avoid the use of toxic chemicals such as cyanide, the latest advance is the use of sodium gold sulfite electrolyte (pH\8) containing sodium sulfite, 40 – 150 g l − 1; gold sulfite, 1 – 30 g l − 1; buffering and conducting salts, 5 – 150 g l − 1 and other additives. Prior to the discharge of gold plating bath effluents (drag out solutions) in the environment,
they are given suitable chemical treatment to remove the complexing agents (such as cyanides, citrate, sulfite, etc.) and to bring down the pH towards neutrality. However, such drag out solutions may contain considerable concentrations of gold (up to 50 mg l − 1, as detected in many Indian jewelry manufacturing units by the authors). Loss of gold in the effluents generated by industries needs attention, because, although the effluents contain gold in low concentrations, the net value of gold lost is significant, if large volumes of effluents generated are taken into consideration. Although gold poisoning is not common and maximum permissible limits for the discharge of gold containing effluents are not available, the toxicity of gold to humans has been reported (Ishida and Orimo, 1994). Due to dwindling resources of gold it is necessary to recover it from waste materials such as industrial effluents. It may be mentioned that at present, the gold plating or jewelry manufacturing industry in developing countries like India operates on a small scale, usually as a business run by an individual or family. Therefore, the amount of effluent generated is not very high (1–10 l/U per week). Under these circumstances, the methods commonly used in industrialized countries, viz. adsorption on ionexchange resins and activated charcoal for treating large volumes of effluents may become economically unattractive. Clearly, there is a need for the development of suitable technologies of gold recovery from solutions, which may be selective, efficient and economical for gold recovery even at a smaller scale. Numerous micro-organisms including algae (Sakaguchi et al., 1981; Ting et al., 1995), bacteria (Charley and Bull, 1979), yeasts (Mowll and Gadd, 1983) and fungi (deRome and Gadd, 1987) are known to accumulate heavy metals actively as well as passively. The biosorption process is a passive physico–chemical interaction between the charged surface groups of micro-organisms and ions in solution, in which living as well as dead organisms can be used. There are many advantages of using biosorbent technology for metal-ion removal. Firstly, large quantities of dead biomass are available as industrial by-products and its use would not only aid in metal removal; but, could
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also help solve the problem of biomass disposal (Paknikar et al., 1993; Puranik et al., 1995; Puranik and Paknikar, 1997). Second, the dead biomass is not affected by toxic chemicals, metals and other unfavorable factors generally associated with the effluents. Third, the biosorbent can be regenerated and reused many times (Gadd, 1990). Moreover, the removal of metals by biosorption can be economically used for industrial effluents that are characterized by high-volume and lowtenor (Brierley et al., 1986a). Much work on biosorption has been focused on fungal cultures, because, fungal cell wall chitin and other polymers such as chitosan, glucans, mannans, metal binding proteins, etc. play an important role in metal binding (Gadd, 1990). Fungi can grow under conditions of acidic pH, tolerate high concentrations of toxic metals and are in general easier to grow and harvest. They are also amenable to genetic manipulations (White and Gadd, 1990). In the light of these observations studies on biosorption of gold were initiated in our laboratory. The present paper deals with the use of biosorbent beads containing biomass of a specially isolated fungal culture, Cladosporium cladosporioides and highlights the commercial importance of the process.
2. Materials and methods
2.1. Culture used, its growth and har6esting of biomass A fungal culture, Cladosporium cladosporioides was selected under an exhaustive isolation and screening program for metal adsorbing fungi. The screening tests indicated that C. cladosporioides could adsorb gold preferentially from acidic solutions. To exploit this property of the culture, it was selected for further studies. The culture was grown in 250 ml Sabouraud’s medium in a 1000 ml capacity Erlenmeyer flask. The flask was incubated at 30°C for 5 days on a rotary shaker (Gallenkamp, UK) at 120 rpm. The log phase culture (250 ml) was inoculated in 4 l Sabouraud’s medium in a 5 l fermenter (B. Braun, Germany). The pH and temperature of the medium were
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maintained at 5.79 0.1 and 30°C, respectively during growth. Sterile air was sparged into the fermenter at the rate of 0.15 l min − 1. The agitation of the medium was carried out at 100 rpm. After 5 days incubation, the medium turned greenish due to the release of a pigment. At this stage, the biomass was harvested by vacuum filtration and washed several times with deionized water to remove the traces of medium constituents.
2.2. Metal solutions Stock metal solutions (1000 mg l − 1 and 1 M) of gold, silver, cobalt, cadmium, chromium, copper, nickel and zinc were prepared by dissolving appropriate quantities of pure analytical grade metal powders or their salts in minimal quantities of nitric/hydrochloric acid and then making up the volumes to 1 l with 1% (v v − 1) nitric or hydrochloric acid. The stock solutions were diluted further with deionized glass distilled water to obtain working solutions of 100 mg l − 1 and 1 mM strength.
2.3. Biosorption of metals by Cladosporium cladosporioides biomass The powdered biomass (1 g) was contacted for 60 min with metal solutions (50 ml, 100 mg l − 1, pH 4) on an orbital shaker at 120 rpm. Metal solutions without the addition of biomass served as negative controls. The solutions were separated from the biomass by filtration and then analyzed for residual metal content on an atomic absorption spectrometer (ATI-UNICAM, UK, Model 929).
2.4. Effect of biomass pretreatment Freshly harvested biomass of C. cladosporioides (10 g wet weight) was treated with 50 ml of the following solutions for 30 min; sodium carbonate (1 M), sodium hydroxide (1 M), hydrochloric acid (1 M), urea (1 M), ammonium sulfate (1 M), Triton-X 100, dimethyl-sulfoxide (100%), ethanol (absolute) and deionized boiling water. The biomass was separated out by filtration and
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washed several times with deionized water to remove the traces of adhering chemicals. It was then dried at 60°C and powdered in a blender. The powdered biomass was used in gold sorption experiments.
cumulative biosorption (mg gold bound per gram dry weight of the biosorbent beads) was determined by contacting the biosorbent beads (2 g) several times with fresh batches of 100 ml gold solution (100 mg l − 1, pH 4) till the biosorbent was saturated with metal ions.
2.5. Granulation of C. cladosporioides biomass A polymeric matrix was obtained from waste poultry feathers by a proprietary process. The polymer by itself did not adsorb metals and served only as a binding material (Paknikar et al., 1995). The polymer was admixed with the C. cladosporioides biomass (in 1:1000 proportion) and rolled out into granules. The diameter of the granules or beads was measured by micrometry using a reflected light optical microscope. Stability of the beads was checked by studying the effect of heat, strong acids and alkalis on the structure of the beads.
2.6. Optimization of parameters for gold biosorption by C. cladosporioides biomass beads To find out the appropriate phase of growth of the culture for maximum gold uptake, 1 ml spore suspension of C. cladosporioides containing 2× 106 spores was inoculated into 100 ml Sabouraud’s medium in 500 ml Erlenmeyer flasks. The flasks were incubated at 30oC on a rotary shaker (120 rpm). Fungal biomass from each of the flasks was harvested at different time intervals, washed with deionized glass distilled water and granulated. It was then contacted with gold solution (100 mg l − 1, pH 4) for 60 min with constant stirring at 120 rpm. For the determination of optimum biosorbent concentration, gold solution (50 ml, 100 mg l − 1, pH 4) was contacted with varying amounts of biosorbent beads, ranging from 0.5 to 4% (w v − 1). For pH experiments, the biosorbent beads were pre-conditioned to the desired pH and then appropriate amount of the beads was contacted with gold solution of varying pH values. Rate of gold uptake was studied by contacting dried biosorbent beads and beads presoaked in distilled water (1 g each) with 50 ml gold solution (100 mg l − 1), for different time intervals (0–90 min). The gold loading capacity or
2.7. Effect of cations, anions and complexing agents on gold biosorption Two-metal mixtures containing equimolar concentrations of 1 mM gold and one of the metals such as cadmium, copper, chromium, zinc and nickel (1 mM final concentration) were prepared. The pH of the mixtures was adjusted to four to keep all the metals in solution. The mixtures (50 ml) were contacted with 1 g granulated biosorbent for 60 min. Solutions consisting of 50 mg l − 1 of gold and one of the following, viz. potassium carbonate (300 mg l − 1), mixture of citric acid and sodium citrate (400 mg l − 1 each), sodium sulfite (900 mg l − 1) and sodium thiosulfate (350 mg l − 1) were prepared (final pH adjusted to four). Biosorbent beads (1 g) were contacted with 50 ml of the solutions for 60 min. The beads were contacted several times in order to determine the gold loading capacity in the presence of the anions.
2.8. Adsorption isotherms Gold solutions of varying concentration (ranging from 0 to 300 mg l − 1) were used to study the effect of initial gold concentration on its adsorption. The pH of all the solutions was adjusted to four. Biosorbent beads (1 g) were contacted with 50 ml of each of the solutions for 1 h on a rotary shaker at 120 rpm. In order to obtain the sorption kinetics data, the metal uptake value (Q) was calculated using the following equation: Q= V(Ci − Cf)/1000m
(1)
where, Q is the metal uptake per unit biosorbent (mg g − 1), V the volume of metal solution (ml), Ci the initial concentration of metal in solution (mg l − 1), Cf the final concentration of metal in solution (mg l − 1) and m is the mass of biosorbent (g). The value of Q thus obtained was used to plot an
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adsorption isotherm according to the Freundlich equation: ln Q= lnk +(1/n)lnCeq
(2)
where, Ceq is the liquid phase concentration of the metal (mg l − 1), and Q the metal uptake per unit biosorbent (mg g − 1).
2.9. Breakthrough cur6es A solution containing 100 mg l − 1 gold (pH 4) was passed in upflow mode through three identical glass columns (length 5.5 cm, internal diameter 1 cm) each containing 1 g biosorbent beads. The void volume of each of the columns after packing the biosorbent was 4 ml. The solution flow rates were adjusted to 0.13, 0.2 and 0.4 ml min − 1 using a programmable peristaltic pump (Ismatec, Switzerland, model MCP 552). Effluents coming out from the columns were analyzed for residual gold content periodically. The sorption kinetics of continuously fed columns were studied by determining the mass transfer coefficients according to Adams-Bohart equation (Jansson-Charrier et al., 1995): ln (Cs/Co) =k × Co× t− k ×No(Z/Uo)
(3)
where, Cs is the concentration of gold in the column effluent (mg l − 1), Co is the initial or inlet gold concentration (mg l − 1), t is the time when effluent sample is drawn (min), No is the volumic metal uptake by the biosorbent (mg l−1), Z is the height of column (m), Uo is the linear flow rate of solution (m min − 1) and k is the mass transfer coefficient (l mg − 1 min − 1).
2.10. Uptake of gold from electroplating effluent An effluent containing gold was procured from an electroplating shop. The proximate chemical analysis of the effluent was — pH (7.5), calcium (376.7 mg l − 1), potassium (1621 mg l − 1), sodium (1775 mg l − 1), gold (46 mg l − 1), chlorides (3550 mg l − 1), cyanide (not detected), carbonate (not detected). For gold biosorption experiments, the effluent was adjusted to pH 4 (initial pH 7.5) by adding 0.1 N hydrochloric acid. Fresh batches of 50 ml
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effluent were contacted with 1 g biosorbent beads and the metal loading capacity was determined as described earlier.
2.11. Packed-bed reactor for gold reco6ery from solution The biosorbent beads (3 g) were packed in a glass column (10 cm length and 1 cm internal diameter). A solution of gold (20 mg l − 1, pH 4) was continuously passed through the column in upflow mode. The retention time of the metal solution was adjusted to 20 min. The effluent was collected after every 6 h and the fractions were analyzed for residual gold content. The gold saturated column was eluted with 0.2% alcoholic solution of sodium cyanide or 1 M thiourea. Gold was recovered from the eluent by electrowinning at a potential difference of 0.8 V and 224 mA current. Electrodes of gold and carbon were used as cathode and anode, respectively.
2.12. Biodegradation of the biosorbent beads Biosorbent beads (1 g) were kept in 100 g garden soil at a depth of 1 cm in a polypropylene container. Plastic templates were used to mark the exact location of the biosorbent beads. In all six replicate containers were kept. Beads from each of these containers were removed at an interval of 30 days, cleaned with distilled water and dried to a constant weight. The differences in the average weights of the beads before and after burying in soil were used to calculate the percentage biodegradation of the beads.
3. Results
3.1. Effect of culture age on gold biosorption Cladosporium cladosporioides was found to produce a green diffusible pigment after 5 days incubation (during late log phase). Biomass harvested before pigment production (4 days incubation) could adsorb B 30% gold. However, the
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biosorption efficiency after pigment production was 80%.
3.4. Optimization of process parameters for batch experiments
3.2. Effect of pretreatments
The amount of biosorbent required for maximum gold biosorption (95%) was 3% (w v − 1) as shown in Fig. 1. Further increase in the amount of biosorbent beads did not increase the biosorption efficiency. The optimum pH for gold biosorption was found to be in the range of pH 2–4 (Fig. 2). A shift from pH 6 to 7 significantly reduced gold uptake and very little uptake occurred at pH values over seven. It was found that during 20 min contact with gold solution, the biosorption efficiency of the dried beads was 30%, while that of the pre-soaked beads was 80% (Fig. 3). The biosorption efficiency of dried beads reached the maximum (90%) and equaled that of the presoaked beads when the contact time was increased to 90 min. The gold loading capacity of the biosorbent beads was 100 mg g − 1.
Among the different pretreatments given to the biomass, only dimethyl sulfoxide treatment for 30 min resulted in an increase in biosorption efficiency of C. cladosporioides from 80 to 92% (Table 1). However, biosorbent beads prepared from the DMSO treated biomass did not show significant increase in the gold loading capacity of the biosorbent.
3.3. Characterization of biomass beads of C. cladosporioides The fungal biosorbent beads obtained by a proprietary process had an average diameter of 2.28 mm and consisted of over 99% fungal biomass. The matrix itself had no contribution to gold binding. The beads were porous in nature because of an intricate network of fungal mycelia. When immersed in water, the beads absorbed water and swelled. The increase in diameter of the granules was 14%. It was found that the granules had good structural stability as evidenced from the resistance to autoclaving at 121°C for 30 min, without loss of gold biosorption efficiency. The beads did not disintegrate in strongly acidic or alkaline solutions.
Table 1 Effect of physical and chemical pretreatments on gold biosorption Treatment
Gold biosorption (%)
Untreated Sodium carbonate Ethanol Dimethyl sulfoxide Sodium chloride Sodium hydroxide Hydrochloric acid Ammonium sulfate Urea Triton X-100 Boiling water
80.00 67.50 70.80 92.02 62.10 64.40 69.64 53.62 57.54 75.54 64.73
3.5. Effect of cations, anions and complexing agents It was observed that the presence of other cations such as cadmium, copper, chromium, zinc and nickel did not adversely affect the gold biosorption process (Fig. 4). Biosorption of gold in the presence of cobalt and silver could not be determined because of precipitation of these metals in the mixture. On the other hand, it was found that the presence of anionic moieties in solutions considerably affected the gold biosorption. In the presence of carbonate, the gold loading capacity of the biosorbent reduced by about 50% from 100 (pure solution of tetrachloroauric acid) to 46 mg g − 1. The gold loading capacity was 22, 11, and 20 mg g − 1 in the presence of citrate, sulfite and thiosulfate, respectively.
3.6. Adsorption isotherm It is evident from Fig. 5 that gold uptake by the biomass beads is directly proportional to the concentration of metal in solution at equilibrium. Fig. 6 shows that gold uptake values could be fitted to the Freundlich isotherm model (r 2 = 0.99).
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Fig. 1. Effect of biomass concentration on gold uptake by C. cladosporioides biosorbent beads. Varying quantities of biosorbent beads (0.5 – 4% w v − 1) were contacted with 50 ml gold solution (100 mg l − 1, pH 4) for 60 min.
3.7. Breakthrough cur6es The results of gold biosorption in column trials are presented in Fig. 7, where a solution of gold was passed through biosorbent columns at different flow rates. It was observed that the gold content in the treated effluent was initially high ( \ 20 mg l − 1), irrespective of the flow rates. The metal content reduced gradually after passing ten bed volumes of gold solution. At the flow rate of 0.13 ml min − 1 a plateau was reached (12 mg l − 1) with no further reduction in residual gold concentration. It was observed that the slope of the breakthrough curve changed with the flow rate of metal solution. This clearly indicated that gold biosorption by C. cladosporioides beads was dependent upon the solution flow rate. At a flow rate of 0.13 ml min − 1, 54 bed volumes of gold solution (100 mg l − 1) could be passed through a column with gold removal efficiency of 85%; whereas, at a flow rate of 0.2 ml min − 1, ten volumes could be passed before the efficiency
dropped below 85%. It was found that the removal efficiency did not exceed 80% at a flow rate of 0.4 ml min − 1, and the efficiency reduced to less than 50% after passing 70 bed volumes of metal solution. It was possible to run the column at 75% efficiency for more than 125 bed volumes at a flow rate of 0.13 ml min − 1 and 30 bed volumes at 0.2 ml min − 1. The calculated mass transfer coefficient (k) at 0.13 ml min − 1 was 5.59× 10 − 6 l mg − 1 min − 1 while at flow rates of 0.2 and 0.4 ml min − 1 it was 2.24 ×10 − 5 and 5.5 × 10 − 5 l mg − 1 min − 1, respectively.
3.8. Gold uptake from electroplating effluent In order to test the applicability of the biosorbent preparation for industrial effluents, gold sorption studies were carried out using an electroplating effluent (containing 46 mg l − 1 gold). In batch experiments the uptake of gold by the biosorbent beads was found to be 55%. The observed gold loading capacity was 36 mg g − 1.
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Fig. 2. Effect of pH on gold biosorption by C. cladosporioides biosorbent beads (initial gold concentration, 100 mg l − 1; biosorbent concentration, 2% w v − 1). Beads conditioned to different pH were contacted with 50 ml gold solution of desired pH values for 60 min.
3.9. Packed-bed reactor
4. Discussion
When a packed-bed reactor was run on a continuous basis (Fig. 8), 20 l of a 20 mg l − 1 gold solution could be passed through the column with loading of 110 mg g − 1 gold. The metal removal efficiency was above 80%. The loaded gold could be eluted in thiourea and cyanide solutions with more than 99% efficiency. However, the beads did not adsorb gold in the second biosorption cycle after thiourea elution. With cyanide desorption method, the beads could be reused at least three times without reducing gold uptake efficiency. Metallic gold was finally recovered from the concentrated eluent by electrowinning.
The selection of C. cladosporioides as a preferentially gold and silver biosorbing culture was based on the preliminary screening experiment. Among all the metals tested such as copper, cobalt, chromium, cadmium, nickel, zinc and silver, only gold and silver were efficiently adsorbed by the fungal biomass. This was probably the manifestation of the intrinsic ability of the culture to adsorb these metals preferentially. None of the other soil isolates tested showed such preferential uptake behavior. These observations underline the importance of a culture isolation and screening program so as to get isolates with high and specific metal sorption ability. The age of culture is known to have a pronounced effect on its biosorption ability (Tsezos, 1990). In the case of C. cladosporioides, the culture produced a green pigment in the medium after 5 days incubation when the culture attained stationary phase of growth. The nature and role of this pigment in metal biosorption are not
3.10. Biodegradation of biosorbent beads It was observed that soil microorganisms actively degraded the biosorbent beads. As seen from Fig. 9, after a slow initial degradation up to 30 days of incubation, the rate of degradation increased exponentially and complete degradation of the beads occurred in about 140 days.
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Fig. 3. Rate of gold uptake by C. cladosporioides biosorbent beads (initial gold concentration, 100 mg l − 1; biosorbent concentration, 2% w v − 1). Beads were contacted with 50 ml gold solution (pH 4) for different time intervals ranging from 10 to 100 min. , Dry beads; , pre-soaked beads.
known. However, from a practical point of view, it serves as an indicator for harvesting the biomass. The cell-wall composition of the culture at this stage probably favored maximum gold biosorption. None of the other fungal isolates studied so far produced the green pigment. The role of chitin as a metal binding site in fungi has been highlighted earlier (Muzzarelli and Tanfani, 1982). In contrast, copper biosorption by Ganoderma lucidium (Muraleedharan and Venkobachar, 1990) was reported to be unrelated to the chitin content of the cell wall. In the present studies, chitin content in the cell wall of C. cladosporioides was found to be 0.1%. Our preliminary studies (data incomplete and hence not shown) on the cell wall characterization showed that the wall constituents were not significantly different from those reported for the genus Cladosporium (Bartnicki-Garcia and Lippman, 1982), with the exception of chitin. These observations indicated that in addition to cell wall components, some of the cellular components might be involved in gold uptake by C. cladosporioides
which might confer selectivity. However, an exhaustive study on the role of cell wall and cellular components using modern techniques such as Xray dispersion analysis, fourier transform infrared spectroscopy, electron spectroscopic and chemical analysis, etc. need to be carried out before drawing any conclusions on these aspects. Since the biosorption process is an electrostatic interaction between charged groups on the microbial surface and the metal ions in solution, one way of altering the surface properties of the biosorbent to increase biosorption is to subject it to different chemical and physical pretreatments (Nakajima et al., 1981; Brady et al., 1994; Ting and Teo, 1994). This results in unmasking or exposing the metal binding groups, adding new metal binding groups or modifying the existing ones. Although DMSO treatment for 30 min resulted in increased percentage biosorption efficiency in batch experiments, the gold loading capacity of the treated biomass remained unaltered. The action of DMSO, therefore, seems unlikely to be an addition of extra binding sites. It
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Fig. 4. Effect of competing ions on biosorption of gold by C. cladosporioides biosorbent beads (initial concentration of all metals including gold, 1 mM; biosorbent concentration, 2% w v − 1). Biosorbent beads were contacted with two-metal mixtures with gold as one of the components (50 ml, pH 4) for 60 min.
is likely that DMSO merely exposed the hidden metal binding groups. Since pretreating the biomass did not increase the gold loading, untreated biomass was used in the further experiments. A naturally occurring polymeric matrix derived from poultry feather keratin was used for the immobilization of C. cladosporioides biomass. The resulting biosorbent beads showed resistance to acids, alkalis, high temperatures and autoclaving. This is an important feature of the biosorbent, since, it could be useful for recovery of precious metals from industrial effluents characterized by acidic or alkaline pH. The biosorption process is actually a surface interaction with very rapid uptake of ions by microbial surfaces. The rapidity of the process makes it a good candidate for use in effluent treatment. The results of batch experiments showed that the rate of gold biosorption was increased by pre-soaking the beads in distilled water. This was reflected in the breakthrough curves, where a higher metal content was detected initially in the treated effluent. The gold concentration reduced progressively as more solution
was passed through the columns to soak the beads. This may be because of the limitation to diffusion of solution in the biosorbent. According to Jansson-Charrier et al. (1995) the adsorption of a metal ion onto a polymer particle proceeds through four steps: (a) diffusion of the metal ion from the bulk solution to the boundary layer; (b) diffusion of the solute from the external layer to the surface of the sorbent; (c) internal diffusion of the metal ions within the micro- or macro-pores, and (d) sorption at the binding site. The rate of diffusion is controlled by the stages (b) and (c) i.e. the external and intraparticular diffusion. Jansson-Charrier et al. (1995) reported that dry gelatin sorbed molybdate with higher efficiency than gelatin soaked in water. In dry, non-soaked gelatin, diffusion was not a limiting factor and that the progressive soaking of the polymer favored mass transfer. Pre-soaking the polymer resulted in the production of diffusion barriers. However, diffusion was a complex mechanism with quasi constant kinetics of metal adsorption on soaked particles. The dry gelatin exhibited a rapid initial sorption followed by a rather slow uptake. The same authors also reported that hy-
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Fig. 5. Effect of initial gold concentration (5–300 mg l − 1) on uptake by C. cladosporioides biosorbent beads (biosorbent concentration, 2% w v − 1). The beads were contacted with 50 ml of gold solution (pH 4) of varying concentrations for 60 min.
dration pre-treatment of sheepskin leather by using acetone as well as further soaking reduced the equilibrium time and increased molybdate sorption rates. The slower uptake of gold by dry C. cladosporioides biomass beads is indicative of the fact that the beads take some time to swell in the aqueous medium and expose a larger binding surface. An important aspect of wastewater treatment for the removal of metallic residues is the pH of the solution. Solution pH influences metal biosorption in two ways, viz. it determines the availability of the metal in soluble form for biosorption; and it dictates the overall surface charge of the biosorbent material, which in turn influences metal uptake behavior. Most of the metals precipitate at neutral to alkaline pH that makes them unavailable for biosorption. On the other hand, in highly acidic solutions, a large number of H + and H3O + ions may compete with metal cations for binding sites on the biosorbent (Townsley and Ross, 1986). Gold biosorption by C. cladosporioides occurred maximally under acidic conditions. In another
set of experiments (data not shown) it was observed that silver biosorption by C. cladosporioides had pH optima in the alkaline range. Thus, gold and silver biosorption processes can be made selective by altering the solution pH. The presence of other cations in solution is known to affect the biosorption of desired metals in two ways. The cations either compete for available binding sites on the biosorbent, thus inhibiting biosorption of the metal under study; or they may enhance biosorption by taking part in the formation of co-ordination compounds, complexes or nuclei for the adsorption of a particular metal (Ting and Teo, 1994). Thus, the presence of other cations in an effluent has an important bearing on the selection of an appropriate biosorbent and the designing of an effluent treatment plant. The property of selective metal uptake by C. cladosporioides was confirmed when the culture showed preferential uptake of gold in the presence of other heavy metal cations such as cadmium, copper, nickel and zinc. The cations did not alter the gold uptake efficiency of the biosorbent.
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Fig. 6. Freundlich adsorption isotherm for gold biosorption by C. cladosporioides beads (initial gold concentration, 10 – 300 mg l − 1; biosorbent concentration, 2% w v − 1). The beads were contacted with 50 ml of gold solution (pH 4) of varying concentrations for 60 min.
The different anionic constituents and complexing agents in the gold solution affected the uptake capacity of gold. The adverse effect of sodium thiosulfate was most pronounced as seen from the reduction in loading capacity from 100 to 11 mg g − 1. This effect was expected, since thiosulfate is a known gold complexing agent used for gold extraction. Citrate and sulfite are other metal complexing agents and are used in some electroplating baths. However, these chemicals along with cyanide are destroyed during the oxidation treatment procedure followed before disposal of the effluents. The use of activated charcoal as an adsorbent, is known to be a highly economical method in gold recovery. The gold loading capacity of activated charcoal used in commercial mining operations is 1.7 – 17 mg g − 1 (Jackson, 1986). Green and Potgeiter (1984) reported loading capacities of up to 6 mg g − 1 on weak-base anion exchange resins like Amberlite XAD-1. Gold loading of 2.9 mg g − 1 was reported for IRA 400 UC, a strong-base resin, whereas the weak base
resin DU A7 loaded gold to the extent of 1.95 mg g − 1 (Mehmet and Te Riele, 1984). The gold loading capacity of C. cladosporioides beads reduced to 36 mg g − 1 (from 100 mg g − 1 in case of gold solution) when an electroplating effluent was used. Although, free cyanide was not present in the effluent used in the present studies, it was possible that some of the gold might be complexed with cyanide and these complexes might be refractory to sorption. It is also possible that presence of high concentrations of alkali metals such as sodium, potassium and calcium and anions like chlorides and carbonates that do not normally interfere with metal biosorption might have adversely affected the sorption efficiency by exerting a cumulative effect. It must, however, be mentioned that the observed gold loading capacity of C. cladosporioides beads (36 mg g − 1) was much higher (: 2–20 folds) than that reported for activated charcoal and ion exchange resins. In addition, the biosorbent beads were found to be selective for gold uptake. The
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Fig. 7. Breakthrough curves for gold biosorption. Gold solution (100 mg l − 1; pH 4) was passed at different flow rates through 1 g beads which were packed in a column of length 5.5 cm and 1 cm internal diameter. () Flow rate, 0.13 ml min − 1; () flow rate, 0.2 ml min − 1; ( ) flow rate, 0.4 ml min − 1.
reported gold loading achieved by MRA granules in the AMT-BIOCLAIM process was to the tune of 390 mg g − 1 (Brierley et al., 1986b) but the gold uptake was not preferential. The effluents emanating from gold plating units and printed circuit board manufacturing units typically contain gold in the concentration range of 1 –100 mg l − 1. In this range, gold uptake by C. cladosporioides increased with the initial concentration as long as binding sites were free. Thus, the process was chemically equilibrated and saturable. The adsorption isotherm obtained in batch experiment was consistent with the Freundlich adsorption isotherm model (Freundlich, 1926). This indicated an exponential distribution of adsorption site energies, characteristic of heterogeneous surfaces, and an immobile adsorption. The dynamic removal of gold was studied using continuously fed columns filled with C. cladosporioides beads. Since biosorption depends on the period of contact between metal ions and the biosorbent surface, the flow rate of metal solution through the biosorbent column affects metal re-
moval efficiency. At higher flow rates for example, the contact phase would be reduced resulting in early breakthrough and less metal uptake. At lower flow rates large amount of mixing or axial dispersion would occur, thereby, increasing the metal uptake (Sag et al., 1995). In the present study, a similar effect of flow rates on the gold biosorption was observed. Thus, at lower flow rates, a large volume of metal solution could be passed through the column with maximum metal removal. As the metal binding sites are progressively occupied, the effluent coming out of the column would show increasing amounts of unadsorbed metal. The saturated biosorbent column could then be replaced with a new column or it could be stripped of the adsorbed metal by desorption for further use. Vincent et al. (1995) reported that an increase in the height of column increases metal biosorption because of greater time of contact and more intraparticular diffusion. In the present work, although the height of the columns was not varied, different contact times (10, 20, and 30 min) were
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Fig. 8. Schematic representation of packed-bed system for gold biosorption and recovery from solutions.
used. The calculated mass transfer coefficients (k) at these retention times were 5.5×10 − 5, 2.24 × 10 − 5, and 5.59× 10 − 6 l mg − 1 min − 1, respectively. The increase in gold biosorption by C. cladosporioides at lower flow rates could be explained on the basis of greater time of contact in the bed reactor that might make intraparticular diffusion more effective. The final recovery of adsorbed gold from the biosorbent beads could be carried out using any one or a combination of the commonly used physical and chemical processes. Solutions of cyanide, thiourea and thiosulfate could be employed for the extraction of gold so as to get a highly concentrated solution of gold. Metallic gold could then be obtained by electrolytic recovery or electrowinning. Alternatively, the gold in solution could be recovered by zinc precipitation method. However, this method generates zinc cyanide solution which might require proper treatment before disposal. It must be emphasized that the use of cyanide may not be permitted by local statutory agencies. The choice of the method, therefore, would largely depend on such considerations. Incineration of the biosorbent beads could also be a method of choice for gold recovery if the economics permit.
The work described so far conclusively points to the potential use of Cladosporium cladosporioides in gold recovery from industrial discharges. Preferential biosorption, tolerance to pH fluctuations and high metal loading capacity are the key features of this biosorbent. Good mechanical strength of the granulated product, ease of handling, high porosity, reduced risk of column blockage, biosorbent regeneration ability and cheap availability of the immobilization matrix make the biosorption process practicable at large scale. The biosorbent granules are easily bio-degradable and hence environmentfriendly.
Acknowledgements The authors thank the Director, ARI for the laboratory facilities provided. The authors are also thankful to M/s Deval Electroplaters, Pune for providing electroplating effluents during the course of these investigations. This work was carried out under a Council of Scientific and Industrial Research, New Delhi, sponsored project no. 38 (0847) 93/EMR-II.
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Fig. 9. Biodegradation of C. cladosporioides biosorbent beads. One gram of beads were kept in soil at a depth of 1 cm. Degradation of the beads was monitored as a function of the dry weight of beads measured at different time intervals.
References Bartnicki-Garcia, S., Lippman, E., 1982. Fungal cell wall composition. In: Laskin, A.I., Lechevalier, H.A. (Eds.), Handbook of Microbiology. CRC Press, Boca Raton, FL, pp. 229 – 252. Brady, D., Stoll, A.D., Starke, L., Duncan, J.R., 1994. Chemical and enzymatic extraction of heavy metal binding polymers from isolated cell-walls of Saccharomyces cere6isiae. Biotechnol. Bioeng. 44, 297–302. Brierley, J.A., Goyak, G.M., Brierley, C.L., 1986a. Considerations for commercial use of natural products for metal recovery. In: Eccles, H., Hunt, S. (Eds.), Immobilization of Ions by Biosorption. Horwood, Chichester, pp. 105–117. Brierley, J.A., Brierley, C.L., Goyak, G.M., 1986b. AMTBIOCLAIM: a new wastewater treatment and metal recovery technology. In: Lawrence, R.W., Branion, R.M.R., Ebner, H.G. (Eds.), Fundamental and Applied Biohydrometallurgy. Elsevier, pp. 291–303. Charley, R.C., Bull, A.T., 1979. Bioaccumulation of silver by a multispecies community of bacteria. Arch. Microbiol. 123, 239 – 244. deRome, L., Gadd, G.M., 1987. Copper adsorption by Rhizopus arrhizus, Cladosporium resinae, and Penicillium italicum. Appl. Microbiol. Biotechnol. 26, 84–90. Freundlich, H., 1926. Colloid and Capillary Chemistry. Methuen, London. Gadd, G.M., 1990. Fungi and yeasts for metal accumulation. In: Ehrlich, H.L., Brierley, C.L. (Eds.), Microbial Mineral Recovery. McGraw-Hill, New York, pp. 249–275.
Green, B.R., Potgeiter, A.H., 1984. Unconventional weak-base anion exchange resins, useful for the extraction of metals, especially gold. In: Naden, D., Streat, M. (Eds.), Ion Exchange Technology. Ellis Horwood, Chichester, UK, pp. 626 – 636. Goldie, W., 1974. Gold Electroplating. Electrochemical Publications, Scotland. Ishida, K., Orimo, H., 1994. Gold. In: Seiler, H.G., Sigel, A., Sigel, H. (Eds.), Handbook of Metals in Clinical and Analytical Chemistry. Marcel Dekker, New York, pp. 387 – 397. Jackson, E., 1986. Hydrometallurgical Extraction and Reclamation. Ellis Horwood, Chichester, UK. Jansson-Charrier, M., Guibal, E., Surjous, R., Le Cloirec, P., 1995. Continuous removal of uranium by biosorption onto chitosan: application to an industrial effluent. In: Jerez, C.A., Vargas, T., Toledo, H., Wiertz, J.V. (Eds.), Biohydrometallurgical Processing, vol. II. University of Chile, Santiago, Chile, pp. 267 – 276. Mehmet, A., Te Riele, W.A.M., 1984. The recovery of gold from cyanide liquors in a counter current contactor using ion exchange resin. In: Naden, D., Streat, M. (Eds.), Ion Exchange Technology. Ellis Horwood, Chichester, UK, pp. 637 – 652. Mowll, J.L., Gadd, G.M., 1983. Zinc uptake and toxicity in the yeasts Sporobolomyces roseus and Saccharomyces cere6isiae. J. Gen. Microbiol. 129, 3421 – 3425. Muraleedharan, T.R., Venkobachar, C., 1990. Mechanisms of biosorption of copper (II) by Ganoderma lucidium. Biotechnol. Bioeng. 35, 320 – 325.
136
A.V. Pethkar, K.M. Paknikar / Journal of Biotechnology 63 (1998) 121–136
Muzzarelli, R.A., Tanfani, F., 1982. The chelating ability of chitinous materials from Aspergillus niger, Streptomyces, Mucor rouxii, Phycomyces blaskeseanus and Choanephora cucurbitarum. In: Mirano, S., Tokura, S. (Eds.), Chitin and Chitosan. Japanese Society of Chitin and Chitosan, Tottori, pp. 183 – 186. Nakajima, A., Horikoshi, T., Sakaguchi, T., 1981. Studies on the accumulation of heavy metal elements in biological systems. XVII: selective accumulation of heavy metal ions by Chlorella regularis. Eur. J. Appl. Microbiol. Biotechnol. 12, 76 – 83. Paknikar, K.M., Palnitkar, U.S., Puranik, P.R., 1993. Biosorption of metals from solution by mycelial waste of Penicillium chrysogenum. In: Torma, A.C., Apel, M.L., Brierley, C.L. (Eds.), Biohydrometallurgical Technologies. The Minerals, Metals and Materials Society, Warrendale, PA, pp. 229 – 235. Paknikar, K.M., Pethkar, A.V., Vernekar, J.V., 1995. Indian Patent pending. Puranik, P.R., Chabukswar, N.S., Paknikar, K.M., 1995. Cadmium biosorption by Streptomyces pimprina waste biomass. Appl. Microbiol. Biotechnol. 43, 1118–1121. Puranik, P.R., Paknikar, K.M., 1997. Biosorption of lead and zinc from solutions using Strepto6erticillium cinnamoneum waste biomass. J. Biotechnol. 55, 113–124. Sag, Y., Nourbakhsh, N., Aksu, Z., Kutsal, T., 1995. Com-
.
parison of Ca-alginate and immobilized Z. ramigera as sorbents for copper (II) removal. Process Biochem. 30 (2), 175 – 181. Sakaguchi, T., Nakajima, A., Horikoshi, T., 1981. Studies on the accumulation of heavy metal elements in biological systems: accumulation of molybdenum by green microalgae. Eur. J. Appl. Microbiol. Biotechnol. 12, 84 – 89. Ting, Y.P., Teo, W.K., 1994. Uptake of cadmium and zinc by yeast: effects of co-metal ions and physical/chemical treatments. Bioresour. Technol. 50, 113 – 117. Ting, Y.P., Teo, W.K., Soh, C.Y., 1995. Gold uptake by Chlorella 6ulgaris. J. Appl. Phycol. 7, 97 – 100. Townsley, C.C., Ross, I.S., 1986. Copper uptake in Aspergillus niger during batch growth and in non-growing mycelial suspension. Exp. Mycol. 10, 281 – 288. Tsezos, M., 1990. Engineering aspects of metal binding by biomass. In: Ehrlich, H.L., Brierley, C.L. (Eds.), Microbial Mineral Recovery. McGraw-Hill, New York, pp. 325 – 339. Vincent, T., Guibal, E., Roussy, J., Le Cloirec, P., 1995. Flocculation, precipitation and adsorption of molybdenum ions onto biopolymers. In: Jerez, C.A., Vargas, T., Toledo, H., Wiertz, J.V. (Eds.), Biohydrometallurgical Processing, vol. II. University of Chile, Santiago, Chile, pp. 257 – 266. White, C., Gadd, G.M., 1990. Biosorption of metallic residues by fungal biomass. J. Chem. Technol. Biotechnol. 49, 331 – 343.
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