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Biosorption of silver ions by processed Aspergillus niger biomass

Biosorption of silver ions by processed Aspergillus niger biomass
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  BIOTECHNOLOGY LETTERS Volume 17 No.5 May 1995) pp.551-556 Received as revised l Oth March BIOSORPTION OF SILVER IONS BY PROCESSED SPERGILLUS NIGER BIOMASS By NASEEM AKTHAR, Md., SIVARAMA SASTRY, K and MARUTHI MOHAN, P* Department of Biochemistry, Osmania University, Hydetabad-500 007, INDIA. SUMMARY An alkali treated A. niger biomass was found to efficiently sequester silver ions from dilute as well as concentrated solutions (2.5-1000 ppm Ag+), with an ability to bind it to a level of upto 10% of dry weight. Biosorption of silver ions was not influenced by pH between 5-7. The bound Ag + could be fully desorbed by dilute HNO~ and the biosorbent regenerated by washing with Ca2+/Mg 2÷ solution. This biosorbent is unique in that the mechanism of metal ion sorption has been found to be exclusively by stoichiometric exchange with Ca 2÷ and Mg 2÷ of the biosorbent. INTRODUCTION Fungal biomass is f'mding increasing application in current biotechnology procedures for the clean up of polluted effluents and for recovery of precious metal ions from ore leachings (Gadd, 1990, 1993; Volesky, 1994; Siegel et al., 1990). The latter assumes economic importance in view of the continuing demand for such metal ions and the progressive depletion of material resources. Competitive effects exerted by calcium and magnesium ions decrease the efficacy of conventional ion exchange resins in sorption of metal ions, especially from dilute solutions. Fungal, algal and bacterial biomass (live or inactivated) have been utilized for metal ions sorption (Kumar et al., 1992; Volesky, 1987, 1990; Gadd, 1992~ Beveridge, 1989; Strandberg et al., 1981; Goddard and Bull 1989; Bedeil and Darnall 1990). Microorganisms bind metal ions by any one (or more) of several known mechanisms which include adsorption and inorganic precipitation complexation, ion exchange or active transport (Wainwright, 1992). Asperglllus niger had earlier been examined for Ag + recovery and although good mycelial accumulation had been achieved, efficient desorption of Ag + fi'om tile 551  biomass had not been possible (Wainwright, 1992). In several other studies also, reviewed by Hutchins et al., (1986) microbial biomass had been utilized for leaching ofAg + from ores by different microorganisms but, in none of these cases, had reversible sorption ofAg ÷ by biomass been feasible. In the present study, we show that the alkali washed mycelium of Neurospora crassa, Fusarium oxysporium and (particularly) Aspergillus niger provides a reusable biosorbent which can efficiently abstract silver ions even from dilute solutions. The Ag + ions have been shown to be bound by stoichiometric exchange of Ag + for Ca2 /Mg 2 of the matrix. Following stripping of silver ions, the biosorbent can be fully regenerated by treating with Ca2+/Mg 2+ solution. MATERIALS AND METHODS Neurospora crassa (FGSC# 4200) and Fusarium oxysporium (FGSC# 6607) were obtained from the Fungal Genetics Stock Centre, Kansas City, U.S.A. The Aspergillus niger strain used herein was a laboratory isolate selected for its ability to grow on agar plates containig a toxic concentration ofmetalion (10 mM Cd2+). Growth was for 72 h at 30°C with shaking at 100 rpm in a Labline Environmental Shaker in 1L flasks containing 500 mL of a basal medium described by Kumar et al., (1992). The mycelia, -Aspergillus niger grows like beaded structure with a yield of 20g fresh wt/ flask -, and both Neurospora and Fusarium with a yield of 10g fresh wt/flask) were harvested by filtration through cheese cloth, washed with distilled water and processed by treatment with 5% KOH at 100°C for 15 rain and then the biomass was washed off extensively with water to neutral pH and stored with 0.1% aqueous sodium azide at 4°C. When needed, aliquots were removed and pressed dry between folds of filter paper to determine fresh weights (5g fresh weight of the biosorbent was equal to lg dry weight). Unless otherwise specified fresh weights have been the basis for experimental procedures. To determine the Ag + binding capacities of the biosorbents, in each case, 2g lots were suspended in 25ml of silver solution (1 mg/ml) in 100 ml conical flasks and silver sorption was allowed for 60 rain with shaking at 100 rpm in a rotary shaker at 30°C. Thereafter, the mycelial biomass was separated by centrifugation and the left over silver in solution was determined by Atomic Absorption Spectrophotometry (AAS Perkin-Elmer model No. 2380) to compare Ag + binding abilities. In view of the results obtained, in further experiments the processed biomass from Aspergillus niger was invariably employed. The ability to bind Ag + from dilute solution was examined by tying the biosorbent (2g) in a cheese cloth bag, dipped into 2L of a magnetically stirred solution of 2.5 ttg Ag+/ml. Aliquots were removed at constant intervals of time (0-240 min) and Ag + remaining was determined by AAS. The feasibility of sorption of Ag ÷ from dilute as well as concentrated solutions was studied by suspending 2g biosorbent in 50 ml silver solution (either 10 ~g/ml or 1000 ~tg Ag+/ml), with constant shaking at 100 rpm, at 30°C, the unbound metal was analysed by AAS. 552  When required, the Ca 2+ and Mg z+ released into the suspension medium was also quantitated by AAS. The effect ofpH on the sorption of silver was studied by taking 2g lots of treated mycefium extensively washed with acetate buffers of desired pH (50 mM sodium acetate adjusted to pH 4-7 with acetic acid or NaOH as required) and examining their ability to bind silver under conditions similar to those indicated in Table 1. Regeneration of the biomass, after elution of the bound Ag + (after sorption from ling Ag*/mL solution) with 0.1N nitric acid and subsequent water washing, was performed in three different ways: 1) distilled water, 2) with 5% KOH or 3)with 0.1M CaC12 + 0.1M MgSO 4 for 15 rain. In each case, after treating the biomass, it was washed to neutrality with distilled water. Five cycles ofAg + binding, elution and regeneration were carried out and the sorptive capacity of the biomass was examined afresh. RESULTS AND DISCUSSION A comparison ofAg + binding capacity (Table 1) revealed that alkali processed biomass of Aspergillus niger was more efficient than that from Neurospora or Fusarium. Further, it was able to sequester Ag + efficiently from dilute solution (Fig 1). It needs to be noted that the data of Fig 1 represent the ability of treated mycelium to accumulate and remove quantitatively Ag + present in a large volume of a stirred solution even when suspended in the same (tied into a cheese cloth bag) within 3-4 h. Table 1. Biosorption of silver by fungal biosorbents. Biosorbent from Amount of Ag* bound (mg/g dry wt) Aspergillus niger Neurospora crassa Fusarium oxysporium 98.75 68.25 57.50 Biosorbents (2g fresh wt) were suspended in 25 mL solution containing 1 mg/mL silver ions. After 1 h incubation as described in the text the left over metal and bound silver released by 0.1N HNO 3 were seperately estimated by AAS. The biosorbents were dried in oven at 70oC overnight to obtain dry weights. When treated mycelium is suspended in Ag + solution of different concentration, under the conditions employed (Fig 2), near total Ag + sorption is achieved from dilute solution (10 ppm) within 60 rain. Even when the Ag + concentration is higher (1000 ppm) Ag + sorption is rapid and the unbound Ag + corresponding to that left out in solution is a consequence of saturation of the mass of mycelium available. 553  0 o ~ ..., ,.., ~ ~-~.~ ~ --, r-~, o o o m~ ~0 0 0 0 g~ 0 | o o ........ , "Z " ~ ~x~'~\\~\\\x o : ~ ~ ~ o o ~ ~ ~ ° / f . = ......................... i o ~ £" _~" o o 554  Studies (data not shown) on the effect ofpH on Ag + abstraction showed that this was near maximal between pH 5-7, being only 5% more at pH 7 (than at pH 6); it decreased below pH 6, falling to about 30% of maximal at pH 4. The binding ofAg + as shown by Table 2 was attended by a stoichiometric release of Ca 2+ and Mg 2+ ions together (the sum of the two released being equal to Ag + bound in molar terms, as expected on the basis of the univalency of the Ag + ion). This equivalence established the mechanism ofAg + sorption as being quantitatively due to exchange with (Ca 2+ + Mg 2+) ions of the biosorbent. Table 2. Silver binding and release of Ca 2÷ and Mg 2+ by A niger biosorbent. ttmole of Ag + ~tmoles released Conc./25 mL Total Ag + bound Ca 2+ Mg 2+ (Ca 2+ + Mg 2+) 30 29.9 10.6 6.6 17.2 53 50.9 16.0 12.8 28.8 100 76.7 20.9 16.4 37.3 200 158.7 43.4 37.0 80.4 Incubation was carried out as described in the legend of Table 1 and the left over Ag ÷ and the release of Ca 2+ and Mg 2+ in the solution was analysed by AAS. This is further borne out by two other features. Firstly, as indicated by the data of Fig 3, regeneration of the biosorbent is total and reversible, as expected since Ca2++Mg 2+ solution(s) are best used for regeneration (in the sense that sorptive ability is retained over at least five cycles of use-regeneration). Other regenerative procedures are significantly less efficient. Secondly, the metal (Ag +) binding ability of the biosorbent is reduced by nearly 80% on modification ofcarboxyl groups by treatment with a water soluble carbodiimide and ethylene diamine (data not presented) and the importance ofcarboxyl groups in the exchange mechanism was clearly indicated by the altered IR spectra following carboxyl group modification. In Bacillus subtilis (Beveridge and Murray 1980) cellwall carboxyl groups have been implicated as major sites for metal deposition and in some seaweeds the same groups have been suggested as being possibly the main metal sequestering sites (Majidi et al., 1990), though in neither case has metal binding been exclusively assigned to carboxylic groups. Heavy metal biosortion by inactive biomass from certain organisms Rhi:opus arrhizus, Mucor miehei and Penicillium chrysogenum) had been "improved" by calcium saturation ofbiomass (Fourest et al., 1994). However, one difference herein is that sorption of Ni 2+, Zn 2+, Cd 2+ and Pb 2÷ in these instances is not by exchange solely 555
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