Biotechnology Letters Voi ii No 4 Received as revised March 7

275-280

(1989)

SELECTIVE ACCUMULATION OF SILVER BY FUNGI

Pighi L, Pfunpel T', Schi-ner F Institut fiir Mikrobiologie der Universifiit Iunsbruek TechnlkerstraBe 25, A-6020 Inn.~bruek,Austria

SUMMARY: A Phoma sp., tolerating 1 retool/1 Ag +, and 32 other fungi from a culture collection (not selected for their metal tolerance), were tested for their ability to accumulate silver, cadmium, copper, nickel and lead ions from aqueous solutions. Silver was accumulated selectively. Bivalent ions Cu, Cd, Ni and Pb were partially released after about 50 rnln~ whereas the monovalent Ag ion remained bound. The selectivity of the Phorna sp. for silver developed in the late linear growth phase and might be connected with the occurenee of a slimy exopolymer. The mean accumulation of the five heavy element ions by 32 strains of fungi could not be correlated with the ionic radii of the ions.

INTRODUCTION:

Two fundamentally different mechanisms are responsible for the accumulation of heavy element ions by microorganisms: firstly intracellular uptake which can be active (different transport systems) and/or passive (diffusion) and secondly adsorption to the outer structures of the cell (cell wall, capsule, slime) ( Kelly et al., 1979). Both mechanisms were detected during the accumulation of silver by fungi. Byrne et al (1979) found an intracellular accumulation of silver in several mushrooms, mainly Agaricaceae, up to a maximum of 133 I~g/g dry weight. For other heavy elements similiar accumulation values are mentioned (Byrne, et al., 1979; Gadd and White, 1984; Mohan, et al., 1984; Mowll and Gadd, 1983; Tyler, 1982):e.g. Cu: 0.5 - 240 I~g/gdry weight, Cd: 1 - 140 l~g/gdry weight, Ni: 100 - 2500 I~g/gdry weight, Pb: 0.5 - 6.4 l~g/gdry weight.

The often very selective intracellular accumulation is linked with physiological processes and the microorganisms therefore have to tolerate heavy metal solutions. It is an additional disadvantage that bound metal cannot be removed from the cell without its destruction. With regard to possible technical utilization, the more efficient extracellular adsorption (shorter time, higher capacity, repeated use of biomass) is more interesting. Rhizopus arrhizus biomass was able to accumulate a maximum of 50 mg Ag +/g dry wt. (Tobin et al., 1984); the authors found a linear correlation between the accumulation capacity and the ionic radius of 10 of the 15 tested elements, only Cr(m) and the alkaline earth metals deviated markedly from the calculated regression line. Under special conditions erystallisatioa can enhance accumulation capacity so that the metal content in dry weight amounts to 50% (Kuyucak and Volesky, 1988). In examin;ng the effects of external factors on silver accumulation

275

by Phoma sp. Piimpel and Sehlnner (1986) found a maximal capacity of about 20 mg/g dry wt. Between 4* and 80"C the process is nearly independent of temperature; as to the optimum pH, a slight preference for the neutral range was observed. The present study should give an insight into the accumulation pattern of imperfect fungi; the primary purpose was to determine how fungi react with a mixture of heavy metals, and special attention was paid to silver. MATERIALS AND METHODS:

Org~ni.~ms and culture conditions: The following microorganisms were used: - strain PT 35, Phoma sp., isolated by Piimpel and Schlnner (1986) 32 straln.~ of soil fungi, selected at random from the culture coUeetion of the Institute for Microbiology at Iunsbruek (Tab. 1). -

From stock cultures grown on oatmeal agar, Czapek Dox broth (free of ehioride) was inoculated and then incubated at 25"C. After 7 days the mycelium was separated by filtration and repeatedly washed with distilled water. 9 g of the wet biomass were used to determine the percentage of dry weight while the rest was available for the accumulation tests. Accumulation capacity and accumulation selectivity test: In order to inhibit complexation, unbuffered solutions of the following metal salts were used: AgNO3, Cd(NO3)2, Ni(NO3)2, CuSO4, PbSO4, all at 100 i~mol/1. Although all metals were accumulated after about 3h the added biomass (4 g wet weight) remained in the metal solution (300 ml, 25~ shaken) for a maximum of 12h. Fungus and solution were then separated by centrifugation (5 mlnj 5000g) and the accumulated amount of the specific metal was determined by measuring the concentration in the supernatant by flame atomie absorption spectrometry (Perkin Elmer 2380). A digestion of the washed fungal pellet by means of HNO 3 and heat showed the same result and that is why the easier and more rapid method was preferred.

RESULTS AND DISCUSSION: The preliminary tests of the Phorna strain P'I" 35 (Piimpel and Schlnner, 1986) concentrated on the time pattern of the accumulation with regard to five elements (silver, cadmium, copper, nickel, lead). Two substantial differences were established between the accumulation of silver and that of the other elements. Firstly, the eapaeity for silver was markedly higher (not only in g/g but also in tool/g) and secondly, the time pattern was different: silver accumulation can be expressed by the exponential equation Y = a + b * exp (-t / c) ;a represents the equilibrium concentration, b the initial silver concentration, c the time constant and t the elapsed time. For the other elements a maximum absorption is reached after about 20 to 30 minutes followed by a slow partial release (Fig 1). Since the mycelinm survived the tests we suggest this was due to a physiological process. Galun et a1.(1987) found it advantageous to pretreat Penicillium biomass with 5% formaldehyde (and thereby kill the fungus) to inhibit the loss of bound nickel. Our hypothesis is that the stress increased fermentation with liberation of organic acids (Hoffmann, et al., 1976), whose eomplexation effects are to be taken into consideration.

276

Comparing the accumulation capacity of the tested fimgus PT 35 for individual metals (100 tzmolA) and for a mixture of them (each 100 l~molA) the fotlow~g was observed: in the multielement solution'the accumulation capacity was decreased for silver and cadmium but increased for copper and lead. Probably copper and nickel, strong complexing agents (Irving Williams series) and even silver modify the conformation of cell wall polymers and thereby expose or mask binding sites. (Fig. 2). The total m o u n t of accumulated ions was 689 Izmol/g dry wt with individual metals and 777 ~mol/g dry wt with the mixture. This indicates that there might be selective binding sites for silver, which are hardly used by the other tested ions. As shown in Fig 3 the referred selectivity is decisively dependent on the age of the culture. An almost equally high accumulation capacity (about 25 l~mollg dry wt. for all five elements) was recorded in mycelittm which was stored for several weeks. Only after the fresh fungal biomass had been cultured for some days in nutrient broth did it develop selectivity for silver which reached a maximum on the 7~ day, when the culture was in the late linear growth phase. The selective binding site for silver appears to be active only in fully developed cell walls and the role of a slimy exopolymer produced by the fungus is under further investigation. The next question which arose was, whether the selectivity was a specific attribute of PT 35 or a common one of soil fungi. When the accumulation properties of 32 strains of our culture collection were compared (Tab 1), only one species (Doratomyces purpureofuscus) was found to prefer lead to silver. The mean accumulation capacity of a!l 32 strains, taken at an ionic concentration of 100 p.mol/l, with individual ions, could not be correlated with the ionic radii. Only silver, the largest ion, was accumulated the most (Tab. 2). By contrast Tobin et al. (1984) found a rather good correlation with 10 of the 15 tested elements when testing Rhizopus arrhizus biomass. When checking the accumulation capacity of 11 fungi Nakajima and Sakaguchi (1986) found almost the same values for Cu, Cd, Ni, and Pb, but observed a selective accumulation af uranium to a maximum amount of 380 ~mot/g dry wt. Since 9 species of the genus Penicillium were represented in this study, their average accumulation capacity was compared with that of the other species tested: no significant difference was detected (Fig 4), indicating that accumulation properties are connected with a species rather than with a genus.

277

strain

selectivity

accumulated

Ag [~mol/g] Absidia orchidis Altematia sp. Arthrobotrys superba Aspergillus clavaUts Aspergillus sp. Botrytis cinerea Chaetomium globosum Ch@osporium pseudomerdarium Cladosporium cladosporioides Colletotrichium dematium Doratomyces purpureofuscus Fusarium roseum Gliocladium roseum Mammaria echinobotryoides Metarrhizium an~opliae Mortierella sp. Penicillium caseicolum Penicillium chrysogenum Penicillium citrinum Penicillium claviforrne Penicillium dupontii Penicillium frequentans Penicillium fimiculosum Penicillium roquefortii Penicillium thomii Phoma betae Phoma glomerata Phomasp. Rhizopus nigricans Scopulariopsis brevicaulis Scopulariopsis carbonaria Tontlomyces sp.

Ag > Ca = Pb > Ni > Cd Ag > Cu > Pb = Ni > Cd Ag > Cu > Ni > Pb > Cd Ag > Cu > Ni > Pb = Cd Ag > Cu > P b > N i > C d Ag > Cu > Pb > Ni > Cd Ag > Cu > Pb > Ni > Cd Ag > Pb > Cu > Ni > Cd Ag > Cu > Pb > Ni > Cd Ag > Cu > Pb > Ni > Cd Pb > Ag > Cu > Ni > Cd Ag > Pb > C u > N i > Cd Ag > Cu > Cd > Ni = Pb Ag > Cu > Pb = Ni > Cd Ag > Cu > Cd > Ni > Pb Ag > Cu > Pb > Ni > Cd Ag > Cu > Pb > Ni > Cd Ag > Cu > Pb > Cd > Ni Ag > C'u > Pb > Ni > Cd Ag > Cu > Ni > Pb = Cd Ag > C u > Pb > N i > Cd Ag > Cu > Pb > Ni > Cd Ag > Cu > Pb > Cd > Ni Ag > Cu > Ni > Pb > Cd Ag > Cu > Ni > Pb = Cd Ag > Pb > Cu > Ni > Cd A g > Pb > Cu > Ni > Cd Ag>Cu>Pb>Ni>Cd Ag > Pb > Cu > Ni > Cd Ag > Pb > Cu > Ni > Cd Ag > Cu > Ni > Pb > Cd Ag > Cu > Pb > Ni > Cd

157 127 242 208 244 458 200 268 171 178 276 207 296 250 402 276 181 247 162 172 407 209 147 112 124 224 208 244 389 153 132 209

Tab 1: Selectivity of 32 strains for silver, cadmium, copper, nickel and lead. The accumulation capacity for Ag is shown for an initial silver concentration of 100 p,moFl.

Ion

ionic radius

[A] Ag + Cu + + Pb + + Ni + + Cd + +

1.26 0.72 1.20 0.69 0.97

accumulated metal [p,mol/g dry wt] max mean s.dev 458 163 303 88 50

224 95 79 39 15

86 31 66 21 11

Tab. 2: Accumtflation capacity (maximum, mean valu~ and standard deviation) of 32 strain~ of fungi taken at an initial concentration of 100 l~mol/l. The ionic radius is not correlated with the accumulated amount.

278

-7 5oo!

r---i Mycelium stored for 2 weeks at 4"C Myee|ium 5 days pregrown

100Cop~r

Mycelium7 days pregrown

400.

80' V

7

60-

4o)

Silv~

I I I I

300200-

o

-

100-

M"

0 0 ,<

I I

0

0

100

50 ~me

Ag

150

[mini

Accumulation from o mixture of metals

"6

71

200.

/3 Jq

c

Pb

300'

"~ 400-

E :r 300-

Ni

o 9 Penicilllumsp. 9 the other species tested

5oo. r - - ' l Accumulation of |ndividuol metals

-6

Cu

Fig 2 : Heavy metal accumulation by PT 35. Accumulation of individual ions and of a mixture of them.

Fig 1 : Decreasing metal concentration in solution (300 ml) with strain PT 35 (60 mg dry wt.).Silver and copper are compared.

I~

Cd

Accumulated metol

C 0

o 200-

0

1009

E 100(J o

o o
0

Ag

Cd

Cu

Ni

O,

Pb

Ag

Cu

Pb

Ni

Cd

Accumulated metal

Accumulated metal

Fig 3 : Heavy metal accumulation by FI" 35 myeefium of different ages.

Fig 4 : Heavy metal accumulation by 32 strains of fungi resp. 9 Penicilliura sp.. Mean values and standard deviations are shown.

279

REFERENCES Byrne, A.R., Dermelj, M., and Vakselj, T. (1979). Chemosphere 10, 815-821. Gadd, G.M. and White, C. (1984). J Gen Microbiol 131, 1875-1879. Galun, M., Galun, E., Siegel, B.Z., Keller, P., Lehr, H., Siegel, and S.M., (1987). Water, Air and Soll Pollution 33, 359-371. Hoffmann~ G.M., Nienhaus, F., gt al. (1976). Lehrbuch der Phytomedlzin, Berlin, Hamburg: Paul Parey, p. 267 Kelly, D.P., Norris, P.R., and Brierlgy, C.L. (1979). Microbial Technology : current state.Symposium of the society for general microbiology 29, 263-308. Kuyucak, N. and Volesky B. (1988). Biotechnology Letters 10, 137-142 Mohan, P.M., Rudra, M.P.P., and Sastry, K.S. (1984). Current Microbiology 10, 125-128. Mowll, J.L. and Gadd, G.M. (1983). J Gen Microbiol 130, 279-284. Nakajima, A. and Sakaguchi, T. (1986). Appl Microbiol Biotechno124, 59-64. Piimpel, T. and Schinner, F. (1986). Appl Microbiol Biotechnol 24, 244-247. Tobin, J.M., Cooper, D.G., and Neufeld, R.J. (1984). Appl Environ Microbiol 47, 821-824. Tyler, G. (1982). Chemospherr 11/11, 1141-1146.

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