Cu, Zn, Cd, Pb, Fe, Ni, Ag, Th, Ra and u by fungal biomass has been observed to varying extents. Fungal biosolption largely depends on parameters such as pH ...
Bioresource
0960-8524(95)00072-O
ELSEVIER
Technology 53 (1995) 195-206 Elsevier Science Limited Printed in Great Britain 0960-X524/95/$29.00
FUNGAL BIOSORPTION - AN ALTERNATIVE TREATMENT OPTION FOR HEAVY METAL BEARING WASTEWATERS: A REVIEW A. Kapoor & T. Viraraghavan* Faculty of Engineering, University of Regina, Regina, Saskatchewan,
Canada, S4S 0A2
(Received 8 August 1994; revised version received 8 August 1994; accepted 6 May 1995) Abstract
membrane processes have limited their use in heavymetal removal. Adsorption on activated carbon is a recognized method for the removal of heavy metals from wastewater. The high cost of activated carbon limits its use in adsorption. A search for a low-cost and easily available adsorbent has led to the investigation of materials of agricultural and biological origin, along with industrial byproducts, as potential metal sorbents. The variety of materials tested as metal adsorbents includes Girdish coal, crushed coconut shell, peat, bark, straw, waste tyre rubber and human hair. Recent developments in the field of environmental biotechnology include the search for microorganisms as sorbents for heavy metals. Bacteria, fungi, yeast and algae can remove heavy metals and radio nuclides from aqueous solutions in substantial quantities (Brierley, 1990; Gadd, 1988; 1992; Muraleedharan et al., 1991). The uptake of heavy metals by biomass can take place by an active mode (dependent on the metabolic activity) known as bioaccumulation or by a passive mode (sorption and/or complexation) termed as biosorption. Shumate and Strandberg (1985) defined biosorption as “a non-directed physico-chemical interaction that may occur between metal/radio nuclide species and the cellular compounds of biological species”. Fungi and yeasts accumulate micronutrients, such as Cu, Zn and Mn, and non-nutrient metals, like U, Ni, Cd, Sn, Hg, in amounts higher than the nutritional requirement (Gadd, 1986). The potential of fungal biomass as adsorbents for the removal of heavy metals and radio nuclides from polluted waters was recognized by Shumate et al. (1978) and Jilek et al. (1975). This paper reviews the removal of heavy metals and radio nuclides by fungi (Aspergillus spp., Mucor spp., Rhizopus spp. and Penicillium spp.) and yeast (Saccharomyces spp.) from aqueous solutions.
The common jilamentous fungi can sorb heavy metals from aqueous solutions, The sorption of heavy metals, Cu, Zn, Cd, Pb, Fe, Ni, Ag, Th, Ra and u by fungal biomass has been observed to varying extents. Fungal biosolption largely depends on parameters such as pH, metal ion and biomass concentration, physical or chemical pre-treatment of biomass, presence of van’ous ligands in solution, and to a limited extent on temperature. Fungal biosorption pegorrns well in comparison to sorption on commercial ion-exchange resins, activated carbon, and metal oxides. Limited data indicate the potential for regenerating the biomass. The cell-wall fraction of biomass plays an important role in the sorption of heavy metals. The mechanisms of biosorption are understood only to a limited extent. The potential of fungal biomass as sorbents is indicated by the available data, and more research and development of the fungal biosorption technology is recommended. Key words:
Biosorption, adsorption, fungi, yeast and regeneration, review.
heavy metals,
INTRODUCTION Rapid industrialization has led to increased disposal of heavy metals and radio nuclides into the environment. Removal of heavy metals and radio nuclides from metal-bearing wastewater is usually achieved by physico-chemical processes before discharging the effluents into natural water-body systems. Physicochemical processes in use for heavy-metal removal from wastewater include precipitation, coagulation, a reduction process, ion exchange, membrane processes (such as ultrafiltration, electrodialysis and reverse osmosis) and adsorption (Beszedits, 1983; Dean et al., 1972). Conventional treatment technologies like precipitation and coagulation become less effective and more expensive when metal concentrations are in the range of l-100 mg/l. High costs, process complexity and low removal efficiency of
SOURCES OF BIOMASS Fungi and yeasts are easy to grow, produce high yields of biomass and at the same time can be manipulated genetically and morphologically. The
*Author to whom correspondence should be addressed. 195
A. Kapoor; 7: Viraraghavan
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fungal organisms are widely used in a variety of large-scale industrial fermentation processes. For example, strains of Aspergillus are used in the production of ferrichrome, kojic acid, gallic acid, itaconic acid, citric acid and enzymes like amylases, glucose isomerase, pectinase, lipases and glucanases; while Saccharomyces cerevisiae is used in the food and beverage industries. The biomass can be cheaply and easily procured in rather substantial quantities, as a byproduct from the established industrial fermentation processes, for the biosorption of heavy metals and radio nuclides. The use of biomass as an adsorbent for heavy-metal pollution control can generate revenue for industries presently wasting the biomass and at the same time ease the burden of disposal costs associated with the waste biomass produced. Alternatively, the biomass can also be grown using unsophisticated fermentation techniques and inexpensive growth media (Kuyucak, 1990). METAL ION UPTAKE The presence of heavy metals affects the metabolic activities of fungal and yeast cultures, and can affect commercial fermentation processes. The effect of the presence of heavy metals on the yield of commercial fermentation processes created interest in relating the behavior of fungi to the presence of heavy metals (Clark et al., 1966). Results from such studies led to a concept of using fungi and yeasts for the removal of heavy metals from waste streams. The toxicity of heavy metals on the growth of fungi is well known and will not be the part of this review. The models used to describe the sorption phenomenon and removal of various metals by fungi are reviewed in the following sections. BIOSORPTION EQUILIBRIA The available literature indicates that equilibria of biosorption of heavy metals and organic compounds follow an adsorption-type isotherm (Rome & Gadd, 1987; Huang et al., 1991; Kiff & Little, 1986; Mullen et al., 1992; Tsezos & Volesky, 1981; Yakubu & Dudeney, 1986). The degree of biosorption of a metal ion on a biosorbent has been found to be a function of the equilibrium metal-ion concentration in solution at constant pH and temperature conditions. The single-solute adsorption isotherm models of Langmuir, Freundlich and Brunauer-EmmettTeller (BET) have been shown to describe the biosorption equilibrium. The Langmuir model can be described as (Weber, 1972) X
-_= m
energy or net enthalpy of biosorption; b = amount of metal ion biosorbed per unit weight of biomass; C, = equilibrium concentration of metal ion in solution after biosorption. The Langmuir model is based on the assumption that maximum adsorption occurs when a saturated monolayer of solute molecules is present on the adsorbent surface, and the energy of adsorption is constant and that there is no migration of adsorbate molecules in the surface plane. The Freundlich isotherm has the form (Weber, 1972) Q = KC;“’ where
Q = metal uptake capacity of biomass; equilibrium constant indicative of biosorptive uptake capacity; y1= biosorption equilibmetal-ion C, = equilibrium rium constant; concentration. The Freundlich model is basically empirical, and was developed for heterogeneous surfaces. The model is a useful means of data description. The BET isotherm represents the multi-layer adsorption at the adsorbent surface and assumes that a Langmuir isotherm applies to each layer (Weber, 1972):
K = biosorption
C, (Cs -C,)
=L+($g)@) BQ”
C, = saturation concentration of the metal ion; Q” = amount adsorbed per unit weight of biomass for monolayer biosorption; B = constant relating to the energy of interaction with the surface. The adsorption models described above were developed for gas adsorption on surfaces. The application of these models to complex biological system may not be able to explain the biosorption behavior. The applicability of the models should be considered as a mathematical representation of the biosorption equilibrium over a given metal-ion concentration range. The mechanistic conclusions from the good fit of the models alone should be avoided. In spite of the above limitations, these models can provide information on metal-uptake capacities and differences in metal uptake between various species. Scatchard plots have also been used to describe the biosorption equilibria (Huang et al., 1990). The model was developed to describe the attractions of proteins for small molecules and ions (Scatchard, 1949). The metal-ion interaction with the binding sites on the cell surface can be described by an association constant, K, according to the following equation: M+XoMX
abC,
K=
l+bC,
where x/m = amount of metal ion biosorbed weight of biomass; a = constant related
[Mx Pfl[xl
per unit to the
where M is the metal ion in consideration,
X is the
Metal adsorption by fungi: a review
number of binding sites on the biomass surface and MX is the metal-ion biosorbed on biomass. The plot of amount of bound metal ion on biomass divided by equilibrium metal-ion concentration versus the amount of bound metal ion gives a typical Scatchard plot. The equation can be represented as follows: M-X
M. =K(xo-Mw ‘
where A4, and X,, are the equilibrium metal-ion concentration and the concentraton of potential binding sites. The slope of the plot gives -K and the abscissa intercept gives X,,. The total number of binding sites may be extrapolated from data in which the complete saturation of the biomass binding sites is not observed. The Scatchard plots have been used to represent the binding of ligands to macromolecules (Dahlquist, 1978). Table 1 presents the biosorptive capacities of various fungal and yeast (living and dead cells) species for different metals. METAL UPTAKE BY LIVING CELLS Penicillium
can remove a variety of heavy metals from aqueous solutions. Spores of Penicillium italicum were shown to accumulate copper (Somers, 1963). The uptake was believed to be an ionexchange reaction. Penicillium spinulosum has been shown to remove copper, gold, zinc, cadmium and manganese (Townsley et al., 1986a; Ross & Townsley, 1986). The metal uptake by non-growing mycelia reached an equilibrium in 60-120 min. The uptake of heavy metals by fungal cells was usually very rapid and about 90% uptake was achieved in 10 min. The metal accumulation of growing cells varied with the cell age. The maximum metal uptake took place during the lag period or the early stages of growth and declined as cultures reached a stationary phase. Aspergillus nigel; P spinulosum and Trichoderma viride showed a similar uptake pattern (Townsley & Ross, 1985; 1986; Townsley et al., 19866). The reduction in metal-uptake pattern with growth may be due to a drop in pH during growth, but a reduction in metal uptake where pH is maintained constant can be explained by the changes in cell-wall composition with growth and the release of metabolites that bind with the metal ions. Copper uptake by living S. cerevisiae yeast cells was biphasic, consisting of an initial rapid surface binding of copper ions, followed by a second, slower, intracellular uptake of copper. The initial rapid uptake phase was completed in 5 s and the second, slower phase lasted for 150 min. Cadmium (II) and Pb (II) uptake was observed to be through surface binding only (Huang et al., 1990). Copper uptake by S. cerevisiae was observed to be slightly higher in the presence of glucose, indicative of an intracellular uptake mechanism (Gadd et al., 1988). May (1984) observed
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higher copper uptake by live and freeze-dried, aerobically grown, S. cerevisiae than by anaerobically grown cells. The higher uptake under aerobic conditions could be due to the presence of an active metallothionein. The copper-binding protein removal of copper from aqueous solution by Penicillium italicum has been described by the Freundlich and Langmuir isotherms (Rome & Gadd, 1987); for R. arrhizus the copper removal was described by BET isotherms (Table 1). The metal uptake by yeast for a range of metal ions did not follow the Langmuir or Freundlich isotherms and depended on cell density (Itoh et al., 1975). The uptake of mercury by S. cerevisiae was described by the Langmuir model for the initial phase; this was followed by a transition phase and then a multilayer or penetration phase was observed for metal-ion uptake (Brown et al., 1974). The uptake of metals by living cells also depends on contact time, pH of the metal solution, culture conditions, initial metal-ion concentraton and the concentration of cells in aqueous solutions (Galun et al., 1987; Siegel et al., 1987; Kiff & Little, 1986; Kurek et al., 1982; Gadd et al., 1988). The uptake of Ni, Zn, Cd and Pb by the mycelium of Penicillium digitatum was highly pH-sensitive and was inhibited below pH 3 (Galun et al., 1987). Huang et al. (1988) also observed that cadmium biosorption on various fungal strains was pH sensitive. Aspergillus oryzae, Fusarium solani and Candida utilis were found to perform better in the acidic range. The change in the sorption capacity with pH can be explained on the basis of proton-competitive adsorption reactions (Huang et al., 1991). Higher uptake of cadmium was observed at lower biomass concentrations of Aspergillus oryzae, Aspergillus niger, Mucor racemosus, Penicillium chlysogenum and Trichoderma viride (Kiff & Little, 1986; Kurek et al., 1982). The lower uptake
at higher biomass concentrations can be attributed to the electrostatic interactions of the functional groups at the cell surfaces. The cells at higher concentrations in suspension attach to each other, thus lowering the cell surface area in contact with the solution. The growth conditions also affect the metal uptake of the biomass. Strandberg et al. (1981) observed S. cerevisiae cultures grown on a synthetic medium had a higher uranium uptake rate than cultures grown on a rich, organic medium, while the uranium uptake rate was found to be the same for cultures grown under aerobic or anaerobic conditions. Treen-Sears et al. (1984a) observed that growth, uptake capacity and biosorptive yield (biox uptake capacity) were concentration mass enhanced by the addition of mineral salts to the growth media. Growth media control the cell-wall chemistry (formation of various functional groups on the cell surface) and can lead to increased metal uptake capacity. Mortierella ramannianc, Rhizopus sexualis, R. stolonifer, Zygorhynchus heterogamus and Z. moelleri,
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Table 1. Biosorption of metal ions by various fungal species Organism
Element and experimental details
Aspergillus niger Non-growing Growing
Cu, 35 mg/l, 1 h Cd, 10 mg/l, 1 h Au, 10 to 1000 mg/l Ag, 107 mgil, 2 h Cu, 635 mg/l, 2 h La, 138 m&l, 2 h U, 71.4 mgil, 4 h U, 10 m&l, 1 h
A. oryzae
Zn, 200 mgjl” Cu, 6.35 mgil Cd, 1.1 mg/l, 3 h Cd, 1 h U, 30 mgjl” Th, 30 mg/l”
A. terreus Penicillium spinulosum Non-growing Growing, midlinear phase Non-growing Growing, lag period Non-growing Growing, midlinear phase Penicillium notatum
Penicillium sp.
Penicillium chtysogenum Penicillium janthinellum Penicillium sp. Penicillium chrysogenum Penicillium chrysogenum Rhizopus arrhizus
Rhizopus arrhizus
“The equilibrium
References
Townsley and Ross (1986) Cu, 2.5 mg/l Cu, 2.5 mgil
A. niger
Saccharomyces
Isotherm applicability
Biosorption capacity (mgig)
cerevisiae
metal-ion
Cd, Cd, Cu, Cu, Zn, Zn,
2.5 2.5 2.5 2.5 2.5 2.5
mgil, mgil, mg/l, mg/l, mgil, mg/l
2 h 12 h 2h 24 h 2 h
cu Zn Cd Al Sn Pb Pb, 50 mgIl,2 h U, 30 m&l” U, 10 mg/l, 1 h U 100 mgil, 18 h Th, 30 mg/l” Pb, 90 m&l, 24 h Cr Mn cu Zn Cd Hg Pb U Ag Cu, 19 mg/l Th, 30 mg/l” Th, 696 mgil, 0.5 h Cd, 0.5-50 mg/l, I h Cd, 22.4 mgil, 1 h U, 10 mg/l, 1 h U, 57 mg/l Th, 696 m&l”, 30 min Cu, 3.2 mgil, 3 h Cd, 200 mgil”, 45 min concentration.
1.9 5.0 7.22 3.74 170 22.3 1.8 7.5 215 80.7
Freundlich Freundlich Freundlich Freundlich -
17.6 13.8 2.2 12.8 10 60
Langmuir Langmuir Langmuir Langmuir Langmuir
1.5 0.4 2.4 3.6 1.3 0.2
and Langmuir
Yakubu and Dudeney (1986) Horikoshi et al. (1981) May (1984) Huang et al. (1991) Huang et al. (1988a) Kiff and Little (1986) and Freundlich and Freundlich
Tsezos and Volesky (198 1)
Townsley and Ross (1986)
-
Ross and Townsley (1986) Town&y et al. ( 1986)
-
Siegel et al. (1983)
80 23 5.0 53 61 5.0 6.1 70 52.7 1.4 142 116 31 12 16 20 30 58 104 195 54 17.1 185 97 2.4-31.2 8.9 33.8 150 119 1.9 71
Rao et al. (1993) Kurek et al. (1982) Kuyuack & Volesky (1988) Mullen et al. (1992)
Langmuir
and Freundlich
Langmuir
and Freundlich
-
Siegel et al. (1986) Tsezos and Volesky ( I98 1) Horikoshi et al. (1981) Galun et al. (I 983) Tsezos and Volesky (1981) Niu et ~1. (1993) Tobin et al. (1984)
BET Langmuir -
Scatchard Langmuir
and Frcundlich
Rome and Gadd (1987) Tsezos and Volesky (1981) Gadd et al. (1988) Gadd and Mow11 (I 983) Norris and Kelly (1977) Horikoshi et al. (1981) Strandberg et al. (1981) Gadd et al. (1988) Huang et al. (1990) Volesky et al. (1993)
Metal adsorption by fungi: a review Aspergillus niger, Mucor racemosus, Penicillium chrysogenum and Tnkhoderma viride were able to remove cadmium from aqueous solutions (Azab et al., 1990; Kurek et al., 1982; Ross & Townsley, 1986). Aspergillus terrus and Mucor ramanniunus exhibited higher sorption capacities for cadmium than activated charcoal and ion-exchange resins (Azab & Peterson, 1989). The Langmuir model explained the sorption of cadmium on Aspergillus oryzae (Kiff & Little, 1986). The fungal cells of A. niger, Mucor rouxxi and R. arrhizus were found to take up precious metals like gold and silver (Mullen et al., 1992; Gee & Dudeney, 1988; Townsley et al., 1986a; Kuyucak & Volesky, 1988). Biosorption of metal ions primarily occurs by surface binding, including ion-exchange reactions and complexation with the functional groups present on the cell surface. Various functional groups believed to be involved in metal binding include carboxyl, amine, hydroxyl, phosphate and sulfhydryl groups (Strandberg et al., 1981). The removal of silver by A. niger was believed to be also a result of precipitation reactions at the cell wall surface. Electron-dense aggregates were observed to accumulate at and within the fungal cells, as revealed by the energy dispersive X-ray spectroscopy (Mullen et al., 1992). Gold has been found to crystallize on the algal surface, forming hexagonal and trigonal laminae (Gee & Dudeney, 1988). Similar reactions may also be partly responsible for the uptake of gold by fungal cells. It is likely that for such depositions on the cell surface the biosorption equilibria may be described by the BET model. Research has been focused on the removal of radioactive metals like uranium, thorium, strontium and radium from waste process-streams by sorption on microorganisms. Zajic and Chiu (1972) isolated fungal strains of Penicillium from wastewater, which exhibited significant growth in media containing salts of uranium, strontium, platinum and titanium. The uranium uptake was dependent on the culture age. The 5-day-old cultures were twice as effective as 15-day-old cultures. The reasons for the change in uptake pattern of uranium with culture age are not very clear. A possible explanation is that change in the cell-surface chemistry and morphology with age contributed to higher uptake. Living cells of Aspergillis, Pusarium, Mucor, Penicillium and Rhizopus can also take up uranium from aqueous solutions (Nakajima & Sakaguchi, 1986; Galun et al., 1983a;b). METAL UPTAKE BY DEAD CELLS The biosorptive capacity of dead fungal cells has been studied extensively in comparison to living cells. The biosorptive capacity of dead cells may be greater, equivalent to or less than that of living cells. Use of dead biomass in industrial applications offers certain advantages over living cells. Systems using living cells are likely to be more sensitive to metal-
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ion concentration (toxicity effects) and adverse operating conditions (pH and temperature). Furthermore, constant nutrient supply is required for systems using living cells (increased operating cost for waste streams devoid of nutrients) and recovery of metals and regeneration of biosorbent is more complicated for living cells. The dead biomass can be procured from industrial sources as a waste product from established fermentation processes. Cells can be killed by physical treatment methods using heat treatment (Siegel et al., 1986; 1987; Galun et al., 1983a; Townsley et al., 1986a), autoclaving, and vacuum drying (Huang et al., 1988a; Tobin et al., 1984) or chemicals like acids, alkalis, detergents (Azab & Peterson, 1989; Brady et al., 1994; Rao et al., 1993; Huang et al., 1988a; Muzzarelli et al., 1980a;b; 1981; Wales & Sagar, 1990; Tsezos & Volesky, 1981; Ross & Townsley, 1986; Gadd et al., 1988) or other organic chemicals like formaldehyde (Huang et al., 1988b), or by mechanical disruption (Tsezos & Volesky, 1981; Yakubu & Dudeney, 1986). Pretreatment methods have usually shown an increase in the metal sorption capacity for a variety of fungal species. The alkali pretreatment was observed to be most effective in increasing the biosorptive capacity of fungal biomass. The alkali treatment (usually with sodium hydroxide) of fungal biomass for 4-6 h at 95-100°C deacetylates chitin present in the cell wall to form chitosan-glucan complexes with higher affinity for metal ions (Muzzarelli, 1980a;b). Tobin et al. (1984) investigated the uptake of 17 metal species by heat-killed Rhizopus arrhizus at a pH of 4.0. A linear relationship was observed between maximum biosorptive capacity of a metal ion by biomass and ionic radii of the various cations. Higher uptake capacity was observed for larger ions, the exceptions being Cr3+ and the alkali-metal ions. Alkali-metal ions were not prone to biosorption, due to the lack of ability of these metal ions to form complexes with the ligand groups present on the fungal cell surface. Exceptionally high uptakes for uranium and thorium were observed for Rhizopus and Aspergillus (in excess of 180 mg/g) (White & Gadd, 1990; Tsezos & Volesky, 1981; Yakubu & Dudeney, 1986; Treen-Sears et al., 1984b). Uranium biosorption on Rhizopus, Aspergillus and Mucor was described using Freundlich and Langmuir isotherms (Tsezos & Volesky, 1981; Guibal et al., 1992). Removals were higher than for activated-carbon and studied. Optimum removals ion-exchange-resins were achieved in the pH range of 4-5, and removals were substantially reduced at a pH of 2.5. Uranium removals by Penicillium were more or less the same in the pH range of 2.5-9.5 (Galun et al., 1983b). This showed that there were differences in the behavior of fungal cell-wall surfaces. The complex behavior of fungal cell-wall surfaces was further substantiated by the fact that for Rhizopus, uranium uptake was reduced by the presence of Fe3+ and
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A. Kapoor; T. Viraraghavan
Zn’+, but for Penicillium, only Fe”+ inhibited uranium sorption (Tsezos & Volesky, 1981; Galun et al., 1984; Siegel et al., 1986). Uranium removals improved with an increase in temperature from 20 to 50°C (Strandberg et al., 1981; Tsezos & Volesky, 1981). Penicillium can remove radium, strontium and cesium (Tsezos & Keller, 1983; Tsezos et al., 1986; Rome & Gadd, 1991). The optimum removal for radium was observed in the pH range of 7-9. Radium uptake was mildly affected by the presence of calcium but strongly affected by the presence of magnesium and barium. Penicillium biomass was found to be approximately 14 times more effective than activated carbon (F-400). The metal-binding capacity of Penicillium was observed to follow the series Fe >Cu, Zn, Ni>Cd, Pb>UO, (Galun et al., 1987). For Rhizopus, the series was U02>Pb>CD> Zn >Cu (Tobin et al., 1984) and for Aspergillus the series was Fe”+ > UO, > Cu > Zn (Yakubu & Dudeney, 1986). The differences in the metal-binding capacities can be attributed solely to the variations in the characteristics of the fungal cell-wall. Huang et al. (1988a) observed that for Cd (II) removal, various adsorbents were effective in the following order: hydroxyapatite > Nuchar SA =A. oyzae =S. cerevisiae > Dothan sandy soil >Al,O, > Filtrasorb 400. The presence of anions also affects the biosorptive capacity of fungal biomass. The metal uptake was shown to be reduced by the presence of ethylenediamine tetraacetate (EDTA), sulfate, chloride, phosphate carbonate and glutamate ions (Zhou & Kiff, 1991; Tobin et al., 1987; Tsezos & Noh, 1984). The biosorption of copper, lanthanum, uranium, silver, cadmium and lead was inhibited most severely by the presence of EDTA and for uranium the carbonate ions also considerably suppressed the biosorption. EDTA chelates the metal ions, forming metal-EDTA complexes. The stability constants of metal-ion-EDTA complexes are very high. If the stability constants of metal-ligand complexes are greater than stability constants for metal-biosorption sites on the cell-wall surface, the biosorption can be expected to be reduced considerably. Thus anion complexation decreases biosorption of complexed metal. Thus the presence of anions in actual wastestreams can reduce the biosorptive capacity of fungal biomass. As a result, effects of the presence of both anions and competing cations should be included in process-design considerations. The biosorptive equilibrium, as described by single-solute models, may not represent the true equilibrium of metals for the actual waste streams. For a complex waste stream the biosorptive equilibrium can be best described by the application of competitive adsorption models (Murali & Aylmore, 1983; Bajracharya & Vigneswaran, 1990; Digiano et al., 1978; Yonge & Keinath, 1986; Susarla et al., 1992) and chemical equilibrium models capable of simulating equilib-
concentrations of species in rium metal multi-metal-multi-ligand mixtures (Morel & Morgan, 1972; Westall et al., 1986; Perrin & Sayce, 1967). The small particle size and low strength of fungal cells can cause difficulties in the separation of biomass and treated effluent. As a result, industrial applications of fungal biosorption for removal and recovery of metal ions from solutions may prefer an immobilized or pelletized biomass. Foam biomasssupport particles, sand, paper or textile-making fibers and polymers have been successfully used for immobilization in the removal of metal from aqueous solutions (Tobin et al., 1993; Zhou & Kiff, 1991; Kiff & Little, 1986; Huang et al., 1990; Tsezos & Deutschmann, 1990). Use of flotation for separation of metal-loaded biomass from aqueous suspensions can overcome the need for using immobilized biomass. At present, only limited data are available on the use of flotation for separation of fungal biosolutions (Zouboulis & sorbents from aqueous Matis, 1993). REGENERATION AND ELUTION OF METALS FROM LOADED FUNGAL BIOMASS The use of fungal biomass as a potential biosorbent depends not only on the biosorptive capacity, but also on how well the biomass can be regenerated and used again. To this end, Galun et al. (1983~) and Tsezos (1984) screened various elutants for the recovery of adsorbed uranium from Rhizopus and Penicillium biomass, respectively. Sulfuric and hydrochloric acids were effective in the recovery of sorbed uranium, but substantially damaged the biomass and reduced the uranium biosorption capacity. Sulfate ions in the elutant were reported to impart crystallinity to the cell-wall chitin network and shifted the desorption equilibria towards the solid phase, thus reducing the elution efficiency (Tsezos, 1984). Sodium bicarbonate was found to be most effective elutant. The optimum conditions for elution systems were reported to exceed the solid to liquid ratio of 12O:l (mgiml) for 1 N NaHC03 for complete uranium recovery. Galun et al. (1983~) observed that the adsorbed uranium was desorbed in excess of 90% by (NH,),CO, and NaHC03, but 0.1 M EDTA eluted only 61% of the adsorbed uranium. The uptake capacity of uranium increased by 200% for subsequent uptakes when desorbing agents were 0.1 M EDTA and carbonate ions. Penicillium biomass loaded with Ni, Cu, Zn and Cd can be effectively eluted using 0.1 N NaOH. Elution with 0.1 N NaOH and washing to pH 5.5 to 6.0 resulted in an approximately 2-6-fold increase in mycelial uptake during the second uptake-cycle. Such an increase in metalremoval capacity for adsorbents other than biological ones is not very common. No significant changes in biosorption of Ni, Cu and Zn were observed when HCl was used alone.
Metal adsorption by fungi: a review
Huang et al. (1988a) observed that Cd (II)-loaded A. oryzae can be effectively regenerated using a strong acid. For eight adsorption-desorption runs, 35 mg of Cd (II) per gram of dry weight of A. oryzae was removed, while activated carbon (Nuchar SA) removed only 14 mg of Cd (II) per gram of dry weight in five cycles.
BIOSORPTION MECHANISM The metal uptake by fungal biomass takes place by two basic processes. The first is by living organisms, where the metal uptake is dependent on the metabolic activity. The second process involves metal uptake by dead and living cells as a result of the chemical functional groups of the cell and, in particular, the cell wall. It should be noted that the metal uptake by the second process may also be involved during the metabolism-dependent metal uptake of growing cells (Gadd, 1986). The cell wall of the fungi is the first to come into contact with metal ions in solution, where the metals can be deposited on the surface or within the cellwall structure before interacting with the cytoplasmic material or the other cellular parts. In extreme cases, for the living cells, intracellular uptake may take place due to the increased permeability as a result of cell-wall rupture and subsequent exposure of the metal-binding sites (Gadd, 1990). The metal uptake by the cell wall has been broadly based on two mechanisms: uptake directed by functional groups like phosphate, carboxyl, amine and phosphate diester species of these compounds. The second uptake mechanism results from physicochemical inorganic interactions directed by adsorption phenomena. The removal mechanisms for radionuclides result from the combination of the above two processes, while for other heavy metals, the first process seems to play an important role. Tobin et al. (1984) reported that the metal uptake was independent of the ionic charge or electrostatic strength and was influenced by the ionic radius. The carboxylate and/or phosphate ligands were proposed to be actively involved along with the hydroxy and amide functional groups, which would form relatively weak bonds with metal ions. Treen-Sears (1986) also reported that uptake of metals depended on the ratio of phosphate to carboxyl residues. It was also indicated that ion exchange may be the principal mechanism for metal sequestering during the rapid initial uptake phase. Fungal cell walls contain chitin and chitosan. Chitin and chitosan have been shown to sequester metal ions (Muzzarelli, 1972; Tsezos, 1983). Chitin and chitosan contents of the fungal cell wall can change during growth of mycelia (Blumenthal & Roseman, 1957; Aronson & Machlis, 1959; Bartnicki-Garcia & Nickerson, 1962; Farkas, 1980) and this can account for the variations in the metal-uptake capacity with the cell age.
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Uranium has been shown to be deposited throughout the cell wall of non-living R. arrhizus cells exposed to uranium solutions. IR spectroscopy revealed the sequestered uranium to be associated with the nitrogen of the chitin monomer N-acetyl-Dglucosamine (NAGI). The pure chitin had a very low uranium uptake (6 mg/g). Based on experimental results, Tsezos and Volesky (1982~) proposed a three-step process to describe the biosorption of uranium by non-living cells of R. arrhizus. The first process involves the formation of a complex between the dissolved uranium ionic species and the chitin network present in the cell wall. The complex forms between the uranium and the amine-nitrogen of the chitin crystallates. The second process involves the adsorption of additional uranium by the chitin network close to that complexed by the chitin nitrogen. Thus, the complexed uranium could be acting as nucleation sites for further deposition of uranium. The third process is the hydrolysis of the uraniumchitin complex formed by the first process and the precipitation of the hydrolysis product in the cell wall. Tsezos and Volesky (1982b) observed a different removal mechanism for biosorption of thorium. The first process involves the formation of a coordinated complex between thorium and nitrogen of the cell-wall chitin. Pure chitin exhibited a capacity of 8 mg/g. The biosorptive uptake capacity of thorium by R. arrhizus was found to be greater than 170 mgig, thus suggesting other processes to be involved apart from the complexation by chitin. The second process involved the adsorption of hydrolyzed thorium ions by the outer layers of the R. arrhizus cell wall. Tsezos (1983) and Tsezos and Volesky (1982a) suggested that uranium forms a complex with the chitin nitrogen and a free radical (hydroxyl group) was suggested to participate in the uranyl ion coordination to nitrogen. The presence of CU*+ and Fe2’ reduced the uptake of uranium, possibly due to the competition for the nitrogen sites of the chitin crystalline network. This resulted in a reduced number of nucleation sites created as a result of the first mechanism. The interactions of metals with proteins is well known (Spiro, 1981) and can also be involved in biosorption of metals. Muraleedharan and Venkobachar (1990) showed that proteins in Ganodemza lucidurn, a macro-fungus, do not play any substantial role by themselves in copper (II) uptake. It was also indicated that chitin did not play a significant role in copper uptake by G. lucidum. EPR spectra of G. lucidurn, the chitin fraction of G. lucidum and the fraction devoid of chitin, indicated the presence of a free radical, which seemed to be present in a very stable cell-wall matrix. Tsezos & Mattar (1986) and Muzzarelli et al. (1979) indicated the presence of this radical to be associated with chitin nitrogen, in contrast to the observations of Muraleedharan and Venkobachar (1990). Muraleedharan and Venkoba-
A. Kapool; 7: Viraraghavan
202
char (1990) further indicated that the free radical present on the biosorbent did not take part in the metal uptake, and the cellular matrix trapping this free radical opened up upon metal uptake. The exposed cell-wall matrix thus freely interacted with metal ions. The recent data suggest the structural polysaccharides of G. lucidum to be the main site of interaction. The biosorption site is believed to be oxygen dominated and the majority of the metal taken up was exchanged with calcium and hydrogen present on the cell wall (Muraleedharan et al., 1994). Siegel (1990) suggested that biosorption of UO, ion by various purified cell wall polymers is in the following order: chitin > cellulose phosphate > carboxymethyl cellulose > cellulose; and the difference between the highest and lowest is only 20%. It was further shown that metal ion-uncharged molecule interplay can also play an important role in the biosorption process, which has often been overlooked in the past. To date, the biosorption mechanism of metal ions by fungal biomass has been studied largely in relation to chitin, its deacetylated derivative, chitosan, and cellulose. The fungal cell walls also contain glycans, proteins, lipids, polyuronides and melanin. The role played by these cell-wall fractions and structural polysaccharides is not fully understood, and needs to be studied in greater detail. APPLICATION
1,000 mmobAzed !hlWpUS --_--, rrhim
P /
Activated Carbon (Netier and
100
Hughes,
1984)
10
1
0. I
‘,
Peat (Chen et al, 1991))
Hydrous oxide gel of alummium (Kinrub&& et al, 1976)
Muur TOWX (Mullen et al, 1992)
001
0.001
0 0001
0 00001 0.3
300
30
3
Equilibrium Concentration,
Fig. 1.
Comparison
of adsorption adsorbents.
mg/L
of copper on various
TO PRACTICE
A number of factors govern the application of an adsorbent to be used in practice. The important ones include: (a) the effectiveness in removing pollutants; (b) the availability of the adsorbent; (c) the cost of the adsorbent; (d) the regeneration of the adsorbent; and (e) the ease with which the adsorbent can be used. These issues are addressed below. The data for copper uptake on fungi are compared with similar data for several other adsorbents in Fig. 1. Figure 1 presents only a gross comparison, as details of the experiments, such as the ionic strength, equilibrium time and pH, were different for the various systems. The fungal biomass appears to provide an equal or better adsorbent surface for copper than do other adsorbents. It can be seen from Fig. 1 that activated carbon is also out-performed by the fungal biomass. The data on the regeneration of the adsorbents also play an important role in determining the overall effectiveness of adsorbents. It has been shown that fungal biomass can be regenerated relatively easily. Fungal cells are relatively small particles with low density, low mechanical strength and rigidity. The use of immobilized biomass systems offers a practical approach for the treatment of metal-bearing wastewaters. Various techniques are available for the immobilization of fungal biomass (Anderson, 1983). Studies utilizing immobilized fungal biomass have been discussed earlier in the paper.
Table 2. Cost comparison
Source As a byproduct from industrial fermentation processes Biomass cultured for biosorption purposes only” Activated carbon” Ion-exchange resins”.”
for various
adsorbents
Price of biosorbent Includes transportation and drying costs $1-5 per kg $2.0-55 per kg $13-30 per kg
” After Kuyucak (1990).
“Nalco Canada Inc. The source of raw biomass and the cost of immobilization (if required) are the important factors in determining the overall cost of the biosorbent material. The approximate costs of the fungal biomass, ion-exchange resin and activated carbon are shown in Table 2. The cost of immobilization is often considered to be the expensive step in the biosorbent preparation. Surprisingly, the cost of immobilization is not very high, as biomass does not require a large quantity of support material. Tsezos and Deutschmann (1990) used approximately 10-20s (as cell weight) of the support material for immobilization of Rhizopus arrhizus. The procurement of the raw biomass is one of the most important factors in determining the overall cost of the biosorbent material.
Metal adsorption by fungi: a review Table 3. Comparison
of various
metal-removal
technologies
Properties
Technology Concentration dependence Biosorption Hydroxide ppt. Sulfide ppt. Ion exchange Evaporation Reverse osmosis Activated carbon Adsorption
PH
203
(Brierley et al., 1986; Kuyucak,
1990)
of each technology
Suspended solids
Effluent cont. mg/l
Regeneration
Sludge generation
yes no no yes yes no
yes no no some yes some
yes yes yes no yes no
