Biotech. Adv. Vol. 12, pp.

647-652,1994
Copyright © 1994 Elsevier Science Ltd
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0734-9750(94)00012-3

MICROBIAL BIOSORPTION OF METALS: POTENTIAL IN THE

TREATMENT OF METAL POLLUTION

A. P. McHALE 1 and S. McHALE2

1 School of Applied Biological and Chemical Sciences, University of Ulster, Coleraine, Co.
Londonderry, BT52 1SA, Northern Ireland
2 BioResources (Ireland) Ltd, 1 Ardagh Court, Blackrock, Dublin, Republic of Ireland

ABSTRACT

The phenomenon of metal biosorption by microorganisms has been thoroughly documented.

Although this phenomenon is exhibited by both living and non-living forms of biomass, the
purpose of this chapter will be to review biosorption by the latter. In addition, the application of
various technological processes required for exploitation of this phenomenon in waste treatment
will be examined.

KEYWORDS

Fungal biomass; Non-living; Biosorbent; Metals; Poluution

INTRODUCTION

The existence of heavy metals and/or radionuclides in the environment, whether derived from
natural or anthropological activities, represents a very significant and long-term environmental
hazard. Increasing public awareness of the problems associated with heavy metal/radionuclide
contamination has given rise to substantial changes in legislation governing the disposal of
those materials. When these materials are introduced into the environment it has been noted that
biological activity plays an important natural role in their sequestration and immobilization
(Beveridge, 1989; Lovley et al., 1991). If one analyses this naturally occurring beneficial
activity, it is found that microorganisms play a very significant role and it has been proposed
that this phenomenon might be exploited in biotechnological processes relating to pollution
control (Gadd and White, 1993).

In studying biosorption exhibited by isolated pure cultures of microorganisms in the laboratory

it has been shown that no single mechanism is responsible for metal uptake. The term
"adsorption" implies binding of a material to the surface of an adsorbent, while the term
"absorption" usually implies penetration of material through the surface to an inner matrix.
Since the up-take of metals/radionuclides by microbial biomass appears to involve combinations
of the above. The term "biosorption" is most commonly used to describe the process.

Although most living and non-living forms of microbial biomass exhibit heavy
metal/radionuclide up-take, the ability of non-living fungal biomass, has received considerable

647
648 A.P. McHALEand S. McHALE

attention since it is produced as a waste material from several major industrial fermentation
processes (Volesky et al., 1993; Norris and Kelly, 1977). It has been suggested that four
broad areas of application for this type of biosorbent material may be identified and these
include; (1) detoxification of metal-bearing waste waters; (2) decontamination of radioactive
waste waters, (3) recovery of metals from ore processing solution and (4)
concentration/recovery of strategic/rare metals from sea-water. The purpose of this paper will
be to review the forms of biomass which might be used in applied processes, consider the
mechanisms by which the material functions and finally to examine the application of current
process technologies which might be employed to facilitate commercial exploitation of the
overall phenomenon.

BIOSORPTION OF HEAVY METALS BY NON-LIVING FUNGAL BIOMASS.

In considering metal biosorption by fungal biornass, or indeed any other form of biomass, the
desired attribute of the biosorbent, for applied purposes, will be an ability to take up metals
from very dilute solutions. In most cases this is determined by isotherm analysis and usually
expressed as the biosorptive capacity (mg. of metal taken up/g. dry weight of biomass) at a
known residual or equilibrium concentration. The most commonly used models applied to
biosorption of metals by biomass include those used to describe monolayer binding such as the
Langmuir and Freundlich models (Langmiur, 1918; Fmendlich, 1926). However, those forms
of data analysis are restricted by assuming monolayer interaction with the biosorbent. In certain
circumstances the data may fit more accurately to the BET (Bmnauer-Emmett-Teller) model in
which the isotherms reflect multi-layer binding to the biosorbent (Brunauer et al., 1938).
Application of these models to binding or uptake studies allows a comparison between
calculated and observed biosorptive capacities.

Over the past 10-15 years it has been demonstrated that an enormous number of biosorbents
from an even more diverse range of fungi exhibit biosorption phenomena. However, if
biomass is to be considered for use as a biosorbent in commercial applications, then existing
commercial fermentations would tend to be the ideal material source. In taking the above into
consideration, the list of microorganisms for use in commercial applications becomes somewhat
less diverse. In general, it has been demonstrated that fungal biomass has a very high affinity
for metals such as uranium. It has been shown that biomass derived from commercial
fermentations involving the use of organisms such as Aspergillus niger, A. oryzae,
Penicillium chrysogenum and Saccharomyces cerevisiae may be capable of binding up to
200mg. uranium / g. dry weight of biomass (Gadd, 1990) from solutions at concentrations as
low as 0.4rnM. Recently it has been demonstrated that the filamentous fungus Talaromyces
emersonii CBS 814.70 has a maximum biosorption capacity of 240mg. uranium/g, dry weight
biomass (Bengtsson et al., 1994). Binding of other metals such as copper, lead and cadmium,
considered as a result of their pollution potential is widely reported in the literature. Again, a
large number of biomass sources have been shown to be capable of efficient uptake of these
metals. However, availability from commercial fermentations tends to restrict the list. In
general terms the overall maximum biosorption capacities for these metals are not as high as
those quoted for uranium. Recently it has been shown that waste yeast from a Canadian
brewery was capable of binding 70 mg of cadmium/g dry weight of biomass although this
degree of accumulation resulted from both metabolic-dependent and independent processes
(Volesky et al., 1993). Biomass from the filamentous fungus Rhizopus arrhizus has been
shown to be capable of binding up to 104 mg. of lead/g, dry weight of biomass (Tobin et al.,
1984).

In studying biosorption of metals by fungal biomass it has also been demonstrated that a degree
of selectivity with respect to the metal ion bound may be achieved (Gadd, 1990). It has been
demonstrated that metal cations such as Fe2+, Fe 3+, Zn2+, Cu2+, Mn2+, Mg2+ and Ca 2+ all
interfere with the ability of biomass, derived from R. arrhizus, to take up uranium (Tzesos and
 MICROBIALBIOSORPTIONOF METALS 649

Volesky, 1982; Galun et al., 1984). This observation, although not unexpected, does have
important consequences when considering the use of a biosorbent in naturally occurring
heterogeneous waste water streams.
With respect to the influence ofpH on biosorption of metals by fungal biomass, the effects are
varied. Maximum uptake of radium by P. chrysogenum was shown to occur at pH 7, whilst
little or no binding was observed at pH 2.0 (Tzesos and Keller, 1983). Again these
observations become highly relevant in considering biomass for practical use and this is
illustrated by the following example. Recently it has been demonstrated that brewery yeast may
be used to select metals from arsenopyrite leachates. Upon evaluation of the efficiency of metal
binding under various conditions it was found that 63-97% of the various cations in the leachate
were removed by the biomass at pH 5.6. However, at pH 1.2 only 30% of the metals were
removed (Larsson et al., 1992).

Although, in general, the interaction of metals with fungal biomass appears to be extremely
rapid, temperature does appear to play an important role. It has been shown that uptake of
uranium by Saccharomyces cerevisiae and Penicillium spp. increased when the temperature was
increased from 30oC to 50oC (Shumate et al., 1978; Galun et al., 1983) although uptake by
other forms of biomass appears to be temperature independent (Marques et al., 1991). Studies
in our own laboratory have demonstrated that uptake of uranium by Talaromyces emersonii
biomass remains unchanged between 15-60oC, although a significant decrease in uptake
capacity was observed at 5oC (Bengtsson et al., 1994). Although many suggested reasons for
this temperature-dependent variation in metal biosorption exist, the important point to note is the
relevance this bears to commercial applications of the technology.

STUDIES ON METAL-BIOMASS INTERACTIONS

As mentioned above, both living and non-living fungal biomass have the ability to take up
heavy metals and radionuclides. For a variety of reasons, the biosorptive capacity of dead
biomass may be greater, equivalent to or less than that of living biomass. Variations in the
uptake capacity of the biomass may depend upon the manner in which the microbial material is
pretreated prior to use. In any case, use of dead fungal biomass is somewhat analogous to
using conventional ion-exchange resins. For industrial applications the use of non-living
biomass is advantageous in that the phenomenon is immune to toxicity effects of industrial
waste water streams and it may be employed in systems already devised and in routine use (ion-
exchange). Although the use of non-living biomass has been compared with conventional ion-
exchange matrices, it should be noted that biosorption by biomass-based matrices is usually
much greater that adsorption by ion-exchange technology.

As already mentioned the efficiency of binding of the relevant component to the biomass is
greatly influenced by the manner in which that biomass is treated prior to use. Pretreatment
may consist of initially heat treating the material, usually to achieve product stability. The
material may then be reduced to a powdered form. It has been shown that heat treatment of
Penicillium sp. biomass resulted in an initial increase in the uptake rate, but had no overall effect
on the maximum biosorptive capacity (Siegel et aL, 1986). In effect, any pretreatment modality
which affects the cell wall character will, in turn affect the nature of uptake.

In earlier investigations concerned with the ehiciditation of some mechanism for binding of
metals to fungal biomass, it was suggested that uptake of uranium by biomass, derived from
Rhizopus arrhizus, resulted from initial interaction with amine nitrogens of chitin in order to
form coordination complexes (Tzesos and Voleskey, 1982). It was also suggested that
subsequent precipitation around the initially formed centres occurred as a result of changes in
the influences of hydration. Data derived from electron microscopical studies of uranium
binding to the fungal biomass demonstrated a relatively high concentration of the bound material
in the cells walls and it was possible to see microcrystals of uranium concentrated around those
650 A.P. McHALEand S. McHALE

cells walls (Strandberg et al., 1981). It was therefore concluded that cell wall components
played a major role in binding of uranium to the biomass. Studies like those described above
prompted an examination of the ability of isolated cell wall components to take up metals. Of
those components, chitin received most attention and it has been demonstrated that removal of
chitin from biomass results in a decrease in the ability of the latter to take up metal species
(Tzesos, 1986). Other materials isolated from fungal cell walls and implicated in the
biosorption phenomenon include mannans, glucans, phosphomannans, melanins and chitosan
(Gadd, 1991; Rizzo et al., 1992). In general terms binding of metals to the cell walls would
appear to result from complex interactions involving ion-exchange, van der Waals attractions
and formation of coordinate complexes.

Although fungal biomass cell walls and their components have received much attention in the
past, interactions with intracellular components should not be ignored. Fungal biomass
contains a wide variety of non-specific metal chelators and these would include intracellular
proteins, nucleic acids, microfibril components and lipophilic components. Access to
intracellular spaces by the relevant sorbate would of course be paramount in considering the
above listed components as contributors to overall metal uptake. In very recent studies in our
laboratory, we have demonstrated that exposure of non-livingforms of biomass, derived from a
thermotolerant yeast, to short and intense electric pulses increased the initial rate of binding of
uranium to the biosorbent (unpublished results). We would suggest that the application of the
electric pulses increased access of the sorbate to intracellular components. In addition to the
non-specific, intracellular metal binding components it is also important to note that fungi are
capable of producing molecules with very high affinities for certain types of metals. These
include the cadystins or small, highly specific, metal chelating peptides produced by the yeast,
Schizosaccharomyces pombe (Hayashi et al., 1992). The yeast Kluyveromyces marxianus has
been shown to produced a small metallothionein protein which confers resistance to toxic
concentrations of heavy metals (Yazgan et al., 1993). While both of the examples given above
might be considered to bear major relevance to active binding of metals by living forms of
biomass, their obvious existence and potential participation in metal binding, should not be
ruled out as significant contributors to the overall phenomenon of biosorption.

PROCESS TECHNOLOGY ASSOCIATED WITH EXPLOITATION OF

BIOSORPTION BY NON-LIVING FUNGAL BIOMASS.

Although non-living fungal biomass has been shown to have greater metal uptake capacities
than conventionally used ion-exchange resins, its use is hindered by problems associated with
the physical character of the material. These include heterogenous particle size and reduced
mechanical strength. In general terms these problems contribute to difficulties associated with
separation of the biomass from effluents and this, in turn, contributes to limitations in process
design. However, many of these problems may be overcome by using either cross-linking or
immobilization technologies. Variations in the use of cross-linking techniques employing
agents such as aldehydes, formaldehyde/urea and vinyl sulfones, have resulted in the
production of biosorbent matrices with modified functionality (Holan et al., 1993). In some
cases filamentous fungi grow naturally in pellet form and it has been suggested that Aspergillus
pellets may be used directly as metal biosorbents (Yakubu and Dudeney, 1986). However
these pellets do appear to break up with continued use and fragments give rise to flow
restrictions in continuous-flow contact vessels. Perhaps combining natural pellet formation
with chemical cross-linking techniques may prove worthy of future investigation.

Examples of immobilization of fungal biomass in polymer based matrices for use as metal
biosorbents include Aspergillus oryzae on reticulated foam particles (Kiff and Little, 1986) and
immobilizationofSaccharomycescerevisiae in alginate (Larsson etaL, 1992). Immobilization
of biomass in alginate-based systems is of particular interest since the alginate itself is an active
biosorbent. Alginate is also widely used as an immobilization matrix in commercial
fermentations.
 MICROBIALBIOSORPTIONOF METALS 651

With respect to plant design for use with biosorbents, it would appear that a dependence upon
continuous flow technology is desirable. In most cases the biosorbent is placed in either fixed
bed or fluidized bed reactor systems (Gadd and White, 1993) and these form the basis for most
proposed commercial processes. With respect to using immobilized fungal biomass, it has been
reported that immobilized particles (0.7 - 1.2mm diameter) of Rhizopus arrhizus effected
complete uranium removal from uranium-ore bioleaching solutions containing less than 300mg
of uranium per litre. Particles were shown to maintain full loading capacity over multiple
cyclings of the material (Tzesos et al., 1989). It is important to note at this stage that if the
biosorbent is to be used in a continuous or semi-continuous system then some form of
regeneration mechanism is necessary. These methods of regeneration must obviously be
relatively non-destructive for most bioremediative processes. Although the methods for
regeneration are many, the process usually involves recharging the systems by washing in acid,
base or exposure to chelating agents followed by chemical recovery of the metal (Gadd and
White, 1993). In systems where recovery of a strategic/rare metal may be the object of the
exercise, then simple incineration of the biomass-metal complex has been proposed (Brierley et
al., 1985). This may also prove to be the only alternative when the metal is bound tightly to
intracellular components.

CONCLUSION
While it has been demonstrated widely in the literature that non-living fungal biomass represents
an efficient source material for use in bioremediative processes, the development of matrices
suitable for commercial exploitation will require significant advances in matrix design and
process design technologies. For commercial purposes matrix design must be flexible in order
to achieve adaptation of a single biomass source. This, in turn, would lead to flexibility with
respect to matrix specificity and application. Regeneration of the matrix would appear to
impinge strongly upon process design and this is borne out by previous "commercial"
experiences with processes such as the ATM-BioclaimTM system (VistaTech Partnership Ltd.,
Salt Lake City, Ut., USA). Relatively recent reports suggest that mixtures of microorganisms
may represent a more realistic approach to the design of biosorbent systems, although this
would tend to further complicate characterization of such biosorption systems (Sulka et al.,
1993). Of course the application of modern molecular genetics to many microorganisms may,
in the future, influence greatly, the specificity of biosorbents particularly if applied to synthesis
of metal-specific peptides (Hayashi et al., 1992) or cell wall polymer composition. In any
event, the above mentioned advances will only serve to adapt what is already known to be a
vast, naturally occurring bioremediative potential and application of the developing technology
will be dependent upon acceptance by open-minded industrial and legislative bodies.

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