J. Chem. T echnol. Biotechnol.
1998, 71, 356È367
Extended Summaries
Biochemistry of Bioremediation by Fungi
T he following are summaries of papers presented at a meeting organised by Dr Alan Bunch of the Industrial Biochemistry
and Biotechnology Group of the Biochemical Society and Professor Chris Bucke of the SCI Biotechnology Group, held at
Belgrave Square on 21 May 1997.
Introduction
Christopher Bucke
Fungal Biotechnology Group, School of Biological and Health Sci-
ences, University of Westminster, 115 New Cavendish Street, London
W1M 8JS, UK
Bioremediation, in the broadest sense, is the normal
ecological role of fungi. After plant materials have been
comminuted by “shreddersÏ in the environment, such as
insects and crustacea, fungi take over the degradation
process. It is estimated that leaf litter on the forest Ñoor
may contain 1 kilometre of fungal hyphae per gram.
This indicates the ability of fungi to invade their
environment, a valuable attribute in the development of
bioremediation processes based on fungal biocatalysts.
In spite of this, fungi have not yet been used on any
signiÐcant scale in bioremediation, in fact during the
early stages of the development of this meeting the
phrase “A resource under-utilisedÏ was appended to the
title. Reasons for this neglect of a potentially valuable
resource are obscure and one objective in organising the
meeting was to attempt to clarify this stage of a†airs.
Fungi are widely used in industry : the obvious
example is the production of penicillin but there are
many less-apparent but important fungal products, in
particular enzymes. Many more than 50% of the
enzymes used in large-scale processes such as glucose
syrup production, fruit juice and vegetable oil extrac-
tion, fat modiÐcation, animal feed modiÐcation and,
more recently, pulp and paper production are of fungal
origin. Technology for the very large-scale culture of Ðl-
amentous strains, mainly Penicillium and Aspergillus
species, is readily available. Although the fungi grow
relatively slowly compared with bacteria, they provide
advantages in ease of downstream processing and in
handling waste biomass. The great majority of fungal
356
( 1998 SCI. J. Chem. T echnol. Biotechnol. 0268-2575/98/$17.50.
enzymes used on the commercial scale are produced
extracellularly, as are the enzymes of relevance to bio-
remediation. However, the Penicillium and Aspergillus
strains used by enzyme and antibiotic producers are the
highly developed thoroughbreds of the fungal world :
the wood-rotting, lignolytic species such as Phanero-
chaete chrysosporium, Coriolus versicolor and Bjerkan-
dera spp. grow more slowly and consequently
production of their extracellular enzymes will be rela-
tively costly. A solution to this problem would be the
cloning of the key lignolytic enzymes into the familiar
species : the feasibility of this has been demonstrated in
the cloning and expression of the lignin peroxidase
genes from Phanerochaete chrysosporium into Asper-
gillus niger.1 A great deal of process development is still
required to make the appropriate enzymes from the lig-
nocellulytic fungi available at reasonable costs. There is
little sign of UK Research Council funding being
devoted to research into the appropriate process devel-
opment, indeed it may be premature to embark on
extensive process development because the number of
fungal strains which have been investigated as bio-
catalysts or sources of biocatalysts for bioremediation is
small. Once a speciÐc bioremediation task has been
identiÐed a case may be made for intensive screening for
novel strains with properties superior to those currently
available.
It is questionable whether it is appropriate to con-
sider isolated enzymes for serious bioremediation :
speakers at this meeting showed clearly that consortia
of enzymes are needed to break down lignocellulose and
recalcitrant pollutants such as polyaromatic hydrocar-
bons. Combinations of enzymes with di†ering charac-
teristics such as pH and temperature optima and
longevity are notoriously difficult and expensive to use
e†ectively when isolated from the organisms that pro-
duced them. The obvious approach is to grow fungi in
the environment containing the pollutant or to extract
Printed in Great Britain
Extended Summaries
the pollutant and contact the extract with the growing
fungal culture : speakers provide examples of both
approaches.
In nature microbes rarely exist as pure cultures : the
kilometre of hyphae in the gram of leaf litter will consist
of several species, yet there are few examples of the
application of mixed cultures, making use of the meta-
bolic versatility of several species to degrade pollutants.
This is an important point which was revealed at this
meeting particularly in the answers to questions from
the audience : in several of the cases described materials
such as PAH and pesticides are not completely mineral-
ised by single species in vitro. It is probable that mixed
cultures will have greater ability to mineralise these
materials. It has been established (e.g. Ref. 2) that
contact between the mycelia of di†erent species inÑu-
ences the activity of oxidative enzymes in the mycelial
environment.
The fascination of fungi to the biochemist is their
great metabolic versatility. The relatively few species
mentioned in this meeting have many unusual proper-
ties. Many other potentially valuable enzymes or
enzyme complexes may await discovery.
REFERENCES
1. Almeida-Vara, E., Expression of the lipH8 gene of Phanero-
chaete chrysosporium in Aspergillus niger and Penicillium
frequentans. PhD thesis, University of Westminster,
November, 1995.
2. Griffith, G. S., Rayner, A. D. M. & Wildman, H. G.,
InterspeciÐc interaction and mycelial morphogenesis of
Hypholoma fasciculare (Agaricaceae). Nova Hedwigia, 59
(1994) 47È74.
Breakdown of Plant Polymers by Fungi and Their
Potential for Use in Bioremediation
Christine S. Evans, Robert G. Veness & Millie Ullah
Fungal Biotechnology Group, University of Westminster, 115 New
Cavendish Street, London W1M 8JS, UK
The major plant polymers of wood are cellulose, hemi-
cellulose and lignin. Relatively few micro-organisms are
able to degrade lignocellulose. The most e†ective are
the wood-rotting Basidiomycetes particularly the white-
rot fungi which degrade all the components of the cell
wall. Brown-rot fungi degrade the cellulose and hemi-
cellulose, leaving the lignin modiÐed but essentially
intact. Lignin is the most recalcitrant component of
wood.
White-rot fungi are a highly specialised group of
organisms. They belong to the Basidiomycetes which
357
include all the higher fungi that are characterised by
their sexual fruiting bodies, the mushrooms, toadstools
and brackets. The vegetative fungal hyphae cause deg-
radation within the wood structure by penetrating into
the wood cells attacking the lignocellulose polymers.1,2
During later stages of degradation the wood becomes
pale in colour as a result of the selective breakdown of
lignin exposing cellulose. Examples of the most studied
white-rot species are Phanerochaete chrysosporium,
Coriolus versicolor and some Pleurotus and Phlebia
strains.
The structure of lignocellulose in wood is complex.
Cellulose Ðbres which are composed of b-1,4-linked
glucose molecules are laid down in parallel chains in the
secondary wall layers of the wood cell wall. Di†erent
layers have the Ðbres in a di†erent orientation to
provide greater strength. Bound to the cellulose Ðbres
by hydrogen bonds are the shorter hemicellulose
branched chains composed of glucose backbones with
xylose side chains. Additional sugars present are galac-
tose, arabinose, mannose and fucose. Pectins and pro-
teins are also interlinked. Surrounding and
interdispersed amongst the cellulosic components is
lignin, a three-dimensional polymer produced by free-
radical polymerisation of phenylpropanoid monomeric
units. A wide variety of bond linkages occur within the
lignin polymer. Lignin is NatureÏs ultimate recalcitrant
molecule, as such a unique polymer with no regular
repeating bond pattern is a poor substrate for biotrans-
formation.3
White-rot fungi have evolved mechanisms to mineral-
ise lignin. The process of degradation of both cellulose
and lignin is initiated by secretion by the fungal hyphae
of small molecular mass mediators. Such molecules as
oxalic acid, hydrogen peroxide and veratryl alcohol,
produced by the hyphae, are mobile di†usible molecules
that play an essential role in preparing the substrates
for enzyme attack.4 Oxalic acid has multiple roles, such
as lowering the pH, chelating calcium from calcium
pectate and chelating manganese for reactions of
manganese-dependent peroxidases.5 Hydrogen peroxide
gives rise to oxygen free radicals by interaction with
Fe(II) in the wood cell walls. Free radicals readily cause
fragmentation of both lignin and cellulose. Di†usion of
radical cations of veratryl alcohol, arising from the cata-
lytic reactions of veratryl alcohol, hydrogen peroxide
and lignin peroxidase, enable transfer of the reactive
radicals to lignin.
However it is the speciÐc complement of lignocellu-
lolytic enzymes that is secreted by white-rot fungi that
distinguishes them from other litter and detritus-
degrading organisms. The predominent lignolytic
enzymes are lignin-peroxidases, manganese-dependent
peroxidases, polyphenol oxidases, and aryl alcohol oxi-
dases which interact synergistically to e†ect degrada-
tion. Di†erent white-rot fungi produce them in varying
amounts. Nitrogen or carbon limitation stimulates pro-