TERM PAPER

MICROBIAL PHYSIOLOGY
AND
METABOLI
SM.

WHITE ROT
SUBMITTED BY:
 SHASHI SHARMA
(RP8003B15)

INTRODUCTION

Background

One of the major environmental problems facing the world today is the
contamination of soil, water, and air by toxic chemicals. Eighty billion
pounds of hazardous organopollutants are produced annually and only 10%
of these are disposed of safely. Certain hazardous compounds, such as
polycyclic aromatic hydrocarbons (PAH), pentachlorophenols (PCP),
polychlorinated biphenyls (PCB), 1,1,1-trichloro-2,2-bis(4-
chlorophenyl)ethane (DDT), benzene, toluene, ethylbenzene, and xylene
(BTEX), and trinitrotoluene (TNT) are persistent in the environment and are
known to have carcinogenic and mutanogenic effects. It has cost
approximately $1 trillion to decontaminate toxic waste sites in the United
States using traditional waste disposal methods such as incineration and
landfilling. Due to the magnitude of this problem and the lack of a reasonable
solution, a rapid, cost-effective, ecologically responsible method of cleanup is
greatly needed. One growing mechanism of decontamination that may fit
these requirements is bioremediation. Utilizing microorganisms to degrade
toxic organopollutants is an efficient, and economical approach. The motive
is to apply these techniques in situ under field conditions on a large scale. I
will present here the use of microorganisms ( white-rot fungi) in degrading
some of the top pollutants throughout the world and the mechanisms involved
in this process.

Characterizing organopollutants
There are several classes of chemicals that have been targeted by the USEPA as
priority pollutants due to their toxic effects on the environment and human health.
Six of these include PAH, PCP, PCB, DDT, BTEX, and TNT.
PAHs are recalcitrant environmental contaminants that are generated from the
burning of fossil fuels, coal mining, oil drilling, and wood burning. They are not
water soluble, favoring sorption to soil organic matter. Because of this they have a
tendency to bioaccumulate in natural systems. They consist of 2 or more fused
aromatic rings in linear, angular, or clustered arrangements. Clustered and
angularly arranged ring structures are more stable than linear arrangements,
making them less biodegradable. Compounds with more than 3 rings are typically
extremely toxic to microorganisms and, therefore, very difficult to break down into
mineralizable substrates.
PCPs have been used as wide spectrum pesticides and wood preservatives
throughout the world. They are currently banned in most countries, however soil
contamination continues to be a problem. PCP is toxic to most organisms at
concentrations of 50ppm but some contaminated sites have concentrations greater
than 1600ppm, making them very difficult to biodegrade. PCPs are also
hydrophobic with low water solubility, increasing their persistence in the
environment.
DDT is an organochloride insecticide that was banned in the United States over 30
years ago. This chemical is persistent in the environment, however, biomagnifiying
through the food chain. Toxic effects and population declines at higher trophic
levels due to DDT have been recorded, with some studies finding stable residues in
air, water, soil, sediment, fish and birds more than 10 years after it was banned.
PCBs were used extensively until 1993 in dielectric and hydraulic fluids, flame
retardants, plasticizers, solvent extenders, textiles and printing. Estimates of total
production of this volatile, bioaccumulative toxin range from 1.3-2 million tons
worldwide.
BTEX compounds are components of gasoline and aviation fuels that are
carcinogenic and neurotoxic to most organism . They enter the environment
primarily through leaking into soil, sediment, or water from underground storage
tanks and pipelines.
TNT contamination is a major problem at many military complexes, with over
900,000 kg produced annually in the United States alone. It is toxic to most
organisms at 50ppm but some sites have concentrations of 4,000-12,000ppm.
Currently, incineration is the most effective and common remediation practice, but
this is extremely costly, in terms of dollars and energy used. All of these chemical
compounds pose a significant threat to the health and vitality of the earth system
and a sustainable method of detoxification is key.

Bioremediation

Bioremediation is defined as the application of biological processes to the treatment

of pollution. Most research within the field of bioremediation has focused on
bacteria, with fungal bioremediation (mycoremediation) attracting interest just
within the past two decades. The toxicity of many of the above- named pollutants
limits natural attenuation by bacteria, but white rot fungi can withstand toxic levels
of most organopollutant. White rot fungi is a physiological grouping of fungi that
can degrade lignin (and lignin- like substances). Four main genera of white rot
fungi have shown potential for bioremediation: Phanerochaete, Trametes,
Bjerkandera, and Pleurotu. These fungi cannot use lignin as a source of energy,
however, and instead require substrates such as cellulose or other carbon sources.
Thus, carbon sources such as corncobs, straw, and sawdust can be easily used to
enhance degradation rates by these organisms at polluted sites. Also, The
branching, filamentous mode of fungal growth allows for more efficient colonization
and exploration of contaminated soil. The main mechanism of biodegradation
employed by this group of fungi, however, is the lignin degradation system of
enzymes. These extracellular lignin modifying enzymes (LMEs) have very low
substrate specificity so they are able to mineralize a wide range of highly
recalcitrant organopollutants that are structurally similar to lignin. The fact that
these fungal enzymes work extracellularly allows them to access many of the non-
polar, non-soluble toxic compounds that intracellular processes (such as
cytochrome P450) cannot. The three main LMEs are lignin peroxidase, Mn-
dependent peroxidase, and laccase. All three of these enzyme groups are stimulated
by nutrient limitation. They are most effective at degrading lignin and lignin- like
substances when certain nutrient levels, primarily nitrogen, are low. Conversely,
activities of these enzymes are completely suppressed in media containing high
levels of nitrogen. This characteristic is advantageous for the fungi inhabiting
highly contaminated sites with very low productivity due to toxic levels of
organopollutants. Lignin peroxidase is a glycosylated heme protein that catalyzes
hydrogen peroxide-dependent oxidation of lignin-related aromatic compounds. They
have a higher redox potential than most peroxidases and so are able to oxidize a
wide range of chemicals, including some non-phenolic aromatic compounds. Mn-
dependent peroxidase also requires hydrogen peroxide to oxidize Mn2+ to Mn3+.
The Mn3+ state of the enzyme then mediates the oxidation of phenolic substrates.
Laccase, a multicopper oxidase enzyme, is the primary enzyme involved in the
degradation process. It was first described in 1883, making it one of the oldest
enzymes ever described. It uses dioxygen as an oxidant, reducing it to water and it
has the ability to catalyze the oxidation of a wide range of dihydroxy and diamino
aromatic compounds). It is most stable at a pH of 5-6 and temperature of 45°C.
However, this enzyme is still active at pH
levels as low as 4 and as high as 7. This is
beneficial in contaminated field sites with
very low pH levels. The mechanism of
biodegradation depends in part, on the
compound being degraded, but there are
some consistent steps in the process
regardless of the substrate. The ligninolytic
enzymes in white rot fungi catalyze the
degradation of pollutants by using a non-
specific free radical mechanism. When an
electron is added or removed from the
ground state of a chemical it becomes
highly reactive, allowing it to give or take
electrons from other chemicals. This
provides the basis for the non-specificity of
the enzymes and the ability of the enzymes
to degrade xenobiotics, chemicals that have never been encountered in nature. The
main reactions that are catalyzed by the ligninolytic enzymes include
depolymerization, demethoxylation, decarboxylation, hydroxylation and aromatic
ring opening. Many of these reactions result in oxygen activation, creating radicals
that perpetuate oxidation of the organopollutants. Once the peroxidases have
opened the aromatic ring structures by way of introducing oxygen, other more
common species of fungi and bacteria can mineralize the products intracellularly
into products such as CO2 and other benign compounds.

WHITE ROT FUNGI

White rot fungus is a bioremediation technology. Wood-rotting enzymes in white rot
fungus degrade a variety of pollutants. Treatment involves mixing soil with fungus
and a suitable substrate such as wood chips. White rot fungus has been tested in situ
(i.e., in place) and in an above-ground bio-reactor. Moisturized air on wood chips is
used in a reactor for biodegradation. This system is similar to composting, except
that white-rot fungus works best in a nitrogen-limited environment

One group of fungi, Phanerochaete chyrsosporium , or white-rot fungus, produces

a family of enzymes called lignin peroxidases, or ligninases, which have extensive
biodegrative properties..

White rot fungi produce extracellular phenol oxidases and can decompose lignin
efficiently. Elfvingia applanata has been successfully applied for the bioconversion
of bisphenol A, suggesting the usefulness of white rot fungi for bioremediation. In
order to attain real bioremediation, the recycling of eco molecules in the ecosystem
is important. The sophisticated symbiotic system of white rot fungi of the genera
Termitomyces with fungus growing termites is an attractive example of an efficient
natural system to be studied, in which cooperation between termites and fungi
accomplishes efficient decomposition of lignin and complete biorecycling of plant
litter

Lignocellulose is the predominant component of woody plant and dead plant

materials, and the most abundant biomass on earth. Lignin is a heterogenous and
irregular arrangement of phenyl propanoid polymer that resists chemical or
enzymatic degradation to protect cellulose. It has been thought that lignin
degradation is a rate-limiting step of carbon recycling. Microbial activity of
metabolizing lignin and its components is one of the plausible evolutional origins
for the degrading pathway of aromatic xenobiotics and/or environmental pollutants,
such as PCB and dioxin. For applied and environmental science, it is important to
understand the nature of the decomposition and biorecycling of lignocellulose by
responsible organisms.

Basidiomycetes, which cause white rot decay, are able to degrade lignin in wood.
These fungi are called white rot fungi. Lignin degradation by white rot fungi has
been extensively studied, and results revealed that three kinds of extracellular
phenoloxidases, namely, lignin peroxidase (LiP), manganese peroxidase (MnP) and
laccase (Lac), are responsible for initiating the depolymerization of lignin.1) The
expression pattern of these enzymes depends on the organisms: some secret LiP and
MnP (no Lac), whereas others secrete MnP and Lac (no LiP). In addition to lignin,
white rot fungi are able to degrade a variety of environmentally persistent
pollutants, such as chlorinated aromatic compounds, heterocyclic aromatic
hydrocarbons, various dyes and synthetic high polymers. Probably, this
degradability of white rot fungi is due to the strong oxidative activity and the low
substrate specificity of their ligninolytic enzymes. Thus, white rot fungi and their
enzymes are thought to be useful not only in some industrial processes like
biopulping and biobreaching but also in bioremediation.
EXPERIMENTS CONDUCTED:

Photographs of Basidiomes of white rot fungi.

1. Phanerochaete chrysosporium
2. Phlebia subserialis
There have been many experiments performed in the last few years to test the
degradation capabilities of white rot fungi. They are almost exclusively conducted in
laboratory, providing information about the structure, function and potential uses of
ligninolytic enzymes in bioremedation. Overall, they have succeeded in showing that
white rot fungi, and specifically, the ligninolytic enzyme system, can efficiently
remove
most organopollutants from the soil in laboratory conditions. Cajthaml et al (2002)
looked at the ability of purified enzymes to degrade several PAHs (anthracene,
phenanthrene, pyrene, fluoranthene). They found in their in vitro studies that the
ligninolytic fungi extensively degrade these compounds through both intra- and
extracellular processes. After 50 days of incubation of a culture solution there was
less
than 1% of anthracene remaining, less than 5% phenanthrene remaining, 32%
fluoranthene remaining and 7% pyrene remaining. No toxic intermediates
accumulated during the incubation and the polarity and solubility of the products
increased, something that would increase the likelihood of intracellular metabolism
by native microorganisms but could cause problems for ground water
contamination if metabolizing organisms are not present.

Levin et al (2003) tested the biodegradation of two PAHs, nitrobenzene and

anthracene, by a white rot basidiomycete, Trametes trogii. They found that the
fungus was tolerant to extremely high levels of these compounds (250-500ppm) and
it was able to metabolize 90-97% of highly concentrated anthracene and
nitrobenzene, respectively. During the primary phase of fungal growth
(trophophasic phase) and 100% of the compounds during the secondary phase of
growth (idiophasic phase). They found high of laccase and Mn-dependent
peroxidase during both the trophophasic and idiophasic growth stages in the fungal
cultures, suggesting that the enzymes are responsible for PAH degradation. They
suggested that complete degradation of PAHs by most white rot fungi likely involve
cytochrome P450 enzymes as well as ligninolytic enzymes. The presence of
cytochrome P450 and ligninolytic systems in most white rot fungi supports this
hypothesis. Another study aimed at testing removal capabilities by white rot fungi
was conducted with TNT-contaminated soil from military complexes (Boopathy,
2000). This study analyzed two different technologies used to clean up contaminated
soils: a soil slurry reactor and in situ bioremedation. The TNT concentrations in
their soil samples were 4000-12,000mg/kg, much higher than the detection limit
(0.5mg/kg). The soil slurry reactor involved mixing the contaminated soil with
molasses (carbon source), water, and oxygen and maintaining a constant
temperature (22°C) for two weeks. The in situ bioremediation technique involved
placing the soil in glass columns and infusing them with different nutrient solutions,
depending on their treatment (molasses, succinate, yeast extract water control).
Both techniques removed the TNT from the soil, but at different rates. The soil slurry
reactor removed all of the TNT (to below detection limits) within three months while
the bioremediation method took nearly 12 months to decrease TNT levels to
detection limits. Despite the time advantage of the soil slurry reactor, it is a not a
wholly attractive technique because it is relatively expensive. On the other hand, the
in situ soil column is much less expensive and easier to implement.
One bioremedation technique that has gained recognition within the past few years
is the application of spent-mushroom compost (SMC) to contaminated soil. This
method utilizes the high levels of laccase and Mn-dependent peroxidase and high
carbon contents in SMC to breakdown complex organopollutants. There are 5kg of
SMC generated for every 1kg of edible mushrooms produced, which equaled 40
Megatons of SMC. Putting this compost to use is a great recycling tool and
potentially a sustainable approach to bioremediation. Law et al (2003) used SMC
from Pleurotus pulmonarius to treat a PCP-contaminated water system. After 2 days
of incubating contaminated water samples with various concentrations of SMC, they
found that 5% SMC removed 89% of low concentration PCP samples. This was due
primarily to biodegradation by ligninolytic enzymes (70%) and biosorption by the
SMC (19%). Another study found that 5% SMC can completely remove certain PAH
compounds from contaminated soil samples after 2 days (Lau et al, 2003). These
laboratory studies provide evidence that SMC has great potential in bioremediating
heavily contaminated sites. The degradation of an endocrine disruptor, bisphenol A
(BPA), by a white rot fungus, Elfvingia applanata , which is known in Japan as
“kofuki-saruno- koshikake” mushroom.
 Decolorization of lignin and degradation of aromatic rings in lignin were
observed in the culture indicating that E. applanata is a strong lignin-
degrading white rot fungus.

Under ligininolytic condition, E. applanata secreted MnP and Lac but not

LiP.

The molecular cloning of the gene encoding MnP of E. applanata has been
reported and the production of MnP was induced at the transcriptional level
by the presence of MnII and aromatic compounds like 2,5-xylidine. When the
culture supernatant was incubated with BPA, the conversion of BPA was
observed, showing the usefulness of E. applanata for bioremediation. The
presence of either veratryl alcohol or 2,6-dimethoxyphenol in the culture
stimulated the conversion of BPA.
.

The addition of hydrogen peroxide (1mM) in the reaction mixture stimulated

the conversion only slightly, suggesting a sufficient endogenous supply of
hydrogen peroxide
in the culture. Higher concentrations of hydrogen peroxide inhibited the
conversion. The putative pathway of the conversion of BPA by MnP is shown
in Fig.
In order to attain real bioremediation, the degradation products should be
effectively mineralized and reutilized or recycled in the ecosystem. As shown in the
case of E. applanata, white rot fungi and the enzymes produced by them are useful
as primary agents in bioremediation. It is considered beneficial to learn from the
most efficient natural systems in which white rot fungi are involved in recycling
lignocellulose. A symbiotic relationship between termites and fungi is such an
attractive example of an efficient natural system. Termites (Isoptera) are extremely
abundant in tropical terrestrial ecosystems, and play important roles in biorecycling
of plant litter. The so-called fungus-growing termites belong to an evolutionary
related group of termites (Termitidae, Macrotermitinae) and are abundant in Asian
and African tropics.4) They consume more than 90% of dry wood in some arid
tropical areas and directly mineralize up to 20%of the net primary production in
wetter savannas. This group of termites has an interesting symbiotic relationship
with basidiomycete fungi of the genus Termitomyces, such that they cultivate the
symbiotic fungi within their nests. In some types of fungus-growing termites, the
“termite mushroom”, the fruiting body of Termitomyces, blooms seasonally from
termite nests. The termite mushroom is unique in nature, growing from only the
termite nests.

There have been several suggestions for the roles of the symbiotic fungi in termite
nutrition:
• decomposition of lignin;
• supply of cellulase and xylanase to work synergistically with the enzymes
produced by the termite; and
• concentration of nutrients such as nitrogen for the termite.

Many works are involved in studies of the second suggestion, also known as the
“acquired enzyme hypothesis. But some researchers questioned a part of this
hypothesis. Since an endogenouscellulase, an enzyme produced by the termite, has
recently been recognized in wood- and litter-feeding termites. It is difficult to make
generalizations on the significance of the “acquired” fungal cellulase in cellulose
digestion in fungus growing termites. The sophisticated and well-coordinated
cooperation between
the termites and the fungi enables efficient utilization of lignocellulose. The so-
called old workers forage outside the nest and collect plant litter. In the nest, young
workers masticate and ingest the collected plant litter which passes rapidly through
the termite gut without digestion. The resulting fecal pellets (primary feces) are
pressed together to form a sponge-like structure (called fungus comb). The
symbiotic fungi grow on the comb-like matrices of the fungus comb. They form
mycelia and white round and asexual conidial structures called fungus nodules. It
has been reported that the lignin content progressively decreased as the fungus
comb matured.

It has also been shown that in vitro digestibility of cellulose in a matured fungus
comb was approximately 3-fold higher than that in a newly formed one. The fungus
nodules are usually consumed by young workers, whereas the old senescent combs
are consumed by old workers to produce final feces. However, final feces are rarely
found in the nest of fungus-growing termites, suggesting the highly efficient
decomposition and the complete biorecycling of plant litter. These observations
support the finding that symbiotic fungi have the ability to degrade lignin, which
makes cellulose more easily degraded by the cellulase produced by the termite.
We are now studying the extracellular phenoloxidases produced by a symbiotic
fungus, Termitomyces albuminosus. Two genes encoding MnP (tam1 and 2) were
identified, both of which have essential amino acid residues for peroxidase activity
and MnII ion binding whereas the residue for veratryl alcohol oxidation was not
observed. These features suggestthat both genes encode typical MnP. The mRNA
level of the tam2 was higher than that of tam1, and interestingly, the expression of
tam2 was not affected by the presence of MnII in the culture medium in spite of the
induction of MnP by MnII in general. Moreover, we have found a novel peroxidase
(designated as TAP) in the culture supernatant. TAP was able to oxidize phenolic
compounds in the presence of hydrogen peroxide. However, MnII was not required
in the reaction and veratryl alcohol was not oxidized. TAP has some interesting
characteristics with respect to the optimum pH and the optimum concentration of
hydrogen peroxide, and we are now characterizing them in detail. We have
cultivated a number of the symbiotic fungi from the fungus nodules of various
termite species. Molecular phylogenetic analysis of the cultivated fungi is in
progress. Identification of fungal species in the combs without cultivation is
probably advantageous to avoid some biases introduced during their cultivation.
These phylogenetic analyses are beneficial for understanding the selection and
evolution of the associations of symbiotic fungi with fungus-growing termites.
Moreover, we are now studying the lignin-degrading enzymes of the cultivates,
particularly laccase, because laccase is the major enzyme exhibiting ligninolytic
activity in the fungus combs. The characterization of ligninolytic activity of the
symbiotic fungi is important for understanding the nature of this symbiotic
relationship, which is most successful in biorecycling the naturally occurring
ecomolecule, lignocellulose.

MECHANISM OF BIO DEGRADATION

The first published report on the degradation of chemicals by white rot fungi
demonstrated that these fungi degrade DDT, PCB, Lindane, dioxin, and benzo[a]-
pyrene. That initial publication not only demonstrated the nonspecificity of the
fungus but also showed its ability to oxidize highly chlorinated chemicals. Such
highly chlorinated chemicals are electron deficient due to the high electronegativity
of the chlorine. These chemicals must therefore be reduced before they can be
oxidized.
White rot fungus synthesizes and secretes a substrate for its own peroxidase
enzymes: veratryl alcohol (3,4-dimethoxybenzyl alcohol). The partially oxidized
veratryl alcohol (the cation free radical) then oxidizes other chemicals that are not
directly oxidized by the peroxidase enzymes.

To complete further degradation, the fungus produces organic acids that also inhibit
veratryl alcohol oxidation.

Why would the fungus produce a substrate and an inhibitor of its own enzymes?

Further research demonstrated that oxalate, the major organic acid synthesized by
white rot fungi, is also an excellent inhibitor of lignin peroxidases. Oxalate is easily
oxidized to carbon dioxide, thus its oxidation is essentially irreversible. However,
the oxidation of oxalate is a two-electron oxidation, whereas the reduction of the
veratryl alcohol cation radical consumes only one electron. Therefore, the odd
electron left in oxalate is available for other reductions. A number of electron
acceptors can thus be reduced by lignin peroxidases provided with hydrogen
peroxide, veratryl alcohol (which is now called a "mediator"), and oxalate (which
has been termed the "donor"). The reductive dechlorination of carbon tetrachloride
(a highly oxidized chemical) to the trichloromethyl radical was demonstrated with
this system. The reductive dechlorination process was accomplished using a
peroxidase, thus opening up a new field of investigation into the role of lignin
peroxidases in the metabolism of chemicals requiring reductive dechlorination.

Molecular oxygen can also be reduced by the oxalate radical to give superoxide, a
species of oxygen that is useful for either oxidations or reductions. At low pH,
superoxide is an excellent oxidant. Alternatively, superoxide is a good reductant,
especially at higher pH, for reductive dechlorinations.

Superoxide is also useful to generate another powerful oxidant, the hydroxyl

radical. In the presence of transition metals, such as iron, superoxide can catalyze
the generation of hydroxyl radicals by a sequence of reactions called the Haber-
Weiss reaction.

Because white rot fungi also produce hydrogen peroxide, it may not be necessary to
dismutate superoxide to produce hydrogen peroxide. In such a case, the hydroxyl
radical is simply produced by the last reaction in the sequence using Fe2+ and
hydrogen peroxide, Fenton's reagent.
The production of oxygen radicals by white rot fungi had been suggested earlier by
several investigators. However, the source of oxygen radicals remained unknown.
Now the involvement in the biodegradation of environmental pollutants must be
elucidated. Polyaromatic hydrocarbons present a case in point. Some of these
chemicals, such as in coal tar and creosote, have oxidation potentials too high to be
oxidized by the lignin peroxidases. However, polyaromatic hydrocarbons are
oxidized by the fungi, and there is evidence that the lignin peroxidases are indeed
involved in their oxidation. Perhaps polyaromatic hydrocarbons are being oxidized
by the hydroxyl radicals.

These mechanisms may also explain why the peroxidases catalyze the
depolymerization of lignin rather than polymerization, as well as dehalogenation
rather than halogenation. The presence of reductants would help form reduced, not
polymerized, products of radicals.

LIMITATIONS AND CONCERNS

A major limitation of white-rot fungus is its sensitivity to biological process

operations. It has been observed that the fungus does not grow well in suspended
cell systems, enzyme induction is negatively affected by mixing action; and the
ability of the fungus to effectively attach itself to fixed media is poor. High
concentrations of TNT in the contaminated media, toxicity inhibition, chemical
sorption, and competition with indigenous microbes also can limit applicability. The
transformations by white-rot fungus are known to be slow and the full potential of
its catabolic activity has not been reported widely in the field.

High contaminant concentrations and heavy metals in the soil may be toxic to the
fungus. The degradation of contaminants may not be sufficient to meet clean up
levels. Low ambient temperatures can decrease biodegradation rates. Competition
from native bacterial populations and sorption of the contaminant may limit
effectiveness. Fungi are susceptible to water stress. Little is known of the ability of
the white rot fungus to compete with other forms of fungi.
TECHNOLOGY TRANSFER
Interest in the application of white rot fungi for the bioremediation of hazardous
waste sites is growing. Researchers at Utah State University have patented the
application of white rot fungi for biodegradation of many environmental pollutants,
and additional patents are pending for other chemicals treatable by the fungi. Other
laboratories, agencies, and environmental restoration companies are also
investigating this technology and other potential applications. Field trials under the
EPA SITE program are being conducted under the auspices of the Risk Reduction
Engineering Laboratory in Cincinnati, Ohio.

The technology, however, must not only prove feasible in the field, but it must also
be cost effective compared with traditional or alternative solutions. White rot fungi
are ubiquitous and are constantly recycling carbon fixed within lignin of plants.

The application of white rot fungi is expected to be relatively economical; the fungi
can be grown on a number of inexpensive agricultural or forest wastes such as corn
cobs and sawdust. The fungi inoculum can also be mass produced by current
techniques used to produce other fungal spawn. In the quest for economical and
ecologically sound methods for environmental remediation, the use of white rot
fungi may not be such a rotten idea.

FIELD APPLICATIONS

A major concern associated with the budding field of bioremediation is the

applicability of the laboratory research to actual large-scale contaminated field
sites. The majority of the research on fungal performance has been conducted on
autoclaved soil or on synthetic media. While the results overwhelmingly show white
rot fungi to be efficient and successful at degrading highly toxic, complex
organopollutants in these conditions, the results may not be as significant when all
the natural environmental variables are taken into consideration (native soil fauna,
temperature, moisture, pH) (Reddy and Mathew, 2001; Hestbjerg et al, 2003). Very
few studies actually test biodegradative capabilities under field conditions. One
problem with field application deals with the strict growth conditions required for
most white rot fungi. For example, Phanerochaete chrysosporium, a major
bioremediator, has very high temperature requirements (30-37°C) for growth and
ligninolytic enzyme production (Hestbjerg et al, 2003). Many white rot fungi have
low competitive capabilities in the environment, as well. However, certain genera
may prove useful for field application. Species of Pleurotus (major genera used for
edible mushroom production and, hence, SMC) have much lower temperature
requirements for growth and enzyme production and are less affected by native soil
organisms than most other fungal species (Hestbjerg et al, 2003). Another major
difficulty encountered when performing field studies are the complications
surrounding chemical transformations by other soil microorganisms. Determining
the chemical transformations from the fungi of interest has proven to be extremely
tricky in field conditions. Radiolabelling pollutants to determine their fates is one
potential solution, but it is confounded by high rates of sorption to organic matter
and may not accurately represent field conditions since it is adding ‘fresh’ pollutant
to the soil community (Reddy and Mathew, 2001). Results from field studies,
however basic, are extremely valuable for directing future research and for
demonstrating complications that arise when bioremediation is applied at a large
scale. The base of knowledge on bioremediation capabilities of white rot fungi is
growing rapidly from laboratory studies so the next step is to utilize this pool of
information in an exploratory way in the field. Considering the serious
consequences on human and ecosystem health that some of the above- mentioned
contaminants create, the sooner we find a set of preliminary sustainable solutions,
the better. White rot fungi may play a large role in this search, providing an
environmentally- friendly, economical approach that we are really just beginning to
understand.

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