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
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
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:
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.
.
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.
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.
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.
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
REFERENCES