SEPA
United States
Environmental Protection
Agency
Hazardous Waste Engineering
Research Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/M-87/012 June 1987
ENVIRONMENTAL
RESEARCH BRIEF
Biodegradation of Halogenated Hydrocarbons
Steven D. Aust* and John A. Bumpus*
Introduction and Rationale
This research brief describes investigations conducted
under a cooperative agreement between the U.S.
Environmental Protection Agency and the Department of
Biochemistry of Michigan State University. The investi-
gations focus on the use of the white rot fungus
Phanerochaete chrysosporium to degrade persistent
environmental pollutants. This naturally occurring fungus
is able to degrade lignin via a very non-specific and non-
stereoselective mechanism. Eiecause of this non-specific
mechanism and because lignin itself is a very difficult-
to-degrade compound, this research investigated the lignin
degrading effects of this microorganism to degrade many
synthetic and environmentally persistent organic
pollutants.
Microbial degradation of contaminated material using
microorganisms and/or microbial enzymes in appropriate
waste treatment systems is an effective and economical
method for the destruction of many hazardous organic
pollutants. However, some compounds are resistant to
microbial degradation. Typically, polyaromatic hydrocar-
bons, such as benzo[a]pyrene. and halogenated aromatic
and aliphatic compounds such as DDT and Lindane,
respectively, are included in this group of resistant
chemicals Because of their lipophilic nature, environmen-
tally persistent organic pollutants often accumulate in the
food chain in the body fat of animals, occupying higher
trophic levels at concentrations that are often toxic,
mutagenic and/or carcinogenic. Still other compounds are
teratogenic or otherwise interfere with reproduction. Thus,
in order for microbial treatment systems to be effective
in the destruction of these more persistent compounds.
"Center for the Study of Active Oxygen in Biology and Medicine, Department
of Biochemistry, Michigan State University, East Lansing, Ml 48824
microorganisms must be found or developed which can
degrade these chemicals.
One strategy in the search for microorganisms capable
of degrading environmentally persistent synthetic
compounds is to identify and study microorganisms that
degrade recalcitrant naturally occurring compounds. P.
chrysosporium was selected for study because it degrades
lignin, a naturally occurring organic compound that is
extremely difficult to degrade. P. chrysosporium degrades
lignin during idophasic metabolism (induced by nutrient
nitrogen, sulfur or carbohydrate starvation) by secreting
a family of unique HjOz requiring hemeproteins (ligni-
nases) that are able to catalyze the oxidative depolymer-
ization of the insoluble lignin polymer. The soluble
depolymerization products are then absorbed by the cell
and metabolized to Krebs cycle intermediates and,
ultimately, to carbon dioxide to complete the minerali-
zation process.
The lignin degrading system of this fungus appears to
possess a number of characteristics which make it
particularly suited for use in biodegradation processes.
First, the lignin degrading system is able to cleave many
types of carbon-carbon and carbon-oxygen bonds which
comprise the lignin molecule. Furthermore, bond cleavage
occurs regardless of the conformation of chiral carbons.
Thus, the lignin degrading system is non-stereoselective
as well as non-specific. Second, the non-specific and non-
stereoselective nature of the lignin degrading system
appears to be due, at least in part, to a free radical
mechanism in which low molecular weight carbon-
centered free radicals or other active species serve as
secondary oxidants which may catalyze lignin depolymer-
ization or oxidation of other compounds at sites that are
remote from the active site of the enzyme. Such a free
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radical mechanism would have a profound effect on the
biodegradation of organic pollutants if operative in the
degradation of these compounds. Typically, degradative
enzymes must possess very high affinites (low Km) in
order for biodegradation to continue until the chemical
is essentially gone. A free radical mechanism would,
theoretically, allow complete conversion of a substrate
to an oxidized product by a single enzyme. This is not
possible with enzymes exhibiting normal Michaelis-
Menton kinetics. Third, lignin is a large water-insoluble
polymer. Thus, by definition, enzymes capable of
catalyzing oxidative depolymerization of lignin must be
able to attach extracellular, insoluble substrates. This is
important because many environmental pollutants are
poorly soluble in water and are usually quite tenaciously
bound to organic substances in soil, making their
absorption by organisms quite difficult This limitation
may be circumvented in this case because the enzymes
and their activator (H202) are secreted. Fourth, the lignin
degrading system is induced under nutrient (nitrogen,
carbohydrate or sulfur) deficient conditions. Unlike many
other biodegradative systems, substrate (i.e., lignin) is not
required to be present to induce the biosynthesis of
enzymes required for its biodegradation. By analogy,
synthesis of lignin degrading enzymes which also attack
xenobiotics would not require prior exposure of the
microorganism to the xenobiotic in question, nor would
enzyme synthesis be expected to be repressed when
levels of the xenobiotic reached low concentrations Fifth,
many microorganisms possess the ability to catalyze only
partial degradation of environmentally persistent
compounds. Only the exceptional microorganism has the
ability to catalyze the initial oxidation of an environmen-
tally persistent compound as well as; all of the steps in
its degratory pathway to carbon dioxide, the ultimate
microbial degradation product in aerobic systems Thus,
the study of P. chrysosporium, a microorganism which
possessed a non-specific degratory system able to
degrade lignin to carbon dioxide, was of great interest
Procedures and Experimental Approach
A major objective of this study was to deter mine if nutrient
nitrogen deficient cultures of P. chrysosporium could
degrade a wide variety of structurally different com-
pounds. Although degradation may btt measured in many
ways, mineralization of 14C-labeled organic pollutants was
selected as the technique-of-choice to screen compounds
for their ability to be degraded by this fungus This
technique was chosen because mineralization not only
demonstrates that the compound is degraded, but that
degradation proceeds to 14CO2. Mineralization also
implies that intermediate degradation products are also
degraded. A minor drawback to the use of this technique
is the fact that mineralization is a minimal measure of
biodegradation, representing only that amount of the
compound that is completely degraded to carbon dioxide.
In fact, degradation, as measured by disappearance of
chemical would be expected to be greater than the amount
mineralized until substantial amounts of all intermediates
are also degraded. For use in more detailed biodegradation
studies of this fungus, three model compounds were
selected for specific reasons: (1) Because it is well studied
and acknowledged as a persistent environmental
pollutant, DDT was used to study pollutant disappearance
and metabolite formation. DDT was also used in studies
designed to optimize degradation; (2) Pentachlorophenol
(PCP) is toxic to many fungi, including P. chrysosporium.
Therefore, this compound was selected for use in toxicity
studies and in biodegradation studies in which PCP was
supplied at various concentrations; (3) The water soluble
triphenylmethane dye, crystal violet (hexamethylpararo-
saniline), was selected for use in biodegradation studies
utilizing purified enzymes.
Results
Mineralization Studies
To date, twenty-one 14C-labeled compounds have been
assayed for their ability to be degraded to 14C02 by P.
chrysosporium (Table 1). These studies demonstrated that
a wide range of structurally diverse compounds were
mineralized by this fungus. These studies also show that
certain structural features of these compounds affect their
biodegradability. Thus, the following generalizations can
be made: (1) Chlorination inhibits, but does not prevent
mineralization. This phenomenon was seen for the
benzoic acid/p-chlorobenzoic acid and biphenyl/polych-
lorinated biphenyl (Aroclors 1242 and 1254) pairs in
which the chlorinated compound was always mineralized
more slowly than the unchlorinated analog; (2) The
presence of a substituent other than chlorine appears
to be necessary for the mineralization of chlorinated
aromatic compounds. Hexachlorobenzene (HCB), a
perchlorinated aromatic compound, did not appear to be
mineralized by this fungus. However, pentachlorophenol,
a compound differing from HCB only by the presence of
a hydroxyl group was relatively quickly mineralized.
Similarly, the fact that PCBs were mineralized demon-
strated that hydrogen atom substituents are sufficient to
allow the initial oxidation of the organic pollutant in
question; (3) Chlorinated aliphatic compounds were also
mineralized by this fungus. Lindane and Chlordane were
mineralized at rates comparable to those observed for
the chlorinated aromatic compounds that were miner-
alized Mirex, however, a perchlorinated compound, was
poorly mineralized. Similarly, Atrazine, a widely used
herbicide was also resistant to mineralization.
Involvement of the Lignin Degrading System
Three experiments have confirmed that the lignin
degrading system of this fungus is responsible, at least
in part, for the biodegradation of organic pollutants. First,
mineralization experiments demonstrated that the
temporal onset, time course, and disappearance of 14C-
PCP and 14C-DDT mineralization was similar to those
observed for 14C-lignin mineralization, suggesting that all
three mineralizations were carried out by the same
system. Second, mineralization of 14C-DDT and 14C-PCP
was promoted in nutrient nitrogen deficient cultures
whereas it was suppressed in nutrient nitrogen sufficient
cultures. This is the same pattern of promotion and
suppression of mineralization as was seen for 14C-lignin.
Third, direct evidence for the involvement of the lignin
degrading system was provided by studies in which it
was shown that the HzOjrequiring ligninases, isolated
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Table 1.
Compound
Twenty-one
Degradable to
14C-labeled Compounds
''Czby/5. chrysosporium
Percent Mineralized
m 30 Days
Hexachlorobenzene
Atrazine
3,4,3',4'-Tetrachlorobiphenyl
Naphthalene
Mirex
DDE
2,3,7,8-TCDD
2-Methyl naphthalene
p-Chlorobenzoic Acid
DDT
Chlordane
Phenanthrene
Benzo(a)Pyrene
Aroclor 1254
Lindane
Aroclor 1242
Benzoic Acid
Biphenyl
Pentachlorophenol
p-Cresol
Methoxychlor
<1"
<1
2*
2
3'
4*
4*
7
8
4-8
10
12
13
14
15
17
24
32
41
42
49
*60 Days
from the fungus, were also capable of oxidizing crystal
violet.
Model Compound Studies
DDT Degradation
More detailed biodegradation studies were performed
using DDT as a model compound in order to thoroughly
document the extensive biodegradation of an acknowl-
edged environmentally hazardous and persistent organic
pollutant. Substrate disappearance studies demonstrated
that approximately 50% of the DDT initially present was
metabolized during the first 30 days of incubation in
nutrient nitrogen starved cultures of P. chrysosporium
Disappearance appeared to be linear for the first 18 days
of incubation after which the rate of degradation gradually
declined for the rest of the thirty-day incubation period.
Glucose, which was used as a growth substrate, was
depleted after thirty days of incubation. Supplemental (56
mM) glucose added after 31 and 61 days of incubation
resulted in substantial increased metabolism of DDT. After
90 days of incubation and two additions of glucose, less
than 1 % of the DDT originally present (1.7 ppm) was still
detectable.
Biodegradation in some experiments was eilso documented
by mass balance analysis which demonstrated the
presence of polar metabolites. For example, in one
experiment, cultures of P. chrysosporium which had been
incubated with 14C-DDT for 12 days, were extracted with
hexane, followed by acidification to pH 2.0 with HCI, and
extraction with methylene chloride. In these studies, 71%
of the recovered radioactivity was shown to be present
in the hexane fraction, while 14% and 7% were present
in the acidic methylene chloride extract and the aqueous
fractions, respectively, demonstrating the formation of
polar and water soluble metabolites. In this study, 8% of
the 14C-DDT was mineralized and less than 0.1% was
incorporated into insoluble portions of the mycelium after
1 2 days of incubation. The total mass recovery was 92%.
Metabolite formation was documented in hexane extracts
of cultures obtained after various incubation times. These
studies demonstrated that ODD was the predominant and,
indeed, the only metabolite formed during the first three
days of incubation. Between day 3 and day 6, the
concentration of ODD began to decline and continued to
decline for the duration of the 30-day incubation period.
After day 3, the DDT metabolites dicofol (2,2,2-trichloro-
1,1 bis(4-chlorophenyl)ethanol), DBP (4,4'-dichlorobenzo-
phenone), FW-152 (2,2-dichloro-1,1 -bis(4-chloro-
phenyl)ethanol) and two unidentified metabolites, were
observed in hexane extracts of cultures incubated with
DDT. In addition, two unidentified metabolites were
present in acidified methylene chloride extracts. It is
important to note that neither DDE nor ODD accumulated
in nutrient nitrogen deficient cultures of P. chrysosporium.
Although ODD was identified as an intermediate, it too
was metabolized.
POP Degradation and Toxicity Studies
In order to be of use in waste treatment systems, a
microorganism must be able to survive in the presence
of the organopollutants it is degrading. The problem is
compounded by the fact that many organic pollutants have
enjoyed widespread use precisely because of their
fungicidal or bacteriocidal ability. Pentachlorophenol was
selected for study because of its acknowledged fungicidal
ability. Mineralization studies performed at low (33 ppb)
concentrations showed that PCP was relatively quickly
degraded by P. chrysosporium. Studies using high
concentrations of PCP were hampered by the lethality of
PCP at concentrations higher than four ppm in cultures
initiated with spores. This toxicity problem was overcome
by allowing the fungus to grow in culture for six days
during which time a mycelial mat was formed. At this
time concentrations of PCP up to at least 100 ppm were
not lethal and were degraded. Although PCP at these
higher concentrations suppressed respiration, as mea-
sured by 14CO2 evolution from 14C-glucose, they did not
stop PCP degradation from occurring. It is noted that the
water solubility of PCP at acid pH is well below 100 ppm.
Thus, this concentration must be regarded as the nominal
concentration in the incubation rather than the amount
of PCP in true solution. Similar toxicity problems were
overcome in studies using crystal violet but DDT showed
no apparent toxicity at a nominal concentration of 330
ppm.
Crystal Violet Oxidation Studies
During the course of this study, it became apparent that
the H2C>2 requiring lignin degrading enzymes (collectively
known as ligninases) played a role in organic pollutant
degradation. Some of these enzymes were purified and
their ability to oxidize crystal violet was determined. Unlike
most of the compounds examined in this study, crystal
violet is very water soluble. Additionally, its enzymatic
biodegradation can be easily assayed spectrophoto-
metrically.
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Our studies with one of the purified ligninase enzymes
indicated that crystal violet was oxidized to form a red
compound that comigrated with trimethylpararosaniline
during thin layer chromatography. This red compound was
in turn, oxided to an unidentified colorless product. These
studies also confirmed the fact that the initial oxidation
of organic pollutants was accomplished by extracellular
ligninases. Furthermore, we demonstrated that oxidation
proceeded until the substrate (i.e. crystal violet) was no
longer detectable.
Effect of Growth Substrate on Mineralization of
Xenobiotics
Mineralization of lignin and of the xenobiotics examined
in this study required the presence of a growth substrate.
Typically, glucose (56 mM) was used as the growth
substrate of choice. Studies in which the glucose
concentration was varied between 23 mM and 112 mM
showed that, in general, the rate and extent of miner-
alization increased with increasing glucose concentration.
An exception to this generalization, however, was noted
when the fungus was grown on 23 mM glucose At this
concentration, the greatest initial rate of mineralization
was observed. However, the rate of mineralization quickly
declined as the concentration of glucose became limiting.
In addition to the fact that biodegradation of organic
pollutants requires the presence of a growth substrate,
there are a number of other factors concerning growth
substrates which are likely to affect biodegradation. For
example, it was shown that growth substrates differed
in their ability to support or enhance mineralization. In
these studies glucose, fructose, mannose, manitol and
glycerol all supported growth and mineralized 80.7, 1 57.7,
125.7, 48.3 and 47.5 pmoles, respectively of the 1.25
nanomoles of 14C-DDT originally present during 30 days
of incubation. Polyethylene glycol (PE!G-4000), glycine and
benzoic acid did not support growth of the fungus or
mineralization of 14C-DDT. Carbohydrate polymers may
also serve as growth substrates. The extent of mineral-
ization, after 30 days of incubation, was increased
approximately twofold, relative to glucose, when an
equivalent amount of cellulose was used as a growth
substrate. Cellulose, an insoluble polymer, is a growth
substrate for this fungus in nature Furthermore, when
grown on cellulose, it is possible that the fungus is better
able to regulate the availability and utilization of glucose
for more efficient growth and mineralization of organic
pollutants.
Hydrogen peroxide is a required cofactor/activator of
ligninases. When glucose serves as growth substrate, the
glucose oxidase system is a major source of hydrogen
peroxide. However, it is known that wood rotting fungi
possess a number of carbohydrate oxidases and other
enzymes that are able to produce H2C>2. Thus, the relative
ability of a growth substrate to support mineralization of
organic pollutants may be dependent upon the ability of
the microorganisms to generate H?02 from the growth
substrate. This, of course, assumes that H2C»2 availability
is rate limiting. The precise role of H2O in degradation
and the optimal conditions for growth and degradation
are topics of continuing research.
Biodegradation of Organic Pollutants on Solid
Matrices
Preliminary studies in which 14C-labeled substrates were
adsorbed onto selected soils and sediments were
performed in order to determine if this microorganism
could be used in the decontamination of such materials.
Initial studies showed that PCP mineralization was not
inhibited when PCP was adsorbed onto washed sea sand.
However, when adsorbed onto top soil or peat, substantial
inhibition of mineralization was observed. In other studies,
mineralization of Aroclor 1 242, Aroclor 1 254, benzo(a)py-
rene, and Lindane was found to occur only very slowly
when the compounds were adsorbed onto washed sea
sand However, in other studies, substantial (40%)
amounts of 14C-naphthalene was mineralized following its
addition to coal tar contaminated soils. These studies
indicate that the solid matrix to which an organic pollutant
is adsorbed will influence mineralization of the compound.
Some solid matrices may cause an inhibition of miner-
alization while others may even accelerate mineralization.
In any case the properties and effects of the solid matrix
to which an organic pollutant is adsorbed must be
considered in any future decontamination studies using
this fungus.
Conclusions
• Based on a mineralization assay, it was shown that
P. chrysosporium is able to degrade a broad spectrum
of structurally diverse, environmentally persistent
organic pollutants.
• Studies using 14C-DDT as a model compound demon-
strated that this compound was extensively degraded
by the fungus. Degradation was studied by DDT
disappearance, metabolite formation and disappear-
ance, mass balance analysis, and 14C-DDT minerali-
zation studies.
• The lignin degrading system appears to be responsible,
at least in part, for the non-specific biodegradative
ability of the fungus. Indirect evidence for this comes
from studies which showed that, like lignin mineral-
ization, mineralization of organic pollutants was
promoted in nutrient nitrogen deficient cultures
whereas mineralization was suppressed in nutrient
nitrogen sufficient cultures. Studies of crystal violet
oxidation by purified ligninases provided direct evidence
that the lignin degrading system is also able to degrade
some organic pollutants.
• Toxicity studies showed that the model compounds, PCP
and crystal violet, prevented growth when the fungus
was grown from spores at concentrations of four and
five ppm, respectively. However, toxicity could be
circumvented by allowing the fungal cultures to grow
for six days before addition of the compounds to be
degraded.
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Recommendations
Because of its ability to degrade a wide variety of typically
difficult-to-degrade organic pollutants, it may be possible
to use P. chrysosporium and/or its extracellular ligni-
nase(s), in selected waste treatment systems. However,
a number of concerns require additional research before
this technology can be applied in practical waste treatment
systems.
To date, most biodegradative applications using this fungus
have been hampered by the fact thai: the extracellular
ligninases are secreted in very small amounts. Thus, a
straight forward approach to increasing the biodegradative
potential of this fungus would be to increase the product ion
of the extracellular ligninases. Three approaches are
recommended. First, strain development studies should
be pursued to select strains that are hyper producers of
ligninases. An especially attractive approach would entail
development of strains in which ligninase production was
"uncoupled" from the requirement for nutrient starvation.
Second, culture conditions should be optimized for
ligninase production since some succesis in this area has
already been achieved. Third, gene cloning techniques
should be used to create microorganisms capable of
synthesizing and secreting large amounts of these
enzymes.
Research focusing on the use of P. chrysosporium in the
biodegradation of contaminated solid matrices (soils and
sediments, for example) should be pursued. Preliminary
studies show that organic pollutants adsorbed to some
solid matrices are mineralized by this fungus but at rates
that appear to be lower than those observed in aqueous
culture. Also, some sediments appear 10 prevent miner-
alization altogether. In contrast, substantial mineralization
(40%) of 14C-napthalene was observed in coal tar
contaminated soils. Thus, a major goal cif future research
should focus on what factors promote and what factors
inhibit biodegradation in different solid matrices.
Another area deserving of special attention centers on
the availability of H2C>2. Since many transition metal
complexes possess catalase type activity, H2O2 may be
limiting in systems heavily contaminated with transition
metals. The use of transition metal chelators to inhibit
catalase activity may be effective and their use in such
systems should be explored. Secondly, it is important to
consider the substrates for H2C>2 production. The substrate
should neither repress ligninase synthesis nor produce
excess H2O2 which would inactivate the ligninases. An
ideal substrate might therefore be a complex carbohydrate
that would supply a constant but appropriate concentration
of substrate for the oxidase.
Organic pollutants which are adsorbed cm solid matrices
may, in some cases, be refractory to biodegradation
because they are inaccessible to enzymatic attack or
otherwise have a low bioavailability. Thus, future work
should include studies aimed at increasing the bioavail-
abilty of these compounds. The use of selected detergents
as solubilizing agents is one recommended approach.
Most research in the field of microbial degradation of
organic pollutants has been performed at the level of the
cell. Except for a few notable exceptions, little is known
about the basic biochemistry and enzymology of these
microbial processes. During the past four years much has
been learned concerning the overall biodegradative
abilities of P. chrysosporium. Similarly, much has been
learned concerning the enzyme systems involved in
biodegradation. For exmaple, it is now known that the
ligninases are responsible for the initial oxidation of, at
least, some organic pollutants. However, many of the
biochemical and enzymological questions concerning
biodegradation remain to be answered. This area of basic
research should be pursued in order to provide a firm
understanding of these processes.
Publications
The following publications describe research supported by
Cooperative Agreement CR811464 between the United
States Environmental Protection Agency and Michigan
State University.
Bumpus, J. A., Tien, M., Wright, D. and Aust, S. D. (1 985),
"Oxidation of Persistent Environmental Pollutants by a
White Rot Fungus," Science 228, 1434-1436.
Bumpus, J. A., Tien, M., Wright, D. and Aust, S. D.,
"Biodegradation of Environmental Pollutants by the White
Rot Fungus Phanerochaete chrysosporium," Symposium
Proceedings, USEPA Eleventh Annual Research
Symposium on Toxic Waste Disposal, April 1985,
Cincinnati, OH, EPA/600/9-85/028, pp. 120-126.
Bumpus, J. A. and Aust, S. D., "Studies on the
Biodegradation of Organopollutants by a White Rot
Fungus," International Conference on New Frontiers for
Hazardous Waste Management, September 1985,
Pittsburgh, PA, EPA/600/9-85/025, pp. 404-410.
Bumpus, J. A. and S. D. Aust (1986) "Biodegradation of
Environmental Pollutants by the White Rot Fungus
Phanerochaete chrysosporium: Involvement of the Lignin
Degrading System," BioEssays (In Press).
Bumpus, J. A. and S. D. Aust (1986) "Biological Oxidations
by Enzymes from a White Rot Fungus," AlChE Symposium
Publication (In Press).
Bumpus, J. A. and S. D. Aust (1986) "Biodegradation of
Chlorinated Organic Compounds by Phanerochaete
chrysosporium, A Wood Rotting Fungus," ACS Symposium
Publication (In Press).
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Environmental Protection
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Center tor Environmental Research
Information
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