ENVIRONMENTAL

RESEARCH BRIEF

&EPA

UnlWd States

Environmental Protection

Agency -----

Research and Development

Hazardous Waste Engineering

Research Laboratory

Cincinnati OH 45268

EPA/600/M-87/012 June 1987

Biodegradation of Halogenated Hydrocarbons

Steven D. Aust* and John A. Bumpus*

Introduction and Rationalle

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 investigations 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 nonstereoselective mechanism. Because of this non-specific

mechanism and because lignin itself is a very difficultto-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 hydrocarbons, such as benzo[a]pyrene, and halo!Jenated aromatic

and aliphatic compounds sLich as DDT and Lindane,

respectively, are included in this group of resistant

chemicals. Because of their lipophilic nature, environmentally 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 othE~r 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 Unive,rsity, East Lansing, MI 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 H20 2 requiring hemeproteins (Iigninases) that are able to catalyze the oxidative depolymerization of the insol uble 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 mineralization 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 nonstereoselective nature of the lignin degrading system

appears to be due, at least in part, to a free radical

mechanism in which low molecular weight carboncentered free radicals or other active species serve as

secondary oxidants which may catalyze lignin depolymerization or oxidation of other compounds at sites that are

remote from the active site of the enzyme. Such a free
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 frele radical mechanism would,

theoretically, allow complE~te 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 (H 20 2) 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 Hnvironmentally persistent

compounds. Only the exceptional microorganism has the

ability to catalyze the initial oxidation of an environmentally 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. chrysospo!ium, 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 determine if nutrient

nitrogen deficient cultures of P. chrysosporium could

degrade a wide variety of structurally different compounds. Although degradation may bE'! measured in many

ways, mineralization of 14C-labeled or~lanic 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 14C02. 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 tha n 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 81 persistent environmental

pollutant, DDT was used to study pollutant disappearance

2

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 (hexamethylpararosaniline), 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. Th us, 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/polychlorinated 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 demonstrated 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 mineialized. 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,

rn i nera Iization experi ments demonstrated that the

temporal onset, time course, and disappearance of 14C_

PCP and 14C_00T mineralization was si milar to those

observed for 14C-lignin mineralization, suggesting that all

three mineralizations were carried out by the same

system. Second, mineralization of 14C_00T 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 H20 2requiring ligninases, isolated
Table 1. Twenty-one 14C-labeled Compounds

Degradable to 14C 2 by P. chrysosporium

polar and water sol uble 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

12 days of incubation. The total mass recovery was 92%.

Model Compound Studies

DDT Degradation

from the fungus, were also capable of oxidizing crystal

violet.

More detailed biodegradation studies were performed

using DDT as a model compound in order to thoroughly

document the extensive biodegradation of an acknowledged environmentally hazardous and pHrsistent organic

pollutant. Substrate disappearance studil3s 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 degrcldation 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.

PCP 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 measured by 14C02 evolution from 14C-glucose, they did not

stop PCP degradation from occurring. his noted that the

water sol ubility 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.

Metabolite formation was documented in hexane extracts

of cultures obtained after various incubation times. These

studies demonstrated that DDD was the predominant and,

indeed, the only metabolite formed duri'ng the first three

days of incubation. Between day 3 and day 6, the

concentration of DDD began to decline and continued to

decline for the duration of the 3D-day incubation period.

After day 3, the DDT metabolites dicofol (2,2,2-trichloro1, 1-·bis(4-chlorophenyl)ethanol), DBP (4,4'-dichlorobenzophenone), FW-152 (2,2-dichloro-1, 1-bis(4-chlorophenyl)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 DDD accumulated

in nutrient nitrogen deficient cultures of P. chrysosporium.

Although DDD was identified as an intermediate, it too

was metabolized.

<1*

<1

2*

2

3*

4*

4*

7

8

4-8

10

12

13

14

15

17

24

32

41

42

49

Percent Mineralized

Compound In 30 Days

Hexachlorobenzene

Atrazine

3,4.3',4' -Tetrach lorobiphenyl

Naphthalene

Mirex

DOE

2,3,7,8-TCDD

2-Methyl na phtha lene

p-Chlorobenzoic Acid

DDT

Chlordane

Phenanthrene

Benzo(a)Pyrene

Aroclor 1254

Lindane

Aroclor 1242

Benzoic Acid

Biphenyl

Pentachlorophenol

p-Cresol

Methoxychlor

*60 Days

Biodegradation in some experiments was also documented

by mass balance analysis which demonstrated the

presence of polar metabolitos. For example, in one

experiment, cultures of P. chrysosporium which had been

incubated with 14C-DDT for 12 days, Wert3 extracted with

hexane, followed by acidification to pH 2.0 with HCI, and

extraction with methylene chlol'ide. 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

Crystal Violet Oxidation Studies

During the course of this study, it became apparent that

the H20 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 sol uble. Additionally, its enzymatic

biodegradation can be easily assayed spectrophotometrically.

3
Our studies with one of the purified ligninase enzymes

indicated that crystal violt:H was oXidized to form a red

compound that comigrated with trimethylpararosaniline

during thin layer chromatoqraphy. 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 Mmeralization 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 mineralization increased with increasing glucose concentration.

An exception to this generalization, however, was not8d

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 biodeuradation of organic

pollutants requires the presence of a growth substrate,

there are a number of other factors concerning growth

substrates which are likeily 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 80.7,157.7,

125.7, 48.3 and 47.5 pmoles, respectively of the 1.25

nanomoles of 14C-00T originally present during 30 days

of incubation. Polyethylene glycol (PEG-4000), glycine and

benzoic acid did not support growth of the fungus or

mineralization of 14C-00T. Carbohydrate polymers may

also serve as growth substrates. The extent of mineralization, 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 insol uble 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 minEHalization of organic

pollutants.

Hydrogen peroxide is a required cofactor/activator of

ligninases. When glucose serves as ,growth substrate, the

gl ucose 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 H2 0 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 gl3nerate H 20 2 from the growth

substrate. This, of course, assumes that H20 2 availability

is rate limiting. The precise role of H20 in degradation

and the optimal conditions for growth and degradation

are topics of continuing research.

4

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 1242, Aroclor 1254, benzo(a)pyrene, and Lindane was found to occur only very slowly

when the compounds were adsorbed onto washed sea

sa nd . However, in other studies, substa ntial (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 mineralization 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-DOT as a model compound demonstrated that this compound was extensively degraded

by the fungus. Degradation was studied by DDT

disappearance, metabolite formation and disappearance, mass balance analysis, and 14C-DOT mineralization 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 mineralization, 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.
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 ligninase(s). in selected waste tl'eatment 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 th13 fact thalt 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 production

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 whllch ligninase production was

"uncoupled" from the requirement for nutrient starvation.

Second. culture conditions should be optimized for

ligninase production since some success 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. chr)/sosporium in the

biodegradation of contaminated solid matrices (soi/s and

sediments, for example) should be pursued. Preliml/lary

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 to prevent mIneralization altogether. In contrast, substantial mineralization

(40%) of 14C-napthalene was observed in coal tar

contaminated soils. Thus, a major goal of 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 H2 0 2. Since many transition metal

complexes possess catalase type activity, H20 2 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 H20 2 production. The substrate

should neither repress ligninase synthesis nor produce

excess H20 2 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 on 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 bioavailabilty of these compounds. The use of sel,ected detergents

as solubilizing agents is one recommendE!d 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. (1985),

"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," AIChE 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).

5
United States

Environmental Protection

Agency

Official Business

Penalty for Private Use $300

EPA/600/M-87/012

Cpntt~r for Environmental Research

InformatIOn

CinCinnati OH 4S268