Develooifient of Water and Soil Treatment
Technolocjy Based on the Utilization of a
White-Rot, Wood Rotting punqus
(U.S.) Environmental Protection Agency
Cincinnati, OH
Aug 88
I
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EPA/600/D-88/U3
August 1988
THE DEVELOPMENT OF WATER AND SOIL TREATMENT TECHNOLOGY
BASED ON THE UTILI7.VTION OF A WHITE-ROT, WOOD ROTTING FUNGUS
By
John A. Claser
United States Environmental Protection Agency
Hazardous Waste Engineering Research Laboratory
Cincinnati, Ohio 45268
HAZARDOUS WASTE KN<". FNT.ERI'Jn RESEARCH LABORATORY
OFFICE OF RESEARCH AM) DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION ACENCY
CINCINNATI, OH 45268
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TECHNICAL REPORT DATA
fffeftr rt*d tmtnttneni on it* mnr*t btfon comfttring}
• REPORT NO.
EPA/600/D-88/143
i.
4. TITLE AND SUBTITLE
THE DEVELOPMENT OF WATER AND SOIL TREATMENT TECHNOLOGY
BASED ON THE UTILIZATION OF A WHITE-ROT, WOOD ROTTING
f-'-Jhlilli
7. AUj«O*tS)
Jo?in A. Glaser
B. PERFORMING ORGANIZATION NAME ANO ADDRESS
Hazardous Waste Engineering Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
12. SPONSORING AGENCY NAME ANO ADC
Hazardous Waste Engineering
U. S. Environmental Proteci
Cincinnati, OH 45268
>RESS
j Research Laboratory
;ion Agency
IS. SUPPLEMENTARY NOTES
16. A8ST»ACT
The wood rotting fut
selected as a candidate t
waste organic conslstuenl
this species Is attribute
llgnln rapidly. Its ablH
ture optimum. Based on 1
Investigators speculated
aromatic organic constlti
with the polychlorlnated
and other chlorinated cor
exceptional degradatlve i
the fungus Is a secondary
absence of certain nutrlc
i».
1 DESCRIPTORS
L.R4CUMENT-S ACCESSION ftO. nf.
* »o O - ~ o o .?, ; 7-i MS
S. REPORT DATE
August 1988
•, PERFORMING ORGANIZATION CODE
•.PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
13. TYPE 0« REPORT ANO PERIOD COVERED
14. SPONSORIiW AGENCY CODE
EPA/600/12
igus, Phanerochaete chrysosporlum has been
ipecles co be used as a degrader of hazardous
:s found In liquids and soils. The selection of
ible to Its rapid growth, Its ability to degrade
Lty to asexually multiply, and Its high tempera-
:he fungus' ability to degrade llgnln several
that the fungus should be able Co degrade
tents found In hazardous waste. Early studies
blphenyl mixture Arochlor 125*, DDT, Llndane
itamlnants Indicated that the fungus may have
ibllltles. The llgnln degrading ability of
r metabolic cycle that Is controlled by the
•nts.
KEY WORDS ANO DOCUMENT ANALYSIS
b.lOENTlFIERS/OPEN ENDED TERMS C. COSATI Fttld/Gioup
-
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
•19 SECURITY CLASS (THaRlportl 71. NO. OF PAGES
i UNCLASSIFIED 15
' 70 SECURITY CLASS (T*U ptf*l 77. PRICE
i UNCLASSIFIED yC£*>/*?#T"
IfA Fm 704-1 <•«.. 4-77) P«t»iOo» COITIO-
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
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THE DEVELOPMENT OF WATER AND SOIL TREATMENT TECHNOLOGY
BASED ON THE UTILIZATION OF A WHITE-ROT, WOOD ROTTING FJNCUS
John A. Claser. United States EnvtrDnmental Protection
Agency, Hazardous Waste Engineering Rese.irch Laboratory,
Cincinnati, Ohio.
INTRODUCTION
The detoxification of hazardous waste Is becoming an
Important objective In the reduction of risk associated with
such waste materials. Detoxification refers to the conversion
of a toxicant to Innocuous metabolites; It does not necessarily
mean that the target substrate has been mineralized. Minerali-
zation Is the conversion of toxicant substrates to carbon
dioxide and Inorganic products. The potential of biological
means to detoxify hazardous waste Is beginning to be realized
through recent technology developments. Greater envlrjnmental
compatibility and potentially lower cost are significant Induce-
ments permitting biological treatment technology to assume a
more competitive status for site cleanup. In spite of these
very promising aspects, biological detoxification must be recog-
nized as a fledgling technology having excellent credentials In
the areas of municipal and Industrial wastes but underdeveloped
for the treatment of mixtures of more toxic and persistent chem-
icals found as components of hazardous waste sites.
A major hazardous waste problem confronting authorities In
the United States Is the waste associated with the wood treat-
ment Industry. Depending on the age of a facility, the accumu-
lated waste can be derived from a mixture of at least three
technologies. Historically, creosote treatment was followed by
pentachlorophenol which was replaced with copper chrontated
arsenlte. Each of these technologies present Its special con-
ditions for cleanup. Creosote, derived from coal tar produc-
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tlon, usually contained a host of compounds ranging from
straight aromatic compounds to polyaroreatlc species Including
smaller quantities of aromatic nitrogen bases and an array of
phenolic compounds, 'entachlorophenol Is a potent fungicide
leading to Its selection for wood preservation technology. The
analysis of wastes derived from the pentachlorophenol technology
have Identified other potential toxic components. For our cur-
rent development efforts, we have narrowly focussed on a signif-
icant portion of the waste Including the major contributors that
are polycycllc aromatic compounds and phenols. It Is necessary
to limit the scope to permit the treatment objective to be
achleveable.
BACTERIA VERSUS FUNGI
Microorganisms (both bacteria and fungi) a^e kno-.rn to
possess a variety of detoxification skills, associated with the
utilization of new sources of energy (for Instance xenoblotics)
and the need to survive (I). M.'iny bacteria can accomplish
simple transformations on organic substrates but often fall to
complete the conversion of toxicant substrate to carbon dioxide.
The use of bacterial communities recognizes these deficiencies
through the combined use of many species where the abilities of
one species supplants the Inadequacies of another. Since the
collective action of these communities Is Important to treatment
success, It Is Important to protect them from environmental
effects that may adversely affect the communities.
Fungi have not been Investigated to any extent for use as
degraders of waste materials until recently (2). Sewage treat-
ment operations steered clear of filamentous fungi due to pro-
cessing problems and the possibility that such fungi may be
pathogenic. Exceptions to these generalization do exist. Some
sixty years ago Falck and Haag reported the ability of wood
rotting fungi to degrade phenols (3). A wood rotting basldlo-
mycetes, Tranetes versicolor, was studied, twenty-five years
ago, spectrophotometrlcally Irr an attempt to quantify Its
degrading ability (4).
A llgnlnolytlc fungus, Phanerochaete chrysosporium charac-
terized by fast growth and easy reproductive cycles degrades an
Increasing list of hazardous waste conslstuents under laboratory
conditions. This ability to degrade hazardous pollutants
appears to correlate well with the fungus' ability to degrade
llgnln, a complex natural polymer composed of phenylpropane
units that Is resistant to decay by most organisms.
Some of the more common substructures of llgnln, 1,2-aryl
dlethers, alkyl sldechalns, and connected aryl systems, resemble
the chemical structure of many persistent organic compounds
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contaminating the environment. The remarkable similarity In
structures offered a connection for several investigators to
pursue application of a white rot fungus, P_«_ chrysosporlum to
the blodegradation of hazardous waste constituents (5). The
early findings of Aust (6) and Eaton (7), have propelled the
area of application of the white rot fungus, P^ chrysosporlum to
the detoxification of hazardous waste constituents significantly.
Comparison of enzyme activity between the extracellular enzymes
of P. chrysosporturn and other peroxldases point out that the
fungus* extracellular enzymes are among the most powerful bio-
logical oxidation systems known.
TTPES OF WOOD ROTTING FUNGI
There are some 1600 different wood rotting fungi known.
These organisms are divided into three main categories: soft
rot, white and brown rotting fungi. The identification of white
or brown does not refer to the growing appearance of the fungi
but rather to the residue left after the fungus has Infested a
suitable host. The brown coloration characteristic of a brown
rot, wood rotting fungus is attributed to Incompletely degraded
llgnln and can be contrasted with the white rot fungi that have
more complete capabilities to degrade llgnln.
P._ chrysosporlum is a filamentous, white, wood rotting
fungus and has been typed to be a member of the Rymenomycetes
subclass of Basidlomycetes (8). Fungi are eukaryotlc, ie. they
possess a nuclear meabrane and as microorganisms are considered
to be plantlike without chlorophyll having no photosynthetlc
abilities (9).
WOOD ROOTING FUNGI AS CARBON STRUCTURE DEGRADERS
White rot fungi are primary wood degraders in nature (10).
The naturally occurring polymers of cellulose and llgnln are
degraded by these fungi forming the major sources of carbon to
assist fungal growth. Of the two general polymers, lignln, a
structural component of wood, is by far the more difficult to
degrade due to Its composition as a heteropolymer formed from
the cross linking of three precursor elnnamyl alcohols (11). Of
necessity, the fungus must Se able to switch its ability degrade
these various polymers as the concentration of polymer varies
with the composition of the wood. This ability for P_^
chrysosporium is controlled by the absence of certain nutrients.
Nitrogen deficiency Is generally used to Induce this secondary
metabolic cycle of llgnln utilization.
3
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THE IMPORTANCE OF EXTRACELLULAR ENZYMES
The enzyme systems responsible for the Initial attack on
llgnln require unusual abilities due to the complexity and
resistance of the llgnln structure. The 600-1000 k-dalton size
range for llgnln Is far too large to enter the cells of micro-
organisms by known transport systems. An enzyme system permit-
ting the microorganism to overcome this limitation must be
extracellular, non-specific (due to the heterogeneity and large
molecular weight of the substrate), and not susceptible to
protease destruction. Analogies with other blopolymers degrad-
ing extracellular, non-specific (due to the heterogeneity and
large systems fall since these other systems are hydrolytlc and
specific (11).
Llgnln degradation Is viewed as being accomplished In two
distinct compartments: extracellular and Intracellular. The
extracellular llgnln degrading enzymes serve to fragment llgnln
Into pieces that can be assimilated by the fungus. This model
stresses the Importance of the Individual enzyme's activity and
function. Little Is known of the Intracellular enzyme com-
ponent* that complete the conversion of the llgnln fragments
Into carbon dioxide.
Among the more Important reactions In llgnln breakdown by
P. ehrysosporium arc the cleavage of llgnln alkyl sldechalns,
ring demethylatlon, and ring cleavage. The alkyl sldechaln
cleavage Is catalyzed by a hemoproteln llgnlnase. Hydrogen
peroxide Is consumed Ir. this reaction with corresponding changes
In the enzyme absorption spectrum during catalysis attributable
to resting states and transient Intermediates Indicating a per-
oxfdatlve mechanism. Stolchlometrles of product formation as
well as hydrogen peroxide and oxygen uptake are consistent with
a radical pathway (12). These results established the one-
•slectroo oxldatlve mechanism as the primary extracellular oxi-
datlve pathway for P«_ chrysosporluc.
DEGRADATION STUDIES OF WASTE CONSTITUENTS
14
Radloresplrometrlc studies of the degradation of [U- C]
pentachlorophenol In aqueous media Indicated that the substrate
was rapidly converted to carbon dioxide. Extracellular enzyme
studies showed that pentachlorophenol is converted to the 1,4-
tetrachlorobenzoqulnone by the fungus (13). The qulnone is
difficult to quantify due to Its propensity to form charge
transfer complexes with cellular materials. Further elucidation
of the metabolic pathway Is in progress. Several aromatic
hydrocarbons, benzo[a]anthracene, pyrene. anthracene,
benso[a]pyrene and pyrelene, (potential constituents of
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creosote), were converted to carbon dioxide by the fungus In
liquid culture (14,15). This latter finding serves to differ-
entiate the fungus fro» bacterial species since few bacteria
have the ability to utilise the higher Molecular weight aromatic
polycycllcs.
LIFE CYCLE OP PHAHEROCHAETE CHRTSOSPORIUM
To adequately harness the striking abilities of the wood
rotting fungi. It Is necessary to understand their life cycle to
benefit the optimisation of the. treatment process. The life
cycle of Hymenomycetes fungi is characterised by many structures
formed during vegetative, sexual, and asexual reproductive
phases. The thallus Is the basic vegetative body of the fungus
(1).
Growth occurs in all directions rather than from an apical
point. The thallus mainly functions to asslat the absorption,
assimilation, and accumulation of food. Reproductive bodies
develop using the thallus as a base and the thallus plays a
reproductive role by becoming a reproductive structure Itself.
The fungal mycelium Is a mass of Interwoven filamentous
hyphae usually submerged In growth medium. The mycelllum passes
through three distinct stages of development. The vegetative
phase Is the longest and dominant growth phase. The highest
concentration of extracellular enzymes are secreted during the
vegetative puase. Eventually the tissues of the tertiary
mycelium differentiate Into fruiting bodies that are shed
depending on environmental conditions. Asexual reproduction can
occur anytime during the vegetative growth phase. Pj^
chrysosporlum produces asexual spores prollfically and at all
stages of the life cycle (16).
ENZYME ACTIVITY STUDIES
It has been shown that P. chrysosporlum produces at least
ten extracellular hemoprotelns and roughly half have llgninase
activity (17). The enzyme component designated H8 has been used
by several researchers to characterize the llgninase activity.
Depending on culture conditions, H8 can be displaced as the
major component In favor of H2. The extracellular hemoprotelns
have distinct amlno acid sequences hence they are separate gene
products and not merely degradation products of a single pre-
cursor. The heme components H3-HS have manganese peroxidase
activity. This three component fraction catalyzes a hydrogen
peroxide-dependent oxidation of Nn(Il) to Mn(lll) but lacks the
specificity of 118 to cleave the alkyl sidechalns. Two reportB
(18,19) of soluble manganese Ion acceleration on this fraction
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prompted further Inspection of the enzymatic activity of these
peroxldases. The current status of this research Is unclear
with respect to the reported accelerations, spectral contamina-
tion has been observed that contributes to a complex situation
for rate data Interpretation (20).
The heterogeneity of the various extracellular proteins
produced by P. chrysosporlum points to possible functional
differences aaong then Important to pollutant degradation.
Presently there are attempts to uncover substrate specificities
where Information Indicates that they say exist. Oxidation of
aro*atlc substrates by peroxldases leads to the formation of
cation radicals that may be sufficiently stable to diffuse some
distance from the active site of the ensyae. These cation
radical Intermediates can be viewed as possible "oxldant" Inter-
mediaries leading to the oxidation of other substrates at sites
remote to the enzyme and fungal hyphae.
The ability of the llgnlnase H8 to oxidise polynuclear
hydrocarbons has been related to the Lonlsatlon potential of
these compounds. When pyrene Is used as a substrate with H8
both pyrene-l,6-dlone and pyrene-l,8-dlone are the major prod-
ucts. In similar fashion, anthracene I* converted to anthra-
qulnone and bens[a]anthracene yields 7,l2-benz[a]anthraqulnone
(14). Both the pyrene-l,6-dlone and pyrene-l,8-dlone are muta-
genlc by the Ames test. When the compounds are presented as
substrates to the fungus these dlones do not accumulate.
Dlbenzodloxln and 2-chlorodlbenzodloxln are oxidised by the
H8 llgnlnase In the presence of hydrogen peroxide (14). Only
determination of radical cation Intermediates by flow cell ESR
studies was made for these compounds without any product Identi-
fication. Current work Is directed to determine whether chlori-
nated aromatlcs are substrates (21,22). Chlorinated phenols
have been found to be very suitable substrate*, and product
Identification Is under way (13). The Individual llgnlnases
have been assayed for their ability to oxidize 2,4,6-trlchloro-
phenol. The activities determined In this process are In order
of magnitude lower than those for the degradation of llgnln.
Considering, the chemical processes Involved, this Is not too
surprising. In the case of one extracellular enzyme, llgnln
degrading activity was found to Inadequately predict the ability
to oxidize 2,4,6-trlchlorophenol.
DETOXir[CATION TECHNOLOGY DEVELOPMENTS
Water Treatment
A water treatment process (MyCoR - Nycellal Color Removal)
using P. chrysosporlum Is under Investigation at the bench and
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Is slated for scale up (23,24). Based on the organism's ability
to degrade polynuclear aromatic compounds especially the multlr-
Ing compounds that may be degraded by bacterial species slowly,
the first application of this technology will be the treatment
of waste derived fro* wood treating waste sites. The patented
reactor (25) Is a specially designed rotating biological con-
tactor that utilizes ?._ chrysosporlum as the biological species
for treatment. Optimal growth conditions In the reactor for the
fungus are 40°C, a pH of 4.5 and a 100 percent oxygen atmos-
phere. Since the fungus does not have the same means to adhere
to a surface as do bacterial species, the reactor design Is
modified to permit attachment of the mycellal mass to the
plates. This reactor has been used to treat pretreated gasifi-
cation wastewater for simple color removal with hydraulic
retention times of one day, showing 122 color removal for raw
wastewater (26). Color removal was found to be dependent on
Initial color concentrations and active fungal decolorlzatlon
lifetimes. Recent results derived from the bench scale opera-
tion of this technology rhow that this reactor will degrade 250
ppm pentachlorophenol In water to 5 ppm In 8 hr (27). Pink
water associated with munitions production Is adequately
treated. Degradation of 2,4,6-trlnltrotoluene and 2,4-dlnltro-
toluene In concentrations up to 150 ppm occurs In 24 hr (27).
In both cases several sequential doses of the original concen-
tration of contaminant are removed to the same extent. .These
results Indicate that metabolism of substrate Is occurring and
not merely absorption onto the mycellil mat. Plans to scale up
this technology In the next twelve months Include pilot scale
operations to treat surrogate waste. Once operational condi-
tions are established leachate derived from an actual wood
treating site will be treated. Preliminary designs for this
treatment at full scale call for parallel treatment trains.
Soil Treatment (28.29)
The general success of solution biodegradatlon studies with
the fungus stimulated speculation that this microorganism may be
an appropriate candidate for the treatment of contaminated
soils. Attempts to Innoculate environmental matrices with non-
native microorganisms have met with varying degrees of success
(30). The elucidation of optimal practices leading to success-
ful Innoculatlon of contaminated environmental materials remains
to be discovered. At the outset of this research, P^
chrysosporlum was not known to Inhabit the soil. Due to this
general lack of knowledge of the habitat, a rather cautious
research effort was engaged to determine the ability of the
fungus to Inhabit and thrive In the soil. Recent research has
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assessed Che effects of selected soil types, temperatures, pH,
and water potentials on the growth of the fungus In sterile and
non-sterile soils. Three well characterised soils (two topsolls
and a subsoil) were used In this work. Blomass accumulations as
well as growth habit of P^ chrysosporlum were greatly Influenced
by soil type. Soil nitrogen content appears to be the primary
factor responsible for differences In fungal growth In the three
studied soils. Growth was strongly and positively correlated
with nitrogen content. This factor, therefore, appears to play
a major role In mediating the growth of the fungus In the soil,
and is easily controlled by nitrogen supplementation.
Increasing the soil water potential from -1.5 MPa to 0.03
MPa resulted In greatly Increased growth of P.^ chrysosporlum.
Research data suggest that fungal growth might benefit from soil
water potentials greater than -0.03 MPa. Hater potential Is
another easily controlled soil factor (31).
Early work Indicated that P_._ chrysos?orlu« did not grow
well In non-sterile soils; this may be attributable In part due
to ineffective competition with the"Indigenous mlcroflora.
These results were anticipated since the soil Is not the normal
habitat of P._ chrysosporlum. Lately, It has been found that
growth within the soil can be accomplished through the use of
larger quantities of Inoculum.
The white rot fungus grows over a wide range of tempera-
tures. Growth has been assessed from IC-3?°C. No growth was
observed at 10 C whereas growth significantly Increases with
temperature from 15 to 30 C. No significant difference In
growth was recorded between 30 to 39°C. Soil temperatures
under field conditions can be controlled by selecting the normal
warm months and by soil solarIzatIon.
The Initial application of the fungus to sell treatment Is
the remediation of wood treating sites. Target pollutants Iden-
tified for treatment at these sites are pentachlorophenol and
the major aromatic hydrocarbon contaminants found In creosote
(napthalene, anthracene, and phenanthrene). Creosote has been
extensively characterized and minor constituents of creosote
will be added to the dosing mlzture as the work progresses when
<*eemed necessary (32). The degradatlve ability of the fungus In
the soil will be evaluated through the measurement of evolved
carbon dioxide and material balances will be derived by the
measurement of parent compound disappearance and the determina-
tion of Identity and quantity of metabolic Intermediates.
..Initial experiments monitoring the mineralization of
[U- C] pentachlorophenol In the three soils was somewhat dis-
appointing. Roughly 51 conversion to carbon dioxide was ob-
served observed for the fungus In soil cultures at 39°C and
under an atmosphere of 1001 oxygen. This low conversion Is to
8
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be contrasted with liquid culture mineralization of SOX.
Clearly, the degradation In the soil Is more complex than we
first expected. Ve are currently gaining a sore complete knowl-
edge of the Metabolite chemistry of pentachlorophenol and how
this chemistry Is modified In the soil. There are a variety of
reasonable explanations for the observed behavior of the fungus
In the soil. Ve reserve further discussion until sufficient
supplemental Information Is gathered to clarify the overall
picture. In conclusion, this fungal system has several features
that continue to support Its use as a degrader of recalcitrant
xenoblotlcs found In contaminated soil. These aollltles Include
the following: 1) the llgnln degrading system of the fungus has
a broad substrate specificity, 2) the degrading ability Is
Induced by nitrogen starvation and, 3) the rate and extent of
degradation Is dependent on the amount of carbohydrates avail-
able to the fungus for energy production. Assessment of the
amounts and fates of residual pentachlorophenol and Its bio-
transformed products will lead to a better understanding of the
degradatlve ability of P_^ chrysosporlum leading to oxidation of
the phenol to carbon dioxide or Incorporation Into soil organic
constituents.
Future research In the soil application will Include small
scale treatment of selected pollutants at environmentally sig-
nificant concentrations, the evaluation of amendments on primary
and secondary metabolism, and the delivery of oxygen within the
soil to the growing fungus. Ancillary Investigations will In-
clude the development of analytical procedures to assay the
fungal growth within the soil, the Importance of soil steriliza-
tion to growth of the fungus, and Inoculum development.
ACKNOWLEDGED NTS
The research disclosed In this report Is the combined
efforts of several groups supported by the U.S. Environmental
Protection Agency.
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19. Passcsynskl, A., Hunyh, V.-B. and Crawford, R., 'Comparison
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J.A., "Oxidation of Persistent Aromatic Pollutants by Lignin
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of the 13th Annual Hazardous Waste Symposium, EPA/600/9-
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Ohio, 1987. pp. 522.
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of Aromatic Pollutants by Phanerochaete chrysosporium
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Hazardous Waste, ed. J.A. Claser, Lewis Publishers, Inc.,
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29. Lamar, R.T., Larsen, M.J., Kirk, T.K., and Glaser, J.A.,
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