Biodegradation of Polycyclic Aromatic Hydrocarbons by Phanerochaete Chrysosporium


PB93-191591
Biodegradation of Polycyclic Aromatic Hydrocarbons by
Phanerochaete chrysosporium1
Utah State Univ., Logan
Prepared for:
Environmental Protection Agency, Cincinnati, OH
1989
U.S. DEPARTMENT OF COMMERCE

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TECHNICAL REPORT DATA
/Pleas* rteti /niiructtont on tht rtvtru btjort compltt
1 REPORT NO 2
EPA/600/J-93/162
3 PB93-19 159 1
t title ano subtitle
Biodegradation of Polycyclic Aromatic Hydrocarbons by
Phanerochaete chrysosporium
9 REPORT DATE
6 PERFORMING ORGANIZATION cooe
7 AUThOR(S)
John A. Bumpus
8 PERFORMING ORGANIZATION REPORT NO
9 PERFORMING ORGANIZATION NAME ANO AOORESS
Biology Dept and the Biotechnology Center
Utah State University
Logan, UT
io program element no
11 CONTRACT/GRANT NO
CK-814448
12 SPONSORING AGENCY NAME ANO AOORESS
Risk Reduction Engineering Laboratory—Cincinnati , OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati , OH 45268
13 TYPE OF REPORT ANO PERlOO COVERED
Journal Artirip
14 SPONSORING AGENCV COOE
EPA/bOO/14
is supplementary notes Project Officer = Pasquale Sferra (5!
Environmental Microbiology, Vol. 55(1 ): 154-158, January
3) 569-7618; Applied and
989
18 ABSTRACT
The ability of the white rot fungus Phanerochaete chrysosporiurn to degrade
polycyclic aromatic hydrocarbons (PAHs) that are present in anthracene oil (a distil-
lation product obatined from coal iar) was demonstrated. Analysis by capillary gas
chromatography and hi gh-nerforinance liquid chromatography showed that at least 22
PAHs, including all of the most abundant PAH components present in anthracene oil,
underwent 70 to 100. disappearance during 27 days of incubation with nutrient nitrogen-
limited cultures of this fungus. Because phenanthrene is the most abundant PAH present
ly^anthracene oil, this PAH was selected for further study. In experiments in which
[ C]phenanthrene was incubated with cultures of P. chrysosporium containing anthracene
oil for 27 da^s, it was shown that 7.7* of the recovered radiolabeled carbon originally
present in [' Cjphenanthrene was metabolized to C0„ and 25.2% was recovered from the
aqueous fraction, while 56.1 and 11.0'.J were recovered from the methylene chloride and
^rticulate fractions, respectively. High-performance liquid chromatography of the
C-1 a beled material present in the methylene chloride fraction revealed that most
(91.9',) of this material was composed of polar rr.etabol i ties of [ C]phenanthrene.
These results suggest that this microorganism may be useful for the decontamination of
sites in the environment contaminated with PAHs.
17 KEY WORDS AND OOCUMENT ANALYSIS
i DESCRIPTORS
b IDENTIFIERS/OPEN ENOEO TERMS
C. COSATi Field Croup

biodegradation
polycyclic aromatic
hydrocarbons
phanerochaete
chrysosporium

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RELEASE TO PUBLIC
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UNCLASSIFIED
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CPA form 2220-1 (R»» 4-77) pkcviOui COi tiOn i t c HOL C T (

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M'lS i« JulHorut< 10 rep'OHutl ind itli ftn
icport PermiulM tax further repioduclion
muU bi obtamtd Iron tM cojy"«M o»m>
Am II I) am) Hn\ikosmi m m Mi< hohioi tx,\ J.in IW/ p l<4-|l<4.ll<:>(i: 00 0
C'opvnghl • I "WW \meiu.in Sonet v l«>i Miuohiologv
Biodegradation of Polycyclic Aromatic Hydrocarbons by
Plumerochaete chrysosporium
JOHN A HUMI'US
Hn>lin;\ /)<¦/»(// lint ill mill llif Hinti i li>inhn;\ ( ciiici . Ultili S lute llimcr\it\. I o^an. Ultih \porium to degrade pol>cvclic aromatic hvdrocar-
bons (I'Mls) that ure present in unthruicnc oil (a distillation product obtained from coal tar) was
demonstrated Allah sis h\ capillars gas chromatography and high-performance liquid chromatography
showed that at least 22 I'Mls. including all or thi- must abundant PAII components present in anthracene oil,
underwent 71) to MMlty disappearance during 27 davs of incubation with nutrient nitrogen-limited cultures of
this fundus, liccausc phenanlhreiie is the most abundant I'AII present in anthracene oil, this HAII was selected
for further stud\. In experiments in which | ,4( Iphcnunthrcnc was incubated with cultures of P chryso\ponum
containing anthracene ml lor 27 da\s. it was shown that 7,lr/< of the reco\ered radiolabeled carbon originally
present in | l'*C|phciiaiilhrene was metabolized to ,J( (), and 25 l'/< was recovered front the aqueous fraction,
while 56 I and ll.(lfi were recovered from the meth\lciie chloride and particulate fractions, respectively
High-pcrformancc liquid chrmnutography of the l4(-labeled material present in the meth\lene chloride
fraction revealed that most (VI.S)% ) of this material was composed of polar mctabolities of | uC|phenanthrene
I hese results suggest that this microorganism may he useful for the decontamination of sites in the environment
contaminated with I'Mls.
EPA/600/J—93/162
Vol No 1
PB93-191591
l'ol\c\ cIn. arnm.itic Ind local Kins 11* MIs) .no vvidespiead
cm iinniiK-ni.il pollutants I lie contamination ol an I lt)l. -soil
l^l licshwater tsinl.iv.0 water and gioundwatci I III. and
maiine environments ill h\ I'Mls li.is been shown Ol
concern to public health is the tact thai mam I' Mis or then
metabolites arc mutagenic, caicinogciiic oi both
\pparentlv the ahilitv ol micioorganisiiis lo degrade
I'Mls is a (unction ol the number ol aiom.itic lings in the
compound lor example niicrooig.inisms have been iso-
laied that are able to use naphthalene biphenvl antliiacene.
and phen.inlhiene as their .ole c.itbon source (V) However,
there are no reports ol miciooiganisnis that are able to use
PA Ms cunt.lining Unit or moie lings as their sole carbon
source (9) I his is not to sa\ that P Mis containing moie than
lour rings are not degiaded since there are numeious
reports ol niicrooiganisnis thai .ue able to metabolize lour-
and live-ring compounds provided that anothei carbon
source is present (9|
Recent studies have shown that the while rot lungus
I'luinvrtx liiicte t In \	is able Ic> degrade a wide
variety ol \enobiotic compounds including P\Hs tocarbon
dioxide (2 4-X II. IS) I he biodegradation ol these com-
pounds has been shown lo be dependent on the lignin-
degruding system ol this niicrooiganism and the initial
oxidation ol several P-MIs has been icporied to be cataly /ed
by ligninases isolated lioni /' < In \ so\i>mmm < H 14)
lo date most biodegiadatioii expeliments (the studies ol
polychlorinated biphenvl degradation I 111 and the degrada-
tion of phenolic wasies ol paper pulping processes |16| aie
exceptions) with this niicrooiganism h.ive locusc-d on its
ability lo degrade pure compounds Unfortunately, chemic;1..
contamination by mixiuies ol chemicals is much more com-
mon than contamination b> a single compound Thus, in
order lor hiodegradative processes to be ol pr.icltc.il use,
they must be able lo degrade all or most ol the individual
components ol these mixtures
Anthracene oil is a complex mixture ol PAHs that is
obtained by the Iractional distillation ol coal tar (1^) It is
similar in composition l< creosote and has been used in the
environment as a wood | .-servative In the present study it
was demonstiated that i < hr\ \<>\i>onm>i is able lo degrade
P-MIs present in unthiaccnc oil
MA I KKIAI.S AM) MKTMODS
Fungi. /' < In v stnportiiin BKM-F-1767 was obtained Ironi
the lorest Products Laboratory. U S Department ol Agri-
culture Madison. Wis I he lungus was maintained on malt
agai slants at room temperature until use Subcultures were
routinely made every W to M) days
Chemicals. I'hcnanlhrene, anthracene, i aphtiialcnc. acc-
naphthene. lUiorene, 1-methyllluorcne. (luoranthene py-
rene. and ben/o|w|lluorene were purchased I rum Aldrich
Chemical Co . Inc 'Milwaukee. Wis ) Standard Reference
Material 1W (SRM-1597). a partially characterised rcler-
ence mixture ol PAHs, was purch -.ed from the National
Pure.in ol Standards (Ciailhersbur, Md ) Anthracene oil
was a gilt Irom Rulgerswerke Akucngesellschafi (Castrop-
Rauxel. Federal Republic of Germany) Partial characteriza-
tion ol this material by the gas chromatographic (GC) and
mass spectroscopic (MS) technique*- described below re-
vealed that the major PAH components present in this
sample were diben/.ofuran (26 mg/g). fluorene (54 mg/g)
diben/othiophene (9 mg/g). phonanthrenc (172 r.g/g). anthra-
cene (32 mg/g). carba/ole (IS mg/g), 4//-cyclopenla|llluorene. ucd 1-methylpyrene PAH concen-
trations were calculated on the basis of the chromatographic

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Vol << 11)1 (ik \l) \ 11( IN ()l I'OI V( YC I IC M«>M\IK HYDKOCAKHONS 1«
peak area h> using a sianc'.ud curve loi each I' Ml It should
he noted th.il conccntiations were <..ili.iil.iiod onlv loi those
PAHs lor vv hi*, h I hoi o was .1 coiiesponding compound ol
known concentration present in SKM-I-"C Iphenan-
thrcne ill) niC 1 mmoli was puichasecl I10111 I'.ithlindci I ah-
oraiories. Inc I St I ouis Mo 1
Culture conditions. /' < lu\ \n\i>nimi>i was ineiihaicd in the
null lent mtiogcn-limilcd liquid ciilluie medium (10 ml) de-
scribed previouslv tXi .it in 2^0-ml Whealon hollies
equipped with lids wuh .1 I ellon se.il I his uiltuie medium is
.1 moditk.ihon ol th.it dc'sciihed hv I enn .unl Knk (12)
Cultures weie established hs inoculating the medium with
spoies .is described picviouslv (17i \ tot.il ol It) jil ol
anthracene oil 1*0 1111; mil 111 acetone .md llHi 11C 1 i22t>(HHt
elpmlol |''( Iphen.uithiene lin K> 2 jil ol acetone) weie .aided
to v. till 11 res ol /' i In \\n\/i.inio;i llowcvei because this
.iniount 1t1n.1l 1.0m.c11l1.1l 1011 so Mig lilei 1 ol anlhiaeciic oil
was lelh.il to /' (In \ \ii\/>i 111 inn when .iv.li.lcel on d.i\ (I
I.11	It 111 es weie allowed to tmu loi ' dav s hcloie the addition
ol anthiacenc oil ,nid |"( Iphen.intlii .iK I his pioeoduic
circumvented the ktli.il elleets ol .mthi.it.ene oil Dunne tlK
lust > d.i\s ol incubation uilluies weie allowed to giow 111
.in almosphcic ol .111 \ltu * J.n* the ciilluics weie lliished
vs 11 h oxvgen aiiel scaled loi I Ik iciii,undei ol the incubation
penod 1.1 tol.il ol Ml dav si
Mass hjlanu' analvMs I ollowing niclibation ol |1'( Iplicn-
aiilhrenc in initiii.nl nilingcn-hniilcil culluies ol /' iluwu-
\iniiiinii kiiltuies weie acielilicd with 2 diops ol coiicen-
II.tied	hvdiochloiic .iv.id .uid homogcni/cd in .1 I'oiiei-
I l\eh|em tissue hoinoee ni/ei equipped with 1 lellon pestle
idu I'oiil) I he homogenized m.iien.il was then cxiiaelcd
w uh ihiee Ml-1111 poilions ol me lh \ leiie chloiide thesc tinee
piiilions wcic stihsei|iieiiiK pooled following melhvlene
cllloildc c'Xllacllon tile niVcclllllll w.is se p.H .lie d 11 villi I lie
aqueous traction he tilli.ilion thiough class eeool S.ilelv
Sole e I 111 nil Ke se.ile ll Plod dels I lite I ll.illoll.il I Ol p Moll III
I'tospeel III 1 w.is then .idiled lo I ml poitions ol the
melhvlene ehlonde .uul .iqueous liaclioiis .uiel to the e'lilue
imcchuni tiaciion Kadioactiviiv w.is deteimined In iiquid
sv. nil 1ll.1t iv>n speeliomeli \ with .1 liquid scintillation spec-
liomelei tmoilel I S-\N01 llcckmaii Insii unients liie I ill -
leilon ( .1I1I 1 I he II niimhei technique w.is used lo com
pens.ite loi quelleh 1111:
I he .iinoiinl ol ''C (). cv ol e e'el I10111 |"( Iphcn.tnlhitne 111
e lilt III es ot /' t III \ Ml s/h >1 linn w.is ile te I lllllleel 111 sep.1l.1le
expcrinicnls b\ iimiii: eiilluie eondilions ide'iilie.il lo those
described .ihove c\cepl lh.il ciiltuie bottles weie equipped
with gas-exchange m.uulolds .ind wcie llushcd Willi oxvgen
.il *-d.iv intcivals dining the "C (). evolution assav Del.ills
ol this prueeduie have been desctihed previouslv (X 171
111*1 ('. High-pci toimancc liquid c hioniaiogiaphic (HI'I C I
analv sis of | "C Iplieii.tulhiciic .uilhi.iccuc oil .1i1d1ndiMilu.il
I'\M si.inel.irds w.is peiloimcd h\ using .111 lll'l C svstein
(S\stem (mid (ii.ulient lleckm.ini equipped Willi .1 ic-
versed-ph.ise column t4 (1 bs 1 so mm 01 4 (1 b\ 2MI mm
V^I)\C 2lll I I's 1 lie Sep .1 r.i lions (jinup llespen.i
C .1I1I 1 I he lll'l C eluiion pioui.nn consisted ol isoci.ilic
elution lor the Itrst s nun with .1 1 mixture ol .icetomtrile
m w.iter During the ne\t ^ nun the concenir.ition ol .iceto-
niirile w.is uicre.ised line.uls 10 .leetomlnle .md w.is
m.unt.lined .it this coiKenti.iiion lor Ml nun the llow i.iie
w.is 2 ml mm I he reiention times ol I' Ml st.uid.uds ,ind ol
the components ol .mlhr.ieene oil were momtoied .il 2^4 11111
In .1 i\pic.il e\perimeni. etiliuies ol /' i/mim/m/i/iiii were
eMr.icled .is described .ihiwe I he nieilulene chloride ex-
tracts were ev.ipor.iled undei .1 penile sire.1111 ol .iryon and
ledissoKeel 111 It) nil ol .icelonitrile lwent> microhteis ol
this m.iien.il was then used lor HI'l.C analysis Percent
disappe.nance was calculated h\ comparing peak areas h>
usiny a digil.il integrator (model H90A Hewlett-Packard
Co Palo \lto. C alil ) Metabolite lormation I rum |"C|
phenaiilhiene was monitored by liquid scintillation spec-
lionietrs ol the lll'l ( tractions In these experiments the
niellnlene chloride llaction was eeaporaled and ledissoleed
111 ll ^ nil ol aecioniirilc Iwentv nuiinliieis ol this m.nen I
was then used loi HI'I C analysis 1 tactions ll ml) were
collected 111 scintillation vials S.ilel> Solve till mil w.is
added lo each llaction and ladioactiv it> was determined be
liquid scintillation spectrometrs
(iC-MS anakses weie peilormed on the same samples
that weie used loi lll'l I excepl thai the samples wcic
leduccd 111 voliinie to *0 1 lo achieve this s m| ol Ihe
sample was e\apoialeil undei a jientle slieaill ol aigon to
1(MI (il in a s-inl vial lKeacti-M.il I'lcTcc Chemical Co
Kocklo-d 111 I Ihe exact volume was then detcimmcd In
usine a ltKI-(il sMinj;e I I lie Hamilton Co Keno Ncv 1
(ontiols weie Healed 111 the same mannei ()ne-micioliici
poitions weie llien used loi (iC MS analvsts CiC -MS aii.il-
v-.es weie pel loi nied hv usini; a gas 1 hi omatoi:i aph tsciies
l-l(KI V.111. in 111st 1 u me ill si equipped w it h an ion 11 ap del ee loi
(M \l "'IHKI I inmganl Ihe loni/ation voltage was 7|> eV
Sepaialion ol P Mis was achieved on a column III 2s 111111 In
ill in l)H ^ I \ W Scientific I In isoihcinial eluiion loi 2 mm
at 1INI C lollowed bv .111 uiciease ol 4 C nun unl il a tempo
atuie ol 27(1 C was achieved and this was lollowed hv
isiiiheim.il eluiion at 27(1 ( Im Is mm I he tempeiaime ol
the 1111 e'clinn poit was VHI C and helium ll ml mm 1 was ihe
camel eas Iteeaiise tile mass spec 11 a ol sevei.il I'Mis .11 e
v 11111 a 11 x indistinguishable the lin.tl stitictui.il assignments ol
seveial I'MIs 11-mel h\Iphenanlhi ene and 2-nielln Iphenaii-
thiene loi example 1 weie made 011 the basis ol companion
ol 1 etcnlutii limes ol these compounds with those ol com-
pounds piesent in SKM-lsv7 I'eicent recoveiv was calcu-
lated loi each I'Ml 011 the basis ol peak area hv using a
vt.uid,ud ciuve generated loi each l'\H (except loi two
melln Ihiphenv I congeners) bs using SKM-1W I'ecause ihe
amotinls ol two met hv Ihiphenv I congeners 111 anthracene oil
exceeded the amounts ol these compounds lound in SKM-
|sy7 the peiccnt tecoveis ol these compounds was calcu-
lated diiectlv 1111111 1 he peak aiea hv assuming that ihe (>C
ic spouse lac loi was lineal
RUNI I IS
1 lie initial 1 estills obtained bv HI'I C indicated that onlv 2
\ 2. 2. 26 and 2(P» nl the lluorene pheniinlhrene anthra-
cene earha/ole lltioiaiilhene. and pviene iniliallv present
tespeclivelv lem.uned in ctiltuies ol /' 1 hr\ sin/miium alter
27 da\s ol inciihalion lilala not shown) Also, hen/o
|nllluniene 1-melhvllluorene and :icenaphthalcnc were de-
graded to below the level ol detectiihility However, because
some P.Mls were inaelequatclv separ.ited h> HPI.C and
bee in se I'All 1dcnl1tic.il ion was dependent solelv 011 conu-
gr.it 1011 with authentic standards, subsequent analyses by
(iC -MS weie peiloimcd (I able 1)
Ihe results obtained bv CiC-MS analyses showed that 22
I'All components present 111 anthracene oil underwent ex-
tensive disappearance during 27 days ol incuhalion in nu-
trient nilrogen-hmited cultures ol /' 1 lir\ >1111111 lo
control lor nonhiolo^ical loss, conirol experiments were
peiloimcd 111 which anihracene oil was added to cultuics ol
/' i lir\ \nv/»o/mm. which were Ihen immediately extracted

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IV. BUMPUS
Am Knvirdn Mil KOBIOI
I MM I 1 Disappearance ol PAH iom|«s>nenls ol anthracene ml alter uicuh.ilion with nutrient nitrogen-limited
liquid klllllircs Ol /' ( On Miyportinii
'i Kciumtv Irom sonliols and lll-d.i\ old iiiliuris
I ompound Imol nil
(it iiliniion iniu


ui r i
hr\ \i>\/iiiriniti in

(mini
( onlrol
I"
( unirol 2"
i \pi r
1 \pi 2
Dihenzoluran 1 16X)
IS K)\
49 .*
21
46 *
11
<1
- 1
Huorcne t 1661
17 S7
7< I
iy
X7 i
12
•'l
Nl)'
1
Nl>'
Melh)Ihiphenv 1 (16X1 tunidennlied congener)
1H
Ml •
IX
r
26
Melh)Ihiphenv 1 (16X) Himdenlilied congenei)
1H "
41 •
n
47 i
26
Nl)
Nl)
y//-Xanlhene l 1X21
IK XS
XII I
h
71
2U
M>
Nl)
Melh)Idiben/oluran ilX2l lunidenlilii'd congenu)
14 2<
XX •.
ih
72 -
16
*»
Nl)
l-MelhvItluorene lIXlll
21 U^
l)7 •
12
77 :
1<
Nl)
SI)
Diben/olhiophenc ilX4i
22 <>
1(U •
y
41 •
A
26

Khenanlhrene117X1
2' -t<
1(H) 1
1(1
X7 i
12
4
4
\nlhraicne 117X1
21 N>
122 •-
14
1111 i
IX
1
i
( arbazole 1 lh7l
24 7(1
111 •
n
X<> :
11
•>

'•Methv Iphenanihrene (142)
2 17
11(1 JL
HI
4(1 •
1(1
ll
11
All-C \clopenta|i/i /lphenaiuhuiK i l'lii
2(> SI
Xl< •
X
71 :
14

1
11
4- and or v-Methv Iphen.niihiene i l^i
2h VX
110 :
12
4<, _•
14
1<
1 luor.inlhciK
Hi <2
1(IX •
111
42 I
14
21
2X
I'vrene 120-1
H 71
1111 •
7
X7 •.
H
11
III
I'hi in Inaphth.ilcni i^IUi
M 7<
•
11
72 •

m:
SI)
Men/o|ii|lUk>reni. i^l'o
u s:
>». :
7
(.1 •.
11
4
7
Ken/olAltliiorene i21<»
14
1UX •
12
*X :
1<

*
l-XlelhvIpvrene i21hi
n ii^
ItU :
2X
lM) :
211
M)
1
111
Hen/|(i|anihr.iii'ne i22Si
iy ui
120 •
211
XII •
1(1
14
' \nthr.inm oil (Mi liu r i .is nkk.l it* Ui J.o old tttil ik ill niiio^i n lunik J s u hurts ol 1' i In \ \it\i>tntwii C ulmri's I hen i vlr.u led .mil .in.il\ / v. J loi
l> Mis h\ (il MS
" Anlhr.uini ml I *0 iiij: lik 11 «.is .ulikd |<> i he mltim medium i ihI iikuKiI v. tl iinJn I he sunk londilions .is ihosi Ji mi ibid loi niilrienl nuio^m linukd iiiliuu s
ol /' I hr\i.»/>,irimii isn liKilnnk i I I ulluii midiiun dsn lOiil.uikd 11^1 I i7ii iii|! JXI iiilluici in inluhil mismhi.il fiimih ( ultuic nudiuni ».is ihm (Mi.kIoI
..ml .iiulwal ku I'\ll> h\ lit Ms
\nlhi in in- oil imi Mii; Mil i » is .iddid ki 1 il i\ old null urn milium Innilid iiilliiii's ol /' . /m s. .s;>.	( ulliin's um ihm ivn.uiid an J .in il\ /id loi
I' Uls hi (il MS
' M> Sol ill In k J
and analvzcd lor indmdu.il I'\H components (control ll In
other control experiments. anthracene oil was incubated
w ith sterile medium and Hgt'l, lor 27 da)s prior lo extraction
and analvsis Iconiiol 2l I he results I I able ll indicate thai,
in general. recoveries in most control experiments ranged
between 70 and UKK ^ lor individual PAHs whereas in
experiments in which anihracene oil was incubated with /'
t lirwinpuriiim recoveries ol individual PAHs ranged be-
tween (I and 2X'i In some cases substantial nonbiological
disappearance ol individual PAHs did occur Howcvei. even
in manv ol these cases the amount ol biodegradalion ob-
served was siibst.iiiii.ill> greater than ihe nonbiologic.il dis-
appearance observ ed in either control H>r example. onl> 46
- 15 and 4^ j. 2VJ recover) was observed in the two
controls lor diben/oluian disappearance, whereas in exper-
iments in which anthracene oil was incubated with I'
ihr\\i)\pi>riinu. less than Y'i ol the diben/oluran onginall)
present was lound alter 27 da)s ol incubation It should also
be noted that because ol very low -ecoveries in some control
experiments it was not possible lo assess by CiC'-MS the
biodegradalion ol a number ol PAH components present in
anthracene oil ll was particular^ difficult lo assa) those
P\Hs that are know n to have rel.itivelv high vapor pressures
le g . naphthalene, acenaphthene. and biphenyl) and man)
ol those that were present in only trace umounls (e g .
per)lene. triphen)lene. chrysene. acenaphthylene. ben-
/o|\p(iriiiin containing anthracene oil 1*0
mg/liler) Ihe arrow indicates the point al which supplemental
glucose (*6 mM) was added lo each culture Kach point represents
Ihe mean of four determinations ± the standard deviation Ihe
width ol the error bars is less than the diiimeler of Hie circles

-------
Vol <5 1989
IUODKiKADA I ION Oh POLYCYCUC AROMATIC HYDROCARBONS H7
2000
0
1900
500
I'll) NAN"! Ilk) Nl
?0
40	SO	40
10
F roction
H(i 2 MI'I ( oluiinn piiilik'x nl |"l Ipln.-ihinlhieno Mmo nun-
h.ilnwi i \i .inJ "l m .i niillnliiH' ihlnridc i.ul nl .i miirienl
mitogen limned ii'liul of /' i hi ui«/wimn lh.il was iiKuhaleil
«nh |"I IphiiianihreiH Ini 2~ il.iw in thv. pimnu nl .nilhi.iuno nil
(Ml nit: 11(ci i i li i
mineralization It \houlJ he noted thai the NC" label in
| l4C|phenanthrene was located exclusivek in ihe C-9 posi-
tion (1 ig 2l Ihus it v..m onlv be stated with ccrt.tintx lh.il
this carbon atom is mineralized However it should also
he noted th.it mineralization ol the C-Y carbon in
|l4C Iphen.inlhrene would requnc cleavage ol the middle
aromatic ring
Mass balance experiments were performed alter
|I4C Iphen.inlhrene was ini.uh.iied lor 27 days in a nutrient
miro^en-limiled culture ol /' < /n\ wifumim containing M)
my ol anthracene oil per liter I he total radioactivity recov-
ered was 81 ol that which was initiallv present 01 this.
25 2*"/ was water soluble ^6 l'f was lound in the methylene
chloride traction. 7 T<< was evolved as l4C0;. and 11 (Y4
was present in the nncelium In control experiments in
which | ,4C|phenanthrcne was added to culture medium and
extracted immediately. greater than SW/J of the radioactivity
was lound in the methylene chloride Ir.iclion. and when the
|l4C|phen«.Hhrene was added to 30-day-old cultures. 97.10f
of the radi i.ictiMiy was lound in the methylene chloride
fraction, while 1 SOv was lound in the mycelium and 1 W<
was found in the aqueous traction
When the methylene chloride traction was analyzed h>
HPLC (Fig 2l. 91 V/f ol the radiolabeled material present in
this fraction was lound to be polar melabolities of |I4C]-
phenanthrene. and the amount ol residual | l4C|phenan-
ihrene present (calculated based on radiochemical datal was
found to be only 4 y/< of thai which was present initially and
was in close agreement with that found in disappearance
studies (i e . 4 to VA of phenanthrene remaining)
DISCUSSION
Ihese results indicate that the wood-rotting lungus I'
c lu\st>\i><>iiwn is able to extensively degrade many ol the
PAH components ol anthracene oil Degradation ol the PaH
components ol anthracene oil was demonstrated by sub-
sir.ite disappe.il.ince and corroborative data demonstrated
that the iadiolabeled carbon ol |1'(. Iphen.inlhrene which
was ih: predominant component ol this mixture, was oxi-
dized to "CO, Furthermore, mass balance analysis and
HPLC experiments showed that | IJC|phenanthrene was
convened to nunc polar and waier-soluble metabolites
I	hat /' i /m wnpniinni was able to degrade a wide varietv
ill PAHs is consistent with previously published reports that
ha\c shown thai, in addition to |l4C|phenanlhrene. this
lungus is able to degrade |l4C|2-melhylnaphlhalenc. |I4C|
hiphenvl and |l4( |henzo|«|p\rene lo l4CO; (6 X 1X> It is
significant thai lo dale no PAH has been reported lo he
compleielv lesisi.uit to degradation b\ /' < hr\ wm/jumhui
II	has been shown lhal the abilils ol I' < Ii/iwm/iu/iii/ii to
degrade a wide vanctv ol struclurallv diverse organic com-
pounds. including a number ol I'AHs. is dependent, ai least
in part on the lignin-degrading system ol this lungus that is
cxpiessed under nutrieni-limiting conditions (2. 4-X 11. H
14. 181 Ihc lignm-degrading swem ol this lungus includes
a lanuly ol lignin peroxidases, or ligninases as they are
comnionls known, which cal.ilv/e the initial oxidalive depo-
hmcri/ation ol lignin (14) Ol particular interest are the
findings ol Haemmerli el al lH) who have shown that a
purified ligninasc is also able lo catalyze I hi initial oxidation
ol ben/o|(/|p\rene lo Ihc corresponding 1.6-. V6-. and 6 12-
hcnzo|cV(14) 1 hi>. is significant since
horseiadish peroxidase does not oxidize PAHs with ioniza-
tion potentials greater than 7 H eV and it has been suggested
that the intermediate oxidation states ol ligiunase H-8 (i e
compounds 1 and II) may he more electropositive li e .
belter oxidants) than corresponding intermediates in classi-
cal peroxidases (14) II (rue. this would mean lhal the ability
ol /' (lir\un/iornim to degrade such a broad spectrum of
environmentally persistent organic compounds may simply
be a lunction ol the ability of ligmnase to catalyze the initial
tand olten most dilhcult) oxidation of these compounds
Although Hammel el al (14) have shown lhat ligmnase
H-X does not have ihe ability to oxidize PAHs with ioniza-
tion potentials greater than 7 56 eV. 1 have shown here thai
|14C|phenanihrene. a compound with an ionization potential
greater than 8 0 eV. is oxidized to 1JCO; in nutrient nitrogen-
limited cultures ol H < lirvuispuriitiii These results demon-
strate lhat the fungus has the ability to oxidize PAHs with
ionization potentials higher than 7.56 eV and suggest that
lungal enzymes, possibly other ligninases, with oxidizing
abilities even greater than those of ligmnase H-8 may be
present in this microorganism.
Because of Us ability lo degrade a wide variety of organo-
pollutants, it has been proposed lhat P. ihr\\o\i»mum may

-------
nx HUMi'iis
Afl'l Hnvikon Mk KOHIOI
he usclul in some w.tsic treatment systems I he develop-
meni ;ind siud\ ol sWems designed U> tio.it PAM-uwl.im-
in.itcd soil .ind w.iicr vuth this lun^us aic cmrcnll> in
progress
A( KNOWI I IX.MI-MS
I hlN llM.lllh VV.|\ MippOl till In HHipCl.lIlM' .IJill' 'llll'lll ( K
X1444X 111hi\ ih, M.i/.hdons W.isti I npmi'imj: I .ihoi.ilorv (Hliie
ol Kese.irih and Detelopiiuni I S limioniiunt.il 1'ioliiiion
\t:eni\ ( inunnali Ohm I' K Siena pioieil olliiei
1 ih.ink Ici11 M.iuyti.in loi e\p«.ii sciiit.iiul .ismsI.hu.c ll.nn
liroik I.'in I'.ilik .mil J it it IKi.iik loi lcihnn.il avsistame and
Siocn I) \iim loi icuiunif lhe m.iniiMiipt
I 11 h K \ I. Kl- (IIM)
1	Andilmaii, J II . and I 1- Snndnrass l4>74 Iniidciin .nul
M|:nilii.iiKe nl polwiiiili.n .iioni.ilu li\ilioi.uhons in ilu watei
in\ironnicn! Cm Ko I imi.ni ( oiniol 5 ii'J-M
2	Arjniand. M . jnd II Sandirniann. Jr ll'X* Minuali/ation ol
i hloi oanilinc lijiiun Hilling.ill s .nul til liei i till iio.inili Hi s h\ the
u hili 101 liiiijiiis I'limn tin lim h i lit \ .< >*t'i" nnn J \pii I i md
( hem .V* 1II«-IIIM>
' llosurl. I . jnd K Hartha W4 I hi l.ili ol pciioluiin in soil
eios\sicnis p 4U_i7* In K \l Ml is led i I'lliolunn iiimo
hioloyv M.ii Mill.hi Ni» Wik
4 Huinpus. J. A.. and S I) \unI PM* Sliuliis on Ihi huHk^ia-
d.ilion ol oitMnopollul.ini* h\ .i wink inl limpis p 41(4—41(1 In
Proicidiii|:s ol the lniiin.iiion.il ( onleiciKe loi Sew I lonticis
loi lla/.udoiis U.,vii M.iii.ijiiinuii 1 l'\HIINkMl!< I S In
\ ironnienl.il 1'ioieilion \ucno ^ .Islington 1)1
^ Humpus. J . and S. I) \usl 1W? liiiiilcpad.tlion ol cn\i
ionincni.il pollutants h\ ihi while ml lun^us I'limn un hm n
* lit \	iih i .1 \ i iiient ol I lit* lijjnin ili^i.iJin^ ssstcni
lliol ssa\s ft |iw>- |7(l
h llumpus, J \ . and s I) Xusl I'IXT Miiki.iIi/.iIioii ol icialu
train enMionnieni.il pollutants h\ ,i while ml 1 linens p 14h-
1M Pi m. ceilings ol ilie Vilion.il I onleiciKi on H.iaiiiIoiis
Wastes and Il.i/.ndoiiN M.ileu.iK Ha/aidous M.ilen.iK ( ontiol
Kese.iiih InsliKiti Silui Spnnj: \ld
7 Humpus. J \.M lirii. I> S Nriuht. undS I) \usl. P>X<
UuHJegr.id.il.on ol eimronnienl.il pollutants tu ihi while lot
fungus I'limn t*n Inn n < hi \irmnt p 12(1-12^ In Innnei.i
lion .ind lie.ilinenl ol h.i/.irdous w.isic proieednnis ol ihe
elevenlh .mmi.il rcM'.mh symposiuni H'A.'NKW-X^'02X U S
I imroninenl.il Proleuion Ayemy. W.ishinylon I) C
X llumpu\, J. A., M. lien, I). Wrluhl, und S. I) Ausl. W
Ovid.ilion ol persistenl cn\iriinmcnl.il pollui.inis h\ .i while roi
lun(!iis Sileme 22X. 14 <4-14'h
l> C irnitliu. t'. h. 11>KI \roni.iln hjdroe.nbonv niel.iholiMii In
h.iileri.i lun(!i .ind .ilji.ie Ke\ Himheni IomioI J^21-*'»|
1(1	J. M.. M. A. I.tvko, und I J. Knilp. VP') Soiim
idenlihi.ilion .ind .illoi.ilion ol polMiin.le.il .irom.ilii. h>dioi..ii
hon lonipounds in (he New York City ,ieruMil niilhods .nul
.ipplii.ilioi.'* p 2(M-21^ In P W Jones .ind P l.elxr led I
I'oKnuile.ir .iioni.itii h\droi.irbons Ann -\ihoi Snenie I'uh
lislieis Ann Aihor Muh
11	Kuiiui. I). ( . 1W< Miner.ili/.ilion ol pol>ihlonn.iled hiphunN
I'lnunii'i Iniih i In \ \n\pnriiim .i littnimilWu lnnj;iis In
/\me Muroh lethnol 7.1V4—)
12	Kim. I'.. und I K Kirk 1W I ipiiiiohlu s\skin ol I'limn
mi Inn it i lin \tI\ fh>tmm mhihilion h\ o-phlh.il.ili \iiti Mi
nohiol 12.1 UI7-MW
H lljiiiiinirh. S. I) . M S A. IaiviIj. I) Sanj;ljrd. and A
Huhtir I'iKh ()\id.ition ol hen/o|.i|p\iene h\ i\ii.nilliil.u
li|!iun.isis ol I'limn nn Inn It i lawm/tninim ici.ilnl .iliohul
¦ind si.ihiliix ol li^nin.ise J Muil L hem 26l.(ilXHMilAll
14 llanimrl, K K. It Kal\anurumun. und I. K Kirk llJM>
()\id.ilion ol pohiMln .irom.ilii h\dioi.irhons .nul dihen/o|p|
iln>\m> h\ I'lmnt nn Inn h t lir\\it\iniiiiiin J Hiol (hem 261
Kiy4X-lW<2
1* IIjwIo, (J. (.. 1^71 Ihe londensed thcimc.il dnlion.m Ml)
eil p M> V'.in Nosti.ind Keinhold I o Sew Voik
l(i llmnh. \ . H . II.-iii C hunjj. I. \\ . Jiivee. und I K Kirk IW>
Deihlorin.ition ol ihloni-oi^.inn.s h\ a while lot limpis lappi
J 6X W-KI2
17 Kirk, I. K.. I-. .Sihull/, \\ . J. C »nn«ir>, 1. I-. I.iirin/. and J <.
/.iikus. l'J77 Inllueme ol v.nilurc p.ir.imeteis on h)iiiin mel.iho
Iimii h\ I'lnnn mi Inn h t lir\ Misfitimm \rih Mmohiol 117
277-2X<
IX Sunclurd, 1) . M. S. A. I.iimiIu. und A Hrihtrr WXh Kok ol
eMr.nellul.ir ligninases in hiodegr.id.ilion ol hen/o|.i|p>ieni h\
I'lniiirini Inn lr i lin \tt\iniriiini I n/> nic Miuoh lnhnol X
2IW-212
IV lien. M . and I. K Kirk. NX* I ij;nin-dei:i.idiin: en/\nu Iroin
the h>nnnoi»\iclc I'limn rm Inn h < lir\hh/miimh Hinds Su
erne 22l.hM-W>l

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DATE:

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