Degradation of 3,3′,4,4′-tetrachlorobiphenyl by selected white rot fungi

Chemospher¢,VoL 28, No. 6, pp. 1127-1134, 1994 Cop~ght O 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved (3045-6535/94 $6.00+0.00

Pergamon

O045-6535(94)EOO46-V

DEGRADATION

OF

3 , 3 ' , 4 , 4 ' - T E T R A C H L O R O B I P H E N Y L BY

S E L E C T E D W H I T E R O T FUNGI

B.R.M.

*Institute Academy

Vyas*,

V.

~a%ekX*,

of Microbiology,

of Sciences,

and Z I n s t i t u t e

142 20 Prague

Radioohemlstry,

M. M a t u c h a 2 and M. Bubner ~

Research Federal

of Experimental

4, The Czech

Centre

Rossendorf,

Republic

Botany,

Republic, D-01314

Czech

3Institute

of

Dresden,

of G e r m a n y

(Received in Germany 29 Novembe~1993; accepted 4 January 1994)

ABSTRACT

N-limited

stationary

chrysosporlum,

Trametss

1.393

± 0.353

(0.301

0.004

(0.002 ± 0.0008)

cultures

of

versicolor,

± 0.023)

the

t 0.061

% of the o r i g i n a l l y

3,3',4,4'-tetrachloro[U-t4C]biphenyl

(PCB

PCB

by

77

degradation

aqueous,

organic

organic pattern

soluble

was

soluble,

biomass

(intracellular)

of d i s t r i b u t i o n

C. polysona at higher d iffe r e d

followed

77)

pattern

polysona

mineralized

± 0.010),

and 0.015

30.14

during

4

associated,

nmol

was

the

dose

in

The

nmol)

extent

partitioning

± of of

into

(intracellular)

four weeks

similar

lower

(513.7

weeks.

aqueous

After

Phanerochaete

fungi

x4C-radioactivity

of r a d i o a c t i v i t y

in the d i s t r i b u t i o n

(0.112

supplied

fractions.

dose but not at

rot

Coriolopsis

and

and 0.398

white

incubation

and the

P. chrysosporium and

of PCB 77.

T. versicolor

of radiolabel.

INTRODUCTION

PCBs

are

properties develop has

a method

initiated White

also shown

inert,

a wide

chemically

attribute

towards

stable

their

for the a b a t e m e n t

intensive

rot fungi variety

studies

known

at least

soil

but m o s t l y

to degrade

of aromatic

to be attributed,

of

and h i g h l y

toxic

recalcitrance

and water employing

lignin,

were

organopollutants in part, ]127

to the

substances

[1][2].

The

pollution bacteria

found

[3][4].

ligninolytic

need

to

caused

by PCBs

(see e.g.

[2]).

to be able This

and these

dire

to degrade

ability

has been

peroxidases

[5].

1128

P.

chrFsosporium

[3] [5] [7]. by

other

higher

But

there

white

number

resistant degradation

of

and

distribution mycelial

with

chlorine

atoms

per

radiolabel

(aqueous

the

individual

PCB

77 s p i k e d .

white

and

-

species

and w a s

Chemicals.

Microorganisms ME-446

(ATCC

also

Coriolopsis

maintained

on

ma!t

triplicates

and

set-up

in d a r k flask

at 28

°C.

cultures

Cultures

agar

slants

were

was

in 2

grown

malt

(I

flask inlet

mg/mL) (Table and

consisted

Table

or

20 ~ L

i) . C o t t o n outlet

of

i.

uninoculated

I II

AnaZysis (75

mL/min,

one week;

the

were

and

°C.

(I and

Trametes

A]]

at al.

in

concentration

of

II)

i) w e r e

by mildly

solution

for

with

were

1

shake

% glucose.

wJth of

3,3',4,

preparation

( 0 o 4 4 m g / m L ) , was

rubber

in

incubated

I0 d old

spiked

of e t h a n ~ l i c

changed

and

containing

before

III w e r e

cultures

homogenizing

medium

II s o l u t i o n

sealed

chrysosporium

(in p r e s s ) ,

stoppers

used per

{fitted

paraffJn

film.

Abiotic

and

doses

of

preparations PCB

used

in the

degradation

77/flask

with

control

(uCJ/mmoi}

nmo!

33 11450

513.7 30.!4

of ~4 C-radioactivity. immediately

~g 150 8.8

All

after

head-space

air

the

studies

814000 766000

flasks

PCB-spiking containing

were and

3 6

flushed

then

evolved

3.3',4,4'-

spiked o~ day

dpm/flask

displaced

(Table

versicolor

f~ngal

[9]

characteristics

activity

30 mgn)

and

differed

flasks.

tetrachloro[U-14C]biphenyl Sp.

the

T.

final

organic),

Phanerochaete

319.36,

air

150 u L

stoppers

Radiochemical

PCB 77 Preparation

4

of p r e p a r a t i o n

vents),

and

The

METHODS

extract

under

4'-tetrachloro[U-i4C]biphenyl; I

at

to V y a s prepared

%

by

that

77.

homogenization,

influenced

conditions.

polysona

according

Inoculum

grown

showed PCB

(aqueous

after

more

investigated

e t a / . [8] .

to K l 6 t z e r

and c u l t i v a t i o n

34351),

are g e n e r a l l y

results

3,3',4,4'-tetrachloro[U-14C]bipheny!

according

(4 and

to m i n e r a l i z e

and

AND

of PCBs

3,3',4,4'-tetrachloro[U-

The

extracellular

before

MATERIALS

synthesized

able

degradation

therefore

congener

fungi.

also

as 14COz ,

organic)

fungal

PCB

We

conditions

of c h l o r i n a t i o n

structure)

[2][6][7].

rot

are

degree

biphenyl

resistant

polysona

C,

of

a

ligninolytio

pertaining

higher

degradation

selected

under

literature

PCBs

such

by

of

PCBs

fungi.

microbial

'4C]biphenyl

versicolor

is p a u c i t y

rot of

to

mineralizes

with

at an

14COz

oxygen

interval

was

bubbled

of in

1129

i0

m L of 2 M e t h a n o l a m i n e

*4C-radioactivity. and

extracted

various

All

according

fractions

Tri-Carb

2500TR

technique

with

Fig

i.

rot

fungi

Scheme

was

estimated

liquid

for

the with

flasks

to the

quench

as 1 4 C O 2 - t r a p

harvested

scheme using

shown Bray

after

in Fig.

i.

scintillation

analyzer,

I with

counted of

cocktail

using

the

for

incubation

14C-radioactivity

of the

radiolabel

in

on a P a c k a r d

quench-monitoring

in the

3,3',4,4'-tetrachloro[U-14C]biphenyl

of the

of w h i t e

I

dist. H 2 0 < washing

Biomass I ext. w i t h EA s o n i c a t i o n ( 1 0 mL,

EA

cultures

(PCB 77)

flask

I

I Filtrate(--

and

4 weeks

compensation.

fractionation

Content

ext.

were

scintillation

automatic

spiked

in m e t h y l c e l l o s o l v e

the

30 m i n

x3)

l i

i

i

Aqueous

EA ext

EA ext

$2

s3

S1

i

Biomass

l homogenized

I

ext. w i t h EA son,cation (I0 mL, 30 min x3) i

EA ext - ethyl a c e t a t e e x t r a c t s e s t i m a t i o n of L 4 C - r a d i o a c t i v i t y

dried

RESULTS

P.

chrysosporium,

PCB

77

cultures the

excepting of

PCBs

nmol

combustion

(Fig.

of

fhat the

Aqueous $5

anhydrous

i

Biomass combustion

NazSO4

were

1]sed for

the

DISCUSSION

T.

versicolor mineralized

albeit when

at

cultures

low

and

were

spiked

3,3',4,4'-

different with

r~tes.

30.14

nmol

2, Tabl ~ 2).

recovered

in the

ranged

is d i f f i c u l t

range

suggest

77)

higher

mycelium,

in the s a m e

experiments upon

513.7

raOioacfivity

recovery

~t is

(PCB

of PCB 77 w a s

than w i t h

Total

AND

C. p o l y s o n a and

tetrachloro[u-14C]biphenyl Mineralization

over

1

1

EA ext $4

from

[7],

as r e p o r t e ~ additional

different 50-80

recovery by Thomas

%. we et

radioactivity

mycelium.

Distribution

fractions

(Fig.

Considering achieved a~.

(ca. of

was

[7]. O u r

10%)

could

th~

I)

of

fact

the that

appreciable; preliminary be

recovered

~4C-radJoactivi~y

~n

1130

5O

12

--~-

~0 ~0

A

C.polysona

~ 10

o x40.

P~chn/sosporium 0 •

8"

~ ~ 0

6-

~

4

-,-t

0 -,-'1

~0 3Q)5 -,-I

T.

~25' ~ ~20o.. 15:>4J 0

~10~;~ 5

O) U ~ 0~ 0 -,--I

2"

00

Flg.

5

10

15 days

20

25

30

5

10

15

20 clays

2. M i n e r a l i z a t i o n of 3 , 3 ' , 4 , 4 ' - t e t r a c h l o r o [ U - * 4 C ] b l p h e n y l

(A)higher

(513.7 nmol/flask)

and

(B)lower

{30.14 nmol/flask)

the s t a t i o n a r y cultures of the white rot fungi c u l t i v a t e d Arrow indicates

25

30

35

(PCB 77) at its

concentration

by

in N - l i m i t e d medium.

the time of PCB 77 spiking

~O

r~O ~,-~ 0 4J X

8'

--A-7 nmol

7

0 • 6 -,.4 ~o r.-.I ~n. 5'

.o

4

~ -,-.I ~. 3

~-,~ ~u

2

,.-..I ~

0

o

5

1o

Is

20

3o

35

days Flg.

3.

cultivated

M i n e r a l i z a t i o n of PCB 77 by the s t a t i o n a r y cultures of In

N-limited

medium spiked with 513.7

3,3',4,4'-tetrachloro[U-14C]biphenyl

or

30.14

C.

polFsona

nmol/flask

of

respectlvel 7 on day 3 or 6 of incubation

1131

Table

2. D i s t r i b u t i o n

rot

fungi

nmol

or

on the

after

30.14 third

of r a d i o a c t i v i t y

4 weeks

nmol

of

in the s t a t i o n a r y

incubation

in N - l i m i t e d

d a y of

PCB 77 (nmol)

Coriolopsis polysona

513.7

30.14

513.7

30.14

Control

-

3.

3,3'4,4'-)

Influence on

its

Concentration of TCB" (nM) 40 1884 5300 32106

of

513.7

(~a)

Intracellular Aqueous $5

%a Recovered

53.75 ±0.47

8.23 ±4.79

0.03 ±0.006

71.24 ±3.82

3.63 ±0.44

75.22 ±7.40

7.54 ±3.40

0.35 ±0.05

86.08 ±5.65

2.55 ±0.74

17.61 ±4.16

40.30 ±4.84

7.9 ±3.09

0.25 ±0.02

68.03 ~5.21

1.393 ±0.351

3.47 ±0.68

23.32 ±7.15

14.75 ±7.88

16.21 ±3.08

1.27 ±0.17

61.31 ±9.36

0.112 ±0.020

0.44 ±0.14

1.59 Z0.6

25.84 ±14.70

37.37 ±4.88

1.5 ±0.5

62.56 ±15.19

0.378 ±0.062

2.14 ±0.i0

9.82 ±1.32

23.11 ±9.69

25.03 ±2.32

1.36 ±0.87

60.39 ±10.22

0.000

0.60

67.59

-

0.002 ±0.002

0.28 ±0.01

71.07 ±0.95

-

CO2

Organic $2

0.0019 ±0.0008

0.94 ±0.5

11.14 ±1.29

0.015 ±0.014

0.62 ±0.08

0.301 ±0.023

of the r a d i o a c t i v i t y not determined

Table

with

respectively

Organic $4

Aqueous S1

513.7 30.14

"%

the w h i t e

Mycelial

30.14

Trametes versioolor

spiked

(PCB 77)

of 1 4 C - r a d i o a c t i v i t y

Extracellular

513.7

Phanerochaete chrysosporium

of

incubation Distribution

White rot fungi

medium

3,3',4,4'-tetraohloro[U-'4C]biphenyl

or s i x t h

cultures

originally

by

Position of CI- a t o m s on b l p h e n y l 2,2',3,3' 3,3',4,4' 2,2',3,3' 3,3',4,4'

-

-

68.19

-

71.34 ±0.96

applied

concentration

degradation

S3

of

tetraohlorobiphenyl

(2,2'3,3'-

P. chrysosporium t 4 CO± (%b)

Aqueous ,4C (%b)

3.92 1.39 0.48 0.30

25.07 4.74 3.89 2.75

Referenc~

Thomas eta!. Present work T h o m a s et al. Present work

(1993) (1993)

"TCB tetrachlorobiphenyl b v a l u e s r e c a l c u l a t e d as % of the total r a d i o a c t i v i t y a p p l i e d e v a l u e s are c o m p a r a b l e s i n c e the d u r a t i o n of e x p e r i m e n t s in the c o n c e r n e d i n v e s t i g a t i o n s are s i m i l a r .

or

1132

different

fractions

incubation

in the p r e s e n c e

PCBs b i n d the

native

of

the white

to the m y c e l i u m w i t h (non-modified)

R a d i o a c t i v i t y w h i c h did addition

rot fungal

of PCB 77

to the native

PCB

not b i n d

high affinity

[7].

77

molecules

rendered water-soluble

fraction

2).

This

fraction)

are d e g r a d e d

constitute

extracellular

p r o d u c t s m a y be to the 14CO2

further

(Sl)

in the size of the S2

These

The

in

lost

PCB m o l e c u l e s

d e g r a d a t i o n process. cell

represents

to that

modified

rendered water-soluble.

taken up by the

mycellum.

w h i c h have

Changes

corresponded the

represents

the

too,

of d e g r a d a t i o n .

that

p a r t of the

ultimately

($2

reactions

water-soluble

and m i n e r a l i z e d

r e p r e s e n t s m o l e c u l e s w h i c h have b e e n s u b j e c t e d

assimilated

homogenization 77 m o l e c u l e s

as

of

cellular

the biomass.

w h i c h have b e e n

fraction

may

be

Fractions

$4,

$5 and '4CO2

reactions

and

to

ones

of

contributing

evolution.

S4 f r a c t i o n and

indicated

$3 f r a c t i o n

($2 fraction)

the m o d i f i e d

in the p r o c e s s

four weeks

2.

bound

to the m y c e l i u m

of r a d i o a c t i v i t y (Table

after

in Table

PCB 77 m o l e c u l e s

mycelium binding properties

cultures

is s u m m a r i z e d

largely

of PCB

degradation

material

could

$5

together

taking

for

place

the

represented

On

the

be

released

fraction constitutes

e x t e n s i v e l y oxidized. responsible

77 d e g r a d a t i o n .

reactions

that

Water-soluble

to d e g r a d a t i o n

Further

degradation

evolved

radiolabel

the

basis

of this as COz .

a m o u n t of i n t r a c e l l u l a r

of

extracellular

upon the PCB

these

facts,

amount of

and i n t r a c e l l u l a r

can be

quantified.

C. polysona resembled

cultures mineralized

those

P.

of

PCB

chrysosporium

77 p o o r l y at cultures

in

d i s t r i b u t i o n pattern.

PCB 77 m i n e r a l i z a t i o n

improved

cultures

were

with

2,3).

precisely

in

spiked

the size of

(SI) w h i c h was of PCB 77

of

lower at h i g h e r

aqueous

in the m y c e l i a l 77 results

Moreover, responsible

fraction

and

the

lower

These

facts

are

of w a t e r - s o l u b l e

than

at the

but

*4C-radioactivity

significantly when

products

which

are

the

mirrored radiolabel

lower c o n c e n t r a t i o n

therefore

in

general,

P. chrysosporium c u l t u r e s

fraction

in

and the PCB

77

and d e c r e a s e

the d e g r a d a t i o n

leading

toxic

of PCB

to the f o r m a t i o n

not only to the enzyme

c h l o r i n e - b e a r i n g biphenyl

larger amount

lower m i n e r a l i z a t i o n

that

lipophylic

increase

to '4CO2 .

c o n c e n t r a t i o n m a y be of

PCB m i n e r a l i z e d

in aqueous

It s u g g e s t s less

and u l t i m a t e l y

the d e g r a d a t i o n

a m o u n t of

concentration,

increase

(Table 2,3).

metabolism

and

At

accompanied with

higher

for

cellular

degradation

With

(Fig

concentration

influenced

into m e t a b o l i t e s

of w a t e r - s o l u b l e

fraction,

of PCB fraction.

m i n e r a l i z a t i o n was

the

dose

i n t r a c e l l u l a r pool

the

dose

(Table 2).

Concentration size

lower the

its h i g h e r

resulting

in

of radiolabel

lower was

system

ring but also rates

to

of PCB 77

found as mycelial

(Table 2). decrease

in the size of S3 s i m u l t a n e o u s l y

1133

with

the

increase

increased These

in

that

intraoellular

data

suggest

intracellular

of

$4

and

degradation

that

at

degradation

S5

of PCB

innocuous

reactions

at 77

lower

at

concentration

its

lower

concentrations

contributed

implied

concentration.

of

the

significantly

to

pollutant, the

overall

mineralization. T.

versJcoJor

polysona

in the

Changes

distribution

in the

significant

significantly

distribution

with

C.

of

PCB

common

the

three

all

radiolabel

could

be

of w a t e r - s o l u b l e in

77.

degradation

most

part

is an

These ability been

Our

process.

understanding higher

degree

Though also

other

about

in t h e i r

of of

77

homogenizing

was

T.

vers~color.

These

concentration)

% of

the b i o m a s s .

the b i o m a s s

by

T.

not so

both

appeared

50

with

of

at,

the m y c e l i u m verslcolor

C.

fractions.

were

versicolor

to T.

that

the

and

mineralize

the

of

the w h i t e

the

rot

the to be

the b o u n d The

higher

amount

than

facts

that

suggested

versico~or

to d e g r a d e

PCBs

for

the

steps

of

degradation

rot

in t h e i r

fungi

have

in c o n t a m i n a t e d

soil

also

of PCB

pollutant

differ

White

of P C B s

the m e c h a n i s m s respective

fungi

PCB 77.

concentration

Investigating

rate-limiting

i~ this rot

paper fungi

biodegrar~ation

identification

T.

in

after

in d i f f e r e n t

t4C-radioactivity

and

degradation

may of

influence~

confribute PCB

the using

to the

congeners

with

chlorination.

white

the

of PCB

capacity

that

concentrations

as

chrz~o~porium

process.

to d e g r a d e

showed

well

higher

demonstrated

to d i f f e r

degradation innocuous

intracellular

data

radiolabel the

fluid

(at

P.

of

however,

only

from

of

associated

culture PCB 77

strategy

shown

[I0].

of

results

and

Binding

extracted

the e x t r a c e l l u l a r

as

fungi,

radiolabe!

that

pattern pattern

polysona

concentrations to

differed

of

it

is d e m o n s t r a t e d

are

able

pathway.

intermediate

that b e s i d e s

to m i n e r a l i z e This

will

PCBs, be

no

P.

chrysosporium

information

studied

exists

nn the b a s i s

of

metabolites.

ACKNOWLEDGEMENT

UNESCO

Fellow~hip

research

work

and M a t u c h a

was

in the

to

B.R.M.

supported frame

of

Vyas

by

~s g r a t e f u l l y

the G r a n t

Scientific

III/35

acknowledged.

awarded

Co-operation

jointly

between

FRG

Part

to Prs. and The

of

the

Bubner Czech

Republic.

REFERENCES

[i]

Hardman,

D.J.

Cr~t.

Biotechnol.

Rev.

(1991)

Biotransformation II:

1-40.

of

ha!ogenated

compounds.

1134

[2]

Chaudhry,

G.R.

and Chapalamadugu,

organic compounds.

[3]

Bumpus,

Microbiol.

J.A., Tien,

persistant

S.

Rev.

M., Wright,

environmental

(1991)

Biodegradation

of h a l o g e n a t e d

55: 59-79.

D. and Aust,

S.D.

(1985)

Oxidation

pollutants by a white rot fungus.

Science

of 228:

1434-1436.

[4]

Field,

J.A.,

Screening

de Jong,

for

xenobiotics.

[5]

Bumpus,

FiebJg,

and by

involvement

[6]

ligninolytic

[7]

R.,

Schulze,

D.R.,

bipheny]

KlOtzer,

(1986)

rot

Lett.

P.,

April

[93

Vyas,

K.S.

BioEssays

Slawinski,

and Georgiou, rot fungus

M.

Heise,

K.-H.

(G. Bernhard

Ed.)

G.

M.

Dellweg,

H.

in contaminated

Mineralization

Phanerochaete

of

chrysosporium.

M.

(1992)

Synthese

Jahresbericht

Forschungszentrum

1991,

Rossendorf,

1992.

B.R.M.,

chrysospori'~m.

Sa~ek,

6: 166-170.

and

(1993)

and Matucha,

pp.41-42,

Voic,

J. and Sa~ek,

p r o d u c t i o n of m a n g a n e s e peroxidase

[i0]

environmental

chrysosporium:

40: 1395-1402.

D., Bubner,

92-08

of

Phanerochaete

und Analyse yon 3,3',4,4 - t e t r a c h l o r o [ U - ~ 4 C ] b i p h s n y l . FZR

of

15: 93-98.

Carswell,

Bioeng.

(1993)

biodegradation

Biodegradation

fungus

Erlemann,

and PCBs by the white

Biotechnol.

[8]

D.,

the

d e g r a d a t i o n of p o l y c h l o r i n a t e d biphenyls

Biotechnol.

Thomas,

S.D.

white

to

J.A.M.

II: 44-49.

of the iignin degradation system.

(1993) Microbial soil.

Aust,

the

G. and de Bont,

fungi applicable

Trends Biotechnol.

J.A.

pollutants

E., Feijoo-Costa,

(Folia Microbiol.

V., Volfov~,

Degradation

O., Erbanov~,

V. Effects

temperature

on

the

and lignin peroxidase by Phanerochaete

in press).

P., Vyas,

of PCBs by white rot fungi,

ufilizing yeasts and bacteria.

of

B.R.M.

and Ma~ucha,

methy]otrophic

Biotechnol.

Lett.

and

15: 521-526.

M.

(1993)

hydrocarbon