Agric. Biol. Chem.

, 52 (ll), 2885-2891, 1988 2885

Isolation and Characterization of a Mixed Culture

That Degrades Polychlorinated Biphenyls
Kazuhide Kimbara, Toshiyuki Hashimoto, Masao Fukuda,
Takao Koana,* Masamichi Takagi, Michio Oishi**
and Keiji Yano
Department of Agricultural Chemistry, The University of Tokyo,
Bunkyo-ku, Tokyo 113, Japan
* Biotechnology Department, Industrial Research Institute Japan,
1201 Takada, Kashiwa, Chiba 277, Japan
**Institute of Applied Microbiology, The University of Tokyo,
Bunkyo-ku, Tokyo 113, Japan

Received June 10, 1988

A mixed culture composed of two Pseudomonas strains, designated as KKL101and KKS102,

was isolated from soil. This mixed culture had an enhanced ability to degrade various polychlori-
nated biphenyls (PCBs) which include highly chlorinated components. They did not grow
individually on the mineral salts medium supplemented with a highly chlorinated PCB (PCB48, a
mixture of mainly tetrachlorobiphenyl) and biphenyl. When the spent medium of KKL101 was
added to the washed cell preparation ofKKS102, however, the latter grew on these carbon sources,
producing yellow compounds which were identified as metabolic intermediates of the carbon
sources, biphenyl and PCBs. These results suggest that KKL101 produces a growth factor(s)
essential for KKS102 to grow on PCBs and that the growth of KKL101 is supported by the
metabolic intermediates produced by KKS102. It appears that these two bacterial strains have a
symbiotic relationship. From the analysis of the degradation products of various PCB congeners, it
was found that strain KKS102degrades a wide range of PCBs which have been considered to be
refractory to biological degradation.

Polychlorinated biphenyls (PCBs) are en- mixed cultures10'14~18) has been reported,
vironmental pollutants which are distributed suggesting that PCBs-degrading bacteria are
widely, like 2,2-bis(p-chlorophenyl)ethane and widely distributed in the environment. Bac-
1 ,2,3,4,5,6-hexachlorocyclohexane. Chlorines terial degradation of PCBs was reviewed
can be substituted at ten positions in the in detail by Furukawa19) and Bedard.10'20'21)
biphenyl molecules, hence 209 different com- According to them, highly chlorinated conge-
pounds can be theoretically produced. How-
ners (more than four chlorines per molecule)
ever, approximately half of them have been are highly recalcitrant to microbial degrada-
analytically detected so far. Although PCBs tion. This is consistent with the fact that most
are chemically inert and stable, there are a of PCBsnowpresent in the environment have
number of reports of microbial degradation four or more chlorines per molecule. The
of PCBs. Ahmedand Focht first reported the resistance of highly chlorinated PCBs to the
degradation of several monochloro-
degradation and di-
by bacteria poses a formidable
chloro-biphenyls by two species of Achromo- problem to clean up the PCB-polluted envi-
bacter.1] Since then, the degradation of PCBs ronment by bacteria. To circumvent this, we
by a number of microorganisms such as Aci- examined whether mixed cultures of bacteria
netobactor,2 ~1] Alcaligenes^*'8^ Bacillus^ have an enhanced ability to degrade highly
Pseudomonas 1A0 ~13) Streptococcus1"* and some chlorinated PCBs which are normally resist-
2886 K. Kimbara et al.

ant to biological degradation. Classification of bacterial strains. Bacterial flagella were

In this report, we describe screening of stained as described previously.22* The GCcontent anal-
mixed cultures of bacteria which are capable of ysis of DNAand identification of quinone-type of the
cells from the pure cultures were done as described
degrading highly chlorinated PCBs and the previously.23 24)
isolation of one culture which consists ofa pair
of bacterial strains, both Pseudomonas species. Assay for the degradation of PCBcongeners. Stock
The roles played by these two strains in the solutions of each PCB congener were prepared at a
degradation of PCBs are discussed. concentration of 0.5mg/ml in ethylacetate. One ml of a
PCBstock solution was placed in a 42-ml test tube and
ethylacetate was evaporated on a water bath at 70°C with
MATERIALS AND METHODS the help of an aspirator. A halfml of spent medium of one
strain was then added into the test tube, followed by
Enrichment culture. Mixed cultures were isolated from addition of the mineral salts medium (4.5ml) containing
various soil and water samples obtained from industrial cells of other strain corresponding to 50 fi\ culture grown
regions in Tokyo and Kawasaki City. For the enrichment to the absorbance 1.0 at 660nm. The mixture was in-
of desired bacteria, six different media were used. They cubated for 7 days at 30°C under aerobic conditions.
were mineral salts media containing 1 mg/ml of one of the Before and after the incubation, 1 ml of the culture was
following carbon sources; biphenyl, 4-chlorobiphenyl, removed and acidified to pH 1.0 with concentrated HC1.
PCB42 (PCBs mixture, mainly trichlorobiphenyls), PCB48 PCBsand someof their metabolites were then extracted
(PCBs mixture, mainly tetrachlorobiphenyls), a mixture of with 1 ml of ethylacetate. Trimethylsilyl derivatives of
biphenyl and PCB42 (each 1 mg/ml), and a mixture of some of the metabolites were prepared by addition of a
biphenyl and PCB48 (each 1 mg/ml). The mineral salts small volume of Af-methyl-N-trimethylsilyltrifluoroacet-
medium was composed of KH2PO4(1.7g/1), Na2HPO4 amide (MSTFA) to the samples and subsequent incuba-
(9.8g/1), (NH4)2SO4 (1.0g/1), MgSO4-7H2O (0.1g/1), tion (60°C, 1 hr) after evaporation of ethylacetate under
FeSO4-7H2O (0.95mg/1), MgO (10.75mg/1), CaCO3 a gentle stream of nitrogen gas.
(2.0mg/l), ZnSO4-7H2O (1.44mg/1), CuSO4-5H2O
PCBcongeners were analyzed by gas chromatography
(0.25 mg/1), CoSO4-7H2O (0.28 mg/1), H3BO3 (0.06mg/1) (GC) using a model 263-30 (Hitachi Ltd.) with a 63Ni-
and cone. HC1 (51.3^1/1). The phosphate buffered electron capture detector. The column used in the analysis
saline was composed of Na2HPO4-12H2O (2.19g/1), was a glass column (2m by 3mminner diameter) packed
Na2HPO4- 12H2O (0.61 g/1), pH 7.2 and NaCl (8.0g/1). with silicon OV17 (at 2% Chromosorb WAWDMCS
Soil and water samples were suspended in phosphate- 80/100 mesh). Columnoperating conditions were as fol-
buffered saline, agitated for several minutes and then lows. The injection and detection temperature was 250°C.
rested for about one minute. The resultant supernatant The columntemperature was 205°C. Nitrogen was used as
(100/^1) was added to 10ml of each of the six media the carrier gas at a flow rate of 50ml/min. Trimethylsilyl
described above in a test tube and incubated for a week at derivatives of some PCBmetabolites were analyzed by
30°C under aerobic conditions. When cells grew, judged gas chromatography-mass spectometry (GC-MS) using a
by the increase of turbidity, the medium was replaced by model JMS DX303 (JEOL Ltd.). The column used in
fresh mediumwhosecarbon sources were a mixture of the analysis was an OV-1 capillary column (25mx0.25
biphenyl and PCB48. This replacement procedure was mm inner diameter). Column operating conditions were
repeated several times. as follows. The column temperature during GCwas in-
creased from 120 to 250°C at a rate of 16°C/min. The
Isolation of pure cultures and reconstitution of mixed electron impact MSwas measured at a 70-eV ionization
cultures. The most active PCB-degrading mixed culture potential, 300-/iA trap current, and 200°C ion source
(sample No. 10) was selected for further study. This temperature.
culture was grown aerobically at 30°C in 100ml of the
mineral salts medium containing biphenyl and PCB48 Chemicals. Monochlorobiphenyl isomers and PCB
(each 1mg/ml). At mid log phase, cells were plated on congeners were purchased from Gasukuro KogyoInc.,
DLGagar after appropriate dilution and incubated at Tokyo. Biphenyl, 4-chlorobenzoate, PCB42 and PCB48
30°C. The DLGagar was composed of Bactotryptone were purchased from Tokyo Chemical Industry Co.,
(3.3 g/1), yeast extract (1.7 g/1), NaCl (5 g/1), glucose (5 g/1), Tokyo. A/^-Methyl-A^-trimethylsilyltrifluoroacetamide
and 1.5% agar. Cells from isolated colonies were grown in (MSTFA) was obtained from Nakarai Chemicals, Ltd.,
10ml of the DLGmedium. Following a wash with phos- Kyoto.
phate buffered saline, the two individual strains were
mixed together and added to 10ml of the mineral salt
medium containing biphenyl and PCB48 (each 1 mg/ml).
 A Mixed Culture to Degrade PCBs 2887

RESULTS Table I. Cell Growth and Degradation

OF BlPHENYL (BP) PLUS PCB48 IN

Isolation of mixed cultures Reconstituted Cultures

Using the enrichment method described in Washed cell
Filtered Change of
Materials and Methods, we obtained 10 dif- preparation spent Growth color to
ferent mixed cultures which degraded PCB48 medium yellow
in the presence of biphenyl. It took approx- KKL101
imately two months to establish reproducible KKS102
KKL1O1 KKS102
growth rates of these mixed cultures. Among KKS102 KKL101

+
the 10 mixed cultures, one (sample No. 10)
showed the highest growth rate on these car-
bon sources, and could also grow on only Cells from a pure culture of one of the two strains were
grown in 10ml of the DLGmedium. After centrifugation
PCB48as the sole carbon source, although the (4,000 xg for lOmin at 4°C), the supernatant was filtered
growth on PCB48 was slower. Light yellow through a 0.20/mi-pore-sized membrane filter and the
filtrate was used as spent medium. The cell pellet after
compounds whoseabsorption maximum was centrifugation was washed several times with phosphate-
at about 400nm appeared in the culture me- buffered saline, suspended in 9.5ml of the mineral salts
dium. The yellow compounds gradually disap- medium, and transferred to a 42-ml test tube. To this test
peared during further cultivation. These yel- tube, lOmg of a mixture of biphenyl and PCB48, and
low compounds might be ring raeta-cleavage 0.5 ml ofDLG medium (top two lines) or of spent medium
intermediates of PCBs as described previ- obtained from a culture of the other strain (bottom two
ously.1'5'8* lines) were added. The incubation was done at 30°C. The
cell growth was assayed by counting the number of
colonies appeared on DLGplates and the color of the
Isolation of two bacterial species from the mixed culture was recorded.
culture and classification of the bacteria
Two types of colonies with apparently dif- carbon sources (Table I), When these two
ferent growth rates appeared after plating strains were mixed, however, growth of the
sample No. 10 on a DLG agar plate. The culture was observed with the formation of
bacterium which grew faster was designated as yellow compounds. It was found that the
KKL101and the other which grew slower was growth rate and biphenyl/PCB degradation
designated as KKS102. These two bacteria were dependent on the mixing ratio of the two
were both Gram-negative, motile, rod-shaped strains in the reconstituted mixed culture; the
organisms with polar flagella. They were higher the ratio of the culture volume of
catalase-positive, oxidase-positive, and show- KKS102to thatofKKL101 upto 10to 1, the
ed an oxidative reaction in the Hugh-Leifson more reproducible the growth of the cells and
test. Nitrate reduction was observed with production of yellow compounds.
KKS102. A fluorescent pigment was observed
on a colony of KKL101. The GC content of The roles of the two strains in the degradation of
the DNA was found to be 64.1mol% for PCBs
KKL101, and 67.0mol% for KKS102. The To investigate the roles played by the two
type of quinone was found to be Q9 for cell cultures in PCBsdegradation, the cultures
KKL101 and Q8 for KKS102. According to were reconstituted with a cell preparation and
the standard taxonomic and chemotaxonomic a filtered spent mediumof each culture. As
procedures, both of them were Pseudomonas
sp. KKL101 was further identified as Pseu-
shown in Table I, addition of the spent me-
dium of KKL101to the washed cell prepara-
tion of KKS102 resulted in the growth of
domonasfluorescens.
These two Pseudomonas strains did not
KKS102 producing yellow compounds. On the
grow individually in the mineral salts medium other hand, addition of the spent mediumof
with a mixture of biphenyl and PCB48 as KKS102 to the washed cell preparation of
zooo K. Kimbara et al.

KKL101 did not. These results suggest that PCB congeners by KKS102 in the presence
KKS102plays a major role in the degradation of the spent medium of KKL101. Monochlo-
of PCBs and that the metabolic intermediates robiphenyl isomers were quickly degraded
supports the growth of KKL101. It is also without the accmulation of intermediate
suggested that KKL101 provides a growth compounds. PCBs with more than two chlori-
factor(s) essential for the growth of KKS102 nes were not completely degraded after 7 days
on PCBs. The unknownsubstance(s) produced and accumulation of some intermediates were
by KKL101 was hydrophilic, non-dialyzable, observed. Most of PCBs with two or three
and precipitable in 5% perchloric acid. It was chlorines were degraded to colorless interme-
stable at 100°C for 30min and at 121°C for diate compounds. Most of PCBswith four or
15min at the neutral pH, and at 65°C for five chlorines were degraded to yellow inter-
30min at pH 2.0 and 9.0. mediate compounds. PCBs including 2,5,2'-
and 2,4,2'-trichlorobiphenyl, 2,3,4,5-, 2,3,5,6-,
Degradation of PCB congeners by KKS102 2,6,2/,6/- and 3,4,3',4'-tetrachlorobiphenyl,
Table II shows the degradation of various and 2,3,4,2/,5/- and 2,4,5,2',5'-pentachloro-
biphenyl were degraded slowly. 2,6-Dichlo-
Table II. Degradation of PCB Isomers by robiphenyl and 2,3,6-trichlorobiphenyl were
Pseudomonas sp. KKS102
previously reported to be quite resistant to
Residual
Intermediate degradation.3*4'19* However, 90% of2,6-dichlo-
PCBcongeners compounds
(chlorine positions) PCBs robiphenyl and 50% of 2,3,6-trichlorobiphenyl
accumulated were degraded after 7 days of incubation
2-, 3- or4- and some metabolic intermediates accumulat-
+
2,3- ++ ed. Furthermore, extensive degradation of
+
2,6- ++ some tetrachlorobiphenyls to yellow interme-
+
2,2'- ++
+
2,4'- ++
diates was also observed in this culture. These
+
4,4'- ++ results suggest that the strain KKS102 can
+
2,3,6- ++
++ degrade a wider range of PCB congeners
2,4,5- ++
++ than the strains reported in the literature.
2,4,6- ++
++ When KKS102 was cultured for one day
2,5,2'- +
++
2,4,2'- + with 4,4/-dichlorobiphenyl as a sole carbon
+
2,3,4,5- +
+
+
source, only one major peak was detected in
2,3,5,6-
2,3,2/,3/-
+
++
the GC analysis of the culture medium. This
2,4,2/,4/- ++ compound was further analyzed by GC-MS,
2,4,3/,4/-
++
++ and the result is shown in Fig. 1 (A). In Fig. 1
2,5,2/,5/-
++
++ (B), the GC-MSprofile of authentic 4-chloro-
2,6,2/,6/-
3,4,3/,4/-
++
+
benzoic acid is shown. These data indicate that
++
+
the major metabolite of 4,4/-dichlorobiphenyl
2,3,4,2/,5/-
++
2,4,5,2',5/- + is 4-chlorobenzoic acid. Furthermore, GC-MS
2,3,4,5,6- ++ data of the yellow compoundwhich accumu-
lated within one day of the incubation of
Test tubes were coated with each 0.5 mg of either of the KKS102 with 4,4/-dichlorobiphenyl indicate it
PCB congeners described above. To each of them, 0.5 ml
offiltered spent medium of the strain KKL101,the 4.5 ml to be the mem-cleavage metabolic intermediate
of the 2,3-dihydroxy compound (data not
of mineral salts medium containing washed cell prepara-
tion of the strain KKS102were added. After incubation shown). These data indicate that the meta-
for 7 days the amounts of the residual PCBcongeners and bolic pathway of 4,4/-dichlorobiphenyl and
intermediate compoundsaccumulatedin the mediumwere most of the other PCBcongeners in the strain
analyzed. The amounts were calculated from the GCpeak KKS102 are the same oxidative route as de-
areas and presented as follows: -, not detected; +,
0.25mg or less; ++, 0.25mg or more. scribed in many other bacteria.
 A Mixed Culture to Degrade PCBs 2889

Fig. 1. Mass Spectra of the Major Intermediate Metabolite of 4,4'-Dichlorobiphenyl by Strain KKS102
(A) and of the Authentic 4-Chlorobenzoic Acid (B).
KKS102was cultured in the presence of a cultured mediumof KKL101with 4,4/-dichlorobiphenyl as a
carbon source. After incubation for one day, the intermediate metabolites in the cultured mediumwere treated
with Af-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) to form trimethylsilyl (TMS) derivatives before
GC-MSanalysis. The spectrum of the TMSderivative of the major compound is shown in (A), and that of the
TMSderivative of the authentic 4-chlorobenzoic acid is shown in (B).

DISCUSSION systems have been knownin

In soil, microorganisms form a variety of
communities. When soil is contaminated
recalcitrant molecules such as PCB congeners,
microbe communities in the area may evolve
to acquire abilities to use such molecules as
carbon sources. Examples of such community
of several herbicides25
the degradation
26) and chlorobenzoic
acids.27)
by Many studies have been done to isolate
strains which degraded PCBs, but most of
them did not pay attention to the mixed
culture systems in which two or more strains
were involved in degradation of PCBs. There-
2890 K. Kimbara et al.

fore, for screening and isolation of microor- The result of GC-MSanalysis indicates that
ganisms to degrade PCBsof highly chlorinated it degrades 4,4/-dichlorobiphenyl to the rneta-
congeners, we focused on mixed microbe cul- cleavage metabolic intermediate of a 2,3-di-
tures which efficiently degrade PCBs. Among hydroxy compound (a yellow compound), and
dozens of mixed cultures capable of degrading then to 4-chlorobenzoic acid, suggesting that
PCBs, which were found in soil obtained the raeta-cleavage pathway is the major route
around gas stations or from industrial regions for KKS102 to degrade various PCB conge-
in Tokyo or KawasakiCity, one culture (sam- ners, as reported for manyother bacteria.
ple No. 10) which showed the highest growth Acknowledgments. The authors wish to thank Pro-
rate on biphenyl and PCBswas selected and fessor Kuraishi of Tokyo University of Agriculture
studied further. The mixed culture degraded and Technology for his help and advice as to the ta-
PCBsand the degradation rate was stimulated xonomical identification of the isolated bacteria. Helpful
by the addition of biphenyl in the cultivating discussion by Mr. K. Tanemura, Mr. N. Nishikawa, and
medium. the other membersof the Engineering Research Institute
Two Pseudomonas strains, designated as of the Tokyo Electric Power Co. are greatly acknowledg-
KKL101and KKS102 were isolated from this ed. This work was partly supported by a fund from the
mixed culture. KKS102 apparently is the ma- Tokyo Electric Power Co.
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