Bacterial degradation of 4-chlorobiphenyl

A novel strain of Pseudomonas capable of utilizing 4-chlorobiphenyl as sole carbon and energy source is disclosed. The bacterium identified as Pseudomonas MB86 is shown to degrade 4-chlorobiphenyl to 4'-chloroacetophenone and other metabolites.

TECHNICAL FIELD 
This invention relates to degradation of 4-chlorobiphenyl by a strain of 
Pseudomonas bacteria. The novel strain of bacteria, Pseudomonas MB86, is 
capable of utilizing 4-chlorobiphenyl. 
BACKGROUND 
Polychlorinated biphenyls (PCBs) represent a class of toxic xenobiotic 
chemicals that are distributed throughout the biosphere. Over the past 
several years, PCBs have received ever-increasing attention due to 
concerns about their toxicity and potential carcinogenicity. PCBs are 
produced by direct chlorination of biphenyl. Due to the large number of 
hydrogen atoms present on the biphenyl nucleus, many different chlorinated 
species (termed "congeners.revreaction.) are possible. As many as 210 
congeners of the PCBs could be theoretically produced [Furukawa, 
Biodegradation and Detoxification of Environmental Pollutants, p. 44-57 
(CRC Press 1982)]; however, due to steric restrictions, only about half 
this number are actually observed. Therefore, PCBs are mixtures of a 
variety of chlorine-substituted biphenyl molecules. 
PCBs have been widely used industrially due largely to their thermal 
stability and flame retardance. Such characteristics encouraged PCB use in 
transformer oils and in other high-temperature applications. Until the 
early 1970's PCBs were found in various pesticide formulations. PCBs have 
also been used in plasticizers, heat transfer and capacitor systems, 
surface coatings, printing inks, carbonless duplication paper, and waxes. 
While industrial use of PCB has been sharply restricted significant 
quantities of PCBs are still being released into the environment from 
waste dumps and failures of old electrical equipment. PCB contamination 
has been observed in drinking water, wastewater, foods, and especially in 
fish. 
PCBs are lipophilic. They accumulate and are bioenhanced in fatty tissues 
[Furukawa supra; Jacobson et al., Develop. Psychol., 20:523-532, 
(1984)]The physical effects of polychlorinated biphenyls vary from 
mammals, to birds, to humans. Mammals exposed to these chemicals exhibit 
marked changes in the liver [Fishbein, Ann. Rev. Pharmocol., 14:139-156, 
(1974)], including lesions, fatty infiltration, centrolobular atrophy 
necrosis, and liver cell enlargement. In the case of rats, hyaline 
degeneration, are evidence of such exposure. Among the adverse physical 
effects exhibited in birds, kidney damage, fluid around the heart, 
intestinal hemorrhage, and reduced spleen size, have been observed [See 
Rehfeld et al., Poultry Sci. 50:1090-1096, (1971)]. Dermatitis from 
surface exposure can occur in both mammals and birds [Voss et al., 
Toxicol. Appl. Pharmacol., 17:656-658 (1971)]. In addition to toxicity, 
PCBs may be carcinogenic and mutagenic (Fishbein, supra). 
PCBs are not easily removed by natural microbial populations. In 1978, 
Furukawa et al., Applied Environ. Micro., 35:223-227 studied the 
biodegradability of several isomers of polychlorinated biphenyls. They 
found that as chlorine substitution increased, degradability decreased. An 
isomer with four or more chlorines was not easily degraded. The position 
of the chlorine was also important. Ortho positioning of two chlorines on 
a single ring greatly inhibited degradation. If all the chlorines were on 
the same ring, degradation occurred at a faster rate than for isomers with 
the same number of chlorines spread over both rings. It was noted that 
ring fission usually took place on the lesser or non-chlorinated ring. 
These results suggested that PCBs could be utilized by bacteria, but only 
in certain isomeric forms. The products of such degradation were usually 
chlorinated. Subsequent studies have verified these observations. 
Considering the environmental importance of PCBs and the hazards they pose, 
numerous investigators have been examining biological detoxification 
systems to deal with PCBs. One way to decipher the complexities of highly 
chlorinated isomers is to look at lesser chlorinated isomers. One of these 
which has been studied is 4-chlorobiphenyl. 
4-chlorobiphenyl is one of the three monochlorinated isomers that can 
result from the chlorination of biphenyl. As with the other biphenyls, it 
is very insoluble in water, but freely soluble in a variety of organic 
solvents. Pure 4-chlorobiphenyl is crystalline, white to off-white in 
color. It is classified as an irritant. 
Unlike the highly chlorinated biphenyls, a number of microorganisms have 
been identified that can degrade 4-chlorobiphenyl. Wallnofer and 
Englehardt Chemosphere, 2:69-73 (1973) isolated a soil fungus, Rhizopus 
japonicus, which hydroxylated 4-chlorobiphenyl at the 4' position. Other 
intermediates were not noted. In 1973, Ahmed and Focht, Can. J. Microbiol, 
19:47-52 discovered a species of Achromobacter which produced 
4-chlorobenzoic acid from 4-chlorobiphenyl. A study by Masse et al. Appl. 
Environ. Micro., 47:947-951 (1984) also described the presence of 
4-chlorobenzoate as a major metabolite of 4-chlorobiphenyl. 
In 1982, Sylvestre and Fauteux J. Gen. Appl. Micro., 28:61-72, reported a 
facultative anaerobe able to utilize 4-chlorobiphenyl. Up until that time, 
only strict aerobes were reported to degrade the lesser chlorinated PCB 
isomers. This organism was tentatively identified as a member of the 
bacterial group IVe. As with other 4-chlorohiphenyl degraders, 
4-chlorobenzoic acid accumulated in the culture media. 
Sylvestre et al. Appl. Microbiol. Biotechnol., 21:192-195 (1985) reported 
that a two-membered bacterial culture was able to degrade both 
4-chlorobiphenyl and 4-chlorobenzoate rapidly. An axenic culture was able 
to degrade 4-chlorobiphenyl alone, with a 45% decrease in substrate 
remaining after eight days. When incubated with a 4-chlorobenzoate 
degrader, 99% of the 4-chlorobiphenyl was degraded over the same time 
frame. However, no organism, to date, has been isolated which is able to 
grow on 4-chlorobiphenyl and degrade 4-chlorobiphenyl to 
4'-chloroacetophenone and other metabolites. 
SUMMARY OF INVENTION 
We have discovered a bacteria of the genus Pseudomonas which is capable of 
degrading 4-chlorobiphenyl. The bacteria identified as Pseudomonas MB86 
can utilize 4-chlorobiphenyl. 
The present invention comprises a biologically pure strain of the genus 
Pseudomonas having the characteristics of ATCC Deposit No. 53728 and the 
mutations thereof. The present invention can be used to degrade 
4-chlorobiphenyl by cultivating a media containing 4-chlorobiphenyl with 
an effective amount of Pseudomonas MB86 cells. Pseudomonas MB86 is capable 
of sustaining growth in medium concentrations of 4-chlorobiphenyl from 
about 10 mg/50 ml to about 100 mg/50 ml. Preferably the organism is 
capable of sustaining growth in medium concentrations of 4-chlorobiphenyl 
up to 50 mg/50 ml.

DETAILED DESCRIPTION OF THE INVENTION 
A novel strain of Pseudomonas desiqnated as Pseudomonas MB8 6 capable of 
degrading 4-chlorobiphenyl and producing 4'-chloroacetophenone as a major 
metabolite was obtained and characterized using the materials and methods 
described below. 
CULTURE MEDIA 
A defined mineral salts medium was prepared from stock solutions of ten 
times concentrated buffer and ten times concentrated trace metals 
solutions. The stock buffer solution contained 42.50 g K.sub.2 
HPO.sub.4.3H.sub.20, 10.00 g NaH.sub.2 PO.sub.4.H.sub.2 O and 20.00 g 
NH.sub.4 Cl per liter of distilled water. The stock trace metals solution 
contained 1.00 g nitrilotriacetic acid, 2.00 g MgSO.sub.4.7H.sub.2 O, 0.12 
g FeSO.sub.4.7H.sub.2 O, 0.03 g MnSO.sub.4.H.sub.2 O, 0.03 g 
ZnSO.sub.4.H.sub.2 O, and 0.01 g CaCl.sub.2.5H.sub.2 O per liter of 
distilled water. One liter of growth medium consisted of 800 ml of 
distilled, deionized water and 100 ml of each of the 10X stock solution. 
The pH was adjusted to 7.45 with 6N NaOH. The resulting solution 
constituted basal medium. 
After autoclaving, 4-chlorobiphenyl (1 mg/ml) was added directly and the 
medium was inoculated with Pseudomonas MB86 (a loopful of culture when 
taken from plates; 1 ml to 50 mls from liquid cultures--depending on 
culture size 50 ml up to 1000 ml. In addition to use as a broth, Noble 
agar (Difco) was added at 15.00 g per liter to the defined medium for 
substrate-free plates. 4-chlorobiphenyl crystals (approximately 10 mg) 
were placed in the lid of petri plates. Vaporization of the crystals 
provided a source of carbon and energy for Pseudomonas MB86 streaked onto 
the agar surface. Other carbon sources (yeast extract, 4-chlorobenzoate, 
etc.) were added directly to the medium as required (yeast extracts 0.25%; 
4-chlorobenzoate 0.025%, 0.050%; 3-chlorobenzoate 0.025%, 0.050%). 
Subsequently, these plates were utilized for screening the organism's 
ability to utilize specific substrates as sources of carbon for growth and 
to maintain Pseudomonas MB86. 
The same basal medium was used to prepare liquid or plated media containing 
alternate carbon sources such as yeast extract, 3-chlorobenzoate, 
4-chlorobenzoate, 3-5,dichlorobenzoate, 4-hydroxybenzoate and sodium 
benzoate. Those volatile aromatic compounds that were insoluble in water, 
such as liquid 2-chlorobiphenyl (2-3 drops), liquid 3-chlorobiphenyl (2-3 
drops), solid, crystalline biphenyl (10 mg up to 30 mg), and liquid 
4'-chloroacetophenone (2-3 drops) were provided as carbon sources by 
placing the above indicated amounts into cotton-filled Durham tubes taped 
to the inside of the plate lid (for liquid compounds). Solid, volatile 
substrates (5-10 mg) were placed in the lid, as for 4-chlorobiphenyl. Most 
incubations were performed at 30.degree. C, with liquids placed on a 
rotary shaker at 200 rpm. 
MEDIA USED FOR GENUS IDENTIFICATION 
Preliminary screenings of cultural characteristics of the bacterium were 
accomplished utilizing Klinger Triple Sugar Iron agar, SIMS media, Eosin 
Methylene Blue plates. Final identification was performed utilizing an 
OxiFerm tube from Roche Labs. (A control was run using a known culture of 
Pseudomonas aeruginosa to ensure that the Oxiferm tubes were running 
correctly.) Other tests were done utilizing Pseudomonas A and B agar, 
DNase plates, blood agar, and glucose, sucrose, and lactose broths 
obtained from the University of Minnesota microbiology preparatory labs. 
To determine oxygen requirements, yeast extract plates (0.25% yeast 
extract into the defined mineral salts medium listed previously) were 
inoculated and incubated aerobically, in increased CO.sub.2 (candle jar), 
and anaerobically (BBL-GasPak). 
TEMPERATURE SCREENING 
Pseudomonas MB86 was examined for its optimal growth temperature using 
0.25% yeast extract and 4-chlorobiphenyl broth and plates. Cultures were 
run in duplicate at 20.degree. C., 30.degree. C, 37.degree. C., and 
41.degree. C. 
FREEZING AND STORAGE OF CULTURES 
After purification by repeated streaking on yeast extract plates, one liter 
of the organism was grown to mid-log phase in 0.25% yeast extract. Cells 
were concentrated by centrifugation for fifteen minutes at 8000.times.g in 
sterile bottles. After decanting the supernatant solution, 10 ml of fresh, 
sterile, 0.25% yeast extract were added and the cells resuspended. To each 
of 13 sterile plastic vials was added 0.75 ml of the cell suspension and 
0.25 ml sterile glycerol. The cultures were then frozen at -70.degree. C. 
for use as stocks. 
PH MEASUREMENTS 
pH measurements were carried out using a Fischer model 825MP digital 
pH/millivolt meter or Corning model #120 pH meter. 
TURBIDITY MEASUREMENTS 
Measurements of cell growth in yeast extract broths were performed using a 
Beckman DU quartz spectrophotometer by determining absorbance of culture 
fluids at 600 nm. When cultures were grown on 4-chlorobiphenyl as the sole 
carbon and energy source, turbidity was determined using a Hewlett-Packard 
diode array spectrophotometer model #8482. Spectra of the compounds 
present in such cultures were also obtained with this instrument. 
GROWTH CURVES 
For determination of growth rate in 0.25% yeast extract, several colonies 
from a stock plate were inoculated into yeast extract broth which was 
incubated with shaking for approximately 18 hours. One ml of this culture 
was then added to each of two similar flasks. Samples were taken every 
thirty minutes and their optical densities at 600 nm determined. During 
logarithmic growth phase, five samples were taken for determination of 
viable counts. Samples were diluted into sterile basal medium and the 
10.sup.31 6 through 10.sup.-8 dilutions were plated in triplicate. 
For determination of growth kinetics in 4-chlorobiphenyl broth, several 
colonies were taken from a stock plate and inoculated into a flask of 
basal medium containing 4-chlorobiphenyl as the only growth substrate. 
After three days of incubation, one ml of the initial culture was added to 
each of two flasks of the same medium. Samples were taken every 24 hours 
over a period of seven days for determination of absorbance at 600 nm. Due 
to production of colored compounds, the samples were also spun for five 
minutes in a Beckman microfuge to remove cells, and the supernatant 
solutions read again at 600 nm to allow correction for absorption due to 
organic compounds. Viability counts also were made at various times during 
the course of the experiment. 
TIME COURSE STUDIES FOR PRODUCTION OF METABOLITES 
Pseudomonas MB86 was inoculated into each of 14 flasks containing 50 ml of 
basal medium and 50 mg of 4-chlorobiphenyl. Seven additional flasks 
contained the substrate, but were not inoculated. Each day, two inoculated 
flasks and one uninoculated flask were sacrificed. Observations were made 
on their turbidity (A.sub.600), purity of the culture, color, and pH. 
Exhaustive extractions into ethyl ether were performed, as described 
below, and the extracts analyzed by gas-chromatography (GC). 
QUALITATIVE AND QUANTITATIVE EXTRACTIONS 
Whole culture extractions were performed using ethyl ether (amounts 
approximately equaled the total volume of the culture when totally 
extracted). Samples (total cultures were sacrificed) at neutral pH were 
extracted for four minutes with vigorous shaking. The water phases were 
removed, acidified to pH&lt;2 (10% H.sub.2 SO.sub.4) and re-extracted with 
ethyl ether. Both extractants were dried for several minutes over 
anhydrous sodium sulfate and filtered through Whatman #11 filter paper. 
After evaporation of the ether at 40.degree. C. to near dryness, the 
samples were taken up into acetone and dispensed into GC vials which were 
capped and stored in the dark at 4.degree. C. until analyzed (usually not 
stored more than 3 days). Standard solutions of various expected and/or 
related compounds (4-chlorobenzoate, acetophenone, 4'-chloroacetophenone, 
etc.) were prepared by dissolving the compounds in acetone (10 mg/ml). 
Following tentative identification of some gas-chromatography peaks 
considered indicative of 4-chlorobiphenyl metabolites of interest, 
rigorous quantitive extractions were performed. Here each culture was 
first acidified to pH&lt;2 (10% H.sub.2 SO.sub.4), extracted three times into 
ethyl ether for four minutes each time (vigorous shaking for four 
minutes), concentrated by evaporation, and taken up into 1 ml of acetone 
containing an internal standard (60 ppm, 2'-chloroacetophenone). Such a 
technique was utilized for time course as well as one time studies. 
Solutions were stored in the dark at 4.degree. C. until analyzed. 
In an attempt to isolate multi-milligram amounts of 4-chlorobiphenyl 
metabolites, 1L cultures were extracted exhaustively with ether. This was 
done to insure that as much of the products as possible were extracted. 
After purification by preparative TLC described herein, unknown 
metabolites were analyzed by gas-chromatography/mass-spectroscopy (GC/MS). 
This method was later altered to separate base/neutrals from acidic 
compounds. The 1L culture was filtered to remove excess 4-chlorobiphenyl. 
Approximately 50 g of sodium bicarbonate were added to the culture. This 
was followed by exhaustive extraction into ethyl ether. The water phase 
was then acidified to pH 2 with 10% H.sub.2 SO.sub.4, exhaustively 
extracted into ether, and back extracted with 5% NaHCO.sub.3 to remove any 
base/neutrals that may have been present. After removal of ether by 
evaporation, the residues were taken up into 20 ml of ethyl ether and 
examined using analytical thin-layer chromatography (TLC) and/or 
preparative TLC. Organic compounds were located on TLC plates by 
observation under ultraviolet light (254 nm). Spots or bands were scraped 
from the plates and compounds eluted from the silica gel with a small 
amount (10-20 ml) of ether or acetone. 
THIN LAYER CHROMATOGRAPHY (TLC). 
Initially, TLC silica gel sheets containing a fluorescent indicator were 
utilized. They were developed with a solvent mixture containing: toluene: 
ethyl acetate: formic acid (85:15:1 by volume). Analytical chromatograms 
were sprayed with Gibbs' reagent (.05% N-2,6-trichloro-p-benzoquinone 
imine in ethanol for detection of phenols and with 10.4% 
2,4-dinitrophenylhydrazine in 2N HC1 for detection of ketones. 
TLC analytical and larger preparative plates were employed for the 
isolation of 4-chlorobiphenyl metabolites. The solvent used here was 
toluene: ethylacetate: formic acid (90:9:1, volume/volume). Bands of 
organic compounds were located by observing their quenching of plate 
fluorescence under 254 nm light. 
GAS-CHROMATOGRAPHY 
Analyses of metabolites in ether extracts of culture media also were 
performed using a Hewlett-Packard gas-chromatograph model #5790A. A 5% 
phenyl methyl silica capillary column was employed with a flame ionization 
detector (FID) for peak detection. Hydrogen was the carrier gas. A 
temperature program was used, with a range of 80.degree. C. to 300.degree. 
C., with an increase of 8.degree. C./minute. Injector temperature was 
225.degree. C. With rare exceptions, these conditions yielded peaks that 
were well-defined and well-separated. 
SAMPLE PREATION FOR MASS-SPECTROSCOPY (MS) 
Cultures were exhaustively extracted into ethyl ether and the ether extract 
then concentrated by evaporation at 40.degree. C. The organic residues 
were taken up with 3 ml of ethyl ether. Using a Pasteur pipette, drawn out 
to a capillary, dissolved residues were loaded onto TLC preparative 
plates. Standards of 4'-chloroacetophenone and 4-chlorobiphenyl were 
spotted, one on each side of the plate. The plates were then developed in 
the appropriate solvent (see above). After drying the plates, separated 
compounds, seen as bands, were scraped from the plates into individual 
beakers where the silica gel was first suspended in acetone and then 
filtered. An aliquot of the filtrate was then spotted onto an analytical 
plate to examine the purity of each extracted compound. If judged pure 
(one spot), the sample was evaporated to dryness and taken up into 2 ml of 
fresh acetone and sent out for mass-spectral analysis. If the sample was 
judged to be impure (multiple spots), preparative TLC was repeated. 
GLC/MASS-SPECTROSCOPY 
Following establishment of proper separation conditions using the 
Hewlett-Packard GC, some samples were analyzed on a Krator GLC/MS 25 
mass-spectrometer/gas-chromatograph. Ionization energy was set at 70eV. 
Samples which were thought to contain carboxylic acid moieties were 
derivatized with trimethylsilane (TMS) prior to GLC/MS analysis. 
SAMPLE PREATION FOR NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (NMR) OF 
4'-CHLOROACETOPHENONE 
Cultures were extracted as above into ethyl ether. After evaporation of the 
ether, samples were taken up into 3 ml of ethyl ether, streaked onto a TLC 
preparative plate, and chromatographed. Several bands were observed, the 
band that corresponded with the 4'-chloroacetophenone standard was 
removed, suspended in ethyl ether, and filtered through a fritted glass 
filter. The filtrate was then evaporated to dryness, redissolved in 2 ml 
of deuterated chloroform (CDCl.sub.3), and evaporated to dryness again. 
One ml of CDCl.sub.3 was added, the solution placed into a 10 mm NMR tube, 
and sent out for analysis by NMR spectroscopy. Standard compounds were 
prepared at 100 mM concentration in ether (1 ml), evaporated to dryness, 
and taken up in 1 ml of CDCl.sub.3. Samples were examined at 300 mHz with 
TMS as a reference using the spectrometer facilities in the Department of 
Biochemistry, St. Paul (Model, Nicollet NT-300 WB). 
ENZKYME ASSAYS 
One liter of cells grown to approximately midlogarithmic phase on 
4-chlorobiphenyl and one liter grown to late mid-logarithmic phase on 
0.25% yeast extract were harvested by centrifugation, washed with cold 
buffer (phosphate, pH 7.0, 50 mM), resuspended in a small volume of cold 
buffer, and lysed by passing through a pre-chilled French Press at 20,000 
psi. The lysed cell suspension was centrifuged at 20,000.times.g at 
4.degree. C. for fifteen minutes using a Sorvall centrifuge. The enzyme 
extracts were kept on ice and assayed immediately for their abilities to 
catalyze oxygen consumption when presented with 4-phenylpryocathechol 
(Kodak Chemicals). Assays were run in a thermostatic chamber equipped with 
a Clarke electrode (Yellow Springs Instruments). Assay mixtures contained 
1 ml of buffer (50 mM PO.sub.4,pH 7.0), 100ul of cell-free extract (lmg 
protein) and 5 umole of 4-phenylpyrocatechol. The reaction was initiated 
by addition of substrate. Oxygen uptake was corrected for uptake observed 
with cell extract alone. 
PLASMID MINIPREPS 
To examine bacterial cells for the presence of plasmids, the alkaline lysis 
method of Ish-Horowitz (Maniatis, et al., Molecular Cloning, p. 368-369 
(Spring Harbor Laboratory Publications (1982)) was employed. Agarose gels 
were 0.7% and each well contained 10 ul of the prepared sample. Lambda DNA 
restricted with HindIII and with BstEII served as molecular markers. 
Escherichia coli mini-preps containing pBR322 served as a control to 
insure the gel electrophoresed properly. Eletrophoresis took place at 25V 
overnight. The gel was then stained with 0.0001% ethidium bromide for 45 
to 60 minutes, rinsed with distilled water, and photographed with a 
Polaroid camera using a UV filter. 
MUTAGENESIS AND PLASMID CURING 
Fifty microliters of a mid-logarithmic phase culture of the bacterium were 
added to each of ten test tubes containing 0.25% yeast extract and one of 
the following agents: acridine orange, novabiocin, mitomycin C, or 
ethidium bromide. Tubes containing the latter two compounds were wrapped 
tightly in foil and incubated in the dark to prevent photoinactivation of 
the curing/mutagenic agent. Controls, containing only yeast extract, were 
also used. Cultures were checked each day for growth and transferred to a 
new series of test tubes of the same medium and curing agent, except that 
curing agents were added at slightly increased concentrations at each 
transfer. After several passages, dilution platings were done in 
triplicate on 0.25% yeast extract agar. Once colonies appeared on plates, 
replica plating was done to determine if any colonies had become unable to 
utilize 4-chlorobiphenyl as a growth substrate. Suspected cured or mutant 
cell lines were patched from master plates to a new yeast extract plate 
and to a plate containing 4-chlorobiphenyl as the sole carbon and energy 
source. After incubation, cell lines that failed to grow on the 
4-chlorobiphenyl plates were streaked onto another yeast extract plate and 
subsequently inoculated into 4-chlorobiphenyl broths. Failure to grow in 
such a broth was considered proof of inability to utilize the substrate. 
ELECTRON MICROSCOPY 
Bacterial cells were placed on a paladiumcoated electron microscope grid 
and negatively stained with phosphotungstic acid by standard procedures. 
Stained cells were viewed and photographed using a Phillips 201 
transmission electron microscope. These facilities were located at the 
University of Minnesota, Department of Biology, St. Paul, Minnesota. 
CHEMICALS 
Chemicals were purchased as follows: acetophenone, 2'-chloroacetophenone, 
3'-chloroacetophenone, 4'-chloroacetophenone, 3-chlorobenzoic acid, 
4-chlorobenzoic acid, and 3,5-dichlorobenzoic acid from the Aldrich 
Chemical Co.; 4-chlorobiphenyl from Pfaltz and Bauer, recrystallized from 
absolute ethanol; 2-chlorobiphenyl from Pfaltz and Bauer; 3-chlorobiphenyl 
from Alfa Products; biphenyl from Mallinckrodt; 4-hydroxybenzoate from 
Eastman; 4-phenylpyrocatechol from Kodak. 
ISOLATION OF PSEUDOMONAS MB86. 
Pseudomonas MB86 was isolated from soil contaminated with creosote and 
pentachlorophenol, obtained in an industrial area of southern New 
Brighton, Minn. 
Approximately one gram of the contaminated soil was added to each of three, 
125 ml Erlenmeyer flasks containing 50 ml of basal medium containing 0.05% 
(weight/volume) 4-chlorobenzoate. These flasks were incubated on a shaker 
at 30.degree. C. for one week. Microscopic analysis at this time revealed 
a variety of microorganisms. One hundred microliters of each enrichment 
were spread onto each of three agar plates containing 0.05% 
4-chlorobenzoate. Three agar plates containing basal medium, but no growth 
substrates were inoculated with 100 ul of the 4-chlorobenzoate enrichment. 
Approximately 10.0 mg of 4-chlorobiphenyl were placed in the lids of each 
of these plates Colonies that appeared on these plates were then 
restreaked several times on 1% yeast extract agar plates for purification. 
Pure cultures obtained from yeast extract plates after 2-3 days were then 
streaked onto plates with 4-chlorobiphenyl as the sole carbon source and 
incubated for 4-10 days. 
After 4 days, one bacterium, later identified as Pseudomonas MB86, produced 
a characteristic yellow/brown color on 4-chlorobiphenyl medium. The plates 
also had a sweet smell. Colonies were very small and opaque on the minimal 
medium. On yeast extract the bacterium produced light yellow colonies. 
Once purified, the bacterium was grown in quantity, concentrated, and 
frozen for stock cultures using the materials and methods described 
herein. 
IDENTIFICATION AND CHARACTERIZATION OF THE 4-CHLOROBIPHENYL DEGRADING 
BACTERIUM 
The purified 4-chlorobiphenyl medium isolate described above was 
characterized for morphologic and physiologic traits as described below. 
Morphology: rod shaped, motile with single polar flagellum at the cell 
terminus 
Gram Stain: negative 
Physiology: positive with respect to catalase and oxidase; non-fermentive 
as indicated by triple sugar iron media 
The bacterium was inoculated into an OxiFerm tube (Roche Labs), as was a 
known Pseudomonas species (P. aeruginosa). After 24 and 48 hours various 
tests in the tubes were scored as indicated in Table 1 below: 
TABLE 1 
__________________________________________________________________________ 
Anaerobic Aerobic 
Organism 
Oxidase 
(Dextrose) 
Arginine 
N.sub.2 Gas 
H.sub.2 Gas 
Indole 
Xylose 
(Dextrose) 
Urea 
Citrate 
__________________________________________________________________________ 
MB86 + - - - - - - - - - 
Control + - + - - - + - + - 
(P. aeruginosa) 
__________________________________________________________________________ 
Results from 24 hour incubation of OxiFerm tubes. 
(+) = positive reaction growth 
(-) = no reaction, no growth 
To further characterize the organism's ability to grow on various compounds 
other media prepared as described herein were utilized and growth 
observations are reported in Table 2 below: 
TABLE 2 
______________________________________ 
Medium Observations 
______________________________________ 
Biphenyl (p) scant growth; (l) scant growth 
2-chlorobiphenyl 
(p) no growth; (l) no growth 
3-chlorobiphenyl 
(p) growth; (l) ND 
4-chlorobiphenyl 
(p) growth, yellow/brown pigment; 
(l) same as (p) 
3-chlorobenzoate 
(p) no growth; (l) no growth 
3,5-dichlorobenzoate 
(p) no growth; (l) no growth 
p-hydroxybenzoate 
(p) ND; (l) no growth 
benzoate (p) ND; (l) no growth 
4'-chloroacetophenone 
(p) no growth; (l) no growth 
______________________________________ 
Results from media inoculated with Pseudomonas MB86. 
(p) = plate; 
(l) = broth. 
Based on the above morphologic and physiologic traits the purified 
4-chlorobiphenyl degrading bacteria isolated from the creosote and 
pentachlorophenol contaminated soil described herein was determined to be 
a strain of Peudomonas appropriately classified as Psudomonas MB86. The 
purified culture of Psudomonas MB86 has been deposited with the American 
Type Culture Collection, Rockville, Md., and has been assigned ATTC No. 
53728. 
GROWTH CURVE OF PSUDOMONAS MB86 IN 0.25% YEAST EXTRACT BROTH. 
In order to gain information pertaining to the organism's generation time 
on a rich carbon source, cell densities of 2 freshly inoculated 0.25% 
yeast extract cultures were measured over an eight hour period. As can be 
seen in FIG. 1 (data points represent average values of duplicate flasks), 
growth progressed in the expected three phase pattern of lag, log, and 
stationary phases. Death phase was not observed in this time period. Cell 
doubling time was determined from the growth curve during logarithmic 
phase and shown to be approximately two hours. Viable counts revealed that 
during logarithmic phase, cell densities of approximately 10.sup.9 cells 
per ml were obtained. 
GROWTH CURVE OF PSUDOMONAS MB86 ON 4-CHLOROBIPHENYL 
To determine the rate at which Pseudomonas MB86 grew on 4-chlorobiphenyl as 
the sole carbon and energy source, flasks were inoculated in duplicate 
with 4-chlorobiphenyl-induced cells (1 ml per 50 ml of medium). Samples 
were taken every 24 hours for turbidity measurements. As seen in FIG. 2 
(data points represent average values of duplicate flasks), the doubling 
time increased substantially from that observed for growth in 0.25% yeast 
extract. No lag phase was apparent, but both the logarithmic and 
stationary phases were observed. Cell doubling time was approximately 14 
hours and viable counts revealed approximately 10.sup.8 cells per ml at 
midlogarithmic phase. 
During growth on 4-chlorobiphenyl, colored compounds were produced. To 
determine if these compounds affected readings at 600 nm, samples were 
centrifuged for five minutes in a Beckman microfuge and the absorbance of 
the supernatant solution at 600 nm determined. Absorbance values were 
recorded and substracted from turbidity readings. (FIG. 2). Also, it 
appeared that the appearance of color in culture fluid coincided with cell 
growth. 
IDENTIFICATION OF 4'-CHLOROACETOPHENONE 
During growth of Pseudomonas MB86 in the presence of 4-chlorobiphenyl, a 
very sweet smell was noted. The odor faded with time. Gas-chromatography 
of ether extracts of culture fluids revealed several peaks indicative of 
4-chlorobiphenyl metabolites. A major peak had the same retention time as 
known 4'-chloroacetophenone. As shown in FIG. 4, 
gas-chromatography/mass-spectroscopy of the unknown revealed an 
essentially identical fragmentation pattern as that seen for known 
4'-chloroacetophenone run as a standard (FIG. 3). The metabolite was 
extracted from larger culture volumes (1L), purified by preparative TLC 
and analyzed by NMR spectroscopy. As seen in FIGS. 5 and 6, the purified 
metabolite had an NMR spectrum identical to known 4'-chloroacetophenone. 
These experiments confirmed that Pseudomo as MB86 produced 
4'-chloroacetophenone. 
PRELIMINARY IDENTIFICATION OF ADDITIONAL METABOLITES 
Preparative TLC revealed, in addition to bands for 4-chlorobiphenyl and 
4'-chloroacetophenone, an additional well-defined band. This metabolite 
was isolated and prepared for GLC/MS analysis. GLC indicated the presence 
of several compounds. Two of these were initially identified as 
2-hydroxy,2-[4'-chlorophenyl]ethane and 4-chlorobenzoic acid. As shown in 
FIGS. 7-10, upon comparison to known standards, identification was 
confirmed. In the case of the 4-chlorobenzoic acid, a TMS derivitized 
sample was compared with a TMS derivitized standard. Another chlorinated 
compound was found and was consistent with the structure. 
##STR1## 
Mass spectral data (FIG. 11) indicated a molecular ion (M+) of 170, and an 
M+(--CH.sub.2 OH) of 139, with a subsequent fragmentation pattern 
identical to the M+(--CH.sub.3) of 4'-chloroacetophenone. This compound 
was tentatively identifed as 2-oxo, 2-[4'-chlorophenyl] ethanol. 
TIME COURSE STUDIES 
Data from a seven day growth experiment of Pseudomonas MB86 on 
4-chlorobiphenyl confirmed the importance of 4'-chloroacetophenone as a 
4-chlorobiphenyl metabolite. GC analyses indicated a decline in 
4-chlorobiphenyl concentration over time, with a concurrent increase in 
4'-chloroacetophenone concentration. As shown in FIG. 12, (data points 
represent average values obtained after normalization to the internal 
standard 2-chloroacetophenone) in several cases at day 6 this production 
of 4'-chloroacetophenone peaked and began to decline to day 7. 
An interesting aspect of these time course experiments was the predictable 
color change that occurred. Twenty-four to thirty-six hours after 
inoculation, the cultures exhibited a lilac color. Spectrophotometric 
analysis revealed a peak in the range of 532 nm. Upon acidification at 
pH&lt;2 with 10% H.sub.2 SO.sub.4, this color disappeared. Upon addition of 
base (6N NaOH) to pH&gt;10, no change was noted. The compound was quickly 
reduced (decolorized) with a small amount of sodium borohydride and easily 
reoxidized (color recovered) with 3% H.sub.2 O.sub.2. These data suggest 
the presence of a quinone. 
Within 48 hours, the cultures exhibited a golden color. This gradually 
darkened over time until at seven days the cultures took on an olive 
color. Heavy inoculation of the organism from a stock plate produced a 
brilliant yellow color within 24 hours. Upon acidification to pH&lt;2 10% 
H.sub.2 SO.sub.4, the color bleached. As seen in FIG. 13, in alkaline 
conditions of pH&gt;10, the color intensified. This indicates the presence of 
a ring fission product such as muconic semialdehyde (Gibson, Science, 
161:1093-1097 (1968); Furukawa and Matsumura, J. Agric. Food Chemistry, 
24:251-256(1976)). As seen further in FIG. 13, spectral analysis at 
neutral, acidic, and basic pH's revealed a shift in wavelength. 
PLASMID PREPS. 
As seen in FIG. 14, electrophoresis of alkaline "miniprep" lysates of 
Pseudomonas MB86 showed five or six DNA bands. This pattern was 
reproducible. These bands may represent individual plasmids, or different 
forms of a plasmid. 
MUTAGENESIS AND PLASMID CURING 
The organism failed to show viability in as little as 1 ug/ml of mitomycin 
C. Growth in acridine orange was noted, but upon subculturing viability 
was lost. The organism was successfully subcultured through increasing 
amounts of ethidium bromide (EtBr) (up to 50 ppm) and Novobiocin (up to 21 
ppm). Replica plating of cells surviving subculturing through five 
transfers in media containing these curing agents/mutagens revealed the 
presence of two potential mutants or cured strains from the ETBr series 
and seven from the Novobiocin series. Approximately 110 colonies were 
screened from both series. Further testing of the potential mutants 
revealed only five that could not grow on 4-chlorobiphenyl. All five were 
from the Novobiocin series.