World J Microbiol Biotechnol (2006)

DOI 10.1007/s11274-006-9179-4

Aerobic degradation of highly chlorinated polychlorobiphenyls

by a marine bacterium, Pseudomonas CH07

F
Jaysankar De N. Ramaiah A. Sarkar

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PR
Received: 1 December 2005 / Accepted: 7 April 2006
 Springer Science+Business Media B.V. 2006

1
2 Abstract Hitherto, aerobic degradation of polychlo- PCB congeners can thus be produced. Due to their 24
3 rinated biphenyls (PCBs) has been reported to be non-flammability, chemical stability, high boiling point 25
4 limited to the less chlorinated biphenyls. We report and electrical insulating properties, PCBs have been 26
5
6
7
8
here a marine mercury-resistant bacterium, Pseudo-
monas CH07 (NRRL B-30604) which was capable of
degrading a variety of highly chlorinated congeners of
PCBs from the technical mixture Clophen A-50. Of the
ED
used for hundreds of industrial and commercial pur-
poses (Pieper 2005). PCBs are considered potential
carcinogens because some mixtures have been shown
to increase the development of hepatic tumors in rats
27
28
29
30
9 two most toxic coplanar PCBs present in Clophen (Safe 1984; Kimbrough 1987; Pieper 2005). PCB con- 31
10 A-50, one coplanar pentachloro congener CB-126 and taminations of drinking water, sediments, wastewater, 32
CT
11 one toxic sterically hindered heptachloro congener foods, and aquatic organisms have been documented 33
12 CB-181 were found to be degraded completely and the (Boon et al. 1985). Estuaries and the marine sediments 34
13 other coplanar tetrachloro congener CB-77 was are the ultimate global sinks for worldwide accumula- 35
14 degraded by more than 40% within 40 h by this tions of PCBs (Berkaw et al. 1996). The toxicity of 36
15 microorganism. The apparent absence of bphC in this different congeners of PCBs varies according to the 37
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16 bacterium leads to the proposal of a different mecha- chlorine substitution at different positions of the 38
17 nism for degradation of PCBs. biphenyl ring and the physical effects of PCBs vary 39
from mammals, to birds, to humans. It has been 40
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18 Keywords Pseudomonas CH07 Æ PCBs Æ Clophen observed that DNA strand-breaks occur in various 41
19 A-50 Æ CB-126 Æ CB-181 Æ Aerobic degradation species of marine organisms due to interaction with 42
polychlorinated biphenyl contaminants having coplan- 43
arity (Dunn et al. 1987; Everaarts et al. 1994). 44
20 Introduction PCBs have affected our environment through acci- 45
dents, spills in industrial facilities and mismanagement 46
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21 Polychlorinated biphenyls (PCBs) are a class of com- of storage or disposal areas (Nagayama 1976; Chen 47
22 pounds where the aromatic biphenyl rings carry from 1 et al. 1980). The Environmental Protection Agency 48
23 to 10 chlorine atoms and, theoretically 209 different (EPA) regulates the disposal of PCBs through the 49
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Toxic Substance Control Act (TSCA) which requires 50

that the PCBs be disposed of in accordance with the 51
methods regulated by the EPA. Incineration is the 52
J. De Æ N. Ramaiah Æ A. Sarkar
National Institute of Oceanography, Dona Paula, standard method of destruction for PCBs and the only 53
Goa 403 004, India one approved for the removal of PCBs from the soil 54
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and sediment (Blake 1994). Regulation provides scope 55

J. De (&)
for alternative methods like biodegradation/bioreme- 56
Graduate School of Kuroshio Science (GRAKUS), Kochi
University, Nankoku, Kochi 783-8502, Japan diation that could demonstrate the destruction of PCBs 57
e-mail: jaysankarde@yahoo.com equivalent to incineration. The microbial degradation 58

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59 of PCBs has been extensively studied in recent years. using ABI sequencing instrument (ABI PRISOM 106
60 The genetic organization of the biphenyl catabolic model no. 3700). The sequence was submitted online, 107
61 genes has been elucidated in various groups of micro- checked for the match using FASTA program (http:// 108
62 organisms, their structures have been analyzed with www.ebl.ac.uk) and was deposited with GenBank 109
63 respect to their evolutionary relationships, and new (accession numbers: DQ377451–DQ377452). 110
64 information on mobile elements has become available.
65 111

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Key enzymes, specifically biphenyl 2,3-dioxygenases, Growth pattern of CH07
66 have been intensively characterized, structure/se-
67 quence relationships have been determined and The growth of the isolate was determined by moni- 112

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68 enzymes optimized for PCB transformation. However, toring its OD following its inoculation into SWNB. In 113
69 due to the complex metabolic network responsible for brief, 50 ll of a 24 h old culture of CH07 was inocu- 114
70 PCBs degradation, optimizing degradation by a single lated into two 250-ml flasks containing SWNB (100 ml) 115

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71 bacterial species is necessarily limited (Pieper 2005). with Clophen A-50 added to them to a final concen- 116
72 The bacterium CH07 has shown the potential to tration of 100 mg/l (ppm). A control without any PCBs 117
73 clean up several other toxic chemicals including high was included. The flasks were incubated on a rotary 118

PR
74 concentrations of phenol, Cd, Pb, and Hg. Such mul- shaker (200 rev/min) at room temperature (ca. 119
75 tiple resistances make this bacterium a worthy candi- 28 – 2 C) for 120 h. 120
76 date for use in bioremediation of mixed wastes (De The attenuance (OD660) of the cultures was mea- 121
77 et al. 2003). This bacterium was checked for its sured every 12 h. Cell numbers were determined by 122
78 potential to degrade different congeners of PCBs from plating aliquot samples after proper dilution with 123
79 the technical mixture Clophen A-50 and was found to sterile 50% seawater. Log values of cell numbers were 124
80 125
81
degrade many highly chlorinated congeners convinc-
ingly within just 40 h.
ED
plotted to draw growth curves (Fig. 1).

Culture media and degradation experiment 126

82 Materials and methods A defined seawater nutrient broth (SWNB) medium 127
containing beef extract 3 g l)1, peptic digest of animal 128
CT
83 Isolation of microorganism tissue 5 g l)1 was used. One liter of medium contained 129
500 ml seawater and 500 ml distilled water and the 130
84 Several mercury-resistant Pseudomonas aeruginosa final pH was adjusted to 7 using 0.1 M NaOH. 131
85 like organisms (PALO) were isolated using Cetrimide Required amount of stock solution (10 g/l in hexane) 132
86 agar medium amended with 50 ppm Hg (as HgCl2) of Clophen A-50 (Bayer, Lot no. 16572) was added 133
87
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from water samples collected from a contaminated into the sterilized medium in sterile condition to 134
88 coastal site in the east coast of India. A bacterium achieve a final concentration of 100 ppm. Immediately 135
89 designated as Pseudomonas CH07 was found to after adding the stock solution of PCBs, the hexane 136
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90 degrade several congeners of PCBs containing five or was evaporated out by gently swirling the flasks in 137
91 more-chlorine atoms on the biphenyl ring from the sterile condition. Sterilized glycerol was added to the 138
92 technical mixture of PCBs, Clophen A-50 (Beyer,
93 Germany).
12
94 Identification
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95
log cell no ml-1

The isolate was characterized biochemically and was 10

96 identified by 16S rDNA sequence analysis. Overnight
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97 grown cultures were used for extraction of the genomic

98 DNA and the extraction was done using the Necleo- 8
control
99 spin Extract (Macherey Nagel, Germany) and the test
100 quality of the product was checked on 0.8% TAE
101 agarose gel. The sequencing primers used were (for-
6
102
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ward primer 8–27: 5¢AGAGTTTGATCCTGG 0 24 48 72 96

103 CTCAG 3¢ (Weisberg et al. 1991) and reverse primer time (h)
104 1492: 5¢GGTTACCTTACGACTT 3¢ (Reysenbach Fig. 1 Growth curve of CH07 with Clophen A-50 (100 ppm) and
105 et al. 1992). Sequencing (Sanger et al. 1977) was done without Clophen A-50 in 50% seawater nutrient broth

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139 medium in a 1:1 ratio of stock solution, glycerol. Forty Table 1 Biochemical characteristics of CH07
140 (40) ll of 24 h old broth culture of CH07 was Test Response Test Response
141 added in two replicates of 20 ml of test medium
142 (SWNB + ClophenA-50) and normal SWNB (without Gram stain )ve Nitrate reduction ++
Shape Very small rod H2S )a
143 any addition of Clophen A-50). Controls in duplicate Motility +b Indole )
144 were also maintained without the addition of the Pigment Fluorescent green Urease )

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145 organism in one set and with killed bacterial cells in Oxidase Slow reaction Growth on
146 another set at room temperature (ca. 28 C). At vari- OF No reaction Citrate +
Gelatinase + Sucrose Alkaline
147 ous predecided intervals of time, the samples were

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Catalase ++c Mannitol Acidic
148 taken out aseptically and prepared for GC analysis. Lipase ) Rhamnose Acidic
149 The comparison of degradation of PCBs was done with Starch hydrolysis + Arabinose Alkaline
150 the control without added bacteria and test condition Arginine + Streptomycin ++

O
Omithin ) Tetracycline ++
151 with the live bacterial cells. The possibility of absorp- VP ) Demechlocyclin ++
152 tion of PCBs onto the cells was checked by comparing MR ) Kanamycin ++
153 the control with killed bacterial cells and test condi-

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a
Negative response (no growth)
154 tions with live bacterial cells. b
Positive response (slight or moderate growth)
c
Very good response (good growth)
155 Analysis of PCBs

156 The PCBs were extracted following a standardized B-30604 as an integral part of the US patent no. 184
157 method. The samples were analyzed by GLC (Varian 6544773 (Sarkar et al. 2003). 185
158
159
160
161
GC-3380) coupled with an electron capture detector
(ECD) and an autosampler 8200. A capillary column
VA-5 (30 m · 0.25 mm) was employed with ECD for
peak detection. Argon with 5% methane was used as
ED
Growth pattern

It was clearly evident that there was no appreciable

change or effect of PCBs on the growth of CH07
186

187
188
162 the carrier gas. The injector temperature was fixed at
163 250 C. The analysis of PCBs was calibrated using the (Fig. 1). The bacterium had attained the exponential 189
CT
164 standards for the individual congeners of PCBs. phase within 12 h of growth in the presence of PCBs in 190
the growth medium, akin to the growth in the control 191
165 Identification of breakdown pathway flask. 192

166 The isolate was streaked on M9 (Sambrook et al. 1989) Analysis of PCB 193
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167 agar plates adding a biphenyl crystal on the lid as the

168 sole source of carbon and sealed with parafilm. As a It was observed that Clophen A-50 was sufficiently 194
169 control, the M9 agar plate was supplemented with degraded by CH07. Among the different congeners of 195
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170 yeast extract as carbon source. After 24 h of incubation PCBs present in Clophen A-50 14 chlorobiphenyls 196
171 in the dark, the colonies were sprayed with 2,3-di- were degraded to varying degrees (Table 2). 197
172 hydroxybiphenyl (2,3-OHBP) an intermediate in the Of the three most toxic coplanar PCBs (CB-77, CB- 198
173 biphenyl degradation pathway to see if there was any 126 and CB-169), this organism degraded one of them 199
174 appearance of yellow meta cleavage product (2-hy- (CB-126) completely in about 40 h (Fig. 2). 200
175 droxy-6-oxo-6-phenylhexa-2,4-dienoic acid; HOPDA) The other coplanar PCB, CB-77 (3,3¢, 4,4¢-Tetra- 201
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176 which would appear because of the production of 2,3- chlorobiphenyl) was also degraded substantially within 202
177 dihydroxibiphenyl-dioxygenase by the bphC gene. a short period of 40 h. One heptachlorobiphenyl, CB- 203
181 (2,2¢,3,4,4¢,5,6) was degraded completely within 204
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40 h (Table 3). 205

178 Results Two asymmetric di-ortho chlorinated biphenyls viz. 206
2,2¢,4,5,5¢-pentachlorobiphenyl and 2,3¢,4,4¢,6-penta- 207
179 Identification of the microorganism chlorobiphenyl were degraded to 20.19% (20.19 – 0.6) 208
and 19.66% (19.66 – 0.5) respectively. One congener 209
CB-116 (2,3,4,5,6-pentachlorobiphenyl) with all the 210
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180 The microorganism CH07 was purified and was iden-

181 tified from the biochemical test as a Pseudomonas chlorines substituted on one of the aromatic rings 211
182 aeruginosa (Table 1) which was further confirmed by being highly polar was degraded to only 20.04% 212
183 16S rDNA analysis. The culture is deposited as NRRL (Table 2). 213

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Table 2 Molecular formulae of PCBs in Clophen A-50 and their percent (mean – SD) degradation by CH07
Chlorobiphenyls Molecular Retention PCBs at PCBs at Degradation
formula time (min) 0 h (ng/ml) 40 h (ng/ml) of PCBs (%)

CB-101 (2,2¢,4,5,5¢) C12H5Cl5 19.564 18.17 14.50 20.19 – 0.6

CB-119 (2,3¢,4,4¢,6) C12H5Cl5 19.886 8.07 6.48 19.66 – 0.5
CB-97 (2,2¢,3¢,4,5) C12H5Cl5 20.892 8.17 6.57 19.69 – 1.2

F
CB-116 (2,3,4,5,6) C12H5Cl5 21.211 10.09 8.06 20.04 – 0.9
CB-77 (3,3¢,4,4¢) C12H6Cl4 21.823 53.37 40.42 24.25 – 1.7
CB-151 2,2¢,3,5,5¢,6) C12H4Cl6 22.595 2.04 1.28 37.32 – 0.6

O
CB-118 (2,3¢,4,4¢,5) C12H5Cl5 23.400 1.31 0.77 40.72 – 2.5
CB-105 (2,3,3¢,4,4¢) C12H5Cl5 24.830 17.54 9.29 46.69 – 1.3
CB-141 (2,2¢,3,4,5,5¢) C12H4Cl6 25.449 3.57 1.59 55.38 – 2.4
CB-138 (2,2¢,3,4,4¢,5¢) C12H4Cl6 25.819 1.62 0.71 55.97 – 1.6

O
CB-126 (3,3¢,4,4¢,5) C12H5Cl5 26.658 2.75 00.00 100 – 0.00
CB-128 (2,2¢,3,3¢,4,4¢) C12H4Cl6 27.702 5.02 1.79 64.33 – 2.3
CB-181 (2,2¢,3,4,4¢,5,6) C12H3Cl7 29.219 2.87 00.00 100 – 0.00
CB-180 (2,2¢,3,4,4¢,5,5¢) C12H3Cl7 30.484 1.64 0.63 61.33 – 1.7

PR
Identification of breakdown pathways 219

The marine MRB strain CH07 did not grow on M9 220

ED
medium which had biphenyl crystals as the soul source
of carbon whereas it grew normally on the M9 agar
plates supplemented with yeast extract as carbon
source. The colonies on the latter medium when
221
222
223
224
sprayed with 2,3-dihydroxybiphenyl (2,3-OHBP) were 225
226
CT
negative for metacleavage product (2-hydroxy-6-oxo-6-
phenylhexa-2,4-dienoic acid) indicating the non-func- 227
tioning or total absence of 2,3-dihydroxibiphenyl 228
dioxygenase by bphC gene. Subsequently, there was no 229
production of the yellow meta cleavage product 2-hy- 230
droxy-6-oxo-6-phenylhexa-2, 4-dienoic acid after 231
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spraying the colonies with 2,3-dihydroxybiphenyl. 232

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Discussion 233

Some PCB congeners have been found to be trans- 234

formed by both anaerobic and aerobic bacteria 235
(Bradley et al. 1996; Kimbara et al. 1999). Degradation 236
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of mono-, di- and tri-chlorinated biphenyl is relatively 237

common (Bedard et al. 1987). Some strains like Pseu- 238
domonas LB400 (Gibson et al. 1993), Alcaligenes 239
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Fig. 2 Gas chromatograms showing PCB degradation (upper

eutrophus H850 (Bedard et al. 1987), Corynebacterium 240
panel represents ‘‘0’’ h and lower one represent ‘‘40’’ h) by CH07
MB1 (Williams et al 1997) and Acinetobacter P6 241
(Kohler et al. 1988) are exceptional PCB degraders. 242
The specificity of the dioxygenases in these organisms 243
214 As the control with the dead cells did not show any differs greatly. Strains like P6 and MB1 are particularly 244
215 active against double para-chlorinated PCBs. Strains 245
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remarkable decrease of PCBs from the growth med-

216 ium, it was quite obvious that the PCBs were indeed H850 and LB400 preferentially express a 3,4-dioxy- 246
217 biodegraded, not absorbed and/or adsorbed by the genase, forming a cis-dihydrodiol form 2,5,2¢,6¢-tetra- 247
218 cells. chlorobiphenyl, which is degraded faster by H850 than 248

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Table 3 Structural characteristics of PCBs degraded by CH07

Chlorobiphenyls Molecular formula Mol. wt. Cl (%) Structures

CB-101 (2,2¢,4,5,5¢-Pentachloro) C12H5Cl5 254.5 69.74 Cl Cl

Cl

F
Cl Cl

CB-119 (2,3¢,4,4¢,6-Pentachloro) C12H5Cl5 254.5 69.74 Cl Cl

O
Cl Cl

Cl

O
CB-97 (2,2¢,3¢,4,5-Pentachloro) C12H5Cl5 254.5 69.74 Cl Cl Cl

Cl

PR
Cl

CB-116 (2,3,4,5,6-Pentachloro) C12H5Cl5 254.5 69.74 Cl Cl

Cl

Cl Cl

CB-77 (3,3¢,4,4¢-Tetrachloro) C12H6Cl4

ED 220 64.54

Cl
Cl Cl

Cl

CB-151 (2,2¢,3,5,5¢,6-Hexachloro) C12H4Cl6 289 73.70 Cl Cl Cl

CT

Cl Cl Cl

CB-118 (2,3¢,4,4¢,5-Pentachloro) C2H5Cl5 254.5 69.74 Cl Cl

E

Cl Cl

Cl
RR

CB-105 (2,3,3¢,4,4¢-Pentachloro) C12H5Cl5 254.5 69.74 Cl Cl Cl

Cl Cl

CB-141 (2,2¢,3,4,5,5¢-Hexachloro) C12H4Cl6 289 73.70 Cl Cl Cl

O

Cl

Cl Cl
NC

CB-138 (2,2¢,3,4,4¢,5¢-Hexachloro) C12H4Cl6 289 73.70 Cl Cl Cl

Cl Cl

Cl
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Table 3 Continued
Chlorobiphenyls Molecular formula Mol. wt. Cl (%) Structures

CB-126 (3,3¢,4,4¢,5-Pentachloro) C12H5Cl5 254.5 69.74 Cl Cl

Cl Cl

F
Cl

CB-128 (2,2¢,3,3¢,4,4¢-Hexachloro) C12H4Cl6 289 73.70 Cl Cl Cl Cl

O
Cl Cl

O
CB-181 2,2¢,3,4,4¢,5,6-Heptachloro) C12H3Cl7 323.5 76.81 Cl Cl Cl

Cl Cl

PR
Cl Cl

CB-180 (2,2¢,3,4,4¢,5,5¢-Heptachloro) C12H3Cl7 323.5 76.81 Cl Cl Cl

Cl Cl

Cl Cl

249
250
PCBs with an unchlorinated ring (Bedard et al. 1987).
The chlorobenzoic acids are not further degraded by
ED Because aerobes are only capable of degrading lower
chlorinated PCBs (4 or less chlorines per biphenyl), it is
278
279
251 most PCB-degrading bacteria. An exception is the important that the anaerobes grow well, transforming 280
252 281
CT
degradation of 2,3¢-dichlorobiphenyl by LB400. It is as many of the higher chlorinated PCBs as possible to
253 generally believed that biodegradation of PCBs lower chlorinated PCBs. Several factors affect the 282
254 decreases with the increase in chlorine substitution growth of bacteria in presence of toxic PCBs. Briefly, 283
255 (Furukawa et al. 1979). Generally, for mixtures of they are the chemical structure and composition of the 284
256 lower chlorinated PCBs, sufficient degradation may be PCB waste, the initial concentration, the solubility 285
257 achieved using a one-stage procedure involving only of the PCBs in the media, and the environmental 286
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258 aerobes. There is a danger, however, if higher chlori- conditions (temperature, pH, salinity, redox potential, 287
259 nated PCBs are left intact because they are generally available carbon, and presence of co-contaminants). 288
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260 more bioaccumulative than the less chlorinated PCBs Bioremediation technology (BT) has countless 289
261 (Unterman 1996). Additionally, there are the highly advantages and is a highly attractive alternative for 290
262 toxic congeners referred to as ‘‘dioxin-like’’, containing dealing with PCBs due to the high costs of transpor- 291
263 chlorine at the two para positions (4 on the biphenyl tation, incineration and other procedures of remedia- 292
264 ring) and at least two chlorines at the meta positions (3 tion that currently exist. BT can be used to treat low 293
265 or 5 position). The lack of ortho groups allows the concentration of contaminants; prevent physical and 294
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266 atoms in these congeners to line up in a single plane chemical treatment; maintain alteration of the con- 295
267 (referred to as coplanar PCBs), that makes them taminated area to a minimum; eliminate long term 296
268 especially toxic. responsibility and destroy organic contaminants. It 297
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269 A combination of anaerobic followed by aerobic does not generate secondary waste and can be com- 298
270 biodegradation has been found to maximize the bined with other treatment technologies for highly 299
271 removal of chlorine. Significance of aerobic degrada- complex mixed waste. BT is cost-effective (Churchill 300
272 tion of PCBs can be explained and understood in the et al. 1995; McGraph 1995; Sturman 1995). PCBs do 301
273 following way. The most critical stage of the two-step, present a challenge to bioremediation, as they are 302
274 anaerobic to aerobic process is getting the anaerobic highly stable. Nevertheless, the availability and evolu- 303
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275 bacteria to proliferate at a new site, cometabolizing the tion of potential cost saving possibilities makes BT 304
276 higher chlorinated PCBs (greater than four chlorines worthy of consideration when assessing/choosing 305
277 per biphenyl). treatment options. 306

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307 The marine bacterium, CH07 (Pseudomonas aeru- Bradley CA, Kearns RD, Wood PP, Black WE (1996) Degra- 345
308 ginosa) is capable of degrading PCBs of different dation of polyhalogenated biphenyl compounds with white- 346
rot fungus grown on sugar beet pulp. US Patent 5,583,041 347
309 configurations. This is the first conclusive demonstra- Chen PH, Wong CK, Rappe C, Nygren M (1980) Environ Health 348
310 tion of an aerobic microbial process involving a marine Persp 59:59 349
311 bacterium that degrades highly chlorinated PCBs Churchill PF, Dudley RJ, Churchill SA (1995) Waste Manage 350
312 contained in Clophen A-50. The extent of degradation 15:371 351
De J, Ramaiah N, Mesquita A, Verlekar XN (2003) Mar 352

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313 of different congeners of PCBs in presence of other Biotechnol 5:185 353
314 chlorobiphenyls and with varying degree of polarity Dunn BP, Black JJ, Maccubbin A (1987) Cancer Res 47:6543 354
315 and stereochemical asymmetry is a clear indication Everaarts JM, Sleiderink HM, Besten PJD, Halbrook RS, Shu- 355

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316 bacterial strains such as CH07, isolated from marine gart LR (1994) Environ Health Persp 102:37 356
Furukawa K, Tomizuka N, Kamibayashi A (1979) Appl Environ 357
317 environments can be used effectively for their detoxi- Microbiol 38:301 358
318 fication. Most importantly, highly chlorinated congen- 359

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Gibson DT, Cruden DL, Haddock JD, Zylstra GJ, Brand JM
319 ers, CB-180 and CB-181 were found to be degraded (1993) J Bacteriol 175:6735 360
320 sufficiently. However, the inability of the organism to Kimbara K, Shimura M, Hatta T, Kiyohara H (1999) Method for 361
degrading polychlorinated biphenyls and novel microor- 362
321 grow on biphenyl as the sole carbon source indicates

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ganism. US Patent 5,989,896 363
322 that the organism was unable to use biphenyl as sole Kimbrough RD (1987) Annu Rev Pharmacol Toxicol 27:87 364
323 carbon and energy source. Absence of the bphC Kohler HP, Kohler-Staub D, Focht DD (1988) Appl Environ 365
324 product 2,3-dihydroxybiphenyl dioxygenase indicates Microbiol 54:1940 366
McGraph R (1995) Pollut Eng 27:34 367
325 the absence of the complete bph operon and opens the Nagayama L, Kuratsune M, Masuda Y (1976) Bull Environ 368
326 possibility of different pathways of PCBs degradation, Contam Toxicol 15:9 369
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328
329
330
331
Acknowledgements We thank Dr. Shetye, Director, NIO, Drs.
D. Chandramohan and S.W.A. Naqvi for their support and
facilitation. Critical reviews of two anonymous reviewers are
gratefully acknowledged. De acknowledges CSIR for the SRF
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332 grant (31/26/75/2002 EMR-I) and UGC-DAAD short-term
333 scholarship. This is NIO contribution number xxx. Sanger F, Nicklen S, Coulson AR (1977) Proc Natl Acad Sci 377
USA 74:5463 378
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Sarkar A, De J, Ramaiah N (2003) Microbial process for deg- 379
radation of PCBS in Clophen A-50 using a novel marine 380
334 References microorganism, Pseudomonas CH07. US Patent 6,544,773 381
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335 Bedard DL, Haberl ML, May RJ, Brennan MJ (1987) Appl JH (1995) J Contam Hydrol 19:171 383
336 Environ Microbiol 53:1103 Unterman R (1996) In: Crawford RL, Crawford DL (eds) Bio- 384
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337 Berkaw M, Sowers KR, May HD (1996) Appl Environ Microbiol remediation: principles and applications. Cambridge Uni- 385
338 62:2534 versity Press, Great Britain, pp 209–253. ISBN 0521470412 386
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nated and polycyclic aromatic hydrocarbon compounds. Bacteriol 173:697

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Article No. : 9179 h LE h TYPESET
MS Code : WIBI966 4 CP
h 4 DISK
h