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47 Degradation of polychlorinated biphenyls by two species of Achromobacter1 M. Ahmed and D. D. Focht C>eptrtmtnt of Soil Science tod Atrtctdtural Engineering, University of Californio, Riverside, California Accepted September 1 lf 1972 Aimed, M.. end D. D. Focht. 1973. Degradation of polychlorinetod biphenyls by (wo species otAekromobtcter. Can. J. Microbiol. 19:47-52. Two species of AcSromobactef were isolated from sewage effluent using biphenyl (BP) and p^hloro- biphenyt 0*CB) respectively as sole carbon sources. Achromobacter BP grown on biphenyl accumulated a product with an ultraviolet absorption maximum at 257 nm which could not be identified. Washed cell suspension* of both isoUtos oxidized biphenyl, o-phenylphenol, phenylpyruvatc, catechol, p-ch!orobi- phenyi evchlorobiphenyl, o-chlorobiphenyt, o.o'-dichlorobiphcnyl, and p,p'-dichlorobiphenyl. Both bobMl produced mere cleavage products by fission of the benzene ring. However, spectral characteristics of degradation products from respective substrates were different between the two isolates, indicating divergent degradation pathways. Benzoic and p-chlorobcruoic adds were produced from the degrada tion of BP and pCB, respectively, by Achromobacter pCB. Chloride was not produced by either isolate during the degradation of all etiloroblphenyia tested including the growth of Achromobacter pCB on jhdiiorobiphenyL ____________ Ahmed, M.,etD. D. Focht. 197). Degradation of polychlorinated biphenyls by two species of Achromo bacter. Can. 1. Microbiol. 19: 47-52. Deux espices d`Achromobacter ftirent Isoldes da faffluent de boues tfigout en utilisant le bipMnyl (BP) ct le p-chlorobiphinyl (pCB) respectivement, comme seules sources de carbone. VAchromobacter BP, qot s'est diveloppi tur le biphenyl, aocumule un produit non WemiW oui a une absorption maxi mum da Tuliravioletie 4 257 nm. Des suspensions de cellules Lavies des deux isolats oxydent le bipMnyl, Tortho pMnylphdnol, le phtnylpyniyate, le catechol, le ponr-chforobiphdnyl, le mrio-chiorobiph4nyl, rrfiko-chlorob<ph6nyl, I'orrto.orrto'dichlorobipheny) et le pare.pans'-dichlorobipMnyl. Les deux isolats pnduisent des composes h clivage mtta par fission du noyau benzine. Cepcndant, les caractiristiques spectrales des produits de digradation des aubstratt respective! diffirent pour les deux isolats. ce qui ksdique alnti des chemins de digradation divergents. Les acides benzolque et porixhlorobenzolque aont dea produits de la degradation du BP et du pCB, respectivement par VAchromobacter pCB. Le cfckfre n'est pas produit par aueun des isolats au court de la digradation de tous let chlorobiphinyls testis y oomprit la crousance Achromobacter pCB tur p-chlorobiphinyL (Traduit par le journal] Introduction PoiycUorinated biphenyls (PCB't) have been found to b widespread pollutants throughout the world (11,14,16) and have only recently been assessed for their environmental impact though they have had widespread commercial use in several hundred products since the 1920's. PCB't are used in plastics, wrapping paper, carbon paper, printing inks, paints, resins, tires, cooling systems, and as stabilizers in pesticide sprays. Because they have excellent fire-retardant proper ties, they are chiefly used for incorporation into electrical wires. PCB's have very similar properties to 1,1,1trichloro 2,2-bis(/>-ch1orophenyl)ethane (DPI) and the persistent chlorinated hydrocarbon in secticides. In fact, many of the DDT-resjdoe studies before 1969 may be invalid because analytical techniques for the most part were not able to distinguish between DDT and PCB bomera. PCB's have been shown to be toxic to fiErtMl J--7,1971 birds (14,19) and mammals (15), and are powerfal inducers of steroid hydroxylases as is 1,1dichloro-2,2-bis(p-chlorophcnyl)ethene (DDE), a . DDT metabolite. PCB accumulations in livers and eggs of birds have been observed to be as high as 900 ppm (14). Fire-retardant properties of the biphenyl mole cule increase with increasing chlorine substitu tion. It is unfortunately axiomatic that biodegra dation of aromatic compounds decreases as chlorine substitution increases. If it were com mercially possible and desirable to make PCB's that would not pose a potential environmental hazard (i.e. biphenyls containing less chlorine atoms), we would need to know the effect of the number of chlorine substituents and their position upon biodegradation. The knowledge of their degradative pathway is also important in assessing potential environmental contamination. No investigation, to our knowledge, has been nude on the biodegradation of these compounds, although Lunt and Evans (13) showed that bi phenyl was degraded by a soil bacterium to DSW 032775 u CAM. I MCKOMOL VOL. It. ItT) phenylpyruvate. It it our intention to provide tome meaningful data in tbit regard using bac teria from sewage as models of the biodegradativa process in nature. Materials and Methods Cultural Methods Two types of organisms used in this investigation were isolated from sewage effluent by elective culture with biphenyl (BP) and p-chlorobiphenyi (pCB) u sole carbon aouroes in t basal salt solution (6). Biphenyl or pCB in 0.1% concentration was included as a carbon source. The isolates were transferred several times to fresh and pure cultures were obtained by streaking onto 2% agar plates containing the respective carbon source (BP or pCB). The isolates were maintained by periodic transfers to sterile agar media. Biphenyl and fCB isolates were grown in 200-mi volumes of media contained in 500-mJ flasks. The cultures were incubated at 2S*C on a reciprocating shaker for 36 and 66 h. respectively, to achieve thdr stationary phases of growth. Manometry Oxygen uptake was determined at 30*C using a Gilson ropirometer (Gilson Medical Electronics, Inc., Middle ton, Wisconsin). Retting cell suspensions were prepared from cultures grown at reported above. The becheria wen harvested by centrifugation at 5000 g for 10 min. The cells were washed three times in cold 0.2 M (pH 7.0) phosphate buffer and suspended in the same buffer. The luspensions were adjusted such that a 23-fold dilution of the cell suspension in an II-mm tube had an optical density (O.D.) of 0.64 at 325 mm. The substrates used in this investigation were water- insoluble. They were dissolved in ethanol to obtain a Anal concentnttion of 10 unvote*/ml; 0.2 ml of test substrate (2|unolet) were introduced into the main compartment of the respiration flask. The ethanol was evaporated, 2.5 ml of 0.02 M pH 7D phosphate btrffar was added, and 0.3 ml of the cell suspension (O.D. I6c0) was placed in the side arms. The oxygen uptake was corrected for endogenous respiration. Chemicali Chemicals were purchased from the following sources: phenylpyruvtc acid and catechol from Nutritional . Biochemical! (Cleveland, Ohio); rrt-chlorobiphcnyl MTB). p-cfclofobiphenyt (pCB), py-dichlorobiphenyl (y-DCB), and py-dichlorobiphcnyt (p,p'-DCB) from Chomkal Procurement Labs., Inc., (College Point. New York); 4-phenylcstcchoi, -phenylphenot, sod p-cMonw benaoic acid from Eastman Kodak Co., (Rochester, New York); o-chkwodiphcnyl (oCB) from K it K Labs., Inc.. (PUinview, New York); biphenyl from Aldrich Chemical Co* Inc., (Milwaukee, Wisconsin). Analytical Techniques Ultraviolet (uv.) spectral analysis was performed with a Beckman Dll-G Recording Spectrophotometer, infra red (i.r.) spectra of Kllr pellets were obtained with a Ptrkin-Elmer 621 Infrared Spectrophotometer, oust spectra were obtained with a Finitigu 1015 S/L Mats Spectrometer, and nuclear magnetic resonance (un.t.) spectra were obtained with a Varian T-6 apparatus. Chloride was assayed by addition of acidified AgNOj to supernatant material! (6). pCB isolate was grown on BP and pCB separately. To isolate the final products of degradation, the culture medium was centrifuged to obtain clear supernatant solutions. The supernatants were acidified with 1 N acetic acid. A compound was precipitated from the culture supernatant of pCB on acidification, whereas no precipitate was formed from the BP supernatant. The precipitate was washed twice with distilled water and dissolved in ethanol. Light brown crystals were formed on evaporation of the alcohol. The crystals were purified by crystallization with chloroform. The product was identified by uv., i.r* n.m.r* and mass spectral analyses. Acidified BP supernatant was extracted with diethyl ether (6). The crystalline material obtained on evapora tion of the ether was recrystallized twice from ethanol. The compound was identified by uv, and i.r. spectral analytes. Results Both isolates were nonmolile, short, gram negative rods that produced acid with no gag from glucose, produced neither acid nor gas Cron) lactose and maltose, produced no indole, did not hydrolyze starch, and wefe catalase-positive. Methyl red, Vogues-Proskauer, and cytochrome, oxidase tests were positive, and nitrates were no( reduced by the BP isolate, while the oppositg effects were noted with the pCB isolate witlj nitrate being oxidized to nitrite. According tq standard taxonomic methods (17), they were boitj identified os species of Achromobacter. Growth of the Organisms Biphenyl isolate (Achromobacter BP) grew well with BP as the sole carbon source, pCB was cometabolized and did not support growth. At regular intervals, samples were taken from grow ing BP cultures, and the supernatants were ana lyzed spectrophotometrically. The original spec trum of BP (Xaut - 246 nm) had completely disappeared during the early phase of growth (12-14 h). A light yellow-colored compound with an absorption maximum at 400 ntn ap peared in the growth medium. The spectral max imum shifted to 412 then 432 nm with time, and the yellow color of the medium changed to bright yellow. The color and absorption peak a| 432 nm were abolished on acidification, and a new peak at 335 nm was formed. The origina absorption peak and color reappeared when thg medium was made neutral or alkaline. As tha growth progressed, the yellow color faded an<j DSW 032776 STLCOPCB4016738 AHMED AND POCKT: *IPHFNYL DBCHADATION SY ACHKOMOSACTF.* 49 finally disappeared from the growth medium. This color loss coincided with a rapid growth rate. Finally, a product with an absorption peak at 257 nm accumulated in the medium. p-Chlorobiphenyl isolate (Achromobacter pCB) grew slowly in basal salts solution with pCB as the sole carbon source. To obtain an idea of intermediates being formed during growth of this isolate on pCB, samples were withdrawn at regu lar intervals and assayed spectrophotometrically. Analogous to BP degradation, the original absorption peak of pCB (Xmi " 235 nm) dis appeared, and a new absorption peak at 400 nm was formed with concomitant appearance of a light yellow color. This absorption peak was replaced by another peak at 412 nm, which shifted to 432 nm with lime as the yellow color intensified. On further incubation, the inter mediate with peak absorbance at 432 nm dis appeared, and a new intermediate with a uv. absorption maximum at 235 nm accumulated. The bright yellow color gradually faded with time. Achromobacter pCB grew well on BP. Absorp tion peaks at 400, 412, and 432 nm and a yellow color were formed during growth as in the case Fta. i. Rates of oxygen uptake by washed cell mis* pensions of biphenyl-frown Achromobacter BP meuboUn| various substrates. All rates ara corrected for endogenous respiration. (O), biphenyl; (A), a-phcnylphenol; (), catechol; 19). pbenyipynjvais. 0 30 SO 90 IZO ISO ISO TIME (minutes) Fso. 2. Rates of oxygen uptake by washed ceU sus pensions of p-chlorobiphenyl-frown Achromobacter pCB metabolizing various substrates. Alt rates are corrected for endogenous respiration. (O), p-Chtorobiphertyl; (A), e-pbenylpbenol; (Y), catechol; (), phenylpyruvate; (O) biphenyl. irfY TABLE ! Oxygen uptake and abaorptioa maxima of the intermediate* formed after tha degradation of wferant substrates by BP and fCB hoUtes "T - rr BPiaotete pCB isolate SutMtrat* Oteygen uptake, |MB0lft Abaorptioa maxima of products, am Oxygen uptake, nmoles Absorption maxima of substrate*, nm Bfehanyt a-Phenylphatsoi S3sr~ 4^-te.hn. 7.5 257 f.O 2.9 3177 M0 1.5 375 (Mydtaxymuoonte samtekMtyd*) 9 7.0 230 6.0 1.5 313 3.0 375 (Mqrdroxytnuoonic samiaktehyde) 9 DSW 03277? STLCOPCB4016739 JO CAM L MtCtOaSM. VOL. . im of pCB degradation by this isohte. The final product of degradation of BP had a uv. absorptioa peak at 230 nm. The appearance of a bright yellow color during growth of both isolates on their respective sub strates, its disappearance on acidification, and its reappearance in basic solutions indicated a meta cleavage of the benzene nucleus. Characterization ofDegradation Products Formed from Achromobacter pCB The mass spectrum of the compound with uv. absorption at 235 nm showed a parent ion peak at m/ 156, and the intensity of the parent + 2 peak (about 33%) strongly indicated the presence Fn. 3. lutes of oxygen uptake by washed cefl me* MwioBi of biphenyl-grown Actwmoimtier BP comettboiirini various substrates. AH rates we corrected for endogenous respiration. (Ok p-Chlorobipbenyl; (A), e-cfalorobiphcnyl; (O), m-cfclorobiphenyl; (), 4,4'dichlorobiphenyl; (), 2^-didilorobipbaqrL of a single chlorine atom in the molecule; the base peaks at m/e 111 and 113 occurring in a 3:1 ratio indicated fragmentation of COOH. Nuclear magnetic resonance spectra established that chlorine was at the para position. The compound was unequivocally identified as p-chlorobenzoic acid upon further observations showing that the uv. and i.r. spectra of the isolated compound were identical with an authentic sample. The compound with uv. spectrum at 230 nm had identical uv. and i.r. spectra with authentic benzoic acid. Manometry Initial experiments showed that both isolates grown on BP and pCB respectively were simul taneously adapted to the utilization of a variety of substrates. The oxidation of BP. o-phenylphenol, phenylpyruvate, and catechol was studied using washed cell suspensions of BP and pCB isolates. These compounds were all oxidized without lag. The results are given in Figs. 1 and 2 and Table l. Figure 2 also shows the oxidation of pCB by the pCB isolate. On completion of the experiments as deter mined by the return of respiration rate to that of endogenous, the flask contents were withdrawn and centrifuged to obtain clear supernatant solu tions. The supernatant solutions were assayed spectrophotometrically to determine the extent of substrate degradation and to detect any inter mediate accumulated. The absorption maxima obtained after the degradation of the different substrates are also given in Table 1. Catechol was oxidized by both isolates with concomitant formation of a yellow-colored substance having maximal absorption at 375 nm (neutral or alka line) and 317 nm (acid). These peaks are at the TABLE 2 OxygM uptake and ibeorpdee maxima of the intermediate* formed after the degradation of various cMprinaied biphenyl* by BP and pCB isolates . BP Mate febslrale Oxygen upwlre. pmoies Aheaeption maxima of products, nm x-CWocobiphenyl e-Cbtorobiphenyl fjr'-Dtchlorobi phenyl e^-Dichlorobiphenyl boWVlHWUwneMM. S.O $.0 3.0 4.5 OS 237 400 and 377* 400 and 263* 257 400 pCB isolate Oxygen uptake, tunom Abiorptioo maxima of products, tun 6.5 235 4.0 270 S.O 235 3.0 235 0.5 400 OSlni 032778 STLCOPCB4016740 AHMED AND POCHT: MTHENYL DEGRADATION IV ACHROMO*ACTE* 31 fame wavelength as reported for 2-hydroxymutonic semialdehyde (2, 4, 7, 12) indicating that loth organisms possessed the enzyme capable or opening the aromatic ring by melt cleavage. Achromobacler BP would not grow on pCB, iCB, oCB. o,o'*DCB, and p^-DCB, but washed cell suspensions of this isolate grown on BP comelabolized these substrates with uptake of oxygen as given in Fig. 3 and Table Z Admmcbacter pCB when grown on pCB oome tabolized oCB, mCB. pjt-DCB, and with the consumption of oxygen as shown in Fig. 4 and Table Z Absorption maxima obtained from the eon* tents of manomctric flasks after the completion of degradation of the above noted compounds as* also given in Table Z Although not all the intermediates obtained were characterized, a product obtained after degradation ofp./'-DCB ated to be p-chlorobenzoic acid aa it was rprecipitated with acid and had the same uv. absorption peak as an authentic sample. The results dearly establish that biphenyl and jMchlorobiphenyi are rapidly degraded by both cultures of Achrwnobacttr on the basis of growth studies, manometric data, and spectrophotomctric analyses of resting cell and culture super* The weB documented observations that substitution decreases biodegradation is illustrated in our study. Achromobaatr BP grew only on BP and comctabolizcd the monoand di-chlorobiphcnyls; Aehromobactcr pCB grew belter on BP than on pCB and cometabo lized mCB, oCB, and the dichlorobiphcnyls. The positive identification of benzoic and p-chlorobenzoic adds as degradation products formed from biphenyl and pCB respectively and the likelihood of p-chlorobenzoic acid being formed from pjt'-DCB strongly suggests the occurrence of a common degradation pathway for Achromabaettr pCB in accordance with Horvath and Alexander's (8) explanation for cometabolism. On the basis of these findings, a hypothetical degradation pathway is given in Fig. 5 in accordance with well established cata bolic pathways for aromatic hydrocarbons (2,7). It if interesting that this pathway is analogous to that reported by Focht and Alexander (S) for the degradation of diphenylmethanes. The for mation of a catechol would most likely occur at the 2J rather than the 3,4 positions since p,p'DCB is blocked by a chlorine substituent in the 4 position and is more rapidly oxidized than o/S-DCB. Furthermore, 4-phenyIcatechol is not oxidized by the bacterium. The immediate and rapid oxygen uptake on o-phenylphenol may suggest the participation of a mixed function oxidase as opposed to a dioxygenase in the oxida tion of the ring. Inasmuch as the bacterium pos sesses a meta cleaving enzyme and "meta cleavage products" are observed in resting cell and culture supernatants, it ia unlikely that ortho cleavage occurs. Cleavage of the hypothetical catechol would moat likely occur between carbons 1 and 2 fface cleavage between 3 and 4 (with p.p'-DCB) * Ihp. 4. Rates of oeygai uptaks brwubsdedj kd pMhau of achJonUplMrl posn AArvfwhdSr SCB eowmbollnna vanoua substrates. AB rates srs corrected fce mdoetnous respiretioa. (Ah eOileroMptwayt; |6l i^chtoobij>henylj (), M>-dkhlowhiphsnyl; DSW 032779 a CAM I. MKftOKOL. VOL. 1*. ini would generate an acyl chloride, a compound that deoompoces spontaneously in water to liberate chloride--an ion not found in our studio. Furthermore, cleavage of unaubatituted ring* of pCB and BP at the 3,4 position is un likely since aldehydes were never detected from culture or resting cell supernatants. The degradation of biphenyl compounds by Achromobaaer BP follows a pathway different from the other isolate as noted by the appearance of different degradation products (Tables 1 and 2). This difference may account for the failure of the isolate to use pCB as a growth substrate. The accumulation of p-chlorobenzoic acid from degradation ofpCB by Achromobaaer pCA further indicates the preferential degradation of the untubsututed to the chlorinated ring without release of chloride. Although p-chlorobenzoic acid was apparently refractory to further degra dation by the pCB isolate, several workers have indicated the cometabolism of monochlorobenzoic acids by different species of microorgan isms (1, 9, 10,1ft). The absence of chloride in reaction mixtures of both isolates (indicating the accumulation of chlorinated degradation products) strongly sugfests that the failure of both isolates to mineralize the chlorobiphenyls to CQi, HjO, and HQ is due to the lack of or inhibition of enzymes effecting dehalogenation. Acknowledgment The authors thank Dr. William Fenical for performing mass spectral and n.m.r. analyses. This investigation was supported by U.S. Public Health Service Environmental Sciences Training Grant No. ES 00084-05. I. Cain. R. I., E. K. Tranter, slut J. A. Dajuuh. 1961. 77m utilization of some halogeiiatcd aromatic acids by Nocardie. Oxidation and metabolism. Kodwm. J. 1M: 211-227. X Daouy. S. 1971. Catabolism of aromatic eomi pounds by microorganisms. Adv. Microbial Phytisjj, 3. Daolev, S., P. J. Chapman, D. J. Ciison, and .. . Wooo. 1964. Degradation of the benzene nucleus I bacteria. Nature (London), 202: 775-778. 4. Daoizy, S., W. C. Evans, and D. W. Ribbons. 196(1 New pathways in the oxidative metabolism of aromatic compounds by microorganisms. Nature (London), 188 : 560-566. 5. Focht, D. D., and M. Alexander. 1970. Bacterial degradation of diphenylmethane. a DDT model substrate. Appl. Microbiol. 20: 608-611. 6. Focht. D. D., and M. Alexander. 1971. Aerobic comeutbolism of DDT analogues by Hydrogeno- monas sp. Agric. Food Chem. 19: 20-22. ~- 7. Girson, D. T. 1968. Microbial degradation of aromatic compounds. Science (Washington), 161: 1093-1097. I. Horvath, R. S., and M. Alexander. 1970. Come tabolism: a technique for the accumulation of biochemical products. Can. J. Microbiol. 16: 1131 1132- 9. Horvath, R. S., and M. Alexander. 1970. Come tabolism of m-chlorobcruoate by an Anhrobacter. Appl. Microbiol. 20 : 254-258. 10. Huohes, D. E. 1965. The metabolism of halogen- substituted benzoic acids by Pteudotttotuis fluorescent. Biochcm. J. 96: 181-188. 11. Koeman, J. H., M. C. Ten Noe ver De Brauw, and R. H. De Vos. 1969. Chlorinated biphenyls in fish, mussels and birds from the River Rhine and the Netherlands Coastal area. Nature (London), 221: 1126-1)28. IX Kohma, Y., N. Itada, and O. Hayaiski. 1961. Metapyrocatcchase: a new catechol-cleaving enzyme. J. Biol. Chem. 236: 2223-2227. 13. Lunt, D., and W. C. Evans. 1970. The microbial metabolism of biphenyl. Biochem. J. 118: 54p-55p. 14. Paeirr, I., D. J. Jeffries, and N. W. Moore. 1970. Polychlorinated biphenyls in wild birds in Britain and their avian toxicity. Environ. Pollut. 1: 3-26. 15. Riseirouoh, R. W., P. Reiche. D. B. Peakall, S. O. Hersman, and M. N. Kirven. 1968. Poly, chlorinated biphenyls in the global ecosystems. Nature (London), 220: 1098-1102. 16. Schmidt, T. T., R. W. Rjsebrouch, and F. Gress. 1971. Input of polychlorinated biphenyls intq California coastal waters from urban sewage out falls. Bull. Environ. Contsm. Toxicol. 6: 235-243. 17. Skerman, V. B. D. 1967. Guide to the idcntiticaiioi) of the genera of bacteria. 2nd cd. 1ft. Walker, N,, and D. Harris. 1970. Metabolism of 3-chlorobenzoic acid by Atotobactrr species. Soil Biol. Biochem. 2: 27-32. 19. Wildish, D. J. 1970. The toxicity of polychlorinaied biphenyls (PCB) in sea water to Cranutwus onanicus. BulL Environ. Conum. Toxicol. 5: 202-204. DSW 032780 STLCOPCB4016742