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o 41 Degradation of polychlorinated biphenyls by two species of Achromobacter' M, Ahmed and D. D. Focht Department of Soli Science and Agricultural Engineering, University of California, Riverside, California Accepted September 11,1972 Ahmto, M., and D. D. Focht. 1973. Degradation of polychlorinated biphenyls by two species of Achro. mobacter. Can. J. Microbiol. 19: 47-52. Two species of Achromobacter were isolated from sewage effluent using biphenyl (BP) and p-chloro- bipbeny! (/>CB) respectively as sole carbon sources. Achromobactcr BP grown on biphenyl accumulated a product with an ultraviolet absorption maximum at 257 nm which could not be identified. Washed cell suspensions of both isolates oxidized biphenyl, o-phcnylphcnol, phcnylpyruvate, catechol, /i-chlorobi- phenyl, m-chtorobiphcnyl, o-chlorobiphenyl, o.o'-dichlorobiphcnyl, and />,p'-dichlorobiphcnyl. Both isolates produced nicta 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 r-chlorobcnzoic acids were produced from the degrada tion of BP and pCIi, respectively, by Achromobactcr pCB. Chloride was not produced by either isolate during the degradation of all chlorobiphenyls tested including the growth of Achromobacter pCB on p-chlorobiphcnyl. _________ Ahmed, M.,etD.D. Foctrr. 1973. Degradation of polychlorinated biphenyls by two species of Achromo bacter. Can. J. Microbiol. 19: 47-52. Deux espiccs A'Achromobacter furent Isoldes de l'affluent de boucs d'6gout en utilisant le biphdnyl (BF) el le p-chlorobiphenyl (/>CB) rcspcctivemcnt, comnte seulcs sources de carbonc. VAchromobacter ' BP, qui s'est ddveloppd sur le biphdnyl, accumulc un produit non identify qui a unc absorption maxi mum de rultraviolctte h 257 nm. Dcs suspensions de cellules )aves dcs deux isolats oxydent le biphdnyl, Vorlho phdnylphdnol, le phdnylpvruvate, le catdchol, le poru-ch lorobiphenyl, le mcm-chlorobiphinyi, I'ozMo-chlorobipMnyl, 1'orrAojOrWio'dichlorobiphinyl et le para.porn'-dichlorobiphdnyl. Lcs deux isolats produisenl des composes 6 clivage mila par fission du noyau benzine. Cepcndant, lcs caractiristiques spoet rales dcs produifs de digradation des substrats respcctivcs different pour lcs deux isolats, cc qui Indique ainsi des chemins de digradation divergents. Les acidcs benzolque et pora-chlorobcnzolque sont det produits de la digradation du BP et du pCB, respcctivcment par 1*Achromobacter pCB. Le chlorc n'est pas produit par aucun des isolats au cours de la degradation de tous lcs chlorobiphinyls testis y compris la croissance &Achromobacter pCB sur p-chlorobiphinyl. [Traduit par le journal] Introduction Polychlorinated biphenyls (PCB's) have been found to be 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's 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 elcctrical'wircs. . PCB's have very fcimilar properties to 1,1,1- trichloro 2,2-bis(/M;hlorophenyI)ethane (DDT) and the persistent chlorinated hydrocarbon in secticides. In fact, many of the DDT-residue studies before 1968 may be invalid because analytical techniques for the most part were not able to distinguish between DDT and PCB isomers. PCB's have been shown to be toxic to JRccdvcd June 7,1972. birds (14,19) and mammals (15), and are power ful inducers of steroid hydroxylases as is 1,1dichloro-2,2-bis(p-chlorophenyl)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 made on the biodegradation of these compounds, although Lunt and Evans (13) showed that bi phenyl was degraded by a soil bacterium to , mmm DSW 029876 STLCOPCB4013838 f*M ^ m&mm tiMhWrrfrtr*1 48 CAN, J. MICROBIOL. VOL. 19, !*71 phenylpyruvate. It is our intention to provide some meaningful data in this regard using bac teria from sewage as models of the biodegrada- tive 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-chlorobiphcnyl (pCB) as sole carbon sources in a 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 media, 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 pCB isolates were grown in 200-ml volumes of media contained in 500-mi flasks. The cultures were incubated at 28C on a reciprocating shaker for 36 and 66 h, respectively, to achieve their stationary phases of growth. Manometry Oxygen uptake was determined at 30C using a Gilson respirometer (Gilson Medical Electronics, Inc., Middle ton, Wisconsin). Resting cell suspensions were prepared from cultures grown os reported above. The bacteria were harvested by centrifugation at 5000 for 10 min. The cells were washed three times in cold 0.2 M (pi I 7.0) phosphate buffer and suspended in the same buffer. The suspensions were adjusted such that a 25-fold dilution of the cell suspension in an 18-mm tube had an optical density (O.D.) of 0.64 at 525 mm. The substrates used in this investigation were water- insoluble. They were dissolved in ethanol to obtain a final concentration of lOpmoles/mf; 0.2 ml or test substrate (2 pmoles) were introduced into the main compartment of the respiration flask. The ethanol was evaporated, 2.5 ml of 0.02 M pH 7.0 phosphate buffer was added, and 0.5 ml of the cell suspension (O.D. 16.0) was placed in the side arms. The oxygen uptake was corrected for endogenous respiration. ' Chemicals Chemicals were purchased from the following sources: phenylpyruvlc acid and catechol from Nutritional Biochemicals (Cleveland, Ohio); m-chlorobiphenyl (mCB), p-chlorobiphcnyl (pCB), o.o'-dichlorobiphenyl (o.o'-DCB). and />,p'-dichlorobiphcnyl (p.p'-DCB) from Chemical Procurement Labs.. Inc., (College Point, New York); 4-phcnyIcatcchol, o-phcnylphcnol, and p-chlorobenzoic acid from Eastman Kodak Co., (Rochester, New York); o-chlorodiphcnyl (oCB) from K & K Labs., Inc., (Plainview, New York); biphenyl from Aldrich Chemical Co., Inc., (Milwaukee, Wisconsin). Analytical techniques Ultraviolet (uv.) spectral analysis was performed with a Beckman DB-G Recording Spectrophotometer, infra red (i.r.) spectra of KBr pellets were obtained with a Perkin-Elmcr 621 Infrared Spectrophotometer, mass spectra were obtained with a Finnigan 1015 S/L Mass Spectrometer, and nuclear magnetic resonance (n.m.r.) spectra were obtained with a Varian T-6 apparatus. Chloride was assayed by addition of acidified AgNOj to supernatant materials (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 front ethanol. The compound was identified by uv. and Lr. spectral analyses. Results Both isolates were nonmotile, short, gram negative rods that produced acid with no gas from glucose, produced neither acid nor gas from lactose and maltose, produced no indole, did not hydrolyze starch, and wcfe catalase-positive. Methyl red, Vogues-Proskauer, and cytochromeoxidase tests were positive, and nitrates were not reduced by the BP isolate, while the opposite effects were noted with the pCB isolate with nitrate being oxidized to nitrite. According to standard taxonomic methods (17), they were both identified as 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 (Xmax * 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 nin 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 at 432 nm were abolished on acidification, and a new peak at 335 nm was formed. The original absorption peak and color reappeared when the medium was made neutral or alkaline. As the growth progressed, the yellow color faded and QSW 02987? STLCOPCB4013839 AHMED AND FOCHT: BIPHPNYL DEGRADATION BY ACHB0M0BAC1EA 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. />-Ch!orobiphenyl isolate (Achromobacfer/>CB) 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 spcctrophotomctrically. Analogous to BP degradation, the original absorption peak of pCB (kmx 255 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 time 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. Achromobacfer 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 l i ' j.i ' Fio. 1. Rates of oxygen uptake by washed cell sus pensions of biphenyl-grown Achromobacfer BP metabo lizing various substrates. All rates are corrected for endogenous respiration. (O), biphenyl; (A), o-pticnylphenol; (), catechol; (), phenylpyruvate Fio. 2. Rates of oxygen uptake by washed cell sus pensions of p-chlorobiphenyl-grown Achromobacfer pCB metabolizing various substrates. All rates are corrected for endogenous respiration. (O), p-Chlorobiphcnyl; (A), <?-phenylphenol; (V), catechol; (9), phenylpyruvate; (O) biphenyl. TABLE I Oxygen uptake and absorption maxima or the intermediates formed after the degradation of different substrates by BP and pCB isolates -- ...... ......... .................... BP Isolate pCB isolate Substrate Oxyfeu uptake. pmoic* Absorption maxima of products, nm Oxygen uptake, limolcs Absorption maxima of substrates, nm Biphenyl o-Phcnylphcnol Phenylpyruvate Catechol 4-Phenylcatecho! 7.5 257 6.0 --- 2.0 .' 317, 260 3.5 375 7.0 230 6.0 1.5 ' 315 3.0 375 (2-hydroxymucotdc emWdehyde) -. * (2-hydroxymu con Ic semialdchyde) 0-- 0- --- 'I t ^ : DSW 029878 STLCOPCB4013840 O afeted* CAN. J. MICROBIOL. VOL. 19, 17J of pCB degradation by this isolate. The final of a single chlorine atom in the molecule; the product of degradation of BP had a uv. absorp base peaks at mje 111 and 113 occurring in a 3:1 tion peak at 230 nm. ratio indicated fragmentation of COOH. Nuclear The appearance of a bright yellow color during magnetic resonance spectra established that growth of both isolates on their respective sub chlorine was at the para position. The compound strates, its disappearance on acidification, and was unequivocally identified as p-chlorobenzoic its reappearance in basic solutions indicated a acid upon further observations showing that the meta cleavage of the benzene nucleus. uv. and i.r. spectra of the isolated compound 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/e 156, and the intensity of the parent -f 2 peak (about 33%) strongly indicated the presence 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-phenyl- phenol, 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 I. 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 6pectrophotomctricalty to determine the extent of substrate degradation and to detect any inter CO 90 <20 TIME (minuter) mediate accumulated. The absorption maxima obtained after the degradation of the different Fk>. 3. Rates of oxygen uptake by washed cell sus pensions of biphenyl-grown Achromobacter BP come-. Sabolizing various substrates. All rales are corrected for endogenous respiration. (OX p-Chlorobiphenyl; (A), #-chlorobiphenyl; (O), m-cnlorobiphenyl; (T), 4,4'- dichlorobiphenyl; (), 2^'-diclilorobiphenyl. 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 - Oaygen uptake and absorption maxima of the intermediates formed after the degradation of various chlorinated biphenyls by BP and pCB isolates BP isolate Substrate Oxygen uptake, nmoles Absorption maxima of products, nm /-Chlorobiphcnyl m-Chlorobiphcnyt -Chlorobiphenyl /,p'-Dich lorobiphenyl .o'-Dkhlorobiphenyl order of eppcmoce, nepeMtotr. 5.0 5.0 5.0 4.5 0.5 257 400 and 377* 400 and 265* 257 400 />CB isolate Oxygen uptake, pmoles Absorption maxima of products, nm 6.5 235 4.0 270 5.0 235 3.0 235 0.5 400 DSW 029879 STLCOPCB4013841 AHMED AND FOCHT: BIPHENYL DEGRADATION BY ACHROMOBACTER 5J same wavelength as reported for 2-hydroxymuconic scmialdchydc (2, 4, 7, 12) indicating that both organisms possessed the enzyme capable of opening the aromatic ring by meta cleavage. Achromobactcr BP would not grow on pCB, mCB, oCB, o.o'-DCB, and p,p'-DCB, but washed cell suspensions of this isolate grown on BP cometabolized these substrates with uptake of oxygen as given in Fig. 3 and Table 2. Achromobactcr pCB when grown on pCB cometabolized oCB, mCB, p,p'-DCB, and o,o'-DCB with tire consumption of oxygen as shown in Fig. 4 and Table 2. Absorption maxima obtained from the contents of manometric flasks after the completion of degradation of the above noted compounds ' Are also given in Table 2. Although not all the Intermediates obtained were characterized, a product obtained after degradation of p,p'-DCB appeared to be p-chtorobenzoic acid as it was also'precipitated with acid and had the same uv. absorption peak as an authentic sample. . Discussion The results clearly establish that biphenyl and p-chlorobiphenyl are rapidly degraded by both cultures of Achromobacter on the basis of growth studies, manometric data, and spectrophotometric analyses of resting cell and culture supernatants. The well documented observations that chlorine substitution decreases biodegradation is illustrated in our study. Achromobacter BP grew only on BP and cometabolized the monoand di-chlorobiphcnyls; Achromobacter pCB grew better on BP than on pCB and comctabolized mCB, oCB, and the dichlorobiphenyls. The positive identification of benzoic and p-chlorobcnzoic acids as degradation products formed from biphenyl and pCB respectively and the likelihood of p-chlorobcnzoic acid being formed from p,p'-DCB strongly suggests the occurrence of a common degradation pathway for Achromobacter 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 catabolic pathways for aromatic hydrocarbons (2,7). It is interesting that this pathway is analogous to that reported by Focht and Alexander (5) for the degradation of diphenylmcthanes. The for- mation of a catechol would most likely occur at the 2,3 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,o'-DCB. Furthermore, 4-phenylcatcchol 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 oxidatjon rjng inasmuch as the bacterium pos- gesses a meta cleaving enzyme and "meta cleavage products" are observed in resting cell and culture supernatants, it is unlikely that ortho cleavage 1 t' 'j{ : nwi>iir upr <iiH j'iiimi miifRiiwi mm DSW 029880 STLCOPCB4013842 52 CAN, I, MICROBIOL, VOL. I, 1971 would generate an acyl chloride, a compound that decomposes spontaneously in water to liberate chloride--an ion not found in our studies. Furthermore, cleavage of unsubstituted rings 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 Achromobactcr 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 Achromobacier pCB further indicates the preferential degradation of the unsubstituted 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 monochloro- bcnzoic acids by different species of microorgan isms (1,9, 10. 18). The absence of chloride in reaction mixtures of both isolates (indicating the accumulation of chlorinated degradation products) strongly sug gests that the failure of both isolates to mineralize the chtorobiphenyls to C02, H20, and HC1 is due to the lack of or inhibition of enzymes effecting dchalogenation. - 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. 1. Cain, R. B., E. K. Tranter, and J. A. Darrah. 1968. The utilization of tome halogenated aromatic acids by Nocardia. Oxidation and metabolism. Biochcm. J. 106:211-227. 2. Daoley, S. 1971. Catabolism of aromatic com pounds by microorganisms. Adv. Microbial Physiol. 6: 1-46. 3. Daoley, S., P. J. Chapman, D. J. Gibson, and J. M. Wood. 1964. Degradation of (he benzene nucleus by bacleria. Nature (London), 202: 775-778. 4. Daoley, S., W. C. Evans, and D. W. Ribbons. 1960. 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 comctabolism of DDT analogues by Hydrogenomonas sp. Agric. Food Chem. 19: 20-22. 7. Gibson, D. T. 1968. 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