Document R0gy9x8z2nmaqy3jj782D2ra

47 ViS^radatioK of polychlorinated biphenyls by two species of Achromobactcr1 M. Amu15 and D. D. Focht /V/v;> e tent of S. 'il Science awl Agricultural Engineering, University of California, Riverside, California Accepted September II, 1972 Ahmi r>. M.. and D. IX 1 ihiit. 1973. Degradation of polychlorinated biphenyls by two species otAchromebaenr. Can. J. Microbiol. 19: 47-52. Two species of Achromobactcr were isolated from sewage effluent using biphenyl (BP) and p-chloro- biphcnyl (/>CB3 ivspcclhcly as sole carbon sources. Achromobactcr BP grown on biphenyl n. cumulated a product "Oh_an .old absorption maximum at 257 nm which could not Iv identiti.-d \\ a-.ti.-.t. ,t| suspcri' of N't'-! isolates oxtdired biplienyl, o ('hemIphonol. pbemlpMu\ate. cal.stiol. . I<1.<. phenvi. --^ alor.'-' :-!vn\I. o-chtowhiphcnsl. i\.''-ili.lil.'tobiphenx 1. and dii hloiobiph. m I t.*iIt isolates pr.viuced ' a clc.ix.ige products b\ fission of the Ivii.viv ring. I lowever. spistial dial a. I. 'I . of degradation pi .'ducts from respective substrates were different between the two isolates, nidi, umi: divcrpeiti degradation pathways. Benzoic and /i-chlorohcnzoic acids were produced from the degrada tion of BP and /( It, respectively, by Achromobactcr pCB. Chloride was not produced by either isolate during the degradation of all chlorobiphcnyls tested including the growth of Achromobactcr pCB on /r-chkvohiphcnvl. A m; ii. Si., et D. 13. Focht. 1973. Degradation of polychlorinated biphenyls by two species of Achromo-"-.cter. Can. J. Microbiol. 19: 47-52. Deux espeges d'Achromobactcr furent isolces de I'afflucnt de boucs d'egout cn ulilisant Ic biphdnyl i'P) et le p-chlorobiphenyl (pCB) rcspeclivement, comme sculcs sources de carbonc. L'Achromobactcr BP. qui s'est devcloppc sur le biphenyl, accumule un produit non klcnlific qui a unc absorption maxi- mum de 1'ultraviolcuc 4 257 nm. Des suspensions de cellules lav6cs dcs deux isolats oxydenl Ic biphenyl, Vortho phenylphcnol, le phdnylpyruvatc, le catechol, le pura-chlorohiphcnyl, le nteto-chlorobiphdnyj, l'or//;o-chlorobiphcn\ l, ri7rr/w,or//io'dichlorobiph6nyl et le /7r,para'-dichlorobiph6nyl. Lcs deux isolats produisent des com [roses 4 clivage meta par fission du noyau benzine. Cependant, tes caraclcrisliqucs spcctrales des produits de degradation dcs substrats rcspcctivcs different pour les deux isolats, cc qui indique ainsi des chcmins de degradation divergents. Les acides benzolque et para-chlorobcnz.otquc sont des produits de la degradation du BP et du pCB, rcspeclivement par 1'Achromobacter pCB. Le chlore n'est pas produit par aucun des isolats au cours de la degradation de tous les chlorobiphdnyls testds y compris la croissance A'Achromobacter pCB sur p-chlorobiphcnyl. [Traduit par le journal] Introduction PoIychlorin.Fcd biphenyls (PCB's) have been found to be widespread pollutants throughout the world (II. 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 tire used in plastics, wrapping paper, carbon paper, printing inks, paints, resins, tires, cooling v stems, 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-bi>(/>-chIorophenyl)ethane (DDT) nd 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. PCo s have been shown to be toxic to 1Received June 7,1972. birds (14, 19) and mammals (15), and are power ful inducers of steroid hydroxylases as is 1,1dichloro-2,2-bis(/)-chlorophenyl)ethcnc (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 . ;. ` _ { l f ' DSW 346180 _\ jf : mmm STLCOPCB4084471 48 CAN. J. MICROBIOL. VOL. 19, 1973 phenylpyruvatc. It is our intention to provide Spectrometer, and nuclear magnetic resonance (n.m.r.) sonic meaningful data in this regard using bac teria from sewage as models of the biodegradative process in nature. 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 Materials and Methods medium was centrifuged to obtain clear supernatant solutions. The supernatants were acidified with I N Cultural Methods acetic acid. A compound was precipitated from the Two types of organisms used in this investigation were culture supernatant of pCB on acidification, whereas no isolated from sewage effluent by elective culture with precipitate was formed from the BP supernatant. The ; biphenyl (BP) and p-chlorobiphcnyl (pCB) as sole carbon precipitate was washed twice with distilled water and ' sources in a basal salt solution (6). Biphenyl or pCB in dissolved in ethanol. Light brown crystals were formed . 0.1r', concentration was included as a carbon source. on evaporation or the alcohol. The crystals were purified . The isolates were transferred several times to fresh by crystallization with chloroform. The product was media, and pure cultures were obtained by streaking identified by uv., i.r., n.m.r., and mass spectral analyses. . onto 2% agar plates containing the respective carbon Acidified BP supernatant was extracted with diethyl ; source (BP or /jCB). The isolates were maintained by ether (6). The crystalline material obtained on evapora periodic transfers to sterile agar media. Biphenyl and tion of the ether was recrystallized twice from ethanol. ; pCB isolates were grown in 200-ml volumes of media The compound was identified by uv. and i.r. spectral , contained in 500-ml flasks. The cultures were incubated analyses. at 28C on a reciprocating shaker for 36 and 66 h. J i i respectively, to achieve their stationary phases of growth. Results j : Manometry Both isolates were nonmolile, short, gram- - Oxygen uptake was determined at 30C using a Gilson negative rods that produced acid with no gas respirometer (Gilson Medical Electronics, Inc., Middle ton, Wisconsin). Resting cell suspensions were prepared from cultures grown as reported above. The bacteria were 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 from glucose, produced neither acid nor gas from lactose and maltose, produced no indole, did not hydrolyze starch, and were catalase-positive. Methyl red, Vogues-Proskauer, and cytochromeoxidase tests were positive, and nitrates were not , 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 (his investigation were waterinsoluble. They were dissolved in ethanol to obtain a reduced by the BP isolate, while the opposite effects were noted with the pCB isolatewith nitrate being oxidized to nitrite.According to standard taxonomic methods (17), they were both j } [ ! final concentration of 10 nmoles/ml; 0.2 ml of lest identified as species of Achromobacter. 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. 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 Chemicals Chemicals were purchased from the following sources: ing BP cultures, and the supernatants were ana phcnylpyruvic acid and catechol from Nutritional lyzed spectrophotometrically. The original spec Biochcmicals (Cleveland, Ohio); m-chlorobiphenyl trum of BP (kmax = 246 nm) had completely ^(mCB), p-chlorobiphenyl (pCB), o,o'-dichlorobiphcnyl disappeared during the early phase of growth 3 (o.o'-DCB), and p,p'-dichlorobiphenyl (p,p'-DCB) from Chemical Procurement Labs., Inc., (College Point, New (12-14 h). A light yellow-colored compound York); 4-phcnylcatechol, o-phenylphenol, and p-chloro- with an absorption maximum at 400 nm ap benzoic acid from Eastman Kodak Co., (Rochester, New peared in the growth medium. The spectral max- > York); o-chlorodiphenyl (oCB) from K & K Labs., Inc., (Plainview, New York); biphenyl from Aldrich Chemical Co., Inc., (Milwaukee, Wisconsin). 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 ' i Analytical Techniques 432 nm were abolished on acidification, and a Ultraviolet (uv.) spectral analysis was performed with new peak at 335 nm was formed. The original a Beckman DB-G Recording Spectrophotometer, infra red (i.r.) spectra of KBr pellets were obtained with a absorption peak and color reappeared when the Perkin-Elmer 621 Infrared Spectrophotometer, mass medium was made neutral or alkaline. As the j spectra were obtained with a Finnigan 1015 S/L Mass growth progressed, the yellow color faded and , HPR9P DSW 346181 STLCOPCB4084472 AHMED AND POCHT: BIPIIF.NYL DEGRADATION BY ACIIROMOBACTER 49 finally disappeared from the growth medium. was formed with concomitant appearance of a This color loss coincided with a rapid growth light yellow color. This absorption peak was rate. Finally, a product with an absorption peak replaced by another peak at 412 nm, which at 257 nm accumulated in the medium. shifted to 432 nm with time as the yellow color p-Chlorobiphcnyl isolate (AchromobaclcrpCB) intensified. On further incubation, the inter grew slowly in basal salts solution with />CB as mediate with peak absorbance at 432 nm dis the sole carbon source. To obtain an idea of appeared, and a new intermediate with a uv. intermediates being formed during growth of this absorption maximum at 235 nm accumulated. isolate on/?CB, samples were withdrawn at regu The bright yellow color gradually faded with lar intervals and assayed spectrophotometrically. time. t Analogous to BP degradation, the original Achromobacter pCB grew well on BP. Absorp absorption peak of pCB (Xmax = 255 nm) dis tion peaks at 400, 412, and 432 nm and a yellow appeared, and a new absorption peak at 400 nm color were formed during growth as in the case f i I i i * )i fi Fig. 1. Rates of oxygen uptake by washed cell sus pensions of biphenyl-grown Achromobacter BP metabo lizing various substrates. Alt rates are corrected for endogenous respiration. (O), biphenyl; (A), o-phenyl- phenol; (Y), catechol; (), phenylpyruvate. Fig. 2. Rates of oxygen uptake by washed cell sus pensions of /7-chlorobiphcnyl-grown Achromobacter pCB metabolizing various substrates. All rates are corrected for endogenous respiration. (O), p-Chlorobi- phenyl; (A), o-phenylphenol; (Y), catechol; (), phenylpyruvate; (O) biphenyl. TABLE 1 Oxygen uptake and absorption maxima of the intermediates formed after the degradation of different substrates by BP and pCB isolates Substrate Biphenyl o-Phcnylphenol Phenylpyruvate Catechol 4-Phenylcatechol BP isolate Oxygen uptake, pmoles Absorption maxima of products, nm 7.5 257 6.0 -- 2.0 317, 260 3.5 375 (2-hydroxymuconic semialdehyde) 0-- pCB isolate Oxygen uptake, pmoles Absorption maxima of substrates, nm 7.0 230 6.0 -- 1.5 315 3.0 375 (2-hydroxymuconic semialdehyde) 0-- i DSW 346182 STLCOPCB4084473 50 CAN. J. MICROnlOL. VOL. 19, 1973 of />CB 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 m je 111 and 113 occurring in a 3:1 tion peak at 230 nm, ratio indicated fragmentation ofCOOM. 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-chlorobcnzoic 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 I were identical with an authentic sample. The Characterization ofDegradation Products Formed compound with uv. spectrum at 230 nm had ii i from Achromobacter pCB The mass spectrum of the compound with uv. absorption at 235 nm showed a parent ion peak identical uv. and i.r. spectra with authentic benzoic acid. - S i j at m je 156, and the intensity of the parent -f 2 Manometry ! peak (about 33%) strongly indicated the presence Initial experiments showed that both isolates ; grown on BP and pCB respectively were simul- tancously adapted to the utilization of a variety ; of substrates. The oxidation of BP, o-phcnyl- phenol, phcnylpyruvatc, and catechol was | studied using washed cell suspensions of BP and i pCB isolates. These compounds were all oxidized without lag. The results are given in Figs. 1 and 2 and Table 1. Figure 2 also shows the oxidation ; of pCB by the pCB isolate. , On completion of the experiments as deter- 1 mined by the return of respiration rate to that of 1 endogenous, the flask contents were withdrawn and centrifuged to obtain clear supernatant solu- j 5 tions. The supernatant solutions were assayed | spectrophotometrically to determine the extent } of substrate degradation and to detect any inter- j mediate accumulated. The absorption maxima - obtained after the degradation of the different [ Fig. 3. Rates of oxygen uptake by washed cell sus pensions of biphenyl-grown Achromobacter BP come tabolizing various substrates. All rates are corrected for endogenous respiration. (O). p-Chlorobiphenyl; (A), o-chlorobiphenyl; (O), m-chlorobi phenyl; (y), 4,4'dichlorobiphenyl; (), 2,2'-dichlorobiphenyl. 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 alkaline) and 317 nm (acid). These peaks are at the j TABLE 2 Oxygen uptake and absorption maxima of the intermediates formed after the degradation of various chlorinated biphenyls by BP and pCB isolates BP isolate pCB isolate Absorption Absorption Oxygen uptake, maxima of Oxygen uptake, maxima of Substrate gmoles products, nm pmoles products, nm ' />-Chlorobiphenyl 5.0 257 6.5 235 m-Chlorobiphcnyl 5.0 400 and 377 4.0 270 o-Chlorobiphenyl 5.0 400 and 265- 5.0 235 jj,p'-Dichlorobiphenyl 4.5 257 3.0 235 o,o'-Dichlorobipheny! 0.5 400 0.5 400 In order of appearance, respectively. STLCOPCB4084474 AHMED AND FOCHT: BIPHENYL DEGRADATION BY ACHROMOBACTER 51 e same wavelength as reported for 2-hydroxymu- is illustrated in our study. Achromobacter BP 1 conic scmialdchyde (2, 4, 7, 12) indicating that grew only on BP and cometabolized the mono- x both organisms possessed the enzyme capable of and di-chlorobiphcnyls; Achromobacter pCB ;t opening the aromatic ring by meta cleavage. grew better on BP than on pCB and comctabo d Achromobacter BP would not grow on pCB, lized mCB, oCB, and the dichlorobiphenyls. c /nCB, oCB, 0,o'-DCB, and p,p'-DCB, but The positive identification of benzoic and e washed cell suspensions of this isolate grown on p-chlorobenzoic acids as degradation products d BP comctabolized these substrates with uptake formed from biphenyl and pCB respectively and e of oxygen as given in Fig. 3 and Table 2. the likelihood of p-chlorobenzoic acid being i Achromobacter pCB when grown on pCB comc formed from p,p'-DCB strongly suggests the tabolized oCB, wCB, p,p'-DCB, and o,o'-DCB occurrence of a common degradation pathway with the consumption of oxygen as shown in for Achromobacter pCB in accordance with Fig. 4 and Table 2. Horvath and Alexander's (8) explanation for s Absorption maxima obtained from the con cometabolism. On the basis of these findings, a tents of manometric flasks after the completion hypothetical degradation pathway is given in / of degradation of the above noted compounds Fig. 5 in accordance with well established cata are also given in Table 2. Although not all the bolic pathways for aromatic hydrocarbons (2, 7). intermediates obtained were characterized, a It is interesting that this pathway is analogous 1 product obtained after degradation of p,p'-DCB to that reported by Focht and Alexander (5) for 1 appeared to be p-chlorobenzoic acid as it was the degradation of diphenylmethanes. The for l also precipitated with acid and had the same uv. mation of a catechol would most likely occur at i absorption peak as an authentic sample. the 2,3 rather than the 3,4 positions since p,p'- DCB is blocked by a chlorine substituent in the Discussion 4 position and is more rapidly oxidized than 0,o'-DCB. Furthermore, 4-phenylcatechol is not i The results clearly establish that biphenyl and oxidized by the bacterium. The immediate and p-chlorobiphenyl are rapidly degraded by both rapid oxygen uptake on o-phenylphenol may cultures of Achromobacter on the basis of growth suggest the participation of a mixed function studies, manometric data, and spectrophoto- oxidase as opposed to a dioxygenase in the oxida metric analyses of resting cell and culture super tion of the ring. Inasmuch as the bacterium pos natants. The well documented observations that sesses a meta cleaving enzyme and "meta cleavage chlorine substitution decreases biodegradation products" are observed in resting cell and culture supernatants, it is unlikely that ortho cleavage occurs. Cleavage of the hypothetical catechol would most likely occur between carbons 1 and 2 since cleavage between 3 and 4 (with p,p'-DCB) R' I Fig. 4. Rates of oxygen uptake by washed cell sus pensions of p-chlorobi phenyl-grown Achromobacter pCB cometabolizing various substrates. All rates are corrected for endogenous respiration. (A), o-Chlorobiphcnyl; (O), m-chlorobiphenyl; (), 4,4'-dichlorobiphenyl; (), 2,2'-dichlorobiphenyl. Fio. 5. Flypothetical pathway of biphenyl (R,R' = H), p-chlorobiphenyl (R = H, R' = Cl), and pp'-dichloro- biphenyl (R,R' = Cl) degradation by Achromobacter pCB. DSW 346184 STLCOPCB4084475 52 CAN. J MICROBIOL. VOl.. 19. 1973 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 ;>CB 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 Achromobacier 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 /vchlorobenzoic acid from degradation of/>CB by Achromobacier pCB further indicates the preferential degradation of the unsubstituted to the chlorinated ring without release of chloride. Although /7-chIorobenzoic 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, 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 chlorobiphenyls to C02, H20, and HC1 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. 1. Cain, R. B., E. K. Tranter, and J. A. Darrah. ^ 1968. The utilization of some halogenated aromatic acids by Nocardia. Oxidation and metabolism. Biochem. J. 106: 211-227. 2. Dagi.iy. S. 1971. Catabolism of aromatic com pounds by microorganisms. Adv. Microbial Physiol. 6: 1-46. 3. Dagi.ey, S., I'. J. Chapman, D. J. Giiison, and J. M. Wooi>. 1964. Degradation of the benzene nucleus by bacteria. Nature (London), 202: 775-778. 4. Daglfy, S., W. C. Evans, and D. W. Kiihions. I960. New pathways in the oxidative metabolism of aromatic compounds by microorganisms. Nature (London), 188: 560-566. 5. Loot it, D. D., and M. Alexander. 1970. Bacterial degradation of diphcnylmcthanc, a DDT model substrate. Appl. Microbiol. 20: 608-611. 6. Foe11r, D. D., and M. Alexander. 1971. Aerobic cometabolism of DDT analogues by Hydrogenonionas sp. Agric. Food Chcm. 19: 20-22. 7. Giiison, D. T. 1968. Microbial degradation of aromatic compounds. Science (Washington), 161: 1093-1097. 8. Horvath, R. S., and M. Alexander. 1970. Come tabolism: a technique for the accumulation of biochemical products. Can. J. Microbiol. 16: 1 Hi ll 32. 9. Horvath, R. S., and M. Alexander. 1970. Come tabolism of w-chlorobcnzoate by an Arthrobacter. Appl. Microbiol. 20: 254-258. 10. Hughes, D. E. 1965. The metabolism of halogen- substituted benzoic acids by Pseudomonas fluorescens. 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: 1126-1128. 12. Kojima, Y., N. Itada, and O. Hayaishi. 1961. Metapyrocatcchasc: a new catechol-cleaving enzyme. J. Biol. Chcm. 236: 2223-2227. 13. Lunt, D., and W. C. Evans. 1970. The microbial metabolism of biphenyl. Biochcm. J. 118: 54p-55p. 14. Prestt, I., D. J. Jefferies, and N. W. Moore. 1970. Polychlorinated biphenyls in wild birds in Britain and their avian toxicity. Environ. Pollut. 1: 3-26. 15. Risebrough. R. W., P. Reiche, D. B. Peakall, S. G. Hersman, and M. N. Kirven. 1968. Poly chlorinated biphenyls in the global ecosystems. Nature (London), 220: 1098-1102. 16. Schmidt, T. T., R. W. Risebrough, and F. Gress. 1971. Input of polychlorinated biphenyls into California coastal waters from urban sewage out falls. Bull. Environ. Contam. Toxicol. 6: 235-243. 17. Skerman, V. B. D. 1967. Guide to the identification of the genera of bacteria. 2nd ed. 18. Walker, N., and D. Harris. 1970. Metabolism of 3-chlorobenzoic acid by Azotobacter species. Soil Biol. Biochem. 2: 27-32. 19. Wildish, D. J. 1970. The toxicity of polychlorinated biphenyls (PCB) in sea water to Grammarus oceanicus. Bull. Environ. Contam. Toxicol. 5: 202-204. t t i j I j Na bacte has-p and unkn small may techr. of or Th (1), ? the d an a i mierc >Re 2A| well at 3Prt logic. STLCOPCB4084476