APPLID AND ENVIRONMENTAL MICROBIOLOGY, May 1981, p. 1177-11830099-2240/81/051177-07$02.00/0

Vol. 41, No. 5

Degradation of Phthalic Acids by Denitrifying, Mixed Culturesof Bacteria

R. PAUL AFTRING,t BRUCE E. CHALKER,: AND BARRIE F. TAYLOR*Rosenstiel School ofMarine and Atmospheric Science, University ofMiami, Miami, Florida 33149

Received 17 November 1980/Accepted 2 March 1981

Mixed cultures of bacteria, enriched from aquatic sediments, grew anaerobicallyon all three isomers of phthalic acid. Each culture grew anaerobically on only oneisomer and also grew aerobically on the same isomer. Pure cultures were isolatedfrom the phthalic acid (o-phthalic acid) and isophthalic acid (m-phthalic acid)enrichments that grew aerobically on phthalic and isophthalic acids. Cell suspen-sion experiments indicated that protocatechuate is an intermediate of aerobiccatabolism. Pure cultures which grew aerobically on terephthalic acid (p-phthalicacid) could not be isolated from the enrichments, and neither could pure culturesthat grew anaerobically on any of the isomers. Cell suspension experimentssuggested that separate pathways exist for the aerobic and anaerobic oxidation ofphthalic acids. Each enrichment culture used only one phthalic acid isomer underanaerobic conditions, but all isomers were simultaneously adapted for the anaer-obic catabolism of benzoate. Cells grown anaerobically on a phthalic acid imme-diately attacked the isomer under anaerobic conditions, whereas there was a lagbefore aerobic breakdown occurred, and, for phthalic and terephthalic acids,chloramphenicol stopped aerobic adaptation but had no effect on anaerobiccatabolism. This work suggests that phthalic acids are biodegradable in anaerobicenvironments.

Diesters of o-phthalic acid (hereafter calledphthalic acid) are industrially important chem-icals used mainly in the manufacture of plastics.Their production has increased enormouslysince World War II, and they deserve the de-scription of ubiquitous environmental contami-nants (1, 22). m-Phthalic acid (hereafter calledisophthalic acid) and p-phthalic acid (hereaftercalled terephthalic acid) are manufactured insmaller quantities than is phthalic acid, but theyhave significant industrial uses; terephthalicacid, for example, is used to make Dacron (1).There is especial concem about the environmen-tal fate and effects of phthalic acid esters onorganisms (1, 2, 7, 20, 22). Phthalic acid estersare aerobically degraded by bacteria (10, 17-19),but little is known about their catabolism in theabsence of oxygen. In an isolated report, theanaerobic catabolism of dibutyl phthalate wasobserved in a freshwater hydrosoil system (15).Esterases involved in the initial attack onphthalic acid esters have no requirement foroxygen and, in fact, anaerobic conditions have

t Present address: Department of Medicine, Division ofEndocrinology, Metabolism and Nutrition, Medical Universityof South Carolina, Charleston, SC 29043.

t Present address: Australian Institute of Marine Science,Townesville, M.S.O., Queensland 4810, Australia.

been used to show the accumulation of phthalicacid from monobutyl phthalate by a Nocardiasp. (9). Thus, a principal question about phthalicacid esters catabolism is the anaerobic fate ofthe aromatic ring. This is a relevant concern,since phthalic acid esters, like other hydrophobiccompounds, are probably adsorbed to particu-late matter and deposited in sediments (16). Thecurrent communication indicates that all threeisomers of phthalic acid can be degraded anaer-obically by bacteria.

MATERIAILS AND MErHODSCultural methods. Cultures which grew on

phthalic acids were obtained by elective enrichment,using sediment samples from the Miami and Missis-sippi rivers (freshwater sources) and Biscayne Bay,Fla. (marine source). Cultures were incubated at 30°Cin 18-ml screw cap tubes filled with medium. The basalmarine medium was: NaCl, 13.35 g; MgCl2-6H20, 2.6g; MgSO4.7H20, 3.45 g; KC1, 0.165 g; CaCl2.2H20,0.735 g; NH4Cl, 0.3 g; yeast extract (Difco Laboratories,Detroit, Mich.), 0.25 g; NaNO3, 2.12 g; KH2PO4 plusK2HPO4 (as a 5% solution, pH 7.5), 0.5 g; trace metalsolution (23), 10 ml; and distilled water to 1 liter. Inthe freshwater basal medium, the MgSO4 and KCIwere omitted, and the following changes were made:NaCl, 1.0 g; MgCl2.6H20, 0.2 g; and CaCl2*2H20, 0.02g. Phthalic acids and other substrates were added to10 mM, with the exception of acetate (40 mM). Agar

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1178 AFTRING, CHALKER, AND TAYLOR

(1.5%; Difco) was used to solidify media. An enrichedmedium, used to isolate bacteria from mixed cultures,consisted of the basal medium plus 0.1% yeast extract,0.1% dextrose, 0.1% CH3COONa.3H20, and 0.8% nu-trient broth (Difco).

Batch cultures were grown either anaerobically inscrew cap bottles (180 or 250 ml) filled with mediumor aerobically in 500-ml Erlenmeyer flasks containing100 ml of medium. NaNO3 was omitted from theaerobic medium, and the flasks were incubated withrotary shaking at 150 to 200 rpm. Cells were harvestedby centrifugation (12,000 x g, 10 min; 150C), washed,and suspended to a Klett reading of about 1,000 units(red filter; 640- to 700-nm transmission). The suspen-sion medium was 200 mM NaCl-50 mM MgSO4 plus10 mM KCl for marine cultures and either 0.05 Mphosphate buffer (pH 7.5) or 20 mM NaCl plus 1 mMMgCl2 for freshwater cultures.Respirometry. Aerobic respiration was measured

at 300C either manometrically, with a Warburg ap-paratus (Gilson Medical Electronics, Middleton,Wisc.), or polarographically, with a Clark-type elec-trode (Yellow Springs Instrument Co., Yellow Springs,Ohio). In the latter method, the re-ction vessel con-tained 0.1 or 0.2 ml of cell suspension diluted to 5 mlwith 20mM NaCl-50mM MgSO4-10mM KCl-50mMtris(hydroxymethyl)aminomethane (Tris)-hydrochlo-ride buffer (pH 7.5) for marine cultures and 0.05 Mphosphate buffer (pH 7.5) for freshwater cultures.After endogenous activity was recorded for 5 to 10min, 1 or 2 p,mol of substrate was added. Constant-volume manometry was used to measure aerobic andanaerobic respiration with nitrate or nitrite, and thegases consumed or evolved were assumed to be O2 andN2, respectively. The Warburg flasks contained: Tris-hydrochloride or phosphate buffer (pH 7.5), 50 pmol;cell suspension (1,000 Klett units), 1.0 ml (aerobic) or2.0 ml (anaerobic); NaNO2 or NaNO3, 25 to 50,mol;substrate, 2 to 10,umol; 20% KOH, 0.1 ml (center well);and water to 3.0 ml. In anaerobic incubations, theflasks were gassed for 10 min with N2 before themanometers were closed. After temperature equilibra-tion, the substrates (and NaNO2 or NaNO3 in anaer-obic experiments) were added from the side arms atzero time. Nitrite production was sometimes deter-mined by adding 0.1 ml of 1 M sulfamic acid from aside arm at the end of the incubation (4). C02 produc-tion was measured by omitting KOH from the centerwelLs and tipping in 0.2 ml of 4 N H2SO4 at the end ofthe experiment (29).Chromatography. Aqueous samples were acidi-

fied and extracted with diethyl ether for analysis bythin-layer chromatography or gas-liquid chromatog-raphy. Silica gel sheets (Eastman Kodak, Rochester,N.Y.) were used for thin-layer chromatography witha solvent system of benzene-acetic acid (8:2) or ben-zene-dioxane-acetic acid (45:5:2). Spots on thin-layerchromatography plates were visualized with ultravi-olet light. For gas-liquid chromatography, the etherealextracts were treated with diazomethane generatedfrom nitrosomethyl urea, and the methyl esters wereseparated and quantified with a gas chromatograph(model 2700; Varian Associates, Walnut Creek, Calif.)with flame ionization detectors. Initially, Poropak Q(80/100 mesh), in a stainless steel column (183 by 0.3-

APPL. ENVIRON. MICROBIOL.

cm outer diameter), at 2400C with a carrier gas (N2)flow rate of 65 ml/min, was used to separate methyl-ated aromatic compounds. In later work, 3% SE-30 on100/200 Varaport 30 in a stainless-steel column (183cm by 0.3-cm outside diameter), was used with acarrier gas flow rate of 30 ml/min. The column tem-perature was kept at 900C for 3 min and then raisedto 140°C at a rate of 20°C/min. Standards for gas-liquid chromatography were prepared by addingknown amounts of a compound to endogenous (nosubstrate) systems and then extracting and methylat-ing, as with the experimental samples.

Later, aromatic compounds were quantified in cul-ture media, after centrifugation to remove bacteria, byreverse-phase, high-pressure liquid chromatography.A Laboratory Data Control (Riviera Beach, Fla.) chro-matograph with a column (20 cm by 0.4-cm insidediameter) containing PXS 10/25 ODS (Whatman Inc.,Clifton, N.J.) was used. The solvent system was meth-anol-1% aqueous acetic acid (2:8) at a flow rate of 0.8ml/min with an ultraviolet detector (254 and 280 nm).Other methods. Phthalic acid utilization was

sometimes determined by the decrease in absorbancyat 278 nm. Standard tests were used for bacterialidentification (6, 26), and flagella were stained by themethod of Mayfield and Inniss (21). The biuret reac-tion, performed on intact cells with bovine serumalbumin as the standard (14), was used to convertKlett units into protein contents. Nitrite was deter-mined colorimetrically by diazotization with sulfanilicacid and coupling with a-naphthylamine (27).

RESULTSPhthalic acid catabolism. Mixed cultures

which grew anaerobically with denitrification onphthalic acid were easily enriched and main-tained. Two cultures, termed ON-7 and MO,obtained from the marine sediments of BiscayneBay, were studied in detail. Only phthalic acid,of the isomeric phthalic acids, supported anaer-obic growth. Aerobically, the ON-7 consortiumwas restricted to the ortho isomer, but the MOculture grew on either phthalic acid or iso-phthalic acid. Under aerobic conditions, bothcultures seemed to metabolize phthalic acid viaprotocatechuate. For example, cells of cultureON-7 grown aerobically on phthalic acid imme-diately oxidized phthalic acid and protocatechu-ate at high initial rates compared with benzoateand catechol (Fig. 1B). Also, chloramphenicolprevented the adaptation to benzoate and cate-chol, but it had no effect on the aerobic rates ofoxidation of phthalic acid and protocatechuate.Cell suspensions of culture MO also oxidizedprotocatechuate, but not benzoate or catechol,when grown aerobically on either phthalic orisophthalic acids.

Anaerobically grown cells of culture ON-7 rap-idly metabolized phthalic and benzoic acids, butnot protocatechuate and catechol, under anaer-obic conditions (Fig. 1A). Cell suspensions of

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PHTHALIC ACID DEGRADATION 1179

0 60 120 0 60 120TIME MIN

FIG. 1. Oxidation of aromatic compounds by cul-ture ON-7 grown either anaerobically (A) or aerobi-cally (B) onphthalic acid. Warburg flasks containedin 3 ml: cells, 3.7mg ofprotein (A) or 1.7mg ofprotein(B); Tris-hydrochloride buffer (pH 7.5), 50 pmol; sub-strate, 5 (A) or 10 (B) pmol; KNO3 (A only), 50 Wmol;20% KOH (center wells [A and B]), 0.1 ml. Endoge-nous (no substrate) rates were subtracted. Symbols:0, phthalic acid; 0, benzoate; A, catechol; A, proto-catechuate.

either culture grown anaerobically on phthalicacid immediately oxidized phthalic and benzoicacids with nitrate, but there was a significant lagbefore aerobic breakdown proceeded. Chloram-phenicol had no effect on the anaerobic metab-olism of phthalic acid by anaerobically growncells of culture ON-7, but it did prevent adaptiveresponse under aerobic conditions (Fig. 2).Chloramphenicol did not affect the aerobic oxi-dation of acetate by these cells, indicating thatit interfered with the production of enzymes ofaerobic aromatic catabolism and not those ofaerobic respiration.

Phthalic acid oxidation by nitrate can be de-scribed by the equation: C8H604 + 6 KNO3 = 8C02 + 3 N2 + 6 KOH.Asuming that the gas evolved was N2, ON-7

cell suspensions produced over 50% of the cal-culated amount of N2 from nitrate (Table 1). N2evolution rates by ON-7 from nitrite, unlikethose from nitrate, were not stimulated byphthalic acid, and the anaerobic consumption ofphthalic and benzoic acids was much more ef-fective with nitrate than with nitrite (Table 2).Despite repeated attempts, pure cultures whichgrew anaerobically on phthalic acid by nitraterespiration were not isolated from ON-7 or MO.However, organisms which grew aerobically on

phthalic acid were obtained from the mixedcultures. Each isolate was a short, fat rod andwas gram negative, oxidase negative, and cata-lase positive; neither fermented glucose but nei-ther grew well aerobically on glucose. The isolate

from the ON-7 culture was nonmotile, was clas-sified as an Acinetobacter sp., and was desig-nated Acinetobacter sp. strain OP-1. The orga-

nism from the MO culture was motile with a

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FIG. 2. Effect of chloramphenicol on the aerobicand anaerobic oxidation ofphthalic acid by cultureON-7 grown anaerobically on phthalate. Warburgflasks contained in 3ml: cells, 4.2 mgofprotein; Tris-hydrochloride buffer (pH 7.5), 50 pmot; chloramphen-icol (when present), 300 pg; phthalic acid, 10 Mmol;KNO3 (anaerobic flasks only), 50 Wmol; 20% KOH(center wells), 0.1 ml. Endogenous rates (no substrate)were subtracted. Symbols: 0, anaerobic; 0, anaerobicwith chloramphenicol; 4 aerobic; A, aerobic withchloramphenicol.

TABLE 1. Phthalic acid utilization and N2evolution during anaerobic catabolism ofphthalic

acid by culture ON- 7grown anaerobically onphthalic acid0

Nitrate added N2 evolvedb Phthalic acid used(jMol) (pmol) (pmol)12.5 4.8 2.125 10.2 6.137.5 14.3 8.150 17.0 10.0

'Warburg flasks contained in 3 ml: cells, 4.2 mg ofprotein; Tris-hydrochloride buffer (pH 7.5), 50,umol;KNO3, as shown; phthalic acid, 15 iLmol; sulfamic acid,100 tmol; 20% KOH (center wells), 0.1 ml. The flaskswere incubated until gas evolution equaled the endog-enous rate, and then sulfamic acid was tipped into themain compartment to stop the reaction. Nitrite accu-mulation was negligible, and residual phthalic acid wasdetermined by the absorbance at 278 nm after centrif-ugation of the flask contents to remove intact cells.

b Gas assumed to be N2. Endogenous values sub-tracted (no phthalic acid added).

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1180 AFTRING, CHALKER, AND TAYLOR

TABLE 2. Anaerobic utilization ofphthalic andbenzoic acids by culture ON- 7grown anaerobically

on phthalic acid, in the presence of nitrate ornitritea

Respiratory electron Substrate used (,umol)acceptor Phthalic acid Benzoate

Nitrite 0.6 2.3Nitrate 8.9 9.5

a Warburg flasks contained in 3 ml: cells, 4.0 mg ofprotein; Tris-hydrochloride buffer (pH 7.5), 50 umol;NaNO2 or NaNO3, 50 ,umol; benzoate or phthalic acid,10 pmol; sulfamic acid, 100 ,umol; 20% KOH (centerwells), 0.1 ml. The flasks were incubated for 6 h at30°C before the sulfamic acid was tipped in. Residualsubstrate was assayed by gas-liquid chromatography.

polar flagellum, was tentatively identified as aPseudomonas sp., even though it lacked oxidaseactivity, and was termed Pseudomonas sp.strain MO-1. Four more pure cultures of bacteriawere isolated by plating ON-7 onto the enrichedmedium and, after incubation and growth, se-lecting different colonial types. None of theseisolates grew on phthalic acid, but two grew withdenitrification on the enriched medium. Com-binations of Acinetobacter sp. strain OP-1(which reduced nitrate to nitrite but did notdenitrify) and the denitrifiers did not developanaerobically on a phthalic acid-nitrate medium.Terephthalic acid catabolism. Only mixed

cultures were obtained which grew with denitri-fication on terephthalic acid. Two consortia werederived from the freshwater sediments of theMississippi and Miami rivers; they were namedTN-1 and FT, respectively. Neither culture grewon the other phthalic acid isomers, and bothwere even difficult to maintain on terephthalicacid unless frequently subcultured. Aerobic cul-tures grew for one transfer only on terephthalicacid. Also, it was imnpossible to isolate fromeither consortium pure cultures which grew onterephthalic acid by either aerobic or anaerobicrespiration.

In the presence of nitrate, TN-1 cell suspen-sions grown anaerobically on terephthalic acidrapidly metabolized terephthalic and benzoicacids but not protocatechuate or catechol (Fig.3). The same cells attacked all four compoundsunder aerobic conditions but only after a lagperiod (Fig. 3). Similar results were obtainedwith culture FT, except that cells grown anaer-obically never adapted to protocatechuate uponaerobic incubation. Cells grown aerobically onterephthalic acid oxidized benzoate and catecholbut not protocatechuate. Chloramphenicol pre-vented the aerobic adaptation to, but not anaer-obic use of, terephthalic acid by cells of TN-1grown anaerobically on terephthalic acid (Fig.

APPL. ENVIRON. MICROBIOL.

4). The complete utilization of terephthalic andbenzoic acids under anaerobic conditions wasconfirmed by high-pressure liquid chromatog-raphy of residual flask contents from respirom-etry experiments with culture FT.

Despite numerous attempts, pure cultureswhich grew on terephthalic acid, either aerobi-cally or anaerobically, were not isolated fromthe terephthalic acid-degrading consortia. Atleast seven different bacteria, distinguished bynutritional characteristics, were present in theTN-1 culture, but the consortium could not bereconstituted from the isolates. Culture TN-1also contained conspicuous numbers of motilespiral-shaped bacteria when grown anaerobi-cally on terephthalic acid. The spirilla were notevident during anaerobic growth on other sub-strates and were impossible to isolate in pureculture.Isophthalic acid catabolism. An associa-

tion of bacteria which grew on isophthalic acidwas isolated from a marine sediment. The en-richment culture, termed MM, developed anaer-obically on isophthalic acid but not on the otherphthalic acid isomers, whereas aerobically, it

0 60 120 180TIME MIN

FIG. 3. Aerobic and anaerobic metabolism of ar-omatic compounds by culture TN-I grown anaerobi-cally on terephthalic acid. Warburg flasks containedin 3 ml: cells, 3.7 mg ofprotein; Tris-hydrochloridebuffer (pH 7.5), 50 pmol; substrate, 10 pmol; KNO3(anaerobic flasks only), 50 jmol; 20% KOH (centerwells), 0.1 ml. Endogenous rates were subtracted.Symbols: 0, terephthalic acid; *, benzoate; A, pro-tocatechuate; A, catechol.

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PHTHALIC ACID DEGRADATION 1181

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FIG. 4. Effect of chloramphenicol on the aerobicand anaerobic oxidation of terephthalic acid by cul-ture TN-1 grown anaerobically on terephthalic acid.Warburg flasks contained in 3 ml: cells, 3.4 mg ofprotein; Tris-hydrochloride buffer (pH 7.5), 50 pnol;chloramphenicol, 300 pg; terephthalic acid, 10 gnol;KNO3 (anaerobic flasks only), 50 gmol; 20% KOH(center wells), 0.1 ml. Endogenous rates were sub-tracted. Symbols: 0, anaerobic; 0, anaerobic withchloramphenicol; A, aerobic; A, aerobic with chlor-amphenicoL

grew on either isophthalic or phthalic acid. Purecultures of bacteria were isolated which grewaerobically on either isophthalic or phthalicacid, and, even though each organism only usedone isomer, they had similar morphological andphysiological characteristics. Both were short,nonmotile, gram negative, oxidase negative, andcatalase positive and did not ferment glucose.They were probably species ofAcinetobacter. Itwas impossible to isolate a pure culture whichgrew anaerobically by nitrate respiration onisophthalic acid; therefore, anaerobic catabolismwas investigated with the mixed culture. Cellsuspensions ofMM, grown anaerobically on iso-phthalic acid, showed immediately increasedrates of N2 evolution with either isophthalic orbenzoic acid (Fig. 5). High-pressure liquid chro-matography of the flask contents, after the N2evolution rates had returned to the endogenouslevel, showed that substrate utilization was com-plete. Cells grown anaerobically but incubatedaerobically showed sigmoidal 02 uptake rateswith isophthalic or benzoic acid, rather than thelinear rates of N2 evolution in anaerobic systems(Fig. 5). During the anaerobic breakdown ofisophthalic acid, about 50% of the theoreticalamounts were N2 and C02 produced (Table 3).

DISCUSSIONThe catabolism of phthalic and isophthalic

acids by some of the pure and mixed cultures inthis study proceeded via protocatechuate.

Phthalic acid conversion to protocatechuate isprevalent among a variety of bacteria (11, 17,

a 25). Isophthalic acid metabolism has receivedw less attention, but there is a report of its aerobic

breakdown via protocatechuate (17). The micro-bial oxidation of terephthalic acid, like that of

W isophthalic acid, has not been well studied, butz there is evidence that it is also converted toa protocatechuate (11, 17). Engelhardt et al. (11)

isolated three strains of Nocardia spp. which,when grown aerobically on terephthalic acid,

z oxidized both protocatechuate and catechol.Protocatechuate was the true intermediate, andthe enzymes for catechol oxidation were coinci-dentally induced. The mixed culture (FT), how-ever, oxidized catechol but not protocatechuate

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TIME MINFIG. 5. Aerobic and anaerobic metabolism of iso-

phthalic acid by cultureMMgrown anaerobically onisophthalic acid. Warburg flasks contained in 3 ml:ceUs, 3.1 mg ofprotein (aerobic) or 6.8 mg ofprotein(anaerobic); Tris-hydrochloride buffer (pH 7.5), 50pmol; isophthalic acid, 10 pmol; KNO3 (anaerobicflasks), 50 pmol; 20% KOH (center wells), 0.1 ml.Endogenous rates were subtracted. Symbols: 0, aero-bic isophthalic acid; *, anaerobic isophthalic acid;A, aerobic benzoate; A, anaerobic benzoate.

TABLE 3. Isophthalic acid utilization, N2 evolution,and CO2 production during anaerobic catabolism of

isophthalic acid by culture MMgrownanaerobicaly on isophthalate'

Substrate N2 evolved CO2 evolved(pmol) (AMol)

None 4.2 9.5Isophthalate 8.8 20.9

a Warburg flasks contained in 3 ml: cells, 4.6 mg ofprotein; Tris-hydrochloride buffer (pH 7.5), 50 umol;KNO3, 30 ,imol; isophthalic acid, 3 tmol. The centerwells of some flasks contained 0.1 ml of 20% KOH, butfor C02 determinations, the KOH was omitted fromsome flasks. The flasks were incubated until gas evo-lution rates equaled the endogenous rates, and then0.2 ml of 4 N H2S04 was tipped in from the side armsof flasks used in the C02 determinations.

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1182 AFTRING, CHALKER, AND TAYLOR

when grown on terephthalic acid; therefore, an-other route for the aerobic catabolism of tere-phthalic acid may exist.The importance of anaerobic microorganisms

in the global carbon cycle and their role in thecatabolism of aromatic compounds has recentlybeen emphasized (12). Anaerobic breakdownlinked to nitrate respiration has been shown forvarious aromatic compounds but not for thephthalic acids (3, 28, 30). The present resultssuggest that specific pathways exist for the an-aerobic catabolism of phthalic acids. Cells ofmixed cultures grown anaerobically on aphthalic acid immediately metabolized theirgrowth substrate with nitrate, whereas with cul-tures grown aerobically, there was a lag periodbefore metabolism proceeded. Also, chloram-phenicol prevented the adaptation to aerobiccatabolism, but had no effect on the anaerobicrate of dissimilation of the phthalic acids. Fur-ther evidence for distinct degradative pathwaysis provided by the fact that aerobic intermedi-ates, e.g., catechol and protocatechuate, werenot used under anaerobic conditions by anaero-bically grown cells. It was not determined whymixed cultures were necessary for the break-down of the phthalic acids under anaerobic con-ditions. Presumably, symbiotic relationships areinvolved in which growth factors are supplied ortoxic (or inhibitory) metabolites are removed (3,5). Nitrite, which inhibited aromatic utilizationby culture ON-7, is a possible inhibitory sub-stance (24). The presence of spiral-shaped or-ganisms in culture TN-1, when grown on tere-phthalate-nitrate medium, is reminiscent of theobservation of Bakker (3), who detected signifi-cant numbers of spirilla in mixed cultures grow-ing with denitrification on phenol. Pure culturesisolated from the enrichments were rod-shapedand grew only slowly on phenol-nitrate medium.Although the mixed cultures were restricted

to metabolism of the phthalic acid isomer uponwhich they were enriched, they all attackedbenzoate under anaerobic conditions after an-aerobic growth on the phthalic acid. This sug-gests specific decarboxylations for the phthalicacid isomers, followed by a common pathwayfor benzoate, presumably a reductive route (12).More work is needed to test this hypothesis,because benzoate is often metabolized by bac-teria grown anaerobically on aromatic com-pounds (8, 13, 28), and the enzymes for its break-down may be coincidentally induced.

In conclusion, all three isomeric phthalic acidsare potentially biodegradable in both aerobicand anaerobic environments. Previous studiesshowed aerobic breakdown, but until this report,susceptibility to attack under anaerobic condi-tions had not been proven.

ACKNOWVLEDGMENTSWe thank Sandy Lee, Gary Mortoro, John Paul, and Dennis

Taylor for their contributions to this work.Financial support was provided by Public Health Service

grants ES00994 and GM20172-06 from the National Institutesof Health and by the Louis A. Reitmeister Foundation.

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3. Bakker, G. 1977. Anaerobic degradation of aromatic com-pounds in the presence of nitrate. FEMS Microbiol.Lett. 1:103-108.

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10. Engelhardt, G., P. R. Wallnofer, and 0. Hutzinger.1975. The microbial metabolism of di-n-butyl phthalateand related dialkyl phthalates. Bull. Environ. Con-tamin. Toxicol. 13:342-347.

11. Engelhardt, G., P. R. Wallnofer, H. G. Rast, and F.Fiedler. 1976. Metabolism of o-phthalic acid by differ-ent gram-negative and gram-positive soil bacteria. Arch.Microbiol. 109:109-114.

12. Evans, W. C. 1977. Biochemistry of the bacterial catab-olism of aromatic compounds in anaerobic environ-ments. Nature (London) 270:17-20.

13. Healy, J. B., Jr., L. Y. Young, and M. Reinhard. 1980.Methanogenic decomposition of ferulic acid, a modellignin derivative. Appl. Environ. Microbiol. 39:436-444.

14. Herbert, D., P. J. Phipps, and R. E. Strange. 1971.Chemical analysis of microbial cells, p. 209-344. In J.R. Norris and D. W. Ribbons (ed.), Methods in micro-biology, vol. 5B. Academic Press, Inc., New York.

15. Johnson, B. T., and W. Lulves. 1975. Biodegradation ofdi-n-butyl phthalate and di-2-ethylhexyl phthalate infreshwater hydrosoil. J. Fish. Res. Board Can. 32:333-339.

16. Karickhoff, S. W., D. S. Brown, and T. A. Scott. 1979.Sorption of hydrophobic pollutants on natural sedi-ments. Water Res. 13:241-248.

17. Keyser, P., B. G. Pujar, R. W. Eaton, and D. W.Ribbons. 1976. Biodegradation of the phthalates andtheir esters by bacteria. Environ. Health Perspect. 18:159-166.

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