Vol. 48, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1984, p. 102-1070099-2240/84/070102-06$02.00I0Copyright © 1984, American Society for Microbiology

Anaerobic Degradation of 2-Aminobenzoate (Anthranilic Acid) byDenitrifying Bacteria

KONSTANTIN BRAUN'* AND DAVID T. GIBSON2

Lehrstuhl fir Chemische Mikrobiologie, Universitdt Gesamthoschschule Wluppertal, D-5600 Wulppe1rtal, Feder-al Republicof Germany,' and Center for Applied Microbiology and Department of Microbiology, The University of Texas at Alustin,

Austin, Texas 787122

Received 30 January 1984/Accepted 2 April 1984

In the presence of oxygen many aminoaromatic compounds polymerize to form recalcitrant macromolecules.To circumvent undesirable oxidation reactions, the anaerobic biodegradation of a simple member of this classof compounds was investigated. Two strains of bacteria were isolated which degrade 2-aminobenzoateanaerobically under denitrifying conditions, with nitrate as the terminal electron acceptor. Both organisms,which were assigned to the genus Pseudomonas, oxidized 2-aminobenzoate completely to CO2 and NH4+.Nitrate was reduced to nitrite. When nitrate was depleted from the growth medium the accumulated nitrite wasreduced to nitrogen. The results establish a model system for the anaerobic, rapid, and complete oxidation ofan aminoaromatic compound.

Nitro- and aminoaromatic compounds are produced inlarge amounts by chemical industries. Many of these arexenobiotic compounds that can cause severe problems in theenvironment (12). In addition, aminoaromatics as well asdegradation products formed from nitroaromatics easilypolymerize in the presence of oxygen to persistent macro-molecules (6). This is prevented by an anoxic atmosphere,and these conditions promote the reduction of nitroaroma-tics and azo dyes to amino compounds (19, 31). The biodeg-radation of these chemicals should be favored by anaerobicconditions that will, first, promote the formation of a singlegroup of compounds, i.e., aminoaromatics, from a variety ofnitrogen-containing xenobiotics and, second, prevent poly-merization reactions. Thus, anaerobic enrichment condi-tions would enable organisms to activate preexisting degra-dation pathways for aminoaromatics undisturbed bychemical modification of these compounds. Anaerobic deg-radation of aromatic compounds by pure cultures is unlikelyto occur by fermentation. On thermodynamic grounds anaer-obic catabolism is dependent on the presence of symbioticorganisms, such as methanogenic bacteria (11, 21), on thepresence of certain structural elements of the compounds,such as polyhydroxy-substituted benzene nuclei (16, 24), oron the ability to use external electron acceptors, such assulfate (23) or nitrate (28).To initiate studies on the degradation of nitrogen-contain-

ing aromatic compounds, we have chosen to investigate theanaerobic degradation of a simple aminoaromatic com-pound, 2-aminobenzoate (anthranilic acid). We also chose touse nitrate as an electron acceptor in our studies. Underthese conditions several aromatic compounds can easily bemetabolized (1, 9). We report the anaerobic degradation of 2-aminobenzoate and, for comparison, benzoate by strains ofdenitrifying bacteria of the genus Pseudomonas.

MATERIALS AND METHODSMedium and cultivation. Media were made anaerobic

according to standard procedures (15). Reducing agentswere omitted, except where noted. Air was replaced by

* Corresponding author.

argon. Liquid cultures were kept in Hungate tubes (BellcoGlass, Inc.). Volumes of >10 ml were grown in glass bottlessealed with rubber stoppers. The mineral medium wasadapted from reference 28 and contained, in moles per liter(except where noted): KH2PO4-K2HPO4 buffer (pH 8.0),0.04; NH4Cl, 0.005 (omitted when 2-aminobenzoate wassubstrate); MgSO4, 0.0004; CaCl2, 0.00025; KNO3, 0.02;substrate (carboxylic acids were neutralized with NaOH),0.005 (except where noted); trace element solution (22)without H3B03, 10 ml; agar (where needed), 20 g. Stockcultures were transferred every day (10%, vol/vol, inoculum)and kept at 30°C. Experiments were performed at 37°C.

Determination of growth parameters. (i) Preparation ofsamples. Samples (0.6 ml) of the medium were taken with a1.0-ml syringe. Optical density at 578 nm (Zeiss PL4 pho-tometer) and pH were determined where necessary. Eachsample was centrifuged, and to 0.4 ml of the clear superna-tant 0.04 ml of 0.05 M 3-hydroxybenzoate in 1% (wt/vol)H3PO4 was added as an internal standard for high-pressureliquid chromatography determinations. This sample was alsoused for ammonium determination. If the CO, content of themedium was to be determined, a second sample (1.0 ml) wastransferred into a 4.0-ml glass bottle, sealed with a siliconeseptum, and 0.1 ml of 6 N HCI was added for quantitativeCO2 evolution.Gaseous samples were withdrawn for analysis by gas

chromatography with a 0.250-ml gas-tight syringe (PrecisionSampling) which was valve locked before removal from thetightly sealed culture flask. Thus, an isobaric precise propor-tion of the known headspace volume could be analyzed.Hence, calculation of gas concentrations gave correct val-ues, irrespective of the pressure changes within the head-space volume of the culture flask.

(ii) Ammonium (NH4+). Ammonium ion concentration wasdetermined colorimetrically by the Berthelot procedure asdescribed by Kaplan (17). Culture supernatant solutions (0.3ml) were added to 5.0 ml of a solution containing phenol(0.106 M) and Na2Fe(CN)5NO (0.00017 M). The resultingsolution was added to 5.0 ml of 0.125 M NaOH whichcontained NaOCl (0.011 M). The reaction mixture wasincubated at 37°C for 30 min, and the absorption at 546 nmwas determined. Standard curves were linear up to 0.002 MNH4+. Samples were diluted when necessary.

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ANAEROBIC DEGRADATION OF AMINOBENZOATE 103

(iii) Nitrate (NO3-), nitrite (NO2-), 2-aminobenzoate, andbenzoate determinations. NO3, NO,- 2-aminobenzoate,and benzoate were analyzed by high-pressure liquid chroma-tography on a Waters model 440 high-pressure liquid chro-matograph equipped with a Schoeffel GM 770 detector. A C8reversed-phase column (130-mm length, 4-mm internal diam-eter; Lichrosorb RP8, 5-,m particles; Knauer GmbH) wasused to separate the individual compounds which weredetected at 215 nm. The column was eluted isocratically atroom temperature with a solvent that contained acetonitrile(20%), water (80%), and H3PO4 (0.1%). The flow rate was1.0 ml/min. Under these conditions nitrate was not retainedby the column. However, nitrite, 2-aminobenzoate, andbenzoate were well separated. Quantitation of individualcomponents, except for N03-, was based on 3-hydroxyben-zoate as the internal standard. Quantitation of N037, whichgave a nonlinear response with increasing concentrations,was based on a separate calibration curve. Peak elution andcapacity factors, k' (26), were as follows: N03 , 0 (notretained); NO2-, 0.92; 3-hydroxybenzoate (internal stan-dard), 2.17; 2-aminobenzoate, 3.40; benzoate, 5.41.

(iv) Oxygen (02), nitrogen (N2), and carbon dioxide (CO2).Analysis of 0,, N2, and CO, was performed by gas chroma-tography with a Perkin-Elmer 900 gas chromatographequipped with a hot-wire detector. Temperatures were asfollows: injector, 100°C; oven, 70°C; detector, 150°C. Thebridge current was 125 mV. Oxygen and nitrogen wereseparated on a 3-m molecular-sieve column (0.5 nm, 30 to 60mesh; Perkin-Elmer OSO.91). Nitrogen and carbon dioxidewere separated on a 4-m silica gel column (Perkin-ElmerOS0.93). The total concentration of CO, was calculatedfrom the amounts determined in the medium and the atmo-sphere. Quantitation was based on standard curves that wereregistered with each run.The lowest detection limits of gases were as follows

(approximate values): CO2(dissolved), 0.0002 M; CO2(g), 0.1%(vol/vol; in this report equivalent to 0.00006 M); N2(g), 0.03%(vol/vol; in this report equivalent to 0.00002 M); 02(g). 0.02%(vol/vol; in this report equivalent to 0.00001 M).

Characterization of organisms. Cell morphology was ex-amined in detail by electron microscopy with negativelystained cells according to procedures described previously(29). Gram stains were performed by the Hucker method asdescribed by Bartholomew (3), except that fixation of cellswas done with methanol (18). Staphylococcus alureius andEscherichia coli served as controls. For confirmation of theGram stain the KOH test was performed (13). Oxidaseactivity with tetramethyl-p-phenylenediamine dihydrochlor-ide, catalase activity, and the oxidation-fermentation testwith fructose were performed as described previously (25).Temperature requirements for growth were determined witha temperature gradient incubator (Toyo Kagaku SangyoCo.). Influence of pH was determined with two differ-ent buffer systems: KH2PO4 supplemented with varyingamounts of K,HPO4 (final concentration, 0.04 M) orKH2PO4 (0.04 M) supplemented with Na2CO3 (0.004 to 0.047M).

Chemicals. Chemicals were reagent grade or the purestavailable. 1-Cyclohexenecarboxylic acid was obtained fromAlfa, Europe (through Ventron Inc.).

RESULTSEnrichment and isolation of organisms. Enrichment of

organisms was performed in Hungate tubes containing 10 mlof anaerobic mineral medium. Each tube was inoculatedwith 1.0 ml of soil or sediment samples and maintained at

37°C. When media became turbid (between 2 and 7 days),1.0-ml samples were inoculated into fresh medium. Afterseveral transfers cells were streaked on mineral agar plateswhich were incubated either aerobically or anaerobically(anaerobic jar, argon atmosphere). Isolated colonies grownunder anaerobic conditions immediately resumed anaerobicgrowth when transferred to anaerobic liquid media (Hungatetube, 10 ml of mineral medium, argon atmosphere). Howev-er, colonies grown aerobically failed to grow under anaero-bic conditions. Instead, readaptation to nitrate respirationwas necessary. This was achieved by transferring colonies toadaptation tubes which contained limiting amounts of air(Hungate tube, 10 ml of mineral medium, air atmosphere).The amount of air (6.5 ml) was not enough to allow completeaerobic degradation of substrates. However, readaptation tonitrate respiration occurred, and cells grew well despite theinsufficient amount of oxygen.

After several aerobic transfers to mineral agar and readap-tation to anaerobic growth in liquid media, cells were platedaerobically on 0.8% nutrient broth (Difco Laboratories)agar. Isolated colonies were grown anaerobically in mineralmedia and then reisolated on 3% caso bouillon (E. MerckAG; equivalent to Trypticase soy broth [BBL MicrobiologySystems]) agar. Colonies obtained were small (1 to 2 mm indiameter), circular, convex, with an entire margin, beigecolored, and creamy in texture. Each pure culture was ableto grow anaerobically in mineral media.Three strains were isolated from different sources with

different substrates. They were designated as strains KB650(flower bed soil, United States; benzoate-nitrate); KB740(creek sediment, United States; 2-aminobenzoate-nitrate),and KB820 (compost, Federal Republic of Germany; 2-aminobenzoate-nitrate).

Characterization of strains. Cells of all strains were rodswith rounded ends (1.5- to 3.0-pRm length, 0.8-,um diameter)which were motile by one subpolar inserted flagellum (Fig.1). Spores were not observed. All strains were gram negativeand KOH positive. Oxidase and catalase reactions werepositive. Metabolism was strictly oxidative, with oxygen ornitrate serving as the electron acceptor. With the exceptionof strain KB650, which did not grow on sugars (Table 1),oxidative oxidation-fermentation tests were observed withstrains KB740 and KB820. Acid was formed from fructoseby both organisms. The results obtained indicate that allstrains belong to the genus Pseudomonas.

Nitrate was essential for anaerobic growth and could notbe replaced by nitrite. Ammonium was not necessary forgrowth with benzoate, and nitrate was utilized for bothassimilatory and dissimilatory metabolism. The mean gener-ation times calculated for Pseudomonas sp. strains KB650(benzoate-nitrate), KB740 (2-aminobenzoate-nitrate), andKB820 (2-aminobenzoate-nitrate) were 2.6, 3.8, and 4.4 h,respectively. Optimal growth conditions were 37°C and pH7.8. For strain KB740, no growth was observed at 41°C andonly slight growth occurred at pH 7.0 (Fig. 2).

It is of interest to note that fresh enrichment cultures wereseverely inhibited by relatively low concentrations of NH4+(0.01 M), although this sensitivity decreased with the age ofthe cultures. In addition, the degradation of 2-aminoben-zoate by Pseudomonas sp. strain KB740 was completelyinhibited by the presence of yeast extract (0.1%, wt/vol).Thus, enrichment experiments which utilize complex media(4) or mineral salts media at neutral pH or high NH4+concentrations or both may fail to yield the desired organ-isms. Such conditions are often used in enrichment cultureexperiments (14).

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104 BRAUN AND GIBSON

FIG. 1. Morphology of negatively stained cells: (A) Pseudomonas sp. strain KB740 (bar, 1 p.m); (B) Pseudomonas sp. strain KB650 (bar,0.2 ,um); (C) Pseudomonas sp. strain KB820 (bar, 0.2 ,um). Details show subpolar flagellar insertion (B) and pili (C). Fractures in the cell wall(B) are artifacts due to preparation of the specimen.

All three Pseudomonas sp. strains grew anaerobicallywith a variety of substrates when nitrate was present in thegrowth medium (Table 1). Aerobic growth was observedwith benzoate and 2-aminobenzoate. However, no growthwas observed under fermentative conditions, i.e., in theabsence of either 02 or N03 . Adipate, 1-cyclohexenecar-boxylate, and pimelate, which have been detected as inter-mediates in the anaerobic degradation of aromatic acids (9),were also utilized as growth substrates. In contrast, catecholand protocatechuate, which are central intermediates in theaerobic degradation of a variety of aromatic compounds,were not utilized.

Anaerobic growth on 2-aminobenzoate. Figure 3 shows thegrowth and anaerobic degradation of 2-aminobenzoate byPseudomonas sp. strain KB740. Similar results were ob-tained with strains KB650 and KB820. One mole of 2-aminobenzoate was converted to 0.4 mol of NH4' and 5 molof CO2. Respiration occurred in two stages, with nitratebeing converted to nitrite followed by the conversion ofnitrite to nitrogen. The second stage never occurred beforethe medium was totally depleted of nitrate. If excess nitrate

was added to the growth medium the cells grew by N03--NO2- respiration with little or no production of nitrogen.

Role of molecular oxygen in anaerobic degradation ofbenzoate and 2-aminobenzoate. Trace amounts of oxygen (upto 0.03 mol per mol of substrate) were always detected insamples taken from growing cultures. This was probably dueto trace amounts of air entering the needle during transfer ofthe syringe from the sample to the injector. However, todefinitively exclude the involvement of oxygen in the degra-dation of benzoate and 2-aminobenzoate, strict anaerobicconditions (Hungate tube, 10 ml of mineral medium supple-mented with 0.0002 M palladium black, hydrogen atmo-sphere) were utilized. Under these conditions resazurinewas decolorized, indicating strict anaerobiosis (15), and botharomatic substrates were degraded. The growth-inhibitoryeffects of sulfides, which are usually used as reducing agents(7), precluded their use in the growth medium. The resultsobtained clearly showed that the anaerobic degradation ofbenzoate and 2-aminobenzoate by Psetudomonas sp. strainsKB650, KB740, and KB820 did not require molecular oxy-gen.

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ANAEROBIC DEGRADATION OF AMINOBENZOATE 105

TABLE 1. Growth substrates utilized b)strains KB650, KB740, and I

Electron Substrateaacceptor

Nitrate Benzoate2-Aminobenzoate3-Hydroxybenzoate4-Hydroxybenzoate2-Carboxylbenzoate (phthalate)

CatecholProtocatechuate1-CyclohexenecarboxylateAdipatePimelate

Formate (0.02 M)Acetate (0.02 M)Propionate (0.01 M)ButyrateCaproateDL-LactateDL-MalateFumarateSuccinateGlucoseFructoseSucroseMaltoseAcetoinAcetoneNutrient broth (0.8%, wt/vol)Caso bouillon (3%, wt/vol)Yeast extract (0.1%, wt/vol)

Oxygen Benzoate2-Aminobenzoate

y Pseudomonas sp. phthalate and glucose. These substrates were not utilized byKB820 Pseudomonas sp. strain KB820. Sugars were not utilized by

Strain Pseudomonas sp. strain KB650, which was isolated byvirtue of its ability to grow with benzoate under denitrifyingKB650 KB740 KB820 conditions. All three isolates appear to resemble Pseudomo-

+ + + nas sp. strain PN1, which is able to grow anaerobically with- + + aromatic compounds by nitrate respiration (28).+ + + The identification of strains KB650, KB740, and KB820 as(+) + + members of the genus Pseudomonas was based on a variety

of criteria. These included morphology, Gram stain, andpositive oxidase and catalase reactions. They are strictly

- _ _ oxidative organisms that can grow either aerobically or+ + + anaerobically with oxygen and nitrate as the respective+ + + terminal electron acceptors. The presence of a subpolar+ + + flagellum is not in accordance with the generally accepted

definition of the genus Pseudomonas (5, 27). However,+ + + Pseudomonas flava (8) and Pselidomonas carboxydovorans+ + + (20) both have subpolar flagella.+ + + It has been reported that oxygen is necessary for the+ + + degradation of aromatic compounds by denitrifying bacteria+ + + (4, 10). Our results clearly show that this is not the case. At+ + + least 1 mol of oxygen per mol of benzoate or 2-aminoben-+ + + zoate would be required for degradation. The highest possi-+ + + ble oxygen consumption observed in our experiments was- (+) - 0.03 mol per mol of benzoate or 2-aminobenzoate. In addi-- + + tion, degradation of both aromatic acids was observed under

+ + strict anaerobic conditions when oxygen was completely+ + + absent from the culture medium.+ + + Enrichment for denitrifying biodegraders may be dis-+ + + turbed by even slightly improper growth conditions. This+ + + had to be concluded from the results presented here. Espe-+ + + cially the presence of complex components, even in low

+ + +ND" + +

+ +

+ + +

+ + + 100-

Nonec Benzoate (0.02 M)

Fructose (0.02 M)Nutrient broth (0.8%, wt/vol)

a All substrates were 0.005 M, except where noted. Compounds not listedthat did not support growth of all three strains under anaerobic conditions inthe presence of nitrate were: 3- and 4-aminobenzoate; 2-, 3-, and 4-nitroben-zoate; 2-hydroxybenzoate (salicylate); 2-, 3-, and 4-methylbenzoate; 2-. 3-,and 4-methoxybenzoate; 3-carboxylbenzoate (isophthalate), 4-carboxylben-zoate (terephthalate); pyrogallol; methanol (0.02 M), ethanol (0.02 M), pro-panol (0.01 M), butanol; ribose, rhamnose, lactose; gluconate, mannitol;diacetyl, 2,3-butanediol.

b ND, Not determined.c Fermentation conditions.

DISCUSSIONDenitrifying bacteria that degrade benzoate under anaero-

bic conditions have been isolated from a variety of sources(28, 30). This communication represents the first report ofdenitrifying organisms that can degrade 2-aminobenzoate.Such organisms are probably ubiquitous in the environmentsince Pseudomonas sp. strains KB740 and KB820 were

isolated from quite different sources. Both strains were

similar in terms of morphology and the spectrum of sub-strates that would support growth. However, Pseudomonassp. strain KB740 was not as rich in pili as strain KB820. Inaddition, strain KB740 could grow anaerobically with

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FIG. 2. Effect of temperature and pH on Pseudomonas sp. strainKB740. Cells were grown anaerobically in 10 ml of mineral mediumin Hungate tubes. Data are mean values of percent optical densityreached and percent substrate utilized after 20 h of growth. Opti-mum values have been indicated. Symbols: *, temperature depen-dence (two experiments); 0, pH dependence (KH2PO4-K2HPO4buffer); 0, pH dependence (KH2PO4-Na2CO3 buffer).

FructoseNutrient broth (0.8%, wt/vol)Caso bouillon (3%, wt/vol)

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106 BRAUN AND GIBSON

concentrations (e.g., 0.1% yeast extract) (4), or a neutral pHmight totally inhibit the desired metabolic reactions.The anaerobic degradation of benzoate has been studied in

detail (9). However, little is known about the anaerobicbreakdown of 2-aminobenzoate (2). The theoretical reac-tion for complete degradation is as follows: 1.0C7H702N +1.8H20 + 5.6NO3-* 7.0C02 + 1.ONH4+ + 2.8N2 +6.60H-. Thus, Pseudomonas sp. strains KB740 and KB820were able to oxidize 2-aminobenzoate completely in the

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FIG. 3. Anaerobic degradation of 2-aminobenzoate in the pres-ence of nitrate by Pseudomonas sp. strain KB740. Cells (5% [vol/vol] inoculum) were grown anaerobically in 50 ml of mineral mediumsupplemented with 0.005 M NH4Cl in a 100-ml bottle at 37°C. CO.,NH4', N02-, and optical density were corrected for values of thefreshly inoculated medium. Symbols: 0, 2-aminobenzoate; 0, CO2;A, NH4; V, NO37; *, NO27; *, N2, *, optical density.

absence of oxygen if nitrate was present as an electronacceptor. The only detectable products were CO2, NH4',and N2.The results presented establish a model anaerobic system

for the rapid and complete oxidation of an aminoaromaticcompound by oxygen-insensitive bacteria. Studies in pro-gress are directed towards elucidating the reactions involvedin the anaerobic degradation of 2-aminobenzoate and otheramino- and nitroaromatic compounds.

ACKNOWLEDGMENTS

This work was supported by grant F-440 from The Robert A.Welch Foundation. K.B. was supported by a stipend from theDeutsche Forschungsgemeinschaft.We thank H.-J. Knackmuss for providing laboratory space and

equipment for part of these studies, F. Mayer for the electronmicrographs, M. Bachmann and K.-H. Engesser for assistance withclassification of the strains, and D. Claus for discussions of theresults. We also thank J. R. Andreesen and H. Hippe for criticaldiscussions and Roberta DeAngelis for typing the manuscript.

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