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JOURNAL OF BACTERIOLOGY Vol. 87, No. 4, p. 910-919 April, 1964 Copyright © 1964 American Society for Microbiology Printed in U.S.A. MICROBIAL METABOLISM OF AROMATIC COMPOUNDS I. DECOMPOSITION OF PHENOLIC COMPOUNDS AND AROMATIC HYDROCARBONS BY PHENOL-ADAPTED BACTERIA HENRY H. TABAK, CECIL W. CHAMBERS, AND PAUL W. KABLER Microbiology Section, Basic and Applied Sciences Branch, Division of Water Supply and Pollution Control, Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio Received for publication 19 December 1963 ABSTRACT TABAK, HENRY H. (Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio), CECIL W. CHAMBERS, AND PAUL W. KABLER. Microbial metabolism of aromatic carbon compounds. I. Decomposition of phenolic compounds and aro- matic hydrocarbons by phenol-adapted bacteria. J. Bacteriol. 87:910-919. 1964.-Bacteria from soil and related environments were selected or adapted to metabolize phenol, hydroxy phenols, nitro- phenols, chlorophenols, methylphenols, alkyl- phenols, and arylphenols when cultured in mineral salts media with the specific substrate as the sole source of carbon. A phenol-adapted culture (sub- strate-induced enzyme synthesis proven) was challenged in respirometric tests with 104 related compounds; probable significant oxidative ac- tivity occurred with 65. Dihydric phenols were generally oxidized; trihydric phenols were not. Cresols and dimethylphenols were oxidized; add- ing a chloro group increased resistance. Benzoic and hydroxybenzoic acids were oxidized; sulfo- nated, methoxylated, nitro, and chlorobenzoic acids were not; m-toluic acid was utilized but not the o- and p-isomers. Benzaldehyde and p-hy- droxybenzaldehyde were oxidized. In general, nitro- and chloro-substituted compounds and the benzenes were difficult to oxidize. The dissimilation of phenols and aromatic hydrocarbons by bacteria has been reported by many workers. Results of early studies indicating that microorganisms are able to degrade ring compounds were reported by Buddin (1914), Sen Gupta (1921), Dooren de Jong (1926), Tattersfield (1928), and Gray and Thornton (1928). Stanier (1947) described his technique of simultaneous adaptation for determining meta- bolic pathways in the bio-oxidation of aromatic compounds and also showed that fluorescent pseudomonads attack many aromatic compounds (Stanier, 1948). Subsequent studies on the mechanism, optimal conditions for degradation, and the intermediate products of the metabolism of aromatic compounds by microorganisms were reviewed by Happold (1950). ZoBell (1946, 1950), in reviewing literature relating to the action of microorganisms on hydrocarbons, mentioned numerous phenolic compounds and hydrocarbons oxidized by bacteria, and the sources and types of organisms involved. Few studies have been made concerning the effect of chemical structure on the ability of bacteria adapted to a given aromatic compound to oxidize related compounds. Czekalowski and Skarzynski (1948) investigated the relationship between chemical structure and the use of aromatic compounds by one phenol-tolerant strain of Achromobacter, and Kramer and Doetsch (1950) used Achromobacter, Micrococcus, and Vibrio species that are capable of utilizing phe- nols to study their ability to grow on related aromatic compounds. The purpose of this investigation was to determine the ability of specifically adapted bacteria to degrade phenol and substituted phenols, and to study the relationship between the chemical structure of phenol derivatives and cyclic hydrocarbons and their susceptibility to decomposition by organisms adapted to related aromatic compounds. MATERIALS AND METHODS Selection and adaptation of bacteria to utilize phenol and substituted phenols. Organisms were obtained from garden soil, compost, river mud, and sediment from a waste lagoon of a petroleum refinery catalytic cracking plant. Methods used in selecting or adapting organisms to degrade these compounds were soil perfusion, continuous- feed activated sludge, primary enrichment in flasks on a shaker, and enrichment in batch-type 910
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Page 1: Microbial Metabolism of Aromatic Compounds

JOURNAL OF BACTERIOLOGYVol. 87, No. 4, p. 910-919 April, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

MICROBIAL METABOLISM OF AROMATIC COMPOUNDS

I. DECOMPOSITION OF PHENOLIC COMPOUNDS AND AROMATIC HYDROCARBONSBY PHENOL-ADAPTED BACTERIA

HENRY H. TABAK, CECIL W. CHAMBERS, AND PAUL W. KABLERMicrobiology Section, Basic and Applied Sciences Branch, Division of Water Supply and

Pollution Control, Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio

Received for publication 19 December 1963

ABSTRACT

TABAK, HENRY H. (Robert A. Taft SanitaryEngineering Center, Cincinnati, Ohio), CECILW. CHAMBERS, AND PAUL W. KABLER. Microbialmetabolism of aromatic carbon compounds. I.Decomposition of phenolic compounds and aro-matic hydrocarbons by phenol-adapted bacteria. J.Bacteriol. 87:910-919. 1964.-Bacteria from soiland related environments were selected or adaptedto metabolize phenol, hydroxy phenols, nitro-phenols, chlorophenols, methylphenols, alkyl-phenols, and arylphenols when cultured in mineralsalts media with the specific substrate as the solesource of carbon. A phenol-adapted culture (sub-strate-induced enzyme synthesis proven) waschallenged in respirometric tests with 104 relatedcompounds; probable significant oxidative ac-tivity occurred with 65. Dihydric phenols weregenerally oxidized; trihydric phenols were not.Cresols and dimethylphenols were oxidized; add-ing a chloro group increased resistance. Benzoicand hydroxybenzoic acids were oxidized; sulfo-nated, methoxylated, nitro, and chlorobenzoicacids were not; m-toluic acid was utilized but notthe o- and p-isomers. Benzaldehyde and p-hy-droxybenzaldehyde were oxidized. In general,nitro- and chloro-substituted compounds and thebenzenes were difficult to oxidize.

The dissimilation of phenols and aromatichydrocarbons by bacteria has been reported bymany workers. Results of early studies indicatingthat microorganisms are able to degrade ringcompounds were reported by Buddin (1914),Sen Gupta (1921), Dooren de Jong (1926),Tattersfield (1928), and Gray and Thornton(1928). Stanier (1947) described his technique ofsimultaneous adaptation for determining meta-bolic pathways in the bio-oxidation of aromaticcompounds and also showed that fluorescentpseudomonads attack many aromatic compounds(Stanier, 1948). Subsequent studies on the

mechanism, optimal conditions for degradation,and the intermediate products of the metabolismof aromatic compounds by microorganisms werereviewed by Happold (1950). ZoBell (1946,1950), in reviewing literature relating to theaction of microorganisms on hydrocarbons,mentioned numerous phenolic compounds andhydrocarbons oxidized by bacteria, and thesources and types of organisms involved.Few studies have been made concerning the

effect of chemical structure on the ability ofbacteria adapted to a given aromatic compoundto oxidize related compounds. Czekalowski andSkarzynski (1948) investigated the relationshipbetween chemical structure and the use ofaromatic compounds by one phenol-tolerantstrain of Achromobacter, and Kramer and Doetsch(1950) used Achromobacter, Micrococcus, andVibrio species that are capable of utilizing phe-nols to study their ability to grow on relatedaromatic compounds.The purpose of this investigation was to

determine the ability of specifically adaptedbacteria to degrade phenol and substitutedphenols, and to study the relationship betweenthe chemical structure of phenol derivatives andcyclic hydrocarbons and their susceptibility todecomposition by organisms adapted to relatedaromatic compounds.

MATERIALS AND METHODS

Selection and adaptation of bacteria to utilizephenol and substituted phenols. Organisms wereobtained from garden soil, compost, river mud,and sediment from a waste lagoon of a petroleumrefinery catalytic cracking plant. Methods usedin selecting or adapting organisms to degradethese compounds were soil perfusion, continuous-feed activated sludge, primary enrichment inflasks on a shaker, and enrichment in batch-type

910

Page 2: Microbial Metabolism of Aromatic Compounds

METABOLISM OF PHENOL-ADAPTED BACTERIA

fermentors. In all instances, the material con-taining organisms subjected to preliminary en-richment was eventually inoculated in baffledErlenmeyer flasks containing 50 ml of Gray andThornton's (1928) mineral salts medium towhich 0.025 ,ug of vitamin B12 had been added.The specific plhenolic compound served as thesole source of carbon. The medium was preparedby aseptically adding concentrates to steriledistilled water. Final pH wvas 7.0 to 7.2. Allcultures were incubated at room temperature onan orbital shaker.

Subcultures in the same medium were madeperiodically for several weeks or months. Alltraces of organic inutrients as debris carried inthe original inocultum were rapidly lost, and allnonbacterial forms soon disappeared. As soon asthe oxidative capacity of the selected bacteriacould be (lemonstrated in media containing alow concentiatioin of the phenolic substrate, 2 to10 ml of this material were subcultured intomedia containing progressively increasing con-centrations of the same compound. The increasein concentration of the substrate throughout theculture enrichmllent leriod ranged froin 100 ppmto a maximum of 500 ppm. Comp)ounds used wereEastman Grade (highest purity chemicals suitablefor reagent use) or Eastman Practical Grade(suitable for most laboratory syntheses). Oc-casionally, comnparable grades from other sourceswere use(.

Identifcation of organisnms. The mixed cultureswere streaked on Tryptose Agar, TrypticaseSoy Agar, yeast extract-agar, Rhizobiumasl agar,and egg albumin-agar. T'he pure cultures isolatedfrom these plates were examined for motility;reaction to Gram stain; ability to ferment lactose,glucose, sucrose, salicin, rhainnose, (luleitol,mannitol, Imaltose, and xylose; capacity to

produce hydrogen sulfide, amnmonia, indole, andurease; and ability to convert nitrates to nitrites.Their reaction in litmus milk and abilitv toliquefy gelatin and hydrolyze starch were alsodetermined. The presence of pseudomnonadstrains was confirmedl by the use of the cyto-chrome oxidase test (Gaby and Hadley, 1957;Gaby and Free, 1958).

Determination of the amouint of phenolic sub-strate remainin,g. At approlpriate intervals (luringincubation, contents of flasks were restored to theoriginal volume. For analysis of remailing phe-nolic conpoundl, solids were separated by cen-

trifugation; the supernatant fluid was filteredthrough a Whatman 50 filter paper. With nitro-phenols, degradation was determined spectropho-tometrically by measuring the intensity of theyellow color, characteristic of these compounds,at 400 m,u in alkaline solution at a pH higher than7.0 (Gundersen and Jensen, 1956), and bydetermining the amount of organic nitrogenconverted into nitrite-nitrogen, one of theintermediate products of nitrophenol metabolism(Rider and iIellon, 1946). The 4-aminoanti-pyrine method (Ettinger, Ruchhoft, and Lishka,1951; Mlohler and Jacob, 1957) was used spectro-photometrically to determine residual concentra-tions of phenols other than nitrophenols. Thecolor absorbance of aqueous solutions and chloroform extracts was determined at wavelengths of460 and 510 m,u, respectively. The solutions oftest compounds from the respective uninoculatedcontrol flasks were also analyzed to v-erify thatany loss of substrate in the test flask was not dueto volatilization loss or chemical oxidation. Tofollow the increase in oxidative ability of organ-isms and Inake l)eriodic determinations of therate of dissimilation of )henols, the 4-amino-antipyrine test was modified for use as a semi-micro-method. This test required smnall aruountsof medium and was useful as a rapid screen testfor most of the non-nitrogenous compounds used.

lfanometric studies. The purpose of the respiro-metric work was to determine whether organismsthat, had been adalted to utilize phenol couldimmediately (legrade structurally related com-pounds to which they had not been adapted.The formation of adaptive enzymes to metabolizephenol was indicated by a marked lag in oxygenul)take in reslirometric tests with phenol whenorganisms were grown in nutrient broth. Stand-ard techniques described by IUmbreit, lBurris,and Stauffer (1959) were used.

Phenol-adapted bacteria were inoculated inGrav and Thornton's liquid mineral salts mediumcontaining 300 ppmn of phenol as the only sourceof carbon, and incubated on a shaker at roomtemperature for 16 hr. The cells were then re-moved bv centrifugation, washed several timeswith buffered dilution water (American PublicHealth Association, 1960), stored overnight at 5 Cin the same buffer, aerated 3 to 4 hr, removed bycentrifugation, an(d resuspended in 0.067 Mphosphate buffer at pH 7.2 (Clark, 1920). EachWarburg flask contained an appropriate amount

VOL. 87, 1964 911

Page 3: Microbial Metabolism of Aromatic Compounds

TABAK, CHAMBERS, AND KABLER

TABLE 1. Frequency of occurrence and distribution of bacteria degrading phenolic conpounds

Generic grouping of culture*Classes of compounds Sources of culturesdegraded

Pseudomonas Achromobacter Flavobacterium Xanthomonas

Nitrophenols Garden soil, compost, and river 23 3 14 3mud

Chlorophenols Sediment from petroleum re- 37 3 3 0finery wastes lagoon

Cresols Sediment from petroleum re- 48 8 4 0finery wastes lagoon, gardensoil, and compost

Phenol Sediment from petroleum re- 12 1 1 0finery wastes lagoon

Alkyl phenols Garden soil, compost, and re- 8 0 1 1finery lagoon sediment

Aryl phenols Garden soil, compost, and re- 11 4 1 0finery lagoon sediment

Hydroxy Garden soil, compost, and re- 13 6 1 0phenols finery lagoon sediment

Per cent occur- 73.8 12.1 12.1 1.93rencet

* Figures represent number of cultures, each having different biochemical or other characteristics,isolated from media in which a compound in the class indicated served as the sole source of carbon.

t Per cent of a total of 206 isolations falling in the respective genera indicated.

of 0.067 M buffer at pH 7.2 and 0.5 ml of cellsuspension in the main compartment, 0.2 ml of10% KOH solution in the center well, and anamount of stock solution of substrate in the sidearm necessary to produce the desired test con-centration. The total volume of reagents and cellsuspension added to a flask was 3.2 ml. A flaskcontaining substrate without cell suspension wasincluded for each compound to control chemicaloxidation, along with an endogenous control anda test with phenol plus cells to confirm that theorganisms used in the test had a normally highcapacity to utilize phenol. The uniformity ofoxygen uptake in the phenol controls indicatedthere was little variation in the activity of thecell suspensions used in different experiments. Allflasks were incubated in a Warburg water bathat 30 C and shaken at a speed of 68 strokes permin. The gas phase was air. Results of 10-minobservations of 02 uptake were averaged foreach successive 30-min interval.

Chemical analysis of residual substrate. When-ever the observed oxygen uptake appeared to besignificant, the centrifuged supernatant fluidfrom the respirometric test was analyzed forresidual substrate. Phenols were analyzed bythe same methods used during culture enrich-

ment. Nitro-aromatic compounds other thannitrophenols were analyzed by the methods ofPorter (1955) and Heotis and Cavett (1959).

RESULTS

Degradation of phenol and substituted phenols bycultural methods. Bacteria utilizing phenoliccompounds were obtained from all environ-ments sampled. Table 1 shows the original sourcesof the bacteria and their frequency of occurrence.In the case of the culture utilizing catechol, con-tinued subculture produced a pure culture. Inall other instances, enrichment methods yieldedmixed cultures of species common to all samplesources. These bacteria were all gram-negative,nonsporulating, aerobic, small to medium sizedrods. Optimal growth occurred at 20 to 25 C, orat 35 C. Both motile and nonmotile forms werefound. On the basis of colony and cell morphol-ogy, motility, Gram's stain, pigment production,and biochemical tests, the following genera weredistinguished: Pseudomonas, Flavobacterium,Achromobacter, and Xanthomonas. These generaare noted for borderline biochemical reactions,and the findings in this investigation confirmprevious reports (Davey, 1961; Haynes, 1951;Rhodes, 1959) regarding the difficulty of defining

912 J. BACTERIOL.

Page 4: Microbial Metabolism of Aromatic Compounds

VIETABOLISM OF PHENOL-ADAPTED BACTERIA

these species by any arbitrary selection ofmorphological and biochemical characters. Datashowing the ability of these bacteria to degradetheir respective parent substrates are presentedin Table 2.

It should be noted that during incubation ofnitrophenol-containing culture, the yellow color,characteristic of nitrophenols and chloronitro-phenols, progressively faded and finally disap-peared from the medium. The color loss coincidedwith the appearance of abundant growth anddisappearance of the substrate from the medium.With the culture enriched on 2,4, 6-trinitro-phenol, instead of the fading observed withother nitrophenols the color changed from yellowto orange-red; this color persisted in flasks con-taining the higher concentrations but graduallydisappeared in the lower concentrations. Thereaction of the medium changed from pH 7.2 to6.3, and the yellow color was not restored byreadjusting to pH 7.2.Manometric studies with phenol-adapted bac-

teria. The relationship between molecular struc-ture and ease of degradation was evaluated on abasis of tests with a total of 104 compounds in the

TABLE 2. Time required for bacteria toutilize 95% of parent substrate

Compounds degraded in*

1 to 2 days 3 to 6 days 7 to 10 days

Phenol o-Nitrophenol 2,4-Dinitro-m-Nitrophenol phenol

Catechol p-Nitrophenol 2,6-Dimethyl-Resorcinol 2,4,6-Trinitro- phenol, 200Quinol phenol, 250 ppmPhloroglucinol ppm 2,4-Dichloro-o-Cresol 2-Chloro-4- phenol, 200m-Cresol nitrophenol ppmp-Cresol 2,6-Dichloro- 2,4,6-Tri-

4-nitrophenol chlorophenolmn-Chloro-phenol, 150ppm

p-Chlorophenolo-Phenyl-

phenol, 100ppm

Thymol, 150ppm

* Initial concentration was 300 ppm unless in-dicated otherwise.

90 120 150 ISOTIME IN MINUTES

FIG. 1. Oxidation of hydroxyphenol derivatives.

following classes: phenols, benzyl alcohols, ben-zoic acids, benzaldehydes, benzenes, cyclohexane,and heterocyclic compounds. The results ob-tained with substituted phenols indicated that thephenol-adapted culture had a significant degreeof ability to oxidize many of these compounds,while other compounds were relatively resistant.There was no measurable biological oxygen con-sumption with pyrogallol, the only compoundshowing a significant oxygen uptake in the chemi-cal oxidation control.

Results presented in Fig. 1 indicate that allhydroxyphenols except phloroglucinol and pyro-gallol were degraded; Fig. 2 shows that the oxygenuptake with aminophenols, alkylphenols, aryl-phenols, and chloronitrophenols varied from 50to 150 ,uliters. The activity with cresols washigher than with phenol, and oreinol, thymol,and the dimethylphenols were less susceptible todissimilation than was phenol (Fig. 3). Thepresence of a chlorine atom increased the resist-ance of phenol and methylphenols (Fig. 4), andthis resistance to decomposition was likewiseobserved with nitro-aromatic compounds (Fig.5). Degradation of benzoic and other aromaticacids depended on the nature of the groups

VOL. 87, 1964 913

Page 5: Microbial Metabolism of Aromatic Compounds

CONCENTRATI ON

TABAK, CHAMBERS, AND KABLER

60 90 120 150TIME IN MINUTES

FIG. 2. Oxidation of alkyl, aryl, chloro-nitro, andamflinophenol derivatives.

OF ALL COMPOUNDS 100ppm

m-CRESOL

-CRESOL

PHENOL

tpCRESOL

50-

°°- -| /3,4-DiMETHYLPENOL -

150-|2,4-DIMETHYLPHENOL

/CIINOL DIMETHYLPHENOL-M E

50 THYMOL

30 60 90 120 150TIME IN MINUTES

I80 210

FIG. 3. Oxidation of cresols and other methyl-phenol derivatives.

J. BACTERIOL.

FIG. 4. Oxidation of chlorophenols and chloro.methylphenols.

FIG. 5. Oxidation of nitro aromatic compounds.

914

400 _

0

.4

I-

i 2m

Z 2C

0X0

11

I'll

Page 6: Microbial Metabolism of Aromatic Compounds

METABOLISM OF PHENOL-ADAPTED BACTERIA

attached to the aromatic ring (Fig. 6). Benzoicacid and the hydroxybenzoic, shikimic, andamino acids had sizable oxygen uptake rates,whereas nitro, chloro, and methoxylated benzoicacids, and benzenesulfonic acids were relativelyresistant to decomposition. Activity was highwith benzaldehyde and p-hydroxybenzaldehyde(Fig. 7), moderate with the methoxylated benzal-dehyde, vanillin, and low with the nitrobenzalde-hydes and an amide, benzamide. None of thebenzenes was rapidly oxidized. High resistance tooxidation was observed with benzene, chloro-benzenes, and nitrobenzenes, whereas nitro-toluenes exhibited a steady but low oxygen up-take (Fig. 7). Although a measurable degree ofactivity was observed with aniline, the nitro-anilines were less susceptible to oxidation. Therewas little oxygen uptake with either benzylalcohol or DL-a-methylbenzyl alcohol, but aheterocyclic compound, quinoline, exhibited sig-nificant activity.

Quantitative chemical determinations. Analysesat the conclusion of the respirometric tests indi-cated a definite correlation between oxygen con-sumption and the depletion of the phenolicsubstrate. The correlation became more apparentas the oxygen uptake increased, and this reduced

TIME IN MINUTES

FIG. 6. Oxidation of benzoic and other acids.

en IVL ,.owcvA 1,3,5-TRINITROBENZENE * p-HYDROXYBENZ-

|^A m-DINITROBENZENE ALDEHYDE-J I50 a BENZENE * BENZALDEHYDE-e |a* 1,3,5-TRICHLOROCENZENE o VANILLIN0 | o NITROBENZENE A ANILINE

A m-NITROBENZ-u 125 ALDEHYDE.z al o p-NITROIBENZ-

ALDEHYDE. BENZAMIDE

z 1000

075

50

25

60 20o ISO 0 60 20o tooTIME IN MINUTES

FIG. 7. Oxidationt of benzenes, anilines, andbenzaldehydes.

the possibility that adsorption of substrate bythe organisms was a significant factor in removalof the test compound. Data showing the correla-tion between oxygen uptake and disappearanceof substrate are presented in Table 3. Compoundsresistant to decomposition are presented in Table4.

DISCUSSIONA considerable number of the enriched cultures

have been screened for adaptive enzyme forma-tion. In all instances, an immediate and rapidoxygen uptake was observed in respirometrictests with the compound used in the originalenrichment of the culture if the cells were har-vested from a medium containing the same sub-strate as the sole source of carbon. A marked lagin oxygen consumption was noted in paralleltests when the bacteria were grown in nutrientbroth. These findings indicate adaptive enzymeformation (Stanier, 1948).The results obtained indicate that pseudo-

monads occur in a variety of natural environ-ments and readily adapt to utilize phenol deriva-tives. Similar trends were reported by Parr,Evans, and Evans (1959), Simpson and Evans

915,Voi.. 87, 1964

Page 7: Microbial Metabolism of Aromatic Compounds

TABAK, CHAMBERS, AND KABLER

TABLE 3. Relationship between 02 uptake anddecomposition of substrate

Test concn Amt of 02consumed*

Test compound (endogenousInitial Loss corrected)ppm ppm_~~~~~~~P P

Phienol ..................Phenol ..................Phenol ..................Catechol ................Resorcinol ..............Quinol ..................Phloroglucinol..,m-Chlorophenol .........p-Chlorophenol .........2, 4-Dichlorophenol .....2, 6-Dichlorophenol ......2, 4,6-Trichlorophenol ...o-Cresol ................mit-Cresol ................p-Cresol ................2, 6-Dimethylphenol.3, 5-Dimethylphenol.2, 4-Dimethylphenol.3, 4-Dimet,hylphenol.OI Cinol .............Thymol .................

6-Chloro-mii-cresol. ....(;-Chloro-2-niethylphenol4-Chloro-2-rnetlhylplhenol4-C hloro-3i-methylphenolo-Nitrophenol ...........mz-Nitrophenol ..........p-Nitrophenol ...........2, 4-Dinitrophenol .......2,6-Dinitrophenol .......2,4, 6-Trinitrophenol ....4, 6-Dinitro-o-cresol.....2,4, 6-Trinitroresorcinol.2,4, 6-Trinitro-mn-cresol.4-Chloro-2-nitrophenol.2-Chlloro-4-nitrophenol.2, 6-Dichloro-4-nitIro-phenol ................

m-Dinitrobenzene.p-Dinitrobenzene.m-Nitroaniline ..........2, 4-Dinitroaniline .....in -Nitrobenzaldehyde ....3,5-Dinitrobenzoic acid.

1008060

1(010010060100100601001001001001001001001001001001(0808080(6O1001001(06060100100606010060

1(0100100100100100100

9979599798863

506618357097979769378190364451375046493932198

2860138

647

9252031392713

Mllters319252186255252149126680463956

4174573064070126189724881669011348655466512231614

12351

(1953), Durham (1957), Rogoff and Wender(1959), i\Iarr and Stone (1961), and Davey(1961).

Differences in resistance to degradation werenoted within each of several well-defined groupsof phenol derivatives as well as between theseclasses of comiipounds as a whole. This relation-ship was also apparently affected by the positionof a group) on the ring and by the size and com-plexity of the substituents.The results obtained with substituted phenols

indicate the possibility of a relationship betweenstructure an(d susceptibility to bacterial degrada-tion. The l)resence of more than two hydroxylgroups on the ring appeared to increase resist-ance to decomposition, e.g., phloroglucinol andpyrogallol, while dihydric l)henols seem to beoxidized to about the same or a slightly lesserdegree than phenol. Similar resuilts were reportedby Happold and Key (1932), Evans (1947),Czekalowski and Skarzynski (1948), IKramer andDoetsch (1950), Stanier (1950), Stanier et al.(1950), and Kilby (1951). Addition of a nitrogroup to the ring markedly increased the resist-ance to degradation, and none of the nitrophenols,with the exception of o- and rn-nitrophenol and2, 4-dinitrophenol, had an ap))reciable oxygen up-take. Simple nitrophenols were degraded by bac-teria, whereas those with more complex groupswere resistant. These trends are similar to thosereported by Kramer and D)oetsch (1950) andCzekalowski and Skarzynski (1948). Chlorosub-stitution also increased resistance to degradation,dichlorophenols being more refractory thanmonochlorophenols. Adding a methyl groupreduced resistance, as was shown by the highlevel of activity with cresols and some dimeth-ylphenols. The effect of position of substi-tution on the ring was illustrated with cresolsand dimethylphenols. With cresols there issome indication that substitution of a methylgroul) in the para l)osition resulted in a higherinitial oxygen uptake rate but a lower totaloxygen uptake than with the ortho and metaisomers. The same effect on total oxygein uptake,but not on the rate, was observedl with para-substituted dimethylphenols where a high oxygenuptake wvas observed with 2, 4, - and 3, 4-di-methylphenol while the activity was low-er withdimethylphenols having methyl groups in otherpositions. All monochloromethylphenols had ameasurable oxygen uptake, whereas dichloro-

* Based on 180 min results with all exceptorcinol; if endogenous rate was reached sooner,result is that obtained at corresponding time.

916 J. BACTERIOL.

Page 8: Microbial Metabolism of Aromatic Compounds

7METABOLISM OF PHENOL-ADAPTED BACTERIA

TABLE 4. Compounds resistant to degradation*

Pheinol derivativesPhloroglucinolPyrogallolD)imethyldihydroresorcinol2, 6-Dichlorophenol2, 6-Dinitrothymol2, 6-Dinitro-o-cresol2, 4, 6-Triunitrophenol2,4, 6-Trinitro-m-cresol2,4, 6-Tri nitroresorcinol4, 6-Dichloro-nm-cresol2, 6-Dichloro-4-nitrophenol2, 4-Dibroomophenolm-Phenoxyphenol

Berizaldehydesm-Nitrobenzaldehydep-Nitrobenzaldehyde

Benizene

Benzoic acidso-Nitrobenzoic acidm-Nitrobenzoic acidp-Nitrobenzoic acid2,5-Dinitrobenzoic acid3,4-Dinitrobenzoic acid3,5-Dinitrobenzoic acid3,5-Dinitrosalicylic acid2,4,6-Trinitrobenzoic acido-Chlorobenzoic acidmi-Chlorobenzoic acido-Toluic acidp-Toluic acidSyringic acidVanillic acid

Benzensulfonic acidsBenzenesulfonic acidp-Toluenesulfonic acid

Benzene derivativesChlorobenzeneo-Dichlorobenzene1, 3, 5-TrichlorobenzeneNitrobenzenep-Dinitrobenzenep-Nitroanilinep-Phenylenediamine

Benzyl alcoholsBeuizyl alcoholD-Mlethylbenzyl alcohol

Miscellaneous compoundsPhthalic acidDL-MIandelic acidAnthranilic acidTannic acidBenzamideCyclohexanol

* Compounds having a maximal endogenous corrected 02 uptake value below 40 Aliters.

methylphenols were more resistant. This indi-cates that dichlorosubstitution increased theresistance of both chlorophenols and chloro-methylphenols. Adding a methoxyl or a phenoxylgroup to the ring also increased resistance.

Differences in oxygen uptake related to molecu-lar structure and substitution were also observedwith benzoic acids. Activity with benzoic acidwas higher than with its dihydroxy derivatives,and the latter had greater activity than did thetrihydroxy benzoic acids. In all instances, thehydroxybenzoic acids were more susceptible todegradation than other substituted benzoic acids.The influence of positional substitution wasdemonstrated by methylbenzoic acids becausem-toluic acid was more readily oxidized than theortho and para derivatives. With methoxylatedbenzoic acids, increasing the number of methoxylgroups on the ring apparently interferes withoxidation, syringic acid being more resistant todegradation than vanillic acid. The presence ofmore than one carboxyl group significantly re-duced the rate of oxygen uptake by the benzoicacids. In comparing the susceptibility of benzoicand hydroxybenzoic acids with that of nitro-benzoic acids, the number of compounds testedappears to be sufficient to provide some basis forconcluding that the former were readily oxidizedby phenol-adapted bacteria and the latter wereextremely resistant. Similar trends were reportedby Kramer and Doetsch (1950), Stanier (1948,

1950), Evans (1947), Evans, Parr, and Evans(1949), Evans et al. (1951), Cain (1958), andCain, Ribbons, and Evans (1961).With p-hydroxybenzaldehyde, replacement

of the hydroxyl by a nitro group blocked theactivity. This was demonstrated by the resist-ance of p-nitrobenzaldehyde to decomposition.The resistance of benzamide indicated that thepresence of a CONH2 group also increased theresistance.

Results obtained with benzene and its chloroderivatives, regardless of the number or positionof the chlorosubstitutions, indicated that, ingeneral, they were resistant to degradation. Theeffect of a nitro group was significant becausesuch compounds as nitrobenzene and rn- andp-dinitrobenzene showed little activity, particu-larly the mononitrobenzene whose activity wasbelow the endogenous level. The resistance of thenitrobenzenes to degradation seemed to decreaseas the number of nitro groups on the benzenering increased. Neither the methyl nor aminogroup appeared to interfere completely withoxidation of benzene, because both aniline andtoluene were oxidized to a limited degree. Sub-stitution of a nitro group on the benzene ring ofaniline markedly increased the resistance, aswas demonstrated with the nitroanilines, p-and m-nitroaniline. There appeared to be aslight difference in oxidizability between thenitroanilines, which increased in the order of

Voi,. 87, 1964 917

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TABAK, CHAMBERS, AND KABLER

ni-, p-, o-. Results obtained with an aminoaniline,p-phenylenediamine, indicate that adding a

second amino group to the benzene moleculeincreases resistance to oxidation.

LITERATURE CITED

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BUDDIN, W. 1914. Partial sterilization of soil byvolatile and nonvolatile antiseptics. J. Agr.Sci. 6:417-455.

CAIN, Rt. B. 1958. The microbial metabolism ofnitro-aromatic compounds. J. Gen. Micro-biol. 19:1-14.

CAIN, R. B., D. W. RIBBONS, AND W. C. EVANS.1961. The metabolism of protocatechuiic acidby certain microorganisms. Biochem. J.79:312-316.

CLARK, W. M. 1920. The determination of hy-drogen ions. The Williams and Wilkins Co.,Baltimore.

CZEKALOWSKI, J. W., AND B. SKARZYNSKI. 1948.The breakdown of phenols and related com-

pounds by bacteria. J. Gen. Microbiol. 2:231-238.

DAVEY, B. B. 1961. Some phenol decomposingstrains of Pseudomonas. J. Appl. Bacteriol.24 :78-82.

DOOREN DE JONG, L. E. DEN. 1926. Bijdrage tot dekennis van het mineralisatieproces. Thesis,Technisehe Hoogeschool te Delft. Nijgh &Van Ditmar's Uitgevers-Mij, Rotterdam.

DURHAM, N. N. 1957. Effect of structurally relatedcompounds on the oxidation of p-amino-benzoic acid by Pseudomonas fluorescens. J.Bacteriol. 73:612-615.

ETTINGER, M. B., C. C. RUCHHOFT, AND R. J.LISHKA. 1951. Sensitive 4-aminoantipyrinemethod for phenolic compounds. Anal. Chem.23 :1783-1788.

EVANS, W. C. 1947. Oxidation of phenol andbenzoic acid by some soil bacteria. Biochem.J. 41:373-382.

EVANS, R. A., W. H. PARR, AND W. C. EVANS.1949. The bacterial oxidation of aromaticcompounds. Biochem. J. 44:viii.

EVANS, W. C., B. S. W. SMITH, R. P. LINSTEAD,AND J. A. ELVIDGE. 1951. Chemistry of theoxidative metabolism of certain aromaticcompounds by microorganisms. Nature168 :772-775.

GABY, W. L., AND E. FREE. 1958. Differentialdiagnosis of Pseudomonas-like microorgan-

isms in the clinical laboratory. J. Bacteriol.76:442-444.

GABY, W. L., AND C. HADLEY. 1957. Practicallaboratory test for the identification of Pseu-dontonas aeruginosa. J. Bacteriol. 74:356-358.

GRAY, P. H. H., AND H. G. THORNTON. 1928. Soilbacteria that decompose certain aromaticcompounds. Zentr. Bakteriol. Parasitenk.Abt. II 73:74.

GUNDERSON, K., AND H. L. JENSEN. 1956. A soilbacterium decomposing organic nitrophlenols.Acta Agr. Scand. 6:1.

HAPPOLD, F. C., AND A. KEY. 1932. The bacterialpurification of gas-works liquors. The actionof the liquors on the bacterial flora of sewage.J. Hyg. 32:573-580.

HAPPOLD, F. C. 1950. Biological oxidation ofaromatic rings. Biochem. Soc. Symp. (Cam-bridge, Engl.) No. 5.

HAYNES, W. C. 1951. Pseudoinonas aeruginosa-itscharacterization and identification. J. Gen.Microbiol. 5:939-950.

HEOTIS, J. P., AND J. W. CAVETT. 1959. Colorreaction for determination of some meta-dinitro aromatic compounds. Anal. Chem.31:1977-1978.

KILBY, B. A. 1951. The formation of a beta-ketoadipic acid by bacterial fission of aro-matic rings. Biochem. J. 49:67-674.

KRAMER, N., AND R. H. DOETSCH. 1950. Thegrowth of phenol-utilizing bacteria on aro-matic carbon sources. Arch. Biochem. 26:401-405.

MARR, E. K., AND R. W. STONE. 1961. Bacterialoxidation of benzene. J. Bacteriol. 81:425-430.

MOHLER, E. F., JR., AND L. N. JACOB. 1957. De-termination of phenolic compounds in waterand industrial waste waters. Comparison ofanalytical methods. Anal. Chem. 29:1369-1374.

PARR, W. H., R. A. EVANS, AND W. C. EVANS.1949. The mechanism of the bacterial oxida-tion of certain aromatic compounds andproperties of a cell-free enzyme system whichaccomplishes ring cleavage. Biochem. J.46 :xxix.

PORTER, C. C. 1955. Color reaction for determina-tion of some aromatic nitro compounds. Anal.Chem. 27:804-807.

RHODES, M. E. 1959. The characterization ofPseudomonas fluorescens. J. Gen. Microbiol.21:221-263.

RIDER, B. F., AND M. G. MELLON. 1946. Colori-metric determination of nitrites. Ind. Eng.Chem. Anal. Ed. 18:96-99.

ROGOFF, M. H., AND I. WENDER. 1959. Methyl-naphthalene oxidations by pseudomonads.J. Bacteriol. 77:783-788.

918 J. BACTERI OL.

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METABOLISAI OF PHENOL-ADAPTED BACTERIA

SEN GUPTA, N. N. 1921. Dephenolization in soil.J. Agr. Sci. 2:136-158.

SIMPSON, J. R., AND W. C. EVANS. 1953. Themetabolism of nitrophenols by certain bac-teria. Biochem. J. 55:xxiv.

STANIER, R. Y. 1947. Simultaneous adaptation:a new technique for the study of metabolicpathways. J. Bacteriol. 54:339-348.

STANIER, R. Y. 1948. The oxidation of aromaticcompounds by fluorescent pseudomonads. JBacteriol. 55:477-494.

STANIER, R. Y. 1950. The bacterial oxidation ofaromatic compounds. IV. Studies on themechanisms of enzymatic degradation ofprotocatechuic acid. J. Bacteriol. 59:527-532.

STANIER, R. Y., B. P. SLEEPER, AI. TSUCHIDA,

AND D. L. MACDONALD. 1950. The bacterialoxidation of aromatic compounds. III. Theenzymatic oxidation of catechol and proto-catechuic acid to beta-ketoadipic acid. J.Bacteriol. 59:137-151.

TATTERSFIELD, F. 1928. The decomposition ofnaphthalene in the soil and the effect upon itsinsecticide action. Ann. Appl. Biol. 15:57-80.

UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF-FER. 1959. Manometric techniques. BurgessPublishing Co., Minneapolis.

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hydrocarbons. Bacteriol. Rev. 10:1-49.ZoBELL, C. W. 1950. Assimilation of hydrocarbons

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