DITERMINAL OXIDATION OF LONG-CHAIN ALKANES BY BACTERIA1

A. S. KESTER AND J. W. FOSTERDepartment of Microbiology, The University of Texas, Austin, Texas

Received for publication 19 November 1962

ABSTRACTKESTER, A. S. (The University of Texas,

Austin) AND J. W. FOSTER. Diterminal oxidationof long-chain alkanes by bacteria. J. Bacteriol.85:859-869. 1963.-A corynebacterial organismcapable of growing in mineral salts with in-dividual pure alkanes as carbon sources producesa series of acids from the CI0-C14 alkanes. Theyhave been isolated in pure form and identifiedas monoic, w-hydroxy monoic, and dioic acidscontaining the same number of carbon atomsas the substrate alkane. Oxidation took placeat both terminal methyl groups-"diterminaloxidation." Appropriate labeling experimentsindicate that omega oxidation of fatty acidsoccurs in this organism and that an oxygenationwith 02 occurs.

last few years and has recently been collectedand codified (Fuhs, 1961; Foster, 1962a, b).It is, therefore, unnecessary to review the litera-ture in detail here, except as it pertains to pointsspecifically made in this paper.

Microbial oxidation of aliphatic alkanes isbelieved to occur by a monoterminal oxidation,i.e., the oxygenation of one of the terminalmethyl groups yielding as the first stable productthe homologous primary alcohol. In some cases,this is further oxidized to the homologous fattyacid, which accumulates according to the follow-ing sequence (Stewart et al., 1959; Leadbetterand Foster, 1960; Proctor, 1960; Heydeman,1960; Webley, Duff, and Farmer, 1956; Gholsonand Coon, 1960, Heringa, Huybregste, andvan der Linden, 1961; Azoulay and Senez, 1960;Seeler, 1962):

One of the fundamental aspects of microbialutilization of hydrocarbons concerns the site ofprimary attack on the hydrocarbon molecule.In the case of the saturated long-chain alkanes,this question is especially interesting becauseof the multiple potential points of attack, thesimilarity of the multiple methylene groups,and the identical terminal methyl groups. In-formation on these points has mounted in the

I A preliminary note on this subject has beenpublished (Kester and Foster, Bacteriol. Proc.,p. 168, 1960).

RCH2CH3 -- RCH2CH20H -> RCH2COOH

In addition to the universal monoterminalmechanism, there appears to be a concomitantalpha oxidation, at least in the case of propane,n-butane, n-pentane, and n-hexane (Leadbetterand Foster, 1960; Lukins, 1962; Lowery, personalcommunication). The stable product most com-monly arising first by this mechanism is thehomologous methyl ketone, whose formation hasbeen viewed as a variation of monoterminaloxidation involving a free radical equilibrium(Leadbetter and Foster, 1960; Foster, 1962a, b):

RCH2CH3 -- RCH2CH2 2 RCHCH3

102 {02

RCH2CH20H RCHOHCH3

I ~~~~~~~~~~~~~IIRCH2COOH RCCH3

0

Terminaloxidation

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Alphaoxidation

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KESTER AND FOSTER

All of the alkane oxidations hitherto describedare of one or the other of those two types. Inthe case of aliphatic alkanes, it is clear that bothterminal methyl groups are indistinguishable.However, the terminal methyl group remainingafter one has been oxygenated must, in a mannerof speaking, be accorded a form of recognition,since in all previously described instances itremains unattacked subsequent to the oxygena-tion of the opposing terminal methyl carbon.An interesting case of methyl recognition hasbeen described by Thijsse and van der Linden(1961) for the oxidation of 2-methylhexane byPseudomonas aeruginosa. This organism selec-tively attacks a single methyl group, but adifferent one on different molecules of the sub-strate. A mixture of 5-methylhexanoic and2-methylhexanoic acids was produced from2-methylhexane. Thus, although the organismcannot make a strict distinction between oneend of the isoalkane molecule and the other,once any particular molecule is breached at aterminal methyl the other terminal methylsremained unattacked. In this respect, the iso-alkane oxidation is similar to n-alkane oxidationin discriminating between methyls on any onealkane molecule.The present paper deals with an organism

which is unable to discriminate between theends of long-chain n-alkanes. Both terminalmethyl groups are oxidized without change inthe carbon skeleton. This process is called"diterminal oxidation."

MATERIALS AND METHODS

Organism and culture methods. Most of theexperiments were performed with a pink bac-terium, strain 7E1C, tentatively assigned to thegenus Corynebacterium. This organism wasisolated from soil in propane-mineral salts en-richment cultures. In this work, "mineral salts"refers to the salts solution "L" of Leadbetterand Foster (1958). The individual hydrocarbonsused were 99 mole % pure (Phillips PetroleumCo., Bartlesville, Okla.). In some cases, e.g.,n-hexadecane, the highest purity commerciallyavailable within reasonable cost is 95%(Humphrey-Wilkinson Co., North Haven, Conn.).Production of the various acids in amountssuitable for isolation was conducted in 2-literErlenmeyer flasks containing 250 ml of mineralsalts solution and 4 to 10 ml of an individual

liquid hydrocarbon as the sole substrate. Fromtwo to ten flasks were employed in various ex-periments. It proved unnecessary to sterilizethe hydrocarbons, although the salts solution wasalways autoclaved. Microbial contaminationwas rarely a practical limitation in this extremelyselective environment. A satisfactory state ofculture purity was established by routine platingson nutrient agar.The cotton-plugged flasks were incubated on

a reciprocal shaker for 6 days at 37 C. Whengaseous hydrocarbons were used as substrates,suction flasks were employed for culture vessels.The top was sealed with a rubber stopper, andthe appropriate gas phase was introduced throughthe side arm after partial evacuation of the airin the flask. The final gas mixture was 50% air-50% gaseous hydrocarbon. The side arms weresealed with a screw clamp immediately aftergassing.

Extraction of acids. The whole culture wasacidified to pH 1.0 with H2SO4 and extractedtwice in a separatory funnel with successiveequivalent volumes of diethyl ether. The etherhad been washed with 2 N NaOH to remove anyacids, especially acetic, which might have beenpresent. The combined ether extracts were thenwashed by shaking with 10 ml of distilled water,and the clear extract was concentrated to drynessin an air stream at room temperature. Thecrude residue was dried in vacuo over P205.Paper chromotography. Ether solutions con-

taining 30, 60, or 90 ,ug of total acid were spottedon Whatman no. 1 filter paper. Three differentsolvent systems were used, in the ascendingtechnique: solvent A, 95% ethanol and con-centrated NH40H (100:1; v/v); solvent B,n-propanol and concentrated NH40H (7:3;v/v); solvent C, n-butanol and 1.5 N NH40H(1:1; v/v). Solvent A proved to be most usefulin this work. The papers were dried in a currentof air in a hood, and acid spots were developedby spraying with bromothymol blue indicatorsolution.

Partition column chromotography. A number ofpartition chromotography methods with silicicacid columns were tested. A modification of theMarvel and Rands (1950) method was found tobe the best. The particular procedures had to bealtered according to the acids being separatedand isolated; details are given in connectionwith the respective experiments described in

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VOL. 85, 1963 DITERMINAL OXIDATION OF LONG-CHAIN ALKANES

the next section. Acids in the various eluentfractions were titrated with dilute, standardNaOH. A small amount of water was added tothe tubes before titration; alcoholic phenol-phthalein was the indicator.

Chemical methods. Fractions containing apredominant acid were pooled and concentratedin vacuo, and the acid was recovered by crystal-lization. The isolated acids were recrystallizedto constant melting point. Melting point de-terminations were done with capillary tubes.The temperature was raised at the rate of 1 to2 C per min for the last 10 C. Identification ofeach pure acid was made by cochromatographyon paper, by molecular weight determinations(Rast), by neutralization equivalent, by ele-mentary analysis, and by comparison of theinfrared-absorption spectrum of the bacterialproduct with that of an authentic compound.A Baird model 4-55 recording spectrophotometerwas used.

Isotopic procedures. Oxygen rich in 018 wasprepared by electrolysis of H2018 (6.25 atom %excess 018) obtained from the Weizmann In-stitute of Science, Rehevoth, Israel. Productsto be analyzed for 018 content were convertedto CO2 by the reductive combustion procedureof Rittenberg and Ponticorvo (1956), and theisotopic composition of the CO2 was determinedin a Consolidated type 21-102 mass spectrometer.Decanoic-1-C'4 was purchased from the Nuclear-Chicago Corp., Des Plaines, Ill. Radioactivecarbon measurements were made with a thin-window Geiger-Muller tube in a Tracerlab "64"scaler and were based on infinitely thin layers ofsamples.

RESULTS

Growth substrates. Originally isolated as apropane utilizer, Corynebacterium 7E1C iscapable of growing on a large variety of non-hydrocarbon as well as hydrocarbon substrates.It requires no exogenous growth factors, normore than a single organic compound for growthin the mineral salts solution. It is, however, ableto grow luxuriantly in conventional complexmedia such as nutrient broth, yeast extract, etc.Among the nonhydrocarbon substances utilizablefor growth were glucose, fructose, maltose, gal-actose, sucrose, mannose, ribose, mannitol, sor-bitol, citrate, a-ketoglutarate, succinate, andmalate. A variety of primary alcohols and fatty

acids, intermediates in the oxidation of alkanes,could also serve as sole sources of carbon. Cory-nebacterium 7E1C did not grow autotrophicallywith H2, C02, and O2, as was the case with severalother hydrocarbon utilizers (Dworkin andFoster, 1958).

Hydrocarbons. Good growth was made at theexpense of each n-alkane in the series C3 to C18;C15 and C17 were not available for testing.Growth was not observed in methane or ethane;alkanes with more than 18 carbons were nottested. Of a variety of aliphatic alkenes tested,growth was obtained only on long-chain members,e.g., dodecene-1, tetradecene-1, hexadecene-1,and octadecene-1. Olefins not supporting growthincluded ethylene, propylene, isobutylene, bu-tene-2 (cis), and butene-2 (trans).

Of several methyl-substituted isoalkanestested, only isopentane, 2-methylpentane, and2, 2-dimethylbutane were utilized for growth.A series of 23 haloalkanes were likewise testedas sole carbon sources; those supporting growthwere 1-chlorooctane, 1-chlorododecane, 1-chloro-hexadecane, and 1-chlorooctadecane.

In general, the longer chain compounds werefound to be more susceptible to attack than theshorter chain compounds. Optimal growth wasobtained in 3 days on the CI0 to C16 compounds,whereas it took 5 to 6 days on the shorter chaincompounds.

Acid formation. Total acid formation fromindividual hydrocarbons (C5 through C18) wasdetermined by titration of the crude acid etherextracts. Significant acid formation was obtainedfrom all of the hydrocarbons. Highest yieldswere found in the C1l, C12, and C13 substrates.In this experiment, 0.322 meq of acid, a maxi-mum, was produced in the tridecane culture.From a comparison with a large number of otherbacteria, it was apparent that strain 7E1C wasexceptional in oxidizing hydrocarbons incom-pletely, accumulating acids. A series of physi-ological experiments testing the effect of variousenvirQnmental factors on acid accumulationresulted in one noteworthy finding. A fivefoldincrease in the level of phosphate employed inthe growth medium decidedly enhanced acidproduction. Thus, in 5 days a dodecane cultureyielded 0.45 meq per 100 ml of medium; withthe usual (low) phosphate level, the yield wasabout one-third of that figure.

Identification of acids. Most of the acids re-

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covered from the C1o to C16 hydrocarbon cul-tures were nonvolatile and were solid at room

temperature. The reverse was true of the acidsproduced from the shorter chain hydrocarbons.They were volatile and liquid and did not giveany indication of being unusual fermentationproducts. The acids from the C10 to C16 hydro-carbons appeared from the outset to be unusual,and these were studied in detail.

Tables 1 and 2 summarize the data concerninga number of crystalline acids which were formedfrom various long-chain alkanes. Except for11-formylundecanoic acid, authentic specimensof which were unobtainable, conclusive identitywas established by comparisons of the infrared-absorption spectra of the bacterial and theauthentic acids (Fig. 1).A few words are warranted about the methods

of isolation in particular cases. Three acids were

observed in the paper chromatograms of theether extracts from the decane cultures. Thecrude acids were added to silicic acid columnsby absorbing them on a small amount of water-

saturated silicic acid, and elution was effectedwith benzol (benzene, 90%) followed byn-butanol-benzol mixtures. n-Decanoic acidcame off the column in the eluting solventfront. The 10-hydroxydecanoic acid was elutedwith benzol containing 0.5% n-butanol. Afterall of the hydroxy acid had been eluted, the1,10-decanedioic acid was eluted with benzolcontaining 3 to 5% n-butanol. The dioic acidwas recrystallized from ethyl acetate; the hy-droxy acid was recrystallized from a mixtureof benzene and petroleum ether. The lack offacilities for low-temperature crystallization ofdecanoic acid necessitated determination of itspurity by paper chromatography and subsequentidentification by infrared-absorption spectrumin chloroform.The problem of purification of the do-

decanedioic acid produced from dodecane was

much simplified in that the dioic acid could becrystallized directly from the mixture of acidsdissolved in benzene. Paper chromatography ofthe mixture revealed two spots, the one with

TABLE 1. Chemical properties of acids isolated from growth cultures of Corynebacterium7E1C on long-chain alkanes

Melting point Molecular Elementary

Acid wt (Rast) analysisBacterial Authentic Mixedproduct acid Found Theory Found Theory

c c c ~~~~~~%NoFrom n-decane cultures

(n-Decanoic*)10-Hydroxydecanoic 74-76 73-74.5 73-75 178.5 188 C, 63.64 63.8

H, 10.87 10.61 ,10-Decanedioic. 130.5-133 130-133 130-133 241 201

From n-dodecane cultures3-Hydroxydodecanoic... 54-56 t 204 216 C, 66.68 66.6

H, 11.25 11.111-Formylundecanoic. 64-66 t - 222 214 C, 67.19 67.3

H, 10.40 10.41,12-Dodecanedioic. 126-127.5 127-128 126.8-127.8 238 230 C, 62.88 62.6

H, 9.88 9.5612-Hydroxydodecanoic. 82-84 t 201 216 C, 67.11 66.6

H, 11.18 11.1From n-tridecane cultures

1,13-Tridecanedioic 111-114 112-115 111.5-113.5 C, 63.89 63.95From n-tetradecane cultures H, 10.67 9.83

1,14-Tetradecanedioic... 124-125.5 123.5-125 123-125

* Crystalline material not obtained. Identity established by RF values. The bacterial product be-haved the same as authentic decanoic, and a mixture of the two was unresolvable on paper chromato-grams. Identity was verified by comparison of infrared spectra of the authentic and bacterial products.

t Authentic sample not available.

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VOL. 85, 1963 DITERMINAL OXIDATION OF LONG-CHAIN ALKANES

TABLE 2. RF values of acids isolatcultures of Corynebacterium

long-chain alkanes

AcidA

1, 1O-Decanedioic acidAuthentic acid........ 0.17

Bacterial product... 0.21

Mixed acidst....... 0.20

1, 12-Dodecanedioic acidAuthentic acid........ 0.27

Bacterial product.... 0.27

Mixed acids........... 0.28

1 ,14-Tetradecanedioicacid

Authentic acid........ 0.31

Bacterial product... 0.30

Mixed acids........... 0.32Decanoic acidAuthentic acid. 0.79

Bacterial product 0.77

10-Hydroxydecanoic acidBacterial product.... 0.69

11-Formylundecanoic acidAuthentic acid ........ 0.78Bacterial product.... 0.78

Mixed acids........... 0.77

8-Hydroxydodecanoic acidBacterial product..... 0.68

* A, ethanol-NH,OH (100:1, v/vNH40H (7:3, v/v); C, butanol-(1:1, v/v).

t Mixed acids refers to mixturiacid and bacterial product.

the lower RF value being dode(The faster moving spot proved toacids; one was a keto acid whic]2, 4-dinitrophenylhydrazine, andwere hydroxy acids. The keto acito be an aldo acid since it gave a

test. Additional data recorded instrongly support the identity o

1l-formylundecanoic acid. Onetwo acids in the fast-moving spoacid with the hydroxyl group in a

than terminal, and it may be 3-h3acid. -Its hydroxy nature is dedfollowing facts. (i) It either lact4merizes by forming an intermolecuon heating gives a positive hyd(ii) Elementary analysis is consie

ted from growth conclusion. (iii) The infrared absorption spectrum7EIC on shows a strong absorption maximum at 2.86 M.

The third acid in the fast-moving spot has aSolvent* melting point closely approximating the litera-

B C ture value for 12-hydroxydodecanoic acid.Elementary analysis and the molecular weightdetermination support this conclusion.

0.52 0.46 The aldo acid was separated from the hydroxy0.56 0.55 acids on a silicic acid column using a mobile0.55 0.48 phase consisting of a solution of benzol and

n-hexane (1:1). Both hydroxy acids came off0.64 0.47 the column in the same eluate fractions but0.64 0.47 could be purified by fractional crystallization.0.64 0.45 From the tridecane cultures, two acids were

obtained, as revealed by paper chromatography0.75 0.22 of the acid ether extracts. These were separated0.75 0.23 on a silicic acid column. The dioic acid was0.76 0.24 recrystallized from benzene and identified by the

usual criteria. The other acid was probably0.86 0.80 tridecanoic acid, as judged by its movement on0.86 0.81 paper. It was not characterized further.

The acid produced from tetradecane as a sub-0.78 0.65 strate was recrystallized five times from benzene,0.83 0.70 after which its identity was verified by com-0.83 0.63 parison of its infrared-absorption spectrum with0.85 0.65 that of an authentic specimen.

Pathways of oxidation. The three acids formed0.77 0.57 in n-decane growth cultures, namely, decanoic,

10-hydroxydecanoic, and 1, 10-decanedioic acids,r); B, propanol- represent an oxidation sequence which suggests-1.5 N NH40H they are part of a pathway of oxidation of this

hydrocarbon and, further, that the analogouscompounds represent intermediates in the oxida-tion of the other long-chain alkanes utilized by

canedi ac.

Corynebacterium 7E1C.canedioic aci. It seems plausible that the oxidation occursconsist of three via the monoic acid to the w-hydroxy acid andh reacted with thence to the dioic acid. If this were the case,the othertwo the organism might be expected to use decanoic

pd s considered acid for growth, and also to produce decanedioic

postlve Schaff acid from it. However, decanoic acid did not

Tables 1and 2 serve as a growth substrate for strain 7E1C,of this acid as presumably because of a limited permeabilityofthe her of the cell to the exogenous acid. However, as

t is a hydroxy seen below, the impermeability cannot be ab-position other solute. Numerous attempts were made futilely

ydroxydecanoic to convert decanoic acid to decanedioic acid inuced from the growth cultures and in resting-cell suspensions.onizes or poly- Tests for the formation of the homologous dioicilar ester which acids from propionic, undecanoic, and dodecanoicIroxamate test. acids, respectively, were likewise negative.stent with this Time course. Figure 2 shows the course of acid

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864 YKESTER AND FOSTER J. BACTERIOL.

. ......

a6

I~~~~~~~~~tf~~~~~~~~~~~1ilf~~~~~~~~~~~~~~~~~~~~~......I4c d...

4e ....f......T~~~~~~f

C:~~~~FIG.1.InfraredabsorptIon spectra of acIds produced from long chasn alkanes byCorynebacterium~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...7EC()Deaoi ci:dakcuv, uhetc cd;lgh ure bcera pouc choofr sltin)

pelets. (Theserared photographsiofsethe original chrts.) fo ln-hi llae yCryeatru

chromatydogrpydrealc cdtatdduring thdeaedfirst 3 cd akcre uhni ai;lgtcreatraprdays theracides)consistedredominatelyacdpofuefo oeae(ettvl dniida -yrxyddecanoic.and 112-hDroxdecanedoic acid. ihOnuthe, auhniai;dr 0uv,bceilpout(Bfoureth) fda,1decan aedoic acid;wastlloter purve- uhni cdpeuv,bceilpout Bdominats) aci,1buTethraecwsanediincreased;upopor-£uv,atetcai;lwrcuv,bceilpoutBpeltion ofTheshyrox phtgand othediicaids.nBy cathe.)ffthrndixtays,thnprpotiooodecanoictai .Pae 0.25acidhroadtdereashed,aldthato lhduroxydtecanics3dacid rhemie abisoutisthedsame,omandtatelyofthdicaoicacid1increaseanicacd.On theseeth3iht,an715-ninrth days,tedecanoic anid was-hydroxydecanoicacmidsawer presenbt onlyeia n minoreaamounts,-whereas the hdioi acidbecae theo ajoacids. Onththeteth and elevethdays,teporionly ecni1,0 0.2acidhaddecreased,thato2f314hy6rox8d9c10o1c

dcndioic acidi coudeeaetced.OthsenthesethreulsnAYninthdiate,that decanoic and 10-hydroxydecanoic FG .Ai rdcindrn rwho oye

acides are inemdiocatiesaminthe faormatid.OnofbceimE1on-dae.Teskeulrs

1, 10-decanedioic acid. contained 4 ml of substrate in 250 ml of mineral salts-T -.1 .- -.f -

medium in 2-liter flasks.Labeled precursor. To a 36-hr decane culture of

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VOL. 85, 1963 DITERMINAL OXIDATION OF LONG-CHAIN ALKANES

strain 7E1C, 50 ,uc of decanoic-1-C54 acid were

added. The culture was further incubated forcontinued growth at the expense of the decane,which was always present in excess. The steam-volatile acid fraction was removed, and the non-

volatile acids were separated on a silicic acidpartition column prepared according to Smith(1960). Individual acids were recrystallized toconstant specific radioactivity. This was achievedby two recrystallizations in each case. Chemicalpurity of the final acid products was confirmedby paper chromatography and melting points.The steam volatile acid was identified as aceticacid by paper chromatograms. Each of the acidswas radioactive: acetic (Na salt), 242 counts per

min per mg; 10-hydroxydecanoic, 57 counts per

min per mg; and 1: 10-decanedioic, 204 counts per

min per mg. Barring the remote possibility thatthe labeling was derived from radioactive im-purities in the commercial labeled decanoate(which was not further purified in this work),these results indicate that decanoic acid, or an

"active" derivative, was the precursor of theradioactive acids. The unequal labeling in theacid products could be explained in various ways,

but this fact is not germane to the main point.Volatile acids. Almost invariably, short-chain

fatty acids were produced along with the long-chain acids in cultures growing at the expense oflong-chain alkanes. These steam-volatile acidstended to accumulate most when the oxygen

TABLE 3. RFp values of steam-volatile acids producedfrom dodecane and tridecane by

Corynebacterium 7EIC

Solvent system*Acid

A B C

Acid from dodecane cul-turest ................ 0.32 0.14

Authentic acetic acidt... 0.32 0.14Acids from tridecane

cultures .............. 0.26, 0.40, 0.10,0.38- 0.49 0.14

Authentic formic acidt. 0.23 0.37 0.09Authentic acetic acidt... 0.26 0.39 0.10Authentic propionic

acidt .............. 0.39 0.49 0.14Authentic butyric acidt. 0.46 0.58 0.26

* A, ethanol-NH40H (100:1; v/v); B, propanol-NH40H (7:3; v/v); C, butanol-NH40H (1.5 N;1:1; V/V).

t As ammonium salts.

i As sodium salts.

TABLE 4. Incorporation of molecular oxygen in acidsproduced from long-chain alkanes by

Corynebacterium 7EIC

018 in OxygenSubstrate Acid initial acid* derived

phase from 02

Dodecane 1,12-Dode- 2.16 0.047 2.18canedioic

Dodecane 1,12-Dode- 2.22 0.022 0.99canedioic

Decane 10-Hydroxy- 3.50 0.727 20.8decanoic

* Normal distribution of 018 in bacterial 1,12-dodecanedioic acid was determined (by analysis)as 0.202%. Results expressed as atom % excess.

supply was limited, as in a rubber-stopperedflask. Stoppered flasks in which the respiratoryCO2 was continuously absorbed by means ofKOH pellets in a small beaker suspended in-ternally usually showed a conspicuous increasein volatile acid accumulation. From substratehydrocarbons with even numbers of carbonatoms, acetic acid was the only steam-volatileacid found. From substrate hydrocarbons withodd numbers of carbon atoms, the steam-volatileacid fraction consisted of a mixture of acetic andpropionic acids (Table 3). The identity of theacetic and propionic acids was further verifiedby Duclaux distillation of the respective acidsseparated by means of partition columnchromatography. The Duclaux values were prac-tically identical with those of authentic aceticand propionic acids measured under the sameconditions.

Isotopic oxygen. The evidence is definite thatoxidation of long-chain and short-chain alkanesby certain bacteria involves the incorporation ofmolecular oxygen, probably as the initial oxida-tion step. Documented instances of this are few(Stewart et al., 1959; Leadbetter and Foster,1959), and the view does exist (Senez andAzoulay, 1961; Chouteau, Azoulay, and Senez,1962; also Foster, 1962a, b, for literature) thatthe alkane molecule may be dehydrogenativelybreached in the absence of molecular oxygen. Theready isolation of pure acid products from alkaneoxidation by Corynebacterium 7E1C made itdesirable to secure additional experimental resultswith labeled oxygen.The experiments were done in 2- or 4-liter suc-

tion flasks containing 250 or 500 ml of culture

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KESTER AND FOSTER

grown with propane as the substrate. The long-chain alkanes were added and the air was evacu-ated and replaced with N2 containing 80% 02enriched with 018. A sample of the gas phase wasremoved immediately after final gassing for massspectrophotometric determination of 018 content.After 2 to 5 days of incubation on the shakingmachine, the acids were isolated and purifiedas described earlier; the isotopic compositionsare listed in Table 4. The 018 content of thedecanedioic acid obtained in two independentexperiments was very low. But there was a de-cided incorporation of molecular oxygen in the10-hydroxydecanoic acid.

DISCUSSIONThe experiments reported here make it prob-

able that the diterminal oxidation of long-chainalkanes without rupture of the carbon chains inreality is composed of two stages: (i) mono-terminal oxidation of the alkane to the corre-sponding fatty acid, and (ii) omega oxidation ofthe fatty acid. Thus, the attack on the hydro-carbon is basically two successive monoterminaloxidations and is, therefore, consistent with otherinstances of alkane oxidation and appears to be ageneral rule. Corynebacterium 7E1C is distin-guished from all previously described alkaneoxidizers by its inability to discriminate betweenthe terminal methyl groups, so that it attacksthe second methyl group after the first methylgroup has been attacked. Previous evidenceindicated that oxidation of one methyl groupautomatically protected the omega group fromattack without breaking down the carbon chain.It is not possible to decide whether omega oxida-tion requires the fatty acid or only the primaryalcohol. Since the enzyme is a terminal methyl"oxygenase," the nature of the oxidized carbonat the remote distal end very likely has no in-fluence on the omega methyl oxidation.Omega oxidation of fatty acids has not been

demonstrated heretofore in microorganisms,although it has long been known in mammaliansystems. Verkade et al. (1933), Verkade andvan de Lee (1934), and Verkade (1938) recoveredthe corresponding dioic acids in the urine ofhuman subjects fed esterified fatty acids (seealso Dueul, 1957, for literature), and Robbins(1961) obtained cell-free extracts from hog liverwhich oxidized decanoic acid to 1, 10-decanedioicacid. We have not found dioic acids producedfrom alkanes with fewer than ten carbon atoms,

although this may merely mean they did notaccumulate in detectable amounts. However,T. S. Robinson (Manchester University,England) has found suberic acid (1,8-octanedioicacid) in a bacterial culture with n-octane as thesole carbon source (T. S. Robinson and N. A.Hall, personal communication), and the processmay yet be found to extend to even shorter hydro-carbons. The results of a limited survey we havemade on a variety of alkane-utilizing bacteriaindicate that the accumulation of diacids is notrare among such bacteria. It is possible that otherbacteria oxidize dioic acids as fast as they areformed from hydrocarbons and that the capacityfor diterminal oxidation may be of wider dis-tribution than is apparent.

Long-chain dioic acids can serve as the solesubstrates for growth of Corynebacteriunm 7E1C,and a problem to be decided is whether dioateformation is essential for assimilation of thehydrocarbon substrate or is merely a secondaryand minor pathway. In mammalian systems,dioic acids are utilized via a "bi-lateral" betaoxidation (Verkade, 1938); this subject has notbeen examined in strain 7E1C.

This organism also produces the monoic acidscorresponding to the alkanes undergoing oxida-tion, and there is excellent evidence that theintermediate fatty acids are further broken downby alkane utilizers via beta oxidation (Azoulayand Senez, 1960; Webley et al., 1956). Evidencesimilar to that obtained by Webley et al. (1956)favors beta oxidation of the monoic acid as themain degradation pathway in strain 7E1C. Fromdodecane only acetic acid was found, and fromtridecane a mixture of acetic and propionic acids.This is what would be expected from beta oxida-tion of monoic acids with even and odd numbersof carbon atoms, respectively. If the dioic acidshad undergone beta oxidation, acetic, but notpropionic acid, would be expected from sub-strates containing either even or odd numbers ofcarbons. Also, shorter chain length dioic acidswith even and odd numbers of carbons shouldbe formed. None of the latter has been detectedin the large number of analyses we have made.More direct evidence for beta oxidation via

the monoic acid pathway is the isolation of 3-hydroxydodecanoic acid from cultures grown ondodecane.

All in all, the monoic acid appears to be thefocal point of alkane oxidation in strain 7E1C,with the common beta oxidation accounting for

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VOL. 85, 1963 DITERMINAL OXIDATION OF LONG-CHAIN ALKANES

the main flow of carbon; omega oxidation to thedioic acid seems to be a minor and seeminglyunessential pathway. In fact, dioic acid formationmay well be merely a metabolic accident in thelife of strain 7E1C. The following scheme sum-marizes these points:

Rates of purely chemical exchanges betweenwater and compounds of this nature under theconditions of the experiments are such that anonbiological mechanism is unlikely in thepresent case (Cohn and Urey, 1938), althougha hydrogen ion-catalyzed exchange during puri-

RCH2CH302

RCH2CH20HAlkane Primary alcohol

It Main PathwayRCH2COOH beta oxidationMonoic acid

I Minor Pathway

HOH2C(CHDnCOOH -2-- HOOC(CH2)nCOOHw-Hydroxymonoic acid Dioic acid

Ibeta oxidation

Oxygen"8 incorporation. The data in Table 4make it clear that an oxygenase type of mecha-nism incorporating molecular oxygen is operativein strain 7E1C, and that a mechanism similar tothat described previously (Stewart et al., 1959;Leadbetter and Foster, 1959) is involved.

It is almost certain that hydroxyl groups areprecursors of carboxyl groups in this system.Therefore, the nonlabeling of the dodecanoicacid (Table 4) needs explaining. Those resultssuggest that the carboxyl group of the 10-hy-droxydecanoic acid is also unlabeled and that allof the excess 018 probably is located in the hy-droxy group.

Using that assumption, how does the labeledhydroxy group lose its isotope in becoming acarboxyl group, and how can one account for the018 content of the hydroxyl group being less thanthe theoretical 100% if it is derived from isotopic02? Both of these apparent discrepancies couldhave a logical explanation in exchange reactionswherebv the initially incorporated 018 atombecomes replaced with 016 atoms derived fromwater. Between the alcohol and the acid, thefollowing reactions may be assumed:

I II

fication is a possibility that cannot be ignored.More likely is an enzyme-catalyzed exchangeespecially involving the hydration reaction (II).As Stewart et al. (1959) have shown in theirester coccus, the acid would consist at most of50% 018, the other 50% being 016, according tothe formula at top of page 868.

If one assumes that the hydration reaction isfaster than the dehydrogenation reaction, or thatthe equilibrium constant for the former reactionis higher than the latter, the observed non-labeling pattern of the carboxyl groups of thebacterial acids is logically accounted for.

If one assumes, for the same reasons, that thelabeling of the 10-hydroxydecanoate (Table 4)is entirely in the hydroxyl group, then 3 X 20.8or 62.3% of the hydroxyl oxygen was derivedfrom 02. To account for this being less than100%, either of two things could have occurred.Least likely, the primary alcohol could be formedfrom the alkane by two independent mechanisms,one an oxygenase reaction incorporating oneatom of oxygen from 02 and the other a differentreaction whereby the oxygen of water participatesin a primary oxidation reaction.

III

H H H 0:T2H FHOH F2H/

R-C-OH =--=I RC=O ===R(R-C-OH) -H R-C

H OH OH

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AcidPrimary alcohol Aldehyde

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KESTER AND FOSTER

H HH06H , ( -2H

C=I (R-C-o1H) - I R-C=0'8

O'6H 0'F6H

The second and more likely possibility is thebreaching of the hydrocarbon molecule by anoxygenase adding one atom of oxygen from 02,followed by a chemical or biochemical exchangewith the oxygen of water. The 018 actually foundsimply represents the molecules of the primaryoxidation product which escaped the exchangereaction.

ACKNOWLEDGMENTS

This work was supported in part by grantsfrom the National Science Foundation, theNational Institutes of Health, and the Office ofNaval Research.

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