APPIED AND ENVIRONMENTAL MICROBIOLOGY, July 1977, P. 60-68Copyright 0 1977 American Society for Microbiology

Vol. 34, No. 1Printed in U.S.A.

Numerical Taxonomy and Ecology of Petroleum-DegradingBacteria

B. AUSTIN, J. J. CALOMIRIS,1 J. D. WALKER,2 AND R. R. COLWELL*Department of Microbiology, University of Maryland, College Park, Maryland 20742

Received for publication 15 February 1977

A total of 99 strains of petroleum-degrading bacteria isolated from Chesa-peake Bay water and sediment were identified by using numerical taxonomyprocedures. The isolates, together with 33 reference cultures, were examined for48 biochemical, cultural, morphological, and physiological characters. The datawere analyzed by computer, using both the simple matching and the Jaccardcoefficients. Clustering was achieved by the unweighted average linkagemethod. From the sorted similarity matrix and dendrogram, 14 phenetic groups,comprising 85 ofthe petroleum-degrading bacteria, were defined at the 80 to 85%similarity level. These groups were identified as actinomycetes (mycelial forms,four clusters), coryneforms, Enterobacteriaceae, Klebsiella aerogenes, Micrococ-cus spp. (two clusters), Nocardia species (two clusters), Pseudomonas spp. (twoclusters), and Sphaerotilus natans. It is concluded that the degradation ofpetroleum is accomplished by a diverse range of bacterial taxa, some of whichwere isolated only at given sampling stations and, more specifically, fromsediment collected at a given station.

Despite numerous studies undertaken to ex-amine the structure and metabolism of petro-leum-degrading bacteria, the taxonomy ofthese organisms has been comparatively ne-glected. Petroleum-degrading bacteria occurextensively in the aquatic environment andhave been found in sediment and seawater col-lected from temperate, tropical, and arcticzones (32). Microbial degradation of petroleumis influenced by a number of factors, includingseason, history of previous exposure of thegiven environment to oil, temperature, sedi-ment type, and medium used for the isolation ofthe organisms (4, 7). The method used for isola-tion of petroleum-degrading bacteria has astrong influence on the number and types ofbacteria recovered; for example, Sohngen (25),in 1913, observed colonies of mycobacteria onmineral agar exposed to hydrocarbon vapor,whereas in liquid culture designed to isolatehydrocarbon-utilizing bacteria, pseudomonadsare generally found (4, 28). In the study re-ported here, petroleum-degrading bacteria iso-lated from samples of oil-polluted and unpol-luted water and sediment have been classifiedby using numerical taxonomic procedures. Theresults of the analyses have proved useful in

I Present address: Mail Stop 236-5, NASA/AMES, Mof-fett Field, CA 94035.

2 Present address: Environment Technology Center,Martin Marietta Corp., Baltimore, MD 21227.

elucidating ecological aspects of petroleum deg-radation in the estuarine environment.

MATERIALS AND METHODSIsolation and maintenance of strains. Bacterial

strains used in this study were isolated from sedi-ment and water samples collected in Colgate Creekin Baltimore Harbor, an oil-contaminated site, andfrom Eastern Bay and Poole's Island, both nonpol-luted areas of Chesapeake Bay. Water temperaturesat the time of samplings were inthe range 14 to 25°C.The samples were collected aseptically, using a

Niskin sterile bag sampler (General Oceanics Inc.,Miami, Fla.) and a Ponar grab sampler (WildlifeSupply Co., Saginaw, Mich.) for water and sedi-ment, respectively. The samples were diluted andinoculated into petroleum media immediately aftercollection. Liquid enrichment cultures were pre-pared by inoculating 100 ml of a sterile salts solutionsupplemented with nitrate and phosphate (29, 31)with 1.0 ml of a 10-2 dilution of sediment or with 1.0ml of water, and overlaying it with 1.0 ml of a 20-weight motor oil. Direct plating of the same sedi-ment and water samples was accomplished by usingthis liquid salts medium to which agar (Difco Labo-ratories, Detroit, Mich.) was added to a final concen-tration of2% (wt/vol). The oil medium and its prepa-ration have been described elsewhere (29, 30).

After incubation at 15°C for 21 days, colonies ap-pearing on the oil agar and 0.1 ml of appropriatedilutions of the liquid cultures were streakedonto GTYEA (glucose-tryptone-yeast extract agar,Difco). After incubation at 15°C for 21 -days, theGTYEA plates were examined, and colonies were

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selected for further study. Isolates were examinedfor purity by alternatively transferring inocula intoESWYE broth (estuarine salts solution [4] contain-ing 0.15% [wt/vol] yeast extract) and onto GTYEA.This procedure was repeated three times. Orga-nisms were stored at 5°C on GTYEA slants overlaidwith sterile mineral oil. The sources of the petro-leum-degrading strains used in this study are listedin Table 1.

Bacterial strains isolated from Chesapeake Bayduring October 1972 through September 1973 andreference strains were examined for ability to utilizemixed hydrocarbon substrate (29). After purifica-tion, each isolate was transferred to basal mediumbroth. After incubation for 4 to 8 h at 15°C, ca. 102 to103 cells (final concentration) were added to a 5-mloil-salts broth in a 16- by 125-mm test tube. Eachculture, as well as uninoculated controls (triplicate),was overlayered with sterile 1% (wt/vol) mixed hy-drocarbon substrate. The test culture and controltubes were incubated, without shaking, at 25°C for30 days.On day 30, each culture was mixed thoroughly

with a Vortex mixer and allowed to stand for severalminutes to stabilize the culture, after which 3 mlwas transferred to a cuvette to measure absorbance(at 600 nm) and pH. The transfer pipette was rinsedby drawing up 2 ml of n-hexane and depositing then-hexane in the original culture tube. The 2 ml ofn-hexane was removed, and the original culture wasextracted twice more, each time with 2 ml of n-hexane. After absorbancy and pH were measured,the pH meter probe was rinsed by pipetting 2 ml ofn-hexane into the cuvette. The n-hexane was re-moved, and the culture in the cuvette was extractedwith an additional 2 ml of n-hexane. All portions ofn-hexane were combined, and any remaining waterwas removed by drying over Na2SO4. The anhydrousextracts were transferred to a vial and stored underN2 at -20°C until gas-liquid chromatography analy-sis was accomplished.

Before gas-liquid chromatography analysis, 0.1ml of 1-dodecanol and/or 0.1 ml of indan was addedto each extract, serving as the internal standard.Chromatographs were obtained on a Shimadzumodel GC-4BMPF gas chromatograph equippedwith a single-flame ionization detector. A glass col-umn (3 by 1,500 mm) packed with 3% OV-1 on 80/100-mesh Shimalite was used. The carrier gas, ni-trogen, was run through the column at a rate of 40ml/min. Temperature was programmed from 50 to300°C at 5°C/min, and individual peak areas werequantified by using a Hewlett-Packard model 3373B

TABLE 1. Sources ofpetroleum-degrading strainsincluded in this study

No. of strains isolated from:

Sample type Polluted wa- Unpolluted waterter (Colgate

Creek) Eastern Bay Poole's Island

Water 16 2 0Sediment 54 18 9

integrator. Column efficiency and detector response,measured with a mixture of C14-C20 saturated andunsaturated hydrocarbons (Applied Science mixtureno. 19251), yielded a relative error of less than 2.5%for all components.

Percent hydrocarbon remaining was calculatedusing the following formula, 100 x [(peak area ofhydrocarbon X for culture Y/peak area of hydrocar-bon X for control)/(peak area of internal standardfor culture Y/peak area of internal standard forcontrol)], whereX represents one ofthe 15 hydrocar-bons. The internal standard was 1-dodecanol and/orindan.The ability of the cultures to degrade petroleum

was confirmed as follows. The strains were inocu-lated into 5-ml portions of oil-salts overlaid with 1%(wt/vol) mixed hydrocarbon substrate (29, 30). Afterincubation at 25°C for 28 days, petroleum degrada-tion was recorded as the presence ofgrowth in the oillayer and turbidity in the liquid phase.

In addition, growth in crude oil or the hydrocar-bon mixture was recorded by measuring protein inthe liquid culture by the modified Folin method ofLowry et al. (19). Positive tubes were those contain-ing at least 50 ,ug/ml, a protein concentration signif-icantly higher than that ofthe inoculum. Utilizationof the crude oil or hydrocarbon mixture was deter-mined by measuring weight loss (in milligrams),compared with sterile controls. Thus, utilization ofthe substrate was measured, as well as cell yield, toverify the degradation of hydrocarbon. The cultureswere also examined for lipolytic activity. The resultsof the growth and utilization studies are being pre-pared for publication separately.

Reference strains. In addition to the freshly iso-lated strains of petroleum-degrading bacteria, 33cultures representing a diverse range of taxa wereincluded in the study, serving as reference cultures.These included: Acinetobacter calcoaceticus

(ATCC 15308); A. iwoffi (ATCC 15309); Bacillus cer-eus subsp. mycoides (ATCC 6462); B. subtilis (ATCC6051); Corynebacterium poinsettiae (ATCC 9069); En-terobacter aerogenes (ATCC 13048); Erwinia herbi-cola (ATCC 12287); Escherichia coli (ATCC 11775);Flavobacterium spp., group llb (NCTC 10795) andgroup llf (NCTC 10798); Klebsiella aerogenes(NCTC 8172); Leucothrix mucor (ATCC 25107); Mi-crococcus luteus (ATCC 4698); M. roseus (ATCC418); Moraxelha osloensis (ATCCQ 19976); Nocardiaasteroides (ATCC 14759); N. corallina (ATCC 4273);N. otitidis-caviarum (ATCC 14629); Pseudomonasacidovorans (ATCC 17438); P. aeruginosa (ATCC10145); P. fluorescens (ATCC 13525); P. maltophilia(ATCC 13637); P. pseudoalcaligenes (ATCC 17440);P. putida (ATCC 12633); Rhizobium leguminosarum(ATCC 10004); R. meliloti (ATCC 4399); Serratiamarcescens (ATCC 13880); Sphaerotilus natans(ATCC 15291); Staphylococcus epidermidis (ATCC14990); S. lactis (ATCC 15306); Staphylococcus spp.(ATCC 15365); Streptomyces griseus (ATCC 23345);and Vibrio parahaemolyticus (ATCC 17082).

Numerical taxonomy. Each strain was examinedfor 48-unit characters. All media were inoculatedwith cultures incubated for 5 days at 15°C on

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GTYEA. The taxonomic tests were repeated wheninconclusive results were obtained.Micromorphology and staining reactions. Iso-

lates, grown in ESWYE broth, were examined after18 h. Motility was determined by examination ofwet-mount preparations under a phase-contrast mi-croscope. Gram reaction and cell morphology wererecorded from observations of heat-fixed smearsstained by the Gram reaction (2). The presence ofsoluble, nonsoluble, and fluorescent pigments wasdetermined by examining 24- to 48-h cultures indaylight and under ultraviolet light.

Biochemical characters. Where possible, testswere performed by using the multipoint inoculationprocedure (18).

Catalase activity was determined by the additionof hydrogen peroxide to 24-h cultures, and a positiveresponse was recorded when effervescence of oxygenresulted. Oxidase activity was detected by using themethod of Kovacs (13).

Fermentative and oxidative metabolism of glu-cose was determined by using the method of Hughand Leifson (12). The tubes were examined for acidand gas production at 1, 2, 7, and 14 days afterinoculation. Nitrate and nitrite reduction was re-corded from nitrate broth (Difco), using the methoddescribed by Cowan and Steel (8). Methyl red andVoges-Proskauer tests were performed by usingDifco medium amended with NaCl (1%, wt/vol) andexamined after 5 days by the method described byCowan and Steel (8).

Substrate utilization. Casein hydrolysis was de-termined by using GTYEA medium with skim milk(5%, wt/vol), following the procedure of Sizemoreand Stevenson (23). Gelatin, starch, and Tween 20and Tween 80 hydrolysis were measured accordingto the methods of Frazier (10), Cowan and Steel (8),and Sierra (22), respectively. Tubes containing ureabroth (Difco), to which NaCl (1%, wt/vol) was added,were examined 28 days after inoculation for an alka-line reaction, indicative of urea hydrolysis.

Utilization of substrates as sole carbon sources.The ability to utilize D-arabinose, ethanol, n-fruc-tose, D-galactose, lactose, maltose, sucrose, and D-(+ )-xylose was tested, using basal oxidation-fer-mentation medium (Difco) to which the carbonsources were added to a final concentration of 1%(wt/vol). Acid production was scored as positive.

Utilization of substrate as sole nitrogen source.The utilization ofNH4H2PO4 (1%, wt/vol) was scoredby the presence of an acid reaction.

Coding of data. The characters were coded "1" forpositive or present, "0" for negative or absent, and"9" for noncomparable or not applicable. The final nx t matrix contained 132 strains and 48 characters.Computer analyses. The data were analyzed by

using the simple matching coefficient (26), whichincludes both positive and negative matches, andthe Jaccard coefficient (24), which excludes negativematches. Clustering was achieved by means of theunweighted average linkage method (26), and asorted similarity matrix and dendrogram were con-structed. Programs used included the GTP2 andUMDTAXON 3 programs, written for the IBM 370/165 and the Univac 1108 computers, respectively.

RESULTS

Clustering of strains. A total of 85 (85%) ofthe petroleum-degrading bacteria and 4 refer-ence cultures, K. aerogenes, N. asteroides, N.corallina, and S. natans, were recovered in the12 clusters defined at the 80 to 85% similaritylevel (Fig. 1 and 2). Five clusters (1, 2, 3, 4, and10) comprised 54 strains (54% of the total), withthe remaining 31 strains distributed among 8smaller clusters. Cluster 4 was divided into twosubgroups (4a and 4b) at the 90% similaritylevel (Fig. 1). The composition and shared char-acters of these groups are given in Table 2. Allof the clusters defined by using the SSM (simplematrix) coefficient (Fig. 1) were recognizable inthe analysis prepared with the SJ (Jaccard)coefficient (Fig. 2), but at a lower similaritylevel. It is important when comparing a widespectrum of bacterial types, such as will occurin the natural environment, to be aware of thepossibility of negative matches resulting inhigh similarity values. Thus, the SJ coefficientproved to be more useful in discriminating clus-ters of related strains when ecological conclu-sions were intended. Since the outcome of theSj analysis was similar to that obtained byusing the SsmI coefficient, the full results forboth analyses are not presented here; instead, ashaded diagram, representing a sorted similar-ity matrix obtained from the analysis with theSSM coefficient (Fig. 1), and a simplified dendro-gram of the analysis with the SJ coefficient(Fig. 2) are presented.

Identification and description of the phe-netic groups. Relationships among the groupsof petroleum-degrading bacteria can be seen inFig. 2. The characters of the groups are listed inTable 2. Four phena were readily identified,since the reference cultures were included inthe groups. These were K. aerogenes (phenon7), N. asteroides (phenon 9), N. corallina(phenon 10), and S. natans (phenon 8). Theremaining eight phenetic groups were identi-fied by matching their descriptions with thoseprovided in Bergey's Manual of DeterminativeBacteriology (3) (Table 3). The most numerousof the petroleum-degrading bacteria were thepseudomonads, which were recovered in twophena (4a, 4b, and 5) and accounted for 19% ofthe strains. However, the intergroup similaritywas low (<70%), indicating separate-speciesstatus (6). Seven additional strains (Fig. 2) alsopossessed the general characteristics of Pseu-domonas (27) but could not be assigned to anyof the clusters defined in this study.The actinomycetes and related organisms

comprised the second most numerous group ofpetroleum-degrading bacteria. The mycelial ac-

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64 AUSTIN ET AL.

PHENON PRESUMPTIVEIDENTIFICATION

NO. OFSTRAINS

APPL. ENVIRON. MICROBIOL.

SIMILARITY (%)

Coryneform 9Coryneform

9 * Aiocardio osteroides 210 * N corol/no 11

* N oti/dis - caviorum*Streptomnyces griseus* Corynebocterlum polnset/ioe* Micrococcus luteus* M roseus* Staphylococcus spp.*S oct/is* S epidermrdis

3 Micrococcus spp. 102 Micrococcus spp o_5 Pseudomonos spp 4

* P ocidovorons* P fluorescens

4 b Pseudomonos spp 940 Pseudomonos spp 6

* P ml/tophlio* P pseudoolco/igenesPseuvdomonos sppPseudomonos sppPseudomonos spp.Pseudomonos sppPseudomonas spp.

* P pu/idaPseuldomonas sppPseudomonos sppUnidentified Gram negative rod

* P aeruginoso* Enterobocter oerogenes* Escherichto co/i

7 * Klebsie/la oerogenes 3Enterobacterioceae

6 Enterobacterlaceae 3Unidentif ied Gram negative rod

* Erwinio herbicola* Serrotia marcescens* Leucothrix mucor

13 Actinomycetes (myceliol form) 714 Actinomycetes (mycetial form) 511 Actinomycetes (mycelial form) 312 Actinomycetes (mycelial form) 68 * Sphoeroti/us notaons 2

* Baci/us subti/is* Bocillus cereus var. mycoides* Morose//a os/lensls* Rhizobium leguminosorum* R mo/i/oti* Vibrio parahoemolyticusUnidentified Gram negative rodUnidentified Gram negative rod

* F/avoboc/riuvm spp., Group It b* F/ovoboceriurm spp., Group It f* Acine/obacter /woff* A ca/cooceicus

t00 80 60 40 20* Reference Culture

FIG. 2. Simplified dendrogram, prepared by using the S, coefficient and unweighted average linkagemethod of clustering.

tinomycetes clustered in four phena (Fig. 2),which were all morphologically similar tomembers of the genus Streptomyces. Only N.asteroides and N. corallina were identified atthe species level.Micrococcus isolates, which together com-

prised the third most numerous group of petro-leum-degrading bacteria, were recovered in twoclusters. Although both clusters were distinct,all strains identified as Micrococcus were

spherical in morphology, gram positive, andoxidative in their utilization of glucose.General properties of the petroleum-de-

grading bacteria. Just over half of the strains(63%) were pigmented, and, ofthese, most wereeither orange or yellow, although a few (3%)possessed a red pigmentation on solid medium.The majority of the strains were gram-negativerods, but, of the gram-positive component, 20%were either filmentous or mycelial and 5% were

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TAXONOMY AND ECOLOGY OF PETROLEUM DEGRADERS 65

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66 AUSTIN ET AL.

acid fast. Single cells were most commonly ob-served (60%), and a further 10% were predomi-nantly paired. Motility was observed in 32% ofthe cultures, and, of these, the majority were

gram-negative rods.The biochemical reactions of the petroleum

degraders, as a whole, were not uniform, withmost of the organisms being catalase positive(98%) and capable of reducing nitrate (74%),although only 42% could further reduce nitrite.Approximately half (49%) of the strains metab-olized glucose oxidatively, 26% were fermenta-tive, and the remaining organisms did not growin the test media. Oxidase was produced by20% of the strains, and 35% oxidized ethanol.Some of the carbon sources were utilized by a

wide range of organisms, including fructose(50%), glycerol (51%), and maltose (51%). Incontrast, arabinose (10%), sucrose (28%), andxylose (22%) were utilized by only a restrictedrange of microorganisms. It was observed that,

TABLE 3. Identification ofpetroleum-degradingbacteria, as indicated by relationship to referencestrains and comparison with descriptions given inBergey's Manual ofDeterminative Bacteriology (3)

IsolatesTaxonomic group identified

(%)

Pseudomonas spp....................... 27Enterobacteriaceae ........................ 6Micrococcus spp. ........................ 20Coryneforms ........................ 10Actinomycetes (mycelial forms) ....... ..... 21Nocardia spp. (N. asteroides, N. corallina) . 11S. natans......................... 1Unidentified strains ....................... 4

APPL. ENVIRON. MICROBIOL.

of the compounds tested, only the lipids in theforms of Tween 20 (92%) and Tween 80 (78%)were readily utilized. Organisms showing thesereactions were distributed among many taxa,and no pattern of relatedness based on thesereactions was discernible.The petroleum-degrading bacteria examined

in this study were capable of growth at both lowand high temperatures. At the lower tempera-ture range, 52% produced colonies on agar at5°C, whereas, at 37°C, more than 78% grew; andeven at 43°C, growth occurred in approximately50% of the cultures. Clearly, the petroleum de-graders are capable of tolerating wide tempera-ture fluctuations, well outside the range likelyto occur in the natural environment, i.e., inChesapeake Bay. Only 32% of the isolates grewin the presence of 7% (wt/vol) NaCl. Chesa-peake Bay waters are estuarine, well below thesalinity of seawater, which is ca. 35%o. Thus, a

halotolerant microbial flora would not be ex-

pected, as was observed in the salt tolerancetest.

It is interesting that, of the reference cul-tures, A. calcoaceticus, L. mucor, N. aster-oides, N. corallina, N. otitidis-caviarum, P.aeruginosa, R. leguminosarum, and R. meli-loti utilized a mixed hydrocarbon substrate invitro and that representatives of two taxa (N.asteroides and N. corallina) were associatedwith a similar role in Chesapeake Bay.

Distribution of phenetic groups with re-

spect to geographical location. A high propor-

tion of the phenetic groups were each specific toa given sampling station within a single geo-

graphical site and, more specifically, to the sed-iment at that station (Table 4). Ofthe 14 phena,

TABLE 4. Distribution ofphenetic groups among the three geographical locations and two sample types

Phenon Percentage of strains included in the study

Colgate Creek Eastern Bay Poole's IslandNo. No. of strains Name

Water Sediment (sediment) (sediment)

1 9 Coryneform 33 45 22 0

2 10 Micrococcus spp. 0 100 0 03 10 Micrococcus spp. 0 50 0 504a 6 Pseudomonas spp. 0 100 0 04b 9 Pseudomonas spp. 0 66 23 115 4 Pseudomonas spp. 0 100 0 0

Enterobacteriaceae 33 67 0 07 3 K. aerogenes 0 100 0 08 2 S. natans 0 100 0 09 11 N. asteroides 0 0 100 010 3 N. corallina 0 70 30 0

11 3 Mycelial actinomycetes 0 53 67 0

12 6 Mycelial actinomycetes 33 17 33 1713 7 Mycelial actinomycetes 0 70 30 014 5 Mycelial actinomycetes 0 80 20 0

Total no. of phena 3 13 8 3

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TAXONOMY AND ECOLOGY OF PETROLEUM DEGRADERS

6 were specific to the sediment ofeither ColgateCreek or Eastern Bay. Another 5 phena wereisolated from sediment samples collected atmore than one station, for example, Micrococ-cus spp., N. corallina, and three clusters ofmycelial actinomycetes. No clusters were spe-cific to water, although strains of coryneforms,Enterobacteriaceae, and mycelial actinomy-cetes were present in the water samples col-lected from Colgate Creek. Neither water col-lected at Eastern Bay nor that collected atPoole's Island contributed petroleum degraderssubsequently recovered in the clusters. It isinteresting to note that Colgate Creek, the pol-luted station, yielded the widest range of bacte-rial taxa containing petroleum-degradingstrains, i.e., 13 phena, whereas the EasternBay and Poole's Island samples contained rep-resentatives of 8 and 3 phena, respectively.

DISCUSSION

In a preliminary report published elsewhere(1), microorganisms growing on an oil-basedmedium were purified and subjected to numeri-cal taxonomy analysis. In this report, a morerigorous definition of petroleum degradationwas applied, and those strains clearly utilizingoil or petroleum hydrocarbons for growth weresubjected to taxonomic analysis. From the re-sults reported in this study, it is concluded, asin the earlier study, that degradation of petro-leum in the aquatic environment is accom-plished by a wide range of bacterial taxa. Manyof the taxa described here have also been iso-lated from water and sediment samples col-lected in Chesapeake Bay in the earlier studiescarried out in our laboratory. Unusual divers-ity in the composition of petroleum biode-graders from a single habitat was not found.Pseudomonas spp. are commonly recognized

as being capable of degrading hydrocarbons. Itis not surprising, therefore, that a number ofPseudomonas strains capable of degrading pe-troleum should be isolated from ChesapeakeBay, particularly from areas receiving petro-leum in waste discharges. Pseudomonas spp.have been noted to be competent in degradingn-paraffins (11, 16). Unfortunately, all too oftenthe methods used to identify petroleum-degrad-ing bacteria are not reported in sufficient detailin the literature. Polyakova, in 1962 (21), usedthe keys of Krasil'nikov (14) to conclude thatPseudomonas spp. were the most numerous ofthe hydrocarbon-utilizing bacteria, and thesespecies were reported to occur in such remotesites as Neva Bay in the U.S.S.R.The actinomycetes, which include coryne-

forms, Nocardia species, as well as the mycelialforms, have been recognized as petroleum de-graders (9, 11, 19, 21); yet, it appears that theseorganisms are less abundant in ChesapeakeBay water and sediment than would be ex-pected from studies carried out in other naturalenvironments. Mycobacterium spp., in particu-lar, have been reported to be capable of degrad-ing petroleum (11, 21), yet none were isolatedfrom Chesapeake Bay water or sediment in thecourse of these studies. No doubt, more specificenrichment procedures will be needed to isolatemycobacteria from natural waters if they are,indeed, present. It is noteworthy that N. coral-lina, strains of which were recorded in thisstudy, have been reported to degrade aromaticcompounds, for example, benzyl alcohol (11). Inthe natural environment, however, it appearsthat such degradation is temperature depend-ent (20).Micrococcus spp. comprised the third most

numerous group of petroleum degraders identi-fied in this study. Micrococci can degrade C7 toC,8 chain hydrocarbons (9, 11, 21). In fact, thereseems to be some degree of specificity in thetypes of hydrocarbons degraded by given bacte-rial species, and it is conceivable that differenttaxa may degrade certain components of theoil, with the overall effect being a measurabledegradation of a crude oil or similar petroleumsubstrate (20). However, the specific roles ofindividual taxa in the overall process of petro-leum degradation remain to be defined morefully.Few studies have suggested an ecologically

important role for the Enterobacteriaceae, ex-cept in connection with plant or animal patho-genic processes. Yet, in this study, K. aero-genes and an unidentified species of the Entero-bacteriaceae were isolated and subsequentlyfound to utilize petroleum. It is not surprising,however, that members of the Enterobacteria-ceae capable of degrading petroleum shouldhave been recovered in large numbers fromChesapeake Bay at a site receiving domesticsewage effluent.Apart from one report (21) that Pseudomonas

and Mycobacterium spp. were more numerousthan 6ther taxa in the aquatic environment, atleast with respect to their ability to degradehydrocarbons, it was not until 1974 that it wassuggested that certain petroleum-degradingbacteria may be specific to given ecologicalniches (20). In the latter case, bacteria capableof degrading petroleum that had been isolatedfrom Atlantic Ocean water and sediment werepredominantly pseudomonads. The pseudomo-nads were further differentiated into subgroups

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68 AUSTIN ET AL.

showing specificity for type of sample, i.e., wa-ter or sediment.The identification and classification of petro-

leum-degrading bacteria reported here are be-ing further studied in our laboratory. Severalrepresentatives, including the hypothetical me-dian organisms (17) of each of the taxa identi-fied on the basis of the characters and computa-tions used in this study, have been selected forfurther analysis by numerical taxonomy. Thesestrains have been subjected to more extensivetesting so that a polythetic taxonomic analysis(5) can be done. Thus, a more precise descrip-tion of the species of bacteria found to be capa-ble of degrading petroleum and, hopefully,probabilistic identification matrices (15) can beprepared to assist other investigators workingon problems of microbial degradation of petro-leum.

ACKNOWLEDGMENTSThis research was supported by National Science Foun-

dation Grants BMS-72-02227-A03 and OCE-75-02635-AO1and Contract no. N00014-75-C-0340 between the Office ofNaval Research, Department of the Navy, and the Univer-sity of Maryland. The computer time for this project wassupported through the facilities of the Computer ScienceCenter of the University of Maryland.

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