Microbiol Rev. 1981 Atlas 180 209

1981, 45(1):180. Microbiol. Rev.

R M Atlas environmental perspective.petroleum hydrocarbons: an Microbial degradation of

onhttp://mmbr.asm.org/content/45/1/180.citatifound at: Updated information and services can be

These include:

CONTENT ALERTS

more»alerts (when new articles cite this article), Receive: RSS Feeds, eTOCs, free email

http://mmbr.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:

on February 29, 2012 by guest

http://mm

br.asm.org/

Dow

nloaded from

http://mmbr.asm.org/cgi/alerts

http://mmbr.asm.org/

MICROBIOLoGICAL REVIEWS, March 1981, p. 180-209 Vol. 45, No. 10146-0749/81/010180-30$02.00/0

Microbial Degradation of Petroleum Hydrocarbons: anEnvironmental Perspective

RONALD M. ATLASDepartment of Biology, University of Louisville, Louisville, Kentucky 40292

INTRODUCTION 180CHEMISTRY OF PETROLEUM BIODEGRADATION . 180

Degradation of Individual Hydrocarbons ...... 180Degradation of Hydrocarbons Within Petroleum Mixtures ..... 182

TAXONOMIC RELATIONSHIPS OF HYDROCARBON-UTILIZING MICRO-ORGANISMS .............................. ... .. 184

DISTRIBUTION OF HYDROCARBON-UTILIZING MICROORGANISMS ...... 186ENVIRONMENTAL FACTORS INFLUENCING BIODEGRADATION OFPETCROLEUM HYDROCARBONS ........................................... 189

PHYSICAL STATE OF OIL POLLUTANTS ................................... 189TEMPERATURE .................................................. 190NUTRIENTlS ..........192.............................192OXYGEN .......... 194SALINITY AND PRESSURE .................... 195CASE HISTORIES ..................... 196CONCLUSIONS ..................... 198LITERATURE CITED .................... 199

INTRODUCTIONIn 1946, Claude E. ZoBell reviewed the action

of microorganisms on hydrocarbons (298). Herecognized that many microorganisms have theability to utilize hydrocarbons as sole sources ofenergy and carbon and that such microorga-nisms are widely distributed in nature. He fur-ther recognized that the microbial utilization ofhydrocarbons was highly dependent on thechemical nature of the compounds within thepetroleum mixture and on environmental deter-minants.Twenty-one years after ZoBell's classic re-

view, the supertanker Torrey Canyon sank inthe English Channel. With this incident, theattention of the scientific community was dra-matically focused on the problems of oil pollu-tion. After this event, several studies were initi-ated on the fate of petroleum in various ecosys-tems. The expansion of petroleum developmentinto new frontiers, such as deep offshore watersand ice-dominated Arctic environments, and theapparently inevitable spillages which occur dur-ing routine operations and as a consequence ofacute accidents have maintained a high researchinterest in this field.The chemically and biologically induced

changes in the composition of a polluting petro-leum hydrocarbon mixture are known collec-tively as weathering. Microbial degradationplays a major role in the weathering process.Biodegradation of petroleum in natural ecosys-tems is complex. The evolution of the hydrocar-

bon mixture depends on the nature of the oil, onthe nature of the microbial community, and ona variety of environmental factors which influ-ence microbial activities.

Attention has been focused on marine envi-ronments since the world's oceans are the largestand ultimate receptors of hydrocarbon pollu-tants. Most previous reviews concerning the mi-crobiology of petroleum pollutants have beenconcerned with the marine environment (6, 15,24, 34, 76, 87, 107-110, 161a, 165, 209, 267, 298,300, 301, 304). This review expands the scope toinclude consideration of the fate of petroleumhydrocarbons in freshwater and soil ecosystems.It also discusses several case histories relevantto the role of microbial degradation in determin-ing the fate of petroleum pollutants from majoroil spills.

CHEMISTRY OF PETROLEUMBIODEGRADATION

Degradation of Individual HydrocarbonsPetroleum is an extremely complex mixture of

hydrocarbons. From the hundreds of individualcomponents, several classes, based on relatedstructures, can be recognized. The petroleummixture can be fractionated by silica gel chro-matography into a saturate or aliphatic fraction,an aromatic fraction, and an asphaltic or polarfraction (48). Several studies have been per-formed to determine the metabolic pathways fordegradation of these compounds, and there havebeen a number of reviews on this subject (103,

180


http://mm

br.asm.org/

Dow

nloaded from


VOL. 45, 1981 BIODEGRADAT]

112, 113, 122, 123, 144, 190, 198, 209, 216, 217,222, 237, 259, 266, 268).Hydrocarbons within the saturate fraction in-

clude n-alkanes, branched alkanes, and cycloal-kanes (naphthenes). The n-alkanes are generallyconsidered the most readily degraded compo-nents in a petroleum mixture (91, 169, 197, 265,298). Biodegradation of n-alkanes with molecu-lar weights up to n-C44 have been demonstrated(137). The biodegradation of n-alkanes normallyproceeds by a monoterminal attack; usually aprimary alcohol is formed followed by an alde-hyde and a monocarboxylic acid (112, 113, 198,201, 228, 268, 270, 299). Further degradation ofthe carboxylic acid proceeds by fl-oxidation withthe subsequent formation of two-carbon-unitshorter fatty acids and acetyl coenzyme A, witheventual liberation of C02. Fatty acids, some ofwhich are toxic, have been found to accumulateduring hydrocarbon biodegradation (16, 172).Omega (diterminal) oxidation also has been re-ported (162). Subterminal oxidation sometimesoccurs, with formation of a secondary alcoholand subsequent ketone, but this does not appearto be the primary metabolic pathway utilized bymost n-alkane-utilizing microorganisms (190). Anew pathway recently was elucidated by W. R.Finnerty (personal communication), who foundthat an Acinetobacter species can split a hydro-carbon at the number 10 position, forming hy-droxy acids. The initial steps appear to involveterminal attack to form a carboxylic acid, sub-terminal dehydrogenation at the number 10 po-sition to form an unsaturated acid, and splittingof the carbon chain to form a hydroxy acid andan alcohol.Highly branched isoprenoid alkanes, such as

pristane, have been found to undergo omegaoxidation, with formation of dicarboxylic acidsas the major degradative pathway (199, 222,223). Methyl branching generally increases theresistance of hydrocarbons to microbial attack(105, 197, 222, 241). Schaeffer et al. (241), forexample, found that terminal branching inhibitsbiodegradation of hydrocarbons. Methylbranching at the beta position (anteiso-termi-nus) blocks ,f-oxidation, requiring an additionalstrategy, such as alpha oxidation (35, 188), om-ega oxidation (222), or beta alkyl group removal(57, 248).Cycloalkanes are particularly resistant to mi-

crobial attack (103, 213, 217, 259, 266). Complexalicycic compounds, such as hopanes (tripenta-cyclic compounds), are among the most persist-ent components of petroleum spillages in theenvironment (18). There have been several re-ports of the direct oxidative and co-oxidativedegradation of both substituted and unsubsti-tuted cycloalkanes. The microbial metabolism

ION OF PETROLEUM HYDROCARBONS 181

of cyclic hydrocarbons and related compoundshas been reviewed by Perry (27). Up to six-membered condensed ring structures have beenreported to be subject to microbial degradation(73, 281). Several unsubstituted cycloalkanes,including condensed cycloalkanes, have been re-ported to be substrates for co-oxidation withformation of a ketone or alcohol (35-37, 217).Once oxygenated, degradation can proceed withring cleavage. Degradation of substituted cy-cloalkanes appears to occur more readily thanthe degradation of the unsubstituted forms, par-ticularly if there is an n-alkane substituent ofadequate chain length (217, 256). In such cases,microbial attack normally occurs first on thesubstituted portion, leading to an intermediateproduct of cyclohexane carboxylic acid or a re-lated compound. A novel pathway for the deg-radation of cylohexane carboxylic acid involvesformation of an aromatic intermediate (217) fol-lowed by cleavage of the aromatic ring structure.The degradation of aromatic hydrocarbons

has been reviewed by Gibson and others (85,122, 123, 125-127, 144, 237). The bacterial deg-radation of aromatic compounds normally in-volves the formation of a diol followed by cleav-age and formation of a diacid such as cis,cis-muconic acid. In contrast, oxidation of aromatichydrocarbons in eucaryotic organisms has beenfound to form a trans-diol. For example, fungihave been shown to oxidize naphthalene to formtrans- 1,2 - dihy _y- 1, 2 -dihydronaphthalene(59, 60, 66, 106). The results indicate that onlyone atom of molecular oxygen is incorporatedinto the aromatic nucleus, as has been found formammalian aryl hydrocarbon hydroxylase sys-tems. Cerniglia and Gibson (62,63) and Cernigliaet al. (65, 66) have investigated the fungal oxi-dation of polynuclear aromatic hydrocarbons.They found evidence for formation of trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene by Cun-ninghamelUa elegans from the oxidation ofbenzo(a)pyrene. Cerniglia and Gibson (61) andCerniglia et al. (64, 68, 69) also investigated themetabolism of naphthalene by cyanobacteria.They found that naphthalene was oxidized inthe light but not in the dark. Scenedesmusstrains also were shown to utilize n-heptadecanein the light (mixotrophic growth), but were un-able to utilize this alkane in the dark (191). Themajor product formed by Agmenellum and Os-cillatoria species was 1-naphthol (61). Theseorganisms also formed cis-1,2-dihydroxy-1,2-dihydronaphthalene and 4-hydroxy-1-tetralene(68). These results suggest that cyanobacteriahave a variety of mechanisms for initiating theoxidation of naphthalene. The Oscillatoria spe-cies also has been found to oxidize biphenyl,indicating that a wider range of aromatic hydro-


http://mm

br.asm.org/

Dow

nloaded from


182 ATLAS

carbons are subject to oxidation by cyanobac-teria (69).

Light aromatic hydrocarbons are subject toevaporation and to microbial degradation in adissolved state (163, 164). Extensive methyl sub-stitution can inhibit initial oxidation (18, 85).Initial enzymatic attack may be on the alkylsubstituent or, alternatively, directly on the ring(123). Condensed ring aromatic structures aresubject to microbial degradation by a similarmetabolic pathway as monocycic structures (85,96, 124, 302); condensed ring aromatic hydrocar-bons, however, are relatively resistant to enzy-matic attack; for example, Lee and Ryan (180)found that biodegradation rates were over 1,000times higher for naphthalene than for benzopy-renes. Structures with four or more condensedrings have been shown to be attacked, in somecases, by co-oxidation or as a result of commen-salism (30, 85, 124, 275, 284, 286).The metabolic pathways for the degradation

of asphaltic components of petroleum are prob-ably least well understood. These are complexstructures which are difficult to analyze withcurrent chemical methodology. The degradationof various sulfur-containing components of pe-troleum has been examined (151, 176, 208, 286,296), but no uniform degradative pathway, com-parable to the pathways established for aliphaticand aromatic hydrocarbons, has yet emerged forthe asphaltic petroleum components. Advancesin determining degradative pathways for as-phaltic petroleum components are dependent onimproved chemical analytical methodology. Theelucidation of the biochemical fate of asphalticpetroleum compounds is a major challenge forfuture research on petroleum biodegradation.Another important future research need in-

volves determining the importance in the envi-ronment of the various pathways for hydrocar-bon biodegradation. It is clear that various bio-chemical strategies exist for the microbial utili-zation of petroleum hydrocarbons. What re-mains to be done is to detect intermediate prod-ucts in natural environments that receive petro-leum hydrocarbons to determine which path-ways are actively used by microbial populationsin natural ecosystems. It is likely, but as yetunproven, that different pathways will be activeunder different conditions, e.g., at different hy-drocarbon concentrations.

Degradation of Hydrocarbons WithinPetroleum Mixtures

The qualitative hydrocarbon content of thepetroleum mixture influences the degradabilityof individual hydrocarbon components. Walkeret al. (287) examined the susceptibility to micro-

MICROBIOL. REV.

bial degradation of hydrocarbons in weatheredcrude and fuel oils. They reported far less deg-radation in a heavy no. 6 fuel oil (Bunker C oil)than in a light no. 2 fuel oil (heating oil anddiesel fuel) and less degradation in a heavy Ku-wait crude oil than in a light south Louisianacrude oil. They reported major differences in thesusceptibility to degradation of each of the com-ponents (identical compounds) within the con-text of the different hydrocarbon mixtures of theoils tested.

Mulkins-Phillips and Stewart (206) found thatn-alkanes within a Venezuelan crude oil weredegraded less than the same n-alkanes within anArabian crude oil. Westlake et al. (292) exam-ined the effect of crude oil composition on petro-leum biodegradation. The ability of mixed mi-crobial populations to utilize the hydrocarbonsin four crude oils as the sole carbon source wasfound to depend not only on the composition ofthe unsaturated fraction but also on that of theasphaltic fraction. Using an oil which lacked anormal n-alkane component, they demonstratedthat the aromatic fraction of oil was capable ofsustaining bacterial growth. Horowitz et al. (148)used the technique of sequential enrichment toisolate organisms which could utilize progres-sively more complex (i.e., resistant to microbialdegradation) compounds.

Several investigators have examined the po-tential activities of hydrocarbon-degrading bac-teria by using 14C-radiolabeled hydrocarbons.Caparello and LaRock (58) described an enrich-ment method for quantifying the activity of hy-drocarbon-oxidizing bacteria in water and sedi-ment that used ['4C]hexadecane. They foundthat the hydrocarbon-oxidizing potential of en-vironmental samples reflects the hydrocarbonburden of the area and the ability of the indig-enous microorganisms to utilize hydrocarbons.Walker and Colwell (279) observed that rates ofmineralization were greater for hexadecane thanfor naphthalene, which were greater than thosefor toluene, which were greater than those forcyclohexane. Greater rates of uptake and min-eralization were observed for bacteria and sam-ples collected from an oil-polluted harbor thanfor samples from a relatively unpolluted region.They reported turnover times of 15 and 60 minfor the polluted and unpolluted areas, respec-tively, using ['4C]hexadecane. Roubal and Atlas(239) found that biodegradation potentials fol-low the order hexadecane > naphthalene >>pristane > benzanthracene. Lee (179) found thatalkanes and low-molecular-weight aromatics(benzene, toluene, naphthalene, and methyl-naphthalene) were degraded to C02 by micro-organisms in river water, but that higher-molec-ular-weight aromatics were relatively resistant


http://mm

br.asm.org/

Dow

nloaded from


BIODEGRADATION OF PETROLEUM HYDROCARBONS 183

to microbial degradation. Herbes and Schwall(140) found that polyaromatic hydrocarbonturnover times in petroleum-contaminated sed-iments increased from 7.1 h for naphthalene to400 h for anthracene, 10,000 h for benz(a)-anthracene, and more than 30,000 h for benz(a)-pyrene. Polynuclear aromatic compoundstended to be only partially, rather than com-pletely, degraded to C02.Two processes which need be considered in

the metabolism of petroleum hydrocarbons areco-oxidation and sparing. Both processes canoccur within the context of a petroleum spillage.LePetit and Tagger (186), for example, foundthat acetate, an internediate product in hydro-carbon biodegradation, reduced the utilizationof hexadecane. A diauxic phenomenon has beenreported for the degradation of pristane, inwhich pristane was not degraded in the presenceof hexadecane (223). The basis for this sparingeffect was not defined, and it is not knownwhether this is an example of classical cataboliterepression. Similar sparing effects undoubtedlyoccur for other hydrocarbons. Such diauxic phe-nomena do not alter the metabolic pathways ofdegradation, but rather determine whether theenzymes necessary for metabolic attack of aparticular hydrocarbon are produced or active.These sparing effects have a marked influenceon the persistence of particular hydrocarbonswithin a petroleum mixture and thus on theevolution of the weathered petroleum hydrocar-bon mixture.The phenomenon of co-oxidation has been

referred to several times in this section. Com-pounds which otherwise would not be degradedcan be enzymatically attacked within the petro-leum mixture due to the abilities of the individ-ual microorganisms to grow on other hydrocar-bons within the oil (150). A petroleum hydrocar-bon mixture, with its multitude of potential pri-mary substrates, provides an excellent chemicalenvironment in which co-oxidation can occur.Many complex branched and cyclic hydrocar-bons undoubtedly are removed as environmen-tal contaminants after oil spills as a result of co-oxidation (217, 230, 231). Jamison et al. (155)found that the degradation of hydrocarbonswithin a high-octane gasoline was not in agree-ment with the degradation of individual hydro-carbons by pure cultures. They concluded thatco-oxidation played a major role in the degra-dation of the hydrocarbon mixture within gaso-line. Horowitz and Atlas (145) found, using chro-matographic and mass spectral analysis, thatresidual oils recovered after exposure in Arcticcoastal waters contained similar percentages ofthe individual components in classes of hydro-carbons regardless ofthe amount of degradation,

indicating that most hydrocarbon componentsof the oil were being degraded at similar rates.This study is in contrast to most, which showpreferential utilization of n-alkane hydrocar-bons. Co-oxidation was hypothesized to be re-sponsible for the degradation of a number ofcompounds in the oil to account for the similarrates of disappearance of compounds which arenormally easily degraded and those which arenornally resistant. Herbes and Schwall (140)also postulated that co-oxidation led to the ac-cumulation of relatively large amounts of par-tially oxidized products of polynuclear aromatichydrocarbon degradation in sediments and onlylimited amounts of CO2 production. Assessingthe role of co-oxidation in natural environmentsis difficult since multiple microbial populationsare present. In the above-mentioned mixed-pop-ulation studies, synergism could be an alterna-tive hypothesis to explain the observed results.Future studies are needed to clarify the role ofco-oxidation in determining the fate of petro-leum hydrocarbons in natural ecosystems.An interesting and as yet unexplained, but

consistent, process which occurs during the bi-odegradation of petroleum hydrocarbons is theenrichment of compounds within the "unre-solved envelope" which occurs during gas chro-matographic analysis of petroleum hydrocar-bons. This envelope is due to a mixture of severalcompounds which are not resolved into individ-ually defined peaks even by glass capillary gaschromatography. Since these compounds cannotcurrently be analytically resolved, they cannotbe identified. It has been hypothesized that,during the biodegradation of petroleum hydro-carbons, microorganisms are producing (synthe-sizing) hydrocarbons of different molecularweights or chemical structures. Walker and Col-well (278) found that a wax was produced duringmicrobial degradation of Altamont crude oil butnot during abiotic weathering of oil. The high-boiling n-alkanes in the wax were associatedwith microbial degradation of the oil and ap-peared to be similar to components of tar ballsfound in the open ocean. The possible produc-tion of such high-molecular-weight alkanes dur-ing petroleum biodegradation also has been re-ported by several other investigators (226, 245).The biochemical mechanism for formation ofsuch hydrocarbons during petroleum biodegra-dation is unknown. Sexstone et al. (251, 252)found that oil biodegradation in tundra soils wasaccompanied by accumulation of polar lipoidalcompounds in the soil column that were notpresent in fresh oil and were not detected inunoiled soils; the identities of the compounds,however, were not determined. Jobson et al.(158) reported an increase in the polar nitrogen-

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


MICROBIOL. REV.

sulfur-oxygen fraction during oil biodegradationin soil.The role of microorganisms in producing com-

plex products from hydrocarbon metabolismwhich may persist in the environment requiresfurther investigation. Of particular importanceis the possible involvement of microorganisms inthe formation of tar balls. The synthesis of com-plex high-molecular-weight hydrocarbons wouldsuggest that microorganisms can play a role inprolonging the impact of petroleum pollutantsas well as in abating the impact of such environ-mental contaminants through biodegradative re-moval. It is difficult to separate the importanceof photochemical and biochemical processes inthe formation ofoxygenated and polymeric com-pounds in the environment. It is apparent thatthe fate of the component hydrocarbons in amixture of the complexity of petroleum is ex-tremely complicated and requires further re-search efforts.

TAXONOMIC RELATIONSHEPS OFHYDROCARBON-UTILIZING

MICROORGANISMS

The ability to degrade petroleum hydrocar-bons is not restricted to a few microbial genera;a diverse group of bacteria and fungi have beenshown to have this ability. ZoBell (298) in hisreview noted that more than 100 species repre-senting 30 microbial genera had been shown tobe capable of utilizing hydrocarbons. In a pre-vious review, Bartha and Atlas (34) listed 22genera of bacteria, 1 algal genus, and 14 generaof fungi which had been demonstrated to containmembers which utilize petroleum hydrocarbons;all of these microorganisms had been isolatedfrom an aquatic environment. The most impor-tant (based on frequency of isolation) genera ofhydrocarbon utilizers in aquatic environmentswere Pseudomonas, Achromobacter, Arthro-bacter, Micrococcus, Nocardia, Vibrio, Acine-tobacter, Brevibacterium, Corynebacterium,Flavobacterium, Candida, Rhodotorula, andSporobolomyces (34). Bacteria and yeasts ap-pear to be the prevalent hydrocarbon degradersin aquatic ecosystems. In polluted freshwaterecosystems, bacteria, yeasts, and filamentousfungi all appear to be important hydrocarbondegraders (81). Jones and Eddington (161) foundthat isolates representing 11 genera of fungi and6 genera ofbacteria were the dominant microbialgenera responsible for hydrocarbon oxidation insoil samples. They found that fungi played animportant role in the hydrocarbon-oxidizing ac-tivities of the soil samples. Cerniglia and Perry(67) found that several fungi (Penicillium andCunninghamella spp.) exhibited greater hydro-

carbon biodegradation than bacteria (Flavobac-terium, Brevibacterium, andArthrobacter spp.).Recent studies continue to expand the list ofmicrobial species which have been demonstratedto be capable of degrading petroleum hydrocar-bons. In one such study, Davies and Westlake(92) examined 60 fungal isolates for their abilityto grow on n-tetradecane, toluene, naphtha-lene, and seven crude oils of various composi-tions. Forty cultures, including 28 soil isolates,could grow on one or more of the crude oils. Thegenera most frequently isolated from soils werethose producing abundant small conidia, e.g.,Penicillium and Verticillium spp. Oil-degradingstrains of Beauveria bassiana, Mortieriellaspp., Phoma spp., Scolecobasidium obovatum,and Tolypocladium inflatum also were isolated.Walker et al. (272) compared the abilities of

bacteria and fungi to degrade hydrocarbons. Thefollowing genera were included in their study:Candida, Sporobolomyces, Hansenula, Aureo-basidium, Rhodotorula, Cladosporium, Peni-cillium, Aspergillus, Pseudomonas, Vibrio, Aci-netobacter, Leucothrix, Nocardia, and Rhizo-bium. Bacteria and yeasts showed decreasingabilities to degrade alkanes with increasing chainlength. Filamentous fungi did not exhibit pref-erential degradation for particular chain lengths.Patterns of degradation, i.e., which hydrocar-bons could be utilized, were similar for bacteriaand fungi, but there was considerable variabilityamong individual isolates.Komagata et al. (177) examined almost 500

yeasts for their ability to degrade hydrocarbonsand found 56 that could utilize hydrocarbons,almost all of which were in the genus Candida.The fernentation industry has considered usinghydrocarbon-utilizing Candida species for pro-ducing single-cell protein. Ahearn and co-work-ers (2, 79) have examined yeasts that can utilizehydrocarbons and have isolated strains of Can-dida, Rhodosporidium, Rhodotorula, Saccha-romyces, Sporobolomyces, and Trichosporon,which are capable of doing so. Cladosporiumresinae has been isolated from soil (82, 273) andhas repeatedly been found as a contaminant ofjet fuels (29, 80, 142, 143). The organism cangrow on petroleum hydrocarbons and createsproblems in the aircraft industry by clogging fuellines.Nyns et al. (210) examined the "taxonomic

value" of the property of fungi to assimilatehydrocarbons, i.e., whether the ability of fungito utilize hydrocarbons was a useful diagnostictest for defining different fungal genera or spe-cies. They found that the ability to utilize hy-drocarbons occurred mainly in two orders, theMucorales and the Monilales. They found thatAspergillus and Penicillium are rich in hydro-

184 ATLAS


http://mm

br.asm.org/

Dow

nloaded from



carbon-assimilating strains. They concludedthat the property of assimilating hydrocarbonsis relatively rare and that it is a property ofindividual strains and not necessarily a charac-teristic of particular species or related taxa.Lianos and Kjoller (187) examined changes infungal populations in soil after oil waste appli-cation. They found that oil application favoredgrowth of Graphium and Paecilomyces. In theirstudy, strains of Graphium, Fusarium, Penicil-lium, Paecilomyces, Acremonium, Mortierella,Gliocladium, Trichoderma, and Sphaeropsi-dales were found to be important groups of soilfungi capable of utilizing crude oil hydrocarbons.In a similar study, Jensen (157) studied thebacterial flora of soil after application of oilywaste and found that the most important speciesof oil degraders belonged to the genera Arthro-bacter and Pseudomonas.

Cundell and Traxler (89) studied 15 isolatesfrom an asphaltic flow near a natural seepage atCape Simpson, Alaska. The isolates were psy-chrotrophic and utilized paraffinic, aromatic,and asphaltic petroleum components. The iso-lates belonged to the bacterial genera Pseudom-onas, Brevibacterium, Spirillum, Xantho-monas, Alcaligenes, and Arthrobacter. Innorthwest Atlantic coastal waters and sediment,Mulkins-Phillips and Stewart (205) reportedfinding hydrocarbon-utilizing bacteria of thegenera Nocardia, Pseudomonas, Flavobacter-ium, Vibrio, and Achromobacter.Walker et al. (285) isolated Vibrio, Pseudom-

onas, and Acinetobacter species from oil-con-taminated sediment and Pseudomonas and cor-yneform species from oil-free sedinent. Micro-organiss from the oil-free sediment producedgreater quantities of polar compounds (asphal-tics) after degradation, whereas microorganismsfrom the oil-contaminated sediment providedgreater degradation of saturated and aromatichydrocarbons. Walker et al. (283) also examinedbacteria from water and sediment for their abil-ity to degrade petroleum. Water samples con-tained a greater variety of bacterial species ca-pable of degrading petroleum than sedimentsamples. Cultures from both water and sedimentcontained Pseudomonas and Acinetobacter spe-cies. Bacteria present in the water samplesyielded significantly greater degradation oftwo-, three-, four-, and five-ring cycloalkanesand mono-, di-, tri-, tetra-, and penta-aromaticscompared with bacteria from sedinent samples.Both temperature and chemical composition

ofa crude oil have been shown to have a selectiveinfluence on the genera of hydrocarbon utilizers.Cook and Westlake (78) isolated Achromobac-ter, Alcaligenes, Flavobacterium, and Cyto-phaga at 40C on a substrate of Prudhoe Bay

crude oil; Acinetobacter, Pseudomonas, and un-identified gram-negative cocci at 40C on a sub-strate of Atkinson Point crude oil; Flavobacte-rium, Cytophaga, Pseudomonas, and Xantho-monas at 4°C with Norman Wells crude oil assubstrate; and Alcaligenes and Pseudomonason Lost Horse crude oil at 4°C. At 300C, themajor genera isolated on Prudhoe Bay crude oilwere Achromobacter, Arthrobacter, and Pseu-domonas; on Atkinson Point crude oil, the majorgenera were Achromobacter, Alcaligenes, andXanthomonas; on Norman Wells crude oil, themajor genera were Acinetobacter, Arthrobacter,Xanthomonas, and other gram-negative rods;and on Lost Horse crude oil, they were Achro-mobacter, Acinetobacter, and Pseudomonas.

Several thermophilic hydrocarbon-utilizingbacteria have been isolated, including species ofThermomicrobium and other, yet unidentifiedgenera (200). Both gram-negative and gram-pos-itive thermophilic bacteria have been demon-strated to be capable of hydrocarbon utilization.Some isolated thermophiles are obligate hydro-carbon utilizers and cannot grow on other carbonsources. The possible existence of obligate hy-drocarbon utilizers is intriguing but perplexing,since the biochemical degradative pathways in-dicate that hydrocarbon utilizers must also becapable of metabolizing fatty acids and alcohols.A large number of Pseudomonas species have

been isolated which are capable of utilizing pe-troleum hydrocarbons. The genetics and enzy-mology of hydrocarbon degradation by Pseu-domonas species has been extensively studied(70, 71, 104, 116, 294). The genetic informationfor hydrocarbon degradation in these organismsgenerally has been found to occur on plasmids.Pseudomonas species have been used for geneticengineering, and the first successful test case inthe United States to determine whether geneti-cally engineered microorganisms can be pat-ented involved a hydrocarbon-utilizing Pseu-domonas which was "created" by Chakrabarty(98).Numerical taxonomy has been used to exam-

ine petroleum degrading bacteria (26,27). Austinet al. (26) examined 99 strains of petroleum-degrading bacteria, isolated from ChesapeakeBay water and sediment, by numerical taxon-omy procedures. Eighty-five percent of the pe-troleum-degrading bacteria examined in thisstudy were defined at the 80 to 85% similaritylevel within 14 phenetic groups. The groups wereidentified as actinomycetes (mycelial forms, fourclusters), coryneforms, Enterobacteriaceae,Klebsiella aerogenes, Micrococcus spp. (twoclusters), Nocardia spp. (two clusters), Pseu-domonas spp. (two clusters), and Sphaerotilusnatans. It was concluded that degradation of

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


MICROBIOL. REV.

petroleum is accomplished by a diverse range ofbacterial taxa. Of particular note was the findingthat some enteric bacteria can utilize petroleumhydrocarbons; the suggestion has been madethat some of these enteric bacteria may haveacquired this ability through plasmid transfer.Some cyanobacteria and algae have been

found to be capable ofhydrocarbon degradation.Walker et al. (282) described a hydrocarbon-utilizing achlorophyllous strain of the alga Pro-totheca. Cerniglia et al. (64) tested nine cyano-bacteria, five green algae, one red alga, onebrown alga, and two diatoms for their ability tooxidize naphthalene. They found that Oscilla-toria spp., Microcoleus sp., Anabaena spp., Ag-menellum sp., Coccochloris sp., Nostoc sp., Aph-anocapsa sp., Chlorella spp., Dunaliella sp.,Chlamydomonas sp., Ulva sp., Cylindrethecasp., Amphora sp., Porphyridium sp., and Peta-lonia all were capable of oxidizing naphthalene.Their results indicate that the ability to oxidizearomatic hydrocarbons is widely distributedamong the cyanobacteria and algae.

It is now abundantly clear that the ability toutilize hydrocarbons is widely distributed amongdiverse microbial populations. Hydrocarbons arenaturally occurring organic compounds, and it isnot surprising that microorganisms have evolvedthe ability to utilize these compounds. Whennatural ecosystems are contaminated with pe-troleum hydrocarbons, the indigenous microbialcommunities are likely to contain microbial pop-ulations of differing taxonomic relationshipswhich are capable of degrading the contaminat-ing hydrocarbons.

DISTRIBUTION OF HYDROCARBON-UTILIZING MICROORGANISMS

Hydrocarbon-degrading bacteria and fungi arewidely distributed in marine, freshwater, andsoil habitats. The literature on actual numbersof hydrocarbon utilizers is confusing because ofmethodological differences used to enumeratepetroleum-degrading microorganisms. A num-ber of investigators have used hydrocarbons in-corporated into an agar-based medium (13, 147,149, 250, 258). This approach has been criticized(9, 74, 202, 277); in some cases, a high correlationhas been found between growth on agar mediacontaining hydrocarbons as the sole carbonsource and the ability to rigorously demonstratehydrocarbon utilization of isolates from thesemedia in liquid culture; in other studies, only alow percentage of isolates from agar-based me-dia could be demonstrated to be capable ofhydrocarbon utilization. The inclusion oforganiccontaminants in agar media and the growth ofoligotrophic bacteria probably result in the

counting of non-hydrocarbon utilizers in somecases when plate counts are used for enumera-ting hydrocarbon utilizers.The use of silica gel as a solidifying agent has

been shown to improve the reliability of proce-dures for enumerating hydrocarbon utilizers(246). Walker and Colwell (277) reported that amedium containing 0.5% oil and 0.003% phenolred was best for enumerating petroleum-degrad-ing microorganisms. They also found that addi-tion of Amphotericin B permitted selective iso-lation of hydrocarbon-utilizing bacteria and thataddition of either streptomycin or tetracyclinepermitted selective isolation of yeasts and fungi.Washing the inoculum to remove contaminatingorganic compounds did not improve the recov-ery of petroleum degraders in this study. Theseauthors specifically recommended the use of asilica gel-oil medium for enumerating petro-leum-degrading microorganisms; they also sug-gested that counts of petroleum degraders beexpressed as a percentage of the total populationrather than as total numbers of petroleum de-graders per se.Buckley et al. (51) characterized the distribu-

tion of microorganisms in an estuary relative toambient hydrocarbon concentrations. Althoughcounts were performed on non-hydrocarbon-based media, at all but two stations most of thespecies isolated were able to grow on hydrocar-bons, indicating that the ability to utilize hydro-carbons is widespread, even in environments notsubjected to high levels of hydrocarbon pollu-tion. Crow et al. (86) examined the distributionof hydrocarbon utilizers in surface ocean layersand in the underlying water column. They foundthat populations of hydrocarbonoclastic micro-organisms occurred in concentrations 10 to 100times greater in the surface layer than at a 10-cm depth.

Mulkins-Phillips and Stewart (205) examinedthe distribution of hydrocarbon-utilizing bacte-ria in northwestern Atlantic waters and coastalsediments. The fraction of the total hetero-trophic bacteria represented by the hydrocarbonutilizers ranged up to 100%, depending on thearea's previous history of oil spillage; most val-ues were less than 10%. They found that thelocation, numbers, and variety of the microbialhydrocarbon utilizers illustrated their ubiquityand that the broad enzymatic capacity for hy-drocarbon degradation indicated the microbialpotential for removal or conversion of oil in theenvironments examined. The presence of hydro-carbon-utilizing microorganisms was demon-strated in sediments and adjacent waters takenfrom Bermuda, Canadian Northwest Atlantic,and eastern Canadian Arctic marine shorelines.Bunch and Harland (53) found that numbers

186 ATLAS


http://mm

br.asm.org/

Dow

nloaded from



of hydrocarbon utilizers occurred in similar con-centrations in Arctic and temperate marine sam-ples; i.e., quantitative differences in the distri-bution of hydrocarbon utilizers were relativelyunimportant over large geographic distances. In-deed, hydrocarbon utilizers have been found tobe widely distributed even in cold marine eco-systems (8, 88, 90, 234, 235, 260, 277).Most-probable-number (MPN) procedures

have been suggested as a substitute for platecount procedures for enumerating hydrocarbon-utilizing microorganisms, since such procedureseliminate the need for a solidifying agent andpermit direct assessment of the ability to ac-tually utilize hydrocarbons (9, 74). The use ofliquid media for MPN procedures permits re-moval of trace organic contaminants and allowsfor the chemical definition of a medium with ahydrocarbon as the sole source of carbon. Enu-meration methods which incorporate the speci-ficity for counting only hydrocarbon utilizersand which eliminate the problem of countingorganisms growing on other trace organic con-taminants represents a significant improvementin the accuracy with which numbers of hydro-carbon utilizers can be determined. Higashiharaet al. (141) reported that plate counts, usingeither agar or silica gel solidifying agents, wereunsuitable for enumerating hydrocarbon-utiliz-ing microorganisms since many marine bacteriagrow and produce microcolonies even on smallamounts of organic matter. They recommendedthe use of an MPN procedure, with hydrocar-bons as the source of carbon and trace amountsof yeast extract for necessary growth factors, foraccurate enumerations of microbial populationswhich degrade hydrocarbons in marine environ-ments. Mills et al. (202) compared several mediadesigned for use in an MPN determination ofpetroleum-degrading microorganisms. The bestresults, i.e., largest numbers, were obtained witha buffered (32 mM phosphate) liquid mediumcontaining 1% hydrocarbon substrate. In thisstudy, turbidity was used as the criterion forestablishing positive results. Counts of petro-leum degraders obtained with a liquid mediumand an MPN procedure are usually higher thanthose obtained on silica gel medium with oiladded as the carbon source.

14C-radiolabeled hydrocarbons have been usedin MPN procedures for determining the distri-bution of hydrocarbon-utilizing microorganisms.Atlas (9) has described a technique that uses["C]hexadecane-spiked crude oil to enumeratepetroleum-degrading microorganisms. Thismethod uses the conversion of a radiolabeledhydrocarbon to radiolabeled carbon dioxide forestablishing positive results in the MPN proce-dure. Placing the radiolabeled hydrocarbon

within a crude oil mimics the availability ofhydrocarbons to the microbial community, aswould occur in an actual oil spill. Lehmicke etal. (181) used low concentrations of radiolabeledhydrocarbons in MPN determinations; in theirstudies, the concentrations of radiolabeled hy-drocarbons were adjusted to reflect actual con-centrations which might be present in solubleform.Roubal and Atlas (238) studied the distribu-

tion of hydrocarbon-utilizing microorganisms inAlaskan Continental Shelf regions, using anMPN procedure based on the mineralization of"C-labeled hydrocarbons. They reported thathydrocarbon utilizers were ubiquitously distrib-uted, with no significant overall concentrationdifferences between Arctic and subarctic sam-pling regions nor between surface water andsediment samples. There were, however, signifi-cant seasonal differences in numbers of hydro-carbon utilizers. In a study in a temperate region,Raritan Bay, N.J., Atlas and Bartha (13), usingoil-agar plate enumerations, also found thatnumbers of hydrocarbon-utilizing microorga-nisms were lower during winter than summer.Walker and Colwell (280) similarly found sea-sonal variations in numbers of hydrocarbon uti-lizers in Chesapeake Bay.

It is clear from a number of studies that thedistribution of hydrocarbon-utilizing microor-ganisms reflects the historical exposure of theenvironment to hydrocarbons. A large numberof laboratory studies have demonstrated sizableincreases in populations ofhydrocarbon-utilizingmicroorganisms when environmental samplesare exposed to petroleum hydrocarbons (11, 56,93, 166, 218, 225, 256, 263, 304).Mironov (203) and Mironov and Lebed (204)

found highly elevated populations of hydrocar-bon-utilizing microorganisms in the oil tankershipping channels of the Indian Ocean and theBlack Sea. Polyakova (224) found high numbersof hydrocarbon-oxidizing microorganisms inNeva Bay, U.S.S.R., in association with petro-leum inputs. ZoBell and Prokop (305) reportedthat numbers of hydrocarbon utilizers in sedi-ment of Baritaria Bay, La., were correlated withsources of oil pollutants. Similarly, Atlas andBartha (13) for Raritan Bay and Colwell et al.(77) and Walker and Colwell (276) for Chesa-peake Bay found that distributions of hydrocar-bon utilizers correlated highly with sources of oilpollutants entering the bays. The distribution ofhydrocarbon utilizers within Cook Inlet was alsopositively correlated with the occurrence of hy-drocarbons in the environment (238). LePetit etal. (185) reported that bacteria utilizing a gas-oilas the sole carbon source represented 10% of theheterotrophic bacteria in the area of a refinery

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


MICROBIOL. REV.

effluent compared with 4% in an area not di-rectly polluted by hydrocarbons. The degrada-tion potential was highest in areas of chronicdischarge (261).

Several studies have shown a rise in popula-tions of hydrocarbon-utilizing microorganismsafter oil spills. Kator and Herwig (167) foundthat, within a few days after spillage of SouthLouisiana crude oil in a coastal estuary in Vir-ginia, levels of petroleum-degrading bacteriarose by several orders of magnitude. The ele-vated levels of hydrocarbon utilizers were main-tained for over 1 year. Raymond et al. (229)found significant increases in hydrocarbon-uti-lizing microorganisms in soils receiving hydro-carbons; increased populations were maintainedthroughout the year. Pinholt et al. (221) exam-ined the microbial changes during oil decompo-sition in soil. They found an increase from 60 to82% in oil-utilizing fungi and an increase from 3to 50% in oil-degrading bacteria after a fuel oilspill. Oppenheimer et al. (214) found a tendencytoward higher ratios of hydrocarbon-utilizingbacteria to total viable heterotrophs in the activeEkofisk oil field of the North Sea, probably dueto the occurrence of hydrocarbons in the sedi-ments of this region. Gunkel et al. (135) con-firmed the occurrence of high numbers of hydro-carbon-utilizing microorganisms in the vicinityof the North Sea oil fields and found a highcorrelation between concentrations of hydrocar-bons and oil-utilizing bacteria in the North Sea.High numbers of fungi have been found in

association with the Cape Simpson, Alaska, oilseeps (31). Numbers of filamentous fungi 0.2 mfrom the edge of the seep were reported to bethree times higher than those 50 m from theseep; bacterial populations in ponds in contactwith the Cape Simpson oil seeps were found tobe higher than in unstressed ponds; bacterialpopulations in soils adjacent to the asphalticsections of the seeps were higher than those 50m from the seep.In experimental field studies in the Arctic,

Atlas and co-workers have found large increasesin hydrocarbon-utilizing microorganisms in ma-rine (8, 20, 21, 24, 25, 147), freshwater (25, 146),and soil (250, 253) ecosystems; concentrations ofhydrocarbon-utilizing microorganisms havebeen found to rise rapidly and dramatically inresponse to acute inputs of petroleum hydrocar-bons. Bergstein and Vestal (38), however, founda lack of elevated microbial populations in anoil-treated tundra pond unless phosphate alsowas added. Horowitz and Atlas (146), using acontinuous-flow-through model system, foundlarge increases and shifts to a high percentage ofhydrocarbon utilizers in Arctic coastal waterwhen nitrogen and phosphorus were added to

oil slicks. Sexstone and Atlas (250) found thataddition of crude oil to Arctic tundra soils re-sulted in large increases in total numbers ofheterotrophs and of oil-utilizing microorganisms.The response of microbial populations to con-taminating oil was found to depend on soil typeand depth. Increases in microbial populations insubsurface soils paralleled downward migrationof the oil (249).Sparrow et al. (257) found a rise in oil-utilizing

bacterial populations in taiga soils which wereexperimentally contaminated with hot PrudhoeBay crude oil. Studies in the Swan Hills area ofnorth-central Alberta, Canada, by Cook andWestlake (78) showed slightly increased bacte-rial populations 308 and 433 days after treatmentwith Swan Hills oil at an application rate of 6.5liters/m2. Increases in numbers of bacteria weresignificantly higher when the plots were alsotreated with urea-phosphate fertilizer. Similarresults were obtained at Norman Wells 321 and416 days after treatment with 6.5 liters of Nor-man Wells crude per m2. As with the Swan Hillsspill, slight increases in bacterial numbers oc-curred when oil alone was added, and signifi-cantly higher increases occurred when fertilizerwas also added.Gunkel (133, 134) reported that populations of

hydrocarbon utilizers were elevated in sedimentsaffected by the Torrey Canyon spill. Stewartand Marks (258) found higher numbers of hy-drocarbon utilizers in sediment affected by theArrow spill in Chedabucto Bay, Nova Scotia; 5years after the spill, only a few sites examinedhad significant concentrations of residual petro-leum and elevated counts of hydrocarbon utiliz-ers. Significantly elevated numbers of hydrocar-bon utilizers (several orders of magnitude abovenormal) were found after the Amoco Cadiz spillin Brittany (19) and the XTOC-I well blowoutin the Bay of Campeche, Gulf of Mexico (23). Inthe case of the Amoco Cadiz spill, the numbersof hydrocarbon utilizers in intertidal sedimentswere positively correlated with the degree ofhydrocarbon contamination; during recoveryafter the spillage, the numbers of hydrocarbonutilizers returned at most sites to backgroundlevels as the oil disappeared due to biodegrada-tive removal. Counts of hydrocarbon utilizersassociated with an oil-in-water emulsion(mousse) from the XTOC-I well blowout werethree to five orders of magnitude higher than insurface water samples not contaminated with oil(23). In the sediment of an Arctic lake that hadbeen contaminated with a leaded refined gaso-line, populations of hydrocarbon-utilizing micro-organisms were found to be significantly ele-vated within a few hours of the spill (146)through 1 year after the spill (147). This degree

188 ATLAS


http://mm

br.asm.org/

Dow

nloaded from



of elevation in numbers of microbial hydrocar-bon utilizers corresponded with the degree ofcontamination.

In general, population levels of hydrocarbonutilizers and their proportions within the micro-bial community appear to be a sensitive index ofenvironmental exposure to hydrocarbons. In un-polluted ecosystems, hydrocarbon utilizers gen-erally constitute less than 0.1% of the microbialcommunity; in oil-polluted ecosystems, they canconstitute up to 100% of the viable microorga-nisms. The degree of elevation above unpollutedcompared reference sites appears to quantita-tively reflect the degree or extent of exposure ofthat ecosystem to hydrocarbon contaminants.

ENVIRONMENTAL FACTORSINFLUENCING BIODEGRADATION OF

PETROLEUM HYDROCARBONSThe fate of petroleum hydrocarbons in the

environment is largely determined by abioticfactors which influence the weathering, includ-ing biodegradation of the oil. Factors which in-fluence rates of microbial growth and enzymaticactivities affect the rates ofpetroleum hydrocar-bon biodegradation. The persistence of petro-leum pollutants depends on the quantity andquality of the hydrocarbon mixture and on theproperties of the affected ecosystem. In one en-vironment petroleum hydrocarbons can persistindefinitely, whereas under another set of con-ditions the same hydrocarbons can be com-pletely biodegraded within a relatively few hoursor days.

PHYSICAL STATE OF OILPOLLUTANTS

The physical state of petroleum hydrocarbonshas a marked effect on their biodegradation. Atvery low concentrations hydrocarbons are solu-ble in water, but most oil spill incidents releasepetroleum hydrocarbons in concentrations far inexcess of the solubility limits (46, 115, 139, 195).The degree of spreading determines in part thesurface area of oil available for microbial colo-nization by hydrocarbon-degrading microorga-nisms; in aquatic systems, the oil normallyspreads, forming a thin slick (39). The degree ofspreading is reduced at low temperatures be-cause of the viscosity of the oil. In soils, petro-leum hydrocarbons are absorbed by plant mat-ter and soil particles, limiting its spreading.Wodzinsky and LaRocca (295) found that liq-

uid aromatic hydrocarbons were utilized by bac-teria at the water-hydrocarbon interface butthat solid aromatic hydrocarbons were not me-tabolized. At 300C diphenylmethane is a liquidand could be degraded, but at 200C the solid

form of diphenylmethane could not be utilizedby a Pseudomonas sp. They also found thatnaphthalene could not be utilized in the solidform but could be utilized if dissolved in a liquidhydrocarbon. Atlas (unpublished data) similarlyfound that hexadecane supported only marginalbacterial growth at 5°C when the compound wasin the solid form, but if hexadecane was dis-solved in another liquid hydrocarbon or crudeoil, extensive degradation of the liquid hexadec-ane occurred at 50C. The role of temperature indetermining the physical state of a hydrocarbonand the influence of the physical state on ratesof microbial hydrocarbon degradation are ap-parent in these studies.

Hydrocarbon-degrading microorganisms actmainly at the oil-water interface. Hydrocarbon-degrading microorganisms can be observedgrowing over the entire surface of an oil droplet;growth does not appear to occur within oil drop-lets in the absence of entrained water. Availa-bility of increased surface area should acceleratebiodegradation (117, 118). Not only is the oilmade more readily available to microorganisms,but movement of emulsion droplets through awater column makes oxygen and nutrients morereadily available to microorganisms.

In aquatic ecosystems, oil normally formsemulsions. This has been termed "pseudosolu-bilization" of the oil (136). The water-in-oilemulsion which occurs in seawater after oil spillsis referred to as "chocolate mousse" or simply"mousse." The processes involved in the fonna-tion of mousse have been examined by a numberof investigators (40, 54, 95). Mousse is chemicallyand physically heterogeneous. Photooxidation(54) and microbial oxidation (40) have been re-ported to play a role in mousse formation underdifferent environmental conditions; both abioticand microbial processes appear to be capable ofinitiating mousse formation under appropriateenvironmental conditions. In some cases, a fineemulsion is formed with small droplets ofmousse. In these cases, the hydrocarbons in themousse probably are more susceptible to micro-bial degradation, and their fate is similar to thatof "dissolved" hydrocarbons. Mousse can alsorefer to large accumulations of emulsified oil in"globs" up to 1 m in diameter. Such large"mousse plates" have limited surface areas, andhydrocarbons internal to the mousse may bespared from microbial degradation. Davis andGibbs (94) found that large accumulations of"mousse)" weathered extremely slowly with nonet loss of hydrocarbons over 2 years. Atlas etal. (23) proposed that degradation of hydrocar-bons released into the Gulf of Mexico by theXTOC-I blowout was limited in part by thephysical properties of the mousse accumula-

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


190 ATLAS

tions. Colwell et al. (75) postulated that degra-dation of oil from the Metula spill was restrictedby the formation of tar balls and aggregates ofoil which restricted accessibility of the hydro-carbons to microorganisms. Microbial degrada-tion was ineffective when oil was deposited onthe beach and subsequently buried or when theoil formed asphalt layers or tar balls.

Dispersants have been used to treat oil spills.In some cases the use of toxic dispersants prob-ably has resulted in greater ecological impactthan the oil spill itself; such was the case in theTorrey Canyon incident (83, 255). Some disper-sants may contain chemicals which are inhibi-tory to microorganisms. Without toxicity, how-ever, dispersion can enhance petroleum biodeg-radation. Mulkins-Phillips and Stewart (207)found that some dispersants enhanced n-alkanedegradation in crude oil, but that other disper-sants had no effect. Gatellier et al. (117, 118) andRobichaux and Myrick (236) likewise found thatsome dispersants inhibited hydrocarbon-oxidiz-ing populations, whereas others enhanced hy-drocarbon-degrading microorganisms. Atlas andBartha (14) tested several dispersants and foundthat all increased the rate but not the extent ofhydrocarbon mineralization.A number of hydrocarbon-degrading micro-

organisms produce emulsifying agents (1, 131,233, 298). Some of these bioemulsifiers havebeen considered for use in cleaning oil storagetanks, such as on supertankers (136). Reisfeld etal. (233) have studied an Arthrobacter strainwhich extensively emulsifies oil when growingon hydrocarbons. Zajic and co-workers (297)have characterized the emulsifying agents pro-duced by strains of Pseudomonas and Coryne-bacterium. In some cases, the emulsifying agentsappear to be fatty acids or derivatives of fattyacids; in other cases, more complex polymers arethe active emulsifying agents. Although the pro-duction of emulsifying agents should increasethe susceptibility of hydrocarbons in an oil tomicrobial degradation, microbial strains whicheffectively emulsify oil often do not extensivelydegrade the hydrocarbons in the oil. It is notclear yet why extensive emulsification does notpermit greater hydrocarbon degradation bythese organisms.

After extensive weathering, petroleum hydro-carbons often occur in the environment as tarballs. Hydrocarbons in tar are quite resistant tomicrobial degradation. Many of the hydrocar-bons in tar have chemical structures which arenot readily attacked by microbial enzymes. Thesurface area-to-volume ratio of a tar ball is notfavorable for microbial growth on this insolublesubstrate. Tar balls often accumulate on beacheswhere microbial activities are limited by avail-

MICROBIOL. REV.

able water, which is needed to support microbialgrowth and enzymatic hydrocarbon-degradingactivities.From the point of view of microbial hydrocar-

bon degradation, dissolution and emulsificationof hydrocarbons appear to have a positive effecton degradation rates. If there are no adversetoxic effects, dispersion of oil should acceleratemicrobial hydrocarbon degradation. This is animportant consideration when determiningwhether dispersants should be added to oil spills.Increased toxicity must remain, however, a ma-jor concern when considering the use of suchchemical dispersants.

TEMPERATURE

Hydrocarbon biodegradation can occur over awide range of temperatures, and psychrotrophic,mesophilic, and thermophilic hydrocarbon-uti-lizing microorganisms have been isolated. ZoBell(303) and Traxler (263) reported on hydrocarbondegradation at below 0°C; Klug and Markovetz(174, 175) and Mateles et al. (192) reported onhydrocarbon degradation at 70°C.Temperature can have a marked effect on the

rates of hydrocarbon degradation. The effects oftemperature on the physical state of hydrocar-bons was discussed in the previous section.ZoBell (301) found that hydrocarbon degrada-tion was over an order of magnitude faster at250C than at 5°C. Very low rates of hydrocarbonutilization were found by Gunkel (132) at lowwater temperatures. Ludzack and Kinkead (189)found that motor oil was rapidly oxidized at20°C but not at 5°C. Mulkins-Phillips and Stew-art (206) found that, 9 months after the spillageof Bunker C fuel oil into Chedabucto Bay, thebacterial populations isolated from contami-nated areas showed rates of degradation at 5°Cthat were 21 to 70% less during 14 days ofincubation than during 7 days at 10°C.There are seasonal shifts in the composition

of the microbial community which can be re-flected in the rates of hydrocarbon metabolismat a given temperature. Atlas and Bartha (13)found that higher numbers of hydrocarbon uti-lizers capable of growth at 50C were present inRaritan Bay, N.J., during winter than duringother seasons. Rates of hydrocarbon mineraliza-tion measured at 5°C were significantly higherin water samples collected in winter than insummer. The evidence suggests a seasonal shiftto a microbial community capable of low-tem-perature hydrocarbon degradation.Gibbs and Davis (120) studied the degradation

of oil in beach gravel at temperatures from 6 to260C. They found a Qio (6 to 160C) of 3.3 and a

Qlo (11 to 210C) of 2.05. The average Qio value


http://mm

br.asm.org/

Dow

nloaded from



of 2.7 was the same as the value found in otherstudies, by Gibbs et al. (121), on the effects oftemperature on the degradation of oil in seawa-ter. Atlas and Bartha (10) found a Qlo of approx-imately 4, using seawater over a temperaturerange of 5 to 200C.

Atlas and Bartha (10) found that the effectsof temperature differ, depending on the hydro-carbon composition ofa petroleum mixture. Lowtemperatures retard the rates of volatilization oflow-molecular-weight hydrocarbons, some ofwhich are toxic to microorganisms. The presenceof such toxic components was found to delay theonset of oil biodegradation at low temperatures(10). In a subsequent study, Atlas (5) examinedthe biodegradability of seven different crude oilsand found biodegradation to be highly depend-ent on the composition and on incubation tem-perature. At 200C, lighter oils had greater abioticlosses and were more susceptible to biodegra-dation than heavier oils; rates of oil mineraliza-

tion for the heavier oils were significantly lowerat 200C than for the lighter ones. The light crudeoils, however, had toxic volatile componentswhich evaporated only slowly, inhibiting micro-bial degradation of these oils at 100C. A signifi-cant lag phase before the onset of hydrocarbonbiodegradation was found for the lighter oils. Notoxic volatile fraction was associated with theheavy oils tested. Paraffinic, aromatic, and as-phaltic fractions were subject to biodegradation.Some preference was shown for paraffin degra-dation, especially at low temperatures. Horowitzand Atlas (145) found that during sujmmer, inArctic surface waters, different structural classesof hydrocarbons were degraded at similar rates.They postulated that at low temperatures co-metabolism played an important role in deter-mining the rates of disappearance of hydrocar-bons in the mixture.Walker and Colwell (274), using a model pe-

troleum incubated with estuarine water col-lected during winter, found that slower but moreextensive biodegradation occurred at 00C thanat higher temperatures. Decreased toxicity ofhydrocarbons at lower temperatures was hy-pothesized to explain the more extensive growthat the lower temperature.Ward and Brock (289) studied the influence

of environmental factors on the rates of hydro-carbon oxidation in temperate lakes. Rates ofhydrocarbon oxidation were assessed by usingthe conversion of 14C-radiolabeled hexadecaneto 1'CO2. They found that a lag phase precededhydrocarbon oxidation and that the length ofthe lag phase depended on population density or

on factors influencing growth rate. Hydrocarbonoxidation was coincident with growth and was

presumed to occur only under conditions of de-

velopment ofindigenous hydrocarbon-degradingmicroorganisms. They found that hydrocarbon-degrading microorganisms persisted during theyear, but that there were seasonal variations inthe rates of hydrocarbon oxidation. Rates ofpetroleum hydrocarbon biodegradation werecorrelated with temperature. During winter,spring, and fall, temperature was a major limit-ing factor. Dibble and Bartha (102) found thatthe rates of disappearance ofhydrocarbons froman oil-contaminated field in New Jersey showeda definite correlation with mean monthly tem-perature.

Arhelger et al. (4) compared Arctic andsubarctic hydrocarbon biodegradation. In situ[14C]dodecane oxidation rates based on 14CO2production were: Port Valdez, 0.7 g/liter per day;Chukchi Sea, 0.5 g/liter per day; and ArcticOcean, 0.001 g/liter per day. This study indicatesthat rates of hydrocarbon degradation show adefinite climatic shift and are lower in the ArcticOcean than in more southerly Alaskan regions.

Atlas et al. (7, 21) examined the degradationof Prudhoe Bay crude oil in Arctic marine ice,water, and sediment ecosystems. Petroleum hy-drocarbons were degraded slowly. They foundthat ice greatly restricted losses of light hydro-carbons and that biodegradation of oil on thesurface of ice or under sea ice was negligible.They concluded that petroleum hydrocarbonswill remain in cold Arctic ecosystems for longperiods of time after oil contamination. In thesestudies, however, temperature was not specifi-cally elucidated as a major factor limiting hydro-carbon degradation, except as it related to theoccurrence of ice.

Colwell et al. (75) reported greater degrada-tion of Metula crude oil at 30C than at 220Cwith mixed microbial cultures in beach sandsamples; when 0.1% oil was added, 48% of theadded hydrocarbons were degraded at an incu-bation temperature of 30C, compared with only21% degraded at 220C with cultures adapted atthe same temperatures as the incubation tem-perature. They found that under in situ condi-tions oil degradation proceeded slowly, but con-cluded that temperature does not seem to be thelimiting factor for petroleum degradation in theAntarctic marine ecosystem affected by the Me-tula spill.A number of studies have been conducted on

the fate of oil in cold Arctic soils. Sexstone andcolleagues (251-253) have reported very longpersistence times for oil in tundra soils. It ap-pears that degradation of hydrocarbons ceasesduring winter when tundra soils are frozen.Westlake and colleagues (78, 158, 292) foundthat the microbial populations in northern soilswere able to degrade hydrocarbons at the am-

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


192 ATLAS

bient temperatures found during the warmerseasons. Several aspects of these studies werediscussed earlier in this review.

It is apparent that the influence of tempera-ture on hydrocarbon degradation is more com-plex than simple consideration of Qlo values.The effects of temperature are interactive withother factors, such as the quality of the hydro-carbon mixture and the composition of the mi-crobial community. Hydrocarbon biodegrada-tion can occur at the low temperatures (<50C)that characterize most of the ecosystems whichare likely to be contaminated by oil spills. Tem-perature often is not the major limiting factorfor hydrocarbon degradation in the environmentexcept as it relates to other factors such as thephysical state of the oil or whether liquid wateris available for microbial growth. Concern mustbe expressed, however, regarding the rates ofmicrobial oil degradation in Arctic and subarcticregions. These are areas of new petroleum de-velopment and the data gathered to date suggestthat rates of microbial degradation in these coldecosystems may not be adequate to rapidly re-move hydrocarbon contaminants.

NUTRIENTS

There is some confusion and considerable ap-parent conflict in the literature regarding thelimitation of petroleum biodegradation by avail-able concentrations of nitrogen and phosphorusin seawater. Several investigators (12, 32, 109,111, 132, 183, 184) have reported that concentra-tions of available nitrogen and phosphorus inseawater are severely limiting to microbial hy-drocarbon degradation. Other investigators(173), however, have reached the opposite con-clusion, i.e., that nitrogen and phosphorus arenot limiting in seawater. The difference in resultsis paradoxical and appears to be based onwhether the studies are aimed at assessing thebiodegradation of hydrocarbons within an oilslick or the biodegradation of soluble hydrocar-bons. When considering an oil slick, there is amass of carbon available for microbial growthwithin a limited area. Since microorganisms re-quire nitrogen and phosphorus for incorporationinto biomass, the availability of these nutrientswithin the same area as the hydrocarbons iscritical. Extensive mixing can occur in turbulentseas, but in many cases the supply of nitrogenand phosphorus is dependent on diffusion to theoil slick. Rates of diffusion may be inadequateto supply sufficient nitrogen and phosphorus toestablish optimal C/N and C/P ratios for micro-bial growth and metabolism. Researchers ex-amining the fate of large oil spills have thusproperly concluded in many cases that concen-trations of N and P are limiting with respect to

rates ofhydrocarbon biodegradation. When con-sidering soluble hydrocarbons, nitrogen andphosphorus are probably not limiting since thesolubility of the hydrocarbons is so low as topreclude establishment of an unfavorable C/Nor C/P ratio. Investigators considering the fateof low-level discharges of hydrocarbons (solublehydrocarbons) have, thus, properly concludedthat available nutrient concentrations are ade-quate to support hydrocarbon biodegradation.

Floodgate (111), in considering the limitationsof nutrients to biodegradation of hydrocarbonsin the sea, proposed the concept of determiningthe "nitrogen demand," analogous to the con-cept of biochemical oxygen demand. Based onKuwait crude oil at 14°C, the nitrogen demandwas found to be 4 umol of nitrogen per ng of oil.Bridie and Bos (47) found that addition of 3.2mg of ammonium nitrogen and 0.6 mg of phos-phate permitted maximal rates of degradation ofKuwait crude in seawater at a concentration of70 mg of oil per liter. Atlas and Bartha (12)found that concentrations of 1 mg of nitrogenand 0.07 mg of phosphorus per liter supportedmaximal degradation of Sweden crude oil inNew Jersey coastal seawater at a concentrationof8 g of oil per liter. Reisfeld et al. (233) reportedoptimal concentrations ofnitrogen and phospho-rus of 11 and 2 mg per liter for biodegradation of1 g ofIranian crude oil per liter in Mediterraneanseawater.

Colwell et al. (75) concluded that Metula oilis degraded slowly in the marine environment,most probably because of limitations imposedby the relatively low concentrations of nitrogenand phosphorus available in seawater.Ward and Brock (289) reported that although

temperature was the main limiting factor muchof the year, during summer nutrient deficiencieslimited oil biodegradation in temperate lakes.Higher rates of oil biodegradation could be ob-tained by addition of nitrogen and phosphorus.High rates of hydrocarbon degradation werefound only during 1 month of the year whentemperature and nutrient supplies were optimal.They concluded that environmental factors lim-ited hydrocarbon biodegradation, but that avail-ability of hydrocarbon-utilizing microorganismswithin the indigenous microbial community wasnot a limiting factor.

LePetit and N'Guyen (184) found that theartificial stimulation of bacterial hydrocarbondegradation requires the addition of phosphorusto seawater. They reported optimal concentra-tions ofphosphorus to support hydrocarbon deg-radation of between 2 x 10-4 and 8 x 10' M forseawater and between 1.5 x 10-3 and 3 x 10-3Mfor coastal waters receiving a significant supplyof fresh water. Inhibition of bacterial develop-ment was observed with higher phosphate con-

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



centrations. Gibbs (119) calculated that 1 m3 ofIrish Sea water provides sufficient nitrogen todegrade 30 g of oil per year at summer temper-atures and 11 g of oil per year at winter temper-atures.

Bergstein and Vestal (38) studied the biodeg-radation of crude oil in Arctic tundra ponds.They concluded that oleophilic fertilizer mayprovide a useful tool to enhance the biodegra-dation of crude oil spilled on such oligotrophicwaters. Without addition of nitrogen and phos-phorus, hydrocarbon biodegradation was lim-ited. Atlas and Bartha (17) described an oleo-philic nitrogen and phosphorus fertilizer whichcould overcome limitations ofnitrogen and phos-phorus in seawater and stimulate petroleum bi-odegradation in seawater. The fertilizer consist-ing of paraffinized urea and octylphosphate sup-ported degradation of oil in seawater. OptimalC/N and C/P ratios were 10:1 and 100:1, respec-tively. In conjunction with the U.S. Office ofNaval Research, they obtained a patent for useof fertilizers for stimulating oil degradation inseawater (33).

Olivieri et al. (212) described a slow-releasefertilizer containing paraffin-supported magne-sium ammonium phosphate as the active ingre-dient for stimulating petroleum biodegradation.They reported that the biodegradation of Sarircrude oil in seawater was considerably enhancedby addition of the paraffin-supported fertilizer.After 21 days, 63% of the oil had disappearedwhen fertilizer was added compared with 40% ina control area. Kator et al. (168) suggested theuse of paraffinized ammonium and phosphatesalts for enhancing oil biodegradation in seawa-ter. Raymond et al. (232) were able to stimulatethe microbial degradation of hydrocarbons incontaminated groundwaters by procedureswhich included the addition of nitrogen andphosphorus nutrients.Dibble and Bartha (99) examined the effect of

iron on the biodegradation of petroleum in sea-water. Biodegradation of south Louisiana crudeoil and the effects of nitrogen, phosphorus, andiron supplements on this process were comparedin polluted and relatively clean littoral seawatercollected along the New Jersey coast. Withoutsupplements, the biodegradation of south Louis-iana crude oil was negligible in both seawatersamples. Addition of nitrogen and phosphorusallowed very rapid biodegradation; up to 73% ofthe oil was degraded within 3 days in pollutedseawater. Total iron in the seawater sample washigh (5.2 mM), and the addition of iron did notincrease biodegradation rates. In less pollutedand less iron-rich (1.2 mM Fe) seawater samples,biodegradation of south Louisiana crude oil wasconsiderably slower (21% in 3 days), and additionof chelated iron had a stimulating effect. Ferric

octoate was shown to have a stimulating effecton south Louisiana crude oil biodegradation,similar to that of chelated iron. Ferric octoate incombination with paraffinized urea and octyl-phosphate is suitable for treatment of floatingoil slicks. The authors concluded that spills ofsouth Louisiana crude oil and similar oils can becleaned up rapidly and efficiently by stimulatedbiodegradation, provided that water tempera-tures are favorable.Dibble and Bartha (100) examined the effect

of environmental factors on the biodegradationof oil sludge. They conducted a laboratory studyaimed at evaluating and optimizing the environ-mental factors of land fanning, i.e., disposal bybiodegradation in soil of oily sludges generatedin the refining of crude oil. They found that oilsludge biodegradation was optimal at a soil wa-ter-holding capacity of 30 to 90%, a pH of 7.5 to7.8, a C/N ratio of 60:1, and a C/P ratio of 800:1. Optimal temperatures were 20°C or above.They reported that an application rate of5% (byweight) oil sludge hydrocarbon to soil, i.e.,100,000 liters/hectare, gave a good compromisebetween high biodegradation rates and efficientland use and resulted in the best overall biodeg-radation rate of oil hydrocarbon classes. Fre-quent small applications resulted in higher bio-degradation rates than single large applications.Fedorak and Westlake (personal communica-

tion) found that, without added nutrients, aro-matic hydrocarbons were more readily attackedthan saturated hydrocarbons by soil and marinemicrobes; addition of nitrogen and phosphorusnutrients stimulated degradation of saturatedhydrocarbons more than of aromatic hydrocar-bons.Westlake et al. (292) examined the in situ

degradation of oil in a soil of the boreal region ofthe northwest territories of Canada. Where fer-tilizer containing nitrogen and phosphorus wasapplied to the oil, there was a rapid increase inbacterial numbers, but no increase in fungalpropagules. This was followed by a rapid disap-pearance of n-alkanes and isoprenoids and acontinuous loss of weight of saturated com-pounds in the recovered oil. The seeding of oilslick plots with oil-degrading bacteria had noeffect on the composition of the recovered oil.Jobson et al. (159) similarly found that nitrogenand phosphorus addition stimulated hydrocar-bon degradation in oil applied to soil but thatseeding did not stimulate degradation. Hunt etal. (152) found that fertilizer application tosubarctic soils enhanced microbial hydrocarbondegradation. They found, however, in laboratorytests that nitrogen addition caused an initialnegative response in microbial activity whichwas followed by enhanced biodegradation; mi-

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


MICROBIOL. REV.

crobial activity also responded positively tophosphorus addition.Raymond et al. (229) studied oil biodegrada-

tion in soil. Greater oil degradation was found insoils receiving fertilizer application and rototill-ing than in untreated soils. They did not findany leaching of hydrocarbons into groundwater.Dibble and Bartha (101) studied the leachingaspect of oil sludge biodegradation in soil. Theyadded fertilizer to oil sludges in soils and exam-ined the leachate for phosphate and undegradedhydrocarbons. There was a modest increase intotal organic carbon in the leachate, presumablydue to hydrocarbon biodegradation, but no un-degraded hydrocarbons or phosphorus was re-covered in the leachate. The results support theconcept that oil sludge application to soil can beused for biodegradative removal of these mate-rials.The above studies indicate that the available

concentrations of nitrogen and phosphorus se-verely limit the extent of hydrocarbon degrada-tion after most major oil spills. Rates of nutrientreplenishment generally are inadequate to sup-port rapid biodegradation of large quantities ofoil. The addition of nitrogen- and phosphorus-containing fertilizers can be used to stimulatemicrobial hydrocarbon degradation.

OXYGENAs with nutrients, there has been controversy

over whether oxygen is absolutely required forhydrocarbon biodegradation or whether hydro-carbons are subject to anaerobic degradation.The current evidence supports the view thatanaerobic degradation by microorganisms atbest proceeds at negligible rates in nature (28,288). The existence of microorganisms which arecapable of anaerobic hydrocarbon metabolismhas not, however, been excluded. In fact, therehave been several reports of isolated microor-ganisms which are capable of alkane dehydro-genation (72, 153, 215, 247, 264) under anaerobicconditions. These organisms have an enzymaticmechanism which should permit addition of wa-ter across the double bond, forming a secondaryalcohol, and therefore permit anaerobic growth.Although there have been preliminary reports(264) on the ability of isolated organisms to growon n-alkanes anaerobically, these findings gen-erally have not been adequately repeated uponfurther testing (R. W. Traxler, personal com-munication). In the case of the Pseudomonasstrain studied by Senez and Azoulay (247), theorganisms consumed oxygen when growing onheptane even though it had an n-heptane de-hydrogenase enzyme.There have been few reports on the anaerobic

degradation of hydrocarbons in natural ecosys-

tems (28, 48, 49, 220, 305). These reports sug-gested that nitrate or sulfate could serve as analternate electron acceptor during anaerobic res-piration using hydrocarbon substrates. Thismechanism has not been biochemically con-firmed for hydrocarbon utilization in pure cul-tures, and the criteria used for assessing anaer-obic hydrocarbon degradation in the above-nien-tioned studies generally were inadequate to es-tablish definitive results; either there was a lackof exhaustive evidence for the complete exclu-sion of oxygen or there was a lack of chemicalevidence needed to establish that hydrocarbonswere in fact degraded. In the study by Sheltonand Hunter (254), there was an 11% decrease inhexane-extractable material under anaerobicconditions compared with only 4% under aerobicconditions in oiled sediments during 30 weeks ofincubation. They concluded, however, that therapid loss of aliphatic hydrocarbons under an-aerobic conditions could not be accounted for bymicrobial degradation.Hambrick et al. (138) found that, at pH values

between 5 and 8, mineralization ofhydrocarbonsin estuarine sediments was highly dependent onoxygen availability. Rates of hydrocarbon deg-radation decreased with decreasing oxygen re-duction potential, i.e., with increasing anaero-biosis. They concluded that hydrocarbons wouldpersist in reduced sediments for longer periodsof time than would hydrocarbon contaminantsin aerated surface layers. Some mineralizationof alkanes (about 10 to 20%) was reported during35 days of incubation under anaerobic condi-tions, but mineralization of naphthalene wasinsignificant (about 0.4% under these incubationconditions). Naphthalene mineralization in-creased from 0.6 to 22.6% when the redox poten-tial was gradually increased from -220 mV to+130 mV over an additional 35-day incubationperiod (97). Ward and Brock (290) similarlyfound that hexadecane was rapidly mineralizedin freshwater lake sediments under aerobic con-ditions but that almost no hydrocarbon min-eralization occurred under anaerobic conditions.Addition of nitrate and sulfate, in this study,failed to increase hydrocarbon mineralizationunder anaerobic conditions.

In a recent study, Ward et al. (288) comparedrates of hydrocarbon oxidation in sediments af-fected by the Amoco Cadiz spillage under aero-bic and anaerobic conditions. With 14C-labeledhydrocarbons, 14CO2 production from hepta-decane and toluene, but not from hexadecane,was found during anaerobic incubation. Meth-anogenesis could be demonstrated in these tests,indicating rigorous anaerobic conditions. Al-though measurable degradation rates under an-aerobic conditions were found, rates of 14CO2

194 ATLAS


http://mm

br.asm.org/

Dow

nloaded from



production were orders of magnitude lower un-der anaerobic than under aerobic conditions. Inthe absence of oxygen, less than 5% of addedhydrocarbon was oxidized to "4CO2 during 233days compared with over 20% during 14 daysunder aerobic conditions. In this study, petro-leum was found in a relatively unweathered statein anaerobic sediments oiled by the AmocoCadiz spill, indicating that hydrocarbons are in-deed preserved from microbial attack under an-aerobic conditions in the environment.The importance of oxygen for hydrocarbon

degradation is indicated by the fact that themajor degradative pathways for both saturatedand aromatic hydrocarbons, discussed earlier,involve oxygenases and molecular oxygen. Thetheoretical oxygen demand is 3.5 g of oil oxidizedper g of oxygen (111, 301). ZoBell (301) calcu-lated that the dissolved oxygen in 3.2 x 105 litersof seawater therefore would be required for thecomplete oxidation of 1 liter of oil. Within anoxicbasins, the hypolimnion of stratified lakes andbenthic sediments, oxygen may severely limitbiodegradation.Johnston (160) examined the consumption of

oxygen in sand columns containing Kuwaitcrude oil. The oxygen concentration in the in-terstitial water decreased rapidly. The mean rateof oxygen consumption over 4 months was 0.45g/m2 per day at 100C, corresponding to an oildegradation rate of 90 mg of oil/M2 per day.Biodegradation of oil in sediments has beenfound to be stimulated by bioturbation (128,178). The introduction of oxygen by burrowinganimals such as polychaete worms is apparentlyvery important in determining the rate of bio-degradation of hydrocarbons in oil-contami-nated sediments.Jamison et al. (154) used forced aeration to

supply oxygen for hydrocarbon biodegradationin a groundwater supply which had been con-minated by gasoline. Nutrient addition with-

out aeration failed to stimulate biodegradation,but when both nutrients and oxygen were sup-

plied, it was estimated that up to 1,000 barrelsof gasoline was removed by stimulated microbialdegradation. Such manipulations to supply ox-

ygen probably are not feasible in open systemswhere natural forces such as wind and wave

action will have to be relied upon for turbulentmixing and resupply of oxygen to support bio-degradation of oil.

Regardless of whether hydrocarbon degrada-tion can occur at all under anaerobic conditions,the environmental importance of anaerobic hy-drocarbon biodegradation can be discounted.Rapid biodegradation of hydrocarbons does notoccur in anaerobic environments. Hydrocarbonswhich enter anaerobic environments such as an-

oxic sediments are well preserved and persistindefinitely as environmental contaminants.

SALiNITY AND PRESSUREThe influence of several other environmental

factors on hydrocarbon biodegradation has beenstudied. Typically these factors are specific fea-tures of particular ecosystems such as salinelakes or deep seas (high hydrostatic pressure),which represent specialized environments thatmay be contaminated by petroleum hydrocar-bons.Ward and Brock (291) examined hydrocarbon

biodegradation in hypersaline environments.When hydrocarbons were added to natural sam-ples of various salinities (from 3.3 to 28.4%) fromsalt evaporation ponds of Great Salt Lake, Utah,rates of metabolism of these compounds de-creased as salinity increased. Rate limitationsdid not appear to relate to low oxygen levels orto availability of organic nutrient. Gas chro-matographic examination of hexane-solublecomponents of tar samples from natural seeps atRozel Point in Great Salt Lake demonstrated noevidence of biological oxidation of isoprenoidalkanes which are subject to degradation in nor-mal environments. Attempts to enrich for micro-organisms, in saline waters, able to use mineraloil as a sole source of carbon and energy weresuccessful below, but not above, approximately20% salinity. The study strongly suggests a gen-eral reduction ofmetabolic rate at extreme salin-ities and raises doubts about the biodegradationof hydrocarbons in hypersaline environments.Hydrocarbon pollutants in the world's oceans

may eventually sink; some petroleum compo-nents may contaminate deep benthic zones. Mi-crobial degradation oforganic matter in the deepsea has been found to be greatly restricted (156).Hydrocarbons do not appear to be an exception.Schwarz et al. (242-244) examined the growthand utilization of hydrocarbons at ambient andin situ pressure for deep-sea bacteria. The rateof hydrocarbon utilization under high pressureand ambient temperatures was found to be sig-nificantly less than rates found under conditionsof ambient temperatures and atmospheric pres-sure. Whereas 94% of hexadecane was utilizedwithin 8 weeks at 1 bar, at 500 bars it took 40weeks for similar degradation. It appears that oilwhich enters deep-ocean environments will bedegraded very slowly and persist for long periodsof time.

CASE HISTORIESThe fate of petroleum hydrocarbons in the

environment from various actual environmentaloil contamination incidents has now been ex-

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


MICROBIOL. REV.

amined. It is extremely complex to study theweathering of a mixture such as petroleum innatural, variable environments. Patchiness of oildistribution and uncertainty about localized en-vironmental variations make definitive scientificconclusions difficult to reach. Determiningquantitatively the specific role of microorga-nisms in the fate of polluting oil is difficult, butchanges in an environmentally contaminating oilcan be viewed in light of the enzymatic degra-dative capacity of the indigenous hydrocarbon-degrading microbial populations. Environmentalfactors known to influence rates of microbialhydrocarbon degradation can be examined toestimate limitations of the biodegradative con-tribution to the removal ofpetroleum pollutants.

In February 1970, the tanker Arrow ranaground and spilled a large portion of its 108,000-barrel cargo into Chedabucto Bay, Nova Scotia.An estimated 300 km of shoreline was affected.Rashid (227) described the changes in the oil 3.5years after the spillage. Degradation of oil de-pended largely on environmental factors, espe-cially wave energy. Degradation was greatest inhigh-wave-energy environments and lowest inprotected embayment areas. In the high-energyenvironments, there was a substantial loss of n-alkanes, which was believed to be due to micro-bial degradation. Presumably, oxygen and nutri-ents replenished by wave-driven mixing permit-ted more extensive degradation. Six years afterthe spill, it was impossible to estimate theamount of oil remaining in Chedabucto Bayfrom the spillage due to the patchy distributionof the oil, contributions of more recent spillages,and the absence of adequate control sites (170).

In January 1973, the Irish Stardust ranaground near Vancouver Island, B.C. Approxi-mately 180 metric tons of fuel oil was spilled.Cretney et al. (84) examined the long-term fateof the heavy fuel oil from the spill that contam-inated a British Columbia, Canada, coastal bay.They reported that biodegradation accountedfor almost complete removal ofn-alkanes duringthe first year after the spill. Pristane and phy-tane were biodegraded more slowly, but werealmost completely gone after 4 years. The non-n-alkane components of the C2s-to-C3o range ap-peared to be the most resistant to degradationof all the components resolved by gas chroma-tography.During March 1971, a pipeline rupture al-

lowed JP-4 jet fuel and no. 2 fuel oil to enter theintertidal zone of a cove at Searsport, Maine.The spill was approximately 13 metric tons, butonly a fraction of that amount probably enteredthe cove. Mayo et al. (194) examined the weath-ering characteristics of petroleum hydrocarbonsdeposited in fine-clay marine sediments of the

cove. They found that petroleum residues iso-lated from the spill gave the appearance ofweathering particularly slowly in the cold anoxicsediment. In 1976, they found that the averagearea contained roughly 20% less hydrocarbonthan in 1971, when the spill occurred. At anumber of sites, there appeared to be no declinein gross hydrocarbon concentrations and essen-tially no weathering of the aliphatic portions ofthe petroleum residues. The data indicated thatalthough microbial degradation of the aliphaticlinear chain systems had a measurable impacton the residues contained in upland sediments,this action was greatly suppressed in the residuesabsorbed on the anoxic cold clay silt of the cove.Microbial degradation apparently played a roleduring transport of the oil from the upland spilllocation to the marine sediment, but within themarine sediments rates of microbial degradationmust have been near zero. It is likely that a lackof available oxygen in the contaminated sedi-ments severely limited rates of biodegradation.The tanker Metula grounded in the Straits of

Magellan in August 1974. Approximately 46,000metric tons of oil was lost, contaminating a coldmarine environment. Colwell et al. (75) exam-ined the biodegradation of petroleum from theMetula spill in the Straits of Magellan region.They found from biodegradation studies that oildegradation under in situ conditions proceededrelatively slowly, with marked persistence ofMetula oil in the Straits of Magellan 2 yearsafter the spill. They reported that the slow ratesof oil degradation most probably were due tolimitations imposed by relatively low concentra-tions of nitrogen and phosphorus available inseawater, as well as restricted accessibility todegradable compounds within aggregated oils ortar balls. Temperature did not seem to be alimiting factor for petroleum degradation in thecold marine environment. There was an indige-nous cold-adapted microbial community capableof utilizing hydrocarbons. Microbial degradationwas not effective in attacking buried oil or oilthat had formed asphalt layers on beaches. Mi-crobial action may have contributed signifi-cantly to the formation of polar material andcontributed to the extensive removal of aliphatichydrocarbons in favorable environments. It wasconcluded that the oil from the Metula spillwould persist for a long period of time.Two major spillages of no. 2 fuel oil into

Buzzards Bay, Mass., have been studied. TheFlorida created the West Falmouth spill in 1969,and the Bouchard was the source of a secondspill in 1974. Blumer et al. (41-43) examined thedisappearance of oil from the West Falmouthspill. They found that the disappearance of pe-troleum hydrocarbons was slow and that bacte-

196 ATLAS


http://mm

br.asm.org/

Dow

nloaded from



rial degradation contributed to the removal of n-paraffins. Teal et al. (262) examined the aro-matic hydrocarbons contaminating the sedi-ments of Buzzards Bay resulting from both spil-lages. Microbial degradation was believed tocontribute to the disappearance ofnaphthaleneswith zero to three alkyl substituents and phen-anthrenes with zero to two substituents fromsurface sediments. The more substituted aro-matics decreased relatively less and probablywere more resistant to biodegradation.

Pierce et al. (220) examined the persistenceand biodegradation of fuel oil on an estuarinebeach which came from the spillage of 90,000 gal(ca. 342,000 liters) of no. 6 fuel oil into Narra-gansett Bay, R.I., in 1973. The concentrations ofhydrocarbons in the midtide region declined si-multaneously with an increase in populations ofhydrocarbon-utilizing bacteria. During the win-ter months, hydrocarbon biodegradation was ap-parent at rates of less than 1 ,ug of hydrocarbonper g (dry weight) of sediment per day. Mc-Auliffe et al. (196) examined the fate of 65,000barrels of crude oil spilled in 1970 from a Chev-ron platform 11 miles (ca. 17.6 km) east of theMississippi River delta. They found that only1% of the oil entered the sediments; much of theoil dissipated. One week after the spill, there wasevidence for biodegradation of the oil in thesediment as shown by an alteration in the ratioof n-paraffins to isoprenoid hydrocarbons.Within 1 year, most of the oil was gone and rapidbiodegradation appeared to contribute to theremoval of contaminating hydrocarbons.

Atlas et al. (21) studied petroleum biodegra-dation in various coastal Arctic ecosystemswhich had been experimentally contaminatedwith Prudhoe crude oil. Hydrocarbon biodegra-dation potentials were lower in ice than in wateror sediment. Natural rates of degradation wereslow, and maximal losses from experimental oilspills were less than 50% during the Arctic sum-mer due to combined abiotic and biodegradativelosses. Rates of biodegradation were found to belimited by temperature and concentration ofavailable nitrogen and phosphorus. Residual oilhad similar percentages of hydrocarbon classesas fresh oil; i.e., biodegradation of all oil com-ponent classes, including paraffinic and aromaticfractions, apparently proceeded at similar rates.In March 1977, there was a spill from the Poto-mac into the ice-laden waters of Melville Bay inthe northeastern part of Baffin Bay, off westernGreenland. About 107,000 gal (ca. 406,600 liters)of Bunker C fuel oil was lost. The fate of the oilwas investigated by a team of scientists (129).Biodegradation of the oil at the low water tem-peratures was found to proceed very slowly if atall. There was no significant increase in numbers

of hydrocarbon utilizers within a few weeks afterthe spill. During this period there was also al-most no change in the C17/pristane ratio in theoil, indicating that biodegradation was not oc-curring at a significant rate.The spill of the supertanker Amoco Cadiz in

March 1978 resulted in the largest oil spill tothat date. In excess of 190,000 metric tons of oilwas released into the marine environment dur-ing 2 weeks. A variety of intertidal sites off theBrittany coast was affected. Aminot (3) exam-ined the fate of the oil in the water colunmbefore reaching the shoreline. He found a deple-tion of N, P, and 02 in the water column beneaththe oil, which apparently resulted from micro-bial degradation of petroleum hydrocarbons.The in situ deficits of N, P, and 02 converted toa hydrocarbon biodegradation rate of 0.03 mg ofoil degraded per liter per day in the water col-umn under the oil. Aminot estimated that 9,000metric tons of oil was biodegraded in the watercolumn during the 2 weeks following the spill.The fate of the Amoco Cadiz oil within theintertidal zone was studied by several investi-gators (18, 19, 44, 55, 269, 288). Microbial deg-radation appears to have played a very impor-tant role in the weathering of oil stranded withinthe littoral zone. Atlas and Bronner (19) esti-mated a biodegradation rate of 0.5 ,ug of hydro-carbon per g (dry weight) of sediment per daywithin the affected interidal zone. The onset ofextensive changes in the oil appears to haveoccurred more rapidly after the wreck than wasanticipated, extensive biodegradation even pre-ceding complete evaporation and dissolution ofvolatile aromatics (18, 55); there was a rapidchange in the n-alkane/isoprenoid hydrocarbonratio within days to weeks. The isoprenoid al-kanes, C27 to C31 n-alkanes, hopanes, alkylateddibenzothiophenes, and alkylated phenan-threnes were the classes of hydrocarbons mostresistant to biodegradation. Despite the rapidrates of biodegradation, the magnitude of thespill was such that the oil will persist within thelittoral zone for a prolonged period. Oil that wasburied, oil within anoxic sediments, and oilwithin embayments appears to be most persist-ent (18, 44, 280). Conditions which enhance aer-ation and resupply nutrients, such as high-en-ergy wave action, favor biodegradation.The magnitude of the Amoco Cadiz spill was

surpassed by the spill from the IXTOC-I wellblowout. In June 1979, oil began spilling into theBay ofCampeche, GulfofMexico. The oil flowedfor 10 months before the well was capped. Someof the oil washed onto the coastal beaches ofTexas, but for the most part the current carriedthe oil away from U.S. waters. The oil from theIXTOC-I well formed a mousse. Boehm and

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


MICROBIOL. REV.

Fiest (45) found little evidence for biologicalweathering of the hydrocarbons in the mousse.Atlas and co-workers (22, 23) found that biodeg-radation of mousse was greatly restricted, prob-ably due to nutrient limitations and limited sur-face area for microbial attack. During a 6-monthlaboratory incubation under simulated naturalconditions, 2 to 5% of the mousse (73) was con-verted to CO2. Despite favorable temperaturesand high populations ofhydrocarbon utilizers inassociation with the mousse, changes in n-al-kane/isoprenoid ratios took months rather thandays to weeks. The contribution of biodegrada-tion to weathering of oil from the IXTOC-I wellwas notably slower and of less magnitude thanwas found for the Amoco Cadiz. Pfaender andco-workers (52, 219) examined the degradationof hydrocarbons within the water column af-fected by the IXTOC-I oil. They found relativelyrapid turnover times for hydrocarbons whichhad become dissolved in the water column.Rates of degradation ranged from 0.01 to 44pugof aliphatic hydrocarbon respired per liter per hwith turnover times of 30 to 266 h.In contrast to the cited studies on large marine

oil spills, there have been few studies on fresh-water oil spills. Hydrocarbon contamination offreshwater ecosystems occurs frequently, but thespillages are generally of small magnitude. Un-less a special resource such as a drinking watersupply is contaminated, such "minor" spillages

are often neglected. Jamison et al. (154,155) didexamine the degradation ofgasoline in a contam-inated groundwater supply. They used stimu-lated biodegradation to enhance removal of hy-drocarbons from the contaminated water supply.Roubal et al. (240) followed the disappearanceof hydrocarbons from the Ohio River after a

major spillage of gasoline. They found that thehydrocarbons were rapidly removed. The micro-bial community was found to be capable of con-tributing to the disappearance of the contami-nating hydrcarbons; the biodegradative poten-tial was capable ofresponding within 1 to 2 days.Horowitz and Atlas (146) examined the fate of55,000 gal (ca. 209,000 liters) of gasoline whichhad contaminated an Arctic lake that served asa drinking water supply. In situ measurement ofgasoline degradation showed that, if untreated,sediment retained even "volatile" light hydro-carbons. Nutrient addition was found to enhancebiodegradative losses.

Several studies have examined the fate of oilin soil ecosystems. Some of these studies in-volved experimental contamination of soil toexamine the feasibility of using land farmng for

removal of oily wastes (100, 114, 130, 171, 182,193, 229). Concern has been expressed about theleaching of oil applied to soil into groundwater

supplies..There have been some reports on mo-bilization of oil into the soil column (271), but inmost cases there has been little evidence forsignificant downward leaching of oil (100, 229).Kincannon (171) applied residual oil from a re-finery tank, Bunker C fuel oil, and a waxy raffi-nate to soils and found a degradation rate of 8.3m3/4 x 103 m2 per month. Francke and Clark(114) rerorted a degradation rate of 11.9 m3/4x 103 m per month for used crankcase oil ap-plied to soil. Raymond et al. (229) conductedextensive field tests to examine the optimal con-ditions for oil degradation in soil. They foundthat rates of degradation did not exceed 2.4 i3/4 x 103 m2 per month.

Dibble and Bartha (102) examined the reha-bilitation of a New Jersey wheat field which hadbeen c t with approximately 1.9 mil-lion liters of kerosene over 1.5 hectares. A reha-bilitation program consisting of liming, fertiliz-ing, and frequent tilling was initiated, and thedecrease ofhydrocarbon contaminantswas mon-itored for a 2-year period. During the 2 years ofthe study, the hydrocarbon content of the sur-face oil decreased to an insigificant level. Sea-sonal differences were found in the rate of hy-drocarbon disappearance. Within 1 year afterthe spillage, the field returned to a near-normalproductive state. Odu (211) reported evidencefor microbial degradation of oil spilled on asandy soil in Nigeria from an oil well blowout.Several Arctic terrestial oil spills have beenexamined. Cook and Westlake (78) found evi-dence for extensive utilization of n-alkanes inoils applied in the Norman Wells area of thenorthwest territories and in the Swan Hill areaof northern Alberta. They also found evidencefor biodegradation of oil of the Nipisi spill innorthern Alberta. The spil was on a agnuimbog. Sexstone et al. (252), in contrast, foundevidence for greatly restricted rates of biodeg-radation in northern soils. They found that hy-drocarbons were still present in soils at FishCreek, Alaska, 28 years after contamination byspillage of refined oil.

CONCLUSIONS

The rates of biodegradation of hydrocarbonsfrom oil spills appear to be highly dependent onlocalized environmental conditions. It is appar-ent that the microbial degradation of oil pollu-tants is a complex process and that environmen-tal factors have a great influence on the fate ofspilled oil. The fate of many components inpetroleum, the degradative pathways which areactive in the environment, the importance of co-oxidation in natural ecosystems, and the role ofmicroorganisms in forming persistent environ-

198 ATLAS


http://mm

br.asm.org/

Dow

nloaded from



mental contaminants from hydrocarbons suchas the compounds found in tar balls are unknownand require future research. Although a numberof rate-limiting factors have been elucidated, theinteractive nature of microorganisms, oil, andenvironment still is not completely understood,and further examination of case histories is nec-essary to improve predictive understanding ofthe fate of oil pollutants in the environment andthe role of microorganisms in biodegradativeenvironmental decontamination. With an under-standing of the microbial hydrocarbon degrada-tion process in the environment, it should bepossible to develop models for predicting thefate of hydrocarbon pollutants and to developstrategies for utilizing microbial hydrocarbon-degrading activities for the removal of hydrocar-bons from contaminated ecosystems.

LITERATURE CITED1. Abbott, B. J., and W. G. Gledhill. 1971. The

extracellular accumulation of metabolic prod-ucts by hydrocarbon-degrading microorga-nisms. Adv. Appl. Microbiol. 14:249-388.

2. Ahearn, D. G., S. P. Meyers, and P. G. Stand-ard. 1971. The role of yeasts in the decompo-sition of oils in marine environments. Dev. Ind.Microbiol. 12:126-134.

3. Aminot, A. 1980. Mise en 6vidence et 6stimationquantitative de la biod6gradation in situ deshydrocarbures de L'Amoco Cadiz. In Proceed-ings of the International Symposium on theAmoco Cadiz: Fates and Effects ofthe Oil Spill.Centre Oceanologique de Bretagne, Brest,F rance, in press.

4. Arhelger, S. D., B. R. Robertson, and D. K.Button. 1977. Arctic hydrocarbon biodegra-dation, p. 270-275. In D. Wolfe (ed.), Fate andeffects of petroleum hydrocarbons in marineecosystems and organisms. Pergamon Press,Inc., Elmsford, N.Y.

5. Atlas, R. M. 1975. Effects of temperature andcrude oil composition on petroleum biodegra-dation. Appl. Microbiol. 30:396-403.

6. Atas, R. M. 1977. Stimulated petroleum biodeg-radation. Crit. Rev. MicrobioL 5:371-386.

7. Atlas, R. M. 1977. Studies on petroleum biodeg-radation in the Arctic, p. 261-269. In D. Wolfe(ed.), Fate and effects of petroleum hydrocar-bons in marine ecosystems and organisms. Per-gamon Press, Inc., Elmsford, N.Y.

8. Atlas, R. M. 1978. An assessment of the biodeg-radation of petroleum in the Arctic, p. 86-90.In M. W. Loutit and J. A. R. Miles (ed.),Microbial ecology. Springer-Verlag, Berlin.

9. Atlas, R. M. 1978. Measurement of hydrocarbonbiodegradation potentials and enumeration ofhydrocarbon utilizing microorganisms using 14Cradiolabelled spiked crude oil, p. 196-204. In J.W. Costerton and R. R. Colwell (ed.), Nativeaquatic bacteria: enumeration, activity andecology. ASTM-STP 695. American Societyfor Testing and Materials, Philadelphia.

10. Atlas, R. M., and R. Bartha. 1972. Biodegra-

dation of petroleum in seawater at low temper-atures. Can. J. Microbiol. 18:1851-1855.

11. Atlas, R. M., and R. Bartha. 1972. Degradationand mineralization of petroleum by two bacte-ria isolated from coastal water. Biotechnol.Bioeng. 14:297-308.

12. Atlas, R. M., and R. Bartha. 1972. Degradationand mineralization of petroleum in seawater:limitation by nitrogen and phosphorus. Bio-technol. Bioeng. 14:309-317.

13. Atlas, R. M., and R Bartha. 1973. Abundance,distribution and oil biodegradation potential ofmicroorganisms in Raritan Bay. Environ. Pol-lut. 4:291-300.

14. Atlas, R. M., and R. Bartha 1973. Effects ofsome commercial oil herders, dispersants andbacterial inocula on biodegradation of oil inseawater, p. 283-289. In D. G. Ahearn and S.P. Meyers (ed.), The microbial degradation ofoil pollutants. Publication no. LSU-SG-73-01.Center for Wetland Resources, Louisiana StateUniversity, Baton Rouge.

15. Atlas, R. M., and R. Bartha. 1973. Fate andeffects of oil pollution in the marine environ-ment. Residue Rev. 49:49-85.

16. Atlas, R. M., and R. Bartha. 1973. Inhibitionby fatty acids of the biodegradation of petro-leum. Antonie van Leeuwenhoek J. Microbiol.Serol. 39:257-271.

17. Atlas, R. M., and R. Bartha. 1973. Stimulatedbiodegradation of oil slicks using oleophilic fer-tilizers. Environ. Sci. Technol. 7:538-541.

18. Atlas, R. M., P. D. Boehm, and J. A. Calder.1981. Chemical and biological weathering of oilfrom the Amoco Cadiz oil spillage in the littoralzone. Estuarine Coastal Mar. Sci., in press.

19. Atlas, RK M., and A. Bronner. 1980. Microbialhydrocarbon degradation within intertidalzones impacted by the Amoco Cadiz oil spil-lage. In Proceedings of the International Sym-posium on the Amoco Cadiz: Fates and Effectsof the Oil Spill. Centre Oceanologique de Bre-tagne, Brest, France, in press.

20. Atlas, R. M., and M. Busdosh. 1976. Microbialdegradation of petroleum in the Arctic, p. 79-86. In J. M. Sharpley and A. M. Kaplan (ed.),Proceedings of the Third Intemational Biodeg-radation Symposium. Applied Science Publish-ers, Ltd., London.

21. Atlas, R. M., A. Horowitz, and M. Busdosh.1978. Prudhoe crude oil in arctic marine ice,water, and sediment ecosystems: degradationand interactions with microbial and benthiccommunities. J. Fish. Res. Board Can. 35:585-590.

22. Atlas, R. M., G. Roubal, A. Bronner, and J.Haines. 1980. Microbial degradation of hydro-carbons in mousse from IXTOC-I. In Proceed-ings of Conference on Researcher/PierceIXTOC-I Cruises. National Oceanographic andAtmospheric Administration, AOML, Miami,in press.

23. Atlas, R. M., G. Roubal, and J. Haines. 1980.Biodegradation of hydrocarbons in moussefrom the IXTOC-I well blowout. In Proceed-ings of Symposium on Assessment of the En-

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


MICROBIOL. REV.

vironmental Impact of Accidental Oil Spills inthe Oceans. Presented before the Division ofEnvironmental Chemistry of the AmericanChemical Society, 24-29 August 1980, SanFrancisco. Ann Arbor Science Publications,Inc., Ann Arbor, Mich.

24. Atlas, R. M., E. A. Schofield. 1975. Petroleumbiodegradation in the Arctic, p. 183-198. In A.W. Bourquin, D. G. Ahearn, and S. P. Meyers(ed.), Impact of the use of microorganisms onthe aquatic environment. U.S. EnvironmentalProtection Agency, Corvallis, Ore.

25. Atlas, R. M., E. A. Schofield, F. A. Morelli,and R. E. Cameron. 1976. Interactions ofmicroorganisms and petroleum in the Arctic.Environ. Pollut. 10:35-44.

26. Austin, B., J. J. Calomiris, J. D. Walker, andR. R. Colwell. 1977. Numerical taxonomy andecology of petroleum degrading bacteria. Appl.Environ. Microbiol. 34:60-68.

27. Austin, B., R. R. Colwell, J. D. Walker, andJ. J. Calomiris. 1977. The application of nu-merical taxonomy to the study of petroleumdegrading bacteria isolated -from the aquaticenvironment. Dev. Ind. Microbiol. 18:685-696.

28. Bailey, N. J. L., A. M. Jobson, and M. A.Rogers. 1973. Bacterial degradation of crudeoil: comparison of field and experimental data.Chem. Geol. 11:203-221.

29. Bailey, C. A., and M. E. May. 1979. Evaluationof microbiological test kits for hydrocarbon fuelsystems. Appl. Environ. Microbiol. 37:871-877.

30. Barnsley, E. A. 1975. The bacterial degradationof fluoranthene and benzo(a)pyrene. Can. J.Microbiol. 21:1004-1008.

31. Barsdate, R. M. 1973. Ecologic changes in anArctic tundra pond following exposure to crudeoil, p. 52. In Impact of oil resource developmenton northern plant communities. OccasionalPublications of Northern Life no. 1. Instituteof Arctic Biology, University of Alaska, Fair-banks.

32. Bartha, R., and R. M. Atlas. 1973. Biodegra-dation of oil in seawater: limiting factors andartificial stimulation, p. 147-152. In D. G.Ahearn and S. P. Meyers (ed.), The microbialdegradation of oil pollutants. Publication no.LSU-SG-73-01. Center for Wetland Resources,Louisiana State University, Baton Rouge.

33. Bartha, R., and R. M. Atlas. 1976. Biodegra-dation of oil on water surfaces. U.S. patent3,939,127, May 25, 1976.

34. Bartha, R., and R. M. Atlas. 1977. The micro-biology of aquatic oil spills. Adv. Appl. Micro-biol. 22:225-266.

35. Beam, H. W., and J. J. Perry. 1973. Co-metab-olism as a factor in microbial degradation ofcycloparaffinic hydrocarbons. Arch. Mikrobiol.91:87-90.

36. Beam, H. W., and J. J. Perry. 1974. Microbialdegradation and assimilation of n-alkyl-substi-tuted cycloparaffins. J. Bacteriol. 118:394-399.

37. Beam, H. W., and J. J. Perry. 1974. Microbialdegradation of cycloparaffinic hydrocarbons

via co-metabolism and commensalism. J. Gen.Microbiol. 82:163-169.

38. Bergstein, P. E., and J. R. Vestal. 1978. Crudeoil biodegradation in Arctic tundra ponds. Arc-tic 31:158-169.

39. Berridge, S. A., R. A. Dean, R. G. Fallows,and A. Fish. 1968. The properties of persistentoils at sea, p. 2-11. In P. Hepple (ed.), Scientificaspects of pollution of the sea by oil. Instituteof Petroleum, London.

40. Berridge, S. A., M. T. Thew, and A. G. Lor-iston-Clarke. 1968. The formation and stabil-ity of emulsions of water in crude petroleumand similar stocks, p. 35-59. In P. Hepple (ed.),Scientific aspects of pollution of the sea by oil.Institute of Petroleum, London.

41. Blumer, M., M. Ehrhardt, and J. H. Jones.1972. The environmental fate ofstranded crudeoil. Deep Sea Res. 20:239-259.

42. Blumer, M., and J. Sass. 1972. Indigenous andpetroleum-derived hydrocarbons in a pollutedsediment. Mar. Pollut. Bull. 3:92-94.

43. Blumer, M., and J. Sass. 1972. Oil pollution:persistence and degradation of spilled fuel oil.Science 176:1120-1122.

44. Boehm, P. D., and D. L. Fiest. 1980. Compar-ative weathering patterns of hydrocarbonsfrom the Amoco Cadiz oil spill observed at avariety of coastal environments. In Proceed-ings of the International Symposium on theAmoco Cadiz: Fates and Effects of the Oil Spill.Centre Oceanologique de Bretagne, Brest,France, in press.

45. Boehm, P. D., and D. L. Fiest. 1980. Aspects ofthe transport of petroleum hydrocarbons tothe offshore benthos during the IXTOC-Iblowout in the Bay of Campeche. In Proceed-ings of Conference on Researcher/PierceIXTOC-I Cruises. National Oceanographic andAtmospheric Administration, AOML, Miami,in press.

46. Boylan, D. B., and B. W. Tripp. 1971. Deter-mination of hydrocarbons in seawater extractsof crude oil and crude oil fractions. Nature(London) 230:44-47.

47. Bridie, A. L., and J. Bos. 1971. Biological deg-radation of mineral oil in seawater. J. Inst. Pet.London 57:270-277.

48. Brown, D. W., L. S. Ramos, A. J. Fiedman,and W. D. Macleod. 1969. Analysis of tracelevels of petroleum hydrocarbons in marinesediments using a solvent-slurry extractionprocedure, p. 161-167. In Trace organic analy-sis: a new frontier in analytical chemistry. Spe-cial publication no. 519. National Bureau ofStandards, Washington, D.C.

49. Brown, L. R., W. E. Phillips, G. S. Pabst, andC. M. Ladner. 1969. Physical, chemical andmicrobiological changes occurring during deg-radation of oil in aquatic and brackish waterenvironments. Presented at the American So-ciety of Mechanical Engineers Annual WinterMeeting, 16-20 November, Los Angeles, Calif.

50. Brown, L. R., and R. G. Tischer. 1969. The

200 ATLAS


http://mm

br.asm.org/

Dow

nloaded from



decomposition of petroleum products in ournatural waters. Water Resources Research In-stitute, Mississippi State University, State Col-lege.

51. Buckley, E. N., R. B. Jonas, and F. K. Pfaen-der. 1976. Characterization of microbial iso-lates from an esturarine ecosystem: relation-ship of hydrocarbon utilization to ambient hy-drocarbon concentrations. Appl. Environ. Mi-crobiol. 32:232-237.

52. Buckley, E. N., and F. K. Pfaender. 1980.Response of the pelagic microbial communityto oil from the IXTOC-I blowout: II. Modelecosystem studies. In Proceedings of Confer-ence on Researcher/Pierce IXTOC-I Cruises.National Oceanographic and Atmospheric Ad-ministration, AOML, Miami, in press.

53. Bunch, J. N., and R C. Harland. 1976. Biodeg-radation of crude petroleum by the indigenousmicrobial flora of the Beaufort Sea. BeaufortSea Technical Report 10. Environment Can-ada, Victoria, B.C.

54. Burwood, R., and G. C. Speers. 1974. Photo-oxidation as a factor in the environmental dis-persal of crude oil. Estuarine Coastal Mar. Sci.2:117-135.

55. Calder, J. A., and P. D. Boehm. 1980. Yearstudy of weathering processes acting on theAmoco Cadiz oil spill. In Proceedings of theInternational Symposium on the Amoco Cadiz:Fates and Effects of the Oil Spill. CentreOceanologique de Bretagne, Brest, France, inpress.

56. Calomiris, J. J., B. Austin, J. D. Walker, andR. R. Colwell. 1976. Enrichment for estuarinepetroleum-degrading bacteria using liquid andsolid media. J. Appl. BacterioL 42:135-144.

57. Cantwell, S. G., E. P. Lau, D. S. Watt, and R.R. Fall. 1978. Biodegradation of acyclic iso-prenoids by Pseudomonas species. J. Bacteriol.135:324-333.

58. Caparello, D. M., and P. A. Larock. 1975. Aradioisotope assay for the quantification of hy-drocarbon biodegradation potential in environ-mental samples. Microb. Ecol. 2:28-42.

59. Cerniglia, C. E., and D. T. Gibson. 1977. Me-tabolism of naphthalene by Cunninghamellaelegans. Appl. Environ. Microbiol. 34:363-370.

60. Cerniglia, C. E., and D. T. Gibson. 1978. Me-tabolism of naphthalene by cell extracts ofCunninghameeUa elegans. Arch. Biochem. Bio-phys. 186:121-127.

61. Cerniglia, C. E., and D. T. Gibson. 1979. Algaloxidation of aromatic hydrocarbons: formationof 1-naphthol from naphthalene by Agmenel-lum quadruplication, strain PR-6. Biochem.Biophys. Res. Commun. 88:50-58.

62. Cerniglia, C. E., and D. T. Gibson. 1980. Fun-gal oxidation of benzo(a)pyrene and (+)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene: evi-dence for the formation of a benzo(a)pyrene7,8-diol-9,10-epoxide. J. Biol. Chem. 225:5159-5163.

63. Cerniglia, C. E., and D. T. Gibson. 1980. Fun-

gal oxidation of (±)-9,10 dihydroxy-9,10-dihy-drobenzo(a)pyrene: formation of diastereo-metic benzo(a)pyrene 9,10-diol 7,8-epoxides.Proc. Natl. Acad. Sci. U.S.A. 77:4554-4558.

64. Cerniglia, C. E., D. T. Gibson, and C. vanBaalen. 1980. Oxidation of naphthalene bycyanobacteria and microalgae. J. Gen. Micro-biol. 116:495-500.

65. Cerniglia, C. E., R. L. Hebert, R. H. Dodge,P. J. Szanszlo, and D. T. Gibson. 1978.Some approaches to studies on the degradationof aromatic hydrocarbons by fungi, p. 360-369.In A. L. Bourquin and P. H. Pritchard (ed.),Microbial degradation of pollutants in marineenvironments. EPA-600/9-79-012. Environ-mental Research Laboratory, Gulf Breeze, Fla.

66. Cerniglia, C. E., R L. Hebert, P. J. Szaniszlo,and D. T. Gibson. 1978. Fungal transforma-tion of naphthalene. Arch Microbiol. 117:135-143.

67. Cerniglia, C. E., and J. J. Perry. 1973. Crudeoil degradation by microorganisms isolatedfrom the marine environment. Z. AlUg. Mikro-biol. 13:299-306.

68. Cerniglia, C. E., C. van Baalen, and D. T.Gibson. 1980. Metabolism of naphthalene bythe cyanobacterium Oscillatoria sp., strainJCM. J. Gen. Microbiol. 116:485-494.

69. Cerniglia, C. E., C. van Baalen, and D. T.Gibson. 1980. Oxidation of bi-phenyl by thecyanobacterium Oscillatoria sp., strain JCM.Arch. Microbiol., in press.

70. Chakrabarty, A. M. 1972. Genetic basis of thebiodegradation of salicylate in Pseudomonas.J. Bacteriol. 112:815-823.

71. Chakrabarty, A. M., G. Chou, and L. C. Gun-salus. 1973. Genetic regulation of octane dis-similation plasmid in Pseudomonas. Proc.Natl. Acad. Sci. U.S.A. 70:1137-1140.

72. Chouteau, J., E. Azoulay, and J. C. Senez.1962. Anaerobic formation ofn hept-l-ene fromn heptane by resting cells of Pseudomonasaeruginosa. Nature (London) 194:576-578.

73. Cobet, A. B., and H. E. Guard. 1973. Effect ofa bunker fuel on the beach bacterial flora, p.815-819. In Proceedings of Joint Conferenceon Prevention and Control of Oil Spills. Amer-ican Petroleum Institute, Washington, D.C.

74. Colwell, R R 1978. Enumeration of specificpopulations by the most-probable-number(MPN) method, p. 56-61. In J. W. Costertonand R. R. Colwell (ed.), Native aquatic bacte-na: enumeration, activity and ecology. ASTM-STP 695. American Society for Testing andMaterials, Philadelphia.

75. Colwell, R RK, A. L Mills, J. D. Walker, P.Garcia-Tello, and V. Campos-P. 1978. Mi-crobial ecology studies of the Metula spill inthe Straits of Magellan. J. Fish. Res. BoardCan. 35:573-580.

76. Colwell, RK R., and J. D. Walker. 1977. Eco-logical aspects of microbial degradation of pe-troleum in the marine environment. Crit. Rev.Microbiol. 5:423-445.

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


MICROBIOL. REV.

77. Colwell, R. R., J. D. Walker, and J. D. Nelson,Jr. 1973. Microbial ecology and the problem ofpetroleum degradation in Chesapeake Bay, p.185-197. In D. G. Ahearn and S. P. Meyers(ed.), The microbial degradation of oil pol-lutants. Publication no. LSU-SG-73-01. Centerfor Wetland Resources, Louisiana State Uni-versity, Baton Rouge.

78. Cook, F. D., and D. W. S. Westlake. 1974.Microbiological degradation of northern crudeoils. Environmental-Social Committee; North-ern Pipelines, Task Force on Northern Oil De-velopment, report no. 74-1. Catalog no. R72-12774. Information Canada, Ottawa.

79. Cook, W. L, J. K. Massey, and D. G. Ahearn.1973. The degradation of crude oil by yeastsand its effects on Lesbistes reticulatus, p. 279-282. In D. G. Ahearn and S. P. Meyers (ed.),The microbial degradation of oil pollutants.Publication no. LSU-SG-73-01. Center forWetland Resources, Louisiana State Univer-sity, Baton Rouge.

80. Cooney, J. J., P. Edmonds, and Q. M. Bren-ner. 1968. Growth and survival of fuel isolatesin hydrocarbon-fuel emulsions. Appl. Micro-biol. 16:569-571.

81. Cooney, J. J., and R. J. Summers. 1976. Hy-drocarbon-using microorganisms in threefreshwater ecosystems, p. 141-156. In J. M.Sharpley and A. M. Kaplan (ed.), Proceedingsof the Third International BiodegradationSymposium. Applied Science Publishers, Ltd.,London.

82. Cooney, J. J., and J. D. Walker. 1973. Hydro-carbon utilization by Cladosporium resinae, p.25-32. In D. G. Ahearn and S. P. Meyers (ed.),The microbial degradation of oil pollutants.Publication no. LSU-SG-73-01. Center forWetland Resources, Louisiana State Univer-sity, Baton Rouge.

83. Cowell, E. B. (ed.). 1971. The ecological effectsof oil pollution on littoral communities. Ap-plied Science Publishers, Ltd., London.

84. Cretney, W. I., C. S. Wong, D. R. Green, andC. A. Bawden. 1978. Long-term fate of aheavy fuel oil in a spill-contaminated coastalbay. J. Fish. Res. Board Can. 35:521-527.

85. Cripps, R. E., and R. J. Watkinson. 1978.Polycyclic aromatic hydrocarbons: metabolismand environmental aspects, p. 113-134. In J. R.Watkinson (ed.), Developments in biodegra-dation of hydrocarbons-i. Applied SciencePublishers, Ltd., London.

86. Crow, S. A., W. L. Cook, D. G. Ahearn, andA. W. Bourquin. 1976. Microbial populationsin coastal surface slicks, p. 93-98. In J. M.Sharpley and A. M. Kaplan (ed.), Proceedingsof the Third International BiodegradationSymposium. Applied Science Publishers, Ltd.,London.

87. Crow, S. A., S. P. Meyers, and D. G. Ahearn.1974. Microbiological aspects ofpetroleum deg-radation in the aquatic environment. Mer 12:37-54.

88. Cundell, A. M., and R. W. Traxler. 1973. The

isolation and characterization of hydrocarbon-utilizing bacteria from Chedabucto Bay, NovaScotia, p. 421-426. In Proceedings of JointConference on Prevention and Control of OilSpills. American Petroleum Institute, Wash-ington, D.C.

89. Cundell, A. M., and R. W. Traxler. 1974. Hy-drocarbon degrading bacteria associated withArctic oil seeps. Dev. Ind. Microbiol. 15:250-255.

90. Cundell, A. M., and R. W. Traxler. 1976. Psy-chrophilic hydrocarbon-degrading bacteriafrom Narragansett Bay, Rhode Island, U.S.A.Mater. Org. 11:1-17.

91. Davies, J. A., and D. E. Hughes. 1968. Thebiochemistry and microbiology ofcrude oil deg-radation, p. 139-144. In J. D. Carthy and D. R.Arthur (ed.), The biological effects of oil pol-lution on littoral communities (supplement toField Stud., vol. 2). E. W. Classey, Ltd., Hamp-ton, Middesex, England.

92. Davies, J. S., and D. W. S. Westlake. 1979.Crude oil utilization by fungi. Can. J. Microbiol.25:146-156.

93. Davis, J. B. 1956. Microbial decomposition ofhydrocarbons. Ind. Eng. Chem. 48:1444-1448.

94. Davis, S. J., and C. F. Gibbs. 1975. The effectof weathering on a crude oil residue exposed atsea. Water Res. 9:275-285.

95. Dean, R. A. 1968. The chemistry of crude oils inrelation to their spillage on the sea, p. 1-6. InJ. D. Carthy and D. R. Arthur (ed.), Thebiological effects of oil pollution on littoralcommunities (supplement to Field Stud., vol.2). E. W. Classey, Ltd., Hampton, Middlesex,England.

96. Dean-Raymond, D., and R. Bartha. 1975. Bi-odegradation of some polynuclear aromatic pe-troleum components by marine bacteria. Dev.Ind. Microbiol. 16:97-110.

97. Delaune, R. D., G. A. Hambrick m, and W.H. Patrick, Jr. 1980. Degradation of hydro-carbons in oxidized and reduced sediments.Mar. Pollut. Bull. 11:103-106.

98. Diamond v. Chakrabarty. 1980. 48 U.S.L.W.4714 (U.S. June 16, 1980).

99. Dibble, J. T., and R. Bartha. 1976. The effectof iron on the biodegradation of petroleum inseawater. Appl. Environ. Microbiol. 31:544-550.

100. Dibble, J. T., and R. Bartha. 1979. Effect ofenvironmental parameters on the biodegrada-tion of oil sludge. Appl. Environ. Microbiol. 37:729-739.

101. Dibble, J. T., and R. Bartha. 1979. Leachingaspects of oil sludge biodegradation in soil. SoilSci. 127:365-370.

102. Dibble, J. T., and R. Bartha. 1979. Rehabili-tation of oil-inundated agricultural land: a casehistory. Soil Sci. 128:56-60.

103. Donoghue, N. A., M. Griffin, D. G. Norris,and P. W. Trudgill. 1976. The microbial me-tabolism of cyclohexane and related com-pounds, p. 43-56. In J. M. Sharpley and A. M.Kaplan (ed.),' Proceedings of the Third Inter-

202 ATLAS


http://mm

br.asm.org/

Dow

nloaded from


BIODEGRADATION OF PETROLEUMW HYDROCARBONS 203

national Biodegradation Symposium. AppliedScience Publishers, Ltd., London.

104. Dunn, N. W., and L. C. Gunsalus. 1973. Trans-missible plasnid coding early enzymes ofnaph-thalene oxidation in Pseudomonas putida. J.Bacteriol. 114:974-979.

105. FalL R. R., J. L. Brown, and T. L Schaeffer.1979. Enzyme recruitment allows the biodeg-radation of recalcitrant branched hydrocar-bons by Pseudomonas citronellolis. Appl. En-viron. Microbiol. 38:715-722.

106. Ferris, J. P., L H. MacDonald, M. A. Patrie,and M. A. Martin. 1976. Aryl hydrocarbonhydroxylase activity in the fungus Cunning-hamella bainierii. Evidence of the presence ofcytochrome P450. Arch. Biochem. Biophys.175:443-452.

107. Floodgate, G. D. 1972. Biodegradation of hydro-carbons in the sea, p. 153-171. In R. Mitchell(ed.), Water pollution microbiology. JohnWiley & Sons, Inc., New York.

108. Floodgate, G. D. 1972. Microbial degradation ofoiL Mar. Pollut. Bull. 3:41-43.

109. Floodgate, G. D. 1973. A threnody concerningthe biodegradation of oil in natural waters, p.17-24. In D. G. Ahearn and S. P. Meyers (ed.),The microbial degradation of oil pollutants.Publication no. LSU-SG-73-01. Center forWetland Resources, Louisiana State Univer-sity, Baton Rouge.

110. Floodgate, G. D. 1976. Oil biodegradation in theoceans, p. 87-92. In J. M. Sharpley and A. M.Kaplan (ed.), Proceedings of the Third Inter-national Biodegradation Symposium. AppliedScience Publishers, Ltd., London.

111. Floodgate, G. D. 1979. Nutrient limitation, p.107-118. In A. W. Bourquin and P. H. Prit-chard (ed.), Proceedings of workshop, Micro-bial Degradation of Pollutants in Marine En-vironments. EPA-66019-79-012. Environmen-tal Research Laboratory, Gulf Breeze, Fla.

112. Foster, J. W. 1962. Bacterial oxidation of hydro-carbons, p. 241-271. In 0. Hiyashi (ed.), Oxy-genases. Academic Press, Inc., New York.

113. Foster, J. W. 1962. Hydrocarbons as substratesfor microorganisms. Antonie van LeeuwenhoekJ. Microbiol. Serol. 28:241-274.

114. Francke, H. C., and F. E. Clark. 1974. Disposalof oil wastes by microbial assimilation. ReportY-1934. U.S. Atomic Energy Commission,Washington, D.C.

115. Frankenfeld, J. W. 1973. Factors governing thefate of oil at sea; variations in the amounts andtypes of dissolved or dispersed materials duringthe weathering process, p. 485-495. In Proceed-ings of Joint Conference on Prevention andControl of Oil Spills. American Petroleum In-stitute, Washington, D.C.

116. Friello, D. A., J. R. Mylroie, and A. M. Chak-rabarty. 1976. Use of genetically engineeredmulti-plasmid microorganisms for rapid deg-radation of fuel hydrocarbons, p. 205-214. In J.M. Sharpley and A. M. Kaplan (ed.), Proceed-ings of the Third International Biodegradation

Symposium. Applied Science Publishers, Ltd.,London.

117. Gatellier, C. R. 1971. Les facteurs limitant labiodegradation des hydrocarbures dansl'epuration des eaux. Chim. Ind. (Paris) 104:2283-2289.

118. Gatellier, C. R., J. L Oudin, P. Fusey, J. C.Lacase, and M. L. Priou. 1973. Experimentalecosystems to measure fate of oil spills dis-persed by surface active products, p. 497-507.In Proceedings of Joint Conference on Preven-tion and Control of Oil Spills. American Petro-leum Institute, Washington, D.C.

119. Gibbs, C. F. 1975. Quantitative studies in marinebiodegradation of oil. I. Nutrient limitation at14°C. Proc. R. Soc. London Ser. B 188:61-82.

120. Gibbs, C. F., and S. J. Davis. 1976. The rate ofmicrobial degradation of oil in a beach gravelcolumn. Microb. Ecol. 3:55-64.

121. Gibbs, C. F., K. B. Pugh, and A. R. Andrews.1975. Quantitative studies in marine biodegra-dation of oil. II. Effects of temperature. Proc.R. Soc. London Ser. B 188:83-94.

122. Gibson, D. T. 1968. Microbial degradation ofaromatic compounds. Science 161:1093-1097.

123. Gibson, D. T. 1971. The microbial oxidation ofaromatic hydrocarbons. Crit. Rev. Microbiol.1:199-223.

124. Gibson, D. T. 1975. Oxidation of the carcinogensbenzo(a)pyrene and benzo(a)anthracene to di-hydrodiols by a bacterium. Science 189:295-297.

125. Gibson, D. T. 1976. Microbial degradation ofpolycyclic aromatic hydrocarbons, p. 57-66. InJ. M. Sharpley and A. M. Kaplan (ed.), Pro-ceedings of the Third International Biodegra-dation Symposium. Applied Science Publish-ers, Ltd., London.

126. Gibson, D. T. 1977. Biodegradation of aromaticpetroleum hydrocarbons, p. 36-46. In D. Wolfe(ed.), Fate and effects of petroleum hydrocar-bons in marine ecosystems and organisms. Per-gamon Press, Inc., New York.

127. Gibson, D. T., and W. K. Yeh. 1973. Microbialdegradation of aromatic hydrocarbons, p. 33-38. In D. G. Ahearn and S. P. Meyers (ed.),The microbial degradation of oil pollutants.Publication no. LSU-SG-73-01. Center forWetland Resources, Louisiana State Univer-sity, Baton Rouge.

128. Gordon, D. C., J. Dale, and P. D. Keizer. 1978.Importance of sediment working by the de-posit-feeding polychaete Arenicola marina onthe weathering rate of sediment-bound oil. J.Fish. Res. Board Can. 35:591-603.

129. Grouse, P. L., J. S. Mattson, and H. Petersen(ed.). 1979. USNS Potomac oil spill MelvilleBay, Greenland, 5 August 1977. NOAA-S/T79-202. U.S. Department of Commerce, Wash-ington, D.C.

130. Gudin, C., and W. J. Syratt. 1975. Biologicalaspects of land rehabilitation following hydro-carbon contamination. Environ. Pollut. 8:107-112.

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


MICROBIOL. REV.

131. Guire, P. E., J. D. Friede, and R. K. Gholson.1973. Production and characterization of emul-sifying factors from hydrocarbonoclastic yeastand bacteria, p. 229-231. In D. G. Ahearn andS. P. Meyers (ed.), The microbial degradationof oil pollutants. Publication no. LSU-SG-73-01. Center for Wetland Resources, LouisianaState University, Baton Rouge.

132. Gunkel, W. 1967. Experimentell-okologischeUntersuchungen uber die limitierenden Fak-toren des mikrobiellen Olabbaues in marinenMilieu. Helgol. Wiss. Meeresunters. 15:210-224.

133. Gunkel, W. 1968. Bacteriological investigationsof oil-polluted sediments from the Cornishcoast following the "Torrey Canyon" disaster,p. 151-158. In J. D. Carthy and D. R. Arthur(ed.), The biological effects of oil pollution onlittoral communities (supplement to FieldStud., vol. 2). E. W. Classey, Ltd., Hampton,Middlesex, England.

134. Gunkel, W. 1968. Bacteriological investigationsof oil-polluted sediments from the Cornishcoast following the Torrey Canyon disaster.Helgol. Wiss. Meeresunters. 17:151-158.

135. Gunkel, W., G. Gassmann, C. H. Oppenhei-mer, and I. Dundas. 1980. Preliminary resultsof baseline studies of hydrocarbons and bacte-ria in the North Sea: 1975, 1976 and 1977, p.223-247. In Ponencias del Simposio Interna-tional en: Resistencia a los Antibiosis y Micro-biologia marina. Santiago de Compostela,Spain.

136. Gutnick, D. L, and E. Rosenberg. 1977. Oiltankers and pollution: a microbiological ap-proach. Annu. Rev. Microbiol. 31:379-396.

137. Haines, J. R., and M. Alexander. 1974. Micro-bial degradation of high-molecular weight al-kanes. Appl. Microbiol. 28:1084-1085.

138. Hambrick, G. A., m, R. DeLaune, and W. H.Patrick, Jr. 1980. Effect of estuarine sedimentpH and oxidation-reduction potential on mi-crobial hydrocarbon degradation. Appl. Envi-ron. Microbiol. 40:365-369.

139. Harrison, W., M. A. Winnik, P. T. Y. Kwong,and D. Mackay. 1975. Crude oil spills. Dis-appearance of aromatic and aliphatic compo-nents from small sea-surface slicks. Environ.Sci. Technol. 9:231-234.

140. Herbes, S. E., and L. R. Schwall. 1978. Micro-bial transformation of polycyclic aromatic hy-drocarbons in pristane and petroleum-contam-inated sediments. Appl. Environ. Microbiol.35:306-316.

141. Higashihara, T., A. Sato, and U. Simidu.1978. An MPN method for the enumeration ofmarine hydrocarbon degrading bacteria. Bull.Jpn. Soc. Sci. Fish. 44:1127-1134.

142. Hill, E. C. 1978. Biodegradation of hydrocarbon-based products in industrial use, p. 201-226. InJ. R. Watkinson (ed.), Developments in bio-degradation of hydrocarbons-1. Applied Sci-ence Publishers, Ltd., London.

143. Hill, E. C., and A. R. Thomas. 1976. Microbio-logical aspects of supersonic aircraft fuel, p.

157-174. In J. M. Sharpley and A. M. Kaplan(ed.), Proceedings of the Third InternationalBiodegradation Symposium. Applied SciencePublishers, Ltd., London.

144. Hopper, D. J. 1978. Microbial degradation ofaromatic hydrocarbons, p. 85-112. In J. R.Watkinson (ed.), Developments in biodegra-dation of hydrocarbons-i. Applied SciencePublishers, Ltd., London.

145. Horowitz, A., and R. M. Atlas. 1977. Continu-ous open flow-through system as a model foroil degradation in the Arctic Ocean. Appl. En-viron. Microbiol. 33:647-653.

146. Horowitz, A., and R. M. Atlas. 1977. Responseof microorganisms to an accidental gasolinespillage in an Arctic freshwater ecosystem.Appl. Environ. Microbiol. 33:1252-1258.

147. Horowitz, A., and R. M. Atlas. 1978. Crude oildegradation in the Arctic: changes in bacterialpopulations and oil composition during oneyear exposure in a model system. Dev. Ind.Microbiol. 19:517-522.

148. Horowitz, A., D. Gutnick, and E. Rosenberg.1975. Sequential growth of bacteria on crudeoil. Appl. Microbiol. 30:10-19.

149. Horowitz, A., A. Sexstone, and R. M. Atlas.1978. Hydrocarbons and microbial activities insediment of an Arctic lake one year after con-tamination with leaded gasoline. Arctic 31:180-191.

150. Horvath, R. S. 1972. Microbial co-metabolismand the degradation of organic compounds innature. Bacteriol. Rev. 36:146-155.

151. Hou, C. T., and A. I. Laskin. 1976. Microbialconversion of dibenzothiophene. Dev. Ind. Mi-crobiol. 17:351-362.

152. Hunt, P. G., W. E. Rickard, F. J. Deneke, F.R. Koutz, and R. P. Murrmann. 1973. Ter-restrial oil spills in Alaska: environmental ef-fects and recovery, p. 733-740. In Proceedingsof the Joint Conference on Prevention andControl of Oil Spills. American Petroleum In-stitute, Washington, D.C.

153. Iizuka, H., M. Ilida, and S. Fujita. 1969. For-mation of n-decene-1 from n-decane by restingcells of C. rugosa. Z. Allg. Mikrobiol. 9:223-226.

154. Jamison, V. M., R. L. Raymond, and J. 0.Hudson, Jr. 1975. Biodegradation of high-oc-tane gasoline in groundwater. Dev. Ind. Micro-biol. 16:305-312.

155. Jamison, V. M., R. L. Raymond, and J. 0.Hudson. 1976. Biodegradation of high-octanegasoline, p. 187-196. In J. M. Sharpley and A.M. Kaplan (ed.), Proceedings of the Third In-ternational Biodegradation Symposium. Ap-plied Science Publishers, Ltd., London.

156. Jannasch, H. W., K. Eimhjellen, C. 0. Wir-sen, and A. Farmaian. 1971. Microbial deg-radation of organic matter in the deep sea.Science 171:672-675.

157. Jensen, V. 1975. Bacterial flora of soil afterapplication of oily waste. Oikos 26:152-158.

158. Jobson, A., F. D. Cook, and D. W. S.

204 ATLAS


http://mm

br.asm.org/

Dow

nloaded from



Westlake. 1972. Microbial utilization of crudeoil. Appl. Microbiol. 23:1082-1089.

159. Jobson, A., M. McLaughlin, F. D. Cook, andD. W. S. Westlake. 1974. Effect of amend-ments on the microbial utilization of oil appliedto soil. Appl. Microbiol. 27:166-171.

160. Johnston, R. 1970. The decomposition of crudeoil residues in sand columns. J. Mar. Biol.Assoc. U.K. 50:925-937.

161. Jones, J. G., and M. A. Eddington. 1968. Anecological survey of hydrocarbon-oxidizing mi-croorganisms. J. Gen. Microbiol. 52:381-390.

161a.Jordan, R. E., and J. R. Payne. 1980. Fate andweathering of petroleum spills in the marineenvironment. Ann Arbor Science Publications,Inc., Ann Arbor, Mich.

162. Jurtehuk, P., and G. E. Cardini. 1971. Themechanism of hydrocarbon oxidation by a Co-rynebacterium species. Crit. Rev. Microbiol. 1:239-289.

163. Kappeler, Th., and K. Wuhrmann. 1978. Mi-crobial degradation of the water-soluble frac-tion of gas oil-I. Water Res. 12:327-334.

164. Kappeler, Th., and K. Wuhrmann. 1978. Mi-crobial degradation of the water-soluble frac-tion of gas oil-IH. Bioassays with pure strains.Water Res. 12:335-342.

165. Karrick, N. 1977. Alterations in petroleum re-sulting from physicochemical and microbiolog-ical factors, p. 225-299. In D. C. Malins (ed.),Effects of petroleum on Arctic and subarcticmarine environments and organisms, vol. 1.Nature and fate of petroleum. Academic Press,Inc., New York.

166. Kator, H. 1973. Utilization of crude oil hydro-carbons by mixed cultures of marine bacteria,p. 47-66. In D. G. Ahearn and S. P. Meyers(ed.), The microbial degradation of oil pol-lutants. Publication no. LSU-SG-73-01. Centerfor Wetland Resources, Louisiana State Uni-versity, Baton Rouge.

167. Kator, H., and R. Herwig. 1977. Microbial re-sponses after two experimental oil spills in aneastern coastal plain estuarine ecosystem, p.517-522. In Proceedings of the 1977 Oil SpillConference. American Institute of Petroleum,Washington, D.C.

168. Kator, H., R. Miget, and C. H. Oppenheimer.1972. Utilization of paraffin hydrocarbons incrude oil by mixed cultures of marine bacteria.Paper no. SPE 4206. Symposium on Environ-mental Conservation. Society ofPetroleum En-gineers, Dallas, Tex.

169. Kator, H., C. H. Oppenheimer, and R. J.Miget. 1971. Microbial degradation of a Louis-iana crude oil in closed flasks and under simu-lated field conditions, p. 287-296. In Joint Con-ference on Prevention and Control of Oil Spills.American Petroleum Institute, Washington,D.C.

170. Keizer, P. D., T. P. Ahern, J. Dale, and J. H.Vandermeulen. 1978. Residues of Bunker Coil in Chedabucto Bay, Nova Scotia, 6 yearsafter the Arrow spill. J. Fish Res. Board Can.35:528-535.

171. Kincannon, C. B. 1972. Oily waste disposal bysoil cultivation process. EPA-R2-72-100. U.S.Environmental Protection Agency, Washing-ton, D.C.

172. King, D. H., and J. J. Perry. 1975. The originof fatty acids in the hydrocarbon-utilizing mi-croorganism, Mycobacterium vaccae. Can. J.Microbiol. 21:85-89.

173. Kinney, P. J., D. K. Button, and D. M. Schell.1969. Kinetics of dissipation and biodegrada-tion of crude oil in Alaska's Cook Inlet, p. 333-340. In Proceedings of 1969 Joint Conferenceon Prevention and Control of Oil Spills. Amer-ican Petroleum Institute, Washington, D.C.

174. Klug, M. J., and A. J. Markovetz. 1967. Deg-radation of hydrocarbons by members of thegenus Candida. J. Bacteriol. 93:1847-1852.

175. Klug, M. J., and A. J. Markovetz. 1967. Ther-mophilic bacteria isolated on n-tetradecane.Nature (London) 215:1082-1083.

176. Kodama, K., S. Nakatani, K. Umehara, K.Shimizu, Y. Minoda, and K. Yamada. 1970.Microbial conversion of petro-sulfur com-pounds. m. Isolation and identification ofproducts from dibenzothiophene. Agric. Biol.Chem. 34:1320-1324.

177. Komagata, K., T. Nakase, and N. Katsuya.1964. Aimilation of hydrocarbons by yeasts.I. Preliminary screening. J. Gen. Appl. Micro-biol. 10:313-321.

178. Lee, R. F. 1977. Accumulation and turnover ofpetroleum hydrocarbons in marine organisms,p. 60-70. In D. A. Wolfe (ed.), Fate and effectsof petroleum hydrocarbons in marine orga-nisms and ewsystems. Pergamon Press, Inc.,New York.

179. Lee, R. F. 1977. Fate of petroleum componentsin estuarine waters of the Southeastern UnitedStates, p. 611-616. In Proceedings of the 1977Oil Spill Conference. American Petroleum In-stitute, Washington, D.C.

180. Lee, R F., and C. Ryan. 1976. Biodegradationof petroleum hydrocarbons by marine mi-crobes, p. 119-126. In J. M. Sharpley and A.M. Kaplan (ed.), Proceedings of the Third In-ternational Biodegradation Symposium. Ap-plied Science Publishers, Ltd., London.

181. Lehmicke, L. G., R. J. Williams, and R. LCrawford. 1979. '4C-most-probable-numbermethod for enumeration of active hetero-trophic microorganisms in natural waters.Appl. Environ. Microbiol. 38:644-649.

182. Lehtomake, M., and S. Niemela. 1975. Improv-ing microbial degradation of oil in soil. Ambio4:126-129.

183. LePetit, J., and M. H. Barthelemy. 1968. Leshydrocarbures en mer le probleme del'epuration des zones littorales par les micro-organismes. Ann. Inst. Pasteur Paris 114:149-158.

184. LePetit, J., and M.-H. N'Guyen. 1976. Besoinsen phosphore des bacteries metabolisant leshydrocarbures en mer. Can. J. Microbiol. 22:1364-1373.

185. LePetit, J., M.-H. N'Guyen, and S. Tagger.

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


206 ATLAS

1977. Quelques donnees sur l'ecologie d'unezone marine littorales recevant les rejets d'uneraffinerie de petrole. Environ. Pollut. 13:41-56.

186. LePetit, J., and S. Tagger. 1976. D6gradationdes hydrocarbures en presence d'autres sub-stances organiques par des bacteries isol6es del'eau de mer. Can. J. Microbiol. 22:1654-1657.

187. Llanos, C., and A. Kioller. 1976. Changes inthe flora of soil fungi following oil waste appli-cation. Oikos 27:337-382.

188. Lough, A. K. 1973. The chemistry and biochem-istry of phylenic, pristanic and related acids.Prog. Chem. Fats Other Lipids 14:1-48.

189. Ludzack, F. L., and D. Kinkead. 1956. Persist-ence of oily wastes in polluted water underaerobic conditions. Ind. Eng. Chem. 48:263-267.

190. Markovetz, A. J. 1971. Subterminal oxidationof aliphatic hydrocarbons by microorganisms.Crit. Rev. Microbiol. 1:225-237.

191. Masters, M. J., and J. E. Zajic. 1971. Myxo-trophic growth of algae on hydrocarbon sub-strates. Dev. Ind. Microbiol. 12:77-86.

192. Mateles, R I., J. N. Baruah, and S. R. Tan-nenbaum. 1967. Growth ofa thermophilic bac-teria on hydrocarbons: a new source of single-cell protein. Science 157:1322-1323.

193. Maunder, B. R., and J. S. Waid. 1973. Disposalof waste oil by land spreading, p. 142-160. InProceedings of the Pollution Research Confer-ence, 20-21 June 1973, Wairakei, New Zealand.Information series no. 97. New Zealand De-partment of Scientific and Industrial Research,Wellington.

194. Mayo, D. W., D. S. Page, J. Cooley, E. Solen-son, F. Bradley, E. S. Gilfillan, and S. A.Hanson. 1978. Weathering characteristics ofpetroleum hydrocarbons deposited in fine claymarine sediments, Searsport, Maine. J. FishRes. Board Can. 35:552-562.

195. MeAuHiffe, C. 1966. Solubility in water of paraf-fin, cycloparaffin, olefin, acetylene, cycloolefin,and aromatic hydrocarbons. J. Phys. Chem.70:1267-1275.

196. McAuliffe, C. D., A. E. Smalley, R. D.Groover, W. M. Welsh, W. S. Pickle, andG. E. Jones. 1975. The Chevron Main PassBlock 41 oil spill: chemical and biological in-vestigations, p. 555-566. In Proceedings 1975Conference on Prevention and Control of OilPollution. American Petroleum Institute,Washington, D.C.

197. McKenna, E. J., and R. E. Kallio. 1964. Hydro-carbon structure: its effect on bacterial utiliza-tion of alkanes, p. 1-14. In H. Heukelian andN. C. Dondero (ed.), Principles and applica-tions in aquatic microbiology. John Wiley &Sons, Inc., New York.

198. McKenna, E. J., and R. E. Kallio. 1965. Thebiology of hydrocarbons. Annu. Rev. Micro-biol. 19:183-208.

199. McKenna, E. J., and R. E. Kaillo. 1971. Micro-bial metabolism of the isoprenoid alkane, pris-tane. Proc. Natl. Acad. Sci. U.S.A. 68:1552-1554.

MICROBIOL. REV.

200. Merkel, G. J., S. S. Stapleton, and J. J. Perry.1978. Isolation and peptidoglycan of gram-neg-ative hydrocarbon-utilizing thermophilic bac-teria. J. Gen. Microbiol. 109:141-148.

201. Miller, T. L., and M. J. Johnson. 1966. Utili-zation of normal alkanes by yeasts. Biotechnol.Bioeng. 8:549-565.

202. Mills, A. L, C. Breuil, and R. R. Colwell. 1978.Enumeration of petroleum-degrading marineand estuarine microorganisms by the most-probable-number method. Can. J. Microbiol.24:552-557.

203. Mironov, O. G. 1970. Role of microorganismsgrowing on oil in the self-purification and in-dication of oil pollution in the sea. Oceanology10:650-656.

204. Mironov, O. G., and A. A. Lebed. 1972. Hydro-carbon-oxidizing microorganisms in the NorthAtlantic. Hydrobiol. J. 8:71-74.

205. Mulkins-Phillips, G. J., and J. E. Stewart.1974. Distribution of hydrocarbon-utilizingbacteria in northwestern Atlantic waters andcoastal sediments. Can. J. Microbiol. 20:955-962.

206. Mulkins-Phillips, G. J., and J. E. Stewart.1974. Effect of environmental parameters onbacterial degradation of Bunker C oil, crudeoils, and hydrocarbons. Appl. Microbiol. 28:915-922.

207. Mulkins-Phillips, G. J., and J. E. Stewart.1974. Effect of four dispersants on biodegra-dation and growth of bacteria on crude oil.Appl. Microbiol. 28:547-552.

208. Nakatani, S., T. Akasaki, K. Kodama, Y.Minoda, and K. Yamada. 1968. Microbialconversion of petro-sulfur compounds. II. Cul-ture conditions of dibenzothiophene-utilizingbacteria. Agric. Biol. Chem. 32:1205-1211.

209. National Academy of Sciences. 1975. Petro-leum in the marine environment. NationalAcademy of Sciences, Washington, D.C.

210. Nyns, E. J., I. P. Auquiere, and A. L Wiaux.1968. Taxonomic value of property of fungi toassimilate hydrocarbons. Antonie van Leeu-wenhoek J. Microbiol. Serol. 34:441-457.

211. Odu, C. T. L. 1972. Microbiology of soils contam-inated with petroleum hydrocarbons. I. Extentof contamination and some soil and microbialproperties after contamination. J. Inst. Pet.London 58:201-208.

212. Olivieri, R., P. Bacchin, A. Robertiello, N.Oddo, L. Degen, and A. Tonolo. 1976. Mi-crobial degradation of oil spills enhanced by aslow-release fertilizer. Appl. Environ. Micro-biol. 31:629-64.

213. Ooyama, J., and J. W. Foster. 1965. Bacterialoxidation of cycloparaffinic hydrocarbons. An-tonie van Leeuwenhoek J. Microbiol. Serol. 31:45-65.

214. Oppenheimer, C. H., W. Gunkel, and G.Gassman. 1977. Microorganisms and hydro-carbons in the North Sea during July-August1975, p. 593-610. In Proceedings of the 1977 OilSpill Conference. American Petroleum Insti-tute, Washington, D.C.


http://mm

br.asm.org/

Dow

nloaded from



215. Parekh, V. R., R. W. Trailer, and J. M. So.bek. 1977. n-ALkane oxidation enzymes of aPseudomonad. Appl. Environ. Microbiol. 33:881-884.

216. Perry, J. J. 1977. Microbial metabolism of cyclichydrocarbons and related compounds. Crit.Rev. Microbiol. 5:387-412.

217. Perry, J. J. 1979. Microbial cooxidations involv-ing hydrocarbons. Microbiol. Rev. 43:59-72.

218. Perry, J. J., and C. E. Cerniglia. 1973. Studieson the degradation of petroleum by filamen-tous fungi, p. 89-94. In D. G. Ahearn and S. P.Meyers (ed.), The microbial degradation of oilpollutants. Publication no. LSU-SG-73-01.Center for Wetland Resources, Louisiana StateUniversity, Baton Rouge.

219. Pfaender, F. K., E. N. Buckley, and R. Fer-guson. 1980. Response of the pelagic microbialcommunity to oil from the IXTOC-I blowout:I. In situ studies. Proceedings of the Confer-ence on Researcher/Pierce IXTOC-I Cniises.National Oceanographic and Atmospheric Ad-ministration, AOML, Miami, in press.

220. Pierce, R. H., A. M. Cundell, and R. W. Trax-ler. 1975. Persistence and biodegradation ofspilled residual fuel oil on an estuarine beach.Appl. Microbiol. 29:646-652.

221. Pinholt, Y., S. Struwe, and A. Kjoller. 1979.Microbial changes during oil decomposition insoil. Holarctic Ecol. 2:195-200.

222. Pirnik, M. P. 1977. Microbial oxidation ofmethylbranched alkanes. Crit. Rev. Microbiol. 5:413-422.

223. Pirnik, M. P., R. M. Atlas, and R. Bartha.1974. Hydrocarbon metabolism by Brevibac-terium erythrogenes:. normal and branched al-kanes. J. Bacteriol. 119:868-878.

224. Polyakova, L N. 1962. Distribution of hydrocar-bons in water of Neva Bay. Mikrobiologiya 31:1076-1081.

225. Pritchard, P. H., and T. J. Starr. 1973. Micro-bial degradation of oil and hydrocarbons incontinuous culture, p. 39-46. In D. G. Ahearnand S. P. Meyers (ed.), The microbial degra-dation of oil pollutants. Publication no. LSU-SG-73-01. Center for Wetland Resources,Louisiana State University, Baton Rouge.

226. Pritchard, P. H., R. M. Ventullo, and J. M.Sufita. 1976. The microbial degradation ofdiesel oil in multistage continuous culture sys-tems, p. 67-78. In J. M. Sharpley and A. M.Kaplan (ed.), Proceedings of the Third Inter-national Biodegradation Symposium. AppliedScience Publishers, Ltd., London.

227. Rashid, MI A. 1974. Degradation of Bunker Coil under different coastal environments ofChedabucto Bay, Nova Scotia. EstuarineCoastal Mar. Sci. 2:137-144.

228. Ratledge, C. 1978. Degradation of aliphatic hy-drocarbons, p. 1-46. In J. R. Watkinson (ed.),Developments in biodegradation of hydrocar-bons-1. Applied Science Publishers, Ltd., Lon-don.

229. Raymond, R. L., J. 0. Hudson, and V. W.

Jamison. 1976. Oil degradation in soil. Appl.Environ. Microbiol. 31:522-535.

230. Raymond, R. L, and V. W. Jamison. 1971.Biochemical activities of Nocardia. Adv. Appl.Microbiol. 14:93-122.

231. Raymond, R. L, V. M. Jamison, and J. 0.Hudson. 1967. Microbial hydrocarbon co-oxi-dation. I. Oxidation of mono- and dicyclic hy-drocarbons by soil isolates of the genus Nocar-dia. Appl. Microbiol. 15:857-865.

232. Raymond, R. L, V. W. Jamison, and J. 0.Hudson. 1976. Beneficial stimulation of bac-terial activity in ground waters containing pe-troleum products, p. 319-327. In Water 1976.American Institute of Chemical Engineers,New York.

233. Reisfeld, A., E. Rosenberg, and D. Gutnick.1972. Microbial degradation of oil: factors af-fecting oil dispersion in seawater by mixed andpure cultures. Appl. Microbiol. 24:363-368.

234. Robertson, B., S. Arhelger, P. J. Kinney, andD. K. Button. 1973. Hydrocarbon biodegra-dation in Alaskan waters, p. 171-184. In D. G.Ahearn and S. P. Meyers (ed.), The microbialdegradation of oil pollutants. Publication no.LSU-SG-73-01. Center for Wetland Resources,Louisiana State University, Baton Rouge.

235. Robertson, B. R., S. D. Arhelger, RI A. T.Law, and D. K. Button. 1973. Hydrocarbonbiodegradation, p. 449-479. In D. W. Hood, W.E. Shiels, and E. J. Kelley (ed.), Environmentalstudies of Port Valdez. Occasional publicationno. 3. University of Alaska, Institute of MarineScience, Fairbanks.

236. Robichaux, T. J., and H. N. Myrick. 1972.Chemical enhancement of the biodegradationof crude oil pollutants. J. Petrol. Technol. 24:16-20.

237. Rogoff, M. H. 1961. Oxidation of aromatic com-pounds by bacteria. Adv. Appl. Microbiol. 3:193-221.

238. RoubaL G., and R. M. Atlas. 1978. Distributionof hydrocarbon-utilizing microorganisms andhydrocarbon biodegradation potentials inAlaska continental shelf areas. Appl. Environ.Microbiol. 35:897-905.

239. Roubal, G., and R. M. Atlas. 1979. Hydrocar-bon biodegradation in Cook Inlet, Alaska. Dev.Ind. Microbiol. 20:498-502.

240. Roubal, G., A. Horowitz, and R. M. Atlas.1979. Disappearance ofhydrocarbons followinga major gasoline spill in the Ohio River. Dev.Ind. Microbiol. 20:503-507.

241. Schaeffer, T. L., S. G. Cantwell, J. L. Brown,D. S. Watt, and R. RI FalL 1979. Microbialgrowth on hydrocarbons: terminal branchinginhibits biodegradation. Appl. Environ. Micro-biol. 38:742-746.

242. Schwarz, J. R., J. D. Walker, and R. R. Col-wefl. 1974. Growth of deep-sea bacteria onhydrocarbons at ambient and in situ pressure.Dev. Ind. Microbiol. 15:239-249.

243. Schwarz, J. R., J. D. Walker, and R. R. Col-well. 1974. Hydrocarbon degradation at am-

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


MICROBIOL. REV.

bient and in situ pressure. Appl. Microbiol. 28:982-986.

244. Schwarz, J. R., J. D. Walker, and R. R. Col-well. 1975. Deep-sea bacteria: growth and uti-lization of n-hexadecane at in situ temperatureand pressure. Can. J. Microbiol. 21:682-687.

245. Seesman, P. A., J. D. Walker, and R. R.Colwell. 1976. Biodegradation of oil by marinemicroorganisms at potential off-shore drillingsites. Dev. Ind. Microbiol. 17:293-297.

246. Seki, H. 1976. Method for estimating the decom-position of hexadecane in the marine environ-ment. Appl. Environ. Microbiol. 31:439-441.

247. Senez, J. C., and E. Azoulay. 1961. Dehyrogen-ation of paraffinic hydrocarbons by resting cellsand cell free extracts of Pseudomonas aerugi-nosa. Biochim. Biophys. Acta 47:307-316.

248. Seubert, W., and E. Fass. 1964. Untersuchun-gen uber den bakteriellen Abbau von Isopre-noiden. V. Der Mechanismus des Isoprenoid-Abbaues. Biochem. Z. 341:35-44.

249. Sexstone, A. J., and R. M. Atlas. 1977. Mobil-ity and biodegradability of crude oil in Arctictundra soils. Dev. Ind. Microbiol. 18:673-684.

250. Sexstone, A. J., and R. M. Atlas. 1977. Re-sponse of populations in arctic tundra soils tocrude oil. Can. J. Microbiol. 23:1327-1333.

251. Sexstone, A. J., and R. M. Atlas. 1978. Per-sistence of oil in tundra soils. Dev. Ind. Micro-biol. 19:507-515.

252. Sexstone, A., K. Everett, T. Jenkins, and R.M. Atlas. 1978. Fate of crude and refined oilsin north slope soils. Arctic 31:339-347.

253. Sexstone, A., P. Gustin, and R. M. Atlas. 1978.Long-term interactions of microorganisms andPrudhoe Bay crude oil in tundra soils at Bar-row, Alaska. Arctic 31:348-354.

254. Shelton, T. B., and J. V. Hunter. 1975. Anaer-obic decomposition of oil in bottom sediments.J. Water Pollut. Control Fed. 47:2256-2270.

255. Smith, J. E. 1968. "Torrey Canyon" pollutionand marine life. Cambridge University Press,England.

256. Soli, G. 1973. Marine hydrocarbonoclastic bac-teria: types and range of oil degradation, p.141-146. In D. G. Ahearn and S. P. Meyers(ed.), The microbial degradation of oil pol-lutants. Publication no. LSU-SG-73-01. Centerfor Wetland Resources, Louisiana State Uni-versity, Baton Rouge.

257. Sparrow, E. B., C. V. Davenport, and R. C.Gordon. 1978. Response of microorganisms tohot crude oil spills on a subarctic taiga soil.Arctic 31:324-338.

258. Stewart, J. E., and L. J. Marks. 1978. Distri-bution and abundance of hydrocarbon-utilizingbacteria in sediments of Chedabucto Bay,Nova Scotia, in 1976. J. Fish Res. Board Can.36:581-584.

259. Stirling, L. A., R. J. Watkinson, and I. J.Higgins. 1977. Microbial metabolism of ali-cyclic hydrocarbons: isolation and propertiesof a cyclohexane-degrading bacterium. J. Gen.Microbiol. 99:119-125.

260. Tagger, S., L. Deveze, and J. LePetit. 1976.The conditions for biodegradation of petro-

leum hydrocarbons at sea. Mar. Pollut. Bull. 7:172-174.

261. Tagger, S., L. Deveze, and J. LePetit. 1979.Sur L'epuration biologique d'une zone littoralemarine affectee par des rejets d'hydrocarbures.Environ. Pollut. 18:275-288.

262. Teal, J. M., K. Burns, and J. Farrington.1978. Analyses of aromatic hydrocarbons inintertidal sediments resulting from two spills ofNo. 2 fuel oil in Buzzards Bay, Massachusetts.J. Fish Res. Board Can. 35:510-520.

263. Traxler, R. W. 1973. Bacterial degradation ofpetroleum materials in low temperature ma-rine environments, p. 163-170. In D. G. Ahearnand S. P. Meyers (ed.), The microbial degra-dation of oil pollutants. Publication no. LSU-SG-73-01. Center for Wetland Resources,Louisiana State University, Baton Rouge.

264. Traxler, R. W., and J. M. Bernard. 1969. Theutilization of n-alkanes by Pseudomonasaeruginosa under conditions of anaerobiosis.Int. Biodeterior. Bull. 5:21-25.

265. Treccani, V. 1964. Microbial degradation of hy-drocarbons. Prog. Ind. Microbiol. 4:3-33.

266. Trudgill, P. W. 1978. Microbial degradation ofalicycic hydrocarbons, p. 47-84. In J. R. Wat-kinson (ed.), Developments in biodegradationof hydrocarbons-1. Applied Science Publishers,Ltd., London.

267. Van der Linden, A. C. 1978. Degradation of oilin the marine environment, p. 165-200. In J. R.Watkinson (ed.), Developments in biodegra-dation of hydrocarbons-1. Applied SciencePublishers, Ltd., London.

268. Van der Linden, A. C., and G. J. E. Thijsse.1965. The mechanisms of microbial oxidationsof petroleum hydrocarbons. Adv. Enzymol. 27:469-546.

269. Vandermeulen, J. H., and R. W. Traxler.1980. Hydrocarbon-utilizing microbial activityin marsh, mudflat and sandy sediments fromnorth Brittany. In Proceedings of The Inter-national Symposium on the Amoco Cadiz:Fates and Effects of the Oil Spill. CentreOceanologique de Bretagne, Brest, France, inpress.

270. Van Eyk, J., and T. J. Bartels. 1968. Paraffinoxidation in Pseudomonas aeruginosa. I. In-duction of paraffin oxidation. J. Bacteriol. 96:706-712.

271. Verstraete, W., R. Vanlooke, R deBorger,and A. Verlinde. 1975. Modelling of thebreakdown and the mobilization of hydrocar-bons in unsaturated soil layers, p. 98-112. In J.M. Sharpley and A. M. Kaplan (ed.), Proceed-ings of the Third Intemational BiodegradationSymposium. Applied Science Publishers, Ltd.,London.

272. Walker, J. D., H. F. Austin, and R. R. Colwell.1975. Utilization of mixed hydrocarbon sub-strate by petroleum-degrading microorga-nisms. J. Gen. Appl. Microbiol. 21:27-39.

273. Walker, J. D., L. Cofone, Jr., and J. J. Coo-ney. 1973. Microbial petroleum degradation:the role of Cladosporium resinae, p. 821-825.In Proceedings of Joint Conference on Preven-

208 ATLAS


http://mm

br.asm.org/

Dow

nloaded from



tion and Control of Oil Spills. American Petro-leum Institute, Washington, D.C.

274. Walker, J. D., and R. R. Colwell. 1974. Micro-bial degradation of model petroleum at lowtemperatures. Microb. Ecol. 1:63-95.

275. Walker, J. D., and R. R. Colwell. 1974. Micro-bial petroleum degradation: use of mixed hy-drocarbon substrates. Appl. Microbiol. 27:1053-1060.

276. Walker, J. D., and R. R. Colwell. 1975. Someeffects of petroleum on estuarine and marinemicroorganisms. Can. J. Microbiol. 21:305-313.

277. Walker, J. D., and R. R. Colwell. 1976. Enu-meration of petroleum-degrading microorga-nisms. Appl. Environ. Microbiol. 31:198-207.

278. Walker, J. D., and R. R. Colwell. 1976. Long-chain n-alkanes occurring during microbialdegradation of petroleum. Can. J. Microbiol.22:886-891.

279. Walker, J. D., and R. R. Colwell. 1976. Mea-suring the potential activity of hydrocarbon-degrading bacteria. Appl. Environ. Microbiol.31:189-197.

280. Walker, J. D., and R. R. Colwell. 1976. Petro-leum: degradation by estuarine microorga-nisms, p. 197-204. In J. M. Sharpley and A. M.Kaplan (ed.), Proceedings of the Third Inter-national Biodegradation Symposium. AppliedScience Publishers, Ltd., London.

281. Walker, J. D., R. R. Colwell, and L. Petrakis.1975. Bacterial degradation of motor oil. J.Water Pollut. Control Fed. 47:2058-2066.

282. Walker, J. D., R. R. Colwell, and L Petrakis.1975. Degradation of petroleum by an alga,Prototheca zopfii. Appl. Microbiol. 30:79-81.

283. Walker, J. D., R. R. Colwell, and L Petrakis.1975. Evaluation of petroleum-degrading po-tential of bacteria from water and sediment.Appl. Microbiol. 30:1036-1039.

284. Walker, J. D., R. R. Colwell, and L. Petrakis.1975. Microbial petroleum degradation: appli-cation of computerized mass spectrometry.Can. J. Microbiol. 21:1760-1767.

285. Walker, J. D., R. R. Colwell, and L. Petrakis.1976. Biodegradation of petroleum by Chesa-peake Bay sediment bacteria. Can. J. Micro-biol. 22:423-428.

286. Walker, J. D., R. R. Colwell, and L Petrakis.1976. Biodegradation rates of components ofpetroleum. Can. J. Microbiol. 22:1209-1213.

287. Walker, J. D., L. Petrakis, and R. R. Colwell.1976. Comparison of the biodegradability ofcrude and fuel oils. Can. J. Microbiol. 22:598-602.

288. Ward, D., R. M. Atlas, P. D. Boehm, and J.A. Calder. 1980. Microbial biodegradation andthe chemical evolution of Amoco Cadiz oilpollutants. Ambio 9:277-283.

289. Ward, D. M., and T. D. Brock. 1976. Environ-mental factors influencing the rate of hydro-carbon oxidation in temperate lakes. Appl. En-viron. Microbiol. 31:764-772.

290. Ward, D. M., and T. D. Brock. 1978. Anaerobicmetabolism of hexadecane in marine sedi-

ments. Geomicrobiol. J. 1:1-9.291. Ward, D. M., and T. D. Brock. 1978. Hydrocar-

bon biodegradation in hypersaline environ-ments. Appl. Environ. Microbiol. 35:353-359.

292. Westlake, D. W. S., A. M. Jobson, and F. D.Cook. 1978. In situ degradation of oil in a soilof the boreal region of the Northwest Territo-ries. Can. J. Microbiol. 24:254-260.

293. Westlake, D. W. S., A. Jobson, R. Philippe,and F. D. Cook. 1974. Biodegradability andcrude oil composition. Can. J. Microbiol. 20:915-928.

294. Wifliams, P. A. 1978. Microbial genetics relatingto hydrocarbon degradation, p. 135-164. In J.R. Watkinson (ed.), Developments in biodeg-radation of hydrocarbons-1. Applied SciencePublishers, Ltd., London.

295. Wodzinsky, R. S., and D. LaRocca. 1977. Bac-terial growth kinetics on diphenylmethane andnaphthalene-heptamethylnonane. Appl. Envi-ron. Microbiol. 33:660-665.

296. Yamada, K., Y. Monoda, K. Komada, S. Nak-atani, and T. Akasaki. 1968. Microbial con-version of petro-sulfur compounds. I. Isolationand identification of dibenzothiophene-utiliz-ing bacteria. Agric. Biol. Chem. 32:840-845.

297. Zajic, J. E., B. Supplisson, and B. Volesky.1974. Bacterial degradation and emulsificationof No. 6 fuel oil. Environ. Sci. Technol. 8:664-668.

298. ZoBell, C. E. 1946. Action of microorganisms onhydrocarbons. Bacteriol. Rev. 10:1-49.

299. ZoBell, C. E. 1950. Assimilation of hydrocarbonsby microorganisms. Adv. Enzymol. 10:443-486.

300. ZoBell, C. E. 1964. The occurrence, effects andfate of oil polluting the sea. Adv. Water Pollut.Res. 3:85-118.

301. ZoBell, C. E. 1969. Microbial modification ofcrude oil in the sea, p. 317-326. In Proceedingsof Joint Conference on Prevention and Controlof Oil Spills. American Petroleum Institute,Washington, D.C.

302. ZoBell, C. E. 1971. Sources and biodegradationof carcinogenic hydrocarbons, p. 441-451. InProceedings of Joint Conference on Preventionand Control of Oil Spills. American PetroleumInstitute, Washington, D.C.

303. ZoBell, C. E. 1973. Bacterial degradation of min-eral oils at low temperatures, p. 153-161. In D.G. Ahearn and S. P. Meyers (ed.), The micro-bial degradation of oil pollutants. Publicationno. LSU-SG-73-01, Center for Wetland Re-sources, Louisiana State University, BatonRouge.

304. ZoBeli, C. E. 1973. Microbial degradation of oil:present status, problems and perspectives, p.3-16. In D. G. Ahearn and S. P. Meyers (ed.),The microbial degradation of oil pollutants.Publication no. LSU-SG-73-01. Center forWetland Resources, Louisiana State Univer-sity, Baton Rouge.

305. ZoBell, C. E., and J. F. Prokop. 1966. Microbialoxidation of mineral oils in Barataria Bay bot-tom deposits. Z. Allg. Mikrobiol. 6:143-162.

VOL. 45, 1981


http://mm

br.asm.org/

Dow

nloaded from


Documents

Microbiol Rev. 1981 Atlas 180 209