Vol. 54, No. 9APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1988, p. 2197-22020099-2240/88/092197-06$02.00/0Copyright © 1988, American Society for Microbiology

Biodegradation of Nitriles in Shale OilJACKIE AISLABIE AND RONALD M. ATLAS*

Department ofBiology, University of Louisville, Louisville, Kentucky 40292

Received 8 March 1988/Accepted 1 June 1988

Enrichment cultures were obtained, after prolonged incubation on a shale oil as the sole source of nitrogen,that selectively degraded nitriles. Capillary gas chromatographic analyses showed that the mixed microbialpopulations in the enrichments degraded the homologous series of aliphatic nitriles but not the aliphatichydrocarbons, aromatic hydrocarbons, or heterocyclic-nitrogen compounds found in this oil. Time coursestudies showed that lighter nitriles were removed more rapidly than higher-molecular-weight nitriles. APseudomonasfluorescens strain isolated from an enrichment, which was able to completely utilize the individualnitriles undecyl cyanide and undecanenitrile as sole sources of carbon and nitrogen, was unable to attackstearonitrile when provided alone as the growth substrate. A P. aeruginosa strain, also isolated from one of theenrichments, used nitriles but not aliphatic or aromatic hydrocarbons when the oil was used as a sole nitrogensource. However, when the shale oil was used as the sole source of carbon, aliphatic hydrocarbons in additionto nitriles were degraded but aromatic hydrocarbons were still not attacked by this P. aeruginosa strain.

Unlike oils from petroleum reservoirs, oils produced fromshale deposits have high concentrations of nitrogen- andsulfur-containing compounds. The nitrogen contents of shaleoils, for example, range from 0.5 to 2.1%, which is consid-erably higher than that in crude oil (average value, 0.094%)(29). These nonhydrocarbon components preclude the directuse of these oils as a fuel in combustion engines because theywould produce unacceptable levels of nitrogen and sulfuroxide air pollutants. Additionally, the nitrogen componentsof shale oil act as a poison for refinery catalysts and, togetherwith the oxygen-containing compounds that occur in theseoils, cause poor storage stability of the oil. Nitrogen andsulfur compounds can be removed from shale oils by hydro-treatment, but such high-temperature, high-pressure proc-essing is costly.

Biotechnology potentially could reduce the cost of upgrad-ing shale oil to a useful fuel if microorganisms could selec-tively remove the nitrogen- and sulfur-containing com-pounds without attacking the hydrocarbon components ofthe oil. The ability of microorganisms to attack nitrogen andsulfur components of oils has received some attention (10-12, 15, 22). Such studies are important because of theincreasing worldwide need to utilize alternative fuel sources,such as coal-derived liquids and shale oils, and the conse-quent need to reduce the nitrogen and sulfur content of thesematerials at reasonable cost. In this report we are concernedwith the ability of microbes to remove one class of nitrogen-containing compounds, the nitriles, from a shale oil.

MATERIALS AND METHODS

Enrichment cultures. Several soil sources contaminatedwith petroleum, including soils from oily-sludge landfarmingand drill cores, were used as sources of inocula for enrich-ment cultures. Culture conditions were established to enrichfor microbial populations capable of using the nitrogen-containing compounds in shale oil. Ten grams of each soilwas added to 100 ml of a nitrogen-free medium containing (ingrams per liter): MgSO4, 0.2; CaCl2, 0.02; KH2PO4, 1;K2HPO4, 1; FeCl3, 0.05. The pH was adjusted to 7.5, and themedium was then autoclaved. After sterilization the medium

* Corresponding author.

was amended with a sterile glucose solution to give a finalconcentration of 0.1% (wt/vol) and shale oil to give aconcentration of 1% (wt/vol). The shale oil was thus the solesource of nitrogen but not the sole source of carbon for theseenrichments. The shale oil used in these enrichment cultureswas the middle cut of a raw shale oil (250 to 400°C boilingrange) produced at the Taciuk Processor Pilot Plant bypyrolysis of shale from the Stuart Deposit, Queensland,Australia (3). This shale oil contains 0.87% Kjeldahl nitrogen(analysis provided by Galbraith Laboratories, Knoxville,Tenn.), with the nitrogen found in aliphatic amines, nitriles,and heterocyclic nitrogen compounds (quinolines, isoquino-lines, pyridines, pyrroles, and carbazoles) (analysis providedby Southern Pacific Petroleum, Sydney, Australia). Approx-imately half of the nitrogen is contained in the heterocycliccompounds, and the remainder occurs primarily as aliphaticnitriles and aliphatic amines. The enrichments were incu-bated at 28°C on a rotary shaker at 200 rpm. The enrichmentcultures have been maintained by monthly transfers intofresh medium.

Biodegradation of shale oil components. After at least eightsubcultures to ensure maintenance of growth on medium inwhich the shale oil was the sole source of nitrogen but notcarbon, enrichment cultures showing emulsification andvisible changes in the oil were analyzed to determine whichcompounds, if any, in the shale oil had been biodegraded.For these analyses, the enrichment cultures were incubatedfor 28 days at 28°C on a rotary shaker at 200 rpm in the samemedium used for the initial enrichments. Sterile controlswere included to distinguish nonbiological changes in the oilfrom those due to biodegradation. The residual oil wasrecovered by extraction with methylene chloride and ana-lyzed as described below. In addition to these experiments,in which glucose was supplied as a supplemental carbonsource, subcultures were inoculated into medium in whichsuccinate (0.1%) was provided instead of glucose as analternate source of carbon.

Rates of nitrile biodegradation. Of the various enrichmentcultures that were capable of using the shale oil as a sourceof nitrogen, one was selected for time course biodegradationstudies. This enrichment culture came from a subsurfaceAlberta tar sands drill core. Portions (5 ml) from the enrich-ment were added to replicate flasks containing 100 ml of the

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nitrogen-free medium described previously supplementedwith glucose (0.1%) and shale oil (1 ml). Incubation was at28°C on a rotary shaker at 200 rpm. Controls containingshale oil and 100 ml of sterile mineral medium were incu-bated under identical conditions. The controls served toaccount for any substrate loss due to volatilization or othernonbiological loss. Periodically during the 45-day incubationperiod, the residual oil from duplicate control and inoculatedflasks was extracted and analyzed for nitrile biodegradationas described below.

Biodegradation of nitriles by pure cultures. One of theenrichments was subcultured onto undecanenitrile at a con-centration of 0.5% (vol/vol) in Bushnell-Haas broth (DifcoLaboratories). Growth in the broth was monitored by ob-serving turbidity and by plating onto 0.1 strength tryptic soybroth solidified with 2% purified agar. A bacterial strain wasisolated from the undecanenitrile broth platings and identi-fied by using Rapid NFT Strips (API Systems S.A.) as astrain of Pseudomonasfluorescens. The ability of this strainto grow on cyanide (0.1%) as the sole nitrogen source waschecked. This strain also was tested for growth on undeca-nenitrile, undecyl cyanide, and stearonitrile (Aldrich Chem-ical Co.) as the sole source of nitrogen and carbon at 0.1%(wt/vol) in nitrogen-free medium. After 7 days of incubationwith the aliphatic nitriles as substrates, hexamethylbenzene(HMB) was added as an internal standard, and the nitrileswere recovered from the broth by extraction with methylenechloride. The ability to utilize these individual nitriles as solenitrogen and carbon sources was determined by gas-liquidchromatography with a flame ionization detector and thechromatographic conditions described below. The strain ofP. fluorescens was also inoculated into the same mediumused initially for enrichment culture with 1% shale oil as thesole source of nitrogen and 0.1% glucose as an alternativecarbon source.A bacterial strain was also isolated directly from the

original enrichment by plating onto 0.1 strength Trypticasesoy broth (BBL Microbiology Systems) solidified with puri-fied agar (Difco). It was identified as a strain of Pseudomo-nas aeruginosa by using the Rapid NFT Strips. This strainwas grown in nitrogen-free medium with 0.1% glucose and1% shale oil (identical conditions as the parent enrichmentculture with the shale oil as the sole nitrogen but not the solecarbon source) or Bushnell-Haas medium with 0.3% shale oil(shale oil as the sole carbon but not the sole nitrogen source).Seven-day-old cultures were subcultured (10% inoculum)into fresh medium with the same composition. After 14 daysof incubation at 28°C with shaking at 200 rpm, the residual oilwas recovered from both the glucose-containing and glu-cose-free media and analyzed as described below.

Analyses of oil fractions. Prior to extraction, HMB wasadded to each culture flask as an internal standard. Themedia were extracted with methylene chloride (Burdick andJackson) at neutral pH, and the oil was concentrated byusing a rotary evaporator. The oil was fractionated by amodification of the column chromatographic method de-scribed by Fedorak and Westlake (10). In this procedure thechromatography column was developed sequentially with 50ml of pentane (MCB Manufacturing Chemists), 50 ml ofbenzene (Burdick and Jackson), and 70 ml of chloroform(Fisher Scientific), to give the saturate, aromatic, and nitrile-containing fractions, respectively. In some cases the hetero-cyclic nitrogen components were separated from the oil byacid extraction by the procedure of Bett et al. (5) prior tofractionation.

After the solvent volume had been reduced under vacuum,

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A

17

19

B

FIG. 1. GC tracings of nitrile fractions recovered after 28 days ofincubation from a sterile control (A) and an enrichment culture (B).The profile of the nitriles in the sterile control after this incubationperiod was identical to that for the initial oil, indicating no abioticdegradation of these nitriles. The internal standard was quinoline(Q). The chain lengths of the n-nitriles are indicated. The unsaturat-ed nitriles occurred just before the n-nitriles of corresponding chainlengths.

the fractions were analyzed by capillary gas-liquid chroma-tography. An Ultra 2-coated fused silica column (25 m by 0.2mm; Hewlett Packard) was used for compound separation.The operational parameters were: injection port at 240°C;detector at 320°C; column temperature, 100°C isothermal for5 min, 5°C/min increase to 290°C, isothermal at 290NC for 20min; helium as carrier and makeup gas. The saturate andaromatic fractions were analyzed with a Hewlett Packardmodel 5840 gas chromatograph (GC) with a flame ionizationdetector; the nitrile and heterocyclic nitrogen fractions wereanalyzed with a nitrogen-specific detector.

RESULTS

Biodegradation of shale oil components. GC analyses of theoil fractions recovered from the broth enrichment cultures inwhich the shale oil was the sole nitrogen source and glucosewas an alternative carbon source showed selective microbialattack on the aliphatic nitrile compounds from the shale oil(Fig. 1). The same preferential attack on the nitrile fractionwas observed when succinate was used instead of glucose asthe alternative carbon source.

Analyses of the nitrile fraction of the Stuart shale oil usedin these experiments was found to contain a series ofn-nitriles from C7 to C30 with a maxima at C12 and unsatu-rated nitriles as identified by GC and mass spectrometricanalyses. Some of these unsaturated nitriles are probably

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singly unsaturated alkylnitriles with the double bond at theopposite end from that of the nitrile group, as reported byHarvey et al. (17). The concentrations of the unsaturatednitriles were less than those of the saturated nitriles ofcomparable chain lengths, with the ratio of unsaturated tosaturated nitrile getting progressively smaller with increasingchain length above C12. The concentrations of these nitrileswere essentially unchanged in the sterile controls after 28days of incubation compared with the nitrile fraction at thebeginning of the experiment. In the enrichment cultures,when visible changes in the oil were detected, however,there was extensive removal of all the saturated and unsat-urated nitriles. The extent of removal varied among thevarious enrichment cultures.

In contrast to the nitrile fraction, there was no evidence ofattack on the heterocyclic nitrogen compounds or on thealiphatic and aromatic hydrocarbons (Fig. 2). The concen-trations of the unsubstituted and alkyl-substituted quinolinesand isoquinolines, which were found to be the dominantcompounds in the heterocyclic nitrogen-containing fraction,as well as the pyridines and carbazoles, which were found tobe minor components of this fraction, were the same in theoil fractions recovered from all the enrichment cultures as inthose from the sterile controls. Likewise, the concentrationsof aliphatic hydrocarbons, which included a series of n-alkanes and 1-alkenes in the range from C10 to C30 with amaximum at C17, and aromatic hydrocarbons, which in-cluded two and three-ring polynuclear aromatic hydrocar-bons (1- and 2-methylnaphthalene, fluorene, phenanthrene,anthracene, and dibenzothiophene), were the same in thefractions recovered after 28 days of incubation with theenrichment cultures as in those from the sterile controls.

Rates of nitrile biodegradation. The time course studyindicated that the nitriles were rapidly degraded by themicrobial populations in the mixed enrichment culture (Fig.3). There was sequential utilization of nitriles, with lower-molecular-weight nitriles being degraded before higher-mo-lecular-weight nitriles. As an example of this point, after 3days, 100% of the C7 to C12 nitriles had been degraded, butthere was limited attack on C13 to C15 nitriles and no attackon C16 to C20 nitriles; however, after 45 days of incubation,all C7 to C14 nitriles and more than 90% of the C15 to C20nitriles were removed from the oil (Table 1). The shorternitriles were thus degraded preferentially over the higher-molecular-weight nitriles. The apparent faster attack on theunsaturated than on the saturated nitriles may be due to thefact that the relative concentrations of unsaturated nitrileswere higher in the lower-molecular-weight range wherepreferential degradation occurred, but the results clearlyindicate that the unsaturated nitriles were degraded at leastas rapidly as the saturated nitriles of identical chain lengths.

Biodegradation of nitriles by pure cultures. The P. fluores-cens strain isolated from the enrichment culture was able touse the pure aliphatic nitrile compound undecanenitrile asthe sole source of carbon and nitrogen. Cell numbers in-creased from the inoculum of 106 per ml to over 108 per ml incultures inoculated with this strain when undecanenitrilewas the sole source of carbon and nitrogen. This bacteriumalso could grow on cyanide.

After 1 week of incubation of this strain of P. fluorescenswith either undecanenitrile, undecyl cyanide, or stearonitrileas the sole source of carbon and nitrogen, the undecaneni-trile and undecyl cyanide could no longer be detected (100%degradation), but stearonitrile had not been degraded. Thispreferential degradation of the lower-molecular-weight ni-

triles is consistent with the observations in the mixed enrich-ment cultures.When this strain of P. fluorescens was inoculated into the

same medium used initially for enrichment culture with 1%shale oil as the sole source of nitrogen and 0.1% glucose asan alternative carbon source, it preferentially removed thelower-molecular-weight nitriles. During 7 days of incubationthe organism degraded unsaturated nitriles in the range fromC7 to C13 and saturated nitriles in the range from C7 to C12;there was no removal of unsaturated nitriles above C13 orsaturated nitriles above C12 from the shale oil by thisbacterial strain.The P. aeruginosa strain isolated from one of the enrich-

ments used nitriles but not aliphatic or aromatic hydrocar-bons when the oil was used as a sole nitrogen source andglucose was provided as an alternative carbon source.However, when the shale oil was used as the sole source ofcarbon, aliphatic hydrocarbons were degraded in addition tonitriles (Fig. 4). Significant quantities of prist-1-ene andprist-2-ene remained, indicating that branched alkanes werenot attacked by this organism. Aromatic hydrocarbons werenot attacked by this P. aeruginosa strain either when theshale oil served as the sole nitrogen source or when it wasused as the sole carbon source.

DISCUSSION

In contrast to petroleum, shale oils contain significantconcentrations of nitrogen (29). In many shale oils, a sub-stantial proportion of the organic nitrogen is contained inlinear alkyl nitriles (9, 16, 17, 23, 24), which are compoundsnot found in petroleum. Evans et al. (9) report that thenitriles found in shale oils are formed during the pyrolysis ofthe shale by the reaction of carboxylic acids and ammonialiberated from minerals, such as the ammonium feldsparbuddingtonite, which is present in the shale; if neithercarboxylic acids nor ammonia is present, nitriles are notformed in the product oil.

Various studies have examined the abilities of microor-ganisms, both fungi and bacteria, to degrade nitriles (1, 2, 4,7, 8, 13, 14, 18-21, 27, 28, 30, 31). Several of these studiesfocused on the microbial degradation of aromatic nitrilesbecause several herbicides, such as bromoxynil, ioxynil, anddichlobenil, contain such aromatic nitriles (4, 13, 14, 18, 21,27). Others have considered the potential use of microbialnitrile conversions in industrial processes (6) and in waste-water treatment (28). The degradation pathway of aliphaticnitriles has been found in these studies to proceed via thecorresponding amide to the fatty acid plus ammonia by theaction of a nitrilase and an amidase, respectively (1, 2). Noneof these studies examined the abilities of microorganisms todegrade nitriles within complex mixtures such as shale oil.Our findings indicate that nitriles can be selectively re-

moved from shale oil by various mixed enrichment cultureswhen the oil is provided as the sole nitrogen source and analternative carbon source is provided, such as glucose or

succinate. Although 50% of the nitrogen in shale oil iscontained in the heterocyclic nitrogen compounds, thesecomplex aromatic compounds were not attacked under theconditions employed in this study. Heterocyclic nitrogen-containing compounds found in this oil have been reportedto be subject to biodegradation (10, 25), and we have alsoobserved degradation of these compounds when the acid-extractable fraction of the oil that contains these basicnitrogen heterocyclic compounds is used as a source ofcarbon for enrichment cultures (unpublished results). Also,

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A

BQ

HMB

14

13

B

2223 24 25 26 27 28

F~~~~~~

P

2MN M

FIG. 2. GC tracings of heterocyclic nitrogen (A), aliphatic hydrocarbon (B), and aromatic hydrocarbon (C) fractions recovered after 28days of incubation from an enrichment culture. The profiles for each of the fractions were identical to those for the sterile control and theinitial oil, indicating no abiotic losses or biodegradation of the compounds in these fractions. In chromatogram A, the internal standard was7,8-benzoquinoline (BQ); quinoline (Q) and isoquinoline (IQ) were identified, and the ranges of C1 to C4 derivatives of quinoline andisoquinoline are shown. In chromatogram B, the internal standard was HMB. The series of n-alkanes is shown. The n-alkanes were precededby a series of 1-alkenes. In chromatogram C, the internal standard was HMB. Labeled peaks are 1-methylnaphthalene (1-MN),2-methylnaphthalene (2-MN), fluorene (F), and phenanthrene (P).

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10090

28070 /

.2 60 -

0040

01 3 713 28 45

Time (days)FIG. 3. Time course of nitrile degradation by the mixed popula-

tions of an enrichment culture. Symbols: 0, degradation of thesaturated nitriles; l, degradation of unsaturated nitriles.

unlike the study on carbazole biodegradation in a crude oilby Fedorak and Westlake (10), in which degradation of theheterocyclic nitrogen compounds occurred together withdegradation of aliphatic and aromatic hydrocarbons, theenrichments obtained in the current study did not attack anyhydrocarbons (alkanes, alkenes, or condensed aromatics)under conditions in which nitriles were attacked. As in theenrichment cultures, isolated strains showed preferentialattack on the lower-molecular-weight nitriles. Similar pref-erential attack on alkanes has been reported for somemicrobial strains (26). Interestingly, the P. aeruginosa iso-late has the ability to degrade alkanes, but does so only when

TABLE 1. Removal of nitriles from Stuart shale oil duringincubation with a nitrile-degrading enrichment culture

% Nitrile removal at incubation time (days):Nitrilea

1 3 7 14 28 45

n-C7 100 100 100 100 100 100Unsat. C7 100 100 100 100 100 100n-C8 100 100 100 100 100 100Unsat. C8 100 100 100 100 100 100n-Cs 100 100 100 100 100 100Unsat. C9 100 100 100 100 100 100

n-Cl. 100 100 100 100 100 100Unsat. C10 100 100 100 100 100 100n-Cl 16 100 100 100 100 100Unsat. Cl >90 100 100 100 100 100n-C12 0 100 100 100 100 100Unsat. C12 >90 100 100 100 100 100n-C13 0 45 100 100 100 100Unsat. C13 0 >90 100 100 100 100n-CI4 0 46 >90 100 100 100Unsat. C14 0 58 100 100 100 100n-C15 0 24 72 >90 >90 >90Unsat. Cl1 0 31 100 100 100 100n-C16 0 0 33 >90 >90 >90Unsat. C16 0 0 65 >90 100 100n-C17 0 0 10 54 84 >90Unsat. C17 0 0 30 >90 >90 >90n-C18 0 0 0 50 72 >90Unsat. C18 0 0 0 >90 >90 >90n-Cl9 0 0 0 44 62 >90n-C20 0 0 0 37 64 >90

a Unsat., Unsaturated.

A

B

2P

FIG. 4. GC tracings of nitrile (A) and aliphatic hydrocarbon (B)fractions recovered after 14 days of incubation with a strain of P.aeruginosa on carbon-free medium. The remaining peaks in thealiphatic hydrocarbon fraction were branched alkenes and alkanes,including prist-1-ene (1P) and prist-2-ene (2P). For comparison withundegraded nitrile fraction, see Fig. 1A, and for comparison withundegraded aliphatic hydrocarbon fraction, see Fig. 2B.

the shale oil serves as the sole source of carbon; when thereis an alternative carbon source and the shale oil serves onlyas the sole nitrogen source, alkanes are spared from attackwhile nitriles are degraded. This selective removal capacityis critical for upgrading of shale oil or other synthetic fuelsbecause of the need to retain the caloric value of thehydrocarbons within the fuel.

ACKNOWLEDGMENTS

This study was supported by Southern Pacific Petroleum, Sydney,Australia.We thank Southern Pacific Petroleum for supplying the shale oil

and technical information, Don Westlake and Phil Fedorak forassistance in analytical design, and Harrel Hurst for performingmass spectral analyses.

LITERATURE CITED1. Asano, Y., K. Fujishiro, Y. Tani, and H. Yamada. 1982. Ali-

phatic nitrile hydratase from Arthrobacter sp. J-1. Purificationand characterization. Agric. Biol. Chem. 46:1165-1174.

2. Asano, Y., M. Tachibana, Y. Tani, and H. Yamada. 1982.Purification and characterization of amidase which participatesin nitrile degradation. Agric. Biol. Chem. 46:1175-1181.

3. Baker, L. A., and B. C. Wright. 1987. Current developments inthe Stuart Oil Shale Project, p. 23-25. In Proceedings of the 4thAustralian Workshop on Oil Shale, Brisbane, Australia. CSIRODivision of Energy Chemistry, Menai, Australia.

VOL. 54, 1988

4 11 . OOA a,-,, -.

on January 2, 2019 by guesthttp://aem

.asm.org/

Dow

nloaded from

2202 AISLABIE AND ATLAS

4. Bandyopadhyay, A. K., T. Nagasawa, Y. Asano, K. Fujishiro, Y.Tani, and H. Yamada. 1986. Purification and characterization ofbenzoriitrilases from Arthrobacter sp. strain J-1. Appl. Environ.Microbiol. 51:302-306.

5. Bett, G., T. G. Harvey, T. W. Matheson, and K. C. Pratt. 1983.Determination of polar compounds in Rundle shale oil. Fuel 62:1445-1454.

6. Cheetham, P. J. S. 1987. Screening for novel biocatalysts.Enzyme Microb. Technol. 9:194-213.

7. Collins, P. A., and C. J. Knowles. 1983. The utilization of nitrilesand amides by Nocardia rhodochrous. J. Gen. Microbiol. 129:711-718.

8. DiGeronimo, M. J., and A. D. Antoine. 1976. Metabolism ofacetonitrile and propionitrile by Nocardia rhodochrous LL1100-21. Appl. Environ. Microbiol. 31:900-906.

9. Evans, E. J., B. D. Batts, N. W. Cant, and J. W. Smith. 1985.The origin of nitriles in shale oil. Org. Geochem. 8:367-374.

10. Fedorak, P. M., and D. W. S. Westlake. 1984. Microbialdegradation of alkyl carbazoles in Norman Wells crude oil.Appl. Environ. Microbiol. 47:858-862.

11. Finnerty, W. R. 1982. Microbial desulfurization and denitroge-nation of fossil fuels, p. 883-890. In R. F. Hill (ed.), Energytechnology: proceedings of the ninth energy technology confer-ence, Washington, D.C. Government Institutes, Inc., Washing-ton, D.C.

12. Finnerty, W. R., K. Shockley, and H. Attaway. 1982. Microbialdesulfurization and denitrogenation of hydrocarbons, p. 83-91.In J. E. Zajic, D. C. Cooper, T. R. Jack, and N. Kosaric (ed.),Microbial enhanced oil recovery. PennWell Books, Tulsa, Okla.

13. Harper, D. B. 1977. Microbial metabolism of aromatic nitriles.Biochem. J. 165:309-319.

14. Harper, D. B. 1977. Fungal degradation of aromatic nitriles.Biochem. J. 167:685-692.

15. Hartdegen, F. J., J. M. Coburn, and R. L. Roberts. 1984.Microbial desulfurization of petroleum. Chem. Eng. Prog. 80:63-67.

16. Harvey, T. G., T. W. Matheson, and K. C. Pratt. 1984. Chemicalclass separation of organics in shale oil by thin-layer chroma-tography. Anal. Chem. 56:1277-1281.

17. Harvey, T. G., T. W. Matheson, and K. C. Pratt. 1986. Extrac-tion and identification of nitrile compounds in Rundle shale oil,

p. 241-244. In Proceedings of the 3rd Australian Workshop onOil Shale, Lucas Heights, Australia. CSIRO Division of EnergyChemistry, Menai, Australia.

18. Jallageas, J.-C., A. Arnaud, and P. Galzy. 1980. Bioconversionof nitriles and their applications. Adv. Biochem. Eng. 12:1-32.

19. Kuwahara, M., H. Yanase, Y. Ishida, and Y. Kikuchi. 1980.Metabolism of aliphatic nitriles in Fusarium solani. J. Ferment.Technol. 58:573-578.

20. Linton, E. A., and C. J. Knowles. 1986. Utilization of aliphaticamides and nitriles by Nocardia rhodochrous LL100-21. J. Gen.Microbiol. 132:1493-1501.

21. McBride, K. E., J. W. Kenny, and D. M. Stalker. 1986.Metabolism of the herbicide Bromoxynil by Klebsiella pneumo-niae subsp. ozaenae. Appl. Environ. Microbiol. 52:325-330.

22. Monticello, D. J., and W. R. Finnerty. 1985. Microbial desulfu-rization of fossil fuels. Annu. Rev. Microbiol. 39:371-389.

23. Regtop, R. A., P. T. Crisp, and J. Ellis. 1982. Chemicalcharacterization of shale oil from Rundle, Australia. Fuel 61:185-192.

24. Rovere, C. E., P. T. Crisp, J. Ellis, and P. D. Bolton. 1983.Chemical characterization of shale oil from Condor, Australia.Fuel 62:1273-1281.

25. Shukla, 0. P. 1986. Microbial transformation of quinoline by aPseudomonas sp. Appl. Environ. Microbiol. 51:1332-1342.

26. Singer, M. E., and W. R. Finnerty. 1984. Microbial metabolismof straight and branched alkanes, p. 1-59. In R. M. Atlas (ed.),Petroleum microbiology. Macmillan, New York.

27. Stalker, D. M., and K. E. McBride. 1987. Cloning and expres-sion in Escherichia coli of a Klebsiella ozaenae plasmid-bornegene encoding a nitrilase specific for the herbicide Bromoxynil.J. Bacteriol. 169:955-960.

28. Thiery, A., M. Maestracci, A. Arnaud, and P. Galzy. 1986.Nitriles as growth substrates for Brevibacterium sp. R312 andits mutant M2. Zentralbl. Mikrobiol. 141:575-582.

29. Tissot, B. P., and D. H. Welte. 1984. Petroleum formation andoccurrence. Springer-Verlag, New York.

30. Yamada, H., Y. Asasno, T. Hino, and Y. Tani. 1979. Microbialutilization of acrylonitrile. J. Ferment. Technol. 57:8-14.

31. Yanase, H., T. Sakai, and K. Tonomura. 1985. Microbial me-tabolism of nitriles in Pseudomonas sp. J. Ferment. Technol.63:193-198.

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