See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/23718391Characterization of monoterpenebiotransformation in two pseudomonadsARTICLEinJOURNAL OF APPLIED MICROBIOLOGY JANUARY 2009Impact Factor: 2.39 DOI: 10.1111/j.1365-2672.2008.03923.x Source: PubMedCITATIONS25DOWNLOADS32VIEWS964 AUTHORS, INCLUDING:Juliano Lemos BicasFederal University of So Joo del-Rei20 PUBLICATIONS 172 CITATIONS SEE PROFILEpierre FONTANILLEUniversit Blaise Pascal - Clermont-Ferrand II28 PUBLICATIONS 279 CITATIONS SEE PROFILEChristian LarrocheUniversit Blaise Pascal - Clermont-Ferrand II95 PUBLICATIONS 1,551 CITATIONS SEE PROFILEAvailable from: Christian LarrocheRetrieved on: 02 August 2015ORI GI NALARTI CLECharacterizationofmonoterpenebiotransformationintwopseudomonadsJ.L.Bicas1,2,P.Fontanille2,G.M.Pastore1andC.Larroche21Laborato riodeBioaromas,DepartamentodeCie nciadeAlimentosFEA,UniversidadeEstadual deCampinas,RuaMonteiroLobato,Campinas-SP,Brasil2LaboratoiredeGe nieChimiqueetBiochimiquePolytechClermont-Ferrand,Universite BlaisePascal,Aubie` reCedex,FranceIntroductionTerpenes are secondary metabolites of plants that areproduced, inpart, as a defense against micro-organismsand insects, in addition to their pollinator-attractiveproperties (Gershenzon and Dudareva 2007). The sim-pler terpenes (mono- andsesquiterpenes) andterpenoidsare the major constituents of essential oils and arewidely usedinthe avour industry. Theymaybeincor-poratedinthe formulationof foods, cosmetics, hygieneand household products, acting not only as avourings(Bauer etal. 2001),but also asantibacterialagents(Grifnetal. 1999; Bakkali etal. 2007). Moreover, somefunctio-nalized terpenes also present bioactivity against certaintypes of tumour cells (Crowell 1999; Junetal. 2006). Inthis context, the interest in such compounds is con-stantlygrowing.R-(+)-limoneneanda-pinenearethemost widespreadmonoterpenes innature. Owingtotheir abundance andrelatively lowcost, these two monoterpenes have beenused as the main precursors for the synthesis of theiroxygenated counterparts. Oxygenation reactions may bechemicallycatalysed, buttheuseofbiotechnological toolstoperformthemhasbeenthoroughlyinvestigatedinthelast years, as they proceedunder mildconditions, havehighregio-andenantioselectivities, donot generatetoxicwaste and the products obtained can be labelled asnatural(Serraetal.2005).Keywordsa-pinene, b-pinene,biphasicsystem,limonene,Pseudomonas.CorrespondenceJulianoLemosBicas,Laborato riodeBioaromas,DepartamentodeCie nciadeAlimentosFEA-UNICAMP.RuaMonteiroLobato80,13083-862,Campinas-SP,Brazil.E-mail:jlbicas@gmail.com2008 0374:received:3March2008,revised5June2008andaccepted6June2008doi:10.1111/j.1365-2672.2008.03923.xAbstractAims: Tostudythemetabolicproleof PseudomonasrhodesiaeandPseudomo-nas uorescens in waterorganic solvent systems using terpene substrates forbothgrowthandbiotransformationprocessesandtodeterminetheaerobicoranaerobicstatusofthesedegradationpathways.Materials and Methods: Substrates frompinene (a-pinene, a-pinene oxide,b-pinene, b-pineneoxide, turpentine)andlimonene(limonene, limonene-1,2-oxide, orangepeel oil)familiesweretested. Forthebioconversion, theterpene-grownbiomass was concentratedandusedeither as wholecells or as acrudeenzymaticextract.Conclusion: Pseudomonas rhodesiae was the most suitable biocatalyst for theproduction ofisonovalal from a-pineneoxide and did notmetabolize limonene.Pseudomonas uorescens wasamoreversatilemicro-organismandmetabolizedlimonene in two different ways. The rst (anaerobic, cofactor-independent,noninducible) allowedlimonene eliminationby synthesizing a-terpineol. Thesecond(aerobic, cofactor-dependent)involvedlimonene-1,2-oxideas aninter-mediate for energy production through a b-oxidation process.Signicance and Impact of the Study: Enzymatic isomerization of b- toa-pinene was described for the rst time for both strains. Alpha-terpineolproduction by P.uorescens was very efcient and appeared as a promisingalternativeforthecommercialproductionofthisbioavour.JournalofAppliedMicrobiologyISSN1364-50722008TheAuthorsJournal compilation 2008TheSocietyforAppliedMicrobiology,Journal ofAppliedMicrobiology105(2008)19912001 1991The microbial transformation of terpenes started 4050yearsago, whensoil pseudomonads wereusedforthedegradation of limonene (Dhavalikar and Bhattacharyya1966; Dhavalikar etal. 1966), a- andb-pinenes (Shuklaand Bhattacharyya 1968; Shukla etal. 1968). Fungal-mediatedoxidationofterpeneswasdescribedinthesameperiod, after anAspergillus niger capable of metabolizinga-pinene to oxygenated products was selected amongdifferent moulds (Bhattacharyya etal. 1960; Prema andBhattacharyya1962a,b).The mechanismof a-pinene degradationdescribedinthe rst studies passed through limonene and perillylalcohol toformisopropyl pimelicacid, besidesfourotherpossiblepathways(ShuklaandBhattacharyya1968).Someyears later, other authors described another a-pinenedegradationpathwaypassingbylimonenebut withotherintermediate acids (Gibbon and Pirt 1971; Tudroszenetal. 1977). A third metabolic route (pathway 1a inFig.1), avariationof thelast, was proposedfor Pseudo-monas uorescens NCIMB11671, which was capable ofcompletelydegradinga-pinene(Best etal. 1987). Asimi-larpathwaywasalsoevidencedforNocardiasp. (Grifthsetal. 1987a,b). Further reports suggest a differentdynamic for this pathway, explaining the formation ofnovalal by isomerizationof isonovalal (Zornetal. 2004;Larocheetal. 2006; pathway1binFig. 1). Studyingthisprocessmoreindepth, anoptimizedmethodforisonova-lal production from a-pinene oxide by Pseudomonasrhodesiae has been developed (Fontanille etal. 2002;Fontanille and Larroche 2003). More recently, Yoo andDay(2002) reportedanothera-pinenedegradationpath-way for Pseudomonas PIN. This novel route integratesa-pinene, b-pinene, limoneneandp-cymeneandleadstotheformationofperillicacid, cumicacidand a-terpineol.The degradation pathways for b-pinene have not yetbeen fully elucidated. It is known that some A. nigerstrains are capableof transformingit intoa-terpineol asthemainproduct(Toniazzoetal. 2005; Rozenbaumetal.2006). Different hydroxylated products have also beenidentied for Botrytis cinerea (Farooq etal. 2002) andArmillariellamellea(Draczyn skaetal. 1985; pathways 17and18inFig.1). Forbacteria, acommonrouteforlimo-nene, a- and b-pinene has already been described forBacillus pallidus BR425. Inthis case, myrtenol andpino-carveol are the mainproducts fromb-pinene (Savithiryetal. 1998). Conversely, thefermentationof b-pinenebyPseudomonas PL resulted in different products (ShuklaandBhattacharyya1968; pathway20inFig. 1). Themainmetabolicpathways fora- andb-pinenearesummarizedinFig.1.It is observedthat limoneneiscommonlyfoundasanintermediate in the metabolism of a- and b-pinene.Therefore, it is possibletoassumethat, for somemicro-organisms, these twosubstrates may alsofollowa path-waysimilartothatobservedforlimonene. Maro sticaandPastore (2007a) have recently reviewed the mainmetabolicroutesforlimoneneandFig. 2illustratesitssixdifferent degradationpathways, whichare: (i) oxidationof carbon7toperillyl compounds; (ii)ringdoublebondepoxidation, followed by the corresponding diol forma-tionandits oxidation; (iii) carbon6oxidationtoformcarveol, carvone and dihydrocarvone; (iv) carbon 8hydroxylationtodirectlyforma-terpineol; (v) oxidationof carbon 3 to formisopiperitenol and isopiperitenoneand(vi)8,9doublebondepoxidationtoformlimonene-8,9-epoxide. Other papers describe the catabolism ofacyclic monoterpenes (Forster-Fromme etal. 2006 andreferences therein), the degradation of monoterpenes inanaerobic conditions (Harder and Probian 1995) andreviewthe biotransformation of limonene (Duetz etal.2003) andother terpenes (VanDer Werf etal. 1997; DeCarvalhoandDaFonseca2006).The twostrains testedinthis study, P.rhodesiae CIP107491 andP.uorescens NCIMB11671, are recognizedfortheirabilitytousea-pineneassolecarbonsourceandtoconvert it intodifferent oxygenatedterpenoids. How-ever, theinformationontheutilizationof otherterpenesislimited. Therefore, differentterpenesourcesweretestedas sole carbonsources andas substrates for biotransfor-mationofthesemicro-organisms.MaterialsandmethodsMicro-organismsandchemicalsThetwostrains employedinthis workwereP.rhodesiaeCIP107491 and P.uorescens NCIMB 11671. Alpha-pinene (Acros, 97%purity), ())-b-pinene (Fluka, >99%Figure1Mainmetabolicpathwaysofa-andb-pinenedescribedintheliterature.Pathways:1areportedbyBestetal.(1987)andGrifthsetal.(1987a,b); 1bbyLarocheetal. (2006); Zornetal. (2004); 24and1315bySchrader (2007); 5byAgrawal etal. (1999); Agrawal andJoseph(2000a,b);Draczyn skaetal.(1985);PremaandBhattacharyya(1962a);Rozenbaumetal.(2006);VanDyketal.(1998);andWrightetal.(1986);6byGibbonandPirt (1971); Savithiryetal. (1998); Tudroszenetal. (1977) andYooandDay(2002); 7byChatterjeeetal. (1999); Draczyn skaetal.(1985);Rozenbaumetal.(2006)andWrightetal.(1986);810,21and22bySavithiryetal.(1998);11and20byShuklaandBhattachar-yya(1968)andShuklaetal.(1968);12byDraczyn skaetal.(1985);PremaandBhattacharyya(1962a)andWrightetal.(1986);16byRozenbaumetal. (2006) andToniazzoetal. (2005); 17by Farooqetal. (2002); 18by Draczyn skaetal. (1985); 19by VanDyketal. (1998) and23bySavithiryetal.(1998)andYooandDay(2002).ThewidearrowsrepresentthemetabolismofpinenesbyPseudomonasrhodesiaeandPseudomo-nasuorescensandthedasharrowscorrespondtotheisomerizationdescribedinthisstudy.Degradationofmonoterpenes J.L.Bicasetal.1992 Journal compilation 2008TheSocietyforAppliedMicrobiology,Journal ofAppliedMicrobiology105(2008)199120012008TheAuthorsCarvoneCarveolIsonovalal Isonovalic acidThujone -pinene oxide Dimethylpentanoic acid Novalal Novalic acidBorneolVerbenone Verbenol6-hydroxy--pinene9-hydroxy- -pinene 4,5-dihydroxy--pinene Limonene-terpineol 2,3-dihydroxy--pinene4-hydroxy--pinene-6-one -pinene -pinene4-hydroxy--pinene-6-onePinocamphone PinocarveolMyrtenoltrans-sobrerolPinocarvoneMyrtenalPinocamphone7-hydroxy--terpineol BorneolOleuropeic acid4-hydroxyphellandric acid(from -pinene)4-hydroxydihydrophellandric acid(from -pinene)-isopropyl pimelic acidFig. 2 15514+ + or+1b1a1a1a,b1a1a,b 1a,b +++671011 12 13 1617182021222319COOHCH2OHCH2OHCH2OCOOHCOOHOHOHOHOHOHOHOHOHHOOOOOOOCOOHOHCOOHOHOHOHOHOHOHOHCHO COOHCOOHCOOH CHOOHHO OO432OHOOHJ.L.Bicasetal. Degradationofmonoterpenes2008TheAuthorsJournal compilation 2008TheSocietyforAppliedMicrobiology,Journal ofAppliedMicrobiology105(2008)19912001 1993purity), R-(+)-limonene (Fluka, 98% purity), ())-a-pinene oxide (Aldrich, 97%purity), b-pinene oxide(Acros, mixture of cis trans isomers, 75%purity), (+)-limonene-1,2-oxide(Aldrich, mixtureofcis transisomers,97%purity) and(+)-carvone (Acros, 98%purity) wereusedas substrates inn-hexadecane(SDS, 99%purity) asorganic phase. The complexmonoterpene mixtures, tur-pentine and orange peel oil, obtained in the Brazilianmarket,werealsotested.PreculturepreparationThreefullloopsofa24-h-oldcultureonapetridishweretransferredtoa500ml conical askthat contained10gof carbon source (sodiumlactate for P.rhodesiae andglucose for P.uorescens), 025g of (NH)4SO4, 5ml ofHutner solution (Fontanille 2002), 10ml of solution A(65gof K2HPO4, 828gof KH2PO4in250ml distilledwater) and 235ml of distilled water. The asks wereincubatedat 30Cand200revmin)1for 24h, toreachanopticaldensitycloseto40at600nm(OD600).CellculturesTwentymillilitresof thepreculturewereasepticallytrans-ferred to a 500ml conical ask with the same culturemediumas described before. However, the sole carbonsourceconsistedof 05gof oneof theterpenesubstratesPerillyl alcohol perillyl-CoALimonene-1,2-oxide Limonene-1,2-diol 1-hydroxy-2-oxolimonene3-isopropenyl-6-oxoheptanoate 3-isopropenyl-6-oxoheptanyl-CoACarveol-teripenol Isopiperitenone IsopiperitenolLimonene-8,9-oxide-oxidation123456Carvone DihydrocarvonePerillyl aldehyde Perillic acidCH2OHOOHO O HOOHOH OOOHOHOOOOOSCoAOCOH COOHCOSCoAFigure2Thesixmainmetabolicpathwaysforlimonene(VanDerWerfetal.1999;Maro sticaandPastore2007a).ThewidearrowsrepresentthemetabolismoflimonenebyPseudomonasuorescensdescribedinthisstudy.Degradationofmonoterpenes J.L.Bicasetal.1994 Journal compilation 2008TheSocietyforAppliedMicrobiology,Journal ofAppliedMicrobiology105(2008)199120012008TheAuthorsdilutedin125mlofn-hexadecane. Theaskswereleftat30Cand200revmin)1for24h.RecoveryofthebiomassAfter centrifuging the culture at 2600g for 10min, thesupernatantwaseliminatedandtheresultingbiomasswasresuspendedin25ml of20mmoll)1ofphosphatebufferpH75. This biomass was either useddirectly(biotrans-formationwithfreshcells)orwasfrozen()18C)forthetrials using crude enzymatic extracts. The biomass con-centrationwasdeterminedindirectlybytheOD600of themedium, throughthefollowingexperimental correlations:P.rhodesiae (Fontanille etal. 2002): biomass (gramperlitre of the medium)=041 OD600; P.ourescens: bio-mass(gramperlitreofthemedium)=035 OD600.ProductionofthecrudeenzymaticextractsThe concentratedbiomass of P.rhodesiae (see precedingparagraph) was thawedandtreatedfor 1hat 30Cand200revmin)1with 6%(v v) diethyl ether (Fontanilleand Larroche 2003). The P.uorescens strain employedin this study has shown a relatively high resistance tocell permeabilization, in comparison with P.rhodesiae.Hence, the standard protocol used for this last micro-organism allowed 7090% of the initial biomass toremainviable. The cells were thendisruptedbyathree-cycle passage in a two-stage high-pressure homogenizer(model APV2000; APVSystems, Alberstlund, Denmark),using 1000bars in the rst stage and 100bars in thesecond. This methodology was able to reduce the nalnumber of viable cell counts to5%of the initial value.The suspensionresultingfromthis operationwas mem-brane ltered(045 lm) toremove the remainingviablecells.BiotransformationprocedureTwenty-ve millilitres of the concentrated biomass andthe same volume of n-hexadecane were transferredtoa250ml conical ask. Thesubstratewas addedtoreachanalconcentrationof40gl)1ofn-hexadecane. Theaskswereincubatedat30Cand200revmin)1. Sampleswereperiodically takenfromthe organic andaqueous phasestofollowsubstrate consumptionandproduct formation.Theorganicphasewas directlyinjected(1 ll) inthegaschromatographwhiletheaqueous phasehadtobeacidi-ed(20 llml)1concentratedsulfuricacid)andextractedwiththesamevolumeof etherhexane(1:1, v v)beforetheorganiclayerwasinjected(1 ll)inthegaschromato-graph. Theprocedureforthecrudeenzymaticextractwassimilar. Experiments were carried out in triplicate andconcentrationsmeasuredat agiventimealwaysgavedif-ferenceslessthat10%.AnalyticalconditionsThe products obtainedwere analysedina HP5890gaschromatograph with a ame ionization detector (GC-FID). A SBP-5 (Supelco) capillary column of 30m 032mm 025 lmidwas employed. Nitrogenwas thecarrier gas, witha constant pressure inthe headof thecolumnof 08bar andsplit ratioof 1:5. The tempera-ture programme usedwas as follows: initial temperatureof80Cfor5min, risingat20Cmin)1until 200C, thenheld for 5min. The temperatures in the injector anddetector were both 250C. The quantication was per-formedusing10ml of heptadecane(Fluka, 98%purity)per litre of n-hexadecane as internal standard and theyieldwascalculatedastheratiooftheamountofproductrecovered(g)tothemassofsubstrateconsumed(g). TheproductsobtainedwereidentiedbyaHP6890gaschro-matographcoupledwithaHP5973massselectivedetec-tor (GC-MS). Heliumwas the carrier gas andthe splitratiowas1:5. Thecapillarycolumnandthetemperatureprogrammewasthesameasbefore. TheMSsystemoper-ated with an electron impact of 70eV, an accelerationvoltageof 11kVandanemissioncurrent of 35 lA. Thetemperaturesof thequadrupole, theionicsourceandtheGC-MSinterface were 150C, 230Cand280C, respec-tively. The isomers obtained, i.e. novalal andisonovalal,wereidentiedonthebasis of their retentiontimes andmass spectra. Anexample of a GCanalysis is showninFig.3.ResultsCharacterizationofthecomplexsubstratesTurpentineandorangepeel oil arecheapandwidespreadterpene sources andcouldbe involvedinbioconversionprocesses, either as inducing agents for biocatalyst pro-ductionorasprecursorsforthereactionitself.Asforanyotheragroindustrial product, theircomposi-tion may vary depending on seasonal conditions. Thecompositionof turpentineandorangepeel oil, showninTable1, was determined by GC-FIDand GC-MS. Theconcentration of the main compound in each mixturewasconsistentwithalreadyreportedvalues(Table1).UtilizationoftheterpenesubstratesforbacterialgrowthThetwomicro-organismswerersttestedfortheirabilitytogrowonterpenesubstrates as solecarbonandenergysources. The growthwas stoppedafter 24hbecause theJ.L.Bicasetal. Degradationofmonoterpenes2008TheAuthorsJournal compilation 2008TheSocietyforAppliedMicrobiology,Journal ofAppliedMicrobiology105(2008)19912001 1995biomassobtainedatthistimewasagoodindicatoroftheability of a givensubstrate tosupport bacterial growth.The utilization of a given terpene indicated that ametabolicpathwayinvolvingit was actuallyactiveinthecorrespondingbacterialstrain.It canbe observedinTable2that a-pinene, b-pineneand a-pineneoxideweresubstratesforbothmicro-organ-isms. Asexpected, turpentinewasalsoasubstrateforthetwobacteria, probably operating as ana-pinene source.Theotherconstituentsofturpentinewerenottoxictothebacteria, at least at the concentrationassayed. Limoneneand b-pineneoxideweresubstratesonlyforP.uorescens,while limonene-1,2-oxide and orange peel oil weresubstrates fornoneof thetwostrains. All thedegradableterpenesubstrates producedbiomass withconcentrationsrangingfrom03to18gl)1andthesewereusuallylowerfortheepoxides(Table2).BiocatalyticactivityofPseudomonasrhodesiaeandPseudomonasuorescensTodenethemetabolicpathwaysthroughwhichtheter-penesubstrates weredegradedandtondactivities thatcouldbe exploited at a preparative scale, bioconversion0 2 4 6 8 10 12 14Time (min)FID signalNovalalIsonovalal-pinene-pineneInternal standard-pinene oxideSolventFigure3Example ofagas-chromatographicanalysis:bioconversionof b-pinenebyfreshcellsofPseudomonasrhodesiaegrownonb-pinene.Experimental conditionsaredescribedinTable3.Table1Mainconstituentsof turpentineandorangepeel oil usedinthisstudyComponentProportion(%,peakareaingaschromatography) Reportedvalues(%)Turpentinea-pinene 671 6070(Baueretal.2001);4389(Coppenetal.1998)Camphene 206R-(+)-limonene 46Others 77Orangepeel oilR-(+)-limonene 877 8397(Shaw1979);943(Maro sticaandPastore2007b)Cis-andtrans-limonene-1,2-oxide36Carvone 16Others 71Table2UtilizationoftheterpenesubstratesbyPseudomonasrhode-siae and Pseudomonas uorescens for growth. The inoculumconsistedof aculturegrownonamediumwith025gof (NH)4SO4,5ml of Hutner solution(Fontanille2002), 10ml of solutionA(65gof K2HPO4, 828gof KH2PO4in250ml of distilledwater), 1gofNa-lactate(P.rhodeisae)or1gofglucose(P.uorescens)and235mlof distilledwater. Cell growthwas developedinthesame mediumusing05gof theterpenesubstratein125ml of n-hexadecaneassolecarbonsourceTerpenesubstrateGrowthafter24hP.rhodesiae P.uorescensa-pin +(09) +(17)b-pin +(18) +(16)Lim ) +(14)a-pinox +(03) +(08)b-pinox ) +(09)Limox ) )Turp +(11) +(18)OPO ) )a-pin,a-pinene;b-pin,())-b-pinene;lim,R-(+)-limonene;a-pinox,())-a-pinene oxide; b-pin ox, b-pinene oxide; limox, (+)-limonene-1,2-oxide; Turp, turpentine; OPO, orange peel oil; +, growth; ), nogrowth.Numbersinparenthesesaretheapproximatenal biomassconcentra-tion(gl)1).Degradationofmonoterpenes J.L.Bicasetal.1996 Journal compilation 2008TheSocietyforAppliedMicrobiology,Journal ofAppliedMicrobiology105(2008)199120012008TheAuthorsexperiments were carried out with either concentratedfreshcells or crude enzymatic extracts. The latter couldbeconsideredasaformof thebiocatalyst unabletoper-formcofactor-dependent reactions. Results obtained arepresented in Table3, with the main products accumu-lated after 24h of bioconversion and their respectiveapproximate yields. When product accumulation andsubstrate consumption were very low, the parameteramountproducedwaschosenasamorereliableoption.Alphaand b-pineneThe results showninTable3indicate that bothprecur-sorswereconsumedandall theproductsaccumulatedbybothstrainswerebasicallyfromthesamemetabolicpath-way (pathway 1b in Fig. 1). When a fresh biomass ofP.rhodesiae grown on a-pinene was used to converta-pinene, 3,4-dimethyl pentanoic acid (DMPA) was themajor product accumulatedafter 24h. Thesamefeaturewas observedforfreshP.uorescens cells, but withloweryields. Alpha-pinene oxide, the rst intermediate ina-pinenedegradation, allowedtheaccumulationofisono-valal, novalal, novalic acid and traces of DMPA withP.rhodesiae, while isonovalal couldnot be detectedwithP.uorescens. The behaviour of the crude enzymaticextracts (Table3) demonstrated that, for both strains,epoxidation of a-pinene was a cofactor-dependent pro-cess,whilealdehydesynthesisfromtheepoxidewascofac-tor-independent.Thebioconversionofb-pinenebyfreshcellsof P.rho-desiaeandP.uorescens preliminarilygrownonthesamecarbon source resulted in the formation of a-pinene,DMPAandtraces of intermediates showninpathway1binFig.1(Fig.3). Thesamereactionwiththecrudeenzy-matic extracts of bothstrains gave only a lowa-pineneaccumulation, not observed in the control experiments(Table3).Whenb-pinene oxide was usedas substrate, the pro-ducts obtainedwere always the same as those foundinthe control experiments (without biomass). This beha-viour was evidencedfor all kinds of biomass andstrainsTable3Mainproductsaccumulatedinthebioconversionofthea-pinene,b-pineneandlimonenefamiliesbyPseudomonasrhodesiaeandPseu-domonasuorescens. Theculturegrownonaterpenesubstrateassolecarbonsource(24hat 30Cand200revmin)1) wasconcentrated10timesandtheresultingbiomasswaseither usedfreshor asacrudeenzymaticextract (1hat 30Cand200revmin)1, inthepresenceof 6%ether,forP.rhodesiae,orthreecyclepassageinatwo-stageAPVSystemshomogenizer,using1000barintherststageand100barinthesec-ond,forP.uorescens,beforebeingused).Thebioconversiontrialsconsistedofa250ml conical asklledwith25ml ofconcentratedbiomass,25ml ofn-hexadecaneand1gofterpenesubstrateincubatedfor24hat30Cand200revmin)1Terpenesubstrates Productsaccumulatedafter24hCellgrowth BioconversionP.rhodesiae P.uorescensFreshCrudeenzymaticextract FreshCrudeenzymaticextracta-pin a-pin DMPA(10) 0 DMPA(2) 0a-pin a-pinox Isonov(6),nov(11),novac(44) Isonov(80) Nov(48),novac(18) Isonov(66)a-pinox a-pinox Isonov(47),nov(7),novac(2) Nov(10),novac(17) Turp Turp* DMPA(8) 0 a-pinox(3),nov(6) 0Turp a-pinox Isonov(13),nov(24),novac(35) Isonov(75) b-pin b-pin a-pin(14),DMPA(7) a-pin(04) a-pin(20),DMPA(1) a-pin(03)b-pin b-pinox Autodegrad Autodegrad Autodegrad Autodegradb-pinox b-pinox Autodegrad AutodegradLim Lim a-ter(50) a-ter(91)Lim OPO ** a-ter(05),Limdiol (02)Lim Limox Limdiol (27),HOL(77) Limdiol (05)0, noproducts; , not done; a-pin, a-pinene; a-pinox, a-pineneoxide; a-ter, a-terpineol; b-pin, ())-b-pinene; b-pinox, b-pineneoxide; auto-degrad,thesameproductsobtainedinthecontrol experiments(inactivebiomass);DMPA,3,4-dimethyl pentanoicacid;HOL,1-hydroxy-2-oxolimo-nene; isonov, isonovalal; lim, R-(+)-limonene; limdiol, limonene-1,2-diol; limox, limonene-1,2-oxide; nov, novalal; nov ac, novalic acid; OPO,orangepeel oil;turp,turpentine.Numbersinparenthesesaretheapproximateyield(%).*only a-pineneismetabolizedinturpentine,whiletheotherconstituentsarenotconsumed.After6hbioconversion.Productconcentration(gl)1)after24h(toolowsubstrateconsumptionandproductformationtocalculateareliableyield).Nogrowthonthecarbonsource.Limoneneandpartoflimonene-1,2-oxidearemetabolizedinorangepeel oil.**Orangepeel oil permeabilizesthebiomass.J.L.Bicasetal. Degradationofmonoterpenes2008TheAuthorsJournal compilation 2008TheSocietyforAppliedMicrobiology,Journal ofAppliedMicrobiology105(2008)19912001 1997usedinthis work(Table3). It was thus concludedthatthemainphenomenonwasaprecursorauto-oxidationintheaqueousphase, whichtookplacewithout anysigni-cantbiocatalyticactivity.LimonenePseudomonas rhodesiae didnot growonlimoneneas thesole carbon source, it was thus not considered for thelimonene bioconversion trials shown in Table3. Forthe freshcells andcrude enzymatic extract of P.uores-cens biomass, thesolemetaboliteaccumulatedwasa-ter-pineol, the yield being higher with the extract. Theproductionobtainedinthis process, around1011gl)1of n-hexadecane after 3040hat a maximumrate of c.1gl)1h)1(datanot shown), was infact, tothebest ofour knowledge, thehighest alreadyreportedfor thebio-productionof a-terpineol.Interestingly, it was noticed that whole cells of thismicro-organismwere alsoable toaccumulate limonene-1,2-diol and 1-hydroxy-2-oxolimonene fromlimonene-1,2-oxide, aprocesspracticallyinoperativewiththecrudeextract(Table3).TurpentineandorangepeeloilThebioconversionof turpentine(sourceof a-pinene) byfreshP.rhodesiaecells grownonthesamesubstrategavealmost thesameproleas that observedfor theconver-sionof purea-pinene. Thisresult demonstratedthat thisessential oil was also able to act as a cell inducer fora-pinene degradation. The other major constituents ofturpentine(camphene, limonene)werenevermetabolized(Table3). Thesubstitutionof a-pinenebyturpentineforthe cell growth inthe process of isonovalal production(FontanilleandLarroche2003)resultedinsimilarresultsto that obtained in the standard process, i.e. maximalconcentrationof c. 80gl)1obtained after 4h, with aninitial maximumrateofc. 40gl)1h)1andayieldof7580%(datanotshown).It was not possible to performthe bioconversion oforangepeel oil withfreshP.uorescens biomass because,after a short period(