Research Article Isolation and Characterization of ...downloads.hindawi.com/journals/bmri/2015/929424.pdfResearch Article Isolation and Characterization of Hydrocarbon-Degrading Yeast

Research ArticleIsolation and Characterization of Hydrocarbon-Degrading YeastStrains from Petroleum Contaminated Industrial Wastewater

Boutheina Gargouri Najla Mhiri Fatma Karray Fathi Aloui and Sami Sayadi

Laboratoire des Bioprocedes Environnementaux Centre de Biotechnologie de Sfax Universite of Sfax BP 1177 3018 Sfax Tunisia

Correspondence should be addressed to Boutheina Gargouri boutheina fssyahoofr

Received 13 November 2014 Revised 28 March 2015 Accepted 5 April 2015

Academic Editor J P A Hettiaratchi

Copyright copy 2015 Boutheina Gargouri et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Two yeast strains are enriched and isolated from industrial refinery wastewater These strains were observed for their abilityto utilize several classes of petroleum hydrocarbons substrates such as n-alkanes and aromatic hydrocarbons as a sole carbonsource Phylogenetic analysis based on the D1D2 variable domain and the ITS-region sequences indicated that strains HC1 andHC4 were members of the genera Candida and Trichosporon respectively The mechanism of hydrocarbon uptaking by yeastCandida andTrichosporonhas been studied bymeans of the kinetic analysis of hydrocarbons-degrading yeasts growth and substrateassimilation Biodegradation capacity and biomass quantity were daily measured during twelve days by gravimetric analysis andgas chromatography coupled with mass spectrometry techniques Removal of n-alkanes indicated a strong ability of hydrocarbonbiodegradation by the isolated yeast strains These two strains grew on long-chain n-alkane diesel oil and crude oil but failed togrow on short-chain n-alkane and aromatic hydrocarbons Growth measurement attributes of the isolates using n-hexadecanediesel oil and crude oil as substrates showed that strain HC1 had better degradation for hydrocarbon substrates than strain HC4In conclusion these yeast strains can be useful for the bioremediation process and decreasing petroleum pollution in wastewatercontaminated with petroleum hydrocarbons

1 Introduction

Soil and water contamination are frequently caused by oiland oil-related compounds Strategies for controlling envi-ronmental contamination by petroleum and its derivativeshave been the subject of various studies over the pastthree decades When a spillage occurs the first action is toremove the oily phase by mechanical or physicochemicalmeans through the application of surfactants in order todisperse the oil layer Although a variety of physicochemicaltechniques are available for the clean-up of water surfacesome interest in the use of microbial biodegradative activityis growing [1] Biodegradation is an alternative that has beenused to eliminate or minimise the effects of pollutants byusing microorganisms which have biodegradation potential[2] In these environments organic pollutants frequentlyoccur when mixed with other synthetic or natural organiccompounds Microorganisms potentiality pointed out in

the literature as degradation agents of several compoundsindicate that biodegradation is one of the most promisingalternative of biological treatments to reduce the environ-mental impact caused by oil spills Moreover it is known thatthe main microorganisms consuming petroleum hydrocar-bons are bacteria yeasts and fungi [3]

Therefore it is necessary to understand how the biodegra-dation of the polluting compounds is affected by the presenceof alternate substrates It is also necessary to study themicrobial degradation of crude oil as an environmentallyfriendly way of cleaning up oil-polluted areas

The first step in such studies is to isolate and identifythemicroorganisms from contaminated soil and water whichare capable of crude oil degradation Among many studiesconducted on microbial biodegradation of oil-related con-taminants more than 80 are devoted to bacterial biodegra-dation Bacteria are the most studied microorganisms andtheir participation during hydrocarbon mineralization in

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 929424 11 pageshttpdxdoiorg1011552015929424

2 BioMed Research International

water has been studied by many authors [4] However notonly are bacteria capable of hydrocarbon biodegradation butalso yeasts isolated from hydrocarbon contaminated sitesdisplay the ability to use oil-related compounds [5 6] Asa matter of fact a wide variety of yeasts and filamentousfungi are capable of utilizing hydrocarbons Yeasts capable ofdegrading hydrocarbons include genera of Yarrowia lipolyt-ica Candida tropicalis Candida albicans and DebaryomyceshanseniiThe alkane-utilizing yeastY lipolytica degrades veryefficiently hydrophobic substrates (HS) such as triglycerides(fats oils) alkanes and fatty acids It has been observed thatY lipolytica is the predominant yeast form during alkanedegradation and emulsifier production [7]

The study of hydrocarbon biodegradation by nativemicroorganisms is of extreme ecological importance Theactual contribution of yeasts in the biodegradation of hydro-carbons in the environment may be more important thanthat previously expected considering the metabolic diver-sity demonstrated for yeasts [8] However bioavailabilityof hydrophobic organic compounds to microorganisms isgenerally a limiting step during the biodegradation process[9] Hydrophobic interactions play an important role in theadherence of microorganisms to a wide variety of surfaces[10] In particular the hydrophobic nature of the bacterialsurface has been cited as a key factor in cells growth onwater-insoluble hydrophobic substrates such as hydrocarbon[11 12] To enter cells HS must interact with the cell surfaceTwo hypotheses have been formulated to explain this step ofpoorly water-miscible substrates transport into microor-ganisms (i) the compounds can be solubilised (or pseu-dosolubilised) in the presence of surface-active compounds(surfactant-mediated transport) or (ii) they can adheredirectly to the cell wall (direct interfacial transport) [13] Theaforementioned yeast species can produce surfactants duringgrowth on HS A correlation has been observed betweencells and HS adhesion induction and the increase in apolarproperties of the cell surface [13] Aerobic degradation ofalkanes can be carried out by twomajor types of enzymes thealkane monooxygenase (also known as alkane hydroxylaseor AlkB) and certain cytochrome P450 systems

In a screening program carried out in our laboratory weisolated yeast strains from petroleum contaminated wastew-ater effluents that are able to utilize hydrocarbons The use ofnaturally occurring yeasts represents a potentially promisingmeans of destroying polluting chemicals in wastewater treat-ment systems and soil Therefore the objective of the presentwork was to isolate identify and characterize n-alkane usingyeasts capable of degrading petroleum hydrocarbons in orderto employ yeasts strains in the future as a biodegrader ofpolluted environmentThe hydrocarbon degradation activityand cell hydrophobicity were also investigated in relation ton-alkane utilization

2 Material and Methods

21 Isolation and Characterization of Yeast Isolates Thehydrocarbons-degradingmicrobial consortiumwas enriched

from petrochemical industrial wastewater using a continu-ously stirred tank-reactorCSTR [14] Twoof the best-growingyeast strains were isolated from the hydrocarbons-degradingmicrobial consortium The yeast strains were isolated byusing a selective enrichment culture and a single-colonyisolation technique The selected yeasts were identified inthe present study by routine morphological microbiologicaland biochemical methods following directions of the latestedition of Bergeyrsquos Manual [15]

22 Culture Conditions Yeast isolates were cultivated in250ml Erlenmeyer flasks containing 50ml of mineralmedium (MM) The pH was measured using the Cyberscan500 pH meter The pH was adjusted by using 1N standardNaOH and 1N standard HCl (Merck Germany) The min-eral medium used contained per litre 34 g K

2HPO4 43 g

KH2PO4 03 g MgCl

2sdot2H2O and 1 g (NH

4)2SO4 which was

supplemented with 1ml of a trace element solution [16] Themedia used for the yeast growthwere sterilized by autoclavingat 121∘C for 20 minutes Petroleum compounds and deriva-tives were added as carbon source to the sterilized MM at thedesired concentrations A liquid culturewas started by addinga loop full of cells from a standard agar plate into a 250mlErlenmeyer flask containing a 50ml medium Flasks wereinoculated with 1ml cultures pregrown Then isolates wereincubated at 30∘C under agitation (180 rpm) for 10ndash15 days(depending on the strain) Samples were taken at intervalsfrom shake flasks The isolated yeast was also grown onPotato Dextrose Agar (PDA) in order to observe largercolonies for better study Growth was assessed by measuringthe optical density (OD) at 600 nm Three independentexperiments were carried out for each yeast strain

23 Biomass Growth during the Biodegradation ProcessBiomass concentration was estimated from the absorbance ofappropriately diluted culture medium at 600 nm (Ultrospec3300 pro UVVisible) according to the predetermined cor-relation between optical density and dry weight of biomassDuring the biodegradation assays the number of viable yeastswas measured by counting of heterotrophic microbes in Petridishes expressing the result as colony-forming units (CFU)per ml With a sterile pipette 1ml of sample was taken fromthe culture and a series of 1 10 dilutions made in NaCl solu-tion at 09 Each dilution was analysed in duplicate 1ml ofsample to be analysed was placed on Petri dishesThen 20mlof previously sterilized culture medium was poured onto thePetri dishes tempered at 60∘C and gently stirred to completethe homogenization The mixture was cooled until completesolidification and then incubated at 30∘C for 72 h Thetotal number of microorganisms was determined by mul-tiplying the number of CFU by the corresponding dilutionfactor The count was made in an automatic colony counter(Countermat Flash IUL Instruments) All experiments wereperformed in triplicate with appropriate controls

24 Cell Surface Hydrophobicity Test Cell surface hydropho-bicity was measured by microbial adherence to hexadecaneaccording to themethod of Rosenberg et al [17] Yeast strains

BioMed Research International 3

were grown in 50ml basal medium containing hexadecane(15) or glucose (20mM) as sole carbon source Yeastcells were washed twice and resuspended in PUM bufferpH 71 (222 g K

2HPO4sdot3H2O 726 g KH

2PO4 18 g urea

02 g MgSO4sdot7H2O and distilled water of 1000ml) to an

initial absorbance at 550 nm of 05ndash06 The cell suspension(12ml) with hexadecane (02ml) was vortexed in a test tubevigorously for 2min and left at room temperature for 1 hThe optical density of the bottom aqueous phase was thenmeasured at 550 nm Hydrophobicity was expressed as thepercentage of adherence to the hydrocarbon which wascalculated using the following

Hydrophobicity

= 100times(1minus OD of the aqueous phaseOD of the initial cell suspension

)

(1)

Hydrophobicity of the yeasts grown on glucose was thereference of hydrophobicity

25 Tests of Hydrocarbon Tolerance and BiodegradationStudies In order to determine the degradation of n-alkanes present in the hydrocarbon-rich wastewater by theselected yeast isolate the total petroleumhydrocarbon (TPH)was determined by the gravimetric weight loss technique[18] The analyses of hydrocarbons were carried out afterdichloromethane extraction The aqueous phase sample wasremoved and put in a sealed flask for subsequent analysisThen it was concentrated to approximately 3ml using arotary evaporator under reduced pressure in a waterbath Afterwards it was dissolved in equal volume ofdichloromethane and further cleaned through a columnfilled with florisil (SUPELCLEAN LC-FLORISIL USA) andthen analyzed by gas chromatography mass spectrometryapparatus After the evaporation of the solvent the amount ofresidual TPH was determined by gravimetric methods afterdichloromethane evaporation by simple distillation at 60∘C[18] Hydrocarbon content was determined using an extrac-tion of hydrocarbon according to the standardmethod for oilgravimetric determinationThe degree of biodegradationwascalculated as [1minus(119883119900minus1198831)119883119900] 100 [] where119883119900 is initialamount of hydrocarbon and 1198831 is amount of hydrocarbonafter biodegradation

The ability of the selected strains to utilize hydrocarbonsas the sole carbon source was tested by growing yeasts strainsin MM with different hydrocarbons in separate flasks Thebiodegradation assay was carried out over 12 days in six250ml Erlenmeyer flasks (corresponding to 0 4 6 9 and 12days of experiment resp) with each flask containing 50ml ofthe mineral medium and 10 of acclimated inoculumunder aseptic conditions For the induction and hydrocar-bon degradation studies using nonproliferating cells strainsHC1 and HC4 were grown in Erlenmeyer flasks containing50ml MM with 1 hydrocarbon compounds as the solecarbon and energy source and were incubated in a rotaryshaker (180 rpm) at 30∘C The cells were washed twice withsterile 50mM potassium phosphate buffer pH 76 Growthon individual substrate was removed periodically to assess

the concentration of following changes in optical density at600 nm of washed cells against biotic (without a substrate)and sterile (without bacteria) controls and tested for its biore-mediation capacity in situ mesocosm to degrade multiplesubstrates after 12-day incubation period

26 Hydrocarbon Analysis The evaluation of the hydrocar-bons biodegradation of the contaminated wastewater effluentby yeast strains was carried out during the process of 12days using gas chromatography with mass spectrometryGCMS After 12 days of incubation the undegraded alkanehydrocarbon residue was extracted twice with equal vol-umes of dichloromethane and the aromatic residue withtoluene One microlitre of the alkane fraction (dissolved indichloromethane) was analyzed by gas chromatography anal-ysisGCMS (ShimadzuModel 5975B inertMSD)using aHP-5MS chromatographic column (5 phenyl Methyl Siloxane)of size 30m times 025mm times 025 120583m The temperature wasprogrammed to vary linearly from 70∘C to 230∘C at the rate of20∘C minminus1 then from 230∘C to 300∘C at 40∘C minminus1 and10min at 300∘C Samples of 10 120583l were injected into theGCMS operating in the splitless mode with an injectortemperature of 250∘C and Helium was the carrier gas

Similarly the aromatic fraction was dissolved in tolueneand 1ml was analyzed by GCMS using a 30-m long HP-5MS (025mm film thickness) column During analysis theinjector and detector of GCMS were maintained at 300∘Cand the oven temperature was programmed to rise from150∘C to 300∘C with an increase of 5∘C per minute and thenheld at 300∘C for 5min

The highest resolution chromatographic peaks werescanned to find their corresponding mass fragmentationprofile Compounds were characterized based on similaritiesbetween their mass spectrum and those presented by WileyCompounds Library Control peak-areas were used as apoint of reference for the remaining compounds (100) inthe untreated system Sample peak-areas were reported asa percentage of control peak-areas Individual compoundspresent in the alkane and aromatic fractions were determinedbymatching the retention timeswith authentic standards (then-alkanes C8 C10 C11 C12 C13 C14 C17 C18 C19 C20C21 C22 C23 C24 C25 C28 and C30 and the aromatichydrocarbon standards fluoranthene pyrene phenanthreneand anthracene were obtained from Sigma-Aldrich USA)

27 Identification and Phylogenetic Affiliation of theYeast Strains Genomic DNA of the strains HC1 andHC4 was isolated using a protocol described by Masneuf-Pomarede et al (2007) [19] The primers ITS1 (51015840-TCCGTAGGTGAACCTGCGG-31015840) and ITS4 (51015840-TCC-TCCGCTTATTGATATGC-31015840) described by White et al(1990) [20] were used to amplify the 58S ITS region ofstrain HC4 The D1D2 domain of 26S rDNA gene of strainHC1 was amplified under the same conditions with primersNL1 (51015840-GCATATCAATAAGCGGAGGAAAAG-31015840) andNL4 (51015840-GGTCCGTGTTTCAAGACGG-31015840) [21] The PCRreaction was performed on a Thermocycler GeneAmp PCRSystem 9700 (Applied Biosystems) in a 50120583l reactionmixture


containing 5x GoTaq reaction buffer (Promega) 025mMeach dNTP 02 120583M each primer 50 ng DNA template and25UGoTaq DNA polymerase (Promega)The PCR programwas as follows denatured by heating for 2min at 94∘C andsubjected to 30 cycles for 30 s at 94∘C 45 s at 58∘C and 1min45 s at 72∘C this was followed by a final elongation step for10min at 72∘C PCR products were purified with illustraGFX PCR DNA and Gel Band Purification kit (AmershamBiosciences GE Healthcare) according to the manufacturerrsquosprotocol DNA sequencing of PCR products was performedusing a BigDye Terminator v31 Cycle Sequencing Kit onthe ABI PRISM 3100-Avant Genetic Analyser (AppliedBiosystems) Target sequences were analyzed using BLASTonline (httpsblastncbinlmnihgov) [22] The relatedsequences were collected and aligned using the MUSCLEsoftware [23 24] A phylogenetic tree was constructed usingthe neighbor-joining method in MEGA version 41 Yeastspecies were identified based on the phylogenetic analysisresults [25] The topology of the distance tree was tested byresampling data with 1000 bootstraps to provide confidenceestimates Nucleotide sequences obtained in this work weredeposited in the NCBI GenBank data library

3 Results and Discussion

31 Isolation and Screening of Hydrocarbons-DegradingMicroorganisms To isolate different hydrocarbon-degradingyeasts enrichment culture methods were used according toprotocol described inMaterial andMethodsThe enrichmentprocedure for obtaining contaminated wastewater hydrocar-bon-degrading microbes was performed in multiplecycles to ensure that the microbes which were obtained atthe end of the enrichment cycle were capable of utilizingthe petroleum compounds rather than just tolerating itFive yeast strains were isolated from enrichment culturesthat were established at 30∘C for 2 weeks Two of theisolated strains that showed higher growth rate on crude oilwere selected from the five isolates for further studyThese isolates showed a varying degradation profile for thetotal petroleum hydrocarbon [TPH] of the petroleumcontaminated wastewater It varied from 295 to 95(Figure 1) In our present work we report that yeasts Candidatropicalis and Trichosporon asahii could efficiently degradetotal petroleum hydrocarbon removal about 97 and 95respectively over a period of 20 days According to manyauthors bacteria have been described as being more efficienthydrocarbon degraders than yeast or at least that bacteriaare more commonly used as a test microorganism [26ndash28]Many other investigators have reported the involvement ofbacteria and yeast in crude oil biodegradation [12 29] Onthe contrary there is scanty information that yeasts are betterhydrocarbon degraders than bacteria [11 30] For that reasongrowth ability of two selected strains (HC1 and HC4) wastested in an enrichment liquid medium

32 Phylogenetic Analyses of the Yeast-Degrading Hydro-carbon To analyse the phylogenetic position of the yeast

0

20

40

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80

100

0 5 10 15 20Time (days)

HC1HC4

Tota

l pet

role

um h

ydro

carb

on (

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Figure 1 Biodegradation efficiency of yeast strains

isolates the 26S rRNA gene of isolate HC1 and the 58S-ITS noncoding regions of the rDNA of the isolate HC4 wereamplified sequenced and compared with those availablein the GenBank nucleotides sequences database Percentagesimilarity values were obtained after pairwise alignment ofthe sequences of the 16S rDNA of the yeast strains and EMBLdatabase sequences The sequences giving the highest scoreswere retried to construct the phylogenetics dendogramsSequence comparison demonstrated the affiliation of thestrain HC1 to the genus Candida The phylogenetic analysisbased on the D1D2 sequences showed that strain HC1belongs to ascomycetous yeasts and indicated that strain HC1is closely associated with the speciesof Candida tropicalis inFigure 2(a) The determined sequences displayed similaritiesof 994 99 988 and 99 to Candida sojae Candidatetrigidarum Candida maltosa and Candida neerlandicarespectively [31 32]

Identification of the yeast strain HC4 was performedby amplifying the 58-ITS rRNA Compared to the 58-ITSrDNA sequence analysis it was observed that the strain HC4has identical sequence to Trichosporon asahii CBS 2479TBasidiomycetous yeast HC4 strain was clustered closelywith Trichosporon asahii and showed also more than 99similarity between the 16s rRNA gene sequences with the Tri-chosporon astreoides and Trichosporon coremiiforme Basid-iomycetous yeast HC4 also shared similarity of about 99799 and 998 identities with Trichosporon faecale Tri-chosporon japonicum and Trichosporon aquatile respectively[33] Phylogenetic relationships of the sequence of 58S ITSregion of the yeast strain HC4 are shown in Figure 2(b) andplaced this isolate in the Ovoides clade of the genus Tri-chosporon The nucleotide sequences for the two yeast strains(HC1 andHC4) determined in this study have been depositedin the GenBank under the accession numbers JN088216 andJN088217 respectively Candida tropicalis and Trichosporonasahii are awell-known species ofmicroorganisms to degradehydrocarbons and have been used widely for petroleumremediation [34 35]


100

100

94

86

89

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48

63

63

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8998

002

Strain HC1

Candida neerlandica NRRL Y-27057T (AF245404)Candida frijolesensis NRRL Y-48062T (EF120597)

Candida viswanathii NRRL Y-6660T (U45752)

Candida lodderae NRRL Y-17317T (U45755)Candida tetrigidarum NRRL Y-48142T (EF120599)

Candida sojae NRRL Y-17909T (U7107)

Candida tropicalis NRRL Y-12968T (U45749)Candida maltosa NRRL Y-17677T (U45745)

Candida parapsilosis NRRL Y-12969T (U45754)Candida hyderabadensis NRRL Y-27953T (AM159100)

Candida albicans NRRL Y-12983T (U45776)Candida dubliniensis NRRL Y-17841T (U57685)

Candida corydalis NRRL Y-27910T (DQ655679)Candida ergastensis NRRL Y-17652T (U45746)

Saccharomyces cerevisiae NRRL Y-12632T (AY048154)

(a)

99

50

100

97

78

61

100

46

41

005

66

Trichosporon astreoides CBS 2481T (AF444416)

Trichosporon coremiiforme CBS 2482T (AF444434)

Trichosporon japonicum CBS 8641T (AF444473)

Trichosporon aquatile CBS 5973T (AF410475)

Trichosporon faecale CBS 4828T (AF444419)

Trichosporon ovoides CBS 7556T (AF444439)

Trichosporon inkin CBS 5585T (AF444420)

Trichosporon asahii CBS 2479T (AY0553818)

Trichosporon cutaneum CBS 2466T (AF444325)

Trichosporon moniliiforme

Trichosporon multisporum CBS 2495T (AF414695)

Trichosporon laibachii CBS 5790T (AF444421)

Trichosporon dulcitum CBS 8257T (AF444428)

Trichosporon gracile CBS 8189T (AF444440)

Tsuchiyaea wingfieldii CBS 7118T (AF444327)

Strain HC4

(b)

Figure 2 Phylogenetic dendogram data showing the taxonomic location of strains HC1 and HC4 (a) Neighbour-joining phylogeneticdendogram based on sequences of the D1D2 domain of the 26S rRNA gene of HC1 and related taxa Bootstrap values are given at thenodes Scale bar represents the substitution percentage Saccharomyces cerevisiae NRRL Y-12632T was used as outgroup GenBank accessionnumbers follow species name in parenthesis (b) Phylogenetic dendogram obtained by neighbour-joining analysis of the 58S-ITS sequenceof HC4 and related taxa Bootstrap values determined from 100 replicates are shown at branch nodes Scale bar represents the substitutionpercentage The outgroup we used was Tsuchiyaea wingfieldii CBS 7118T GenBank accession numbers follow species name in parenthesis

33 Cell Surface Hydrophobicity Results for cell surfacehydrophobicity when the strains were grown on hexadecaneand glucose are shown in Figure 3 As it can be seen cellhydrophobicity of tested strains remained unchanged during

growth on glucose Cell hydrophobicity reached approxi-mately 78 and 854 with Trichosporon and Candida strainsrespectively During our studies and for both yeast strainswe noticed that the maximal increase in hydrophobicity is


0102030405060708090

1 2 3 4 5 6 7 8 9 10Time (days)

GlucoseHC1HC4

Hyd

roph

obic

ity (

)

Figure 3 Hydrophobicity of yeast strains isolates during growth inmineral medium with 15 hexadecane or glucose as a sole carbonsource ◻ after growth on glucose ◼ after growth on hexadecaneValues are the mean of three separate experiments plusmn sd

correlated with the maximum biodegradation of hexadecane(or TPH) A direct relationship was found between both cellhydrophobicity of yeast strains and petroleum hydrocarbonbiodegradation (Figures 1ndash3) Based on data obtained fromgrowth and cell hydrophobicity cell surface hydrophobicitycould be proposed as the most probable mechanism forhexadecane uptake by the tested yeast strains Increase incell hydrophobicity by both yeast strains during growthon hexadecane indicated that the strains could employbiosurfactant-mediated alkane uptake In fact Prabhu andPhale suggest that a biosurfactant production and a cell sur-face hydrophobicity play an important role in hydrocarbonuptake [36] In this case the cell contact with hydrophobiccompounds is a requirement because the first step in aromaticor aliphatic hydrocarbon degradation is the introduction ofmolecular oxygen into the molecules by cell-associated oxy-genases [37] Cell hydrophobicity can be considered one ofthe important factors controlling hydrocarbon assimilationThe yeast strain Trichosporon asahii isolated from petroleumcontaminated soil in India by Chandran and Das producedbiosurfactant inmineral saltmedia containing diesel oil as thecarbon source and degraded diesel oil (95) over a period of10 days [34] An extracellular bioemulsifier was isolated fromCandida tropicalis and this composed stable hexadecane-in-water emulsions [38]

34 Microbial Growth on Hydrocarbon Compounds Theextent of oil-related compounds degradation and the timefor complete hydrocarbon compound degradation varied asa function of yeast strains In addition to their capacity togrow on hexadecane strains can also grow on some refinerysubproduct such as crude oil diesel fuel and lubricating oil(Figure 4)Hexadecanewas the best substrate to support yeaststrains growth However the growth of strain belonging tothe genus Trichosporon in the presence of diesel fuel showedextended long degradation time (Figure 4(b)) Diesel oil

is a medium distillate of petroleum containing n-alkanesbranched alkanes and small concentrations of aromaticpolycyclic compounds Yeast strains were able of utilizing awide range of hydrocarbons with a preference for alkaneswith intermediate carbon chain lengths Measurement ofgrowth attributes of the isolates using n-hexadecane dieseloil and crude oil as substrates showed that the yeast strainswere better while using of hydrocarbon substrates Thusthe biodegradation of mixtures of substrates is an importantway in biological treatment of petroleum contaminatedwastewater In fact petroleum is found typically as complexmixtures in the environment containing distinct petroleumconstituents such as alkanes (linear branched and cyclic)PAHs heterocyclic aromatic compounds and asphaltenesYeasts are relatively rarely isolated from crude oil or hydro-carbon sources Moreover yeasts are capable of using a widerange of different carbon sources [29] though only fewstudies have been carried out on the potential use of yeast onoil-contaminated sites [39]

The decreased growth pattern in short chain could beattributed to the toxicity of these alkanes to cell membranes[40] On the other hand while growth on long chain alkanescould be limited by decreased bioavailability of the substratethe ability of the yeast isolates to use long chain 119899-alkanesin preference to shorter-chain ones below C8ndashC10 couldbe attributed to reduced toxicity and higher growth yieldsobtainable from those substrates Substrates growth couldeither be due to the constitutive nature of hydrocarbon assim-ilating capabilities in the isolates or reflect the adaptationof the yeast strains due to previous exposure to exogenoushydrocarbons It may also indicate the ability of the yeaststrains to emulsify hydrocarbons which is a major factor forhydrocarbon uptake and assimilation

These results were confirmed also by viable cells enumer-ation indicating positive growth with the three substratesshown in Figure 5These findings suggest that the capacity todegrade crude oil by yeast strains is higher than that by dieselfuel However the major compound of diesel fuel is aromatichydrocarbons These compounds are more recalcitrant thanaliphatic hydrocarbon present in crude oil and lubricating oil[40]

35 Degradation of the Alkane and Aromatic Fractions byYeast Strains The degradation of the alkane and aromatichydrocarbons by Candida and Trichosporon species wasanalyzed by gas chromatography coupled to mass spectrom-etry (GCMS) From Figure 6 the strains depicted efficientutilization of the alkane fraction compared to the untreatedcontrol Analysis by GCMS revealed that both isolates werecapable of degrading the aliphatic fractions which showedthat all the detectable hydrocarbons peaks were completelyutilized within 12 days by the two yeast isolates The isolateyeast strains were capable of degrading the entire range of n-alkanes on the medium containing petroleum hydrocarbonsfrom undecane (C11) to hexacosane (C26) as the sole carbonsource and energy The hydrocarbon utilization pattern ofyeast strains indicated that the growth was less on shortchain alkanes like octane decane and undecane However


0

05

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0 2 4 6 8 10 12Time (days)

DecaneHexadecanePetroleum

DieselControl

DO

(600

nm)

(a) Candida tropicalis

DecaneHexadecane

Petroleum

005

115

225

3

0 2 4 6 8 10 12Time (days)

DieselControl

DO

(600

nm)

(b) Trichosporon asahii

Figure 4 Growth of selected yeasts on mineral medium with some alkanes (1 wv) as the sole carbon source with respect to uninoculatedcontrols over 12 days of degradation (a) Candida tropicalis (b) Trichosporon asahii

35

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0 2 4 6 8 10 12Time (days)


DieselControl

Log(

CFU

mlminus

1 )


354

455

556

657

0 2 4 6 8 10 12Time (days)

DecaneHexadecane

Petroleum

DieselControl

Log(

CFU

mlminus

1 )


Figure 5 Yeast cell count during 12 days (a) Candida tropicalis (b) Trichosporon asahii Each point represents mean plusmn SE of triple assays

it started increasing from dodecane with a maximum growthbeing observed on heptadecane octadecane and nonadecaneafter which the growth was again comparatively reduced witheicosane tricosane tetracosane and hexacosane (Figure 6)Both short and long chain hydrocarbons of the industrialwastewater refinery were highly susceptible to be attackedby the yeast strains probably due to the fact that theyhave a very efficient degradative enzyme system The strainsalso showed considerable utilization of components fromaromatic fraction as depicted in Figure 7While Candida andTrichosporon species were capable of efficient degradationof alkanes as the sole carbon source they were unableto completely degrade aromatic hydrocarbons as the solecarbon source Aromatic compounds proved to be resistantto biodegradation and we observed that Trichosporon canassimilate phenanthrene anthracene and fluoranthene by47 73 and 314 respectively within 7 days

The yeast strain Candida species HC1 was capable ofdegrading a mixture of low and high molecular weight of

aromatic hydrocarbons and a degradation efficiency wasobserved with anthracene (51) and fluoranthene (74) atthe end of 7 days

Aromatic hydrocarbons are known to be more resis-tant to biodegradation than aliphatic compounds and areoften a serious problem during bioremediation processes[18 41] However the n-alkanes are the most biodegradablepetroleum hydrocarbons and are attacked by more micro-bial species than aromatic or naphthenic compounds Theoxidation of alkanes was considerably more profound thanthat of aromatic hydrocarbons This finding is in agreementwith other reports where hydrocarbon-degrading microbeshave shown preferential degradation of alkanes to aromaticsandNSO-asphaltene fractions [18 42] However as expectedthe results obtained in the laboratory may not scale up tothe natural environment thus we must analyze the behaviorof these strains in mesocosm experiments to determine howthey may act in the natural environment for the bioremedia-tion of hydrocarbon-polluted wastewater


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e

times105

(a) Candida after 12 days of degradation

(b) Trichosporon after 12 days of degradation

Figure 6 Gas chromatogram showing the biodegradation of alkane fraction of rich-hydrocarbon wastewater from Tunisian refinery byCandida tropicalis (a) and Trichosporon asahii (b) compared to the control

4 Conclusion

Our study focused on the yeast strains isolated from enrich-ment culture of the contaminated industrial effluent fromTunisian petroleum refinery wastewater identified as Can-dida tropicalis and Trichosporon asahii which developedadaptationmechanisms to survive in the harsh environmentsof that refinery A direct relationship was found betweenboth cell hydrophobicity of the yeast strains and crude oilbiodegradation These strains have high levels of crude oil

degradation and sufficient growth on some aliphatic hydro-carbons In this study the isolated yeasts have been shownto degrade a wide range of hydrocarbons and completelymetabolize n-alkanes On the other hand they were unable tocompletely degrade aromatic hydrocarbons (phenanthreneanthracene fluoranthene and pyrene) as the sole carbonsource From the data presented in this study it can be con-cluded that the investigated strainsCandida andTrichosporoncould be considered as good prospects for their application inbioremediation of hydrocarbon contaminated environment


600 800 1000 1200 1400 1600 1800 20005

152535455565758595

TimeAb

unda

nce

600 800 1000 1200 1400 1600 1800 2000Time

600 800 1000 1200 1400 1600 1800 2000Time

Control

(a) Candida after 7 days of incubation

(b) Trichosporon after 7 days of incubation

PhenanthreneAnthracene

Pyrene

Fluoranthenetimes105

5152535455565758595

Abun

danc

e

times105

5152535455565758595

Abun

danc

e

times105

Figure 7 Gas chromatogram showing the biotransformation of the aromatic fraction of rich-hydrocarbonwastewater fromTunisian refineryby Candida tropicalis (a) and Trichosporon asahii (b) compared to the control

and improvement of hydrocarbon removing treatment ofindustrial wastewater

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are grateful to Dr Mehdi Triki for his pricelesshelpful comments and for having proofread the manuscript

References

[1] M P Maila and T E Cloete ldquoThe use of biological activitiesto monitor the removal of fuel contaminantsmdashperspective for

monitoring hydrocarbon contamination a reviewrdquo Interna-tional Biodeterioration and Biodegradation vol 55 no 1 pp 1ndash82005

[2] C J Berry S Story D J Altman et al ldquoBiological treatment ofpetroleum in radiologically contaminated soilrdquo in Remediationof Hazardous Waste in the Subsurface Bridging Flask and FieldC J Clark II and A Stephenson Lindner Eds pp 87ndash103American Chemical Society 2006

[3] J D VanHamme A Singh andO PWard ldquoRecent advances inpetroleum microbiologyrdquo Microbiology and Molecular BiologyReviews vol 67 no 4 pp 503ndash549 2003

[4] A-M Tanase R Ionescu I Chiciudean T Vassu and I StoicaldquoCharacterization of hydrocarbon-degrading bacterial strainsisolated from oil-polluted soilrdquo International BiodeteriorationampBiodegradation vol 84 pp 150ndash154 2013

[5] P O Okerentugba and O U Ezeronye ldquoPetroleum degradingpotentials of single and mixed microbial cultures isolated from


rivers and refinery effluent in Nigeriardquo African Journal ofBiotechnology vol 2 no 9 pp 312ndash319 2003

[6] J F T Spencer A L R de Spencer and C Laluce ldquoNon-conventional yeastsrdquo Applied Microbiology and Biotechnologyvol 58 no 2 pp 147ndash156 2002

[7] M Hassanshahian H Tebyanian and S Cappello ldquoIsolationand characterization of two crude oil-degrading yeast strainsYarrowia lipolytica PG-20 and PG-32 from the Persian GulfrdquoMarine Pollution Bulletin vol 64 no 7 pp 1386ndash1391 2012

[8] N Sood and B Lal ldquoIsolation of a novel yeast strain Can-dida digboiensis TERI ASN6 capable of degrading petroleumhydrocarbons in acidic conditionsrdquo Journal of EnvironmentalManagement vol 90 no 5 pp 1728ndash1736 2009

[9] L-M Whang P-W G Liu C-C Ma and S-S ChengldquoApplication of rhamnolipid and surfactin for enhanced dieselbiodegradationmdasheffects of pH and ammonium additionrdquo Jour-nal of Hazardous Materials vol 164 no 2-3 pp 1045ndash10502009

[10] S Mnif M Chamkha and S Sayadi ldquoIsolation and charac-terization of Halomonas sp strain C2SS100 a hydrocarbon-degrading bacterium under hypersaline conditionsrdquo Journal ofApplied Microbiology vol 107 no 3 pp 785ndash794 2009

[11] M O Ilori S A Adebusoye and A C Ojo ldquoIsolation andcharacterization of hydrocarbon-degrading and biosurfactant-producing yeast strains obtained from a polluted lagoon waterrdquoWorld Journal of Microbiology and Biotechnology vol 24 no 11pp 2539ndash2545 2008

[12] S Chakraborty and S Mukherji ldquoSurface hydrophobicity ofpetroleum hydrocarbon degrading Burkholderia strains andtheir interactions with NAPLs and surfacesrdquo Colloids andSurfaces B Biointerfaces vol 78 no 1 pp 101ndash108 2010

[13] S Zinjarde M Apte P Mohite and A R Kumar ldquoYarrowia li-polytica and pollutants interactions and applicationsrdquo Biotech-nology Advances vol 32 pp 920ndash933 2014

[14] B Gargouri F Karray N Mhiri F Aloui and S SayadildquoApplication of a continuously stirred tank bioreactor (CSTR)for bioremediation of hydrocarbon-rich industrial wastewatereffluentsrdquo Journal of Hazardous Materials vol 189 no 1-2 pp427ndash434 2011

[15] Bergeyrsquos Manual of Determinative Bacteriology vol 87 Williamsand Wilkins Baltimore Md USA 9th edition 1994

[16] S Abdelkafi S Sayadi Z B Ali Gam L Casalot and M LabatldquoBioconversion of ferulic acid to vanillic acid by Halomonaselongata isolated from table-olive fermentationrdquo FEMS Micro-biology Letters vol 262 no 1 pp 115ndash120 2006

[17] M Rosenberg D Gutnick and E Rosenberg ldquoAdherence ofbacteria to hydrocarbons a simple method for measuring cell-surface hydrophobicityrdquo FEMS Microbiology Letters vol 9 no1 pp 29ndash33 1980

[18] S Mishra J Jyot R C Kuhad and B Lal ldquoIn situ bioreme-diation potential of an oily sludge-degrading bacterial consor-tiumrdquo Current Microbiology vol 43 no 5 pp 328ndash335 2001

[19] I Masneuf-Pomarede C Le Jeune P Durrens M Lollier MAigle and D Dubourdieu ldquoMolecular typing of wine yeaststrains Saccharomyces bayanus var uvarum using microsatellitemarkersrdquo Systematic and AppliedMicrobiology vol 30 no 1 pp75ndash82 2007

[20] T J White T Bruns S Lee and J Taylor ldquoAmplificationand direct sequencing of fungi ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innis D H Gelfand J J Sninsky and T

J White Eds pp 315ndash322 Academic Press San Diego CalifUSA 1990

[21] K OrsquoDonnell ldquoFusarium and its near relativesrdquo in The FungalHolomorph Mitotic Meiotic and Pleomorphic Speciation inFungal Systematics D R Reynolds and J W Taylor Eds pp225ndash233 CAB International Wallingford UK 1993

[22] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[23] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[24] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[25] K Tamura J Dudley M Nei and S Kumar ldquoMEGA4 Molec-ular Evolutionary Genetics Analysis (MEGA) software version40rdquo Molecular Biology and Evolution vol 24 no 8 pp 1596ndash1599 2007

[26] S Cappello R Denaro M Genovese L Giuliano and M MYakimov ldquoPredominant growth of Alcanivorax during experi-ments on lsquooil spill bioremediationrsquo in mesocosmsrdquoMicrobiolog-ical Research vol 162 no 2 pp 185ndash190 2007

[27] R Margesin C Moertelmaier and J Mair ldquoLow-temperaturebiodegradation of petroleum hydrocarbons (n-alkanes phenolanthracene pyrene) by four actinobacterial strainsrdquo Interna-tional Biodeterioration and Biodegradation vol 84 pp 185ndash1912013

[28] K S M Rahman I M Banat J Thahira-Rahman and P Lak-shmanaperumalsamy ldquoTowards efficient crude oil degradationby a mixed bacterial consortiumrdquo Bioresource Technology vol85 no 3 pp 257ndash261 2002

[29] C Schmitz I Goebel S Wagner A Vomberg and U KlinnerldquoCompetition between n-alkane-assimilating yeasts and bac-teria during colonization of sandy soil microcosmsrdquo AppliedMicrobiology and Biotechnology vol 54 no 1 pp 126ndash132 2000

[30] A E-L Hesham S A Alamri S Khan M E Mahmoud andH M Mahmoud ldquoIsolation and molecular genetic character-ization of a yeast strain able to degrade petroleum polycyclicaromatic hydrocarbonsrdquoAfrican Journal of Biotechnology vol 8no 10 pp 2218ndash2223 2009

[31] C P Kurtzman C J Robnett and D Yarrow ldquoTwo newanamorphic yeasts Candida germanica and Candida neer-landicardquo Antonie van Leeuwenhoek vol 80 no 1 pp 77ndash832001

[32] S-O Suh N H Nguyen and M Blackwell ldquoYeasts isolatedfrom plant-associated beetles and other insects seven novelCandida species near Candida albicansrdquo FEMS Yeast Researchvol 8 no 1 pp 88ndash102 2008

[33] G Scorzetti J W Fell A Fonseca and A Statzell-TallmanldquoSystematics of basidiomycetous yeasts a comparison of largesubunit D1D2 and internal transcribed spacer rDNA regionsrdquoFEMS Yeast Research vol 2 no 4 pp 495ndash517 2002

[34] P Chandran and N Das ldquoBiosurfactant production and dieseloil degradation by yeast species Trichosporon asahii isolatedfrom petroleum hydrocarbon contaminated soilrdquo InternationalJournal of Engineering Science and Technology vol 2 pp 6942ndash6953 2010

[35] P Chandran and N Das ldquoDegradation of diesel oil by immo-bilized Candida tropicalis and biofilm formed on gravelsrdquoBiodegradation vol 22 no 6 pp 1181ndash1189 2011


[36] Y Prabhu and P S Phale ldquoBiodegradation of phenanthrene byPseudomonas sp strain PP2 novel metabolic pathway role ofbiosurfactant and cell surface hydrophobicity in hydrocarbonassimilationrdquo Applied Microbiology and Biotechnology vol 61no 4 pp 342ndash351 2003

[37] C D Coimbra R D Rufino J M Luna and L A SarubboldquoStudies of the cell surface properties of candida species andrelation to the production of biosurfactants for environmentalapplicationsrdquo Current Microbiology vol 58 no 3 pp 245ndash2512009

[38] C D Coimbra R D Rufino J M Luna and L A SarubboldquoStudies of the cell surface properties of Candida species andrelation to the production of biosurfactants for environmentalapplicationsrdquo Current Microbiology vol 58 no 3 pp 245ndash2512009

[39] M Yang and S Zheng ldquoPollutant removal-oriented yeastbiomass production from high-organic-strength industrialwastewater a reviewrdquo Biomass and Bioenergy vol 64 pp 356ndash362 2014

[40] G C Okpokwasili and S C Amanchukwu ldquoPetroleum hydro-carbon degradation by Candida speciesrdquo Environment Interna-tional vol 14 no 3 pp 243ndash247 1988

[41] P Wongsa M Tanaka A Ueno M Hasanuzzaman I Yumotoand H Okuyama ldquoIsolation and characterization of novelstrains of Pseudomonas aeruginosa and Serratia marcescenspossessing high efficiency to degrade gasoline kerosene dieseloil and lubricating oilrdquo Current Microbiology vol 49 no 6 pp415ndash422 2004

[42] B Gargouri F Aloui and S Sayadi ldquoReduction of petroleumhydrocarbons content from an engine oil refinery wastewaterusing a continuous stirred tank reactormonitored by spectrom-etry toolsrdquo Journal of Chemical Technology and Biotechnologyvol 87 no 2 pp 238ndash243 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


water has been studied by many authors [4] However notonly are bacteria capable of hydrocarbon biodegradation butalso yeasts isolated from hydrocarbon contaminated sitesdisplay the ability to use oil-related compounds [5 6] Asa matter of fact a wide variety of yeasts and filamentousfungi are capable of utilizing hydrocarbons Yeasts capable ofdegrading hydrocarbons include genera of Yarrowia lipolyt-ica Candida tropicalis Candida albicans and DebaryomyceshanseniiThe alkane-utilizing yeastY lipolytica degrades veryefficiently hydrophobic substrates (HS) such as triglycerides(fats oils) alkanes and fatty acids It has been observed thatY lipolytica is the predominant yeast form during alkanedegradation and emulsifier production [7]

The study of hydrocarbon biodegradation by nativemicroorganisms is of extreme ecological importance Theactual contribution of yeasts in the biodegradation of hydro-carbons in the environment may be more important thanthat previously expected considering the metabolic diver-sity demonstrated for yeasts [8] However bioavailabilityof hydrophobic organic compounds to microorganisms isgenerally a limiting step during the biodegradation process[9] Hydrophobic interactions play an important role in theadherence of microorganisms to a wide variety of surfaces[10] In particular the hydrophobic nature of the bacterialsurface has been cited as a key factor in cells growth onwater-insoluble hydrophobic substrates such as hydrocarbon[11 12] To enter cells HS must interact with the cell surfaceTwo hypotheses have been formulated to explain this step ofpoorly water-miscible substrates transport into microor-ganisms (i) the compounds can be solubilised (or pseu-dosolubilised) in the presence of surface-active compounds(surfactant-mediated transport) or (ii) they can adheredirectly to the cell wall (direct interfacial transport) [13] Theaforementioned yeast species can produce surfactants duringgrowth on HS A correlation has been observed betweencells and HS adhesion induction and the increase in apolarproperties of the cell surface [13] Aerobic degradation ofalkanes can be carried out by twomajor types of enzymes thealkane monooxygenase (also known as alkane hydroxylaseor AlkB) and certain cytochrome P450 systems

In a screening program carried out in our laboratory weisolated yeast strains from petroleum contaminated wastew-ater effluents that are able to utilize hydrocarbons The use ofnaturally occurring yeasts represents a potentially promisingmeans of destroying polluting chemicals in wastewater treat-ment systems and soil Therefore the objective of the presentwork was to isolate identify and characterize n-alkane usingyeasts capable of degrading petroleum hydrocarbons in orderto employ yeasts strains in the future as a biodegrader ofpolluted environmentThe hydrocarbon degradation activityand cell hydrophobicity were also investigated in relation ton-alkane utilization

2 Material and Methods

21 Isolation and Characterization of Yeast Isolates Thehydrocarbons-degradingmicrobial consortiumwas enriched

from petrochemical industrial wastewater using a continu-ously stirred tank-reactorCSTR [14] Twoof the best-growingyeast strains were isolated from the hydrocarbons-degradingmicrobial consortium The yeast strains were isolated byusing a selective enrichment culture and a single-colonyisolation technique The selected yeasts were identified inthe present study by routine morphological microbiologicaland biochemical methods following directions of the latestedition of Bergeyrsquos Manual [15]

22 Culture Conditions Yeast isolates were cultivated in250ml Erlenmeyer flasks containing 50ml of mineralmedium (MM) The pH was measured using the Cyberscan500 pH meter The pH was adjusted by using 1N standardNaOH and 1N standard HCl (Merck Germany) The min-eral medium used contained per litre 34 g K

2HPO4 43 g

KH2PO4 03 g MgCl

2sdot2H2O and 1 g (NH

4)2SO4 which was

supplemented with 1ml of a trace element solution [16] Themedia used for the yeast growthwere sterilized by autoclavingat 121∘C for 20 minutes Petroleum compounds and deriva-tives were added as carbon source to the sterilized MM at thedesired concentrations A liquid culturewas started by addinga loop full of cells from a standard agar plate into a 250mlErlenmeyer flask containing a 50ml medium Flasks wereinoculated with 1ml cultures pregrown Then isolates wereincubated at 30∘C under agitation (180 rpm) for 10ndash15 days(depending on the strain) Samples were taken at intervalsfrom shake flasks The isolated yeast was also grown onPotato Dextrose Agar (PDA) in order to observe largercolonies for better study Growth was assessed by measuringthe optical density (OD) at 600 nm Three independentexperiments were carried out for each yeast strain

23 Biomass Growth during the Biodegradation ProcessBiomass concentration was estimated from the absorbance ofappropriately diluted culture medium at 600 nm (Ultrospec3300 pro UVVisible) according to the predetermined cor-relation between optical density and dry weight of biomassDuring the biodegradation assays the number of viable yeastswas measured by counting of heterotrophic microbes in Petridishes expressing the result as colony-forming units (CFU)per ml With a sterile pipette 1ml of sample was taken fromthe culture and a series of 1 10 dilutions made in NaCl solu-tion at 09 Each dilution was analysed in duplicate 1ml ofsample to be analysed was placed on Petri dishesThen 20mlof previously sterilized culture medium was poured onto thePetri dishes tempered at 60∘C and gently stirred to completethe homogenization The mixture was cooled until completesolidification and then incubated at 30∘C for 72 h Thetotal number of microorganisms was determined by mul-tiplying the number of CFU by the corresponding dilutionfactor The count was made in an automatic colony counter(Countermat Flash IUL Instruments) All experiments wereperformed in triplicate with appropriate controls

24 Cell Surface Hydrophobicity Test Cell surface hydropho-bicity was measured by microbial adherence to hexadecaneaccording to themethod of Rosenberg et al [17] Yeast strains




2PO4 18 g urea



Hydrophobicity


)

(1)














0

20

40

60

80

100

0 5 10 15 20Time (days)

HC1HC4

Tota

l pet

role

um h

ydro

carb

on (

)





100

100

94

86

89

70

48

63

63

61

56

8998

002

Strain HC1










(a)

99

50

100

97

78

61

100

46

41

005

66
















Strain HC4

(b)





0102030405060708090

1 2 3 4 5 6 7 8 9 10Time (days)

GlucoseHC1HC4

Hyd

roph

obic

ity (

)









0

05

1

15

2

0 2 4 6 8 10 12Time (days)


DieselControl

DO

(600

nm)


DecaneHexadecane

Petroleum

005

115

225

3

0 2 4 6 8 10 12Time (days)

DieselControl

DO

(600

nm)



35

4

45

5

55

6

0 2 4 6 8 10 12Time (days)


DieselControl

Log(

CFU

mlminus

1 )


354

455

556

657

0 2 4 6 8 10 12Time (days)

DecaneHexadecane

Petroleum

DieselControl

Log(

CFU

mlminus

1 )








400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

26

1014182226

Time

Time

Abun

danc

e

26

1014182226

Abun

danc

e

Control

Dod

ecan

eU

ndec

ane

Pent

adec

ane

Hex

adec

ane

Hep

tade

cane

Oct

adec

ane

Eico

sane Doc

osan

e

Hex

acos

ane

Hex

acos

ane

Hen

eico

sane

Oct

acos

ane

Tetr

adec

ane

Non

adec

ane

Cyclo

hexa

deca

ne

Cyclo

hexa

deca

ne

Cyclo

tetr

acos

ane

Tric

osan

e

Trid

ecan

e

times105

times105

26

1014182226

Abun

danc

e

times105




4 Conclusion




600 800 1000 1200 1400 1600 1800 20005

152535455565758595

TimeAb

unda

nce

600 800 1000 1200 1400 1600 1800 2000Time

600 800 1000 1200 1400 1600 1800 2000Time

Control




Pyrene


5152535455565758595

Abun

danc

e

times105

5152535455565758595

Abun

danc

e

times105





Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2PO4 18 g urea



Hydrophobicity


)

(1)














0

20

40

60

80

100

0 5 10 15 20Time (days)

HC1HC4

Tota

l pet

role

um h

ydro

carb

on (

)





100

100

94

86

89

70

48

63

63

61

56

8998

002

Strain HC1










(a)

99

50

100

97

78

61

100

46

41

005

66
















Strain HC4

(b)





0102030405060708090

1 2 3 4 5 6 7 8 9 10Time (days)

GlucoseHC1HC4

Hyd

roph

obic

ity (

)









0

05

1

15

2

0 2 4 6 8 10 12Time (days)


DieselControl

DO

(600

nm)


DecaneHexadecane

Petroleum

005

115

225

3

0 2 4 6 8 10 12Time (days)

DieselControl

DO

(600

nm)



35

4

45

5

55

6

0 2 4 6 8 10 12Time (days)


DieselControl

Log(

CFU

mlminus

1 )


354

455

556

657

0 2 4 6 8 10 12Time (days)

DecaneHexadecane

Petroleum

DieselControl

Log(

CFU

mlminus

1 )








400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

26

1014182226

Time

Time

Abun

danc

e

26

1014182226

Abun

danc

e

Control

Dod

ecan

eU

ndec

ane

Pent

adec

ane

Hex

adec

ane

Hep

tade

cane

Oct

adec

ane

Eico

sane Doc

osan

e

Hex

acos

ane

Hex

acos

ane

Hen

eico

sane

Oct

acos

ane

Tetr

adec

ane

Non

adec

ane

Cyclo

hexa

deca

ne

Cyclo

hexa

deca

ne

Cyclo

tetr

acos

ane

Tric

osan

e

Trid

ecan

e

times105

times105

26

1014182226

Abun

danc

e

times105




4 Conclusion




600 800 1000 1200 1400 1600 1800 20005

152535455565758595

TimeAb

unda

nce

600 800 1000 1200 1400 1600 1800 2000Time

600 800 1000 1200 1400 1600 1800 2000Time

Control




Pyrene


5152535455565758595

Abun

danc

e

times105

5152535455565758595

Abun

danc

e

times105





Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






0

20

40

60

80

100

0 5 10 15 20Time (days)

HC1HC4

Tota

l pet

role

um h

ydro

carb

on (

)





100

100

94

86

89

70

48

63

63

61

56

8998

002

Strain HC1










(a)

99

50

100

97

78

61

100

46

41

005

66
















Strain HC4

(b)





0102030405060708090

1 2 3 4 5 6 7 8 9 10Time (days)

GlucoseHC1HC4

Hyd

roph

obic

ity (

)









0

05

1

15

2

0 2 4 6 8 10 12Time (days)


DieselControl

DO

(600

nm)


DecaneHexadecane

Petroleum

005

115

225

3

0 2 4 6 8 10 12Time (days)

DieselControl

DO

(600

nm)



35

4

45

5

55

6

0 2 4 6 8 10 12Time (days)


DieselControl

Log(

CFU

mlminus

1 )


354

455

556

657

0 2 4 6 8 10 12Time (days)

DecaneHexadecane

Petroleum

DieselControl

Log(

CFU

mlminus

1 )








400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

26

1014182226

Time

Time

Abun

danc

e

26

1014182226

Abun

danc

e

Control

Dod

ecan

eU

ndec

ane

Pent

adec

ane

Hex

adec

ane

Hep

tade

cane

Oct

adec

ane

Eico

sane Doc

osan

e

Hex

acos

ane

Hex

acos

ane

Hen

eico

sane

Oct

acos

ane

Tetr

adec

ane

Non

adec

ane

Cyclo

hexa

deca

ne

Cyclo

hexa

deca

ne

Cyclo

tetr

acos

ane

Tric

osan

e

Trid

ecan

e

times105

times105

26

1014182226

Abun

danc

e

times105




4 Conclusion




600 800 1000 1200 1400 1600 1800 20005

152535455565758595

TimeAb

unda

nce

600 800 1000 1200 1400 1600 1800 2000Time

600 800 1000 1200 1400 1600 1800 2000Time

Control




Pyrene


5152535455565758595

Abun

danc

e

times105

5152535455565758595

Abun

danc

e

times105





Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100

100

94

86

89

70

48

63

63

61

56

8998

002

Strain HC1










(a)

99

50

100

97

78

61

100

46

41

005

66
















Strain HC4

(b)





0102030405060708090

1 2 3 4 5 6 7 8 9 10Time (days)

GlucoseHC1HC4

Hyd

roph

obic

ity (

)









0

05

1

15

2

0 2 4 6 8 10 12Time (days)


DieselControl

DO

(600

nm)


DecaneHexadecane

Petroleum

005

115

225

3

0 2 4 6 8 10 12Time (days)

DieselControl

DO

(600

nm)



35

4

45

5

55

6

0 2 4 6 8 10 12Time (days)


DieselControl

Log(

CFU

mlminus

1 )


354

455

556

657

0 2 4 6 8 10 12Time (days)

DecaneHexadecane

Petroleum

DieselControl

Log(

CFU

mlminus

1 )








400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

26

1014182226

Time

Time

Abun

danc

e

26

1014182226

Abun

danc

e

Control

Dod

ecan

eU

ndec

ane

Pent

adec

ane

Hex

adec

ane

Hep

tade

cane

Oct

adec

ane

Eico

sane Doc

osan

e

Hex

acos

ane

Hex

acos

ane

Hen

eico

sane

Oct

acos

ane

Tetr

adec

ane

Non

adec

ane

Cyclo

hexa

deca

ne

Cyclo

hexa

deca

ne

Cyclo

tetr

acos

ane

Tric

osan

e

Trid

ecan

e

times105

times105

26

1014182226

Abun

danc

e

times105




4 Conclusion




600 800 1000 1200 1400 1600 1800 20005

152535455565758595

TimeAb

unda

nce

600 800 1000 1200 1400 1600 1800 2000Time

600 800 1000 1200 1400 1600 1800 2000Time

Control




Pyrene


5152535455565758595

Abun

danc

e

times105

5152535455565758595

Abun

danc

e

times105





Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0102030405060708090

1 2 3 4 5 6 7 8 9 10Time (days)

GlucoseHC1HC4

Hyd

roph

obic

ity (

)









0

05

1

15

2

0 2 4 6 8 10 12Time (days)


DieselControl

DO

(600

nm)


DecaneHexadecane

Petroleum

005

115

225

3

0 2 4 6 8 10 12Time (days)

DieselControl

DO

(600

nm)



35

4

45

5

55

6

0 2 4 6 8 10 12Time (days)


DieselControl

Log(

CFU

mlminus

1 )


354

455

556

657

0 2 4 6 8 10 12Time (days)

DecaneHexadecane

Petroleum

DieselControl

Log(

CFU

mlminus

1 )








400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

26

1014182226

Time

Time

Abun

danc

e

26

1014182226

Abun

danc

e

Control

Dod

ecan

eU

ndec

ane

Pent

adec

ane

Hex

adec

ane

Hep

tade

cane

Oct

adec

ane

Eico

sane Doc

osan

e

Hex

acos

ane

Hex

acos

ane

Hen

eico

sane

Oct

acos

ane

Tetr

adec

ane

Non

adec

ane

Cyclo

hexa

deca

ne

Cyclo

hexa

deca

ne

Cyclo

tetr

acos

ane

Tric

osan

e

Trid

ecan

e

times105

times105

26

1014182226

Abun

danc

e

times105




4 Conclusion




600 800 1000 1200 1400 1600 1800 20005

152535455565758595

TimeAb

unda

nce

600 800 1000 1200 1400 1600 1800 2000Time

600 800 1000 1200 1400 1600 1800 2000Time

Control




Pyrene


5152535455565758595

Abun

danc

e

times105

5152535455565758595

Abun

danc

e

times105





Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

05

1

15

2

0 2 4 6 8 10 12Time (days)


DieselControl

DO

(600

nm)


DecaneHexadecane

Petroleum

005

115

225

3

0 2 4 6 8 10 12Time (days)

DieselControl

DO

(600

nm)



35

4

45

5

55

6

0 2 4 6 8 10 12Time (days)


DieselControl

Log(

CFU

mlminus

1 )


354

455

556

657

0 2 4 6 8 10 12Time (days)

DecaneHexadecane

Petroleum

DieselControl

Log(

CFU

mlminus

1 )








400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

26

1014182226

Time

Time

Abun

danc

e

26

1014182226

Abun

danc

e

Control

Dod

ecan

eU

ndec

ane

Pent

adec

ane

Hex

adec

ane

Hep

tade

cane

Oct

adec

ane

Eico

sane Doc

osan

e

Hex

acos

ane

Hex

acos

ane

Hen

eico

sane

Oct

acos

ane

Tetr

adec

ane

Non

adec

ane

Cyclo

hexa

deca

ne

Cyclo

hexa

deca

ne

Cyclo

tetr

acos

ane

Tric

osan

e

Trid

ecan

e

times105

times105

26

1014182226

Abun

danc

e

times105




4 Conclusion




600 800 1000 1200 1400 1600 1800 20005

152535455565758595

TimeAb

unda

nce

600 800 1000 1200 1400 1600 1800 2000Time

600 800 1000 1200 1400 1600 1800 2000Time

Control




Pyrene


5152535455565758595

Abun

danc

e

times105

5152535455565758595

Abun

danc

e

times105





Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

400

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

500

600

700

800

900

100

0

110

0

120

0

130

0

140

0

150

0

26

1014182226

Time

Time

Abun

danc

e

26

1014182226

Abun

danc

e

Control

Dod

ecan

eU

ndec

ane

Pent

adec

ane

Hex

adec

ane

Hep

tade

cane

Oct

adec

ane

Eico

sane Doc

osan

e

Hex

acos

ane

Hex

acos

ane

Hen

eico

sane

Oct

acos

ane

Tetr

adec

ane

Non

adec

ane

Cyclo

hexa

deca

ne

Cyclo

hexa

deca

ne

Cyclo

tetr

acos

ane

Tric

osan

e

Trid

ecan

e

times105

times105

26

1014182226

Abun

danc

e

times105




4 Conclusion




600 800 1000 1200 1400 1600 1800 20005

152535455565758595

TimeAb

unda

nce

600 800 1000 1200 1400 1600 1800 2000Time

600 800 1000 1200 1400 1600 1800 2000Time

Control




Pyrene


5152535455565758595

Abun

danc

e

times105

5152535455565758595

Abun

danc

e

times105





Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


600 800 1000 1200 1400 1600 1800 20005

152535455565758595

TimeAb

unda

nce

600 800 1000 1200 1400 1600 1800 2000Time

600 800 1000 1200 1400 1600 1800 2000Time

Control




Pyrene


5152535455565758595

Abun

danc

e

times105

5152535455565758595

Abun

danc

e

times105





Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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