1297

Available online at www.sciencedirect.com

Journal of Hazardous Materials 154 (2008) 96104

Biodegradation pattern of hydrocarbons frg mJ.Lfa y M5, U9onal drgenti

er 2002007

Abstract

The biode x resbioreactor using a microbial consortium in seawater medium. Experiments with initial concentrations of 0.18 and 0.53% (v/v) of bilge waste werecarried out. In order to study the biodegradation kinetics, the mass of n-alkanes, resolved hydrocarbons and unresolved complex mixture (UCM)hydrocarbons were assessed by gas chromatography (GC). Emulsification was detected in both experiments, possibly linked to the n-alkanesdepletion, with differences in emulsification start times and extents according to the initial hydrocarbon concentration. Both facts influenced thehydrocarbon biodegradation kinetics. A sequential biodegradation of n-alkanes and UMC was found for the higher hydrocarbon content. Being theformer growhydrocarbonbiodegradatbiodegradabthe biodegra 2007 Else

Keywords: B

1. Introdu

In recento managecarbons. Bbecause ofdation of oistudies. Neto the toxieffluents, wance with ebiodegrada[4].

CorresponE-mail ad

0304-3894/$doi:10.1016/jth associated, while UCM biodegradation was a non-growing process showing enzymatic-type biodegradation kinetics. For the lowerconcentration, simultaneous biodegradation of n-alkanes and UMC were found before emulsification. Nevertheless, certain UCM

ion was observed after the medium emulsification. According to the observed kinetics, three main types of hydrocarbons (n-alkanes,le UCM and recalcitrant UCM) were found adequate to represent the multicomponent substrate (bilge waste) for future modelling ofdation process.vier B.V. All rights reserved.

iodegradation; Bilge waste; Fuel oil residue; UCM; Emulsification

ction

t years, various technologies have emerged in orderoil residues and effluents contaminated with hydro-ioremediation is one of the most extensively usedits low cost and high efficiency [1,2]. The biodegra-l and its derived products has been the focus of manyvertheless, an important concern still remains duecity of their constituents [3], the large amounts ofaters and soils that need to be remediated in compli-nvironmental standards, and the need to enhance thetion of the more recalcitrant petroleum compounds

ding author. Tel.: +54 2965 451024; fax: +54 2965 451543.dress: [email protected] (M.L. Nievas).

Oil and derived product residues are complex mixtures ofthousands of compounds, with a high proportion of hydrocar-bons, which posses different solubility and microbial resistanceto biodegradation [3,5]. Diesel and fuel oils constitute the mainfuels for cargo vehicles and ships, and they are generally blendsof medium distillate and residual petroleum cuts. Gas chro-matographic analysis of these fuels usually shows, in additionto the resolved compounds, an elevation of the baseline whichis named unresolved complex mixture (UCM). This portion ofthe fuel, UCM, is mainly composed by branched and cyclicaliphatic hydrocarbons and aromatic hydrocarbons [6], whichusually show the greatest resistance to biodegradation [5,6]. Inagreement with this, UCM is used as an indicator of petrogenicenvironmental inputs due to its large persistence after accidentalor chronic oil spills [7].

Aerobic petroleum biodegradation is a multiphase reactionwhere microorganisms, oxygen, water soluble mineral salts, and

see front matter 2007 Elsevier B.V. All rights reserved..jhazmat.2007.09.112residue by an emulsifier-producinM.L. Nievas a,, M.G. Commendatore a,

a Centro Nacional Patagonico, Unidad de Investigacion de OceanograQumica y Contaminacion de Aguas, CONICET, Bv. Brown 282

b Departamento de Ingeniera Qumica, PLAPIQUI, Universidad NaciKm 7, 8000, Baha Blanca, A

Received 1 July 2007; received in revised form 27 SeptembAvailable online 2 October

gradation of a hazardous waste (bilge waste), a fuel oil-type compleom a fuel oil-type complexicrobial consortium

. Esteves a, V. Bucala beteorologa, Laboratorio de Oceanografa120ACF, Puerto Madryn, Argentinael Sur, CONICET, Camino La Carrindangana

7; accepted 27 September 2007

idue from normal ship operations, was studied in a batch

M.L. Nievas et al. / Journal of Hazardous Materials 154 (2008) 96104 97

low-soluble substrates are generally involved [8]. Thus, masstransfer processes are critical in the hydrocarbon biodegrada-tion kineticinterfacial tbecoming htion. Biosuhave lower[10]. Toxiction have[2,12], butbiosurfactatems of n-residues [1of the simuon the biod[17].

In the presidue (shan emulsifiBilge wastewater and hthe main coics and unUCM biodeprocess, e.gcitrant comwaste biodemulsificaton hydrocabiodegradabon biodegcompoundscould be usprocesses.

2. Materia

2.1. Waste

The reswas obtaidownloadeMadryn, Awaste oilyresidue, wa[1820]. HBWOP indetail in Scolumn to[20]. Hydr(expressed(being 89%136; andUCM. RTHdensity at 1content werespectivel

2.2. Microorganisms and culture medium

OProorgment

as

, w[20]m)

iquidions.

48-= 0.rs ol

ssay

aysseaw

nic ned wtedn (2men

con

was

). Aerforf thrpmme

at 2or thtivelpt ins wo peydrand

odeglso warbomicrs conntinuw co

to quifierm lo

naly

hydred

23].m (w

wats [1,9]. Surfactants act by decreasing the surface andension and by stabilizing oilwater emulsion [10,11],ydrocarbon compounds bioavailable for biodegrada-

rfactants are preferred to chemical surfactants as theytoxicity and shorter persistence in the environmentor inhibitory effects on hydrocarbon biodegrada-

been observed with the addition of biosurfactantsseveral reports also focus on the positive effect ofnt addition on biodegradation in biphasic liquid sys-alkanes [13], PAHs [14], oil [15] and hydrocarbon6]. However, little information exists on the effectltaneous production of surfactants and/or emulsifiersegradation pattern of complex hydrocarbon mixtures

resent study the biodegradation of a fuel oil-typeip bilge waste produced in normal ship operations) byer-producing microbial consortium was investigated.is a hazardous waste composed by a mixture of sea-ydrocarbon residue, where n-alkanes and UCM arenstituents. Knowledge of the biodegradation kinet-

derstanding the effects of in situ emulsification ongradation is necessary to improve the biodegradation. by reducing the time required to biodegrade recal-pounds. The aim of this study was to investigate bilgeegradation kinetics and understand the effects of theion produced during the biodegradation experimentrbon bioavailability, in consequence, on bilge wastetion. In addition, the study focused on the hydrocar-radation pattern, in order to identify the key types of

in the multicomponent hydrocarbons mixture thated to model bilge waste hydrocarbon biodegradation

ls and methods

idue used as substrate in the biodegradation assaysned by gravitational separation of bilge wasted from ships that moored at the port of Puertorgentina. The floating organic phase named bilgephase (BWOP), a partially weathered fuel oil-types previously physically and chemically characterizedydrocarbon analyses were performed by dissolvingn-hexane and treating with silica gel (see more

ection 2.4). BWOP was also fractioned in aluminaquantified aliphatic and aromatic hydrocarbons

ocarbon-concentrations found by GC analysis wereas g kg1 BWOP): total hydrocarbons (TH), 542aliphatics and 11% aromatics); resolved TH (RTH),

UCM, 406. TH represents the sum of RTH andcontained 75 g kg1 BWOP of n-alkanes. BWOP

5 C, kinematic viscosity (KV) at 40 C and waterre 863 kg m3, 1.995 105 m2 s1 and 15% (v/v),y, determined as described by Nievas et al. [19].

BWof micenrichBWOPsortiumtested(160 rpfresh lconditwith aOD450transfe

2.3. A

AsssterileinorgapreparplemensolutioExperiBWOPBWOP15 minalso p(v/v) oat 250culturetaineddays frespecwas kesampletimes tyses. Hculturebon biwere a

hydrocphasecarbonThe coand flometerhumidmediu

2.4. A

Aperformdure [mediutillatefrom a bilge waste dumping pool was used as sourceanisms. A microbial consortium was obtained by anprocedure, using a mineral medium and 1% (v/v)

the only carbon and energy source [20]. The con-hose emulsifier production capacity was previously, was mechanically stirred using an orbital shakerat 25 C. Cultures were transferred twice a week in

medium (1%, v/v) and then incubated in the sameInoculums used in the present study were prepared

h culture by washing and resuspending the cell at1 [20]. Consortia used as inoculums were at least 10d since the start of the enrichment procedure.

conditions

were carried out in a 3-l bioreactor using 2 l ofater mineral medium (SWM), supplemented withutrients and 0.2 g l1 yeast extract [19]. SWM wasith natural filtered seawater (through 0.45m) sup-with 1 g l1 NH4NO3 and 4 ml l1 of phosphate

0 g l1 Na2HPO47H2O and 4 g l1 NaH2PO4H2O).ts were performed with two levels of initial sterilecentration at 0.53 and 0.18% (v/v) of the SWM.sterilized in autoclave in a sealed container (121 C,control experiment without BWOP addition was

med. The inoculum suspension was added at 1%e SWM in each experiment. Cultures were stirred, aerated at 0.5 vvm (volume of air per volume ofdium per minute), and the temperature was main-5 C. Experiments were carried out for 14 and 10e high and the low initial BWOP concentrations,

y. Culture pH, monitored online with an electrode,the range 78 with 0.5N NaOH. Duplicate culture

ere withdrawn through a liquid sampler at differentrform biomass, turbidity and superficial tension anal-ocarbon content was measured in the batch liquidin the exhaust air of the bioreactor to assess hydrocar-radation efficiency. Duplicate 20 ml liquid samplesithdrawn using a syringe at each sampling time for

n analysis. The exhaust air was sampled by solidoextraction (SPME) to determine evaporated hydro-centration as it was previously described [19,21,22].ous airflow rate was controlled by means of pressurentroller valves and was measured by a bubble-flow-antify the hydrocarbons evaporation rate. A sterile

was used before the air inlet to the bioreactor to avoidss by evaporation.

tical methods

ocarbon degrading bacteria count (HDC) wasby the most probable number (MPN) proce-The liquid medium used consisted of a mineralith the following composition in g l1 of dis-

er: NaCl, 24.0; MgSO47H2O, 1.0; KCl, 0.7;

98 M.L. Nievas et al. / Journal of Hazardous Materials 154 (2008) 96104

KH2PO4, 2.0; Na2HPO4, 3.0; NH4NO3, 1.0) with 1 ml l1of a trace metal solution (with the following composition inmg l1 of10; MnClNiCl26H2Petroleumwas sterilizstrate. Plat20 C for 3also perforusing a ricseawater):extract, 3. PComparisoby CochranHewlett Pamedium wtrifugationof extracellthe supernDuNouy tebons in thepreviouslyfied to pHn-hexane.anhydrous24 h) silicaextracts we5 min and tpaper Whatrated undewere quantnal standarwere determevaporationa non-equiconcentratias describelated by mvolumetriccarbons (Twas obtainexperiment

3. Results

3.1. Micro

Heterotrand biodegtial BWOPreached arocultures wihigher thansubstrate (between da

Heter450) (dEmptyrationv/v).

ial csou

e co), anitialwn iC r

th cucon

ncen

e BWwas

t coas

icallificargan

ment8% (hydrocarbon-degrading microorganisms to grow in theedium used to determine HC in the assays. BWOP couldntain some toxic compounds, such us heavy metals, thate responsible of lower heterotrophic microorganism con-

tions observed for the 0.53% (v/v) BWOP experiment witht of those of 0.18% (v/v) one. A metal analysis performedlge water sample, withdrawn from the same source that

used in this work, showed the presence of Cr(III), Cu,and Zn. These metals come probably from the ship bilgegine corrosion, as the exhausted lubricant oils are usuallyed in the bilge wastes.bidity increased to approximately 1 absorbance unit (AU)first day, for the 0.53% (v/v) BWOP assay (Fig. 2a). Forest BWOP concentration experiment, the same turbidityas reached after approximately 2.5 days (Fig. 3a). Then

OD450 increases occurred about 1 and 2.5 days reachingvalue of around 16 and 5 AU for initial concentrations ofd 0.18% (v/v) BWOP, respectively (Figs. 2a and 3a). Ondistillate water: FeSO47H2O, 200; ZnSO47H2O,24H2O, 3; CoCl26H2O, 20; CuCl22H2O, 1;O, 2; Na2MoO42H2O, 500; H3BO3, 30 [24]).from Comodoro Rivadavia provided by Shell Capsa,ed in autoclave (121 C for 15 min) and used as sub-es were counted after incubation between 16 andweeks. Heterotrophic microbial counts (HC) were

med with the same procedure as that for HDC, buth culture medium that contained (in g l1 of naturalglucose, 3; yeast extract, 2.5; meat peptone, 5; meatlates were counted at 72 h of incubation at 1620 C.n between MPN results were performed as described[25]. Medium turbidity was measured at 450 nm in a

ckard 8452A spectrophotometer. Samples of cultureere separated from cells and hydrocarbons by cen-at 16,000 g for 15 min to determine the productionular biosurfactant. Surface tension measurements inatant were performed using the ring method on ansiometer at standard room temperature. Hydrocar-liquid culture medium were determined by GC as

described [18]. Briefly, 20 ml samples were acidi-< 2 with 6N HCl and extracted thrice with 20 ml ofThen, the hexane extracts were pooled, dried overNa2SO4, and treated with activated (at 200 C duringgel to remove polar compounds. The dried hexanere shaken with silica gel in a magnetic stirrer byhen silica gel was removed by filtering using a filtertman No. 40. The hydrocarbon extract was concen-r N2 flow, and analyzed by GC-FID. Hydrocarbonsified using the n-alkanes homologous series as exter-ds. The masses of TH, RTH, UCM and n-alkanesined at different culture times. Hydrocarbon loss byfrom the bioreactor was quantified by SPME using

librium quantitation method [21,22]. Hydrocarbonon in the bioreactor air outlet stream was quantifiedd by Nievas et al. [19]. Evaporation rate was calcu-ultiplying the hydrocarbon air concentration by theair flow rate. Total evaporated mass of the hydro-

H, RTH, UCM and n-alkanes) as a function of timeed by the integration of the evaporation rate over theal time.

and discussion

bial growth and emulsication

ophic count (HC) and culture turbidity for the controlradation experiments with 0.53 and 0.18% (v/v) ini-content are shown in Fig. 1. For all experiments HCund 108 MPN ml1 in approximately 0.8 day. Then

th BWOP showed HC values between 2- and 10-foldthat of the control experiment without hydrocarbon

Fig. 1), being significantly greater than the controlys 2 and 6 (p < 0.05). This fact suggested that the

Fig. 1.ity (ODassays.concent0.18% (

microbcarbon

Timizationwith inare shothat HDfor boBWOPism coFor thHDChighesisms w(statista signmicrooexperiof 0.1some

rich malso cocould bcentrarespecin a biBWOPFe, Niand endispos

Turin thethe lowlevel wsharpa final0.53 anotrophic microbial count (HC, MPN ml1) (squares) and turbid-iamonds) time course of the BWOP biodegradation and controlsymbols, control without BWOP; filled symbols, initial BWOP0.53% (v/v); semi-filled symbols, initial BWOP concentration

onsortium used hydrocarbons and yeast extract asrces.

urse of HDC, pH, turbidity, NaOH (used for neutral-d hydrocarbons content for the biodegradation assaysconcentrations of 0.53 and 0.18% (v/v) of BWOP

n Figs. 2 and 3, respectively. Figs. 2a and 3a indicateeached approximately 2 108 MPN ml1 after 1 dayltures, showing the culture with 0.53% (v/v) initialtent the highest hydrocarbon-degrading microorgan-tration between 2 and 3 days (9 108 MPN ml1).OP concentration of 0.18% (v/v), the maximum

approximately 3 108 MPN ml1 at 2.5 days. Thencentration of hydrocarbon-degrading microorgan-found for the highest BWOP concentration testedy greater than the lowest BWOP concentration, atnce level of 10%). Contrarily lower heterotrophicism growth was observed for the 0.53% (v/v) BWOP(p < 0.10 from days 4 to 9) compared with that

v/v) one (Fig. 1), probably due to the inability of


Fig. 2. Time course of the 0.53% (v/v) initial BWOP concentration biodegradation assay. (a) () HDC (MPN ml1), () turbidity (OD450), and () amount ofNaOH added to maintain pH between 7 and 8 (meq alkali); (b) () TH (mg), () UCM (mg), () RTH (mg), and () n-alkanes (mg); (c) detail of HDC, turbidity,UCM and n-alkanes in the 04.5 days period.

Fig. 3. Time course of the 0.18% (v/v) initial BWOP concentration biodegradation assay. (a) () HDC (MPN ml1), () turbidity (OD450), and () pH; (b) () TH(mg), () UCM (mg), () RTH (mg), and () n-alkanes (mg); (c) detail of HDC, turbidity, UCM and n-alkanes in the 06 days period.


the other hand, culture turbidity in the control assay increasedin agreement with the microbial count to a maximum value ofOD450 = 0.up to the enconcentratiwith OD450.18% (v/vand opticalp < 0.01, bein each exdays (R2 >cated that,to microbia0.65 (n = 1whole duracated thatassociatedemulsificatprevious st[20]. Furthhigh and threspectivelbons concereinforcingby hydrocastarted aftein agreemeproduced w[10,11]. Suthe assaysfrom the incentage errconcentratitures in thtension, cobiosurfactaganisms (ua high moBWOP feremulsificatface tensiodifferent inbeing latercentrationbioemulsifithe nitrogenitrogen soprevious anhydrocarbo(1 g l1 NH

Acidificon hydrocassay, it wthe rangetion with tun = 17, p Rsed aimil70%

65%an 6trant32 at day 2, and then remained lower than this valued of the experiment at day 14 (Fig. 1). For both BWOPons, HC and HDC showed initially linear correlation0 in the range of 01 AU. For the cases of 0.53 and) BWOP, good linear correlations between biomassdensity were verified up to 1 day (R2 > 0.84, n = 6,ing n the number of experimental points regressed

periment over the mentioned time period) and 2.50.84, n = 9, p < 0.01), respectively. These results indi-for both cultures, turbidity initially augmented duel growth. However, R2 decreased to values lower to

4, p < 0.05) for both experiments when data of thetion of the assays were correlated. This fact indi-

the observed sharp turbidity increases could not bewith microbial growth, suggesting that hydrocarbonsion could be responsible of them, as it was found in audy where the same microbial consortium was usedermore, the final turbidity values (16 and 5 AU for thee low initial BWOP concentrations, Figs. 2a and 3a,y) were in good agreement with the initial hydrocar-ntration of the cultures (approximately a 3:1 ratio),the hypothesis that turbidity increases were causedrbons emulsification. The sharp turbidity increasesr the end of the microbial exponential growth phase,nt with reports that state that emulsifiers are usuallyhen cultures reach the stationary phase of growth

rface tension of the culture medium supernatant alongshowed little change (data not shown), decreasingitial value of 70 to 50 and 60 mN m1 (relative per-or less than 13%) for the high and low initial BWOPons, respectively, at the end of the experiments. Cul-e present study showed a low reduction in surfacempared with that found when low molecular weightnts are produced by hydrocarbon-degrading microor-p to 30 mN m1) [10,26]. This fact suggests that

lecular weight emulsifier was produced during thementation, as these types of compounds show highion capabilities without extensively lowering the sur-n [11,14]. The emulsification start point occurred atcubation times for the BWOP concentrations tested,for the culture with the lowest hydrocarbons con-

(Figs. 2a and 3a). Some authors have reported thater production starts when a nutrient is depleted, e.g.n source [10,27]. Although in the present study theurce was not monitored during the experiments, aalysis in a biodegradation experiment with similarns biodegradation extent and initial nutrient content4NO3) showed that it was not nitrogen limited [28].ation of the reaction medium found was dependentarbon concentration. For the 0.53% (v/v) BWOPas necessary to add alkali to maintain the pH in

78, showing the acid production a linear correla-rbidity for the whole experiment duration (R2 = 0.98,0.01) (Fig. 2a). For the low BWOP concentration,ed slightly from 8.0 to 7.5 (Fig. 3a) until the high-

sary towere n

for theassay,aroundculturewas foity (R2mediupoundreactoan acidhigheshydrocthe aciment othat woil biosix-badroppethat sathe pHspite oFoghton cru

was grthe obof ammby thehydrocwas en

and 0.1neededBWOPtion wthe prediffereBWOPspecififor thewith thfound

3.2. H

Foran imvaluesalkaneexpresto be saroundof 57less threcalcip the pH in the range 78, while OD450 and pHively linear correlated (R2 = 0.86, n = 14, p < 0.01)ole experiment duration (10 days). For the controlout BWOP, the pH gradually decreased from 8 toat the end of the experiment (data not shown), thetralization was not necessary. No linear correlationfor the control experiment between pH and turbid-005, n = 12, p < 0.95). These results indicate that theulsification can be related to the release of acid com-ich depends of the hydrocarbon concentration in thes. 2a and 3a), suggesting that the emulsifier could hasructure. Experimental observation indicated that thedification and turbidity were found for the highestns concentration. Another possible explanation foration found could be related with the nutrient amend-e culture medium. Foght et al. [29] have reportedNH4NO3 was used as nitrogen source in a crudeadation experiment in a seawater medium (using al-strains consortium) the pH of the culture medium

gnificantly (to less than 6). These authors reportede-hydrocarbon-degrading strains were responsible ofp, when they grew in the presence of ammonium. Inacidification mechanism was not clearly established,[29] stated that acidification was not linked to growthl, as it was also observed when the same consortiumin glucose as sole carbon source. In the present studyd acidification could be explained by the presenceum as nitrogen source, as previously was reportedthors. However, pH drop seems to be linked to thens amount, due to the poorly buffered medium usedh to keep the pH medium above 7.0 for the control(v/v) BWOP experiments, while 6 meq of alkali wereeep the pH near neutrality (7.08.0) for the highesttent experiment (Figs. 2a and 3a). If the acidifica-used by the saturated-degrading strains growing ine of ammonium as reported by Foght et al. [29], theobserved in the medium acidification between both

ays could be attributable to a highest growth of thismponent of the hydrocarbon-degrading consortium% (v/v) BWOP experiment. This fact is in agreementdrocarbon-degrading microorganism concentrationshe 0.18 and 0.53% (v/v) BWOP assays.

carbon biodegradation extent

BWOP levels, all the hydrocarbon types showedant concentration reduction from their initiale extent of biodegradation followed the order n-TH > UCM (Table 1). The residual hydrocarbons,s percentage of the initial hydrocarbons, were foundar for both experiments (Table 1). TH reduction of

and TH biodegradation efficiencies in the rangewere achieved for both BWOP concentrations indays (Table 1). Some UCM compounds remainedto the microorganism action (approximately 30%


Table 1Extent and efficiency of biodegradation in assays using a bioemulsifier-producing microbial consortium with two different BWOP initial concentrations

Hydrocarbon 0.

Inm

m

n-Alkanesd 2RTHd 3UCMd 13THd 16

Values are me cate hthat was 21%

a In the liqub Values arec Percentagd Values co he ini

of the initiaresidues froand lubricain this studcosity (KVexpected foexhaust luberties of pafinding a nity and log2 105 met al. [31],expected. Tof TH remthat recalciBWOP we

3.3. Hydro

Hydrocashown inbiodegradations; howefound.

For thedecreasedn-alkanes aAlkanes dichange madegraded avalue (Figinitial UCafter day34.5% of ttion n-alkamicrobial(9 108 Mextinguishestrate for hythe other h

that cnes dtratiationed foed pioavaed hf inds in

on pnism: thrudodro4,26mene thity mullynt freightionulsifit fortype 0.53% (v/v) BWOP initial concentrationInitialmassa

Evaporatedmass

Residualmassa

Biodegradationefficiencyb

mg mg %c mg %c %c

603 27 4.4 24 4.1 91.5 (2 days)956 90 9.4 118 12.4 78.2 (6 days)

3158 137 4.3 1089 34.5 61.2 (6 days)4115 231 5.6 1208 29.3 65.0 (6 days)

an of determination in duplicate samples. Relative percentage errors for dupli.id culture medium.calculated at the assay time indicated in parenthesis.

e referred to the initial mass of the same hydrocarbon type.rrespond to the time indicated in the biodegradation efficiency column, except t

l TH for both experiments). BWOP is a mixture ofm hydrocarbon products used in ships, mainly fuelsnt oils. Chemical composition of the BWOP usedy matched with fuel oil #2, but the kinematic vis-) value observed was five times greater than the oner this fuel [19,30] indicating that BWOP includedricant oil. Haus et al. [31] correlated physical prop-

raffinic base oils with their intrinsic biodegradability,egative linear relationship between %biodegradabil-(KV). The BWOP kinematic viscosity was about2 s1; according to the correlation obtained by Hausfor this KV value 6070% biodegradability can behis result is in good agreement with the percentage

aining in the present study (about 30%), suggestingtrant branched hydrocarbons from lubricant oils inre probably resistant to microbial attack.

carbons biodegradation pattern

rbons time course during biodegradation assays isFigs. 2b and 3b. n-Alkanes, RTH, UCM and THtion were observed for both initial BWOP concentra-ver, differences in the biodegradation patterns were

0.53% initial BWOP experiment TH (UCM + RTH)

and 4,n-alkaconcen

degraddegraddegradbons benhancrates ochangeadhesimechadation(b) psewithin[1,11,1ing ferdescribsolubilis not fdiffereular winteracbioemExcepin two stages (Fig. 2b), influenced firstly by thend secondly by UCM biodegradation patterns. n-sappeared in around 2 days, while UCM did notrkedly during the first 3 days. UCM was quicklyfter medium emulsification reached its maximum. 2a and b), showing a reduction of 57% of theM between days 3 and 4. Subsequent reduction6 was almost negligible, remaining undegradedhe initial UCM. For this initial BWOP concentra-nes degradation agreed with hydrocarbon-degradinggrowth, that showed its maximum concentrationPN ml1) by the time that n-alkanes were almostd. This fact suggests that n-alkanes were used as sub-drocarbon-degrading microbial growth (Fig. 2c). On

and, UCM biodegradation produced between days 3

graded witfor the 0.5obtained fAlkanes biand b), wh3 (Fig. 4boutlet air sfrom C9 tothe liquid s

UCM bdelay withbiodegradaas diesel oiintrinsic dipounds. Ge18% (v/v) BWOP initial concentrationitialassa

Evaporatedmass

Residualmassa

Biodegradationefficiencyb

g mg %c mg %c %c

00 28 14.1 7 3.6 82.4 (3 days)80 62 16.4 43 11.4 72.1 (5.7 days)07 120 9.2 490 37.5 53.3 (5.7 days)84 182 10.8 534 31.7 57.5 (5.7 days)ydrocarbon masses were less than 11%, except for the sample

tial mass.

onstitute a hydrocarbon mass three times greater thanegraded, did not cause an increase in the microbialon. (Fig. 2c). UCM showed non-growth associated

kinetics [1]. This fact suggests that UCM wasllowing a different mechanism fromn-alkanes, beingrobably after emulsification increases the hydrocar-ilability. Surfactants and emulsifiers have frequentlyydrocarbon aqueous solubility and biodegradationividual hydrocarbon compounds, usually producinghydrocarbons bioavailability and in microorganism

roperties to hydrophobic interfaces [1,14,26]. Threes are accepted for microbial hydrocarbon biodegra-ough utilization of (a) soluble organic molecules,solubilized hydrocarbons by surfactants (substratesplets less than 1m) and (c) stabilized emulsions]. As the surface tension showed a low reduction dur-tation, the last mechanism seems to be adequate toe observed UCM biodegradation. Increasing apparent

echanism by high molecular weight bioemulsifiersunderstood [14,32]. However, it was shown that it wasom the micellar mechanism described for low molec-t biosurfactants, and that, probably, multimoleculars could occur between hydrophobic regions of theer molecules and the hydrophobic substrates [14,32].the n-alkanes, the rest of the RTH were biode-h similar pattern as UCM (Fig. 2b). Fig. 4 shows,3% BWOP assay, the TH chromatographic profilesrom liquid and air samples at different times. n-odegradation occurred in the first 2 days (Fig. 4aile the highest UCM reduction was found after dayand c). The evaporated compounds, detected in thetream, comprised resolved and UCM hydrocarbonsC16, showing complementary profiles with those ofamples (compare right and left graphs of Fig. 4).iodegradation, with less preference and with a time

respect to n-alkanes, has been reported in othertion studies of different hydrocarbon mixtures suchl [3335]. This behavior was usually attributed to thefficulty of microorganisms to biodegrade these com-erdink et al. [33] consider that the probable cause for


Fig. 4. TH chblock (ac) cosamples in the(b and e), 3.1Pr: pristane; P

the delay iity of lineasystem invsortium, di0.53% (v/voccurred afthat bioavawas respon

Hydrocaest BWOPn-alkanes aperiod (Figobserved fromatographic profiles from the biodegradation assay with 0.53% (v/v) BWOP initrresponds to the same amount of liquid samples, being the y-axis scales the same soexhaust air of the bioreactor, being the y-axis scales the same so they can be directly

days; and third file (c and f), 14.0 days. Only qualitative comparisons can be done bh: phytane; UCM: unresolved complex mixture.

n branched-alkanes degradation is the higher affin-r n-alkanes for one or more of the enzymes in theolved in diesel oil fermentation by a microbial con-smissing the hypothesis of diauxic utilization. For the) BWOP experiment, the higher UCM biodegradationter emulsifier production (Fig. 2). This fact suggestsilability, and not intrinsic biodegradation resistance,sible for the observed UCM biodegradation delay.rbons in the experiment carried out with the low-concentration (0.18%, v/v) showed simultaneous

nd UCM biodegradation during the 03 days time. 3b), in contrast with the sequential biodegradationor the 0.53% (v/v) BWOP concentration. However

after emulsably followenhances ththat the bihydrophobculture acctration addmetabolitesally to the0.18% (v/vhydrocarboof the 0.53%sifier was pial concentration at different times. X-axis, time in minutes. Leftthey can be directly compared. Right block (df) corresponds tocompared. First file (a and d), 0.08 day (initial time); second file

etween air and liquid samples. Cx: n-alkane of x carbon number;

ification the UCM degradation rate increases, prob-ing a mechanism mediated by the emulsifier whiche UCM bioavailability. Kim et al. [27] have reportedoemulsifier MEL-SY16, produced at expenses ofic substrate, reached higher concentration in theording the higher hydrophobic substrate concen-ed to the reaction medium. In addition secondary(as bioemulsifiers) are usually produced proportion-biomass [36]. In the experiments carried out with) BWOP both initial hydrocarbons concentration andn-degrading microorganisms were lower than those

(v/v) BWOP experiment, suggesting that the emul-roduced in less concentration. This also agreed with


the lower turbidity found in the 0.18% (v/v) BWOP experiment.These facts could be responsible for the delay observed in emul-sification foconsequenc

Despitebiodegradaexperimentemulsificatsimultaneofact suggesfier producto producesule, whendroplets [11n-alkanes,mer whichpotent emuof n-alkanepattern obstion in BW

4. Conclu

Surfactato enhancebic composimultaneoplex hydroused for bemulsificattial hydrochydrocarbobe linked tthe higherBWOP inin-alkanes aciated procfollowing ation was liconcentratibiodegradasification, awas observematicallyand operatebons can bto the obseable UCM,obtained reconcentratiterns for loobserved.

Acknowled

This wotigaciones

Nacional de Promocion Cientfica y Tecnologica (ANPCyT) ofArgentina. We thank Dr. Pablo Yorio for his help in improving

t.

nce

Alexss, Sa. Van

crobio. Ka

ycycl920

Kordadiatiomerc

(1997. Atl

ntal p. Fry

ved c. TechWangntificaRosenroleu92) 3. Ch

solid99) 7. Desrcial p. Rontechn. Fog

ion ocrobiohang

a PsecrobioBarkaubilizemulsAbalodegrasencedegra. Osurfa

ge wa. Vanonas

wth oviron.L. Nia elues,entin

L. Niedegramun

L. Niedificay, Int.. Barair st101.r the lower hydrocarbon content experiment, and ine on the hydrocarbons degradation kinetics.of the differences found on n-alkanes and UCM

tion pattern for both BWOP concentrations tested,al observations indicated that the start of theion in the cultures (turbidity increases) occurredusly with n-alkanes depletion (Figs. 2c and 3c). Thists that depletion of n-alkanes triggered the emulsi-tion. Acinetobacter calcaoceticus has been reportedthe bioemulsifier Emulsan, a cell bound minicap-growing on n-alkanes attached to the oil interface]. When hydrocarbons droplets became exhausted of

cells detached from interface, releasing the biopoly-remains attached to the hydrocarbons and acts as alsifier. The Emulsan production was a consequencebiodegradation. A similar situation could explain theerved for n-alkanes biodegradation and emulsifica-OP fermentations.

sions

nt and emulsifier addition has often been usedd bioavailability and biodegradation of hydropho-unds; however little is known about the effect ofus emulsifier production on biodegradation of com-carbon mixtures. A emulsifier-producing consortiumiodegradation of BWOP showed differences in theion start time and extent, according to the ini-arbon concentration used. Both facts influenced then biodegradation kinetics. Emulsification appears too the n-alkanes depletion and occurred earlier forhydrocarbon concentration tested. For 0.53% (v/v)tial concentration, a sequential biodegradation ofnd UMC was found, being the former a growth asso-ess and the later a non-growth associated one, likelyemulsifier mediated mechanism where biodegrada-

mited by hydrocarbon accessibility. For the loweron experiment (0.18%, v/v, BWOP) simultaneoustion of n-alkanes and UMC were found before emul-lthough a final and important UCM biodegradationed after this step. These results are useful to math-model BWOP biodegradation, in order to designan effluent treatment bioreactor. BWOP hydrocar-

e characterized by three compound types accordingrved biodegradation kinetics: n-alkanes, biodegrad-and UCM that remained undegraded. In view of thesult, further investigations are needed to explore theon range over which the biodegradation kinetic pat-w and high initial hydrocarbon concentrations are

gments

rk was supported by the Consejo Nacional de Inves-Cientficas y Tecnicas (CONICET) and the Agencia

the tex

Refere

[1] M.Pre

[2] J.Dmi

[3] R.Apol205

[4] A.me

com

48[5] R.M

me

[6] G.SsolSci

[7] W.ide

[8] E.Pet(19

[9] D.Hof(19

[10] J.Dme

[11] E.ZBio

[12] J.MdatMi

[13] Y. ZbyMi

[14] T.solbio

[15] A.biopreBio

[16] N.LBiobil

[17] J.DdomgroEn

[18] M.parbuqArg

[19] M.Biocom

[20] M.mo

nit[21] R.J

in92s

ander, Biodegradation and Bioremediation, second ed., Academicn Diego, 1999.Hamme, A. Singh, O.P. Ward, Recent advances in petroleum

logy, Microbiol. Mol. Biol. Rev. 67 (2003) 503549.naly, S. Harayama, Biodegradation of high-molecular-weightic aromatic hydrocarbons by bacteria, J. Bacteriol. 182 (2000)67., P. Santas, A. Tenente, R. Santas, Petroleum hydrocarbon biore-n: sampling and analytical techniques, in situ treatments andial microorganisms currently used, Appl. Microbiol. Biotechnol.) 677686.as, Microbial degradation of petroleum hydrocarbons: an environ-erspective, Microbiol. Rev. 45 (1981) 180209.singer, R.B. Gaines, L. Xu, C.M. Reddy, Resolving the unre-omplex mixture in petroleum contaminated sediments, Environ.nol. 37 (2003) 16531662., M.F. Fingas, Development of oil hydrocarbon fingerprinting andtion techniques, Mar. Poll. Bull. 47 (2003) 423452.berg, R. Legman, A. Kushmaro, R. Taube, E. Adler, E. Ron,

m bioremediationa multiphase problem, Biodegradation 337350.oi, K. Hori, Y. Tanji, H. Unno, Microbial degradation kineticsalkane dissolved in nondegradable oil phase, Biochem. Eng. J. 3178.ai, I.M. Banat, Microbial production of surfactant and their com-otential, Microbiol. Mol. Biol. Rev. 61 (1997) 4764., E. Rosenberg, Biosurfactant and oil bioremediation, Curr. Opin.ol. 13 (2002) 249252.ht, D.L. Gutnik, D.W.S. Westlake, Effect of emulsan on biodegra-f crude oil by pure and mixed bacterial cultures, Appl. Environ.l. 55 (1989) 3642., R.M. Miller, Enhanced octadecane dispersion and biodegradationudomonas rhamnolipid surfactant (biosurfactant), Appl. Environ.l. 58 (1992) 32763282.y, S. Navon-Venezia, E.Z. Ron, E. Rosenberg, Enhancement ofation and biodegradation of polyaromatic hydrocarbons by theifier Alasan, Appl. Environ. Microbiol. 65 (1999) 26972702.s, M. Vinas, J. Sabate, M.A. Manresa, A.M. Solanas, Enhanceddation of Casablanca crude oil by microbial consortium inof rhamnolipid produced by Pseudomonas aeruginosa AT10,

dation 15 (2004) 249260.livera, M.G. Commendatore, A.C. Moran, J.L. Esteves,ctant-enhanced degradation of residual hydrocarbons from shipste, J. Ind. Microbiol. Biotechnol. 25 (2000) 7073.Hamme, O.P. Ward, Physical and metabolic interactions of Pseu-sp. strain JA5-B45 and Rhodococcus sp. strain F9-D79 during

n crude oil and effect of a chemical surfactant on them, Appl.Microbiol. 67 (2001) 48744879.

evas, Diseno, simulacion y optimizacion de un reactor biologicotratamiento de efluentes organicos provenientes de sentinas dePh.D. Thesis, Universidad Nacional del Sur, Baha Blanca,a, 2006.vas, M.G. Commendatore, N.L. Olivera, J.L. Esteves, V. Bucala,dation of bilge waste from Patagonia with an indigenous microbiality, Bioresour. Technol. 97 (2006) 22802290.vas, M.G. Commendatore, J.L. Esteves, V. Bucala, Effect of pHtion on bilge waste biodegradation by a native microbial commu-Biodeterior. Biodegrad. 56 (2005) 151157.telt, B.W. Zilkowski, Nonequilibrium quantitation of volatilesreams by solid phase microextraction, Anal. Chem. 71 (1999)


[22] R.J. Bartelt, B.W. Zilkowski, Airflow rate in quantitation of volatilesin air streams by solid-phase microextraction, Anal. Chem. 72 (2000)39493955.

[23] J.R. Haines, B.A. Wienn, E.L. Holder, K.L. Strohmeier, R.T. Herrington,A.D. Venosa, Measurement of hydrocarbon-degrading microbial popula-tions by a 96 well plate most-probable-number procedure, J. Ind. Microbiol.Biotechnol. 16 (1996) 3641.

[24] L.E. Rice, B.B. Hemmingsen, Enumeration of hydrocarbon-degrading bac-teria, in: E. Sheehan (Ed.), Methods in Biotechnology: BioremediationProtocols, Humana Press, New Jersey, 1997 (Chapter 8).

[25] W.G. Cochran, Estimation of bacterial densities by means of the MostProbable Number, Biometrics 6 (1950) 105116.

[26] M. Bouchez-Natali, H. Rakatozafy, Y.-Y. Leveau, J.-P. Vandecasteele,Diversity of bacterial strains degrading hexadecane in relation to the modeof substrate uptake, J. Appl. Microbiol. 86 (1999) 421428.

[27] H.S. Kim, J.W. Jeon, B.H. Kim, C.Y. Ahn, H.M. Oh, B.D. Yoon, Extracel-lular production of a glycolipid biosurfactant, mannosylerythritol lipid, byCandida sp. SY16 using fed-batch fermentation, Appl. Microbiol. Biotech-nol. 70 (2006) 391396.

[28] N.L. Olivera, Biorremediacion de sistemas contaminados con hidro-carburos, Ph.D. Thesis, Universidad Nacional del Sur, Baha Blanca,Argentina, 2000.

[29] J. Foght, K. Semple, C. Gauthier, D.W.S. Westlake, S. Blenkinsopp, G.Sergy, Z. Wang, M. Fingas, Effect of nitrogen source on biodegradation ofcrude oil by a defined bacterial consortium incubated under cold, marineconditions, Environ. Technol. 20 (1999) 839849.

[30] ASTM D396, Standard Specification for Fuel Oils, ASTM International,PA, 2005.

[31] F. Haus, J. German, G.-A. Junter, Viscosity properties of mineral paraffinicbase oils as a key factor in their primary biodegradability, Biodegradation11 (2000) 365369.

[32] T.R. Neu, Significance of bacterial surface-active compounds in interactionof bacteria with interfaces, Microbiol. Rev. 60 (1996) 151166.

[33] M.J. Geerdink, M.C.M. van Loosdrecht, K.Ch.A.M. Luyben, Biodegrad-ability of diesel oil, Biodegradation 7 (1996) 7381.

[34] R. Marchal, S. Penet, F. Solano-Serena, J.-P. Vandecasteele, Gasolineand diesel oil biodegradation, Oil Gas Sci. Technol. 58 (2003) 441448.

[35] S. Penet, R. Marchal, A. Sghir, F. Monot, Biodegradation of hydrocarboncuts used for diesel oil formulation, Appl. Microbiol. Biotechnol. 66 (2004)4047.

[36] J.E. Bailey, D.F. Ollis, Biochemical Engineering Fundamentals, second ed.,McGraw-Hill, New York, 1986, pp. 1984.

Biodegradation pattern of hydrocarbons from a fuel oil-type complex residue by an emulsifier-producing microbial consortiumIntroductionMaterials and methodsWasteMicroorganisms and culture mediumAssay conditionsAnalytical methods

Results and discussionMicrobial growth and emulsificationHydrocarbon biodegradation extentHydrocarbons biodegradation pattern

ConclusionsAcknowledgmentsReferences

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