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International Biodeterioration & Biodegradation 56 (2005) 143–150

www.elsevier.com/locate/ibiod

Anthracene biodegradation by Pseudomonas sp. isolated from apetrochemical sludge landfarming site

Rodrigo J.S. Jacquesa, Eder C. Santosa, Fatima M. Bentoa, Maria C.R. Peralbab,Pedro A. Selbacha, Enilson L.S. Saa, Flavio A.O. Camargoa,�

aDepartment of Soil Science, Faculty of Agronomy, Federal University of Rio Grande do Sul, Porto Alegre, RS,

Brazil 7712 Bento Gonc-alves Ave 91540-000, BrazilbDepartment of Organic Chemistry, Institute of Chemistry, Federal University of Rio Grande do Sul. Porto Alegre, RS,

Brazil 9500 Bento Gonc-alves Ave 91540-000, Brazil

Received 1 April 2005; received in revised form 1 June 2005; accepted 20 June 2005

Available online 2 September 2005

Abstract

Anthracene is a polycyclic aromatic hydrocarbon (PAH) that presents a high pollution potential and health risk and has been

used as a model for degradation studies on PAHs because of its relative toxicity. This study aimed to evaluate anthracene

degradation by Pseudomonas sp. isolated from a 14-year-old petrochemical sludge landfarming site. Three isolates were selected

from 26 by the best growth in anthracene and two of them were identified by 16S rRNA gene sequencing as Ps. aeruginosa and Ps.

citronellolis. They showed better growth at pH 7.0 and 30 1C in medium containing up to 2 g anthracene L�1. They were also able to

grow in medium containing phenanthrene, pyrene, gasoline and diesel oil. Analysis of anthracene degradation estimated by gas

chromatography showed that Ps. aeruginosa isolate 312A had the highest rate of degradation (3.90 mg L�1 day�1), degrading 71%

of the anthracene added to the medium (250mg L�1) after 48 days. Ps. citronellolis 222A showed an intermediate level of

degradation (51%), but Ps. aeruginosa 332C degraded only 24.4%. Isolate 312A was also responsible for the highest phenanthrene

and pyrene degradation after 48 days. In order to establish the mechanisms involved in the PAH degradation, surfactant production

by the isolates was assessed by an emulsification index and reduction of the surface tension in the mineral medium free of cells.

Emulsification was not detected, indicating that the isolates did not produce high molecular weight surfactant, although reduction in

surface tension indicated production of low molecular weight surfactant compounds. The medium containing Ps. citronellolis 222A

showed the highest reduction in surface tension, which could increase anthracene bioavailability for biodegradation. To our

knowledge, this is the first report concerning increase of anthracene degradation by surfactants produced by Ps. citronellolis.

However, the highest degradation rate shown by Ps. aeruginosa 312A was not related to surfactant production, indicating that some

other mechanism could be involved in anthracene degradation. The Pseudomonas isolates may be useful for the study of PAH

degradation and for bioremediation purposes.

r 2005 Published by Elsevier Ltd.

Keywords: Ps. aeruginosa; Ps. citronellolis; Biosurfactant; Phenanthrene; Pyrene

1. Introduction

Anthracene is one among more than 100 polycyclicaromatic hydrocarbons (PAHs) and was chosen for the

e front matter r 2005 Published by Elsevier Ltd.

iod.2005.06.005

ing author. Tel.: +5551 3316 6035;

6 6040.

ess: fcamargo@ufrgs.br (F.A.O. Camargo).

study of PAH degradation because of its relativetoxicity. These chemical compounds show a skeletonof carbon and hydrogen atoms arranged in two or morearomatic rings of low solubility that makes themresistant to nucleophilic attack (Johnsen et al., 2005).PAH exposure occurs by inhalation, ingestion anddermal contact and PAHs are highly lipid-soluble andquickly absorbed by the gastro-intestinal tract of

ARTICLE IN PRESSR.J.S. Jacques et al. / International Biodeterioration & Biodegradation 56 (2005) 143–150144

mammals. PAH metabolism in the human bodyproduces epoxide compounds with mutagenic andcarcinogenic properties, cases of lung, intestine, liver,pancreas and skin cancer being reported (Samanta et al.,2002). PAHs are widespread in the environment, beingfound in air, water and soil. They are formed naturallyduring the incomplete combustion of organic matter orby many anthropogenic activities, such as the petro-chemical industry and oil refining. In these activities,PAHs are present in the effluent as complex mixtures oflow bioavailability that are highly limiting for conven-tional remediation techniques. Bioremediation is not anew strategy for PAH removal (Guieysse et al., 2004),but most of the results of in situ remediation show asmall rate of degradation. This could be associated withthe microorganisms’ inability to degrade, to lowsolubility of the contaminant and to specific nutrientlimitation of the biota (Boopathy, 2000).

PAH bioremediation approaches used in soils includetillage, nitrogen and phosphorus addition and microbeinoculation (Abbondanzi et al., 2004). However, theseapproaches do not include the solution to the problemof unavailability of those compounds to microbialdegradation. Bioavailability is determined by the rateof substrate mass transfer into microbial cells relative totheir intrinsic catabolic and excretion activity (Johnsenet al., 2005). PAH-degrading bacteria may enhance thebioavailability of PAHs by excreting biosurfactants(Johnsen and Karlson, 2004). Biosurfactants are sur-face-active molecules that have both hydrophobic andhydrophilic domains and are capable of lowering thesurface tension and the interfacial tension of the growthmedium. They can also emulsify hydrophobic com-pounds, form stable emulsions and increase PAHsolubility and consequently bioavailabity in the envir-onment (Cameotra and Bollag, 2003).

The main objective of this study of bacterial isolationfrom a 14-year-old petrochemical sludge landfarmingsite was based on their selective growth in anthracene asthe sole carbon source and establishing optimumconditions for anthracene biodegradation.

2. Materials and methods

2.1. Site description and anthracene-degrading bacteria

isolation

Soil samples were collected aseptically from a layer0–30 cm deep at a 14-year-old landfarming site at theeffluent treatment plant of petrochemical industries andan oil refinery. Landfarming cells of 15,000 m2 each werebuilt in 1989 for the disposal, at different rates, of theeffluent treatment plant sludge, containing a complexmixture of PAHs, including anthracene, phenathreneand pyrene. For the isolation of anthracene-degrading

bacteria, soil samples (1 g) were added to Tannermineral medium (Shuttleworth and Cerniglia, 1996),containing anthracene (Mercks) at a concentration of250 mg L�1. Mineral medium (MM) used was composedof (g L�1 deionized water) 0.04 CaCl2 � 2H2O; 0.1KH2PO4; 0.8 NaCl; 1.0 NH4Cl; 0.2 MgSO4 � 7H2O;0.1 KCl. Micronutrients used were (mg L�1) 0.1CoCl2 � 6H2O; 0.425 MnCl2 � 4H2O; 0.05 ZnCl2; 0.015CuSO4 � 5H2O; 0.01 NiCl2 � 6H2O; 0.01 Na2MoO4 �

2H2O; 0.01 Na2SeO4 � 2H2O. The pH was adjusted to7.0 with either HCl or NaOH. The medium containingsoil and anthracene was incubated at 30 1C with orbitalshaking (150 rpm). A 1mL aliquot was transferred every8 days to the same sterile medium and incubated underthe same conditions. After eight transfers, 1 mL wasdiluted in saline solution, plated on nutrient agar(peptone, meat extract and agar at concentrations of5.0, 3.0 and 15 gL�1, respectively) and incubated for24 h at 30 1C in dark. Bacterial colonies were purified onthe same medium.

2.2. Selection of anthracene-degrading isolates

Bacterial isolates were obtained that grew in the mediumcontaining anthracene. The most efficient were selected bythe best growth, using for comparison growth kineticsparameters. Growth rate (k), generation time (G) andgeneration number (N) were computed using the equa-tions: k ¼ N=T ; G ¼ T=N and N ¼ ðlog CFU0�

log CFU1Þ=0:301, respectively. For kinetic experiments,each isolate was added to three Erlenmeyer flaskscontaining 50mL MM and 250mganthraceneL�1 andincubated for 96h at 30 1C in an orbital shaker (150 rpm).Every 12h, 1mL medium was diluted and plated on agarmedium and incubated for 24h at 30 1C in dark. Colonieswere directly counted and expressed as CFUmL�1.

2.3. Identification of anthracene-degrading isolates

The bacterial isolates selected were identified by 16SrRNA sequencing as described by Camargo et al.(2003). They were grown on MM agar containing250mganthraceneL�1 during 2 days at 30 1C. Bacterialcolonies were then suspended in nuclease-free water.DNA was extracted from the suspension according to themethod described by Asubel et al. (1997). Coloniessuspended in a mixture of TE buffer, SDS (10%) andproteinase K were incubated for 1 h at 37 1C. NaCl (5M)and CTAB/NaCl solution (4.1 g NaCl and 10gN-cetyl-N.N.N.-trimethylammonium bromide (CTAB)in100mL pre-warmed distilled water) were added andincubated for 10min at 65 1C. The solution was extractedwith 780mL chloroform–isoamyl alcohol (24:1), centri-fuged for 5min and the aqueous phase further extractedwith an equal volume of phenol–chloroform–isoamylalcohol (25:24:1). After centrifugation for 5min, DNA

ARTICLE IN PRESSR.J.S. Jacques et al. / International Biodeterioration & Biodegradation 56 (2005) 143–150 145

present in the aqueous phase was precipitated with 0.6vol. isopropanol and the precipitate washed with 70%ethanol. The DNA pellet was dried using a lyophilizerand resuspended in nuclease-free water. Universalbacterial primers corresponding to Escherichia coli

positions 27F and 519R were used for PCR amplificationof the 16S rRNA gene. A PCR master mix (PromegaM7502, Madison, WI) was used according to themanufacturer’s instructions. Genomic DNA template(1mL) was amplified using a 35-cycle PCR (initialdenaturation, 95 1C for 3min; subsequent denaturation,95 1C for 1min; annealing temperature, 55 1C for 1min;extension temperature, 72 1C for 1min; final extension,72 1C for 5min). The PCR product was analyzed on 2%agarose gel and purified using a QIAEX II gel extractionkit (QIAGEN Valencia, CA) according to the manufac-turer’s instructions. Briefly, the gel slice was suspended inbuffer QXI and the QIAEXII suspension was added andmixed by vortexing. The suspension was incubated at50 1C for 10min, centrifuged for 30 s and the pelletwashed twice with buffer QXI and once with buffer PE.After air drying for 15min, the DNA was eluted withnuclease-free water. DNA cycle sequencing was per-formed using a BigDye terminator kit (Applied Biosys-tems, Foster City, CA) and an Applied Biosystems ABI3100 genetic analyzer, MEGABLAST (Altschul et al.,1997) was used for homology searching.

2.4. Growth conditions

The influence of pH, temperature, anthracene con-centration and some hydrocarbons on the growth ofselected isolates was assessed using MM, three repli-cates. The autoclaved medium was adjusted to pH 5.0,6.0, 7.0, 8.0, 9.0 and 10.0 using predetermined amountsof filter-sterilized (0.22 m) 1 M HCl or 1M NaOH andincubated at 30 1C. The incubation temperatures usedwere 20, 25, 30, 35 and 40 1C and the anthraceneconcentrations were 0, 125, 250, 500, 1000 and2000 mg L�1. The effect of other hydrocarbons as solecarbon sources on growth of the isolates was evaluatedusing phenanthrene, pyrene, gasoline, diesel oil, ben-zene, toluene, xylene and ethylbenzene at a concentra-tion of 250 mg L�1 MM. The inocula, containing 5% ofthe total volume, were sampled in the logarithmic phasecultures in MM broth and incubated for 24 h withorbital shaking (150 rpm). After incubation for 24 h,1mL medium was diluted and 0.1 mL of 10�2, 10�3 and10�4 dilutions were plated on nutrient agar andincubated for 24 h at 30 1C in dark and colonies weredirectly counted and expressed as CFU mL�1.

2.5. PAH biodegradation

The efficiency of the selected isolates in anthra-cene, phenanthrene and pyrene degradation was indivi-

dually evaluated by gas chromatography (CG–MS).Isolates were inoculated in 50 mL MM containing250 mg PAHs L�1 (six replicates) and incubated at30 1C for 48 days with orbital shaking (150 rpm). Threereplicates were used for enumeration of bacteria(CFU mL�1). After incubation, the pH was reduced to2.0 in each flask containing MM, PAH and isolates,followed by three extractions with 20 mL dichloro-methane (EM Sciences, 499.8%) in a separationfunnel. Excess water was removed by adding sodiumsulfate (Shuttleworth and Cerniglia, 1996). Extractedmaterial was quantified in a gas chromatograph–massspectrometer (Agilent, Palo Alto, CA), GC model6890, mass selective detector model 5973 and a30 m� 0.25 mm� 0.25 mm DB5–5% phenyl methylsiloxane capillary column. The injector and transfer linetemperature were set at 290 1C, and the temperatureprogram was as follows: 1min at 40 1C, ramp to 220 1Cat 6 1C min�1, maintaining isotherm for1 min and rampto 300 1C at 15 1C min�1. A 0.2 mL aliquot was injectedat a split rate of 1:50. The mass selective detector wasoperated in the scan mode to obtain data for identifica-tion of hydrocarbon components. For internal control,deuterated phenanthrene was added to the sample at thesame PAH concentration (250 mg L�1) before injectioninto the chromatograph.

2.6. Surfactant detection

The surfactant produced by the selected isolates wasestimated by emulsification index and by the reductionof surface tension. The isolates were added to MM(50 mL) containing 250 mg anthracene L�1 (three repli-cates) and incubated at 30 1C for 48 days with orbitalshaking (150 rpm). Evaluation was performed by thepresence and absence of cells in the MM (cells wereremoved by centrifugation at 10,000 rpm for 30 min at4 1C). A 2 mL aliquot of MM was mixed with diesel oilin a PyrexTM glass tube (100 mm� 15 mm) using avortex for 2 min. After that, tubes were rested for 24 hbefore the volume and stability of the emulsion weremeasured. The surface tension of the supernatant wasmeasured after sample equilibration (1 h at 25 1C) usinga Lecont Du Nouy tensiometer. Distilled water was usedas control (69.2mN m�1).

3. Results and discussion

3.1. Anthracene-degrading bacteria isolation and

selection

When after 64 days of successive transfers undercontrolled conditions, 26 preselected bacterial isolateswere grown in MM with 250 mg anthracene L�1 as thesole carbon source (Fig. 1), morphological parameters

ARTICLE IN PRESSR.J.S. Jacques et al. / International Biodeterioration & Biodegradation 56 (2005) 143–150146

such as size, shape, color, bord and colony texture wereused for preliminary selection and identification.Twenty-three isolates were rod-shaped of which 18 wereGram negative. The remaining three isolates were cocci,two being Gram negative.

All 26 isolates grew well in MM containing anthra-cene, with the isolates yielding the largest and smallestnumbers of cells after incubation for 96 h being,respectively, 332C and 231B (Fig. 1). A new selectionwas made among the isolates, using kinetic parametersfor measuring the growth performance based on thesame initial inocula for all isolates and after incubationfor 96 h (Table 1). Isolates 222A, 312A and 332C werethe only ones that showed cell numbers above12 Log CFU as well as the highest generation time andnumber. These isolates were incubated for 600 hshowing a logarithmic increase in cell number until48 h followed by a linear growth (pseudo-stationaryphase) until the end of the experiment (Fig. 2). Theinitiation of pseudo-stationary growth by the isolates(before pseudo-stationary phase) was not related toinorganic nutrient limitation or accumulation of toxic

Time, hours0 12 24 36 48 60 72 84 96

Log

CF

U m

L-1

0

2

4

6

8

10

12

14 222A312A332C231B312E331A

Fig. 1. Growth of anthracene-degrading isolates with highest and

lowest growth rates at 30 1C in MM containing 250mg anthraceneL�1

(data are the mean of three replicates).

Table 1

Growth kinetics parameters of anthracene-degrading isolates with highest an

250mganthraceneL�1

Isolates LogCFU0 Log CFU1

222A 1.9070.06 12.3170.13

312A 2.6670.08 12.6370.13

332C 2.2670.08 12.8270.11

231B 1.0070.02 7.0070.11

312E 1.0070.04 7.2670.08

331A 2.3270.05 8.0470.11

Data are mean7SD of three replicates.

LogCFU0, initial CFUmL�1; LogCFU1, final CFUmL�1; K, growth rate;

substances since the isolates grew in the same mediumand were cultured under the same conditions withglucose replacing anthracene as an equal amountof carbon. The growth curves of the selected isolates(Fig. 1) are characteristically from bacteria growingunder shaking using PAH crystals as the sole carbonsource in amounts exceeding the aqueous solubility inthe MM. In the initial phase, exponential growthwas based on anthracene dissolved in the aqueousmedium, close to or at the maximum concentration. Inthis phase, population growth was controlled only bymetabolic activity, and not by anthracene availability,with a high biomass increasing as verified by Wicket al. (2001). Exponential growth ceased when anthra-cene consumption by the isolates exceeded the anthra-cene dissolution rate. Isolates reached a pseudo-lineargrowth phase when limited physically by the maximumdissolution of anthracene which is converted in cells.In the pseudo-stationary phase, the anthracene con-sumption of individual cells reached the maintenancelevel and consequently the growth ceased (Johnsenet al., 2005).

d lowest growth rates at 30 1C over 96 h in mineral medium containing

K G N

0.3170.009 3.2470.09 10.4170.14

0.3270.004 3.1570.04 9.9770.09

0.3270.005 3.1470.10 10.5670.13

0.2170.004 4.8270.08 6.0070.09

0.2270.004 4.6270.05 6.2670.08

0.2070.002 5.0570.08 5.7270.07

G, generation time; N, generation number.

Time, hours

0 100 200 300 400 500 600

log

CF

U m

L-1

0

1

2

3

4

5

6

7

8

222A312A332A

Fig. 2. Growth of Pseudomonas isolates in MM containing

250mg anthracene L�1. Data are mean of three replicates; error bars

represent SD.

ARTICLE IN PRESS

Temperature, °C

20 25 30 35 40

Log

CF

U m

L-1

0.0

4.0

5.0

6.0

7.0

222A312A332A

Fig. 3. Effect of temperature on growth of Pseudomonas isolates in

MM containing 250mg anthraceneL�1. Data are mean of three

replicates; error bars represent SD.

R.J.S. Jacques et al. / International Biodeterioration & Biodegradation 56 (2005) 143–150 147

Compounds like anthracene have low aqueoussolubility, which makes enzymic action of degradersdifficult. However, many microorganisms have somemechanisms that increase PAH availability. Amongthem, biosurfactant production possesses an uptakesystem with high affinity for PAH (increasing thedissolution flux of the substrate) and reduction of thedistance between cell and substrate via structures thatbind on the cell surface (Cameotra and Bollag, 2003). Toevaluate the existence of some mechanisms involved inthe capture of anthracene by the cell, biosurfactantproduction was verified by the reduction in surfacetension of the medium free of cells after growing inanthracene. Previously, results (data not shown) indi-cated that the selected isolates did not producesurfactant after incubation for 96 h. It is possible thatthe period of 4 days was not long enough to inducebiosurfactant production.

3.2. Anthracene-degrading isolates identification and

culture conditions

The three isolates were identified by the partial genesequencing of 16S rRNA, and all isolates belonged tothe genus Pseudomonas. This genus is one of the moststudied and reported as a PAH degrader as well as ofother organic recalcitrant pollutants (Zhang et al.,2004). Two isolates were identified as Ps. aeruginosa,i.e., isolates 312A (accession no. AY162139, 100%similarity) and 332C (AY631241, 84% similarity). Thisspecies has been isolated from places contaminated withhydrocarbons, and for which many authors havereported PAH degradation associated with biosurfac-tant production (Hwang and Cutright, 2002; Garcia-Junco et al., 2003; Straube et al., 2003). Although thereare few citations, the other species identified, Ps.

Citronellolis, has shown ability to degrade recalcitrantorganic compounds like poly(cis�1

� 4 isoprene), anatural polymer in the latex produced by Hevea

brasiliensis (Bode et al., 2000). When Bhattacharya etal. (2003) isolated 150 hydrocarbon-degrading bacteriafrom contaminated soils in India, Ps. citronellolis wasthe predominant species with 29 isolates showing abilityto degrade aliphatic and aromatic fractions present inoil sludge.

When, in order to determine the best growthconditions for the selected isolates, temperature, pH,anthracene concentration and hydrocarbon sourceswere evaluated, isolates showed similar behavior inresponse to temperature. The largest number of cells wasfound at 30 1C. Ps. citronellolis 222A showed the bestgrowth performance at 25 1C, but at 30 1C growth wasnot significantly greater than for the other two isolatestested (Fig. 3). Temperature influences biodegradationby increasing both microbial activity and PAH solubility(and hence PAH bioavailability), but above 30 1C

numbers were lower, indicating that the adverse effectof high temperature on the cell is more important thanthe increase in substrate availability. Since pH is aselective environmental factor affecting microbialdiversity and activity, controlling enzyme activity,transport process and nutrient solubility (Wong et al.,2002), its effect on the three isolates growing on250 mg anthracene L�1 as the sole carbon source wasassessed (Fig. 4). Ps. aeruginosa 312A showed thehighest log cell number of the cells at pH 7.0. Apartfrom this observation, the isolates showed similargrowth over the range pH 5.0–9.0. This could be takenas an indicator of good adjustment to this factor andconsequently of advantage in using those isolates forremediation. Wong et al. (2002) observed that pH 5.5affected phenanthrene degradation, but the growth ofthe isolates over the pH range 5.5–7.5 was notsignificantly different. The Burkholderia cocovenenans

BU-3 strain isolated by these authors could degradephenanthrene at a comparable or even faster rate thanother reported strains under neutral pH.

Evaluation of the effect of anthracene concentra-tion (Fig. 5) revealed that growth was strong at100 mg anthracene L�1 but, although cell counts in-creased with concentration up to 500 mg L�1, therewas no further increase at higher concentrations.However, cell growth was not inhibited at anyconcentration even after 24 h. Based on these results,250 mg anthracene L�1 was chosen for the degradationtests. In evaluation of other hydrocarbons as the solecarbon source after 24 h, the isolates grew well on MMcontaining phenanthrene, pyrene, gasoline and diesel oil(data not shown), but benzene, toluene, xylene andethylbenzene caused death of all three isolates. Thebiocidal action of the BTEX compounds on themicrobial cell is related to membrane solubilization.

ARTICLE IN PRESSR.J.S. Jacques et al. / International Biodeterioration & Biodegradation 56 (2005) 143–150148

Presumably BTEX present in gasoline and diesel oil hadno negative effect on these hydrocarbons.

As well as growing in medium containing phenan-threne and pyrene, quantification by gas chromatogra-phy after 48 days showed that the isolates degraded

pH

5 6 7 8 9 10

Log

CF

U m

L-1

0.0

4.0

5.0

6.0

7.0

8.0

222A312A332A

Fig. 4. Effect of pH on growth of Pseudomonas isolates in MM

containing 250mg anthraceneL�1. Data are mean of three replicates;

error bars represent SD.

Anthracene, mg L-1

0 250 500 750 1000 1250 1500 1750 2000

Log

CF

U m

L-1

0.0

3.0

4.0

5.0

6.0

7.0

222A312A332A

Fig. 5. Effect of anthracene concentration on growth of Psedomonas

isolates in MM. Data are mean of three replicates; error bars represent

SD.

Table 2

Degradation of the PAHs phenanthrene and pyrene by selected Pseudomonas

in mineral medium containing 250mgPAHL�1

Isolates Phenanthrene

Final concentration

(mgL�1)

Degradation (%) Degradation ra

(mgL�1 day�1)

222A 102.471.23 48.271.2 3.0870.09

312A 135.672.05 31.472.0 2.3870.12

332C 132.170.98 33.271.4 2.4570.07

Data are mean7SD of three replicates.

those PAHs (Table 2). There was greater degradation ofphenanthrene than pyrene (Table 2), the latter being acompound with four aromatic rings and a highermolecular weight. Ps. citronellolis 222A was the mostactive strain against both PAHs. Ps. aeruginosa 312Aand 332C were similar to each other in their ability todegrade phenanthrene and pyrene in soil. Hwang andCutright (2002) reported that bioaugmentation with Ps.

aeruginosa achieved a substantial increase on the totalbiodegradation of phenanthrene and pyrene in soil.

3.3. Anthracene biodegradation and surfactant

production

Anthracene was degraded to a greater extent thanphenanthrene and pyrene by the three isolates (Table 3),possibly because it had been used as the sole carbonsource during the earlier enrichment process. Onaverage, anthracene degradation by the Ps. citronellolis

isolate 222A was around 56% (Table 3, Fig. 6) after 48days, but Ps. aeruginosa 312A had the highest rate ofdegradation (3.90mg L�1 day�1) and degraded 71% ofthe anthracene. The other Ps. aeruginosa isolate (332C)degraded approx. 24.4%. Zhang et al. (2004) reportedthat a Pseudomonas isolate Wphe1 completely degradedphenanthrene and naphthalene but not anthracene.Only a Paraccocus sp. isolate Ophe1 was capable ofdegrading anthracene. However, the degradation rate ofthe Paraccocus sp. isolate was 7.8 times smaller thanthe degradation rate observed with our Pseudomonas

isolate 222A.When surfactant production was assessed, emulsifica-

tion was not detected, indicating that the isolates do notproduce high molecular weight surfactants as bioemul-sificants responsible by the formation of micro-emulsionon the MM. Despite this, reduction in surface tensionwas observed with all three isolates indicating theproduction of low molecular weight surfactant com-pounds (Cameotra and Bollag, 2003). The surfacetension of the MM used was 69.2 mN m�1, very closeto that of distilled water. The medium from Ps.

citronellolis 222A caused the greatest reduction withthe two Ps. aeruginosa at only 10.8 mN m�1. Ability to

isolates estimated by gas chromatography after incubation for 48 days

Pyrene

te Final concentration

(mgL�1)

Degradation (%) Degradation rate

(mgL�1 day�1)

133.873.05 36.872.2 2.4270.08

160.075.48 24.871.3 1.8770.06

154.872.33 26.973.4 1.9870.06

ARTICLE IN PRESS

Table 3

Degradation of anthracene and surfactant production by selected Pseudomonas isolates after incubation for 48 days in mineral medium containing

250mganthraceneL�1

Isolates Final concentration (mgL�1) Degradation (%) Degradation rate (mgL�1 day�1) Surface tension (mNm�1)

222A 96.472.33 56.571.3 3.2070.13 36.070.9

312A 62.871.89 71.172.6 3.9070.09 58.371.3

332C 167.675.63 24.471.4 1.7270.06 58.071.6

Data are mean7SD of three replicates.

Fig. 6. Chromatograms showing relative to initial anthracene concentration (a) degradation of the PAH by Ps. citronellolis 222A (b), Ps. aeruginosa

312A (c), and Ps. aeruginosa 332C (d). Deuterated phenanthrene was used as internal control.

R.J.S. Jacques et al. / International Biodeterioration & Biodegradation 56 (2005) 143–150 149

reduce surface tension, shown by the isolate 222A, couldinfluence the anthracene degradation. The bioavailabil-ity and biodegradation of PAHs can be increased withsurfactants. It has been reported that improved degra-dation is more significant when surfactants produced byPs. aeruginosa are added (Wong et al., 2004). To ourknowledge, the use of surfactants to increase anthracenedegradation by Ps. citronellolis has not been reported inthe literature. Zhang et al. (1997) showed that phenan-threne solubility in MM increased from 0.7 to 35 mg L�1

in the presence of a surfactant produced by apseudomonad increasing the mineralization rate. Amicroorganism is considered an efficient surfactantproducer if the surface tension could be reduced toaround 30–35 mN m�1 (Desai and Banat, 1997). So, it ispossible to view Ps. citronellolis 222A as a goodproducer. However, Bacillus subtilis produces a lipopep-tide surfactant, surfactin, that can reduce surface tensionof an aqueous solution to 27 mN m�1 (Mulligan, 2005).Although Ps. aeruginosa 312A showed the highestdegree of anthracene reduction (71.%) it produced onlya small reduction in surface tension (Table 3). Johnsen

and Karlson (2004) evaluated the strategy for increasingthe bioavailability of 22 PAHs by surfactant production,the authors considered formation of biofilms on thesurface of the PAH crystals and the production ofextracellular polymeric substances. It is possible that instrain 312A some of these mechanisms increase anthra-cene degradation.

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