ResearchArticle Methylobacterium populi VP2: Plant Growth ......ResearchArticle Methylobacterium...

Research ArticleMethylobacterium populi VP2 Plant Growth-PromotingBacterium Isolated from a Highly Polluted Environment forPolycyclic Aromatic Hydrocarbon (PAH) Biodegradation

Valeria Ventorino1 Filomena Sannino1 Alessandro Piccolo1

Valeria Cafaro2 Rita Carotenuto2 and Olimpia Pepe1

1 DIA-Dipartimento di Agraria Universita degli Studi di Napoli Federico II Via Universita 100 80055 Portici Italy2 Dipartimento di Biologia Universita degli Studi di Napoli Federico II Complesso Universitario Monte S AngeloVia Cintia 4 80126 Napoli Italy

Correspondence should be addressed to Olimpia Pepe olipepeuninait

Received 25 March 2014 Revised 28 May 2014 Accepted 10 July 2014 Published 3 August 2014

Academic Editor Wen-Jun Li

Copyright copy 2014 Valeria Ventorino 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

The use of microorganisms to accelerate the natural detoxification processes of toxic substances in the soil represents an alternativeecofriendly and low-cost method of environmental remediation compared to harmful incineration and chemical treatmentsFourteen strains able to grow on minimal selective medium with a complex mixture of different classes of xenobiotic compoundsas the sole carbon source were isolated from the soil of the ex-industrial site ACNA (Aziende Chimiche Nazionali Associate)in Cengio (Savona Italy) The best putative degrading isolate Methylobacterium populi VP2 was identified using a polyphasicapproach on the basis of its phenotypic biochemical and molecular characterisation Moreover this strain also showed multipleplant growth promotion activities it was able to produce indole-3-acetic acid (IAA) and siderophores solubilise phosphate andproduce a biofilm in the presence of phenanthrene and alleviate phenanthrene stress in tomato seeds This is the first report onthe simultaneous occurrence of the PAH-degrading ability by Methylobacterium populi and its multiple plant growth-promotingactivities Therefore the selected indigenous strain which is naturally present in highly contaminated soils is good candidate forplant growth promotion and is capable of biodegrading xenobiotic organic compounds to remediate contaminated soil alone andorsoil associated with plants

1 Introduction

The accumulation of large amounts of persistent chemicalsin the soil from urban industrial and agricultural activitiesis an increasingly important worldwide problem In Italythe ex-industrial site of ACNA (Aziende Chimiche NazionaliAssociate) in Cengio (Savona Italy) was contaminated bydifferent classes of organic compounds and is on the list ofnational priorities for environmental reclamation

Among the most common pollutants in contaminatedenvironments polycyclic aromatic hydrocarbons (PAHs) arefused-ring aromatic compounds that exist in nature as a

result of anthropogenic polluting events such as waste com-bustion industrial treatment and petroleum processing [1]PAHs have been recognised to be mutagenic carcinogenicand cytotoxic to humans and animals [2] therefore theirpresence in contaminated soils represents a significant riskto the environment due to their intrinsic chemical stabilitylower solubility and high recalcitrance to different types ofdegradation [3] For this reason the United States Environ-mental Protection Agency (US EPA) considers PAHs to bepriority pollutants for remediation

Numerous physical and chemical technologies such assoil washing have been developed to remove PAHs from the

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 931793 11 pageshttpdxdoiorg1011552014931793

2 The Scientific World Journal

environment and to remediate contaminated soils Howeverthese methods are expensive and only partially effective[4] In this context bioremediation appears to be a validenvironmentally friendly and cost-competitive approach forthe clean-up of PAH-polluted sites and to achieve perma-nent detoxification of those sites This appealing technologyis based on natural degradation carried out by in situstimulation of indigenous microorganisms present in thepolluted soils [5 6] andor by the inoculation of selectedmicroorganisms that are able to remove the contaminants bymineralising the PAHs andor forming metabolites that areless toxic than the original pollutants [7] In fact bacteriahave evolved several mechanisms to overcome the very lowwater solubility of PAHs such as bioemulsifier productionmembrane carriers and the ability to grow in biofilms onhydrophobic surfaces [8] In particular biofilms are assem-blages of surface-associated microbial cells that are enclosedin an extracellular polymeric matrix made primarily of apolysaccharide material [9] They are considered to be theprevailing microbial life form in most environments [10 11]and allow the bacteria to enjoy a number of advantagessuch as increased resistance to antimicrobial agents [12ndash15]However the use of strains isolated from other environmentsis not always effective because the efficiency of the inoculumdepends on several factors including the site conditions theindigenous microbial populations and the type concentra-tion bioavailability and toxicity of the pollutants present inthe soil [16] In addition when microorganisms are addedthe duration of assessment and biological process efficiencydepend on the evolution of the bacterial communities interms of composition and catabolic activity [17]

Recently the use of plant growth-promoting rhizobacte-ria (PGPR) associated with plants for environmental remedi-ation has emerged as a promising field although very fewfieldstudies have been performed [18] PGPR are able to benefitplant development using awide range ofmechanisms includ-ing synthesising compounds to promote plant growth andorincreasing the uptake of nutrients and acting as biocontrolagents by suppressing plant pathogens in the rhizosphereIn addition plant growth-promoting microorganisms couldcontribute to the remediation process via multiple modes ofaction [19] because these microorganisms can both degradeandor mineralise organic xenobiotic compounds allowingthem to serve directly as contaminant degraders [20] or incombination with plants [18 21] In fact synergistic actionof both the rhizosphere microorganisms and the plants canlead to increased availability of hydrophobic compoundsaffecting their removal andor degradation [22] In additionduringmicrobe-assisted phytoremediation the plant growth-promoting microbial strains could increase the tolerance ofplants to contaminants both enhancing plant germinationand root biomass accumulation through the biosynthesisof phytohormones [23] and degrading the xenobiotic com-pounds before they negatively impact the plants [24]

On the basis of these considerations indigenous plantgrowth-promoting microbial strains were isolated charac-terised and selected fromhighly contaminated environmentsto evaluate their potential use in bioremediation andor amicrobe-assisted phytoremediation project

2 Materials and Methods

21 Soil Description The soil sample was collected from thesite of ACNA (Aziende Chimiche Nazionali Associate) anindustrial area of Cengio (near Savona) in northern ItalyThe site was extremely polluted due to the irregular disposalof organic and inorganic contaminants on the surface andlower soil horizons since 1882 The pollution of the area hasintensified since the manufacturing of a range of organiccolorants that began in 1939 In 1999 due to the seriouscontamination of the surrounding soils and waters theACNA site was included in the list of national prioritiesfor environmental remediation The chemical and physicalproperties of the soil were previously reported by Conte etal [25]

22 Soil Contaminants Contaminated soil aqueous extract(CSAE) was obtained as previously reported [26] Brieflyaccording to this method a Soxhlet extraction system underreflux with an acetone119899-hexane (50 50 v v) mixture wasemployed The organic extracts were dried by rotavaporand dissolved in an acetonewater solution (5 95 v v)Then solid phase extraction (SPE) was performed Finallythe elutes were analysed through GCMS to evaluate theconcentration of the contaminants The soil contaminantsidentified in the GCMS chromatograms were previouslyreported [26]

23 Microbial Isolation and Enrichment Methods The soilsamples (20 g) were shaken for 30min in 180mL of quarter-strength Ringerrsquos solution (Oxoid Milan Italy) containing0324 g of tetrasodium pyrophosphate to detach the bacteriafrom the soil particles After the soil particles were allowedto settle for 15min the solution was diluted tenfold in seriesPotential PAH-degrading microorganisms were isolated byspreading the serial dilutions on a minimal selective solidmedium (MSSM) containing 1 natural nutrients (soilextract obtained from freshly collected meadow soil) [27]05 contaminated soil aqueous extract (CSAE) obtained asabove reported and 15 agar bacteriological (Oxoid) Theplates were incubated at 28∘C for 15 days

The isolated colonies that were able to grow by streakingon aminimal solidmedium containing 15CSAE as the solecarbon source were further selected

A serial enrichment strategy was carried out that inoc-ulated the selected microbial isolates in a minimal selectiveliquid medium (MSLM) containing increasing concentra-tions of CSAE (up to 50) with or without the addition ofnutrients (1 soil extract) The growth of the isolates in theliquid culturewas determined by a spread plate countmethodusing MSSM containing the same amount of CSAE (up to50)

24 Identification of the Selected Putative PAH-DegradingMicroorganisms The putative selected PAH-degrading bac-terial strains were identified using a polyphasic approach onthe basis of their phenotypic biochemical and molecularcharacterisation

The Scientific World Journal 3

A colony morphology analysis was carried out observingthe shape edge dimension elevation consistency and colourof the cells Catalase activity was detected by evaluatingthe effervescence production after dissolving the colony in3 hydrogen peroxide Oxidase activity was detected usingoxidase strips (Oxoid) A Gram reaction was performedusing the KOH test as described by Halebian et al [28] Themicromorphology of the cells was observed using an opticmicroscope (Eclipse E200 Nikon)

A carbon source utilisation test was performed usinga standard protocol as described by Green and Bousfield[29] and different carbon sources (fructose methane methy-lamine ethanol cyanate thiocyanate glucose and citrate)

Molecular identification was performed by sequencingthe 16S rRNA gene The total genomic DNA was extractedand purified by the InstaGene Matrix (Bio-Rad LaboratoriesMilan Italy) according to the supplierrsquos recommendationsApproximately 50 ng of DNA was used as a template for thePCR assay

The synthetic oligonucleotide primers fD1 (51015840-AGAGTT-TGATCCTGGCTCAG-31015840) and rD1 (51015840-AAGGAGGTG-ATCCAGCC-31015840) were used to amplify the 16S rRNA gene[30] The PCR mixture was prepared as reported by Alfonzoet al [31] The PCR conditions were performed as describedby Palomba et al [32]

The presence of the PCR products was verified byagarose (15wtvol) gel electrophoresis at 100V for 1 h andthe products were purified using a QIAquick gel extrac-tion kit (Qiagen SpA Milan Italy) and sequenced TheDNA sequences were determined and analysed as previ-ously reported [33] and they were compared to the Gen-Bank nucleotide data library using the Blast software atthe National Centre of Biotechnology Information website(httpwwwncbinlmnihgovBlastcgi) [34]

The sequence obtained has been deposited in the Gen-Bank database under accession number KF955558

Multiple nucleotide alignments of the nearly full-length16S rRNA gene of the bacterial strain and 32 type strainswere carried out using the Clustal W program [35] from theMEGA version 40 [36]The nucleotide sequences of the typestrains were retrieved from the Ribosomal Database Project(RDP httprdpcmemsuedu) The phylogenetic tree wasinferred using the neighbor-joiningmethodwith amaximumcomposite likelihood model in the MEGA4 program withbootstrap values based on 1000 replications

25 Biofilm Analysis The biofilm formation was analysed inM9 medium [37] supplemented with low-viscosity paraffin(LVP) as an organic hydrophobic phase Phenanthrene (PHE)dissolved in LVP was used as the model PAH compound totest the ability of the strain to respond to the presence ofaromatic carbon sources

The bacterial strain was grown inM9medium containing05 methanol (M9MeOH medium) as the carbon sourceThe cultures were incubated for 72 h at 28∘C and then dilutedin 10mL of M9MeOHmedium until they reached an opticaldensity of 001 OD

600 nm The cultures were supplementedwith 400120583L of low-viscosity paraffin (LVP) or 8mg of PHEdissolved in 400120583L of low-viscosity paraffin (LVPPHE) and

incubated in 50mLpolypropylene tubes at 28∘Cunder orbitalshaking (220 rpm) The control samples were prepared andincubated as described above in the absence of bacteria After96 h of incubation 500120583L of a mediumoil drop emulsionswere collected frozen at minus20∘C thawed and centrifuged at14000timesg for 10min at 4∘C to separate the mixtures into fourphases a cell pellet (planktonic cells) an aqueous phase adisk of gelatinous (not homogeneous) material and an oilphase

26 Cell Surface Hydrophobicity Test and Emulsification Prop-erties The ability of the bacterial cells to adhere to thehydrocarbons was used as a measure of their cell surfacehydrophobicity (CSH)The assay was performed as describedby Mattos-Guaraldi et al [38] with some modificationsBriefly the strain was grown inM9MeOHmediumwith andwithout LVP or LVPPHE as described above The controlsamples were prepared and incubated in the absence of theorganic phase The cultures were incubated for 72 h at 28∘Cunder orbital shaking (220 rpm) The water phase (3mL) ofeach culture was collected and centrifuged at 5000timesg for20min at 4∘C to separate the cells and the supernatant Thecells were washed twice in 10mM phosphate buffer pH 70and suspended in fresh M9 medium at a final optical densityat 600 nm of approximately 1 OD

600 nm The cell suspensionabsorbance at 600 nm was measured (119860) To test the CSH01mL of LVP was added to 1mL of the cell suspension andthe sample was vortexed vigorously for 2min The phaseswere allowed to separate for 15min at room temperatureTheoptical density at 600 nm of the cells remaining in suspensionin the water phase (119861) was then measured Hydrophobicity(CSH index) was calculated as the proportion of cells thatwere excluded from the water phase determined by thefollowing equation [(119860 minus 119861)119860] times 100 When the CSHindex was le20 the strains were considered hydrophilicstrains with CSH values ranging between 20 and 50 wereconsideredmoderately hydrophobic and highly hydrophobicstrains had CSH values ge50

To test the emulsification properties of the free cellmedium the supernatants were further centrifuged at18000timesg for 30min at 4∘C and used without further purifi-cation Typically 1mL of the supernatants and 1mL of LVPwere mixed vigorously for 2min in glass tubes (5 times 100mm)The emulsions were allowed to stand for 24 h at roomtemperatureThe emulsification index (119864

24) was calculated by

the following equation (height of emulsified layerheight ofthe total oil phase) times 100 An 119864

24value of 0 indicated no

emulsification whereas an 11986424value of 100 indicated 100

emulsification

27 Phenanthrene Degradation The hydrocarbon degrada-tion experiments were carried out in M9 medium sup-plemented with PHE crystals Typically 10mL of the M9medium was supplemented with 2mg of PHE dissolved in100 120583L of acetone to reach a final concentration of 200mg Lminus1After 30min at room temperature to allow for acetone volatil-isation the cultures were inoculated with 50ndash100 120583L aliquotsof cells grown inM9MeOHmedium to reach an optical den-sity of O D

600 nmsim001 Thecultures were incubated at 28∘C


under orbital shaking (220 rpm) The control samples wereprepared and incubated as described above in the absenceof bacteria After 20 days the samples were freeze-thawedand PHE was extracted with 5mL of hexane by vortexingthe mixtures for 5min The water and organic phases wereseparated by centrifugation at 18000timesg for 30min at 4∘CThe supernatant organic phase was diluted in hexane and thePHE concentration was determined spectrophotometricallyby measuring the absorbance at 250 nm A calibration curvewas constructedwith knownPHEconcentrationsThe abioticsamples were used to calculate the percentage of PHEdegradation

28 Evaluation of Plant Growth-Promoting Activities Theproduction of indole acetic acid (IAA) was quantified usingthe Salkowski colorimetric assay Briefly the strains wereinoculated in 5mL of Nutrient Broth (Oxoid) with and with-out L-tryptophan (2mg Lminus1 Sigma-Aldrich Milan Italy)incubated at 28∘C for 7 days and centrifuged at 6500 rpm for15min One millilitre of Salkowskirsquos reagent (2mL of 05MFeCl3with 49mL of 35 [vv] HClO

4) was added to the

supernatant (1mL) and incubated for 30ndash60min at roomtemperatureThe IAAconcentrationwas determined by spec-troscopic absorbance measurements at 530 nm according tothe standard curve

The Chrome Azurol S (CAS) agar assay was used toevaluate the ability to produce siderophores as described bySilva-Stenico et al [39] After 7 days of incubation at 28∘Can orange or yellow halo around the colony indicates theproduction of siderophores by the microorganisms

The ability to solubilise phosphates was assayed byinoculating the strains on Pikovskayarsquos agar (05 g Lminus1 ofyeast extract 10 g Lminus1 of dextrose 08 g Lminus1 of Ca

3(HPO

4)

05 g Lminus1 of (NH4)2SO4 02 g Lminus1 of KCl 01 g Lminus1 of MgSO

4

00001 g Lminus1 of MnSO4 00001 g Lminus1 of FeSO

4 and 15 g Lminus1

of agar bacteriological) [40] After incubating the plates at28∘C for 7 days the capacity of phosphate solubilisation wasindicated by clear zones around the colonies

Ammonia productionwas tested by inoculating the strainin 5mL of tryptone water (2) and incubating it at 28∘Cfor 7 days The presence of ammonia was detected by thedevelopment of a yellow-orange colour after adding a fewdrops of Nesslerrsquos reagent (Sigma-Aldrich) to the brothculture

All the tests were performed in triplicate

29 Plant Germination Test The surface of the tomato(Lycopersicon esculentum L var Jordan) seeds was sterilisedwith ethanol (70) for 10min and 25 (vv) sodiumhypochlorite with 005 Tween-20 for 10min followed bythree or more rinses in sterile H

2OThe seeds were planted in

sterile magenta boxes containing sterile sand irrigated withM9 medium that was supplemented with soil extract (10)with or without PHE (200mg Lminus1) The bacterial strain VP2was inoculated in magenta boxes to evaluate its ability toimprove the seedsrsquo germination under stress conditions Thestrain was grown at 28∘C for 48 h in M9MeOH (10mL)Then the culture was inoculated (1) in splashboard flasks

(200mL) containing M9MeOH and incubated under shak-ing for 48 h in aerobic conditions The cells were then har-vested by centrifugation washed and suspended in quarter-strength Ringerrsquos solution (Oxoid) until achieving microbialcounts of approximately 5 times 108 CFUmLminus1 (counting cham-berThoma 002 depth Hawksley UK 05ndash1 of the McFarlandscale) Finally the VP2 strain was inoculated in magentaboxes to reach a microbial concentration of approximately 1times 106 cells gminus1 The control tests were prepared in the absenceof PHE and the inoculum

The plants were cultured in a growth chamber undercontrolled conditions (constant temperature of 21 plusmn 1∘Cdarklight cycle of 168 h dminus1) for 21 days Every 7 days thegerminated seeds were counted which was defined as thegermination rate All the tests were performed in triplicate

3 Results

31 Isolation and Identification of Potential PAH-DegradingMicroorganisms A total of 14 indigenous microbial isolatesthat were able to grow in MSSM with CSAE (05) wereobtained from soil that was contaminated by different classesof organic compounds (PAHs) Further biotechnologicalinvestigations allowed for the selection of the VP2 isolateas the best putative PAH-degrading strain able to grow onMSSM with 15 CSAE as the sole carbon source

The ability of this strain to grow in the highly contami-nated habitat was enhanced using a serial enrichment strat-egy in minimal selective broth media (MSBM) containingincreasing concentrations of CSAE The bacterial strain wasable to grow in up to 50CSAEwith andwithout the additionof nutrients In fact after 7 days of incubation at 25∘CVP2 showed an increase of approximately 2 LogCFUmLminus1(721 plusmn 012 Log CFU mLminus1) compared to the beginning ofthe experiment (483 plusmn 013 Log CFU mLminus1) in the presenceof nutrients In the absence of nutrients the strain showed asmaller increase of up to 568plusmn010LogCFUmLminus1Moreoverthe selected VP2 strain was then identified using a polyphasicapproach on the basis of its phenotypic and molecularcharacterisation The VP2 colonies were creamy pink andcomposed of Gram-negative rod-shaped cells single or inrosettes Moreover this strain was strictly aerobic and testedpositive in the catalase and oxidase tests

The nearly full-length gene sequence (1409 bp) of thisbacterial strain showed an identity of 99 with differentMethylobacterium species using Blast software A phyloge-netic tree was constructed that included 32 type strains ofMethylobacterium speciesThe results of the neighbor-joininganalysis of the 16S rRNA sequences of the 32 type strains andVP2 are shown in the dendrogram depicted in Figure 1 Theclosest relatives of strain VP2 were Methylobacterium pop-uli Methylobacterium thiocyanatum and Methylobacteriumrhodesianum

BecauseMethylobacterium species are closely related andit is difficult to identify new isolates at the species levelbiochemical characterisation was performed to ascertain theidentity of strain VP2 VP2 was able to use fructose methanemethylamine and ethanol as its sole carbon sources but it


Methylobacterium thiocyanatum ALLSCN-P (U58018)

Methylobacterium populi BJ001 (AY251818)

Methylobacterium zatmanii DSM 5688 (AB175647)

Methylobacterium podarium FM4 (AF514774)

Methylobacterium suomiense NCIMB 13778 (AB175645)

Methylobacterium aminovorans JCM 8240 (AB175629)

Methylobacterium extorquens JCM 2802 (D32224)

Methylobacterium salsuginis MR (EF015478)

Methylobacterium rhodinum DSM 2163 (AB175644)

Methylobacterium isbiliense AR24 (AJ888239)

Methylobacterium nodulans ORS 2060 (AF220763)

Methylobacterium variabile GR3 (AJ851087)

Methylobacterium platani (EF426729)

Methylobacterium aquaticum GR16 (AJ635303)

Methylobacterium adhaesivum AR27 (AM040156)

Methylobacterium organophilum ATCC 27886 (AB175638)

Methylobacterium marchantiae JT1 (FJ157976)

Methylobacterium jeotgali S2R03-9 (DQ471331)

Methylobacterium persicinum 002-165 (AB252202)

Methylobacterium aerolatum 5413S-11 (EF174498)

Methylobacterium komagatae 002-079 (AB252201)

Methylobacterium gregans 002-074 (AB252200)

Methylobacterium hispanicum GP34 (AJ635304)

Methylobacterium brachiatum B0021 (AB175649)

Methylobacterium mesophilicum DSM 1708 (AB175636)

Methylobacterium phyllosphaerae CBMB27 (EF126746)

Methylobacterium fujisawaense DSM 5686 (AJ250801)

Methylobacterium oryzae CBMB20 (AY683045)

Methylobacterium tardum RB677 (AB252208)

Methylobacterium radiotolerans JCM 2831 (D32227)

0005

96

70

38

40

92

84

66

59

49

43

100

100

95

94

72

84

95

94

99

100

93

9697

36

66

48

56

34

Methylobacterium rhodesianum DSM 5687 (AB175642)

Methylobacterium iners 5317S-33 (EF174497)

Methylobacterium spp VP2 (KF955558)

Figure 1 A phylogenetic tree representing the relationship of the 16S rRNA gene sequences of strain VP2 and the type strains ofMethylobacterium sequences fromRDP Bootstrap values (expressed as percentages of 1000 replications) are given at the nodesThe sequenceaccession numbers used for the phylogenetic analysis are shown in parentheses following the species nameThe scale bar estimates the numberof substitutions per site

was unable to metabolise cyanate thiocyanate glucose andcitrate as shown byM populiBJ001T (Table 1) In contrastMthiocyanatum was able to use cyanate thiocyanate glucoseand citrate andM rhodesianum was unable to use methaneThe metabolic profile of VP2 was the same as that shown byM populi BJ001T and differed from theM thiocyanatum and

M rhodesianum profiles Based on these results the strainVP2 appears to be more similar toMethylobacterium populi

32 Biofilm Formation on Hydrophobic Surfaces To assessthe ability of M populi VP2 to grow in a sessile form onhydrophobic surfaces biofilm growth was performed in an


Table 1 Carbon source utilisation in strain VP2M populi BJ001T [41]M thiocyanatum [42] andM rhodesianum [41]

Carbon source Strain VP2 M populi BJ001T M thiocyanatum M rhodesianumFructose + + ND +Methane + + ND minus

Methylamine + + + +Ethanol + + ND +Cyanate minus minus + NDThiocyanate minus minus + NDGlucose minus minus + minus

Citrate minus minus + minus

+ growth minus no growth ND not determined

M9MeOH reach medium broth in the presence of LVP asthe hydrophobic phase To investigate the influence of PAHson biofilm formation PHE was dissolved in LVP M populiVP2 was able to form biofilms on the hydrophobic phasein the absence of PHE As shown in Figure 2 the culturesgrown in the presence of LVPPHE (Figure 2(a)) or LVP (datanot shown) induced a stable emulsification of the oil phasethat was completely covered with the extracellularmatrix andcells Freeze-thawing of the emulsified cultures led to thebreaking of the biofilm and the releasing of the oil phaseA disk of gelatinous (not homogeneous) material betweenthe aqueous and oil phase was observed (Figure 2(b)) Thismaterial could be constructed with the extracellular matrixthat was produced from M populi VP2 during its growth inthe presence of the hydrophobic phase

33 Cell Surface Hydrophobicity The propensity to grow asa biofilm on hydrophobic surfaces has been often relatedto the hydrophobicity of the bacterial cell surface which isbelieved to be predictive of biofilm formation However theCSH test highlighted the fact thatM populiVP2 did not showa propensity to adhere to LVP used as a hydrophobic phaseThe CSH index value determined for the bacteria grown inthe M9MeOH medium was 17 which was very similarto the value calculated in the presence of LVP (CSH = 2)suggesting that the presence of LVP alone was unable tochange the hydrophobic properties of the cell surface of Mpopuli VP2 It is worth noting that a significant increase ofhydrophobicity was observed in the presence of LVP-PHE(CSH = 12) This finding suggests that the cell surface ismodified in the presence of hydrocarbons such as PHE Thisfeature could be related to the uptake of this hydrophobicsubstrate during cell growth

Furthermore the emulsification test of LVP that wascarried out with the culture supernatants allowed for theformation of coalescent drops and a homogeneous oil phasewas separated from the water phase in a few minutes (119864

24=

0) A similar behaviour was observed when fresh mediumbroths were tested thus supporting the inability of the strainto produce bioemulsifiers in all the growth conditions tested

34 Phenanthrene Degradation PHE degradation was inves-tigated in liquid cultures supplemented with PHEmicrocrys-tals The amount of the aromatic hydrocarbon was measured

LVPBiofilm

Collapsedbiofilm

(a) (b)

Figure 2 Analysis of theMethylobacterium populi VP2 biofilm thatformed on LVPPHEThe sample was collected after 96 h of growthin M9MeOH liquid broth supplemented with LVP-dissolvedphenanthrene (a) the top phase represents LVP-phenanthreneembedded in a large amount of extracellular polymeric matrixharvested at the end of the growth (b) the top phase representsLVP-phenanthrene released after freeze-thawing and separation ofthe phases by centrifugation the extracellular matrix (collapsedbiofilm) forms a disk between the organic phase (top) and waterphase (bottom)

using a spectrophotometer and showed that M populi VP2degraded approximately 27 of the PHE in 20 days

35 Characterisation of Plant Growth Promotion ActivitiesThe selected strainM populi VP2 was also evaluated for itspotential plant growth promotion activities

The colorimetric assay showed the ability of this strain toproduce IAA In fact after 7 days of incubation the strainaccumulated the phytohormone in amounts ranging from138 plusmn 007 to 527 plusmn 005mgLminus1 in the absence and in thepresence of L-tryptophan respectively (Table 2)

Moreover VP2 was able to solubilise phosphate andto produce siderophores although in low quantities Inparticular the CAS agar assay showed the ability of the strainto produce siderophores after incubation a little yellow halo(14plusmn003mm) developed around the colony that was grownunder iron-limiting conditions In addition the M populiVP2 developed a clear halo with a diameter of 28 plusmn 006mm


Table 2 Plant growth-promoting activities ofMethylobacterium populi VP2

Microbial strain IAAa production (NB)(mg Lminus1 plusmn SD)

IAAb production (NB + TRP)(mg Lminus1 plusmn SD)

Siderophoresproduction

Phosphatesolubilization

Ammoniaproduction

M populi VP2 138 plusmn 007 527 plusmn 005 14 plusmn 003mm 28 plusmn 006mm mdashaIAA production in Nutrient Broth without L-tryptophanbIAA production in Nutrient Broth supplemented with L-tryptophan

Table 3 The effect of phenanthrene (PHE) andM populi VP2 on the germination rate () of tomato seedsa

Time (days) Controlb M populi VP2c PHEd PHE +M populi VP2e

7 d 1481 plusmn 641A 3703 plusmn 641A 0 plusmn 0C 0 plusmn 0C

14 d 1481 plusmn 641A 8889 plusmn 1111B 0 plusmn 0C 1852 plusmn 641A

21 d 1481 plusmn 641A 9259 plusmn 641B 0 plusmn 0C 7778 plusmn 1111BaThe values represent the means plusmn SD of three replicates of independent experiments Different letters after the values indicate significant differences (119875 le 001119905 test)bSeeds irrigated with M9 medium supplemented with soil extract (10)cSeeds irrigated with M9 medium supplemented with soil extract (10) and inoculated withM populi VP2 (106 cells gminus1)dSeeds irrigated with M9 medium supplemented with soil extract (10) and phenanthrene (200mg Lminus1)eSeeds irrigated with M9 medium supplemented with soil extract (10) and phenanthrene (200mg Lminus1) and inoculated withM populi VP2 (106 cells gminus1)

on a Pikovskayarsquos agar plate (Table 2) showing a weak abilityto solubilise phosphate

The strain VP2was not able to produce ammonia becauseno yellow-orange colour appeared in the broth culture afterthe addition of Nesslerrsquos reagent (Table 2)

36 Plant Test To determine whether the methylotrophicbacterial strain could stimulate seed germination the strainVP2 was inoculated in tomato seeds with and withoutPHE M populi VP2 significantly increased the growth anddevelopment of the seedlings both in the presence andin the absence of PHE compared to the non-inoculatedplants (Table 3) In fact after 14 days of inoculation theseeds showed a germination rate of 8889 plusmn 1111 whilethe non-inoculated controls showed a germination rate of1481 plusmn 641 after 7 days the control rate remained con-stant throughout the experiment (Table 3) Moreover in thepresence of PHE VP2 alleviated PHE phytotoxicity in thetomato seeds While no seeds germinated in the presence ofPHE after 7 days in the magenta box that was inoculatedwith M populi VP2 or in the non-inoculated control thatwas treated with PHE after 14 days the germination rate inthe magenta box was approximately 18 and increased up to7778 plusmn 1111 by the end of the experiment (after 21 days)This value can be compared to that achieved in the seedsinoculated with M populi VP2 and in the seeds that werenot treated with PHE (Table 3) No control seeds that wereexposed to PHE stress were able to germinate

4 Discussion

The use of microorganisms is the least expensive and mostecofriendlymethod used to remediate highly polluted soil Inthis study indigenous microbial strains able to use differentclasses of polluted organic compounds as their sole carbonsource were isolated characterised and selected for theirpotential use in the bioremediation of highly contaminatedsites

Among all the isolates the strain VP2 that was grown inMSSM with 15 CSAE was also able to grow in the presenceof up to 50CSAE To describe this strain at the species levelit was characterised at the molecular and phenotypic levelsby a polyphasic approach Because this strain was closelyrelated to different Methylobacterium species based on thenucleotide sequences of the 16S rRNAgene a biochemical testwas performed as described by Van Aken et al [41] Whencomparing the results of the metabolic profile of the VP2strain with literature data [41 42] theMethylobacterium sppVP2 strain appears to bemore similar to theM populi speciesbecause it was able to use methane [41]

M populi VP2 was selected as the best putative PAH-degrading strain There are many studies that describe thecapacity of several bacteria to mineralise or to degrade thehydrocarbons Bodour et al [43] and Andreoni et al [17]isolated and identified a phenanthrene-degrading culture thatcontained representative strains belonging to Methylobac-terium sp Recently the biodegradative potential of methy-lotrophic bacteria toward different polluted compounds suchas explosive methyl tert-butyl ether (MTBE) and PAHshas been reported [44ndash46] An interesting finding from thecurrent study is that the bacterial species M populi had notpreviously been reported to be PAH-degrading M populiVP2 was also able to grow with PHE as its sole carbon andenergy source with a decrease of 27 of the PHE in 20days The bacterial strategy utilised to grow on hydrophobiccompounds is often related to their ability to form biofilmsand produce bioemulsifiers to increase the water solubil-ity of the hydrophobic compounds [47] M populi VP2was unable to produce bioemulsifiers when grown in theM9MeOH medium in the presence of LVP or LVP-PHEthus indicating that emulsifying PHE is not the mechanismfor the efficient uptakedegradation of PHE in this strainHowever M populi VP2 that was grown in the M9MeOHmedium in the presence of LVP and LVP-PHE exhibitedthe ability to form biofilms on the hydrophobic phase thatwas not related to the LVP-PHE the emulsifying oil phase


during growth Similar behaviour was also observed for aNovosphingobium sp strain [37] that was able to emulsifydiesel oil by homogeneously coating oil drops with biofilmsTo the best of our knowledge the biofilm formation ofMethylobacterium on liquid organic phases has not yet beenreported even thoughMethylobacterium strains are known tobe able to grow in biofilms on living and non-living surfaces[48ndash52] Because a crucial event in biofilm formation is theadhesion of the bacteria to a surface and the hydrophobicityof the microbial surface plays a critical role in the adherenceof the bacteria [47] we investigated the hydrophobic featuresof M populi VP2 cell surface The assays performed usingLVP as a hydrophobic phase highlighted the fact that Mpopuli VP2 cell surface was essentially hydrophilic with CSHindex values lt20 However the CSH values increased from17ndash2 in the absence of PHE up to 12 when PHE wasdissolved in LVP This finding suggests that the cell surfacecan undergo changes that aremost likely related to the uptakeof PHE into the cellsTherefore the ability ofM populiVP2 toform biofilms on hydrophobic surfaces is not related to CSHbut it might be the only opportunity for this strain to interactwith hydrophobic substrates because it is unable to produceemulsifiers

However the correlation between CSH and biofilm for-mation has been found to be dependent on the specific straininvolved Bacterial cell hydrophobicity seems to have littleor no influence on biofilm formation in Methylobacteriumsp andMycobacterium mucogenicum that were isolated fromdrinking water [50] or on Staphylococcus epidermidis [53]and Candida strains [54] however a positive correlation wasobserved in 40 clinical Stenotrophomonas maltophilia strainstested [55] as well as in corynebacteria that was isolated aspart of the natural flora of human skin [56]

Methylobacterium sp has also been shown to inter-act symbiotically with different plant species of agronomicimportance [57] through biofilm formation M extorquenswas endowed with extensive plant-independent biofilm for-mation whereas the biofilm formation ofM mesophilicum isinduced by the plant root [51]

M populi VP2 showed multiple plant growth-promotingtraits In fact the use ofmicroorganismswith these character-istics is becoming increasingly attractive because they can beused in combination with plants to remediate contaminatedenvironments Several studies have been conducted on thebioremediation of organic contaminants by PGPR Huang etal [23] reported that the removal of PAHs by phytoremedi-ation with the use of PGPR was more rapid and completethan without PGPR because they likely played an importantrole in increasing plant tolerance to PAHs and promotingplant growth under stress In fact soil microorganisms canaffect plant growth through the production of hormonessiderophores and ammonia in addition to improving theplantsrsquo mineral supply of phosphorous

Different studies have reported plant growth-promotingabilities in aerobic methylotrophic bacteria They are con-sidered to be phytosymbionts and can participate in plantdevelopment via the biosynthesis of phytohormones nitro-gen fixation or by the suppression of ethylene biosynthesisin plants [58] although these activities have never been

reported in M populi species In fact a clear variabilityamongMethylobacterium strains in their capacity to producethe phytohormone IAA has been demonstrated by Omeret al [59] who reported that only 3 of 16 methylotrophicisolates tested produced IAA under the chosen conditionsThe strain M populi VP2 that was obtained in this studypresented several desirable features for PGPR The best ofthese beneficial features can be observed by the synthesis ofIAA This hormone was used because it is the most commonand most efficient among auxins According to Omer andcoworkers [59] the production of IAA increased in the pres-ence of L-tryptophan resulting in a value almost four timesgreater In fact this amino acid is a precursor of IAA andoccurs in plant exudates stimulating the synthesis of auxinsin aerobic methylotrophic bacteria [60] However severaltryptophan-independent pathways have been described thatwould explain the production of IAA by M populi VP2 inthe absence of this precursor [61] In addition the strain VP2demonstrated other plant growth-promoting activities it wasable to solubilise phosphate and to produce siderophoresTheability of microorganisms to convert insoluble phosphorusto a soluble form is a promising attribute for the selectionof bacteria capable of increasing available phosphorus in therhizosphere [61] The observed ability of strain M populiVP2 to solubilise calcium phosphate should be related to aprocess of acidification due to the production of organic acidsandor the secretion of H+ or chelating agents as reportedby Anastasio et al [40] Jayashree et al [62] describedthe ability of 13 pink-pigmented facultative methylotrophicstrains belonging to the genusMethylobacterium to solubilisephosphate they demonstrated a phosphate solubilisationindex ranging from 11 cm to 27 cmMoreover they observeda significant decline in the pH level coupled with an increasein acidity indicating that medium acidification is responsiblefor P-solubilisation

Another plant growth-promoting activity of M populiVP2was the production of siderophores which can influenceplant growth by binding Fe3+ and making the iron lessavailable for other microorganisms in the rhizosphere suchas plant pathogens [63 64] Members of the genus Methy-lobacterium which are frequently isolated as endophytesfrom citrus plants are also able to produce siderophores[65] Simionato et al [66] reported and characterised highproduction of siderophores by M mesophilicum (AR515and AR516 strains) and M extorquens (AR162 strain)Gholizadeh and Kohnehrouz [67] investigated the presenceof Fe-efficientMethylobacterium symbiosis inCelosia cristata

In addition the bacterial strain VP2 was found to havesignificant and consistent stimulatory effects on tomato seedgermination both in the presence and in the absence of PHEcontamination Madhaiyan and coworkers [68] reportedthe ability of pink-pigmented facultative methylotrophicbacteria (PPFMs) belonging to the genus Methylobacteriumto promote seed germination and plant growth as wellas to induce systematic resistance in rice Moreover thephytotoxicity of PAHs such as PHE is well recognised inaddition to their inhibitory effects on seed germination andthe growth of seedlings of different plants [69ndash72] In factPAH stress can exert adverse effects on growth and the


photosynthetic process in addition to causing morphologydeformation and changes in enzyme activities [73ndash76] Inparticular Wei and coworkers [72] reported that germi-nation energy and the germination rate decreased withincreasing PHE concentrations in wheat in particular thegermination rate was 625 with 005mgmLminus1 PHE and wassignificantly reduced by 68 at high PHE concentrations(02mgmLminus1) In our study the germination rate of thetomato seeds was completely inhibited by a PHE concen-tration of 02mgmLminus1 but this effect was alleviated by Mpopuli VP2 inoculation which led to a germination rate of778

5 Conclusions

Theeffectiveness of the selective ecological strategy employedin this study allowed for the isolation of indigenous strainsthat are naturally present in highly contaminated soils Thestrain M populi VP2 demonstrated multiple plant growthpromotion activities because it was able to produce IAA andsiderophores and solubilise phosphate interestingly VP2wasable to produce biofilms in the presence of phenanthreneand alleviated PHE stress in tomato seeds To our knowledgethis is first report describing the simultaneous occurrence ofmultiple plant growth-promoting activities and the potentialbiodegradation of xenobiotic organic compounds of indus-trial origin in M populi VP2 Therefore this selected straincould be a good candidate to employ in bioremediationstrategies to remediate contaminated agricultural soil andmay be useful in microbe-assisted phytoremediation

Conflict of Interests

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

Acknowledgments

This work was supported in part by Research ProjectldquoImplementation of Eco-Compatible Protocols for Agri-cultural Soil Remediation in Litorale Domizio-Agro Aver-sano NIPSrdquo (LIFE11ENVIT275mdashECOREMED) and byResearch project BIOFECTORmdashldquoResource Preservation byApplication of BIOefFECTORs in European Crop Produc-tionrdquo Collaborative Project Medium Scale Grant Agreementno 312117

References

[1] S K Samanta O V Singh and R K Jain ldquoPolycyclic aromatichydrocarbons environmental pollution and bioremediationrdquoTrends in Biotechnology vol 20 no 6 pp 243ndash248 2002

[2] H Sato and Y Aoki ldquoMutagenesis by environmental pollutantsand bio-monitoring of environmental mutagensrdquo Current DrugMetabolism vol 3 no 3 pp 311ndash319 2002

[3] M Alexander Biodegradation and Bioremediation AcademicPress San Diego Calif USA 1999

[4] W Pimda and S Bunnag ldquoBiodegradation of used motor oil bysingle and mixed cultures of cyanobacteriardquo African Journal ofBiotechnology vol 11 no 37 pp 9074ndash9078 2012

[5] R Margesin and F Schinner ldquoEfficiency of indigenous andinoculated cold-adapted soil microorganisms for biodegrada-tion of diesel oil in alpine soilsrdquo Applied and EnvironmentalMicrobiology vol 63 no 7 pp 2660ndash2664 1997

[6] W L Straube J Jones-Meehan P H Pritchard and W RJones ldquoBench-scale optimization of bioaugmentation strategiesfor treatment of soils contaminated with high molecular weightpolyaromatic hydrocarbonsrdquoResources Conservation and Recy-cling vol 27 no 1-2 pp 27ndash37 1999

[7] D L JohnsonK LMaguireD RAnderson and S PMcGrathldquoEnhanced dissipation of chrysene in planted soil the impact ofa rhizobial inoculumrdquo Soil Biology and Biochemistry vol 36 no1 pp 33ndash38 2004

[8] R J Grosser M Friedrich D M Ward and W P InskeepldquoEffect of model sorptive phases on phenanthrene biodegrada-tion different enrichment conditions influence bioavailabilityand selection of phenanthrene-degrading isolatesrdquo Applied andEnvironmentalMicrobiology vol 66 no 7 pp 2695ndash2702 2000

[9] I W Sutherland ldquoBiofilm exopolysaccharides a strong andsticky frameworkrdquoMicrobiology vol 147 no 1 pp 3ndash9 2001

[10] M E Davey and G A OrsquoToole ldquoMicrobial biofilms fromecology to molecular geneticsrdquo Microbiology and MolecularBiology Reviews vol 64 no 4 pp 847ndash867 2000

[11] R M Donlan ldquoBiofilms microbial life on surfacesrdquo EmergingInfectious Diseases vol 8 no 9 pp 881ndash890 2002

[12] J W Costerton P S Stewart and E P Greenberg ldquoBacterialbiofilms a common cause of persistent infectionsrdquo Science vol284 no 5418 pp 1318ndash1322 1999

[13] R M Donlan and J W Costerton ldquoBiofilms survival mecha-nisms of clinically relevant microorganismsrdquo Clinical Microbi-ology Reviews vol 15 no 2 pp 167ndash193 2002

[14] R Patel ldquoBiofilms and antimicrobial resistancerdquo ClinicalOrthopaedics and Related Research no 437 pp 41ndash47 2005

[15] L Chen and Y Wen ldquoThe role of bacterial biofilm in persistentinfections and control strategiesrdquo International Journal of OralScience vol 3 no 2 pp 66ndash73 2011

[16] M Megharaj B Ramakrishnan K Venkateswarlu N Sethu-nathan and R Naidu ldquoBioremediation approaches for organicpollutants a critical perspectiverdquo Environment Internationalvol 37 no 8 pp 1362ndash1375 2011

[17] V Andreoni L Cavalca M A Rao et al ldquoBacterial communi-ties and enzyme activities of PAHs polluted soilsrdquoChemospherevol 57 no 5 pp 401ndash412 2004

[18] X Zhuang J Chen H Shim and Z Bai ldquoNew advancesin plant growth-promoting rhizobacteria for bioremediationrdquoEnvironment International vol 33 no 3 pp 406ndash413 2007

[19] M A Amezcua-Allieri A Rodrıguez-Dorantes andJ Melendez-Estrada ldquoThe use of biostimulation andbioaugmentation to remove phenanthrene from soilrdquoInternational Journal of Oil Gas and Coal Technology vol3 no 1 pp 39ndash59 2010

[20] S Cherian and M M Oliveira ldquoTransgenic plants in phytore-mediation Recent advances and new possibilitiesrdquo Environ-mental Science and Technology vol 39 no 24 pp 9377ndash93902005

[21] W Liu J Sun L Ding Y Luo M Chen and C TangldquoRhizobacteria (Pseudomonas sp SB) assist phytoremediationof oily-sludge-contaminated soil by tall fescue (Testuca arundi-nacea L)rdquo Plant and Soil vol 371 no 1-2 pp 533ndash542 2013


[22] M Mackova B Vrchotova K Francova et al ldquoBiotransforma-tion of PCBs by plants and bacteria e consequences of plant-microbe interactionsrdquo European Journal of Soil Biology vol 43no 4 pp 233ndash241 2007

[23] X Huang Y El-Alawi D M Penrose B R Glick and BM Greenberg ldquoA multi-process phytoremediation system forremoval of polycyclic aromatic hydrocarbons from contami-nated soilsrdquo Environmental Pollution vol 130 no 3 pp 465ndash476 2004

[24] L Liu C Jiang X Liu J Wu J Han and S Liu ldquoPlant-microbeassociation for rhizoremediation of chloronitroaromatic pol-lutants with Comamonas sp strain CNB-1rdquo EnvironmentalMicrobiology vol 9 no 2 pp 465ndash473 2007

[25] P Conte A Agretto R Spaccini and A Piccolo ldquoSoil reme-diation humic acids as natural surfactants in the washings ofhighly contaminated soilsrdquo Environmental Pollution vol 135no 3 pp 515ndash522 2005

[26] F Sannino R Spaccini D Savy andA Piccolo ldquoRemediation ofhighly contaminated soils from an industrial site by employinga combined treatment with exogeneous humic substances andoxidative biomimetic catalysisrdquo Journal of HazardousMaterialsvol 261 pp 55ndash62 2013

[27] O Pepe V Ventorino and G Blaiotta ldquoDynamic of func-tional microbial groups during mesophilic composting of agro-industrial wastes and free-living (N

2

)-fixing bacteria applica-tionrdquoWaste Management vol 33 no 7 pp 1616ndash1625 2013

[28] S Halebian B Harris S M Finegold and R D Rolfe ldquoRapidmethod that aids in distinguishing gram-positive from gram-negative anaerobic bacteriardquo Journal of Clinical Microbiologyvol 13 no 3 pp 444ndash448 1981

[29] P N Green and I J Bousfield ldquoA taxonomic study of someGram-negative facultatively methylotrophic bacteriardquoMicrobi-ology vol 128 no 3 pp 623ndash638 1982

[30] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991

[31] A Alfonzo S Lo Piccolo G Conigliaro V Ventorino SBurruano and G Moschetti ldquoAntifungal peptides produced byBacillus amyloliquefaciens AG1 active against grapevine fungalpathogensrdquoAnnals ofMicrobiology vol 62 no 4 pp 1593ndash15992012

[32] S Palomba G Blaiotta V Ventorino A Saccone and O PepeldquoMicrobial characterization of sourdough for sweet baked prod-ucts in the Campania region (southern Italy) by a polyphasicapproachrdquo Annals of Microbiology vol 61 no 2 pp 307ndash3142011

[33] O Pepe S Palomba L Sannino et al ldquoCharacterization inthe archaeological excavation site of heterotrophic bacteria andfungi of deteriorated wall painting of Herculaneum in ItalyrdquoJournal of Environmental Biology vol 32 no 2 pp 241ndash2502011

[34] 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

[35] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[36] K Tamura J Dudley M Nei and S Kumar ldquoMEGA4 molec-ular evolutionary genetics analysis (MEGA) software version

40rdquo Molecular Biology and Evolution vol 24 no 8 pp 1596ndash1599 2007

[37] E Notomista F Pennacchio V Cafaro et al ldquoThe MarineIsolate Novosphingobium sp PP1Y Shows Specific Adaptationto Use the Aromatic Fraction of Fuels as the Sole Carbon andEnergy Sourcerdquo Microbial Ecology vol 61 no 3 pp 582ndash5942011

[38] A L Mattos-Guaraldi L C D Formiga and A F B AndradeldquoCell surface hydrophobicity of sucrose fermenting and non-fermenting Corynebacterium diphtheriae strains evaluated bydifferent methodsrdquo Current Microbiology vol 38 no 1 pp 37ndash42 1999

[39] M E Silva-Stenico F T H Pacheco J L M Rodrigues ECarrilho and M T Siu ldquoGrowth and siderophore productionof Xylella fastidiosa under iron-limited conditionsrdquo Microbio-logical Research vol 160 no 4 pp 429ndash436 2005

[40] M Anastasio O Pepe T Cirillo S Palomba G Blaiotta andF Villani ldquoSelection and use of phytate -degrading LAB toimprove cereal-based products bymineral solubilization duringdough fermentationrdquo Journal of Food Science vol 75 no 1 ppM28ndashM35 2010

[41] B Van Aken C M Peres S L Doty J M Yoon and JL Schnoor ldquoMethylobacterium populi sp nov a novel aer-obic pink-pigmented facultatively methylotrophic methane-utilizing bacterium isolated frompoplar trees (Populus deltoidesx nigra DN34)rdquo International Journal of Systematic and Evolu-tionary Microbiology vol 54 no 4 pp 1191ndash1196 2004

[42] A P Wood D P Kelly I R McDonald et al ldquoA novelpink-pigmented facultative methylotroph Methylobacteriumthiocyanatum sp nov capable of growth on thiocyanate orcyanate as sole nitrogen sourcesrdquo Archives of Microbiology vol169 no 2 pp 148ndash158 1998

[43] A A Bodour J Wang M L Brusseau and R M MaierldquoTemporal change in culturable phenanthrene degraders inresponse to long-term exposure to phenanthrene in a soilcolumn systemrdquo Environmental Microbiology vol 5 no 10 pp888ndash895 2003

[44] B Van Aken J M Yoon and J L Schnoor ldquoBiodegradation ofnitro-substituted explosives 246-trinitrotoluene hexahydro-135-trinitro-135-triazine and octahydro-1357-tet-ranitro-135-tetrazocine by a phytosymbiotic Methylobacteriumsp associated with poplar tissues (Populus deltoidestimes nigraDN34)rdquo Applied and Environmental Microbiology vol 70 no1 pp 508ndash517 2004

[45] J Li and J Gu ldquoComplete degradation of dimethyl isophthalaterequires the biochemical cooperation between Klebsiella oxy-toca Sc and Methylobacterium mesophilicum Sr Isolated fromWetland sedimentrdquo Science of the Total Environment vol 380no 1ndash3 pp 181ndash187 2007

[46] B Lalevic V Raicevic D Kikovic et al ldquoBiodegradationof MTBE by bacteria isolated from oil hydrocarbonsmdashcontaminated environmentsrdquo International Journal of Environ-mental Research vol 6 no 1 pp 81ndash86 2012

[47] M Rosenberg and S Kjelleberg ldquoHydrophobic interactions inbacterial adhesionrdquo Advances in Microbial Ecology vol 9 pp353ndash393 1986

[48] V Anesti J Vohra S Goonetilleka et al ldquoMolecular detectionand isolation of facultativelymethylotrophic bacteria includingMethylobacterium podarium sp nov from the human footmicroflorardquo Environmental Microbiology vol 6 no 8 pp 820ndash830 2004


[49] V Anesti I R McDonald M Ramaswamy W G Wade DP Kelly and A P Wood ldquoIsolation and molecular detectionof methylotrophic bacteria occurring in the human mouthrdquoEnvironmental Microbiology vol 7 no 8 pp 1227ndash1238 2005

[50] L C SimoesM Simoes andM J Vieira ldquoAdhesion and biofilmformation on polystyrene by drinking water-isolated bacteriardquoAntonie van Leeuwenhoek vol 98 no 3 pp 317ndash329 2010

[51] P B Rossetto M N Dourado M C Quecine et al ldquoSpecificplant induced biofilm formation in Methylobacterium speciesrdquoBrazilian Journal of Microbiology vol 42 no 3 pp 878ndash8832011

[52] T Yano H Kubota J Hanai J Hitomi and H Tokuda ldquoStresstolerance ofMethylobacteriumbiofilms in bathroomsrdquoMicrobesand Environments vol 28 no 1 pp 87ndash95 2013

[53] N Cerca G B Pier M Vilanova R Oliveira and J AzeredoldquoQuantitative analysis of adhesion and biofilm formation onhydrophilic and hydrophobic surfaces of clinical isolates ofStaphylococcus epidermidisrdquoResearch inMicrobiology vol 156no 4 pp 506ndash514 2005

[54] L J Douglas ldquoCandida biofilms and their role in infectionrdquoTrends in Microbiology vol 11 no 1 pp 30ndash36 2003

[55] A Pompilio R Piccolomini C Picciani D DrsquoAntonio VSavini and G Di Bonaventura ldquoFactors associated with adher-ence to and biofilm formation on polystyrene by Stenotro-phomonas maltophilia The role of cell surface hydrophobicityand motilityrdquo FEMS Microbiology Letters vol 287 no 1 pp 41ndash47 2008

[56] A K Kwaszewska A Brewczynska and E M SzewczykldquoHydrophobicity and biofilm formation of lipophilic skincorynebacteriardquo Polish Journal ofMicrobiology vol 55 no 3 pp189ndash193 2006

[57] M Madhaiyan S Poonguzhali S P Sundaram and T SaldquoA new insight into foliar applied methanol influencing phyl-loplane methylotrophic dynamics and growth promotion ofcotton (Gossypium hirsutum L) and sugarcane (Saccharumofficinarum L)rdquo Environmental and Experimental Botany vol57 no 1-2 pp 168ndash176 2006

[58] D N Fedorov N V Doronina and Y A Trotsenko ldquoCloningand characterization of indolepyruvate decarboxylase fromMethylobacterium extorquens AM1rdquo Biochemistry vol 75 no12 pp 1435ndash1443 2010

[59] Z S Omer R Tombolini A Broberg and B GerhardsonldquoIndole-3-acetic acid production by pink-pigmented facultativemethylotrophic bacteriardquo Plant Growth Regulation vol 43 no1 pp 93ndash96 2004

[60] E G Ivanova N V Doronina and Y A Trotsenko ldquoAerobicmethylobacteria are capable of synthesizing auxinsrdquoMicrobiol-ogy vol 70 no 4 pp 392ndash397 2001

[61] C M Ribeiro and E J B N Cardoso ldquoIsolation selectionand characterization of root-associated growth promoting bac-teria in Brazil Pine (Araucaria angustifolia)rdquo MicrobiologicalResearch vol 167 no 2 pp 69ndash78 2012

[62] S Jayashree P Vadivukkarasi K Anand Y Kato and SSeshadri ldquoEvaluation of pink-pigmented facultative methy-lotrophic bacteria for phosphate solubilizationrdquo Archives ofMicrobiology vol 193 no 8 pp 543ndash552 2011

[63] Z A Siddiqui ldquoPGPR prospective biocontrol agents of plantphatogensrdquo in PGPR Biocontrol and Biofertilization Z A Sid-diqui Ed pp 111ndash142 Springer Dordrecht The Netherlands2006

[64] G K Sahu and S S Sindhu ldquoDisease control and plantgrowth promotion of green gram by siderophore producing

Pseudomonas sprdquo Research Journal of Microbiology vol 6 no10 pp 735ndash749 2011

[65] P T Lacava M E Silva-Stenico W L Araujo et al ldquoDetec-tion of siderophores in endophytic bacteria Methylobacteriumspp associated with Xylella fastidiosa subsp paucardquo PesquisaAgropecuaria Brasileira vol 43 no 4 pp 521ndash528 2008

[66] A V C Simionato C Simo A Cifuentes et al ldquoCapillaryelectrophoresis-mass spectrometry of citrus endophytic bacte-ria siderophoresrdquo Electrophoresis vol 27 no 13 pp 2567ndash25742006

[67] A Gholizadeh and B B Kohnehrouz ldquoAmolecular evidence forthe presence of methylobacterial-type Fe siderophore receptorin Celosia cristatardquo Plant Soil and Environment vol 56 no 3pp 133ndash138 2010

[68] MMadhaiyan S PoonguzhaliM Senthilkumar et al ldquoGrowthpromotion and induction of systemic resistance in rice cultivarCo-47 (Oryza sativa L) by Methylobacterium spprdquo BotanicalBulletin of Academia Sinica vol 45 no 4 pp 315ndash324 2004

[69] M Kummerova and E Kmentova ldquoPhotoinduced toxicity offluoranthene on germination and early development of plantseedlingrdquo Chemosphere vol 56 no 4 pp 387ndash393 2004

[70] Y Song P Gong Q Zhou and T Sun ldquoPhytotoxicity assess-ment of phenanthrene pyrene and their mixtures by a soil-based seedling emergence testrdquo Journal of Environmental Sci-ences vol 17 no 4 pp 580ndash583 2005

[71] M J Smith T H Flowers H J Duncan and J Alder ldquoEffectsof polycyclic aromatic hydrocarbons on germination and sub-sequent growth of grasses and legumes in freshly contaminatedsoil and soil with aged PAHs residuesrdquo Environmental Pollutionvol 141 no 3 pp 519ndash525 2006

[72] H Wei S Song H Tian and T Liu ldquoEffects of phenanthreneon seed germination and some physiological activities of wheatseedlingrdquo Compets Rendus Biologies vol 337 no 2 pp 95ndash1002014

[73] XMei D Lin Y Xu YWu and Y Tu ldquoEffects of phenanthreneon chemical composition and enzyme activity in fresh tealeavesrdquo Food Chemistry vol 115 no 2 pp 569ndash573 2009

[74] I Oguntimehin F Eissa and H Sakugawa ldquoNegative effectsof fluoranthene on the ecophysiology of tomato plants (Lycop-ersicon esculentum Mill) fluoranthene mists negatively affectedtomato plantsrdquo Chemosphere vol 78 no 7 pp 877ndash884 2010

[75] G J Ahammed H-L Yuan J O Ogweno et al ldquoBrassi-nosteroid alleviates phenanthrene and pyrene phytotoxicityby increasing detoxification activity and photosynthesis intomatordquo Chemosphere vol 86 no 5 pp 546ndash555 2012

[76] G J Ahammed C Gao J O Ogweno et al ldquoBrassinosteroidsinduce plant tolerance against phenanthrene by enhancingdegradation and detoxification in Solanum lycopersicum LrdquoEcotoxicology and Environmental Safety vol 80 pp 28ndash36 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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Zoology


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GenomicsInternational Journal of

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Microbiology


environment and to remediate contaminated soils Howeverthese methods are expensive and only partially effective[4] In this context bioremediation appears to be a validenvironmentally friendly and cost-competitive approach forthe clean-up of PAH-polluted sites and to achieve perma-nent detoxification of those sites This appealing technologyis based on natural degradation carried out by in situstimulation of indigenous microorganisms present in thepolluted soils [5 6] andor by the inoculation of selectedmicroorganisms that are able to remove the contaminants bymineralising the PAHs andor forming metabolites that areless toxic than the original pollutants [7] In fact bacteriahave evolved several mechanisms to overcome the very lowwater solubility of PAHs such as bioemulsifier productionmembrane carriers and the ability to grow in biofilms onhydrophobic surfaces [8] In particular biofilms are assem-blages of surface-associated microbial cells that are enclosedin an extracellular polymeric matrix made primarily of apolysaccharide material [9] They are considered to be theprevailing microbial life form in most environments [10 11]and allow the bacteria to enjoy a number of advantagessuch as increased resistance to antimicrobial agents [12ndash15]However the use of strains isolated from other environmentsis not always effective because the efficiency of the inoculumdepends on several factors including the site conditions theindigenous microbial populations and the type concentra-tion bioavailability and toxicity of the pollutants present inthe soil [16] In addition when microorganisms are addedthe duration of assessment and biological process efficiencydepend on the evolution of the bacterial communities interms of composition and catabolic activity [17]

Recently the use of plant growth-promoting rhizobacte-ria (PGPR) associated with plants for environmental remedi-ation has emerged as a promising field although very fewfieldstudies have been performed [18] PGPR are able to benefitplant development using awide range ofmechanisms includ-ing synthesising compounds to promote plant growth andorincreasing the uptake of nutrients and acting as biocontrolagents by suppressing plant pathogens in the rhizosphereIn addition plant growth-promoting microorganisms couldcontribute to the remediation process via multiple modes ofaction [19] because these microorganisms can both degradeandor mineralise organic xenobiotic compounds allowingthem to serve directly as contaminant degraders [20] or incombination with plants [18 21] In fact synergistic actionof both the rhizosphere microorganisms and the plants canlead to increased availability of hydrophobic compoundsaffecting their removal andor degradation [22] In additionduringmicrobe-assisted phytoremediation the plant growth-promoting microbial strains could increase the tolerance ofplants to contaminants both enhancing plant germinationand root biomass accumulation through the biosynthesisof phytohormones [23] and degrading the xenobiotic com-pounds before they negatively impact the plants [24]

On the basis of these considerations indigenous plantgrowth-promoting microbial strains were isolated charac-terised and selected fromhighly contaminated environmentsto evaluate their potential use in bioremediation andor amicrobe-assisted phytoremediation project

2 Materials and Methods

21 Soil Description The soil sample was collected from thesite of ACNA (Aziende Chimiche Nazionali Associate) anindustrial area of Cengio (near Savona) in northern ItalyThe site was extremely polluted due to the irregular disposalof organic and inorganic contaminants on the surface andlower soil horizons since 1882 The pollution of the area hasintensified since the manufacturing of a range of organiccolorants that began in 1939 In 1999 due to the seriouscontamination of the surrounding soils and waters theACNA site was included in the list of national prioritiesfor environmental remediation The chemical and physicalproperties of the soil were previously reported by Conte etal [25]

22 Soil Contaminants Contaminated soil aqueous extract(CSAE) was obtained as previously reported [26] Brieflyaccording to this method a Soxhlet extraction system underreflux with an acetone119899-hexane (50 50 v v) mixture wasemployed The organic extracts were dried by rotavaporand dissolved in an acetonewater solution (5 95 v v)Then solid phase extraction (SPE) was performed Finallythe elutes were analysed through GCMS to evaluate theconcentration of the contaminants The soil contaminantsidentified in the GCMS chromatograms were previouslyreported [26]

23 Microbial Isolation and Enrichment Methods The soilsamples (20 g) were shaken for 30min in 180mL of quarter-strength Ringerrsquos solution (Oxoid Milan Italy) containing0324 g of tetrasodium pyrophosphate to detach the bacteriafrom the soil particles After the soil particles were allowedto settle for 15min the solution was diluted tenfold in seriesPotential PAH-degrading microorganisms were isolated byspreading the serial dilutions on a minimal selective solidmedium (MSSM) containing 1 natural nutrients (soilextract obtained from freshly collected meadow soil) [27]05 contaminated soil aqueous extract (CSAE) obtained asabove reported and 15 agar bacteriological (Oxoid) Theplates were incubated at 28∘C for 15 days

The isolated colonies that were able to grow by streakingon aminimal solidmedium containing 15CSAE as the solecarbon source were further selected

A serial enrichment strategy was carried out that inoc-ulated the selected microbial isolates in a minimal selectiveliquid medium (MSLM) containing increasing concentra-tions of CSAE (up to 50) with or without the addition ofnutrients (1 soil extract) The growth of the isolates in theliquid culturewas determined by a spread plate countmethodusing MSSM containing the same amount of CSAE (up to50)

24 Identification of the Selected Putative PAH-DegradingMicroorganisms The putative selected PAH-degrading bac-terial strains were identified using a polyphasic approach onthe basis of their phenotypic biochemical and molecularcharacterisation




















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Zoology





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Zoology





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Advances in

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Volume 2014




Enzyme Research



Microbiology




4) was added to the




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3 Results




































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96

70

38

40

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Volume 2014




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