RESEARCH ARTICLE

Influence of salinity and temperature on the activityof biosurfactants by polychaete-associated isolates

Carmen Rizzo & Luigi Michaud & Christoph Syldatk &

Rudolf Hausmann & Emilio De Domenico &

Angelina Lo Giudice

Received: 2 August 2013 /Accepted: 17 October 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Influence of different parameters on biosurfactant(BS) activity was carried out on strains that were isolated fromthe polychaetesMegalomma claparedei , Sabella spallanzaniiand Branchiomma luctuosum and additional 30 strains thatwere previously identified as potential BS producers fromcrude oil enrichments of the same polychaete specimens.The selection of BS-producing strains from polychaete naturalsamples was carried out by using standard screening tests. TheBS activity by each isolate was evaluated for the effect ofsalinity and temperature on emulsion production and surfacetension reduction, during incubation in mineral mediumsupplemented with tetradecane or diesel oil. All isolatesshowed a similar time course of BS activity, and the latterwas more influenced by salinity rather than temperature.Some of the BS producers belonged to genera that have not

(i.e. Citricoccus , Cellulophaga , Tenacibaculum andMaribacter ) or have poorly been (Psychrobacter , Vibrio ,and Pseudoalteromonas) reported as able to produce BSs.This is remarkable as some of them have previously beendetected in hydrocarbon-enriched samples. Results confirmthat filter-feeding polychaetes are an efficient source for theisolation of BS producers.

Keywords Biosurfactants . Filter-feeding organisms .

Salinity . Temperature .Megalomma claparedei . Sabellaspallanzanii . Branchiomma luctuosum

Introduction

Biosurfactants (BSs) have gained renewed interest in recentyears mostly because of their biodegradability and reducedtoxicity compared with synthetic surfactants (Satpute et al.2010; Pacwa-Płociniczak et al. 2011). In this context,biological matrices have been rarely considered for theisolation of BS-producing bacteria (Gandhimathi et al. 2009;Kiran et al. 2010; Rizzo et al. 2013) that have been mostlyobtained from hydrocarbon-contaminated water or soil.Sabellids (Polychaeta: Annelida) are sedentary worms thatare able to filter large volumes of waters to collect particlesand bacteria suspended in the bulk water for their foodrequirements (Licciano et al. 2005, 2007). They canpotentially accumulate different kinds of contaminants (suchas heavy metals, hydrocarbons and organochlorinatedcompounds), both in the soluble and in the particulate phases,from the environment. Thus, it is expected that associatedbacteria have to cope with the presence of contaminants inthe host tissues.

Hydrocarbons or other water-insoluble substrates areknown to induce BS production in many microorganisms(Radwan and Sorkhoh 1993), and in some cases, it was

Responsible editor: Robert Duran

Electronic supplementary material The online version of this article(doi:10.1007/s11356-013-2259-8) contains supplementary material,which is available to authorized users.

C. Rizzo : L. Michaud : E. De Domenico :A. Lo GiudiceDepartment of Biological and Environmental Sciences, University ofMessina, Viale Ferdinando Stagno d’Alcontrès 31, 98166 Messina,Italy

C. SyldatkInstitute of Process Engineering in Life Sciences, Section II:Technical Biology, Karlsruhe Institute of Technology (KIT),Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany

R. HausmannInstitute of Food Science and Biotechnology, Section BioprocessEngineering, University of Hohenheim, 70593 Stuttgart, Germany

A. Lo Giudice (*)Department of Biological and Environmental Sciences (DISBA),Università di Messina, Viale F. Stagno d’Alcontrès, 98166 Messina,Italye-mail: alogiudice@unime.it

Environ Sci Pollut ResDOI 10.1007/s11356-013-2259-8

observed that BS production started only when the solublecarbon source was consumed and the hydrocarbon wasavailable (Banat et al. 1991; Banat 1995).

In some cases, temperature causes alteration in thecomposition of the BS (Syldatk et al. 1985), while saltconcentration affects BS production depending on its effecton cellular activity (Abu-Ruwaida et al. 1991).

The kinetics of BS activity may either display a growth-associated or a stationary phase-associated character. In somecases, the addition of precursors to the growth medium hasbeen shown to enhance the BS production rate (Tulloch et al.1962; Margaritis et al. 1979; Cooper and Paddock 1983;Stuwer et al. 1987; Lee and Kim 1993).

Enhancement of BS activity by microorganisms wasobserved widely for bacteria isolated from terrestrialenvironment or from water and sediment samples (Haddadet al. 2009; Khopade et al. 2012a, b). However, there is only avery limited number of studies dealing with bacteria isolatedfrom marine benthic organisms (Gandhimathi et al. 2009;Kiran et al. 2010).

The aim of the present work was the study of the BS-production efficiency under different growth conditions bybacterial strains which were isolated from both polychaetespecimens and enrichment cultures.

Materials and methods

Study area

Lake Faro is a small coastal pond (0.263 km2), located at thenortheastern corner of Sicily, Italy (coordinates, 38°16′ N,15°38′ E), which features a funnel-shaped profile, with a steepsloping bottom that declines to a central basin reaching a depthof 29 m. The lake is connected via a shallow channel to theStraits of Messina, which separates the island of Sicily fromthe Italian peninsula. Another channel, which is silted up mostof the time, is artificially opened for a few days during thehottest summer period establishing a communication with theTyrrhenian Sea to allow water circulation into the lake (Saccàet al. 2008). Lake Faro is a meromictic basin, with a salinitythat seasonally varies from 34 to 38. The lake is alsocharacterised by anoxic and sulfidric waters.

Collection and preliminary treatment of samples

Sampling was performed as previously described (Rizzo et al.2013). Briefly, all samples were collected within a 10-m radiusin the Lake Faro at depths ranging from 0.6 to 0.8 m.Conductivity, temperature, pH and oxygen saturation valueswere measured on site using a portable multiparametric probe(CTD YSI 6600V2). Temperature and salinity values were

15.4 °C and 37.78, while pH and dissolved oxygenconcentration were 8.61 and 8.95 mg/L, respectively.

Adult specimens of the polychaete annelids Sabellaspallanzanii (Gmelin, 1791), Branchiomma luctuosum(Grube, 1870) and Megalomma claparedei (Gravier, 1906)were aseptically collected and immediately washedwith filter-sterilised natural seawater to remove transient and looselyattached bacteria and/or debris. Specimens were then placedinto individual sterile plastic bags containing filter-sterilisednatural seawater.

All samples were transported directly to the laboratory at4 °C for microbiological processing (within 2 h aftersampling).

Bacterial strains

Immediately upon return to the laboratory, organisms werewashed several times with filter-sterilised seawater andhomogenised in ice for 9 s by using Ultraturrax. Polychaetehomogenates were serially diluted by using filter-sterilisedseawater and 100 μL of each dilution was spread-plated intwo replicates on solidified ONR7a (1.5 % agar, w /v )(Dyksterhouse et al. 1995) that was supplied withhydrocarburic substrates as vapour by placing a sterile filterpaper disc containing 0.5 mL of filter-sterilised crude oil(ONR7a-C; Arabian Light, Sigma-Aldrich) or crystals ofpolyaromatic hydrocarbons (ONR7a-PAH; mixture of pyrene,phenanthrene, fluoranthen; Sigma-Aldrich) or biphenyl(ONR7a-BP, Sigma-Aldrich) in the Petri dish lid. Inoculatedsubstrate-free media in addition to sterile hydrocarbon-containing medium served as negative controls (Lo Giudiceet al. 2010). Plates were incubated at 28 °C for 15 days.Colonies were randomly selected from agar plates, pickedand subcultured three times under the same conditions.

Selection of BS-producing strains

A preliminary screening procedure was carried out to selectisolates for further analyses. Isolates were inoculated inmarine broth (pH 7–7.5; Difco) and incubated for 1 week at25 °C. At regular intervals of 48 and 120 h screening testswere performed as described below. When necessary (seebelow), cultures were preliminary centrifuged at 4,700 rpmfor 20 min at 4 °C, and only the obtained supernatants wereused. For each test, uninoculated medium was treated exactlyas the cultures and then used as negative control.

Emulsifying activity A 2-mL portion of the sample from eachculture was added to 2 mL of kerosene (Petroleum ether,Panreac) as test-oil and the mixture was vigorously vortexedfor 2 min. After 24 h, the emulsification index (E24) wascalculated by dividing the measured height of the emulsion

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layer by the total height of the mixture and multiplying by 100(Satpute et al. 2008).

Surface tension measurement The surface tension of the cell-free supernatant was determined with a digital tensiometerK10T (Krüss, Hamburg, Germany) by using Wilhelmy Platemethod (Tuleva et al. 2005).

Haemolytic activity Each culture was inoculated on thesurface of blood agar plates (BA; Sriram et al. 2011) andincubated for 48–72 h at 37 °C. Plates were then observedfor the presence of clearing halos around the spots, indicativeof BS production (Mulligan et al. 1984; Youssef et al. 2004).

Blue agar plate method The cetyltrimethylammoniumbromide (CTAB) agar plate method is a rapid screeningmethod for the detection of anionic BSs (Siegmund andWagner 1991). Mineral salts agar medium was prepared witha carbon source (glucose 2 %, w /v ) and CTAB(0.5 mg mL−1)-methylene blue (0.2 mg mL−1). Aliquots(5 μL) of each culture were spotted on the surface of CTABagar plates, incubated at 30 °C for 24–48 h, and then stored at4 °C for further colour development (24 h). A dark blue haloaround the colony was considered a positive result.

Thin-layer chromatography BS molecules were priorextracted as follows. Aliquots (1 mL) of cell-freesupernatants were acidified with 85 % phosphoric acid(final concentration 1 %, v /v ) to adjust pH of supernatantof about 2–3. BSs were extracted twice with ethyl acetate(Sigma-Aldrich) 1:1.25 by vigorous vortexing for 2 min.Then 1 mL of the upper phase was transferred two times toa 2-mL tube, and the ethyl acetate was evaporated at roomtemperature (Syldatk et al. 1985; Schenk et al. 1995;Hörmann et al. 2010).

The extract was characterised using analytical thin-layerchromatography (TLC), carried out on silica gel plates(stationary phase). One-millilitre aliquot of each crude BSextract was concentrated, resuspended in 10 μL of ethylacetate and separated on a silica gel plate using chloroform-methanol-acetic acid (65:15:2; Romil, Sps) as developingsolvent system with different colour-developing reagents.The sugar moieties were stained with anisaldehyde(anisaldehyde:sulphuric acid/glacial acetic acid, 0.5:1:50;Carlo Erba reagents; Romil, SpS) (Anandaraj andThivakaran 2010). The spots on TLC plate were developedby heating with a fan. The chromatograms of the extracts werecompared with the TLC pattern of a mixture of rhamnolipidswhich was prepared from Jeneil JBR 425 (JeneilBiosurfactants Company, Saukville, USA). For the standard,85 μL of JBR 425 were suspended in 1 mL of 0.1 M sodiumphosphate buffer, pH 7, and acidified with 10 μL ofconcentrated phosphoric acid. This mixture was extracted

with 1.333 mL of ethyl acetate and this ethyl acetate phasewas applied as TLC standard.

Strains who gave an E24 of ≥50 % and showed interestingspots on TLC plates were selected as potential positive strainsfor BS production, and deeply studied with further analyses.

PCR amplification of 16S rRNA genes

Single colonies of each strain were lysed by heating at 95 °Cfor 10 min. Amplification of 16S rRNA gene was performedwith a thermocycler (Mastercycler GeneAmp PCR-System9700 Applied Biosystem, USA) using Bacteria-specificprimer 27F (5′-AGA GTT TGA TC(AC) TGG CTC AG-3′)and primer 1385R (5′-CGG TGT GT(AG) CAA GGC CC-3′)(Rizzo et al. 2013). The reaction mixtures were assembled at0 °C and contained 5 μL DNA, 1 μL of each forward andreverse primer (10 μM), 0.5 μL of dNTP mix (10 mM; GEHealthcare, Buckinghamshire, UK), 2.5 μL of reaction buffer10× (containing 15 mM of MgCl2), 0.125 μL of polymerase(5 U mL−1; Hot Star Taq™ Qiagen, Hilden, Germany) andsterile distilled DNA-free water to a final volume of 25 μL.Negative controls for DNA extraction and PCR setup(reaction mixture without a DNA template) were also usedin every PCR run. The PCR program was as follows: a firststep of 15 min at 95 °C for the polymerase activation,followed by 30 cycles of 1 min at 94 °C for denaturation,annealing phase of 1 min at 55 °C, elongation phase of 1 minand 30 s at 72 °C, followed by a final elongation at 10 min at72 °C.

The results of the amplification reactions were analysed byagarose gel electrophoresis (1 %, w /v ) in TAE buffer (0.04 MTris-acetate, 0.02 M acetic acid and 0.001 M EDTA),containing 1 μg mL−1 of ethidium bromide.

Sequencing and analysis of 16S rRNA genes

Sequencing was carried out at the GATC Biotech Laboratory(Konstanz, Germany). Next relatives of isolates weredetermined by comparison to 16S rRNA gene sequences inthe NCBI GenBank and the EMBL databases using BLAST,and the ‘Seqmatch’ and ‘Classifier’ programs of theRibosomal Database Project II (http://rdp.cme.msu.edu/)(Altschul et al. 1997).

Sequences were further aligned using the program ClustalW (Thompson et al. 1994) to the most similar orthologoussequences retrieved from database. Each alignment waschecked manually, corrected and then analysed using theneighbour-joining method (Saitou and Nei 1987) accordingto the model of Jukes–Cantor distances. A Phylogenetic treewas constructed using the Molecular Evolutionary GeneticsAnalysis 5 software (Kumar et al. 1993). The robustness of theinferred trees was evaluated by 400 bootstrap re-samplings.

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Nucleotide sequence accession numbers

Nucleotide sequences have been deposited in the NCBIGenBank database under the accession numbers KF032912-KF032929.

Improvement analyses

Improvement analyses were carried out on strains that wereisolated from M. claparedei , S. spallanzanii and B.luctuosum , and additional 30 strains that were previouslyobtained from crude oil enrichments of the same polychaetespecimens and then selected using the same screeningprocedures reported above (Table S1; Rizzo et al. 2013).

Selection of optimal medium and carbon source for bacterialgrowth

The first tested parameter was the carbon source. All isolateswere inoculated in two different mineral media, Bushnell Haas(BH; Difco) and mineral salt medium (MSM; Bodour et al.2004) which were amended with diesel oil or tetradecane (seebelow). The composition of the MSM was as follows (finalvolume 1 L): NaNO3, 2.5 g MgSO4·7H2O, 0.4 g; NaCl, 1 g;KCl, 1 g; CaCl2·2H2O, 0.05 g; and H3PO4 (85 %), 10 mL;1 mL solution B, composed as follows (final volume 100mL):FeSO4·7H2O, 50 mg; ZnSO4·7H2O, 150 mg; MnSO4·H2O,150 mg; H3BO3, 30 mg, CaCl2·6H2O, 15 mg; CuSO4·5H2O,15 mg; and NaMo2O4·2H2O, 10 mg.

Both media are based on different ion concentrations. InBH and MSM, the nitrogen source is provided in the form ofammonium nitrate and sodium nitrate, respectively, while thephosphor is provided as phosphate mono/potassium, andphosphoric acid. At each medium NaCl was added to a finalconcentration of 30 g L−1. Both media were sterilised byautoclaving at 121 °C for 15 min.

Isolates were cultured in BH and MSM and incubated at25 °C under shaking (120 rpm) with two different carbonsources (diesel oil or tetradecane, 2 %, v /v ), to test both theinfluence of the mineral composition and the carbon source onthe bacterial growth. Each culture was monitored for twoweeks at intervals of 48 h, by measuring the optical densityat 580 nm (OD580) with a spectrophotometer (UV-mini-1240,Shimadzu).

Influence of NaCl concentration and temperature on BSactivity

Kinetics studies were carried out in 250 mL shake flask byinoculating bacterial isolates in 60mL of the preferred mineralmedium (BH or MSM) supplemented with the optimalhydrocarbon source (tetradecane or diesel oil). Cultures wereincubated in a rotary shaker at two different temperatures (15

and 25 °C) and salinity values (0 and 3 % NaCl, w /v ) tostudy the influence of these physical parameters on BSactivity. Each culture was constantly monitored for about15 days at 48 h intervals by measuring E 24 detection(Satpute et al. 2008) and surface tension measurement asit was described above.

Additionally, a preliminary emulsification test wascarried out in to establish the emulsification ability of theisolates. Briefly, an aliquot (500 μL) of each culture wasvortexed for 2 min with an equal amount of kerosene(although it can be replaced with other hydrocarboncompounds; 1:1). After about 1 min stabilisation the ratiobetween the emulsion layer and the total culture height wasmeasured and expressed as a percentage (Christova et al.2004).

Results

Preliminary selection of BS-producing strains

A total of 96 strains were directly isolated from polychaetespecimens (38, 30 and 28 from B. luctuosum , S. spallanzaniiand M. claparedei , respectively). The first preliminaryscreening allowed selecting 18 isolates as potential BS-producers (Table 1). The majority of them were obtained fromS. spallanzanii (nine isolates) and B. luctuosum (sevenisolates). The E 24 index ranged from 10 to 76 % (S.spallanzanii strain (Ss)91 after 48 and 120 h incubation,respectively), with values that resulted generally higher aftera 120 h incubation. No direct correlation was observedbetween optical density and E24 values. All the producedemulsions remained stable up to 1 month.

Surface tension generally remained stable during all thescreening period with values that ranged from 54.2 (B.luctuosum strain (Bl)52 after 48 h incubation) to 68.6 mN/m(Bl39 after 48 h incubation).

On TLC plates, yellow-orange spots were observed afterstaining with anisaldehyde. All examined extracts showed asimilar profile on TLC plates. The retention factor values of allspots ranged from 0.7 to 0.76.

Phylogenetic identification of BS-producing strains

The comparative sequence analysis of isolates indicated thatthe 18 potential BS producers were closely related to knownbacteria (16S rRNA gene similarity, ≥97 %). Isolates weremainly affiliated to the Cytophaga–Flavobacteria–B a c t e ro i d e t e s g r o u p o f B a c t e ro i d e t e s a n dGammaproteobacteria (seven isolates per group). Threeisolates were affiliated to the Actinobacteria and only one tothe Firmicutes (Table 2). In particular, among theBacteroidetes, four strains were affiliated to Cellulophaga

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spp. (Ss85, Ss88, Ss91 and M. claparedei strain (Mc)108),one to Tenacibaculum sp. (Mc99), and two to Maribacterspp. (Ss71 and Ss79). The Gammaproteobacteria resulted

mainly affiliated to the genus Pseudoalteromonas (Ss89,Ss86, Bl46 and Bl65). The genera Psychrobacter (Bl39) andVibrio (Bl49) were represented each by a single isolate.

Table 1 Results from thepreliminary screening for the 18isolates that were selected aspotential biosurfactant producers

E2448 emulsifying activity after a

48-h incubation, E24120

emulsifying activity after a 120-hincubation, ST48 surface tensionafter a 48-h incubation, ST120

surface tension after a 120-hincubation, TLC thin-layerchromatography, Bl B, luctuosumstrain, Ss S. spallanzanii strain,McM. claparedei strain, #ONR7a

C ONR7a plus crude oil, ONR7a

PAH ONR7a plus a mixture ofpyrene, phenanthrene andfluoranthen, ONR7a BP ONR7a

plus biphenyl, ONR7a, ONR7a

without carbon source

Strain E24 (%) ST (mN/m) TLC Origin Isolation medium

E2448 E24

120 ST48 ST120

Bl39 – 60±3.54 68.6 64.5 + B. luctuosum ONR7a BP

Bl46 – 59.2±0.57 58.2 58.2 + B. luctuosum ONR7a

Bl49 – 62.9±2.05 57.7 56.8 + B. luctuosum ONR7a PAH

Bl52 15±3.54 66.6±1.13 54.2 57.2 + B. luctuosum ONR7a PAH

Bl54 43.3±1.20 63.3±1.20 59.7 59.1 + B. luctuosum ONR7a PAH

Bl55 17.7±1.63 60±3.54 58.9 58.7 + B. luctuosum ONR7a PAH

Bl65 20±3.54 56.6±2.40 57.2 57.8 + B. luctuosum ONR7a C

Ss67 30±3.54 56.6±2.40 56.2 58.4 + S. spallanzanii ONR7a

Ss71 12.5±1.77 59.2±0.57 60.7 58.2 + S. spallanzanii ONR7a

Ss76 70±3.54 63.3±1.20 62.5 64.7 + S. spallanzanii ONR7a

Ss79 52±2.12 20±3.54 55.2 55.8 + S. spallanzanii ONR7a PAH

Ss85 12.5±5.30 60±3.54 57.6 60.1 + S. spallanzanii ONR7a BP

Ss86 55.2±0.14 50±3.54 67.2 68.4 + S. spallanzanii ONR7a BP

Ss88 60±3.54 18.5±1.08 56.4 62.5 + S. spallanzanii ONR7a C

Ss89 62.8±1.56 23.3±3.20 62.3 72.7 + S. spallanzanii ONR7a C

Ss91 10±1.41 76±1.41 68 64.3 + S. spallanzanii ONR7a C

Mc99 50±3.54 73±1.00 59.5 59.1 + M. claparedei ONR7a

Mc108 64±1.71 59.2±0.4 59.1 59.7 + M. claparedei ONR7a C

Table 2 Phylogenetic affiliation of isolates from Branchiomma luctuosum , Sabella spallanzanii and Megalomma claparedei

Phylum or class Strain Origin Next relative by GenBank alignment (AN, organism) Sim (%)

GAM Bl39 B. luctuosum JF273871, Psychrobacter sp. TB2 99

Bl46 B. luctuosum JN578479, Pseudoalteromonas sp. H9 99

Ss86 S. spallanzanii NR_026218, Pseudoalteromonas sp. strain IAM 12927 99

Ss89 S. spallanzanii EU195931, Pseudoalteromonas sp. P102 98

Bl65 B. luctuosum FR744867, Pseudoalteromonas sp. A2B10 98

Bl49 B. luctuosum JQ083317, Vibrio sp. strain FA97 99

Ss76 S. spallanzanii EU195925, Alteromonadaceae bacterium P120 16S 99

BAC Ss71 S. spallanzanii AB526333, Maribacter sp. JAM-BA06 99

Ss79 S. spallanzanii AB526333, Maribacter sp. JAM-BA06 98

Ss85 S. spallanzanii AB681016, Cellulophaga sp. 99

Ss88 S. spallanzanii AB681016, Cellulophaga sp. 99

Ss91 S. spallanzanii AB681016, Cellulophaga sp. 97

Mc108 M. claparedei AB681016, Cellulophaga sp. 99

Mc99 M. claparedei AM746477, Tenacibaculum sp. 98

ACT Bl52 B. luctuosum EU305672, Citricoccus sp. FS24 99

Bl54 B. luctuosum EU305672, Citricoccus sp. FS24 98

Bl55 B. luctuosum EU305672, Citricoccus sp. FS24 98

FIR Ss67 S. spallanzanii JN251751, Staphylococcus sp. CRP7 99

ACT Actinobacteria , FIR Firmicutes , GAM Gammaproteobacteria , BAC CFB group of Bacteroidetes , AN accession number, Sim similarity, Ss S.spallanzanii strain, Bl B. luctuosum strain, Mc M. claparedei strain

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Strains grouping within the Actinobacteria were all affiliatedto the genus Citricoccus , while the only strain belonging tothe Firmicutes was found to be affiliated to the genusStaphylococcus (Ss67).

Selection of optimal medium and carbon source for bacterialgrowth

With regards to the 30 isolates from enrichment cultures(listed in Table S1), based on OD580 measures, among theCFB group of Bacteroidetes members of the genera Joostellaand Cellulophaga showed a good growth on both media andcarbon sources, although Cellulophaga members seemed toprefer diesel oil as a carbon source (Table 3).

Among theGammaproteobacteria , Pseudomonas spp. A6and A14 and Idiomarina sp. A19 showed satisfactory growthin all growth conditions. Conversely, Pseudomonas spp. A18and A45 preferred diesel oil as a carbon source, in bothmineral media. Alcanivorax spp. A52 and A53 optimallygrew in BH in the presence of tetradecane. Cobetia sp. A20preferred diesel oil as carbon source in both culture media,whileMarinobacter sp. A1 achieved optimal values of opticaldensity only during incubation in BH supplemented withtetradecane.

Among the Alphaproteobacteria , Thalassospira spp. A46and A57 did not show satisfactory growth in the presence ofhydrocarbons. Pseudovibrio sp. A27 showed betterperformance when grown on diesel oil. Cohaesibacter sp.A25 recorded adequate growth in all incubation conditions.

With regards to the 18 isolates from polychaete specimens,a scarce ability to grow in the presence of hydrocarbons wasgenerally observed (Table 3). Strains generally seemed toprefer BH as optimal medium, except for Pseudoalteromonassp. Bl46 and Cellulophaga sp. Ss85, which grew only inMSM supplemented with diesel oil.

Except for Tenacibaculum sp. Mc99 that grew well in BHin the presence of both hydrocarbon sources, all strainsgenerally seemed to prefer diesel oil for their growth.

In particular, any growth was recorded for Vibrio sp. Bl49,Psychrobacter sp. Bl39 and Citricoccus spp. Bl52 and Bl55on tetradecane. Conversely, Citricoccus sp. Bl54 was able togrow only in BH in the presence of tetradecane.

In case of optimal growth in both media and carbonsources, those conditions, at which individual strains reachedfaster the exponential phase, were chosen.

Influence of NaCl concentration and temperature on BSactivity

To investigate the influence of NaCl and temperature on BSactivity, isolates were grown in the optimal medium in thepresence of the preferred carbon source. The BS activity wasdetermined by monitoring the production of emulsions and

stable emulsions, and the surface tension reduction. All thetested strains positively responded to each test. However, onlyresults obtained from strain belonging to genera that havebeen poorly reported in literature in relation to BS production,or results that highlight a great strength or speediness ofinterfacial activity will be showed (Fig. 1a–f).

Overall, the BS activity was mainly influenced by theconcentration of NaCl (3 %, w /v ) in the culture medium,whereas temperature appeared to have minor influence on it.

With regards to the 30 isolates from enrichment cultures,among the Bacteroidetes the BS production by Joostellaaffiliates was analysed in BH amended with tetradecane,except for the strains Joostella spp. A9 and A22, incubatedin the presence of diesel oil. Joostella spp. A15, A24, A29 andA30 were not able to produce stable emulsions or emulsionsin the absence of NaCl. Conversely, they showed emulsifyingactivity in the presence of salt, reaching E24 values that rangedfrom 12.5 (Joostella sp. A15, 25 °C after 48 h) to 72.5 %(Joostella sp. A30, 25 °C after 240 h). Joostella spp. A3, A8,A9 and A22 were able to produce low percentages of stableemulsion in the absence of NaCl, but the value increased in thepresence of salt. Among Joostella affiliates, the strain A8gave the best results (Fig. 1a). It recorded the maximum valueof E24 (72.5 %) after 240 h incubation at 15 °C with NaCl inthe medium, compared with a minimum value of 5% obtainedfor the same isolate after 192 h incubation at 25 °C and 0 %NaCl. The same strain strongly reduced the surface tension(35.6 units from 67.1 to 31.5 mN/m) when growing at 15 °Cwith NaCl in the medium, followed by Joostella spp. A3 andA22 that determined a reduction of the surface tension of33.65 (from 66.5 to 32.85 mN/m) and 21.45 units (from51.5 to 30.05 mN/m), respectively, during incubation at15 °C in the presence of NaCl. Finally, Joostella spp. A15,A24, A29 and A30 strongly reduced the surface tension(>20mN/m) when incubated at 25 °C in the presence of NaCl.

Joostella sp. A22 grew well at all growth conditions,although it appeared to be more rapid and effective in thepresence of salt. The emulsifying capacity appeared after 48 hat 25 °C, with a maximum E24 value of 54%, and after 96 h at15 °C, with a maximum of 55 % and the maximum surfacetension reduction.

Contrary to Joostella spp., isolates that belonged to thegenus Cellulophaga did not provide promising results duringthe second step of the work, as it showed an inadequategrowth in all tested conditions. All strains recorded low andsporadic values of emulsion during the incubation period.Exception was Cellulophaga sp. A55 that producedemulsions and stable emulsions in the presence of NaCl, andreduced the surface tension of 19.3 units (from 50.5 to31.2 mN/m).

Among the Gammaproteobacteria , the efficiency ofMarinobacter sp. A1 (Fig. 1b) was greater at 15 °C in theabsence of NaCl, although the optical density values were

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Table 3 Growth values obtained for isolates from enrichment cultures and polychaete specimens

Source Strain Origin Culture medium

BH T BH D MSM T MSM D

Enrichments Cohaesibacter sp. A25 Mc-E + + + + + + + + + + +

Pseudovibrio sp. A27 Mc-E + + + + − + + +

Thalassospira sp. A46 Ss-E − − + −Thalassospira sp. A57 Bl-E − + + − −Alcanivorax sp. A52 Bl-E + + − + + −Alcanivorax sp. A53 Bl-E + + + + + + +

Cobetia sp. A20 Mc-E − + + + − + + +

Marinobacter sp. A1 Mc-E + + + + − −Idiomarina sp. A19 Mc-E + + + + + + + + + + + +

Pseudomonas sp. A6 Mc-E + + + + + + + + +

Pseudomonas sp. A14 Mc-E + + + + + + + + + +

Pseudomonas sp. A18 Mc-E + + + + − + +

Pseudomonas sp. A45 Ss-E − + + + − + +

Joostella sp. A2 Mc-E + + + + + + − + + +

Joostella sp. A3 Mc-E + + + + + + + + + + +

Joostella sp. A8 Mc-E + + + + + + + + + + + +

Joostella sp. A9 Mc-E + + + + + + + + + + +

Joostella sp. A11 Mc-E + + + + + + − + +

Joostella sp. A15 Mc-E + + + + + + − + + +

Joostella sp. A17 Mc-E − + + − + +

Joostella sp. A22 Mc-E − + − + + +

Joostella sp. A24 Mc-E + + + + + + + + + + +

Joostella sp. A29 Mc-E + + + + + + + + +

Joostella sp. A30 Mc-E + + + + + + + + + + + +

Joostella sp. A32 Mc-E − + + + − + +

Cellulophaga sp. A49 Bl-E + + + + +

Cellulophaga sp. A50 Bl-E − + − + + +

Cellulophaga sp. A51 Bl-E − + + + − + +

Cellulophaga sp. A55 Bl-E − + + + − + +

Cellulophaga sp. A60 Bl-E + + + + + + + + + +

Polychaetes Pseudoalteromonas sp. Bl46 B. luctuosum − − − + +

Pseudoalteromonas sp. Bl65 B. luctuosum + + + + + + − −Pseudoalteromonas sp. Ss86 S. spallanzanii − + + − −Pseudoalteromonas sp. Ss89 S. spallanzanii + + + − − −Psychrobacter sp. Bl39 B. luctuosum − + + + − + +

Vibrio sp. Bl49 B. luctuosum + + + + + + +

Alteromonadaceae sp. Ss76 S. spallanzanii − + + + − + +

Cellulophaga sp. Ss85 S. spallanzanii − − − + + +

Cellulophaga sp. Ss91 S. spallanzanii + + − −Cellulophaga sp. Mc108 M. claparedei + − − −Cellulophaga sp. Ss88 S. spallanzanii − + − −Maribacter sp. Ss71 S. spallanzanii + + + + + + − −Maribacter sp. Ss79 S. spallanzanii + + + − + + + −Tenacibaculum sp. Mc99 M. claparedei + + + + + + + −Citricoccus sp. Bl52 B. luctuosum ++ ++ − −Citricoccus sp. Bl54 B. luctuosum + + + + + + − −Citricoccus sp. Bl55 B. luctuosum − + + − + +

Staphylococcus sp. Ss67 S. spallanzanii + + + + + + − + +

MC-E enrichment of Megalomma claparadei , Bl-E enrichment of Branchiomma luctuosum , Ss-E enrichment of Sabella spallanzanii , Mc M.claparedei strain, Bl B. luctuosum strain, Ss S. spallanzanii strain, BH T Bushnell Haas broth plus tetradecane, BH D Bushnell Haas broth plus dieseloil,MSM T mineral salt medium plus tetradecane,MSMD mineral salt medium plus diesel oil, ‘+++’OD580>0.5, ‘++’OD580=0.3–0.5, + OD580=0.2–0.3, ‘−’ no growth

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lower than those obtained at 25 °C. It produced the highestvalue of emulsion in the absence of NaCl (E24 index 52.5% at15 °C and 25 °C after 288 and 240 h incubation) in BHamended with diesel oil. Surface tension was reduced from57.5 to 36.85 mN/m and from 50.5 to 30.5 mN/m duringincubation at 15 and 25 °C, respectively. An average E24

value of 14.15 % was obtained during growth at 25 °C inthe presence of NaCl.

The BS activity by Pseudomonas spp. A6, A14, A45 (inincubation in BH supplemented with tetradecane) and A18(BH plus diesel oil) was influenced by the addition of NaCl inthe medium. Among the Pseudomonas isolates, A18 wasstrongly influenced by temperature. Only in the presence ofNaCl at 25 °C it showed values of stable emulsion higher than50 % and a good reduction of surface tension. The highestvalues of stable emulsion index were obtained forPseudomonas sp. A45, with an E24 of 68.3 % after 240 h at15 and 25 °C in the presence of NaCl (Fig. 1c) and A18, withanE24 of 55.15% after 144 h at 25 °C in the presence of NaCl.The surface tension was further reduced by Pseudomonas sp.A45, with a difference between the initial of 52.5 mN/m andfinal value of 20.5 mN/m, under incubation at 25 °C withaddition of NaCl, followed by Pseudomonas sp. A14 with areduction of 28 units under the same conditions.

Finally, Alcanivorax sp. A52 and Alcanivorax sp. A53(Fig. 1d) did not grow in the absence of salt, while in thepresence of it they produced stable emulsion values higherthan 50 % and reduced the surface tension of approximately20 units during incubation in BH supplemented withtetradecane.

Cobetia sp. A20 was incubated in BH supplemented withdiesel oil, and showed better results at 25 °C, with themaximum emulsion and stable emulsion values in thepresence of NaCl, while the maximum reduction of surfacetension was recorded in the absence of salt at 15 °C with areduction from 55 to 24.14 mN/m.

The strain Idiomarina sp. A19 (in BH amended withtetradecane) showed emulsifying capacity after 336 h ofincubation at 25 °C in the presence of NaCl, and created astable emulsion with an E24 index of 36.65 %, and a surfacetension reduction of 30.05 units, from 72.65 to 42.6 mN/m.

Among the Alphaproteobacteria , Thalassospira sp. A46proved to be more able in producing stable emulsions in thepresence of NaCl after 144 h of incubation at 15 °C(maximum E 24 of 70 %) and 25 °C (maximum E 24 of48.3 %) in BH supplemented with tetradecane. The valuesof surface tension were greatly reduced, from 67 to 32.65 mN/m during incubation at 15 °C, and from 70.5 to 31.65 mN/m at25 °C. Temperature did not influence considerably theemulsion production and stabilisation.

Cohaesibacter sp. A25 produced emulsions and stableemulsions only in the presence of NaCl (incubated in BHsupplemented with tetradecane), but achieved E24 of nearly

50 % only during incubation at 25 °C, when the highestsurface tension reduction was recorded (from 60.65 to33.95 mN/m).

Pseudovibrio sp. A27 grew more rapidly at 25 °C, withhigher values of optical density, by reducing also the surfacetension and showing greater emulsifying capacity at lowertemperature. It showed positive responses to the enhancmentstudy, with a range of stable emulsions between 17.5 and22.5% during growth on BH plus tetradecane, in the presenceof NaCl and incubation at 15 °C (optimal temperature). Thereduction of the surface tension was of 36.75 units comparedto initial time. No growth was recorded in the absence of saltin the culture medium.

Among the strains isolated from natural samples, only fewpositively responded to the enhancement tests. Among theGammaproteobacteria , Pseudoalteromonas sp. Ss86 wasthe most promising producer with maximum values of E24

at 25 °C in the absence of NaCl (49.8%), and a surface tensionreduction from 54.5 to 28.5 mN/m at the same conditions (inBH amended with tetradecane). Pseudoalteromonas sp. Bl65achieved an E24 of 20 % in the presence of salt at 15 °C, but itdid not show other remarkable results for BS production.

The absence of salt in the culture medium was a limitingaspect at 15 °C for Vibrio sp. Bl49 (Fig. 1e) which wasincubated in BH supplemented with tetradecane. Suchstrain showed a performance that resulted higher in thepresence of NaCl at 25 °C with a maximum E 24 value of51.65 % after 192 h of incubation, and a surface tensionreduction of more than 20 units. Anyway, at 25 °C itshowed production both in the absence and presence ofsalt.

Among the CFB group of Bacteroidetes , Tenacibaculumsp. Mc99 and Cellulophaga sp. Mc108 (Fig. 1f) showed mostremarkable results in BH amended with tetradecane. Inparticular, the BS activity of such strains was significantlyinfluenced by salinity, as they were able to produce higher

�Fig. 1 a Joostella sp. A8. Effect of salinity (NaCl, 0 and 3 %) on BSactivity and bacterial growth during incubation under optimal conditions:BH added with tetradecane (2 %) at 15 (a ) and 25 °C (b ); bMarinobacter sp. A1. Effect of salinity (NaCl, 0 and 3 %) on BSactivity and bacterial growth during incubation under optimalconditions: BH added with diesel oil (2 %) at 15 (a) and 25 °C (b); cPseudomonas sp. A45. Effect of salinity (NaCl, 0 and 3%) onBS activityand bacterial growth during incubation under optimal conditions: BHadded with tetradecane (2 %) at 15 (a) and 25 °C (b); d Alcanivorax sp.A53. Effect of salinity (NaCl, 0 and 3 %) on BS activity and bacterialgrowth during incubation under optimal conditions: BH added withtetradecane (2 %) at 15 (a) and 25 °C (b); e Vibrio sp. Bl49. Effect ofsalinity (NaCl, 0 and 3 %) on BS activity and bacterial growth duringincubation under optimal conditions: BH added with tetradecane (2 %) at15 (a) and 25 °C (b); f Cellulophaga sp. Mc108. Effect of salinity (NaCl,0 and 3 %) on BS activity and bacterial growth during incubation underoptimal conditions: BH added with tetradecane (2 %) at 15 (a) and 25 °C(b). Note the different scales

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Fig. 1 (continued)

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Fig. 1 (continued)

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percentages (up to 50 %) only in the presence of NaCl.Tenacibaculum sp. Mc99 showed ability to form emulsionsand stable emulsions at both temperatures (maximum E24,62.5 %; 25 °C after 192 h of growth), whereas it did notrecord significant reductions of surface tension.Cellulophagasp. strain Mc108 had a similar response to salinity variation,but the emulsifying capacity assumed an increasing profilewhen the strain was incubated at 15 °C, and a decreasingprofile when incubated at 25 °C. Temperature did not affectthe surface tension reduction.

Finally, among Gram-positive isolates (Firmicutes andActinobacteria affiliates) Staphylococcus sp. Ss67 gavepositive results only at 15 °C in the presence of NaCl, andachieved a maximum E 24 value (57 %) after 192 h ofincubation in BH plus tetradecane. Staphylococcus sp. Ss67did n0t show promising results, if not exclusively at 15 °C inthe presence of salt, while in the other conditions showed nogrowth.

Strains affiliated to the generaCitricoccus andMaribacter,in addition to strains Ss76 among the Alteromonadaceae andPsychrobacter sp. Bl39, did not show remarkable results forBS production.

Figure 2 shows more clearly the effect of salinity andtemperature on E24 maximum value obtained for isolates thatwere representative of each phylum and derived from bothenrichment cultures and natural samples. All strains producedhigher stable emulsion percentages in the presence of salt, sohighlighting the strong influence of NaCl concentration(Fig. 2a). Exceptions were Cobetia sp. A20 and Vibrio sp.Bl49, which were able to produce stable emulsions duringincubation at both NaCl concentrations. Moreover, thedifferent results obtained for Marinobacter sp. A1 arehighlighted, as it appears from the E24 value obtained in theabsence of salt.

About the effect of temperature, the figure shows that onlyin some cases (e.g.Cellulophaga sp. A55 and Staphylococcussp. Ss67) temperature influenced the production of stableemulsion by the isolates (Fig. 2b).

Course of BS activity

The course of BS activity was expressed as the relationshipbetween the capacities of selected isolates to create stableemulsions and emulsifying activity. This course duringincubation under optimum conditions is shown in Fig. 3.The course of BS activity showed a similar trend without adirect correspondence between the optical density andemulsions or E24. On the basis of this, depending on the timerequired for emulsification and stable emulsion production,the kinetics was expressed in terms of ratio E24/emulsion. Oneobserved course type displayed by most strains and the profileobtained for Joostella sp. A8 is shown as an example inFig. 3a. The profile of the ratio E24/emulsions had a very

low value at the initial stage (when only emulsions wereproduced, not accompanied by stable emulsions), and themaximum E24/emulsions ratio (i.e. about 4.88, with a valueof E24 of 61 % and a value of emulsions of 13 %) appearedafter 192 h of incubation. Then stable emulsions disappearedand the ratio decreased.

The second course type displayed is well exemplified byAlcanivorax sp. A52, which produced emulsions and stableemulsions simultaneously, by achieving the maximum E24/emulsions ratio (E24 of 27.5 % and emulsion percentage of12.5 %) as the beginning of the emulsion appearance after192 h of incubation (Fig. 3b).

In the case ofVibrio sp. Bl49 and Tenacibaculum sp.Mc99(Fig. 3c, d, respectively) the E24/emulsions ratio started from0 at the beginning, when the strains started to emulsify, and nostable emulsions were produced. Then the stable emulsionsoccurred and the ratio E24/emulsions increased with highervalues of 2.20 (E24 of 47.5 % and emulsion percentage of21.5 %) and 2.84 (E24≈48.3 %; emulsion percentage≈17 %),respectively, for Vibrio sp. Bl49 and Tenacibaculum sp.Mc99, after 240 h of incubation.

As an example, the E24/emulsions ratios at 15 and 25 °C inthe absence of NaCl for Marinobacter sp. A1 are shown inFig. 4. During incubation at 15 °C the maximum E 24/emulsions ratio (i.e. 0.72, with a value of E24 of 52.5 % anda value of emulsions of 72.5%) was recorded. The same strainshowed the maximum value of optical density after 288 h (i.e.0.609) during incubation at 25 °C, in concomitance with themaximum E24/emulsions ratio (i.e. 0.91, of E24 of 21 %;emulsification ability of 23 %).

The surface tension reduction was differently related to theemulsifying ability of the strains. In some cases higheremulsion values corresponded to a greater lowering of surfacetension, as in the case of Pseudoalteromonas sp. Ss86 andVibrio sp. Bl49. In other cases, despite some strains showedemulsifying activity, they did not generally reduce the surfacetension (e.g. Tenacibaculum sp. Mc99 at 25 °C and 3 %NaCl). In the absence of emulsifying activity, the surfacetension generally assumed values more or less stable, withoutundergoing a considerable reduction. Cellulophaga sp.Mc108 and Vibrio sp. Bl49 were exceptions. The formerwas capable of reducing surface tension of 19.3 mN/m at15 °C in the absence of NaCl, and the latter reduced thesurface tension of 32.4 units although it did not showemulsifying capacity.

Discussion

Biological matrices have been rarely considered for theisolation of BS-producing bacteria (Gandhimathi et al. 2009;Kiran et al. 2010; Rizzo et al. 2013) that have been mostlyobtained from hydrocarbon-contaminated water or soil. In this

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paper we propose the use of sabellids, on the base of theirgreat filtration rate, and consequent accumulation ofcontaminants in their apparatus.

Isolates from natural samples were preliminarily screenedfor BS production as previously reported by Rizzo et al.(2013) for strains obtained from polychaete crude oilenrichments. Selected isolates showed a strong emulsifyingactivity which was not accompanied by a surface tension

reduction. The majority of isolates gave negative results inthe use of CTAB and BA tests, confirming their use as anintegration of the screening procedure (Mulligan et al. 1984;Rizzo et al. 2013). The TLC assay generally confirmed theresults obtained from the E24 index detection. Yellow-orangespots, which positively stained with anisaldehyde reagent,indicate carbohydrate components in the BS molecules(Satpute et al. 2010; Ellaiah et al. 2002).

Fig. 2 Effect of NaCl concentration (a) and temperature (b) on E24 index that was produced by representative strains. Please note that only E24

maximum value obtained from each strain is reported here. E24<10 % was considered as null

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Interestingly, isolates were affiliated to bacterial genera(e.g. Citricoccus , Cellulophaga , Tenacibaculum ,Psychrobacter, Vibrio , Pseudoalteromonas and Maribacter)that have been never or poorly reported in relation to BSproduction. The genera Cellulophaga , Vibrio andPseudoalteromonas have been found in hydrocarbon-contaminated environments, or reported in relation tohydrocarbon biodegradation (Hedlund and Staley 2001;Gutierrez et al. 2013; Rizzo et al. 2013). However, only thegenus Pseudoalteromonas has been reported in relation to BSproduction. In addition, Pseudoalteromonas species arefrequently found in association with eukaryotic hosts inthe marine environment and many of them producebiologically active metabolites (e.g. extracellularpolysaccharides) (Holmström and Kjelleberg 1999). Thestructure of an acidic O-specific polysaccharide from themarine bacterium Cellulophaga baltica was reported byTomshich et al. (2007), while Perepelov et al. (2007)isolated a pseudoaminic acid-containing O-specificpolysaccharide from C. fucicola .

Several parameters such as carbon source, concentration ofions, temperature and pH could influence the BS activity(Atlas 1981; Ristau and Wagner 1983; Lotfabad et al. 2009)from qualitative and quantitative point of view. Informationon BS activity enhancement are available for bacteria isolatedfrom water and sediment samples (Haddad et al. 2009;Salehizadeh and Mohammadizad 2009; Khopade et al.2012a, b). Cho et al. (2011) determined the optimal conditionsfor the production of BS by Pseudoalteromonas sp. HK-3,originating from oil-spilled areas. On the other hand, similarinvestigations on the influence of abiotic factors on BSproduction from strains isolated from biological matrices havebeen poorly treated (Kiran et al. 2010).

In this context, the optimal conditions for the BSs activityby 18 bacterial strains which were directly isolated fromspecimens of the polychaetes S. spallanzanii , B. luctuosumand M. claparedei throughout this study, and additional 30strains that were previously obtained from crude oilenrichments carried out using homogenates of the sameorganisms (Rizzo et al. 2013) were determined. In particular,

Fig. 3 E24/emulsion profile in relation to OD580 and time during incubation under optimal conditions. a Joostella sp. A8, b Alcanivorax sp. A52, cVibrio sp. Bl49 and d Tenacibaculum sp. Mc99

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the optimal conditions for BS activity by polychaete-associated bacteria were investigated by considering theinfluence of the culture medium (MSM or BH) and carbonsource (diesel oil or tetradecane), in addition to salinity (NaCl,0 or 3 %) and temperature (15 or 25 °C) values. The efficiencyin BS activity was evaluated by measuring surface tension,emulsification ability and stable emulsion production.

All isolates deriving from the enrichment cultures wereable to grow in the presence of hydrocarbons under at leastone incubation condition. This finding is particularlyinteresting as microorganisms growing on hydrocarbonsfrequently produce BSs with emulsifying activity (Karanthet al. 1999). Conversely, strains that were isolated from naturalsamples generally showed a lower ability to grow in thepresence of hydrocarbons.

Results obtained from our experiments provided evidencethat surface tension reducing activity and emulsification of themost of isolates was significantly dependent on NaClconcentration, which strongly affected bacterial growth, ratherthan by temperature. It is not to be excluded that the differentBS efficiency values in the presence of salt could bedependent on an increase/decrease of BS amount, enhancedcellular activity and changes in BS composition (Syldatk et al.1985; Abu-Ruwaida et al. 1991).

Overall, isolates from enrichment cultures were morepromising. Among them, the genera Joostella andAlcanivorax gave better results, by showing a generaloptimum in the presence of NaCl during incubation at 15

and 25 °C, respectively. In particular, Alcanivorax strains(A52 and A53) recorded the higher E24 index percentage(between 70 and 80 %). Some strains, such as Pseudomonasisolates, recorded heterogeneous behaviours that wereprobably species-specific. However, all strains fromenrichment cultures showed better results in the presence ofsalt. It could be argued that salinity represents a limiting factorfor BS production by strains. On the other hand, temperaturewas not a limiting factor, but affected only the bacterial speedand efficiency of emulsifying activity and surface tensionreduction.

The only exception was represented by Marinobacter sp.A1, which allowed recording the best responses at 15 °C andin the absence of NaCl. This condition may be associated witha kinetic production in limiting conditions of growth: this hasbeen extensively demonstrated for example for Pseudomonasaeruginosa with an overproduction of BSs under limitedconditions of nitrogen and iron (Guerra-Santos et al. 1984;Mulligan and Gibbs 1989).

Among strains that were isolated from natural samples,Tenacibaculum sp. Mc99 and Cellulophaga sp. Mc108showed better results, with optimum of production duringincubation at 15 °C in the presence of salt (E24≈50 %, surfacetension of about 20 mN/m). The optimum of BS activity forVibrio was found during incubation at 25 °C in the presence ofNaCl (E24≈51 %, surface tension reduction≈20 units). So,also in case of isolates from natural samples, the BS activitypattern reflected a strong dependence on salt concentration.

Fig. 4 Marinobacter sp. A1effect of salinity (NaCl, 0 and3 %) on BS activity and bacterialgrowth during incubation underoptimal conditions: BH addedwith diesel oil (2 %) at 15 (a) and25 °C (b). Note the differentscales

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Nevertheless, they expressed a lower ability than isolates fromenrichment cultures.

The relationship between emulsification and stableemulsion has been highlighted and used as a ratio byconsidering that the emulsion stability could designate theBS strength (Satpute et al. 2010) or its concentration (Walteret al. 2010).

All isolates considered in this study showed a good abilityto produce emulsions in rich medium, without a remarkablereduction of surface tension (Rizzo et al. 2013). However,some of them were able to reduce surface tension duringincubation in mineral medium supplemented withhydrocarbon. This finding could suggest a considerableincreasing of interface activity from strains during stressfulconditions with complex carbon sources.

Surface-active compounds produced by microorganismsare of two main types: those that reduce surface tension atthe air–water interface (BSs) and those that reduce theinterfacial tension between immiscible liquids or at thesolid–liquid interface (bioemulsifiers) (Karanth et al. 1999).Rosenberg and Ron (1999) divided BSs into low-molecular-mass molecules (glycolipids, lipopeptides, flavolipids,corynimycolic acids and phospholipids), which efficientlyreduce surface and interfacial tension at the air/water, and highmolecular-mass polymers (polymeric and particulatesurfactants), which are more effective as emulsion-stabilisingagents, but not reduce the surface tension and exhibitconsiderable substrate specificity (Dastgheib et al. 2008;Salihu et al. 2009). For this reason, differences in interfacialactivities observed during the two screening procedures(Rizzo et al. 2013) could suggest a too low amount of BSsduring incubation in rich medium and an intensification ofproductivity in mineral medium with hydrocarbons.Otherwise, this finding could be explained by assuming adifferent chemical nature of the surface-active compounds infunction of the carbon source.

In conclusion, obtained results suggest that it could beinteresting to improve the capacity of each benthicorganisms-associated strain to produce BSs, by carrying outquantitative and qualitative analyses on the BS molecules.Moreover, it might be interesting to further investigate theBS activity for each strain associated with filter feedingorganisms, assaying this capability even under the influenceof other factors, such as the nitrogen source or pH. Finally, toprove a correlation between such parameters and theirinfluence on BS amount it could be necessary quantifyingthe amount of crude produced BS. In this way, it wouldcomplete a framework of knowledge necessary for an efficientand safe application.

Acknowledgements Rizzo C. wishes to thank all the colleagues at theKarlsruher Institut für Technologie (KIT), Germany, for assistance andsupport during her stay in their lab. The authors also thank Dr. Andrea

Cosentino for the taxonomic identification of Polychaetes. This researchwas supported by grants from the Doctoral School ‘Dottorato di Ricercain Scienze Ambientali: Ambiente Marino e Risorse’ of the University ofMessina, Italy.

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