Influence of salinity and temperature on the activity of ...

Influence of salinity and temperature on the activity of biosurfactants by polychaeteassociated isolates Carmen Rizzo, Luigi Michaud, Christoph Syldatk, Rudolf Hausmann, Emilio De Domenico & Angelina Lo Giudice Environmental Science and Pollution Research ISSN 0944-1344 Volume 21 Number 4 Environ Sci Pollut Res (2014) 21:2988-3004 DOI 10.1007/s11356-013-2259-8

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Author's personal copy Environ Sci Pollut Res (2014) 21:2988–3004 DOI 10.1007/s11356-013-2259-8

RESEARCH ARTICLE

Influence of salinity and temperature on the activity of 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 / Published online: 30 October 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Influence of different parameters on biosurfactant (BS) activity was carried out on strains that were isolated from the polychaetes Megalomma claparedei, Sabella spallanzanii and Branchiomma luctuosum and additional 30 strains that were previously identified as potential BS producers from crude oil enrichments of the same polychaete specimens. The selection of BS-producing strains from polychaete natural samples was carried out by using standard screening tests. The BS activity by each isolate was evaluated for the effect of salinity and temperature on emulsion production and surface tension reduction, during incubation in mineral medium supplemented with tetradecane or diesel oil. All isolates showed a similar time course of BS activity, and the latter was more influenced by salinity rather than temperature. Some of the BS producers belonged to genera that have not 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 Giudice Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d’Alcontrès 31, 98166 Messina, Italy C. Syldatk Institute of Process Engineering in Life Sciences, Section II: Technical Biology, Karlsruhe Institute of Technology (KIT), Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany R. Hausmann Institute of Food Science and Biotechnology, Section Bioprocess Engineering, 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, Italy e-mail: [email protected]

(i.e. Citricoccus , Cellulophaga , Tenacibaculum and Maribacter ) or have poorly been (Psychrobacter, Vibrio , and Pseudoalteromonas) reported as able to produce BSs. This is remarkable as some of them have previously been detected in hydrocarbon-enriched samples. Results confirm that filter-feeding polychaetes are an efficient source for the isolation of BS producers. Keywords Biosurfactants . Filter-feeding organisms . Salinity . Temperature . Megalomma claparedei . Sabella spallanzanii . Branchiomma luctuosum

Introduction Biosurfactants (BSs) have gained renewed interest in recent years mostly because of their biodegradability and reduced toxicity compared with synthetic surfactants (Satpute et al. 2010; Pacwa-Płociniczak et al. 2011). In this context, biological matrices have been rarely considered for the isolation of BS-producing bacteria (Gandhimathi et al. 2009; Kiran et al. 2010; Rizzo et al. 2013) that have been mostly obtained from hydrocarbon-contaminated water or soil. Sabellids (Polychaeta: Annelida) are sedentary worms that are able to filter large volumes of waters to collect particles and bacteria suspended in the bulk water for their food requirements (Licciano et al. 2005, 2007). They can potentially accumulate different kinds of contaminants (such as heavy metals, hydrocarbons and organochlorinated compounds), both in the soluble and in the particulate phases, from the environment. Thus, it is expected that associated bacteria have to cope with the presence of contaminants in the host tissues. Hydrocarbons or other water-insoluble substrates are known to induce BS production in many microorganisms (Radwan and Sorkhoh 1993), and in some cases, it was

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observed that BS production started only when the soluble carbon source was consumed and the hydrocarbon was available (Banat et al. 1991; Banat 1995). In some cases, temperature causes alteration in the composition of the BS (Syldatk et al. 1985), while salt concentration affects BS production depending on its effect on cellular activity (Abu-Ruwaida et al. 1991). The kinetics of BS activity may either display a growthassociated or a stationary phase-associated character. In some cases, the addition of precursors to the growth medium has been 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 was observed widely for bacteria isolated from terrestrial environment or from water and sediment samples (Haddad et al. 2009; Khopade et al. 2012a, b). However, there is only a very limited number of studies dealing with bacteria isolated from marine benthic organisms (Gandhimathi et al. 2009; Kiran et al. 2010). The aim of the present work was the study of the BSproduction efficiency under different growth conditions by bacterial strains which were isolated from both polychaete specimens and enrichment cultures.

Materials and methods Study area Lake Faro is a small coastal pond (0.263 km2), located at the northeastern corner of Sicily, Italy (coordinates, 38°16′ N, 15°38′ E), which features a funnel-shaped profile, with a steep sloping bottom that declines to a central basin reaching a depth of 29 m. The lake is connected via a shallow channel to the Straits of Messina, which separates the island of Sicily from the Italian peninsula. Another channel, which is silted up most of the time, is artificially opened for a few days during the hottest summer period establishing a communication with the Tyrrhenian Sea to allow water circulation into the lake (Saccà et al. 2008). Lake Faro is a meromictic basin, with a salinity that seasonally varies from 34 to 38. The lake is also characterised 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 radius in the Lake Faro at depths ranging from 0.6 to 0.8 m. Conductivity, temperature, pH and oxygen saturation values were measured on site using a portable multiparametric probe (CTD YSI 6600V2). Temperature and salinity values were

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15.4 °C and 37.78, while pH and dissolved oxygen concentration were 8.61 and 8.95 mg/L, respectively. Adult specimens of the polychaete annelids Sabella spallanzanii (Gmelin, 1791), Branchiomma luctuosum (Grube, 1870) and Megalomma claparedei (Gravier, 1906) were aseptically collected and immediately washed with filtersterilised natural seawater to remove transient and loosely attached bacteria and/or debris. Specimens were then placed into individual sterile plastic bags containing filter-sterilised natural seawater. All samples were transported directly to the laboratory at 4 °C for microbiological processing (within 2 h after sampling). Bacterial strains Immediately upon return to the laboratory, organisms were washed several times with filter-sterilised seawater and homogenised in ice for 9 s by using Ultraturrax. Polychaete homogenates were serially diluted by using filter-sterilised seawater and 100 μL of each dilution was spread-plated in two replicates on solidified ONR7a (1.5 % agar, w /v ) (Dyksterhouse et al. 1995) that was supplied with hydrocarburic substrates as vapour by placing a sterile filter paper disc containing 0.5 mL of filter-sterilised crude oil (ONR7a-C; Arabian Light, Sigma-Aldrich) or crystals of polyaromatic hydrocarbons (ONR7a-PAH; mixture of pyrene, phenanthrene, fluoranthen; Sigma-Aldrich) or biphenyl (ONR7a-BP, Sigma-Aldrich) in the Petri dish lid. Inoculated substrate-free media in addition to sterile hydrocarboncontaining medium served as negative controls (Lo Giudice et al. 2010). Plates were incubated at 28 °C for 15 days. Colonies were randomly selected from agar plates, picked and subcultured three times under the same conditions. Selection of BS-producing strains A preliminary screening procedure was carried out to select isolates for further analyses. Isolates were inoculated in marine broth (pH 7–7.5; Difco) and incubated for 1 week at 25 °C. At regular intervals of 48 and 120 h screening tests were performed as described below. When necessary (see below), cultures were preliminary centrifuged at 4,700 rpm for 20 min at 4 °C, and only the obtained supernatants were used. For each test, uninoculated medium was treated exactly as the cultures and then used as negative control. Emulsifying activity A 2-mL portion of the sample from each culture was added to 2 mL of kerosene (Petroleum ether, Panreac) as test-oil and the mixture was vigorously vortexed for 2 min. After 24 h, the emulsification index (E 24) was calculated 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 cellfree supernatant was determined with a digital tensiometer K10T (Krüss, Hamburg, Germany) by using Wilhelmy Plate method (Tuleva et al. 2005).

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with 1.333 mL of ethyl acetate and this ethyl acetate phase was applied as TLC standard. Strains who gave an E 24 of ≥50 % and showed interesting spots on TLC plates were selected as potential positive strains for BS production, and deeply studied with further analyses.

PCR amplification of 16S rRNA genes Haemolytic activity Each culture was inoculated on the surface of blood agar plates (BA; Sriram et al. 2011) and incubated for 48–72 h at 37 °C. Plates were then observed for the presence of clearing halos around the spots, indicative of BS production (Mulligan et al. 1984; Youssef et al. 2004). Blue agar plate method The cetyltrimethylammonium bromide (CTAB) agar plate method is a rapid screening method for the detection of anionic BSs (Siegmund and Wagner 1991). Mineral salts agar medium was prepared with a 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 CTAB agar plates, incubated at 30 °C for 24–48 h, and then stored at 4 °C for further colour development (24 h). A dark blue halo around the colony was considered a positive result. Thin-layer chromatography BS molecules were prior extracted as follows. Aliquots (1 mL) of cell-free supernatants were acidified with 85 % phosphoric acid (final concentration 1 %, v /v ) to adjust pH of supernatant of 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 to a 2-mL tube, and the ethyl acetate was evaporated at room temperature (Syldatk et al. 1985; Schenk et al. 1995; Hörmann et al. 2010). The extract was characterised using analytical thin-layer chromatography (TLC), carried out on silica gel plates (stationary phase). One-millilitre aliquot of each crude BS extract was concentrated, resuspended in 10 μL of ethyl acetate and separated on a silica gel plate using chloroformmethanol-acetic acid (65:15:2; Romil, Sps) as developing solvent 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 and Thivakaran 2010). The spots on TLC plate were developed by heating with a fan. The chromatograms of the extracts were compared with the TLC pattern of a mixture of rhamnolipids which was prepared from Jeneil JBR 425 (Jeneil Biosurfactants Company, Saukville, USA). For the standard, 85 μL of JBR 425 were suspended in 1 mL of 0.1 M sodium phosphate buffer, pH 7, and acidified with 10 μL of concentrated phosphoric acid. This mixture was extracted

Single colonies of each strain were lysed by heating at 95 °C for 10 min. Amplification of 16S rRNA gene was performed with a thermocycler (Mastercycler GeneAmp PCR-System 9700 Applied Biosystem, USA) using Bacteria-specific primer 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 at 0 °C and contained 5 μL DNA, 1 μL of each forward and reverse primer (10 μM), 0.5 μL of dNTP mix (10 mM; GE Healthcare, Buckinghamshire, UK), 2.5 μL of reaction buffer 10× (containing 15 mM of MgCl2), 0.125 μL of polymerase (5 U mL−1; Hot Star Taq™ Qiagen, Hilden, Germany) and sterile 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 used in every PCR run. The PCR program was as follows: a first step 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 min and 30 s at 72 °C, followed by a final elongation at 10 min at 72 °C. The results of the amplification reactions were analysed by agarose gel electrophoresis (1 %, w/v) in TAE buffer (0.04 M Tris-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 were determined by comparison to 16S rRNA gene sequences in the NCBI GenBank and the EMBL databases using BLAST, and the ‘Seqmatch’ and ‘Classifier’ programs of the Ribosomal Database Project II (http://rdp.cme.msu.edu/) (Altschul et al. 1997). Sequences were further aligned using the program Clustal W (Thompson et al. 1994) to the most similar orthologous sequences retrieved from database. Each alignment was checked manually, corrected and then analysed using the neighbour-joining method (Saitou and Nei 1987) according to the model of Jukes–Cantor distances. A Phylogenetic tree was constructed using the Molecular Evolutionary Genetics Analysis 5 software (Kumar et al. 1993). The robustness of the inferred trees was evaluated by 400 bootstrap re-samplings.

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Nucleotide sequence accession numbers Nucleotide sequences have been deposited in the NCBI GenBank database under the accession numbers KF032912KF032929. Improvement analyses Improvement analyses were carried out on strains that were isolated from M. claparedei , S. spallanzanii and B. luctuosum , and additional 30 strains that were previously obtained from crude oil enrichments of the same polychaete specimens and then selected using the same screening procedures reported above (Table S1; Rizzo et al. 2013).

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and 25 °C) and salinity values (0 and 3 % NaCl, w /v ) to study the influence of these physical parameters on BS activity. Each culture was constantly monitored for about 15 days at 48 h intervals by measuring E 24 detection (Satpute et al. 2008) and surface tension measurement as it was described above. Additionally, a preliminary emulsification test was carried out in to establish the emulsification ability of the isolates. Briefly, an aliquot (500 μL) of each culture was vortexed for 2 min with an equal amount of kerosene (although it can be replaced with other hydrocarbon compounds; 1:1). After about 1 min stabilisation the ratio between the emulsion layer and the total culture height was measured and expressed as a percentage (Christova et al. 2004).

Selection of optimal medium and carbon source for bacterial growth Results The first tested parameter was the carbon source. All isolates were 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 (see below). The composition of the MSM was as follows (final volume 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 100 mL): 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. In BH and MSM, the nitrogen source is provided in the form of ammonium nitrate and sodium nitrate, respectively, while the phosphor is provided as phosphate mono/potassium, and phosphoric acid. At each medium NaCl was added to a final concentration of 30 g L−1. Both media were sterilised by autoclaving at 121 °C for 15 min. Isolates were cultured in BH and MSM and incubated at 25 °C under shaking (120 rpm) with two different carbon sources (diesel oil or tetradecane, 2 %, v/v), to test both the influence of the mineral composition and the carbon source on the bacterial growth. Each culture was monitored for two weeks at intervals of 48 h, by measuring the optical density at 580 nm (OD580) with a spectrophotometer (UV-mini-1240, Shimadzu). Influence of NaCl concentration and temperature on BS activity Kinetics studies were carried out in 250 mL shake flask by inoculating bacterial isolates in 60 mL of the preferred mineral medium (BH or MSM) supplemented with the optimal hydrocarbon source (tetradecane or diesel oil). Cultures were incubated in a rotary shaker at two different temperatures (15

Preliminary selection of BS-producing strains A total of 96 strains were directly isolated from polychaete specimens (38, 30 and 28 from B. luctuosum , S. spallanzanii and M. claparedei , respectively). The first preliminary screening allowed selecting 18 isolates as potential BSproducers (Table 1). The majority of them were obtained from S. spallanzanii (nine isolates) and B. luctuosum (seven isolates). 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 after a 120 h incubation. No direct correlation was observed between optical density and E 24 values. All the produced emulsions remained stable up to 1 month. Surface tension generally remained stable during all the screening 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 after staining with anisaldehyde. All examined extracts showed a similar profile on TLC plates. The retention factor values of all spots ranged from 0.7 to 0.76. Phylogenetic identification of BS-producing strains The comparative sequence analysis of isolates indicated that the 18 potential BS producers were closely related to known bacteria (16S rRNA gene similarity, ≥97 %). Isolates were mainly affiliated to the Cytophaga–Flavobacteria– Bacteroidetes gr oup of Bacteroidetes and Gammaproteobacteria (seven isolates per group). Three isolates were affiliated to the Actinobacteria and only one to the Firmicutes (Table 2). In particular, among the Bacteroidetes, four strains were affiliated to Cellulophaga

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Table 1 Results from the preliminary screening for the 18 isolates that were selected as potential biosurfactant producers

E 24 48 emulsifying activity after a 48-h incubation, E 24 120 emulsifying activity after a 120-h incubation, ST 48 surface tension after a 48-h incubation, ST 120 surface tension after a 120-h incubation, TLC thin-layer chromatography, Bl B, luctuosum strain, Ss S. spallanzanii strain, Mc M. claparedei strain, #ONR 7a C ONR7a plus crude oil, ONR 7a PAH ONR7a plus a mixture of pyrene, phenanthrene and fluoranthen, ONR 7a BP ONR7a plus biphenyl, ONR 7a , ONR7a without carbon source

Strain

E 24 (%) E 2448

Bl39

–

Bl46 Bl49 Bl52 Bl54 Bl55 Bl65 Ss67 Ss71 Ss76 Ss79 Ss85 Ss86 Ss88 Ss89 Ss91 Mc99 Mc108

– – 15±3.54 43.3±1.20 17.7±1.63 20±3.54 30±3.54 12.5±1.77 70±3.54 52±2.12 12.5±5.30 55.2±0.14 60±3.54 62.8±1.56 10±1.41 50±3.54 64±1.71

ST (mN/m) E 24120

TLC

Origin

Isolation medium

ST48

ST120

60±3.54

68.6

64.5

+

B. luctuosum

ONR7a BP

59.2±0.57 62.9±2.05 66.6±1.13 63.3±1.20 60±3.54 56.6±2.40 56.6±2.40 59.2±0.57 63.3±1.20 20±3.54 60±3.54 50±3.54 18.5±1.08 23.3±3.20 76±1.41 73±1.00 59.2±0.4

58.2 57.7 54.2 59.7 58.9 57.2 56.2 60.7 62.5 55.2 57.6 67.2 56.4 62.3 68 59.5 59.1

58.2 56.8 57.2 59.1 58.7 57.8 58.4 58.2 64.7 55.8 60.1 68.4 62.5 72.7 64.3 59.1 59.7

+ + + + + + + + + + + + + + + + +

B. luctuosum B. luctuosum B. luctuosum B. luctuosum B. luctuosum B. luctuosum S. spallanzanii S. spallanzanii S. spallanzanii S. spallanzanii S. spallanzanii S. spallanzanii S. spallanzanii S. spallanzanii S. spallanzanii M. claparedei M. claparedei

ONR7a ONR7a PAH ONR7a PAH ONR7a PAH ONR7a PAH ONR7a C ONR7a ONR7a ONR7a ONR7a PAH ONR7a BP ONR7a BP ONR7a C ONR7a C ONR7a C ONR7a ONR7a C

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

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

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 Bl46 Ss86 Ss89

B. luctuosum B. luctuosum S. spallanzanii S. spallanzanii

JF273871, Psychrobacter sp. TB2 JN578479, Pseudoalteromonas sp. H9 NR_026218, Pseudoalteromonas sp. strain IAM 12927 EU195931, Pseudoalteromonas sp. P102

99 99 99 98

Bl65 Bl49 Ss76 Ss71 Ss79 Ss85 Ss88 Ss91 Mc108 Mc99 Bl52 Bl54 Bl55 Ss67

B. luctuosum B. luctuosum S. spallanzanii S. spallanzanii S. spallanzanii S. spallanzanii S. spallanzanii S. spallanzanii M. claparedei M. claparedei B. luctuosum B. luctuosum B. luctuosum S. spallanzanii

FR744867, Pseudoalteromonas sp. A2B10 JQ083317, Vibrio sp. strain FA97 EU195925, Alteromonadaceae bacterium P120 16S AB526333, Maribacter sp. JAM-BA06 AB526333, Maribacter sp. JAM-BA06 AB681016, Cellulophaga sp. AB681016, Cellulophaga sp. AB681016, Cellulophaga sp. AB681016, Cellulophaga sp. AM746477, Tenacibaculum sp. EU305672, Citricoccus sp. FS24 EU305672, Citricoccus sp. FS24 EU305672, Citricoccus sp. FS24 JN251751, Staphylococcus sp. CRP7

98 99 99 99 98 99 99 97 99 98 99 98 98 99

BAC

ACT

FIR

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 affiliated to the genus Citricoccus, while the only strain belonging to the Firmicutes was found to be affiliated to the genus Staphylococcus (Ss67). Selection of optimal medium and carbon source for bacterial growth With regards to the 30 isolates from enrichment cultures (listed in Table S1), based on OD580 measures, among the CFB group of Bacteroidetes members of the genera Joostella and Cellulophaga showed a good growth on both media and carbon sources, although Cellulophaga members seemed to prefer diesel oil as a carbon source (Table 3). Among the Gammaproteobacteria, Pseudomonas spp. A6 and A14 and Idiomarina sp. A19 showed satisfactory growth in all growth conditions. Conversely, Pseudomonas spp. A18 and A45 preferred diesel oil as a carbon source, in both mineral media. Alcanivorax spp. A52 and A53 optimally grew in BH in the presence of tetradecane. Cobetia sp. A20 preferred diesel oil as carbon source in both culture media, while Marinobacter sp. A1 achieved optimal values of optical density only during incubation in BH supplemented with tetradecane. Among the Alphaproteobacteria, Thalassospira spp. A46 and A57 did not show satisfactory growth in the presence of hydrocarbons. Pseudovibrio sp. A27 showed better performance 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 was generally observed (Table 3). Strains generally seemed to prefer BH as optimal medium, except for Pseudoalteromonas sp. Bl46 and Cellulophaga sp. Ss85, which grew only in MSM supplemented with diesel oil. Except for Tenacibaculum sp. Mc99 that grew well in BH in the presence of both hydrocarbon sources, all strains generally 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 Bl55 on tetradecane. Conversely, Citricoccus sp. Bl54 was able to grow only in BH in the presence of tetradecane. In case of optimal growth in both media and carbon sources, those conditions, at which individual strains reached faster the exponential phase, were chosen. Influence of NaCl concentration and temperature on BS activity To investigate the influence of NaCl and temperature on BS activity, isolates were grown in the optimal medium in the presence of the preferred carbon source. The BS activity was determined by monitoring the production of emulsions and

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stable emulsions, and the surface tension reduction. All the tested strains positively responded to each test. However, only results obtained from strain belonging to genera that have been poorly reported in literature in relation to BS production, or results that highlight a great strength or speediness of interfacial activity will be showed (Fig. 1a–f). Overall, the BS activity was mainly influenced by the concentration 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 Joostella affiliates was analysed in BH amended with tetradecane, except for the strains Joostella spp. A9 and A22, incubated in the presence of diesel oil. Joostella spp. A15, A24, A29 and A30 were not able to produce stable emulsions or emulsions in the absence of NaCl. Conversely, they showed emulsifying activity in the presence of salt, reaching E 24 values that ranged from 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 stable emulsion in the absence of NaCl, but the value increased in the presence of salt. Among Joostella affiliates, the strain A8 gave the best results (Fig. 1a). It recorded the maximum value of E 24 (72.5 %) after 240 h incubation at 15 °C with NaCl in the medium, compared with a minimum value of 5 % obtained for 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 °C with NaCl in the medium, followed by Joostella spp. A3 and A22 that determined a reduction of the surface tension of 33.65 (from 66.5 to 32.85 mN/m) and 21.45 units (from 51.5 to 30.05 mN/m), respectively, during incubation at 15 °C in the presence of NaCl. Finally, Joostella spp. A15, A24, A29 and A30 strongly reduced the surface tension (>20 mN/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 the presence of salt. The emulsifying capacity appeared after 48 h at 25 °C, with a maximum E 24 value of 54 %, and after 96 h at 15 °C, with a maximum of 55 % and the maximum surface tension reduction. Contrary to Joostella spp., isolates that belonged to the genus Cellulophaga did not provide promising results during the second step of the work, as it showed an inadequate growth in all tested conditions. All strains recorded low and sporadic values of emulsion during the incubation period. Exception was Cellulophaga sp. A55 that produced emulsions and stable emulsions in the presence of NaCl, and reduced the surface tension of 19.3 units (from 50.5 to 31.2 mN/m). Among the Gammaproteobacteria , the efficiency of Marinobacter sp. A1 (Fig. 1b) was greater at 15 °C in the absence 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

Enrichments

Polychaetes

Strain

Cohaesibacter sp. A25 Pseudovibrio sp. A27 Thalassospira sp. A46 Thalassospira sp. A57 Alcanivorax sp. A52 Alcanivorax sp. A53 Cobetia sp. A20 Marinobacter sp. A1 Idiomarina sp. A19 Pseudomonas sp. A6 Pseudomonas sp. A14 Pseudomonas sp. A18 Pseudomonas sp. A45 Joostella sp. A2 Joostella sp. A3 Joostella sp. A8 Joostella sp. A9 Joostella sp. A11 Joostella sp. A15 Joostella sp. A17 Joostella sp. A22 Joostella sp. A24 Joostella sp. A29 Joostella sp. A30 Joostella sp. A32 Cellulophaga sp. A49 Cellulophaga sp. A50 Cellulophaga sp. A51 Cellulophaga sp. A55 Cellulophaga sp. A60 Pseudoalteromonas sp. Bl46 Pseudoalteromonas sp. Bl65 Pseudoalteromonas sp. Ss86 Pseudoalteromonas sp. Ss89 Psychrobacter sp. Bl39 Vibrio sp. Bl49 Alteromonadaceae sp. Ss76 Cellulophaga sp. Ss85 Cellulophaga sp. Ss91 Cellulophaga sp. Mc108 Cellulophaga sp. Ss88 Maribacter sp. Ss71 Maribacter sp. Ss79 Tenacibaculum sp. Mc99 Citricoccus sp. Bl52 Citricoccus sp. Bl54 Citricoccus sp. Bl55 Staphylococcus sp. Ss67

Origin

Mc-E Mc-E Ss-E Bl-E Bl-E Bl-E Mc-E Mc-E Mc-E Mc-E Mc-E Mc-E Ss-E Mc-E Mc-E Mc-E Mc-E Mc-E Mc-E Mc-E Mc-E Mc-E Mc-E Mc-E Mc-E Bl-E Bl-E Bl-E Bl-E Bl-E B. luctuosum B. luctuosum S. spallanzanii S. spallanzanii B. luctuosum B. luctuosum S. spallanzanii S. spallanzanii S. spallanzanii M. claparedei S. spallanzanii S. spallanzanii S. spallanzanii M. claparedei B. luctuosum B. luctuosum B. luctuosum S. spallanzanii

Culture medium BH T

BH D

MSM T

MSM D

+++ ++ − − ++ ++ − +++ +++ +++ +++ + − +++ +++ +++ +++ +++ +++ − − +++ +++ +++ − + − − − + − +++ − +++ − + − −

+++ ++ − ++ − ++ +++ + +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + +++ + +++ +++ + + +++ +++ +++ − +++ ++ − +++ ++ +++ − ++ − + +++ − +++ ++ +++ ++ +++

+++ − + − ++ ++ − − +++ +++ +++ − − − +++ +++ +++ − − − − +++ +++ +++ − + − − − +++ − − − − − ++ − − − − − − +++ + − − − −

++ +++ − − − + +++ − +++ + + ++ ++ +++ ++ +++ ++ ++ +++ ++ +++ ++ ++ +++ ++ ++ +++ ++ ++ +++ ++ − − − ++ ++ ++ +++ − − − − − − − − ++ ++

+ − +++ +++ +++ ++ +++ − +++

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 diesel oil, MSM T mineral salt medium plus tetradecane, MSM D 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 highest value of emulsion in the absence of NaCl (E 24 index 52.5 % at 15 °C and 25 °C after 288 and 240 h incubation) in BH amended with diesel oil. Surface tension was reduced from 57.5 to 36.85 mN/m and from 50.5 to 30.5 mN/m during incubation at 15 and 25 °C, respectively. An average E 24 value of 14.15 % was obtained during growth at 25 °C in the presence of NaCl. The BS activity by Pseudomonas spp. A6, A14, A45 (in incubation in BH supplemented with tetradecane) and A18 (BH plus diesel oil) was influenced by the addition of NaCl in the medium. Among the Pseudomonas isolates, A18 was strongly influenced by temperature. Only in the presence of NaCl at 25 °C it showed values of stable emulsion higher than 50 % and a good reduction of surface tension. The highest values of stable emulsion index were obtained for Pseudomonas sp. A45, with an E 24 of 68.3 % after 240 h at 15 and 25 °C in the presence of NaCl (Fig. 1c) and A18, with an E 24 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 and final value of 20.5 mN/m, under incubation at 25 °C with addition of NaCl, followed by Pseudomonas sp. A14 with a reduction 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 the presence of it they produced stable emulsion values higher than 50 % and reduced the surface tension of approximately 20 units during incubation in BH supplemented with tetradecane. Cobetia sp. A20 was incubated in BH supplemented with diesel oil, and showed better results at 25 °C, with the maximum emulsion and stable emulsion values in the presence of NaCl, while the maximum reduction of surface tension was recorded in the absence of salt at 15 °C with a reduction from 55 to 24.14 mN/m. The strain Idiomarina sp. A19 (in BH amended with tetradecane) showed emulsifying capacity after 336 h of incubation at 25 °C in the presence of NaCl, and created a stable emulsion with an E 24 index of 36.65 %, and a surface tension reduction of 30.05 units, from 72.65 to 42.6 mN/m. Among the Alphaproteobacteria, Thalassospira sp. A46 proved to be more able in producing stable emulsions in the presence of NaCl after 144 h of incubation at 15 °C (maximum E 24 of 70 %) and 25 °C (maximum E 24 of 48.3 %) in BH supplemented with tetradecane. The values of 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 at 25 °C. Temperature did not influence considerably the emulsion production and stabilisation. Cohaesibacter sp. A25 produced emulsions and stable emulsions only in the presence of NaCl (incubated in BH supplemented with tetradecane), but achieved E 24 of nearly

2995 Fig. 1 a Joostella sp. A8. Effect of salinity (NaCl, 0 and 3 %) on BS„ activity and bacterial growth during incubation under optimal conditions: BH added with tetradecane (2 %) at 15 (a ) and 25 °C (b ); b Marinobacter sp. A1. Effect of salinity (NaCl, 0 and 3 %) on BS activity and bacterial growth during incubation under optimal conditions: BH added with diesel oil (2 %) at 15 (a) and 25 °C (b); c Pseudomonas sp. A45. Effect of salinity (NaCl, 0 and 3 %) on BS activity and bacterial growth during incubation under optimal conditions: BH added 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 bacterial growth during incubation under optimal conditions: BH added with tetradecane (2 %) at 15 (a) and 25 °C (b); e Vibrio sp. Bl49. Effect of salinity (NaCl, 0 and 3 %) on BS activity and bacterial growth during incubation under optimal conditions: BH added with tetradecane (2 %) at 15 (a) and 25 °C (b); f Cellulophaga sp. Mc108. Effect of salinity (NaCl, 0 and 3 %) on BS activity and bacterial growth during incubation under optimal conditions: BH added with tetradecane (2 %) at 15 (a) and 25 °C (b). Note the different scales

50 % only during incubation at 25 °C, when the highest surface tension reduction was recorded (from 60.65 to 33.95 mN/m). Pseudovibrio sp. A27 grew more rapidly at 25 °C, with higher values of optical density, by reducing also the surface tension and showing greater emulsifying capacity at lower temperature. It showed positive responses to the enhancment study, with a range of stable emulsions between 17.5 and 22.5 % during growth on BH plus tetradecane, in the presence of NaCl and incubation at 15 °C (optimal temperature). The reduction of the surface tension was of 36.75 units compared to initial time. No growth was recorded in the absence of salt in the culture medium. Among the strains isolated from natural samples, only few positively responded to the enhancement tests. Among the Gammaproteobacteria , Pseudoalteromonas sp. Ss86 was the most promising producer with maximum values of E 24 at 25 °C in the absence of NaCl (49.8 %), and a surface tension reduction from 54.5 to 28.5 mN/m at the same conditions (in BH amended with tetradecane). Pseudoalteromonas sp. Bl65 achieved an E 24 of 20 % in the presence of salt at 15 °C, but it did not show other remarkable results for BS production. The absence of salt in the culture medium was a limiting aspect at 15 °C for Vibrio sp. Bl49 (Fig. 1e) which was incubated in BH supplemented with tetradecane. Such strain showed a performance that resulted higher in the presence of NaCl at 25 °C with a maximum E 24 value of 51.65 % after 192 h of incubation, and a surface tension reduction of more than 20 units. Anyway, at 25 °C it showed production both in the absence and presence of salt. Among the CFB group of Bacteroidetes, Tenacibaculum sp. Mc99 and Cellulophaga sp. Mc108 (Fig. 1f) showed most remarkable results in BH amended with tetradecane. In particular, the BS activity of such strains was significantly influenced by salinity, as they were able to produce higher

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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 emulsions and stable emulsions at both temperatures (maximum E 24, 62.5 %; 25 °C after 192 h of growth), whereas it did not record significant reductions of surface tension. Cellulophaga sp. strain Mc108 had a similar response to salinity variation, but the emulsifying capacity assumed an increasing profile when the strain was incubated at 15 °C, and a decreasing profile when incubated at 25 °C. Temperature did not affect the surface tension reduction. Finally, among Gram-positive isolates (Firmicutes and Actinobacteria affiliates) Staphylococcus sp. Ss67 gave positive results only at 15 °C in the presence of NaCl, and achieved a maximum E 24 value (57 %) after 192 h of incubation in BH plus tetradecane. Staphylococcus sp. Ss67 did n0t show promising results, if not exclusively at 15 °C in the presence of salt, while in the other conditions showed no growth. Strains affiliated to the genera Citricoccus and Maribacter, in addition to strains Ss76 among the Alteromonadaceae and Psychrobacter sp. Bl39, did not show remarkable results for BS production. Figure 2 shows more clearly the effect of salinity and temperature on E 24 maximum value obtained for isolates that were representative of each phylum and derived from both enrichment cultures and natural samples. All strains produced higher stable emulsion percentages in the presence of salt, so highlighting the strong influence of NaCl concentration (Fig. 2a). Exceptions were Cobetia sp. A20 and Vibrio sp. Bl49, which were able to produce stable emulsions during incubation at both NaCl concentrations. Moreover, the different results obtained for Marinobacter sp. A1 are highlighted, as it appears from the E 24 value obtained in the absence of salt. About the effect of temperature, the figure shows that only in some cases (e.g. Cellulophaga sp. A55 and Staphylococcus sp. Ss67) temperature influenced the production of stable emulsion by the isolates (Fig. 2b). Course of BS activity The course of BS activity was expressed as the relationship between the capacities of selected isolates to create stable emulsions and emulsifying activity. This course during incubation under optimum conditions is shown in Fig. 3. The course of BS activity showed a similar trend without a direct correspondence between the optical density and emulsions or E 24. On the basis of this, depending on the time required for emulsification and stable emulsion production, the kinetics was expressed in terms of ratio E 24/emulsion. One observed course type displayed by most strains and the profile obtained for Joostella sp. A8 is shown as an example in Fig. 3a. The profile of the ratio E 24/emulsions had a very

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low value at the initial stage (when only emulsions were produced, not accompanied by stable emulsions), and the maximum E 24/emulsions ratio (i.e. about 4.88, with a value of E 24 of 61 % and a value of emulsions of 13 %) appeared after 192 h of incubation. Then stable emulsions disappeared and the ratio decreased. The second course type displayed is well exemplified by Alcanivorax sp. A52, which produced emulsions and stable emulsions simultaneously, by achieving the maximum E 24/ emulsions ratio (E 24 of 27.5 % and emulsion percentage of 12.5 %) as the beginning of the emulsion appearance after 192 h of incubation (Fig. 3b). In the case of Vibrio sp. Bl49 and Tenacibaculum sp. Mc99 (Fig. 3c, d, respectively) the E 24/emulsions ratio started from 0 at the beginning, when the strains started to emulsify, and no stable emulsions were produced. Then the stable emulsions occurred and the ratio E 24/emulsions increased with higher values of 2.20 (E 24 of 47.5 % and emulsion percentage of 21.5 %) and 2.84 (E 24 ≈48.3 %; emulsion percentage≈17 %), respectively, for Vibrio sp. Bl49 and Tenacibaculum sp. Mc99, after 240 h of incubation. As an example, the E 24/emulsions ratios at 15 and 25 °C in the absence of NaCl for Marinobacter sp. A1 are shown in Fig. 4. During incubation at 15 °C the maximum E 24/ emulsions ratio (i.e. 0.72, with a value of E 24 of 52.5 % and a value of emulsions of 72.5 %) was recorded. The same strain showed the maximum value of optical density after 288 h (i.e. 0.609) during incubation at 25 °C, in concomitance with the maximum E 24/emulsions ratio (i.e. 0.91, of E 24 of 21 %; emulsification ability of 23 %). The surface tension reduction was differently related to the emulsifying ability of the strains. In some cases higher emulsion values corresponded to a greater lowering of surface tension, as in the case of Pseudoalteromonas sp. Ss86 and Vibrio sp. Bl49. In other cases, despite some strains showed emulsifying activity, they did not generally reduce the surface tension (e.g. Tenacibaculum sp. Mc99 at 25 °C and 3 % NaCl). In the absence of emulsifying activity, the surface tension generally assumed values more or less stable, without undergoing a considerable reduction. Cellulophaga sp. Mc108 and Vibrio sp. Bl49 were exceptions. The former was capable of reducing surface tension of 19.3 mN/m at 15 °C in the absence of NaCl, and the latter reduced the surface tension of 32.4 units although it did not show emulsifying capacity.

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

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Fig. 2 Effect of NaCl concentration (a) and temperature (b) on E 24 index that was produced by representative strains. Please note that only E 24 maximum value obtained from each strain is reported here. E 24