Isolation and characterization of Halomonas sp. strain C2SS100, a ...

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tion on solid medium. Aliquots (100 ll) of 10. )1 to 10. )10 dilutions were plated onto agar basal medium with crude oil (1% v⁄v) as carbon source. The plates were ...

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Isolation and characterization of Halomonas sp. strain C2SS100, a hydrocarbon-degrading bacterium under hypersaline conditions S. Mnif, M. Chamkha and S. Sayadi Laboratoire des Bioproce´de´s, Poˆle d’Excellence Re´gional AUF, (PER-LBP), Centre de Biotechnologie de Sfax, Sfax, Tunisie

Keywords biodegradation, crude oil, Halomonas sp., halotolerant, hydrocarbons, production water. Correspondence Mohamed Chamkha, Laboratoire des Bioproce´de´s, Poˆle d’Excellence Re´gional AUF, (PER-LBP), Centre de Biotechnologie de Sfax, B.P. ‘1177’, 3018 Sfax, Tunisie. E-mail: [email protected]

2008 ⁄ 1271: received 23 July 2008, revised and accepted 13 January 2009 doi:10.1111/j.1365-2672.2009.04251.x

Abstract Aims: To isolate and characterize an efficient hydrocarbon-degrading bacterium under hypersaline conditions, from a Tunisian off-shore oil field. Methods and Results: Production water collected from ‘Sercina’ petroleum reservoir, located near the Kerkennah island, Tunisia, was used for the screening of halotolerant or halophilic bacteria able to degrade crude oil. Bacterial strain C2SS100 was isolated after enrichment on crude oil, in the presence of 100 g l)1 NaCl and at 37C. This strain was aerobic, Gram-negative, rod-shaped, motile, oxidase + and catalase +. Phenotypic characters and phylogenetic analysis based on the 16S rRNA gene of the isolate C2SS100 showed that it was related to members of the Halomonas genus. The degradation of several compounds present in crude oil was confirmed by GC–MS analysis. The use of refined petroleum products such as diesel fuel and lubricating oil as sole carbon source, under the same conditions of temperature and salinity, showed that significant amounts of these heterogenic compounds could be degraded. Strain C2SS100 was able to degrade hexadecane (C16). During growth on hexadecane, cells surface hydrophobicity and emulsifying activity increased indicating the production of biosurfactant by strain C2SS100. Conclusions: A halotolerant bacterial strain Halomonas sp. C2SS100 was isolated from production water of an oil field, after enrichment on crude oil. This strain is able to degrade hydrocarbons efficiently. The mode of hydrocarbon uptake is realized by the production of a biosurfactant which enhances the solubility of hydrocarbons and renders them more accessible for biodegradation. Significance and Impact of the Study: The biodegradation potential of the Halomonas sp. strain C2SS100 gives it an advantage for possibly application on bioremediation of water, hydrocarbon-contaminated sites under high-salinity level.

Introduction During exploration, production, refining, transport and storage of petroleum and petroleum products, some accidental spill could be occur. This had as consequence a contamination of several soil area and sea water which had a very bad effect on the fauna and flora (Margesin and Shinner 2001). In order to control the environmental risks caused by petroleum products, various new regulations have been introduced and research focusing on

remediation of contaminated soils has increased (Chaineau et al. 2003). Bioremediation has a great potential as an alternative method for the restoration of contaminated sites. The use of natural micro-organisms was the first mechanism by which hydrocarbons could be eliminated from contaminated sites (Wongsa et al. 2004; Das and Mukherjee 2007). The presence of micro-organisms with the catabolic potential to degrade the target pollutant is essential. Many efforts were provided to isolate aerobic micro-organisms which can degrade various compounds

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of hydrocarbons such as bacterial strains belonging to the genera Pseudomonas, Mycobacterium, Sphingomonas and Arthrobacter (Kelley et al. 1990; Sutherland et al. 1995). Moreover, it is important to understand the complex interdependences between the micro-organism degrading the pollutant and the factors limiting and ⁄ or supporting the process of biological breakdown, such as biological availability, toxicity, temperature, pH, salinity, pressure and nutrients (Vogel 1996; Yang et al. 2000; Kato et al. 2001). Production waters collected from petroleum reservoirs, which can contain halophilic and thermophilic bacteria, constitute a promising biotope for the isolation of bacterial strains with high degradation potential under extreme conditions of salinities and temperatures (Nazina et al. 2005; Da Cunha et al. 2006). The low-aqueous solubility of hydrocarbons including aliphatics, mono- and polycyclic-aromatics, increases their recalcitrance to bacteria and limits their biodegradation. Addition of surfactants increases the availability of these compounds to microbes and renders them more accessible for microbial enzyme systems (Iwabuchi et al. 2002; Banat et al. 2004). Some bacterial strains have the capacity to produce biosurfactants such as Pseudomonas aeruginosa, Halomonas sp., Rhodococcus sp. and Bacillus subtilis (Olivera et al. 2000; Pepi et al. 2005; Das and Mukherjee 2007; Peng et al. 2007). To our knowledge, reports on the degradation of hydrocarbons under hypersaline conditions are still few (De Carvalho and Da Fonseca 2005; Li et al. 2006). In the present study, we isolated and characterized from the production water of a Tunisian off-shore oil field, a hydrocarbon-degrading bacterium under hypersaline conditions. Materials and methods Chemicals Complex hydrocarbons including crude oil, diesel fuel and lubricating oil were obtained from ‘Thyna Petroleum Services’ (TPS) and Shell companies (Sfax, Tunisia). Simple hydrocarbons used as carbon sources were octane, decane, hexadecane, methylcyclopentane, methylcyclohexane, heptamethylnonane, benzene, toluene, ethylbenzene, m-, o- and p-xylenes, naphthalene, carbazole, anthracene and phenanthrene. The products were purchased from Sigma-Aldrich. Culture media The basal medium contained 0Æ5 g KH2PO4, 0Æ4 g NH4Cl, 0Æ33 g MgCl2 6H2O, 0Æ05 g CaCl2 2 H2O, 0Æ5 g yeast 786

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extract and 1 ml trace-element solution (Widdel Pfennig 1981) per litre of distilled water. The pH adjusted to 7 with 10 mol l)1 KOH solution. medium was sterilized by autoclaving at 121C 20 min. Crude oil was added at 1% (v ⁄ v), as carbon energy source.

and was The for and

Enrichment and isolation procedure ‘Sercina’ petroleum reservoir is an off-shore reservoir, located near the Kerkennah island, Tunisia. It is a geothermal oil field with a temperature of approximately 80C. The production water was collected on December 2006, from a marine pipeline of about 30 km, and had a temperature of 25C, a salinity of 71 g l)1 and a pH of 7Æ8. Production water was used for the screening of aerobic bacterial strains degrading hydrocarbons under saline conditions. Four enrichment cultures named S1, S2, S3 and S4 with NaCl concentrations of 10, 50, 100 and 150 g l)1, respectively, were realized by the addition of 1 ml production water to 49 ml basal medium containing crude oil (1% v ⁄ v). Enrichment culture was sub-cultured under aerobic conditions (150 rev min)1) and at 37C until the degradation of crude oil. The degradation of crude oil was studied by measuring OD at 600 nm, according to Barathi and Vasudevan (2001), comparing emulsion evolution and GC–MS analysis. Activity was only absent in the enrichment culture at 150 g l)1 NaCl. Enrichment culture at 100 g l)1 NaCl was used for isolation on solid medium. Aliquots (100 ll) of 10)1 to 10)10 dilutions were plated onto agar basal medium with crude oil (1% v ⁄ v) as carbon source. The plates were incubated at 37C, under aerobic conditions for 3 days, until colony formation. Five Single colonies were picked and used for screening. Individual colonies were purified by repeated streaking on agar basal medium containing 1% (v ⁄ v) crude oil. The purity of the strains, their shape and motility were analysed using a phase contrast microscope OLYMPUS BX50. Strain C2SS100 was chosen for further investigation because it showed the maximum crude oil biodegradation potentiality in the basal medium, without yeast extract addition, and in the presence of 100 g l)1 NaCl. Growth of strain C2SS100 Growth studies were performed in flask cultures containing 50 ml basal medium with 100 g l)1 NaCl, under agitation of 150 rev min)1 and at 37C. All experiments were performed in duplicate with an inoculum size of 3% (v ⁄ v), which had been sub-cultured at least once under the same conditions. Growth of C2SS100 on hydrocarbons was verified in liquid medium by measuring of OD

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at 600 nm, by GC–MS analysis and by determination of Colonies Forming Units (CFU) by plating onto LuriaBertani agar medium containing 100 g l)1 NaCl (LB: 10 g l)1 tryptone, 5 g l)1 yeast extract and 100 g l)1 NaCl) at different times of culture. The results were expressed in log CFU ml)1. In order to enhance the degradation of hydrocarbons present in crude oil, Tween 80 (0Æ05% v ⁄ v) was added to the culture medium as a synthetic surfactant, as described previously (Franzetti et al. 2008). Characterization of strain C2SS100 The NaCl concentration range for growth of strain C2SS100 was determined by weighing in flasks different amounts of NaCl prior to dispensing 50 ml basal medium to obtain the desired NaCl concentration (from 0 to 20% w ⁄ v). Optical density at 600 nm was followed as a measurement of growth, and data from the mid-log phase growth of the strain were selected for calculating the specific growth rates. The optimization of growth temperature was done by culture incubation at temperature range between 0 and 55C. The pH of the medium was adjusted with 5 mol l)1 HCl or 10 mol l)1 to obtain a range between 4 and 12. The Gram reaction was determined using the BioMe´rieux Gram stain Kit according to the manufacturer’s instructions. Catalase activity was determined by bubble production in 3% (v ⁄ v) hydrogen-peroxide solution. Oxidase activity was determined by oxidation of 1% p-aminodimethylaniline oxalate. Diesel fuel and lubricating oil were added at a concentration of 1% (v ⁄ v) and the growth curves for each compound were determined after an adaptation time of the strain for growing on these compounds. For adaptation, strain C2SS100 was cultivated three times on basal medium supplemented with diesel fuel or lubricating oil before being cultured on fresh new basal medium containing each of the substrates. Simple hydrocarbons were added directly into flasks containing 50 ml basal medium, without yeast extract added, in order to study the ability of strain C2SS100 to degrade these compounds. The aliphatic hydrocarbons including octane, decane and hexadecane, were used at a concentration of 0Æ5% (v ⁄ v). The cycloalcane hydrocarbons including methylcyclopentane and methylcyclohexane were used at 0Æ5% (v ⁄ v). The following aromatic hydrocarbons: anthracene, phenanthrene and carbazole were added at 200 mg l)1 and naphthalene was added at 300 mg l)1. An increase in the OD600 nm in substrate-containing cultures, compared with control tubes lacking substrates, was considered as positive growth.

A hydrocarbon degrading Halomonas sp.

16S rRNA sequencing and phylogenetic analysis The 16S rRNA gene of the isolate C2SS100 was amplified by PCR using a Stratagene PCR system (Robocycler gradient 96) with GoTaq DNA polymerase (Promega, Madison, WI, USA) as described previously (Chamkha et al. 2002). The universal primers Fd1 and Rd1 (Fd1, 5¢-AGAGTTTGATCCTGGCTCAG-3¢; Rd1, 5¢-AAGGAG G-TGATCCAGCC-3¢) (Weisburg et al. 1991) were used to obtain a PCR product of approximately 1Æ5 kb corresponding to base position 8-1542, based on Escherichia coli numbering of the 16S rDNA gene (Winker and Woese 1991). Sequence data were imported into the sequence editor Bioedit version 5.0.9 (Hall 1999). The full sequence was aligned using the RDP Sequence Aligner Program (Maidak et al. 2001). The consensus sequence was manually verified to conform to the 16S rRNA secondary structure model (Winker and Woese 1991). Sequences used in the phylogenetic analysis were obtained from the RDP and GenBank databases (Benson et al. 1999; Maidak et al. 2001). Positions of sequence and alignment ambiguity were omitted and pairwise evolutionary distances were calculated using the method of Jukes and Cantor (1969). A dendrogram was constructed using the neighbour-joining method (Saitou and Nei 1987). Confidence in the dendrogram topology was determined using 100-bootstrapped trees (Felsenstein 1985). GC–MS analysis Samples (50 ml) of culture C2SS100 containing hydrocarbons and an abiotic control were extracted three times with dichloromethane (DCM). The organic fraction was evaporated, dissolved in equal volume of DCM and then analysed by gas-chromatography mass-spectrometry. GC– MS was performed with a Hewelett-Packard model 6890N chromatograph apparatus equipped with a capillary Hewelett-Packard HP-5 column (length, 30 m; internal diameter, 250 lm; film thickness, 0Æ25 lm). The carrier gas was helium used at a flow rate of 1 ml min)1. The temperature was first set at 70C for 2 min and was increased to 230C at 20C min)1, then to 300C at 40C min)1 and finally set at 300C for 10 min. For detection of intermediates, extraction was performed three times with hexane. The combined organic phase was evaporated to dryness and then dissolved in 5 ml hexane. 200 ll of the organic extract were derivatized using 170 ll BSTFA (bis (trimethylsilyl)-trifluoroacetamide containing trimethylchlorosilane 1%; Sigma) and 30 ll pyridine. One microlitre of the solution obtained was analysed by GC–MS using the same program as described above.

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Cell surface hydrophobicity and emulsifying activity Cell surface hydrophobicity of strain C2SS100 was measured by a bacterial adhesion to hexadecane according to the method of Rosenberg et al. (1980). Strain C2SS100 was grown in 50 ml basal medium containing hexadecane (0Æ5% v ⁄ v) or glucose (20 mmol l)1) as sole carbon source. Bacterial cells were washed twice and resuspended in PUM buffer, pH 7Æ1 (22Æ2 g K2HPO4 3 H2O, 7Æ26 g KH2PO4, 1Æ8 g urea, 0Æ2 g MgSO4 7 H2O and 1 l distilled water) to an initial absorbance at 550 nm of 0Æ5–0Æ6. Hexadecane (0Æ2 ml) was added to 1Æ2 ml of the cell suspension. The solution obtained was dispensed into a round-bottom test tube (i.d. 10 mm) and was vortexed vigorously for 2 min. After allowing 1 h at room temperature for the phases to separate, the lower aqueous phase was carefully removed with a Pasteur pipette and its turbidity at 550 nm measured. Hydrophobicity was expressed as the percentage of adherence to the hydrocarbon that was calculated as follows: 100· (1 - OD of the aqueous phase ⁄ OD of the initial cell suspension). Emulsifying activity was determined according to a method described by Berg et al. (1990). Sample (0Æ2 ml) of C2SS100 culture in the presence of (0Æ5% v ⁄ v) hexadecane, as carbon source, was mixed with 0Æ5 ml of TM buffer (20 mmol l)1 Tris–HCl buffer, pH 7Æ0 and 10 mmol l)1 MgSO4), then 0Æ1 ml of hexadecane was added. The test tube was vortexed for 1 min and the turbidity of the aqueous phase was measured at 550 nm after incubation for 1 min at room temperature. One unit of emulsifying activity was defined as the amount of the emulsifier required to obtain an increase in absorbance of 1Æ0 OD unit.

Results Isolation of halophilic crude oil degrading bacterium All enrichment cultures initiated in basal medium containing 1% (v ⁄ v) crude oil as the carbon and energy source, except enrichment S4 (150 g l)1 NaCl), became turbid and the dark layer of crude oil became clear, indicating the degradation of this substrate. Enrichment culture S3, in the presence of 100 g l)1 NaCl, showed the presence of aerobic bacteria with diverse cellular morphologies and then was used for the isolation of pure bacterial strains in solid media. Five colonies were picked and a pure culture designed C2SS100 was selected. The selection of the strain was based on its high capacity to degrade crude oil (1% v ⁄ v), in solid and liquid media, without yeast extract added and in the presence of 100 g l)1 NaCl. The strain C2SS100 was used for further characterization. Characterization of strain C2SS100 Bacterial strain C2SS100 was aerobic, Gram-negative, rodshaped, motile, nonsporulated bacterium, oxidase + and catalase +. The cells were found both singly and double. Colonies of the strain were small, yellow-cream and smooth and 1–2 mm in diameter after overnight culture at 37C. Strain C2SS100 was able to grow over a temperature range of 30–45C with optimum growth at 37C. The NaCl concentration range for growth was between 0 and 150 g l)1, with an optimum at 50–80 g l)1 NaCl. Crude oil degradation activity was maintained on basal medium only until 140 g l)1. The study of the strain

0·01

Halomonas pantelleriensis DSM 9661T (X93493)

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Halomonas muralis DSM 14789T (AJ276807)

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Halomonas lutea YIM 91125T (EF674852)

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Strain C2SS100 (EU601735) Halomonas anticariensis LMG 22089T (AY489405)

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Halomonas taeanensis BH 539T (AY671975) Halomonas gudaonensis LMG 23610T (DQ421808) Halomonas campaniensis DSM 15293T (AJ515365)

56 83

Halomonas salina ATCC 94509T (AJ243447)

100 Halomonas ventosae CECT 5797T (AY268080) Modicisalibacter tunisiensis CCUG 52917T (DQ641495)

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Figure 1 Phylogenetic tree based on 1039 unambiguous nucleotides of the 16S rRNA sequence, constructed by the neighbourjoining method, showing the position of strain C2SS100 among related members of the genus Halomonas. Reference type-strain organisms are included and sequence accession numbers are given in parentheses. Bootstrap values, expressed as percentage of 100 replications, are shown in branching points. Bar, 1 substitution in 100 nt.

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A hydrocarbon degrading Halomonas sp.

Table 1 Differential phenotypic characteristics of strain C2SS100 and other related type strains of the genus Halomonas

Characteristics Morphology Pigmentation NaCl range (%, w ⁄ v) NaCl optimum (%, w ⁄ v) pH range Temperature range (C) Temperature optimum (C) Oxidase Catalase Growth on Glucose Fructose Mannitol Mannose Lactose Cellobiose Lactate Pyruvate Origin

Halomonas sp. C2SS100

Halomonas lutea YIM 91125T (Wang et al. 2008)

Halomonas muralis DSM 14789T (Heyrman et al. 2002)

Halomonas taeanensis DSM 16463T (Lee et al. 2005)

Halomonas anticariensis LMG 22089T (Martı´nez-Ca´novas et al. 2004)

Short rods Yellow-cream 0–15 5–8 5Æ5–9 30–45 37 + + +

Short rods Orange 1–20 5–10 5–9 4–45 37 + + +

Rods Cream 0–15 2Æ5–10 5–10 10–35 25–35 + + +

Rods Cream 1–25 10–12 7–10 10–45 35 + + +

Rods Cream 0Æ5–15 7Æ5 6–9 20–45 ND + + +

+ + – – – + + Oilfield production water, Tunisia

+ + + + + ND + Salt lake, north-west China.

– ND +⁄+ – ND ND Walls and murals of the SaintCatherine Chapel, Austria

+ + w+ + ND ND ND Solar saltern at Taean, Korea

+ + + – – – ND Saline-wetland wildfowl reserve, southern Spain

+, positive; ), negative; w+, weakly positive; + ⁄ ), growth is variable; ND, not determined.

growth in the presence of some carbohydrates at a concentration of 20 mmol l)1 showed that it fermented glucose, fructose, maltose, sucrose, mannitol and glycerol but not lactose, sorbose and cellobiose. Strain C2SS100 used also formate (10 mmol l)1), lactate (10 mmol l)1), pyruvate (10 mmol l)1), decanoic acid (5 mmol l)1) and decanol (5 mmol l)1). To analyse the phylogenetic position, the 16S rRNA gene sequence of strain C2SS100

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(comprising 1039 nt) was determined, and a dendrogram based on 1039 unambiguous nt was constructed (Fig. 1). The 16S rRNA gene sequence of strain C2SS100 was deposited in the GenBank database under accession number EU601735. The phylogenetic analysis indicated that strain C2SS100 is closely related to the members of the genus Halomonas with Halomonas lutea (EF674852) (Wang et al. 2008) being the most closely related species (98Æ2% 16S rRNA gene sequence similarity). Differential phenotypic characteristics of strain C2SS100 and its closest neighbours in the genus Halomonas are given in Table 1.

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Crude oil biodegradation

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C2SS100 was able to degrade crude oil (1% v ⁄ v) in basal liquid and solid media, in the presence of 100 g l)1 NaCl. The growth of the strain on crude oil was followed by measuring the OD600 nm at different culture’s time (Fig. 2). GC–MS analysis showed that strain C2SS100 was active on the total aliphatic hydrocarbons present in crude oil (C11–C22) after 30 days of incubation (Fig. 3). This result was confirmed by diminution or total disappearance of the correspondent peak of each compound, as shown in

OD (600 nm)

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Figure 2 Growth of Halomonas strain C2SS100 on (r) crude oil (1% v ⁄ v), ( ) crude oil (1% v ⁄ v) + Tween 80 (0Æ05%) and ( ) Tween 80 (0Æ05%).

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Counts 190 000 180 000 170 000 160 000 150 000 140 000 130 000 C22 C21 C20 C19 C18 C17 C16 C15 C14 C13 C11

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Fig. 4. The strain C2SS100 degrades higher than 90% of crude oil present in the culture after 4 weeks incubation (Fig. 4). Addition of Tween 80 showed a significant acceleration of crude oil degradation. In fact, the growth curve of C2SS100 showed that stationary phase was reached for about 3 days only in the presence of Tween 80, whereas in the absence of this surfactant, 1 week was required (Fig. 2). The crude oil toxicity for growth of the strain C2SS100 was also studied, by measuring the strain growth at a crude oil concentration range between 0 and 40% (v ⁄ v). Strain C2SS100 can grow even in presence of 20% (v ⁄ v) crude oil. The optimum growth was observed in the presence of 2% (v ⁄ v) of crude oil. At 40% (v ⁄ v) crude oil became toxic and inhibited the growth of strain C2SS100 (data not shown). 790

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Figure 3 GC–MS profiles of crude oil remaining in basal medium after aerobic incubation, without (a) and with (b) strain C2SS100, at 37C for 4 weeks. C11 to C22 indicate n-alcanes with the number of carbon atoms from 11 to 22.

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% remaining of petroleum hydrocarbons

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C20

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C18

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190 000 180 000 170 000 160 000 150 000 140 000 130 000 120 000 110 000 100 000 90 000 80 000 70 000 60 000 50 000 40 000 30 000 20 000 10 000

80 60 40 20 0 Abiotic control

4 days

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30 days

Figure 4 Biodegradation of crude oil by strain C2SS100. Cells were cultivated in basal medium supplemented with 1% (v ⁄ v) crude oil.

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Residual hexadecane (%)

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OD (600 nm)

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Emulsifying activity (U ml–1)

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Figure 6 Evolution of emulsifying activity during degradation of hexadecane by strain C2SS100. ( ): Emulsifying activity. (r): Percentage of residual hexadecane.

(b) 10

log (CFU) ml

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Figure 5 Growth of strain C2SS100 on (r) crude oil (1% v ⁄ v), ( ) diesel fuel (1% v ⁄ v) and ( ) lubricating oil (1% v ⁄ v) determined by measuring the optical density at 600 nm (a) and by bacterial cell counts (b).

Growth on diesel fuel and lubricating oil In addition to its capacity to grow on crude oil, strain C2SS100 can grow on some refinery crude oil sub-products such as diesel fuel and lubricating oil, in the presence of 100 g l)1 NaCl (Fig. 5a,b). Crude oil was the best substrate to support bacterial growth, and strain C2SS100 reached the stationary phase within 1 week. In contrast, the growth of C2SS100 in the presence of lubricating oil and diesel fuel showed extended lag times, and approximately 10 days were required to reach stationary phase (Fig 5a). These results were confirmed also by enumeration of viable cells, indicating positive growth with the three substrates (Fig. 5b). These findings suggest that strain C2SS100 had the substrate preference as follows: crude oil > lubricating oil > diesel fuel (Fig. 5a,b). Biodegradation potential of individual hydrocarbons Bacterial strain C2SS100 was tested for its ability to grow on a variety of simple hydrocarbons, as sole carbon and energy source, in the presence of 100 g l)1 NaCl. The

strain was able to degrade hexadecane (0Æ5% v ⁄ v), an aliphatic hydrocarbon with 16 carbon atoms (Fig. 6). It was also able to degrade carbazole (200 mg l)1), an aromatic heterocyclic hydrocarbon, however, activity was weak. Tween 80 addition enhances this activity (data not shown). However, strain C2SS100 was not capable of degrading short aliphatic chain hydrocarbons such as octane (C8) and decane (C10). It was not also able to grow in the presence of two methylated cyclic structures, methylcyclopentane and methylcyclohexane, even after 12 days incubation. C2SS100 was able to grow aerobically at 37C, in the basal medium containing 100 g l)1 NaCl and supplemented with 0Æ5% (v ⁄ v) hexadecane as the sole carbon and energy source. The concentration of hexadecane in the culture decreased until substrate utilization was complete. Hexadecane was completely degraded after 12 days incubation (Fig. 6). After 6 days incubation, GC– MS profile showed the formation of a new peak corresponding to hexadecanoic acid. The new peak of this intermediate was maintained even at 8 days incubation but disappeared after 10 days incubation. During growth on hexadecane, emulsifying activity and cell surface hydrophobicity were increased (Figs 6 and 7). This result suggests that the strain has the capacity to produce biosurfactant that enhance hydrocarbon degradation. Discussion To carry out the bioremediation of hydrocarbon contaminated sites, it is very important to isolate and select bacteria possessing a high capacity to degrade many components of petroleum products. Many aerobic and nonhalophilic bacterial strains which have a degradation potential of hydrocarbons were isolated from different origins. These bacteria belong to the genera Pseudomonas,

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50 Cell hydrophobicity (%)

45 40 35 30 25 20 15 10 5 0

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100 120 140 160 180 200 Time (h)

Figure 7 Hydrophobicity of bacterial strain C2SS100 during growth in basal medium with 0Æ5% (v ⁄ v) hexadecane ( ) or 20 mmol l)1 (v ⁄ v) glucose (r) as a sole carbon source.

Bacillus, Dietzia and Mycobacterium (Peng et al. 2007). It have been reported that the majority of these hydrocarbon-degrading bacteria could not utilize crude oil if the medium contained even 30 g l)1 NaCl (Kapley et al. 1999). In fact, there is an inverse relationship between salinity and hydrocarbon biodegradation (Kumar et al. 2007). Nonetheless, there are several reports about halophilic micro-organisms able to oxidize petroleum hydrocarbons (Dı´az et al. 2002), even in the presence of 30% (w ⁄ v) NaCl (Margesin and Shinner 2001). A crude oil-degrading Streptomyces albiaxialis, and an n-alkane (C10–C30)-degrading Halobacterium have been described (Kuznetsov et al. 1992). An approach to extend the capacity of efficient hydrocarbon degraders for biodegradation in saline environments has been performed by impairing the phenotype of osmotolerance to a crude oil-degrading consortium, consisting of four Pseudomonas strains (Kapley et al. 1999). The E. Coli pro-U (an important osmoregulatory locus) operon was sub-cloned and expressed into each member of the consortium. The transformed organisms could grow only in the presence of 6% (w ⁄ v) NaCl. However, strain C2SS100 is able to degrade crude oil in the presence of 100 g l)1 NaCl. This strain, isolated from a production water of a petroleum reservoir after enrichment on crude oil, is an aerobic, mesophilic, extremely halotolerant, motile, Gram-negative, rod shaped, nonsporulated bacterium and phylogenetically is closely related to members of the Halomonas genus. An aerobic, moderately halophilic bacterium, strain Lit2, belonging to a new genera of Modicisalibacter was isolated previously from a Tunisian oil field waterinjection (Ben Ali Gam et al. 2007). An aerobic, thermophilic and halotolerant Geobacillus bacterium, strain C5, was isolated from another Tunisian high-temperature oil field. This strain C5 was able to degrade tyrosol, a wide 792

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range of other aromatic compounds, diesel and crude oil (Chamkha et al. 2008). Aerobic bacteria were isolated from oil fields and other subsurface horizons with temperatures from 20 to 70C. They come with injection waters, drilling solution or, probably due to the natural hydrodynamics of subsurface waters (Nazina et al. 2005). Other studies showed that Halomonas species can degrade complex hydrocarbons such as diesel fuel and crude oil under psychrotrophic conditions (Pepi et al. 2005). However, to our knowledge, no previous study has been reported the crude oil degradation under hypersaline conditions by Halomonas strains. Oil-oxidizing micro-organisms have been sampled in various regions of Siberia and used in strain associations, which degrade n-alkanes of oil from various fields by 64–92% after 6 days of growth in a wide temperature range. The combination of two hydrocarbon-degrading strains Pseudomonas sp. KL-1 and Yarrowia lipolytica NF5-1 (1:1) involved the biodegradation of 92% of crude oil (Andreeva et al. 2007). Strain C2SS100 has also the capability to degrade other crude oil refinery sub-products such as diesel fuel and lubricating oil in the presence of 100 g l)1 NaCl. The capacity to degrade crude oil by strain C2SS100 is higher than its capacity to degrade lubricating oil and diesel fuel. Crude oil constitutes the natural carbon source in natural environment of this strain. All components in crude oil are highly reduced by C2SS100. This strain particularly degrades n-alkane from C11 to C22 almost completely after 30 days of incubation. Lubricating oil also containing minor n-alkane of C10–C20 and major unidentified long chain from C20 to C35. However, the major component of diesel fuel is aromatic hydrocarbons including toluene (C7), dimethylbenzene (C8) and trimethylbenzene (C9). These compounds are more recalcitrant than aliphatic hydrocarbons present in crude oil and lubricating oil (Wongsa et al. 2004). Addition of the synthetic surfactant Tween 80 accelerates the degradation rate of crude oil by strain C2SS100. In fact, surfactant has the capacity to emulsify hydrocarbons and renders them more accessible for bacteria biodegradation (Calvo et al. 2004). Hexadecane biodegradation by strain C2SS100 was quantified by GC–MS analysis. Results showed that the totality of hexadecane was removed by the strain for about 12 day’s culture at the same condition of salinity (100 g l)1 NaCl). The utilization of hexadecane by strain C2SS100 suggested that the degradation pathway proceed via monoterminal oxidation with the formation of three central intermediates which are respectively: hexadecanol, hexadecanaldehyde and hexadecanoic acid (Kato et al. 2001). However, only hexadecanoic acid was detected in the culture medium. During growth on hexadecane, strain C2SS100 produces an emulsifying agent, which facilitates the assimilation of

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hydrocarbons. This observation suggests that surfactants ⁄ emulsifiers promote the degradation of petroleum components (Olivera et al. 2000). The mode of action of these compounds is on promoting cell surface hydrophobicity and emulsifying activity during growth on hexadecane and enhancing the bioavailability of this insoluble compound for bacterial biodegradation. In conclusion, we isolated and characterized from a production water of a Tunisian off-shore oil field, a Halomonas sp. strain C2SS100 after enrichment on crude oil under saline conditions. This strain is capable of degrading some aliphatic hydrocarbons present in crude oil efficiently. The halophilic character of this bacterium could add further advantages for its use in marine and saline environments-oil bioremediation. Acknowledgement This work was supported by a grant provided by the Tunisian Ministry of Higher Education, Scientific Research and Technology, the French Research Institute for Development (Program JEAI ‘BIOAROMEXT’) and the CMCU (Comite´ Mixte de la Coope´ration Universitaire Tuniso-Franc¸aise). Thanks to ‘Thyna Petroleum Services’ for providing the samples used in this study. We thank Ms Feten Bennour Mrad for her technical assistance in GC–MS. References Andreeva, I.S., Emel’yanova, E.K., Ol’kin, S.E., Reznikova, I.K., Zagrebel’nyi, S.N. and Repin, V.E. (2007) Consumption of hydrocarbons by psychrotolerant degrader strains. Appl Biochem Microbiol 43, 201–206. Banat, I.M., Marchant, R. and Rahman, T.J. (2004) Geobacillus debilis sp. nov., a novel obligately thermophilic bacterium isolated from a cool soil environment, and reassignment of Bacillus pallidus to Geobacillus pallidus comb. nov. Int J Syst Evol Microbiol 54, 2197–2201. Barathi, S. and Vasudevan, N. (2001) Utilization of petroleum hydrocarbons by Pseudomonas fluorescens isolated from a petroleum-contaminated soil. Environ Int 26, 413–416. Ben Ali Gam, Z., Abdelkafi, S., Casalot, L., Tholozan, J.L., Oueslati, R. and Labat, M. (2007) Modicisalibacter tunisiensis gen. nov., sp. nov., an aerobic, moderately halophilic bacterium isolated from an oilfield-water injection sample, and emended description of the family Halomonadaceae Franzmann et al. 1989 emend Dobson and Franzmann 1996 emend. Ntougias et al. 2007. Int J Syst Evol Microbiol 57, 2307–2313. Benson, D.A., Boguski, M.S., Lipman, D.J., Oullette, B.F.F., Rapp, B.A. and Wheeler, D.L. (1999) GenBank. Nucleic Acids Res 27, 12–17.

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