Molecular characterization of biosurfactant producing marine bacterium isolated from hydrocarbon-contaminated soil using 16S rRNA gene sequencing

2.1 Isolation of biosurfactant-producing marine bacteria by enrichment method

The sediment samples were collected from 6 spots with the designation SAN1, ADY2, KLM3, PRG4, TRP5 and KTR6 in and around the coastal line of Chennai harbor having an exposure history of crude oil, white oil and furnace oil since 1972 when the first oil terminal was commissioned. Sediments were collected in sterile polypropylene containers and transported to the laboratory within 2 h and stored at 4 °C until further process. Four samples were collected in different areas of each spot, and all four samples were pooled to make a single sample. The samples collected were shade dried, sieved and homogenized. Enrichment was done in minimal salt medium (MSM) with 2% diesel as the sole source of carbon in shaking condition at 150 rpm with constant transfer to fresh medium at an interval of 7 days for 3 weeks. Then the samples were spread plated on nutrient agar (Difco, USA) (pH 7.2 ± 0.2) prepared in sterilized seawater and incubated at 28 °C ± 2 for 1–2 days. After incubation, morphologically different colonies were selected and purified by repeated streaking and stored as slant cultures at 4–8 °C and glycerol stock (30%) at −80 °C for further studies (Maneerat and Phetrong, 2007).

2.2 Biosurfactant activity assays

2.3 Identification of strains and DNA extraction

Preliminary identification of the isolate was carried out using gram staining, motility and biochemical tests using conventional bacterial culture identification methods. The single pure colonies of the bacterial isolates were grown in Luria-Bertani (LB) broth overnight at 28 °C. 2 ml of the culture was centrifuged at 5000 rpm for 5 min and pellet was suspended in 200 µl of TE (pH = 8) containing RNase (50 ng /ml), then 400 µl of lysis buffer was added followed by mixed well and incubated for 15 min at 37 °C with intermittent shaking for every 5 min. Immediately chloroform and isoamylalcohol in the ratio (24:1) was taken and mixed by inversion. Tubes were centrifuged at 10000 rpm for 5 min, supernatant was transferred carefully to another microcentrifuge tube. To the supernatant, 0.1 vol 3 M sodium acetate (pH = 5.2) and 0.6 vol isopropanol were added, mixed well by inversion and kept in the ice for 10 min followed by centrifugation at 1000 rpm for 10 min. The pellet was washed with 70% ethanol with gentle shaking and centrifuged at 10000 rpm for 3 min. Supernatant was removed and pellet was air dried. Extracted DNA was visualized using 0.8% agarose gel electrophoresis and images were documented.

Using 16S ribosomal RNA gene specific universal primers 27F 5′-AGA GTT TGA TCC TGG CTC AG-3′ and 1492R 5′-GGT TAC CTT GTT ACG ACT T-3′ (Sigma), the 16S rRNA gene was amplified with 50 μl reaction mixture containing 1X reaction buffer (10 mM Tris [pH 8.3], 50 mM KCl and 1.5 mM MgCl₂), 200 μl dNTPs, 0.05 U Taq DNA polymerase enzyme (Sigma, USA), 0.5 μM of each primer and 1 ng template DNA. The thermal cycling conditions were: 5 min at 94 °C for initial denaturation; 31 cycles of 30 s at 95 °C, 1 min at 54 °C, 2 min at 72 °C, and a final extension for 5 min at 72 °C. The amplification reaction was performed with a thermal cycler (MyCycler, Bio-Rad, USA) and the PCR amplicons (approximately 1500 bp) were resolved by electrophoresis in 1% (w/v) agarose gel to confirm the expected size of the product.

2.4 Phylogenetic analysis

The 16 rRNA gene sequence obtained in this study was aligned with the sequences published in NCBI. Aligned sequences were edited and phylogenetic tree was constructed using MEGA6 software. The phylogenetic tree for relationship among strains was constructed by the Maximum Liklihood Method with a boot-strap of 500 using Kimura-2 parameter.

3.1 Enrichment and isolation of the bacterial isolates

Hydrocarbon contaminated sediment samples were collected from six different sites in and around the coastal line of Chennai harbour and designated as SAN1, ADY2, KLM3, PRG4, TRP5 and KTR6. They were kept for enrichment in Mineral Salt Medium (MSM) with 2% diesel as the sole source of carbon at 28 °C ± 2 for 21 days with constant shaking. Enriched samples were serially diluted and spread plated on nutrient agar amended with diesel oil. Morphologically seven distinct colonies (flat, large, irregular, opaque, light green pigmented, and non-pigmented) were selected, pure cultures were obtained and designated as SAN1a, ADY2a, ADY2b, KLM3c, PRG4a, TRP5a, KTR6b. The seven isolates were further subjected to screening of biosurfactants.

3.2 Screening for biosurfactant production

In the present study all the isolates were subjected to different types of screening methods given by Satpute et al. (2008) Among seven bacterial isolates, only four isolates (ADY2b, KTR6b, KLM3c and PRG4a) produced β-haemolysis. The remaining isolates did not show any haemolysis and the results were tabulated (Table. 1). Blood haemolysis has been chosen to be the preliminary screening method for biosurfactant production (Ghayyomi Jazeh et al., 2012). Safary et al. (2010) reported that while the other microbial products such as virulence factors lyse blood agar, the biosurfactants that are poorly diffusible will not lyse blood cells. However, blood agar lysis screening can be used as a rapid screening method, in which samples with the positive result are subsequently subjected to biosurfactant-activity tests in liquid media. In this study the isolates ADY2b, KLM3c, PRG4a and KTR6b that had shown complete haemolysis were subjected to further analysis for biosurfactant production.

The micro-organisms which produce biosurfactant can only displace the oil. The drop collapse test and oil displacement tests are indicative of the surface and wetting activities (Youssef et al., 2004). Oil displacement test is an indirect measurement of surface activity of a sample tested against diesel oil, a larger diameter represents a higher surface activity of the testing solution (Rodrigues et al., 2006). In oil displacement test all the four isolates, that had shown blood haemolysis, displaced the oil and produced zones showing various diameters and the results were tabulated (Table 1). The isolate ADY2b produced a clear zone of 3.7 cm showing better activity than the strain CpA 1 isolated by Safary et al. that had shown a zone of 3.1 cm. The halo zones formed by culture supernatants of the four isolates that were measured in centimetres are given graphically (Fig. 2). In microplate assay (drop collapse test) isolate ADY2b was the only strain to give positivity which is an indicative of biosurfactant production (Table 1 & Fig. 1a). The growth of bacteria on hydrocarbons is usually accompanied by the production of surfactants that helps in the adherence of the cells to oil droplets (Rosenberg and Rosenberg, 1981).

Close

Fig. 1

(a) Showing microplate test & (b) Emulsification Index (E₂₄).

Export to PPT

Close

Fig. 2

Showing the zone size produced by bacterial isolates in the oil displacement test.

Export to PPT

Emulsification index determines the productivity of bioemulsifiers (Bonilla et al., 2005). Thus, by examining the emulsification units, it is possible to select a potent biosurfactant/bioemulsifier producer and this is one of the important and effective assays for screening the biosurfactant/bioemulsifier producers as reported by Satpute et al. (2008). Strain ADY2b had shown better emulsification index of about 58.33% (Table 1 & Fig. 1b) when compared with that of Bacillus subtilis (20%) and Bacillus cereus (30%) on diesel oil reported by Jayasree et al. (2011). The emulsification index (E24) is given in Table 1. Since ADY2b had shown promising results for all the tests performed for biosurfactant production, this isolate was further subjected to microscopical, biochemical and molecular characterization.

3.3 Identification of the potent isolate

Microscopic and biochemical studies on the isolate ADY2b indicated that it was Gram-negative, motile, non-spore forming rod and had shown positive for catalase and oxidase production. Based on the above results, this strain was preliminarily identified as Pseudomonas sp. The genomic DNA of ADY2b (approximately 4–5 Mb) was extracted and confirmed through agarose gel electrophoresis. The 16S rRNA of the strain was amplified and sequenced. Sequencing results showed the gene size of 1441 base pairs. Sequence alignment of the 16S rRNA gene of the strain and Data base search revealed 98% similarity to Pseudomonas mendocina by BLAST analysis and the identified gene was submitted in the GenBank (Accession Number-KF378590). The phylogenetic tree was constructed with the data obtained through the BLAST results (Fig. 3). In the present study, the bacterial strain ADY2b is identified as Pseudomonas mendocina ADY2b which throws light on the fact that Pseudomonas species are the major group of organisms colonizing the hydrocarbon contaminated sites in the environment as reported by Sneha et al. (2012).

Close

Fig. 3

Phylogenetic tree showing the position of the bacterial isolate ADY2b within the Pseudomonas group, constructed with Maximum-Likelihood Method.

Export to PPT