Characterization of Five Psychrotolerant Alcanivorax spp. Strains Isolated from Antarctica

Abstract

Five psychrotolerant Alcanivorax spp. strains were isolated from Antarctic coastal waters. Strains were screened for molecular and physiological properties and analyzed regarding their growth capacity. Partial 16S rDNA, alk-B1, and P450 gene sequencing was performed. Biolog EcoPlates and the API 20E test were used to evaluate metabolic and biochemical profiles. Bacterial growth in sodium acetate was determined at 4, 15, 20, and 25 °C to evaluate the optimal temperature. Furthermore, the ability of each strain to grow in a hydrocarbon mixture at 4 and 25 °C was assayed. Biosurfactant production tests (drop-collapse and oil spreading) and emulsification activity tests (E₂₄) were also performed. Concerning results of partial gene sequencing (16S rDNA, alk-B1, and P450), a high similarity of the isolates with the same genes isolated from other Alcanivorax spp. strains was observed. The metabolic profiles obtained by Biolog assays showed no significant differences in the isolates compared to the Alcanivorax borkumensis wild type. The results of biodegradative tests showed their capability to grow at different temperatures. All strains showed biosurfactant production and emulsification activity. Our findings underline the importance to proceed in the isolation and characterization of Antarctic hydrocarbon-degrading bacterial strains since their biotechnological and environmental applications could be useful even for pollution remediation in polar areas.

Keywords: Alcanivorax spp.; Antarctica; biodegradation; molecular analyses; physiological analyses.

Figure 1

Rooted phylogenetic tree clustered by neighbour-joining of maximum likelihood values showing affiliation of complete bacterial 16S rRNA gene sequences to closest related sequences from Alcanivorax spp. Isolates are indicated in bold-type. Percentages of 1000-bootstrap resampling that supported the branching orders in each analysis are shown above or near the relevant nodes. All trees were rooted and outgrouped with 16S rRNA gene sequences of a Bacillus subtilis (AB192294). Vertical lines indicate evolutionary distance; each scale bar length corresponds to 0.05 fixed point mutations per sequence position.

Figure 2

Rooted phylogenetic tree clustered by neighbour-joining of maximum likelihood values showing gene sequences to closest related sequences from Alcanivorax spp. Isolates are indicated in bold-type. Percentages of 1000-bootstrap resampling that supported the branching orders in each analysis are shown above or near the relevant nodes. All trees were rooted and outgrouped with alk-B1 gene sequences of Novosphingobium sp. PCY alkane monooxygenase gene (KJ650249). Vertical lines indicate evolutionary distance; each scale bar length corresponds to 0.05 fixed point mutations per sequence position.

Figure 3

Rooted phylogenetic tree clustered by neighbour-joining of maximum likelihood values showing affiliation of P450 gene sequences to closest related sequences from Alcanivorax spp. Isolates are indicated in bold-type. Percentages of 1000-bootstrap resampling that supported the branching orders in each analysis are shown above or near the relevant nodes. All trees were rooted and outgrouped with P450 gene sequences of Novosphingobium sp. SB32149 gene for P450 (LC214353). Vertical lines indicate evolutionary distance; each scale bar length corresponds to 0.05 fixed point mutations per sequence position.

Figure 4

Growth curves at 4 (light blue line), 15 (blue line), 20 (orange line), and 25 °C (red line) for a/ANT5a (a), b/ANT6 (b), b/ANT8 (c), b/ANT10 (d), b/ANT11 (e) Alcanivorax strains in ONR7a medium amended with sodium acetate (1% w/v).

Figure 5

Growth curves at 4 °C (light blue line) and 25 °C (red line) for a/ANT5a (a), b/ANT6 (b), b/ANT8 (c), b/ANT10 (d), b/ANT11 (e) Alcanivorax strains in ONR7a medium amended with hydrocarbon mixture (PierE1, Dansk Crude Oil, 1% w/v).