Culturable hydrocarbonoclastic marine bacterial isolates from Indonesian seawater in the Lombok Strait and Indian Ocean

Abstract

Purpose: The study aims to isolate the culturable marine bacteria and to assess their potential as the bioremediation agent for petroleum hydrocarbons contamination in marine environment.

Methods: Bacteria isolates were obtained by repetitive streaks to obtain purified bacteria on Zobell marine agar plates before further analysis and culture through direct visualization on agar plates. Identification were conducted using 16S rDNA sequence which are compared using NCBI BLAST and, combined with phenotypic and phylogenetic data. The potential use of the selected bacteria was tested by culturing them with two carbon sources i.e., glucose and crude oil.

Result: Fifty-one culturable marine hydrocarbonoclastic bacteria were isolated from the Lombok Strait (LS-3, LS-13, LS-14, LS-15, LS-16 and LS-20) and Indian Ocean (IO-1, IO-6, IO-8, IO-19, IO-24 and IO-25). Twelve isolates were found to degrade crude oil efficiently at a >2% concentration and to grow with crude oil as their sole carbon and energy source. These 12 strains belong to the genus Bacillus, which is well known to produce surface active agents, and the oil displacement assay indicated the production of these agents by these strains. Within the genera Bacillus, five species (Bacillus flexus, B. methylotrophicus, B. aquimaris, B. horikoshii, and B. thioparans) were represented by the 12 identified strains.

Conclusion: Selected strains from the Lombok Strait and Indian Ocean were capable of degrading crude oil (2% v/v) by 43.9-71.9% over 14 days. These results are important for marine bioremediation in Indonesia, which often faces risks of oil spill contamination and disaster.

Keywords: Earth sciences; Environmental science; Microbiology.

Fig. 1

Sampling locations for the in situ study. A. Indian Ocean. B. Lombok Strait.

Fig. 2

Example oil displacement assays. Positive results (A, B, C, D) and a negative result (E). A = IO-4, B=IO-25, C = LS-20, D = LS-13, E = IO-30.

Fig. 3

Bacterial growth kinetics of IO isolates on glucose as a carbon source using Lineweaver-Burk transformation.

Fig. 4

Bacterial growth kinetics of IO isolates on crude oil (2% v/v) as a carbon source using Lineweaver-Burk transformation.

Fig. 5

Crude oil remaining (%) after degradation by selected strains adapted to 2% crude oil. Biodegradation tests were conducted in triplicate.

Fig. 6

Phylogenetic tree. The history of evolution was inferred using the neighbor-joining method. The percentage of trees that replicate the associated taxa are clustered together in the bootstrap (1,000 replicates) and displayed at the branches. Evolutionary distances were calculated using Kimura's method. This analysis was performed using a 16-nucleotide sequence. All positions containing gaps or missing data were eliminated. A total of 511 positions are in the final dataset. Evolution analysis was performed using MEGA5.