Diversity and Hydrocarbon-Degrading Potential of Deep-Sea Microbial Community from the Mid-Atlantic Ridge, South of the Azores (North Atlantic Ocean)

Abstract

Deep-sea sediments (DSS) are one of the largest biotopes on Earth and host a surprisingly diverse microbial community. The harsh conditions of this cold environment lower the rate of natural attenuation, allowing the petroleum pollutants to persist for a long time in deep marine sediments raising problematic environmental concerns. The present work aims to contribute to the study of DSS microbial resources as biotechnological tools for bioremediation of petroleum hydrocarbon polluted environments. Four deep-sea sediment samples were collected in the Mid-Atlantic Ridge, south of the Azores (North Atlantic Ocean). Their autochthonous microbial diversity was investigated by 16S rRNA metabarcoding analysis. In addition, a total of 26 deep-sea bacteria strains with the ability to utilize crude oil as their sole carbon and energy source were isolated from the DSS samples. Eight of them were selected for a novel hydrocarbonoclastic-bacterial consortium and their potential to degrade petroleum hydrocarbons was tested in a bioremediation experiment. Bioaugmentation treatments (with inoculum pre-grown either in sodium acetate or petroleum) showed an increase in degradation of the hydrocarbons comparatively to natural attenuation. Our results provide new insights into deep-ocean oil spill bioremediation by applying DSS hydrocarbon-degrading consortium in lab-scale microcosm to simulate an oil spill in natural seawater.

Keywords: 16S rRNA gene; bioremediation; deep-sea; microbial consortia; next-generation sequencing; petroleum hydrocarbons.

Figure 1

Relative abundance of prokaryotic phyla in deep-sea samples detected by NGS. Other <1% groups the phyla with abundance lower than 1%; Unassigned_No_Relative: groups the phyla without any close relatives.

Figure 2

Venn diagram showing the low abundant microbial community detected in the four deep-sea sediment samples highlighting those detected in all samples. Percentage values correspond to the average abundance of the genera in the 4 samples. The bar plot represents the number of low abundance taxa within each site.

Figure 3

Phylogenetic tree (16S rRNA gene) of strains isolated. Maximum Likelihood analysis was performed in MEGA 7 with 53 strains and their GenBank nearest neighbours. The tree was generated using 1412 bp and 1000 bootstraps. Bootstrap values (%) are represented on tree branches (values below 60% have not been displayed). Numbers in parenthesis correspond to GenBank accession numbers.

Figure 4

Visual inspection of oil degradation in the beginning (T0) and end (T15) of the bioremediation experiments in controlled laboratory conditions. The four treatments are natural attenuation (a), biostimulation (b), bioaugmentation with inoculum pre-grown in petroleum (c) and bioaugmentation with inoculum pre-grown in acetate (d).

Figure 5

Hydrocarbon-degrading microorganisms estimated by most probable number (log₁₀ MPN, mean and standard deviation, n = 3) of the bioremediation experiments. MPN tests were carried out at the first (T0) and last (T15) days of the experiments. * Methodological saturation. NA: natural attenuation; BS: biostimulation; BAp: bioaugmentation with inoculum pre-grown in petroleum; BAa: bioaugmentation with inoculum pre-grown in acetate.

Figure 6

Total Petroleum Hydrocarbon degradation observed at the end of the 15-day experiment (mean and standard deviation, n = 3). T15 + NA: natural attenuation; T15 + BS: biostimulation; T15 + BAp: bioaugmentation with inoculum pre-grown in petroleum; T15 + BAa: bioaugmentation with inoculum pre-grown in acetate.