Effects of Dispersants and Biosurfactants on Crude-Oil Biodegradation and Bacterial Community Succession

Abstract

This study evaluated the effects of three commercial dispersants (Finasol OSR 52, Slickgone NS, Superdispersant 25) and three biosurfactants (rhamnolipid, trehalolipid, sophorolipid) in crude-oil seawater microcosms. We analysed the crucial early bacterial response (1 and 3 days). In contrast, most analyses miss this key period and instead focus on later time points after oil and dispersant addition. By focusing on the early stage, we show that dispersants and biosurfactants, which reduce the interfacial surface tension of oil and water, significantly increase the abundance of hydrocarbon-degrading bacteria, and the rate of hydrocarbon biodegradation, within 24 h. A succession of obligate hydrocarbonoclastic bacteria (OHCB), driven by metabolite niche partitioning, is demonstrated. Importantly, this succession has revealed how the OHCB Oleispira, hitherto considered to be a psychrophile, can dominate in the early stages of oil-spill response (1 and 3 days), outcompeting all other OHCB, at the relatively high temperature of 16 °C. Additionally, we demonstrate how some dispersants or biosurfactants can select for specific bacterial genera, especially the biosurfactant rhamnolipid, which appears to provide an advantageous compatibility with Pseudomonas, a genus in which some species synthesize rhamnolipid in the presence of hydrocarbons.

Keywords: OHCB; Oleispira; Pseudomonas; bacteria; biosurfactants; dispersants; hydrocarbons; oil.

Figure 1

Total alkane concentrations (mean ± SE, n = 2) at day 1. Blue (upper) percentages (and asterisks) represent degradation in comparison to the oil-only control. Black (lower) percentages (and asterisks) represent degradation in comparison to starting concentration (“Time Zero”). Dispersant and biosurfactant codes are as follows: SP = sophorolipid, RL = rhamnolipid, TH = trehalolipid, SD25 = Superdispersant 25, F52 = Finasol OSR 52, and SG = Slickgone NS. *** p < 0.001, ** p < 0.01, and * p < 0.05.

Figure 2

Bacterial 16S rRNA gene abundance (mean ± SE, n = 3) over a 21 day period for total bacteria (A) and at day 1 only, for total bacteria and proportion (percentage figures) of obligate hydrocarbonoclastic bacteria (B). OHCB sequences are defined as from the genera: Alcanivorax, Cycloclasticus, Oleibacter, Oleispira, Thalassolituus. All microcosms contained seawater, nutrients, and oil (NSO-1 0.1% v/v), and some also contained additional dispersants or biosurfactants (0.005% v/v; 20:1 ratio of oil). Dispersant and biosurfactant codes are as follows: SP = sophorolipid, RL = rhamnolipid, TH = trehalolipid, SD25 = Superdispersant 25, F52 = Finasol OSR 52, and SG = Slickgone NS. Asterisks above percentage figures indicate significant differences in mean OHCB relative abundance compared to the oil-only control (*** p < 0.001).

Figure 3

Unrooted Neighbour-Joining phylogeny based on 16S rRNA gene sequences from representative bacterial ASVs which increased in relative abundance in microcosms containing seawater, nutrients, oil (NSO-1 0.1% v/v), and additional dispersants or biosurfactants (0.005% v/v; 20:1 ratio to oil) compared with oil-only. ASV sequences are aligned with known hydrocarbon-degrading bacteria and closest relatives; bootstrap values >70 displayed (1000 iterations). Evolutionary distances computed by Maximum Composite Likelihood protocol (using the Tamura-Nei model (Tamura and Nei, 1993)), sum of branch length = 1.42. Analysis involved 85 nucleotide sequences (including 29 ASVs and 66 related strains), with a total of 258 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. Percentage figures in parentheses next to individual ASVs display the relative abundance of that sequence within the assigned genus. Horizontal bars (right of clades) represent the proportion of sequences assigned to ASVs within the clade in the different treatments (see colour key) over all time points (excluding day zero). Percentage figures next to horizontal bars show the maximum relative abundance reached for ASVs within that clade in any treatment at any time point (excluding day zero; see additional figures for treatment-specific bacterial succession). Vertical bars (right of percentage figures) represent OHCB (black), genera where some isolates have grown on hydrocarbons (grey), and genera that have increased in relative abundance in oil-contaminated environments, but no isolates have been shown to degrade hydrocarbons (white).

Figure 4

Relative abundance (% of the bacterial community; mean ± SE, n = 3) of 16S rRNA gene sequences within ASVs assigned to obligate hydrocarbonoclastic bacteria (A), and bacteria that were abundant (and are often associated with hydrocarbon degradation; see Supplementary Table S2) (B), over a 21 day period. All microcosms contained seawater, nutrients, and oil (NSO-1 0.1% v/v), and some also contained additional dispersants or biosurfactants (0.005% v/v; 20:1 ratio of oil). Dispersant and biosurfactant codes are as follows: SP = sophorolipid, RL = rhamnolipid, TH = trehalolipid, SD25 = Superdispersant 25, and F52 = Finasol OSR 52, SG = Slickgone NS.

Figure 5

Network modules based on clustering of a SPIEC-EASI co-occurrence network formed of ASVs from bacterial genera associated with hydrocarbon degradation. Each point represents a single ASV, which are coloured according to their identity at the genus level (RDP classifier confidence >0.8). Blue edges represent positive co-occurrences between ASVs across the experiment, whereas red edges show negative co-occurrences.

Figure 6

Relative abundances of modules based on clustering of a SPIEC-EASI co-occurrence network of ASVs from bacterial genera associated with hydrocarbon degradation. The total relative abundance (as a proportion of bacterial genera associated with hydrocarbon degradation) of ASVs in each module across the course of the experiment, based on counts of 16S rRNA sequences. All microcosms contained seawater, nutrients, and oil (NSO-1 0.1% v/v), and some also contained additional dispersants or biosurfactants (0.005% v/v; 20:1 ratio of oil). Dispersant and biosurfactant codes are as follows: SP = sophorolipid, RL = rhamnolipid, TH = trehalolipid, SD25 = Superdispersant 25, F52 = Finasol OSR 52, and SG = Slickgone NS.