Gelatinizing oil in water and its removal via bacteria inhabiting the gels

Abstract

When crude oil samples were shaken (200 rpm) in seawater samples from the Arabian Gulf at 30 °C, usually oil-gels were produced spontaneously leaving the water quite clear. The gelators could probably be based on cholesteryl derivatives. Microscopic examination of the established gels revealed nanofibrellar structures similar to those described by earlier workers for artificially synthesized gelators. Communities of bacteria including prosthetic and stalked members as well as oil-degrading bacteria were recorded in such gels. Chemical analysis revealed that 88.5% of the oil entrapped by gelation was biodegraded after 40 days at 30 °C. Individual bacterial species isolated from the oil-gels biodegraded in batch cultures between 17.8 and 33.3% of the oil added at time zero in 12 days at 30 °C. Gelation is a promising approach, not only for clean, physical removal of oil spilled in aquatic habitats, as so far suggested, but also in its effective microbiological biodegradation, as the current study revealed.

Figure 1

Natural oil-gelation in shaken (200 rpm) water samples. (a,b,c) Gels established after 4 days in Kuwait Towers (KT), Fintas (F) and tap water (TW) samples, respectively. (d,e,f) Gels established in the same samples after 40 days. (g,h,i) Gels established in the same samples after 4 months. Those results were obtained when previously autoclaved (Au) and unautoclaved (Un) water samples were used. (j) Gel established in Khiran (Kh) water, but first after 40 days. (k,l) Gels established after 40 days in Kuwait Towers water, note how clear the seawater was left after removing the gel. (m,n) Gels established after only 24 hours in Kuwait Towers waters inoculated with 1 g pieces of previously established gels.

Figure 2

TLC-Analysis of lipid classes in total lipids. Sorbent: Silica Gel G. Running solvent: Hexane, diethyl ether, acetic acid, 85:15:1, by vol. Visualization: Phosphomolybdic acid and 50% H₂SO₄, at 120 °C. 1, Standard cholesterol; 2, standard oleic acid; 3, crude oil extract; 4&5, oil-gel extracts; 6, standard methyl oleate; 7, mixture of the standard samples; 8, fish extract. Spot identities: ST, sterols; FA, fatty acids; TG, triglycerides; ME, methyl esters of fatty acids; ST, steryl esters; HC, hydrocarbons.

Figure 3

Light (a–d) and scanning electron (e–l) micrographs of 40 day oil-gel established in Fintas seawater sample. The light micrographs revealed the nanofibrillary structures (a,d) and the prosthetic (b, arrow) and stalked (c, arrow) bacteria. The prosthetic bacteria belonging to genus Hyphomonas showed the main bodies of the reproductive cells as well as the prosthecae and progenies. The stalked bacterial cells belonging to the genus Maricaulis had relatively long stalks. The scanning electron microscopic images showed the formation of nanofibrillar structures both in the previously autoclaved and unautoclaved samples and reveal bacterial cells in the unautoclaved (i–l) but not in the previously autoclaved (e–h) water samples.

Figure 4

Culture-dependent analysis of bacterial communities associated with established oil-gels. Phylogenetic tree of 16S rRNA genes of the isolated bacteria. Values shown on each node of the phylogenetic tree are bootstrap values. A total of 2,000 bootstrap replicates were performed.

Figure 5

Culture-independent analysis of bacterial communities associated with established oil-gels. (a) DGGE Profile of 16S rRNA-gene amplicons in total genomic DNA extract of the 4 months old gel established in Fintas seawater. (b) DGGE Profiles of 16S rRNA-gene amplicons in total genomic DNA extracted from gels established for 40 days in water samples from different sites. (c) Phylogenetic tree based on 16S rRNA-gene partial sequences, showing the phylogeny of bacterial bands from both DGGE gels.

Figure 6

GLC Profiles of hydrocarbons recovered from an established gel and from oil-containing batch cultures supporting hydrocarbonoclastic bacteria isolated from the gels. (a) Crude oil at time zero. (b) Oil recovered from the gel established after 4 months in Fintas seawater. (c) Oil recovered from Alcanivorax venustensis culture batch. (d) Oil recovered from Alcanivorax jadensis culture batch. (e) Oil recovered from Alcanivorax borkumensis culture batch. (f) Oil recovered from Thalassospira australica culture batch. (g) Oil recovered from Kocuria flava culture batch. (h) Oil recovered from Blastomonas natatoria culture. Values on the individual profiles are those of the hydrocarbon consumption values, they were means of 3 replicates ± standard deviation values.