Biosurfactant Production in Sub-Oxic Conditions Detected in Hydrocarbon-Degrading Isolates from Marine and Estuarine Sediments

Abstract

Hydrocarbon bioremediation in anoxic sediment layers is still challenging not only because it involves metabolic pathways with lower energy yields but also because the production of biosurfactants that contribute to the dispersion of the pollutant is limited by oxygen availability. This work aims at screening populations of culturable hydrocarbonoclastic and biosurfactant (BSF) producing bacteria from deep sub-seafloor sediments (mud volcanos from Gulf of Cadiz) and estuarine sub-surface sediments (Ria de Aveiro) for strains with potential to operate in sub-oxic conditions. Isolates were retrieved from anaerobic selective cultures in which crude oil was provided as sole carbon source and different supplements were provided as electron acceptors. Twelve representative isolates were obtained from selective cultures with deep-sea and estuary sediments, six from each. These were identified by sequencing of 16S rRNA gene fragments belonging to Pseudomonas, Bacillus, Ochrobactrum, Brevundimonas, Psychrobacter, Staphylococcus, Marinobacter and Curtobacterium genera. BSF production by the isolates was tested by atomized oil assay, surface tension measurement and determination of the emulsification index. All isolates were able to produce BSFs under aerobic and anaerobic conditions, except for isolate DS27 which only produced BSF under aerobic conditions. These isolates presented potential to be applied in bioremediation or microbial enhanced oil recovery strategies under conditions of oxygen limitation. For the first time, members of Ochrobactrum, Brevundimonas, Psychrobacter, Staphylococcus, Marinobacter and Curtobacterium genera are described as anaerobic producers of BSFs. microorganisms have been identified as capable of producing BSF in anaerobiosis, with most of these belonging to the Bacillus and Pseudomonas genera [8].Hydrocarbonoclastic bacteria are characterized as being able to metabolize one or more PHs by using them as carbon and energy sources, usually preferring these substrates over others [9][10][11]. These bacteria are ubiquitous and can be found both in aerobic and anaerobic environments [12][13][14]. Natural anaerobic biodegradation of PHs occurs primarily in deep subsurface oil reservoirs [15], in the deep ocean near natural seeps [16,17] and in most microaerobic or anaerobic environments contaminated with PHs [18][19][20].While most of the deep-sea is characterized by low energy, productivity and biological activity rates [21], metabolic activity and biomass is higher in locations where seepage of PHs and other compounds occurs from within deep sediment layers to the seabed, such as hydrothermal vents and cold seeps [22,23]. Cold seeps, including mud volcanoes (MVs), are geologically diverse ecosystems associated with emissions of methane, other PHs and, often, reduced sulfate from the subsurface [24]. Deep-sea sediments, which are often anoxic below a thin upper layer [25], are known sinks for aliphatic and aromatic hydrocarbons [26,27]. Furthermore, bacterial communities from deep-sea surface sedi...

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