Alcanivorax Borkumensis SK2 Research Articles

High-quality physiology of Alcanivorax borkumensis SK2 producing glycolipids enables efficient stirred-tank bioreactor cultivation

Glycine-glucolipid, a glycolipid, is natively synthesized by the marine bacterium Alcanivorax borkumensis SK2. A. borkumensis is a Gram-negative, non-motile, aerobic, halophilic, rod-shaped γ-proteobacterium, classified as an obligate hydrocarbonoclastic bacterium. Naturally, this bacterium exists in low cell numbers in unpolluted marine environments, but during oil spills, the cell number significantly increases and can account for up to 90% of the microbial community responsible for oil degradation. This growth surge is attributed to two remarkable abilities: hydrocarbon degradation and membrane-associated biosurfactant production. This study aimed to characterize and enhance the growth and biosurfactant production of A. borkumensis, which initially exhibited poor growth in the previously published ONR7a, a defined salt medium. Various online analytic tools for monitoring growth were employed to optimize the published medium, leading to improved growth rates and elongated growth on pyruvate as a carbon source. The modified medium was supplemented with different carbon sources to stimulate glycine-glucolipid production. Pyruvate, acetate, and various hydrophobic carbon sources were utilized for glycolipid production. Growth was monitored via online determined oxygen transfer rate in shake flasks, while a recently published hyphenated HPLC-MS method was used for glycine-glucolipid analytics. To transfer into 3 L stirred-tank bioreactor, aerated batch fermentations were conducted using n-tetradecane and acetate as carbon sources. The challenge of foam formation was overcome using bubble-free membrane aeration with acetate as the carbon source. In conclusion, the growth kinetics of A. borkumensis and glycine-glucolipid production were significantly improved, while reaching product titers relevant for applications remains a challenge.

The effect of Alcanivorax borkumensis SK2, a hydrocarbon-metabolising organism, on gas holdup in a 4-phase bubble column bioprocess.

To design bioprocesses utilising hydrocarbon-metabolising organisms (HMO) as biocatalysts, the effect of the organism on the hydrodynamics of bubble column reactor (BCR), such as gas holdup, needs to be investigated. Therefore, this study investigates the first use of an HMO, Alcanivorax borkumensis SK2, as a solid phase in the operation and hydrodynamics of a BCR. The study investigated the gas holdup in 3-phase and 4-phase systems in a BCR under ranges of superficial gas velocities (UG) from 1 to 3cm/s, hydrocarbon (chain length C13-21) concentrations (HC) of 0, 5, and 10% v/v and microbial concentrations (MC) of 0, 0.35, 0.6g/l. The results indicated that UG was the most significant parameter, as gas holdup increases linearly with increasing UG from 1 to 3cm/s. Furthermore, the addition of hydrocarbons into the air-deionized water -SK2 system showed the highest increase in the gas holdup, particularly at high UG (above 2cm/s). The solids (yeast, cornflour, and SK2) phases had differing effects on gas holdup, potentially due to the difference in surface activity. In this work, SK2 addition caused a reduction in the fluid surface tension in the bioprocess which therefore resulted in an increase in the gas holdup in BCR. This work builds upon previous investigations in optimising the hydrodynamics for bubble column hydrocarbon bioprocesses for the application of alkane bioactivation.

Marine Bioremediation Using Alcanivorax Borkumensis SK2 as A Waste Prevention Oil Industry on the Tunda Island to the Impact of Flood in Banten Region

Marine pollution in the water triggers many problems, one of them is in the area of Tunda Island, Banten, Indonesia. Waste pollution that occurs in the area occurs due to oil drilling activities conducted by Pertamina. As result, such drilling waste pollutes the environment of Tunda Island. The distance between residential areas and oil rigs is about 18 to 20 miles, which will take 2 hours to complete. As a result of the waste, many octopuses on Tunda Island were found drunk and dead. It will be more dangerous if the waste sticks to mangrove seedlings, in mangrove trunks and in seagrass beds, when exposed to sunlight it will melt because the gravel waste is pitch black and can burn. As the world's largest island nation, 60% of Indonesia's 265 million people depend on one-system waters. Toxic chemicals enter the oceans every day from industrial waste. This problem can lead to the risk of flood impacts for the environment affected by industrial waste pollution. Marine cleanliness plays an important role in realizing the prosperity of land and marine ecosystems. Efforts can be made with bioremediation that is recovery with the help of live bacteria such as Alcanivorax borkumensis SK2 which is γ-proteobacterium of the sea that can be used to clean contaminated environments. This research aims to prevent industrial waste pollution and prevent the risk of flood impacts in Indonesia, especially in Tunda Island, Banten. Various literature explains that A. borkumensis SK2 is able to digest linear and branched alkanes commonly found in crude oil. Bacteria grow naturally in polluted areas, when added with inorganic nitrogen and phosphorus, they can accelerate the cleaning process to 2 -5 years faster than natural processes for 5 - 10 years. Therefore, the application of the use of A. borkumensis SK2 has the potential to be applied in Indonesia to overcome the problem of marine pollution.

Corksorb Enhances Alkane Degradation by Hydrocarbonoclastic Bacteria.

Biosorbent materials are effective in the removal of spilled oil from water, but their effect on hydrocarbonoclastic bacteria is not known. Here, we show that corksorb, a cork-based biosorbent, enhances growth and alkane degradation by Rhodococcus opacus B4 (Ro) and Alcanivorax borkumensis SK2 (Ab). Ro and Ab degraded 96 ± 1% and 72 ± 2%, respectively, of a mixture of n-alkanes (2 g L–1) in the presence of corksorb. These values represent an increase of 6 and 24%, respectively, relative to the assays without corksorb. The biosorbent also increased the growth of Ab by 51%. However, no significant changes were detected in the expression of genes involved in alkane uptake and degradation in the presence of corksorb relative to the control without the biosorbent. Nevertheless, transcriptomics analysis revealed an increased expression of rRNA and tRNA coding genes, which confirms the higher metabolic activity of Ab in the presence of corksorb. The effect of corksorb is not related to the release of soluble stimulating compounds, but rather to the presence of the biosorbent, which was shown to be essential. Indeed, scanning electron microscopy images and downregulation of pili formation coding genes, which are involved in cell mobility, suggest that cell attachment on corksorb is a determinant for the improved activity. Furthermore, the existence of native alkane-degrading bacteria in corksorb was revealed, which may assist in situ bioremediation. Hence, the use of corksorb in marine oil spills may induce a combined effect of sorption and stimulated biodegradation, with high potential for enhancing in situ bioremediation processes.

Production of High Purity Biosurfactants Using Heavy Oil Residues as Carbon Source

Typically, oil pollution cleanup procedures following first response actions include dispersion. Crude oil is biodegradable, and its bioavailability can be increased when dispersed into very fine droplets by means of chemical surfactants. Although their use is widely spread in many applications, the latter may prove toxic, depending on the extent of use. The use of biological means, such as bioremediation and biosurfactants, has emerged over the past years as a very promising ‘green’ alternative technology. Biosurfactants (BSs) are amphiphilic molecules produced by microorganisms during biodegradation, thus increasing the bioavailability of the organic pollutants. It is their biodegradability and low toxicity that render BSs as a very promising alternative to the synthetic ones. Alcanivorax borkumensis SK2 strain ability to produce BSs, without any impurities from the substrate, was investigated. The biosurfactant production was scaled up by means of a sequencing batch reactor (SBR) and a heavy oil residue substrate as the carbon source. The product is free from substrate impurities, and its efficiency is tested on oil bioremediation in the marine environment. The product’s dispersion efficiency was determined by the baffled flask test. The production method proposed can have a significant impact to the market, given the ever-increasing demand for ecologically friendly, reliable, commercially viable and economically competitive environmental cleanup techniques.

Sequencing batch airlift reactors (SBAR): a suitable technology for treatment and valorization of mineral oil wastewaters towards lipids production

Produced water (PW) and spent oil-based wastewaters are some of the largest mineral oil wastewaters produced. Due to the high toxicity of hydrocarbons, several countries set stringent discharge limits and its treatment is compulsory before discharge. In this work, biological treatment of mineral oil wastewaters coupled with the production of bacterial lipids is demonstrated in sequential batch airlift reactors (SBAR). Two SBAR (2 L working volume) were used for treatment of PW and lubricant-based wastewater (LW), inoculated with Alcanivorax borkumensis SK2 (SBARAb+PW) and Rhodococcus opacus B4 (SBARR.o+LW), respectively. A total petroleum hydrocarbon removal (TPH) efficiency up to 96% and 80% were achieved for SBARAb+PW and SBARR.o+LW, respectively. Intracellular lipids production in SBARAb+PW increased when lower TPH/N ratios and higher feast stage duration were applied (up to 0.74 g g-1 cell dry weight (CDW)), whereas in SBARR.o+LW higher lipids production was observed for higher TPH/N ratios (0.94 g g-1 in CDW). Triacylglycerols (TAG) were the main intracellular lipid accumulated in both SBARAb+PW and SBARR.o+LW operations, while wax ester (WE) production was only observed extracellularly in the SBARAb+PW.

Genomic features and copper biosorption potential of a new Alcanivorax sp. VBW004 isolated from the shallow hydrothermal vent (Azores, Portugal)

A new Alcanivorax sp. VBW004 was isolated from a shallow hydrothermal vent in Azores Island, Portugal. In this study, we determined VBW004 was resistant to copper. This strain showed maximum tolerance of copper concentrations up to 600 μg/mL. Based on 16S rRNA gene sequencing and phylogeny revealed that this strain was more closely related to Alcanivorax borkumensis SK2. We sequenced the genome of this strain that consist of 3.8 Mb size with a G + C content of 58.4 %. In addition, digital DNA-DNA hybridizations (dDDH) and the average nucleotide identities (ANI) analysis between Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9 revealed that Alcanivorax sp. VBW004 belongs to new species. Functional annotation revealed that the genome acquired multiple copper resistance encoding genes that could assist VBW004 to respond to high Cu toxicity. Our results from biosorption analysis presumed that the VBW004 is an ecologically important bacterium that could be useful for copper bioremediation.

Characterization of an alkaline esterase from an enriched metagenomic library derived from an oil-spill area

A novel esterase gene (est7S) was cloned from an enriched metagenomic library derived from an oil-spill area. The gene encoded a protein of 505 amino acids, and the molecular mass of the Est7S was estimated to be 54,512 Da with no signal peptide. Est7S showed the highest identity of 40% to an esterase from a sludge metagenome compared to the characterized enzymes with their properties, although it showed 99% identity to a carboxylesterase in the genome sequence of Alcanivorax borkumensis SK2. Est7S had catalytic triad residues, Ser183, Glu312, and His420, and the GESAG motif in most family VII lipolytic enzymes. Est7S was purified from the crude extract of clone SM7 using Sephacryl S-200 HR and HiTrap Q column chromatographies. The purified Est7S was optimally active at 50 ℃ and pH 10.0. Est7S showed a high specific activity of 366.7 U/mg protein. It preferred short length esters, particularly p-nitrophenyl acetate, efficiently hydrolyzed R- and S-enantiomers of methyl-3-hydroxy-2-methylpropionate, and glyceryl tributyrate. These properties of Est7S may provide potential merits in biotechnological applications such as detergent and paper processing under alkaline conditions.

Membrane Fatty Acid Composition and Cell Surface Hydrophobicity of Marine Hydrocarbonoclastic Alcanivorax borkumensis SK2 Grown on Diesel, Biodiesel and Rapeseed Oil as Carbon Sources.

The marine hydrocarbonoclastic bacterium Alcanivorax borkumensis is well known for its ability to successfully degrade various mixtures of n-alkanes occurring in marine oil spills. For effective growth on these compounds, the bacteria possess the unique capability not only to incorporate but also to modify fatty intermediates derived from the alkane degradation pathway. High efficiency of both these processes provides better competitiveness for a single bacteria species among hydrocarbon degraders. To examine the efficiency of A. borkumensis to cope with different sources of fatty acid intermediates, we studied the growth rates and membrane fatty acid patterns of this bacterium cultivated on diesel, biodiesel and rapeseed oil as carbon and energy source. Obtained results revealed significant differences in both parameters depending on growth substrate. Highest growth rates were observed with biodiesel, while growth rates on rapeseed oil and diesel were lower than on the standard reference compound (hexadecane). The most remarkable observation is that cells grown on rapeseed oil, biodiesel, and diesel showed significant amounts of the two polyunsaturated fatty acids linoleic acid and linolenic acid in their membrane. By direct incorporation of these external fatty acids, the bacteria save energy allowing them to degrade those pollutants in a more efficient way. Such fast adaptation may increase resilience of A. borkumensis and allow them to strive and maintain populations in more complex hydrocarbon degrading microbial communities.

The Wax–Liquid Transition Modulates Hydrocarbon Respiration Rates in Alcanivorax borkumensis SK2

Marine hydrocarbon biodegradation is an important environmental process conducted by microbes and modulated by oceanographic conditions. Following up on the patterns of petroleum hydrocarbon biodegradation observed after the Deepwater Horizon disaster, we measured respiration rates for the obligate alkane-degrading bacterium, Alcanivorax borkumensis SK2, to assess the relationship between hydrocarbon respiration rates and the phase, wax versus liquid, of the substrate. Using a matrix of temperatures (20, 25, 30, 35, and 40 °C) and n-alkanes (n-C14, n-C15, n-C16, n-C17, n-C18, n-C19, and n-C20) for which each temperature gap spans the phase transition point of an n-alkane, we demonstrate that the n-alkane respiration rate decreases substantially when the substrate is in the wax versus liquid phase. The observed effect spans the full experimental temperature range. Subsequent experimentation with only wax-phase n-C19 indicates that the availability of surface area modulates the n-alkane respiration rate and...

Polycaprolactone-based scaffold for oil-selective sorption and improvement of bacteria activity for bioremediation of polluted water: Porous PCL system obtained by leaching melt mixed PCL/PEG/NaCl composites: Oil uptake performance and bioremediation efficiency

A novel floatable and biodegradable sponge for the selective absorption of oil from water and potentially useful as cell carrier for bioremediation treatments was prepared in polycaprolactone (PCL). The eco-friendly process for fabricating the PCL sponge does not involve either synthetic routes or organic solvents, thus minimizing environmental hazard. In particular, the 3D porous materials have been prepared by mixing in the melt the polymer matrix with two water-soluble porogen agents (NaCl and PEG) and thereafter leaching the obtained PCL/NaCl/PEG composites in water. The PCL sponges here proposed are capable to remove different types of oily pollutants (up to 500wt%), and were successfully tested as carrier materials of hydrocarbon(HC)-degrading bacteria for bioremediation. The bioremediation efficiency of the scaffold-bacteria system was analyzed on crude oil and n-alkanes using two highly performant HC-degrading bacterial strains: the marine hydrocarbonoclastic model strain Alcanivorax borkumensis SK2 and the soil long-chain n-alkane degrader Nocardia sp. SoB.Morphological analysis highlighted a high capacity of adhesion and proliferation of bacterial cells within the whole interconnected three-dimensional structure. HC degradation rates, evaluated by GC-FID analysis, demonstrated the higher degrading performance of immobilized-cells if compared with conventional submerged liquid culture.

Valorization of lubricant-based wastewater for bacterial neutral lipids production: Growth-linked biosynthesis

Lipids produced by microorganisms are currently of great interest as raw material for either biofuels or oleochemicals production. Significant biosynthesis of neutral lipids, such as triacylglycerol (TAG) and wax esters (WE) are thought to be limited to a few strains. Hydrocarbonoclastic bacteria (HCB), key players in bioremediation of hydrocarbon contaminated ecosystems, are among this group of strains. Hydrocarbon rich wastewaters have been overlooked concerning their potential as raw material for microbial lipids production. In this study, lubricant-based wastewater was fed, as sole carbon source, to two HCB representative wild strains: Alcanivorax borkumensis SK2, and Rhodococcus opacus PD630. Neutral lipid production was observed with both strains cultivated under uncontrolled conditions of pH and dissolved oxygen. A. borkumensis SK2 was further investigated in a pH- and OD-controlled fermenter. Different phases were assessed separately in terms of lipids production and alkanes removal. The maximum TAG production rate occurred during stationary phase (4 mg-TAG/L h). The maximum production rate of WE-like compounds was 15 mg/L h, and was observed during exponential growth phase. Hydrocarbons removal was 97% of the gas chromatography (GC) resolved straight-chain alkanes. The maximum removal rate was observed during exponential growth phase (6 mg-alkanes/L h). This investigation proposes a novel approach for the management of lubricant waste oil, aiming at its conversion into valuable lipids. The feasibility of the concept is demonstrated under low salt (0.3%) and saline (3.3%) conditions, and presents clues for its technological development, since growth associated oil production opens the possibility for establishing continuous fermentation processes.

Osmotic Stress Confers Enhanced Cell Integrity to Hydrostatic Pressure but Impairs Growth in Alcanivorax borkumensis SK2.

Alcanivorax is a hydrocarbonoclastic genus dominating oil spills worldwide. While its presence has been detected in oil-polluted seawaters, marine sediment and salt marshes under ambient pressure, its presence in deep-sea oil-contaminated environments is negligible. Recent laboratory studies highlighted the piezosensitive nature of some Alcanivorax species, whose growth yields are highly impacted by mild hydrostatic pressures (HPs). In the present study, osmotic stress was used as a tool to increase HP resistance in the type strain Alcanivorax borkumensis SK2. Control cultures grown under standard conditions of salinity and osmotic pressure with respect to seawater (35.6 ppt or 1136 mOsm kg-1, respectively) were compared with cultures subjected to hypo- and hyperosmosis (330 and 1720 mOsm kg-1, or 18 and 62 ppt in salinity, equivalent to brackish and brine waters, respectively), under atmospheric or increased HP (0.1 and 10 MPa). Osmotic stress had a remarkably positive impact on cell metabolic activity in terms of CO2 production (thus, oil bioremediation) and O2 respiration under hyperosmosis, as acclimation to high salinity enhanced cell activity under 10 MPa by a factor of 10. Both osmotic shocks significantly enhanced cell protection by reducing membrane damage under HP, with cell integrities close to 100% under hyposmosis. The latter was likely due to intracellular water-reclamation as no trace of the piezolyte ectoine was found, contrary to hyperosmosis. Notably, ectoine production was equivalent at 0.1 MPa in hyperosmosis-acclimated cells and at 10 MPa under isosmotic conditions. While stimulating cell metabolism and enhancing cell integrity, osmotic stress had always a negative impact on culture growth and performance. No net growth was observed during 4-days incubation tests, and CO2:O2 ratios and pH values indicated that culture performance in terms of hydrocarbon degradation was lowered by the effects of osmotic stress alone or combined with increased HP. These findings confirm the piezosensitive nature of A. borkumensis, which lacks proper resistance mechanisms to improve its metabolic efficiency under increased HP, thus explaining its limited role in oil-polluted deep-sea environments.

Comparison the effects of bioaugmentation versus biostimulation on marine microbial community by PCR–DGGE: A mesocosm scale

In order to better understand the effects of biostimulation and bioaugmentation processes on a marine microbial community, three different mesocosm experiments were planned. Natural seawater (10.000L) was artificially polluted with crude oil (1L) and (1) inorganic nutrients (Biostimulating Mesocosm, BM), (2) inorganic nutrients and an inoculum of Alcanivorax borkumensis SK2 (Single Bioaugmentation Mesocosm, SBM), (3) inorganic nutrients and inoculums of A. borkumensis SK2 and Thalassolituus oleivorans MIL-1 (Consortium Bioaugmentation Mesocosm, CBM). During the experimental period (20days), samples were taken from each mesocosm and the community structure was analyzed by PCR–DGGE. The 16S rRNA gene DGGE banding patterns and sequence analysis demonstrated that biostimulation had the lowest effect on microbial biodiversity in the mesocosms; however, the biodiversity of the marine microbial community dramatically decreased in the CBM (Shannon index was 0.6 in T3). The community structures among the three mesocosms were also markedly different, and major bacteria derived from DGGE bands were related to uncultured Gamma Proteobacteria. The biodegradation results show that the Single Bioaugmentation Mesocosm (SBM) system had the highest percentage of degradation (95%) in comparison to the BM mesocosm (80%) and CBM (70%).

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium.

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Alcanivorax borkumensis produces an extracellular siderophore in iron-limitation condition maintaining the hydrocarbon-degradation efficiency

Obligate marine hydrocarbonoclastic bacteria possess genetic and physiological features to use hydrocarbons as sole source of carbon and to compete for the uptake of nutrients in usually nutrient-depleted marine habitats. In the present work we have studied the siderophore-based iron uptake systems in Alcanivorax borkumensis SK2 and their functioning during biodegradation of an aliphatic hydrocarbon, tetradecane, under iron limitation conditions. The antiSMASH analysis of SK2 genome revealed the presence of two different putative operons of siderophore synthetases. Search for the predicted core structures indicated that one siderophore is clearly affiliated to the family of complex oligopeptidic siderophores possessing an Orn-Ser-Orn carboxyl motif whereas the second one is likely to belong to the family of SA (salicylic acid)–based siderophores.Analyzing the supernatant of SK2 culture, an extracellular siderophore was identified and its structure was resolved. Thus, along with the recently described membrane-associated amphiphilic tetrapeptidic siderophore amphibactin, strain SK2 additionally produces an extracellular type of iron-chelating molecule with structural similarity to pseudomonins. Comparative Q-PCR analysis of siderophore synthetases demonstrated their significant up-regulation in iron-depleted medium. Different expression patterns were recorded for two operons during the early and late exponential phases of growth, suggesting a different function of these two siderophores under iron-depleted conditions.

High-cell-density cultivation of recombinant Escherichia coli, purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase

We have recently described the biocatalytic characterization of a self-sufficent biosynthetic alkane hydroxylase based on CYP153A13a from Alcanivorax borkumensis SK2 (thereafter A13-Red). Despite remarkable regio- and chemo-selectivity, A13-Red suffers of a difficult-to-reproduce expression and moderate operational stability. In this study, we focused our efforts on the production of A13-Red using high-cell-density cultivation (HCDC) of recombinant Escherichia coli. We achieved 455 mg (5,000 nmol) of functional enzyme per liter of culture. Tight control of cultivation parameters rendered the whole process highly reproducible compared with flask cultivations. We optimized the purification of the biocatalyst that can be performed in either two or three steps depending on the application needed to afford A13-Red up to 95 % homogeneous. We investigated different reaction conditions and found that the total turnover numbers of A13-Red during the in vitro hydroxylation of n-octane could reach up to 3,250 to produce 1-octanol (1.6 mM) over a period of 78 h.

Development of bioreporter assays for the detection of bioavailability of long‐chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2

Long-chain alkanes are a major component of crude oil and therefore potentially good indicators of hydrocarbon spills. Here we present a set of new bacterial bioreporters and assays that allow to detect long-chain alkanes. These reporters are based on the regulatory protein AlkS and the alkB1 promoter from Alcanivorax borkumensis SK2, a widespread alkane degrader in marine habitats. Escherichia coli cells with the reporter construct reacted strongly to octane in short-term (6 h) aqueous suspension assays but very slightly only to tetradecane, in line with what is expected from its low water solubility. In contrast, long-term assays (up to 5 days) with A. borkumensis bioreporters showed strong induction with tetradecane and crude oil. Gel-immobilized A. borkumensis reporter cells were used to demonstrate tetradecane and crude oil bioavailability at a distance from a source. Alcanivorax borkumensis bioreporters induced fivefold more rapid and more strongly when allowed physical contact with the oil phase in standing flask assays, suggesting a major contribution of adhered cells to the overall reporter signal. Using the flask assays we further demonstrated the effect of oleophilic nutrients and biosurfactants on oil availability and degradation by A. borkumensis. The fluorescence signal from flask assays could easily be captured with a normal digital camera, making such tests feasible to be carried out on, e.g. marine oil responder vessels in case of oil accidents.

Transcriptional profiling of the marine oil-degrading bacterium Alcanivorax borkumensis during growth on n-alkanes

The marine oil-degrading bacterium Alcanivorax borkumensis SK2 has attracted significant interest due to its hydrocarbonoclastic lifestyle, its alkane-centered metabolism, and for playing an important ecological role in cleaning up marine oil spills. In this study, we used microarray technology to characterize the transcriptional responses of A. borkumensis to n-hexadecane exposure as opposed to pyruvate, which led to the identification of a total of 220 differentially expressed genes, with 109 genes being upregulated and 111 genes being downregulated. Among the genes upregulated on alkanes are systems predicted to be involved in the terminal oxidation of alkanes, biofilm formation, signal transduction, and regulation.

Neutral lipid production in Alcanivorax borkumensis SK2 and other marine hydrocarbonoclastic bacteria

AbstractTriacylglycerols (TAG) and wax esters (WE) constitute together with polyhydroxyalkanoates (PHA) the major storage lipophilic compounds in prokaryotes. Recently, the production of neutral lipids such as TAG and WE has been reported in species of the genus Alcanivorax, which belongs to the group of obligate hydrocarbonoclastic bacteria (OHCB). We analyzed the production of such neutral lipids by different marine hydrocarbonoclastic bacteria growing on pyruvate or hexadecane as sole carbon source, and compared it to other bacteria such as Rhodococcus opacus strain PD630 and Acinetobacter baylyi strain ADP1, which are two well‐studied strains for production of neutral lipids. Alcanivorax borkumensis SK2 synthesized mainly TAG when cells are cultivated on pyruvate, while biosynthesis and accumulation of WE was mainly observed in cells growing on hexadecane. Alcanivorax jadensis T9 synthesized and accumulated mainly WE if cells were cultivated with hexadecane, while both TAG and WE were observed if cells were cultivated with pyruvate as sole carbon source, respectively. Predominantly production as well as export of WE was observed in Marinobacter hydrocarbonoclasticus SP17 growing on pyruvate or hexadecane as sole carbon source. The chemical structures of TAG and WE produced by A. borkumensis SK2 were analyzed by gas chromatography and/or mass spectrometry, and first studies to investigate the influence of different C/N ratio (7, 50 or 150) on the production of neutral lipids were performed.