Project description: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.
Project description: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.
Project description:The marine hydrocarbonoclastic bacterium Alcanivorax borkumensis is able to degrade mixtures of n-alkanes as they occur in marine oil spills. However, investigations of growth behavior and physiology of these bacteria when cultivated with n-alkanes of different chain lengths (C6 to C30) as the substrates are still lacking. Growth rates increased with increasing alkane chain length up to a maximum between C12 and C19, with no evident difference between even- and odd-numbered chain lengths, before decreasing with chain lengths greater than C19. Surface hydrophobicity of alkane-grown cells, assessed by determination of the water contact angles, showed a similar pattern, with maximum values associated with growth rates on alkanes with chain lengths between C11 and C19 and significantly lower values for cells grown on pyruvate. A. borkumensis was found to incorporate and modify the fatty acid intermediates generated by the corresponding n-alkane degradation pathway. Cells grown on distinct n-alkanes proved that A. borkumensis is able to not only incorporate but also modify fatty acid intermediates derived from the alkane degradation pathway. Comparing cells grown on pyruvate with those cultivated on hexadecane in terms of their tolerance toward two groups of toxic organic compounds, chlorophenols and alkanols, representing intensely studied organic compounds, revealed similar tolerances toward chlorophenols, whereas the toxicities of different n-alkanols were significantly reduced when hexadecane was used as a carbon source. As one adaptive mechanism of A. borkumensis to these toxic organic solvents, the activity of cis-trans isomerization of unsaturated fatty acids was proven. These findings could be verified by a detailed transcriptomic comparison between cultures grown on hexadecane and pyruvate and including solvent stress caused by the addition of 1-octanol as the most toxic intermediate of n-alkane degradation.
Project description:Alcanivorax borkumensis is a ubiquitous marine bacterium that utilizes alkanes as a sole carbon source. We observed two phenotypes in the A. borkumensis SK2 type strain: rough (R) and smooth (S) types. The S type exhibited lower motility and higher polysaccharide production than the R type. Full genome sequencing revealed a mutation in the S type involved in cyclic-di-GMP production. The present results suggest that higher c-di-GMP levels in the S type control the biofilm forming behavior of this bacterium in a manner commensurate with other Gram-negative bacteria.
Project description:The potential of the folic acid biosynthesis pathway as a target for the development of antibiotics has been clinically validated. However, many pathogens have developed resistance to these antibiotics, prompting a re-evaluation of potential drug targets within the pathway. The ydaH gene of Alcanivorax borkumensis encodes an integral membrane protein of the AbgT family of transporters for which no structural information was available. Here we report the crystal structure of A. borkumensis YdaH, revealing a dimeric molecule with an architecture distinct from other families of transporters. YdaH is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins that suggest a plausible pathway for substrate transport. Further analyses also suggest that YdaH could act as an antibiotic efflux pump and mediate bacterial resistance to sulfonamide antimetabolite drugs.
Project description:This project addresses the question of impact of oil spills on lung health. Specifically the project will address the general hypothesis, which is upon oil/dispersant respiratory exposure there will be a higher carcinogenic potential of lung tissue.
Project description:A novel biosurfactant was discovered to be synthesized by the marine bacterium Alcanivorax borkumensis in 1992. This bacterium is abundant in marine environments affected by oil spills, where it helps to degrade alkanes and, under such conditions, produces a glycine-glucolipid biosurfactant. The biosurfactant enhances the bacterium's attachment to oil droplets and facilitates the uptake of hydrocarbons. Due to its useful properties expected, there is interest in the biotechnological production of this biosurfactant. To support this effort analytically, a method combining reversed-phase high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) was developed, allowing the separation and identification of glycine-glucolipid congeners. Accurate mass, retention time, and characteristic fragmentation pattern were utilized for species assignment. In addition, charged-aerosol detection (CAD) was employed to enable absolute quantification without authentic standards. The methodology was used to investigate the glycine-glucolipid production by A. borkumensis SK2 using different carbon sources. Mass spectrometry allowed us to identify congeners with varying chain lengths (C6-C12) and degrees of unsaturation (0-1 double bonds) in the incorporated 3-hydroxy-alkanoic acids, some previously unknown. Quantification using CAD revealed that the titer was approximately twice as high when grown with hexadecane as with pyruvate (49 mg/L versus 22 mg/L). The main congener for both carbon sources was glc-40:0-gly, accounting for 64% with pyruvate and 85% with hexadecane as sole carbon source. With the here presented analytical suit, complex and varying glycolipids can be identified, characterized, and quantified, as here exemplarily shown for the interesting glycine-glucolipid of A. borkumensis.