Degradation and enhanced oil recovery potential of Alcanivorax borkumensis through production of bio-enzyme and bio-surfactant.

Microbial enhanced oil recovery (EOR) has become the focus of oilfield research due to its low cost, environmental friendliness and sustainability. The degradation and EOR capacity of A. borkumensis through the production of bio-enzyme and bio-surfactant were first investigated in this study. The total protein concentration, acetylcholinesterase, esterase, lipase, alkane hydroxylase activity, surface tension, and emulsification index (EI) were determined at different culture times. The bio-surfactant was identified as glycolipid compound, and the yield was 2.6 ± 0.2 g/L. The nC12 and nC13 of crude oil were completely degraded, and more than 40.0 % of nC14-nC24 was degraded by by A. borkumensis. The results of the microscopic etching model displacement and core flooding experiments showed that emulsification was the main mechanism of EOR. A. borkumensis enhanced the recovery rate by 20.2 %. This study offers novel insights for the development of environmentally friendly and efficient oil fields.

Alcanivorax bacteria as important polypropylene degraders in mesopelagic environments.

IMPORTANCE: PP biodegradation has not been clearly shown (it has been uncertain whether the PP structure is actually biodegraded or not). This is the first report on the obvious biodegradation of PP. At the same time, this study shows that Alcanivorax bacteria could be major degraders of PP in mesopelagic environments. Moreover, PP biodegradation has been investigated by using solid PP as the sole carbon source. However, this study shows that PP would not be used as a sole carbon and energy source. Our data thus provide very important and key knowledge for PP bioremediation.

Alcanivorax borkumensis biofilms enhance oil degradation by interfacial tubulation.

During the consumption of alkanes, Alcanivorax borkumensis will form a biofilm around an oil droplet, but the role this plays during degradation remains unclear. We identified a shift in biofilm morphology that depends on adaptation to oil consumption: Longer exposure leads to the appearance of dendritic biofilms optimized for oil consumption effected through tubulation of the interface. In situ microfluidic tracking enabled us to correlate tubulation to localized defects in the interfacial cell ordering. We demonstrate control over droplet deformation by using confinement to position defects, inducing dimpling in the droplets. We developed a model that elucidates biofilm morphology, linking tubulation to decreased interfacial tension and increased cell hydrophobicity.

Bacteria stretch and bend oil to feed their appetite.

Microbes reshape oil droplets to speed biodegradation.

Beyond oil degradation: enzymatic potential of Alcanivorax to degrade natural and synthetic polyesters.

Pristine marine environments are highly oligotrophic ecosystems populated by well-established specialized microbial communities. Nevertheless, during oil spills, low-abundant hydrocarbonoclastic bacteria bloom and rapidly prevail over the marine microbiota. The genus Alcanivorax is one of the most abundant and well-studied organisms for oil degradation. While highly successful under polluted conditions due to its specialized oil-degrading metabolism, it is unknown how they persist in these environments during pristine conditions. Here, we show that part of the Alcanivorax genus, as well as oils, has an enormous potential for biodegrading aliphatic polyesters thanks to a unique and abundantly secreted alpha/beta hydrolase. The heterologous overexpression of this esterase proved a remarkable ability to hydrolyse both natural and synthetic polyesters. Our findings contribute to (i) better understand the ecology of Alcanivorax in its natural environment, where natural polyesters such as polyhydroxyalkanoates (PHA) are produced by a large fraction of the community and, hence, an accessible source of carbon and energy used by the organism in order to persist, (ii) highlight the potential of Alcanivorax to clear marine environments from polyester materials of anthropogenic origin as well as oils, and (iii) the discovery of a new versatile esterase with a high biotechnological potential.

Nanoshell Assembly for Magnet-Responsive Oil-Degrading Bacteria.

The modified polyelectrolyte-magnetite nanocoating was applied to functionalize the cell walls of oil decomposing bacteria Alcanivorax borkumensis. Cationic coacervate of poly(allylamine) and 20 nm iron oxide nanoparticles allowed for a rapid single-step encapsulation process exploiting electrostatic interaction with bacteria surfaces. The bacteria were covered with rough 70-100-nm-thick shells of magnetite loosely bound to the surface through polycations. This encapsulation allowed for external manipulations of A. borkumensis with magnetic field, as demonstrated by magnetically facilitated cell displacement on the agar substrate. Magnetic coating was naturally removed after multiple cell proliferations providing next generations of the cell in the native nonmagnetic form. The discharged biosurfactant vesicles indicating the bacterial functionality (150 ± 50 nm lipid micelles) were visualized with atomic force microscopy in the bacterial biofilms.

Microbial oil-degradation under mild hydrostatic pressure (10 MPa): which pathways are impacted in piezosensitive hydrocarbonoclastic bacteria?

Oil spills represent an overwhelming carbon input to the marine environment that immediately impacts the sea surface ecosystem. Microbial communities degrading the oil fraction that eventually sinks to the seafloor must also deal with hydrostatic pressure, which linearly increases with depth. Piezosensitive hydrocarbonoclastic bacteria are ideal candidates to elucidate impaired pathways following oil spills at low depth. In the present paper, we tested two strains of the ubiquitous Alcanivorax genus, namely A. jadensis KS_339 and A. dieselolei KS_293, which is known to rapidly grow after oil spills. Strains were subjected to atmospheric and mild pressure (0.1, 5 and 10 MPa, corresponding to a depth of 0, 500 and 1000 m, respectively) providing n-dodecane as sole carbon source. Pressures equal to 5 and 10 MPa significantly lowered growth yields of both strains. However, in strain KS_293 grown at 10 MPa CO2 production per cell was not affected, cell integrity was preserved and PO4(3-) uptake increased. Analysis of its transcriptome revealed that 95% of its genes were downregulated. Increased transcription involved protein synthesis, energy generation and respiration pathways. Interplay between these factors may play a key role in shaping the structure of microbial communities developed after oil spills at low depth and limit their bioremediation potential.

Hydrocarbon-Degrading Bacteria Exhibit a Species-Specific Response to Dispersed Oil while Moderating Ecotoxicity.

The Deepwater Horizon blowout in April 2010 represented the largest accidental marine oil spill and the largest release of chemical dispersants into the environment to date. While dispersant application may provide numerous benefits to oil spill response efforts, the impacts of dispersants and potential synergistic effects with crude oil on individual hydrocarbon-degrading bacteria are poorly understood. In this study, two environmentally relevant species of hydrocarbon-degrading bacteria were utilized to quantify the response to Macondo crude oil and Corexit 9500A-dispersed oil in terms of bacterial growth and oil degradation potential. In addition, specific hydrocarbon compounds were quantified in the dissolved phase of the medium and linked to ecotoxicity using a U.S. Environmental Protection Agency (EPA)-approved rotifer assay. Bacterial treatment significantly and drastically reduced the toxicity associated with dispersed oil (increasing the 50% lethal concentration [LC50] by 215%). The growth and crude oil degradation potential of Acinetobacter were inhibited by Corexit by 34% and 40%, respectively; conversely, Corexit significantly enhanced the growth of Alcanivorax by 10% relative to that in undispersed oil. Furthermore, both bacterial strains were shown to grow with Corexit as the sole carbon and energy source. Hydrocarbon-degrading bacterial species demonstrate a unique response to dispersed oil compared to their response to crude oil, with potentially opposing effects on toxicity. While some species have the potential to enhance the toxicity of crude oil by producing biosurfactants, the same bacteria may reduce the toxicity associated with dispersed oil through degradation or sequestration.

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.

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.

Interaction of Alcanivorax borkumensis with a Surfactant Decorated Oil-Water Interface.

Alcanivorax borkumensis is a hydrocarbon degrading bacterium linked to oil degradation around oil spill sites. It is known to be a surface bacterium leading to substantial interaction with the oil-water interface. Because of its abundance in oil spill regions, it has great potential to be used actively in oil spill remediation. Dispersants are thought to be important in the creation of oil-in-water emulsions that are meant to aid in the biodegradation process by bacteria. Although it is likely that some sort of dispersant will be used again in the case of another oil spill, to date, no studies have shown the impact of dispersants on the bacteria population. Corexit 9500 was the main dispersant used during the Deepwater Horizon oil spill, but little is known about its effect on the bacteria community. We built an experimental platform to quantitatively measure the transient growth of Alcanivorax borkumensis at the interface of oil and water. To our knowledge, this is the first study of how A. borkumensis interacts with a surfactant decorated oil-water interface. We use COREXIT EC9500A, cetylytrimethylamonium bromide, dioctyl sulfosuccinate sodium salt, l-α-phosphatidylcholine, sodium dodecyl sulfate, and Tween 20 to investigate the impact of dispersants on Alcanivorax borkumensis. We assess the impact of these dispersants on the growth rate, lag time, and maximum concentration of Alcanivorax borkumensis. We show that the charge, structure, and surface activity of these surfactants greatly impact the growth of A. borkumensis. Our results indicated that out of the surfactants tested only Tween 20 assists Acanivorax borkumensis growth. The results of this study will be important in the decision of dispersant use in the future.

Quick stimulation of Alcanivorax sp. by bioemulsificant EPS2003 on microcosm oil spill simulation

Oil spill microcosms experiments were carried out to evaluate the effect of bioemulsificant exopolysaccharide (EPS2003) on quick stimulation of hydrocarbonoclastic bacteria. Early hours of oil spill, were stimulated using an experimental seawater microcosm, supplemented with crude oil and EPS2003 (SW+OIL+EPS2003); this system was monitored for 2 days and compared to control microcosm (only oil-polluted seawater, SW+OIL). Determination of bacterial abundance, heterotrophic cultivable and hydrocarbon-degrading bacteria were carried out. Community composition of marine bacterioplankton was determined by 16S rRNA gene clone libraries. Data obtained indicated that bioemulsificant addition stimulated an increase of total bacterial abundance and, in particular, selection of bacteria related to Alcanivorax genus; confirming that EPS2003 could be used for the dispersion of oil slicks and could stimulate the selection of marine hydrocarbon degraders thus increasing bioremediation process.

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.

Amphiphilic siderophore production by oil-associating microbes.

The Deepwater Horizon oil spill in 2010 released an unprecedented amount of oil into the ocean waters of the Gulf of Mexico. As a consequence, bioremediation by oil-degrading microbes has been a topic of increased focus. One factor limiting the rate of hydrocarbon degradation by microbial communities is the availability of necessary nutrients, including iron. The siderophores produced from two Vibrio spp. isolated from the Gulf of Mexico following the Deepwater Horizon oil spill, along with the well-studied oil-degrading microbe, Alcanivorax borkumensis SK2, are studied under iron-limiting conditions. Here we report the amphiphilic amphibactin siderophores produced by the oil-associated bacteria, Vibrio sp. S1B, Vibrio sp. S2A and Alcanivorax borkumensis SK2. These findings provide insight into oil-associating microbial iron acquisition.

Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: a mesocosm simulation study.

Bioaugmentation (amendment with selected bacterial strains) and/or biostimulation (nutrients addition and/or air supply) are relatively new fields in environmental microbiology for preventing pollution and cleanup contamination. In this study, the efficiency of application of bioaugmentation/biostimulation treatments, for recovery of crude oil-polluted seawater, was evaluated. Three different series of experiments were performed in a "Mesocosm Facility" (10.000 L). Natural seawater was artificially polluted with crude oil (1000 ppm) and was amended with inorganic nutrients (Mesocosm 1, M1), inorganic nutrient and an inoculum of Alcanivorax borkumensis SK2(T) (Mesocosm 2, M2) and inorganic nutrient and an inoculum of A. borkumensis SK2(T) and Thalassolituus oleivorans MIL-1(T) (Mesocosm 3, M3), respectively. During the experimental period (20 days) bacterial abundance (DAPI count), culturable heterotrophic bacteria (CFU count), MPN, microbial metabolic activity [Biochemical Oxygen Demand and enzymatic activity (leucine aminopeptidase LAP, ß-glucosidase BG, alkaline phosphatase AP)] and quali-, quantitative analysis of the composition of total extracted and resolved hydrocarbons and their derivates (TERHCs) were carried out. The microbiological and physiological analysis of marine microbial community found during the three different biostimulation and bioaugmentation assays performed in mesocosms show that the load of crude oil increases total microbial abundance, inhibits the activity of some enzymes such as LAP while stimulates both AP and BG activities. The biodegradation results show that bioaugmentation with A. borkumensis SK2(T) alone is able to produce the highest percentage of degradation (95%) in comparison with the biostimulation treatment (80%) and bioaugmentation using an Alcanivorax-Thalassolituus bacterial consortium (70%). This result highlights the reduced biodegradation capability of the consortium used in this study, suggesting an unfavourable interaction between the two bacterial genera.

[Microbial potential for cleaning the oiled iron scale].

The possibility of using microorganisms to clean oiled iron scale of metallurgical production was investigated with the goal of recuperation. A stable microbial association growing on mineral oil as the sole carbon source was isolated from a sample from oiled iron scale taken directly from a metallurgical plant. For microbial cultures isolated from this association, the taxonomic position, as well as their morphological and cultural characteristics, were determined. The microorganisms belonged to the genera Luteimonas, Alcanivorax, Flavobacterium, and Pseudomonas. Microbial associations oxidizing mineral oil were found to contain some microorganisms incapable of its utilization, which stimulated the hydrocarbon-oxidizing microflora. Application of the isolates, as well as of the strains from microbial collections, resulted in a 58% decrease in residual oil content in treated samples of the oiled iron scale.

Quick stimulation of Alcanivorax sp. by bioemulsificant EPS2003 on microcosm oil spill simulation.

Oil spill microcosms experiments were carried out to evaluate the effect of bioemulsificant exopolysaccharide (EPS2003) on quick stimulation of hydrocarbonoclastic bacteria. Early hours of oil spill, were stimulated using an experimental seawater microcosm, supplemented with crude oil and EPS2003 (SW+OIL+EPS2003); this system was monitored for 2 days and compared to control microcosm (only oil-polluted seawater, SW+OIL). Determination of bacterial abundance, heterotrophic cultivable and hydrocarbon-degrading bacteria were carried out. Community composition of marine bacterioplankton was determined by 16S rRNA gene clone libraries. Data obtained indicated that bioemulsificant addition stimulated an increase of total bacterial abundance and, in particular, selection of bacteria related to Alcanivorax genus; confirming that EPS2003 could be used for the dispersion of oil slicks and could stimulate the selection of marine hydrocarbon degraders thus increasing bioremediation process.

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.

Composition and dynamics of biostimulated indigenous oil-degrading microbial consortia from the Irish, North and Mediterranean Seas: a mesocosm study.

Diversity of indigenous microbial consortia and natural occurrence of obligate hydrocarbon-degrading bacteria (OHCB) are of central importance for efficient bioremediation techniques. To investigate the microbial population dynamics and composition of oil-degrading consortia, we have established a series of identical oil-degrading mesocosms at three different locations, Bangor (Menai Straits, Irish Sea), Helgoland (North Sea) and Messina (Messina Straits, Mediterranean Sea). Changes in microbial community composition in response to oil spiking, nutrient amendment and filtration were assessed by ARISA and DGGE fingerprinting and 16Sr RNA gene library analysis. Bacterial and protozoan cell numbers were quantified by fluorescence microscopy. Very similar microbial population sizes and dynamics, together with key oil-degrading microorganisms, for example, Alcanivorax borkumensis, were observed at all three sites; however, the composition of microbial communities was largely site specific and included variability in relative abundance of OHCB. Reduction in protozoan grazing had little effect on prokaryotic cell numbers but did lead to a decrease in the percentage of A. borkumensis 16S rRNA genes detected in clone libraries. These results underline the complexity of marine oil-degrading microbial communities and cast further doubt on the feasibility of bioaugmentation practices for use in a broad range of geographical locations.