Populations of heavy fuel oil-degrading marine microbial community in presence of oil sorbent materials

Aims:  To investigate the feasibility of applying sorbent material X-Oil^® in marine oil spill mitigation and to survey the interactions of oil, bacteria and sorbent.
Methods and Results:  In a series of microcosms, 25 different treatments including nutrient amendment, bioaugmentation with Alcanivorax borkumensis and application of sorbent were tested. Microbial community dynamics were analysed by DNA fingerprinting methods, RISA and DGGE. Results of this study showed that the microbial communities in microcosms with highly active biodegradation were strongly selected in favour of A. borkumensis . Oxygen consumption measurements in microcosms and gas chromatography of oil samples indicated the fast and intense depletion of linear alkanes as well as high oxygen consumption within 1 week followed by consequent slower degradation of branched and polyaromatic hydrocarbons.
Conclusion:  Under given conditions, A. borkumensis was an essential organism for biodegradation, dominating the biofilm microbial community formation and was the reason of emulsification.
Significance and Impact of the Study:  This study strongly emphasizes the pivotal importance of A. borkumensis as an essential organism in the initial steps of marine hydrocarbon degradation. Interaction with the sorbent material X-Oil^® proved to be neutral to beneficial for biodegradation and also promoted the growth of yet unknown micro-organisms.     

Alcanivorax which prevails in oil-contaminated seawater exhibits broad substrate specificity for alkane degradation

Alcanivorax is an alkane-degrading marine bacterium which propagates and becomes predominant in crude-oil-containing seawater when nitrogen and phosphorus nutrients are supplemented. In order to understand why Alcanivorax overcomes other bacteria under such cultural conditions, competition experiments between Alcanivorax indigenous to seawater and the exogenous alkane-degrading marine bacterium, Acinetobacter venetianus strain T4, were conducted. When oil-containing seawater supplemented with nitrogen and phosphorus nutrients was inoculated with A. venetianus strain T4, this bacterium was the dominant population at the early stage of culture. However, its density began to decrease after day 6, and Alcanivorax predominated in the culture after day 20. The crude-oil-degrading profiles of both bacteria were therefore investigated. Alcanivorax borkumensis strain ST-T1 isolated from the Sea of Japan exhibited higher ability to degrade branched alkanes (pristane and phytane) than A. venetianus strain T4. It seems that this higher ability of Alcanivorax to degrade branched alkanes allowed this bacterium to predominate in oil-containing seawater. It is known that some marine zooplanktons produce pristane and Alcanivorax may play a major role in the biodegradation of pristane in seawater.     

Genome sequence completed of Alcanivorax borkumensis, a hydrocarbon-degrading bacterium that plays a global role in oil removal from marine systems

In this paper, we provide background to the genome sequencing project of Alcanivorax borkumensis, which is a marine bacterium that uses exclusively petroleum oil hydrocarbons as sources of carbon and energy (therefore designated "hydrocarbonoclastic"). It is found in low numbers in all oceans of the world and in high numbers in oil-contaminated waters. Its ubiquity and unusual physiology suggest it is globally important in the removal of hydrocarbons from polluted marine systems. A functional genomics analysis of Alcanivorax borkumensis strain SK2 was recently initiated, and its genome sequence has just been completed. Annotation of the genome, metabolome modelling, and functional genomics, will soon reveal important insights into the genomic basis of the properties and physiology of this fascinating and globally important bacterium.     

Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation-related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.     

Proteomic insights into metabolic adaptations in Alcanivorax borkumensis induced by alkane utilization

Alcanivorax borkumensis is a ubiquitous marine petroleum oil-degrading bacterium with an unusual physiology specialized for alkane metabolism. This "hydrocarbonoclastic" bacterium degrades an exceptionally broad range of alkane hydrocarbons but few other substrates. The proteomic analysis presented here reveals metabolic features of the hydrocarbonoclastic lifestyle. Specifically, hexadecane-grown and pyruvate-grown cells differed in the expression of 97 cytoplasmic and membrane-associated proteins whose genes appeared to be components of 46 putative operon structures. Membrane proteins up-regulated in alkane-grown cells included three enzyme systems able to convert alkanes via terminal oxidation to fatty acids, namely, enzymes encoded by the well-known alkB1 gene cluster and two new alkane hydroxylating systems, a P450 cytochrome monooxygenase and a putative flavin-binding monooxygenase, and enzymes mediating beta-oxidation of fatty acids. Cytoplasmic proteins up-regulated in hexadecane-grown cells reflect a central metabolism based on a fatty acid diet, namely, enzymes of the glyoxylate bypass and of the gluconeogenesis pathway, able to provide key metabolic intermediates, like phosphoenolpyruvate, from fatty acids. They also include enzymes for synthesis of riboflavin and of unsaturated fatty acids and cardiolipin, which presumably reflect membrane restructuring required for membranes to adapt to perturbations induced by the massive influx of alkane oxidation enzymes. Ancillary functions up-regulated included the lipoprotein releasing system (Lol), presumably associated with biosurfactant release, and polyhydroxyalkanoate synthesis enzymes associated with carbon storage under conditions of carbon surfeit. The existence of three different alkane-oxidizing systems is consistent with the broad range of oil hydrocarbons degraded by A. borkumensis and its ecological success in oil-contaminated marine habitats.     

Niche-specificity factors of a marine oil-degrading bacterium Alcanivorax borkumensis SK2

Alcanivorax borkumensis strain SK2 is a cosmopolitan hydrocarbonoclastic marine bacterium, with a specialized metabolism adapted to the degradation of petroleum oil hydrocarbons. Transposon mutagenesis was used for functional genome analysis of Alcanivorax SK2 to reveal the genetic basis of other environmentally relevant phenotypes, such as biofilm formation, adaptation to UV exposure, and to growth at either low temperature or high salinity. Forty-eight relevant transposon mutants deficient in any one of these environmentally responsive functions were isolated, and the corresponding genes interrupted by the mini-Tn 5 element were sequenced using inverse PCR. Several cross connections between different phenotypes (e.g. biofilm and UV stress; biofilm and UV and osmoadaptation) on signal transduction level have been revealed, pointing at complex and tightly controlled cellular interactions involving oxygen as a primary messenger and cyclic-di-GMP as a secondary messenger required for Alcanivorax responses to environmental stresses. These results provide insights into bacterial function in a complex marine environment.     

Rubredoxin reductase from Alcanivorax borkumensis: expression and characterization

Oil pollution is an environmental problem of increasing importance. Alcanivorax borkumensis, with a high potential for biotechnological applications, is a key marine hydrocarbonoclastic bacterium and plays a critical role in the bioremediation of oil-polluted marine systems. In oil degrading bacteria, the first step of alkane degradation is catalyzed by a monooxygenase. The reducing electrons are tunneled from NAD(P)H via rubredoxin, one of the most primitive metalloproteins, to the hydroxylase. Rubredoxin reductase is a flavoprotein catalyzing the reduction of rubredoxin. There are two rubredoxin genes, alkG and rubA, in A. borkumensis genome. In this work, the genes encoding rubredoxin reductase (ABO_0162, rubB) and AlkG(ABO_2708, alkG) were cloned and functionally overexpressed in E. coli. Our results demonstrate that RubB could reduce AlkG, therefore compensating for the absence of AlkT, also a rubredoxin reductase, missing in A. borkumensis SK2 genome. These results will increase our knowledge concerning biological alkane degradation and will lead us to design more efficient biotransformation and bioremediation systems.     

Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2

Alcanivorax is an alkane-degrading marine bacterium which propagates and becomes predominant in crude-oil-containing seawater when nitrogen and phosphorus nutrients are supplemented. To identify the genes responsible for alkane degradation in this organism, two putative genes for alkane hydroxylases were cloned from Alcanivorax borkumensis SK2. They were named alkB1 and alkB2. These genes were subsequently disrupted in A. borkumensis SK2, and the growth phenotypes of the disruptants were examined. The results indicate that the alkB1 gene is responsible for the degradation of short-chain n-alkanes. A double mutant defective in both alkB1 and alkB2 was still able to grow on medium-chain n-alkanes, indicating that genes other than alkB1 and alkB2 are also involved in n-alkane hydroxylation by A. borkumensis SK2.     

Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases

Selection experiments and protein engineering were used to identify an amino acid position in integral membrane alkane hydroxylases (AHs) that determines whether long-chain-length alkanes can be hydroxylated by these enzymes. First, substrate range mutants of the Pseudomonas putida GPo1 and Alcanivorax borkumensis AP1 medium-chain-length AHs were obtained by selection experiments with a specially constructed host. In all mutants able to oxidize alkanes longer than C13, W55 (in the case of P. putida AlkB) or W58 (in the case of A. borkumensis AlkB1) had changed to a much less bulky amino acid, usually serine or cysteine. The corresponding position in AHs from other bacteria that oxidize alkanes longer than C13 is occupied by a less bulky hydrophobic residue (A, V, L, or I). Site-directed mutagenesis of this position in the Mycobacterium tuberculosis H37Rv AH, which oxidizes C10 to C16 alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C10 and C11 alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.     

Global features of the Alcanivorax borkumensis SK2 genome

The global feature of the completely sequenced Alcanivorax borkumensis SK2 type strain chromosome is its symmetry and homogeneity. The origin and terminus of replication are located opposite to each other in the chromosome and are discerned with high signal to noise ratios by maximal oligonucleotide usage biases on the leading and lagging strand. Genomic DNA structure is rather uniform throughout the chromosome with respect to intrinsic curvature, position preference or base stacking energy. The orthologs and paralogs of A. borkumensis genes with the highest sequence homology were found in most cases among γ-Proteobacteria, with Acinetobacter and P. aeruginosa as closest relatives. A. borkumensis shares a similar oligonucleotide usage and promoter structure with the Pseudomonadales. A comparatively low number of only 18 genome islands with atypical oligonucleotide usage was detected in the A. borkumensis chromosome. The gene clusters that confer the assimilation of aliphatic hydrocarbons, are localized in two genome islands which were probably acquired from an ancestor of the Yersinia lineage, whereas the alk genes of Pseudomonas putida still exhibit the typical Alcanivorax oligonucleotide signature indicating a complex evolution of this major hydrocarbonoclastic trait.     

Microbial consortia in mesocosm bioremediation trial using oil sorbents, slow-release fertilizer and bioaugmentation

An experimental prototype oil boom including oil sorbents, slow-release fertilizers and biomass of the marine oil-degrading bacterium, Alcanivorax borkumensis , was applied for sorption and degradation of heavy fuel oil in a 500-L mesocosm experiment. Fingerprinting of DNA and small subunit rRNA samples for microbial activity conducted to study the changes in microbial communities of both the water body and on the oil sorbent surface showed the prevalence of A. borkumensis on the surface of the oil sorbent. Growth of this obligate oil-degrading bacterium on immobilized oil coincided with a 30-fold increase in total respiration. A number of DNA and RNA signatures of aromatic hydrocarbon-degrading bacteria were detected both in samples of water body and on oil sorbent. Ultimately, the heavy fuel oil in this mesocosm study was effectively removed from the water body. This is the first study to successfully investigate the fate of oil-degrading microbial consortia in an experimental prototype for a bioremediation strategy in offshore, coastal or ship-bound oil spill mitigation using a combination of mechanical and biotechnological techniques.     

Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2

A novel mutant of the marine oil-degrading bacterium Alcanivorax borkumensis SK2, containing a mini-Tn5 transposon disrupting a "tesB-like" acyl-coenzyme A (CoA) thioesterase gene, was found to hyperproduce polyhydroxyalkanoates (PHA), resulting in the extracellular deposition of this biotechnologically important polymer when grown on alkanes. The tesB-like gene encodes a distinct novel enzyme activity, which acts exclusively on hydroxylated acyl-CoAs and thus represents a hydroxyacyl-CoA-specific thioesterase. Inactivation of this enzyme results in the rechanneling of CoA-activated hydroxylated fatty acids, the cellular intermediates of alkane degradation, towards PHA production. These findings may open up new avenues for the development of simplified biotechnological processes for the production of PHA as a raw material for the production of bioplastics.     

Central role of dynamic tidal biofilms dominated by aerobic hydrocarbonoclastic bacteria and diatoms in the biodegradation of hydrocarbons in coastal mudflats

Mudflats and salt marshes are habitats at the interface of aquatic and terrestrial systems that provide valuable services to ecosystems. Therefore, it is important to determine how catastrophic incidents, such as oil spills, influence the microbial communities in sediment that are pivotal to the function of the ecosystem and to identify the oil-degrading microbes that mitigate damage to the ecosystem. In this study, an oil spill was simulated by use of a tidal chamber containing intact diatom-dominated sediment cores from a temperate mudflat. Changes in the composition of bacteria and diatoms from both the sediment and tidal biofilms that had detached from the sediment surface were monitored as a function of hydrocarbon removal. The hydrocarbon concentration in the upper 1.5 cm of sediments decreased by 78% over 21 days, with at least 60% being attributed to biodegradation. Most phylotypes were minimally perturbed by the addition of oil, but at day 21, there was a 10-fold increase in the amount of cyanobacteria in the oiled sediment. Throughout the experiment, phylotypes associated with the aerobic degradation of hydrocarbons, including polycyclic aromatic hydrocarbons (PAHs) (Cycloclasticus) and alkanes (Alcanivorax, Oleibacter, and Oceanospirillales strain ME113), substantively increased in oiled mesocosms, collectively representing 2% of the pyrosequences in the oiled sediments at day 21. Tidal biofilms from oiled cores at day 22, however, consisted mostly of phylotypes related to Alcanivorax borkumensis (49% of clones), Oceanospirillales strain ME113 (11% of clones), and diatoms (14% of clones). Thus, aerobic hydrocarbon biodegradation is most likely to be the main mechanism of attenuation of crude oil in the early weeks of an oil spill, with tidal biofilms representing zones of high hydrocarbon-degrading activity.     

Analysis of storage lipid accumulation in Alcanivorax borkumensis: Evidence for alternative triacylglycerol biosynthesis routes in bacteria

Marine hydrocarbonoclastic bacteria, like Alcanivorax borkumensis, play a globally important role in bioremediation of petroleum oil contamination in marine ecosystems. Accumulation of storage lipids, serving as endogenous carbon and energy sources during starvation periods, might be a potential adaptation mechanism for coping with nutrient limitation, which is a frequent stress factor challenging those bacteria in their natural marine habitats. Here we report on the analysis of storage lipid biosynthesis in A. borkumensis strain SK2. Triacylglycerols (TAGs) and wax esters (WEs), but not poly(hydroxyalkanoic acids), are the principal storage lipids present in this and other hydrocarbonoclastic bacterial species. Although so far assumed to be a characteristic restricted to gram-positive actinomycetes, substantial accumulation of TAGs corresponding to a fatty acid content of more than 23% of the cellular dry weight is the first characteristic of large-scale de novo TAG biosynthesis in a gram-negative bacterium. The acyltransferase AtfA1 (ABO_2742) exhibiting wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) activity plays a key role in both TAG and WE biosynthesis, whereas AtfA2 (ABO_1804) was dispensable for storage lipid formation. However, reduced but still substantial residual TAG levels in atfA1 and atfA2 knockout mutants compellingly indicate the existence of a yet unknown WS/DGAT-independent alternative TAG biosynthesis route. Storage lipids of A. borkumensis were enriched in saturated fatty acids and accumulated as insoluble intracytoplasmic inclusions exhibiting great structural variety. Storage lipid accumulation provided only a slight growth advantage during short-term starvation periods but was not required for maintaining viability and long-term persistence during extended starvation phases.     

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.     

Determining the identity and roles of oil-metabolizing marine bacteria from the Thames estuary, UK

Crude oil is a complex mixture of different hydrocarbons. While diverse bacterial communities can degrade oil, the specific roles of individual members within such communities remain unclear. To identify the key bacterial taxa involved in aerobic degradation of specific hydrocarbons, microcosm experiments were established using seawater from Stanford le Hope, Thames estuary, UK, adjacent to a major oil refinery. In all microcosms, hydrocarbon degradation was significant within 10 weeks, ranging from > 99% of low-molecular-weight alkanes (C(10)-C(18)), 41-84% of high-molecular-weight alkanes (C(20)-C(32)) and pristane, and 32-88% of polycyclic aromatic hydrocarbons (PAHs). Analysis of 16S rRNA sequences from clone libraries and denaturing gradient gel electrophoresis (DGGE) indicated that, except when incubated with fluorene, PAH-degrading communities were dominated by Cycloclasticus. Moreover, PAH-degrading communities were distinct from those in microcosms containing alkanes. Degradation of the branched alkane, pristane, was carried out almost exclusively by Alcanivorax. Bacteria related to Thalassolituus oleivorans (99-100% identity) were the dominant known alkane degraders in n-alkane (C(12)-C(32)) microcosms, while Roseobacter-related bacteria were also consistently found in these microcosms. However, in contrast to previous studies, Thalassolituus, rather than Alcanivorax, was dominant in crude oil-enriched microcosms. The communities in n-decane microcosms differed from those in microcosms supplemented with less volatile alkanes, with a phylogenetically distinct species of Thalassolituus out-competing T. oleivorans. These data suggest that the diversity and importance of the genus Thalassolituus is greater than previously established. Overall, these experiments demonstrate how degradation of different petroleum hydrocarbons is partitioned between different bacterial taxa, which together as a community can remediate petroleum hydrocarbon-impacted estuarine environments.     

Obligate oil-degrading marine bacteria

Over the past few years, a new and ecophysiologically unusual group of marine hydrocarbon-degrading bacteria - the obligate hydrocarbonoclastic bacteria (OHCB) - has been recognized and shown to play a significant role in the biological removal of petroleum hydrocarbons from polluted marine waters. The introduction of oil or oil constituents into seawater leads to successive blooms of a relatively limited number of indigenous marine bacterial genera--Alcanivorax, Marinobacter, Thallassolituus, Cycloclasticus, Oleispira and a few others (the OHCB)--which are present at low or undetectable levels before the polluting event. The types of OHCB that bloom depend on the latitude/temperature, salinity, redox and other prevailing physical-chemical factors. These blooms result in the rapid degradation of many oil constituents, a process that can be accelerated further by supplementation with limiting nutrients. Genome sequencing and functional genomic analysis of Alcanivorax borkumensis, the paradigm of OHCB, has provided significant insights into the genomic basis of the efficiency and versatility of its hydrocarbon utilization, the metabolic routes underlying its special hydrocarbon diet, and its ecological success. These and other studies have revealed the potential of OHCB for multiple biotechnological applications that include not only oil pollution mitigation, but also biopolymer production and biocatalysis.     

Efficacy of intervention strategies for bioremediation of crude oil in marine systems and effects on indigenous hydrocarbonoclastic bacteria

There is little information on how different strategies for the bioremediation of marine oil spills influence the key indigenous hydrocarbon-degrading bacteria (hydrocarbonoclastic bacteria, HCB), and hence their remediation efficacy. Therefore, we have used quantitative polymerase chain reaction to analyse changes in concentrations of HCB in response to intervention strategies applied to experimental microcosms. Biostimulation with nutrients (N and P) produced no measurable increase in either biodegradation or concentration of HCB within the first 5 days, but after 15 days there was a significant increase (29%; P < 0.05) in degradation of n-alkanes, and an increase of one order of magnitude in concentration of Thalassolituus (to 10(7) cells ml(-1)). Rhamnolipid bioemulsifier additions alone had little effect on biodegradation, but, in combination with nutrient additions, provoked a significant increase: 59% (P < 0.05) more n-alkane degradation by 5 days than was achieved with nutrient additions alone. The very low Alcanivorax cell concentrations in the microcosms were hardly influenced by addition of nutrients or bioemulsifier, but strongly increased after their combined addition, reflecting the synergistic action of the two types of biostimulatory agents. Bioaugmentation with Thalassolituus positively influenced hydrocarbon degradation only during the initial 5 days and only of the n-alkane fraction. Bioaugmentation with Alcanivorax was clearly much more effective, resulting in 73% greater degradation of n-alkanes, 59% of branched alkanes, and 28% of polynuclear aromatic hydrocarbons, in the first 5 days than that obtained through nutrient addition alone (P < 0.01). Enhanced degradation due to augmentation with Alcanivorax continued throughout the 30-day period of the experiment. In addition to providing insight into the factors limiting oil biodegradation over time, and the competition and synergism between HCB, these results add weight to the use of bioaugmentation in oil pollution mitigation strategies.     

Whole‐cell bacterial bioreporter for actively searching and sensing of alkanes and oil spills

Acinetobacter baylyi ADP1 was found to tolerate seawater and have a special ability of adhering to an oil–water interface of 10–80 µm emulsified mineral and crude oil droplets. These properties make ADP1 an ideal bacterial chassis for constructing bioreporters that are able to actively search and sense oil spill in water and soils. Acinetobacter baylyi bioreporter ADPWH_alk was developed and applied to the detection of alkanes and alkenes in water, seawater and soils. Bioreporter ADPWH_alk was able to detect a broad range of alkanes and alkenes with carbon chain length from C7 to C36. So far, ADPWH_alk is the only bioreporter that is able to detect alkane with carbon chain length greater than C18. This bioreporter responded to the alkanes in about 30 min and it was independent to the cell growth phase because of two point mutations in alkM promoter recognized by alkane regulatory protein ALKR. ADPWH_alk was applied to detect mineral oil, Brent, Chestnut and Sirri crude oils in water and seawater in the range 0.1–100 mg l^?1, showing that the bioreporter oil detection was semi‐quantitative. This study demonstrates that ADPWH_alk is a rapid, sensitive and semi‐quantitative bioreporter that can be useful for environmental monitoring and assessment of oil spills in seawater and soils.