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Appl Environ Microbiol. 2010 May; 76(9): 3057–3060.
Published online 2010 March 19. doi:  10.1128/AEM.02810-09
PMCID: PMC2863424

Anaerobic Oxidation of Fatty Acids and Alkenes by the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus[down-pointing small open triangle]


Archaeoglobus fulgidus oxidizes fatty acids (C4 to C18) and n-alk-1-enes (C12:1 to C21:1) in the presence of thiosulfate as a terminal electron acceptor. End products of metabolism were CO2 and sulfide. Growth on perdeuterated hexadecene yielded C15- to C17-labeled fatty acids as metabolites, thus confirming the ability of A. fulgidus to oxidize alkyl chains.

Many studies have shown that under anaerobiosis, hydrocarbon oxidation can be coupled to sulfate reduction (28), and several sulfate-reducing bacteria have been reported to oxidize n-alkanes and/or n-alkenes (13, 15). Most of these strains are mesophilic, except Desulfothermus naphthae strain TD3, which oxidizes alkanes at 55 to 65°C (23). However, in deep hot environments (e.g., oil reservoirs), it has been established that oil biodegradation could occur at temperatures up to 85 to 90°C (3). Until now, few hyperthermophilic sulfate-reducing microorganisms growing at temperatures higher than 80°C have been isolated from oil field environments. They include the genera Desulfotomaculum, Thermodesulfobacterium, and Archaeoglobus (20), but these microorganisms have never been tested for their ability to oxidize hydrocarbons. Among the Archaea, members of the genus Archaeoglobus are well represented in deep environments and quite widespread in marine (e.g., hydrothermal vents) and terrestrial (hydrothermal systems and oil reservoirs) hot environments (5, 6, 16, 19, 26, 27). Interestingly, the analysis of the complete genome of Archaeoglobus fulgidus strain VC-16 revealed the presence of β-oxidation genes (18). However, its ability to degrade a variety of hydrocarbons and organic acids has been hypothesized (18) but never demonstrated. In the present study, experiments were conducted to elucidate whether A. fulgidus VC-16 can oxidize n-alkanes or more oxidized compounds (n-alkenes and fatty acids) in the presence of thiosulfate or sulfate as a terminal electron acceptor.

Growth of A. fulgidus on long alkyl chains (fatty acids, n-alkanes, and n-alk-1-enes).

Archaeoglobus fulgidus strain VC-16 (DSM 4304), isolated from a terrestrial heated sea floor at Vulcano, Italy (27), was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). This strain was cultivated on medium described by Zellner et al. (30) and modified as followed (g per liter unless indicated): NH4Cl, 0.3; KH2PO4, 0.3; K2HPO4, 0.3; KCl, 0.1; CaCl2·2H2O, 0.1; NaCl, 18; Na2S2O3, 2.37 (15 mM); yeast extract, 0.1; Fe2SO4·7H2O, 1.42 mg; NiSO4·6H2O, 1.6 mg; Na2WO4·2H2O, 38 μg; Na2SeO3·5H2O, 3 μg; l-cysteine hydrochloride, 0.5; resazurin, 1 mg; and trace element solution (4), 10 ml·liter−1. The pH of the medium was adjusted to 7.0. The medium was prepared anoxically, and the headspace of the tubes and bottles was filled with N2-CO2 (4:1). Following sterilization, the medium was amended with Na2S·9H2O (0.4 g·liter−1), NaHCO3 (2.0 g·liter−1), MgCl2·6H2O (3.0 g·liter−1), and 10 ml·liter−1 of a vitamin solution (29). Cultures were inoculated at 10% (vol/vol). For growth tests with hydrocarbons, a preculture on octanoate was used as an inoculum. The inoculum was washed with substrate-free medium and concentrated four times before use.

A. fulgidus was cultivated with fatty acids (2 mM), n-alkanes (1.2 mM), or n-alk-1-enes (1.2 mM) as an energy source. Cultures were grown at 70°C in tubes (Bellco) containing 10 ml of medium and sealed with butyl rubber stoppers. Tubes containing hydrocarbons were incubated upside down. Anaerobic oxidation of substrates was followed by measurement of dissolved sulfide production (8) and thiosulfate or sulfate consumption (ionic chromatography after sulfide removal by precipitation with zinc carbonate [50 g·liter−1]). All cultures were performed in triplicate and compared to control cultures without substrate.

A. fulgidus was able to grow on a wide range of short- to long-chain fatty acids: butyrate, valerate, octanoate, nonanoate, palmitate, and stearate (results not shown). Sulfide production with fatty acids was in the range of 10.5 to 14.2 mM (soluble sulfide) after 7 weeks of incubation, whereas sulfide production in control cultures without a substrate reached only 1.8 mM. Although growth of A. fulgidus (DSM 8774) on short-chain fatty acids, such as valerate, in the presence of hydrogen has already been suggested (5), this is the first demonstration that the hyperthermophilic, sulfate-reducing archaeon A. fulgidus is able to use valerate but also longer-chain fatty acids (C8 to C18) as the sole energy source. Interestingly, the same ability was reported for its close hyperthermophilic relative Geoglobus ahangari, known to grow exclusively by reducing Fe(III) (17a).

The growth of Archaeoglobus fulgidus on some aliphatic saturated (dodecane, hexadecane, and pristane) and unsaturated (dodec-1-ene and hexadec-1-ene) hydrocarbons was also tested. After 1 month of incubation, based on soluble sulfide production and thiosulfate consumption, alkenes but not alkanes were shown to be oxidized (data not reported). Growth of A. fulgidus on individual alk-1-ene was further tested with compounds ranging from C12 to C21. After 24 weeks of incubation, thiosulfate reduction to sulfide appeared clearly higher in cultures with alkenes than in control cultures incubated under the same temperature condition without a substrate (Fig. (Fig.1a).1a). Moreover, all these cultures could be subcultured on the corresponding alkenes, thus proving efficient growth of A. fulgidus on these substrates (Fig. (Fig.1b).1b). This demonstrated that A. fulgidus strain VC-16 is able to grow on C12 to C21 n-alk-1-enes as a unique source of energy. Growth of A. fulgidus on n-alk-1-enes was also demonstrated in the presence of sulfate (15 mM) instead of thiosulfate as a terminal electron acceptor.

FIG. 1.
Thiosulfate consumption after 24 weeks of incubation at 70°C of A. fulgidus strain VC-16 with C12 to C21 n-alk-1-enes. (a) First cultures; (b) corresponding subcultures. Control cultures were incubated without alkenes at the same temperature.

Quantitative degradation of hexadec-1-ene.

Quantitative growth experiments were carried out in tubes sealed with Teflon-coated rubber stoppers (West Pharmaceutical Services) containing 15 ml of medium and 5.5 μl of hexadec-1-ene (1.27 mM). Total sulfide production was determined after alkalinization of the culture by adding 0.3 ml KOH (10 M). The hexadecene concentration was determined by gas chromatography after extraction with heptane using pristane as an internal standard. Three replicate assays together with three biotic (without hydrocarbon but inoculated) and three abiotic (with hydrocarbon but not inoculated) controls were incubated for 17 weeks under the same temperature condition before analysis. Growth of A. fulgidus was demonstrated through the consumption of 0.17 mM hexadec-1-ene and the simultaneous production of 4 mM sulfide. Proportionally, 23.52 mM sulfide would have been produced from 1 mM hexadec-1-ene, which is close to the values expected from the following equation:

equation M1

Hydrocarbon depletion and sulfide production were not observed in incubated abiotic controls.

Identification of perdeuterated hexadecene-derived fatty acids.

A. fulgidus was grown in 450 ml of anoxic medium with labeled hexadecene (perdeuterated d32-hexadec-1-ene, 0.55 mM, synthesized from perdeuterated palmitic acid [Euriso-top] as described by Grossi et al. [14]). Cells were collected by filtration through glass microfiber filters (GF/B; Whatman) and treated with 1 M KOH in methanol-water (1:1 [vol/vol]). Saponifiable (acid) lipids were extracted from the acidified solution (9). Extracts were silylated [bis(trimethylsilyl)trifluoroacetanamide-pyridine (1:1, vol/vol)] and analyzed by gas chromatography-mass spectrometry (GC-MS) using a Finnigan Voyager MD800 mass spectrometer coupled to a 6890HP gas chromatograph equipped with a DB5MS capillary column (30 m by 0.25 mm; film thickness, 0.25 μm). Growth of A. fulgidus on d32-hexadec-1-ene yielded deuterated fatty acids (Fig. (Fig.22 and and3).3). Isotopomers which differed by one or two deuterium atoms were formed but did not yield distinct GC peaks. Structural identification of the main isotopomers (d29-15:0, d29-16:0, and d31-17:0 fatty acids) was achieved by careful selection of mass spectra using mass chromatograms of selected ions and by comparison with previously reported mass spectra (7, 9). The number of deuterium atoms in each fatty acid was deduced from the molecular ion (M+) and from the ion corresponding to the loss of one methyl from the TMS group (M-15). These deuterated fatty acids were not detected in incubated abiotic controls (with hydrocarbon but not inoculated [Fig. [Fig.2]),2]), further demonstrating that A. fulgidus strain VC-16 is able to oxidize n-alkenes anaerobically.

FIG. 2.
Partial mass chromatograms (m/z 76 + 119 + 135) of total cellular fatty acids (silylated) of a culture of A. fulgidus strain VC-16 incubated with d32-hexadec-1-ene at 70°C (top) and of a corresponding incubated abiotic control ...
FIG. 3.
Mass spectra and tentative structure of silylated d29-pentadecanoic acid (a), d29-hexadecanoic acid (b), or d31-heptadecanoic acid (c) from a culture of A. fulgidus strain VC-16 incubated with d32-hexadec-1-ene.

Previous studies have reported the biodegradation of n-alkenes by mesophilic sulfate-reducing bacteria (namely, strains Hxd3 [1], Pnd3 [2], AK-01 [25], Desulfatibacillum aliphaticivorans strain CV2803 [11], Desulfatibacillum alkenivorans strain PF2803 [12], and Desulfatiferula olefinivorans strain LM2801 [10]). To our knowledge, this is the first report of the anaerobic oxidation of hydrocarbons, specifically n-alkenes, by a member of the domain Archaea and at a temperature as high as 70°C. Different initial reactions of anaerobic alk-1-ene oxidation by D. aliphaticivorans strain CV2803 have been proposed (14). This bacterium oxidized alkenes into either branched (methyl- or ethyl-) or linear fatty acids by addition of organic carbon or by hydroxylation of the double bond, respectively. In the present case, the formation of d29-16:0 fatty acid from d32-hexadec-1-ene may suggest that A. fulgidus also hydroxylated the alkene double bond at C-1. However, the loss of three deuterium atoms between the alkene substrate and the corresponding fatty acid (Fig. (Fig.3b)3b) is not in agreement with the results obtained with D. aliphaticivorans strain CV2803, which induced the loss of only two deuterium atoms for the same oxidation step (14). The present results may be explained either by hydrogen/deuterium exchanges at high temperature and/or by distinct mechanisms of alk-1-ene oxidation between A. fulgidus strain VC-16 and D. aliphaticivorans strain CV2803. The involvement of a distinct mechanism in A. fulgidus strain VC-16 is further suggested by the formation of C-odd d29-15:0 and d31-17:0 fatty acids, which could not result from chain shortening (i.e., β-oxidation) or elongation of d29-16:0 fatty acid. The formation of C-even and C-odd fatty acids from C-odd and C-even alkanes, respectively, has been demonstrated in the mesophilic sulfate-reducing bacterium strain Hxd3 (24). The mechanism proposed includes subterminal carboxylation with inorganic carbon at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms, resulting in a fatty acid one carbon shorter than the original alkane but which can subsequently be elongated (24). A similar mechanism could explain the transformation of d32-hexadec-1-ene to d29-15:0 fatty acid by A. fulgidus strain VC-16 but would not support the presence of two additional deuterium atoms in d31-17:0 fatty acid (Fig. (Fig.3c).3c). Alternative mechanisms can be envisaged for the oxidation of alk-1-enes by strain VC-16, including the addition of an organic carbon unit distinct from the one involved in alk-1-ene oxidation by D. aliphaticivorans strain CV2803 (14) or the epoxidation of the alkene double bond. These speculative mechanisms still require further investigation.

Enzymes involved in the activation of alkenes have not yet been defined. Johnson et al. (17) isolated and characterized a Mo-Fe-S-containing enzyme from Azoarcus sp. strain EB1 that was able to mediate, in the absence of oxygen, the hydroxylation of a branched alkene. This enzyme is a heterotrimer composed of a molybdopterin-binding subunit (EbdA), a [4Fe-4S] cluster binding subunit (EbdB), and a membrane anchor subunit that would bind a b-type heme. In addition to these structural genes, the operon would also include a chaperonin-like protein-encoding gene (ebdD) (see Fig. S4 in the supplemental material) (21). Genome analysis of A. fulgidus reveals the presence of a gene cluster encoding a molybdopterin oxidoreductase, exhibiting significant homology with the ebdABCD operon from Azoarcus sp. (21) (see Fig. S4 in the supplemental material). Therefore, the A. fulgidus multisubunit Mo-containing enzyme could be involved in the hydroxylation of the double bond of alk-1-enes, but such a mechanism still needs to be demonstrated.

Based on physiologic and metabolic results, we here provide direct evidence that A. fulgidus is able to grow on mid- to long-chain fatty acids and to form linear fatty acids from n-alk-1-enes, although the complete metabolic pathways of alkene degradation by A. fulgidus still need to be further characterized.

Supplementary Material

[Supplemental material]


This work was partly supported by a French CNRS-INSU grant (BIOHYDEX EC2CO-MicrobiEn project).


[down-pointing small open triangle]Published ahead of print on 19 March 2010.

Supplemental material for this article may be found at


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