INTRODUCTION

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Alkanes are major components of petroleum fuels, which can be commonly found in contaminated environments, and numerous studies on their biodegradability have been conducted. Earlier investigations concentrated on processes occurring under aerobic condition (4), whereby the initial attack of the aliphatic hydrocarbon chain is generally mediated by monooxygenases. Detailed studies on the enzymology and genetics of specific systems are also well documented (26, 28). In contrast, little is known about the biodegradation of alkanes under anoxic conditions, where oxygen-initiated reactions cannot occur. Cases of alkane degradation under different reducing conditions were sparsely reported in enrichment or microcosm studies over the past decades (6, 8, 10, 11, 15, 16, 27). While degradation by pure cultures of Desulfovibrio under sulfate-reducing conditions has been reported in the early literature (9, 18, 22), these cultures were not preserved and have not been further studied (2). Even now, little is known about the degrading organisms and the degradation mechanism(s). The scarcity of pure bacterial cultures available for detailed studies, the generally slow anaerobic processes, and the requirement for more elaborate microbiological techniques may have hindered the progress in this area.

A key publication by Aeckersberg et al. in 1991 (2) reported the isolation of a sulfate-reducing bacterium which, under rigorous conditions, was shown to degrade and grow on alkanes under strictly anoxic conditions. Later, Rueter et al. (23) reported the isolation of a new type of thermophilic, sulfate-reducing bacterium from the sediment of Guaymas Basin (Gulf of California, Mexico) and demonstrated its ability to perform anaerobic alkane degradation. These reports have again generated interest in this area, and the acquisition of the pure isolates opens opportunities for more detailed investigations on the degradation processes. In this paper, we report the isolation and characterization of a novel alkane-degrading, sulfate-reducing bacterium.

	MATERIALS AND METHODS

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Initial culturing conditions. The alkane-degrading bacterial strain AK-01 was isolated from the active sulfate-reducing enrichment cultures established previously with petroleum-contaminated sediments collected from the Arthur Kill, N.Y., a hydrocarbon-impacted intertidal waterway between Staten Island and New Jersey (24). The medium used for maintaining the enrichment cultures and isolating the bacterium contained, per liter of deionized water, the following: NaCl, 23 g; KCl, 1.3 g; MgCl₂ · 6H₂O, 1 g; CaCl₂ · 2H₂O, 0.1 g; NH₄Cl, 0.5 g; KH₂PO₄, 0.2 g; Na₂SO₄, 1.42 g; NaHCO₃, 2.5 g; Na₂S · 9H₂O, 0.5 g; vitamin solution, 1 ml; and trace element solution, 5 ml. The vitamin solution was modified from that of Widdel and Bak (31) and contained, per liter of deionized water, the following: vitamin B₁₂, 1 mg; D-(+)-biotin, 20 mg; folic acid, 20 mg; nicotinic acid, 50 mg; p-aminobenzoic acid, 50 mg; calcium D-(+)-pantothenate, 50 mg; pyridoxine HCl, 100 mg; riboflavin, 50 mg; thiamine, 50 mg; and thioctic acid, 50 mg (final pH adjusted to 7). The trace element solution contained, per liter of 0.01 N HCl, the following: CoCl₂ · 6H₂O, 6 g; CuCl₂, 30 mg; FeCl₂ · 4H₂O, 0.3 g; H₃BO₃, 1.14 g; MnCl₂ · 4H₂O, 4 g; Na₂MoO₄ · 2H₂O, 0.5 g; NiCl₂ · 6H₂O, 0.3 g; ZnCl₂, 0.42 g. All of the above medium components except NaHCO₃, Na₂S · 9H₂O, and the vitamin solution were added to deionized water and then deoxygenated by bubbling with a stream of N₂-CO₂ gas (70:30) for 1 h. The medium was dispensed into serum bottles, crimp-sealed with butyl rubber stoppers, and autoclaved. A filter-sterilized stock solution containing 1 M NaHCO₃ and 67 mM Na₂S · 9H₂O was then added to the autoclaved medium at 3% (vol/vol). The vitamin solution was also filter sterilized and added to the medium. The final pH of the medium was about 6.8. Medium for the serial dilution procedures was further amended with 0.1 g of yeast extract per liter.

Pure culture growth and maintenance. After the pure culture of strain AK-01 was obtained, the medium used for routine maintenance and most characterization experiments was the same as the one described above, except that another trace element solution modified from that of Widdel and Bak (31) was used. Each liter of medium received 1 ml of the modified trace element solution, which contained, per liter of 0.1 N HCl, the following: CoCl₂ · 6H₂O, 190 mg; CuCl₂ · 2H₂O, 2 mg; FeCl₂ · 4H₂O, 1.5 g; H₃BO₃, 6 mg; MnCl₂ · 4H₂O, 100 mg; Na₂MoO₄ · 2H₂O, 36 mg; NiCl₂ · 6H₂O, 24 mg; and ZnCl₂, 70 mg. Yeast extract (0.1 g/liter) was also added except when stated otherwise. In addition, the pH and NaCl concentration of this mineral salt medium were varied to test for their effects on growth of the strain on hexadecane. The pH was finally set at 7.0 (by addition of sodium hydroxide) and the NaCl concentration was set at 10 g/liter after these conditions were shown to support optimal growth. The incubation temperature was 30°C except when stated otherwise. Growth was monitored routinely by measuring sulfate loss instead of increase in optical density (OD) over time due to interference from water-insoluble substrates like alkanes and long-chain fatty acids. The sulfate reduction rates thus determined were used to estimate the doubling times of the bacterium cultured under specific conditions. (The direct correlation between the amount of sulfate loss and cell growth was confirmed by a supplementary experiment which simultaneously monitored the OD₅₄₀ and sulfate concentration of a culture of AK-01 grown on octanoate, a water-soluble fatty acid.)

Isolation. Initial enrichments established as described previously (24) were subcultured by transferring a 5% inoculum to fresh medium with hexadecane (0.1 ml per 50 ml of medium) as the sole carbon source. Growth of the subcultures, as indicated by sulfate reduction, was consistently observed upon refeeding with sulfate when it was exhausted (with hexadecane in excess). The subcultures were then serially diluted 10-fold into tubes containing medium plus hexadecane (0.1 ml per 15 ml of medium) and yeast extract (0.1 g/liter). Utilization of hexadecane was indicated by sulfate loss and increase in cell number in the culture medium. Sulfate loss of less than 3% of the total and no cell growth were observed in the tubes with yeast extract only (no hexadecane). In each dilution series, the most highly diluted medium showing growth was further serially diluted in the same manner. After three consecutive series of dilution, taking place over 1 year, cultures apparently with a single morphotype (short rods) were obtained. The purity of the culture was verified by subculturing it into the mineral salt medium containing 5 mM lactate, pyruvate, formate, or butyrate and other complex media including tryptic soy broth (TSB; Difco Laboratories, Detroit, Mich.) brain heart infusion (Difco) and nutrient broth (Difco) prepared under anoxic conditions. (Agar shake tubes containing hexadecane as the substrate were also inoculated with cultures obtained by serial dilution, but no visible colony was found over 6 months of incubation.)

Cell surface hydrophobicity. The cell surface hydrophobicity of AK-01 was determined by an adaptation of the bacterial adherence to hydrocarbons test (21). AK-01 was cultured on hexadecane for 1 month to the stationary phase. Cells were collected by centrifugation, resuspended in about 2 ml of the residual medium to give a concentrated suspension, and used without further washing. Various amounts of the cell concentrate were then added to sealed glass tubes, each containing 4 ml of the mineral salt medium under anoxic conditions. The diluted cell suspensions in the tubes had initial OD₅₄₀ of 0.3 to 0.6 as determined with a spectrophotometer (Spectronic 20; Bausch & Lomb, Rochester, N.Y.). To each tube, 1 ml of hexadecane (previously deoxygenated by bubbling with N₂) was then added. The tubes were vortexed for 2 min and allowed to stand for 30 min for phase separation. The OD₅₄₀ of the aqueous phase of each tube was again measured. A hydrophobicity index, defined as the percentage of total cells that adhered to hexadecane, was calculated for each tube as follows: 100% × (initial OD₅₄₀  final OD₅₄₀)/initial OD₅₄₀.

Detection of desulfoviridin. The presence of desulfoviridin in strain AK-01 was tested by the method of Postgate (20). Cells of AK-01 grown on octanoate (2 mM) were collected by centrifugation, resuspended in deionized water, and lysed by adding drops of 2 N NaOH. The cell lysate was then examined under UV light (365 nm) for red fluorescence. Cultures of Desulfovibrio gigas and Desulfobacter curvatus were used as positive and negative controls, respectively.

G+C content. The G+C content of the DNA of AK-01 was determined by a high-pressure liquid chromatography (HPLC) method modified from that of Mesbah et al. (14) with salmon sperm DNA as a standard. Eppendorf tubes each containing 50 µl of DNA sample (80 µg per ml) were heated in a boiling-water bath for 2 min and then immediately chilled in an ice bath. To each sample, 100 µl of sodium acetate buffer (30 mM; pH 5.3), 10 µl of zinc sulfate (20 mM), and 6 µl of P1 nuclease (1 mg per ml of sodium acetate buffer; equivalent to 340 U per ml) were added. All tubes of the reaction mixture were first incubated at 37°C for 2.5 h. After that, 10 µl of calf intestinal mucosa alkaline phosphatase (100 U per ml of 0.1 M Tris buffer [pH 8.1]) was added and the tubes were further incubated for 6 h. After incubation, the debris was spun down at 13,500 × g for 10 min in a microcentrifuge and the supernatant was stored frozen at 20°C until analyzed by HPLC.

Utilization of electron donors and acceptors. Utilization of electron donors for growth by strain AK-01 was tested by adding the compounds to the mineral salt medium and then inoculating it with cultures of strain AK-01 pregrown on hexadecane or fatty acids. Sodium salts of fumarate (5 mM), lactate (5 mM), malate (5 mM), pyruvate (5 mM), succinate (5 mM), formate (5 mM), acetate (5 mM), propionate (5 mM), isobutyrate (4 mM), 2-methylbutyrate (4 mM), 3-methylbutyrate (4 mM), 2-methylheptanoate (2 mM), benzoate (2 mM), and phenylacetate (2 mM) were added as electron donors. Their concentrations were monitored by HPLC with detection by UV absorption (see "Chemical analysis" below). Butyrate (1 mM), pentanoate (1 mM), hexanoate (1 mM), heptanoate (0.5 mM), octanoate (0.5 mM), nonanoate (0.5 mM), and decanoate (0.5 mM) were monitored by extracting the compounds from acidified samples of the culture media with pentane and analyzing them by gas chromatography with flame ionization detection (GC-FID; see "Chemical analysis" below). Water-insoluble fatty acids of C₁₁ to C₁₆ (0.25 mM each) were extracted from the whole culture (100 ml) with hexane after acidification. The extracts were then evaporated dry and methylated by being heated at 80°C in a mixture of methanol and 6 N HCl (volume ratio, 1:1.18) for 10 min. The resulting fatty acid methyl esters were extracted with hexane and analyzed by GC-FID. Autotrophic growth with hydrogen as the electron donor was tested by preparing cultures in yeast extract-free medium and pressurizing the headspace with hydrogen gas (100%). Growth was monitored by measuring sulfate losses in the culture media by ion chromatography (IC). Autoclaved controls were established for all experiments described above.

16S rRNA gene sequence determination and phylogenetic analysis. Strain AK-01 was cultured in mineral salt medium (with hexadecane or butyrate) and TSB (supplemented with 1 mM decanoate). Genomic DNA samples were prepared independently from cultures grown under the three different nutritional conditions by density gradient centrifugation in cesium chloride (19). Strain Hxd3 was grown on stearate, and its genomic DNA was extracted by a miniprep method (25).

Mineralization of [1-¹⁴C]hexadecane. Strain AK-01 grown on hexadecane was inoculated into fresh medium at 20% (vol/vol) to make a master culture that was 100 ml. Aliquots of 25 ml were then anaerobically dispensed into 30-ml serum bottles, which were sealed with Teflon-coated butyl rubber stoppers and aluminum crimp seals. For sterile controls, a 100-ml culture grown on hexadecane was autoclaved and replicates of the sterilized cultures were prepared as described above. [1-¹⁴C]hexadecane (Amersham, Arlington Heights, Ill.) was dissolved in unlabeled hexadecane to 0.084 µCi/µl, of which 2 µl was added to the experimental cultures through a microliter syringe. To each of the sterile controls, 2 µl of labeled hexadecane at 0.125 µCi/µl was added. All cultures were shaken (200 rpm) for 1 h to disperse the hexadecane in the medium and then incubated at 30°C in the dark without shaking. After 78 days of incubation, the entire contents of each bottle were acidified with HCl and purged with N₂ gas for 10 min. The purged gas from the cultures was directed to a series of three vials of Oxosol ¹⁴C scintillation cocktail (National Diagnostics, Atlanta, Ga.), in which the liberated ¹⁴CO₂ was trapped. After purging, 2 ml of the acidified culture suspension was added to 10 ml of ReadySafe scintillation cocktail (Beckman Instruments, Inc., Fullerton, Calif.). All the vials were counted in a scintillation counter (model LS 5000 TD; Beckman).

Utilization of hexadecane and hexadecanoate. The doubling times of AK-01 grown on hexadecane, octanoate and hexadecanoate were estimated by determining the sulfate reduction rates in these cultures. Cultures containing 20 µl of hexadecane per 100 ml of medium (0.68 mmol/liter), 2.5 mM sodium octanoate, or 0.6 mM sodium hexadecanoate as the growth substrate were prepared and monitored for sulfate loss by IC.

Chemical analysis. HPLC analysis was performed on a Beckman System Gold liquid chromatograph with detection by UV absorption. Nonaromatic compounds including fumaric, lactic, malic, pyruvic, succinic, formic, acetic, propionic, isobutyric, 2-methylbutyric, and 3-methylbutyric acids were analyzed on a ion-exchange column (ROA; 300 by 7.8 mm; particle size, 8 µm; Phenomenex, Torrance, Calif.) with 0.005 N H₂SO₄ as the mobile phase (flow rate, 0.6 ml/min) and UV absorption at 210 nm. Benzoic and phenylacetic acids were analyzed on a reverse-phase column (Ultrasphere ODS C₁₈; 250 by 4.6 mm; particle size, 5 µm; Beckman) with methanol-water-acetic acid (60:38:2) as the mobile phase (flow rate, 1 ml/min) and UV absorption at 254 nm. Nucleosides in the digested DNA sample for G+C content determination was analyzed on the same reverse-phase column but with a mobile phase containing 12% methanol in triethylamine phosphate (20 mM; pH 5.1). The flow rate was set at 1.5 ml per min, and UV absorption was measured at 254 nm.

Nucleotide sequence accession numbers. The determined 16S rRNA gene sequences of strains AK-01 and Hxd3 were submitted to GenBank under the accession numbers AF141328 and AF141881, respectively.

	RESULTS

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Isolation of strain AK-01. Active consortia enriched on hexadecane under sulfate-reducing conditions had been previously established, and their ability to degrade hexadecane by coupling to sulfate reduction was demonstrated (24). The consortia were serially diluted in mineral salt medium with hexadecane under sulfate-reducing conditions, and growth was observed as sulfate reduction and as an increase in cell number in tubes at a dilution of 10⁷ or 10⁸. Cultures with apparently a single morphotype were obtained after three consecutive dilution series. The purity of the culture was tested by subculturing it in the sulfate-containing mineral salt medium amended with lactate, pyruvate, formate, or butyrate (growth substrates commonly used by a variety of sulfate-reducing bacteria). Ample growth on formate and butyrate and poor growth on lactate and pyruvate were observed. Only one morphotype appeared with each substrate, and it appeared similar to that grown on hexadecane. In addition, when the putative pure culture was inoculated into complex media (TSB, brain heart infusion broth, and nutrient broth), no bacterial growth of any kind was observed after 1 month of incubation, supporting the purity of the hexadecane-degrading culture. The sequences of the 16S rRNA genes amplified from genomic DNA extracted from cultures grown in three different media (the mineral salt medium with hexadecane, the mineral salt medium with butyrate, and TSB with decanoate) were also compared. The sequences determined for the three different cultures contained about 900 bp (with the 27f-907r primer set) and were identical. These results led us to conclude that a pure culture was obtained.

Morphology. Cells of strain AK-01 are short rods about 1 to 1.5 µm long and 0.5 µm in diameter when grown on hexadecane (Fig. 1). Clumps of dividing cells are commonly seen to cluster closely around droplets of hexadecane under phase-contrast microscopy. On the other hand, cells found dispersed in the aqueous medium do not appear to be actively dividing. When grown on favorable substrates like butyrate and hexadecanoate, cells were typically longer (1.5 to 2 µm), with long unicellular filaments occurring occasionally during log phase of growth. After reaching the late stationary phase, the cells appeared to change from rods to spheres. The cells stained gram negative and showed no motility or sporulation at any stage of growth. Small refractile granules were occasionally seen inside cells (in about 1 or 2 of 10 cells), suggesting the occurrence of storage materials like poly--hydroxyalkanoates.

View larger version (97K): [in this window] [in a new window]   FIG. 1.   Transmission electron photomicrograph of strain AK-01 grown on hexadecane (negatively stained with uranyl acetate). Bar, 1 µm.

Physiology and other characteristics. Strain AK-01 is mesophilic, with an optimal growth temperature at 26 to 28°C when cultured on hexadecane and 33 to 35°C when cultured on octanoate. The pH optimum for growth on hexadecane is 6.9 to 7.0. Growth on hexadecane was observed at NaCl concentrations of 1 to 60 g/liter, with the optimum at 10 g/liter. (The site from which it was isolated has a salinity of about 20 ppt.) When cultured at 30°C in medium adjusted to the optimal pH and NaCl concentration, doubling times were 3.0, 1.2, and 0.8 days with hexadecane (0.68 mmol/liter), octanoate (2.5 mM), and hexadecanoate (0.6 mmol/liter) as growth substrates, respectively.

Phylogenetic analysis. The PCR products obtained by amplification of the 16S rRNA gene of strain AK-01 with the primer set of 27f and SRB-R3 contained approximately 1,500 bp. Those of strain Hxd3 obtained with the primer set of 27f and SRB-R1 contained approximately 1,400 bp. The results that turned up in the similarity search were predominantly 16S rRNA sequences of bacteria that belong to the delta subdivision of the class Proteobacteria. A phylogenetic tree showing the relationship between strains AK-01, Hxd3, TD3 and other selected bacteria is shown in Fig. 2. As illustrated, AK-01 clearly belongs to the delta subdivision of the class Proteobacteria and is most closely related to the type species of the genera Desulfosarcina, Desulfonema, and Desulfococcus. Evolutionary distances (calculated by the Kimura two-parameter method) between strain AK-01 and Desulfosarcina variabilis, Desulfonema limicola, Desulfococcus multivorans, strain Hxd3, and strain TD3 are 9.11, 10.39, 9.35, 10.53, and 21.55 substitutions per 100 bp, respectively.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Phylogenetic relationship between the three alkane-degrading strains AK-01, Hxd3, and TD3 and other bacteria in the class Proteobacteria based on 16S rRNA sequence. The tree was constructed from approximately 1,300 aligned bases; T, type species in the genus; *, the 10 bacteria with their 16S rRNA sequences most similar to that of strain AK-01 according to the FASTA search.

Degradation of hexadecane coupled to sulfate reduction. Mineralization of hexadecane to carbon dioxide by strain AK-01 was demonstrated with [1-¹⁴C]hexadecane. As shown in Fig. 3, about 40% of the added ¹⁴C was recovered as ¹⁴CO₂ in the experimental cultures after 78 days of incubation. Less than 1% of the added radioactivity was recovered as ¹⁴CO₂ in the autoclaved controls. Recoveries of the total added radioactivity were about 80%. The incomplete recovery of radioactivity may be due to the long period of incubation needed for this experiment. Despite the presence of their Teflon coating, some sorption of [1-¹⁴C]hexadecane by the rubber stoppers may have occurred. Nonetheless, these results indicate that mineralization to carbon dioxide does take place.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Formation of 14CO2 in autoclaved and active cultures of strain AK-01 fed with [1-14C]hexadecane after 78 days of incubation. Results are means of triplicate determinations, and error bars represent one standard deviation.

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	DISCUSSION

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We have isolated and characterized a sulfate-reducing strain, AK-01, which is able to degrade alkanes under strict anoxic conditions. Its characterization includes both phenotypic and phylogenetic studies, as recommended for the taxonomic examination of the class Proteobacteria (17). Table 2 summarizes a number of phenotypic characteristics of strain AK-01 which are taxonomically important for the gram-negative sulfate-reducing bacteria and are useful for determining the relationship of AK-01 to the other sulfate reducers. Strains Hxd3 and TD3, two of the recently reported alkane-degrading sulfate-reducing bacteria (1, 3, 23), and the type species of key genera in the gram-negative sulfate-reducing bacteria (31) are included for comparison with the new strain. As shown in Table 2, strain AK-01 is different in several aspects from the genus Desulfovibrio in the suggested family Desulfovibrionaceae (31). Desulfovibrio species typically contain desulfoviridin; grow well on lactate, fumarate, and malate but cannot grow on fatty acids; and are incomplete oxidizers of organic substrates. Desulfomicrobium species also characteristically grow well on lactate and malate, incompletely oxidize substrates, and do not utilize fatty acids for growth. In contrast, strain AK-01 does not contain desulfoviridin, grows poorly on lactate and the dicarboxylic acids, but grows very well on fatty acids. Also, substrate oxidation is generally complete.

Strain AK-01 is also physiologically different from the genera Desulfobulbus, Desulfobacter, and Desulfobotulus of the suggested family Desulfobacteriaceae (31). Desulfobulbus species characteristically grow well on lactate and propionate by incomplete oxidation to acetate but cannot utilize any other fatty acids. Desulfobacter species grow effectively on acetate by complete oxidation but do not utilize formate and other fatty acids with longer chains. Desulfobotulus sapovorans, the only species of the genus, utilizes fatty acids (C₄ to C₁₆) and lactate with incomplete oxidation to acetate and does not grow on hydrogen, formate, or acetate. In contrast, strain AK-01 is distinctively different from the three genera in these key taxonomic characteristics (see Table 2).

Strain AK-01 is nutritionally similar to species in the genera Desulfoarculus, Desulfobacterium, Desulfococcus, Desulfonema, and Desulfosarcina. AK-01, along with members of these genera, grows well on fatty acids (C₄ and above) and is able to utilize formate, acetate, and a variety of dicarboxylic acids. However, AK-01 is indeed morphologically different from the Desulfonema species, which form multicellular filaments, and Desulfosarcina variabilis, which forms cell packets at some growth stage.

The results of the phylogenetic analysis based on 16S rRNA sequences generally agree with the phenotypic characterization but have revealed further details. The 16S rRNA phylogeny (Fig. 2) shows that strain AK-01 is distantly related to the genera in the family Desulfovibrionaceae. Although the strain is phenotypically similar to the genera Desulfoarculus, Desulfobacterium, Desulfococcus, Desulfonema, and Desulfosarcina, this phylogenetic analysis places it closest to the last three genera. In addition, the phylogenetic distances between strain AK-01 and the type species of those three genera (between 9.11 and 10.39) are in the same range as those between the type species themselves (between 9.27 and 10.46). Whether AK-01 should be designated a genus, however, remains to be seen.

Strain AK-01 and the other two alkane-degrading sulfate reducers can also be compared phenotypically and phylogenetically. As shown in Table 2, strains TD3 and AK-01 are both fatty acid utilizers. However, unlike AK-01, TD3 cannot utilize hydrogen, formate, acetate, or propionate as an electron donor. In addition, TD3 is thermophilic while AK-01 is a mesophile. The range of alkanes used by TD3 is also somewhat different from those used by strain AK-01 (Table 1). The 16S rRNA phylogeny places strain TD3 distant from strains AK-01 and Hxd3 (Fig. 2). This is consistent with phylogenetic analyses conducted in other studies, which suggest that the thermophile isolated from a hydrothermally active environment represents a new type of sulfate-reducing bacteria (3, 23).

Strains Hxd3 and AK-01 are both fatty acid utilizers, complete oxidizers (Table 2), and mesophiles and are able to utilize a similar range of alkanes (Table 1). Phylogenetic distance based on 16S rRNA sequences also shows that strain AK-01 is more closely related to strain Hxd3 than to TD3 (Fig. 2). On the other hand, strain Hxd3 is unable to use hydrogen and formate as electron donors. Growth of Hxd3 on the fatty acids from C₄ to C₈ is poor, in contrast to that of AK-01. Its G+C content of 63% is also substantially higher than that of AK-01 (57%).

An ability to degrade alkanes by strain AK-01 is demonstrated by the formation of radiolabeled CO₂ from [1-¹⁴C]hexadecane. Utilization of alkanes for growth is shown by cell mass production (as protein produced) upon degradation of hexadecane. The coupling of alkane oxidation to sulfate reduction is indicated by sulfate loss concomitant with hexadecane degradation and the dependence of degradation activity on sulfate reduction. In addition, the amount of sulfate reduced upon hexadecane degradation is reasonably reflected by the stoichiometric equations. The measured ratio of sulfate loss per mole of hexadecane is up to 89% of the predicted ratio when cell mass production is also considered. In previous reports on other anaerobic alkane degraders, the stoichiometric ratio determined for strains Hxd3 and TD3 was 91% of that predicted for complete oxidation of hexadecane (1) and 97% of that predicted for complete oxidation of decane (23).

Although the stoichiometric ratios determined for both hexadecane and hexadecanoate oxidation by strain AK-01 are similar, growth on hexadecane is about four times slower than is growth on hexadecanoate. Similarly, growth of strain Hxd3 on hexadecane is also much slower than that on hexadecanoate and octadecanoate (1, 2). The low water solubility of hexadecane (5.2 × 10⁵ mg/liter) may limit its availability, and growth may be impeded by the limited surface area of the hydrophobic hexadecane droplets available for direct contact with the bacterial cells. This is also supported by the observation that actively dividing cells of strain AK-01 are mostly found clumped closely around hexadecane droplets rather than in suspension.

The initial reaction(s) for the oxidation of alkanes might also be a rate-limiting factor for utilization of alkanes. It was observed that cellular fatty acids of strain AK-01 were predominantly C-even when the alkane substrates were C-even and were predominantly C-odd when the alkanes were C-odd (data not shown). The strong impact of the carbon numbers of alkane substrates on those of the predominant cellular fatty acids, although manifested in a different way, can also be observed on strain Hxd3 (3). These observations strongly suggest that alkanes are anaerobically oxidized to fatty acids and are directly assimilated into cellular lipids. The much slower growth of both strains on alkanes than on long-chain fatty acids may therefore be caused by a rate-limiting step(s) in the initial reaction(s) for the anaerobic oxidation of alkanes to fatty acids. The lower growth yield on hexadecane than on hexadecanoate also suggests that the initial reaction(s) may be energy expending.

We report the isolation and characterization of a novel alkane-degrading bacterial strain. As evidenced by this study and those published in the last few years (2, 6, 8, 13, 23), anaerobic alkane degradation by sulfate reducers may be a more widespread phenomenon than was previously thought. Further studies with these pure cultures should provide interesting insights into potentially novel mechanisms for anaerobic alkane metabolism.