Initial reactions in anaerobic alkane degradation by a sulfate reducer, strain AK-01

Abstract

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, isolated from a petroleum-contaminated sediment was studied to elucidate its mechanism of alkane metabolism. Total cellular fatty acids of AK-01 were predominantly C even when it was grown on C-even alkanes and were predominantly C odd when grown on C-odd alkanes, suggesting that the bacterium anaerobically oxidizes alkanes to fatty acids. Among these fatty acids, some 2-, 4-, and 6-methylated fatty acids were specifically found only when AK-01 was grown on alkanes, and their chain lengths always correlated with those of the alkanes. When [1,2-(13)C(2)]hexadecane or perdeuterated pentadecane was used as the growth substrate, (13)C-labeled 2-Me-16:0, 4-Me-18:0, and 6-Me-20:0 fatty acids or deuterated 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0 fatty acids were recovered, respectively, confirming that these monomethylated fatty acids were alkane derived. Examination of the (13)C-labeled 2-, 4-, and 6-methylated fatty acids by mass spectrometry showed that each of them contained two (13)C atoms, located at the methyl group and the adjacent carbon, thus indicating that the methyl group was the original terminal carbon of the [1, 2-(13)C(2)]hexadecane. For perdeuterated pentadecane, the presence of three deuterium atoms, on the methyl group and its adjacent carbon, in each of the deuterated 2-, 4-, and 6-methylated fatty acids further supported the hypothesis that the methyl group was the terminal carbon of the alkane. Thus, exogenous carbon appears to be initially added to an alkane subterminally at the C-2 position such that the original terminal carbon of the alkane becomes a methyl group on the subsequently formed fatty acid. The carbon addition reaction, however, does not appear to be a direct carboxylation of inorganic bicarbonate. A pathway for anaerobic metabolism of alkanes by strain AK-01 is proposed.

FIG. 1

Gas chromatograms (GC-FID) showing the fatty acid profiles of strain AK-01 grown on pentadecane (a) and hexadecane (b). The n-saturated fatty acids identified by their retention times are annotated. C-odd fatty acids are underlined.

FIG. 2

Relative abundance of the total cellular fatty acids of strain AK-01 grown on tetradecane (a), pentadecane (b), hexadecane (c), and heptadecane (d). Only those which can be identified and comprise more than 1% of the total are shown. C-even fatty acids are represented by black bars, and C-odd fatty acids are represented by white bars. The 2-methyl, 4-methyl, and 6-methyl fatty acids are denoted by the numbers 2, 4, and 6, respectively. (The 2-Me-17:0 fatty acid is also shown, though it constitutes only 0.7% of the total.) No fatty acid was observed in the sterile controls.

FIG. 3

Mass spectra of the methyl esters of the unlabeled (a) and ¹³C-labeled (b) 2-Me-16:0 fatty acids recovered from cultures of strain AK-01 grown on unlabeled hexadecane and [1,2-¹³C₂]hexadecane, respectively. Chemical structures of the fatty acids represented by the mass spectra are shown as insets. The key diagnostic ion peaks are annotated with their m/z values. Structural compositions of the ion fragments represented by the annotated peaks are delineated. Intersection with a dotted line indicates a point of bond cleavage, and the ion fragment formed contains only the part of the molecule on the left side of the dotted line. (Note: the mass spectrum for the ¹³C-labeled 2-Me-16:0 fatty acid indicates the coexistence of two isotopmers.)

FIG. 4

Ion fragments represented by the peaks at m/z = 241 for the unlabeled 2-Me-16:0 fatty acid methyl ester (a) (see Fig. 3a) and m/z = 241 (b) and m/z = 243 (c) for the ¹³C-labeled 2-Me-16:0 fatty acid methyl ester (see Fig. 3b). These ion fragments are formed by the elimination of the bracketed segment of the molecule and a hydrogen, followed by the rearrangement of the remaining parts (12, 33, 37a).

FIG. 5

Mass spectra of the methyl esters of the unlabeled (a) and ¹³C-labeled (b) 4-Me-18:0 fatty acids recovered from cultures of strain AK-01 grown on unlabeled hexadecane and [1,2-¹³C₂]hexadecane, respectively. Chemical structures of the fatty acids represented by the mass spectra are shown as insets. (Note: the mass spectrum for the ¹³C-labeled 4-Me-18:0 fatty acid indicates the coexistence of two isotopmers.)

FIG. 6

Mass spectra of the methyl esters of the unlabeled (a) and deuterated (b) 2-Me-15:0 fatty acids recovered from cultures of strain AK-01 grown on unlabeled and perdeuterated pentadecane, respectively. Chemical structures of the fatty acids represented by the mass spectra are shown as insets. Interpretation of the mass spectra shows that there are three deuterium atoms, located on C-2 and the 2-methyl group of the deuterated 2-Me-15:0 fatty acid. The locations of all three deuterium atoms on the 2-methyl group as shown in Fig. 6b represent one possible arrangement.

FIG. 7

Proposed pathway for anaerobic alkane metabolism by strain AK-01. (The original alkane atoms are boldfaced, and the major pathway is indicated by bold arrows.)