The results of incubation of 2H-tetradecane with soil in the presence of the biocide, Kathon®, is shown in Figure 2. The mode of action of Kathon® is to inhibit respiration causing bacterial, fungal, and yeast cell death (Supelco, 1997). The biocide essentially stops the reaction confirming that the observed loss of the target compounds is the result of biological activity.

FIGURE 2. Degradation of 2H-tetradecane in the presence of local garden soil and the biocide Kathon®.


Investigation of Metabolic Products.
To look for metabolic products, soil samples subjected to 16 days of incubation were extracted, resolved into polar lipid fractions by thin layer chromatography and reacted to form methyl esters of any fatty acids present. The methylation reaction used converts both free fatty acids and those found in complex lipids, such as glycerides or waxes, into their methyl esters.

Analysis of the polar lipid fractions showed a range of normal fatty acid methyl esters from about 12 to 20 carbons in length in samples for both control and test reactors. These most likely represent the fatty acid component of the polar lipids arising from the microbial population.

In three of the polar lipid fractions from the reactors spiked with the target compounds and exposed to air at room temperature, compounds identified as 2H-tetradecanoic acid-d27 methyl ester and phenyldodecanoic acid methyl ester were found. The mass spectrum and gas chromatographic retention time of the deuterated metabolite matched that of authentic 2H-tetradecanoic acid-d27 methyl ester available commercially. The spectra of the metabolite and the authentic material are shown in Figure 3.

Authentic phenyl dodecanoic acid was not available commercially, but the mass spectrum of the phenyl-labeled metabolite possessed all of the expected elements including ions at 91 and 105 mass units characteristic of alkyl benzenes, m/z 74 typical of methyl esters, m/z 290 corresponding to the molecular weight of the compound and m/z 258 corresponding to the loss of methanol from the molecular ion. The mass spectrum was also closely similar to authentic purchased samples of the related homologues phenyldecanoic acid methyl ester and phenyl hexanoic methyl ester.

These metabolites were not detected in the control reactor which contained the same soil and peat mix without the target compounds. No fatty alcohols, fatty aldehydes or water soluble metabolites that could be traced to the starting material were detected in any of the reactors.

The nature of the polar lipids in which the metabolites were found was not firmly established, but the mobility of the material on the thin layer plates would be consistent with phosphatidyl ethanolamine or phosphatidyl glycerol. Fatty acids produced from n-alkanes by bacteria in culture have been shown to be incorporated into cellular glycerolipids including phosphatidyl ethanolamine and phosphatidyl glycerol (Makula and Finnerty 1972).

The detection of metabolic products possessing the labels present in the starting material, and their incorporation into higher glyceride structures confirms that the removal of the starting material from the reactors is the result of biological action. This is also supported by our ability to stop the reaction using a commercially available biocide.

FIGURE 3. Mass spectra of 2H-tetradecanoic acid-d27 methyl ester derived from 2H-tetradecane in the lab scale reactors and authentic 2H-tetradecanoic acid-d27 methyl ester.

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