Relationship Between Compounds and Metabolites.
The results of the initial reactor experiments establish that both phenyl-labeled and fully deuterated alkane analogues are oxidized to the corresponding fatty acids and incorporated into higher lipids by micro-organisms in soil. In the following experiments, the kinetics of the appearance of these metabolites were investigated.

To track the appearance of the metabolites over time, samples of soil were taken over a three week period from the aerated reactors incubated at 4 ºC and 25 ºC and nitrogen-flushed reactor incubated at 25 ºC. The total lipid extract of each was methylated and analyzed by GCMS. The results, summarized in Figure 4, show that under aerobic conditions at room temperature, the metabolite 2H-tetradecanoic acid-d27, formed from the degradation of 2H-tetradecane-d30, increases in concentration to a maximum of about 3 ppm after 150 hours then decreases. This trend has been observed in other studies of the rate of appearance of metabolites (Beller et al 1995) and may indicate further conversion of the metabolite. It is commonly accepted that fatty acids undergo beta-oxidation in bacteria, yeasts and fungi expected to be present in soil (Atlas 1981). We found no trace of chain-shortened fatty acids possessing deuterium labels in any of the samples from this study.

Much lower amounts of 2H-tetradecanoic acid-d27 were formed in the nitrogen-flushed reactor in the first 400 hours. This is consistent with the assumption that oxygen is required for the conversion of alkanes to fatty acids by microorganisms in soil (Bartha 1986). There are reports of the biological degradation of petroleum hydrocarbons occurring under anaerobic conditions (Caldwell et al, 1999, Fuhr, 1985) but, in this case we cannot rule out that the metabolism that occurred under a nitrogen atmosphere may also have been due to incomplete purging or to trace amounts of oxygen leaking into the reactor.

Neither metabolite was detected in the soil from the 4 ºC reactor. This is consistent with reports that metabolism of alkanes is greatly slowed at low temperatures (Bartha 1986, Pollard et al 1995). This result is also of interest because it implies that preservation of field samples at refrigerator temperatures may prevent biological degradation for three weeks or more, assuming that the target molecules behave in a similar manner to diesel fuel.

The overall yield of the metabolites, taken at the time point at which the maximum concentrations occurred, did not exceed 1% of the total amount of starting material on a molar basis. The total accumulation of the deuterated fatty acid reached about 2.5% of the total fatty acid content of the samples at this point. These levels of incorporation are low compared to reports of the behaviour of bacteria in culture. It has been reported (Makula and Finnerty 1968) that Micrococcus cerificans grown on n-tetradecane in culture produced tetradecanoic acid that accumulated in the biomass until it represented the main fatty acid. These cultures also produced the corresponding alcohol. This was not observed in our experiments. The rates of fatty acid production found in our experiments may underestimate the true rate if subsequent metabolism is occurring, however, we take the generally low yield to indicate that the bulk of the starting material is mineralized to carbon dioxide.

FIGURE 4: The Rates of Formation of 2H-Tetradecanoic and Phenyldodecanoic Acids at 4 ºC, 25 ºC, and Under Nitrogen Degradation in the Presence of Diesel Fuel.


To assess the suitability of 2H tetradecane and phenyldodecane as markers for the degradation of diesel fuel, reactors containing soil (200g) were spiked with the two target compounds at concentrations of 1300 µg/g and with diesel fuel at a concentration of 13, 000 µg/g. This level of fuel was chosen as it is typical of moderately contaminated sites and allows adequate distribution of the fuel in the soil reactor without separation of free product. The reactors (in triplicate) were incubated at 25ºC and lost moisture replenished as before. Duplicate samples (2 grams each) were collected daily for 4 weeks and stored frozen until analysis.

The results, illustrated in Figure 5, show that the degradation of the diesel fuel occurred at a uniform rate for the first 2 weeks, then stopped after about three quarters of the fuel had been consumed. The time lag noted in earlier experiments during the initial 24-48 hours was also seen in these experiments. The target compounds, 2H-tetradecane and phenyldodecane degraded at a uniform rate in the presence of diesel fuel in our reactors.

FIGURE 5: The Rates of Degradation for Diesel Fuel, 2H-Tetradecanoic and Phenyldodecanoic Acids at Room Temperature

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