A denitrifying bacterium, designated strain Ti, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain Ti. Greater than 50% of the toluene carbon was oxidized to C02, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain Ti was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 ,umol min-' liter-' (56 nmol min-' mg of protein-) in a cell suspension. Strain Ti was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain Ti. This transformation was dependent on the degradation of toluene.

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Strain Tl is a facultative bacterium that is capable of anaerobic toluene degradation under denitrifying conditions. While 80% of the carbon from toluene is either oxidized to carbon dioxide or assimilated into cellular carbon, a significant portion of the remainder is transformed into two dead-end metabolites. These metabolites were produced simultaneous to the mineralization of toluene and were identified as benzylsuccinic acid and benzylfumaric acid. Identification was based on comparison of mass spectra of the methyl esters of the metabolites and authentic compounds that were chemically synthesized. Strain Tl is also capable of o-xylene transformation during growth on toluene. o-Xylene does not serve as a source of carbon and is not mineralized. Rather, it is transformed to analogous dead-end metabolites, (2-methylbenzyl)-succinic acid and (2-methylbenzyl)-fumaric acid. o-Xylene transformation also occurred during growth on succinic acid, which suggests that attack of the methyl group by succinyl-coenzyme A is a key reaction in this transformation. We reason that the main pathway for toluene oxidation to carbon dioxide involves a mechanism similar to that for the formation of the metabolites and involves an attack of the methyl group of toluene by acetyl-coenzyme A. Strain Ti is a denitrifying bacterium that has been shown to oxidize toluene under anaerobic conditions primarily to carbon dioxide and biomass (8). This organism is also capable of o-xylene transformation during growth on toluene; however, o-xylene cannot serve as a sole source of energy or cellular carbon. Metabolites were formed from both toluene and o-xylene during their transformation and were not further metabolized. These dead-end metabolites were determined not to be benzyl alcohol, benzaldehyde, benzoic acid, cresol, hydroxybenzyl alcohol, hydroxybenzaldehyde, hydroxybenzoic acid, methylbenzyl alcohol, tolualdehyde, or toluic acid (8). Since the metabolites were not further degraded, it was reasoned that they were not intermediates in the pathway of toluene oxidation to carbon dioxide. However, since a significant portion of toluene carbon was transformed to these metabolites, investigation of their formation was warranted. The toluene-dependent transformation of o-xylene is significant because o-xylene is relatively recalcitrant to biodegradation under both aerobic (2) and anaerobic (8) conditions. However, incomplete transformations have a potentially negative side since the metabolites formed may be more toxic than the original chemical that was the source of contamination. Therefore, identification of these metabolites can provide information on their toxicity, their formation, and how to initiate their mineralization.

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Seven different sources of inocula that included sediments, contaminated soils, groundwater, process effluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, and xylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion of toluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene was correlated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene were observed in these initial enrichment cultures. However, m-xylene was degraded by a subculture that was incubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for the degradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greater than 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of these compounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradative capabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.

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We have previously defined in situ biogeochemical transformation as the biogenic formation of reactive minerals that are capable of abiotically degrading chlorinated solvents such as trichloroethene without accumulation of degradation products such as vinyl chloride (AFCEE et al. ). This process has been implemented in biowalls used to intercept contaminated groundwater. Abiotic patterns of contaminant degradation were observed at Altus Air Force Base (AFB) and in an associated column study, but not at other sites including Dover AFB. These abiotic patterns were associated with biogenic formation of reactive iron sulfide minerals. Iron sulfides in the form of small individual grains, coatings on magnetite, and sulfur‐deficient pyrite framboids were observed in samples collected from the Altus AFB biowalls and one of the EPA columns. Larger iron sulfide grains coated with oxide layers were observed in samples collected from Dover AFB. Altus AFB and the EPA column differed from Dover AFB in that groundwater flow at Dover AFB was relatively slow and potentially reversing. High volumetric sulfate consumption rates, an abiotic pattern of trichloroethene (TCE) degradation, and the formation of small, high surface area iron sulfide particles were associated with relatively high rates of TCE removal via an abiotic pattern. Geochemical modeling demonstrated that iron monosulfides such as mackinawite were near saturation, and iron disulfides such as pyrite were supersaturated at all sites. This environmental condition can be supportive of nucleation of small particles rather than crystal growth leading to larger particles. When nucleation is dominant, small, high surface area, and reactive particles result. When crystal growth dominates the crystals are larger and have lower specific surface area and reactivity. These results taken together suggest that creation of a dynamic environment can promote biogeochemical transformation based on generation of reactive iron sulfides.

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