The aromatic hydrocarbons toluene and benzene were anaerobically transformed by mixed methanogenic cultures derived from ferulic acid-degrading sewage sludge enrichments. In most experiments, toluene or benzene was the only semicontinuously supplied carbon and energy source in the defined mineral medium. No exogenous electron acceptors other than CO2 were present. The cultures were fed 1.5 to 30 mM unlabeled or 14C-labeled aromatic substrates (ring-labeled toluene and benzene or methyl-labeled toluene). Gas production from unlabeled substrates and 14C activity distribution in products from the labeled substrates were monitored over a period of 60 days. At least 50% of the substrates were converted to CO2 and methane (>60%). A high percentage of 14CO2 was recovered from the methyl group-labeled toluene, suggesting nearly complete conversion of the methyl group to CO2 and not to methane. However, a low percentage of 14Co2 was produced from ring-labeled toluene or from benzene, indicating incomplete conversion of the ring carbon to CO2. Anaerobic transformation pathways for unlabeled toluene and benzene were studied with the help of gas chromatography-mass spectrometry. The intermediates detected are consistent with both toluene and benzene degradation via initial oxidation by ring hydroxylation or methyl oxidation (toluene), which would result in the production of phenol, cresols, or aromatic alcohol. Additional reactions, such as demethylation and ring reduction, are also possible. Tentative transformation sequences based upon the intermediates detected are discussed.

Toluene and o-xylene were completely mineralized to stoichiometric amounts of carbon dioxide, methane, and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was creosote-contaminated sediment from Pensacola, Fla. The adaptation periods before the onset of degradation were long (100 to 120 days for toluene degradation and 200 to 255 days for o-xylene). Successive transfers of the toluene-and o-xylene-degrading cultures remained active. Cell density in the cultures progressively increased over 2 to 3 years to stabilize at approximately 109 cells per ml. Degradation of toluene and o-xylene in stable mixed methanogenic cultures followed Monod kinetics, with inhibition noted at substrate concentrations above about 700 FM for o-xylene and 1,800 FM for toluene. The cultures degraded toluene or o-xylene but did not degrade m-xylene, p-xylene, benzene, ethylbenzene, or naphthalene. The degradative activity was retained after pasteurization or after starvation for 1 year. Degradation of toluene and o-xylene was inhibited by the alternate electron acceptors oxygen, nitrate, and sulfate. Degradation was also inhibited by the addition of preferred substrates such as acetate, H2, propionate, methanol, acetone, glucose, amino acids, fatty acids, peptone, and yeast extract. These data suggest that the presence of natural organic substrates or cocontaminants may inhibit anaerobic degradation of pollutants such as toluene and o-xylene at contaminated sites. Soil, sediment, and groundwater are frequently contaminated with petroleum products as a result of leaks in underground storage tanks, improper disposal techniques, and inadvertent spills. Of the many constituents of petroleum, the nonoxygenated, homocyclic aromatic compounds (including benzene, toluene, xylenes, and ethylbenzene) are of particular concern because they are confirmed or suspected carcinogens, even at very low concentrations (6). These compounds are relatively water soluble compared with other components of petroleum and thus frequently migrate through groundwater systems to contaminate drinking water supplies far removed from the actual spill (5, 29). The microbial degradation of compounds such as toluene and benzene under aerobic conditions has been studied in great detail (13, 30); in contrast, the fate of homocyclic aromatic compounds in anaerobic environments is very poorly understood. Until the mid-1980s, it was generally believed that monoaromatic compounds were recalcitrant to degradation under anaerobic conditions. Certain monoaromatic hydrocarbons, most frequently toluene, have since been shown to be degraded by microorganisms under denitrifying (8, 11, 20, 21, 25, 36), iron-reducing (22, 23), sulfate-reducing (2, 10, 17) and methanogenic (16, 32, 33, 34) conditions. We report here the enrichment and maintenance of a mixed culture derived from contaminated aquifer sediments that specifically degrades toluene and o-xylene under methanogenic conditions. The culture has been maintained with toluene or...

Toluene and benzene were anaerobically transformed and eventually mineralized in mixed methanogenic cultures. However, the source of oxygen for the initial oxidation step had been unknown, owing to the presence of both methanol and water. No exogenous electron acceptors other than carbon dioxide, toluene, and benzene were present in the defined mineral medium. Through the use of 180-labeled water, the oxygen incorporated into the monoaromatic compounds was shown to come from water. The cresol from the toluene and the phenol from the benzene contained up to 8% 180 label after incubation in 9% 180-labeled medium. Gas chromatography-mass spectrometry was used to detect the 180-labeled aromatic metabolites.

Benzene was mineralized to CO2 by aquifer-derived microorganisms under strictly anaerobic conditions. The degradation occurred in microcosms containing gasoline-contaminated subsurface sediment from Seal Beach, California, and anaerobic, sulfide-reduced defined mineral medium supplemented with 20 mM sulfate. Benzene, at initial concentrations ranging from 40 to 200 microM, was depleted in all microcosms and more than 90% of 14C-labeled benzene was mineralized to 14CO2.

A denitrifying Pseudomonas sp. (strain KC) capable of transforming carbon tetrachloride (CT) was isolated from groundwater aquifer solids. Major products of the transformation of "4C-labeled CT by Pseudomonas strain KC under denitrification conditions were "4CO2 and an unidentified water-soluble fraction. Little or no chloroform was produced. Addition of dissolved trace metals, notably, ferrous iron and cobalt, to the growth medium appeared to enhance growth of Pseudomonas strain KC while inhibiting transformation of CT. It is hypothesized that transformation of CT by this organism is associated with the mechanism of trace-metal scavenging. Microbial capabilities for dehalogenation are widely distributed in nature. This activity could potentially be exploited for in situ bioremediation of contaminated groundwater. The use of denitrifying organisms would be advantageous for aquifer bioremediation because, unlike oxygen, nitrate and nitrous oxide are highly soluble in water and easily added. Furthermore, nitrate is already present in many groundwaters because of the widespread use of fertilizers. Bouwer and McCarty (2) demonstrated that carbon tetrachloride (CT) and brominated trihalomethanes are transformed under mixed-culture denitrifying conditions. To date, however, there are no reports of pure-culture denitrifiers capable of haloaliphatic transformations. This has led some to speculate that nondenitrifying "secondary" organisms are responsible for biotransformations observed in mixed cultures under denitrification conditions (5). To assess the possibility that subsurface denitrifiers might possess fortuitous dehalogenating capability, several denitrifying enrichments were prepared with aquifer solids as the source of microorganisms. Aquifer materials were obtained from sites in Seal Beach, Calif.; Moffett Field, Calif.; and Savannah River, Ga. These enrichments were screened for their activity toward CT. This screening culminated in the successful isolation of Pseudomonas sp. strain KC, an organism that rapidly and completely degrades CT, with carbon dioxide as its major end product. Several nondenitrifying organisms are also known to transform CT, but the products (chloroform and dichloromethane) are undesirable, and the rates of transformation can be slow. The metabolic basis for the transformation of CT by Pseudomonas strain KC was explored as this transformation may suggest a means for controlling reductive transformations in biological systems so as to produce innocuous products. MATERIALS AND METHODS Chemicals. CT (99+% pure) was obtained from Aldrich Chemical Co., Milwaukee, Wis. Chloroform (CF; Baker analyzed; Photrex grade 99.5% pure) was obtained from J.