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.
The formation of radicals from carbon tetrachloride (CT) is often invoked to explain the product distribution resulting from its transformation. Radicals formed by reduction of CT presumably react with constituents of the surrounding milieu to give the observed product distribution. The patterns of transformation observed in this work were consistent with such a hypothesis. In cultures of Escherichia coli K-12, the pathways and rates of CT transformation were dependent on the electron acceptor condition of the media. Use of oxygen and nitrate as electron acceptors generally prevented CT metabolism. At low oxygen levels (-1%), however, transformation of [14C]CT to '4Co2 and attachment to cell material did occur, in accord with reports of CT fate in mammalian cell cultures. Under fumarate-respiring conditions, ['4C]CT was recovered as '4Co2, chloroform, and a nonvolatile fraction. In contrast, fermenting conditions resulted in more chloroform, more cell-bound '4C, and almost no 04C02. Rates of transformation of CT were faster under fermenting conditions than under fumarate-respiring conditions. Transformation rates also decreased over time, suggesting the gradual exhaustion of transformation activity. This loss was modeled with a simple exponential decay term.
Anaerobic microbial transformation of a polynuclear nitrogen heterocycle, acridine, was studied in laboratory microcosms with three different inocula: a stabilized, mixed culture growing on ferulic acid that was originally enriched from anaerobic sewage sludge, and sulfate‐reducing and methanogenic aquifer materials from two sites at a groundwater aquifer contaminated by landfill leachate. Acridine degradation was investigated under methanogenic, denitrifying and sulfate‐reducing conditions at concentrations of 1 to 6 μg/ml. Substrate degradation was followed using two standard analytical techniques (HPLC and GC‐MS) and a new, in situ remote fiber spectroscopic (RFS) technique. This RFS was used successfully to follow changes in concentration of acridine with time, which indicates the technique has a significant potential for monitoring the degradation process in environmental media. Acridine was degraded extensively in one to three weeks under each of the conditions studied. A range of heterocyclic, homocyclic aromatic and aliphatic intermediates was identified by GC‐MS analyses. On the basis of these compounds, a tentative route of anaerobic acridine transformation is proposed that begins with oxidation and proceeds through the common degradative route for oxidized aromatic compounds.
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