The degradation of pyrene, a polycyclic aromatic hydrocarbon containing four aromatic rings, by pure cultures of a Mycobacterium sp. was studied. Over 60% of [14C]pyrene was mineralized to CO2 after 96 h of incubation at 24°C. High-pressure liquid chromatography analyses showed the presence of one major and at least six other metabolites that accounted for 95% of the total organic-extractable '4C-labeled residues. Analyses by UV, infrared, mass, and nuclear magnetic resonance spectrometry and gas chromatography identified both pyrene cisand trans-4,5-dihydrodiols and pyrenol as initial microbial ring-oxidation products of pyrene. The major metabolite, 4-phenanthroic acid, and 4-hydroxyperinaphthenone and cinnamic and phthalic acids were identified as ring fission products. 1802 studies showed that the formation of cisand trans-4,5-dihydrodiols were catalyzed by dioxygenase and monooxygenase enzymes, respectively. This is the first report of the chemical pathway for the microbial catabolism of pyrene.

Microbiological analyses of sediments located near a point source for petrogenic chemicals resulted in the isolation of a pyrene-mineralizing bacterium. This isolate was identified as a Mycobacterium sp. on the basis of its cellular and colony morphology, gram-positive and strong acid-fast reactions, diagnostic biochemical tests, 66.6% G+C content of the DNA, and high-molecular-weight mycolic acids (C58 to C64). The mycobacterium mineralized pyrene when grown in a mineral salts medium supplemented with nutrients but was unable to utilize pyrene as a sole source of carbon and energy. The mycobacterium grew well at 24 and 30°C and minimally at 35°C. No growth was observed at 5 or 42°C. The mycobacterium grew well at salt concentrations up to 4%. Pyrene-induced Mycobacterium cultures mineralized 5% of the pyrene after 6 h and reached a maximum of 48% mineralization within 72 h. Treatment of induced and noninduced cultures with chloramphenicol showed that pyrene-degrading enzymes were inducible in this Mycobacterium sp. This bacterium could also mineralize other polycyclic aromatic hydrocarbons and alkyl-and nitro-substituted polycyclic aromatic hydrocarbons including naphthalene, phenanthrene, fluoranthene, 3-methylcholanthrene, 1-nitropyrene, and 6-nitrochrysene. This is the first report of a bacterium able to extensively mineralize pyrene and other polycyclic aromatic hydrocarbons containing four aromatic rings.

Microbiological analyses of sediments chronically exposed to petrogenic hydrocarbons resulted in the isolation of a gram-positive, rod-shaped bacterium which mineralized naphthalene (59.5% of the original amount), phenanthrene (50.9%), fluoranthene (89.7%), pyrene (63.0%), 1-nitropyrene (12.3%), 3-methylcholanthrene (1.6%), and 6-nitrochrysene (2.0%) to carbon dioxide when grown for 2 weeks in pure culture with organic nutrients. The bacterium tolerated salt concentrations up to 4% and grew well at 24 to 30°C. The use of this bacterium may be an attractive alternative to existing physicochemical methods for the remediation of polycyclic aromatic hydrocarbons in the environment.

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37؇C and sterile artificial seawater at 20 and 37؇C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death. MATERIALS AND METHODS Bacterial strains and preparation of inocula. The E. coli K-12 strain used was the standard prototrophic wild-type strain W3110 (1). For the studies requiring a plasmid-bearing strain, strain W3110 was transformed with the plasmid pBR322 (2). This particular plasmid was used to permit the inclusion of ampicillin and tetracycline in the direct viable count (DVC) method and to facilitate the PCR measurements employed. Fresh cultures of the strains W3110 and W3110(pBR322) that had been grown for 14 h in Luria-Bertani (LB) medium [plus ampicillin and tetracycline in the case of W3110(pBR322)] were washed with sterile 0.9% saline, adjusted to the desired cell concentration, and added to the water microcosms in 10 ml of inocula or to the soil microcosms in 3 ml of inocula. Media and chemicals. Levine eosin methylene blue (EMB) agar, tryptone, yeast extract, brain heart infusion medium, and Bacto-agar were obtained from Difco Laboratories (Detroit, Mich.). Cycloheximide, nalidixic acid, acridine orange, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT), and glycine betaine were obtained from Sigma Chemical Co. (St. Louis, Mo.). LB medium (35) was used to grow the strains; LB agar was LB medium with 15 g of Bacto-agar per liter. Plating of nonsterile soil suspensions on LB agar gave low total counts, mainly because the colonies were often obscured by mats of fungal growth and because of the tendency of many of the soil bacteria to swarm. Use of soil extract (SEC) agar gave higher total counts, and inclusion of cycloheximide prevented both fungal growth and bacterial swarming. Soil extract was prepared by suspending 1 kg of soil and 0.5 g of calcium carbonate in 1 liter of distilled water. The suspension was autoclaved for 60 min...

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