The use of molecular techniques to evaluate the survival of a microorganism that was injected into an aquifer.

A phenol-degrading methanogenic enrichment was successfully immobilzed in agar as shown by the stoichiometric conversion of phenol to CH4 and CO2. The enrichment contained members of three physiological groups necessary for the syntrophic mineralization of phenol: a phenol-oxidizing bacterium, a Methanothrixlike bacterium, and an H2-utilizing methanogen. The immobilization technique resulted in the cells being embedded in a long, thin agar strand (1 mm in diameter by 2 to 50 cm in length) that resembled spaghetti. Immobilization had three effects as shown by a comparative kinetic analysis of phenol degradation by free versus immobilized cells. (i) The maximum rate of degradation was reduced from 14.8 to 10.0 ,ug of phenol per h; (ii) the apparent Km for the overall reaction was reduced from 90 to 46 ,ug of phenol per ml, probably because of the retention of acetate, H2 and CO2 in the proximity of immobilized methanogens; and (iii) the cells were protected from substrate inhibition caused by high concentrations of phenol, which increased the apparent Ki value from 900 to 1,725 ,ug of phenol per ml. Estimates for the kinetic parameters Ki, Ki, and Vmax were used in a modified substrate inhibition model that simulated rates of phenol degradation for given phenol concentrations. The simulated rates were in close agreement with experimentally derived rates for both stimulatory and inhibitory concentrations of phenol.

Pseudomonas cepacia G4 degrades trichloroethylene (TCE) via a degradation pathway for aromatic compounds which is induced by substrates such as phenol and tryptophan. P. cepacia G4 5223 PR1 (PRI) is a TnS insertion mutant which constitutively expresses the toluene ortho-monooxygenase responsible for TCE degradation. In groundwater microcosms, phenol-induced strain G4 and noninduced strain PR1 degraded TCE (20 and 50 ,uM) to nondetectable levels (<0.1 pM) within 24 h at densities of 108 cells per ml; at lower densities, degradation of TCE was not observed after 48 h. In aquifer sediment microcosms, TCE was reduced from 60 to <0.1 FLM within 24 h at 5 x 108 PR1 organisms per g (wet weight) of sediment and from 60 to 26 FM over a period of 10 weeks at 5 x 107 PR1 organisms per g. Viable G4 and PR1 cells decreased from approximately 107 to 104 per g over the 10-week period.

A PCR primer set and an internal probe that are specific for Pseudomonas sp. strain B13, a 3-chlorobenzoatemetabolizing strain, were developed. Using this primer set and probe, we were able to detect Pseudomonas sp. strain B13 DNA sequences in DNA extracted from aquifer samples 14.5 months after Pseudomonas sp. strain B13 had been injected into a sand and gravel aquifer. This primer set and probe were also used to analyze isolates from 3-chlorobenzoate enrichments of the aquifer samples by Southern blot analysis. Hybridization of Southern blots with the Pseudomonas sp. strain B13-specific probe and a catabolic probe in conjunction with restriction fragment length polymorphism (RFLP) analysis of ribosome genes was used to determine that

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