M.L. Krumme | USGS Science

USGS1994

Use of molecular techniques to evaluate the survival of a microorganism injected into an aquifer

A PCR primer set and an internal probe that are specific for Pseudomonas sp. strain B13, a 3-chlorobenzoate-metabolizing 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 viable Pseudomonas sp. strain B13 persisted in this environment. We isolated a new 3-chlorobenzoate-degrading strain from one of these enrichment cultures. The B13-specific probe does not hybridize to DNA from this isolate. The new strain could be the result of gene exchange between Pseudomonas sp. strain B13 and an indigenous bacterium. This speculation is based on an RFLP pattern of ribosome genes that differs from that of Pseudomonas sp. strain B13, the fact that identically sized restriction fragments hybridized to the catabolic gene probe, and the absence of any enrichable 3-chlorobenzoate-degrading strains in the aquifer prior to inoculation.

Massachusetts↗

USGS1993

Degradation of trichloroethylene by Pseudomonas cepacia G4 and the constitutive mutant strain G4 5223 PR1 in aquifer microcosms

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 (PR1) is a Tn5 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 μM) to nondetectable levels (< 0.1 μM) within 24 h at densities of 10 8 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 μM within 24 h at 5 x 10 8 PR1 organisms per g (wet weight) of sediment and from 60 to 26 μM over a period of 10 weeks at 5 x 10 7 PR1 organisms per g. Viable G4 and PR1 cells decreased from approximately 10 7 to 10 4 per g over the 10-week period.

Applied and Environmental Microbiology↗