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

Technical Report · Fri Oct 01 04:00:00 EDT 1993

DOI:https://doi.org/10.2172/10103098· OSTI ID:10103098

Thiem, S M; Tiedje, J M ^[1]; Krumme, M L ^[2]; Smith, R L ^[3]

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1. Michigan State Univ., East Lansing, MI (United States)
2. Gesellschaft fuer Biotechnologische Forschung mbH, Braunschweig (Germany)
3. Geological Survey, Boulder, CO (United States)

An anonymous polymerase chain reaction (PCR) primer set and an internal probe were developed that are specific for Pseudomonas sp. strain B13, a 3-chlorobenzoate-metabolizing strain. Using this primer set and probe, we were able to detect Pseudomonas sp. strain B13 DNA sequences in DNA extracted from aquifer samples fourteen and a half months after Pseudomonas sp. strain B13 had been injected into a sand and gravel aquifer on Cape Cod. This primer-set and probe was 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 were used determine that viable Pseudomanas sp. strain B13 persisted in this environment. We also isolated a new 3-chlorobenzoate-degrading strain from one of these enrichment cultures. The B-13 specific probe does not hybridize to DNA from this isolate. The new strain could be are indigenous 3-chlorobenzoate degrader or result from gene exchange between Pseudomonas sp. strain B13 and an indigenous bacterium. The later interpretation is supported by an RFLP pattern of ribosome genes that differs from that of Pseudomonas sp. strain B13, identically sized restriction fragments hybridizing to the catabolic gene probe for the B13 chlorobenzoate pathway, and the absence of any enrichable 3-chlorobenzoate degrading strains in the aquifer prior to inoculation.

• Research Organization: Michigan State Univ., East Lansing, MI (United States)
• Sponsoring Organization: USDOE, Washington, DC (United States)
• DOE Contract Number: FG02-89ER60848
• OSTI ID: 10103098
• Report Number(s): DOE/ER/60848--3; ON: DE94001645
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
BIOCHEMISTRY
BIODEGRADATION
GROUND WATER
MICROBIOLOGY
POLLUTANTS
SAMPLING
WATER POLLUTION