Environmental Engineering Reference
In-Depth Information
5.4 Materials and methods
5.4.1 Laboratory evaluations
5.4.1.1 Molecular ecology
A number of chloroethene sites were used to provide source material for
evaluation. Microcosm experiments were conducted to evaluate sediment,
soil, and aquifer materials for their potential to reductively dechlorinate PCE
and other chlorinated ethenes under anaerobic conditions and to assess the
ability of various enhancing agents to stimulate PCE dechlorination. Eleven
freshwater sediments, four marine sediments, five soils, and seven aquifer
samples were used to construct microcosms containing 10 to 15% (v/v)
sample under anaerobic conditions with either anaerobic mineral medium
or phosphate-buffered saline (PBS) and amended with PCE. Dechlorination
of PCE was monitored by gas chromatography (GC) under conditions that
can resolve ethene and each of the chlorinated ethenes. The enrichments that
completely dechlorinated PCE were further enriched on the less chlorinated
ethenes, cis-DCE, and VC, separately. The resulting subcultures completely
dechlorinated their respective chlorinated ethene and were maintained for
more than 10 transfers in basal salt mineral medium.
Subcultures were subjected to an analysis of the 16S rRNA genes by
denaturing gradient gel electrophoresis (DGGE) and terminal restriction
fragment length polymorphism (T-RFLP). The changes in community struc-
ture were evaluated. This technique is performed by polymerase chain reac-
tion (PCR) amplification of 16S rRNA genes from DNA extracted from
a microbial community in which one of the primers is labeled with a
fluorescent molecule. The resulting fluorescently labeled PCR product is then
subject to restriction enzyme digests. The fragments are resolved using an
automated DNA sequencer, which allows for the detection of only the flu-
orescently labeled terminal fragments. The resolved terminal fragments pro-
vide a fingerprint of the community and an estimate of the number of
ribotypes in a community. The Sage's site material was subjected to direct
and postamplification ribosomal gene probing with probes developed from
known PCE-dechlorinating bacteria.
5.4.1.2 Ethanol toxicity
Soil samples were collected from the former dry cleaner site in accordance
with methods described in the published literature, ensuring sample integ-
rity. Microcosms were prepared in triplicate in glass serum bottles fitted with
Teflon™-coated rubber septa with approximately 50 g of soil. The amount
of soil used for the microcosms was dictated by the quantity required for
analyzing the phospholipid fatty acid (PLFA) profiles of the samples. The
liquid phase in each microcosm consisted of Byrd's Mill medium (BMM),
ethanol, and other additives, such as KNO
3
, Na
2
SO
4
, or PCE, depending
upon the electron acceptor condition selected for the particular experiment.
Byrd's Mill medium was made with Byrd's Mill water (Byrd's Mill Spring,
