Environmental Engineering Reference
In-Depth Information
Schematic Representation of a Microcosm Experiment
PCB contaminated soil (20 g)
Serial dilutions
Luria-Bertani Medium
Extraction
and Analysis
Luria-Bertani Medium with
Rifampicin 50
μ
g/ml
Whole cell PCR for the
fcb
genes
Figure 6.11
Microcosm experiment.
6.4.1.5 Survival and activity of GEM RHA1(
fcb
) in nonsterile soil
We investigated the survival and metabolic activity of the recombinant PCB
degrader RHA1(pRHD34::
fcb
) in nonsterile soil microcosms (Figure 6.11).
The loamy sand soil (84.6% sand, 12.1% silt, and 3.4% clay) with 3.5% organic
matter, pH 7.6, was passed through a 4-mm mesh sieve and stored at 4ºC
until used; it originated from a noncontaminated area adjacent to PCB-con-
taminated soils at Picatinny Arsenal, NJ (28 miles northwest of New York
City). Prior to inoculation, indigenous bacterial populations were assessed
by staining with 5-(4,6-dichlorotriazine-2-yl) aminofluoroscein (DTAF) fol-
lowed by epifluorescence microscopy (Bloem, 1995). Total bacterial counts
of the soil before inoculation averaged to 9.9 × 10
8
. Because we could not
distinguish our strain by color and colony morphology in comparison to the
indigenous bacteria, we selected a rifampicin-resistant mutant for tracking
our recombinant strain. No indigenous bacteria grew on this medium. Spon-
taneous rifampicin (Rif+) mutants of the RHA1(pRHD34::
fcb
) were isolated
according to Smith and Tiedje (1980).
RHA1(pRHD34::
fcb
)Rif cells were grown on 4-CBA and, after washing
twice (50 m
M
phosphate buffer, pH 7.0), were resuspended in mineral K1
medium (Tsoi et al., 1999) and inoculated into 20 g of soil amended with 100
ppm 4-CB, at a final density of 10
4
cells/g soil and 30% moisture content.
Results of this study have been summarized in Rodrigues et al. (2001).
As shown in Figure 6.12, recombinant organisms grew in both sterile and
