BIOAUGMENTATION FOR AEROBIC DEGRADATION OF CIS-1,2-DICHLOROETHENE - Bioaugmentation for Groundwater Remediation - page 202

was not sufficiently unique to facilitate the design of strain-specific primers. Plasmid-encoded
genes were avoided as targets because they are less stable than chromosomal genes, and
targeting plasmid-encoded genes in qPCR assays can result in errors in quantification (Wang
et al., 2004 ). Primers were designed to avoid similarity with known sequences in the NCBI
database. In addition, the annealing temperature of the primers was optimized to avoid
non-specific amplification.
The absolute quantification of JS666 in soil was verified by comparison with plate counts
and direct-microscopic counts. Samples of serially diluted cultures were applied to 0.5 g of soil
obtained from the Savannah River Site (SRS) in Aiken, South Carolina. SRS soil was selected
from an aerobic plume in which cis- DCE had accumulated and persisted - presumably due to
incomplete reductive dechlorination. DNA was extracted from duplicate soil samples amended
with JS666, and the copy number of target genes (isocitrate lyase) was quantified using
duplicate qPCR reactions. Copy number from qPCR positively correlated with cell counts
from plating and viability staining. The largest source of variability in the qPCR assay was
between the different soil extractions. The optimum assay conditions require 100-fold dilution
to minimize the effect of inhibitors and provide a conservative minimum detection limit of 10 5
cells per gram of soil. Minimum detection limits could be improved by using a 50-fold dilution
of DNA extract.
In addition to monitoring the progress of bioaugmentation by tracking the bioaugmentation
agent in the subsurface, the qPCR assay for JS666 could be combined with assays for other
dechlorinators to evaluate the potential for natural attenuation via aerobic cis- DCE oxidation or
reductive dechlorination at contaminated sites.

7.2.6 Development of Strategy for Growth of Inocula
Microcosm studies indicated that 10 5 cells/milliliter (mL) is an effective inoculum level to
stimulate cis- DCE degradation by JS666 (Giddings et al., 2010 b). This cell density is similar
to the range of recommended inoculum levels for anaerobic bioaugmentation with Dehalo-
coccoides spp. cultures - ca. 10 4 -10 6 cells/mL (Steffan et al., 2010 ). Therefore, bioaugmenta-
tion with JS666 in the field will require generating sufficient biomass to treat large

contaminated sites.
To achieve high biomass levels necessary for inoculation in the field, it is desirable to grow
JS666 on a substrate other than cis- DCE. cis- DCE in relatively pure form (without impurities
such as chloroform) is expensive, and cis- DCE's toxicity to JS666 limits the concentrations to
which the cultures can be exposed. It is imperative, however, that the ability to grow on cis- DCE
is not lost during growth on the alternative substrate. Several potential alternative substrates
were screened for their ability to generate biomass and maintain induction of cis- DCE
degradation enzymes (S. Nishino and J.C. Spain, Georgia Institute of Technology, unpub-
lished). 2-Chloroethanol did not support growth. Cells grown on succinate or acetate that
were subsequently amended with cis- DCE showed more growth than cis- DCE-only controls,
but reduced specific activity toward cis- DCE.
The use of ethanol as a cosubstrate was evaluated by growing dense suspensions of cells at
different ratios of cis- DCE to ethanol. After the degradation of cis- DCE began, cells were
harvested by centrifugation and suspended into cultures with cis- DCE as the only carbon
source. A 3:2 (volume to volume [v:v]) ratio of cis- DCE to ethanol resulted in the generation
of the most biomass and the fastest cis- DCE degradation. However, when rates were norma-
lized to biomass, the cis- DCE control without ethanol had the highest specific activity for
cis- DCE degradation. The results indicate that the use of ethanol, succinate, and acetate as
cosubstrates increases the amount of biomass but causes a reduction in cis- DCE degradation
performance. They are also more likely to support growth of contaminants.