PRODUCTION AND HANDLING OF DEHALOCOCCOIDES BIOAUGMENTATION CULTURES - Bioaugmentation for Groundwater Remediation - page 104

Dhc -containing cultures are consortia and Dhc are difficult to grow in pure culture,
enumerating Dhc in mixed cultures typically requires use of qPCR methods. Several Dhc -
specific qPCR assays and PCR primer sequences have been described (Hendrickson et al., 2002 ;
Ritalahti et al., 2006 ). Molecular tools for quantifying Dhc are described elsewhere in this
volume (Chapter 6 ), and the methods used at Shaw are described in Vainberg et al. ( 2009 ).
qPCR assays should be performed on each batch of culture produced, but for routine microbial
growth monitoring, OD can sometimes be used, provided enough preliminary work is per-
formed to understand the relationship between total cell density and Dhc concentration at
different stages of culture growth (Vainberg et al, 2009 ). Typical final Dhc concentrations in
some cultures produced by Shaw are shown in Table 3.2 .

3.4.3 Specific Activity

Specific activity is a measure of the amount of target contaminant that can be degraded per
unit of culture within a given time. Measuring the specific activity of both PCE (Figure 3.9a )
and cis -DCE (Figure 3.9b ) degradation is important because most cultures have multiple
dechlorinator populations, some of which can degrade DCE to ethene and others that degrade
PCE to only TCE or cis -DCE. Furthermore, qPCR analysis does not allow differentiation
between live and dead Dhc cells, so even with high Dhc numbers, the degradative activity of
a culture could be low.
Specific activity can be measured in terms of Dhc numbers or protein concentration, but
using the total DWT of washed cells as a standard is preferable for commercial production of
SDC-9 TM . Because the cultures are mixtures, DWT measurements allow assessments of the
ratio of Dhc to non- Dhc organisms. For example, low Dhc numbers with high DWT can indicate
that culture production has led to an imbalance in the relative amount of Dhc to non- Dhc
organisms in the culture. Likewise, high DWT-based specific activity indicates that the culture

a b

12 12

cis -DCE

PCE
TCE
cis -DCE
VC

VC from cis -DCE

10

10

8 8

6

6

4

4

2

2

0

0

0 20 40 60 80 100 120 0 20 40 60 80 100 120

T ( T (

Figure 3.9. Dechlorination assay to monitor the specific activity of an SDC-9 TM culture. The PCE
(a) and cis-DCE (b) degradation rates are measured by using bottle assays to evaluate the specific
activity of the cultures for QA/QC. The incubation temperature was 28 o C, the Dhc concentration
was 1.4 3 10 12 /L, the OD (550) was 1.6, and the DWT was 0.65 g/L.