PRODUCTION AND HANDLING OF DEHALOCOCCOIDES BIOAUGMENTATION CULTURES - Bioaugmentation for Groundwater Remediation - page 94

to determine specific activity. When the specific PCE and cis -DCE dechlorination activity
reaches 1.3-1.7 mg/h g of dry weight (DWT), a continuous feed of neat PCE/TCE is initiated
at a rate of 0.18-0.25 microliter ( m L)/h L as the

culture cell density and dechlorination activity increases. The culture is grown for 13-15 days
until an OD 550

L. This rate is increased to 0.9-1.2 m L/h

0.7-1.1 or 10 10 -10 11 Dhc /L is achieved. Higher Dhc concentrations can be
obtained by extending the growth period for up to 35 days.

3.2.2.3 4,000-L Scale
Growth of the cultures in the 4,000-L reactor (working volume 3,200 L) is performed
essentially as described for the 750-L reactor, but because the 4,000-L reactor does not have an
impeller, the cells are continuously suspended by using a centrifugal pump that circulates the
culture medium. To provide effective distribution of relatively high amounts of added PCE/
TCE (up to 40 mL initially and then continuously up to 21 mL/h) in the reactor medium, these
chemicals are added directly to the centrifugal pump where they are mixed with a high flow of
recirculating medium from the reactor.
The PCE feed is supplied by using either an ISMATEC high precision multichannel pump
(Model C.P 78023-02, Cole Parmer, Vernon Hills, Illinois) or a syringe pump and 100-mL gas
tight glass syringes. To supply the TCE feed, which has four times higher vapor pressure than
PCE (57.9 millimeters of mercury [mm Hg] and 17.8 mm Hg, respectively) and cannot be added
accurately by using a syringe pump, the ISMATEC high precision multichannel pump is used.
The 4,000-L reactor is chemically sterilized by using NaOH and a clean in place system. The
culture medium in the 4,000-L reactor is not sterilized. Substrate feeding and other parameters
are as described for the 750-L reactor. The reactor is inoculated with either culture from the
750-L reactor or refrigerated concentrated cell stocks, but in each case under strict anaerobic
conditions. The inoculum volume is calculated to achieve an initial Dhc concentration of
approximately 10 8 -10 9 Dhc /L.
During the initial growth phase with continuous or periodic PCE feed, the cis -DCE and
VC dechlorination rate is lower than the rate of PCE dechlorination; this results in a rapid
accumulation of cis -DCE and VC in the reactor (Figure 3.1a ). After 1-2 days of growth,
however, even with continuous PCE feed, the concentrations of cis -DCE and VC begin to
decline rapidly and continuous feeding of PCE can resume. This may suggest that it takes
longer to induce cis -DCE and VC degradation genes than PCE degradation genes, that organ-
isms in the consortium that degrade PCE to cis -DCE initially grow faster than organisms that
degrade cis -DCE and VC, or that a combination of both of these factors creates this affect.

3.3 FULL-SCALE PRODUCTION RESULTS
Examples of large-scale production of the SDC-9 TM consortium in a 4,000-L reactor
(culture volumes of 2,500 L and 3,200 L, respectively) are presented in Figure 3.1b and c .
Figure 3.1b shows the growth of a culture inoculated with a culture transferred directly from
the 750-L reactor without refrigeration or storage, and Figure 3.1c shows the growth of a culture
inoculated with a similar concentrated culture that had been stored for 19 days at 4-6 C. The
data show a slight difference in the lag phase observed before the start of log phase growth.
The lag phase varied from 2 days for the culture directly inoculated from the 750-L reactor to
about 5 days for stored culture. During production-scale and research applications, Dhc -
containing consortia are typically grown with lactate as an electron donor and PCE as an
electron acceptor. Other electron donors or electron donor mixtures, however, have been used
successfully, at least for small-scale production. For example, early studies with the KB-1