PRODUCTION AND HANDLING OF DEHALOCOCCOIDES BIOAUGMENTATION CULTURES - Bioaugmentation for Groundwater Remediation - page 91

solvent remediation, however, presents many unique challenges to practitioners. First, the
cultures are consortia, meaning that the success of the culturing process requires maintaining
many different bacterial strains, even some that may not be identified. Likewise, growth of
dehalogenating organisms, such as members of the genera Dehalococcoides and Dehalobac-
ter , requires the contribution of other consortium members that provide electron donor (H 2 )
and other growth nutrients (e.g., corrinoids) for the dehalogenating microbes.
Growth of bacterial inocula can be performed in continuous mode where the culture is
continually grown and harvested from a flow-through reactor, in fed-batch mode where a
culture is grown in a vessel and harvested and then another culture begun, or in a hybrid of the
two whereby the culture is maintained in a reactor until a volume of culture is harvested and
then replaced with fresh medium.
The primary advantage of a continuous growth system is that the culture remains at a
relatively high cell density and specific activity through the culturing process (Stafford, 1986 ).
This technique is typified by the operation of a chemostat where the growth medium continu-
ously flows into and out of a reactor and the feed rate is balanced against the growth rate of the
culture. Theoretically, continuous cultures allow the cell population to grow indefinitely in an
unchanging environment. This continuous growth technique is likely rare for the production of
bioaugmentation cultures because of the sporadic demand for cultures and because of the need
to maintain anaerobic conditions. Use of this method would require that the produced cultures
be continuously collected and stored until use, and media fed into the reactor would have to be
made anaerobic. In addition, continuous culturing requires a more complicated control system
(to balance growth rate and dilution rate) and installation of additional equipment (e.g., tanks
and pumps) that can hold and continuously supply anaerobic medium to the reactor and to
collect and handle the produced culture.
The continuous culture technique may be useful for some on-site applications where the
culture is grown with contaminated groundwater fed into the reactor and the effluent is used as
an aquifer inoculum (Fam et al., 2004 ). The approach would require sufficiently high ground-
water contamination to maintain growth of the organisms because adding chlorinated solvents
for growth could result in further contamination of the aquifer.
A more likely approach for producing cultures for bioaugmentation is a semi-continuous
process whereby the culture is maintained in the reactor until needed and then some of the
culture is harvested. The harvested volume would then be replaced with fresh medium and
growth would continue. This approach is common in research laboratories that maintain
cultures for study. The primary advantage of this technique is that cell growth must replace
only the volume of culture removed. For example, if one half of the culture is harvested,
a single doubling of the remaining culture will replace the cells removed. This process may be
most suitable for cases where demand for the culture is high, and medium is regularly removed
from the culture and replaced with fresh medium.
The primary disadvantages of the semi-continuous culture method is that the cultures are
typically maintained in a stationary growth phase in the reactor and specific activity of the
culture can be reduced relative to that of actively growing and reproducing cells. In addition,
long-term continuous growth or prolonged maintenance of a culture in the reactor vessel can
lead to the accumulation of toxic metabolites that affect culture activity, survival, or perfor-
mance. In fact, many semi-continuous microbial growth processes are designed to produce the
accumulating toxic product, for example, ethanol. Extended maintenance of cultures in
reactors, during either continuous or semi-continuous growth, is rare in industrial applications
because it commonly leads to an accumulation of mutations that ultimately results in strain
degeneration (Dykhuizen and Hartl, 1983 ; Harder et al., 1977 ; Heineken and O'Conner, 1972 ).
Furthermore, long-term maintenance of a mixed culture reactor can result in population