PRODUCTION AND HANDLING OF DEHALOCOCCOIDES BIOAUGMENTATION CULTURES - Bioaugmentation for Groundwater Remediation - page 96

culture suggested a growth benefit if the culture was grown with a mixture of methanol,
ethanol, acetate and lactate (Duhamel et al., 2002 ) presumably because the electron donors
are utilized at different rates or because they support different groups of microbes in the
culture.
Testing of different substrates including lactate, ethanol, methanol and citrate revealed
that the SDC-9 TM consortium was able to utilize all of these substrates, but sufficient degrada-
tion activity and bacterial growth rate is achieved with lactate as the electron donor substrate
(data not shown). Experience at Shaw Environmental, Inc. (Shaw) has shown that it is easier to
control substrate feeding rates by using a single primary electron donor such as sodium lactate.
Even with a single electron donor/hydrogen source like lactate, utilization of the substrate by
the consortium leads to production of a complex mixture of metabolites, primarily VFAs, which
can themselves act as electron donors/hydrogen sources for the culture. Balancing the concen-
tration of a mixture of electron donors and mixtures of electron acceptors (i.e., PCE and PCE
daughter products) during large-scale bacterial culturing adds increased complexity to process
optimization. Although PCE is used as a primary electron acceptor, similar results, in terms of
specific activity and final cell densities, have been obtained when TCE is used as an electron
acceptor to grow SDC-9 TM .
For routine process monitoring, the OD of reactor samples is measured (Figure 3.1b and c ).
During the initial lag phase, the OD of the consortium increases about threefold due primarily
to the rapid growth of non- Dhc organisms in the consortium on the added YE. A similar rapid
increase in non- Dhc organisms, and OD, also is observed if a high concentration of lactate
(5-12 millimolar [mM]) is added to the medium at the beginning of the culture process (data not
shown), despite the lag in Dhc growth. These results demonstrate that, at least during the early
stages of cell growth, OD measurements are not a good indicator of Dhc concentration in the
culture, and more advanced measurements like quantitative polymerase chain reaction (qPCR;
L¨ffler et al., 2000 ; Ritalahti et al., 2006 ) are needed to effectively estimate Dhc numbers in the
culture.
Following the lag phase, and after lactate is fed continuously at a low rate to generate low
levels of hydrogen ( < 20 nanomolar [nM]), the Dhc concentration begins to increase exponen-
tially (i.e., log phase growth) and reaches about 10 9 -10 10 cells/L. During this period of growth,
the culture OD is correlated with the growth of Dhc culture. These results suggest that during
certain periods of the cell growth process, measurements of OD may be useful for estimating
Dhc levels in the reactor and to automate the control of the culturing process.
The OD of the cultures typically stabilizes after approximately 10 days, but exponential
growth of Dhc continues until approximately day 24 (Figure 3.1b and c ). These results suggest
that non- Dhc microorganisms in the consortium initially grow much faster than Dhc . During
this early growth period, Dhc represent a relatively low proportion of the total bacterial
population of the culture, but during extended growth the relative abundance of Dhc in the
culture increases (Figure 3.2 ).
During the initial stages of 3,200-L cell culturing (to day 25), a maximum Dhc concentra-
tion of ~10 11 Dhc /L is achieved in the reactor, even though growth substrates are still present in
the culture broth (Figure 3.1a ). However, Dhc concentrations in the reactor can be increased
~10-fold by the addition of YE as a nutrient source. The exact role of the YE is not known, but
its addition also revives the growth of non- Dhc organisms in the consortium as reflected in a
rapid increase in culture OD (Figure 3.1c ). Because the RAMM medium used at Shaw does not
contain sodium sulfide or other sulfur-containing salts, it is possible that the YE provides a
needed source of sulfur for the cultures. One g/L of YE provides ~5 mg/L sulfur and 0.48 mg/L
iron. YE also could provide a needed source of amino acids and/or precursors for the production