DEHALOCOCCOIDES AND REDUCTIVE DECHLORINATION OF CHLORINATED SOLVENTS - Bioaugmentation for Groundwater Remediation - page 56

Dhc cells are among the smallest bacteria described, and the volume of an individual cell is
roughly 30-fold lower than that of an average bacterial cell (based on an average E. coli cell volume
of 0.64 m 3 ) (Duhamel et al., 2004 ;Kubitschek, 1990 ). The small

and disc-shaped size has implications for light microscopic observation because cells suspended
between the glass slide and the cover slip tumble end over end. The highest resolution a light
microscope achieves is 0.2

m 3 and a Dhc cell volume of 0.02 m m

m, which explains why Dhc cells are visible or “disappear”, depending
on their orientation in the light path. Inexperienced researchers can get easily frustrated when
attempting microscopic observation because Dhc cells are difficult to distinguish from debris and
some training is required to identify Dhc cells. Staining with fluorescent DNA-binding dyes like
acridine orange or 4',6-diamidino-2-phenylindole (DAPI) facilitates microscopic observation and
enumeration of Dhc cells (He et al ., 2003b ).
The small size and disc shape of Dhc cells also serves to maximize the surface area-to-
volume ratio, which can aid in scavenging scarce substrates like haloorganics and hydrogen.
The small cell size also implies that Dhc cell titers can actually be quite high (10 6 -10 8 cells per
mL), even when little biomass is present. Turbidity of Dhc pure cultures is generally very low
and optical density measurements are not applicable for monitoring growth. Growth of Dhc
pure cultures is best monitored by qPCR (see below) or by microscopic counts after staining the
cells with a fluorescent dye and filtration onto black polycarbonate membranes (He et al.,
2003b ; Sanford et al., 2007 ).
Dhc are strictly hydrogenotrophic (i.e., they require hydrogen as the electron donor) and
cannot use organic compounds to derive reducing equivalents for reductive dechlorination.
These findings apply to all known Dhc and also to the few characterized affiliated dechlorina-
tors included in the phylogenetic tree shown in Figure 2.3 . Apparently, all organisms in this
particular branch of the Chloroflexi are adapted to strictly hydrogenotrophic metabolism, using
particular aliphatic and aromatic halogenated organic compounds as electron acceptors. Dhc
dechlorination in mixed cultures is supported by organic compounds provided as the sole source
of reducing equivalents due to the presence of (syntrophic) fermenters that generate hydrogen.
In contrast, pure Dhc cultures depend on hydrogen as electron donor, which must be supplied to
the culture vessels. Reductive dechlorination is a thermodynamically favorable process and
accordingly, hydrogen consumption threshold concentrations of < 1 nanomolar (nM) were
reported (L¨ffler et al., 1999 ;L¨ffler and Sanford, 2005 ; Smatlak and Gossett, 1996 ; Yang
and McCarty, 1998 ). It is practical to feed cultures with a 5-10 fold excess of hydrogen required
to achieve complete dechlorination of the total amount of chlorinated electron acceptor added.
Each Dhc strain has a unique complement of RDase genes and the range of halogenated
substrates used is strain-specific. The range of carbon sources has not been established but
all described Dhc isolates can synthesize their macromolecules from acetate provided in the
growth medium. Dhc genome analysis suggests that several genes encoding enzymes of
the acetyl-coenzyme A pathway for CO 2 fixation are present, but some key components
responsible for the reduction of CO 2 to CO are missing (Seshadri et al., 2005 ). Apparently,
Dhc are not able to grow autotrophically and require a reduced organic compound (i.e., acetate)
as a carbon source. Dhc are typically grown in bicarbonate-buffered medium, and a few reports
suggest that CO 2 enhances growth (M¨ller et al., 2004 ), consistent with the typical pathway for
acetate assimilation in which acetyl-CoA is reductively carboxylated to pyruvate.
The RDases, which are the key components of Dhc energy metabolism, contain cobalamin
(vitamin B 12 ) as a cofactor. As discussed above, Dhc cannot synthesize vitamin B 12 . Instead,
Dhc possess genes encoding for corrinoid salvage pathways including corrinoid uptake and
modification (Seshadri et al., 2005 ), so vitamin B 12 must be added to Dhc growth media,
typically at concentrations of 25-50

m

m g per liter to support reductive dechlorination and growth

(He et al., 2007 ).