BIOAUGMENTATION WITH PSEUDOMONAS STUTZERI KC FOR CARBON TETRACHLORIDE REMEDIATION - Bioaugmentation for Groundwater Remediation - page 254

using 14 CCl 4 also were encouraging, with approximately 55% conversion to carbon dioxide
(CO 2 ) and the balance present as uncharacterized nonvolatile material. The precise chemical
mechanism of activation and the resulting transformation pathway was of interest in that it
could give insight into the chemical nature of the nonvolatile fraction and the identities of any
intermediates produced, and the impacts of local site geochemistry on the products that would
be formed in situ .
The chemistry of the dechlorination carried out by strain KC was expected to entail an
initial mechanism of activating carbon-chlorine bonds, followed by decomposition of reactive
intermediates to stable products. To understand the overall process, it was necessary to identify
the active agent responsible for the initiating step and the type of decomposition pathway that
ensued. A reductive mechanism was suspected due to the fact that CT is largely unreactive
toward nucleophiles, as a result of its symmetrical and enveloping arrangement of electroneg-
ative chlorine atoms (Jeffers et al., 1989 ; Kriegman-King and Reinhard, 1992 ; Schaik, 1983 ).
An important early finding regarding the relevant chemistry was that copper (Cu) was
required in trace amounts for bacterial transformation (Tatara et al., 1993 ). That data indicated
a transition metal-dependent process, but exactly how copper was involved (i.e., whether it
affected the production of the active agent or was an integral part of it) was not evident until
the active agent was identified. Studies of the decomposition pathway were the first to shed
light on the overall process. Those data came from experiments aimed at determining whether
one- or two-electron initiating mechanisms were involved.
9.3.1 Pathway of PDTC-Promoted CT Dechlorination
Either a one- or a two-electron mechanism would produce reactive intermediates that could
decompose to CO 2 , either spontaneously or through a combination of spontaneous and
enzymatic steps. A one-electron mechanism was expected to result in trichloromethyl radical,
and perhaps phosgene intermediates (Asmus et al., 1985 ), whereas a two-electron mechanism
was expected to produce dichlorocarbene (Asmus et al., 1985 ; Criddle et al., 1990 ). Experiments
aimed at trapping electrophilic intermediates such as phosgene utilized nucleophilic agents
(cysteine, N,N 0 -dimethylethylenediamine). Those experiments succeeded in substantially alter-
ing the products of CT transformation whereas an agent known to react with dichlorocarbene
(dimethylbutene) did not (Lewis and Crawford, 1995 ). The CT carbon atom was bound to a
sulfur atom in the resulting products indicating that a sulfur-containing compound played a
role in the reaction. Once PDTC was identified as the active agent, its chemical synthesis
became possible, as well as its use in cell-free pathway characterization experiments.
The observation that trace amounts of copper were required for optimal CT transformation
by bacterial cultures (Tatara et al., 1993 ) was explained by the fact that the Cu:PDTC complex is
the active agent of dechlorination (Lewis et al., 2001 ). Nanomolar concentrations of copper
were sufficient for effective dechlorination, indicating that Cu(II) is catalytic and effective at
concentrations likely to be found within contaminated aquifers. Additional experiments
allowed trapping of trichloromethyl radical derived from CT when exposed to the Cu-PDTC
complex (Lewis et al., 2001 ).
The proposed pathway (Figure 9.2 ) includes the following steps: (1) one-electron reductive
elimination of chlorine (as chloride) to produce trichloromethyl radical and a sulfur-centered
PDTC radical, (2) condensation of trichloromethyl radical and a sulfur-centered PDTC radical
to form the first carbon-sulfur bond involving the CT carbon atom (trichloromethyl thioester
intermediate), (3) hydrolysis of the thioester intermediate to give trichloromethanethiol and a
carboxylate derived from PDTC, (4) rearrangement ( gem elimination) of trichlromethanethiol