BIOAUGMENTATION FOR THE IN SITU AEROBIC COMETABOLISM OF CHLORINATED SOLVENTS - Bioaugmentation for Groundwater Remediation - page 216

( 1996 ), for example, developed a culture of methanotrophic bacteria for bioaugmenation that
had a high PHB content.
Along with physicochemical, geological and hydrological parameters, the feasibility of
in situ bioremediation at any given contaminated site is dependent upon the capacity of the
indigenous microbial population to degrade the compound(s) of interest (Jenal-Wanner and
McCarty, 1997 ). When the indigenous microbial population is not effective at a given site,
bioaugmentation might be used (Steffan et al., 1999 ). Bioaugmentation involves injections of
desired exogenous microorganisms along with required nutrients directly into the contaminated
zone. For bioaugmentation, there are two distinct methods (Steffan et al., 1999 ). The first
method is to add the microorganisms to complement or replace the native microbial population.
The goal of this approach is to achieve prolonged survival and growth of the added organisms
and sustained degradation of the target pollutants. The second bioaugmentation method is to
add large numbers of degradative bacteria to a contaminated site as biocatalysts to degrade a
significant amount of target contaminant before becoming inactive or perishing, in which case
the long-term survival and growth of an active microbial population are not required.
The most widely studied microbial process used for aerobic cometabolism is the oxidation
of CAHs by methanotrophs, which are microorganisms that grow on methane and which
require methane monooxygenase enzyme (MMO) for the initial transformation of methane
to methanol (Arp et al., 2001 ). MMO also initiates oxidation of CAHs such as TCE. TCE
oxidation is initiated through the formation of an epoxide, with an oxygen inserted across the
carbon-carbon double bond. Epoxides are unstable in aqueous solutions and break down
rapidly. In the subsurface, it is possible to stimulate microorganisms possessing soluble
MMO (sMMO) and particulate MMO (pMMO). Microbes expressing sMMO are typically
stimulated under conditions of limited copper. Studies of CAH transformation typically show
faster rates of transformation are achieved when sMMO is expressed compared to pMMO
(Alvarez-Cohen and Speitel, 2001 ). When type I methanotrophs that possess pMMO are
predominately stimulated in situ , limited TCE transformation has been observed (Baker

et al., 2001 ).
The other microbial system that has been studied in great detail is microorganisms that are
stimulated on phenol or toluene where toluene monooxygenase (TMO) or toluene dioxygenase
(TDO) is expressed. The TMO enzymes are responsible for the addition of a hydroxyl group to
the ring structure of toluene or phenol or the methyl group to initiate the oxidation of toluene.
Microbes can possess o -, p -or m -TMOs depending on the location at which the hydroxyl
substitution occurs. Different TMOs exhibit different rates of TCE cometabolism (see reviews
of Alvarez-Cohen and Speitel [ 2001 ] and Arp et al. [ 2001 ]).
Aerobic cometabolism is best suited for in situ remediation for CAH contamination at
concentrations of approximately 1 milligram per liter (mg/L) or less, but well above the drinking
water standard for most of the contaminants. Compounds for which aerobic cometabolism
has been evaluated in laboratory and field studies include the chlorinated ethenes (TCE, cis -
1,2-dichloroethene [ cis -DCE], trans -1,2-dichloroethene [ trans -DCE], 1,1-dichlorothene [1,1-DCE],
and vinyl chloride [VC]); the chlorinated ethanes (1,1,1-trichloroethane [1,1,1-TCA] and the lower
chlorinated ethane isomers); and the chlorinated methanes (chloroform [CF] and the lower
chlorinated methanes) (Semprini, 1997 ). Perchloroethene (PCE) is not susceptible to aerobic
cometabolic transformation (Semprini, 1997 ).
Stimulating indigenous microorganisms through primary substrate addition has been
the most commonly applied form of in situ aerobic cometabolism. Pilot-scale field studies
have demonstrated the potential for stimulating indigenous methane-utilizing microorganisms
(Semprini et al., 1990 , 1991 ), phenol-utilizing organisms (Hopkins et al., 1993a , b ) and toluene-
utilizing organisms (Hopkins and McCarty, 1995 ). Large-scale demonstrations of the