BIOAUGMENTATION FOR AEROBIC DEGRADATION OF CIS-1,2-DICHLOROETHENE - Bioaugmentation for Groundwater Remediation - page 204

JS666 inoculum should come from an active culture grown on cis- DCE as sole carbon/
energy source. Where possible, the use of several inoculum levels (10 7 ,10 6 , and 10 5 cells/mL) is
recommended in microcosm studies, to assist in determining necessary bioaugmentation levels
in the field. Minimum inoculum levels should become standardized after more field experience
with the technology.

7.3.2 Previous Experiences with Microcosm Assessment

The potential of JS666 as a bioaugmentation agent was initially assayed in microcosm
studies (Giddings et al., 2010 b). JS666 survival and activity were assessed using subsurface
materials from five sites: Savannah River Site (SRS), South Carolina; Robins Air Force Base
(AFB), Georgia; Hill AFB, Utah; Fort Lewis, Washington; and an Aerojet facility near Sacra-
mento, California. Microcosm experiments were performed under what could be considered
ideal conditions (i.e., pH buffered and amended with nutrients), and then systematically
challenged with inhospitable conditions and other potential barriers. cis- DCE degradation
was monitored, and because the organism would later be used in field tests of bioaugmentation,
the molecular probe (based on the isocitrate-lyase gene of JS666) was applied to track JS666
within some microcosms as a test of the probe's efficacy as well as survival of JS666.
Additionally, microcosms were constructed using two dilutions of primary sewage effluent -
unautoclaved (contributing both complex organic substrates as well as competing and/or
predatory microbes), and autoclaved (thus contributing only complex organics).
In buffered, neutral pHmicrocosms constructed from all five site materials, cis- DCE at high
concentration (ca. 60 mg/L) was degraded completely within 10-15 days when inoculated with
JS666 at 7 10 6 cells/mL. Without inoculation of JS666, no significant cis- DCE degradation was

observed. Studies were undertaken to determine effective inoculum size, using three levels (1X,
0.1X, and 0.01X) where 1X corresponds to the 7

10 6 cells/mL concentration with SRS soil. In

microcosms constructed of SRS soil MSM, cis- DCE was depleted in about 20 days at 1X, and

was about 50% depleted in 60 days at both 0.1X and 0.01X inoculum levels. With a more realistic
initial cis- DCE concentration (0.6 mg/L), complete degradation was observed in about 5 days at
1X and 0.1X, and in about 20 days at the 0.01X inoculum level. The results suggest that 10 5 cells/
mL is a reasonable minimum inoculum level for field application.
As a rigorous test of both microbial competition/predation, and of the presence of
alternative substrates, studies were conducted in which municipal primary sewage effluent
was added to SRS-soil microcosms along with JS666. Without JS666 addition, no significant
degradation of cis- DCE occurred. All JS666-inoculated microcosms prepared with either 1% or
10% primary effluent were able to degrade 60 mg/L cis- DCE, regardless of the initial inocula-
tion level. The lower inoculum level required more time to degrade the cis- DCE.
This demonstrates that even in the presence of a mixture of alternative (and most likely
preferable) carbon sources and competing/predatory microbes, JS666 is able to degrade large
amounts of cis- DCE.

7.4 FIELD DEMONSTRATION
7.4.1 Test Site Selection

The ideal site for bioaugmentation with JS666 would have the following characteristics:

Concentrations of cis- DCE in ground water greater than 300 m g/L to serve as a growth

substrate for JS666;