BIOAUGMENTATION FOR AEROBIC DEGRADATION OF CIS-1,2-DICHLOROETHENE - Bioaugmentation for Groundwater Remediation - page 203

Acetonitrile (MeCN) is a good growth substrate for JS666. Rates of cis- DCE degradation
were slower in cultures containing MeCN and cis- DCE. However, after cells were harvested
and transferred to fresh medium with cis- DCE as the only carbon source, the specific activity
of MeCN cis- DCE-grown cultures was the same as that of cis- DCE only cultures. In

contrast, cells grown on MeCN without cis- DCE were only half as active when transferred to
medium with cis- DCE as the sole carbon source. Thus, MeCN can be used as a cosubstrate to
generate large amounts of JS666 biomass without sacrificing specific activity. Cyclohexanone
also supported growth of JS666 without the loss of ability to degrade cis- DCE. The growth
strategy using cyclohexanone alternating with cis- DCE routinely yielded cultures with OD 600
> 1.0 in a 66-L reactor. Both substrates supported less growth of contaminants in the cultures
than did succinate or ethanol. To attain such high densities, it was necessary to periodically
exchange the medium (using cross-flow filtration), because the accumulation of chloride
otherwise inhibits JS666.
Several methods were investigated for long-term storage of cells, including use of cryo-
protectants such as glycerol or DMSO, lyophilization, and flash-freezing in liquid nitrogen.
cis- DCE degradation activity was best restored from cell pellets stored at 80 C without

cryoprotectant. Liquid cultures also could be stored with cis- DCE as the sole carbon source at
4 C for up to 14 days without significant loss of activity.

7.3 MICROCOSM ASSESSMENT OF SITE-SUITABILITY
Bioaugmentation with JS666 for aerobic degradation of cis- DCE is a technology in its
nascent stage. Therefore, the relative lack of experience with JS666 under different site
conditions makes it prudent to conduct microcosm assessment of site suitability. With greater
experience, this step may become less important, and decisions regarding suitability may be
reasonably made based on site physical and biogeochemical parameters.

7.3.1 Microcosm Preparation

Microcosm studies should be conducted in 160-mL serum bottles with ambient-air head-
space, sealed with Teflon TM -lined, butyl-rubber septa and aluminum crimps. It is best if
microcosms are incubated at the subsurface temperature of the prospective site, under agita-
tion (to enhance aeration from the headspace), and in the dark. Where possible, microcosms
should be prepared with both soil (50 g dry mass) and groundwater (in volumes such that
groundwater 50 mL) from the prospective site. If

soil samples are not available, microcosms can be conducted with groundwater only. Care
should be taken to avoid cross-contamination of microcosms with materials from different site
locations, otherwise interpretation could be compromised.
Microcosms of the following types should be included: (1) native (i.e., neither pH-adjusted,
nor amended with buffer or nutrients); (2) pH-adjusted (to neutral pH with either HCl or
sodium hydroxide [NaOH]; but not buffered); and (3) buffer/nutrient amended (i.e., with 5 mL
of 10X-concentrated MSM). Each of the above should be done in both inoculated and
uninoculated (control) versions.
After the microcosms are prepared (but before inoculation), they should be allowed to
equilibrate under agitation for at least 6 hours (h) prior to volatile organic compound (VOC)
analysis in headspace samples. cis- DCE should be added to achieve concentrations similar to
levels at the site; however, concentrations above 0.5 mg/L are recommended for analytical
precision, and below 50 mg/L for toxicity avoidance.

soil water any liquid amendments ¼