Biomedical Engineering Reference
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natural biodegradation may be occurring. The overall fraction (F) of MTBE remaining (assuming
fractionation is constant) can be approximated by the equations for d
13
2
Candd
H:
d
13
C
plume
d
13
C
source
¼ e
C
ln
ð
F
C
Þ
(Eq. 10.1)
d
2
H
plume
d
2
H
source
¼ e
H
ln
ð
F
H
Þ
(Eq. 10.2)
e
H
refer to the enrichment factors for
13
C and
2
H.
“Source” and “plume” isotopes ratios can be substituted for bioactive zones and inactive
zones in biobarriers or initial and final values, respectively, in microcosm tests. In studies on
MTBE isotope fractionation, a difference in enrichment of
where
e
C
and
13
Cand
2
H has been observed under
aerobic and anaerobic conditions. Aerobic pure cultures of the PM1 organism (Gray et al.,
2002
)or
aquifer sediment microcosms from different sites (Gray et al.,
2002
;Hunkeleretal.,
2001
;Lesser
et al.,
2008
) have shown that
d
d
H
varied from -29 to -66.
In a field study of an aerobic biobarrier, Lesser et al. (
2008
) found little significant
difference in the enrichment of either
e
C
varied little from -1.5 to -2.4 while
e
2
H -MTBE in bioaugmented zones compared
to those from a zone unaffected by biostimulation (oxygen addition) or bioaugmentation. In
contrast, anaerobic studies of MTBE biodegradation indicated that
13
Cor
d
d
C
varied from -8.1 to 15.6
for sediment microcosms and anaerobic aquifers (Kolhatkar et al.,
2002
; Kuder et al.,
2005
;
Somsamak et al.,
2005
; Zwank et al.,
2005
). It is important to note that under these anaerobic
conditions, MTBE is transformed to TBA. In this respect, Zwank et al. (
2005
) showed the
apparent large enrichment observed for MTBE in
e
13
C is due to a kinetic isotope positional
effect of the enzymatic cleavage of the O-CH
3
group of MTBE. Under aerobic conditions, the
ether is essentially degraded beyond TBA and little or no carbon enrichment is observed.
Stable isotopes also can be used to identify whether labeled carbon from MTBE or TBA is
being incorporated into microbial biomass within the subsurface (Busch-Harris et al.,
2008
). In
this method, “biotraps” are labeled with
13
C-MTBE or TBA, and then suspended in a well. The
traps also contain activated carbon beads, and the indigenous bacteria colonize these beads, and
are exposed to the labeled compound(s). After some period of time, the traps are retrieved and the
cells in the biotraps are analyzed for
13
C that has been incorporated into the cell lipids, providing
evidence that
in situ
biodegradation is occurring under the prevailing environmental conditions.
Of course, there are potential artifacts that should be considered when using such methods,
notably that the microenvironment within a well may differ from that within the aquifer.
In summary, laboratory and field data based on stable isotope analyses, including
d
13
C
fractionation of MTBE and stable isotope analyses of lipids from bacteria recovered from biotraps,
may provide powerful evidence for extensive natural and enhanced bioattenuation, both in
“aerobic” or “anaerobic” aquifers. However, such data should be used with appropriate caution.
d
10.4 BIOAUGMENTATION PILOT TESTING
10.4.1 Bioaugmentation with Direct Degraders (MC-100 and SC-100)
MTBE biotreatment technologies have been in development since the early 1990s. Shell
Development (now Shell Global Solutions) researchers identified and enriched a mixed culture
(MC-100) capable of completely degrading MTBE to carbon dioxide and water (Salanitro et al.,
2000
). In the late 1990s, a single MTBE-degrading organism (SC-100) was isolated from the
mixed culture. The bench-scale studies and ability to produce the MTBE-degrading cultures at a
large enough scale led to pilot-scale studies conducted collaboratively between Shell Global
Solutions (then Equilon Enterprises, LLC), Arizona State University (ASU) and the Naval
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