BIOAUGMENTATION FOR MTBE REMEDIATION - Bioaugmentation for Groundwater Remediation - page 289

Table 10.1. (continued)
Method Feature Advantages Disadvantages

5. Selective isotope
probing techniques

In situ method
for demonstrating
substrate specific
biodegradation
13 C incorporation
into phospholipid
fatty acids,
dissolved organic
carbon or DNA

In situ analysis
reflects actual site
conditions
Unambiguous
demonstration of
biodegradation

Expensive analyses required
Requires 30-45 days
incubation time
Requires substrate addition
to wells

Note: CoA-coenzyme A
a Other pitfalls of any method given is the analysis of small and few sediment/groundwater samples and the spatial
variability (horizontal and vertical) affecting the assessment of microbial function, activity, presence of degraders and
enzymes in aquifer plumes

from MTBE-degraders (Table 10.1 ), and these can be used to amplify and identify specific
sequences of MTBE-degraders from environmental samples. Such probes include those for
Methylibrium petroleiphilum PM1 (Nakatsu et al., 2006 ), Rhodococcus aetherivorans (Good-
fellow et al., 2004 ), Mycobacterium austroafricanum IFP 2012 (Francois et al ., 2002 ) and
Hydrogenophaga flava ENV735 (Hatzinger et al., 2001 ). These probes also may be used to
estimate MTBE decay rates from their copy numbers in deoxyribonucleic acid (DNA) extracts
of soil samples. Serious drawbacks of this method are: (1) these specific organisms may not be
present in all aquifer sediments where MTBE may be degrading; (2) bacterial cell numbers may
be too low for amplification; and (3) other unknown ether-degraders may be present and
responsible for the bioattenuation.
More recently, oligonucleotide probes have become available for identifying enzymes
responsible for MTBE and TBA metabolism in pure cultures (Method 5, Table 10.1 ). For
example, transcriptosome microarray analysis of enzyme expression in PM1 cells grown on
MTBE (enzyme-induced) and ethanol (non-inducing substrate) by Hristova et al. ( 2003 )
suggests that: (1) an MTBE monoxygenase is involved in the initial oxidation of the -O-
CH 3 group of MTBE to TBA, and (2) a TBA hydroxylase converts TBA to HIBA. Rohwerder
et al. ( 2006 ) have shown that in strain L108, a HIBA cobalamin-dependent mutase carries out
the transformation of HIBA to 3-hydroxybutyrate. These three major enzymes represent key
transformation steps required for complete mineralization of MTBE to carbon dioxide (CO 2 ) .
Again, the presence of these enzyme genes in aquifer sediment samples may correlate with
decay rates observed in bioaugmented and biostimulated active zones; however, the mere
presence of these genes may not indicate that they are fully functional in the metabolism
of MTBE.
Another diagnostic analytical tool used for assessing MTBE biodegradation in sediment
microcosms, monitored aquifers and bioactive zones of biobarriers is compound specific stable
isotope analysis (CSIA) (Method 6, Table 10.1 ). This technique measures the abundance of 13 C/ 12 C
and 2 H/ 1 H in MTBE molecules relative to those in the international standards for carbonate
( 13/12 C) and ocean water ( 2/1 H), respectively, and are expressed as

13
Cand

2

Hpermil( oo or
parts per thousand). In principle, the lighter atoms ( 12 Cand 1 H) are enzymatically attacked
preferentially at some rate relative to the heavier atoms ( 13 C/ 2 H). Differences in the enrichment
of 13/12 C/ 2/1 H in the remaining MTBE (e.g., residual ether) after significant biodegradation in
microcosms or groundwater samples from bioactive zones in an aquifer, would be indicative of
metabolism. The same isotope differences can be applied to the MTBE plumes where “lighter”
sources of

d d

13 Cand 2 H are different from the “heavier” isotopes remaining downstream where

d d