Microbial Degradation of Methyl Tert-Butyl Ether and Tert Butyl Alcohol in The Subsurface

Journal of Contaminant Hydrology 70 (2004) 173 – 203
www.elsevier.com/locate/jconhyd
Microbial degradation of methyl tert-butyl ether and

tert-butyl alcohol in the subsurface
Torsten C. Schmidt a,*, Mario Schirmer b,
Holger Weiß c, Stefan B. Haderlein a
a
Environmental Mineralogy, Center for Applied Geoscience, Eberhard-Karls-University of Tuebingen,
Wilhelmstr. 56, D-72074 Tuebingen, Germany
b
Department of Hydrogeology, UFZ Centre for Environmental Research Leipzig-Halle,
Theodor-Lieser-Str. 4, D-06120 Halle (Saale), Germany
c
Interdisciplinary Department of Industrial and Mining Landscapes,
UFZ Centre for Environmental Research Leipzig-Halle, Permoserstr. 15, D-04318 Leipzig, Germany
Received 26 November 2002; received in revised form 29 August 2003; accepted 18 September 2003
Abstract
The fate of fuel oxygenates such as methyl tert-butyl ether (MTBE) in the subsurface is governed
by their degradability under various redox conditions. The key intermediate in degradation of MTBE
and ethyl tert-butyl ether (ETBE) is tert-butyl alcohol (TBA) which was often found as accumulating
intermediate or dead-end product in lab studies using microcosms or isolated cell suspensions. This
review discusses in detail the thermodynamics of the degradation processes utilizing various terminal
electron acceptors, and the aerobic degradation pathways of MTBE and TBA. It summarizes the
present knowledge on MTBE and TBA degradation gained from either microcosm or pure culture
studies and emphasizes the potential of compound-specific isotope analysis (CSIA) for identification
and quantification of degradation processes of slowly biodegradable pollutants such as MTBE and
TBA.
Microcosm studies demonstrated that MTBE and TBA may be biodegradable under oxic and
nearly all anoxic conditions, although results of various studies are often contradictory, which
suggests that site-specific conditions are important parameters. So far, TBA degradation has not been
shown under methanogenic conditions and it is currently widely accepted that TBA is a recalcitrant
dead-end product of MTBE under these conditions. Reliable in situ degradation rates for MTBE and
TBA under various geochemical conditions are not yet available. Furthermore, degradation pathways
under anoxic conditions have not yet been elucidated. All pure cultures capable of MTBE or TBA
degradation isolated so far use oxygen as terminal electron acceptor.
* Corresponding author. Tel.: +49-7071-297-31-47; fax: +49-7071-29-51-39.

E-mail address: torsten.schmidt@uni-tuebingen.de (T.C. Schmidt).
0169-7722/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconhyd.2003.09.001
174 T.C. Schmidt et al. / Journal of Contaminant Hydrology 70 (2004) 173–203
In general, compared with hydrocarbons present in gasoline, fuel oxygenates biodegrade much
slower, if at all. The presence of MTBE and related compounds in groundwater therefore frequently
limits the use of in situ biodegradation as remediation option at gasoline-contaminated sites. Though
degradation of MTBE and TBA in field studies has been reported under oxic conditions, there is
hardly any evidence of substantial degradation in the absence of oxygen. The increasing availability
of field data from CSIA will foster our understanding and may even allow the quantification of
degradation of these recalcitrant compounds. Such information will help to elucidate the crucial
factors of site-specific biogeochemical conditions that govern the capability of intrinsic oxygenate
degradation.
D 2003 Elsevier B.V. All rights reserved.
Keywords: MTBE; TBA; Biodegradation; Thermodynamics; Isotope analysis; CSIA
1. Introduction
The fuel oxygenate methyl tert-butyl ether (MTBE) is one of the organic chemicals
with the highest production volume worldwide. In 1999, about 21 million tons were
produced globally, with 3.3 million tons in the European Union (Krayer von Krauss and
Harremoës, 2001). In Europe, other fuel oxygenates such as ethyl tert-butyl ether (ETBE)
and tert-amyl methyl ether (TAME) are also used in substantial amounts (Schmidt et al.,
2002b). The occurrence and fate of these compounds, in particular MTBE, in the
environment continues to be a major scientific and political issue. Groundwater is the
environmental compartment of most concern with regard to contamination by MTBE and
related compounds. Over only a few years of intense use, MTBE has become one of the
most frequently detected groundwater pollutants in the US (Baehr et al., 1999; Squillace et
al., 1996), and similar findings are emerging in Europe (Klinger et al., 2002; Schmidt et
al., 2002b). The poor state of fuel storage systems led to more than 400,000 leaking
underground storage tank sites in the US identified by the US EPA since 1988 (Small et
al., 2002). In Europe, although in some countries stricter storage facility regulations have
been implemented as early as the 1970s, there is also a growing number of reports of
point-source releases of gasoline containing MTBE (Hansen et al., 2002; Schmidt et al.,
2002b).
A key point in the appraisal of the environmental behavior of MTBE and related
compounds is their degradability in subsurface systems. Accordingly, a large number of
field and laboratory studies have been carried out on the subject over the last 10 years.
Most studies on MTBE degradation have been done in the US, while ETBE has been
mainly investigated in France, where it has been used commercially for several years
(Kharoune et al., 2001).
The key intermediate of MTBE and ETBE in degradation and in vivo metabolism
studies is tert-butyl alcohol (TBA) that is formed via cleavage of the ether bond. The
microbial degradation of TBA has mostly been studied in conjunction with degradation of
dialkyl ethers.
The degradation of fuel oxygenates and in particular of MTBE has been reviewed
several times over the last years (Deeb et al., 2000b, 2002; Fayolle et al., 2001; Prince,
T.C. Schmidt et al. / Journal of Contaminant Hydrology 70 (2004) 173–203 175
2000). However, the emphasis of all reviews was on MTBE, while TBA degradation only
played a minor role and anaerobic degradation was not well covered.
The scope of this review is therefore to (i) highlight the role of TBA as co-contaminant
and intermediate in MTBE degradation, (ii) to discuss the degradation of MTBE and TBA,
in particular under anoxic conditions, and (iii) to present the potential of using stable
isotope techniques for assessing the fate of slowly degrading compounds such as MTBE
and TBA. Outside the scope of this review are the numerous field studies that have shown
the presence and persistence or disappearance of MTBE at contaminated sites based solely
on concentration measurements in the field. Such studies seldomly provide insights in the
conditions necessary for or mechanisms of degradation. References are with a few
exceptions restricted to peer-reviewed articles. However, there is also a large number of
proceeding papers dealing with MTBE from conference series such as the yearly
Petroleum Hydrocarbons and Organic Chemicals in Groundwater conference (see http://
www.ngwa.org/) and the various Battelle bioremediation conferences (see http://
www.battelle.org/conferences/default.stm), all of which have recently had special sessions
on MTBE research.
2. Sources of tert-butyl alcohol in groundwater
TBA is the key intermediate in the degradation of several dialkyl ethers used as fuel
oxygenates, namely MTBE and ETBE. However, besides MTBE degradation, partitioning
of TBA present in gasoline may also contribute to elevated concentrations in groundwater
at contaminated sites. TBA may occur in gasoline due to several reasons. In a study of
New Jersey gasoline, it was found that TBA was present in five out of six investigated
samples at up to 11% of the MTBE concentration. The authors concluded that TBA was
added intentionally to the fuel since TBA impurities in MTBE should be below 2%
(Kramer and Douthit, 2000). TBA was also found in seven investigated gasoline samples
from Switzerland, but at lower concentrations which could be explained as MTBE
impurities (Zwank et al., 2002). In both studies, aqueous phase concentrations of TBA
in batch equilibrium experiments reached the high mg/l to low g/l range. For Germany, the
use of tert-butyl alcohol in mixtures with methanol as fuel oxygenate has been reported
(Schmidt et al., 2002b). The formation of branched alcohols such as isopropanol and sec-
butanol in gasoline due to oxidative transformations of olefins has been recently shown
(Zanier and Jäckle, 2000). This investigation did not include TBA but presumably TBA
could be formed via the same mechanism. In contrast to MTBE, which is almost
exclusively used as a fuel oxygenate, TBA is also a widely used solvent and intermediate
in industrial processes (Clark, 2002).
Despite the various sources of TBA, the environmental behavior and fate of TBA has
been hardly studied so far, although in contrast to MTBE it is a known animal carcinogen
(Cirvello et al., 1995). In California, a drinking water action level for TBA of 12 Ag/l has
recently been set because of its anticipated human toxicity (Kane et al., 2001). One of the
reasons for the lack of attention paid to TBA contaminations is the difficult analysis of this
compound at trace levels (Schmidt et al., 2001). Nevertheless, some field investigations
have reported the presence of TBA at accidental spill or leakage sites (Kolhatkar et al.,
2000, 2002; Kramer and Douthit, 2000; Landmeyer et al., 1998; Zwank et al., 2002),
sometimes at concentrations higher than MTBE.
3. Physico-chemical data and thermodynamic considerations
Environmentally relevant physico-chemical data for MTBE, TBA and proven or

postulated degradation intermediates of both compounds are summarized in Table 1.
MTBE and TBA differ in several relevant parameters. The Henry’s Law constant of TBA
is 40 times lower, and thus, the transfer from (surface) water to air is less pronounced. The
fuel – water partition coefficient Kfw of TBA is 70 times lower than the one of MTBE
leading to a more efficient transfer of TBA from a non-aqueous phase liquid (NAPL) to
water (Zwank et al., 2002). However, evaporative losses from gasoline as indicated by
Kaf -values should be similar for both compounds. The frequently cited log (Koc) value for
TBA of 1.57 is based on an inappropriate extrapolation of a single-parameter linear free
energy relationship derived for other compound classes. Therefore, a multi-parameter
approach (Poole and Poole, 1999) was used instead that resulted in the much more
reasonable value (log (Koc) = 0.41) included in Table 1. For some of the other MTBE
metabolites, only few of the pertinent physico-chemical data are available. Most of the
compounds are highly water-soluble, and will evaporate from water or partition into
octanol or organic matter only to a small degree. Some are acidic and will be present as
their conjugated bases at natural pH.
The standard free energies of reaction DrG0 for the degradation of MTBE and TBA
under oxic and various anoxic conditions are summarized in Table 2a. Reactions are all
written as an oxidation of MTBE or TBA to bicarbonate to allow a comparison with
previously published data (Finneran and Lovley, 2001). The two half reactions are by
convention written as reductions (Pankow, 1991):
5 HCO þ
3 þ 30 e þ 35 H W C5 H12 O þ 14 H2 O pe0 ¼ 2:94 ð1Þ
MTBE
and
4 HCO þ
3 þ 24 e þ 28 H W C4 H10 O þ 11 H2 O pe0 ¼ 3:22 ð2Þ
TBA
The pe values of both reactions are highly dependent on the pH of the aqueous solution.
The standard free energy of reaction DrG0 consists of an enthalpic and an entropic term:
DG ¼ DH T DS ð3Þ
with T being the temperature in Kelvin [K].
A comparison of reaction enthalpies, DrH 0, and reaction entropies, DrS 0, in Table 2a
shows that for oxic, nitrate reducing and methanogenic conditions the enthalpic contri-
bution to the free energy of reaction dominates whereas for the oxidation of MTBE and
TBA under sulfidogenic conditions the entropic contribution is more important. These data
are valid at standard conditions, i.e., pH = 0. Since such a low pH is not environmentally
relevant, DrG0(w) values were calculated for pH = 7 and compared with the data given by
Table 1
Physico-chemical properties of MTBE, TBA and their potential degradation products
Substance Methyl t-butyl t-butyl 2-Methyl- 2-Hydroxy Isopropyl Methacrylic 2,3-Dihydroxy- Acetone Hydroxy- Pyruvic Lactic
t-butyl alcohol formate 2-hydroxy- isobutyric alcohol acid 2-methyl acetone acid acid
ether 1-propanol acid propionic acid
T.C. Schmidt et al. / Journal of Contaminant Hydrology 70 (2004) 173–203

Abbreviation MTBE TBA TBF MHP HIBA IPA MA DHMPA AC HAC PA LA
CAS No. 1634-04-4 75-65-0 762-75-4 558-43-0 594-61-6 67-63-0 79-41-4 21620-60-0 67-64-1 116-09-6 127-17-3 50-21-5
Commercially available? yes yes yes no yes yes yes no yes yes yes yes
Molecular weight [g/mol] 88.15 74.12 102.13 90.12 104.11 60.10 86.09 120.11 58.08 74.08 88.06 90.08
Boiling point, Tb [jC] 55.2 82.4 83 178 212 82.2 163 359a 56.0 145.5 165 decomp.
Density [kg l 1] 0.744 0.791 0.879 nd nd 0.789 1.015 nd 0.786 1.08 1.265 nd
Vapor pressure, p0 [mbar] 332 56 nd 0.460a 0.012 61 1.32 1.8E 06a 309 3.93 1.29a 0.11
Water solubility, 48 complete 17.4b nd complete complete 89 nd complete complete complete complete
cwsat [g l 1]
Henry’s law constant, 5.9E 04 1.4E 05 5.9E 04b nd 1.5E 07 7.9E 06 3.9E 07 nd 4.0E 05 7.7E 06 nd 1.1E 07
KH [atm m3 mol 1]
log KOW 1.24 0.35 1a 0.99a 0.36 0.05 0.93 1.58a 0.24 0.78 1.06a 0.72
log KOC 1.05 0.41c 1.91a 0.84a 0a,d 0.23c 0a,d 0a,d 0.32c 0.95a 0a,d 0a,d
Fuel – water partition 16e 0.24e nd nd nd nd nd nd 0.26f nd nd nd
coefficient, Kfw
Air – fuel partition 1.5E 03 2.4E 03 nd nd nd nd nd nd 6.3E 03 nd nd nd
coefficient, Kafg
Dissociation constant, – 19.2 – nd 3.61 nd 4.65 3.78a 20 13.1a 2.39/2.5 3.86
pKa
All data at 25 jC; Reference (MTBE, TBA, IPA): (Schmidt et al., 2001), Reference (remaining compounds): Syracuse Research Physical Property database (free access
under http://esc.syrres.com), nd: no data available.
a
Values calculated with Advanced Chemistry Development (ACD) Software Solaris V4.67 were extracted from Chemical Abstracts using SciFinder Scholar.
b
Estimated data from Church et al. (1999).
c
Calculated according to Poole and Poole (1999), Eq. (9).
d
Values at pH 7.
e
Data from Zwank et al. (2002).
f
Measured by us using the standard addition method described in Schmidt et al. (2002a) and the analytical method described in Zwank et al. (2002).
g
Calculated from (KH/RT)/Kfw.
177
178
Table 2a
Calculated free energy yields of complete MTBE and TBA oxidation with various terminal electron acceptors under standard conditions
Compound Acceptor Reactants Products DrH 0 in DrS 0 in DrG0 in DrG0(w) in DrG0(w) in

kJ mol 1 J K 1 mol 1 kJ mol 1 kJ mol 1 kJ mol 1a
MTBE oxygen C5H12O + 7.5 O2(aq) 5 HCO3 + H2O + 5H
+
3343 571.9 3172 3372 –
MTBE nitrate C5H12O + 6 NO3 +H
+
5
HCO3 + 3 N2(aq) + 3044b 166.4b 3039 2999 3054.8
4 H2O
MTBE nitrate C5H12O + 3.75 NO 3 + 5 HCO +
3 + 3.75 NH4 2078 125.3 2040 1941 1951.2
2.5 H+ + 2.75 H2O
MTBE iron(III) C5H12O + 5 HCO3 + 30 Fe
2+
+ na na 2237 1066c 347.4
30 (am)Fe(OH)3(s) + 55 H+ 76 H2O
MTBE sulfate C5H12O + 3.75 SO24 + 5 HCO3 + 3.75 H2S(aq) + 170.0 640.1 362.0 262.1 275.2
2.5 H+ H2O
MTBE methanogenesis C5H12O + 2.75 H2O 3.75 CH4(aq) + 1.25 HCO 3 + 97.47 29.68 88.6 138.5 238.7
1.25 H+
TBA oxygen C4H10O + 6 O2(aq) 4 HCO3 + H2O + 4 H
+
2624 420.8 2499 2659
TBA nitrate C4H10O + 4.8 NO3 + 4 HCO3 + 2.4 N 2(aq) + 2386b 169.9b 2392 2360 –
0.8 H+ 3.4 H2O
TBA nitrate C4H10O + 3 NO3 + 4 HCO3 + 3 NH4
+
1613 63.52 1594 1514 –
2 H+ + 2 H2O
TBA iron(III) C4H10O + 4 HCO3 + 24 Fe
2+
+ na na 1751 814.4c –
24 (am)Fe(OH)3(s) + 44 H+ 61 H2O
TBA sulfate C4H10O + 3 SO42 + 2 H+ 4 HCO3 + 3 H2S(aq) + 86.28 548.8 250.8 170.9 –
H2O
TBA methanogenesis C4H10O + 2 H2O 3 CH4(aq) + HCO3 +H
+
28.26 12.98 32.1 72.06 –
DrG0(w) referring to pH 7, DrG 0, DrH 0 and DrS 0 values were calculated from Gf0, Hf0 and S 0 data, respectively, taken from Stumm and Morgan (1996), for conversion of
Gf0(N2(g)) to Gf0(N2(aq)), the Henry’s Law Constant of N2 given in Wilhelm et al. (1977) was used (0.00065 bar kg mol 1); na: not available.
a
Data from Finneran and Lovley (2001).
b
Using Hf0 and S 0 data for N2(g).
c
Following Stumm and Morgan (1996), the concentration of Fe2 + was set to 10 6 mol/l.
Finneran and Lovley (2001). These two datasets agree reasonably well with the exception
of free energy values for iron reducing conditions. In this case, Finneran and Lovley used a
Gf0 value of 688.35 kJ/mol for ferrihydrite (Finneran, 2002) whereas here the value
given by Stumm and Morgan (1996) ( 699 kJ/mol) was used. Furthermore, a concen-
tration of dissolved free Fe2 + of 10 6 mol/l instead of 1 mol/l was used for our calculation
as suggested by Stumm and Morgan (1996). Nevertheless, the conclusion remains the
same: at equilibrium, the degradation of both compounds, TBA and MTBE, will take place
under all environmentally relevant standard redox conditions. There is no thermodynamic
rationale for an accumulation of TBA since the degradation of TBA yields nearly as much
energy as the degradation of MTBE. It has to be kept in mind, though, that these
calculations only account for the thermodynamic feasibility of a reaction but not for kinetic
limitations that may lead to imperceptibly slow reaction rates.
Table 2b
Calculated free energy yields of complete MTBE and TBA oxidation with various terminal electron acceptors
under typical conditions found at contaminated sitesa
Compound Acceptor Concentration of reactants and DrG in DrG0(w) in
products in mol/l or atm kJ mol 1 kJ mol 1
MTBE oxygen p(O2) = 0.21, pH = 7, 3411 3372
[MTBE] = 10 3, [HCO 3 ] = 10
3
MTBE oxygen p(O2) = 0.002, pH = 7, [MTBE] = 10 3, 3325 3372

[HCO 3 ] = 10
3
(microaerophilic)
MTBE nitrate p(N2) = 0.77, [NO 3 ] = 10
4
, pH = 7, 2932 2999
[MTBE] = 10 , [HCO3 ] = 10 3
3
MTBE nitrate [NO3 ] = 10

4
, [NH +4] = 10 3, pH = 7, 1988 1941
3
[MTBE] = 10 , [HCO 3 ] = 10
3
MTBE iron(III) [Fe2 +] = 10 6, pH = 7, 1135 1066b

[MTBE] = 10 3, [HCO 3 ] = 10
3
MTBE sulfate [SO42 ] = 10 2, p(H2S) = 10 2, pH = 7, 330.6 262.1

[MTBE] = 10 3, [HCO 3 ] = 10
3
MTBE methanogenesis [HCO 3 ] = 10 3,

[CH 4 ] = 10 4
, 228.4 138.5
pH = 7, [MTBE] = 10 3
TBA oxygen p(O2) = 0.21, pH = 7, 2675 2659
[TBA] = 10 5, [HCO 3 ] = 10
3
TBA oxygen p(O2) = 0.002, pH = 7, [TBA] = 10 5, 2606 2659

[HCO 3 ] = 10
3
(microaerophilic)
TBA nitrate p(N2) = 0.77, [NO 3 ] = 10
4
, pH = 7, 2292 2360
[TBA] = 10 , [HCO3 ] = 10 3
5
TBA nitrate [NO3 ] = 10

4
, [NH +4] = 10 3, pH = 7, 1537 1514
5
[TBA] = 10 , [HCO 3 ] = 10
3
TBA iron(III) [Fe2 +] = 10 6, pH = 7, 854.4 814.4b

[TBA] = 10 5, [HCO 3 ] = 10
3
TBA sulfate [SO42 ] = 10 2, p(H2S) = 10 2, pH = 7, 210.8 170.9

[TBA] = 10 5, [HCO 3 ] = 10
3
TBA methanogenesis [HCO 3 ] = 10

3
, [CH4] = 10 4, 129.1 72.06
pH = 7, [TBA] = 10 5
a
If no data from currently investigated sites was available, values given in Stumm and Morgan (1996) and
Bradley et al. (2001a,c) were used. For water and solids, the activities on a mole fraction scale are used (these are
1 in good approximation).
b
Fe2+ set to 106 mol/l.
In order to examine if typical concentrations of oxidants and reductants found at a

contaminated site change the thermodynamics of the redox reactions considerably,
corresponding DrG values for all reactions were calculated. Table 2b summarizes the
concentrations of oxidants and reductants used for the calculations and the resulting DrG
values. For a direct comparison, the DrG0(w) values in Table 2a are also included. It can be
inferred that the concentrations of oxidants and reductants have only a minor influence on
the free energies of reaction. Thus, DrG0(w) or even DrG0 values might generally be used.
The only exception is the oxidation of MTBE or TBA under iron-reducing conditions
because the reduction of ferrihydrite is rather sensitive to both pH and concentration of
dissolved Fe2 + (see Table 2a). However, an analysis of sensitivity towards concentrations
of involved species shows that regardless of the terminal electron acceptor the variation of
milieu parameters in a realistic range does not yield positive DrG values, i.e., the reaction
should be thermodynamically feasible under all prevailing conditions.
The calculations for a microaerophilic case show that the oxygen concentration does
hardly influence the free energy of reaction. This indicates that from a thermodynamic
viewpoint even traces of oxygen that cannot be measured by routine methods would favor
aerobic degradation. It must, however, be emphasized that the actual concentrations of all
species in bacteria microenvironments are highly variable and generally not known at the
field scale. Thus, we can only infer from such data that thermodynamics do not prohibit
degradation of MTBE and TBA at typical field conditions.
4. Degradation of MTBE and TBA in microcosms
Table 3 gives a comprehensive overview of microcosm studies using collected sediment

or soil material. However, when comparing results of the various studies one needs to keep
in mind that site-specific conditions may substantially influence the capability of MTBE or
TBA degradation in microcosms. For example, Bradley et al. (2001a) point out that the
effect of redox conditions on contaminant degradation in surface-water sediments and
aquifer sediments is similar but that degradation rates are usually substantially lower in
aquifer sediments.
The approach to quantify substrate degradation in all studies was to measure MTBE or
TBA loss or mineralization (mostly using radiolabeled MTBE or TBA) over the time
period of the experiment. Degradation rates were rarely reported. TBA as a substrate was
only used in few studies, thus information on TBA degradation is often obtained from the
removal of TBA during MTBE mineralization. However, one needs to carefully check if
adequate analytical methods were employed for the determination of TBA at rather low
concentrations. In particular, the widely used Headspace-GC-FID methods (e.g., Zoeckler
et al., 2003) are not suitable because of the very low Henry’s law constant of TBA. When
interpreting results from studies using [14C]MTBE it must be pointed out that the presence
of small amounts of [14C]TBA must not be taken as proof of MTBE degradation since 3–
4% [14C]TBA are present in commercial [14C]MTBE (Bradley et al., 2001a,b) (see also
discussion of TBA sources in groundwater above).
In studies under anoxic conditions, it is important to ensure that no oxygen traces
are present in the system because even small amounts of oxygen from a thermody-
namic viewpoint might lead to preferential consumption of oxygen as terminal electron

acceptor (see above). Such studies should therefore support the assumption of the
dominant electron acceptor by providing information on (i) the redox indicator used
(e.g., resazurin) that will indicate the presence of oxygen, (ii) the reducing agent used
to ensure that traces of oxygen will be removed, and/or (iii) oxygen demand
calculations or electron balances that show the insignificance of aerobic metabolism
in overall transformation. In Table 3, it is marked if and how the studies under anoxic
conditions fulfill at least one of these requirements.
Except for studies utilizing 14C-labelled compounds, mass balances or electron
balances were hardly reported. It is thus often difficult to unequivocally assign the
disappearance of the substrate to a certain electron acceptor. This is a general problem
when using microcosm studies utilizing (often contaminated) field material. In these cases,
the total amount of available electron donors can hardly be estimated, and living controls
without substrate addition that could be used to quantify the consumption of electron
acceptors due to other organic compounds in the sediments were with few exceptions not
used (or reported). If other possible electron donors are present in comparable concen-
trations to the substrate, the consumption of a specific terminal electron acceptor is not
conclusive evidence for the utilization of this electron acceptor in the oxidation of the
substrate. This is especially a problem in studies with radiolabeled MTBE and TBA under
anoxic conditions, since the concentration of radioactive substrate is kept as low as
possible. For example, in Bradley et al. (2001b) the degradation of MTBE under
denitrifying conditions was reported. The electron balance based on nitrate consumption
in microcosms from two sites, however, shows that only 2.5% to 6% of the consumed
nitrate can be explained by MTBE mineralization.
On the other hand, the substrate concentrations should not be too high to avoid
inhibitory effects by poisoning microorganisms as was shown for BTEX compounds
(Morasch et al., 2001).
MTBE and TBA degradation has been reported in the presence of all environmentally
relevant terminal electron acceptors, however, except for oxic conditions, results are
controversial in literature or very limited studies have been carried out so far. Under
methanogenic conditions, TBA degradation has not been observed so far, and it is
currently widely accepted that TBA is an accumulating dead-end product of MTBE
degradation under such conditions. The following section highlights important findings of
microcosm studies on MTBE and TBA biodegradation and shows how different or even
contradictory reported results are.
4.1. Laboratory microcosm studies
Under oxic conditions, the overwhelming majority of studies has reported degrada-
tion of MTBE and TBA (see Table 3). However, several studies found a dependence
of intrinsic degradation potential on the origin of the collected sediments (Borden et
al., 1997; Kane et al., 2001; Zoeckler et al., 2003). Kane et al. (2001) found MTBE
degradation only in two out of four contaminated sediments. In one of the two active
microcosms, an inhibition of MTBE degradation by water-soluble gasoline components
was found and TBA accumulation rates also varied substantially between the two. This
182
Table 3
Degradation of MTBE and TBA in microcosm studies
Assumed Sample Methoda Duration of Spiked MTBE degr. TBA TBA degr./ Anoxic Reference

terminal experiment MTBE (extent) accumulation extent conditions
electron in d in Amol (extent)b assured?
acceptor
14
Oxygen Surface-water C 166 0.08 yes none yes/ni – (Bradley et al.,
sediment (58 – 90% m) after 166 d 2001a)
14
Surface-water C 50 ca. 0.1 yes (66% m) ni ni – (Bradley et al.,
sediment 2001c)
14
Surface-water C 105 0.03 yes ni yes – (Bradley et al.,
sediment (0 – 70% m) (70% m/27 d) 1999)
0.08 (TBA)
14
Surface-water C 198 0.11 (TBA) ni ni yes – (Bradley et al.,
sediment (99% m) 2002)
Aquifer GC 263 5 yes/no yes yes/ni – (Borden et al.,
sediment (29 – 52%), (max. 17% 1997)
no further of MTBE)
MTBE degr.
after 93 d, rate:
0 – 0.001 d 1
14
Aquifer C, GC 80 0.2 yes/99% m none yes/ni – (Landmeyer
sediment after 80 d et al., 2001)
14
Aquifer C, GC up to 35 0.23 dependent on dep. on sed. yes/no – (Kane et al.,
sediment sediment (up to 50%) (rate < MTBE) 2001)
Aquifer y13C, GC up to 40 3–8 yes (>99%) yes/no yes – (Hunkeler
sediment (0 – 50%) (rate < MTBE) et al., 2001)
Aquifer GC up to 666 0.5 – 2 yes yes/no yes – (Schirmer
sediment (0.04 – 0.07 d 1) (0 – 20%) (0.12 d 1) et al., 2003)
Aquifer GC up to 140 ca. 0.6 yes (0.022 – ni ni – (Zoeckler
sediment 0.065 d 1), et al., 2003)
lag phase
20 – 33 d, no degr.
in previously

uncontam. aquifer
14
Nitrate Surface-water C 166 0.08 yes none yes/ni not stringent (Bradley et al.,
sediment (23 – 75% m) after 166 d 2001a)
14
Surface-water C 77 0.1 yes (25% m) no yes not stringent (Bradley et al.,
sediment 2001b)
14
Surface-water C 198 0.11 ni ni yes not stringent (Bradley et al.,
sediment (TBA) (49% m) 2002)
Estuarine GC 1160 55 – 110 no – ni indicator (Somsamak
sediment et al., 2001)
Soil GC 280 ca. 15 no – yes reductant (Yeh and
ca. 18 Novak, 1994)
(TBA)
Aquifer GC 85 8.5 no – ni indicator, (Mormile et al.,
sediment reductant, 1994)
electron
balance
Aquifer GC 263 5 no – ni indicator, (Borden et al.,
glove box
14
Sulfate Surface-water C 166 0.08 yes yes/no yes/no/ni elevated (Bradley et al.,
sediment (9 – 20% m) (1 – 9%) conc. sulfide 2001a)
14
Surface-water C 198 0.11 ni ni yes elevated (Bradley et al.,
sediment (TBA) (5% m/198 d) conc. sulfide 2002)
Estuarine GC 1160 + 293 55 – 110 yes yes/st no indicator, (Somsamak
sediment e-balance et al., 2001)
Soil GC 280 ca. 15 no – yes/no reductant (Yeh and
ca. 18 Novak, 1994)
(TBA)
(continued on next page)
183
184
Table 3 (continued)
Assumed Sample Methoda Duration of Spiked MTBE degr. TBA TBA degr./ Anoxic Reference
terminal experiment MTBE (extent) accumulation extent conditions
electron in d in Amol (extent)b assured?

acceptor
Aquifer GC 244 8.5 no – ni indicator, (Mormile et al.,
electron
balance
14
Fe(III) Surface-water C 166 0.08 yes/no (14% m) yes/no (4%) yes/no/ni elevated (Bradley et al.,
sediment conc. Fe(II) 2001a)
14
Surface-water C 198 0.11 (TBA) ni ni no elevated (Bradley et al.,
sediment conc. Fe(II) 2002)
14
(not excluding Aquifer C up to 825 18 yes no yes/(>25% elevated (Finneran and
sulfate sediment and 22 (TBA) (19 – 30% m in 63 d) conc. Fe(II) Lovley, 2001)
reduction and Surface-water 0.1 (14C) in 130 d
methanogenesis) sediment after 300d
preincubation)

14
Mn(IV) Surface-water C 166 0.08 yes/no (11% m) ni ni elevated (Bradley et al.,
sediment conc. Mn(II) 2001a)
14
Surface-water C 198 0.11 (TBA) ni ni yes (75% m) elevated (Bradley et al.,
sediment conc. Mn(II) 2002)
14
Methanogenic Surface-water C 166 0.08 yes/no (10%, yes (4 – 7%)c ni production of (Bradley et al.,
14
(other sediment 0 – 2% m) CH4 2001a)
14
e-acceptors Surface-water C 77 0.1 yes/no (19%) yes (7%)d no production of (Bradley et al.,
14
likely) sediment CH4 2001b)
14
Surface-water C 198 0.11 (TBA) ni ni no production of (Bradley et al.,
14
sediment CH4 2002)
(not excluding Soil GC 280 ca. 15 no – yes reductant (Yeh and
sulfate ca. 18 Novak, 1994)
reduction) (TBA)

Aquifer GC 250 8.5 no – no production of (Suflita and
sediment CH4, electron Mormile, 1993)
balance
Aquifer and GC 152 8.5 yes/no (1 out of yes/st no indicator, (Mormile et al.,
surface-water 3, 54%) reductant, 1994)
sediment electron
balance
Aquifer GC 263 5 no – ni indicator, (Borden et al.,
glove box
14
Surface-water C 105 0.03 no – no production of (Bradley et al.,
14
sediment 0.08 (TBA) CH4 1999)
Aquifer GC 490 0.9 – 1.8 yes no ni glove box (Wilson et al.,
sediment (degrad. rates: 2000)
3.02 F 0.52
year 1 with
added BTEX)
3.5 F 0.65
year 1 without
added BTEX,
lag phase of
at least 175 d
ni: not investigated or not reported, m: mineralization, d: days, st: stoichiometric formation of TBA, yes/no: either border-case classification or different results in
replicates.
a
Methods used were gas chromatography (GC), radiometric detection (14C) and isotopic analysis of carbon (d13C). % m refers to the relative amount of 14CO2 evolved
from the substrate.
b
Note that [14C]TBA is present at about 3 – 4% in [14C]MTBE (Bradley et al., 2001a,b).
c
Corrected for 4% initial [14C]TBA content.
d
Corrected for 3% initial [14C]TBA content.
185
led to the authors’ conclusion that ‘‘caution is warranted for generalizations about
[aerobic] in situ MTBE degradation’’. In a survey of 11 surface-water sediments,
MTBE degradation under oxic conditions was also highly variable (5% to 66%
mineralization after 50 days) but independent of the contamination history (Bradley
et al., 2001c). In contrast, in two studies (Borden et al., 1997; Zoeckler et al., 2003),
MTBE degradation was limited to sediments previously contaminated with MTBE.
Two studies reported cease of MTBE degradation in first-generation microcosms after
30– 55% removal (Borden et al., 1997; Schirmer et al., 2003). Borden et al. postulated
that this was due to the depletion of unknown cosubstrate(s) that allowed cometabolic
degradation. However, in other studies, a surplus of electron donors in the form of
high organic matter content of the sediment (Bradley et al., 1999) or of easily
degradable substrates (Kane et al., 2001) was found to impede or retard MTBE
degradation.
Transient accumulation of TBA (up to 50%) was found in several studies. However,
TBA was never reported to be recalcitrant under oxic conditions and TBA mineral-
ization using radiolabeled TBA as substrate was also shown (Bradley et al., 2002). In
contrast to most other studies, Bradley et al. (2002) concluded from their parallel
investigations of [14C]MTBE and [14C]TBA with the same sediment, that TBA is
degraded more rapidly than MTBE and that TBA is unlikely to accumulate.
Under anoxic (not further specified) conditions, TBA degradation in soil was reported
as early as 1989 (Hickman and Novak, 1989). However, TBA was not degraded in all
soil layers and degradation was substantially slower than for methanol or phenol that
were studied at the same time. Reported zero-order degradation rates were 0.1 – 0.3 mg
l 1 day 1. Furthermore, TBA degradation required an acclimation period of 75– 200
days, which the authors attributed to preferential degradation of other carbon sources in
natural soils that were easier to use as electron donor. Confirming these results, a
subsequent study has shown that TBA degradation was impeded in the presence of
easier degradable substrates. This was also found for MTBE and ETBE (Yeh and
Novak, 1994).
Under (proposed) denitrifying conditions, MTBE and TBA degradation was only
reported by one group using [14C]MTBE and [14C]TBA (Bradley et al., 2001a,b, 2002)
whereas several other studies reported no degradation of MTBE even after very long
acclimation periods (Borden et al., 1997; Mormile et al., 1994; Somsamak et al., 2001;
Yeh and Novak, 1994). The degradation of TBA under denitrifying conditions was
confirmed in one other study (Yeh and Novak, 1994). In the reports of substantial MTBE
mineralization in nitrate-amended microcosms, no accumulation of [14C]TBA was found
(Bradley et al., 2001a,b).
Under sulfidogenic conditions, two studies had shown no degradation of MTBE
(Mormile et al., 1994; Yeh and Novak, 1994) whereas in recent studies substantial
MTBE degradation was found (Bradley et al., 2001a; Somsamak et al., 2001).
Compared with denitrifying conditions, Bradley et al. (2001a, 2002) found consistently
lower mineralization rates for [14C]MTBE and [14C]TBA. The degradation of TBA
under sulfidogenic conditions has not been unambiguously shown yet. Somsamak et al.
(2001) found a stoichiometric accumulation of TBA that suggested TBA degradation to
be rate-limiting in overall removal of MTBE under sulfidogenic conditions.
Under Fe(III) reducing conditions, mineralization of [14C]MTBE has been shown by

two studies (Bradley et al., 2001a; Finneran and Lovley, 2001). In contrast to these
findings, another recent study did not find degradation of MTBE under Fe(III) reducing
conditions (Somsamak et al., 2001). In some cases, addition of humic substances or
anthraquinone-2,6-disulfonate (AQDS) stimulated the degradation of MTBE, and an
electron shuttle mechanism between Fe(III) oxides and Fe(III)-reducing microorganisms
as suggested by previous studies was invoked as explanation (Finneran and Lovley, 2001).
Substantial TBA mineralization was only observed in one study and even occurred
without lag phase whereas MTBE degradation required an adaptation period of 250 days
(Finneran and Lovley, 2001). However, in this study the terminal electron accepting
process in the sediments was not clear due to concomitant sulfate reducing, Fe(III)
reducing and methanogenic conditions. Bradley et al. (2002) suggested that TBA
degradation in the study by Finneran and Lovley was more likely attributable to
sulfidogenic activity. Bradley et al. (2001a) also reported on the sediment-specific
response to amendments with Fe(III) with only one out of three sediments developing
substantial Fe(III) reduction activity, while in the other two methanogenic activity was still
dominant.
Under Mn(IV) reducing conditions, two studies by Bradley et al. have shown
mineralization of [14C]MTBE (Bradley et al., 2001a) and [14C]TBA (Bradley et al.,
2002). However, as for Fe(III) reducing conditions, only in some of the Mn(IV) amended
microcosms strict Mn(IV) reducing conditions developed and only in these cases,
mineralization occurred.
Under methanogenic conditions, there are controversial reports on the biodegradability
of MTBE. In several studies, no degradation was observed (Borden et al., 1997; Bradley et
al., 1999; Somsamak et al., 2001; Suflita and Mormile, 1993; Yeh and Novak, 1994). In
contrast, Wilson et al. (2000) observed MTBE degradation in methanogenic microcosms
and determined pseudo first-order degradation rates of 3.0 F 0.52 year 1 with added
alkylbenzenes and 3.5 F 0.65 year 1 without added alkylbenzenes. In subsequent studies,
utilizing similar methanogenic microcosms that confirmed degradation of MTBE, TBA
was found as a dead-end product (Kolhatkar et al., 2002; Kuder et al., 2002). Mormile et
al. (1994) reported 50% disappearance (9.6 to 4.4 mg l 1) of MTBE in one of three
replicates of microcosms prepared from river-bed sediments after 152 days with stoichio-
metric formation of TBA. TBA was not further degraded. Bradley et al. observed relatively
small losses of MTBE but no significant mineralization (Bradley et al., 2001a) and no
degradation of [14C]TBA (Bradley et al., 2002). Thus, in contrast to MTBE degradation,
there is general consensus that TBA is recalcitrant under methanogenic conditions. The
only study showing TBA degradation so far was from an unamended anoxic soil where
sulfate reduction and methanogenesis were both taking place (Yeh and Novak, 1994).
4.2. Combined laboratory and field studies
Under oxic conditions, Landmeyer et al. (2001) found a rather rapid degradation of
MTBE with no detectable TBA as intermediate in microcosms prepared with sediments
from a contaminated site and a follow-up field study at this site. However, no degradation
rate for MTBE was reported.
From extensive field data at the oxic Borden aquifer site, Schirmer et al. calculated a
degradation rate for MTBE of 0.44 year 1 assuming a pseudo first-order reaction, which
corresponds to a half-life of 580 days (Schirmer and Barker, 1998, Schirmer et al.,
1999). In these studies, the extent of all other possible attenuation processes for MTBE
was estimated and could not explain substantial MTBE removal. TBA was not found at
the field site, which the authors attributed to a more rapid degradation of TBA compared
with MTBE under Borden field conditions. This was confirmed in a subsequent study
with oxic microcosms containing either MTBE or TBA as substrate (Schirmer et al.,
2003). However, in soil column studies carried out simultaneously, TBA and MTBE
degradation rates were found to be similar (i.e., 0.27 and 0.26 year 1, respectively),
probably due to replenishment of TBA via degradation of the continuously added
MTBE. Such conditions might be typical for continuous sources of MTBE in the
environment (e.g., from storage tanks leaking over extended periods of time) whereas
the microcosms as well as the slug injection at the Borden site (Schirmer and Barker,
1998) represented a discontinuous source (e.g., from gasoline accidentally spilled during
transport).
Wilson et al. (2000) reported pseudo first-order degradation rates for MTBE of 2.2 to
5.0 year 1 from field data at another contaminated site under conditions predominantly
methanogenic. Methane concentrations were used to quantify MTBE attenuation due to
dilution/dispersion and only the excess removal was assigned to biodegradation. TBA was
frequently found at the site, but with few exceptions concentrations were much lower than
those of MTBE, which disagrees with lab results that generally found TBA to be
recalcitrant under methanogenic conditions. This might indicate that besides methano-
genesis other electron acceptors such as Fe(III) or sulfate have been utilized or that
microaerophilic conditions were important at the site.
At this point, it should be emphasized that there is not enough known about the crucial
biogeochemical factors that limit degradation of MTBE and TBA on the field scale in
order to infer general conclusions. For example, although it is generally accepted that
degradation of MTBE under anoxic conditions is much slower than under oxic conditions
or MTBE is even recalcitrant, the only available degradation rates derived from field
studies indicate the opposite: for methanogenic conditions a 5– 10 times higher rate than
for oxic conditions has been found.
5. Aerobic degradation of MTBE and TBA by pure and mixed microbial cultures
Table 4 gives a comprehensive overview of published studies with pure and mixed
cultures of MTBE- and TBA-degrading microorganisms. All of these studies have
employed aerobic cultures. No pure culture of a microorganism able to degrade either
MTBE or TBA using another terminal electron acceptor than oxygen has been identified
so far. Two homoacetogens, Acetobacterium woodi and Eubacterium limosum, that were
capable of degrading several unbranched dialkylethers under anerobic conditions could
not degrade MTBE (Mormile et al., 1994). Degradation rates included in Table 4 were all
normalized per mass of protein to facilitate a comparison. The variability of the reported
rates is very high and covers the range from 0.1 to 50 nmol min 1 (mg protein) 1, and
there is no apparent difference between rates for cometabolic degradation and for
microorganisms that utilize MTBE as sole substrate. In general, such rates should be
used and interpreted with care since they often differ substantially in repetitive experi-
ments with the same organism.
5.1. Cometabolic degradation
Steffan et al. (1997) were the first to study in detail several organisms for their
ability to cometabolically degrade not only MTBE, ETBE, TAME but also TBA.
Emphasis of the study was on propane oxidizers that were able to degrade cometaboli-
cally all fuel oxygenates when grown on propane. Other growth substrates led to less
efficient degradation. In general, TBA was the most recalcitrant of the four co-
substrates studied. Confirming these results, TBA was found in other degradation
studies with MTBE in substantial or even stoichiometric amounts (Corcho et al., 2000;
Hardison et al., 1997; Hernandez-Perez et al., 2001; Liu et al., 2001), before TBA was
degraded.
Cometabolic degradation of MTBE and TBA was shown for a variety of micro-
organisms growing on alkanes and isoalkanes, whereas most organisms grown on
aromatic compounds seem to be incapable of MTBE degradation (Deeb and Alvarez-
Cohen, 2000; Hyman and O’Reilly, 1999). It was therefore suggested that the presence of
alkanes or isoalkanes in polluted aquifers could support MTBE and TBA degradation
(Hyman et al., 2000). Liu et al. (2001) recently investigated the ability of three different
strains for MTBE and TBA degradation in the sub-mg/l range. One of the three strains,
Arthrobacter, was able to degrade MTBE and TBA when grown on butane, but not when
grown on other substrates (glucose, 1-butanol, tryptose phosphate broth). TBA degrada-
tion rates were much lower than MTBE degradation rates. The authors reported increasing
inhibition of MTBE degradation by increasing TBA concentrations. Furthermore, they
speculated that further metabolites might be toxic for the organism. The accumulation of
TBA might therefore lead to a decrease in MTBE degradation potential.
5.2. MTBE and/or TBA as sole carbon and energy source
A number of organisms have been described that can grow on MTBE or TBA as sole
carbon and energy source (see Table 4). In some cases, growth is limited to one of these
substances as substrate (e.g., Piveteau et al., 2001). Generally, growth of these organisms
with MTBE or TBA as substrate is slow and growth yields are low (Hanson et al., 1999;
Hatzinger et al., 2001; Mo et al., 1997; Salanitro et al., 1994).
Several studies with mixed cultures enriched from activated sludge able to utilize
MTBE and/or TBA as substrate have been described (Park and Cowan, 1997; Salanitro
et al., 1994). This includes the BC-1 consortium which was the first culture reported to
degrade MTBE (Salanitro et al., 1994). In all of these cultures, TBA was degraded
slower than MTBE, resulting in a transient accumulation of TBA.
The organism studied most extensively is a h-proteobacterium strain PM1 that was first
isolated by Hanson et al. (1999) from a microbial consortium in a compost biofilter. At the
conditions given, PM1 was able to mineralize 46% of MTBE within 120 h. TBA
Table 4
Degradation of MTBE and TBA with pure aerobic cultures/strains
Culture/Strain Substrate Method MTBE TBA TBA Reference
degradation formation/ degradation
rate extent rate (nmol min 1
(nmol min 1 (mg protein) 1)a
(mg protein) 1)a
14
Mixed enrichment MTBE C, GC 13 yes/max. 6.3 (Salanitro
culture BC-1 40% et al., 1994)
14
Methylobacterium MTBE/TBA C, GC yes (28% over ni ni (Mo et al.,
mesophilicum MTBE/TBA 2 weeks), 1997)
Rhodococcus sp. MTBE/TBA inhibition by
Arthrobacter rilicis TBA, IPA,
acetone,
butyl formate
14
Rubrivivax MTBE C, GC yes ni ni (Hanson et al.,
gelatinosus (46% m/5d) 1999)
PM1 (Leptothrix 19 n yes (>MTBE ?) (Deeb et al.,
branch of b- 2000a)
Proteobacteria) inhibition ni ni (Deeb et al.,
by BTEX 2001)
Methylotrophic TBA GC no no 16 (Piveteau
bacterium et al., 2001)
CIP I-205
(Burkholderia
cepacia)
14
Mixed enrichment MTBE C, GC 2.7 – 20 yes/ni yes (= MTBE) (Fortin
culture F et al., 2001)
14
Hydrogenophaga MTBE/TBA C, GC 46 yes/st yes (< MTBE) (Steffan
flava ENV 735 et al., 2000)
yes/ni inhibition by (Hatzinger
formaldehyde et al., 2001)
Mycobacterium MTBE/TBA GC 9 – 20 yes/st 8 – 10 inhibition (Francois
austroafricanum inhibition by by acetone et al., 2002,
IFP 2012 acetone 2003)
and TBF
Mixed enrichment ETBE/ GC 0.08 no 1.4 (Kharoune
culture MTBE/TBA et al., 2002)
14
ENV425 Propane C, GC 4.6 (28 jC) yes/st 1.6 (28 jC) (Steffan
(Nocardia et al., 1997)
strain)
ENV421 Propane 9.2 (28 jC) yes/ni 2.4 (28 jC)
ENV420 Propane yes (rate nd) ni yes (rate nd)
JOB5 Propane yes (rate nd) yes/ni yes (rate nd)
(Mycobacterium
vaccae)
P. putida CAM Camphor 0.4 yes/st ni
P. mendocina KR1 Toluene no no ni
P. oleovorans Gpo1 Alkane (?) no no ni
Rhodococcus Aromatics (?) no no ni
rhodochrous 116
Table 4 (continued)
Culture/Strain Substrate Method MTBE TBA TBA Reference
degradation formation/ degradation
rate extent rate (nmol min 1
(nmol min 1 (mg protein) 1)a
(mg protein) 1)a
JOB5 n-/iso- GC 17 – 21 yes/ni yes ( < MTBE) (Hyman et al.,
(Mycobacterium alkanes 1998)
vaccae) propane GC 24.4 yes/ni 10.4 (Smith et al.,
2003)
Xanthobacter n-/iso- GC 51 yes ( < MTBE) (Hyman and
alkanes O’Reilly,
1999)
P. mendocina 4
Alcaligenes 15
eutrophus
Filamentous Butane GC 0.35 yes/25% yes (rate nd) (Hardison
fungus et al., 1997)
Graphium sp.
Pseudomonas Pentane GC 3.9 ni 0.95 (Garnier
aeruginosa (20% in et al., 1999)
3 days)
14
Arthrobacter Butane C, GC 6.8 (inhib. yes/55% 0.002 (Liu et al.,
by TBA) ( < MTBE) 2001)
Streptomyces griseus no
Methylosinus no
trichosporium
Gordonia terrae Ethanol GC 14 yes/st no (Hernandez-
(now: Rhodococcus Perez et al.,
rubber IFP2007) 2001)
ni: not investigated, nd: not determined, st: stoichiometric formation of TBA.
a
Most rates taken from Fayolle et al. (2001).
degradation was not studied in this and subsequent studies using the same organism (Deeb
et al., 2000a, 2001). Deeb et al. (2001) observed severe inhibition of MTBE degradation
by several BTEX compounds, indicating that some microorganisms capable of MTBE
degradation will not do so efficiently until residual BTEX concentrations at a contami-
nated site are substantially reduced. In combination with the slow degradation of MTBE
inhibition might explain the frequently found extended plumes of MTBE in groundwater.
Unfortunately, the effect of BTEX on TBA degradation has not been studied. In another
study utilizing PM1, TBA was not found (Church et al., 2000). Deeb et al. therefore
conclude that TBA is not accumulated during biotransformation of MTBE by PM1 due to
a higher degradation rate of TBA compared with MTBE (Deeb et al., 2000b) and attribute
this to two different monooxygenases involved in the degradation pathways (Deeb et al.,
2000a). However, degradation rates for TBA have not yet been determined for PM1.
The occurrence of autochthonous bacteria similar to PM1 in groundwater at several
sites contaminated with MTBE was shown with phylogenetic analysis based on 16S rDNA
(Hristova et al., 2003; Kane et al., 2001). Using microcosms with sediment from one
contaminated site, it was shown that there is a correlation between cell density of PM1 like
bacteria and MTBE degradation (Hristova et al., 2003). Hristova et al. (2001) have also
been able to correlate PM1 density and MTBE degradation in a field-scale bioaugmenta-
tion study by a TaqMan PCR method, thus providing evidence for the capability of in situ
degradation of MTBE by this strain.
TBA accumulation was not found with a mixed bacterial consortium that contained
PM1-like microorganisms (Sedran et al., 2002). This study investigated the effect of
BTEX on degradation of MTBE and TBA by a mixed consortium and found no effect for
MTBE and a slight degradation lag phase for TBA. However, even in the presence of
BTEX, TBA degraded faster than MTBE. The authors attributed the difference in results
compared with the pure strain PM1, to the ability of the consortium to overcome metabolic
inhibition.
Hatzinger et al. (2001) investigated degradation of both MTBE and TBA with a
hydrogen-oxidizing bacterium Hydrogenophaga flava. They found transient accumulation
of TBA though the organism was able to degrade TBA. Substrate inhibition studies with
several potential inhibitors showed different results for both compounds, implying that
separate genes control MTBE and TBA degradation in this organism. Most importantly,
TBA degradation was inhibited by rather low concentrations of formaldehyde, another
proposed metabolite of MTBE during degradation (see Fig. 1 and discussion below).
Fig. 1. Proposed degradation pathways for MTBE under oxic conditions. Solid arrows: directly observed
transformations, dashed arrows: transformation products found but mechanism unclear. Tert-butoxy methanol is
marked in brackets since it has not yet been possible to observe this likely but very reactive intermediate.
Francois et al. (2002) recently isolated a methylotrophic bacterium (identified as

Mycobacterium austroafricanum) from activated sludge that can grow either on TBA or
MTBE. Again, TBA accumulated during MTBE degradation and was only degradable in a
mixture at reduced MTBE concentrations. Degradation rates determined with resting cells
where of similar order of magnitude for both compounds.
Another methylotrophic bacterium (Burkholderia cepacia) investigated by the same
group has been shown to grow on TBA but not on MTBE (Piveteau et al., 2001). This
organism has shown the highest degradation rate for TBA so far [16 nmol min 1 (mg
protein) 1].
6. Degradation pathways under oxic conditions
All studies of MTBE and TBA degradation pathways have employed pure aerobic
cultures. MTBE degradation was always found to occur via TBA as the key intermediate
(Church et al., 2000; Steffan et al., 1997). The formation of TBA from MTBE is depicted
in Fig. 1. Tert-butoxymethanol has been proposed as an intermediate in the oxidation of
MTBE but not yet been observed directly. Such hemiacetals are unstable and dismutate to
give the corresponding carbonyl and alcohol compounds (here: formaldehyde and tert-
butyl alcohol). The formation of formaldehyde has hardly been studied and only been
reported once (Hardison et al., 1997). In several studies, tert-butyl formate (TBF) was
observed as an intermediate and it was suggested that tert-butoxymethanol might
preferentially be oxidized rapidly to yield TBF (Hyman et al., 1998) and formic acid,
thus excluding the formation of formaldehyde. However, as for formaldehyde the presence
of formic acid has been reported only once (Hernandez-Perez et al., 2001). This might be
due to difficulties in analysis of these compounds in low aqueous concentrations or
because of the rapid utilization of the C1 compounds by the studied strains (Francois et al.,
2002). In other studies, TBF has not been found which might be due to either the direct
formation of TBA via dismutation or a rapid hydrolysis of TBF to TBA (Church et al.,
1999). Despite these uncertainties in the degradation pathway of MTBE, there is no
evidence of a degradation pathway of MTBE that does not involve TBA as key
intermediate.
The work of Steffan et al. (1997) still provides the most detailed information on the
degradation pathway of TBA. 2-Methyl-2-hydroxy-1-propanol (MHP) and 2-hydroxy
isobutyric acid (HIBA) were identified as degradation intermediates of TBA, however, the
further metabolism of HIBA could not be revealed. The authors suggested three possible
pathways for further degradation. Key intermediates of these pathways are (i) methacrylic
acid, (ii) 2,3-dihydroxy-2-methyl propionic acid and lactic acid, (iii) isopropanol, acetone,
hydroxyacetone, and pyruvic acid. The proposed degradation pathways are depicted in
Fig. 2. The investigated strains grew on all of these intermediates albeit slowly. Church et
al. (2000) also proposed the acetone pathway for strain PM1, although no TBA
degradation intermediates were observed. The formation of HIBA has recently been
confirmed by Francois et al. (2002) for another pure culture, M. austroafricanum IFP
2012. They did not find further degradation intermediates but suggested the acetone
pathway because the same protein patterns were found in cells growing on MTBE, TBA or
Fig. 2. Proposed degradation pathways for TBA (under oxic conditions). Solid arrows: observed transformations,
dashed arrows: hypothesized transformations. Note that the monooxygenase responsible for TBA oxidation is
currently believed to be different from the MTBE degrading enzyme for strains PM1 and ENV 735 but to be the
same for strain IFP 2012.
HIBA on the one hand and IPA or acetone on the other hand. Deeb et al. (2000b)
summarized studies on TBA metabolism in vivo which have shown that acetone is a major
intermediate in addition to MHP and HIBA. They concluded that the acetone pathway is
the most likely in microbial degradation. In contrast, Piveteau et al. (2001) proposed the
pathway involving lactate for a methylotrophic bacterium able to degrade TBA but not
MTBE. This strain was not able to grow on the intermediates in the acetone pathway
(isopropanol, acetone, hydroxyacetone) or on methacrylic acid. All of these studies
exclusively deal with aerobic degradation pathways. Degradation under anoxic conditions
also yields TBA, possibly by enzymes that may cleave the ether bond in the absence of
molecular oxygen (Kolhatkar et al., 2002). However, in part due to the lack of pure
anaerobic cultures of MTBE or TBA degraders, detailed studies of anaerobic degradation
pathways have not been carried out so far.
Detailed studies of MTBE and TBA degradation mechanisms by advanced oxidation

processes using either UV/H2O2 (Stefan et al., 2000) or ozone/H2O2 (Acero et al., 2001)
revealed further potential metabolites. Primary degradation products of MTBE included
tertbutyl formate, tert-butyl alcohol, 2-methoxy-2-methyl propionaldehyde, acetone,
methyl acetate, and formaldehyde. Primary intermediates during TBA degradation were
acetone, formaldehyde and hydroxy-isobutyraldehyde. Further intermediates included
pyruvaldehyde, formic acid, acetic acid, oxalic acid, pyruvic acid, HIBA, and hydrox-
yacetone. Some of the latter were also found in a study of acetone degradation by the same
group (Stefan and Bolton, 1999). Whether such intermediates also result from microbial
degradation is not known at the moment.
7. Compound-specific stable isotope studies
The rate-determining step in the biodegradation of a compound is often accompanied

by a kinetic isotope effect that leads to a relative depletion of the compound fraction
containing lower mass isotopes because they are preferentially degraded (Galimov, 1985).
Compound-specific stable isotope analysis (CSIA) yields data of the isotope signature of a
single compound relative to an international standard that are often expressed as d values
in per mil:

Rx Rreference
dx ¼ 1000½x ð4Þ
Rreference
where Rx and Rreference are the ratios of the heavy isotope to the light isotope (e.g.,
13 12
C/ C or D/H) in the compound x and an international standard, respectively. The
isotope fractionation between two compounds, e.g., a substrate and its degradation
product, can be expressed either with the fractionation factor a or the enrichment factor e
according to:
Rproduct 103 dp þ 1
apr ¼ ¼ 3 ð5Þ
Rreactant 10 dr þ 1
and

Rproduct
epr ¼ 1 1000 ¼ ða 1Þ1000½x ð6Þ
Rreactant
where r = reactant and p = product and Rreactant and Rproduct are the ratios of the heavy
isotope to the light isotope in the substrate and the degradation product, respectively,
that appear in an infinitely short period of time (Clark and Fritz, 1997). It must be
pointed out that sometimes inverse definitions of a and e, i.e., the ratio Rreactant/Rproduct,
are used, therefore care must be taken in the comparison of values from various
references.
The enrichment factor e or the fractionation factor a is usually determined using a

relation of substrate concentration change and isotope fractionation that is assumed to
follow a Rayleigh type equation (Clark and Fritz, 1997):
Rt
¼ f ða1Þ ð7Þ
R0
and

Rt e ½xt
ln ¼ ða 1Þlnf ¼ ln ð8Þ
R0 1000 ½x0
which yields (Mariotti et al., 1981):

dx;t þ 1000 ½xt
1000ln ¼ eln ð9Þ
dx;0 þ 1000 ½x0
where Rt and R0 are the ratios of the heavy isotope to the light isotope in the reactant at
time t = 0 and t, respectively, dx,0 and dx,t are the same isotope ratios expressed in the d
notation, f is the remaining fraction of the reactant at time t and [x0] and [xt] are the
concentrations of reactant x at time t = 0 and t, respectively. For carbon enrichment factors
typically obtained during (bio)degradation (jej < 20x), a simplified Rayleigh equation
can be used
½xt
d13 Cx;t ¼ d13 Cx;0 þ eln : ð10Þ
½x0
In the last few years, the use of stable isotope techniques has been established as an
invaluable tool for the investigation of degradation processes, in particular regarding the
ratio of 13C/12C (Schmidt et al., in press). Most of the reported studies have focused on
BTEX (Ahad et al., 2000; Meckenstock et al., 1999) or chlorinated hydrocarbons (Bloom
et al., 2000; Slater et al., 2001).
Investigations of the isotopic composition of MTBE in various gasolines and neat
standards yielded d13C values ranging from 28.1xto 31.7x(Gray et al., 2002;
Smallwood et al., 2001, 2002; Zwank et al., 2003). This rather narrow range is
surprising if one considers that MTBE is manufactured by three different processes
(Smallwood et al., 2002), but may reflect the small number of analyses published to
date. Equilibrium partitioning of MTBE from an organic phase (e.g., spilled gasoline) to
water does not lead to a significant shift in isotopic composition (Hunkeler et al., 2001;
Smallwood et al., 2002). For d2H, only values for aqueous MTBE standards are
available that range from 100xto 117x(Gray et al., 2002). For TBA, no such
data are available yet.
Isotopic effects during aerobic degradation of MTBE have recently been studied in
microcosms from contaminated sites and with strain PM1 (Gray et al., 2002; Hunkeler et
al., 2001). Hunkeler et al. determined C-isotopic enrichment factors e of 1.5 F 0.06x
to 2.0 F 0.05xfor MTBE. Enrichment factors in microcosms with MTBE as energy
and carbon source and in cometabolic experiments with 3-methylpentane as substrate did
not differ significantly. In the cometabolic experiment, isotopic enrichment during TBA
degradation was also observed and yielded 4.2 F 0.07x. Gray et al. obtained similar
carbon enrichment factors for MTBE: 1.5 F 0.1xto 1.8 F 0.1xfor a microcosm
from a different contaminated site, and 2.0 F 0.1xto 2.4 F 0.3xfor MTBE
degradation by strain PM1. The corresponding hydrogen isotopic enrichment factors
were: 29 F 4x and 66 F 3x in microcosm duplicates, and 33 F 5xto
37 F 4xin the PM1 experiment. The large variations between the microcosms reflect
the larger uncertainties in such systems compared with pure strain studies. For example,
the microorganism community will not be exactly the same in duplicates which may
influence the obtained e values. However, it is not clear why this effect was only observed
in hydrogen but not in carbon isotopic analysis. The authors conclude that carbon isotope
analysis is best suited for quantification of biodegradation at a contaminated site, whereas
the large hydrogen isotopic fractionation enables a more sensitive identification of in situ
biodegradation.
Recently, Kolhatkar et al. (2002) have shown the first data on isotopic fractionation
under anoxic (presumably methanogenic) conditions. They reported fractionation in 12
samples from a contaminated site and obtained a carbon enrichment factor e of
8.1 F 0.85x. In anoxic microcosms prepared with aquifer sediment from this site,
they obtained a similar factor of 9.2 F 5.0x. The authors attribute the large scatter
(high 95% confidence intervals) in comparison with the previous studies to the fact that
independent sacrificial microcosms were used which enabled slightly different bacterial
communities to develop in each serum bottle. As for the oxic systems, TBA was the
principal intermediate that accumulated in the microcosms. The clear difference in isotopic
enrichment factors e for carbon obtained under oxic and anoxic conditions has two
important implications. First, obviously, the fractionating step under anoxic conditions is
the attack of MTBE by enzymes other than monooxygenases. Secondly, one should be
able to distinguish aerobic and anaerobic degradation of MTBE in the field if other
processes such as dilution do not bias the resulting e. A subsequent study by Kuder et al.
(2002) has shown a rather good correlation of d13C and d2H data for MTBE at three
contaminated, anoxic sites. Enrichment factors e for carbon ranged from 7.4x to
10.2x, enrichment factors for hydrogen were 11.4x, which is significantly lower
than reported for oxic systems (Gray et al., 2002). Use of hydrogen isotopes was hampered
by the wide variation of d2H in the source MTBE (>50x). TBA was also included in both
studies of anoxic sites and no significant fractionation was observed. This might be due to
two counteracting trends that always complicate the application of CSIA to degradation
intermediates. Isotopically lighter TBA formed during MTBE degradation may offset
isotopically heavier TBA that remains when TBA is further degraded.
The few studies published so far have shown the potential of compound specific
isotope analysis for the identification and quantification of degradation processes of
MTBE and related compounds. In addition to the measurement of d2H and d13C, the
compound-specific determination of d18O values is now possible with commercial
instruments. Analysis of d18O might be useful for a further elucidation of degradation
mechanisms for ethers and alcohols because a substantial fractionation should only be
obtained in cases where the carbon – oxygen bond is affected in the rate-determining
step.
8. Conclusions
Regardless of environmental conditions, natural attenuation of MTBE and TBA in the

subsurface is slow compared to other gasoline-derived hydrocarbons. However, several
studies proved MTBE degradation in the field, either under natural oxic conditions (Magar
et al., 2002; Schirmer and Barker, 1998; Schirmer et al., 1999) or after supplying oxygen
(Landmeyer et al., 2001; Wilson et al., 2002) and, in some cases, additional inoculation of
microorganisms (Salanitro et al., 2000). In these studies, TBA was not found in substantial
amounts after depletion of MTBE. Under anoxic conditions that are commonly found at
contaminated sites, MTBE degradation has been shown only a few times in the field. Most
of these studies evidenced TBA accumulation in the absence of molecular oxygen
(Kolhatkar et al., 2002; Kuder et al., 2002).
Due to the difficulty of TBA analysis at trace concentrations, many field studies lack
the analysis of TBA at sites contaminated with MTBE. However, this information is not
only essential for the evaluation of MTBE biodegradation, but also to assess whether
TBA was present in gasoline itself. TBA has been found to be as recalcitrant as MTBE
in the subsurface and can be of even more concern due to its higher toxicity. Depending
on biogeochemical conditions, TBA instead of MTBE might therefore be an appropriate
parameter to evaluate the success of natural attenuation approaches and remediation
efforts. In such cases, it is mandatory to monitor TBA concentrations at contaminated
sites in order to determine if TBA is still present or even accumulates under the chosen
remediation conditions. The analysis of carbon and/or hydrogen isotope signatures in
MTBE and TBA degradation experiments will foster our understanding of the degra-
dation of these recalcitrant compounds. Since biodegradation of MTBE can be hindered
in the presence of other gasoline components such as BTEX, inhibitory effects are also
conceivable and should be studied for TBA. A prerequisite for such research is the
establishment of analytical methods that can determine not only the content of MTBE
and TBA but also the TBA degradation products that have generally been neglected
so far.
Acknowledgements
We acknowledge the financial support of UFZ Centre for Environmental Research
Leipzig-Halle for carrying out this work. We thank R. Meckenstock, B. Morasch and L.
Zwank for critical comments on the manuscript, and Phil Gschwend and an anonymous
reviewer for their helpful reviews all of which improved this paper substantially.
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Microbial Degradation of Methyl Tert-Butyl Ether and Tert Butyl Alcohol in The Subsurface

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Journal of Contaminant Hydrology 70 (2004) 173 – 203

Microbial degradation of methyl tert-butyl ether and

* Corresponding author. Tel.: +49-7071-297-31-47; fax: +49-7071-29-51-39.

Keywords: MTBE; TBA; Biodegradation; Thermodynamics; Isotope analysis; CSIA

2. Sources of tert-butyl alcohol in groundwater

3. Physico-chemical data and thermodynamic considerations

Environmentally relevant physico-chemical data for MTBE, TBA and proven or

T.C. Schmidt et al. / Journal of Contaminant Hydrology 70 (2004) 173–203

T.C. Schmidt et al. / Journal of Contaminant Hydrology 70 (2004) 173–203

MTBE oxygen p(O2) = 0.002, pH = 7, [MTBE] = 10 3, 3325 3372

MTBE nitrate [NO3 ] = 10

MTBE iron(III) [Fe2 +] = 10 6, pH = 7, 1135 1066b

MTBE sulfate [SO42 ] = 10 2, p(H2S) = 10 2, pH = 7, 330.6 262.1

MTBE methanogenesis [HCO 3 ] = 10 3,

TBA oxygen p(O2) = 0.002, pH = 7, [TBA] = 10 5, 2606 2659

TBA nitrate [NO3 ] = 10

TBA iron(III) [Fe2 +] = 10 6, pH = 7, 854.4 814.4b

TBA sulfate [SO42 ] = 10 2, p(H2S) = 10 2, pH = 7, 210.8 170.9

TBA methanogenesis [HCO 3 ] = 10

In order to examine if typical concentrations of oxidants and reductants found at a

4. Degradation of MTBE and TBA in microcosms

Table 3 gives a comprehensive overview of microcosm studies using collected sediment

namic viewpoint might lead to preferential consumption of oxygen as terminal electron

4.1. Laboratory microcosm studies

T.C. Schmidt et al. / Journal of Contaminant Hydrology 70 (2004) 173–203

T.C. Schmidt et al. / Journal of Contaminant Hydrology 70 (2004) 173–203

T.C. Schmidt et al. / Journal of Contaminant Hydrology 70 (2004) 173–203

Estuarine GC 1160 55 – 110 no – ni indicator (Somsamak

T.C. Schmidt et al. / Journal of Contaminant Hydrology 70 (2004) 173–203

Under Fe(III) reducing conditions, mineralization of [14C]MTBE has been shown by

4.2. Combined laboratory and field studies

5.1. Cometabolic degradation

5.2. MTBE and/or TBA as sole carbon and energy source

Francois et al. (2002) recently isolated a methylotrophic bacterium (identified as

6. Degradation pathways under oxic conditions

Detailed studies of MTBE and TBA degradation mechanisms by advanced oxidation

7. Compound-specific stable isotope studies

The rate-determining step in the biodegradation of a compound is often accompanied

The enrichment factor e or the fractionation factor a is usually determined using a

which yields (Mariotti et al., 1981):

Regardless of environmental conditions, natural attenuation of MTBE and TBA in the

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