Carbon isotopic fractionation during anaerobic biotransformation of methyl tert-butyl ether and tert-amyl methyl ether.

Abstract

The fuel oxygenate methyl tert-butyl ether (MTBE) has been frequently detected in groundwater and surface water. Since contaminated sites are often subsurface, anaerobic degradation of MTBE will likely be significant for remediation. As traditional approaches to evaluate biodegradation generally involve laboratory microcosm studies which require time and resources, innovative approaches are needed to demonstrate active in situ biodegradation of MTBE. This study was conducted to gather information at the laboratory level to evaluate the potential of applying carbon isotope fractionation as an indicator for in situ biodegradation of the fuel oxygenates MTBE and tert-amyl methyl ether (TAME). In this study, MTBE utilization was observed in a methanogenic sediment microcosm after a lengthy lag period of about 400 days. MTBE utilization was sustained upon refeeding and subculturing. tert-Butyl alcohol (TBA) was found to accumulate after propagation of cultures. The MTBE-grown cultures also utilized TAME and produced tert-amyl alcohol (TAA). The detection of TBA and TAA indicated that ether bond cleavage was the initial step in degradation for both compounds. Carbon isotope fractionation during anaerobic MTBE and TAME degradation was studied, and isotopic enrichment factors (epsilon) with 95% confidence intervals of -15.6 +/-4.1% and -13.7+/-4.5% were estimated for anaerobic MTBE and TAME degradation, respectively. Addition of 2-bromoethanesulfonic acid, an inhibitor of methanogenesis, substantially prolonged the lag period before transformation, but did not influence carbon isotope fractionation. Our experiment provided strong evidence of significant carbon isotope fractionation during anaerobic MTBE and TAME degradation, demonstrating that this technique can be used as an indicator for in situ MTBE and TAME degradation.

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Carbon Isotopic Fractionation during
Anaerobic Biotransformation of
Methyl tert-Butyl Ether and
tert-Amyl Methyl Ether
P I Y A P A W N S O M S A M A K ,†
H A N S H . R I C H N O W ,‡A N D
M A X M . H A ¨ G G B L O M *, †
Department of Biochemistry and Microbiology and
Biotechnology Center for Agriculture and the Environment,
Rutgers, The State University of New Jersey, New Brunswick,
New Jersey 08901, and Department of Bioremediation, Center
for Environmental Research (UFZ), Permoserstrasse 15,
D-04318 Leipzig, Germany
The fuel oxygenate methyl tert-butyl ether (MTBE) has
beenfrequentlydetectedingroundwaterandsurfacewater.
Since contaminated sites are often subsurface, anaerobic
degradation of MTBE will likely be significant for
remediation. As traditional approaches to evaluate
biodegradation generally involve laboratory microcosm
studies which require time and resources, innovative
approaches are needed to demonstrate active in situ
biodegradation of MTBE. This study was conducted to
gather information at the laboratory level to evaluate the
potential of applying carbon isotope fractionation as an
indicator for in situ biodegradation of the fuel oxygenates
MTBE and tert-amyl methyl ether (TAME). In this study,
MTBE utilization was observed in a methanogenic sediment
microcosm after a lengthy lag period of about 400 days.
MTBE utilization was sustained upon refeeding and
subculturing.tert-Butylalcohol(TBA)wasfoundtoaccumulate
after propagation of cultures. The MTBE-grown cultures
also utilized TAME and produced tert-amyl alcohol (TAA).
The detection of TBA and TAA indicated that ether
bond cleavage was the initial step in degradation for both
compounds. Carbon isotope fractionation during anaerobic
MTBE and TAME degradation was studied, and isotopic
enrichmentfactors(?)with95%confidenceintervalsof-15.6
( 4.1‰ and -13.7 ( 4.5‰ were estimated for anaerobic
MTBE and TAME degradation, respectively. Addition of
2-bromoethanesulfonicacid,aninhibitorofmethanogenesis,
substantiallyprolongedthelagperiodbeforetransformation,
but did not influence carbon isotope fractionation. Our
experiment provided strong evidence of significant carbon
isotope fractionation during anaerobic MTBE and TAME
degradation, demonstrating that this technique can be used
as an indicator for in situ MTBE and TAME degradation.
Introduction
Methyl tert-butyl ether (MTBE) is a synthetic compound
producedalmostexclusivelyforuseingasolineasanoctane
enhancerandlatelyasafueloxygenatetoreduceatmospheric
concentrationsofcarbonmonoxideandozoneinaccordance
with the United States Clean Air Act Amendments of 1990.
Several other chemicals have also been used as fuel oxygen-
ates,includingethyltert-butylether(ETBE),tert-amylmethyl
ether (TAME), diisopropyl ether (DIPE), tert-butyl alcohol
(TBA), methanol, and ethanol (1). Because of its low cost,
ease of production, and favorable transfer and blending
characteristics, MTBE is the most commonly used fuel
oxygenate (2-4). MTBE is currently the focus of public
concern,particularlyintheUnitedStates,asMTBEhasbeen
detectedingroundwaterandsurfacewateracrosstheUnited
States (5, 6). MTBE has a very low taste and odor threshold,
andthusevensmallquantitiesofMTBEwillaffectthequality
of drinking water. There is also concern about its possible
risk to human health, which is still inconclusive so far.
Generally, contaminants can be naturally attenuated by
various processes, including volatilization, adsorption, dis-
persion, hydrolysis, and biodegradation. Unlike other gaso-
line components, such as BTEX compounds (benzene,
toluene,ethylbenzene,o-,m-,p-xylene),MTBEisverywater-
soluble, and it tends to partition from gasoline to the water
phase. Once dissolved in water, the relatively low Henry’s
law constant of MTBE does not lead to significant losses by
partitioningintothegasphase.TherelativelylowKocofMTBE
implies that its movement is minimally retarded by soil
particles,thusallowingMTBEplumestotravelatalmostthe
same velocity as the groundwater stream. The reduction of
MTBEmassbyphysicalprocessesingroundwaterisprobably
insignificant, because volatilization in aquifers is not very
efficient and the hydrolysis of MTBE at almost neutral pH
values is very slow (7). Therefore, bioremediation may play
asignificantroleinmassreductionofMTBEatcontaminated
sites.
AlthoughearlyreportsindicatedthatMTBEwasresistant
to biodegradation, aerobic MTBE biodegradation has been
clearly demonstrated (see refs 8-10 for reviews) along with
the biodegradation of other structurally related fuel oxygen-
atessuchasTAME,ETBE,andTBA.Recently,MTBEhasalso
been shown to be biodegradable anaerobically under meth-
anogenic (11, 12), denitrifying (13), iron(III)-reducing (14),
and sulfate-reducing (15) conditions. TBA is often detected
asanintermediateofMTBEbiodegradation,suggestingthat
cleavageoftheetherbondistheinitialstepinthedegradation
pathway. Under both aerobic and anaerobic conditions, the
slowdegradationofTBAindicatedbyanenrichmentofthese
componentssuggeststhatthedegradationofthemetabolite
is a crucial step in MTBE mineralization.
Anaerobic MTBE degradation is extremely important for
natural attenuation as a remediation option, since MTBE-
contaminatedsitesareoftensubsurfacewithlimitedoxygen
available for biodegradation. Moreover, cocontamination
with a mixture of gasoline hydrocarbons leads to a rapid
consumption of oxygen in aquifers. Although MTBE deg-
radationhasbeenconclusivelydemonstratedbothinlabor-
atory studies and at field sites, evaluation of in situ bio-
degradationisauniquechallenge,inparticularwhennatural
attenuation is taken into account as a remediation option.
Thegeneralstrategytoconfirminsitubiodegradationshould
include three types of evidence: (i) documented loss of
contaminants from the site, (ii) laboratory assays showing
that microorganisms in site samples have the potential to
transform the contaminants under the expected site condi-
tions, and (iii) one or more lines of evidence showing that
the biodegradation potential is actually realized in the field
(16).Consideringthetimeandresourcesrequired,innovative

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approaches are needed to demonstrate active ongoing
biodegradation of MTBE as well as to evaluate the effective-
ness of various cleanup technologies applied in the field.
Isotope fractionation has been reported for biodegrada-
tion of environmental contaminants, such as petroleum
hydrocarbons(17-22)andchlorinatedcompounds(23-26),
and may serve as an indicator for in situ biodegradation.
Compound-specific stable carbon isotope analysis has
been proposed as a method for verifying in situ MTBE
biodegradation (27-29). Hunkeler et al. (27) demonstrated
that partitioning between environmental media revealed a
very low carbon isotope fractionation in comparison to
carbon isotope fractionation measured during biodegrada-
tion of MTBE in aquifer sediment microcosms. Isotope
enrichment factors of -1.5‰ to -2.4‰ were estimated for
aerobicMTBEdegradation(27,28).Incontrast,muchlarger
isotope enrichment factors were obtained for anaerobic
MTBE degradation; however, the specific degradation con-
ditionssuchaselectronacceptorswerenotdocumented(29).
To quantify in situ biodegradation, isotope fractionation
factors representative for the biogeochemical conditions
governing in situ biodegradation are essential. Therefore,
the extent of isotope fractionation in particular degradation
pathways should be investigated before quantification of in
situ biodegradation.
To date, only one study has reported carbon isotopic
fractionation during anaerobic MTBE degradation (29), and
it remains to be determined whether different microbial
communitieswillproducesimilardegreesofcarbonisotope
fractionation. Since anaerobic biodegradation is significant
forremediationofMTBE-contaminatedsites,thisstudywas
conducted to gather information at the laboratory level to
evaluate the potential of applying carbon isotope fraction-
ationasanindicatorforinsitubiodegradationofMTBEand
TAME. The objective of this study was to examine carbon
isotope fractionation during MTBE and TAME degradation
byanaerobicenrichmentculturesandtoestimatetheisotope
enrichment factor for these processes.
Experimental Section
EstablishmentofMethanogenicMTBE-DegradingCultures.
The methanogenic cultures investigated in this study were
established from anaerobic microcosms using a sediment
inoculum from the Arthur Kill inlet, an intertidal strait
betweenNewJerseyandStatenIsland,NY.Themicrocosms
were set up to examine the potential of indigenous micro-
organisms to biodegrade MTBE. The microcosms were
established in anaerobic medium as previously described
(15), without the addition of terminal electron acceptors
(exceptforcarbonate).Sedimentslurries(1:10v/vsediment)
weredividedintoaliquotsinserumvialscappedwithTeflon-
coated rubber stoppers and crimped with aluminum seals.
Each vial contained 50 mL of slurry and had a 10 mL
headspace of N2/CO2(70:30 v/v) passed over hot reduced
copper filings to remove traces of O2. Sterile controls were
autoclaved three times on consecutive days before the
experiment was initiated. MTBE (Aldrich, Milwaukee, WI)
was added to all enrichments and autoclaved controls to a
final concentration of 100 mg L-1. Relatively high substrate
levelswereusedtoenrichforanaerobicorganismsthatcould
utilize the fuel oxygenate as a source of carbon and energy.
Strictanaerobicmicrobialtechniqueswereusedthroughout
all experimental manipulations. Microcosms were set up in
triplicate and incubated under static conditions in the dark
at 28 °C. Substrate concentrations were monitored periodi-
cally. Active cultures were refed and monitored for the
depletionofsubstrate.Finally,activecultureswererepeatedly
transferred (1:5 v/v) to fresh medium to establish stable
cultures utilizing MTBE as the sole carbon source.
Experimental Setup. For the isotope fractionation ex-
periments,thesixth-generationcultures(approximately10-5
dilutionoftheoriginalmicrocosm)wereused.Thetransferred
cultures were fed once with 10 mg L-1MTBE to establish
activity, after which the supernatant liquid was discarded
and 75 mL of fresh methanogenic medium was added. This
enrichment slurry was used as the inoculum. A total of 15
serum vials were prepared for each live treatment. Each vial
received1mLofenrichmentslurryand9mLofmethanogenic
medium. The serum vials were capped with gray Teflon-
lined butyl rubber septa and crimped with aluminum seals.
Anaerobic MTBE and TAME solutions were prepared by
adding MTBE or TAME (Aldrich) neat to anaerobic medium
to a final concentration of 120 mg L-1. A 2 mL sample of
anaerobicMTBEorTAMEsolutionwasaddedtoyieldafinal
substrate concentration of 20 mg L-1. Abiotic controls were
also prepared the same way, except that 1 mL of anaerobic
mediumwasusedinsteadofenrichmentslurry.Afteraddition
of substrate, all live cultures and sterile control vials were
shaken for 30 min at 100 rpm and allowed to settle for 12 h,
after which a 1 mL liquid sample was taken. Two vials of
each treatment were sacrificed immediately by addition of
1 mL of saturated NaCl solution and adjusted to pH 1 by
additionof3MHCl.Substrateconcentrationsinallremaining
live cultures and abiotic controls were measured by head-
space analysis. Thereafter, the remaining live cultures and
sterilecontrolvialswereincubatedat28°Cwithoutshaking.
The headspace concentration of each vial was monitored
periodically, and the biodegradation of MTBE and TAME
was initially evaluated by comparing the headspace con-
centrationtotheinitialconcentrationofthatrespectivevial.
At different stages of substrate utilization, cultures were
sacrificed. Before the addition of NaCl solution and HCl, a
1 mL liquid sample was taken and analyzed immediately by
GC injection for the concentration of substrate remaining
and intermediates formed. The sacrificed vials were then
stored at -20 °C for later carbon isotope analysis.
InhibitionofAnaerobicMTBEDegradationbyBES.Live
cultures and abiotic controls were prepared as described
above. Anaerobic 2-bromomethanesulfonic acid (BES) as a
selective inhibitor of methanogenesis was added from an
anaerobicstocksolutiontoeachvialtoafinalconcentration
of 20 mM (30).
Analytical Methods. The concentration of MTBE and
TAME was determined with a static headspace method. A
100µLheadspacesamplewasanalyzedforMTBEandTAME
with a Hewlett-Packard 5890 gas chromatograph equipped
witha0.53mm×30mDB1column(J&WScientific,Folsom,
CA) and a flame ionization detector. The GC column
temperature was first held at 35 °C for 3 min, increased to
120 °C at a rate of 5 °C min-1, and then held for 1 min. In
addition, the concentrations of MTBE and TAME were
confirmedbydirectinjectionofa1µLaqueoussampleusing
the same instrument and temperature program. Intermedi-
ates were identified by comparison of their retention times
toauthenticstandards.Standardcurvesforeachcompound
were generated with aqueous standards of 1, 5, 10, and 25
mgL-1.Detectionlimitswere0.5mgL-1forMTBEandTAME,
and1.0mgL-1forTBAandtert-amylalcohol(TAA).Methane
was detected by headspace analysis using the same instru-
mentdescribedaboveatanoventemperatureheldconstant
at 35 °C. Because the volume of methane produced was too
low to be accurately measured (no overpressure), methane
concentrations during MTBE degradation were estimated
on the basis of the peak area from headspace injection.
Stable Carbon Isotope Analysis. Stable isotope analyses
wereconductedattheStableIsotopeLaboratoryoftheCenter
forEnvironmentalResearch(UFZ),Leipzig-Halle.Thesystem
consisted of a gas chromatograph (6890 series, Agilent
Technology) coupled with a combustion interface (Thermo

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Finnigan GC-combustion III, Bremen, Germany) and a
FinniganMAT252isotoperatiomassspectrometer(Thermo-
Finnigan,Bremen).TheorganicsubstancesintheCGeffluent
wereoxidizedtoCO2onaCuO/Ni/Ptcatalystheldat960°C.
A Poraplot Q column (0.32 mm × 25 m, Chrompack, The
Netherlands) was used for separation. Helium at a flow rate
of 1.5 mL min-1was used as the carrier gas. The GC
temperature program was held at 150 °C for 15 min, then
increased to 220 °C at a rate of 3 °C min-1, and then held for
10 min isothermally. Samples were injected in split mode
with a split ratio 1:1 into a hot injector held at 220 °C.
Headspaceinjectionvolumesrangedfrom0.2to1mLbased
on the concentration of MTBE determined previously. Each
sample was analyzed at least in triplicate.
The direct headspace method had a detection limit of
approximately 4 mg L-1for MTBE and 6 mg L-1for TAME.
The carbon isotopic compositions (R) are reported as δ
notationinpartsperthousand(denotedas‰)enrichments
or depletions relative to a standard (V-PDB; Vienna Pee Dee
Belemnite standard) of known composition. δ values of
carbon were calculated as follows:
where Rsample and Rstandard represent13C/12C ratios of the
sampleandtheinternationalstandard.Thedirectheadspace
analysis of standard MTBE and TAME had a mean isotope
composition of -30.5 ( 0.4 ‰ (n ) 8) and -22.3 ( 1.7 ‰
(n ) 8), respectively.
Results
EnrichmentofMethanogenicMTBE-DegradingConsortia.
MTBE degradation profiles of the original methanogenic
microcosms established with intertidal sediment are shown
in Figure 1. With an initial concentration of 108.4 ( 3.9 mg
L-1, there was no evidence of any substrate utilization
observed during the first 300 days of incubation. However,
on day 390, the MTBE concentration in one live microcosm
was decreased by 20%. The MTBE concentration in that
particularenrichmentculturegraduallydecreasedovertime,
and complete depletion was observed after 550 days of
incubation. TBA, the common MTBE degradation interme-
diate, was not detected by GC analysis of aqueous samples.
Even though comparable levels of methane were detected
inallthreereplicates(datanotshown),nodepletionofMTBE
in comparison to sterile controls was observed in the other
two microcosms. About 20% depletion of the MTBE con-
centration due to abiotic processes or losses was observed
in autoclaved controls over 550 days.
After depletion of MTBE in the one active microcosm it
was respiked with 20 mg L-1MTBE to confirm utilization.
Depletion of MTBE was faster compared to the initial spike,
and MTBE was removed below the detection limit of 0.5 mg
L-1within 44 days (Figure 2). A TBA concentration of 6.6 mg
L-1was measured on day 44. The microcosm was fed with
20 mg L-1MTBE two more times. During the experiment
TBA accumulated to a concentration of 32.7 mg L-1after the
third addition of MTBE. The detection of TBA confirmed
anaerobicMTBEbiotransformation.Afterthethirdspike,10
mL of the microcosm slurry was transferred into 40 mL of
fresh methanogenic medium and spiked with MTBE. After
establishment of activity, these culture transfers (1:5) were
sequentiallyrepeated,andMTBEwasfedasthesolesubstrate
throughout the course of the subsequent incubation. The
enrichmentsmaintainedtheircapabilitytotransformMTBE.
The headspace methane concentration increased as MTBE
utilization proceeded, and TBA was accumulated in sto-
ichiometric amounts to MTBE utilized in more diluted
enrichments (data not shown).
MTBE/TAMEDegradationProfiles.Thesixthgeneration
ofMTBE-growncultures(approximately10-5dilutionofthe
original microcosm) were tested for their capability to
biodegrade TAME, as well as for the role of methanogenesis
in MTBE and TAME biodegradation. Individual 12 mL
cultures were established and spiked with MTBE or TAME
with or without BES to inhibit methanogenesis. Headspace
substrate concentrations were monitored over time, and
culturesweresacrificedatdifferentstagesofbiodegradation
for analysis. With an average initial MTBE concentration of
20.5(0.8mgL-1,MTBEdegradationinlivecultureswithout
BES proceeded after a lag period of 30 days (Figure 3a). In
the experiments with BES, minimal MTBE degradation was
observed during the first 120 days of incubation (Figure 3a).
From an average initial MTBE concentration of 20.6 ( 0.9
mg L-1, between 10% and 90% MTBE transformation was
observedatday160.TheremainingoflivecultureswithBES
weresacrificedonday185withanMTBEconcentrationrange
from 0.9 to 14.3 mg L-1. MTBE depletion in abiotic controls
was less than 10% at the end of the experiments on day 185.
As MTBE was utilized, with and without BES, stoichiometric
amounts of TBA accumulated (Figure 4a). Without BES,
headspacemethaneconcentrationsincreasedcorresponding
to MTBE utilized, while methane was not detectable in
cultures with BES, indicating that methanogenesis was
inhibited (Figure 4a).
Figure 3b shows TAME degradation profiles by MTBE-
grown methanogenic cultures with and without BES. Under
methanogenic conditions, TAME utilization began after a
lag period of about 7-15 days (Figure 3b). TAME concentra-
tionsinallliveculturesdecreasedovertimefromanaverage
initial concentration of 20.4 ( 1.0 mg L-1. In the presence
of BES, TAME utilization began after 30 days of incubation.
TAME concentrations in these cultures were not monitored
FIGURE 1. Anaerobic MTBE loss in three replicates (solid tilted
squares, replicate 1; solid triangles, replicate 2; solid circles,
replicate 3) of intertidal sediment microcosms without amendment
oftheexternalelectronacceptor.TheinitialconcentrationofMTBE
added was ca. 100 mg L-1. Autoclaved control data are the means
of triplicate analyses (open circles).
δ(13C) (‰) ) (Rsample/Rstandard- 1) × 1000
FIGURE 2. Transformation of MTBE (solid squares) to TBA (open
squares) in the active microcosm upon respiking with MTBE. The
arrow indicates addition of MTBE.

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between day 59 and day 165. On day 165, all of the cultures
were sacrificed. TAME concentrations in most vials were
below 2 mg L-1except for three cultures with TAME
concentrations of 5.8, 7.8, and 8.0 mg L-1. TAA was detected
as the TAME transformation product both in the presence
and in the absence of BES, with concentrations accounting
for 50-80% of TAME utilized (Figure 4b). Without BES,
headspacemethaneconcentrationsincreasedcorresponding
totheamountofTAMEutilized,whilemethanewasdetected
at relatively low concentrations in a few cultures with BES,
indicating that methanogenesis was almost completely
inhibited (Figure 4b).
Isotope Fractionation during Anaerobic Biotransfor-
mation of MTBE and TAME. Individual cultures from the
previous experiment were sacrificed at different stages of
substrate utilization. All live samples with MTBE concentra-
tionsgreaterthan4mg/L-1wereanalyzedforcarbonisotope
composition. The δ(13C) of the MTBE standard was -30.5 (
0.4‰, and the mean δ(13C) of two sample vials sacrificed on
day 0 was -29.0 ( 0.3‰. In live cultures, δ(13C) of MTBE
increased as biodegradation proceeded. A total shift of
δ(13C) of 24.4‰ was observed at 77.8% MTBE degradation,
indicating a strong enrichment of13C in the residual MTBE
fraction. The mean δ(13C) of MTBE of two abiotic control
vials collected on day 125 (-29.0 ( 0.1‰) was comparable
to the initial values of both live cultures (-29.0 ( 0.3‰) and
abiotic controls (-29.1 ( 0.1‰). Because the differences of
δ(13C) of MTBE in abiotic controls at the beginning and the
endoftheexperimentwereminimal,theisotoperatioswere
not analyzed for other abiotic controls collected during the
course of the experiment.
The mean δ(13C) values of TAME in two live cultures and
two abiotic vials sacrificed on day 0 were -19.2 ( 0.5‰ and
-20.1(0.4‰,respectively.Thevalueswereslightlyenriched
in13C compared to the carbon isotope composition of the
TAME standard (-22.3 ( 1.7‰). In the live cultures an
enrichment of13C in the residual fraction during TAME
biodegradation was observed. The isotope composition of
TAMEwasenrichedin13Cto-8.1‰and-5.1‰after50.3%
and 64.0% TAME utilization, respectively. A reliable deter-
mination of the isotope composition of residual TAME at a
more advanced stage of transformation was not possible.
ThemeanisotopecompositionofTAMEoftwoabioticcontrol
vials collected at the end of the experiment was -18.4 (
0.3‰.
With the addition of BES to inhibit methanogenesis, the
transformationsofMTBEandTAMEwerealsoaccompanied
by an enrichment of13C in the residual parent substrates. At
72.1% MTBE degradation, a δ(13C) value of -8.1‰ was
measured in the residual MTBE, which gives a total shift of
21.5‰comparedtotheinitialisotopecomposition.Analysis
of δ(13C) of TAME in live enrichments also demonstrated
strong enrichment of13C in the residual TAME fraction.
A total shift of 15.1‰ was observed in live enrichments at
72.6% TAME utilization.
Estimation of the Isotopic Fractionation Factor (R) and
IsotopicEnrichmentFactor(?).Calculationswerebasedon
the Rayleigh equation Rt/R0) (ct/c0)(1/R-1)for a closed system
(31, 32), where R is the isotope ratio, C is the concentration,
and the index (0, t) describes the incubation time at the
beginning (0) and during the reaction time (t) of the
experiment. When ln(Rt/R0) versus ln(ct/c0) is plotted (Figure
5), the kinetic isotope fractionation factor (R) and isotopic
enrichment factor (?) can be determined from the slope of
the curve (b), with b ) 1/R - 1 and ? ) 1000b. Isotope ratios
(Rt/R0) were determined from the equation Rt/R0) (δt/1000
+ 1)/(δ0/1000 + 1). Linear regression was used to estimate
the slope of each data set. The enrichment factors of the
individual anaerobic MTBE and TAME degradation were
-15.6‰ and -13.7‰, respectively. The addition of BES to
inhibitmethanogenesisinparallelbatchexperimentsresulted
in enrichment factors of -14.6‰ and -11.2‰, which are
slightly lower compared to those of the fractionation experi-
ments without BES addition.
FIGURE 3. (a) MTBE concentrations in live cultures with MTBE
only(solidsquares),MTBE+BES(solidcircles),andabioticcontrols
(opensquares).(b)TAMEconcentrationsinlivecultureswithTAME
only(solidtriangles),TAME+BES(solidtiltedsquares),andabiotic
controls(opencircles).Errorbarsrepresentthestandarddeviation,
n ) 5.
FIGURE 4. Concentrations of headspace methane (solid symbols)
and tertiary alcohol (open symbols) at different stages of (a) MTBE
and(b)TAMEdegradationinlivecultureswithMTBEonly(squares),
MTBE + BES (circles), TAME only (triangles), and TAME + BES
(tilted squares).

Page 5

The intrinsic isotope fractionation factor Rintrinsic and
isotope enrichment factor ?intrinsicwere also calculated from
the equation Rintrinsic) 1/[(n/R) - (n - 1)] and the related
?intrinsic)(1/Rintrinsic-1)×1000(33).Theequationstakeinto
account the number of carbon atoms (n) which do not
undergo isotope fractionation. This approach allows the
comparison of isotope fractionation of organic compounds
with different numbers of carbon atoms. The isotopic
enrichment factor (?) and the intrinsic enrichment factor
(?intrinsic) estimated for each treatment are summarized in
Table 1.
Discussion
The prospect of using stable isotope fractionation as a tool
to demonstrate in situ MTBE degradation has received
increased attention. Only a few studies, however, have been
reportedsofar.Hunkeleretal.(27)demonstratedthatcarbon
isotope fractionation during partitioning between environ-
mental compartments was small in comparison to carbon
isotope fractionation measured during aerobic biodegrada-
tion. Aerobic degradation experiments with MTBE as the
onlysubstrateandacometabolictransformationexperiment
with 3-methylpentane yielded similar isotope enrichment
factors (-1.52 ( 0.06‰ to -1.97 ( 0.05‰). Gray et al. (28)
reported carbon isotope enrichment factors of -2.0 ( 0.1‰
to -2.4 ( 0.3‰ and -1.5 ( 0.1‰ to -1.8 ( 0.1‰ during
aerobic biodegradation of MTBE by a bacterial pure culture
andamixedconsortium,respectively.Thecarbonenrichment
factors obtained in those two studies show small variability
associated with the cleavage of the methyl group under
aerobic conditions.
In contrast, much stronger enrichment factors were
observed during anaerobic MTBE and TAME degradation.
Ourstudypresentsevidenceofsignificant13Cenrichmentin
residual MTBE and TAME during anaerobic biodegradation
duetothefasterreactionratesoflightercomparedtoheavier
isotopomers. Isotopic enrichment factors (?) with a 95%
confidenceintervalof-15.6(4.1‰and-13.7(4.5‰were
estimated for anaerobic MTBE and TAME degradation,
respectively.Kolhatkaretal.(29)foundanenrichmentfactor
forMTBEdegradationinanundefinedanaerobicmicrocosm
of-9.16(5.0‰,whichisslightlylowerbutassociatedwith
a high uncertainty. Considering the upper limit of the
uncertainty, it is similar to the factors found in our experi-
ments. Nevertheless, our observation and previous results
strongly indicate that carbon isotope analysis has the
potential to demonstrate active anaerobic MTBE and TAME
biodegradation,especiallyinsubsurfaceenvironmentswhere
oxygenislimited.Asmallcarbonisotopeenrichment(<2‰)
will often be difficult to use as a suitable indicator of in situ
biodegradation (34); however, the anaerobic in situ degrada-
tionofMTBEandTAMEcanobviouslybetracedwithahigher
sensitivity by means of isotope fractionation.
The original methanogenic enrichments were obtained
frommicrocosmsusingintertidalsedimentsastheinoculum.
Previousmicrocosmstudiesofsedimentsfromthesamesite
demonstratedbiotransformationofMTBEandTAMEtoTBA
and TAA, respectively (15). In the previous report, biotrans-
formation was observed only under sulfate-reducing condi-
tions in one of two replicates, with no MTBE or TAME
depletion observed in methanogenic microcosms after over
1160 days. This may be due to the fact that sediments were
acquired at different times and did not come from exactly
the same location. Nonetheless, the observation of MTBE
degradation in only one of three replicate methanogenic
enrichments in this study, as well as a similar observation
reported elsewhere (11, 15), suggests that the size of the
anaerobic MTBE-degrading population is very small. In our
present study, MTBE and TAME were utilized by MTBE-
FIGURE 5. Double logarithmic plot according to the Rayleigh equation of the isotopic composition versus the residual concentration of
substrates: (a) MTBE, (b) MTBE + BES, (c) TAME, (d) TAME + BES. Solid symbols represent live cultures. Open symbols represent abiotic
controls.
TABLE 1. Carbon Fractionation Factors (r),aCarbon
Enrichment Factors (E) with a 95% Confidence Interval,band
Intrinsic Carbon Enrichment Factors (EIntrinsic) for MTBE and
TAME Biotransformation by Methanogenic Cultures
rE (‰)
Eintrinsic
R2
MTBE,
methanogenic
MTBE + BES
TAME,
methanogenic
TAME + BES
1.0159 ( 0.0015 -15.6 ( 4.1 -78.4 0.9662
1.0148 ( 0.0023 -14.6 ( 5.2 -73.0 0.8634
1.0139 ( 0.0011 -13.7 ( 4.5 -82.2 0.9885
1.0113 ( 0.0010 -11.2 ( 3.2 -67.2 0.9759
aWith an uncertainty of (1σ.b95% confidence interval obtained
from regression analysis of the Rayleigh equation.