Methyl tert-butyl Ether (MTBE)

Carbon and hydrogen isotope effects have been observed during the biodegradation of MTBE.  Isotope effects differ based on the redox conditions and the specific microorganisms and pathways involved.

  • Anaerobic Biodegradation:  Demonstrating that MTBE biodegradation is occurring under the predominantly anaerobic conditions observed at many gasoline impacted sites can be an important component of monitored natural attenuation (MNA).  To date, anaerobic MTBE utilizing microorganisms have not been isolated in pure culture.  However, CSIA provide can provide conclusive evidence of anaerobic biodegradation of MTBE.  In anaerobic enrichment cultures, Kuder et al. (2005) measured carbon isotope enrichment factors on the order of -11.9 to -14.1‰ and hydrogen isotope enrichment factors of -11 to -21‰.
  • Aerobic Biodegradation:  Carbon isotope fractionation has been observed during aerobic biodegradation of MTBE but enrichment factors are different based on the specific microorganisms and pathways involved.  For example, significant carbon (ε = -2.0 to -2.4‰) and hydrogen (ε = -33 to -37‰) isotope fractionation have been measured for MTBE utilizing microorganisms including Methylibium petroleiphilum PM1 (Gray et al. 2002).  With Mycobacterium pure cultures that oxidize MTBE via an alkane monooxygenase (AlkB), carbon isotope fractionation is observed (ε = -2.5 to -2.6‰) but hydrogen isotopic fractionation is insignificant (Rosell et al. 2012).  For other aerobic organisms like Aquincola tertiaricarbonis L108 and Rhodococcus ruber IFP 2001 thought to employ an ethB-type monooxygenase, carbon isotope effects are minor and hydrogen isotopic fractionation is insignificant (Rosell et al. 2012).

For aerobic biodegradation of TBA in microcosm studies, Hunkeler et al. (2001) measured a carbon isotope enrichment factor of -4.2‰.

  • Chemical Oxidation:  Chemical oxidation of MTBE by permanganate (ε = -5.5‰) and Fenton-like reagents (ε = -1.4‰) results in significant carbon isotope fractionation (Rosell et al. 2012).  Hydrogen isotopic enrichment factors of -109‰ and -31‰ were also reported for permanganate and Fenton-like oxidation, respectively.

2D-CSIA: Two dimensional compound specific isotope analysis (2D-CSIA) or multi-dimensional CSIA is simply the analysis of the isotope ratios of multiple elements (e.g. 13C/12C, 2H/1H, or 37Cl/35Cl).  For MTBE, 2D-CSIA using both carbon and hydrogen can provide insight into MTBE biodegradation and is particularly valuable with low extent of biodegradation.  For the 2D-CSIA approach, δH vs δ13C plots are constructed.  The slope of the line is Λ which is often characteristic of specific degradation mechanisms.  As pointed out by Rosell et al. (2012) however, 2D-CSIA alone cannot distinguish aerobic and anaerobic biodegradation.

Also Consider Stable Isotope Probing:  Stable isotope probing (SIP) is an innovative method to track the environmental fate of a 13C “labeled” contaminant like benzene to conclusively determine if biodegradation is occurring.  The 13C label serves much like a tracer.  If biodegradation is occurring the 13C label will be detected in the end products of biodegradation – biomass and dissolved inorganic carbon (CO2).  CSIA on the other hand is based on analysis of the 13C/12C ratio of the contaminant itself.

Also Consider CENSUS qPCR or QuantArray-Petro:  CSIA can be performed in conjunction with qPCR or QuantArray analyses to quantify functional genes involved in the aerobic biodegradation of MTBE.


Gray, J.R., G. Lacrampe-Couloume, D. Gandhi, K.M. Scow, R.D. Wilson, D.M. Mackay, and B. Sherwood Lollar. 2002. Carbon and Hydrogen Isotopic Fractionation during Biodegradation of Methyl tert-Butyl Ether. Environmental Science & Technology 36 no. 9: 1931-1938.

Hunkeler, D., B.J. Butler, R. Aravena, and J.F. Barker. 2001. Monitoring Biodegradation of Methyl tert-Butyl Ether (MTBE) Using Compound-Specific Carbon Isotope Analysis. Environmental Science & Technology 35 no. 4: 676-681.

Kuder, T., J.T. Wilson, P. Kaiser, R. Kolhatkar, P. Philp, and J. Allen. 2005. Enrichment of Stable Carbon and Hydrogen Isotopes during Anaerobic Biodegradation of MTBE:  Microcosm and Field Evidence. Environmental Science & Technology 39 no. 1: 213-220.

Rosell, M., R. Gonzalez-Olmos, T. Rohwerder, K. Rusevova, A. Georgi, F.-D. Kopinke, and H.H. Richnow. 2012. Critical Evaluation of the 2D-CSIA Scheme for Distinguishing Fuel Oxygenate Degradation Reaction Mechanisms. Environmental Science & Technology 46 no. 9: 4757-4766.