CENSUS – Gasoline – MTBE and TBA Table

Detect and quantify bacteria responsible for biodegradation of Gasoline – MTBE and TBA

In the early 1990s, methyl tert-butyl ether (MTBE) was added to gasoline at concentrations from 11% to 15% (v/v).  With increased use, MTBE has become one of the most commonly detected groundwater contaminants at sites impacted by petroleum products.  The compound tert-butyl alcohol (TBA) was present as an impurity in the commercial grade MTBE used in reformulated gasoline.  As described below however, TBA is also an intermediate in the degradation of MTBE and is frequently detected in impacted groundwater at concentrations far exceeding the contributions of the original gasoline.  This net accumulation of TBA can indicate slow or incomplete biodegradation of MTBE under existing site conditions.

Biodegradation of MTBE and TBA

Aerobic biodegradation of MTBE and the intermediate TBA has been extensively studied in the laboratory and documented in the field.  However, Methylibium petroleiphilum PM1 remains one of the few bacterial cultures isolated to date that utilizes MTBE and TBA as growth supporting substrates. The key steps in aerobic MTBE/TBA biodegradation by strain PM1 is the initial oxidation mediated by a monooxygenase and the continued biodegradation of TBA catalyzed by TBA hydroxylase.  The net result is complete oxidation to CO2 without accumulation of TBA.  Anaerobic biodegradation of MTBE has been observed but as yet is poorly understood.  Furthermore, while complete mineralization of MTBE has been noted, significant accumulation of TBA particularly under methanogenic conditions is also frequently reported.  Taken as a whole, available field evidence suggests that complete biodegradation of MTBE and TBA under anaerobic conditions is likely to be quite site specific.

Biodegradation of ETBE and other Fuel Oxygenates

While MTBE may be the most common, additional fuel oxygenates including ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) were also employed to increase combustion efficiency and decrease exhaust emissions particularly outside the US.  Bacteria, primarily of the genera Rhodococcus  and Gordonia, capable of utilizing ETBE as a growth supporting substrate have been isolated.  Aerobic ETBE biodegradation is initiated by a cytochrome P450 monooxygenase (ethB).  As with many of the monooxygenases, ethB exhibits relaxed specificity and will co-oxidize MTBE and TAME to TBA and tert-amyl alcohol (TAA), respectively.  Most ETBE utilizing isolates grow on the ethoxy group releasing TBA which is not further degraded.  The notated exception is Aquincola teriaricarbonis L108 which constitutively expresses ethB and possesses pathways for complete mineralization of ETBE, MTBE, and TAME.

CENSUS Targets for Aerobic Gasoline – MTBE and TBA

The following table describes the individual CENSUS targets, their imporatance in evaluating aerobic MTBE and TBA biodegradation, and provides guidelines for integrating CENSUS results into routine groundwater monitoring for common corrective actions.



MI Code 

  Relevance / Data Interpretation

MTBE utilizing PM1 PM1 Targets Methylibium petroleiphilum PM1, one of the few bacteria isolated that is capable of growth on MTBE.  Indicative of the potential for aerobic MTBE biodegradation.
TBA Funtional Gene TBA Targets the functional gene which catalyzes the continued biodegradation of TBA. Since aerobic biodegradation of ETBE by most known strains produces TBA as a metabolite, monitor TBA monooxygenase to evaluate the potential for continued biodegradation of TBA.
ethB ETH Targets the functional gene encoding a P450 cytochrome monooxygenase responsible for initiating aerobic biodegradation of ETBE.  Also capable of co-oxidation of MTBE and TAME.
HIBA Mutase
HCM A key step in the aerobic metabolism of TBA is the conversion of 2-hydroxyisobutyrate (HIBA) to 3-hydroxybutyrate (3HB) by the HIBA mutase enzyme (HCM).  Along with TBA monooxygenase, HCM is consistently associated with TBA metabolizing bacteria and in laboratory studies, disruption of the HCM gene eliminated the ability to grow on TBA establishing the critical role HCM plays in metabolism of compounds with a tert-butyl group.  In fact, at least one leading researcher considers the HCM mutase reaction as the defining reaction in aerobic MTBE/TBA metabolism.