Benzene, Toluene, Ethylbenzene and Xylenes (BTEX)

Carbon and hydrogen isotope effects have been observed during the biodegradation of BTEX (see Meckenstock et al. (2004) for a review).  Carbon isotope enrichment factors differ considerably between biodegradation pathways and in general are smaller than observed for chlorinated hydrocarbons.  While important, the more subtle carbon isotope effects during BTEX biodegradation mean that biodegradation must be more extensive in order to observe significant carbon isotope fractionation.

In general, hydrogen isotope effects are much larger than carbon isotope effects (Hunkeler et al. 2001) and a number of studies have suggested that 2 dimensional CSIA (2D-CSIA) using both carbon and hydrogen may be the best approach for identifying BTEX biodegradation and elucidating biodegradation pathways (Fischer et al. 2008; Hunkeler et al. 2001; Mancini et al. 2008; Mancini et al. 2003).

Stable isotope probing (SIP), a technique using a synthesized 13C labeled contaminant, is also a strong option for demonstrating BTEX biodegradation particularly in source areas.

  • Anaerobic Biodegradation:  Demonstrating that benzene biodegradation is occurring under the predominantly anaerobic conditions observed at many petroleum hydrocarbon sites is often critical in gaining approval for monitored natural attenuation (MNA).  CSIA provide can provide conclusive evidence of biodegradation of benzene as well as toluene, ethylbenzene and xylenes.

For benzene biodegradation under nitrate-reducing, iron-reducing, sulfate-reducing, and methanogenic conditions, carbon isotope enrichment factors range from -1.9 to -3.6‰ (Fischer et al. 2008; Mancini et al. 2003).  Reported hydrogen isotope enrichment factors are on the order of -59‰ and -79‰ under methanogenic and sulfate-reducing conditions, respectively.

Significant carbon isotope fractionation is also observed during anaerobic biodegradation of toluene, ethylbenzene and xylenes although enrichment factors ε do vary notably (-0.5 to -3.2‰).  Likewise, anaerobic biodegradation of toluene results in significant hydrogen isotope fractionation but reported 2H isotope enrichment factors also vary considerably (Morasch et al. 2001; Ward et al. 2000)

  • Aerobic Biodegradation:  Carbon isotope fractionation has been observed during aerobic biodegradation of BTEX but can be a minor effect depending on the biodegradation pathway (Hunkeler et al. 2001; Morasch et al. 2001).  In general, substantial carbon isotope effects are observed during TEX biodegradation that proceeds via oxidation of the methyl group (TOL – toluene/xylene monooxygenase).  Dioxygenase (TOD) and monoxygenase (RMO, RDEG, PHE) attack at the aromatic ring usually results in only minor carbon isotope fractionation.

Hydrogen isotope effects are greater than carbon isotope effects during aerobic BTEX biodegradation.  As with carbon isotope effects, 2H enrichment factors during oxidation of the methyl group (TOL) are still larger but significant 2H fractionation is observed during other aerobic BTEX biodegradation pathways (Hunkeler et al. 2001; Morasch et al. 2002).

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 BTEX, a number of studies have suggested that 2 dimensional CSIA (2D-CSIA) using both carbon and hydrogen may be the best approach for identifying BTEX biodegradation and elucidating biodegradation pathways (Fischer et al. 2008; Hunkeler et al. 2001; Mancini et al. 2008; Mancini et al. 2003).  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.  For example, (Fischer et al. 2008) observed that although ranges overlapped, Λ values for aerobic biodegradation via TOD were <2, between 7 and 9 for monohydroxylation (RMO, RDEG, PHE) and >17 for 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.

SIP rather than CSIA should be strongly considered as an option to document biodegradation in a source area where contaminant dissolution from a residual NAPL could potentially mask isotopic enrichment effects measured in CSIA.

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 and anaerobic biodegradation of BTEX.  Characterization of the microbial community in conjunction with CSIA may be particularly valuable given the differences in the reported isotopic enrichment factors for the various biodegradation mechanisms.


Fischer, A., I. Herklotz, S. Herrmann, M. Thullner, S.A.B. Weelink, A.J.M. Stams, M. Schlömann, H.-H. Richnow, and C. Vogt. 2008. Combined Carbon and Hydrogen Isotope Fractionation Investigations for Elucidating Benzene Biodegradation Pathways. Environmental Science & Technology 42 no. 12: 4356-4363.

Hunkeler, D., N. Andersen, R. Aravena, S.M. Bernasconi, and B.J. Butler. 2001. Hydrogen and carbon isotope fractionation during aerobic biodegradation of benzene. Environmental Science & Technology 35 no. 17: 3462-3467.

Mancini, S.A., C.E. Devine, M. Elsner, M.E. Nandi, A.C. Ulrich, E.A. Edwards, and B. Sherwood Lollar. 2008. Isotopic Evidence Suggests Different Initial Reaction Mechanisms for Anaerobic Benzene Biodegradation. Environmental Science & Technology 42 no. 22: 8290-8296.

Mancini, S.A., A.C. Ulrich, G. Lacrampe-Couloume, B. Sleep, E.A. Edwards, and B.S. Lollar. 2003. Carbon and Hydrogen Isotopic Fractionation during Anaerobic Biodegradation of Benzene. Applied and Environmental Microbiology 69 no. 1: 191-198.

Meckenstock, R.U., B. Morasch, C. Griebler, and H.H. Richnow. 2004. Stable isotope fractionation analysis as a tool to monitor biodegradation in contaminated aquifers. Journal of Contaminant Hydrology 75 no. 3–4: 215-255.

Morasch, B., H.H. Richnow, B. Schink, and R.U. Meckenstock. 2001. Stable Hydrogen and Carbon Isotope Fractionation during Microbial Toluene Degradation: Mechanistic and Environmental Aspects. Applied and Environmental Microbiology 67 no. 10: 4842-4849.

Morasch, B., H.H. Richnow, B. Schink, A. Vieth, and R.U. Meckenstock. 2002. Carbon and Hydrogen Stable Isotope Fractionation during Aerobic Bacterial Degradation of Aromatic Hydrocarbons. Applied and Environmental Microbiology 68 no. 10: 5191-5194.

Ward, J.A.M., J.M.E. Ahad, G. Lacrampe-Couloume, G.F. Slater, E.A. Edwards, and B.S. Lollar. 2000. Hydrogen Isotope Fractionation during Methanogenic Degradation of Toluene:  Potential for Direct Verification of Bioremediation. Environmental Science & Technology 34 no. 21: 4577-4581.