Analysis of Carbon and Hydrogen Stable Isotope Ratios of Phenolic Compounds: Method Development and Biodegradation Applications

Phenolic compounds are priority water pollutants of both natural and anthropogenic origins. Distinguishing the sources and degradation pathways of phenolic compounds in the environment is essential for sustainable water management. In this study, we have developed and validated methods based on gas chromatography–isotope ratio mass spectrometry for analysis of hydrogen and carbon isotope ratios of phenol and cresols. For carbon stable isotope analysis, solid-phase extraction was applied, whereas liquid–liquid extraction, followed by derivatization with trifluoroacetic anhydride, was used for hydrogen stable isotope analysis. The methods were verified in biodegradation experiments using p-cresol and phenol as substrates. Methyl group oxidation of p-cresol by Pseudomonas pseudoalcaligenes and Aromatoleum aromaticum resulted in the strong hydrogen (−76 ± 2 and −103 ± 2 mUr, respectively) and varying carbon isotopic fractionation (−2.1 ± 0.1 and −1.0 ± 0.1 mUr, respectively), leading to distinguishable two-dimensional plots for hydrogen versus carbon isotope ratios, indicating slightly different enzymatic reactions. For anaerobic phenol degradation by Desulfosarcina cetonica, the absence of hydrogen isotopic fractionation indicates adenosine triphosphate-dependent carboxylation as the initial step of the biodegradation pathway. The methods developed in this study are useful to detect biological degradation of phenolic compounds in highly contaminated aqueous environmental systems.