Monitoring Microbial Mineralization Using Reverse Stable Isotope Labeling Analysis by Mid-Infrared Laser Spectroscopy

Assessing the biodegradation of organic compounds is a frequent question in environmental science. Here, we present a sensitive, inexpensive, and simple approach to monitor microbial mineralization using reverse stable isotope labeling analysis (RIL) of dissolved inorganic carbon (DIC). The medium for the biodegradation assay contains regular organic compounds and ¹³C-labeled DIC with ¹³C atom fractions (x(¹³C)_DIC) higher than natural abundance (typically 2-50%). The produced CO₂ (x(¹³C) ≈ 1.11%) gradually dilutes the initial x(¹³C)_DIC allowing to quantify microbial mineralization using mass-balance calculations. For ¹³C-enriched CO₂ samples, a newly developed isotope ratio mid-infrared spectrometer was introduced with a precision of x(¹³C) < 0.006%. As an example for extremely difficult and slowly degradable compounds, CO₂ production was close to the theoretical stoichiometry for anaerobic naphthalene degradation by a sulfate-reducing enrichment culture. Furthermore, we could measure the aerobic degradation of dissolved organic carbon (DOC) adsorbed to granular activated carbon in a drinking water production plant, which cannot be labeled with ¹³C. Thus, the RIL approach can be applied to sensitively monitor biodegradation of various organic compounds under anoxic or oxic conditions.