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

Abstract

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.

Quantification of microbial degradation activities in biological activated carbon filters by reverse stable isotope labelling

“…Carbon isotope ratios of CO 2 were measured as described previously (Dong et al 2017). Briefly, liquid samples (0.5 mL) were taken with a syringe through the stopper of the incubation bottles and injected into closed 12 mL Labco Exetainer vials (Labco Limited, Lampeter, Wales, UK.).…”

“…The vials were pre-filled with 85% phosphoric acid (50 µL) and flushed with CO 2 -free synthetic air (CO 2 < 0.1 ppm, Air Liquide, Oberhausen, Germany). Then, the liberated CO 2 gas in the headspace of these samples was analyzed using a Thermo Fisher Delta Ray CO 2 Isotope Ratio Infrared Spectrometer (IRIS) with Universal Reference Interface (URI) Connect (Dong et al 2017; Fischer and Lopez 2016). IRIS is capable of measuring both carbon and oxygen isotope compositions of CO 2 by comparing various CO 2 isotopologues of the sample in air (i.e.…”

“…CO 2 production was calculated from changes in carbon isotope ratios based on isotope mass balance consideration (Eq. 2) (Dong et al 2017; Schulte et al 2019). where is the initial amount of total inorganic carbon in the system [mmol], is the CO 2 released from microbial mineralization of DOC [mmol], is the final carbon isotope ratio of the total inorganic carbon in the system, is the initial carbon isotope ratio of total inorganic carbon in the system, is the carbon isotope ratio of CO 2 released by microbial production [assumed to be 1.1% based on the abundance of 13 C in nature (Aelion et al 2009)], is the amount of inorganic carbon in the added bicarbonate solution [mmol], is the amount of inorganic carbon in the inoculum [mmol], is the amount of inorganic carbon in the headspace [mmol].…”

“…Dong et al (2017) presented a simple and cost-effective method for evaluating microbial mineralization of any kind of organic carbon to CO 2 by reverse stable isotope labelling (RIL). The method is based on adding 13 C-labelled bicarbonate buffer to the incubation medium at a molar ratio of approximately ten atom percent.…”

“…Since the degradation of non-labelled organic carbon leads to the evolution of 12 CO 2 , the 13 C-stable isotope ratio of the total inorganic carbon (TIC) in the system drops accordingly. Microbial mineralization rates can thus be derived following mass-balance calculations (Dong et al 2017; Schulte et al 2019). The advantage of the reverse stable isotope labelling lies in the high sensitivity and precision of 13 CO 2 / 12 CO 2 measurements with stable isotope ratio analysis allowing for the detection of very small amounts of CO 2 evolution.…”