Incorporation of Ambient Water-H into the C-Bonded H Pool of Bacteria during Substrate-Specific Metabolism

Abstract

The stable isotope ratios of C-bonded H (δ2Hn values) can be used to locate soil samples for forensic purposes because of their close correlation with the δ2H values of precipitation. Post-sampling bacterial activity might change the δ2Hn values via glycolysis. We tested to which degree C-bonded H is replaced by H from ambient water under favorable and unfavorable growing conditions. We provided two heterotrophic bacteria (Bacillus atrophaeus, Escherichia coli) with glucose (favorable) or lysine (unfavorable) under aerobic conditions. We assessed the H incorporation from ambient water via 2H labeling. We found that the H incorporation into bacterial biomass in the glucose treatment was 79 ± 5.9% (B. atrophaeus) and 43 ± 3.0% (E. coli), likely as a consequence of glycolysis and conservation of the δ2H value in the anabolic mode of the tricarboxylic acid (TCA) cycle. Differences between species were possibly related with different compositions of metabolite mixtures. The bacteria did hardly grow with lysine while respiration continued, and we found no H incorporation because the catabolic mode of the TCA cycle, which was active when the bacteria grew on lysine, is associated with CO2 release and a complete cleavage of former C–2H bonds. Our results support the glycolysis pathway as a mechanism underlying the incorporation of ambient-water H into the C-bonded H pool of bacteria. Stressful conditions forcing bacteria into a catabolism-dominated metabolism disable the incorporation of ambient-water H, and δ2Hn values can be applied to identify the origin of soil samples in forensics.

Deuterium Isotope Probing: a potential game-changer in assessing chemical persistency in soil

“…In contrast, microorganisms utilize water-H next to the substrate-or necromass-H for biosynthesis (32,33), so the direct incorporation of D from a D-labeled substrate into microbial biomass may be low depending on the active metabolic pathway. While substrate-H bound to O, S or N is generally considered exchangeable, also H from more stable C-H bonds can be eventually exchanged with H from ambient water after enzymatic bond cleavage by microorganisms in redox reactions (17,33,34).…”

Equilibrium isotope fractionation factors of H exchange between steam and soil clay fractions

“…[2][3][4][5] The latter can be reached by closing the samples in airtight tin capsules under an Ar atmosphere, which require less mechanical force for crimping than Ag capsules. [23][24][25] For samples with a high χ e value, the calculated δ 2 H n values respond sensitively to variations in the α ex-w value, because the α ex-w value influences both the slope and the intercept of the regression line of δ 2 H t values of the studied sample on the δ 2 H w values used for the exchange (Equation [1]). 8,13,19,22,26 Thus, the knowledge of a sample-specific α ex-w value might improve the measurement of δ 28 and a minor contribution of rockderived clay minerals that were not formed under the current climatic conditions.…”

“…In the hygroscopic clay minerals, in which not only adsorbed water contributes to the exchangeable H pool but also hydroxyl‐H at the mineral edges, which cannot be removed by drying, 2 there is a particular risk of exchanging H with ambient atmospheric water vapor after steam equilibration, which needs to be avoided 2–5 . The latter can be reached by closing the samples in airtight tin capsules under an Ar atmosphere, which require less mechanical force for crimping than Ag capsules 23–25 . For samples with a high χ e value, the calculated δ 2 H n values respond sensitively to variations in the α ex‐w value, because the α ex‐w value influences both the slope and the intercept of the regression line of δ 2 H t values of the studied sample on the δ 2 H w values used for the exchange (Equation []) 8,13,19,22,26 .…”