Tracing the assimilation of organic compounds using δ13C analysis of unique amino acids in the bacterial peptidoglycan cell wall
Abstract Stable isotope analysis of bacterial nucleic acids can be used to trace carbon that is assimilated and respired by the bacterioplankton in aquatic ecosystems. However, in sediment and soil environments humic acids co-extract with the nucleic acids, resulting in inaccurate isotope analysis. In this study we have examined the use of amino acids found in bacterial cell walls as biomarkers to trace carbon sources that support growth. In the development of the method, peptidoglycan from laboratory grown Pseudomonas sp. was hydrolyzed to amino acids. Stable carbon isotope ratios (δ13C) were analyzed with a dual mass spectrometer, ion trap and isotope ratio, equipped with a gas chromatograph sample inlet (GC/ITMS/IRMS). Comparisons of δ13C values of whole cells, cell wall peptidoglycan and the amino acids d -alanine and diaminopimelic acid from the cell wall were made using different carbon substrates and through different stages of growth to determine isotopic fractionation of these compounds. The δ13C values of whole cells, peptidoglycan and d -alanine and the substrate sources (glucose, glutamic acid, isoleucine, lysine, phenylalanine) were similar. The δ13C values of the d -alanine were within 0.5‰ of the substrate. In comparison, diaminopimelic acid was enriched in 13C by 10.3‰ relative to the whole cells, peptidoglycan and substrate. Additional laboratory experiments also demonstrated that the δ13C of d -alanine did not vary significantly relative to the whole cell and substrate through different growth stages. Stable carbon isotope analysis of the bacterial amino acids was determined at two field locations, water from Santa Rosa Sound, Florida, a humic rich estuarine ecosystem, and jet-fuel contaminated soils of Tyndall Air Force Base, Florida. d -alanine and diaminopimelic acid were isolated from these water and soil samples and the amino acids were analyzed for purity after extraction and for their δ13C values relative to organic matter in the environments. In the Santa Rosa Sound the δ13C value of d -alanine was −27.6±0.6‰. This value is in the range of δ13C values of bacteria and organic matter previously measured in the system, −24.0 to −27.0‰. The δ13C value of d -alanine in soil samples from Tyndall Air Force Base was −20.5±1.7‰ (n=4) similar to ranges of values measured for spilled jet-fuel and CO2 respired from the soil at this site. Results from this study demonstrate that d -alanine can be used as a biomarker for analysis of carbon sources that are assimilated by bacteria in soils and sediments.