Tracing the assimilation of organic compounds using δ13C analysis of unique amino acids in the bacterial peptidoglycan cell wall

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 (delta(13)C) Were analyzed with a dual mass spectrometer, ion trap and isotope ratio, equipped with a gas chromatograph sample inlet (GC/ITMS/IRMS). Comparisons of delta(13)C 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 delta(13)C values of whole cells, peptidoglycan and D-alanine and the substrate sources (glucose, glutamic acid, isoleucine, lysine, phenylalanine) were similar. The delta(13)C values of the D-alanine were within 0.5 parts per thousand of the substrate. In comparison, diaminopimelic acid was enriched in C-13 by 10.3 parts per thousand relative to the whole cells, peptidoglycan and substrate. Additional laboratory experiments also demonstrated that the delta(13)C 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 delta(13)C values relative to organic matter in the environments. In the Santa Rosa Sound the delta(13)C value of D-alanine was -27.6 +/- 0.6 parts per thousand. This value is in the range of delta(13)C values of bacteria and organic matter previously measured in the system, -24.0 to -27.0 parts per thousand. The delta(13)C value of D-alanine in soil samples from Tyndall Air Force Base was -20.5 +/- 1.7 parts per thousand (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. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.