Downloaded from www.microbiologyresearch.org by IP: 54.242.161.225 On: Mon, 07 Mar 2016 18:45:13 Journal of General Microbiology (1984), 130, 1051-1059. Printed in Great Britain 1051 Comparison of Gas-liquid and High-performance Liquid Chromatography, and Mass Spectrometry, for the Detection of Organic Acids in Culture Media of A zospirillum bvasilense and Desulfovibvio desulfuvicans By W. HSIAO-TSU LOH,t NICHOLAS SANTOR0,S ROBERT H. MILLER$ AND OLLI H. TUOVINEN* Department of Microbiology, The Ohio State University, Columbus, Ohio 43210, USA (Received 5 September 1983) Gas-liquid chromatography (GLC) and high-performance liquid chromatography (HPLC) were used to separate organic acids in spent culture media. Organic acid components were identified on the basis of their retention times and confirmed by gas chromatography/mass spectrometry (GLC/MS). The sensitivity of the analytical procedures varied considerably with each organic acid. GLC analysis of spent culture media esterified by the H,SO,/methanol procedure failed to detect fumarate, malate and lactate. The BF,/methanol derivatization was particularly sensitive to pyruvate and lactate. GLC/MS, using the BF,/methanol technique, was insensitive to acetate. Cation-exchange HPLC, using a Bio-Rad acid analysis column, did not require prior derivatization of the sample, and proved to be a rapid and non-destructive assay technique for organic acids. This procedure was used to analyse the spent culture media of aerobically grown Azospirillum brasilense and those of Desulfovibrio desulfuricans grown on pyruvate fermentatively or with sulphate as the electron acceptor. Organic acid profiles of the spent culture media obtained by cation-exchange HPLC analysis were compared with those obtained by GLC analysis using both the H,SO,/methanol and the BF,/methanol esterification procedures. INTRODUCTION The study of metabolic pathways in bacteria often requires an analysis of the end products of sugar catabolism. During anaerobic metabolism, these acid end products are usually fermentation acids, although tricarboxylic acid (TCA) cycle intermediates may also accumulate. A variety of methods have been developed for the separation of these metabolites, including silica gel chromatography, gas-liquid chromatography (GLC), and high-performance liquid chromatography (HPLC). Organic acids were initially separated by silica gel chromatography on a chloroform/5 % (v/v) tert-butanol gradient (Frohman et al., 1951). To increase the sensitivity of the assay, erythrocytes were incubated with a 14C-labelled substrate (Dajani & Orten, 1958). The radioactive products of catabolism were collected and identified as tricarboxylic acids on the basis of previously determined elution rates and quantified by liquid scintillation counting. Other workers have subsequently applied GLC in the detection and identification of organic acids. The major drawback to this approach is the need for a derivatization step, without which some organic acids are insufficiently stable and volatile for GLC analysis. In addition, formic acid responds poorly to flame ionization detection (FID) without prior derivatization (Lambert f Present address : Department of Biology, University of Virginia, Charlottesville, Virginia 22901, USA. $, Present address : Department of Microbiology, University of Illinois, Urbana, Illinois 61801, USA. 0 Present address : Department of Soil Sciences, North Carolina State University, Raleigh, North Carolina 27650, USA. Abbreviations: FID, flame ionization detection/detector ; TCD, thermal conductivity detection/detector 0022-1287/84/0001-1468 $02.00 0 1984 SGM