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Applied and Environmental Microbiology, March 2004, p. 1583-1592, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1583-1592.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

High-Throughput Metabolic Fingerprinting of Legume Silage Fermentations via Fourier Transform Infrared Spectroscopy and Chemometrics

Helen E. Johnson,1,2* David Broadhurst,1 Douglas B. Kell,1,{dagger} Michael K. Theodorou,2 Roger J. Merry,2 and Gareth W. Griffith1

Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD,1 The Institute of Grassland and Environmental Research, Aberystwyth, Ceredigion SY23 3EB, United Kingdom2

Received 14 August 2003/ Accepted 10 December 2003

Silage quality is typically assessed by the measurement of several individual parameters, including pH, lactic acid, acetic acid, bacterial numbers, and protein content. The objective of this study was to use a holistic metabolic fingerprinting approach, combining a high-throughput microtiter plate-based fermentation system with Fourier transform infrared (FT-IR) spectroscopy, to obtain a snapshot of the sample metabolome (typically low-molecular-weight compounds) at a given time. The aim was to study the dynamics of red clover or grass silage fermentations in response to various inoculants incorporating lactic acid bacteria (LAB). The hyperspectral multivariate datasets generated by FT-IR spectroscopy are difficult to interpret visually, so chemometrics methods were used to deconvolute the data. Two-phase principal component-discriminant function analysis allowed discrimination between herbage types and different LAB inoculants and modeling of fermentation dynamics over time. Further analysis of FT-IR spectra by the use of genetic algorithms to identify the underlying biochemical differences between treatments revealed that the amide I and amide II regions (wavenumbers of 1,550 to 1,750 cm-1) of the spectra were most frequently selected (reflecting changes in proteins and free amino acids) in comparisons between control and inoculant-treated fermentations. This corresponds to the known importance of rapid fermentation for the efficient conservation of forage proteins.

* Corresponding author. Mailing address: Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Ceredigion SY23 3DD, United Kingdom. Phone: 44 (0) 1970 622353. Fax: 44 (0) 1970 622307. E-mail: hej{at}aber.ac.uk.

{dagger} Present address: Department of Chemistry, UMIST, Manchester M60 1QD, United Kingdom.

Applied and Environmental Microbiology, March 2004, p. 1583-1592, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1583-1592.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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