ORIGINAL PAPER Rapid reagentless quantification of alginate biosynthesis in Pseudomonas fluorescens bacteria mutants using FT-IR spectroscopy coupled to multivariate partial least squares regression Elon Correa & Håvard Sletta & David I. Ellis & Sunniva Hoel & Helga Ertesvåg & Trond E. Ellingsen & Svein Valla & Royston Goodacre Received: 24 November 2011 / Revised: 14 March 2012 / Accepted: 19 April 2012 / Published online: 15 May 2012 # Springer-Verlag 2012 Abstract Alginate is an important medical and commercial product and currently is isolated from seaweeds. Certain microorganisms also produce alginate and these polymers have the potential to replace seaweed alginates in some applications, mainly because such production will allow much better and more reproducible control of critical qual- itative polymer properties. The research conducted here presents the development of a new approach to this problem by analysing a transposon insertion mutant library con- structed in an alginate-producing derivative of the Pseudo- monas fluorescens strain SBW25. The procedure is based on the non-destructive and reagent-free method of Fourier transform infrared (FT-IR) spectroscopy which is used to generate a complex biochemical infrared fingerprint of the medium after bacterial growth. First, we investigate the potential differences caused by the growth media fructose and glycerol on the bacterial phenotype and alginate syn- thesis in 193 selected P. fluorescens mutants and show that clear phenotypic differences are observed in the infrared fingerprints. In order to quantify the level of the alginate we also report the construction and interpretation of multi- variate partial least squares regression models which were able to quantify alginate levels successfully with typical normalized root-mean-square error in predictions of only approximately 14 %. We have demonstrated that this high- throughput approach can be implemented in alginate screens and we believe that this FT-IR spectroscopic methodology, when combined with the most appropriate chemometrics, could easily be modified for the quantification of other valuable microbial products and play a valuable screening role for synthetic biology. Keywords Pseudomonas fluorescens . Alginate . Alginate acetylation . FT-IR spectroscopy . Partial least squares regression Introduction Alginates are linear polysaccharides composed of mannur- onic acid and guluronic acid residues produced by brown algae and bacteria such as Azotobacter vinelandii and some species of Pseudomonas. These polysaccharides are used in several large-scale industrial processes, such as food addi- tives, and are also used in the pharmaceutical and medical sectors [1, 2]. Even though the synthesis of alginate is common among the Pseudomonas species, commercially Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-6068-6) contains supplementary material, which is available to authorized users. E. Correa (*) : D. I. Ellis : R. Goodacre School of Chemistry, Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7ND, UK e-mail: elon.correa@manchester.ac.uk H. Sletta : S. Hoel : T. E. Ellingsen Department of Biotechnology, SINTEF Materials and Chemistry, SINTEF, 7465 Trondheim, Norway H. Ertesvåg : S. Valla Department of Biotechnology, Norwegian University of Science and Technology (NTNU), Sem Sælandsvei 6/8, 7491 Trondheim, Norway Anal Bioanal Chem (2012) 403:2591–2599 DOI 10.1007/s00216-012-6068-6