ORIGINAL ARTICLE Development of a minimal growth medium for Lactobacillus plantarum A. Wegkamp 1,2 , B. Teusink 1,2 , W.M. de Vos 1,3 and E.J. Smid 1,2 1 TI Food and Nutrition, Nieuwe Kanaal, Wageningen, The Netherlands 2 NIZO food research, Kernhemseweg, Ede, The Netherlands 3 Laboratory of Microbiology, Wageningen University, Dreijenplein, Wageningen, The Netherlands Introduction Lactic acid bacteria have been used unintentionally in food fermentations for thousands of years, principally for preventing spoilage of food products like meat, vegetables, fruits and milk. Lactic acid bacteria convert carbohydrates rapidly into mainly lactic acid but also other organics acids, which cause acidification of the food product. Pro- longed cultivation of these bacteria in artisanal and indus- trial settings resulted in adaptation to these man-made niches. Analysis of the genetic diversity between two dairy strains and one plant-derived Lactococcus lactis strain showed that several genes needed for growth on plants were absent in dairy species (van Hylckama Vlieg et al. 2006; Wegmann et al. 2007). In general, lactic acid bacte- ria have multiple amino acid and vitamin auxotrophies, and it is believed that evolution on rich and complex media resulted in selection of specific auxotrophies which are closely correlated to their biotope (Bringel 1998). The lactic acid bacterium Lactobacillus plantarum WCFS1 fer- ments several types of complex substrates ranging from plant material (Tamminen et al. 2004) to liquid media Keywords folate production, genome-scale metabolic model, lactic acid bacteria, Lactobacillus plantarum, minimal medium. Correspondence Eddy J. Smid, NIZO food research, Kernhemseweg 2, PO Box 20, 6710 BA, Ede, The Netherlands. E-mail: Eddy.smid@nizo.nl Present address: A. Wegkamp, Department of Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam. 2009 ⁄ 0855: received 1 June 2009, revised 2 September 2009 and accepted 28 September 2009 doi:10.1111/j.1472-765X.2009.02752.x Abstract Aim: A medium with minimal requirements for the growth of Lactobacillus plantarum WCFS was developed. The composition of the minimal medium was compared to a genome-scale metabolic model of L. plantarum. Methods and Results: By repetitive single omission experiments, two minimal media were developed: PMM5 (true minimal medium) and PMM7 [a pseudo- minimal medium, supporting proper biomass formation of 350 mg l )1 dry weight (DW)]. The specific growth rate of L. plantarum on PMM7 was found to be 50% and 63% lower when compared to growth on established growth media (chemically defined medium and MRS, respectively). Using a genome- scale metabolic model of L. plantarum, it was predicted that PMM5 and PMM7 would not support the growth of L. plantarum. This is because the bio- synthesis of para-aminobenzoic acid (pABA) was predicted to be essential for growth. The discrepancy in simulated growth and experimental growth on PMM7 was further investigated for pABA; a molecule which plays an impor- tant role in folate production. The growth performance and folate production were determined on PMM7 in the presence and absence of pABA. It was found that a 12 000-fold reduction in folate pools exerted no influence on formation of biomass or growth rate of L. plantarum cultures when grown in the absence of pABA. Conclusion: Largely reduced folate production pools do not have an effect on the growth of L. plantarum, showing that L. plantarum makes folate in a large excess. Significance and Impact of the study: These experiments illustrate the impor- tance of combining genome-scale metabolic models with growth experiments on minimal media. Letters in Applied Microbiology ISSN 0266-8254 ª 2009 TI Food and Nutrition, Wageningen, the Netherlands Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 50 (2010) 57–64 57