Lactobacillus ghanensis sp. nov., a motile lactic acid bacterium isolated from Ghanaian cocoa fermentations Dennis S. Nielsen, 1 Ulrich Schillinger, 2 Charles M. A. P. Franz, 2 Jose ´ Bresciani, 3 Wisdom Amoa-Awua, 4 Wilhelm H. Holzapfel 2 and Mogens Jakobsen 1 Correspondence Dennis S. Nielsen dn@life.ku.dk 1 Department of Food Science, Food Microbiology, Centre for Advanced Food Studies (LMC), Faculty of Life Sciences, Copenhagen University, Rolighedsvej 30, 1958 Frederiksberg C, Denmark 2 Institute for Hygiene and Toxicology, Federal Research Centre for Nutrition and Food, Karlsruhe, Germany 3 Department of Ecology, Faculty of Life Sciences, Copenhagen University, Copenhagen, Denmark 4 CSIR – Food Research Institute, Accra, Ghana Three Gram-positive, catalase-negative, motile, rod-shaped strains, designated L486, L489 T and L499, were isolated from fermenting cocoa. These organisms produced DL-lactic acid from glucose without gas formation. Ammonia was not produced from arginine. Acid was produced from amygdalin, D-cellobiose, aesculin, D-fructose, D-glucose, D-galactose, D-mannitol, D-mannose, N-acetylglucosamine, L-rhamnose, sucrose, salicin and D-trehalose. The cell walls contained peptidoglycan of the D-meso-diaminopimelic acid type. A 16S rRNA gene sequence analysis revealed that the isolates belong phylogenetically to the genus Lactobacillus and are closely related to Lactobacillus nagelii, Lactobacillus vini and Lactobacillus satsumensis. Low DNA–DNA reassociation values were obtained between the isolates and the phylogenetically closest neighbours. On the basis of the genetic and phenotypic results, the isolates are considered to represent a novel species, for which the name Lactobacillus ghanensis is proposed. The type strain is L489 T (=DSM 18630 T =CCUG 53453 T ). Cocoa beans, the principal raw material of chocolate, have to be fermented, dried and roasted to produce the character- istic cocoa flavour and taste. The fermentation of cocoa is a microbiologically complex process involving the activities of yeasts, lactic acid bacteria and acetic acid bacteria (Schwan & Wheals, 2004). During an investigation of the micro-organisms involved in the fermentation of cocoa beans, a number of isolates with unusual properties were isolated from MRS agar and tentatively identified as Lacto- bacillus species (Nielsen et al., 2007). This study presents the morphological, biochemical and molecular characterization of three of these isolates, designated L486, L489 T and L499. Strains L486, L489 T , L499 and the reference strains Lacto- bacillus nagelii DSM 13675 T , Lactobacillus satsumensis DSM 16230 T and Lactobacillus vini DSM 20605 T were grown in MRS broth (Merck) at 30 u C for 2–3 days; for long-term storage, 20 % glycerol was added and the cultures were stored at 280 u C. The cell morphologies of cultures grown overnight in MRS broth (30 u C) were determined using phase-contrast micro- scopy and scanning electron microscopy. For the latter, 30 ml culture diluted 10-fold with sterile MilliQ water was filtered through a polycarbonate filter (pore size, 0.2 mm), exposed to osmium vapour for 1 h and coated with gold– palladium. Cells were observed in a FEI Quanta 200 scan- ning electron microscope at 15 kV. The cells were rod- shaped (0.7–0.861.4–2.5 mm), occurring either singly, in pairs or in short chains of three to four cells (Fig. 1). The cells were observed to be highly motile under the phase- contrast microscope. Scanning electron microscopy showed the presence of peritrichous flagella (Fig. 1). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains L486, L489 T and L499 are DQ867003, DQ523489 and DQ867004, respectively. Maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showing the phylogenetic position of strains L486, L489 T and L499 within the L. casei cluster are available as supplementary figures with the online version of this article. 1468 64811 G 2007 IUMS Printed in Great Britain International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1468–1472 DOI 10.1099/ijs.0.64811-0