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Applied and Environmental Microbiology, March 2007, p. 1809-1824, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.02189-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana{triangledown}

Nicholas Camu,1 Tom De Winter,1 Kristof Verbrugghe,1 Ilse Cleenwerck,2 Peter Vandamme,2 Jemmy S. Takrama,3 Marc Vancanneyt,2 and Luc De Vuyst1*

Research Group of Industrial Microbiology and Food Biotechnology, Department of Applied Biological Sciences and Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium,1 BCCM/LMG Bacteria Collection and Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium,2 Cocoa Research Institute of Ghana, New Tafo, Akim, Ghana3

Received 18 September 2006/ Accepted 20 January 2007

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as "Weissella ghanaensis," was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named "Acetobacter senegalensis" (A. tropicalis-like) and "Acetobacter ghanaensis" (A. syzygii-like).

* Corresponding author. Mailing address: Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium. Phone: 32 2 6293245. Fax: 32 2 6292720. E-mail: ldvuyst{at}vub.ac.be.

{triangledown} Published ahead of print on 2 February 2007.

Applied and Environmental Microbiology, March 2007, p. 1809-1824, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.02189-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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