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Applied and Environmental Microbiology, February 2007, p. 825-837, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.02378-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Microbial Community Structure and Dynamics of Dark Fire-Cured Tobacco Fermentation ^,

Michele Di Giacomo,¹ Marianna Paolino,¹ Daniele Silvestro,¹ Giovanni Vigliotta,² Francesco Imperi,^3,4 Paolo Visca,^3,4 Pietro Alifano,² and Dino Parente^1*

British American Tobacco Italia, via Cinthia, 80126 Naples, Italy,¹ Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università di Lecce, via Monteroni, 73100 Lecce, Italy,² Dipartimento di Biologia, Università Roma Tre, viale Marconi, 00146 Rome, Italy,³ Unità di Microbiologia Molecolare, Istituto Nazionale per le Malattie Infettive I.R.C.C.S. "Lazzaro Spallanzani," via Portuense, 00149 Rome, Italy⁴

Received 9 October 2006/ Accepted 20 November 2006

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.

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* Corresponding author. Mailing address: British American Tobacco, R&D, via Cinthia 45, 80126 Naples, Italy. Phone: 39 081674051. Fax: 39 081674090. E-mail: dparente{at}unina.it.

Published ahead of print on 1 December 2006.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, February 2007, p. 825-837, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.02378-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.