Microbial community structure and dynamics of dark fire-cured tobacco fermentation

Abstract

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.

FIG. 1.

Microbiological profile of tobacco fermentation as determined by plate counts of major microbial groups. Symbols: ▪, total aerobic bacteria; ⧫, endospore-forming bacteria; •, lactic acid bacteria; ▴, yeasts. Shuffling of the tobacco bulk was performed on days 5, 10, and 15, as shown by arrows.

FIG. 2.

DGGE analysis of the eubacterial community during tobacco fermentation. DGGE fingerprints are of the V3 region of the 16S rRNA gene amplified with primers 341-FGC and 518-R from DNA samples extracted at different times, as indicated. The formamide-urea denaturing gradient ranged from 30% to 55%. Bands excised and successfully identified by sequencing (Table 3) are reported as continuous lines and sequentially numbered from top to bottom. Dotted lines show the bands for which the reamplification step failed. Shuffling of the tobacco bulk was performed on days 5, 10, and 15.

FIG. 3.

SSCP analysis of the microbial community during tobacco fermentation. (A) Profile with the Bacteria-specific primer pair Com1-F/Com2-R. (B) Profile with the Eukarya-specific primer pair FUN18S1-F/FUN18S2-R. The day of sampling is indicated above each lane. Arrows mark the position of sequentially numbered bands excised and identified by DNA sequencing (Table 3). The four groups of bands TOB-B-S7 to -9, TOB-B-S10 to -12, TOB-B-S13 to -15, and TOB-B-S16 to -18 (A, right) could not be resolved by SSCP, and the corresponding sequences were determined by RFLP analysis as detailed in the text. B, left lane, shows the profile obtained with DNA extracted from D. hansenii TOB-Y7 cultures (Table 5), used as an internal control.

FIG. 4.

Phylogenetic relationships inferred from the alignment of partial 16S rRNA gene sequences (nt 123 to 1333 relative to the E. coli rrnB gene; see Materials and Methods for details) of 29 Bacillaceae isolated from fermenting tobacco and 34 reference strains of representative species belonging to the genera Bacillus, Brevibacillus, Geobacillus, Paenibacillus, Virgibacillus, and Lactobacillus. GenBank accession numbers of reference sequences are reported. The T superscript stands for type strain. The tree was constructed using the Kimura two-parameter algorithm and the neighbor-joining method. The bar indicates 1% sequence divergence. Bootstrap values (expressed as percentages of 1,000 replications) are reported at each node. Species are abbreviated as follows: B., Bacillus; Br., Brevibacillus; G., Geobacillus; P., Paenibacillus; V., Virgibacillus; L., Lactobacillus.