Changes in physicochemical properties and microbial community succession during leaf stacking fermentation

Abstract

Leaf stacking fermentation involves enzymatic actions of many microorganisms and is an efficient and environmentally benign process for degrading macromolecular organic compounds. We investigated the dynamics of metabolite profiles, bacterial and fungal communities and their interactions during fermentation using cigar leaves from three geographic regions. The results showed that the contents of total sugar, reducing sugar, starch, cellulose, lignin, pectin, polyphenol and protein in cigar tobacco leaves was significantly decreased during fermentation. Notably, the furfural, neophytadiene, pyridine, benzyl alcohol, geranylacetone, 3-hydroxy-2-butanone, N-hexanal, 3-Methyl-1-butanol and 2,3-pentanedione were important features volatile aroma compounds during fermentation. The α-diversity of fungi and bacteria initially increased and then decreased during fermentation. An analysis of variance showed that microbial diversity was influenced by fermentation stages and growing locations, in which the all stages had greater impacts on α- and β-diversity than all regions. Microbiome profiling had identified several core bacteria including Sphingomonas, Bacillus, Staphylococcus, Pseudomonas, Ralstonia, Massilia and Fibrobacter. Fungal biomarkers included Aspergillus, Penicillium, Fusarium, Cladosporium and Trichomonascus. Interestingly, the molecular ecological networks showed that the core taxa had significant correlations with metabolic enzymes and physicochemical properties; bacteria and fungi jointly participated in the carbohydrate and nitrogen compound degrading and volatile aroma compound chemosynthesis processes during fermentation. These studies provide insights into the coupling of material conversion and microbial community succession during leaf fermentation.

Keywords: Cigar Tobacco leaf; Fermentation; Microbial community, physicochemical property, material conversion.

Fig. 1

Overview of the cigar tobacco leaf fermentation processing experiments

Fig. 2

Changes in conventional chemical components and physical properties in cigar tobacco leaves during fermentation. The values in the Figure are the mean value of four biological replicates sourced from three locations (n = 12, P < 0.05)

Fig. 3

Multivariate statistical analysis of volatile aroma compounds (VACs) during fermentation of cigar tobacco leaves. (A) Score scatter plot for the principal component analysis (PCA) model (PE). (B) Hierarchical clustering analysis and heatmap visualization of VAC profiles at different fermentation stages of PE cigar tobacco leaves. (C) Important characteristics of different locations (DH, PE and LC) before and after fermentation identified by OPLS-DA. The colored boxes on the right indicate the relative concentrations of the VACs in each group under study. (D) The potential markers responsible for the aroma differences among DH, PE and LC. The screening criteria were VIP > 1 and FC > 1.

Fig. 4

The changing trend of fungal and bacterial alpha diversity (Shannon index) during fermentation (A). The PCA graphs of the fungal community and the bacterial community at different fermentation stages and from different locations (B)

Fig. 5

The relative abundance of core microbiota at different fermentation stages. The top 10 relative abundances are shown. A, B, and C represent the relative abundance of core bacterial communities in PE, LC, and DH, respectively. D, E, and F represent the relative abundance of core fungal communities in PE, LC, and DH, respectively

Fig. 6

The core taxa can distinguish the fermentation stages of cigar tobacco leaves. Classification of random forest models of the fermentation stage of the core taxa of PE bacteria (A) and fungi (B). The important features (top 20) based on Mean Decrease Gini (MDG) of random forest models of the core taxa of PE bacteria (C) and fungi (D)

Fig. 7

Network analysis based on the cooccurrence of volatile components (A, B), physicochemical properties (C, D), metabolic enzymes (E, F) and the bacterial and fungal communities. Purple vee nodes represent volatile components. Grey ellipse nodes represent microbial members. Direct connections between nodes indicate strong correlations (Pearson correlation coefficient, P < 0.05). Red lines represent positive interactions between nodes, and blue lines represent negative interactions. The sizes of vee nodes represent the interconnected degree. The sizes of the circle nodes represent the average relative abundances of bacteria and fungi