Microbiota Dynamics Associated with Environmental Conditions and Potential Roles of Cellulolytic Communities in Traditional Chinese Cereal Starter Solid-State Fermentation

Abstract

Traditional Chinese solid-state fermented cereal starters contain highly complex microbial communities and enzymes. Very little is known, however, about the microbial dynamics related to environmental conditions, and cellulolytic communities have never been proposed to exist during cereal starter fermentation. In this study, we performed Illumina MiSeq sequencing combined with PCR-denaturing gradient gel electrophoresis to investigate microbiota, coupled with clone library construction to trace cellulolytic communities in both fermentation stages. A succession of microbial assemblages was observed during the fermentation of starters. Lactobacillales and Saccharomycetales dominated the initial stages, with a continuous decline in relative abundance. However, thermotolerant and drought-resistant Bacillales, Eurotiales, and Mucorales were considerably accelerated during the heating stages, and these organisms dominated until the end of fermentation. Enterobacteriales were consistently ubiquitous throughout the process. For the cellulolytic communities, only the genera Sanguibacter, Beutenbergia, Agrobacterium, and Erwinia dominated the initial fermentation stages. In contrast, stages at high incubation temperature induced the appearance and dominance of Bacillus, Aspergillus, and Mucor. The enzymatic dynamics of amylase and glucoamylase also showed a similar trend, with the activities clearly increased in the first 7 days and subsequently decreased until the end of fermentation. Furthermore, β-glucosidase activity continuously and significantly increased during the fermentation process. Evidently, cellulolytic potential can adapt to environmental conditions by changes in the community structure during the fermentation of starters.

FIG 1

Dynamics of physicochemical characteristics during the cereal starter fermentation process. (a) Changes in incubation temperature and corresponding moisture content. Stage 3, heating stage; stage 4, high-temperature stage; stages 5 and 6, cooling stages. (b) Change in pH. (c) Changes in starch and reducing sugar (calculated as glucose) contents.

FIG 2

Dynamics of amylase and glucoamylase (a) as well as CMCase and β-glucosidase (b) activities during the cereal starter fermentation process.

FIG 3

Dynamics of 16S/18S rRNA gene (a) and β-glucosidase gene (b) abundances during the cereal starter fermentation process. Specific primers were used in qPCR: GH1, family 1 β-glucosidase genes from bacteria and fungi; GH3E, family 3 β-glucosidase genes from fungi.

FIG 4

Analysis of cellulolytic communities targeting β-glucosidase genes by clone library construction. (a to c) Distribution and relative abundance of cellulolytic communities targeting family 1 β-glucosidase genes from bacteria and fungi at the order (a) and genus (b) levels and NJ phylogenetic tree based on partial family 1 β-glucosidase genes from bacteria and fungi and reference sequences from GenBank (c). ⬥, specific dominant species on day 1; ●, specific dominant species on day 3; ■, specific dominant species on days 1 and 3; ▼, specific dominant species from day 7 to 30; ▲, specific dominant species from day 13 to 30. (d to f) Distribution and relative abundance of cellulolytic communities targeting family 3 β-glucosidase genes from fungi at the order (d) and genus (e) levels (family 3 β-glucosidase genes from the fungi cannot be amplified on days 1 and 3) and NJ phylogenetic tree based on partial family 3 β-glucosidase genes from fungi and reference sequences from GenBank (f). ♦, specific dominant species on day 7; ■, specific dominant species from day 19 to 30; ⭑, specific dominant species from day 7 to 30.

FIG 5

Dynamics of relative abundances of the major bacterial orders (a) and genera (b) belonging to Enterobacteriales, Lactobacillales, and Bacillales and fungal orders (c) and genera (d) belonging to Saccharomycetales, Eurotiales, and Mucorales in samples as obtained by Illumina MiSeq sequencing targeting the V4 regions of the 16S and 18S rRNA genes, respectively. The abundance is presented as of percentage of total effective bacterial sequences. The abundances of bacterial “other” orders and genera were <1% in all samples; a more detailed overview of bacterial “other” orders is shown in Fig. S8 in the supplemental material. Fungal “other” orders were hardly detected in samples, and the abundances of fungal “other” genera were 0.04% in all samples; a more detailed overview of bacterial “other” genera is shown in Fig. S9 in the supplemental material.

FIG 6

PCoA/PCA of microbial communities in samples. (a) Weighted UniFrac distance PCoA of bacterial communities in samples as obtained by Illumina MiSeq sequencing; (b) Bray-Curtis distance PCoA of fungal communities in samples as obtained by Illumina MiSeq sequencing; (c and d) PCA of DGGE profiles of partial 16S rRNA genes from bacteria (c) and 18S rRNA genes from fungi (d) in samples.