Phylogenetic Analysis of a Spontaneous Cocoa Bean Fermentation Metagenome Reveals New Insights into Its Bacterial and Fungal Community Diversity

Abstract

This is the first report on the phylogenetic analysis of the community diversity of a single spontaneous cocoa bean box fermentation sample through a metagenomic approach involving 454 pyrosequencing. Several sequence-based and composition-based taxonomic profiling tools were used and evaluated to avoid software-dependent results and their outcome was validated by comparison with previously obtained culture-dependent and culture-independent data. Overall, this approach revealed a wider bacterial (mainly γ-Proteobacteria) and fungal diversity than previously found. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. In addition, bacteriophage-related sequences were found. The bacterial diversity depended partially on the methods used, as composition-based methods predicted a wider diversity than sequence-based methods, and as classification methods based solely on phylogenetic marker genes predicted a more restricted diversity compared with methods that took all reads into account. The metagenomic sequencing analysis identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing species. Also, the presence of occasional members of the cocoa bean fermentation process was revealed (such as Erwinia tasmaniensis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides, and Oenococcus oeni). Furthermore, the sequence reads associated with viral communities were of a restricted diversity, dominated by Myoviridae and Siphoviridae, and reflecting Lactobacillus as the dominant host. To conclude, an accurate overview of all members of a cocoa bean fermentation process sample was revealed, indicating the superiority of metagenomic sequencing over previously used techniques.

Effect of Input Production against Quality of Cocoa Beans

“…This is due to the fact that the quality of beans greatly affects both the quality of final products and market price for cocoa and cocoa-related commodities. High quality cocoa beans must also be fermented, cleaned and dried (Sulistyowati and Wahyudi, 1999;Schwan and Wheals, 2004;Illeghems et al, 2012;Trognitz et al, 2013). Furthermore, physical characteristics, chemical and organoleptic factors affect the quality of cocoa beans (Wahyudi et al, 2008).…”

Oxidation of Metabolites Highlights the Microbial Interactions and Role of Acetobacter pasteurianus during Cocoa Bean Fermentation

“…Their succession during cocoa bean fermentation is important for the required metabolic activities in the fermenting cocoa pulp-bean mass, although the occurrence of these microbial communities may overlap (2,3,(5)(6)(7). Yeast species involved in cocoa bean fermentation mainly encompass Saccharomyces cerevisiae and Hanseniaspora guilliermondii, H. opuntiae, and H. uvarum, which are responsible for pectinolysis of the cocoa pulp and the production of ethanol from carbohydrates (mainly glucose) (8)(9)(10)(11)(12)(13)(14)(15). LAB species involved in cocoa bean fermentation, in particular Lactobacillus fermentum, consume carbohydrates (glucose and fructose) and citric acid and produce lactic acid, acetic acid, and mannitol (6,7,12,(16)(17)(18)(19).…”

“…Yeast species involved in cocoa bean fermentation mainly encompass Saccharomyces cerevisiae and Hanseniaspora guilliermondii, H. opuntiae, and H. uvarum, which are responsible for pectinolysis of the cocoa pulp and the production of ethanol from carbohydrates (mainly glucose) (8)(9)(10)(11)(12)(13)(14)(15). LAB species involved in cocoa bean fermentation, in particular Lactobacillus fermentum, consume carbohydrates (glucose and fructose) and citric acid and produce lactic acid, acetic acid, and mannitol (6,7,12,(16)(17)(18)(19). Concerning AAB, mainly Acetobacter species are found, which oxidize ethanol (produced by yeast) and lactic acid (produced by LAB) into acetic acid (6,7,12,15,17,18,20,21).…”

Virtual metagenome reconstruction from 16S rRNA gene sequences

“…We downloaded metagenome data of the cocoa bean fermentation samples 21 from NCBI SRA (http://www.ncbi.nlm.nih.gov/ sra/). We assembled the reads using Celera Assembler version 7.0 (http://www.jcvi.…”

“…Recently, Papalexandratou et al 20 published a metagenome analysis of cocoa bean fermentation samples determined by pyrosequencing technique. A DGGE analysis of the same samples was also previously performed by the same group 21 . We therefore compared the virtual metagenome reconstructed by our method and the real metagenome from the same cocoa bean fermentation samples.…”