Identifying Microbial Community Structure and Ecological Trends Contributing to Variation in Post-Fermented Theobroma cacao using Shotgun Metagenomics

The fermentation of Theobroma cacao is the most important factor in the final quality and flavor of their resulting chocolate product. While cacao is grown in over 50 countries worldwide, there is limited information identifying how the varied environment may impact the growth of microbial diversity during fermentation. The purpose of this study is to identify microbial communities of post-fermented cocoa beans from a wide range of geographic locations. We investigated correlations between their abundances and the environment in which they are fermented. Genomic DNA was extracted from 25 samples of dried, fermented cocoa beans and shotgun metagenomic sequencing was performed using the Illumina NovaSeq X Plus Sequencer. Taxonomic results recorded 13,445 species Operational Taxonomic Units (OTUs) and 3,376 genus OTUs throughout all samples, including high abundances for Acetobacter pasteurianus, Acetobacter oryzoeni, and Alkalihalobacillus clausii. OTU data was used to calculate several alpha and beta diversity metrics. Results show high levels of microbial diversity across samples in South America, and lower levels of diversity across samples from Asia. Canonical Correlation Analysis was conducted to identify correlations between Alpha Diversity and environmental factors, in which precipitation and mean annual temperature had correlations above 0.95. Generalized Linear Models were performed to identify correlations between environmental factors and microbe species, in which 8 species correlated with precipitation, 5 species correlated with mean annual temperature, 4 species correlated with elevation, 7 species correlated with soil pH, and 6 species correlated with distance from the equator. The results of this study give insight into the correlation between microbial communities and environmental factors, providing cacao farmers the opportunity to optimize their fermentation and produce a favorable product.