Metagenomic Analysis of Potential Microbes in Microbial Fermentation and Flavour Formation of Ghanaian Cocoa Beans
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Date
2015-08
Authors
Agyirifo, Daniel Sakyi
Journal Title
Journal ISSN
Volume Title
Publisher
Kenyatta University
Abstract
Production of cocoa contributes significantly to poverty reduction and generates
huge foreign exchange earnings for national development. Fermentation of cocoa
beans is the most critical stage in production of cocoa products such as chocolates
and its derivatives. Chocolate acquires its desired colour and flavour during
fermentation through the activities of a variety of microorganisms including
yeasts, lactic acid and acetic acid bacteria. There is a limited understanding of the
complex response of microbial diversity during cocoa bean fermentation.
Traditional methods for elucidating microbial diversity are time consuming and
they focus on the detection of a single microbe or a panel of microbes. However,
molecular methods such as whole genome shotgun metagenomics approach
sequences DNA directly from the environment bypassing the need for culturing.
The aim of the present study was to investigate microbial communities in the
cocoa bean fermentation heap using a culture-independent approach to elucidate
microbial diversity, structure, functional annotation and mapping unto metabolic
pathways. Genomic DNA was purified from four samples of cocoa bean
fermentation heap using a modified CTAB method followed by library
preparation. Sequence data was generated on Illumina Hiseq 2000 paired-end
technology (Macrogen Inc). Normalisation and quality control was performed
using FASTQC vO.ll.2. Paired-end sequencing of libraries generated 46
gigabytes (GB) (462,844,468 reads) of data. Quality filtering retained
373,289,474 reads (approximately 38 GB). Sequences were assembled by
iterative deBruijn graph algorithm using SOAPdenov02 with an N50 of 2,017
base pairs. Bacterial taxa assignment, rarefaction analysis, and the Shannon
alpha-diversity index were calculated using the Metagenome Rapid Annotation
using Subsystem Technology (MG-RAST) server. Taxonomic analysis based on
genes predicted from the metagenome identified a high percentage of bacteria
(88.75%), yeast (7.19%), and bacteriophages (1%) from the cocoa microbiome.
The cocoa microbiome was dominated by the following genera: Lactobacillus
(20%), Gluconacetobacter (9%), Acetobacter (7%) and Gluconobacter (6%). The
mean species diversity, measured by Shannon alpha-diversity index, was
estimated at 142.81. Assignment of metagenomic sequences to SEED database
categories at 97% sequence similarity identified a genetic profile characteristic
of heterotrophic lactic acid fermentation of carbohydrates and aromatic amino
acids. Metabolism of aromatic compounds, amino acids and their derivatives and
carbohydrates represented 0.7%, 6.2% and 9.6% respectively of the microbiome.
Genome coverage of the mapped reads was observed at 2.0X, 1.7X and 0.2X for
the recruited A. pasteurianus, Gluconobacter oxydans and L. plantarum
respectively. Overall, these results provide insights into the cocoa microbiome,
identifying fermentation processes carried out broadly by complex microbial
communities and metabolic pathways encoding aromatic compounds such as
phenylacetaldehdye, butanediol, acetoin, theobromine and luteolin that are
required for flavour and aroma production. The results obtained will help develop
targeted inoculations to produce desired chocolate flavour or targeted metabolic
pathways for the selection of microbes for good aroma and flavour compounds formation
Description
A thesis submitted in fulfilment of the requirements for the award of the degree of doctor of philosophy in the department of biochemistry and biotechnology in the school of pure and applied sciences of Kenyatta University, August 2015