BACKGROUND
lactic acid bacteria  are gram-positive bacteria that are key players in the majority of food fermentation ecosystems  <cit> .
the genus lactobacillus represents the largest group among the lab, encompassing more than  <dig> species  <cit> .
as this genus possesses a wide metabolic diversity, lactobacilli occur and have been used as functional starter cultures in a wide variety of fermented food products, including cheese, fermented plant-derived foods, fermented meats, wine and beer production, and sourdoughs  <cit> .
furthermore, lactobacilli play an important role during the cocoa bean fermentation process  <cit> .
at the onset of the cocoa bean fermentation process, a wide lab species diversity is often present .
however, only a restricted number of lactobacillus species predominate this fermentation process, consisting mainly of  strictly heterofermentative lactobacillus fermentum  and  facultative heterofermentative lactobacillus plantarum .
it has been proposed that l. plantarum is the dominant member at the onset of the cocoa bean fermentation process, while l. fermentum dominates in later stages  <cit> .
this is in accordance with the cocoa-specific functional roles that have been associated with these two species .
homolactic fermentation of cocoa pulp-bean mass carbohydrates such as glucose and fructose, resulting from the hydrolysis of sucrose present in the cocoa pulp-bean mass by pulp or yeast invertase, results in lactic acid, while heterolactic fermentation gives lactic acid, acetic acid and/or ethanol, and carbon dioxide  <cit> .
fructose and citric acid, present in the cocoa pulp, are also used as alternative external electron acceptors by heterofermentative lab, enhancing their competitiveness and resulting in the production of mannitol and succinate, or lactate and flavor-active compounds such as  <dig> -butanediol or acetoin, respectively .
to better understand the exact role of cocoa-derived microorganisms, their whole-genome sequence can be investigated, as has been done for the cocoa-specific acetobacter pasteurianus 386b strain  <cit> .
whole-genome sequencing of particular strains of the lab species l. fermentum and l. plantarum originating from other sources  has been performed previously, thereby elucidating a plethora of specific functionalities and niche adaptations.
for instance, genome sequence analysis of particular l. fermentum strains has revealed adaptations to specific host systems, such as the presence of adhesins  <cit>  and probiotic properties .
further, comparative genomics has enabled insights into the genomic features that are unique for l. fermentum strains  <cit> .
genome sequencing of particular l. plantarum strains elucidated niche adaptations  <cit> , probiotic properties , prophage occurrence , general metabolic properties , and bacteriocin production  <cit> .
therefore, it can be expected that whole-genome sequence analysis of the cocoa-derived l. fermentum  <dig> and l. plantarum  <dig> strains, isolates from a spontaneous ghanaian cocoa bean fermentation process and proposed as interesting functional starter culture strains  <cit> , will provide detailed insight into their genetic potential, metabolic capacities and functionalities as well as cocoa niche adaptations.
next to their central carbohydrate metabolism, their proteolytic system and amino acid conversion pathways have to be analysed, as these are important functionalities of lab in various food fermentation ecosystems .
RESULTS
<dig> pyrosequencing, genome sequence assembly, and annotation
<dig> pyrosequencing of the genomic dna of l. fermentum  <dig> and l. plantarum  <dig> yielded  <dig>  reads with a median length of  <dig> base pairs  and  <dig>  reads with a median length of 416 bp, respectively .
the reads obtained from the genomic dna of l. fermentum  <dig> were assembled into  <dig> contigs  with a 104-fold depth of coverage, whereas the reads obtained from the genomic dna of l. plantarum  <dig> were assembled into  <dig> contigs  with a 65-fold depth of coverage.
furthermore, the genome sequences were estimated to be  <dig>  million bp  with a guanine plus cytosine  content of  <dig>  % for l. fermentum  <dig> and  <dig>  mb with a g+c content of  <dig>  % for l. plantarum  <dig>  for l. fermentum  <dig>  these data were in accordance with other members of this species .
however, the genome sequence size of l. plantarum  <dig> was larger compared with other members of this species, generally being around  <dig>  mb  <cit> , indicating that the genome sequence of l. plantarum  <dig> encompassed one or more plasmids, which is common for this species  <cit> .
indeed, two genes encoding a plasmid replication initiator  were found, located on a  <dig> -bp  and a  <dig> -bp  contig.
also, several contigs showed high sequence identity with the plasmid-specific patric database, providing further evidence for the presence of plasmids.
by automated annotation of the contigs obtained,  <dig>  and  <dig>  protein-encoding sequences  were found in the genome sequences of l. fermentum  <dig> and l. plantarum  <dig>  respectively.
other relevant features deduced from the genome sequences are summarised in table  <dig> table  <dig> general features of the reads, contigs, and draft genome sequences of lactobacillus fermentum  <dig> and l. plantarum 80
estimated genome size a
number of rrna operonsb
aestimation according to newbler
bestimation based on contig coverage
genome architecture and genetic potential
the genome sequences of l. fermentum  <dig> and l. plantarum  <dig> did not contain virulence factors, antibiotic resistance genes, or clustered regularly interspaced short palindromic repeat  sequences, confirming their applicability for food fermentation processes.
one prophage-related genomic region was found in the genome sequence of l. fermentum  <dig>  and three such regions were found in the genome sequence of l. plantarum  <dig> .
although these regions were identified by phast analysis as ‘intact prophage’, these phage-associated regions might not be active or inducible, as has been reported before in lab  <cit> .
further, genes involved in bacteriocin biosynthesis were not found in the genome sequence of l. fermentum  <dig>  the genome sequence of l. plantarum  <dig> contained a plantaricin  locus  on a  <dig> -bp contig , comprising all genes needed for the production of this bacteriocin  <cit> .
blastn analysis of this contig using the plasmid-specific patric database revealed that this plantaricin is chromosomally encoded in l. plantarum  <dig>  this plantaricin locus was similar to that of l. plantarum wcfs <dig>  <cit> , including a transport operon , all genes encoding a two-peptide bacteriocin jk , a regulatory operon , a putative operon , and genes encoding a putative toxin-antitoxin system .
lactobacillus plantarum  <dig> did not contain the genes encoding the two-peptide bacteriocin ef , which were present in the pln locus of l. plantarum wcfs <dig>  instead, genes encoding putative proteins were found in the pln locus of l. plantarum  <dig> .
production of plantaricin jk might enable l. plantarum  <dig> to outcompete other l. plantarum species present in the cocoa bean fermentation process, as this bacteriocin inhibits mainly the species l. plantarum  <cit> .
the genome sequence of l. fermentum  <dig> contained a 14-kb exopolysaccharide biosynthesis gene cluster , including a gene encoding a dextransucrase , indicating that this strain is able to produce extracellular homopolysaccharides of the dextran type, possibly to protect itself.fig.
<dig> genetic map of the pln loci of lactobacillus plantarum  <dig> and l. plantarum wcfs <dig>  for l. plantarum  <dig>  a partial sequence of the  <dig> -bp contig encoding the pln locus  is shown.
for l. plantarum wcfs <dig>  the chromosomal region containing the pln genes lp_0400-lp_ <dig> is shown
concerning adaptation to stress conditions, both genome sequences contained genes encoding an atp synthase , which enables regulation of the internal ph  <cit> .
further, the heat-shock operon hrca-grpe-dnakj as well as several other heat-shock protein-encoding genes were found in both genome sequences .
also, cold-shock protein-encoding genes as well as genes encoding clp atpases and proteases, which are involved in stress responses  <cit> , were found in both genome sequences .
these cold-shock proteins might be part of the stress adaptation system of l. plantarum strains, as previously suggested for l. plantarum wcfs <dig>  <cit> .
finally, the genome sequence of l. plantarum  <dig> contained a kata gene  encoding a heme-dependent catalase, which protects the cell from oxidative stress  <cit> .table  <dig> mechanisms of lactobacillus fermentum  <dig> and lactobacillus plantarum  <dig> involved in stress responses.
the locus tags are depicted for each mechanism as well as the gene names when appropriate
heat-shock operon hrca-grpe-dnakj
metabolic pathway reconstruction
pathway analysis of lactobacillus fermentum 222
central carbohydrate metabolism
all genes encoding the enzymes involved in the heterolactic fermentation pathway, responsible for the production of lactic acid, acetic acid and/or ethanol, were retrieved in the genome sequence of l. fermentum  <dig> .
further, different carbohydrate transport mechanisms were found, such as phosphoenolpyruvate -dependent sugar phosphotransferase systems  and permeases.
pyruvate, originating from the heterolactic fermentation or the citrate metabolism , may be used by l. fermentum  <dig> for nad+ regeneration by production of diacetyl and/or acetoin, as the genes encoding α-acetolactate synthase , α-acetolactate decarboxylase ,  <dig> -butanediol dehydrogenase , and diacetyl reductase  were all found in the genome sequence .
alternatively, pyruvate may be converted into acetyl-coa by the pyruvate dehydrogenase complex, with acetate or ethanol as the final products.
finally, a gene encoding pyruvate oxidase  was found, enabling this strain to convert pyruvate into carbon dioxide and acetyl phosphate when oxygen is available.
this requires reactive oxygen species tolerance, for which a gene encoding a thiol peroxidase  was found.fig.
<dig> metabolic pathways of lactobacillus fermentum  <dig> and lactobacillus plantarum  <dig>  the names of the genes that were present in both genomes are depicted in blue, those that were present in l. fermentum  <dig> only are depicted in yellow, and those that were present in l. plantarum  <dig> only are depicted in grey.
metabolites involved in the homo- and heterolactic fermentation pathways are depicted in green, those involved in pyruvate metabolism are depicted in purple, those involved in citrate metabolism are depicted in black, those involved in the proteolytic system and amino acid conversion pathways are depicted in orange, those involved in the arginine deiminase pathway are depicted in red, and those involved in the respiratory machinery are depicted in dark red.
enzymes and locus tags that were associated with the gene names are listed in additional file  <dig>  dhap dihydroxyacetone phosphate, pep phosphoenolpyruvate, bcaa, branched-chain amino acid, araa aromatic amino acid, gaba γ-aminobutyric acid, tpp thiamine pyrophosphate
a citrate operon, involved in the conversion of citric acid  <cit> , was found in l. fermentum  <dig> , consisting of genes coding for a citrate lyase and its regulator , an oxaloacetate decarboxylating malate dehydrogenase , a putative citrate transporter , and an accessory protein .
in comparison with the two citrate operons that are encoded in the genome sequence of l. fermentum f- <dig>  the only l. fermentum strain for which the whole-genome sequence is available harbouring citrate operons, the citrate operon of l. fermentum  <dig> showed differences with the ones of l. fermentum f- <dig>  indeed, the first citrate operon of l. fermentum f- <dig> seemed to be incomplete, as the mae gene was missing and the second operon had an atypical citrate transporter  that was also found in l. fermentum  <dig>  <cit> , which is unlikely to be specific for citrate based on the sequence annotation of this gene.
as such, the gene encoding this transporter was neither homologous to the one present in the citrate operon of l. fermentum  <dig> nor to the one in the other citrate operon of l. fermentum f- <dig>  besides the citrate transporter present in the citrate operon, two additional genes encoding a putative citrate transporter  were found in the genome sequence of l. fermentum  <dig>  yet, not all genes encoding the enzymes to convert oxaloacetate  into succinate were found, because only genes encoding fumarase  and fumarate reductase  were present, while a gene encoding malate dehydrogenase was missing.fig.
<dig> organisation of the citrate operons in lactobacillus fermentum  <dig>  l. fermentum f- <dig>  and l. plantarum  <dig>  the appropriate genomic regions encoding the proteins involved in citrate uptake and conversion are shown for each genome sequence.
for l. fermentum f- <dig>  both citrate operons are depicted.
flanking genes outside the citrate operon are shown in black
pathways that enable the use of oxygen or glycerol as alternative external electron acceptors were not found in the genome sequence of l. fermentum  <dig>  although this strain is able to use fructose as an alternative external electron acceptor  <cit>  and the genome sequence contained a gene coding for a fructose permease , a gene encoding a mannitol dehydrogenase could not be found.
however, a fructose-specific pep-dependent pts  was present, which is associated with mannitol production from fructose in homofermentative lab  <cit> .
based on the genome sequence annotation, no further evidence was found that this transporter was involved in mannitol production.
proteolytic system and amino acid conversion pathways
the genome sequence of l. fermentum  <dig> harboured genes encoding a peptide transporter  and several amino acid atp-binding cassette  transporters specific for glutamine , methionine , and several other amino acids that could not be further specified based on the genome sequence annotation .
further, a plethora of genes encoding peptidases were retrieved, such as pepf and pepo encoding endopeptidases, pepi, pepp, pepq, and pepx encoding proline-specific peptidases, pepd and pepv encoding dipeptidases, pept encoding a tripeptidase, and pepc, pepn, and pepm encoding aminopeptidases .
the acquisition of a large variety of peptidases by lactobacillales is related to their adaptation to nutritionally rich environments  <cit> , which might also be the case for members of the cocoa bean fermentation process, as cocoa pulp consists of  <dig> – <dig>  % proteins  <cit> .
aspartate transaminase-encoding genes were found , enabling l. fermentum  <dig> to produce oxaloacetate from aspartate.
this oxaloacetate might contribute to the pyruvate metabolism through the pathway associated with citrate metabolism .
indeed, it has been shown for other lactobacillus species that diacetyl and acetoin can be produced through aspartate catabolism  <cit> .
genes encoding aspartase , fumarase , and succinate dehydrogenase  were found as well, offering an alternative pathway for aspartate catabolism .
this pathway may contribute to nad+ regeneration, and, therefore, offer a more efficient degradation of carbohydrates  <cit> .
genes encoding the transaminases arat and bcat, which catalyse the hydrolysis of branched-chain amino acids as well as aromatic amino acids, were retrieved .
the concomitant α-oxoacids produced might be further reduced to hydroxyacids by the 2-hydroxyacid dehydrogenases  present, thereby contributing to nad+ regeneration and thus allowing more efficient energy production through heterofermentation, as hypothesised before  <cit> .
genes associated with the conversion of α-oxoacids by oxidative decarboxylation into carboxylic acids or by decarboxylation into aldehydes were not found.
neither were enzymes involved in decarboxylation of histidine and aromatic amino acids found, the latter indicating that l. fermentum  <dig> is unable of biogenic amine production.
further, evidence was found for the presence of other amino acid conversion pathways, such as the occurrence of genes encoding cystathionine β-lyase  that is involved in methionine catabolism, serine deaminase  that enables the production of pyruvate and ammonia from serine, and serine hydroxymethyltransferase  that is involved in the production of the flavour compound acetaldehyde .
the genome sequence of l. fermentum  <dig> contained an operon encoding the arginine deiminase  pathway, consisting of genes encoding arginine deiminase , ornithine transcarbamoyltransferase , carbamate kinase , an arginine/ornithine antiporter , and a transaminase .
this pathway might provide l. fermentum  <dig> with additional energy, provided through amino acid conversions, as well as protection against acid stress conditions  <cit>  and has been shown to be of importance for competitiveness in food fermentation processes .
further, a gene encoding ornithine cyclodeaminase  was present, enabling direct biosynthesis of proline from ornithine, thereby producing ammonia .
the production of additional ammonia may further protect against acid stress, whereas proline can act as an osmotic protectant  <cit> .
pathway analysis of lactobacillus plantarum 80
central carbohydrate metabolism
the genome sequence of l. plantarum  <dig> contained all genes encoding the enzymes involved in the homo- and heterolactic fermentation pathways  as well as various genes encoding carbohydrate uptake systems, enabling this strain to switch between both metabolic pathways, depending on the energy source available.
metabolic pathway analysis revealed that pyruvate can be converted into acetyl-coa by a pyruvate dehydrogenase complex  or by pyruvate-formate lyase , the latter yielding formate .
further, a gene encoding pyruvate oxidase  was present, enabling l. plantarum  <dig> to convert pyruvate into carbon dioxide and acetyl phosphate under oxic conditions.
genes encoding α-acetolactate synthase  and α-acetolactate decarboxylase  were found, but diacetyl reductase- or  <dig> -butanediol dehydrogenase-encoding genes were absent, indicating that acetoin would be the only flavour compound that can be formed out of pyruvate by l. plantarum  <dig>  although  <dig> -butanediol nor acetoin were found during monoculture fermentations with l. plantarum  <dig> in cocoa pulp simulation medium for lab  <cit> .
genes associated with uptake and conversion mechanisms of citrate, such as a gene encoding citrate lyase , oxaloacetate decarboxylating malate dehydrogenase , a citrate transporter , and accessory proteins , were all retrieved in the genome sequence of l. plantarum  <dig> .
however, these were not clustered together , as genes encoding transposases , an abc transporter , a fumarase  and a fumarate reductase  were found between them.
further, a gene encoding a malate dehydrogenase  was found, which might enable l. plantarum  <dig> to use citrate as an alternative external electron acceptor by converting citrate into succinate by the reductive tricarboxylic acid cycle, next to its ability to convert citrate into pyruvate, which has been shown experimentally  <cit> .
lactobacillus plantarum  <dig> might be able to use oxygen as an alternative external electron acceptor, as a nadh-dependent oxidase-encoding gene  was found.
genes associated with the ability to use glycerol or fructose as alternative external electron acceptors were absent.
the genome sequence of l. plantarum  <dig> harboured genes encoding a respiration machinery, which is an energetically favourable metabolic pathway that leads to less oxidative and acid stress in lab  <cit> .
indeed, genes encoding a nadh-dependent dehydrogenase, a bd-type cytochrome , an atp synthase , and a heme-dependent catalase  were found .
although several l. plantarum strains possess a similar electron transport chain  <cit> , there are no experimental data indicating that l. plantarum  <dig> could perform aerobic respiration when provided with exogenous sources of heme and menaquinones.
proteolytic system and amino acid conversion pathways
genes encoding extracellular proteases were not found in the genome sequence of l. plantarum  <dig>  which seems to be a general feature for l. plantarum strains  <cit> .
genes encoding a dtpt proton motive force-dependent peptide transporter and an opp transporter  were found as well as peptidase-encoding genes, including the genes pepd, pepe, and pepv encoding dipeptidases, the genes pepn and pepc encoding aminopeptidases, the genes pepx, pepr, and pepp encoding proline-specific peptidases, and the genes pepf, pepq, pepo, and pepr encoding endopeptidases .
this elaborate peptide uptake and degradation machinery might be an important mechanism for l. plantarum  <dig> to thrive in the protein-containing fermenting cocoa pulp-bean mass.
further, several genes encoding various amino acid transporters were present, such as livabcde, enabling l. plantarum  <dig> to take up a variety of amino acids from the environment.
the genome sequence of l. plantarum  <dig> contained genes encoding an asparaginase , an aspartate transaminase , and an aspartase .
this pathway enables production of oxaloacetate and/or fumarate, which can subsequently be used for energy production and/or nad+ regeneration by the enzymes encoded in the citrate operon .
although a gene encoding glutamate decarboxylase was found , this pathway was unlikely to be operational, as the genes encoding the glutamate-γ-aminobutyric acid antiporter  and the transcriptional regulator  were not found, which are required for glutamate decarboxylation activity in other lab  <cit> .
a gene encoding the α-oxoglutarate-dependent aminotransferase bcat was found in the genome sequence of l. plantarum  <dig>  enabling the production of α-oxoacids from branched-chain amino acids.
further degradation of the α-oxoacids into hydroxyacids by this strain is feasible, as the genome sequence harboured a gene encoding a 2-hydroxyacid dehydrogenase .
enzymes involved in the conversion of α-oxoacids by oxidative decarboxylation into carboxylic acids or by decarboxylation into aldehydes were not found in the genome sequence.
further, the aromatic aminotransferase arat was present in the genome sequence, which enables l. plantarum  <dig> to transaminate aromatic amino acids.
in addition, aromatic amino acid decarboxylation enzymes were not found, indicating that l. plantarum  <dig> is not able to produce biogenic amines.
genes encoding cystathionine β-lyase  and cysteine kinase  were found in the same locus, which indicated that the methionine and cysteine catabolic pathways of l. plantarum  <dig> are linked, as has been shown previously for other lab  <cit> .
further, genes associated with the catabolism of serine and threonine, i.e., a serine deaminase  and a serine hydroxymethyltransferase , were retrieved in the genome sequence of l. plantarum  <dig> .
genes involved in histidine decarboxylation or the adi pathway were absent.
comparative genome sequence analysis
comparative genomics of lactobacillus fermentum 222
the genome sequence of l. fermentum  <dig> harboured  <dig> strain-specific genes, i.e., genes without a homolog in other known l. fermentum genome sequences .
most of these genes encoded hypothetical proteins or were disrupted, although some had a predicted function.
the strain-specific genes included two putative citrate transporters  and a gene cluster  containing a diacetyl reductase-encoding gene .
the complete cit gene cluster that was not interrupted by other genes or transposable elements  and the occurrence of two unique additional  citrate transporters might be the result of specific adaptations that enable this strain to consume citrate in an efficient way during the cocoa bean fermentation process  <cit> .
further, a gene cluster involved in amino acid uptake and catabolism, consisting of genes coding for an amino acid transporter , an aspartase , an amino acid permease , and an aminotransferase  was unique for l. fermentum  <dig>  providing evidence that amino acid conversion pathways might be an important feature for functional starter culture strains that have to improve the cocoa bean fermentation process.
the core genome of the species l. fermentum, based on eleven complete and draft genome sequences available , consisted of  <dig> genes.
the pan-genome of the species l. fermentum was found to encompass  <dig>  genes and seemed to be open, i.e., the addition of more strains in the comparative analysis would result in a larger pan-genome  <cit> .
the latter result was in agreement with the fact that this species has been found in various environments, including mammalian body sites  <cit>  and food  environments  <cit> .
a phylogenetic tree based on the core gene sequences revealed that l. fermentum  <dig> was not grouped together with any other l. fermentum strain , which might indicate its specific adaptation to the cocoa bean fermentation ecosystem.fig.
<dig> phylogenetic tree based on all core gene sequences of lactobacillus fermentum available.
multiple sequence alignments of concatenated core gene sequences were calculated using the edgar framework, including plasmid sequences.
the genome sequence of l. plantarum  <dig> was used as an outgroup
comparative genomics of lactobacillus plantarum
a total of  <dig> strain-specific genes were found in the genome sequence of l. plantarum  <dig> compared to the currently available l. plantarum genome sequences .
among these strain-specific genes was a gene cluster  consisting of the pts-encoding sorabcde genes, a dehydrogenase-encoding gene , and a gene encoding the transcriptional regulator .
in lactobacillus casei, this gene cluster is involved in transport of sorbose and subsequent production of fructose 6-phosphate, which can be channeled into the glycolytic pathway  <cit> .
this mechanism can also be used to transport fructose, as shown for l. casei and klebsiella pneumoniae  <cit> .
these genes might thus offer l. plantarum  <dig> unique features to be able to dominate the fructose-containing cocoa pulp-bean mass during cocoa bean fermentation.
furthermore, a unique cluster of four genes was found, consisting of an oxidoreductase , a ribose 5-phosphate isomerase , a fructose-specific pts , and a transcriptional regulator .
these traits underline the fructophilic character of l. plantarum  <dig> during cocoa bean fermentation processes  <cit> .
based on a comparative analysis of 25 l. plantarum genome sequences , the core- and pan-genome of this species encompassed  <dig> and  <dig>  genes, respectively.
construction of a phylogenetic tree based on core gene sequences only  revealed that two strains  were not part of a larger cluster that contained all other strains.
these two strains originated both from fermented food products  <cit> .
although the phylogenetic distance between the strains was small , the cocoa bean fermentation isolate l. plantarum  <dig> was grouped together with l. plantarum lp <dig>  an italian wine isolate  <cit> .
the latter strain possesses the adi pathway  <cit>  and is tolerant to wine fermentation conditions, such as high sulphite concentrations  <cit> .fig.
<dig> phylogenetic tree based on all core gene sequences of lactobacillus plantarum available.
multiple sequence alignments of concatenated core gene sequences were calculated using the edgar framework, including plasmid sequences.
the genome sequence of l. fermentum  <dig> was used as an outgroup
CONCLUSIONS
genomics-based pathway reconstruction of the carbohydrate metabolism and amino acid conversion mechanisms of candidate functional lab starter culture strains for the cocoa bean fermentation process revealed important insights into their lifestyle, niche adaptations, and metabolic capacities.
although some studies report on comparative genomics of lab  <cit> , this is the first extensive  genome sequence analysis of these species, revealing their core- and pan-genome.
identification of the genetic potential of cocoa-derived candidate functional starter cultures obtained from this and other studies  <cit>  in metagenomic data sets will allow further insights into the contribution of functional starter culture strains to cocoa bean fermentation processes  <cit> .
this information is crucial for an improved and rationalised selection of functional starter culture strains for the cocoa bean fermentation process.

 lactic acid bacteria are gram-positive bacteria that are key players in the majority of food fermentation ecosystems. the genus lactobacillus represents the largest group among the lab, encompassing more than species. as this genus possesses a wide metabolic diversity, lactobacilli occur and have been used as functional starter cultures in a wide variety of fermented food products, including cheese, fermented plant-derived foods, fermented meats, wine and beer production, and sourdoughs. furthermore, lactobacilli play an important role during the cocoa bean fermentation process. at the onset of the cocoa bean fermentation process, a wide lab species diversity is often present. however, only a restricted number of lactobacillus species predominate this fermentation process, consisting mainly of strictly heterofermentative lactobacillus fermentum and facultative heterofermentative lactobacillus plantarum. it has been proposed that l. plantarum is the dominant member at the onset of the cocoa bean fermentation process, while l. fermentum dominates in later stages. this is in accordance with the cocoa-specific functional roles that have been associated with these two species. homolactic fermentation of cocoa pulp-bean mass carbohydrates such as glucose and fructose, resulting from the hydrolysis of sucrose present in the cocoa pulp-bean mass by pulp or yeast invertase, results in lactic acid, while heterolactic fermentation gives lactic acid, acetic acid and/or ethanol, and carbon dioxide. fructose and citric acid, present in the cocoa pulp, are also used as alternative external electron acceptors by heterofermentative lab, enhancing their competitiveness and resulting in the production of mannitol and succinate, or lactate and flavor-active compounds such as -butanediol or acetoin, respectively. to better understand the exact role of cocoa-derived microorganisms, their whole-genome sequence can be investigated, as has been done for the cocoa-specific acetobacter pasteurianus 386b strain. whole-genome sequencing of particular strains of the lab species l. fermentum and l. plantarum originating from other sources has been performed previously, thereby elucidating a plethora of specific functionalities and niche adaptations. for instance, genome sequence analysis of particular l. fermentum strains has revealed adaptations to specific host systems, such as the presence of adhesins and probiotic properties. further, comparative genomics has enabled insights into the genomic features that are unique for l. fermentum strains. genome sequencing of particular l. plantarum strains elucidated niche adaptations, probiotic properties, prophage occurrence, general metabolic properties, and bacteriocin production. therefore, it can be expected that whole-genome sequence analysis of the cocoa-derived l. fermentum and l. plantarum strains, isolates from a spontaneous ghanaian cocoa bean fermentation process and proposed as interesting functional starter culture strains, will provide detailed insight into their genetic potential, metabolic capacities and functionalities as well as cocoa niche adaptations. next to their central carbohydrate metabolism, their proteolytic system and amino acid conversion pathways have to be analysed, as these are important functionalities of lab in various food fermentation ecosystems.  pyrosequencing, genome sequence assembly, and annotation pyrosequencing of the genomic dna of l. fermentum and l. plantarum yielded reads with a median length of base pairs and reads with a median length of 416 bp, respectively. the reads obtained from the genomic dna of l. fermentum were assembled into contigs with a 104-fold depth of coverage, whereas the reads obtained from the genomic dna of l. plantarum were assembled into contigs with a 65-fold depth of coverage. furthermore, the genome sequences were estimated to be million bp with a guanine plus cytosine content of  % for l. fermentum and  mb with a g+c content of  % for l. plantarum for l. fermentum these data were in accordance with other members of this species. however, the genome sequence size of l. plantarum was larger compared with other members of this species, generally being around  mb, indicating that the genome sequence of l. plantarum encompassed one or more plasmids, which is common for this species. indeed, two genes encoding a plasmid replication initiator were found, located on a -bp and a -bp contig. also, several contigs showed high sequence identity with the plasmid-specific patric database, providing further evidence for the presence of plasmids. by automated annotation of the contigs obtained, and protein-encoding sequences were found in the genome sequences of l. fermentum and l. plantarum respectively. other relevant features deduced from the genome sequences are summarised in table  table general features of the reads, contigs, and draft genome sequences of lactobacillus fermentum and l. plantarum 80  estimated genome size a  number of rrna operonsb aestimation according to newbler bestimation based on contig coverage genome architecture and genetic potential the genome sequences of l. fermentum and l. plantarum did not contain virulence factors, antibiotic resistance genes, or clustered regularly interspaced short palindromic repeat sequences, confirming their applicability for food fermentation processes. one prophage-related genomic region was found in the genome sequence of l. fermentum and three such regions were found in the genome sequence of l. plantarum. although these regions were identified by phast analysis as ‘intact prophage’, these phage-associated regions might not be active or inducible, as has been reported before in lab. further, genes involved in bacteriocin biosynthesis were not found in the genome sequence of l. fermentum the genome sequence of l. plantarum contained a plantaricin locus on a -bp contig, comprising all genes needed for the production of this bacteriocin. blastn analysis of this contig using the plasmid-specific patric database revealed that this plantaricin is chromosomally encoded in l. plantarum this plantaricin locus was similar to that of l. plantarum wcfs, including a transport operon, all genes encoding a two-peptide bacteriocin jk, a regulatory operon, a putative operon, and genes encoding a putative toxin-antitoxin system. lactobacillus plantarum did not contain the genes encoding the two-peptide bacteriocin ef, which were present in the pln locus of l. plantarum wcfs instead, genes encoding putative proteins were found in the pln locus of l. plantarum. production of plantaricin jk might enable l. plantarum to outcompete other l. plantarum species present in the cocoa bean fermentation process, as this bacteriocin inhibits mainly the species l. plantarum. the genome sequence of l. fermentum contained a 14-kb exopolysaccharide biosynthesis gene cluster, including a gene encoding a dextransucrase, indicating that this strain is able to produce extracellular homopolysaccharides of the dextran type, possibly to protect itself.fig. genetic map of the pln loci of lactobacillus plantarum and l. plantarum wcfs for l. plantarum a partial sequence of the -bp contig encoding the pln locus is shown. for l. plantarum wcfs the chromosomal region containing the pln genes lp_0400-lp_ is shown concerning adaptation to stress conditions, both genome sequences contained genes encoding an atp synthase, which enables regulation of the internal ph. further, the heat-shock operon hrca-grpe-dnakj as well as several other heat-shock protein-encoding genes were found in both genome sequences. also, cold-shock protein-encoding genes as well as genes encoding clp atpases and proteases, which are involved in stress responses, were found in both genome sequences. these cold-shock proteins might be part of the stress adaptation system of l. plantarum strains, as previously suggested for l. plantarum wcfs. finally, the genome sequence of l. plantarum contained a kata gene encoding a heme-dependent catalase, which protects the cell from oxidative stress.table mechanisms of lactobacillus fermentum and lactobacillus plantarum involved in stress responses. the locus tags are depicted for each mechanism as well as the gene names when appropriate heat-shock operon hrca-grpe-dnakj metabolic pathway reconstruction pathway analysis of lactobacillus fermentum 222 central carbohydrate metabolism all genes encoding the enzymes involved in the heterolactic fermentation pathway, responsible for the production of lactic acid, acetic acid and/or ethanol, were retrieved in the genome sequence of l. fermentum. further, different carbohydrate transport mechanisms were found, such as phosphoenolpyruvate -dependent sugar phosphotransferase systems and permeases. pyruvate, originating from the heterolactic fermentation or the citrate metabolism, may be used by l. fermentum for nad+ regeneration by production of diacetyl and/or acetoin, as the genes encoding α-acetolactate synthase, α-acetolactate decarboxylase, -butanediol dehydrogenase, and diacetyl reductase were all found in the genome sequence. alternatively, pyruvate may be converted into acetyl-coa by the pyruvate dehydrogenase complex, with acetate or ethanol as the final products. finally, a gene encoding pyruvate oxidase was found, enabling this strain to convert pyruvate into carbon dioxide and acetyl phosphate when oxygen is available. this requires reactive oxygen species tolerance, for which a gene encoding a thiol peroxidase was found.fig. metabolic pathways of lactobacillus fermentum and lactobacillus plantarum the names of the genes that were present in both genomes are depicted in blue, those that were present in l. fermentum only are depicted in yellow, and those that were present in l. plantarum only are depicted in grey. metabolites involved in the homo- and heterolactic fermentation pathways are depicted in green, those involved in pyruvate metabolism are depicted in purple, those involved in citrate metabolism are depicted in black, those involved in the proteolytic system and amino acid conversion pathways are depicted in orange, those involved in the arginine deiminase pathway are depicted in red, and those involved in the respiratory machinery are depicted in dark red. enzymes and locus tags that were associated with the gene names are listed in additional file dhap dihydroxyacetone phosphate, pep phosphoenolpyruvate, bcaa, branched-chain amino acid, araa aromatic amino acid, gaba γ-aminobutyric acid, tpp thiamine pyrophosphate a citrate operon, involved in the conversion of citric acid, was found in l. fermentum, consisting of genes coding for a citrate lyase and its regulator, an oxaloacetate decarboxylating malate dehydrogenase, a putative citrate transporter, and an accessory protein. in comparison with the two citrate operons that are encoded in the genome sequence of l. fermentum f- the only l. fermentum strain for which the whole-genome sequence is available harbouring citrate operons, the citrate operon of l. fermentum showed differences with the ones of l. fermentum f- indeed, the first citrate operon of l. fermentum f- seemed to be incomplete, as the mae gene was missing and the second operon had an atypical citrate transporter that was also found in l. fermentum, which is unlikely to be specific for citrate based on the sequence annotation of this gene. as such, the gene encoding this transporter was neither homologous to the one present in the citrate operon of l. fermentum nor to the one in the other citrate operon of l. fermentum f- besides the citrate transporter present in the citrate operon, two additional genes encoding a putative citrate transporter were found in the genome sequence of l. fermentum yet, not all genes encoding the enzymes to convert oxaloacetate into succinate were found, because only genes encoding fumarase and fumarate reductase were present, while a gene encoding malate dehydrogenase was missing.fig. organisation of the citrate operons in lactobacillus fermentum l. fermentum f- and l. plantarum the appropriate genomic regions encoding the proteins involved in citrate uptake and conversion are shown for each genome sequence. for l. fermentum f- both citrate operons are depicted. flanking genes outside the citrate operon are shown in black pathways that enable the use of oxygen or glycerol as alternative external electron acceptors were not found in the genome sequence of l. fermentum although this strain is able to use fructose as an alternative external electron acceptor and the genome sequence contained a gene coding for a fructose permease, a gene encoding a mannitol dehydrogenase could not be found. however, a fructose-specific pep-dependent pts was present, which is associated with mannitol production from fructose in homofermentative lab. based on the genome sequence annotation, no further evidence was found that this transporter was involved in mannitol production. proteolytic system and amino acid conversion pathways the genome sequence of l. fermentum harboured genes encoding a peptide transporter and several amino acid atp-binding cassette transporters specific for glutamine, methionine, and several other amino acids that could not be further specified based on the genome sequence annotation. further, a plethora of genes encoding peptidases were retrieved, such as pepf and pepo encoding endopeptidases, pepi, pepp, pepq, and pepx encoding proline-specific peptidases, pepd and pepv encoding dipeptidases, pept encoding a tripeptidase, and pepc, pepn, and pepm encoding aminopeptidases. the acquisition of a large variety of peptidases by lactobacillales is related to their adaptation to nutritionally rich environments, which might also be the case for members of the cocoa bean fermentation process, as cocoa pulp consists of –  % proteins. aspartate transaminase-encoding genes were found, enabling l. fermentum to produce oxaloacetate from aspartate. this oxaloacetate might contribute to the pyruvate metabolism through the pathway associated with citrate metabolism. indeed, it has been shown for other lactobacillus species that diacetyl and acetoin can be produced through aspartate catabolism. genes encoding aspartase, fumarase, and succinate dehydrogenase were found as well, offering an alternative pathway for aspartate catabolism. this pathway may contribute to nad+ regeneration, and, therefore, offer a more efficient degradation of carbohydrates. genes encoding the transaminases arat and bcat, which catalyse the hydrolysis of branched-chain amino acids as well as aromatic amino acids, were retrieved. the concomitant α-oxoacids produced might be further reduced to hydroxyacids by the 2-hydroxyacid dehydrogenases present, thereby contributing to nad+ regeneration and thus allowing more efficient energy production through heterofermentation, as hypothesised before. genes associated with the conversion of α-oxoacids by oxidative decarboxylation into carboxylic acids or by decarboxylation into aldehydes were not found. neither were enzymes involved in decarboxylation of histidine and aromatic amino acids found, the latter indicating that l. fermentum is unable of biogenic amine production. further, evidence was found for the presence of other amino acid conversion pathways, such as the occurrence of genes encoding cystathionine β-lyase that is involved in methionine catabolism, serine deaminase that enables the production of pyruvate and ammonia from serine, and serine hydroxymethyltransferase that is involved in the production of the flavour compound acetaldehyde. the genome sequence of l. fermentum contained an operon encoding the arginine deiminase pathway, consisting of genes encoding arginine deiminase, ornithine transcarbamoyltransferase, carbamate kinase, an arginine/ornithine antiporter, and a transaminase. this pathway might provide l. fermentum with additional energy, provided through amino acid conversions, as well as protection against acid stress conditions and has been shown to be of importance for competitiveness in food fermentation processes. further, a gene encoding ornithine cyclodeaminase was present, enabling direct biosynthesis of proline from ornithine, thereby producing ammonia. the production of additional ammonia may further protect against acid stress, whereas proline can act as an osmotic protectant. pathway analysis of lactobacillus plantarum 80 central carbohydrate metabolism the genome sequence of l. plantarum contained all genes encoding the enzymes involved in the homo- and heterolactic fermentation pathways as well as various genes encoding carbohydrate uptake systems, enabling this strain to switch between both metabolic pathways, depending on the energy source available. metabolic pathway analysis revealed that pyruvate can be converted into acetyl-coa by a pyruvate dehydrogenase complex or by pyruvate-formate lyase, the latter yielding formate. further, a gene encoding pyruvate oxidase was present, enabling l. plantarum to convert pyruvate into carbon dioxide and acetyl phosphate under oxic conditions. genes encoding α-acetolactate synthase and α-acetolactate decarboxylase were found, but diacetyl reductase- or -butanediol dehydrogenase-encoding genes were absent, indicating that acetoin would be the only flavour compound that can be formed out of pyruvate by l. plantarum although -butanediol nor acetoin were found during monoculture fermentations with l. plantarum in cocoa pulp simulation medium for lab. genes associated with uptake and conversion mechanisms of citrate, such as a gene encoding citrate lyase, oxaloacetate decarboxylating malate dehydrogenase, a citrate transporter, and accessory proteins, were all retrieved in the genome sequence of l. plantarum. however, these were not clustered together, as genes encoding transposases, an abc transporter, a fumarase and a fumarate reductase were found between them. further, a gene encoding a malate dehydrogenase was found, which might enable l. plantarum to use citrate as an alternative external electron acceptor by converting citrate into succinate by the reductive tricarboxylic acid cycle, next to its ability to convert citrate into pyruvate, which has been shown experimentally. lactobacillus plantarum might be able to use oxygen as an alternative external electron acceptor, as a nadh-dependent oxidase-encoding gene was found. genes associated with the ability to use glycerol or fructose as alternative external electron acceptors were absent. the genome sequence of l. plantarum harboured genes encoding a respiration machinery, which is an energetically favourable metabolic pathway that leads to less oxidative and acid stress in lab. indeed, genes encoding a nadh-dependent dehydrogenase, a bd-type cytochrome, an atp synthase, and a heme-dependent catalase were found. although several l. plantarum strains possess a similar electron transport chain, there are no experimental data indicating that l. plantarum could perform aerobic respiration when provided with exogenous sources of heme and menaquinones. proteolytic system and amino acid conversion pathways genes encoding extracellular proteases were not found in the genome sequence of l. plantarum which seems to be a general feature for l. plantarum strains. genes encoding a dtpt proton motive force-dependent peptide transporter and an opp transporter were found as well as peptidase-encoding genes, including the genes pepd, pepe, and pepv encoding dipeptidases, the genes pepn and pepc encoding aminopeptidases, the genes pepx, pepr, and pepp encoding proline-specific peptidases, and the genes pepf, pepq, pepo, and pepr encoding endopeptidases. this elaborate peptide uptake and degradation machinery might be an important mechanism for l. plantarum to thrive in the protein-containing fermenting cocoa pulp-bean mass. further, several genes encoding various amino acid transporters were present, such as livabcde, enabling l. plantarum to take up a variety of amino acids from the environment. the genome sequence of l. plantarum contained genes encoding an asparaginase, an aspartate transaminase, and an aspartase. this pathway enables production of oxaloacetate and/or fumarate, which can subsequently be used for energy production and/or nad+ regeneration by the enzymes encoded in the citrate operon. although a gene encoding glutamate decarboxylase was found, this pathway was unlikely to be operational, as the genes encoding the glutamate-γ-aminobutyric acid antiporter and the transcriptional regulator were not found, which are required for glutamate decarboxylation activity in other lab. a gene encoding the α-oxoglutarate-dependent aminotransferase bcat was found in the genome sequence of l. plantarum enabling the production of α-oxoacids from branched-chain amino acids. further degradation of the α-oxoacids into hydroxyacids by this strain is feasible, as the genome sequence harboured a gene encoding a 2-hydroxyacid dehydrogenase. enzymes involved in the conversion of α-oxoacids by oxidative decarboxylation into carboxylic acids or by decarboxylation into aldehydes were not found in the genome sequence. further, the aromatic aminotransferase arat was present in the genome sequence, which enables l. plantarum to transaminate aromatic amino acids. in addition, aromatic amino acid decarboxylation enzymes were not found, indicating that l. plantarum is not able to produce biogenic amines. genes encoding cystathionine β-lyase and cysteine kinase were found in the same locus, which indicated that the methionine and cysteine catabolic pathways of l. plantarum are linked, as has been shown previously for other lab. further, genes associated with the catabolism of serine and threonine, i.e., a serine deaminase and a serine hydroxymethyltransferase, were retrieved in the genome sequence of l. plantarum. genes involved in histidine decarboxylation or the adi pathway were absent. comparative genome sequence analysis comparative genomics of lactobacillus fermentum 222 the genome sequence of l. fermentum harboured strain-specific genes, i.e., genes without a homolog in other known l. fermentum genome sequences. most of these genes encoded hypothetical proteins or were disrupted, although some had a predicted function. the strain-specific genes included two putative citrate transporters and a gene cluster containing a diacetyl reductase-encoding gene. the complete cit gene cluster that was not interrupted by other genes or transposable elements and the occurrence of two unique additional citrate transporters might be the result of specific adaptations that enable this strain to consume citrate in an efficient way during the cocoa bean fermentation process. further, a gene cluster involved in amino acid uptake and catabolism, consisting of genes coding for an amino acid transporter, an aspartase, an amino acid permease, and an aminotransferase was unique for l. fermentum providing evidence that amino acid conversion pathways might be an important feature for functional starter culture strains that have to improve the cocoa bean fermentation process. the core genome of the species l. fermentum, based on eleven complete and draft genome sequences available, consisted of genes. the pan-genome of the species l. fermentum was found to encompass genes and seemed to be open, i.e., the addition of more strains in the comparative analysis would result in a larger pan-genome. the latter result was in agreement with the fact that this species has been found in various environments, including mammalian body sites and food environments. a phylogenetic tree based on the core gene sequences revealed that l. fermentum was not grouped together with any other l. fermentum strain, which might indicate its specific adaptation to the cocoa bean fermentation ecosystem.fig. phylogenetic tree based on all core gene sequences of lactobacillus fermentum available. multiple sequence alignments of concatenated core gene sequences were calculated using the edgar framework, including plasmid sequences. the genome sequence of l. plantarum was used as an outgroup comparative genomics of lactobacillus plantarum a total of strain-specific genes were found in the genome sequence of l. plantarum compared to the currently available l. plantarum genome sequences. among these strain-specific genes was a gene cluster consisting of the pts-encoding sorabcde genes, a dehydrogenase-encoding gene, and a gene encoding the transcriptional regulator. in lactobacillus casei, this gene cluster is involved in transport of sorbose and subsequent production of fructose 6-phosphate, which can be channeled into the glycolytic pathway. this mechanism can also be used to transport fructose, as shown for l. casei and klebsiella pneumoniae. these genes might thus offer l. plantarum unique features to be able to dominate the fructose-containing cocoa pulp-bean mass during cocoa bean fermentation. furthermore, a unique cluster of four genes was found, consisting of an oxidoreductase, a ribose 5-phosphate isomerase, a fructose-specific pts, and a transcriptional regulator. these traits underline the fructophilic character of l. plantarum during cocoa bean fermentation processes. based on a comparative analysis of 25 l. plantarum genome sequences, the core- and pan-genome of this species encompassed and genes, respectively. construction of a phylogenetic tree based on core gene sequences only revealed that two strains were not part of a larger cluster that contained all other strains. these two strains originated both from fermented food products. although the phylogenetic distance between the strains was small, the cocoa bean fermentation isolate l. plantarum was grouped together with l. plantarum lp an italian wine isolate. the latter strain possesses the adi pathway and is tolerant to wine fermentation conditions, such as high sulphite concentrations.fig. phylogenetic tree based on all core gene sequences of lactobacillus plantarum available. multiple sequence alignments of concatenated core gene sequences were calculated using the edgar framework, including plasmid sequences. the genome sequence of l. fermentum was used as an outgroup  genomics-based pathway reconstruction of the carbohydrate metabolism and amino acid conversion mechanisms of candidate functional lab starter culture strains for the cocoa bean fermentation process revealed important insights into their lifestyle, niche adaptations, and metabolic capacities. although some studies report on comparative genomics of lab, this is the first extensive genome sequence analysis of these species, revealing their core- and pan-genome. identification of the genetic potential of cocoa-derived candidate functional starter cultures obtained from this and other studies in metagenomic data sets will allow further insights into the contribution of functional starter culture strains to cocoa bean fermentation processes. this information is crucial for an improved and rationalised selection of functional starter culture strains for the cocoa bean fermentation process. 
further, in-depth functional analysis revealed the capacities of these strains associated with carbohydrate and amino acid metabolism, such as the ability to use alternative external electron acceptors, the presence of an extended pyruvate metabolism, and the occurrence of several amino acid conversion pathways. in-depth genome sequence analysis of the candidate functional starter culture strains l. fermentum and l. plantarum revealed their metabolic capacities, niche adaptations and functionalities that enable them to dominate the cocoa bean fermentation process. lactobacillus fermentum and lactobacillus plantarum isolates from a spontaneous ghanaian cocoa bean fermentation process, proved to be interesting functional starter culture strains for cocoa bean fermentations. a comparative genome sequence analysis using publicly available genome sequences of strains of the species l. plantarum and l. fermentum revealed unique features of both strains studied. lactobacillus fermentum is a thermotolerant strain, able to dominate the fermentation process, thereby converting citrate and producing mannitol. lactobacillus plantarum is an acid-tolerant and facultative heterofermentative strain that is competitive during cocoa bean fermentation processes. in this study, whole-genome sequencing and comparative genome analysis was used to investigate the mechanisms of these strains to dominate the cocoa bean fermentation process. further, these results offered insights into the cocoa bean fermentation ecosystem as a whole and will facilitate the selection of appropriate starter culture strains for controlled cocoa bean fermentation processes. indeed, l. fermentum possessed genes encoding additional citrate transporters and enzymes involved in amino acid conversions, whereas l. plantarum is the only member of this species that harboured a gene cluster involved in uptake and consumption of fructose and/or sorbose.
 lactobacillus fermentum and lactobacillus plantarum isolates from a spontaneous ghanaian cocoa bean fermentation process, proved to be interesting functional starter culture strains for cocoa bean fermentations. lactobacillus fermentum is a thermotolerant strain, able to dominate the fermentation process, thereby converting citrate and producing mannitol. lactobacillus plantarum is an acid-tolerant and facultative heterofermentative strain that is competitive during cocoa bean fermentation processes. in this study, whole-genome sequencing and comparative genome analysis was used to investigate the mechanisms of these strains to dominate the cocoa bean fermentation process.  through functional annotation and analysis of the high-coverage contigs obtained through pyrosequencing, plantaricin production was predicted for l. plantarum for l. fermentum genes encoding a complete arginine deiminase pathway were attributed. further, in-depth functional analysis revealed the capacities of these strains associated with carbohydrate and amino acid metabolism, such as the ability to use alternative external electron acceptors, the presence of an extended pyruvate metabolism, and the occurrence of several amino acid conversion pathways. a comparative genome sequence analysis using publicly available genome sequences of strains of the species l. plantarum and l. fermentum revealed unique features of both strains studied. indeed, l. fermentum possessed genes encoding additional citrate transporters and enzymes involved in amino acid conversions, whereas l. plantarum is the only member of this species that harboured a gene cluster involved in uptake and consumption of fructose and/or sorbose.  in-depth genome sequence analysis of the candidate functional starter culture strains l. fermentum and l. plantarum revealed their metabolic capacities, niche adaptations and functionalities that enable them to dominate the cocoa bean fermentation process. further, these results offered insights into the cocoa bean fermentation ecosystem as a whole and will facilitate the selection of appropriate starter culture strains for controlled cocoa bean fermentation processes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords lactobacillus fermentumlactobacillus plantarumcocoa bean fermentation pyrosequencingfunctional starter culturesresearch council of the vrije universiteit brusselᅟde vuyst luc http://dx.doi.org/ /501100003130fonds wetenschappelijk onderzoekᅟweckx stefan http://dx.doi.org/ /501100003130hercules foundationᅟde vuyst luc http://dx.doi.org/ /501100003132agentschap voor innovatie door wetenschap en technologie101089illeghems koen issue-copyright-statement© the author 2015 