International Journal of Food Microbiology 394 (2023) 110163

Contents lists available at ScienceDirect

International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

New insights into the role of key microorganisms and wooden barrels
during lambic beer fermentation and maturation
Louise Vermote a, 1, Jonas De Roos a, 1, Margo Cnockaert b, Peter Vandamme b, Stefan Weckx a,
Luc De Vuyst a, *
a
Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Department of Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050
Brussels, Belgium
b
Laboratory for Microbiology, Department of Biochemistry and Microbiology, Ghent University, K.L. Ledganckstraat 35, B-9000 Ghent, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: Belgian lambic beers are still produced through traditional craftsmanship. They rely on a spontaneous fermen­
Lambic beer tation and maturation process that is entirely carried out in wooden barrels. The latter are used repetitively and
Spontaneous fermentation may introduce some batch-to-batch variability. The present systematic and multiphasic study dealt with two
Wooden barrels
parallel lambic beer productions carried out in nearly identical wooden barrels making use of the same cooled
Shotgun metagenomic sequencing
wort. It encompassed a microbiological and metabolomic approach. Further, a taxonomic classification and
Metagenome-assembled genomes
Functional analysis metagenome-assembled genome (MAG) investigation was based on shotgun metagenomics. These investigations
Acetobacter lambici provided new insights into the role of these wooden barrels and key microorganisms for this process. Indeed,
besides their role in traditionality, the wooden barrels likely helped in establishing the stable microbial
ecosystem of lambic beer fermentation and maturation by acting as an inoculation source of the necessary mi­
croorganisms, thereby minimizing batch-to-batch variations. They further provided a microaerobic environment,
which aided in achieving the desirable succession of the different microbial communities for a successful lambic
beer production process. Moreover, these conditions prevented excessive growth of acetic acid bacteria and,
therefore, uncontrolled production of acetic acid and acetoin, which may lead to flavor deviations in lambic beer.
Concerning the role of less studied key microorganisms for lambic beer production, it was shown that the
Acetobacter lambici MAG contained several acid tolerance mechanisms toward the harsh environment of maturing
lambic beer, whereas genes related to sucrose and maltose/maltooligosaccharide consumption and the glyox­
ylate shunt were absent. Further, a Pediococcus damnosus MAG possessed a gene encoding ferulic acid decar­
boxylase, possibly contributing to 4-vinyl compound production, as well as several genes, likely plasmid-based,
related to hop resistance and biogenic amine production. Finally, contigs related to Dekkera bruxellensis and
Brettanomyces custersianus did not possess genes involved in glycerol production, emphasizing the need for
alternative external electron acceptors for redox balancing.

1. Introduction
Beer productions carried out on an industrial scale generally make
use of yeast starter cultures to produce stable and consistent end-
products (Bokulich and Bamforth, 2013). However, certain beers are
still produced through a spontaneous fermentation and maturation
process, such as particular acidic ales. Belgium is particularly known for
its traditional lambic and lambic-based beers, which are produced
without the intended inoculation of yeasts or bacteria (Bongaerts et al.,
2021; Bouchez and De Vuyst, 2022; De Roos and De Vuyst, 2019;
Pothakos et al., 2016; Spitaels et al., 2017; Verachtert and Derdelinckx,
2005). These Belgian acidic beers get increasingly popular worldwide as
trademarks of traditional craftsmanship. They possess a unique flavor
profile, which is composed of lactic acid, acetic acid, ethyl esters (in
particular ethyl lactate and ethyl acetate), acetoin, and phenolic com­
pounds (in particular 4-ethylphenol and 4-ethylguaiacol) that are
important for the fresh acidic, sharp acidic, fruity, buttery, and Brett-
flavor notes, respectively (Bongaerts et al., 2021; Bouchez and De

* Corresponding author.
E-mail address: luc.de.vuyst@vub.be (L. De Vuyst).
1
Equal contribution

https://doi.org/10.1016/j.ijfoodmicro.2023.110163
Received 6 October 2022; Received in revised form 24 February 2023; Accepted 26 February 2023
Available online 6 March 2023
0168-1605/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
L. Vermote et al.
Vuyst, 2022; De Keersmaecker, 1996; Dysvik et al., 2020; Pothakos
et al., 2016; Steensels et al., 2015).
Traditional Belgian lambic beers are produced through the sponta­
neous fermentation of an aqueous mixture of barley malt, unmalted
wheat, and aged dry hops in horizontal wooden barrels (Bongaerts et al.,
2021; De Keersmaecker, 1996; De Roos and De Vuyst, 2019; Spitaels
et al., 2017). This fermentation and maturation process can proceed up
to three years and is characterized by a microbial succession, acting
during four different phases and comprising different yeast and bacterial
species, in particular Saccharomyces cerevisiae, Saccharomyces kudriav­
zevii, Acetobacter lambici, Pediococcus damnosus, Dekkera bruxellensis,
and/or Brettanomyces custersianus (Bongaerts et al., 2021; De Roos et al.,
2018a, 2018b; Spitaels et al., 2017; Van Oevelen et al., 1977; Verachtert
and Iserentant, 1995). The lambic beer wort that was first boiled is
cooled down in an open coolship overnight, which allows its inoculation
with microorganisms present in the surrounding air. This is followed by
fermentation and maturation of the cooled wort in wooden barrels
(casks or foeders), which further serve the traditionality of the process
(Bongaerts et al., 2021; De Roos et al., 2019). Wooden casks are involved
in the maturation of several alcoholic beverages, such as (port)wine,
whisky and cider, to allow some oxygen ingress as well as flavor for­
mation by extracting typical wood compounds, such as polyphenols and
tannins (Canas et al., 2016; del Alamo-Sanza and Nevares, 2014; Del
Campo et al., 2003; Guzzon et al., 2011; White et al., 2017). However, it
remains largely unknown why wooden barrels are traditionally used
during lambic beer production (Spitaels et al., 2017). Moreover, wooden
surfaces display a microbial risk, as they are difficult to sanitize due to
their physical inertness and porosity. Alternatively, it has been shown
that the resident microbiota, present on the interior barrel surfaces,
plays an inoculation and functional role during spontaneous lambic beer
productions (Bongaerts et al., 2021; De Roos et al., 2019; Oelofse et al.,
2008; Spitaels et al., 2017). For instance, P. damnosus, D. bruxellensis,
and Dekkera anomala, which are considered as key microorganisms for
successful lambic beer productions, have been isolated from the interior
surfaces of wooden lambic beer barrels and studied intensively (Bon­
gaerts et al., 2021; Bouchez and De Vuyst, 2022; Spitaels et al., 2015).
The role of air-borne acetic acid bacteria (AAB), such as Acetobacter
lambici, is less studied. This AAB species was originally isolated from a
maturing lambic beer wort and has not been found in other niches up to
now (Spitaels et al., 2014a, 2014b). Although the whole-genome
sequence of A. lambici has been published (Sombolestani et al., 2020),
no in-depth investigation of its genetic potential was performed so far.
Further, high-throughput, partial 16S rRNA gene amplicon
sequencing of whole-community DNA from swab samples of the inside
wooden surfaces of lambic beer barrels after extensive barrel cleaning
shows the presence of fermentation- and maturation-related microor­
ganisms (De Roos et al., 2019). Since the same wooden barrels are used
several times for lambic beer batch productions and are only cleaned
superficially with high-pressurized water in between consecutive pro­
duction batches, these fermentation- and maturation-related microor­
ganisms presumably originate from previous lambic beer productions
(De Roos et al., 2019; Spitaels et al., 2015). Besides cleaning with high-
pressurized water, sulphur dioxide is commonly used as a sanitation
technique for the barrels (Barata et al., 2013; De Roos et al., 2019;
Guzzon et al., 2011; Oelofse et al., 2008). As it is known that some yeast
species enter a reversible, viable but non-culturable (VBNC) state after
sulphur dioxide treatments, it is likely that some of these microorgan­
isms survive the entire cleaning procedure, whether or not in a VBNC
state, and act as an additional inoculation source, besides the house
microbiota of the brewery air and brewery equipment (Bongaerts et al.,
2021; Bokulich et al., 2012, 2015; De Roos et al., 2019; Spitaels et al.,
2015).
A straightforward procedure to assess all microorganisms present,
including VBNC ones, encompasses culture-independent, DNA-based
analyses. Until now, only one study applied amplicon-based high-
throughput sequencing of whole-community DNA during a fermentation
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International Journal of Food Microbiology 394 (2023) 110163
and maturation process of lambic beer wort that was carried out in a
single cask (De Roos et al., 2018a). This study targeted both bacteria and
fungi and allowed microbial identifications at genus level. In addition, a
shotgun metagenomic analysis of the same fermentation and maturation
process has allowed mapping of the microbial diversity at species level.
This further indicated the functional potential of the microorganisms
found, allowing to link substrate consumption and metabolite produc­
tion to specific microbial groups or species during lambic beer produc­
tion (De Roos et al., 2020). Whereas these two studies aimed to use state-
of-the-art DNA-based methodologies to investigate the microbiota
involved in the fermentation and maturation of lambic beer wort in a
single wooden barrel, it is unclear which variability the use of different
wooden barrels of the same type add to lambic beer production pro­
cesses originating from the same coolship batch.
The present study aimed at a further determination of the influence
of individual wooden barrels on lambic beer production processes.
Therefore, two parallel lambic beer productions using nearly identical
wooden barrels of wine origin were carried out in a Belgian traditional
lambic brewery and were started using wort from the same coolship
batch. These productions were monitored temporally by means of a
systematic and multiphasic analysis approach. This consisted of a high-
throughput microbiological analysis, in casu molecular identification of
isolates picked from selective agar media, and shotgun metagenomics of
whole-community DNA, in combination with an extended metabolite
target analysis. This study further examined the genetic potential of
A. lambici, P. damnosus, D. bruxellensis, and B. custersianus.
2. Materials and methods
2.1. Lambic beer production and sampling
In April 2015, two lambic beer production processes were started in a
Belgian, traditional lambic brewery, located in the Senne river valley
southwest of Brussels. Lambic beer wort of 12.5◦ P was prepared ac­
cording to the brewer’s recipe (consisting of water, barley malt,
unmalted wheat, and overaged hop), manually acidified with lactic acid
after wort boiling, and cooled overnight in a coolship open to the sur­
rounding air. The wort was then chilled to 4 ◦ C and transferred to two
nearly identical 620-L oak casks of the same type (further referred to as
casks 1 and 2) for fermentation and maturation, hence representing
biological duplicates. The oak casks originated from a wine producer
and were already used several times to produce lambic beer in the
brewery. Before their use, the oak casks were cleaned and treated as
described previously (De Roos et al., 2018b). The casks were not closed
airtightly and were located next to each other in a cellar at ambient
temperature (ranging from 12 ◦ C to 21 ◦ C due to seasonal effects). These
two parallel lambic beer productions were monitored as a function of
time by withdrawing samples of 100 mL or 200 mL (metagenomic
samples) from the cooled wort before its transfer to the wooden casks,
further referred to as the time point 0 h, and from the fermenting wort
and maturing beer after 1 h; 1 and 3 days; 1, 2, 6, 9, and 16 weeks; and 6,
12 (time point with volumetric adjustment of the liquid levels in the
casks), 18, 24, and 30 months. Aseptic sampling was performed through
the cork-plugged bunghole of the wooden casks at the middle of the
fermenting wort and maturing beer, as described previously (De Roos
et al., 2018b). Part of the samples were chilled; another part was frozen
at − 20 ◦ C. The chilled samples were further handled for microbiological
plating and to obtain cell-free supernatants for metabolite target anal­
ysis, as described previously (De Roos et al., 2018b). The frozen samples
served for metagenomic DNA extraction.
2.2. Determination of temperature, pH, and apparent attenuation
The temperature, pH, and apparent attenuation were measured on
site, immediately after sampling, as described previously (De Roos et al.,
2018b).
L. Vermote et al.
2.3. Analysis of the microbial community dynamics and species diversity
2.3.1. Culture-dependent analysis
2.3.1.1. Selective plating and culturing for colony enumeration and iso­
lation. Four selective agar media supplemented with the appropriate
antibiotics were used for colony enumeration. This was based on dilu­
tion series of the original samples. The counts were expressed as log of
colony-forming units (CFU)/mL. The agar media also served for colony
isolation (for subsequent identification) of the presumptive lactic acid
bacteria (LAB; modified de Man-Rogosa-Sharpe or mMRS agar medium),
AAB (modified deoxycholate-mannitol-sorbitol or mDMS agar medium),
yeasts (yeast extract-peptone-glucose or YPG agar medium), and
cycloheximide-resistant yeasts (YPG agar medium supplemented with
cycloheximide or YPGc agar medium), as described previously (Papal­
exandratou et al., 2013; De Roos et al., 2018a, 2018b). Additionally,
100 μL of each dilution was plated on violet red-bile-glucose (VRBG)
agar medium (Oxoid, Basingstoke, Hampshire, UK), supplemented with
5 ppm of amphotericin B and 200 ppm of cycloheximide, for the
enumeration of presumptive enterobacteria during the first days of
fermentation after aerobic incubation of the plates at 30 ◦ C for 7 days.
2.3.1.2. Dereplication and identification of microbial isolates by MALDI-
TOF MS. The microbial isolates were sub-cultivated twice on their
respective agar media for dereplication and identification purposes. The
resulting third-generation colonies were cultivated in their respective
liquid media, stored at − 80 ◦ C, and further used for matrix-assisted laser
desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)
fingerprinting, as described previously (De Roos et al., 2018b). Minor
modifications included that the final cell pellets were resuspended in 40
μL of 70 % (v/v) formic acid (Merck, Darmstadt, Germany) and vortexed
thoroughly. Also, 40 μL of acetonitrile (Merck) was added and the
contents were mixed. After centrifugation (21,000 ×g, 3 min, 4 ◦ C), 1 μL
of these solutions was spotted in duplicate on a MBT Biotarget 96
stainless steel plate (Bruker, Billerica, MA, USA) and overlaid with 1 μL
of matrix solution, consisting of 5 mg/mL of α-cyano-4-hydroxycin­
namic acid in water:acetonitrile:trifluoroacetic acid (48:50:2). The mass
spectra were measured by means of a Microflex MALDI-TOF mass
spectrometer (Bruker) using built-in software, and the spectra of indi­
vidual isolates were dereplicated into clusters using BioNumerics
(version 7.5; Applied Maths, Sint-Martens-Latem, Belgium). Represen­
tative isolates from each cluster were identified through comparative
gene sequence analysis, as described previously (De Roos et al., 2018a).
2.3.2. Culture-independent analysis
2.3.2.1. Metagenomic DNA extraction. Cell pellets obtained from the
samples taken at 0 h, 3 days; 1, 6, 9, and 16 weeks; and 18 and 30
months were used for metagenomic DNA extraction, which was based on
enzymatic lysis, chemical treatment and mechanical disruption, as
described previously (Vermote et al., 2018), with some modifications.
After the lysis steps, a phenol/chloroform/isoamyl alcohol (Merck) pu­
rification was applied, followed by an RNase A treatment on the aqueous
phase (55 U; Thermo Fisher Scientific, Waltham, MA, USA) at 37 ◦ C for
10 min. The DNA was further purified using a DNeasy Blood and Tissue
Kit (Qiagen, Hilden, Germany), following the manufacturer’s in­
structions and applying an elution with 100 μL of nuclease-free water
(VWR International, Darmstadt, Germany). The DNA purity was
assessed with a NanoDrop 2000 spectrophotometer and the concentra­
tion was measured with a Qubit 2.0 fluorometer using a Qubit dsDNA HS
Assay Kit (all from Thermo Fisher Scientific).
2.3.2.2. Shotgun metagenomic sequencing. The metagenomic DNA
extracted was used for the construction of DNA fragment libraries, with
an intended fragment length of 350 bp, as described previously
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International Journal of Food Microbiology 394 (2023) 110163
(Vermote et al., 2018). The DNA fragment libraries were sequenced
using an Ion Torrent Personal Genome Machine (PGM) with an Ion PGM
Hi-Q View Sequencing Kit (Thermo Fisher Scientific). For each of the 15
samples sequenced, one Ion 316 Chip v2 BC (Thermo Fisher Scientific)
was used, following the manufacturer’s instructions, which resulted in
15 data sets. All 15 metagenomic data sets are accessible at the European
Nucleotide Archive of the European Bioinformatics Institute (ENA/EBI;
Hixton, UK) under the study accession number PRJEB53155.
2.3.2.3. Bioinformatics analysis
2.3.2.3.1. Metagenomic sequence reads quality trimming. The quality
of the metagenomic sequence reads obtained was assessed using FastQC
(version 0.10.1; Andrews, 2018), followed by a quality trimming step
with prinseq-lite (version 0.20.2; Schmieder and Edwards, 2011), as
described previously (Vermote et al., 2018). This resulted in a set of
50,819,736 quality-trimmed metagenomic sequence reads (MSRs), ac­
counting for 11.7 Gbp, ranging from 3,030,740 to 3,864,756 MSRs and
from 641.4 to 1016.9 Mbp per sample. All bioinformatic analysis steps
described below were performed on all these MSR data sets using the
same databases.
2.3.2.3.2. Taxonomic classification at genus level. The taxonomic
composition of all samples was assessed in a software- and database-
independent manner (Illeghems et al., 2012), using four classification
tools, namely blastn (Altschul et al., 1990), DIAMOND (Buchfink et al.,
2015), Kaiju (Menzel et al., 2016), and Kraken 2 (Wood et al., 2019), as
described previously (Verce et al., 2021). The results of Kraken 2 will be
further referred to as Kraken 2-nt for the classification at nucleotide
level and Kraken 2-aa for the classification at amino acid level. All an­
alyses were performed with databases constructed or downloaded
before the reclassification of the Lactobacillus genus (Zheng et al., 2020).
2.3.2.3.3. Taxonomic classification at species level based on fragment
recruitment plotting. Fragment recruitment plotting (FRP) was per­
formed as described previously (Verce et al., 2019), using an in-house
database containing a representative genome sequence (if available) of
each species of the genera detected in at least one of the samples with at
least one taxonomic classification method and with a minimum relative
abundance of 0.1 %. This in-house database was extended with a
representative genome sequence (if available) of species of the genera
found with the culture-dependent analysis and the genera detected in a
previous metagenomics-based study on a lambic beer production from
the same brewery (De Roos et al., 2020).
2.3.2.3.4. Statistical analysis. Rstudio software (Rstudio Team,
2020) was used for all statistical analyses, using the tidyverse (Wickham
et al., 2019), ggrepel (Slowikowski, 2020), vegan (Oksanen et al., 2019),
and RVAideMemoire (Hervé, 2021) packages. A data set based on the
relative abundance of species obtained by FRP was used for the statis­
tical analyses. Principal component analysis (PCA) was performed to
identify patterns between the wort sample before transfer to the casks
and the time-dependent fermenting wort/maturing beer samples of cask
1 and cask 2. Intra-sample diversity (alpha-diversity) was assessed by
calculating the Simpson (diversity) and Pielou (evenness) indices
(Mouillot and Leprêtre, 1999) and inter-sample diversity (beta-di­
versity) was assessed to determine the differences between casks 1 and 2
by permutational multivariate analysis of variance (PERMANOVA),
based on Bray-Curtis dissimilarity scores (Anderson, 2017).
2.3.2.3.5. Co-assembly of metagenomes, contig binning, and contig
annotation. The 15 MSR data sets were co-assembled using MEGAHIT
(version 1.2.9; Li et al., 2015) with a minimum contig length set at 1000
bp. The resulting set of contigs was imported into anvi’o (version 6.2;
Eren et al., 2015), only considering contigs of at least 2500 bp. Each
MSR data set was mapped to those contigs using Bowtie 2 to build up a
profile database (version 2.3.5.1; Langmead and Salzberg, 2012). The
taxonomic classification of the contigs was determined by classifying the
predicted genes from anvi’o using Kaiju by means of the database
mentioned above used for taxonomic analysis. The contigs were binned
L. Vermote et al.
using CONCOCT (version 1.1.0; Alneberg et al., 2014). The bins ob­
tained were imported into anvi’o and manually refined based on the
sequence composition and differences in coverage, reported complete­
ness, and contamination statistics. After refinement, only bins with a
high completion (> 75 %) and low contamination level (< 10 %) were
considered and the taxonomy assigned was checked with minimap2
(version 2.11; Li, 2018), using the database used for FRP.
The most complete metagenome-assembled genome (MAG), namely
that of A. lambici, was compared with the genome of A. lambici LMG
27439T [National Center for Biotechnology Information (NCBI) acces­
sion number GCF_011516885.1; Sombolestani et al., 2020] to calculate
the average nucleotide identity (ANI) value using OrthoANI (Lee et al.,
2016) with the default settings. Contigs belonging to bins assigned to
bacterial genera were annotated with Prokka (version 1.14.6; Seemann,
2014), using the options “metagenome” and “adding gene features”, and
with an e-value cut-off of 1 × 10− 20. Contigs belonging to bins assigned
to fungal genera were annotated with Maker2 (version 2.31.10; Holt and
Yandell, 2011), as described previously (Verce et al., 2021). A targeted
search among the high completion bins and bins derived thereof during
a manual refinement process was performed to find metabolic pathways
or genes relevant for lambic beer production. If a particular gene was not
found, a sequence alignment was performed manually, using an avail­
able amino acid sequence for the particular gene, to possibly link hy­
pothetical proteins to a function.
2.4. Metabolite target analysis of the substrate and metabolite dynamics
The cell-free supernatants were used to determine substrate and
metabolite concentrations in the fermenting wort and maturing beer. All
samples were both prepared and analyzed in triplicate.
2.4.1. Determination of simple carbohydrate and maltooligosaccharide
concentrations.
High-performance anion exchange chromatography coupled to
pulsed amperometric detection (HPAEC-PAD) with internal standardi­
zation was applied, making use of an ICS3000 chromatograph (Dionex,
Sunnyvale, CA, USA), equipped with a Carbopac™ PA10 column (Dio­
nex) to determine the concentrations of fructose, glucose, maltose, and
sucrose, or a Carbopac™ PA100 column (Dionex) to determine the
concentrations of maltotriose, maltotetraose, maltopentaose, malto­
hexaose, and maltoheptaose, as described previously (De Roos et al.,
2018a, 2018b).
2.4.2. Determination of ethanol and short-chain fatty acid concentrations
High-performance liquid chromatography with refractive index
detection (HPLC-RI), applying external calibration, was applied to
determine the concentrations of ethanol, acetic acid, propionic acid, and
butyric acid, as described previously (De Roos et al., 2018a, 2018b).
Therefore, a Waters Alliance 2695 HPLC chromatograph (Waters, Mil­
ford, MA, USA), equipped with an ICSep ICE ORH-801 column (Trans­
genomic North America, Omaha, NE, USA) and coupled to a W410 RI
detector (Waters), was used.
2.4.3. Determination of lactic acid stereoisomer concentrations
HPLC with ultraviolet detection (HPLC-UV) and external calibration
was applied to determine the concentrations of D-lactic acid and L-lactic
acid, as described previously (De Roos et al., 2018a). Therefore, a Wa­
ters Alliance 2695 HPLC chromatograph (Waters), equipped with a
Shodex Orpak CRX-853 column (Showa Denko, Tokyo, Japan) and
coupled to a UV detector operating at 253 nm (Waters), was used.
2.4.4. Determination of volatile organic compound concentrations
Gas chromatography with flame ionization detection (GC-FID),
applying internal standardization, was applied to determine the con­
centrations of 2,3-butanediol, 2,3-butanedione, acetoin, ethyl acetate,
ethyl lactate, isoamyl acetate, (iso)butyric acid, (iso)valeric acid, and
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International Journal of Food Microbiology 394 (2023) 110163
methyl-1-butanol, as described previously (De Roos et al., 2018a,
2018b). Therefore, a Focus gas chromatograph (Interscience, Breda, The
Netherlands), equipped with a Stabilwax-DA column (Restek, Belle­
fonte, PA, USA) coupled to a FID-80 detector (Interscience), was used.
2.4.5. Determination of other organic acid concentrations
Ultra-performance liquid chromatography with tandem mass spec­
trometric detection (UPLC-MS/MS), applying external calibration, was
applied to determine the concentrations of citric acid, gluconic acid,
malic acid, and succinic acid, as described previously (De Roos et al.,
2018a, 2018b). Therefore, an Acquity system chromatograph (Waters),
equipped with a HSS T3 column (Waters) and coupled to a triple
quadrupole (TQ) tandem mass spectrometer with a Zspray™ electro­
spray ionization source in negative ionization mode (Waters), was used.
2.4.6. Determination of biogenic amine concentrations
UPLC-MS/MS, using multiple reaction monitoring and internal
standardization, was applied to determine the concentrations of agma­
tine, cadaverine, histamine, 2-phenylethylamine, putrescine, trypt­
amine, and tyramine, as described previously (De Roos et al., 2018a).
Therefore, an Acquity chromatograph, equipped with a HSS T3 column
coupled to a TQ tandem mass spectrometer with a Zspray™ electrospray
ionization source used in positive ionization mode (Waters), was used.
2.4.7. Statistical analysis
A Spearman correlation analysis (p < 0.05) was performed between
the metabolites measured and the main microbial species detected with
FRP, using the Hmisc package (version 4.6–0; Harrell, 2021). The
calculated correlation was visualized and hierarchically clustered using
the ComplexHeatmap package (version 2.6.2; Gu et al., 2016).
3. Results
3.1. Course of temperature, pH, and apparent attenuation
Two nearly identical oak casks of the same type were filled with
lambic beer wort coming from the same brew with a temperature, pH,
and apparent attenuation of 13.7 ◦ C, 4.3, and 12.5◦ P, respectively. The
fermenting wort and maturing beer displayed comparable temperature,
pH, and apparent attenuation profiles in the two casks (Fig. 1A). The
temperature inside the casks followed the seasonal changes of the
temperature in the cellar. During the first week of fermentation, the pH
of the fermenting wort in both casks decreased from 4.3 to 3.9, after
which the pH remained constant. From week 6 onwards, the pH
continued to drop to values below 3.6. The apparent attenuation of the
fermenting wort rapidly decreased during the first six weeks of
fermentation, followed by a slow decrease later on in the production
process.
3.2. Culture-dependent microbial community dynamics and species
diversity
The lambic beer production processes in both nearly identical oak
casks examined displayed four phases, namely an initial or enterobac­
terial fermentation phase, a main or alcoholic fermentation phase, an
acidification phase, and a maturation phase (Figs. 1B, C, and 2). The
presence of colony counts of presumptive LAB (mMRS), enterobacteria
(VRBG), AAB (mDMS), yeasts (YPG), and cycloheximide-resistant yeasts
(YPGc) before the transfer of the cooled wort to the oak casks indicated
that the starting lambic beer wort was spontaneously inoculated over­
night during the coolship step and by the house microbiota of the
brewery equipment used.
During the short enterobacterial and AAB phase (first two weeks),
Enterobacter species were the most prevalent bacteria, exceeding log 5.0
(CFU/mL) on VRBG agar medium, in the fermenting wort of both casks.
The same bacterial species were isolated from both VRBG and mMRS
L. Vermote et al. International Journal of Food Microbiology 394 (2023) 110163

Fig. 1. (A) Course of the physicochemical parameters temperature, pH, and apparent attenuation during 30-month lambic beer production processes carried out in
two nearly identical oak casks [cask 1 (light coloured) and cask 2 (dark coloured)]. (B, C) Counts of presumptive yeasts, cycloheximide-resistant yeasts, acetic acid
bacteria, lactic acid bacteria and enterobacteria, expressed as log (CFU/mL), as plated on yeast extract-peptone-glucose (YPG) agar medium, YPG agar medium
supplemented with cycloheximide (YPGc), modified deoxycholate-mannitol-sorbitol (mDMS) agar medium, modified de Man-Rogosa-Sharpe (mMRS) agar medium
and violet red-bile-glucose (VRBG) agar medium, respectively, during 30-month lambic beer production processes carried out in two nearly identical oak casks [cask
1 (B), cask 2 (C)]. The four characteristic phases of a lambic beer production process are indicated at the top of the graphs. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

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L. Vermote et al. International Journal of Food Microbiology 394 (2023) 110163

Fig. 2. Community dynamics of the presumptive acetic acid bacteria (AAB) species, as determined on modified deoxycholate-mannitol-sorbitol (mDMS) agar
medium, lactic acid bacteria (LAB) species, as determined on modified de Man-Rogosa-Sharpe (mMRS) agar medium, yeast species, as determined on yeast extract-
peptone-glucose (YPG) agar medium, and cycloheximide-resistant yeast species, as determined on YPG agar medium supplemented with cycloheximide (YPGc agar
medium), during 30-month lambic beer production processes carried out in two nearly identical oak casks [cask 1 (top graphs), cask 2 (bottom graphs)]. The number
of isolates picked and identified is given between brackets at the bottom line of the graphs. If no isolates (indicated as 0) were picked, the bacterial counts were < log
2.0 (CFU/mL). The following species were identified on mDMS: Acetobacter cerevisiae (Genbank accession number KF537430.1), Gluconobacter japonicus
(NR_041445.1), Acetobacter indonesiensis (NR_028616.1), Yarrowia lipolytica (NR_111212.1), Acetobacter fabarum (HG329536.1), Kosakonia sacchari (NR_118333.1),
Gluconobacter cerevisiae (HG329585.1), Gluconobacter oxydans (FN391653.1), Acetobacter lambici (HG329531.1), and Dekkera bruxellensis (KY103321.1). The
following species were identified on mMRS: Enterobacter species (CP017184.1, CP017183.1, and NR_117547.1), Carnobacterium maltaromaticum (LC065032.1),
A. lambici (HG329531.1), Pediococcus damnosus (NR_042087.1), Lacticaseibacillus paracasei (NR_025880.1), Gluconobacter liquefaciens (FN391626.1), Gluconobacter
nephelii (HG329579.1), Gluconobacter cerinus (FN391644.1), Serratia liquefaciens (CP006253.1), A. cerevisiae (KF537430.1), A. fabarum (HG329536.1), Acetobacter
persici (KF537423.1), A. indonesiensis (NR_028616.1), Acetobacter lovaniensis (NR_113551.1), G. oxydans (FN391653.1), G. cerevisiae (HG329585.1), and Debar­
yomyces hansenii (KY103230.1). The following species were identified on YPG: Hanseniaspora uvarum (KY103558.1), Saccharomyces cerevisiae (KC881067.1), Pichia
kudriavzevii (AY939808.1), D. hansenii (KY103230.1), Saccharomyces kudriavzevii (NR_111355.1), Pichia fermentans (NR_130688.1), A. cerevisiae (KF537430.1),
D. bruxellensis (KY103321.1), Millerozyma farinosa (NR_111254.1), Pichia membranifaciens (NR_111195.1), and Brettanomyces custersianus (NR_138184.1). The
following species were identified on YPGc: H. uvarum (KY103558.1), A. lambici (HG329531.1), Ogataea pini (NR_138175.1), D. bruxellensis (KY103321.1), Dekkera
anomala (KY103306.1), Candida lundiana (NR_137645.1), and B. custersianus (NR_138184.1).

agar media. A first peak of AAB was found during this initial fermen­
tation phase in both casks as well, up to almost log 5.0 (CFU/mL), for
which Acetobacter cerevisiae and Acetobacter fabarum were most preva­
lent. During the initial stages of the fermentation processes (after 24 h
till 9 weeks), high counts of both cycloheximide-resistant yeasts, up to
almost log 6.0 (CFU/mL), among which Hanseniaspora uvarum was most
prevalent, and cycloheximide-sensitive yeasts, above log 6.0 (CFU/mL),
among which S. cerevisiae was most prevalent, were found in the fer­
menting wort of both casks. Both H. uvarum and S. cerevisiae were pre­
sent in nearly equal counts, up to almost log 6.0 (CFU/mL), in the
fermenting wort of both oak casks during the first day of fermentation.
The numbers of cycloheximide-resistant yeasts reached a maximum of
log 5.8 (CFU/mL) after 24 h of fermentation, before they gradually
decreased to counts below log 2.0 (CFU/mL) after 6 weeks of fermen­
tation. The ratio of Saccharomyces species to H. uvarum increased to a
value higher than 1:1 after 24 h in the fermenting wort of both oak casks.
The most prevalent yeast species shifted from S. cerevisiae to
S. kudriavzevii after 9 weeks of fermentation. Low counts of between log
2.0 (CFU/mL) and log 4.0 (CFU/mL) of these Saccharomyces species
were found until month 6 of the production processes.
The LAB counts increased when the counts of the yeasts decreased
and the temperature exceeded 15 ◦ C, which marked the start of the
acidification phase (Fig. 2). LAB, all identified as P. damnosus, were
isolated from week 6 (cask 1) and 9 (cask 2) until month 6, which gave
counts up to almost log 7.0 (CFU/mL) and log 5.0 (CFU/mL) in the
fermenting wort, respectively.
Oxidative yeasts, such as Brettanomyces and Pichia species, co-
occurred in the maturing beer of both oak casks from month 6 on­
wards, which marked the start of the maturation phase. Acetobacter
lambici was the prevalent AAB species that could be isolated from month
13 onwards, when Saccharomyces species were absent. This indicated an
increase in oxygen availability due to lower carbon dioxide production
by the yeasts. However, the AAB counts fluctuated as a function of time
and were relatively low, which indicated still limited oxygen availability
in the oak casks. The counts of the AAB decreased when the yeast counts
were high, from month 13 and month 18 onwards in the maturing beer
of casks 1 and 2, respectively.
3.3. Shotgun metagenomic analysis
3.3.1. Taxonomic classification at genus level using all metagenomic
sequence reads (MSRs)
The MSRs obtained were used for taxonomic classification at genus
level using blastn, DIAMOND, Kaiju, and Kraken 2, to obtain software-
and database-independent results, and showed similar trends for the
fermenting wort of both casks (Fig. 3A). The 0-h sample included the
genera Acinetobacter, Arcobacter, Chryseobacterium, Flavobacterium,
Janthinobacterium, Lactococcus, and Pseudomonas, which were not found
in subsequent time points during these fermentation and maturation
processes. Fungi (such as Mucor, Penicillium, Rhizopus, and Trichococcus)

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L. Vermote et al. International Journal of Food Microbiology 394 (2023) 110163

Fig. 3. Taxonomic classification of the metagenomic sequence reads during 30-month lambic beer production processes carried out in two nearly identical oak casks.
The 0-h sample on the top of the figure represents the cooled lambic beer wort before its transfer to the wooden casks. (A) Genus level identification using blastn with
the NCBI nt database (nt), DIAMOND with the NCBI nr database (Diam), Kaiju with a customized database of bacterial, archaeal, and lower eukaryotic protein
sequences (Kaiju), and Kraken2 with a customized database of bacterial, archaeal, and lower eukaryotic nucleotide (K2nt) or protein (K2aa) sequences. The category
“Minorities” represents all genera present with a relative abundance below 1.0 % for all methods used. The category “Higher than genus” represents all taxonomic
levels assigned above genus level. The category “Unassigned” represents reads that were not assigned to any taxonomic level. The category “No hits” includes all
reads that could not be classified at all. (B) Species level identification, using fragment recruitment plotting with a custom-made database containing genera pre­
viously assigned through taxonomic classification at genus level [cask 1 (left) and cask 2 (right)].

were found, depending on the taxonomic tool used. The genera Gluco­
nobacter, Hanseniaspora, and Saccharomyces were the only ones detected
in the 0-h sample that were also present upon fermentation and matu­
ration. For the fermenting wort and maturing beer in both casks, the
yeast genera Saccharomyces, Hanseniaspora, Brettanomyces, Ogataea, and
Pichia, and the bacterial genera Acetobacter, Gluconobacter, Pediococcus,
and Lactobacillus were found as the main genera, i.e., having a relative
abundance of at least 1 %, although many other genera were present in
lower relative abundances. Overall, the percentage of MSRs that could
be assigned at genus level ranged from 75 % for the samples taken at the
start of the fermentation processes until 15 % for the 18- and 30-month
samples.
3.3.2. Taxonomic classification at species level based on fragment
recruitment plotting
MSRs were aligned to an in-house database containing the genome
sequence of 3662 microbial species, corresponding to 175 genera, also
taking into account the new taxonomy for Lactobacillus (Zheng et al.,
2020). Overall, 95 % of the MSRs could be classified with FRP for all
time points.
For the 0-h sample, many species from various genera were found,
even when applying a MSR ANI cut-off of at least 95 % (Table 1). The
largest number of reads, namely 30.33 %, aligned to Mucor circinelloides,
with a MSR ANI value of 94.34 %. This suggests that a closely related
Mucor species was present in the sample, for which there was no genome
sequence available in the database used. In decreasing order of relative
abundance, H. uvarum, S. cerevisiae, Penicillium roqueforti, Acinetobacter

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L. Vermote et al. International Journal of Food Microbiology 394 (2023) 110163

Table 1 and P. damnosus were found, albeit at lower relative abundances.

List of microbial species identified in the metagenome from the 0-h sample, this In the fermenting wort and maturing beer of cask 2, similar trends
is the sample before the wort was transferred to the barrels, based on fragment were seen (Fig. 3B), although at day 3, A. cerevisiae (0.07 % of all reads)
recruitment plotting. Only (sub)species with a metagenomic sequence read and Acetobacter malorum (0.16 %) were found. Saccharomyces cerevisiae
average nucleotide identity (MSR ANI) of >95 % are listed. (*) Species with a recruited most of the MSRs (67.54–54.93 %) from day 3 until week 9,
high percentage of reads assigned but with an MSR ANI score lower than 95 %.
and P. damnosus was only found starting from week 9. At month 18, next
Species % of reads Species % of reads to D. bruxellensis, also A. lambici was found. At month 30, >90 % of the
assigned assigned
MSRs aligned to B. custersianus.
Mucor circinelloides 30.33* Lelliottia amnigena 0.12
Hanseniaspora uvarum 3.50 Buttiauxella gaviniae 0.11 3.3.3. Statistical analysis
Saccharomyces 3.46 Carnobacterium inhibens 0.10
Considering all fermenting wort samples, a PCA of the relative spe­
cerevisiae subsp. inhibens
Penicillium roqueforti 2.28 Carnobacterium inhibens 0.10 cies abundance based on FRP resulted in two principal components
subsp. gilichinskyi (PCs) that together covered 86.8 % of the total variance (Fig. 4A). PC1
Acinetobacter guillouiae 1.63 Saccharomyces bayanus 0.09 was characterized by negative loadings for most species found in the 0-h
Lactococcus raffinolactis 1.53 Penicillium biforme 0.09
sample and positive loadings for S. kudriavzevii, S. cerevisiae,
Geotrichum candidum 0.67 Penicillium carneum 0.09
Chryseobacterium bovis 0.58 Penicillium camemberti 0.09 P. damnosus, S. uvarum, D. bruxellensis, B. custersianus, A. lambici, and
Flavobacterium 0.55 Carnobacterium viridans 0.09 D. anomala. PC2 was characterized by negative loadings for H. uvarum,
hibernum Gluconobacter oxydans, Gluconobacter japonicus, S. cerevisiae, Saccharo­
Yarrowia lipolytica 0.51 Buttiauxella agrestis 0.09 myces bayanus, A. fabarum, A. cerevisiae, Pichia fermentans, S. uvarum,
Trichococcus 0.50 Candida sake 0.08
Clavispora lusitaniae, and S. kudriavzevii and positive loadings for
flocculiformis
Pseudomonas saxonica 0.46 Rahnella variigena 0.08 D. bruxellensis, B. custersianus, P. damnosus, and A. lambici. The 0-h
Penicillium solitum 0.46 Acetobacter cerevisiae 0.08 sample was separated from the other samples and associated with a
Pseudomonas veronii 0.45 Buttiauxella noackiae 0.08 negative loading of PC1 and an almost neutral loading of PC2. The
Enterobacter soli 0.44 Acetobacter malorum 0.08
fermenting wort samples from the casks were associated with slightly
Wickerhamomyces 0.42 Chryseobacterium 0.08
anomalus balustinum
positive loadings of PC1 and varying loadings of PC2, forming a vertical
Pseudomonas rhodesiae 0.42 Carnobacterium 0.07 line, and those from months 18 and 30 fell together. For this reason, a
maltaromaticum second PCA was performed with only the fermenting wort samples from
Saccharomyces 0.39 Cellulosimicrobium 0.07 casks 1 and 2. The first two PCs obtained covered together 54.7 % of the
kudriavzevii aquatile
total variance (Fig. 4B). PC1 was characterized by positive loadings of
Clavispora lusitaniae 0.34 Saccharomyces uvarum 0.07
Pichia membranifaciens 0.34 Kregervanrija fluxuum 0.07 C. lusitaniae, H. uvarum, Pi. fermentans, S. cerevisiae, G. oxydans,
Pseudomonas helleri 0.27 Pseudomonas simiae 0.06 G. japonicus, Enterobacter soli, A. cerevisiae, S. uvarum, and S. bayanus.
Gluconacetobacter 0.27 Pseudomonas putida 0.06 PC2 was characterized by positive loadings of A. fabarum, S. bayanus,
liquefaciens
G. japonicus, A. cerevisiae, and G. oxydans and negative loadings of
Gluconobacter japonicus 0.22 Yersinia enterocolitica 0.05
subsp. enterocolitica
S. kudriavzevii, Wickerhamomyces anomalus, S. uvarum, Pe. roqueforti, and
Pichia fermentans 0.22 Cellulosimicrobium 0.05 S. cerevisiae. Except for the 3-day sample from cask 1, which was asso­
terreum ciated with positive loadings of PC1 and positive loadings of PC2, all
Torulaspora delbrueckii 0.21 Cellulosimicrobium funkei 0.05 other fermenting wort samples were associated with either positive
Duganella zoogloeoides 0.21 Pediococcus damnosus 0.05
loadings of PC1 and negative loadings of PC2 or negative loadings of
Penicillium paneum 0.20 Acetobacter fabarum 0.05
Paraburkholderia 0.20 Dekkera bruxellensis 0.05 PC1 and positive loadings of PC2. All fermenting wort samples of the
tropica two casks did not differ much regarding the PC combination, except for
Flavobacterium 0.19 Yersinia enterocolitica 0.04 the 3-day and 9-week samples.
sinopsychrotolerans subsp. paleartica The intra-sample (alpha) diversity showed that the highest diversity
Sphingobacterium 0.18 Acetobacter indonesiensis 0.04
siyangense
and highest evenness was associated with the 0-h sample (Table 2). The
Acinetobacter albensis 0.18 Carnobacterium jeotgali 0.04 1-week and 6-week fermenting wort samples of cask 1 had slightly lower
Cellulosimicrobium 0.15 Kazachstania unispora 0.03 values for diversity and evenness, followed by the 6-week and 9-week
cellulans samples of cask 2. The 18-month sample of cask 1 and the 30-month
Penicillium 0.15 Enterobacter ludwigii 0.03
sample of cask 2 had a very low diversity and low evenness. The inter-
fuscoglaucum
Pseudomonas carnis 0.14 Sphingomonas 0.03 sample diversity showed no significant differences (p < 0.05) between
paucimobilis the two casks.

3.3.4. Co-assembly of metagenomes, contig binning, and contig annotation

guillouiae, Lactococcus raffinolactis, Geotrichum candidum, Chrys­
eobacterium bovis, Flavobacterium hibernum, Yarrowia lipolytica, and Tri­
chococcus flocculiformis were found in the 0-h sample, ranging from 3.50
% to 0.50 % of the MSRs.
In the fermenting wort and maturing beer of cask 1, most MSRs
aligned to S. cerevisiae at day 3, week 1, and week 6 (Fig. 3B), followed
by S. kudriavzevii and H. uvarum, both displaying lower relative abun­
dances. At day 3, A. cerevisiae (0.15 % of all reads) and A. fabarum (0.39
%) were found. At week 1, MSRs aligned to Saccharomyces uvarum, albeit
at a low relative abundance. From week 6 onward, P. damnosus
occurred, having the highest relative abundance at weeks 9 and 16.
Hanseniaspora uvarum, S. cerevisiae, and S. kudriavzevii were found until
week 16. At month 18, >90 % of the MSRs aligned to D. bruxellensis,
after which this shifted to B. custersianus, with >80 % of the MSRs
aligned at month 30. At that time point, also A. lambici, D. bruxellensis,
Assembly and subsequent binning with CONCOCT of the 15 MSR
data sets resulted in 24 bins; after manual refinement, 42 bins were
obtained, of which 7 bins had a completion of minimum 75 % and a
contamination of <10 % (Table 3). Two of these bins were related to
bacteria and five to yeasts. Two bins, Bin_23_A and Bin_18_A_1, could be
considered as high-quality MAGs (> 90 % completion and < 5 %
contamination). Using minimap2, Bin_23_A could be identified as
A. lambici and Bin_18_A_1 as P. damnosus. Because of the high comple­
tion and low contamination of Bin_23_A, the contigs were compared to
the genome of A. lambici LMG 27439T, resulting in an ANI value of
99.92 %. Minimap2 was also used to check the identity of the other bins,
as represented in Table 3.
3.3.4.1. Acetobacter lambici MAG (Bin_23_A). In the A. lambici MAG, no

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L. Vermote et al. International Journal of Food Microbiology 394 (2023) 110163

Fig. 4. Statistical analysis of the metagenomic sequence data of the lambic beer fermentation and maturation process samples analyzed, obtained during 30-month
lambic beer production processes carried out in two nearly identical oak casks. (A, B) Principal component analysis of the taxonomic composition based on the
relative abundances of all species detected by fragment recruitment plotting (FRP). Samples are colour- and shape-coded according to the time point and sample type,
respectively. (A) All wort samples, including the 0-h sample (cooled lambic beer wort before its transfer to the casks). (B) Only the fermenting wort samples. (C)
Hierarchical clustering analysis of the Spearman correlation values (p < 0.05) obtained by comparing the main microbial species, determined by FRP, and the
concentrations of the substrates consumed and metabolites produced. Positive correlations are represented in blue and negative correlations in red; the intensity of
the colour represents the degree of correlation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)

genes for the conversion of sucrose or maltose/maltooligosaccharides
were found, neither with Prokka nor with a manual search using blastp
(Fig. 5). Yet, some genes encoding maltooligosyl trehalose synthase and
maltooligosyl trehalase were found in other, not complete bins, on
contigs identified with minimap2 as A. malorum and A. cerevisiae,
respectively. All genes coding for the enzymes involved in the Embden-
Meyerhof-Parnas (EMP) pathway, except for phosphoglucose isomerase
and 6-phosphofructokinase, and for the enzymes involved in the pentose
phosphate pathway (PPP) were found. Also, a gene coding for pyruvate
phosphate dikinase was found. Genes coding for pyrroloquinoline
quinone-dependent membrane-bound alcohol and aldehyde de­
hydrogenases (all subunits) as well as for cytoplasmic NADH-dependent
dehydrogenases were found. Genes coding for all subunits of NAD(P)
transhydrogenase, both terminal ubiquinol oxidases (cytochrome bd
quinol oxidase and cytochrome bo3 ubiquinol oxidase) and ATP syn­
thase, were found. For NADH:quinone oxidoreductase, not all genes
encoding its subunits were found. In addition, genes encoding enzymes
involved in the production of (R)-acetoin and (S)-acetoin from pyruvate
via α-acetolactate and the further oxidation of acetoin into acetaldehyde
and acetyl-CoA were found. Genes responsible for pyruvate decarbox­
ylase and the pyruvate dehydrogenase complex, converting pyruvate
into acetaldehyde or acetyl-CoA, respectively, were present. Genes
encoding acetyl-CoA synthetase, phosphotransacetylase, and acetate
kinase were found, catalyzing the reversible conversion of acetate into
acetyl-CoA, acetyl-CoA into acetyl phosphate, and acetyl phosphate into
acetate, respectively. All genes involved in a modified tricarboxylic acid
(TCA) cycle were found, with genes coding for succinyl-CoA:acetate
CoA-transferase, instead of succinyl-CoA synthetase, converting
succinyl-CoA and acetate into succinate and acetyl-CoA, and for malate:
quinone oxidoreductase, instead of malate dehydrogenase, converting
malate into oxaloacetate. All genes coding for the 2-methylcitrate cycle
were found; however, no genes of the glyoxylate shunt were found. A
gene coding for the NAD-dependent malic enzyme was found, respon­
sible for the conversion of malate into pyruvate. A gene coding for a

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L. Vermote et al. International Journal of Food Microbiology 394 (2023) 110163

Table 2 acetyl esterase Aes (two copies; HAMAP annotation MF_01958), which
Alpha diversity indexes based on the relative abundances of species detected produces short-chain fatty acid esters (acyl chains containing up to eight
using fragment recruitment plotting in the different lambic beer fermentation carbon atoms), and an esterase YbfF (UniProt accession number
and maturation process samples obtained during 30-month lambic beer pro­ P75736), displaying an esterase activity toward palmitoyl-CoA and
duction processes carried out in two nearly identical oak casks. The Simpson (D) malonyl-CoA.
and Pielou (Je) indices were calculated for all samples to determine their di­
versity and evenness, respectively. C1, cask 1; C2, cask 2.
3.3.4.2. Pediococcus damnosus MAG (Bin_18_A_1). The P. damnosus
Sample Simpson (D) Pielou (Je)
MAG had genes coding for sucrose 6-phosphate hydrolase and maltose
0h 0.93 0.79 phosphorylase, allowing to use sucrose and maltose, respectively
C1 3 d 0.43 0.37 (Fig. 6). In this MAG, genes encoding all enzymes involved in both the
C1 1 w 0.55 0.47
homo- and heterolactic fermentation pathways were found, as well as
C1 6 w 0.64 0.62
C1 9 w 0.30 0.36 genes coding for D-lactate dehydrogenase and L-lactate dehydrogenase,
C1 16 w 0.16 0.20 allowing to convert pyruvate into D-lactate and L-lactate, respectively.
C1 18 m 0.02 0.04 Genes involved in the conversion of pyruvate into acetyl phosphate and
C1 30 m 0.20 0.28
its subsequent conversion into either acetate or ethanol were found. Also
C2 3 d 0.37 0.31
C2 1 w 0.37 0.33 a gene coding for the malolactic enzyme was found, allowing to convert
C2 6 w 0.51 0.42 S-malate into L-lactate. A ferulic acid decarboxylase gene was found,
C2 9 w 0.53 0.43 enabling the conversion of ferulic acid and p-coumaric acid into 4-vinyl
C2 16 w 0.23 0.32 guaiacol and 4-vinyl phenol, respectively. Further, in a bin constructed
C2 18 m 0.26 0.41
during manual refinement of Bin_18, also genes encoding enzymes
C2 30 m 0.02 0.04
involved in the production of (R)-acetoin and (S)-acetoin from pyruvate
via α-acetolactate were found in the constituting contigs identified as
P. damnosus. This bin also contained genes coding for tyrosine decar­
Table 3
boxylase [98.55 % identity and 99.20 % similarity to the Tetragenococcus
Bins obtained after contig binning with CONCOCT and two rounds of manual
halophilus protein (NCBI accession number WP_031944088.1); contig
refinement using anvi’o. The taxonomy shown here was determined with Kaiju
within the anvi’o pipeline used. Only bins with at least 75 % completion and identified as Pediococcus cellicola] and histidine decarboxylase [100 %
<10 % contamination were considered. identity to the Lentilactobacillus hilgardii protein (UniProt accession
number Q5DLT9); contig identified as Pediococcus parvulus], catalyzing
Metagenomic bin Size Completion Contamination Taxonomy
(Kaiju) (Mbp) (%) (%) (minimap2)
the conversion of tyrosine into tyramine and histidine into histamine,
respectively. Also, genes coding for the hop resistance proteins HorA
Bin_23_A 2.61 97.18 1.41 Acetobacter
[99.66 % identity and 99.83 % similarity to the Pediococcus claussenii
Acetobacter lambici
Bin_18_A_1 1.87 94.37 1.41 P. damnosus protein (UniProt accession number G8PFG1)], HorB [100.00 % identity
Pediococcus to the Loigolactobacillus backii protein (UniProt accession number
damnosus A4UX85)], HorC [98.83 % identity and 99.30 % similarity to the Levi­
Bin_5_A_1 11.37 87.95 2.41 D. bruxellensis
lactobacillus brevis protein (UniProt accession number Q6I7K2)], and
Brettanomyces
bruxellensis
HitA [99.78 % identity and 100.00 % similarity to the L. brevis protein
Bin_7_A 9.99 85.54 2.41 Brettanomyces (UniProt accession number Q93V04)] were found in this bin, all on the
Brettanomyces custersianus constituting contigs identified as P. damnosus, as well as a gene coding
bruxellensis for a putative glucan synthase [97.88 % identity and 98.94 % similarity
Bin_13_A_1 9.75 81.93 9.64 S. cerevisiae,
to the P. damnosus protein (UniProt accession number Q336K4); contig
Saccharomyces S. kudriavzevii
cerevisiae x identified as P. claussenii].
Saccharomyces
kudriavzevii 3.3.4.3. Yeast bins (Bin_2_A, Bin_4_A_1, Bin_5_A_1, Bin_7_A, and Bin
Bin_4_A_1 10.25 77.11 9.64 Saccharomyces
13_A_1). The yeast bins differed in predicted carbohydrate usage
Unknown cerevisiae
Saccharomyces (Fig. 7). In Bin_5_A_1, identified as D. bruxellensis, a gene encoding
Bin_2_A 8.52 78.31 2.41 H. uvarum maltodextrin phosphorylase was found, allowing to convert maltooli­
Hanseniaspora gosaccharides into glucose 6-phosphate via glucose 1-phosphate. In Bin
uvarum 13_A_1, identified as S. kudriavzevii, and in a bin constructed during the
refinement of Bin 4_A (contigs identified as S. cerevisiae), genes coding
glycerol uptake transporter was found as well as all genes to convert for a maltase that can hydrolyze maltose, maltotriose and sucrose, and
glycerol into dihydroxyacetone phosphate, or vice versa. No genes cod­ an invertase, converting sucrose into glucose and fructose, were found.
ing for lactate dehydrogenase were found, although genes coding for a In the S. cerevisiae bin (Bin_4_A_1), a gene encoding an α-glucosidase,
lactate permease and a lactate utilization protein were found. which can hydrolyze maltose, was found. Both Saccharomyces bins also
The A. lambici MAG was also searched for several genes known to be possessed a gene coding for a sporulation-specific glucoamylase, capable
responsible for acid tolerance in AAB. A gene coding for an acetic acid of degrading glycogen, starch, maltotriose and maltose into glucose, as
resistance ABC transporter ATP-binding protein Uup was found and this well as a gene coding for a polygalacturonase, a pectinolytic enzyme. In
gene had also 82.09 % identity and 92.04 % similarity to the acetic acid the D. bruxellensis bin (Bin_5_A_1), a gene coding for a putative glycosyl
resistance ABC transporter AatA of Acetobacter pasteurianus NBRC 3188 transferase [97.14 % identity and 98.45 % similarity to the
(UniProt accession number A0A401WXJ5). Also, the genes coding for D. bruxellensis AWRI1499 protein (NCBI accession number EIF48743)]
urea carboxylase [97.66 % identity and 99.07 % similarity to the was found, indicating the possible breakdown of β-glycosides, such as
A. fabarum protein (UniProt accession number A0A269XV37)] and the β-1,3-glucan laminarin. Another β-glucosidase that can assimilate
allophanate hydrolase responsible for the conversion of urea into cellobiose, gentiobiose, and arbutin showed a sequence identity of only
ammonia were found. Finally, a gene coding for N5-carbox­ 66.91 % and a similarity of 81.84 % to the D. bruxellensis AWRI1499
yaminoimidazole ribonucleotide (N5-CAIR) mutase, involved in purine protein (NCBI accession number EIF45415). One gene in Bin_2_A,
biosynthesis, was found. The MAG also possessed genes coding for an identified as H. uvarum, showed 51.24 % and 56.50 % identity and

10
L. Vermote et al. International Journal of Food Microbiology 394 (2023) 110163

Fig. 5. Overview of the main catabolic pathways reconstructed with genes found in Bin_23_A, identified as Acetobacter lambici, through functional analysis of
metagenomic contigs derived from whole-community DNA of samples from lambic beer fermentation and maturation processes carried out in two nearly identical
oak casks. Catabolic reactions are indicated with black arrows; dashed arrows depict multiple reactions. Chemical conversions are indicated with red arrows. TCA,
tricarboxylic acid. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

68.88 % and 71.75 % similarity with these putative glycosyl transferase
and β-glucosidase genes, respectively, hinting to the presence of another
β-glucosidase gene in this bin. Almost all yeast bins (Bin_2_A, Bin_5_A_1,
and Bin_13_A_1) contained all genes involved in the EMP pathway and
PPP. Although the S. cerevisiae bin (Bin_4_A_1) was missing a gene
coding for fructose bisphosphate aldolase in the EMP pathway, this gene
was found in a bin obtained during refinement (contig identified as
Saccharomyces paradoxus). In Bin_7_A, identified as B. custersianus, the
gene encoding ribose 5-phosphate epimerase in the PPP was missing. All
yeast bins contained all genes allowing to convert pyruvate into acet­
aldehyde, α-acetolactate, and (R)-acetoin by multifunctional pyruvate
decarboxylase; however, the H. uvarum bin was the only bin that did not
possess genes encoding diacetyl reductase, converting diacetyl into (R)-
acetoin, and its subsequent conversion into (R,R)-butanediol by (R,R)-
butanediol reductase. All yeast bins possessed genes to further convert
acetaldehyde into ethanol or acetate and subsequently acetate into
acetyl-CoA to enter the TCA cycle. Also, a gene coding for malate de­
hydrogenase, converting malate into pyruvate, was found in all yeast
bins. The H. uvarum bin was the only bin missing genes involved in the
glyoxylate cycle. The H. uvarum (Bin_2_A) and the S. kudriavzevii
(Bin_13_A_1) bins were the only bins containing all genes encoding all
subunits of the pyruvate dehydrogenase complex, converting pyruvate
into acetyl-CoA. In the Saccharomyces bins (Bin_4_A_1 and Bin_13_A_1), a
gene coding for ferulic acid decarboxylase, converting ferulic acid and p-
coumaric acid into 4-vinyl guaiacol and 4-vinyl phenol, respectively,
was found. In the D. bruxellensis bin (Bin_5_A_1), a gene coding for vinyl
phenol reductase [98.70 % identity and similarity to the D. bruxellensis
AWRI1499 protein (UniProt accession number I2JWC1)] was found,
converting the latter metabolites into 4-ethyl guaiacol and 4-ethyl
phenol, respectively, but also known to have a superoxide dismutase
activity. In the other yeast bins (Bin_2_A, Bin_4_A_1, Bin_7_A, and
Bin_13_A_1), the gene coding for superoxide dismutase was found with
identities of 64.10 %, 73.86 %, 88.96 %, and 73.86 % and similarities of
77.56 %, 83.66 %, 92.86 %, and 84.79 %, respectively. All yeast bins
possessed a gene coding for an NAD-dependent glycerol 3-phosphate
dehydrogenase; however, in the Brettanomyces/Dekkera bins
(Bin_5_A_1 and Bin_7_A), no gene coding for glycerol 3-phosphate
phosphatase was found. The S. kudriavzevii (Bin_13_A_1) and Brettano­
myces/Dekkera bins (Bin_5_A_1 and Bin_7_A) possessed a gene coding for
glycerol kinase and a quinol-dependent glycerol 3-phosphate dehydro­
genase, which is involved in the degradation of glycerol. The
S. kudriavzevii bin (Bin_13_A_1) harbored also a gene coding for a glyc­
erol uptake/efflux facilitator protein.
3.4. Substrate consumption and metabolite production dynamics
Unless stated otherwise, the concentrations and profiles of the sub­
strates and metabolites measured were comparable for the fermenting
wort and maturing beer of the two oak casks of the lambic beer pro­
duction processes examined.
Glucose (7.3 g/L), fructose (2.0 g/L), sucrose (7.0 g/L), maltose
(55.0 g/L), maltotriose (12.0 g/L), and higher maltooligosaccharides

11
L. Vermote et al.
Fig. 6. Overview of the main catabolic pathways reconstructed with genes found in Bin_18_A_1, identified as Pediococcus damnosus, through functional analysis of
metagenomic contigs derived from whole-community DNA of samples from lambic beer fermentation and maturation processes carried out in two nearly identical
oak casks. Catabolic reactions are indicated with black arrows; dashed arrows depict multiple reactions. Chemical conversions are indicated with red arrows.
Catabolic reactions in grey were found in contigs of related bins that were identified as P. damnosus. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
(6.3 g/L) were the most abundant carbohydrates present in the initial
lambic beer wort (Fig. 8A and B). Also organic acids were present,
namely lactic acid (1.2 g/L), malic acid (180 mg/L), citric acid (170 mg/
L), and gluconic acid (53 mg/L) (Fig. 8A and C). No short-chain or
branched-chain fatty acids were found during the short enterobacterial
and AAB phase, indicating limited growth of this background micro­
biota. A sequential consumption of the mono- and disaccharides took
place (Fig. 8A), with a nearly complete depletion of the mono-, di-, and
trisaccharides as follows. The depletion of sucrose in the fermenting
wort after three days of fermentation was followed by the depletion of
glucose and fructose after two weeks, which corresponded with the high
prevalence of S. cerevisiae. A continuous degradation of maltose
occurred until it was nearly completely exhausted after week 9. The
same degradation profile was followed by maltotriose, which indicated
the ability of the strains of the Saccharomyces species present to take up
and consume simultaneously maltose and maltotriose, although small
concentrations of maltotriose remained present until month 13. Coin­
ciding with the low concentrations of both maltose and maltotriose, the
counts belonging to Saccharomyces species decreased. From week 2
onwards, a simultaneous degradation of the higher maltooligo­
saccharides (from maltotetraose to maltoheptaose) took place.
During the first seven weeks of the lambic beer production processes
examined, the yeast-associated metabolites ethanol, methyl-1-butanol,
and succinic acid were produced (Fig. 8A, C, and D). Acetic acid was
barely produced in the fermenting wort and maturing beer of both oak
casks, which reflected the low counts of the AAB present. Only during
the final year of the maturation process, which coincided with the
growth of AAB and Brettanomyces species, acetic acid was produced up
to 1000 mg/L and 400 mg/L in the maturing beer of casks 1 and 2,
respectively. The differences in acetic acid concentrations indicated
differences in the AAB counts because of oxygen availability, likely
depending on barrel porosity. Lactic acid was produced from weeks 2
and 9 onwards in the fermenting wort of casks 1 and 2, respectively. The
12
International Journal of Food Microbiology 394 (2023) 110163
continuous production of lactic acid in the maturing beer of casks 1 and
2, even when no LAB were found, reflected the presence of LAB at low
counts, below log 2.0 (CFU/mL), throughout the entire maturation
phase. D-lactic acid and L-lactic acid were both produced in a nearly
equimolar ratio. Malic acid was rapidly depleted after the occurrence of
LAB at week 6 in the fermenting wort of cask 1 and week 9 in cask 2,
which coincided with the production of lactic acid and thus indicated
malolactic fermentation (Fig. 8A and C). During the final year of
maturation, when all other substrates were exhausted or present in very
low concentrations, citric acid was metabolized, probably by the yeasts
present (Fig. 8C). The concentration of gluconic acid that was present
from the start of the fermentation remained stable throughout the
lambic beer production processes performed.
The most abundant esters of lambic beer, ethyl lactate and ethyl
acetate, were produced from week 2 onwards, when ethanol was present
abundantly (Fig. 8A and C). In the fermenting wort of both oak casks,
maximal concentrations of over 50 mg/L of ethyl lactate were reached
after 6 weeks. Maximal concentrations of over 300 mg/L and around
200 mg/L of ethyl acetate were found in the maturing beer of casks 1 and
2 after 30 months, respectively. Isoamyl acetate was not found. Acetoin
production was negligible in the fermenting wort of both casks, indi­
cating minimal AAB growth or oxidation.
Some biogenic amines were present at low concentrations in the
initial lambic beer wort (Fig. 8D). It concerned agmatine (12 mg/L),
putrescine (6 mg/L), and cadaverine (2 mg/L). The concentrations of
those biogenic amines remained stable upon fermentation. Histamine
and tyramine were produced mainly during the acidification phase in
the fermenting wort of both oak casks, which coincided with the pres­
ence of LAB. The concentrations of histamine were around 20 mg/L in
the fermenting wort of casks 1 and 2. The most prevalent biogenic
amine, tyramine, was still present in the fermenting wort of both casks at
the end of the maturation phase. It reached concentrations between 40
and 50 mg/L in the maturing beer. Phenylethylamine and tryptamine
L. Vermote et al. International Journal of Food Microbiology 394 (2023) 110163

Fig. 7. Overview of the main catabolic pathways reconstructed with genes found in fungal bins through functional analysis of metagenomic contigs derived from
whole-community DNA samples from lambic beer fermentation and maturation processes carried out in two nearly identical oak casks. Pathways found in Bin_5_A_1,
Bin_7_A, Bin_2_A, Bin_4_A_1, and Bin_13_A_1, identified as Dekkera bruxellensis, Brettanomyces custersianus, Hanseniaspora uvarum, Saccharomyces cerevisiae, and
Saccharomyces kudriavzevii, respectively, are displayed. Catabolic reactions present in all bins are indicated with black arrows; dashed arrows depict multiple re­
actions. Catabolic reactions that were not present in all bins are indicated with purple arrows. Reactions represented by genes present at low identities are indicated
with brown arrows. Chemical conversions are indicated with red arrows. TCA, tricarboxylic acid. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

were not found.
A Spearman correlation analysis showed two clusters, one consisting
of the yeast species present during the enterobacterial and main
fermentation phases and another consisting of the species present during
the acidification and maturation phases (Fig. 4C). The carbohydrates
and malic acid correlated positively with the first cluster (correlation
values ranging from 0.56 to 0.99), whereas acetoin and ethyl acetate
displayed a positive correlation with P. damnosus (correlation values of
0.73 and 0.76, respectively). Methyl 1-butanol, ethyl lactate, acetic acid,
histamine, tyramine, lactic acid, ethanol, and succinic acid were posi­
tively correlated with the second cluster (correlation values ranging
from 0.54 to 0.89).
4. Discussion
Lambic beer productions have been examined thoroughly, in
particular regarding their microbiology, using culture-dependent tech­
niques (De Roos et al., 2018a, 2018b; Spitaels et al., 2014b, 2015), and
to a lesser extent culture-independently, either through amplicon-based
high-throughput sequencing (De Roos et al., 2018a, 2019) or shotgun
metagenomics (De Roos et al., 2020). Some of those studies have com­
bined the microbiological assessment with a thorough metabolomic
analysis (De Roos et al., 2018a, 2018b, 2019, 2020). Although the
shotgun metagenomic sequencing approach of the present study showed
similar trends as the culture-dependent investigations, it unraveled
additional features. First, methodologically, taxonomic analysis of the
MSRs using FRP, allowing MSR-based taxonomic assignments at species
level, showed that more reads could be assigned compared with a series
of software tools and databases used that only allowed taxonomic as­
signments at genus level. The remaining unassigned MSRs could
represent species without a sequenced genome and/or novel species. In
earlier work, FRP has proven to be successful to identify MSRs at species
level and even to unravel the presence of novel species, as was the case
for Acetobacter sicerae in water kefir samples (Verce et al., 2019, 2020).
Second, functional analysis of two high-quality bacterial MAGs and five
yeast bins obtained after manual refinement showed the presence of
genes that have not been shown before, for instance those coding for
ferulic acid decarboxylase (P. damnosus) and maltodextrin phosphory­
lase (D. bruxellensis) that are of importance in the maturation of lambic
beer. Finally, microbial growth and gene prediction could be backed up
by metabolite target analysis.
The present study further contributed to the unravelling of the role of
wooden casks in the spontaneous inoculation of the wort and maturing
beer during lambic beer production. Although shotgun metagenomics
revealed that the cooled wort had the highest diversity and evenness, its
harboring species did not contribute to the wort fermentation and beer
maturation in the casks, as many of these species were solely present at
the coolship stage. The present multiphasic study of two parallel lambic
beer production processes, starting from the same wort and carried out
in nearly identical oak casks of wine production origin, showed that the
wooden barrels did have an impact on the production process from a
microbiological point of view. The four characteristic phases of a lambic
beer production process occurred as described before (De Roos et al.,
2018a), with similar microbial community, substrate consumption, and
metabolite production dynamics, and typical characteristics, including
malolactic fermentation performed by P. damnosus and simultaneous
degradation of maltooligosaccharides by yeasts. Besides these generally
encountered characteristics, some specific differences with previously
studied lambic beer production processes in the same brewery were
found, possibly highlighting that a combination of different coolship

13
L. Vermote et al.
Fig. 8. Concentrations of monosaccharides, disaccharides, lactic acid, and ethanol (A); maltooligosaccharides (maltotriose, M3; maltotetraose, M4; maltopentaose,
M5; maltohexaose, M6; and maltoheptaose, M7) and acetic acid (B); other organic acids, ethyl acetate, and ethyl lactate (C); and acetoin, higher alcohols, and
biogenic amines (D) during 30-month lambic beer production processes carried out in two nearly identical oak casks (cask 1 and cask 2). The four characteristic
phases of a lambic beer production process are indicated at the top of the graphs.
batches and the casks used may influence lambic beer production. AAB
generally grow during both the enterobacterial and main fermentation
phases, depending on the oxygen availability (De Roos et al., 2018b). In
the fermenting wort of both oak casks of the present study, culture-
dependent analysis showed that the AAB that grew during the entero­
bacterial phase mostly belonged to the species A. fabarum and
A. cerevisiae, as opposed to A. orientalis as the most prevalent AAB spe­
cies during the first AAB peak of the fermenting wort in a previous study
(De Roos et al., 2018b). Although the type strains of A. fabarum,
A. cerevisiae, and A. orientalis were isolated from different niches, strains
of these three species have been isolated from lambic beer productions
before (Cleenwerck et al., 2002, 2008; Lisdiyanti et al., 2001; Spitaels
et al., 2015). As these three AAB species are relatively distinct, the risk
for misidentifications is minimal. The shotgun metagenomic analysis of
the present study identified A. cerevisiae and A. fabarum during the main
fermentation phase of cask 1 and A. cerevisiae and A. malorum (phylo­
genetically closely related to A. cerevisiae) in that of cask 2. Hence, the
identity of the AAB species prevailing during the enterobacterial and
AAB phase of a lambic beer production process may vary between
production batches. Moreover, their presence seemed to be mostly
dependent on the environmental inoculation during the overnight
cooling of the wort and the use of non-sterile brewery equipment instead
of potential inoculation of AAB species residing in the wood of the
barrels. The maturing lambic beer in the oak casks used in the present
study displayed a delayed and limited second booming of AAB, mainly of
the species A. lambici, likely due to a limited oxygen availability as a
result of the low porosity of the casks. The oxygen availability and,
hence, the growth of AAB during lambic beer production processes
seems to be a combined effect of barrel characteristics (porosity, head­
space, and surface to volume ratio), and the presence of specific yeast
species that cause carbon dioxide production and pellicle formation (De
Roos et al., 2018b). The production of carbon dioxide, which causes
14
International Journal of Food Microbiology 394 (2023) 110163
oxygen to be flushed out, is mostly dependent on the presence of
Saccharomyces species, whereas pellicle formation is mostly determined
by Brettanomyces and Pichia species. Besides regulating the oxygen
availability, wooden barrels harbor resident microorganisms and can
thus act as an additional microbial inoculation source, in particular of
LAB and yeasts (del Alamo-Sanza and Nevares, 2014; De Roos et al.,
2019). This additional inoculation helps to diminish batch-to-batch
variations concerning fermentation profile, thereby outcompeting un­
desirable microorganisms (De Roos et al., 2018b). The fact that barrels
act as an additional inoculation source was further strengthened by the
presence of highly similar microorganisms in the present study and a
previous one carried out in the same brewery (De Roos et al., 2018b).
The additional inoculation caused by wooden surfaces has also been
reported for spontaneous cider fermentations, for which the use of
traditional wooden presses versus pneumatic stainless steel presses and
stainless steel fermentors shows a profound influence on the production
process (Del Campo et al., 2003; Morrissey et al., 2004). In addition, it is
known, for example, that the heterogenous microbial communities that
inhabit the internal wine barrel surfaces include Brettanomyces species
(Barata et al., 2013; Fugelsang and Edwards, 2007). These yeast species
are of importance during lambic beer maturation (Bongaerts et al., 2021;
Bouchez and De Vuyst, 2022; De Roos and De Vuyst, 2019; Spitaels
et al., 2017). Moreover, due to their physical inertness and porosity,
wooden surfaces are not easy to sanitize (De Roos et al., 2019; Guzzon
et al., 2011; Oelofse et al., 2008; Spitaels et al., 2017). The higher the
porosity of the barrels, the better the conditions for survival and biofilm
formation by microorganisms are. Therefore, wooden barrels, with their
highly abundant pores and cracks, permit a good substrate for biofilm
formation and a protective barrier for microorganisms (Guzzon et al.,
2011; Swaffield and Scott, 1995; Swaffield et al., 1997), which are likely
of importance for later inoculation of the contents of the barrel (De Roos
et al., 2019).
L. Vermote et al.
Several new insights into the genetic potential of typical members of
lambic beer fermentation and maturation processes were obtained based
on the metagenomic bin analysis. Several of the genes related to acetic
acid tolerance, identified in the A. lambici MAG, were common for
Acetobacter species to allow their survival in an acid environment, such
as the conversion of acetate into acetyl-CoA by different enzymatic re­
actions, an acetic acid resistance ABC transporter, the conversion of urea
into ammonia, the 2-methylcitrate cycle, and a N5-CAIR mutase (Ill­
eghems et al., 2013; Lynch et al., 2019; Nakano et al., 2006; Yang et al.,
2019). The A. lambici MAG further confirmed the uptake and conversion
of lactate and glycerol as carbon sources (Pelicaen et al., 2019) as well as
a pyruvate phosphate dikinase as the driving force of gluconeogenesis in
AAB (Adler et al., 2014). Although predicted before in an Acetobacter bin
that was composed of different Acetobacter contigs, among which some
belonging to A. pasteurianus (De Roos et al., 2020), maltose/maltooli­
gosaccharide consumption by A. lambici was probably not possible based
on the current metagenomic analysis. Yet, a maltooligosyl trehalose
synthase and maltooligosyl trehalase could be attributed to A. malorum
and A. cerevisiae, respectively, which may reflect their presence in a
larger Acetobacter bin that was found in a previous study (De Roos et al.,
2020). The former two AAB species were present in the initial lambic
beer wort as well as during the enterobacterial fermentation phase,
shown both culture-dependently and -independently. Furthermore, su­
crose consumption by A. lambici did not seem to be possible, as the
concomitant gene was absent in the MAG. The gene encoding a putative
glucan synthase and hop resistance proteins in a refined bin related to
the P. damnosus MAG was in accordance with the strain’s ropy pheno­
type (Walling et al., 2005) and resistance to hop (Bergsveinson et al.,
2015; Garcia-Garcia et al., 2017). These genes are plasmid-based
(Bergsveinson et al., 2015; Garcia-Garcia et al., 2017; Walling et al.,
2005). Therefore, they were possibly removed during the manual
curation of the P. damnosus MAG. Indeed, binning tools can have
problems with plasmids, as plasmids replicate autonomously, resulting
in deviating relative abundance profiles (Beaulaurier et al., 2017). The
sequence composition profiles and GC contents can also differ (Beau­
laurier et al., 2017; Shintani et al., 2015). As the genes coding for his­
tidine decarboxylase, tyrosine decarboxylase, and enzymes involved in
the production of (R)-acetoin and (S)-acetoin from pyruvate were
located on the same bin, on contigs identified as P. damnosus or other
Pediococcus species, they could be plasmid-based as well (Shintani et al.,
2015). All these genes were also detected during a previous meta­
genomic study of lambic beer, but they were not associated with a
plasmid location because another binning strategy was used (De Roos
et al., 2020). The presence of genes coding for ferulic acid decarboxylase
indicated potential 4-vinyl compound production by P. damnosus, next
to their production by Saccharomyces species. However, previously, this
gene has also been shown in D. bruxellensis but not in S. kudriavzevii (De
Roos et al., 2020). The degradation of starch, maltotriose, and maltose
could be attributed to a sporulation-specific glucoamylase of the
Saccharomyces species, whereas maltooligosaccharide phosphorylase
could be responsible for maltooligosaccharide usage by D. bruxellensis,
as all the concomitant genes were present in the respective bins. The
absence of a gene encoding glycerol 3-phosphate phosphatase in the
D. bruxellensis and B. custersianus bins indicated that glycerol could not
be produced, proving the need to use alternative external electron ac­
ceptors, such as 4-vinyl compounds and acetoin to recuperate NAD+
(Bongaerts et al., 2021; Steensels et al., 2015). The Acetobacter lambici
MAG and H. uvarum bin were not equipped with a glyoxylate cycle,
which indicated its species specificity within these genera (Illeghems
et al., 2013; Pelicaen et al., 2022; Sakurai et al., 2013; Seixas et al.,
2019). As has been shown for D. bruxellensis strains from wine, the
presence of two β-glucosidase genes could be responsible for the hy­
drolysis of β-1,3 (laminarin) and likely β-1,4 (cellobiose) and β-1,6
bonds (gentiobiose), respectively. Furthermore, this may have an in­
fluence on the flavoring capability of D. bruxellensis in lambic beer
(Crauwels et al., 2014, 2015). Although mainly ascribed to
15
International Journal of Food Microbiology 394 (2023) 110163
enterobacterial growth, MAG-originating genes related to amino acid
decarboxylation linked P. damnosus with biogenic amine formation,
which supported previous findings (De Roos et al., 2018a). Nevertheless,
the biogenic amine concentrations were well below the health-
acceptable concentrations that have been issued by the panel on bio­
logical hazards of the European food safety authority for fermented
foods (Biohaz, 2011).
In conclusion, the present study showed that a systematic and
multiphasic analysis approach, encompassing classical microbiology,
metabolite target analysis, and shotgun metagenomics could further
unravel the secrets of lambic beer production. The casks used likely
helped in establishing a stable microbiota of yeasts, LAB, and AAB, by
acting as an additional inoculation source of the necessary microor­
ganisms, as the cooled wort transferred to the casks was still lacking
most fermentation and maturation microorganisms, thereby minimizing
batch-to-batch variations. Further, given the stable microbiota obtained,
it is obvious that the oak casks used provided a desired microaerobic
environment to achieve appropriate microbial community dynamics for
the fermentation and maturation of lambic beer wort. In particular, they
likely helped in preventing excessive growth of AAB and, therefore,
excessive production of acetic acid and acetoin, of which high concen­
trations might cause flavor deviations in lambic beer. A functional
analysis showed that the A. lambici MAG did not possess genes related to
sucrose and maltose/maltooligosaccharide consumption and the
glyoxylate shunt, but harbored genes involved in several acid tolerance
mechanisms. Several typical traits, such as hop resistance, ropy pheno­
type, and biogenic amine production, of P. damnosus were probably
plasmid-based and the P. damnosus MAG also possessed a ferulic acid
decarboxylase, indirectly contributing to lambic beer flavor formation.
The lack of a gene coding for glycerol 3-phosphate phosphatase in the
D. bruxellensis and B. custersianus bins indicated that glycerol could not
be produced, emphasizing the need for alternative external electron
acceptors for redox balancing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This work was financially supported by the Research Council of the
Vrije Universiteit Brussel (SRP7, IOF2442, and IOF3017 projects), the
Hercules Foundation (projects UABR09004 and UAB13002), and the
KMO-Portefeuille (projects 2014KMO084991, 2015KMO091056,
2016KMO149170, and 2017KMO112091, in collaboration with the
brewery Oud Beersel). Part of the computational resources and services
were provided by the Vrije Universiteit Brussel, the Flemish Super­
computer Centre (VSC), and the Research Foundation – Flanders (FWO-
Vlaanderen). LV and JDR were the recipients of a PhD fellowship of the
Vrije Universiteit Brussel. We thank ing. Wim Borremans for providing
technical assistance.
References
Adler, P., Frey, L.J., Berger, A., Bolten, C.J., Hansen, C.E., Wittmann, C., 2014. The key to
acetate: metabolic fluxes of acetic acid bacteria under cocoa pulp fermentation-
simulating conditions. Appl. Environ. Microbiol. 80, 4702–4716.
Alneberg, J., Bjarnason, B.S., de Bruijn, I., Schirmer, M., Quick, J., Ijaz, U.Z., Lahti, L.,
Loman, N.J., Andersson, A.F., Quince, C., 2014. Binning metagenomic contigs by
coverage and composition. Nat. Methods 11, 1144–1146.
L. Vermote et al.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local
alignment search tool. J. Mol. Biol. 215, 403–410.
Anderson, M.J., 2017. Permutational multivariate analysis of variance (PERMANOVA).
Available online:. In: Balakrishnan, N., Colton, T., Everitt, B., Piegorsch, W.,
Ruggeri, F., Teugels, J.L. (Eds.), Wiley StatsRef: Statistics Reference Online,
pp. 1–15. https://doi.org/10.1002/9781118445112.stat07841.
Andrews, S., 2018. FastQC: a quality control tool for high throughput sequence data.
Available online: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
Barata, A., Laureano, P., D’Antuono, I., Martorell, P., Stender, H., Malfeito-Ferreira, M.,
Querol, A., Loureiro, V., 2013. Enumeration and identification of 4-ethylphenol
producing yeasts recovered from the wood of wine aging barriques after different
sanitation treatments. J. Food Res. 2, 140–149.
Beaulaurier, J., Zhu, S., Diekus, G., Mogno, I., Zhang, X.-S., Davis-Richardson, A.,
Canepa, R., Triplett, E.W., Faith, J.J., Sebra, R., Schadt, E.E., Fang, G., 2017.
Metagenomic binning and association of plasmids with bacterial host genomes using
DNA methylation. Nat. Biotechnol. 36, 61–69.
Bergsveinson, J., Baecker, N., Pittet, V., Ziola, B., 2015. Role of plasmids in lactobacillus
brevis BSO 464 hop tolerance and beer spoilage. Appl. Environ. Microbiol. 81,
1234–1241.
Biohaz, 2011. European Food Safety Authority (EFSA) Panel on Biological Hazards.
Scientific opinion on risk based control of biogenic amine formation in fermented
foods. Available online: EFSA J. 9, 2393. https://doi.org/10.2903/j.efsa.2011.2393
www.efsa.europa.eu/efsajournal.
Bokulich, N.A., Bamforth, C.W., 2013. The microbiology of malting and brewing.
Microbiol. Mol. Biol. Rev. 77, 157–172.
Bokulich, N.A., Bamforth, C.W., Mills, D.A., 2012. Brewhouse-resident microbiota are
responsible for multi-stage fermentation of american coolship ale. PLoS One 7,
e35507.
Bokulich, N.A., Bergsveinson, J., Ziola, B., Mills, D.A., 2015. Mapping microbial
ecosystems and spoilage-gene flow in breweries highlights patterns of contamination
and resistance. elife 4, e04634.
Bongaerts, D., De Roos, J., De Vuyst, L., 2021. Technological and environmental features
determine the uniqueness of the lambic beer microbiota and production process.
Appl. Environ. Microbiol. 87 e00612-21.
Bouchez, A., De Vuyst, L., 2022. Acetic acid bacteria in sour beer production: friend or
foe? Front. Microbiol. 13, 957167.
Buchfink, B., Xie, C., Huson, D.H., 2015. Fast and sensitive protein alignment using
DIAMOND. Nat. Methods 12, 59–60.
Canas, S., Caldeira, I., Anjos, O., Lino, J., Soares, A., Belchior, A.P., 2016.
Physicochemical and sensory evaluation of wine brandies aged using oak and
chestnut wood simultaneously in wooden barrels and in stainless steel tanks with
staves. Int. J. Food Sci. Technol. 51, 2537–2545.
Cleenwerck, I., González, Á., Camu, N., Engelbeen, K., De Vos, P., De Vuyst, L., 2008.
Acetobacter fabarum sp. nov., an acetic acid bacterium from a ghanaian cocoa bean
heap fermentation. Int. J. Syst. Evol. Microbiol. 58, 2180–2185.
Cleenwerck, I., Vandemeulebroecke, K., Janssens, D., Swings, J., 2002. Re-examination
of the genus acetobacter, with descriptions of acetobacter cerevisiae sp. nov. and
Acetobacter malorum sp. nov. Int. J. Syst. Evol. Microbiol. 52, 1551–1558.
Crauwels, S., Van Assche, A., de Jonge, R., Borneman, A.R., Verreth, C., Troels, P., De
Samblanx, G., Marchal, K., Van de Peer, Y., Willems, K.A., Verstrepen, K.J.,
Curtin, C.D., Lievens, B., 2015. Comparative phenomics and targeted use of
genomics reveals variation in carbon and nitrogen assimilation among different
brettanomyces bruxellensis strains. Appl. Microbiol. Biotechnol. 99, 9123–9134.
Crauwels, S., Zhu, B., Busschaert, P., De Samblanx, G., Marchal, K., Willems, K.A.,
Verstrepen, K.J., Lievens, B., 2014. Assessing genetic diversity among brettanomyces
yeasts by DNA fingerprinting and whole-genome sequencing. Appl. Environ.
Microbiol. 80, 4398–4413.
De Keersmaecker, J., 1996. The mystery of lambic beer. Sci. Am. 275, 375–385.
del Alamo-Sanza, M., Nevares, I., 2014. Recent advances in the evaluation of the oxygen
transfer rate in oak barrels. J. Agric. Food Chem. 62, 8892–8899.
Del Campo, G., Santos, J., Berregi, I., Velasco, S., Ibarburu, I., Duenas, M., Irastorza, A.,
2003. Ciders produced by two types of presses and fermented in stainless steel and
wooden vats. J. Inst. Brew. 109, 342–348.
De Roos, J., De Vuyst, L., 2019. Microbial acidification, alcoholization, and aroma
production during spontaneous lambic beer production. J. Sci. Food Agric. 99,
25–38.
De Roos, J., Vandamme, P., De Vuyst, L., 2018a. Wort substrate consumption and
metabolite production during lambic beer fermentation and maturation explain the
successive growth of specific bacterial and yeast species. Front. Microbiol. 9, 2763.
De Roos, J., Van Der Veken, D., De Vuyst, L., 2019. The interior surfaces of wooden
barrels are an additional microbial inoculation source for lambic beer production.
Appl. Environ. Microbiol. 85 e02226-18.
De Roos, J., Verce, M., Aerts, M., Vandamme, P., De Vuyst, L., 2018b. Temporal and
spatial distribution of the acetic acid bacterium communities throughout the wooden
casks used for the fermentation and maturation of lambic beer underlines their
functional role. Appl. Environ. Microbiol. 84 e02846-17.
De Roos, J., Verce, M., Weckx, S., De Vuyst, L., 2020. Temporal shotgun metagenomics
revealed the potential metabolic capabilities of specific microorganisms during
lambic beer production. Front. Microbiol. 11, 1692.
Dysvik, A., Leanti La Rosa, S., De Rouck, G., Rukke, E.-O., Westereng, B., Wicklund, T.,
2020. Microbial dynamics in traditional and modern sour beer production. Appl.
Environ. Microbiol. 86 e00566-20.
Eren, A.M., Esen, Ö.C., Quince, C., Vineis, J.H., Morrison, H.G., Sogin, M.L., Delmont, T.
O., 2015. Anvi’o : an advanced analysis and visualization platform for ‘omics data.
PeerJ 3, e1319.
16
International Journal of Food Microbiology 394 (2023) 110163
Fugelsang, K.C., Edwards, G.C., 2007. Wine Microbiology: Practical Applications and
Procedures, Second edition. Springer, Boston, USA. 394 pp.
Garcia-Garcia, J.H., Damas-Buenrostro, L.C., Cabada-Amaya, J.C., Elias-Santos, M.,
Pereyra-Alférez, B., 2017. Pediococcus damnosus strains isolated from a brewery
environment carry the horA gene. J. Inst. Brew. 123, 77–80.
Gu, Z., Eils, R., Schlesner, M., 2016. Complex heatmaps reveal patterns and correlations
in multidimensional genomic data. Bioinformatics 32, 2847–2849.
Guzzon, R., Widmann, G., Malacarne, M., Nardin, T., Nicolini, G., Larcher, R., 2011.
Survey of the yeast population inside wine barrels and the effects of certain
techniques in preventing microbial spoilage. Eur. Food Res. Technol. 233, 285–291.
Harrell, F.E., 2021. Hmisc: Harrell miscellaneous. R package version 4.6-0. Available
online: https://CRAN.R-project.org/package=Hmisc.
Hervé, M., 2021. RVAideMemoire: testing and plotting procedures for biostatistics. R
package version 0.9.79. Available online. https://CRAN.R-project.org/package
=RVAideMemoire.
Holt, C., Yandell, M., 2011. MAKER2: an annotation pipeline and genome-database
management tool for second-generation genome projects. BMC Bioinform. 12, 491.
Illeghems, K., De Vuyst, L., Papalexandratou, Z., Weckx, S., 2012. Phylogenetic analysis
of a spontaneous cocoa bean fermentation metagenome reveals new insights into its
bacterial and fungal community diversity. PLoS One 7, e38040.
Illeghems, K., De Vuyst, L., Weckx, S., 2013. Complete sequence and comparative
analysis of acetobacter pasteurianus 368B, a strain well-adapted to the cocoa bean
fermentation ecosystem. BMC Gen. 14, 526.
Langmead, B., Salzberg, S.L., 2012. Fast gapped-read alignment with bowtie 2. Nat.
Methods 9, 357–359.
Lee, I., Kim, Y.O., Park, S.-C., Chun, J., 2016. OrthoANI: an improved algorithm and
software for calculating average nucleotide identity. Int. J. Syst. Evol. Microbiol. 66,
1100–1103.
Li, H., 2018. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34,
3094–3100.
Li, D., Liu, C.M., Luo, R., Sadakane, K., Lam, T.W., 2015. MEGAHIT: an ultra-fast single-
node solution for large and complex metagenomics assembly via succinct de bruijn
graph. Bioinformatics 31, 1674–1676.
Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T., Komagata, K., 2001.
Identification of acetobacter strains isolated from indonesian sources, and proposals
of acetobacter syzygii sp. nov., acetobacter cibinongensis sp. Nov., and acetobacter
orientalis sp. nov. J. Gen. Appl. Microbiol. 47, 119–131.
Lynch, K.M., Zannini, E., Wilkinson, S., Daenen, L., Arendt, E.K., 2019. Physiology of
acetic acid bacteria and their role in vinegar and fermented beverages. Compr. Rev.
Food Sci. Food Saf. 18, 587–625.
Menzel, P., Ng, K.L., Krogh, A., 2016. Fast and sensitive taxonomic classification for
metagenomics with kaiju. Nat. Commun. 7, 1–9.
Morrissey, W.F., Davenport, B., Querol, A., Dobson, A.D., 2004. The role of indigenous
yeasts in traditional irish cider fermentations. J. Appl. Microbiol. 97, 647–655.
Mouillot, D., Leprêtre, A., 1999. A comparison of species diversity estimators. Res. Popul.
Ecol. 41, 203–215.
Nakano, S., Fukaya, M., Horinouchi, S., 2006. Putative ABC transporter responsible for
acetic acid resistance in acetobacter aceti. Appl. Environ. Microbiol. 72, 497–505.
Oelofse, A., Pretorius, I.S., du Toit, M., 2008. Significance of brettanomyces and dekkera
during winemaking: a synoptic review. S. Afr. J. Enol. Vitic. 29, 128–144.
Oksanen, J., Blanchet, F.G., Friendly, M., Kindt, R., Legendre, P., McGlinn, D.,
Minchin, P.R., O’Hara, R.B., Simpson, G.L., Solymos, P., Stevens, M.H.H., Szoecs, E.,
Wagner, H., 2019. Vegan community ecology package. R package version 2.5-4.
Available online: https://CRAN.R-project.org/package=vegan.
Papalexandratou, Z., Lefeber, T., Bahrim, B., Lee, O.S., Daniel, H.-M., De Vuyst, L., 2013.
Hanseniaspora opuntiae, Saccharomyces cerevisiae, lactobacillus fermentum, and
acetobacter pasteurianus predominate during well-performed malaysian cocoa bean
box fermentations, underlining the importance of these microbial species for a
successful cocoa bean fermentation process. Food Microbiol. 35, 73–85.
Pelicaen, R., Gonze, D., Teusink, B., De Vuyst, L., Weckx, S., 2019. Reconstruction of
acetobacter pasteurianus 386B, a candidate functional starter culture for cocoa bean
fermentation. Front. Microbiol. 10, 2801.
Pelicaen, R., Weckx, S., Gonze, D., De Vuyst, L., 2022. Application of comparative
genomics of acetobacter species facilitates genome-scale metabolic reconstruction of
the acetobacter ghanensis LMG 23848T and acetobacter senegalensis 108B cocoa
strains. Front. Microbiol. 13, 1060160.
Pothakos, V., Illeghems, K., Laureys, D., Spitaels, F., Vandamme, P., De Vuyst, L., 2016.
Acetic acid bacteria in fermented food and beverage ecosystems. In: Matsushita, K.,
Toyama, H., Tonouchi, N., Okamoto-Kainuma, A. (Eds.), Acetic Acid Bacteria:
Ecology and Physiology. Springer, Tokyo, Japan, pp. 73–99.
RStudio Team, 2020. RStudio: integrated development for R. Available online. RStudio,
PBC, Boston, MA, USA. http://www.rstudio.com.
Sakurai, K., Yamazaki, S., Ishii, M., Igarashi, Y., Arai, H., 2013. Role of the glyoxylate
pathway in acetic acid production by acetobacter aceti. J. Biosci. Bioeng. 115,
32–36.
Schmieder, R., Edwards, R., 2011. Quality control and preprocessing of metagenomic
datasets. Bioinformatics 27, 863–864.
Seemann, T., 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30,
2068–2069.
Seixas, I., Barbosa, C., Mendes-Faia, A., Güldener, U., Tenreiro, R., Mendes-Ferreira, A.,
Mira, N.P., 2019. Genome sequence of the non-conventional wine yeast
Hanseniaspora guilliermondii UTAD222 unveils relevant traits of this species and of
the hanseniaspora genus in the context of wine fermentation. DNA Res. 26, 67–83.
Shintani, M., Sanchez, Z.K., Kimbara, K., 2015. Genomics of microbial plasmids:
classification and identification based on replication and transfer systems and host
taxonomy. Front. Microbiol. 6, 242.
L. Vermote et al.
Slowikowski, K., 2020. ggrepel: automatically position non-overlapping text labels with
‘ggplot2’. R package version 0.8.2. Available online: https://CRAN.R-project.
org/package=ggrepel.
Sombolestani, A.S., Cleenwerck, I., Cnockaert, M., Borremans, W., Wieme, A.D., De
Vuyst, L., Vandamme, P., 2020. Novel acetic acid bacteria from cider fermentations:
acetobacter conturbans sp. nov. and acetobacter fallax sp. nov. Int. J. Syst. Evol.
Microbiol. 70, 6163–6171.
Spitaels, F., Li, L., Wieme, A., Balzarini, T., Cleenwerck, I., Van Landschoot, A., De
Vuyst, L., Vandamme, P., 2014a. Acetobacter lambici sp. Nov., isolated from
fermenting lambic beer. Int. J. Syst. Evol. Microbiol. 64, 1083–1089.
Spitaels, F., Wieme, A.D., Janssens, M., Aerts, M., Daniel, H.-M., Van Landschoot, A., De
Vuyst, L., Vandamme, P., 2014b. The microbial diversity of traditional
spontaneously fermented lambic beer. PLoS One 9, e95384.
Spitaels, F., Wieme, A.D., Janssens, M., Aerts, M., Van Landschoot, A., De Vuyst, L.,
Vandamme, P., 2015. The microbial diversity of an industrially produced lambic
beer shares members of a traditionally produced one and reveals a core microbiota
for lambic beer fermentation. Food Microbiol. 49, 23–32.
Spitaels, F., Wieme, A.D., Snauwaert, I., De Vuyst, L., Vandamme, P., 2017. Microbial
ecology of traditional beer fermentations. In: Bokulich, N., Bamforth, C. (Eds.),
Brewing Microbiology: Current Research, Omics and Microbial Ecology. Caister
Academic Press, Poole, UK, pp. 179–196.
Steensels, J., Daenen, L., Malcorps, P., Derdelinckx, G., Verachtert, H., Verstrepen, K.J.,
2015. Brettanomyces yeasts – from spoilage organisms to valuable contributors to
industrial fermentations. Int. J. Food Microbiol. 206, 24–38.
Swaffield, C.H., Scott, J.A., 1995. Existence and development of natural microbial
populations in wooden storage vats used for alcoholic cider maturation. J. Am. Soc.
Brew. Chem. 53, 117–120.
Swaffield, C.H., Scott, J.A., Jarvis, B., 1997. Observations on the microbial ecology of
traditional alcoholic cider storage vats. Food Microbiol. 14, 353–361.
Van Oevelen, D., Spaepen, M., Timmermans, P., Verachtert, H., 1977. Microbiological
aspects of spontaneous wort fermentation in the production of lambic and gueuze.
J. Inst. Brew. 83, 356–360.
Verachtert, H., Derdelinckx, G., 2005. Acidic beers: enjoyable reminiscences of the past.
Cerevisia 30, 38–47.
Verachtert, H., Iserentant, D., 1995. Properties of belgian acidic beers and their
microflora. I. The production of gueuze and related refreshing acid beers. Cerevisia
20, 37–41.
17
International Journal of Food Microbiology 394 (2023) 110163
Verce, M., De Vuyst, L., Weckx, S., 2019. Shotgun metagenomics of a water kefir
fermentation ecosystem reveals a novel oenococcus species. Front. Microbiol. 10,
479.
Verce, M., De Vuyst, L., Weckx, S., 2020. The metagenome-assembled genome of
candidatus oenococcus aquikefiri from water kefir represents the species oenococcus
sicerae. Food Microbiol. 88, 103402.
Verce, M., Schoonejans, J., Hernandez Aguirre, C., Molina-Bravo, R., De Vuyst, L.,
Weckx, S., 2021. A combined metagenomics and metatranscriptomics approach to
unravel costa rican cocoa box fermentation processes reveals yet unreported
microbial species and functionalities. Front. Microbiol. 12, 641185.
Vermote, L., Verce, M., De Vuyst, L., Weckx, S., 2018. Amplicon and shotgun
metagenomic sequencing indicates that microbial ecosystems present in cheese
brines reflect environmental inoculation during the cheese production process. Int.
Dairy J. 87, 44–53.
Walling, E., Gindreau, E., Lonvaud-Funel, A., 2005. A putative glucan synthase gene dps
detected in exopolysaccharide-producing pediococcus damnosus and oenococcus
oeni strains isolated from wine and cider. Int. J. Food Microbiol. 98, 53–62.
White, B., Smyth, M.R., Lunte, C.E., 2017. Determination of phenolic acids in a range of
irish whiskies, including single pot stills and aged single malts, using capillary
electrophoresis with field amplified sample stacking. Anal. Methods 9, 1248–1252.
Wickham, H., Averick, M., Bryan, J., Chang, W., McGowan, L.D., François, R.,
Grolemund, G., Hayes, A., Henry, L., Hester, J., Kuhn, M., Pedersen, T.L., Miller, E.,
Bache, S.M., Müller, K., Ooms, J., Robinson, D., Seidel, D.P., Spinu, V., Takahashi, K.,
Vaughan, D., Wilke, C., Woo, K., Yutani, H., 2019. Welcome to the tidyverse. J. Open
Source Softw. 4, 1686.
Wood, D.E., Lu, J., Langmead, B., 2019. Improved metagenomic analysis with kraken 2.
Genome Biol. 20, 257.
Yang, H., Yu, Y., Fu, C., Chen, F., 2019. Bacterial acid resistance toward organic weak
acid revealed by RNA-seq transcriptomic analysis in acetobacter pasteurianus. Front.
Microbiol. 10, 1616.
Zheng, J., Wittouck, S., Salvetti, E., Franz, C.M.A.P., Harris, H.M.B., Mattarelli, P.,
O’Toole, P.W., Pot, B., Vandamme, P., Walter, J., Watanabe, K., Wuyts, S., Felis, G.
E., Gänzle, M.G., Lebeer, S., 2020. A taxonomic note on the genus lactobacillus:
description of 23 novel genera, emended description of the genus lactobacillus
beijerinck 1901, and union of lactobacillaceae and leuconostocaceae. Int. J. Syst.
Evol. Microbiol. 70, 2782–2858.