Microecological Succession in Flanders Red Ale
Comparative Vessel Construction in an Integrated Microbial Milieu
and its Implications for Appropriate Stylistic
Development and Accurate
Nicholas Andrew Bokulich
Hampshire College Division III Examination, May 2008
Committee: Chris Jarvis, Jason Tor
The influence of fermentation vessel material on the microbial ecology and sensory
profile of a Flanders Red Ale fermented with a commercial mixed yeast-bacteria inoculum was
investigated. Identical batches were fermented in glass, steel, plastic and oak vessels and trends
in microbial growth, pH and titrateable acidity were monitored over a seven month period. At the
end of this period, GCMS paired with controlled sensory analysis were performed to determine
differences in volatile composition and stylistic adherence. The yeast population was best
supported within the barrel and lactic acid bacteria within the steel fermentor; the beer acidified
within the barrel significantly faster than the other vessels, however. GCMS and sensory analysis
determined the barrel fermentation to be the most complex and appealing of the four, though the
steel was considered a better example of the style. The most appropriate flavor development of
this style ale seems to require a fermentation within wood, though a steel fermentor with the use
of controlled inocula can produce an excellent example of the style. Traditional blending is a
necessary aspect to conserve for proper crafting of this style ale
Deep within the Northwestern Flanders region of Belgium a peculiar sort of ale is
produced and consumed that is quite unlike any other of the modern age, known in America
most commonly as Flanders Red Ale (FRA). Rich, malty and fruity, it can resemble some other
Belgian beer styles (if any generalization might be made) but, due to an extended secondary
fermentation and aging process in oak casks harboring a community of wild yeast and bacteria,
this beer takes on a delicate tartness and layered aromatic complexity reminiscent of an aged
Burgundy wine. This unique product and its methods of production may be reminiscent of older
brewing traditionsI, before the introduction of stainless steel and pure yeast cultures (44).
It is the semi-spontaneous fermentation of FRA and the unique community of microbes
responsible for this process that so distinguish it from other alcoholic fermentations. Otherwise
the brewing schedule and primary fermentation of a FRA are quite typical, compared to other
In particular, FRA may hearken back to the origins of London Porter, traditionally a blend of an aged, soured Old
Ale, a fresher Mild Ale and a darker, alcoholic ale, generally the brewery‘s strongest, stoutest offering, at the time
known as a Stout. Rodenbach Klassiek, a blend of the young, light sour ale directly from the secondary fermentation
and the Grand Cru, the higher gravity, oak aged product, underlines this correlation. Alexandr Rodenbach, the
founder, studied brewing in Northern England in the early nineteenth century prior to establishing his own brewery
and likely adopted the methods which he learned to the production of his own brew. (10, 69)
beers. A base of light malt, supplemented with low levels of a dark, aromatic caramel malt and
10-12% of maize adjunct, is mashed using a semi-decoction method through the addition of the
boiled adjunct. It is boiled for ~1.5 hours with the addition of bitter Belgian hops (typically
varieties of Northern Brewer, Brewers Gold, Target or Yeoman) to bring the beer to 14-16
European bittering units (EBU). It is then clarified and cooled (10). A proprietary strain
composed mostly of Saccharomyces cerevisiae is harvested and serially re-inoculated into
successive batches. FRA normally undergoes only brief primary (7 days at 16-21°C) and
secondary fermentations (4-5 weeks at 15-21°C) in stainless steel vessels, before an extensive
tertiary fermentation (20-24 months) aging in giant oak casks (44). Saccharomyces performs the
primary stage, alcoholic fermentation, consuming most wort mono-, di- and trisaccharides and
oxygen to produce ethanol, carbon dioxide, and a variety of secondary metabolites, such as esters
and other flavor compounds; by quickly consuming the majority of directly utilizable carbon
(saccharides) and making the solution rather alcoholic and anaerobic, they make the environment
much more stringent, restricting the growth of undesirable contaminants, while selecting for
others. What makes the repitched yeast slurry of FRA so special is that it contains a mixture of
―contaminant‖ lactic acid bacteria (LAB), primarily Lactobacillus and Pediococcus spp., which
are carried over to each successive batch and, under anaerobic conditions optimal to their growth,
are responsible for the production of lactic acid and subsequent acidification of the beer (44, 80).
From here the fermentation profile of FRA better resembles that of lambic or gueuze (which are
produced by completely spontaneous fermentations), demonstrating periods of ecological
succession dominated by the different yeast and bacteria (76), though, unlike lambic, it is
transferred (‗racked‘) to a secondary and then a tertiary fermentation in oak (a similarly
important process in winemaking), where the beer is further modified by the activity of wild
microbes resident in the wood (though not detectable within the inoculant; 44). This process of
racking is an important feature that distinguishes FRA from lambic and gueuze, removing the
young beer from the lees of (primarily) dead and dormant Saccharomyces, which otherwise
autolyse, releasing such intracellular components as enzymes (e.g. esterases), fatty acids and
amino acids into the beer, providing nutriment to the other microbes present (35, 58) and
transforming the organoleptic profile considerably (79). The trends of microbial succession
demonstrated in FRA (44), lambic (76), ciders (47, 71) and wines (28, 33, 54, 56, 59) are central
to the typical flavor development of these beverages and imbalances in the microbial community
will directly effect an imbalance in the compositional and thus complete sensory profile, through
insufficient or excessive production of esters, fatty acids, phenolics, organic acids, alcohols and
other components, disturbing not only the aromatic character but also the physical properties of
Especially relevant to the tertiary fermentation are the wild yeast of Brettanomyces spp.,
(teleomorph Dekkera) generally regarded with dread as a contaminant of beer (32), wine (57)
and softdrinks (38), primarily due to their production of acetic acid (13, 14), tetrahydropyridines
(―mousy‖ off-flavor; 66) and volatile ethylphenols, which are responsible for the typical ―brett‖
charactersII (12); they are, however, crucial to the production of FRA (44) and lambic (76). In a
bygone age, Brettanomyces was even considered essential to the fermentation of a proper old
English Stock/Export beer (3).III It is non-spore-forming and displays a range of morphologies,
from ovoid to elongated and ogive (pointed-end), often forming pseudomycelia and branched
chains. Several species have been identified, all isolated from different alcoholic fermentations
(32), B. bruxellensis, B. lambicus being the primary strains in the fermentation of FRA and
lambic (44, 76); differences between growth rate, metabolic breadth and secondary metabolite
production (11, 12) could be important to the spoilage potential of each species (or, inversely,
their contribution to the flavor development of FRA) but little work has been done on the effect
of different strains on the organoleptic profile of fermented beverages. This yeast demonstrates
Custer‘s effect (negative Pasteur effect), exhibiting greater fermentative ability under aerobic
conditions than under anaerobic conditions (32). This yeast displays particularly slow growth in
culture and cannot compete with Saccharomyces for simple sugars present in wort (1), especially
under conditions of anaerobiosis (14) and alcohol stress (45); even if slow to reproduce, it
exhibits extreme long-term resistance to environmental (i.e. ethanol, acidity) stress (64) and
nutrient limitation (2) and plays a dominant role in the later stages of lambic (76), wine (57) and
FRA (44), likely because it is capable of hydrolyzing residual starches and polysaccharides
Primarily 4-vinylphenol (4VP) and 4-vinylguaicol (4VG) (pharmaceutical, wet plaster), derived from enzymatic
decarboxylation of hydrocinnamic acids by cinnamate decarboxylase; and 4-ethylguaiacol (smoke, bacon) and 4-
ethylphenol (animal, horse sweat, leather, barnyard), formed by the activity of vinylphenol reductase on 4VG and
4VP (26). Hydrocinnamic acids and other phenolic acids (which can be biotransformed similarly, though the
products are of less libation-spoiling notoriety) are associated with plant cell walls and are extracted during
mashing/maceration in the production of beer (Vanbeneden et al., 2008) and wine (12).
Before rigorous, modern sanitation techniques and the advent of stainless steel fermentation vessels,
Brettanomyces. was likely unavoidable in most any ale, especially those aged in wood, which we might observe to
be its preferred habitat. (44, 56)
present in wort (63) and released by spontaneous hydrolysis of hemicellulose in wooden
fermentation vessels (23), including cellobiose (8, 79); this release of polysaccharides and the
diffusion of oxygen through the wood may explain why in wine fermentation Brettanomyces
seems favored by aging in oak (56). Under anaerobic (i.e. in steel, glass vessels) and semi-
aerobic (i.e. plastic, wooden vessels) conditions, as typical of beer or wine fermentation,
Brettanomyces produces alcohol and low levels of acetic acid (or higher levels corresponding to
the level of aerobiosis; 13) and a range of secondary metabolites, such as volatile phenols (12),
acetaldehyde (14), tetrahydropyridines (66), ethyl esters (68), fatty acids (67) and, through the
expression of α- and β-glucosidases, may release otherwise non-volatile, glycosidically-bound
flavor compounds from fruits, herbs (i.e. hops) or spices, generating unique flavors (19, 79).
Much of the volatile composition of lambic is thought to be developed by Brettanomyces (68,
76) and, though nothing is known of the volatile composition of FRA, it can, by analogy, be
assumed that species of Brettanomyces also play a central role in the flavor development of this
beer, as they form a stable population with Pediococcus in the later stages of FRA fermentation
(44), as observed in lambic (76).
Pediococci are gram positive, homofermentative, lactic acid-forming cocci that divide to
form pairs or tetrads along two planes (61). These bacteria are nutritionally fastidious and
microaerobic (37) but commonly found to be beer spoilage bacteria, especially strains of
Pediococcus damnosus, which displays particular resistance to the harsh conditions of beer: low
pH, ethanol and the addition of antimicrobial hop compounds (61). These bacteria spoil beer by
producing, in addition to lactic acid, copious amounts of diacetyl (60) and some strains produce
exopolysaccharides composed largely of a β –D-glucan which raise the viscosity of the beer and
can create an unpleasant ―ropiness‖ (83). These bacteria can withstand high alcohol (14%) and
low pH (>3.8) conditions and, as a contaminant, can effect super-attenuation of beer by the
fermentation of dextrins and starch (3, 37); α- and β-glycosidases isolated from spoilage and
oenological strains of Pediococcus are responsible for the hydrolysis of different
oligosaccharides and starches, as well as the release of glycosidically-bound flavor compounds
(34). In wines these bacteria are part of the community of LAB responsible for malolactic
fermentation (MLF) (27) and seem to be capable of producing volatile phenols such as
vinylphenols (12) and vanillin (7) from hydrocinnamic precursors. Both P. damnosus and P.
parvalus could be isolated from FRA (44), in which they form, as observed in lambic (76), a
stable ecology with Brett in the later stages of the fermentations. The repetition of this ecological
pattern is probably not a coincidence: it has been suggested that Brett and Pediococcus interact
synergistically within the barrel to hydrolyze dextrins and starches (44) and exopolysaccharide
―rope‖ spoilage is eliminated in the presence of Brettanomyces (77).
The genus Lactobacillus is the largest of all LAB, with species involved in the production
of a variety of fermented foods and beverages. Physiologically diverse, the genus is
characterized by gram positive, lactic acid producing rods. They are facultative anaerobes,
preferring the absence of oxygen and exhibit generally fastidious growth habits (37). The few
species that are capable of growth in beer are the most frequently encountered beer spoilage
organisms (60), though also found in and exerting a positive fermentative influence in such
beverages as FRA (44), wine (27), whisky (74) and dolo and pito, two West African sorghum
beers (62). Interestingly, lactobacilli seem to play a minor role in lambic, Pediococcus being the
prime souring agent (76). L. brevis, the most prevalent beer-spoiling lactobacillus, grows
optimally around 30°C at pH 4-6 and displays a rude resistance to hop compounds.
Heterofermentative, it can utilize a range of substrates, producing lactic acid, diacetyl and a
range of other organic acids and volatile compounds (60), and superattenuate beer through the
hydrolysis of dextrins and starch (20). Lactobacilli play important roles in MLF in wine (42) and
some strains have been shown to be capable of producing volatile phenols, especially
vinylphenols, from hydrocinnamic precursors, though one strain, L. plantarum, has also
displayed moderate ethylphenol production (12), a trait fairly unique to Brettanomyces (65). The
presence of LAB during MLF in toasted barrels has also been shown to increase the
concentrations of certain volatile wood-derived components, such as vanillin and isoeugenol (21,
22), though the conversion of these volatiles from their phenolic precursors has proven limited in
vitro (7). α- and β-glycosidases produced by certain strains of Lactobacillus can hydrolyze
glycosidically bound aromatic compounds (mostly plant-derived), imparting a unique flavor to
the beverage (34). The presence of different LAB has also been shown to influence the retention
of aroma compounds (especially hydrophobic volatiles) in liquid emulsions through direct and
indirect interaction (43), suggesting that beverages (and foods) produced in the presence of a
healthy population of these strains would develop and conserve a more complex organoleptic
profile. Several species of Lactobacillus have been isolated from FRA: the homofermentative
strains L. delbrueckii, L. bulgaricus, L. casei, L. plantarum and L. cornyniformis and the
heterofermentative L. brevis. These bacteria are important for the acidification and flavor typical
of FRA (44).
Following brief primary (7 days at 16-21°C) and secondary (4-5 weeks at 15-21°C)
fermentations in stainless steel vessels, a nascent FRA spends the vast majority of its
development aging in a tertiary fermentation (20-24 months) in giant oak casks. These casks
permit the diffusion of some oxygen into the beer, influencing the growth of different microbial
populations within; as oxygen slowly diffuses through the wood, creating a concentration
gradient, the microbes colonize the porous structure of the wood (72), perhaps forming a distinct
population gradient corresponding to oxygen requirement/tolerance; developed over great spans
of time and dependent upon the structure of the wood, this intricate microecology may be
impossible to support under different fermentation conditions and recreated with mixed inocula.
Additionally, even though these casks are used and re-used, compounds present in the oak or
formed by charringIV may influence these microbial populations. It has been demonstrated that
certain Brettanomyces spp. can utilize cellobiose as a carbon source (8, 63, 79) and that strains of
LAB may metabolize vanillins and other oak compounds into desirable flavor attributes (21, 42).
Certain aspects of the process of malo-lactic fermentation, well studied in wineV, may even be
relevant to the aging and appropriate flavor development of FRA. Studies of malolactic
fermentation in wine (21, 22) and cider (24) suggest that this process and, analogously, growth of
the LAB responsible are stimulated by fermentation in oak as opposed to steel vessels. The age
of these vessels may affect the microbial flora within, which may either establish themselves or
find the conditions of new or used barrels more or less favorable (41). Additionally, compounds
(i.e. organic acids, tannin hydrolysates, phenolic aldehydes, lactones) derived directly from the
Charring is a standard cooperage practice whereby the wood is cured by the application of controlled heat.
Different temperatures and exposure times are indicated for barrels intended for different alcoholic beverages.
Charring releases a variety of aromatic aldehydes, such as vanillin and syringaldehyde, from the degradation of
lignin. The charred surface also absorbs undesirable flavor congeners such as sulphur compounds from the aging
libation as desireable components are released (16).
Malolactic fermentation is a secondary fermentation observed in wine 2-4 weeks following the primary (alcoholic)
fermentation. It is performed by ethanol and acid resistant LAB, particularly Oenococcus oeni but also species of
Pediococcus and Lactobacillus, which suddenly bloom to populations of 10 7-108 CFU during this period, and is
basically the biotransformation (actually a decarboxylation, not a fermentative process; 42) of L-malic to L-lactic
acid, which produces CO2, reduces acidity and increases the pH (27). The metabolism of citric acid to acetoinic
compounds (i.e. diacetyl) is also involved, as well as the production of methylglyoxal and other flavor compounds;
vegetative aromas pass over to fruity aromas, astringency is reduced, color stabilized, and the ―strong green taste of
malic acid is replaced by the less aggressive taste of lactic acid‖ (42). For a review of this process as moderated by
LAB, see 40. In sum, malolactic fermentation is not only characterized by this reaction but also as a phase of
oenomicrobiological succession by LAB. Has not this phenomenon, then, already been observed in lambic (76) and
wood itself may be important to the flavor of FRA; such compounds have been shown to be
relevant to the organoleptic development, activity and stability of wine (9, 23, 31) and whisky
(53; 16), as well as flavor release during tasting (17, 18). Certain oak-derived compounds are
diminished by repeated use of the barrel (9) but studies have found comparable volatile
constitution and correspondent organoleptic profiles in wines aged in once-used and new barrels
after MLF (22, 31). Oak casks are difficult and expensive to produce and maintain, however,
and the establishment of stable resident microflora within new barrels is a variable and elusive
process, as opposed to modern cylindro-conical steel fermentors, which are more affordable to
furnish, clean and maintain and may be sterilized, allowing for the generation of consistent
batches for some fermentations, with the use of regulated inocula (73). Thus the significance of
oak becomes a central question regarding the production of FRA, particularly to brewers and
homebrewers attempting to recreate this special style outside of Flanders—where the indigenous
microflora colonize the wood spontaneously—necessitating the use of commercial strains of
inocula, which inevitably represent a considerably less subtle microbial community.
The influence of fermentation vessel construction on the microbial succession and
organoleptic characteristics of FRA was studied through the fermentation of an identical FRA
wort in four separate vessels constructed of different, commonly used materials (oak barrel, glass
carboy, HDPE plastic, stainless steel keg), using a commercial mixed inoculant. The microbial
growth and succession was monitored over seven months by regular plate counts on selective
media and with microscopy. PCR and sequencing of 16S rDNA was also used to identify an
uninoculated bacterium isolated from the barrel. The pH and titrateable acidities were regularly
tested in order to detect acid production in the beer, an obvious physiochemical characteristic of
an acid ale, which demonstrates the parallel growth and activity of acid producing bacteria
(primarily LAB). To the author‘s knowledge, the volatile composition of FRA has never been
studied; the complete organoleptic characters of the four separate batches were examined by gas
chromatography mass spectrometry (GCMS) paired with controlled sensory evaluation.
Materials & Methods
1. Generation of Red Ale
As the primary cylindro-conical fermentor had a maximum capacity of only 14 gallons, two
separate batches of 10 gal each were produced and blended evenly within four separate
fermentation vessels (thus 5 gallons per), producing homogeneity between the green beer
entering each vesselVI, though necessitating a delay in sampling until blending was performedVII.
1.1. Grist Composition and Hop Load
The net grain bill consisted of 41lb German two row pils malt, 17lb German Vienna malt, 2lb
cararoma, 1.5lb caramunich and 3lb flaked maize, totaling 64.5lb. The net hop load, with boil
durations (see Boiling Schedule) as follow: .5oz whole Magnum flowers, 165 min; 1oz whole
East Kent Golding flowers, 165 min; 2oz whole Saaz flowers, 15 min. This is the sum total from
two separate ten gallon mashes and a five gallon supplementary batch. Though the grist and hop
additions varied slightly between mashes to accommodate for the market availability of certain
specialty grains and hop shortages, all brewing schedules were performed identically and all wort
produced was distributed evenly between the four fermentation vessels. All grains were stored at
21°C (70°F) in a dry, dark environment before use and only milled immediately before mashing.
All hops were obtained vacuum packed and stored at 12°C (54°F) in darkness before use.
1.2. Mash Schedule
All mashes were done on a ―Brew-Magic‖ system (SABCO) according to the equipment
protocol provided. The grain, freshly ground, was placed within the mash tun and covered with
water, heated to ~49°C (120°F), to a depth of ~1 inch above the grain bed by slowly back-filling
by siphon from the sparge tank. The mash was heated to 65.5°C (150°F) in the mash tun and
maintained at this temperature for 2 hr by recirculation through the internal heating unit. At this
point, recirculation was ceased and the wort was lautered to the kettle by siphon, whilst sparging
with water heated to ~74°C (165°F).
1.3. Boiling Schedule and Cooling
The blending also standardized the green beer, so that small but important differences in batches, such as SG, and
others more difficult to measure, such as IBUs and chemical composition (of saccharides, amino acids, terpenoids
and other color and flavor compounds, especially maillard intermediates), which are directly related to SG, would
not alter the fermentation performance in any vessel.
It may be assumed that, prior to the blending of the two batches, any samples extracted would be irrelevant to the
main body of data, being representative of a partial and uneven wort, difficult to synthesize in communion with
other data to provide a complete picture relative to samples taken post-blending. I realize now, however, that these
data points could have been valuable to detecting populations present within the primary fermentation, influencing
pH and titrateable acidity even if irrelative to the trend data.
For each 10 gal batch produced, 12 gal of sweet wort were brought to a slow, rolling boil and
maintained so for 165 min, additions of whole hop flowers made as described above. This
resulted in 10 gallons of green beer yielded per boil, which was immediately siphoned through a
Therminator (Blichmann) heat exchanger into the primary fermentor, cooling the beer to ~21°C
(70°F). Pure oxygen was then diffused into the beer through an oxygen stone at 10 psi for 30 sec
and the beer was pitched with 100ml Belgian Sour Mix I WLP655 (White Labs). This yeast-
bacterial mix is reported to contain strains of Saccharomyces, Brettanomyces, Lactobacillus and
Pediococcus; therefore, for our purposes, we may assume that organisms detected and observed
to display characteristics of these genera may be considered to be such, whereas those displaying
aberrant characteristics may be considered mutants or contaminants. The original gravity (OG)
of the first batch was 1.040 (target gravity 1.050). To compensate for the apparent reduced mash
efficiency, the grain bill of the second mash was proportionately altered to obtain the target
gravity of 1.060, resulting in a true OG of 1.050, as intended, after blending.
The first primary fermentation lasted 5 days before equal division into the four secondary
fermentation vessels: a clean used 6 gal glass carboy, a new 5 gal barrel constructed of French
oak (Quercus sessiliflora), a clean used 6 gal food-safe plastic ―brewer‘s bucket,‖ and a clean
used 5 gal stainless steel keg. The barrel was sanitized by storing filled with sodium
metabisulfite before use; the other vessels were cleaned and sterilized with iodophor prior to use.
The second primary fermentation lasted 8 daysVIII before even division into the four
secondary fermentation vessels, producing identically blended wort. The specific gravity (SG) of
each beer was recorded at this time, designated as ―week 1‖. At week 8 a five gallon
―supplementary‖ 5 gal batch was produced and evenly distributed between the fermentation
vessels to accommodate for volume lost to sampling and evaporation, especially in the barrel.
The ambient temperature was monitored regularly and remained within 21-24°C (70-75°F)
throughout the entire fermentation. Finishing gravity (FG) was determined when sensory
analysis was performed at 25 weeks.
As the first batch produced was of such low gravity and the second of slightly higher gravity to compensate,
different primary fermentation durations were accorded to each batch to allow for significant attenuation before
racking to the secondary without leaving the primaries to ferment any longer than necessary so that blending, and
thus accurate monitoring, could occur in a timely fashion.
The physical parameters of the different fermentation vessels are outlined in table 1. The
glass carboy and the plastic bucket are clearly of similar shape, having circular bases of the same
radius and straight sides, though the bucket is sealed with a fitted, rubber-lined lid and has more
headspace above the beer, whereas the glass tapers to a bottleneck at the top and has less
headspace. The barrel is not so dissimilar in shape, though the sides bulge so that the radius at
the center of the barrel is ~17cm. The barrel is stored on its side, however, and kept nearly full,
as beer will gradually evaporate from the container through the porous sides; therefore, the
surface area at the liquid-air interface and the surface area above the beer are constantly
changing; there was very little headspace in the barrel, however, as it was topped off to
compensate for evaporation and sampling. The shape of the liquid columns in these three vessels
were similar (considering radial dimension and height, in spite of the configuration of the barrel)
but that of the steel was taller and of less basal area, resulting in less exposure to the headspace
and there was less headspace overall.
2. Sampling Method
All samples, unless otherwise indicated, were collected from the liquid within 1cm of the
liquid-air interface. This was done in a meticulously standardized manner by which a sterile
serological pipette was penetrated through the forming pellicle (gently pushing aside this dense
layer as needed, so as to minimize pellicle intake) at the same regular sampling site, lowered to
the desired depth so as to avoid the pellicle and, after a pause of 5-10 seconds, the desired
volume was withdrawn.IX,X
The choice of sample site from a mixed culture fermentation is difficult; The microbes present are likely to be
dispersed over a range of residential zones, primarily the pellicle, different fractions throughout the fluid column, the
lees or adhered to/colonized within the surface of the vessel. Different microbes would be likely to display different
preferences for these regions and even to display different preferences in the different vessels and at different times
during the fermentation and still their activity, viability and significance to the fermentation are to be questioned
even if they are prevalent within the zone examined. The upper liquid fraction, just below the pellicle, was chosen as
a sort of ‗compromise‘ site; the inhabitance patterns of different microbes in any fermentation is undocumented but,
in general, yeast and bacteria are observed to rise to the top of an active fermentation. Bacteria demand more
speculation, however, for, even if they tend to remain suspended at random throughout a fermentation, as opposed to
displaying a preference for biofilm formation,X we may expect that they will reproduce and form their densest
populations at the sites of greatest nutrient concentration, and so it may be that they localize to the lees, where
sampling and standardized quantification of viable cells (as this is not a suspension, a stratified emulsion, rather)
would be difficult.
Biofilms and pellicles are not formed by all organisms involved in the fermentation, however, and are often
formed in response to certain environmental stimuli, such as oxygen, and would, as a solid composed of microbes,
proteins, polysaccharides and other materials, be difficult to quantify in an especially resultative manner. Hence the
3. Microbial Analysis
The growth and population of the individual microbes were tracked by aliquoting 10 or 100
µl of serially diluted beer samples onto selective media agar plates. Malt agar medium (30g/L
malt extract, 1% agarose), treated with 50 mg/L chlortetracycline (Sigma) to prevent the growth
of bacteria and incubated aerobically at 25°C for 3 days, was used to count the total yeast
population. Lysine medium (Sigma) (66g/L), incubated aerobically at 25°C for 3 daysXI was
used for the quantification of non-Saccharomyces yeast (presumably consisting entirely of
Brettanomyces spp.). These lysine medium plates were augmented with 50 mg/L
chlortetracycline if significant bacterial growth was observed on the plates. MRS medium
(Oxoid) (55g/L, 1% agarose), incubated anaerobically in a vinyl glove box (Coy) under
H2/N2/CO2 (7:80:13, v/v) atmosphere at room temperature for 3-5 days, was used to count lactic
acid bacteria. It was assumed (and microscopically confirmed) that the bacteria growing within
this period were primarily of Lactobacillus spp., whereas the slower growing pediococci were
grown on MRS+ plates (MRS medium with 1.75ml/L of 2-phenylethanol [Sigma]) added to
control the growth of lactobacilli and counted after anaerobic incubation at 25°C for 10 days.
After the allotted growth time had elapsed, colony forming units (CFU) were counted on plates
exhibiting 30-300 colonies. Phase contrast microscopy was used to approximate the identity of
colony forming microorganisms based on morphologyXII. Populations of Saccharomyces were
quantified as the number of CFUs forming on malt agar minus the CFUs forming on the
corresponding lysine medium plate. Phase contrast microscopy was frequently used to visually
examine qualitatively the microbial populations present in different regions of each fermentation
The populations of pellicle forming organisms were examined at weeks 6 and 7, when the
pellicles in each fermentation vessel were observed to have reached peak robustness. This was
done by extracting a portion of pellicle and suspending the solids in 1ml of water with vigorous
agitation. The weight of the liquid was taken before and after to determine the total weight of the
additional need to avoid the inclusion of pellicle particles in the samples taken, as their dense, generally exclusive
community could significantly skew growth counts.
It was noted that this medium supported equally robust and numerous growth when autoclaved (rather than
boiled) and not adjusted to a pH of 5.0 using lactic acid, as is directed by the product information provided.
Though Saccharomyces and Brettanomyces yeasts are sometimes undifferentiable by morphological
identification, they and the bacteria Lactobacillus and Pediococcus, tend to be readily distinguishable.
pellicle suspended. These suspensions were serially diluted and plated on selective agar media as
described above. Results were quantified as CFU/g of pellicle. The majority of the pellicles were
composed of Brettanomyces spp. with minor bacterial portions, so the readout of this data is
difficult, either providing a view of the percentage of the different populations comprising the
pellicle or of pellicle density, as it is composed not only of cells displaying a range of sizes but
potentially also of exopolysaccharides and proteins.
4. Physiochemical Analysis
Titrateable acidity was tested by diluting 0.5 ml of beer in 9.5 ml of DI water and titrating to
pH 8.2 (as detected by two drops of a phenylphthalein indicator, which turns the solution a
charming rosé at this pH, the threshold at which it changes molecular conformation) with 0.1N
NaOH. ApproximateXIII titrateable acidity (g/L of lactic acid) was calculated by the following
T.A. (g/L) = titer NaOH (ml) * 18 (g/ml2)
Regular pH measurements were performed with an IQ125 electronic pH indicator (IQ
Scientific Instruments) calibrated to pH 7 and pH 4 prior to use. Gravity readings were taken
using a hydrometer for original gravity (OG), specific gravity (SG) when the batches were
blended at the start of the secondary fermentation and for the finishing gravity (FG) at 25 weeks.
5. 16S PCR/Sequencing
An unknown bacterium observed in the barrel fermentation was isolated by serial dilution
and plating on MRS agar, as described above. A selected colony was suspended in MRS broth
and replated on MRS agar. A colony grown on this plate was removed, resuspended in MRS
Lactic acid should be the predominant acid in the solution, though a range of other acids (especially acetic acid)
are likely present. Titrateable acidity, which measures total acidity, is based upon the molar ratio of NaOH to acid X
in a homogenous acid solution (as well as the number of protons released into solution by this acid); thus the molar
ratio is skewed by the variable (if minor) presence of other acids, making this an approximate measure.
broth, incubated overnight at 37°C and confirmed by microscopy to be composed only of the
organism of interest (determined by motility as described in the results).
5.2. DNA Extraction
To extract DNA, first the overnight culture was vortexed and 1.5ml was removed and
centrifuged for 2.5 minutes at 13,000g. The supernatant was decanted and 100 μl of lysis buffer
(100mM Tris, 30mM EDTA, .5% SDS [wt/vol]) was added to the pelleted cells. This was
incubated at 100°C for 10 minutes. 100 μl of potassium acetate (3M) was added to the lysate and
incubated on ice for 60min. This was centrifuged at max speed (16,110g) for 5 min and the
supernatant transferred carefully to a new tube, to which was added an equal volume of
isopropanol and again centrifuged for 5 min at max speed. The supernatant was decanted and
500 μl of 70% EtOH added to the pellet. This was centrifuged at max speed for 20 min at 4°C,
the supernatant removed and the pellet dried in the tube inside a laminar flow hood. The pellet
was resuspended in 100 μl of TE/8 (10mM Tris, 1mM EDTA, pH 8.0) and stored at -20°C.
5.3. 16S PCR
PCR was performed using the primers 8F (AGA GTT TGA TCC TGG CTC AG) and 1492R
(ACG GCT ACC TTG TTA CGA CTT) universal bacterial16S rDNA. The PCR master mix
consisted of 0.2 mM dNTP, 0.2 μM of each primer and 50 ng of template DNA prepared in
REDTaq ReadyMix PCR Reaction Mix with MgCl2 (Sigma), brought to a final volume of 50 μl
with sterile DI H2O. The PCR thermocycler (Applied Biosystems GeneAmp PCR System 2700)
program consisted of a 95º C hot start (3 min), 35 cycles consisting of a denaturation step at 95º
C (40 s), annealing at 49º C (45 s) and extension at 72º C (1min), then a final extension at 72º C
(7min). PCR product was analyzed by electrophoresis on a 1% agarose gel in 1X LB buffer with
ethidium bromide (10 mg/l) run at a voltage of 226V for 20 minutes before visualization under
5.4. Amplicon Purification/Sequencing
The PCR product was purified on a QIAquick PCR Purification Kit (Qiagen) using the
protocol provided. The final elution was sent off for automated DNA sequencing at the
Genomics & Bioinformatics Facility at the University of Massachusetts, Amherst with the
primers 8F (AGAGTTTGATCCTGGCTCAG), 786F (CGAAAGCGTGGGAGCAAACAGG)
and 1492R (ACGGCTACCTTGTTACGACTT).
Freshly withdrawn beer samples (12ml) were centrifuged for 5 minutes at 1,000g/min and the
top 10ml carefully withdrawn. To this was added 3ml of a 20μM solution of butylated
hydroxytoluene (Sigma), added as an internal standard, in spectrometry grade pentanes (Acros).
The mixtures were vigorously shaken and allowed to settle as the organic phase separates from
the aqueous and viscous intermediate layers. The organic layer was removed and dried with
sodium sulfate. The extracts were analyzed using a Hewlett-Packard HP6890 gas
chromatography system coupled with a Hewlett-Packard HP5973 mass selective detector. 3 μl of
the extracts were injected in splitless mode into a HP-5 (30.0m x 0.32mm x 0.25μm) 5% phenyl
methyl siloxane column (Agilent). The operating conditions were programmed as follow: The
oven temperature was held at 40°C for 2.5 minutes and then brought to 250°C at a rate of
10°C/min; injector temperature 175°C; carrier gas (He) flow rate 2.9ml/min at 7.72 psi; scanning
rate 50-550 a.m.u.s; electron energy was 69.9eV. The identification of compounds was based on
their mass spectra and retention times, as compared by Agilent G1701 MSD Productivity
Chemstation Software version D.03.xx and confirmed by manual analysis. GC/MS was
performed in triplicate for each sample and the relative concentrations of each volatile compound
determined by the average of the three runs adjusted by the relative chromatograph peak areas of
the internal standard.
7. Sensory Evaluation
After 25 weeks of secondary fermentation, portions were racked from each sample into 22oz
bottles with 1 tsp dextrose and left to ferment further for 12 days at 21-24°C (70-75°F). These
bottles were then chilled to 12°C (54°F) and poured into clean, identical standard pint glasses at
21°C (69°F) and served to a panel consisting of four experienced tasters in a single blind/mute
tasting. The tasters were asked to judge each beer based on the BJCP beer scoresheet
(http://www.bjcp.org/SCP_BeerScoreSheet.pdf) and on ten different key characteristics,
corresponding to the compounds selected for comparison by GCMS analysis, and scored on a 0-
5 scale, 0 being not detected, 5 being prominent. These characteristics were mild fruits, tropical
fruits, grapes, vinous, red fruits, rancid, almond, rose, animal sweat and spice. Elaborated
descriptions of these flavor precepts were provided on the scoring sheet (Appendix A).
2° SG FG Dimensions r/h Surf. A Pellicle D (mm) /V (cm3) , Week: Pop. %
(cm2) 6 16 20 S B L P
G 1.010 1.006 15.24 30.1 729.66 0.1/7.297 0.5/36.49 1/72.97 ND 100 ND ND
B 1.008 1.007 13.97 40.64 825.8* 5/412.9 5/412.9 8/660.64 ND 80.0 11.1 8.9
P 1.006 1.004 15.24 45.72 729.66 0.2/14.59 0.1/7.297 0.2/14.59 23.4 76.6 ND ND
S 1.008 1.005 12.7 50.8 506.71 2/101.3 0.2/10.13 1/50.67 14.0 22.6 19.9 43.5
Table 1. Selected statistics of the fermentation vessels. The OG of the blended, undivided wort was 1.050. Gravity
readings were taken when the wort was blended in the secondary fermentation vessels at day 13 (2° SG) and the FG
when samples were withdrawn for sensory analysis at week 25. Dimensions (radius/total height; all measurements
given in cm) and pellicle depth (D) are provided and the resultant surface area (A) is calculated (A=πr2). From this
the total pellicle volume (V) can be derived (A x D) for different periods during the fermentation. The percentages of
the different microbes comprising the total population at week 6 are calculated at the right (fig.5). (Vessels: G =
glass, B = barrel, P = wood, S = steel; Populations: S = Saccharomyces B = Brettanomyces P = Pediococcus L =
Lactobacillus; ND=None Detected). *Note that the barrel, resting on its side, has a variable surface area,
dependent upon the depth of the ale therein and is not calculated by the equation for the area of a circle; this
surface area is a rough average, the vertical headspace between the pellicle and the bung most often measuring 3-
1. Physiochemical Analysis
Immediately following the blending of the beers in the secondary fermentation vessels (after
8 and 13 days of primary fermentation for the separate batches), the specific gravities of the four
beers had each dropped from the original gravity of 1.050 to 1.010 or below (table 1). 25 weeks
of secondary fermentation hardly resulted in a further reduction of gravity, least of all in the
barrel, which finished at a specific gravity of 1.007. The titrateable acidity (TA) and pH of each
beer was tested regularly to monitor acid production, which correlates to the growth of different
microorganisms as both a product and environmental factor and thus reports/predicts microbial
activity in each vessel. Regression trend lines were calculated to demonstrate the progress of pH
(dashed lines) and TA (solid lines) over time. The pH of the glass, plastic and steel fermentors
did not change throughout the secondary fermentation, hovering around 4.5 (fig.1). The pH in the
barrel slowly descended to 4.0 by week 16 and did not significantly change thereafter. The TA
did not rise significantly in any vessel between weeks 1 and 9, then suddenly rose around 2g/L in
each vessel at week 10, and very slowly growing thereafter in the glass, steel and plastic, likely
in response to the wort addition at week 8. The TA of the barrel rose sharply, reaching an
approximate value of 11g/L of lactic acid by week 20, more than double that of the other vessels.
The TA in the glass and plastic was almost parallel, leveling off around 5g/L lactic acid by week
11; the TA of the steel continued to rise after this time, though, to approximately 6g/L lactic acid
by week 25. It should be noted that each vessel was topped off with 1.25 gal of fresh wort at the
beginning of week 9 to compensate for sampling and evaporation (especially in the barrel). This
was done before samples were taken, which explains the decrease in TA, but seems to have
subsequently resulted in a sudden increase in TA in each vessel around week 10.
4 TA B
3.5 TA P
g/L Lactic Acid
2 pH P
1.5 pH S
0 5 10 15 20 25
Fig. 1. TA and pH. Titrateable acidity was tested by titration with 0.1N NaOH to pH 8.2 as indicated by
phenylphthalein. pH was determined using an electronic indicator (IQ Scientific Instruments). pH data correspond
to the right Y axis and TA data to the left. Regression trends were calculated to demonstrate changes in pH (dashed
line) and TA (solid line) over time for each vessel. The addition of fresh wort at week 9 is indicated by the arrow.
(G=glass: blue ♦; B=barrel: red ■; P=plastic: yellow ▲; S=steel: green X).
2. Microbial Analysis
The processes of microbial succession in the four fermentation vessels are outlined in figures
2 and 3. In each vessel, Saccharomyces yeast were present in high number during the first
several weeks of the secondary fermentation (fig. 2). Their population in the plastic bucket at
week 2 was over ten times that of the other vessels (3.9X106CFU/ml) before falling by week 3
(2.9X105CFU/ml) to approximately match those of the glass and steel fermentors (2.2X105 and
5.3X105CFU/ml, respectively). The glass and steel fermentors displayed similar growth patterns,
multiplying between weeks 2 and 3 before returning at week 4 to populations similar to those
counted in week 2. The population of Saccharomyces in the barrel was roughly similar to that of
the glass and steel at week 2 (5X104CFU/ml) but, by week 3, exceeded that in all other
containers by a factor of 2 to 5 (1.28X106CFU/ml); this extended to week 4, at which time the
population was more than 10 times that of the glass and plastic and 5 times that of the steel. By
week 7 no Saccharomyces could be detected by culturing from any of the vessels, though
diminishing quantities of large, granulated cells, typical of senescent Saccharomyces, were
observed by microscopy, before disappearing entirely by week 9. Observations made between
weeks 14-16 detected more such granular, ovoid cells in each vessel, again in low number,
though this number had increased to ~10% of the total yeast observed in the steel keg. At weeks
19 and 20 these yeast comprised 50% of the total yeast observed in beer drawn from the steel keg,
though only ~25% of the total at week 23. These observations correspond with counts taken on
malt agar plates; no Saccharomyces were detected weeks 7 through 16, when these yeast
reappeared on the malt medium in moderate number (8.6X102CFU/ml). At week 20 these yeast
(1.6X103CFU/ml) did comprise approximately 50% of the total yeast (Brett: 2.4X103CFU/ml),
as observed microscopically. Following their secondary peak population (2.8X103CFU/ml) at
week 18, the population of these yeast plateaued and then slowly declined again between weeks
23 and 25. Although yeast morphologically resembling Saccharomyces were observed
periodically in some of the other vessels, especially the plastic, in which these yeast appeared to
comprise ~20% of the total yeast population by microscopic analysis at week 23, they were not
detected by culturing methods.
0 5 10 15 20 25
Fig. 2. Growth of Saccharomyces. From one week following racking the beer to the secondary fermentation, the
growth of Saccharomyces yeast was monitored by culturing serially diluted beer samples extracted from each
fermentation vessel on malt agar plates. These were incubated aerobically at 25°C for 3 days before colony forming
units were counted. (G=glass, B=barrel, P=plastic, S=steel)
In the first weeks of the secondary fermentation, Brettanomyces appeared in each vessel,
quickly overtaking the Saccharomyces populations. None of these yeast were detected at week 1
but suddenly spiked at week 2, reaching populations of between 2.4X105 (glass) and 9.3X105
(barrel) cells/ml at week 3 (fig. 3). In the weeks following this, no data regarding the growth of
Brettanomyces were obtained until week 13, though they were detected in high number in the
rapidly developing pellicles. At week 13 the population in the glass (3.24X106CFU/ml) more
than doubled that in the barrel (1.83X106CFU/ml) and tripled that in the plastic
(1.23X106CFU/ml); the population in the steel was well below this (2.52X105CFU/ml). This
may have been due to misread data (see discussion), as the population in the glass diminished by
nearly a factor of 100 by week 16 (4.1X104CFU/ml), around which it hovered for the remainder
of the period during which it was monitored. The population in the barrel remained
approximately as numerous, though, slowly increasing and only beginning to decline at week 20,
whereas the populations in the other vessels had been in decline since week 13; thus
Brettanomyces in the barrel displayed a sustained growth phase, a delayed death phase and a
population 5 (week 16 plastic) to 50 (week 16 glass) times as numerous as that in the other
vessels throughout this period. The plastic displayed similarly enhanced growth—5-10 times that
in the glass and steel weeks 13-18 (assuming misread data for the glass week 13)—but no delay
of the death phase, as the population began to decline from week 13 until relatively matching the
populations of the glass and steel at week 20. Except for week 13, the populations of Brett within
the steel and glass vessels remained similar throughout the fermentation through week 20 and
slowly declined in the range of 2.5X105 and 2.4X104CFU/ml (steel) between weeks 13 and 20.
At week 23 the population in the barrel continued to decrease (3.0X105CFU/ml) and the steel
remained in relatively low number (3.75X104CFU/ml) but the glass and plastic suddenly
increased dramatically, multiplying by approximately a factor of 70 in parallel. During this time,
the pellicles in these containers were entering a new phase of growth, thickening and beginning
to heal after a period of decline from weeks 13-18) and microscopic observations found that the
yeast populations in these vessels had increased from three weeks before and were markedly
greater than those in the barrel and steel. At week 25, however, these populations declined again,
as the pellicles in these vessels ceased to grow, and the stable population of the barrel was once
again the most numerous. The population in the steel also remained approximately stable, 1/10 as
numerous as that of the other vessels (6.3X104CFU/ml).
0 5 10 15 20 25
Fig. 3. Growth of Brettanomyces. From one week following racking the beer to the secondary fermentation, the
growth of Brettanomyces yeast was monitored by culturing serially diluted beer samples extracted from each
fermentation vessel on lysine medium plates. These were incubated aerobically at 25°C for 3 days before colony
forming units were counted. (G=glass, B=barrel, P=plastic, S=steel)
Bacteria were observed in abundance in the beers during the first weeks of the secondary
fermentation and initially cultured in high number on selective media plates but consistent counts
were unsuccessful as the population first fluctuated so rapidly and then became difficult to
culture or disappeared from the beer later in the fermentation. Some counts were successfully
taken from the wooden and steel vessels, however, as they exhibited cultureable bacterial growth
(and observable activity) after week 10 (as well as during the tumultuous early time points). In
particular, a certain motile rod first observed in the barrel after week 6 was readily cultured on all
growth media and so consistent counts were performed. At week 1, rod-form bacteria
(presumably lactobacilli) were detected in the wooden and steel vessels at 8.0X103 and
3.7X104CFU/ml, respectively (fig.4). Cocci were also detected in the wooden and steel vessels at
2.9X104 and 2.5X104CFU/ml, respectively. At week 2, cocci grew on MRS+ plates from the
steel and plastic only, at numbers of 3.8X104 and 6.4X104CFU/ml, respectively, but lactobacilli
were only cultured from the plastic at 2.9X106CFU/ml. At week 3, 1.3X106CFU/ml of cocci
were counted in the steel. At week 6, a large number of lactobacilli (1.46X108CFU/ml) and
pediococci (3.19X108CFU/ml) were counted in the pellicle of the steel keg; a somewhat smaller
number (9.11X106 lactobacilli and 6.69X107CFU/ml pediococci) were counted in the pellicle of
the barrel at week 6 (fig.6). After this point, both cocci and rods were observed in each vessel.
After week 8, however, their numbers dwindled and they were rarely observed in any of the
vessels, and none were successfully cultured on selective media. After week 16, however,
pediococci were observed at an apparently higher number in the steel and they were finally
counted week 18 and subsequent weeks between 1.0X103 and 1.1X104CFU/ml. Rods also
reappeared at weeks 20-25 growing on all growth media agar from the steel, though no growth
was optically confirmed in the beer itself. Motile rods continued to be counted in the barrel,
peaking at week 16 (7.2X106CFU/ml) before slowly declining. In figure 4 both motile and non-
motile bacilli counted in each vessel are plotted in the same series for the barrel, though those
detected at week 1 are likely lactobacilli and thereafter Acetobacter.
1.00E+04 S rods
1.00E+03 B Pedio
0 5 10 15 20 25
Fig. 4. Bacterial growth. . From one week following racking the beer to the secondary fermentation, the growth of
Pediococcus and Lactobacillus spp. were monitored by culturing serially diluted beer samples extracted from each
fermentation vessel on MRS+ and MRS agar, respectively. These were incubated anaerobically at 25°C or 37°C for
3-10 days before colony forming units were counted. (B=barrel, P=plastic, S=steel)
These data seem to correlate with microscopic observations performed regularly throughout
the fermentation; a sudden bloom of yeasts displaying morphologies typical of Brettanomyces
(fig.5) overtook the declining populations of Saccharomyces-like yeast in the first weeks of the
secondary fermentation, accompanied by many cocci and rods. By week 9 these were the only
yeasts observed in the vessels and the cocci and rods had nearly disappeared, except for many
small, motile rods observed only the barrel. Pellicles began to form over this period (see below).
Initially the majority of these yeasts displayed the irregular ellipsoidal and elongated
morphotypes in each of the vessels, very few filamentous forms and pseudohyphae observed.
After week 8, however, the populations in the different vessels exhibited diverse mixtures of
phenotypes. In general the yeast became smaller and more irregular in each vessel and the
ellipsoidal/elongated population passed over to a strongly elongated/filamentous population
around week 9 and pseudohyphae were often observed in each vessel. The pellicles became
denser and fuller during this period. Starting at week 13, however, fewer pseudohyphae were
seen and the populations became less dense, less filamentous and more elongated and even
smaller over the following weeks and the pellicles in the glass, plastic and steel vessels began to
thin, healing less from sampling damage and even breaking apart in the plastic and steel. Though
each vessel followed this general trend, marked differences distinguished each vessel. In the
glass and plastic, yeast tended to be larger, thicker and exhibit more ellipsoidal/elongated forms
than the barrel and steel, which tended to be more irregular, smaller, thinner and exhibit more
filamentous and dark, ―salsicciaform‖ yeasts. The barrel and steel keg also contained more
apiculate, ogive and abnormal yeast. After week 12, disperse cocci and rods were irregularly
seen in each vessel (except the glass, in which only cocci were observed, rarely), though much
less so in the barrel, as it was still populated by the motile rods, making distinction between
―swimmers‖ and ―non-swimmers‖ difficult. Over the following weeks, however, the population
density of motile rods decreased and nonmotile rods and cocci became apparent in the barrel,
though in low number. The bacteria remained in very low number in the other vessels, especially
the glass and plastic, and the rods showed no signs of growth, though clusters and tetrads of
cocci were observed and their population in the steel keg seemed to increase, in parallel with
their reappearance on agar plates.
Fig. 5. Typical morphotypes of Brettanomyces as observed in FRA in four different fermentation vessels . Clockwise
from top right: Filamentous/pseudohyphal (P7); small, dark salsicciaform (S7); Irregular elongated, large
salsiccioid (P14); “gourdform” elongated composing pellicle (G7); Irregular ellipsoidal: ogive and apiculate (P7
lysine medium); Elongated (P7). All images are of samples taken from the different fermentation vessels at different
times, as indicated in parentheses; Bar = 10 μm. (G=glass, B=barrel, P=plastic, S=steel.)
The pellicles formed in each vessel also displayed distinct characteristics. These remained
consistent throughout the fermentation and a parallel growth trend was established in common
between each vessel. By week 5, the pellicles formed in each vessel and continued to grow until
week 12. They were eggshell white and were primarily composed of filamentous and
pseudohyphal yeast and many rods and cocci in the barrel and steel keg, though less so in the
plastic and glass. The pellicles thinned between Weeks 13 and 18 (January-February), except in
the barrel, which became less foamy white and more gummy and yellow. The others healed from
sampling scars less and began to break apart. At this time, the pellicles were composed of fewer
filamentous yeast and more ellipsoidal and elongated yeast. Following this was another phase of
growth and the pellicles began to thicken and heal once more but a common trend ceased to be
apparent. The pellicle in the glass was a powdery matte white in the first phase and uniformly
formed of long, filamentous yeast, giving way to large, elongated yeast as it began to thin after
week 13. After week 18 it demonstrated robust regrowth, thickening and healing rapidly and
again becoming matte white, though composed of small, clear ellipsoidal and large elongated
yeasts. Bacteria were almost never detected in this layer. In the barrel, it formed a thick, gummy,
―elephant hide‖-like layer thicker than 1cm at certain points. Though it followed the common
trend, in the barrel this layer tended to be composed of more filamentous and pseudohyphal yeast.
It was mottled beige and ochre and a filmy, yellow substratum was found between the thick,
hide-like superstratum. This layer was composed almost entirely of motile rods and
ovoid/ellipsoidal yeast. With time, the population of the pellicle became composed more of
smaller irregular and salsiccioid yeast, as seen in the beer during that time. In the plastic, the
pellicle formed a very thin, papery, white layer during the first weeks. Opposite the barrel, but
similar to the glass, the composition of the pellicle in the plastic leant to a majority of large,
irregular ellipsoidal, gourd-form yeast. After thinning and beginning to break up, it formed again
as a hazy, latex-like, filmy layer around week 20 and composed primarily of slightly smaller,
elongated, gourd-form yeast. Bacteria were rarely detected in this layer after week 9. The pellicle
in the steel keg was rough, solid, opaque tan and ropy by week 6. It was composed of
filamentous yeast and many rods and cocci during this time. After week 9 it began to heal less
from sample scarring and proceeded to thin and break up. At this time more elongated yeasts
were seen than filamentous, following the general pattern, but many Saccharomyces were also
seen in the dissolving pellicle, as in the beer itself. After being so degraded and broken to be
described as ―pitiful‖ at week 18, it suddenly rebounded by week 20, forming a stable, elastic
layer, matte white speckled with yellow. It may be noted that, as with the barrel, the white and
yellow zones exhibited starkly different microbial compositions; whereas the white layer was
almost entirely elongated, light salsiccioid yeast, the yellow layer was a roughly even mixture of
bacterial cocci, clustered Saccharomyces-like yeast, filamentous yeast and small, dark salsiccioid
G6 B6 P6 S6
Fig. 6. Microbial composition of the pellicles at week 6. Sections of pellicle were removed from each vessel with a
sterile loop and placed in pre-weighed milliliters of sterile H2O. These were disassociated and suspended by
agitation, serially diluted and plated on selective media as described for the quantification of microbial growth (figs.
1, 2; Methods 3). The units of the resulting data are in CFU per gram of pellicle.
The physical dimensions (thickness, volume) of the pellicles in each vessel at different points
in the fermentation are presented in table 1, along with their relative microbial compositions at
week 6 (% total volume, calculated from table 1 and fig. 6). The pellicles in the glass and plastic
vessels tended to be thinner, less voluminous and more homogenous, containing no cultureable
bacteria at week 6 and very few afterwards. The pellicles of the barrel and steel keg were thicker
and more voluminous (especially the barrel) and composed of a mixture of organisms. It is
interesting to note that the total populations of the glass carboy, the barrel and the plastic bucket
are relatively similar whereas that of the steel keg, though a fraction of the volume of the barrel,
is 8 times as populous per gram and considerably more evenly diverse.
3. 16S PCR/Sequencing
After 6 weeks of fermentation a motile, ellipsoidal rod, approximately 1 x 1-3μm, occurring
singly or in pairs, was observed in the barrel. This bacterium grew in a facultatively aerobic
fashion and formed small (usually <1mm), rough, round colonies, translucent on lysine medium
and slightly hazy on MRS agar and malt agar. Analysis of the 16S rDNA sequence revealed that
this bacterium is in the genus Acetobacter, most likely of the species A. lovaniensis. This species
is distinguished by previous studies as a catalase positive, oxidase negative, gram negative rod
(0.4-1.0 X 1.5-2.0µm), generally motile by peritrichous flagella; most strains produce acid from
ethanol, D-glucose, D-mannose, D-galactose, L-arabinose and D-xylose (though not D-fructose,
glycerol, trehalose, sucrose, maltose, lactose or starch, among other sugars), oxidize acetic acid
and lactic acid to CO2 and H2O and utilize primarily the Q-9 ubiquinone system (39). It was
originally isolated from soil in Belgium (15).
Fig. 7. Relative volatile concentrations as determined by GCMS. Samples of FRA drawn from each vessel were
extracted in pentanes containing 20μM butylated hydroxytoluene as an internal standard and injected into a
Hewlett-Packard HP6890 gas chromatography system programmed as described above. Results were obtained in
triplicate and the average relative concentrations, determined by the internal standard, are adjusted to fractions of
the most abundant sample, set at a value of 1; standard deviation is indicated by the error bars.(G=glass, B=barrel,
Samples of beer were withdrawn from each of the four fermentation vessels and extracted in
pentanes for GCMS analysis, as described above. Of the volatile compounds detected, eleven
were selected for comparison, based upon their relevance as flavor compounds and absolute
abundance as detected by gas chromatography (APPENDIX B). The concentrations of these
compounds were not quantified but relative concentrations (relative between the four samples)
were determined by the use of a butylated hydroxytoluene internal standard. The esters isoamyl
acetate (―mild fruit,‖ banana, pear), ethyl hexanoate (tropical fruit) and diethyl succinate (vinous)
were most abundant in the barrel, especially isoamyl acetate, which was at least twice as
abundant in the barrel as in the other vessels, and diethyl succinate, none of which was detected
in the glass (fig.7). The concentrations of the fatty acid ester ethyl hexanoate in the glass, plastic
and steel vessels were all around 70% that of the barrel. The other fatty acid esters detected,
ethyl octanoate (―red fruit,‖ apple)and ethyl decanoate (grape, apple), were most abundant in the
steel vessel, followed by an 80% relative concentration in the barrel, then the glass and finally
the plastic, less than 50% maximum relative concentration. The fatty acid octanoic acid (rancid)
was also detected in each sample but only in significant abundance in the glass (exhibiting about
as much absolute abundance as ethyl octanoate, data not shown). Benzaldehyde (bitter almond)
was also significantly more abundant in the glass than in the other vessels, followed by the
plastic (60%), the barrel (40%) and finally the steel (30%). 4EG and 4EPII seem to be in greatest
concentration in the glass and plastic, significantly so at least in the case of 4EP. 2-phenylethanol
(rose) was found to be most abundant in the glass and barrel, the plastic and steel exhibiting
around 80% relative to these.
5. Sensory Analysis
Sensory analysis was performed on the four beers produced by this experiment to determine
the stylistic accuracy (table 2) and, in conjunction with GCMS data, the influence of vessel
construction upon the organoleptic profile of FRA (fig.8). The beer fermented in the steel vessel
was given the highest average score of each beer according to the BJCP Beer Scoresheet,
followed shortly by the barrel (table 2); those fermented in the glass and plastic were scored at
least 11.5 points less (of 50 points). The glass and plastic produced mild, sweet, low-acid beers;
those from the barrel and steel were fruitier, more complex, more acidic and adhering better to
the stylistic guidelines overall. The barrel in particular had a sharp, acidic character, rich
fruitiness and vanilla/oaken characteristics, as well as the most crimson in color. The acetic
character was slightly too high and sharp and the beer thusly lost a few points in the flavor
category. The steel was the only carbonated beer of the four and was distinguished by a
refreshing, light lactic character and bright fruitiness. Three of four judges remarked that this
beer was noticeably more alcoholic than the rest in aroma, flavor and overall impression. The
plastic was hazy, losing appearance points, though the rest were brilliantly clear. It was also
darker in color and had a particularly mellow, round flavor, reminiscent of raisins, prunes or
other dark, dried fruits and one taster noticed a distinctly kiwi-like flavor but this beer was less
fruity and three of four judges considered it to be the least interesting. The beer from the glass
vessel was mellow, lightly fruity, very lightly acidic and drinkable, though considered dull. It
had a nose of animal sweat and solvent.
Aroma Appearance Flavor Mouthfeel Overall Impression Score
Crisp, fruity, Brilliant clarity; Mild, malty, light Flat, smooth, low Mild, round, fruity,
G acetone, caramel, bourbon;, no acid, green, plum, body; no carb.; very drinkable if dull, very low
solvent, horse head/carb. cherry, cardboard low acidity acidity
Acetone, grape, Brilliant clarity; dark Acetic, balsamic, red Flat, med. Body, Acetic; complex, full,
B champagne, red, oxidized copper; no cherry, black cherry, round, buttery, full; profound, oaky, like
wine vinegar head/carb. grape, plum, pear, no carb.; sharp, dry, Rodenbach Grand Cru;
rose, vanilla, oak. sour, coating favorite
Cider, musty, Hazy; deep copper, Sweet, dark fruit, Mild, med.-low body, Sticky, sweet, dark, mildly
P barn, sweet, murky; no head/carb. malt, raisin, cassis, round, dull, flat, interesting and pleasant but
black cherry mild, dull, kiwi, dried sticky; no carb.; no dull
fruit, paper acidity
Fruity, Pepper, Brilliant clarity; new Alcohol, fruit, Low body; low carb,, Crisp, pleasant, rich,
S green apple, penny, caramel; no pepper, cherry, grape, full.; light acidity, refreshing, most alcoholic,
apple, cherry head/carb. cassis, phenol, rich, crisp, dry; refreshing fruity, spicy; like Monk‘s
pie, alcohols light citrus Sour Red
Table 2. Summary of analysis/comparison of FRA via BJCP Beer Scoresheet. Compiled descriptions, summary and
scores for FRA produced in four fermentation vessels of different composition judged by four experienced tasters
using the BJCP Beer Scoresheet. The scores provided are averages and are a total measure of stylistic accuracy
(determined by BJCP Guidelines; 70) decided by the points accorded each parameter.
(http://www.bjcp.org/SCP_BeerScoreSheet.pdf). (G=glass, B=barrel, P=plastic, S=steel; carb.=carbonation).
According to the tasting precept scoresheet (Appendix A), the ale produced in the barrel had
the most complex character, spread across all 10 flavor precepts except for almond, and the
plastic the most diminished, especially in the rose and mild fruit categories (fig.8). In particular,
significantly higher levels of grape, vinous and red fruit-like characters were detected in the
barrel, each scoring at least ~3 (described as apparent on the scoresheet). Plastic and steel both
scored ~2 for grape and ~2.5 for vinous; they scored 1.5 and 2, respectively, for red fruit. Glass
only had faint notes of grape and red fruit, though an apparent vinous quality. Notes of rose and
mild fruit (pear, banana) were also detected in the barrel, followed within one point by the glass
and steel (i.e. faint detection opposed to modest detection). Modest notes of spice were noted in
the steel and barrel though only faintly in the glass and plastic; animal sweat was slightly more
detectable in the glass and plastic (.5 points or less). Significantly more almond was present in
the glass, only faintly detected in the steel and plastic and almost none in the barrel. A faint to
modest note of tropical fruit was described in all vessels. A faint rancid character was noticed in
the barrel and plastic, though hardly in the glass or steel.
Spice 3 Tropical Fruit
Animal Sweat Grapes G
Almond Red Fruit
Fig.8. Comparison of specific organoleptic profiles. FRAs fermented in the four different fermentation vessels were
scored and compared by a four-judge panel according to ten selected flavor precepts corresponding to compounds
analyzed by GCMS (Appendix B). Judges were asked to score each beer on a scale of 0-5, a score of 0 denoting
negative detection and 5 a dominant characteristic (Appendix A); the averages of the four scores are reported above.
(G=glass, B=barrel, P=plastic, S=steel).
As reported by Martens et al. (1997), the general trend of fermentation of FRA seems to
involve a week-long primary dominated by Saccharomyces and LAB, resulting in a swift decline
in fermentable sugars and a slight increase in acidity. In the initial weeks of the secondary
fermentation, these yeast and bacteria decline in population. In the tertiary fermentation, as wild
yeast of the genus Brettanomyces appear in abundance, LAB flourish anew. After the initial
bloom, the population of both LAB and Brettanomyces spp. decline and stabilize around 103-105
cells/ml for the remainder of the fermentation, as lactobacilli disappear and Pediococcus emerge
as the predominant LAB. This general fermentation profile is conserved in the four fermentation
vessels in this experiment. The major difference is in populations of LAB: after an apparent
initial bloom, these bacteria all but disappeared from the sampling point (summit of fluid column,
below pellicle) in each vessel and the reemergence of Pediococcus was only observed in the steel
vessel. This may be explained by the diffusion of oxygen in each vessel. The semi-permeable
walls of both the wooden and plastic vessels permit the diffusion of oxygen, which hinders the
growth of the fastidious Pediococcus, but the steel and glass are impermeable, cultivating a less
aerobic environment; the glass vessel, however, having a greater surface area and larger
headspace, as well as being closed by a rubber stopper and water trap (semi-permeable) as
opposed to the air-tight fittings of the keg, would be exposed to more oxygen than the steel
vessel, especially considering that the regular sampling times would dilute the CO2 headspace
with oxygen. Therefore the steel keg, being exposed to very light doses of oxygen absorbed from
the headspace, may approximate the dissolved oxygen concentrations of the massive (reducing
surface area by volume and thus oxygen diffusion), oldXIV oaken tuns traditionally used for the
fermentation of FRA, promoting the growth of LAB. Martens (1997) further proposes that a
fermentation in wood, selecting for Pediococcus, enhances the growth of Brettanomyces by
allowing the symbiotic interaction of these organisms, degrading residual starch and dextrins.
Correlating to this hypothesis, the yeast of the genus Brettanomyces do indeed seem favored by
the conditions in the barrel, nearly 10 times as populous in this vessel as in the other vessels at
most points throughout the fermentation, though Pediococcus was rarely observed in significant
number. The absence of this bacterium may be due to a number of factors (see below) but, in any
case, some feature of the barrel fermentation is obviously enhancing the growth of
Brettanomyces; this may be in part due to the semi-porous nature of the wood, providing this
yeast with oxygen, stimulating their growth, as the population in the semi-porous plastic bucket
also demonstrated enhanced growth at weeks 13 through 18, though another factor must be
responsible for the remarkably sustained, fervid growth of the Brettanomyces within the barrel.
This may be due to the hydrolysis of starch, dextrins and other compounds released from the
wood, with or without the aid of Pediococcus. Though enumerated for only a few weeks in the
beginning of the fermentation, Saccharomyces was similarly favored within the wood and plastic,
compared to the glass and steel, suggesting again that the semi-porosity of these materials
promotes the growth of yeast. Under the actual semi-spontaneous conditions by which true FRA
is produced, wild yeast other than Saccharomyces spp. and Brettanomyces spp. would not be
excluded from sharing in this boon and may subtly contribute to the character of FRA as they do
As a barrel ages, the pores of the wood grain begin to seal and clog with solids, diminishing gas diffusion.
in the spontaneous fermentations of lambic (76) and wine (54; 56); indeed some wild yeasts such
as Candida spp. have been detected in the earliest stages of FRA fermentation (44) but the
influences of such yeasts on the flavor of FRA and other fermented beverages have yet to be
Though the yeast populations within the steel keg were of lesser number than those in the
other vessels, it must be noted that a second bloom of yeast displaying characteristics typical of
Saccharomyces occurred only in the steel. This partially explains the diminished numbers of
Brettanomyces yeast in this vessel compared to the glass (which should be similarly anaerobic
aside from the hypothesis that the beer fermenting within the carboy absorbed more oxygen from
the headspace, which would better support the yeast community), as a portion of the physical and
metabolic niche of Brettanomyces was occupied by this yeast. In other words, a very limited
biomass may be supported by the physical dimensions of the fermentation vessel (6) and the
availability, composition and concentrations of nutriment contained therein would further restrict
the rate of consumption, growth and sustenance of different members of the microbial
community. Just as Brettanomyces spp. can cause stuck fermentation in wine through substrate
competition with Saccharomyces during alcoholic fermentation (6), in the steel keg this very
same interaction may be working in reverse in this later stage of fermentation, which is usually
dominated by Brettanomyces. Considering the low gravity of the wort at this time (1.005), this
may have taken the form of a quasi-parasitic antagonism, whereby the Saccharomyces is
consuming the oligosaccharides released from starches and dextrins hydrolyzed by the activity
Brettanomyces. The yeast may even be producing its own glucosidases, in which case it is most
likely not the inoculated strain of S. cerevisiae, rather a wild strain of Saccharomyces or another
yeast. An origin as an environmental contaminant would explain why this reemergence was
unique to the keg. Another explanation would be that the greater conditions of aerobiosis in the
other vessels so promoted Brettanomyces growth that other yeasts were ―crowded out‖ and had
not the opportunity for growth in the face of such competition late in the fermentation. Even an
amensal interaction could have been responsible, such as if the Brettanomyces yeasts (or
contaminants) in the other vessels were creating toxins or killer factors, limiting the growth of
other yeasts. Brettanomyces can withstand a great degree of environmental stress, such as low
pH, high acidity and high ethanol concentrations, whereas Saccharomyces yeast and most
contaminating yeasts are sensitive to such toxins as ethanol and organic acids, especially in
combination, as in a sour ale (64). The low pH and high acidity in the barrel may explain the
absence of Saccharomyces-like yeast in the late fermentation but these conditions were very
similar in the glass, plastic and steel (fig.1) and cannot account for the difference between these
vessels. The differences in bacterial growth may, however, as the second bloom of
Saccharomyces-like yeast in the steel vessel paralleled the reemergence of LAB; these bacteria
may have succored these yeast through the excretion of growth-stimulating long chain fatty acids,
essentially reviving a stuck fermentation (67; see below).
Not only were Saccharomyces-like yeast not cultured from the glass or plastic vessels in
the late fermentation, as in the steel, but the yeast in these fermentors present tended to be
homogenously large (3X7µm) and elongated, as opposed to the vastly mixed morphotypes,
primarily of small, dark, salsicciaform yeast, observed in the barrel and in the keg (fig.5).
Different environmental conditions, such as carbon and nitrogen sources, oxygen, or
environmental stressors may affect the distribution of morphotypes displayed by some yeasts
(50; 51). The pH of the steel was the same as that of the glass and plastic (~4.5) and the TA was
not significantly greater (~6g/L steel; ~4.5g/L glass, plastic, week 25), though the barrel had both
significantly lower pH (~4.0) and higher TA (~11g/L, week 25). Some other aspect of the glass
and plastic vessels is altering the mixed fermentation dynamic. Fusel alcohols can induce
pseudohyphal formation in some yeast (25) but no fusel alcohols were detected by the GCMS, as
most are too volatile and do not separate and become overshadowed by the extraction solvent
(see below). Polymorphism in the genus Brettanomyces is not well documented; perhaps other
compounds, such as the esters determined by GCMS to be most abundant in the barrel and steel,
could invoke such changes. These could, moreover, be entirely different species of
Brettanomyces displaying diverse morphologies, selected by the conditions present in these
vessels. Bacteria also tended to be cultured only from the steel and barrel and so a distinct pattern
of population diversity forms. It might not be a common condition, per se, that the plastic and
glass fermentors share, however, rather a similarity of differences from the barrel and steel: that
these latter vessels, through two different means (supporting elements of the wood, increased
anaerobic conditions of the steel) sustain healthy populations of bacteria whereas the former
vessels, lacking these conditions, select against the bacteria. Yeast-bacterial interactions,
orchestrated by the conditions present in the steel and in the barrel, may be influencing or
supporting the diversity and/or morphological characteristics of the yeast. If certain species of
Brettanomyces perform best in conjunction with a healthy bacterial community, as Martens
suggests (1997), whereas other species retain ―independence,‖ the glass and plastic vessels
would obviously select accordingly. The barrel is the only vessel that can sustain a microbial
population within its walls, though, and in the steel the population of Brettanomyces, if not that
of the bacteria, would likely decline in population through successive batches without repeat
inoculation of commercial strains.
The culturing of LAB in this experiment proved difficult and, as no significant difference
in pH or TA was observed in the steel vessel, in which growth was observed, and as the acidity
was perceptively increasing in each vessel, the cell counts may have been due to the location of
the sampling site. Perhaps the anaerobic conditions within the keg allowed the localization of
more cocci to the surface of the fermentation, whence all samples were drawn, whereas oxygen
diffusing into the headspace of the other vessels dissuaded the growth or localization of LAB to
this stratum. Moreover, the generally fastidious nature of the bacteria, the greater aerobic
conditions of the smaller fermentation vessels (versus those normally used for FRA
fermentation) and the harsh conditions of the fermenting ale may have lulled the bacteria into a
state of non-cultureable viability, in which the cells are metabolically active but cease growth
and thus are not detected by culture-dependent methods (i.e. plating) (46). The greater growth of
yeasts within the glass, barrel and plastic may also be causing the bacteria to flocculate or
coflocculate to the bottom of the vessel, away from the sampling point (52). TA, then, may be
used as a much better indicator of bacterial activity. Interestingly, though LAB were cultured
from the keg in high number (1.0X103-105CFU/ml) after week 16, the pH and TA readings taken
at this time were not significantly different from those of the glass and plastic. By week 25 the
acidity of the steel (~6g/L) may have exceeded that of the other two (~4.5g/L), suggesting
moderately enhanced growth of LAB. Presumably, these bacteria were active in the primary
fermentation, for the beers already possessed a very mild lactic acid aroma when racked to the
secondary fermentors, and so the first readings, taken after the beers had already reached primary
attenuation,XV were likely elevated above the original acidity of the wort, and continued to
slowly increase thence. The sudden increase of acidity at week 10, after a wort addition at week
8, demonstrates the immediate responsiveness of the LAB to an available carbon source. That
The limit of attenuation reached by Saccharomyces, e.g. after most of the mono-, di- and trisaccharides present in
the wort have been consumed (3).
only the TA of the barrel continued to increase significantly beyond this point may be due to
activity of Brettanomyces and LAB consuming sugars and other useable substrates released from
the oak (21, 23), organic acids leached from the wood (17) and concentrated by evaporation (4-
5% total volume/year; 48) or the growth of the Acetobacter found swimming within the oak.
The Acetobacter isolated from the barrel was not advertised as an intentional component
of the inoculum and may have originated as an airborne environmental contaminant, as the
aseptic conditions under which the vessels are opened for sampling are not absolute: the
fermentation vessels are large and cumbersome and thus cannot be used under a flame or laminar
flow hood and their openings, which are thusly sterilized by ethanol, are large and often must be
kept open for up to a minute while samples are being drawn; it is a wonder, then, that the steel
was not contaminated first, its aperture being the largest, as well as the most difficult to sterilize,
obstructed by an array of metal parts comprising the lever lock and keg handles. Another source
of this contaminant could be from the wood itself, as certain bacteria and moulds are wont to
inhabit the surfaces of unused barrels, including species of acetic acid bacteria (55). In any case,
Acetobacter contamination occurs later in the fermentation of commercially produced FRA and
contributes to the overall profile of the beer (44) though their premature appearance may have
negatively influenced this ale and their singular presence in the oak certainly skews the
significance of the results. For example, the differences in TA and pH may be due to the
production of acetate, which would influence the microbial community and the organoleptic
profile: LAB growth would be inhibited by the low pH, extracellular enzymatic efficacy would
be altered and ethanol would be oxidized to acetate. As a regular tier of the microbial succession
of FRA, however, it may be that these alterations are stylistically normal and therefore, that their
population, not supported by the conditions (i.e. oxygen) present in the other fermentors (60),
signify a divergence from the normal fermentation profile by each of the other vessels. This
statement softens the impact of this contaminant on the significance of the data but can hardly be
accepted completely: the barrel used in this experiment is considerably less voluminous that the
gigantic oaken tuns in which FRA is usually fermented, increasing surface area by volume and
thus oxygen diffusion, supporting the premature growth of acetic acid bacteria; emerging so
early in the fermentation, before late-phase conditions such as high ethanol concentration, low
pH and low gravity can check their growth, these bacteria would be considered a spoiling
contaminant. After seven months of fermentation, however, the acetic character contributed by
this bacterium is only slightly in excess and, seeing as its population proceeded to decline after
week 16, further activity may be negligible.
The GCMS data uncover interesting differences in the volatile compositions of each of
the experimental beers, shedding further light on the processes of microbial succession within the
consortium. The relative concentrations of the esters isoamyl acetate (banana, pear), diethyl
succinate (floral, vinous) and ethyl hexanoate (pineapple, citrusy, sour apple) were significantly
enhanced within the barrel compared to the other vessels. The biosynthesis of esters by S.
cerevisiae is performed by a variety of enzymes which condense two substrates, an alcohol and
acyl-Coenzyme A (acyl-CoA), such as the alcohol acetyltransferases (AATase), responsible for
the formation of alcohol acetates, the most abundant and important esters in beer; the major
limiting factors to this reaction are substrate concentration and strain-specific expression of the
associated genes and any effect on these factors will modulate ester production downstream (82).
Factors affecting ester synthesis include wort composition, temperature and AATase (or related
gene) expression, which is both strain-dependent and modulated by external factors such as
oxygen and unsaturated fatty acids (81). The original wort composition and ambient conditions
(i.e. temperature) were identical for the four fermentation vessels and so significant differences
in sugar, nitrogen and lipid contents may not be expected, unless if uneven microbial growth and
autolysis contributed AATase-repressing fatty acids to the fermentation. Oxygen also represses
AATase expression but is also necessary for yeast growth and fatty acid catabolysis; whereas a
highly oxygenated wort would hinder ester production, insufficient oxygenation would limit
yeast growth, ultimately reducing total ester production (81). The concentration of dissolved
oxygen likely differed between each vessel, as discussed previously. Therefore, the meager
quantity of isoamyl acetate in the plastic may be due to the constant permission of excess oxygen,
downregulating AATase expression, whereas the barrel may admit just enough to enhance yeast
growth without accumulating to unacceptable levels. The glass and steel, having the least yeast
growth, produced fewer esters, though without the oxygen inhibition experienced in the plastic.
Furthermore, the selection of different species of yeast and bacteria by the diverse conditions of
the fermentors would alter ester formation (as well as that of other flavor compounds) according
to the species-specific regulation of the genes involved (esterases, metabolic enzymes). For
example, the robust growth of Brettanomyces, in particular, may explain the high relative
concentrations of certain aroma compounds in the barrel. The plastic vessel, though exhibiting
the second greatest population of Brettanomyces, has a low ester profile due to esterase
inhibition by oxygen (while stimulating yeast growth).
The concentrations of 4EP and 4EG, which are only produced in significant quantity by
Brettanomyces spp. (12, 65), are highest in the glass and plastic, despite the lesser growth of
these yeast in these vessels compared to that in the barrel. These data suggest that the conditions
within these vessels promote the production of these phenolic compounds. This may be due to
strain-specific conversion of these compounds from their phenolic acid precursors; different
strains of Brettanomyces may exhibit different potentials for the formation of 4EG and 4EP (78)
lending further evidence to the hypothesis that the patterns of similarity in yeast morphology and
4EP/4EG concentration in the glass and plastic versus the barrel and steel may correlate to the
presence of entirely different strains of Brettanomyces in these fermentors.
The biosynthesis of 2-phenylethanol and benzaldehyde in these beers seems to display
signs of similar strain-specific regulation. The pattern of abundance of these compounds, both
products of phenylalanine degradation, differed from each other and from that of the selected
esters, fatty acids and 4EG and 4EP and may be partially explained by environmental conditions.
Unfortunately, the conversion pathways of these compounds by most or all of the microbes
relevant to FRA fermentation remain unclear. Under the conditions of FRA fermentation, the
production of 2-phenylethanol may result either from Ehrlich pathway catabolism of
phenylalanine (29) or release from glycosidically bound compounds (36). Garavaglia et al.
(2007) found that the bioconversion of 2-phenylethanol by the enological yeast strain
Klyuveromyces marxianus was influenced by pH, temperature, medium composition and
increased oxygen. Garde-Cerdan and Ancin-Azpilicueta (2006a) found that this alcohol was
produced in much higher concentration in wines fermented entirely spontaneously, i.e. that wild
yeast convert/release this compound at an exceptionally high rate and thus, in the absence of the
competition of S. cerevisiae, more is present as more substrate is left to the conversion of wild
yeast. The temperature and wort compositon of the vessels were identical (though compounds,
such as sugars, released from the wood of the barrel may influence reaction rates) but the pH,
oxygenation and microbial communities differed considerably. The glass carboy and the barrel
had the greatest concentrations of 2-phenylethanol. The glass had lowest populations of
Saccharomyces in the early fermentation, along with the steel, as indicated by fig.1 and by its
relatively high SG (1.010) at the start of the secondary fermentation (table 1), but did not
experience a reemergence of this yeast and also hypothetically contained more dissolved oxygen
than the steel. The plastic fermentor had a very high initial population of Saccharomyces (fig.2),
which, it seems, consumed the majority of wort nutrients (including nitrogen, though this is
indicated by its low SG of 1.060; table 1), leaving fewer to the wild yeast. The barrel, in spite of
its sustained Saccharomyces growth in the early weeks of the fermentation (fig.2) and lower pH,
produced nearly as much of this alcohol as the glass vessel from the sheer mass of the
Brettanomyces population, further enhanced by modest levels of oxygen diffusion. The steel
experienced two phases of Saccharomyces competition, the lowest population of Brettanomyces
and hypothetically very low levels of oxygen, resulting in the lowest yield of 2-phenylethanol.
Nierop Groot and de Bont (1998) report that benzaldehyde is yielded by a non-enzymatic, Cu
(II)-catalyzed oxidation of phenylpyruvate, the product of phenylalanine transamination, in L.
plantarum cellular extracts; they found the rate of this reaction to occur proportionally to
temperature (optimum 37°C), pH (8.0), cation (especially copper) availability and oxygenation.
Once again, temperature and pH were not diversifying factors, though oxygen was. Cation
residues in the vessels may have been significant to this delicate reaction but were not measured
and are unpredictable. The glass contained considerably higher concentrations of benzaldehyde
than the other vessels: the plastic exhibited 60% relative concentration and the barrel and steel
less than 40%. The plastic/glass enhancement seems to suggest a strain dependency to the rates
of phenylpyruvate bioconversion or bioelimination, the dominant yeast strain in these fermentors
being either particularly adept at the transamination of phenylalanine or particularly poor at its
decarboxylation, leaving a bottlenecked excess ripe for clearance via chemical oxidation to
benzaldehyde. The semi-permeable plastic bucket would be expected to excel in this oxygen-
dependent reaction but the research examining this reaction has all been performed in vitro and
therefore oxygen may exert a different influence in vivo. Additionally, similar to 2-phenylethanol,
wild yeast may well convert more benzaldehyde in the absence of Saccharomyces competition,
as phenylpyruvate would be the first catabolic product. The glass, being the least attenuated by
this yeast, would leave the most substrate to Brettanomyces consumption, resulting in an even
greater pool of phenylpyruvate for further degradation as compared to the well-attenuated
plastic: having less substrate, the yeast would uptake less phenylalanine at a slower rate, convert
less phenylpyruvate and thus provide less substrate for chemical oxidation to benzaldehyde.
Yeast and bacteria exhibit species-dependent differences in fatty acid metabolism, yeast
generally producing more medium-chain fatty acids (MCFA) (C6-C12) and bacteria tending
toward longer chain fatty acids. Brettanomyces spp. have been shown to produce high quantities
of octanoic (C8) and decanoic (C10) acids (especially the latter) in comparison to Saccharomyces
spp., (67), which release primarily octanoic acid and lesser quantities of hexanoic (C6) and
decanoic acids (4). MCFAs seem to be released by yeast when fatty acid biosynthesis and thus
cell growth is ceased under anaerobiosis; this event is associated with stuck fermentation, which
may be relieved by the addition of higher fatty acids, reinitiating fatty acid chain elongation (4).
The higher chain fatty acids produced by bacteria in pure culture are consumed by yeast in the
mixed fermentation of lambic, stimulating growth, evincing the benefits of a healthy microbial
consortium (67). Another fate of MCFAs in wort is esterification, producing a more highly
volatile aromatic compound. The reason for this reaction is unclear but it has been proposed as a
means of recycling free CoA from the surfeit of acyl-CoA left by the termination of fatty acid
elongation at the incession of anaerobiosis (5). The greater concentration of ethyl hexanoate in
the barrel, compared to the ~80% relative concentration detected in all of the other vessels, is
clearly due to some condition unique to the barrel; this may be an indirect influence, such as the
enhanced growth of Brettanomyces in this vessel, the pH or acidity, or a direct factor related to
the wood, such as the semi-aerobic environment or presence of compounds leached from the oak.
As the concentration of this compound is similar in the plastic, glass and steel, it is likely that the
enzyme catalyzing the production of this ester is not as sensitive to oxygen as other esterases.
Ethyl octanoate and ethyl decanoate, on the other hand, are most abundant in the steel, ~80% as
abundant in the barrel, then the glass (65% and 50% maxima, respectively), and least of all in the
plastic (50% and 30% maxima, respectively). The conservation of this pattern suggests that these
compounds are produced by the same or similar enzymes at similar rates, equilibria and ambient
conditions, though the lower pH of the barrel may be inhibiting the production of these esters.
This enzyme also seems highly sensitive to oxygen, hence the low relative concentrations in the
plastic. Why the steel has so much more than all of the other vessels, then, is likely due to the
anaerobic environment and the late bloom of Saccharomyces-like yeast. More, and better, data
regarding fatty acid concentrations would be useful to understand what relative quantities of
MCFAs are being esterified under the variable conditions of the fermentors in order to elucidate
this process. For now, highly variable data seem to indicate a much higher concentration of
octanoic acid in the glass carboy, compared to the other fermentors, and other MCFAs were not
detected under the GCMS conditions used.
A range of other compounds, such as ethyl lactate, ethyl acetate and phenylethyl acetate,
important flavor compounds in lambic (75), were not detected in these ales. This may be due to
the extraction and GCMS protocols. Pentanes may be too non-polar to extract many of the
compounds present and overshadows the most volatile compounds present in the beer, which
exhibit the same retention time. The use of headspace sampling, a longer GC column and/or
different running conditions may better separate highly volatile compounds (such as ethyl
acetate) from the extraction solvent for adequate detection. Additionally, this may be due to the
immaturity of these experimental ales, which are meant to age for periods of nearly three years.
During this period the flavor of such a beer changes dramatically as the beer acidifies and flavor
compounds are enzymatically and chemically formed and hydrolyzed as they approach
equilibrium (68). Young as the beers examined in this experiment are, and containing so little
lactic acid, it is not surprising that ethyl lactate has not formed to detectable levels, nor more
subtle but influential flavor congeners.
Sensory evaluation is an analysis of the ultimate product of fermentation as it is presented
to and perceived by the consumer and therefore of pinnacle importance to brewer. Olfacto-
gustatory perception is a phenomenon so multivariate to be beyond complete experimental
quantification and prediction, though connection of sensory analysis to GCMS, TA and growth
studies completes the perspective of the product as being directly influenced by the chemical
composition, which is, in turn, modulated by microbial activity and ambient conditions. By
evaluating these beers, the effect of experimental variation (i.e. of differential vessel construction
on the organoleptic profile of FRA) is experienced directly. The analysis of these beers using the
BJCP Beer Scoresheet determined that the steel fermentor produced the most stylistically
accurate beer, though the judges unanimously agreed that the barrel-fermented ale was their
favorite of the four. This is partially due to the steel being the only carbonated beerXVI;
carbonation is of pivotal importance not only to mouthfeel but also to aromatic and flavor
retention/release and as counterpoint to the sweet, bitter and sour modalities. Therefore, the ale
fermented in the steel retained style points in each category lost by the other ales, though the
Likely due to the residual Saccharomyces population; the other vessels would either require more time for
tertiary fermentation in the bottle or the addition of priming yeast during bottling.
barrel was the most flavorful, profound and complex; this beer contained the greatest breadth and
balance of phenolic, fruity, vinous and floral notes, as well as flavors derived from the wood,
such as vanilla, oak and spice. The beers from the glass and plastic not only scored poorly,
lacking the fruity tang of the style, while overexpressing phenolic off flavors, but were also
considered dull, though drinkable, overall. The plastic exhibited some curious traits: it was not
only darker in color (though not red, rather an orange, ―deep copper‖) but also possessing a deep,
round flavor, principally of dark, dried fruits; one judge also described paper and another kiwi.
These traits are typical of aged, dark beers and signify thorough oxidization of the beer, further
evidence of the high permeability of the HDPE plastic vessel. The kiwi flavor, alternatively, may
be a key to the flavor of phthaleins detected only this beer by GCMS (data not shown),
plasticizing agents likely leached from the plastic walls of the fermentor. Interestingly, two
judges, commenting independently in the overall impression category, compared the barrel and
steel-fermented ales to Rodenbach Grand Cru and Monk‘s Sour Red Ale, respectively, two
commercial varieties exemplifying the dual polarity of FRA: a heavy, oaken, vinous, dark, sweet
and sour beer opposed to a light, fruity, effervescent, tangy one. Further aging is required to
determine the effects of fermentation vessel selection on the flavor development of a fully
mature FRA but these data already distinguish major differences between these ales, which are
unlikely to reduce to convergence with time. After completion, careful blending, as is traditional
to achieve maximal flavor balance in FRA, may well be appropriate to reduce the acerbic
qualities of the barrel whilst utilizing its fruity, oaken complexity unmatched by the other vessels.
The comparison of the sensory analysis and GCMS data of the different beers uncovered
interesting differences between absolute volatile abundance and physical perception. None of the
volatile compounds examined by GCMS were quantified, only relative concentrations given, so
it is unknown whether the concentrations of these compounds exceed their individual flavor
thresholds, though the phenomenon of perception is far more complex than threshold values:
different concentrations will be perceived as entirely different flavors and synergize with other
volatiles and non-volatiles present, even at sub-threshold levels, coalescing to a novel flavor. The
predominance of certain aromas in the beers may also overshadow more subtle ones and change
the overall profile, as will a multitude of whimsical physical conditions, such as association and
retention with non-volatile compounds, oral pH, temperature, carbonation, lingual motility,
breathing rate, mood and individual perceptions. Moreover, individual interpretation of tasting
session prompts, intersections of precept descriptors and the ambiguity of scalar score
descriptions (e.g. of prominence versus dominance) may result in confusion between such
flavors as rancid and horse sweat or red fruit, grapes, and tropical fruit. Moreover, any beer
changes during bottle refermentation and so the volatile compositions may have altered between
the GCMS analysis and tasting session. Any number of such physical and mental variables may
have resulted in the disparities between taster perception and the resulting contradictions with
GCMS data. For example, the ambiguous, broad descriptor vinous, correlated here to the
abundance of diethyl succinate, is almost as apparent in the glass, in which none was detected, as
in the barrel, which actually contained the greatest relative concentration of this compound.
Similarly, spice was most apparent in the steel and barrel, whereas the plastic and glass
contained slightly more of the corollary component, 4EG. This may be due to such oak
compounds (structurally similar to 4EG) as guaiacol (smoke), isoeugenol (clove, wood) and
eugenol (clove) in the oak, or the contrasting carbonation in the steel, being perceived as spice.
Animal sweat, however, was a character detected slightly more in the plastic and glass,
correlating to the higher relative concentrations of 4EP in these vessels. The barrel and plastic
were also described as the most rancid (though only faintly so), the flavor appended to octanoic
acid, which was in significantly greater abundance in the glass; again, the oak-derived
compounds or acetic acid present in the barrel and the oxidization compounds or phthaleins in
the plastic may be contributing a flavor perceived as such. The respective descriptors of the fatty
acid esters ethyl octanoate and ethyl decanoate, red fruit and grapes, were most apparent in the
barrel, followed by both the plastic and steel, and least of all in the glass, which differs widely
from the abundance of these compounds as determined by GCMS: The steel had the greatest
relative concentration of both these compounds, followed by ~80% in the steel, ~50-65% in the
glass and ~30-50% in the plastic. This analysis may have been skewed by such other volatiles as
ethyl acetate, probably most abundant in the barrel (through analogy to the similarly regulated
ester, isoamyl acetate, as well as according to scoresheet comparisons), or oxidization products
present in the plastic, in which these esters may well not even exceed their flavor thresholds
(considering their relative abundance), as they rarely do in beers other than lambic (75) and
probably FRA. Tropical fruit, similarly perceived in each vessel, though slightly more in the
steel, is the intended descriptor for ethyl hexanoate, which was most abundant in the barrel and
~70-80% the relative concentration in the other vessels. This is probably an accurate correlation,
as the concentrations are so close in each vessel that the greater abundance in the barrel may not
be resulting in a stronger detection. The vessel differences in concentrations of other volatiles
and their corresponding flavors, such as benzaldehyde (almond), isoamyl acetate (mild fruit) and
2-phenylethanol (rose), seem to correlate appropriately. This may because these compounds
have such distinct aromas or that their concentrations, as detected by GCMS, are so starkly
contrasting that these nuances distinguish themselves at once in the vessels of maximal
abundance. The complex chemical compositions of these beers and the pluripotent subtleties of
olfactory phenomena skew the correlation of chemical abundance (GCMS data) and perception;
these disparities cannot even be briefed here but the tasting data may be taken candidly as the
purest form of analysis, that is, just as the beer encounters the tongue.
These beers must continue to be aged and analyzed for another thirteen months, thereby
matching the minimal maturation time allotted most commercial FRAs; in time, it may become
apparent that the differences in growth and organoleptic profiles vessel to vessel are temporary.
Over an extensive maturation period the chemical properties of an ale will approach equilibrium:
metabolically active cells will continue to alter the medium, whereas dead cells will lyse,
releasing esterases and other enzymes, as well as more substrate for conversion; compounds
produced in abundance, such as esters, will reduce to appropriate levels, whereas others,
produced by inefficient enzymes, under restrictive conditions or by non-enzymatic reactions will
increase to above threshold values. Some such compounds are the products of other compounds
that gradually accumulate over time, such as ethyl lactate, from the condensation of ethanol and
lactic acid. In some ways these are the true distinguishing features of FRA not versed by this
experiment. For the time being, however, the product is viewed as an immediate, unequilibrated,
raw substance, measured according to extremes of growth and organolepticism, even if the
extreme would ultimately be unacceptable. Each vessel, then, defines its own extreme: the barrel,
with its semi-permeable, porous, substrate-enriched walls harbors and nurtures both yeast and
bacteria within, resulting in the strongest ester profile; the plastic, while stimulating yeast growth
with the constant diffusion of oxygen, stalls bacterial growth and ester production, resulting in an
homogenous microcommunity and excessive oxidization of the beer; the steel, enhancing
bacterial growth under anaerobiosis, indirectly supports a mixed population of yeast and a
balanced but fruitier profile; the glass excels at mediocrity: it is neither too aerobic nor too
anaerobic and lacks the beneficial aspects of the wood, resulting in an homogenous yeast
community and restrained organolepsis. It must also be considered that the barrel is the only
vessel capable of sustaining a resident microbial community: with the use of other fermentors, a
regular inoculant must be either maintained or purchased, in which case the consistency of
performance and population diversity of commercial inocula must also be considered.
Additionally, Martens (1997) suggests that Brettanomyces spp. disappeared from traditional beer
fermentations parallel to the demise of the wooden fermentor principally because they relied
upon association with Pediococcus, selected for by the porous structure of the wood. Although
the steel vessel promotes the growth of LAB, theoretically to the benefit of Brettanomyces, in
opposition to the glass and plastic vessels, in which bacterial growth was poor, both of these
populations originated from the inoculant and may well disappear in successive batches without
repeat inoculation. The consistency of commercial inocula and the regularity of these
fermentation profiles themselves may not be perfect. Blending some proportion of the beer
fermented in steel (enhanced bacterial growth) with that fermented in wood (enhanced yeast
growth) may, then, yield the optimal result, exactly as FRA is produced in Belgium today. This
would not only suppress flaws and beautifully balance the organoleptic profile but is probably
vital to the consistent reproduction of this beer.
To this day, FRA is the product of a traditional brewing process featuring an extensive
aging within oak, specifically within the west Flanders region of Belgium. This highlights two
unique features of the fermentation on FRA which are not only the most important but the least
reproducible: the massive barrels in which this beer is aged contribute a delicate semi-aerobic
environment that likely harbors wild yeast and bacteria; Belgian wild yeast and bacteria which
cannot quite be approximated by laboratory strains and a fermentation in modern cylindroconical
steel vessels. In this experiment, the fermentation within the barrel was shown to support the
greatest population of Brettanomyces yeast, responsible for many of the flavors typical of FRA;
LAB growth was observed at periods but rarely detected by culture-dependent methods. The
fermentation in the steel vessel supported a healthy population of bacteria and Saccharomyces
though far fewer Brettanomyces than the barrel. The glass and plastic vessels held only moderate
populations of yeasts and very few bacteria. The barrel was also much more acidic than the other
vessels, possibly due to the premature presence of a species of Acetobacter. The slightly
increased TA in the steel vessel suggests swifter acidification than the glass and plastic. The
barrel had the most complex organoleptic profile, although, due to minor flaws, scored as
slightly less stylistically accurate as the steel; the glass and plastic were both scored as
significantly less stylistic. Continued aging would likely suppress these flaws, as would careful
blending. These data rely upon the use of a commercial inoculant, however, and only the barrel
would likely conserve this microecological profile through successive fermentations reinoculated
from the lees of the previous batch. Therefore this fermentation either requires the hygienic
maintenance of functional oaken fermentation vats or of regulated microbial inoculants. More
research must be done comparing the efficacy and stability of commercial inoculants versus
traditional semi-spontaneous fermentation over the successive generation of multiple batches.
I would like to thank Chris Jarvis and Jason Tor for all of their support and
encouragement and Rayane Moreira for her advice and help using and interpreting the GCMS. I
would also like to thank Phil Lorenz for his aid brewing and analyzing the Flanders Red Ale
during the autumn of 2007 and Erin Eggleston for her help with 16S PCR and sequencing.
1. Abbot DA, HynesSH, Ingledew WM. Growth Rates of Dekkera/Brettanomyces Yeasts Hinder their Ability to Compete with
Saccharomyces cerevisiae in Batch Corn Mash Fermentations. Applied Microbiology and Biotechnology, 66: 641–647, 2005.
2. Aguilar-Uscangna MG, Delia ML, Strehaiano P. Nutritional Requirements of Brettanomyces bruxellensis: Growth and Physiology in
Batch and Chemostat Cultures. Canadian Journal of Microbiology, 46: 1046-1050, 2000.
3. Andrews J, Gilliland RB. Super-Attenuation of Beer: A Study of Three Organisms Capable of Causing Abnormal Attenuations. Journal
of the Institute of Brewing, 58: 189-196, 1952.
4. Bardi L, Cocito C, Marzona M. Saccharomyces cerevisiae Cell Fatty Acid Composition and Release during Fermentation without
Aeration and in Absence of Exogenous Lipids. International Journal of Food Microbiology, 47: 133–140, 1999.
5. Bardi L, Crivelli C, Marzona M. Esterase Activity and Release of Ethyl Esters of Medium-Chain Fatty Acids by Saccharomyces
cerevisiae during Anaerobic Growth. Can. J. Microbiol. 44(12): 1171–1176, 1998.
6. Bisson LF. Stuck and Sluggish Fermentations. American Journal of Enology and Viticulture, 50: 107-119, 1999.
7. Bloem A, Bertrand A, Lonvaud-Funel A, de Revel G. Vanillin Production from Simple Phenols by Wine-Associated Lactic Acid
Bacteria. Letters in Applied Microbiology 44: 62–67, 2007.
8. Blondin B, Ratomahenina R, Arnaud A, Galzy P. A Study of Cellobiose Fermentation by a Dekkera Strain. Biotechnology and
Bioengineering, 24: 2031-2037, 1982.
9. Boidron JN, Chatonnet P, Pons M. Influence du Bois sur Certaines Substances Odorantes des Vins. Connaissance du vigne et du vin,
22(4): 275-294, 1988.
10. Bouckaert, Peter. ―Brewery Rodenbach: Brewing Sour Ales.‖ http://hbd.org/brewery/library/Rodnbch.html
11. Castro-Martinez C, Escudero-Abarca BI, Gomez Rodriguez J, Hayward-Jones PM, Aguilar-Uscanga MG. Effect of Physical Factors on
Acetic Acid Production in Brettanomyces Strains. Journal of Food Process Engineering, 28: 133–143. 2005.
12. Chatonnet P, Dubourdieu D, Boidron JN. Influence of Brettanomyces/Dekkera sp. Yeasts and Lactic Acid Bacteria on the Ethylphenol
Content of Red Wines. American Journal of Enology and Viticulture, 46(4):463-468, 1995.
13. Ciani M, Ferraro L. Role of Oxygen on Acetic Acid Production by Brettanomyces/Dekkera in Winemaking. J Sci Food Agric, 75, 489-
14. Ciani M, Maccarelli F, Fatichenti F. Growth and Fermentation Behaviour of Brettanomyces/Dekkera Yeasts under Different Conditions
of Aerobiosis. World Journal of Microbiology & Biotechnology, 19(4): 419-422, 2003.
15. Cleenwerck I, Vandemeulebroecke K, Janssens D, Swings J. Re-Examination of the Genus Acetobacter, with Descriptions of
Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. International Journal of Systematic and Evolutionary Microbiology,
52: 1551–1558, 2002.
16. Clyne J, Conner JM, Paterson A, Piggott JR. The Effect of Cask Charring on Scotch Whisky Maturation. International Journal of Food
Science and Technology, 28: 69-81, 1993.
17. Conner JM, Paterson A, Birkmyre L, Piggott JR. Role of Organic Acids in Maturation of Distilled Spirits in Oak Casks. Journal of the
Institute of Brewing, 105(5): 287-291, 1999a.
18. Conner JM, Paterson A, Piggott JR. Realease of Distillate Flavour Compounds in Scotch Malt Whisky. J Sci Food Agric, 79: 1015-1020,
19. Daenen L, Saison D, Sterckx F, Delvaux FR, Verachtert H, Derdelinckx G. Screening and Evaluation of the Glucoside Hydrolase
Activity in Saccharomyces and Brettanomyces Brewing Yeasts. Journal of Applied Microbiology, 104(2): 478-88, 2008.
20. De Cort S, Shantha Kumara HMC, Verachtert H. Localization and Characterization of α-Glucosidase Activity in Lactobacillus brevis.
Applied and Environmental Microbiologz, 60(9): 3074-3078, 1994.
21. de Revel G, Bloem A, Augustin M, Lonvaud-Funel A, Bertrand A. Interaction of Oenococcus oeni and oak wood compounds. Food
Microbiology, 22: 569–575, 2005..
22. de Revel G, Martin N, Pripis-Nicolau L, Lonvaud-Funel A, Bertrand A. Contribution to the Knowledge of Malolactic Fermentation
Influence on Wine Aroma. J. Agric. Food Chem., 47: 4003-4008, 1999.
23. del Alamo M, Bernal JL, del Nozal MJ, Gomez-Cardoves C. Red Wine Aging in Oak Barrels: Evolution of the Monosaccharide Content.
Food Chemistry 71: 189-193, 2000.
24. del Campo G, Santos JI, Berregi I, Velasco S, Ibarburu I, Dueñas MT, Irastorza A. Ciders Produced by Two Types of Presses and
Fermented in Stainless Steel and Wooden Vats. Journal of the Institute of Brewing, 109(4): 342–348, 2003.
25. Dickinson JR. ‗Fusel‘ Alcohols Induce Hyphal-like Extensions and Pseudohyphal Formation in Yeasts. Microbiology UK, 142: 1391-
26. Edlin DAN, Narbad A, Dickinson JR, Lloyd D. The Biotransformation of Simple Phenolic Compounds by Brettanomyces anomalus.
FEMS Microbiology Letters, 125: 311-316, 1995.
27. Fleet G. Microorganisms in Food Ecosystems. International Journal of Food Microbiology, 50:101–117, 1999.
28. Fleet G. Yeast interactions and wine flavour. International Journal of Food Microbiology, 86: 11 – 22, 2003.
29. Garavaglia J, Hickmann Flores S, Mara Pizzolato T, do Carmo Peralba M, Ayub M. Bioconversion of L-Phenylalanine into 2-
Phenylethanol by Kluyveromyces marxianus in Grape Must Cultures. World Journal of Microbiology and Biotechnology, 23:1273–
30. Garde Cerdan T, Ancin-Azpilicueta C. Contribution of Wild Yeasts to the formation of Volatile Compounds in Inoculated Wine
Fermentations. European Food Research and Technology, 222: 15-25, 2006a.
31. Garde Cerdan T, Ancin-Azpilicueta C. Effect of Oak Barrel Type on the Volatile Composition of Wine: Storage Time Optimization.
LWT, 39: 199–205, 2006b.
32. Gilliland RB. Brettanomyces. I. Occurrence, Characteristics, and Effects on Beer Flavour. Journal of the Institute of Brewing, 67: 256-
33. Gonzalez SS, Barrio E, Querol A. Molecular Identification and Characterization of Wine Yeasts Isolated from Tenerife (Canary Island,
Spain). Journal of Applied Microbiology, 102: 1018–1025, 2007.
34. Grimaldi A, Bartowsky E, Jiranek V. Screening of Lactobacillus spp. and Pediococcus spp. for Glycosidase Activities that are Important
in Oenology. Journal of Applied Microbiology, 99: 1061–1069, 2005.
35. Guilloux-Benatier M, Chassagne D, Alexandre H, Charpentier D, Feuillat M. Influence of Yeast Autolysis After Alcoholic Fermentation
on the Development of Brettanomyces/Dekkera in Wine. . Journal Internationale des Sciences de la Vigne et du Vin, 35(3): 157-164,
36. Hayashi S, Yagi K, Ishikawa T, Kawasaki M, Asai T, Picone J, Turnbull C, Hiratake J, Sakata K, Takada M, Ogawad K, Watanabe N.
Emission of 2-Phenylethanol from its b-D-glucopyranoside and the Biogenesis of these Compounds from [2H8] L-Phenylalanine in
Rose Flowers. Tetrahedron, 60: 7005–7013, 2004.
37. Jespersen L, Jakobsen M. Specific Spoilage Organisms in Breweries and Laboratory Media for their Detection. International Journal of
Food Microbiology, 33: 139-155, 1996.
38. Kolfschoten GA, Yarrow D. Brettanomyces naardenensis, a New Yeast from Soft Drinks. Antonie Leeuwenhoek, 36: 458-460, 1970.
39. Lisdiyanti P, Kawasaki H, Seki T, Yamada Y, Uchimura T, Komagata K. Systematic Study of the Genus Acetobacter with Descriptions
of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov.,
Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis (Carr 1958) comb. nov. Journal of General and
Applied Microbiology, 46: 147–165, 2000.
40. Liu SQ. Malolactic Fermentation in Wine – Beyond Deacidification. Journal of Applied Microbiology, 92: 589–601, 2002.
41. Lonvaud-Funel A, Renouf V. Incidence Microbiologique de l‘usage de barriques neuves et/ou de barriques usagees. Revue Francais
d‘Oenologie, 211: 10-14, 2005.
42. Lonvaud-Funel A. Lactic Acid Bacteria in the Quality Improvement and Depreciation of Wine. Antonie van Leewenhoek, 76: 317-331,
43. Ly MH, Covarrubias-Cervantes M, Dury-Brun C, Bordet S, Voilley A, Le TM, Belin JM, Wache Y. Retention of Aroma Compounds by
Lactic Acid Bacteria in Model Food Media. Food Hydrocolloids, 22: 211–217, 2008.
44. Martens H, Iserentant D, Verachtert H. Microbial Aspects of a Mixed Yeast-Bacterial Fermentation in the Production of a Special
Belgian Ale. Journal of the Institute of Brewing, 103: 85-91, 1997.
45. Medawar W, Strehaiano P, Delia ML. Yeast growth: lag phase modelling in alcoholic media. Food Microbiology, 20: 527–532, 2003.
46. Millet V, Lonvaud-Funel A. The Viable but Non-Culturable State of Wine Micro-Organisms during Storage. Letters in Applied
Microbiology, 30: 136–141, 2000.
47. Morrissey WF, Davenport B, Querol A, Dobson ADW. The Role of Indigenous Yeasts in Traditional Irish Cider Fermentations. Journal
of Applied Microbiology, 97: 647–655, 2004.
48. Moutounet M, Mazauric JP, Saint-Pierre B, Hanocq JF. Gaseous Exchange in Wines Stored in Barrels. J. Sci. Tech. Tonnellerie, 4: 131-
49. Nierop Groot MN, De Bont JAM. Conversion of Phenylalanine to Benzaldehyde Initiated by an Aminotransferase in Lactobacillus
plantarum. Applied and Environmental Microbiology, 64(8): 3009–3013, 1998.
50. O‘Shea DG, Walsh PK, Morphological Characterization of the Dimorphic Yeast KIuyveromyces marxianus var. marxianus NRRLy2415
by Semi-Automated Image Analysis. Biotechnology and Bioengineering, 51: 679-690 (1996).
51. O‘Shea DG, Walsh PK, The effect of culture conditions on the morphology of the dimorphic yeast Kluyveromyces marxianus var.
marxianus NRRLy2415: a study incorporating image analysis. Appl Microbiol Biotechnol, 53: 316-322, 2000.
52. Peng X, Sun J, Iserentant D, Michiels C, Verachtert H. Flocculation and coflocculation of bacteria by yeasts. Appl Microbiol
Biotechnol, 55: 777–781, 2001.
53. Piggott JR, Conner JM, Paterson A, Clyne J. Effects on Scotch Whisky Composition and Flavour of Maturation in Oak Casks with
Varying Histories. International Journal of Food Science and Technology, 28: 303-318, 1993.
54. Renouf V, Claisse O, Lonvaud-Funel A. Inventory and Monitoring of Wine Microbial Consortia. Applied Microbiology and
Biotechnology, 75: 149-164, 2007a.
55. Renouf V, Claisse O, Miot-Sertier C, Perello MC, de Revel G, Lonvaud-Funel A. Etude de l‘Ecosysteme Microbien Present a la Surface
des Barriques Utilisees lors de la Vinification. Sciences Alimentes, 26(5): 427-445, 2006a.
56. Renouf V, Falcou M, Miot-Sertier C, Perello MC,. De Revel G, Lonvaud-Funel A. Interactions between Brettanomyces bruxellensis and
other yeast species during the initial stages of winemaking. Journal of Applied Microbiology, 100: 1208–1219, 2006b.
57. Renouf V, Lonvaud-Funel A, Coulon J. The Origin of Brettanomyces bruxellensis in Wines: A Review. . Journal Internationale des
Sciences de la Vigne et du Vin, 41(3): 161-174, 2007b.
58. Renouf V, Lonvaud-Funel A. Racking are Key Stages for the Microbial Stabilization of Wines. Journal Internationale des Sciences de la
Vigne et du Vin, 38: 219-224, 2004.
59. Renouf V, Miot-Sertier C, Strehaiano P, Lonvaud-Funel A. The Wine Microbial Consortium: A Real Terroir Characteristic. . Journal
Internationale des Sciences de la Vigne et du Vin, 40(4): 209-216, 2006c.
60. Sakamoto K, Konings W. Beer Spoilage Bacteria and Hop Resistance. International Journal of Food Microbiology, 89: 105– 124, 2003.
61. Satokari R, Mattila-Sandholm T, Suihko ML. Identification of Pediococci by Ribotyping. Journal of Applied Microbiology, 88: 260–265,
62. Sawadogo-Lignani H, Lei V, Diawara B, Nielsen DS, Moller PL, Traore AS, Jakobsen M. The Biodiversity of Predominant Lactic Acid
Bacteria in Dolo and Pito Wort for the Production of Sorghum Beer. Journal of Applied Microbiology, 103: 765–777, 2007.
63. Shantha Kumara HMC, De Cort S, Verachtert H. Localization and Characterization of α-Glucosidase Activity in Brettanomyces
lambicus. Applied and Environmental Microbiologz, 69(8): 2352-2358, 1993.
64. Shantha Kumara HMC, Verachtert H. Identification of Lambic Superattenuating Micro-organisms by the Use of Selective Antibiotics.
Journal of the Institute of Brewing, 97: 181-185, 1991.
65. Shinohara T, Kubodera S, Yanagida F. Distribution of Phenolic Yeasts and Production of Phenolic Off-Flavors in Wine Fermentation.
Journal of Bioscience and Bioengineering, 90(1): 90-97, 2000.
66. Snowdon EM, Bowyer MC, Grbin PR, Bowyer PK. Mousy Off-Flavor: A Review. Journal of Agricultural and Food Chemistry, 54:
67. Spaepen M, Van Oevelen D, Verachtert H. Fatty Acids and Esters Produced During the Spontaneous Fermentation of Lambic and
Gueuze. Journal of the Institute of Brewing, 84: 278-282, 1978.
68. Spaepen M, Verachtert H. Esterase Activity in the Genus Brettanomyces. Journal of the Institute of Brewing, 88: 11-17, 1982.
69. Sparrow J. Wild Brews: Beer Beyond the Influence of Brewer‘s Yeast. Boulder, CO: Brewer‘s Publications, 2005.
70. Strong G, Bach R, Garofalo P, Hall M, Houseman D, Tumarkin M. 2008 BJCP Style Guidelines for Beer, Mead and Cider.
71. Suarez Valles B, Pando Bedrinana R, Fernandez Tascon N, Querol Simon A, Rodriguez Madrera R. Yeast Species Associated with the
Spontaneous Fermentation of Cider. Food Microbiology 24: 25–31, 2007.
72. Swaffield CH, Scott JA, Jarvis B. Observations on the microbial ecology of traditional alcoholic cider storage vats. Food Microbiology,
14: 353–361, 1997.
73. Swaffield CH, Scott JA. Existence and Development of Natural Microbial Populations in Wooden Storage Vats Used for Alcoholic Cider
Maturation. J Am Soc Brew Chem, 53(3): 117-120, 1995.
74. van Beek S, Priest FG. Evolution of the Lactic Acid Bacterial Community during Malt Whisky Fermentation: a Polyphasic Study.
Applied and Environmental Microbiology, 68(1): 297-305, 2002.
75. Van Oevelen D, de l‘Escaille F, Verachtert H. Synthesis of Aroma Components During the Spontaneous Fermentation of Lambic and
Gueuze. Journal of the Institute of Brewing, 82: 322-326, 1976.
76. Van Oevelen D, Spaepen M, Timmermans P, Verachtert H. Microbial Aspects of Spontaneous Wort Fermentation in the Production of
Lambic and Gueuze. Journal of the Institute of Brewing, 83: 356-360, 1977.
77. Van Oevelen D, Verachtert H. Slime Production by Brewery Strains of Pediococcus Cerevisiae. Journal of the American Society of
Brewing Chemists, 37: 34-37, 1979
78. Vandebenden N, Gils F, Delvaux F, Delvaux FR. Formation of 4-Vinyl and 4-Ethyl Derivatives from Hydroxycinnamic Acids:
Occurrence of Volatile Phenolic Flavour Compounds in Beer and Distribution of Pad1-Activity among Brewing Yeasts. Food
Chemistry, 107: 221–230, 2008.
79. Vanderhaegen B, Neven H, Coghe S, Verstrepen KJ, Derdelinckx G, Verachtert H. Bioflavoring and Beer Refermentation. Spplied
Microbiology and Biotechnology, 62: 140-150, 2003.
80. Verachtert, H, personal communication, February 28, 2008.
81. Verstrepen K, Derdelinckx G, Dufour JP, Winderickx J, Thevelein JM, Pretorius IS Delvaux FR. Flavor-Active Esters: Adding
Fruitiness to Beer. Journal of Bioscience and Bioengineering, .96(2): 110-118, 2003a.
82. Verstrepen K, Van Laere SDM, Vanderhaegen BMP, Derdelinckx G, Dufour JP, Pretorius IS, Winderickx J, Thevelein JM, Delvaux FR.
Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range
of Volatile Esters. Applied and Environmental Microbiology, 69(9): 5228–5237, 2003b.
83. Walling E, Gindreau E, Lonvaud-Funel A. La Biosynthèse d‘Exopolysaccharide par des Souches de Pediococcus damnosus Isolées du
Vin : Mise au Point d‘Outils Moléculaires de Détection. Lait, 81: 289–300, 2001.
A. FRA Flavor Precept Score Card
Please score the prominence of the listed flavors for each of the beers presented according to the
Not present 0 Faint 1 Modest 2 Apparent 3 Prominent 4 Dominant 5
Beer 1 Beer 2 Beer 3 Beer 4 Beer 5 Beer 6
Apple, waxy, dark, sweet
Wine, fermented fruits
Waxy, sweet, bright fruits
Funky, goaty, fatty, rank
Soft, mellow, apricot
Floral, hyacinth, honey
Horse blanket, band-aids
Smoke, bacon, clove
B. Compounds Selected for GCMS Analysis
Correspond ordinally (from left to right) with the flavor precepts presented vertically in Appendix A.
isoamyl acetate: pear, banana ethyl hexanoate: pineapple, wax, green banana ethyl decanoate: grape, apple
diethyl succinate: wine, fruit ethyl octanoate: fruit, fat, wax, sweet octanoic acid: fatty, rancid, sour, fruity, goat
benzaldehyde: almond 2-phenylethanol: floral, rose, hyacinth, honey
4-ethylphenol: animal sweat, horse blanket, medicinal 4-ethylguaiacol: smoke, bacon, spice, clove