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The Horace Brown Medal Lecture:

Forty Years of Brewing Research

Graham G. Stewart1,2

J. Inst. Brew. 115(1), 329, 2009

Horace Brown spent fifty years conducting brewing research in
Burton-on-Trent, Dublin and London. His contributions were
remarkable and his focus was to solve practical brewing problems by employing and developing fundamental scientific principles. He studied all aspects of the brewing process including
raw materials, wort preparation, fermentation, yeast and beer
stability. As a number of previous presenters of the Horace
Brown Lecture have discussed Browns achievements in detail,
the focus of this paper is a review of the brewing research that
has been conducted by the author and his colleagues during the
past forty years. Similar to Horace Brown, fundamental research
has been employed to solve brewing problems. Research studies
that are discussed in this review paper include reasons for premature flocculation of ale strains resulting in wort underattenuation including mechanisms of co-flocculation and pure strain
flocculation, storage procedures for yeast cultures prior to propagation, studies on the genetic manipulation of brewers yeast
strains with an emphasis on the FLO1 gene, spheroplast fusion
and the respiratory deficient (petite) mutation, the uptake and
metabolism of wort sugars and amino acids, the influence of
wort density on fermentation characteristics and beer flavour and
stability, and finally, the contribution that high gravity brewing
has on brewing capacity, fermentation efficiency and beer
quality and stability.

formation, wort composition and beer analysis. His contributions were remarkable and his focus was to solve
practical brewing problems by employing and developing
fundamental scientific principles. A number of previous
recipients of the Horace Brown Medal have reviewed
Browns achievements in detail14,73,105. However, a brief
summary here of Browns research is appropriate.
In 1916, the London Section of the Institute of Brewing presented Horace Brown with a portrait of himself
(Fig. 1) as an expression of the affection, esteem, and
homage of the Members of the Institute. In reply, Brown
presented a lecture15 entitled: Reminiscences of Fifty
Years Experience of the Application of Scientific Methods to Brewing Practice. Brown began his presentation
with the following statement: The origin of this paper is
the result of a retrospect and a process of mental stock-

Key words: Co-flocculation, flavour, flocculation, genetic manipulation, high gravity brewing, petite mutation, propagation,
stability, wort clarity and composition, yeast management.

The Horace Brown Medal commemorates Horace
Tabberer Brown (1848-1925), one of the founding fathers of the Institute of Brewing. Although largely selftaught, Horace Brown was a true polymath who left his
mark on virtually all areas of science as applied to brewing, in a career which lasted over 50 years. His research
work considered barley germination, beer microbiology,
water composition, oxygen and fermentation, beer haze
1 International

Centre for Brewing and Distilling, Heriot-Watt

University, Riccarton, Edinburgh, EH14 4AS, Scotland.
2 Present address: G.G. Stewart Associates, Brook House, Caerphilly
Business Park, Caerphilly, Wales, CF83 3GS.
Corresponding author. E-Mail:
This lecture was presented at the IBD Africa Section Convention,
March 2, 2009.
Publication no. G-2009-0225-581
2009 The Institute of Brewing & Distilling

Fig. 1. Horace Tabberer Brown.

VOL. 115, NO. 1, 2009

taking, supported by the fact that I have been closely

connected for half-a-century with one of the most interesting industries in the world. Brown discussed his research
activities in Burton-on-Trent, Dublin and London and that
his focus was to solve practical brewing problems by
employing and developing fundamental scientific principles. His studies on all aspects of the brewing process,
including raw materials, wort preparation, fermentation
and beer stability, were reviewed.
At the end of this wide ranging lecture (which was
published as a 91-page paper in the Journal of the Institute
of Brewing)15 Brown expressed personal sentiments that
are, in part, relevant today. Brown stated that he was
deeply disappointed that more had not been achieved to
exploit science in brewing. He blamed two factors the
lack of education of brewers and the short-term vision of
brewery directors. He concluded Those who are really
the responsible heads of manufacturing businesses seldom
have any desire to know anything of the inner meaning of
their processes or the scientific principles which underline
them. One has to wonder if Brown overstated the problem that prevailed at the time. A great deal of fundamental
research in brewing and related industries occurred in the
second half of the nineteenth century. It will be discussed
in this paper that a great deal of relevant research has been
conducted since 1916. However, one cannot help wondering if the industry has come full circle and its interest in
learning more about basic scientific principles is waning!
Most of the period of Browns research coincided with
the industrial revolution. Consequently, brewing (as in
many other industries) was being converted from a cottage craft into a major industry and with this conversion
came the need for scientific understanding. The brewing
industry became the centre of much scientific research
and it is interesting to note that, at this time, four Fellows
of the Royal Society (including Horace Brown) were employed by breweries in the United Kingdom118. This same
phenomenon was also occurring in other countries. For
instance in Denmark, J.C. Jacobsen, who founded the
Carlsberg Brewing Company in 1847 (naming it after his
son Carl), stated that, The person who has the most
thorough knowledge in chemistry, and supporting sciences, together with the necessary technical proficiency
and insight, will be Europes foremost brewer in the coming generation. This led to the establishing of the Carlsberg Foundation in 1876, which became, and still is, the
home of a number of distinguished scientists59.
This paper is largely (but not exclusively) a discussion
of personal experiences with brewing research, mainly
with yeast, during the past forty years, working, first of
all, for twenty-five years in the R&D Department of the
Labatt Brewing Company Limited and subsequently in
the International Centre for Brewing and Distilling at
Heriot-Watt University. However, interest in yeast research began as a PhD student at Bath University when,
as with many other research workers, yeast proved to be a
suitable and inexpensive micro-organism for biochemical
(and later molecular biological) research. Indeed, it was
purchased in the mid-1960s from the local bakers yeast
factory for 4 shillings (approximately 0.20)/pound. The
focus of this paper is to discuss (similar to Horace Brown)
how basic research has been employed to answer practical


brewing questions. Although most of the research discussed in this paper has been conducted at Labatt and
Heriot-Watt University, relevant research by other groups
has been included. All of the research discussed was initially published elsewhere, mainly in peer-reviewed journals.


For the past 50 years and longer, the brewing industry
has been going through a period of seeking scientific reasons for empirical knowledge, and fermentation and yeast
research have been no exception to this generalisation.
Efforts have been concentrated on what the cell does and
how it does it. The focus has been to discover the reasons
behind the empirical facts of yeast growth, fermentation
and flocculation. Early research concentrated upon biochemical and physiological aspects. During the past 25
years, this has been expanded to include consideration of
genetic and molecular biological aspects together with a
greater understanding of the influence of the genotype on
a cultures physiology and function. In addition, 30 years
ago131 it was believed that brewers yeast strains would be
genetically in vitro constructed and particularly employed
for specific brewing purposes. However, for reasons that
will be discussed later, although specific strains have been
developed by genetic manipulation, none of them have
been used for brewing132.
Over the years, considerable efforts have been devoted
to a study of the biochemistry and genetics of brewers
yeast (and other industrial yeast strains)130. The objectives
of these studies have been two fold: (1) to learn more
about the biochemical and genetic make-up of brewing
yeast strains; and (2) to improve the overall performance
of such strains, with particular emphasis being placed on
broader substrate utilization capabilities, increased ethanol production, improved stress tolerance to environmental conditions such as high osmotic pressure, ethanol,
temperature, salt and physical shear and to understand the
mechanism(s) of flocculation.
Understanding the substrate specificity of brewing and
other industrial yeast strains is a major objective of many
zymologists. Progress in achieving these objectives has
been impeded for several reasons, which include the fact
that such strains employed in the production of beer and
distilled products, are not readily amenable to genetic
manipulation by classical techniques. There are many
methods that can be employed in genetic research and
development of industrial yeast strains. These methods,
which will be discussed in greater detail later in this document130, include hybridisation, mutation and selection,
spheroplast (or protoplast) fusion and transformation, and
associated with it, DNA transformation and associated
gene manipulation techniques. Industrial yeast strains are
often polyploid or even aneuploid and, as a consequence,
do not possess a mating type, have a low degree of sporulation and spore viability, rendering genetic analysis of
such strains difficult but not impossible154. However, modifications to the incubation temperature and presporulation carbon source have significantly increased sporogenic
capabilities of industrial strains, thereby facilitating recovery of viable spores and thus providing an opportunity to

manipulate such strains by classical (hybridisation) techniques5,6.

The contribution of the Carlsberg Foundation to brewing research has already been discussed. One of their notable scientists was Emil Christian Hansen (Fig. 2). He
successfully isolated four strains of bottom fermenting
yeast from the Carlsberg lager yeast culture. He studied
them from the standpoint of brewery performance, and
only one of these strains proved to be suitable for beer
fermentation. The strain, described as Carlsberg Yeast
No.1 was introduced into the Carlsberg Brewery for use
on a production scale on 13 May 1883 and pure strain
brewing of lager can be said to have commenced from this
date59. Due to the origin of Carlsberg Yeast No.1 it was
named Saccharomyces carlsbergensis Hansen 1883.
This grouping of lager yeasts is now known as Saccharomyces pastorianus91.
Hansen soon found that it was too tedious and inconvenient for the laboratory to regularly furnish the Carlsberg Brewery with pure cultures and it would be easier to
obtain a specific apparatus for this purpose. With the assistance of the coppersmith, W.E. Jansen, Hansen started
to construct such an apparatus. By the beginning of 1886,
the apparatus was working effectively in the Carlsberg
Brewery. Jansen began to sell the apparatus and Heineken
was one of the first breweries to purchase this equipment.
As a result of Hansen and Jansens work, the practice
of employing a pure strain in lager production was soon
adopted by breweries all over the world, particularly in
the United States. Ale-producing regions however, met
this radical innovation with severe opposition! The
method was merely regarded as a means of reducing
infection by wild yeasts and bacteria. In 1959 it was reported60 that of 39 ale yeast cultures in use commercially
in Britain, 12 contained a single strain, 16 had two major

Fig. 2. Emil Christian Hansen.

strains and the rest contained three or more yeast strains.

These ale strains are taxonomically classified as part of
the species Saccharomyces cerevisiae91.
In the 1960s and 1970s ale consumption was popular
in Canada. In 1970, it was 60% of the beer produced in
Ontario. However, similar to the situation in Britain, the
ale yeast cultures employed were largely uncharacterised.
The Labatt ale culture possessed top cropping properties
and exhibited intermittent premature flocculation characteristics resulting in underfermented wort with residual
sugars (mainly maltotriose details later). This problem
was exacerbated during high gravity brewing trials. It was
of interest to enumerate the number of strains in this ale
culture and characterise them. One of the most suitable
methods available in the late 1960s for this purpose was
the Giant Colony Morphology Method106. This method
involved inoculating the yeast culture onto solid media
and examining the colonial morphology that developed
after incubating under standard conditions for at least
three weeks at 18C. It has been found that gelatin, as the
solidifying matrix, tends to enhance the distinctive features of the colonial morphology to a greater extent than
agar. It is usual to find that malt-wort, corn steep liquor,
fruit and vegetable infusions induce greater colony differentiation than complete media based on nutrients from
animal sources or defined synthetic media. This method
has, however, one major shortcoming (as well as the
prolonged incubation period necessary at below ambient
temperature). It gives no information on the value of a
brewing strain for brewing purposes, to quote Cook26 who
stated in his address to the European Brewery Convention
Congress in 1969: It is important to realize that this procedure (i.e., the giant colony procedure) is rather like taking photographs of those in the hall. The photographs
would enable us to identify individuals elsewhere but
would tell us nothing of their performance as maltsters,
brewers and scientists. However, although the ale brewing strains exhibit characteristic colonial morphologies
when grown on wort gelatin media; lager strains exhibit
similar, uncharacteristic, and somewhat uniform morphology, strain to strain.
Analysis of the Labatt ale cultures strain composition
showed that two morphologically different colony types
(Fig. 3) were present. On isolation, both colony types
proved to be stable respiratory sufficient separate strains
of the species Saccharomyces cerevisiae and they were

Fig. 3. Giant colony morphologies of Labatt ale yeast strains

LAB A/69 (left colony) and LAB B/69 (right colony).
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Fig. 4. Co-flocculation cylinder wort fermentation test.

Fig. 5. Co-flocculation Helms sedimentation test.

coded LAB A/69 and LAB B/69. Both strains, when

cultured alone in wort, were non-flocculent during all
phases of growth. However, when cultured together in
wort in a 1:1 ratio, the culture was flocculent in the later
stages of a wort fermentation and sedimented out of suspension (Fig. 4). Also, when stationary phase cells of the
two strains were mixed together in a 1:1 ratio in the
presence of calcium ions at pH 4.5 (a variation of the
Helms sedimentation test57) flocs immediately began to
appear and a very flocculent culture resulted that sedimented out of suspension (Fig. 5). This type of behaviour,
where two yeast strains are non-flocculent alone but
flocculent when mixed together, has been termed coflocculation137,128. Co-flocculation has also been termed
mutual aggregation and mutual flocculation42,170.
To date, co-flocculation has only been observed with
ale strains, and there are no reports of co-flocculation
between non-flocculent lager yeast strains. Another type
of co-flocculation reaction which has been described is
where an ale yeast strain has the ability to aggregate and
co-sediment with contaminating bacteria such as Hafnia
protea, Lactobacillus brevis, Pediococcus sp. and Lactobacillus sp170,176.
It was decided that the two strain composition of the
Labatt production ale strain was undesirable, particularly
because of its tendency for pre-mature flocculation and
wort under-attenuation, resulting in failure to meet the
beers alcohol specification (this is prior to the introduction of high gravity brewing on a production basis). Production trials with LAB A/69 strain were conducted in
both lower (sales) (12Plato) and higher gravity (16Plato)
wort. This strain proved to be capable of successfully
fermenting both wort gravities but, because of its nonflocculent property, required centrifugation to harvest the
culture for both clarification and re-use. This strain has

been employed for ale production by Labatt with high

gravity worts for the past twenty-five years.


Pure strain flocculation

As well as co-flocculation between two or more brewers yeast strains, the flocculation characteristics of individual strains are also important. As previously discussed,
the flocculation property, or conversely, lack of flocculation, of a particular culture (strain) is one of the major
factors when considering important characteristics during
brewing and other ethanol fermentations. Unfortunately, a
certain degree of confusion has arisen by the use of the
term flocculation in the scientific literature to describe
different phenomena in yeast behaviour. Specifically, flocculation, as it applies to brewers yeast, is the phenomenon wherein yeast cells adhere in clumps and either sediment from the medium in which they are suspended or rise
to the mediums surface149. This definition excludes
other forms of aggregation particularly that of clumpy
growth or chain formation148 (Fig. 6). This non-segregation of daughter and mother cells during growth has
sometimes erroneously been referred to as flocculation.
Chain formation only occurs with some ale strains and
has not been observed in lager strains to the same extent.
The term non-flocculation applies to the lack of cell
aggregation and, consequently, a much slower separation
of (dispersed) yeast cells from a static liquid medium.
Flocculation usually occurs in the absence of cell division
(but not always) during late logarithmic and stationary
growth phases and only under rather circumscribed environmental conditions, involving specific yeast cell surface
components (proteins and carbohydrates) and the interaction of calcium ions (details later). Although yeast separation often occurs by sedimentation (bottom cropping), as
already described in the discussion of co-flocculation, it

Fig. 6. Flocculation in Saccharomyces cerevisiae.

Fig. 8. EDTA wash deflocculation.

Fig. 7. Water wash deflocculation.

may also be by flotation because of cell aggregates entrapping CO2 bubbles. These are top-cropping ale brewing
Calcium adsorption
The importance of calcium ions during yeast flocculation cannot be over emphasised147. With many flocculent
strains, the calcium can be removed from the yeast cell
wall as a result of washing with deionised water and the
culture will become reversibly non-flocculent156. If calcium is then added to this de-flocculated culture the cells
become flocculent again (Fig. 7). Some flocculent strains
are not de-flocculated by washing with water, the cells
need to be treated with a solution of a chelating agent
such as EDTA followed by washing with water to remove

the EDTA. This treatment will de-flocculate these cultures

and the flocculation phenotype will be restored upon addition of calcium ions (Fig. 8).
It has been proposed72 that flocculent yeast strains are
able to adsorb considerably more Ca++ ion than non-flocculent strains. It has also been suggested that cell walls
isolated from flocculent cultures bind more Ca++ ions than
walls isolated from non-flocculent cultures. Employing
radiolabelled Ca45, studies have been conducted to compare the calcium binding ability of a number of ale and
lager flocculent and non-flocculent brewers yeast cultures150,151. When the final calcium uptake of each culture
was analysed (Table I), it was clear that no direct correlation existed between the total calcium adsorbed and
flocculation. There is strain-to-strain variation in calcium
binding, and furthermore this variation does not correlate
with flocculation and non-flocculation, when comparing
one strain to another. However, with the knowledge that
VOL. 115, NO. 1, 2009

Table I. Total calcium adsorbed by flocculent and non-flocculent yeast

Yeast culture


Calcium adsorbed (mg/mg

of dry weight yeast)




Fig. 9. Schematic diagram of the architecture of the yeast cell

wall. Components such as mannoprotein are found distributed
throughout the entire wall and therefore the layered arrangement
shows zones of enrichment.

Table II. Calcium removed from flocculent and non-flocculent cultures

during de-flocculation washings.

Yeast culture


Total calcium washed off

yeast (mg/100 mg of dry
weight yeast)




many flocculent yeast cultures can be de-flocculated by

washing with deionised water (Fig. 7), if one could correlate the amount of calcium washed off a yeast culture
with the visible loss of flocculation, an improved perspective of calcium binding behaviour in yeast and its relationship to flocculation might be obtained.
To test this hypothesis, aliquots of flocculent and nonflocculent yeast suspensions were taken after 120 min
incubation with the Ca45 solution and centrifuged. The
yeast pellet was washed four times with 2 mL of deionised water pH 4.0 for 15 sec on a Vortex mixer and the
activity of each centrifuged supernatant determined with a
scintillation counter. Using standard curves relating calcium concentration to radioactivity, the amount of calcium removed with each washing was determined. The
first wash did not de-flocculate the flocculent yeast cultures, but did remove loosely adhering calcium around
and in the interstitial spaces between the yeast cells. This
source of calcium should be relatively the same percentage of total calcium bound for each yeast culture (Table I)
and is in all probability not related to flocculation, since
the visible observation of flocculation did not disappear
during this first wash.
The subsequent washings gradually removed any observable flocculation. The sum of the calcium removed in
washings 2 to 4 were expressed as a percentage of the
total calcium removed during washing. When the results
were expressed in these terms (Table II) for flocculent and
non-flocculent cultures, the flocculent cultures bound 28
to 40% more calcium after four water washings than nonflocculent cultures. As would be expected, there is strainto-strain variation in calcium adsorption. This variation is
in all likelihood a reflection of diversities in cell wall


structure from strain to strain. In addition, this strain-tostrain variation in calcium adsorption per se does not correlate with the flocculation phenotype, when comparing
one strain to another. The only meaningful measure of
calcium behaviour that correlated with flocculation was
the ease with which calcium washed off the cell and this
coincided with the visible loss of flocculation.
Yeast flocculation and cell surface fimbriae
The yeast cell wall is a complex structure consisting of
mannan, glucan, protein, chitin, lipid and a number of
other compounds (Fig. 9)66. Flocculation requires the
presence of cell surface proteins and mannan receptors80.
If these are not available, masked, blocked, inhibited or
denatured, flocculation cannot occur. Onset of flocculation is an aspect of the subject where there is significant
commercial interest, but about which little is understood.
The ideal brewing strain is one which in a typical fermentation, without the use of a centrifuge, remains in suspension as fermenting single cells until close to the end of
fermentation, when the wort sugars and most amino acids
have been utilised (details later) and the vicinal diketones
(diacetyl, etc.) are reduced. Only then will the culture rapidly flocculate and settle out of suspension to be harvested
and re-pitched into a subsequent fermentation126,172. What
signals the onset of flocculation? This is still an unanswered question162.
Electron microscopy of flocculent and non-flocculent
brewers yeast cultures shadowed with tungsten oxide has
revealed that flocculent cultures possess a hairy outer
surface (called fimbriae), whereas, non-flocculent cultures
do not contain cell surface fimbriae (Fig. 10)40. This
observation has been re-confirmed by recent studies125,163.
There are a number of treatments that will result in deflocculation of flocculent cultures that contain fimbriae
prior to treatment. Treatments include protease and shear
in a blender, both of which result in irreversible loss of
flocculation and these cultures no longer contain cell surface fimbriae. It is interesting to note that when these deflocculated fimbriae-less cultures were recultured in wort,
they became flocculent again when they entered late logarithmic growth phase and they also once again contained
cell surface fimbriae.

Fig. 10. Electron photomicrographs of Saccharomyces cerevisiae flocculent and non-flocculent shadow cast with
tungsten oxide.

Genetic control of yeast flocculation

Genetic studies on yeast flocculation began over 50
years ago. The first publication on this subject was by
Pomper and Burkholder97 who reported crossing a haploid
culture that possessed a dispersed character (non-flocculent) with a haploid culture that possessed a non-disperse character (flocculent). The disperse character
was reported to be dominant over the non-disperse character. In the early 1950s, Gilliland and Thorne independently carried out extensive studies on the genetics of yeast
flocculation. Gilliland49 studied two non-brewing strains
of S. cerevisiae which differed only in their flocculation
properties. It was proposed that a single gene was responsible for the flocculence phenotype. Thorne166 confirmed
Gillilands studies and demonstrated that flocculence was
an inherited characteristic and that flocculence was dominant over non-flocculence.
As has already been discussed, and will be described in
greater detail later, genetic work involving brewers yeast
strains is fraught with difficulties because of their frequent
triploid, polyploid, or aneuploid nature147. Consequently,
studies have focussed on haploid and diploid flocculent
and non-flocculent strains. The flocculation characteristic
studied was reversible and induced by calcium ions. The
flocculent haploid strain (coded 169) was of opposite
mating type to the non-flocculent haploid strain (coded
168). These two strains were mated using micromanipulation techniques and the resulting diploid hybrid
(169/168) was found to be flocculent, confirming previous
findings that the flocculent character was dominant and
Tetrad analysis of spores isolated from asci of the
169/168 hybrid revealed that the dominant flocculence of
strain 169 was controlled by a single gene locus (i.e. 2:2
segregation). This gene has been coded FLO1. The next

question was the location of the gene on one of the 16

chromosomes of Saccharomyces. A detailed discussion of
the chromosome mapping procedures employed in this
study is beyond the scope of this paper. Suffice to say, it
has been shown that FLO1 is located on Chromosome I,
33 cM from the centromere on the right hand side of the
The mapping of the FLO1 gene employed traditional
gene mapping techniques (mating, sporulation, micromanipulation, tetrad analysis, etc.). Today novel genetic techniques have been developed, the principle of which is the
sequencing of the Saccharomyces genome76. This has expanded our knowledge of the genetic control of yeast
flocculation. Flocculation genes identified to date include
FLO1, FLO2, flo3, FLO4, FLO5, flo6, FLO7, FLO8,
FLO9, FLO10, FLO11 and MUC1165. Of all the flocculation genes identified, FLO1 is the most extensively studied and perhaps the most important and capable of conferring flocculation when transformed into non-flocculent
S. cerevisiae strains165.


The development of yeast propagation techniques by
Hansen and Jansen has already been discussed. Of equal
importance to the actual scale-up facilities and procedures
is the maintenance of the stock yeast cultures between
propagations. This became even more important with the
advent of the use of ale yeast single strains, along with
lager strains. The long term preservation of a brewing
yeast strain requires that not only is optimal survival important, but it is imperative that no change in the character
of the yeast strain occurs. Many yeast strains are difficult
to maintain in a stable state and long term preservation by
VOL. 115, NO. 1, 2009

lyophilisation (freeze drying), which has proven useful for

mycelial fungi and bacteria64, has been found to give poor
results with brewing yeast strains65. Storage studies have
been conducted with a number of ale and lager brewing
strains108. The following storage conditions were investigated:
Low temperature [70C refrigeration or liquid nitrogen (196C)];
Lyophilisation (freeze drying);
Storage in distilled water;
Storage under oil;
Repeated direct transfer on culture media (subcultured
once a week for two years);
Long term storage at 21C on solid nutrient medium
subcultured every six months;
Long term storage at 4C on solid nutrient medium
subcultured every six months.
After a two year storage period, wort fermentation tests
including fermentation rate and wort sugar uptake efficiency, flocculation characteristics, sporulation ability,
formation of respiratory deficient colonies and ease of
survival were conducted and the results compared to the
characteristics of the stored control culture. Low temperature storage appears to be the storage method of choice, if
cost and availability of the appropriate equipment is not a
significant factor. Cultures stored either at 70C or in
liquid nitrogen168 had the lowest death rate and were the
easiest to revitalise. Also, the degree of flocculation, wort
fermentation properties, sporulation ability and proportion
of respiratory deficient mutants present were all unaffected by this storage method. Storage at 4C on nutrient
agar slopes, subcultured every 6 months, was the next
method of preference to low temperature storage. Lyophilisation and other storage methods revealed yeast instability, which varied from strain to strain. Currently, many
breweries store their strains (or contract store them) at
70C. Routine subculturing of cultures on solid media
every six months is less desirable, but a very cost effective
storage method. Lyophilisation of brewers yeast cultures
should be avoided!

The behaviour, performance and quality of a yeast
strain is influenced by two sets of determining factors, collectively called nature-nurture effects. The nurture effects
are all the environmental factors (i.e. the phenotypes), to
which the yeast is subjected from pitching onwards. On
the other hand, the nature influence is the genetic makeup (i.e. the genotype) of a particular yeast strain.
It has already been stated in this paper that the expectation that genetically manipulated yeast strains would be
employed in brewing has not been achieved. In 1986 we
stated132: The use of manipulated yeast strains in brewing
will become commonplace within the next decade with
yeast strains specifically bred for such characteristics as
extra-cellular amylases, -glucanases, proteinases, -glucosidase production, pentose and lactose utilisation, carbon catabolite repression and production of a plethora of
heterologous proteins. There is no doubt that prior to the


introduction of such strains at the production level, the

environmental and legal impact of such a move will have
to be assessed. Over 20 years later, genetically manipulated brewers yeast strains are still not employed commercially, due in large part because of opposition from
public opinion. Whether this will change, only time will
tell! Nevertheless, genetic techniques have been used to
study the genetic composition and function of such
There are a number of methods that can be employed
in the genetic research and development of brewers134
and related yeast strains55. Classical approaches to strain
improvement include mutation and selection7, screening
and cross-breeding (hybridisation)146,48. The use of hybridisation to map FLO1 on Chromosome I of the Saccharomyces genome has already been described. Mutation
is any change that alters the structure of the DNA molecule, thus modifying the genetic material. The mutagenised strains often no longer exhibit many desirable
properties of the parent strain and exhibit slower growth
rates and produce undesirable taste and aroma compounds
during fermentation145. Mutagenesis is seldom employed
with brewing strains due to their polyploid/aneuploid nature.
Spontaneous yeast mutations are a common occurrence
throughout the growth and fermentation cycle, but they
are usually recessive, due to functional loss of a single
gene56. Because of the aneuploid or polyploid nature of
most strains, the dominant gene will function adequately
in the strain, as it will be phenotypically normal. Only if
the mutation takes place in all complementary genes will
the recessive character be expressed. However, if the mutation weakens the yeast, the mutated strain will be unable
to compete and will soon be outgrown by the non-mutated
yeast population. The characteristics that are routinely encountered resulting from mutations that are harmful to a
wort fermentation are:
The tendency of yeast strains to mutate from flocculent
to non-flocculent133;
The loss of ability to ferment maltotriose111;
The presence of respiratory deficient mutants85.
The respiratory deficient (RD) or petite mutation is
the most frequently identified mutant found in brewing
yeast strains. This mutant arises spontaneously when a
segment of the DNA in the mitochondria becomes defective to form a flawed mitochondrial genome. The mitochondria are then unable to synthesise certain proteins.
This type of mutation is also called the petite mutation
because colonies of such a mutant are usually much
smaller than respiratory sufficient (RS) colonies (also
called grande) (Fig. 11). The RD mutation usually
occurs at frequencies of between 0.5 and 5% of the population, but in some strains, levels as high as 50% have
been reported121. RD mutants can also occur as a result of
deficiencies in nuclear DNA, but these are much rarer.
Deficiencies in mitochondrial function result in diminished ability to function aerobically and as a result these
yeasts are unable to metabolise non-fermentable carbon
sources such as lactate, glycerol or ethanol (Fig. 12).
Many phenotypic effects occur as a result of this mutation
and include alteration in sugar uptake (particularly maltose and maltotriose), by-product formation and subse-

quent metabolism (for example, diacetyl), and intolerance

to stress factors such as ethanol, osmotic pressure and
temperature. Also, further to the discussion of storage
and preservation of stock yeast cultures, RD mutants are
difficult to store and liquid nitrogen and 70C refrigeration have both been found to be the most effective storage
methods108. Flocculation, cell wall and plasma membrane
structure, and cellular morphology are affected by this RD
Beer produced with a yeast culture that is RD is likely
to have flavour defects and fermentation problems. For
example, beer produced from these mutants contained elevated levels of diacetyl and higher alcohols122. Wort fermentation rates were slower, higher dead cell counts were
observed and biomass production and flocculation ability
were reduced45,51.

Fig. 11. Respiratory sufficient and respiratory deficient mutants

triphenyl tetrazolium chloride overlay.

The advent of the new biotechnology has stimulated

the development of novel methods of genetic manipulation141 including spheroplast (protoplast) fusion and
recombinant DNA. Spheroplast fusion is a technique that
can be employed in the genetic manipulation of brewers
yeast strains110. The method does not depend on ploidy
and mating type and consequently has great applicability
to such strains because of their polyploid nature and
absence of mating type characteristics. Details of the
procedure can be found elsewhere155. Examples of
successful fusions with commercial brewing and related
strains are:
The construction of a brewing yeast with amylolytic
activity by fusion of S. cerevisiae and S. diastaticus113;
A polyploid strain capable of high ethanol production
by fusion of a flocculent strain with sak yeasts90 and
Construction of strains with improved osmotolerance
by fusion of S. diastaticus and S. rouxii (an osmotolerant yeast strain)112,123.
Although spheroplast fusion is an extremely efficient
technique, it relies mainly on trial and error and is not
specific enough to modify strains in a predictable manner.
The fusion product is nearly always very different from
both the original fusion partners, because the genome of
both strains becomes integrated. Spheroplast fusion has
been found to be a viable technique when flavour of the
final product is not critical. For example, fusion products
were constructed that could survive high osmotic pressure, elevated fermentation temperatures (ca>40C) and
showed increased ethanol tolerance152. Such strains are
being used successfully in the industrial alcohol industry,
but produce beer with unsatisfactory beer flavour/taste
Recombinant DNA techniques can also be used to
make thousands of copies of the same DNA molecule to
amplify DNA, thus generating sufficient DNA for various
kinds of experiments or analysis. Although the author and
his colleagues have not employed these techniques to improve brewers yeast strains, other groups55 have success-

Fig. 12. Growth of respiratory sufficient (RS) and respiratory deficit (RD) cultures on fermentable (glucose) and nonfermentable (lactate) carbon sources.
VOL. 115, NO. 1, 2009


fully conducted such experiments to improve brewers

yeast strains. Examples include:
Glucoamylase activity from the fungus Aspergillus
Glucanase activity from the bacterium Bacillus subtilis, the fungus Trichoderma reesii and from barley;
-Acetolactate decarboxylase activity from the bacteria Enterobacter aerogenes and Acetobacter spp. for
diacetyl control;
Extracellular protease for chill-proofing beer; and
Modification of the yeasts flocculation properties.
The future prospects for the use of recombinant DNA
with brewers and distillers yeast in the industry are unclear. It is surprising that recombinant brewers yeasts are
not commercially in use today, in both brewing and
distilling, but public opinion does not support their use.
Since the early days of yeast genetic manipulation there
has been considerable activity, which has been succinctly
described by Boulton and Quain10. Permission was
granted over a decade ago from the appropriate authorities
in the United Kingdom for the use of a bakers yeast
strain that is genetically manipulated for more rapid maltose use, leading to enhanced baking properties, and for a
brewing strain cloned with DNA from S. diastaticus, that
secretes glucoamylase to utilise wort dextrins and produce
low calorie beer48.
The sequencing of the S. cerevisiae genome began in
1989 and was completed with the publication of the
sequence in 199650. Although a major achievement, the
DNA sequence of S. cerevisiae is relatively small, with a
genome size of 13.5 Mb compared to the genome of man
with a genome size of the order of 3500 Mb88.
Recently, the Australian Wine Research Institute, in
collaboration with the Australian Genome Research Facility, has sequenced the DNA of a wine yeast strain9. It is
interesting to note that this study took approximately six
months to complete. The first yeast strain to be sequenced
involved 70 laboratories, took 10 years and cost millions
of dollars!
The sequencing of the yeast genome, in conjunction
with gene expression analysis, has enabled the identification of genes that have altered gene expression patterns in
response to stressful environmental conditions18. Alltech,
in collaboration with Heriot-Watt University, has studied
the inhibitory effects of the stress factors most commonly
encountered during alcoholic fermentation of corn mashes53. The key stress related yeast genes with different resistance levels to environmental stress have been assessed.
A stress model has been developed to assess yeast stress
resistance and evaluate the suitability of a specific strain
for use in industrial ethanol fermentations52. This stress
model could potentially be used for screening candidate
yeast strains for relative stress resistance in the fuel
ethanol industry, and other fermentation industries, where
yeast encounters similar stresses.


Compared to other media employed for the production
of fermentation alcohol, both industrial and potable, wort


Fig. 13. John Pierce and Margaret Jones.

is by far the most complex. As a consequence of this,

when yeast is pitched into wort it is introduced into an
intricate environment because it consists of simple sugars,
dextrins, amino acids, peptides, proteins, vitamins, ions
such as zinc, magnesium, manganese, calcium, sodium
and potassium, nucleic acids and other constituents too
numerous to mention. One of the major advances in brewing science, during the past 40 years, has been the elucidation of the mechanisms by which the yeast cell utilises,
in an orderly manner, the plethora of wort nutrients.
Reference has already been made to the uptake of wort
sugars, in the context of premature flocculation of coflocculent ale cultures, resulting in residual maltotriose in
the fermented wort. Quantitative determination of wort
sugars has evolved during the past 40 years. The initial
experiments employed paper and thin layer chromatography95, followed by gas chromatography (GC) which
involved pre-derivatisation to make the sugar volatile
when injected into the GC94. Current methods for wort
sugar determination employ high performance liquid
chromatography (HPLC)89. Similar to wort sugars, the
determination of the 19 amino acids in wort has also
developed over time. The pioneering studies on wort
amino acid composition and uptake by yeast were conducted by Jones and Pierce62 (Fig. 13) who employed
rudimentary liquid chromatography, compared to todays
HPLC techniques, however, they still established the fundamentals of the order of amino acid uptake.
Wort sugar uptake
Wort contains the sugars sucrose, glucose, fructose,
maltose and maltotriose together with dextrin material. In
the normal situation, brewing yeasts are capable of utilising sucrose, glucose, fructose, maltose and maltotriose,
in this approximate sequence (or priority), although some
degree of overlap does occur, leaving maltotetraose and

Fig. 16. Structure of D-glucose and 2-deoxy-D-glucose.

Fig. 14. Order of uptake of sugars by yeast from wort.
Table III. Typical sugar spectrum of wort.
Percent composition


Fig. 15. Uptake of sugars by the yeast cell.

the other dextrins unfermented36 (Fig. 14). Brewers yeast

is also capable of utilising sugars such as galactose, but
not lactose, unless it is hydrolysed to its constituent
monosaccharides31,32. In addition, there is a closely related
species (regarded as the same species by some) to S. cerevisiae, designated as S. diastaticus. This yeast species
produces an extracellular glucoamylase that is capable of
hydrolysing wort dextrins to glucose, a sugar that is easily
Maltose and maltotriose are the major sugars in brewers wort (Table III), and as a consequence, a brewing
yeasts ability to use these two sugars is vital and depends
upon the correct genetic complement153. Brewers yeast
cultures possess independent uptake mechanisms (maltose
and maltotriose permease) to transport these two sugars
across the cell membrane into the cell119. Once inside the
cell, both sugars are hydrolysed to glucose units by the glucosidase system (Fig. 15). The transport, hydrolysis
and fermentation of maltose are particularly important in
beer production, in Scotch whisky production and in
baking, since maltose is the major component of brewing

wort, spirit mash in Scotland and wheat dough. Maltose

fermentation by Saccharomyces sp. requires the presence
in the genome of one of five unlinked MAL loci, each
consisting of three genes encoding the structural gene for
-glucosidase (maltase) (MALS), maltose permease
(MALT) and an activator (MALR), whose product coordinately regulates the expression of the -glucosidase
and permease genes41. The expression of MALS and
MALT is regulated by maltose induction and repressed by
Indeed, a major limiting factor in the fermentation of
wort is the repressing influence of glucose (and possibly
fructose) upon maltose and maltotriose uptake. Only
when approximately 50% of the wort glucose has been
taken up by the yeast cells will the uptake of maltose
commence (Fig. 14). This is yeast strain and wort composition dependent38,39. In other words, in most strains of
S. cerevisiae and related species, maltose utilization is
subject to control by carbon catabolite repression115. In a
similar manner, the presence of glucose will repress the
production of glucoamylase by S. diastaticus, thereby inhibiting the hydrolysis of wort dextrins and starch75. Repression of this nature has a negative effect on overall
fermentation rate.
Studies have also been conducted where glucose is
added to fermenting wort when the yeast strain is metabolising maltose and has already taken up all of the available
wort glucose. The added glucose caused inhibition (repression) of the maltose uptake. Once this glucose had
been taken up by the yeast culture, the metabolism of
maltose recommenced43,44,146,147.
In order to try to overcome this repression, the glucose
analogue, 2-deoxy-glucose (2-DOG) has been successfully employed for the selective isolation of spontaneous
mutants of yeasts114,167 and other fungi4 (Fig. 16). These
mutants were derepressed for the production of carbohydrate-hydrolysing enzymes employing this non-metabolisable glucose analogue. Derepressed mutants of brewing
and other industrial strains have been isolated that are able
to metabolise wort maltose and maltotriose in the presence of glucose (Fig. 17). Fermentation and ethanol formation rates in 12Plato wort were also increased in the 2DOG mutants, when compared with the parental strain. In
addition, 2-DOG starch mutants of S. diastaticus have
been isolated that exhibited increased fermentation ability
in brewers wort, cassava and corn mash171.
VOL. 115, NO. 1, 2009


In order to illustrate the phenomenon of derepression

and the effect of 2-DOG mutants on overall fermentation
rate of a brewers wort, several strains of S. cerevisiae
have been studied63,143. In all instances, the presence of
glucose derepressed the uptake of maltose. Numerous stable 2-DOG mutants were found to be capable of utilising
maltose in the presence of significant concentrations of
glucose82,83 (Fig. 17).
All of our studies with 2-DOG mutants discussed
above were conducted with ale and distilling yeast strains.
We were unable to isolate 2-DOG mutants from the lager
yeast strains that were screened. This is the major reason
why large scale trials were not conducted with 2-DOG
mutants in a country (Canada), which by the late 1980s,
was predominantly a lager producer. Recently, a major

Spanish brewing company, with university collaborators,

has re-examined the use of 2-DOG mutants to ferment
25Plato wort, this time with a lager yeast strain96. Stable
2-DOG mutants of their lager yeast strain were isolated
and their fermentation characteristics in 25Plato wort,
using 2 L EBC tubes, were assessed at 13C. Improved
fermentation capacity, where wort glucose did not repress
maltose uptake, was achieved without changes in the beer
flavour profile.
Uptake of wort maltose and maltotriose
differences and similarities between ale
and lager yeast strains
A number of ale and lager yeast strains have been
employed in order to explore the mechanisms of maltose
and maltotriose uptake from wort. A 16Plato all malt
wort was used in a 30 L static fermentation (Fig. 18).
Under these conditions, lager strains utilised maltotriose
more efficiently than ale strains, whereas maltose utilisation efficiency was not dependent on the type of brewing
strain employed158,177. Although strain to strain variations
were observed, this supports the proposition, already discussed, that maltotriose and maltose possess independent,
but closely linked, uptake (permease) systems178. In addition, this consistent difference between ale and lager
strains supports the observation93 that ale strains appear to
have greater difficulties completely fermenting wort (particularly high gravity wort) than lager strains.
Free amino nitrogen in wort and beer

Fig. 17. Degree of Plato reduction (A) on ethanol production (B)

by an ale brewing strain and its 2-DOG derepressed variants.

The individual wort amino acids, ammonium ions and

small peptides (di- and tripeptides) are known collectively
as free amino nitrogen (FAN)103. FAN is believed to be a
good index for potential yeast growth and fermentation
efficiency164. Adequate levels of FAN in wort ensure efficient yeast cell growth and hence, a desirable fermentation performance84. FAN is only a general measurement
and a blunt instrument for setting wort and, ultimately,
malt specifications. The objective of this study was to elucidate the role of different nitrogen wort components on
yeast fermentation.
Static fermentations were conducted in 2 L cylinders
using 15Plato wort containing 30% very high maltose

Fig. 18. Maltotriose (A) and maltose (B) uptake profiles from 16Plato wort 30 L static fermentation at 15C.


syrup (VHMS), supplemented with zinc sulphate (0.2

mg/L). A lager and an ale yeast strain were employed for
fermentation at 13C and 20C respectively. Samples
were taken throughout the fermentations and yeast in suspension, specific gravity, total FAN (Fig. 19), ammonia,
individual amino acids, di and tripeptide levels and
proteinase activity determined69 (Fig. 20). A novel method
for the determination of di- and tripeptides was employed70. Following yeast removal and protein precipitation, the samples were filtered through an ultra-filtration
membrane (molecular weight exclusion of 500 Daltons)
and hydrolysis followed by HPLC was employed to determine the resulting amino acids.
The results confirmed Jones and Pierces findings62 that
amino acid uptake can be divided into four groups (Table
IV) (Fig. 21) with amino acid uptake completed, with the
exception of proline, within the first 48 h of fermentation.
Peptide removal commenced during the first 19 h of

fermentation, increased between 1924 h of fermentation

and between 2467 h of fermentation decreased gradually
(Fig. 22). The important finding is that yeast fermentation
activity does not cease when wort FAN is depleted70. During fermentation, oligopeptides are produced as a result of
larger peptide hydrolysis due to protease excretion/secretion (Fig. 23). Both lager and ale yeast strains can simultaneously use amino acids and small peptides as sources
of assimilable nitrogen. The implications of yeast protease
secretion on beer foam stability, particularly during high
gravity brewing, will be discussed later.

Fig. 21. Amino acid absorption pattern for a typical ale yeast
strain with a 15Plato wort. , Group A; M, group B; f, group C;
z, proline.

Fig. 19. Fermentation profile of a typical lager yeast strain with

15Plato wort. , FAN; M, S.G.; f, pH; z, number of suspended

Fig. 22. Total nitrogen absorption profile for a typical ale yeast
strain during fermentation of a 15Plato wort. , Total
oligopeptides; M, proline; f, total ammonia; z, total amino

Fig. 20. Total nitrogen fermentation absorption profile for a

typical lager yeast strain with 15Plato wort. , Total
oligopeptides; M, proline; f, total ammonia; z, total amino
Table IV. The order of wort amino acid uptake during fermentation.
Group A: fast
Glutamic acid
Aspartic acid

Group B:

Group C: slow

Group D:
little or no

Fig. 23. Proteinase activity for ale and lager yeast strains during
fermentation of a 15Plato wort. M, Proteinase lager activity; ,
proteinase ale activity.
VOL. 115, NO. 1, 2009


Fig. 24. The ICBD 2 hL pilot brewery.


The level of wort solids (trub) carried over from cereal
mashing and grape crushing has been the subject of
considerable debate in the production of beer, whisky and
Wort solids influence:
The removal of CO2 from solution during fermentation
by acting as nucleation sites. Removal of CO2 from
solution can occur with no suspended particles but requires a great deal of energy68.
Solids confer nutritive value to the wort. The rate of
fermentation is faster in the presence of insoluble material. Wort solids are generally associated with higher
levels of fatty acids71.
In some instances, yeast cells are able to attach themselves to wort solids and display enhanced growth because of the concentration of nutrients at the particle
In order to assess the influence of wort clarity on ester
formation (ethyl acetate and isoamyl acetate), 15Plato
wort was produced in the ICBD 2 hL pilot plant (Fig. 24)
and a number of wort types fermented in 1 L volumes in 2
L cylinders at 27C employing the S. cerevisiae M
strain a distilling strain obtained from Kerry Biosciences, Menstrie, Scotland. Carbon dioxide evolution rates
during fermentation and ethyl acetate and isoamyl acetate
concentrations at the end of fermentation were determined129. The following wort types were studied:


Table V. Concentration of carbon dioxide present during fermentation.

Cloudy wort
Clear wort
Clear wort plus diatomaceous earth
Clear wort plus bentonite

24 hours (g/L)

48 hours (g/L)



Cloudy wort
Clear wort
Clear wort plus 0.2 g/L diatomaceous earth (DE)
Clear wort plus DE and 5.5 mg/L C16:1 fatty acid
Clear wort plus 0.2g/L bentonite.
The concentration of CO2 during fermentation of the
15Plato wort types was determined (Table V). Cloudy
wort, containing trub, and wort with DE acted as nucleation sites and increased CO2 evolution out of the wort.
Clear wort and wort plus bentonite did not function as
nucleation sites and consequently CO2 remained in the
fermenting medium to a much greater extent. Why was
there a difference between trub, DE and bentonite? In
order to visualise each type of particle and obtain data on
their surface characteristics, environmental scanning electron microscopy (ESEM) was conducted (Fig. 25). In DE,
there was a heterogeneous mix of particle shapes and
sizes. The surface of most of the particles had an extremely porous structure, as would be expected. The micrograph of the cloudy wort solids showed a mix of
different structures, again porous in nature. Bentonite had
a more homogeneous structure. In addition, it possessed a

Fig. 25. Environmental scanning electron microscopy (ESEM) of different solid materials.
Table VI. Concentration of ethyl acetate and isoamyl acetate following
160 h fermentation of different wort types.

Cloudy wort
Clear wort
Clear wort plus diatomaceous
earth and C16:1 fatty acid

Ethyl acetate

Isoamyl acetate





much different surface topology. It did not appear to

possess the same porous nature as DE or wort solids.
Ester levels are also influenced by the particle concentration and type in wort. Ethyl acetate and isoamyl acetate
concentrations are high in cloudy wort and DE containing
C16:1 fatty acid (Table VI). This reflects the fact that
unsaturated fatty acids, that would be absorbed onto wort
trub, induce the synthesis of esters74. The influence of
mashing conditions under controlled circumstances has
been studied using the ICBD 2 hL pilot brewery157. Mashing conditions influence wort solid material levels. These
solids enhance the wort fatty acid composition, which results in increased yeast growth and viability. This in turn,
results in decreased ester and increased higher alcohol
concentrations in the fermented wort.


During the past 30 years, process optimization and increased efficiencies have been priorities for many brewing
companies worldwide138. Process intensification has become part of this endeavour and has focussed on reduced
capital expenditure142, increased fermentation rates and

final attenuation37, high quality yeast viability and vitality17, decreased maturation times92, more efficient stabilisation and filtration140, enhanced beer quality and stability135 and high gravity brewing81.
Various studies with high gravity worts have already
been discussed in this paper. High gravity brewing employs wort at higher than normal concentration, and consequently requires dilution with water (usually de-oxygenated) at a later stage in processing81. By this means, increased production demands can be met without expanding brewing, fermenting and storage facilities.
The use of high gravity brewing,
followed by appropriate dilution
Dilution of high gravity wort, before or after fermentation, requires that the water employed be given special
treatment. The specifications of the treatment procedure
will vary depending on the dilution point81. The longer the
fermented wort is maintained undiluted, the greater the
capacity efficiency. Consequently, most breweries add
water to the concentrated beer immediately prior to the
final polishing filter. Dilution at this point requires that
the water is specially treated to secure biological purity
and chemical consistency. Most importantly, the dissolved
oxygen (DO) content of the dilution water must be reduced to approximately 50100 g/L (DO), in order to
enhance beer flavour stability.
High gravity brewing began in the United States in the
early 1960s and spread throughout North America, Australia and South Africa. Taxation and regulation difficulties impeded its implementation in a number of European
countries. Regulation problems have now largely been
overcome and high gravity brewing can now be implemented worldwide without financial penalties. Nevertheless, the impact on the flavour and foam stability of cerVOL. 115, NO. 1, 2009


tain product types at high gravity remains a concern and

challenge to some breweries.
There are a number of advantages and disadvantages to
this process. The advantages can be summarised as follows81:
Increased brewing capacity, more efficient use of existing plant facilities reduced capital expenditure;
Reduced energy (heating, refrigeration, etc.), labour,
cleaning and effluent costs;
Improved beer physical and flavour stability;
More alcohol per unit of fermentable extract because
of reduced yeast growth and consequently more of the
wort sugars being converted to alcohol;
High gravity worts may contain higher adjunct rates;
Although largely anecdotal, beers produced from high
gravity worts are often rated smoother in taste; and
High gravity brewing offers greater flexibility in product type. From one mother liquid a number of products can be brewed as a result of dilution and/or use of
malt and hop extracts, syrups and stabilisers.
Although these advantages are significant, as with
most important processes, high gravity brewing has a
number of disadvantages142:
Due to the more concentrated mash (increased ratio of
carbohydrate to water), there is decreased brewhouse
material efficiency and reduced kettle hop utilisation.
This problem can be alleviated by the use of modern
mash filters58 (instead of a lauter tun), kettle syrups127
and/or hop extracts107;
Decreased foam stability (head retention)11,30 details
There can be difficulty in achieving beer flavour match
compared to comparable lower gravity beers1,169,175.
The effects of high gravity wort on ester formation and
the influence of wort spectra (ratio of glucose to maltose) will be discussed later. However, flavour problems with high gravity brewed beers have been exaggerated and adjustments to the process can make a
difference. Particularly important is the yeast pitching
rate and the wort dissolved oxygen level at the beginning of fermentation86. As a rule of thumb, 1 million/mL viable yeast cells are pitched for every wort
Plato (i.e., a 16Plato wort would be pitched with 16
million cells/mL) and 1 ppm dissolved oxygen (DO)
for every wort Plato (i.e., a 16Plato wort would require 16 ppm DO) at the beginning of fermentation;
High gravity worts can influence yeast performance
with effects apparent upon fermentation and flocculation101. The increased osmotic pressure, elevated alcohol concentration and modified nutrient balance, all
have a profound influence on yeast performance during
the fermentation of high gravity worts details later.
Another negative effect of high gravity worts on yeast
performance concerns the number of generations
(yeast cycles) that can be fermented by a single yeast
culture. The number of cycles that can be employed is
reduced with increasing wort gravity. The use of the
Labatt lager yeast strain exhibited the following specifications:
<20 Cycles (generations)
15 Cycles


12 Cycles
8 Cycles
Significant strain to strain variation has been observed
and, although there are exceptions, it would appear that,
in general, ale strains are more susceptible than lager
strains to repeated re-pitching in high gravity wort
Influence of high gravity worts
on yeast viability
When yeast is first pitched into high gravity wort,
passive diffusion of water out of the cell occurs and this
results in a decrease in cell viability (determined by
methylene blue and methylene violet staining). Figures 26
and 27 illustrate experiments with 12 and 20Plato worts
fermented with a lager and an ale yeast strain. Cell viability decreased in both strains within the first 24 h of
fermentation99. However, the decrease in viability was
exacerbated with the 20Plato compared to the 12Plato
wort. However, with both yeast types (which are representative of a number of lager and ale strains studied99
data not shown) the viability recovered later in the fermentation. In addition, for reasons that are unclear, ale
strains maintained higher viability than lager strains102.

Fig. 26. Effect of wort gravity on the viability of a lager yeast

strain. Viability determined by the methylene blue staining

Fig. 27. Effect of wort gravity on the viability of an ale yeast

strain. Viability determined by the methylene blue staining

Effects of stress on yeast intracellular storage

Saccharomyces and related species, including brewers
yeast strains, contain two major intracellular storage carbohydrates trehalose and glycogen. Trehalose is a disaccharide containing glucose units (Fig. 28). It protects the
cell against stress (for example, osmotic pressure, ethanol,
high and low temperature and desiccation)87. It has been
correlated with cell survival under adverse conditions and

is also an important stress indicator in brewing yeast cultures during high gravity wort fermentation (Figs. 29 and
30). There was rapid synthesis of trehalose in 20Plato
wort during the first 24 h of fermentation. As the cultures
acclimatised to the stress conditions imposed by this wort,
the intracellular trehalose levels decreased. It is interesting
to note that lager strains maintained higher trehalose levels than ale strains99.
Glycogen is an intracellular glucose polysaccharide
with a structure similar to starch consisting of 1,4 linkages with 1,6 branch points (Fig. 31). It is the major
reserve energy storage material in yeast cells and many
other organisms (including humans). Glycogen accumulates in yeast under nutrient limiting conditions. It has a
role in providing carbon and energy for the maintenance
of cellular activities104. During the first 68 h of wort
fermentation there is rapid utilisation of intracellular glycogen (Fig. 32). This utilisation is directly proportional to
the synthesis of lipids [mainly unsaturated fatty acids
(UFA) and sterols (ergosterol)]. These lipids are employed
by the cells to produce de novo membrane material during
cell division. Once cell division begins to decrease, glyco-

Fig. 28. Structure of trehalose.

Fig. 31. Structure of glycogen.

Fig. 29. Effect of wort gravity on trehalose metabolism in a
lager yeast strain.

Fig. 30. Effect of wort gravity on trehalose metabolism in an ale

yeast strain.

Fig. 32. Intracellular concentrations of glycogen and lipids in a

lager yeast strain during fermentation of a 15Plato wort.
VOL. 115, NO. 1, 2009


Fig. 33. Effect of wort gravity on vacuole size with an ale yeast strain.

Fig. 34. Changes in the vacuolar morphology during high gravity wort fermentations with an ale yeast strain.

gen accumulates. It is important that maximum levels of

intracellular glycogen are present in the yeast culture
when it is harvested for storage, prior to being re-pitched
into a subsequent wort fermentation. It is critical that glycogen levels in yeast are conserved during storage because depleted glycogen levels will lead to incomplete
Another form of stress imposed upon brewers yeast is
the process of acid washing. It has been shown to be an
effective procedure to remove bacterial contaminants from
yeast slurries2. The physiological condition of the yeast
and various environmental factors have been shown to
affect the resistance of brewers yeast to acid washing
conditions124. One such environmental condition is high
gravity brewing35. Acid washing adversely affected yeast
viability from a 20Plato wort fermentation, whereas
yeast from a 12Plato wort fermentation were not affected
to the same extent34. Strain variations were observed be20


tween lager yeast strains in their resistance to high gravity

conditions and acid washing33. The resistance to acid
washing was also influenced by storage conditions, with
yeast that was stored poorly having the lowest yeast
viability. Yeast management procedures must be optimised when repitching yeast from high gravity fermentation to ensure that the yeast is in good physiological
condition and can maintain its resistance to acid washing.
Yeast morphological changes induced
by high gravity worts
The yeast vacuole is an oval, intracellular body, which
exists as a single organelle or with several distinct
compartments and occupies approximately one third of
the volume of the cell. It acts as a reservoir for storage
nutrients and specific enzymes100. Its volume changes
with growth phase and environmental conditions. Yeast
cells modify their vacuolar volume by increasing in size

Fig. 35. Effect of wort gravity on the cell surface morphology of an ale yeast strain.

12Plato wort. Those fermenting the low gravity wort

exhibited a much smoother surface structure98.
Effect of high gravity brewing
on beer foam stability

Fig. 36. Changes in hydrophobic polypeptide levels from kettle

full to final beer. Final beers diluted to 4.5% abv.

Fig. 37. Changes in hydrophobic polypeptide levels during the

fermentation of low and high gravity worts. Final beers diluted
to 4.5% abv.

during the fermentation of 20Plato wort compared to the

same yeast strain fermenting 12Plato wort (Figs. 33 and
34)98. As well as changes in vacuolar size, high gravity
wort modifies the cell surface morphology of a yeast
culture (Fig. 35). Viewed with a scanning electron microscope, cells fermenting a 20Plato wort had a very uneven
cell surface topology compared to cells fermenting a

Beers brewed at higher gravities, followed by dilution,

have poorer foam stability compared to similar beers
brewed at lower gravities37. It is known that specific hydrophobic polypeptides play an important role in foam
formation and stability3. Also important are isoalpha
acids, metal ions and melanoidins. The level of hydrophobic polypeptides has been determined throughout the
brewing and fermentation of high gravity (20Plato) and
low gravity (10Plato) worts. During brewing, there is a
proportionately greater loss of hydrophobic polypeptides
with the 20Plato wort than with the 10Plato counterpart16 (Fig. 36). When the high gravity beer was diluted to
4.5% alcohol by volume, equivalent to the low gravity
beer, it contained a level of hydrophobic polypeptides that
was less than 50% of the level produced in a low gravity
beer (Fig. 37).
The head retention of the diluted high gravity brewed
beer was less than that of the low gravity brewed beer28.
This difference was apparent when both beers were
poured into 100 mL measuring cylinders and the time
taken for foam collapse determined. Two minutes after
pouring, there was very little foam or cling with the
diluted high gravity beer sample, but substantial foam and
cling remained on the low gravity beer sample (Fig. 38).
Four minutes after pouring, differences in the foam stability of the two beer types were even more exacerbated
(Fig. 39).
Fermentation is a key stage where hydrophobic polypeptides are lost (Fig. 37). At least three factors would
account for this loss27. Firstly, losses occur due to fermenter foaming. During wort fermentation, a high gradient of hydrophobic polypeptides towards the surface was
shown to occur12. This enhances adhesion of foam-active
compounds to the side of the fermenter vessel during
transfer to the conditioning vessel. Secondly, foam-positive hydrophobic polypeptides are lost as a result of hot
VOL. 115, NO. 1, 2009


Table VII. Influence of wort gravity on beer ester levels.





Ethanol (v/v)
Ethyl acetate (mg/L)
Isoamyl acetate (mg/L)

Table VIII. Percentage viability of brewing yeast strains after 96 h

fermentation of synthetic mediaa.

Fig. 38. Beer foam collapse characteristics 2 min after pouring.

Beer on left produced with low gravity wort (10Plato). Beer on
right produced with high (20Plato) gravity wort.

Ale 1
Ale 2
Ale 3
Lager 1
Lager 2
Lager 3






yeast extract 4% sugar medium. Methylene blue and

methylene violet stains employed.

Table IX. Vitality of brewing yeast strains after a 96 h fermentation of

synthetic mediaa.
Ale 1
Ale 2
Ale 3
Lager 1
Lager 2
Lager 3

Fig. 39. Beer foam collapse characteristics 4 min after pouring.

Beer on left produced with low gravity wort (10Plato). Beer on
right produced with high (20Plato) gravity wort.





yeast extract 4% sugar medium. Acidification power test.

During high gravity wort fermentations, increased stress

on the yeast, in the form of elevated osmotic pressure and
ethanol concentration, appears to have stimulated the secretion of proteinase A into the wort during fermentation13.
Influence of wort sugar spectrum
and gravity on ester formation

Fig. 40. The effect of low (10Plato) and high (20Plato) worts
on proteinase A release by yeast during fermentation.

and cold break formation12. Due to increasing polyphenol

levels in high gravity worts, a disproportionately greater
amount of hydrophobic polypeptides are lost in high gravity worts due to hot and cold break precipitation, compared to lower gravity wort. Finally, yeast secretes proteolytic enzymes into the fermenting wort and these
appear to have a negative effect on the foam stability of
finished beer due to polypeptide degradation that occurs
during fermentation and storage. Proteinase A increased
throughout fermentation (Fig. 40)29. Higher amounts of
proteinase A were released during a 20Plato wort fermentation compared to the 10Plato wort fermentation.


It has already been briefly discussed that one of the

disadvantages of this process is that fermentation of high
gravity worts induces the production of disproportionately
high levels of esters (Table VII)37. Varying the wort sugar
source has been reported173 to modify the level of many
metabolites, including esters, although reasons for these
differences are unclear. Entry of the hexose sugars, glucose and fructose, into the yeast cell is facilitated by the
same protein system, although utilisation of glucose
occurs more quickly than fructose, when the two sugars
are fermented separately, possibly due to the differing
affinities of the sugars for the transporter8. It has already
been discussed that the disaccharide maltose in wort is
internalised by the cell only when 4050% of the glucose
has been removed from the wort38 and occurs via an active
transport system, whereas the uptake of glucose and fructose is by passive transport173.
Initially 4% glucose and maltose in a synthetic medium (yeast extract peptone medium) were fermented
separately with shaking at 21C, in order to eliminate any
possible inhibition of sugar uptake and the production of
ethyl acetate and isoamyl acetate was monitored173. The
fermentation performance of three ale and three lager
brewing yeast strains employed in this study was similar.
Tables VIII and IX show the viabilities (determined by
methylene blue staining) and vitalities (determined by the

Table X. Ethyl acetate and isoamyl acetate produced by brewing yeast

strains during the fermentation of a synthetic mediaa.
Isoamyl Acetate (mg/L)

Ethyl Acetate (mg/L)

Ale 1
Ale 2
Ale 3
Lager 1
Lager 2
Lager 3
a Peptone









yeast extract 4% sugar medium

Table XI. Percent sugar composition of brewing syrups.


Maltose syrup

Very high maltose

syrup (VHMS)



Fig. 42. Ethyl acetate concentration (mg/L) in fermenting worts

of differing gravities and sugar composition. , 12Plato (30%
MS); , 20Plato (30% MS); S, 20Plato (30% VHMS).

Fig. 43. Isoamyl acetate concentration (mg/L) in fermenting

worts of differing gravities and sugar composition. , 12Plato
(30% MS); , 20Plato (30% MS); S, 20Plato (30% VHMS).

Fig. 41. Wort sugar profiles of 12Plato and 20Plato worts

containing either 30% (w/v) maltose syrup or 30% (w/v) very
high maltose syrup.

acidification power test) of the cells, respectively, following four days of fermentation. For all six strains studied,
cells cultured in maltose consistently had higher viabilities and enhanced vitalities compared to their glucose cultured counterparts. Reasons for these differences are not
immediately apparent. It may be the result of slower
initial uptake rates of maltose compared to glucose and
consequent reduced growth rates. In addition, the fact that
maltose uptake occurs by active transport and glucose by
passive transport is no doubt relevant.
Despite the apparent sturdiness of the maltose grown
cells, the production of ethyl acetate and isoamyl acetate
was lower than in the glucose grown cells (Table X). The
lower levels of ester production, with maltose as the substrate, could be due to a number of reasons. It is possible
that fermentation with maltose inhibits the transport of
esters out of the cell, perhaps by modifying the plasma
membrane, thus giving the impression that fewer esters
are produced. However, in the light of the increased
viability and vitality of the maltose grown cells, this is unlikely. Another possibility is that maltose metabolism produces lower levels of acetyl-CoA, which has been suggested as resulting in fewer esters due to a lack of

intermediate metabolites. It has also been proposed that

ester production is linked to lipid metabolism144. If this is
the case, or if for some reason maltose metabolism
produces fewer toxic fatty acids, it would be reasonable to
assume that reduced toxic fatty acids would be produced
in wort containing elevated levels of maltose120.
It is generally agreed that a reduction in ester levels
particularly ethyl acetate and isoamyl acetate from high
gravity brewed beers, would be welcome. In order to
study the influence of maltose and glucose levels in high
gravity worts, two 20Plato worts were prepared, one containing 30% maltose syrup (MS) and the other containing
30% very high maltose (VHMS) syrup. The sugar composition of the two brewing syrups are shown in Table XI. In
addition, a 12Plato wort containing 70% (w/v) maltose
syrup (MS) was prepared and used as a control. The sugar
spectra of the three worts is shown in Fig. 41. The maltose
plus maltotriose concentration in the 20Plato VHMS
wort had increased compared to the 20Plato MS wort
with a corresponding decrease in the concentration of glucose plus fructose.
The three worts were fermented in the ICBD 2 hL pilot
brewery with a lager yeast strain at 13C, and the concentrations of ethyl acetate and isoamyl acetate determined
throughout the fermentation (Figs. 42 and 43). The profiles were similar for both esters. The concentration of
both esters in the 20Plato (MS) fermented wort was
twice that observed in the 12Plato (MS) fermented wort.
However, the ester concentration in the 20Plato (VHMS)
was approximately 25% reduced compared to the
VOL. 115, NO. 1, 2009


Table XII. Percentage viability of an ale and a lager brewing strain after
fermentation in 12Plato and 20Plato wortsa.









a Methylene

blue and methylene violet stains employed.

(55) syrup.
c Very high maltose (70) syrup.
b Maltose

Table XIII. Vitality of an ale and a lager brewing strain after

fermentation in 12Plato and 20Plato wortsa.









a Acidification

power test.
(55) syrup.
c Very high maltose (70) syrup.
b Maltose

20Plato (MS) wort174. This confirms the findings employing synthetic media with single sugars, that maltose
fermentations produce less ethyl acetate and isoamyl acetate than glucose fermentations. In addition, similar to the
synthetic media fermentations, the wort with elevated
concentrations of maltose produced yeast with higher viabilities than the wort containing lower levels of maltose139
(Tables XII and XIII).


Recent applications of fluorescent analytical methods
for assessing yeast performance and quality utilise novel
analytical instruments. Specifically, confocal imaging and
flow cytometry are beginning to be invaluable techniques
in the brewing research laboratory but are still underused.
Studies to date have focussed on the implementation of
fluorescence optical methods and laser scanning confocal
microscopy for monitoring yeast performance during high
gravity wort fermentation79 and to examine hydrodynamic
stresses160 imposed by yeast centrifugation20.
Confocal microscopy
A number of physiological parameters and cell compounds in yeast cells (glycogen, neutral lipids, trehalose,
bud scars, DNA and intracellular proteinases) have been
successfully visualised with the aid of highly specific
fluorochromes. In particular, the expression and sub cellular localisation of proteinase A during yeast fermentation
has been studied employing a green fluorescent protein
clone117. These studies confirm previous findings13, using
the Kondo et al. assay67 method for proteinase A, which
demonstrated that this enzyme is located primarily in the
yeast vacuole, but under stress conditions significant activity appears in the cell cytoplasm. As previously described in this paper, the cells sorting mechanism that
targets proteinase A to the vacuole is partially failing un24


der stress conditions (for example, high gravity wort fermentations) and proteinase A remains in the cytoplasm or
is excreted into the environment. Laser scanning confocal
microscopy as a qualitative tool, in conjunction with flow
cytometry61,177 details later, is a powerful tool to gain
insight into the response of yeast cells to the changing
environmental conditions occurring during fermentation.
Flow cytometry
Flow cytometry is a technology that simultaneously
measures then analyses multiple physical characteristics
of single particles, usually cells, as they flow in a fluid
stream through a beam of light. It can assess yeast physical and chemical characteristics based on cell size, relative granularity and fluorescence. Flow cytometry methods have been developed to measure cell viability,
damaged cells, intracellular pH(pHi), mannan residues21
and intracellular glycogen and trehalose22.
An example of the application of flow cytometry in
brewing research is the recently conducted study of disc
stack centrifuge operating parameters and their impact on
yeast physiology24. Modern centrifuges produce forces in
excess of 10,000 times the earths gravitational force,
achieving solid separation in seconds with reduced equipment volume. Centrifuges have a number of applications
in a brewery and can be used for:
Cropping of non-flocculent yeast cultures at the end of
primary fermentation which may then be re-pitched
into a subsequent fermentation;
Reducing the yeast quantity from green beer before
the start of secondary fermentation;
Beer recovery from cropped yeast;
Separation of the hot break after wort boiling;
Removal of cold break and yeast at the end of maturation.
This study has concentrated on the effect that centrifugation has on yeast which is intended to be re-pitched.
The passage of yeast through a centrifuge exposes cells to
mechanical and hydrodynamic shear stresses161. We have
shown23 that these stresses can cause a decrease in cell
viability and flocculation, cell wall damage, increased extracellular proteinase A (PrA) levels, hazier beers and
reduced foam stability. In these studies, a commercial ale
yeast was subjected to differing operating conditions during centrifugation with 56 hL/h Westfalia Separator (Fig.
44). Yeast cultures, following centrifugation, were analysed using flow cytometry techniques. Cell viability and
intracellular pH decreased due to the processing conditions encountered during yeast cropping with a centrifuge.
A relationship has been established whereby yeast cell
wall mannan, an unfilterable haze constituent, as a function of G-force and centrifugation cycles is released from
the cell wall while concurrently, particle sizes between
0.52.8 mm and beer haze increased. Furthermore, yeast
intracellular glycogen and trehalose levels were depleted
as a result of centrifugation. During these experiments,
the centrifuge was operated under conditions similar to
those encountered during commercial production. It is important for yeast management systems that utilize centrifuges, that they are designed and operated properly, to
minimize negative consequences on beer quality and stability19. Recent investigations found that implementation

on fermentation performance and beer quality and


Fig. 44. A 5-6 hL/h Westfalia centrifuge.

of newer model centrifuges and redesigned pipe work

improved green beer quality.
Flow cytometry analytical methods have also been
used to assess yeast physiological status during high and
low gravity wort fermentation. Analyses such as cell viability, damaged cells, pHi and intracellular glycogen and
trehalose content, were conducted. The results demonstrate that high gravity fermentations, compared to low
gravity fermentations, initially contain increased numbers
of damaged cells and lower glycogen and trehalose levels,
confirming the presence of stressed cells.

Although Horace Brown conducted his fifty years of
research over one hundred years ago, the results of his
studies are still apparent. The award that has been established in his name is to recognise eminent services on the
scientific or technical side of the fermentation industries,
has to date recognised 24 brewing scientists who have
made considerable contributions to our understanding of
the brewing process and to the development of beer quality and stability. The studies discussed in this paper have
attempted to solve fermentation questions with the application of fundamental research principles. Areas that are
discussed include: yeast flocculation, yeast management
and storage methods, genetic manipulation of yeast, wort
sugar and amino acid uptake and the influence of wort
composition (particularly wort clarity and concentration)

Many people and organisations have contributed to the research studies described in this paper and are gratefully acknowledged. Much of the research described has been conducted as
part of studies for PhD degrees. Fourteen such theses are cited.
In addition, many post doctoral scientists have made considerable contributions to the research effort at both Labatt and
Heriot-Watt University. Also visiting scientists from Argentina,
Australia, Brazil, Canada, Croatia, China, Finland, Germany,
Jamaica, Japan, Slovenia, South Africa, Turkey, the United
Kingdom and the United States, have contributed to the research
programme. In Canada, as well as both financial and moral
support from Labatt, the Canadian Federal Government provided invaluable grants and advice. Thanks are specifically due
to: Agriculture Canada, the National Research Council of
Canada (NRC), and Natural and Engineering Sciences Research
Council (NESRC) for their support. In Heriot-Watt, a number of
industry organisations have supported the research effort particularly: Brewery Research International (BRi), the Brewing, Food
and Beverage Industry Suppliers Association (BFBi), the British
Brewery and Pub Association (BBPA)(including the IBD Grants
Committee), the Scotch Whisky Association (SWA), the Scotch
Whisky Research Institute (SWRI), and the companies who have
supported the research programme include: Alltech, Asahi
Limited, Briggs of Burton, British Sugar, Charlton United Brewers, Coopers Brewery, Coors Brewing (including Bass), Corn
Products, Diageo (including Guinness and their Spirits Division), Ineos Silicas, Lion Nathan, Miller Brewing, Scottish
Courage, South African Breweries, Steiner Hops, Suntory Limited and Wessex Grain (Green Spirit).
During my forty years in the brewing industry, I have been
supported by a large number of excellent scientists. Administrators and secretaries have also been an invaluable part of this
endeavour. Worthy of particular mention and thanks are Karen
Smith (who was my secretary at Labatt for thirteen years) and
Anne Anstruther (who was my secretary at Heriot-Watt University for three years immediately prior to my retiral).
Lastly, but by no means least, I have been supported by Inge
Russell since 1970. Her encouragement, enthusiasm, friendship
and critical approach cannot be over emphasised.
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(Manuscript accepted for publication February 2009)

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