Brewery microbiology eighth century

1. J. lust. Brew., January-February, 1977, Vol. 83, pp. 9-14
THE HORACE BROWN MEMORIAL LECTURE*
A CENTURY OF BREWING MICROBIOLOGY
By C. Rainbow
The First Phase: Pasteur and Horace Brown
Even the most partisan of us in our industry scarcely
appreciate the great contribution that fermentation science in
general, and brewing science in particular, has made to the
science of Microbiology, so that it is appropriate to reflect
that Pasteur's great work on fermentation simultaneously
revolutionised general, medical and industrial aspects of the
subject.
Pasteur's work provoked much criticism from the more
conventional scientists of his day, especially from the promin
ent chemist Licbig, who denied that living cells intervened in
the phenomena of fermentation and putrefaction, which he
regarded as resulting from the decomposition of chemically
unstable 'albuminous' molecules, It says much for Horace
Brown that his mind was receptive and original enough to
take Pasteur's broad view of the close connection between the
outcome of studies on fermentation and those on preventive
medicine. Only four years after he had joined Worthington's
Brewery on 1 January 1866 as a junior brewer, he had so
grasped the essential scientific truthfulness of Pasteur's con
clusions and so thrust aside current prejudices, that he was
already applying Pasteur's practices to brewing microbiology.
Speaking to the London Section in 1916, he recalls 'I had just
turned to microscopic methods when Pasteur's Studies on Wine
came into my hands in 1870. The immediate effect was that
of a ray of light piercing the darkness and illuminating a new
path into the unknown'. Thus, although we usually associate
with Brown his classical work on starch, it is with him as a
microbiologist and as the brewing disciple of Pasteur that any
survey of brewing microbiology must start.
A hundred years ago, the microbial spoilage of beer was
causing great financial losses. In Burton, the large stocks of
ale brewed between October and May for sale in the warm
months after brewing had ceased were especially prone to
spoilage by a bacterium causing, first a 'silky' turbidity, and
then lactic acidification. By 1871, Brown knew the life history
and effect of the causative organism, later named Saccltaro-
bacillus pastorianus. Brown's experimental difficulties were
great: lacking techniques, which are today commonplace, he
had to devise his own methods of isolation and staining, based
on those of Pasteur. He also devised, as a method of detecting
spoilage organisms and predicting spoilage by them, the
'forcing' method still in use today.
At this time, other types of beer spoilage were being studied
by Pasteur and Brown. In Etudes stir le Vin (1866) and the
subordinate work Etudes stir le Vinaigre (1868), Pasteur des
cribed acetification, ascribing it to the film-forming organism,
Mycoderma aceti, which he recognized as the agent by which
atmospheric oxygen was transferred to alcohol in the form
ation of acetic acid. Furthermore, in his Mycoderma vini, he
recognized those acetic acid bacteria which could 'over-
oxidize' acetic acid into carbon dioxide and water, thus making
an important basic observation in the field of chemical
microbiology. As we shall see later, ability or failure to 'over-
oxidize' continues to provide us with a fundamental criterion
for distinguishing the genera Acetobacter and Acetomonas.
For his part, Brown, ever reacting sensitively to all that was
most progressive in experiment and theory, recognized the
importance of the work of Buchner (1897), who first showed
that cell-free yeast juice could bring about alcoholic ferment
ation. Thereby he clearly established the enzymic nature of the
fermentation process and finally resolved the Liebig-Pasteur
controversy.
Both Pasteur and Brown were familiar with the phenomenon
of 'ropiness'. They recognized the causative organism as a
• Presented at a meeting of the Institute of Brewing held in the
Royal Institution, London, on Monday, 18 October 1976.
'viscous ferment', but they seem not to have proceeded far in
its study. Brown also knew the role of 'wild' yeasts in produc
ing beer 'frets', which, in his experience, caused more beer
spoilage than did bacteria.
As a method of controlling microbial spoilage, Pasteur
ultimately rejected that of adding harmless antiseptics, such as
sulphite, in favour of the heat treatment now universally
known as pasteurization, the invention of which illustrates
just one facet of Pasteur's practical genius. At the laboratory
level, Brown's 'forcing' test had also the touch of genius,
although he himself recognized its shortcomings when applied
to predict the behaviour in cask of beer infected with those
yeasts responsible for secondary 'frets'.
E. C. Hansen
To the late 19th century belongs another great name in
brewing microbiology, E. C. Hansen, who, in the Carlsberg
Laboratory, worked on the yeasts in all their environments.
He evolved the criteria by which even morphologically similar
yeasts could be distinguished by their modes of forming
endospores and their abilities to ferment individual sugars.
Essential to his studies, he devised techniques of isolating
single cells and thus of producing pure cultures, the applica
tions of which in commercial brewing he was first to realize.
He isolated strains of Saccharomyces pastorianus and S.
ellipsoideus from a spoiled lager and demonstrated their role
in rendering beer turbid. Hansen bequeathed to us our
knowledge of the practical management of pure yeasts and his
name is perpetuated in the names of our culture species, S.
cerevisiae Hansen and S. carlsbergensis Hansen.
Taxonomists in Difficulty and in Doubt
By about 1900, we had reached the point marking the end of
the great classical studies of brewing microbiology. This period
of rapid advance and accumulation of a mass of undigested
knowledge perforce brought with it untidiness, confusion and
the need for a sound filing system.
Among the matters worse confounded, it might be expected
that microbial classification would be one. Unicellular micro
bial forms, like yeasts and bacteria, arc difficult, even today,
to classify. They lack the richness of morphological diversifica
tion characteristic of the vegetative and sexual forms of the
higher plants on which Linnaeus based his great classification.
In microbiology, much more reliance must be placed on
colony forms, but more especially on biochemical characters,
information on which was not to hand in the late 19th Century.
As an early example of taxonomic confusion, we may note
how the name Mycoderma, now applied as a specific name
only to certain yeast-like organisms, was once applied to film-
forming organisms, regardless of whether they were yeast-like
or bacterial. Again, the superficial resemblance of the tetrad
groups of brewers' 'sarcina' to the octads of the aerobic cocci
of true sarcinae, led to their erroneous name and concealed
their nature as true lactic acid bacteria. To this Hanscn's
authority connived. Although Pasteur, in Etudes sur la Biere
(1876) did not commit himself taxonomically (he called the
organism Ferment no. 7), Hansen (1879) gave the name
Sarcina, which persisted some 60 years, despite Balcke (1884),
who gave it the generic name Pediococcus, now accepted,
despite some vicissitudes, of which more anon.
Nor were the taxonomic niches occupied by the lactic beer
spoilages rods and by the acetic acid bacteria understood.
Rod-like bacteria were often given vague or incorrect desig-
nations like BacteriumorBacillus. In Etudessurla Biere, Pasteur
called his lactic spoilers 'bacilles des bicrcs tourndes' and it was
not until 1892 tha the special properties of lactic rods were
signified by van Laer's naming them Saccharobacillus pastori-

2. 10 RAINBOW! HORACE BROWN MEMORIAL LECTURE [J. Inst. Brew.
anus. Thereafter, their status as true members of Beijerinck's
(1901) genus Lactobacillus was attained rather slowly during
the two decades 1920-1940. Even Shimwell, who became pre
eminent in this field, was still using the name Saccharobacillus
in 1937; but, by 1941, he had completely adopted the correct
classification as that of a heterofermentative rod in the genus
Lactobacillus.
The vicissitudes of classification of the acetic acid bacteria
are scarcely less involved. Pasteur used Persoon's (1822) name
Mycoderma, but it was supplanted by several others, including
especially Bacterium applied by A. J. Brown (1886) to the
cellulose-pellicle-forming organism then called B. xylinum.
Ultimately, their distinctive property of ability to oxidize
ethanol to acetic acid led to the adoption of the generic name
Acetobacter, first used in 1898 by Beijerinck.
Yeast Taxonomy
During the past century, yeast nomenclature has been less
bedevilled by changes than has that of brewery bacteria. For
this, we can be grateful that brewing yeasts and many of the
beer spoilage yeasts possess the property, most valuable for
clear classification, of ability to form ascospores. Schwann
(1839) observed yeast endospores and Reess, working between
1868 and 1870, described them in many species, showing them
to be ascospores like those developed by certain of the lower
Ascomycetes. In 1870, he suggested that the name Saccharo-
myces, previously used by Meyen (1837) for any budding
yeasts, should be applied only to the spore-forming yeasts.
Hence arose the modern concept of the genus Saccharomyces
(Meyen) Reess.
Changes of name at the specific level and, not surprisingly
at the generic level with the non-sporing yeasts, have given
more difficulty. A recent example of the former is the view,
expressed in Lodder's monograph, The Yeasts (1970), that S,
carlsbergensis should be included in the species S. ttvarum.
There are numerous examples of changes of generic name,
made in order to correct, in the light of new information,
erroneous classification of the asporogenous, so-called 'torula'
yeasts. The generic name Torula was banished by taxonomists
many years ago from yeast nomenclature and now Mycoderma
has lost generic significance, many of its species of brewing
interest having been found more appropriate places in the
asporogenous genera Candida, Cryptococcus, Torulopsis and
Modotorula. Of special interest, are certain 'torula' yeasts
which have had to be allotted to sporogenous genera as a result
of discoveries of sexual features in them. Thus, matings of
M. cerevisiae have proven its taxonomic relationship with
Hansenula anomala, and some strains of Candida mycoderma
have been transferred to the genus Pichia because they have
recently been observed to form spores.
I will conclude my comments on yeast classification by
paying tribute to the painstaking studies of the school of
workers at Delft and their associates, recorded in a series of
authoritative monographs, starting with that of Stelling-
Dekker (1931) and culminating in Lodder's recent edited work
of nearly 1400 pages, The Yeasts (970).
J. L. Shimwell's Work on Brewery Bacteria
The inter-war years saw the isolation and precise description
of many strains of lactic and acetic acid bacteria. Prominent
among those responsible were the late Professor T. K. Walker
and his colleagues Kulka and Tosic, working especially on
acetic acid bacteria, and J. L. Shimwell, whose work encom
passed all types of brewery bacteria and to whom we largely
owe the present orderly state of their classification. Much of
hs work appeared in our Journal for 30 years onwards from
1935.
As well as recognizing beer spoilage lactic rods as hetero
fermentative strains of Lactobacillus, he brought final convic
tion that 'brewer's sarcina' was not a sarcina, but a true homo-
fermentative lactic coccus, which he proposed to include in
the genus Streptococcus. Although present practice is to
classify 'brewer's sarcina' in the lactic genus Pediococcus,
Shimwell had nevertheless rectified a basic error. He also
showed how the phenomenon of 'ropiness' could be caused
both by lactic pediococci and by certain capsule-forming
acetic acid bacteria; how the causative organisms could be
'distinguished) and how the proneness of beer to spoilage by
one or other of the rope-formers could be predetermined by
its carbohydrate composition (Shimwell, 1936; 1947; 1948).
ShimwelPs work on the acetic acid bacteria was even more
extensive. He was quick to support the view of Leifson (1954)
that there were two types of acetic acid bacteria, differing in
the number and disposition of their flagella and that these types
corresponded to two groups, long recognized by their respec
tive abilities to oxidize acetic acid itself. This distinction was
also shown by my co-workers and myself (Rainbow, 1966) to
be paralleled on nutritional and biochemical criteria. That
there are two genera of acetic acid bacteria is now accepted.
Acetobacter includes all species which can oxidize acetic acid,
while Acetomonas (Gtuconobacter according to some authori
ties) provides for those which cannot do so. According to
Shimwell (1960), the acetomonads are the more dangerous as
beer spoilers. They are also industrially important in that they
have been applied to produce gluconate and 2- and 5-oxo-
gluconates from glucose, dihydroxyacetone from glycerol, and
sorbose (an intermediate in the synthesis of ascorbic acid) from
sorbitol. By contrast, the strains applied in commercial vinegar
brewing are acetobacters.
Their work on the acetic acid bacteria led Shimwell & Carr
(1960) to comment on their 'great mutability—even great by
bacterial standards' and ultimately to the conclusion that 'it
seems to follow that 'species' of bacteria are virtually unclassi-
fiable and that even the conception of a genus should be on a
broader basis than is often the case at present'. This is not
necessarily a defeatist view: rather it is a recognition of the
great rate at which evolutionary changes take place in bac
teria, a fact which demands a radically different approach to
bacterial classification than to that of higher organisms, whose
much slower processes of evolution permit us to freeze
considerable periods of time for the convenience of rigorous
classification: it is permissible for Acer and Acacia, but not
for Acetobacter.'
In addition to his major contributions to the bacteriology
of brewery lactic and acetic acid bacteria, we owe to Shimwell
(1937) our first definitive description of Zymomonas anaerobia
and to Shimwell & Grimes (1936) that of Flavobacterium
proteus (later called Obesumhacterium proteum). The names of
both these bacteria have undergone changes which we have
seen to reflect the intrinsic difficulties of bacterial classification.
Shimwell made great contributions to brewing bacteriology
and, although he was not primaily an innovator, he has left
the subject projected within an orderly framework, while such
studies as those on ropiness have contributed valuably to
technical and scientific knowledge of beer spoilage.
Microbial Nutrition and Metabolism
Nutrition.—The years 1860-1930 saw the completion of a
period of descriptive biology in the field of brewing micro
biology. The second quarter of the present century saw the
rapid advance in knowledge of the biochemistry of micro
organisms, not least of those inhabiting the brewery.
Important for these advances was an understanding of
minimal requirements for nutrition, so that micro-organisms
could be studied during growth in media of precisely known
composition. In 1901, Wildiers had found that he could not
cultivate yeast on defined medium unless small amounts of
extracts of natural materials were added. Thus arose his
concept of 'bios', i.e. substances required in trace quantities
in the medium for the growth of yeast and, as we now know,
other micro-organisms. Interest in the growth factors became
worldwide in the 1920s, especially as their identity with
vitamins of the B complex, essential for human and animal
nutrition, became evident. An early advance on the 'bios'

3. Vol. 83, 1977] RAINBOW: HORACE BROWN MEMORIAL LECTURE II
front was the identification of bios I as mcso-inositol by
Eastcott (1928). Even more important was the isolation of
II mg of the methyl ester of D-biotin (bios 11B) from 250 kg
of dried egg yolk by Kogl & Tonnis (1936) and the synthesis
of pantothenic acid (bios HA) by Williams & Major (1940).
The scale and excellence of the work of Kogl & Tonnis were
readily appreciated by Dr Bishop and myself. We were
attempting, in one of the Insitutc's research projects at Birm
ingham University, to isolate biotin from a modest 40 kg of
dried egg yolk, when Kogl & Tonnis forestalled us. However,
we showed lhat our strain of brewer's yeast needed exogenous
sources of biotin, pantothenate and inositol for growth and
that, when minute concentrations of these nutrilities were
added, it would grow on a simple medium containing glucose,
mineral salts and an ammonium salt as sole source of nitrogen
(Rainbow, 1939). Subsequent observations indicate that this
account of minimal nutritional requirements needs only minor
qualification to be applicable in general to brewer's yeasts, most
of which appear to require for growth added biotin, i.e. they
cannot synthesize their own requirements of this substance.
Additionally, some strains must be supplied with D-pantothen-
ate and/or inositol. Brewing yeasts rarely show absolute
requirements for other growth factors, although 1 found two
strains for which added p-aminobenzoatc was essential
(Rainbow, 1948) and many strains are stimulated during the
early stages of growth on defined medium by supplying
thiamin (vitamin B,) and pyridoxin (vitamin B»).
It is now common knowledge that the significance of most
microbial growth factors and vitamins of the B complex lies
in their being parts of the molecules of coenzymes: this fact
illustrates well how discoveries in microbiology illuminate
biochemical phenomena in higher plants and animals and
vice versa.
Between 1937 and 1949, R. S. W. Thome of the Institute's
research team in Birmingham published important work show
ing that, during its growth in brewer's wort, yeast drew
chiefly on free amino acids for its nitrogenous nutrients and
that, on defined medium, complex mixtures of amino acids
supported better growth and fermentation than single amino
acids or simplex mixtures of them. He found evidence that
yeast obtained about 50% of its nitrogen requirements from
wort by assimilating amino acids intact and about 40% by
their deamination. The remaining approximately 10% he
suggested, on less convincing grounds, might arise from a
metabolic process, shown by Stickland (1934; 1935) to occur
in certain anaerobic bacteria and to involve the mutual
oxidation and reduction of certain pairs of amino acids, by
which their amino nitrogen became available as ammonia for
assimilation. Thome's view of nitrogen assimilation is still
acceptable, except as regards the contribution of the Stickland
reaction. Lewis & Rainbow (1965) failed to find evidence that
the yeast cell could assimilate nitrogen by this means and
considered that Thome's observations on the point were
capable of another explanation. Possibly, any nitrogen
assimilation neither ascribable to that of intact amino acids,
nor to Ehrlich deamination, might result from the assimilation
of small peptides. This point remains to be clarified, our
knowledge of the molecular size and amino acid composition
of those wort peptides assimilable by yeast being inadequate
and worthy of further study.
Other nutritional studies in the environment of brewer's
wort by Jones & Pierce (1964) have shown that yeast takes up
individual amino acids sequentially. If the same is true for
individual wort carbohydrates, as work by Phillips (1955)
suggests, it may well be that the technological implications of
wort carbohydrate composition for the attainment of sound
brewery fermentations, adequate attenuations and consistency
of beer flavour are insufficiently appreciated. Indeed, these
implications may be the more important when consideration
is given to the observations of Lewis, PhafT and their co-
workers in the 1960s on 'shock excretion'. This phenomenon
is characterized by the loss of amino acids and nucleotide
material from yeast cells on suspending them in glucose
solutions. Its significance in brewing is not known but, since
mature cells arc more affected by it than are young cells, it
would be of particular interest to assess its effects on yeast at
pitching into worts of different compositions.
The minimal nutritional requirements of the chief groups of
beer spoilage bacteria have also been elucidated in the past two
decades. At Birmingham University, my colleagues and I
showed that beer spoilage lactobacilli are nutritionally as
fastidious as their cousins in the dairy, needing exogenous
supplies of most a-amino acids, several growth factors of the
vitamin B complex and one or more purine and pyrimidine
bases. This implies that well attenuated beers, i.e. those whose
nutritional status has been depleted the most by vigorous
yeast growth and fermentation, should be the least prone to
lactic spoilage and might even resist such spoilage completely
if one or more of the nutrilitics essential for the growth of
lactobacilli were completely removed by the yeast.
Here it may be interjected that the nutritional exactingness
of lactic acid bacteria, which has been applied since 1939 as
an analytical tool in the microbiological assay of vitamins in
foodstuffs, was applied from 1943 onwards by Hopkins and
his co-workers (1943-48), Tullo & Stringer (1945) and Norris
and his co-workers (1945-53) to the materials of brewing and
to beer itself, revealing inter alia that beer in the diet makes an
appreciable contribution to the human intake ofsome vitamins
of the B complex.
In contrast to the lactic acid bacteria, the acetic acid bacteria
are nutritionally less exacting. Indeed, my team at Birmingham
showed that most acetobacters grew on simple, defined lactate-
ammonia-salts medium, without added growth factors or
amino acids, while acctamonads grew on a defined glucose-
salts medium only if certain growth factors and glutamate, or
certain substances biochemically related to it, were supplied
(Rainbow & Mitson, 1953; Brown & Rainbow, 1956; Cooksey
& Rainbow, 1962; Williams & Rainbow, 1964).
Metabolism.—Since the early years of this century, much
effort has been devoted to elucidating the mechanism of
glycolysis, the biochemical sequence, occurring in yeast,
muscle and in many other cells, by which hexose carbohydrate
is broken down primarily to pyruvate and thence to cthanol
(in yeast), lactatc(in muscle and homofermentative lactic acid
bacteria), or to other products, depending on the cells con
cerned. Simultaneously with these catabolic changes, energy
in the form of the high-energy phosphate bonds of adenosine
triphosphate (ATP) becomes available to the cells. In this lies
the biological significance of glycolysis.
An essential preliminary to the elucidation of the sequence
was Buchner's discovery in 1897 that fermentation could be
brought about by a cell-free yeast juice. Next in the trail of
detection came the observations of Harden & Young (1905;
1906) that, during fermentations with yeast juice, added
inorganic phosphate disappeared rapidly and became con
verted into a form no longer precipitated by magnesium. They
concluded that living yeast converted inorganic phosphate
into an organic form. Simultaneously in Russia, Ivanov (1905;
1906) isolated from fermenting solutions a compound he
erroneously thought to be a triose phosphate, but which
Young (1907) showed to be a hexosc diphosphatc. Harden's
classical monograph on Alcoholic Fermentation (1911;
reprinted 1932) gives an authentic account of this work. The
importance of these discoveries can readily be appreciated
now we know that the fermentation of glucose proceeds to
the pyruvate stage entirely via phosphorylated intermediates,
to which Harden & Young's work provided the vital clue.
The detailed elucidation of the pathway was perhaps the
most notable achievement of pre-World War II biochemists.
To quote Florkin (1975), it 'resulted from a convergence of
sudies of ... alcoholic fermentation and muscle glycolysis
which were not suspected ... to be based on the same meta
bolic scheme'. The process of elucidation took place between

4. 12 RAINBOW. HORACE BROWN MEMORIAL LECTURE [J. Inst. Brew.
1911, when Ncuberg & Wastcnson recognized pyruvate as a
product of glycolysis, and 1939, when the labile ester 1,3-
diphosphoglycerate was isolated as a glycolytic intermediate
by Negclein & Bromel. Embdcn, Meyerhof and Parnas made
outstanding contributions to these studies and the glycolytic
pathway is now appropriately known as the Embdcn-
Meyerhof-Parnas (EMP) pathway. The experimental methods
applied ranged from the chemical trapping of intermediates
(e.g., aldehyde and pyruvate) to the more sophisticated
promotion of their accumulation by preventing their further
transformation, either by changing the medium, or by dialys-
ing, or by adding enzyme inhibitors.
In matters of nitrogen metabolism of interest to brewers,
we must go back in time to Ehrlich, who, in a series of papers
published between 1906 and 1912, showed that the growth of
yeast on certain amino acids led, by deamination and decarb-
oxylation, to the formation of the fusel alcohols characteristic
of fermentation. Ehrlich's results have stood the test of time
well: they were confirmed and extended by Thome (1937),
during his work in the Institute's Research Laboratories at
Birmingham. Only Ehrlich's speculation that the nitrogen
from the amino acids became available to yeast as ammonia
has undergone amendment, for we now know it to involve
the enzymic transfer (transamination) of the amino group of a
participating amino acid to 2-oxoglutarate: L-glutamate is
thereby synthesized and used by the cell and the 2-oxo-acid
corresponding to the dcaminated amino acid is formed
(SentheShanmuganathan & Elsden, 1958). This 2-oxo-acid is
then decarboxylated to an aldehyde, which, in turn, is reduced
to the fusel alcohol and excreted into the beer.
Our knowledge of the metabolism of all cells owes much to
studies of fermentation and one of the critical discoveries on
which modern experimental biochemistry is based was the
first preparation of cell-free enzymes, in the form of yeast
juice, by Buchner (1897), to which I have already referred.
As well as demonstrating the enzymic nature of fermentation
and resolving the Pasteur-Liebig controversy, he bequeathed
to us a biochemical tool of inestimable value in metabolic
studies—the cell-free, enzymically active extract through which
individual enzymes and enzyme systems could be studied
outside the living cell.
As examples of such studies in the brewing context I will
quote work by my colleagues and myself at Birmingham
University. We used cell extracts to show that the differences
between acetobacters and acctomonads, which I have already
mentioned, were reflected in their respective enzyme contents,
the former possessing all the enzymes necessary to operate
the tricarboxylic acid cycle, whereas the acetomonads did not
(Williams & Rainbow, 1964). Again, Wood & myself (1961)
showed that cells of beer spoilage lactobacilli possessed a
remarkable means of metabolizing maltose, by cleaving it
phosphorolytically by the enzyme maltose phosphoryiase to
glucose and /3-glucose 1-phosphate, not, be it noted, to the
familiar a-anomcr. The 0-ester then undergoes further
metabolism, while the glucose is largely discarded by the cell.
The system is quite distinct from the hydrolytic cleavage of
maltose to two molecules of glucose. Indeed, it seems almost
unique in biological systems, having been demonstrated previ
ously only in the human pathogen, Neisseria meningitidis (the
meningococcus). The biological significance of this enzyme
makes interesting speculation: in beer lactobacilli, it may have
evolved in response to long cultivation in fermenting wort and
beer, both maltose-containing media. Certainly, possession of
the system endows beer lactobacilli with the biological advan
tage of saving, for each molecule of maltose cleaved, a mole
cule of ATP, which must otherwise be expended for the initial
phosphorylation of glucose to enable it to enter the energy-
yielding metabolic cycle. On the other hand, the system is
prodigal ofcarbohydrate raw material, since the unphosphory-
lated glucose half-molecule derived from each maltose mole
cule is substantially discarded by the cell.
Beer lactobacilli contain another unusual enzyme system by
which L-arginine is metabolized (Rainbow, 1975). In this, the
arginine dihydrolasc system, arginine is first cleaved by
arginine deaminasc to ammonia and L-citrullinc: in turn, in
the presence of orthophosphatc, the citrulline is converted
enzymically to L-ornithine and carbamoyl phosphate, which,
in the presence of a specific kinase, donates its high-energy-
linked phosphate to adenosine diphosphate (ADP) to effect
the resynthesis of ATP. This system, already known to occur
in certain lactic cocci (Knivctt, 1954), thus seems to be an
example of the biological use of a nitrogen compound as
distinct from a carbohydrate, as a source of energy.
While these examples of studies with cell-free extracts
involve brewery micro-organisms, such examples can be
multiplied ten-thousand-fold from all fields of biochemistry.
Nevertheless, I venture to suggest that, compared with some
other microbial forms, studies of yeast metabolism by this, or
by other techniques, are a little neglected. For example, less
effort seems to have been expended on aspects of the detailed
metabolism of brewer's yeast than on those of Escherichia coli,
which, for all its suitability for metabolic studies, cannot be
compared in technological importance with brewer's yeast.
Aspects of Modern Yeast Biology
Having reached the present day in my review, I would like
to refer to two subjects, which, in so far as they concern yeast,
are modern in origin. They concern genetics and immuno-
logical response.
Yeast genetics developed only after Winge (1935) had shown
that the life cycle of Saccharomyces eUipsoideus had haploid
and diploid phases. With the subsequent demonstration by
Winge & Laustscn (1939) that Mendelian segregation occurred
in another yeast species, Saccharomycodes ludwigii, it became
apparent that yeasts were subject to genetic changes based on
sexuality equally with other organisms. Since Winge, yeast
genetics has proved a fruitful field ofacademic study, especially
for C.C. and G. Lindegren over the years 1945-1960. How
ever, despite technologically orientated studies by Fowell
(1951). Gilliland (1951; 1953) and Lindegren (1956), yeast
genetics Ijas proved disappointing in its possible applications
to brewing, and hybrid yeasts with improved brewing proper
ties, such as Johnston (1965) produced at B.I.R.F., seem rarely
to have been applied in the industry. Possible reasons for this
may include (1) the conservative attitude of the brewing
industry; (2) inability to define exactly the properties desirable
in yeast for a given brewery situation; (3) practical difficulties
of hybridization; and (4) flavour problems incidental to the
application of an otherwise improved yeast. Nevertheless, it is
feasible to breed hybrid yeasts with desirable properties, or
lacking undesirable ones. For example, since Thome and
Gilliland independently in 1951 showed that flocculence was a
genetically controlled character, it should be possible to breed
yeasts combining, say, the properties of high flocculence and
desirable flavour characteristics, suitable for continuous tower
operation.
Hybridization is not the only genetical approach to the
generation of desirable yeasts. The genetical substance (dcoxy-
ribonucleic acid, DNA) can be mutated by several means,
including radiation and chemical mutagens. Such mutation is
usually, but not invariably, destructive, so that the mutants
lack some enzyme possessed by the parent. This presents the
opportunity to generate new yeasts, especially those lacking
undesirable properties, among which the production of
excessive hydrogen sulphide, diacetyl or esters immediately
springs to mind as relevant to brewing problems. Like
hybridization, this approach to the production of improved
brewing yeasts has received some, although to my mind
insufficient, attention. Among other as yet untried means to
induce genetical changes are the processes known as trans
formation and transduction. In the former, a small piece of
exogenous genetic material, extracted from a donor cell, is
introducedas part'of a free DNA'particle into a receptor cell.
This process has been known since 1944, when O. T. Avcry

5. Vol. 83, 1977] RAINBOW: HORACE BROWN MEMORIAL LECTURE 13
and his colleagues transformed pneumococcal types by this
means. In transduction, discovered by Zinder & Lederberg
in 19S2, bacterial viruses (phages) are used as vectors for trans
ferring bacterial genes from one cell line to another. Whether
either of these processes could succeed, or, with transduction,
be even possible to apply to yeasts, is not known, but the
attempt might be worthwhile.
Immunological response, induced in animals by the injection
of foreign proteins or certain polysaccharides, has been
applied in medicine and veterinary science for many years,
but the realization that it could serve the brewer did not
occur until Grabar (19S7) applied it to study the fate of
barley proteins during the brewing process. Later, it was
realized that, because of its sensitivity and high specificity,
this response could revolutionize our methods of detecting and
enumerating 'wild' yeasts in the presence of overwhelming
numbers of culture yeasts and, indeed, provide us, for the
first time, with a satisfactory method for this purpose. When
injected into animals, by virtue of the proteins and poly
saccharides situated at, or near, its cell surface, yeast induces
the immune response, so that sera prepared from such yeast-
injected animals can be used as antiseral reagents, reacting
specifically with the injected yeast. On the basis of their
immunological reactions, distinction can often be made
between culture and contaminant yeasts, provided those
immune responses common to both culture and contaminant
yeasts are first eliminated by cross absorption. In the 1960s,
work by Campbell, the Assistant Editor of our Journal, and
his colleagues, by Japanese workers led by Tsuchiya (1961),
by Kockova-Kratochvilova (1964) in Czechoslovakia and by
Richards & Cowland (1967) at B.I.R.F. has provided us
methods for detecting and enumerating 'wild' yeasts with
specificity and sensitivity scarcely imaginable 20 years ago.
The Future
In conclusion, I wish to refer to, but not attempt to predict,
the future.
At present, we can say that the descriptive biology and
much of the fundamental physiology and biochemistry of
brewery micro-organisms has been done. I feel that, although
beer spoilage bacteria may continue to provide excellent
material for fundamental biochemical study and continue to
surprise us by throwing up unusual metabolic features, such
study is unlikely to be of immediate value to the brewer, who
will do better so to continue to improve standards of aseptic
operation that microbial beer spoilage will pass away from
experience, much as have the human plagues of bygone days.
However, blinkered though they may be, my eyes do see areas
in which microbiological advances would benefit fundamental
science and brewing technology alike.
First, I have already referred to the need to study the metab
olism of the yeast cell in all its most subtle detail. We need
more information about the enzymic make-up of the yeast
cell, the genetic control of that make-up, the quantitative
interplay of its metabolic pathways and the changes (he latter
undergo in response to changes in wort composition and dur
ing the growth cycle, not only in conventional batch ferment
ation, but also in the scarcely explored environment of such
continuous systems as tower fermentation. Such studies could
hardly fail to benefit technology by creating greater under
standing of fermentation, that most complex of all the brewing
processes (if malting be excluded). Hence we should acquire
greater ability to control fermentation and thereby the com
position of its subtle and complex mixture of end-products,
which determines the flavour and character of beer.
Secondly, I will repeat what I said earlier that I believe it
would be worthwhile to discover whether 'shock excretion' is
significant in brewery fermentations, more particularly during
the lag phase which immediately succeeds pitching.
In the more purely biological field, one may ask whether
the recent discoveries of 'killer' yeasts will lead to practical
applications. In another biological area, I wonder whether
mixtures of selected pure cultures might not be preferable to
single-strain pure cultures, in providing an equilibrium of
synergistic strains more resistant to the disturbing influence,
on population statistics, of chance contaminants. In talking
to some of my colleagues, I have spoken of this concept as one
of 'biological buffering', loosely analogous to the familiar
ionic buffering. As a hypothesis, it might explain the apparent
enduring stability of some brewery non-pure cultures. In
practice, if suitable 'mixed pure cultures' could be prepared,
they would render less frequent the periodic replacement of
pure cultures in the brewery and tend towards greater consist
ency in fermentation and in products. Thus, 1 visualize the
completion of the circle from 'natural' pure cultures, through
single-strain pure cultures, to deliberately contrived mixtures
of strains.
Probably, these and other ideas will already have occurred
to some members of my audience. I hope they will press them
forward with enthusiasm and find encouragement when they
do so. The Brewing Industry, concerned as it is with a multi
plicity of scientific disciplines, continues to provide a rich
field for scientific and technological research. The microbio
logical contribution to that research will not only continue to
serve the Industry well, but it will continue to enrich and be
enriched by all branches of microbiology, just as (as I hope
my lecture will have shown) it has done for the past 100 years.
And, in doing so, may it produce others as outstanding as
Horace T. Brown.
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