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 BrownEven the most partisan of us in our industry scarcelyappreciate the great contribution that fermentation science ingeneral, and brewing science in particular, has made to thescience of Microbiology, so that it is appropriate to reflect

that Pasteur's great work on fermentation simultaneouslyrevolutionised general, medical and industrial aspects of thesubject.

Pasteur's work provoked much criticism from the moreconventional scientists of his day, especially from the prominent chemist Licbig, who denied that living cells intervened inthe phenomena of fermentation and putrefaction, which heregarded as resulting from the decomposition of chemicallyunstable 'albuminous' molecules, It says much for Horace

Brown that his mind was receptive and original enough totake Pasteur's broad view of the close connection between theoutcome of studies on fermentation and those on preventive

medicine. Only four years after he had joined Worthington'sBrewery on 1 January 1866 as a junior brewer, he had sograsped the essential scientific truthfulness of Pasteur's conclusions 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 justturned to microscopic methods when Pasteur's Studies on Winecame into my hands in 1870. The immediate effect was that

of a ray of light piercing the darkness and illuminating a newpath into the unknown'. Thus, although we usually associatewith Brown his classical work on starch, it is with him as a

microbiologist and as the brewing disciple of Pasteur that anysurvey of brewing microbiology must start.

A hundred years ago, the microbial spoilage of beer wascausing great financial losses. In Burton, the large stocks of

ale brewed between October and May for sale in the warmmonths after brewing had ceased were especially prone tospoilage by a bacterium causing, first a 'silky' turbidity, and

then lactic acidification. By 1871, Brown knew the life historyand effect of the causative organism, later named Saccltaro-bacillus pastorianus. Brown's experimental difficulties were

great: lacking techniques, which are today commonplace, hehad to devise his own methods of isolation and staining, basedon 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 studiedby Pasteur and Brown. In Etudes stir le Vin (1866) and thesubordinate work Etudes stir le Vinaigre (1868), Pasteur described acetification, ascribing it to the film-forming organism,

Mycoderma aceti, which he recognized as the agent by whichatmospheric oxygen was transferred to alcohol in the formation of acetic acid. Furthermore, in his Mycoderma vini, herecognized those acetic acid bacteria which could 'over-

oxidize' acetic acid into carbon dioxide and water, thus makingan 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 criterionfor distinguishing the genera Acetobacter and Acetomonas.

For his part, Brown, ever reacting sensitively to all that wasmost progressive in experiment and theory, recognized the

importance of the work of Buchner (1897), who first showedthat cell-free yeast juice could bring about alcoholic fermentation. Thereby he clearly established the enzymic nature of thefermentation process and finally resolved the Liebig-Pasteurcontroversy.

Both Pasteur and Brown were familiar with the phenomenonof 'ropiness'. They recognized the causative organism as a

Presented at a meeting of the Institute of Brewing held in theRoyal Institution, London, on Monday, 18 October 1976.

'viscous ferment', but they seem not to have proceeded far inits study. Brown also knew the role of 'wild' yeasts in producing beer 'frets', which, in his experience, caused more beerspoilage than did bacteria.

As a method of controlling microbial spoilage, Pasteurultimately rejected that of adding harmless antiseptics, such assulphite, in favour of the heat treatment now universally

known as pasteurization, the invention of which illustratesjust 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 thoseyeasts responsible for secondary 'frets'.

E. C. HansenTo the late 19th century belongs another great name inbrewing microbiology, E. C. Hansen, who, in the CarlsbergLaboratory, worked on the yeasts in all their environments.He evolved the criteria by which even morphologically similaryeasts could be distinguished by their modes of formingendospores and their abilities to ferment individual sugars.Essential to his studies, he devised techniques of isolatingsingle 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 ourknowledge 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 DoubtBy about 1900, we had reached the point marking the end ofthe great classical studies of brewing microbiology. This periodof rapid advance and accumulation of a mass of undigestedknowledge perforce brought with it untidiness, confusion and

the need for a sound filing system.Among the matters worse confounded, it might be expectedthat microbial classification would be one. Unicellular microbial forms, like yeasts and bacteria, arc difficult, even today,to classify. They lack the richness of morphological diversification characteristic of the vegetative and sexual forms of thehigher plants on which Linnaeus based his great classification.

In microbiology, much more reliance must be placed oncolony 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 notehow 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-likeor bacterial. Again, the superficial resemblance of the tetradgroups of brewers' 'sarcina' to the octads of the aerobic cocci

of true sarcinae, led to their erroneous name and concealedtheir nature as true lactic acid bacteria. To this Hanscn'sauthority connived. Although Pasteur, in Etudes sur la Biere

(1876) did not commit himself taxonomically (he called theorganism Ferment no. 7), Hansen (1879) gave the nameSarcina, 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 beerspoilages rods and by the acetic acid bacteria understood.

Rod-like bacteria were often given vague or incorrect desig-nations like Bacteriumor Bacillus. In Etudessurla Biere, Pasteurcalled his lactic spoilers 'bacilles des bicrcs tourndes' and it was

not until 1892 tha the special properties of lactic rods weresignified by van Laer's naming them Saccharobacillus pastori-

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 duringthe two decades 1920-1940. Even Shimwell, who became preeminent in this field, was still using the name Saccharobacillusin 1937; but, by 1941, he had completely adopted the correctclassification as that of a heterofermentative rod in the genus

Lactobacillus.The vicissitudes of classification of the acetic acid bacteriaare scarcely less involved. Pasteur used Persoon's (1822) name

Mycoderma, but it was supplanted by several others, includingespecially Bacterium applied by A. J. Brown (1886) to the

cellulose-pellicle-forming organism then called B. xylinum.Ultimately, their distinctive property of ability to oxidizeethanol to acetic acid led to the adoption of the generic nameAcetobacter, first used in 1898 by Beijerinck.

Yeast TaxonomyDuring the past century, yeast nomenclature has been lessbedevilled by changes than has that of brewery bacteria. For

this, we can be grateful that brewing yeasts and many of thebeer spoilage yeasts possess the property, most valuable forclear classification, of ability to form ascospores. Schwann

(1839) observed yeast endospores and Reess, working between1868 and 1870, described them in many species, showing themto be ascospores like those developed by certain of the lowerAscomycetes. In 1870, he suggested that the name Saccharo-

myces, previously used by Meyen (1837) for any buddingyeasts, 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 surprisinglyat the generic level with the non-sporing yeasts, have givenmore 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 Mycodermahas lost generic significance, many of its species of brewinginterest having been found more appropriate places in theasporogenous genera Candida, Cryptococcus, Torulopsis andModotorula. Of special interest, are certain 'torula' yeastswhich have had to be allotted to sporogenous genera as a result

of discoveries of sexual features in them. Thus, matings ofM. cerevisiae have proven its taxonomic relationship withHansenula anomala, and some strains of Candida mycodermahave been transferred to the genus Pichia because they have

recently been observed to form spores.I will conclude my comments on yeast classification bypaying tribute to the painstaking studies of the school ofworkers at Delft and their associates, recorded in a series ofauthoritative monographs, starting with that of Stelling-

Dekker (1931) and culminating in Lodder's recent edited workof nearly 1400 pages, The Yeasts (\970).

J. L. Shimwell's Work on Brewery BacteriaThe inter-war years saw the isolation and precise descriptionof many strains of lactic and acetic acid bacteria. Prominentamong those responsible were the late Professor T. K. Walkerand his colleagues Kulka and Tosic, working especially onacetic acid bacteria, and J. L. Shimwell, whose work encom

passed all types of brewery bacteria and to whom we largelyowe the present orderly state of their classification. Much ofhs work appeared in our Journal for 30 years onwards from

1935.As well as recognizing beer spoilage lactic rods as heterofermentative strains of Lactobacillus, he brought final conviction 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 alsoshowed how the phenomenon of 'ropiness' could be caused

both by lactic pediococci and by certain capsule-formingacetic 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 byits carbohydrate composition (Shimwell, 1936; 1947; 1948).

ShimwelPs work on the acetic acid bacteria was even moreextensive. He was quick to support the view of Leifson (1954)that there were two types of acetic acid bacteria, differing inthe numberanddisposition of their flagella and that these typescorresponded to two groups, long recognized by their respec

tive abilities to oxidize acetic acid itself. This distinction wasalso shown by my co-workers and myself (Rainbow, 1966) to

be paralleled on nutritional and biochemical criteria. Thatthere 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 authorities) provides for those which cannot do so. According toShimwell (1960), the acetomonads are the more dangerous asbeer 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, andsorbose (an intermediate in the synthesis ofascorbic acid) fromsorbitol. 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 mutabilityeven great bybacterial standards' and ultimately to the conclusion that 'itseems to follow that 'species' of bacteria are virtually unclassi-

fiable and that even the conception of a genus should be on abroader basis than is often the case at present'. This is notnecessarily a defeatist view: rather it is a recognition of thegreat rate at which evolutionary changes take place in bac

teria, a fact which demands a radically different approach tobacterial classification than to that of higher organisms, whosemuch slower processes of evolution permit us to freezeconsiderable periods of time for the convenience of rigorousclassification: it is permissible for Acer and Acacia, but not

for Acetobacter.'In addition to his major contributions to the bacteriologyof brewery lactic and acetic acid bacteria, we owe to Shimwell(1937) our first definitive description ofZymomonas anaerobia

and to Shimwell & Grimes (1936) that of Flavobacteriumproteus (later called Obesumhacterium proteum). The names of

both these bacteria have undergone changes which we haveseen to reflect the intrinsic difficulties of bacterial classification.

Shimwell made great contributions to brewing bacteriologyand, although he was not primaily an innovator, he has left

the subject projected within an orderly framework, while suchstudies as those on ropiness have contributed valuably totechnical and scientific knowledge of beer spoilage.

Microbial Nutrition and MetabolismNutrition.The years 1860-1930 saw the completion of aperiod of descriptive biology in the field of brewing microbiology. The second quarter of the present century saw therapid advance in knowledge of the biochemistry of microorganisms, not least of those inhabiting the brewery.

Important for these advances was an understanding ofminimal requirements for nutrition, so that micro-organismscould be studied during growth in media of precisely knowncomposition. In 1901, Wildiers had found that he could notcultivate yeast on defined medium unless small amounts ofextracts of natural materials were added. Thus arose hisconcept 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 becameworldwide in the 1920s, especially as their identity withvitamins of the B complex, essential for human and animal

nutrition, became evident. An early advance on the 'bios'

Vol. 83, 1977] RAINBOW: HORACE BROWN MEMORIAL LECTURE II

front was the identification of bios I as mcso-inositol byEastcott (1928). Even more important was the isolation of

II mg of the methyl ester of D-biotin (bios 11B) from 250 kgof dried egg yolk by Kogl & Tonnis (1936) and the synthesisof pantothenic acid (bios HA) by Williams & Major (1940).The scale and excellence of the work of Kogl & Tonnis werereadily appreciated by Dr Bishop and myself. We wereattempting, in one of the Insitutc's research projects at Birmingham University, to isolate biotin from a modest 40 kg ofdried egg yolk, when Kogl & Tonnis forestalled us. However,we showed lhat our strain of brewer's yeast needed exogenoussources of biotin, pantothenate and inositol for growth and

that, when minute concentrations of these nutrilities wereadded, 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 thisaccount of minimal nutritional requirements needs only minorqualification to beapplicable in general to brewer's yeasts, most

of which appear to require for growth added biotin, i.e. theycannot synthesize their own requirements of this substance.

Additionally, some strains must be supplied with D-pantothen-ate and/or inositol. Brewing yeasts rarely show absoluterequirements for other growth factors, although 1 found twostrains for which added p-aminobenzoatc was essential

(Rainbow, 1948) and many strains are stimulated during theearly stages of growth on defined medium by supplying

thiamin (vitamin B,) and pyridoxin (vitamin B).It is now common knowledge that the significance of mostmicrobial growth factors and vitamins of the B complex liesin their being parts of the molecules of coenzymes: this factillustrates well how discoveries in microbiology illuminatebiochemical phenomena in higher plants and animals andvice versa.

Between 1937 and 1949, R. S. W. Thome of the Institute'sresearch team in Birmingham published important work show

ing that, during its growth in brewer's wort, yeast drewchiefly on free amino acids for its nitrogenous nutrients and

that, on defined medium, complex mixtures of amino acidssupported better growth and fermentation than single aminoacids or simplex mixtures of them. He found evidence thatyeast obtained about 50% of its nitrogen requirements fromwort by assimilating amino acids intact and about 40% by

their deamination. The remaining approximately 10% hesuggested, on less convincing grounds, might arise from ametabolic process, shown by Stickland (1934; 1935) to occur

in certain anaerobic bacteria and to involve the mutualoxidation and reduction of certain pairs of amino acids, bywhich their amino nitrogen became available as ammonia forassimilation. Thome's view of nitrogen assimilation is still

acceptable, except as regards the contribution of the Sticklandreaction. Lewis & Rainbow (1965) failed to find evidence thatthe yeast cell could assimilate nitrogen by this means andconsidered that Thome's observations on the point werecapable of another explanation. Possibly, any nitrogenassimilation neither ascribable to that of intact amino acids,

nor to Ehrlich deamination, might result from the assimilationof small peptides. This point remains to be clarified, ourknowledge of the molecular size and amino acid compositionof those wort peptides assimilable by yeast being inadequateand worthy of further study.

Other nutritional studies in the environment of brewer'swort by Jones & Pierce (1964) have shown that yeast takes upindividual 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 ofwort carbohydrate composition for the attainment of sound

brewery fermentations, adequate attenuations and consistencyof beer flavour are insufficiently appreciated. Indeed, theseimplications may be the more important when considerationis given to the observations of Lewis, PhafT and their co-workers in the 1960s on 'shock excretion'. This phenomenonis characterized by the loss of amino acids and nucleotide

material from yeast cells on suspending them in glucosesolutions. Its significance in brewing is not known but, since

mature cells arc more affected by it than are young cells, itwould be of particular interest to assess its effects on yeast atpitching into worts of different compositions.

The minimal nutritional requirements of the chief groups ofbeer spoilage bacteria have also been elucidated in the past twodecades. At Birmingham University, my colleagues and Ishowed that beer spoilage lactobacilli are nutritionally asfastidious as their cousins in the dairy, needing exogenoussupplies of most a-amino acids, several growth factors of thevitamin B complex and one or more purine and pyrimidine

bases. This implies that well attenuated beers, i.e. those whosenutritional status has been depleted the most by vigorousyeast growth and fermentation, should be the least prone to

lactic spoilage and might even resist such spoilage completelyif 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 exactingnessof lactic acid bacteria, which has been applied since 1939 asan analytical tool in the microbiological assay of vitamins infoodstuffs, was applied from 1943 onwards by Hopkins andhis co-workers (1943-48), Tullo & Stringer (1945) and Norrisand his co-workers (1945-53) to the materials of brewing andto beer itself, revealing inter alia that beer in the diet makes anappreciable contribution to the human intake ofsome vitaminsof the B complex.

In contrast to the lactic acid bacteria, the acetic acid bacteriaare nutritionally less exacting. Indeed, my team at Birminghamshowed that most acetobacters grew on simple, defined lactate-

ammonia-salts medium, without added growth factors oramino acids, while acctamonads grew on a defined glucose-salts medium only if certain growth factors and glutamate, orcertain 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, mucheffort has been devoted to elucidating the mechanism of

glycolysis, the biochemical sequence, occurring in yeast,muscle and in many other cells, by which hexose carbohydrateis 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 concerned. Simultaneously with these catabolic changes, energy

in the form of the high-energy phosphate bonds of adenosinetriphosphate (ATP) becomes available to the cells. In this lies

the biological significance of glycolysis.An essential preliminary to the elucidation of the sequencewas Buchner's discovery in 1897 that fermentation could bebrought about by a cell-free yeast juice. Next in the trail ofdetection came the observations of Harden & Young (1905;

1906) that, during fermentations with yeast juice, addedinorganic phosphate disappeared rapidly and became converted into a form no longer precipitated by magnesium. Theyconcluded that living yeast converted inorganic phosphate

into an organic form. Simultaneously in Russia, Ivanov (1905;1906) isolated from fermenting solutions a compound heerroneously thought to be a triose phosphate, but which

Young (1907) showed to be a hexosc diphosphatc. Harden'sclassical monograph on Alcoholic Fermentation (1911;

reprinted 1932) gives an authentic account of this work. Theimportance of these discoveries can readily be appreciatednow we know that the fermentation of glucose proceeds tothe pyruvate stage entirely via phosphorylated intermediates,

to which Harden & Young's work provided the vital clue.The detailed elucidation of the pathway was perhaps themost notable achievement of pre-World War II biochemists.To quote Florkin (1975), it 'resulted from a convergence ofsudies of ... alcoholic fermentation and muscle glycolysis

which were not suspected ... to be based on the same metabolic scheme'. The process of elucidation took place between

12 RAINBOW. HORACE BROWN MEMORIAL LECTURE [J. Inst. Brew.

1911, when Ncuberg & Wastcnson recognized pyruvate as aproduct of glycolysis, and 1939, when the labile ester 1,3-diphosphoglycerate was isolated as a glycolytic intermediateby Negclein & Bromel. Embdcn, Meyerhof and Parnas madeoutstanding contributions to these studies and the glycolytic

pathway is now appropriately known as the Embdcn-Meyerhof-Parnas (EMP) pathway. The experimental methodsapplied ranged from the chemical trapping of intermediates(e.g., aldehyde and pyruvate) to the more sophisticated

promotion of their accumulation by preventing their furthertransformation, 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 paperspublished between 1906 and 1912, showed that the growth ofyeast on certain amino acids led, by deamination and decarb-

oxylation, to the formation of the fusel alcohols characteristicof fermentation. Ehrlich's results have stood the test of timewell: 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 nitrogenfrom the amino acids became available to yeast as ammonia

has undergone amendment, for we now know it to involvethe enzymic transfer (transamination) of the amino group of a

participating amino acid to 2-oxoglutarate: L-glutamate isthereby synthesized and used by the cell and the 2-oxo-acidcorresponding to the dcaminated amino acid is formed(SentheShanmuganathan & Elsden, 1958). This 2-oxo-acid is

then decarboxylated to an aldehyde, which, in turn, is reducedto the fusel alcohol and excreted into the beer.

Our knowledge of the metabolism of all cells owes much tostudies of fermentation and one of the critical discoveries onwhich modern experimental biochemistry is based was the

first preparation of cell-free enzymes, in the form of yeastjuice, by Buchner (1897), to which I have already referred.

As well as demonstrating the enzymic nature of fermentationand resolving the Pasteur-Liebig controversy, he bequeathedto us a biochemical tool of inestimable value in metabolicstudiesthe cell-free, enzymically active extract through whichindividual enzymes and enzyme systems could be studiedoutside the living cell.

As examples of such studies in the brewing context I willquote work by my colleagues and myself at Birmingham

University. We used cell extracts to show that the differencesbetween acetobacters and acctomonads, which I have alreadymentioned, were reflected in their respective enzyme contents,the former possessing all the enzymes necessary to operatethe tricarboxylic acid cycle, whereas the acetomonads did not(Williams & Rainbow, 1964). Again, Wood & myself (1961)

showed that cells of beer spoilage lactobacilli possessed aremarkable means of metabolizing maltose, by cleaving itphosphorolytically by the enzyme maltose phosphoryiase toglucose and /3-glucose 1-phosphate, not, be it noted, to thefamiliar 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 almostunique in biological systems, having been demonstrated previously only in the human pathogen, Neisseria meningitidis (themeningococcus). The biological significance of this enzymemakes interesting speculation: in beer lactobacilli, it may haveevolved in response to long cultivation in fermenting wort and

beer, both maltose-containing media. Certainly, possession ofthe system endows beer lactobacilli with the biological advan

tage of saving, for each molecule of maltose cleaved, a molecule 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 molecule is substantially discarded by the cell.

Beer lactobacilli contain another unusual enzyme system by

which L-arginine is metabolized (Rainbow, 1975). In this, thearginine dihydrolasc system, arginine is first cleaved by

arginine deaminasc to ammonia and L-citrullinc: in turn, inthe presence of orthophosphatc, the citrulline is convertedenzymically 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 effectthe resynthesis of ATP. This system, already known to occurin certain lactic cocci (Knivctt, 1954), thus seems to be anexample of the biological use of a nitrogen compound asdistinct from a carbohydrate, as a source of energy.

While these examples of studies with cell-free extractsinvolve brewery micro-organisms, such examples can bemultiplied ten-thousand-fold from all fields of biochemistry.Nevertheless, I venture to suggest that, compared with someother microbial forms, studies of yeast metabolism by this, orby other techniques, are a little neglected. For example, lesseffort seems to have been expended on aspects of the detailed

metabolism of brewer's yeast than on those ofEscherichia coli,which, for all its suitability for metabolic studies, cannot becompared in technological importance with brewer's yeast.

Aspects of Modern Yeast BiologyHaving reached the present day in my review, I would liketo 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 shownthat the life cycle of Saccharomyces eUipsoideus had haploidand diploid phases. With the subsequent demonstration by

Winge & Laustscn (1939) that Mendelian segregation occurredin another yeast species, Saccharomycodes ludwigii, it becameapparent that yeasts were subject to genetic changes based onsexuality equally with other organisms. Since Winge, yeastgenetics has proved a fruitful field ofacademic study, especiallyfor C.C. and G. Lindegren over the years 1945-1960. However, despite technologically orientated studies by Fowell(1951). Gilliland (1951; 1953) and Lindegren (1956), yeastgenetics Ijas proved disappointing in its possible applications

to brewing, and hybrid yeasts with improved brewing properties, such as Johnston (1965) produced at B.I.R.F., seem rarelyto have been applied in the industry. Possible reasons for thismay include (1) the conservative attitude of the brewingindustry; (2) inability to define exactly the properties desirablein yeast for a given brewery situation; (3) practical difficultiesof hybridization; and (4) flavour problems incidental to theapplication of an otherwise improved yeast. Nevertheless, it is

feasible to breed hybrid yeasts with desirable properties, orlacking undesirable ones. For example, since Thome andGilliland independently in 1951 showed that flocculence was agenetically controlled character, it should be possible to breedyeasts combining, say, the properties of high flocculence anddesirable flavour characteristics, suitable for continuous toweroperation.

Hybridization is not the only genetical approach to thegeneration of desirable yeasts. The genetical substance (dcoxy-ribonucleic acid, DNA) can be mutated by several means,

including radiation and chemical mutagens. Such mutation isusually, but not invariably, destructive, so that the mutantslack some enzyme possessed by the parent. This presents theopportunity to generate new yeasts, especially those lacking

undesirable properties, among which the production ofexcessive hydrogen sulphide, diacetyl or esters immediatelysprings to mind as relevant to brewing problems. Like

hybridization, this approach to the production of improvedbrewing yeasts has received some, although to my mindinsufficient, attention. Among other as yet untried means toinduce genetical changes are the processes known as transformation and transduction. In the former, a small piece ofexogenous 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

Vol. 83, 1977] RAINBOW: HORACE BROWN MEMORIAL LECTURE 13

and his colleagues transformed pneumococcal types by thismeans. In transduction, discovered by Zinder & Lederbergin 19S2, bacterial viruses (phages) are used as vectors for transferring bacterial genes from one cell line to another. Whethereither of these processes could succeed, or, with transduction,

be even possible to apply to yeasts, is not known, but theattempt might be worthwhile.

Immunological response, induced in animals by the injectionof 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 notoccur until Grabar (19S7) applied it to study the fate ofbarley proteins during the brewing process. Later, it wasrealized that, because of its sensitivity and high specificity,this response could revolutionize our methods ofdetecting andenumerating 'wild' yeasts in the presence of overwhelming

numbers of culture yeasts and, indeed, provide us, for thefirst time, with a satisfactory method for this purpose. Wheninjected into animals, by virtue of the proteins and polysaccharides situated at, or near, its cell surface, yeast inducesthe immune response, so that sera prepared from such yeast-

injected animals can be used as antiseral reagents, reactingspecifically with the injected yeast. On the basis of their

immunological reactions, distinction can often be madebetween culture and contaminant yeasts, provided thoseimmune responses common to both culture and contaminantyeasts are first eliminated by cross absorption. In the 1960s,

work by Campbell, the Assistant Editor of our Journal, andhis colleagues, by Japanese workers led by Tsuchiya (1961),by Kockova-Kratochvilova (1964) in Czechoslovakia and byRichards & Cowland (1967) at B.I.R.F. has provided usmethods for detecting and enumerating 'wild' yeasts withspecificity and sensitivity scarcely imaginable 20 years ago.

The FutureIn conclusion, I wish to refer to, but not attempt to predict,the future.

At present, we can say that the descriptive biology andmuch of the fundamental physiology and biochemistry ofbrewery micro-organisms has been done. I feel that, althoughbeer spoilage bacteria may continue to provide excellentmaterial for fundamental biochemical study and continue tosurprise us by throwing up unusual metabolic features, suchstudy is unlikely to be of immediate value to the brewer, who

will do better so to continue to improve standards of asepticoperation that microbial beer spoilage will pass away fromexperience, much as have the human plagues of bygone days.

However, blinkered though they may be, my eyes do see areasin which microbiological advances would benefit fundamentalscience and brewing technology alike.

First, I have already referred to the need to study the metabolism of the yeast cell in all its most subtle detail. We needmore information about the enzymic make-up of the yeastcell, the genetic control of that make-up, the quantitative

interplay of its metabolic pathways and the changes (he latterundergo in response to changes in wort composition and dur

ing the growth cycle, not only in conventional batch fermentation, but also in the scarcely explored environment of suchcontinuous systems as tower fermentation. Such studies could

hardly fail to benefit technology by creating greater understanding of fermentation, that most complex of all the brewingprocesses (if malting be excluded). Hence we should acquire

greater ability to control fermentation and thereby the composition 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 itwould be worthwhile to discover whether 'shock excretion' is

significant in brewery fermentations, more particularly duringthe lag phase which immediately succeeds pitching.

In the more purely biological field, one may ask whetherthe recent discoveries of 'killer' yeasts will lead to practicalapplications. In another biological area, I wonder whether

mixtures of selected pure cultures might not be preferable tosingle-strain pure cultures, in providing an equilibrium ofsynergistic strains more resistant to the disturbing influence,

on population statistics, of chance contaminants. In talkingto some of my colleagues, I have spoken of this concept as oneof 'biological buffering', loosely analogous to the familiar

ionic buffering. As a hypothesis, it might explain the apparentenduring 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 ofpure cultures in the brewery and tend towards greater consistency in fermentation and in products. Thus, 1 visualize thecompletion of the circle from 'natural' pure cultures, through

single-strain pure cultures, to deliberately contrived mixturesof strains.

Probably, these and other ideas will already have occurredto some members of my audience. I hope they will press themforward with enthusiasm and find encouragement when theydo so. The Brewing Industry, concerned as it is with a multi

plicity of scientific disciplines, continues to provide a richfield for scientific and technological research. The microbiological contribution to that research will not only continue toserve the Industry well, but it will continue to enrich and beenriched 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.

References

Avery, O. T., MacLeod, C. M. & McCarty, M., Journal ofExperimental Medicine, 1944,79, 137.Iialckc, J., Wochenschrift fur Brauerei, 1884,1, 257.Bcijerinck, M. W. (1898; 1901). Cited in Bergey's Manual of

Determinative Bacteriology, 8th edition, Baltimore: Williams &Wilkins, 1974, pp. 276, 576.Brown, A. J., Journal of the Chemical Society, 1886, 49, 432.Brown, G. D. & Rainbow, C, Journal of General Microbiology,

1956,15,61.Brown, H. T., Journal of(he Institute ofBrewing, 1916, 22, 285.Buchner, E., Bericht der Deulschen Chemischen Gesellscltaft, 1897,

30, 1110.Campbell, I., Proceedings of the European Brewery Convention

Congress, Madrid, 1967, 145.Campbell, 1. & Allen, A. M., Journal of the Institute of Brewing,

1964,70,316.Campbell, I. & Brudzynski, A., Journal ofthe Institute ofBrewing,

1966, 72, 556.Cooksey, K. E. & Rainbow, C, Journal of General Microbiology,

\962,21, 135.Eastcott, E. V., Journal of Physical Chemistry, 1928, 32, 1094.

Ehrlich, F., Bericht der Deutschen Chemischen Gesellschaft, 1906,39, 4072; 1907, 40, 1027, 2538; 1911, 44, 139; 1912, 45, 883.Florkin, M., Comprehensive Biochemistry, Vol. 31: A History of

Biochemistry, Elsevicr, Amsterdam and London, 1975, p. 148.Fowell, R. R., Journal of the Institute of Brewing, 1951, 57, 180.Gilliland, R. B., Proceedings ofthe European Brewery Convention

Congress, Brighton, 1951, 35.Gilliland, R. B., Proceedings of the European Brewery Convention

Congress, Nice, 1953, 121.Grabar, P. Proceedings of the European Brewery Convention

Congress, Copenhagen, 1957, 147.Hansen, E. C. Cited in Bergey's Manual of Determinative Bac

teriology, 8th edition, Baltimore: Williams & Wilkins, 1974,p. 514.Harden, A. & Young, W. J., Proceedings ofthe Chemical Society,

1905,21, 189.Harden, A. & Young, W. J., Proceedings of the Royal Society

(London), Series B, 1906, 77, 405.Harden, A., Alcoholic Fermentation, London: 1911 (4th ed.

London, 1932).Hopkins, R. H. & Pcnnington, R. J., Journal of the Institute of

Brewing, 1947,53,251.Hopkins, R. H. & Wiener, S., Journal of the Institute of Brewing,

1944,50, 124; 1945,51,34.Hopkins, R. H., Wiener, S., & Rainbow, C, Journal of the

Institute of Brewing, 1948, 54, 264.Ivanov, L., cited in Florkin, M., Comprehensive Biochemistry,

Vol 31: A History of Biochemistry, Elsevier, Amsterdam, 1975,p. 49.Johnston, J. R., Journal ofthe Institute of Brewing, 1965, 71, 130;

135.

14 RAINBOW: HORACE BROWN MEMORIAL LECTURE [}. lnst. Brew.

Jones, M. & Pierce, J. S., Journal ofthe Institute ofBrewing, 1964,70, 307.Knivctt, V. A., BiochemicalJournal, 1954, 56, 602; 606.Kogl, F. & Tonnis, B., Zeitsclirift fur Physiologischc Chemie,

1936, 242, 43.Leifson, E., Antonie van Leeuwenhoek, 1954, 20, 102.Lewis, M. J. & PhaflT, H. J., Proceedings of the American Society

of Brewing Chemists, 1963, 114; 123.Lewis, M. J. & PhalT, H. J., Journal of Bacteriology, 1964, 87,

1389; 1965,89,960.Lewis, M. J. & Rainbow, C, Journal of the Institute of Brewing,

1965,71, 150.Lindcgrcn, C. C, Proceedings of the American Society of Brewing

Chemists, 1956, 165.Loddcr, J. (ed), The Yeasts: A Taxonomic Study, 2nd edition,

Amsterdam and London: North Holland, 1970.Meyen, J., cited in Lodder, 1970, p. 555.Negelein, P. & Bromcl, H., Bioeltemische Zeitschrift, 1939, 301,

135; 1939,303,132.Neuberg, C. & Wastenson, H., cited in Florkin, 1975, p. 69.Norris, F. W., Journal of the Institute ofBrewing, 1945, 51, 38.

Norris, F. W., Wallerstein Laboratories Communications, 1950,13, 141.Norris, F. W. & Lyncs, K. J., Journal of the Institute of Brewing,

1948, 54, 150; 207.Pasteur, L., Etudes sur le Vin, Paris, 1866.Pasteur, L., Etudes sur le Vinaigre, Paris, 1868.Pasteur, L., Etudes sur la Biere, Paris: Gauthicr-Villars, 1876.

Pcrsoon, C. H., Mycologia Europea 1822,1, 96.Phillips, A. W., Journal of the Institute of Brewing, 1955, 61, 122.

Rainbow, C, Journal of the Institute ofBrewing, 1939,45, 533.Rainbow, C, Nature, London, 1948, 162, 572.

Rainbow, C, Wallerstein Laboratories Communications, 1966, 29,Rainbow, C, In Lactic Acid Bacteria in Beverages and Food,

edited by Carr, J. C, Cutting, C. V. & Whiting, G. C,Academic Press, London, New York and San Francisco, 1975,

p. 149.Rainbow, C. & Mitson, G. W., Journal of General Microbiology,

1953, 9, 371.Reess, M., cited in Loddcr, 1970, p. 555.

Richards, M. & Cowland, T. W., Journal of the Institute ofBrewing, 1967,73,552.Schwann, T., Annalen Physilc und Chemie, 1839, 41, 184.SenthcShanmuganathan, S. & Elsden, S. R., Biochemical Journal,

1958, 69, 210.Shimwell, J. L., Journal ofthe Institute of Brewing, 1936, 42, 585;

1937,43, 507; 1947,53, 280; 1948,54. 237.Shimwell, J. L., Wallerstein Laboratories Communications, 1941,

4,41.Shimwcll, J. L., Brewers' Digest, July 1960, 38.Shimwell, J. L. & Carr, J. G., Antonie van Leeuwenhoek, 1960, 26,

383.Shimwell, J. L. & Grimes, M., Journal ofthe Institute of Brewing,

1936, 42, 348.Stclling-Dekkcr, N. M., Die Sporogenen Hefen, Amsterdam, 1931.

Stickland, L. H., Biochemical Journal, 1934, 28, 1746; 1935, 29,288; 889.Sandula, J., Kockova-Kratochvilova, A. & Zamccnikova, M.,

Brauwissenschaft, 1964, 17, 130.Thome, R. S. W., Journal of the Institute of Brewing, 1937, 43,

288; 1941, 47, 255; 1944, 50, 186; 1945, 51, 6; 1946, 52, 5;1949,55,201.Thome, R. S. W., Proceedings of the European Brewery Conven

tion Congress, Brighton, 1951, 21.Tsuchiya, T., Fukazawa, Y. & Yamase, Y., Japanese Journal of

Microbiology, 1961, 5, 417.Tullo, J. W. & Stringer, W. J., Journal of the Institute of Brewing,

1945, 51, 86.Wildiers, E., La Cellule, 1901, 18, 311.Williams, P. J. le B. & Rainbow, C, Journal of General Micro

biology, 1964, 35, 237.Williams, R. J. & Major, R. T., Science, New York, 1940,91, 246.Winge, 0., Comptes Rendus des Travaux du Laboratoire Carlsberg,

1935, 21, 77.Winge, 0. & Laustsen, O., Comptes Rendus des Travaux du

Laboratoire Carlsberg, 1939, 22, 357.Wood, B. J. B. & Rainbow, C, Biochemical Journal, 1961, 78,

204.Young, W. J., Proceedings of the Chemical Society, 1907, 23, 65.Zindcr, N. D. & Lederbcrg, J., Journal of Bacteriology, 1952, 69,

679.