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IN

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PRACTICAL STUDIES

IN

FERMENTATION



BEING



CONTRIBUTIONS TO THE LIFE HISTORY
OF MICRO-ORGANISMS



BY



EMIL CHR. HANSEN, PH.D.

PROFESSOR AND DIRECTOR AT THE CARLSBERG PHYSIOLOGICAL LABORATORY,
COPENHAGEN



TRANSLATED BY

ALEX. K. MILLER, Pn.D., F.I.C., F.C.S.

AND REVISED BY THE AUTHOR





E. & F. N. SPON, 125 STRAND

flcfo gorfc:
SPON & CHAMBERLAIN, 12 CORTLANDT STREET

1896



Bo noufct man selects fearping inntoiiwals, sofos tl&eir seeus, am again
ttieir barping offspring;. "But t!je initial Variation on to^jic^ man
toorfcg, ann toit!jout tot)ict) $e can Bo nothing, i^ caussen Bp ssUg^jt c|jange0
in ttie conoitionss of life. CHARLES DARWIN.

JBot^ing gibe0 t^e scientific ini) estimator greater pleasure t^an to mafee
uciu Ui0cot)erie0 5 Ttt 'iji0 joj> (0 renottliktJ tu^en Ijief ot>0erfcation0 prolic to
liabe a Birect application in practical life* Louis PASTEUR.




PREFACE



EXPERIMENTAL studies on the micro-organisms readily lead
to practical problems relating on the one hand to medicine
and on the other to industry. The theoretical and practical
problems in this field go hand in hand, and are frequently
inseparable. This has also been the case with my investi-
gations, as is seen in the first of them which appeared in
1878, and still more distinctly in the series published since
1 88 1 under the common title 'Recherches sur la physiologic
et la morphologic des ferments alcooliques.' Some of my
researches are mainly of theoretical interest, whilst others
have a more direct practical bearing, and according to
whether the one or the other side predominates, they acquire
importance for one or the other of the two classes of readers
for whom they are written namely, scientific investigators
who look for theoretical deductions, and practical men who
wish to work in accordance with rational principles and
thereby to obtain a material gain. These considerations
induced me to publish my investigations in two series since
1888, the theoretical studies appearing, as before, under the
title given above, whilst those having a direct practical bear-
ing were published in a new series.

The investigations brought together in this book treat in
the main of the great questions of the circulation in nature of



vi FERMENT A TION.

the alcoholic fungi, their relationship to the diseases of beer,
the pure cultivation of yeast and the employment of sys-
tematically selected species and races. The main point is the
reform which I succeeded in introducing into the brewing
industry twelve years ago, and which has since found its way
into the other branches of the colossal industry in which the
cultivation of alcoholic ferments plays an important part,
including distilleries, pressed yeast factories, and the wine,
cider and fruit-wine industries. My work appeals, however,
not only to those practically engaged in the fermentation
industries, to technologists and chemists, but also to biolo-
gists, and I have, therefore, given it the additional title
* Contributions to the Life History of Micro-Organisms.'

This English edition is a translation of the new edition
of my ' Untersuchungen aus der Praxis der Garungsindustrie.'
Some additions have, however, been made here and there,
and the book thus contains also an account of my most
recent investigations. It consists of a series of treatises
which have been published at different times. Some of these
have been more or less remodelled, whilst others have been
reproduced in the same form in which they were originally
published ; the latter can be recognised by the dates which
are printed below the titles ; and where it has been necessary
to make any addition, this has been done in a foot-note.

At the time when I commenced my studies on the yeast
fungi and their fermentations, the practice of starting new
hypotheses was much in vogue ; the journals contained
abundant discussions concerning different possibilities, but
a rigorous enquiry was avoided, and no account was taken
of what was actually known and what was mere surmise.
The problem in this field was, therefore, in the first place to



PREFACE. vii

apply strict method, and in the place of conjectures to sub-
stitute experimental investigations and accurate demonstra-
tions. It was this view of the matter which led to both my
practical and theoretical investigations.

The new ideas which I brought forward in my practical
studies in fermentation were at first favourably received by a
few only of my colleagues, but were, on the contrary, opposed
by most of them. I am glad to be able to state here that
some of my former opponents may now be counted amongst
the most active supporters of my work. I regard this as the
greatest tribute which could be paid to it.

Notwithstanding the success which in different countries
attended my endeavours at reform, I had in reality to fight
an unbroken battle for its progress ; every step had to be
gained by a struggle, and it is this which has to a large
extent put a characteristic stamp upon the following re-
searches. A great incitement to me in this case, as always,
has been the desire to contribute to that literature, the object
of which is to prove to the outside world that we in Denmark
earnestly take our share of the work of progress, and that,
notwithstanding all political reverses, our little nation is still
able to develop and carry out independent scientific research.

In conclusion I have much pleasure in expressing my best
thanks to Dr. MILLER for the great interest and care which
he has bestowed upon the translation of my work.

EMIL CHR. HANSEN.

CARLSBERG LABORATORIUM, COPENHAGEN :
June 1895.







lt&



CONTENTS



CHAPTER I.

PURE CULTURES OF SYSTEMATICALLY SELECTED
YEASTS IN THE FERMENTATION INDUSTRIES.

PAGE

1. WHEREIN THE NEW ADVANCE CONSISTS I

Pitching yeast before and after introduction of the reform . . . . I

Three periods of conflict .. .. .. .. .. .. 2

2. MY METHODS OF PURE CULTIVATION 5

My first pure cultures were prepared by dilution method in liquid

medium .. .. .. .. .. .. .. .. 5

Koch's plate culture in nutrient gelatine . . . . . . . . 7

My single-cell culture in moist chamber .. .. .. .. 8

Brefeld's methods ,, .. .. .. .. .. .. 9

The methods of pure culture are only technical expedients .. .. 10

3. THE CONTRIBUTIONS OF PREVIOUS INVESTIGATORS .. .. io

The new science of the organisms of fermentation was founded by

Cagniard Latour, Schwann, Turpin and Kutzing .. .. .. io

Pasteur's ' Etudes sur la biere ' II

The reasons why Pasteur's method for the purification of yeast by

means of tartaric acid found no application in practice .. .. 12

Different views concerning yeast .. .. .. .. .. 12

4. THE PRACTICAL RESULTS WHICH HAVE BEEN ACHIEVED .. 14

Jacobsen's objections .. .. .. .. .. .. .. 14

The "diseases" produed by alcoholic ferments in the Tuborg and

Old Carlsberg Breweries .. .. .. .. .. . 14

My first experiments with pure cultivated yeast in Old Carlsberg .. 15
Are special secondary yeasts necessary to bring about the desired

fermentation? .. .. .. .. .. .. .. .. 16

Scientists who from the beginning have worked for my reform . . 17

The advantages of the reform .. .. .. .. .. .. 18

Misunderstandings and mistakes .. .. .. .. .. 19

Reasons for advising against the employment of mixed yeasts . . 22
The question of abolishing the open coolers could only be considered

after the yeast question had been solved . . . . . . . . 23



FERMENTATION.

PAGE

THE PREPARATION OF PURE-CULTIVATED YEASTS ON A

LARGE SCALE 25

Preliminary work . . . . . . . . . . . . . . . . 25

The sample to be tested for wild yeast is best taken at the end of
the primary fermentation, and the sample to be used in the pre-
paration of the desired pure culture of the brewery yeast is best

taken at the commencement of the primary fermentation .. .. 25

Temporary variation .. .. .. .. .. .. .. 28

Duration of vitality of yeast cells in wort, cane sugar solution and

in filter paper . . . . . . . . . . . . . . . . 29

Experience shows that a solution of cane sugar affords a good medium

for the preservation of yeasts at the ordinary room- temperature .. 30

My Old Method 31

The sterilisation of wort in glass and metal vessels .. .. .. 3 1

The thorough aeration of the wort is of especial importance . . 36

Manipulations with pure yeast in the fermenting cellar . . . . 38

The Pure Yeast Apparatus 40

Description of the separate parts and instructions for the employment

of the apparatus devised by Kiihle and myself .. .. .. 40

Various modifications of the apparatus for low fermentation . . 58

For high fermentation . . . . . . . . . . . . . . 62

Cost of the apparatus 63

Filters 64

Chamberland's porcelain filters . . . . . . . . . . . . 65

Schroder and Dusch's cotton- wool filters .. .. .. .. 66

The filters of the pure yeast apparatus . . . . . . . . 66

Their efficiency . . . . . . . . . . . . . . . . 68

The Introduction of the Yeast into the Propagating Apparatus and its

Transport .. .. .. .. .. .. .. .. 7

How the yeast is introduced into the apparatus 70

Laboratories in which my methods are carried out .. .. .. 7 1

Course for students in Carlsberg laboratory .. .. .. .. 7 1

Methods for transmitting pure cultivated yeasts . . . . . . 7 2



CHAPTER II.

RESEARCHES ON YEASTS.

I. CHARACTERISTICS OF THE SACCHAROMYCETES 77

Development and germination of spores j film-formation ; form of
the cells under certain conditions of cultivation ; behaviour with
regard to the carbohydrates .. .. .. .. .. .. 77

Difference between the spore contents of the culture yeasts as com-
pared with the wild yeasts .. .. .. .. .. .. 78

Recent investigations on the origin of the Saccharomycetes .. .. 81



CONTENTS. xi

PAGE

2. HIGH AND LOW YEASTS 82

Pasteur's and Reess's views . . . . . . . . , . . . 82

My experiments undertaken since 1884 .. .. .. .. 83

3. INVESTIGATIONS ON LOW-FERMENTATION YEASTS WHICH

HAVE BEEN TESTED IN PRACTICE 85

The names Sacch. cerevisice> Sacch. cllipsoideus, &c., do not represent

single fixed species but groups of species and varieties . . . . 85

The Carlsberg bottom yeasts Nos. I and 2 86

4. ON VARIATION 92

Transformations of a temporary nature . . . . . . . . 92

Transformations which under the most varied conditions of culture

are permanent, and transmitted from generation to generation . . 99

5. MAIN RESULT IO2



CHAPTER III.

THE PRACTICAL EXAMINATION OF BEER IN
THE STORAGE CASKS WITH REFERENCE
TO ITS STABILITY.

GENERAL , .. 103

THE METHOD AND THE EXPERIMENTS 10$



CHAPTER IV.

THE TECHNICAL ANALYSIS OF AIR AND WATER
FOR MICRO-ORGANISMS.

1. THE PRACTICAL RESULTS OF MY AIR ANALYSES .. .. no

2. THE PRINCIPLES OF ZYMOTECHNIC WATER ANALYSES .. 113

Comparison of the results obtained by Koch's gelatin plate

method with those obtained by my flask method .. .. 114
The only use of the hygienic method in the brewery is for testing

the filters 120

Wort bacteria and the common water bacteria 121

Description of my method .. .. .. .. .. .. 123

Its employment in the brewery 126



xii FERMENT A TION.



CHAPTER V.

WHAT IS THE PURE YEAST OF PASTEUR f



1. The old method which I made use of in 1880 for the preparation

of my pure cultures of Sacch. apiculatus .. .. .. 130

Duclaux's statement as to the efficiency of Pasteur's method of

pure culture 132

Theoretical objections raised by Miquel, Jb'rgensen, Denamur
and myself ., .. .. .. .. .. .. .. 133

Duclaux's lectures at the Brewers' Congresses at Paris and Lille

in 1889 and 1890 135

Velten's attacks against my method of pure culture .. .. 136

His statement that Pasteur's method is the right one for purify-
ing brewers' yeast .. .. .. .. .. .. .. 136

The experiments of Messrs. Jacobsen at Old and New Carlsberg 137

2. Pasteur's method of pure culture is tested 138

The first four experiments .. .. .. .. .. .. 139

Results 141

The fifth and sixth experiments were specially carried out in order

to test Velten's statements 142

In Pasteur's method, as recommended by Velten and Duclaux,
there is no certainty of obtaining a pure culture, and with
regard to brewery yeast it even brings about a suppression of
the desired "culture" yeast, whilst the disease yeasts present
are even caused to develop more strongly .. .. .. 145

New objections raised by Velten in 1891 146

Jorgensen's investigations .. .. .. .. .. .. 147

The new objections are disproved by the seventh and eighth
experiments .. .. .. .. .. .. .. 148

3. The objections to the employment of tartaric acid apply also to

hydrofluoric acid and other antiseptics 149

Cultivation in a nutrient solution containing tartaric acid in the
analysis of yeast 151

4. What Pasteur meant by a pure brewery yeast 153

Pasteur's work and my work rest upon two quite different stand-
points ' 154



CONTENTS. xiii



CHAPTER VI.

INVESTIGATIONS ON THE "DISEASES" OF BEER,
PRODUCED BY ALCOHOLIC FERMENTS.

PAGE

1. INTRODUCTION 156

2. HOW THE DOCTRINE OF " DISEASES " IN FERMENTED LIQUIDS

WAS GRADUALLY DEVELOPED 157

Needham's and Spallanzani's investigations on spontaneous genera-
tion 158

Scheele discovers the method of preserving vinegar by heat .. 158
Chaptal and the first indication of the relationship of the diseases

of wine and micro-organisms .. .. .. .. .. 159

The method of heating is further developed by Appert, and is

employed also for the preservation of wine and beer .. .. 159

Franz Schulze, Theodor Schwann and Cagniard Latour .. .. 162

Kutzing discovers an acetic acid bacterium .. 163

Turpin's theory of fermentation .. .. .. .. .. 164

Schroder and Dusch's cotton- wool filters for sterilising air .. .. 164

Bail 165

Pasteur's investigations on lactic fermentation and spontaneous

generation .. .. .. .. .. .. .. .. 166

On acetic fermentation 167

On wine 169

Reess 169

Holzner's, Lintner's and Engel's publications on Sacch. exiguus .. 170

Cohn, Cienkowski, Harz 171

Pasteur's * Etudes sur la biere ' 172

Lintner, sen., on "light yeast" .. .. .. .. .. 177

Nageli 178

Discussions on the degeneration and transformation of brewery yeast 179

The standpoint of the French school in 1883 181

Koch's school .. .. .. .. .. .. .. .. 182

Fitz, Thausing 182

3. MY INVESTIGATIONS 184

Problem and Method .. .. .. .. .. .. .. .. 184

Yeast Turbidity in Beer caused by Sacch. ellipsoideus II. and Sacch.

Pastorianus HI, 188

Main result ,. .. .. .. .. .. .. .. 200

Saccharomyces exiguus .. .. .. ., .. .. .. 201

Main result . . . . . . . . . . . . . . . . 204

Disagreeable Odour and Taste produced in Beer by Sacch. Pastorianus I. 205

Main result .. .. .. .. .. .. .. .. 210

Literature from 1883 to the present day 213



xiv FERMENT A TION.

PAGE

Whence come the Disease Yeasts ? .. .. .. .. .. .. 215

The circulation of Sacch. apiculatus in nature .. .. .. .. 215

Place of development and winter habitat of the true Saccharomycetes 21.7

Open coolers . . . . . . . . . . . . . . . . 220

Cask deposits, and the old-fashioned custom of getting a change of

pitching yeast .. .. .. .. .. .. .. .. 221

Mixtures of Different Species of Brewery Yeasts .. .. .. .. 221

Main result .. .. .. .. .. .. 226

Mycoderma cerevisitz .. .. .. .. .. .. 227

Main result .. .. .. 231



CHAPTER VII.

ON THE PRESENT POSITION OF MY SYSTEM OF
PURE YEAST CULTURE.

1. THE OBJECT OF THIS REVIEW 232

2. LOW-FERMENTATION BREWERIES ..234

3. HIGH FERMENTATION BREWERIES .. 240

4. DISTILLERIES AND YEAST FACTORIES 252

5. WINE, CIDER AND FRUIT-JUICE FERMENTATIONS 258

6. RETROSPECT AND CONCLUDING REMARKS 268



INDEX 273



Of THX -<^V

r7BRSITTl

\, r.r^^<v^;.i-

FERMENTATION



CHAPTER I.

PURE CULTURES OF SYSTEMATICALLY SELECTED
YEASTS IN THE FERMENTATION INDUSTRIES.

i. WHEREIN THE NEW ADVANCE CONSISTS.

IT will be known to many readers that, in the year 1883 I
succeeded in introducing pure cultures of systematically
selected yeasts in the brewing industry. My first experiments
were carried out in the famous Old Carlsberg Brewery at
Copenhagen. When I commenced work in this direction,
the yeast question was everywhere a perfect enigma ; it was
the weakest point in brewing. When difficulties occurred, a
change of yeast was introduced from another brewery, and
frequently the yeasts from several breweries were mixed.
Sometimes a good result was obtained in this way, some-
times also a bad one, and often the result was worse than
that which induced the brewer to try a change of yeast. In
all cases he was working completely in the dark ; in short, he
did not know in the least what he was introducing into the
wort. At that time it was not possible to do more than at
most detect whether the pitching yeast was contaminated with
bacteria and mould fungi ; it was, however, very frequently
found that such an examination led to the assumption that a
yeast was good, but which nevertheless gave a bad result. I
was thus naturally led to the opinion that the secret must

B



2 PURE CULTURES OF

lie in the yeast cells themselves, and that these apparently
similar cells might possibly belong to different species. It
was from this starting point that my investigations on the
Saccharomycetes gradually developed. The practical results
to which they led were in the first place a new analytical
method, and the certain demonstration that some of the com-
monest and most serious diseases of beer, such as yeast
turbidity and objectionable changes in flavour, were caused not
by bacteria but by certain species of yeast.

It was only after this had been proved by exact experi-
ments, both in the laboratory and on a large scale in the
brewery, that it was evident that it was not sufficient to
purify the yeast from bacteria and mould fungi, but that the
question must be treated from a quite different point of view.
And when I further showed that there are also different
species of good brewery yeast which produce beers of
different character, it also became clear that the pitching yeast
should consist only of a single species, namely, that best suited
to the brewery in question.

A short preliminary account of how this reform was
effected and might be carried further, was published in the
'Zeitschrift f. d. ges. Brauwesen' for 1884. The first detailed
description, however, appeared in 1888, in the first German
edition of this work. In some respects it is as well that it
was not published earlier ; I had in the meantime gained
greater experience, and was thereby in a position to sum-
marise the most important publications which had appeared
in the interval, and which threw light on my work, partly
in support of it and partly against it. It would be out of
place here to quote these treatises ; they are published in the
journals relating to the fermentation industries, and especially
in the brewing journals, all the leading zymotechnologists
having added their contributions. A list of the most im-
portant authors will be found in the first German editions
of this book.



SYSTEMATICALLY SELECTED YEASTS. 3

It was only to be expected that my work would meet
with marked opposition. This was especially the case at
three different points. The first and severest struggle which I
had to encounter was with the founder of the laboratory, the
late Captain J. C. Jacobsen. He, in fact, regarded my efforts
as misdirected, and for a time tried to oppose them. But
as soon as he recognised that my discoveries might become
of great importance, he at once decided to recommend them,
although not always in a manner which I could approve
(1883-1884). The experiments which I conducted at that
time, in order to convince Jacobsen, became of fundamental
importance for the full development of the question, and they
will therefore be described in detail in the following pages.

Shortly afterwards the dispute arose with Prof. Delbriick
and his followers in Berlin. My opponents started with the
view that a botanical treatment of the yeast question could
not lead to the desired end, and that my methods were not
suited to the practical conditions of the brewery. They did
not content themselves, however, with a verbal dispute, but
experimented themselves. By means of these investigations,
and some new ones published by myself and my associates,
my opponents gradually changed their views, and they have
also openly stated so. In 1889 my colleagues at the Berlin
Station recognised and adopted my methods in their pro-
gramme, and awarded me their diploma as honorary member.
Amongst the marks of honour accorded to my work, I valued
this especially, as showing that the dispute had been carried
on without personal animosity.

The third important attack was from the French school,
at the head of which are the brewer Velten of Marseilles,
well known through Pasteur's ' Etudes sur la biere/ and
Prof. Duclaux, Member of the Institute of France, and
Director of Pasteur's Institute at Paris. Some other pupils
of the French school sided with the above. Velten com-
menced his attack in 1886, and this was taken up by Duclaux

B 2









4 PURE CULTURES OF

in 1889. (I am glad to be able to add that Pasteur took no
part in these attacks ; on the contrary, he publicly recognised
my work, in that at the end of 1886 he caused the award to
be made to it of the gold medal of the Societd d'Encourage-
ment pour PIndustrie Nationale.) The dispute has not yet
terminated. Velten constantly maintains that I was com-
pletely wrong in introducing into the brewery a pitching
yeast consisting only of a single race. A refutation of his
assertions will be found in the following pages. Prof. Duclaux,
whose sympathies were at first in the same direction as
Velten's. subsequently abandoned in some degree the latter's
views, and at the Brewers' Congress at Lille in 1890, he even
emphasised the fact that the yeast question had, as regards
low fermentation, been solved by my investigations. With
regard to high fermentation, he will still have nothing to do
with my system, and argues in favour of the introduction of
the old methods of Pasteur. The future will show that he is
wrong.

L. Aubry, the director of the scientific station for brewing
at Munich, has expressed himself as follows on this question
('La Gazette du Brasseur,' 1890, p. 79) : "Although we have
to deal mainly with the preparation of pure cultures of bottom
yeasts, it does not infrequently happen that we are asked for
pure top yeasts, and we have already introduced pure cultures
of such yeasts into practice and with good results. The
method which we have employed, in the case of both top
yeast and bottom yeast, has always been Hansen's method ;
we never make use of Pasteur's method. In the present
position of science, no one who is concerned with the pre-
paration of pure yeast will employ any other than Hansen's
method. The hostility which Hansen's efforts have aroused
is much to be deplored. We all recognise, and Hansen no



Online LibraryEmil Christian HansenPractical studies in fermentation; being contributions to the life history of micro-organisms → online text (page 1 of 25)