Table Of ContentSTUDIES ON THE METABOLISM OF THE YEAST, HAhSKNULA ANOMAIA (HANSKH) SXBCftT
Dissertation
Presented in Partial Fulfillment of the Requirements
for the Derree Doctor of philosophy in the
Graduate School of the Ohio State
University
By
THOMAS DALE J5100K,,, B ,, M. Sc.
*•«•»*»*** * * • • ■ • • • * •
The Ohio State University
19£2
Approved bys
Adviser
ACKNOWLEDGEMENTS
I would, like to thank especially my adviser Dr- W. D. Gray for
his interest and invaluable counsel and assistance during the course
of this work. Thanks are also due to Dr. G. L. Stahly and Dr. B. S.
Meyer for reading the manuscript and offering many valuable criticisms.
I also wish to thank the Graduate School of the Ohio State University
for granting me a University Fellowship which was used to complete
this work.
i
809381
TABLE OF CONTENTS
1. Introduction - - - - - ----- _ _ _ _ _ _ _ _ _ _ _ ^
2. Materials and Methods - - - - - - - - - -- _ _ _ _ _ _ _ _ o
3- Results and Discussion
Formation of Storage Products in Grovdnr Ceils - - - - - - 1|
Respiratory Studies with Washed Suspensions - - - - - -- 22
I4. Summary and Conclusions _ _ _ _ -— __ 3$
5. Literature Cited _ _ _ _ _ _ _ _ — __ jp?
ii
STUDIES ON THE METABOLISM OF THE YEAST, HANSENULA ANOMALA (HANSEN) SYDOW
INTRODUCTION
In recent years there has been considerable interest in the
metabolism of Hansenula anomala (Hansen) Sydow. This yeast is of
particular interest because of its ability to produce large amounts of
ethyl acetate. Some physiological aspects of this ester synthesis
were first investigated by Gray (191*9) • He was the first to actually
show that the ester formed ethyl acetate, and he studied the effect of
various environmental factors on ester synthesis. Later Peel (1951)
and Tabachnik (1951) made further studies on some biochemical aspects
of this synthesis, and Tabachnik in particular has done much to deduce
some of the overall steps leading to synthesis. Kuehner (1951) has
studied the effect of added vitamins on the amount of ester synthesized,
and Hughes (1952) has identified some of the acidic metabolic products
produced by H. anomala. In an earlier work, Usami (19U2) studied the
respiration of many alcohols and several acids. Wickerham (1951) has
recently monographed the genus in a considerable extension over the
earlier work of Bedford (19U2). Wickerham1s work may prove to be an
excellent model for further study in the taxonomy of the yeasts. He
has designated H. anomala the type species for the genus.
Most of the work on H. anomala has been directed towards the
special problem of ester synthesis and toward the study of other
water-soluble metabolites and many of the more general aspects of
glucose metabolism have not been studied. It was felt that a study of
other aspects of glucose dissimilation was warranted. In particular,
)
1
2
the problem of lipid synthesis from carbohydrate has been stressed, and
preliminary work on the mechanism of glucose dissimilation and its fate
in the yeast cell have been carried out.
MATERIALS AND METHODS
In most of this work, the yeast has been cultured 011 a medium
containing 7 per cent glucose, 0.6 per cent and 0.7 per cent
Difco Yeast Extract. For certain experiments the glucose concentration
has been varied. In certain work a synthetic medium has been used
consisting of:
Glucose 70 g.
KH2F0l 1.5 g-
MgS0j^*7Hg0 0.5 g.
Nitrogen'source 5 P.*
Vitamins, in ug/liter: riboflavin, 100;
pyridoxinhydrochloride, 200; thiamin
hydrochloride, 200; niacin, 200; biotia
methyl ester, 0.2; calcium pantothenate,
200; inositol, 1000; choline, 100;
p-aminobe nzoic acid, 100.
Trace, elements in mg/liter: Mn++,. 0.01;
Zn++, 0.07; 8, 0.01; Cu*'*’, 0.01; Fe++,
0.05; Mo, 0.01.
This medium is a modification of a medium used by Kuehner (1951)*
When cells were grown for respiration studies they were cultured in
the above Yeast Extract medium with 5 per cent glucose.
In preliminary experiments it was found that rapid growth and
good cell and lipid yields were obtained if the yeast cells were
cultured in liter flasks containing 100 ml. of medium per flask.
These flasks were inoculated with a loopful from a 2k hr. liquid
culture which had been inoculated from an agar slant. In all work
the yeast cells were incubated at room temperature which varied
between 2k-280 C.
At the end of the incubation period the cells were harvested by
centrifugation at 2000 r.p.m. Since H. anomala produces a film of
pseudo-nyceliar cells of light weight on the surface of the liquid,
some of these lighter cells would not spin down, but their contribution
to the total weight of material was negligible and they were ignored.
The cells were washed into weighed petri dishes and dried to constant
weight at k5° C.
Lipid analyses were made by a method described by Usdin (1951)
which is identical with that of Fan et al. (19k9)» The dried cells
were hydrolyzed for 2 hr. in 1 ft HC1 in boiling water, filtered,
washed from the filter paper, dried, and extracted with absolute ether
in a micro-Soxhlet apparatus for 2k hr. At the end of this time the
ether was evaporated from the extraction flask and the flask weighed
to determine the amount of lipid. It must be stressed that in this
work "lipid" signifies total ether extractables of the water insoluble
material remaining after preliminary hydrolysis. Carbohydrate analyses
were made by taking the filtrate of the acid hydrolysis, neutralizing,
and determining the reducing sugar present. The amount of carbohydrate
present is expressed as glucose. Reducing sugar analyses were made by
the method of Stiles et al. (1926).
For the respiration measurements standard manometric techniques
were used (Umbreit et al., 19U9)• The cells for this work were
obtained by centrifuging from 2k hour cultures, washing three times in
I
distilled water, and resuspending in 0.02M phosphate buffer. Since
the endogenous respiration of unstarved cells was rather high, the
h
cells were aerated for two hours in buffer to deplete stored reserves
and reduce the endogenous respiration to a low value. If the cells
were not used immediately after starving they were stored in the
refrigerator. Sometimes cells that were refrigerated for 2ij.-L8 hr.
were used, as it was found that their respiratory activity was not
appreciably diminished. Ail respiratory studies were carried out at
28° G. at a shaking rate of 110 cycles per minute. Further details
of experimental set-ups will be found in the section Results and
Discussion. Aerobic CO2 production was determined by the "direct"
method of Warburg (Umbreit _et al., 19R9)• Unless otherwise stated the
respiratory work was done with yeast at pH 6.0.
RESULTS AMD DISCUSSIOU
Formation of Storage Products in Growing Cells.
The major water-soluble products formed by H. anomala have been
identified by Gray (I9li9), Peel (1951), Tabachnik (1951) and Hughes
(1952). They are ethanol, ethyl acetate, acetic acid, acetaldehyde,
and an unidentified non-volatile acid. The relative amounts formed
of these various products varies with the conditions of growth: pH,
aeration, nitrogen source, and others. In growing cultures there are
a large number of various oxidative and synthetic mechanisms competing
for the supplied carbohydrate and small changes in environmental
conditions sometimes have a rather large effect in determining which
of the various competing mechanisms will predominate. If there is a
minimal amount of .carbohydrate present initially, certain mechanisms
will be active in utilizing this material and others will not operate.
5
However, in an excess of carbohydrate, other mechanisms will also play
a part in carbohydrate transformations, and a different fermentation
picture will prevail.
Relationship betv/een glucose concentration and lipid synthesis.—
Figures 1-3 present data representing a time course study of glucose
utilization by H. anomala in various concentrations of sugar. Hi. . 1
presents the dry weight data for a typical experiment. Although there
was considerable fluctuation, it is evident that in general there was an
initial rapid rise in dry weight during the early part of the fermenta
tion, followed by a more gradual increase during the later stages until
all of the sugar in the flask was utilized. In the lowest concentration
of glucose (ca. 1 per cent), maximum dry weight was attained early (5
i
^ days) and after this maximum, the dry weight decreased slightly,
presumably because of oxidation of stored food reserves. In the higher
glucose concentrations (13 per cent and 17 per cent), no maximum value
was. obtained, as the dry weight continued to rise throughout the entire
time period (21 days). Presumably there was still some available carbon
compounds left in the medium at the end of this period (although all
glucose was gone, undoubtedly some water-soluble compounds: ethanol,
ethyl acetate, acetic acid, were still present), and it was these carbon
compounds that were now being utilised in dry weight production.
Pig. 2 represents graphically the glucose utilized at various time
intervals after inoculation in different sugar concentrations. In the
lowest concentration (1 per cent) all of the sugar was utilized within
»
two days. In general, the higher the concentration, the longer the time
interval until all of the sugar was utilized. In the 13 per cent and 17
ro
o
G
.R 00
A
M
I %
o.
S 3 %
0>
7 %
1 3 %
p. o
I 7%
A
o
/
k>
2 4 6 8 10 12 14 16 18 20 22
D A Y S
Fig. 1. Dry weight production at various incubation times and at various glucose
concentrations. Dry weight values are for 100 c.c. of medium. Glucose concentrations in per
cent are listed on the graph.
G
R
A
M
S
3%
7 %
1 3 %
17%
D A Y S
Fig. 2. Grams of glucose utilized from various initial glucose concentrations at
various time periods.' Ordinates are grams of glucose utilized in 100 c.c. of medium.
