Citation
The declining rate of ethanol production during batch fermentation by Saccharomyces cerevisiae

Material Information

Title:
The declining rate of ethanol production during batch fermentation by Saccharomyces cerevisiae
Creator:
Dombek, Kenneth Michael, 1959-
Publisher:
[s.n.]
Publication Date:
Language:
English
Physical Description:
ix, 166 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Alcohols ( jstor )
Batch fermentation ( jstor )
Broths ( jstor )
Cell growth ( jstor )
Enzymes ( jstor )
Ethanol ( jstor )
Fermentation ( jstor )
Magnesium ( jstor )
Nucleotides ( jstor )
Yeasts ( jstor )
Alcohol ( lcsh )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Fermentation ( lcsh )
Microbiology and Cell Science thesis Ph.D
Saccharomyces cerevisiae ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1987.
Bibliography:
Bibliography: leaves 152-165.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Kenneth Michael Dombek.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
001055994 ( ALEPH )
18649850 ( OCLC )
AFD9533 ( NOTIS )
AA00004839_00001 ( sobekcm )

Downloads

This item has the following downloads:


Full Text












THE DECLINING RATE OF ETHANOL PRODUCTION
DURING BATCH FERMENTATION BY SACCHAROMYCES CEREVISIAE










BY

KENNETH MICHAEL DOMBEK


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1987















ACKNOWLEDGMENTS

The ideas presented in these studies could not have

been developed without the encouragement and patience of my

major advisor, Dr. Neal Ingram. I am greatly indebted to

him for sharing with me his knowledge and expertise. I also

would like to express my gratitude to the other members of

my committee, Dr. Allen, Dr. Farrah, Dr. Gander and Dr.

Preston, for their contributions during preparation and

review of this manuscript. Similarly, thanks are due to my

colleague and friend, Dr. Yehia Osman, for his many

suggestions which were helpful in completing this work and

to the rest of the Microbiology and Cell Science Department

for their part in my graduate education. Finally, I would

like to thank my parents for their love and support while I

pursued this study.
















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS............... ......................... ii

LIST OF TABLES.......................................... v

LIST OF FIGURES...................................... .. vi

ABSTRACT...................................................... viii

CHAPTERS

I GENERAL INTRODUCTION............................ 1

II CHARACTERIZATION OF THE DECLINING RATES OF
GROWTH AND ETHANOL PRODUCTION DURING BATCH
FERMENTATION BY S. CEREVISIAE KD2............... 12

Introduction..................... ............. 12
Materials and Methods........................... 14
Results........................................... 18
Discussion...................................... 27

III NUTRIENT LIMITATION AS A BASIS FOR THE
APPARENT TOXICITY OF LOW LEVELS OF ETHANOL
DURING BATCH FERMENTATION....................... 30

Introduction.............. ........ ............. 30
Materials and Methods........................... 30
Results................................... ......36
Discussion............... ....................... 50

IV MAGNESIUM LIMITATION AND ITS ROLE IN THE
APPARENT TOXICITY OF ETHANOL DURING YEAST
FERMENTATION....... ................ ............ 56

Introduction. .................................. 56
Materials and Methods........................... 59
Results............................ ..... ....... 62
Discussion.... .............. ................ 80

V GLYCOLYTIC ENZYMES AND INTERNAL pH.............. 84

Introduction................................... 84

iii










Materials and Methods........................... 86
Results........................................... 89
Discussion........................... ........... 103

VI PHOSPHORYLATED GLYCOLYTIC INTERMEDIATES AND
NUCLEOTIDES............... ..................... 113

Introduction................ ................. 113
Materials and Methods........................... 116
Results ......................................... 125
Discussion...................................... 139

VII SUMMARY AND FUTURE DIRECTIONS................... 146

BIBLIOGRAPHY................... ........................ 152

BIOGRAPHICAL SKETCH.. .................................. 166















LIST OF TABLES


Page


Table 1.


Table 2.


Table 3.


Table 4.


Table 5.


Effects of ethanol and fermentation medium
composition on fermentation rate..............

Intracellular and extracellular ethanol
concentrations under various conditions.......

Effect of growth in broths of different
composition on fermentation rate.............

Effect of nutrient supplementation on growth
of S. cerevisiae KD2..........................

Specific activities of glycolytic enzymes at
the peak of fermentative activity (12 h) and
after a 50% decline (24 h) ....................















LIST OF FIGURES


Page


Figure 1.




Figure 2.


Figure 3.


Figure 4.


Figure 5.


Figure 6.


Figure 7.


Figure 8.


Figure 9.


Figure 10.



Figure 11.



Figure 12.


Growth and ethanol production by S.
cerevisiae KD2 during a typical batch
fermentation in YEPD medium containing 20%
glucose.......................................

Growth rate of S. cerevisiae KD2 in the
presence of ethanol..........................

Rate of fermentation in the presence of
ethanol .................................. ...

Determination of intracellular ethanol
concentration................................

Inhibition of fermentation rate of 12-h and
24-h cells by added ethanol..................

Dose-response of cell growth to added
magnesium....................................

Magnesium levels in broth and cells during
the course of batch fermentation.............

Effect of magnesium addition on cell growth
and fermentation..............................

Effect of added magnesium on the rate of
fermentation.................................

Effect of magnesium supplementation on the
inhibition of fermentation rate by added
ethanol ................................. ...

Effect of ethanol removal on the
fermentative activity of cells grown in YEPD
medium containing 0.5 mM MgSO4..............

Effects of ethanol exposure on the
fermentative activities of 12- and 24-h
cells.......................................









Figure 13.



Figure 14.



Figure 15.

Figure 16.


Figure 17.


Figure 18.


Figure 19.


Figure 20.


Changes in the levels of glycolytic and
alcohologenic enzymes during batch
fermentation with 20% glucose................

Changes in intracellular pH and membrane
energization during batch fermentation of
20% glucose..................................


97



101


Effects of added ethanol on A pH........... 104


A typical thin layer chromatogram of a
fermentation sample extract.................

Changes in [32P]-labelled cellular
metabolites during batch fermentation.......


120


126


Comparison of nucleotide levels found in the
cells with those found in the fermentation
broth ....................................... 131


Intracellular concentration of nicotinamide
nucleotides during batch fermentation.......

Intracellular concentration of adenine
nucleotides and energy charge during batch
fermentation................................


133



136


vii















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


THE DECLINING RATE OF ETHANOL PRODUCTION
DURING BATCH FERMENTATION BY SACCHAROMYCES CEREVISIAE

By

Kenneth Michael Dombek

August 1987

Chairman: Lonnie O. Ingram
Major Department: Microbiology and Cell Science

As Saccharomyces cerevisiae ferments 20% glucose to

ethanol in batch culture, the rate of this conversion

declines. Previous studies assumed that ethanol caused this

inhibition because added ethanol repressed both yeast growth

and alcohol production. However, produced ethanol appeared

to be a more potent inhibitor than added ethanol. Also,

removal of ethanol from fermenting yeast cells did not fully

alleviate this inhibition. The yeast cell envelope was

freely permeable to ethanol and, thus, the intracellular

accumulation of ethanol to levels high enough to inhibit

glycolytic enzymes appeared unlikely. A nutrient limitation

for magnesium in complex fermentation medium was identified

as being partially responsible for declining rates of

alcohol production. Supplementation of broth with magnesium

prevented much of the decline but did not entirely eliminate

viii









it. Since the levels of glycolytic and alcohologenic

enzymes remained high and internal pH was maintained near

neutrality, inactivation of enzymes or lowered levels of in

vivo activity due to acidification of the cell cytoplasm

appear unlikely. As yeast cells produced ethanol, they did

change physiologically, however, becoming more resistant to

inhibition of fermentation by added ethanol and to ethanol-

induced decreases in ApH. Initially, the intracellular

levels of phosphorylated glycolytic intermediates decreased

as fermentation rate was declining. These results suggested

that the rates of glucose uptake and/or phosphorylation were

slowing relative to carbon flux through the rest of the

pathway. Declining glycolytic intermediate levels probably

were not due to inhibition of glycolytic enzymes by

declining levels of nicotinamide nucleotides. Initially

during fermentation, ATP levels decreased by 60%, while AMP

increased by 900%. One possible explanation for the decline

in glycolytic intermediates and the corresponding decrease

in fermentation rate is that the increased level of AMP

inhibits glucose phosphorylation which may slow the rate of

glucose uptake. In this study, the roles of inhibition by

ethanol, nutrient limitation, and physiological changes in

decreasing the rate of fermentation have been defined and

characterized. Each of these factors appears to be

partially responsible for the decline in ethanol production

by S. cerevisiae during batch fermentation.















CHAPTER I
GENERAL INTRODUCTION


Even before 2000 BC, Saccharomyces cerevisiae was used

to ferment malted barley and wheat into a type of beer-bread

in Mesopotamia (Corran, 1975). The Babylonians and

Egyptians adapted this fermentation process to the

production of a high alcohol beer from many different sugar

sources. In Greece, grape juice was a popular sugar source

for fermentation to make wine. By the middle ages, wine and

ale were among the only beverages available that were not

contaminated with disease-causing agents. The ethanol

produced during their fermentation acted as a preservative.

Even after the advent of refrigeration and sterile packaging

procedures, liquors derived from yeast fermentation remained

as popular beverages.

Recently, uses of ethanol other than for beverages have

received much attention. Because of the growing concern

about protecting the environment from pollution and the

increasing dependence of the United States economy on

imported oil, ethanol has been adopted as both a gasoline

additive and an alternative energy source. Almost 95% of

the global ethanol produced is the product of sugar

fermented by S. cerevisiae.











Despite the obvious importance of ethanol production by

S. cerevisiae, the physiological constraints which limit the

rate of ethanol production are not fully understood.

Identification of these constraints represents an important

step toward the development of improved organisms and

process conditions for more rapid ethanol production. Such

improvements could increase the ethanol-production capacity

of existing fermentation plants and reduce the cost of

future facilities.

Some of the initial research on alcoholic fermentation

took place in the 1830s. Independently, Cagniard-Latour,

Schawn and Kutzing described the microscopic structure of

yeast cells and initiated studies on the role of yeast in

fermentation (Schlenk, 1985). Twenty years later, Pasteur

expanded these findings and performed the first decisive

experiments showing that yeast cells were the living

entities responsible for fermenting sugar to ethanol

(Pasteur, 1860). He then went on to describe the effect of

oxygen on yeast fermentation, an effect which has since

become known as the "Pasteur effect" (Pasteur, 1861).

Biochemistry had its beginnings with the work of Buchner who

showed that the proteinaceous material in yeast juice was

capable of converting sugar to ethanol (Buchner, 1897). In

the decades that followed this discovery, the chemical

nature of alcoholic fermentation and the enzymes responsible











for the conversion which Pasteur had described 50 years

earlier were characterized (Fraenkel, 1982).

Even before these early studies on alcoholic

fermentation, it had been observed that yeast stopped

growing and fermenting before all of the sugar in the

fermentation broth had been utilized. In one of the

earliest investigations of this phenomenon, Brown examined

the influence of various environmental conditions on the

rate of growth of S. cerevisiae (Brown, 1905). The addition

of ethanol to growth medium, indeed, did inhibit yeast

reproduction. However, inhibition occurred only at a much

higher ethanol concentration than was observed to have

accumulated at the point during the fermentation when yeast

growth had ceased. This was the first indication that the

presence of ethanol may not be the only factor involved in

the premature termination of carbohydrate fermentation by

yeast.

The studies of Brown were complemented by the work of

Richards (1928) who showed that removing ethanol produced

during growth and maintaining a constant nutrient supply

allowed yeast cell multiplication to continue almost

indefinitely. Recently, the vacuum fermentation experiments

conducted by Cysewski and Wilke (1977) and Maiorella et al.

(1983) have corroborated and extended this finding. Boiling

off the ethanol as it was produced from a continuous culture

under reduced pressure, increased achievable cell densities











and fermentation rates. Because such investigations have

shown that ethanol does inhibit yeast growth and

fermentation, many studies have dealt with characterizing

these inhibitory effects (Brown et al., 1981; Hoppe and

Hansford, 1982; Jones and Greenfield, 1985; Lafon-Lafourcade

and Ribereau-Gayon, 1984; Vega et al., 1987).

The inhibitory effect of ethanol on the rate of sugar

conversion to ethanol was first quantitated by Rahn (1929).

He demonstrated an inverse relationship between the amount

of fermentation product, including ethanol, added to the

medium and the rate of its production by determining the

amount of heat evolved when sucrose was converted to

ethanol. Also, as ethanol accumulated in the medium, the

rate of fermentation declined. This represents some of the

original evidence that ethanol inhibits alcohol production

by yeast.

Recent studies also have attempted to quantitatively

describe the inhibitory effect of ethanol on product

formation during yeast fermentation (Luong, 1985; van Uden,

1985). Holtzberg et al. (1967) examined grape juice

fermentation by S. cerevisiae var. elipsoideus and

calculated an inverse linear relationship between the rate

of alcohol production and the amount of ethanol in the

juice. Similar findings were made by Ghose and Tyagi (1979)

for the batch fermentation of cellulose hydrolysate by S.

cerevisiae NRL y-132, however, the constants in the equation











relating the rate of alcohol production to the amount of

ethanol in the fermentation broth were slightly different.

A glucose-limited continuous culture of a respiratory-

deficient baker's yeast was found by Aiba et al. (1968) to

exhibit an exponential decrease in rate of alcohol formation

as increasing concentrations of ethanol were added to the

fermentation broth. They also demonstrated that ethanol

acted as a non-competitive inhibitor of alcohol formation.

Both batch and continuous culture fermentations of S.

cerevisiae ATCC No. 4126 in a synthetic medium were shown by

Bazua and Wilke (1977) to exhibit kinetics of ethanol

inhibition entirely different than had been described

previously. In each case, similar models were constructed

for growth in the presence of ethanol. The large variety of

kinetic models suggests that many factors, such as strain

variations, environmental conditions and nutritional state,

also may have important roles in the inhibition of growth

and alcohol production during yeast fermentation.

Strains of yeast able to grow and ferment in the

presence of higher concentrations of ethanol may be capable

of producing larger amounts of alcohol at faster rates.

Thus, many investigations have examined the mechanism of

ethanol tolerance in yeast (Casey and Ingledew, 1986; Ingram

and Buttke, 1984; Ingram et al., 1986; Moulin et al., 1984).

Initial studies on the alcohol tolerance of yeast were

performed by Gray (1941). Various ethanol producing species











of yeast, including several different strains of S.

cerevisiae, were grouped according to their ability to

utilize glucose in the presence of a series of ethanol

concentrations. The ethanol tolerance trait was not

characteristic of any specific genus or species since

different strains of the same species varied in their

tolerance. Yeast strains with differing ethanol tolerances

also had different cellular compositions (Gray, 1948).

Strains of lower alcohol tolerance contained higher amounts

of carbohydrate and lipid than did the more tolerant ones.

The studies of Troyer (1953) confirmed the results of

Gray and further examined the relationship between growth

and glucose utilization. Alcohol tolerant strains of yeast

exhibited increased growth in parallel with increased rates

of glucose utilization over less tolerant strains. During

yeast fermentation, the initial effect of ethanol added to

the medium was to decrease the total number of cells formed

followed by a corresponding decrease in glucose utilization.

The manner in which ethanol inhibits glucose

utilization by S. cerevisiae was examined by Gray and Sova

(1956). The ability of ethanol to inhibit glucose

utilization was not a specific property of this fermentation

product but rather a property shared by a class of

substances, short chain aliphatic alcohols. The potency of

normal alcohols as inhibitors of glucose utilization was

related to their chain length and primary alcohols were











found to be more inhibitory than secondary or tertiary

alcohols. This suggested that the mode of action of ethanol

may involve a hydrophobic site. Numerous investigations

have described the detrimental effects of a variety of

hydrophobic compounds, including alcohols, on the function

of many different types of cells and membranes (Barondes et

al., 1979; Eaton et al., 1982; Hayashida and Ohta, 1978;

Leao and van Uden, 1982b; Lenaz et al., 1978; Seeman, 1972).

The inhibitory potential of an alcohol on D-xylose transport

of S. cerevisiae was correlated directly with the lipid-

buffer partition coefficient of that alcohol by Leao and van

Uden. Hayashida and Ohta reported that ethanol promoted

leakage of ultraviolet-absorbing material from yeast cells,

an ethanol induced compromise of membrane barrier function.

Since ethanol alters the physical state of both artificial

and biological membranes (Chin and Goldstein, 1977; Dombek

and Ingram, 1984; Janoff and Miller, 1982; Rowe, 1983;

Vanderkooi et al., 1977), one site of ethanol action may be

the cell cytoplasmic membrane.

The cytoplasmic membrane of S. cerevisiae contains

about 50% protein and 40% lipid by dry weight (Hunter and

Rose, 1971). It has a unique lipid composition, containing

phospholipids with no polyunsaturated fatty acids like those

found in prokaryotes and large proportions of

phosphatidylcholine and sterols like those found in

eukaryotic cells (Ingram and Buttke, 1984). As much as 80%











of the fatty-acyl residues are unsaturated and as much as 6%

of the membrane dry weight consists of sterols, mainly

ergosterol (Hunter and Rose, 1971). The synthesis of

unsaturated fatty acids from saturated acids requires an

oxygen-dependent desaturase enzyme and the synthesis of

ergosterol from squalene requires oxygen-dependent

peroxidation and demethylation reactions (Henry, 1982).

When cultured under anaerobic conditions, S. cerevisiae

exhibits a requirement for both unsaturated fatty acids and

ergosterol in its nutrient supply for growth (Andreasen and

Stier, 1953; Andreasen and Stier, 1954).

Taking advantage of this anaerobically induced

nutritional requirement, the laboratory group of Rose

selectively enriched plasma membranes of S. cerevisiae by up

to 60% with an individual unsaturated fatty-acid or by up to

70% with a particular sterol (Hossack and Rose, 1976).

Using yeast with altered plasma membrane lipid composition,

they showed that cells enriched in ergosterol and linoleyl

residues remained viable for a longer period of time than

cells enriched in other sterols and oleyl residues when

exposed to ethanol (Thomas et al., 1978). Ethanol was also

less inhibitory to growth and solute accumulation when

plasma membrane lipids were enriched similarly (Thomas and

Rose, 1979). Because unsaturated fatty acids and

ergosterol appear to protect yeast cells from the inhibitory

effects of ethanol, it is not surprising that S. cerevisiae











has been shown to alter its plasma membrane lipid

composition when grown in the presence of ethanol (Beaven et

al., 1982). The amount of unsaturated fatty-acyl chains in

the phospholipids rose with increasing amounts of added

ethanol. The proportion of oleyl residues increased by 100%

with a corresponding decrease in the proportion of palmitic

residues in the presence of 1.5 M ethanol. In light of the

previous studies on yeast cells containing plasma membranes

enriched in unsaturated fatty-acyl residues, this change in

lipid composition may be an adaptive response to growth in

the presence of ethanol. As ethanol accumulated during

normal growth, however, the lipid fatty-acyl composition

became more saturated, probably the result of decreasing

oxygen tension in the medium.

Since enrichment of the plasma membrane with

unsaturated fatty-acyl residues and ergosterol protects

yeast cells from ethanol inhibition and the plasma membrane

fatty-acyl composition becomes more saturated during growth,

addition of unsaturated lipid supplements to the

fermentation broth might be expected to enhance the

fermentation rate and final ethanol yield from fermentable

substrates. Studies of sake fermentation by Hayashida have

shown that addition of proteolipid containing linoleyl

fatty-acyl residues to synthetic medium promotes the

formation of over 20% ethanol (Hayashida et al., 1974).

Sake yeast normally only produce this high amount of ethanol











under the very specialized conditions of the sake

fermentation. Koji mold proteolipid, found in sake mash,

also enhanced yeast growth, survival and fermentative

activity (Hayashida et al., 1975). This proteolipid,

isolated from Aspergillus oryzae, was shown to contain a

high percentage of phosphatidylcholine with linoleic acid

comprising the major portion of its fatty-acyl residues

(Hayashida et al., 1976). Supplementation with proteolipid

actually increased the proportion of phosphatidylcholine and

linoleyl fatty-acyl residues in the sake yeast plasma

membrane (Hayashida and Ohta, 1978). The

phosphatidylcholine promoted yeast growth and fermentative

activity, while addition of ergosterol-oleate increased

survivability in the presence of ethanol (Hayashida and

Ohta, 1980). Many confirming studies have shown that

addition of unsaturated lipids improves the fermentative

productivity of yeast (Damiano and Wang, 1985; Janssens et

al., 1983; Lafon-Lafourcade et al., 1979; Ohta and

Hayashida, 1983; Watson, 1982).

Although much is known about the effects of ethanol on

various aspects of yeast fermentation and about the

involvement of the plasma membrane in mediating many of

these effects, very little is known about the factors which

cause the rate of fermentation to decline as ethanol

accumulates in the medium. The following studies have

examined this phenomenon in more detail to determine the









11

role of ethanol produced during fermentation in causing this

decline in ethanol production rate. Other possible causes

of the decreasing rate of ethanol production during batch

fermentation, such as nutrient limitation, also were

studied. Finally, the physiological changes which accompany

the declining fermentation rate were characterized in order

to better understand the constraints that limit the rate at

which S. cerevisiae produces ethanol.















CHAPTER II
CHARACTERIZATION OF THE DECLINING RATES OF GROWTH AND
ETHANOL PRODUCTION DURING BATCH FERMENTATION BY
SACCHAROMYCES CEREVISIAE KD2


Introduction

Yeast metabolize sugar via Embden-Meyerhof glycolysis

to produce ethanol as the major reduced product of

fermentation. As ethanol accumulates in the fermentation

broth, both the rate of growth and alcohol production

declines (Ingram and Buttke, 1984; Luong, 1985; van Uden,

1985). The potency of ethanol as an inhibitor of yeast

growth and fermentation, however, differs in various species

(Gray, 1941). Not all strains of Saccharomyces cerevisiae

have equal abilities to grow and ferment in the presence of

added ethanol.

Environmental factors, such as temperature, also have a

key role in determining the potency of ethanol as an

inhibitor (Casey and Ingledew, 1986; Jones et al., 1981; van

Uden, 1985). Elevating the temperature reduced the maximum

ethanol yield of wine fermentations (Hohl and Cruess, 1936).

Both high and low temperatures decreased the ability of S.

cerevisiae to grow in the presence of ethanol (Loureiro and

van Uden, 1982; Sa-Correia and van Uden, 1983). Sa-Correia

and van Uden (1983) have shown that the temperature of











maximum ethanol tolerance for growth of S. cerevisiae is

between 280C and 300C. Similarly, the ability to survive in

the presence of ethanol decreased with increasing

temperature (Casey and Ingledew, 1986; Leao and van Uden,

1982a; Nagodawithana et al., 1974). Ethanol also enhanced

the thermal death rate of S. cerevisiae (van Uden and da

Cruz Duarte, 1981). In contrast, the rate of sugar

conversion became more resistant to ethanol inhibition as

the fermentation temperature was raised to 450C (Brown and

Oliver, 1982). Alcohol production proceeds at an

accelerated pace at the higher temperatures. When

optimizing the conditions for carrying out a fermentation

process, a compromise between these competing factors must

be reached.

Other environmental factors which affect the ability of

yeast to tolerate ethanol include the sugar and oxygen

concentrations in the fermentation broth (Jones et al.,

1981). Glucose concentrations above 14% decreased the

ability of S. cerevisiae to convert the sugar to ethanol

(Gray, 1945). This inhibition occurred as the cells began

to undergo plasmolysis and probably was caused by the

osmotic effects of these high amounts of glucose on the

yeast. Osmotic pressure also has an adverse effect on yeast

cell viability during fermentation (Panchal and Stewart,

1980). These effects of high sugar concentrations appear to

be synergistic with the inhibitory effects of ethanol











(Kunkee and Amerine, 1968; Moulin et al., 1980). A small

amount of oxygen increases the ethanol tolerance of S.

cerevisiae because it is required for the synthesis of

unsaturated lipids and ergosterol (Henry, 1982). These

lipids protect anaerobically grown yeast cells from ethanol-

induced growth inhibition (Ingram and Buttke, 1984). Under

continuous culture conditions, trace amounts of oxygen were

shown to decrease the ethanol inhibition of growth without

significantly affecting the ethanol yield per amount of

substrate consumed (Hoppe and Hansford, 1984).

Because strain differences and environmental factors

influence the ability of yeast to grow and ferment in the

presence of ethanol, it was necessary to characterize these

processes in the organism chosen for this study. This

organism was a genetically undefined petite brewery yeast,

S. cerevisiae KD2. Decreasing growth and alcohol production

rates as ethanol accumulated during batch fermentations were

characterized. Also, the effect of ethanol added to the

growth medium on the rates of growth and alcohol production

was examined.

Materials and Methods

Yeast Strains

The principal organism used in these studies was S.

cerevisiae KD2, a petite mutant of strain CC3 (G.G. Stewart,

Labatts Brewery, London, Canada). It was derived from the

parent strain by selection for the inability to form











colonies on lactate agar plates (Ogur and St. John, 1956)

after growth for 3 days in broth containing 6 mM MnCl2

(Putrament et al., 1973). It has been speculated that

manganese induces mutations by interacting with the

manganese-sensitive mitochondrial DNA polymerase causing

error-prone replication of the mitochondrial DNA. Strain

KD2 did not grow on glycerol containing medium or reduce

2,3,5-triphenyltetrazoleum chloride (Ogur et al., 1957). It

also lacked the cytochrome a+a3 absorbance bands at 600 nm

and 440 nm and the cytochrome b bands at 560 nm and 530 nm.

A petite strain was chosen for this study because growth and

fermentation have been shown to be almost identical both

anaerobically and aerobically, eliminating the need to

perform experiments under anaerobic conditions (Loureiro-

Dias and Arrabaca, 1982). In some studies, S.cerevisiae CC3

and S. cerevisiae A10 p (NRRL Y-12707) were used for

comparison. The latter strain was provided generously by

N.J. Alexander (Northern Regional Research Center, U.S.

Department of Agriculture, Peoria, Ill.).

Growth Conditions

All organisms were grown on YEPD medium which contained

5 g/liter yeast extract, 10 g/liter peptone and 200 g/liter

glucose as described by Leao and van Uden (1982a). The

medium was adjusted to pH 5.0 with 2.0 N HCl prior to

autoclaving. Solid medium for culture maintenance consisted

of YEPD broth containing 1.5% agar.











Batch fermentations were carried out in 250-mi tissue

culture spinner bottles (Bellco Glass, Inc., Vineland,

N.J.), immersed in a 300C water bath and agitated at

150 rpm. Culture bottles were fitted with water-trapped

exit ports for the escape of carbon dioxide and sampling

ports for the removal of culture by syringe. Growth was

allowed to proceed under conditions of self-induced

anaerobiosis. Inocula were prepared by transferring cells

from a slant to a test tube containing 10 ml of YEPD broth.

Cells were incubated at 30C for 36 h without agitation and

diluted 1:40 into 300 ml of fresh YEPD in a spinner bottle.

This culture was incubated for approximately 12 h until an

optical density at 550 nm of 3.5 (1.3 mg of cell protein per

ml) was reached. Fermentations were started by diluting the

12-h culture 1:100 into 300 ml of growth medium.

Preparation of Fermentation Samples for Analysis

Fermentation samples were centrifuged at 10,000 x g for

0.5 min. The supernatant was removed and saved by freezing

at -200C. Cells were washed once in 50 mM KH2PO4 buffer (pH

5.0), and the pellets were saved for further analysis by

freezing at -200C.

Analytical Methods

Ethanol was measured by gas-liquid chromatography as

described by Goel and Pamment (1984) with 2% (vol/vol)

acetone as an internal standard. Glucose was initially

determined with the glucose oxidase procedure (Raabo and











Terkildsen, 1960) using the Glucostat reagents supplied by

the Sigma Chemical Company (St. Louis, Mo.). In later

experiments, glucose was measured with a YSI model 27

glucose analyzer (YSI, Yellow Springs, Oh.) Cell mass was

measured as optical density at 550 nm with a Bausch and Lomb

Spectronic 70 spectrophotometer and as total cell protein by

the method of Lowry et al. (1951) as described by Layne

(1957).

Respirometry Measurements

Samples were pipetted into Warburg flasks and

equilibrated for 10 min at 300C. During the first 5 min of

the equilibration period, the flasks were flushed with

nitrogen gas. Rates of CO, production were measured with a

differential respirometer (Gilson, Middleton, Wis.). These

values were used to calculate fermentation rates as pmoles

of CO2 evolved per mg of cell protein. The rate of CO,

evolution was independent of sample volume, up to 4 ml, and

linearly increased with cell concentration, up to 5 mg cell

protein per ml.

Chemicals

Yeast extract, peptone and agar were obtained from

Difco Laboratories, Detroit, Mich. Glucose and other

biochemicals were obtained from Sigma Chemical Co. Acetone

and inorganic salts were purchased from Fisher Scientific

Company, Orlando, Fla. Absolute ethanol was supplied by

AAPER Alcohol and Chemical Co., Shelbyville, Ky. Gas











chromatography supplies were obtained from Supelco,

Bellefonte, Pa.

Results

Batch Fermentation by S. cerevisiae KD2

A typical batch fermentation profile of S. cerevisiae

KD2 in YEPD medium is shown in figure 1. Glucose conversion

essentially was completed after 60 h under these conditions

with the production of between 12 and 13% (vol/vol) ethanol.

Cell protein stopped increasing after 24 h at 2.4 mg per ml

medium, although the optical density at 550 nm of this

culture continued to rise for an additional 12 h period

(data not shown). Nearly identical profiles were obtained

in medium supplemented with Tween 80 (5 g/liter), linoleate

(45 mg/liter) and ergosterol (30 mg/liter). Similar

profiles also were obtained by the addition of small amounts

of 10 N KOH during the course of fermentation using a pH

stat to maintain the pH of the growth medium at 5.0.

Likewise, batch fermentations of S. cerevisiae CC3, the

parental grande strain, were indistinguishable from those of

strain KD2.

Inhibition of Growth Rate by Ethanol

Although it is not obvious from figure 1, the growth

rate of S. cerevisiae KD2 decreases as ethanol accumulates

in the fermentation broth (Fig. 2). Using the data from

batch fermentations, rates of growth were calculated as the

increase in cell protein over a 1.6 h period at the various































Figure 1. Growth and ethanol production by S.
cerevisiae KD2 during a typical batch
fermentation in YEPD medium containing
20% glucose. Symbols: O cell
protein (mg/ml culture); ,
glucose; Q ethanol.
























I0.0

5.0


I a I I I I 1


0 10 20 30
TIME


40
(h)


50 60


I0.0

5.0
0


I-
r

1.0
0
-4
0.5 m
z

o.
3

3
O. I"

0.05


- 0.01
70


1.0

J 0.5
U)
0
0
-J

* 0.I

0.05


0.01


























Figure 2.


Growth rate of S. cerevisiae KD2 in
the presence of ethanol. Rates were
measured from batch culture
experiments as the increase in cell
protein during a 1.6 h interval around
the time point sampled. These are
plotted as a function of ethanol
accumulated in the medium during
fermentation. Growth rates in YEPD
medium containing different
concentrations of ethanol were
calculated as the exponential increase
in cell mass per h as measured by
optical density at 550 nm. These are
plotted as a function of ethanol added
to the medium. Symbols: 0, growth
rate during batch fermentation; U ,
effect of added ethanol on growth
rate.


















































ETHANOL (% V/V)


0.40


0.30 1


0.201


0. 10


0.00


I I 2 I I I I 7
0 I 2 3 4 5 6 7











times sampled during the fermentations. Growth rate

decreased exponentially as ethanol accumulated in the

medium. The concentration of ethanol that had been produced

at half the maximum observed growth rate was

1.08% (vol/vol).

The addition of ethanol to fermentation broth also

decreased growth rate (Fig. 2). Cells inoculated into broth

containing increasing concentrations of ethanol were

inhibited in a dose-dependent manner. This inhibition was

linear above concentrations of 2% (vol/vol). The amount of

ethanol required to decrease the growth rate by 50% was

4.66% (vol/vol). Thus, four times more ethanol was required

to decrease growth rate by one-half than was produced when

growth rate had declined by this same fraction.

Inhibition of Fermentation Rate by Ethanol

The rate of alcohol production per mg cell protein was

calculated in a fashion analogous to the growth rate data.

These fermentation rates are shown as a function of average

ethanol accumulated in the medium in figure 3. Fermentation

rates also were determined by manometry using samples from

batch fermentations with excellent agreement for samples

taken 12 h and later. Identical plots were obtained with S.

cerevisiae CC3, the grande parent strain of KD2. The trends

observed were similar for cells grown with and without lipid

supplements and for cells grown in a pH stat where the pH of

the medium was held at 5.0. The fermentative activity of






















Figure 3. Rate of fermentation in the presence
of ethanol. Fermentative activity was
determined from batch culture
experiments as the increase in ethanol
concentration over 1.6-h time
intervals divided by the average
cellular protein concentration in the
medium during that time interval. It
is plotted as a function of ethanol
accumulated in the growth medium and
is expressed as umoles ethanol
produced per h per mg cell protein.
The effect of added ethanol on the
activity of cells at their highest
measured rate of fermentation, 12 h
after inoculation, is included for
comparison. Ethanol was added
directly to fermentation samples and
fermentation rates were measured by
respirometry. These are expressed as
umoles CO, produced per h per mg cell
protein and are plotted as a function
of total ethanol in the medium.
Symbols: *, fermentation rate
during batch fermentation; ,
effect of added ethanol on the
fermentation rate of 12-h cells.

































> 40-
I-\


< 30

> *
I-
S20
w


10 -



0
0 2 4 6 8 10 12
ETHANOL (% V/V)











cells exhibited a biphasic decline as a function of

accumulated ethanol. An initial decline in fermentation

rate occurred during the accumulation of 3.7% (vol/vol)

ethanol with a 50% loss of activity. This was followed by a

more gradual decline in fermentation rate with approximately

20% of the original activity remaining after the production

12% (vol/vol) ethanol. The fermentation rate of S.

cerevisiae AI0 po, a respiratory-deficient haploid

laboratory yeast strain, also declined as ethanol

accumulated in the medium (data not shown). As with S.

cerevisiae KD2, a 50% decrease in fermentation rate was

observed after the accumulation of 3.5% (vol/vol) ethanol.

However, the maximum rate of fermentation was lower,

40 pmoles CO2 per h per mg protein compared to 50 for strain

KD2 and greater than 90% of the maximum fermentation rate

was lost by the time 6.5% (vol/vol) ethanol had accumulated.

Unlike ethanol accumulated during fermentation, the

addition of low concentrations of ethanol to rapidly

fermenting cells 12 h after inoculation did not result in a

large decline in fermentative activity (Fig. 3). Ethanol

caused a dose-dependent linear decline in activity.

Fermentation was inhibited only 12% by the addition of 3.7%

(vol/vol) ethanol and 8.5% (vol/vol) added ethanol was

required to cause 50% inhibition.











Discussion

Because the ability of ethanol to inhibit alcohol

production varies with the yeast strain and fermentation

conditions employed (Jones et al., 1981), the effect of

ethanol on these processes in the yeast strain chosen for

these studies of alcohol production was characterized. The

observed decline in both growth and fermentation rates was

similar under a variety of culture conditions for S.

cerevisiae KD2. This decline also was seen with other

strains of S. cerevisiae, but strain KD2 maintained equal or

greater rates and accumulated more ethanol during

fermentation. Identical fermentation profiles of strain KD2

and its grande parent strain suggest that the manganese

treatment used to obtain respiratory-deficient cells, in

itself, was not responsible for the observed decline in rate

of growth and alcohol production. Induction of respiratory-

deficiency has been observed to decrease ethanol tolerance

in some petite strains of yeast (Aguilera and Benitez, 1985;

Esser et al., 1982) while increasing the fermentation rate

and ethanol tolerance of other strains (Bacilia et al.,

1978; Moulin et al., 1981). Differences between wild-type

strains, the harshness of the mutagenic treatments and the

limited numbers of mutants screened may be responsible for

these contradictory reports.

The brewery yeast strain used in most of these studies

exhibited an exponential decrease in growth rate (Fig. 2)











and a biphasic decrease in rate of alcohol production

(Fig. 3) as ethanol accumulated in the fermentation broth.

The exponentially falling growth rate was similar to that

observed by Aiba et al. (1968). However, the biphasic

decrease in ethanol production fits both an exponential

model and a linear model describing a combination of two

events. In batch fermentations by strain KD2, one cellular

site may be much more sensitive to the accumulation of

ethanol than another. Thus, a biphasic inhibition profile

occurs with the more sensitive site being characterized by

the line with the steepest slope. Fermentation rate

declined by 50% after the accumulation of about three and

one-half times more ethanol than was accumulated when growth

rate decreased by a similar amount (Fig. 2 and Fig. 3).

Clearly, ethanol added to the fermentation broth

linearly decreased both the rate of cell growth and alcohol

production as previously described (Brown et al., 1981;

Moulin et al., 1984). This contrasts with the results of

Luong (1985) who reported that S. cerevisiae ATCC 4126

exhibited non-linear decreases in growth and fermentation

rates under anaerobic conditions. As with the accumulation

of ethanol, growth rate was inhibited more than fermentation

rate, but was only about two-fold more sensitive.

Growth rate decreased faster than fermentation rate as

ethanol accumulated in the medium and as increasing amounts

of ethanol were added exogenously to the medium. Thus, the











actual alcohol production machinery is more resistant to

ethanol inhibition than is cell growth (Brown et al., 1981;

Luong, 1985). The magnitude of the observed inhibition,

however, was greater for endogenously produced ethanol than

for exogenously added ethanol for both processes in

agreement with previous reports (Moulin et al., 1984; Novak

et al., 1981). These results suggest that the mere presence

of ethanol may not be entirely sufficient to account for the

observed decline in fermentation rates. In the following

studies, the causal role of ethanol in the decreasing

fermentation rate was chosen for more detailed examination.

The role of factors other than ethanol also will be studied

in order to account more fully for this observed decrease in

rate of ethanol production.















CHAPTER III
NUTRIENT LIMITATION AS A BASIS FOR THE APPARENT TOXICITY OF
LOW LEVELS OF ETHANOL DURING BATCH FERMENTATION


Introduction

As has already been described for Saccharomyces

cerevisiae KD2, the rate of alcohol production per unit cell

mass decreases substantially during batch fermentations as

ethanol accumulates in the medium (Fig. 3). This decrease

has been attributed to the inhibitory effects of ethanol by

most researchers (Aiba et al., 1968; Bazua and Wilke, 1977;

Ghose and Tyagi, 1979; Luong, 1985; Millar et al., 1982;

Moulin et al., 1984; Rahn, 1929). However, recent studies

by Casey et al. (1983, 1984) have provided evidence that

nutrient limitation, in addition to ethanol accumulation, is

also an important factor limiting the rate of fermentation

during high-gravity brewing. The following studies examine

the role of ethanol in limiting the rate of alcohol

production and provide evidence that nutrient limitation is

an additional factor which contributes to the initial

decline in fermentative activity.

Materials and Methods

Organism and Growth Conditions

The organism used in these studies was Saccharomyces

cerevisiae KD2, a petite derivative of strain CC3 (G.G.

30











Stewart, Labatts Brewery, London, Canada). This organism

was grown in YEPD medium as described in chapter II.

Fermentations were carried out at 300C in spinner bottles

designed for tissue culture, also as described in

chapter II. "Conditioned broth" refers to medium in which

cells have been allowed to grow for 12 or 24 h and have been

removed by centrifugation. This broth was sterilized by

filtration.

Analytical Methods

Cell mass was monitored by measuring optical density at

550 nm using a Bausch and Lomb Spectronic 70

spectrophotometer. Total cell protein was determined using

the method of Layne (1957). Cell viability was measured

with the methylene blue dye exclusion procedure of Trevors

et al. (1983). Ethanol was determined using gas

chromatography as described by Goel and Pamment (1984).

Rates of fermentation were measured as the rate of CO2

production at 300C under a nitrogen atmosphere using

respirometry as described in chapter II.

Measurement of the Intracellular Ethanol Concentration

The procedure used to determine the intracellular

concentration of ethanol in actively fermenting yeast cells

is illustrated in figure 4. Cells from batch fermentations

were concentrated by centrifugation (10,000 x g, 2 min,

ambient temperature) and resuspended in the same medium to a

density of 50 mg of cell protein per ml. [14C]sorbitol












a)


0)Q) 00
M 04 0)
'g 0 0H 4)
ni <- o 4-1
p ^ r-l M
tP Pt 0 PC C0

t( >tH U 0 0
*> r-1 r-4 *ri >
rO 0 0 0 r- +
Co r-4 > 4) r-4



44 0
-cqr-I9a)k




CO 0 ) >
0 4 r-1 4- 0 ()
4 4. (0 0 4) 0) M 4r
+- 35 '0 4+4J



S) o ) >



.4 ,r- 0 Q)O
S-l 0 -4J rl (0



S4 ) 0 4 -
O} O M
r-I -p 4 H 0 P 0 4J
0 Q) 0 r :3 z C





C E- 3 H a 0
id 0 0




a) 1 m () 0 U)
S1d 4.) 0 --4 0
Q) (r --4 p 4V 0
o *, O P 0


3 c4 +) C nl
r-4 4) TI -H 0),3




r-l M 0 M 4- 4
Q) 3 U >i C Q) Q)





U4 *rd 4- Q $-I r0





0H OOq (o
r44 j r-0-4

4a) E a4 4) :30 V
*0 ) W 4(0 0 E-l 0
4-1 ) E-i (o r-l r-
C 0 0 O 3d
pl C H r -l r-l r
WP -r rul () 4 0 ()
(10 4P ) r-l L M




c41 u V 0








tr
*ri
44


















o
4-




0I


ce
emd


C0=
Q* -
CL
a,



O/1
SI
o,\
*SI
** P I^


0


|o I f


iv x C /
* 00 s
0 E 0
o E
6 I) V
'-I


- r-
o w



0 "J









0
I






c C m
0rL
US

w .
0D


C
0


c.)


4-.

E S
0


c '
C. O


5.x
4- 0.

* *
.4-. U_
";C


C
0
.4-

E
* s-
4.h
0


Z
- j
0
z







-JS





I-
z.
Oe


c I
r J











(specific activity, 50 pCi/mmole) was added to a 1-ml

suspension at a final activity of 42 nCi/ml. The suspension

was mixed for 10 sec using a vortex mixer, and 0.1 ml

samples were transferred to Whatman 3MM filter paper disks

(3 cm) for sorbitol measurements and to sample vials

containing 0.1 ml perchloric acid for subsequent alcohol

determinations. The remaining suspension was centrifuged

immediately at 10,000 x g for 30 sec in a microcentrifuge.

Supernatant samples of 0.1 ml then were transferred to 3MM

filter paper disks and to sample vials containing 0.1 ml of

0.58 M perchloric acid for the measurement of ethanol.

Filter disks were air dried at 800C before the addition of

scintillation fluid. The radioactivity of these samples was

measured using a Beckman model 8000 scintillation

spectrometer. Total cell volume was estimated as the

difference in ["4C]sorbitol counts between the suspension

and the supernatant.

A correction was made for the volume of total cell

solids included in the sorbitol-based estimate of cell

volume (Fig. 4b). This was done in a separate experiment to

ensure sufficient time for equilibration of tritiated water.

Control experiments were performed to confirm that tritiated

water had reached equilibrium after 5 min and that the

sorbitol did not leak into the cells during this period.

Tritiated water and [1C]sorbitol (specific activity,

50 pCi/mmole) were added to concentrated cell suspensions











(50 to 100 mg of cell protein per ml) at a final activity of

2 pCi/ml and 84 nCi/ml, respectively. After 5 min, 0.1-ml

samples were pipetted directly into scintillation fluid for

aqueous samples. The cell solid volume was calculated as

the difference between the tritiated water counts in the

suspension and the supernatant samples. The fraction of the

total cell volume occupied by solids was computed as

follows:


1 (3Hsu8/SHup)
V. = (1)
1 (14C-u.14CB U)


where V, is the fraction of solid volume, sus is the

suspension and sup is the supernatant fraction. Typically,

the cell solid volume represented 20 to 25% of the sorbitol-

excluded volume. The intracellular water content decreased

from 2.23 ul per mg cell protein 12 h after inoculation to

0.83 pl per mg cell protein by 48 h.

The intracellular concentration of ethanol was computed

based on the aqueous cell volume, i.e., the sorbitol-

excluded volume minus the solid volume. This was calculated

by assuming that the amount of ethanol in the suspension is

equal to the intracellular concentration of ethanol times

the aqueous cell volume plus the concentration of ethanol in












the supernatant times the supernatant volume as follows:

14C 14Cu
8US BUS
EsuS = Ecell (1 ) (1 V) + Eup (---- (2)
14CS14U
1 sup 1 sup


where Eus is the ethanol concentration in the suspension,

Eup is the ethanol concentration in the supernatant and

Ecell is the ethanol concentration within the aqueous cell

volume.

Chemicals

Complex medium components and agar were purchased from

Difco Laboratories, Detroit, Mich. Glucose and other

biochemicals were obtained from Sigma Chemical Co., St.

Louis, Mo. Inorganic salts were purchased from Fisher

Scientific Company, Orlando, Fla. Absolute ethanol was

supplied by AAPER Alcohol and Chemical Co., Shelbyville, Ky.

Radioactive compounds were purchased from New England

Nuclear, Boston, Mass. Gas chromatography supplies were

obtained from Supelco, Bellefonte, Pa.

Results

Effect of Ethanol Removal on Fermentation Rate

These studies have focused on two time points during

batch fermentation, 12-h and 24-h, to investigate the

possible reasons for the initial drop in fermentative

activity. To minimize possible variability arising from

inoculum differences, autoclaving, etc., 12-h cells have

been operationally defined as those which have increased in











cell mass 100-fold after inoculation. Typically, these

samples contain 1.2 to 1.3% (vol/vol) ethanol and 1.3 mg

cell protein per ml culture medium. Cells which have

produced 5.0 to 5.6% (vol/vol) ethanol, in addition to any

ethanol that may have been present in the original medium,

were operationally defined as 24-h cells. Typically, these

samples contained 2.6 mg cell protein per ml culture medium.

Table 1 shows the effects of ethanol removal on the

fermentation rates of 12-h and 24-h cells. This activity of

12-h cells was much higher than that of 24-h cells. Ethanol

removal by suspension in fresh broth had little effect on

the activity of 12-h cells and did not result in a

significant increase in the fermentation rate of 24-h cells.

Similarly, suspension in conditioned broth from 12-h

fermentations, containing 1.1% (vol/vol) ethanol, did not

affect fermentation rate. Suspension of cells in the 24-h

conditioned broth, containing 5.6% (vol/vol) ethanol,

reduced the fermentative activity of 12-h cells but had less

effect on the activity of 24-h cells. Removal of volatile

medium components from the 24-h conditioned broth eliminated

its inhibitory effect on the fermentation rate of 12-h cells

but did not result in a significant increase in activity of

the 24-h cells. The addition of ethanol to the 24-h

conditioned broth restored its ability to repress the

fermentation rate of 12-h cells, indicating that ethanol was
















Table 1. Effects of ethanol and fermentation medium
composition on fermentation rate


Rate of Fermentationa
(pmoles CO2 per h per mg protein (SD))
Assay medium

12-h cells 24-h cells


Original broth 36.3 (2.4) 16.5 (2.6)

Fresh broth 39.5 (2.3) 20.3 (2.5)

Conditioned broth 38.9 (1.9) 22.7 (5.7)
(12-h, 1.1% (vol/vol)
ethanol)

Conditioned broth 22.8 (0.6) 16.1 (2.0)
(24-h, 5.6% (vol/vol)
ethanol)

Conditioned broth 34.9 (0.8) 17.6 (2.0)
(24-h, volatiles removed
under vacuum)

Conditioned broth 21.9 (0.2) 15.7 (1.0)
(24-h, volatiles removed
under vacuum,
reconstituted to give
5.6% (vol/vol) ethanol)


a Cells from 12-h and 24-h batch fermentations were
harvested by centrifugation at ambient temperature and
suspended to their original volume in various broths. Where
indicated, volatiles were removed from conditioned broth by
vacuum distillation at 550C, reducing the volume by two-
thirds. The broth then was reconstituted with distilled
water or distilled water plus ethanol. Fermentation rates
were measured by respirometry. Averages and standard
deviations (SD) represent the results from three separate
batch fermentations.











the principle volatile component responsible for this

inhibition.

Cell Viability and Overcrowding Effects on Fermentation Rate

A trivial possibility for the failure of 24-h cells to

recover activity after suspension in broth lacking ethanol

would be the presence of large numbers of dead cells.

However, based on methylene blue dye exclusion, over 90% of

the yeast cells appeared active and intact at 24 h. Another

trivial possibility for the failure of 24-h cells to recover

activity after suspension in fresh medium is that by 24 h,

the cells are so crowded that they can no longer efficiently

take up nutrients and glucose for conversion to ethanol.

This possibility was addressed by suspending 24-h cells in

fresh medium at different cell concentrations and measuring

the rate of CO, production by respirometry. The rate of CO,

production increased linearly with increasing cell

concentrations. At a cell concentration comparable to that

of 12-h cells, the fermentation rate of 24-h cells was only

half that of 12-h cells (data not shown).

Intracellular Ethanol Concentration

The failure of 24-h cells to recover activity after

suspension in fresh medium could be caused by the failure of

the suspension procedure to effectively remove the

intracellular ethanol or by the accumulation of large

amounts of intracellular ethanol that could permanently

damage the fermentative capacity of the cells. To explore











these possibilities, the intracellular and extracellular

concentrations of ethanol were measured at 12 h and 24 h

during batch fermentations (Table 2). The external ethanol

concentrations in these suspensions at the time of sampling

were 1.2% (vol/vol) and 5.0% (vol/vol), respectively, before

concentrating the cells. The level of extracellular ethanol

measured in the concentrated cell suspension was slightly

higher than the starting culture reflecting the rapid

metabolism of cells during the less than 3 min period of

cell concentration and sampling. In all cases, the

calculated intracellular ethanol concentration was lower

than or equivalent to the extracellular ethanol

concentration.

To confirm that the higher amounts of ethanol in the

concentrated cell suspension resulted from rapid metabolism,

a potent inhibitor of enolase (Warburg and Christian, 1941)

and of fermentation, potassium fluoride, was added before

cell concentration. Previously, 50 mM potassium fluoride

was determined to cause immediate cessation of CO, evolution

(data not shown). In both 12-h and 24-h fermentation

samples, the addition of fluoride prevented the increase in

extracellular ethanol during cell concentration and sampling

(Table 2).

The removal of ethanol by suspension of cell pellets in

fresh medium lacking ethanol substantially decreased the

intracellular ethanol concentration (Table 2). Regardless






















Table 2. Intracellular and extracellular ethanol
concentrations under various conditions


Ethanol concentration (% vol/vol) (SD) in
different media


Fresh
Sample Native Native Fresh + 10%
+ KF ethanol


12b
Int 1.9 (0.4) 1.4 (0.2) 0.6 (0.1) 6.9 (1.5)

Ext 1.7 (0.1) 1.3 (0.1) 0.4 (0.1) 10.7 (1.1)

24b
Int 3.4 (0.7) 3.7 (0.7) 0.7 (0.1) 8.1 (1.3)

Ext 5.5 (0.1) 5.1 (0.1) 0.5 (0.1) 9.7 (0.1)


a Three or more independent determinations. Native refers
to the broth in the batch fermentation with or without added
KF (50 mM). Fresh refers to sterile, unused medium with or
without added ethanol (10% (vol/vol)).

b Age of batch fermentation. Int and Ext refer to the
intracellular and extracellular ethanol concentrations,
respectively.











of the cell age and original ethanol concentration, the

intracellular ethanol concentration was found to be 0.6 to

0.7% (vol/vol) after ethanol removal. These values were

somewhat higher than anticipated and appeared to be due to

ethanol production by continued metabolism during suspension

and sampling. The inclusion of potassium fluoride during

harvesting and suspension in fresh medium resulted in a very

low internal and external ethanol concentration (0.06%

(vol/vol)), consistent with dilution of the cell pellet

volume with fresh medium.

In an analogous fashion, the failure of exogenously

supplied ethanol to raise the internal ethanol concentration

of 24-h cells to a level equivalent with that of cells

during fermentative alcohol production could provide an

explanation for the apparent resistance of 24-h cells to the

inhibitory effects of added ethanol (Table 1). Samples

taken after 48 h and processed to determine the

intracellular ethanol concentration contained approximately

11.2% (vol/vol) (SD 1.0) ethanol in the fermentation broth.

The intracellular ethanol concentration of these cells was

8.2% (vol/vol) (SD 1.7). Suspension of 24-h cells in broth

containing 10% (vol/vol) ethanol resulted in an increase in

the intracellular ethanol concentration to 8.1% (vol/vol)

(SD 1.3). These values indicate that the addition of

ethanol to 24-h cells increased the intracellular ethanol

concentration to the level found in cells during batch











fermentation. Thus, on the time scale of the ethanol

removal experiments, 24-h cells appear to be freely

permeable to ethanol added to the fermentation broth.

Effect of Added Ethanol on the Fermentation Rate of 12-h and
24-h Cells

The sensitivity of 12-h and 24-h cells to inhibition of

fermentative activity by added ethanol is illustrated in

figure 5. The fermentation rate of 24-h cells was

approximately one-half that of 12-h cells when assayed in

fresh broth lacking ethanol. Both types of cells were

insensitive to ethanol concentrations up to 2% (vol/vol)

after which they exhibited a dose-dependent linear decline

in activity up to between 12 and 14% (vol/vol) ethanol.

When plotted as a percentage of maximal rate, 24-h cells

appeared slightly more resistant, 50% inhibition at 8.3%

(vol/vol) ethanol as compared with 7.4% for 12-h cells.

Effect of Medium Composition During Growth on the
Fermentation Rate of 12-h and 24-h Cells

The slight differences in sensitivity to inhibition by

ethanol and the failure of ethanol removal to increase

fermentation rates suggest that the reduced activity of 24-h

cells may be primarily due to physiological changes in the

cells rather than to the immediate presence of ethanol.

Several experiments were performed to identify possible

causes of the physiological changes which may be involved

(Table 3). In these experiments, cells were grown under a

variety of conditions, harvested by centrifugation at











(,C

of


0 4J r3
* 90


4- < G
) CQ) 4-
() '4 c.


Q) 0c U

(0 -H4 4 H
rdW WU)


Q C) I
f -H (d ( r-

1Q) () 0
U 'd --HO

I 4 ,Q


Sa ,

4- E4-
I 3rl a
4 0 r- r-
4-4Al 0 0
0 tpiW i r
0 z 0 0
Q) 44 0 4 4
Srl 4-
rd 4J 0

Q) 4- ) Z3
'-I 0) rc 9
C 0 0 0


0) 0) H


4 > -ri 4J
p E) r>-I



0 C 0 0



*I r-H 4 0) 4-J r-
r. (U) 0 HM 4 1)
HO U P 0 0




Q)


*O
brz
,QMM ,C
*HH 09H
0000 0
HC)OMOO:
ekrd
tou

tPS~











*
m


NOIlJ.VN3NaI3- JO- 31Va


(D







z
CCO

O
-J

I-
O








CO N
OD CD qt CCJ

31V8t "lVVlXVV4 %



(D

T N





O


-o
z

I
It




0 0 0 0 0
CO CO ^- C


0
0
c


0


oC r- r N -





















Sco
.QQ

H H
(M LnI
i-l r-I


00

* 4
IAH


.0
In




0'4-1





00
Wd
o o




HV
^.~1.


OH

0 r-
H*
VO 0


0)
4-1







0
-rw

) 0 0


41 H I 4 o
0 0 0 W0

0 0 4r-4 4


es rl c Oc
4 0 0 > x0
v> -r C ) C



*O 10 -P X! (-
40 -*H 0 4-)NM 4-
P(0 0 A 0 *0 c-
a! U t-44i U.i r. u



S%4 0 H
*O P COO 0
tUUi HUUXXU
(M *4
-1
r-l N ^


0
4-1)

Q)0






0
r-l
HO

4-)


4 -


O 0
>0
\4

>X
4-


Io\ 4






-q, -H
(N3


0 0
0) Q)
4- 4-)

0 0


ao

r-4i r-i
04 ft4




o o
(0




0 0
4 *4-)
Q 0)

X4 r. -
4 rH -4-) r-H
0 0 0 0 0
> C p> 0)
XI N0 .X r.

0> 0 >
C 0) C--
O JO V
04)cr 4 4) CM U4> 0
M-*H 3*- 0

CC PC H

14 4- I )
CM *'H 4
HW N-H


















00










*e
LO CO









cO










co
















H


0

4.)
*H



4 4-)



4J 4.)
C-im
00U
10 0
4J (4
* ox
*z U LO


0n9



cd -
0 4
CP 0 Q
Q) W
G tp >


0 -0
*O

X Q -4-


4 0 g








P ,r-44
4. -H







-rl 4) 0W
0- U) 4













to o0
(i 0) z
4.), 0 0
V40 )
So H
Q) 0 0>0





P0 Q0) (0
S4W rC-




0 <-1 0
0 0 )-1
a o





U-H ()U
U O C





S0 0
000
0 > (-

X *0-H 0







*H 1 +I




(0 (O '-











ambient temperature and suspended in fresh medium lacking

ethanol to measure the rate of fermentation under standard

conditions. In all experiments, the fermentation rate of

cells used as inoculum to start these batch fermentations

were included as controls.

Experiment 1 examined the possibility that the

physiological changes in 12-h cells to produce 24-h cells

were due to growth in the presence of ethanol. Batch

fermentations in which 5% (vol/vol) ethanol was added prior

to inoculation were allowed to grow to the same cell mass as

12-h control cells, 1.2 mg cell protein per ml fermentation

broth. The fermentation rate of these cells grown in the

presence of added ethanol was only slightly lower than that

of control cells grown for 12-h in the absence of ethanol.

This indicated that exposure to 5% (vol/vol) ethanol during

growth was not sufficient to account for most of the

observed reduction in fermentation rate.

The possibility that growth in the presence of ethanol

and other fermentation products may be responsible for the

reduction in fermentation rate was examined in experiment 2.

Cultures were inoculated into bottles containing filter-

sterilized conditioned broth which had been supplemented

with 5 g/L yeast extract and enough glucose to increase the

concentration in the broth back to 20%. Conditioned broth

from 12-h cultures contained 1.2% (vol/vol) ethanol and

broth from 24-h cultures contained 4.5% (vol/vol) ethanol.











These fermentations were allowed to proceed until the

culture cell density reached the state defined as 12-h

cells, approximately 1.3 mg protein per ml broth. Cells

grown in the supplemented 12-h conditioned broth fermented

at rates equal to those of control cells. The fermentation

rate of cells grown in the supplemented 24-h broth was

lower, but was at least twice that of the 24-h control.

After allowing these fermentations to continue until 5%

(vol/vol) ethanol had been produced in addition to that

present at the time of inoculation, the fermentation rate of

both types of "24-h" cells were similar to that of the

control cells. Thus, the decline in rate of fermentation

observed after the production of 5% (vol/vol) ethanol is not

due entirely to the accumulation of ethanol and/or other

stable inhibitors in the fermentation broth.

The last possibility examined in order to understand

the reasons for the decline in fermentation rate of cells

after 24 h was the effect of nutrient limitation. Neither

12-h conditioned broth nor 24-h conditioned broth

supplemented with glucose supported vigorous growth of

strain KD2 following reinoculation (Table 3, experiment 3).

In experiment 2, the addition of yeast extract restored the

ability of conditioned broth to support growth, promoting

fermentation rates equivalent to the control. Cells grown

in broth containing 25 g/L yeast extract, 5-fold greater

than that of control broth, exhibited fermentation rates











equivalent to control cells after 12 h. These were twice as

high as control cells after the production of 5% (vol/vol)

ethanol in approximately 24 h (Table 3, experiment 4).

Discussion

Previous studies have shown that the rate of alcohol

production by yeast per unit cell mass decreases as ethanol

accumulates during fermentation (Holtzberg et al., 1967;

Navarro and Durand, 1978; Strehaiano and Goma, 1983). Most

of these studies have attributed this reduction in

fermentation rate to adverse effects of ethanol (Ingram and

Buttke, 1984; Maiorella et al., 1983; Millar et al., 1982;

Moulin et al., 1984). In fact, the possible accumulation of

high intracellular concentrations of ethanol in S.

cerevisiae and its involvement in the inhibition of growth

and fermentation have been the subject of considerable

controversy. Previous studies have shown that the growth

and fermentation rates of S. cerevisiae are much less

sensitive to inhibition by added ethanol than is inferred by

the decrease in alcohol production and growth rates which

accompany the accumulation of ethanol during fermentation

(Ingram and Buttke, 1984; Moulin et al., 1984; Nagodawithana

and Steinkraus, 1976; Navarro and Durand, 1978; Novak et

al., 1981). To explain this anomaly, it has been proposed

that the leakage of ethanol from yeast cells is, in some

way, limited by the permeability of the plasma membrane.

This would result in the accumulation of high cytosolic











levels of ethanol during rapid fermentation. Addition of

exogenous ethanol would not readily duplicate this

condition. However, the ethanol retention hypothesis is not

supported by direct measurements of intracellular ethanol

concentrations during fermentation.

A series of attempts to measure the intracellular

concentration of ethanol have resulted in conflicting data.

Problems in experimental design associated with the

measurement of a small, rapidly produced metabolite, such as

ethanol, contribute to these differences. The conflicting

reports result from two basic problems. First, measurements

of ethanol concentrations in the pellets of rapidly

fermenting cells result in calculated intracellular

concentrations of ethanol which are often several-fold

higher than those of the surrounding medium (Navarro and

Durand, 1978; Novak et al., 1981; Panchal and Stewart,

1980). This was, in large part, due to the continued

production of ethanol by the cells in pellets during

centrifugation and processing (Dasari et al., 1984). The

acuteness of this problem also would be expected to decrease

as fermentation rate and substrate levels declined during

batch fermentations. The reduction in apparent

intracellular/extracellular ratios of ethanol observed by

Beaven et al. (1982) during the latter stages of

fermentation supports this idea. Dasari et al. (1984)

demonstrated that precooling the culture significantly











reduced the error introduced by continued ethanol production

during cell harvesting. However, cooling may introduce

other potential problems associated with temperature-

induced changes in the organization of and permeability

properties of the plasma membrane.

The second type of experimental problem associated with

measurements of internal ethanol involves washing of the

yeast cells. Experimental designs which included washing of

cells (Nagodawithana and Steinkraus, 1976; Panchal and

Stewart, 1980) before estimation of ethanol resulted in

lower apparent intracellular ethanol concentrations than do

unwashed samples (Beaven et al., 1982; Dasari et al., 1984).

Beaven et al. (1982) clearly showed that even minimal

washing leaches most of the intracellular ethanol from the

cells. These two experimental designs, measurement of

ethanol in a metabolically active cell pellet and washing,

each introduce errors which change the calculated values of

intracellular ethanol in opposite ways.

Recent studies by Guijarro and Lagunas (1984) have

employed a procedure which eliminated these two basic

problems in experimental design by using glass fiber filters

to rapidly harvest cells. With this method, extracellularly

added ["4C]ethanol rapidly equilibrated with the

intracellular environment, indicating that the plasma

membrane is freely permeable to ethanol. However, this

still does not answer the question of the true intracellular











ethanol concentration in yeast cells during active

fermentation and ethanol production.

In the studies presented in this chapter, the

intracellular ethanol concentration was estimated by an

independent method using cells that were actively producing

ethanol in suspension culture. The results obtained using

this method confirm the reports by Beaven et al. (1982) and

Guijarro and Lagunas (1984) which indicated that yeast cells

are freely permeable to ethanol. In addition, these results

provide direct evidence that the intracellular concentration

of ethanol produced during fermentation is not several-fold

higher than that of the surrounding medium as proposed

previously (Beaven et al., 1982; Nagodawithana and

Steinkraus, 1976; Novak et al., 1981; Strehaiano and Goma,

1983). Identical conclusions were reached by Dasari et al.

(1985) using high cell density fermentations which allowed

rapid processing of the cells for analysis. There does not

appear to be any problem associated with the efficient

diffusion of ethanol from yeast cells into the environment

during fermentation. Thus, it is unlikely that the

retention of unusually high intracellular ethanol

concentrations contributes toward the decrease in

fermentative activity of S. cerevisiae during fermentation.

Recently, Casey et al. (1983, 1984) have shown that

yeast nutritional requirements limit fermentative activity

in high gravity brewing. Supplementing worts with yeast











extract and lipids substantially improved fermentation rates

and reduced the time required to complete the fermentation.

The studies reported in this chapter using a yeast

extract/peptone-based fermentation broth also illustrate

this point and provide further support for the hypothesis

that nutritional deficiencies, in addition to accumulated

ethanol, also are responsible for the initial decline in

fermentation activity during the accumulation of low levels

of ethanol.

The reduced fermentation rate of cells after the

production of approximately 5% (vol/vol) ethanol appears to

result from the combination of a small inhibitory effect of

ethanol and physiological changes in the cells. These

physiological changes were not induced by growth in the

presence of 5% (vol/vol) added ethanol or by growth in the

presence of ethanol along with other natural fermentation

products. Conditioned broth was deficient in nutrients

provided by yeast extract and supported very little growth.

The addition of 5 g/L of yeast extract restored the ability

of this spent broth to support vigorous growth and

fermentation. By further increasing the concentration of

yeast extract to 25 g/L in the growth medium, the decline in

fermentative activity associated with the initial production

of 5% (vol/vol) ethanol was partially prevented. These

results support the hypothesis that physiological changes in

the cells caused by nutrient limitation are major factors in









55

the initial 50% decline in fermentative activity. Further

studies will include identification of this limiting

nutrient and, upon supplementation, characterization of its

effect on growth and fermentation.















CHAPTER IV
MAGNESIUM LIMITATION AND ITS ROLE IN THE APPARENT TOXICITY
OF ETHANOL DURING YEAST FERMENTATION


Introduction

The rate of ethanol production by Saccharomyces spp.

decreases in batch fermentations as alcohol accumulates in

the medium (Moulin et al., 1984; Rahn, 1929; Strehaiano and

Goma, 1983). The onset of this decline in fermentative

activity occurs at very low ethanol concentrations, often

less than 3% (vol/vol). Since ethanol has been shown to

inhibit fermentation (Brown et al., 1981; Cysewski and

Wilke, 1977; Gray, 1941), it generally has been accepted

that this accumulation of ethanol is responsible for the

progressive decline in fermentative activity (Bazua and

Wilke, 1977; Ghose and Tyagi, 1979; Luong, 1985). However,

the extent of inhibition by exogenously added ethanol is

less than would be predicted by the decline in fermentation

rate which normally occurs during the fermentative

accumulation of ethanol (Fig. 3).

Further studies have attempted to define the

mechanisms) of ethanol inhibition of fermentation and to

reconcile the failure of added ethanol to inhibit

fermentation to the extent observed during the fermentative

accumulation of ethanol. Early experiments provided

56











evidence that the intracellular concentration of ethanol was

much higher than that of the surrounding medium during

fermentation (Nagodawithana and Steinkraus, 1976; Navarro

and Durand, 1978; Panchal and Stewart, 1980), a condition

not readily duplicated by exogenously added ethanol.

However, these early data can be explained by problems in

the measurement of internal ethanol concentrations (Dasari

et al., 1984). Several research groups have developed

independent methods which demonstrated that ethanol is

freely permeable in Saccharomyces spp. and that the

intracellular concentration of this metabolic product is the

same as that in the surrounding fermentation broth (Dasari

et al., 1985; Guijarro and Lagunas, 1984; Table 2).

Additional studies have investigated the sensitivity of

glycolytic enzymes and alcohologenic enzymes to in vitro

inhibition by ethanol. Millar et al. (1982) have shown that

these enzymes are stable in ethanol concentrations higher

than 20% (vol/vol). The two enzymes most sensitive to

inhibition by ethanol were pyruvate decarboxylase and

phosphoglycerate kinase. Both, however, retained, 50% of

maximal activity in the presence of over 12% (vol/vol)

ethanol, the final alcohol concentration achieved by the

complete fermentation of 200 g of glucose per L of broth.

Similarly, Larue et al. (1984) concluded that the cessation

of alcohol production during stuck fermentations was not due











to ethanol inhibition of alcohol dehydrogenase and

hexokinase activities.

Casey et al. (1984) have reported that nutrient

limitation is a major factor restricting the ethanol

productivity of high-gravity fermentations. Anaerobically

cultured yeasts are known to have a nutritional requirement

for ergosterol and unsaturated lipids (Hossack and Rose,

1976; Nes et al., 1978; Proudlock et al., 1968).

Unsaturated lipids have been shown to increase biomass,

alcohol production and ethanol durability of yeast cells

during anaerobic fermentation (Ingram and Buttke, 1984;

Janssens et al., 1983; Lafon-Lafourcade et al., 1979; Thomas

et al., 1978). A variety of lipid-protein complexes and

nutrient supplements, ranging from albumin-ergosterol-

monoolein to soy flour and yeast extract, also have been

shown to yield increased rates of alcohol production and

higher final ethanol concentrations (Damiano and Wang, 1985;

Hayashida et al., 1976; Lafon-Lafourcade et al., 1979; Ohta

and Hayashida, 1983).

The studies described in chapter III suggest that the

initial decline in fermentative activity during batch

fermentation of 20% glucose is not caused by the presence of

ethanol or by growth in the presence of 5% (vol/vol)

ethanol. These studies indicated that a components) of

yeast extract was limiting cell growth and that this

limitation contributed to the early loss of fermentative











activity. The results presented in this chapter identify

magnesium as the limiting component of yeast extract and

demonstrate that when this nutrient limitation is relieved,

a dramatic decrease in the time required for total

conversion of glucose to ethanol is achieved. This decrease

in time required for the completion of fermentation resulted

from a delay in the onset of stationary phase which

increased the total cell number during that part of

fermentation in which over 90% of the ethanol is produced.

Materials and Methods

Organisms and Growth Conditions

The principal organism used in these studies was

Saccharomyces cerevisiae KD2, described in chapter II. In

addition, S. cerevisiae CC3, S. cerevisiae A10 (NRRL Y-

12707) and S. sake (NRRL Y-11572) were used for comparison

in some experiments. The latter two strains generously were

provided by N.J. Alexander (Northern Regional Research

Center, U.S. Department of Agriculture, Peoria, Ill.). All

organisms were grown in YEPD broth and maintained on YEPD

agar, as stated in chapter II. Batch fermentations also

were carried out as described in chapter II.

Preparation of Fermentation Samples for Analysis

Fermentation samples were centrifuged at 10,000 x g for

0.5 min. The supernatant was removed and saved by freezing

at -200C. Cells were washed once in 50 mM KH2PO4 buffer (pH











5.0) and the pellets were saved for further analysis by

freezing at -200C.

Preparation of Glucose-Reconstituted Medium for Growth
Experiments

Batch fermentations were allowed to reach an optical

density at 550 nm of 3.5. Cells were removed by

centrifugation in a Sorvall RC-2B centrifuge at 10,000 x g

for 2 min. The amount of ethanol in the supernatant was

determined and used to estimate the amount of glucose needed

to reconstitute the medium to a concentration of 20%. This

glucose-reconstituted medium was sterilized by vacuum

filtration with 0.45 pm Metricel membrane filters (Gelman

Sciences Inc., Ann Arbor, Mich.).

Preparation of Ashed Medium Components

Yeast extract (20 g) and peptone (30 g) were burned

over a gas burner for 5 h in a porcelain crucible. After

being transferred to a muffle furnace, the medium components

were ashed at 6000C for 72 h. The yeast extract ash was

suspended in 40 ml of deionized water and the peptone ash

was suspended in 30 ml of deionized water. These aqueous

suspensions of ash were adjusted to pH 5.0 with concentrated

HC1 and sterilized by autoclaving.

Nutrient Supplementation Growth Experiments

Nutrient supplements were added to culture tubes

containing 5 ml fresh YEPD medium or glucose-reconstituted

medium and a 1% by volume inoculum (initial optical density

at 550 nm of 0.035). Culture tubes were incubated at 30C











and agitated (30 rpm) in a Rototorque culture rotator (Cole-

Parmer, Chicago, Ill.).

Medium Analyses

Ethanol and glucose were measured as described in

chapter II. The magnesium concentration of the medium was

measured with the 60 Second Magnesium reagents purchased

from American Monitor Corporation, Indianapolis, Ind., as

described by Osman and Ingram (1985).

Cellular Analyses and Respirometry Measurements

Cell mass and total cell protein were measured as

described in chapter II. To determine the amount of

intracellular magnesium, yeast cells were washed once in

50 mM KH2PO4 buffer (pH 5.0) and the cell pellets were

stored frozen at -200C until analyzed. These yeast pellets

contained 1 to 3 mg of cell protein and were permeabilized

by incubation in a boiling-water bath for 1.5 min. The

resulting debris was suspended in 1 ml of 50 mM KH2PO4

buffer (pH 5.0) and then pelleted. The supernatant was

analyzed for magnesium as described above. Respirometry

measurements were made as described in chapter II and

fermentation rates were calculated from these values as

pmoles of CO2 produced per h per mg cell protein.

Viable-Cell Determinations

Cell numbers were determined microscopically with a

Petroff-Hausser counting chamber. Viable-cell counts were











determined by the methylene blue staining procedure of Mills

(1941).

Chemicals

Yeast extract, peptone and agar were obtained from

Difco Laboratories, Detroit, Mich. Glucose and other

biochemicals were obtained from Sigma Chemical Co., St.

Louis, Mo. Magnesium sulfate and other inorganic salts were

purchased from Fisher Scientific Company, Orlando, Fla.

Absolute ethanol was supplied by AAPER Alcohol and Chemical

Co., Shelbyville, Ky. Gas chromatography supplies were

obtained from Supelco, Bellefonte, Pa.

Results

Effect of Nutrient Supplements on Growth in Glucose-
Reconstituted Medium

As shown in Tables 3 and 4, fermentation broth in which

S. cerevisiae KD2 had grown for 12 h supported very little

further growth and limited the fermentative activity of

strain KD2 even after supplementation with glucose (glucose-

reconstituted medium). At this stage of fermentation (1.2%

(vol/vol) accumulated ethanol), ethanol production was at

its maximum rate (50 pmoles/h per mg protein). This time

point also marked the end of exponential growth (Fig. 1),

indicating either a nutrient limited state or the presence

of an inhibitor.

The addition of yeast extract and peptone at the

original medium concentration restored the ability of the

used medium to support growth at 71% and 54% of the control





















Table 4. Effect of nutrient supplementation
S. cerevisiae KD2


on growth of


Optical Density % of
Medium Supplement at 550 nm after control (SD)
48 h (SD)


YEPD None 13.8 (1.5) 100

12-ha None 1.25 (0.48) 9.1 (4.0)

12-ha Yeast extract (5 g/L) 9.77 (0.75) 71 (9)
12-ha Peptone (10 g/L) 7.43 (0.35) 54 (6)
12-ha Ashed yeast extract 9.70 (0.40) 70 (8)
12-ha Ashed peptoneb 3.47 (0.20) 25 (3)

12-ha Trace minerals 1.88 (0.20) 14 (2)
12-ha KH2PO4 (7.3 mM) 1.49 (0.17) 11 (2)
12-ha (NH4)2SO4 (7.6 mM) 1.60 (0.35) 12 (3)
12-ha MgSO4 (2 mM) 12.7 (0.1) 92 (10)
12-ha MgCl2 (2 mM) 12.1 (0.5) 88 (10)
12-ha CaC12 (2 mM) 1.55 (0.14) 11 (2)
12-ha Na2SO4 (2 mM) 1.62 (0.20) 12 (2)


a Medium isolated from a batch fermentation after 12 h of
yeast growth and supplemented to 20% with glucose.

b An amount of ashed yeast extract equivalent to 5 g of
whole yeast extract per L or an amount of ashed peptone
equivalent to 10 g of whole peptone per L.


c As described by Wickersham (1951).











level, respectively (Table 4). These results indicate that

nutrient limitation rather than the presence of an inhibitor

was responsible for the inability of the used medium to

support further yeast growth. Vitamin supplements also were

tested and did not promote growth in this glucose-

reconstituted medium (data not shown).

The organic components of yeast extract and peptone are

both diverse and complex. Before embarking on a

fractionation of these, the inorganic constituents were

tested after ashing. Supplementation with ashed yeast

extract was as effective as with whole yeast extract, while

ashed peptone was only half as effective as whole peptone.

These results suggested that an inorganic component of YEPD

medium was the principle factor limiting growth.

The inorganic constituents of a mineral-based minimal

medium were tested to determine which ions were limiting

(Table 4). The addition of potassium, ammonium, sodium,

calcium, phosphate, sulfate and a trace mineral mixture

described by Wickersham (1951) did not promote growth in

glucose-reconstituted medium. Only magnesium salts were

effective as nutrient supplements, allowing growth

equivalent to 90% of the control in fresh YEPD medium.

The dose-response of growth to added magnesium, yeast

extract and ashed yeast extract is shown in figure 6. Yeast

extract contained 27 moles of magnesium per g. This value

was used to calculate the appropriate amount of whole and




























Figure 6.


Dose-response of cell growth to added
magnesium. Magnesium values represent
the amount of magnesium contained in
the added nutrient supplement. Error
bars represent the average standard
deviation for each experiment.
Symbols; U whole yeast extract
added to glucose-reconstituted, used
medium; 0 ashed yeast extract
added to glucose-reconstituted, used
medium; 0 MgSO4 added to
glucose-reconstituted, used medium;
0 MgSO4 added to fresh YEPD broth.
















18

16


12I


0 0.2 0.4
MAGNESIUM


1.0


0.6 0.8
(mMolar)











ashed yeast extract to be added. Whole yeast extract, ashed

yeast extract and MgSO4 gave similar dose-responses.

However, at concentrations below 0.2 mM, MgSO4 appeared to

be a better supplement. Magnesium sulfate-supplemented

fresh YEPD medium also was plotted for comparison. Maximum

growth occurred at added magnesium concentrations above

0.2 mM. A MgSO4 concentration of 0.5 mM was chosen for

subsequent fermentation studies because growth at this

concentration was no longer limited by an inadequate supply

of magnesium.

To confirm that magnesium indeed was limiting in YEPD

medium, the magnesium content of cells and the surrounding

broth was determined at various times during batch

fermentation (Fig. 7). The magnesium content of the cells

reached a maximum of 130 nmoles/mg cell protein at 12 h,

rapidly declining to 48 nmoles/mg cell protein by 24 h and

remaining at this lower level throughout the final period of

fermentation. In the medium, the magnesium content fell to

less than 0.05 mM by 24 h and remained constant until

fermentation had been completed. Thus, the decline in

magnesium content per mg cell protein observed after 12 h

appears to result from continued cell growth after near

depletion of the magnesium in the surrounding broth.

Supplementing the broth with 0.5 mM magnesium resulted in

the peak accumulation of higher levels of magnesium

(200 nmoles/mg cell protein) at 12 h, followed by a decline












0
0o

c O
4 .
a) CO

C 4-) 0 T
0) r-4 -H 0
:3 4-) Wo

(4-4a

S4J Q)
,CQ 0

to G 4-
(0 -H 4 0)



r oP
C O)
4-1 0 U 9 4
O H -r i .-j (0
+J (0 *H
a)0 u > )w
0 40 0

UMO9
P 0 P 04 r:





,--i : 0
0 O0 V5



* ( Q) W

m -- r- 0
c0 0 0-H

r- (I 0 4-)
0e a



0 44 a -





-H O
r-ri r4 a)




00 w 0





*Hr 0 0
4a 1 a) 0

3-H -
Sx- O 0p
4-n) M 0o
0 4C -) En
(D Q) Q) 0


.n (0 4c




















r4










































0 0 0 0 0 0
SOD CD CM
- 0 0 0 0
(J|IOANW) HIOUS NI INIS3NOVM


I I i0


0

(uiolOjd 6w/calowu)


0 0
0 In
VnlS3N9\m yvIrm-1


0 A
O4











to about 130 nmoles of magnesium per mg cell protein after

24 h. Magnesium-supplemented cultures maintained a higher

level of cellular magnesium throughout fermentation than

cultures grown in unsupplemented YEPD medium.

Magnesium-Limited Growth of Other Yeast Strains

Three other yeast strains were investigated to

determine whether magnesium also limited their growth: S.

cerevisiae CC3 (parent organism), S. cerevisiae A10 and S.

sake. Glucose-reconstituted medium was prepared for each of

these strains. Cultures were inoculated into their

respective glucose-reconstituted medium with and without

added magnesium (0.5 mM) and incubated for 48 h on a

rotator. S. cerevisiae CC3 and S. sake exhibited magnesium-

dependent growth almost identical to that reported for

strain KD2. The optical density at 550 nm was 8.3 to 9.4

after 48 h with added magnesium and 0.5 without added

magnesium. S. cerevisiae A10 grew poorly in its glucose-

reconstituted medium, with an optical density at 550 nm of

1.0 to 1.2 after 48 h for both control and supplemented

cultures. All three strains reached a similar cell density

in fresh YEPD medium (optical density at 550 nm of 14.2 to

15.2). These results indicate that the magnesium limitation

observed in strain KD2 was not caused by the petite mutation

and was not limited to strain CC3 and its derivatives.

However, additional factors were clearly involved with S.

cerevisiae A10.











Effect of Magnesium Supplementation of Batch Fermentation

The effects of supplementing YEPD medium with 0.5 mM

MgSO4 on batch fermentation are illustrated in figure 8.

The production of cell mass as measured by cellular protein

is shown in figure 8A. Supplementation with magnesium

prolonged the exponential rise in cellular protein, allowing

a 53% increase in cell mass over that of the control within

18 h after inoculation. The addition of magnesium also

increased the rate at which glucose was consumed and ethanol

was produced (Fig. 8B and 8C). After 30 h of incubation,

magnesium-supplemented cultures had produced one-third more

ethanol than the controls. The conversion of glucose to

ethanol was complete after 48 h in magnesium-supplemented

cultures, but required 72 h in control YEPD broth. The

final yield of ethanol was essentially identical for both

magnesium-supplemented and control cultures, 12.7% (vol/vol)

(98% of theoretical maximum yield).

Effect of Magnesium Supplementation on Rate of Fermentation

Samples were removed from magnesium-supplemented and

control fermentations at various times during batch

fermentation. Ethanol concentration, cell protein and CO,

evolution of unwashed cells were measured. Figure 9 shows

the fermentation rate as a function of accumulated ethanol.

Both control and magnesium-supplemented cultures exhibited

the same maximum rate of fermentation at 1% (vol/vol)

ethanol. However, magnesium-supplemented cultures











o0
0) -H
..PC
4-) 4-1
4d--) (d
P O.P
0 a)


(1) 4(4
- .0O

> 0) 4-
E nl C
C r. ( 0
0 p0
CP to 0) tP
M0 r1 C
1- ) r-4 )
4-i) 0)4)






40 0p





o *4



rzI40i 0
0) (1)






J OC)
zo0
rC 4-)
oJ C '4i




-0 0) 0
000


N >4
*H -r 0) ,
In rl4J 4-)
0- 0 )H-H



0 (1) t
0)O 0
O-O0) )c





44 -P 00


(44 M 0 C 0)
*P i 01






cO




&-H










































N 0


(A/A%) IONVH13


(A/5%) 3SOonslO


(lW/8w) NI310id 1130






























Figure 9.


Effect of added magnesium on the rate
of fermentation. Fermentation rates
were measured by respirometry of
unwashed cells immediately after
sampling and are plotted as a function
of accumulated ethanol for four
separate batch fermentations. Closed
symbols represent cultures
supplemented with 0.5 mM MgSO4 and
open symbols represent control
fermentations in YEPD broth alone.
















60-



I--
x 40
Z E



z- O
Wo


E
0o o



0 '

0 2 4 6 8 10 12
ETHANOL (%v/v)











maintained a higher rate of fermentation as ethanol

accumulated during the completion of the batch fermentation.

This rate was 40% higher than that of control cells after

the accumulation of 8% (vol/vol) ethanol. The fermentation

rate of both supplemented and unsupplemented cultures fell

precipitously at about 12.5% (vol/vol) ethanol, coincident

with exhaustion of glucose.

Effect of Magnesium Addition on Cell Viability

The percentage of viable cells in both magnesium-

supplemented and unsupplemented batches remained greater

than 90% for the first 48 h of the fermentation. Glucose

was exhausted at this time in supplemented cultures and the

percentage of viable cells began to decrease, reaching 58%

by 72 h. The unsupplemented batches consumed glucose more

slowly and maintained high viability (>90%) until between 60

and 72 h, the time at which glucose was exhausted.

Effect of Ethanol on the Fermentation Rate of Magnesium-
Supplemented Cultures

Two points during fermentation were chosen at which to

compare cells grown with and without added magnesium. These

were the same two points described in chapter III as 12-h

and 24-h cells. Cells at 12 h were still undergoing

exponential growth and cells at 24 h were in early

stationary phase.

Figure 10A shows the dose-response of fermentative

activity of the younger cells plotted as a function of total

ethanol concentration endogenouss plus added). Magnesium-











>1

Q) () o

4aO 10 c0
4-M

OH O1O OP
*-i 0 4. 4Ji 0 0
(d 0) QU) A




> -V 0 -4
-4 (L) +4J A
.)' -,-

-0 44. 94 ,

r



Sa)4 -. 0-
>0 d 4 0



C Or -4-
**HQ U l
*H-- ) A3 F. 0)


C -0H o r
0 )0 1- 4 p




















e-
,o H
1 U) CO
0l to0 O
C 4 rr 0 U
U) -aHO e
0 0) 1 4)
-H W) 04-)


g> > *
ar M MI OO
a0im r .oo
P4 A > > >-




m' 0 r-i (0
0 -A 0 W 4-)

P 4-) > c C

(OUN rt3

0 0 44
a) c w ra r-i a)




U410 Q)1 (Z r:







-.4



FZ4











































0 0 0 0 0 0
o co cNj
(oIeJ IPi!u! %) ,lAlAIlOV 3AIlViN31IN3_


0 0 0 0 0
o0 CO v N C
(alej I!l!u! %) A111AIOV 3AIlViN3VIU3-


WDZ

I
I-
CO


78


-i
0
Z
"l-

I-
Lw











supplemented and unsupplemented cells had a similar initial

fermentation rate, about 57 moles of CO2 produced per h per

mg cell protein and exhibited identical dose-response

curves. A concentration of 7.6% (vol/vol) ethanol resulted

in 50% inhibition of fermentative activity.

Figure 10B shows the effect of ethanol on the

fermentation rate of the older cells. Supplemented cells

had an initial fermentation rate of 27.4 pmoles of CO2

produced per h per mg cell protein with an SD of 0.5, while

unsupplemented cells exhibited a significantly lower rate,

22.9 pmoles of CO2 produced per h per mg cell protein with

an SD of 1.2. The fermentation rate of cells from

supplemented cultures was always higher than that of control

cells. The amount of ethanol present in the supplemented

culture was 3.0% (vol/vol) higher than in the unsupplemented

culture when compared at equal fermentation rates. The

fermentation rate of magnesium-supplemented and control

fermentations exhibited linear dose-responses to ethanol

addition. A measure of the sensitivity of fermentation rate

to ethanol is the slope of the dose-response curve. The

slope for supplemented batches was -0.17 with an SD of 0.02,

while that of the controls was -0.14 with an SD of 0.01.

The differences in these slopes (Fig. 10B) are suggestive,

but do not conclusively demonstrate that supplementation

with magnesium reduced the sensitivity of fermentation in

older cells to inhibition by ethanol.











Discussion

The decline in fermentation rate that begins at low

alcohol concentrations does not seem to be exclusively

caused by the immediate presence of ethanol, by growth in

the presence of ethanol or by cell death (Chapter III).

This decline appears to be related in part to a magnesium

deficiency, although other factors are involved also. In

yeast extract-peptone-based medium, a magnesium deficiency

is developed which limits cellular growth and the rate of

carbohydrate conversion into ethanol. The addition of

magnesium to batch fermentations prolonged exponential

growth, allowing greater accumulation of cell mass without

affecting cell viability. In addition, cells in magnesium-

supplemented cultures maintained a higher fermentation rate

as ethanol accumulated. These two factors, increased cell

mass plus higher fermentation rate, combined to reduce the

time required for the conversion of 20% glucose to ethanol

by one-third in magnesium-supplemented cultures.

During batch fermentation, yeast cells concentrated

magnesium from the medium. In unsupplemented cultures,

magnesium uptake stopped at the end of exponential growth.

At this point, the concentration of magnesium in the medium

was 48 pM, within the range of Km values reported for

magnesium transport by microorganisms (Jasper and Silver,

1977). The end of exponential growth also coincided with

the beginning of the decline in fermentative activity. In











magnesium-supplemented cultures, higher levels of

intracellular magnesium were achieved early in fermentation

and decreased to a lesser extent than observed in

unsupplemented cultures. Magnesium-supplemented cultures

had 6.5 times more magnesium in the medium at the end of

exponential growth than did unsupplemented cultures. Thus,

the magnesium supply of the supplemented culture appears to

be adequate for growth and other factors are limiting the

fermentative ability of the yeasts under these conditions.

The ubiquitous role of magnesium in cellular processes

is well documented (Jasper and Silver, 1977). Magnesium

constitutes a major portion of the cellular cations, mostly

bound in structures such as ribosomes and the cell envelope.

The free cation concentration, however, may play a more

direct role in regulating overall cellular metabolism and

cell division (Walker and Duffus, 1980). Many of the

enzymes that function in DNA replication, transcription and

translation require magnesium for activity. In fermentation

pathways, magnesium is a required cofactor and nucleotide

counter-ion in many reactions. Magnesium levels typically

are maintained at mmolar intracellular concentrations and it

is not surprising that this cation is a limiting nutrient

during high-gravity fermentations.

Previous studies have demonstrated that the inhibition

of fermentation by added ethanol in Zymomonas mobilis is

primarily due to ethanol-induced leakage, particularly of











magnesium (Osman and Ingram, 1985). The addition of

magnesium salts at 0.5 mM substantially reversed the

inhibitory effects of up to 13% (vol/vol) ethanol. Although

analogous studies have not been performed with S.

cerevisiae, it is likely that ethanol also increases the

leakage of small molecules in this organism.

Casey et al. (1984) also have reported that nutrient

limitation is an important factor in limiting the

productivity of a fermentation. Supplementation of high-

gravity brewing wort (containing up to 31% dissolved solids)

with yeast extract, ergosterol and oleic acid allowed the

production of 16.2% (vol/vol) ethanol by brewers' yeast.

Higher rates of alcohol production primarily resulted from

an increase in cell mass associated with nutrient-

supplemented fermentations and did not appear to include an

increase in the resistance of fermentation rate to ethanol.

Addition of nutrients in the form of soy flour to

fermentation broth has been shown to increase the

fermentative productivity of both S. cerevisiae (Damiano and

Wang, 1985) and Z. mobilis (Ju et al., 1983). Viegas et al.

(1985) also reported that soy flour addition to a yeast

extract-based medium containing 30 to 40% glucose enhanced

the rate of ethanol production by S. bayanus. Again,

supplementation led to an increase in cell concentration.

It was further demonstrated that the aqueous fraction of soy

flour, rather than the lipid fraction, contained the











components beneficial for fermentation. This aqueous

fraction would have included inorganic ions, such as

magnesium. Indeed, it is possible that many of the complex

nutrient additives used to increase ethanol production also

are correcting an inorganic ion deficiency.

The causes of the progressive decline in fermentative

activity which is observed as ethanol accumulates during

batch fermentation appear to be much more complicated than

expected. The results presented in this chapter indicate

that direct ethanol inhibition is only partially

responsible. A nutrient limitation for magnesium also

appears to be partially responsible. With abundant

magnesium, only a 50% further increase in cell mass was

observed, indicating that another factors) becomes limiting

for growth and fermentation at this point. Indeed, a

complete understanding of the biochemical basis for the

decline in fermentation rate in yeasts may require

determination of the factors responsible for the termination

of exponential growth and the associated physiological and

enzymatic changes.















CHAPTER V
GLYCOLYTIC ENZYMES AND INTERNAL pH


Introduction

Saccharomyces cerevisiae is capable of very rapid rates

of glycolysis and ethanol production under optimal

conditions, producing over 50 pmoles of ethanol per h per mg

of cell protein (Fig. 9). However, this high rate is

maintained for only a brief period during batch fermentation

and declines progressively as ethanol accumulates in the

surrounding broth (Casey and Ingledew, 1986; Ingram and

Buttke, 1984; Moulin et al., 1984). Earlier studies have

identified a requirement for lipids (Beaven et al., 1982;

Casey et al., 1984; Thomas et al., 1978) or molecular oxygen

for lipid biosynthesis (Andreasen and Stier, 1954; Buttke et

al., 1980; Buttke and Pyle, 1982) in many fermentation

broths as being essential for the maintenance of high

fermentative activity. Magnesium is an essential cofactor

for many of the glycolytic enzymes and has been identified

also as a limiting nutrient in fermentation broth containing

peptone and yeast extract (Chapter IV). Supplying these

nutritional needs reduces but does not eliminate the decline

in fermentative activity during batch fermentation (Fig. 9).











The basis for the decline in fermentation rate is not

fully understood. Since the addition of ethanol to cells in

batch cultures and in chemostats causes a dose-dependent

inhibition of ethanol production (Casey and Ingledew, 1986;

Fig. 10), most investigations have focused on ethanol as the

inhibitory agent (Casey and Ingledew, 1986; Ingram and

Buttke, 1984; Millar et al., 1982). Ethanol is known to

alter membrane permeability and disrupt membrane function in

a variety of biological systems (Casey and Ingledew, 1986;

Ingram and Buttke, 1984). In yeast, ethanol causes an

increase in hydrogen ion flux across the plasma membrane of

cells suspended in water (Cartwright et al., 1986). This

increased hydrogen ion flux has been proposed as being

responsible for the ethanol-induced decline in transport

rates observed under similar conditions (Beaven et al.,

1982; Leao and van Uden, 1982b, 1984a, 1984b).

Evidence has been accumulating which indicates that the

presence of ethanol may not be the only factor responsible

for the decline in fermentative activity. The replacement

of fermentative broth containing ethanol with fresh medium

lacking ethanol did not immediately restore fermentative

activity (Table 1). In a comprehensive study, Millar et al.

(1982) demonstrated that ethanol concentrations below

12% (vol/vol) do not denature glycolytic enzymes or cause

appreciable inhibition of activity in vitro under substrate-

saturating conditions. Since ethanol does not accumulate











within yeast cells, but rapidly diffuses across the cell

membrane (Dasari et al., 1985; Guijarro and Lagunas, 1984;

Table 2), direct inhibition of glycolytic enzymes by

intracellular ethanol is unlikely during fermentations which

produce 12% (vol/vol) ethanol or less.

In this chapter, changes in the amounts of glycolytic

and alcohologenic enzymes, and internal pH and membrane

energization have been examined as possible physiological

causes for the decline in fermentative activity during batch

fermentations of 20% glucose in a yeast extract-peptone-

based medium supplemented with magnesium.

Materials and Methods

Organism and Growth Conditions

The petite yeast strain characterized in chapter II, S.

cerevisiae KD2, was used in the studies presented in this

chapter. This yeast was grown in the complex medium also

described in chapter II, except that the medium was

supplemented with 0.5 mM MgSO4, as described in chapter IV.

Batch fermentations were carried out as outlined in

chapter II.

Analytical Methods

Cell mass, glucose, ethanol and fermentation rates all

were determined by the methods discussed in chapter II.

Cell protein was measured as described by Lowry et al.

(1951). The protein content of cell extracts was

quantitated using the method of Bradford (1976). Bovine











serum albumin served as the protein standard for both

methods.

Enzyme Analyses

Activities of glycolytic and alcohologenic enzymes were

determined in 2-ml samples removed at various times during

batch fermentation. Cells were harvested by centrifugation

at 10,000 x g for 30 sec at 40C and washed in an equal

volume of 50 mM potassium phosphate buffer (pH 7.4). All

subsequent steps were carried out at 40C. The pellet was

suspended in the same buffer containing 2 mM mercaptoethanol

and 2 mM EDTA, and disrupted with 0.1-mm glass beads using a

Mini-Bead Beater (Biospec Products, Bartlesville, Okla.).

Five 1-min periods of disruption, each were followed by 5-

min periods of cooling on ice. Cell debris was removed by

centrifugation at 10,000 x g for 5 min, and the supernatant

was assayed immediately for enzymatic activities. Only two

enzymes at a time were assayed in each batch fermentation

experiment to avoid problems which could result from storage

of cells or extracts.

Pyruvate decarboxylase and all glycolytic enzymes were

assayed spectrophotometrically by the methods of Maitra and

Lobo (1971) as modified by Clifton et al. (1978). All

enzymes were assayed under substrate-saturating conditions

except triose phosphate isomerase, which was assayed with

1 mM substrate. The amounts of coupling enzymes were

adjusted as needed to ensure a linear reaction rate.











Alcohol dehydrogenase was assayed by measuring the oxidation

of ethanol as described by Maitra and Lobo (1971), but using

a buffer at pH 8.7 containing 75 mM sodium pyrophosphate,

75 mM semicarbazide hydrochloride and 21 mM glycine (Bernt

and Gutman, 1971).

Determination of Internal pH and Membrane Energization

The measurements of internal pH and A were

performed using 7-[14C]benzoic acid and [3H-

phenyl]tetraphenylphosphonium bromide, respectively.

Protocols were similar to those described by Cartwright et

al. (1986) except that cells were incubated in their native

growth medium rather than distilled water and 0.4-pm pore

size polycarbonate filters were used instead of mixed

cellulose ester filters. Cell volumes were determined as

described in chapter III. As a control for adventitious

binding of radioactive compounds, cells were permeabilized

with a combination of ethanol, toluene and Triton X-100 as

described by Salmon (1984), washed with 50 mM phosphate

buffer, resuspended in native broth and processed. This

treatment resulted in a complete collapse of A pH and loss

of membrane potential. Calculations were performed as

described by Rottenberg (1979).

Materials

Yeast extract, peptone and agar were obtained from

Difco Laboratories, Detroit, Mich. Glucose, coupling

enzymes, coenzymes and substrates were purchased from Sigma











Chemical Co., St. Louis, Mo. Inorganic salts were obtained

from Fisher Scientific Co., Orlando, Fla. Absolute ethanol

was supplied by AAPER Alcohol and Chemical Co., Shelbyville,

Ky. Radioactive compounds were purchased from New England

Nuclear Corp., Boston, Mass.

Results

Reversibility of the Decline in Fermentative Activity

In chapter IV, the ability of magnesium supplementation

to partially relieve the decline in fermentative activity

associated with the accumulation of ethanol was

demonstrated. After supplementation, immediately reversible

inhibition by accumulated ethanol may be responsible for the

remaining decrease in fermentation rate. To investigate

this possibility, cells were removed at various times during

batch fermentation and the rate of ethanol production per mg

cell protein was determined (Fig. 11). Cells were most

active at the earliest times measured, 12 h, and

fermentation rate declined by 50% when 6.5% (vol/vol)

ethanol had accumulated after 24 h. Approximately 40% of

the fermentative activity was retained after the

accumulation of 10% (vol/vol) ethanol with 30 g glucose

per L remaining in the fermentation broth. The abrupt,

final decline in activity reflects the near-complete

exhaustion of glucose. Removal of ethanol from cells by

washing and suspending in fresh medium resulted in only a

modest increase in fermentative activity in all but the




























Figure 11. Effect of ethanol removal on the
fermentative activity of cells grown
in YEPD medium containing 0.5 mM
MgSO4. Cells were sampled during
batch fermentation and were either
untreated or washed once and then
suspended in fresh medium containing
20% glucose. The fermentation rate of
these samples were measured
immediately by respirometry. Symbols:
0 activity measured in native
broth; 0 activity measured after
cells were suspended in fresh medium.
























60



/

cr 40 4
z E



tW "20

J\

=L
O I\
2 6 10 14
ETHANOL (%V/V)




Full Text
41
Table 2. Intracellular and extracellular ethanol
concentrations under various conditions
Ethanol concentration (% vol/vol) (SD) in
different media3
Fresh
Sample Native Native Fresh + 10%
+ KF ethanol
12b
Int
1.9
(0.4)
1.4
(0.2)
0.6
(0.1)
6.9
(1.5)
Ext
1.7
(0.1)
1.3
(0.1)
0.4
(0.1)
10.7
(1.1)
b
Int
3.4
(0.7)
3.7
(0.7)
0.7
(0.1)
8.1
(1.3)
Ext
5.5
(0.1)
5.1
(0.1)
0.5
(0.1)
9.7
(0.1)
3 Three or more independent determinations. Native refers
to the broth in the batch fermentation with or without added
KF (50 mM). Fresh refers to sterile, unused medium with or
without added ethanol (10% (vol/vol)).
b Age of batch fermentation. Int and Ext refer to the
intracellular and extracellular ethanol concentrations,
respectively.


149
including carrier molecules and hexose-phosphorylating
proteins, or a decrease in transport rate brought on by
membrane lipid changes during fermentation. A constant rate
of facilitated diffusion would suggest that inactivation or
membrane lipid changes may not play an important role in
lowering the levels of glycolytic intermediates. Rather,
inhibition of the hexose kinase reactions by the large
increase in AMP levels and the decrease in ATP concentration
during fermentation may be a more reasonable explanation
(Fig. 20). The hexokinase activity of S. cerevisiae KD2 in
the presence of varying ATP and AMP concentrations could be
examined to confirm an inhibitory effect of high levels of
AMP on the glucose phosphorylating activity of this yeast
strain in vitro. Subsequent studies also might include
determining the origin of the AMP that accumulates during
fermentation and examining its general role in regulating
metabolism as cell growth slows.
From the data presented in this study, a hypothetical
model describing the cellular changes that occur as
fermentation rate declines can be formulated. As yeast
cells ferment 20% glucose, approximately 1% (vol/vol)
ethanol is produced during exponential growth (Fig. 1). As
the rate of growth slows, so does macromolecular synthesis
(Boucherie, 1985; Chapman and Atkinson, 1977). The
physiological character of the yeast cells changes with
changes in macromolecular composition (Busturia and Lagunas,


THE DECLINING RATE OF ETHANOL PRODUCTION
DURING BATCH FERMENTATION BY SACCHAROMYCES CEREVISIAE
BY
KENNETH MICHAEL DOMBEK
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA


Figure 5. Inhibition of fermentation rate of 12-h and 24-h cells by added ethanol.
Cells were harvested by centrifugation and suspended in fresh medium
containing various concentrations of ethanol. (A) Decrease in fermentation
rate as a function of added ethanol. (B) Inhibition as a percentage decrease
of the control rate lacking ethanol. Symbols: 12-h cells; 24-h
cells.


128
then glycolytic intermediates should accumulate as
fermentation rate declines. By 18 h, however, the amount of
total phosphorylated intermediates declined by 50% (Fig.
17A), indicating that the glycolytic capacity of the cells
had increased faster than their carbon uptake capacity.
Alternatively, decreasing rates of carbon uptake with a
constant glycolytic capacity could account for this
observation. The level of intermediates began to increase
by 24 h. By 42 h, just before glucose in the medium was
exhausted, the level increased back to that at 12 h. The
increasing levels of intermediates after 18 h suggests that
by this time, one or more of the glycolytic enzyme
activities are restricting carbon flow through the pathway.
In figure 17B, total glycolytic intermediates are
divided into hexose phosphates and trise phosphates to
examine whether the increase in total intermediates resulted
from restricted flow through the hexose or trise parts of
the pathway. Consistent with the observed decrease in total
intermediates, the amount of hexose phosphates dropped by
50% and the level of trise phosphates fell by 30% after
18 h. By 36 h, the level of trise phosphates gradually
rose to 130% of the 12 h value while the level of hexose
phosphates remained low, at about 50% of the 12 h value. As
the level of trise phosphates rapidly fell after 36 h, the
level of hexose phosphates increased to 120% of the 12 h
value. These observations suggest that carbon flow through


122
Thompson (1978). Their migration positions are marked on
the chromatogram in figure 16 by the small arrows. Finally,
fermentation extracts that had been dried at 4C under
vacuum to remove the ethanol from the sample were treated
with various glycolytic enzymes. Samples were suspended in
the enzyme buffer described by Maitra and Lobo (1971) and
treated with pyruvate kinase and fructose 1,6-bisphosphate
aldolase at room temperature for 15 min. Reaction mixtures
were those described by Clifton et al. (1978). Untreated
and treated samples were chromatographed under identical
conditions. Aldolase treatment caused the disappearance of
a portion of the spot identified as hexose phosphates and
increased the intensity of the trise phosphate spot.
Pyruvate kinase treatment had little or no effect on the
intensity of the inorganic phosphate-phosphoenolpyruvate
spot, however, a portion of the large spot associated with
nucleotides increased in intensity. This suggests that the
phosphoenolpyruvate spot was poorly separated from the
inorganic phosphate spot (Fig. 16) and that ATP migrated
with the nucleotide phosphate spot. Thus, measurements of
phosphoenolpyruvate were not included in values reported for
total phosphorylated glycolytic intermediates and trise
phosphates.
Quantitation of Adenylate and Nicotinamide Nucleotides in
Fermentation Extracts
Amounts of the individual adenine nucleotides in the
fermentation extracts were determined by firefly luciferase


58
to ethanol inhibition of alcohol dehydrogenase and
hexokinase activities.
Casey et al. (1984) have reported that nutrient
limitation is a major factor restricting the ethanol
productivity of high-gravity fermentations. Anaerobically
cultured yeasts are known to have a nutritional requirement
for ergosterol and unsaturated lipids (Hossack and Rose,
1976; Nes et al.. 1978; Proudlock et al.. 1968).
Unsaturated lipids have been shown to increase biomass,
alcohol production and ethanol durability of yeast cells
during anaerobic fermentation (Ingram and Buttke, 1984;
Janssens et al.. 1983; Lafon-Lafourcade et al.. 1979; Thomas
et al.. 1978). A variety of lipid-protein complexes and
nutrient supplements, ranging from albumin-ergosterol-
monoolein to soy flour and yeast extract, also have been
shown to yield increased rates of alcohol production and
higher final ethanol concentrations (Damiano and Wang, 1985;
Hayashida et al.. 1976; Lafon-Lafourcade et al.. 1979; Ohta
and Hayashida, 1983).
The studies described in chapter III suggest that the
initial decline in fermentative activity during batch
fermentation of 20% glucose is not caused by the presence of
ethanol or by growth in the presence of 5% (vol/vol)
ethanol. These studies indicated that a component(s) of
yeast extract was limiting cell growth and that this
limitation contributed to the early loss of fermentative


Ill
membrane becomes more energized as ethanol accumulates in
the fermentation broth.
Cells from the later stages of fermentation were more
resistant to inhibition by ethanol and to the disruptive
effects of ethanol on membrane integrity as measured by
proton leakage. A greater resistance to the ethanol-induced
decrease in A pH may result from membranes with stronger
barrier properties or from ion pumps that become less
sensitive to inhibition by ethanol as fermentation
progresses. During batch fermentation, yeast cells may be
undergoing progressive adaptations to accumulated ethanol.
Changes in lipid composition of yeast cell membranes have
been observed in response to accumulated ethanol and have
been proposed as an important factor involved in such
adaptation (Beaven et al.. 1982; Casey and Ingledew, 1986;
Ingram and Buttke, 1984). Alterations in lipid biosynthesis
when yeast cell growth stops also change the membrane
phospholipid composition (Homann et al.. 1987). In light of
the results reported in chapter III, these growth phase-
dependent lipid changes may be the actual cause of ethanol-
resistant A pH rather than growth in the presence of
ethanol.
Although the internal pH remained almost neutral and
the activities of glycolytic and alcohologenic enzymes
assayed in vitro remained high during batch fermentation,
the in vivo activities of these enzymes within the cell


39
the principle volatile component responsible for this
inhibition.
Cell Viability and Overcrowding Effects on Fermentation Rate
A trivial possibility for the failure of 24-h cells to
recover activity after suspension in broth lacking ethanol
would be the presence of large numbers of dead cells.
However, based on methylene blue dye exclusion, over 90% of
the yeast cells appeared active and intact at 24 h. Another
trivial possibility for the failure of 24-h cells to recover
activity after suspension in fresh medium is that by 24 h,
the cells are so crowded that they can no longer efficiently
take up nutrients and glucose for conversion to ethanol.
This possibility was addressed by suspending 24-h cells in
fresh medium at different cell concentrations and measuring
the rate of C02 production by respirometry. The rate of C02
production increased linearly with increasing cell
concentrations. At a cell concentration comparable to that
of 12-h cells, the fermentation rate of 24-h cells was only
half that of 12-h cells (data not shown).
Intracellular Ethanol Concentration
The failure of 24-h cells to recover activity after
suspension in fresh medium could be caused by the failure of
the suspension procedure to effectively remove the
intracellular ethanol or by the accumulation of large
amounts of intracellular ethanol that could permanently
damage the fermentative capacity of the cells. To explore


4
and fermentation rates. Because such investigations have
shown that ethanol does inhibit yeast growth and
fermentation, many studies have dealt with characterizing
these inhibitory effects (Brown et al.. 1981; Hoppe and
Hansford, 1982? Jones and Greenfield, 1985; Lafon-Lafourcade
and Ribereau-Gayon, 1984; Vega et al. 1987).
The inhibitory effect of ethanol on the rate of sugar
conversion to ethanol was first quantitated by Rahn (1929).
He demonstrated an inverse relationship between the amount
of fermentation product, including ethanol, added to the
medium and the rate of its production by determining the
amount of heat evolved when sucrose was converted to
ethanol. Also, as ethanol accumulated in the medium, the
rate of fermentation declined. This represents some of the
original evidence that ethanol inhibits alcohol production
by yeast.
Recent studies also have attempted to quantitatively
describe the inhibitory effect of ethanol on product
formation during yeast fermentation (Luong, 1985; van Uden,
1985). Holtzberg et al. (1967) examined grape juice
fermentation by S. cerevisiae var. elipsoideus and
calculated an inverse linear relationship between the rate
of alcohol production and the amount of ethanol in the
juice. Similar findings were made by Ghose and Tyagi (1979)
for the batch fermentation of cellulose hydrolysate by S.
cerevisiae NRL y-132, however, the constants in the equation


103
of yeast suspended in water to hydrogen ions (Cartwright et
al.. 1986). To further examine this point, the effect of
added ethanol on the internal pH of cells from various
stages of fermentation was measured (Fig. 15). Ethanol
concentrations of 15% (vol/vol) or above were required to
cause a measurable decrease in internal pH. The addition of
20% (vol/vol) ethanol to 12-h and 24-h cells caused a
complete collapse of A pH. The A pH of cells from 36-h
and 48-h cultures was considerably less affected by 20%
(vol/vol) added ethanol, consistent with adaptation of the
older cells.
Discussion
The fermentation of glucose to ethanol represents a
series of coordinated enzymatic reactions. This process is
internally balancing and thermodynamically favorable
provided that cellular enzymes consume the net ATP generated
from substrate-level phosphorylation. The requirements for
this process include glucose, functional enzymes, coenzymes
(NAD+, thiamine pyrophosphate, ADP, ATP), cofactors (Mg2+,
Zn2+) appropriate internal pH, a functional membrane to
maintain the concentration of reactants and enzymes, and a
glucose uptake system. Indeed, fermentation can proceed
well in concentrated preparations of disrupted cells
(Harden, 1923; Welch and Scopes, 1985).
The rate of glycolysis in viable yeast cells, however,
declines as ethanol accumulates during batch fermentation.


115
The cell cytoplasm also contains many effector
molecules which modulate the activities of enzymes in
response to changes in extracellular environmental
conditions. The activities of some of the yeast glycolytic
enzymes are known to be controlled by allosteric effector
molecules and the availability of cofactors and coenzymes
(Sols et al.. 1971). Glucose phosphorylation by hexokinases
I and II and glucokinase requires ATP and Mg2+ (Colowick,
1973) and glyceraldehyde-3-phosphate dehydrogenase requires
NAD+ for activity (Harris and Waters, 1976). Pyruvate
kinase activity is regulated by the availability of fructose
1,6-bisphosphate (Hess et al.. 1966; Maitra and Lobo, 1977)
and requires ADP, K+ and Mg2+ (Kayne, 1973).
Phosphofructokinase, which synthesizes fructose 1,6-
bisphosphate, has many allosteric regulators. These include
fructose 2,6-bisphosphate (Francois et al.. 1984), ammonium
and citrate (Bloxham and Lardy, 1973; Uyeda, 1979). This
enzyme activity also is modulated by energy charge with ATP
acting as a strong inhibitor, and AMP and fructose-6-
phosphate reversing the inhibition (Betz and Moore, 1967).
If the availability of required cofactors, coenzymes and
allosteric activators becomes limited or if large amounts of
inhibitors build up intracellularly, then the rate of
glycolysis would be expected to decrease.
One method of determining which part of the
fermentative pathway is restricting carbon flow to ethanol


the initial 50% decline in fermentative activity. Further
studies will include identification of this limiting
nutrient and, upon supplementation, characterization of its
effect on growth and fermentation.


163
Pena, A., G. Cinco, A. Gomez-Puyou and M. Tuena. 1972.
Effect of the pH of the incubation medium on glycolysis
and respiration in Saccharomvces cerevisiae. Arch.
Biochem. Biophys. 153:413-425.
Proudlock, J.W., L.W. Wheeldon, D.J. Jollow and A.W.
Linnane. 1968. Role of sterols in Saccharomvces
cerevisiae. Biochim. Biophys. Acta 152:434-437.
Putrament, A., H. Baranowska and W. Prazmo. 1973. Induction
by manganese of mitochondrial antibiotic resistance
mutations in yeast. Mol. Gen. Genet. 126:357-366.
Raabo, E. and T.C. Terkildson. 1960. On the enzymatic
determination of blood glucose. Scand. J. Lab. Invest.
12:402-182.
Rahn, 0. 1929. The decreasing rate of fermentation. J.
Bacteriol. 18:207-226.
Richards, O.W. 1928. Potentially unlimited multiplication of
yeast with constant environment and the limiting of growth
by changing environment. J. Gen. Physiol. 11:525-538.
Rottenberg, H. 1979. The measurement of membrane potential
and pH in cells, organelles and vesicles. Methods
Enzymol. 55:547-569.
Rowe, E.S. 1983. Lipid chain length and temperature
dependence of ethanol-phosphatidylcholine interactions.
Biochemistry 22:3299-3305.
Rudolph, F.B. and H.J. Fromm. 1971. Computer simulation
studies with yeast hexokinase and additional evidence for
the random bi bi mechanism. J. Biol. Chem. 246:6611-6619.
Sa-Correia, I. and N. van Uden. 1983. Temperature profiles
of ethanol tolerance: effects of ethanol on the minimum
and the maximum temperatures for growth of the yeasts
Saccharomvces cerevisiae and Kluvveromvces fraqilis.
Biotechnol. Bioeng. 25:1665-1667.
Salmon, M. 1984. Application of the technique of cellular
permeabilization to the study of enzymatic activities of
Saccharomvces cerevisiae in continuous alcoholic
fermentation. Biotechnol. Lett. 6:43-48.
Schlenk, F. 1985. Early research on fermentation A story
of missed opportunities. Trends in Biochem. Sci. 10:252-
254.


Terkildsen, 1960) using the Glucostat reagents supplied by
the Sigma Chemical Company (St. Louis, Mo.). In later
experiments, glucose was measured with a YSI model 27
glucose analyzer (YSI, Yellow Springs, Oh.) Cell mass was
measured as optical density at 550 nm with a Bausch and Lomb
Spectronic 70 spectrophotometer and as total cell protein by
the method of Lowry et al. (1951) as described by Layne
(1957) .
Respirometrv Measurements
Samples were pipetted into Warburg flasks and
equilibrated for 10 min at 30C. During the first 5 min of
the equilibration period, the flasks were flushed with
nitrogen gas. Rates of C02 production were measured with a
differential respirometer (Gilson, Middleton, Wis.). These
values were used to calculate fermentation rates as pmoles
of C02 evolved per mg of cell protein. The rate of C02
evolution was independent of sample volume, up to 4 ml, and
linearly increased with cell concentration, up to 5 mg cell
protein per ml.
Chemicals
Yeast extract, peptone and agar were obtained from
Difco Laboratories, Detroit, Mich. Glucose and other
biochemicals were obtained from Sigma Chemical Co. Acetone
and inorganic salts were purchased from Fisher Scientific
Company, Orlando, Fla. Absolute ethanol was supplied by
AAPER Alcohol and Chemical Co., Shelbyville, Ky. Gas


FERMENTATION RATE
o
(pmoles C02/h per mg protein)
ro $>.
o o
CD
o
T
VO


161
Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall.
1951. Protein measurement with the folin phenol reagent.
J. Biol. Chem. 193:265-275.
Luong, J.H.T. 1985. Kinetics of ethanol inhibition in
alcohol fermentation. Biotechnol. Bioeng. 27:280-285.
Maiorella, B., H.W. Blanch and C.R. Wilke. 1983. By-product
inhibition effects on ethanol fermentation by
Saccharomvces cerevisiae. Biotechnol. Bioeng. 25:103-121.
Maitra, P.K. and Z. Lobo. 1971. A kinetic study of
glycolytic enzyme synthesis in yeast. J. Biol. Chem.
246:475-488.
Maitra, P.K. and Z. Lobo. 1977. Yeast pyruvate kinase: A
mutant form catalytically insensitive to fructose 1,6-
bisphosphate. Eur. J. Biochem. 78:353-360.
Millar, D.G., K. Griffiths-Smith, E. Algar and R.K. Scopes.
1982. Activity and stability of glycolytic enzymes in the
presence of ethanol. Biotechnol. Lett. 4:601-606.
Mills, D.R. 1941. Differential staining of living and dead
yeast cells. Food Res. 6:361-371.
Moulin, G., H. Boze and P. Galzy. 1980. Inhibition of
alcoholic fermentation by substrate and ethanol.
Biotechnol. Bioeng. 22:2375-2381.
Moulin, G., H. Boze and P. Galzy. 1981. A comparative study
of the inhibitory effect of ethanol and substrates on the
fermentation rate of parent and a respiratory-deficient
mutant. Biotechnol. Lett. 3:351-356.
Moulin, G., H. Boze and P. Galzy. 1984. Inhibition of
alcoholic fermentation. Biotechnol. Genet. Eng. Rev.
2:365-382.
Nagodawithana, T.W., C. Castellano and K.H. Steinkraus.
1974. Effect of dissolved oxygen, temperature, initial
cell count and sugar concentration on the viability of
Saccharomvces cerevisiae in rapid fermentations. Appl.
Env. Microbiol. 28:383-391.
Nagodawithana, T.W. and K.H. Steinkraus. 1976. Influence of
the rate of ethanol production and accumulation on the
viability of Saccharomyces cerevisiae in "rapid
fermentation". Appl. Environ. Microbiol. 31:158-162.


Figure 6. Dose-response of cell growth to added
magnesium. Magnesium values represent
the amount of magnesium contained in
the added nutrient supplement. Error
bars represent the average standard
deviation for each experiment.
Symbols; whole yeast extract
added to glucose-reconstituted, used
medium; O ashed yeast extract
added to glucose-reconstituted, used
medium; # MgS04 added to
glucose-reconstituted, used medium;
MgS04 added to fresh YEPD broth.


160
Lagunas, R., C. Dominguez, A. Busturia and M.J. Saez. 1982.
Mechanisms of appearance of the Pasteur effect in
Saccharomvces cerevisiae: Inactivation of the sugar
transport systems. J. Bacteriol. 152:19-25.
Larue, F., S. Lafon-Lafourcade and P. Ribereau-Gayon. 1984.
Relationship between the inhibition of alcoholic
fermentation by Saccharomvces cerevisiae and the
activities of hexokinase and alcohol dehydrogenase.
Biotechnol Lett. 6:687-692.
Layne, E. 1957. Spectrophotometric and turbidimetric methods
for measuring proteins. Methods Enzymol. 3:447-454.
Leao, C. and N. van Uden. 1982a. Effects of ethanol and
other alkanols on the kinetics and the activation
parameters of thermal death in Saccharomvces cerevisiae.
Biotechnol. Bioeng. 24:1581-1590.
Leao, C. and N. van Uden. 1982b. Effects of ethanol and
other alkanols on the glucose transport system of
Saccharomvces cerevisiae. Biotechnol. Bioeng. 24:2601-
2604.
Leao, C. and N. van Uden. 1984a. Effects of ethanol and
other alkanols on passive proton influx in the yeast
Saccharomvces cerevisiae. Biochim. Biophys. Acta 774:43-
48.
Leao, C. and N. van Uden. 1984b. Effects of ethanol and
other alkanols on the general amino acid permease of
Saccharomvces cerevisiae. Biotechnol. Bioeng. 26:403-405.
Lenaz, G., G. Curatola, L. Mazzanti and G. Parenti-
Castelli. 1978. Biophysical studies on agents affecting
the state of membrane lipids: Biochemical and
pharmacological implications. Mol. Cell. Biochem. 22:3-32.
Lopez, S. and J.M. Gancedo. 1979. Effect of metabolic
conditions on protein turnover in yeast. Biochem. J.
178:769-776.
Loureiro-Dias, M.C. and J.D. Arrabaca. 1982. Flow
microcalorimetry of a respiratory-deficient mutant of
Saccharomvces cerevisiae. Z. Allg. Mikrobiol. 22:199-122.
Loureiro, V. and N. van Uden. 1982. Effects of ethanol on
the maximum temperature for growth of Saccharomvces
cerevisiae: A model. Biotechnol. Bioeng. 24:1881-1884.


83
components beneficial for fermentation. This aqueous
fraction would have included inorganic ions, such as
magnesium. Indeed, it is possible that many of the complex
nutrient additives used to increase ethanol production also
are correcting an inorganic ion deficiency.
The causes of the progressive decline in fermentative
activity which is observed as ethanol accumulates during
batch fermentation appear to be much more complicated than
expected. The results presented in this chapter indicate
that direct ethanol inhibition is only partially
responsible. A nutrient limitation for magnesium also
appears to be partially responsible. With abundant
magnesium, only a 50% further increase in cell mass was
observed, indicating that another factor(s) becomes limiting
for growth and fermentation at this point. Indeed, a
complete understanding of the biochemical basis for the
decline in fermentation rate in yeasts may require
determination of the factors responsible for the termination
of exponential growth and the associated physiological and
enzymatic changes.


LIST OF TABLES
Page
Table 1. Effects of ethanol and fermentation medium
composition on fermentation rate 38
Table 2. Intracellular and extracellular ethanol
concentrations under various conditions 41
Table 3. Effect of growth in broths of different
composition on fermentation rate 46
Table 4. Effect of nutrient supplementation on growth
of S. cerevisiae KD2 63
Table 5. Specific activities of glycolytic enzymes at
the peak of fermentative activity (12 h) and
after a 50% decline (24 h) 96
v


82
magnesium (Osman and Ingram, 1985). The addition of
magnesium salts at 0.5 mM substantially reversed the
inhibitory effects of up to 13% (vol/vol) ethanol. Although
analogous studies have not been performed with S.
cerevisiae. it is likely that ethanol also increases the
leakage of small molecules in this organism.
Casey et al. (1984) also have reported that nutrient
limitation is an important factor in limiting the
productivity of a fermentation. Supplementation of high-
gravity brewing wort (containing up to 31% dissolved solids)
with yeast extract, ergosterol and oleic acid allowed the
production of 16.2% (vol/vol) ethanol by brewers' yeast.
Higher rates of alcohol production primarily resulted from
an increase in cell mass associated with nutrient-
supplemented fermentations and did not appear to include an
increase in the resistance of fermentation rate to ethanol.
Addition of nutrients in the form of soy flour to
fermentation broth has been shown to increase the
fermentative productivity of both S. cerevisiae (Damiano and
Wang, 1985) and Z. mobilis (Ju et al.. 1983). Viegas et al.
(1985) also reported that soy flour addition to a yeast
extract-based medium containing 30 to 40% glucose enhanced
the rate of ethanol production by S. bayanus. Again,
supplementation led to an increase in cell concentration.
It was further demonstrated that the aqueous fraction of soy
flour, rather than the lipid fraction, contained the


These fermentations were allowed to proceed until the
culture cell density reached the state defined as 12-h
cells, approximately 1.3 mg protein per ml broth. Cells
grown in the supplemented 12-h conditioned broth fermented
at rates equal to those of control cells. The fermentation
rate of cells grown in the supplemented 24-h broth was
lower, but was at least twice that of the 24-h control.
After allowing these fermentations to continue until 5%
(vol/vol) ethanol had been produced in addition to that
present at the time of inoculation, the fermentation rate of
both types of "24-h" cells were similar to that of the
control cells. Thus, the decline in rate of fermentation
observed after the production of 5% (vol/vol) ethanol is not
due entirely to the accumulation of ethanol and/or other
stable inhibitors in the fermentation broth.
The last possibility examined in order to understand
the reasons for the decline in fermentation rate of cells
after 24 h was the effect of nutrient limitation. Neither
12-h conditioned broth nor 24-h conditioned broth
supplemented with glucose supported vigorous growth of
strain KD2 following reinoculation (Table 3, experiment 3).
In experiment 2, the addition of yeast extract restored the
ability of conditioned broth to support growth, promoting
fermentation rates equivalent to the control. Cells grown
in broth containing 25 g/L yeast extract, 5-fold greater
than that of control broth, exhibited fermentation rates


18
chromatography supplies were obtained from Supelco,
Bellefonte, Pa.
Results
Batch Fermentation by S. cerevisiae KD2
A typical batch fermentation profile of S. cerevisiae
KD2 in YEPD medium is shown in figure 1. Glucose conversion
essentially was completed after 60 h under these conditions
with the production of between 12 and 13% (vol/vol) ethanol.
Cell protein stopped increasing after 24 h at 2.4 mg per ml
medium, although the optical density at 550 nm of this
culture continued to rise for an additional 12 h period
(data not shown). Nearly identical profiles were obtained
in medium supplemented with Tween 80 (5 g/liter), linoleate
(45 mg/liter) and ergosterol (30 mg/liter). Similar
profiles also were obtained by the addition of small amounts
of 10 N KOH during the course of fermentation using a pH
stat to maintain the pH of the growth medium at 5.0.
Likewise, batch fermentations of S. cerevisiae CC3, the
parental grande strain, were indistinguishable from those of
strain KD2.
Inhibition of Growth Rate by Ethanol
Although it is not obvious from figure 1, the growth
rate of S. cerevisiae KD2 decreases as ethanol accumulates
in the fermentation broth (Fig. 2). Using the data from
batch fermentations, rates of growth were calculated as the
increase in cell protein over a 1.6 h period at the various


Figure 1.
Growth and ethanol production by S.
cerevisiae KD2 during a typical batch
fermentation in YEPD medium containing
20% glucose. Symbols: Q cell
protein (mg/ml culture); £ ,
glucose; ethanol.


Figure 18. Comparison of nucleotide levels found
in the cells with those found in the
fermentation broth. Values are
plotted as counts per min per ml of
fermentation extract for direct
comparison. The counts in the whole
fermentation samples were corrected
for counts found in the broth alone.
Qualitatively similar results were
obtained from three independent batch
fermentations. Symbols: cell-
associated nucleotide counts; ,
nucleotide counts found in the medium.


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
/V
- k
//
Lonnie 0. Ingram
Chairman, Processor of
Microbiology and Cell Science
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Charles M. Allen
Professor of Biochemistry
and Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
s
fi. F
JL
Samuel R. Farrah
Associate Professor of
Microbiology and Cell Science


138
had dropped by 90% and remained low until the glucose in the
medium was depleted. When the fermentation was completed,
ATP increased to a level 2.5 times that at 12 h. This
suggests that as the carbon source is depleted, the ATP
utilizing pathways of the cell become inactive and the
little ATP synthesized during this period accumulates.
While ATP levels decreased by 90% after 36 h, a
dramatic increase in the level of AMP was detected by 24 h
(Fig. 20A). The intracellular concentration of AMP
increased by 5 times the 12-h value at 18 h and by 10 times
the 12-h value at 24 h. This rising AMP concentration could
not be accounted for by decreasing levels of ADP or ATP.
After 24 h, the level of AMP remained rather constant at
7 mM until the end of the fermentation when it dropped to
only 4 times the 12-h value. The intracellular
concentration of ADP fluctuated around 2 mM throughout the
duration of the fermentation. The level of total adenine
nucleotides increased from 12 to 18 h concomitant with the
increase in cell protein due to growth. When cell growth
ceased after 18 h, the intracellular adenine nucleotide
concentration remained constant at 9 mM until the end of
fermentation.
Because the intracellular concentration of ATP
initially remained constant and then fell by 90%, and the
level of AMP dramatically increased early in the
fermentation (Fig. 20A), the adenylate energy charge showed


15
colonies on lactate agar plates (Ogur and St. John, 1956)
after growth for 3 days in broth containing 6 mM MnCl2
(Putrament et al.. 1973). It has been speculated that
manganese induces mutations by interacting with the
manganese-sensitive mitochondrial DNA polymerase causing
error-prone replication of the mitochondrial DNA. Strain
KD2 did not grow on glycerol containing medium or reduce
2,3,5-triphenyltetrazoleum chloride (Ogur et al.. 1957). It
also lacked the cytochrome a+a3 absorbance bands at 600 nm
and 440 nm and the cytochrome b bands at 560 nm and 530 nm.
A petite strain was chosen for this study because growth and
fermentation have been shown to be almost identical both
anaerobically and aerobically, eliminating the need to
perform experiments under anaerobic conditions (Loureiro-
Dias and Arrabaca, 1982). In some studies, S.cerevisiae CC3
and S. cerevisiae A10 0 (NRRL Y-12707) were used for
comparison. The latter strain was provided generously by
N.J. Alexander (Northern Regional Research Center, U.S.
Department of Agriculture, Peoria, Ill.).
Growth Conditions
All organisms were grown on YEPD medium which contained
5 g/liter yeast extract, 10 g/liter peptone and 200 g/liter
glucose as described by Leao and van Uden (1982a). The
medium was adjusted to pH 5.0 with 2.0 N HCl prior to
autoclaving. Solid medium for culture maintenance consisted
of YEPD broth containing 1.5% agar.


35
(50 to 100 mg of cell protein per ml) at a final activity of
2 pCi/ml and 84 nCi/ml, respectively. After 5 min, 0.1-ml
samples were pipetted directly into scintillation fluid for
aqueous samples. The cell solid volume was calculated as
the difference between the tritiated water counts in the
suspension and the supernatant samples. The fraction of the
total cell volume occupied by solids was computed as
follows:
1 (3HSU8/3Hsup)
V8 = (1)
i (14c8U8/14c8up)
where Vs is the fraction of solid volume, sus is the
suspension and sup is the supernatant fraction. Typically,
the cell solid volume represented 20 to 25% of the sorbitol-
excluded volume. The intracellular water content decreased
from 2.23 pi per mg cell protein 12 h after inoculation to
0.83 pi per mg cell protein by 48 h.
The intracellular concentration of ethanol was computed
based on the aqueous cell volume, i.e., the sorbitol-
excluded volume minus the solid volume. This was calculated
by assuming that the amount of ethanol in the suspension is
equal to the intracellular concentration of ethanol times
the aqueous cell volume plus the concentration of ethanol in


Materials and Methods 86
Results 89
Discussion 103
VI PHOSPHORYLATED GLYCOLYTIC INTERMEDIATES AND
NUCLEOTIDES 113
Introduction 113
Materials and Methods 116
Results 125
Discussion 139
VII SUMMARY AND FUTURE DIRECTIONS 14 6
BIBLIOGRAPHY 152
BIOGRAPHICAL SKETCH 166
iv


Figure 3. Rate of fermentation in the presence
of ethanol-. Fermentative activity was
determined from batch culture
experiments as the increase in ethanol
concentration over 1.6-h time
intervals divided by the average
cellular protein concentration in the
medium during that time interval. It
is plotted as a function of ethanol
accumulated in the growth medium and
is expressed as umoles ethanol
produced per h per mg cell protein.
The effect of added ethanol on the
activity of cells at their highest
measured rate of fermentation, 12 h
after inoculation, is included for
comparison. Ethanol was added
directly to fermentation samples and
fermentation rates were measured by
respirometry. These are expressed as
umoles C02 produced per h per mg cell
protein and are plotted as a function
of total ethanol in the medium.
Symbols: £, fermentation rate
during batch fermentation; ,
effect of added ethanol on the
fermentation rate of 12-h cells.


Figure 7.
Magnesium levels in broth and cells during the course of batch fermentation.
(A) Intracellular magnesium. (B) Magnesium concentration in the culture
broth. Results have been plotted for four separate batch fermentations.
Closed symbols represent fermentations supplemented with 0.5 mM MgSC>4 and open
symbols represent control fermentations in YEPD broth alone.


32
Figure 17. Changes in [ P]-labelled cellular metabolites during batch fermentation.
Spots identified as shown in figure 16 were marked, scraped and counted. For
reasons specified in the text, values for total phosphorylated glycolytic
intermediates and trise phosphates did not include measurements of
phosphoenolpyruvate. Chromatograms of fermentation broth extracts also were
run and the counts in the medium were subtracted from the counts in the whole
fermentation extracts. Values for intermediate levels were calculated as
counts per min per mg cell protein. Fermentation rates were measured as umoles
CC>2 evolved per h per mg cell protein. All values are plotted as a percentage
of the value at 12 h to facilitate comparison. Qualitatively similar results
were obtained in duplicate batch fermentations. (A) Rates of glycolysis
( # ) and total phosphorylated glycolytic intermediates ( O ) (B) Hexose
phosphates ( ), trise phosphates ( Q ) and total nucleotides ( ).


Figure 14.
Changes in intracellular pH and membrane energization during batch
fermentation of 20% glucose. Error bars represent the average standard
deviation for determinations from three separate batch fermentations. (A)
Intracellular pH. Symbols: internal pH; external pH; ,
A pH. (B) Membrane energization. Symbols: Ayj; ApH;
, proton motive force.


23
times sampled during the fermentations. Growth rate
decreased exponentially as ethanol accumulated in the
medium. The concentration of ethanol that had been produced
at half the maximum observed growth rate was
1.08% (vol/vol).
The addition of ethanol to fermentation broth also
decreased growth rate (Fig. 2). Cells inoculated into broth
containing increasing concentrations of ethanol were
inhibited in a dose-dependent manner. This inhibition was
linear above concentrations of 2% (vol/vol). The amount of
ethanol required to decrease the growth rate by 50% was
4.66% (vol/vol). Thus, four times more ethanol was required
to decrease growth rate by one-half than was produced when
growth rate had declined by this same fraction.
Inhibition of Fermentation Rate by Ethanol
The rate of alcohol production per mg cell protein was
calculated in a fashion analogous to the growth rate data.
These fermentation rates are shown as a function of average
ethanol accumulated in the medium in figure 3. Fermentation
rates also were determined by manometry using samples from
batch fermentations with excellent agreement for samples
taken 12 h and later. Identical plots were obtained with S.
cerevisiae CC3, the grande parent strain of KD2. The trends
observed were similar for cells grown with and without lipid
supplements and for cells grown in a pH stat where the pH of
the medium was held at 5.0. The fermentative activity of


Figure 19. Intracellular concentration of
nicotinamide nucleotides during batch
fermentation. The values plotted have
been corrected for nicotinamide
nucleotides detected in the medium.
Error bars represent the average
standard deviation for three
independent batch fermentations.
Symbols: # NADH; O, NAD+; ,
total nicotinamide nucleotides.


11
role of ethanol produced during fermentation in causing this
decline in ethanol production rate. Other possible causes
of the decreasing rate of ethanol production during batch
fermentation, such as nutrient limitation, also were
studied. Finally, the physiological changes which accompany
the declining fermentation rate were characterized in order
to better understand the constraints that limit the rate at
which S. cerevisiae produces ethanol.


actual alcohol production machinery is more resistant to
ethanol inhibition than is cell growth (Brown et al.. 1981;
Luong, 1985) The magnitude of the observed inhibition,
however, was greater for endogenously produced ethanol than
for exogenously added ethanol for both processes in
agreement with previous reports (Moulin et al.. 1984; Novak
et al.. 1981). These results suggest that the mere presence
of ethanol may not be entirely sufficient to account for the
observed decline in fermentation rates. In the following
studies, the causal role of ethanol in the decreasing
fermentation rate was chosen for more detailed examination.
The role of factors other than ethanol also will be studied
in order to account more fully for this observed decrease in
rate of ethanol production.


53
ethanol concentration in yeast cells during active
fermentation and ethanol production.
In the studies presented in this chapter, the
intracellular ethanol concentration was estimated by an
independent method using cells that were actively producing
ethanol in suspension culture. The results obtained using
this method confirm the reports by Beaven et al (1982) and
Guijarro and Lagunas (1984) which indicated that yeast cells
are freely permeable to ethanol. In addition, these results
provide direct evidence that the intracellular concentration
of ethanol produced during fermentation is not several-fold
higher than that of the surrounding medium as proposed
previously (Beaven et al.. 1982; Nagodawithana and
Steinkraus, 1976; Novak et al.. 1981; Strehaiano and Goma,
1983) Identical conclusions were reached by Dasari et al.
(1985) using high cell density fermentations which allowed
rapid processing of the cells for analysis. There does not
appear to be any problem associated with the efficient
diffusion of ethanol from yeast cells into the environment
during fermentation. Thus, it is unlikely that the
retention of unusually high intracellular ethanol
concentrations contributes toward the decrease in
fermentative activity of S. cerevisiae during fermentation.
Recently, Casey et al. (1983, 1984) have shown that
yeast nutritional requirements limit fermentative activity
in high gravity brewing. Supplementing worts with yeast


28
and a biphasic decrease in rate of alcohol production
(Fig. 3) as ethanol accumulated in the fermentation broth.
The exponentially falling growth rate was similar to that
observed by Aiba et al. (1968). However, the biphasic
decrease in ethanol production fits both an exponential
model and a linear model describing a combination of two
events. In batch fermentations by strain KD2, one cellular
site may be much more sensitive to the accumulation of
ethanol than another. Thus, a biphasic inhibition profile
occurs with the more sensitive site being characterized by
the line with the steepest slope. Fermentation rate
declined by 50% after the accumulation of about three and
one-half times more ethanol than was accumulated when growth
rate decreased by a similar amount (Fig. 2 and Fig. 3).
Clearly, ethanol added to the fermentation broth
linearly decreased both the rate of cell growth and alcohol
production as previously described (Brown et al.. 1981;
Moulin et al.. 1984). This contrasts with the results of
Luong (1985) who reported that S. cerevisiae ATCC 4126
exhibited non-linear decreases in growth and fermentation
rates under anaerobic conditions. As with the accumulation
of ethanol, growth rate was inhibited more than fermentation
rate, but was only about two-fold more sensitive.
Growth rate decreased faster than fermentation rate as
ethanol accumulated in the medium and as increasing amounts
of ethanol were added exogenously to the medium. Thus, the


124
addition of monitoring reagent. Luminescence of samples
treated with alcohol dehydrogenase represents the combined
amounts of NAD+ and NADH present in the samples. Amounts of
NAD+ were calculated as the difference in concentration
between samples exposed to alcohol dehydrogenase and
untreated samples. As with the firefly bioluminescence
assay, ethanol is known to inhibit the reaction. To correct
for this inhibition, standards were prepared containing
amounts of ethanol identical to that found in the samples.
Fermentation extracts used for nicotinamide determinations
were stored and handled in a manner identical to that
described for the adenine nucleotide determinations.
Analysis of Batch Fermentation Samples
Batch fermentations were monitored by following the
increase in optical density at 550 nm of the culture and the
amounts of cellular protein and ethanol in the broth. These
measurements were made as described previously in
chapter II. Fermentation rates were measured by
respirometry also as described in chapter II. Cell volume
measurements were made according to the method discussed in
chapter III.
Chemicals
Complex medium components and agar were purchased from
Difco Laboratories, Detroit, Mich. Glucose, enzymes and
other biochemicals were supplied by Sigma Chemical Co., St.
Louis, Mo. Inorganic salts were obtained from Fisher




UNIVERSITY OF FLORIDA
V> i V" "" "'i mi m
3 1262 08554 1596


Figure 9. Effect of added magnesium on the rate
of fermentation. Fermentation rates
were measured by respirometry of
unwashed cells immediately after
sampling and are plotted as a function
of accumulated ethanol for four
separate batch fermentations. Closed
symbols represent cultures
supplemented with 0.5 mM MgS04 and
open symbols represent control
fermentations in YEPD broth alone.


144
of ATP (Fig. 20A) may inhibit the in vivo activity of the
hexokinases and, as a result, glucose uptake. This
accumulation of AMP may be the result of RNA degradation
(Chapman and Atkinson, 1977) which can act as an ATPase to
convert ATP to AMP as cells enter stationary phase.
Alternatively, the changes in membrane lipid composition
that occur as growth slows (Hunter and Rose, 1972) and cells
enter stationary phase (Homann et al.. 1987) may decrease
the activity of the transport system. Evidence to support
this hypothesis was supplied by Thomas and Rose (1979) who
showed that changes in plasma membrane lipid composition
affect solute uptake by S. cerevisiae.
In addition to the activity of the hexose uptake
system, the amounts of transport molecules and hexose
phosphorylating enzymes are also important determinants of
glucose uptake rate. In chapter V, the amount of hexose
phosphorylating enzymes assayed as hexokinase were shown to
remain high throughout fermentation (Table 4 and Fig. 13).
However, Busturia and Lagunas (1986) observed that under
nitrogen-limiting growth conditions, the glucose uptake
systems of S. cerevisiae were inactivated and this
inactivation required the utilization of a fermentable
carbohydrate by the cells. During nitrogen starvation, the
fermentation rate of S. cerevisiae decreases in a manner
similar to that described in the present study. Likewise,
glucose uptake was inactivated when protein synthesis was


PERCENTAGE OF
PERCENTAGE OF
VALUE AT 12 h
LZ T


10
under the very specialized conditions of the sake
fermentation. Koji mold proteolipid, found in sake mash,
also enhanced yeast growth, survival and fermentative
activity (Hayashida et al.. 1975). This proteolipid,
isolated from Aspergillus orvzae. was shown to contain a
high percentage of phosphatidylcholine with linoleic acid
comprising the major portion of its fatty-acyl residues
(Hayashida et al.. 1976). Supplementation with proteolipid
actually increased the proportion of phosphatidylcholine and
linoleyl fatty-acyl residues in the sake yeast plasma
membrane (Hayashida and Ohta, 1978). The
phosphatidylcholine promoted yeast growth and fermentative
activity, while addition of ergosterol-oleate increased
survivability in the presence of ethanol (Hayashida and
Ohta, 1980). Many confirming studies have shown that
addition of unsaturated lipids improves the fermentative
productivity of yeast (Damiano and Wang, 1985; Janssens et
al.. 1983; Lafon-Lafourcade et al.. 1979; Ohta and
Hayashida, 1983; Watson, 1982).
Although much is known about the effects of ethanol on
various aspects of yeast fermentation and about the
involvement of the plasma membrane in mediating many of
these effects, very little is known about the factors which
cause the rate of fermentation to decline as ethanol
accumulates in the medium. The following studies have
examined this phenomenon in more detail to determine the


130
This decrease in total nucleotide phosphate could have
resulted from increased leakage or excretion of nucleotides
from the cells during fermentation. In figure 18, the
amount of nucleotide phosphate measured as counts per min
per ml of fermentation broth without or with cells
(corrected for counts in the culture broth) was plotted as
fermentation progressed. The nucleotide phosphate counts in
the fermentation broth remained constant and low throughout
the duration of the fermentation even as the cell-associated
nucleotide phosphate levels decreased. These results
suggest that cellular processes other than nucleotide
leakage or excretion were responsible for the dramatic
decrease in nucleotide phosphate.
Nicotinamide Nucleotides
Glyceraldehyde-3-phosphate dehydrogenase requires NAD+
and alcohol dehydrogenase requires NADH during the process
of converting glucose to ethanol. If the intracellular
concentration of either of these nucleotides follow the same
dramatic decline as total nucleotide phosphate (Fig. 17B),
then the increasing pool of trise phosphates may result
from decreasing activity of either of these enzymes in vivo.
In figure 19 is plotted the intracellular concentrations of
nicotinamide nucleotides throughout fermentation, as
determined by bacterial luminescence. The intracellular
concentration of NADH remained low at 0.11 mM and did not
decrease after 12 h. Thus, alcohol dehydrogenase should


110
significantly the A pH of yeast cells suspended in fresh
medium. Only at ethanol concentrations above 15% (vol/vol)
is the A pH lowered. Although enhanced ion leakage also
may occur in fermentation broth, the maintenance of a high
internal pH in broth containing ethanol indicates that such
leakage must be offset by the action of hydrogen ion pumps,
such as ATPases. Pena et al. (1972) proposed a similar
hypothesis to explain the conversion of ATP to ADP and
inorganic phosphate observed when the external pH of their
strain of S. cerevisiae in buffer was raised from 4.0 to
7.5.
During fermentation, A pH increased to 3.36 and A ijj
decreased to -68.8 mV as the pH of the medium decreased to
3.5 (Fig. 13). This gradually lowered the proton motive
force to -156 mV. Similarly, De La Pena et al. (1982)
reported that the proton motive force of yeast cells
suspended in a series of buffers with the pH decreasing to
4.0 became more negative. Cartwright et al. (1986) was not
able to detect a membrane potential when 6% (vol/vol)
ethanol was added to an aqueous suspension of early
stationary-phase cells. This plus the decrease in A pH
observed when ethanol was added to identical cell
suspensions gradually made the proton motive force more
positive. This report is in contrast with the data
presented in figure 14, which indicates that the plasma


27
Discussion
Because the ability of ethanol to inhibit alcohol
production varies with the yeast strain and fermentation
conditions employed (Jones et al.. 1981), the effect of
ethanol on these processes in the yeast strain chosen for
these studies of alcohol production was characterized. The
observed decline in both growth and fermentation rates was
similar under a variety of culture conditions for S.
cerevisiae KD2. This decline also was seen with other
strains of S. cerevisiae. but strain KD2 maintained equal or
greater rates and accumulated more ethanol during
fermentation. Identical fermentation profiles of strain KD2
and its grande parent strain suggest that the manganese
treatment used to obtain respiratory-deficient cells, in
itself, was not responsible for the observed decline in rate
of growth and alcohol production. Induction of respiratory-
deficiency has been observed to decrease ethanol tolerance
in some petite strains of yeast (Aguilera and Benitez, 1985;
Esser et al.. 1982) while increasing the fermentation rate
and ethanol tolerance of other strains (Bacilia et al..
1978; Moulin et al.. 1981). Differences between wild-type
strains, the harshness of the mutagenic treatments and the
limited numbers of mutants screened may be responsible for
these contradictory reports.
The brewery yeast strain used in most of these studies
exhibited an exponential decrease in growth rate (Fig. 2)


164
Seeman, P. 1972. The membrane actions of anaesthetics and
tranquilizers. Pharmacol. Rev. 24:583-655.
Sols, A., C. Gancedo and G. Dla Fuente. 1971. Energy-
yielding metabolism in yeasts, p. 271-307. In A.H. Rose
and J.S. Harrison (ed.), the yeasts, vol. 2. Academic
Press, New York.
Srivastava, D.K. and S.A. Bernhard. 1986. Enzyme-enzyme
interactions and the regulation of metabolic reaction
pathways. Curr. Topics Cell. Regul. 28:1-68.
Strehaiano, P. and G. Goma. 1983. Effects of initial
substrate concentration on two wine yeasts. Relation
between glucose sensitivity and ethanol inhibition. Am. J.
Enol. Vitic. 34:1-5.
Thomas, D.S., J.A. Hossack and A.H. Rose. 1978. Plasma-
membrane lipid composition and ethanol tolerance in
Saccharomvces cerevisiae. Arch. Microbiol. 117:239-245.
Thomas, D.S. and A.H. Rose. 1979. Inhibitory effect of
ethanol on growth and solute accumulation by Saccharomvces
cerevisiae as affected by plasma-membrane lipid
composition. Arch. Microbiol. 122:49-55.
Thompson, J. 1978. In vivo regulation of glycolysis and
characterization of sugar:phosphotransferase systems in
Streptococcus lactis. J. Bacteriol. 136:465-476.
Tompa, P., J. Bar and J. Batke. 1986. Interaction of enzymes
involved in triosephosphate metabolism-comparison of yeast
and rabbit muscle cytoplasmic systems. Eur. J. Biochem.
159:117-124.
Trevors, J.T., R.L. Merrick, I. Russell and G.G. Stewart.
1983. A comparison of methods for assessing yeast
viability. Biotechnol. Lett. 2:131-134.
Troyer, J.R. 1953. A relationship between cell
multiplication and alcohol tolerance in yeasts. Mycologia
45:20-39.
Uyeda, K. 1979. Phosphofructokinase. Adv. Enzymol. 48:193-
244 .
Vanderkooi, J.M., R. Landesberg, H. Selick and G.G.
McDonald. 1977. Interaction of general anesthetics with
phospholipid vesicles and biological membranes. Biochim.
Biophys. Acta 464:1-16.


Table 3-continued
4. Nutrient limitation II
Control 48.8 46.1 (0.6) 13.5 (0.2)
5X yeast extract 44.6 (1.3) 28.2 (0.4)
a Experimental designs are described in more detail in the text. Fermentations
were carried out in various media. Cells were harvested by centrifugation at
ambient temperature and suspended to original volume in fresh broth, YEPD medium,
immediately prior to measurement of fermentation rate by respirometry.
Not determined.


Figure 20. Intracellular concentration of adenine nucleotides and energy charge during
batch fermentation. The values plotted have been corrected for adenine
nucleotides detected in the medium. Error bars represent the average standard
deviation for three independent batch fermentations. (A) Adenine nucleotides
during batch fermentation. Symbols: O ATP; O ADP; AMP;
# total adenine nucleotides. (B) Changes in energy charge during batch
fermentation. Energy charge was calculated as described by Ball and Atkinson
(1975).



PAGE 2

7+( '(&/,1,1* 5$7( 2) (7+$12/ 352'8&7,21 '85,1* %$7&+ )(50(17$7,21 %< 6$&&+$520<&(6 &(5(9,6,$( %< .(11(7+ 0,&+$(/ '20%(. $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( 6&+22/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 3

$&.12:/('*0(176 7KH LGHDV SUHVHQWHG LQ WKHVH VWXGLHV FRXOG QRW KDYH EHHQ GHYHORSHG ZLWKRXW WKH HQFRXUDJHPHQW DQG SDWLHQFH RI P\ PDMRU DGYLVRU 'U 1HDO ,QJUDP DP JUHDWO\ LQGHEWHG WR KLP IRU VKDULQJ ZLWK PH KLV NQRZOHGJH DQG H[SHUWLVH DOVR ZRXOG OLNH WR H[SUHVV P\ JUDWLWXGH WR WKH RWKHU PHPEHUV RI P\ FRPPLWWHH 'U $OOHQ 'U )DUUDK 'U *DQGHU DQG 'U 3UHVWRQ IRU WKHLU FRQWULEXWLRQV GXULQJ SUHSDUDWLRQ DQG UHYLHZ RI WKLV PDQXVFULSW 6LPLODUO\ WKDQNV DUH GXH WR P\ FROOHDJXH DQG IULHQG 'U
PAGE 4

7$%/( 2) &217(176 3DJH $&.12:/('*0(176 LL /,67 2) 7$%/(6 9 /,67 2) ),*85(6 YL $%675$&7 YLLL &+$37(56 *(1(5$/ ,1752'8&7,21 ,,&+$5$&7(5,=$7,21 2) 7+( '(&/,1,1* 5$7(6 2) *52:7+ $1' (7+$12/ 352'8&7,21 '85,1* %$7&+ )(50(17$7,21 %< 6 &(5(9,6,$( .' ,QWURGXFWLRQ 0DWHULDOV DQG 0HWKRGV 5HVXOWV 'LVFXVVLRQ ,,,1875,(17 /,0,7$7,21 $6 $ %$6,6 )25 7+( $33$5(17 72;,&,7< 2) /2: /(9(/6 2) (7+$12/ '85,1* %$7&+ )(50(17$7,21 ,QWURGXFWLRQ 0DWHULDOV DQG 0HWKRGV 5HVXOWV 'LVFXVVLRQ ,90$*1(6,80 /,0,7$7,21 $1' ,76 52/( ,1 7+( $33$5(17 72;,&,7< 2) (7+$12/ '85,1* <($67 )(50(17$7,21 ,QWURGXFWLRQ 0DWHULDOV DQG 0HWKRGV 5HVXOWV 'LVFXVVLRQ 9*/<&2/<7,& (1=<0(6 $1' ,17(51$/ S+ ,QWURGXFWLRQ LLL

PAGE 5

0DWHULDOV DQG 0HWKRGV 5HVXOWV 'LVFXVVLRQ 9, 3+263+25
PAGE 6

/,67 2) 7$%/(6 3DJH 7DEOH (IIHFWV RI HWKDQRO DQG IHUPHQWDWLRQ PHGLXP FRPSRVLWLRQ RQ IHUPHQWDWLRQ UDWH 7DEOH ,QWUDFHOOXODU DQG H[WUDFHOOXODU HWKDQRO FRQFHQWUDWLRQV XQGHU YDULRXV FRQGLWLRQV 7DEOH (IIHFW RI JURZWK LQ EURWKV RI GLIIHUHQW FRPSRVLWLRQ RQ IHUPHQWDWLRQ UDWH 7DEOH (IIHFW RI QXWULHQW VXSSOHPHQWDWLRQ RQ JURZWK RI 6 FHUHYLVLDH .' 7DEOH 6SHFLILF DFWLYLWLHV RI JO\FRO\WLF HQ]\PHV DW WKH SHDN RI IHUPHQWDWLYH DFWLYLW\ Kf DQG DIWHU D b GHFOLQH Kf Y

PAGE 7

/,67 2) ),*85(6 3DJH )LJXUH *URZWK DQG HWKDQRO SURGXFWLRQ E\ 6 FHUHYLVLDH .' GXULQJ D W\SLFDO EDWFK IHUPHQWDWLRQ LQ <(3' PHGLXP FRQWDLQLQJ b JOXFRVH )LJXUH *URZWK UDWH RI 6 FHUHYLVLDH .' LQ WKH SUHVHQFH RI HWKDQRO )LJXUH 5DWH RI IHUPHQWDWLRQ LQ WKH SUHVHQFH RI HWKDQRO )LJXUH 'HWHUPLQDWLRQ RI LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQ )LJXUH ,QKLELWLRQ RI IHUPHQWDWLRQ UDWH RI K DQG K FHOOV E\ DGGHG HWKDQRO )LJXUH 'RVHUHVSRQVH RI FHOO JURZWK WR DGGHG PDJQHVLXP )LJXUH 0DJQHVLXP OHYHOV LQ EURWK DQG FHOOV GXULQJ WKH FRXUVH RI EDWFK IHUPHQWDWLRQ )LJXUH (IIHFW RI PDJQHVLXP DGGLWLRQ RQ FHOO JURZWK DQG IHUPHQWDWLRQ )LJXUH (IIHFW RI DGGHG PDJQHVLXP RQ WKH UDWH RI IHUPHQWDWLRQ )LJXUH (IIHFW RI PDJQHVLXP VXSSOHPHQWDWLRQ RQ WKH LQKLELWLRQ RI IHUPHQWDWLRQ UDWH E\ DGGHG HWKDQRO )LJXUH (IIHFW RI HWKDQRO UHPRYDO RQ WKH IHUPHQWDWLYH DFWLYLW\ RI FHOOV JURZQ LQ <(3' PHGLXP FRQWDLQLQJ P0 0J6 )LJXUH (IIHFWV RI HWKDQRO H[SRVXUH RQ WKH IHUPHQWDWLYH DFWLYLWLHV RI DQG K FHOOV YL

PAGE 8

)LJXUH &KDQJHV LQ WKH OHYHOV RI JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV GXULQJ EDWFK IHUPHQWDWLRQ ZLWK b JOXFRVH )LJXUH &KDQJHV LQ LQWUDFHOOXODU S+ DQG PHPEUDQH HQHUJL]DWLRQ GXULQJ EDWFK IHUPHQWDWLRQ RI b JOXFRVH )LJXUH (IIHFWV RI DGGHG HWKDQRO RQ $ S+ )LJXUH $ W\SLFDO WKLQ OD\HU FKURPDWRJUDP RI D IHUPHQWDWLRQ VDPSOH H[WUDFW )LJXUH &KDQJHV LQ >3@ODEHOOHG FHOOXODU PHWDEROLWHV GXULQJ EDWFK IHUPHQWDWLRQ )LJXUH &RPSDULVRQ RI QXFOHRWLGH OHYHOV IRXQG LQ WKH FHOOV ZLWK WKRVH IRXQG LQ WKH IHUPHQWDWLRQ EURWK )LJXUH ,QWUDFHOOXODU FRQFHQWUDWLRQ RI QLFRWLQDPLGH QXFOHRWLGHV GXULQJ EDWFK IHUPHQWDWLRQ )LJXUH ,QWUDFHOOXODU FRQFHQWUDWLRQ RI DGHQLQH QXFOHRWLGHV DQG HQHUJ\ FKDUJH GXULQJ EDWFK IHUPHQWDWLRQ YLL

PAGE 9

$EVWUDFW RI 'LVVHUWDWLRQ 3UHVHQWHG WR WKH *UDGXDWH 6FKRRO RI WKH 8QLYHUVLW\ RI )ORULGD LQ 3DUWLDO )XOILOOPHQW RI WKH 5HTXLUHPHQWV IRU WKH 'HJUHH RI 'RFWRU RI 3KLORVRSK\ 7+( '(&/,1,1* 5$7( 2) (7+$12/ 352'8&7,21 '85,1* %$7&+ )(50(17$7,21 %< 6$&&+$520<&(6 &(5(9,6,$( %\ .HQQHWK 0LFKDHO 'RPEHN $XJXVW &KDLUPDQ /RQQLH ,QJUDP 0DMRU 'HSDUWPHQW 0LFURELRORJ\ DQG &HOO 6FLHQFH $V 6DFFKDURPYFHV FHUHYLVLDH IHUPHQWV b JOXFRVH WR HWKDQRO LQ EDWFK FXOWXUH WKH UDWH RI WKLV FRQYHUVLRQ GHFOLQHV 3UHYLRXV VWXGLHV DVVXPHG WKDW HWKDQRO FDXVHG WKLV LQKLELWLRQ EHFDXVH DGGHG HWKDQRO UHSUHVVHG ERWK \HDVW JURZWK DQG DOFRKRO SURGXFWLRQ +RZHYHU SURGXFHG HWKDQRO DSSHDUHG WR EH D PRUH SRWHQW LQKLELWRU WKDQ DGGHG HWKDQRO $OVR UHPRYDO RI HWKDQRO IURP IHUPHQWLQJ \HDVW FHOOV GLG QRW IXOO\ DOOHYLDWH WKLV LQKLELWLRQ 7KH \HDVW FHOO HQYHORSH ZDV IUHHO\ SHUPHDEOH WR HWKDQRO DQG WKXV WKH LQWUDFHOOXODU DFFXPXODWLRQ RI HWKDQRO WR OHYHOV KLJK HQRXJK WR LQKLELW JO\FRO\WLF HQ]\PHV DSSHDUHG XQOLNHO\ $ QXWULHQW OLPLWDWLRQ IRU PDJQHVLXP LQ FRPSOH[ IHUPHQWDWLRQ PHGLXP ZDV LGHQWLILHG DV EHLQJ SDUWLDOO\ UHVSRQVLEOH IRU GHFOLQLQJ UDWHV RI DOFRKRO SURGXFWLRQ 6XSSOHPHQWDWLRQ RI EURWK ZLWK PDJQHVLXP SUHYHQWHG PXFK RI WKH GHFOLQH EXW GLG QRW HQWLUHO\ HOLPLQDWH YLLL

PAGE 10

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b ZKLOH $03 LQFUHDVHG E\ b 2QH SRVVLEOH H[SODQDWLRQ IRU WKH GHFOLQH LQ JO\FRO\WLF LQWHUPHGLDWHV DQG WKH FRUUHVSRQGLQJ GHFUHDVH LQ IHUPHQWDWLRQ UDWH LV WKDW WKH LQFUHDVHG OHYHO RI $03 LQKLELWV JOXFRVH SKRVSKRU\ODWLRQ ZKLFK PD\ VORZ WKH UDWH RI JOXFRVH XSWDNH ,Q WKLV VWXG\ WKH UROHV RI LQKLELWLRQ E\ HWKDQRO QXWULHQW OLPLWDWLRQ DQG SK\VLRORJLFDO FKDQJHV LQ GHFUHDVLQJ WKH UDWH RI IHUPHQWDWLRQ KDYH EHHQ GHILQHG DQG FKDUDFWHUL]HG (DFK RI WKHVH IDFWRUV DSSHDUV WR EH SDUWLDOO\ UHVSRQVLEOH IRU WKH GHFOLQH LQ HWKDQRO SURGXFWLRQ E\ 6 FHUHYLVLDH GXULQJ EDWFK IHUPHQWDWLRQ ,;

PAGE 11

&+$37(5 *(1(5$/ ,1752'8&7,21 (YHQ EHIRUH %& 6DFFKDURP\FHV FHUHYLVLDH ZDV XVHG WR IHUPHQW PDOWHG EDUOH\ DQG ZKHDW LQWR D W\SH RI EHHUEUHDG LQ 0HVRSRWDPLD &RUUDQ f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b RI WKH JOREDO HWKDQRO SURGXFHG LV WKH SURGXFW RI VXJDU IHUPHQWHG E\ 6 FHUHYLVLDH

PAGE 12

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f 7ZHQW\ \HDUV ODWHU 3DVWHXU H[SDQGHG WKHVH ILQGLQJV DQG SHUIRUPHG WKH ILUVW GHFLVLYH H[SHULPHQWV VKRZLQJ WKDW \HDVW FHOOV ZHUH WKH OLYLQJ HQWLWLHV UHVSRQVLEOH IRU IHUPHQWLQJ VXJDU WR HWKDQRO 3DVWHXU f +H WKHQ ZHQW RQ WR GHVFULEH WKH HIIHFW RI R[\JHQ RQ \HDVW IHUPHQWDWLRQ DQ HIIHFW ZKLFK KDV VLQFH EHFRPH NQRZQ DV WKH 3DVWHXU HIIHFW 3DVWHXU f %LRFKHPLVWU\ KDG LWV EHJLQQLQJV ZLWK WKH ZRUN RI %XFKQHU ZKR VKRZHG WKDW WKH SURWHLQDFHRXV PDWHULDO LQ \HDVW MXLFH ZDV FDSDEOH RI FRQYHUWLQJ VXJDU WR HWKDQRO %XFKQHU f ,Q WKH GHFDGHV WKDW IROORZHG WKLV GLVFRYHU\ WKH FKHPLFDO QDWXUH RI DOFRKROLF IHUPHQWDWLRQ DQG WKH HQ]\PHV UHVSRQVLEOH

PAGE 13

IRU WKH FRQYHUVLRQ ZKLFK 3DVWHXU KDG GHVFULEHG \HDUV HDUOLHU ZHUH FKDUDFWHUL]HG )UDHQNHO f (YHQ EHIRUH WKHVH HDUO\ VWXGLHV RQ DOFRKROLF IHUPHQWDWLRQ LW KDG EHHQ REVHUYHG WKDW \HDVW VWRSSHG JURZLQJ DQG IHUPHQWLQJ EHIRUH DOO RI WKH VXJDU LQ WKH IHUPHQWDWLRQ EURWK KDG EHHQ XWLOL]HG ,Q RQH RI WKH HDUOLHVW LQYHVWLJDWLRQV RI WKLV SKHQRPHQRQ %URZQ H[DPLQHG WKH LQIOXHQFH RI YDULRXV HQYLURQPHQWDO FRQGLWLRQV RQ WKH UDWH RI JURZWK RI 6 FHUHYLVLDH %URZQ f 7KH DGGLWLRQ RI HWKDQRO WR JURZWK PHGLXP LQGHHG GLG LQKLELW \HDVW UHSURGXFWLRQ +RZHYHU LQKLELWLRQ RFFXUUHG RQO\ DW D PXFK KLJKHU HWKDQRO FRQFHQWUDWLRQ WKDQ ZDV REVHUYHG WR KDYH DFFXPXODWHG DW WKH SRLQW GXULQJ WKH IHUPHQWDWLRQ ZKHQ \HDVW JURZWK KDG FHDVHG 7KLV ZDV WKH ILUVW LQGLFDWLRQ WKDW WKH SUHVHQFH RI HWKDQRO PD\ QRW EH WKH RQO\ IDFWRU LQYROYHG LQ WKH SUHPDWXUH WHUPLQDWLRQ RI FDUERK\GUDWH IHUPHQWDWLRQ E\ \HDVW 7KH VWXGLHV RI %URZQ ZHUH FRPSOHPHQWHG E\ WKH ZRUN RI 5LFKDUGV f ZKR VKRZHG WKDW UHPRYLQJ HWKDQRO SURGXFHG GXULQJ JURZWK DQG PDLQWDLQLQJ D FRQVWDQW QXWULHQW VXSSO\ DOORZHG \HDVW FHOO PXOWLSOLFDWLRQ WR FRQWLQXH DOPRVW LQGHILQLWHO\ 5HFHQWO\ WKH YDFXXP IHUPHQWDWLRQ H[SHULPHQWV FRQGXFWHG E\ &\VHZVNL DQG :LONH f DQG 0DLRUHOOD HW DO f KDYH FRUURERUDWHG DQG H[WHQGHG WKLV ILQGLQJ %RLOLQJ RII WKH HWKDQRO DV LW ZDV SURGXFHG IURP D FRQWLQXRXV FXOWXUH XQGHU UHGXFHG SUHVVXUH LQFUHDVHG DFKLHYDEOH FHOO GHQVLWLHV

PAGE 14

DQG IHUPHQWDWLRQ UDWHV %HFDXVH VXFK LQYHVWLJDWLRQV KDYH VKRZQ WKDW HWKDQRO GRHV LQKLELW \HDVW JURZWK DQG IHUPHQWDWLRQ PDQ\ VWXGLHV KDYH GHDOW ZLWK FKDUDFWHUL]LQJ WKHVH LQKLELWRU\ HIIHFWV %URZQ HW DO +RSSH DQG +DQVIRUG -RQHV DQG *UHHQILHOG /DIRQ/DIRXUFDGH DQG 5LEHUHDX*D\RQ 9HJD HW DO f 7KH LQKLELWRU\ HIIHFW RI HWKDQRO RQ WKH UDWH RI VXJDU FRQYHUVLRQ WR HWKDQRO ZDV ILUVW TXDQWLWDWHG E\ 5DKQ f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f +ROW]EHUJ HW DO f H[DPLQHG JUDSH MXLFH IHUPHQWDWLRQ E\ 6 FHUHYLVLDH YDU HOLSVRLGHXV DQG FDOFXODWHG DQ LQYHUVH OLQHDU UHODWLRQVKLS EHWZHHQ WKH UDWH RI DOFRKRO SURGXFWLRQ DQG WKH DPRXQW RI HWKDQRO LQ WKH MXLFH 6LPLODU ILQGLQJV ZHUH PDGH E\ *KRVH DQG 7\DJL f IRU WKH EDWFK IHUPHQWDWLRQ RI FHOOXORVH K\GURO\VDWH E\ 6 FHUHYLVLDH 15/ \ KRZHYHU WKH FRQVWDQWV LQ WKH HTXDWLRQ

PAGE 15

UHODWLQJ WKH UDWH RI DOFRKRO SURGXFWLRQ WR WKH DPRXQW RI HWKDQRO LQ WKH IHUPHQWDWLRQ EURWK ZHUH VOLJKWO\ GLIIHUHQW $ JOXFRVHOLPLWHG FRQWLQXRXV FXOWXUH RI D UHVSLUDWRU\ GHILFLHQW EDNHUnV \HDVW ZDV IRXQG E\ $LED HW DO f WR H[KLELW DQ H[SRQHQWLDO GHFUHDVH LQ UDWH RI DOFRKRO IRUPDWLRQ DV LQFUHDVLQJ FRQFHQWUDWLRQV RI HWKDQRO ZHUH DGGHG WR WKH IHUPHQWDWLRQ EURWK 7KH\ DOVR GHPRQVWUDWHG WKDW HWKDQRO DFWHG DV D QRQFRPSHWLWLYH LQKLELWRU RI DOFRKRO IRUPDWLRQ %RWK EDWFK DQG FRQWLQXRXV FXOWXUH IHUPHQWDWLRQV RI 6 FHUHYLVLDH $7&& 1R LQ D V\QWKHWLF PHGLXP ZHUH VKRZQ E\ %D]XD DQG :LONH f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f ,QLWLDO VWXGLHV RQ WKH DOFRKRO WROHUDQFH RI \HDVW ZHUH SHUIRUPHG E\ *UD\ f 9DULRXV HWKDQRO SURGXFLQJ VSHFLHV

PAGE 16

RI \HDVW LQFOXGLQJ VHYHUDO GLIIHUHQW VWUDLQV RI 6 FHUHYLVLDH ZHUH JURXSHG DFFRUGLQJ WR WKHLU DELOLW\ WR XWLOL]H JOXFRVH LQ WKH SUHVHQFH RI D VHULHV RI HWKDQRO FRQFHQWUDWLRQV 7KH HWKDQRO WROHUDQFH WUDLW ZDV QRW FKDUDFWHULVWLF RI DQ\ VSHFLILF JHQXV RU VSHFLHV VLQFH GLIIHUHQW VWUDLQV RI WKH VDPH VSHFLHV YDULHG LQ WKHLU WROHUDQFH
PAGE 17

IRXQG WR EH PRUH LQKLELWRU\ WKDQ VHFRQGDU\ RU WHUWLDU\ DOFRKROV 7KLV VXJJHVWHG WKDW WKH PRGH RI DFWLRQ RI HWKDQRO PD\ LQYROYH D K\GURSKRELF VLWH 1XPHURXV LQYHVWLJDWLRQV KDYH GHVFULEHG WKH GHWULPHQWDO HIIHFWV RI D YDULHW\ RI K\GURSKRELF FRPSRXQGV LQFOXGLQJ DOFRKROV RQ WKH IXQFWLRQ RI PDQ\ GLIIHUHQW W\SHV RI FHOOV DQG PHPEUDQHV %DURQGHV HW DO (DWRQ HW DO +D\DVKLGD DQG 2KWD /HDR DQG YDQ 8GHQ E /HQD] HW DO 6HHPDQ f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f RQH VLWH RI HWKDQRO DFWLRQ PD\ EH WKH FHOO F\WRSODVPLF PHPEUDQH 7KH F\WRSODVPLF PHPEUDQH RI 6 FHUHYLVLDH FRQWDLQV DERXW b SURWHLQ DQG b OLSLG E\ GU\ ZHLJKW +XQWHU DQG 5RVH f ,W KDV D XQLTXH OLSLG FRPSRVLWLRQ FRQWDLQLQJ SKRVSKROLSLGV ZLWK QR SRO\XQVDWXUDWHG IDWW\ DFLGV OLNH WKRVH IRXQG LQ SURNDU\RWHV DQG ODUJH SURSRUWLRQV RI SKRVSKDWLG\OFKROLQH DQG VWHUROV OLNH WKRVH IRXQG LQ HXNDU\RWLF FHOOV ,QJUDP DQG %XWWNH f $V PXFK DV b

PAGE 18

RI WKH IDWW\DF\O UHVLGXHV DUH XQVDWXUDWHG DQG DV PXFK DV b RI WKH PHPEUDQH GU\ ZHLJKW FRQVLVWV RI VWHUROV PDLQO\ HUJRVWHURO +XQWHU DQG 5RVH f 7KH V\QWKHVLV RI XQVDWXUDWHG IDWW\ DFLGV IURP VDWXUDWHG DFLGV UHTXLUHV DQ R[\JHQGHSHQGHQW GHVDWXUDVH HQ]\PH DQG WKH V\QWKHVLV RI HUJRVWHURO IURP VTXDOHQH UHTXLUHV R[\JHQGHSHQGHQW SHUR[LGDWLRQ DQG GHPHWK\ODWLRQ UHDFWLRQV +HQU\ f :KHQ FXOWXUHG XQGHU DQDHURELF FRQGLWLRQV 6 FHUHYLVLDH H[KLELWV D UHTXLUHPHQW IRU ERWK XQVDWXUDWHG IDWW\ DFLGV DQG HUJRVWHURO LQ LWV QXWULHQW VXSSO\ IRU JURZWK $QGUHDVHQ DQG 6WLHU $QGUHDVHQ DQG 6WLHU f 7DNLQJ DGYDQWDJH RI WKLV DQDHURELFDOO\ LQGXFHG QXWULWLRQDO UHTXLUHPHQW WKH ODERUDWRU\ JURXS RI 5RVH VHOHFWLYHO\ HQULFKHG SODVPD PHPEUDQHV RI 6 FHUHYLVLDH E\ XS WR b ZLWK DQ LQGLYLGXDO XQVDWXUDWHG IDWW\DFLG RU E\ XS WR b ZLWK D SDUWLFXODU VWHURO +RVVDFN DQG 5RVH f 8VLQJ \HDVW ZLWK DOWHUHG SODVPD PHPEUDQH OLSLG FRPSRVLWLRQ WKH\ VKRZHG WKDW FHOOV HQULFKHG LQ HUJRVWHURO DQG OLQROH\O UHVLGXHV UHPDLQHG YLDEOH IRU D ORQJHU SHULRG RI WLPH WKDQ FHOOV HQULFKHG LQ RWKHU VWHUROV DQG ROH\O UHVLGXHV ZKHQ H[SRVHG WR HWKDQRO 7KRPDV HW DO f (WKDQRO ZDV DOVR OHVV LQKLELWRU\ WR JURZWK DQG VROXWH DFFXPXODWLRQ ZKHQ SODVPD PHPEUDQH OLSLGV ZHUH HQULFKHG VLPLODUO\ 7KRPDV DQG 5RVH f %HFDXVH XQVDWXUDWHG IDWW\ DFLGV DQG HUJRVWHURO DSSHDU WR SURWHFW \HDVW FHOOV IURP WKH LQKLELWRU\ HIIHFWV RI HWKDQRO LW LV QRW VXUSULVLQJ WKDW 6 FHUHYLVLDH

PAGE 19

KDV EHHQ VKRZQ WR DOWHU LWV SODVPD PHPEUDQH OLSLG FRPSRVLWLRQ ZKHQ JURZQ LQ WKH SUHVHQFH RI HWKDQRO %HDYHQ HW DO f 7KH DPRXQW RI XQVDWXUDWHG IDWW\DF\O FKDLQV LQ WKH SKRVSKROLSLGV URVH ZLWK LQFUHDVLQJ DPRXQWV RI DGGHG HWKDQRO 7KH SURSRUWLRQ RI ROH\O UHVLGXHV LQFUHDVHG E\ b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b HWKDQRO +D\DVKLGD HW DO f 6DNH \HDVW QRUPDOO\ RQO\ SURGXFH WKLV KLJK DPRXQW RI HWKDQRO

PAGE 20

XQGHU WKH YHU\ VSHFLDOL]HG FRQGLWLRQV RI WKH VDNH IHUPHQWDWLRQ .RML PROG SURWHROLSLG IRXQG LQ VDNH PDVK DOVR HQKDQFHG \HDVW JURZWK VXUYLYDO DQG IHUPHQWDWLYH DFWLYLW\ +D\DVKLGD HW DO f 7KLV SURWHROLSLG LVRODWHG IURP $VSHUJLOOXV RUY]DH ZDV VKRZQ WR FRQWDLQ D KLJK SHUFHQWDJH RI SKRVSKDWLG\OFKROLQH ZLWK OLQROHLF DFLG FRPSULVLQJ WKH PDMRU SRUWLRQ RI LWV IDWW\DF\O UHVLGXHV +D\DVKLGD HW DO f 6XSSOHPHQWDWLRQ ZLWK SURWHROLSLG DFWXDOO\ LQFUHDVHG WKH SURSRUWLRQ RI SKRVSKDWLG\OFKROLQH DQG OLQROH\O IDWW\DF\O UHVLGXHV LQ WKH VDNH \HDVW SODVPD PHPEUDQH +D\DVKLGD DQG 2KWD f 7KH SKRVSKDWLG\OFKROLQH SURPRWHG \HDVW JURZWK DQG IHUPHQWDWLYH DFWLYLW\ ZKLOH DGGLWLRQ RI HUJRVWHUROROHDWH LQFUHDVHG VXUYLYDELOLW\ LQ WKH SUHVHQFH RI HWKDQRO +D\DVKLGD DQG 2KWD f 0DQ\ FRQILUPLQJ VWXGLHV KDYH VKRZQ WKDW DGGLWLRQ RI XQVDWXUDWHG OLSLGV LPSURYHV WKH IHUPHQWDWLYH SURGXFWLYLW\ RI \HDVW 'DPLDQR DQG :DQJ -DQVVHQV HW DO /DIRQ/DIRXUFDGH HW DO 2KWD DQG +D\DVKLGD :DWVRQ f $OWKRXJK PXFK LV NQRZQ DERXW WKH HIIHFWV RI HWKDQRO RQ YDULRXV DVSHFWV RI \HDVW IHUPHQWDWLRQ DQG DERXW WKH LQYROYHPHQW RI WKH SODVPD PHPEUDQH LQ PHGLDWLQJ PDQ\ RI WKHVH HIIHFWV YHU\ OLWWOH LV NQRZQ DERXW WKH IDFWRUV ZKLFK FDXVH WKH UDWH RI IHUPHQWDWLRQ WR GHFOLQH DV HWKDQRO DFFXPXODWHV LQ WKH PHGLXP 7KH IROORZLQJ VWXGLHV KDYH H[DPLQHG WKLV SKHQRPHQRQ LQ PRUH GHWDLO WR GHWHUPLQH WKH

PAGE 21

UROH RI HWKDQRO SURGXFHG GXULQJ IHUPHQWDWLRQ LQ FDXVLQJ WKLV GHFOLQH LQ HWKDQRO SURGXFWLRQ UDWH 2WKHU SRVVLEOH FDXVHV RI WKH GHFUHDVLQJ UDWH RI HWKDQRO SURGXFWLRQ GXULQJ EDWFK IHUPHQWDWLRQ VXFK DV QXWULHQW OLPLWDWLRQ DOVR ZHUH VWXGLHG )LQDOO\ WKH SK\VLRORJLFDO FKDQJHV ZKLFK DFFRPSDQ\ WKH GHFOLQLQJ IHUPHQWDWLRQ UDWH ZHUH FKDUDFWHUL]HG LQ RUGHU WR EHWWHU XQGHUVWDQG WKH FRQVWUDLQWV WKDW OLPLW WKH UDWH DW ZKLFK 6 FHUHYLVLDH SURGXFHV HWKDQRO

PAGE 22

&+$37(5 ,, &+$5$&7(5,=$7,21 2) 7+( '(&/,1,1* 5$7(6 2) *52:7+ $1' (7+$12/ 352'8&7,21 '85,1* %$7&+ )(50(17$7,21 %< 6$&&+$520<&(6 &(5(9,6,$( .' ,QWURGXFWLRQ
PAGE 23

PD[LPXP HWKDQRO WROHUDQFH IRU JURZWK RI 6 FHUHYLVLDH LV EHWZHHQ r& DQG r& 6LPLODUO\ WKH DELOLW\ WR VXUYLYH LQ WKH SUHVHQFH RI HWKDQRO GHFUHDVHG ZLWK LQFUHDVLQJ WHPSHUDWXUH &DVH\ DQG ,QJOHGHZ /HDR DQG YDQ 8GHQ D 1DJRGDZLWKDQD HW DO f (WKDQRO DOVR HQKDQFHG WKH WKHUPDO GHDWK UDWH RI 6 FHUHYLVLDH YDQ 8GHQ DQG GD &UX] 'XDUWH f ,Q FRQWUDVW WKH UDWH RI VXJDU FRQYHUVLRQ EHFDPH PRUH UHVLVWDQW WR HWKDQRO LQKLELWLRQ DV WKH IHUPHQWDWLRQ WHPSHUDWXUH ZDV UDLVHG WR r& %URZQ DQG 2OLYHU f $OFRKRO SURGXFWLRQ SURFHHGV DW DQ DFFHOHUDWHG SDFH DW WKH KLJKHU WHPSHUDWXUHV :KHQ RSWLPL]LQJ WKH FRQGLWLRQV IRU FDUU\LQJ RXW D IHUPHQWDWLRQ SURFHVV D FRPSURPLVH EHWZHHQ WKHVH FRPSHWLQJ IDFWRUV PXVW EH UHDFKHG 2WKHU HQYLURQPHQWDO IDFWRUV ZKLFK DIIHFW WKH DELOLW\ RI \HDVW WR WROHUDWH HWKDQRO LQFOXGH WKH VXJDU DQG R[\JHQ FRQFHQWUDWLRQV LQ WKH IHUPHQWDWLRQ EURWK -RQHV HW DO f *OXFRVH FRQFHQWUDWLRQV DERYH b GHFUHDVHG WKH DELOLW\ RI 6 FHUHYLVLDH WR FRQYHUW WKH VXJDU WR HWKDQRO *UD\ f 7KLV LQKLELWLRQ RFFXUUHG DV WKH FHOOV EHJDQ WR XQGHUJR SODVPRO\VLV DQG SUREDEO\ ZDV FDXVHG E\ WKH RVPRWLF HIIHFWV RI WKHVH KLJK DPRXQWV RI JOXFRVH RQ WKH \HDVW 2VPRWLF SUHVVXUH DOVR KDV DQ DGYHUVH HIIHFW RQ \HDVW FHOO YLDELOLW\ GXULQJ IHUPHQWDWLRQ 3DQFKDO DQG 6WHZDUW f 7KHVH HIIHFWV RI KLJK VXJDU FRQFHQWUDWLRQV DSSHDU WR EH V\QHUJLVWLF ZLWK WKH LQKLELWRU\ HIIHFWV RI HWKDQRO

PAGE 24

.XQNHH DQG $PHULQH 0RXOLQ HW DO f $ VPDOO DPRXQW RI R[\JHQ LQFUHDVHV WKH HWKDQRO WROHUDQFH RI 6 FHUHYLVLDH EHFDXVH LW LV UHTXLUHG IRU WKH V\QWKHVLV RI XQVDWXUDWHG OLSLGV DQG HUJRVWHURO +HQU\ f 7KHVH OLSLGV SURWHFW DQDHURELFDOO\ JURZQ \HDVW FHOOV IURP HWKDQRO LQGXFHG JURZWK LQKLELWLRQ ,QJUDP DQG %XWWNH f 8QGHU FRQWLQXRXV FXOWXUH FRQGLWLRQV WUDFH DPRXQWV RI R[\JHQ ZHUH VKRZQ WR GHFUHDVH WKH HWKDQRO LQKLELWLRQ RI JURZWK ZLWKRXW VLJQLILFDQWO\ DIIHFWLQJ WKH HWKDQRO \LHOG SHU DPRXQW RI VXEVWUDWH FRQVXPHG +RSSH DQG +DQVIRUG f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
PAGE 25

FRORQLHV RQ ODFWDWH DJDU SODWHV 2JXU DQG 6W -RKQ f DIWHU JURZWK IRU GD\V LQ EURWK FRQWDLQLQJ P0 0Q&O 3XWUDPHQW HW DO f ,W KDV EHHQ VSHFXODWHG WKDW PDQJDQHVH LQGXFHV PXWDWLRQV E\ LQWHUDFWLQJ ZLWK WKH PDQJDQHVHVHQVLWLYH PLWRFKRQGULDO '1$ SRO\PHUDVH FDXVLQJ HUURUSURQH UHSOLFDWLRQ RI WKH PLWRFKRQGULDO '1$ 6WUDLQ .' GLG QRW JURZ RQ JO\FHURO FRQWDLQLQJ PHGLXP RU UHGXFH WULSKHQ\OWHWUD]ROHXP FKORULGH 2JXU HW DO f ,W DOVR ODFNHG WKH F\WRFKURPH DD DEVRUEDQFH EDQGV DW QP DQG QP DQG WKH F\WRFKURPH E EDQGV DW QP DQG QP $ SHWLWH VWUDLQ ZDV FKRVHQ IRU WKLV VWXG\ EHFDXVH JURZWK DQG IHUPHQWDWLRQ KDYH EHHQ VKRZQ WR EH DOPRVW LGHQWLFDO ERWK DQDHURELFDOO\ DQG DHURELFDOO\ HOLPLQDWLQJ WKH QHHG WR SHUIRUP H[SHULPHQWV XQGHU DQDHURELF FRQGLWLRQV /RXUHLUR 'LDV DQG $UUDEDFD f ,Q VRPH VWXGLHV 6FHUHYLVLDH && DQG 6 FHUHYLVLDH $ 155/
PAGE 26

%DWFK IHUPHQWDWLRQV ZHUH FDUULHG RXW LQ PO WLVVXH FXOWXUH VSLQQHU ERWWOHV %OLFR *ODVV ,QF 9LQHODQG 1-f LPPHUVHG LQ D r& ZDWHU EDWK DQG DJLWDWHG DW USP &XOWXUH ERWWOHV ZHUH ILWWHG ZLWK ZDWHUWUDSSHG H[LW SRUWV IRU WKH HVFDSH RI FDUERQ GLR[LGH DQG VDPSOLQJ SRUWV IRU WKH UHPRYDO RI FXOWXUH E\ V\ULQJH *URZWK ZDV DOORZHG WR SURFHHG XQGHU FRQGLWLRQV RI VHOILQGXFHG DQDHURELRVLV ,QRFXOD ZHUH SUHSDUHG E\ WUDQVIHUULQJ FHOOV IURP D VODQW WR D WHVW WXEH FRQWDLQLQJ PO RI <(3' EURWK &HOOV ZHUH LQFXEDWHG DW r& IRU K ZLWKRXW DJLWDWLRQ DQG GLOXWHG LQWR PO RI IUAVK <(3' LQ D VSLQQHU ERWWOH 7KLV FXOWXUH ZDV LQFXEDWHG IRU DSSUR[LPDWHO\ K XQWLO DQ RSWLFDO GHQVLW\ DW QP RI PJ RI FHOO SURWHLQ SHU POf ZDV UHDFKHG )HUPHQWDWLRQV ZHUH VWDUWHG E\ GLOXWLQJ WKH K FXOWXUH LQWR PO RI JURZWK PHGLXP 3UHSDUDWLRQ RI )HUPHQWDWLRQ 6DPSOHV IRU $QDO\VLV )HUPHQWDWLRQ VDPSOHV ZHUH FHQWULIXJHG DW [ J IRU 2 PLQ 7KH VXSHUQDWDQW ZDV UHPRYHG DQG VDYHG E\ IUHH]LQJ DW r& &HOOV ZHUH ZDVKHG RQFH LQ P0 .+3 EXIIHU S+ f DQG WKH SHOOHWV ZHUH VDYHG IRU IXUWKHU DQDO\VLV E\ IUHH]LQJ DW r& $QDO\WLFDO 0HWKRGV (WKDQRO ZDV PHDVXUHG E\ JDVOLTXLG FKURPDWRJUDSK\ DV GHVFULEHG E\ *RHO DQG 3DPPHQW f ZLWK b YROYROf DFHWRQH DV DQ LQWHUQDO VWDQGDUG *OXFRVH ZDV LQLWLDOO\ GHWHUPLQHG ZLWK WKH JOXFRVH R[LGDVH SURFHGXUH 5DDER DQG

PAGE 27

7HUNLOGVHQ f XVLQJ WKH *OXFRVWDW UHDJHQWV VXSSOLHG E\ WKH 6LJPD &KHPLFDO &RPSDQ\ 6W /RXLV 0Rf ,Q ODWHU H[SHULPHQWV JOXFRVH ZDV PHDVXUHG ZLWK D <6, PRGHO JOXFRVH DQDO\]HU <6,
PAGE 28

FKURPDWRJUDSK\ VXSSOLHV ZHUH REWDLQHG IURP 6XSHOFR %HOOHIRQWH 3D 5HVXOWV %DWFK )HUPHQWDWLRQ E\ 6 FHUHYLVLDH .' $ W\SLFDO EDWFK IHUPHQWDWLRQ SURILOH RI 6 FHUHYLVLDH .' LQ <(3' PHGLXP LV VKRZQ LQ ILJXUH *OXFRVH FRQYHUVLRQ HVVHQWLDOO\ ZDV FRPSOHWHG DIWHU K XQGHU WKHVH FRQGLWLRQV ZLWK WKH SURGXFWLRQ RI EHWZHHQ DQG b YROYROf HWKDQRO &HOO SURWHLQ VWRSSHG LQFUHDVLQJ DIWHU K DW PJ SHU PO PHGLXP DOWKRXJK WKH RSWLFDO GHQVLW\ DW QP RI WKLV FXOWXUH FRQWLQXHG WR ULVH IRU DQ DGGLWLRQDO K SHULRG GDWD QRW VKRZQf 1HDUO\ LGHQWLFDO SURILOHV ZHUH REWDLQHG LQ PHGLXP VXSSOHPHQWHG ZLWK 7ZHHQ JOLWHUf OLQROHDWH PJOLWHUf DQG HUJRVWHURO PJOLWHUf 6LPLODU SURILOHV DOVR ZHUH REWDLQHG E\ WKH DGGLWLRQ RI VPDOO DPRXQWV RI 1 .2+ GXULQJ WKH FRXUVH RI IHUPHQWDWLRQ XVLQJ D S+ VWDW WR PDLQWDLQ WKH S+ RI WKH JURZWK PHGLXP DW /LNHZLVH EDWFK IHUPHQWDWLRQV RI 6 FHUHYLVLDH && WKH SDUHQWDO JUDQGH VWUDLQ ZHUH LQGLVWLQJXLVKDEOH IURP WKRVH RI VWUDLQ .' ,QKLELWLRQ RI *URZWK 5DWH E\ (WKDQRO $OWKRXJK LW LV QRW REYLRXV IURP ILJXUH WKH JURZWK UDWH RI 6 FHUHYLVLDH .' GHFUHDVHV DV HWKDQRO DFFXPXODWHV LQ WKH IHUPHQWDWLRQ EURWK )LJ f 8VLQJ WKH GDWD IURP EDWFK IHUPHQWDWLRQV UDWHV RI JURZWK ZHUH FDOFXODWHG DV WKH LQFUHDVH LQ FHOO SURWHLQ RYHU D K SHULRG DW WKH YDULRXV

PAGE 29

)LJXUH *URZWK DQG HWKDQRO SURGXFWLRQ E\ 6 FHUHYLVLDH .' GXULQJ D W\SLFDO EDWFK IHUPHQWDWLRQ LQ <(3' PHGLXP FRQWDLQLQJ b JOXFRVH 6\PEROV 4 FHOO SURWHLQ PJPO FXOWXUHf e JOXFRVH Â’ HWKDQRO

PAGE 30

(7+$12/ bYYf RU */8&26( bZYf Â’ &(// 3527(,1 PJPOf

PAGE 31

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‘ HIIHFW RI DGGHG HWKDQRO RQ JURZWK UDWH

PAGE 32

*52:7+ 5$7( Krf 1-

PAGE 33

WLPHV VDPSOHG GXULQJ WKH IHUPHQWDWLRQV *URZWK UDWH GHFUHDVHG H[SRQHQWLDOO\ DV HWKDQRO DFFXPXODWHG LQ WKH PHGLXP 7KH FRQFHQWUDWLRQ RI HWKDQRO WKDW KDG EHHQ SURGXFHG DW KDOI WKH PD[LPXP REVHUYHG JURZWK UDWH ZDV b YROYROf 7KH DGGLWLRQ RI HWKDQRO WR IHUPHQWDWLRQ EURWK DOVR GHFUHDVHG JURZWK UDWH )LJ f &HOOV LQRFXODWHG LQWR EURWK FRQWDLQLQJ LQFUHDVLQJ FRQFHQWUDWLRQV RI HWKDQRO ZHUH LQKLELWHG LQ D GRVHGHSHQGHQW PDQQHU 7KLV LQKLELWLRQ ZDV OLQHDU DERYH FRQFHQWUDWLRQV RI b YROYROf 7KH DPRXQW RI HWKDQRO UHTXLUHG WR GHFUHDVH WKH JURZWK UDWH E\ b ZDV b YROYROf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

PAGE 34

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e IHUPHQWDWLRQ UDWH GXULQJ EDWFK IHUPHQWDWLRQ ‘ HIIHFW RI DGGHG HWKDQRO RQ WKH IHUPHQWDWLRQ UDWH RI K FHOOV

PAGE 35

R )(50(17$7,9( $&7,9,7< HQ 2 f§ UR FM A R R R R 1-

PAGE 36

FHOOV H[KLELWHG D ELSKDVLF GHFOLQH DV D IXQFWLRQ RI DFFXPXODWHG HWKDQRO $Q LQLWLDO GHFOLQH LQ IHUPHQWDWLRQ UDWH RFFXUUHG GXULQJ WKH DFFXPXODWLRQ RI b YROYROf HWKDQRO ZLWK D b ORVV RI DFWLYLW\ 7KLV ZDV IROORZHG E\ D PRUH JUDGXDO GHFOLQH LQ IHUPHQWDWLRQ UDWH ZLWK DSSUR[LPDWHO\ b RI WKH RULJLQDO DFWLYLW\ UHPDLQLQJ DIWHU WKH SURGXFWLRQ b YROYROf HWKDQRO 7KH IHUPHQWDWLRQ UDWH RI 6 FHUHYLVLDH $ Sr D UHVSLUDWRU\GHILFLHQW KDSORLG ODERUDWRU\ \HDVW VWUDLQ DOVR GHFOLQHG DV HWKDQRO DFFXPXODWHG LQ WKH PHGLXP GDWD QRW VKRZQf $V ZLWK 6 FHUHYLVLDH .' D b GHFUHDVH LQ IHUPHQWDWLRQ UDWH ZDV REVHUYHG DIWHU WKH DFFXPXODWLRQ RI b YROYROf HWKDQRO +RZHYHU WKH PD[LPXP UDWH RI IHUPHQWDWLRQ ZDV ORZHU MDPOHV & SHU K SHU PJ SURWHLQ FRPSDUHG WR IRU VWUDLQ .' DQG JUHDWHU WKDQ b RI WKH PD[LPXP IHUPHQWDWLRQ UDWH ZDV ORVW E\ WKH WLPH b YROYROf HWKDQRO KDG DFFXPXODWHG 8QOLNH HWKDQRO DFFXPXODWHG GXULQJ IHUPHQWDWLRQ WKH DGGLWLRQ RI ORZ FRQFHQWUDWLRQV RI HWKDQRO WR UDSLGO\ IHUPHQWLQJ FHOOV K DIWHU LQRFXODWLRQ GLG QRW UHVXOW LQ D ODUJH GHFOLQH LQ IHUPHQWDWLYH DFWLYLW\ )LJ f (WKDQRO FDXVHG D GRVHGHSHQGHQW OLQHDU GHFOLQH LQ DFWLYLW\ )HUPHQWDWLRQ ZDV LQKLELWHG RQO\ b E\ WKH DGGLWLRQ RI b YROYROf HWKDQRO DQG b YROYROf DGGHG HWKDQRO ZDV UHTXLUHG WR FDXVH b LQKLELWLRQ

PAGE 37

'LVFXVVLRQ %HFDXVH WKH DELOLW\ RI HWKDQRO WR LQKLELW DOFRKRO SURGXFWLRQ YDULHV ZLWK WKH \HDVW VWUDLQ DQG IHUPHQWDWLRQ FRQGLWLRQV HPSOR\HG -RQHV HW DO f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f ZKLOH LQFUHDVLQJ WKH IHUPHQWDWLRQ UDWH DQG HWKDQRO WROHUDQFH RI RWKHU VWUDLQV %DFLOLD HW DO 0RXOLQ HW DO f 'LIIHUHQFHV EHWZHHQ ZLOGW\SH VWUDLQV WKH KDUVKQHVV RI WKH PXWDJHQLF WUHDWPHQWV DQG WKH OLPLWHG QXPEHUV RI PXWDQWV VFUHHQHG PD\ EH UHVSRQVLEOH IRU WKHVH FRQWUDGLFWRU\ UHSRUWV 7KH EUHZHU\ \HDVW VWUDLQ XVHG LQ PRVW RI WKHVH VWXGLHV H[KLELWHG DQ H[SRQHQWLDO GHFUHDVH LQ JURZWK UDWH )LJ f

PAGE 38

DQG D ELSKDVLF GHFUHDVH LQ UDWH RI DOFRKRO SURGXFWLRQ )LJ f DV HWKDQRO DFFXPXODWHG LQ WKH IHUPHQWDWLRQ EURWK 7KH H[SRQHQWLDOO\ IDOOLQJ JURZWK UDWH ZDV VLPLODU WR WKDW REVHUYHG E\ $LED HW DO f +RZHYHU WKH ELSKDVLF GHFUHDVH LQ HWKDQRO SURGXFWLRQ ILWV ERWK DQ H[SRQHQWLDO PRGHO DQG D OLQHDU PRGHO GHVFULELQJ D FRPELQDWLRQ RI WZR HYHQWV ,Q EDWFK IHUPHQWDWLRQV E\ VWUDLQ .' RQH FHOOXODU VLWH PD\ EH PXFK PRUH VHQVLWLYH WR WKH DFFXPXODWLRQ RI HWKDQRO WKDQ DQRWKHU 7KXV D ELSKDVLF LQKLELWLRQ SURILOH RFFXUV ZLWK WKH PRUH VHQVLWLYH VLWH EHLQJ FKDUDFWHUL]HG E\ WKH OLQH ZLWK WKH VWHHSHVW VORSH )HUPHQWDWLRQ UDWH GHFOLQHG E\ b DIWHU WKH DFFXPXODWLRQ RI DERXW WKUHH DQG RQHKDOI WLPHV PRUH HWKDQRO WKDQ ZDV DFFXPXODWHG ZKHQ JURZWK UDWH GHFUHDVHG E\ D VLPLODU DPRXQW )LJ DQG )LJ f &OHDUO\ HWKDQRO DGGHG WR WKH IHUPHQWDWLRQ EURWK OLQHDUO\ GHFUHDVHG ERWK WKH UDWH RI FHOO JURZWK DQG DOFRKRO SURGXFWLRQ DV SUHYLRXVO\ GHVFULEHG %URZQ HW DO 0RXOLQ HW DO f 7KLV FRQWUDVWV ZLWK WKH UHVXOWV RI /XRQJ f ZKR UHSRUWHG WKDW 6 FHUHYLVLDH $7&& H[KLELWHG QRQOLQHDU GHFUHDVHV LQ JURZWK DQG IHUPHQWDWLRQ UDWHV XQGHU DQDHURELF FRQGLWLRQV $V ZLWK WKH DFFXPXODWLRQ RI HWKDQRO JURZWK UDWH ZDV LQKLELWHG PRUH WKDQ IHUPHQWDWLRQ UDWH EXW ZDV RQO\ DERXW WZRIROG PRUH VHQVLWLYH *URZWK UDWH GHFUHDVHG IDVWHU WKDQ IHUPHQWDWLRQ UDWH DV HWKDQRO DFFXPXODWHG LQ WKH PHGLXP DQG DV LQFUHDVLQJ DPRXQWV RI HWKDQRO ZHUH DGGHG H[RJHQRXVO\ WR WKH PHGLXP 7KXV WKH

PAGE 39

DFWXDO DOFRKRO SURGXFWLRQ PDFKLQHU\ LV PRUH UHVLVWDQW WR HWKDQRO LQKLELWLRQ WKDQ LV FHOO JURZWK %URZQ HW DO /XRQJ f 7KH PDJQLWXGH RI WKH REVHUYHG LQKLELWLRQ KRZHYHU ZDV JUHDWHU IRU HQGRJHQRXVO\ SURGXFHG HWKDQRO WKDQ IRU H[RJHQRXVO\ DGGHG HWKDQRO IRU ERWK SURFHVVHV LQ DJUHHPHQW ZLWK SUHYLRXV UHSRUWV 0RXOLQ HW DO 1RYDN HW DO f 7KHVH UHVXOWV VXJJHVW WKDW WKH PHUH SUHVHQFH RI HWKDQRO PD\ QRW EH HQWLUHO\ VXIILFLHQW WR DFFRXQW IRU WKH REVHUYHG GHFOLQH LQ IHUPHQWDWLRQ UDWHV ,Q WKH IROORZLQJ VWXGLHV WKH FDXVDO UROH RI HWKDQRO LQ WKH GHFUHDVLQJ IHUPHQWDWLRQ UDWH ZDV FKRVHQ IRU PRUH GHWDLOHG H[DPLQDWLRQ 7KH UROH RI IDFWRUV RWKHU WKDQ HWKDQRO DOVR ZLOO EH VWXGLHG LQ RUGHU WR DFFRXQW PRUH IXOO\ IRU WKLV REVHUYHG GHFUHDVH LQ UDWH RI HWKDQRO SURGXFWLRQ

PAGE 40

&+$37(5 ,,, 1875,(17 /,0,7$7,21 $6 $ %$6,6 )25 7+( $33$5(17 72;,&,7< 2) /2: /(9(/6 2) (7+$12/ '85,1* %$7&+ )(50(17$7,21 ,QWURGXFWLRQ $V KDV DOUHDG\ EHHQ GHVFULEHG IRU 6DFFKDURPYFHV FHUHYLVLDH .' WKH UDWH RI DOFRKRO SURGXFWLRQ SHU XQLW FHOO PDVV GHFUHDVHV VXEVWDQWLDOO\ GXULQJ EDWFK IHUPHQWDWLRQV DV HWKDQRO DFFXPXODWHV LQ WKH PHGLXP )LJ f 7KLV GHFUHDVH KDV EHHQ DWWULEXWHG WR WKH LQKLELWRU\ HIIHFWV RI HWKDQRO E\ PRVW UHVHDUFKHUV $LED HW DO %D]XD DQG :LONH *KRVH DQG 7\DJL /XRQJ 0LOODU HW DO 0RXOLQ HW DO 5DKQ f +RZHYHU UHFHQW VWXGLHV E\ &DVH\ HW DO f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

PAGE 41

6WHZDUW /DEDWWV %UHZHU\ /RQGRQ &DQDGDf 7KLV RUJDQLVP ZDV JURZQ LQ <(3' PHGLXP DV GHVFULEHG LQ FKDSWHU ,, )HUPHQWDWLRQV ZHUH FDUULHG RXW DW r& LQ VSLQQHU ERWWOHV GHVLJQHG IRU WLVVXH FXOWXUH DOVR DV GHVFULEHG LQ FKDSWHU ,, &RQGLWLRQHG EURWK UHIHUV WR PHGLXP LQ ZKLFK FHOOV KDYH EHHQ DOORZHG WR JURZ IRU RU K DQG KDYH EHHQ UHPRYHG E\ FHQWULIXJDWLRQ 7KLV EURWK ZDV VWHULOL]HG E\ ILOWUDWLRQ $QDO\WLFDO 0HWKRGV &HOO PDVV ZDV PRQLWRUHG E\ PHDVXULQJ RSWLFDO GHQVLW\ DW QP XVLQJ D %DXVFK DQG /RPE 6SHFWURQLF VSHFWURSKRWRPHWHU 7RWDO FHOO SURWHLQ ZDV GHWHUPLQHG XVLQJ WKH PHWKRG RI /D\QH f &HOO YLDELOLW\ ZDV PHDVXUHG ZLWK WKH PHWK\OHQH EOXH G\H H[FOXVLRQ SURFHGXUH RI 7UHYRUV HW DO f (WKDQRO ZDV GHWHUPLQHG XVLQJ JDV FKURPDWRJUDSK\ DV GHVFULEHG E\ *RHO DQG 3DPPHQW f 5DWHV RI IHUPHQWDWLRQ ZHUH PHDVXUHG DV WKH UDWH RI & SURGXFWLRQ DW r& XQGHU D QLWURJHQ DWPRVSKHUH XVLQJ UHVSLURPHWU\ DV GHVFULEHG LQ FKDSWHU ,, 0HDVXUHPHQW RI WKH ,QWUDFHOOXODU (WKDQRO &RQFHQWUDWLRQ 7KH SURFHGXUH XVHG WR GHWHUPLQH WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI HWKDQRO LQ DFWLYHO\ IHUPHQWLQJ \HDVW FHOOV LV LOOXVWUDWHG LQ ILJXUH &HOOV IURP EDWFK IHUPHQWDWLRQV ZHUH FRQFHQWUDWHG E\ FHQWULIXJDWLRQ [ J PLQ DPELHQW WHPSHUDWXUHf DQG UHVXVSHQGHG LQ WKH VDPH PHGLXP WR D GHQVLW\ RI PJ RI FHOO SURWHLQ SHU PO >&@ VRUELWRO

PAGE 42

)LJXUH 'HWHUPLQDWLRQ RI LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQ $f )ORZ GLDJUDP LOOXVWUDWLQJ WKH SURFHGXUH XVHG WR GHWHUPLQH WKH DPRXQW RI HWKDQRO SUHVHQW ZLWKLQ FHOOV 7KLV PHWKRG RYHUHVWLPDWHV WKH DFWXDO LQWUDFHOOXODU YROXPH DFFHVVLEOH WR HWKDQRO E\ LQFOXGLQJ WKH FHOO YROXPH RFFXSLHG E\ VROLGV %f )ORZ GLDJUDP LOOXVWUDWLQJ WKH SURFHGXUH XVHG WR GHWHUPLQH WKH YROXPH RFFXSLHG E\ FHOO VROLGV 7KLV GHWHUPLQDWLRQ ZDV XVHG WR SURYLGH D FRUUHFWLRQ IDFWRU IRU WKH FDOFXODWLRQ RI HWKDQRO FRQFHQWUDWLRQ LQ WKH DTXHRXV FHOO YROXPH

PAGE 43

-?n ,175$&(//8/$5 (7+$12/ '(7(50,1$7,21 &RQFHQWUDWHG &HOO 6XVSHQVLRQ n &6RUELWRO 9RUWH[ VHF L 6DPSOH 6XVSHQVLRQ &6RUELWRO 'HWHUPLQDWLRQ &HQWULIXJH (WKDQRO 'HWHUPLQDWLRQ & &XLf R [ J VHFf (8f L 6DPSOH 6XSHUQDWDQW &f§6RUELWRO 'HWHUPLQDWLRQ : (WKDQRO 'HWHUPLQDWLRQ &(683f % &255(&7,21 )25 &(// 62/,'6 &RQFHQWUDWHG &HOO 6XVSHQVLRQ + &6RUELWRO 0L[ DQG (TXLOLEUDWH PLQ L 6DPSOH 6XVSHQVLRQ + DQG &f§6RUELWRO 'HWHUPLQDWLRQ FKXf &XEf &HQWULIXJH [ J VHFf 6DPSOH 6XSHUQDWDQW + DQG &f§6RUELWRO 'HWHUPLQDWLRQ Y QVXSI nnVXSrn 8! 2-

PAGE 44

VSHFLILF DFWLYLW\ S&LPPROHf ZDV DGGHG WR D PO VXVSHQVLRQ DW D ILQDO DFWLYLW\ RI Q&LPO 7KH VXVSHQVLRQ ZDV PL[HG IRU VHF XVLQJ D YRUWH[ PL[HU DQG PO VDPSOHV ZHUH WUDQVIHUUHG WR :KDWPDQ 00 ILOWHU SDSHU GLVNV FPf IRU VRUELWRO PHDVXUHPHQWV DQG WR VDPSOH YLDOV FRQWDLQLQJ PO SHUFKORULF DFLG IRU VXEVHTXHQW DOFRKRO GHWHUPLQDWLRQV 7KH UHPDLQLQJ VXVSHQVLRQ ZDV FHQWULIXJHG LPPHGLDWHO\ DW [ J IRU VHF LQ D PLFURFHQWULIXJH 6XSHUQDWDQW VDPSOHV RI PO WKHQ ZHUH WUDQVIHUUHG WR 00 ILOWHU SDSHU GLVNV DQG WR VDPSOH YLDOV FRQWDLQLQJ PO RI 0 SHUFKORULF DFLG IRU WKH PHDVXUHPHQW RI HWKDQRO )LOWHU GLVNV ZHUH DLU GULHG DW r& EHIRUH WKH DGGLWLRQ RI VFLQWLOODWLRQ IOXLG 7KH UDGLRDFWLYLW\ RI WKHVH VDPSOHV ZDV PHDVXUHG XVLQJ D %HFNPDQ PRGHO VFLQWLOODWLRQ VSHFWURPHWHU 7RWDO FHOO YROXPH ZDV HVWLPDWHG DV WKH GLIIHUHQFH LQ > &@VRUELWRO FRXQWV EHWZHHQ WKH VXVSHQVLRQ DQG WKH VXSHUQDWDQW $ FRUUHFWLRQ ZDV PDGH IRU WKH YROXPH RI WRWDO FHOO VROLGV LQFOXGHG LQ WKH VRUELWROEDVHG HVWLPDWH RI FHOO YROXPH )LJ Ef 7KLV ZDV GRQH LQ D VHSDUDWH H[SHULPHQW WR HQVXUH VXIILFLHQW WLPH IRU HTXLOLEUDWLRQ RI WULWLDWHG ZDWHU &RQWURO H[SHULPHQWV ZHUH SHUIRUPHG WR FRQILUP WKDW WULWLDWHG ZDWHU KDG UHDFKHG HTXLOLEULXP DIWHU PLQ DQG WKDW WKH VRUELWRO GLG QRW OHDN LQWR WKH FHOOV GXULQJ WKLV SHULRG 7ULWLDWHG ZDWHU DQG > &@ VRUELWRO VSHFLILF DFWLYLW\ S&LPPROHf ZHUH DGGHG WR FRQFHQWUDWHG FHOO VXVSHQVLRQV

PAGE 45

WR PJ RI FHOO SURWHLQ SHU POf DW D ILQDO DFWLYLW\ RI S&LPO DQG Q&LPO UHVSHFWLYHO\ $IWHU PLQ PO VDPSOHV ZHUH SLSHWWHG GLUHFWO\ LQWR VFLQWLOODWLRQ IOXLG IRU DTXHRXV VDPSOHV 7KH FHOO VROLG YROXPH ZDV FDOFXODWHG DV WKH GLIIHUHQFH EHWZHHQ WKH WULWLDWHG ZDWHU FRXQWV LQ WKH VXVSHQVLRQ DQG WKH VXSHUQDWDQW VDPSOHV 7KH IUDFWLRQ RI WKH WRWDO FHOO YROXPH RFFXSLHG E\ VROLGV ZDV FRPSXWHG DV IROORZV +68+VXSf 9 f L F8FXSf ZKHUH 9V LV WKH IUDFWLRQ RI VROLG YROXPH VXV LV WKH VXVSHQVLRQ DQG VXS LV WKH VXSHUQDWDQW IUDFWLRQ 7\SLFDOO\ WKH FHOO VROLG YROXPH UHSUHVHQWHG WR b RI WKH VRUELWRO H[FOXGHG YROXPH 7KH LQWUDFHOOXODU ZDWHU FRQWHQW GHFUHDVHG IURP SL SHU PJ FHOO SURWHLQ K DIWHU LQRFXODWLRQ WR SL SHU PJ FHOO SURWHLQ E\ K 7KH LQWUDFHOOXODU FRQFHQWUDWLRQ RI HWKDQRO ZDV FRPSXWHG EDVHG RQ WKH DTXHRXV FHOO YROXPH LH WKH VRUELWRO H[FOXGHG YROXPH PLQXV WKH VROLG YROXPH 7KLV ZDV FDOFXODWHG E\ DVVXPLQJ WKDW WKH DPRXQW RI HWKDQRO LQ WKH VXVSHQVLRQ LV HTXDO WR WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI HWKDQRO WLPHV WKH DTXHRXV FHOO YROXPH SOXV WKH FRQFHQWUDWLRQ RI HWKDQRO LQ

PAGE 46

WKH VXSHUQDWDQW WLPHV WKH VXSHUQDWDQW YROXPH DV IROORZV S S A8 A (VXV (HH,, f 9f (VXS f f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

PAGE 47

FHOO PDVV IROG DIWHU LQRFXODWLRQ 7\SLFDOO\ WKHVH VDPSOHV FRQWDLQ WR b YROYROf HWKDQRO DQG PJ FHOO SURWHLQ SHU PO FXOWXUH PHGLXP &HOOV ZKLFK KDYH SURGXFHG WR b YROYROf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b YROYROf HWKDQRO GLG QRW DIIHFW IHUPHQWDWLRQ UDWH 6XVSHQVLRQ RI FHOOV LQ WKH K FRQGLWLRQHG EURWK FRQWDLQLQJ b YROYROf HWKDQRO UHGXFHG WKH IHUPHQWDWLYH DFWLYLW\ RI K FHOOV EXW KDG OHVV HIIHFW RQ WKH DFWLYLW\ RI K FHOOV 5HPRYDO RI YRODWLOH PHGLXP FRPSRQHQWV IURP WKH K FRQGLWLRQHG EURWK HOLPLQDWHG LWV LQKLELWRU\ HIIHFW RQ WKH IHUPHQWDWLRQ UDWH RI K FHOOV EXW GLG QRW UHVXOW LQ D VLJQLILFDQW LQFUHDVH LQ DFWLYLW\ RI WKH K FHOOV 7KH DGGLWLRQ RI HWKDQRO WR WKH K FRQGLWLRQHG EURWK UHVWRUHG LWV DELOLW\ WR UHSUHVV WKH IHUPHQWDWLRQ UDWH RI K FHOOV LQGLFDWLQJ WKDW HWKDQRO ZDV

PAGE 48

7DEOH (IIHFWV RI HWKDQRO DQG IHUPHQWDWLRQ PHGLXP FRPSRVLWLRQ RQ IHUPHQWDWLRQ UDWH $VVD\ PHGLXP 5DWH RI SPROHV & SHU K )HUPHQWDWLRQ SHU PJ SURWHLQ 6'ff K FHOOV K FHOOV 2ULJLQDO EURWK f f )UHVK EURWK f f &RQGLWLRQHG EURWK K b YROYROf HWKDQROf f f &RQGLWLRQHG EURWK K b YROYROf HWKDQROf f f &RQGLWLRQHG EURWK K YRODWLOHV XQGHU YDFXXPf UHPRYHG f f &RQGLWLRQHG EURWK K YRODWLOHV UHPRYHG XQGHU YDFXXP UHFRQVWLWXWHG WR JLYH b YROYROf HWKDQROf f f &HOOV IURP K DQG K EDWFK IHUPHQWDWLRQV ZHUH KDUYHVWHG E\ FHQWULIXJDWLRQ DW DPELHQW WHPSHUDWXUH DQG VXVSHQGHG WR WKHLU RULJLQDO YROXPH LQ YDULRXV EURWKV :KHUH LQGLFDWHG YRODWLOHV ZHUH UHPRYHG IURP FRQGLWLRQHG EURWK E\ YDFXXP GLVWLOODWLRQ DW r& UHGXFLQJ WKH YROXPH E\ WZR WKLUGV 7KH EURWK WKHQ ZDV UHFRQVWLWXWHG ZLWK GLVWLOOHG ZDWHU RU GLVWLOOHG ZDWHU SOXV HWKDQRO )HUPHQWDWLRQ UDWHV ZHUH PHDVXUHG E\ UHVSLURPHWU\ $YHUDJHV DQG VWDQGDUG GHYLDWLRQV 6'f UHSUHVHQW WKH UHVXOWV IURP WKUHH VHSDUDWH EDWFK IHUPHQWDWLRQV

PAGE 49

WKH SULQFLSOH YRODWLOH FRPSRQHQW UHVSRQVLEOH IRU WKLV LQKLELWLRQ &HOO 9LDELOLW\ DQG 2YHUFURZGLQJ (IIHFWV RQ )HUPHQWDWLRQ 5DWH $ WULYLDO SRVVLELOLW\ IRU WKH IDLOXUH RI K FHOOV WR UHFRYHU DFWLYLW\ DIWHU VXVSHQVLRQ LQ EURWK ODFNLQJ HWKDQRO ZRXOG EH WKH SUHVHQFH RI ODUJH QXPEHUV RI GHDG FHOOV +RZHYHU EDVHG RQ PHWK\OHQH EOXH G\H H[FOXVLRQ RYHU b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f ,QWUDFHOOXODU (WKDQRO &RQFHQWUDWLRQ 7KH IDLOXUH RI K FHOOV WR UHFRYHU DFWLYLW\ DIWHU VXVSHQVLRQ LQ IUHVK PHGLXP FRXOG EH FDXVHG E\ WKH IDLOXUH RI WKH VXVSHQVLRQ SURFHGXUH WR HIIHFWLYHO\ UHPRYH WKH LQWUDFHOOXODU HWKDQRO RU E\ WKH DFFXPXODWLRQ RI ODUJH DPRXQWV RI LQWUDFHOOXODU HWKDQRO WKDW FRXOG SHUPDQHQWO\ GDPDJH WKH IHUPHQWDWLYH FDSDFLW\ RI WKH FHOOV 7R H[SORUH

PAGE 50

WKHVH SRVVLELOLWLHV WKH LQWUDFHOOXODU DQG H[WUDFHOOXODU FRQFHQWUDWLRQV RI HWKDQRO ZHUH PHDVXUHG DW K DQG K GXULQJ EDWFK IHUPHQWDWLRQV 7DEOH f 7KH H[WHUQDO HWKDQRO FRQFHQWUDWLRQV LQ WKHVH VXVSHQVLRQV DW WKH WLPH RI VDPSOLQJ ZHUH b YROYROf DQG b YROYROf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f DQG RI IHUPHQWDWLRQ SRWDVVLXP IOXRULGH ZDV DGGHG EHIRUH FHOO FRQFHQWUDWLRQ 3UHYLRXVO\ P0 SRWDVVLXP IOXRULGH ZDV GHWHUPLQHG WR FDXVH LPPHGLDWH FHVVDWLRQ RI & HYROXWLRQ GDWD QRW VKRZQf ,Q ERWK K DQG K IHUPHQWDWLRQ VDPSOHV WKH DGGLWLRQ RI IOXRULGH SUHYHQWHG WKH LQFUHDVH LQ H[WUDFHOOXODU HWKDQRO GXULQJ FHOO FRQFHQWUDWLRQ DQG VDPSOLQJ 7DEOH f 7KH UHPRYDO RI HWKDQRO E\ VXVSHQVLRQ RI FHOO SHOOHWV LQ IUHVK PHGLXP ODFNLQJ HWKDQRO VXEVWDQWLDOO\ GHFUHDVHG WKH LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQ 7DEOH f 5HJDUGOHVV

PAGE 51

7DEOH ,QWUDFHOOXODU DQG H[WUDFHOOXODU HWKDQRO FRQFHQWUDWLRQV XQGHU YDULRXV FRQGLWLRQV (WKDQRO FRQFHQWUDWLRQ b YROYROf 6'f LQ GLIIHUHQW PHGLD )UHVK 6DPSOH 1DWLYH 1DWLYH )UHVK b .) HWKDQRO E ,QW f f f f ([W f f f f E ,QW f f f f ([W f f f f 7KUHH RU PRUH LQGHSHQGHQW GHWHUPLQDWLRQV 1DWLYH UHIHUV WR WKH EURWK LQ WKH EDWFK IHUPHQWDWLRQ ZLWK RU ZLWKRXW DGGHG .) P0f )UHVK UHIHUV WR VWHULOH XQXVHG PHGLXP ZLWK RU ZLWKRXW DGGHG HWKDQRO b YROYROff E $JH RI EDWFK IHUPHQWDWLRQ ,QW DQG ([W UHIHU WR WKH LQWUDFHOOXODU DQG H[WUDFHOOXODU HWKDQRO FRQFHQWUDWLRQV UHVSHFWLYHO\

PAGE 52

RI WKH FHOO DJH DQG RULJLQDO HWKDQRO FRQFHQWUDWLRQ WKH LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQ ZDV IRXQG WR EH WR b YROYROf DIWHU HWKDQRO UHPRYDO 7KHVH YDOXHV ZHUH VRPHZKDW KLJKHU WKDQ DQWLFLSDWHG DQG DSSHDUHG WR EH GXH WR HWKDQRO SURGXFWLRQ E\ FRQWLQXHG PHWDEROLVP GXULQJ VXVSHQVLRQ DQG VDPSOLQJ 7KH LQFOXVLRQ RI SRWDVVLXP IOXRULGH GXULQJ KDUYHVWLQJ DQG VXVSHQVLRQ LQ IUHVK PHGLXP UHVXOWHG LQ D YHU\ ORZ LQWHUQDO DQG H[WHUQDO HWKDQRO FRQFHQWUDWLRQ b YROYROff FRQVLVWHQW ZLWK GLOXWLRQ RI WKH FHOO SHOOHW YROXPH ZLWK IUHVK PHGLXP ,Q DQ DQDORJRXV IDVKLRQ WKH IDLOXUH RI H[RJHQRXVO\ VXSSOLHG HWKDQRO WR UDLVH WKH LQWHUQDO HWKDQRO FRQFHQWUDWLRQ RI K FHOOV WR D OHYHO HTXLYDOHQW ZLWK WKDW RI FHOOV GXULQJ IHUPHQWDWLYH DOFRKRO SURGXFWLRQ FRXOG SURYLGH DQ H[SODQDWLRQ IRU WKH DSSDUHQW UHVLVWDQFH RI K FHOOV WR WKH LQKLELWRU\ HIIHFWV RI DGGHG HWKDQRO 7DEOH f 6DPSOHV WDNHQ DIWHU K DQG SURFHVVHG WR GHWHUPLQH WKH LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQ FRQWDLQHG DSSUR[LPDWHO\ b YROYROf 6' f HWKDQRO LQ WKH IHUPHQWDWLRQ EURWK 7KH LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQ RI WKHVH FHOOV ZDV b YROYROf 6' f 6XVSHQVLRQ RI K FHOOV LQ EURWK FRQWDLQLQJ b YROYROf HWKDQRO UHVXOWHG LQ DQ LQFUHDVH LQ WKH LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQ WR b YROYROf 6' f 7KHVH YDOXHV LQGLFDWH WKDW WKH DGGLWLRQ RI HWKDQRO WR K FHOOV LQFUHDVHG WKH LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQ WR WKH OHYHO IRXQG LQ FHOOV GXULQJ EDWFK

PAGE 53

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b YROYROf DIWHU ZKLFK WKH\ H[KLELWHG D GRVHGHSHQGHQW OLQHDU GHFOLQH LQ DFWLYLW\ XS WR EHWZHHQ DQG b YROYROf HWKDQRO :KHQ SORWWHG DV D SHUFHQWDJH RI PD[LPDO UDWH K FHOOV DSSHDUHG VOLJKWO\ PRUH UHVLVWDQW b LQKLELWLRQ DW b YROYROf HWKDQRO DV FRPSDUHG ZLWK b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f ,Q WKHVH H[SHULPHQWV FHOOV ZHUH JURZQ XQGHU D YDULHW\ RI FRQGLWLRQV KDUYHVWHG E\ FHQWULIXJDWLRQ DW

PAGE 54

)LJXUH ,QKLELWLRQ RI IHUPHQWDWLRQ UDWH RI K DQG K FHOOV E\ DGGHG HWKDQRO &HOOV ZHUH KDUYHVWHG E\ FHQWULIXJDWLRQ DQG VXVSHQGHG LQ IUHVK PHGLXP FRQWDLQLQJ YDULRXV FRQFHQWUDWLRQV RI HWKDQRO $f 'HFUHDVH LQ IHUPHQWDWLRQ UDWH DV D IXQFWLRQ RI DGGHG HWKDQRO %f ,QKLELWLRQ DV D SHUFHQWDJH GHFUHDVH RI WKH FRQWURO UDWH ODFNLQJ HWKDQRO 6\PEROV f K FHOOV ‘ K FHOOV

PAGE 55

(7+$12/ &21& b99f (7+$12/ &212 b99f 5$7( 2) )(50(17$7,21

PAGE 56

7DEOH (IIHFW RI JURZWK LQ EURWKV RI GLIIHUHQW FRPSRVLWLRQ RQ IHUPHQWDWLRQ UDWH ([SHULPHQW D SPROHV &&! ,QRFXOXP *URZWK ZLWK DGGHG HWKDQRO &RQWURO b YROYROf HWKDQRO ,QKLELWRU SURGXFWLRQ &RQWURO &RQGLWLRQHG EURWK K b YROYROf HWKDQRO VXSSOHPHQWHG ZLWK \HDVW H[WUDFW DQG JOXFRVHf &RQGLWLRQHG EURWK K b YROYROf HWKDQRO VXSSOHPHQWHG ZLWK \HDVW H[WUDFW DQG JOXFRVHf )HUPHQWDWLRQ 5DWH SURGXFHGK SHU PJ SURWHLQf K FHOOV K FHOOV f 1'E f 1'E f f f f f f 1XWULHQW OLPLWDWLRQ &RQWURO &RQGLWLRQHG EURWK K b YROYROf HWKDQRO VXSSOHPHQWHG ZLWK JOXFRVH DORQHf &RQGLWLRQHG EURWK K b YROYROf HWKDQRO VXSSOHPHQWHG ZLWK JOXFRVH DORQHf f f GRXEOLQJV DIWHU K GRXEOLQJV DIWHU K

PAGE 57

7DEOH FRQWLQXHG 1XWULHQW OLPLWDWLRQ ,, &RQWURO f f ; \HDVW H[WUDFW f f D ([SHULPHQWDO GHVLJQV DUH GHVFULEHG LQ PRUH GHWDLO LQ WKH WH[W )HUPHQWDWLRQV ZHUH FDUULHG RXW LQ YDULRXV PHGLD &HOOV ZHUH KDUYHVWHG E\ FHQWULIXJDWLRQ DW DPELHQW WHPSHUDWXUH DQG VXVSHQGHG WR RULJLQDO YROXPH LQ IUHVK EURWK <(3' PHGLXP LPPHGLDWHO\ SULRU WR PHDVXUHPHQW RI IHUPHQWDWLRQ UDWH E\ UHVSLURPHWU\ 1RW GHWHUPLQHG

PAGE 58

DPELHQW WHPSHUDWXUH DQG VXVSHQGHG LQ IUHVK PHGLXP ODFNLQJ HWKDQRO WR PHDVXUH WKH UDWH RI IHUPHQWDWLRQ XQGHU VWDQGDUG FRQGLWLRQV ,Q DOO H[SHULPHQWV WKH IHUPHQWDWLRQ UDWH RI FHOOV XVHG DV LQRFXOXP WR VWDUW WKHVH EDWFK IHUPHQWDWLRQV ZHUH LQFOXGHG DV FRQWUROV ([SHULPHQW H[DPLQHG WKH SRVVLELOLW\ WKDW WKH SK\VLRORJLFDO FKDQJHV LQ K FHOOV WR SURGXFH K FHOOV ZHUH GXH WR JURZWK LQ WKH SUHVHQFH RI HWKDQRO %DWFK IHUPHQWDWLRQV LQ ZKLFK b YROYROf HWKDQRO ZDV DGGHG SULRU WR LQRFXODWLRQ ZHUH DOORZHG WR JURZ WR WKH VDPH FHOO PDVV DV K FRQWURO FHOOV PJ FHOO SURWHLQ SHU PO IHUPHQWDWLRQ EURWK 7KH IHUPHQWDWLRQ UDWH RI WKHVH FHOOV JURZQ LQ WKH SUHVHQFH RI DGGHG HWKDQRO ZDV RQO\ VOLJKWO\ ORZHU WKDQ WKDW RI FRQWURO FHOOV JURZQ IRU K LQ WKH DEVHQFH RI HWKDQRO 7KLV LQGLFDWHG WKDW H[SRVXUH WR b YROYROf HWKDQRO GXULQJ JURZWK ZDV QRW VXIILFLHQW WR DFFRXQW IRU PRVW RI WKH REVHUYHG UHGXFWLRQ LQ IHUPHQWDWLRQ UDWH 7KH SRVVLELOLW\ WKDW JURZWK LQ WKH SUHVHQFH RI HWKDQRO DQG RWKHU IHUPHQWDWLRQ SURGXFWV PD\ EH UHVSRQVLEOH IRU WKH UHGXFWLRQ LQ IHUPHQWDWLRQ UDWH ZDV H[DPLQHG LQ H[SHULPHQW &XOWXUHV ZHUH LQRFXODWHG LQWR ERWWOHV FRQWDLQLQJ ILOWHU VWHULOL]HG FRQGLWLRQHG EURWK ZKLFK KDG EHHQ VXSSOHPHQWHG ZLWK J/ \HDVW H[WUDFW DQG HQRXJK JOXFRVH WR LQFUHDVH WKH FRQFHQWUDWLRQ LQ WKH EURWK EDFN WR b &RQGLWLRQHG EURWK IURP K FXOWXUHV FRQWDLQHG b YROYROf HWKDQRO DQG EURWK IURP K FXOWXUHV FRQWDLQHG b YROYROf HWKDQRO

PAGE 59

7KHVH IHUPHQWDWLRQV ZHUH DOORZHG WR SURFHHG XQWLO WKH FXOWXUH FHOO GHQVLW\ UHDFKHG WKH VWDWH GHILQHG DV K FHOOV DSSUR[LPDWHO\ PJ SURWHLQ SHU PO EURWK &HOOV JURZQ LQ WKH VXSSOHPHQWHG K FRQGLWLRQHG EURWK IHUPHQWHG DW UDWHV HTXDO WR WKRVH RI FRQWURO FHOOV 7KH IHUPHQWDWLRQ UDWH RI FHOOV JURZQ LQ WKH VXSSOHPHQWHG K EURWK ZDV ORZHU EXW ZDV DW OHDVW WZLFH WKDW RI WKH K FRQWURO $IWHU DOORZLQJ WKHVH IHUPHQWDWLRQV WR FRQWLQXH XQWLO b YROYROf HWKDQRO KDG EHHQ SURGXFHG LQ DGGLWLRQ WR WKDW SUHVHQW DW WKH WLPH RI LQRFXODWLRQ WKH IHUPHQWDWLRQ UDWH RI ERWK W\SHV RI K FHOOV ZHUH VLPLODU WR WKDW RI WKH FRQWURO FHOOV 7KXV WKH GHFOLQH LQ UDWH RI IHUPHQWDWLRQ REVHUYHG DIWHU WKH SURGXFWLRQ RI b YROYROf HWKDQRO LV QRW GXH HQWLUHO\ WR WKH DFFXPXODWLRQ RI HWKDQRO DQGRU RWKHU VWDEOH LQKLELWRUV LQ WKH IHUPHQWDWLRQ EURWK 7KH ODVW SRVVLELOLW\ H[DPLQHG LQ RUGHU WR XQGHUVWDQG WKH UHDVRQV IRU WKH GHFOLQH LQ IHUPHQWDWLRQ UDWH RI FHOOV DIWHU K ZDV WKH HIIHFW RI QXWULHQW OLPLWDWLRQ 1HLWKHU K FRQGLWLRQHG EURWK QRU K FRQGLWLRQHG EURWK VXSSOHPHQWHG ZLWK JOXFRVH VXSSRUWHG YLJRURXV JURZWK RI VWUDLQ .' IROORZLQJ UHLQRFXODWLRQ 7DEOH H[SHULPHQW f ,Q H[SHULPHQW WKH DGGLWLRQ RI \HDVW H[WUDFW UHVWRUHG WKH DELOLW\ RI FRQGLWLRQHG EURWK WR VXSSRUW JURZWK SURPRWLQJ IHUPHQWDWLRQ UDWHV HTXLYDOHQW WR WKH FRQWURO &HOOV JURZQ LQ EURWK FRQWDLQLQJ J/ \HDVW H[WUDFW IROG JUHDWHU WKDQ WKDW RI FRQWURO EURWK H[KLELWHG IHUPHQWDWLRQ UDWHV

PAGE 60

HTXLYDOHQW WR FRQWURO FHOOV DIWHU K 7KHVH ZHUH WZLFH DV KLJK DV FRQWURO FHOOV DIWHU WKH SURGXFWLRQ RI b YROYROf HWKDQRO LQ DSSUR[LPDWHO\ K 7DEOH H[SHULPHQW f 'LVFXVVLRQ 3UHYLRXV VWXGLHV KDYH VKRZQ WKDW WKH UDWH RI DOFRKRO SURGXFWLRQ E\ \HDVW SHU XQLW FHOO PDVV GHFUHDVHV DV HWKDQRO DFFXPXODWHV GXULQJ IHUPHQWDWLRQ +ROW]EHUJ HW DO 1DYDUUR DQG 'XUDQG 6WUHKDLDQR DQG *RPD f 0RVW RI WKHVH VWXGLHV KDYH DWWULEXWHG WKLV UHGXFWLRQ LQ IHUPHQWDWLRQ UDWH WR DGYHUVH HIIHFWV RI HWKDQRO ,QJUDP DQG %XWWNH 0DLRUHOOD HW DO 0LOODU HW DO 0RXOLQ HW DO f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f 7R H[SODLQ WKLV DQRPDO\ LW KDV EHHQ SURSRVHG WKDW WKH OHDNDJH RI HWKDQRO IURP \HDVW FHOOV LV LQ VRPH ZD\ OLPLWHG E\ WKH SHUPHDELOLW\ RI WKH SODVPD PHPEUDQH 7KLV ZRXOG UHVXOW LQ WKH DFFXPXODWLRQ RI KLJK F\WRVROLF

PAGE 61

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f 7KLV ZDV LQ ODUJH SDUW GXH WR WKH FRQWLQXHG SURGXFWLRQ RI HWKDQRO E\ WKH FHOOV LQ SHOOHWV GXULQJ FHQWULIXJDWLRQ DQG SURFHVVLQJ 'DVDUL HW DO f 7KH DFXWHQHVV RI WKLV SUREOHP DOVR ZRXOG EH H[SHFWHG WR GHFUHDVH DV IHUPHQWDWLRQ UDWH DQG VXEVWUDWH OHYHOV GHFOLQHG GXULQJ EDWFK IHUPHQWDWLRQV 7KH UHGXFWLRQ LQ DSSDUHQW LQWUDFHOOXODUH[WUDFHOOXODU UDWLRV RI HWKDQRO REVHUYHG E\ %HDYHQ HW DO f GXULQJ WKH ODWWHU VWDJHV RI IHUPHQWDWLRQ VXSSRUWV WKLV LGHD 'DVDUL HW DO f GHPRQVWUDWHG WKDW SUHFRROLQJ WKH FXOWXUH VLJQLILFDQWO\

PAGE 62

UHGXFHG WKH HUURU LQWURGXFHG E\ FRQWLQXHG HWKDQRO SURGXFWLRQ GXULQJ FHOO KDUYHVWLQJ +RZHYHU FRROLQJ PD\ LQWURGXFH RWKHU SRWHQWLDO SUREOHPV DVVRFLDWHG ZLWK WHPSHUDWXUH LQGXFHG FKDQJHV LQ WKH RUJDQL]DWLRQ RI DQG SHUPHDELOLW\ SURSHUWLHV RI WKH SODVPD PHPEUDQH 7KH VHFRQG W\SH RI H[SHULPHQWDO SUREOHP DVVRFLDWHG ZLWK PHDVXUHPHQWV RI LQWHUQDO HWKDQRO LQYROYHV ZDVKLQJ RI WKH \HDVW FHOOV ([SHULPHQWDO GHVLJQV ZKLFK LQFOXGHG ZDVKLQJ RI FHOOV 1DJRGDZLWKDQD DQG 6WHLQNUDXV 3DQFKDO DQG 6WHZDUW f EHIRUH HVWLPDWLRQ RI HWKDQRO UHVXOWHG LQ ORZHU DSSDUHQW LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQV WKDQ GR XQZDVKHG VDPSOHV %HDYHQ HW DO 'DVDUL HW DO f %HDYHQ HW DO f FOHDUO\ VKRZHG WKDW HYHQ PLQLPDO ZDVKLQJ OHDFKHV PRVW RI WKH LQWUDFHOOXODU HWKDQRO IURP WKH FHOOV 7KHVH WZR H[SHULPHQWDO GHVLJQV PHDVXUHPHQW RI HWKDQRO LQ D PHWDEROLFDOO\ DFWLYH FHOO SHOOHW DQG ZDVKLQJ HDFK LQWURGXFH HUURUV ZKLFK FKDQJH WKH FDOFXODWHG YDOXHV RI LQWUDFHOOXODU HWKDQRO LQ RSSRVLWH ZD\V 5HFHQW VWXGLHV E\ *XLMDUUR DQG /DJXQDV f KDYH HPSOR\HG D SURFHGXUH ZKLFK HOLPLQDWHG WKHVH WZR EDVLF SUREOHPV LQ H[SHULPHQWDO GHVLJQ E\ XVLQJ JODVV ILEHU ILOWHUV WR UDSLGO\ KDUYHVW FHOOV :LWK WKLV PHWKRG H[WUDFHOOXODUO\ DGGHG >&@ HWKDQRO UDSLGO\ HTXLOLEUDWHG ZLWK WKH LQWUDFHOOXODU HQYLURQPHQW LQGLFDWLQJ WKDW WKH SODVPD PHPEUDQH LV IUHHO\ SHUPHDEOH WR HWKDQRO +RZHYHU WKLV VWLOO GRHV QRW DQVZHU WKH TXHVWLRQ RI WKH WUXH LQWUDFHOOXODU

PAGE 63

HWKDQRO FRQFHQWUDWLRQ LQ \HDVW FHOOV GXULQJ DFWLYH IHUPHQWDWLRQ DQG HWKDQRO SURGXFWLRQ ,Q WKH VWXGLHV SUHVHQWHG LQ WKLV FKDSWHU WKH LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQ ZDV HVWLPDWHG E\ DQ LQGHSHQGHQW PHWKRG XVLQJ FHOOV WKDW ZHUH DFWLYHO\ SURGXFLQJ HWKDQRO LQ VXVSHQVLRQ FXOWXUH 7KH UHVXOWV REWDLQHG XVLQJ WKLV PHWKRG FRQILUP WKH UHSRUWV E\ %HDYHQ HW DO‘ f DQG *XLMDUUR DQG /DJXQDV f ZKLFK LQGLFDWHG WKDW \HDVW FHOOV DUH IUHHO\ SHUPHDEOH WR HWKDQRO ,Q DGGLWLRQ WKHVH UHVXOWV SURYLGH GLUHFW HYLGHQFH WKDW WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI HWKDQRO SURGXFHG GXULQJ IHUPHQWDWLRQ LV QRW VHYHUDOIROG KLJKHU WKDQ WKDW RI WKH VXUURXQGLQJ PHGLXP DV SURSRVHG SUHYLRXVO\ %HDYHQ HW DO 1DJRGDZLWKDQD DQG 6WHLQNUDXV 1RYDN HW DO 6WUHKDLDQR DQG *RPD f ,GHQWLFDO FRQFOXVLRQV ZHUH UHDFKHG E\ 'DVDUL HW DO f XVLQJ KLJK FHOO GHQVLW\ IHUPHQWDWLRQV ZKLFK DOORZHG UDSLG SURFHVVLQJ RI WKH FHOOV IRU DQDO\VLV 7KHUH GRHV QRW DSSHDU WR EH DQ\ SUREOHP DVVRFLDWHG ZLWK WKH HIILFLHQW GLIIXVLRQ RI HWKDQRO IURP \HDVW FHOOV LQWR WKH HQYLURQPHQW GXULQJ IHUPHQWDWLRQ 7KXV LW LV XQOLNHO\ WKDW WKH UHWHQWLRQ RI XQXVXDOO\ KLJK LQWUDFHOOXODU HWKDQRO FRQFHQWUDWLRQV FRQWULEXWHV WRZDUG WKH GHFUHDVH LQ IHUPHQWDWLYH DFWLYLW\ RI 6 FHUHYLVLDH GXULQJ IHUPHQWDWLRQ 5HFHQWO\ &DVH\ HW DO f KDYH VKRZQ WKDW \HDVW QXWULWLRQDO UHTXLUHPHQWV OLPLW IHUPHQWDWLYH DFWLYLW\ LQ KLJK JUDYLW\ EUHZLQJ 6XSSOHPHQWLQJ ZRUWV ZLWK \HDVW

PAGE 64

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b YROYROf HWKDQRO DSSHDUV WR UHVXOW IURP WKH FRPELQDWLRQ RI D VPDOO LQKLELWRU\ HIIHFW RI HWKDQRO DQG SK\VLRORJLFDO FKDQJHV LQ WKH FHOOV 7KHVH SK\VLRORJLFDO FKDQJHV ZHUH QRW LQGXFHG E\ JURZWK LQ WKH SUHVHQFH RI b YROYROf DGGHG HWKDQRO RU E\ JURZWK LQ WKH SUHVHQFH RI HWKDQRO DORQJ ZLWK RWKHU QDWXUDO IHUPHQWDWLRQ SURGXFWV &RQGLWLRQHG EURWK ZDV GHILFLHQW LQ QXWULHQWV SURYLGHG E\ \HDVW H[WUDFW DQG VXSSRUWHG YHU\ OLWWOH JURZWK 7KH DGGLWLRQ RI J/ RI \HDVW H[WUDFW UHVWRUHG WKH DELOLW\ RI WKLV VSHQW EURWK WR VXSSRUW YLJRURXV JURZWK DQG IHUPHQWDWLRQ %\ IXUWKHU LQFUHDVLQJ WKH FRQFHQWUDWLRQ RI \HDVW H[WUDFW WR J/ LQ WKH JURZWK PHGLXP WKH GHFOLQH LQ IHUPHQWDWLYH DFWLYLW\ DVVRFLDWHG ZLWK WKH LQLWLDO SURGXFWLRQ RI b YROYROf HWKDQRO ZDV SDUWLDOO\ SUHYHQWHG 7KHVH UHVXOWV VXSSRUW WKH K\SRWKHVLV WKDW SK\VLRORJLFDO FKDQJHV LQ WKH FHOOV FDXVHG E\ QXWULHQW OLPLWDWLRQ DUH PDMRU IDFWRUV LQ

PAGE 65

WKH LQLWLDO b GHFOLQH LQ IHUPHQWDWLYH DFWLYLW\ )XUWKHU VWXGLHV ZLOO LQFOXGH LGHQWLILFDWLRQ RI WKLV OLPLWLQJ QXWULHQW DQG XSRQ VXSSOHPHQWDWLRQ FKDUDFWHUL]DWLRQ RI LWV HIIHFW RQ JURZWK DQG IHUPHQWDWLRQ

PAGE 66

&+$37(5 ,9 0$*1(6,80 /,0,7$7,21 $1' ,76 52/( ,1 7+( $33$5(17 72;,&,7< 2) (7+$12/ '85,1* <($67 )(50(17$7,21 ,QWURGXFWLRQ 7KH UDWH RI HWKDQRO SURGXFWLRQ E\ 6DFFKDURPYFHV VSS GHFUHDVHV LQ EDWFK IHUPHQWDWLRQV DV DOFRKRO DFFXPXODWHV LQ WKH PHGLXP 0RXOLQ HW DO 5DKQ 6WUHKDLDQR DQG *RPD f 7KH RQVHW RI WKLV GHFOLQH LQ IHUPHQWDWLYH DFWLYLW\ RFFXUV DW YHU\ ORZ HWKDQRO FRQFHQWUDWLRQV RIWHQ OHVV WKDQ b YROYROf 6LQFH HWKDQRO KDV EHHQ VKRZQ WR LQKLELW IHUPHQWDWLRQ %URZQ HW DO &\VHZVNL DQG :LONH *UD\ f LW JHQHUDOO\ KDV EHHQ DFFHSWHG WKDW WKLV DFFXPXODWLRQ RI HWKDQRO LV UHVSRQVLEOH IRU WKH SURJUHVVLYH GHFOLQH LQ IHUPHQWDWLYH DFWLYLW\ %D]XD DQG :LONH *KRVH DQG 7\DJL /XRQJ f +RZHYHU WKH H[WHQW RI LQKLELWLRQ E\ H[RJHQRXVO\ DGGHG HWKDQRO LV OHVV WKDQ ZRXOG EH SUHGLFWHG E\ WKH GHFOLQH LQ IHUPHQWDWLRQ UDWH ZKLFK QRUPDOO\ RFFXUV GXULQJ WKH IHUPHQWDWLYH DFFXPXODWLRQ RI HWKDQRO )LJ f )XUWKHU VWXGLHV KDYH DWWHPSWHG WR GHILQH WKH PHFKDQLVPVf RI HWKDQRO LQKLELWLRQ RI IHUPHQWDWLRQ DQG WR UHFRQFLOH WKH IDLOXUH RI DGGHG HWKDQRO WR LQKLELW IHUPHQWDWLRQ WR WKH H[WHQW REVHUYHG GXULQJ WKH IHUPHQWDWLYH DFFXPXODWLRQ RI HWKDQRO (DUO\ H[SHULPHQWV SURYLGHG

PAGE 67

HYLGHQFH WKDW WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI HWKDQRO ZDV PXFK KLJKHU WKDQ WKDW RI WKH VXUURXQGLQJ PHGLXP GXULQJ IHUPHQWDWLRQ 1DJRGDZLWKDQD DQG 6WHLQNUDXV 1DYDUUR DQG 'XUDQG 3DQFKDO DQG 6WHZDUW f D FRQGLWLRQ QRW UHDGLO\ GXSOLFDWHG E\ H[RJHQRXVO\ DGGHG HWKDQRO +RZHYHU WKHVH HDUO\ GDWD FDQ EH H[SODLQHG E\ SUREOHPV LQ WKH PHDVXUHPHQW RI LQWHUQDO HWKDQRO FRQFHQWUDWLRQV 'DVDUL HW DO f 6HYHUDO UHVHDUFK JURXSV KDYH GHYHORSHG LQGHSHQGHQW PHWKRGV ZKLFK GHPRQVWUDWHG WKDW HWKDQRO LV IUHHO\ SHUPHDEOH LQ 6DFFKDURPYFHV VSS DQG WKDW WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI WKLV PHWDEROLF SURGXFW LV WKH VDPH DV WKDW LQ WKH VXUURXQGLQJ IHUPHQWDWLRQ EURWK 'DVDUL HW DO *XLMDUUR DQG /DJXQDV 7DEOH f $GGLWLRQDO VWXGLHV KDYH LQYHVWLJDWHG WKH VHQVLWLYLW\ RI JO\FRO\WLF HQ]\PHV DQG DOFRKRORJHQLF HQ]\PHV WR LQ YLWUR LQKLELWLRQ E\ HWKDQRO 0LOODU HW DO f KDYH VKRZQ WKDW WKHVH HQ]\PHV DUH VWDEOH LQ HWKDQRO FRQFHQWUDWLRQV KLJKHU WKDQ b YROYROf 7KH WZR HQ]\PHV PRVW VHQVLWLYH WR LQKLELWLRQ E\ HWKDQRO ZHUH S\UXYDWH GHFDUER[\ODVH DQG SKRVSKRJO\FHUDWH NLQDVH %RWK KRZHYHU UHWDLQHG b RI PD[LPDO DFWLYLW\ LQ WKH SUHVHQFH RI RYHU b YROYROf HWKDQRO WKH ILQDO DOFRKRO FRQFHQWUDWLRQ DFKLHYHG E\ WKH FRPSOHWH IHUPHQWDWLRQ RI J RI JOXFRVH SHU / RI EURWK 6LPLODUO\ /DUXH HW DO f FRQFOXGHG WKDW WKH FHVVDWLRQ RI DOFRKRO SURGXFWLRQ GXULQJ VWXFN IHUPHQWDWLRQV ZDV QRW GXH

PAGE 68

WR HWKDQRO LQKLELWLRQ RI DOFRKRO GHK\GURJHQDVH DQG KH[RNLQDVH DFWLYLWLHV &DVH\ HW DO f KDYH UHSRUWHG WKDW QXWULHQW OLPLWDWLRQ LV D PDMRU IDFWRU UHVWULFWLQJ WKH HWKDQRO SURGXFWLYLW\ RI KLJKJUDYLW\ IHUPHQWDWLRQV $QDHURELFDOO\ FXOWXUHG \HDVWV DUH NQRZQ WR KDYH D QXWULWLRQDO UHTXLUHPHQW IRU HUJRVWHURO DQG XQVDWXUDWHG OLSLGV +RVVDFN DQG 5RVH 1HV HW DO 3URXGORFN HW DO f 8QVDWXUDWHG OLSLGV KDYH EHHQ VKRZQ WR LQFUHDVH ELRPDVV DOFRKRO SURGXFWLRQ DQG HWKDQRO GXUDELOLW\ RI \HDVW FHOOV GXULQJ DQDHURELF IHUPHQWDWLRQ ,QJUDP DQG %XWWNH -DQVVHQV HW DO /DIRQ/DIRXUFDGH HW DO 7KRPDV HW DO f $ YDULHW\ RI OLSLGSURWHLQ FRPSOH[HV DQG QXWULHQW VXSSOHPHQWV UDQJLQJ IURP DOEXPLQHUJRVWHURO PRQRROHLQ WR VR\ IORXU DQG \HDVW H[WUDFW DOVR KDYH EHHQ VKRZQ WR \LHOG LQFUHDVHG UDWHV RI DOFRKRO SURGXFWLRQ DQG KLJKHU ILQDO HWKDQRO FRQFHQWUDWLRQV 'DPLDQR DQG :DQJ +D\DVKLGD HW DO /DIRQ/DIRXUFDGH HW DO 2KWD DQG +D\DVKLGD f 7KH VWXGLHV GHVFULEHG LQ FKDSWHU ,,, VXJJHVW WKDW WKH LQLWLDO GHFOLQH LQ IHUPHQWDWLYH DFWLYLW\ GXULQJ EDWFK IHUPHQWDWLRQ RI b JOXFRVH LV QRW FDXVHG E\ WKH SUHVHQFH RI HWKDQRO RU E\ JURZWK LQ WKH SUHVHQFH RI b YROYROf HWKDQRO 7KHVH VWXGLHV LQGLFDWHG WKDW D FRPSRQHQWVf RI \HDVW H[WUDFW ZDV OLPLWLQJ FHOO JURZWK DQG WKDW WKLV OLPLWDWLRQ FRQWULEXWHG WR WKH HDUO\ ORVV RI IHUPHQWDWLYH

PAGE 69

DFWLYLW\ 7KH UHVXOWV SUHVHQWHG LQ WKLV FKDSWHU LGHQWLI\ PDJQHVLXP DV WKH OLPLWLQJ FRPSRQHQW RI \HDVW H[WUDFW DQG GHPRQVWUDWH WKDW ZKHQ WKLV QXWULHQW OLPLWDWLRQ LV UHOLHYHG D GUDPDWLF GHFUHDVH LQ WKH WLPH UHTXLUHG IRU WRWDO FRQYHUVLRQ RI JOXFRVH WR HWKDQRO LV DFKLHYHG 7KLV GHFUHDVH LQ WLPH UHTXLUHG IRU WKH FRPSOHWLRQ RI IHUPHQWDWLRQ UHVXOWHG IURP D GHOD\ LQ WKH RQVHW RI VWDWLRQDU\ SKDVH ZKLFK LQFUHDVHG WKH WRWDO FHOO QXPEHU GXULQJ WKDW SDUW RI IHUPHQWDWLRQ LQ ZKLFK RYHU b RI WKH HWKDQRO LV SURGXFHG 0DWHULDOV DQG 0HWKRGV 2UJDQLVPV DQG *URZWK &RQGLWLRQV 7KH SULQFLSDO RUJDQLVP XVHG LQ WKHVH VWXGLHV ZDV 6DFFKDURPYFHV FHUHYLVLDH .' GHVFULEHG LQ FKDSWHU ,, ,Q DGGLWLRQ 6 FHUHYLVLDH && 6 FHUHYLVLDH $ 155/ < f DQG 6 VDNH 155/
PAGE 70

f DQG WKH SHOOHWV ZHUH VDYHG IRU IXUWKHU DQDO\VLV E\ IUHH]LQJ DW r& 3UHSDUDWLRQ RI *OXFRVH5HFRQVWLWXWHG 0HGLXP IRU *URZWK ([SHULPHQWV %DWFK IHUPHQWDWLRQV ZHUH DOORZHG WR UHDFK DQ RSWLFDO GHQVLW\ DW QP RI &HOOV ZHUH UHPRYHG E\ FHQWULIXJDWLRQ LQ D 6RUYDOO 5&% FHQWULIXJH DW [ J IRU PLQ 7KH DPRXQW RI HWKDQRO LQ WKH VXSHUQDWDQW ZDV GHWHUPLQHG DQG XVHG WR HVWLPDWH WKH DPRXQW RI JOXFRVH QHHGHG WR UHFRQVWLWXWH WKH PHGLXP WR D FRQFHQWUDWLRQ RI b 7KLV JOXFRVHUHFRQVWLWXWHG PHGLXP ZDV VWHULOL]HG E\ YDFXXP ILOWUDWLRQ ZLWK SP 0HWULFHO PHPEUDQH ILOWHUV *HOPDQ 6FLHQFHV ,QF $QQ $UERU 0LFKf 3UHSDUDWLRQ RI $VKHG 0HGLXP &RPSRQHQWV
PAGE 71

DQG DJLWDWHG USPf LQ D 5RWRWRUTXH FXOWXUH URWDWRU &ROH 3DUPHU &KLFDJR ,OOfr 0HGLXP $QDO\VHV (WKDQRO DQG JOXFRVH ZHUH PHDVXUHG DV GHVFULEHG LQ FKDSWHU ,, 7KH PDJQHVLXP FRQFHQWUDWLRQ RI WKH PHGLXP ZDV PHDVXUHG ZLWK WKH 6HFRQG 0DJQHVLXP UHDJHQWV SXUFKDVHG IURP $PHULFDQ 0RQLWRU &RUSRUDWLRQ ,QGLDQDSROLV ,QG DV GHVFULEHG E\ 2VPDQ DQG ,QJUDP f &HOOXODU $QDO\VHV DQG 5HVSLURPHWUY 0HDVXUHPHQWV &HOO PDVV DQG WRWDO FHOO SURWHLQ ZHUH PHDVXUHG DV GHVFULEHG LQ FKDSWHU ,, 7R GHWHUPLQH WKH DPRXQW RI LQWUDFHOOXODU PDJQHVLXP \HDVW FHOOV ZHUH ZDVKHG RQFH LQ P0 .+3 EXIIHU S+ f DQG WKH FHOO SHOOHWV ZHUH VWRUHG IUR]HQ DW r& XQWLO DQDO\]HG 7KHVH \HDVW SHOOHWV FRQWDLQHG WR PJ RI FHOO SURWHLQ DQG ZHUH SHUPHDELOL]HG E\ LQFXEDWLRQ LQ D ERLOLQJZDWHU EDWK IRU PLQ 7KH UHVXOWLQJ GHEULV ZDV VXVSHQGHG LQ PO RI P0 .+3 EXIIHU S+ f DQG WKHQ SHOOHWHG 7KH VXSHUQDWDQW ZDV DQDO\]HG IRU PDJQHVLXP DV GHVFULEHG DERYH 5HVSLURPHWU\ PHDVXUHPHQWV ZHUH PDGH DV GHVFULEHG LQ FKDSWHU ,, DQG IHUPHQWDWLRQ UDWHV ZHUH FDOFXODWHG IURP WKHVH YDOXHV DV SPROHV RI & SURGXFHG SHU K SHU PJ FHOO SURWHLQ 9LDEOH&HOO 'HWHUPLQDWLRQV &HOO QXPEHUV ZHUH GHWHUPLQHG PLFURVFRSLFDOO\ ZLWK D 3HWURII+DXVVHU FRXQWLQJ FKDPEHU 9LDEOHFHOO FRXQWV ZHUH

PAGE 72

GHWHUPLQHG E\ WKH PHWK\OHQH EOXH VWDLQLQJ SURFHGXUH RI 0LOOV f &KHPLFDOV
PAGE 73

7DEOH (IIHFW RI QXWULHQW VXSSOHPHQWDWLRQ RQ JURZWK RI 6 FHUHYLVLDH .' 0HGLXP 6XSSOHPHQW 2SWLFDO 'HQVLW\ DW QP DIWHU K 6'f b RI FRQWURO 6'f <(3' 1RQH f KD 1RQH f f KD
PAGE 74

OHYHO UHVSHFWLYHO\ 7DEOH f 7KHVH UHVXOWV LQGLFDWH WKDW QXWULHQW OLPLWDWLRQ UDWKHU WKDQ WKH SUHVHQFH RI DQ LQKLELWRU ZDV UHVSRQVLEOH IRU WKH LQDELOLW\ RI WKH XVHG PHGLXP WR VXSSRUW IXUWKHU \HDVW JURZWK 9LWDPLQ VXSSOHPHQWV DOVR ZHUH WHVWHG DQG GLG QRW SURPRWH JURZWK LQ WKLV JOXFRVH UHFRQVWLWXWHG PHGLXP GDWD QRW VKRZQf 7KH RUJDQLF FRPSRQHQWV RI \HDVW H[WUDFW DQG SHSWRQH DUH ERWK GLYHUVH DQG FRPSOH[ %HIRUH HPEDUNLQJ RQ D IUDFWLRQDWLRQ RI WKHVH WKH LQRUJDQLF FRQVWLWXHQWV ZHUH WHVWHG DIWHU DVKLQJ 6XSSOHPHQWDWLRQ ZLWK DVKHG \HDVW H[WUDFW ZDV DV HIIHFWLYH DV ZLWK ZKROH \HDVW H[WUDFW ZKLOH DVKHG SHSWRQH ZDV RQO\ KDOI DV HIIHFWLYH DV ZKROH SHSWRQH 7KHVH UHVXOWV VXJJHVWHG WKDW DQ LQRUJDQLF FRPSRQHQW RI <(3' PHGLXP ZDV WKH SULQFLSOH IDFWRU OLPLWLQJ JURZWK 7KH LQRUJDQLF FRQVWLWXHQWV RI D PLQHUDOEDVHG PLQLPDO PHGLXP ZHUH WHVWHG WR GHWHUPLQH ZKLFK LRQV ZHUH OLPLWLQJ 7DEOH f 7KH DGGLWLRQ RI SRWDVVLXP DPPRQLXP VRGLXP FDOFLXP SKRVSKDWH VXOIDWH DQG D WUDFH PLQHUDO PL[WXUH GHVFULEHG E\ :LFNHUVKDP f GLG QRW SURPRWH JURZWK LQ JOXFRVHUHFRQVWLWXWHG PHGLXP 2QO\ PDJQHVLXP VDOWV ZHUH HIIHFWLYH DV QXWULHQW VXSSOHPHQWV DOORZLQJ JURZWK HTXLYDOHQW WR b RI WKH FRQWURO LQ IUHVK <(3' PHGLXP 7KH GRVHUHVSRQVH RI JURZWK WR DGGHG PDJQHVLXP \HDVW H[WUDFW DQG DVKHG \HDVW H[WUDFW LV VKRZQ LQ ILJXUH
PAGE 75

)LJXUH 'RVHUHVSRQVH RI FHOO JURZWK WR DGGHG PDJQHVLXP 0DJQHVLXP YDOXHV UHSUHVHQW WKH DPRXQW RI PDJQHVLXP FRQWDLQHG LQ WKH DGGHG QXWULHQW VXSSOHPHQW (UURU EDUV UHSUHVHQW WKH DYHUDJH VWDQGDUG GHYLDWLRQ IRU HDFK H[SHULPHQW 6\PEROV ‘ ZKROH \HDVW H[WUDFW DGGHG WR JOXFRVHUHFRQVWLWXWHG XVHG PHGLXP 2 DVKHG \HDVW H[WUDFW DGGHG WR JOXFRVHUHFRQVWLWXWHG XVHG PHGLXP 0J6 DGGHG WR JOXFRVHUHFRQVWLWXWHG XVHG PHGLXP ’ 0J6 DGGHG WR IUHVK <(3' EURWK

PAGE 77

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f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f DW K IROORZHG E\ D GHFOLQH

PAGE 78

)LJXUH 0DJQHVLXP OHYHOV LQ EURWK DQG FHOOV GXULQJ WKH FRXUVH RI EDWFK IHUPHQWDWLRQ $f ,QWUDFHOOXODU PDJQHVLXP %f 0DJQHVLXP FRQFHQWUDWLRQ LQ WKH FXOWXUH EURWK 5HVXOWV KDYH EHHQ SORWWHG IRU IRXU VHSDUDWH EDWFK IHUPHQWDWLRQV &ORVHG V\PEROV UHSUHVHQW IHUPHQWDWLRQV VXSSOHPHQWHG ZLWK P0 0J6&! DQG RSHQ V\PEROV UHSUHVHQW FRQWURO IHUPHQWDWLRQV LQ <(3' EURWK DORQH

PAGE 79

7,0( Kf 7,0( Kf

PAGE 80

WR DERXW QPROHV RI PDJQHVLXP SHU PJ FHOO SURWHLQ DIWHU K 0DJQHVLXPVXSSOHPHQWHG FXOWXUHV PDLQWDLQHG D KLJKHU OHYHO RI FHOOXODU PDJQHVLXP WKURXJKRXW IHUPHQWDWLRQ WKDQ FXOWXUHV JURZQ LQ XQVXSSOHPHQWHG <(3' PHGLXP 0DJQHVLXP/LPLWHG *URZWK RI 2WKHU
PAGE 81

(IIHFW RI 0DJQHVLXP 6XSSOHPHQWDWLRQ RI %DWFK )HUPHQWDWLRQ 7KH HIIHFWV RI VXSSOHPHQWLQJ <(3' PHGLXP ZLWK P0 0J6 RQ EDWFK IHUPHQWDWLRQ DUH LOOXVWUDWHG LQ ILJXUH 7KH SURGXFWLRQ RI FHOO PDVV DV PHDVXUHG E\ FHOOXODU SURWHLQ LV VKRZQ LQ ILJXUH $ 6XSSOHPHQWDWLRQ ZLWK PDJQHVLXP SURORQJHG WKH H[SRQHQWLDO ULVH LQ FHOOXODU SURWHLQ DOORZLQJ D b LQFUHDVH LQ FHOO PDVV RYHU WKDW RI WKH FRQWURO ZLWKLQ K DIWHU LQRFXODWLRQ 7KH DGGLWLRQ RI PDJQHVLXP DOVR LQFUHDVHG WKH UDWH DW ZKLFK JOXFRVH ZDV FRQVXPHG DQG HWKDQRO ZDV SURGXFHG )LJ % DQG &f $IWHU K RI LQFXEDWLRQ PDJQHVLXPVXSSOHPHQWHG FXOWXUHV KDG SURGXFHG RQHWKLUG PRUH HWKDQRO WKDQ WKH FRQWUROV 7KH FRQYHUVLRQ RI JOXFRVH WR HWKDQRO ZDV FRPSOHWH DIWHU K LQ PDJQHVLXPVXSSOHPHQWHG FXOWXUHV EXW UHTXLUHG K LQ FRQWURO <(3' EURWK 7KH ILQDO \LHOG RI HWKDQRO ZDV HVVHQWLDOO\ LGHQWLFDO IRU ERWK PDJQHVLXPVXSSOHPHQWHG DQG FRQWURO FXOWXUHV b YROYROf b RI WKHRUHWLFDO PD[LPXP \LHOGf (IIHFW RI 0DJQHVLXP 6XSSOHPHQWDWLRQ RQ 5DWH RI )HUPHQWDWLRQ 6DPSOHV ZHUH UHPRYHG IURP PDJQHVLXPVXSSOHPHQWHG DQG FRQWURO IHUPHQWDWLRQV DW YDULRXV WLPHV GXULQJ EDWFK IHUPHQWDWLRQ (WKDQRO FRQFHQWUDWLRQ FHOO SURWHLQ DQG & HYROXWLRQ RI XQZDVKHG FHOOV ZHUH PHDVXUHG )LJXUH VKRZV WKH IHUPHQWDWLRQ UDWH DV D IXQFWLRQ RI DFFXPXODWHG HWKDQRO %RWK FRQWURO DQG PDJQHVLXPVXSSOHPHQWHG FXOWXUHV H[KLELWHG WKH VDPH PD[LPXP UDWH RI IHUPHQWDWLRQ DW b YROYROf HWKDQRO +RZHYHU PDJQHVLXPVXSSOHPHQWHG FXOWXUHV

PAGE 82

)LJXUH (IIHFW RI PDJQHVLXP DGGLWLRQ RQ FHOO JURZWK DQG IHUPHQWDWLRQ $f *URZWK %f *OXFRVH XWLOL]DWLRQ &f (WKDQRO SURGXFWLRQ 5HVXOWV KDYH EHHQ SORWWHG IRU IRXU VHSDUDWH EDWFK IHUPHQWDWLRQV &ORVHG V\PEROV UHSUHVHQW IHUPHQWDWLRQV VXSSOHPHQWHG ZLWK P0 0J62 DQG RSHQ V\PEROV UHSUHVHQW FRQWURO IHUPHQWDWLRQV LQ <(3' EURWK DORQH

PAGE 83

7,0( 7,0( Kf 7,0( &(// 3527(,1 PJPOf f§ UR 2L R E E E (7+$12/ bYYf $ 2f &' 2

PAGE 84

)LJXUH (IIHFW RI DGGHG PDJQHVLXP RQ WKH UDWH RI IHUPHQWDWLRQ )HUPHQWDWLRQ UDWHV ZHUH PHDVXUHG E\ UHVSLURPHWU\ RI XQZDVKHG FHOOV LPPHGLDWHO\ DIWHU VDPSOLQJ DQG DUH SORWWHG DV D IXQFWLRQ RI DFFXPXODWHG HWKDQRO IRU IRXU VHSDUDWH EDWFK IHUPHQWDWLRQV &ORVHG V\PEROV UHSUHVHQW FXOWXUHV VXSSOHPHQWHG ZLWK P0 0J6 DQG RSHQ V\PEROV UHSUHVHQW FRQWURO IHUPHQWDWLRQV LQ <(3' EURWK DORQH

PAGE 85

)(50(17$7,21 5$7( MPROHV &K SHU PJ SURWHLQf UR A Gf R R R R nM /Q

PAGE 86

PDLQWDLQHG D KLJKHU UDWH RI IHUPHQWDWLRQ DV HWKDQRO DFFXPXODWHG GXULQJ WKH FRPSOHWLRQ RI WKH EDWFK IHUPHQWDWLRQ 7KLV UDWH ZDV b KLJKHU WKDQ WKDW RI FRQWURO FHOOV DIWHU WKH DFFXPXODWLRQ RI b YROYROf HWKDQRO 7KH IHUPHQWDWLRQ UDWH RI ERWK VXSSOHPHQWHG DQG XQVXSSOHPHQWHG FXOWXUHV IHOO SUHFLSLWRXVO\ DW DERXW b YROYROf HWKDQRO FRLQFLGHQW ZLWK H[KDXVWLRQ RI JOXFRVH (IIHFW RI 0DJQHVLXP $GGLWLRQ RQ &HOO 9LDELOLW\ 7KH SHUFHQWDJH RI YLDEOH FHOOV LQ ERWK PDJQHVLXP VXSSOHPHQWHG DQG XQVXSSOHPHQWHG EDWFKHV UHPDLQHG JUHDWHU WKDQ b IRU WKH ILUVW K RI WKH IHUPHQWDWLRQ *OXFRVH ZDV H[KDXVWHG DW WKLV WLPH LQ VXSSOHPHQWHG FXOWXUHV DQG WKH SHUFHQWDJH RI YLDEOH FHOOV EHJDQ WR GHFUHDVH UHDFKLQJ b E\ K 7KH XQVXSSOHPHQWHG EDWFKHV FRQVXPHG JOXFRVH PRUH VORZO\ DQG PDLQWDLQHG KLJK YLDELOLW\ !bf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f 0DJQHVLXP

PAGE 87

)LJXUH (IIHFW RI PDJQHVLXP VXSSOHPHQWDWLRQ RQ WKH LQKLELWLRQ RI IHUPHQWDWLRQ UDWH E\ DGGHG HWKDQRO (UURU EDUV UHSUHVHQW DYHUDJH VWDQGDUG GHYLDWLRQV IRU WKUHH VHSDUDWH GHWHUPLQDWLRQV $f 6DPSOHV IURP EDWFK IHUPHQWDWLRQV WKDW KDG DFFXPXODWHG b YROYROf HWKDQRO %f 6DPSOHV IURP EDWFK IHUPHQWDWLRQV WKDW KDG DFFXPXODWHG b YROYROf HWKDQRO 6\PEROV f P0 0J62 VXSSOHPHQWHG IHUPHQWDWLRQV 2 FRQWURO IHUPHQWDWLRQV LQ <(3' EURWK DORQH

PAGE 88

(7+$12/ bYYf (7+$12/ bYYf )(50(17$7,9( $&7,9,7< b LQLWLDO UDWHf UR P &' R R R R R R R )(50(17$7,9( $&7,9,7< b LQLWLDO UDWHf UR HQ RV R 2 2 2 2 2 2 /

PAGE 89

VXSSOHPHQWHG DQG XQVXSSOHPHQWHG FHOOV KDG D VLPLODU LQLWLDO IHUPHQWDWLRQ UDWH DERXW SPROHV RI & SURGXFHG SHU K SHU PJ FHOO SURWHLQ DQG H[KLELWHG LGHQWLFDO GRVHUHVSRQVH FXUYHV $ FRQFHQWUDWLRQ RI b YROYROf HWKDQRO UHVXOWHG LQ b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b YROYROf KLJKHU WKDQ LQ WKH XQVXSSOHPHQWHG FXOWXUH ZKHQ FRPSDUHG DW HTXDO IHUPHQWDWLRQ UDWHV 7KH IHUPHQWDWLRQ UDWH RI PDJQHVLXPVXSSOHPHQWHG DQG FRQWURO IHUPHQWDWLRQV H[KLELWHG OLQHDU GRVHUHVSRQVHV WR HWKDQRO DGGLWLRQ $ PHDVXUH RI WKH VHQVLWLYLW\ RI IHUPHQWDWLRQ UDWH WR HWKDQRO LV WKH VORSH RI WKH GRVHUHVSRQVH FXUYH 7KH VORSH IRU VXSSOHPHQWHG EDWFKHV ZDV ZLWK DQ 6' RI ZKLOH WKDW RI WKH FRQWUROV ZDV ZLWK DQ 6' RI 7KH GLIIHUHQFHV LQ WKHVH VORSHV )LJ %f DUH VXJJHVWLYH EXW GR QRW FRQFOXVLYHO\ GHPRQVWUDWH WKDW VXSSOHPHQWDWLRQ ZLWK PDJQHVLXP UHGXFHG WKH VHQVLWLYLW\ RI IHUPHQWDWLRQ LQ ROGHU FHOOV WR LQKLELWLRQ E\ HWKDQRO

PAGE 90

'LVFXVVLRQ 7KH GHFOLQH LQ IHUPHQWDWLRQ UDWH WKDW EHJLQV DW ORZ DOFRKRO FRQFHQWUDWLRQV GRHV QRW VHHP WR EH H[FOXVLYHO\ FDXVHG E\ WKH LPPHGLDWH SUHVHQFH RI HWKDQRO E\ JURZWK LQ WKH SUHVHQFH RI HWKDQRO RU E\ FHOO GHDWK &KDSWHU ,,,f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b JOXFRVH WR HWKDQRO E\ RQHWKLUG LQ PDJQHVLXPVXSSOHPHQWHG FXOWXUHV 'XULQJ EDWFK IHUPHQWDWLRQ \HDVW FHOOV FRQFHQWUDWHG PDJQHVLXP IURP WKH PHGLXP ,Q XQVXSSOHPHQWHG FXOWXUHV PDJQHVLXP XSWDNH VWRSSHG DW WKH HQG RI H[SRQHQWLDO JURZWK $W WKLV SRLQW WKH FRQFHQWUDWLRQ RI PDJQHVLXP LQ WKH PHGLXP ZDV S0 ZLWKLQ WKH UDQJH RI YDOXHV UHSRUWHG IRU PDJQHVLXP WUDQVSRUW E\ PLFURRUJDQLVPV -DVSHU DQG 6LOYHU f 7KH HQG RI H[SRQHQWLDO JURZWK DOVR FRLQFLGHG ZLWK WKH EHJLQQLQJ RI WKH GHFOLQH LQ IHUPHQWDWLYH DFWLYLW\ ,Q

PAGE 91

PDJQHVLXPVXSSOHPHQWHG FXOWXUHV KLJKHU OHYHOV RI LQWUDFHOOXODU PDJQHVLXP ZHUH DFKLHYHG HDUO\ LQ IHUPHQWDWLRQ DQG GHFUHDVHG WR D OHVVHU H[WHQW WKDQ REVHUYHG LQ XQVXSSOHPHQWHG FXOWXUHV 0DJQHVLXPVXSSOHPHQWHG FXOWXUHV KDG WLPHV PRUH PDJQHVLXP LQ WKH PHGLXP DW WKH HQG RI H[SRQHQWLDO JURZWK WKDQ GLG XQVXSSOHPHQWHG FXOWXUHV 7KXV WKH PDJQHVLXP VXSSO\ RI WKH VXSSOHPHQWHG FXOWXUH DSSHDUV WR EH DGHTXDWH IRU JURZWK DQG RWKHU IDFWRUV DUH OLPLWLQJ WKH IHUPHQWDWLYH DELOLW\ RI WKH \HDVWV XQGHU WKHVH FRQGLWLRQV 7KH XELTXLWRXV UROH RI PDJQHVLXP LQ FHOOXODU SURFHVVHV LV ZHOO GRFXPHQWHG -DVSHU DQG 6LOYHU f 0DJQHVLXP FRQVWLWXWHV D PDMRU SRUWLRQ RI WKH FHOOXODU FDWLRQV PRVWO\ ERXQG LQ VWUXFWXUHV VXFK DV ULERVRPHV DQG WKH FHOO HQYHORSH 7KH IUHH FDWLRQ FRQFHQWUDWLRQ KRZHYHU PD\ SOD\ D PRUH GLUHFW UROH LQ UHJXODWLQJ RYHUDOO FHOOXODU PHWDEROLVP DQG FHOO GLYLVLRQ :DONHU DQG 'XIIXV f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

PAGE 92

PDJQHVLXP 2VPDQ DQG ,QJUDP f 7KH DGGLWLRQ RI PDJQHVLXP VDOWV DW P0 VXEVWDQWLDOO\ UHYHUVHG WKH LQKLELWRU\ HIIHFWV RI XS WR b YROYROf HWKDQRO $OWKRXJK DQDORJRXV VWXGLHV KDYH QRW EHHQ SHUIRUPHG ZLWK 6 FHUHYLVLDH LW LV OLNHO\ WKDW HWKDQRO DOVR LQFUHDVHV WKH OHDNDJH RI VPDOO PROHFXOHV LQ WKLV RUJDQLVP &DVH\ HW DO f DOVR KDYH UHSRUWHG WKDW QXWULHQW OLPLWDWLRQ LV DQ LPSRUWDQW IDFWRU LQ OLPLWLQJ WKH SURGXFWLYLW\ RI D IHUPHQWDWLRQ 6XSSOHPHQWDWLRQ RI KLJK JUDYLW\ EUHZLQJ ZRUW FRQWDLQLQJ XS WR b GLVVROYHG VROLGVf ZLWK \HDVW H[WUDFW HUJRVWHURO DQG ROHLF DFLG DOORZHG WKH SURGXFWLRQ RI b YROYROf HWKDQRO E\ EUHZHUVn \HDVW +LJKHU UDWHV RI DOFRKRO SURGXFWLRQ SULPDULO\ UHVXOWHG IURP DQ LQFUHDVH LQ FHOO PDVV DVVRFLDWHG ZLWK QXWULHQW VXSSOHPHQWHG IHUPHQWDWLRQV DQG GLG QRW DSSHDU WR LQFOXGH DQ LQFUHDVH LQ WKH UHVLVWDQFH RI IHUPHQWDWLRQ UDWH WR HWKDQRO $GGLWLRQ RI QXWULHQWV LQ WKH IRUP RI VR\ IORXU WR IHUPHQWDWLRQ EURWK KDV EHHQ VKRZQ WR LQFUHDVH WKH IHUPHQWDWLYH SURGXFWLYLW\ RI ERWK 6 FHUHYLVLDH 'DPLDQR DQG :DQJ f DQG = PRELOLV -X HW DO f 9LHJDV HW DO f DOVR UHSRUWHG WKDW VR\ IORXU DGGLWLRQ WR D \HDVW H[WUDFWEDVHG PHGLXP FRQWDLQLQJ WR b JOXFRVH HQKDQFHG WKH UDWH RI HWKDQRO SURGXFWLRQ E\ 6 ED\DQXV $JDLQ VXSSOHPHQWDWLRQ OHG WR DQ LQFUHDVH LQ FHOO FRQFHQWUDWLRQ ,W ZDV IXUWKHU GHPRQVWUDWHG WKDW WKH DTXHRXV IUDFWLRQ RI VR\ IORXU UDWKHU WKDQ WKH OLSLG IUDFWLRQ FRQWDLQHG WKH

PAGE 93

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b IXUWKHU LQFUHDVH LQ FHOO PDVV ZDV REVHUYHG LQGLFDWLQJ WKDW DQRWKHU IDFWRUVf EHFRPHV OLPLWLQJ IRU JURZWK DQG IHUPHQWDWLRQ DW WKLV SRLQW ,QGHHG D FRPSOHWH XQGHUVWDQGLQJ RI WKH ELRFKHPLFDO EDVLV IRU WKH GHFOLQH LQ IHUPHQWDWLRQ UDWH LQ \HDVWV PD\ UHTXLUH GHWHUPLQDWLRQ RI WKH IDFWRUV UHVSRQVLEOH IRU WKH WHUPLQDWLRQ RI H[SRQHQWLDO JURZWK DQG WKH DVVRFLDWHG SK\VLRORJLFDO DQG HQ]\PDWLF FKDQJHV

PAGE 94

&+$37(5 9 */<&2/<7,& (1=<0(6 $1' ,17(51$/ S+ ,QWURGXFWLRQ 6DFFKDURPYFHV FHUHYLVLDH LV FDSDEOH RI YHU\ UDSLG UDWHV RI JO\FRO\VLV DQG HWKDQRO SURGXFWLRQ XQGHU RSWLPDO FRQGLWLRQV SURGXFLQJ RYHU SPROHV RI HWKDQRO SHU K SHU PJ RI FHOO SURWHLQ )LJ f +RZHYHU WKLV KLJK UDWH LV PDLQWDLQHG IRU RQO\ D EULHI SHULRG GXULQJ EDWFK IHUPHQWDWLRQ DQG GHFOLQHV SURJUHVVLYHO\ DV HWKDQRO DFFXPXODWHV LQ WKH VXUURXQGLQJ EURWK &DVH\ DQG ,QJOHGHZ ,QJUDP DQG %XWWNH 0RXOLQ HW DO f (DUOLHU VWXGLHV KDYH LGHQWLILHG D UHTXLUHPHQW IRU OLSLGV %HDYHQ HW DO &DVH\ HW DO 7KRPDV HW DO f RU PROHFXODU R[\JHQ IRU OLSLG ELRV\QWKHVLV $QGUHDVHQ DQG 6WLHU %XWWNH HW DO %XWWNH DQG 3\OH f LQ PDQ\ IHUPHQWDWLRQ EURWKV DV EHLQJ HVVHQWLDO IRU WKH PDLQWHQDQFH RI KLJK IHUPHQWDWLYH DFWLYLW\ 0DJQHVLXP LV DQ HVVHQWLDO FRIDFWRU IRU PDQ\ RI WKH JO\FRO\WLF HQ]\PHV DQG KDV EHHQ LGHQWLILHG DOVR DV D OLPLWLQJ QXWULHQW LQ IHUPHQWDWLRQ EURWK FRQWDLQLQJ SHSWRQH DQG \HDVW H[WUDFW &KDSWHU ,9f 6XSSO\LQJ WKHVH QXWULWLRQDO QHHGV UHGXFHV EXW GRHV QRW HOLPLQDWH WKH GHFOLQH LQ IHUPHQWDWLYH DFWLYLW\ GXULQJ EDWFK IHUPHQWDWLRQ )LJ f

PAGE 95

7KH EDVLV IRU WKH GHFOLQH LQ IHUPHQWDWLRQ UDWH LV QRW IXOO\ XQGHUVWRRG 6LQFH WKH DGGLWLRQ RI HWKDQRO WR FHOOV LQ EDWFK FXOWXUHV DQG LQ FKHPRVWDWV FDXVHV D GRVHGHSHQGHQW LQKLELWLRQ RI HWKDQRO SURGXFWLRQ &DVH\ DQG ,QJOHGHZ )LJ f PRVW LQYHVWLJDWLRQV KDYH IRFXVHG RQ HWKDQRO DV WKH LQKLELWRU\ DJHQW &DVH\ DQG ,QJOHGHZ ,QJUDP DQG %XWWNH 0LOODU HW DO f (WKDQRO LV NQRZQ WR DOWHU PHPEUDQH SHUPHDELOLW\ DQG GLVUXSW PHPEUDQH IXQFWLRQ LQ D YDULHW\ RI ELRORJLFDO V\VWHPV &DVH\ DQG ,QJOHGHZ ,QJUDP DQG %XWWNH f ,Q \HDVW HWKDQRO FDXVHV DQ LQFUHDVH LQ K\GURJHQ LRQ IOX[ DFURVV WKH SODVPD PHPEUDQH RI FHOOV VXVSHQGHG LQ ZDWHU &DUWZULJKW HW DO f 7KLV LQFUHDVHG K\GURJHQ LRQ IOX[ KDV EHHQ SURSRVHG DV EHLQJ UHVSRQVLEOH IRU WKH HWKDQROLQGXFHG GHFOLQH LQ WUDQVSRUW UDWHV REVHUYHG XQGHU VLPLODU FRQGLWLRQV %HDYHQ HW DO /HDR DQG YDQ 8GHQ E D Ef (YLGHQFH KDV EHHQ DFFXPXODWLQJ ZKLFK LQGLFDWHV WKDW WKH SUHVHQFH RI HWKDQRO PD\ QRW EH WKH RQO\ IDFWRU UHVSRQVLEOH IRU WKH GHFOLQH LQ IHUPHQWDWLYH DFWLYLW\ 7KH UHSODFHPHQW RI IHUPHQWDWLYH EURWK FRQWDLQLQJ HWKDQRO ZLWK IUHVK PHGLXP ODFNLQJ HWKDQRO GLG QRW LPPHGLDWHO\ UHVWRUH IHUPHQWDWLYH DFWLYLW\ 7DEOH f ,Q D FRPSUHKHQVLYH VWXG\ 0LOODU HW DO f GHPRQVWUDWHG WKDW HWKDQRO FRQFHQWUDWLRQV EHORZ b YROYROf GR QRW GHQDWXUH JO\FRO\WLF HQ]\PHV RU FDXVH DSSUHFLDEOH LQKLELWLRQ RI DFWLYLW\ LQ YLWUR XQGHU VXEVWUDWH VDWXUDWLQJ FRQGLWLRQV 6LQFH HWKDQRO GRHV QRW DFFXPXODWH

PAGE 96

ZLWKLQ \HDVW FHOOV EXW UDSLGO\ GLIIXVHV DFURVV WKH FHOO PHPEUDQH 'DVDUL HW DO *XLMDUUR DQG /DJXQDV 7DEOH f GLUHFW LQKLELWLRQ RI JO\FRO\WLF HQ]\PHV E\ LQWUDFHOOXODU HWKDQRO LV XQOLNHO\ GXULQJ IHUPHQWDWLRQV ZKLFK SURGXFH b YROYROf HWKDQRO RU OHVV ,Q WKLV FKDSWHU FKDQJHV LQ WKH DPRXQWV RI JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV DQG LQWHUQDO S+ DQG PHPEUDQH HQHUJL]DWLRQ KDYH EHHQ H[DPLQHG DV SRVVLEOH SK\VLRORJLFDO FDXVHV IRU WKH GHFOLQH LQ IHUPHQWDWLYH DFWLYLW\ GXULQJ EDWFK IHUPHQWDWLRQV RI b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f 7KH SURWHLQ FRQWHQW RI FHOO H[WUDFWV ZDV TXDQWLWDWHG XVLQJ WKH PHWKRG RI %UDGIRUG f %RYLQH

PAGE 97

VHUXP DOEXPLQ VHUYHG DV WKH SURWHLQ VWDQGDUG IRU ERWK PHWKRGV (Q]\PH $QDO\VHV $FWLYLWLHV RI JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV ZHUH GHWHUPLQHG LQ PO VDPSOHV UHPRYHG DW YDULRXV WLPHV GXULQJ EDWFK IHUPHQWDWLRQ &HOOV ZHUH KDUYHVWHG E\ FHQWULIXJDWLRQ DW [ J IRU VHF DW r& DQG ZDVKHG LQ DQ HTXDO YROXPH RI P0 SRWDVVLXP SKRVSKDWH EXIIHU S+ f $OO VXEVHTXHQW VWHSV ZHUH FDUULHG RXW DW r& 7KH SHOOHW ZDV VXVSHQGHG LQ WKH VDPH EXIIHU FRQWDLQLQJ P0 PHUFDSWRHWKDQRO DQG P0 ('7$ DQG GLVUXSWHG ZLWK PP JODVV EHDGV XVLQJ D 0LQL%HDG %HDWHU %LRVSHF 3URGXFWV %DUWOHVYLOOH 2NODf )LYH PLQ SHULRGV RI GLVUXSWLRQ HDFK ZHUH IROORZHG E\ PLQ SHULRGV RI FRROLQJ RQ LFH &HOO GHEULV ZDV UHPRYHG E\ FHQWULIXJDWLRQ DW [ J IRU PLQ DQG WKH VXSHUQDWDQW ZDV DVVD\HG LPPHGLDWHO\ IRU HQ]\PDWLF DFWLYLWLHV 2QO\ WZR HQ]\PHV DW D WLPH ZHUH DVVD\HG LQ HDFK EDWFK IHUPHQWDWLRQ H[SHULPHQW WR DYRLG SUREOHPV ZKLFK FRXOG UHVXOW IURP VWRUDJH RI FHOOV RU H[WUDFWV 3\UXYDWH GHFDUER[\ODVH DQG DOO JO\FRO\WLF HQ]\PHV ZHUH DVVD\HG VSHFWURSKRWRPHWULFDOO\ E\ WKH PHWKRGV RI 0DLWUD DQG /RER f DV PRGLILHG E\ &OLIWRQ HW DO f $OO HQ]\PHV ZHUH DVVD\HG XQGHU VXEVWUDWHVDWXUDWLQJ FRQGLWLRQV H[FHSW WULVH SKRVSKDWH LVRPHUDVH ZKLFK ZDV DVVD\HG ZLWK P0 VXEVWUDWH 7KH DPRXQWV RI FRXSOLQJ HQ]\PHV ZHUH DGMXVWHG DV QHHGHG WR HQVXUH D OLQHDU UHDFWLRQ UDWH

PAGE 98

$OFRKRO GHK\GURJHQDVH ZDV DVVD\HG E\ PHDVXULQJ WKH R[LGDWLRQ RI HWKDQRO DV GHVFULEHG E\ 0DLWUD DQG /RER f EXW XVLQJ D EXIIHU DW S+ FRQWDLQLQJ P0 VRGLXP S\URSKRVSKDWH P0 VHPLFDUED]LGH K\GURFKORULGH DQG P0 JO\FLQH %HUQW DQG *XWPDQ f 'HWHUPLQDWLRQ RI ,QWHUQDO S+ DQG 0HPEUDQH (QHUJL]DWLRQ 7KH PHDVXUHPHQWV RI LQWHUQDO S+ DQG $ ,I ZHUH SHUIRUPHG XVLQJ > &@EHQ]RLF DFLG DQG >+ SKHQ\O@WHWUDSKHQ\OSKRVSKRQLXP EURPLGH UHVSHFWLYHO\ 3URWRFROV ZHUH VLPLODU WR WKRVH GHVFULEHG E\ &DUWZULJKW HW DO f H[FHSW WKDW FHOOV ZHUH LQFXEDWHG LQ WKHLU QDWLYH JURZWK PHGLXP UDWKHU WKDQ GLVWLOOHG ZDWHU DQG SP SRUH VL]H SRO\FDUERQDWH ILOWHUV ZHUH XVHG LQVWHDG RI PL[HG FHOOXORVH HVWHU ILOWHUV &HOO YROXPHV ZHUH GHWHUPLQHG DV GHVFULEHG LQ FKDSWHU ,,, $V D FRQWURO IRU DGYHQWLWLRXV ELQGLQJ RI UDGLRDFWLYH FRPSRXQGV FHOOV ZHUH SHUPHDELOL]HG ZLWK D FRPELQDWLRQ RI HWKDQRO WROXHQH DQG 7ULWRQ ; DV GHVFULEHG E\ 6DOPRQ f ZDVKHG ZLWK P0 SKRVSKDWH EXIIHU UHVXVSHQGHG LQ QDWLYH EURWK DQG SURFHVVHG 7KLV WUHDWPHQW UHVXOWHG LQ D FRPSOHWH FROODSVH RI $ S+ DQG ORVV RI PHPEUDQH SRWHQWLDO &DOFXODWLRQV ZHUH SHUIRUPHG DV GHVFULEHG E\ 5RWWHQEHUJ f 0DWHULDOV
PAGE 99

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f &HOOV ZHUH PRVW DFWLYH DW WKH HDUOLHVW WLPHV PHDVXUHG K DQG IHUPHQWDWLRQ UDWH GHFOLQHG E\ b ZKHQ b YROYROf HWKDQRO KDG DFFXPXODWHG DIWHU K $SSUR[LPDWHO\ b RI WKH IHUPHQWDWLYH DFWLYLW\ ZDV UHWDLQHG DIWHU WKH DFFXPXODWLRQ RI b YROYROf HWKDQRO ZLWK J JOXFRVH SHU / UHPDLQLQJ LQ WKH IHUPHQWDWLRQ EURWK 7KH DEUXSW ILQDO GHFOLQH LQ DFWLYLW\ UHIOHFWV WKH QHDUFRPSOHWH H[KDXVWLRQ RI JOXFRVH 5HPRYDO RI HWKDQRO IURP FHOOV E\ ZDVKLQJ DQG VXVSHQGLQJ LQ IUHVK PHGLXP UHVXOWHG LQ RQO\ D PRGHVW LQFUHDVH LQ IHUPHQWDWLYH DFWLYLW\ LQ DOO EXW WKH

PAGE 100

)LJXUH (IIHFW RI HWKDQRO UHPRYDO RQ WKH IHUPHQWDWLYH DFWLYLW\ RI FHOOV JURZQ LQ <(3' PHGLXP FRQWDLQLQJ P0 0J6 &HOOV ZHUH VDPSOHG GXULQJ EDWFK IHUPHQWDWLRQ DQG ZHUH HLWKHU XQWUHDWHG RU ZDVKHG RQFH DQG WKHQ VXVSHQGHG LQ IUHVK PHGLXP FRQWDLQLQJ b JOXFRVH 7KH IHUPHQWDWLRQ UDWH RI WKHVH VDPSOHV ZHUH PHDVXUHG LPPHGLDWHO\ E\ UHVSLURPHWU\ 6\PEROV b DFWLYLW\ PHDVXUHG LQ QDWLYH EURWK 2 DFWLYLW\ PHDVXUHG DIWHU FHOOV ZHUH VXVSHQGHG LQ IUHVK PHGLXP

PAGE 101

)(50(17$7,21 5$7( R SPROHV &K SHU PJ SURWHLQf UR R R &' R 7 92

PAGE 102

KLJKHVW OHYHO RI DFFXPXODWHG HWKDQRO 7KH DSSDUHQW LQFUHDVH LQ DFWLYLW\ LQ WKH FHOOV ZKLFK KDG DFFXPXODWHG b YROYROf HWKDQRO ZDV SULPDULO\ GXH WR WKH UHVWRUDWLRQ RI IHUPHQWDEOH VXEVWUDWH ,Q D FRQWURO H[SHULPHQW WKH LQKLELWLRQ RI HWKDQRO SURGXFWLRQ E\ DGGHG HWKDQRO DQG WKH UHYHUVLELOLW\ RI WKLV LQKLELWLRQ E\ ZDVKLQJ ZDV LQYHVWLJDWHG &HOOV ZHUH KDUYHVWHG DQG VXVSHQGHG LQ IUHVK PHGLXP FRQWDLQLQJ YDULRXV FRQFHQWUDWLRQV RI HWKDQRO &HOOV DW K DIWHU LQRFXODWLRQ )LJ $f ZHUH PRUH DFWLYH DQG PRUH VHQVLWLYH WR LQKLELWLRQ E\ DGGHG HWKDQRO WKDQ FHOOV VDPSOHG DIWHU K RI IHUPHQWDWLRQ )LJ %f (WKDQRO FDXVHG D SURJUHVVLYH GRVHGHSHQGHQW LQKLELWLRQ RI IHUPHQWDWLRQ LQ ERWK ZLWK WKLV LQKLELWLRQ EHLQJ LPPHGLDWH DQG FRPSOHWH ZLWKLQ WKH ILUVW PLQ RI H[SRVXUH 1R IXUWKHU GHFOLQH LQ DFWLYLW\ ZDV REVHUYHG GXULQJ D VXEVHJXHQW K RI LQFXEDWLRQ DW r& ZLWK b YROYROf DGGHG HWKDQRO GDWD QRW VKRZQf 7KH FRQFHQWUDWLRQV RI HWKDQRO UHTXLUHG WR LQKLELW b RI WKH PD[LPXP REVHUYHG IHUPHQWDWLRQ UDWH ZHUH DQG b YROYROf UHVSHFWLYHO\ IRU K DQG K FHOOV 7KH LQKLELWLRQ RI IHUPHQWDWLRQ FDXVHG E\ H[SRVXUH WR FRQFHQWUDWLRQV RI HWKDQRO DERYH b YROYROf IRU PLQ ZDV RQO\ SDUWLDOO\ UHYHUVHG E\ VXVSHQVLRQ LQ IUHVK PHGLXP ODFNLQJ HWKDQRO )LJ f LQGLFDWLQJ WKDW H[SRVXUH WR HWKDQRO GDPDJHG WKH FHOOV LQ VRPH ZD\ $JDLQ K FHOOV DSSHDUHG PRUH VHQVLWLYH WR HWKDQRO GDPDJH WKDQ K FHOOV

PAGE 103

)LJXUH (IIHFWV RI HWKDQRO H[SRVXUH RQ WKH IHUPHQWDWLYH DFWLYLWLHV RI K DQG K FHOOV )HUPHQWDWLYH DFWLYLW\ ZDV PHDVXUH DIWHU VDPSOH SUHSDUDWLRQ E\ UHVSLURPHWU\ &HOOV ZHUH KDUYHVWHG DIWHU K $f RU K %f DQG VXVSHQGHG LQ IUHVK PHGLXP FRQWDLQLQJ YDULRXV FRQFHQWUDWLRQV RI JWKDQRO 7KHLU IHUPHQWDWLYH DFWLYLW\ ZDV PHDVXUHG DIWHU PLQ DW r& $ SDUDOOHO VHW RI VDPSOHV ZDV H[SRVHG WR HWKDQRO IRU PLQ KDUYHVWHG E\ FHQWULIXJDWLRQ ZDVKHG RQFH DQG VXVSHQGHG LQ IUHVK PHGLXP ODFNLQJ HWKDQRO (UURU EDUV GHQRWH WKH DYHUDJH VWDQGDUG GHYLDWLRQ IRU WKUHH VHSDUDWH EDWFK IHUPHQWDWLRQV 6\PEROV p FHOOV LQ WKH SUHVHQFH RI DGGHG HWKDQRO 2 FHOOV H[SRVHG WR HWKDQRO DQG VXVSHQGHG LQ IUHVK PHGLXP

PAGE 104

)(50(17$7,21 5$7( )(50(17$7,21 5$7( SPROHV &2MK SHU PJ SURWHLQf V

PAGE 105

/RQJHU LQFXEDWLRQ SHULRGV ZLWK b YROYROf HWKDQRO UHVXOWHG LQ D GHFUHDVH LQ DFWLYLW\ UHFRYHUDEOH E\ ZDVKLQJ ZLWK RQO\ b RI WKH RULJLQDO DFWLYLW\ UHFRYHUDEOH DIWHU K &KDQJHV LQ WKH /HYHOV RI *O\FRO\WLF DQG $OFRKRORTHQLF (Q]\PHV 'XULQJ %DWFK )HUPHQWDWLRQ 7KH VSHFLILF DFWLYLWLHV RI WKH HQ]\PHV LQYROYHG LQ DOFRKRO SURGXFWLRQ XQGHU VXEVWUDWHVDWXUDWLQJ FRQGLWLRQV DUH OLVWHG LQ 7DEOH IRU FHOOV KDUYHVWHG DIWHU K WKH PRVW DFWLYH VWDJH RI IHUPHQWDWLRQf DQG K b PD[LPDO DFWLYLW\f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b DQG WZR KDG LQFUHDVHG )LJXUH $ LOOXVWUDWHV WKH FKDQJHV LQ WKH VSHFLILF DFWLYLWLHV IRU WKHVH HQ]\PHV WKURXJKRXW EDWFK IHUPHQWDWLRQ UHODWLYH WR WKDW RI

PAGE 106

7DEOH 6SHFLILF DFWLYLWLHV RI JO\FRO\WLF HQ]\PHV DW WKH SHDN RI IHUPHQWDWLYH DFWLYLW\ Kf DQG DIWHU D b GHFOLQH Kf 6SHFLILF DFWLYLW\ SPROHVPLQ SHU (Q]\PH PJ SURWHLQf6'fD K FHOOV K FHOOV *O\FRO\WLF IOX[ KH[RVHfE +H[RNLQDVH f f 3KRVSKRJOXFRVH LVRPHUDVH f f 3KRVSKRIUXFWRNLQDVH f f )UXFWRVH ELVSKRVSKDWH f f DOGRODVH *O\FRO\WLF IOX[ WULVHfE 7ULVH SKRVSKDWH LVRPHUDVH f f *O\FHUDOGHK\GHSKRVSKDWH f f GHK\GURJHQDVH 3KRVSKRJO\FHUDWH NLQDVH f f 3KRVSKRJO\FHUDWH PXWDVH f f (QRODVH f f 3\UXYDWH NLQDVH f f 3\UXYDWH GHFDUER[\ODVH f f $OFRKRO GHK\GURJHQDVH f f D 6DPSOHV ZHUH UHPRYHG IURP EDWFK IHUPHQWDWLRQV DIWHU DQG K &HOOV ZHUH PHFKDQLFDOO\ GLVUXSWHG DQG WKH H[WUDFWV ZHUH DVVD\HG XQGHU VXEVWUDWHVDWXUDWLQJ FRQGLWLRQV 6WDQGDUG GHYLDWLRQV DUH EDVHG XSRQ GHWHUPLQDWLRQV IURP WKUHH VHSDUDWH EDWFK IHUPHQWDWLRQV E *O\FRO\WLF IOX[ IRU KH[RVH DQG WULVH LQWHUPHGLDWHV ZDV HVWLPDWHG IURP PHDVXUHPHQWV RI IHUPHQWDWLRQ UDWH

PAGE 107

)LJXUH &KDQJHV LQ WKH OHYHOV RI JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV GXULQJ EDWFK IHUPHQWDWLRQ ZLWK b JOXFRVH &HOOV ZHUH UHPRYHG DW YDULRXV VWDJHV RI IHUPHQWDWLRQ DQG GLVUXSWHG 7KH DFWLYLWLHV RI LQGLYLGXDO HQ]\PHV ZHUH GHWHUPLQHG XQGHU VXEVWUDWHVDWXUDWLQJ FRQGLWLRQV 9DOXHV DUH H[SUHVVHG UHODWLYH WR K FHOOV WKH WLPH DW ZKLFK WKH KLJKHVW DFWLYLW\ SHU PJ FHOO SURWHLQ ZDV REVHUYHG (UURU EDUV UHSUHVHQW DQ DYHUDJH VWDQGDUG GHYLDWLRQ IRU GHWHUPLQDWLRQV IURP WKUHH VHSDUDWH EDWFK IHUPHQWDWLRQV $f &KDQJHV LQ WKH VSHFLILF DFWLYLWLHV RI UHSUHVHQWDWLYH HQ]\PHV %f &KDQJHV LQ WKH DFWLYLWLHV RI UHSUHVHQWDWLYH HQ]\PHV SHU PO RI FXOWXUH $QDORJRXV SORWV RI WKH FKDQJHV LQ IHUPHQWDWLRQ UDWH $ DQG %f DQG WKH FKDQJHV LQ WKH DPRXQW RI VROXEOH FHOO SURWHLQ % RQO\f KDYH EHHQ LQFOXGHG IRU FRPSDULVRQ 6\PEROV $ SKRVSKRJOXFRPXWDVH ’ JO\FHUDOGHK\GHSKRVSKDWH GHK\GURJHQDVH ‘ WULVH SKRVSKDWH LVRPHUDVH 4 SKRVSKRIUXFWRNLQDVH JO\FRO\VLV Z VROXEOH FHOO SURWHLQ

PAGE 108

3(5&(17$*( 2) 63(&,),& $&7,9,7< $7 K A RR UR R R R R R R R 3(5&(17$*( 2) $&7,9,7< 3(5 0/ $7 K I2 f &' R R R R R R R R

PAGE 109

K FHOOV bf $Q DQDORJRXV SORW RI IHUPHQWDWLRQ UDWH LV LQFOXGHG IRU FRPSDULVRQ 1RQH RI WKH VSHFLILF DFWLYLWLHV GHFOLQHG GUDPDWLFDOO\ GXULQJ EDWFK IHUPHQWDWLRQ $W WLPHV EH\RQG K ZLWK WKH H[FHSWLRQV QRWHG DERYH DOO HQ]\PHV ZHUH LQ H[FHVV RI WKH PHDVXUHG IHUPHQWDWLRQ UDWHV 3KRVSKRIUXFWRNLQDVH GHFOLQHG WR WKH JUHDWHVW H[WHQW ZLWK D b GURS LQ DFWLYLW\ DIWHU K 7KH VSHFLILF DFWLYLWLHV RI SKRVSKRJOXFRPXWDVH b LQFUHDVHf KH[RNLQDVH b LQFUHDVH QRW VKRZQf DQG HQRODVH b LQFUHDVH QRW VKRZQf LQFUHDVHG E\ PRUH WKDQ b $OO RWKHU HQ]\PHV ZLWK JO\FHUDOGHK\GHSKRVSKDWH GHK\GURJHQDVH DV D W\SLFDO UHSUHVHQWDWLYH H[KLELWHG VLPLODU LQFUHDVHV RI XS WR b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f KH[RNLQDVH QRW VKRZQf DQG JO\FHUDOGHK\GHSKRVSKDWH GHK\GURJHQDVH LQFUHDVHG PRUH UDSLGO\ WKDQ VROXEOH FHOO SURWHLQ FRQVLVWHQW ZLWK WKH REVHUYHG LQFUHDVHV LQ VSHFLILF

PAGE 110

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f 6LQFH WKH S+ RSWLPD IRU JO\FRO\WLF HQ]\PHV DUH QHDU RU DERYH QHXWUDOLW\ &OLIWRQ HW DO f WKH IDLOXUH RI 6 FHUHYLVLDH WR PDLQWDLQ D ODUJH $ S+ GXULQJ WKH DFFXPXODWLRQ RI HWKDQRO FRXOG H[SODLQ WKH UDSLG GHFOLQH LQ WKH IHUPHQWDWLYH DFWLYLWLHV RI FHOOV GHVSLWH WKH DEXQGDQFH RI JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV +RZHYHU WKLV WKHRU\ LV QRW VXSSRUWHG E\ WKH GDWD SUHVHQWHG LQ ILJXUH 7KH $ S+ RI \HDVW FHOOV LQFUHDVHV ZLWK WKH GHFUHDVH LQ H[WHUQDO S+ PDLQWDLQLQJ D UHODWLYHO\ FRQVWDQW LQWHUQDO S+ RI EHWZHHQ DQG WKURXJKRXW EDWFK IHUPHQWDWLRQ )LJ $f 6LPLODUO\ $ DOVR LQFUHDVHG GXULQJ EDWFK IHUPHQWDWLRQ UHVXOWLQJ LQ DQ RYHUDOO LQFUHDVH LQ SURWRQ PRWLYH IRUFH )LJ %f 7KHVH UHVXOWV ZHUH VRPHZKDW VXUSULVLQJ VLQFH SUHYLRXV ZRUNHUV KDYH VKRZQ WKDW HWKDQRO LQFUHDVHV WKH SHUPHDELOLW\

PAGE 111

)LJXUH &KDQJHV LQ LQWUDFHOOXODU S+ DQG PHPEUDQH HQHUJL]DWLRQ GXULQJ EDWFK IHUPHQWDWLRQ RI b JOXFRVH (UURU EDUV UHSUHVHQW WKH DYHUDJH VWDQGDUG GHYLDWLRQ IRU GHWHUPLQDWLRQV IURP WKUHH VHSDUDWH EDWFK IHUPHQWDWLRQV $f ,QWUDFHOOXODU S+ 6\PEROV p LQWHUQDO S+ £ H[WHUQDO S+ ‘ $ S+ %f 0HPEUDQH HQHUJL]DWLRQ 6\PEROV f $\M ‘ $S+ SURWRQ PRWLYH IRUFH

PAGE 112

$M DQG 352721 027,9( )25&( PYf r R R R Wa R P A Q +G

PAGE 113

RI \HDVW VXVSHQGHG LQ ZDWHU WR K\GURJHQ LRQV &DUWZULJKW HW DO f 7R IXUWKHU H[DPLQH WKLV SRLQW WKH HIIHFW RI DGGHG HWKDQRO RQ WKH LQWHUQDO S+ RI FHOOV IURP YDULRXV VWDJHV RI IHUPHQWDWLRQ ZDV PHDVXUHG )LJ f (WKDQRO FRQFHQWUDWLRQV RI b YROYROf RU DERYH ZHUH UHTXLUHG WR FDXVH D PHDVXUDEOH GHFUHDVH LQ LQWHUQDO S+ 7KH DGGLWLRQ RI b YROYROf HWKDQRO WR K DQG K FHOOV FDXVHG D FRPSOHWH FROODSVH RI $ S+ 7KH $ S+ RI FHOOV IURP K DQG K FXOWXUHV ZDV FRQVLGHUDEO\ OHVV DIIHFWHG E\ b YROYROf DGGHG HWKDQRO FRQVLVWHQW ZLWK DGDSWDWLRQ RI WKH ROGHU FHOOV 'LVFXVVLRQ 7KH IHUPHQWDWLRQ RI JOXFRVH WR HWKDQRO UHSUHVHQWV D VHULHV RI FRRUGLQDWHG HQ]\PDWLF UHDFWLRQV 7KLV SURFHVV LV LQWHUQDOO\ EDODQFLQJ DQG WKHUPRG\QDPLFDOO\ IDYRUDEOH SURYLGHG WKDW FHOOXODU HQ]\PHV FRQVXPH WKH QHW $73 JHQHUDWHG IURP VXEVWUDWHOHYHO SKRVSKRU\ODWLRQ 7KH UHTXLUHPHQWV IRU WKLV SURFHVV LQFOXGH JOXFRVH IXQFWLRQDO HQ]\PHV FRHQ]\PHV 1$' WKLDPLQH S\URSKRVSKDWH $'3 $73f FRIDFWRUV 0J =Qf DSSURSULDWH LQWHUQDO S+ D IXQFWLRQDO PHPEUDQH WR PDLQWDLQ WKH FRQFHQWUDWLRQ RI UHDFWDQWV DQG HQ]\PHV DQG D JOXFRVH XSWDNH V\VWHP ,QGHHG IHUPHQWDWLRQ FDQ SURFHHG ZHOO LQ FRQFHQWUDWHG SUHSDUDWLRQV RI GLVUXSWHG FHOOV +DUGHQ :HOFK DQG 6FRSHV f 7KH UDWH RI JO\FRO\VLV LQ YLDEOH \HDVW FHOOV KRZHYHU GHFOLQHV DV HWKDQRO DFFXPXODWHV GXULQJ EDWFK IHUPHQWDWLRQ

PAGE 114

)LJXUH (IIHFWV RI DGGHG HWKDQRO RQ $S+ &HOOV ZHUH UHPRYHG DW YDULRXV VWDJHV RI EDWFK IHUPHQWDWLRQ LQGLFDWHG RQ JUDSKf KDUYHVWHG DQG VXVSHQGHG LQ IUHVK PHGLXP FRQWDLQLQJ YDULRXV FRQFHQWUDWLRQV RI HWKDQRO 6DPSOHV ZHUH LQFXEDWHG DW r& IRU PLQ DQG S+ ZDV GHWHUPLQHG

PAGE 115

$ S+

PAGE 116

7ZR QXWULWLRQDO IDFWRUV KDYH EHHQ LGHQWLILHG SUHYLRXVO\ ZKLFK UHGXFHG EXW GR QRW HOLPLQDWH WKH HWKDQRODVVRFLDWHG GHFOLQH LQ DFWLYLW\ &DVH\ DQG ,QJOHGHZ &KDSWHU ,9f 7KH UHVXOWV SUHVHQWHG LQ WKLV FKDSWHU ZLWK DGGHG DQG DFFXPXODWHG HWKDQRO FRQILUP WKH UHVXOWV RI FKDSWHU ,,, LQGLFDWLQJ WKDW SK\VLRORJLFDO FKDQJHV LQFOXGLQJ HWKDQRO GDPDJH LQ DGGLWLRQ WR DQ LPPHGLDWHO\ UHYHUVLEOH HIIHFW RI HWKDQRO DSSHDU UHVSRQVLEOH $GGHG HWKDQRO LQKLELWHG IHUPHQWDWLRQ EXW ZDVKLQJ GLG QRW UHVWRUH IXOO DFWLYLW\ )LJ f 6LPLODUO\ WKH UHSODFHPHQW RI HWKDQROFRQWDLQLQJ EURWK IURP WKH PLGGOH WR ODWWHU VWDJHV RI IHUPHQWDWLRQ ZLWK IUHVK PHGLXP GLG QRW LPPHGLDWHO\ UHVWRUH IXOO IHUPHQWDWLYH DFWLYLW\ 7KH H[SRVXUH RI FHOOV WR HWKDQRO LQ VRPH ZD\ GDPDJHG WKHLU DELOLW\ WR SURGXFH HWKDQRO DQG WKH H[WHQW RI WKLV GDPDJH DSSHDUV UHODWHG WR HWKDQRO FRQFHQWUDWLRQ DJH RI WKH FHOOV DQG GXUDWLRQ RI WKH H[SRVXUH &KDQJHV LQ LQWHUQDO S+ DQG LQ DPRXQWV RIn WKH LQGLYLGXDO JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV ZHUH H[DPLQHG DV SRVVLEOH UHDVRQV IRU GHFUHDVHG HWKDQRO SURGXFWLYLW\ GXULQJ EDWFK IHUPHQWDWLRQ 7KH DPRXQWV RI JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV UHPDLQHG KLJK WKURXJKRXW IHUPHQWDWLRQ ,Q JHQHUDO WKHVH DPRXQWV ZHUH PRUH WKDQ DGHTXDWH WR PDLQWDLQ WKH REVHUYHG IHUPHQWDWLRQ UDWHV /DUXH HW DO f UHSRUWHG VLPLODU REVHUYDWLRQV FRQFHUQLQJ KH[RNLQDVH DQG DOFRKRO GHK\GURJHQDVH DFWLYLWLHV GXULQJ VLPXODWHG ZLQH IHUPHQWDWLRQV 8QGHU JOXFRVHOLPLWHG JURZWK FRQGLWLRQV

PAGE 117

%RXFKHULH f UHSRUWHG WKDW HQRODVH KH[RNLQDVH DQG JO\FHUDOGHK\GHSKRVSKDWH GHK\GURJHQDVH V\QWKHVLV FRQWLQXHG DIWHU FHOO JURZWK HQGHG (YHQ WKRXJK WKH IHUPHQWDWLRQV VWXGLHG LQ WKLV FKDSWHU ZHUH IDU IURP JOXFRVHOLPLWHG WKH UHVXOWV LQGLFDWH WKDW VRPH RI WKH JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV LQFOXGLQJ HQRODVH KH[RNLQDVH DQG JO\FHUDOGHK\GHSKRVSKDWH PD\ EH SUHIHUHQWLDOO\ V\QWKHVL]HG GXULQJ VWDWLRQDU\ SKDVH ,W ZDV VXJJHVWHG E\ %RXFKHULH f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f (QWLDQ HW DO f DOVR UHSRUWHG DQG IROG LQFUHDVHV LQ S\UXYDWH NLQDVH DQG S\UXYDWH GHFDUER[\ODVH DFWLYLWLHV UHVSHFWLYHO\ RQO\ K DIWHU DGGLWLRQ RI JOXFRVH WR HWKDQROJURZQ FHOOV 6LQFH JO\FRO\WLF HQ]\PHV HDFK DFFRXQW IRU EHWZHHQ WR b RI WKH WRWDO VROXEOH FHOO SURWHLQ GXULQJ JURZWK RQ JOXFRVH )UDHQNHO f D ODUJH LQFUHDVH LQ HQ]\PH VSHFLILF DFWLYLW\ PD\ QRW EH SRVVLEOH ,QFUHDVLQJ DPRXQWV RI

PAGE 118

JO\FRO\WLF HQ]\PHV XSRQ JOXFRVH DGGLWLRQ WR GHUHSUHVVHG \HDVW FHOOV DOVR ZHUH FRUUHODWHG ZLWK LQFUHDVHG UDWHV RI JO\FRO\WLF DFWLYLW\ (QWLDQ HW DO f 7KLV FRQWUDVWV ZLWK WKH SUHVHQW VWXG\ ZKLFK VKRZV DQ LQFUHDVH LQ PRVW RI WKH HQ]\PH DFWLYLWLHV ZKLOH WKH UDWH RI JO\FRO\VLV GHFOLQHV )LJ f 2QO\ GXULQJ WKH ODWWHU VWDJHV RI IHUPHQWDWLRQ ZDV D PRGHVW ORVV RI WRWDO DFWLYLW\ SHU PO REVHUYHG LQGLFDWLQJ D ORZ WXUQRYHU UDWH IRU WKHVH HQ]\PHV DIWHU WKH UDWH RI IHUPHQWDWLRQ KDG GHFOLQHG E\ DW OHDVW b 7KLV LV FRQVLVWHQW ZLWK SUHYLRXV UHSRUWV WKDW PRVW HQ]\PHV RI 6 FHUHYLVLDH UHWDLQ WKHLU DFWLYLWLHV XQGHU D YDULHW\ RI PHWDEROLF FRQGLWLRQV LQFOXGLQJ FDUERQ DQG SKRVSKDWH OLPLWDWLRQ +DOYRUVRQ /DJXQDV HW DO /RSH] DQG *DQFHGR f *O\FRO\WLF HQ]\PHV FRPSUL]H D ODUJH SRUWLRQ RI WKH VROXEOH FHOO SURWHLQ 6ULYDVWDYD DQG %HUQKDUG f IDYRULQJ WKH IRUPDWLRQ RI SURWHLQSURWHLQ LQWHUDFWLRQV %DQXHORV DQG *DQFHGR f WKDW SUREDEO\ GR QRW RFFXU ZKHQ GLOXWH FHOO H[WUDFWV DUH DVVD\HG IRU LQ YLWUR HQ]\PH DFWLYLWLHV (YLGHQFH VXSSRUWLQJ WKLV WKHRU\ LQFOXGHV WKH REVHUYDWLRQ RI %DQXHORV DQG *DQFHGR f WKDW WKH RI HQRODVH IRU SKRVSKRJO\FHUDWH ZDV IROG ORZHU LQ SHUPHDELOL]HG FHOOV WKDQ LQ FHOO H[WUDFWV 7RPSD HW DO f VKRZHG WKDW DOGRODVH FDQ VSHFLILFDOO\ FRPSOH[ ZLWK SKRVSKRIUXFWRNLQDVH DQG JO\FHUDOGHK\GHSKRVSKDWH GHK\GURJHQDVH LQ YLWUR SURYLGLQJ IXUWKHU HYLGHQFH WKDW O

PAGE 119

JO\FRO\WLF HQ]\PHV FDQ IRUP VSHFLILF HQ]\PDWLF FRPSOH[HV WKDW PD\ IXQFWLRQ LQ VXEVWUDWH FKDQQHOLQJ 7KH UROH RI SURWHLQSURWHLQ LQWHUDFWLRQV DOVR KDV EHHQ LPSOLFDWHG LQ PRGXODWLQJ WKH DOORVWHULF UHVSRQVH RI SKRVSKRIUXFWRNLQDVH WR $73 $UDJRQ DQG 6DQFKH] f 7KH IHUPHQWDWLYH DFWLYLW\ RI FHOOV H[SRVHG WR b YROYROf HWKDQRO WKDW LV QRW UHFRYHUDEOH E\ ZDVKLQJ )LJ f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b YROYROf HWKDQRO 7KLV ODWWHU REVHUYDWLRQ ZDV FRQWUDU\ WR H[SHFWDWLRQ EDVHG XSRQ HDUOLHU H[SHULPHQWV ZLWK FHOOV VXVSHQGHG LQ ZDWHU &DUWZULJKW HW DO f 7KHVH HDUOLHU VWXGLHV KDG GHPRQVWUDWHG WKDW HWKDQRO HQKDQFHG WKH OHDNDJH RI SURWRQV DQG RWKHU LRQV &DUWZULJKW HW DO /HDR DQG YDQ 8GHQ Df 7KH F\WRSODVP ZDV DFLGLILHG EHORZ WKH RSWLPDO S+ IRU JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV )LJXUH FOHDUO\ VKRZV WKDW DW FRQFHQWUDWLRQV IRXQG LQ WKH PHGLXP GXULQJ WKH IHUPHQWDWLRQ RI b JOXFRVH DGGHG HWKDQRO GRHV QRW DIIHFW

PAGE 120

VLJQLILFDQWO\ WKH $ S+ RI \HDVW FHOOV VXVSHQGHG LQ IUHVK PHGLXP 2QO\ DW HWKDQRO FRQFHQWUDWLRQV DERYH b YROYROf LV WKH $ S+ ORZHUHG $OWKRXJK HQKDQFHG LRQ OHDNDJH DOVR PD\ RFFXU LQ IHUPHQWDWLRQ EURWK WKH PDLQWHQDQFH RI D KLJK LQWHUQDO S+ LQ EURWK FRQWDLQLQJ HWKDQRO LQGLFDWHV WKDW VXFK OHDNDJH PXVW EH RIIVHW E\ WKH DFWLRQ RI K\GURJHQ LRQ SXPSV VXFK DV $73DVHV 3HQD HW DO f SURSRVHG D VLPLODU K\SRWKHVLV WR H[SODLQ WKH FRQYHUVLRQ RI $73 WR $'3 DQG LQRUJDQLF SKRVSKDWH REVHUYHG ZKHQ WKH H[WHUQDO S+ RI WKHLU VWUDLQ RI 6 FHUHYLVLDH LQ EXIIHU ZDV UDLVHG IURP WR 'XULQJ IHUPHQWDWLRQ $ S+ LQFUHDVHG WR DQG $ LMM GHFUHDVHG WR P9 DV WKH S+ RI WKH PHGLXP GHFUHDVHG WR )LJ f 7KLV JUDGXDOO\ ORZHUHG WKH SURWRQ PRWLYH IRUFH WR P9 6LPLODUO\ 'H /D 3HQD HW DO f UHSRUWHG WKDW WKH SURWRQ PRWLYH IRUFH RI \HDVW FHOOV VXVSHQGHG LQ D VHULHV RI EXIIHUV ZLWK WKH S+ GHFUHDVLQJ WR EHFDPH PRUH QHJDWLYH &DUWZULJKW HW DO f ZDV QRW DEOH WR GHWHFW D PHPEUDQH SRWHQWLDO ZKHQ b YROYROf HWKDQRO ZDV DGGHG WR DQ DTXHRXV VXVSHQVLRQ RI HDUO\ VWDWLRQDU\SKDVH FHOOV 7KLV SOXV WKH GHFUHDVH LQ $ S+ REVHUYHG ZKHQ HWKDQRO ZDV DGGHG WR LGHQWLFDO FHOO VXVSHQVLRQV JUDGXDOO\ PDGH WKH SURWRQ PRWLYH IRUFH PRUH SRVLWLYH 7KLV UHSRUW LV LQ FRQWUDVW ZLWK WKH GDWD SUHVHQWHG LQ ILJXUH ZKLFK LQGLFDWHV WKDW WKH SODVPD

PAGE 121

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f $OWHUDWLRQV LQ OLSLG ELRV\QWKHVLV ZKHQ \HDVW FHOO JURZWK VWRSV DOVR FKDQJH WKH PHPEUDQH SKRVSKROLSLG FRPSRVLWLRQ +RPDQQ HW DO f ,Q OLJKW RI WKH UHVXOWV UHSRUWHG LQ FKDSWHU ,,, WKHVH JURZWK SKDVH GHSHQGHQW OLSLG FKDQJHV PD\ EH WKH DFWXDO FDXVH RI HWKDQRO UHVLVWDQW $ S+ UDWKHU WKDQ JURZWK LQ WKH SUHVHQFH RI HWKDQRO $OWKRXJK WKH LQWHUQDO S+ UHPDLQHG DOPRVW QHXWUDO DQG WKH DFWLYLWLHV RI JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV DVVD\HG LQ YLWUR UHPDLQHG KLJK GXULQJ EDWFK IHUPHQWDWLRQ WKH LQ YLYR DFWLYLWLHV RI WKHVH HQ]\PHV ZLWKLQ WKH FHOO

PAGE 122

FDQQRW EH DFFXUDWHO\ SUHGLFWHG 7KH DFWLYLWLHV RI VRPH RI WKHVH DUH VXEMHFW WR PRGXODWLRQ E\ DOORVWHULF HIIHFWRUV LQ DGGLWLRQ WR FRQVWUDLQWV LPSRVHG E\ WKH DYDLODELOLW\ RI LQGLYLGXDO VXEVWUDWHV FRIDFWRUV DQG FRHQ]\PHV )UDHQNHO f ,Q WKH QH[W FKDSWHU WKH OHYHOV RI VRPH RI WKHVH ORZPROHFXODUZHLJKW LQWUDFHOOXODU FRQVWLWXHQWV ZLOO EH H[DPLQHG WR GHWHUPLQH ZKDW FRQVWUDLQWV RQ WKH JO\FRO\WLF SDWKZD\ PD\ EH UHVSRQVLEOH IRU WKH GHFOLQLQJ UDWH RI HWKDQRO SURGXFWLRQ GXULQJ \HDVW IHUPHQWDWLRQ

PAGE 123

&+$37(5 9, 3+263+25
PAGE 124

,Q FKDSWHU 9 WKH DPRXQW RI HDFK JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PH ZDV PHDVXUHG DV IHUPHQWDWLRQ SURJUHVVHG 7DEOH DQG )LJ f 7KH OHYHOV RI WKHVH HQ]\PHV LQ JHQHUDO UHPDLQHG KLJK DQG ZHUH VXIILFLHQW WR DFFRXQW IRU WKH REVHUYHG UDWHV RI HWKDQRO SURGXFWLRQ WKURXJKRXW IHUPHQWDWLRQ 7KH DPRXQWV RI JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV ZHUH PHDVXUHG DV VSHFLILF DFWLYLWLHV LQ YLWUR XQGHU VXEVWUDWH VDWXUDWLQJ FRQGLWLRQV 7KH DFWXDO LQ YLYR DFWLYLW\ RI WKHVH HQ]\PHV KRZHYHU LV VXEMHFW WR PRGXODWLRQ E\ WKH F\WRSODVPLF HQYLURQPHQW RI WKH \HDVW FHOO $V JOXFRVH LV FRQYHUWHG WR HWKDQRO WKH IHUPHQWDWLRQ EURWK LV DFLGLILHG )LJ f ,I WKH LQWUDFHOOXODU S+ VLJQLILFDQWO\ GHFUHDVHG GXULQJ WKLV DFLGLILFDWLRQ WKHQ WKH LQ YLYR DFWLYLWLHV RI WKH JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV DOVR VKRXOG KDYH GHFUHDVHG +RZHYHU \HDVW FHOOV VXVSHQGHG LQ WKHLU QDWLYH PHGLXP KHOG D FRQVWDQW LQWUDFHOOXODU S+ WKURXJKRXW IHUPHQWDWLRQ )LJ f DQG ZHUH DEOH WR PDLQWDLQ D KLJK $ S+ LQ FRQFHQWUDWLRQV RI HWKDQRO JUHDWHU WKDQ WKH DPRXQW DWWDLQDEOH IURP WKH FRPSOHWH FRQYHUVLRQ RI b JOXFRVH )LJ f 7KHVH UHVXOWV ZHUH XQH[SHFWHG DQG FRQWUDU\ WR ZKDW KDG EHHQ REVHUYHG IRU \HDVW FHOOV VXVSHQGHG LQ ZDWHU &DUWZULJKW HW DO /HDR DQG YDQ 8GHQ Df (WKDQRO VWLOO PD\ FDXVH SURWRQOHDNDJH EXW FHOOV LQ QDWLYH EURWK PD\ FRSH ZLWK WKLV SHUPHDELOL]LQJ HIIHFW SUHVXPDEO\ E\ DFWLYDWLQJ LRQ SXPSV VXFK DV $73DVHV 3HQD HW DO f

PAGE 125

7KH FHOO F\WRSODVP DOVR FRQWDLQV PDQ\ HIIHFWRU PROHFXOHV ZKLFK PRGXODWH WKH DFWLYLWLHV RI HQ]\PHV LQ UHVSRQVH WR FKDQJHV LQ H[WUDFHOOXODU HQYLURQPHQWDO FRQGLWLRQV 7KH DFWLYLWLHV RI VRPH RI WKH \HDVW JO\FRO\WLF HQ]\PHV DUH NQRZQ WR EH FRQWUROOHG E\ DOORVWHULF HIIHFWRU PROHFXOHV DQG WKH DYDLODELOLW\ RI FRIDFWRUV DQG FRHQ]\PHV 6ROV HW DO f *OXFRVH SKRVSKRU\ODWLRQ E\ KH[RNLQDVHV DQG ,, DQG JOXFRNLQDVH UHTXLUHV $73 DQG 0J &RORZLFN f DQG JO\FHUDOGHK\GHSKRVSKDWH GHK\GURJHQDVH UHTXLUHV 1$' IRU DFWLYLW\ +DUULV DQG :DWHUV f 3\UXYDWH NLQDVH DFWLYLW\ LV UHJXODWHG E\ WKH DYDLODELOLW\ RI IUXFWRVH ELVSKRVSKDWH +HVV HW DO 0DLWUD DQG /RER f DQG UHTXLUHV $'3 DQG 0J .D\QH f 3KRVSKRIUXFWRNLQDVH ZKLFK V\QWKHVL]HV IUXFWRVH ELVSKRVSKDWH KDV PDQ\ DOORVWHULF UHJXODWRUV 7KHVH LQFOXGH IUXFWRVH ELVSKRVSKDWH )UDQFRLV HW DO f DPPRQLXP DQG FLWUDWH %OR[KDP DQG /DUG\ 8\HGD f 7KLV HQ]\PH DFWLYLW\ DOVR LV PRGXODWHG E\ HQHUJ\ FKDUJH ZLWK $73 DFWLQJ DV D VWURQJ LQKLELWRU DQG $03 DQG IUXFWRVH SKRVSKDWH UHYHUVLQJ WKH LQKLELWLRQ %HW] DQG 0RRUH f ,I WKH DYDLODELOLW\ RI UHTXLUHG FRIDFWRUV FRHQ]\PHV DQG DOORVWHULF DFWLYDWRUV EHFRPHV OLPLWHG RU LI ODUJH DPRXQWV RI LQKLELWRUV EXLOG XS LQWUDFHOOXODUO\ WKHQ WKH UDWH RI JO\FRO\VLV ZRXOG EH H[SHFWHG WR GHFUHDVH 2QH PHWKRG RI GHWHUPLQLQJ ZKLFK SDUW RI WKH IHUPHQWDWLYH SDWKZD\ LV UHVWULFWLQJ FDUERQ IORZ WR HWKDQRO

PAGE 126

LV WR TXDQWLWDWH WKH OHYHOV RI JO\FRO\WLF LQWHUPHGLDWHV DV IHUPHQWDWLRQ SURJUHVVHV ,QFUHDVLQJ OHYHOV RI DQ LQGLYLGXDO LQWHUPHGLDWH PD\ UHIOHFW DQ LPEDODQFH EHWZHHQ WKH V\QWKHVLV DQG XWLOL]DWLRQ RI WKDW LQWHUPHGLDWH 7KLV ZDV GHPRQVWUDWHG E\ &LULDF\ DQG %UHLWHQEDFK f ZKR VKRZHG WKDW PXWDQWV RI 6 FHUHYLVLDH PLVVLQJ RQH RI WKH JO\FRO\WLF HQ]\PH DFWLYLWLHV DFFXPXODWHG WKH JO\FRO\WLF LQWHUPHGLDWH ZKLFK ZDV VXEVWUDWH IRU WKH PLVVLQJ DFWLYLW\ 6LPLODUO\ HOHYDWHG OHYHOV RI KH[RVH SKRVSKDWHV ZHUH GHWHFWHG E\ 3105 LQ H[WUDFWV RI FHOOV ODFNLQJ HLWKHU SKRVSKRJOXFRVH LVRPHUDVH RU SKRVSKRIUXFWRNLQDVH DFWLYLW\ 1DYRQ HW DO f ,Q WKLV FKDSWHU WKH DPRXQWV RI SKRVSKRU\ODWHG JO\FRO\WLF LQWHUPHGLDWHV DQG WRWDO QXFOHRWLGHV GXULQJ WKH IHUPHQWDWLRQ RI b JOXFRVH ZHUH TXDQWLWDWHG LQ RUGHU WR GHWHUPLQH ZKLFK SDUWVf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

PAGE 127

3UHSDUDWLRQ RI U3 3&AODEHOOHG 3@RUWKRSKRVSKDWH ZDV DGGHG PO RI LQRFXOXP 7KLV FXOWXUH ZDV VWLUUHG XQGHU VHPLDQDHURELF FRQGLWLRQV ZKLOH LQFXEDWLQJ LQ D ZDWHUEDWK DW r& %HJLQQLQJ K DIWHU LQRFXODWLRQ VDPSOHV ZHUH WDNHQ DW K LQWHUYDOV IRU WKH SUHSDUDWLRQ RI IHUPHQWDWLRQ H[WUDFWV ,Q D SDUDOOHO H[SHULPHQW FHOOV ZHUH LQRFXODWHG LQWR XQODEHOOHG PHGLXP DQG VDPSOHV ZHUH WDNHQ IRU FHOO SURWHLQ DQG HWKDQRO GHWHUPLQDWLRQV 3UHSDUDWLRQ RI )HUPHQWDWLRQ ([WUDFWV 6DPSOHV WDNHQ IURP EDWFK IHUPHQWDWLRQV ZHUH H[WUDFWHG XVLQJ D PHWKRG VLPLODU WR WKDW GHVFULEHG E\ +ROPVHQ HW DO f &HOOV ZHUH LQDFWLYDWHG E\ SLSHWWLQJ PO RI IHUPHQWDWLRQ VDPSOH LQWR D PLFURFHQWULIXJH WXEH FRQWDLQLQJ PO RI b YROYROf HWKDQRO DQG P0 ('7$ SUHFRROHG WR r& 7KH HWKDQRO('7$ H[WUDFWLRQ VROXWLRQ ZDV SUHSDUHG IUHVK EHIRUH HDFK XVH WR LQVXUH WKDW WKH ('7$ GLG QRW SUHFLSLWDWH 7KH VDPSOHV WKHQ ZHUH YRUWH[HG UDSLGO\ IRU VHF LPPHGLDWHO\ SODFHG LQ DQ LFHEDWK DQG DOORZHG WR H[WUDFW IRU PLQ DW r& (YHU\ PLQ GXULQJ WKLV H[WUDFWLRQ SHULRG WKH VDPSOHV ZHUH YRUWH[HG YLJRURXVO\ 7KH UHPDLQLQJ FHOO GHEULV ZDV SHOOHWHG E\ FHQWULIXJDWLRQ DW [ J IRU PLQ DW r& 7KH FHOO H[WUDFWV ZHUH VDYHG IRU IXUWKHU DQDO\VLV E\ IUHH]LQJ DW r& $W WKH VDPH WLPH

PAGE 128

WKDW WKH FHOO H[WUDFWV ZHUH EHLQJ SUHSDUHG PO RI IHUPHQWDWLRQ VDPSOH ZDV FHQWULIXJHG DW [ J IRU VHF WR SHOOHW WKH FHOOV (WKDQRO('7$ H[WUDFWV RI WKH VXSHUQDWDQW IHUPHQWDWLRQ EURWK ODFNLQJ FHOOV ZHUH SUHSDUHG DV GHVFULEHG IRU ZKROH IHUPHQWDWLRQ VDPSOHV 7KH HIIHFWLYHQHVV RI WKH HWKDQRO('7$ H[WUDFWLRQ SURFHGXUH ZDV FRPSDUHG ZLWK WKDW RI WKH IRUPLF DFLG PHWKRG GHVFULEHG E\ %RFKQHU DQG $PHV f DQG WKH SHUFKORULF DFLG PHWKRG RI %DOO DQG $WNLQVRQ f ,GHQWLFDO IHUPHQWDWLRQ VDPSOHV ODEHOOHG ZLWK >3@3n ZHUH H[WUDFWHG E\ HDFK PHWKRG DQG PO RI HDFK H[WUDFW ZDV VSRWWHG RQWR :KDWPDQ 00 ILOWHU SDSHU GLVNV $IWHU GU\LQJ WKH ILOWHU GLVNV ZHUH FRXQWHG LQ D %HFNPDQ PRGHO VFLQWLOODWLRQ FRXQWHU 7KH IRUPLF DFLG SURFHGXUH H[WUDFWHG OHVV WKDQ KDOI RI WKH FRXQWV H[WUDFWHG E\ WKH SHUFKORULF DFLG SURFHGXUH ZKLOH WKH HWKDQRO('7$ SURFHGXUH H[WUDFWHG b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

PAGE 129

$QDO\VLV RI U3 32AODEHOOHG )HUPHQWDWLRQ ([WUDFWV ([WUDFWV RI >3@ 3ODEHOOHG IHUPHQWDWLRQ VDPSOHV ZHUH DQDO\]HG E\ WZRGLPHQVLRQDO WKLQ OD\HU FKURPDWRJUDSK\ XVLQJ WKH VROYHQW V\VWHPV GHVFULEHG E\ +ROPVHQ HW DO f $OLJXRWV RI H[WUDFW FRQWDLQLQJ DSSUR[LPDWHO\ FSP HDFK ZHUH VSRWWHG RQWR $QDOWHFK &HOOXORVH 01 FP [ FPf WKLQ OD\HU FKURPDWRJUDSK\ SODWHV )LVKHU 6FLHQWLILF &RPSDQ\ 2UODQGR )ODf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f DQG WHQWDWLYH LGHQWLILFDWLRQV ZHUH PDGH 7KHQ DXWKHQWLF VWDQGDUGV RI JO\FRO\WLF LQWHUPHGLDWHV ZHUH FKURPDWRJUDSKHG XQGHU WKH VDPH FRQGLWLRQV DV WKH IHUPHQWDWLRQ H[WUDFWV 6WDQGDUGV ZHUH YLVXDOL]HG E\ LPPHUVLQJ WKH WKLQ OD\HU SODWHV LQ D EDWK FRQWDLQLQJ )H DQG VXOIRVDOLF\OLF DFLG DV GHVFULEHG E\

PAGE 130

)LJXUH $ W\SLFDO WKLQ OD\HU FKURPDWRJUDP RI D IHUPHQWDWLRQ VDPSOH H[WUDFW &HOOV ZHUH LQRFXODWHG LQWR <(3' PHGLXP FRQWDLQLQJ X&LPO >3@ RUWKRSKRVSKDWH $W K D IHUPHQWDWLRQ VDPSOH ZDV WDNHQ DQG H[WUDFWHG ZLWK WKH HWKDQRO('7$ VROXWLRQ DV GHVFULEHG LQ WKH WH[W $SSUR[LPDWHO\ FRXQWV RI H[WUDFW ZHUH VSRWWHG RQWR WKH RULJLQ DQG WKH FKURPDWRJUDP ZDV GHYHORSHG LQ WZR GLPHQVLRQV 6SRWV ZHUH YLVXDOL]HG E\ DXWRUDGLRJUDSK\ DQG LGHQWLILHG DV GHVFULEHG LQ WKH WH[W $EEUHYLDWLRQV 3c LQRUJDQLF SKRVSKDWH 3(3 SKRVSKRHQROS\UXYDWH *3 JO\FHURSKRVSKDWH 3* SKRVSKRJO\FHUDWH 3* SKRVSKRJO\FHUDWH *$3 JO\FHUDOGHK\GH SKRVSKDWH )3 IUXFWRVH SKRVSKDWH *3 JOXFRVHSKRVSKDWH )O3 IUXFWRVH ELVSKRVSKDWH $73 DGHQRVLQH WULSKRVSKDWH $'3 DGHQRVLQH GLSKRVSKDWH 73 WULVH SKRVSKDWHV +3 KH[RVH SKRVSKDWHV 1;3 WRWDO FHOOXODU QXFOHRWLGHV

PAGE 131

)LUVW 'LPHQVLRQ 6HFRQG 'LPHQVLRQ

PAGE 132

7KRPSVRQ f 7KHLU PLJUDWLRQ SRVLWLRQV DUH PDUNHG RQ WKH FKURPDWRJUDP LQ ILJXUH E\ WKH VPDOO DUURZV )LQDOO\ IHUPHQWDWLRQ H[WUDFWV WKDW KDG EHHQ GULHG DW r& XQGHU YDFXXP WR UHPRYH WKH HWKDQRO IURP WKH VDPSOH ZHUH WUHDWHG ZLWK YDULRXV JO\FRO\WLF HQ]\PHV 6DPSOHV ZHUH VXVSHQGHG LQ WKH HQ]\PH EXIIHU GHVFULEHG E\ 0DLWUD DQG /RER f DQG WUHDWHG ZLWK S\UXYDWH NLQDVH DQG IUXFWRVH ELVSKRVSKDWH DOGRODVH DW URRP WHPSHUDWXUH IRU PLQ 5HDFWLRQ PL[WXUHV ZHUH WKRVH GHVFULEHG E\ &OLIWRQ HW DO f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f DQG WKDW $73 PLJUDWHG ZLWK WKH QXFOHRWLGH SKRVSKDWH VSRW 7KXV PHDVXUHPHQWV RI SKRVSKRHQROS\UXYDWH ZHUH QRW LQFOXGHG LQ YDOXHV UHSRUWHG IRU WRWDO SKRVSKRU\ODWHG JO\FRO\WLF LQWHUPHGLDWHV DQG WULVH SKRVSKDWHV 4XDQWLWDWLRQ RI $GHQ\ODWH DQG 1LFRWLQDPLGH 1XFOHRWLGHV LQ )HUPHQWDWLRQ ([WUDFWV $PRXQWV RI WKH LQGLYLGXDO DGHQLQH QXFOHRWLGHV LQ WKH IHUPHQWDWLRQ H[WUDFWV ZHUH GHWHUPLQHG E\ ILUHIO\ OXFLIHUDVH

PAGE 133

ELROXPLQHVFHQFH 7KH UHDJHQWV IRU WKLV DVVD\ ZHUH REWDLQHG IURP /.% *DLWKHUVEXUJ 0Gf 6DPSOHV ZHUH SUHSDUHG IRU DQDO\VLV HVVHQWLDOO\ DV GHVFULEHG E\ %DOO DQG $WNLQVRQ f H[FHSW WKDW RQO\ XQLW RI DGHQ\ODWH NLQDVH ZDV XVHG LQ WKH FRQYHUVLRQ RI $03 WR $73 6LQFH HWKDQRO KDV EHHQ UHSRUWHG WR LQKLELW WKH OXFLIHUDVH UHDFWLRQ %DOO DQG $WNLQVRQ f DOO VWDQGDUGV FRQWDLQHG OHYHOV RI HWKDQRO LGHQWLFDO WR WKDW IRXQG LQ WKH VDPSOHV +RZHYHU VDPSOHV XVHG IRU DGHQLQH QXFOHRWLGH GHWHUPLQDWLRQV ZHUH VWRUHG DW r& WKDZHG RQ LFH DQG QRW UHIUR]HQ IRU IXUWKHU GHWHUPLQDWLRQV RQFH WKDZHG /XPLQHVFHQFH ZDV PHDVXUHG XVLQJ D %HFNPDQ VFLQWLOODWLRQ VSHFWURPHWHU ZLWK FRLQFLGHQFH FRXQWLQJ VZLWFKHG RII 1LFRWLQDPLGH QXFOHRWLGHV ZHUH GHWHUPLQHG E\ EDFWHULDO OXFLIHUDVH ELROXPLQHVFHQFH 7KH UHDJHQWV IRU WKLV DVVD\ DOVR ZHUH SXUFKDVHG IURP /.% *DLWKHUVEXUJ 0Gf DQG OXPLQHVFHQFH ZDV PHDVXUHG DV GHVFULEHG DERYH 7KH DVVD\ SURFHGXUH XVHG ZDV VLPLODU WR WKDW GHVFULEHG E\ .DUS HW DO f DQG SHUIRUPHG DW URRP WHPSHUDWXUH $ SL DOLTXRW RI 1$'+ PRQLWRULQJ UHDJHQW ZDV DGGHG WR SL RI 0 SRWDVVLXP SKRVSKDWH EXIIHU DW S+ 7KHQ WKH EDFNJURXQG OXPLQHVFHQFH ZDV PHDVXUHG ,PPHGLDWHO\ DIWHU WKH DGGLWLRQ RI SL RI IHUPHQWDWLRQ H[WUDFW HPLVVLRQ GXH WR 1$'+ ZDV PHDVXUHG ,Q RUGHU WR GHWHUPLQH WKH DPRXQW RI 1$' LQ VDPSOHV SL RI D PJPO DTXHRXV VROXWLRQ RI DOFRKRO GHK\GURJHQDVH ZDV V\ULQJHG LQWR WKH DVVD\ EXIIHU EHIRUH WKH

PAGE 134

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

PAGE 135

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f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b RI WKH IHUPHQWDWLYH DFWLYLW\ REVHUYHG DW K ZDV ORVW $V WKH JOXFRVH LQ WKH PHGLXP ZDV H[KDXVWHG E\ K WKH UDWH RI IHUPHQWDWLRQ GURSSHG E\ RYHU b ,I WKH DFWLYLW\ RI RQH RI WKH JO\FRO\WLF HQ]\PHV FRQVWULFWV FDUERQ IORZ WR HWKDQRO

PAGE 136

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f 5DWHV RI JO\FRO\VLV f DQG WRWDO SKRVSKRU\ODWHG JO\FRO\WLF LQWHUPHGLDWHV 2 ff %f +H[RVH SKRVSKDWHV k f WULVH SKRVSKDWHV 4 f DQG WRWDO QXFOHRWLGHV Â’ f

PAGE 137

3(5&(17$*( 2) 3(5&(17$*( 2) 9$/8( $7 K /= 7

PAGE 138

WKHQ JO\FRO\WLF LQWHUPHGLDWHV VKRXOG DFFXPXODWH DV IHUPHQWDWLRQ UDWH GHFOLQHV %\ K KRZHYHU WKH DPRXQW RI WRWDO SKRVSKRU\ODWHG LQWHUPHGLDWHV GHFOLQHG E\ b )LJ $f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b DQG WKH OHYHO RI WULVH SKRVSKDWHV IHOO E\ b DIWHU K %\ K WKH OHYHO RI WULVH SKRVSKDWHV JUDGXDOO\ URVH WR b RI WKH K YDOXH ZKLOH WKH OHYHO RI KH[RVH SKRVSKDWHV UHPDLQHG ORZ DW DERXW b RI WKH K YDOXH $V WKH OHYHO RI WULVH SKRVSKDWHV UDSLGO\ IHOO DIWHU K WKH OHYHO RI KH[RVH SKRVSKDWHV LQFUHDVHG WR b RI WKH K YDOXH 7KHVH REVHUYDWLRQV VXJJHVW WKDW FDUERQ IORZ WKURXJK

PAGE 139

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f DQG SUREDEO\ DOVR LQFOXGHG QXFOHRWLGH LQWHUPHGLDWHV IURP ELRV\QWKHWLF SURFHVVHV DQG RWKHU PXOWLSO\ SKRVSKRU\ODWHG PROHFXOHV LQ DGGLWLRQ WR DGHQLQH DQG QLFRWLQDPLGH QXFOHRWLGHV ,Q ILJXUH % WKHVH YDOXHV DUH SORWWHG DV IHUPHQWDWLRQ SURJUHVVHV %\ K WKH DPRXQW RI SKRVSKDWH LQ WKH WRWDO QXFOHRWLGH SRRO GHFUHDVHG E\ b DQG E\ K LW GURSSHG E\ b IURP WKH OHYHO REVHUYHG DW K 7RWDO QXFOHRWLGH SKRVSKDWH UHPDLQHG ORZ XQWLO WKH HQG RI IHUPHQWDWLRQ

PAGE 140

7KLV GHFUHDVH LQ WRWDO QXFOHRWLGH SKRVSKDWH FRXOG KDYH UHVXOWHG IURP LQFUHDVHG OHDNDJH RU H[FUHWLRQ RI QXFOHRWLGHV IURP WKH FHOOV GXULQJ IHUPHQWDWLRQ ,Q ILJXUH WKH DPRXQW RI QXFOHRWLGH SKRVSKDWH PHDVXUHG DV FRXQWV SHU PLQ SHU PO RI IHUPHQWDWLRQ EURWK ZLWKRXW RU ZLWK FHOOV FRUUHFWHG IRU FRXQWV LQ WKH FXOWXUH EURWKf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f WKHQ WKH LQFUHDVLQJ SRRO RI WULVH SKRVSKDWHV PD\ UHVXOW IURP GHFUHDVLQJ DFWLYLW\ RI HLWKHU RI WKHVH HQ]\PHV LQ YLYR ,Q ILJXUH LV SORWWHG WKH LQWUDFHOOXODU FRQFHQWUDWLRQV RI QLFRWLQDPLGH QXFOHRWLGHV WKURXJKRXW IHUPHQWDWLRQ DV GHWHUPLQHG E\ EDFWHULDO OXPLQHVFHQFH 7KH LQWUDFHOOXODU FRQFHQWUDWLRQ RI 1$'+ UHPDLQHG ORZ DW P0 DQG GLG QRW GHFUHDVH DIWHU K 7KXV DOFRKRO GHK\GURJHQDVH VKRXOG

PAGE 141

)LJXUH &RPSDULVRQ RI QXFOHRWLGH OHYHOV IRXQG LQ WKH FHOOV ZLWK WKRVH IRXQG LQ WKH IHUPHQWDWLRQ EURWK 9DOXHV DUH SORWWHG DV FRXQWV SHU PLQ SHU PO RI IHUPHQWDWLRQ H[WUDFW IRU GLUHFW FRPSDULVRQ 7KH FRXQWV LQ WKH ZKROH IHUPHQWDWLRQ VDPSOHV ZHUH FRUUHFWHG IRU FRXQWV IRXQG LQ WKH EURWK DORQH 4XDOLWDWLYHO\ VLPLODU UHVXOWV ZHUH REWDLQHG IURP WKUHH LQGHSHQGHQW EDWFK IHUPHQWDWLRQV 6\PEROV ’ FHOO DVVRFLDWHG QXFOHRWLGH FRXQWV ‘ QXFOHRWLGH FRXQWV IRXQG LQ WKH PHGLXP

PAGE 142

18&/(27,'( 3+263+$7( ; &RXQWV SHU PLQ SHU POf 2 f§rf I2 *2 f f f f R R R R

PAGE 143

)LJXUH ,QWUDFHOOXODU FRQFHQWUDWLRQ RI QLFRWLQDPLGH QXFOHRWLGHV GXULQJ EDWFK IHUPHQWDWLRQ 7KH YDOXHV SORWWHG KDYH EHHQ FRUUHFWHG IRU QLFRWLQDPLGH QXFOHRWLGHV GHWHFWHG LQ WKH PHGLXP (UURU EDUV UHSUHVHQW WKH DYHUDJH VWDQGDUG GHYLDWLRQ IRU WKUHH LQGHSHQGHQW EDWFK IHUPHQWDWLRQV 6\PEROV 1$'+ 2 1$' Â’ WRWDO QLFRWLQDPLGH QXFOHRWLGHV

PAGE 144

1,&27,1$0,'( 18&/(27,'(6 P0f

PAGE 145

KDYH UHPDLQHG YHU\ DFWLYH LQ YLYR 7KH OHYHO RI 1$' DOVR GLG QRW GHFUHDVH EXW IOXFWXDWHG DERXW P0 IRU WKH ILUVW K RI WKH IHUPHQWDWLRQ :LWK WKH H[KDXVWLRQ RI JOXFRVH LQ WKH PHGLXP E\ WKH HQG RI IHUPHQWDWLRQ WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI 1$' LQFUHDVHG WR DOPRVW P0 %HFDXVH WKH OHYHOV RI 1$'+ FRPSDUHG WR 1$' ZHUH VR ORZ WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI WRWDO QLFRWLQDPLGH QXFOHRWLGHV PLPLFNHG WKH VDPH SDWWHUQ DV WKH OHYHOV RI 1$' 7KXV WKH LQWUDFHOOXODU FRQFHQWUDWLRQV RI 1$' DQG 1$'+ GLG QRW IROORZ WKH GHFOLQH LQ WRWDO QXFOHRWLGH SKRVSKDWH DQG SUREDEO\ ZHUH QRW UHVSRQVLEOH IRU WKH LQFUHDVHG OHYHOV RI WULVH LQWHUPHGLDWHV 2Q WKH FRQWUDU\ WKH LQWUDFHOOXODU OHYHOV RI 1$'+ DQG 1$' VKRXOG KDYH DFFHOHUDWHG JO\FHUDOGHK\GHSKRVSKDWH GHK\GURJHQDVH DFWLYLW\ $GHQLQH 1XFOHRWLGHV DQG (QHUJ\ &KDUJH $73 LV UHTXLUHG IRU WKH SKRVSKRU\ODWLRQ RI KH[RVHV DV WKH LQLWLDO VWHS WKDW FKDQQHOV KH[RVHV LQWR WKH JO\FRO\WLF SDWKZD\ 'HSOHWLRQ RI $73 GXULQJ IHUPHQWDWLRQ FRXOG EH UHVSRQVLEOH IRU WKH LQLWLDO GHFOLQH LQ JO\FRO\WLF LQWHUPHGLDWHV )LJ f E\ UHVWULFWLQJ FDUERQ IORZ LQWR WKH SDWKZD\ $V D UHVXOW WKH UDWH RI IHUPHQWDWLRQ ZRXOG GHFUHDVH ,Q ILJXUH $ WKH LQWUDFHOOXODU FRQFHQWUDWLRQV RI DGHQLQH QXFOHRWLGHV DV GHWHUPLQHG E\ ILUHIO\ OXFLIHUDVH OXPLQHVFHQFH DUH SORWWHG DV IHUPHQWDWLRQ SURJUHVVHV )URP WR K WKH OHYHO RI $73 UHPDLQHG FRQVWDQW DW DERXW P0 $IWHU K WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI $73

PAGE 146

)LJXUH ,QWUDFHOOXODU FRQFHQWUDWLRQ RI DGHQLQH QXFOHRWLGHV DQG HQHUJ\ FKDUJH GXULQJ EDWFK IHUPHQWDWLRQ 7KH YDOXHV SORWWHG KDYH EHHQ FRUUHFWHG IRU DGHQLQH QXFOHRWLGHV GHWHFWHG LQ WKH PHGLXP (UURU EDUV UHSUHVHQW WKH DYHUDJH VWDQGDUG GHYLDWLRQ IRU WKUHH LQGHSHQGHQW EDWFK IHUPHQWDWLRQV $f $GHQLQH QXFOHRWLGHV GXULQJ EDWFK IHUPHQWDWLRQ 6\PEROV 2 $73 2 $'3 Â’ $03 WRWDO DGHQLQH QXFOHRWLGHV %f &KDQJHV LQ HQHUJ\ FKDUJH GXULQJ EDWFK IHUPHQWDWLRQ (QHUJ\ FKDUJH ZDV FDOFXODWHG DV GHVFULEHG E\ %DOO DQG $WNLQVRQ f

PAGE 147

$'(1,1( 18&/(27,'(6 P0f $'(1
PAGE 148

KDG GURSSHG E\ b DQG UHPDLQHG ORZ XQWLO WKH JOXFRVH LQ WKH PHGLXP ZDV GHSOHWHG :KHQ WKH IHUPHQWDWLRQ ZDV FRPSOHWHG $73 LQFUHDVHG WR D OHYHO WLPHV WKDW DW K 7KLV VXJJHVWV WKDW DV WKH FDUERQ VRXUFH LV GHSOHWHG WKH $73 XWLOL]LQJ SDWKZD\V RI WKH FHOO EHFRPH LQDFWLYH DQG WKH OLWWOH $73 V\QWKHVL]HG GXULQJ WKLV SHULRG DFFXPXODWHV :KLOH $73 OHYHOV GHFUHDVHG E\ b DIWHU K D GUDPDWLF LQFUHDVH LQ WKH OHYHO RI $03 ZDV GHWHFWHG E\ K )LJ $f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b DQG WKH OHYHO RI $03 GUDPDWLFDOO\ LQFUHDVHG HDUO\ LQ WKH IHUPHQWDWLRQ )LJ $f WKH DGHQ\ODWH HQHUJ\ FKDUJH VKRZHG

PAGE 149

D ODUJH GHFUHDVH DIWHU K )LJ %f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b JOXFRVH LQ FRPSOH[ PHGLXP XQGHU VHOILQGXFHG DQDHURELRVLV WKH UDWH RI DOFRKRO SURGXFWLRQ GHFOLQHV DV HWKDQRO DFFXPXODWHV LQ WKH IHUPHQWDWLRQ EURWK )LJ f 7KH VWXGLHV SUHVHQWHG LQ FKDSWHU ,,, VKRZ WKDW HWKDQRO LV QRW WKH RQO\ IDFWRU FRQWULEXWLQJ WR WKLV UHGXFWLRQ LQ IHUPHQWDWLRQ UDWH $ QXWULWLRQDO GHILFLHQF\ IRU PDJQHVLXP DOVR ZDV GHPRQVWUDWHG WR EH UHVSRQVLEOH &KDSWHU ,9f (OLPLQDWLQJ WKLV QXWULWLRQDO GHILFLHQF\ SDUWLDOO\ UHVWRUHG KLJKHU IHUPHQWDWLRQ UDWHV EXW GLG QRW SUHYHQW WKH LQLWLDO GHFOLQH LQ DFWLYLW\ )LJ f 7KH \HDVW FHOOV DSSHDUHG WR KDYH

PAGE 150

FKDQJHG SK\VLRORJLFDOO\ DV WKH IHUPHQWDWLRQ SURJUHVVHG )LJ DQG )LJ f 9DULDWLRQV LQ WKH DPRXQWV RI JO\FRO\WLF HQ]\PHV DQG LQWHUQDO S+ ZHUH H[DPLQHG LQ FKDSWHU 9 DV SRWHQWLDO SK\VLRORJLFDO FKDQJHV ZKLFK PLJKW EH UHVSRQVLEOH IRU WKH REVHUYHG GHFOLQH LQ IHUPHQWDWLRQ UDWH 7KH OHYHOV RI JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV LQFOXGLQJ KH[RNLQDVH PHDVXUHG LQ YLWUR UHPDLQHG KLJK WKURXJKRXW IHUPHQWDWLRQ )LJ $f 7KH DFWLYLW\ RI WKHVH HQ]\PHV LQ YLYR KRZHYHU LV PRGXODWHG E\ WKH LQWUDFHOOXODU HQYLURQPHQW 6ROV HW DO f 2QH VXFK HQYLURQPHQWDO IDFWRU LV S+ 7KURXJKRXW IHUPHQWDWLRQ WKH LQWUDFHOOXODU S+ UHPDLQHG FORVH WR QHXWUDOLW\ )LJ $f QHDU WKH S+ RSWLPXP IRU PDQ\ RI WKH JO\FRO\WLF DQG DOFRKRORJHQLF HQ]\PHV 7KH ODUJH GHFOLQH LQ LQWUDFHOOXODU $73 GXULQJ IHUPHQWDWLRQ )LJ $f VXJJHVWV WKDW $73DVHV DUH DFWLYHO\ H[FUHWLQJ SURWRQV LQ DQ DWWHPSW WR FRXQWHUDFW WKH SHUPHDELOL]LQJ HIIHFWV RI HWKDQRO DFFXPXODWLRQ 3HQD HW DO f ,QFOXGHG LQ WKH LQWUDFHOOXODU HQYLURQPHQW DUH VXEVWUDWH FRIDFWRU FRHQ]\PH DQG HIIHFWRU PROHFXOHV ZKLFK DOVR FDQ PRGXODWH WKH DFWLYLWLHV RI WKH JO\FRO\WLF HQ]\PHV 7KH VWXGLHV SUHVHQWHG LQ WKLV FKDSWHU H[DPLQH WKH FKDQJHV LQ WKH LQWUDFHOOXODU OHYHOV RI JO\FRO\WLF LQWHUPHGLDWHV DQG QXFOHRWLGHV $V \HDVW FHOO JURZWK VORZHG GXULQJ EDWFK IHUPHQWDWLRQ )LJ $f WKH LQWUDFHOOXODU OHYHO RI QXFOHRWLGH SKRVSKDWH GUDPDWLFDOO\ GHFOLQHG E\ PRUH WKDQ b

PAGE 151

)LJ f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f ,Q WKH SUHVHQW VWXG\ DV JO\FRO\WLF IOX[ GHFUHDVHG GXULQJ IHUPHQWDWLRQ LQLWLDOO\ WKH OHYHOV RI SKRVSKRU\ODWHG JO\FRO\WLF LQWHUPHGLDWHV DOVR GHFUHDVHG 7KHVH UHVXOWV DUH FRQVLVWHQW ZLWK WKRVH RI 'HQ +ROODQGHU HW DO f ZKR REVHUYHG ORZHU OHYHOV RI JO\FRO\WLF LQWHUPHGLDWHV LQ DQDHURELFDOO\ JURZQ FHOOV WKDQ LQ DHURELFDOO\ JURZQ FHOOV $QDHURELFDOO\ JURZQ FHOOV KDYH D KLJKHU UDWH RI JOXFRVH XWLOL]DWLRQ OLNH K FHOOV WKDQ DHURELFDOO\ JURZQ FHOOV OLNH K RU ROGHU FHOOV 6LPLODU REVHUYDWLRQV ZHUH PDGH E\ )UDQFR HW DO f IRU WKH JOXFRVHSKRVSKDWH DQG IUXFWRVH ELVSKRVSKDWH OHYHOV RI EDNHUVn \HDVW IHUPHQWLQJ b JOXFRVH 6LQFH WKH DPRXQWV RI JO\FRO\WLF HQ]\PHV GLG QRW IDOO )LJ f WKLV GHFUHDVH ZDV

PAGE 152

QRW GXH WR D GHFOLQH LQ JO\FRO\WLF FDSDFLW\ $V WKH HQHUJ\ FKDUJH GUDPDWLFDOO\ IHOO WKH UDWHV RI SKRVSKRIUXFWRNLQDVH %HW] DQG 0RRUH f DQG S\UXYDWH NLQDVH VKRXOG LQFUHDVH .D\QH f DFWLYDWLQJ FDUERQ IORZ WKURXJK JO\FRO\VLV $ PRUH SODXVLEOH H[SODQDWLRQ IRU WKH GHFOLQH LQ JO\FRO\WLF LQWHUPHGLDWHV LV WKDW WKH UDWH RI FDUERQ LQSXW LQWR WKH SDWKZD\ PD\ KDYH GHFUHDVHG ZLWK UHVSHFW WR JO\FRO\WLF FDSDFLW\ 6LPLODU SURSRVDOV KDYH EHHQ PDGH WR H[SODLQ WKH 3DVWHXU HIIHFW LQ \HDVW %HFNHU DQG %HW] 'HQ +ROODQGHU HW DO f 7KH LQFUHDVLQJ OHYHO RI WULVH SKRVSKDWHV DORQJ ZLWK WKH FRQVWDQW ORZ OHYHO RI KH[RVH SKRVSKDWHV IURP WR K GXULQJ IHUPHQWDWLRQ )LJ %f VXJJHVWV WKDW WKH WULVH SDUW RI WKH JO\FRO\WLF SDWKZD\ ZDV UHVWULFWLQJ FDUERQ IORZ WR HWKDQRO 'HFOLQLQJ JO\FHUDOGHK\GHSKRVSKDWH GHK\GURJHQDVH DFWLYLW\ LQ YLYR SUREDEO\ ZDV QRW UHVSRQVLEOH IRU WKLV FRQVWULFWLRQ FRQVLGHULQJ WKH KLJK OHYHOV RI 1$' FRPSDUHG WR 1$'+ WKURXJKRXW IHUPHQWDWLRQ )LJ f 6LQFH WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI 1$'+ GLG QRW GHFUHDVH DV IHUPHQWDWLRQ UDWH GHFOLQHG D IDOO LQ DOFRKRO GHK\GURJHQDVH DFWLYLW\ LQ YLYR DOVR GRHV QRW DSSHDU WR EH D OLNHO\ FDXVH 5DWKHU WKH ORZ OHYHO RI KH[RVH SKRVSKDWHV PD\ KDYH ORZHUHG WKH DFWLYLW\ RI S\UXYDWH NLQDVH E\ GHFUHDVLQJ WKH DYDLODELOLW\ RI IUXFWRVH ELVSKRVSKDWH D SRWHQW DFWLYDWRU RI WKLV HQ]\PH +HVV HW DO f 7KLV UHVWULFWLRQ FRXOG OHDG WR DQ DFFXPXODWLRQ RI LQWHUPHGLDWHV

PAGE 153

HDUOLHU LQ WKH SDWKZD\ XQWLO KH[RVH SKRVSKDWHV LQFUHDVH UDLVLQJ WKH OHYHOV RI IUXFWRVH ELVSKRVSKDWH $OWHUQDWLYHO\ WKH DFWLYLW\ RI IUXFWRVH ELVSKRVSKDWH DOGRODVH PD\ KDYH GHFUHDVHG DOORZLQJ WKH DFFXPXODWLRQ RI LWV VXEVWUDWH 7KHQ WKH OHYHOV RI WULVH SKRVSKDWHV EHJDQ WR GHFOLQH )LJ %f SUREDEO\ GXH WR LQFUHDVHG DFWLYLW\ RI S\UXYDWH NLQDVH 7KH UDWH RI FDUERQ LQSXW LQWR JO\FRO\VLV LV GHWHUPLQHG E\ WKH DPRXQWV DQG WKH DFWLYLWLHV RI WKH KH[RVH SKRVSKRU\ODWLQJ HQ]\PHV DQG WKH WUDQVSRUW FDUULHU PROHFXOHV LQYROYHG LQ JOXFRVH XSWDNH %HFNHU DQG %HW] f +H[RVH FDUERQ LV FKDQQHOOHG LQWR WKLV SDWKZD\ E\ VXJDU WUDQVSRUW FDUULHUV ORFDWHG LQ WKH FHOO PHPEUDQH )UDQ]XVRII DQG &LULOOR f 7KH DFWLYLW\ RI WKHVH FDUULHU PROHFXOHV VHHPV WR EH FRQWUROOHG E\ WKH PHWDEROLF VWDWH RI WKH FHOO (GG\ f +H[RVH NLQDVHV KDYH EHHQ LPSOLFDWHG LQ UHJXODWLQJ JOXFRVH XSWDNH LQ 6 FHUHYLVLDH %LVVRQ DQG )UDHQNHO %LVVRQ DQG )UDHQNHO %XVWXULD DQG /DJXQDV f 0XWDQWV GHILFLHQW LQ ERWK KH[RNLQDVHV DQG JOXFRNLQDVH ODFN WKH KLJK DIILQLW\ JOXFRVH XSWDNH V\VWHP A RI P0f ZKLOH UHWDLQLQJ WKH ORZ DIILQLW\ V\VWHP LA RI P0f 7KH VWXGLHV RI 1RDW HW DO f DQG 5XGROSK DQG )URPP f LQGLFDWH WKDW $03 DFWV DV D FRPSHWLWLYH LQKLELWRU RI $73 LQ WKH KH[RNLQDVH UHDFWLRQ 7KXV WKH IROG LQFUHDVH LQ WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI $03 IURP K WR K GXULQJ IHUPHQWDWLRQ ZLWK WKH GHFUHDVLQJ OHYHO

PAGE 154

RI $73 )LJ $f PD\ LQKLELW WKH LQ YLYR DFWLYLW\ RI WKH KH[RNLQDVHV DQG DV D UHVXOW JOXFRVH XSWDNH 7KLV DFFXPXODWLRQ RI $03 PD\ EH WKH UHVXOW RI 51$ GHJUDGDWLRQ &KDSPDQ DQG $WNLQVRQ f ZKLFK FDQ DFW DV DQ $73DVH WR FRQYHUW $73 WR $03 DV FHOOV HQWHU VWDWLRQDU\ SKDVH $OWHUQDWLYHO\ WKH FKDQJHV LQ PHPEUDQH OLSLG FRPSRVLWLRQ WKDW RFFXU DV JURZWK VORZV +XQWHU DQG 5RVH f DQG FHOOV HQWHU VWDWLRQDU\ SKDVH +RPDQQ HW DO f PD\ GHFUHDVH WKH DFWLYLW\ RI WKH WUDQVSRUW V\VWHP (YLGHQFH WR VXSSRUW WKLV K\SRWKHVLV ZDV VXSSOLHG E\ 7KRPDV DQG 5RVH f ZKR VKRZHG WKDW FKDQJHV LQ SODVPD PHPEUDQH OLSLG FRPSRVLWLRQ DIIHFW VROXWH XSWDNH E\ 6 FHUHYLVLDH ,Q DGGLWLRQ WR WKH DFWLYLW\ RI WKH KH[RVH XSWDNH V\VWHP WKH DPRXQWV RI WUDQVSRUW PROHFXOHV DQG KH[RVH SKRVSKRU\ODWLQJ HQ]\PHV DUH DOVR LPSRUWDQW GHWHUPLQDQWV RI JOXFRVH XSWDNH UDWH ,Q FKDSWHU 9 WKH DPRXQW RI KH[RVH SKRVSKRU\ODWLQJ HQ]\PHV DVVD\HG DV KH[RNLQDVH ZHUH VKRZQ WR UHPDLQ KLJK WKURXJKRXW IHUPHQWDWLRQ 7DEOH DQG )LJ f +RZHYHU %XVWXULD DQG /DJXQDV f REVHUYHG WKDW XQGHU QLWURJHQOLPLWLQJ JURZWK FRQGLWLRQV WKH JOXFRVH XSWDNH V\VWHPV RI 6 FHUHYLVLDH ZHUH LQDFWLYDWHG DQG WKLV LQDFWLYDWLRQ UHTXLUHG WKH XWLOL]DWLRQ RI D IHUPHQWDEOH FDUERK\GUDWH E\ WKH FHOOV 'XULQJ QLWURJHQ VWDUYDWLRQ WKH IHUPHQWDWLRQ UDWH RI 6 FHUHYLVLDH GHFUHDVHV LQ D PDQQHU VLPLODU WR WKDW GHVFULEHG LQ WKH SUHVHQW VWXG\ /LNHZLVH JOXFRVH XSWDNH ZDV LQDFWLYDWHG ZKHQ SURWHLQ V\QWKHVLV ZDV

PAGE 155

LQKLELWHG LQ WKH SUHVHQFH RI D IHUPHQWDEOH FDUERQ VRXUFH $V FHOO PXOWLSOLFDWLRQ GHFUHDVHV ZKHQ WKH \HDVW FHOOV HQWHU VWDWLRQDU\ SKDVH WKH UDWH RI SURWHLQ V\QWKHVLV DOVR GHFOLQHV %RXFKHULH f 7KXV WKH DPRXQWV RI WKH JOXFRVH WUDQVSRUW PROHFXOHV PD\ IDOO DV WKH \HDVW FHOOV HQWHU VWDWLRQDU\ SKDVH GXH WR LQDFWLYDWLRQ 7KH LQKLELWLRQ RI JOXFRVH WUDQVSRUW RU SKRVSKRU\ODWLRQ LV WKH OLNHO\ FDXVH RI WKH LQLWLDO GHFOLQH LQ JO\FRO\WLF LQWHUPHGLDWH OHYHOV DQG WKH UHVXOWLQJ b GHFUHDVH LQ IHUPHQWDWLYH DFWLYLW\ VHHQ DIWHU K )LJ $f 7KLV GHFUHDVH LQ JO\FRO\WLF IOX[ WKHQ LV PDQLIHVWHG DV D GHFOLQH LQ WKH UDWH RI DOFRKRO SURGXFWLRQ DV HWKDQRO DFFXPXODWHV GXULQJ IHUPHQWDWLRQ

PAGE 156

&+$37(5 9,, &21&/86,216 $1' )8785( ',5(&7,216 7KH VWXGLHV SUHVHQWHG KHUH H[DPLQH WKH UDWH RI JOXFRVH FRQYHUVLRQ WR HWKDQRO GXULQJ EDWFK IHUPHQWDWLRQ E\ 6DFFKDURPYFHV FHUHYLVLDH DQG LWV GHFOLQH DV DOFRKRO DFFXPXODWHV LQ WKH PHGLXP )LJ f 0RVW SUHYLRXV VWXGLHV KDYH DWWULEXWHG WKLV GHFOLQH LQ DOFRKRO SURGXFWLRQ UDWH WR WKH SUHVHQFH RI HWKDQRO LQ WKH IHUPHQWDWLRQ EURWK ,QJUDP DQG %XWWNH 0RXOLQ HW DO f ,Q WKH SUHVHQW VWXG\ WKLV SKHQRPHQRQ KDV EHHQ FKDUDFWHUL]HG XVLQJ D UHVSLUDWRU\GHILFLHQW VWUDLQ RI EUHZHU\ \HDVW IHUPHQWLQJ b JOXFRVH DQG PDQ\ SRWHQWLDO FDXVHV RI WKH GHFOLQLQJ IHUPHQWDWLRQ UDWH KDYH EHHQ H[DPLQHG )URP WKLV ZRUN D QXPEHU RI LPSRUWDQW IHDWXUHV RI DOFRKROLF IHUPHQWDWLRQ E\ 6 FHUHYLVLDH KDYH EHHQ HOXFLGDWHG $f (YHQ WKRXJK WKH UDWHV RI IHUPHQWDWLRQ DQG JURZWK GHFOLQHG DV HWKDQRO DFFXPXODWHG DQG ZKHQ HWKDQRO ZDV DGGHG WR IHUPHQWDWLRQ EURWK )LJ DQG )LJ f WKH DSSDUHQW LQKLELWLRQ E\ DFFXPXODWHG HWKDQRO ZDV JUHDWHU WKDQ LQKLELWLRQ E\ DGGHG HWKDQRO %f 5HPRYDO RI HWKDQRO IURP \HDVW FHOOV GLG QRW IXOO\ UHVWRUH IHUPHQWDWLRQ UDWH 7DEOH DQG )LJ f DQG JURZWK LQ WKH SUHVHQFH RI DGGHG HWKDQRO GLG QRW H[WHQVLYHO\ LQKLELW IHUPHQWDWLRQ UDWH 7DEOH f 7KHVH UHVXOWV VXJJHVW WKDW DFFXPXODWLRQ RI

PAGE 157

HWKDQRO LQ WKH IHUPHQWDWLRQ EURWK PD\ QRW EH WKH RQO\ FDXVH RI GHFOLQLQJ IHUPHQWDWLRQ UDWH &f (WKDQRO IUHHO\ SHUPHDWHG WKH \HDVW FHOO HQYHORSH 7DEOH f DQG GLG QRW UHDFK LQWUDFHOOXODU FRQFHQWUDWLRQV VXIILFLHQW WR LQKLELW WKH DFWLYLW\ RI JO\FRO\WLF HQ]\PHV 0LOODU HW DO f GXULQJ IHUPHQWDWLRQ 'f ,Q D FRPSOH[ PHGLXP FRQWDLQLQJ D KLJK DPRXQW RI IHUPHQWDEOH FDUERK\GUDWH WKH GHFOLQH LQ IHUPHQWDWLRQ UDWH ZDV FDXVHG LQ SDUW E\ D QXWULHQW OLPLWDWLRQ &KDSWHU ,,,f 7KLV OLPLWDWLRQ LQ D \HDVW H[WUDFWSHSWRQH EDVHG PHGLXP ZDV IRU PDJQHVLXP &KDSWHU ,9f KRZHYHU VXSSOHPHQWDWLRQ ZLWK PDJQHVLXP GLG QRW HQWLUHO\ HOLPLQDWH WKH GHFOLQH LQ IHUPHQWDWLRQ UDWH )LJ f /LPLWDWLRQV IRU RWKHU QXWULHQWV DIWHU PDJQHVLXP VXSSOHPHQWDWLRQ DOVR PD\ RFFXU DOWKRXJK QRQH KDYH EHHQ LGHQWLILHG \HW (f 7KH UHPDLQLQJ GHFOLQH LQ IHUPHQWDWLRQ UDWH ZDV QRW GXH WR LQDFWLYDWLRQ RI JO\FRO\WLF HQ]\PHV WR WKH LQDELOLW\ WR PDLQWDLQ D QHXWUDO LQWHUQDO S+ RU WR D GHFUHDVH LQ WKH LQWUDFHOOXODU OHYHOV RI QXFOHRWLGHV )f 7KH \HDVW FHOOV FKDQJHG SK\VLRORJLFDOO\ DV JOXFRVH ZDV FRQYHUWHG WR HWKDQRO 2OGHU FHOOV ZHUH PRUH UHVLVWDQW WR HWKDQROLQGXFHG LQKLELWLRQ RI IHUPHQWDWLRQ UDWH )LJ DQG )LJ f DQG WR HWKDQROLQGXFHG GHFUHDVH LQ $ S+ )LJ f WKDQ \RXQJHU FHOOV ,QWUDFHOOXODU FRQFHQWUDWLRQV RI $73 IHOO DQG $03 URVH GUDPDWLFDOO\ /HYHOV RI ERWK KH[RVH SKRVSKDWHV DQG WULVH SKRVSKDWH LQWHUPHGLDWHV LQLWLDOO\ IHOO )LJ f VXJJHVWLQJ WKDW WKH LQLWLDO GHFOLQH LQ

PAGE 158

IHUPHQWDWLRQ UDWH PD\ UHVXOW IURP LQKLELWLRQ DW WKH HDUOLHVW VWDJHV RI JOXFRVH PHWDEROLVP WUDQVSRUW RU DVVRFLDWHG SKRVSKRU\ODWLRQ *f 2QH SRVVLEOH H[SODQDWLRQ IRU D GHFUHDVH LQ KH[RVH XSWDNH FDSDFLW\ FRXOG EH WKH LQKLELWLRQ RI KH[RVH SKRVSKRU\ODWLRQ E\ ORZ OHYHOV RI $73 DQG KLJK OHYHOV RI $03 WKDW DFFXPXODWH GXULQJ IHUPHQWDWLRQ )LJ f DOWKRXJK RWKHU H[SODQDWLRQV DUH DOVR SODXVLEOH &KDSWHU 9,f ,Q WKH ODVW FKDSWHU D GHFOLQH LQ JOXFRVH WUDQVSRUW RU SKRVSKRU\ODWLQJ DFWLYLW\ ZDV VXJJHVWHG WR EH WKH OLNHO\ FDXVH RI WKH LQLWLDO b GHFOLQH LQ IHUPHQWDWLRQ UDWH WKDW UHPDLQHG DIWHU QXWULHQW VXSSOHPHQWDWLRQ 7KH ODUJH LQFUHDVH LQ LQWUDFHOOXODU $03 PD\ FRPSHWLWLYHO\ LQKLELW WKH JOXFRVH SKRVSKRU\ODWLRQ UHDFWLRQV 1RDW HW DO 5XGROSK DQG )URPP f LPSOLFDWHG LQ UHJXODWLQJ KH[RVH XSWDNH %LVVRQ DQG )UDHQNHO %LVVRQ DQG )UDHQNHO %XVWXULD DQG /DJXQDV f RU FKDQJHV LQ PHPEUDQH OLSLG FRPSRVLWLRQ PD\ RFFXU +RPDQQ HW DO +XQWHU DQG 5RVH f ZKLFK FDQ GLUHFWO\ ORZHU WKH UDWH RI JOXFRVH XSWDNH 7KRPDV DQG 5RVH f $OWHUQDWLYHO\ WKH KH[RVH WUDQVSRUW FDUULHU PROHFXOHV PD\ EH LQDFWLYDWHG ZKHQ WKH FHOOV HQWHU VWDWLRQDU\ SKDVH ,Q RUGHU WR GLVWLQJXLVK EHWZHHQ WKHVH K\SRWKHVHV IXUWKHU VWXGLHV QHHG WR EH FRQGXFWHG WR PHDVXUH WKH FDSDFLW\ RI WKH IDFLOLWDWHG GLIIXVLRQ FDUULHU V\VWHP IRU JOXFRVH WKURXJKRXW IHUPHQWDWLRQ $ GHFOLQH LQ WKH UDWH RI IDFLOLWDWHG GLIIXVLRQ EHWZHHQ K DQG K ZRXOG LQGLFDWH HLWKHU DQ LQDFWLYDWLRQ RI WKH KH[RVH XSWDNH V\VWHP

PAGE 159

LQFOXGLQJ FDUULHU PROHFXOHV DQG KH[RVHSKRVSKRU\ODWLQJ SURWHLQV RU D GHFUHDVH LQ WUDQVSRUW UDWH EURXJKW RQ E\ PHPEUDQH OLSLG FKDQJHV GXULQJ IHUPHQWDWLRQ $ FRQVWDQW UDWH RI IDFLOLWDWHG GLIIXVLRQ ZRXOG VXJJHVW WKDW LQDFWLYDWLRQ RU PHPEUDQH OLSLG FKDQJHV PD\ QRW SOD\ DQ LPSRUWDQW UROH LQ ORZHULQJ WKH OHYHOV RI JO\FRO\WLF LQWHUPHGLDWHV 5DWKHU LQKLELWLRQ RI WKH KH[RVH NLQDVH UHDFWLRQV E\ WKH ODUJH LQFUHDVH LQ $03 OHYHOV DQG WKH GHFUHDVH LQ $73 FRQFHQWUDWLRQ GXULQJ IHUPHQWDWLRQ PD\ EH D PRUH UHDVRQDEOH H[SODQDWLRQ )LJ f 7KH KH[RNLQDVH DFWLYLW\ RI 6 FHUHYLVLDH .' LQ WKH SUHVHQFH RI YDU\LQJ $73 DQG $03 FRQFHQWUDWLRQV FRXOG EH H[DPLQHG WR FRQILUP DQ LQKLELWRU\ HIIHFW RI KLJK OHYHOV RI $03 RQ WKH JOXFRVH SKRVSKRU\ODWLQJ DFWLYLW\ RI WKLV \HDVW VWUDLQ LQ YLWUR 6XEVHTXHQW VWXGLHV DOVR PLJKW LQFOXGH GHWHUPLQLQJ WKH RULJLQ RI WKH $03 WKDW DFFXPXODWHV GXULQJ IHUPHQWDWLRQ DQG H[DPLQLQJ LWV JHQHUDO UROH LQ UHJXODWLQJ PHWDEROLVP DV FHOO JURZWK VORZV )URP WKH GDWD SUHVHQWHG LQ WKLV VWXG\ D K\SRWKHWLFDO PRGHO GHVFULELQJ WKH FHOOXODU FKDQJHV WKDW RFFXU DV IHUPHQWDWLRQ UDWH GHFOLQHV FDQ EH IRUPXODWHG $V \HDVW FHOOV IHUPHQW b JOXFRVH DSSUR[LPDWHO\ b YROYROf HWKDQRO LV SURGXFHG GXULQJ H[SRQHQWLDO JURZWK )LJ f $V WKH UDWH RI JURZWK VORZV VR GRHV PDFURPROHFXODU V\QWKHVLV %RXFKHULH &KDSPDQ DQG $WNLQVRQ f 7KH SK\VLRORJLFDO FKDUDFWHU RI WKH \HDVW FHOOV FKDQJHV ZLWK FKDQJHV LQ PDFURPROHFXODU FRPSRVLWLRQ %XVWXULD DQG /DJXQDV

PAGE 160

" 7KRPDV DQG 5RVH f 'HJUDGDWLRQ SURGXFWV RI ELRV\QWKHWLF SURFHVVHV LQFOXGLQJ QXFOHRWLGHV VXFK DV $03 PD\ DFFXPXODWH LQWUDFHOOXODUO\ 7KH FHOO HQYHORSH PD\ EHFRPH PRUH SHUPHDEOH WR SURWRQV DV HWKDQRO DFFXPXODWHV LQ WKH PHGLXP &DUWZULJKW HW DO /HDR DQG YDQ 8GHQ Df DQG WKH FHOOV FRXOG UHVSRQG E\ XWLOL]LQJ $73 WR SXPS RXW SURWRQV YLD SODVPD PHPEUDQHERXQG $73DVHV 3HQD HW DO f 7KH DFFXPXODWLRQ RI $03 DORQJ ZLWK WKHVH ORZHU OHYHOV RI $73 GXH WR $73DVH DFWLRQ FRXOG LQKLELW KH[RVH XSWDNH DQG SKRVSKRU\ODWLRQ 1RDW HW DO 5XGROSK DQG )URPP f 7KHQ WKH WRWDO OHYHO RI JO\FRO\WLF LQWHUPHGLDWHV ZRXOG GHFUHDVH DV SKRVSKRIUXFWRNLQDVH EHFRPHV DFWLYDWHG E\ WKH GHFOLQLQJ $73 WR $03 UDWLR RI WKH FHOOV %HW] DQG 0RRUH f $V LQWUDFHOOXODU OHYHOV RI KH[RVH SKRVSKDWHV GHFOLQH IUXFWRVH ELVSKRVSKDWH D SRWHQW DOORVWHULF DFWLYDWRU RI S\UXYDWH NLQDVH +HVV HW DO .D\QH f DOVR ZRXOG GHFOLQH 7KLV VKRXOG GHFUHDVH WKH LQ YLYR DFWLYLW\ RI S\UXYDWH NLQDVH DQG WULVH SKRVSKDWHV WKHQ FDQ DFFXPXODWH 0DLWUD DQG /RER f $V IHUPHQWDWLRQ SURJUHVVHV WKH DFFXPXODWLRQ RI KH[RVH SKRVSKDWHV PD\ EH GXH WR WKLV GHFUHDVH LQ S\UXYDWH NLQDVH DFWLYLW\ RU DQ LQDFWLYDWLRQ RI IUXFWRVH ELVSKRVSKDWH DOGRODVH 7KHQ IUXFWRVH ELVSKRVSKDWH VKRXOG DFFXPXODWH DQG S\UXYDWH NLQDVH UHJDLQ DFWLYLW\ FDXVLQJ WKH OHYHOV RI WULVH SKRVSKDWHV WR GHFOLQH :LWK WKH SURGXFWLRQ RI b YROYROf HWKDQRO DQG WKH H[KDXVWLRQ RI JOXFRVH LQ WKH

PAGE 161

IHUPHQWDWLRQ EURWK WKH LQWUDFHOOXODU FRQFHQWUDWLRQ RI $73 LQFUHDVHV )LJ f DV $73 GHJUDGLQJ SURFHVVHV SUHVXPDEO\ EHFRPH LQDFWLYDWHG 7KLV LQFUHDVLQJ FRQFHQWUDWLRQ RI $73 DOVR FDQ KHOS WR UHGXFH WKH DFWLYLW\ RI SKRVSKRIUXFWRNLQDVH %OR[KDP DQG +DUG\ 8\HGD f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b JOXFRVH WR HWKDQRO )LQDOO\ SK\VLRORJLFDO FKDQJHV RFFXU GXULQJ WKLV GHFOLQH LQ IHUPHQWDWLRQ UDWH DIWHU QXWULHQW VXSSOHPHQWDWLRQ 7KHVH FKDQJHV DOVR DSSHDU UHVSRQVLEOH IRU WKH GHFOLQH LQ IHUPHQWDWLRQ UDWH KRZHYHU WKHLU H[DFW FDXVH KDV QRW \HW EHHQ HVWDEOLVKHG 'HWHUPLQLQJ WKH RULJLQ RI WKHVH SK\VLRORJLFDO FKDQJHV VKRXOG OHDG WR D EHWWHU XQGHUVWDQGLQJ RI WKH FRQVWUDLQWV ZKLFK OLPLW WKH UDWHV RI JO\FRO\VLV GXULQJ IHUPHQWDWLRQ

PAGE 162

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f ELRFKHPLVWU\ DQG JHQHWLFV RI \HDVWV $FDGHPLF 3UHVV 1HZ
PAGE 163

%D]XD &' DQG &5 :LONH (WKDQRO HIIHFWV RQ WKH NLQHWLFV RI D FRQWLQXRXV IHUPHQWDWLRQ ZLWK 6DFFKDURPYFHV FHUHYLVLDH %LRWHFKQRO %LRHQJ 6\PS %HDYHQ 0& &KDUSHQWLHU DQG $+ 5RVH 3URGXFWLRQ DQG WROHUDQFH RI HWKDQRO LQ UHODWLRQ WR SKRVSKROLSLG IDWW\DFLG FRPSRVLWLRQ LQ 6DFFKDURPYFHV FHUHYLVLDH 1&<& *HQ 0LFURELRO %HFNHU -8 DQG $ %HW] 0HPEUDQH WUDQVSRUW DV FRQWUROOLQJ SDFHPDNHU RI JO\FRO\VLV LQ 6DFFKDURPYFHV FDUOVEHUJHQVLV %LRFKLP %LRSK\V $FWD %HUQW ( DQG *XWPDQ (WKDQRO GHWHUPLQDWLRQ ZLWK DOFRKRO GHK\GURJHQDVH S ,Q +8 %HUJHUPH\HU HGf PHWKRGV RI HQ]\PDWLF DQDO\VLV YRO $FDGHPLF 3UHVV 1HZ
PAGE 164

%URZQ 6: DQG 6* 2OLYHU 7KH HIIHFW RI WHPSHUDWXUH RQ WKH HWKDQRO WROHUDQFH RI WKH \HDVW 6DFFKDURPYFHV XYDUXP %LRWHFKQRO /HWW %URZQ 6: 6* 2OLYHU '() +DUULVRQ DQG 5& 5LJKHODWR (WKDQRO LQKLELWLRQ RI \HDVW JURZWK DQG IHUPHQWDWLRQ 'LIIHUHQFHV LQ WKH PDJQLWXGH DQG FRPSOH[LW\ RI WKH HIIHFW (XU $SSO 0LFURELRO %LRWHFKQRO %XFKQHU ( $ONRKROLVFKH JDKUXQJ RKQH KHIH]HOOHQ %HU 'HXWVFK &KHP *HVHOO %XVWXULD $ DQG 5 /DJXQDV &DWDEROLWH LQDFWLYDWLRQ RI WKH JOXFRVH WUDQVSRUW V\VWHP LQ 6DFFKDURPYFHV FHUHYLVLDH *HQ 0LFURELRO %XWWNH 70 6' -RQHV DQG %ORFK (IIHFW RI VWHURO VLGH FKDLQV RQ JURZWK DQG PHPEUDQH IDWW\ DFLG FRPSRVLWLRQ RI 6DFFKDURPYFHV FHUHYLVLDH %DFWHULRO %XWWNH 70 DQG $/ 3\OH (IIHFWV RI XQVDWXUDWHG IDWW\ DFLG GHSULYDWLRQ RQ QHXWUDO OLSLG V\QWKHVLV LQ 6DFFKDURPYFHV FHUHYLVLDH %DFWHULRO &DUWZULJKW &3 -5 -XURV]HN 0%HDYHQ )06 5XE\ 60) 'H 0RUDOV DQG $+ 5RVH (WKDQRO GLVVLSDWHV WKH SURWRQPRWLYH IRUFH DFURVV WKH SODVPD PHPEUDQH RI 6DFFKDURPYFHV FHUHYLVLDH *HQ 0LFURELRO &DVH\ *3 DQG :0 ,QJOHGHZ (WKDQRO WROHUDQFH LQ \HDVWV &5& &ULW 5HY 0LFURELRO &DVH\ *3 &$ 0DJQXV DQG :0 ,QJOHGHZ +LJK JUDYLW\ EUHZLQJ 1XWULHQW HQKDQFHG SURGXFWLRQ RI KLJK FRQFHQWUDWLRQV RI HWKDQRO E\ EUHZLQJ \HDVW %LRWHFKQRO /HWW &DVH\ *3 &$ 0DJQXV DQG :0 ,QJOHGHZ +LJK JUDYLW\ EUHZLQJ (IIHFWV RI QXWULWLRQ RQ \HDVW FRPSRVLWLRQ IHUPHQWDWLYH DELOLW\ DQG DOFRKRO SURGXFWLRQ $SSO (QY 0LFURELRO &KDSPDQ $* DQG '( $WNLQVRQ $GHQLQH QXFOHRWLGH FRQFHQWUDWLRQV DQG WXUQRYHU UDWHV 7KHLU FRUUHODWLRQ ZLWK ELRORJLFDO DFWLYLW\ LQ EDFWHULD DQG \HDVW $GY 0LFURELDO 3K\VLRO &KLQ -+ DQG '% *ROGVWHLQ 'UXJ WROHUDQFH LQ ELRPHPEUDQHV $ VSLQ ODEHO VWXG\ RI WKH HIIHFWV RI HWKDQRO 6FLHQFH

PAGE 165

&LULDF\ 0 DQG %UHLWHQEDFK 3K\VLRORJLFDO HIIHFWV RI VHYHQ GLIIHUHQW EORFNV LQ JO\FRO\VLV LQ 6DFFKDURPYFHV FHUHYLVLDH %DFWHULRO &OLIWRQ 6% :HLQVWRFN DQG '* )UDHQNHO *O\FRO\VLV PXWDQWV LQ 6DFFKDURPYFHV FHUHYLVLDH *HQHWLFV &RORZLFN 63 7KH KH[RNLQDVHV S ,Q 3' %R\HU HGf WKH HQ]\PHV YRO $FDGHPLF 3UHVV 1HZ
PAGE 166

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f EDVLF OLIH VFLHQFHV 3OHQXP 3UHVV 1HZ
PAGE 167

*UD\ :' 7KH VXJDU WROHUDQFH RI IRXU VWUDLQV RI GLVWLOOHUVn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
PAGE 168

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
PAGE 169

-DQRII $6 DQG .: 0LOOHU $ FULWLFDO DVVHVVPHQW RI WKH OLSLG WKHRULHV RI JHQHUDO DQDHVWKHWLF DFWLRQ S ,Q &KDSPDQ HGf ELRORJLFDO PHPEUDQHV $FDGHPLF 3UHVV 1HZ
PAGE 170

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

PAGE 171

/RZU\ 2+ 15RVHEURXJK $/ )DUU DQG 55DQGDOO 3URWHLQ PHDVXUHPHQW ZLWK WKH IROLQ SKHQRO UHDJHQW %LRO &KHP /XRQJ -+7 .LQHWLFV RI HWKDQRO LQKLELWLRQ LQ DOFRKRO IHUPHQWDWLRQ %LRWHFKQRO %LRHQJ 0DLRUHOOD % +: %ODQFK DQG &5 :LONH %\SURGXFW LQKLELWLRQ HIIHFWV RQ HWKDQRO IHUPHQWDWLRQ E\ 6DFFKDURPYFHV FHUHYLVLDH %LRWHFKQRO %LRHQJ 0DLWUD 3. DQG = /RER $ NLQHWLF VWXG\ RI JO\FRO\WLF HQ]\PH V\QWKHVLV LQ \HDVW %LRO &KHP 0DLWUD 3. DQG = /RER
PAGE 172

1DYRQ 5* 6FKXOPDQ 7
PAGE 173

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

PAGE 174

6HHPDQ 3 7KH PHPEUDQH DFWLRQV RI DQDHVWKHWLFV DQG WUDQTXLOL]HUV 3KDUPDFRO 5HY 6ROV $ & *DQFHGR DQG 'OD )XHQWH (QHUJ\ \LHOGLQJ PHWDEROLVP LQ \HDVWV S ,Q $+ 5RVH DQG -6 +DUULVRQ HGf WKH \HDVWV YRO $FDGHPLF 3UHVV 1HZ
PAGE 175

YDQ 8GHQ 1 (WKDQRO WR[LFLW\ DQG HWKDQRO WROHUDQFH LQ \HDVWV S ,Q *7 7VDR HGf DQQXDO UHSRUWV RQ IHUPHQWDWLRQ SURFHVVHV YRO $FDGHPLF 3UHVV 1HZ
PAGE 176

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

PAGE 177

, FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ 9 f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

PAGE 178

, FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ -RKQ ( *DQGHU 3URIHVVRU RI 0LFURELRORJ\ DQG &HOO 6FLHQFH FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ -£PHV ) 3UHVWRQ 3URIHVVRU RI 0LFURELRORJ\ DQG &HOO 6FLHQFH 7KLV GLVVHUWDWLRQ ZDV VXEPLWWHG WR WKH *UDGXDWH )DFXOW\ RI WKH &ROOHJH RI $JULFXOWXUH DQG WR WKH *UDGXDWH 6FKRRO DQG ZDV DFFHSWHG DV SDUWLDO IXOILOOPHQW RI WKH UHTXLUHPHQWV IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ $XJXVW HJH RI $JULFXOWXUH 'HDQ *UDGXDWH 6FKRRO

PAGE 179

81,9(56,7< 2) )/25,'$ 9! Lf m 9 nL PL P

PAGE 181

=22+

PAGE 182

+


86
within yeast cells, but rapidly diffuses across the cell
membrane (Dasari et al.. 1985; Guijarro and Lagunas, 1984;
Table 2), direct inhibition of glycolytic enzymes by
intracellular ethanol is unlikely during fermentations which
produce 12% (vol/vol) ethanol or less.
In this chapter, changes in the amounts of glycolytic
and alcohologenic enzymes, and internal pH and membrane
energization have been examined as possible physiological
causes for the decline in fermentative activity during batch
fermentations of 20% glucose in a yeast extract-peptone-
based medium supplemented with magnesium.
Materials and Methods
Organism and Growth Conditions
The petite yeast strain characterized in chapter II, S.
cerevisiae KD2, was used in the studies presented in this
chapter. This yeast was grown in the complex medium also
described in chapter II, except that the medium was
supplemented with 0.5 mM MgS04, as described in chapter IV.
Batch fermentations were carried out as outlined in
chapter II.
Analytical Methods
Cell mass, glucose, ethanol and fermentation rates all
were determined by the methods discussed in chapter II.
Cell protein was measured as described by Lowry et al.
(1951). The protein content of cell extracts was
quantitated using the method of Bradford (1976). Bovine


50
equivalent to control cells after 12 h. These were twice as
high as control cells after the production of 5% (vol/vol)
ethanol in approximately 24 h (Table 3, experiment 4).
Discussion
Previous studies have shown that the rate of alcohol
production by yeast per unit cell mass decreases as ethanol
accumulates during fermentation (Holtzberg et al.. 1967;
Navarro and Durand, 1978; Strehaiano and Goma, 1983). Most
of these studies have attributed this reduction in
fermentation rate to adverse effects of ethanol (Ingram and
Buttke, 1984; Maiorella et al.. 1983; Millar et al.. 1982;
Moulin et al.. 1984). In fact, the possible accumulation of
high intracellular concentrations of ethanol in S.
cerevisiae and its involvement in the inhibition of growth
and fermentation have been the subject of considerable
controversy. Previous studies have shown that the growth
and fermentation rates of S. cerevisiae are much less
sensitive to inhibition by added ethanol than is inferred by
the decrease in alcohol production and growth rates which
accompany the accumulation of ethanol during fermentation
(Ingram and Buttke, 1984; Moulin et al.. 1984; Nagodawithana
and Steinkraus, 1976; Navarro and Durand, 1978; Novak et
al.. 1981). To explain this anomaly, it has been proposed
that the leakage of ethanol from yeast cells is, in some
way, limited by the permeability of the plasma membrane.
This would result in the accumulation of high cytosolic


NUCLEOTIDE PHOSPHATE
(10 X Counts per min per ml)
O * fO GO

o o o o
132


157
Gray, W.D. 1945. The sugar tolerance of four strains of
distillers' yeast. J. Bacteriol. 49:445-452.
Gray, W.D. 1948. Further studies on the alcohol tolerance of
yeast: Its relationship to cell storage products. J.
Bacteriol. 55:53-59.
Gray, W.D. and C. Sova. 1956. Relation of molecule size and
structure to alcohol inhibition of glucose utilization by
yeast. J. Bacteriol. 72:349-356.
Guijarro, J.M. and R. Lagunas. 1984. Saccharomvces
cerevisiae does not accumulate ethanol against a
concentration gradient. J. Bacteriol. 160:874-878.
Halvorson, H. 1958. Studies on protein and nucleic acid
turnover in growing cultures of yeast. Biochim. Biophys.
Acta 27:255-266.
Harden, A. 1923. Alcoholic fermentation. Longmans, Green and
Co., New York.
Harris, J.I. and M. Waters. 1976. Glyceraldehyde-3-
phosphate dehydrogenase, p. 1-49. In P.D. Boyer (ed.), the
enzymes, vol. 13. Academic Press, New York.
Hayashida, S., D. Der Feng and M. Hongo. 1974. Function of
the high concentration alcohol-producing factor. Agrie.
Biol. Chem. 38:2001-2006.
Hayashida, S., D. Der Feng and M. Hongo. 1975. Physiological
properties of yeast cells grown in the proteolipid-
supplemented media. Agrie. Biol. Chem. 39:1025-1031.
Hayashida, S., D. Der Feng, K. Ohta, S. Chaitiumvong and M.
Hongo. 1976. Compositions and a role of Aspergillus
orvzae-proteolipid as a high concentration alcohol-
producing factor. Agrie. Biol. Chem. 40:73-78.
Hayashida, S. and K. Ohta. 1978. Cell structure of yeasts
grown anaerobically in Aspergillus orvzae-proteolipid-
supplemented media. Agrie. Biol. Chem. 6:1139-1145.
Hayashida, S. and K. Ohta. 1980. Effects of
phosphatidylcholine or ergosterololeate on physiological
properties of Saccharomvces sake. Agrie. Biol. Chem.
44:2561-2567.
Henry, S.A. 1982. Yeast membrane lipids, p. 101-158. In J.N.
Strathern, E.W. Jones and J.R. Broach (ed.), the molecular
biology of the yeast Saccharomvces: metabolism and gene
expression. Cold Spring Harbor, New York.


2
Despite the obvious importance of ethanol production by
S. cerevisiae. the physiological constraints which limit the
rate of ethanol production are not fully understood.
Identification of these constraints represents an important
step toward the development of improved organisms and
process conditions for more rapid ethanol production. Such
improvements could increase the ethanol-production capacity
of existing fermentation plants and reduce the cost of
future facilities.
Some of the initial research on alcoholic fermentation
took place in the 1830s. Independently, Cagniard-Latour,
Schawn and Kutzing described the microscopic structure of
yeast cells and initiated studies on the role of yeast in
fermentation (Schlenk, 1985). Twenty years later, Pasteur
expanded these findings and performed the first decisive
experiments showing that yeast cells were the living
entities responsible for fermenting sugar to ethanol
(Pasteur, 1860). He then went on to describe the effect of
oxygen on yeast fermentation, an effect which has since
become known as the "Pasteur effect" (Pasteur, 1861).
Biochemistry had its beginnings with the work of Buchner who
showed that the proteinaceous material in yeast juice was
capable of converting sugar to ethanol (Buchner, 1897). In
the decades that followed this discovery, the chemical
nature of alcoholic fermentation and the enzymes responsible


118
that the cell extracts were being prepared, 1.0 ml of
fermentation sample was centrifuged at 10,000 x g for 30 sec
to pellet the cells. Ethanol-EDTA extracts of the
supernatant, fermentation broth lacking cells, were prepared
as described for whole fermentation samples.
The effectiveness of the ethanol-EDTA extraction
procedure was compared with that of the formic acid method
described by Bochner and Ames (1982) and the perchloric acid
method of Ball and Atkinson (1975). Identical fermentation
samples labelled with [32P]P043' were extracted by each
method and 0.1 ml of each extract was spotted onto Whatman
3MM filter paper disks. After drying, the filter disks were
counted in a Beckman model 8000 scintillation counter. The
formic acid procedure extracted less than half of the counts
extracted by the perchloric acid procedure while the
ethanol-EDTA procedure extracted 60% more counts than the
perchloric acid procedure. This higher level of counts
extracted by ethanol may reflect partial solubilization of
phospholipids in addition to the release of small
phosphorylated molecules from cellular metabolic pools. The
ethanol-EDTA procedure was chosen for the present study
because it allowed the simultaneous extraction of both
oxidized and reduced nicotinamide nucleotides. In addition,
the extracts could be analyzed directly without having to
neutralize the pH and without having to remove perchlorate
precipitation.


100
activities of these enzymes during fermentation. With the
exception of phosphoglucomutase, which declined more
rapidly, the rates of decline of the glycolytic enzyme
activities per ml broth paralleled that of bulk proteins,
indicating neither a preferential retention nor degradation
of these central catabolic activities.
Changes in Internal pH and Membrane Energization During
Batch Fermentation
Although ethanol is the principal reduced fermentation
product from the metabolism of glucose by S. cerevisiae.
organic acids also are produced. This lowers the pH of the
fermentation broth from 5.0 at inoculation to 3.5 by 24 h
(Fig. 14A). Since the pH optima for glycolytic enzymes are
near or above neutrality (Clifton et al.. 1978), the failure
of S. cerevisiae to maintain a large A pH during the
accumulation of ethanol could explain the rapid decline in
the fermentative activities of cells despite the abundance
of glycolytic and alcohologenic enzymes. However, this
theory is not supported by the data presented in figure 14.
The A pH of yeast cells increases with the decrease in
external pH, maintaining a relatively constant internal pH
of between 6.7 and 7.0 throughout batch fermentation (Fig.
14A) Similarly, A also increased during batch
fermentation, resulting in an overall increase in proton
motive force (Fig. 14B).
These results were somewhat surprising since previous
workers have shown that ethanol increases the permeability


Figure 13. Changes in the levels of glycolytic and
alcohologenic enzymes during batch
fermentation with 20% glucose 97
Figure 14. Changes in intracellular pH and membrane
energization during batch fermentation of
20% glucose 101
Figure 15. Effects of added ethanol on A pH 104
Figure 16. A typical thin layer chromatogram of a
fermentation sample extract 120
Figure 17. Changes in [32P]-labelled cellular
metabolites during batch fermentation 126
Figure 18. Comparison of nucleotide levels found in the
cells with those found in the fermentation
broth 131
Figure 19. Intracellular concentration of nicotinamide
nucleotides during batch fermentation 133
Figure 20. Intracellular concentration of adenine
nucleotides and energy charge during batch
fermentation 136
vii


34
(specific activity, 50 pCi/mmole) was added to a 1-ml
suspension at a final activity of 42 nCi/ml. The suspension
was mixed for 10 sec using a vortex mixer, and 0.1 ml
samples were transferred to Whatman 3MM filter paper disks
(3 cm) for sorbitol measurements and to sample vials
containing 0.1 ml perchloric acid for subsequent alcohol
determinations. The remaining suspension was centrifuged
immediately at 10,000 x g for 30 sec in a microcentrifuge.
Supernatant samples of 0.1 ml then were transferred to 3MM
filter paper disks and to sample vials containing 0.1 ml of
0.58 M perchloric acid for the measurement of ethanol.
Filter disks were air dried at 80C before the addition of
scintillation fluid. The radioactivity of these samples was
measured using a Beckman model 8000 scintillation
spectrometer. Total cell volume was estimated as the
difference in [ 14C]sorbitol counts between the suspension
and the supernatant.
A correction was made for the volume of total cell
solids included in the sorbitol-based estimate of cell
volume (Fig. 4b). This was done in a separate experiment to
ensure sufficient time for equilibration of tritiated water.
Control experiments were performed to confirm that tritiated
water had reached equilibrium after 5 min and that the
sorbitol did not leak into the cells during this period.
Tritiated water and [ 14C] sorbitol (specific activity,
50 pCi/mmole) were added to concentrated cell suspensions


Figure 8. Effect of magnesium addition on cell growth and fermentation. (A) Growth.
(B) Glucose utilization. (C) Ethanol production. Results have been plotted
for four separate batch fermentations. Closed symbols represent fermentations
supplemented with 0.5 mM MgSO and open symbols represent control
fermentations in YEPD broth alone.


165
van Uden, N. 1985. Ethanol toxicity and ethanol tolerance in
yeasts, p. 11-58. In G.T. Tsao (ed.), annual reports on
fermentation processes, vol. 8. Academic Press, New York.
van Uden, N. and H. da Cruz Duarte. 1981. Effects of ethanol
on the temperature profile of Saccharomvces cerevisiae.
Zeit. Allg. Mikrobiol. 21:743-750.
Vega, J.L., A.R. Navarro, E.C. Clausen and J.L. Gaddy. 1987.
Effects of inoculum size on ethanol inhibition modeling
and other fermentation parameters. Biotechnol. Bioeng.
29:633-638.
Viegas, C.A., I. Sa-Correia and J.M. Novis. 1985. Nutrient-
enhanced production of remarkably high concentrations of
ethanol by Saccharomvces cerevisiae through soy flour
supplementation. Appl. Environ. Microbiol. 50:1333-1335.
Walker, G.M. and J.H. Duffus. 1980. Magnesium ions and the
control of the cell cycle in yeast. J. Cell Sci. 42:329-
356.
Warburg, O. and W. Christian. 1941. Isolierung und
kristalization des garungsferments enolas. Biochem. Z.
310:348-421.
Watson, K. 1982. Unsaturated fatty acid but not ergosterol
is essential for high ethanol production in Saccharomvces.
Biotechnol. Lett. 4:397-402.
Welch, P. and R.K. Scopes. 1985. Studies on cell-free
metabolism: Ethanol production by a yeast glycolytic
system reconstituted from purified enzymes. J. Biotechnol.
2:257-273.
Wickersham, L.J. 1951. Taxonomy of yeasts. USDA Technical
Bulletin No. 1029, p. 1-56. U.S. Department of
Agriculture, Washington, D.C.


156
Eddy, A.A. 1982. Mechanisms of solute transport in selected
eukaryotic microorganisms. Adv. Microbial Physiol. 23:2-
78.
Entian, K.-D., K.-U. Frohlich and D. Mecke. 1984. Regulation
of enzymes and isoenzymes of carbohydrate metabolism in
the yeast Saccharomvces cerevisiae. Biochim. Biophys. Acta
799:181-186.
Esser, K., U. Schmidt and U. Stahl. 1982. Ethanol and
biomass production of wild strains and respiratory
deficient mutants of Saccharomvces cerevisiae under
anaerobic and aerobic conditions. Eur. J. Appl. Microbiol.
Biotechnol. 16:161-164.
Fraenkel, D.G. 1981. The biochemical genetics of glycolysis
in microbes, p. 201-215. In A. Hollaender (ed.), basic
life sciences. Plenum Press, New York.
Fraenkel, D.G. 1982. Carbohydrate metabolism, p. 1-37. In
J.N. Strathern, E.W. Jones and J.R. Broach (ed.), the
molecular biology of the yeast Saccharomvces: metabolism
and gene expression. Cold Spring Harbor, New York.
Franco, C.M.M., J.E. Smith and D.R. Berry. 1984. Changes in
intermediate levels during batch culture of Saccharomvces
cerevisiae. Biotechnol. Lett. 6:803-808.
Francois, J., E. Van Schaftingen and H.-G. Hers. 1984. The
mechanism by which glucose increases fructose 2,6-
bisphosphate concentration in Saccharomvces cerevisiae A
cyclic-AMP-dependent activation of phosphofructokinase 2.
Eur. J. Biochem. 145:187-193.
Franzusoff, A.J. and V.P Cirillo. 1983. Solubilization and
reconstitution of the glucose transport system from
Saccharomvces cerevisiae. Biochim. Biophys. Acta 734:153-
159.
Ghose, T.K. and R.D. Tyagi. 1979. Rapid ethanol fermentation
of cellulose hydrolysate II. Product and substrate
inhibition and optimization of fermentor design.
Biotechnol. Bioeng. 21:1401-1420.
Goel, S.C. and N.B. Pamment. 1984. Direct injection
technigue for gas chromatographic determination of ethanol
and other volatiles in concentrated cell suspensions.
Biotechnol. Lett. 3:177-182.
Gray, W.D. 1941. Studies on the alcohol tolerance of yeasts.
J. Bacteriol. 42:561-574.


112
cannot be accurately predicted. The activities of some of
these are subject to modulation by allosteric effectors in
addition to constraints imposed by the availability of
individual substrates, cofactors and coenzymes (Fraenkel,
1982). In the next chapter, the levels of some of these
low-molecular-weight intracellular constituents will be
examined to determine what constraints on the glycolytic
pathway may be responsible for the declining rate of ethanol
production during yeast fermentation.


108
glycolytic enzymes upon glucose addition to derepressed
yeast cells also were correlated with increased rates of
glycolytic activity (Entian et al.. 1984). This contrasts
with the present study which shows an increase in most of
the enzyme activities while the rate of glycolysis declines
(Fig. 13). Only during the latter stages of fermentation
was a modest loss of total activity per ml observed,
indicating a low turnover rate for these enzymes after the
rate of fermentation had declined by at least 80%. This is
consistent with previous reports that most enzymes of S.
cerevisiae retain their activities under a variety of
metabolic conditions, including carbon and phosphate
limitation (Halvorson, 1958; Lagunas et al.. 1982; Lopez and
Gancedo, 1979).
Glycolytic enzymes comprize a large portion of the
soluble cell protein (Srivastava and Bernhard, 1986)
favoring the formation of protein-protein interactions
(Banuelos and Gancedo, 1978) that probably do not occur when
dilute cell extracts are assayed for in vitro enzyme
activities. Evidence supporting this theory includes the
observation of Banuelos and Gancedo (1978) that the of
enolase for 2-phosphoglycerate was 5-fold lower in
permeabilized cells than in cell extracts. Tompa et al.
(1986) showed that aldolase can specifically complex with
phosphofructokinase and glyceraldehyde-3-phosphate
dehydrogenase in vitro providing further evidence that
l


CHAPTER VII
CONCLUSIONS AND FUTURE DIRECTIONS
The studies presented here examine the rate of glucose
conversion to ethanol during batch fermentation by
Saccharomvces cerevisiae and its decline as alcohol
accumulates in the medium (Fig. 3). Most previous studies
have attributed this decline in alcohol production rate to
the presence of ethanol in the fermentation broth (Ingram
and Buttke, 1984; Moulin et al.. 1984). In the present
study, this phenomenon has been characterized using a
respiratory-deficient strain of brewery yeast fermenting 20%
glucose and many potential causes of the declining
fermentation rate have been examined. From this work, a
number of important features of alcoholic fermentation by S.
cerevisiae have been elucidated. (A) Even though the rates
of fermentation and growth declined as ethanol accumulated
and when ethanol was added to fermentation broth (Fig. 2 and
Fig. 3), the apparent inhibition by accumulated ethanol was
greater than inhibition by added ethanol. (B) Removal of
ethanol from yeast cells did not fully restore fermentation
rate (Table 1 and Fig. 11) and growth in the presence of
added ethanol did not extensively inhibit fermentation rate
(Table 3). These results suggest that accumulation of
146


57
evidence that the intracellular concentration of ethanol was
much higher than that of the surrounding medium during
fermentation (Nagodawithana and Steinkraus, 1976; Navarro
and Durand, 1978; Panchal and Stewart, 1980), a condition
not readily duplicated by exogenously added ethanol.
However, these early data can be explained by problems in
the measurement of internal ethanol concentrations (Dasari
et al., 1984). Several research groups have developed
independent methods which demonstrated that ethanol is
freely permeable in Saccharomvces spp. and that the
intracellular concentration of this metabolic product is the
same as that in the surrounding fermentation broth (Dasari
et al.. 1985; Guijarro and Lagunas, 1984; Table 2).
Additional studies have investigated the sensitivity of
glycolytic enzymes and alcohologenic enzymes to in vitro
inhibition by ethanol. Millar et al. (1982) have shown that
these enzymes are stable in ethanol concentrations higher
than 20% (vol/vol). The two enzymes most sensitive to
inhibition by ethanol were pyruvate decarboxylase and
phosphoglycerate kinase. Both, however, retained, 50% of
maximal activity in the presence of over 12% (vol/vol)
ethanol, the final alcohol concentration achieved by the
complete fermentation of 200 g of glucose per L of broth.
Similarly, Larue et al. (1984) concluded that the cessation
of alcohol production during stuck fermentations was not due


114
In chapter V, the amount of each glycolytic and
alcohologenic enzyme was measured as fermentation progressed
(Table 5 and Fig. 13). The levels of these enzymes, in
general, remained high and were sufficient to account for
the observed rates of ethanol production throughout
fermentation. The amounts of glycolytic and alcohologenic
enzymes were measured as specific activities in vitro under
substrate saturating conditions. The actual in vivo
activity of these enzymes, however, is subject to modulation
by the cytoplasmic environment of the yeast cell.
As glucose is converted to ethanol, the fermentation
broth is acidified (Fig. 14). If the intracellular pH
significantly decreased during this acidification, then the
in vivo activities of the glycolytic and alcohologenic
enzymes also should have decreased. However, yeast cells
suspended in their native medium held a constant
intracellular pH throughout fermentation (Fig. 14) and were
able to maintain a high A pH in concentrations of ethanol
greater than the amount attainable from the complete
conversion of 20% glucose (Fig. 15). These results were
unexpected and contrary to what had been observed for yeast
cells suspended in water (Cartwright et al.. 1986; Leao and
van Uden, 1984a). Ethanol still may cause proton-leakage,
but cells in native broth may cope with this permeabilizing
effect presumably by activating ion pumps, such as ATPases
(Pena et al.. 1972) .


9
has been shown to alter its plasma membrane lipid
composition when grown in the presence of ethanol (Beaven et
al.. 1982). The amount of unsaturated fatty-acyl chains in
the phospholipids rose with increasing amounts of added
ethanol. The proportion of oleyl residues increased by 100%
with a corresponding decrease in the proportion of palmitic
residues in the presence of 1.5 M ethanol. In light of the
previous studies on yeast cells containing plasma membranes
enriched in unsaturated fatty-acyl residues, this change in
lipid composition may be an adaptive response to growth in
the presence of ethanol. As ethanol accumulated during
normal growth, however, the lipid fatty-acyl composition
became more saturated, probably the result of decreasing
oxygen tension in the medium.
Since enrichment of the plasma membrane with
unsaturated fatty-acyl residues and ergosterol protects
yeast cells from ethanol inhibition and the plasma membrane
fatty-acyl composition becomes more saturated during growth,
addition of unsaturated lipid supplements to the
fermentation broth might be expected to enhance the
fermentation rate and final ethanol yield from fermentable
substrates. Studies of sake fermentation by Hayashida have
shown that addition of proteolipid containing linoleyl
fatty-acyl residues to synthetic medium promotes the
formation of over 20% ethanol (Hayashida et al. 1974).
Sake yeast normally only produce this high amount of ethanol


109
glycolytic enzymes can form specific enzymatic complexes
that may function in substrate channeling. The role of
protein-protein interactions also has been implicated in
modulating the allosteric response of phosphofructokinase to
ATP (Aragon and Sanchez, 1985). The fermentative activity
of cells exposed to 10% (vol/vol) ethanol that is not
recoverable by washing (Fig. 12) may be the result of
ethanol disruption of these protein-protein interactions.
Reestablishment of these interactions may not occur
spontaneously and, thus, the glycolytic activity would not
be fully restored immediately after washing.
If the internal cellular pH is not maintained close to
neutrality, then the in vivo activities of the glycolytic
enzymes may be considerably lower than observed in vitro.
The internal pH of the yeast cells, however, was maintained
near neutrality despite acidification of the fermentation
broth and the accumulation of over 12% (vol/vol) ethanol.
This latter observation was contrary to expectation based
upon earlier experiments with cells suspended in water
(Cartwright et al.. 1986). These earlier studies had
demonstrated that ethanol enhanced the leakage of protons
and other ions (Cartwright et al.. 1986; Leao and van Uden,
1984a). The cytoplasm was acidified below the optimal pH
for glycolytic and alcohologenic enzymes. Figure 15 clearly
shows that at concentrations found in the medium during the
fermentation of 20% glucose, added ethanol does not affect


Figure 16. A typical thin layer chromatogram of a
fermentation sample extract. Cells
were inoculated into YEPD medium
containing 20 uCi/ml
[32P] orthophosphate. At 24 h, a
fermentation sample was taken and
extracted with the ethanol-EDTA
solution as described in the text.
Approximately 70,000 counts of extract
were spotted onto the origin and the
chromatogram was developed in two
dimensions. Spots were visualized by
autoradiography and identified as
described in the text. Abbreviations:
P¡, inorganic phosphate; PEP,
phosphoenolpyruvate; GP,
glycerophosphate; 2-PG, 2-
phosphoglycerate; 3-PG, 3-
phosphoglycerate; GAP, glyceraldehyde-
3-phosphate; F6P, fructose-6-
phosphate; G6P, glucose-6-phosphate;
F-l,6-P, fructose 1,6-bisphosphate;
ATP, adenosine triphosphate; ADP,
adenosine diphosphate; TP, trise
phosphates; HP, hexose phosphates;
NXP, total cellular nucleotides.


Figure 4. Determination of intracellular ethanol concentration. (A) Flow diagram
illustrating the procedure used to determine the amount of ethanol present
within cells. This method overestimates the actual intracellular volume
accessible to ethanol by including the cell volume occupied by solids. (B)
Flow diagram illustrating the procedure used to determine the volume occupied
by cell solids. This determination was used to provide a correction factor
for the calculation of ethanol concentration in the aqueous cell volume.


117
Preparation of r32P1 PC^-labelled Yeast Cells
Inoculum was prepared as described in chapter II. To
50 ml of YEPD medium containing 20 pCi/ml
[32P]orthophosphate was added 0.5 ml of inoculum. This
culture was stirred under semi-anaerobic conditions while
incubating in a water-bath at 30C. Beginning 12 h after
inoculation, samples were taken at 6 h intervals for the
preparation of fermentation extracts. In a parallel
experiment, cells were inoculated into unlabelled medium and
samples were taken for cell protein and ethanol
determinations.
Preparation of Fermentation Extracts
Samples taken from batch fermentations were extracted
using a method similar to that described by Holmsen et al.
(1983). Cells were inactivated by pipetting 0.5 ml of
fermentation sample into a microcentrifuge tube containing
0.5 ml of 82% (vol/vol) ethanol and 7.7 mM EDTA precooled to
4C- The ethanol-EDTA extraction solution was prepared
fresh before each use to insure that the EDTA did not
precipitate. The samples then were vortexed rapidly for
10 sec, immediately placed in an ice-bath and allowed to
extract for 30 min at 4C. Every 5 min during this
extraction period the samples were vortexed vigorously. The
remaining cell debris was pelleted by centrifugation at
10,000 x g for 10 min at 4C. The cell extracts were saved
for further analysis by freezing at -70C. At the same time


147
ethanol in the fermentation broth may not be the only cause
of declining fermentation rate. (C) Ethanol freely
permeated the yeast cell envelope (Table 2) and did not
reach intracellular concentrations sufficient to inhibit the
activity of glycolytic enzymes (Millar et al.. 1982) during
fermentation. (D) In a complex medium containing a high
amount of fermentable carbohydrate, the decline in
fermentation rate was caused, in part, by a nutrient-
limitation (Chapter III). This limitation in a yeast
extract-peptone based medium was for magnesium (Chapter IV),
however, supplementation with magnesium did not entirely
eliminate the decline in fermentation rate (Fig. 9).
Limitations for other nutrients after magnesium
supplementation also may occur, although none have been
identified yet. (E) The remaining decline in fermentation
rate was not due to inactivation of glycolytic enzymes, to
the inability to maintain a neutral internal pH or to a
decrease in the intracellular levels of nucleotides.
(F) The yeast cells changed physiologically as glucose was
converted to ethanol. Older cells were more resistant to
ethanol-induced inhibition of fermentation rate (Fig. 5 and
Fig. 12) and to ethanol-induced decrease in A pH (Fig. 15)
than younger cells. Intracellular concentrations of ATP
fell and AMP rose dramatically. Levels of both hexose
phosphates and trise phosphate intermediates initially fell
(Fig. 17) suggesting that the initial decline in




Figure 13. Changes in the levels of glycolytic and alcohologenic enzymes during batch
fermentation with 20% glucose. Cells were removed at various stages of
fermentation and disrupted. The activities of individual enzymes were
determined under substrate-saturating conditions. Values are expressed
relative to 12-h cells, the time at which the highest activity per mg cell
protein was observed. Error bars represent an average standard deviation for
determinations from three separate batch fermentations. (A) Changes in the
specific activities of representative enzymes. (B) Changes in the activities
of representative enzymes per ml of culture. Analogous plots of the changes
in fermentation rate (A and B) and the changes in the amount of soluble cell
protein (B only) have been included for comparison. Symbols: A ,
phosphoglucomutase; glyceraldehyde-3-phosphate dehydrogenase; ,
trise phosphate isomerase; Q phosphofructokinase; 0 glycolysis; w
soluble cell protein.


106
Two nutritional factors have been identified previously
which reduced but do not eliminate the ethanol-associated
decline in activity (Casey and Ingledew, 1986; Chapter IV).
The results presented in this chapter with added and
accumulated ethanol confirm the results of chapter III
indicating that physiological changes, including ethanol
damage, in addition to an immediately reversible effect of
ethanol appear responsible. Added ethanol inhibited
fermentation, but washing did not restore full activity
(Fig. 12). Similarly, the replacement of ethanol-containing
broth from the middle to latter stages of fermentation with
fresh medium did not immediately restore full fermentative
activity. The exposure of cells to ethanol in some way
damaged their ability to produce ethanol and the extent of
this damage appears related to ethanol concentration, age of
the cells and duration of the exposure.
Changes in internal pH and in amounts of' the individual
glycolytic and alcohologenic enzymes were examined as
possible reasons for decreased ethanol productivity during
batch fermentation. The amounts of glycolytic and
alcohologenic enzymes remained high throughout fermentation.
In general, these amounts were more than adequate to
maintain the observed fermentation rates. Larue et al.
(1984) reported similar observations concerning hexokinase
and alcohol dehydrogenase activities during simulated wine
fermentations. Under glucose-limited growth conditions,


13
maximum ethanol tolerance for growth of S. cerevisiae is
between 28C and 30C. Similarly, the ability to survive in
the presence of ethanol decreased with increasing
temperature (Casey and Ingledew, 1986; Leao and van Uden,
1982a; Nagodawithana et al.. 1974). Ethanol also enhanced
the thermal death rate of S. cerevisiae (van Uden and da
Cruz Duarte, 1981). In contrast, the rate of sugar
conversion became more resistant to ethanol inhibition as
the fermentation temperature was raised to 45C (Brown and
Oliver, 1982). Alcohol production proceeds at an
accelerated pace at the higher temperatures. When
optimizing the conditions for carrying out a fermentation
process, a compromise between these competing factors must
be reached.
Other environmental factors which affect the ability of
yeast to tolerate ethanol include the sugar and oxygen
concentrations in the fermentation broth (Jones et al.,
1981). Glucose concentrations above 14% decreased the
ability of S. cerevisiae to convert the sugar to ethanol
(Gray, 1945). This inhibition occurred as the cells began
to undergo plasmolysis and probably was caused by the
osmotic effects of these high amounts of glucose on the
yeast. Osmotic pressure also has an adverse effect on yeast
cell viability during fermentation (Panchal and Stewart,
1980). These effects of high sugar concentrations appear to
be synergistic with the inhibitory effects of ethanol


CHAPTER V
GLYCOLYTIC ENZYMES AND INTERNAL pH
Introduction
Saccharomvces cerevisiae is capable of very rapid rates
of glycolysis and ethanol production under optimal
conditions, producing over 50 pmoles of ethanol per h per mg
of cell protein (Fig. 9). However, this high rate is
maintained for only a brief period during batch fermentation
and declines progressively as ethanol accumulates in the
surrounding broth (Casey and Ingledew, 1986; Ingram and
Buttke, 1984; Moulin et al.. 1984). Earlier studies have
identified a requirement for lipids (Beaven et al.. 1982;
Casey et al.. 1984; Thomas et al.. 1978) or molecular oxygen
for lipid biosynthesis (Andreasen and Stier, 1954; Buttke et
al. 1980; Buttke and Pyle, 1982) in many fermentation
broths as being essential for the maintenance of high
fermentative activity. Magnesium is an essential cofactor
for many of the glycolytic enzymes and has been identified
also as a limiting nutrient in fermentation broth containing
peptone and yeast extract (Chapter IV). Supplying these
nutritional needs reduces but does not eliminate the decline
in fermentative activity during batch fermentation (Fig. 9).
84


119
Analysis of r32P1 PO^-labelled Fermentation Extracts
Extracts of [32P] P04-labelled fermentation samples were
analyzed by two-dimensional thin layer chromatography using
the solvent systems described by Holmsen et al. (1983).
Aliguots of extract containing approximately 70,000 cpm each
were spotted onto Analtech Cellulose MN 300 (20 cm x 20 cm)
thin layer chromatography plates (Fisher Scientific Company,
Orlando, Fla). Plates were developed in the first dimension
and allowed to dry at room temperature before development in
the second dimension. After development in both dimensions
and drying, spots on the thin layer plates were visualized
by autoradiography, marked and scraped into scintillation
vials. After the addition of toluene-based scintillation
cocktail, the radioactivity in the vials was measured using
a Beckman model 8000 scintillation spectrometer.
A typical chromatogram is shown in figure 16. The
areas marked by parentheses were scraped and counted as
groups of phosphorylated intermediates. The identities of
the spots marked on the chromatogram were established in
three ways. A comparison was made with the chromatogram
reported by Holmsen et al. (1983) and tentative
identifications were made. Then, authentic standards of
glycolytic intermediates were chromatographed under the same
conditions as the fermentation extracts. Standards were
visualized by immersing the thin layer plates in a bath
containing Fe3+ and 5-sulfosalicylic acid as described by


CHAPTER III
NUTRIENT LIMITATION AS A BASIS FOR THE APPARENT TOXICITY OF
LOW LEVELS OF ETHANOL DURING BATCH FERMENTATION
Introduction
As has already been described for Saccharomvces
cerevisiae KD2, the rate of alcohol production per unit cell
mass decreases substantially during batch fermentations as
ethanol accumulates in the medium (Fig. 3). This decrease
has been attributed to the inhibitory effects of ethanol by
most researchers (Aiba et al.. 1968; Bazua and Wilke, 1977;
Ghose and Tyagi, 1979; Luong, 1985; Millar et al.. 1982;
Moulin et al.. 1984; Rahn, 1929). However, recent studies
by Casey et al. (1983, 1984) have provided evidence that
nutrient limitation, in addition to ethanol accumulation, is
also an important factor limiting the rate of fermentation
during high-gravity brewing. The following studies examine
the role of ethanol in limiting the rate of alcohol
production and provide evidence that nutrient limitation is
an additional factor which contributes to the initial
decline in fermentative activity.
Materials and Methods
Organism and Growth Conditions
The organism used in these studies was Saccharomvces
cerevisiae KD2, a petite derivative of strain CC3 (G.G.
30


16
Batch fermentations were carried out in 250-ml tissue
culture spinner bottles (Blico Glass, Inc., Vineland,
N.J.), immersed in a 3 0C water bath and agitated at
150 rpm. Culture bottles were fitted with water-trapped
exit ports for the escape of carbon dioxide and sampling
ports for the removal of culture by syringe. Growth was
allowed to proceed under conditions of self-induced
anaerobiosis. Inocula were prepared by transferring cells
from a slant to a test tube containing 10 ml of YEPD broth.
Cells were incubated at 30C for 36 h without agitation and
diluted 1:40 into 300 ml of fr^sh YEPD in a spinner bottle.
This culture was incubated for approximately 12 h until an
optical density at 550 nm of 3.5 (1.3 mg of cell protein per
ml) was reached. Fermentations were started by diluting the
12-h culture 1:100 into 300 ml of growth medium.
Preparation of Fermentation Samples for Analysis
Fermentation samples were centrifuged at 10,000 x g for
O.5 min. The supernatant was removed and saved by freezing
at -20C. Cells were washed once in 50 mM KH2P04 buffer (pH
5.0), and the pellets were saved for further analysis by
freezing at -20C.
Analytical Methods
Ethanol was measured by gas-liquid chromatography as
described by Goel and Pamment (1984) with 2% (vol/vol)
acetone as an internal standard. Glucose was initially
determined with the glucose oxidase procedure (Raabo and


TIME (h) TIME (h)
69


76
maintained a higher rate of fermentation as ethanol
accumulated during the completion of the batch fermentation.
This rate was 40% higher than that of control cells after
the accumulation of 8% (vol/vol) ethanol. The fermentation
rate of both supplemented and unsupplemented cultures fell
precipitously at about 12.5% (vol/vol) ethanol, coincident
with exhaustion of glucose.
Effect of Magnesium Addition on Cell Viability
The percentage of viable cells in both magnesium-
supplemented and unsupplemented batches remained greater
than 90% for the first 48 h of the fermentation. Glucose
was exhausted at this time in supplemented cultures and the
percentage of viable cells began to decrease, reaching 58%
by 72 h. The unsupplemented batches consumed glucose more
slowly and maintained high viability (>90%) until between 60
and 72 h, the time at which glucose was exhausted.
Effect of Ethanol on the Fermentation Rate of Magnesium-
Supplemented Cultures
Two points during fermentation were chosen at which to
compare cells grown with and without added magnesium. These
were the same two points described in chapter III as 12-h
and 24-h cells. Cells at 12 h were still undergoing
exponential growth and cells at 24 h were in early
stationary phase.
Figure 10A shows the dose-response of fermentative
activity of the younger cells plotted as a function of total
ethanol concentration (endogenous plus added). Magnesium-


FERMENTATION RATE
(/jmoles C02/h per mg protein)
ro 4^ d)
o o o o
'-j
Ln


85
The basis for the decline in fermentation rate is not
fully understood. Since the addition of ethanol to cells in
batch cultures and in chemostats causes a dose-dependent
inhibition of ethanol production (Casey and Ingledew, 1986;
Fig. 10), most investigations have focused on ethanol as the
inhibitory agent (Casey and Ingledew, 1986; Ingram and
Buttke, 1984; Millar et al.. 1982). Ethanol is known to
alter membrane permeability and disrupt membrane function in
a variety of biological systems (Casey and Ingledew, 1986;
Ingram and Buttke, 1984). In yeast, ethanol causes an
increase in hydrogen ion flux across the plasma membrane of
cells suspended in water (Cartwright et al.. 1986). This
increased hydrogen ion flux has been proposed as being
responsible for the ethanol-induced decline in transport
rates observed under similar conditions (Beaven et al..
1982; Leao and van Uden, 1982b, 1984a, 1984b).
Evidence has been accumulating which indicates that the
presence of ethanol may not be the only factor responsible
for the decline in fermentative activity. The replacement
of fermentative broth containing ethanol with fresh medium
lacking ethanol did not immediately restore fermentative
activity (Table 1). In a comprehensive study, Millar et al.
(1982) demonstrated that ethanol concentrations below
12% (vol/vol) do not denature glycolytic enzymes or cause
appreciable inhibition of activity in vitro under substrate-
saturating conditions. Since ethanol does not accumulate


NICOTINAMIDE NUCLEOTIDES (mM)
134


38
Table 1. Effects of ethanol and fermentation medium
composition on fermentation rate
Assay medium
Rate of
(pmoles C02 per h
Fermentation3
per mg protein (SD))
12-h
cells
24-h
cells
Original broth
36.3
(2.4)
16.5
(2.6)
Fresh broth
39.5
(2.3)
20.3
(2.5)
Conditioned broth
(12-h, 1.1% (vol/vol)
ethanol)
38.9
(1.9)
22.7
(5.7)
Conditioned broth
(24-h, 5.6% (vol/vol)
ethanol)
22.8
(0.6)
16.1
(2.0)
Conditioned broth
(24-h, volatiles
under vacuum)
removed
34.9
(0.8)
17.6
(2.0)
Conditioned broth
(24-h, volatiles removed
under vacuum,
reconstituted to give
5.6% (vol/vol) ethanol)
21.9
(0.2)
15.7
(1.0)
3 Cells from 12-h and 24-h batch fermentations were
harvested by centrifugation at ambient temperature and
suspended to their original volume in various broths. Where
indicated, volatiles were removed from conditioned broth by
vacuum distillation at 55C, reducing the volume by two-
thirds. The broth then was reconstituted with distilled
water or distilled water plus ethanol. Fermentation rates
were measured by respirometry. Averages and standard
deviations (SD) represent the results from three separate
batch fermentations.


1
162
Navon, G., R.G. Schulman, T. Yamane, T.R. Eccleshall, K.-B.
Lam, J.J. Baronofsky and J. Marmur. 1979. Phosphorus-31
nuclear magnetic resonance studies of wild-type and
glycolytic pathway mutants of Saccharomvces cerevisiae.
Biochemistry 18:4487-4497.
Navarro, J.M. and G. Durand. 1978. Fermentation alcooligue:
Influence de la temperature sur 1'accumulation d'alcool
dans les cellules de leuvre. Ann. Microbiol. Inst. Pasteur
129B:215-224.
Nes, W.R., B.C. Sekula, W.D. Nes and J.H. Alder. 1978. The
functional importance of structural features of ergosterol
in yeast. J. Biol. Chem. 253:6218-6225.
Noat, G., J. Ricard, M. Borel and C. Got. 1970. Kinetic
study of yeast hexokinase Inhibition of the reaction by
magnesium and ATP. Eur. J. Biochem. 13:347-363.
Novak, M., P. Strehaiano, M. Moreno and G. Goma. 1981.
Alcoholic fermentation: On the inhibitory effect of
ethanol. Biotechnol. Bioeng. 23:201-211.
Ogur, M. and R. St. John. 1956. A differential and
diagnostic plate method for population studies of
respiratory deficiency in yeast. J. Bacteriol. 72:500-504.
Ogur, M., R. St. John and S. Nagai. 1957. Tetrazolium
overlay technique for population studies of respiratory
deficiency in yeast. Science 125:928-929.
Ohta, K. and S. Hayashida. 1983. Role of tween 80 and
monoolein in a lipid-sterol-protein complex which enhances
ethanol tolerance of sake yeasts. Appl. Env. Microbiol.
46:821-825.
Osman, Y.A. and L.O. Ingram. 1985. Mechanism of ethanol
inhibition of fermentation in Zvmomonas mobilis CP4. J.
Bacteriol. 164:173-180.
Panchal, C.J. and G.G. Stewart. 1980. The effect of osmotic
pressure on the production and excretion of ethanol and
glycerol by a brewing yeast strain. J. Inst. Brew. 86:207-
210.
Pasteur, L., 1860. Memoire sur la fermentation alcoolique.
Ann. Chim. Phys. 58:323-426.
Pasteur, L. 1861. Influence de l'oxygene sur le development
de la levure et la fermentation alcoolique. Bull. Soc.
Chim. Paris June 28: 79-80.


123
bioluminescence. The reagents for this assay were obtained
from LKB (Gaithersburg, Md.). Samples were prepared for
analysis essentially as described by Ball and Atkinson
(1975) except that only 1 unit of adenylate kinase was used
in the conversion of AMP to ATP. Since ethanol has been
reported to inhibit the luciferase reaction (Ball and
Atkinson, 1975) all standards contained levels of ethanol
identical to that found in the samples. However, samples
used for adenine nucleotide determinations were stored at
-7 0C, thawed on ice and not refrozen for further
determinations once thawed. Luminescence was measured using
a Beckman 8000 scintillation spectrometer with coincidence
counting switched off.
Nicotinamide nucleotides were determined by bacterial
luciferase bioluminescence. The reagents for this assay
also were purchased from LKB (Gaithersburg, Md.) and
luminescence was measured as described above. The assay
procedure used was similar to that described by Karp et al.
(1983) and performed at room temperature. A 20 pi aliquot
of NADH monitoring reagent was added to 60 pi of 0.1 M
potassium phosphate buffer at pH 7.0. Then the background
luminescence was measured. Immediately after the addition
of 20 pi of fermentation extract, emission due to NADH was
measured. In order to determine the amount of NAD+ in
samples, 1 pi of a 1 mg/ml aqueous solution of alcohol
dehydrogenase was syringed into the assay buffer before the


153
Bazua, C.D. and C.R. Wilke. 1977. Ethanol effects on the
kinetics of a continuous fermentation with Saccharomvces
cerevisiae. Biotechnol. Bioeng. Symp. 7:105-118.
Beaven, M.J., C. Charpentier and A.H. Rose. 1982. Production
and tolerance of ethanol in relation to phospholipid
fatty-acid composition in Saccharomvces cerevisiae NCYC
431. J. Gen. Microbiol. 128:1447-1455.
Becker, J.-U. and A. Betz. 1972. Membrane transport as
controlling pacemaker of glycolysis in Saccharomvces
carlsbergensis. Biochim. Biophys. Acta 274:584-597.
Bernt, E. and I. Gutman. 1971. Ethanol determination with
alcohol dehydrogenase, p. 1499. In H.U. Bergermeyer (ed.),
methods of enzymatic analysis, vol. 3. Academic Press, New
York.
Betz, A. and C. Moore. 1967. Fluctuating metabolite levels
in yeast cells and extracts, and the control of
phosphofructokinase activity in vitro. Arch. Biochem.
Biophys. 120:268-273.
Bisson, L.F. and D.G. Fraenkel. 1983. Involvement of kinases
in glucose and fructose uptake by Saccharomvces
cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734.
Bisson, L.F. and D.G. Fraenkel. 1984. Expression of kinase-
dependent glucose uptake in Saccharomvces cerevisiae. J.
Bacteriol. 159:1013-1017.
Bloxham, D.P. and H.A. Hardy. 1973. Phosphofructokinase,
p. 239-278. In P.D. Boyer (ed.), the enzymes, vol. 8.
Academic Press, New York.
Bochner, B.R. and B.W. Ames. 1982. Complete analysis of
cellular nucleotides by two-dimensional thin layer
chromatography. J. Biol. Chem. 257:9759-9769.
Boucherie, H. 1985. Protein synthesis during transition and
stationary phases under glucose limitation in
Saccharomvces cerevisiae. J. Bacteriol. 161:385-392.
Bradford, M.M. 1976. A rapid and sensitive method for the
guantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal. Biochem.
72:248-254.
Brown, A.J. 1905. The influences regulating the reproductive
functions of Saccharomvces cerevisiae. J. Chem. Soc.
87:1395-1412.


CHAPTER II
CHARACTERIZATION OF THE DECLINING RATES OF GROWTH AND
ETHANOL PRODUCTION DURING BATCH FERMENTATION BY
SACCHAROMYCES CEREVISIAE KD2
Introduction
Yeast metabolize sugar via Embden-Meyerhof glycolysis
to produce ethanol as the major reduced product of
fermentation. As ethanol accumulates in the fermentation
broth, both the rate of growth and alcohol production
declines (Ingram and Buttke, 1984; Luong, 1985; van Uden,
1985). The potency of ethanol as an inhibitor of yeast
growth and fermentation, however, differs in various species
(Gray, 1941). Not all strains of Saccharomvces cerevisiae
have equal abilities to grow and ferment in the presence of
added ethanol.
Environmental factors, such as temperature, also have a
key role in determining the potency of ethanol as an
inhibitor (Casey and Ingledew, 1986; Jones et al.. 1981; van
Uden, 1985). Elevating the temperature reduced the maximum
ethanol yield of wine fermentations (Hohl and Cruess, 1936).
Both high and low temperatures decreased the ability of S.
cerevisiae to grow in the presence of ethanol (Loureiro and
van Uden, 1982; Sa-Correia and van Uden, 1983). Sa-Correia
and van Uden (1983) have shown that the temperature of
12


150
1986? Thomas and Rose, 1979). Degradation products of
biosynthetic processes, including nucleotides such as AMP,
may accumulate intracellularly. The cell envelope may
become more permeable to protons as ethanol accumulates in
the medium (Cartwright et al.. 1986; Leao and van Uden,
1984a) and the cells could respond by utilizing ATP to pump
out protons via plasma membrane-bound ATPases (Pena et al..
1972). The accumulation of AMP along with these lower
levels of ATP due to ATPase action could inhibit hexose
uptake and phosphorylation (Noat et al.. 1970; Rudolph and
Fromm, 1971). Then the total level of glycolytic
intermediates would decrease as phosphofructokinase becomes
activated by the declining ATP to AMP ratio of the cells
(Betz and Moore, 1967). As intracellular levels of hexose
phosphates decline, fructose 1,6-bisphosphate, a potent
allosteric activator of pyruvate kinase (Hess et al.. 1966;
Kayne, 1973) also would decline. This should decrease the
in vivo activity of pyruvate kinase and trise phosphates
then can accumulate (Maitra and Lobo, 1977) As
fermentation progresses, the accumulation of hexose
phosphates may be due to this decrease in pyruvate kinase
activity or an inactivation of fructose 1,6-bisphosphate
aldolase. Then fructose 1,6-bisphosphate should accumulate
and pyruvate kinase regain activity causing the levels of
trise phosphates to decline. With the production of
12% (vol/vol) ethanol and the exhaustion of glucose in the


and agitated (30 rpm) in a Rototorque culture rotator (Cole
Parmer, Chicago, Ill.)*
Medium Analyses
Ethanol and glucose were measured as described in
chapter II. The magnesium concentration of the medium was
measured with the 60 Second Magnesium reagents purchased
from American Monitor Corporation, Indianapolis, Ind., as
described by Osman and Ingram (1985).
Cellular Analyses and Respirometrv Measurements
Cell mass and total cell protein were measured as
described in chapter II. To determine the amount of
intracellular magnesium, yeast cells were washed once in
50 mM KH2P04 buffer (pH 5.0) and the cell pellets were
stored frozen at -20C until analyzed. These yeast pellets
contained 1 to 3 mg of cell protein and were permeabilized
by incubation in a boiling-water bath for 1.5 min. The
resulting debris was suspended in 1 ml of 50 mM KH2P04
buffer (pH 5.0) and then pelleted. The supernatant was
analyzed for magnesium as described above. Respirometry
measurements were made as described in chapter II and
fermentation rates were calculated from these values as
pmoles of C02 produced per h per mg cell protein.
Viable-Cell Determinations
Cell numbers were determined microscopically with a
Petroff-Hausser counting chamber. Viable-cell counts were


36
the supernatant times the supernatant volume as follows:
14p 14p
^8U8 ^8118
Esus = EeeII (1 ) (1 V.) + Esup ( ) (2)
14p 14p
^SUp ^8Up
where Esus is the ethanol concentration in the suspension,
Esup is the ethanol concentration in the supernatant and
EcelI is the ethanol concentration within the aqueous cell
volume.
Chemicals
Complex medium components and agar were purchased from
Difco Laboratories, Detroit, Mich. Glucose and other
biochemicals were obtained from Sigma Chemical Co., St.
Louis, Mo. Inorganic salts were purchased from Fisher
Scientific Company, Orlando, Fla. Absolute ethanol was
supplied by AAPER Alcohol and Chemical Co., Shelbyville, Ky.
Radioactive compounds were purchased from New England
Nuclear, Boston, Mass. Gas chromatography supplies were
obtained from Supelco, Bellefonte, Pa.
Results
Effect of Ethanol Removal on Fermentation Rate
These studies have focussed on two time points during
batch fermentation, 12-h and 24-h, to investigate the
possible reasons for the initial drop in fermentative
activity. To minimize possible variability arising from
inoculum differences, autoclaving, etc., 12-h cells have
been operationally defined as those which have increased in


CHAPTER IV
MAGNESIUM LIMITATION AND ITS ROLE IN THE APPARENT TOXICITY
OF ETHANOL DURING YEAST FERMENTATION
Introduction
The rate of ethanol production by Saccharomvces spp.
decreases in batch fermentations as alcohol accumulates in
the medium (Moulin et al.. 1984; Rahn, 1929; Strehaiano and
Goma, 1983). The onset of this decline in fermentative
activity occurs at very low ethanol concentrations, often
less than 3% (vol/vol). Since ethanol has been shown to
inhibit fermentation (Brown et al.. 1981; Cysewski and
Wilke, 1977; Gray, 1941), it generally has been accepted
that this accumulation of ethanol is responsible for the
progressive decline in fermentative activity (Bazua and
Wilke, 1977; Ghose and Tyagi, 1979; Luong, 1985). However,
the extent of inhibition by exogenously added ethanol is
less than would be predicted by the decline in fermentation
rate which normally occurs during the fermentative
accumulation of ethanol (Fig. 3).
Further studies have attempted to define the
mechanism(s) of ethanol inhibition of fermentation and to
reconcile the failure of added ethanol to inhibit
fermentation to the extent observed during the fermentative
accumulation of ethanol. Early experiments provided
56


Figure 12. Effects of ethanol exposure on the fermentative activities of 12-h and 24-h
cells. Fermentative activity was measure after sample preparation by
respirometry. Cells were harvested after 12 h (A) or 24 h (B) and suspended
in fresh medium containing various concentrations of gthanol. Their
fermentative activity was measured after 10 min at 30C. A parallel set of
samples was exposed to ethanol for 10 min, harvested by centrifugation, washed
once and suspended in fresh medium lacking ethanol. Error bars denote the
average standard deviation for three separate batch fermentations. Symbols:
cells in the presence of added ethanol; O cells exposed to
ethanol and suspended in fresh medium.


ADENINE NUCLEOTIDES (mM)
ADENYLATE ENERGY CHARGE
LZ I


125
Scientific Company, Orlando, Fla. Absolute ethanol was
supplied by AAPER Alcohol and Chemical Co., Shelbyville, Ky.
[32P]orthophosphate was obtained from Amersham, Arlington
Heights, Ill. Radioactive compounds for cell volume
measurements were purchased from New England Nuclear,
Boston, Mass. Gas chromatography supplies were purchased
from Supelco, Bellefonte, Pa.
Results
Phosphorylated Glycolytic Intermediates
The declining rate of alcohol production by S.
cerevisiae was reflected in changes in the levels of
glycolytic intermediates during fermentation (Fig. 17).
Fermentative activities were measured by respirometry and
the rates of C02 production per h per mg cell protein were
calculated. These values are plotted in figure 17A as a
percentage of the value observed at 12 h and are compared to
a similar plot of total phosphorylated glycolytic
intermediates. Measurements of glycolytic intermediates
were calculated as counts per min per mg cell protein. The
values for total phosphorylated glycolytic intermediates did
not include measurements of phosphoenolpyruvate for the
reasons mentioned earlier. By 24 h almost 50% of the
fermentative activity observed at 12 h was lost. As the
glucose in the medium was exhausted by 48 h, the rate of
fermentation dropped by over 90%. If the activity of one of
the glycolytic enzymes constricts carbon flow to ethanol,


71
Effect of Magnesium Supplementation of Batch Fermentation
The effects of supplementing YEPD medium with 0.5 mM
MgS04 on batch fermentation are illustrated in figure 8.
The production of cell mass as measured by cellular protein
is shown in figure 8A. Supplementation with magnesium
prolonged the exponential rise in cellular protein, allowing
a 53% increase in cell mass over that of the control within
18 h after inoculation. The addition of magnesium also
increased the rate at which glucose was consumed and ethanol
was produced (Fig. 8B and 8C). After 30 h of incubation,
magnesium-supplemented cultures had produced one-third more
ethanol than the controls. The conversion of glucose to
ethanol was complete after 48 h in magnesium-supplemented
cultures, but required 72 h in control YEPD broth. The
final yield of ethanol was essentially identical for both
magnesium-supplemented and control cultures, 12.7% (vol/vol)
(98% of theoretical maximum yield).
Effect of Magnesium Supplementation on Rate of Fermentation
Samples were removed from magnesium-supplemented and
control fermentations at various times during batch
fermentation. Ethanol concentration, cell protein and C02
evolution of unwashed cells were measured. Figure 9 shows
the fermentation rate as a function of accumulated ethanol.
Both control and magnesium-supplemented cultures exhibited
the same maximum rate of fermentation at 1% (vol/vol)
ethanol. However, magnesium-supplemented cultures


141
(Fig. 17). These nucleotide phosphate values probably
included many unidentified nucleotides and multiply
phosphorylated molecules in addition to adenine and
nicotinamide nucleotides. Thus, even though the
nicotinamide nucleotide levels did not fall and the adenine
nucleotides levels increased, the decline in total
nucleotide phosphate is not surprising. This decline may
reflect a decrease in the intracellular pools of high energy
precusor molecules involved in macromolecular biosynthesis
during growth.
Changes in the intracellular levels of glycolytic
intermediates may indicate which part of the pathway is
restricting carbon flow (Ciriacy and Breitenbach, 1979;
Navon et al.. 1979). In the present study, as glycolytic
flux decreased during fermentation initially, the levels of
phosphorylated glycolytic intermediates also decreased.
These results are consistent with those of Den Hollander et
al. (1986) who observed lower levels of glycolytic
intermediates in anaerobically grown cells than in
aerobically grown cells. Anaerobically grown cells have a
higher rate of glucose utilization, like 12-h cells, than
aerobically grown cells, like 18-h or older cells. Similar
observations were made by Franco et al. (1984) for the
glucose-6-phosphate and fructose 1,6-bisphosphate levels of
bakers' yeast fermenting 5% glucose. Since the amounts of
glycolytic enzymes did not fall (Fig. 13), this decrease was


37
cell mass 100-fold after inoculation. Typically, these
samples contain 1.2 to 1.3% (vol/vol) ethanol and 1.3 mg
cell protein per ml culture medium. Cells which have
produced 5.0 to 5.6% (vol/vol) ethanol, in addition to any
ethanol that may have been present in the original medium,
were operationally defined as 24-h cells. Typically, these
samples contained 2.6 mg cell protein per ml culture medium.
Table 1 shows the effects of ethanol removal on the
fermentation rates of 12-h and 24-h cells. This activity of
12-h cells was much higher than that of 24-h cells. Ethanol
removal by suspension in fresh broth had little effect on
the activity of 12-h cells and did not result in a
significant increase in the fermentation rate of 24-h cells.
Similarly, suspension in conditioned broth from 12-h
fermentations, containing 1.1% (vol/vol) ethanol, did not
affect fermentation rate. Suspension of cells in the 24-h
conditioned broth, containing 5.6% (vol/vol) ethanol,
reduced the fermentative activity of 12-h cells but had less
effect on the activity of 24-h cells. Removal of volatile
medium components from the 24-h conditioned broth eliminated
its inhibitory effect on the fermentation rate of 12-h cells
but did not result in a significant increase in activity of
the 24-h cells. The addition of ethanol to the 24-h
conditioned broth restored its ability to repress the
fermentation rate of 12-h cells, indicating that ethanol was


42
of the cell age and original ethanol concentration, the
intracellular ethanol concentration was found to be 0.6 to
0.7% (vol/vol) after ethanol removal. These values were
somewhat higher than anticipated and appeared to be due to
ethanol production by continued metabolism during suspension
and sampling. The inclusion of potassium fluoride during
harvesting and suspension in fresh medium resulted in a very
low internal and external ethanol concentration (0.06%
(vol/vol)), consistent with dilution of the cell pellet
volume with fresh medium.
In an analogous fashion, the failure of exogenously
supplied ethanol to raise the internal ethanol concentration
of 24-h cells to a level equivalent with that of cells
during fermentative alcohol production could provide an
explanation for the apparent resistance of 24-h cells to the
inhibitory effects of added ethanol (Table 1). Samples
taken after 48 h and processed to determine the
intracellular ethanol concentration contained approximately
11.2% (vol/vol) (SD 1.0) ethanol in the fermentation broth.
The intracellular ethanol concentration of these cells was
8.2% (vol/vol) (SD 1.7). Suspension of 24-h cells in broth
containing 10% (vol/vol) ethanol resulted in an increase in
the intracellular ethanol concentration to 8.1% (vol/vol)
(SD 1.3). These values indicate that the addition of
ethanol to 24-h cells increased the intracellular ethanol
concentration to the level found in cells during batch


96
Table 5. Specific activities of glycolytic enzymes at the
peak of fermentative activity (12 h) and after a
50% decline (24 h)
Specific activity
(pmoles/min per
Enzyme
mg protein)(SD)a
12-h cells
24-h cells
Glycolytic flux (hexose)b
1.0
0.5
Hexokinase
0.84 (0.05)
1.1 (0.1)
Phosphoglucose isomerase
4.2 (0.6)
3.3 (0.1)
Phosphofructokinase
0.64 (0.03)
0.62 (0.03)
Fructose 1,6-bisphosphate
1.4 (0.1)
1.2 (0.1)
aldolase
Glycolytic flux (trise)b
2.0
1.0
Trise phosphate isomerase
110 (2)
97 (2)
Glyceraldehyde-3-phosphate
16 (1)
18 (1)
dehydrogenase
Phosphoglycerate kinase
11 (1)
12 (1)
Phosphoglycerate mutase
6.2 (0.5)
9.0 (0.8)
Enolase
3.0 (0.2)
3.2 (0.4)
Pyruvate kinase
10 (3)
8.4 (1.4)
Pyruvate decarboxylase
1.1 (0.2)
0.92 (0.04)
Alcohol dehydrogenase
4.8 (0.4)
3.8 (0.4)
a Samples were removed from batch fermentations after 12 and
24 h. Cells were mechanically disrupted and the extracts
were assayed under substrate-saturating conditions.
Standard deviations are based upon determinations from three
separate batch fermentations.
b Glycolytic flux for hexose and trise intermediates was
estimated from measurements of fermentation rate.


48
ambient temperature and suspended in fresh medium lacking
ethanol to measure the rate of fermentation under standard
conditions. In all experiments, the fermentation rate of
cells used as inoculum to start these batch fermentations
were included as controls.
Experiment 1 examined the possibility that the
physiological changes in 12-h cells to produce 24-h cells
were due to growth in the presence of ethanol. Batch
fermentations in which 5% (vol/vol) ethanol was added prior
to inoculation were allowed to grow to the same cell mass as
12-h control cells, 1.2 mg cell protein per ml fermentation
broth. The fermentation rate of these cells grown in the
presence of added ethanol was only slightly lower than that
of control cells grown for 12-h in the absence of ethanol.
This indicated that exposure to 5% (vol/vol) ethanol during
growth was not sufficient to account for most of the
observed reduction in fermentation rate.
The possibility that growth in the presence of ethanol
and other fermentation products may be responsible for the
reduction in fermentation rate was examined in experiment 2.
Cultures were inoculated into bottles containing filter-
sterilized conditioned broth which had been supplemented
with 5 g/L yeast extract and enough glucose to increase the
concentration in the broth back to 20%. Conditioned broth
from 12-h cultures contained 1.2% (vol/vol) ethanol and
broth from 24-h cultures contained 4.5% (vol/vol) ethanol.


129
the glycolytic pathway was constricted in the trise part of
the pathway and that as intermediates backed up in the
pathway, this constriction was relieved. Alternatively, a
second constriction in the pathway at the level of the
hexose intermediates may have developed after 36 h,
resulting in the accumulation of these intermediates and
relief of the constricted carbon flow through the trise
portion of the pathway.
Total r32P1-labelled Nucleotides
Since some of the glycolytic enzymes require
nucleotides for activity, a decrease in the availability of
these cofactors could be responsible for the constricted
carbon flow observed earlier. The number of counts per min
in the total nucleotide spot reflects the degree of
phosphorylation of the nucleotide pool as well as the total
pool size. The nucleotide spot was not well defined (Fig.
16) and probably also included nucleotide intermediates from
biosynthetic processes and other multiply phosphorylated
molecules in addition to adenine and nicotinamide
nucleotides. In figure 17B, these values are plotted as
fermentation progresses. By 18 h, the amount of phosphate
in the total nucleotide pool decreased by 70% and by 36 h,
it dropped by 95% from the level observed at 12 h. Total
nucleotide phosphate remained low until the end of
fermentation.


ACKNOWLEDGMENTS
The ideas presented in these studies could not have
been developed without the encouragement and patience of my
major advisor, Dr. Neal Ingram. I am greatly indebted to
him for sharing with me his knowledge and expertise. I also
would like to express my gratitude to the other members of
my committee, Dr. Allen, Dr. Farrah, Dr. Gander and Dr.
Preston, for their contributions during preparation and
review of this manuscript. Similarly, thanks are due to my
colleague and friend, Dr. Yehia Osman, for his many
suggestions which were helpful in completing this work and
to the rest of the Microbiology and Cell Science Department
for their part in my graduate education. Finally, I would
like to thank my parents for their love and support while I
pursued this study.
ii


9ZOOH


40
these possibilities, the intracellular and extracellular
concentrations of ethanol were measured at 12 h and 24 h
during batch fermentations (Table 2). The external ethanol
concentrations in these suspensions at the time of sampling
were 1.2% (vol/vol) and 5.0% (vol/vol), respectively, before
concentrating the cells. The level of extracellular ethanol
measured in the concentrated cell suspension was slightly
higher than the starting culture reflecting the rapid
metabolism of cells during the less than 3 min period of
cell concentration and sampling. In all cases, the
calculated intracellular ethanol concentration was lower
than or equivalent to the extracellular ethanol
concentration.
To confirm that the higher amounts of ethanol in the
concentrated cell suspension resulted from rapid metabolism,
a potent inhibitor of enolase (Warburg and Christian, 1941)
and of fermentation, potassium fluoride, was added before
cell concentration. Previously, 50 mM potassium fluoride
was determined to cause immediate cessation of C02 evolution
(data not shown). In both 12-h and 24-h fermentation
samples, the addition of fluoride prevented the increase in
extracellular ethanol during cell concentration and sampling
(Table 2).
The removal of ethanol by suspension of cell pellets in
fresh medium lacking ethanol substantially decreased the
intracellular ethanol concentration (Table 2). Regardless


-ETHANOL (%v/v) or
-GLUCOSE (%w/v)
20

I 0.0
5.0
1.0
0.5
0.1
0.05
0.01
-CELL PROTEIN (mg/ml)


of yeast, including several different strains of S.
cerevisiae. were grouped according to their ability to
utilize glucose in the presence of a series of ethanol
concentrations. The ethanol tolerance trait was not
characteristic of any specific genus or species since
different strains of the same species varied in their
tolerance. Yeast strains with differing ethanol tolerances
also had different cellular compositions (Gray, 1948).
Strains of lower alcohol tolerance contained higher amounts
of carbohydrate and lipid than did the more tolerant ones.
The studies of Troyer (1953) confirmed the results of
Gray and further examined the relationship between growth
and glucose utilization. Alcohol tolerant strains of yeast
exhibited increased growth in parallel with increased rates
of glucose utilization over less tolerant strains. During
yeast fermentation, the initial effect of ethanol added to
the medium was to decrease the total number of cells formed
followed by a corresponding decrease in glucose utilization.
The manner in which ethanol inhibits glucose
utilization by S. cerevisiae was examined by Gray and Sova
(1956). The ability of ethanol to inhibit glucose
utilization was not a specific property of this fermentation
product but rather a property shared by a class of
substances, short chain aliphatic alcohols. The potency of
normal alcohols as inhibitors of glucose utilization was
related to their chain length and primary alcohols were


139
a large decrease after 24 h (Fig. 20B) At 12 h, the energy-
charge was 0.63, but by 24 h, it had fallen to 0.19. This
energy depleted state should activate glycolytic flux and
promote carbon flow through the pathway. After 30 h, the
energy charge reached a minimum at 0.12 and remained low
until the glucose in the medium was exhausted by 48 h. At
the end of fermentation, the energy charge rose back to the
value observed for 12-h cells. The decrease in AMP
concentration as fermentation ended was accounted for
entirely by the accumulation of ATP. This increase in
energy charge at the end of fermentation suggests that the
ATP utilizing pathways lost activity faster than the ATP
generating pathways.
Discussion
When S. cerevisiae ferments 20% glucose in complex
medium under self-induced anaerobiosis, the rate of alcohol
production declines as ethanol accumulates in the
fermentation broth (Fig. 3). The studies presented in
chapter III show that ethanol is not the only factor
contributing to this reduction in fermentation rate. A
nutritional deficiency for magnesium also was demonstrated
to be responsible (Chapter IV). Eliminating this
nutritional deficiency partially restored higher
fermentation rates, but did not prevent the initial decline
in activity (Fig. 9). The yeast cells appeared to have


CHAPTER VI
PHOSPHORYLATED GLYCOLYTIC INTERMEDIATES AND NUCLEOTIDES
Introduction
The rate of ethanol production decreases as alcohol
accumulates during the fermentation of glucose by
Saccharomvces cerevisiae (Fig. 3). A nutritional deficiency
for magnesium was identified as being partially responsible
for this declining fermentation rate (Chapters III and IV).
Magnesium supplementation attenuated the loss of
fermentation rate as ethanol accumulated but did not
eliminate it (Fig. 9). As the yeast cells converted glucose
to ethanol, their physiological characteristics changed.
Older cells (24-h) were more resistant to inhibition of
fermentative activity by added ethanol than were younger,
more active, cells (12-h) (Fig. 12). In addition, cells
from the latter stages of fermentation were better able to
maintain membrane integrity in the presence of 20% (vol/vol)
ethanol than were younger cells (Fig. 15). Characterizing
the physiological changes which occur during the
fermentation of 20% glucose is an important step towards
understanding the constraints which limit glycolysis and,
ultimately, the rate of ethanol production by S. cerevisiae.
113


TIME 00 TIME (h) TIME 00
CELL PROTEIN (mg/ml)
ro Oi
o b b b
ETHANOL (%v/v)
A O) CD
O


A pH
105


GROWTH RATE (h*1)
NJ


BIBLIOGRAPHY
Aguilera, A. and T. Benitez. 1985. Role of mitochondria in
ethanol tolerance of Saccharomyces cerevisiae. Arch.
Microbiol. 142:389-392.
Aiba, S., M. Shoda and M. Nagatani. 1968. Kinetics of
product inhibition in alcohol fermentation. Biotechnol.
Bioeng. 10:845-864.
Andreasen, A.A. and T.J.B. Stier. 1953. Anaerobic nutrition
of Saccharomyces cerevisiae I. Ergosterol requirement for
growth in a defined medium. J. Cell. Comp. Physiol. 41:23
36.
Andreasen, A.A. and T.J.B. Stier. 1954. Anaerobic nutrition
of Saccharomyces cerevisiae II. Unsaturated fatty acid
requirement for growth in a defined medium. J. Cell. Comp
Physiol. 43:271-281.
Aragon, J.J. and V. Sanchez. 1985. Enzyme concentration
affects the allosteric behavior of yeast
phosphofructokinase. Biochem. Biophys. Res. Comm. 131:849
855.
Bacilia, M., A. Xavier and J. Horii. 1978. Induction of
respiration-deficient mutants of Saccharomyces and
evolution of their efficiency for ethanol production,
p. 577-594. In M. Bacilia, B. Horecker and A. Stoppani
(ed.), biochemistry and genetics of yeasts. Academic
Press, New York.
Ball, W.J. and D.E. Atkinson. 1975. Adenylate energy charge
in Saccharomyces cerevisiae during starvation. J.
Bacteriol. 121:975-982.
Banuelos, M. and C. Gancedo. 1978. In situ study of the
glycolytic pathway in Saccharomyces cerevisiae. Arch.
Microbiol. 117:197-201.
Barondes, S.H., M.E. Traynor, W.T. Schlapfer and P.B.J.
Woodson. 1979. Rapid adaptation to neuronal membrane
effects of ethanol and low temperature: Some speculations
on mechanism. Drug Ale. Depend. 4:155-166.
152


140
changed physiologically as the fermentation progressed (Fig.
12 and Fig. 15).
Variations in the amounts of glycolytic enzymes and
internal pH were examined in chapter V as potential
physiological changes which might be responsible for the
observed decline in fermentation rate. The levels of
glycolytic and alcohologenic enzymes, including hexokinase,
measured in vitro remained high throughout fermentation
(Fig. 13A). The activity of these enzymes in vivo, however,
is modulated by the intracellular environment (Sols et al..
1971). One such environmental factor is pH. Throughout
fermentation, the intracellular pH remained close to
neutrality (Fig. 14A), near the pH optimum for many of the
glycolytic and alcohologenic enzymes. The large decline in
intracellular ATP during fermentation (Fig. 20A) suggests
that ATPases are actively excreting protons in an attempt to
counteract the permeabilizing effects of ethanol
accumulation (Pena et al.. 1972).
Included in the intracellular environment are
substrate, cofactor, coenzyme and effector molecules which
also can modulate the activities of the glycolytic enzymes.
The studies presented in this chapter examine the changes in
the intracellular levels of glycolytic intermediates and
nucleotides. As yeast cell growth slowed during batch
fermentation (Fig. 8A), the intracellular level of
nucleotide phosphate dramatically declined by more than 90%


Table 3. Effect of growth in broths of different composition on
fermentation rate
Experiment
a
(pmoles CC>2
Inoculum
1. Growth with added ethanol
Control 48.5
5% (vol/vol) ethanol
2. Inhibitor production
Control 48.1
Conditioned broth
(12-h, 1.2% (vol/vol) ethanol, supplemented
with yeast extract and glucose)
Conditioned broth
(24-h, 4.5% (vol/vol) ethanol, supplemented
with yeast extract and glucose)
Fermentation Rate
produced/h per mg protein)
12-h
cells
24-h cells
50.7
(1.0)
NDb
44.5
(2.0)
NDb
41.8
(0.1)
12.5 (0.1)
40.7
(1.6)
14.8 (0.3)
33.5
(1.2)
13.6 (0.2)
3.Nutrient limitation I
Control 48.1
Conditioned broth
(12-h, 1.2% (vol/vol) ethanol, supplemented
with glucose alone)
Conditioned broth
(24-h, 4.5% (vol/vol) ethanol, supplemented
with glucose alone)
41.8 (0.1) 12.5 (0.1)
< 5 doublings after 71 h
< 5 doublings after 71 h


it. Since the levels of glycolytic and alcohologenic
enzymes remained high and internal pH was maintained near
neutrality, inactivation of enzymes or lowered levels of in
vivo activity due to acidification of the cell cytoplasm
appear unlikely. As yeast cells produced ethanol, they did
change physiologically, however, becoming more resistant to
inhibition of fermentation by added ethanol and to ethanol-
induced decreases in A pH. Initially, the intracellular
levels of phosphorylated glycolytic intermediates decreased
as fermentation rate was declining. These results suggested
that the rates of glucose uptake and/or phosphorylation were
slowing relative to carbon flux through the rest of the
pathway. Declining glycolytic intermediate levels probably
were not due to inhibition of glycolytic enzymes by
declining levels of nicotinamide nucleotides. Initially
during fermentation, ATP levels decreased by 60%, while AMP
increased by 900%. One possible explanation for the decline
in glycolytic intermediates and the corresponding decrease
in fermentation rate is that the increased level of AMP
inhibits glucose phosphorylation which may slow the rate of
glucose uptake. In this study, the roles of inhibition by
ethanol, nutrient limitation, and physiological changes in
decreasing the rate of fermentation have been defined and
characterized. Each of these factors appears to be
partially responsible for the decline in ethanol production
by S. cerevisiae during batch fermentation.
IX


142
not due to a decline in glycolytic capacity. As the energy
charge dramatically fell, the rates of phosphofructokinase
(Betz and Moore, 1967) and pyruvate kinase should increase
(Kayne, 1973), activating carbon flow through glycolysis. A
more plausible explanation for the decline in glycolytic
intermediates is that the rate of carbon input into the
pathway may have decreased with respect to glycolytic
capacity. Similar proposals have been made to explain the
"Pasteur effect" in yeast (Becker and Betz, 1972; Den
Hollander et al.. 1986).
The increasing level of trise phosphates along with
the constant low level of hexose phosphates from 18 to 36 h
during fermentation (Fig. 17B), suggests that the trise
part of the glycolytic pathway was restricting carbon flow
to ethanol. Declining glyceraldehyde-3-phosphate
dehydrogenase activity in vivo probably was not responsible
for this constriction considering the high levels of NAD+
compared to NADH throughout fermentation (Fig. 19). Since
the intracellular concentration of NADH did not decrease as
fermentation rate declined, a fall in alcohol dehydrogenase
activity in vivo also does not appear to be a likely cause.
Rather, the low level of hexose phosphates may have lowered
the activity of pyruvate kinase by decreasing the
availability of fructose 1,6-bisphosphate, a potent
activator of this enzyme (Hess et al.. 1966). This
restriction could lead to an accumulation of intermediates


116
is to quantitate the levels of glycolytic intermediates as
fermentation progresses. Increasing levels of an individual
intermediate may reflect an imbalance between the synthesis
and utilization of that intermediate. This was demonstrated
by Ciriacy and Breitenbach (1979) who showed that mutants of
S. cerevisiae missing one of the glycolytic enzyme
activities accumulated the glycolytic intermediate which was
substrate for the missing activity. Similarly, elevated
levels of hexose phosphates were detected by 31P-NMR in
extracts of cells lacking either phosphoglucose isomerase or
phosphofructokinase activity (Navon et al.. 1979). In this
chapter, the amounts of phosphorylated glycolytic
intermediates and total nucleotides during the fermentation
of 20% glucose were quantitated in order to determine which
part(s) of the glycolytic pathway limits flow and the rate
of ethanol production. Since the in vivo activities of key
glycolytic enzymes are modulated by the availability and
allosteric actions of adenine and nicotinamide nucleotides,
individual nucleotide levels also were determined.
Materials and Methods
Organism and Growth Conditions
The organism used in these studies was S. cerevisiae
KD2, previously described in chapter II. Growth conditions
and maintenance of this organism were also as described in
chapter II. Batch fermentations were carried out using YEPD
medium as indicated in chapter IV.


66


92
highest level of accumulated ethanol. The apparent increase
in activity in the cells which had accumulated 12.1%
(vol/vol) ethanol was primarily due to the restoration of
fermentable substrate.
In a control experiment, the inhibition of ethanol
production by added ethanol and the reversibility of this
inhibition by washing was investigated. Cells were
harvested and suspended in fresh medium containing various
concentrations of ethanol. Cells at 12 h after inoculation
(Fig. 12A) were more active and more sensitive to inhibition
by added ethanol than cells sampled after 24 h of
fermentation (Fig. 12B). Ethanol caused a progressive,
dose-dependent inhibition of fermentation in both, with this
inhibition being immediate and complete within the first 10
min of exposure. No further decline in activity was
observed during a subseguent 2 h of incubation at 30C with
10% (vol/vol) added ethanol (data not shown). The
concentrations of ethanol required to inhibit 50% of the
maximum observed fermentation rate were 6.5 and 9.0%
(vol/vol), respectively, for 12-h and 24-h cells.
The inhibition of fermentation caused by exposure to
concentrations of ethanol above 5% (vol/vol) for 10 min was
only partially reversed by suspension in fresh medium
lacking ethanol (Fig. 12), indicating that exposure to
ethanol damaged the cells in some way. Again, 12-h cells
appeared more sensitive to ethanol damage than 24-h cells.


148
fermentation rate may result from inhibition at the earliest
stages of glucose metabolism, transport or associated
phosphorylation. (G) One possible explanation for a
decrease in hexose uptake capacity could be the inhibition
of hexose phosphorylation by low levels of ATP and high
levels of AMP that accumulate during fermentation (Fig. 20),
although other explanations are also plausible (Chapter VI) .
In the last chapter, a decline in glucose transport or
phosphorylating activity was suggested to be the likely
cause of the initial 50% decline in fermentation rate that
remained after nutrient supplementation. The large increase
in intracellular AMP may competitively inhibit the glucose
phosphorylation reactions (Noat et al. 1970; Rudolph and
Fromm, 1971) implicated in regulating hexose uptake (Bisson
and Fraenkel, 1984; Bisson and Fraenkel, 1983; Busturia and
Lagunas, 1986) or changes in membrane lipid composition may
occur (Homann et al.. 1987; Hunter and Rose, 1972) which can
directly lower the rate of glucose uptake (Thomas and Rose,
1979). Alternatively, the hexose transport carrier
molecules may be inactivated when the cells enter stationary
phase. In order to distinguish between these hypotheses,
further studies need to be conducted to measure the capacity
of the facilitated diffusion carrier system for glucose
throughout fermentation. A decline in the rate of
facilitated diffusion between 12 h and 24 h would indicate
either an inactivation of the hexose uptake system,


31
Stewart, Labatts Brewery, London, Canada). This organism
was grown in YEPD medium as described in chapter II.
Fermentations were carried out at 30C in spinner bottles
designed for tissue culture, also as described in
chapter II. "Conditioned broth" refers to medium in which
cells have been allowed to grow for 12 or 24 h and have been
removed by centrifugation. This broth was sterilized by
filtration.
Analytical Methods
Cell mass was monitored by measuring optical density at
550 nm using a Bausch and Lomb Spectronic 70
spectrophotometer. Total cell protein was determined using
the method of Layne (1957). Cell viability was measured
with the methylene blue dye exclusion procedure of Trevors
et al. (1983). Ethanol was determined using gas
chromatography as described by Goel and Pamment (1984).
Rates of fermentation were measured as the rate of C02
production at 30C under a nitrogen atmosphere using
respirometry as described in chapter II.
Measurement of the Intracellular Ethanol Concentration
The procedure used to determine the intracellular
concentration of ethanol in actively fermenting yeast cells
is illustrated in figure 4. Cells from batch fermentations
were concentrated by centrifugation (10,000 x g, 2 min,
ambient temperature) and resuspended in the same medium to a
density of 50 mg of cell protein per ml. [14C] sorbitol


70
to about 130 nmoles of magnesium per mg cell protein after
24 h. Magnesium-supplemented cultures maintained a higher
level of cellular magnesium throughout fermentation than
cultures grown in unsupplemented YEPD medium.
Magnesium-Limited Growth of Other Yeast Strains
Three other yeast strains were investigated to
determine whether magnesium also limited their growth: S.
cerevisiae CC3 (parent organism), S. cerevisiae A10 and S.
sake. Glucose-reconstituted medium was prepared for each of
these strains. Cultures were inoculated into their
respective glucose-reconstituted medium with and without
added magnesium (0.5 mM) and incubated for 48 h on a
rotator. S. cerevisiae CC3 and S. sake exhibited magnesium-
dependent growth almost identical to that reported for
strain KD2. The optical density at 550 nm was 8.3 to 9.4
after 48 h with added magnesium and 0.5 without added
magnesium. S. cerevisiae A10 grew poorly in its glucose-
reconstituted medium, with an optical density at 550 nm of
1.0 to 1.2 after 48 h for both control and supplemented
cultures. All three strains reached a similar cell density
in fresh YEPD medium (optical density at 550 nm of 14.2 to
15.2). These results indicate that the magnesium limitation
observed in strain KD2 was not caused by the petite mutation
and was not limited to strain CC3 and its derivatives.
However, additional factors were clearly involved with S.
cerevisiae A10.


levels of ethanol during rapid fermentation. Addition of
exogenous ethanol would not readily duplicate this
condition. However, the ethanol retention hypothesis is not
supported by direct measurements of intracellular ethanol
concentrations during fermentation.
A series of attempts to measure the intracellular
concentration of ethanol have resulted in conflicting data.
Problems in experimental design associated with the
measurement of a small, rapidly produced metabolite, such as
ethanol, contribute to these differences. The conflicting
reports result from two basic problems. First, measurements
of ethanol concentrations in the pellets of rapidly
fermenting cells result in calculated intracellular
concentrations of ethanol which are often several-fold
higher than those of the surrounding medium (Navarro and
Durand, 1978; Novak et al.. 1981; Panchal and Stewart,
1980). This was, in large part, due to the continued
production of ethanol by the cells in pellets during
centrifugation and processing (Dasari et al.. 1984). The
acuteness of this problem also would be expected to decrease
as fermentation rate and substrate levels declined during
batch fermentations. The reduction in apparent
intracellular/extracellular ratios of ethanol observed by
Beaven et al, (1982) during the latter stages of
fermentation supports this idea. Dasari et al. (1984)
demonstrated that precooling the culture significantly


87
serum albumin served as the protein standard for both
methods.
Enzyme Analyses
Activities of glycolytic and alcohologenic enzymes were
determined in 2-ml samples removed at various times during
batch fermentation. Cells were harvested by centrifugation
at 10,000 x g for 30 sec at 4C and washed in an equal
volume of 50 mM potassium phosphate buffer (pH 7.4). All
subsequent steps were carried out at 4C. The pellet was
suspended in the same buffer containing 2 mM mercaptoethanol
and 2 mM EDTA, and disrupted with 0.1-mm glass beads using a
Mini-Bead Beater (Biospec Products, Bartlesville, Okla.).
Five 1-min periods of disruption, each were followed by 5-
min periods of cooling on ice. Cell debris was removed by
centrifugation at 10,000 x g for 5 min, and the supernatant
was assayed immediately for enzymatic activities. Only two
enzymes at a time were assayed in each batch fermentation
experiment to avoid problems which could result from storage
of cells or extracts.
Pyruvate decarboxylase and all glycolytic enzymes were
assayed spectrophotometrically by the methods of Maitra and
Lobo (1971) as modified by Clifton et al. (1978). All
enzymes were assayed under substrate-saturating conditions
except trise phosphate isomerase, which was assayed with
1 mM substrate. The amounts of coupling enzymes were
adjusted as needed to ensure a linear reaction rate.


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES V
LIST OF FIGURES vi
ABSTRACT viii
CHAPTERS
I GENERAL INTRODUCTION 1
IICHARACTERIZATION OF THE DECLINING RATES OF
GROWTH AND ETHANOL PRODUCTION DURING BATCH
FERMENTATION BY S. CEREVISIAE KD2 12
Introduction 12
Materials and Methods 14
Results 18
Discussion 27
IIINUTRIENT LIMITATION AS A BASIS FOR THE
APPARENT TOXICITY OF LOW LEVELS OF ETHANOL
DURING BATCH FERMENTATION 3 0
Introduction 30
Materials and Methods 3 0
Results 36
Discussion 50
IVMAGNESIUM LIMITATION AND ITS ROLE IN THE
APPARENT TOXICITY OF ETHANOL DURING YEAST
FERMENTATION 56
Introduction 56
Materials and Methods 59
Results 62
Discussion 80
VGLYCOLYTIC ENZYMES AND INTERNAL pH 84
Introduction 84
iii


88
Alcohol dehydrogenase was assayed by measuring the oxidation
of ethanol as described by Maitra and Lobo (1971), but using
a buffer at pH 8.7 containing 75 mM sodium pyrophosphate,
75 mM semicarbazide hydrochloride and 21 mM glycine (Bernt
and Gutman, 1971).
Determination of Internal pH and Membrane Energization
The measurements of internal pH and A If! were
performed using 7-[ 14C]benzoic acid and [3H-
phenyl]tetraphenylphosphonium bromide, respectively.
Protocols were similar to those described by Cartwright et
al. (1986) except that cells were incubated in their native
growth medium rather than distilled water and 0.4-pm pore
size polycarbonate filters were used instead of mixed
cellulose ester filters. Cell volumes were determined as
described in chapter III. As a control for adventitious
binding of radioactive compounds, cells were permeabilized
with a combination of ethanol, toluene and Triton X-100 as
described by Salmon (1984), washed with 50 mM phosphate
buffer, resuspended in native broth and processed. This
treatment resulted in a complete collapse of A pH and loss
of membrane potential. Calculations were performed as
described by Rottenberg (1979).
Materials
Yeast extract, peptone and agar were obtained from
Difco Laboratories, Detroit, Mich. Glucose, coupling
enzymes, coenzymes and substrates were purchased from Sigma


FERMENTATION RATE
FERMENTATION RATE
(pmoles COj/h per mg protein)
s


Figure 15. Effects of added ethanol on ApH.
Cells were removed at various stages
of batch fermentation (indicated on
graph), harvested and suspended in
fresh medium containing various
concentrations of ethanol. Samples
were incubated at 30C for 10 min and
pH was determined.


5
relating the rate of alcohol production to the amount of
ethanol in the fermentation broth were slightly different.
A glucose-limited continuous culture of a respiratory-
deficient baker's yeast was found by Aiba et al. (1968) to
exhibit an exponential decrease in rate of alcohol formation
as increasing concentrations of ethanol were added to the
fermentation broth. They also demonstrated that ethanol
acted as a non-competitive inhibitor of alcohol formation.
Both batch and continuous culture fermentations of S.
cerevisiae ATCC No. 4126 in a synthetic medium were shown by
Bazua and Wilke (1977) to exhibit kinetics of ethanol
inhibition entirely different than had been described
previously. In each case, similar models were constructed
for growth in the presence of ethanol. The large variety of
kinetic models suggests that many factors, such as strain
variations, environmental conditions and nutritional state,
also may have important roles in the inhibition of growth
and alcohol production during yeast fermentation.
Strains of yeast able to grow and ferment in the
presence of higher concentrations of ethanol may be capable
of producing larger amounts of alcohol at faster rates.
Thus, many investigations have examined the mechanism of
ethanol tolerance in yeast (Casey and Ingledew, 1986; Ingram
and Buttke, 1984; Ingram et al.. 1986; Moulin et al.. 1984).
Initial studies on the alcohol tolerance of yeast were
performed by Gray (1941). Various ethanol producing species


60
5.0) and the pellets were saved for further analysis by
freezing at -20C.
Preparation of Glucose-Reconstituted Medium for Growth
Experiments
Batch fermentations were allowed to reach an optical
density at 550 nm of 3.5. Cells were removed by
centrifugation in a Sorvall RC-2B centrifuge at 10,000 x g
for 2 min. The amount of ethanol in the supernatant was
determined and used to estimate the amount of glucose needed
to reconstitute the medium to a concentration of 20%. This
glucose-reconstituted medium was sterilized by vacuum
filtration with 0.45 pm Metricel membrane filters (Gelman
Sciences Inc., Ann Arbor, Mich.).
Preparation of Ashed Medium Components
Yeast extract (20 g) and peptone (30 g) were burned
over a gas burner for 5 h in a porcelain crucible. After
being transferred to a muffle furnace, the medium components
were ashed at 600C for 72 h. The yeast extract ash was
suspended in 40 ml of deionized water and the peptone ash
was suspended in 30 ml of deionized water. These aqueous
suspensions of ash were adjusted to pH 5.0 with concentrated
HC1 and sterilized by autoclaving.
Nutrient Supplementation Growth Experiments
Nutrient supplements were added to culture tubes
containing 5 ml fresh YEPD medium or glucose-reconstituted
medium and a 1% by volume inoculum (initial optical density
at 550 nm of 0.035). Culture tubes were incubated at 30C


54
extract and lipids substantially improved fermentation rates
and reduced the time required to complete the fermentation.
The studies reported in this chapter using a yeast
extract/peptone-based fermentation broth also illustrate
this point and provide further support for the hypothesis
that nutritional deficiencies, in addition to accumulated
ethanol, also are responsible for the initial decline in
fermentation activity during the accumulation of low levels
of ethanol.
The reduced fermentation rate of cells after the
production of approximately 5% (vol/vol) ethanol appears to
result from the combination of a small inhibitory effect of
ethanol and physiological changes in the cells. These
physiological changes were not induced by growth in the
presence of 5% (vol/vol) added ethanol or by growth in the
presence of ethanol along with other natural fermentation
products. Conditioned broth was deficient in nutrients
provided by yeast extract and supported very little growth.
The addition of 5 g/L of yeast extract restored the ability
of this spent broth to support vigorous growth and
fermentation. By further increasing the concentration of
yeast extract to 25 g/L in the growth medium, the decline in
fermentative activity associated with the initial production
of 5% (vol/vol) ethanol was partially prevented. These
results support the hypothesis that physiological changes in
the cells caused by nutrient limitation are major factors in


155
Ciriacy, M. and I. Breitenbach. 1979. Physiological effects
of seven different blocks in glycolysis in Saccharomvces
cerevisiae. J. Bacteriol. 139:152-160.
Clifton, D., S.B. Weinstock and D.G. Fraenkel. 1978.
Glycolysis mutants in Saccharomvces cerevisiae. Genetics
88:1-11.
Colowick, S.P. 1973. The hexokinases, p. 1-48. In P.D. Boyer
(ed.), the enzymes, vol. 9. Academic Press, New York.
Corran, H.S. 1975. A history of brewing. David and Charles,
Vancouver.
Cysewski, G.R. and C.R. Wilke. 1977. Rapid ethanol
fermentations using vacuum and cell recycle. Biotechnol.
Bioeng. 19:1125-1143.
Damiano, D. and S.S. Wang. 1985. Improvements in ethanol
concentration and fermentor ethanol productivity in yeast
fermentations using whole soy flour in batch and
continuous recycle systems. Biotechnol. Lett. 7:135-140.
Dasari, G., E. Keshavarz, M.A. Connor and N.B. Pamment.
1985. A reliable method for detecting the intracellular
accumulation of fermentation products: Application to
intracellular ethanol analysis. Biotechnol. Lett. 7:541-
546.
Dasari, G., F. Roddick, M.A. Connor and N.B. Pamment. 1984.
Factors affecting the estimation of intracellular ethanol
concentrations. Biotechnol. Lett. 5:715-720.
De La Pena, P., F. Barros, S. Gascon, S. Ramos and P.S.
Lazo. 1982. The electrochemical proton gradient of
Saccharomvces: The role of potassium. Eur. J. Biochem.
123:447-453.
den Hollander, J.A., K. Ugurbil and R.G. Shulman. 1986. 31P
and 13C NMR studies of intermediates of aerobic and
anaerobic glycolysis in Saccharomvces cerevisiae.
Biochemistry 25:212-219.
Dombek, K.M. and L.O. Ingram. 1984. Effects of ethanol on
the Escherichia coli plasma membrane. J. Bacteriol.
157:233-239.
Eaton, L.C., T.F. Tedder and L.O. Ingram. 1982. Effects of
fatty acid composition on the sensitivity of membrane
functions to ethanol in Escherichia coli. Substan. Ale.
Act./Misuse. 3:77-87.


43
fermentation. Thus, on the time scale of the ethanol
removal experiments, 24-h cells appear to be freely
permeable to ethanol added to the fermentation broth.
Effect of Added Ethanol on the Fermentation Rate of 12-h and
24-h Cells
The sensitivity of 12-h and 24-h cells to inhibition of
fermentative activity by added ethanol is illustrated in
figure 5. The fermentation rate of 24-h cells was
approximately one-half that of 12-h cells when assayed in
fresh broth lacking ethanol. Both types of cells were
insensitive to ethanol concentrations up to 2% (vol/vol)
after which they exhibited a dose-dependent linear decline
in activity up to between 12 and 14% (vol/vol) ethanol.
When plotted as a percentage of maximal rate, 24-h cells
appeared slightly more resistant, 50% inhibition at 8.3%
(vol/vol) ethanol as compared with 7.4% for 12-h cells.
Effect of Medium Composition During Growth on the
Fermentation Rate of 12-h and 24-h Cells
The slight differences in sensitivity to inhibition by
ethanol and the failure of ethanol removal to increase
fermentation rates suggest that the reduced activity of 24-h
cells may be primarily due to physiological changes in the
cells rather than to the immediate presence of ethanol.
Several experiments were performed to identify possible
causes of the physiological changes which may be involved
(Table 3). In these experiments, cells were grown under a
variety of conditions, harvested by centrifugation at


Figure 11. Effect of ethanol removal on the
fermentative activity of cells grown
in YEPD medium containing 0.5 mM
MgS04. Cells were sampled during
batch fermentation and were either
untreated or washed once and then
suspended in fresh medium containing
20% glucose. The fermentation rate of
these samples were measured
immediately by respirometry. Symbols:
% activity measured in native
broth; O activity measured after
cells were suspended in fresh medium.


81
magnesium-supplemented cultures, higher levels of
intracellular magnesium were achieved early in fermentation
and decreased to a lesser extent than observed in
unsupplemented cultures. Magnesium-supplemented cultures
had 6.5 times more magnesium in the medium at the end of
exponential growth than did unsupplemented cultures. Thus,
the magnesium supply of the supplemented culture appears to
be adequate for growth and other factors are limiting the
fermentative ability of the yeasts under these conditions.
The ubiquitous role of magnesium in cellular processes
is well documented (Jasper and Silver, 1977). Magnesium
constitutes a major portion of the cellular cations, mostly
bound in structures such as ribosomes and the cell envelope.
The free cation concentration, however, may play a more
direct role in regulating overall cellular metabolism and
cell division (Walker and Duffus, 1980). Many of the
enzymes that function in DNA replication, transcription and
translation require magnesium for activity. In fermentation
pathways, magnesium is a required cofactor and nucleotide
counter-ion in many reactions. Magnesium levels typically
are maintained at mmolar intracellular concentrations and it
is not surprising that this cation is a limiting nutrient
during high-gravity fermentations.
Previous studies have demonstrated that the inhibition
of fermentation by added ethanol in Zymomonas mobilis is
primarily due to ethanol-induced leakage, particularly of


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
John7 E. Gander
Professor of Microbiology and
Cell Science
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
/
Jmes F. Preston
Professor of Microbiology and
Cell Science
This dissertation was submitted to the Graduate Faculty of
the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
August 1987
/
ege of Agriculture
Dean, Graduate School


107
Boucherie (1985) reported that enolase, hexokinase and
glyceraldehyde-3-phosphate dehydrogenase synthesis continued
after cell growth ended. Even though the fermentations
studied in this chapter were far from glucose-limited, the
results indicate that some of the glycolytic and
alcohologenic enzymes, including enolase, hexokinase and
glyceraldehyde-3-phosphate, may be preferentially
synthesized during stationary phase. It was suggested by
Boucherie (1985) that this is a characteristic physiological
state common to stationary phase nutrient-limited cells.
The increase in enzyme activities observed in the
present study, however, was very low when compared to the
increase in activities reported upon enzyme induction.
Phosphoglucomutase exhibited the largest increase in
activity during fermentation with a maximum rate only 1.9-
fold that at 12 h. Upon addition of glucose and galactose
to acetate-grown cells, 3- and 100-fold increases in the
specific activities of glycolytic enzymes were observed by
Maitra and Lobo (1971). Entian et al. (1984) also reported
2.5- and 4.0-fold increases in pyruvate kinase and pyruvate
decarboxylase activities, respectively, only 4 h after
addition of glucose to ethanol-grown cells. Since
glycolytic enzymes each account for between 0.5 to 10% of
the total soluble cell protein during growth on glucose
(Fraenkel, 1981), a large increase in enzyme specific
activity may not be possible. Increasing amounts of


145
inhibited in the presence of a fermentable carbon source.
As cell multiplication decreases when the yeast cells enter
stationary phase, the rate of protein synthesis also
declines (Boucherie, 1985). Thus, the amounts of the
glucose transport molecules may fall as the yeast cells
enter stationary phase due to inactivation.
The inhibition of glucose transport or phosphorylation
is the likely cause of the initial decline in glycolytic
intermediate levels and the resulting 50% decrease in
fermentative activity seen after 24 h (Fig. 17A). This
decrease in glycolytic flux then is manifested as a decline
in the rate of alcohol production as ethanol accumulates
during fermentation.


154
Brown, S.W. and S.G. Oliver. 1982. The effect of temperature
on the ethanol tolerance of the yeast Saccharomvces
uvarum. Biotechnol. Lett. 4:269-274.
Brown, S.W., S.G Oliver, D.E.F. Harrison and R.C. Righelato.
1981. Ethanol inhibition of yeast growth and fermentation:
Differences in the magnitude and complexity of the effect.
Eur. J. Appl. Microbiol. Biotechnol. 11:151-155.
Buchner, E. 1897. Alkoholische gahrung ohne hefezellen. Ber.
Deutsch. Chem. Gesell. 30:117-124.
Busturia, A. and R. Lagunas. 1986. Catabolite inactivation
of the glucose transport system in Saccharomvces
cerevisiae. J. Gen. Microbiol. 132:379-385.
Buttke, T.M., S.D. Jones and K. Bloch. 1980. Effect of
sterol side chains on growth and membrane fatty acid
composition of Saccharomvces cerevisiae. J. Bacteriol.
144:124-130.
Buttke, T.M. and A.L. Pyle. 1982. Effects of unsaturated
fatty acid deprivation on neutral lipid synthesis in
Saccharomvces cerevisiae. J. Bacteriol. 152:747-756.
Cartwright, C.P., J.R. Juroszek, M.J. Beaven, F.M.S. Ruby,
S.M.F. De Morals and A.H. Rose. 1986. Ethanol dissipates
the proton-motive force across the plasma membrane of
Saccharomvces cerevisiae. J. Gen. Microbiol. 132:369-377.
Casey, G.P. and W.M. Ingledew. 1986. Ethanol tolerance in
yeasts. CRC Crit. Rev. Microbiol. 13:219-280.
Casey, G.P., C.A. Magnus and W.M. Ingledew. 1983. High-
gravity brewing: Nutrient enhanced production of high
concentrations of ethanol by brewing yeast. Biotechnol.
Lett. 6:429-434.
Casey, G.P., C.A. Magnus and W.M. Ingledew. 1984. High-
gravity brewing: Effects of nutrition on yeast
composition, fermentative ability and alcohol production.
Appl. Env. Microbiol. 48:639-646.
Chapman, A.G. and D.E. Atkinson. 1977. Adenine nucleotide
concentrations and turnover rates: Their correlation with
biological activity in bacteria and yeast. Adv. Microbial
Physiol. 15:254-306.
Chin, J.H. and D.B. Goldstein. 1977. Drug tolerance in
biomembranes: A spin label study of the effects of
ethanol. Science 196:684-685.


99
12 h cells (100%). An analogous plot of fermentation rate
is included for comparison. None of the specific activities
declined dramatically during batch fermentation. At times
beyond 12 h, with the exceptions noted above, all enzymes
were in excess of the measured fermentation rates.
Phosphofructokinase declined to the greatest extent with a
20% drop in activity after 48 h. The specific activities of
phosphoglucomutase (100% increase), hexokinase (50%
increase, not shown) and enolase (50% increase, not shown)
increased by more than 20%. All other enzymes, with
glyceraldehyde-3-phosphate dehydrogenase as a typical
representative, exhibited similar increases of up to 20%.
Figure 13B illustrates the changes in the amounts of
these enzymes present per ml of broth relative to that at
12 h. Analogous plots of soluble cell protein and
fermentation rate per ml are included for comparison. The
peak of fermentative activity on a volumetric basis occurred
after 18 h. Although the rate of fermentation declined
beyond 18 h, the activities of all of the glycolytic enzymes
continued to increase until 30 h, the peak in soluble
protein concentration. These increases in activities
roughly paralleled the increase in soluble protein. The
activities of phosphoglucomutase, enolase (not shown),
hexokinase (not shown) and glyceraldehyde-3-phosphate
dehydrogenase increased more rapidly than soluble cell
protein, consistent with the observed increases in specific


BIOGRAPHICAL SKETCH
Kenneth Michael Dombek was born on November 19, 1959, in
Miami, Florida, to Donald and Mary Dombek. He grew up as
the eldest son in a family with three brothers and two
sisters. In Miami, he attended Christopher Columbus High
School where he graduated in 1977. In the fall of that same
year, he moved to Gainesville, Florida, to pursue studies in
science. His undergraduate studies where in chemistry and
he received a Bachelor of Science degree in 1981. Applying
physical-chemical techniques to the study of biological
systems became his main interest at that time. He joined
the Department of Microbiology and Cell Science as a
graduate student of Dr. Neal Ingram in 1981 in order to
pursue this interest. With the help and encouragement of
Dr. Ingram, he received a Master of Science degree in 1982.
Since that time, his scientific interests in the chemical
biology of microorganisms has broadened considerably and
from that curiosity has emerged the studies presented here.
166


26
cells exhibited a biphasic decline as a function of
accumulated ethanol. An initial decline in fermentation
rate occurred during the accumulation of 3.7% (vol/vol)
ethanol with a 50% loss of activity. This was followed by a
more gradual decline in fermentation rate with approximately
20% of the original activity remaining after the production
12% (vol/vol) ethanol. The fermentation rate of S.
cerevisiae A10 p, a respiratory-deficient haploid
laboratory yeast strain, also declined as ethanol
accumulated in the medium (data not shown). As with S.
cerevisiae KD2, a 50% decrease in fermentation rate was
observed after the accumulation of 3.5% (vol/vol) ethanol.
However, the maximum rate of fermentation was lower,
40 jamles C02 per h per mg protein compared to 50 for strain
KD2 and greater than 90% of the maximum fermentation rate
was lost by the time 6.5% (vol/vol) ethanol had accumulated.
Unlike ethanol accumulated during fermentation, the
addition of low concentrations of ethanol to rapidly
fermenting cells 12 h after inoculation did not result in a
large decline in fermentative activity (Fig. 3). Ethanol
caused a dose-dependent linear decline in activity.
Fermentation was inhibited only 12% by the addition of 3.7%
(vol/vol) ethanol and 8.5% (vol/vol) added ethanol was
required to cause 50% inhibition.


62
determined by the methylene blue staining procedure of Mills
(1941).
Chemicals
Yeast extract, peptone and agar were obtained from
Difco Laboratories, Detroit, Mich. Glucose and other
biochemicals were obtained from Sigma Chemical Co., St.
Louis, Mo. Magnesium sulfate and other inorganic salts were
purchased from Fisher Scientific Company, Orlando, Fla.
Absolute ethanol was supplied by AAPER Alcohol and Chemical
Co., Shelbyville, Ky. Gas chromatography supplies were
obtained from Supelco, Bellefonte, Pa.
Results
Effect of Nutrient Supplements on Growth in Glucose-
Reconstituted Medium
As shown in Tables 3 and 4, fermentation broth in which
S. cerevisiae KD2 had grown for 12 h supported very little
further growth and limited the fermentative activity of
strain KD2 even after supplementation with glucose (glucose-
reconstituted medium). At this stage of fermentation (1.2%
(vol/vol) accumulated ethanol), ethanol production was at
its maximum rate (50 pmoles/h per mg protein). This time
point also marked the end of exponential growth (Fig. 1),
indicating either a nutrient limited state or the presence
of an inhibitor.
The addition of yeast extract and peptone at the
original medium concentration restored the ability of the
used medium to support growth at 71% and 54% of the control


activity. The results presented in this chapter identify
magnesium as the limiting component of yeast extract and
demonstrate that when this nutrient limitation is relieved,
a dramatic decrease in the time required for total
conversion of glucose to ethanol is achieved. This decrease
in time required for the completion of fermentation resulted
from a delay in the onset of stationary phase which
increased the total cell number during that part of
fermentation in which over 90% of the ethanol is produced.
Materials and Methods
Organisms and Growth Conditions
The principal organism used in these studies was
Saccharomvces cerevisiae KD2, described in chapter II. In
addition, S. cerevisiae CC3, S. cerevisiae A10 (NRRL Y-
12707) and S. sake (NRRL Y-11572) were used for comparison
in some experiments. The latter two strains generously were
provided by N.J. Alexander (Northern Regional Research
Center, U.S. Department of Agriculture, Peoria, Ill.). All
organisms were grown in YEPD broth and maintained on YEPD
agar, as stated in chapter II. Batch fermentations also
were carried out as described in chapter II.
Preparation of Fermentation Samples for Analysis
Fermentation samples were centrifuged at 10,000 x g for
0.5 min. The supernatant was removed and saved by freezing
at -20C. Cells were washed once in 50 mM KH2P04 buffer (pH


79
supplemented and unsupplemented cells had a similar initial
fermentation rate, about 57 pmoles of C02 produced per h per
mg cell protein and exhibited identical dose-response
curves. A concentration of 7.6% (vol/vol) ethanol resulted
in 50% inhibition of fermentative activity.
Figure 10B shows the effect of ethanol on the
fermentation rate of the older cells. Supplemented cells
had an initial fermentation rate of 27.4 pmoles of C02
produced per h per mg cell protein with an SD of 0.5, while
unsupplemented cells exhibited a significantly lower rate,
22.9 pmoles of C02 produced per h per mg cell protein with
an SD of 1.2. The fermentation rate of cells from
supplemented cultures was always higher than that of control
cells. The amount of ethanol present in the supplemented
culture was 3.0% (vol/vol) higher than in the unsupplemented
culture when compared at equal fermentation rates. The
fermentation rate of magnesium-supplemented and control
fermentations exhibited linear dose-responses to ethanol
addition. A measure of the sensitivity of fermentation rate
to ethanol is the slope of the dose-response curve. The
slope for supplemented batches was -0.17 with an SD of 0.02,
while that of the controls was -0.14 with an SD of 0.01.
The differences in these slopes (Fig. 10B) are suggestive,
but do not conclusively demonstrate that supplementation
with magnesium reduced the sensitivity of fermentation in
older cells to inhibition by ethanol.


ETHANOL (%v/v) ETHANOL (%v/v)
FERMENTATIVE ACTIVITY (% initial rate)
ro m CD o
o o o o o o
FERMENTATIVE ACTIVITY (% initial rate)
ro en os o
O O O O O O
8 L


First Dimension
121
Second Dimension


3
for the conversion which Pasteur had described 50 years
earlier were characterized (Fraenkel, 1982).
Even before these early studies on alcoholic
fermentation, it had been observed that yeast stopped
growing and fermenting before all of the sugar in the
fermentation broth had been utilized. In one of the
earliest investigations of this phenomenon, Brown examined
the influence of various environmental conditions on the
rate of growth of S. cerevisiae (Brown, 1905). The addition
of ethanol to growth medium, indeed, did inhibit yeast
reproduction. However, inhibition occurred only at a much
higher ethanol concentration than was observed to have
accumulated at the point during the fermentation when yeast
growth had ceased. This was the first indication that the
presence of ethanol may not be the only factor involved in
the premature termination of carbohydrate fermentation by
yeast.
The studies of Brown were complemented by the work of
Richards (1928) who showed that removing ethanol produced
during growth and maintaining a constant nutrient supply
allowed yeast cell multiplication to continue almost
indefinitely. Recently, the vacuum fermentation experiments
conducted by Cysewski and Wilke (1977) and Maiorella et al.
(1983) have corroborated and extended this finding. Boiling
off the ethanol as it was produced from a continuous culture
under reduced pressure, increased achievable cell densities


151
fermentation broth, the intracellular concentration of ATP
increases (Fig. 20) as ATP degrading processes presumably
become inactivated. This increasing concentration of ATP
also can help to reduce the activity of phosphofructokinase
(Bloxham and Hardy, 1973; Uyeda, 1979) and the levels of
hexose phosphates should continue to rise.
The studies presented in this paper identify three
possible causes for the declining rate of alcohol production
as ethanol accumulates during fermentation. Firstly,
ethanol does inhibit fermentation rate, however, its
importance has been greatly overemphasized by previous
investigators. Secondly, nutrient limitation also plays a
substantial role in decreasing the rate of fermentation
during the conversion of glucose to ethanol. Nutrient
supplementation partially relieves the decline in
fermentation rate and substantially reduces the time
required for the complete conversion of 20% glucose to
ethanol. Finally, physiological changes occur during this
decline in fermentation rate after nutrient supplementation.
These changes also appear responsible for the decline in
fermentation rate, however, their exact cause has not yet
been established. Determining the origin of these
physiological changes should lead to a better understanding
of the constraints which limit the rates of glycolysis
during fermentation.


H0026


63
Table 4. Effect of nutrient supplementation on growth of
S. cerevisiae KD2
Medium
Supplement
Optical Density
at 550 nm after
48 h (SD)
% of
control (SD)
YEPD
None
13.8 (1.5)
100
12-ha
None
1.25 (0.48)
9.
1 (4.0)
12-ha
Yeast extract (5 g/L)
9.77 (0.75)
71
(9)
12-ha
Peptone (10 g/L)
7.43 (0.35)
54
(6)
12-ha
Ashed yeast extract*3
9.70 (0.40)
70
(8)
12-ha
Ashed peptone*3
3.47 (0.20)
25
(3)
12-ha
Trace minerals0
1.88 (0.20)
14
(2)
12-ha
KH2P04 (7.3 mM)
1.49 (0.17)
11
(2)
12-ha
(NH4)2S04 (7.6 mM)
1.60 (0.35)
12
(3)
12-ha
MgS04 (2 mM)
12.7 (0.1)
92
(10)
12-ha
MgCl2 (2 mM)
12.1 (0.5)
88
(10)
12-ha
CaCl2 (2 mM)
1.55 (0.14)
11
(2)
12-ha
Na2S04 (2 mM)
1.62 (0.20)
12
(2)
a Medium isolated from a batch fermentation after 12 h of
yeast growth and supplemented to 20% with glucose.
b An amount of ashed yeast extract equivalent to 5 g of
whole yeast extract per L or an amount of ashed peptone
equivalent to 10 g of whole peptone per L.
c As described by Wickersham (1951).


67
ashed yeast extract to be added. Whole yeast extract, ashed
yeast extract and MgS04 gave similar dose-responses.
However, at concentrations below 0.2 mM, MgS04 appeared to
be a better supplement. Magnesium sulfate-supplemented
fresh YEPD medium also was plotted for comparison. Maximum
growth occurred at added magnesium concentrations above
0.2 mM. A MgS04 concentration of 0.5 mM was chosen for
subsequent fermentation studies because growth at this
concentration was no longer limited by an inadequate supply
of magnesium.
To confirm that magnesium indeed was limiting in YEPD
medium, the magnesium content of cells and the surrounding
broth was determined at various times during batch
fermentation (Fig. 7). The magnesium content of the cells
reached a maximum of 130 nmoles/mg cell protein at 12 h,
rapidly declining to 48 nmoles/mg cell protein by 24 h and
remaining at this lower level throughout the final period of
fermentation. In the medium, the magnesium content fell to
less than 0.05 mM by 24 h and remained constant until
fermentation had been completed. Thus, the decline in
magnesium content per mg cell protein observed after 12 h
appears to result from continued cell growth after near
depletion of the magnesium in the surrounding broth.
Supplementing the broth with 0.5 mM magnesium resulted in
the peak accumulation of higher levels of magnesium
(200 nmoles/mg cell protein) at 12 h, followed by a decline


LIST OF FIGURES
Page
Figure 1. Growth and ethanol production by S.
cerevisiae KD2 during a typical batch
fermentation in YEPD medium containing 20%
glucose 19
Figure 2. Growth rate of S. cerevisiae KD2 in the
presence of ethanol 21
Figure 3. Rate of fermentation in the presence of
ethanol 24
Figure 4. Determination of intracellular ethanol
concentration 32
Figure 5. Inhibition of fermentation rate of 12-h and
24-h cells by added ethanol 44
Figure 6. Dose-response of cell growth to added
magnesium 65
Figure 7. Magnesium levels in broth and cells during
the course of batch fermentation 68
Figure 8. Effect of magnesium addition on cell growth
and fermentation 7 2
Figure 9. Effect of added magnesium on the rate of
fermentation 74
Figure 10. Effect of magnesium supplementation on the
inhibition of fermentation rate by added
ethanol 77
Figure 11. Effect of ethanol removal on the
fermentative activity of cells grown in YEPD
medium containing 0.5 mM MgS04 90
Figure 12. Effects of ethanol exposure on the
fermentative activities of 12- and 24-h
cells 93
vi


ETHANOL CONC. (%V/V) ETHANOL CONO. (%V/V)
RATE OF FERMENTATION


95
Longer incubation periods with 10% (vol/vol) ethanol
resulted in a decrease in activity recoverable by washing,
with only 60% of the original activity recoverable after
2 h.
Changes in the Levels of Glycolytic and Alcoholoqenic
Enzymes During Batch Fermentation
The specific activities of the enzymes involved in
alcohol production under substrate-saturating conditions are
listed in Table 5 for cells harvested after 12 h (the most
active stage of fermentation) and 24 h (50% maximal
activity) after inoculation. Rates of glycolytic flux,
calculated from rates of C02 evolution, for hexose and
trise intermediates have been included for comparison. The
activities of all but three of these were clearly in excess
of that required to support the measured rates of glycolytic
flux. The exceptions were hexokinase at 12 h, and
phosphofructokinase and pyruvate decarboxylase at both 12
and 24 h. However, the true in vivo activities must be
sufficient to support the measured rates of C02 evolution
except for a small contribution from anabolic processes.
It is of interest to compare the relative activities of
each enzyme at these two times. After 24 h, glycolytic flux
was half that at 12 h while the specific activities of six
enzymes remained unchanged, four had declined by
approximately 20% and two had increased. Figure 13A
illustrates the changes in the specific activities for these
enzymes throughout batch fermentation, relative to that of


J\' INTRACELLULAR ETHANOL
DETERMINATION
Concentrated
Cell Suspension
!'
14
C-Sorbitol
Vortex I 0 sec.
i
Sample Suspension
14,
C-Sorbitol
Determination Centrifuge
Ethanol
Determination
C C,ui) (| o,000 x g, 30 sec.) (E8U8)
i
Sample Supernatant
14CSorbitol
Determination
("(W
Ethanol
Determination
CESUP)
B
CORRECTION FOR CELL SOLIDS
Concentrated
Cell Suspension
H20
14,
C-Sorbitol
Mix and Equilibrate
5 min.
i
Sample Suspension
3H20 and 14CSorbitol
Determination
c3h.u 14C.ub)
Centrifuge
( I 0,000 x g, 30 sec.)
I
Sample Supernatant
3H20 and 14CSorbitol
Determination
v nsupf '-'sup*'
U>
OJ


158
Hess, B., R. Haeckel and K. Brand. 1966. FDP-activation of
yeast pyruvate kinase. Biochem. Biophys. Res. Comm.
24:824-831.
Hohl, L. and W.V. Cruess. 1936. Effect of temperature,
variety of juice and method of increasing sugar content on
maximum alcohol production by Saccharomvces ellipsoideus.
Food Res. 1:405-411.
Holmsen, H., C.A. Dangelmaier and J.-W. N. Akkerman. 1983.
Determination of levels of glycolytic intermediates and
nucleotides in platelets by pulse-labelling with
[32P]orthophosphate. Anal. Biochem. 131:266-272.
Holtzberg, I., R.K. Finn and K.H. Steinkraus. 1967. A
kinetic study of the alcoholic fermentation of grape
juice. Biotechnol. Bioeng. 9:413-427.
Homann, M.J., M.A. Poole, P.M. Gaynor, C.-T. Ho and G.M.
Carman. 1987. Effect of growth phase on phospholipid
biosynthesis in Saccharomvces cerevisiae. J. Bacteriol.
169:533-539.
Hoppe, G.K. and G.S. Hansford. 1982. Ethanol inhibition of
continuous anaerobic yeast growth. Biotechnol. Lett. 4:39-
44.
Hoppe, G.K. and G.S. Hansford. 1984. The effect of micro-
aerobic conditions on continuous ethanol production by
Saccharomvces cerevisiae. Biotechnol. Lett. 6:681-686.
Hossack, J.A. and A.H. Rose. 1976. Fragility of plasma
membranes in Saccharomvces cerevisiae enriched with
different sterols. J. Bacteriol. 127:67-75.
Hunter, K. and A.H. Rose. 1971. Yeast lipids and membranes,
p. 211-270. In A.H. Rose and J.S. Harrison (ed.), the
yeasts, vol. 2. Academic Press, New York.
Hunter, K. and A.H. Rose. 1972. Lipid composition of
Saccharomvces cerevisiae as influenced by growth
temperature. Biochim. Biophys. Acta 260:639-653.
Ingram, L.O. and T.M. Buttke. 1984. Effects of alcohols on
micro-organisms. Adv. Microbial Physiol. 25:254-300.
Ingram, L.O., K.M. Dombek and Y.A. Osman. 1986. A hypothesis
for the evolution of microbial resistance to ethanol. SIM
News. 36:8-11.


CHAPTER I
GENERAL INTRODUCTION
Even before 2000 BC, Saccharomyces cerevisiae was used
to ferment malted barley and wheat into a type of beer-bread
in Mesopotamia (Corran, 1975). The Babylonians and
Egyptians adapted this fermentation process to the
production of a high alcohol beer from many different sugar
sources. In Greece, grape juice was a popular sugar source
for fermentation to make wine. By the middle ages, wine and
ale were among the only beverages available that were not
contaminated with disease-causing agents. The ethanol
produced during their fermentation acted as a preservative.
Even after the advent of refrigeration and sterile packaging
procedures, liquors derived from yeast fermentation remained
as popular beverages.
Recently, uses of ethanol other than for beverages have
received much attention. Because of the growing concern
about protecting the environment from pollution and the
increasing dependence of the United States economy on
imported oil, ethanol has been adopted as both a gasoline
additive and an alternative energy source. Almost 95% of
the global ethanol produced is the product of sugar
fermented by S. cerevisiae.
1


89
Chemical Co., St. Louis, Mo. Inorganic salts were obtained
from Fisher Scientific Co., Orlando, Fla. Absolute ethanol
was supplied by AAPER Alcohol and Chemical Co., Shelbyville,
Ky. Radioactive compounds were purchased from New England
Nuclear Corp., Boston, Mass.
Results
Reversibility of the Decline in Fermentative Activity
In chapter IV, the ability of magnesium supplementation
to partially relieve the decline in fermentative activity
associated with the accumulation of ethanol was
demonstrated. After supplementation, immediately reversible
inhibition by accumulated ethanol may be responsible for the
remaining decrease in fermentation rate. To investigate
this possibility, cells were removed at various times during
batch fermentation and the rate of ethanol production per mg
cell protein was determined (Fig. 11). Cells were most
active at the earliest times measured, 12 h, and
fermentation rate declined by 50% when 6.5% (vol/vol)
ethanol had accumulated after 24 h. Approximately 40% of
the fermentative activity was retained after the
accumulation of 10% (vol/vol) ethanol with 30 g glucose
per L remaining in the fermentation broth. The abrupt,
final decline in activity reflects the near-complete
exhaustion of glucose. Removal of ethanol from cells by
washing and suspending in fresh medium resulted in only a
modest increase in fermentative activity in all but the


PERCENTAGE OF
SPECIFIC ACTIVITY AT 12 h
^ oo ro 05 o
o o o o o o
PERCENTAGE OF
ACTIVITY PER ML AT 12 h
fO
0)
CD
o
o
o
o
o
o
o
o
86


135
have remained very active in vivo. The level of NAD+ also
did not decrease but fluctuated about 1.3 mM for the first
42 h of the fermentation. With the exhaustion of glucose in
the medium by the end of fermentation, the intracellular
concentration of NAD+ increased to almost 5 mM.
Because the levels of NADH compared to NAD+ were so
low, the intracellular concentration of total nicotinamide
nucleotides mimicked the same pattern as the levels of NAD+.
Thus, the intracellular concentrations of NAD+ and NADH did
not follow the decline in total nucleotide phosphate and
probably were not responsible for the increased levels of
trise intermediates. On the contrary, the intracellular
levels of NADH and NAD+ should have accelerated
glyceraldehyde-3-phosphate dehydrogenase activity.
Adenine Nucleotides and Energy Charge
ATP is required for the phosphorylation of hexoses as
the initial step that channels hexoses into the glycolytic
pathway. Depletion of ATP during fermentation could be
responsible for the initial decline in glycolytic
intermediates (Fig. 17) by restricting carbon flow into the
pathway. As a result, the rate of fermentation would
decrease. In figure 20A, the intracellular concentrations
of adenine nucleotides, as determined by firefly luciferase
luminescence, are plotted as fermentation progresses. From
12 to 18 h, the level of ATP remained constant at about
1.8 mM. After 30 h, the intracellular concentration of ATP


143
earlier in the pathway until hexose phosphates increase,
raising the levels of fructose 1,6-bisphosphate.
Alternatively, the activity of fructose 1,6-bisphosphate
aldolase may have decreased, allowing the accumulation of
its substrate. Then, the levels of trise phosphates began
to decline (Fig. 17B), probably due to increased activity of
pyruvate kinase.
The rate of carbon input into glycolysis is determined
by the amounts and the activities of the hexose
phosphorylating enzymes and the transport carrier molecules
involved in glucose uptake (Becker and Betz, 1972). Hexose
carbon is channelled into this pathway by sugar transport
carriers located in the cell membrane (Franzusoff and
Cirillo, 1983) The activity of these carrier molecules
seems to be controlled by the metabolic state of the cell
(Eddy, 1982). Hexose kinases have been implicated in
regulating glucose uptake in S. cerevisiae (Bisson and
Fraenkel, 1983; Bisson and Fraenkel, 1984; Busturia and
Lagunas, 1986). Mutants deficient in both hexokinases and
glucokinase lack the high affinity glucose uptake system (1^
of 1 mM) while retaining the low affinity system (i^ of
20 mM). The studies of Noat et al. (1970) and Rudolph and
Fromm (1971) indicate that AMP acts as a competitive
inhibitor of ATP in the hexokinase reaction. Thus, the 10-
fold increase in the intracellular concentration of AMP from
12 h to 24 h during fermentation with the decreasing level


7
found to be more inhibitory than secondary or tertiary
alcohols. This suggested that the mode of action of ethanol
may involve a hydrophobic site. Numerous investigations
have described the detrimental effects of a variety of
hydrophobic compounds, including alcohols, on the function
of many different types of cells and membranes (Barondes et
al.. 1979; Eaton et al.. 1982; Hayashida and Ohta, 1978;
Leao and van Uden, 1982b; Lenaz et al.. 1978; Seeman, 1972).
The inhibitory potential of an alcohol on D-xylose transport
of S. cerevisiae was correlated directly with the lipid-
buffer partition coefficient of that alcohol by Leao and van
Uden. Hayashida and Ohta reported that ethanol promoted
leakage of ultraviolet-absorbing material from yeast cells,
an ethanol induced compromise of membrane barrier function.
Since ethanol alters the physical state of both artificial
and biological membranes (Chin and Goldstein, 1977; Dombek
and Ingram, 1984; Janoff and Miller, 1982; Rowe, 1983;
Vanderkooi et al.. 1977), one site of ethanol action may be
the cell cytoplasmic membrane.
The cytoplasmic membrane of S. cerevisiae contains
about 50% protein and 40% lipid by dry weight (Hunter and
Rose, 1971). It has a unique lipid composition, containing
phospholipids with no polyunsaturated fatty acids like those
found in prokaryotes and large proportions of
phosphatidylcholine and sterols like those found in
eukaryotic cells (Ingram and Buttke, 1984). As much as 80%


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE DECLINING RATE OF ETHANOL PRODUCTION
DURING BATCH FERMENTATION BY SACCHAROMYCES CEREVISIAE
By
Kenneth Michael Dombek
August 1987
Chairman: Lonnie 0. Ingram
Major Department: Microbiology and Cell Science
As Saccharomvces cerevisiae ferments 20% glucose to
ethanol in batch culture, the rate of this conversion
declines. Previous studies assumed that ethanol caused this
inhibition because added ethanol repressed both yeast growth
and alcohol production. However, produced ethanol appeared
to be a more potent inhibitor than added ethanol. Also,
removal of ethanol from fermenting yeast cells did not fully
alleviate this inhibition. The yeast cell envelope was
freely permeable to ethanol and, thus, the intracellular
accumulation of ethanol to levels high enough to inhibit
glycolytic enzymes appeared unlikely. A nutrient limitation
for magnesium in complex fermentation medium was identified
as being partially responsible for declining rates of
alcohol production. Supplementation of broth with magnesium
prevented much of the decline but did not entirely eliminate
viii


102
Aj and PROTON MOTIVE FORCE
(mv)
o o 0 o
t-~ Hd


159
Janoff, A.S. and K.W. Miller. 1982. A critical assessment of
the lipid theories of general anaesthetic action, p. 417.
In D. Chapman, (ed.), biological membranes. Academic
Press, New York.
Janssens, J.H., N. Burris, A. Woodward and R.B. Bailey.
1983. Lipid-enhanced ethanol production by Kluweromvces
fragilis. Appl. Env. Microbiol. 45:598-602.
Jasper, P. and S. Silver. 1977. Magnesium transport in
microorganisms, p. 7-47. In E.D. Weinberg (ed.),
microorganisms and minerals. Marcel Dekker, Inc., New
York.
Jones, R.P. and P.F. Greenfield. 1985. Replicative
inactivation and metabolic inhibition in yeast ethanol
fermentations. Biotechnol. Lett. 7:223-228.
Jones, R.P., N. Pamment and P.F. Greenfield. 1981. Alcohol
fermentation by yeasts the effect of environmental and
other variables. Proc. Biochem. 16:42-49.
Ju, N., D. Damiano, C.S. Shin, N. Kim and S.S. Wang. 1983.
Continuous ethanol fermentation of Zvmomonas mobilis using
soy flour as a protective agent. Biotechnol. Lett. 5:837-
842 .
Karp, M.T., R.P. Raunio and T.N-E. Lovgren. 1983.
Simultaneous extraction and combined bioluminescent assay
of NAD+ and NADH. Anal. Biochem. 128:175-180.
Kayne, F.J. 1973. Pyruvate kinase, p. 353-382. In P.D. Boyer
(ed.), the enzymes, vol. 8. Academic Press, New York.
Kunkee, R. and M. Amerine. 1968. Sugar and alcohol
stabilization of yeast in sweet wine. Appl. Microbiol.
16:1067-1075.
Lafon-Lafourcade, S., F. Larue and P. Ribereau-Gayon. 1979.
Evidence for the existence of "survival factors" as an
explanation for some peculiarities of yeast growth,
especially in grape must of high sugar concentration.
Appl. Env. Microbiol. 38:1069-1073.
Lafon-Lafourcade, S. and P. Ribereau-Gayon. 1984.
Developments in the microbiology of wine production, p. 1-
45. In M.E. Bushell (ed.), progress in industrial
microbiology, vol. 19. Elsevier, New York.


Figure 2. Growth rate of S. cerevisiae KD2 in
the presence of ethanol. Rates were
measured from batch culture
experiments as the increase in cell
protein during a 1.6 h interval around
the time point sampled. These are
plotted as a function of ethanol
accumulated in the medium during
fermentation. Growth rates in YEPD
medium containing different
concentrations of ethanol were
calculated as the exponential increase
in cell mass per h as measured by
optical density at 550 nm. These are
plotted as a function of ethanol added
to the medium. Symbols: #, growth
rate during batch fermentation; ,
effect of added ethanol on growth
rate.


64
level, respectively (Table 4). These results indicate that
nutrient limitation rather than the presence of an inhibitor
was responsible for the inability of the used medium to
support further yeast growth. Vitamin supplements also were
tested and did not promote growth in this glucose-
reconstituted medium (data not shown).
The organic components of yeast extract and peptone are
both diverse and complex. Before embarking on a
fractionation of these, the inorganic constituents were
tested after ashing. Supplementation with ashed yeast
extract was as effective as with whole yeast extract, while
ashed peptone was only half as effective as whole peptone.
These results suggested that an inorganic component of YEPD
medium was the principle factor limiting growth.
The inorganic constituents of a mineral-based minimal
medium were tested to determine which ions were limiting
(Table 4). The addition of potassium, ammonium, sodium,
calcium, phosphate, sulfate and a trace mineral mixture
described by Wickersham (1951) did not promote growth in
glucose-reconstituted medium. Only magnesium salts were
effective as nutrient supplements, allowing growth
equivalent to 90% of the control in fresh YEPD medium.
The dose-response of growth to added magnesium, yeast
extract and ashed yeast extract is shown in figure 6. Yeast
extract contained 27 pmoles of magnesium per g. This value
was used to calculate the appropriate amount of whole and


Figure 10. Effect of magnesium supplementation on the inhibition of fermentation rate by
added ethanol. Error bars represent average standard deviations for three
separate determinations. (A) Samples from batch fermentations that had
accumulated 1.2% (vol/vol) ethanol. (B) Samples from batch fermentations that
had accumulated 5.5% (vol/vol) ethanol. Symbols: 0.5 mM MgSO -
supplemented fermentations; O control fermentations in YEPD broth alone.


8
of the fatty-acyl residues are unsaturated and as much as 6%
of the membrane dry weight consists of sterols, mainly
ergosterol (Hunter and Rose, 1971). The synthesis of
unsaturated fatty acids from saturated acids requires an
oxygen-dependent desaturase enzyme and the synthesis of
ergosterol from squalene requires oxygen-dependent
peroxidation and demethylation reactions (Henry, 1982).
When cultured under anaerobic conditions, S. cerevisiae
exhibits a requirement for both unsaturated fatty acids and
ergosterol in its nutrient supply for growth (Andreasen and
Stier, 1953; Andreasen and Stier, 1954).
Taking advantage of this anaerobically induced
nutritional requirement, the laboratory group of Rose
selectively enriched plasma membranes of S. cerevisiae by up
to 60% with an individual unsaturated fatty-acid or by up to
70% with a particular sterol (Hossack and Rose, 1976).
Using yeast with altered plasma membrane lipid composition,
they showed that cells enriched in ergosterol and linoleyl
residues remained viable for a longer period of time than
cells enriched in other sterols and oleyl residues when
exposed to ethanol (Thomas et al.. 1978). Ethanol was also
less inhibitory to growth and solute accumulation when
plasma membrane lipids were enriched similarly (Thomas and
Rose, 1979). Because unsaturated fatty acids and
ergosterol appear to protect yeast cells from the inhibitory
effects of ethanol, it is not surprising that S. cerevisiae


o
FERMENTATIVE ACTIVITY
en
O
ro cj ^
o o o o
NJ
(J1


80
Discussion
The decline in fermentation rate that begins at low
alcohol concentrations does not seem to be exclusively-
caused by the immediate presence of ethanol, by growth in
the presence of ethanol or by cell death (Chapter III).
This decline appears to be related in part to a magnesium
deficiency, although other factors are involved also. In
yeast extract-peptone-based medium, a magnesium deficiency
is developed which limits cellular growth and the rate of
carbohydrate conversion into ethanol. The addition of
magnesium to batch fermentations prolonged exponential
growth, allowing greater accumulation of cell mass without
affecting cell viability. In addition, cells in magnesium-
supplemented cultures maintained a higher fermentation rate
as ethanol accumulated. These two factors, increased cell
mass plus higher fermentation rate, combined to reduce the
time required for the conversion of 20% glucose to ethanol
by one-third in magnesium-supplemented cultures.
During batch fermentation, yeast cells concentrated
magnesium from the medium. In unsupplemented cultures,
magnesium uptake stopped at the end of exponential growth.
At this point, the concentration of magnesium in the medium
was 48 pM, within the range of values reported for
magnesium transport by microorganisms (Jasper and Silver,
1977). The end of exponential growth also coincided with
the beginning of the decline in fermentative activity. In


(Kunkee and Amerine, 1968; Moulin et al.. 1980). A small
amount of oxygen increases the ethanol tolerance of S.
cerevisiae because it is required for the synthesis of
unsaturated lipids and ergosterol (Henry, 1982). These
lipids protect anaerobically grown yeast cells from ethanol-
induced growth inhibition (Ingram and Buttke, 1984). Under
continuous culture conditions, trace amounts of oxygen were
shown to decrease the ethanol inhibition of growth without
significantly affecting the ethanol yield per amount of
substrate consumed (Hoppe and Hansford, 1984).
Because strain differences and environmental factors
influence the ability of yeast to grow and ferment in the
presence of ethanol, it was necessary to characterize these
processes in the organism chosen for this study. This
organism was a genetically undefined petite brewery yeast,
S. cerevisiae KD2. Decreasing growth and alcohol production
rates as ethanol accumulated during batch fermentations were
characterized. Also, the effect of ethanol added to the
growth medium on the rates of growth and alcohol production
was examined.
Materials and Methods
Yeast Strains
The principal organism used in these studies was S.
cerevisiae KD2, a petite mutant of strain CC3 (G.G. Stewart,
Labatts Brewery, London, Canada). It was derived from the
parent strain by selection for the inability to form


52
reduced the error introduced by continued ethanol production
during cell harvesting. However, cooling may introduce
other potential problems associated with temperature-
induced changes in the organization of and permeability
properties of the plasma membrane.
The second type of experimental problem associated with
measurements of internal ethanol involves washing of the
yeast cells. Experimental designs which included washing of
cells (Nagodawithana and Steinkraus, 1976; Panchal and
Stewart, 1980) before estimation of ethanol resulted in
lower apparent intracellular ethanol concentrations than do
unwashed samples (Beaven et al.. 1982; Dasari et al.. 1984).
Beaven et al. (1982) clearly showed that even minimal
washing leaches most of the intracellular ethanol from the
cells. These two experimental designs, measurement of
ethanol in a metabolically active cell pellet and washing,
each introduce errors which change the calculated values of
intracellular ethanol in opposite ways.
Recent studies by Guijarro and Lagunas (1984) have
employed a procedure which eliminated these two basic
problems in experimental design by using glass fiber filters
to rapidly harvest cells. With this method, extracellularly
added [14C] ethanol rapidly equilibrated with the
intracellular environment, indicating that the plasma
membrane is freely permeable to ethanol. However, this
still does not answer the question of the true intracellular