CONTINUOUS FERMENTATION OF GRAPE JUICE FOR THE

PRODUCTION OF FORTIFIED SWEET WINE

Thesis submitted for the Degree of

Master of Science

of

The University of New South Wales

by

Robert Leland Cootes, B.Ag. Sc. (Adelaide)

February 1977

DECLARATION

The candidate, Robert Leland Cootes, hereby declares

that none of the work presented in this thesis has

been submitted to any other University or

Institution for a higher degree.

Robert Leland Cootes

ACKNOWLEDGEMENT

i i i

I wish to express my gratitude and appreciation to Dr T.H. Lee, Senior

Lecturer, School of Food Technology, and to Dr P.L. Rogers, Senior

Lecturer, School of Biological Technology (SBT), University of New South

Wales (UNSW) for their advice and guidance during the course of the

research work and on the preparation of this thesis.

I am also indebted to the Directors of S. Smith & Son Pty. Ltd. for

granting study leave and support, which enabled me to carry out this

work.

The assistance of Dr M. Dickson, Head, Biomedical Electron Microscope

Unit, University of New South Wales, Mr R. Doble, Professional Officer,

School of Biological Technology (SBT), Mr S. Preece, Photographer, School of

Chemical Engineering is also gratefully acknowledged.

For translations from Russian I am indebted to Mrs Frieda Guerman,

School of Food Technology, and from German, Tom Babij, School of

Biological Technology, UNSW.

I also wish to thank Mr J. Baker and his staff at S. Smith & Son Pty. Ltd.,

N.S.W. branch, and Chris Smitham for typing the final manuscript of this

thesis.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS iii

TABLE OF CONTENTS iv

SUMMARY v”

1.. INTRODUCTION ix

2. LITERATURE REVIEW 1

2.1 Production of Fortified Sweet Wines 1

2.2 Continuous Fermentation 3

2.21 Theory of Continuous Fermentation 3

2.22 Internal and External Recycle in Continuous Fermentation 7

2.23 Multistage Continuous Fermentation in the Wine Industry 8

2.24 Continuous Fermentation in the Wine Industry 18

2.3 Review of Wine Yeast for Wine Production 24

2.31 Important Characteristics of Yeast in Winemaking 24

2.311 Cytology of Yeast 26

2.312 Hybridization and Mutation in Yeast 28

2.313 Energy-yielding Mechanisms in Yeast 28

2.314 Inhibitors of Yeast and Lactobacilli 29

2.315 Yeast Viability 29

2.316 Selective and Differentiating Solid Media for Yeast 31

2.317 Effect of Vitamins on Yeast Activity 31

2.318 Factors Affecting the Rate of Wine Yeast Fermentations 32

2.3181 Ethanol Concentration 32

2.3182 Yeast Concentration 34

2.3183 pH and Temperature 34

2.3184 Yeast Species and Strain 35

2.3185 Agitation and Carbon Dioxide 36

2.3186 Sugars and Yield of Ethanol 36

2.32 Role of Oxygen and F’gvour Formation in Quality WineProduct^ 3g

V

2.33 Acid Metabolism and Some By-products of Fermentation 42

2.34 Control of Wild Yeast and Bacteria in Fermenting Musts 45

3. EQUIPMENT, MATERIALS AND METHODS 48

3.1 Continuous Fermentation Equipment 48

3.11 Fermentation Vessel 48

3.12 Mixing and Pumping Must 50

3.13 Temperature Control of Fermentation 50

3.14 Constant Parameters pH and Oxygen 52

3.2 Materials 54

3.21 Yeast 54

3.22 Grape Juice 55

3.23 Alcohol Fortification 55

3.3 Methods 57

3.31 Yeast Growth and Preparation of Inoculum 57

3.32 Preparation and Preservation of grape juice 58

3.33 Sterilization of Fermentation Vessel 59

3.34 Estimation of Dry Weight of Yeast 60

3.35 Estimation of Yeast Cell Concentration 60

3.36 Estimation of Total Soluble Solids of Fermenting Must 60

3.37 Estimation of Ethanol Concentration of Fermenting Must 61

3.38 Calculation for Fortification of Fermenting Must 63

3.39 Stabilization of New Wine 63

4. RESULTS 65

4.1 Preliminary Experiments in Continuous Fermentation of GrapeJuice for the Production of Fortified Sweet Wine 65

4.11 Removal of S0^ from Grape Juice 65

4.12 First Preliminary Continuous Fermentation Experiment 66

4.13 Second Preliminary Continuous Fermentation Experiment 67

4.2 Semi-continuous Grape Juice Addition to the Fermenter forthe Production of Fortified Sweet Wine 70

vi

4.3 Continuous Fermentation using Different Strains ofS. ceA2.v4Aajolq. 76

4.4 Control Procedures in Single-stage Continuous Fermentationof Grape Juice for the Production of Fortified Sweet Wine 86

5. DISCUSSION AND CONCLUSIONS 96

6. APPENDIX 102

6.1 Glossary 102

6.2 Symbols 103

6.3 Sample Calculation for Ethanol Production 104

6.4 Schematic Representation of Grape Juice preparation for aContinuous Fermenter 105

.6.5 Removal of Sulphur Dioxide from Grape Juice 106

6.6 Schematic Diagrams of Five Continuous Fermenters(Peynaud, 1967) 108

6.7 Derivation of Productivity Formula 109

6.71 Yeast Productivity 109

6.72 The Ethanol Productivity of the 1 Litre Fermenter 109

6.8 Biomass Determination for Yeast Strain 505 110

6.9 Preparation of Yeast for Electron Microscopy 111

6.10 Identification of Wild Yeast Types 111

6.11 Key to the Yeast Genera 113

6.12 Calculation of Correlation Coefficient 119

7. BIBLIOGRAPHY 120

Fortified sweet wine is made from grape juice in a 1 litre continuous

fermenter. Particular attention is paid to techniques which will be

suitable for commercial application.

The rationale for this study is discussed in the introduction, which

is followed by a review of multistage and single-stage continuous

fermenters in use in four major grape growing areas of the world.

In preliminary experiments the 1 litre laboratory fermenter is fitted

with continuous fortification equipment, and some fermentation control

variables are investigated. Yeast growth rates are between 0.028-0.093 lhr” , and fermenter productivity, as an index of fermentation rate,

is inhibited by high levels of SO^ and ethanol.

A comparison of continuous and semi-continuous grape juice addition

to the fermenter shows that additions of grape juice, twice daily,

do not lower the productivity of the fermenter significantly.

A 10-36% increase in ceil concentration is achieved with a flocculent

yeast strain in the fermenter, so that the specific cell count equilibrium

for a particular yeast strain can be maintained under semi-continuous

grape juice addition.

Five strains of SacchaAomyceA c-OJiAvlsiaa are compared in the 1 litre

fermenter, and wide variations in cell count, growth rate and sensory

quality are apparent.

viii

The fermenter productivity for each of the strains 729, 350, 522, B19

and 505 is 2.5, 1.2, 2.6, 2.7 and 3.4 g EtOH/l/hr respectively. Thirty

six wines have been made in the experiments where yeast strains are

compared, and all wines have been judged commercially acceptable.

The. ethanol concentration in the 1 litre fermenter can be controlled by

varying dilution rate and temperature. For example, a 5°C drop in

fermentation temperature reduces the fermenter productivity by 36%

for strain 729. Fermenter productivity increases 65% following a

change in dilution rate from 0.04 to 0.08 hr"\

Cell growth rate is significantly affected by cell concentration,

rapid temperature changes and ethanol inhibition. Yeast production of7

ethanol per cell per second is between i.4-25x10 molecules EtOH/cel1/sec

for a wide range of the above variables.

The wine making process is discussed in relation to the criterion of

optimum fermenter productivity which often may not be greatly manipulated

for a raw material, such as grape juice. The importance of choosing

economic and appropriate automatic, industrial control systems is also

discussed.

Details of experimental methods are presented and a bibliography of

164 references is listed; 22 figures and 19 tables are included.

ix

]. INTRuDUCTION

Economic and industrial labour considerations have initiated the

redesigning of an Australian wine cellar, which produces 50,000 hi of

fortified sweet wine annually. The cellar was built prior to 1935 and

has operated since then with only minor modifications. For the purpose

of this study, double this production is expected in five years without

a substantial increase in labour input or fermenting capacity. The

tanks that are to be installed to increase the storage capacity must

also be capable of fermenting grape must.

It appeared that some of these requirements could be satisfied by

fermenting grape must in a continuous system. The primary function of

a continuous fermenter is to establish a high yeast cell concentration

in large quantities of fresh must. This enables a higher rate of

fermentation, because the total number of substrate "action sites" is

increased. The production capacity, of the system is greater than

conventional batch fermenters, and therefore the capital outlay is less.

The system is also suited to automatic control, and requires less

operational labour. Furthermore it appeared feasible that good quality-,

fortified sweet wine could be made by continuous fermentation techniques.

The project was designed to provide temperature and dilution rate data

necessary for controlling yeast activity during the production of a

commercially acceptable fortified sweet wine. Similar criteria could

then be used to compare continuous and semi-continuous addition of grape

juice to the fermenter. The fermenter productivity (g EtOH/l/hr) was

to be measured in each experiment with the aim of determining which

yeast fermented grape juice most rapidly under experimental conditions.

X

No attempt was made to create experimental conditions which could not

be achieved or would not be encountered in a corrmercial winery.

Furthermore, no attempt was made to compare batch or continuous

fermentation techniques from a qualitative or economic point of view.

1.

2. LITERATURE REVIEW

*2.1 Production of Fortified Sweet Wines

Before 1967 more fortified wine was produced in Australia than table

wine, but since then the production of table wine has increased more

than threefold, while fortified wine has not shown the same dramatic

increase (Table 1). In 1975, however, the production of fortified

wine and wine for distillation was 1.85 x 106 hi or 52% of Australian

wine production.

Table 1. Australian wine production statistics (from the Australian

Wine Board annual report 1974-75).

Vintage Table wines (including sparkling)

Sherry, dessert and flavoured wines

Wine for Distillation

Total wine production

Average 1945-49 0.09

x 106 hi

0.46 0.69 1.24

Average 1950-54 0.13 0.53 0.75 1.41

Average 1955-59 0.19 0.42 0.70 1.31

Average 1960-64 0.28 0.45 0.83 1.56

Average 1965-69 0.54 0.51 0.86 1.91

1971 0.83 0.50 1.17 2.50

1973 1.18 0.54 0.94 2.66

1975 1.68 0.69 1.16 3.53

Grapes which mature to greater than 22° Brix without weight loss are

preferred for the production of fortified sweet wine. In Australia,

the majority of these grapes are grown in hot climates under irrigation

and include the following varieties:

Muscat Gordo Blanco (Muscat of Alexandria), Brown Muscat, White and Red

Frontignac, Pedro Ximenez, Palomino (Listan), Doradillo, Muscadelle

(incorrectly called Tokay) (Antcliffe, 1976), Sultana (Thompson Seedless),

2.

Semi lion, Grenache, Shiraz and Cabernet Sauvignon. The pH of the finished

wine should be less than 3.4; all of the above varieties can reach high

°Brix and acceptable pH values in good seasons in most grape growing regions

in Australia. Most of these areas, however, often have poor ripening

conditions except for microclimates similar to the Rutherglen and Glenrowan

districts in Victoria. The grapes grown in these districts consistently

achieve greater than 30° Brix without fungal spoilage. Poor quality grapes,

such as those that are rain damaged, low in sugar or badly infected with

downy mildew (Plasmopara sp.), are eventually diverted to wines for

distillation. Crushing and destemming is usually effected with Whitehill

(Garolla type) crushers having capacities up to 100 tonnes/hr. Sulphur

dioxide is always added at the crusher so that the must contains about

100 mg SO^/l. After the addition of 1-5% of yeast starter culture, the

free run juice and pomace are separated by a coarse filter or screen under

gravitation. The pomace can be fermented dry, and the pressings diverted to

wine for distillation to produce fortifying spirit. For the production of

fortified sweet wines, fortifying spirit that must be derived from the

distillation of fermented grape materials is added after fermentation has

lowered the hydrometer reading to about 14.5° Brix. Addition of neutral,

high ethanol spirit to a level of 20% ethanol by volume gives a new hydrometer

reading of about 7° Brix. The fermentation does not stop immediately, however,

and wines are usually reblended prior to preparation for sale, because sweet

cr brown sherries marketed in Australia generally range from 6° to 8° Brix

while liqueur rnuscats are as high as 11° Brix.

3.

2.2 Continuous Fermentation

2.21 Theory of Continuous Fermentation

The concept of continuous fermentation was documented by Semichon (1926),

but the original papers on the theory of continuous fermentation were

published by Monod (1950) and Novick and Szilard (1950 a,b).

Continuous fermentation has now gained wide acceptance as a research tool

and, to a lesser extent, as an industrial process. The most common form

of continuous fermentation vessel consists of a backmix reactor in which

the organism can be grown under the required conditions. Sterile growth

medium is fed continuously into the vessel, and fermented broth,

including the organism (culture), emerges from it at the same rate; the

volume of the vessel is kept constant by an overflow device. As with

all backmix reactors, perfect mixing is assumed, so the culture emerging

is homogeneous with the culture in the vessel and the growth medium

entering the vessel is instantly dispersed. One advantage of continuous

fermentation vs_ batch fermentation for the study of microbial populations

is that growth is occurring under steady state conditions. In batch

fermentation, the levels of substrates and the concentration of cells arid

end products are constantly changing throughout the growth cycle of the

cells. In a continuous fermentation system, however, growth rate and

cell and substrate concentrations can be controlled. Hence the organisms

grow at steady state in a controlled environment under the desired

experimental conditions.

Steady state refers to the culture as a v/hole and although each cell is

in a different stage of growth and metabolic activity, a large number of

cells in a homogeneous environment ensures that sugar uptake and carbon

dioxide and alcohol production remain constant.

4.

The basic theory of continuous fermentation has recently been reviewed

. by Fiechter (1973), Pirt (1975) and Aiba, Humphrey and Millis (1973) and

will be discussed briefly here. Full reviews on all aspects of continuous

fermentation, including its industrial applications, have been published

by Ricica (1973, 1974).

The ratio of the flow rate (F) into and out of the vessel to the volume

of the vessel (V) is defined as the dilution rate (D), which is the

inverse of the mean residence time (tr) of a particle in the vessel,

D = £ = 1 . The rate of washout of the organism from the vessel, if V tr

growth ceased but flow continued, is given by the equation

dx ~ - Dx dt

where x is the concentration of organisms in the vessel.

The growth of micro-organisms is described by the equation

dx = /jx dt

where /j is the specific growth rate of the organisms. Monod (1942)

determined a relationship between the specific growth rate and the

concentration of the essential growth substrate /j = /Jm (^—--)

where un is the maximum specific growth rate, s is the substrate

concentration and Ks is a saturation constant numerically equal to the

substrate concentration at which jj = h /%• For a culture growing in a

continuous fermentation vessel with all substrates in excess, except

a single organic substrate (such as glucose), we can write the following

mass balances,

dx = ;ux - Dx dt

dx = x Corn (tA-.)-D) (Vdt

where s is the concentration of the growth limiting substrate. A mass

balance for the growth limiting substrate can also be written,

5.

ds = D (s0 - s) - px (2)It Y

where sQ is the concentration of substrate in the incoming feed and Y

is the yield constant, defined as the weight of organism formed, divided

by the weight of substrate used over any finite period of growth. If

s0 and D are held constant and D is less than ,um, then values of x and

s exist such that the system is in steady state, ie.,

dx = ds = o (3)dt dt

These steady state values are calculated by solving equations (1) and (2),

x = Y(sq -s) = Y(sq-Ks)

From the mass balance of cells,

dx = jux-Dx "dt

it can be shown at steady state dx = o, that jj = D, ie., the growth rateeft

is equal to the dilution rate.

(4)

(5)

This provides the basis for chemostat control of continuous culture,

where the dilution rate and hence the growth rate is determined by the

addition of medium at a constant rate. The dilution rate must be less

than the maximum growth rate of the organism or washout will occur.

A second important type of control of continuous fermentation is

turbidostat control, in which the cell density of the culture is

monitored in an optical density cell and compared with a set point; as the

organism grows and exceeds the set point, the medium inflow increases

and dilutes the culture, reducing the optical density until the set point

is again reached.

In chemostat control then, the growth rate is fixed and x and s find their

steady state values (u<um due to substrate limitation), whereas with

turbidbstat control x is fixed and p and s find their steady state

values (ju = junl).

The third important type of control of continuous fermentation is product

inhibition of fermentation rate; in wine fermentations ethanol is the

principal product which causes non-competitive (with substrate) inhibition

of the growth of yeast. The growth rate of the organism,

where is the product inhibition constant and P is the product formed,

hence,

Both chemostat and product-!imited control models are applicable to wine

fermentations. In secondary fermentations for the production of sparkling

wine, yeasts with high alcohol tolerance are used and their growth rate

can be substrate limited in a single-stage backmix reactor. However,

in a fermentation for the production of fortified sweet wine growth rate

can be more closely modelled to the product limited system.

The theory of two-stage and multistage continuous fermentation in a product-

limited system has received little attention, and there are no kinetic

data available in the literature. In a chemostat, however, the model is

more simple; for a combination of two identical, homogeneously stirred

chemostats, where the outflow of stage 1, with a concentration of cells x^,

is connected to stage 2 with a concentration x^, the dilution rate no

longer represents the specific growth rate,

e.g., 02 > p?

7.

°2 = >'Z 5

and x2~xl> 1

Thus cell concentration in the first stage can never exceed that of the

latter stages. A multistage system also offers the opportunity to grow

cells in the presence of different amounts of substrate. For example,the

principal sugars in grape musts, glucose and fructose, each have

different rates of utilization by SacckaAomLfceA ctn.zvibicui, and in a

three-stage fermentation, Ough and Amerine (1968) observed a 5%

reduction in fermentation rate (g Et0H/100 ml/hr) between the first

and second stages, despite an increase in viable cell count, and a

further 35.8% in the third stage (Table 2).

A theoretical comparison between batch and continuous fermentation is

useful and the ratio

continuous output (chemostat) = In (y01) + — batch output 0

where x0 is the initial cell concentration, and xm is the maximum cell

concentration in a chemostat. If the time taken (ta) to sterilize and

fill a fermenter is zero, and the yield for a specific organism remains

unchanged, then continuous output = 2.3 batch output. The derivation

and a discussion of this equation is given by Aiba, Humphrey and Mill is

(1973) and Pirt (1975).

2.22 Intprnal and External Recycle in Continuous Fermentation

Pirt (1975) made a kinetic study of three methods of internal recycle

to increase the cell concentration in a fermentation vessel. Firstly,

a filter or screen may be placed over the culture outlet tube, such that

the liquor can pass through without obstruction from cells in the vessel.

8.

Secondly, an organism may be selected that will adhere to sedimentation

baffles in the vessel thus forming a heterogeneous mixture, and finally,

a tall, tower-like fermentation vessel may be used so that only the

bottom portion of the vessel is homogeneously mixed, and the top portion

has a lower concentration of cells due to gravitational settling. The

latter system is more effective if the organism flocculates strongly.

External recycle is achieved by mechanically separating the cells from

the liquor in a centrifuge or sedimentation tank, then pumping a portion

of the concentrated suspension of cells back into the fermentation vessel.

Both separation systems are potential, microbiological contamination

points, and this separation must occur before alcoholic fortification

or cell viability would be reduced. The wine is therefore vulnerable to

oxidation and aerobic spoilage by AceXobacXeA 4pp. and aerobic yeasts,

which would be present in the grape juice but unable to grow during

primary fermentation, because of a low oxidation-reduction potential.

Finally, the installation of an automatic centrifuge or sedimentation

tank is expensive, and the advantages must be carefully evaluated in

terms of improving the productivity of the system as a whole.

2.23 Multistage Continuous Fermentation in the Wine Industry

Multistage, continuous fermentation for the production of sparkling

wines was recommended by Russian scientists Brokhorov and Alekandrovskii

(1952), Kozenko (1953a,b) and Brusilovskii (1957). Further work on

these production techniques by Preobrazhenskii (1961, 1962), Giashvi1i

(1961), Koshev (1961) and Brusilovskii (1963 a,b) was reviewed by

Amerine (1959, 1963). In 1962 the United States of America (U S A)

Patent Office granted Patent No. 3,082,653, entitled "Method of

champagnizing wine in a continuous stream and installation for same",

to Gerasimovich, Merzhanian ari Srusilovskii. The method involved the

9.

continuous addition of base wine and yeast culture through six

hermetically sealed constant pressure tanks, with continuous liqueur

addition after fermentation. The fermenting wine, covered with a

layer of deodorized paraffin oil was held at 14°C initially,

gradually cooled to 9°C in the sixth tank and finally stored at -5°C.

The authors claimed that this process improved productivity without

loss of quality compared to batch fermentations.

Gilyadov (1968) compared continuous and semi-continuous grape juice

addition to a five compartment tower fermenter so that equivalent

dilution rates were achieved by dosing at 0, 1, 3, 6, 12 and 24 hour

intervals. The cost of pump operation was reduced if impulse addition

was adopted, however, as the interval between impulses increased, and

the volume of each dose increased, the rate of fermentation was

reduced. Therefore an equation relating productivity of the installation

Qa, the output of the pump Qp, the number of impulses during 1 hour n

and the time required for each pumping t was tested,

t = Qa.60 ■ m Qp.n .

The equipment described achieved optimum productivity if the number of

impulses per hour n = 12, but no other variables were given. A three

stage system for the continuous fermentation of grape juice,

incorporating modern control equipment necessary for precise operation,

was investigated by Ough and Amerine (1968). The alcohol production

rate and viable yeast count data are shown in table 2. The yeast counts

are similar to those given by Giashvili and Alkanashvili (1971) from a

multistage system and ethanol production rates of 0.88 - 1.55 g EtOH/l/hr

given by Flanzy <Lt at. (1968) for a single-stage continuous

fermentation at 25°C.

Table 2. Alcohol production and live yeast cell concentration in the

three stages of a continuous fermentation system at 21 °C (Arnerine and

Ough, 1968).

Stage EtOH production (g/1/hr)

Viable yeast count (cells/ml x 106)

1 1.03 57.9

2 0.98 80.3

3 0.63 64.5

Martakov (1970) prolonged fermentation by adding fresh grape juice

continuously to fermenting must to maintain the must pool at 8 - 10% v/v

alcohol. Even though the must pool spread to further tanks, the complete

must pool was circulated through each of the vessels in the pool at

regular intervals. Fresh grape must, and theoretically the yeast,

remained actively fermenting for long periods:, consequently each cell

had a high alcohol productivity. The uptake of nitrogenous compounds

from the grape juice was 25-30% less than during conventional

fermentation. Similarly the total yeast biomass was 35% less due to

limited cell reproduction. The oxidation-reduction potential of the

must (E^) was found to be significantly lower and the level of

reductones significantly higher. Reductones were defined by Paul (1963)

as constituents of wine which have reducing properties with respect to

iodine. Martakov (1970) also claimed that the prolonged wine contact

with yeast increased the rate of aging and flavour development, and he

concluded that such prolonged fermentation with the continuous addition

of grape juice was necessary for a good quality wine.

The improved productivity of a multistage, continuous fermentation for

11.

the production of dry wine is reported by Laszlo (1969) and Giashvili

•and Alkanashvili (1971). Within a battery of 12 tanks Giashvili (1961)

made three arbitrary groups to explain varying yeast behaviour. In

the first section, I, there was an accumulation of yeast and 42% of

cells were budding, compared with a maximum of 16% budding in the

third section (III). Desulphitation also occurred in I, while in II,

there was less yeast growth, but higher ethanol productivity. For

example, in stage 1 the average ethanol produced per cell was 1.19 x 10^

molecules of EtOH/cell, and in stages 5 and 11 it was 1.8 and 0.1 x 10^

respectively (Appendix 6.3). However, the respective cell concentrations

in the example given are 38, 71 and 52 x 10° cells/ml, arid the author

gives no explanation for the disappearance of cells from stages 3, 4

and 5. The data presented showed a total acid increase of 0.5 g/1 bet­

ween stages 1 and 5, which was consistent with the observations of

other workers, who have shown an increase in total acid and a corres­

ponding decrease in pH in continuous compared to batch fermentation.

For the duration of the experiment, a total of 15 days, there were no

spoilage organisms reported; however, the author obviously had previous

experience with contaminants, since he stated that the increasing

ethanol concentration depressed contaminant activity so that it did not

influence the quality of the wine. A further observation was made in

that high alcohol and low conditions allowed the selection of yeasts

from successive generations that were more productive than their

parents, but no productivity data was presented to support this claim.

Finally section III contained the highest percentage of yeast in the

stationary phase and dead condition - supporting data was presented.

An extensive chemical and organoleptic analysis for 18 different

experiments was given, including SG, total acid, volatile acid, tartaric

acid, pH, total S02, invert sugar, glycerin, extract, ash, alkalinity,

12.

tannin, total N, P and Fe. The descriptive organoleptic evaluation

was generally favourable and contained some interesting remarks,

for example "reminiscent of European style wine", however, no score

statistics were presented.

Buryan oX at. (1973) presented data to support previous literature

claims that wines made by continuous fermentation under CO2 pressure

were low in E^, higher in reductones and contained less higher alcohols

than wine made by conventional methods. They first showed that

temperature and CO2 could be used to control the specific growth rate = In m? - In mi

F t2 - ti ' ’where mi is the biomass at the beginning and m2 the biomass at the end

of a time period t£ - t^. An over-pressure of 0.5 atmosphere of CO2

was found to limit cell viability, and the pressure was reduced to 0.1

atmosphere for a period of 1 - 2 hr. A reduction in temperature from

25 to 20°C required a pressure decrease to 0.2 atmosphere to achieve a

constant fermentation rate, and continuous stirring improved the

fermentation rate by 150%. The optimum conditions chosen for industrial-1application were a dilution rate of 0.065 hr , continuous stirring at

25°C and incoming grape juice, which had been adjusted to 17% soluble

solids, containing 60 mg/1 free SO2. The CO^ over-pressure chosen at

the Anapa (SSSR) installation was not stated clearly, but the

conclusions drawn from industrial experimentation were that in a

battery of six fermentation tanks in series, the wine produced after

six months aging was better than wine made by the usual method.

Organoleptic evaluation showed an improvement of 3-5% in scoring for, c <( ere

the new wine, although no duplicate data was given. No data was presented

supporting the claim that wine made by the new method contained a lower

concentration of higher alcohols. However, the improved wine quality

13.

was attributed to a lower E^ and higher reductone level, which

, protected the wines from oxidation.

Equipment for continuous fermentation of dry wines (Kovalevsky,

Anoshin and Orehanov, 1973) consisted of eight fermentation tanks

connected in series and surmounted by a horizontal trough which

contained a double archimedian screw; the trough was 24m long and

held marc, but allowed liquid to pass through a perforated screen

into a receival tray. By means of CO2 pressure from fermentation,

fermenting must was intermittently irrigated over grape skins in

the double screw chamber and collected in hermetically sealed fer­

mentation tanks below. The mechanical characteristics of the system

were given in detail, but no data was presented on the quality of

wine produced or in fact if the system was used commercially.

Filippov and Dzhurikyants (1974) discussed a projected apparatus for

the continuous manufacture of champagne, which was installed in 1973

at Odessa's factory, with a projected output of 2.5 x 10^ dozen

bottles or 1,87 x 10^ hi/yr. The apparatus contained between 50 and

60 vessels of varying capacity for storage, stabilization and

continuous control of secondary fermentation; the residence time

for a.quantity of wine was from 3-12 months. A fish glue and

bentonite were used in a jet reactor to increase the reaction efficiency

of agglutination of proteins before continuous sedimentation,

centrifugation and filtration into an enzyme reactor, however, no

specific components were mentioned. Avakiants and Belousova (1965)

reported that in continuous fermentations adequate B - fructofuranosidase

was available for sucrose inversion. Half of the wine was sterile

filtered before liqueuring and yeasting, and the other half was

pasteurized by heating to 60°C, bolding for 48 hr, then cooling to

14.

10 - 15°C, before liqueuring and yeasting. After fermentation the

,wine was stored at 15 - 25°C for 3-12 months then subjected to

heat treatment in a heat exchanger between 40-60°C under 1.6

atmosphere pressure, cooled to -5°C and mixed with an expedition

liqueur before bottling. Dzhurikyjants dt at. (1974) discussed the

detrimental effect of high boiling point (HBP) alcohols on the

quality of champagne made in a series of six vessels. They claimed

that autolysis of yeast in the sixth vessel improved quality and

after the disappearance of sugar, amino acids are the main substrate

for HBP alcohol metabolism; no suggestions were offered to show

how these two supposedly simultaneous effects on wine quality could

be separated.

The use of multistage, continuous fermentation under a flor yeast for

the production of sherry was described by Dzhanpoladyan dt at. (1974).

The conventional method consisted of three vessels two thirds full of

wine at 16% ethanol (v/v), and each six months one third of the wine

was removed from each vessel and replaced by fresh wine or wine from

the proceeding vessel. The final stage, having a larger capacity,

allowed a residence time of up to ten years for any particular wine.

A continuous manufacturing process was then described in which the

first five vessels contained wine under a flor film, and the last two

contained wine on an autolyzed bed of yeast. Table 3 contains some

of the duplicate data comparing the batch method, the continuous flow

method under flor yeast and the continuous flow under flor with

incubation on autolyzed yeast.

Table 3. Chemical and organoleptic evaluation of flor sherry

(Dzhanpoladyan zt at., 1974).

TA* (9/1)

V A*(g/i)

A1dehydes (mg/1)

Esters (mg/1)

Wine yield(i)

Organolepticscore

Batch 3.8 0.53 486 152 3.3 8.4

Continuous 3.6 0.36 574 132 12.5 8.3

Continuous with autolyzation

3.5 0.34 677 166 14.5 8.5

*VA = volatile acidity, TA = titratable acidity

The flow rate of sherry through the continuous system was increased to

give a residence time of two weeks, before the quality fell below that

of the wine produced by the batch system. A further improvement to the

system was claimed by circulating the autolyzate and base wine through

a jet reactor for 20-180 min. This homogenization procedure increased

the acetal content by 40 mg/1 and esters by 13 mg/1 in 20 min, and if

the process was continued for 80 min, the organoleptic ratings improved

by 6%. During prolonged treatment of this kind for three hr, the pH

dropped from 3.7 to 3.54 and the E^ increased from 230 to 310 mV, but

no explanations were given.

Arnerine (1958) and Ough and Amerine (1958) have made semi-continuous

fermentation runs of up to two months with submerged flor cultures.

The system was a true continuous fermentation, though automatic control

of pH and redox potential was not attempted. The experiments on the

submerged culture of a flor-type yeast S. bztlcus employed various

combinations of temperature, rate of gas flow, pressure and agitation;

16.

aldehyde contents of up to 1,000 mg/1 were reported. Acetic and malic

acids, glycerine and alcohol were shown to be carbon sources for the

growth of the yeast. Stirring the fermenter for 25 seconds each hour

and maintaining an air flow of 0.1 litres/min through it gave excellent

yeast growth, and the aldehyde content of the product was maintained

at greater than 500 mg/1. This process was more successful, however,

when conducted on a semi-continuous basis by removing one tenth of

the contents daily. The aldehyde build-up over the period of the

experiment was at least twice as fast as that achieved in the batch

process.

Steinkraus (1972) achieved a rapid fermentation using a flor sherry

yeast, Saccha/iumijcdb ovZ^omu> strain 31, in a column constructed for4

continuous production. Under these conditions the yeast produced 10

molecules of acetaldehyde/cell/sec on a 10% ethanol wine substrate.3 7Although rates of acetaldehyde production that range from 10 to 10

molecules/cell/sec have been observed, the rates are sufficiently

stable for any given set of conditions to permit application of the rates

to the design and manufacture of continuous sherry fermenters (Steinkraus,

1972).

Brusilovskii, Gagarin and Sarishvili (1974) described a new apparatus

for the continuous secondary fermentation of wine for sparkling wine

production which involved a single, cylindrical vessel with concentric,

cylindrical partitions so that each annulus contained the same volume.

The same apparatus was the subject of USA Patent 3,916,775

(Merzhanian oX at., 1975). The vessel was jacketed with a coolant so

that the temperature of fermentation gradually decreased as the wine

moved from the centre to the outside of the vessel through each of seven

17.

cylinders, entering from the top then bottom consecutively. The

«system reduced the quantity of fermenting must remaining in

stationary pockets due to imperfect mixing, compared to that often

encountered in the more conventional series of connected pressure tanks.

The apparatus was also more compact, used comparatively less floor

space, and was more easily maintained and cleaned because there were

fewer pipes and valves. Data was presented to show that there was no

significant difference between the sparkling wine made by conventional

techniques and the new method for sugar, alcohol, E^, reductones,

aldehydes, SO2, VA, total acid total N and HDP algchols, however,

diacetyl was 50% lower in wine from the new apparatus. A three-fold

increase in the ratio of vessel surface area to wine was the main

reason advanced for the significant increase in the fermentation rate

in the cylindrical vessel. There was, however, no sensory evaluation

data given on the sparkling wine produced in this new pilot plant

compared to the conventional method.

Oreshkina at aX. (1974) discussed different techniques used in the

continuors production of tank-fermented champagne and presented data

comparing grape varieties and their contribution to total and amino

acid nitrogen in the resulting wines. The importance of low E^ and

the exclusion of oxygen was emphasized. It appears that high levels

of iron were a problem in some wines and detrimentally affected the

aerobic oxidation potential of the wine. Eight fermentation vessels

in series were recommended, and in the last two tanks wine passed

through ring shaped caps made of polyethylene to retain the yeasts

by adhesion. The wine was thereby enriched with yeast autolyzate,

and this was claimed to improve the flavour of the wine. Classical

and modern methods of sparkling wine production in Czechoslovakia

18.

have been summarized by Drobny (1976), and he indicated that the

, most recent innovation has been the single cylindrical vessel, with

several annular chambers, for the continuous production of both dry

and sweet (6% sugar) sparkling wines. For diabetics, sugar is

replaced with saccharin and sorbitol, and some low alcohol wines with

an added musk flavour are popular. Statistics are provided showing

that the per capita consumption of sparkling wine in Czechoslovakia

has increased from 0.036 in 1955 to 0.36 in 1975. In Australia in

1975 the per capita consumption of champagne and sparkling wine was

1.76.

2.24 Continuous Fermentation in the Wine Industry

The continuous fermentation process for grape juice has been used on a

commercial scale at Vie-Del Grape Products Company since 1949. This

operation was probably based on the Semichon (1926) 1Superquatre' concept,

which had aspects of a continuous fermentation, but contamination was

a serious problem as no methods were available to control the sterility

of the medium. The work showed that by starting the fermentation at

4% ethanol, undesirable microbial activity was virtually eliminated.

Riddell and Nuri (1958) reported that the product made by this process at

Vie-Del was uniformly good in quality, and the turnover time of the

fermenting room had been approximately doubled by changing over to the

continuous fermentation system. Toth and Tengerdy (1952), in a study

of the use of continuous fermentation for wine production, showed that

introduction of the must at a high sugar content and large draw offs

from the initial tanks, gave the highest rate of fermentation and yieldd

of ethanol. They also recommened removing 30% of the fermenter volume

in a batch-wise step each day. If smaller daily additions of grape

juice were made, yeast viability and fermentation rate were significantly

reduced.

19.

Cremaschi (1951) holds a USA Patent on a continuous fermentation

' process, which has been used extensively in Argentina for the

production of red wines. The fermentation tank contained a column of

grape must in progressively advanced stages of fermentation from

bottom to top of the column; skins and pulp from the crushed grapes

floated at the top of the column and seeds fell to the bottom. Crushed

grapes were fed into the bottom portion of the tank and subjected to the

fermenting conditions in the tank. The fermented cap of grape skins and

pulp were withdrawn from the top of the tank by an archimedean screw,

while wine was withdrawn from the uppermost portion of the column of

liquid and grape seeds and other solid matter were drawn from the bottom

of the tank. The process is commendable for its simplicity, however

the possibility that stratification occurred within the column was not

discussed.

Vlillig (1950) described how the above process operated in a winery in

Argentina. The fermenter capacity was 4,545 hi and consisted of a

cylindrical tower with a conical bottom. The tower was 9 m high,

7.3 m diameter and was made of reinforced concrete. At the top of the

unit were circular plows for removing skins. It was reported that an

improved product was being produced with less labour and in less space.

The single fermenter described replaced 30 small open vats that were

used in the batch process. In order to process half a tonne of grapes

the continuous system required 10 hi of fermenter space compared to the

36.3 hi required by the batch method. In the production of red wine,

it was claimed that growth of micro-organisms on the skins was

inhibited as they rose into the zone with the highest alcohol content;

thus, alcohol-tolerant yeasts underwent healthy, uniform fermentation,

and less SO^ was required.

20.

No data was presented to support these claims nor was there any

,indication of the alcohol level required to inhibit undesirable micro­

organisms.

Between 1950 and 1962 little information was published on continuous

fermentation for wine production, however, Amerine (1959) reviewed

several continuous fermentation systems including application to yeast

production (Maxon, 1955; Wiley, 1954; Beesch and Skull, 1956), to

brewing (Wellhoener, 1954; Dunn, 1955), and finally the problems of

automatic control of continuous fermenters (Field and Dunn, 1957;

Denison eX at. , 1958).

A continuous fermenter for making dry red wine has been described by

Ladousse (1962) which saved 45% of the fermenting room space. The

fermenter has been widely used in Europe and is now made in Argeliers

by Societe des ateliers Pujol (Appendix 6.6). The main fermenter consists

of two concentric vessels standing on a common base. The central

vessel is used for regulating temperature and for must removal, as it

is connected to the outer chamber by a perforated screen. The outer

vessel contains seeds at its base, and at the top dry, fermented skins

are removed by an archimedian screw. The vessels have been built with

between 5-150 tonne capacities and are fitted with in-place cooling

jackets. The depth of the cap can be adjusted by recycling fermenting

must over the cap or diverting it into new tanks, hence the residence

time of the marc cap can be controlled. In addition to the saving of

space and lower operating costs, an improvement in quality is claimed

due to the more rapid onset of fermentation by the cultured yeast, and

an earlier onset of the malo-lactic fermentation, which limits infection

by spoilage bacteria. An increase of 0.2% alcohol v/v was claimed as

a result of a lower yeast biomass and the elimination of the lag phase

21.

observed at the commencement of batch fermentation; furthermore the

volume of press wine extracted from the marc was reduced.

Tarantola and Gandini (1966) suggested that fermentation systems such

as the Cremaschi or the Defranceschi (Italy) were really "superquatre"

systems (Semichon, 1926), in that fresh must was introduced into a

dilute alcohol must, which was unfavourable to the growth of Ptclita 4pp.

and Kio&alizfia. 4pp. The fermentation was thus carried on mainly by

S. aoJitvt&iaa, with less frequent isolations of S. ttaLicu4, 5. ovtfiofimt4,

S. ahzvaJU..znt and S. bayanuA.

Guetov (1969) made comparisons between the regular fermentation

procedures for red wine and continuous fermentation. The composition

of the wines, the temperature of fermentation, and the sensory quality

of the wines were not found to be significantly different.

According to Re my (1967) only small quantities of SO^ should be added

to the grape juice prior to entering the continuous fermenter, because

the strong reducing conditions may lead to sulphide formation. Buryan

at. (1973) recommended a high level of 60 mg/1 of free SO^ in the

grape juice prior to continuous fermentation, but made no reference to

sulphide formation. Such a discrepancy may be explained by the use of

different yeasts, some of which are capable of producing sulphides from

sulphite (Wainwright, 1971). Flanzy oX at. (1966) pointed out that the

addition of sulphur dioxide causes the accumulation of acetaldehyde and

decreases the rate of alcoholic fermentation in a continuous fermenter -

this is similar to batch data (Weeks, 1969).

A combined report on continuous fermentation of red grapes in France

22.

described the performance of a number of Ladousse-, Vico- and Padovan-

type fermenters and one Defraceschi unit (Anon., 1970). The units

varied from 800 to 4,000 hi capacity, and fermentations were more rapid

than batch in each of the units. The alcohol concentrations in the

fermenter were from 6 -■ 8%. The wine, maintained at 50 mg/1 SO^, was

monitored for microbial growth, but no significant bacterial contamination

occurred and 85% of the yeasts were S. ceAdvZAZaa. Fermentations at

pH 3.3 resulted in wines with the highest sensory qualities. The main

advantages of the systems described were a reduction in fermentation

time and labour, since the centralization of the operation required

only one technician to operate the equipment. Some taste preference

was shown for wines made in the conventional way, but Brugirard,

Roques and Vignier (1970) considered that their products, derived from

the controlled temperature, continuous fermentation of red grapes,

were superior to those made conventionally.

Peynaud and Guimberteau (1969) observed that good quality table wines

could be made by continuous fermentation; the process benefited by the

choice of a suitable strain of SacchaAomyceA yeast, the rational use of

SO^ and the control of fermentation temperature. A lower level of

methanol resulted from the continuous process, and small increases of

about V/o in total ethanol content were measured. Further reports on

continuous fermenters, including some previously mentioned, and those

of Gelencser and Sarkany (1970) and Bajenov (1969) have been reviewed

by Amerine and Ough (1972) without any new information being presented.

Barre and Combe (1975) described a laboratory scale, single-stage

continuous fermenter, for the production of wine with automatic control

23.

of specific gravity of the fermenting must. Sterile filtered grape

* juice may have been used in the 1 litre fermenter, but this was not clearly

stated. The fermenting must was continuously stirred and the temperature

of fermentation was controlled at 25, 30 and 35°C during the experiment.

The limited data presented showed that a constant sugar concentration

was maintained by changing the dilution rate, despite changes in

temperature and cell viability. No fermenter productivity data or

sensory evaluation of the wines were given. The fermentation was run

continuously for nearly a month without microbial contamination, and at

each temperature, a steady state was achieved and maintained. The

authors emphasized the importance of direct, on-line determination of

sugar or ethanol concentrations in continuous fermentation and then

control by varying the dilution rate. The current method used for

controlling fermentation rate in batch fermentations is by temperature

variation. There is a time lag in fermentation rate response to

temperature variation, and control of dilution rate for regulating

fermentation rate during continuous fermentation is superior to

temperature control for mainti/aning a constant fermentation rate in

batch fermentation. An on-line computer (Rogers, 1976) would be

necessary to estimate the temperature variation required to prevent

over-shooting a set point fermentation rate in a batch fermentation.

Information, such as temperature of must, volume of must, yeast cell

concentration and heat transfer capacity of a particular heat exchanger,

would be necessary for the computer to accurately determine the desired

temperature variation. The necessity for this complicated control

mechanism is essential because of the dynamic nature of yeast activity

in batch fermentation (Fig. 1).

24.

Fig. 1. Changes in yeast and ethanol (E) concentration during batch

fermentation. The rate of fermentation d E_ , is given by the slope ofdt

the ethanol (E) curve. (Reed and Peppier, 1973).

Although continuous fermenters have been used for the production of

wine for several decades, the elucidation of their control has resulted

in a renewed assessment of their use in industry. Ough and Amerine

(1968) stated that continuous fermentation was highly suitable for the

production of dessert wines, and in 1974 Dr A.C. Rice, the director

of research at the Taylor Wine Company Inc., Hammondsport, New York,

predicted an increase in continuous fermentation installations in the

immediate future for still and sparkling wine production.

2.3 Review of Wine Yeast for Wine Production

2.31 Important Characteristics of Yeast in Winemaking

The term 'wine yeast' will be used for species of SacdiaAormjceA, such as

S. ccAeucAf-ae, S. boJxcuA and S. ov^iofimU, which may be grown in pure

culture and added to musts. The term 'wild yeast' will describe yeasts

that occur naturally on grapes and can be found on winery equipment,

and includes all yeasts that may cause instability and off-flavours in

wine or musts. The mycology of yeasts described by Lodder (1970) will

be followed; for example, Giashvili and Alkanashvili (1971) and

Buryan, Kozlovsky and Razuvaev (1975) have used 5. uTrT incorrectly,

25.

and these yeasts will be referred to as S. cdsiav-Usiaz. Flor yeasts

‘ have been variously designated S. beJxcuA and 5. bayarnu (previously

S. chQAQA.Yii>AJ>)./

The yeasts of the SacckaAomycej, genus are strong fermenters and are

usually spheroid, ovoid, ellipsoidal or elongated. They propogate a/

vegetatively by multilateral budding and under suitable conditions can

produce 18-20% ethanol by volume (v/v). Other species of Sacc.kaAonr\yc&>

that produce high concentrations of ethanol are S. ckdvciLieAsL,

S. ovi&osunts, S. acA..dl&acA,e,ns, S. bayavnus and S. battens. Yeast

species that occur frequently in musts include S. hosqa. and S. uva/ium

(previously S. casit&bzHQQ,YiSAJ>). S. kouxLL and 5. moJUJj, are osmophilic

yeasts, which occur occasionally as spoilage organisms in sweet wines.

The cells of Fidvia are short ellipsoid to cylindrical in shape and

propagate by multilateral budding. Fidila mzmbsiandiacie.nA, with a

ceil size of (2.5 - 4.5) x (5-14)/j, occurs in almost all musts; it

does not produce more than 1-2% ethanol, but it readily survives higher

alcohol concentrations and grows readily on the surface of young wines,

where it forms a continuous film.

Yeasts of the genus Hans mala are spherical, ovoid or cylindrical in

shape and propogate by multilateral budding. Hansenula anomala is the

species of particular interest in winemaking, since it may produce up

to 10% ethanol (v/v) and large amounts of ethyl acetate and other

esters.

The fissian yeasts SdUzosacdioAomycaA reproduce vegetatively by

forming a wall across the middle of the elongated cells.

26,

'i The yeasts do not pdroduce high concentrations of ethanol, but

SdvLzoAacck. pombd, cell size (3-5) x (6-16)ju, ferments malic acid4

to ethanol and CO^ (Reed and Peppier, 1973).

KloackeAa are small cells, (2-4.5) x(5-8)/j, pointed, oval or lemon

shaped, which occur abundantly in musts and can dominate the early

phase of natural fermentations to 5-6% ethanol; they can also form

large amounts of volatile acids and esters.

Further discussion on the role of Sacc.haAomtjcodeA, Candida and

To/iulop6i6 species in grape musts is provided by Reed and Peppier (1973).

2.311 Cytology of Yeast

The principal microstructures of a yeast cell are the cell wall,

plasma membrane (plasmalemma or cytoplasmic membrane), nucleus, one

or more vacuoles, mitochondria, polyphosphate granules (volutin),

lipid globules, ribosomes and ground plasma or matrix. The ultra­

structure of yeast cell walls includes a dense net work of microfibrils

or fibrillar mat, a birth scar and numerous bud scars (Streiblova, 1968).

The wall content of S. aojiavdAiaa varies from 6-27% of the cell dry

weight and increases within that range as cells pass from the logarithmic

growth phase to the stationary phase. Upon aging, flocculent yeasts

almost double their wall weight (Mill, 1966; Griffin and MacWilliam,

1969), and they bind twice as much calcium as walls from non-flocculent

strains (Lyons and Hough, 1970). The gross chemical content of the

yeast cell wall is 83% carbohydrate, 10% protein, 3% lipid, 0.45%

sterol, 0.3% ribonucleic acid and 0.04% deoxyribonucleic acid

(Nurminen, Oura and Suomalainen, 1970); some of the components

characterized include glucan, mannan, chitin (1%), glucosanine,

27.

fatty acids, glycerides, phospholipids (0.7%) and phosphate (1%).

.Certain environmental conditions modify cell wall composition, for

example, cells grown in the absence of inosital have weaker cell walls,

contain less mannan, protein and phosphorus, but more glucan and

glucosamine than normal cells (Power and Challinor, 1969). The

ability of the yeast cells to survive freeze-drying is unaffected by

cell wall composition, but pretreatment of the cell with 2-mercaptoethanol

greatly increases the proportion of survivors (Richards and Elliot, 1968).

The appearance of elongated cells during continuous fermentation has

been well documented (Hough and Button, 1972). 5. ceAeuXif.ae cells

show little tendency to elongate in batch fermentations, but elongate

during continuous fermentation under stress situations such as high

pressure or nitrogen limitation. It was also noted that the balance

of thiol and disulphide groupings was modified by adding either sodium

thioglycollate or sodium selenate to the medium. These additions had

the effect of causing elongated cells to bud ovoid ones, even under

conditions of nitrogen limitation. It was also demonstrated that the

mitochondria of ovoid and elongated cells differed in protein-

disulphide reductase activity. McMurrough and Rose (1967) have studied

the composition and structure of cell walls from yeast grown in

continuous culture at different rates and showed that total glucan and

mannan contents of the walls were not greatly affected, however, at

low growth rates the phosphorus and protein content of the wall

increased. It was suggested that with elongated cells grown in a

nitrogen-limiting medium the shape arises from low protein disulphide-

reductase activity and differences in the cell wall composition, both

of which lead to fairly rigid areas of cell wall.

28.

2.312 Hybridization and Mutation in Yeast *

• Although hybridization has the potential for improving yeast for

technological uses, only a few studies have been reported (Kleyn and

Hough, 1971). Wine yeast hybrids of high pectolytic activity and

improved fermentation rate were isolated by Mosiashvili and

Shalutashvi1i (1971). Hybrids of bakers yeast with increased maltase

activity and superior dough performance were mentioned in patents

granted to Burrows and Fowell (1964); after testing 2,000 hybrids,

Lodder and Loggers (1962) found two high-maltase strains which out­

performed conventional bakers yeast. Johnston and Lewis (1976)

record an excellent summary of the techniques used in genetic analysis

of S cqazvi^icLd.

Natural mutations occur in yeast populations, for example, abnormal

cells lacking respiratory ability were observed to be 1% of the yeast

cell population (Ephrussi, 1953), but no mutants of brewers yeast have

proved suitable for commercial use (Reed and Peppier, 1973). A

combination of direct selection pressure and continuous culture was

used to select for altered acid phosphatase activity in S. ceAeufLsffie

(Clarke, 1976), and after 400 generations a mutant was obtained.

Results of this nature indicate that selection by continuous culture

is of limited application and is most likely to select regulating

mutants - mutants with gene amplification and altered enzyme

complements (Wiame and Dubois, 1976).

2.313 Energy-Yielding Mechanisms in Yeast

Yeasts are heterotrophic, facultative anaerobes, which grow in the

absence of air or in the presence of molecular oxygen; the presence

of oxygen induces the change in energy-yielding metabolism from

29.

fermentation to respiration (Pasteur effect, reviewed by Sols,

Gancedo and DelaFuente, 1970). Fermentation is the anaerobic,

energy-releasing transformation of glucose to pyruvic acid in a

series of metabolic, coupled reactions known as the Embden-Meyerhof-

Parnas pathway. Yeast growth per unit of substrate is 5-10 times

more efficient in air than anaerobically, but the cell uses glucose

at a higher rate in fermentation, than by respiration. Glucose

concentration in excess of 5% in the presence of oxygen represses

respiration and allows fermentation (Crabtree Effect).

2.314 Inhibitors of Yeast and Lactobacilli

Diethyl pyrocarbonate (DEPC) is an effective inhibitor of wine yeast

alcohol dehydrogenase at concentrations between 50-100 mg/1

(Reed and Peppier 1973); wild yeasts usually have a lower tolerance,

however, 100-300 mg/1 DEPC is necessary to kill 1actobaci11i and

other bacteria participating in malo-lactic fermentation (Reed and

Peppier, 1973). Sulphur dioxide inhibits yeast growth, but wine

yeasts for use as starter cultures can be fermented in the presence of

200 mg/1 in order to acclimatize them to the microbiocide (Reed and

Peppier, 1973).

2.315 Yeast Viability

Direct cell counts, after staining with equal volumes of yeast and

suspensions of 0.01% methylene blue or 0.2% Congo red, show that stained

cells by either dye are dead (Gleinster, 1973; Suomalainen, Oura and

Nevalainen, 1965). Similarly the ratio of live and dead cells can be

estimated by fluorescent staining with Clayton yellow and acriflavine

hydrochloride and examination under ultraviolet light (Mi 11ipore, 1969).

Live yeasts fluoresce tfith a bright yellow-orange colour, and dead cells

30.

fluoresce faintly with a greenish colour. Kunkee (1976) described

* a similar method using a polychromatic fluorochrome, euchrysine

2 GNX to stain cells under transmitted ultraviolet light, so that

viable cells fluoresce green, and dead cells fluoresce orange.

Yeast cell viability in continuous fermentation is significantly

affected by temperature, ethanol concentration, dissolved oxygen

tension (DOT) and available nitrogen. Some studies of viability

in continuous fermenters have been reviewed by Hough and Buttonr

(1972) - cell concentrations of 50-60x10 /ml have been reported

(Ough and Amerine, 1968; Giashvili and A1kanashvi1i, 1971; Barre and

Combe, 1975). Reduced yeast viability was found by Portno (1968a),

when high concentrations of yeast were maintained by external recycle.

Similarly Nagodawithana, Castellano and Steinkraus (1974) found that

the higher the temperature between 15-30°C, the greater the rate of7

death as initial cell inocula were increased from 1.1x10 tog

7.8 x 10 cells/ml, however, no explanation was given except that

nutrients and oxygen were apparently not limiting. Better retention

of cell viability was obtained as DOT approached 100% at 15°C, but

fermentation rate was lowered above a DOT of 13%. Intracellular

levels of 5 x 10 ^ ethanol molecules/cell were considered normal by

Nagodawithana and Steinkraus (1976), with higher concentrations such

as 2 x 10^ molecules ethanol/cell being accompanied by inactivation

of alcohol dehydrogenase (ADH) and loss of cell viability. Kunkee and

Singh (1975) found that ADH activity was higher in cell-free extracts

of S. c,QA<L\)jj>ia<i (Montrachet Strain, UCD 522) grown aerobically

compared to that from the yeast grown anaerobically. The use of oxygen

to improve cell viability in continuous fermentation has been

successful (Ricketts and Hough, 1961), but Portno (1968b) found that

with high concentrations of yeas-' fhe amount of dissolved oxygen had

31.

little effect on beer production and yeast growth. The detrimental

• effect of aeration of musts during the fermentation of grape juice

was recognized by Flanzy at at. (1966) who found that aeration was

not necessary to maintain a sufficient yeast concentration in a

continuous fermentation experiment that ran for 40 days. Similarly,

Cowland and Maule (1966) were able to maintain S. coAaviAtaa in

steady state for prolonged periods in a continuous fermenter in the

absence of oxygen.

2.316 Selective and Differentiating Solid Media for Yeast

Since yeasts can develop over a wide range of pH, yeast media is

adjusted to pH3.5-4 with lactic acid or hydrochloric acid just before

its distribution to plates or tubes (Reed and Peppier, 1973). No

single medium has been devised for detecting wild yeasts. Wine yeasts

are unable to grow on lysine agar, and this media is generally used

for wild yeast detection; however, S. duutattciJA, a cornnon wild

yeast, does not grow on lysine agar. This yeast may be detected on

the fuchsin - sulphite medium of Brenner at at. (1970), the crystal

violet agar of Kato (1967) or Lin's medium (Lin, 1975). The most

sensitive test for wild yeast contaminants in brewers wort is the

immunofluorescent technique described by Richards (1970).

2*317 Effect of Vitamins on Yeast Activity

Amerine and Ough (1972) in reviewing experiments by Oprya (1969)

stated that the reduction in vitamin content of wine was several times

greater in continuous fermentation compared to batch fermentation; they

postulated that this was due to extra yeast growth. Their conclusion

has been difficult to substantiate from other literature, and it is

apparent that the reverse could be true. Reed and Peppier (1973) showr

that in batch fermentation the maximum cell count is 140-150x10° cells/ml

(Fig.1) and Ough and Amerine (1968) found a maximum of BOxlO6 viable

cells/ml (Table 4) in their continuous fermentations. Oprya (1969)

in fact reported that within some vitamin groups, namely the B

vitamins, there was a 50-70% reduction during continuous fermentation

compared to the level of these vitamins in grape juice. The uptake of

vitamins was maximum between tanks 2 and 4 in a seven tank series; ther

maximum cell count in those tanks was 112 x 10 cells ml. Oprya (1969)

also observed a relationship between the changes in the vitamin and

mineral contents of grape juice and the application of mineral fertilizer

to grape vines. He achieved a doubling of the pantothenic acid and

thiamine levels in wine fermented continuously from grapes grown

under fertilizer supplements. The concentration of vitamins in the

wine was claimed to be reduced by 50-75% with high levels of must SO^,

however, no data were given to support these observations.

2.318 Factors Affecting the Rate of Wine Yeast Fermentations

The following variables are important in influencing the rate of

fermentation: ethanol concentration, pH, temperature, CO^ pressure,

yeast species, Brix of the must and the presence of yeast nutrients

and inhibitors. In addition to the individual effect of each variable,

there are important interactions between variables. For instance, the

inhibiting effect of ethanol on fermentation rate is temperature

dependent, and secondly during a batch fermentation, Brix, ethanol

and viable cell concentrations change markedly. Therefore accurate

information on the effect of each variable on fermentation rate can

only be determined under steady state conditions.

2.3181 Ethanol Concentration

Holzberg, Finn and Steinkraus (1967) found that in a continuous

fermentation system at 21°C a yeast count of 20x10^ cells/ml produced

33.

1.3 g ethanol (EtOH)/l/hr. Ough and Amerine (1968) also worked with

a three stage, continuous fermentation system at 21°C, which produced

less alcohol per litre of fermenting must as the ethanol concentration

increased in successive stages (Table 4).

Table 4. The rate of ethanol production at different ethanol

concentrations in a three-stage continuous fermentation system at

steady state (Ough and Amerine, 1968).

Stage Fermentation rate(g EtOH/l/hr)

Av. ethanol cone.{%) v/v

Viable yeast count (cells/ml x 106)

1 1.03 7.1 57.9

2 0.98 8.8 80.3

3 0.63 11.8 64.3

Table 4 indicates that alcohol production may be independent of small

changes in yeast cell concentration. Nagodawithana oX at. (1974)

observed that the rate of ethanol production per cell gradually de­

creased as the ethanol increased to 12% v/v. At steady state the fer­

mentation rate of a continuous fermenter (g EtOH/1/hr) depends on the

ethanol produced per cell (EtOH/cell/sec), cell count and the dilution

rate. The dilution rate is dependent on the specific growth rate of

yeasts, hence the inhibiting effect of ethanol on growth rate must be

considered in this context. Buryan zt at. (1975) found that the

specific growth rate of yeast under anaerobic conditions decreased

from 0.2-0 hr~^ in a linear manner as ethanol concentration increased

from 0-10%. However, tolerance to ethanol is superior in older cells

(Ough, 1966b), and the death rate of cells is important in considering

growth rate for determination of dilution rate in a continuous fermenter.

34.

An intracellular level of about 5x10^^ EtOH molecules/cell is normal

in rapid fermentations (Nagodawithana and Steinkraus 1976) and this

level does not damage cell viability or ADH activity. However, higher,11intracellular ethanol concentrations of 2 x 10 molecules EtOH/cell

are accompanied by inactivation of ADH and loss of cell viability.

2.3182 Yeast Concentration

In laboratory studies Watson and Hough (1966) demonstrated that over

a wide range of yeast concentrations, the rate of fermentation in

wort for each yeast cell remained constant. Similarly, Steinkraus

(1972) found that within an order of magnitude the rate of 10'7 molecules

EtOH/cell/sec was relatively constant in continuous fermentation of9

grape juice with varying cell counts, including high counts of 10"r

cells/ml. At high cell counts above 80 x 10 cells/ml, however,

fermentation rate decreases as cell viability and growth rate decreases

(Nagodawithana at., 1974). Similarly the rate at which individual

cells grow in continuous fermentation of wort diminishes as the yeast

count increases (Watson and Hough, 1966); no cell concentrations

were given in this study. This yeast count effect is modified by

the concentration of CO^, the agitation rate and the intracellular ethanol

concentration, thus the growth limitation at high cell counts is related

to the excretion and uptake of nutrients, which is controlled by the

selectively permeable, plasma membrane, surrounding the protoplast

(Suomalainen and Oura, 1970).

2-3183 pH and Temperature

The natural pH of grape juice in Australian musts varies between 3.1

and 4.2, and it is well established that fermentation rate is faster

at higher pH values (Fig.2).

35.

Fig.2. Effect of pH on fermentation rate (Reed and Peppier, 1973,

, P 187).

0.1 0.15 0.2

Fermentation rate (°Brix/hr)

Yeast growth rates are also higher at pH 4.0 than at pH 3.0 and batch

fermentation begins earlier at higher pH levels (Ough, 1966 a,b).

The relationship between temperature and fermentation rate is linear

for batch fermentations between 10 - 32°C (Ough, 1964). The effect

of temperature upon continuous fermentation is more complex and has

received little study. Hough and Button (1972) showed that an increase

in temperature between 15-30°C led to a linear increase in beer

production rate in a fully stirred tank. Above 20°C, however, the

increase in yeast growth rate was no longer linear. This effect is

consistent with previous discussion in that, at higher temperatures,

growth rate will be limited by solute and nutrient transport through

the yeast cell wall, but fermentation rate responds to the temperature

change.

2.3184 Yeast Species and Strain

There appears to have been no study comparing the effect of wine yeast

strains on fermentation rate in continuous fermentation, and only yeast

36.

species have been referred to in the literature regarding production

rates in batch fermentations. In beer production, however, Hough and

Button (1972) claimed that strains of S. coAdvL^Zao. that flocculate

and sediment easily accumulate in the fermenter and therefore give the

highest rates of ethanol production.

2.3185 Agitation and Carbon Dioxide

Rice and Helbert (1974) confirmed previous reports in the literature

that agitation significantly increases yeast growth. They concluded

that gently agitated fermentations, either by normal evolution of carbon

dioxide or mechanical means, were supersaturated with CO^ and that the

level of dissolved CO^ was the major factor that affected yeast growth

rate at different levels of agitation. Kunkee and Ough (1966) reported

that carbon dioxide inhibited yeast growth, although only fermentations

under several atmoshperes of pressure showed conclusively that such

inhibition existed. Rice and Helbert (1974) observed that an increase

of one volume of dissolved CO^ reduced growth rate by 28%, and that it

was not pressure per se which caused the decreased yeast growth in

batch fermentations.

2.3186 Sugars and Yield of Ethanol

The following sugars have been isolated and identified in the fruit cf

l;xXc6 vlni&QAa vines; stachyose, raffinose, melibiose, maltose, sucrose,

galactose, glucose and fructose (Amerine and Joslyn, 1970). The two

most important sugars are d-glucose and d-fructose, in approximately

equal amounts at maturity; the remaining sugars together account for

less than 0.5% by weight of the grape must. Fructose is considerably

sweeter than glucose, and most wine yeasts ferment glucose faster than

fructose, hence in a two-stage continuous fermenter a higher ethanol

37.

production rate would be expected in stage one, where glucose whould

be preferentially fermented (Ough and Amerine, 1968).

Pasteur assumed that 48.6g ethanol were evolved from 100 g of glucose,

and actual yields of ethanol in wine fermentations have been close to

this figure (Reed and Peppier, 1973). Several reports describe attempts

to determine alcohol yields from a fermentation, however, estimates of

sugar concentration have been based on total soluble solids or °Brix,

and there have been some ethanol losses due to entrainment with CO^.

In addition, small amounts of ethanol may be obtained from glucosides.

Warkentin and Nury (1963) compared actual ethanol losses in closed

fermenters with losses calculated from physicochemical data and found

good agreement. At 27°C, 0.83% of ethanol was lost by entrainment,

and at 35°C the loss was about twice as great. Ough and Amerine (1968)

found ar overall yield of 48.4% in a three-stage continuous fermentation,

with 44.9% in the first stage, 49.1% in the second and third stages.

An increase of 1% v/v and 0.2% v/v ethanol was reported by Amerine and

Ough (1972) and by Ladousse (1962) respectively in continuous fermenters

compared to batch fermenters for the production of red wine. High

ethanol yields in continuous fermenters have been associated with low

biomass yields and a moreJcomplete fermentation of residual sugars.

Little work has been attempted to explain these industrial observations,

however, fermentation mechanisms have been elucidated by Horecker (1963)

for the reduction of pentoses to yeast metabolisable products. The low

oxidation-reduction potential of must in continuous fermentation is

suitable for the reductive pathways of anaerobic pentose metabolism.

The important pentoses are D-xylose and L-arabinose in polysaccharides

and D-ribose and 2 deoxy-D-ribose in nucleic acids; the end products

of their metabolism are x-ketoglutaric acid, pyruvic acid and L-anabitol.

38.

The activities of the enzymes in relation to the rate of metabolism

• in the glycolytic and pentose phosphate pathways have been discussed

recently by Oura (1976).

2.32 Role of Oxygen and Flavour Formation in Quality Wine Production

Alcoholic felamentation is effected best in the absence of air

(Amerine and Joslyn, 1970) for several reasons. Firstly, fermentative

enzymes secreted by yeast require no oxygen for their activity, and

secondly aeration increases active respiration of sugar by yeasts

and leads to a greater loss of ethanol by evaporation or CO,-,

entrainment. In batch fermentations, however, in order to bring

forward the onset of fermentation, aeration is desirable to promote

the multiplication and vigour of the yeast; generally the oxygen

dissolved in the grape juice after crushing is sufficient to provide

this stimulus to yeast growth. Unfortunately, aeration of grape

juice or fermenting must can detrimentally affect the flavour

precursors and by-products of fermentation. Continuous fermentation

of grape juice provides an opportunity for complete anaerobic

fermentation, since there is no lag period before the onset of

fermentation.

Aeration can be responsible for discolouration, haze formation, loss

of desirable flavour and the presence of undesirable flavours (Amerine

and Joslyn, 1970; Ribereau-Gayon, 1975). Aeration of musts increases

the content of fusel oil acetic acid and acetaldehyde and promotes the

oxidation of catechins by polyphenoloxidase to o-quinones and their

condensation to melanins. Acetaldehyde, a normal by-product of

alcoholic fermentation, may also be formed by enzymatic or aerobic

oxidation of ethanol and conversely, acetaldehyde may be reduced to

ethanol, a reaction which is catalyzed by alcohol dehydrogenase.

Under anaerobic conditions the accumulation of acetaldehyde parallels

the formation of ethanol (Amerine and Ough, 1964) in the early stages

of fermentation and disappears during the latter stages. The

presence of oxygen limits the disappearance of acetaldehyde in the

latter stages of fermentation (Ribereau-Gayon, Peynaud and Lafon, 1956),

and further oxidizes ethanol to acetaldehyde. Acetic acid is formed

by the dismutation (oxidation of one molecule of acetaldehyde to

acetic acid and the simultaneous reduction of another to ethanol) of

acetaldehyde (Joslyn and Dunn, 1938) and by several species of kdatobcioJivi

that oxidize ethanol to acetic acid. Similarly,ethyl acetate is formed

by aerobic yeasts, which oxidise ethanol (Amerine and Joslyn, 1970).

Acetoin (acetyl methylcarbinol) is a normal by-product of yeast

fermentation and can be aerobically oxidized to diacetyl (Amerine and

Ough, 1972), although the bacterial production of diacetyl is more

common as a result of the malolactic fermentation (Rankine, Fornachon

and Bridson, 1969). Higher alcohols are formed by reductive decarboxylation

and transamination of amino acids and also by bacterial fermentation

of carbohydrates. These reactions are quite complex and have been

reviewed by Amerine and Joslyn (1970).

Both enzymic and non-enzymic browning in wines are directly related

to the flavonoid concentration (Peri nt al., 1971); a progressiver

insolubilisation of tannic flavoids and a polymenisation of the non-

tannic flavonoids accompanies the browning reaction. The rate of

browning, however, is apparently controlled by other factors, such as

enzyme concentration, pH, transport phenomena and suspended solids

(White and Ough, 1973). Enzymic oxidase activity is irreversibly

40.

induced by oxygen after grape juice is exposed to air (Lerner, Mayer

and Hare!, 1972).

The reaction mechanisms of grape catechol oxidase activity have been

further elucidated by Lerner and Mayer (1976), Traverso-Rueda and

Singleton (1973) and Ribereau-Gayon (1975).

Paronetto (1966) distinguishes between direct oxidation by oxygen and

enzymic and catalytic oxidation, however, from the data presented

previously, oxygen can induce some enzymic and catalytic oxidation.

Molecular oxygen is a terminal electron acceptor in oxidation processes,

and in the absence of oxygen these are accomplished by some other inorganic

substance, such as a metal ion, nitrate or sulphate (Sols oXal., 1970).

Conversely, the main reducing constituents in musts are yeasts, tannins,

anthocyanins and sulphurous acids (Amerine and Joslyn, 1970), which

together characterize the oxidation-reduction potential of musts and

wines. Paronetto (1966) has presented some data (Table 5) that shows

the relation between oxidation-reduction potential and dissolved oxygen

at different stages in the fermentation of non-sulphited and sulphited

red grape musts.

The oxidation-reduction potential of a must rises rapidly immediately

after crushing and falls again during fermentation (Amerine and Joslyn,

1970). No direct correlation between oxidation-reduction potential in

musts and wine quality was found by Deibner (1957), however, for young

wines a lower potential appeared related to higher quality; during

wine aging the potential varied considerably, and Deibner concluded

that the entire history of a wine must be followed to establish any

correlation between oxidation-reduction potential (E^ ) and sensory quality.

41.

Table 5. The relation between dissolved oxygen and the oxidation

reduction potential in red grape must, with and without sulphite

added (Paronetto, 1966).

Nonsulphited

Time on skins 1 3 6 12

Oxidation-reduction potential (volts) 0.390 0.416 0.436 0.455

Oxygen (mg/1) 3.3 5.2 6.3 7.3

Sulphited

Oxidation-reduction potential (volts) 0.346 0.356 0.376 0.386

Oxygen (mg/1) 0.7 1.0 1.3 1.5

The level of polyphenolic compounds is important in determining the

of wine, and these compounds can be oxidized at high or relatively low

potentials. Thus it is apparent that oxidation-reduction in wine is

complicated and depends on several variables (Ribereau-Gayon, 1963).

The E^ in must fermented continuously was found to be lower than in

batch fermentations (Martakov, 1970; Dzhurikyants oA at., 1974), and

since E^ corresponds to NADH concentration in the yeast cell (Amerine

and Ough, 1972), it is reasonable to assume that higher ethanol

production per cell in continuous fermentation contributes to the change

in E^ noted in the literature.

2.33 Acid Metabolism and Some By-products of Fermentation

Peynaud (1939) reported the formation of acetic, citric;lactic and

succinic acids by 5. during alcoholic fermentation, and

malic and tartaric acid production was demonstrated by Drawert,

Rapp and Ulrich (1965a,b). Although there is some evidence

(Sandegren and Enebo, 1961) that continuous fermentation shows a

greater accumulation of non-volatile acids and hence lower pH than

batch fermentation, this change is not always apparent in the finished

wine. The pH of wine was found by Wejnar (1968) to be regulated by

the tartaric acid content, particularly the ratio of tartaric acid

to potassium content and tartaric acid to the alkalinity of the ash.

Since the alkalinity of the ash is a measure of the potassium acid

bitartrate content, it appeared that the buffer capacity of a wine

was determined largely by the relative concentration of the acid salt.

In addition to these factors, the pH of a wine may be increased by the

secondary bacterial fermentation of malic acid, which according to

Ladousse (1962) is more rapidly induced in a continuous fermenter

under a cap of red grape skins. A detailed review, with 145 references,

of organic acid metabolism of yeasts during fermentation of alcoholic

beverages has been presented by Whiting (1975).

Sandegren and Enebo (1961) found that, compared to batch fermentation,

beer produced by continuous fermentation showed increased levels of

higher alcohols (fusel oil) and sulphydryl compounds. Unfortunately

no comparative data exists for wine made continuously, however, some

observations in this respect can be made. Higher alcohols are by­

products in yeast metabolism of amino and organic acids; some enhance

and others detract from wine quality, depending on their concentration.

Amerine and Joslyn (1970) have shown that higher alcohols increase

43.

throughout fermentation, and Rankine (1967) found considerable

variation between yeasts. Peynaud and Guimberteau (1962) generally

found a greater level of higher alcohols in wines fermented under

anaerobic conditions which may explain the relatively higher levels

of higher alcohols reported in continuous fermentations (Dzhurikyants

eX al., 1974).

It is interesting to note that within the multitude of flavour

compounds recently separated and identified by gas chromotography and

mass spectrometry, the amount of ethyl octoate produced by yeast

during fermentation has been associated with wine quality (du Plessis,

1975), and both grape composition and wine yeast strain affect the

formation of ethyl octoate (Kunkee and Amerine, 1970).

The formation of H^S by wine yeasts has been widely reported in the

literature (Reed and Peppier, 1973; Kunkee and Amerine, 1970), but

the precursors and conditions necessary differ and are sometimes

contradictory. Studies by Wildenradt and Lewis (1969), Anderson and

Howard (1973) and Wainwright (1971) show that in brewers wort, yeast

strain, temperature, metal ions aeration and affect H^S formation

by yeast through the reduction of sulphate, sulphite, sulphur containing

amino acids and peptides. Both methionine and pantothenate decrease

the reduction of sulphate to sulphide, but both also have other effects

on sulphur metabolism. Methionine does not act directly on the

reduction of sulphite to sulphide so it does not always inhibit

sulphide formation, and added methionine can increase the amount of

sulphide formed (Wainwright, 1971). The ATP required for the reduction

of sulphate is supplied by fermentation processes (Dott, Heinzel and

Truper, 1976) and may also coincide with vigorous yeast growth

44.

(Wildenradt and Lewis, 1969; Wainwright, 1971). Thoukis and Stern

* (1962) claim that slow, cold aerobic fermentations encourage the

formation of H^S, however, Reed and Peppier (1973) state that H^S

formation is greatest when the E^ is at a minimum. Eschenbruch and

Bonish (1976) suggest that unknown factors contribute to the control

and regulation of the sulphate reducing systems, the ultimate aim of

which is the sufficient supply of sulphur containing components for

yeast metabolism. They found that some strains of S. ceAzvZAlaa

doubled sulphite production if the initial pH of the medium was

changed from 3.0 to 5.0, while other sulphite producing strains were

not affected. Minarik and Navara (1974) claim that S0^ levels of the

must have no significant effect on sulphate uptake or sulphite

formation, and that SO^-forming yeasts form less sulphide than normal

(non-SO^ forming) yeasts. In comparison Eschenbruch, Haasbroek and

Villiers (1973) found that SO^-forming yeast strains formed H^S fromr35 iboth sulphate and sulphite in experiments using labelled [ SJ sulphate

and sulphite in grape must. The addition of methionine and cysteine

completely suppressed sulphite and sulphide formation from sulphate,

while the formation of sulphide from sulphite was not influenced by

these amino acids, and only some strains were affected by the addition

of sulphite to the must. Further elucidation of the mechanism of

sulphite formation has been discussed by Heinzel and Truper (1976).

They isolated ATP-sulfurylase from two strains of wine yeast and

demonstrated feedback inhibition by adenylsulfate. Although Sandegren

and Enebo (1961) claim that continuous fermentation of brewers wort

showed an increased potential for the formation of sulphydryl compounds,

this is unlikely to be a useful generalization, since yeast strains can

be selected (Rankine, 1963) which are low producers of or do not produce

H^S in particular musts.

45.

The importance of establishing which yeast strains can produce H^S

in grape juice fermentation has been well recognized, and Mueller

(1976) compared five UCD strains of 5. ccAov/LsTae: 51 (Burgundy),

505 (Champagne), 522 (Montrachet), 505 and 586 (Australian strains)

and found that UCD 585 produced the highest level of H^S and UCD 51

the lowest.

2.34 Control of Wild Yeast and Bacteria in Fermenting Musts

Hough and Button (1972) have discussed the likelihood of serious

bacterial and wild yeast infection in a continuous fermenter and

suggested that the nature and number of the wild organisms, and the

part of the plant where infection took pi ace,were the most important

factors. Within the fermenter the wild organisms must be present in

large numbers, or have a considerable biological advantage over the

wine yeast, or if the growth rate of the wild organisms and the

accumulated cells introduced with the grape juice are less than the

dilution rate then washout will occur. Grape juice, stored before

addition to the fermenter, must be protected with microbiocides and

kept as close as possible to 0°C to prevent growth of the wild

organisms. Liquid SO^ added directly to grape juice inhibits the

growth of wild organismsbut the effectiveness of SCU, depends on the

quality of the grapes used. Amerine and Joslyn (1970) recommended that

75 mg/1 SO^ should be added to acid or immature grapes, 112 mg/1 to

mature grapes and 270 mg/1 to over-ripe grapes, which are usually

low in acidity and often mouldy. At pH values between 3 and 3.8,S02

remains dissociated in the SO^” and HSO^" forms (free S0^) and has

then a strong germicidal effect, but as the HS0g~ ion combines with

aldehydes, polysaccharides and organic acids (bound SO^its germicidal

effect is diminished.

46.

The addition of 50-100 mg/1 S0^ inhibits wild yeast, but the sensitivity

' of individual species varies widely, for instance, S. luduoigii and

S. bailii can tolerate high concentrations of SO^, while Kloeakzm

apicuZata and Pidiia membAane.iacie.il6 are sensitive (Reed and Peppier,

1973). In general SO^ inhibits wild bacteria in musts, and although

bound SO2 does not inhibit iacZobaciZluA planiaAum, it does inhibit the

heterofermentative species L. Hil.gaA.ciil and L&uconoAtac meAenteAcZcU.

Furthermore, Fornachon (1963) found that the presence of actively

metobolising yeast cells could be an important factor in encouraging

certain bacteria in any wine containing SO^, as the continuous

production of aldehyde by yeast could prevent the liberation of free

SO^ by bacteria, normally attacking aldehydes bound to SO^. Extensive

reviews of wild yeast and bacteria important in winemaking and their

tolerance to microbiocides have been recently published (Amerine and

Kunkee, 1968; Amerine and Ooslyn, 1970; Amerine at aZ., 1972). Pure

yeast starter cultures can be acclimatized to levels of SO^ between

50-200 mg/1, so that in sound musts of low pH, they have a considerable

advantage over wild organisms. The original Semichon (1926) principle,

that continuously fermenting must, containing over 4% v/v ethanol was

protected from contamination may have been true in 1926, but this has

not been substantiated recently, possibly because some strains of wild

organisms more resistant to 4% v/v ethanol have evolved. Sapis-Domercq

(1969) has provided a comparison of microflora of batch and continuous

fermentations, however, this information is only relevant in the

viticultural region of origin.

There is no single procedure adequate to prevent contaminant growth in

a continuous fermenter, but the aim in controlling their growth is to

47.

place them at a biological disadvantage in the fermenting must, withi

low pH, high alcohol and high microbiocide levels.

Protected microenvironments in grape juice supply lines, valves, stirrer

shaft bearings and imperfect weld joints are potential and often

persistent contamination points. These areas are not effectively

penetrated by chemical microbiocides and must be live steamed regularly

to prevent residual contaminant populations. The most persistent and

microbiocide resistant spoilage organisms are found in storage tanks

and equipment in wineries rather than on grapes before crushing, hence,

winery hygiene is a critical factor in controlling infection by

contaminants.

\

48.

3• EQUIPMENT, MATERIALS AND METHODS

3.1 Continuous Fermentation Equipment

The fermenter was based on the five socket Quickfit adapter MAF 3/52

and the Vibromix El Mixer (Model No. 3405 Edwards Laboratories Pty Ltd,

Smithfield, NSW). The fermentation vessel was a modified, Quickfit wide-

neck, culture vessel, and the assembly, shown in figure 3 was built by

the School of Biological Technology (SBT), University of New South Wales

(UNSW). Special modifications designed by the author for these

experiments include the fortification tube and spirit pump assembly,

the foam escape tube, Brix hydrometer vessel, the coolant reservoir

and pump assembly and the vessel for monitoring dissolved oxygen level

in the grape juice.

3.11 Fermentation Vessel

The fermentation vessel had a working volume of 900 ml, which could be

adjusted by shifting the position of the outlet tube for the partially

fermented must. The five socket adapter MAF 3/52 or lid of the fermenter

v/as attached to the vessel by a flange and clamp assemply. The flange

was washed and smeared with silicone grease, then clamped tightly prior

to sterilization. Steam for sterilization, must and yeast culture were

introduced through the fermenter lid by coarse bore, glass tube pipettes.

The outlet for the partially fermented must was fitted with a foam

escape tube to prevent must hold-up in the fermenter. The foaming

problem was due to two factors - firstly, the wine outlet tube v/as

small and secondly, the partially fermented wine contained 9-12%

soluble solids which produced a foam with high surface tension.

49.

ml $, a >**■*.««teff.fr'.-,:

pt.

f H t $$

m " - sw- ■ '■-■i.-Uiw; ■l-.e- --. „. • - •. ,•; .

PHUH

Fig. 3. Continuous fermentation equipment for the production of

fortified sweet wine in the laboratory. A,1 litre fermentation

vessel; B, five socket Quickfit fermenter lid; C, variable speed

must pump; D, fermenting must mixer; E, Brix hydrometer vessel;

F, fermentation control panel; G, coolant reservoir; H, variable

speed coolant pump; I, wine receiver; J, fortifying spirit pump.

50.

The foam escape tube prevented the blocking of the outlet tube for

' the partially fermented must. A Quickfit condenser type CX6/22 and

a Mackley filter (Microflow Ltd, Hampshire, U.K.) were connected to

the effluent gas outlet.

3.12 Mixing and Pumping Must

The Vibromix El assembly had an air inlet tube in the centre of the

mixer shaft, which was sealed in the lid of the fermenter by a

neoprene diaphragm. The stirrer plate had conical, pierced indentations

which pointed upwards and allowed the yeast culture to be sparged with

air, if high yeast cell concentrations were required. The stirrer

shaft stroke was altered by varying the voltage to the Vibromix, and

during operation the power input to the fermenter was less than one

watt.

Must was supplied to the fermenter by means of a variable speed

(zeromax gearbox made by Gibson Battle and Co. Ltd, Rydalmere, NSW.)

pump (assembled by the SBT) through a silicone tube of medical quality

(J.C. Ludowici and Son Ltd, Castle Hill, NSW). Each time the pump

speed setting was changed, the must flow rate was recalibrated by

collecting the must pumped through the fermenter between samplings,

usually a 12 or 24 hour period.

3.13 Temperature Control of Fermentation

The temperature meter (SBT) used a thermister (Model No. F23B, STC

Cannon Components Pty Ltd, Liverpool, NSW), which fitted into the well

on the side of the fermenter (Fig. 4).

51.

Fig. 4. Fermentation Vessel.

K, outlet tube for partially fermented must; L, thermister well;

M, flange and clamp assembly; pipettes N.O.P, respectively for must

entry, yeast culture entry, and steam entry; Q, foam escape tube;

R, effluent gas outlet and condenser; S, Mackley filter; T, mixer

shaft diaphragm; U, recirculation loop; V, fortifying spirit

reservoir; W, fortification tube; X, pH probe; Y, vessel for

monitoring DOT in grape juice; Z, infra-red reflector lamp.

52.

The well was partly filled with glycerol to ensure liquid contact between

'the thermistor and the fermenter wall. The thermister was a temperature

variable resistor forming one arm in the Wheatstone bridge circuit of

the temperature meter. The temperature meter (Fig. 5) was calibrated

with a mercury thermometer to read between 16 and 32°C.

The temperature of the fermentation was controlled with an Ether Mini

temperature controller type 1990/1 which had a Resistance Thermometer

type P5 sensor (Electro Chemical Engineering, Artarmon, NSW). The

controller was standardised against the temperature meter by the

controller setting knob (Fig. 5). Control over the temperature of

fermentation was maintained with an infra-red reflector lamp

(275 watt, type FJ26, Osram) that was finely controlled by a time-

proportional (or anticipatory) control system. This system prevented

over-shooting of the set temperature. For fermentations 3°C above

ambient temperature, the above control system was adequate, however,

for fermentations at lower temperatures, temperature control required

a cooling system together with the infra-red reflector lamp.

3.14 Constant Parameters pH and 0xygen

The steam sterilizable pH probe (Model No.SS12DY, Jacoby Mitchell and

Co. Pty Ltd, North Rocks, NSW) was set into a B19 Quickfit socket in

the wall of the fermenter (Fig 4) and together with the pH meter

(Dvnaco Model 21A Jacoby Mitchell) (Fig. 5) was calibrated against

standard buffer solutions. The dissolved oxygen tension (DOT) in the

grape juice was monitored with a Johnson-type oxygen probe (Johnson

and Borkowski, 1967) with some modifications (SBT, Fig. 6, Cootes

and Lee, 1977).

53.

The current generated in the probe was amplified (SBT) and read on a

0-100 microamp meter in the control panel (Fig. 5). The meter was

zeroed by placing the probe in boiled grape juice; the meter scale

was then expanded to full deflection to read 100%, when the oxygen

probe was placed in vigorously aerated grape juice.

Fig. 5. Continuous fermentation assembly control panel; a, temperature

meter; b, temperature controller with setting adjustment;

c, dissolved oxygen meter; d, pH meter.

The heat exchange capacity of the recirculation loop (Fig. 4), which

was placed in a cold water bath, was adequate to maintain a constant

temperature fermentation provided the temperature of the coolant (cold

water) was 5°C lower than the fermentation temperature required. The

temperature of the coolant bath was adjusted by changing the coolant

pump speed (variable speed pump, SBT) and the quantity of ice in the

coolant reservoir (Fig. 3).

54.

Fig, 6. Dissolved oxygen probe constructed by the author.

e, lead anode; f, silver cathode; g, teflon membrane; h, electrolyte;

i, glass wool.

3.2 Materials

3,21 Yeast

Five strains of ScLccha/iomycte c.2Ji&vAj>iaz were used in the continuous

fermentation experiments. Each culture was received in good condition

on agar slants; their origins were as follows:

strains 729 and 350 (tort yeast) from the Australian Wine Research

Institute (AWRI); strains 505 (Champagne yeast) and 522 (Montrachet

yeast) from the Department of Viticulture and Enology, University of

California, Davis; and strain B19 from the New York State Agricultural

Experiment Station, Geneva, New York State (Cornell University,

Geneva - CUG).

55.

3.22 Grape Juice

In experiments one to four, Muscat Gordo grape juice from the Barossa

Valley was filtered into the storage vessel (Fig. 7) through D9 filter

pads (Ekwip Industrial Equipment, Paddington, NSW) followed by a 0.2

micron membrane (Sartorius Membranfilter GMBH Guttingen) and held at

2°C until required. The grape juice vessel could be autoclaved after

which it could be filled and emptied by means of the inlet and

branched outlet (Fig. 7) to avoid airborne contamination. In experiments

five to ten, Semilion grape juice was stored in plastic bags (Fig. 8) oand held at -5 C. The plastic bags, commonly used to store wine in the

"Bag in a Box" package, consisted of laminated layers of polyethylene

and polyvinylidene chloride. There was no evidence of damage to the

bag as a result of freezing.

3-23 Alcohol Fortification

Fortifying spirit 95% v/v ethyl alcohol concentration was held in a

500 ml reservoir (Fig. 4) prior to pumping into the wine fortification

tube (Fig 4). The peristaltic pump (Fig. 3, SBT) was controlled by a

one-hour cycle time clock (SBT) to make either 3 or 6 minute-long

injections of fortifying spirit at regular intervals.

56.

Fig. 7. Sterile grape juice vessel and ancillary services; j, Mackley

filter; k, silicone tubing; 1, sterile stainless steel union;

m, 20 1 Pyrex vessel; n, rubber bung; o, live steam; p, condenser

cooling water; q, gas for flame sterilizing.

Fig. 8. Non-sterile grape juice vessel, r, flexible plastic bung;

s, laminated bag; t, rozen grape juice; u, Olympus microscope.

57.

3.3 Methods

3.31 Yeast Growth and Preparation of Inoculumi ■'" 1" ' "T '1 - ^ r n ‘ " r 1 T 1" "

Each yeast culture was subcultured on potato dextrose agar at three-

monthly intervals and stored at 5°C until required. The yeast inoculum

for each fermentation run was prepared as follows:

A 250 ml sample of grape juice was filtered through a D9 pad and a

0.2 micron membrane into a 500 ml inoculation flask (Fig. 9), which was

autoclaved for 10 min to sterilize the juice and remove all free SO^

which would inhibit yeast growth.

After cooling the juice to approximately 26°C, a loop of yeast cells from

the agar slant, warmed to room temperature during the previous 24 hr, was

dispersed in the sterile grape juice; the flask was incubated for 48 hr

at 26°C on a rotary shaking table (SBT, 100 cycles/min) which ensured

oxygen transfer to promote aerobic growth and a high viable cell concentration

in the culture. After 48 hr, the culture lost its brown colour, indicating

rapid yeast growth and the change to fermentative metabolism.

After sterilizing the one litre fermenter, 900 ml of grape juice was pumped

into it and 0.3 ml of 30% Wfl0 added to Semi11 on, and 0.9 ml was added

if Muscat Gordo grape juice was used (Appendix 6.5). The fermenter was

then aerated for 10 min to remove any remaining S0£. This grape juice

was not sterile and therefore could not be aerated further due to the

presence of non-fermentative yeasts and bacteria, which were still viable

after the frozen grape juice had been thawed. After the addition of ther

inoculum, 20-30x10 cell s/ml were expected in the one litre fermentation

vessel. The yeast grew anaerobically until the alcohol concentration

was 4.5% v/v after which time a continuous grape juice feed was begun.

50.

Fig. 9. Inoculation flask, v, cotton wool bung; w, aluminium foil

cover; x, silicone tubing; y, stainless steel union.

3.32 Preparation and Preservation of Grape Juice

Muscat Gordo grape juice, at 25° Brix and pH 3.4, was used for the

preliminary experiments. This juice had been stored at S. Smith &

Son Pty Ltd for three months at 2-7°C in a pressure tank under 500

kilopascals pressure of CO^. The juice contained 330-10 mg/1 SO^

as determined by the method of Rankine and Pocock (1970). The juice,

which was contaminated with yeast and bacteria and had partially browned,

was anaerobically filtered through a 21 cm diam. Sartorius membrane

filter of 0.2 micron pore size into a 20 litre sterile vessel and

then stored at 20C^1°C.

Semi 11 on grapes grown on the Rosemount Estate Vineyard at Denman in the

Upper Hunter Valley were used for the remaining experiments. The must

of 18.5 Brix and pH 3.2 was sulphited, treated with a pectic enzyme

59.

preparation (Klerzyme 500) at the crusher and settled overnight at 12°C.

•The total sulphur dioxide content was adjusted to 120 mg/1 in the clear

juice, and 0.5 g/1 of tartaric acid was added to bring the titratable

acid of the juice to 7.9 g/1. The juice showed no visible haze, and

was subsequently filled into plastic bags (Fig. 8) under CO^ pressure.

The bags were immediately frozen with dry ice and transfered to a -5°C

room. Six months later, when the juice was thawed, there was no

significant change in pH or the S0? level, and the total acid content

had fallen by 0.2 g/1 due to potassium bitartrate deposition.

3.33 Sterilization of Fermentation Vessel

Three important features of the fermentation vessel - it is constructed

of pyrex, all inlets and outlets are silicone tubing and the stainless

steel stirrer shaft enters through a neoprene diaphragm - allow the

whole fermenter, including air inlet ducts and probes, to be sterilized

with live steam. Micro-organisms can produce spores resistant to steam

at 100°C, therefore, the whole apparatus was autoclaved (Model No.

185U334 Atherton Pty Ltd, Frskineville, NSW) for 30 min every six

months.

Before each experiment, the apparatus was live steamed for one hr, and

the following day live steamed for half an hr, just prior to intro­

duction of the grape juice. The pH and oxygen probes were steamed for

only half an hr, and between fermentations were stored in absolute

alcohol. The sampling port on the fermenter, at the base of the cir­

culation tube (Fig. 4), was heated for 10 min with live steam after each

samp!ing.

60.

3.34 Estimation of Dry Weight of Yeast

Pyrex test tubes with 5 ml capacities were baked for 12 hr at 105°C

and fared. Samples of fermenting must (5 ml) were centrifuged at

2,500 rpii. for 5 min in a small bench centrifuge. The cells were

washed twice with distilled water, dried for 24 hr at 105°C and weighed.

3.35 Estimation of Yeast Cell Concentration

SaccfiaAomycdA ccAevxksTac strains 729, 350, 522 and B19 formed light

flocculations which could be dispersed by rapid agitation. Cell

concentrations of these strains were measured by direct cell counting

and spectrophotometrically with a Spectronic 20. Yeast cell concentration3 2was estimated with the Hawks ley haemocytometer slide, which had 2.5x10 mm

counting squares and was 0.1 mm deep. Degassed, fermenting must was

diluted 1:25, read in a Spectronic 20 at 670 mm and introduced under

the haemocytometer covers!ip with a pasteur pipette; yeast cells were

counted in 10 squares under an Olympus Model E microscope. The number

of budding yeast cells was also counted to give an index of the yeast

cell growth status.

3.36 Estimation of Total Soluble Solids of Fermenting Must

The cylindrical vessel containing a floating hydrometer (Fig. 3) was

used as a method for estimating the total soluble solids in the fermen­

tation vessel. The Brix hydrometer was necessary as an indication of

fermentation rate (disappearance of grape sugar) at the beginning of

continuous grape juice feeding, and after a temperature or dilution

rate change. The cylinder containing the hydrometer was as small as

practicable to hold an 8-14° Brix hydrometer. The Brix reading was

temperature corrected (Amerine and Ough, 1974) and used to calculate

the quantity of fortifying spirit to be added to the partially fermented

must.

61.

3.37 Estimation of Ethanol Concentration of Fermenting Must

Samples of must (10-15 ml) were taken at 12 or 24 hr intervals,

degassed and centrifuged; the supernatant was frozen for ethanol

analysis with a Beckman-4 gas chromatograph (Beckman Instruments,

Inc., Fullerton, USA). The operating conditions of the gas

chromatograph were as follows:

Column - stainless steel, 1.8 metres long 2mm internal

diameter, packed with Porapak Q (100-120 mesh),

temperature 180°C

Injection Temperature - 280°C

Detector - hydrogen flame ionization with chamber at 230°C;

hydrogen was burnt in air

Carrier gas - nitrogen 40 ml/min

Sample size - one pi injected by a 1 pi syringe (Scientific Glass

Engineering, Melbourne, Victoria).

Chart recorder - Speedomax W (Leeds and Northup Co., Philadelphia, USA),

Chart speed 2.538 cm/min

Integrator - Disc Model 224 (Disc instruments, Santa Ana, USA).

The length of the integration line printed by the integrator between its

upper and lower limit (Fig 10) v/as proportional to the area under each

ethanol peak; therefore, the ethanol concentration in the sample was

estimated from the length of this line. The chromatogragh was standardized

with a 5% aqueous ethanol solution, and the accuracy of the method was•f- 0.1% ethanol at this concentration. The ethanol concentration of

the fortified wine was also measured by this method after a 1:4 volumetric

dilution. The column was repacked after 12 months operation.

62.

Internal Standard5% Ethanol

WINE WINEA B

l

Integration Line

MINUTES

Fig, 10. Alcohol peaks from the gas chromatograph and the corresponding integration lines for the estimation of the area under the peaks of wine and standard 5% aqueous ethanol samples.

63.

3.38 Calculation for Fortification of Fermenting Must

The fortifying spirit pump and time clock were calibrated to give

3 ml doses of 95% ethanol (EtOH) at regular intervals varying from

1-12 doses per hr. All wine from the fermentation vessel was

measured in a 500 ml measuring cylinder, to give a flow rate (ml/hr)

of partially fermented must. The amount of EtOH to be dosed into the

outflow stream of partially fermented must from the fermenter was

calculated as follows:

ml ethanol /hr = gL.lerm.ent_iaajnustJfcB)(EtOM% in fortifying spirit-A),

where A is the desired ethanol content in the final wine (18.6, in

these experiments) and B is the ethanol content of the fermenting

must at fortification. The final °Brix of the wine depends on the

amount of ethanol added and the initial °Brix of the grape juice,

hence variations in the final °Brix (p.269, Amerine, Berg and Cruess,

1972) occurred directly as the ethanol concentration in the fermenter

changed; therefore it was necessary to adjust the °3rix of the final

wine to achieve the desired style of finished wine.

3.39 Stabilization of New Wine

New wine from the fermenter was stabilized in a 1 litre flask containing

approximately one days yield from the fermenter. After the addition

of 50 mg/1 SO^, the wine was Brix adjusted and fined by the addition

of 5 ml/I of bentonite slurry (80 g/1 in water) and stored at 5°C

until bottling. A stainless steel, 11 cm Sartorious membrane filter

together with wine bottles, were autoclaved for 15 min prior to

bottling, the corks soaked in a 5% SO^ solution and the screw caps soaked

in 95% ethanol for 2 hr.

64,

Fig. 11. New wine, filtration assembly, a, compressed CO^ (food grade);

b, Ekwip laboratory filter (Model 11) and wine receiver; c, Sartorious

Membrane filter housing; d, silicone tubing and filling tube;

e, bottled clarified wine.

The standard wad for the screw caps was neoprene (to enable autoclaving),

but was found to have an undesirable odour and was replaced with a

polyethylene foam wad. The wine was racked off the bentonite lees and

filled under vacuum into the receiver (Fig. 11); the receiver was

pressurised with nitrogen to push the wine through D9 filter pads and

through a 0.2 micron membrane filter into the bottle. Sulphur dioxide

(30 mg/1) was added to the wine at bottling. The bottled wine was

stored at room temperature until tasting.

65.

4. RESULTS

4.1 Preliminary Experiments in Continuous Fermentation of Grape Juice

for the Production of Fortified Sweet Wine

No previous work has been reported on the production of fortified sweet

wine in a laboratory fermenter fitted with continuous fortification

equipment (description of fermenter, Section 3.1), although some data

has been documented in relation to the continuous fermentation of grape

juice (Section 2). Most of the reports concerning the production of

still wines have been general, qualitative reviews of commercial

experience rather than quantitative dissertations using controlled

fermenters. The most recent work on the continuous fermentation of

grape juice was that reported by Barre and Combe (1975), who discussed

a good feedback control system for controlling fermentation rate

(Section 2.24). Consequently,prelimi nary experiments in this study

were designed to establish suitable dilution rates to maintain constant

ethanol concentration in the fermenter with Australian grape juice as

the substrate and several yeasts that are currently used in the wine

1ndustry.

4.11 Removal of SO^ from Grape Juice

Grape juice storage for use in the 1 litre laboratory fermenter has

been discussed briefly in Section 3.32, and as a result of the high levelst

of SO^ required to protect the Muscat Gordo grape juice from oxidation

and microbial spoilage, the juice used for preparation of yeast starter

cultures was desulphited with hydrogen peroxide (Appendix 6.5).

The stoichiometric oxidation of SO^ to the sulphate ion is shown in the

following reactions:

66.

SO 2 + ^ H^SO^

2HS03" + H202 ------ -> 2H2S04 + 2e”

The quantity of hydrogen peroxide required depends on the form or the

oxidation stage of sulphur in its dissociated form in solution. A

series of test additions of H202 was made to the grape juice, and the

residual S02 levels are shown in Table 7.

Table 7. Residual S02 in grape juice after addition of H202-

Grape juice sample

Addition of cone

H202 (ml/1)

Free S02

(mg/1)

Total S0p

(mg/l)

1 - - 330

2 - - 345

3 - - 342

4 - - 338

5 0.1 16 180

6 0.1 32 175

7* 0.3 - 10

8* 0.3 - 3

Q ** 0.3 -

10** 0.3 ~ -

* Samples 7 and 8 were shaken and immediately assayed.

** Samples 9 and 10 were allowed to stand for 10 min after shaking.

4.12 First Preliminary Continuous Fermentation Experiment

An initial preliminary experiment was carried out to test the equipment

and establish a suitable dilution rate for steady state continuous

fermentation.

67.

Ethanol production from sterile, filtered grape juice by AWRI 729 and

•some fermenter variables are presented, in table 8. The DOT in the

grape juice was arbitrarily set between 10-12% saturation or

approximately 1 mg/1 09 at 22°C. The initial dilution rate of 0.12 hr~^

caused the yeast to wash out of the fermenter, and subsequently a

dilution rate of 0.033 hr""^ was used. Direct cell counts and visual

observation indicated no morphological changes of the yeast cells.

Three wines were made from the production of the fermenter at various

intervals during the run, and due to fluctuations in grape juice

addition and overfortification, the ethanol contents of these wines

varied between 14-20.5%v/v EtOH, and Brix readings varied from

5.6-10.7° Brix.

4.13 Second Prelimi nary Continuous Fermentation Experi ment_]

The dilution rate was set in the range 0.015-0.042 hr in the second

preliminary experiment (Table 9) and in an attempt to simulate industrial

conditions, DOT in the grape juice was set at 50-60%. However, from

subsequent calculations based on the normal requirement of yeast

grown aerobically to consume 33 m mole 0^ (Gray, personal communication)

to produce 1 g of cells, it was apparent that grape juice saturated

with oxygen on being added to an active fennentation at 25 ml/hr was

contributing less than 0.2 mg 0p/hr or 0.1% of the oxygen required for

aerobic yeast growth.

The yeast productivity in molecules of ethanol/cell/sec (m EtOH/c/s)

(Appendix 6.71) provides an average ethanol production per cell, assuming

a constant dilution rate and constant ethanol concentration in the

partially fermented must.

68.

Table 8. A preliminary experiment on the continuous production of

fortified sweet wine in a 1 litre fermenter from sterile filtered,

Muscat Gordo grape juice (25°Brix, pH 3.4) and using S.

strain AWRI 729.

Sample

Time (hr)

DOT

(%)

Yeast count

(cells/mlxl0^)

Temp.

(°C)

pH Di1ution

rate (hr~^)

Eton

(%v/v)Fermenter

product!vi ty (g Et0H/l/hr)

48 11.5 80 22.5 3.2 0.033 7.1 1.8

58 11.5 100 21.5 3.2 0.033 - -

70 12.0 110 21.5 3.2 0.033 6.0 1.5

74 12.0 120 21.5 3.2 0.033 5.5 1.4

82 12.0 260 22.0 3.2 - 6,2 -

94 11.5 - 22.5 3.2 - ~ -

106 11.0 260 21.5 3.2 - 6.7 -

118 5.0 120 21.5 3.2 - - -

130 1.0 — 22.0 3.2 - 4.75

Table 9. A preliminary experiment on the continuous production of fortified sweet

wine in a 1 litre fermenter from sterile filtered Gordo grape juice (25° Brix, pH 3.4)

and using S. cerevislae 729.

Samp! etime

(da/s)

DOT Yeast count Temp

{%) (cells/ml xleP) (°C)

pH Dilution rate

(hr'1)

EtO’rl Yeast producti vi ty+

(%v/v) (mEtOH/c/S x 107)

Fermenter producti vi ty

(gEtOH/1/hr)

2 57 128 22.5 3.2 0.042 6 5.65 0.82

3 57 155 22.5 3.2 0.015 7 1.9 0.99

4 56 145 23 3.2 0.018 7 2.4 1.22

5 56 138 23 3.2 0.023 7 3.3 1.22

6 57 130 24 3.2 0.023 6.5 3.3 1.17

7 57 130 22 3.2' 0.023 6.6 3.3 1.19

8 55 128 22.5 3.2 0.023 6.5 3.4 1.18

9 55 130 22.5 3.2 0.023 6.5 3.3 1.17

10 55 138 22 3.2 0.023 6.3 3.3 1.16

11 55 140 22 3.2 0.023 6.5 3.3 1.18

12 54 130 22.5 3.2 0.023 6.3 3.2 1.14

13 54 no 23.5 .3.2 0.023 6.4 3.8 1.15

14 54 no 23.5 3.2 0.024 6.3 3.9 1.19

15 54 no 22.5 3.2 0.024 6.3 4.0 1.2116 54 155 22.5 3.2 0.024 6.2 3.8 1.1617 55 145 22.5 3.2 0.025 6.3 3.1 1.2318 - - - - - - - -

19 55 140 23 3.2 0.025 6.5 3.3 1.2820 55 155 25 3.2 0.024 6.9 3.0 1.3021 54 145 22 3.2 0.024 7.1 3.4 1.3622 54 155 22 3.2 0.025 7.3 3.4 1.4423 54 155 22 3.2 0.024 7.4 3.3 1.3924 55 140 26 3.2 0.024 - - -

25 - 130 26 3.2 0.024 - - -

+ Yeast productivity - molecules EtOH/cell/second x 10?

70.

The fermenter productivity (g EtOH/l/hr, Appendix 6.72) is used as an

,index of fermentation rate (g EtOH/hr).

The second preliminary continuous fermentation (Table 9) was run for

24 days to demonstrate that prolonged fermentation was feasible. Rod

bacteria were detectable after 11 days, and their concentration reached7

10 cells/ml at the end of the fermentation. During the fermentation

the ethanol concentration in the fermenter varied between 6.2-7.4% v/v;

fermenter productivity did not fall significantly after the onset of

the bacterial infection. The infection was believed to have entered

the fermenter in the filtered grape juice, however, no attempt was made

to improve the capacity or design of the filtration equipment, as it

was planned to use unfiltered, fresh grape juice after the 1976 vintage.

4.2 Semi-continuous Grape Juice Addition to the Fermenter for the

Production of Fortified Sweet Wine

The two preliminary experiments demonstrated that the 1 litre continuous

fermenter was suitable for prolonged production (24 days) of fortified

sweet wine. However, previous experience in industry showed that

irregular deliveries of grapes to the winery could lead to prolonged

storage of grape juice prior to addition to the fermenter (Appendix 6.4)

and under such circumstances the juice needs to be refrigerated to

prevent wild organisms from beginning the fermentation. Simple addition

of high levels of SO^ is considered detrimental to quality and an

expensive additional operation, since desulphitation would be necessary

and is not a legal additive to grape juice or wine in Australia.

In order to avoid storage of grape juice and the extra costs of

refrigerating the grape juice to approximately 5°C, semi-continuous

addition of grape juice to the fermenter was considered.

71.

Toth and Tengerdy (1952) reported consecutive draining off and batch

additions of 50-60% of the fermenter volume, but no quantitative

productivity comparisons were made with continuous grape juice addition.

Consequently two experiments were designed to simulate the commercial

situation of unavoidable breaks in the supply of grapes to the winery,

which may arise in traditional vineyard management practice as a result

of manual grape picking by day, or in more mechanised vineyards as a

result of mechanical harvesting at night.

Semi-continuous addition of grape juice to the 1 litre fermenter using

strain 729 was achieved in the first semi-continuous experiment by

blocking the outlet tube from the fermenter, so that the fermenter volume

increased during each 12 hr period between sampling. The equivalent

dilution rates presented (Table 10) do not take into account the

increased residence time of some of the partially fermented must. The

temperature of the fermenting must was maintained at 22°C and the pH,

which was initially 3.4, declined to 3.2 after two days, and then

remained constant.

The second semi-continuous'experiment using strain 350 (Table 11)

incorporated two new design features in addition to those used in the

first semi-continuous experiment. Firstly, semi-continuous grape juice

addition was modified so that grape juice was added in a single dose

at 12 hr intervals. This was achieved by blocking the fermenter outlet,

adding between 300-400 ml of grape juice, then 12 hr later estimating

cell concentration before stopping agitation.

72.

Table 10. Semi-continuous addition of Muscat Gordo grape juice

(25° Brix, pH 3.4) and using S. cerevisiae 729.

Sanpletime(hr)

Yeast count

(cel 1 s/ml xic£)

Temp

(°c)

Equivalentdilution rate

(hr-"1)

Cell growth

(mg/l/hr)

EtOH Yeast productivity

(£v/v) (mEtOH/c/S x 10^)4

Fermenterproducti vi ty

(gEt0H/l/hr)

48 121 22 0.031 189 6.7 5.0 1.66

60 128 22 0.031 204 5.5 3.8 1.36

72 128 22 - - 6.3 - -

84 120 22 0.030 180 6.0 4.3 1.94

. 95 110 22 0.028 157 5.2 3.3 1.10

108 100 22 0.030 152 5.0 3.9 . 1.20

120 100 22 0.029 149 5.2 3.9 1.22

4 Yeast productivity - molecules of ethanol/cell/sec

Secondly, the flocculent yeast S. ceAzviAiaz AWRI 350 was used to allow

internal recycle of cells. After agitation was stopped, large clumps of

cells settled rapidly under gravitation and as a result a low concentration

of cells remained at the top of the fermenter. The fermenting must from

the top of the fermenter was decanted, and the outlet tube was clamped

before commencing the stirrer and further sampling for analysis. In

table 11 the grape juice addition was averaged over the 12 hr period to

give a value for the equivalent dilution rate. Estimates of fermenter

productivity (g EtOH/l/hr) and yeast productivity (molecules EtOH/c/s)

were calculated from the data obtained by sampling the fermenter prior

to decanting. The sample, prior to direct cell count, was diluted 1:50

with distilled water and mechanically agitated for 10 min to disperse

cells - clumps of 3-6 cells remained in most samples after this treatment.

73.

Vigorous agitation of samples led to inaccurate determination of the

percentage of cells budding; these readings were discontinued after

96 hr. No attempt was made to use an alternative method to estimate

% budding cells, as it was presumed that the low growth rate was due

to high cell count and that an increase in growth rate would be easily

observed.

Steady state was eventually achieved with a cell concentration between 6100-120x10 cells/ml, and fermenter productivity of approximately 0.6 g

Et0H/l/hr. It is not valid to make a statistical comparison between

cell counts and productivities in table 11 with those in table 9

because temperature, dilution rate and ethanol concentration in the

fermenter were not comparable. Given these differences, however, it is

important to note that S. strain 350 produced between

2-3x10^ molecules EtOH/cel1/sec compared to 3-4xl0/ molecules EtOH/cel1/sec

produced by strain 729.

The initial cell concentration of strain 350 was attained by aerating the

1 litre fermenter prior to continuous grape juice addition. The bacterial

contamination, measured under a light microscope, using an haemocytometer

slide was higher at the begining of the experiment (4x10 cells/ml) than

after 188 hr (10 cells/ml). Three observations can be made from the

semi-continuous operation of the fermenter. Firstly, anaerobic conditions

and 5-6% v/v ethanol were largely inhibitory for the growth of contaminating

bacteria and their presence did not restrict the ethanol productivity of

the fermenter. Secondly, internal cell recycle was conveniently achieved

by using a flocculating yeast, but high cell concentrations in an anaerobic

fermenter inhibited cell growth, if the cell concentration was above

120xl06 cells/ml.

74.

Table 11. Semi-continuous addition of Muscat Gordo grape juice (25° Brix, pH 3.4)

and using S. cerevisiae 350.

Sample Yeast count

time before decanting

(hr) (cells/ml x10^)

cells

buddi ng(«)

Yeast count Temp Equivalent EtOH Yeast product!vity4 Fermenter

after decanting dilution rate productivity

(cells/ml xlO6) (°C) (hr-1) (*v/v) (irEtOH/c/S x 107) (gEt0H/l/hr)

48 360 32 400 22.0 0.035 6.4 1.8 1.76

60 400 19 440 21.7 0.030 6.7 1.4 1.58

72 355 21 400 21.5 0.035 6.8 1.9 1.88

84 310 17 380 21.2 0.034 6.3 2.0 1.71

96 275 - 310 19.5 0.035 5.7 2.0 1.57

108 245 - 275 20.0 0.030 5.6 2.0 1.30

120 210 - 245 21.7 0.031 5.8 2.2 1.42

136 165 - 210 19.5 0.027 5.4 2.6 1 .15

154 165 - 245 19.0 0,020 5.4 2.0 0.87

178 145 - 175 18.2 0.017 5.4 2.3 0.72

192 145 - 160 19.5 0.016 5.6 1 .9 0.71

216 no - 145 19.5 0.017 5.5 2.3 0.76

240 100 - 135 19.5 0.020 5.5 3.1 0.87

261 120 - 145 20.5 0.012 6.1 1.6 0.57

288 120 - _ 20,5 0.013 6.0 1.7 0.61

4 Yeast productivity - molecules of ethanol/cell/sec

Thirdly, it was possible to make fortified sweet wine of acceptable quality

by semi-continuous, grape juice addition to the fermenter. No attempt was

made to measure the variation in yeast productivity between successive grape

juice additions. It was apparent that the fermentation reached steady state_1

between 164-188 hr with a dilution rate between 0.012-0.013 hr . This is

equivalent to a 76-83 hr retention time for the fermenting must, which is

understandably high since the grape juice contained 330-345 mg/1 SO^.

75.

Eight wines were made during this experiment, and these wines were

preferred when compared to those from previous experiments. The

improvement in quality was attributed to the yeast 350. The wines

retained a richer muscat character and appeared to be more palatable.

In all experiments with Muscat Gordo juice which had been stored

for up to 10 months without freezing and had browned slightly,

continuous fermentation stripped the brown colour from the juice,

producing a straw yellow wine (Fig. 12).

Before Afterfermentation fermentation

Fig. 12 Removal of brown pigment during continuous fermentation of

browned Muscat Gordo grape juice.

Two reasons for the removal of the brown pigment are postulated;

firstly, the strong reducing conditions during continuous fermentation

(low - Martakov, 1970) reduce flavonoids, melanins and the other

coloured compounds (Peri oX at., 1971) and secondly, the coloured

compounds are adsorbed onto the protein and carbohydrates in yeast

cell walls (Fleet, personal communication), which resulted in grey-

76.

brown yeast sediments in the gross lees.

t

4.3 Continuous Fermentation using Different Strains of S. eg

There has been no work reported in the literature concerning the ethanol

productivity of different yeast strains in continuous fermentation,

hence the next series of experiments was designed to compare the

fermentation rates of five strains of S. ceAzv^-lae. in the continuous

fermenter developed for the production of fortified sweet wine.

Previous work (Ough, 1966a5b} on the kinetics of alcoholic fermentation

have been concerned with changing sugar and ethanol concentrations

normally encountered in batch fermentations. Amerine and Joslyn (1970)

have commented that there is an inverse relation between fermentation

rate and flavour quality; this relationship was assessed in these

five experiments. Some of the fermentations were extended to achieve

steady state conditions at different temperatures. The five strains

compared were AWRI 729, 350; UCD 522, 505; and CUG B19. The first

two strains were chosen because they are widely used throughout the

world for still wine production, and 505 is also widely used for

champagne making; 350 or 'port' yeast is used for dessert wine

production in Australia and is recognised as producing a flowery

bouquet character; 729 is the most extensively used wine yeast in

Australia; and B19 was selected by Professor Steinkraus (Cornel Uni­

versity, Geneva, New York) for his experiments in 'rapid' and con­

tinuous fermentations.

The productivity data for the five yeasts are presented in tables

12-16. Strain 505 or ’Champagne' yeast was the only yeast in these

experiments which could not be counted by direct cell count in an

haemocytometer slide under a light microscope.

77.

This became apparent only after the beginning of continuous grape juice addition to the fermenter after which the cells flocculated strongly, consequently an alternative method was immediately adopted.The most accurate method was considered to be determination of yeast dry weight by incubation for 12 hr at 105°C after washing and centrifuging. The cell count was then calculated by assuming that 1 g dry cells/1 was equivalent to 102x10 cells/ml (Appendix 6.8).The low dilution rate for yeast strain B19 after 60 hr (Table 16) was due to fatigue and stretching of the must pump tube.

The fermenter productivity of each yeast strain and the ethanol concentrations attained at 26°C at varying dilution rates without internal recycle are presented in figures 13-15. Lines have been fitted to the data points by visual estimation so that the differences between yeast strains can be easily observed. The range of dilution rates, 0.039-0.095 hr~\ was narrow and resulted from the practical requirement to produce partially fermented must at approximately 4.5% v/v ethanol. If this ethanol concentration had not been maintained the ethanol and grape sugar concentrations would have varied so much that no realistic sensory evaluation of the finished wines could have been made nor would the control of wild organisms have been as effective, for a dilution rate above 0.1 hr~\ that is an average retention time for must in the fermenter of less than 10 hr, one could still expect steady state both in fermenter productivity, cell concentration (which

rmay be as low as 30x10 cells/ml) and ethanol concentration which may have been as low as 3% v/v.

70.

Table 12. Continuous addition of Semillon grape juice (18.5° Brix, pH 3.1) to a 1

litre fermenter using S. cerevisiae strain 729 for the production of fortified

sweet wine.

Sample Yeast count cells Temp

time

(hr) (cell s/ml x 1(f) budding(%) (°C)

Di 1 ution

rate

(hr'1)

Retention

time

(hr)

EtOH Yeast productivity* Fermenter

producti vi ty

(?v/v) (mEtOH/c/S x 107) (gEt0H/l/hr)

48 132 40 25.5 0.041 24 4.9 4.3 1.57

72 167 20 28.0 0.041 24 6.4 4.7 2.04

96 162 32 26.0 0.039 25 6.6 4.2 2.03

120 120 30 26.0 0.060 16 5.4 7.7 2.55

144 120 29 25.8 0.067 15 4.5 7.2 2.38

168 96 20 26.0 0.080 13 4.0 9.4 2.40

192 105 34 26.0 0.080 13 4.1 8.9 2.59

216 115 40 25.5 0.078 13 3.9 7.5 2.40

240 107 32 25.8 0.078 13 4.1 8.5 2.52

264 115 40 26.0 0.080 13 4.2 8.3 2.65

288 78 75 21.5 0.078 13 3.3 9.4 2.03

312 115 25 21.5 0.061 16 4.9 7.4 2.35

4 Yeast productivity - molecules ethanol/cell/sec

If the level of 4.5% v/v is accepted as a desirable level to inhibit wild

organism growth (Section 2.24), then the corresponding dilution rates

for each yeast can be estimated from the graphs at 0.07, 0.052, 0.076,

0.1 and 0.076 respectively (Figs 13-15) for 729, 350, 522, 505 and B19.

Similarly at these dilution rates, which are also the optimal growth

rates for each yeast in must at 4.5% EtOH, expected fermenter productivities

can also be estimated by reference to the fermenter productivity curve at

the above dilution rate for each yeast strain, that is respectively 2.5,

1.2, 2.6, 3.4 and 2.7 g Et0H/l/hr (Figs 13-15).

79.

Table 13. Continuous addition of Semillon grape juice (18.5° Brix,

pH 3.1) to a 1 litre fermenter using S. cerevlsiae strain 350 for the

production of fortified sweet wine.

Samp!e Yeast count ccl 1 s Temp Dilution rate Retention Eton Yeast productivity4 Fermentertine

(hr) (cell s/ml xl Cfi) budding(%) (°C) (hr'1)time

(hr) ("v/v) (mEtOH/c/S x IQ7)product! vi ty

(gE t OH/1 /hr)

48 102 44 26.0 0,067 15 3.4 6.3 1.78

72 48 33 26.5 0.069 15 3.6 14.7 1.84

96 49 65 25.5 0.078 13 3.0 13.S 1.87

120 64 50 26.0 0.078 13 3.7 12.8 2.28

144 42 80 25.5 0.079 13 3.0 16.2 1.87

168 72 58 26.0 0.079 13 3.4 10.7 2.12

192 40 65 26.0 0.079 13 3.3 18.7 2.05

216 68 70 22.0 0.079 13 3.0 10.0 1.87

240 61 55 23.0 0.079 13 3.2 11.9 2.00

264 104 85 22.5 0.078 13 3.3 7.1 2.03

288 96 15 22.0 0,079 13 3.1 7.2 1 .93

312 80 70 18.0 0.053 19 4.4 8.3 1 .84

336 90 60 18.0 0.053 19 3.4 5.7 1 .42

* Yeast productivity - molecules ethanol/cell/sec

Strain 505, a flocculent yeast, has the highest fermenter productivity;

the other flocculent strain 350 (Fig, 16) has the lowest. Strains 729,

522 and B19 have similar fermenter productivities at 26°C, 2.5, 2.6 and

2.7 respectively for dilution rates between 0.07-0.076 and for future

comparisons it is considered that there is no significant difference.

No exacting statistical analysis was applied to the fermenter productivity

data because it was felt that more data points were needed in figures 13-15

before regression lines could be mathematically fitted.

80.

The changes in cell morphology for 505 and 350 during continuous

fermentation are shown in figure 16(a,b). During fermentation prior

to continuous feed of grape juice, the cells were normal or ovoid in

shape (Fig. 16c,d). However, both 505 and 350 were flocculent and

after the commencement of continuous grape juice addition the floes

became larger and more difficult to disperse.

Table 14. Continuous addition of Semillon grape juice (18.5° Brix, pH 3.1) to a 1 litre

fermenter, using S. cerevisiae strain 522 for the production of fortified sweet wine.

Sampletime(hr)

Yeast cells Temp

(cel 1 S/frl x 1 (A) buddinc/X) (°C)

Dil utionrate

(hr'1)

Retentiontime(hr)

EtOH

(%v/v)

Yeast producti vi ty+

(mEtOH/c/S x 107)

Fermenterproducti vi ty

(gEt0H/l/hr)

48 100 - 22.0 0.048 21 4.0 5.5 1.51

72 100 36 21.5 0.048 21 3.9 5.4 1.48

96 90 25 21.5 0.045 22 3.4 5.0 1 .20

120 67 40 22.0 0.069 16 3.6 10.6 1.96

144 40 64 22.0 0.069 16 3.4 16.8 1.85

168 45 30 22.0 0.069 16 4.6 20.2 2.50

192 50 48 22.0 0.069 16 4.4 17.4 2.40

216 115 24 26.0 0.069 16 4.8 8.2 2.61

240 105 32 26.0 0.086 12 4.1 9.6 2.78

264 75 33 26.0 0.086 12 ' 4.0 13.1 2.68

288 62 60 26.5 0.086 12 4.0 15.7 2.68

312 37 56 26.0 0.083 12 4.0 25.4 2.62

+ Yeast productivity - molecules ethanol/cel 1/sec

At the. same time elongation of the cells was evident. Day, Poon and

Stewart (1975) showed an increase in cell wall fimbriae with an

increase in flocculence, and McMurrough and Rose (1967) found an

increase in cell wall phosphorus and protein contents as the cells

elongated. Both of these hypotheses are consistent with the increased

flocculence and elongation found in 350 and 505 in these experiments.

81.

Table 15. Continuous addition of Semillon grape juice (18.5° Brix, pH 3.1) to

q 1 litre fermenter, using 5. cerevisiae strain 505 for the production of

fortified sweet wine.

Sample

time

(hr)

Yeast count Temp 1

(cells/ml xlO6) (°C)

Dilution rate Retention

time

(hr-1) (hr)

EtOH

(2v/v)

Yease prcducti vi ty+

(mEtOH/c/S x in7)

Fermenter

producti vity

(gEt0H/l/hr)

48 65 26 0.078 13 4.9 16.7 2.98

60 56 26 0.072 14 4.6 17.0 2.61

72 70 27 0.072 14 4.6 13.4 2.58

84 90 26 0.095 11 4.6 13.8 2.41

96 130 26 0.093 11 4.6 9.3 3.34

108 140 26 0.094 11 4.6 8.9 3.41

+ Yeast productivity - molecules ethanol/cel 1/sec

Table 16 Continuous addition of Semi 11 on grape juice■ (18.5° Brix, pH 3.1) to a 1 litre

fermenter, using S. cerevisiae strain B1 9 for the production of fortified sweet wine.

Sampl e Yeast count Cells Temp Dilution Retention EtOH Yeast productivity+ Fermenter

time rate timb productivity

(hr) (cells/ml x 1C^) budding(X) (°C) (hH) (hr) (Sv/v) (m/EtOH/c/S x 107) (gE 10H/1/hr)

48 72 5 26.0 0.075 1 3 4.8 14.3 2.84

60 48 50 26.0 0.070 1 4 4.7 19.4 2.57

72 58 60 26.0 0.062 1 6 4.6 14.1 2.25

84 75 56 26.0 0.053 i 9 4.5 9.1 1.88

96 75 52 26.0 0.078 1 3 4.6 13.7 2.83

+ Yease productivity - molecules of ethanol/cel 1/sec

FER

MEN

TER

PROD

UC

TIV

ITY

(g Et

OH

/1/h

r)

82.

DILUTION RATE (hr'1)

Fig. 13. The relation between dilution rate, ethanol concen­

tration and fermenter productivity for 5. strains 729

and 350 at 26°C; 729 - o,©; 350-q,c3 . ©,cs - ethanol concentration;

o, □-fermenter productivity.

ETH

AN

OL(

%v/

v)

83.

Fig. 14. The relation between dilution rate, ethanol concentration

and fermenter productivity for S. ceAeoAUae strains 522 and B19 at

26°C; 522-A,A; B19-o,o. (5,A~ethanol concentration; o,A-

fermenter productivity.

Fig. 15. The relation between dilution rate, ethanol concentration

and fermenter productivity for S. dOJiaviAicLt strain 505 at 26°C;

a -ethanol concentration; □ -fermenter productivity.

1

84.

(a) Strain 505 (x4,000)

(b) Strain 350 (x2,621)

; : S:(c) Strain 522 (x4,000)

fis

(d) Strain 729 (x4,000) j tr

' £Hik%£. 4Fig. 16. Effect of continuous fermentation on the morphology of four

strains of S, cQjiQ.\)Aj>iaa. Strain 350 (b) was photographed by Dr. M. Dixon

under a scanning electron microscope (Stereoscan, Model S4.10, Cambridge

Scientific Instruments Ltd, England); the rigid cell walls of strain 350

enabled air drying and gold coating before photographing (Appendix 6.9).

Strains 505(a), 522(c) and 729(d) were wet mounted cells, photographed

under a Zeitz light microscope using Kodachrome, 25 ASA.

85.

Strain 522 and 729 (Fig. 16c,d) and B19 (not shown) were normal or

ovoid during continuous fermentation and did not flocculate.

In the experiments with 729 and 350, wild bacteria entering the

fermenter with the natural sulphited grape juice were present inr

sufficient numbers (1x10° cell s/ml) to count after 200 hr, however,

they were still difficult to detect at the end of the experiments.

In the experiments with 522, 505 and B19, no bacteria were detected

by direct cell count even though they were expected to be viable

after the grape juice had been thawed (Section3.32). No regular

assays were made to estimate the bacteria status of the fermentations,

because the bacteria were not expected to grow faster than the dilution

rates used and contaminant analysis was undertaken only.if wine quality

v/as detrimentally affected. After 192 hr (Table 12) an unpleasant

H^S-type odour was detected in wines made from Semi lion grape juice

using strain 729. A Lysine medium (Appendix 6.10) was used to isolate

a wild yeast, which was pink in colour - further testing (Appendix 6.11)

indicated that the yeast was a non-fermenter and probably a species

of the genus Rkodoto^ula, It was therefore introduced into the

fermenter with the thawed grape juice and would not have been growing

in the fermenter. It is unlikely therefore to have contributed to the

off odours detected in the wines. Although it is possible that the bacteria

previously mentioned could contribute to the off odour, it is believed

that the odour v/as caused by the yeast S. strain 729; the

off odour did not persist in subsequent wines.

Thirty six wines were made from the five experiments. The volume of

each wine was approximately 1 litre or the output from the fermenter

in one day, depending on the dilution rate.

86.

One sample of each wine, adjusted to 18% v/v ethanol and 4.8°Brix,

has been stored at room temperature for future evaluation, while the

other sample of each wine was tasted by three professional wine

makers, who considered all samples commercially acceptable. Significant

aroma variation between and within experiments was apparent from the

wines prepared. This is contrary to earlier reports in the literature

by Amerine at at, (1972), who stated that wines made by continuous

fermentation were of uniform composition. It is considered that a

change in grape quality, variety or maturity will rapidly affect the

flavour of the product in a continuous fermenter producing fortified

sweet wine. This claim is valid if a relatively small fermenter

capacity is used and the average retention time approaches that

determined in the 1 litre laboratory fermenter, approximately 11-13 hr

(Tables 12-15). Grape juice with a high sugar concentration will have

a longer retention time, conversely if wines made have a higher sugar

requirement, the retention time will be shorter.

4.4 Control Procedures in Single-stage Continuous Fermentation of Grape

Juice for the Production of Fortified Sweet Wine

The previous experiments described in section 4.4 have provided valuable

information on yeast activity and fermenter productivity that will be

discussed here as an introduction to the next experiment, which will

define more accurately the effect of temperature on yeast activity in

continuous fermentation of grape juice.

An increase in the dilution rate at 96 hr after the commencement of the

experiment with strain 729 (Table 12, Fig. 17) reduces the ethanol

concentration in the fermenter, and a decrease in the dilution rate

87.

ooLUorZI—<cccLUD_SUJ i—

3

>- I—t—i

2

1

oJZ ZD\ o i— o ccO-zo•4-JLU ccLUcn z

-----UJz:DCUJLu.

0.08 i C-JZ0.07 LUh-<0.06 cc

zoHH0.05 f—z

J 0.042Q

>- CD

i—i X> COh- O

Q O

C£ LU

RE 130

LU O

SAMPLE TIME (hr)

6.5

5.5

4.5

3.5

100

50

0

>N.>Z5

oz<zh~1U

v__.

CDz>—IoQZCD

C/D_U-ULUC_D

Fig. 17. Effect of continuous addition of Semilion grape juice

(18.5°Brix, pH3.1) using S. coJitviAiaz strain 729 on yeast count (a),

cells budding (©), yeast productivity (v), ethanol (a), dilution

rate (ns), temperature (d), neuter productivity (o).

*yeast. productsmolecules &-VV\anol/cell/sec

88.

S3------ O-—-Q

n —

0.04L

>- o 25

o oC£ UJ

xOE 90

____ -Cr

144 192SAMPLE TIME (hr)

5.0

4.5

4.0

3.5

100

50

0

Fig. 18 Continuous addition of Semi 11 on grape juice (18.5°Brix,

pH 3.1) using S. ce.sio.vjj>lac strain 522; yeast count (a), cells

budding (o), yeast productivity (▼), ethanol (a), dilution rate (a),

temperature (□), fermenter productivity (o).

*yeast productivity - molecules ethanol/cell/sec

CE

LLS

BUD

DIN

G (%)

ET

HAN

OL (%

v/v)

TE

MPE

RAT

UR

E ( C

)

89.

^ 0.095

£ 0.085

0.075 -

SAMPLE TIME (hr)

Fig. 19. The relation between fermenter productivity (o) and

dilution rate ( n) (correlation coefficient r = 0.74, significant

at b% level, 5 degrees of freedom) on the continuous addition of

Semilion grape juice (18.5°Brix, pH 3.1) using S.

strain 505.

FER

MEN

TER

PRO

DU

CTI

VIT

Y (g

Et0H

/l/hr

)

90.

?

niafter 144 hr increased the ethanol concentration, however, there appears

to be no effect on the fermenter productivity. This is due to a

reduction in the percentage of cells budding and the yeast count. In

the experiment with strain 522 (Table 14, Fig. 18), an increase in the20

ethanol concentration occurs between 96 and 116 hr, simultaneous with

an increase in fermenter productivity (g EtOH/l/hr) and again a fall

in yeast count. The increased ethanol concentration limited further

yeast activity, and the fermenter productivity remained relatively

constant. These fluctuations were not apparent in the experiment with

strain 505, and an increase in cell count (Table 15) and fermenter

productivity resulted from an increase in dilution rate (Fig. 19). A

positive correlation (r = 0.74, significant at the-5% level for 5 degrees

of freeciom, Appendix 6.12) was obtained between dilution rate and

fermenter productivity for strain 505. Two observations can be made

from this data - firstly, that yeast activity (growth and ethanol

production) will respond to small changes in dilution rate at steady

state, so that an increase in dilution rate will increase growth rate

and yeast productivity. Secondly, as the dilution rate approached the

optimum yeast activity, there was no significant change in productivity;

this is demonstrated more clearly in figure 14 between dilution rates

0.08-0.09 hr~\ Further increases in dilution rate reduced the ethanol

concentration below that necessary for the wine style being produced.

Figure 19 shows that a change in yeast count of strain 505 is preceeded

by an inverse change in % of cells budding. The same correlation is

shown in tables 12 and 16, which further supports the conclusion from

the data in table 11, that the growth rate of each yeast strain is

critically affected by the cell concentration.

91.

0.04 0.05 0.06 0.07 0.0DILUTION RATE (hr'1)

Fig. 20. Effect of dilution rate on ethanol concentration and

fermenter productivity in continuous fermentation of Semi 11 on grape

juice (18.5°Brix, pH 3.1) using S. strain 729 at; 21°C (□,**),

26°C (A, a); ethanol concentration (b, a); fermenter productivity

( □ , A).

ETH

ANO

L (%v/

v)

92.

These experiments show that in a continuous fermenter without internal' #

recycle the maximum equilibrium cell concentrations in fermenting grape

must containing 4-5% EtOH v/v at 26° are: 120, 104, 115, 140 and

75x10^ cells/ml, respectively, for strains 729, 350, 522, 505 and B19.

Sufficient data is available to demonstrate the effect of a 5°C

temperature change on the ethanol concentration and fermenter

productivity for strain 729 (Fig. 20). Steady state data at 21 and

26°C has been taken from tables 12, 14 and 17, and lines have been

fitted to the data points by visual estimation. A 5°C change in

temperature at a dilution rate of 0.07 hr-"* changes the fermenter

productivity by 0.7 g Et0H/l/hr and the ethanol concentration by 1.4% v/v

It is believed that a similar family of curves could be obtained for

each of the yeasts over a wider range of temperatures and dilution

rates. It is important to note that assays for ethanol were made at

24 hr intervals, and although this is acceptable for overall control

of the fermentations that were stable during a 24 hr period, it provides

no information about point changes in yeast activity immediately after

a temperature change.

An experiment was designed to provide further information on the effect

of temperature on the fermenter productivity of strain 729 in the 1

litre fermenter. The temperature of the fermenter was dropped 11 C,

while the dilution rate was held constant; this drop in temperature

took 2 hr due to the inefficiency of the heat exchange system.

^calculated from dry weights (Appendix 6.8)

s L

93.

Table 17. Effect of temperature on fomentation productivity by $. cerevislae strain

729 in the continuous production of fortified sweet wine with continuous addition

of Semilion grape juice (18.5° Brix, pH 3.1).

Sampletime(hr)

Yeast count.;

(cells/ml x 1 $)

Cells

budding(%)

Temp Dilution rate

(°C) (hr"1)

Retention EtOHtime(hr) (%v/v)

Yeast productivity4

(mEtOH/c/S x 107)

Fermenterproductivity (gEt0H/l/hr)

24 145 42 26 0.058 17.2 5.2 5.9 2.38

36 105 50 26 0.056 17.8 4.8 7.3 2.12

43 96 65 26 0.056 17.8 5.0 8.3 2.21

60 75 39 15 0.059 16.9 3.45 7.8 1.60

72 27 72 15 0.046 21.7 1.86 9.0 0.67

84 57 85 20 0.044 22.7 2.25 4.9 0.78

96 105 64 20 0.048 20.8 3.0 3.9 1.13

108 105 48 20 0.042 23.8 3.2 3.6 1 .06

120 96 57 20 0.055 18.1 3.7 6.0 1.60

-fYeast productivity - molecules of ethanol/cel l/'sec

The effect of the temperature drop on yeast growth and ethanol productivity

is shown in table 17 and figure 21. Within 24 hr the fermenter

productivity had fallen from 2.21 to 0.67 g EtOH/l/hr, and the cell

growth was about half that before the change. It was apparent that the

fermentation would washout despite a small reduction in the dilution

rate, and consequently the temperature was increased to 20°C. The

effect on yeast productivity (molecules EtOH/cel1/sec) was not significant

in the first 24 hr, however, due to the method of analysis, point changes

in ethanol production per cell were not apparent.

94.

>- o> tnh- u

Q OO -t~>O' UJ

ZD XoO E

UJ u

SAMPLE TIME (hr)

5

4

3

2

—! o<cdzb-LU

80'CO2i—i

QCDZDCO

LjJCJ

Fig. 21. Effect of temperature on fermenter productivity by S. ceJizvUiaz

strain 729 in the continuous production of fortified sweet wine with

continuous addition of semilion grape juice (18.5°Brix, pH 3.1), yeast

count (A), cells budding (o), yeast productivity (y), ethanol (a),

dilution rate (n), temperature (□), Fermenter productivity (o).

Yeast productivity - molecules ethanol/cel 1/sec.

95.

The yeast productivity was calculated from the average cell concentra­

tion during the 12 hr period and the total ethanol produced during

that period.

A good correlation (r=0.86) exists between temperature and the

ethanol concentration in the experiment (Fig 21). Tv/elve hr after the

temperature shock, or 48 hr after commencement of continuous must

addition, cells budding had fallen from 65 to 39% and was presumably

stable at this time. The fall in ethanol concentration after 24 hr

was due to a 72% washout of cells, since the remaining cells continued

to produce ethanol though at a slower rate.

The correlation (Appendix 6.12) between fermenter productivity

(g EtOH/l/hr) and temperature (r=0.75) was significant (at 5% level

with 8 degrees freedom); fermenter productivity reduced from 2.21 to

0.67 g EtOH/l/hr in the 24 hr after the temperature shock. The

fermenter productivity recovered to 1.6g EtOH/l/hr within 48 hr after

the temperature was increased to 20°C; furthermore within 24 hr cellg

count returned to 105x10 cells/ml as a result of budding increasing

to as high as 85% of cells counted.

This experiment adequately demonstrates the importance of temperature

control in a continuous fermenter. If a shortage of grape juice in

a winery meant that a continuous fermenter had to be maintained for

several days before resupply of grape juice, then temperature shock

would be more appropriate to slow the fermentation than a slow or

rapid reduction in dilution rate. In the latter case a build up of

ethanol would occur in the fermenter increasing cell death rate, so

that a fermentation recovery would be slow compared to recovery after

temperature shock (Fig. 21).

96,

5. DISCUSSION AND CONCLUSIONS

Each yeast species and strain has a specific growth rate and rate of

ethanol production which can be measured separately in a laboratory

scale continuous fermenter over a range of influencing variables,

such as pH, temperature, ethanol concentration, CO^ pressure,

nutrient status and Brix of the must. In the wine making process

these variables are not always set to optimise yeast growth or

ethanol production rate, but rather to suit quality requirements

for the product. For example,a pH range of 3.1 - 3.6 is desirable

for producing wine with a crisp clean flavour, colour stability and

for reducing bacterial contamination, but this range is not optimum

for yeast activity. In addition, grape juice has a high buffering

capacity, and the pH may not easily be adjusted for optimum yeast

activity without making the wine either too acid or too flat. Similarly,

at the optimum temperature for yeast growth and ethanol production,

the flavour profile of the volatile fermentation by-products may

not be acceptable. Consequently value judgements of wine quality musti

be made before temperature, pH, ethanol concentration and dilution

rate are adjusted for a continuous fermenter.

The preliminary experiments showed that yeast growth and productivity

were decreased by high SO^ and low dilution rates. Low dilution rates

permitted high ethanol concentrations in the fermenter, which further

suppressed fermentation rates, and allowed retention times of up to

80 hr in the fe mien ter.

In batch fermentation Ough (1964) measured fermentation rate by the

disappearance of sugar, however, fermenter productivity (g EtOH/l/hr)

is a more reliable indicator of fermentation rate in a continuous

fermenter.

97.

The low E, during fermentation in a continuous fermenter was demonstrated

by the removal of the brown pigment in Muscat Gordo grape juice.

Continuous and semi-continuous grape juice addition to the fermenter

were compared, and batch additions of grape juice, twice daily, do not

significantly lower the productivity of the fermenter, as only 30% of

the fermenter volume is removed at any time. If single batch additions

to the fermenter are required in a 24 hr period, then the total capacity

of the fermenter must be doubled so that only 30% of the fermenter volume

will be removed. However, 30% removal in 12 hr represents a dilution

rate of 0.025 hr-1 and 30% removal in 24 hr represents a dilution rate

of only 0.013 hr \ and this will correspond to a lower fermenter

productivity. There is no experimental evidence to show that grape juice

additions larger than 30% would not detrimentally affect fermenter

productivity. Toth and Tengerdy (1952) made reference to larger additions,

but no productivity data was presented. The principal reason why additions

larger than 30% detrimentally affect fermenter productivity is that large

quantities of yeast cells are washed out and the ethanol concentration

is diluted, so that contamination by wild organisms is more likely.

The reduction in yeast concentration could be minimized by using a

flocculent strain of yeast which would maintain a high concentration

of cells due to internal recycle. A comparison of the material pro­

cessing systems recommended for commercial operation of the two systems,

batch and continuous addition of must, is shown in appendix 6.6.

Five strains of S. were compared under continuous fermentation

conditions and wide variations in cell concentration and growth rate were

observed, and the productivity of the fermenter with the five strains

ranged from 1.2 - 3.4 g EtOH/l/hr, strain 505 showing the highest

productiwity.

98.

Fermenter productivity at 26°C increased with increasing dilution

rate over a particular range of dilution rates for each yeast and

ethanol concentration in the fermenter decreased.

Eschenbruch oX at. (1973) reported that 729, claimed to be an SO^-

forming strain, could produce sulphide from sulphite and sulphate,

and this study supports those findings, although H^S production was

not measured quantitatively.

In the continuous fermentation system, it was demonstrated that fermenter

productivity could be controlled by temperature and dilution rate.

For yeast strain 729 a change in dulution rate from 0.04 to 0.08 hr ^

increased fermenter productivity from 1.57 to 2.59 g EtOH/l/hr.

Similar effects of dilution rate were demonstrated with strains 350,

522, 505 and B19.

A 5°C change in the temperature of fermentation for strain 729 at a~1dilution rate of 0.07 hr changed the fermenter productivity by 0.7

g Et0H/l/hr, which represents an adjustment of 36% (Fig. 20). Similarly/

an 11°C drop in temperature reduced the fermenter productivity from

2.21 to 0.67 g Et0H/l/hr or a 60% reduction (Fig. 21). This method

for restricting a fermentation is preferred to that of nutrient and

substrate limitation if rapid recovery is required at a later time

(Fig. 21).

The cell growth rate was significantly affected by the cell concentration,

and each yeast strain showed a specific equilibrium cell concentration

at steady state.

99.

It is apparent from this data that ethanol inhibition as a bio­

chemical inhibitor of yeast activity cannot be simply explained.

Hence, more fundamental work must be carried out at the cellular

level to determine the nature of ethanol inhibition in membrane

transport and metabolic systems. Further data is also needed for

each yeast to extend the temperature and fermenter productivity

profiles over wider ranges of dilution rate and temperature.

The control of product formation is the most important, single aspect

of fermenter productivity control in a continuous fermenter with a

high substrate concentration, such as grape juice. This is in

direct comparison to substrate limited systems, where cell growth

rate can be used to control fermenter productivity. Ethanol

concentration in the fermenter with a known dilution rate is the

best index of fermenter productivity, but continuous ethanol

measurement on an industrial scale requires an auto-analyser or a

gas chromatograph with automatic injection. Sophisticated and expensive

equipment of this nature can only be justified in a large winery and

the estimation of ethanol concentration from the loss of grape sugar

by density, specific gravity or refractive index, is normally used.

Stackhouse (1966) described an inline or vessel mounted, critical angle

refractometer with a synthetic diamond prism for the measurement of

soluble solids in brewer's wort. Currently, Anacon (Anacon Inc.,

Ashland, Massachusetts) have similar equipment available, a Model 47

process analyzer. The main disadvantage of a refractometer is that

the prism surface must be cleaned regularly.

100.

Anton Paar (Anton Paar K.G., A-8054 Graz, Austria) are currently

,marketing a density meter, in which the filled, U-shaped sample tube

is electromagnet!'cally excited to vibrate at its natural frequency;

the resonance is compared to a known standard for density calibration.

The main disadvantage with this or any assay procedure external to the

fermentation vessel is contamination, and the necessary sterile

sampling procedures.

Moller (1975) described an instrument called a Platometer, which is

intended for permanent mounting within a fermenter. The Platometer

is a differential pressure meter, automatically compensated for

temperature variations. The principle of the Platometer is that

hydrostatic pressure between two points in a fermenter is a function

of the vertical distance between the points and the density of the

liquid. Two stainless steel diaphragms, mounted 45 cm apart, sense

the pressures and hydraulically transmit them to a transducer, which

converts the pressure differential into anelectronic signal. This

method of total solids estimation appears to be the most convenient

available and is currently cheaper than the previous methods discussed,

costing less than $2,000 to fully automate measurement in six

fermenters. If the °Brix of grape juice entering the fermenter varies

significantly, it is necessary to compensate for these changes if

ethanol concentration is to be accurately estimated with a Platometer,

as constant ethanol concentration in the fermenter is the critical

factor in maintaining a constant fermentation rate. The next step

in automatic control of a continuous fermenter is to compare the

ethanol concentration with a predetermined set point (ie., 4.5%,

depending on the wine style required), and each time the ethanol

101.

concentration in the fermenter rises above the set point the

dilution rate should be increased to compensate. Similarly,if the

ethanol concentration falls below the set point, dilution rate should

be reduced. Barre and Combe (1975) have described a similar system

for control of a 1 litre laboratory continuous fermenter.

102.

6. APPENDIX

6.1 Glossary

Backmix reactor - fermentation vessel in which any

medium addition is instantly dispersed.

Chemostat - backmix culture vessel in which the

growth rate of the organism is limited

by the supply of a substrate nutrient

Culture

(jj < JU ).

- fermentation broth containing an

organism and substrates necessary

for growth.

Fermentation - an organism uses fermentation catabolic

pathways to derive energy for growth -

may be aerobic or anaerobic.

Fermentation productivity - rate of accumulation of ethanol per

unit volume of the fermenter per hour,

g EtOH/l/hr.

Fermentation rate - rate of accumulation of ethanol or

disappearance of grape sugar per hour,

g/hr.

Mass balance equation - technique used to equate two

simultaneous changes in a measurable

variable; e.g., consumption of

glucose by an organism and disappearance

of glucose from the medium feeding the

organism.

Product-limited reactor - backmix reactor in which the growth

rate of the organism is limited by

product inhibition (ju<^m).

103.

Steady state - assumes constant metabolic rate for

by-product formation and cell reproduction,

by averaging specific yeast activities.

Turbidostat - backmix reactor in which the biomass

is controlled by dilution rate

to =

6.2 Symbols1

X s X » X-j " x^» xn - index of cell mass in a chemostat either

cel 1 s/ml or g cel 1/1.

D - dilution rate, the ratio of mediumL _■]

outflow to chemostat volume V, (hr ).

V - chemostat volume (litres).

F - medium outflow (litres).

P - productivity is either product

concentration, volume or weight,

depending on the type of yield being

um

measured (g/1/hr).

- maximum specific growth rate (hr ^).

m - maximum productivity (g/l/hr).

dxdt - rate of change of cell concentration.

Ks - substrate saturation constant.

Y - yield or weight of organism produced

from a weight of substrate (g/g).

s s s ^* o’ nr - substrate concentration (g/1).- substrate concentration (g/1).

104.

6.3 Sample Calculation for Ethanol Production

Table 18 Yeast productivity in an eleven tank continuous fermenter

(Giashvili and Alkanashvili, 1971).

Tank No. 1 2 3 4 5 6 7 8 9 10 11

Sugar consumed (%) 0.8 2.1 2.5 3.0 2.9 1.6 1.4 1.2 1.6 1.5 0.1

Ethanol (%v/v) 0.44 1.16 1.38 1.66 1.61 0.88 0.78 0.67 0.88 0.83 0.05

Yeast count

(cell s/mlxl0G) 38 92 106 75 71 70 65 59 58 52 48

Yeast productivity

(molecul es

EtOH/cellxlO14) 1.19 1.8 0.1

Note. It was assumed that 1.8g sugar produces 1 ml EtOH (Amerine and

molecules EtOHJoslyn 1970) which is equal to 0.79 x 6.0238 x 10

(Appendix 6.7).46

105.

6.4 Schematic Representation of Grape Juice Preparation for a

Continuous Fermenter.

batch addition_ grapes

T enzyme & so2

V'WyI 9raPecrusher

ethanol

continuousfermentercontinuous

rA dejuicerbentonite

continuouspress/\/\/

settling tank r

settling settlingtank

XXX rotating beater

Fig. 22. Comparative schematic diagrams showing different grape

handling procedures for batch and continuous addition of grape

juice to a continuous fermenter.

6.5 Removal of Sulphur Dioxide from Grape Juice

The stoichiometric oxidation of sulphur dioxide to the sulphate ion

is shown in the following reactions.

106.

SO2 + ^2^*2 —* ^2^4............

g mol wt. 64 34

g in soln. 120 x

Solving for x,

x = PMIP = 63_75 g ^

28g = 100 ml H„02 (28% w/w)

63.75g = x

(a)

Solving for y,y = JP,0x|3...75 = 227 ml H ^ (28% w/w)

Hence .227 ml/1 of cone. H^O^ (28% w/w) are required to neutralize

120 mg/1 S02

2HS03~ + H202 — 2H2S04 + 2e~

g mol wt. 162 34

g in soln. 120 x

Solving for xs

x = = 25.18 g H202

28g = 100 ml H202 (28% w/w)

25.18g = y ml

Solving for y,

y = ■100*-^-.1i = 89.9 ml H202 (28% w/w)

Hence 0.089 ml/1 of cone. H202 are required to neutralize 120 mg/1 S02.

107.

The quantity of hydrogen peroxide required depends on the form or the

oxidation state of sulphur in its dissociated form in solution. A

series of test additions of were added to the grape juice and the

residual SO^ levels are shown in Table 7.

108.

6.6 Schematic Diagrams of Five Continuous Fermenters (Peynaud, 1967).

~vn i. _i. 1

“ :r~r. —yLadousse fermenter iwith two

concentric columns

2. Ladousse fermenter constructed with o siri<jle column

3. Defranccschi fermenter shown during recycling period with automa ic sediment discharge system (ricjht)

4 Padovan fermenter with the re­cycling system (right) and sediment discharge (left)

5. V/co fermenter incorporating spritik- fer system for washing the sediment

109.

6* 7 Derivation of Productivity Formula

6.71 Yeast Productivity

Total weight of ethanol produced in 1 hr is:

%EtOH in the fermenter x ml (wine output) x 0.79 g

where ml wine output Dhr"' x 900 and conversion ratio for

EtOH v/v to w/v is 0.79. The number of molecules of EtOH in Ig w/v

is:

6.0238 x 1023 46

ie. Avagadro's number divided by the g molecular wt EtOH. The3

number of seconds in one hr is 3.6 x 10 and yeast count is measured asr

cells/ml x 10 . Hence yeast productivity molecules EtOH/cel1/sec

(m.EtOH/c/s) is:

(%EtOH x D x 900 x 0,79 ) x (6.0238 x 1023)3.6xl03 x 900 x cells/ml x 106 46

Sample calculation for 5% EtOH at 0 = 0.03 hr" , v = 900 ml andr

110 x 10 cell s/ml; yeast productivity is

„ (5 x 0.03 x 900 x 0. 79 ) x (6.0238 x IQ23) molecules EtOH/cel1/sec3.6 x 103 x 900 x 110 x 106 46

= 3.9 x 10^ molecules ethanol/cell/sec.

6.72 The Ethanol Productivity of the 1 litre Fermenter

The total weight of ethanol produced in 1 hr is Et0H% v/v x ml (wine output)

x 0.79 g ie. where ml wine output is Dhr"^ x 900 and conversion ratio for

EtOH v/v to w/v is 0.79. Hence ethanol productivity in g Et0H/l/hr is;

% EtOH x Dhr"1 x 0.79 x 10

where the factor 10 converts % EtOH w/v to Et0H/l w/v.

Sample calculation for 5% EtOH at D = 0.03 hr" :

Fermenter Productivity = 5 x 0.03 x 0.79 x 10 g Et0H/l/hr

1.1 g Et0H/l/hr.

no.

6.8 Biomass Determination for Yeast Strain 505

The method for biomass determination for strain 505 was dry weight

measurement of a 10 ml sample of the fermenting must after washing

twice and centrifuging before incubation at 105°C for 12 hr.

Other methods available were - incubation, after centrifuging,

in a desiccator and acid hydrolysis of the sample, to break up the

floes, prior to counting in an haemocytometer. The second alternative

was chosen to relate yeast dry weights (the method used) to cell count,

however, this method has the inherent problem of prematurely

separating mother and daughter cells and giving an artificially

high cell count. A 10 ml sample of fermenting must was treated with

10, 20 and 30 ml of 0.02M H9S0^ and the 10 ml addition was found most

suitable as some daughter cells were attached to the parent cell

and the floes were broken sufficiently to spread evenly in an

haemocytometer slide. A series of ten duplicates was conducted on

the must at the end of the fermentation and the results are shown

in table 19. The average dry wt, 6.91 mg cel 1 s/10 ml sample or

0.69 g/1, is equivalent to 70 x 10^ cells/ml, hence 1 g cells/1

(dry wt) is equivalent to 102 x 106 cells/ml.

in.

Table 19. A comparison between cell dry weights (incubated at 105°C

for 12 hr) and direct cell count by an haemocytomer slide after acid

hydrolysis (10 ml 0.02M H^SO^ per 10 ml of must) of a yeast floe

in a must sample containing the strain UCD 505.

Samplenumber

Dry weight (mg cel 1/10 ml)

Yeast count

(cells/mlxl0^)

Samplenumber

Dry weight (mg cel 1/10 ml)

Yeast count (cells/mlxlO^

1 7,1 69 6 7.4 78

2 6.7 68 7 7.2 74

3 6.9 70 8 6.6 69

4 7.1 72 9 6.3 77

5 6.9 63 10 6.9 71

6.9 Preparation of Yeast for Electron Microscopy

Yeast cells, which collapse during the usual preparation of samples

for scanning electron microscopy, were dried by critical-point

drying. (Kurtzman, Baker and Smiley, 1974).

6.10 Identification of Wild Yeast Types

All media preparations were standard commercial preparations found

in Difco or Oxoid handbooks and were prepared by staff in the School

of Food Technology, UNSW.

The following media were inoculated with the wild yeast incubated on

lysine medium at 25°C.

112.

a) Sporulation media: V-8 juice agar, Acetate agar, Gorodkowa agar.

Incubate for 2 weeks, checking at 3, 7 and 14 days for spore

formation.

b) Liquid Malt Extract 15%. Check after 3-4 days incubation for

pellicle, ring and sediment formation. Then shake the tube and

examine a wet amount under the microscope. Record the following:

cell morphology (eg spheroidal, ovoidal, elongate, cylindrical,

apiculate, ogival etc.); thallus (cells single, in pairs, chains,

clusters, pseudomycelium, true mycelium etc.). Replace the tube and

examine for pellicle etc. again after 2 weeks.

c) Sugar Fermentation Tubes: glucose, galactose,sucrose, maltose,

lactose, raffinose and melibiose. Observe after one week for gas

production.

d) Carbon Assimilation Auxanogram: Suspend a loopful of yeast from

the agar slant into a tube containing 3.0 ml of sterile water. Pour

into petri dish and mix evenly with melted yeast nitrogen base agar

(this medium has no carbon source). After setting aseptically place

on the agar surface very small discs of filter paper which have been

impregnated with a 20% w/v sugar solution. The sugars tested will

be glucose (control) galactose, sucrose, maltose, lactose, raffinose,

melibiose, melizitose and a methyl-D-glucoside. Incubate and record

growth around the discs.

e) Nitrogen Assimilation Auxanogram. Prepare a plate as for Carbon

Assimilation but use yeast carbon base agar (this medium has no

nitrogen source). After setting, place very small quantities of

peptone, ammonium sulphate, and potassium nitrate on the plate.

Incubate and note growth around each source.

113.

f) Pseudomycelium formation: Inoculate your yeasts in two parallel

lines on a corn meal agar plate. Cover part of the inoculated area

with a sterile cover slip and observe the various morphological

features under the microscope after 5-10 days.

These results were collated and the key to genera was used to identify

the yeast to the genus Rkod.oton.ula 4pp. The assistance of

Dr. Graham Fleet is gratefully acknowledged.

6.11 Key to the Yeast Genera

The key has been prepared in collaboration with Dr. N.J.W. Kreger-van

Rij.

1. a. Vegetative reproduction exclusively by cross wall formation

without constriction

S dvLzoAac.cka/iomyc£A

b. Vegetative reproduction exclusively by cells formed on stalks

StesiigmatomyceA

c. Vegetative reproduction in a different way 2

2. a. Vegetative reproduction by unipolar budding

PttijAoAponuin

b. Vegetative reproduction by bipolar budding 3

c. Vegetative reproduction by multilateral budding; true

mycelium, arthrospores and bal1istospores may also occur 6

3. a. Fermentation absent

S ckizo6£a4 to 6ponton

b. Glucose is fermented 4

4. a. Ascospores are formed 5

b. Ascospores are not formed

KlocckcAa

5.a. Ascospofes cap-shaped

114.

WZckesdiamia

b. Ascospores spherical, warty, brown

Nad^onla

c. Ascospores spherical, smooth, hyaline, conjugating in pairs

in the scus

SacchctAomycodeA

d. Ascospores hat- or helmet-shaped, or seemingly globose with

an indistinct ledge, not conjugating in pairs

HanictvLaAposia

6. a. Ballistospores are formed 7

b. Ba11istospores are not formed 9

7. a. Mycelium with clamp connections

Spotu.diobolLtt>

b. Mycelium may be present, but there are no clamp connections 8

8. a. Bal1istospores asymmetrical; carotenoid pigments

usually produced

SpoAoboloinyces.s

b. Bal1istospores symmetrical; no carotenoid pigments produced

BuZteAai

9. a. Some of the vegetative cells are triangular

. . TAlgonopA-iA

b. Vegetative cells are not triangular 10

10. a. Cells frequently "ogival"; strong acetic acid

production from glucose; characteristic aroma; cells on

malt agar short-lived 11

b. The correlation of characterisecs as mentioned under a.

does not occur 12

11. a. Asospores are formed

Vckke.A a

115.

b. Ascospores are not formed

Bsidt£anomyc&>

12. a. Ascospores are formed 13 •

b. Asocspores are not formed 28

13. a. Nitrate is assimilated 14

b. Nitrate is not assimilated 16

14. a. Hat-shaped ascospores are formed in a globose compartment

at the distal end of a usually tube-shaped ascus

PachyAolzn

b. The ascus has a different shape 15

15. a. Ascospores are spherical with a warty wall

Citatiomycz,6

b. Ascospores are hat- or Saturn-shaped

Hani umita

Endomycop&dA

16. a. The ascus is a sack-like (bursiform) protuberance on a

vegetative cell, spores have a light amber to brown colour

LLpomyceA

b. The ascus is different 17

17. a. An abundance of true mycelium, and budding cells are

produced

Endomyc.op6'U>

b. True mycelium scarce or absent 18

18. a. Ascospores needle-shaped; one or two spores per ascus

MeX6 ctwZkouilcL

b. Ascospores fusiform; more than two spores per ascus 19

116.

c. Ascospores large, oval to cylindrical; growth between o30-40 C and only in complex media with gaseous CO^ present;

occurrence confined to the digestive tract of rabbits and certain

other rodents

SacchaJwmycop&'U

d. Ascospores spherical to oval, warty with an equatorial

ledge

SduoanniomyceA

e. Ascospore different shape 20

19. a. Ascospores with a whip-like appendange

N&mato6potia

b. Ascospores without a whip-like appendage

CocdldicuscLLi,

20. a. Asci easily ruptured, liberating the spores 21

b. Asci not easily ruptured 23

21. a. Ascospores spherical or oval 22

b. Ascospores hat- or Saturn-shaped

Vlckla.

c. Ascospores reniform

KZuyveAomyc.ej>

22. a. Vigorous fermentation of glucose

KluyveAomyczA

b. Fermentation of glucose weak, slow or absent

PZclvia

23. a. Ascospores spherical or oval 24

b. Ascospores large, oblong with obtuse ends

LoddoAomyc.&>

c. Ascospores oblate-ellipsoidal or lentiform: light brown

WZngea

117.

24. a. Vigorous fermentation of glucose 25\

b. Fermentation of glucose slow, weak or absent 26t25. a. Early formation of a pellicle on malt extract

PZchZa

b. No early formation of a pellicle on malt extract

Sac.cJicuwmij£(U>

26. a. Ascospores smooth

PlcJUa

b. Ascospores warty 27

27. a. Conjugation immediately preceding ascus formation

Vz.ban.yomyc.zA

b. No conjugation immediately preceding ascus formation

Plchla

28. a. Multilateral budding on a broad base combined with

septation; no arthrospores

OoAponUdium

b. Multilateral budding, true mycelium and arthrospores

TnUcJioAponon

c. Multilateral budding; true mycelium present or absent,

no arthrospores 29

29. a. Budding cells and true mycelium with dark-coloured

telospores; clamp connections may or may not be present;

pseudomycelium present or absent 30

b. Budding cells; pseudo- and (or) true mycelium may be

formed; dark-coloured teleospores or clamp connections are not

present 31

30. a. Streak culture pink due to carotenoid pigments;

no fermentation

Pkod.oAposiLcU.Luri

118.

If inositol is not assimilated see also

RhodotoAuZa (strains of Rk. giuiiniA)

If inositol is assimilated, see also

CayptococcuA (strains of Oi. in^iamoniniaia)

b. Streak culture not pigmented; fermentation absent

or present

Lcuco A po xidium

31. a. Streak culture pink or yellow due to carotenoid

pigments; no fermentation 32

b. Streak culture not pigmented; fermentation absent

or present 34

32. a. Inositol is assimilated

CfllJptO COCCLiA

b. Inositol is not assimilated 33

33. a. Starch-like compounds are not formed

RkodotoAuZa

Starch-like compounds are formed

Yeast-like forms of Taplv\ina

34. a. Budding cells and pseudomycelium always present;

true mycelium present or absent

Candida

b. Pseudomycelium absent or rudimentary; no true mycelium 35

35. a. Inositol is assimilated; no fermentation

OiyptOCOCCUA

b. Inositol is not assimilated; fermentation present or

absent

ToAuZopAiA

119.

6.12 Calculation of Correlation Coefficient

The correlation (r) between two viariables x, y was calculated

from the following formula,

£y.y( -

( *x)

) (£y -(1^)

n

Degrees of freedom = n-1

Significance levels were read from tables of standard distribution.

120.

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