Applied Microbiology

APPLIED MICROBIOLOGY
VOLUME 2
FOCUS ON BIOTECHNOLOGY
Volume 2
Series Editors
MARCEL HOFMAN
Centre for Veterinary and Agrochemical Research, Tervuren, Belgium
JOZEF ANN
Rega Institute, University of Leuven, Belgium
Volume Editors
ALAIN DURIEUX and JEAN-PAUL SIMON
Institut Meurice, Brussels, Belgium
COLOPHON
Focus on Biotechnology is an open-ended series of reference volumes produced for
Kluwer Academic Publishers BV in co-operation with the Branche Belge de la Socit
de Chimie Industrielle a.s.b.l.
The initiative has been taken in conjunction with the Ninth European Congress on
Biotechnology. ECB9 has been supported by the Commission of the European
Communities, the General Directorate for Technology, Research and Energy of the
Wallonia Region, Belgium and J. Chabert, Minister for Economy of the Brussels Capital
Region.
Applied Microbiology
Volume 2
Edited by
ALAIN DURIEUX and JEAN-PAUL SIMON

Institut Meurice, Brussels, Belgium
KLUWER ACADEMIC PUBLISHERS

NEW YORK / BOSTON / DORDRECHT / LONDON / MOSCOW
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2002 Kluwer Academic Publishers

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EDITORS PREFACE
This book illustrates the major trends in applied microbiology research with immediate
or potential industrial applications. The papers proposed reflect the diversity of the
application fields. New microbial developments have been done as well in the food and
health sectors than in the environmental technology or in the fine chemical production.
All the microbial genera are involved : yeast, fungi and bacteria.
The development of biotechnology in parallel with the industrial microbiology has
enabled the application of microbial diversity to our socio-economical world. The
remarkable properties of microbes, inherent in their genetic and enzymatic material,
allow a wide range of applications that can improve our every day life.
Recent studies for elucidating the molecular basis of the physiological processes in
micro-organisms are essential to improve and to control the metabolic pathways to
overproduce metabolites or enzymes of industrial interest. The genetic engineering is of
course one of the disciplines offering new horizons for the fantastic microbial factory
.
Studies of the culture parameter incidence on the physiology and the morphology
are essential to control the response of the micro-organisms before its successful
exploitation at the industrial scale. For this purpose, fundamental viewpoints are
necessary.
Development of novel approaches to characterise micro-organisms would also
facilitate the understanding of the inherent metabolic diversity of the microbial world, in
terms of adaptation to a wide range of biotopes and establishment of microbial
consortia.
Improvement of selection methods is a crucial step to initiate any research in applied
microbiology. This is necessary both for traditional strains which are used as starter in
the food industry and for more specific strains which are of interest in fine chemical
synthesis and bioremediation. Since the two last decades, in the fermented food and
beverage industry, micro-organisms have been regarded to increase shelf-life and to
improve flavours and nutritional value of food. Antimicrobial activity against pathogens
and spoilage micro-organisms is an important criteria for the selection of a strain as
starter. Beside the development of starters, techniques of foodborne bacterial pathogens
detection are in complete evolution with the introduction of molecular detection and
new typing methods.
The constant apparition of novel applications is the driving force which allows the
constant progress of applied microbiology. Recent development in enzymes production
and discovery of new metabolic pathways related to food or environmental technology
are representative examples .
Editors: A. Durieux and J-P Simon

Brussels March 2000
IN MEMORY
On September 25th, 1999, Professor Charles A. Masschelein died here in Belgium. To
the end, Professor Masschelein remained active in the brewing and biotechnology
industries and had, in fact, just recently returned from visiting a number of African
breweries.
That day, I lost a friend. Moreover, at his moment of passing, I understood that I
had lost my mentor in the field of applied microbiology and the world had lost one of
the truly great scientists of the 20th century.
Twenty years before, I joined Masscheleins department of brewing science at the
Institut Meurice, CERIA. Masschelein was a leader, a dedicated spirit to R&D with an
acutely inquiring mind. He completely devoted himself to the training of people and
transfer of technology from the academic to the industrial world.
Since 1954, C.A. Masscheleins vision, as a Belgian scientist, was to understand the
physiology of micro-organisms in their industrial environment. He taught this idea at
all levels ; to large numbers of students in Europe and internationally, but also to a large
number of engineers already in charge of fermentation plants around the world.
Masscheleins major topic has been the brewery. In this sector he was the defender
of a strict understanding of biochemical and microbiological principles balancing this
with both the reality and progression of the industrial method and the traditional quality
of the final product he knew. For more than 35 years, he held a number of responsible
positions in the European Brewery Convention as well as contributed greatly to brewing
science worldwide.
Biotransformation was always a major driving force for Masschelein. He was able
to introduce this philosophy to R&D in the 1960s and apply the results to processes in
order to use micro-organisms to produce industrially significant materials. For this
reason too, C.A. Masschelein has always been capable of combining new developments
in molecular biology on one hand to new materials and processes on the other. This
challenge was such a reality for him that, when he retired as professor at CERIA, he was
responsible for setting up a company to develop new continuous brewing processes.
In July, 1999, during the Ninth European Congress of Biotechnology, C.A.
Masschelein gave his last lecture delivering his idea concerning the future of applied
microbiology in correlation with new generation of continuous fermentation processes.
As with all great scientists, Masscheleins work will be remembered and referred to for
many decades in the future.
J.P. Simon
TABLE OF CONTENTS
EDITORS PREFACE ....................................................................
IN MEMORY .................................................................................
TABLE OF CONTENTS ...............................................................
PART 1 - STARTERS ................................................................. 11

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED
FOODS ..........................................................................................................................
Rolf Geisen and Paul Frber ....................................................................................
Abstract ...........................................................................................................
1. Introduction ......................................................................................................
2. Penicillium nalgiovense ....................................................................................
2.1. Taxonomic relationships at the molecular level ........................................
2.2. Penicillin production is a common feature of p. nalgiovense ....................
2.3. Heterologous Gene Expression in P . nalgiovense ....................................
2.4. Heterologous Gene Expression in P . nalgiovense 2.4. Cloning of genes
from P . Nalgiovense important for the fermentation process ..........................
3. Penicillium camemberti ....................................................................................
4. Penicillium roqueforti .................................................................................
5. Conclusions ......................................................................................................
References ............................................................................................................
13
13
13
13
15
15
17
20
21
23
25
27
27
STARTERS FOR THE WINE INDUSTRY .............................................................. 31

Aline Lonvaud-Funel ................................................................................................ 31
Abstract ................................................................................................................ 31
1. Introduction ...................................................................................................... 31
2. Yeast starters in winemaking ........................................................................... 32
2.1 The objectives of yeast starters .................................................................. 32
2.2 Properties of yeast used as selective criteria for active dry yeast producers
and winemakers ............................................................................................... 34
2.3 Evaluation of the settlement of active dry yeast during alcoholic
fermentation : ................................................................................................... 37
3. Malolactic starters in winemaking .................................................................... 38
3.1 Indications for use of malolactic starter and description ............................ 39
3
3.2 The influence of lactic acid bacteria starters on wine quality and their
selection ...........................................................................................................
3.3 Efficiency of malolactic starters ................................................................
4. The future of starters for winemaking ..............................................................
5. Conclusion ........................................................................................................
References ............................................................................................................
41
42
43
45
45
PART 2 - PHYSIOLOGY, BIOSYNTHESIS AND METABOLIC

ENGINEERING ........................................................................... 49
METABOLISM AND LYSINE BIOSYNTHESIS CONTROL IN
BREVIBACTERIUM FLAVUM: IMPACT OF STRINGENT RESPONSE IN
BACTERIAL CELLS .................................................................................................. 51
M. Ruklisha, R. Jonina, L. Paegle and G. Petrovica ...................................................... 51
Abstract ................................................................................................................ 51
1. Introduction ....................................................................................................... 51
2. Materials and Methods ..................................................................................... 52
3. Results and Discussion ..................................................................................... 52
4. Conclusions ...................................................................................................... 56
References ............................................................................................................ 57
MOLECULAR BREEDING OF ARMING YEASTS WITH HYDROLYTIC
ENZYMES BY CELL SURFACE ENGINEERING ................................................ 59
Mitsuyoshi Ueda, Toshiyuki Murai, Shouji Takahashi, Motohisa Washida, and Atsuo
Tanaka ....................................................................................................................... 59
Abstract ................................................................................................................ 59
1. Introduction ...................................................................................................... 60
2. Principle of Cell Surface Engineering of Yeast ................................................ 63
3. Display of Amylolytic Enzymes on the Yeast Cell Surface ............................. 65
4. Display of Cellulolytic Enzymes on the Yeast Cell Surface ............................ 67
5. Display of Lipase on the Yeast Cell Surface .................................................... 70
6. Cell Surface Engineering as a Novel Field of Biotechnology .......................... 70
References ............................................................................................................ 71
METABOLIC PATHWAY ANALYSIS OF SACCHAROMYCES CEREVISIAE
....................................................................................................................................... 75
Simon Ostergaard, Lisbeth Olsson and Jens Nielsen ................................................ 75
Abstract ................................................................................................................ 75
1. Introduction ...................................................................................................... 75
2. Metabolic pathway analysis .............................................................................. 76
2.1. Metabolic control analysis ........................................................................ 76
2.2. Metabolic flux analysis ............................................................................. 77
3. Steady-state continuous cultivation an excellent tool for metabolic pathway
analysis ................................................................................................................. 79
4. Metabolic pathway analysis applied to Saccharomyces cerevisiae .................. 80
4
4.1. Kinetic studies of the glycolysis ...............................................................

4.2. Metabolic pathway analysis of the galactose metabolism ........................
Acknowledgements ..............................................................................................
References ............................................................................................................
80
81
85
85
PART 3 - STATE PARAMETERS AND CULTURE CONDITIONS

................. . ................................................................................... 87
EFFECT OF AERATION IN PROPAGATION ON SURFACE PROPERTIES
BREWERS YEAST ....................................................................................................
Andrew Robinson and Susan T. L . Harrison .............................................................
Abstract ................................................................................................................
1. Introduction ......................................................................................................
2. Materials and Methods .....................................................................................
2.1 Propagation conditions ..............................................................................
2.2 Hydrophobicity ..........................................................................................
2.3 Surface charge ............................................................................................
2.4 Flocculation ...............................................................................................
3. Results ..............................................................................................................
3.1 Yield coefficients .......................................................................................
3.2 Cell growth rates ........................................................................................
3.3 Hydrophobicity ..........................................................................................
3.4 Zeta potential .............................................................................................
3.5 Flocculation ...............................................................................................
4. Discussion .........................................................................................................
5. Conclusions ......................................................................................................
Acknowledgements ..............................................................................................
References ............................................................................................................
OF
89
89
89
89
90
90
90
91
92
92
92
92
93
94
95
96
98
98
99
EFFECT OF THE MAIN CULTURE PARAMETERS ON THE GROWTH AND

PRODUCTION COUPLING OF LACTIC ACID BACTERIA ............................ 101
A. Amrane and Y. Prigent ....................................................................................... 101
Abstract .............................................................................................................. 101
1. Introduction .................................................................................................... 101
2. Materials and methods .................................................................................... 102
2.1 Microorganism ......................................................................................... 102
2.2 Media ....................................................................................................... 102
2.3 Fermentors and culture conditions ........................................................... 102
2.4 Analytical methods .................................................................................. 103
3. Results and Discussion ................................................................................... 103
3.1. Preculture conditions .............................................................................. 103
3.2. Nutritional limitations ............................................................................. 105
3.3. Initial lactate additions ............................................................................ 106
4. Conclusions .................................................................................................... 107
Acknowledgements ............................................................................................ 107
5
References ..........................................................................................................
107
PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES

CEREVISIAE ............................................................................................................. 109
F.F. Bauer and I.S . Pretorius ................................................................................... 109
Abstract .............................................................................................................. 109
1. Introduction .................................................................................................... 109
2. Signal transduction in Saccharomyces cerevisiae ........................................... 110
3. Molecular nature of signal transduction processes resulting in pseudohyphal
differentiation ..................................................................................................... 112
3.1. Signal transduction modules .................................................................. 113
3.1.1. Nutrient availability is sensed by permeases ................................... 113
3.1.2. Transmission via receptor associated elements ............................... 114
3.1.3. Intermediate signal transduction modules ....................................... 116
3.2. Transcriptional regulators ....................................................................... 122
3.2.1. Ste12p and Tec1 .............................................................................. 123
3.2.2. Msn1p and Mss11p: Central elements in the pseudohyphal growth
pathway ..................................................................................................... 123
3.2.3. Sfl1p. Sok2p and Flo8p: Factors depending on the cAMP dependent
kinase ........................................................................................................ 124
3.2.4. Other factors .................................................................................... 125
3.3. Effector proteins ..................................................................................... 125
3.3.1. MUC1. a gene encoding a mucin-like protein subjected to complex
transcriptional regulation .......................................................................... 126
3.3.2. Starch degrading enzymes: a direct metabolic link ......................... 127
4 . Scientific and industrial relevance .................................................................. 127
References ................
................................................................................... 129
MICROBIAL PRODUCTION OF THE BIODEGRADABLE POLYESTER
POLY-3-HYDROXYBUTYRATE (PHB) FROM AZOTOBACTER
CHROOCOCCUM 6B: RELATION BETWEEN PHB MOLECULAR WEIGHT.
THERMAL STABILITY AND TENSILE STRENGTH ........................................ 135
Quagliano Javier C. and Miyazaki Silvia S ...
......................... 135
Abstract .............................................................................................................. 135
1.1 Microorganism and culture media ........................................................... 135
1.2 Fermentor experiments ............................................................................ 135
1.3 Extraction and purification procedure ...................................................... 136
1.4 Analytical methods .................................................................................. 136
2. Results and discussion .................................................................................... 136
2.1 Effect of MW on PHB thermal stability .................................................... 136
2.2 Effect of aeration rate on PHB MW .......................................................... 137
2.3 PHB tensile strength () at different MW .................................................
2.4 PHB as a matrix for microencapsulation .................................................
6
138
138
3. Conclusions ....................................................................................................
References ..........................................................................................................
PART 4 - NOVEL APPROACHES TO THE STUDY OF

MICROORGANISMS ................................................................
139
139
141
SHARING OF NUTRITIONAL RESOURCES IN BACTERIAL COMMUNITIES

DETERMINED BY ISOTOPIC RATIO MASS SPECTROMETRY OF
BIOMARKERS .......................................................................................................... 143
Wolf-Rainer Abraham, Christian Hesse, Oliver Pelz, Stefanie Hermann, Michael
Tesar, Edward R. B . Moore, and Kenneth N . Timmis ............................................ 143
1. Introduction .................................................................................................... 143
2. Taxon specific biomarkers .............................................................................. 144
2.1. Polar lipids .............................................................................................. 144
2.2. Outer membrane proteins ........................................................................ 145
3. Isotopic fractionation in microorganisms ....................................................... 146
4. Carbon sharing in a pollutant degrading bacterial community ....................... 147
4.1. Origin and characteristics of the microbial consortium .......................... 147
4.2. Incorporation of [U-13C]-metabolites in microbial biomasses ................ 148
4.3. Substrate competition ............................................................................. 149
4.4. Community physiology of the microbial consortium ............................. 150
5. Outlook ........................................................................................................... 152
Acknowledgement .............................................................................................. 152
References .......................................................................................................... 152
A COMPARISON OF THE MECHANICAL PROPERTIES OF DIFFERENT
BACTERIAL SPECIES ............................................................................................ 155
C . SHIU, Z . ZHANG AND C.R. THOMAS ........................................................... 155
Abstract .............................................................................................................. 155
1. Introduction .................................................................................................... 155
1.1 Relative resistance of different microorganisms to mechanical disruption
.......................................................................................................................
155
1.2 Cell wall structure .................................................................................... 156
1.3 Bacterial biomechanics ............................................................................ 157
1.4 Micromanipulation ................................................................................... 158
2.1 The micromanipulation system ................................................................ 158
2.2 Culture conditions .................................................................................... 159
4. Conclusions and future developments ............................................................ 161
References .......................................................................................................... 162
PART 5 - NOVEL APPLICATIONS ..........................................
163
KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA ........... 165

Nereida Coello and Luis Vidal ................................................................................ 165
Abstract .............................................................................................................. 165
1. Introduction .................................................................................................... 165
2.Isolation, identification and adaptation of feather-degrading microorganisms 166
2.1. Isolation and degradation of feathers by a microbial isolate ...................
166
2.2. Morphological and ultrastructural characteristics of the feather-degrading
isolate ............................................................................................................. 168
3. Microbial growth and feather degradation ...................................................... 168
3.1. Effect of quantity of feathers .................................................................. 168
3.2. Effect of culture temperature on feather degradation and growth of LPB-3
.......................................................................................................................
171
3.3. Kinetic fermentation ............................................................................... 171
4. Industrial applications ..................................................................................... 171
4.1. Fermented feather meal ........................................................................... 171
4.2. Enzymes .................................................................................................. 173
4.3. Pigments ................................................................................................. 173
References .......................................................................................................... 174
COMPARISON OF Pb2+ REMOVAL CHARACTERISTICS BETWEEN
BIOMATERIALS AND NON-BIOMATERIALS ..................................................
Dong Seog Kim and Jung Ho Suh ...........................................................................
Abstract ..............................................................................................................
1. Introduction ....................................................................................................
2. Materials and methods ....................................................................................
2.1. Materials .................................................................................................
2.2. Microorganisms and culture conditions ..................................................
2.3. Pb2+ removal experiment ........................................................................
3. Results and discussion ....................................................................................
3.1. Pb2+ removal characteristics ....................................................................
3.2. Initial Pb2+ removal rate ..........................................................................
4. Conclusions ....................................................................................................
References ..........................................................................................................
177
177
177
177
178
178
178
178
179
179
182
183
183
HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERIA . 185

Gy. Barabs, Gy. Vargha, I. Szab, A. Penyige, J. Szllsi, J. Matk, S.
Damjanovich and T . Hirano .................................................................................... 185
Abstract .............................................................................................................. 185
1.1 Test organisms . oligocarbophylic streptomyces ...................................... 185
1.2 Biomass preparation ................................................................................ 186
1.3 Incorporation of radioactivity from labelled n-Hexadecane into mycelia .

.......................................................................................................................
186
1.4 Fluorescence measurements ..................................................................... 186
1.5 Analysis of fatty acids .............................................................................. 187
1.6 Investigations with GTP analogues .......................................................... 187
3. Conclusion ...................................................................................................... 190
References .......................................................................................................... 190
PART 6 - FOOD SECURITY AND FOOD PRESERVATION ... 191

MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL
PATHOGENS: A REVIEW ...................................................................................... 193
M . Heyndrickx, N . Rijpens and L . Herman ............................................................. 193
Abstract .............................................................................................................. 193
1. Introduction .................................................................................................... 194
2. Characteristics of the foodborne bacterial pathogens ..................................... 194
3. Molecular detection and identification of foodborne bacterial pathogens ...... 198
3.1 Nucleic acid based identification methods ............................................... 198
3.2 The use of virulence genes as target for molecular identification ............ 198
3.3 The use of RRNA genes as target for molecular identification ............... 199
3.4 The use of specific sequences with a known or unknown function as target
for molecular identification ........................................................................... 200
3.5 The available molecular identification systems ....................................... 201
3.6 PCR detection of bacterial pathogens in food products ........................... 203
3.6.1 Influence of food components on PCR performance ....................... 203
3.6.2 Sensitivity and contamination of PCR ............................................. 203
3.6.3 The detection of the viability of cells by DNA based technology .... 204
3.7 Evaluation and validation of DNA based methods .................................. 205
3.8 DNA amplification methods for quantification of foodborne pathogens . 207
4. Molecular typing of foodborne bacterial pathogens ....................................... 208
4.1 Terminology and general information ..................................................... 208
4.1.1 Necessity of bacterial typing of foodborne pathogens ..................... 208
4.1.2 Species-subspecies-variety-clone-strain-isolate ............................... 209
4.1.3 Molecular typing techniques used for bacterial pathogens .............. 210
4.1.4 Analysis of DNA fingerprints .......................................................... 219
4 .. 2 Prospects in molecular typing ................................................................. 220
5. Molecular typing of some specific bacterial foodborne pathogens ............... 221
5.1 Salmonella ............................................................................................... 221
5.2 Campylobacter jejuni ............................................................................... 226
5.3 Listeria monocytogenes .......................................................................... 227
5.4 Escherichia coli 0157 .............................................................................. 228
5.5 Some other foodborne bacterial pathogens .............................................. 229
References .......................................................................................................... 229
BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION ........... 239

Cahill, S.M., Upton, M.E., and McLoughlin. A.J. .................................................. 239
Abstract .............................................................................................................. 239
1. Introduction .................................................................................................... 240
2. Meat preservation ........................................................................................... 241
2.1 Biological fermentation ........................................................................... 241
2.2 Chemical acidification ............................................................................. 243
3. The application of encapsulation technology to meat preservation ................ 243
3.1. The application of encapsulation technology to a microbial fermentation
....................................................................................................................... 243
3.1.1. Encapsulation matrices and the encapsulation process ................... 244
3.1.2. The benefits of meat starter culture encapsulation .......................... 246
3.1.3. Commercial applications ................................................................. 247
3.2. The application of encapsulation technology to chemical acidification . 248
3.2.1. Encapsulation matrices and the encapsulation process ................... 248
3.2.2. The benefits of acidulant encapsulation .......................................... 249
3.2.3 Commercial availability ................................................................... 249
4. Control of emerging pathogens ...................................................................... 250
5. The application of encapsulation technology to bacteriocin delivery ............. 251
5.1 Bacteriocins ............................................................................................. 251
5.2 Nisin ......................................................................................................... 251
5.2.1 Encapsulation of nisin ...................................................................... 252
6. Conclusions and future work .......................................................................... 261
References .......................................................................................................... 261
INDEX .......................................................................................
10
267
Part 1
STARTERS
NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED

FOODS
ROLF GEISEN AND PAUL FRBER
Federal Research Centre for Nutrition
Haid- und Neustr. 9
76131 Karlsruhe
Abstract
The production of a variety of foods include a fermentation step by filamentous fungi.
Nowadays these fermented foods are produced by selected fungal starter cultures
instead of relying on the indigenous flora, which may contain spoilage or
mycotoxinogenic strains. The most important fungal species for food fermentation are
Penicillium nalgiovense for the production of mould fermented meat products, P.
camemberti for the production of white cheeses and P. roqueforti for the production of
blue veined cheeses. Before a fungal strain of these species can be used as a starter
culture for human consumption it must fulfill several requirements. Not all strains
isolated from the food environment and with characteristics suitable for starter cultures
fit to these conditions. In addition also the currently used starter strains possess
undesired properties. On the other hand the modem techniques of molecular biology or
genetics offers various possibilities for screening, characterisation and for specific
improvement of fungal strains. In this article examples for characterisation and
improvement of fungal starter cultures by molecular techniques are described.
1. Introduction
Mould fermented foods play an important role, especially in Asian countries where the
production process for many foods include a fungal fermentation. Fungal species which
are found in Asian type foods belong to different genera: Aspergillus oryzae, A.
glaucus, Rhizopus chinensis, Neurospora intermedia or Monascus purpureus
(Hesseltine, 1983). In contrast to this situation in European countries mainly three
species from the genus Penicillium are used for the production of fermented foods, in
particular Penicillium camemberti for white cheeses, P. roqueforti for blue cheeses and
P. nalgiovense for mould fermented meat products. It has to be mentioned that P.
chrysogenum also can often be isolated from fermented meat products and is sometimes
used as a starter culture for meat products in some countries (Leistner, 1986).
13
A. Durieux and J-P. Simon (eds.), Applied Microbiology, 13-29.
2001 Kluwer Academic Publishers. Printed in the Netherlands.
ROLF GEISEN AND PAUL FARBER
Fungal starter cultures extensively contribute to the flavour and texture formation of the
mould ripened food product. The most important enzymatic activities in this respect are
proteases and lipases (Kinsella and Hwang, 1976). Proteases degrade proteins to
flavour active peptides or amino acids, whereas lipases hydrolyse triacyl glycerins to
free fatty acids and glycerol. The fatty acids can be converted into methylketones by
fungal lipoxygenases that also contribute to the flavour formation. These compounds are
especially important in the ripening of blue cheese (Fox and Law, 1991). But not only
these enzymatic processes play a role in flavour development. It has been shown by
Jacobsen and Hinrichsen (1997) that also volatile compounds produced by the fungus
contribute to this process. Another important feature of the fungal starter culture is the
competition against undesired microorganisms, which would otherwise cover the
surface of the fermented product. These microorganisms are mainly other fungal species
that could lead to a discoloration of the product or the production of undesired
secondary metabolites. In addition the competition of fungal starter cultures agai nst
undesired pathogenic or toxinogenic bacteria is of interest for microbiological safe
production of mould fermented foods. During the fermentation process the pH of the
product raises due to the metabolic activity of the fungus. Lactic acid that may be
produced by accompanying lactic acid bacteria is degraded and also proteins may be
hydrolysed to ammonia by the fungal starter culture. Both processes lead to an increase
in pH during the fermentation which enables the growth of pathogenic or toxinogenic
bacteria like Listeria monocytogenes or Staphylococcus aureus. Of course starter
cultures which would suppress the growth of these undesired bacteria would be
advantageous for improved food safety.
Before a fungal strain can be used as a starter culture it has to fulfil several
requirements (Tab. 1).
Table 1. Requirements for a fungal starter culture
----------------------------------------------------
It should not produce a mycotoxin

It should not produce another undesired secondary metabolite
It should produce the desired flavour changes in the product
It should be adapted to the food product
It should compete against undesired moulds
It should have antibacterial activity against pathogens
---------------------------------------------------Not all the currently used strains fulfil all these requirements. Many species of the genus
Penicillium are able to produce toxic secondary metabolites, the mycotoxins. Even
some species currently used, as starter cultures are able to produce mycotoxins or other
undesired secondary metabolites. For this reason it is important that useful methods
14
NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS
exist, which enables the screening of strains for particular characteristics and that fungal
strains can be optimised according particular requirements.
2. Penicillium nalgiovense
2.1. TAXONOMIC RELATIONSHIPS AT THE MOLECULAR LEVEL
P. nalgiovense was first described by Laxa (1932). The type culture was isolated from a
bohemian cheese. Strains of these species are mainly found on meat and meat products
(Leistner et al, 1989). But they can also be isolated as spoilage organisms from different
cheeses (Lund et al. 1995). P. nalgiovense is not able to produce any of the known
mycotoxins, according to Frisvad (1988). Until recently P. nalgiovense was regarded as
a fungal species with poor capability of producing secondary metabolites.
Because of morphological similarities P. nalgiovense has long been regarded as a
domesticated form of P. chrysogenum, the species which is industrially used for
penicillin production (Samson and van Reenen-Hoekstra, 1988). In a recent publication
Banke et al. reclassified the P. chrysogenum complex (Banke et al, 1997). According to
their results, based on isozyme analysis, the P. chrysogenum complex was separated
into 4 distinct species: P. chrysogenum, P. dipodomyis, P. flavigenum and P.
nalgiovense. Molecular data confirmed the high relationship between P. nalgiovense
and P. chrysogenum (Geisen, 1995). With several strains of P. nalgiovense a RAPD
analysis was performed. This type of analysis is a molecular typing technique, which
was developed by Williams et al. (1990). This technique is a modified PCR approach in
which an arbitrary primer is used. It generates specific patterns if always the same
reaction conditions are used. These patterns are related to the genotype of the analysed
strain. Differences in the pattern reflects differences in the genotype and give
information about the relatedness between different strains. Depending on the primer
used the patterns can be species- (Guthrie et al., 1992), subspecies- (Hamelin et al.,
1993) or even strain specific (Bidouchka et al. 1994). This approach revealed that P.
nalgiovense is a homogenous species, irrespective of the origin of the strains analysed
(Figure 1). The figure shows that all the patterns generated with a random primer were
identical. The same result was derived with a second primer. The only difference in the
pattern exhibited one strain (BFE 61). This was due with all primers used, indicating
that it might be a misclassified strain
To compare P. nalgiovense and P. chrysogenum at the molecular level, the same
approach was carried out with both species. Figure 2 shows the results of this analysis.
They clearly demonstrate, that P. nalgiovense and P. chrysogenum are two distinct
species, because the RAPD patterns of both species are different, however a subsequent
hybridisation analysis, in which the labelled RAPD products of P. nalgiovense were
hybridised to the RAPD-PCR products of P. chrysogenum revealed, that both species
has homologies at the nucleotide sequence level, which indicate the close relationship
between the species.
15
Figure 1: RAPD pattern from different strains of P. nalgiovense generated

with primer aril. Lanes 1 and 2, P. roqueforti BFE54, P. camemberti
BFE224; lanes 3 - 18, P. nalgiovense BFE71, BFE75, BFE55, BFE61,
BFE57, BFE59, BFE67, BFE56, BFE66, BFE65, BFE52, BFE60, BFE69,
BFE64, BFE58, BFE74; lanes 19 and 20 size marker 1 and 2. Fragment
sizes are indicated in kb.
Figure 2: Comparison of the RAPD patterns of P. nalgiovense and P.

chrysogenum. Lanes 1 to 5: P. nalgiovense BFE71, BFE76, P.
chrysogenum DSM 844, DSM 1075, size marker. The fragment sizes are
given in kb.
16
2.2. PENICILLIN PRODUCTION IS A COMMON FEATURE OF P.NALGIOVENSE

Another aspect also indicates the close relationship between both species. P.
chrysogenum is the industrial producer of penicillin and for P. nalgiovense the
capability to produce also penicillin has been described independently by Frber and
Geisen (1994) and Andersen and Frisvad (1994). All strains analysed were able to
produce this secondary metabolite. It was demonstrated by PCR (polymerase chain
reaction), that obviously the genes for penicillin production are very similar at the
nucleotide level between P. chrysogenum and P. nalgiovense. With primers deduced
from the sequences of the three penicillin biosynthetic genes of P. chrysogenum it was
possible to amplify the respective PCR fragments from P. nalgiovense. The PCR
products of both species were identical in length, indicating homology between the
genes (Figure 3).
Figure 3: PCR analysis of the three penicillin biosynthetic genes of P.

nalgiovense (lanes 3 to 5 and 7 to 9) and P. chrysogenum (lanes 11 to 13).
Lanes 3, 7, 11 show the PCR products for the penDE genes, lanes 4, 8, 12
show the PCR products for the pcbC genes and lanes 5, 9, 13 show the
PCR products of the pcbAB genes.
Subsequent sequence analyses of the pcbC gene, the gene coding for isopenicillin
synthetase, revealed that both genes are 94% identical. It is known from the genes of P.
chrysogenum that they are clustered and all located on chromosome 4, the largest
chromosome (Fiero et al. 1993). To analyse this situation in P. nalgiovense an
electrophoretic karyotyping was carried out. This analysis revealed, that like P.
chrysogenum, P. nalgiovense has also 4 chromosomes of high molecular weight, but
17
compared to the chromosomes of P. chrysogenum they are smaller in size resulting in a

total genome size which is considerably smaller than that of P. chrysogenum (26,5 Mb
instead of 34,1 MB). These results again reflect the close relationship between both
organisms, but also demonstrates the distinct differences at the molecular level. A
distinct difference became also obvious when the location of the penicillin biosynthetic
gene cluster have been elucidated. In the case of P. chrysogenum it is located on the
largest chromosome, whereas on the smallest in case of P. nalgiovense.
As mentioned above, one important requirement for a fungal strain used as a starter
culture is the point that it does not produce any undesired secondary metabolite.
Antibiotics like mycotoxins are secondary metabolites produced in the idiophase of the
fungal growth curve. The differentiation of these secondary metabolites in antibiotics
and mycotoxins depend only to the point of view. A fungal starter culture should not
produce any of them. All analysed P. nalgiovense strains obviously were able to
produce the secondary metabolite penicillin, also the strains that are currently used as
fungal starter cultures. As these strains are well adapted to the processing conditions and
have long been used for fermentation purposes. For this reason great experiences exist
concerning the optimal fermentation conditions. It would be advantageous that exactly
these strains would be optimised in a way that they do not produce penicillins. To
achieve this goal the "gene disruption" approach was followed. A gene disruption is a
gene technological method to specifically inactivate an undesired gene within an
organism. In contrast to the inactivation of a gene by mutation with an chemical
mutagene or irradiation only the target gene is inactivated by this approach. This
characteristic is of special interest for the optimisation of strains that are already in use.
With this approach no secondary mutations that may have negative consequences on the
activity of a starter culture can occur. This might be the case with the conventional
mutation/selection approach. The principle of the "gene disruption" approach is shown
in Figure 4.
Figure 4: Scheme of the "gene disruption" approach.
18
There are two important points that must be fulfilled before this method can be used: 1.
a transformation system for the particular organism must be established to be able to
introduce DNA into the microorganism and 2. the gene to be targeted must be available.
If this is the case a central fragment of that gene is cloned into a transformation vector.
This plasmid is than transformed into the fungal strain. The resulting transformants are
screened for activity of the undesired gene. It is highly probable, that some of the
transformants are no longer active in the undesired feature. In that cases the
transforming DNA had integrated into the undesired gene by homologous
recombination. This integration leads to a situation in which the target gene is
duplicated and separated by vector sequences. However both copies of the gene are
inactive, as the first copy is truncated at the 3' end and the second copy is truncated at
the 5' end. This approach was adapted to P. nalgiovense. A transformation system for
that microorganism was available (Geisen, 1989). The central fragment of the penDC
gene was cloned into the transforming vector and introduced into P. nalgiovense. From
the resulting transformants several could be identified, which were no longer able to
produce penicillin. Figure 5 shows the results of the experiment.
Figure 5: Comparison of the antagonistic activity against Micrococcus

luteus of a wild type strain (right) and the gene disrupted transformant
(left)
The transformed strain of P. nalgiovense is no longer able to produce penicillin. As a

strain that is currently used as a starter culture has been used. The non-penicillinogenic
strain for this reason is isogenic to the commonly used strain with the only difference in
respect to penicillin production.
19
2.3 HETEROLOGOUS GENE EXPRESSION IN P. NALGIOVENSE

As mentioned above it is necessary, for microbiological stability of a product, that
fungal starter cultures are able to compete against undesired pathogenic or toxinogenic
microorganisms, which can grow in mould fermented products. However this inhibition
should be caused by "food grade" inhibitory principles. During fermentation unstable
conditions may occur, due to the metabolic activity of the fungal starter culture. The pH
may raise which enables the growth of pathogenic bacteria like Listeria monocytogenes
or Staphylococcus aureus (Marth, 1987). Fungal starter cultures that would be able to
suppress the growth of these organisms would improve the microbiological safety of
these products. Several antagonistic proteins exist, which have GRAS status (generally
recognised as safe) and could improve the antagonistic activity in a food-grade way, if
they would be produced by the fungal starter culture. Examples of these proteins are the
bacteriocins (Stiles, 1994), lysozyme (Bester and Lombard, 1990) or glucose oxidase
(Tiina and Sandholm, 1989). The glucose oxidase (GOD) is an enzyme system that is
widely used on food industry. In the presence of glucose it oxidises glucose to gluconic
acid and hydrogen peroxide. For this reason it is used as a scavenger for traces of
glucose or oxygen in food systems like dried egg or beverages (Reed and Underkofler,
1969). In addition to this activities, the GOD has also inhibitory activities against a
range of pathogenic and toxinogenic bacteria (Tiina and Sandholm, 1989). Mainly the
production of hydrogen peroxide is responsible for antibacterial activity. GOD is active
against L. monocytogenes, Staphylococcus aureus, Clostridium perfringens, Samonella
infantis and Bacillus cereus. This inhibitory activity is dependent on the concentration
of the glucose in the medium. The god gene has been cloned from Aspergillus niger
(Kriechbaum et al. 1989). A. niger also has GRAS status. P. nalgiovense has an
endogenous god gene, however the expression of that gene is weak. For this reason the
endogenous GOD activity of P. nalgiovense is not inhibitory for bacteria. By using the
P. nalgiovense transformation system (Geisen, 1989) the god gene from A. niger was
introduced into P. nalgiovense. Transformed strains could be identified, which leads to
a colour change in a medium containing an pH-indicator, whereas the wild type showed
no colour change under these conditions. This was an indication of increased production
of gluconic acid. Subsequent Southern blotting experiments revealed that all the strains
which exhibited this colour change had copies of the god gene of A. niger integrated
into their genome. An analysis of the GOD enzyme activity with these strains revealed,
that one of the strains had a nearly 6 time and another strain a nearly 3 time higher god
activity than the wild type. With these strains an inhibitory agar diffusion assay was
performed. For that purpose indicator bacteria mixed in soft agar were spread out onto
an agar plate containing glucose and 5 day old colonies of P. nalgiovense strains, either
transformed with the plasmid carrying the god gene or wild type. The plates were
incubated over night. The result of that experiment is shown in Figure 6.
The transformed strains clearly suppressed the growth of the indicator organism S.
aureus. This growth inhibition was dependent on the glucose concentration in the
medium. The higher the concentration, the higher the inhibitory effect. The wild type
showed no antibacterial activity under these conditions indicating that the higher GOD
activities from the transformed strains were obviously responsible for this effect. This
20
inhibition could be observed with S. aureus, L. monocytogenes und S. enterititis as

indicator organisms.
Figure 6: Inhibitory activity of the transformed P. nalgiovense strains (2, 5

and 7 o'clock) compared to the wild type (10 and 12 o'clock). The plates
were covered with 100 l of an overnight culture of S. aureus mixed with
10 ml soft agar.
These results indicate that optimised strains in contrast to the wild type are able to
suppress the growth of undesired bacteria, which may occur in mould fermented foods.
2.4 HETEROLOGOUS GENE EXPRESSION IN P. NALGIOVENSE 2.4. CLONING
OF GENES FROM P. NALGIOVENSE IMPORTANT FOR THE FERMENTATION
PROCESS
It was mentioned above, that especially proteases and lipases play a role in the flavour
formation of the fermented product. The change of proteolytic activity of strains either
by gene amplification or gene inactivation by the gene disruption approach can lead to
strains isogenic to the wild type, but with the differences in the proteolytic activity. This
in term can lead to strains with different fermentation properties. It is known from P.
roqueforti, that this species has different proteolytic systems. It produces extra and
21
intracellular proteases. (Stepaniak et al. 1980). The relative activity between the
particular proteases can differ from strain to strain (Paquet and Gripon, 1980). P.
roqueforti shows its highest proteolytic activity at acidic pH, which is in agreement with
our results, as obviously the acidic protease of P. nalgiovense is the main protease of P.
nalgiovense. From P. roqueforti an aspartic proteinase (Zevaco et al. 1973) and an
metallo proteinase (Gripon and Hermier, 1974) have been characterised. Much less is
known about the proteolytic system of P. nalgiovense. In an attempt to isolate genes for
proteases a gene bank of P. nalgiovense was constructed in an expression plasmid, For
this purpose the chromosomal DNA of P. nalgiovense have been isolated partially,
digested with an restriction enzyme and fragments of 4 - 5 kb were cloned into an E.
coli expression vector. Highly competent cells of E. coli were transformed with the
plasmid gene bank of P. nalgiovense. About 20.000 independent clones of E. coli, each
carrying a particular plasmid were screened for proteolytic activity by growing on
medium containing skim milk. Among these clones one could be identified, which
exhibited proteolytic activity. The detailed analysis of the clone revealed that it
contained a chromosomal DNA fragment from P. nalgiovense of 1.4 kb, which
obviously coded for a protease gene. According to Southern blot experiments the gene
occurs only in a single copy in the genome of P. nalgiovense. To verify that the cloned
fragment indeed codes for a protease gene from P. nalgiovense the plasmid was
modified and reintroduced into the P. nalgiovense wild type strain. From the resulting
transformants one could be identified with highly reduced proteolytic activity (Fig. 7)
Figure 7: Proteolytic activity of the wild type (left) compared to the

transformed strain of P. nalgiovense (right).
Apparently in this strain a gene replacement has taken place. Subsequent Southern Blot
analysis support this possibility (Geisen, 1995). An analysis of the pH optimum of the
cloned protease, by comparing the proteolytic activity of the different strains on media
22
Figure 9: Thin layer chromatogram of the chloroform extractable

secondary metabolites of different mutants of P. camembert (lane 1 to 4,
Cpa2, Cpa3, Cpa4, Cpa5; different mutant strains), the wild type (lane 5)
and purified CPA as standard (lane 6).
The possibility exists that this might be an intermediate of the CPA biosynthetic
pathway, which accumulates just before the mutational block. To analyse this
possibility the mutant strain Cpa2, as well as the wild type strain were grown in the
presence of radioactively labelled 3H-tryptophan, which is a precursor of CPA. The
extracted metabolites of both the wild type and the mutant were separated by twodimensional TLC and subjected to autoradiography. The CPA spot of the wild type
gave a strong signal, however the new metabolite, which occurred in the mutant was not
labelled. The isolated spots were counted in a scintillation counter and the incorporated
radioactivity was quantified. This data show that the new metabolite has the same
background activity than the control. This results indicate that obviously the mutational
block must be located "before" the ligation of the isoprenoid moiety to the amino acid
tryptophane. This means that the mutation should be located in the part of the pathway
in which the isoprenoid moiety is produced. For that reason, the same experiment were
carried out, but in the presence of 14C acetate, which is the direct precursor of the
isoprenoids. The extracted secondary metabolites of the wild type and the mutant Cpa2,
which were grown on the labelled medium, were also subjected to two dimensional
TLC. This time a clear difference in the pattern of the separated labelled secondary
metabolites could be observed (Geisen, 1992). This indicates that a mutation, which
influences the production of isoprenoid precursors, is the reason for the inability of this
strain to produce CPA.
A physiological analysis showed that the mutated strain has the same growth
behaviour than the wild type strain.
24

Table 2: Aristolochene synthases specific PCR reactions with different P. roqueforti
strains
BFE No
PR-toxin
PCR
42
50
53
168
++
169
++
17-
++
172
nd
208
++
209
++
210
++
211
++
215
++
216
219
++
269
++++
270
+++
A better way to screen for PR toxin negative strains, is to look for the presence of
certain genes coding for key enzymes in the biosynthetic pathway of the secondary
metabolite. If strains can be identified, which do not have a gene coding for an enzyme
in the biosynthetic pathway of PR toxin, they should not be able to produce PR toxin
under any condition, if no other metabolic shunt exists. One of the genes for a key
enzyme in the metabolic pathway to PR toxin has been cloned and sequenced (Proctor
26
and Hohn, 1993). It codes for the aristolochene synthase ( ari1 ). Aristolochene is a
precursor in the biosynthesis of PR toxin. The knowledge of the nucleotide sequence of
that gene can be used for a molecular screening method for strains that did not carry the
aril gene by PCR. Strains, which do not carry the ari1 gene, should not be able to
produce PR toxin. For these purpose primer sequences have been deduced from the
published sequence of the ari1 gene and different P. roqueforti strains have been tested
for the presence of the ari1 gene by using these specific primer in a PCR reaction. The
results are shown in Table 2. As can be seen in this table one strain could be identified
by this method, which apparently did not carry an ari1 gene. As it was possible that
only the primer binding sites may have been mutated in that gene, which also would
give rise to a negative PCR reaction, the results were confirmed by dot blot analysis.
This negative strain was also not able to produce PR toxin under the conditions used.
On the other hand another strain could be identified, which also did not produce PR
toxin, however which gave a positive result in the PCR reaction. These results suggest,
that this strain contains either a mutant gene, which is responsible for the non-producing
phenotype, or the strain may produce the toxin under other conditions. In any case the
strain with the negative PCR results should be preferred.
5. Conclusions
Fungal strains should be carefully selected according the specific need of a fermented
product. If it is not possible to find a strain that fulfils all the requirements, it can be
optimised by several methods. Either molecular methods can be used to introduce
additional characteristics to a strain or to inactivate specifically undesired features of a
particular strain. In cases were molecular data are not available, the conventional
mutation/selection approach may be used, however it has to be kept in mind that this
approach is less specific and that secondary mutations with undesired phenotypic
changes may occur. Screening for particular characteristics, based on molecular data is
preferable over conventional phenotypic screening, as the results are more reliable.
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29
STARTERS FOR THE WINE INDUSTRY

ALINE LONVAUD-FUNEL
Facult dnologie - Universit Victor Segalen Bordeaux 2,
351 Cours de la Libration, 33405 TALENCE Cedex
Abstract
Grape must is naturally seeded with yeasts and lactic acid bacteria. Both
microorganisms are needed : yeasts to carry out alcoholic fermentation, and bacteria to
degrade malic acid to lactic acid during malolactic fermentation. Indigenous microflora
is often sufficient. However, use of selected yeast and malolactic starters allow a better
control of the process. Strains are selected for their influence on the sensorial and
hygienic quality of wine.
1. Introduction
Winemaking requires the successive involvement of two microorganisms. Yeasts
ferment the grape must into wine, then lactic acid bacteria complete the malolactic
fermentation. The later fermentation is not obligatory but recommended for optimising
the quality of nearly all red wines and most white wines. Alcoholic fermentation is, of
course, the main microbial event in winemaking. Pasteur was the most famous
microbiologist interested in wine production. The role of yeast in
alcoholic
fermentation was assessed by several authors at the end of the 19th century. Their
observations and experiments were so accurate and judicious that they discovered the
essential proceedings, describing the primary (ethanol) and secondary products of
alcoholic fermentation, the influence of several factors on the fermentation rate and on
the quality of wine. As early as 1895 numerous experiments led one of the oenologists
of this time to write a chapter on the use of selected yeasts for improving wine
fermentation (Brunet, 1894). Modern oenology does not refute this concept. It confirms
and proves that using yeast starters makes alcoholic fermentation safer and faster, thus
conferring to the wine better sensorial quality. Today several dozens of yeast starters are
available and are used intensively in all the wine producing areas in the world.
The situation is somewhat different for lactic acid bacteria that are responsible for
malolactic fermentation. Indeed, this second step of winemaking was recognised as
essential in the 1940s by some oenologists in the Burgundy and Bordeaux areas. It took
more than 20 years in some producing areas to consider that malolactic fermentation
31
A. Durieux and J-P. Simon (eds.), Applied Microbiology, 31 47.
ALINE LONVAUD-FUNEL
was not an accident but a second microbial transformation that improves wine quality.
Usually lactic acid bacteria develop after the end of alcoholic fermentation, i.e. during
and after the decline phase of yeasts. Thus they multiply in a medium which is not only
acidic but which also contains up to 13 % ethanol. They face very harsh conditions. The
addition of selected malolactic starters was first considered in the early 70s. However,
the success of such preparations was much more difficult than for yeast starters,
precisely because of the composition of wine. Therefore, progress has been slowed
down by many drawbacks, so today the state of the art for lactic acid bacteria starters is
approximately the same as it was two decades ago for yeasts. However, due to the
increasing knowledge on the physiology and metabolism of such bacteria, it is expected
that the gap will be soon filled. In the early 90s the first malolactic starter for direct
inoculation of wine was commercialised (Nielsen et al., 1996).
The demand for yeast starters is high both in the new and older wine producing
countries. Yeast producers are still selecting new starters on a wide scale, since the
variety of wines produced all over the world is great, and the potential ability of yeasts
seems greater than as used at present. As for lactic acid bacteria , the knowledge of their
influence on wine is just in its infancy and more needs to be discovered about their
adaptation to the wine medium to increase the success of malolactic starters.
2. Yeast starters in winemaking
After grape crushing, the must is naturally seeded with the microorganisms that are on
the surface of the berries and the cellar equipment. Soon after the grapes have been sent
to the fermentation tank or barrel, alcoholic fermentation normally starts with the
indigenous yeasts, Several yeast species can be isolated on the grapes and thus in the
grape must before fermentation: Candida sp., Kloeckera apiculata, Metschinikovia
pulcherrima, Torulaspora delbruekii, Schizosaccharomyces pombe and Saccharomyces
sp. K. apiculata, T delbrueckii and S. cerevisiae often predominate (Herraiz et al.,
1990). However, most of them quickly disappear except for Saccharomyces cerevisiae
that is the main agent of alcoholic fermentation (Fleet et al, 1984). According to the way
alcoholic fermentation is carried out defects may be induced such as off-flavours,
volatile acidity or even stuck fermentations. This might result in the predominance of
some undesirable yeast strains. The non-Saccharomyces species are mainly involved in
such problems. In addition, some Saccharomyces strains are not welladapted to the
fermentation of sugar-rich medium and cannot complete the whole process. Some also
produce high levels of SO2 and acetic acid. This explains why spontaneous fermentation
can be considered as a hazardous phase. Today yeast starters are exclusively selected
strains of Saccharomyces cerevisiae.
2.1 THE OBJECTIVES OF YEAST STARTERS
The reasons for using yeast starters were established at the end of 19th century. The
experiments consisted in seeding the grape must by actively growing yeasts which were
taken from fermenting musts of good quality. The experimenters observed that the
quality of wine was consequently higher, and fermentation more regular and quick
(Brunet, 1903). In addition, Pasteur (1876) noted that if the same grape must was
32
inoculated by several distinct yeasts, the wines would also be distinct. The present
objectives of winemakers are exactly the same. The only difference between the two
centuries is that the selected strains today are individual clones while they were
mixed starters before, since there was no isolation procedure. However, 100 years
ago, wines were probably obtained by Saccharomyces sp. Now winemakers use single
strain starters in order to control the process more closely.
There are two reasons for using starters. One is to start the alcoholic fermentation
quickly after the harvest. Indeed, in some cases, and preferably at the beginning of the
winemaking the yeast population is too low (less than 104 CFU.ml-1). Multiplication up
to 106 and more takes several days especially if the temperature is low. During this time,
other microorganisms can develop : yeasts with oxidative metabolism and acetic acid
bacteria that take advantage of the presence of oxygen to produce volatile acidity and
many other defects. Thus, inoculation with starters at the concentration of 106 CFU.ml-1
prevents the growth of such microorganisms. The second reason for the winemaker to
use yeast starters is to improve the final phase of alcoholic fermentation. Indeed, grape
musts are so rich in sugar and sometimes so poor in essential nutrients that yeast cannot
survive long enough to ferment all sugars. Stuck fermentation is one of the major
problems in winemaking. The fermentative ability of the yeast cell decreases during
alcoholic fermentation due to its sensitivity to increasing ethanol and fatty acids. Both
compounds are toxic for yeast (Lafon-Lafourcade et al , 1984) and the target of this
effect is the membrane. Oenologists and winemakers have long known that aeration is
essential to improve alcoholic fermentation. This was also explained by the work of
Larue and Lafon-Lafourcade (1989) who demonstrated that the survival of yeast in a
fermenting must depends on the sterol composition of the membrane, which itself
depends on the O2 available during growth. This also explains why yeast starters that
are produced in aerobiosis can respond more efficiently to these conditions than
indigenous yeasts.
The addition to must of selected starters also named active dry yeasts has
become essential in white winemaking. It has replaced the former starters that were
prepared by multiplying yeasts from a spontaneous fermenting wine in highly sulfited
must to prevent bacterial growth. White grape must is not easily fermentable. Indeed,
after crushing it generally undergoes to several operations (decanting, clarification) to
improve its sensorial quality, and these make it poorer in nutrients and in natural
microflora. Therefore, it is necessary to add yeast very soon, once the must has been
clarified or adjusted to the adequate turbidity by addition of light solids (Ollivier et al ,
1987). Active dry yeasts are added to reach 106 CFU.ml-1, i.e. 10 to 15 g.hl-1 of the
commercial preparation. They are added just after the clarification of grape juice, after a
reactivation in grape must and water (v/v ; 1 :1) for 20 minutes at 40C.
Yeast starters are less used, and in fact less necessary in red winemaking. The
operations that follow grape crushing do not deprive the medium of the natural
microflora, since all the parts of the berries skin, pulps and seeds are poured into the
vats. Moreover, the process includes several pumping over phases to extract the wine
colour, and some of these are done with aeration. It is only when the temperature is too
low or if rain has considerably washed the grapes that alcoholic fermentation for red
wines may take several days to start. In these cases active dry yeasts are necessary.
33
ALINE LONVAUD-FUNEL
However, as winemakers are more and more aware of the difficulties encountered in
obtaining the alcoholic fermentation, they also use starters for red wines.
The second reason to use active dry yeasts is to restart uncompleted alcoholic
fermentation. When several g.l-1 of sugar remain unfermented, the wine is not finished
and is exposed to spoilage. Indeed during the active phase of fermentation, yeasts
inhibit bacteria, but since their population decreases and may lead to stuck fermentation,
lactic acid bacteria can develop. This induces one of the major accidents in winemaking
called piqre lactique characterised by the heterolactic fermentation of sugars. The
result is a high volatile acidity. Therefore as soon as alcoholic fermentation slows down
a sufficient population of active yeasts must be added. The commercial active dry yeasts
need a preliminary preparation otherwise they cannot survive, since the medium is high
in ethanol and fatty acids. The reactivation procedure is conducted as follows in a
medium prepared with the wine itself. The alcohol content is reduced to 8-9 % with
water and adjusted to 15 g.l-1 of sugar. After sulfiting at 3 g/hl-1, 20 g.hl-1 of active dry
yeast are added. At 20C growth occurs within several days and the fermentation is
monitored by determination of sugar concentration or density. When the sugar has just
totally disappeared, the yeasts are in the active growing phase. The starter is added at
the concentration of 5-10 % in the wine to be treated. The completion of alcoholic
fermentation still takes several days. The efficiency of such starters can be increased by
the preliminary addition, 24-48 hours before, of yeast cell walls. Lafon-Lafourcade et al
(1984) demonstrated that the preparation obtained from yeast cells by elimination of the
yeast cellular components - i.e. the yeast cell walls enhances yeast survival in
fermenting must. It is now an authorised procedure to prevent or to treat stuck
fermentations. This regulatory property is mainly due to the capacity of cell walls to fix
and to remove yeast inhibitors such as fatty acids.
2.2 PROPERTIES OF YEAST USED AS SELECTIVE CRITERIA FOR ACTIVE
DRY YEAST PRODUCERS AND WINEMAKERS
All the active dry yeasts available share the same basic characters ; i.e. good
multiplication rate, high yield of ethanol production, ability to complete fermentation of
sugars (resistance to ethanol and other toxic compounds such as pesticide residues),
adaptation to low and high temperatures, low production of volatile acidity of ethanol,
sulphur dioxide, sulphur hydrogen, foam, and high glycerol production. In addition, it
is preferable for them to be neutral towards the killer toxins or even producers of the
toxin, and not to be sensitive since they mightbehampered by indigenous killer strains
(Jacobs and van Vuuren, 1991). Finally, such selected strains must withstand the drying
process and recover their properties after storage.
These are the minimum requirements for selected yeast starters. However, among
the dozens of preparations available, one must choose the best adapted to the special
problem to be solved. They can be divided in several categories : quick starter yeasts,
those for restarting stuck alcoholic fermentation, others for prise de mousse of
sparkling wine, some for wines used to make spirits and finally yeasts aimed to develop
regional or aroma typicality. The first are selected for their resistance to the hostile
conditions of wine fermentation. However, more and more work is now underway to
find strains able to develop aromas and to optimise phenolic compound extraction in red
wine. Primeur red wines and white wines are mainly characterised by fruity and
34
floral aromas. Esters, especially shorter chain fatty acid ethyl esters and acetate esters,
are the main volatile components involved in the sensorial quality of wines, together
with carbonyl compounds such as acetaldehyde and diacetyl. They are produced by
yeast metabolisms (Henschke and Jiranek, 1993). The conditions of alcoholic
fermentation, notably temperature and the presence of solids through yeast metabolism
modifications change the final amount of volatile compounds.
However, the strain itself has a real impact (Vasconcelos et al., 1996), e.g. if it
produces two much of one of the volatile components. Some are revealed by yeast
activity on the aroma precursors found in grape berries (Darriet et al , 1991).
Figure 1 : Influence of the yeast strain on the relative intensity of the

Sauvignon aroma (after Darriet et al., 1991)
On the contrary, some yeasts may suppress the variety aromas ; they must be used only
for the fermentation of neutral grape varieties. Among many other compounds, yeast
can produce vinyl phenols from p-coumaric acid and ferulic acid in must. Above a
certain threshold these give the wine pharmaceutical odours. The enzymatic activity,
cinnamate decarboxylase, depends on the yeast strain (Figure 2) (Chatonnet et al, 1993).
The role of yeast in the production of varietal aromas is currently intensively
studied. The involvement of yeast in the release of terpenes which characterise grape
varieties such as muscat, gewrztraminer, riesling, etc.... and in that of norisoprenoids
that are very odorous, is not clear. More is known about the characteristic aroma of
35
ALINE LONVAUD-FUNEL
Sauvignon that appears during alcoholic fermentation. The non-odorous precursors

which have been identified in grape must are cysteine complexes. The aromas are
revealed by the action of a specific -lyase. The activity varies according to the yeast
strain. The S-cysteine conjugates are hydrolysed by yeasts during alcoholic
fermentation (Tominaga et al, 1998). Therefore strains have been selected for their
capacity to reveal the Sauvignon aroma. Other varietal aromas such as gewrztraminer
and manseng are also enhanced.
Figure 2 : Influence of the yeast strain on the vinylphenol concentration of

a white wine (Sauvignon variety) (Chatonnet et al., 1993)
In red wines, the type and amount of polyphenols is very important. An aim of
winemaking is to obtain the highest amounts of the good polyphenols. It has also
been shown that yeast can influence the final composition in phenolic compounds and
thus the wine colour. This study led to the selection of two yeast strains that will be
dried and tested in a pilot-scale study (Arioli et al., 1996).
From such studies, companies producing active dry yeast for the wine industry can
make new preparations. However, winemakers must remember that the wine aroma
precursors are in the must. Therefore, the yeast has to be adapted to the grape must. The
aim is to reveal the aromas without excess, since the overproduction of some
components is undesirable.
36
2.3 EVALUATION OF THE SETTLEMENT OF ACTIVE DRY YEAST DURING

ALCOHOLIC FERMENTATION :
Active dry yeasts are usually added to grape must containing indigenous microflora
originating from the grapes and the cellar equipment. This microflora is usually low in
white grape musts due to several operations aimed to eliminate solid materials from the
medium. The level of microflora is normally higher in red grape musts. However, until
recently it was not easy to assess the efficiency of the starter, i.e. it was difficult to
ascertain whether alcoholic fermentation was done by the starters or by the indigenous
yeast population. Today molecular methods are very reliable tools to monitor and to
assess the role of starters. Several methods can be used to differentiate a Saccharomyces
strain from another. The first method that was developed was the analysis of restriction
patterns generated by hydrolysis of mitochondrial DNA (mtDNA). Oenological strains
were successfully characterised by Dubourdieu et al (1987) and Hallet et al (1988). The
profile of the active dry yeast used as starter is compared to that obtained from the lees
sampled during or after alcoholic fermentation. If there is no extra band, this means that
the starter has grown and has replaced the original yeasts. However, in spite of its
reliability, this method is no longer used because it is unwieldy and time-consuming.
The analysis of karyotypes needs less preparation. It consists in a separation by
pulse field gel electrophoresis of the 16 yeast chromosomes. Since their size varies from
250 to 2500 kbp, this gives a profile for each strain . The preparation of samples is rapid
and easy, but the electrophoresis of such large DNA molecules requires special
electrophoresis equipment. Blondin and Vezhinet (1 988), then Frezier and Dubourdieu
(1991) applied this method for wine yeast strain identification during winemaking. Like
others, Roulland et al (1996) using this method reported that spontaneous alcoholic
fermentation is carried out by one or at the most two dominant strains in wine used to
make Cognac.
Finally PCR (Polymerase chain reaction) using repeated sequences as primers is
another very interesting method for the identification of strains during winemaking
(Ness et al, 1992). It is much more rapid than the other methods and can be used
directly on whole cells. The profile generated by specific amplification can distinguish
most of the strains very easily. Like the mtDNA RFLP profiles and the karyotypes, the
PCR patterns of the yeasts taken in lees are compared to those of the starter. They are
identical if the starter has really taken part in the process. By PCR it is shown that 90 %
of the population is derived from the starter when the profiles are identical.
Usually the starter chosen is added as soon as possible to the grape must to inhibit
and eliminate the indigenous yeasts. The chance of success is 90 % if the ratio
starter/indigenous yeast is 10. Some have recommended successively adding two
different starters in order to enhance wine complexity. A study was undertaken to verify
the role of both starters inoculated in the same vat (Meurgues et al., 1998). Molecular
typing by PCR of the yeasts taken at the end of alcoholic fermentation clearly showed
that the second starter, which was added two days after the first one, had completely
disappeared. As expected, addition at about 6.106 CFU.ml-1 of the second starter, i.e.
three times the normal dose, could not replace the active fermenting population of the
first starter which was about 108 CFU.ml-1. Thus, the addition of selected yeasts during
the course of alcoholic fermentation is of little use.
37
ALINE LONVAUD-FUNEL
Analysis of the diversity of yeast strains during alcoholic fermentation has also
demonstrated that a selected starter may be easily disseminated in a cellar in red wine
winemaking (Frezier and Dubourdieu,1991). While only the first vat filled was
inoculated, the presence of the starter was dominant even in vats filled several days after
and not inoculated (Table I).
Table I : Percentages of yeast strains isolated in fermenting grape musts inoculated or not with active dry
yeasts (after Frezier and Dubourdieu, 1991)
Isolated Vat 1 inoculated

with strain F5
strains
F5
80
F10
0
522M
0
FIb4
10
FIb9
10
FIIIb8
0
Vat 2 inoculated Vat 3 inoculated

Non
with strain F10 with strain 522M inoculated
0
0
60
100
0
40
0
90
0
0
0
0
0
0
0
0
10
0
This means that in red winemaking mere cellar handling is sufficient to disseminate
yeasts. However, the use of massive concentrations of different starters allows their
settlement in several vats. In white winemaking, the conditions and the results may be
similar except when must is fermented in barrels, thus making dissemination less
possible. Progress in the identification of yeast strains has also led to real advances also
for controlling the production of starters themselves. This is obviously very important
since winemakers can now really monitor the role of the starters they have chosen
according to the type of wine.
3. Malolactic starters in winemaking
Lactic acid bacteria must reach the population of 106 CFU.ml-1 in order to start
malolactic fermentation. Depending on the variety, the producing area and the degree of
maturity, the grape must contains about 2 to 8 g.l-1 of malic acid. In most cases all malic
acid must be transformed to lactic acid. The benefit is a softer taste and greater aroma
complexity. However, some white wines only need the partial degradation of malic acid
that results in deacidification while preserving the fruity aromas that normally disappear
with total malolactic fermentation. Bacteria must grow after alcoholic fermentation in
very hostile conditions due to ethanol, fatty acids, other inhibitors and acidic pH.
However, in normal conditions, some days after the yeasts have completely fermented
the sugars, bacteria multiply very quickly from 102-103 CFU.ml.-1 to more than 106
CFU.ml-1. The growth rate depends on the environmental conditions, the adaptation of
the bacteria to the medium and on the yeast responsible for alcoholic fermentation (
Lonvaud-Funel et al, 1988). Like yeasts, the indigenous lactic acid bacteria originate
from grapes and cellar equipment. In the beginning of winemaking up to eight to nine
species are usually identified. However after a few days, a natural selection occurs in
the fermenting must. At the end of alcoholic fermentation, the Oenococcus oeni species
38
is predominant or exclusive, so it is the species most adaptable to wine conditions

(Lonvaud-Funel et al, 1991).
3.1 INDICATIONS FOR USE OF MALOLACTIC STARTER AND DESCRIPTION
The start of malolactic fermentation still remains completely uncontrolled. After
alcoholic fermentation, the winemaker can only watch and wait. Temperature is the only
parameter that may be controlled in order to promote malolactic fermentation. The use
of special equipment to adjust it to about 20C is very common and efficient. The wine
is heated until malic acid degradation starts. However, in spite of temperature control,
malolactic fermentation does not start sometimes due to a very low pH or to unknown
reasons. It is not rare for it to occur several months after alcoholic fermentation,
generally in May-June following the harvest. This proves that bacteria can adapt their
composition and physiology to wine. This of course presents several disadvantages.
Wine cannot be protected from spoilage microorganisms by sulfiting that would prevent
lactic acid bacteria growth. Therefore ageing and maturation may be delayed and
young wines cannot be sold. Moreover, as long as malolactic fermentation has not
begun, the temperature must be maintained even during wintertime, thus creating
additional costs.
The aim of using malolactic starters is to compensate the deficient indigenous
microflora by a high population of lactic acid bacteria. The first experiments with liquid
cultures were for a long time unsuccessful. The bacterial cultures added to the wine
really lost their activity and viability. Later when work on this subject began again, the
objective was to inoculate the wine with very concentrated populations. Since it is
difficult to obtain the multiplication of bacteria in wine, the idea was to add the
necessary level of bacteria (more than 106 CFU.ml-1) in order to do without the growth
phase. The starters were prepared with the species O. oeni. The latter is always used
nowadays in spite of several attempts to use other species such as lactobacilli that can
multiply more easily in industrial conditions.
The concentrated cultures were first frozen, then lyophilised. However, even the
addition of more than 106-107 CFU.ml-1 in wine failed and nearly no malic acid could be
transformed. The first really successful use of malolactic starters was obtained by
Lafon-Lafourcade et al (1983). Their research showed that the survival of lyophilised
bacteria after inoculation could be greatly enhanced by a reactivation step. The
lyophilised preparation was incubated for 1-2 days in a reactivation medium composed
of grape juice diluted in water, enriched with 5 g.l-1 yeast extract and adjusted to pH
4.8. Other reactivation media were later described; all contain yeast extract. The
population during this step is around 1010 CFU.ml-1 and does not increase. Wine is
inoculated with 1 vol. for 1000 vol. with this reactivated culture to reach a viable
population of 107 CFU.ml-1. In these conditions the bacteria survive after inoculation
and malic acid is completely degraded. Thus reactivation allows a better adaptation of
bacteria to wine. Very often such starters are inoculated in small volumes of wine which
are then poured into larger ones, up to the volume of the total vat which needs to be
inoculated, This procedure is also successful but needs more time and care (Gerbaux et
al, 1995). Obviously, in spite of their efficiency, such reactivated starters are not very
convenient for winemakers. They necessitate special attention and good organisation.
39
ALINE LONVAUD-FUNEL
New research in the early 90s was undertaken on the resistance of O. oeni to several
inhibitors such as ethanol and fatty acids. The focus was cell membrane studies since it
was thought that the membrane was the primary target of the inhibitors, which kill the
bacteria. Lonvaud-Funel and Desens (1990), then Garbay and Lonvaud-Funel (1 996)
demonstrated that the cultivation in media added with ethanol or fatty acids or other
stresses such as heat shock induced changes in the membrane composition. The most
striking feature was the overproduction of some membrane proteins and a decrease in
phospholipids. Such stressed cells were also more apt to survive in wine. Following
these results, the first O. oeni lyophilised preparation for direct inoculation in wine
became available in 1993 (Viniflora oenos, Chr. Hansen, Denmark) (Nielsen et al,
1996). The population is about 5-106 CFU.ml-1 and it grows or survives until the
complete transformation of malic acid (Figure 3). A few other preparations are now
available. But it is clear that the production of malolactic starters is much more difficult
than yeast starters for which the winemaker can chose amongst dozens of preparations.
The other problem when using a malolactic starter is the time of inoculation. While
there is no alternative for yeast starters, it has often been suggested that malolactic
starters can be used before, during or after alcoholic fermentation. Of course addition
before alcoholic fermentation avoids the inhibition of bacteria by wine components.
However, as for the indigenous bacteria, survival is readily inhibited by the active
growing yeasts. Moreover, if bacteria survive during alcoholic fermentation, they can
ferment sugar, in competition with yeasts. The real problem is not the loss of ethanol
but the production of volatile acidity which accompanies the heterolactic fermentation
of hexoses and pentoses by O. oeni. In addition, it has clearly been demonstrated that at
the end of alcoholic fermentation, at a time when yeast cells are losing their activity, too
many bacteria (over than 105-106 CFU.ml-1) tend to accelerate the yeast decline phase
Paraskevopoulos (1988). Bacterial enzymatic activity probably contributes to the
hydrolysis of the yeast cell wall. In such conditions the major problem is incomplete
alcoholic fermentation. This frequently occurs naturally when the indigenous bacterial
population grows too early (Ribereau-Gayon et al, 1998). At this time wine still
contains some 5-10 g.l-1 of fermentable sugars, a concentration high enough to produce
volatile acidity. Since the behaviour of the bacteria is completely unpredictable, it is not
recommended to add malolactic starters as long as the sugar have not been completely
fermented.
Figure 3A : Evolution of malic acid in a red wine inoculated or not by a malolactic starter.
(Nielsen et al , 1996)
40
Figure 3B : Evolution of bacterial population in a red wine inoculated or not by a

malolactic starter. (Nielsen et al , 1996)
3.2 THE INFLUENCE OF LACTIC ACID BACTERIA STARTERS ON WINE

QUALITY AND THEIR SELECTION
Since the crucial problem of the survival of malolactic starters in wine has been at least
partially solved, studies now focus on the important question of their influence on wine
quality. Two aspects are now considered : hygiene and the sensory qualities.
For a long time oenologists considered lactic acid bacteria only as the agents of
malic acid degradation. Even if it is the principal biochemical reaction, bacteria also
metabolise many other wine components. The most studied has been citric acid that is
transformed into acetic acid and acetonic compounds. Only about a maximum 0.3 g/l of
citric acid are metabolised but this is enough to induce sensorial changes due to the
production of acetic acid (volatile acidity) and diacetyl which is very aromatic. Only a
few mg/l of the latter gives the wine a buttery aroma. The threshold is dependent on the
wine, and is less for white wines (= 4-5 mg/l) than for red wines (7-8 mg/l). Sensorial
analyses show that some judges prefer buttery wines, while others consider high
diacetyl concentrations as a defect. The other sensorial effects are characterised by the
production of fruity and floral notes. It is not definitively established that they depend
on the dominant bacterial strain. Numerous studies have compared wines inoculated by
distinct bacterial strains and have shown their individual influence (Henick-Kling T.,
1993). However, sensorial analyses rarely show significant differences. Moreover in
such experiments, the repetition of inoculation by a given strain may often give more
sensorial differences than different strains. This point remains unclear and such results
41
ALINE LONVAUD-FUNEL
show that malolactic fermentation has a real influence on wine aroma, but this is less
strain-dependent than alcoholic fermentation.
Other compounds are undesirable such as biogenic amines, which might be
responsible for allergies, and ethyl carbamate that is thought to be carcinogenic. For a
long time only non-malolactic bacteria (all species except O. oeni), and especially
Pediococcus sp. were thought to produce histamine. However O. oeni strains may be
involved. Such strains contain the hdc gene that codes for the histidine decarboxylase.
Surprisingly, it has been shown that many strains have this property (Coton et al,
1998). During and mainly after malolactic fermentation, tyramine and putrescine
(diaminobutane) also increase in some wines. However, until now the bacteria involved
have not been completely characterised. Another observation is that some wines do not
contain any biogenic amine, while others produced from year to year in the same cellar
always contain them. At least for histamine, it is concluded that the indigenous bacterial
flora that is established in the cellar comprises aminoacid decarboxylating strains.
Ethyl carbamate that is produced from precursors such as urea, carbamylphosphate
and citrulline, is also studied by food hygienists. In wine, urea is produced by yeast, and
citrulline is produced by lactic acid bacteria from arginine (Liu et al., 1994). Current
studies aim to study and characterise O. oeni that may utilise the arginine deiminase
pathway and increase the risk of ethyl carbamate in wine.
Therefore, the selection criteria for malolactic fermentation starters are based both
on the capacity of the strain to be prepared at the industrial level for survival in wine
and on some special metabolic pathways. Usually the strains are isolated from wines
during malolactic fermentation. The only species considered is here O. oeni. After
identification the first selection procedure includes sensitivity to ethanol and to pH. The
latter is certainly the most important parameter. Malolactic activity is not a real
selection marker since it is generally high for all strains. The ability to metabolise citric
acid is also a general trait of this species and must be considered as a positive character.
The other property to be considered is the inability to produce biogenic amines. Since
many studies are focusing on sensorial quality and secondary metabolisms, selection
will probably include more criteria in the future.
3.3 EFFICIENCY OF MALOLACTIC STARTERS
Unlike yeast starters, malolactic fermentation starters have been difficult to promote
because in the beginning they were very unreliable. Moreover, in numerous studies,
authors have concluded that they were efficient because malolactic fermentation
occurred after inoculation. Frequently, it was not clear if the malic acid is degraded by
the starter or by the indigenous bacteria, especially when malolactic fermentation starts
long after inoculation. This probably explains why the market for malolactic
fermentation starters is still unsure. Winemakers are not convinced of the effectiveness
of the starter for the above mentioned reasons.
Today, it is possible to verify the settlement and the role of added bacteria with
molecular methods, such as for active dry yeasts. The most relevant method is to
compare the genomic fingerprints of the bacteria taken at the end of malolactic
fermentation to those of the starters. Genomic studies of O. oeni have shown that each
strain can be distinguished from others by the electrophoretic pattern of digested DNA.
Restriction by rare cutting enzymes generates DNA fragments that are well separated by
42
pulse field electrophoresis. Three enzymes are frequently used and are well adapted for
this species. Thus, the procedure consists in the cultivation on solid medium of bacteria
present in wine at the end of malolactic fermentation. The DNA of the whole biomass
(or individual colonies) is analysed. If the starter has really dominated the indigenous
microflora, the profile is strictly similar to that of the lyophilised bacteria. The presence
of additional bands proves that other bacteria were in the wine at the same level and
probably took part in the process (Daniel et al. 1993, Gindreau et al., 1997). Of course,
analyses of samples during the process can indicate whether the starter undergoes a real
competition with the indigenous bacteria. This advance is very new but very promising.
Indeed in none of the previous studies, notably those to analyse the sensorial impact of
starters, could the survival of the starter be assessed. Today, wine scientists have a
suitable method to verify the ability of the starter to induce malolactic fermentation
before the decision is taken to implement fastidious and difficult sensorial and fine
chemical analyses.
After malolactic fermentation sulphur dioxide is added to eliminate as far as possible
all the microorganisms and to protect wine from oxidation. However, in many wines of
relatively high pH, sulphur dioxide reduces its antimicrobial activity and the bacterial
population remains relatively high. Identification to the strain level by fingerprinting
has recently shown an interesting property of malolactic fermentation starters.
Comparison of populations in inoculated and non-inoculated wines proved that the
population remaining after sulfiting did not contain the starter strain at least at a
sufficient level to be detected by DNA fingerprinting. It was composed of the
indigenous lactic microflora (Millet and Lonvaud-Funel, unpublished results). Thus, if
this result was confirmed, it would mean that starters are eliminated soon after
malolactic fermentation. This is very positive, since it means that a given starter would
be very unlikely to be perpetual in the cellar.
4. The future of starters for winemaking
The interest of winemakers for yeasts and bacteria starters is motivated by the potential
they offer to control the fermentation processes. Nowadays, active dry yeasts are widely
used. Winemakers in USA, Canada, South Africa, Australia, New Zealand were the first
users by the mid-1960s and it took about 10 years more for the European producers to
start with yeast inoculation. Malolactic starters came very later and the industrial
preparations for direct inoculation are very recent.
Yeasts are inoculated : i) to shorten the time between the harvest and the active
alcoholic fermentation which is crucial to prevent spoilage, ii) to avoid incomplete
fermentation, iii) more and more, to develop specific aromas. In fact, the application of
elementary oenological rules solves the second point at least. The judicious aeration of
fermenting must provide enough O2 for yeast to synthesise the indispensable sterols it
needs for survival (Lafon-Lafourcade, 1983). Temperature control also minimises the
problems. Nevertheless, it seems that more and more active dry yeasts are widely used
for safety, even if they are not really necessary. While they are indeed almost
indispensable in white wine production because of all the preliminary treatments of the
grape musts, this is not the case for red wines. More and more the principal objective of
the producer is to develop aromas during alcoholic fermentation. Of course, it is highly
43
ALINE LONVAUD-FUNEL
preferable that they are typical aromas characterising if possible not only the grape
variety but also the grape variety in its environment, i.e. marked by the terroir
Even if there is already a considerable choice of starters , much work is still
underway to improve selection. The increasing knowledge of the aroma compounds
revealed during alcoholic fermentation is of course the driving force of this activity.
Typicality related to wine variety is consistently influenced by the dominant strain
during alcoholic fermentation. Therefore, aroma quality could be enhanced by the
selection of the best-adapted strains in view of their enzymatic activities. However,
typicality is also related to many other factors, and the use of a given starter will not
necessarily result in the same wine in different wine areas.
This justifies the increasing attention of yeast selectors for the aromas produced by
candidate strains for starters. As in the early age of wine microbiology, some
oenologists have in recent years proposed using yeast species other than S. cerevisiae.
Indeed it is well known that the indigenous yeast microflora is composed of several
genera such as Candida, Rhodotorula, Brettanomyces, Torulopsis, Harsemaspora, and
Kloeckera. They are in variable proportions in the must and disappear due to their
sensitivity to ethanol and O2 depuration. However, some of them may survive relatively
late during the alcoholic fermentation. Their influence on wine aromas has been
demonstrated (Herraiz et al., 1990). The apiculate yeasts, Kloeckera apiculata and
Hanseniasporia guillermondii, were assayed associated to Saccharomyces cerevisiae
(Romano et al, 1992 ). They were found to greatly influence the composition of wine
and aromas. Thus there is now renewed interest in the non-Saccharomyces microflora,
and active dry yeast producers are studying their potential use. Raguiel et al (1998)
describe the results obtained with a T. delbruekii strain and a strain obtained by fusion
of S. cerevisiae and Schizosaccharomyces pombe characterised by its capacity to
degrade malic acid. While the latter was studied for deacidification, the former was
studied for its property to produce strawberry aroma. The authors conclude that the use
of the T. delbruekii strain mixed in suitable proportions with S. cerevisiae is interesting
and justifies further experiments in various environmental conditions.
Selection of yeasts as starter should also include, strains for deacidification and on
others which retain or even increase the initial acidity of grape must. These mainly
differ by their ability to degrade or produce more or less L-malic acid. The traditional
selection of yeast strains might also be replaced in the future by genetically modified
yeasts. Even if the use of such microorganisms is subjected to controversy several
research teams are already preparing this approach. The first work in the early 90s
concerned the transformation of S. cerevisiae with the malolactic bacterial gene. The
idea was to add to the yeast the malolactic activity which would allow the simultaneous
alcoholic fermentation and malic acid degradation (Ansanay et al., 1993, Denayrolles et
al, 1995, Volschenk et al, 1997 ). Following this research several other functions were
considered for cloning in the wine yeast : higher production of glycerol (Remize et al,
1999), of L-lactic acid (Dequin and Barre, 1994), secretion of proteolytic enzymes
(Lourens and Pretorius, 1996). However, the success and the demand for such yeasts
starters is very unpredictable.
As regards lactic acid bacteria starters, much future work will concern the
adaptability of inoculated bacteria to the stress conditions of wine. Even if great
progress has been made with the starters for direct inoculation, there are still too many
44
unaccountable cases where the best ones fail. Today, the present results on aminoacid
decarboxylation and the production of ethylcarbamate can be added to the conventional
traits for the selection of new starters. Therefore, much more knowledge is necessary
regarding the sensorial influence of lactic acid bacteria at the strain level. If this is
demonstrated and considered relevant, then malolactic starters will also be selected for
these features.
5. Conclusion
The production of quality wine is tightly linked not only to the quality of grapes but also
to the microorganisms that completely change the composition of the medium where
they develop. The fate of grape juice is to promote the growth of yeasts and bacteria.
Alcoholic fermentation and malolactic fermentation spontaneously occur in most cases.
However, fewer winemaking accidents occur now than in the former times by
controlling yeasts and bacteria. Yeast starters are readily available and the choice gets
wider each year. However, the winemaker now has a new challenge: he must choose the
most suitable strain according to the grape must and the type of wine. This is not so
difficult, but there are many examples of bad choice. This is especially true when strains
produce too high concentrations of aromas ; this might finally lead to standardising
wine. It is preferable to choose the strain which express the best the wine typicality in
relation to the grape and its environment. Sometimes the best solution is to use a
neutral strain that does not mark the wine too heavily. The present interest of
researchers for non- Saccharomyces strains is very important since most reports
demonstrate that wine aroma complexity is enhanced by inoculation of such yeasts.
However, mixed cultures even from selected starters may be as difficult to control as
the indigenous microflora.
Finally, the most worrying problem is the dissemination and above all the
remanence of the starters in the cellar. It has already been shown that yeast starters recur
in a given cellar from year to year. Of course this does not prevent to use different
strains efficiently because they are massively added. However, this will surely suppress
the natural biodiversity of grape and wine microflora. At least for the moment, the
danger seems, much lower for lactic acid bacteria starters which seem to be unable to
survive after malolactic fermentation.
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Coton, E., Rollan, G., Bertrand, A., Lonvaud-Funel, A. (1998) Histamine producing lactic acid bacteria in
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Sciences de la Vigne et du Vin 25, 167-174.
Denayrolles, M., Aigle, M., Lonvaud-Funel, A. (1995) Functional expression in Saccharomyces cerevisiae of
Lactobacillus lactis mleS gene encoding the malolactic enzyme. FEMS Microbiology Letters 125, 37-44.
Dequin, S., Barre, P. (1994) Mixed lactic acid-alcoholic fermentation by Saccharomyces cerevisiae expression
the Lactobacillus casei L(+)LDH. Biotechnology 12, 173-177.
Dubourdieu, D., Sokol, A., Zucca, J., Tharlouan, P., Datte, A. and Aigle, M. (1987) Identification des souches
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Fleet, G.H., Lafon-Lafourcade, S. and Riberau-Gayon, P. (1984) Evolution of yeasts and lactic acid bacteria
during fermentation and storage of Bordeaux wines. Applied Environmental Microbiology 48, 10341038.
Frzier, V. and Dubourdieu, D. (1991) Incidence du levurage sur Icologie des souches de Saccharomyces
cerevisiae au cours de la vinification dans deux crus du Bordelais. Journal International de la Vigne et du
Vin 25, 63-70.
Garbay, S. and Lonvaud-Funel, A. (1996) Response of Leuconostoc oenos to environmental changes. J. Appl.
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Gerbaux, V., Cuinier, C., Barrre, C. and Berger, J.L. (1995) Bilan de cinq annes dessais densemencement
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Gindreau, E., Joyeux, A., de Revel, G., Claisse, O., Lonvaud-Funel A. (1997) Evaluation de Itablissement
de levains malolactiques au sein de la microflore bacterienne indigene. Journal International de la Vigne
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Henick-Kling, T. (1993) Malolactic fermentation in Wine Biotechnology , pp. 289-326, G.H. Fleet (eds.),
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Lafon-Lafourcade, S., Carre, E., Lonvaud-Funel, A. and Riberau-Gayon, P. (1983) Induction de la
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Lonvaud-Funel, A., Joyeux, A. and Ledoux, O. (1991) Specific enumeration of lactic acid bacteria in
fermenting grape must and wine colony hybridisation with non-isotopic DNA probes. Journal of Applied
Bacteriology 71, 501-508.
Lonvaud-Funel, A., Masclef, J. Ph., Joyeux, A. et Paraskevopoulos, Y. (1998) Etude des interactions entre
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47
Part 2
PHYSIOLOGY, BIOSYNTHESIS AND METABOLIC
ENGINEERING
METABOLISM AND LYSINE BIOSYNTHESIS CONTROL IN

BREVIBACTERIUM FLAVUM: IMPACT OF STRINGENT RESPONSE IN
BACTERIAL CELLS
M. RUKLISHA*, R. JONINA, L. PAEGLE AND G. PETROVICA
*Corresponding author, Institute of Microbiology and Biotechnology,
University of Latvia, Kronvalda blvd.4, L V 1586, Riga, Latvia; Fax:
(+371) 7 323 065; E-mail: coryne@lanet.lv
Abstract
Parameters affecting lysine biosynthesis by Brevibacterium flavum RC 115 cells under
stringent response conditions (test guanosine 5'-diphosphate 3'-diphosphate
accumulation in cells) caused by threonine limitation were investigated. Experimental
results confirmed that an increase in lysine biosynthesis by this bacterium under
stringent response conditions might be a result of an increase in the intracellular
concentration of NADPH as well as lysine export activity.
1. Introduction
Corynebacterium glutamicum and closely related Brevibacterium flavum species are
widely used for lysine production on an industrial scale. An understanding of the
physiology of amino acid producing bacteria is of special significance to choose the
most suitable environmental conditions in which bacteria will show their maximum
product synthesis rate and yield from the consumed sugars. As has been shown
previously, the decreasing of bacterial growth rate and maintenance of its value below
maximum for an extended period is an essential method to improve the process
productivity under fed-batch cultivation conditions of C. glutamicum and B. flavum
strains (Ruklisha et al., 1992). On the contrary, an increase in the bacterial growth rate
in the exponential growth phase up to maximum was observed to be followed by a
decrease (sometimes even irreversible) in the lysine synthesis activity of cells in the
stationary phase (Ruklisha et al., 1976). An increase in lysine synthesis under growth
limiting conditions was suggested to be a consequence of the stringent control
mechanism mediated via guanosine 5'-diphosphate 3'-diphosphate (ppGpp)
accumulation (Ruklisha et al., 1995).
51
A. Durieux and J-P. Simon (eds.), Applied Microbiology, 51 57.
M.RUKLISHA, R.JONINA, L.PAEGLE AND G.PETROVICA
The aim of the present studies was to investigate the effect of stringent response in B.
flavum RC 115 cells on changes in the concentrations of most relevant internal
metabolites, required for lysine synthesis and bacterial lysine synthesis activity as its
consequence.
2. Materials and Methods
The microorganism used was B. flavum RC 115 - auxotroph for threonine and
methionine (Culture collection of the University of Latvia). Bacteria were cultivated
under batch conditions in fermenter (MBR, Switzerland) on a glucose/corn steep liquor
containing media (Ruklisha et al., 1995). Respiratory activity of the cell culture (QO2)
under fermentation conditions was monitored by oxygen balance method using gasanalysing system to estimate difference between oxygen concentration in the inlet and
outlet gas of fermenter (Baburin et al., 1986). In some experiments bacteria, collected
from fermenter, were recultivated for one hour in flasks on a rotary shaker in slight
modified mineral medium described by Kiss and Stephanopoulos (1991) with 1.5-mM
threonine or without its complementation. Biomass, lysine (as lysine.HCl) and sugar
concentration in the medium, intracellular concentration of ppGpp, as well as
parameters of bacterial physiological activity and lysine biosynthesis (, h-1; qP, g
lysine .g cells-1. h-1 and YP/S, g lysine. g glucose-1) were estimated as described by
(Ruklisha et al., 1995). Pyruvate and NADPH were extracted from cells by the
respective procedures described by Weibel et al., 1974, Matin & Gottschal, 1976 and
measured by enzymatic methods. Intracellular lysine was extracted by a method
proposed by Erdman et al. (1994). Concentration of amino acids in cell extracts or
fermentation medium was assayed by HPLC (Shimadzu C-R4A chromatograph, Japan).
The samples were treated with 10% sulphosalicylic acid in order to remove proteins,
and filtered prior to analysis. The amino acids were quantified by automatic precolumn
derivatisation with o-phtalaldehyde, separated in the column (Nova Pak C 18, Waters,
Milford, Mass., USA) using a buffer/methanol gradient, and detected fluorometrically
using Shimadzu RF-530, fluorescence HPLC monitor (Japan).
3. Results and Discussion
The effect of stringent response on changes in metabolite concentrations in B. flavum
RC 115 cells and lysine biosynthesis was investigated under batch cultivation
conditions. Bacterial respiratory activity profile during fermentation, measured by
oxygen analyser, was applied to test changes in bacterial growth rate and to establish
appropriate intervals of time during the fermentation process to collect samples.
Applying this test, cell culture was collected from fermenter within 10 minutes from the
moment when stringent response in bacterial cells was induced (test: an increase in
intracellular concentration of ppGpp).
The results of the experimental work showed that the lysine biosynthesis of B.
flavum RC 115 cells significantly increased under batch cultivation conditions when
bacterial growth rate decreased below 0.08 h-1 (Fig.1A) as a result of threonine
limitation (Fig 1B). Besides, the lysine yield from consumed sugar in the time interval
52
METABOLISM AND LYSINE BIOSYNTHESIS CONTROL IN BREVIBACTERIUM
18-22 h increased up to 0.55 0.04 g lysine .g glucose-1. The increase in the lysine
synthesis activity of cells was correlated with a sharp increase in ppGpp concentration
in cells (Fig.2A).
Figure 1. Changes in the rate of bacterial growth () and lysine biosynthesis (qP) by B.
flavum RC 115 cells as well as changes in the concentration of threonine in growth medium
during batch cultivation.
Hence, it was assumed that the increase of lysine biosynthesis might be a consequence
of the stringent response in bacterial cells like changes in RNA synthesis and
modification of metabolic pathway functioning, changes in intracellular concentrations
of lysine precursors and others
53
M. RUKLISHA, R. JONINA, L. PAEGLE AND G. PETROVICA
Figure 2. Changes in the bacterial respiratory activity (QO2) and internal concentrations of
ppGpp, pyruvate as well as NADPH in B. flavum RC 115 cells during batch cultivation.
In further experiments, changes in the intracellular concentrations of NADPH and

pyruvate as well as that of lysine biosynthesis by B flavum RC 115 cells under
exponential growth and stringent response conditions were investigated. It was shown
that a stringent response in bacterial cells resulted in a significant accumulation of
NADPH and some increase in the pyruvate concentration in bacterial cells (Fig.2B).
However, no direct correlation between induction of stringent response and changes in
the internal concentration of pyruvate in cells was demonstrated since its internal
concentration during exponential growth increased in parallel with the increase of
bacterial growth rate. Therefore, the increase of the internal concentration of NADPH
and not pyruvate might be considered as a parameter favouring lysine biosynthesis by
B. flavum RC 115 cells under stringent response conditions.
54
METABOLISM AND LYSINE BIOSYNTHESlS CONTROL IN BREVlBACTERIUM
The fact that with the increase of lysine synthesis concomitantly increased synthesis of
other NADPH-consuming amino acids, e.g. valine (Fig.3), proline and isoleucine
(data is not presented), confirmed the possible role of NADPH accumulation in cells on
amino acid biosynthesis under stringent response conditions. It might indicate that the
physiological meaning of the increase of biosynthesis of lysine and other
NADPH-consuming amino acids under stringent response conditions would be
NADP+ regeneration in cells.
Figure 3. Changes in the external concentrations of valine and lysine in the cell culture of
B. flavum RC 115 during batch cultivation.
Changes in the intracellular concentration of lysine and the specific rate of its
extracellular accumulation by B. flavum RC 115 under stringent response conditions
differed. An internal concentration of lysine increased in parallel with the increase of
bacterial growth rate but did not increase further under stringent response conditions
(Fig.4). On the contrary, the extracellular accumulation of lysine under the latter
conditions significantly enhanced (see: Fig. 1A). Hence, it was suggested that, under
stringent conditions, induced by threonine starvation, lysine export from B. flavum RC
115 cells possibly increased. The lysine export with the help of specific excretion
carriers by C. glutamicum had been proved by Erdman et al. (1994). A possible role of
the stringent response on lysine export activity was investigated using cells of different
growth rate values, collected in appropriate moments of batch cultivation. Cell culture
of each sample was divided in two parts; cells were separated from the medium by
centrifugation and transferred in two versions of fresh pre-warmed mineral media (with
or without threonine). Cell culture with an initial concentration of biomass 10 1 g
cells.l-1 were recultivated in shake flasks for one hour. The results of short-term
experiments Metabolism and Lysine Biosynthesis Control in Brevibacterium flavum
showed that lysine as well as valine (the latter data are not presented) were accumulated
in the medium only as a result of threonine limitation (Table 1). The higher was the
55
M. RUKLISHA, R. JONINA, L. PAEGLE AND G. PETROVICA
initial rate of bacterial growth the higher increase in lysine synthesis was achieved
under conditions of threonine limitation.
Figure 4. Changes in the internal concentration of lysine in B. flavum RC 115 cells during
batch cultivation.
Hence the stringent response, induced in B. flavum RC 115 cells by threonine limitation,
probably resulted with the increase in lysine export activity. It might be assumed that a
mechanism exists, which favour increase the export of lysine from bacterial cells under
stringent response conditions. Rapid induction of the export of some amino acids from
bacterial cells as a consequence of stringent response was experimentally proved in
other bacteria. Data obtained by Burkovski et al. (1995) clearly showed that glutamate
export from Escherichia coli cells was induced under stringent response conditions and
this export was rel-A gene controlled. However direct role of the stringent response on
lysine export activity in corynebacteria should be further investigated.
Table 1 Effect of cell incubation in the medium with or without threonine on their specific
lysine synthesis activity (qP) *Cells collected at appropriate times of batch cultivation (see
Fig. IA) **Data represents average means SE of four experiments
Growth rate of initial cells*
(Time of collection, h)
Incubation with .5 mM
threonine
0.18 (6)
0.27 (8)
0.12 (11)
0.08 (1 8)
0.03 (20)
0
0
0
0
0
Incubation without threonine
qP ( g lysine g cells-1 h-1 )**

traces
0.03 0.004
0.07 0.005
0.06 0.005
0.05 0.005
4. Conclusions
The data presented in this study showed that lysine biosynthesis by B. flavum RC 115
cells increased as a consequence of stringent response, induced by threonine limitation.
56
METABOLISM AND LYSINE BIOSYNTHESIS CONTROL IN BREVIBACTERIUM
It was explained as a result of NADPH accumulation in bacterial cells and probably that
of an increase in lysine export activity.
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Kiss, R.D. and Stephaanopoulos, G. (1991) Metabolic activity control of the L-Lysine fermentation by
restrained growth fed batch strategies, Biotechnol Progr 7, 501-509.
Matin, A. and Gottschal, J.C. (1976) Influence of dilution rates on NAD(H) and NADP(H) concentrations and
ratios in a Pseudomonas sp. grown in continuous culture, J. Gen. Microbiol. 94, 333-341.
Ruklisha, M., Ekabsone, M., Viesturs, U., Mezina, G. and Selga,, S. (1976) Control of growth and lysine
synthesis by Brevibacterium sp.22L by variations in medium mixing intensity, Appl. Biochem. &
Microbiol. 2, 4, 518-523 (in Russian).
Ruklisha, M., Shvinka, J. and Viesturs, U. (1992) Biotechnology of Bacrerial Synthesis, Zinatne, Riga (in
Russian); Annex: 1993 (in English).
Ruklisha, M., Viesturs, U. and Labane, L. (1995) Growth control and ppGpp synthesis in Brevibacterium
fravum cells at various medium mixing rates and aeration intensities, Acta Biotechnol. 15, 1, 41 - 48.
Weibel, K.E., Mor, J.-R. and Fiechter, A. (1974) Rapid sampling of yeast cells and automated assay of
adenylate, citrate, pyruvate and glucose-6-phosphate pools, Analyt. Biochem. 58, 208 - 216.
57
MOLECULAR BREEDING OF ARMING YEASTS WITH HYDROLYTIC

ENZYMES BY CELL SURFACE ENGINEERING
MITSUYOSHI UEDA, TOSHIYUKI MURAI, SHOUJI
TAKAHASHI, MOTOHISA WASHIDA, AND ATSUO TANAKA*
Department of Synthetic Chemistry and Biological Chemistry, Graduate
School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 6068501, Japan *Corresponding author. Mailing address: Department of
Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501,
Japan. Phone: +81-75-753-5524, FAX. +81-75-753-5534, E-mail:
atsuo@sbchem. kyoto-u.ac.jp
Abstract
Novel yeast cells armed with biocatalysts - glucoamylase, -amylase, CM-cellulase, glucosidase, and lipase - were constructed by a cell surface engineering system of yeast
Saccharomyces cerevisiae. These surface-engineered yeast cells were termed Arming
yeasts. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal
peptide was fused with the gene encoding the C-terminal half of yeast -agglutinin.
Glucoamylase was shown to be displayed on the cell surface of S. cerevisiae in its
active form, anchored covalently to the cell wall. S. cerevisiae is unable to utilise
starch, while the arming cells could grow on starch as the sole carbon source. For
enhancement of the ability to directly ferment starchy materials by the arming yeast, a
surface-engineered yeast cell displaying two amylolytic enzymes was constructed. The
gene encoding R. oryzae glucoamylase with its own secretion signal peptide and a
truncated fragment of the a-amylase gene from Bacillus stearothermophilus with the
prepro secretion signal sequence of the yeast a-factor, respectively, were fused with the
gene encoding the C-terminal half of the yeast -agglutinin. The arming cell codisplaying glucoamylase and a-amylase could grow faster on starch as the sole carbon
source than the cell displaying only glucoamylase. Furthermore, a novel celluloseutilising yeast cell displaying cellulolytic enzymes in their active forms on the cell
surface of S. cerevisiae was constructed by the cell surface engineering. An arming
yeast co-displaying FI-carboxymethylcellulase (CM-cellulase), one of the endo-type
cellulase, and (3-glucosidase from Aspergillus aculeatus was endowed with the ability of
cellooligosaccharide assimilation, suggesting the possibility that the assimilation of
59
A. Durieux and J-P. Simon (eds.), Applied Microbiology, 59-13.
MITSUYOSHI UEDA et al.
cellulosic materials may be carried out by S. cerevisiae expressing heterologous

cellulase genes on the cell surface. Furthermore, a yeast cell armed with R. oryzae
lipase was also constructed. These idea will be open to all living cells and the technique
will be able to endow them with novel abilities.
1. Introduction
Cell surface is crucial to the life of the cells since it is the interface between the inside
and the outside of the cells. It encloses the cells and maintains essential differences
between the cytosol and extracellular environment. Surface proteins are responsible for
most cell surface functions, serving as cell-cell adhesion molecules, specific receptors,
molecular recognising devices, catalytic enzymes, transport machinery, and so on.
Many surface proteins are bound through noncovalent or covalent interaction to the cell
surface structures. Cells have means of anchoring specific surface proteins and of
confining surface proteins to particular domains of the cell surface structure. Recently,
it has been partially clarified that how these protein molecules are targeted and localised
to the cell surface. Thus, it is possible to remake the functions of cell surface by taking
advantage of the known transport mechanisms of proteins to the cell surface. We have
been developing "Cell Surface Engineering" of yeast based on the techniques of genetic
engineering to endow the cells with novel functions by displaying target proteins on
their cell surface.
The cell surface display system is intended to be applied to bioindustrial processes
because it is required to be safe. In practical use, the most suitable microorganism is
the yeast S. cerevisiae, which has 'generally regarded as safe (GRAS) status and can be
used for food and pharmaceutical production. S. cerevisiae is a useful organism to
develop the cell surface expression system. It is also an advantageous cell as a host for
genetic engineering, since it enables folding and glycosylation of eukaryotic
heterologous proteins expressed and is easy to handle with ample genetic techniques.
Moreover, the yeast can be cultivated to a high cell density with a cheap medium.
S. cerevisiae lacks both amylolytic and cellulolytic activities and is unable to
ferment starchy or cellulosic materials, although they are the most abundant and
utilisable resources of plant origin (Barnett, 1976). In the conventional procedure to
convert starchy materials to ethanol at a high yield, the mash should be cooked at 140180C prior to amylolysis and the energy consumed in this process results in high
production costs (Maiorella, 1985). To save energy in the cooking process, the
development of a non-cooking fermentation system using the enzymes, which
efficiently digest raw starch, has been led. Intensive research has been conducted to
construct further improved starch-utilising systems by introducing heterologous genes
encoding amylolytic enzymes into yeast cells by the genetic engineering for secretive
production of the enzymes. Construction of a yeast cell secreting glucoamylase from
R. oryzae has been attempted for application to fermentation of raw starch without
cooking (Ashikari et al., 1989). Glucoamylase from R. oryzae was an exo-type
amylolytic enzyme cleaving -1,4-linked and -1,6-linked glucose effectively from
starch. The surface-engineered yeast cell displaying glucoamylase on the cell surface
60
MOLECULAR BREEDING OF ARMING YEASTS BY CELL SURFACE ENGINEERING
may be able to saccharify starch by glucoamylase on its cell wall and assimilate the
released glucose to proliferate and ferment (Fig. 1).
Figure 1. A model of cell surface-engineered (arming) S. cerevisiae displaying hydrolytic

enzymes. E: Hydrolytic enzymes.
On the other hand, utilisation of cellulosic materials in fermentation has not been
realised successfully. Cellulose, consisting of glucose units linked together by 1,4glycosidic bonds, is the most abundant carbohydrate in the biosphere. An estimated
synthesis rate of cellulose is approximately 4x107 tons per year. Cellulose is the most
promising renewable carbon source that is available in a large quantity for a long-range
solution to resource problems of energy. However, yeast S. cerevisiae is unable to
utilise cellulosic materials in spite of its versatility in industrial fermentation.
Enzymatic hydrolysis of cellulose has the potential to surmount many of the drawbacks
of acid hydrolysis.
The cellulase system of the anaerobic cellulolytic bacterium,
Clostridium thermocellum, was shown to naturally construct a discrete multi-enzyme
complex located on the cell surface (Lamed et al., 1983; Tokatlidis et al., 1991),
"cellulosomes", which is considered to help C. thermocellum to obtain the source of
carbon and energy efficiently by enzymatic degradation of cellulose on its cell surface.
An attempt to genetically display cellulolytic enzymes in their active form on the cell
surface has been tried to construct a novel cellulose-utilising yeast, S. cerevisiae (Murai
et al., 1997b, 1998b). As one of the target enzymes, carboxymethylcellulase (CMcellulase) from Aspergillus aculeatus classified as endo-1,4--D-glucan glucohydrolase
(endoglucanase) which cleaves the -1,4-glycosidic linkage of cellulose was utilised.
Since CM-cellulase is an endo-type cellulase, enzymatic degradation of cellulose to
glucose requires synergistic hydrolysis by different types of cellulolytic enzymes. It is
predicted that short-chain cellooligosaccharides formed by the endo-action of CMcellulase is converted quickly to glucose by -glucosidase (1,4--D-glucoside
61
glucohydrolase) in A. aculeatus. However, S. cerevisiae lacks -glucosidease activity and

consequently is unable to utilise cellobiose as the carbon source. Thus, to construct a S.
cerevisiae cell which is able to utilise one of cellulosic materials, cellooligosaccharides,
display of -glucosidase, in addition to CM-cellulase, on cell surface of S. cerevisiae is
also necessary.
In this article, our cell surface engineering system of S. cerevisiae is summarised.
Display of hydrolytic enzymes on the cell surface of S. cerevisiae has a great interest
since such yeast strains are expected to contribute to the production of energy and the
bioremediation of environmental pollution.
Figure 2. Structure of -agglutinin and partial amino acid sequence of its C-terminal
region containing GPI anchor attachment signal sequence (A), and the molecular design of
cell surface-displayed enryme (B). A: arrows indicate the predicted cleavage site. The
predicted site is underlined.
62
2. Principle of Cell Surface Engineering of Yeast

To target heterologous proteins to the outmost surface of the glycoprotein layer of the
cell wall, molecular information of -agglutinin was utilised (Murai et al., 1997a). The
anchoring signal of -agglutinin was combined with the signal of the secreted
enzymes using genetic engineering techniques. Fig. 2 shows the general structure of
the gene for cell surface display of an enzyme. The C-terminal half of -agglutinin
(320 amino acid residues) contains a glycosylphosphatidylinositol (GPI) anchor
attachment signal at the C-terminal end, like other cell surface proteins, and is utilised
as an anchoring domain for heterologous proteins since these proteins are covalently
linked with glucan.
The cell wall of S. cerevisiae is mainly composed of mannoproteins and -linked
glucans (Fleet, 1991), and has a bi-layered structure consisting of an internal skeletal
layer of glucan, composed of -1,3- and -1, 6-linked glucose (Manners et al., 1973),
and a fibrillar or brush-like outer layer, which is composed predominantly of
mannoproteins (Horisberger and Vonlanthen, 1977). These proteins are linked to glucan
through covalent bond. Two types of mannoproteins are present in the cell wall of S.
cerevisiae (Klis, 1994; Cid et al., 1995). Mannoproteins loosely associated with the
cell wall through non-covalent bond are extractable with sodium dodecylsulfate (SDS).
The other type of mannoproteins are extractable by glucanase, which are released by p1, 3- or -1, 6-glucanase digestion of the glucan layer of the cell wall but not by SDS
extraction (Fleet and Manners, 1977).
Among these glucanase-extractable
mannoproteins on the cell surface of S. cerevisiae, the mating-type specific agglutinins
(Lipke and Kurjan, 1992) that mediate direct cell-cell adhesion between cells of
opposite mating type during mating, which are supposed to be located on the outermost
surface. Mating type a and cells express a-agglutinin and a-agglutinin, respectively
(Terrance et al., 1987). -Agglutinin is encoded by AG1 gene (Lipke et al., 1989) and
interacts with the binding subunit of the agglutinin complex of a cells (Cappellaro et al.,
1991). A-agglutinin consists of a core subunit encoded by AGA1 gene (Roy et al.,
199 1) that is linked through disulfide bridges to a small binding subunit encoded by a
different gene AGA2 (Cappellaro et al., 1991). The structures of both a-agglutinin and
the core subunit of a-agglutinin are composed of a Secretion signal region, an active
region, a support region rich in serine and threonine, and a putative GPI anchor
attachment signals, and presumably a heavily O-glycosylated form (Wojciechowicz et
al., 1993).
The structure of GPI anchors is highly conserved among molecules from various
organisms (Ferguson and Williams, 1988). The core structure of the yeast GPI anchor
is similar to that found in other eucaryotes (Lipke et al., 1989); ethanolamine
phosphate(6) mannose(1,2) mannose(1,6 )mannose(1,4)glucosamine-(1,6)inositol phospho-lipid (Fig. 3). Many of cell surface proteins in yeast, for example,
Ag1 (Lipke et al., 1989), Aga1 (Roy et al., 1991), Flo1 (Watari et al., 1994), Sed1
(Hardwick et al., 1992), Cwp1, Cwp2, Tip1,Tir1/Srp1 (Van der Vaat et al., 1995), have
GPI anchors, which play important roles for surface expression of cell-surface proteins
and are essential for the viability of yeasts. These glycophospholipid moieties are
covalently attached to the C-termini of proteins and their primary function is to afford
the stable association of proteins with the membrane. GP1-anchored proteins contain
63
hydrophobic peptides at their C termini. After the completion of protein synthesis , the
precursor protein remains anchored in the endoplasmic reticulum (ER) membrane by
the hydrophobic carboxyl-terminal sequence, with the rest of the protein in the ER
lumen. The hydrophobic carboxyl-terminal sequence is cleaved at the
site and
concomitantly replaced with GPI anchor presumably by a transamidase. The
localisation of both -agglutinin and -agglutinin to the cell surface occurs through the
secretory pathway (Tohoyama and Yanagishima, 1987) (Fig, 3). Secreted proteins are
first translocated into the lumen of the ER, and then transported from the ER to the
Golgi apparatus and from there to the plasma membrane in membrane-enclosed vesicles
(Schekman, 1992). Fusion of the Golgi-derived secretory vesicles with the plasma
membrane releases the secreted proteins to the cell exterior. Post-translational
proteolytic modification of precursors of secretory peptides occurs in the late
compartments of the secretory pathway (trans cisternae of the Golgi apparatus and
secretory vesicles). -Agglutinin was proposed to be further transported to the outside
of the plasma membrane through the general secretory pathway in a GPI-anchored form
and then released from the plasma membrane by phosphatidylinositol-specific
phospholipase C (PI-PLC) and transferred to the outmost surface of the cell wall (Lu et
al., 1994).
Figure 3. Transportation of a-agglutinin to cell surface (A) and the structure of GPI
anchor (B), A: ER, endoplasmic reticulum: PI-PLC, phosphatidylinositol-specific
phospholipase C. B: a, ethanolamine phosphate bridge; b, glycan; c, inositol. Man,
Mannose; GlcNH2, glucosamine.
64
3. Display of Amylolytic Enzymes on the Yeast Cell Surface

To construct a novel yeast displaying amylolytic enzymes which hydrolyse starches at
the cell surface, a multi-copy plasmid (pGA11) harbouring the R. oryzae glucoamylase
gene was prepared for expression of glucoamylase/-agglutinin fusion gene containing
the secretion signal sequence of the glucoamylase under the control of the GAPDH
promoter (Fig. 4). When cells were cultivated aerobically with 1 % soluble starch as
the sole carbon source, the cells harbouring the plasmid pGA11 proliferated to reach an
absorbance of about 10, which was the same level as in the case of the culture on 1%
glucose, while no growth was observed with the control cells (Fig. 5). These data show
that the cell surface-anchored glucoamylase reacted sufficiently for the hydrolysis of
starch. Cultivation on an agar plate demonstrated that the cells harbouring the plasmid
pGA11 hydrolysed starch and produced a halo strictly around the colony, while no halo
formation was observed around the cells harbouring the control plasmid, revealing that
the cell surface-engineered cells obtained amylolytic activity due to the expression of
the glucoamylse/-agglutinin fusion gene (Murai et al., 1997a).
Immunofluorescent labelling of cells with anti-glucoamylase IgG showed that cells
expressing the glucoamylase/-agglutinin fusion gene were uniformly labelled,
although the intensity was different from cell to cell, being probably due to differences
in expression levels among the cells (Fig. 6). The localisation of glucoamylase/agglutinin fusion protein on the cell wall was further confirmed by immunoelectron
microscopy.
Thermal stability, optimal temperature, and optimal pH of glucoamylase anchored
on the cell surface were evaluated by comparing with those of the secreted free enzyme
(Ueda et al., 1998). The activity of the anchored glucoamylase was stable in the
temperature range of 0C to 45C. The optimal temperature and the optimal pH of the
anchored glucoamylase was 50C and 4.5, respectively. While no differences on the
thermal stability and the optimal pH were observed between anchored and free
glucoamylases, the optimal temperature of anchored glucoamylase was a little lower
than that of the free enzyme.
To stable express the enzyme on the yeast cell surface, a plasmid pIGA11 to be
integrated into chromosome of S. cerevisiae was constructed (Fig. 4) (Ueda et al.,
1998). The glucoamylase/-agglutinin fusion gene-integrated cells exhibited the
glucoamylase activity on the cell surface as well as the cells harbouring pGA11 and
utilised starch as the sole source of carbon and energy. Mitotic stability of the cells
harbouring pIGA11 was much higher than that of the cells harbouring pGA11.
As the result of the examination whether or not the novel yeast displaying
glucoamylase from R. oryzae on its cell surface can ferment starch directly, it was
suggested that the fermentation efficiency would be enhanced by the addition of aamylase to the medium (Murai et al., 1998a). Furthermore, the enzyme displayed on the
cell surface was regarded as a kind of a renewable catalyst depending on cultivation
conditions. And, bacterial contamination was expected to be prevented, because the
incorporation of glucose, which was liberated on the cell surface, was considered to be
rapid.
65
Figure 4. Structures ofplasmids for displaying of enzymes. pGA11 (A) and plGA1I (B), for
displaying of glucoamylase; pIAA1I (C), for displaying of -amylase; pCMC11 (D), for
displaying of CM-cellulase (CMCase); pBG21 1 (E), for displaying of -glucosidase.
In addition to display glucoamylase, to co-display -amylase from Bacillus

stearothermophilus CU21 (BSTA) (Nakajima et al., 1985) on the cell surface, the
BSTA/C-terminal half of -agglutinin fusion gene (pIAA11) constructed with the same
method as in the case of glucoamylase was integrated into the chromosome of S.
cerevisiae (Fig. 4). In this case, about half of the total -amylase activity was leaked
into the culture supernatant, due to the proteolytic processing of the fusion protein. The
deduced amino acid sequence of BSTA contained one possible processing site for the
Kex2 endopeptidase, Val-Pro-Arg, at the amino acid residues of No. 481 to No. 483.
Kex2, one of the endopeptidases in the secretory process of -mating factor and killer
toxin precursors, exhibits the substrate specificity toward each carboxyl site of Lys-Arg,
Arg-Arg, and Pro-Arg sequences (Mizuno et al., 1989). A Kex2-resistant BSTA/agglutinin fusion protein was designed by eliminating 33 residues from the C-terminus
of BSTA, because this domain was demonstrated not to be essential for the activity
(Vihinen et al., 1994). The cell harbouring this C-terminal truncated BSTA/-agglutinin
fusion gene (pIAA) exhibited -amylase activity only in the cell pellet fraction. On
plate assay, a large halo was detected around the colony of the cells harbouring
pIAA11, while a small halo formation was observed strictly around the cells
66
harbouring pIAA as in the case of the glucoamylase-displaying cells (Murai et al.,

1999).
When the arming cells with both enzymes were cultivated in the medium containing
1% soluble starch as the sole carbon source, the growth of the cells harbouring
pIGA11 and pIAA reached the absorbance of about 10, which was the same level as
in the case of the culture on 1% glucose, although the growth on glucose of these strains
was more rapid. No growth on starch was observed with the control cells and the cells
harbouring only pIAA (Fig. 5). The cell co-displaying glucoamylase and -amylase
could grow faster than the glucoamylase-displaying cells. -Amylase is an
endoglucanase which hydrolyses starch in a random fashion, producing
oligosaccharides. Therefore, the co-operative and sequential reaction probably resulted
in the increased concentration of molecules with non-reducing ends produced from
starch by -amylase, which in turn could serve as substrate molecules for glucoamylase,
thereby increasing the rate of formation of free glucose. Immunofluorescent labelling of
cells with anti-glucoamylase IgG and FITC-goat IgG to rabbit IgG as the second
antibody and the halo formation of the cells harbouring pIGA11 and pIAA11 or
pIAA on a starch-containing plate showed the expression of both glucoamylase and amylase on the cell surface of the same cell.
The fermentation system using the novel cells endowed with a rapid-starch-utilising
ability by displaying two sequential amylolytic enzymes on their cell surface should be
further evaluated in comparison with other systems.
4. Display of Cellulolytic Enzymes on the Yeast Cell Surface
As vectors for co-display of CM-cellulase and -glucosidase on the cell surface,
multi-copy plasmids, pCMC11 and pBG211 (Fig. 4), were constructed (Murai et al.,
1997b; 1998b). The cells harbouring the plasmid pCMC11 and cells harbouring
pCMC11 and pBG211 had the cell-associated CM-cellulase activity, and the cells
harbouring the plasmid pBG2 1 1 and cells harbouring pCMC11 and pBG211 exhibited
the cell-associated -glucosidase activity. Moreover, the cells harbouring the plasmid
pCMC11 and pBG211 showed both CM-cellulase and -glucosidase activities. The
parent cells exhibited neither of the activities. From these results, CM-cellulase and glucosidase proteins were efficiently co-displayed on the cell surface in their active
forms. Immunofluorescence microscopy with FITC-goat IgG to rabbit IgG as the
second antibody confirmed the presence of CM-cellulase protein on the cell surface
(Fig. 6). Anti-CM-cellulase IgG and anti--glucosidase IgG were used as the first
antibodies, Control cells were not labelled with either antibodies, while cells
harbouring pCMC 1 1 were labelled by fluorescence with anti-CM-cellulase IgG. Cells
harbouring pBG211 were also labelled with anti--glucosidase IgG (Murai et al.,
1998b). The cells harbouring pCMC11 and pBG2 1 1 were labelled by fluorescence with
both anti-CM-cellulase IgG and anti-P-glucosidase IgG, indicating that these cells codisplayed the CM-cellulase and -glucosidase proteins on their cell surface (Murai et
al., 1998b).
67
Figure 5. Aerobic cultivation of S. cerevisiae MT8-1 cells harbouring pGA11 (A) and
harbouring various in tegrative glucoamylase and -amylase/-agglutinin fusion genes (B)
in the medium containing starch as the sole source of carbon and energy, and aerobic
cultivation of arming cells in the medium containing cellobiose (C) and
cellooligosaccharides (D) as the sole source of carbon and energy. A, cell growth ( );
starch concentration ( ); ethanol produced ( ); glucoamylase activity in cell pellet (
); glucoamylase activity in culture medium ( ). B, cell growth of the cells harbouring no
plasmid ( ), pIGA11 ( ), pIAA ( ), and pIGA11/pIAA ( ); the concentration of
starch in the culture medium of the cells harbouring no plasmid ( ), pIGA11 ( ), and
pIGA11/pIAA ( ). C and D, symbols for cells: S. cerevisiae MT8-1 ( ); MT8-1
harbouring pCMC11 ( ); MT8-1 harbouring pBG211 ( ); MT8-1 harbouring pCMC11
). C, Cell growth was monitored by absorbance of culture broth at 600
and pBG211 (
nm. D, Cell growth was monitored by counting colonies appeared on plates.
68
Figure 6. Immunofluorescent labelling of arming cells displaying glucoamylase (A, B),

CM-cellulase (C, D) on the cell surface. Nomarsky differential interference micrographs
(A and C) and immunofluorescence micrographs (B and D).
The arming cells with both enzymes were cultivated aerobically in the medium
containing cellobiose as the sole carbon source, and cell growth was monitored (Fig. 5).
Cells harbouring pBG211 and cells harbouring pCMC11 and pBG211 could grow on
cellobiose and reached the absorbance of about 2, which was a little lower than that in
the case of the culture on 1% glucose, while no growth on cellobiose was observed with
the control cells and cells harbouring pCMC11. The arming cells were cultivated
aerobically in the medium supplemented with cellooligosaccharides (approximately
11% (W/W) cellohexaose, 29% (W/W) cellopentaose, 33% (W/W) cellotetraose, 17%
(W/W) cellotriose, 4% (W/W) cellobiose, and less than 1% (W/W) glucose) as the sole
carbon source and the cell growth was monitored by counting colonies appeared on
YPD plates. The cells harbouring pBG211 and cells harbouring pCMC11 and pBG211
could grow on cellooligosaccharides employed, while no growth on
cellooligosaccharides was again observed with control cells and cells harbouring
pCMC11. Since -glucosidase was reported to be capable of degrading
cellooligosaccharides with glucose units of 2 to 6 (Sakamoto et al., 1985), the
breakdown of cellooligosaccharides by -glucosidase seemed to be sufficient to sustain
the growth of the yeast displaying this enzyme. However, the difference between the
growth of the cells harbouring pCMC11 and pBG211 and that of the cells harbouring
pBG211 suggested that the yeast strain co-displaying CM-cellulase and -glucosidase
had the enhanced ability to degrade cellooligosaccharides.
69
Much efforts have been devoted to utilise cellulosic materials by employing S.

cerevisiae and cellulase complex from cellulolytic bacteria (Van Rensburg et al., 1996).
However, these attempts failed because -glucosidase was not expressed. Although
several researches have also been made to express heterologous -glucosidase genes in
S. cerevisiae, cellobiose could not access to the enzyme remained intracellular or the
enzyme was not expressed sufficiently to allow the transformant to grow on cellobiose
(Adam et al., 1995; Kohchi and Toh-e, 1986; Penttila et al., 1984, Raynal and
Guerineau, 1984) . After that, secretive expression of -glucosidase gene in S.
cerevisiae was reported (Cummings and Fowler, 1996), where growth on cellobiose was
not examined. Thus, the results described here are the first step for the assimilation of
cellulosic materials by S. cerevisiae expressing heterologous cellulase genes and also
the first report for assimilation of cellooligosaccharides by yeast and for the
construction of the prototype of cellulosomes (Murai et al., 1998b).
5. Display of Lipase on the Yeast Cell Surface
When lipase of R. oryzae was displayed as mentioned above, little activity was
observed on the cell surface. The reason of this phenomenon seemed to be derived from
the fact that the active site of lipase resides in the C-terminal domain of the molecule.
Fusion of the C-terminus of lipase with the 3'-half of -agglutinin may inhibit the
access of substrates to the active site. Therefore, to insure the free movement of the
lipase molecule, a spacer of an appropriate length was inserted between these two
molecules. This system will be further proved to be effective for the cells to express the
lipase activity on the cell surface, since the length of the spacer seemed to be affected
the enzyme activity.
6. Cell Surface Engineering as a Novel Field of Biotechnology
Schreuder et al. (1993, 1996) reported that they had succeeded in targeting galactosidase from Cyamopsis tetragonoloba seeds, as a reporter enzyme, to the cell
wall of S. cerevisiae. However, the engineered S. cerevisiae cells described here are the
first example of yeast in which proteins were targeted to the cell surface and endowed
the cells with new beneficial properties. These surface-engineered yeast cells were
termed "Arming yeasts" (Anonymous, 1997). The displayed enzymes are also
regarded as a kind of self-immobilised enzymes on the cell surface, this phenomenon
being passed on to daughter cells as long as the genes are retained by the cells. This
display system could turn or remake S. cerevisiae into a novel and attractive
microorganism as a whole-cell biocatalyst by surface expression of various enzymes,
especially when target substrates are not able to be taken up by the cells, and will make
it possible to produce renewable self-immobilised biocatalysts.
Here, the combination of the cell surface display system with endowment of an
additional metabolic reaction to the yeast cells was performed by cell surface
engineering. Thus, the cell surface can be regarded as a new target for giving additional
characteristics of metabolic reactions. This research will open a new frontier of cellular
engineering not only in yeast but also in all living cells. From the viewpoint of
70
biotechnology, the system mentioned here is also regarded as a combination of the

immobilisation of biocatalysts and the genetic engineering. In this system, the cell
surface of yeast was used as a carrier for immobilisation, and the living whole cells
were remade as a cell biocatalyst. This system is expected to have several merits;
the enzyme is readily supplied only by the activation of the promoter and is provided as
naturally immobilised on the cell surface, saving tedious purification and
immobilisation processes. The cell surface engineering will be able to endow all living
cells with novel abilities (Fig. 7) and open a new way in the field of biotechnology.
Figure 7. Application ofarming yeast constructed by cell surface engineering.
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73

SIMON OSTERGAARD, LISBETH OLSSON AND JENS NIELSEN
Center for Process Biotechnology, Department of Biotechnology,
Technical University of Denmark, Building 223, DK-2800 Lyngby,
Denmark.
Abstract
In metabolic engineering, analysis of pathways is central and it involves quantification
of pathway fluxes and how these fluxes are controlled. Therefore metabolic pathway
analysis rely on metabolic control analysis and metabolic flux analysis. This paper
introduces the basic concepts of metabolic pathway analysis that serves as a valuable
analytical tool for examination of cellular metabolism especially in connection with
designing directed genetic changes to achieve specific objectives, e.g. increase flux
toward a product of interest. In the paper, metabolic pathway analysis of the glycolysis
and the galactose metabolism of Saccharomyces cerevisiae are used to illustrate the
power of this analytical tool.
1. Introduction
The multidisciplinary field of metabolic engineering aims at creating and improving
microorganisms for several purposes. The tasks of metabolic engineering can be
classified into the following groups: Extension of substrate range; improvement of
productivity or yield; elimination of by-product formation; improvements of cellular
properties; and extension of product range. When operating in either of these categories,
it is of outmost importance to analyse the cellular metabolism thoroughly in order to
succeed. Metabolic pathway analysis serves as an analytical tool that may help to
identify the most promising targets for metabolic manipulation, and furthermore,
metabolic pathway analysis can also help elucidating metabolic changes of the cell
caused by certain genetic modifications. In this paper we roughly divide metabolic
pathway analysis into two categories that take advantage of two different concepts:
Metabolic control analysis and metabolic flux analysis. The metabolic control analysis
examines perturbations in the enzymatic activities on the systemic metabolic behaviour
to determine which enzyme(s) that exerts control over flux within a given pathway
(Kacser and Burns, 1973; Heinrich and Rapoport, 1974). Thus, the overall aim with this
75
A. Durieux and J-P. Simon (eds.), Applied Microbiology, 7585.
SIMON OSTERGAARD, LISBETH OLSSON, JENS NIELSEN
analysis is to identify the best alternatives for genetic manipulation that may lead to an
increase of overall flux through a given pathway. The principles of metabolic flux
analysis have proven successful as a tool for pathway analysis, since the carbon
distribution within the cell may be evaluated (Stephanopoulos et al., 1998). Hence, the
study of flux control targets the cellular metabolism locally, whereas the metabolic
flux analysis serves as a global cellular approach where the intracellular fluxes of a
given metabolic network are estimated. In this paper the concepts of metabolic pathway
analysis are described. Furthermore, examples of metabolic pathway analysis applied to
the glycolysis and the galactose metabolism of Saccharomyces cerevisiae will be
referred to for demonstrating the use of this analytical tool in metabolic engineering.
2. Metabolic pathway analysis
2.1. METABOLIC CONTROL ANALYSIS
To elucidate which enzyme(s) that controls the flux through a given biochemical route,
it is necessary to quantify the control over the overall flux exerted by the individual
enzymes. The metabolic control analysis aims at calculating the flux control coefficients
(FCCs) of the individual enzymatic steps in a given pathway in order to quantify which
enzyme(s) that is (are) responsible for the control over the flux. The FCCs are
normalised to one and they describe the relative change in steady-state flux (Jk) through
reaction k caused by an infinitesimal change in enzymatic activity (vi) of enzyme i, as
depicted in equation 1 where CiJk designates the flux control coefficient. Thus, the closer
a FCC is to one, the more control over flux is exerted by the respective enzyme.
(1)
The FCCs can directly be obtained from measurements of the flux at various enzymatic
activities or indirectly by calculation of the elasticity coefficients (ij) (equation 2), that
describe the relative change in enzymatic activity (vi) caused by an infinitesimal change
in the concentration of metabolite j. Assuming validity of the flux-control summation
theorem (equation 3) and the flux-control connectivity theorem (equation 4) (Kacser
and Burns, 1973), the elasticity coefficients can be applied for deduction of the FCCs.
(2)
76
(3)
(4)
To determine the FCCs indirectly via calculation of the elasticity coefficients, it is

crucial to obtain detailed knowledge of the enzyme kinetics either in the form of
quantitative information about the elasticity coefficients or in the form of a kinetic
model for the individual enzymatic steps. Each enzymatic reaction included in the
model should be described by a kinetic rate expression that correlates the concentrations
of the substrate(s), the activator(s), and the inhibitor(s) of the enzyme with its specific
enzymatic activity. This correlation will depend on the kinetic parameters of the given
enzyme such as the maximum specific reaction rate (vmax), the affinity constant(s) (Km),
inhibition constants (Ki) and in the case of a reversible reaction also equilibrium
constants between certain metabolites may be included in the kinetic rate expression.
When these kinetic rate expressions are processed as shown in equation 2, and the
intracellular metabolite levels are measured, it is possible to calculate the elasticity
coefficients, and subsequently obtain the FCCs.
2.2. METABOLIC FLUX ANALYSIS
In contrary to the metabolic control analysis that relies on the kinetics of the pathway
examined, the metabolic flux analysis consists of a stoichiometric model where the
metabolic pathway fluxes that span a biochemical network can be estimated by applying
mass balances around the intracellular metabolites of the model i.e. metabolite
balancing, or by determination of the fractional enrichment when using labelled
substrate. By comparison of flux distributions, the metabolic flux analysis can serve as
an analytical tool for identification of physiological changes that arise from changing
the operating conditions or for comparison of the carbon distribution in different
mutants. Thus, the application of the metabolic flux analysis can help investigating the
cellular control of a certain biochemical branch point in order to clarify the rigidity of a
certain pathway node. Furthermore, information regarding alternative pathways may
also be obtained from metabolic flux analysis by including questionable pathways in the
stoichiometric model and then concluding the lack or existence of the given pathway by
virtue of its ability to satisfy the metabolite balances.
For the performance of metabolic flux analysis based on metabolite balancing a
complete stoichiometric model of the cell should be set up which includes J intracellular
reactions comprising K pathway metabolites. The mass balances of the K metabolites
are given in vector notation as depicted in equation 5 where Xmet designates the
concentration of the K metabolites and rmet designates the vector with all the volumetric
net rates of formation of the metabolites in the Jreactions:
77
(5)
Due to the high turnover of the metabolite pools, the change in metabolite
concentrations rapidly adjusts to a new level, and hence, pseudo steady-state may be
assumed (dXmet/dt = 0). Since the metabolite levels generally are very low, the dilution
of the metabolite pool due to biomass growth (described by the term
is
negligible. This assumption is further supported by the fact that the fluxes affecting a
given metabolite pool are significant higher than the effect of dilution. Thus, rmet equals
zero, and as the rmet-vector may be written as the product between the stoichiometric
matrix GT comprising the stoichiometry of the J reactions organised in columns and the
v-vector containing the individual rates of the J reactions, equation 6 forms the basis for
metabolite balancing.
(6)
This equation represents K linear algebraic balances with J unknown parameters equal
to the number of pathway fluxes. This system has J-K degrees of freedom, and
consequently, when J-K reaction rates are measured, it is possible to obtain estimates
for all the pathway fluxes of the v-vector by solving the linear algebraic balances. To do
so, all the measured rates are collected in a new vector, vm, and the residual rates that
are to be calculated, are collected in another vector, vc. Furthermore, the stoichiometric
matrix GT is divided into two sub-matrices GmT and GcT which contain the stoichiometry
of the reactions to be measured and the stoichiometry of the residual reactions,
respectively. Hence, equation 6 may be written as equation 7, and by rearrangements of
this equation, the vector vc containing all the calculated reaction rates of the metabolic
network can be computed as shown in equation 8.
(7)
(8)
The use of metabolite balancing for estimation of intracellular fluxes may serve as a
valuable tool for determination of net fluxes within a metabolic network. Nonetheless,
this approach does not allow for flux determinations of reversible reactions and for the
quantification of flux ratios between biosynthetic pathways leading to the same
metabolite. To get information about the fluxes in these cases it is necessary to apply
labelled substrates, Metabolic flux analysis using labelled substrate approaches the
metabolism from an even further microscopic view compared with metabolite
78
balancing as the pathway fluxes are estimated from balances around each individual
carbon atom in the intracellular metabolites. Hence, the method may also allow for
calculation of the flux ratio between two biosynthetic routes that lead to the same
metabolite, if the two biosynthetic pathways discriminate differently between the
individual carbon atoms (Sonntag et al., 1993). Likewise, the method may also be used
for estimation of reversible fluxes and not only net-fluxes as obtained from metabolite
balancing (Marx et al., 1996; Fiaux et al., 1999). Furthermore, the use of labelled
substrate may also help to elucidate compartmentation to gain information about the
location of certain biosynthetic reactions within the cell (Pasternack et al., 1994). With
additional biochemical knowledge regarding the cellular structure, an extended
metabolic model may be established describing the cellular metabolism even more
accurately. The concepts of metabolic flux analysis using labelled substrate(s) have
been extensively described in Wiechert and de Graaf (1 996).
3. Steady-state continuous cultivation an excellent tool for metabolic pathway
analysis
Having established the structure of a kinetic and/or a stoichiometric model for the
performance of metabolic pathway analysis, it is necessary to obtain values for the
model parameters. For indirectly determination of the FCCs of metabolic control
analysis, the intracellular metabolite levels of the all compounds included in the kinetic
model can be used, and to estimate the flux distribution of the metabolic network
established, the rates included in the vm-vector are needed. For the analysis of microbial
cells it is possible to use submerged fermentation experiments for analysis of the
cellular metabolism. Furthermore, through the use of continuous cultures often
referred to as chemostats it is possible to study the cellular metabolism at a steady
state. By controlling the volumetric feed rate of medium (F) fed to the bioreactor, and
keeping the volume constant by having a simultaneous out-flow of medium from the
bioreactor, the specific growth rate can be controlled at any value, since it equals the
dilution rate D at steady-state. This is observed from the mass-balance of biomass (X)
over the bioreactor as shown in equation 9, i.e. no accumulation of biomass ( dX/dt = 0).
(9)
Furthermore, the volumetric formation rates of the extracellular metabolites (rp) which
may be included in the vm-vector mentioned in section 2.2., are easily obtained when
measuring the concentration of the products in the bioreactor, since the volumetric
formation rates are calculated by multiplication of the dilution rate with the
concentration of the extracellular metabolites at steady-state. This is can be seen from
equation 10 which shows the mass balance of a given product P over the bioreactor
where dP/dt equals zero at steady-state.
79
(10)
Thus, from continuous operated cultivations, input parameters for the metabolic
pathway analysis such as concentrations of extracellular and intracellular metabolites
may be obtained in a highly reproducible fashion under preferable physiological
conditions regarding the specific growth rate. The continuous operated cultivations also
give the possibility to examine dynamic physiological changes of a cellular system
caused by specific perturbations. These perturbations could be obtained from a pulse of
the limiting component that is added to the bioreactor momentarily in order to follow
the cellular response regarding substrate uptake and product formation rates. Also a step
change in dilution rate may be of interest in order to study the dynamics of a culture
when the specific growth rate, consequently, increases or decreases from one level to
another. Furthermore, continuous cultivations may also be used for physiological
studies of metabolic regulation such as glucose control (Klein et al., 1998) exerted on
the metabolism of slow fermentable carbon sources. To establish repressible conditions
within the medium, a high sugar concentration should be obtained which can be
achieved by operation of the bioreactor under nitrogen-limited conditions.
4. Metabolic pathway analysis applied to Saccharomyces cerevisiae
4.1. KINETIC STUDIES OF THE GLYCOLYSIS
The concepts described in the previous sections have been applied to Saccharomyces
cerevisiae which have provided additional information to the understanding of yeast
metabolism. Galazzo and Bailey (1990) established a kinetic model describing the
anaerobic fermentation pathway from glucose to ethanol, glycerol, and polysaccharides
in order to characterise the physiological response caused by a change in the external
pH in suspended and immobilised yeast. In vivo phosphorus-31 nuclear magnetic
resonance measurements for determination of the intracellular concentrations of
substrates and effectors in addition to the kinetic expressions of the model were used for
calculation of the FCCs as described in section 2.1. From this study the authors found
that control over flux was mainly exerted by the glucose uptake in the suspended cells
regardless of the extracellular pH of 4.5 or 5.5 (FCC = 0.83 and FCC = 0.64,
respectively). At the latter pH, the ATPase membrane ion pump also exhibited
considerable control over ethanol production with a FCC of 0.24, since ATP is not
consumed as rapidly by the ATPase at pH 5.5 compared with pH 4.5 in order to
maintain the intracellular pH. In immobilised cells the glucose uptake has a lower
impact, however still considerable, on the ethanol production (FCC = 0.31 and FCC =
0.30 at pH 4.5 and pH 5.5, respectively) compared with suspended cells. The control
over flux in the immobilised cells was mainly exerted by the phosphofructokinase (FCC
= 0.50 and FCC = 0.33 at pH 4.5 and pH 5.5, respectively), but also in these cells the
ATPase exhibited considerable control over ethanol production with a FCC of 0.24
80
when grown at an extracellular pH of 5.5. This study demonstrated the use of metabolic
control analysis for explaining the physiological differences between suspended and
immobilised yeast cells. Hence, metabolic control analysis may be used for
identification of the best alternatives for genetic manipulation but also for description of
metabolic differences in a quantitative fashion.
Another strategy different from metabolic control analysis but also involving kinetic
modelling, was taken for pathway analysis of the glycolysis in S. cerevisiae (Rizzi et al.,
1997; Theobald et al., 1997). The kinetics of the glycolysis was examined in order to
understand the regulation of the yeast metabolism during dynamic conditions. Theobald
et al. (1997) established a rapid sampling technique to measure the dynamic response of
the glycolytic intermediates, co-metabolites, and extracellular metabolites of S.
cerevisiae caused by a physiological perturbation. In order to predict the changes of the
intracellular and extracellular metabolite levels during dynamic conditions, a kinetic
model of the individual enzymatic steps of the glycolysis was set up (Rizzi et al., 1997).
The model comprised rate equations for the individual enzymatic steps of the glycolysis
and these were included in material balances for the individual metabolites, the cometabolites, and the extracellular metabolites. The dynamic response that arose from a
glucose pulse added to a glucose-limited continuous grown culture of S. cerevisiae
operated at a dilution rate of 0.1 h-1, was simulated by solving the material balances for
all these metabolites. To fit the experimental observations of the glucose pulse, the
model was structured into a cytoplasmic and a mitochondrial compartment but with
translocation of the adenine nucleotides. The model was able to simulate the steady state
situation of the glucose-limited continuous cultivation and the transient response of the
metabolite levels after a glucose pulse. The work of Rizzi et al. (1997) and Theobald et
al. (1997) illustrates the use of continuous operated cultivations as a great tool for
kinetic analysis of microorganisms, and furthermore, the studies also demonstrate the
valuable use of mathematical modelling for pathway analysis in order to improve the
understanding of the yeast metabolism.
4.2. METABOLIC PATHWAY ANALYSIS OF THE GALACTOSE METABOLISM
The GAL genes of S. cerevisiae encoding the enzymes of the galactose utilisation
pathway, serve as one of the best studied models of genetic regulation in eukaryotic
systems (reviewed by Johnston and Carlson, 1992, and Melcher, 1997). The GAL genes
are tightly regulated being induced by galactose and strongly repressed by glucose. The
positive tramcriptional activator protein encoded by the GAL4 gene induces the
expression of the structural GAL genes in the presence of galactose. The genes GAL6,
GAL80, and MIG1 encode the proteins responsible for the down-regulation of the
structural GAL genes that encode the enzymes responsible for uptake of extracellular
galactose and subsequent conversion of intracellular galactose to glucose-6-phosphate
(known as the Leloir pathway). GAL2, GAL1, GAL7, GAL10, and GAL5 constitute the
structural GAL genes encoding the following enzymes: galactose permease,
galactokinase, galactose- 1 -phosphate uridylyltransferase, UDP-glucose 4-epimerase,
and phophoglucomutase, respectively, which are shown in Figure 1.
81
Figure I The Leloir pathway containing the enzymes responsible for conversion of
galactose io glucose-6-phosphate See the text for specific nomenclature of the enzymes
The galactose metabolism is interesting to approach with the tools of metabolic pathway
analysis in order to investigate the remarkable physiological differences that exist
between glucose and galactose metabolism. Metabolic control analysis was used for
locally examination of the galactose utilisation pathway (stergaard et al., 1998), and
metabolic flux analysis was carried out to elucidate the global physiological effects on
the entire metabolism caused by deletion of the two regulatory genes GAL80 and MIG1.
To identify which enzymes control flux through the Leloir pathway, the FCCs were
determined indirectly as described in section 2.1., and hence, a kinetic model of the
Leloir pathway was set up followed by calculation of the elasticity coefficients from the
rate expressions of the individual enzymatic reactions. The explicit values for the
elasticity coefficients were obtained from measurements of the intracellular
concentrations of the intermediates of the Leloir pathway from a galactose-limited
continuous cultivation operated at a dilution rate of 0.1 h-1, Thus, the FCCs could be
obtained by use of the summation and the connectivity theorem as described in section
2.1. The only unknown variable of the model was the intracellular galactose
concentration, and hence, the FCCs were computed as a function of the intracellular
galactose concentration as depicted in Figure 2.
According to intracellular measurements of the glucose concentration when the
extracellular concentration was at or below the affinity of the transport system (1-2
mM), the intracellular glucose concentration was less than 0.1 mM (Teusink et al.,
1998). Assuming the same to be the case for the galactose metabolism when the
galactose concentration in the bioreactor was 1 mM, the intracellular galactose
concentration was at or below 0.1 mM. Consequently, from this metabolic control
analysis it was concluded that the control over flux through the Leloir pathway is
mainly exerted by the galactose permease (Gal2) under the physiological conditions
examined. Experimental work is in progress to examine the results of this analysis in
order to gain more insight to the galactose metabolism of yeast.
82
Figure 2 FCC 's calculated as a function of the intracellular galactose concentration.Gal2:

Galactose
permease;
Gall:
Galactokinase;
Gal7:
galactose-1 -phosphate
uridylyltransferase.
The glucose and the galactose metabolism were also studied in nitrogen-limited
continuous cultivations to examine the physiological differences between these two
sugars. It was of interest to investigate the physiological role of the proteins encoded by
the MIG1 and the GAL80 gene which are strongly involved in glucose control exerted
on the galactose metabolism. Mig1 takes part of a protein complex comprising Ssn6,
Tup1, and Mig1 of which the latter directs the complex to a specific consensus motif on
the promoters of the target genes whereby transcription of the target genes does not
occur (Keleher et al., 1992; Treitel and Carlsson, 1995). Mig1-mediated glucose control
not only affects the GAL genes, but also the MAL genes responsible for maltose
utilisation and the SUC genes encoding invertase that hydrolyses sucrose. In contrary,
Gal80 only acts as a negative regulatory protein on the GAL genes by binding to the Cterminal end of Gal4 which prevents transcriptional activation by this protein. The
specific sugar uptake rates of a mig1 gal80 double mutant strain obtained from nitrogenlimited cultivations on glucose and galactose, respectively, were compared with the
corresponding specific sugar uptake rates obtained for the wild type strain (CEN.PK
113-7D).
A stoichiometric matrix of the aerobic yeast metabolism was established to estimate
the flux distributions of the wild type strain and the mig1 gal80 mutant strain from the
data obtained by the nitrogen-limited continuous cultivations on either glucose or
galactose. By measuring the uptake rates of glucose and galactose in addition to the
product formation rates of pyruvate, ethanol, acetate, succinate, glycerol, ammonium,
and the biomass composition, it was possible to compute the vc-vector as described in
section 2.2., and hence, obtain flux distributions over the metabolic network established.
The uptake rates of glucose and galactose of the wild type strain were 4.3 mmol/gDW/h
and 2.7 mmol/gDW/h, respectively, and the corresponding uptake rates of the mig1
83
gal80 mutant strain were 3.4 mmol/gDW/h and 3.8 mmol/gDW/h. Thus, the deletion of
the MIG1 and the GAL80 genes had a remarkable impact on the uptake rates of these
two sugars by decreasing the specific glucose uptake rate and increasing the specific
galactose uptake rate. Some of the fluxes estimated from these four cultivations are
shown in Figure 3.
Figure 3 Flux distributions (given in mmol/gDW/h) obtained for the wild type strain (left)
and the mig1 gal80 mutant strain (right) when grown in nitrogen-limited continuous
cultivations on glucose or galactose (italic). Only selected fluxes are shown for simplicity
The metabolic flux analysis provides valuable information about the cellular control of
the pyruvate branch point. It is observed that the flux entering the TCA-cycle from the
pyruvate node is constant for the two strains examined but it varies between glucose and
galactose. The flux entering the TCA-cycle was 1.6-1.7 mmol/gDW/h when the two
strains were grown on glucose, and 2.1-2.2 mmol/gDW/h for both these two strains
when grown on galactose. Hence, it is concluded that not only growth on glucose results
in overflow metabolism where the residual carbon above the maximum capacity
entering the TCA-cycle is directed towards acetaldehyde formation, and subsequently
ethanol formation, but also galactose exerts a similar response onto the metabolism.
Although both glucose and galactose enter the metabolism at the glucose-6-phosphate
node, the figures of the fluxes entering the TCA-cycle from the pyruvate node show that
the maximum capacity entering the TCA-cycle is dependent on the specific sugar
consumed. Thus, the metabolic flux analysis has demonstrated its potential by
identification and quantification of metabolic changes caused by genetic manipulation,
and this approach enabled us to study the control mechanism around the pyruvate node.
84
Acknowledgements
This work has been partly supported financially by the Danish Programme for Food
Technology II (project 2409) as well as by European Commission Framework IV Cell
Factory (contract BIO-CT95-0 107). Thanks to Kristian 0. Walle for performance of
the nitrogen-limited cultivations on glucose and galactose.
References
Fiaux, J., Anderson, C.I.J., Holmberg, N., Blow, L., Kallio, P.T., Szyperski, T., Bailey, J.E., and Wthrich,
K. (1999) 13C NMR flux ratio analysis of Escherichia coli central carbon metabolism in micro-aerobic
bioprocesses, J. Am. Chem. Soc. 121, 1407-1408.
Galazzo, J.L., and Bailey, J.E. (1990) Fermentation pathway kinetics and metabolic flux control in suspended
and immobilised Saccharomyces cerevisiae, Enzyme Microb. Technol. 12, 162-172.
Heinrich, R., and Rapoport, T.A. (1974) A linear steady state treatment of enzymatic chains. General
properties, control and effector strength, Eur. J. Biochem. 42, 89-95.
Johnston M., and Carlson, M. (1992) Regulation of carbon and phosphate utilisation, in E.W. Jones, J.R.
Pringel, and J. Broach (eds.), The molecular and cellular biology of the yeast Saccharomyces: Gene
expression, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., pp. 193-281.
Kaeser, H., and Burns J.A. (1973) The control offlux, Symp. Soc. Exp. Biol. 27, 65-104.
Keleher, C.A., Redd, M.J., Schultz, J., Carlson, M., and Johnson, A.D. (1992) Ssn6-Tup1 is a general
repressor of transcription in yeast, Cell 68,709-719.
Klein, C.J.L., Olsson, L., and Nielsen, J. (1998) Glucose control in Saccharomyces cerevisiae: the role of
MIG1 in metabolic functions, Microbiology 144,13-24.
Marx, A., de Graaf, A.A., Wiechert, W., Eggeling, L., and Sahm, H. (1996) Determination of the fluxes in the
central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy
combined with metabolite balancing, Biotechnol. Bioeng. 49, 111-129.
Melcher, K. (1997) Galactose metabolism in Saccharomyces cerevisiae: A paradigm for eukaryotic gene
regulation, in F.K. Zimmermann, K.-D. Entian (eds), Yeast sugar metabolism, Technomic Publishing Co.,
Inc. Lancaster, Basel, pp. 235-269.
Pastemack, L.B., Laude, D.A., and Appling, D.R. (1994) 13C NMR analysis of intercompartmental flow of
one-carbon unit into choline and purines in Saccharomyces cerevisiae, Biochemistry 33, 74-82.
Rizzi, M., Bakes, M., Theobald, U., and Reuss, M. (1997) In vivo analysis of dynamics in Saccharomyces
cerevisiae: II. Mathematical model, Biotechnol. Bioeng. 55, 592-608.
Sonntag, K., Eggeling, L., de Graaf, A.A., and Sahm, H. (1993) Flux partitioning in the split pathway of
lysine synthesis in Corynebacterium glutamicum, Eur. J. Biochem. 213, 1325-1331.
Stephanopoulos, G., Aristodou, A., and Nielsen, J. (1998) Metabolic engineering, San Diego: Academic
Press.
Teusink, B., Diderich, J.A., Westerhoff, H.V., Dam, K., Walsh, M.C. (1998) Intracellular glucose
concentration in derepressed yeast cells consuming glucose is high enough to reduce the glucose transport
rate by 50%, J. Bacteriol. 180, 556-562.
Theobald, U., Mailinger, W., Baltes, M., Rizzi, M., and Reuss, M. (1997) In vivo analysis of dynamics in
Saccharomyces cerevisiae: I Experimental observations, Biotechnol. Bioeng. 55, 305-316.
Treitel, M.A., and Carlson, M. (1995) Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator
protein, Proc. Natl. Acad. Sci. USA 92, 3132-3136
Wiechert, W., and de Graaf, A.A. (1996) In vivo stationary flux analysis by 13C labelling experiments, Adv.
Biochem. Eng. Biotechnol. 54, 109-154.
stergaard, S., Olsson, L., and Nielsen, J. (1998) Metabolic control analysis of the Leloir pathway in
Saccharomyces cerevisiae, in BioThermoKinetics In The Post Genomic Era. Proceedings of the 8th
international meeting on Biothermokinetics, held July 2-5 1998 in Fiskebckskil, Sweden, pp. 22-26.
85
Part 3
STATE PARAMETERS AND CULTURE CONDITIONS
EFFECT OF AERATION IN PROPAGATION ON SURFACE PROPERTIES OF

BREWERS YEAST
ANDREW ROBINSON AND SUSAN T. L. HARRISON
Department of Chemical Engineering, University of Cape Town,
Rondebosch, 7701, South Africa.
Abstract
Traditionally, brewers yeast is propagated through a series of poorly aerated batch
fermentation vessels. Acknowledgement of the importance of yeast quality in brewery
fermentation performance and the requirement of adequate oxygen to ensure yeast
quality has prompted the investigation of alternative propagation schemes. Here, the
effect of aeration on yeast production, surface properties and flocculation of 4 brewers
yeast strains (3 flocculent, 1 non-flocculent) have been investigated. The biomass
growth rates and yields of all strains were improved on aeration during the propagation
stage, as expected. In addition, changes were observed in the hydrophobicity and
surface charge of the cell. Aeration lowers the hydrophobicity and increases the total
charge on the cells while also increasing the flocculation capacity of flocculent strains
of yeast. This observation is contradictory to the prediction of the DLVO theory and
suggests the dominance of the lectin mechanism over classical colloidal flocculation
theory. The strain classified as non-flocculent, and measured to be very weakly
flocculent, showed the reduced hydrophobicity and increased charge in the presence of
aeration, however its flocculence decreased in accordance with DLVO theory. This is
consistent with the non-flocculent strain lacking a lectin mechanism of flocculation.
Further studies are required to determine the effect of aeration on fermentation
performance and the effect of altered surface properties and flocculence on brewery
fermentation.
1. Introduction
Brewers yeast is traditionally propagated in a series of batch propagators which
increases the biomass from laboratory flask size to the required quantity for full-scale
brewery fermentation. These propagators are operated under oxygen limitation with
low biomass yields thus requiring a number of propagation stages. This procedure
increases the time required, substrate usage and risk of contamination between each
89
transfer. Aerobic propagation offers a potential advantage over these drawbacks

provided the physical properties, flocculence and fermentative ability of the yeast are
not adversely affected. This paper presents a comparative study of yield coefficients,
propagation rates, the surface properties of hydrophobicity and charge and the yeasts
flocculence for four strains of brewers' yeast propagated under full aerobic and near
anaerobic conditions.
Various authors have studied surface properties of yeast to explain differences in
their physical behaviour of flocculence, with contradictory findings. Amory and
Rouxhet (1988) compared strains belonging to top fermenting Saccharomyces
cerevisiae and bottom fermenting Saccharomyces carlsbergensis, since reclassified as
S. cerevisiae. They observed that the bottom fermenting strains had more negative zeta
potentials than the top fermenting strains. They also found the bottom strains to be less
hydrophobic than the top cropping strains. Bowen and Cook (1989), Bowen et al.
(1992) and Bowen and Ventham (1994) compared differences between bottom
fermenting lager yeast, top cropping ale and bottom cropping ale yeasts. They found
that yeast from top cropping ale strains had higher charge than the yeast from the
bottom ale strains that in turn had more charge than the bottom cropping lager strains.
Dengis et al. (1995) compared the flocculation mechanism of top and bottom cropping
strains of Saccharomyces cerevisiae and found that their charge at the final pH of
fermentation to be similar.
2. Materials and Methods
2.1 PROPAGATION CONDITIONS
Propagation were conducted in autoclavable 2 1 glass vessels with a working volume of
1.5 1, using 16P brewery wort (supplied by South African Breweries, Newlands
brewery) at 18C. Four commercial strains of brewers yeast were used: 3 flocculent
(SAB1, SAB5 & SAB1/96) and one non-flocculent (SAB2). Agitation was provided
with a Rushton impeller operated at 400 rpm. The aerobic propagation was sparged
continuously with sterile air at 1.5 vvm while the near anaerobic propagation was only
aerated prior to inoculation to saturate the wort with oxygen at a concentration of 9.2
mg.1-1. Foaming was controlled by the addition of 2 ml of Antifoam 289 (Sigma) prior
to the start of aeration. Samples were taken to monitor cell concentration, medium
density, cell hydrophobicity and surface charge.
2.2 HYDROPHOBICITY
The hydrophobicity index of the cells was determined by their partitioning between a
hydrocarbon and aqueous phase, based on the method of Smart et al. (1995). The cells
were harvested by centrifugation, washed with distilled water, deflocculated with 2 mM
EDTA and rewashed twice with deionised water. The washed cells were re-suspended
to an OD660 of 0.6 0.75 in a phosphate-urea-MgSO4 (PUM) buffer at pH 7.1. The
PUM buffer consists of 2.22% (w/v) K2HPO4.3H2O, 0.726% (w/v) KH2PO4, 0.18%
(w/v) urea and 0.02% (w/v) MgSO4.7H2O. The optical density of the suspension was
90
AERATION AND SURFACE PROPERTIES OF BREWERS' YEAST
recorded and 2.4 ml was transferred to a wide mouth test tube. 0.2 ml xylene was added
to the test tube. The mixture was vortexed for 2 minutes, and allowed to stand for 15
minutes. The aqueous phase was removed with a Pasteur pipette and its optical density
determined. The hydrophobicity index was calculated as follows.
(1)
where: I = Initial optical density

F = Final optical density
HI = Hydrophobicity Index
2.3 SURFACE CHARGE

Zeta potential of the growing yeast suspensions was measured using a Malvern
ZetaSizer 4. Washed cells were suspended in 20 mM sodium acetate buffer solutions
with pH values between 2.1 and 6.6 prior to zeta potential measurement. The zeta
potential obtained was converted into surface charge density by using the Gouy
Chapman equation (2), which describes the decay in potential around a charged particle
(Hiemenz 1986).
(2)
where: n0
kB
T
Z
e
= molar concentration of the electrolyte

= dielectric constant of solution
= Boltzman constant
= temperature
= valence charge
= charge on the electron
For an aqueous solution at 25 C, this reduces to equation (3).
(3)
where is the surface potential of the yeast (V) and is the surface charge (C.cm-2).
In this study it is recommended that the charge density at the plane of shear, rather than
zeta potential be used as an indicator of the surface charge of particles as this parameter
removes the differences in experimental conditions used by different research groups.
91
2.4 FLOCCULATION
Samples were prepared to measure the extent of flocculation by removing a
homogenous sample from the bioreactor, washing with distilled water, deflocculation
with 2 mM EDTA and washing the yeast twice with deionised water. The washed yeast
was resuspended in 20 mM sodium acetate buffer (pH 4.5) containing 10 mM CaCl2 to
an OD660 in the range 2.3 to 2.4. The cell suspension was placed inside a glass U tube
to which was fixed a square (10 mm) glass section, aligned inside a spectrophotometer.
Air bubbles of approximately 0.5 cm3 were passed through the cell suspension at a rate
of 60 per minute to allow an acclimatisation period and provide the reproducible mixing
required to initiate flocculation. After 5 minutes, the air supply was closed and data
logging of the absorbance started. The cells formed flocs and settled through the light
path. Flocculation was complete within 2 to 4 minutes, after that time the plateau
absorbance value was recorded. Absorbance readings were converted into biomass
concentrations by calibration curves. The final extent of flocculation was reported as
the wt. % of cells removed by flocculation.
3. Results
3.1 YIELD COEFFICIENTS
The biomass yield coefficients were calculated at the end of each propagation and
expressed as the dry mass of cells obtained per mass of total sugars used (g dry weight.g
-1
These values are summarised in Table 1 for both the aerobic and near
carbohydrate ).
anaerobic propagation of the four strains. The increased ratio of aerobic to anaerobic
yields are also presented.
3.2 CELL GROWTH RATES
The rate of increase of cells during propagation was modelled using the logistic
equation (Bailey and Ollis, 1986) (4). The integrated form of the equation (5) providing
the cell concentration profile during propagation from the initial cell concentration, x0,
has two parameters, (ml.cell-1), the inverse of the stationary population concentration
and k (h-1), the rate constant.
(4)
(5)
92
AERATION AND SURFACE PROPERTIES OF BREWERS' YEAST
Since the final population size is known, the logistic equation can be solved to yield the
logistic rate constant. Figure 1 illustrates the prediction of the data by the logistic
equation. The rate constants obtained for each strain are summarised in Table 1. The
ratio of aerobic to anaerobic rate constants is compared.
Table 1.
Comparison of biomass yield coefficients and cell production rates of aerobic
and near anaerobic yeast propagation in high gravity brewers yeast.
Biomass Yield Coefficient
(gdry weigh gcarbohydratge-1)
Strain
SAB5
SAB1
SAB1/96
SAB2
Aerobic
0.139
0.134
0.095
0.117
Anaerobic
0.049
0.051
0.044
0.046
Ratio
(aerobic/
anaerobic)
2.9
2.7
2.1
2.5
Logistic Cell Growth Rate

(h-1)
Aerobic
Anearobic
0.149
0.129
0.111
0.103
0.072
0.059
0.076
0.080
Ratio
(anerobic/
anearobic
1.16
1.08
1.22
0.95
Figure 1.
Cell concentration during aerobic and anaerobic propagation of SAB1 with
logistic equation fit to data.
aerobic data, ______ aerobic logistic fit,
anaerobic data --- - ---- anaerobic logistic fit)
3.3 HYDROPHOBICITY
The cell growth profiles and hydrophobicity of the cell surface during propagation are
shown for the four strains in Figure 2. The hydrophobicity of the cells was found to
increase concomitantly with cell growth under anaerobic conditions. The aerobically
grown yeast were less hydrophobic throughout propagation. Once the cells reached the
stationary phase, no further change in hydrophobicity was observed.
The
hydrophobicity in the stationary phase of propagation is summarised in Table 2.
93
3.4 ZETA POTENTIAL

The zeta potential profiles of all strains measured across the pH range 2.1 to 6.6 varied
with phase of growth to approach a constant charge profile towards the onset of the
stationary phase. This is illustrated for the aerobic and anaerobic propagation of strain
SAB2 in Figure 3a and 3b. The zeta potential of the aerobically grown strains was
generally lower than the anaerobic equivalent, especially under stationary phase
conditions. The iso-electric points for the stationary phase aerobic yeast were generally
below the measured range of the assay, i.e. < pH 2. The charge profiles of the strains in
their stationary phase of propagation are presented in Figures 4a and 4b for the aerobic
and near anaerobic growth condition.
Figure 2.
Increase in cell hydrophobicity with cell growth during propagation of strains
under aerobic and near anaerobic conditions. ((a) SAB5, (b) SAB1, (c) SAB1/96 and (d)
SAB2,
aerobic hydrophobicity, _______ aerobic cell concentration,
anaerobic
hydrophobicity ---------- anaerobic cell concentration).
Figure 3.
Zeta potential profiles for (a) aerobic and (b) anaerobic growth condition of
SAB2 changing smoothly during propagation and reaching a stable profile by the end of
propagation.
pre-exponential, X early-exponential,
late exponential and stationary
phase)
94
AERATION AND SURFACE PROPERTIES OF BREWERS YEAST
Figure 4.
Zeta potential of the four strains of yeast at the end of (a) aerobic (b) and
anaerobic propagation showing the different profiles observed.
SAB1,
SAB2,
SAB1/96 and SAB5).
3.5 FLOCCULATION
A typical absorbance profile for the flocculation assay is shown in Figure 5 illustrating a
change in absorbance from the initial 2.44 to 0.44 after 2 minutes. These absorbencies
correspond to cell concentrations of 97.6 and 3.9 million cells per ml. Based on cell
concentrations the extent of flocculation is 96.4%.
Figure 5.
conditions.
Flocculation assay absorbance profile for SAB5 grown under brewery
The three strains of flocculent yeast (SAB1, SAB1/96 & SAB5) were found to be more
flocculent when grown under aerobic conditions as compared to propagation under
anaerobic conditions. It was noticed that the yeast only became flocculent towards the
end of propagation within the wort medium. This coincides with depletion of sugars,
which may cause inhibition of the lectin binding required for flocculation. Samples
taken during propagation were visually seen to be weakly flocculent in flocculation
buffer until the late exponential phase. This coincided with the flocculation behaviour of
95
the yeasts in situ. The extent of flocculation of yeast harvested in the stationary phase is
summarised in Table 2 for both aerobic and near anaerobic growth conditions. Table 2
includes the zeta potential values at pH 4.5, the same pH as the flocculation buffer, for
reference.
Table 2.
Summary of cell hydrophobicity, surface charge and extent of flocculation
values for aerobic and anaerobic propagation of yeast.
Hydrophobicity Index
Extent of Flocculation
Zeta Potential @ pH 4.5
(%)
(% of cells)
(mV)
Strain
Aerobic
Anaerobic
Aerobic
Anaerobic
Aerobic
Anaerobic
SAB5
2.5
12.4
-8.4(-0.39)
-3.8(-0.17)
96.3
82.4
SAB1
4.5
21.0
-7.3(-0.34)
-3.9(-0.18)
98.6
94.8
SAB1/96
5.5
22.8
-5.8(-0.27)
-3.1(-0.14)
99.1
95.9
SAB2
1.8
25.0
-10.8(-0.50)
-5.8(-0.27)
0.4
4.8
Zeta potentials converted to surface charge values presented in brackets withunits of C.cm-2.
4. Discussion
The biomass yield coefficients of the aerobic propagation were consistently 2 to 3 fold
higher than under anaerobic conditions. In addition, aerobic growth yielded higher
logistic growth rate constants. The increased growth rates and higher yields illustrate
that aerobic propagation is advantageous for the biomass production cycle of yeast in
breweries.
Before exploiting the potential advantage of aerobic propagation, the suitability of
yeast produced by aerobic propagation for anaerobic brewing must be assessed.
Brewing yeast is required to have good fermentative abilities as well as suitable
physical characteristics necessary in the brewing environment. The physical attributes
required include a suitable cell envelope structure and the yeasts ability to flocculate at
the end of fermentation. Here the surface charge, hydrophobicity and flocculation
potential of the commercial strains are assessed as a function of oxygen availability and
growth phase.
All flocculent strains considered in this study were more charged and yet also more
flocculent when grown aerobically over those grown anaerobically. This suggests that
no correlation between lower zeta potential and increased flocculence exists, contrary to
the prediction of the Derjaguin-Landau-Venvey-Overbeek (DLVO) theory. Smit et al.
(1992) showed that flocculation is most effective at a pH of 4.5. This is the approximate
final pH attained in fermentation at which the yeast flocculates and settles out of
suspension. It is therefore instructive to consider the yeasts surface charge at this pH
when studying flocculation. From this study it is concluded that the increased surface
charge associated with the aerobic propagation system does not lower flocculation in
these yeast strains. Previously Amory and Rouxhet (1988), on comparing the surface
charge between top and bottom cropping yeasts, showed the bottom strains to be more
charged (zeta potential of -35 mV, corresponding to a surface charge of -0.12 C.cm-2)
than the top strains (zeta potential of -12 mV and charge of -0.04 C.cm-2) at pH 4.0. In
many of the early studies, zeta potentials were not measured in a defined environment.
As these measurements were taken without any background electrolyte addition, even
96
the smallest leakage of ions from the cells could alter the potential observed
significantly. Bowen and Cooke (1989) determined zeta potential in wort hence surface
charge can not be calculated. Dengis et al. (1995) found surface charges between -0.15
and -0.21 C.cm-2 at pH 5.2 for top and bottom fermenting strains of Saccharomyces
cerevisiae. Bowen and Ventham (1994) reported a decrease in zeta potential and
surface charge during fermentation of top and bottom cropping ale yeast as well as lager
yeast. The bottom ale yeast and lager yeast carried similar charge during fermentation,
decreasing from -12 to -7 mV (-0.39 to -0.23 C.cm-2) on progression from exponential
to stationary phase. The top cropping ale yeast was slightly more charged throughout
fermentation, decreasing from -16 mV to -12 mV (-0.53 to -0.39 C.cm-2). These zeta
potential measurements were made at the pH occurring during fermentation. Smart et
al. (1995) observed a decrease in surface charge at pH 4.0 (-42.6 to -32.5 mV and -1.54
to - 1.12 C.cm-2) when yeast was stored under conditions of nutrient starvation. From
all these investigations, no clear correlation can be drawn between the flocculence or
location of flocculated yeast (top vs. bottom) and the surface charge of the yeast. Van
Hammersveld et al. (1994) calculated the bond strength predicted by the DLVO theory
of flocculation. On comparison, they found the experimentally determined bond
strength to be far higher and concluded that the additional bonding energy resulted from
specific biological interactions. However, the parameters within the theory changed
during fermentation in a manner that favoured flocculation predicted by DLVO. Speers
et al. (1993) concurs that the DLVO theory cannot explain flocculence within flocculent
yeast strains.
The inability of physicochemical interactions at the surface to correlate with
flocculation behaviour is further illustrated by trends in hydrophobicity determined in
this study. Here a greater oxygen availability lowered the hydrophobicity but also
increased the cells flocculence. The hydrophobicity index of the yeast in the near
anaerobic propagation increased during the exponential phase of growth while that of
the aerobic propagation remained relatively low. The increase in hydrophobicity
observed at the end of exponential growth, also seen by Smit et al. (1992), was found to
coincide with the onset of flocculation. Smit et al. inferred that the observed
hydrophobicity was attributed to flocculation proteins (lectins). This was supported by a
reduction in both hydrophobicity and flocculation by protease activity. It can be
concluded from these studies that the yeast lectins may contribute to the cells
hydrophobicity, but this is not the only contributing factor and hydrophobicity alone is
not responsible for flocculation.
The non-flocculent strain (SAB2) showed the similar trend whereby the anaerobic
propagation was less charged while at the same time being more hydrophobic than the
aerobically grown yeast. This strain was observed to have a very low level of
flocculence (<5%) as measured by the flocculation assay used here. For this strain, the
anaerobic propagation condition caused lower charge, higher hydrophobicity and was
more flocculent. This observation is consistent with the prediction of DLVO theory.
Hence it is postulated that the small extent of flocculation found in this yeast strain is a
result of the classical colloidal model while the flocculence observed in the flocculent
strains is governed by lectin binding. The dominance of lectin-mediated flocculation is
indicated.
97
For all strains considered in this study, the inter-strain variation of surface charge profile
and hydrophobicity was smaller than the variation generated by changes in oxygen
supply or the position within the growth phase. This highlights the necessity to
adequately quantify the oxygen availability during growth as well as the position in the
growth cycle at which comparisons are made when comparing these properties for
different strains or classes of yeast, viz. ale vs. lager or top vs. bottom fermenting yeast.
Flocculation in the three flocculent strains studied here appears to be caused by the
lectin/receptor mechanism described by Miki et al. (1982), as reported for other
commercial strains of Saccharomyces cerevisiae. Such flocculation has been shown to
require calcium ions and the sugars mannose and glucose inhibit the formation of flocs.
According to the classification system of Stratford and Assinder (l991), these strains
belong to the NewFlo phenotype owing to the inhibitory action of glucose. Bony et al.
(1998) linked the level of flocculation observed in yeast to the amount of the
flocculation lectin Flop at the surface of the cell. The protein was also detected in the
endoplasmic reticulum, indicating that it was secreted along the protein excretory
pathway. This pathway is known to be partially repressed under anaerobic conditions,
indicating that flocculation may be repressed under anaerobic conditions as shown here.
Although hydrophobicity and surface charge are not the main cause of yeast
flocculation (van Hammersveld et al. 1994), these may play a role. At low levels of
mixing, a sufficiently high surface charge could prevent the close interaction of
individual cells to permit lectin-receptor interactions and so limit flocculation.
5. Conclusions
Aerobic propagation of brewers yeast results in a 2 to 3 fold higher yield of biomass
making it an attractive alternative to traditional oxygen limited propagation of brewers
yeast. Aerobic propagation however does result in yeast with altered surface properties,
namely lower hydrophobicity and greater surface charge. Aerobically propagated
flocculent yeast also shows improved flocculation. Neither hydrophobicity nor surface
charge appear to be directly responsible for this change in flocculation. Because yeast
flocculence affects its fermentative ability and limited oxygen availability restricts the
formation of membrane lipids and sterols, further assessment of fermentation
performance is required to identify if the observed cellular changes affect brewery
fermentation performance.
Acknowledgements
The technical and financial support of The South African Breweries Ltd is gratefully
acknowledged. Further financial support of the NRF, including sponsorship of AR
through a postgraduate bursary is appreciated.
98
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Yeast 7, 557-574.
Van Hamersveld, E.H., van Loosdrecht, M.C.M. and Luyben, K.ch.A.M. (1994) How important is the
physicochemical interactions in the flocculation of yeast cells? Coll. Surf. B: Biointerfaces 2, 165-171.
99
EFFECT OF THE MAIN CULTURE PARAMETERS ON THE GROWTH AND

PRODUCTION COUPLING OF LACTIC ACID BACTERIA
A. AMRANE* AND Y. PRIGENT
Universit de Rennes I, Laboratoire des Procds de Sparation (Unit
associe I.N.R.A.),
Campus de Beaulieu, Bt. 10A, 263 avenue de Gnral Leclerc,
CS 74205, 35042 Rennes Cedex, France
Fax: 33 2 99 84 40 31. E-mail: abdeltif.amrane@univ-rennes1.fr
Abstract
Identification of the main culture parameters for Lactobacillus helveticus growing on
whey supplemented medium was carried out from analysis of their influence on the
growth and production coupling. Seed culture duration (linked to the physiological state
of the inoculated cells), pH control and undissociated lactic acid concentration (the
inhibitory species), as well as nitrogen limitation control this association between
growth and production.
1. Introduction
From the pioneer work of Luedeking and Piret (1959) lactic acid production have been
recognised as partially linked to growth:
(1)
where the constants A and B are the coefficients for growth- and non-growth-associated
production respectively.
However, as previously shown (Amrane and Prigent, 1997), the best way to analyse
growth and production coupling is to plot the amount of lactic acid produced (p - p0) vs.
the biomass formed (x - x0), instead of the graph of the specific production rate as a
function of the specific growth rate. The linear part of this plot corresponded to the
growth-associated production, and its slope was the coefficient for growth-associated
production (parameter A in equation 1), or the product on biomass yield YP/X.
The effect of some culture parameters on growth and production coupling have
been outlined in the available literature. Indeed, contradictory results concerning the
effect of culture pH can be found: according to some authors the uncoupling between
101
A. AMRANE AND Y. PRIGENT
growth and production increase at acidic pH (Roy et al, 1987; Venkatesh et al, 1993),
while Norton et al (1994) have obtained a very low value for the coefficient B for the
non-growth associated production (0.01) at pH 4.3. Moreover, it has also been reported
that nitrogen starvation was involved in this coupling (Turner and Thomas, 1975).
However, systematic analysis of the effect of the main fermentation parameters on
this linking is not at our knowledge available in the bibliography; this study will be the
aim of the present work.
2. Materials and methods
2.1 MICROORGANISM
Lactobacillus helveticus strain milano used throughout this work was kindly supplied by
Dr A. Fur (Even Ltd, Ploudaniel, France). Stock cultures were maintained on 10% (w/v)
skim milk and deep frozen at -16C. As required, these cultures were thawed and
reactivated by two transfers in 10% (w/v) skim milk (42C, 24 h).
2.2 MEDIA
Whey permeate powder (Armor-Proteines, ST Brice, France) was used as a carbon
source; the powder was reconstituted at 57 g l-1, corresponding to a lactose
concentration of 48 g l-1. Before use, the permeate was clarified by a heat / calcium
process (Fauquant et al, 1985): it was supplemented with 3 g l-1 CaCl2, 2H2O, the pH
was settled at 7.3, and the solution was pumped through two heat exchangers at 80 and
16C respectively (mean residence time: 20 s). The solution was left to decant overnight
at 4C, and the supernatant was then supplemented with a range of yeast extract
concentration (2, 5, 10, 20, 30 g l-1) or 20 g l-1 yeast extract and 5 g l-1 of both tryptic
and pancreatic casein peptones (RM medium).
When needed, aliquots of 1 mol l-1 lactic acid was added to RM medium in order to
achieve initial concentrations p0 of 0, 2, 3, and 5 g l-1, corresponding to pH values of
5.90, 4.63, 4.34, and 4.04 respectively. During each run, the pH was maintained at its
initial value by automatic addition of 10 mol l-1NaOH. The same procedure was applied
to a second set of culture medium for which the initial pH was adjusted with
hydrochloric acid instead of lactic acid.
For another set of experiments, RM culture medium was supplemented with 1 M
lactic acid at the required concentration (10,40, 50 g l-1), and then pH was settled at 5.9
by the addition of 10 M NaOH.
2.3 FERMENTORS AND CULTURE CONDITIONS
Batch culture was carried out in a 2-1 fermentor (SET 2M, Inceltech, Toulouse, France),
magnetically stirred (300 rpm), at 42C. The pH was maintained at 5.9 by automatic
addition of 10 M NaOH. The mass of the NaOH solution added for pH control was
continuously recorded, allowing on-line calculation of the quantity of lactate anion
produced at a given pH at each time point; the observed standard deviation was 1 g l-1,
102
CULTURE PARAMETERS, GROWTH AND PRODUCTION COUPLING OF LACTIC ACID BACTERIA
Seed culture was carried out in a 0.25-1 laboratory-designed glass fermentor equipped
with a sterilisable combination glass electrode (Ingold, Paris, France), cotton plug filter,
magnetic stirrer, infrared lamp temperature control (set at 42C), and an aseptic transfer
line.
In addition, the two fermentors were equipped with an aseptic recirculation loop
(Watson-Marlow 50 1 U peristaltic pump; Volumax, Montlouis, France) incorporating a
laboratory-made turbidimeter. As the turbidity was continuously recorded, the total
biomass could be calculated on-line after dry weight calibration; the observed standard
deviation was 10.2 g l-1.
Bacteria were precultivated by inoculating sterile culture medium with 0.8 % (v/v)
of the second skim milk transfer. Then, 1.6 1 of pasteurised culture medium was
inoculated with 0.2 1 seed culture (11% v/v), unless specified, and the reaction
proceeded.
The concentration of total (p) and undissociated (HL) lactic acid was calculated
using the Henderson-Hasselbach equation (pKA = 3.8), the lactate concentration (L-)
and the corresponding pH value:
(2)
and
(3)
2.4 ANALYTICAL METHODS

At the end of both the preculture and culture, the final biomass, lactose, and lactic acid
concentrations were determined as previously described (Amrane and Prigent, 1994).
3. Results and Discussion
3.1. PRECULTURE CONDITIONS
The effect of preculture conditions on the subsequent culture were first considered. As
observed in Fig.1a, the preculture medium seemed to have no effect on the coupling
between growth and production. Indeed, for both cultures carried out on RM medium
and inoculated at 11 % seed level, growth and production were associated until 20-25 g
l-1 lactic acid was produced, i.e. approximately half of the production; in the associated
part of the curve, the same product on biomass yield (YP/X = 4.7, corresponding to the
slope of the curve) was found for both batches. To examine the effect of inoculation
level, another run (2) was carried out on RM medium, but inoculated at 6.3 % (v/v)
103
instead of 11 % (additional pasteurised culture medium was fed to the fermentor to keep
the total volume constant), with cells precultivated on RM medium too. No effect of the
inoculation level appeared at the examination of Fig.1a: as above 20-25 g l-1 lactic acid
was produced by an associated mechanism and YP/X = 4.7.
Figure 1: (a) Product on biomass yield for cells precultivated for approximately 13 h: on
whey supplemented with 5 g l-1 yeast extract and inoculated at 11 % seed level, run 1 (A); on
RM and inoculated at 6.3% seed level, run 2
on RM and inoculated at 11 % seed level,
run 3
; linear fitting (). (b) Product on biomass yield for cells precultivated on RM for
13.3 h: run 3 (0); 7.5 h: run 4
4.7 h: run 5 (A) ; linear fitting for runs 3 (.....), 4 ()
and 5 ().
From Fig.1b, it appeared that the slope YP/X for the plot (p - p0) vs. (x - x0) increased in
the following order for the three preculture duration tested: 7.5 h < 13.3 h < 4.7 h.
Therefore, and by comparison with growth and production kinetics (Amrane and
Prigent, 1998a), product on biomass yield increased when growth and production rates,
as well as maximum cellular concentration, decreased.
104
3.2. NUTRITIONAL LIMITATIONS

As observed in Fig.2, the product on biomass yield displayed a minimum value of 3.7
for 10 g l-1 yeast extract supplementation; as previously demonstrated by derivation of
growth and production parameters with respect to the YE concentration (Amrane and
Prigent, 1998b and 1999), this corresponded to the best performances at minimum cost.
The end of coupled production was arbitrarily defined as the point for which a 3 %
deviation from the oblique asymptotic line was observed. From this, the part played by
an associated mechanism in total production increased continuously with the yeast
extract (YE) supplementation, from 15 % for 5 g l-1 YE to 83 % for 30 g l-1 YE, i.e. an
almost total coupling between growth and production; this had to be brought together
with previous result demonstrating that increasing YE supplementation resulted in a
shift from a nitrogen limitation to a carbon one (Amrane and Prigent, 1998b).
Figure 2: Product on biomass yield for batch cultures of L. helveticus carried out on
clarified whey supplemented with a range ofyeast extract concentrations.
Figure 3: Product on biomass yield for batch cultures of L. helveticus carried out with
various initial lactic acid concentrations p0 (g l-1) : 0
.), 10
), 40
and
50
)
105
3.3. INITIAL LACTATE ADDITIONS

The experiments displayed in Fig.3 were carried out on a largely nitrogen supplemented
medium (RM) to avoid any nitrogen and growth factors limitation. As observed, at a
given pH control (5.9), the proportion of lactic acid produced by an associated
mechanism was not a function of the lactate initially added, about 60 % of the total
production irrespective of the amount of lactic acid initially added; even if for 50 g 1-1 of
initial lactic acid, the critical concentration (83 g l-1, Amrane and Prigent, 1998c) was
achieved before a total lactose exhaustion.
From Fig.3 also, increasing initial lactic acid additions resulted in increasing product
on biomass yield, from 4.2 without initial addition to 15.0 for p0 = 50 g l-1.
Figure 4: Product on biomass yield for batch cultures of L. helveticus at pH

5.90,
4.63,
4.34,
4.04 initially adjusted with (a) lactic acid and (b) hydrochloric acid; ():
linear fitting.
pH control and undissociated lactic acid: As above, the experiments displayed in

Fig.4 were carried out on RM medium to avoid any nutritional limitation. As observed,
106
decreasing pH control and increasing quantity of undissociated lactic acid (the

inhibitory species, Gtje and Gottschalk, 1991) resulted in decreasing amount of lactic
acid produced by an associated mechanism (from 60 % for the optimal pH control, 5.9,
to 17 % for a pH of 4.04 initially adjusted by addition of lactic acid), while the product
on biomass yield increased (from 4.2 for pH = 5.9 to 13.5 for pH = 4.04). Moreover, by
comparing Figs.4a and b, it appeared that growth and production coupling depended
mainly on pH, and to a lesser extent on the undissociated lactic acid concentration.
Indeed, the initial addition of lactic acid to achieve the required pH control resulted only
in a slightly lower proportion of growth-associated production and also a slightly higher
product on biomass yield, if compared to an initial addition of hydrochloric acid to
adjust the pH.
4. Conclusions
For the optimisation of the bioconversion of lactose into lactic acid, high values of the
product on biomass yield YP/X were not a convenient criterion, since they correspond
to a low coupling between growth and production: indeed the fermentation time was
minimal, and the volumetric productivity of the bioreactor was maximal when both
processes were almost totally coupled.
Neither the composition of the seed culture medium nor the inoculation level had
significant effect on YP/X and the percentage of associated production of the subsequent
culture: among the preculture parameters, only the seed culture duration, i.e. the
physiological state of cells, had such an effect.
A high concentration of lactate in the culture medium (50 g l-1) resulted in a large
increase of YP/X when the total lactate concentration was close to the critical one
(83 g 1 1), but it had practically no effect on the coupling.
Acidic culture pHs, leading to high proportions of undissociated lactic acid, resulted
in a large increase of YP/X and a large drop of associated production. A similar
behaviour was observed under conditions of nitrogen limitation (yeast extract
concentration from 2 to 10 g l-1). Therefore it should be pointed out that concentrations
of yeast extract in excess of 10 g l-1 led to a significant increase of YP/X.
Acknowledgements
We wish to thank Ms A. Copeland for correcting this manuscript.
References
Amrane, A. and Prigent, Y. (1994) Mathematical model for lactic acid production from lactose in batch
culture : model development and simulation. Journal of Chemical Technology and Biotechnology 60,
241-246.
Amrane, A. and Prigent, Y. (1997) Growth and lactic acid production coupling for Lactobacillus helveticus
cultivated on supplemented whey: influence of peptidic nitrogen deficiency. Journal of Biotechnology 55,
1-8.
107

Amrane A. and Prigent Y. (1998a) Identification and experimental validation of a criterion allowing
prediction of cellular activity for preculture of lactic acid bacteria. Journal of Fermentation and
Bioengineering 85,328-333.
Amrane A. and Prigent Y. (1998b) Influence of yeast extract concentration on batch cultures of Lactobacillus
helveticus: growth and production coupling. World Journal of Microbiology and Biotechnology 14, 529534.
Amrane A. and Prigent Y. (1998c) Influence of an initial addition of lactic acid on growth, acid production
and their coupling for batch cultures of Lactobacillus helveticus. Bioprocess Engineering 19, 307-312.
Amrane A. and Prigent Y. (1999) Analysis of growth and production coupling for batch cultures of
Lactobacillus helveticus with the help of an unstructured model. Process Biochemistry 34, 1-10,
Fauquant, J., Vico, A., Brul, G. and Maubois, J.-L. (1985) Sweet whey clarification by heat-calcium
aggregation of residual fat material. Lait 65, 1-20 (in French).
Gtje, G. and Gottschalk, G. (1991) Limitation of growth and lactic acid production in batch and continuous
cultures of Lactobacillus helveticus. Applied Microbiology and Biotechnology 34, 446-449.
Luedeking R. and Piret E.L. (1959) A kinetic study of the lactic acid fermentation. Batch process at controlled
pH. Journal of Biochemistry, Microbiology and Technological Engineering 1, 393-412.
Norton S., Lacroix C. and Vuillemard J.C. (1994) Kinetic study of continuous whey permeate fermentation by
immobilised Lactobacillus helveticus for lactic acid production. Enzyme and Microbial Technology 16,
457-466.
Roy, D., Le Duy, A. and Goulef J. (1987) Kinetics of growth and lactic acid production from whey permeate
by Lactobacillus helveticus. Canadian Journal of Chemical Engineering 65, 597-603.
Turner, K. W. and Thomas, T. D. (1975) Uncoupling of growth and acid production in lactic Streptococci.
New Zealand Journal of Dairy. Science Technology 10, 162-167.
Venkatesh, K. V., Okos, M. R. and Wankat, P. C. (1993) Kinetic model of growth and lactic acid production
from lactose by Lactobacillus bulgaricus. Process Biochemistry 28, 23 1-241.
108
PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES

CEREVISIAE
Signal Transduction During Nutrient Limitation
F.F. BAUER AND I.S. PRETORIUS

Institute for Wine Biotechnology, University of Stellenbosch, ZA-7600
Stellenbosch, South Africa
Abstract
Cells of the yeast Saccharomyces cerevisiae can undergo profound molecular,
physiological and morphological modifications in response to a limited supply of
essential nutrients, in particular carbon or nitrogen sources. These include a shift in
transcription patterns, the modification of the cell cycle, a change in budding pattern
and strongly polarised growth. Cells having undergone these modifications do not
separate after cell division is completed and form chains of elongated cells called
pseudohyphae or filaments. Cells growing as filaments are able to invade agar plates
and other substrates, a phenomenon referred to as invasive growth. A network of signal
transduction pathways governs this switch from yeast-like growth to pseudohyphal and
invasive growth. Important elements of this network have been identified, including
nutrient signal-receptors, GTP-binding proteins, components of the pheromonedependent MAP kinase cascade, cAMP, and several transcription factors. In this review,
we summarise our current knowledge in this rapidly progressing field. We focus
particularly on the interactions between several signal transduction modules and on the
different transcription factors, which are regulated by these signalling modules.
1. Introduction
The line between basic and applied research is nowhere more nebulous than in yeast
molecular biology and genetic engineering, and is usually traversed at will in our
laboratory's research on the genetic improvement of industrial yeast strains. One of the
focuses of our yeast strain development programme is to increase the efficiency of
fermentation and processing. By expressing numerous genes encoding extracellular,
polysaccharide-degrading hydrolases in wine, brewing and baking yeast strains, we
109
F F. BAUER AND I.S. PRETORIUS
found that Saccharomyces cerevisiae is able to change its growth pattern

(pseudohyphal/invasive growth, flocculation and vellum formation) when fermentable
sugars progressively become limiting in the growth medium. This observation has
guided our research into fundamental aspects of cellular differentiation,
Cellular adaptations to changes in extracellular parameters have been a central
subject of biological investigation throughout this century. Adaptation requires the
perception (sensing) of the environmental parameter, followed by the transmission of
the information to the relevant compartments of the cell. This process, which will result
in a specific adaptive response to a specific signal, is referred to as signal transduction.
The molecular nature of signalling events hasonly become accessible during the last
two decades. Signal transduction pathways are part of the most essential molecular
attributes of living systems, and the ability to adapt to changing environmental
conditions or the ability to co-ordinate cellular differentiation in a multi-cellular
organism (which relies on the exchange of signals between cells) is essential for
survival. Consequently, most signalling molecules and mechanisms have been well
conserved during evolution, and signalling modules found in S. cerevisiae are very
similar to those found in higher eukaryotic organisms, including mammals (reviewed in
Lim et al., 1996; Widmann et al., 1999; Wilkie and Yokoyama, 1994). This situation
has created new interest in S. cerevisiae as a model system, which allows investigating
complex molecular interactions.
Besides being traditionally the most widely used microorganism in industry,
S. cerevisiae has by now probably become the scientifically best understood of all
organisms, The sequence of the entire genome of S. cerevisiae has been deciphered and
made public in 1996, and a large number of protein-encoding open reading frames
(ORFs) has already been associated with a specific function. The availability of the
entire genome sequence combined with the genetic tractability of S. cerevisiae and the
existence of what is probably the widest range of molecular tools, has lead to an
extraordinarily fast accumulation of new knowledge. In the not so distant future, the
entire functional mapping of the genome and of the proteome of this unicellular
organism will probably be achieved.
This review will mainly focus on some aspects of the complex molecular
interactions that occur during a specific signal transduction event. In the first part of the
review, we provide some general background on S. cerevisiae and introduce the specific
pathway (pseudohyphal differentiation and invasive growth) that has been the focus of
our investigations. In a second part, we detail the complex molecular nature of the
signal transduction events involved in this dimorphic and cell growth adaptation.
Finally, in a last section, we show how the newly gained knowledge has modified our
vision of signal transduction events and how this insight can be of tremendous
importance for future genetic modifications of industrial strains of S. cerevisiae for
biotechnological applications.
2. Signal transduction in Saccharomyces cerevisiae
Like most organisms, S. cerevisiae can perceive two types of extracellular signals:
signals required for intercellular communication (for example hormonal signals like the
pheromones a and ), and signals emanating from the environment. Environmental
110
PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE
signals can be either of a chemical (e.g. the concentration or availability of certain

nutrients) or physical (e.g. temperature and osmotic pressure) nature, and survival
requires a specific response to changes in any of these parameters.
In theory, two possibilities to ensure specific responses to specific signals exist.
Cells would have to either use clearly separated and different signal transduction
pathways for each signal, or use the amplification, differentiation and combinatorial
possibilities of a network of a limited number of cross-talking pathways. Since each
signal has to be integrated to achieve a perfect co-ordination between all cellular
responses to extracellular stimuli, and since the number of potentially important signals
is high, specific and separated transduction pathways for each signal seem a logistical
impossibility. In line with this expectation are recent data, largely obtained through
research on S. cerevisiae, which suggest an increasingly complex picture of
interconnected and cross-talking signalling pathways, relying on a limited amount of
molecular modules (reviewed in Elion, 1995; Pawson, 1995; Levin and Errede, 1995;
Madhani and Fink, 1998).
One of the - at least in a research perspective - most useful phenotypic adaptations
of S. cerevisiae to environmental change is pseudohyphal differentiation, which is also
referred to as the dimorphic switch between yeast-form and filamentous-form (Fig. 1).
This adaptation can be observed in conditions of nutritional limitation and has recently
been rediscovered by molecular biologists (Gimeno et al., 1992). The phenomenon had
been documented by yeast taxonomists and physiologists in the earlier half of the
century, only to be forgotten when molecular biology came to the fore, probably due to
the generalised use of laboratory adapted strains which were unable to implement this
developmental program (reviewed in Kron, 1997).
The environmental signal, which results in the dimorphic transition from yeast-form
to filamentous-form, is the limitation of specific, essential nutrients. Gimeno et al.
(1992) initially described the pathway as an adaptation to nitrogen limitation. However,
later evidence indicates that a limitation in the carbon source or the presence of an
inefficiently used carbon source like starch (Lambrechts et al., 1996a) or the presence of
fusel alcohols (Dickinson, 1996) leads to similar adaptations. Phenotypically, the
process refers to a number of morphogenetic processes, which underlie the
transformation of round or ovoid cells into thin, elongated cells (Figure 1).
Concomitantly, the budding pattern shifts from axial (most haploid strains) or bipolar
(most diploid strains) to unipolar, with all buds emerging from the same pole of the
elongated cells. These cells remain attached to each other and form hyphen-like
structures that grow beyond the perimeter of the colony (Figure 1). Since the process is
triggered by nutritional limitation, it is thought to allow yeast cells to search for
nutrients. The process is not identical for haploid and diploid strains of S. cerevisiae
(Gimeno et al., 1992; Roberts and Fink, 1994). Diploid cells adopt more readily an
elongated, pseudohyphal growth form, and diploid filaments stretch far beyond the
perimeter of the colony. Haploid filaments most frequently are limited to the area below
the colony (Dickinson, 1994; Roberts and Fink, 1994). However, signal transduction
events in both cell types seem to be largely identical. Cells growing in a filamentous
form are able to invade solid substrates like agar, a phenomenon referred to as invasive
growth. However, filamentation is not a prerequisite for invasive growth to occur, and
some strains, which are unable to develop pseudohyphae, are still able to invade the
111
F.F. BAUER AND I.S. PRETORlUS
substrate. The two phenotypes, filamentation and invasive growth, therefore make use
of the same signal transduction pathways, but can be observed independently in some
genetic backgrounds or in specific conditions. In this review, the terms pseudohyphal
differentiation or filamentation will always imply invasive growth, unless otherwise
stated.
Figure 1. Morphology of yeast-type and filament-type yeast cells and observed phenotypic
changes.
As stated above, pseudohyphal differentiation is the result of the complex interaction of

several signal transduction pathways. It requires the perfect co-ordination of essential
cellular processes, including specific modifications of the cell cycle, reorganisation of
the actin cytoskeleton (cell elongation), modification of the cell wall and a change in
budding pattern (Figure 1). The signal transduction network governing this coordination of otherwise apparently unlinked cellular processes is one of the main foci of
this review.
3. Molecular nature of signal transduction processes resulting in pseudohyphal
differentiation
Numerous genes have been, and are still being, identified for playing a role in - or for
influencing directly or indirectly - the events associated with the dimorphic transition of
S. cerevisiae cells. Several systematic genetic approaches using different phenotypic
screens have been implemented to identify such genes. These approaches include the
isolation of genes which (i) induce pseudohyphae formation when present in multiple
copies (Gimeno and Fink; 1994), (ii) lead to elongated cell morphology (Blacketer et
al., 1995) or result in the suppression of filamentous growth when mutated (Msch and
Fink, 1997), or (iii) are able to suppress such mutations (Lorenz and Heitman, 1998a).
112
Other genes have been identified through indirect approaches, for example through their
ability to induce the STA1-3 glucoamylase genes (Lambrechts et al., 1996b), which
were shown to be co-regulated with invasive and pseudohyphal growth. Finally,
numerous genes were linked to the process through general and systematic screening of
phenotypes resulting from mutations within known genes (Radcliffe et al., 1997;
Chandarlapaty and Errede, 1998; Edgington et al., 1999) or because of homologies with
known developmental regulators in other species (Gavrias et al. 1996).
Gene products participating in pseudohyphal differentiation can be divided into four
groups:
Proteins that belong to evolutionarily conserved signal transduction modules,
including plasma membrane receptors and associated heterotrimeric and small
GTP binding proteins forming part of the signal sensing apparatus, as well as
intermediate components of signalling cascades, for example MAP kinase
cascades and second messengers like cAMP (adenosine 3', 5'-cyclic
monophosphate) and their effectors.
Proteins involved in morphogenetic events.
Transcriptional regulators.
Downstream effectors of the signalling pathway.
In the following section, the role of the above-mentioned signal transduction modules
will be discussed in some detail.
3.1. SIGNAL TRANSDUCTION MODULES
3.1.1. Nutrient availability is sensed by permeases
The reduced availability of specific and essential nutrients is the environmental signal
responsible for pseudohyphal differentiation. The molecular sensors of the filamentation
signal therefore have to be able to sense the presence and concentration of those
nutrients. Nutrient sensing in yeast and other organisms has frequently been associated
with nutrient specific transporters, permeases, or homologues thereof (reviewed in
Kruckeberg et al., 1998). The suspicion that permeases could be involved in the
perception of the pseudohyphal differentiation signal was confirmed by Lorenz and
Heitman (1998b). The authors identified the ammonium permease Mep2p to be
responsible for ammonium sensing and to provide the signal resulting in pseudohyphal
differentiation (Lorenz and Heitman, 1998b). Three ammonium permeases with
significant sequence homologies, Mep 1p, Mep2p and Mep3p, have been identified in
S. cerevisiae (Marini et al., 1997). Of the three, Mep2p presents the highest affinity
(KM=1-2 M) for ammonium. The data of Lorenz and Heitman (1998b) suggest that
Mep2p is specifically required for the sensing of ammonium, and does not play any role
in the sensing of other nitrogen sources. A deletion of the MEP2 gene results in cells
unable of filamentous growth in ammonium limited medium. The mep2 strain,
however, is perfectly able to form filaments in response to limitations in other nitrogen
sources like glutamine, asparagine or proline. The deletion does not result in any growth
defects on medium containing ammonium, showing that the two additional ammonium
permeases Mep1p and Mep3p are efficient transporters, but play a less prominent role in
signalling of ammonium availability. The data suggest that yeast cells probably require
113
specific sensors for each of the nitrogen and carbon sources whose limitation results in
pseudohyphal or invasive growth. Several other permeases can be expected to play a
similar role and to specifically signal the presence or limited availability of their
substrates. However, only indirect evidence has been provided so far (Lorenz and
Heitman, 1998b).
No receptor sensing carbon source limitation, and which would be required for
pseudohyphal differentiation, has been identified, but the putative G-protein associated
receptor Gpr1p is a promising candidate (Xue et al., 1998; Yun et al., 1998).
Furthermore, two other putative glucose sensors have recently been identified by Ozcan
et al. (1998). The proteins Snf3p and Rgt2p have homologies with hexose transporters,
but apparently generate an intracellular signal without transporting glucose. As for
Gpr1p, however, no direct evidence for a relation between these sensors and
pseudohyphal growth has yet been presented.
3.1.2. Transmission via receptor associated elements
3.1.2.1. The role of Gpa2p. Gpa2p was initially identified because of strong homology
with -subunits of mammalian heterotrimeric guanine-nucleotide-binding proteins
(Nakafuku et al., 1988). These proteins are well-studied receptor-associated signal
transduction modules consisting of three subunits, , , and (reviewed in Neer, 1995).
Upon activation of the receptor, the a-subunit exchanges GDP for GTP, which results in
the dissociation of the heterotrimer. The signal is passed on by either the a-subunit or
the -subunits, which stay attached to each other.
Based on homology searches, the S. cerevisiae genome sequence revealed the
presence of only two G subunits, Gpa1p and Gpa2p. Gpa1p is the well-studied
a-subunit of the heterotrimeric G-protein associated with the pheromone receptors
Ste2p and Ste3p (reviewed in Kurjan, 1992) and has not been implicated in any other
signalling event so far. Gpa2p, on the other hand, has been implicated in a number of
events, in general related to the control of cAMP levels in the cell (Nakafuku et al.,
1988; Papasavvas et al., 1992; Lorenz and Heitman, 1997; Kbler et al., 1997;
Colombo et al., 1998). Several genetic interactions with the Ras proteins, in particular
the ability of overexpressed GPA2 to suppress the growth defects of temperature
sensitive ras mutants, and the non-viability of a strain carrying deletions of both RAS2
and GPA2 have also been reported (Nakafuku et al., 1988; Papasavvas et al., 1992).
Interestingly, whereas the and subunits of Gpa1p had been easily isolated and have
been well studied, no such subunits associated with Gpa2p have thus so been identified.
GPA2 was more recently shown to play an essential role during pseudohyphal
differentiation in diploids (Lorenz and Heitman, 1997; Kbler et al., 1997). A
gpa2/gpa2 deletion strain is unable - or presents a very reduced ability - to grow in the
filamentous form, whereas a strain carrying a hyperactivated allele (GPA2Gly132Val)
shows enhanced pseudohyphal differentiation even on rich media. A dominant negative
allele (GPA2Gly299Ala) was shown to inhibit pseudohyphal growth. Unpublished data of
our laboratory indicate that Gpa2p affects invasive growth and filamentation similarly
in haploid strains.
Since heterotrimeric G-proteins are receptor-coupled signal transduction modules,
our current knowledge strongly favours the hypothesis that Gpa2p associates with
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receptors like Mep2p to signal nutrient limitation. However, no direct interaction of

Gpa2p with a receptor relevant for filamentation has so far been demonstrated. Genetic
evidence nevertheless suggests that the ammonium transporter Mep2p requires Gpa2p
for signalling (Lorenz and Heitman, l998b). In addition, glucose signalling was also
shown to be dependent on Gpa2p (Colombo et al., 1998). As mentioned previously,
Gpa2p has been shown to be associated with Gpr1p, a putative G protein-coupled
receptor (Xue et al., 1998), and other data suggest that Gpr1p might be involved in the
sensing of glucose and other fermentable sugars (Yun et al., 1998). As stated above, the
relevance of this receptor for pseudohyphal differentiation remains to be established.
Lorenz and Heitman (1997) and Kbler et al. (1997) present evidence that Gpa2p
might transmit the signal through regulation of cAMP levels in the cell. Indeed, a strain
carrying deletions of both gpa2/gpa2 and pde2/pde2, the latter gene encoding the
phosphodiesterase responsible for the cleavage of cAMP, is unable to grow
filamentously, but this phenotype can be suppressed by the addition of extracellular
cAMP. The role of cAMP and the different signal transduction elements associated with
this second messenger molecule is discussed in more detail later in this review.
3.1.2.2. Ras2p regulates filamentation via two separate pathways. The first signal
transduction element to be associated with invasive and pseudohyphal growth was the
small GTP-binding protein, Ras2p. Results presented by Gimeno et al. (1992) showed
that the hyperactivated form of Ras2p, Ras2pVa119, stimulated both pseudohyphal and
invasive growth. Initial experiments suggested that this regulation occurred through the
- at this time - only recognised function of Ras2p, the control of adenylate cyclase
(Cyr1p) activity and consequently the regulation of cAMP concentration within the cell.
Results presented by Ward et al. (1995) strengthened the hypothesis of a cAMP
dependent regulation of the process. Indeed, overexpression of the PDE2 gene
(encoding, as mentioned above, a phosphodiesterase responsible for cAMP degradation)
inhibits pseudohyphal and invasive growth and suppresses the pseudohyphal growth
phenotype of the RAS2Va119 allele.
Concomitantly, Liu et al. (1993) presented evidence for the involvement of elements
of the mating specific MAP kinase cascade, in particular the MEKKK Ste20p, MEKK
Ste11p and MEK Ste7p, and the transcriptional activator Ste12p (Liu et al., 1993;
Roberts and Fink, 1994) in invasive and pseudohyphal growth regulation. The authors
showed that other elements of the pheromone signal transduction cascade, like the
pheromone receptors Ste2p and Ste3p, or the receptor-associated heterotrimeric Gprotein did not have any effect on pseudohyphal differentiation. Initially, no link
between Ras2p and the MAPK cascade was established. Msch et al. (1996) later
presented evidence that the regulation by the MAPK cascade was dependent on Ras2p.
The transmission of the signal via Ras2p and the MAPK cascade was shown to be
cAMP independent, but dependent on the rho-like small G-protein, Cdc42p. The
dominant negative CDC42 allele, CDC42Ala118, is indeed able to block the transmission
of the constitutive filamentation signal created by the hyperactivated RAS2Va119 allele.
Both Ras2p and Cdc42p were shown to act upstream of the MAP kinase cascade
through epistasis analysis. In this case, modification of the activity of cAMP dependent
kinase (PkA) through the use of bcy1 mutants or the overexpression of Tpk1p did not
seem to influence filamentous growth (Msch et al., 1996).
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Ras2p therefore seems to regulate pseudohyphal differentiation both via cAMP

dependent and cAMP independent pathways, as represented in Figure 2 (Msch and
Fink, 1997; Lorenz and Heitman, 1997; Gagiano et al. 1999).
Figure 2. Ras2p dependent pathways leading to pseudohyphal diferentiation.
3.1.3. Intermediate signal transduction modules

Ras2p and Gpa2p appear as two independent signal-transducing elements, both situated
more or less directly downstream of the signal receptor. Some of the data presented in
the previous paragraphs clearly suggest that the signal emanating from these two
elements is further transmitted via both separated and shared signal transduction
modules.
3.1.3.1. CAMP and CAMP-dependent kinase. cAMP is a key signalling molecule in
most, if not all, currently existing organisms. Dependent on organism and cell type, this
second messenger molecule regulates a variety of targets by controlling the activity of
CAMP-dependent protein kinases (cAPK). In S. cerevisiae, cAMP is produced from
ATP by the CYR1 gene encoded adenylyl cyclase, whose activity in turn is controlled
by the two Ras proteins. Whereas it appears clear that basal levels of cAMP are required
for normal cellular growth and cell cycle control, a specific role for cAMP during signal
transduction events in S. cerevisiae has been difficult to pinpoint. Indeed, the only
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specific and significant CAMP-dependent response observed so far is linked to the

sensing of glucose in some conditions (reviewed in Thevelein, 1992).
The involvement of cAMP in the regulation of pseudohyphal differentiation was
suspected when Ras2p was shown to regulate filamentation by Gimeno et al. (1992).
Since then, a number of data have been taken as evidence that cAMP is indeed one of
the major signal transduction components responsible for the establishment of invasive
and pseudohyphal growth patterns, and that both Gpa2p and Ras2p are involved in the
regulation of intracellular cAMP concentrations (Ward et al., 1995; Kebler et al.,
1997; Lorenz and Heitman, 1997, 1998a,b; Robertson and Fink, 1998).
Extracellular cAMP can enhance pseudohyphal differentiation in wild type strains,
and is able to do so at low concentrations in a diploid pde2/pde2 mutant strain. In
addition, extracellular cAMP can suppress the pseudohyphal growth defect of a
gpa2/gpa2 strain in a pde2 background (Lorenz and Heitman, 1997). In the same
manner, the pseudohyphal growth defect of a mep2/mep2 strain can be overcome by
extracellular cAMP (Lorenz and Heitman, 1998b).
As in other organisms, the main, and so far only downstream element directly
regulated by cAMP in S. cerevisiae is the cAMP dependent protein kinase (cAPK),
encoded by three genes, TPK1, TPK2 and TPK3 (Toda et al., 1987) These kinase
isoforms have long been considered to possess largely overlapping functions, since
deletion of any combination of two of the three genes encoding Tpks does not result in
growth defects or reduction in cellular viability (Cannon and Tatchell, 1987; Toda et al.,
1987). The role of Tpks in pseudohyphal development initially seemed doubtful, since
the deletion of BCY1, which encodes the regulatory subunit of cAPK (Matsumoto et al.,
1982), did not show any clear effect on pseudohyphal differentiation (Msch et al.,
1996).
More recently, Robertson and Fink (1 998) were able to show that the three cAMP
dependent protein kinases play different and specific roles in the control of
pseudohyphal differentiation. Diploid tpk2/tpk2 mutant strains fail to grow
filamentously on nitrogen limited media, whereas tpk3/tpk3 mutants or strains that
overexpress TPK2 show enhanced pseudohyphal development in the same conditions.
In conclusion, the authors suggest that Tpk2p is essential for pseudohyphal
development, whereas Tpk1p apparently has no and Tpk3p an inhibiting effect on the
process. Further data, based on 2-hybrid analysis, indicate that Tpk2p, but not Tpk1p or
Tpk3p, associates specifically with the transcription factor Sf11p (discussed in more
detail further on), deletion of which results in enhanced pseudohyphal growth.
It is still unclear how the cAMP signal can be specifically transmitted to a specific
isoform of the cAMP dependent kinase, or how different isoforms can carry out
divergent signalling functions, The pronounced sequence differences in the amino
terminal extension of the three Tpks might direct each kinase to a specific set of target
proteins, as the direct and specific association of Tpk2p with Sf11p suggests (Robertson
and Fink, 1998).
3.1.3.2. The MAP kinase cascade: multidimensional relays station. Whereas the
involvement of cAMP in a nutrient-sensing signal transduction pathway might not seem
surprising, this can not be said about the contribution of several elements of the
fheromone responsive MAP kinase cascade to pseudohyphal differentiation and
invasive growth. (Liu et al., 1993; Roberts and Fink, 1994; Msch et al., 1996). This
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cascade is represented in Figure 3. A MAP kinase signalling module consists of several

protein kinases, which, upon activation, transmit the signal through a series of
phosphorylation events, resulting in the activation of the MAP kinase which directly
regulates various target proteins through phosphorylation (reviewed in Herskowitz,
1995; Levin and Errede, 1995; Madden and Snyder, 1998). The module is
evolutionarily extremely well conserved throughout the eukaryotic kingdom (reviewed
in Widmann et al., 1999).
Figure 3 Comparison between the pheromone response and the pseudohyphal growth
pathways Common elements of the two pathways are in bold italics, whereas elements that
impart MAPK specificity are highlighted by a grey background
The MEKKK Ste20p, MEKK Ste1 1p and MEK Ste7p were shown to be required for
efficient invasive growth, as was the transcription factor Ste12p (Liu et al., 1993). Other
genes which when mutated result in sterile phenotypes like STE50 were also shown to
be required for the regulation of filamentous growth (Ramezani et al., 1998). Deletion
of any of the above-mentioned genes results indeed in strongly reduced invasive and
filamentous growth, whereas hyperactivated alleles (where available) present enhanced
phenotypes for both characteristics.
Epistasis analysis showed that the kinase cascade receives the filamentation signal
via Ras2p and Cdc42p in a cAMP independent manner (Mschet al., 1996).
Several hypotheses, recently reviewed by Madhani and Fink (1998), could explain
how specific signals can result in specific responses while using, at least partially, the
same elements for transmission (Figure 3). Firstly, the MEK Ste7p activates two
different MAP kinases, Fus3p and Kss1p. Cook et al. (1996, 1997) and Madhani et al.
118
(1997) showed that Kss1p is specifically required for pseudohyphal growth, whereas
Fus3p is specific for mating. Both are inhibiting the activity of their specific targets
when inactive (non-phosphorylated), which explains why initial genetic data based on
the study of deletion mutants investigating the pheromone signal transduction suggested
a partially redundant activity for the two kinases (reviewed in Herskowitz, 1995). A
second mechanism to achieve MAP kinase cascade specificity relies on the
combinatorial control of the transcription factor Stel 2p, which has to associate with
Tec1p to activate hyphae specific genes (Gavrias et al., 1996; Madhani and Fink, 1997).
A third potential mechanism to ensure MAP kinase specificity could reside in higher
order association of the different kinases, which would keep particular signal
transduction pathways physically separated and make cross activation impossible. This
possibility is clearly demonstrated by the specific recruitment of the MEKK Stel 1p to
both the mating pheromone pathway-specific scaffold protein Ste5p (Elion, 1995) and
to the HOG MAPK pathway-specific scaffold Pbs2p (Posas and Saito, 1997). Indeed,
besides being required for both the pheromone and pseudohyphal growth pathway,
Stel1p is necessary for the osmotic activation by the osmosensor Sho1p of the HOG
MAP kinase pathway (Posas and Saito, 1997). However, no pseudohyphal responsespecific scaffold protein has yet been identified. MAP kinase associated proteins might
play an additional role in ensuring signalling specificity. The filamentation specific
MAPK Kss1p has been shown to associate with two proteins, Dig1p and Dig2p (Cook
et al., 1997; Tedford et al., 1997). The genes were identified through a 2-hybrid
interaction screen and Dig1p co-immunoprecipitates with Kss1p in the nucleus. A
dig1/dig2 double mutant presents a constitutively invasive phenotype, but neither the
double nor the single mutants present any defect with regard to the pheromone response.
Both proteins also associate with Ste12p, the transcriptional activator regulated by both
MAP kinases Fus3p and Kss1p. The data suggest that Dig2p acts as a negative regulator
of invasive growth, and is directly regulated by Kss 1 p, which, as mentioned above, acts
as a strong repressor of invasive growth in its non-phosphorylated state (Cook et al.,
1997; Madhani et al., 1997). In addition, Bardwell et al. (1998b) show that Kss1p in its
inactive form can directly bind to Ste12p, offering a further possibility for direct
regulation of the protein.
The different elements that might contribute to MAP kinase specificity are
summarised in Figure 3.
The specific contribution of the MAPK cascade to the modifications observed
during pseudohyphal differentiation is unclear. However, both pheromone response and
low nutrient response result in some cellular modifications of a similar nature, in
particular with regard to polarised growth (see following paragraph) and changes in
cell-adhesion properties. Some of these changes might be depending on the common
elements in the two cascades.
3.1.3.3. Morphogenetic factors associate with the MEKKK Ste20p. The major
morphogenetic event observed during filamentation is the strong elongation of the
filament-forming cells. Cell elongation requires polarised growth, which in turn is
dependent on the organisation of the actin cytoskeleton (reviewed in Madden and
Snyder, 1998). Numerous results have been taken as evidence that the morphogenetic
reorganisation of the actin cytoskeleton during pseudohyphal growth is dependent on
the MEKKK Ste20p and its association with the rho-like GTPase, Cdc42p. Results
119
show that both proteins have a more general role in the organisation of polarised growth
during the cell cycle (Evangelista et al., 1997; Leberer et al., 1992, 1997; Leeuw et al.,
1995, 1998; Msch et al., 1996). Thermosensitive mutations in CDC42 result in large,
round, non-budding cells, indicating the requirement for Cdc42p for polarised growth
during bud emergence. Ste20p, on the other hand, directly associates with the actin
cytoskeleton via an adapter protein, Bem1p (Leeuw et al., 1995; reviewed in Madden
and Snyder, 1997). Ste20p also directly binds to Cdc42p, and deletion or mutation of
the Cdc42p-binding site blocks pseudohyphal differentiation (Peter et al., 1996; Leberer
et al., 1997). Further evidence for this more general role of Ste20p is provided by the
fact that the hyperactivated allele of STE11, STE1 1-4, only partially bypasses
filamentation defects of ste20 mutants, whereas mutations in STE11, STE7 or STE12 do
not block cell-elongation in a RAS2Va119 strain (Msch et al., 1996). The morphogenetic
function of Ste20p and Cdc42p could explain at least in part the common requirement
of the MAP kinase cascade for both mating and pseudohyphal differentiation, since in
both cases a strong polarisation of the growth pattern is required, either to produce the
cellular extension called shmoo during mating or to produce elongated cells during
pseudohyphal differentiation. A second phenotypic change associated with both
pathway is the modification of cell-adhesion properties.
Other proteins that probably associate with Ste20p are the S. cerevisiae 14-3-3
proteins, Bmh1p and Bmh2p (Roberts et al., 1997). The authors provide evidence that
these proteins are required specifically for the transmission of the Ras2p dependent
signal to the MAP kinase cascade during pseudohyphal differentiation. However, the
significance of this finding and the exact role of these proteins during signal
transduction are as yet unclear.
3.1.3.4. Cell cycle dependent regulation of filamentous growth. Most responses to
extracellular signals and the associated physiological adaptations can either only be
carried out at specific phases of the cell cycle or result in a direct change in cell cycle
control. All cellular differentiation processes could be expected to influence or be
influenced by the activity of cyclin-dependent kinases (CDK). The central regulatory
CDK in S. cerevisiae is Cdc28p, whose activity determines the passage of most cell
division checkpoints (reviewed in Mendenhall and Hodge, 1998). A second CDK,
Pho85p, has some overlapping functions, and associates with a different set of cyclins
(reviewed in Andrews and Measday, 1998). Several observations indicate that CDKs
are important factors for the regulation and the co-ordination of morphogenesis and
polarised growth (reviewed in Madden and Snyder, 1998). Association of Cdc28 with
G1 phase cyclins Cln1 and Cln2 in particular is required for polarised growth during
bud formation and growth, Overexpression of these two cyclins leads to hyperpolarised
growth and buds with an elongated morphology. A similar morphology can be achieved
by delaying or eliminating the expression of the G2 phase specific cyclins Clb1 and
Clb2.
First evidence for a more direct involvement of Cdc28-CDK in pseudohyphal
differentiation emerged with the finding that cells growing in filaments displayed a
modified cell cycle when compared to yeast-type cells. Kron et al. (1994) reported that
filamentous daughter cells start budding very soon after cytokinesis. No size control,
which normally applies during GI, seems to be implemented, and daughter and mother
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cell bud simultaneously. The G1 delay before Start apparently is eliminated.

Furthermore, pseudohyphal cells have a significantly lengthened G2 phase, during
which cell elongation occurs.
Further indirect evidence was provided by the observation that mutations in some
genes resulting in an increased expression of Cln cyclins showed hyperactivated
invasive and pseudohyphal growth (Oehlen and Cross, 1998a). An example for such a
gene is WH12, a deletion of which results in overexpression of cyclins Cln1p and Cln2p
and a strong pseudohyphal development (Radcliffe et al., 1997).
In a more recent paper, Edgington et al. (1999) suggest a direct link between CDK
alterations and specific pseudohyphal differentiation phenotypes. A strain carrying a
specific mutation in CDC28, cdc28-127, which results in the replacement of cysteine
residue 127 of the kinase by tyrosine, displays most filamentous-form growth
characteristics constitutively. These characteristics include elongated cell shape, bud
site selection and cell cycle modifications leading to simultaneous bud emergence for
mother and daughter cell.
Genes involved in the regulation of Cdc28p activity include ELMI, encoding a
serineithreonine protein kinase which activates another protein kinase, Hs11p. ELM1
was first identified through screening of mutagenised yeast for mutations that would
result in constitutive cell elongation and other filamentation specific characteristics
(Blacketer et al., 1993, 1994). Deletion of ELM1 results in constitutive filamentation. A
second kinase-encoding gene, which results in a similar phenotype when mutated, is
HSL1. Dominant mutations within HSL1 can suppress the constitutive filamentation
phenotype of elm1 mutants, placing Hs11p downstream of Elm1p. Mutant phenotypes of
both elm1 and hsl1 strains can, on the other hand, be suppressed by inactivation of
Swe1p, a third protein kinase situated in the same pathway. The deletion of ELM1 or
HSP1 results in hyperactive Swe1p, which triggers the appearance of some of the
filamentous growth specific phenotypes. Mutations in ELMI, HSL1, or SWE1 have no
influence on the phenotype of the cdc18-127 allele, suggesting that they act upstream of
the kinase. The data are consistent with the existence of a protein kinase cascade that
mediates filamentation by modulating Cdc28p activity. Surprisingly, however, the
effects of the protein kinase cascade on filamentation seem to be limited to haploid
cells. The pathway suggested by the authors is presented in Figure 4.
It is not yet clear how this cell cycle regulating pathway interacts with the various other
signal transduction pathways, which seem more specifically responsible for
pseudohyphal differentiation. Based on a limited amount of genetic data, Edgington et
al. (1999) suggest that the proteins could be acting downstream of the MAP kinase
pathway (Figure 4).
CDK-dependent regulation has been shown to affect other parts of the signal
transduction pathway as well. Oehlen et al. (1998b) show that Cln2p-Cdc28p kinase
represses the pheromone signalling pathway at the level of Ste20p. Since Cln-cyclins
are required for pseudohyphal differentiation as well, a direct influence of the Cln2Cdc28 kinase on Ste20p during this event is equally likely. Similar data were obtained
for Ste11p (Wassmann and Ammerer, 1997).
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Figure 4. Cell-cycle dependent regulation of pseudohyphal differentiation as suggested by

Edgington et al. (1999). This pathway seems specific to haploid cells.
3.2. TRANSCRIPTIONAL REGULATORS

Besides the direct modulation of the activity of pre-existing enzymes, the main
molecular response to extracellular signals resides in the activation or repression of the
transcription of specific target genes. It is therefore not surprising that a large number of
genes whose deletion or overexpression affects the pseudohyphal differentiation
phenotype encode transcriptional regulators.
There are several obvious requirements for transcription factors to be active: It has
to be localised in the nucleus, bind to DNA or associate with DNA-binding proteins,
and has to be able to - directly or indirectly - interact with the basal transcription
apparatus, the RNA polymerase holoenzyme. Each of these requirements, i.e. nuclear
localisation, DNA binding or the interaction with other proteins, can be modulated by
the extracellular signal (reviewed in Hill and Treisman, 1995). In this review, we shall
focus separately on each of the transcription factors that have been shown to contribute
to the filamentous growth phenotype. Several of these factors had previously been
identified for their role in other, sometimes related, cellular processes, underlining again
the interconnected nature of all signalling processes.
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3.2.1. Ste12p and Tec1

Combinatorial control contributes to MAP kinase specificity. Fields and Herskowitz
(1985) identified Ste12p as being required for the expression of cell mating-type
specific genes. In the following years, Ste12p was shown to bind to DNA sequences
that mediate the pheromone response, so-called pheromone response elements or PRES
(Dolan et al., 1989; Errede and Ammerer, 1989; Yuan and Fields, 1991). Additional
data showed Ste 12p to be phosphorylated in a pheromone-dependent manner (Song et
al., 1991). Ste12p was the first transcription factor that was shown to be required for
normal filamentation by S. cerevisiae (Liu et al., 1993). Deletion of STE12 results in
strains with strongly reduced ability to invade the agar or to grow filamentously,
whereas the overexpression from an inducible GAL1 promoter results in enhanced
pseudohyphal growth. Since mating and filamentation are mutually exclusive
developmental pathways, it was suggested that additional factors had to be involved to
impart signal-dependent specificity.
TEC1 was initially isolated by Laloux et al. (1990) for being involved in the
activation of Ty1-mediated gene expression. Gavrias et al. (1996) showed that the
Aspergillus nidulans transcriptional activator AbaA, a gene homologous to TEC1, was
able to induce pseudohyphal differentiation in S. cerevisiae. They consequently
demonstrated that a deletion of TEC1 results in strongly reduced ability of yeast strains
to invade agar. Initial epistasis data either indicated that Ste12p and Tec1p act in
dependent pathways or interact with one another to activate downstream functions. The
latter possibility was confirmed by Madhani and Fink (1997), who showed that Ste12p
was directed to enhancer-elements (FREs for filamentation and invasion response
elements) that were specifically induced in response to the MAP kinase-dependent
filamentation signal. The FRE consists of a Ste12p binding site, similar to the one found
in pheromone-dependent genes, closely associated with a Tec1p binding site. The
binding of Ste12p and Tec1p to this element occurs in a co-operative manner, and the
two proteins are found associated in the same, multimeric protein complex (Baur et al.,
1997; Madhani and Fink, 1997). It is therefore the combination with Tec1p that allows
Ste12p to activate filamentation specific genes. Furthermore, it has been shown that the
unphosphorylated form of Kss1p, which has a repressive effect on filamentation,
directly binds to Ste12p (Bardwell et al., 1998a), giving a further indication on the
mechanism underlying specific activation. The two proteins Dig1p and Dig2p, which
also associate with Ste12p, are required for this Kss1p dependent repression (Tedford et
al., 1997; Bardwell et al., 1998b).
3.2.2. Msn 1p and Mss11p: Central elements in the pseudohyphal growth pathway
MSN1 has first been identified as a multi-copy suppressor of snf1 mutants (MSN1)
(Estruch and Carlson, 1990). Other authors cloned the same gene as FUP1, an enhancer
of iron-limited growth of S. cerevisiae (Eide and Guarente, 1992), as PHD2, a multicopy inducer of pseudohyphal growth (Gimeno and Fink, 1994) and, in our laboratory,
as MSS10, a multi-copy suppressor of the repression exerted by STA10 on the STA1-3
glucoamylase-encoding genes, involved in starch metabolism (Lambrechts et al.,
1996b). MSN1 has been suggested to encode a transcriptional activator since multiple
copies of the gene enhance the transcription of several genes, most of which are
involved in nutrient utilisation. In addition, MSN1 has been shown to activate reporter
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gene expression if fused to the LexA DNA-binding domain (Estruch and Carlson,
1990).
MSS11, like MSN1, was identified as a suppressor of the STA10 dependent
phenotype and was shown to induce STA1-3 encoded glucoamylase expression when
present on a 2 plasmid (Webber et al., 1997). The protein displays homologies with a
number of transcriptional activators and suppressors, such as S. cerevisiae Snf5p,
Ssn6-/Cyc8p and Drosophila NTF-1, and in particular with Flo8p, a protein initially
identified for activating genes involved in flocculation (Kobayashi et al., 1996).
Both MSN1 and MSS11 strongly activate the expression of MUC1, a gene required
for invasive growth and pseudohyphal differentiation, when present in multiple copies
(Lambrechts et al., 1996a; Webber et al., 1997). Recent data by Gagiano et al. (1999)
show that Mss11p is a central factor for the establishment of pseudohyphal and invasive
growth patterns. Genetic evidence indicates that Mss11p is required for both Ras2p
dependent, MAP kinase dependent and independent, signal transduction pathways.
Genetic evidence for Msn1p would suggest that the protein acts in a pathway
downstream of Ras2p, but independent of the MAP kinase cascade. Msn1p requires the
presence of Msn11p in order to induce filamentation, whereas Mss11p is able to induce
the pathway in the absence of Msn1p. Mss11p therefore seems to be situated
downstream of Msn1p (Gagiano et al., 1999).
3.2.3. Sf11p, Sok2p and Flo8p: Factors depending on the CAMP dependent kinase
Three transcriptional regulators apparently controlled by cAPK and regulating
filamentation have been identified, i.e. Sf11p, Sok2p and Flo8p.
SFL1 had initially been cloned as a suppressor of flocculation (Fujita et al., 1989)
since disruption of the gene leads to strong, constitutive flocculation in most strains.
Robertson and Fink (1998) showed that deletion of SFL1 also results in enhanced
pseudohyphal and invasive growth. Sf11p was shown to associate specifically with
Tpk2p, the cAMP dependent protein kinase that specifically activates pseudohyphal
growth. Interestingly, Sf11p has also been shown to interact and associate with the
Srb/mediator proteins, which are part of the RNA polymerase holoenzyme and are
thought to be responsible for transcriptional repression (Song and Carlson, 1998).
Tpk2p therefore has to inactivate Sf11p, which in turn would relieve the repressive
effects of the Srb proteins on the RNA polymerase holoenzyme and allow transcription
of flocculation and pseudohyphal growth specific genes. This mechanism would
provide the first evidence for a direct interaction of the cAMP dependent kinase with
factors associated with the RNA polymerase holoenzyme.
A second putative transcriptional repressor that regulates filamentation is encoded
by the SOK2 gene, which was cloned by Ward and Garret (1 994). The gene was isolated
as a dosage-dependent suppressor of conditional growth defects of
temperature-sensitive cAPK mutants. Ward et al. (1995) presented evidence for the
involvement of Sok2p in cAMP dependent regulation of filamentous growth by Sok2p.
Diploid sok2/sok2 mutants show enhanced pseudohyphal growth, indicating a
repressive activity of Sok2p. Since Sok2p presents important homologies with the
helix-loop-helix DNA-binding motive of Phd1p and other fungal regulators of
morphogenetic processes (Stoldt et al., 1997), it most probably directly represses the
expression of filamentation related genes. Unlike Sf11 p, which directly associates with
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Tpk2p, no such association has yet been found for Sok2p, and its direct regulation by
cAPK has yet to be demonstrated. In addition, the sok2 phenotype presents defects in
the regulation of genes involved in a number of other cAMP dependent metabolic
regulations, including glycogen accumulation (Ward et al., 1995). It therefore behaves
like a general regulator of cAMP dependent cellular responses, and is not specific for
the filamentation response.
A third transcriptional regulator identified as acting downstream of the cAMP signal
and which induces filamentation is Flo8p. Kobayashi et al. (1996) cloned FLO8
because of its ability to induce flocculation. The gene was later shown to be required for
pseudohyphal growth (Liu et al., 1996). Interestingly, the authors presented evidence
that a mutation within this gene was responsible for the inability of most laboratory
strains of S. cerevisiae to undergo the dimorphic switch from yeast-type to
filament-type cells. Through genetic evidence, Rupp et al. (1999) showed that Flo8p
positively activated pseudohyphal differentiation and that this activation was cAMP
dependent. The authors showed that Flo8p activates MUC1 by directly acting on several
sequences within the promoter of this gene.
3.2.4. Other factors
3.2.4.1. Ash1p. Ash1p was first identified for its role in mating type switching, where it
is responsible for the daughter cell specific repression of the HO endonuclease. ASH1
mRNA is localised asymmetrically, close to the tip of large budded cells that have
undergone anaphase (Long et al., 1997; Takizawa et al., 1997). This polarised
localisation explains the asymmetric distribution of Ash1p between mother and
daughter cell and its daughter cell specific effect on mating type switching. Ash1p
contains a zinc-finger-like domain, and binds therefore probably directly to DNA to
regulate gene expression.
Chandarlapaty and Errede (1998) presented data suggesting that Ash1p was required
for pseudohyphal growth. Cells lacking the ASH1 gene did not form filaments, whereas
cells with multiple copies of the gene showed increased filamentation. Similar
observations were made with regard to invasive growth in haploids. Genetic evidence
places Ash1p in a pathway independent of the MAP kinase cascade but downstream of
Ras2p, similar to what was observed for Msn1p (Gagiano et al., 1999).
3.2.4.2. Phd1p. This gene was identified through the screening of a genomic library for
genes that, when present on a multiple copy plasmid, would enhance pseudohyphal and
invasive growth (Gimeno and Fink, 1994). Phd1p showed significant homology with
transcriptional regulators. However, deletion of PHD1 does not affect the ability of
S. cerevisiae to form pseudohyphae or to grow invasively, and its role in pseudohyphal
differentiation is unclear.
3.3, EFFECTOR PROTEINS
No gene has been unequivocally shown to be directly and specifically regulated by
filamentation signal transduction pathways alone. This might seem surprising,
considering the large number of transcriptional regulators, which have been shown to be
125
F.F. BAUER AND I.S. PRETORlUS
required for these pathways, and which have been described in the previous section.
However, these regulators have mostly been cloned for specific roles in processes that
only have an indirect or not yet clearly understood connection with filamentation. For
example, from the above it is clear that a phenomenon like flocculation in liquid media
is closely linked to the filamentation response, and some factors like Flo8p are required
for both. In addition, some obvious phenotypic links between both cellular responses
exist. Both processes for example require modified cell adhesion properties, and both
seem to occur in media with limited amounts of nutrients. However, differences and
similarities between the two events have not yet been fully investigated.
The same is true for signal-dependent processes of polarised growth during both
mating and filamentation. The shared elements in both pathways might at least in part
be involved in aspects of cellular polarisation, but it is as yet not understood how signal
dependent specificity is imparted.
The data presented in this review could suggest that it is the combination of a
number of different factors in a specific context that leads to the implementation of the
filamentation response. If this is the case, specific effector genes and proteins of the
pseudohyphal response pathway should only respond to a specific, finely balanced
combination of all the pathways and factors described above. The gene coming closest
to the above expectation is MUC1/FLO11.
3.3.1. MUC1, a gene encoding a mucin-like protein subjected to complex
transcriptional regulation
MUC1 was first identified by Lambrechts et al. (1996a) as being required for
pseudohyphal differentiation and invasive growth. The gene encodes a mammalian
membrane-bound mucin homologue, with strong structural similarities to dominant
flocculation proteins like Flo1p. The authors showed that deletion of MUC1 results in
strains unable to invade agar or to form filaments, whereas overexpression results in
stronger invasion and filamentation phenotypes. These data were later confirmed by Lo
and Dranginis (1998), who had previously identified the same gene, named FLO11, as
being involved in flocculation (Lo and Dranginis, 1996). The structure of Muc1p
suggests that it is a GPI-anchored cell wall protein, whose N-terminal section probably
reaches outside of the cell wall (Lambrechts et al., 1996a; Lo and Dranginis, 1998).
Particular attention has been paid to the large promoter of MUC1, the largest
promoter identified in S. cerevisiae. Lambrechts et al. (1996a) initially identified MUC1
through the strong sequence homology of this promoter area with the promoters of the
glucoamylase-encoding STA1, 2 and 3 genes. The promoter has been shown to be
responsive to a number of the transcriptional regulators described above, including
Msn1p (Lambrechts et al. 1996a,b), Ste12p/Tec1p (Lo and Dranginis, 1998; Rupp et
al., 1999), Mss11p (Gagiano et al., 1999), and Flo8p (Rupp et al., 1999). MUC1
therefore is regulated by both cAMP dependent and MAPK-dependent pathways, and
could be the first filamentation-specific target gene.
The role of Muc1p during pseudohyphal differentiation has not yet been
unequivocally established. However, based on the structure of the protein and on its cell
wall bound localisation, it is highly probable that it is involved in the regulation of
cell-cell attachment. An alternative hypothesis is that MUC1 would be required for
126
attachment of the cell to a substrate, a step that is thought to be required for efficient
invasion. Both hypotheses are not mutually exclusive.
3.3.2. Starch degrading enzymes: a direct metabolic link
The promoters of the S. cerevisiae STA1-3 genes, encoding starch degrading
glucoamylases, are strongly homologous to the MUC1 promoter (Pretorius et al., 1986;
Lambrechts et al., 1995, 1996a; reviewed in Vivier et al., 1997), and homology extends
over the entire promoter area of more than 3 kb (Gagiano et al., 1999). Experimental
data suggest that the genes are indeed coregulated to a large degree, since the STA genes
were shown to respond to the factors controlling pseudohyphal differentiation, including
elements of the MAP kinase cascade (Gagiano et al., 1999).
The coregulation of starch degradation and pseudohyphal differentiation offers some
interesting evolutionary insights. The STA gene family seems to be a relatively recent
evolutionary acquisition of S. cerevisiae (Lambrechts et al., 1995). Arguments in favour
of this hypothesis are the subtelomeric localisation of the genes, the very limited
sequence divergence between the three STA loci, and the fact that they are only found in
some strains of S. cerevisiae. MUC1, on the other hand, is found in all S. cerevisiae
strains investigated (Carstens et al., 1998), and has a chromosomal position far removed
from the telomers. The STA genes are therefore probably the product of a recombination
combining the promoter and secretion signal sequence of MUC1 with the ORF of the
internal glucoamylase-encoding SGA1 gene. The evolutionary selection of a
filamentation-dependent promoter might offer a real selective advantage to the yeast,
since it could result in an enhanced ability of S. cerevisiae to invade nutrient-rich
compartments in nature. Glucoamylases will be specifically secreted at the growing tip
of the elongated cell during filamentous growth, which could allow the digestion of
polysaccharide-containing barriers (cell walls of plant cells) and consequent penetration
of the filament into new compartments.
4. Scientific and industrial relevance
S. cerevisiae probably is the scientifically best understood of all organisms. The
functional mapping of the genome and of the entire proteom of this unicellular fungus
will be finalised in the not so distant future, including a physical map of protein-protein
interaction. Whether the complete molecular map will provide us with new insights into
the secrets of life itself, a view some will dismiss as reductionist, or whether the
additional knowledge will provide us with "just" a more in-depth understanding of the
molecular machinery of living organisms, remains to be seen.
Already, as pointed out in this review, several new concepts have largely been based
on data obtained through the study of S. cerevisiae. One of these concepts is the
modular nature of signal transduction pathways. A summarised representation of the
different modules and factors involved in pseudohyphal differentiation is given in
Figure 5. This figure illustrates that the response to the limited availability of essential
nutrients is the result of a finely balanced combination of inputs from a number of
independent signalling modules. An outstanding feature of the data presented in this
figure is that they combine three of the major signalling systems that, previously, had
127
been investigated for separate and specific purposes, namely (i) the cell cycle signal
emanating from CDKs, (ii) the nutrient-linked cAMP signal and (iii) the MAPK signal
which might be required for the cell-cell attachment and cell growth-polarisation
processes. This combination of three major pathways again demonstrates that coordination of all cellular compartments and of most molecular processes underlies
cellular differentiation processes.
Figure 5. Summarised signal transduction network of the pseudohyphal growth pathway.
A different perspective can be taken by simply considering the initial phenotypes, which
were used to identify most of the genes mentioned in this review. Some of the genes
were initially identified for their role in the pheromone response, other for phenotypes
linked to flocculation, abnormal cell cycle, abnormal stationary phase entry, starch
degradation, ammonium transport, etc., again demonstrating the interconnected nature
of apparently unlinked events.
How does this fundamental understanding of complex processes impact on the
potential use of yeast in industrial processes? Traditionally, S. cerevisiae has been used
for several types of food and beverage fermentations and for the production of alcohol.
For these traditional uses, industrial yeast has been optimised through centuries of
mostly unaware selection by winemakers, brewers, bakers and other users. This
unconscious optimisation process has lead to a number of S. cerevisiae strains
specifically adapted for specific tasks, be it baking, brewing , winemaking or any other
industrial use. However, from an industrial perspective, every single aspect of yeast
physiology could still be optimised. Fermentation efficiency, alcohol resistance, general
stress resistance, production of specific metabolites without production of additional,
unwanted substances - there probably are as many potential targets for strain
improvement as there are industrial users. It is in this field of strain improvement that
128
recombinant DNA technology and metabolic engineering can provide its most
important contribution.
How can research of the type described in this review contribute to achieve such
strain improvement targets? The answer becomes clear when looking at the results
obtained with recombinant and metabolically engineered strains. These strains can
frequently meet a specific target, be it a higher production of a metabolite (e.g. glycerol)
or of extracellular proteins and enzymes. However, these strains have just as frequently
failed to make an industrial impact. One reason for this failure can be found in the
review above, and resides in the interconnection of all metabolic and signalling
pathways. This interdependence makes it extremely difficult to achieve specifically
improved yeast without modifying several other parameters. These secondary
modifications frequently result in undesirable by-products or have a negative influence
on general yeast physiology. It is therefore essential that we better understand the
underlying network of metabolic and signalling connections, which would allow us to
design mechanisms to avoid metabolic and physiological problems. Such integrated
approaches, which will take into account not only the specific parameter to be modified,
but also all potential consequences on associated or linked pathways, should result in
modified yeast with much higher industrial potential.
Besides this general argument for the scientific and industrial usefulness of the
research outlined in this review, there are several potential industrial benefits directly
associated with the study of pseudohyphal development. Some wanted -or unwantedproperties of industrial yeast strains are indeed directly linked to this pathway. The
aspect retaining most attention is flocculation, which refers to the clumping of cells in
liquid media and to their consequent sedimentation (reviewed in Stratford, 1992).
Flocculation is due to the modification of cell-cell adhesion properties, and is of
importance in several industrial processes, for example during brewing or the
production of sparkling wine (reviewed in Hammond, 1995). The signal transduction
pathways leading to flocculation is similar, if not identical, to the filamentation
pathway, as can be seen by the number of genes shared by both (reviewed in Kron,
1997). Understanding filamentation should therefore allow us to design yeast with
specific flocculation abilities. Other direct applications might reside in the development
of new top-fermenting brewing and sherry yeast. Indeed, the ability of flor yeast to float
on the surface of the substrate (top-ferment) is thought to be due to a modified
implementation of the signal transduction pathways described in this review.
Acknowledgements
We thank the National Research Foundation (NRF) and the South African wine industry
(Winetech) for funding of our research.
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133
MICROBIAL PRODUCTION OF THE BIODEGRADABLE POLYESTER

POLY-3-HYDROXYBUTYRATE (PHB) FROM AZOTOBACTER
CHROOCOCCUM 6B: RELATION BETWEEN PHB MOLECULAR WEIGHT,
THERMAL STABILITY AND TENSILE STRENGTH
QUAGLIANO JAVIER C. AND MIYAZAKI SILVIA S
Facultad de Agronoma, Universidad de Buenos Aires. Av. San Martin
4453 (141 7) Bs.As. Argentina.E-mail:quanliano@,ifeva.edu.ar
Abstract
PHB with a high molecular weight (Mw) was obtained from fermentor cultures of
Azotobacter chroococcum 6B. Mw was significantly affected by aeration rate, being
reduced from 2900 to 110 kDa at 0.25 and 2.5 vvm, respectively. Temperature at which
maximal weight loss was obtained (decomposition temperature) markedly depended on
reached a
Mw , obtaining a 40C span between 100 and 1400 kDa. Tensile strength
value of 215 MPa at a Mw of 1240 kDa, being reduced to nearly 40 MPa at the lower
tested Mw of 445 kDa.
1.1 MICROORGANISM AND CULTURE MEDIA
Azotobacter chroococcum 6B was isolated from rhizospheric soil samples of the
Agronomy Faculty Campus, Buenos Aires, Argentina. Modified productive Burk's
medium (Bm) was utilised, with glucose as carbon source (g l-1): glucose 10,
MgSO4.7H2O 0.4, FeSO4.7H2O 0.012, Na2MoO4.12H2O 0.01, K2HPO4 2, NaCl 0.4
and CaCO3 0.25. (NH4)2SO4 was added at a concentration of 0.1 g l-1 and pH was
adjusted to pH 7.0 with HCl 1N. Stock cultures were maintained at 4C by periodical
transfer on modified productive Burk's medium agar slants.
1.2 FERMENTOR EXPERIMENTS
Batch fermentations were conducted with a 1% (v/v) inoculum of a 36 hours culture in
the same medium and the working volume was 4 litres (Bioflo I, New Brunswick Sci.
Co, USA; Gallemkamp Sanyo, England), both fermentors equipped with two-six blade
135
QUAGLIANO JAVIER C. AND MIYAZAKI SILVIA S.
impellers and baffles. Temperature was controlled at 30C and initial pH was adjusted
to 7.0. The air flow rate was set between 0.25 and 2.5 vvm, Initial C/N ratio (molar
basis) was set between 7.3 and 138 (ammonium sulphate limitation and glucose excess)
and agitation speed was 200 rpm. Batch experiments were finished at 72 h. When
molasses Bm medium was utilised, glucose was replaced by sugar cane molasses (5%
w/v) (Tucumn, Argentina) as carbon source.
1.3 EXTRACTION AND PURIFICATION PROCEDURE
Lyophilised cells were extracted with chloroform during 24 hours in a Soxhlet
apparatus. Chloroform extract (1 50-250 ml) was concentrated by simple distillation to a
final volume of 25 ml. The concentrated PHB was reprecipitated in ethanol cooled in an
ice bath. Reprecipitated biopolymer was filtered, washed with acetone and centrifuged
(1 5 min, 5000 rpm). Purified PHB was dried at 60C until it reached constant weight.
1.4 ANALYTICAL METHODS
Cell growth was monitored by measuring the optical density at 610 nm. Cell dry weight
(cdw) was measured by weighing lyophilised cells harvested from 3 ml culture broth.
Residual glucose in the broth was determined by the enzymatic method using glucose
oxidase/peroxidase (Wiener Lab., Rosario, Argentina). PHB content and its composition
were determined by gas chromatography using 3HB standards (Braunegg et al, 1978).
Mw was measured viscosimetrically and by gel permeation chromatography (GPC)
using five serial Styragel columns of 102, 103, 104, 105 and 106A. Flow rate of
chloroform was 0.5 ml/min. A refractive index detector was used.. Differential scanning
calorimetry (DSC) studies were performed using a Mettler TA9000 calorimeter between
-60 and 250C at 10C/min. Thermogravimetric (TG) measurements were made with a
Mettler TA9900 apparatus from 25 to 800C at 10C/min. Tensile strength was
measured on PHB probes (obtained according to ASTM 1708-84) utilising an Instron
model 1122 tensiometer from slopes of strain-stress curves
2. Results and discussion
2.1 EFFECT OF MW ON PHB THERMAL STABILITY
PHB samples with different Mw obtained at different fermentative conditions were
analysed by measuring the temperature at which maximal weight loss is obtained when
increasing sample temperature in thermogravimetric studies, called decomposition
temperature, Td . This parameter is a measure of the thermal stability of PHB. Other
physical parameters, such as melting temperature (Tm), glass transition temperature (Tg)
and % crystallinity were also analysed. Table 1 summarises the data obtained: it was
shown that an increase in Mw leads to an augmented thermal stability, as samples with a
Mw around 1300-1400 kDa lost 99% of their weight at a temperature 15% higher than
the sample with the lowest Mw experimented (150 kDa). These results showed that
thermal stability is significantly affected by Mw. Tm was not significantly affected, as
expected, as it is a physical constant independent of Mw . Tg did not showed significant
136
MICROBIAL PRODUCTION OF PHB
differences among the analysed samples, confirming that PHB is a semicrystalline

polymer, while % crystallinity was in the range of 50-90%, as generally reported for
PHB (Barham et al. 1984) the differences attributed to different degrees of sample
annealing.
Table 1. Melting point (Tm), glass transition temperature (Tg), decomposition temperature
(Td) and molecular weight (Mw) of PHB samples obtained from strain 6B.
Culture conditions
Tm (C)
Tg (C)
Td (C)
(a)
%crystallin
ity
Mw. 103
(kDa
fixation, 0.5 vvm, 100 rpm
180.9
n.d.
291
59
1420
C/N 7.3, 0.5 vvm, 100 rpm
177.8
3.9
268
82
1330
C/N 29,0.5 vvm, 100 rpm
178.8
4.3
261.7
73
1170
molasses 5%, 0.5 vvm, 100
173.9
n.d.
286
54
1420
C/N 138,2 vvm, 400 rpm
173.0
n.d.
250
55
150
C/N 69,2.5 vvm, 100 rpm
178.2
5.7
n.d.
62
110
rpm
n.d.: not determined. Tg , Tm and % crystallinity obtained from DSC scans. %

crystallinity= Hm pHB (J/gC)/ HmpHB100%crystalline a: Td:temperaturewheremaximal
weight loss is obtained.
2.2 EFFECT OF AERATION RATE ON PHB MW
PHB Mw was significantly affected by aeration rate under fermentor cultivation at a C/N
ratio of 69. Figure 1 depicts the influence of aeration rate on PHB Mw: maximum Mw
(2.900 kDa) was obtained at the lowest aeration rate experimented of 0.25 vvm, while
when increasing aeration rate Mw diminished to 110 kDa at 2.5 vvm. This confirms that
PHB is accumulated under oxygen-deficient conditions, then in such conditions 3hydroxybutyrate monomers are incorporated to the growing PHB chains, consequently
increasing Mw. An increased Mw leads to an improvement of polymer performance, as
lower Mw are not desirable from the point of view of plastics applications, for example,
thermal stability is much lower with low Mw plastics. Also a product of low molecular
mass suffer a sensitive loss of volatile degradation products as temperature is increased
(Scandola et al. 1988).
137
Figure 1. PHB molecular weight (Mw) and PHB content (XPHB) under different aeration
rates under fermentor culture (4 litres) with Burks modified medium (Bm) at a C/N ratio of
69 (mol glucose/mol ammonium sulphate) at 72 h culture. : Mw, o : XPHB (% dry
weight).
2.3 PHB TENSILE STRENGTH () AT DIFFERENT Mw

In an attempt to know how Mw influenced PHB physical properties of mechanical
resistance, tensile strength () was measured on PHB samples. These results,
summarised in Table 2, showed that increased as Mw augmented to 1240 kDa,
probably as a result of the increased intermolecular forces between PHB chains on
higher Mw samples. These results indicated that PHB mechanical resistance depended
significantly on Mw.
Table 2. PHB tensile strength
of samples with different molecular weight (Mw)
(MPa)
38
113
215
Mw (kDa)
445
1000
1240
2.4 PHB AS A MATRIX FOR MICROENCAPSULATION
Applications as a matrix for microencapsulating an enzyme were developed for PHB

obtained from strain 6B. Cellulase was microencapsulated in PHB of three different Mw.
Figure 2 showed that a Mw of 1000 kDa was the best for maintaining a higher relative
cellulase activity (60%) with respect to the PHB samples of 335 and 1350 kDa.,
138
MICROBIAL PRODUCTION OF PHB
suggesting that PHB of 1000 kDa creates the most convenient pore size and
microenvironment for cellulase retention and activity.
Figure 2. Relative activity of cellulase microencapsulated in PHB at different cellulase/PHB

ratios. Left to right: PHB with Mw 1020, 335 and 1350 kDa.
3. Conclusions
From the above results, we concluded that aeration rate should be carefully controlled
under fermentor cultivation of strain 6B for PHB production, as Mw is markedly
affected by dissolved oxygen tension, and consequently by aeration rate. PHB with Mw
higher than 1200-1300 kDa are desirable for obtaining a bioplastic with both high
tensile strength and thermal stability, in order to be successfully processed in industrial
applications.
References
Anderson A.J. and Dawes E.A. 1990. Occurrence, metabolism, metabolic role
and industrial uses of bacterial polyhydroxyalkanoates. Microbiological Reviews 54, 4: 50-472.
Barham P., Keller A., Otun E. and Holmes P. 1984. Crystallisation and morphology of a bacterial
thermoplastic: poly--hydroxybutyrate. J. Mater. Sci. 19: 2781 -2794.
Braunegg G., Sonnleitner B. y Lafferty R. 1978. A rapid gas chromatographic method of the determination of
Poly-6-hydroxybutyric acid in microbial biomass. European J. Appl. Microbiol. Biotechnol. 6:29-37.
Quagliano J. y Miyazaki S. 1997. Effect of aeration and carbon/nitrogen ratio on the molecular mass of the
biodegradable polymer poly--hydroxybutyrate obtained from Azotobacter chroococcum 6B. Appl.
Microbiol. Biotechnol. 47: 662-664.
139

Scandola M., Pizzoli H. y Ceccorulli G. 1988. Viscoelastic and thermal properties of bacterial poly-hydroxybutyrate. Int. J. Biol. Macromol., 10: 373-377.
140
Part 4
NOVEL APPROACHES TO THE STUDY OF
MICROORGANISMS

DETERMINED BY ISOTOPIC RATIO MASS SPECTROMETRY OF
BIOMARKERS
WOLF-RAINER ABRAHAM, CHRISTIAN HESSE, OLIVER
PELZA, STEFANIE HERMANN, MICHAEL TESARB, EDWARD
R. B. MOORE, AND KENNETH N. TIMMIS
GBF - National Research Centre for Biotechnology, Dept. Environmental
Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany;a
present address: ETH Zurich, Institute of Terrestrial Ecology, Schlieren,
Switzerland; b present address: MorphoSys AG, Martinsried/Mnchen,
Germany
1. Introduction
One of the fundamental characteristics of the microbial world is its inherent metabolic
diversity. An extraordinary physiological flexibility, achieved during, at least, 3.5
billion years (Doolittle et al., 1996; Feng et al., 1997; Pace, 1997), allows
microorganisms not only to colonise all habitats which are inhabited by eucaryotes but
also to exploit a wide spectrum of extreme biotopes (e. g., hot springs, salt and soda
lakes, highly-polluted waste waters, etc.) which are inhospitable to higher organisms.
The ubiquitous distribution of bacteria coincides with the observation that
microorganisms, and their natural communities, are of fundamental importance for the
global cycling of organic matter (Pomeroy & Wiebe, 1988). The metabolic diversity is
reflected in the species diversity and, particularly, in the large evolutionary distances
observed between the main phylogenetic lineages of microorganisms (Woese, 1987). It
is currently estimated that probably only 1 % to 10 % of the microorganisms living in
the biosphere are known (i. e., have been isolated and characterised) (Bull et al., 1992).
Moreover, one should recognise that the vast majority of microorganisms present in
environmental samples currently cannot be cultivated in the laboratory. An important
question concerning the cultivable microorganisms arises as to their actual frequencies
and significance in the ecosystem (Amann et al., 1995). The problem is further
complicated due to the fact that, in nature microorganisms live in complex communities
whose compositions vary depending on the environmental conditions. The activities of
individual members of such communities are strongly influenced by the prevailing
conditions in the environment as well as the activities of the other members (Roszak &
Colwell, 1987). The structure and dynamics of natural microbial communities, i. e. the
143
WOLF-RAINER ABRAHAM ET AL.
species compositions, interactions and activities over time and space, is poorly
understood. This is in marked contrast to the wealth of knowledge about the community
structure and dynamics of higher organisms like plants and animals. Due to the problem
of non-culturability of the majority of the bacteria, traditional, cultivation-based
methods of microbiology are often of very limited value (Staley, 1980; Atlas, 1984;
Brock, 1987; Hugenholtz et al., 1998). Rather it is important to develop new
experimental approaches, which do not need cultivation in order to clarify the relations
between structure and function of microbial communities. Several approaches have
been proposed to tackle the basic problems of determining microbial community
structure, most of them based on the molecular analysis of ribosomal RNA or rDNA
(Pace et al., 1986; Hugenholtz & Pace, 1996; Pace, 1996) and some on the analysis of
polar lipids (Tunlid & White, 1992). There is an urgent need for the elucidation of the
critical interactions taking place in microbial communities which determine important
physiological activities, in order to be able to develop a knowledge-based strategy for
the optimisation and control of biotechnological applications in the environment, and to
control their safety. To achieve this long-term goal a novel approach was developed the analysis of biomarkers by isotope ratio mass spectrometry (IRMS) - for tracking the
flux of carbon within microbial consortia. For this approach two components are
needed: i) biomarkers which are specific for bacterial taxa, i. e. genera, families, phyla,
and ii) a calibration of the degree of isotopic fractionation during the microbial
degradation of the substrates and the incorporation of their carbons into the biomarkers.
2. Taxon specific biomarkers
2.1. POLAR LIPIDS
Polar lipids exhibit large structural diversity, an attribute that enables them to be used as
chemotaxonomic criteria. Some lipids are unique to certain taxa, whereas the relative
concentrations of lipids within a taxon provides additional taxonomic resolving power.
The taxonomic utility of lipids is proportional to the quality of the structural information
that is obtained, Hence, in addition to conventional methods of lipid analysis (e.g. 2dimensional thin layer chromatography with selective staining and chemical cleavage,
derivatisation and gas chromatographic (mass spectrometric, GC-MS) analysis of the
component parts), considerable effort is currently being invested in detection/structural
characterisation of lipids (e.g. Fourier transformed infrared spectrometry (FT-IR),
dynamic chemical ionisation and fast atom bombardment (tandem) mass spectrometry
(DCI- and FAB-(MS/)MS), two-dimensional nuclear magnetic resonance spectroscopy
(1H and 13C NMR)).
Because of novel technologies it is more and more possible to analyse intact lipids.
Additional to the already established chromatography mainly the Tandem-MS is used
for the analysis of intact lipids, During the last years we applied this unit successfully
for the study of several lipid mixtures and the structure elucidation of their components.
About 200 glyco-, sulfo- and phospholipids were identified for the first time as natural
products and their structures characterised. Furthermore it was possible to find
144
characteristic biomarkers specific to groups of bacteria and to elucidate their structures

Figure 1) (Abraham et al., 1997; Niepel et al., 1997; Abraham et al., 1998b).
2.2. OUTER MEMBRANE PROTEINS
The application of a newly developed technique (Tesar et al., 1996) allowed us to
identify taxa specific outer membrane proteins. Polyclonal sera were developed for the
identification of group-, respectively species-specific antigens against various
representatives of Pseudomonades and the antisera obtained were further characterised.
Here it was important to find out which sera's were active against a wide spectrum of
antigens within the genus Pseudomonas. The basis were 23 different species of
Pseudomonas which already have been phylogenetically characterised by their 16S
rRNA sequences. With an immunological method called "Westprinting" characteristic
pattern of protein bands have been generated for individual strain, which included
group-specific as well as species-specific features. All tested Pseudomonas sp. revealed
two dominant protein bands in the range of 8 and 18 kDa, which can be used as a genusspecific marker. In combination with the higher molecular proteins it is possible to
make statements about the species, respectively subspecies. A cluster analysis based on
the similarity in the protein profile revealed 5 larger arrangements (around the species
P. aeruginosa, P. oleovorans, P. aureofaciens, P. syringae and P. fluorescens), which
are also seen on the level of the 16S rRNA sequence analysis. To test whether these
results are transferable to other strains of a species 28 different environmental isolates,
which were identified as pseudomonades with the Biolog method were tested and
correctly identified in agreement with other methods. Meanwhile this technique was
successfully applied to other genera as well.
Biomarker are not only characteristic for taxa of bacteria and can be used for their
identification, the isotopic composition of the biomarkers also provide information
about the substrates of these taxa. We understand the term biomarkers as chemical
compounds which are characteristic for bacterial taxa. Although the chemical nature of
a biomarker cannot be limited to a chemical class we focused our studies to polar lipids
and outer membrane proteins and to some extent also to nucleic acids.
Figure 1: Polar lipids useful as biomarkers of bacterial taxa.
145
Figure 1: Continued
3. Isotopic fractionation in microorganisms

Using fatty acids this approach was calibrated in pure culture studies in order to
investigate the relationship between the isotopic composition of certain fatty acids and
that of the bacterial diet. In this manner representative strains of bacteria and fungi
grown in a minimal medium with different carbon sources with known isotopic ratios
were examined. Looking at stable carbon isotopic ratios of palmitic acid which is
common in all higher organisms it was found that all microorganisms vary in their
incorporation of 13C from the substrate into this fatty acid. The rates of isotopic
discrimination in the various substrates and fractions of polar lipids were partially found
to be very different. Especially interesting was a high depletion of 13C of all
microorganisms grown on glucose and in contrast to that the high ranges of isotopic
discrimination of the different organisms grown on mannose. In most cases a significant
146
difference in the isotopic ratio between palmitic acid extracted from glycolipids and that
from phospholipids was observed (Abraham et al., 1998a).
The 13C of bacterial biomass is a parameter in microbial ecology gaining increasing
interest (Boschker et al., 1999). It is used for the identification of substrate utilisation of
bacteria, relying on the observation that bacteria grown on substrates with known
isotopic values produce biomasses which differ in their isotopic ratio of only 2 from
those of their substrates. Our studies determined isotopic differences of palmitic acid of
up to 7 , both depletion and enrichment (Abraham et al., 1998a). We found a much
larger range of isotopic fractionation for amino acids of up to 80 . These ranges were
dependent on the biomarker and the substrate, but also on the bacterial species. From
calibration studies in pure cultures we could derive some rules controlling the depletion
or enrichment of carbon isotopes in the individual biomarkers. This basic new
discoveries of the isotopic discrimination of individual genera of bacteria and fungi may
have a great potential to study the crucial microbial catalysts of the heterotrophic
dominant part of the carbon cycle in natural ecosystems.
4. Carbon sharing in a pollutant degrading bacterial community
4.1. ORIGIN AND CHARACTERISTICS OF THE MICROBIAL CONSORTIUM
From Spittelwasser sediment, a stable bacterial consortium was established growing on
4-chlorosalicylate, an intermediate in the aerobic degradation of 3-chlorodibenzofurane
and 2-chloronaphthalene. The small river Spittelwasser, a tributary of the Elbe river
system near Bitterfeld (in the state of Sachsen-Anhalt, Germany), has served for
decades as an outflow of untreated waste water from the chemical industries
concentrated in this area. Until the recent shut-down of most of these factories, the river
did not contain any detectable eucaryotes but a very broad spectrum of different
procaryotes (Mau & Timmis, 1998). The consortium was grown in a chemostat and fed
with 5 mM 4-chlorosalicylate as the sole source of carbon and energy, with a dilution
rate of 0.16 d-1 and 12C (Faude, 1995). The consortium consisted of four different
bacterial strains identified by 16S rDNA sequence analysis as Pseudomonas sp. MT1,
Empedobacter sp. MT2, Alcaligenes sp. MT3, and Pseudomonas sp. MT4 (Moore et al.,
1996).
To understand the interactions of the bacteria within the consortium it was necessary
not only to identify the different bacteria but also to determine their relative abundance
in the consortium and to monitor them over time. To achieve this goal rabbit antisera
have been produced against all four isolates for immunochemical analysis of the
microbial population and have been tested by different immunological methods.
Applying these antibodies in the immunofluorescence the relative abundance's of the
strains in the chemostat were determined to 84 3 % for Pseudomonas sp. MT1, 1 %
for Empedobacter sp. MT2, 8 4 % for Alcaligenes sp. MT3 and 8 4 % for
Pseudomonas sp. MT4. Furthermore, it was found that the composition of the mixed
culture remained constant over a time period of at least 3 years.
The predominant strain in this consortium, Pseudomonas sp. MT1, grew on 4chlorosalicylate alone tolerating up to 1 mM, above of which cell death occurred.
147
Incubating Pseudomonas sp. MT1 in a chemostat under conditions of the consortium

chemostat led to incomplete mineralisation of 4-chlorosalicylate and the excretion of
degradation metabolites into the culture medium. The accumulation of 4-chlorocatechol,
3-chloro-cis,cis-muconate, and protoanemonin was detected. Protoanemonin, first
isolated from Ranunculaceae (Seegal & Holden, 1945; Didry et al., 1991) and later
observed as a dead-end metabolite of 4-chlorocatechol (Blasco et al., 1995), is known
for its antibiotic activity. Its formation as a metabolic intermediate represents a severe
danger of poisoning for all members of the microbial consortium (Blasco et al., 1997).
Increasing the dilution rate to 0.20 d-1 in the P. sp. MT1 chemostat increased, as well,
the accumulation of 3-chloro- cis,cis- muconate to 4.5 M and that of protoanemonin to
42.7 M after 50 d. Particularly, the concentration of protoanemonin was critical and
caused cell death of P. sp. MT1, expressed by rapidly decreasing cfu, optical density
and substrate conversion. However, contrary to Pseudomonas sp. MT1 the consortium
was able to endure much higher dilution rates, with a maximum rate of 0.80 d-1.
4.2. INCORPORATION OF [U-13C]-METABOLITES IN MICROBIAL BIOMASSES
The hypothesis that protoanemonin is a key intermediate in the degradation of 4chlorosalicylate by P. sp. MT1 was tested by tracing the incorporation of the stable
carbon isotope 13C of 50 nM [U-13C]protoanemonin (i. e. 1000 fold below toxicity) into
biomarker fatty acids of Pseudomonas sp. MT1 . If the degradation of an intermediate is
productive the fatty acids of the degrading microorganism must be enriched in the stable
isotope 13C because of the incorporation of the substrats carbon into the biomass. For
verification of this approach in an experiment, commercially available [U-13C]4chlorocatechol was transformed first to [U-13C]protoanemonin, using a Pseudomonas
sp. MT1 cell-free extract (Blasco et al., 1995), and then pulse-fed into the Pseudomonas
sp. MT1 chemostat. The assimilation activity of the cells was investigated by following
the flow of the tracer into fatty acids of the cellular phospholipids. Fatty acids were
isolated from the biomass of Pseudomonas sp. MT1 and 13C values were measured
using a gas-chromatograph coupled via a combustion interface to an isotopic ratio mass
spectrometer (GC-C-IRMS) (Abrajano et al., 1994). The phospholipid fatty acids were
significantly enriched and the fatty acids C16:0 and C16:1 showed the highest 13Cenrichment found in this experiment. The substrate-utilisation of protoanemonin by
Pseudomonas sp. MT1 was proven by a significant (13C+3.00.5) enrichment of
the isotope 13C in fatty acids C16:1 of Pseudomonas sp. MT1 compared to the unlabeled
control.
Incubation of resting cells of P. sp. MT1 with 1 mM 4-chlorocatechol in sodium
phosphate buffer resulted in the formation of 25 M protoanemonin, 17 M cis,cis-3chloromuconate, and 16 M cis -acetylacrylate. The detection of cis -acetylacrylate
pointed to the further fate of protoanemonin because cis -acetylacrylate supported
growth of Pseudomonas sp. MT1 in minimal medium and resting cells of salicylategrown MT1 cells transformed 1 mM cis-acetylacrylate within 24 h. These results
indicated a conversion of protoanemonin via cis -acetylacrylate in the novel
protoanemonin pathway of Pseudomonas sp. MT1. Recently, it was shown that
protoanemonin can be transformed by the dienelactone hydrolase of Pseudomonas sp.
B13 to cis -acetylacrylate which was further metabolised within the Krebs cycle
148
(Brckmannet al., 1998). Since no activity of cis -dienelactone hydrolase was detected
in the cell extract of Pseudomonas sp. MT1, another, unidentified hydrolase was
assumed to be the responsible transforming enzyme.
4.3. SUBSTRATE COMPETITION
One application of this approach was the incorporation of carbon isotopes from
mixtures of substrates into the individual members of the 4-chlorosalicylate-degrading
consortium. The individual strains grew on a carbon mixture of yeast extract and 4chlorocatechol, the latter of which was enriched with 13C-4-chlorocatechol to 500 for
a better detection of incorporation levels. The isotopic ratios of two fatty acids were
determined for all strains and it was found that Alcaligenes xylosoxidans MT 3
incorporated 4-chlorocatechol the best, while Flavobacterium sp. MT 2 showed almost
no incorporation of this substrate. Both Pseudomonas strains showed intermediate
incorporation levels. This approach will help to elucidate the functional roles of the
individual members of the consortium in the mineralization of 4-chlorosalicylate.
Figure 2: Competing assimilation of 4-chlorocatechol and yeast extract by the four

members of a 4-chlorosalicylate-degrading consortium. The isotopic ratios of the two fatty
acids are shown (C16:17 = 11-hexadecenoic acid, C18:17 = 13-octadecenoic acid).
Alcaligenes xylosoxidans MT 3 showed the highest and Empedobacter sp. MT 2 the lowest
incorporation of 4-chlorocatechol. Note the consistent slight depletion of C18:17
compared to C16:17 in all strains resulting from the different biosynthetic routes of these
fatty acids.
149
4.4. COMMUNITY PHYSIOLOGY OF THE MICROBIAL CONSORTIUM

After the discovery of the degradation of 4-chlorosalicylate via protoanemonin by
Pseudomonas sp. MT1, the question remained open why the consortium could grow
under much higher substrate concentrations and dilution rates than Pseudomonas sp.
MT1 can tolerate or, asking the question the other way around: why are four strains
continuously present in the consortium and not only one? To solve this question the
approach to trace the substrate in Pseudomonas sp. MT1 was used in the study of the
metabolic activity of the consortium. In order to distinguish the individual strains,
immunocapture was applied, using the antibodies generated for the determination of the
abundance of the individual strains.
Using fatty acids C16:1 of the phospholipids as bacteria-specific biomarkers, the
incorporation of 13C from metabolites of 4-chlorosalicylate into bacterial biomass of the
individual strains - separated from the consortium by means of immunocapture - was
determined at different times after the 13C pulse. Pulse-feeding of 0.6 M [U-13C]4chlorocatechol to the consortium chemostat resulted in a 13C-enrichment in whole cells
of the consortium of 13C+24 (6 h). However, the highest 13C-enrichment in C16:1
was found for Alcaligenes sp. MT3 (2.5 h) measuring 13C+1157, while that of
Pseudomonas sp. MT1 was only +126 (6 h) and that of Pseudomonas sp. MT4 was
13C +24 (6 h). The very fast and much higher 13C-enrichments in 16:1 of
Alcaligenes sp. MT3, in relation to that of Pseudomonas sp. MT1 and MT3, indicated
that 4-chlorocatechol, which is partially excreted by Pseudomonas sp. MT1, is
predominantly used as a carbon and energy source by Alcaligenes sp. MT3. The enzyme
activities found in this study were in accordance with the assumption that this strain
assimilates 4-chlorocatechol via the chlorocatechol pathway.
When 1.8 M [U-13C]protoanemonin was pulsed into the chemostat, the 13Cenrichment in whole cells was 45.7 (6 h). The 13C-enrichment of C16:1 of
Pseudomonas sp. MT4 was 13C 17.6 (2.5 h), whereas considerably smaller 13Cenrichments (13C 3.0-4.6) were determined for fatty acids of strains MT1 and MT3
at the early stages (2.5 and 6 h) after the [U-13C]protoanemonin pulse. After 24 h, the
13C-enrichment of fatty acids was 13C 14.9 (Pseudomonas sp. MT1) and 10.8
(MT3). The large and fast 13C-enrichment in C16:1 of Pseudomonas sp. MT4, in
relation to that of Pseudomonas sp. MT1 and MT3, revealed that protoanemonin,
excreted by Pseudomonas sp. MT1, is used as a carbon and energy source
predominantly by Pseudomonas sp. MT4.
Due to its low abundance it was not possible to determine the isotope ratios of fatty
acids from immunocaptured cells of Empedobacter sp. MT2. However, previous 13Ctracer studies of the individual strains of the consortium in pure culture, using unlabeled
yeast extract and 0.2 mM [U-13C]4-chlorocatechol, revealed assimilation of 4chlorocatechol into the biomasses of the strains MT1, MT3 and MT4, but not
Empedobacter sp. MT2 (Pelz et al., 1997) (Figure 2). These data and the low abundance
of the strain suggest that it carries out a role as consumer of cell debris with no active
involvement in the degradation of 4-chlorosalicylate.
Although Pseudomonas sp. MT1 is able to grow alone in a chemostat on 4chlorosalicylate, a stable microbial consortium has been isolated for the degradation of
4-chlorosalicylate. Within the stable consortium, Pseudomonas sp. MT1 is the most
150
abundant strain, which underlines its important functional role as the primary degrader
of 4-chlorosalicylate. Based on the observed high 13C-enrichments in fatty acids of
strains MT3 and MT4 with the metabolites [U-13C]4-chlorocatechol and -protoanemonin
the formation of a consortium can be explained by nutritional interactions of individual
strains. Based on the above results, the functional roles of the consortium members were
assigned (Fig 3). Using immunocapture and IRMS the sharing of substrates within the
microbial community was unravelled and individual community members were shown to
protect the whole consortium from critical intermediates such as 4-chlorocatechol and
protoanemonin by using them as carbon sources and hence, improving the degradation
of 4-chlorosalicylate by Pseudomonas sp. MT1 (Pelz et al., 1999).
Figure 3: Assignment of metabolic functions and interactions of consortium members MT1,

MT3 and MT4 in the 4-chlorosalicylate degrading bacterial community.
151
5. Outlook
The combination of strain-specific antibodies used to immunocapture the individual
strains from the community and biomarker fatty acids used to determine the flux of
carbon into the biomass of the individual strains by means of IRMS proved to be a
powerful tool to study microbial activities in microbial communities. Furthermore we
have shown the existence of a novel pathway for the degradation of 4-chlorosalicylate
via protoanemonin by means of this technique. This was possible because of the high
sensitivity of the IRMS enabling the application of very small amounts of
protoanemonin to the system which do not poison the strains and which do not disturb
the community significantly. Future studies will be directed to the detection of
microbial degraders of poorly degradable pollutants in microbial communities
independent from the need to isolate them. A similar approach was recently reported for
sulphate-reducing bacteria (Boschker et al., 1998). Instead of immunocapture,
biomarkers like polar lipids or outer membrane proteins will be used to trace the flow of
labelled substrates into the biomass of potential degraders.
Since 1950 a huge number of metabolic routes within individual species have been
elucidated. The challenge now is to elucidate metabolic networks in natural biological
assemblages in which metabolites produced via a pathway of one cell type flow to other
cells to enter new pathways. The surprises exposed in the present study are surely only a
mere taste of what is in store! Exploration of metabolic networks in natural assemblages
will ultimately yield an understanding of the functioning of such assemblages as
biological units, of the roles of the component members and of their metabolic,
physiologic and energetic benefits. Furthermore, we will more and more understand
how the assemblage as a unit interacts with its abiotic environment. Such an
understanding will provide the ground rules for interventions to influence
environmental processes and to optimise biotechnological applications in the
environment, such as in situ bioremediation.
Acknowledgement
We thank Dagmar Wenderoth, Tanja Jeschke, Birgit Jung, and Peter Wolff for their
excellent technical assistance. The International Atomic Energy Agency, Vienna
(Austria), is acknowledged for providing free reference materials for the calibration of
the IRMS. This work was supported by a grant of the German Federal Ministry for
Science, Education and Research (Project No. 03 19433C).
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152

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antibiotic. Formation of protoanemonin from 4-chlorocatechol by enzymes of the 3-oxoadipate pathway.
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survival of 4-chlorobiphenyl-cometabolizing microorganisms. Appl. Environ. Microbiol. 63,427-434.
Boschker, H. T. S., Nold, S. C., Wellsbury, P., Bos, D., de Graaf, W., Pel, R., Parkes, R.-J., and Cappenberg,
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Brckmann, M., Blasco, R., Timmis, K. N., and Pieper, D. H. (1998) Detoxification of protoanemonin by
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Bull, A. T., Goodfellow, M., and Slater, J. H. (1992) Biodiversity as a source of innovation in biotechnology.
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154
A COMPARISON OF THE MECHANICAL PROPERTIES OF DIFFERENT

BACTERIAL SPECIES
C. SHIU, Z. ZHANG AND C.R. THOMAS*
Centre for Bioprocess Engineering, School of Chemical Engineering
The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
* corresponding author.
Abstract
Past attempts at direct measurement of the mechanical characteristics of cells have
shown little potential for use with bacteria. However, a technique based on
micromanipulation has now been used to compare the mechanical properties of two
species of bacteria; Gram-negative Escherichia coli and Gram-positive Staphylococcus
epidermis. Gram-negative bacteria have traditionally been considered to be weaker than
their Gram-positive counterparts on the basis of them being more susceptible to
disintegration by mechanical stress. This observation has been corroborated using
micromanipulation, which has directly quantified the differences in cell strength on the
basis of bursting force. The bursting force of rapidly growing S. epidermis was found to
vary from 3 and 34 N with an average value of 13.8 N (standard error 0.8 N). The
corresponding value for continuously growing E. coli (specific growth rate 0.5 h-1) was
3.8 N (standard error 0.4 N).
1. Introduction
1.1 RELATIVE RESISTANCE
MECHANICAL DISRUPTION
OF
DIFFERENT
MICROORGANISMS
TO
The physical properties of cells e.g. cell size, shape and especially wall strength, are
highly species, strain and physiological state specific and will influence the relative
resistance of different microorganisms to disruption, which may be used to indirectly
infer the relative strength of microorganisms (Table 1, Table 2).
155
C. SHIU, Z. ZHANG AND C.R. THOMAS

Table 1: Susceptibility of various kinds of cells in suspension to disintegration by various
methods. A high number indicates that the cells are sensitive. Parenthesis indicates that the
number is uncertain (Edebo, 1969).
Animal cells
Gram - bacilli & cocci
Gram + bacilli
Yeast
Gram + cocci
Spores
Mycelia
a
Liquid-pressing
Freeze-pressing
Agitation
Sonication
7
6
5
4
3
2
(1)a
7
6
4
2.5
2.5
1
5
7
5
(4)
3
(2)
(1)
6
7
6
5
3.5
3.5
2
1
clogging of orifice may occur
The wall thickness of yeast (> 70 nm) is typically greater than that of Gram-positive
cocci (10 - 80 nm) yet the latter would appear to be the more resilient according to
Table 1. This may be because size has been observed to play an important part in
disruption, with larger cells showing increased susceptibility to rupture.
Table 2: Protein release from microorganisms in a high-pressure homogeniser (modified
from Keshavarz et al., 1987).
Microorganism
Rate constant, k
0.41
0.39
0.28
0.23
0.0085
Pseudomonas putida (Gram-negative rod)

Escherichia coli (Gram-negative rod)
Bacillus brevis (Gram-positive rod)
Saccharomyces cerevisiae
Nocardia rhodochrous
The most obvious role of the cell wall is in protecting the cell from mechanical damage,
the structure and composition of which contributes greatly to the strength of individual
bacteria as may' be seen from the fact that Escherichia coli (Gram-negative rod),
although similar in size to Bacillus subtilis (Gram-positive rod), is disrupted more easily
(Table 2). This is most likely due to the relatively thin peptidoglycan layer found in
Gram-negative bacteria (see Section 0).
1.2 CELL WALL STRUCTURE
Prokaryotic cells can be segregated into different groups on the basis of their cell wall
structure. The Archaebacteria possess cell walls but the components and physiology are
vastly different from that of eubacteria. A small fraction of eubacteria, the mollicutes,
lack an outer cell wall and are bounded only by their cytoplasmic membrane (these will
not be considered here). The eubacteria which do possess cell walls can be divided into
two major groups: Gram-positive or Gram-negative, which refer to the manner in which
eubacteria respond to the Gram stain.
Under the electron microscope, the Gram-positive cell wall appears as an amorphous
layer with an average thickness of 30 nm, although this value may vary from between
156
A COMPARISON OF THE MECHANICAL PROPERTIES OF BACTERIAL SPECIES
10 80 nm (Shockman and Barrett, 1983). Unlike Gram-negative bacteria, the

periplasm is practically non-existent as the relatively large osmotic pressure, estimated
at up to 25 atm (Mitchell and Moyle, 1956), found within Gram-positive bacteria forces
the plasma membrane firmly against the cell wall.
By comparison, the cell walls of Gram-negative bacteria are much more complex
than that of Gram-positive bacteria. The cell envelope consists of essentially three
layers: the cytoplasmic membrane, the murein (or peptidoglycan) layer and the outer
membrane. The dual membrane system of Gram-negative bacteria gives rise to the
periplasm, a space 15-20 nm thick comprising between 20-40% of the total cell volume
(reference) which contains a gel-like solution of proteins and in which the murein layer
is found. Peptidoglycan is less abundant in the cell walls of Gram-negative bacteria,
rarely exceeding 5- 10% of the weight of the wall (Lugtenberg and van Alphan, 1983).
1.3 BACTERIAL BIOMECHANICS
A number of researchers have demonstrated the flexibility of the bacterial cell wall
(Mendelson and Thwaites, 1989; Baldwin, 1988; Marquis, 1968; Ou and Marquis,
1970) but perhaps the simplest and most compelling evidence was that presented by
Isaac and Ware (1974) who embedded cells of different organisms in glycerol-gelatine
strips and stretched them on a rack. Spirillum was found to uncoil and stretch reversibly
to a rod three times the length of its original shape. The Gram-positive bacterium, B.
megaterium elongated to one quarter of its original length before becoming detached
from the gel matrix. E. coli showed considerable stretching and deformation - up to 100
% elongation - without detaching from the gel. With the Gram-positive coccus, S.
aureus, no elongation was observed prior to detachment from the gel matrix. These
results would appear to suggest the bacterial cell wall is far from being rigid and nonflexible. With Gram-positive organisms, the thicker murein leads to increased resistance
to stretching while their Gram-negative counterparts show great flexibility in
comparison.
Without a doubt, the small size of individual bacteria has proved to be a
considerable obstacle in the development of a method for directly measuring the cell
mechanical properties. The approach of Thwaites and Mendelson (1985, 1989, 1991)
cleverly side-stepped this difficulty by conceiving a means to produce bacterial thread
up to 1 m long with 50 000 parallel cellular filaments, which can be subjected to similar
techniques applied to textile threads. Tensile tests were performed on bacterial threads
of B. subtilis mutant FJ7 to determine the cell wall properties.
The length and diameter of the filaments were found to depend on the humidity and
unsurprisingly so were the mechanical properties. The tensile strength and modulus of
elasticity at low relative humidity were 300 MPa and 13 GPa respectively.
Corresponding values when extrapolated to 100% humidity were found to be 13 MPa
and 30 MPa. Such values of strength indicated that cells in vivo should theoretically be
able to withstand turgor pressures of up to 2.6 MPa (Thwaites and Surana, 1991). It was
found that the stress/strain curves of unwashed preparations were similar to those with
the residual culture medium removed at roughly 18% higher relative humidity. The
most likely reason for this was that the unwashed cells were encased by the highly
hygroscopic dried medium and were therefore held in a much more humid environment
relative to the bulk atmosphere.
157
In addition, the walls of the B. subtilis mutants were shown to exhibit what was
apparently viscoelastic behaviour since the load depended on the rate of strain and load
relaxation also occurred in the extended state. The time-scale for decay proved to vary
widely but based on the initial rate of decay, was found to occur on the order of minutes
(Thwaites and Mendelson, 1985). Viscoelastic phenomena in cell walls has major
implications for micromanipulation measurements; at high deformation rates, the cells
would appear to be stiffer and more brittle and vice versa (Thwaites and Mendelson,
1991). Such behaviour would need to be accounted for in the development of structural
models.
One major drawback to the technique is that it is limited to filament forming
mutants. Furthermore, the cells were not in their naturally hydrated condition when the
tests were carried out and the humidity has been shown to affect the thread mechanical
properties. Micromanipulation has several advantages over the latter technique in terms
of simplicity, versatility and more importantly, since mechanical properties are likely to
be affected by the external environment, the cells can be measured whilst suspended in
the growth medium.
1.4 MICROMANIPULATION
Micromanipulation is based on a relatively simple concept, which relies on squeezing a
single cell, either between two optic fibre probes or between a single optic fibre and a
slide, until rupture; a force transducer measures the force required. Such work has
already been performed on animal cells (Zhang et al. 1991), yeast cells (Srinorakutara
(1997); Mashmoushy et al., 1998) starch and latex particles. Micromanipulation studies
with filamentous organisms such as fungi are slightly more involved and it has been
more appropriate to determine mechanical properties by extension of the organism until
breaking (Roberts, 1996).
2.1 THE MICROMANIPULATION SYSTEM
The apparatus (fig 1) was based on the rig used by Mashmoushy et al. (1998) for work
with yeast. Cells are squeezed using a 50 m optic fibre probe, one end of which is
attached to a force transducer (Aurora Scientific Inc., Aurora, Canada) with paraffin
wax. The end of the probe which comes into contact with the bacteria is ground down to
leave a tip with a much smaller area, thus minimising alignment errors and incorrect
probe-cell contacts. The transducer is attached to a stage which is driven downwards by
a stepping motor, The force on the probe is measured by the transducer in terms of
voltage, which is converted to give the bursting force. Lighting is provided by a slit
illuminator from the side. Cells are positioned underneath the probe by moving the
stage. When E. coli was being measured, the image was recorded onto video tape for
later image analysis to determine the length of the cell.
158
Fig 1 The micromanipulation system
2.2 CULTURE CONDITIONS

Staphylococcus epidermis ATCC 5885 cells were grown on agar slopes at 30C for 48
hours and then used to inoculate a McCartney bottle containing 10 ml of sterilised
nutrient broth (Oxoid, UK). Following incubation at 30C over 24 hours, the broth was
used to inoculate a 500 ml volume shake flask containing 90 ml of sterilised nutrient
broth to start the batch fermentation. The flask was maintained at 30C and 200 rpm in
an orbital shaker. The progress of the fermentation was followed by taking absorbance
measurements at 6.50 nm. Samples were taken from the rapid growth phase at t = 4, 5
and 6 hours after inoculation.
In preliminary fermentations, E. coli NCIMB 10214 had demonstrated a relatively
short exponential growth phase. Hence, continuous as opposed to batch fermentation
was used to provide physiologically identical samples. Medium of the following
composition was used for E. coli fermentation: (g/l of distilled water): KH2PO4, 13.6;
(NH4)2SO4, 4; MgSO4, 0.2; FeSO4 5x10-4; yeast extract, 5.0 and adjusted to pH 7.0
using 2.0 M KOH solution. Glucose solution at a final concentration of 10 g/l of
medium was made up and sterilised separately and only added to the medium before
inoculation.
Cells of E. coli were incubated at 37C on agar slopes for 24 hours and used to
inoculate a shake flask containing 100 ml of complex medium. The culture was grown
159
overnight for approximately 16 hours at 37C and 200 rpm in an orbital shaker before
being used to seed 1 1 of medium in a 2 1 fermenter. The pH was controlled using 0.5 M
H2SO4 and 2.0 M ammonia solution. Once the cells had reached mid-exponential phase,
the continuous fermentation was started at a dilution rate of 0.5 h-1. Samples were
diluted using distilled water to a concentration of approximately 1 x 107 cells/ml.
Figure 2a and Figure 2b represent typical traces of the bursting response of individual S.
epidermis and E. coli cells respectively. Both traces have a similar form; contact
between the probe and the cell is made at point A after which the cell deforms until
rupture (point B). Following bursting of the cell, there is a decrease in force on the
probe until it reaches the slide at point D, leading to a significant increase in force.
Figure 2: Bursting traces for (a) S. epidermis and (b) E. coli
Plots of bursting force versus (equivalent spherical) diameter for S. epidermis and E.
coli are shown in Figure 3a and Figure 3b respectively. In both cases, the large scatter
observed in bursting force is attributed to biological variation within the culture. Three
separate samples were taken (t = 4, 5 and 6 hours) and tested within one hour of
sampling. Statistical testing using ANOVA indicated a strong probability that the three
samples were from equivalent populations and hence they were treated as such. For cell
diameters ranging from 0.5 to 1.6 m with an average value of 0.92 m (standard error
0.03 m), the bursting force for S. epidermis cells was found to vary from 3 to 34 N
(standard error 0.8 N) with an average value of 13.8 N. Furthermore, simple linear
regression appeared to suggest some relationship between bursting force and cell size,
which was confirmed using an F-test with 95% confidence.
160
Corresponding values for E. coli ranged from 1 to 9 Nwith an average value of 3.6 N
(standard error 0.4 N) for cells with equivalent spherical diameters between 0.7 to 1.8
m with an average value of 1.26 m (standard error 0.05 m). Simple linear regression
showed no relation between the bursting force and equivalent spherical diameter. This
was confirmed with an F-test at the 95% confidence level.
Figure 3: Plots of bursting force vs. (equivalent spherical) diameter for (a) S. epidermis and
(b) E. coli
It is worth mentioning that only a small percentage of E. coli cells demonstrated

bursting behaviour. In other cases, the trace either showed the deformation of the cell
without bursting or nothing was observed. This might be due to rapid relaxation caused
by water loss or viscoelasticity but further experiments will be required to elucidate the
reasons for this phenomenon.
It is significant that the Gram-negative E. coli with the thinner cell wall and lower
internal pressure had a lower average bursting force than the Gram-positive S. epidermis
with the thicker cell wall and higher internal pressure. Differences in cell physiology
could be the underlying cause behind these differences. Although the fact that spheres
are structurally stronger than cylinders could also have some bearing on this result.
4. Conclusions and future developments
The bursting force measurements obtained for S. epidermis and E. coli demonstrate the
usefulness of the micromanipulation technique for obtaining information on the
mechanical properties of bacteria. Linked with a suitable cell model, it should be
possible to estimate other mechanical properties. Biomechanics might be linked to cell
physiology by carrying out micromanipulation in conjunction with experiments to
determine cell wall composition, even using mutants known to lack certain cell wall
components.
161
Bacterial species do not always grow as individual cells but in a range of different
morphologies. Micromanipulation techniques that have been developed for tensile
testing of fungi (Roberts, 1999) and filamentous bacteria (Stocks and Thomas, 1999)
might be adapted to pull apart bacteria that grown in chains of while larger probes might
be used to measure the bursting forces of bacteria that grow in clumps.
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162
Part 5
NOVEL APPLICATIONS
KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA

NEREIDA COELLO AND LUIS VIDAL
Laboratorio de Procesos BiotecnolOgicos. Instituto de Biologla
Experimental. Universidad Central de Venezuela. AP 472 79 Caracas
1041-A, Venezuela.
Abstract
A strain of Kocuria rosea with keratinolytic activity was isolated. This Gram-positive
coccus-shaped bacterium was able to degrade whole feathers in 72 h at 40 C. In batch
culture, the optimum temperature for feather degradation, bacterial growth and protease
secretion was 40 C. Under these conditions, biomass and caseinolytic activity reached
3.2 g/l and 0.15 U/ml, respectively, after 36 h incubation. Extracellular protease
secretion was associated with the exponential growth phase. Feather degradation
reached 51 % in 72 h with a yield on biomass of 0.32 g/g. Enzymatic assay by
polyacrylamide-gelatine gels showed two bands with alkaline proteolytic activities in
the fermentation juice after 72 h of culture.
1. Introduction
The expansion of biotechnology has produced an increasing demand for high-quality
inexpensive microbial growth media. Utilisation of feathers from the poultry processing
industry as a substrate for fermentation might offer an inexpensive alternative for a
microbial method of metabolite production, such as enzymes and unicellular protein can
be achieved provided an efficient utilisation.
Feathers are a poultry by-product rich in protein (mainly keratin) and generated in
very large quantities as a waste product from poultry processing industry. For many
years, they have been the object of nutritional studies in order to incorporate them as
nitrogen supplement in animal foodstuff. This allows poultry industry to take advantage
of their usefulness and eliminates the environmental problem generated. In their native
state, feathers have two important nutritional limitations: an amino acid imbalance and a
poor digestibility (Xiang Lin et al. 1992).
Industrially, a great part of the feather waste is cooked under high pressure and
temperature producing a feather meal, which can be incorporated into poultry foodstuff
as a protein supplement. This product has been evaluated extensively and the results
165
show that it is not very digestible and the destruction of certain amino acids during the
process, such as cysteine, magnifies the imbalance of essential amino acids (Bhargava
& Oneil 1975; Baker et al. 1981).
Thus, there is a growing interest in alternative methods for the treatment of feathers to
improve the nutritional quality of the feather meal and to develop new and useful byproducts from feathers. The nutritional value of the feather meal might be improved by
microbial action, this results in a modification of the structure of keratin, altering the
resistance to the enzymes of the digestive tract. The literature reports that isolates of
Bacillus lichenformis (Williams et al. 1991) and Streptomyces pactum (Bckle et al
1995) grown on feather media were able to modify the digestibility and profiles of the
specific amino acids that were liberated. The bacterial cells incorporated into the
fermented feathers may contribute to some amino acids, hence improving their balance.
Also, feather as a fermentation substrate may be useful in order to obtain other
microbial products (proteolytic enzymes, carotenoid pigments, etc.).
We conducted a study on an isolate from the soil of a poultry-processing plant in
order to determine microbial growth, possible keratinolytic activity and optimum
feather degradation conditions in submerged fermentation. To our knowledge, this is the
first report describing the capacity of Kocuria rosea to degrade feathers.
2.Isolation, identification and adaptation of feather-degrading microorganisms
Isolating microorganisms from nature is the microbiologists first step in screening for
natural microbial products. It is possible to isolate many different microorganisms by
employing enrichment techniques on the appropriate culture medium. One successful
approach for the discovery of new metabolites involves considering the characteristics
of the desired product and process development and using ecological approaches for
isolation and screening. Consideration of the ecological approaches to isolation can
provide a screen with both a large number and a wide variety of microorganisms to
examine for the product of interest. Even though microorganisms are highly adapted,
specific microbial types are associated with different niches within a variety of
ecosystems. Therefore, what is isolated from the defined ecosystem or habitat is a
reflection of the isolation procedures and of the conditions found in nature.
2.1. ISOLATION AND DEGRADATION OF FEATHERS BY A MICROBIAL
ISOLATE
Based on the capacity of some microorganisms to grow in a medium containing feather
powder as the primary organic substrate for supplying carbon and energy a featherdegrading isolate was obtained from soil of a poultry processing plant (Figure 1).
It was found that a culture of the feather-degrading isolate contained microorganisms
exhibiting at least two distinct colony morphologies (red and white) when streaked onto
nutrient agar plates. The white colony contained only rod-shaped bacteria, whereas the
red colony was a coccus-shaped bacteria, which appeared singly and in chains.
Although each type displayed clearing zones when streaked onto the potter-milled
feather agar plates, the red colony produced the more pronounced clearing zones. The
166
isolate, designated as LPB-3, was able to degrade feathers in 72 h at 40 C when isolated

cells were incubated on whole feathers suspended in saline medium.
The keratinolytic activity of LPB-3 isolate was evidenced by visual observation of
feather disintegration, as well as increasing turbidity of the culture due to growing cells
(Figure 2). The bacterium grew aerobically and was catalase positive. Additional
biochemical tests conducted on LPB-3 isolate in our laboratory were confirmed by the
Pasteur Institute of France, which characterised the isolate as Kocuria rosea
Figure 1. Isolation method for feather degrading microorganisms from soil
Figure 2. Degradation of feathers by isolate. The LPB-3 strain was cultivated in assay tubes
with whole feathers suspended in mineral medium (g/l): NH4CI, 0.5; NaCI, 0.5; K2HPO4,
0.3; KH2PO4, 0.4; MgCI2.6H2O, 0.1; 0.1 yeast extract; pH 7.5 at 40Cfor 72 h. A, control;
B, 24 h; C, 48 h; D, 60 h: E, 72 h.
167
NEFEIDA COELLO AND LUIS VIDAL
Only limited information regarding Kocuria rosea is currently available. This Gram
positive micro-organism, which was classified in the past in the genus Micrococcus and
reclassified recently in the genus Kocuria (Stakenbrandt 1999, has been studied mainly
in relation to the structure and properties of the carotenoid pigments it produces
(Cooney 1981; Jagamadham 1991). Until now, the feather degradation capacity of K.
rosea has not been reported in the literature.
Cells of the LPB-3 isolate were grown on whole-feather basal medium, in which the
autoclave treatment time was progressively reduced. The isolate was able to degrade
feathers which had been autoclaved for only 5 min. Attempts to maintain the growth of
the isolate on non-steam-treated feathers were not successful,
2.2. MORPHOLOGICAL AND ULTRASTRUCTURAL CHARACTERISTICS OF
THE FEATHER-DEGRADING ISOLATE
Microscopic observations of the isolate showed a coccus (1.5 m) which appeared
singly and in chains adsorbed mostly to the feathers, although a few microorganisms
swam freely (Figure 3A). The adsorption to the fermentation substrate is part of a
survival strategy of microbes in certain limiting environments in order to improve
feather degradation via proteolytic enzymes secretion. The ultrastructure of K. rosea
was generally similar to that of other Gram-positive bacteria. The cells were separated
from the surrounding medium by a microcapsule and a cell wall with a homogeneous
structure, approximately 20 nm thick (Figure 3B). The cytoplasmic membrane was
closely adjacent to the cell wall and the periplasmic space was poorly discernible. K.
rosea was characterised by a high ribosome density in the cytoplasm and the occurrence
of polyphosphate granules and glycogen accumulation was also observed. The nucleoid,
consisting of DNA fibrils, was located in the centre of the cell and as cell division
began, a septum was formed inside the cells. Transmission electron micrographs
showed in some cells the presence of two asymmetrical septa.
3. Microbial growth and feather degradation
3.1, EFFECT OF QUANTITY OF FEATHERS
Feathers promoted cell biomass production concomitant to feathers degradation, at the
lowest concentration tested (10 g/l). The increase of the concentrations of free amino
groups during the feather fermentation was an evidence that these bacteria produce a
protease(s) capable of digesting keratin of feathers to produce short peptides and amino
acids that can be used as carbon and nitrogen sources for the growth of the microorganism. The concentrations of free amino groups under these conditions are shown in
Figure 4. It was observed that aerobic growth by the isolate LPB-3 on feathers
throughout growth were similar for 20 and 30 g/l. After 96 h of fermentation the final
concentration of free amino groups attained nearly 60 and 35 mmol for 20 and 10 g/l,
respectively. Therefore for the kinetic fermentation studies, 20 g/l of feathers was used.
168
KOCURlA ROSEA AS A NEW FEATHER DEGRADING BACTERIA
Figure 3. [A) Light microscopy of K. rosea cells with Gram-staining. Magnification X

10,000. Single cells and chains ofcoccus-shaped bacteria were adsorbed to the feathers. (B)
Ultrastructure of K. rosea cells. Magnification X 52,000. CW, cell wall: CM, cytoplasmic
membrane: AS, asymmetrical septum: CH, chromatin fibrils: PG, polyphosphate granules.
169
Figure 4. Effect of quantity of feathers. LPB-3 strain was cultivated at 40Cfor 96 h in 500ml Erlenmeyer baffled flasks, containing IO g/l
20 g/l (0) and 30 g/l
scissors-cut
feathers, suspended in mineral medium described earlier shaken at 75 rpm. Control non
inoculated
. Samples were taken and filtered through a Bchnerfunnel with Whatman
paper 4. Afer removing cells by centrifugation supernatant was used for free amino groups
analysis
using
a
ninhydrin
method
described
by
Rosen
(1957).
Figure 5. Growth of Kocuria rosea LPB-3 (O) and free amino groups concentration
at
different temperatures in feather medium (20 g/l). Samples were taken at 72 h filtered
through a Bchner funnel with a Whatman paper 4. Filtrated was used to measure cell mass
density by turbidity (600 nm) andfree amino groups as described earlier.
170
3.2. EFFECT OF CULTURE TEMPERATURE ON FEATHER DEGRADATION

AND GROWTH OF LPB-3
The optimum growth of K. rosea in scissors-cut feathers was obtained at a culturing
temperature of 40 C (Figure 5). Similarly, maximum feather degradation, measured as
concentration of free amino groups occurred at between 35-40 C and reached 43
mmol/l. In both cases, concentration of free amino groups and biomass decreased at
culture temperatures lower than 35 C. For this reason, the optimum temperature chosen
for this mesophilic strain was 40 C. Nevertheless, a psychrotrophic strain of K. rosea
has been reported that growths at lower temperatures (Jagamadham 1996;
Chattopadhyay 1997).
3.3. KINETIC FERMENTATION
At 40 C we found a latent phase of nearly 3 h in batch fermentation, followed by an
exponential growth phase in which K. rosea attained a specific growth rate of 0.17 h-1.
Protease activity was detected in the culture medium throughout growth, with the
maximum activity (0.15 U/ml) associated with maximum biomass (3.1 g/l) 36 h after
inoculation (Figure 6). In the latter case, K. rosea caused up to 51% feather degradation
in 3 days (Figure 6B). This time represents less than the reported numbers in the
literature (at least 5 days) for other microorganisms with keratinolytic activity for
maximum feather hydrolysis in optimum conditions: Streptomyces fradiae (Noval &
Nickerson 1959), Bacillus licheniformis (Williams et al 1990) and Bacillus sp. (Atalo &
Gashe 1993).
Although low residual feathers was found at 96 h, the excess in incubation time was
not practical because this parameter did not decrease significantly and the energy
required for incubation may increase the operational costs. The increase in pH of culture
medium from 7 to 8.5 after 48 h of inoculation (data non shown) was presumably a
result of proteolysis and the release of ammonia following utilisation of amino acids and
soluble peptides as a metabolic fuel for growth and microbial maintenance.
4. Industrial applications
4.1, FERMENTED FEATHER MEAL
Industrially, feather waste is processed into hydrolysed feather meal using high
temperatures and pressure. It is utilised as supplemental source of protein in poultry
foodstuffs. However, the use of hydrolysed feathers meal, as a replacement protein is
limited. Morris and Balloun (1973) observed that the addition of 6 % hydrolysed feather
meal to diets of chickens caused a decreased body weight. Research showed that raw
feathers can be fermented in anaerobiosis with a Bacillus licheniformis strain (Williams
et al, 1991) to produce a fermented feather lysate that can be used as a replacement
protein up to 15 % in poultry foodstuff. The feather lysate obtained with K. rosea is
171
enriched with bacterial biomass up to 10 %, but must be studied in terms of the

digestibility, amino acid profile and bioavailability in order to know more about the
properties of this new product.
Figure 6. Growth -O- (A), proteolytic activity

and residual feathers
(B) of Kocuria
rosea LPB-3 in feather medium (20 g/l) at 40 C for 96 h and 75 rpm agitation. Residual
feathers are expressed as a percentage of dry weight of the residue obtained upon filtration
of the culture contents through a Whatman paper No. 4. Protease activity was assayed by
photometric method (280 nm) using casein as substrate based on the method of Kunitz
(1947) One unit of proteolytic activity was defined as the amount of enzyme which produced
an absorbance increase of 1.0 units within one min.
172
4.2. ENZYMES
Proteolytic enzymes are the most important industrial enzymes because of their wide
applications in the detergent, dairy, pharmaceutical, leather and food industry. Many
alkaline proteases are produced by bacteria and fungi and are also present in
mammalian tissues. Cultures of K. rosea in feather medium stimulate the secretion of at
least two alkaline proteases (Figure 7) with a broad tolerance to temperature (35-70 C)
and high long-term storage stability. These proteases degrade keratin of feathers to
produce short peptides and amino acids that can be used as carbon and nitrogen sources
for the microorganism. They could be useful in the dehairing stage of leather processing
and the formulation of dehairing lotion removers of cosmetic industry. Bovine and
porcine pancreatic trypsin have demonstrated to be effective in reducing the
requirement of lime, shortening the dehairing process time and lowering the waste
treatment costs (Ward, 1985). Thus, extensive genetic and physiological studies must be
carried out for a better understanding of the mechanism that controls keratinase
secretion in the wild-type strain of K. rosea in order to make this protease available for
industrial purposes.
Figure 7. Polyacrylamide-gelatine gels of fermentation juice of Kocuria rosea LPB-3 after

72 h of culture at different pH, A: 10; B: 9; C: 7; D: 5; E: 3.6.
4.3. PIGMENTS
Many microorganisms including algae, fungi and bacteria produce carotenoid pigments.
Industrially, they are used as a food colouring agents (e.g. margarine, cheese, and egg
173
products) and at a lesser extent as oral tanning agents in the cosmetic industry.
Cantaxanthin, a xantophyll carotenoid used as feeding additive together with
astaxanthin, have been used in the poultry industry to improve the colour of chicken and
egg yolk and for muscle pigmentation in farmed salmonids (Elmadfa, 1998).
Cantaxanthin is the major coloured carotenoid pigment biosynthesised in mesophilic K.
rosea strains (Cooney, 198 1). Further research concerning qualitative and quantitative
pigment content and factors influencing carotene biosynthesis in K. rosea must be done
to consider the microbiological pigment processes available.
Acknowledgements
The authors acknowledge Lic. C. Bernal and Dr. A. Bretaa for their helpful
contribution in enzyme gel assay and electron microscopy, respectively. This research
was supported by grants from Interamerican Development Bank and Consejo Nacional
de Ciencia y Tecnologa (BTI-88), and Consejo de Desarrollo Cientfico y Humanstico
of Central University of Venezuela (09- 12-404497).
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degrades various kinds of fibrous proteins. Biotechnology Letters 15, 1115-1156.
Baker, D. H., Blitenthal, R. C., Boebel, K. P., Czrnecki, G. L., Southern, L.L. and Willis, G. M. (1981)
Protein amino-acid evaluation of steam-processed feather meal. Poultry Science 60, 1865-1872.
Bhargava, K.K. and ONeil, J. B. (1975) Composition and utilisation of poultry by-product and hydrolysed
feather meal in broiler diets. Poultry Science 54, 1511-1518.
Bckle, B., Galunsky, B. and Muller, R. (1995) Characterisation of a keratinolytic serine proteinase from
Streptomycespactum DSM 40530. Appl. Environ. Microbiol. 61, 3705-3710
Chattopadhyay, MK., Jagannadham, MV., Vairamani, M., and Shivaji, S. (1997) Carotenoid pigments of an
Antarctic psychrotrophic bacterium Micrococcus roseus: temperature dependant. biosynthesis, structure
and interaction with synthetic membranes. Biochemical and Biophysical Research Communications 239,
85-90.
Cooney, J.J., and Berry, R.A. (1981) Inhibition of carotenoid synthesis in Micrococcus roseus. Canadian
Journal of Microbiology 27, 421-425
Elmadfa, I. and Majchrzak D. (1998) Carotenoids and Vitamin A in fish. ZErnahrungswiss 37, 207-210
Jagannadham, M.V., Narayanan, K., Rao, C.M., and Shivaji, S. (1996) In vivo characteristics and localisation
of carotenoid pigments in psychrotrophic and mesophilic Micrococcus roseus using photoacoustic
spectroscopy. Biochemical and Biophysical Research Communications 227, 221-226.
Jagannadham, MV., Rao, VJ., and Shivaji, S. (1991) The major carotenoid pigment of a psychrotrophic
Micrococcus roseus strain: purification, structure and interaction with synthetic membranes. Journal of
Bacteriology 173, 7911-7917.
Kunitz, M. (1947) Crystalline soybean trypsin inhibitor II. General properties. Journal of General Physiology
30, 291-310.
Noval, J. and Nickerson, W. (1959) Decomposition of native keratin by Streptomyces fradiae. Journal of
Bacteriology 77, 251-263
Rosen, H. (1957). A modified Ninhydrin Colorimetric analysis for amino acids. Archives of Biochemistry and
Biophysics 67, 10-15.
Stackebrandt, E., Koch, C., Gvozdiak, O., and Shumann, P. (1995) Taxonomic dissection of the genus
Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen.
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Ward, O.P (1985). Proteolytic enzymes, in Comprehensive Biotechnology Moo-Young Editor, Pergamon
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Williams, C. M., Richter, C. S., Mackenzie, J. M., Jr. and Shih, J. (1990) Isolation, identification, and
characterisation of a feather-degrading bacterium. Applied and Environmental Microbiology 56, 15091515.
Williams, C. M., Lee, C.G., Garlich, J.D. and Shih, J. (1991). Evaluation of a feather fermentation product,
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Xiang Lin, Chung-Ginn Lee, Ellen S. Casale and Shih, J. (1992) Purification and Characterisation of a
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175
COMPARISON OF PB2+ REMOVAL CHARACTERISTICS BETWEEN

BIOMATERIALS AND NON-BIOMATERIALS
DONG SEOG KIM1 AND JUNG HO SUH2
1
Department of Environmental Science, Catholic University of TaeguHyosung, Kyungbuk 712-702 and 2Department of Industrial Chemistry,
Ulsan College, Ulsan 680-749, Korea.
Abstract
The order of Pb2+ removal capacities in chemical adsorbents were found as ion
exchange resin > zeolite > granular activated carbon (GAC) > powdered activated
carbon (PAC), while in biomass was Aureobasidium pullulans > Saccharomyces
cerevisiae > activated sludge. Although Pb2+ removal capacity (mg Pb2+/g) of the
activated sludge (30.9) was lower than those of ion exchange resin (167.7) and other
pure cultures of A. pullulans (170.4) and S. cerevisiae (95.3), it was higher than those of
other chemical adsorbents such as GAC (26.9), PAC (2.1), and zeolite (30.2). The initial
Pb2+ removal rates in chemical adsorbents were in the order of PAC > GAC > zeolite >
ion exchange resin, while in biomass was A. pullulans > activated sludge > S.
cerevisiae. The initial Pb2+ removal rate of activated sludge was higher than those of
GAC, zeolite, ion exchange resin and S. cerevisiae cells.
1. Introduction
There are numerous reports documenting the capability of pure cultures of bacteria
(Yong and Macaskie, 1997), algae (Leush et al., 1995), and fungi (Suh et al., 1998) to
remove heavy metal ions from solution. According to the report of Shumate and
Strandberg (1985), multi-species communities of bacteria removed silver equal to 32%
of the dry cell weight, which was considerably higher than those exhibited by pure
cultures of Pseudomonas maltophilia, Thiobacillus thiooxidans, and T. ferroxidans. The
important point was thus made that mixed microbial cultures could be more efficient in
removing heavy metal ions than pure cultures.
Removal of heavy metal ions by mixed cultures of activated sludge have been
studied for the last 40 years by many investigators (Ruchoft, 1949; Brown and Lester,
1982; Rudd et al., 1984) to evaluate the possibility for removing a number of heavy
metal ions.
177
DONG SEOG KIM AND JUNG HO SUH
In particular, Pb2+, well recognised for its detrimental effect on the environment where it
accumulates throughout the food web, is generated from mining, metal, dyestuff,
electric, and petroleum industries. Pb2+ was known to be easily removed by
microorganisms. Rossin et al. (1982), for example, pointed out that average heavy metal
ion removal with activated sludge was as low as 1% for Ni2+ and as high as 92% for
Pb2+. Equilibrium metal uptake values using freeze-dried Rhizopus arrhizus increased in
the order of Sr2+ < Mn2+ < Zn2+ < Cd2+ < Cu2+ < Pb2+, and were positively correlated
with the covalent index of the metal ions (Brady and Tobin, 1995). The order of
adsorption for Sargassaumfluitans biomass particles (Leush et al., 1995) was in the order
of Pb2+ > Cd2+ > Cu2+ > Ni2+ > Zn2+.
A major goal of this research is to examine the feasibility of activated sludge on Pb2+
removal, by comparison with two pure cultures (Saccharomyces cerevisiae and
Aureobasidium pullulans) and widely used chemical adsorbents (activated carbons, ion
exchange resin and zeolite).
2.1. MATERIALS
Activated carbon, which is used in a filtration plant, was made with charcoal and the
sizes of granular activated carbon (Sigma C2764, USA) and powdered activated carbon
(Sigma C5260, USA) were 4~8 mesh and 100~400 mesh, respectively. Cation exchange
resin (SK1B) with sulfonyl group was purchased from Sam Yang Co. (Korea). Zeolite
(Z3125) was obtained from Sigma Co. as a powder type, of which diameter was below
10 m. Activated sludge was prepared from the secondary return-sludge from the
municipal wastewater treatment plant.
2.2. MICROORGANISMS AND CULTURE CONDITIONS
Aureobasidium pullulans KFCC (Korean Foundation of Culture Collection) 110245 was
aerobically cultivated with 100 ml medium containing (as g/l) 200, sucrose; 20, yeast
extract; 5, K2HPO4; 2, MgSO4.7H2O; 15, NaNO3 in a rotary-shaker incubator (150 rpm)
at 300 for 72 h. Saccharomyces cerevisiae KCTC (Korean Collection for Type
Cultures) 1199, wasted from the brewery industry, was cultured at 30C for 72 h in 300
ml conical flasks with 100 ml medium composed of (as g/l) 100, glucose; 8.5, yeast
extract; 1.32, NH4Cl; 0.11, MgSO4; 0.06, CaCl2 in a rotary-shaker incubator with 150
rpm. Activated sludge or pure cultures were harvested by centrifugation (3,000 x g, 10
min) and then washed three times with distilled deionised water, and stored at 4 in a
refrigerator until uses in the experiments.
2.3. PB2+ REMOVAL EXPERIMENT
In the case of biomass, the prepared activated sludge or cell suspension was mixed with
an equal volume of the initial concentration of aqueous Pb(NO3)2 solution prepared in
twice the desired concentration. The pH values of the Pb2+ solution, biomass
178
PB2+ REM0VAL CHARACTERISTICS OF BIOMATERIALS AND NON-BIOMATERIALS
suspensions, and the mixture were 3.0-4.0, 5.5-5.7 and 3.5-5.5, respectively. pH
adjustment was not conducted and no spontaneous metal precipitation was observed in
the prepared solutions. The experiments were carried out by employing 50 ml Pb2+
solution and 50 ml activated sludge or cell suspension in 300 ml conical flasks and
shake them on a rotary-shaker incubator at
(150 rpm). Samples of 1.8 ml were
taken at the proper time period and centrifuged immediately (10,000 x g, 10 min). The
Pb2+ concentration in the supernatant was measured by atomic absorption spectrometry
(Perkin Elmer 3300, USA). The dry weight of biomass was obtained after drying at
105| to a constant weight. Removed Pb2+ amounts per dry weight of adsorptive
materials at equilibrium state (q) were calculated from Pb2+ mass balance yield : q (mg
Pb2+/g dry weight) = (Ci - Ce)/m. Where Ci and Ce are the Pb2+ concentrations (mg
Pb2+/l) in the supernatant at the initial and equilibrium state, respectively, and m is the
concentration of the dried activated sludge or cells (mg dry weight/l). The initial Pb2+
removal rate (ri) was measured by calculating the slope from the plot of the removed
Pb2+ amounts per dry weight (mg Pb2+/g dry weight) vs. time (min) at t=0.
3.1. PB2+ REMOVAL CHARACTERISTICS
Fig, 1. Typical time courses of Pb2+ removal by chemical adsorbents under various initial
Pb2+ concentrations: (a) granular activated carbon, (b) powdered activated carbon, (c) ion
exchange resin, (d) zeolite. Initial adsorbent concentrations (g/l) in (a), 1.0; (b), 2.0; (c),
1.0; (d) 1,0,
The order of Pb2+ removal capacity in chemical adsorbents was found as ion exchange
resin > zeolite > granular activated carbon (GAC) > powdered activated carbon (PAC)
(Fig. 1). On the comparison between the results of GAC and PAC, the Pb2+ removal
179
capacity of GAC was three times higher than that of PAC. However, the time required
to reach an equilibrium state with GAC was much longer than with PAC because GAC
has much larger specific surface area and pore diffusion resistance than PAC.
The increase in Pb2+ removal capacity according to the increase of initial Pb2+
concentration in biomass was similar to those of chemical adsorbents (Fig. 2). The Pb2+
removal capacity of the activated sludge was increased only 1.8 times (from 42 mg
Pb2+/g to 74 mg Pb2+/g) even though the initial Pb2+ concentrations increased by 6-fold
(from 37 mg/l to 228 mg/l).
Fig.2. Typical time courses of Pb2+ removal by biomass various initial Pb2+ concentrations:
(a) activated sludge, (b) A, pullulans, (c) S. cerevisiae. Initial biomass concentrations (g/l)
in (a), 1.0; (b). 0.8; (c), 0.8.
An interesting result was obtained by the two selected microorganisms. That is, in our
experimental range, as the initial Pb2+ concentrations were increased accurately 5.7
times (from 49 mg/l to 278 mg/l) and 6 times (from 16 mg/l to 96 mg/l) in A. pullulans
and S. cerevisiae, respectively, the Pb2+ removal capacities were increased about 5.7
times (from 53 mg Pb2+/g to 300 mg Pb2+/g) and 6 times (from 12 mg Pb2+/g to 72 mg
Pb2+/g), respectively. The Pb2+ removal in pure cultures of A. pullulans and S. cerevisiae
gave more favourable result than activated sludge.
Considering Pb2+ removal characteristics, it was found that the time required to
reach an equilibrium state in activated sludge and A. pullulans was independent on the
initial Pb2+ concentrations. But, in S. cerevisiae, the time was increased in response to
180
PB2+ REMOVAL CHARACTERISTICS OF BIOMATERIALS AND NON-BIOMATERIALS
the initial Pb2+ concentration, and was much longer than those of the activated sludge
and A. pullulans. This may be caused by the difference in Pb2+ removal process. In other
words, most of the Pb2+ taken up by S. cerevisiae was deposited in the inner parts of the
cell at equilibrium state and the Pb2+ accumulation mechanism was divided into three
steps, involving metabolism-independent, -dependent and -independent (Suh et al,
1998a). On the contrary, A. pullulans used a different metabolism-independent Pb2+
accumulation process due to the existence of extracellular polymeric substances (EPS)
(Suh et al., 1998b). Therefore, it was suggested that Pb2+ penetration into inner cellular
regions may not occurred in the activated sludge because the trend in Pb2+ removal of
the activated sludge has a resemblance to that of A. pullulans. Nevertheless, the detailed
process is still in doubt because activated sludge is composed of a great number of
organisms and several organic materials such as extracellular polymeric substances and
cell flocs, etc.
When compared the Pb2+ removal capacity of chemical adsorbents with those of
biomass, the Pb2+ removal characteristics of activated sludge and A. pullulans marked
similar process to that of PAC. Moreover, S. cerevisiae had a similar removal process to
that of ion exchange resin. The ion exchange played an important role in the Pb2+
removal in S. cerevisiae but little occur in A. pullulans (data not shown). A similar
result was observed when the initial chemical adsorbents or biomass concentrations
were changed, too (data not shown).
Table 1. Comparison of adsorption models and the
at q10 and q200 between the chemical absorbents and biomass
Materials
Granular
carbon
Powdered
carbon
activated
Model (r2 value)

Langmuir Freundlich
removed Pb2+
amounts
q10* (mg Pb2+/g)
q200 * (mg Pb2+/g)
0.95
0.84
26.0
36.5
0.96
0.90
2.1
17.3
Ion exchange resin
0.92
0.92
167.7
Zeolite
0.99
0.91
30.2
57.7
Activated sludge
0.93
0.98
30.9
68.8
A. pullalans
0.72
0.82
170.4
235.8
activated
95.3
272.7
0.97
0.94
S. cerevisiae
*q10 and q200 represent removal amouts of Pb2+ (mg Pb2+/g absorbent or biomass) at the equilibrium
concentrations of 10 mg/l and 200 mg/l, respectively.
The Pb2+ removal capacity in chemical adsorbents, evaluating by q10, was in the order of
ion exchange resin > zeolite > GAC > PAC, while that of biomass was A. pullulans > S.
cerevisiae > activated sludge. A. pullulans showed remarkably higher Pb2+ removal
capacity than S. cerevisiae in q10 due to the effect of EPS formation. However, the q200
value of S. cerevisiae was slightly higher than that of A. pullulans. The terms of q10 and
q200 were the Pb2+ removal amounts (mg Pb2+/g) at the equilibrium concentrations of 10
mg/l and 200 mg/l, respectively. This interesting phenomenon was observed when the
181
initial Pb2+ concentration was very high or initial biomass concentration was extremely
low. This result may be caused by the difference in Pb2+ removal mechanisms, but
further study is required to reinforce this result.
In general, q10 may be more reasonable than q200 from the practical points of view. The
Pb2+ removal capacity of the activated sludge was relatively lower than those of other
biomass (A. pullulans and S. cerevisiae) or ion exchange resin, but higher than those of
other chemical adsorbents (GAC, PAC, and zeolite). This result implies the possible
application of the activated sludge on Pb2+ removal process.
The Pb2+ removal capacity of the activated sludge was around one to fifth and one to
third as low as those of A. pullulans and S. cerevisiae, respectively. However, the
utilisation of activated sludge on Pb2+ removal can be recommended, taking into
consideration of economical and practical aspects and the stability of operation system.
3.2. INITIAL PB2+ REMOVAL RATE
In heavy metal removal processes, not only the removal capacity but also the initial
removal rates are considered to be very important factors from the practical aspects of
reactor design and process optimisation. The initial Pb2+ removal rate in response to the
variation of initial Pb2+ concentration is shown in Fig. 3. The initial Pb2+ removal rate
was increased as initial Pb2+ concentration increased. However, it was almost
independent of initial Pb2+ concentration over a critical concentration. The Pb2+ removal
rates of activated sludge and A. pullulans were much higher than those of the chemical
adsorbents, but S. cerevisiae was not the case. The order of initial rate of Pb2+ removal
in adsorbents was found as PAC > GAC > zeolite > ion exchange resin (Fig. 3(a)).
Where, the low degree of resistance in pore diffusion of PAC might cause the highest
initial Pb2+ removal rate. Moreover, the ion exchange resin showed the lowest initial
Pb2+ removal rate because ion exchange is here main process rather than physical
adsorption.
Fig. 3. Comparison of the initial Pb2+ removal rates between (a) chemical adsorbents and
(b) biomass: Symbols in (a); (0) GAC,
PAC,
ion exchange resin,
zeolite.
Symbols in (b); (0) activated sludge,
A. pullulans, (A) S. cerevisiae.
182
PB2+ REMOVAL CHARACTERISTICS OF BIOMATERIALS AND NON-BIOMATERIALS
On the other hand, the initial Pb2+ removal rate in biomass was in the order of A.
pullulans > activated sludge > S. cerevisiae, as shown in Fig. 3(b). The Pb2+ removal in
A. pullulans was very rapid, whereas that in S. cerevisiae was very slow. This is, as
mentioned earlier, because Pb2+ removal in A. pullulans was mainly achieved by
adsorption onto EPS around the cell surface and that in S. cerevisiae was caused by the
Pb2+ penetration into the inner cellular region. Therefore, by evaluating as initial Pb2+
removal rate, the activated sludge was placed in the middle of A. pullulans and S.
cerevisiae, and it can be recommended as an useful resource for the removal process of
Pb2+.
4. Conclusions
The comparison of Pb2+ removal characteristics between chemical sorbents (GAC,
PAC, zeolite, ion exchange resin) and biological materials (activated sludge, A.
pullulans, S. cerevisiae) was conducted. The Pb2+ removal capacities of biological
materials were higher than that of chemical materials. And the biological materials
showed the higher initial Pb2+ removal rate than the chemical materials, except the case
of ion exchange resin. Therefore, the biological materials can be applied effectively to
the heavy metal removal process even though the application will be needed more
research works. Especially, activated sludge that is used in municipal wastewater
treatment facility may be easily applied to the continuous heavy metal removal process,
in situ, compared to other biological materials
References
Brady, J.M. and Tobin, J.M. (1995) Binding of hard and soft metal ions to Rhizopus arrhizus biomass.
Enzyme Microb. Technol. 17, 791-796.
Brown, M.L. and Lester, J.N. (1982) Role of bacterial extracellular polymers in metal uptake in pure bacterial
culture and activated sludge-I Effects of metal concentration, Water Res. 16, 1539-1548.
Leusch, A., Holan, Z.R. and Volesky, B. (1995) Biosorption of heavy metals (Cd, Cu, Ni, Pb, Zn) by
chemically-reinforced biomass of marine algae. J Chem. Tech. Biotechnol. 62, 279-288.
Rossin, A.C., Sterritt, R.M. and Lester, J.N. (1982) The influence of process parameters on the removal of
heavy metals in activated sludge. Water, Air, andSoil Pollution 17, 185-198.
Ruchoft, C.C. (1949) The possibilities of disposal of radioactive wastes by biological treatment methods.
Sewage Works J. 21, 877-883.
Rudd, T., Sterritt, R.M. and Lester, J.N. (1984) Complexation of heavy metals by extracellular polymers in
the activated sludge. J Water Pollut. Control Fed. 56, 1260-1268.
Shumate II, S.E. and Strandberg, G.W. (1985) Accumulation of metals by microbial cells, in M. Moo-Young
(ed.), Comprehensive Biotechnology, Pergamon Press, New York, vol. 4, pp. 235 - 247.
Suh, J.H., Kim, D.S., Yun, J.W. and Song, S.K. (1998a) Process of Pb2+ accumulation in Saccharomyces
cerevisiae. Biotechnol. Left. 20, 153-156.
Suh, J.H., Yun, J.W. and Kim, D.S. (1998b) Comparison of Pb2+ accumulation characteristics between live
and dead cells of Saccharomyces cerevisiae and Aureobasidium pullulans. Biotechnol. Lett. 20,247-251.
Yong, P. and Macaskie, L.E. (1997) Removal of lanthanum, uranium and thorium from the citrate complexes
by immobilised cells of Citrobacter sp. in a flow-through reactor: implications for the decontamination of
solutions containing plutonium. Biotechnol. Lett. 19, 251-255.
183
HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERIA

GY. BARABS1, GY. VARGHA1, I. SZAB1, A. PENYIGE1, J.
SZLLSI2, J. MATK2, S. DAMJANOVICH2 AND T. HIRANO3
University Medical School, Departments of Human Genetics1 and
Biophysics & Cell Biology2, H-4012 Debrecen, Hungary;
E-mail: barabas@jaguar.dote. hu, and
The Jikei University, School of Medicine3, Tokyo, Japan
Abstract
Streptomyces strains from Kuwait oil field were isolated by selective inorganic media
containing the oil fractions as sole carbon and energy sources. Hydrocarbon utilisation
was measured by gas-liquid chromatography, and by the use of proper radioactively
labelled oil fraction. Streptomyces strains rapidly used n-alkanes, and incorporated them
to corresponding fatty acids of identical chain length. n-Alkane uptake was markedly
increased by specific GTP-binding protein activators. Fluorescence measurements of the
uptake of the hydrophobic diphenylhexatriene (DPH) showed significant difference
between oil-utilising and non-utilising strains.
1.1 TEST ORGANISMS. OLIGOCARBOPHYLIC STREPTOMYCES
Strains isolated from Kuwait desert oil fields (Barabs et al., 1995) were coded as KCC
(Kuwait Culture Collection). KCC26, KCC28, KCC30 and KCC42 were identified as
Streptomyces plicatus, KCC25 as Streptomyces griseoflavus. Strains KCC18, KCC33,
KCC36 and Khiran30 have not been taxonomically identified yet, they show, however,
the typical Streptomyces morphology in light microscope. The strains, their isolation,
the composition of starch-casein medium and their potential of utilising n-alkanes were
previously described (Barabs et al., 1995). S. griseus 2682 was used as an oil nonutilising control (designated as ,,non-utilising).
185
2001 KIuwer Academic Publishers. Printed in the Netherlands.
GY. BARABS ET AL
1.2 BIOMASS PREPARATION

The biomass samples were obtained by growing the test organisms on suitable
conventional media at 27C. The Streptomyces strain was cultivated on sterile
cellophane sheets covering the surface of starch-casein-agar medium: starch, 10g;
casein (Difco, vitamin free), 0.3g; KNO3, 2g; NaCl, 2g; K2HPO4, 2g; MgSO4.7H2O,
0.05g; CaCO3, 0.02g; FeSO4.7H2O, 0.01g; Bacto agar, 18g; soil extract (from 1% soil
suspension), 100 ml; distilled water to 1000 ml; pH 6.8. The cultures were incubated at
27C for 5 d. The Streptomyces biomass samples were harvested by removing the
cellophane sheets.
For fluorescence measurement of diphenylhexatriene (DPH) uptake, n-hexadecane (C16)
utilising and non-utilising strains were cultivated in filtered soybean liquid medium
described previously for S. griseus 52-1 (Szab et al., 1985). Cells were harvested after
36 h cultivation, washed twice with distilled water, resuspended in inorganic medium
(IM) with the following composition in g per litre: 0.85 NaNO3; 0.56 KH2PO4; 0.86
Na2HPO4; 0.17 K2SO4; 0.37 MgSO4.7H2O; 0.007 CaCl2.6H2O; 0.004 FeIIIEDTA; 2.5
ml of a trace element solution consisting of (g per litre): 2.32 ZnSO4.7H2O; 1.78
MnSO4.4H2O; 0.56 H3BO3; 1.0 CuSO4.5H2O; 0.39 Na2MoO3.2H2O; 0.66 KI; 1.0
EDTA; 0.4 FeSO4.7H2O; 0.004 NiCl2.6H2O.
Incubation was carried out at 27C in a gyratory shaker at 300 rpm. The optical density
of the cell suspension was adjusted to 0.5 at 535 nm and 2% (V/V) of n- hexadecane
(C16) was added to IM.
1.3 INCORPORATION OF RADIOACTIVITY FROM LABELLED
n-HEXADECANE INTO MYCELIA
Strains were cultivated for 48 hours in 5 ml of inorganic medium (IM) supplemented
with 20% of filtered soybean medium (Szab et al., 1985) in 50-ml Erlenmeyer flasks. 2
l of hexadecane-1-14C (105 dpm) was added to each culture at the inoculation. After 48
h of incubation mycelia were pelleted at 3000g. 100 mg aliquots of the mycelia were
hydrolysed with 6 n HCl at 95C for 48 h. The HCl and traces of not metabolised
labelled oil was evaporated. After dissolving the dried remnant material in 200 l water
it was extracted with hexane. The radioactivities of both the water and hexane phases
were measured by scintillation counting.
1.4 FLUORESCENCE MEASUREMENTS
The different potential of C16 utilising and non-utilising strains to take up hydrocarbons
was studied by fluorescence measurements. For this purpose, the hydrophobic
compound diphenylhexatriene (DPH) was selected as the test fluorescent material.
Fresh biomass samples of C16 utilising and non-utilising strains grown in filtered
soybean medium were resuspended in IM than the samples were supplemented with 90
M DPH dissolved in n-hexadecane. Their fluorescence was examined in quartz cells in
a Perkin-Elmer MPF 44B spectrofluorimeter equipped with a thermostatic cell holder.
The DPH fluorescence of cells was excited at 355 nm and the emission was collected at
430 nm. The cell suspensions did not show significant autofluorescence in this
wavelength range. In experiments when the kinetics of DPH uptake was investigated,
186
HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERlA
small aliquots (2ml; n=3) were taken from the culture flask from time to time, washed
and the DPH fluorescence (corrected for light scattering) was determined.
1.5 ANALYSIS OF FATTY ACIDS
The fatty acid determinations were performed as described by Barabs et al., 1995.
1.6 INVESTIGATIONS WITH GTP ANALOGUES
0.5 g aliquots of mycelium precultivated in soy-bean medium for 36h were resuspended
in 25 ml aliquots of inorganic medium containing 10 mg n-hexadecane or n-octadecane.
The submerged cultures were supplemented with various amounts of GTPS or AIF4and incubated at 30C with a shaking frequency of 250 rpm. Biomass was harvested
after 6 hours by centrifugation and the hydrocarbon uptake of the cells was determined
from these samples as described in Barabs et al., 1995.

Streptomyces are soil bacteria occurring even under extreme conditions (hot desert, saltmarsh area, alpine slopes, etc.). They are capable of hydrocarbon uptake and utilisation.
Uptake of oil as followed by incorporated diphenylhexatriene (DPH) from nhexadecane phase of the medium was significantly higher in the oil utilising strain than
in a non-utilising one (Table 1, Radwan et al., 1998). Fluorescence measurements with
DPH showed that the membrane characteristics of hydrocarbon-using strains
significantly differed from that of the non-using ones (Radwan et al., 1998).
Incorporation of the label from radioactive hexadecane into mycelia was also
measured in several oil degrading strains and one non-degrading one (Table 2). During
the 48-hour cultivation with labelled hydrocarbon the highest amount found to be
incorporated (into strain KCC18) was equivalent to about 3% of the total amount of the
radioactivity added to the culture. Other strains incorporated somewhat less amount, and
strain Khiran30, that served as a control, could not incorporate radioactivity.
Table 1 DPH fIuorescence incorporated frorn n-hexadecane phase by oil utilising (KCC 26)
and non-utilising (S griseus) strains
Time
(hour)
DPH fluorescence
(arbitrary units)
KCC 26
S. griseus
0.039
0.010
0.361
0.283
0.65 1
0.324
0.820
0.315
0.795
0.344
0.852
0.308
19
40
62
91
122
182
187
GY. BARABS ET AL
Table 2. Incorporation of the label from hexadecane-l-14C into mycelia of Streptomyces

strains
Strain
KCC42
KCC18
KCC36
KCC33
KCC25
Khiran30
Radioactivity
(dpm)
1956
2985
1719
2070
393
Harvested mycelia were hydrolysed with 6 n HCl and the radioactivity of water-soluble components was
determined. The non-utilising Khiran30 strain was used as control.
Fatty acid analysis showed that the incubation with n-alkanes resulted in an increase of
the fatty acids with chain length equivalent to those of the alkane substrates (Barabs et
al., 1995). The fatty acid 16:1 fraction increased from 17.4 to 29.9 in the presence of nhexadecane. Fatty acids of C18 appeared only in the presence of n-octadecane.
It is known that GTP-binding proteins play essential role in mediating cellular
responses to a wide variety of extracellular signals, such as hormones, growth factors,
neurotransmitters, chemical signals or light (Gilman, 1987, Taylor, 1990). These
proteins transmit signals via a GTP-dependent mechanism to the effector systems
(enzymes or ion channels) and thereby regulate these systems that often control
production of intracellular second messenger molecules. GTP-binding proteins (GBPs)
act as molecular switches, their activation is catalysed by a ligand activated receptor and
deactivation is established by the intrinsic GTPase activity of the GBP. According to
this mechanism, the GTP-bound form is the active complex, which returns to inactive
state by hydrolysing its GTP to GDP. The exchange of GDP to GTP and the rate of
GTP hydrolysis is regulated by specific regulatory proteins (Gilman, 1987, Itoh et al.,
1986, Taylor, 1990).
GTP-binding proteins were reported to be present in S. coelicolor A(3)2 and we have
shown the presence of these proteins in S. griseus and in several other Streptomyces
strains (Itoh et al., 1996, Penyige et al., 1992). Our previous results suggested that in S.
griseus A-factor - a -utyrolactone type autoregulator molecule produced by wild type
S. griseus cells and required for the normal differentiation process and antibiotic
production in the producer strain (Khokhlov et al., 1973) - could activate an intrinsic
GTPase activity present in the cellular membrane of S. griseus NRRL B-2682 (Penyige
et al., 1992).
The study of their role in different cellular processes was greatly enhanced by using
certain reagents, such as AIF4- or GTPS. AlF4- mimics the effect of the -phosphate of
GTP on the inactive GDP-bound form of the protein, GTPS is a non-hydrolysable GTP
analogue (Yatani et al., 1991).
188
HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERIA

Table 3. The effect of AlF4- on n-hexadecane and n -octadecane uptake by oil utilising micro-organisms
Microbial strains
n-alkanes
KCC25
C16
C18
C16
C18
C16
C18
C16
C18
C16
KCC 28
KCC 33
KCC 42
A. nicotiana
M AIF40
7.2
6.3
8.0
5.2
9.2
7.2
7.6
10.3
5.9
50
10.0
ND
9.4
4.9
10.5
ND
12.3
ND
4.9
75
12.5
ND
9.8
5.3
10.9
6.2
ND
12.4
6.7
100
12.0
ND
9.7
7.8
11.8
6.6
13.7
11.2
12.9
125
11.6
ND
9.9
10.2
13.6
8.2
15.7
11.8
10.8
Data are expressed in mg alkane consumed by 1 g fresh biomass at 27C in 6 h

ND: not determined
Arthrobacter nicotiana was used as control strain. In this case the incubation period was 24 h.
Table 4. The effect of GTPS on n -hexadecane uptake by oil utilising micro-organisms
GTPS [g/ml]
0
10
20
50
100
KCC 25
3.2
12.3
12.8
ND
13.4
n-Hexadecane consumed
Arthrobacter nicotiana
1.8
4.2
6.9
9.9
11.1
Data are expressed in mg alkane consumed by 1 g fresh biomass at 27C in 6 h.

ND: not determined
Arthrobacter nicotiana was used as control strain. In this case the incubation period was 24 h.
The results of the effect of GTPS and AIF4- stimulators of GBPs on the uptake of
the hydrocarbon molecules (C16 and C18) are shown in Tables 3 and 4. These show that
the uptake of n-hexadecane (C16) and n-octadecane (C18) from the medium by
hydrocarbon utilising micro-organisms were enhanced by the addition of
and
AIF4- although the rate of uptake was strain specific. Moreover, the magnitude of
uptake was, in most cases, directly proportional to the concentration of these effectors in
the medium. These results suggest that GBPs could fulfil important physiological
functions in Streptomyces strains.
With electron microscopy the hydrocarbon utilising strains were found to be
enriched in large less-electron dense areas as inclusions in the cytoplasm in n-alkane
189
GY. BARABS ET AL
containing media (Radwan et al., 1998). The oil utilising strains also eliminated
hydrocarbons from soil samples artificially impregnated with hydrocarbons in
laboratory experiments. Field experiments for oil bioremediation are in progress.
3. Conclusion
Streptomyces strains isolated from Kuwait oil fields actively utilised oil fractions and
crude oil, either in hydrocarbon containing inorganic media, or in the soil. Since these
strains are typical soil bacteria tolerating extreme conditions (hot desert, alpine slopes,
salt-marsh area) their practical application in bioremediation of oil pollution is
promising.
References
Barabs, Gy., Sorkhoh, N.A., Fardoon, F. and Radwan, S.S. (1995) n-Alkane-utilisation by oligocarbophilic
actinomycete strains from oil-polluted Kuwaiti desert soil. Actinomycetologica 9, 13-18.
Gilman, A. G. (1987) G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56,615-649.
Itoh, H., Kozasa, T., Nagata, S., Nakamura, S., Katada, T., Ui, M., Iwai, S., Ohtsuka, E., Kawasaki, H.,
Suzuki, K. and Kaziro, Y. (1986) Molecular cloning and sequence determination of cDNAs for a subunits
of the guanine nucleotide-binding proteins Gs, Gi and Go from rat brain. Proc. Nail. Acad. Sci. USA 83,
3776-3780.
Itoh, M., Penyige, A., Okamoto, S. and Ochi, K. (1996) Proteins that interact with GTP in Streptomyces
griseus and its possible implication in morphogenesis. FEMS Microbiol. Lett. 135, 311-316.
Khokhlov, A. S., Anisova, L.N., Tovarova, J.J., Kleiner, E.M., Kovalenko, O.S., Krasilnikova, O.S.,
Kornitskaya, E.Y. and Pliner, S.A. (1973) Effect of A-factor on growth of asporogeneous mutant of
Streptomyces griseus, not producing this factor. Z Allg. Microbiol. 13,647-655.
Penyige, A., Vargha, Gy., Ensign, J.C and Barabs, Gy. (1992) The possible role of ADP-ribosylation in
physiological regulation in Streptomyces griseus. Gene 115, 181-185.
Radwan, S.S., Barabs, Gy., Sorkhoh, N.A., Damjanovich, S., Szab, I., Szllsi, J., Matk, J., Penyige, A.,
Hirano, T. and Szab, I.M. (1998) Hydrocarbon uptake by Streptomyces. FEMS Microbiol. Letters 169,
87-94
Szab, I., Benedek, A. and Barabs, Gy. (1985) Possible role of streptomycin released from spore cell wall of
Streptomyces griseus. Appl. Environ. Microbiol. 50, 438-440.
Taylor, C. V. (1990) The role of G proteins in transmembrane signalling. Biochem. J. 272, 1-13.
Yatani, A,, and Brown, A.M. (1991) Mechanism of fluoride activation of G protein-gated muscarinic atrial
K+ channels. J. Biol. Chem. 266,22872-22877.
190
PART 6
FOOD SECURITY AND FOOD PRESERVATION
MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL

PATHOGENS: A REVIEW
M. HEYNDRICKX, N. RIJPENS AND L. HERMAN
Ministry of Small Enterprises, Traders and Agriculture, Centre for
Agricultural Research, Department for Animal Product Quality,
Brusselsesteenweg 3 70, B-9090 Melle, Belgium
Abstract
In most developed countries a sharp increase in foodborne intoxications occurs since the
last decade. Amongst the bacterial pathogens, the incidence rate is highest for
Campylobacter jejuni and Salmonella spp. Nucleid acid based identification and
detection methods have been developed for nearly all bacterial pathogens, based on
probes in hybridisation assays or primers in PCR, NASBA or RT-PCR assays. As
targets for molecular identification, virulence genes, the rRNA gene region or other
specific sequences can be used and several commercialised systems are already
available. For the (direct) detection of pathogens in food products, several problems
may be encountered: PCR inhibition by food components, contamination in sensitive
PCR assays, detection of living as well as dead cells. The latter problem can be solved
by using mRNA as amplification target, but for routine applications the combination of
a short culturing period with a less sensitive PCR is more suitable. Direct quantification
of pathogens is possible with quantitative competitive PCR using an internal standard or
with kinetic quantitative PCR (TaqMan or LightCycler commercial system).
In bacterial typing, distinct types, strains or clones within a pathogenic bacterial
species are differentiated which is important in epidemiological studies of foodborne
outbreaks but also in the from stable to table investigation of the whole food
production chain. Compared to the classical phenotypic typing techniques, molecular
typing techniques have several advantages such as general applicability and a high
discriminatory power. The currently available molecular techniques can be classified
according to their working principle in PCR-mediated typing techniques (RAPD, repPCR), typing techniques combining PCR with restriction analysis (e.g.flaA typing of C.
jejuni), typing techniques based on chromosomal restriction fragment length
polymorphisms (e.g. ribotyping, pulsed field gel electrophoresis or PFGE), typing
techniques combining restriction digestion with selective amplification (AFLP), and
plasmid analysis. Both PFGE and AFLP are proposed as likely candidates for a uniform
definite molecular typing approach using appropriate software for cluster analysis and
193
M. HEYNDRICKX N. RIJPENS AND L. HERMAN
database storing of the fingerprints. For Salmonella, two typing levels can be proposed:
the first important level corresponds with the serovar level and the second level can be
performed by classical phage typing or molecular typing revealing clonal lineage or
strain level. Several molecular techniques have a serovar dependent discriminatory
power with the greatest challenge presented by the highly clonal serovar Salmonella
enteritidis.
1. Introduction
In the last decade a sharp increase in foodborne intoxications has been observed in most
developed countries. This may sound surprising because it is generally assumed that
hygiene at home during cooking and food storage and in manufacturing practices in the
food industry has greatly improved and is of a sufficiently high level in this hightechnology society. This phenomenon may be attributed to several factors such as the
increasing international trade in food and food and feed ingredients, international
tourism, climate changes, demographic changes in the population with a higher number
of elderly people, changing eating habits with a greater proportion of ready-to-eat foods
and mass catering facilities. The general trend is that the zoonoses, i.e. infectious
diseases caused by animal associated microorganisms such as Salmonella, are becoming
a particular increasing problem which is probably associated with the current bioindustrial practice of mass production of animal products (e.g. poultry). In the food
industry, food processors and regulators are asked to establish, control and monitor
critical control points in the plant environment as essential part of the development and
use of a HACCP concept. It is likely that this type of approach will become more
important in the near future as food safety concerns increase in food processing and
distribution systems. Molecular detection and typing methods are powerful tools in the
whole food production chain for quality control and to unravel the prevalence and the
epidemiology of the foodborne pathogens.
In this paper a review is given of the molecular detection, identification and typing
techniques for foodborne bacterial pathogens in the last decade. Their possibilities and
eventually their associated problems, limitations and possible solutions are also
discussed with reference to own observations or those of other researchers. The term
molecular techniques is used here to denote those techniques which are molecular
biology based, and more specific those that make use of the nucleic acids (DNA or
RNA) in the bacterial cell.
2. Characteristics of the foodborne bacterial pathogens
Foodborne intoxications can be caused by viruses, pathogenic bacteria, parasitic
protozoa and nematodes, and by toxins of natural origin (e.g. scombrotoxin caused by
bacterial histamine production in food). Most of the intoxications are of viral or
bacterial nature, The most prevalent bacterial pathogens causing foodborne
intoxications in the developed world are listed in Table 1. This list contains
Gramnegatives, i.e. several representatives of the Enterobacteriaceae (pathogenic
Escherichia coli, Salmonella, Yersinia, Shigella), Aeromonas, Campylobacter and
194
MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS
Vibrio, as well as Grampositives (Clostridium, Staphylococcus, Listeria, Bacillus,

Streptococcus and Enterococcus). Other pathogenic bacterial species occurring in food
can be considered as less important because their associated foodborne intoxications
occur very rarely. Vibrio cholerae serogroup 01 is a major water and foodborne
pathogen in underdeveloped countries because of a lack of elementary hygiene, but is
almost eradicated in the western world. A lot of the minor pathogens are highly related
to the bacterial species of Table 1 (e.g. Campylobacter coli, Vibrio vulnificus, V.
cholerae serogroup non-O 1, enterotoxigenic, enteropathogenic and enteroinvasive E.
coli, Bacillus subtilis). In addition, many bacteria can be considered as putative
pathogens (e.g. Aeromonas caviae and A. sobria, marine Vibrio spp., Plesiomonas
shigelloides, several enteric bacteria such as Citrobacter freundii). Some evidence has
been obtained for a link between the human Crohns disease and Mycobacterium
paratuberculosis occurring in raw and pasteurised milk (Thompson, 1994).
The symptoms of acute foodborne diseases caused by bacterial pathogens (see Table
1) can roughly be divided in gastrointestinal symptoms, either of the upper
gastrointestinal tract (nausea, vomiting) or of the lower gastrointestinal tract (diarrhoea,
abdominal cramps), respiratory symptoms (sore throat), and neurological symptoms
(visual disturbance, vertigo, paralysis). Usually not all of these acute symptoms occur in
any individual with a foodborne infection and also the chronic symptoms and
complications are no inevitable consequences, with the exception of botulism caused by
Clostridium botulinum which in most cases leads to death if not treated with antitoxin.
The course and severity of a foodborne disease is largely influenced by the virulence
and the ingested dose of the bacterial strain involved, and by the fitness and immune
capacity of the infected host. Infants, young children (<5 years), the elderly and
debilitated persons with a compromised immune system (cancer, AIDS and transplant
patients) are particular segments of the population which are more vulnerable to
foodborne intoxications with a higher risk for complications, e.g. Salmonella enteritidis
has a mortality rate of approximately 3.6% in hospital and nursing home outbreaks.
There are large differences between the foodborne pathogens in both frequency of
occurrence and in infective dose (Table 1). Salmonella and Campylobacter are by far
the two pathogens, which are involved in most of the intoxications. The reported cases
of Salmonella infection in Belgium have more than doubled in the period 1986 (6.092
human strains) to 1997 (14.239 human strains). This is still an underestimation because
not all cases of salmonellosis, especially the less severe ones, are reported. Although not
really known to the public, it is estimated that Campylobacter is even outnumbering
Salmonella in this respect, but real data are not available in most countries because of
the more sporadic nature of the disease. Compared to these two pathogens, the
frequency of the other foodborne intoxications is mostly several orders of magnitude
smaller. Nevertheless, two pathogens, Listeria monocytogenes and E. coli 0157:H7,
deserve special attention because they are capable of causing severe disease of a
sometimes fatal nature as has been the case in several recent, large epidemics of the
latter organism in Japan, the USA and Scotland. E. coli 0157:H7, as well as Salmonella
and Shigella spp. are also very infectious organisms (order of magnitude 10 cells) in
comparison with others (B. cereus, C. perfringens, S. aureus, V. parahaemolyticus,
enterococci) which require a much higher infective dose (>105 cells) so that an obvious
spoilage of the food product should be noticeable in most cases.
195
Table I: Foodborne bacterial pathogens, nature of the disease and associated foods
196
Table 1: Continued
: data from the Bad Bug Book (http://vm.cfsan.fda,gov/`mow/intro.htm)

most important foods incriminated are indicated in bold
c: expressed per 105 inhabitants in Belgium. Data from G. Ducoffre (Surveillance van Infectieuze
Aandoeningen door een Netwerk van Laboratoria voor Microbiologie, 1997, Epidemiologische Trends 1983
1996
Wetenschappelijk Instituut Volksgezondheid - Louis Pasteur, Afdeling Epidemiologie, November 1998)
(http://www. iph.fgov. be/epidemio/epinl/plabnl/plabannl/index. htm).
a
197
3. Molecular detection and identification of foodborne bacterial pathogens

3.1 NUCLEIC ACID BASED IDENTIFICATION METHODS
The conventional methodology for the identification of bacterial pathogens is based on
the performance of sets of morphological and biochemical tests. Most of these methods
are laborious and take several days to be completed. Nucleic acid based identification
methods have a lot of advantages compared to these conventional methods. The tests are
quick so that results can be obtained within the same day of the investigation. No pure
culture is necessary and the results are highly reliable. The tests are as well
characterised by a great specificity, offering discrimination of very closely related
species and of pathogenic strains within the same species.
Identification systems using nucleic acids are based on the use of unique
oligonucleotide sequences either as probes in hybridisation assays or as primers for
enzymatic amplification of DNA (PCR) or RNA (NASBA or RT-PCR). Probes and
PCR primers, used for the identification of different foodborne bacterial pathogens, are
extensively reviewed by Olsen et al. (1 995), Feng (1996), Hill (1 996) and Scheu et al.
(1998).
3.2 THE USE OF VIRULENCE GENES AS TARGET FOR MOLECULAR
IDENTIFICATION
For molecular identification different regions of the bacterial DNA can be chosen as
target. For pathogenic organisms virulence determinants are frequently used and often
allow a very specific identification of a bacterial species (differentiation between C.
jejuni and C. coli, Gonzalez et al., 1997) or a serotype (specific identification of S.
enteritidis, Lampel et al., 1996 and of S. enteritidis and S. typhimurium, Soumet et al.,
1999). Some of these PCR identification systems (Gonzalez et al., 1997, Lampel et al.,
1996), however, were not sufficiently validated on field strains and have to be seen
more as an indication than as a real identification (Heyndrickx & Herman, personal
communication)
A lot of these virulence genes are located on plasmid DNA. When possible a
chromosomal localisation is preferred because of the instability of plasmids during lab
manipulations, This is seen for plasmid bearing Yersinia enterocolitica, where loss of
the virulence plasmid during enrichment and isolation complicates the detection of the
pathogen in food products (Bhaduri & Cottrell, 1997).
By PCR targeting virulence loci, pathogenic strains can be discriminated from nonpathogenic strains belonging to the same species, which is important for identification
of pathogenic E. coli and Y. enterocolitica strains. Research on the use of PCR for the
identification of toxin genes (LT I, LT II, ST I and ST II for enterotoxigenic E. coli;
verotoxins, VT1, VT2 and virulence genes, eaeA, hly, virulence plasmid for
enterohaemorrhagic E. coli) is in full expanse (Chen et al., 1998; Tortorello et al., 1998;
Gilgen et al., 1998; Tsen et al., 1998; Radu et al., 1998). For the identification of
pathogenic Yersinia, primers, targeting the chromosomally encoded ail (adhesioninvasion-locus) gene, can be used (Kwaga et al., 1992). Evaluation of these primers
showed no signal for any non-pathogenic Y. enterocolitica strain. The American and
198
European pathogenic strains gave a positive reaction. All non-Y enterocolitica strains
reacted negatively with the exception of some Y. kristensenii strains. The amplicon
obtained for these strains was cloned and sequenced and it was found that the whole
coding region and the 259 bp upstream region of the ail gene as present in European
pathogenic Y. enterocolitica was found in these Y. kristensenii strains (Rijpens et al.,
1999b).
3.3 THE USE OF
IDENTIFICATION
RRNA
GENES
AS
TARGET
FOR
MOLECULAR
For many pathogens rRNA genes are targeted. The rRNA gene contains next to very
conserved parts also variable regions in which specific primers for a certain species can
be chosen. For some pathogens however, the variability is not sufficient for
discriminating particular species and the pathogen may only be identified to the genus
level. This is the case for Brucella (Herman & De Ridder, 1992; Romero et al., 1995)
and Campylobacter where C. jejuni, C. coli and C. lari were be differentiated
(Giesendorf et al., 1992, Linton et al., 1996).
A significant advantage of using rRNA as target is the high copy number (>104/cell)
allowing the use of rRNA probes for in situ hybridisation purposes (Nordentoft et al.,
1997, describing a 23S rRNA probe for Salmonella; Wagner et al., 1998, describing a
16S rRNA probe for L. monocytogenes).
Identification of foodborne pathogens by NASBA applies till now mostly rRNA as
target molecule. In one case the mRNA from the L. monocytogenes hlyA gene was
targeted (Blais et al., 1997). The RNA is amplified through the concerted action of
avian myeloblastosis virus reverse transcriptase (AMV-RT), T7 RNA polymerase and
RNase H (Fig 1). The reaction starts with a non-cyclic phase, in which a downstream
primer containing a tail-sequence of the T7 promoter anneals to the RNA. Through the
action of AMV-RT, cDNA is formed. The RNase H hydrolyses the RNA from the
RNA-DNA hybrid, which results in a single strand of DNA to which the upstream
primer can anneal. The AMV-RT, through its DNA polymerase activity, synthesises a
second DNA strand. The T7 RNA polymerase generates then single-stranded RNA
copies, which can serve as a template in a new cycle. For food pathogens, the
identification of C. jejuni, C. coli and C. lari (Uyttendale et al., 1994, 1995a) and L.
monocytogenes (Uyttendale et al., 199Sb) is described based on 16S rRNA probes. Also
the spacer region between the 16S rRNA and 23S rRNA genes is being used as target
for PCR. The spacer region is showing a considerable variation and is therefore suitable
for species identification. The spacer region offers the possibility to develop multipathogen tests allowing the simultaneous identification of different species. The line
probe assay (LiPA, Innogenetics, Belgium), has been developed for the simultaneous
detection of Listeria spp. and L. monocytogenes (Rijpens et al., 199.5) and recently
extended to all defined Listeria species (Rijpens & Innogenetics N.V., Belgium,
personal communication). The spacer region is amplified using primers targeting quasi
universally conserved sequences at the 3 end of the 16S rRNA gene and the 5 end of
the 23S rRNA gene. The amplification product is used in a reverse hybridisation assay
with a strip where different specific oligonucleotide probes are immobilised as parallel
199
lines (line probe assay or LiPA). Specific identification is achieved by hybridisation of

PCR amplified spacer sequences to the different probes on the strips.
Figure 1. Schematic presentation of the NASBA method. Normal lines represent DNA, wavy
lines represent RNA.
3.4 THE USE OF SPECIFIC SEQUENCES WITH A KNOWN OR UNKNOWN

FUNCTION AS TARGET FOR MOLECULAR IDENTIFICATION
In fewer cases a specific sequence with a known or unknown function is used as target
for the molecular identification. Examples of the use of known genes for PCR are: the
flaA and flaB (flagellin genes) and their spacer region for the identification of C jejuni
and C. coli (Kirk & Rowe, 1994; Wegmller et al., 1993); the IS200 element for
Salmonella (Cano et al., 1993); the IS711 based multiplex-PCR system AMOS allowing
the discrimination of different Brucella spp. (Bricker & Halling, 1994). DNA sequences
with unknown function are used for the identification of Vibrio parahaemolyticus (Lee
et al., 1995) and Salmonella (ST11-ST15 amplicon, Aabo et al., 1993; Tsen et al.,
1994). The full sequence of the ST11-ST15 amplicon is recently published (Rijpens et
al., 1999a).
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3.5 THE AVAILABLE MOLECULAR IDENTIFICATION SYSTEMS

For all important bacterial pathogens, DNA probes and primers are available through
the international literature (see above) and/or through commercialised systems. The
commercialised systems are summarised in Table 2.
Table 2: List of commercialised molecular identification and detection tests for foodborne
bacterial pathogens (taken from Berben, 1998)
The first 2 systems (AccuProbe and Gene-Trak) are using a specific oligonucleotide
probe with the rRNA as target molecule in a hybridisation assay. In the AccuProbe
system the probe is labelled with an acridinium ester and is used in a hybridisation
protection assay. When the DNA probe is hybridised to its target rRNA, the acridinium
is protected from chemical hydrolysis and can react with hydrogen peroxide under basic
conditions, to produce chemiluminescence. If the probe remains unbound, the ester band
201
undergoes hydrolysis and renders the acridinium permanently non-chemiluminescent.

The Gene-Trak system uses a colorimetric DNA hybridisation assay based on a
sandwich hybridisation. The target RNA is hybridised with a capture probe fixed on a
dipstick. The hybridisation is confirmed by a signal-generating probe hybridising to the
rRNA target (Curiale & Klatt, 1990). These hybridisation systems are not using a DNA
or RNA amplification step but are directed at the highly expressed 16S and 23S rRNA.
They require at least 105 to 106 micro-organisms/ml (Mabilat et al., 1996).
Figure 2 Schematic presentation of TaqMan (Applied Biosystems Division of Perkin Elmer,

Foster City, CA) The probe is labelled with a reporter (R) and a quencher (Q) dye
The other 4 systems (Probelia, BAX, GeneSTAR and TaqMan) are using PCR to
specifically amplify the target DNA but differ in their detection system. They provide
all PCR reagents in ready to use reaction mixes, including positive and negative
controls. The Probelia system uses an ELISA detection protocol in a microtiter plate
format. The BAX system detects the amplified product by gel electrophoresis or
temperature dependent fluorescence analysis using SY BR green I (L. monocytogenes:
Stewart & Gendel, 1998; Salmonella: Mrozinski et al., 1998, Bennett et al., 1998; Tige
et al.,1998, E. coli O157:H7, Johnson et al., 1998, Tseng & Ghandi, 1998). In the latter
case the fluorescence is monitored in the PCR tubes during an additional thermal cycle
consisting of a denaturation step and a product annealing step. The GeneSTAR system
uses fluorescent primers for PCR and hybridises the labelled PCR amplicon to
202
microtiter-bound oligonucleotide probes. The TaqMan PCR detection takes advantage

of the endogenous 5, 3-endonuclease activity of Taq DNA polymerase to digest an
internal fluorogenic probe, which is labelled with both a fluorescent reporter dye and a
quencher dye (Fig. 2). A reporter dye is covalently attached to the 5 end of the
fluorogenic probe. A second fluorescent dye, which is capable of quenching the
emission of the reporter dye, is covalently attached to one or more nucleotides
downstream from the reporter. During the amplification of the target, the fluorogenic
probe is displaced by the Taq DNA polymerase and then cleaved, releasing the reporter
dye. The fluorescence emission intensity of the reporter dye increases because it is no
longer quenched by the proximal quenching dye. The increase in fluorescence is
quantitative for the amount of PCR product (Batt 1997, see further).
3.6 PCR DETECTION OF BACTERIAL PATHOGENS IN FOOD PRODUCTS
As already mentioned, PCR opens a lot of possibilities for reliable and quick
identification of bacterial pathogens. By, PCR the DNA target is enzymatically
amplified and therefore could be applied to decrease the bacterial culturing time,
normally applied in the conventional detection methods. It is even possible to directly
detect the pathogen in the food product as is established for e.g. the detection of L.
monocytogenes (Herman et al., 1995) and Brucella (Rijpens et al., 1996) in raw milk.
However, when PCR is applied for the detection of pathogens in food products, some
problems could be encountered.
3.6.1 Influence offood components on PCR performance
Because PCR is an enzymatic reaction, it is not unexpected that a lot of food
components can inhibit the amplification of the DNA target. While PCR is rarely
inhibited when the technique is used for colony confirmation on agarplates, inhibition is
common when PCR is applied to detect pathogens in the enrichment media. The use of
a positive control is therefore indispensable when the methods are applied in routine
laboratories. This positive control could be amplified in separate tubes or could be coamplified with the target DNA. In the last case one has to consider destabilisation of the
system because of competition between the amplification of the positive control DNA
and the target DNA. This is especially the case when the same primer pair is used to
amplify both control DNA and target DNA. Although this competition could be easily
stabilised to confirm colonies on agarplates, this is much more difficult when PCR is
applied for detection in enrichment media (Rijpens et al., 1999a). A lot of problems
with PCR inhibition could be solved by the application of a suitable sample preparation
method (Lantz et al., 1994; Olsen et al., 1995; Hill 1996).
3.6.2 Sensitivity and contamination of PCR

By PCR a very high sensitivity of detection can be obtained when more than 35 cycles
or a two-step PCR protocol were used. Herman et al. (1995) reported a statistical
determined limit between 10 and 5 cfu for the detection of L. monocytogenes in 25 mI
of raw milk when a nested PCR protocol was used. The detection limit of a single 30
cycles PCR was about 1000 times higher.
203
When highly sensitive PCR protocols are used, one has to consider the possibility of
PCR contamination. A good lab hygiene protocol eventually combined with an anticarry over system can overcome most of the problems when a single 30 cycles PCR is
applied. More serious contamination problems can occur when very sensitive PCR
protocols are applied in routine laboratories.
3.6.3 The detection of the viability of cells by DNA based technology
When no or very limited culturing of the bacterial cells occurs, it is not possible to make
a distinction between the detection of living or dead cells when PCR is applied on DNA
(Masters et al., 1994; Herman L., 1997). The efficiency by which dead cells are detected
depends on the way they were killed. DNA could even survive a normal autoclave cycle
(121C for 15 min) in the presence of 0.5 - 2.0 M NaCl (Masters et al., 1998).
Ribosomal RNA would be less stable than DNA and more closely associated with
cellular viability as is shown by a decrease in NASBA signal after antibiotic exposure
and an equal signal for PCR (van der Vliet et al., 1994). However, also for rRNA the
way the cells are killed is important: a decrease in rRNA was observed when the
bacteria were subjected to carbon starvation, heat stress and osmotic stress, no decrease
was seen after cold stress, acetic acid or ethanol treatment (Tolker-Nielsen & Molin,
1996; Tolker-Nielsen et al., 1997). Ribosomal RNA would be a good indicator for
viability under extreme heat inactivation and UV irradiation of cells. At moderate
conditions however signals were still detected 48 h after heat exposure (McKillip et al.,
1998). Many food-processing heat treatments involve moderate conditions indicating
that detection of rRNA may not be associated with viability under these conditions.
Uyttendaele et al. (1997) applied NASBA on the 16S rRNA in an artificial
contamination experiment of poultry with heat killed (10 min at 100C) C. jejuni cells.
False-positive results were still obtained at the last measuring on 12 days after killing of
the cells.
To overcome the problem of the detection of dead bacterial cells the use of
messenger RNA as target for the amplification reaction is investigated. Messenger RNA
can be amplified by RT-PCR or by NASBA. Messenger RNA is associated with
metabolic activity of the cell and is characterised by a short half life (reviewed by
Pedersen et al., 1978; Higgins et al., 1988; Belasco & Higgins, 1988). More than for
PCR the choice of the target is of great importance for the sensitivity and reliability of
the test. Genes with an inducible expression as is the case for some virulence factors
(e.g. the listeriolysin O gene of L. monocytogenes) and regulation proteins (e.g. prfA
gene product as positive regulator factor of several Listeria virulence factors) seem not
to be suitable for RT-PCR detection of the pathogen in food products (Herman L., 1997;
Klein & Juneja, 1997). Because of the inducible expression, the extraction efficiency of
their transcripts is variable. More sensitive results were obtained with RT-PCR targeting
the iap gene coding for p60, a major extracellular protein of L. monocytogenes that is
thought to be associated with invasion of phagocytic cells (Klein & Juneja, 1997). In
this report, the specific detection of viable cells was only investigated after autoclaving
conditions (15 min at 121C). The use of housekeeping genes as the elongation factor as
target for RT-PCR seems reasonable for several reasons (Vaitilingom et al., 1998).
First, the elongation factor may be considered as an appropriate viability marker since
inactivation is a lethal event. Second, the elongation factor gene encodes one of the
204
most abundant proteins, allowing a considerable increase in the sensitivity level. Third,
the function and the primary structure of the gene is conserved between living cells
allowing the modulation of the specificity of detection. Using the elongation factor
messenger as target a sensitive detection (detection level of 10 cells per ml of milk) was
obtained with a specificity for viable cells within about 6 to 8 min after heat treatment
(15 min at 65C) (Vaitilingom et al., 1998). Although these results look promising, a lot
of research will have to be performed before RT-PCR could be used in routine
laboratory practice. The main problem is the reliable and constant destruction of spores
of DNA, which could trouble the correlation with the viability of the cells. Another
problem is the dependence of the sensitivity of the test on the absence of RNA
degrading enzymes during the RNA extraction procedure. This is especially important
because intensive DNase treatments could be necessary for sensitive detection systems
(Rijpens N., personal communication).
For routine application the combination of a short period of culturing (e.g. 16 h
during the night) with a less sensitive PCR detection is most often used to specifically
detect living cells by PCR (see protocols of all commercial PCR systems mentioned in
Table 1). Using the screening/Salmonella BAX method it was estimated that 104 cfu/ml
has to be present in the primary enrichment medium to be detected by PCR (Mrozinski
et al., 1998; Bennett et al., 1998). For the Listeria monocytogenes BAX system the
estimated number was 105- 106cfu/ml (Stewart & Gendel, 1998). Only when highly
contaminated (numbers of 104 to 105cfu/g or ml) products are investigated killed cells
could be detected using these combined protocols. Other advantages of inchding an
enrichment step are the possibility to use less specialised sample preparation methods,
the lower PCR sensitivity which decreases the problems of PCR contamination and the
easier validation of the PCR method which would correlate better with the conventional
method (see further).
3.7 EVALUATION AND VALIDATION OF DNA BASED METHODS

As DNA based methods are used more frequently to detect bacterial pathogens in food
products, there is a great need for a thorough evaluation of the numerous protocols. This
is difficult because the inherent uncertainties of food analysis are coupled with new
variables introduced by the so called rapid methods. Evaluation of these methods is
easiest for these pathogens where the conventional method can be considered as a real
standard to which the results for the rapid methods can be compared. This is the case for
the detection of Salmonella. More problems are encountered when the conventional
detection method lacks sensitivity as is the case for e.g. L. monocytogenes and E. coli.
Artificial inoculation experiments of E. coli O157:H7 in ground beef revealed a
detection rate of 96.5% with the BAX for Screening/E. coli O157:H7 (commercial PCR
method) compared to 39% for the best cultural method (Johnson et al., 1998). Even the
use of a combination of 4 recovery methods yields an unacceptably low recovery rate
(67%). With the Probelia kit for detection of L. monocytogenes in raw milk cheeses a
high sensitivity is reached due to the application of a sensitive PCR (35 cycles). On a
total of 39 raw milk cheeses, 2 cheeses were found positive with the conventional
method, 14 with the Probelia method (Herman L., personal communication).
205
Because the DNA based methods can reach a higher sensitivity in comparison with the
conventional method, it becomes very important to proof the validity of the positive
results and to make sure that no false positives are obtained. This is not so easy because,
unlike cultural methods, which almost always yield a viable pure culture isolate, the
PCR methods require lysis of the bacterial cell. Therefore, viable cells needed for test
confirmation can only be obtained by repeat analysis of the original enrichment medium
using cultural techniques. False positive results can especially be obtained when a very
sensitive PCR is used. This increases the possibility of PCR contamination and the
detection of a small number of killed cells. The absence of PCR contamination is not so
easy to proof. Obtaining a negative PCR control would not be sufficient to exclude the
possibility of a very small contamination during sample preparation and even PCR
preparation. It was experienced that even after plating of 10 ml of the enrichment
culture of L. monocytogenes no strain could be isolated from some PCR positive
samples (Rijpens N., personal communication). Only repeated culturing could validate
the positive result in this case.
For routine applications, the use of less sensitive PCR cycling programs should be
encouraged. This implies, at the time being, a limitation for the use of universal
protocols for detection of different food pathogens in one enrichment. Wang et al.
(1997) reported the possibility to detect up to 13 species of foodborne pathogens in
foods using an universal protocol of enrichment and detection. Such multi-analyte test,
however, implies the use of a very sensitive PCR cycling program (40 cycles) to obtain
a sufficient sensitivity.
In validation experiments, the rapid method is normally directly compared to the
conventional culturing method. Next to naturally contaminated samples different kinds
of spiking experiments are applied. Very often pure well growing bacterial cultures are
used for this purpose. Because bacterial pathogens present in foods are in a more
stressed condition the sensitivities of such experiments may be overestimated. This
could be overcome by an extra validation on naturally contaminated samples. For some
food products (e.g. dairy products for contamination with Salmonella), however, such a
validation is impossible because of the lack of possible samples. Methods could then be
evaluated using artificially stressed bacterial cells. For some bacterial pathogens they
are commercially available. Rijpens et al. (1999a) spiked the different dairy products
with an average of 5.9 stressed S. panama or S. typhimurium cells by using the reference
material prepared at the RIVM (National Institute of Public health and Environment,
Bilthoven, The Netherlands; in t Veld et al., 1996). The results show that, even within
the group of dairy products, a differentiation of methods have to be made depending on
the specific group of products tested. For ice-cream and cheeses made from pasteurised
milk, PCR was applied after 16 h of preenrichment in buffered peptone water (BPW)
using immunomagnetic separation (IMS, using the Dynabeads anti- Salmonella, Dynal,
Oslo, Norway) and alkaline lysis as sample preparation method. For milk powder and
raw milk cheeses, the 16 h preenrichment in BPW was followed by IMS and a 4 h
enrichment in Rappaport-Vassiliadis broth. It is therefore clear that one have to be
careful to apply simplified universal protocols without proper validation on the specific
food products tested.
Because of the need of validation of the DNA based methods a number of validation
schemes have been developed in various countries throughout the world. In Europe
206
there is the Association Franais de Normalisation in France, the European

Microbiological Method Assessment Scheme in the UK and DanVal in Denmark.
However, there have been no microbiological test method assessment and validation
schemes accepted by all countries of Europe. The MicroVal project has developed an
European microbiological method validation and certification scheme. This validation
procedure should become a CEN standard within the next 3 years (Betts and Rentenaar,
1998). In the USA the validation and evaluation scheme of the Association of Official
Analytical Chemists (AOAC) is most widely accepted. Only methods that were
subjected to the rigorous, multi-step, collaborative study process of the AOAC are
approved as standard methods and may be used in official analysis of foods. The BAX
for Screening/Salmonella was the first PCR-based product to receive the AOAC RI
awarded Performance Tested Method status (Mrozinski et al., 1998).
3.8 DNA AMPLIFICATION
FOODBORNE PATHOGENS
METHODS
FOR
QUANTIFICATION
OF
A lack of a simple and reliable method for quantification of the PCR products has partly
hindered the use of PCR in routine food laboratories. Quantification of foodborne
pathogens by PCR is possible by an indirect method based on the Most Probable
Concept (MPN) and by direct quantification of the PCR product.
The MPN concept was developed for estimation of the number of organisms based
on the probability of getting positive and negative results in different dilutions of the
bacterial culture (Cochran W., 1995). MPN-PCR has first been described for
enumeration of specific micro-organisms in soil samples (Picard et al., 1992). The
detection limit in these experiments was quite high, as at least 103 target cells were
needed for positive results. Because of the inefficiency of a PCR of 30 cycles, this
underestimation is also encountered in food samples (Herman L., personal
communication). The application of a nested PCR protocol could improve the PCR
efficiency to about 20 cfu/ml, allowing comparable quantitative results for MPN-PCR
with plate counting methods (Mgntynen et al., 1997).
For direct quantification, quantitative competitive PCR can be used based on the coamplification of the target with a known concentration of an internal standard (IS) in
one reaction tube. In most cases, the IS shares the primer recognition sites with the
specific template. Both template and internal standard must be amplified with the same
efficiency and both end products must be analysed separately. Quantification is
performed by comparing the PCR signal of the specific template with the PCR signals
obtained for the known concentrations of the competitor. Wang & Hong (1999) used an
ELISA-competitive-PCR method to detect and quantify L. monocytogenes in milk
samples. Amplification of the target and the IS was performed in the presence of
fluorescein-dUTP. The labelled PCR products were hybridised with biotinylated probes,
specific for target and IS. The hybrids were bound to streptavidin-coated ELISA plates
to which an alkaline phosphatase-conjugated antibody to fluorescein was added in the
presence of substrate.
Kinetic quantitative PCR is another direct quantification method where the amount
of PCR products is followed during the PCR at several cycles. In the kinetic method, an
internal standard is not mandatory and an external scale is sufficient if the
reproducibility of the PCR is good. The PCR efficiency of each reaction is checked by
207
looking at the slopes of the increase in PCR products for the experimental samples and
for the external scale. If the slopes are parallel quantification is possible. Monitoring
PCR kinetics has become automated by different commercially available systems, The
TaqMan PCR detection system (Fig. 2, Perkin Elmer) depends on the irreversible
cleavage of the probe by the polymerase exonuclease activity. Quantification was
demonstrated for a pure L. monocytogenes culture and proved to be linear over a range
of 50 to 5. 105 cfu of L. monocytogenes (Batt C., 1997). The Lightcycler hybridisation
system (Roche Molecular Biochemicals, Mannheim, Germany) is based on the
hybridisation of 2 independent probes. Resonance energy is transferred from the donor
probe to the acceptor probe resulting in fluorescence emission (Fig. 3). Both
amplification and hybridisation reactions are performed together and the fluorescence is
automatically measured during the process.
Figure. 3. Schematic presentation of the LightCycler hybridisation system (Roche Molecular

Biochemicals, Mannheim. Germany)
4. Molecular typing of foodborne bacterial pathogens

4.1 TERMINOLOGY AND GENERAL INFORMATION
4.1.1 Necessity of bacterial typing of foodborne pathogens
Bacterial typing refers to the differentiation of types, strains or clones in a single
species. Sometimes the term subtyping is also used for techniques, which allow the
further subdivision of distinct types within a species (e.g. biotypes), which is not
necessarily on the strain level. It is not surprising that medically important
microorganisms have been the first candidates for molecular typing (van Belkum,
1994). Typing of bacterial pathogens is necessary in epidemiological studies where
origin, transmission and persistence of pathogenic strains are investigated, and this
applies to nocosomial or hospital related outbreaks as well as to foodborne outbreaks. In
foodborne outbreaks, it is often necessary to distinguish the different types of the
pathogen involved and to link them with the suspected foodstuffs as well as to
discriminate pathogenic strains which are occurring coincidentally but independent
from an epidemic caused by a single strain. This information is of major concern
because it directly affects the preventive and hygienic measures to be implemented.
208
When stable typing data are stored in a database or alternatively, the bacterial isolates
themselves are collected for future typing analysis, the persistence and possible
evolution of certain strains or clones can be followed, which is of importance in the
investigation of the international spread of infectious agents. In the case of zoonotic
pathogens, this typing approach is useful to extend the epidemiological link from the
human infections to the main animal reservoirs (from stable to table) by thorough
investigation of each step in the whole food production chain (environment, farm,
slaughterhouse, distribution, retail, kitchen). In this way, the most critical points for
infection as well as for control or prevention can be identified, but it is also possible to
characterise the strains or clones with a changed or particular pathotype (virulence,
invasion) or which have acquired (higher) resistance to certain disinfecting products
(e.g. hypochlorous acid resistant Salmonella strains, Mokgatla et al., 1998) or
antibiotics (e.g. quinolone resistant Campylobacter, multiresistent Salmonella
typhimurium DT104).
4.1.2 Species-subspecies-variety-clone-strain-isolate
The basic unit of bacterial taxonomy is the species, defined as a group of strains,
including the type strain, sharing at least 70% DNA-DNA relatedness with 5C or less
Tm (with Tm the melting temperature of the hybrid). According to Vandamme et al.
(1996), the bacterial species appears to be an assemblage of isolates which originated
from a common ancestor population in which a steady generation of genetic diversity
resulted in clones with different degrees of recombination, characterised by a certain
degree of phenotypic consistency and by a significant degree of DNA-DNA
hybridisation and over 97% of 16S rDNA sequence homology. The latter authors
advocate the polyphasic taxonomic approach for bacterial classification as first step in
the delineation and description of species, which takes account of all possible variation
on the phenotypic and/or genotypic level. This polyphasic approach gives a higher
guarantee that intra-specific reliable and stable targets (e.g. on the 16S rDNA level) for
molecular identification can be found. Nevertheless, a polyphasic identification will
remain necessary for atypical isolates or isolates from new niches, which may belong to
unknown, related species. It must be stressed that a precise species identification is an
important and necessary step before any typing can be performed. A List of bacterial
names with standing in nomenclature is available on http://wwwsv.cict.fr/bacterio/index.html.
The classification of organisms below the species level is of particular interest for
epidemiology and typing. The only level with nomenclature status is the subspecies
(Wayne et al., 1987), while variety has no official rank. The designation var (e.g.
biovar, pathovar, serovar, phagovar) is commonly given to groups of strains which are
distinguished by certain characters. A strain is the basic working unit in the daily
laboratory practice, where the term denotes a pure culture, either fresh or stored in some
way. In the Bergeys Manual of Systematic Bacteriology, a strain is described as the
descendants of a single isolation in pure culture, and usually made up of a succession
of cultures ultimately derived from an initial single colony. A useful definition for a
strain is also an isolate or a group of isolates exhibiting phenotypic and/or genotypic
traits which are distinctive from those of other isolates of the same species (Struelens
and members of the European Study Group on Epidemiological Markers, 1996). The
209
meaning of the terms strain and isolate are thus synonymous in many cases. The term
clone has a meaning in population biology as denoted by rskov and rskov (1983) as
bacterial cultures isolated independently from different sources, in different locations,
and perhaps at different times, but showing so many identical phenotypic and genetic
traits that the most likely explanation for this identity is a common origin. For bacteria,
clonality has thus a somewhat broader meaning than for eukaryotic organisms, where
clones are definitely genetically identical organisms. In bacterial epidemiology, the
clone concept is of an even more pragmatic nature to denote isolates obtained during
clear-cut outbreaks and exhibiting the same phenotypic and/or genotypic features or to
denote organisms with common features (e.g. multiple antibiotic resistance) from
different geographic locations, so-called epidemic clones. The clonal relatedness
between strains is usually inferred from a numerical analysis of a polyphasic typing
approach combining phenotypic (e.g. multilocus enzyme electrophoresis or MEE of
metabolic enzymes) and genotypic methods (comparative sequence analysis or highresolution typing with pulsed field gel electrophoresis). Many pathogenic species are of
a clonal nature (e.g. Salmonella), whereas in other species genetic recombination occurs
resulting in horizontal gene exchange and/or a mosaic structure of recombined
segments. Because the evidence for clonality in bacteria is relative rather than absolute
(unless the whole genomes of different strains could be sequenced), it may be
recommended to speak only in the sense of clonally related strains or strains of a clonal
lineage or even clone complex when it is to be stated that strains have probably the
same origin as indicated by polyphasic typing.
4.1.3 Molecular typing techniques used for bacterial pathogens
Most if not all of the currently used molecular typing techniques are DNAfingerprinting techniques, i.e. techniques that reveal DNA sequence polymorphisms
within a species by the generation of fingerprints. The advent of molecular techniques
and especially of the PCR technique has revolutionised the approach and possibilities of
bacterial typing. Before that, typing was performed by the classical phenotypic
techniques such as antibiogram typing, biotyping, serotyping, phage typing and
multilocus enzyme electrophoresis (MEE). Many of these classical typing methods,
especially serotyping and phage typing, are still highly used in clinical and public health
laboratories as well as veterinary laboratories to gather timely information which can be
used for epidemiological investigations of foodborne and zoonotic pathogens.
Information of the antibiotic resistance of isolates of some pathogens such as
Salmonella, Campylobacter and enterococci will also be of increasing importance. The
classical typing techniques remain however laborious, require the holding of large sets
of antisera and phages and are therefore only performed in some reference laboratories
per country. Some strains (e.g. auto-agglutinating strains) are not typable with these
techniques, Moreover, it is sometimes difficult to correlate the serotyping and phage
typing data coming from different laboratories (veterinary vs. clinical) to each other for
a certain pathogen such as Salmonella.
Molecular typing offers important advantages such as general applicability (i.e.
typability of all isolates) and the possibility of a higher discriminatory capacity or
resolution which is needed to complement the classical sero- and phage typing when the
above mentioned tasks (4.1.1 & 4.1.2) have to be performed. The fingerprints can also
210
be used for numerical cluster analysis, which can be useful to delineate clonal
relatedness. In several cases, these molecular techniques are characterised by their
simplicity of performance, their reproducibility or their high resolution enabling the
differentiation between individual strains of a species. However, it must be said that not
all these interesting characteristics are automatically combined in one single technique.
In Table 3, a simple and pragmatic classification of the several molecular typing
techniques is given based on their basic working principles. Some indication is also
given of their complexity or simplicity, universal applicability (i.e. typability of all
bacterial species), reproducibility and resolution, which is a somewhat subjective matter
and for the latter sometimes pathogen-dependent.
Table 3: Currently used molecular typing techniques for foodborne pathogens and some of
their characteristics
211
Table 3: Continued
Basic
principle
Molecular typing techniques

(abbreviation)
Further
subdivision
Characteristicsa
Complex.
a
and PCR
plasmid
analysis
plasmid
profiling
plasmid
restriction
(plasmid REA)
(PP)
analysis
N
M
Resolut Reproduc
ion
ibility
Universal
application
V
N
M-H
M-H
: H, high; L, low; M, medium; V, variable; Y, yes; N, No.; Complext., complexity
A more objective indication and comparison of the resolution between different

techniques when applied to a certain pathogen can be obtained with the so-called
discriminatory index (DI) of Hunter and Gaston (1988).
As can be deduced from Table 3, the techniques which are characterised by their high
reproducibility and resolution, are mostly also the most complex and laborious ones
(PFGE and AFLP). In the choice for a certain technique, other factors will also play an
important role, such as the pathogen or case involved, the time and apparatus available
in the laboratory, the kind of information needed and the aim of the typing study (shortor long-term study). Nevertheless, it is generally recommended that several molecular
techniques should be combined.
The techniques are named here by their most commonly used names, which are not
confusing or erroneous. The latter is not always fulfilled since the same names are
sometimes used for different techniques (e.g. ribosomal nucleid acid gene restriction
analysis) or the same techniques bear several names (e.g. arbitrarily primed PCR),
which makes a simple retrieve of the literature sometimes impossible. An extensive
effort to classify and name the whole array of DNA-fingerprinting techniques has been
done by Vaneechoutte (1 996) and we can only strongly recommend the molecular
epidemiologists to name their typing techniques in a consequent manner, for example
based on the guidelines by the latter author or based on a pragmatic consensus. The
several molecular typing techniques most relevant to foodborne bacterial pathogens are
now described in more detail, using their commonly used abbreviation if available (see
Table 3).
4.1.3.1 PCR-mediated typing techniques. The PCR has without any doubt
revolutionised the molecular typing approach of bacteria and has also brought the
possibility of molecular typing within the reach of routine laboratories. A large variety
of PCR mediated typing techniques exists and some of them are listed in Table 3. The
most widely known and used techniques in this category are AP-PCR and RAPD,
developed independently by Welsh and McClelland (1990) and by Williams et al
(1990), respectively. Since there are no fundamental differences between both
techniques and the name RAPD is more frequently used, it is recommended that the
latter name is used throughout. RAPD is based on the random amplification of genome
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fragments lying between arbitrary sequences, which are used as targets for priming,
Usually, only one primer of 10 up to approximately 20 bases long is used in the PCR
working at a low annealing temperature (around 35C, but higher temperatures are also
used) and with a high MgCl2 concentration (up to 4 mM). As a consequence, these
reaction conditions will permit specific as well as non-specific primed amplification of
fragments. Another consequence is the generally low degree of reproducibility of this
kind of molecular typing. A technical review on RAPD typing in microbiology has been
done by Power (1996). The several experimental factors affecting the amplification
reaction and the reproducibility of RAPD have been reviewed by Tyler et al. (1997).
One of the important factors affecting the complexity of the banding pattern is the
primer selection. As a matter of fact, for every organism studied a suitable primer has to
be selected empirically. The most critical factor for reproducibility seems to be the
primer concentration to template DNA concentration. Tyler et al. (1997) have
formulated several recommendations for using arbitrary PCR:
quantify DNA for each organism and each extraction method, and the use of
whole-cell extracts is to be avoided.
primers should be screened for priming ability and reproducibility.
quantify primers for every synthesis reaction.
titrate DNA against primer concentration to reach an ideal primer/template
ratio.
standardise the use of Taq DNA polymerase by maintaining the same supplier,
titrate the Taq DNA polymerase against the primer/template ratio.
standardise the MgCI2 concentration.
use one thermocycler with a standard set of cycling conditions.
run the appropriate control blanks to account for background.
It is now possible to perform RAPD in a commercialised pre-mixed, pre-dispensed
reaction format (Ready-To-GoR RAPD Analysis Beads, Amersham Pharmacia Biotech)
which requires only the addition of template DNA and a primer. This format will help
to fulfil the above recommendations and to increase the inter- and intra-laboratory
reproducibility.
Bacterial genomes contain multiple interspersed repetitive DNA elements (usually
<500 bp) occupying intergenic regions at sites dispersed throughout the whole genome.
These repetitive elements may provide the genomic code necessary for proper
chromosomal structure and function in vivo. Several classes of repetitive elements have
been found in different microbial genomes: polynucleotide sequences, tandem repeats,
short interspersed repetitive sequences (<50 bp) such as the repetitive extragenic
palindromic (REP) element, large interspersed repetitive elements (>50 bp) such as the
enterobacterial repetitive intergenic consensus (ERIC) element, and mosaic repetitive
elements such as the BOX-element. Tandem repeats are polynucleotides occurring in
tandem in a variable number according to the strain. A PCR based on this variable
number of tandem repeats (VNTR) is very useful for typing of unicellular eucaryotes,
but has also typing potential in some bacteria (Frnay et al., 1994). For bacteria, the
REP-, ERIC- and BOX- elements are of greater importance. Genuine REP and ERIC
elements are present in high numbers (e.g. >100 for REP) in the genomes of the
Enterobacteriaceae and REP- and ERIC-like elements have also been demonstrated
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throughout the eubacterial kingdom. They contain a conserved inverted repeat to which
complementary consensus primers have been designed for PCR-typing (Versalovic et
al., 1991). The modular BOX-element is the first repetitive element found in a
Grampositive organism (Streptococcus pneumoniae) consisting of the subunits boxA,
boxB and boxC (Martin et al., 1992). Based on the widespread conservation of the
boxA subunit among diverse bacteria, a similar PCR-typing method has been designed
(Koeuth et al., 1995). Repetitive element primed PCR or rep-PCR is a general term to
collect at present the REP-PCR, ERIC-PCR and BOX-PCR typing methods, based on
the respective repetitive elements. In rep-PCR, amplicons are generated which contain
unique sequence chromosomal segments lying between the repetitive sequences. Unlike
RAPD, rep-PCR is considered not to be arbitrary because known conserved sequences
are used as priming targets at higher annealing temperatures (40C for REP-PCR and
52C for ERIC- and BOX-PCR) (Tyler et al., 1997). However, some authors (e.g.
Giesendorf et al., 1993) use only one of the repetitive element targeting primers at low
annealing temperatures, which resembles more a RAPD typing. We suggest that the
term rep-PCR typing and its different subdivisions should be reserved for the techniques
relying on the original procedures. Recently, some discordant notes have been observed
within the promising proposition of rep-PCR as a stringent PCR-typing approach with
reduced experimental variation and PCR artefacts and with the potential of storing the
patterns in databases for strain and species identification. Gillings and Holley (1997)
concluded that ERIC-PCR, using the standard annealing temperature of 52C, does not
necessarily direct amplification from genuine ERIC sequences, and that this typing
technique applied to non-enterobacterial organisms must be regarded as a highly
reproducible variant of RAPD. A similar observation was made by us (Herman and
Heyndrickx, in press) with REP-PCR applied on the Grampositive UHT-resistant
Bacillus sporothermodurans. For Grampositives, this is not surprising since they do not
contain genuine REP- and ERIC- elements and it is suggested that the BOX-elements
are the preferable targets for these organisms (Tyler et al., 1997). With Salmonella, we
observed small differences (band intensities) in both REP- and ERIC-PCR patterns
between independent PCR-runs, but a considerable variation when primers were used
from 2 different sources (Heyndrickx M., unpublished results). For all rep-PCR
techniques, we recommend to adhere to the same recommendations as for RAPD and to
analyse strains with the same batches of primers and, if possible, within the same PCR
experiment. For a detailed protocol inclusive of the primer sequences for the different
rep-PCR techniques, we refer to http://www.msu.edu/~debruijn/loadr.html. A major
advantage of rep-PCR over RAPD however is the fact that a single primer set targeting
a known conserved and repetitive sequence can be used for both Gramnegative and
Grampositive organisms, whereas in RAPD a suitable primer with enough
discriminatory power has to be selected for any individual organism.
Finally, some PCR techniques aiming at other sequences occurring at multiple sites in
the bacterial genome may also be effective for typing. The 2 most important targets are
the tRNA sequences and the ribosomal spacer regions. tRNA sequences occur in multiple
copies dispersed throughout the genome and contain shared sequence motifs from
which outwardly directed primers can be derived. tRNA-PCR has more potential for
species identification, but in certain cases subgroups corresponding to the variety level
can be delineated (Seal et al., 1992). In many bacterial species multiple copies or alleles
214
of the ribosomal operon are present (e.g. up to 10 copies in Bacillus). The spacer region
between the 16S and the 23S rRNA genes may vary in length between species, between
the rRNA alleles of a species and even between strains of a species as has been
demonstrated convincingly for Clostridium difficile (Grtler, 1993). This variation in
length is in part due to the number and type of tRNA genes present in the spacer. A
universal bacterial identification and typing method is based on the PCR amplification
of the variable length 16S-23S rDNA spacer regions (Grtler and Stanisich, 1996;
Jensen et al., 1993). This typing method has also been called PCR-ribotyping (Kostman
et al., 1992). The typing resolution of the ribosomal spacer based PCR technology can
be enhanced by additional restriction analysis to detect also sequence differences
between the spacers.
4.1.3.2 Typing techniques combining PCR with restriction analysis. After PCR
amplification of certain genes or gene fragments, the amplicons can be digested with
restriction enzymes and the resulting restriction fragments separated by agarose gel
electrophoresis to reveal sequence polymorphisms. This restriction analysis is necessary
because the targeted genes differ only in sequence and (usually) not in length (unless
insertions or deletions have occurred in the gene) between strains of a species. Protein
encoding genes showing intraspecific variability are used for this typing approach. A
good example is the flagellin gene typing of C. jejuni based on the restriction analysis
of the amplified flaA gene (Nachamkin et al., 1993). Since the amplicons are generally
up to 1500 bp long, tetracutter restriction enzymes (i.e. enzymes with a 4-base
recognition sequence) which cut theoretically after each 256 bp, are the best choice to
reveal enough restriction fragments and thus to reveal sequence polymorphisms. Also
enzymes with a longer recognition sequence, which contains only 4 defined bases and
for the rest undetermined bases (e.g. DdeI with the recognition sequence C^TNAG, with
N indicating an undetermined base), are suitable for this purpose. Information
concerning all currently known restriction enzymes is available at the daily updated
Rebase (http://rebase.neb,com/rebase/rebase.html). It must be noted that amplified
ribosomal DNA restriction analysis (ARDRA) is frequently categorised under the
typing techniques with potential of subspecies/strain differentiation. This is erroneous
since this technique relies on the 16S rRNA gene which is conserved amongst bacterial
species and is thus ideally suited for taxonomic and phylogenetic studies, but not for
molecular typing (Heyndrickx et al., 1996).
4.1.3.3 Typing techniques based on chromosomal restriction fragment length
polymorphisms. Before the advent of the PCR methodology, molecular typing was
mainly performed by restriction enzyme digestion of the bacterial chromosome and
subsequent gel electrophoretic separation of the fragments to detect restriction fragment
length polymorphisms (RFLP) by comparing the number and size of restriction
fragments. This technique is also simply called restriction enzyme analysis or REA.
There are 3 main sources for the generation of RFLPs: base substitution within the
restriction recognition sequence, deletions and insertions. Base substitutions affect only
the restriction enzyme(s), which cleaves within the original or mutated recognition
sequence and result in the gain or loss of a restriction fragment(s). If a restriction site is
lost, a new restriction fragment, equal to the sum of the 2 fragments flanking the
previous restriction site is generated; if a restriction site is gained, 2 new restriction
215
fragments which together equal the size of the lost fragment, are generated. Deletions
and insertions affect the RFLP patterns obtained with all restriction enzymes because of
the change in size of a particular restriction fragment corresponding to the size of the
deletion or insertion. For RFLP analysis, hexacutter restriction enzymes (i.e. with a 6base recognition sequence) are normally used, but this still may result in >1000 bands
for a normal bacterial genome of 4x106 bp, which makes separation and interpretation
of the complex banding patterns very difficult. Several approaches (high-size or highfrequency RFLP using tetracutters and low-size RFLP using hexacutters and
polyacrylamide gel electrophoresis, selective restriction fragment hybridisation, pulsed
field gel electrophoresis) have been developed to overcome this problem by reducing
the number of (visible) bands.
In the selective restriction fragment hybridisation (SRFH), the separated
chromosomal restriction fragments are transferred by Southern blotting on
nitrocellulose or on (more durable) nylon filters or membranes by capillary action or by
vacuum. The denatured membrane-bound DNA fragments are hybridised to a probe,
which can be either 32P-radioactive labelled or non-radioactive labelled with a
chemically modified nucleotide containing a hapten (e.g. digoxigenin- or DIG-dUTP of
Boehringer Mannheim, biotin-dATP). Only the fragments hybridising with the probe
are then revealed by autoradiography or with an anti-DIG or -biotin alkaline
phosphatase conjugate combined with a chemiluminescent or chromogenic substrate.
This hybridisation procedure results in simple and easy interpretable SRFH patterns.
Several factors influence the specificity of the hybridisation reaction such as the
stringency of the hybridisation temperature and several critical parameters (e.g. ionic
strength of the hybridisation solution, probe length and % G+C) affecting the Tm of the
hybrids formed. An essential element in SRFH analysis is the type of the probe used,
which can be either a single-stranded DNA or a RNA sequence. SRFH probes can be
categorised in random, directed and reiterated sequence probes (reviewed by Demezas,
1998), with the latter 2 being the most important ones. Reiterated sequence probes
include transposons, insertion sequences (IS) and duplicated genes. SRFH with
virulence/toxin probes, phage probes or IS-probes are very useful in the molecular
typing of certain foodborne pathogens. It must be noted that most of these SRFH
applications are named as RFLP techniques preceded by the name of the probe used,
e.g. IS200-RFLP and
The most widely used SRFH application is based on
probes directed at the conserved ribosomal RNA genes and is named ribotyping
(Grimont and Grimont, 1986) or (erroneously) also restriction analysis of rRNA genes.
Ribotyping is universal applicable but its resolution is dependent on the number and the
spatial distribution of the rRNA copies on the bacterial chromosome. In the first place,
ribotyping is a useful taxonomic and identification technique and also a potential typing
technique for microorganisms containing several ribosomal operons (multiplicity of
rRNA operons in prokaryotes reviewed by Schmidt, 1998). The ribotyping technique
has been automated with the RiboPrintersystem (Qualicon Inc., Wilmington, DE,
USA). Starting from a pure colony, this automated system analyses 32 samples per day
in 4 batches of 8 samples, with the results from the first batch available in 8 hours.
The second approach to reduce the amount of fragments in RFLP analysis is
macrorestriction analysis (MRA) by pulsed field gel electrophoresis (PFGE). This
technique is based on the in situ restriction digestion of the intact unsheared bacterial
216
chromosomes in lysed agarose-embedded cells with so-called rare-cuttingrestriction

enzymes, i.e. hexacutters or octacutters that cut the genome infrequently. This results in
(usually 10 to 30) large chromosomal fragments ranging in size from several kbases up
to several Megabases, which can only be separated by the specialised electrophoresis
technique PFGE. In PFGE, the electric field is not linear but switched with a certain
field angle and shape and pulse time. For an optimum separation of fragments over a
certain molecular weight range, an appropriate ramp of the pulse time intervals has to be
selected for every case. The number and size range of fragments is dependent on the
restriction enzyme used, the % G+C content and the degree of methylation of the
bacterial genome. In practice, it seems that appropriate restriction enzymes have to be
selected for PFGE analysis of each bacterial species and that a combination of different
enzymes is necessary for optimal strain discrimination within the species. An overview
of rare cutting enzymes useful for PFGE analysis of several bacteria is given by
McClelland et al. (1998). The standard protocol for PFGE is time-consuming (up to 6
days), but more rapid procedures for specific organisms (e.g. one day procedure for E.
coli O157:H7, Gautom 1997) or in general (less than 3 days procedure of Matushek et
al., 1996) have been designed without consequences for the PFGE patterns. Guidelines
to interpret macrorestriction patterns have been formulated by Tenover et al. (1995).
Under the condition that isolates obtained from outbreaks spanning relatively short
periods are analysed by PFGE using only one restriction enzyme because of time
considerations, the following categories of isolates can be distinguished on the basis of
the PFGE patterns:
indistinguishable isolates showing no fragment differences and hence no
genetic differences. These isolates all represent the same strain.
closely related isolates showing 2-3 fragment differences consistent with a
single genetic event (point mutation, deletion or insertion). Such variation has
been observed in strains of some species on repeated subculturing or multiple
isolation from the same source.
possibly related isolates showing 4-6 fragment differences consistent with
2 independent genetic events. Such variation has been observed between
isolates collected over longer periods or taken from extended outbreaks, and
can mostly be confirmed by other typing techniques.
unrelated isolates showing 7 fragment differences consistent with 3 genetic
events.
Although these guidelines are in the first place intended to know the epidemiological
relationship of isolates to the outbreak strain, they may be useful in a more general
context when using PFGE as molecular typing tool. It is also advised not to take into
account of the fragments of <40 kb because they are not reproducible comparable. As a
comment on the above guidelines, we believe that the inherent genetic homogeneity of
the bacterial pathogen investigated should also be taken into consideration, which is of
course not facilitating the interpretation of molecular typing patterns. Indeed, there
seems to be a wide span of degree of homogeneity between different microorganisms
with S. enteritidis at the one edge as a very homogeneous, clonal species, S.
typhimurium as a more heterogeneous species, and Helicobacter pylori and
campylobacters in general at the other edge as very heterogeneous species.
217
4.1.3.4 Typing techniques combining restriction digestion with selective amplification

A patented high-resolution technique, which deserves a separate classification, is
AFLP(hereafter referred to as AFLP). Although AFLP should not be used as an
acronym, the name is often used as an abbreviation of amplified fragment length
polymorphism. AFLP has been adapted for bacteria independently by Janssen et al.
(1 996) and Lin et al. (1 996) as follows:
double digestion of the chromosomal DNA with 2 restriction enzymes, a
hexacutter (H), mostly ApaI, EcoRI or HindlII, and a tetracutter (T), mostly
Taq1 or MseI. A correct choice of the enzymes is crucial as this will
predetermine the size and number of restriction fragments and depends on the
% G+C content of the genome. The enzyme combinations EcoRI/MseI and
ApaI/TaqI are the most suited for bacterial genomes with a low and a high %
G+C content, respectively. HindIII is the most suited to digest genomes with a
40-50 mol %G+C.
ligation of restriction halfsite-specific double stranded oligonucleotide linkers
or adapters to both ends of the restriction fragments. The adapters are designed
in such a way that the original restriction sites are not restored after ligation to
the restriction fragments, so that the ligation can be performed in the presence
of the restriction enzymes to prevent fragment-to-fragment ligation.
pre-selective amplification of all H-T fragments with primers complementary
to the sequences of the H- and T-adapters, respectively. In some procedures,
this step is not used and a selective amplification is performed directly.
selective amplification of a subset of the H-T fragments with primers
complementary to the H- and T-adapters and with additionally one or more
selective bases at their 3-end. Under stringent PCR conditions, only the
primers of which the selective base(s) is complementary to the base(s) flanking
the restriction site, will match perfectly and will result in amplification.
Theoretically, this means that per selective base only 1 on the 4 restriction
fragments will be amplified, and thus a selective amplification of restriction
fragments is achieved. The complexity of the AFLP patterns largely depends
on the length and the base composition of the selective bases. Usually one
selective base for both primers is sufficient. The choice of the composition of
the selective base again depends on the % G+C of the bacterial genome, but
also on the complexity and band distribution of the AFLP patterns obtained
with the restriction enzyme combination. In some cases, it may be necessary to
use 1 selective base for one of the primers only or to use 2 selective
nucleotides for one of the primers to achieve an adequate number of evenly
distributed bands. Only the H-complementary primer is labelled, either
radioactive or with a fluorescent dye, to reveal the AFLP patterns by
autoradiography or on an automated sequencing apparatus with laser
fluorescent detection, respectively.
AFLP is applicable in both bacterial taxonomy and typing as demonstrated by its
superior resolution towards the differentiation of highly related strains (Janssen et al.,
1996). Other advantages are the high reproducibility because of the stringent PCR
conditions, and the large possibilities of experimental variation (choice of restriction
enzymes and selective bases) so that optimal settings for each microorganism can be
218
searched. The AFLP technique is now available in a commercial format (AFLP

Microbial Fingerprinting kit of PE Applied Biosystems, Foster City, Ca., USA).
4.1.3.5 Typing techniques based on plasmid analysis. Plasmids were the first targets for
molecular typing starting already in the mid-seventies and they are still very useful for
certain pathogenic bacteria such as Salmonella. Plasmid analysis starts with the specific
isolation of the plasmid DNA which is mostly accomplished by a modified procedure of
the alkaline lysis method, originally described by Kado and Liu (1981). In most rapid
plasmid preparations, chromosomal contamination is difficult to eliminate, but this
should not present a problem for interpretation since the chromosomal DNA appears as
a diffuse single band of the same length for all strains. The most simple technique is
plasmid profiling (PP) in which the size and number of the plasmids present in the
strains is investigated by agarose gel electrophoresis. An E. coli reference strain
harbouring several large plasmids of known size is very useful for exact molecular
weight determination in PP analysis. In some plasmid preparations, the same plasmid
may be present in multiple forms (the native supercoiled form, the open circular form
resulting from a cut in one DNA strand, and the linearised form resulting from a cut in
both DNA strands), each with its own electrophoretic mobility. Especially with smaller
plasmids, this may result in multiple bands for the same plasmid, which results in an
overestimation of the number of plasmids present. Another problem is the instability of
plasmids resulting in the possibility that a certain strain loses one or more of its
plasmids. On the other hand, plasmid DNA may undergo more rapid evolution than
chromosomal DNA because plasmid borne genes do not encode for essential cell
functions. It may thus be expected that plasmid DNA is less subjected to evolutionary
pressure and is more prone to sequence variations. Sequence polymorphisms between
identical sized plasmids are revealed by restriction analysis (plasmid REA).
4.1.4 Analysis of DNA fingerprints.
The DNA fingerprints generated by the various molecular typing techniques can be
visually analysed to distinguish strains with identical patterns, related patterns and
clearly different patterns. This analysis by eye is only possible for strains run on the
same gel, which still may contain several tens of strains. When patterns on different gels
have to be compared, the interpretation becomes more complicated, not only because of
the restrictions in human visual memory, but also because of differences in
electrophoretic mobility between equally sized bands. These differences are sufficient to
make the use of specialised pattern analysis software highly recommendable (e.g. the
state-of the art package GelCompar, Applied Maths, Belgium). All packages allow the
same essential functions: input of the strain information, gel normalisation, cluster
analysis and database generation (Seward et al. 1997). One of the most critical elements
in this computerised analysis is the normalisation step, in which bands of equal size in
the patterns are physically aligned to compensate for inter- and intra-gel inconsistencies
and variations. By normalisation, all gels become compatible with each other which
makes the generation of databases and the cluster analysis of patterns from different
gels possible. By cluster analysis, patterns are grouped in a dendrogram according to
their similarity to each other, which is calculated by the program for every pair of
patterns based on a similarity coefficient. This coefficient can be either based on the
219
presence/absence of bands (Dice and Jaccard coefficient) or on the whole densitometric

curves (Pearson product-moment coefficient). The latter coefficient is more suited for
complex patterns with large differences in band intensities (e.g. RAPD and rep-PCR),
but the Dice coefficient is generally regarded as representing more reliably genetic
relationships between strains on the basis of DNA fingerprints (Nei and Li, 1979) and
hence clonal relationships between strains.
4.2 PROSPECTS IN MOLECULAR TYPING
Molecular typing is a relatively young discipline but has already encountered many
changes and variations on the same theme, in which the PCR technology has played a
major role. It is the prospect that molecular typing of foodborne bacteria will increase
constantly in importance and will generate data, which can be exchanged between
different types of laboratories (veterinary, industrial, and clinical/public health
laboratories) and between countries. On the International Meeting on Bacterial
Epidemiological Markers in 1997 (IMBEM IV, Elsinore, Denmark), PFGE and AFLP
were proposed as the likely candidates for a high-resolution, standardised, definitive
typing approach in which patterns can be reproducibly generated, stored in databases
and exchanged via networks.
In the USA, PulseNet (http://www.cdc.gov/ncidod/dbmd/puIsenet/pulsenet.htm) is
launched as a National Molecular Subtyping Network for Foodborne Disease
Surveillance, permitting rapid comparison of PFGE fingerprints through an electronic
database at the Centers for Disease Control and Prevention (CDC).
Cost and speed are also important parameters for the implementation of a typing
system, which can benefit from new developments or applications. Another fragment
separation technique based on fluorescent labelling and capillary electrophoresis of
DNA fragments (e.g. with the ABI Prism 310 Genetic Analyzer, PE Applied
Biosystems) enables a higher level of automation, flexibility and throughput of samples.
It is also possible to directly reveal sequence polymorphisms (down to the level of point
mutations) with specialised electrophoresis techniques in which a denaturing gradient is
applied in the gel, either chemically in denaturing gradient gel electrophoresis (DGGE),
or in temperature gradient gel electrophoresis (TGGE) or alternatively in temporal
temperature gradient gel electrophoresis (TTGE). Equally sized fragments or amplicons
will stop migrating at different positions and hence will be separated in the denaturing
gradient depending on the melting temperature Tm of their sequence. PCR-TGGE and DGGE analysis is currently used in molecular microbial ecology (reviewed by Muyzer
and Smalla, 1998), but has potential for rapid molecular typing as well.
A further prospect is the deviation of molecular typing by DNA fingerprinting towards
sequencing data and hybridisation pattern data on DNA-chips. Sequence data are still
the preferred type of information because of their absolute nature and it is the challenge
of future molecular typing to identify suitable nucleic acid targets, which show enough
intraspecific variation within a single short sequencing run. DNA chips are high-density
oligonucleotide arrays which are used in fluorescent hybridisation analysis to screen
sequence variation in large sets of (amplified) DNA samples in a high throughput
manner. Simultaneous genotyping and species identification using hybridisation pattern
recognition analysis on DNA-chips has been demonstrated for mycobacteria (Gingeras
220
et al., 1998). It is expected that whole genome sequencing data will provide new targets
(e.g. DNA repeats or genes) for molecular typing.
5.Molecular typing of some specific bacterial foodborne pathogens
5.1 SALMONELLA
From Table 4, it can be deduced that Salmonella has been the subject of numerous
molecular typing studies. It is important to clarify first the rather confusing Salmonella
taxonomy. The genus Salmonella forms a single DNA homology group divided in seven
sub-groups. The seventh group is given the species rank as Salmonella bongori, while
the other 6 groups represent 6 subspecies of one species, which should carry the name
Salmonella choleraesuis (Skerman et al., 1980). Because of confusion with the name of
the serovar Choleraesuis, Le Minor and Popoff (1987) proposed the species name
Salmonella enterica instead, which is now used by almost every author, but has no
standing in nomenclature because not officially accepted by the Judicial Commission of
the International Committee on Systematic Bacteriology. Therefore, Euzby (I 999) has
recently proposed to reject the name Salmonella choleraesuis and to designate
Salmonella enterica as neotype species. The official situation will thus be that
Salmonella contains 2 species, S. bongori and S. enterica, the latter being divided in 6
subspecies (Christensen et al., 1998). S. enterica subsp. enterica is the most important
subspecies containing most of the >2300 serovars. Three serovars of high clinical
importance have been given species rank: S. enteritidis, S. typhimurium and S. typhi.
Other serovars should not be considered as species and should be designated as in the
example: Salmonella enterica subsp. enterica ser. Hadar or (more practically)
Salmonella Hadar.
Two typing levels can be proposed for Salmonella. The serovar level is (and will
remain if only for historical reasons) a very important first typing level for daily
practice. Several molecular typing techniques (see Table 4) such as PCR-ribotyping
(Lagatolla et al., 1996), PCR-RFLP of the ribosomal operon (Shah and Romick, 1997),
ERIC-PCR (Van Lith and Aarts), REP-PCR (Heyndrickx M., unpublished results) and
AFLP (Aarts et al., 1998) seem to provide the possibility of serovar identification and
thus to replace the classical serotyping.
However, it should be noted that some serovars seem to be polyphyletic because of
recombinations in the genes encoding for the O- and H-antigens, which could be
reflected in different fingerprint patterns for some strains of these serovars (Hilton and
Penn, 1998).
221
Table 4: Molecular typing techniques used for foodborne and animal associated
Salmonella serovars and some important literature references for each of them. References
are classified according to the sole typing technique involved or according to the most
studied or (if possible) the most discriminatory typing technique when several techniques
are involved. In the latter case, relevant information on the other typing techniques is given
as well. For more complete information on a specific technique or serovar, we refer to
further studies cited in the references.
222
Table 4: Continued
223
Table 4: Continued
Molecular
typing
technique
Reference
Specific comment
Threlfall et al., 1998
association of S. Anatum outbreak with formula-dried milk by

plasmid profile typing and PFGE
S. enteritidis isolates of human gastro-enteritis linked with eggproducing poultry flocks by plasmid profiles and REA
plasmid REA of Australian S. enteritidis strains
combination of plasmid analysis and PFGE most discriminatory
for S. Choleraesuis strains; see also ribotyping and ISZOO-typing
use of plasmid profiles and IS200-typing for distinction between
epidemic and non-epidemic S. Hadar isolates
discrimination of S. enteritidis poultry isolates with plasmid
profiling, discrimination of S. typhimurium poultry isolates with
ribotyping and IS200-typing; see also ribotyping and IS200-typing
detection of molecular variation in the serotype-specific plasmid
of S. enterifidis by plasmid REA using PstI and SmaI
little or no differentiation amongst S. enteritidis isolates; see also
PFGE
subdivision of same S. enteritidis PFGE type by plasmid profile,
and conversely, same plasmid profile by PFGE; see also PFGE
5 different plasmid profiles amongst S. Dublin field isolates; see
also PFGE
Dorn et al., 1993

Mills et al., 1995
Weide-Botjes et al., 1996
Fantasiaet al., 1997
Millemann et al., 1995
Rankin et al., 1995

Liebisch & Schwarz,
1996a
Suzuki et al., 1995
Liebisch & Schwarz,
1996b
PFGE
Echeita & Usera, 1998

Liebisch & Schwarz,
1996a
Powell et al., 1995
Thong et al., 1998
Murase et al., 1995
Murase et al., 1996

Wegener & Baggesen,
1996
Buchrieser et al., 1997
On & Baggesen, 1997
detection of chromosomal rearrangements in S. typhi by PFGE

with I-CeuI; also ribotyping
superior discriminatory value of PFGE using XbaI, SpeI and NotI
for epidemiologically unrelated S. enteritidis isolates; see also
plasmid profile, ribotyping and 6200-typing
derivation of S. enteritidis PT9a and 7 strains from a PT4 strain
revealed by PFGE and ribotyping; also ribotyping
investigation of S. enteritidis gastro-enteritis outbreak by PFGE
(Xbal, AvrII and SpeI)
evaluation of PFGE (BlnI, XbaI) for epidemiological analysis of
Salmonella outbreaks (S. typhimurium, S. Thompson, S.
enteritidis)
variations in PFGE patterns of S. enteritidis isolates from a food
poisoning
tracing back of aS. Infantis outbreak to a single pig
slaughterhouse and its supplier pig herds by PFGE (XBaI)
subdivision of S. enteritidis PT4 and 8 strains by PFGE using NotI
and XbaI, supporting the hypothesis of a single clone pandemic
determination of 2 principal clonal lines of S. typhimurium in
Denmark by PFGE using XbaI and ribotyping; also ribotyping
224
Table 4: Continued
Molecular
typing
technique
Reference
Specific comment
Boonmar et al., 1998
epidemiological analysis of S. enteritidis isolates from humans

and broilers by PFGE (BlnI and XbaI) and phage typing,
indicating the spread of a genetically identical clone
subdivision of S. Indiana by PFGE using XbaI
derivation of ampicillin-resistant S. enteritidis PT6a from PT4
revealed by PFGE (XbaI) and plasmid analysis
subdivision of S. enteritidis outbreak isolates by PFGE (XbaI and
NotI), plasmid profile and phage type; see also plasmid analysis
PFGE using XbaI, SpeI and Avr II slightly less discriminatory than
ribotyping for S. enteritidis isolates; see also ribotyping
most discrimination between S. Dublin field isolates by PFGE
using XbaI and SpeI; see also plasmid analysis, ribotyping, IS200typing
combination of PFGE (XbaI), RAPD and phage typing for
discrimination of S. enteritidis strains from different origins
Punia et al., 1998

Ridley et al., 1996
Suzuki et al., 1995
Thong et al., 1995
Liebisch & Schwatz,
1996b
Laconcha et al., 1998
PCRribotyping
Lagatolla et al., 1996

Shah & Romick, 1997
rep-PCR
Van Lith & Aarts, 1994

Millemann et al., 1996
Lopez-Molina et al., 1998
Beyer et al., 1998
AFLP
Aarts et al., 1998
specific PCR profile for 7 Salmonella serovars (a.o. S. enteritidis),

spacer region polymorphic for 3 serovars (a.o. S. typhimurium)
restriction analysis (HinfI) of ribosomal spacer regions for
Salmonella subspecies differentiation
typing of Salmonella up to the serotype level with ERIC-PCR
1 and 2 fingerprints obtained respectively for S. enteritidis and S.
typhimurium strains with ERIC-PCR; see also RAPD
ERIC-PCR with 1 primer useful for differentiation between
Salmonella serotypes, not for S. enteritidis phage types; see also
RAPD
REP-PCR and ERIC-PCR (with 1 primer) discriminates epidemic
S. Saintpaul strain from other strains, see also RAPD
Salmonella serotype and phage type identification and
discrimination of strains, previously identified as identical by
other methods
The classical second level is phage typing, but this method has often a too low
discriminatory power for detailed outbreak investigations, a phage typing scheme is
only available for some serovars, and some isolates may be untypable. Being a
phenotypic method, phage typing can not be used for delineating phylogenetic
relationships. Another problem is phage conversion in some serovars, which may
disturb the study of epidemiological relationships (Rankin and Platt, 1995). Several
molecular typing techniques have the potential of second level typing, which
225
corresponds with clonal lineage or strain level (see Table 4). For some techniques, the
discriminatory power or applicability is serovar dependent: e.g. IS200 typing gives the
highest discrimination in S. typhimurium (Olsen et al., 1997), but is not applicable for S.
Hadar (Weide-Botjes et al., 1998b). The demonstrated discriminatory power may also
depend on the set of isolates included: e.g. ribotyping has been reported as giving no or
little differentiation (Liebisch and Schwarz, 1996a) and as being slightly higher
discriminatory than PFGE (Thong et al., 1995) for S. enteritidis. One of the greatest
challenges is the molecular typing of S. enteritidis which is a highly clonal organism,
particularly within certain phage types (e.g. PT4 and 8). Currently, it seems that RAPD
gives the best discriminatory power for S. enteritidis isolates belonging to different or
even the same phage type (e.g. Fadl et al. 1995), provided that a suitable primer is
identified (e.g. Lin et al., 1996). Also PFGE using the enzyme combination XbaI - NotI
- SpeI (Liebisch and Schwarz, 1996a) and plasmid analysis (e.g. Millemann et al., 1995)
have been shown to be useful for this serovar. A combination of different molecular
typing techniques used under optimal conditions and eventually complemented by
phage typing, is highly recommended to trace isolates in epidemiological investigations
(Weide-Botjes et al., 1998a). S. typhimurium isolates usually show more genetic
variation and can be differentiated with several techniques (see Table 4). AFLP using an
EcoRI primer with 2 selective bases enabled phage type identification and
differentiation of strains, which were indistinguishable by other methods (Aarts et al.,
1998).
5.2 CAMPYLOBACTER JEJUNI
C. jejuni is one of the most common causes of sporadic gastro-enteritis with poultry
products being the most important vehicles for infection. C. jejuni is divided in the
subspecies jejuni and doylei, but the former subspecies is the most important one.
Classical typing schemes are the Penner heat-stable and Lior heat-labile serotyping
schemes and biotyping. Several molecular typing techniques have been developed or
evaluated for a higher and universal applicable discrimination of isolates. A PCR-RFLP
technique is based on the sequence heterogeneity of the flagellin gene flaA and is
referred to as flaA typing or profiling (Nachamkin et al., 1993) using mostly the
restriction enzymes DdeI and HinfI (Santesteban et al., 1996). Both the flaA and fla B
genes, occurring in tandem, can also be used as combined target for restriction analysis
(Ayling et al., 1996). Alternatively, a short (150 bp) variable region of theflaA gene
can be used as target in direct sequence analysis for epidemiologic investigations
(Meinersmann et al., 1997). Recently, evidence for intergenomic recombination
betweenflaA genes of different C. jejuni strains as well as intragenomic recombination
between the flaA and flaB genes within a strain has been deduced from mosaic flaA
gene structures, which has as important consequence that flagellin gene typing cannot
be used for long-term epidemiological monitoring and determination of clonal
relationships within Campylobacter populations (Harrington et al., 1997). This problem
could be overcome by combining the polymorphisms determined by PCR-RFLP of
several genetic loci as demonstrated by a multiplex PCR gene fingerprinting method
based on the variable gyrA and pflA genes (Ragimbeau et al., 1998).
It seems that flaA types are conserved across different serotypes and that flaA typing
is less discriminatory than PFGE and thus cannot be used as sole basis for grouping
226
strains (Santesteban et al., 1996). PFGE was also shown to be the most discriminatory
of 3 typing methods (including ribotyping and phage typing) for C. jejuni strains of
different Penner serotypes and within a single heat stable serotype (Gibson et al., 1995),
and the most discriminatory of 4 typing methods (including fatty acid profile typing,
biotyping and serotyping) for C. jejuni and C. coli isolates from abattoirs (Steele et al.
1998). For the DNAse-positive strains of Lior biotype II, formaldehyde fixation of the
cells is necessary to perform PFGE (Gibson et al., 1994). Definition of clonal lineages
within C. jejuni with PFGE must be based on the patterns obtained with at least 2
restriction enzymes (e.g. SmaI and KpnI) (Gibson et al., 1997). PFGE typing of field
isolates from Finnish patients, chicken faecal samples and meat samples (Hnninen et
al., 1998), from Canadian meat processing plants (Steele et al., 1998) and from sporadic
cases of diarrhoeal disease in England (Owen et al., 1997) have all shown a high degree
of genomic diversity within C. jejuni. It is not unlikely that the observed large genetic
variation may at least partly be attributed to genomic instability within single strains as
the result of intragenomic recombinations or natural transformation by non-homologous
DNA and driven by environmental pressures (Wassenaar et al., 1998). In vitro
genotypic variation of C. coli induced by repeated subculturing has already been
observed by PFGE (On, 1998). PFGE patterns must therefore be carefully interpreted
and preferably combined with other (single locus) molecular typing techniques and/or
with numerical analysis in order to evaluate relationships between Campylobacter
strains. The same caution probably also applies to other whole-genome typing
techniques such as RAPD, which has been shown to have an excellent discrimination
ability within different Campylobacter species and Penner serotypes (Hernandez et al.,
1995; Madden et al., 1996). By RAPD typing, possible transmission routes (e.g.
environment, farmers footwear) of Campylobacter infection in well-defined settings
(successive broiler flocks) have been demonstrated (van de Giessen et al., 1998; Payne
et al., 1999). Because Campylobacter does not seem to be of a clonal nature, but rather
consists of genomic mosaics, it may be difficult or impossible to define epidemiological
links by molecular typing in less defined settings (e.g. different farms) as shown by
Weijtens et al. (1997). A SRFH technique with a probe based on the highly conserved
domain (HCD) of the tlpA gene, encoding for a methyl-accepting chemotaxis-like
protein from C. coli, has also been proposed as a molecular typing scheme which is not
likely to be subject to an extensive degree of genetic instability (Gonzalez et al., 1998).
5.3 LISTERIA MONOCYTOGENES
L. monocytogenes causes a rare but severe disease in humans, which is in most if not all
cases caused by industrially processed food with an international distribution (e.g.
cheese). Serotyping is of limited epidemiological value as only 3 serovars (1/2a, 1/2b
and 4b) are causing most of the infections. Because of the large socio-economic impact,
the World Health Organisation (WHO) food safety unit in Geneva mandated in 1990 a
multicenter study on L. monocytogenes subtyping methods. The results of the first phase
are published in a special issue Molecular typing of Listeria of the International
Journal of Food Microbiology (Vol. 32, 1996). The conclusions of this study can be
summarised as follows (Bille and Rocourt, 1996):
227
amongst the phenotypic typing methods, serotyping is a useful first step

method with a good reproducibility for certain serotypes (4b and 1/2a), and
phage typing is most valuable for mass screening, but both methods need
standardization. MEE is reasonably reproducible, but too laborious for large
sets of strains.
ribotyping has a limited discriminatory power, particularly for the serotype 4b
which is responsible for most of the major recent outbreaks, but has the
advantage of an automation of the procedure (Riboprinter) (Swaminathan et
al., 1996).
high-frequency restriction endonuclease typing (REA), which is a variant of
RFLP using high-frequency cutting enzymes (EcoRI, HaeIII, HhaI), shows a
good reproducibility and high discriminatory power, but needs further
standardization and computer analysis for objective interpretation of the
patterns (Gerner-Smidt et al., 1996).
PFGE (using ApaI and SmaI) is highly discriminatory and reproducible and
looks very promising if a computerised analysis of the patterns in association
with a strict standardization of the protocol can be used (Brosch et al., 1996).
RAPD also looks very promising, but several problems of intra- and
interlaboratory reproducibility are encountered (Wemars et al., 1996).
It is expected that this Listeria typing study will serve as a model for other pathogens.
Other studies have also shown the superior discriminatory power of RAPD/AP-PCR
and PFGE for L. monocytogenes strains belonging to several serovars (Louie et al.,
1996; Destro et al., 1996). The importance of silage as a source of listeriosis outbreaks
in ruminants was demonstrated by ribotyping performed with the Riboprinter
(Wiedmann et al., 1996). These authors acknowledge that ribotyping is less
discriminatory than other typing methods such as RAPD, but argue that this is not
necessarily a disadvantage since too sensitive a typing system might detect
epidemiologically irrelevant strain differences based on point mutations and/or DNA
rearrangements. Rep-PCR (REP- and ERIC-PCR) generates L. monocytogenes serotype
specific patterns and within the heterogeneous serotype 1/2a REP-PCR shows a similar
strain discrimination as RAPD (Jersek et al., 1996). It was also observed by rep-PCR
that there is no or only little similarity between L. monocytogenes isolates from humans
and animals and from food, suggesting that only a minor proportion of food strains are
pathogenic (JerSek et al., 1999).
5.4 ESCHERICHIA COLI 0157
The serotype E. coli O157:H7 (motile) and O157:H- (non-motile variant), belonging to
the enterohaemorrhagic E. coli (EHEC) which are a subset of the verocytotoxic E. coli
(VTEC), stands as the type example of the so-called new emerging infectious diseases..
The diversity of strains in cattle and sheep, the major reservoirs of potential pathogenic
VTEC and of E. coli 0157 strains, has been investigated by PFGE (Kudva et al., 1997;
Beutin et al., 1997; Heuvelink et al., 1998). PFGE (using Xba I) has the highest
discrimination for E. coli 0157 (giving the best discrimination) (Grif et al., 1998) and is
therefore highly used in the epidemiological investigation of foodborne outbreaks,
228
which have been linked in many cases to undercooked hamburgers (Barrett et al., 1994).
Ribotyping, on the contrary, is not able to discriminate between E. coli 0157 isolates
(Martin et al., 1996). In the USA, the PulseNet uses a standard PFGE procedure and an
electronic pattern database for E. coli 0157 (Anonymous, 1996). However, in the
interpretation of molecular typing results of E. coli 0157, 2 important phenomena must
be accounted for. Firstly, for the phylogenetically highly related E. coli 0157 isolates
from human infections which seem to belong to a single clone complex, PFGE has its
limitations because epidemiologically unrelated strains may differ in only a few
fragment bands making an unequivocal differentiation between outbreak and nonoutbreak related strains sometimes difficult (Bhm and Karch, 1992). Combination with
another typing method such as RAPD (Birch et al., 1996) or phage typing is therefore
highly advised (Grif et al., 1998). Secondly, E. coli 0157 can undergo rapid genotype
alteration in the course of infection, so-called clonal turnover, which may be caused by
the chromosomal integration or loss of verocytotoxin gene carrying bacteriophages
(Datz et al., 1996), and may result in the appearance of new PFGE patterns. SRFH
techniques with the use of Shiga-like (SLT) or verocytotoxins probes (SLT-RFLP)
(Samadpour, 1995) and bacteriophage probe (-RFLP) (Grimm et al., 1995) have
been shown to be very sensitive methods for interstrain differentiation. A PCR based
typing method based on the E. coli repetitive element IS3 has been developed as a rapid
screening method for the identification of unrelated E. coli O157:H7 isolates
(Thompson et al., 1998).
5.5 SOME OTHER FOODBORNE BACTERIAL PATHOGENS
PFGE (using Sma I) (Liu et al., 1997) and RAPD methods (Nilsson et al., 1998) have
been developed for the discrimination of strains of the sporeformer Bacillus cereus.
The AFLP technique has been extensively evaluated for the genus Aeromonas (Huys et
al., 1996). The combination of different ribotyping procedures (classical ribotyping and
PCR-ribotyping) has been shown useful for strain discrimination in Yersinia
enterocolitica (Lobato et al., 1998).
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238
BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION

CAHILL, S.M., UPTON, M.E., AND MCLOUGHLIN, A.J.
Department of Industrial Microbiology, Ardmore House, University
College Dublin, National University ofIreland, Belfield, Dublin 4,
Ireland
Abstract
The fermentation process has long been used as a method of meat preservation. In order
to eliminate batch to batch variation the fermentation process must be standardised.
This, combined with problems associated with emerging pathogens such as
enterohaemorrhagic Escherichia coli has led to a re-examination of the process of
fermented meat production in order to ensure the production of a consistently high
quality and safe product.
Encapsulation technology can be applied to meat
fermentations with the objectives of enhancing the existing methods of preservation and
in developing novel methods to combat the problem of emerging and re-emerging
pathogens. Encapsulation technology has been shown to be beneficial in the production
of fermented meats by both direct and indirect acidification. Indirect acidification
occurs after the addition of a starter culture to the meat and encapsulation technology
has been observed to enhance its activity upon its addition to meat. The success of
encapsulation would appear to be based on some form of spatial organisation involving
a) protection and b) controlled release. The creation of a microenvironment, which
provides the desired conditions or populations and the physical regulatory systems,
minimises the effect of fluctuations in the macroenvironment and protects the cells from
competition, predation and lysis. Controlled delivery from the microenvironment assist
the cells to adapt to the new macroenvironmental conditions and then release the
adapted cells under regulated conditions. Encapsulated acidulants are already in use in
the United States and their use is on the increase in Europe. Problems such as
discoloration and lack of binding associated with direct acidification can be overcome
by encapsulation, which allows the time and rate of acid release to be controlled.
Emerging pathogens are now challenging the antimicrobial hurdles present in a
fermented meat product. This has led to investigations into enhancing the safety of
existing manufacturing processes for fermented meats. Bacteriocins are antimicrobial
agents, which are naturally produced by lactic acid bacteria. However, their
ineffectiveness towards Gram-negative bacteria and their reduced activity in meat
products has excluded their use until now. Combining bacteriocins, for example nisin,
with other stresses enhances their activity towards Gram-negative pathogens.
239
A. Durieux and J-P. Simon (eds.). Applied Microbiology, 239 266.
Encapsulation in polymer gels facilitates the optimisation of conditions for in situ nisin
production and permits the controlled release of bacteriocins into the
macroenvironment. Therefore, this technology has the potential to facilitate the use of
bacteriocins produced by lactic acid bacteria in meat products as a means of overcoming
potential problems associated with emerging pathogens.
1. Introduction
The deterioration of food products is an inevitable process. However the rate at which
this process occurs is dependent on the food type, composition, formulation, packaging
and storage conditions (Gould, 1995). For centuries meat has been considered as a
highly valued and nutritious food (Shay and Egan, 1992). However, it is due to this
nutritious nature that meat is also a highly perishable product and when stored in air
spoils rapidly as a result of the growth of Gram-negative bacteria (Egan, 1983). Apart
from microbiological spoilage numerous chemical and biochemical changes occur
during the storage of meat, which limit its shelf life. While many foodstuffs, including
meat can now be preserved by refrigeration and freezing, the preservation of food has
long preceded the technical advances, which permit such methods of shelf-life
extension. Examples of the more traditional methods of preservation include heating,
drying, curing, smoking and fermentation. The high sensory and nutritious quality of
the resulting products has meant that, even in the face of modern technology, many of
these methods of preservation have not only survived but also retained a high level of
popularity. For example, pepperoni, a fermented meat product, has an annual
consumption rate of 370 million pounds in weight in the United States (Hinkens et al.,
1996).
While many methods of meat preservation exist, the focus of this article will be the
enhancement, using bioencapsulation technology, of meat preservation techniques based
on fermentation and acidification.
Although meat preservation by microbial
fermentation is among the oldest forms of food preservation (Dillon and Cook, 1994),
dating back to about 1000 BC (Nychas and Arkoudelos, 1990), and also one of the most
successful means of prolonging the shelf-life, new challenges and research opportunities
continually arise. Recent food poisoning outbreaks associated with enterohaemorrhagic
Escherichia coli in fermented meat products (Anonymous, 1995b; Tilden et al., 1996)
have prompted many researchers to further examine the safety of these products and to
search for novel means of enhancing their safety. Also, as in any process, there is a
continual search for technical innovations, which will enhance quality and production
efficiency (Prochaska et al., 1998). Encapsulation, which has been described as an old
yet new technology (Pszczola, 1998) is one means by which advancement can be made
in the area of meat preservation.
Encapsulation technology has been used in the food industry for more than sixty
years with the encapsulation of flavourings by spray drying in the 1930s being
considered the first application of this process (Reineccius, 1995). However, it has only
been adapted slowly and so can still be regarded as a developing technology within the
food sector. This is probably due to the requirement for low cost food grade
encapsulation materials by the food industry. Encapsulation technology in the food
sector is currently growing at a level of 30% annually in the US and one of the leading
240
producers of encapsulated food ingredients reported a doubling of sales in Europe in

1997 (Personal Communication, 1998).
Encapsulation can be defined as the technology of packaging a substrate (solids,
liquids, gases) within another material (Risch, 1995). In the case of bioencapsulation
the material which is packaged is a biologically active agent. The resulting miniature
capsules or packages can range from sub-micron to several millimetres in diameter and
are ideally spherical in shape (Jackson and Lee, 1991). However this shape will depend
on the physical structure of the material being encapsulated. In the encapsulate the
active agent which has been entrapped is termed the core material or the internal phase
while the encapsulating material is referred to as the coating, shell wall material or the
carrier. A large variety of materials can be used as encapsulating agent including
proteins, fats and carbohydrates (Jackson and Lee, 1991). The nature of the material
used is generally dependent on the subsequent use of the encapsulate as well as the
encapsulation and release properties required.
Both encapsulation technology and fermentation technology are each in themselves
quite complex processes. Thus the merging of two such technologies has the potential
to result in interesting developments in the area of meat preservation.
2. Meat preservation
The preservation of meat by acidification or the lowering of pH is currently carried out
using one of two processes:
Biological acidification - the reduction of pH by microbial fermentation. This

is achieved by the addition of lactic acid producing starter cultures to the meat.
The reduction in pH may also be achieved by the activity of the natural lactic
acid producing microflora present in the meat or inoculating the meat with the
product of a successful fermentation.
Chemical acidification - the reduction of the pH of the meat to a level which
will inhibit spoilage and pathogenic bacteria by the addition of a chemical
acidulant such as glucono-delta-lactone. This method of acidification has
developed as a means of overcoming the uncertainties, which are sometimes
associated with a microbial fermentation.
2.1 BIOLOGICAL FERMENTATION
Fermented meat products are defined as meats that are deliberately inoculated to ensure
sufficient acidification and controlled microbial activity to alter the product
characteristics (Bacus, 1986).
Although this technology has been extensively
researched and reviewed (Leistner and Lucke, 1988; Hammes et al., 1990; Shay and
Egan, 1992; Campbell-Platt and Cook, 1995; Ricke and Keton, 1997) there continue to
exist areas for improvement. The general steps in a meat fermentation process are
outlined in Figure 1.
Examination of the steps in the production of a fermented meat product allows the
identification of potential problems in this process (Table 1). While many quality and
safety problems can be overcome by the implementation of a HACCP system, this does
not solve all problems, particularly those associated with the activity of the starter
241
culture. Process control must combine optimisation of the conditions for acid
production with those for drying, flavour and texture development etc. and achieving
this may necessitate a compromise of the conditions. In relation to the starter culture
activity, it is not be possible to control important factors such as strain degeneration,
shelf-life stability and ecological competence by controlling the conditions in the
fermentation process. Encapsulation has been described as a technology which can
solve certain problems which cannot be solved otherwise (Pszczola, 1998) and
therefore its potential in meat preservation, particularly in the area of starter culture
activity deserves some examination.
Figure 1: Outline of the main steps in a meat fermentation process
242

Table 1: Potential problems associated with the meat fermentation process
Potential problems
Microbial load -presence of pathogens
Environmental contamination, growth of
pathogens
-viability and activity
- competition from natural microflora
- microbial load
Uniform distribution of additives in meat
Environmental contamination, Process
control
Process control - temperature, humidity
Process control - temperature, humidity
Step
Meat
Chopping and mixing
Additives - Starter culture
- Spices
Mixing of meat and additives
Filling the casing
Fermentation
Maturation and drying
2.2 CHEMICAL ACIDIFICATION

While the direct addition of organic acids such as lactic acid would appear to be a faster
and more reliable means of preservation than microbial fermentation, their use has been
limited due to their instantaneous reaction with the meat which causes undesirable
colour and texture changes (Shay and Egan, 1992). A more successful alternative has
been the addition of glucono-delta-lactone (GDL) to lower the pH. After addition to the
meat the GDL is hydrolysed to gluconic acid bringing about a reduction in pH (Shay
and Egan, 1992). As the use of GDL does not involve the direct addition of an acid, the
problems associated with discoloration and textural changes are reduced. However, the
extent to which this reduction in undesirable characteristics occurs depends on the rate
at which the GDL is hydrolysed. Furthermore, once hydrolysed to gluconic acid, it can
be further metabolised to acetic acid and carbon dioxide which can add a bitter taste and
gas pockets to the meat (Kneissler et al., 1986). On the other hand, the application of
encapsulation technology to food acids permits the controlled or slow release of these
ingredients thus increasing their usefulness in meat preservation (Dziezak, 1988).
3. The application of encapsulation technology to meat preservation
3.1. THE APPLICATION OF
MICROBIAL FERMENTATION
ENCAPSULATION
TECHNOLOGY
TO
While the natural microflora in a meat system will, under suitable conditions, result in a
fermentation process (Cooke et al., 1987; Gibbs, 1987; Grombas, 1989), the
unreliability of this process has long been recognised. The practice of backslopping,
which involves initiating a new fermentation by the addition of some of the product of a
successful fermentation, was developed as a means of overcoming the uncertainty
(Gibbs, 1987; Grombas, 1989; Nout and Rombouts, 1992). Today, the production or
purchase of specially selected and prepared starter cultures is probably one of the most
important and also expensive parts of the fermentation process since a successful
fermentation is dependent on the addition of a viable and active inoculate. Therefore,
243
the development of the technology to control the ecological competence of starter

cultures in fermented meats is vital. In order to be viable and active it is necessary for
the starter culture to survive the stresses associated with its preservation, storage and
reactivation. Encapsulation or immobilisation of starter cultures has been studied quite
extensively as a means of providing benefits such as cell retention in continuous culture
systems (Prevost and Davies, 1987, 1992), protection from phage (Steenson et al., 1987;
Passos et al., 1994) and improved storage stability (Kim et al., 1988; Sheu and
Marshell, 1993; Sheu et al., 1993; Champagne et al., 1994).
Encapsulation in polymer gels has been examined as a means of ensuring the
ecological competence of meat starter cultures, although studies on the encapsulation of
meat starter cultures have been quite limited (Kearney, 1990; Kearney et al., 1990a, b;
McLoughlin and Champagne, 1994). However, immobilisation of meat starter cultures
has recently been identified as one of the areas in meat fermentation which deserves
further investigation (Ricke and Keeton, 1997; Prochaska et al., 1998). With the aid of
electron microscopy studies on fermented meat products, it has been shown that the
natural microflora, and also the starter culture bacteria, grow in cavities or nests within
the meat (Katsaras and Leistner, 1991). Encapsulation of the starter culture provides a
means of controlling the conditions and the microflora within these nests.
3.1.1. Encapsulation matrices and the encapsulation process.
The encapsulation process involves the trapping of cells or molecules within the
lattices of a polymer gel. The entrapment matrix may be naturally occurring polymers
such as alginate, carrageenan or chitosan or a synthetic polymer such as polyacrylamide
(Woodward, 1988). Naturally occurring polymers are widely used in immobilisation,
with algal polysaccharides being the most popular matrices (Willaert and Baron, 1996).
Alginate, which is a polysaccharide, is most commonly extracted from the brown
seaweed Laminaria hyperborea. Alginate consists of unbranched copolymers of (14)-L-guluronic acid (G) and -(14)-D-mannuronic acid (M) and their relative
proportions (G/M ratio) determines the functional properties of the alginate (Murata et
al., 1993). Gelation of alginate occurs as a result of the binding of divalent cations to
the alginate and this is also accompanied by a conformational change. These cations
bind preferentially to the -(14)-L-guluronic acid; therefore, a high content of Gblocks results in a strong gel (Willaert and Baron, 1996). Calcium alginate beads can be
produced by adding droplets of a sodium alginate solution into a calcium chloride bath
where droplets form gel spheres which maintain their shape as a result of cation
exchange between Na+ and Ca2+. During gelation these gel spheres entrap materials or
cells which were formerly dispersed in the alginate solution within an egg-box type
structure. Immobilisation within alginate beads has been successful because it is a mild
process, which can be carried out quickly, simply and cheaply. The mild process (Figure
2) is also an important factor in that it means that encapsulation does not add additional
stress to the bacteria before they undergo the preservation process. However, this
method also has disadvantages, the most significant of which is the sensitivity of
calcium alginate beads to chelating agents such as phosphate, citrate, lactate and EDTA
(Willaert and Baron, 1996). Nevertheless, alginate in particular, has been focussed upon
as an encapsulation matrix due to the mild nature of the entrapment process and also the
244
status of alginate as a food grade additive (sodium alginate -E401, calcium alginate E404) (Anonymous, 1995a).
Figure 2: The process of cell encapsulation in polymer gel beads
Carrageenans are polysaccharides, which are extracted from red seaweed of which
Chondrus crispus is the main source. There are three main types of carrageenan, kappa
(), lambda () and iota (i) with -carrageenan the most widely used for cell
immobilisation (Willaert and Baron, 1996). Carrageenans are sulphated linear
polysaccharides of D-galactose and 3-6 anhydro-D-galactose with the various
carrageenans differing from one another in their content of 3-6 anhydro-D-galactose and
the number and position of ester groups (Trius and Sebranek, 1996). Gelation can be
achieved by cooling, due to the thermal properties of the gel, or by contact with a
solution of gel inducing reagents such as K+, NH4+, Ca2+, Cu2+, Mg2+, amines or water
miscible organic solvents (Chibata et al., 1987). This is a mild and simple process.
Carrageenan gel networks are formed during cooling by a series of polymer chain
associations to give rise to a 3-D helix framework. This framework is stabilised in the
presence of cations by the formation of additional bonds (Trius and Sebranek, 1996).
carrageenan gelation is dependant on cations but unlike alginate beads, carrageenan
beads are thermally reversible (Willaert and Baron, 1996).
Chitosan is another polysaccharide which is used for immobilisation but it is not as
widely employed a matrix as alginate or carrageenan. Chitosan is a partially
deacetylated chitin, which is formed by reacting chitin with a concentrated alkali
(Vorlop and Klein, 1987). It is a high molecular weight linear polymer consisting of
14 linked glucosamine and has a high nitrogen content (>7% w/w) (Vorlop and Klein,
1987; Willaert and Baron, 1996). Chitosan is soluble in organic acids and to a more
limited extent in mineral acids e.g. HCl. Gelation of chitosan occurs as a result of
245
CAHILL, S.M., UPTON, M.E., ANDMCLOUGHLIN, A.J.
binding with multivalent anions. When chitosan is added dropwise to a cross-linking

solution having a pH < 6 the NH2 groups of chitosan are protonated and an ionic crosslinking occurs. In order to form a true ionotrophic gel the pH of the cross-linking
solution must be less than 6. Bead formation will also occur at a pH greater than 7.5 but
this is merely a precipitation of chitosan as at this pH the chitosan is totally
deprotonated and is water insoluble (Vorlop and Klein, 1987). Chitosan can be crosslinked with low molecular weight counterions such as polyphosphates and also high
molecular weight counterions such as alginate. Cross-linking of chitosan with low
molecular weight counterions results in globules in which cells or molecules are
entrapped in a real network while cross-linking with high molecular weight counterions
results in capsules (Vorlop and Klein, 1987). Also, unlike calcium alginate beads,
ionotrophic chitosan beads are stable to chelators such as phosphate (Willaert and
Baron, 1996).
Entrapping cells in a polymer gel permits greater control of the cell
microenvironment such as the juxtapositioning of cells and nutrients and controlling the
rate of cell release (McLoughlin and Champagne, 1994). Also, gels have the properties
of both liquids and solids, for example a 1% alginate gel contains 99% water and yet it
shows solid characteristics such as a defined shape and resistance to stress
(McLoughlin, 1994). Controlling the level of polymer added permits control over the
characteristics of the encapsulate and so allows the properties of the encapsulate to be
tailored to a certain extent.
3.1.2. The benefits of meat starter culture encapsulation.
The advantages of cell encapsulation can be divided into two main groups:
a) Protection - the provision of optimal conditions for activity and the
protection of the cells from a fluctuating and dynamic macroenvironment
b) Controlled release - controlling the rate of release allows the cells to adapt
to their new environment (McLoughlin, 1994). With regard to meat starter
cultures, the advantages can be defined more specifically as increased stability,
enhanced activity or an increased rate of acidification and protection from
competition and bacteriophage.
3.1.2.1. Enhanced stability of encapsulated starter cultures. The preservation of starter
cultures is necessary to facilitate their storage and transport. The most commonly used
methods of preservation are lyophilisation and freezing. The preservation process
followed by the rehydration or reactivation process subjects the cells to stress, which
can affect their subsequent activity. However, it has been demonstrated that the
encapsulation of the meat starter cultures Lactobacillus plantarum and Pediococcus
pentosaceous in calcium alginate beads, in the presence of cryoprotectants, prior to
freezing or lyophilisation, resulted in a higher level of survival than that observed for
free cells (Kearney, 1990). This increased survival appears to be due to the rate at
which the cells are rehydrated or reactivated. With encapsulated cells rehydration is not
instantaneous and is controlled by the diffusion properties and the actual volume of the
encapsulate thus avoiding the osmotic shock associated with instantaneous hydration
(Prochaska et al., 1998). Thus, the use of encapsulation to create a microenvironment,
246
whose properties are influenced by the presence of polyols and skim milk, can result in
increased survival rates of meat starter cultures (Kearney, 1990).
3.1.2.2. Enhanced activity of encapsulated starter cultures, Encapsulation of meat
starter cultures has also been shown to increase fermentation rates compared to free
cells (Kearney et al., 1990b). This is a consequence of the increased stability provided
by immobilisation (section 3.1.2.1), which subsequently decreases the lag phase of
growth and acidification. The encapsulation matrix also offers protection from other
stresses in the meat macroenvironment such as salts (Kearney et al., 1990a). This is an
important observation particularly in light of the fact that the search for techniques to
control emerging food pathogens may involve the inclusion of additional stresses in the
meat.
3.1.2.3. Protection from bacteriophage. Encapsulation technology has been shown to
protect starter cultures from bacteriophage by means of exclusion of the bacteriophage
from the microenvironment of the encapsulate (Steenson et al., 1987). While
bacteriophage are recognised as a major problem within the dairy industry their role in
meat fermentations appears to be of less consequence (Nes and Sarkeinn, 1984).
However, the fact that entrapment within a matrix protects cells from bacteriophage also
means that the cells are protected from the competitive effects of the natural microflora
present in the meat.
3.1.2.4. Genetic stability. The utilisation of biotechnological approaches as a means of
producing more efficient starter cultures is becoming increasingly important. The usual
approach to gene cloning of the lactic acid bacteria is the development of vectors based
on either cryptic plasmids or heterogeneous plasmids which are resistant to a wide range
of antibiotics (Gasson, 1993). While the development of genetically engineered starter
cultures for use in meat fermentations on a large scale has so far been elusive, work in
this area is ongoing. However, once developed the stability of these strains will be of
great importance. Encapsulation in calcium alginate beads has been shown to increase
the stability of plasmid containing bacteria (ODonnell, 1997).
Therefore,
encapsulation technology has the potential to enhance the biotechnological
developments made in the area of starter culture technology.
3.1.3. Commercial applications.
As yet the advantages of encapsulation of meat starter cultures in alginate beads has not
been exploited at a commercial level. This is probably due in part to the fact that the
technology for the large-scale production of polymer gel encapsulated bacteria is only
evolving, The common entrapment technique, which involves adding the cell
containing alginate solution into a bath of calcium ions in a dropwise manner, is
difficult to perform on a large-scale. However, one engineering company has recently
developed the equipment to carry out this process on a large-scale (Anonymous, 1999).
This may facilitate the large-scale production of polymer gel encapsulated starter
cultures and so enable the commercial exploitation of the technology.
247
Methods of internal gelation, which involve the addition of the calcium source directly
into the alginate solution and controlling the availability of the calcium ions through the
use of calcium chelators (Monshipouri and Rice, 1995) or pH (Poncelet et al., 1992,
1995; Quong et al., 1998) have permitted the development of emulsification or
dispersion methods of encapsulation. This involves the addition of the alginate/calcium
mixture into a bath of oil and applying a shear in order to disperse the gel mixture
throughout the oil, thus producing spherical encapsulates. The use of a calciumchelating agent slows down the gelation process sufficiently to permit the dispersion of
the gel in the oil. The pH method involves the external addition of an acid to reduce the
pH and cause the release of calcium ions from the calcium source present. The acid is
added after the emulsification or dispersion step. While these techniques are suitable
for the large-scale production of alginate beads, an improvement of the encapsulation
technique is still required. The harvesting of the encapsulates from the oil has been
reported to be problematic (Friel, 1998). Also, a broad size distribution of the
encapsulates produced by this method has been observed (Poncelet et al., 1992; 1995).
3.2. THE APPLICATION OF ENCAPSULATION TECHNOLOGY TO CHEMICAL
ACIDIFICATION
In contrast to meat starter cultures, the encapsulation of acidulants has been extensively
studied and commercialised (Marchot, 1993). The application of encapsulation
technology to food acids has been spurred on by the fact that acidification is a valuable
means of food preservation (Davidson, 1997), even though the direct addition of acids
to many foods results in undesirable flavour and colour changes (Shay and Egan, 1992).
Also, the application of acids to a wide range of food products including meat, dairy
products, bakery products, desserts and canned fruits and vegetables (Dziezak, 1988;
Janovsky, 1993; Marchot, 1993) would appear to assure the commercial viability of
encapsulated acids.
3.2.1. Encapsulation matrices and the encapsulation process.
A large variety of materials can be used as the coating or encapsulation matrix and the
selection of the matrix will depend to a certain extent on the properties required by the
encapsulate. Lipids, for example waxes, paraffin, steric acid and oils such as
hydrogenated soya oil, palm oil and cottonseed oil are probably among the most
commonly used encapsulation materials, although there have also been reports on the
entrapment of acids in alginate (Cordray and Huffman, 1985; Ensor et al., 1990;
Siragusa and Dickson, 1992).
While the immobilisation of acids in alginate involves, like free cells, the
crosslinking of the alginate with calcium ions, the process of entrapment within a lipid
matrix is quite different. Some of the processes which have been utilised for the
entrapment of acids in lipids include spray cooling, spray chilling, air suspension
coating and centrifugal-suspension coating (Jackson and Lee, 199 1), techniques which
allow the effective utilisation of "hot melt" technology. Encapsulation protects the acid
from premature reaction with the meat and provides for controlled release when the
necessary conditions are met (Janvsky, 1993). Release of the lipid-encapsulated acids is
248
related to temperature but other mechanisms of release such as that caused by the
gradual intrusion of water are also being investigated.
Spray cooling and spray chilling involve the use of cool air to solidify the
encapsulate (Lamb, 1987). These two processes differ in the encapsulating material
used in that lipids with low melting points (32 42C) are used in spray chilling while
spray cooling utilises lipids with a higher melting point (45 122C) (Dziezak, 1988).
The acid/lipid mixture is extruded through heated nozzles into a low temperature
chamber where the lipid solidifies, entrapping the acid.
While spray chilling and cooling are suitable for liquid acids, air suspension coating
is used to coat or encapsulate a solid core material. The process involves suspending
the solid core material in a fluidised bed of air and spraying with the coating material
(Dziezak, 1988; Kondo, 1989). The thickness of the coating material can be controlled
by the length of time for which the coating is applied.
Centrifugal-suspension coating involves suspending the core particles in the liquid
coating and then introducing them onto a rotating disk (Sparks and Mason, 1990). This
results in the coating material forming a film around the core material. Another process
which has been described for acidulant encapsulation involved plating the particulate
acidulant onto calcium lactate and then coating with a molten edible lipid (Percel and
Perkins, 1985). The product of this process would have controlled release at a higher
temperature.
3.2.2. The benefits of acidulant encapsulation.
The encapsulation of acidulants permits control over the rate and time of acid release.
These are important factors in meat preservation by acidification. During sausage
production the ingredients must be allowed to 'bind' after mixing. This process is
prevented if the pH is reduced too quickly resulting in a product with a crumbly texture,
which is undesirable in a sausage. By encapsulating the acid so that the time of release
is controlled, for example delaying acid release until the meat reaches smokehouse
temperature, means that the use of an organic acid need not compromise sausage
texture.
The second problem with direct acidification is its adverse effect on colour
development. The pH of preserved meats must be carefully controlled in order to allow
the formation of nitroso-hematin pigments, which are responsible for the typical pinkred colour of these meat products (Jackson and Lee, 1990). Encapsulated acids can be
released slowly thus allowing the desired colour pigments to form.
Encapsulated acids have also been reported as a means of overcoming the batch to
batch variation and long processing time which are inherent in the microbial
fermentation process (DeZarn, 1995). Using encapsulated acids it is possible to develop
a formulation or recipe with which a very reproducible pH can be achieved. Also, the
processing time can be drastically reduced compared to a microbial fermentation thus
allowing a higher level of production.
3.2.3 Commercial availability.
The commercial viability of encapsulation is reflected in the number of patent
applications in this area (Pszczola, 1998). Recently Doskocil Food Service Co. in the
United Stated have patented a process for producing dry and semi-dry sausage products
249
using an encapsulated acid (Anonymous, 1998). The process involves the use of an
acidulant, which has been encapsulated in a material with a melting temperature of at
least 90F. Encapsulated acids are available under brand names such as CAP-SHURE
and DURKOTE with some companies also offering the facility whereby they will
tailor-make encapsulates to meet the customers requirements. It must also be noted that
the cost of encapsulates compared to the free form is quite expensive, up to three times
that of the free acid. For example encapsulated lactic acid costs IR7.00/kg compared
to IR1.50/kg to IR2.00/kg for the free form. Therefore, these encapsulates must offer
some unique functionality over the free acid in order to achieve and retain economic
viability.
The application of encapsulation to meat products is not confined to encapsulated
acids. Encapsulated salt is also used in meats (DeZarn, 1995). The use of encapsulated
salt permits the addition of extra salt to meat products without adversely affecting the
shelf life or the texture of the meat.
4, Control of emerging pathogens
Recent food poisoning outbreaks associated with enterohaemorrhagic Escherichia coli
in fermented meat products have raised questions regarding the safety of these
foodstuffs.
In the United Stated this has led to regulatory action requiring
manufacturers of fermented meat products to demonstrate that their process incurs a five
log reduction in E. coli O157:H7 numbers. It has been reported that the traditional
fermentation process is not sufficient to achieve this and currently the only means of
doing so is to incorporate a heat step into the manufacturing process. While
incorporating a heating step will ensure the production of a safe product, taste and
texture may be compromised. As a result, alternative methods of eliminating foodborne
pathogens and ensuring a safe product are being sought.
It is likely that any technique of eliminating foodborne pathogens will also be
detrimental to the starter culture bacteria used in microbial meat fermentation. One way
of overcoming this may be in the use of bacteriocins, antimicrobial agents produced by
lactic acid bacteria. At present only one such agent, nisin, has GUS status
(Anonymous, 1969). Nisin has been applied in the meat industry, but with limited
success. This is probably due to the low solubility of nisin, uneven distribution, heat
sensitivity at neutral pH values, possible binding to meat proteins and antagonising
effects within the meat (De Vuyst and Vandamme, 1994). However there have been
some reports where nisin was found to be effective at reducing numbers of bacteria
attached to meat. Chung et al. (1989) found that nisin had an inhibitory effect on Grampositive bacteria attached to meat. Nisin has also been reported to be successful in
controlling the growth of food spoilage organisms and food pathogens on cooked pork
when used in combination with a modified atmosphere packaging system (Fang and
Lin, 1994). The use of a nisin-producing Streptococcus lactis in reducing bacterial
growth in frankfurters has also been reported (Wang et al., 1986). The application of
nisin as a possible alternative or adjunct to nitrites and nitrates in the preservation of
meats has been suggested (Rayman et al., 1981). This may be an important application
as consumer concerns regarding carcinogenic nitrosamines in cured meats increase.
250
While the successful application of nisin to meat preservation has so far been elusive, as
in the case of direct acidification, encapsulation technology may provide a means of
overcoming this problem.
5. The application of encapsulation technology to bacteriocin delivery
5.1 BACTERIOCINS
Bacteriocins are proteinaceous antimicrobial compounds produced by a wide range of
bacteria but not lethal to the producer organism. The lactic acid bacteria, probably the
most important group of starter culture bacteria, produce a wide range of bacteriocins
and bacteriocin-like substances (De Vuyst and Vandamme, 1994). The demand from
consumers to reduce or remove chemical preservatives from foods has initiated much
interest in the use of naturally occurring metabolites to inhibit the growth of undesirable
microorganisms (De Vuyst and Vandamme, 1994). Bacteriocins produced by lactic
acid bacteria are potential agents for use as natural food preservatives. For example, the
use of bacteriocin producing starter cultures for in situ bacteriocin production may be a
useful means of food preservation and thus the potential exists to use bacteriocins as
natural preservatives in fermented products.
While the use of bacteriocins as natural preservatives appears to be an attractive
option there are a number of factors to be considered relating to their use in fermented
products. Firstly, bacteriocins are generally most effective against bacteria closely
related to the producer organism and therefore their effect on the starter culture must be
considered before using them in fermented products (De Vuyst and Vandamme, 1994).
One means by which this can be overcome is using bacteriocin producing starter
cultures, but this may require the use of genetically manipulated microorganisms.
Secondly, bacteriocins are generally ineffective towards Gram-negative bacteria such as
E. coli and therefore their use can be limited unless they are used in conjunction with
another agent such as a chelator, in order to enhance their activity towards Gramnegatives (Blackburn et al., 1989; Stevens et al., 1991, 1992; Cutter and Siragusa,
1995a, b; Shefet, et al., 1995). Thus, when applying bacteriocins in food preservation
they should be delivered in such a way as to minimise their effect on the naturally
occurring beneficial microflora in both the foods to be fermented and in the consumer
while also having a negative effect on specific pathogens. The complex nature of the
system highlights the need for a very directed delivery system. Encapsulation of the
bacteriocin nisin was studied as a means of modelling a directed delivery system.
While it is recognised that nisin may not be the ideal bacteriocin for application in meat
fermentations, its position as the only bacteriocin with GRAS status (Anonymous,
1988) means that currently it is the only bacteriocin which can be added directly to
foods.
5.2 NISIN
As it is a naturally occurring preservative work to find new applications for nisin is
ongoing. One area of research involves the transfer of the genetic material encoding
251
nisin production and nisin immunity to industrial starter cultures (Hugenholtz and de
Veer, 1991). This would mean that many fermented food products could be protected
by nisin produced in situ without the fermentation being adversely affected. Extending
the spectrum of nisin activity to Gram-negative bacteria is also being examined. The
application of nisin in combination with food grade chelating agents is reported to
increase the inhibitory activity and inhibitory spectrum of nisin (Blackburn et al., 1989;
Stevens et al., 1991, 1992; Cutter and Siragusa, 1995a, b; Shefet et al., 1995). The use
of nisin in combination with other bacteriocins such as pediocin AcH has been reported
to demonstrate greater antibacterial activity against a greater number of Gram-positive
bacteria (Hanlin et al., 1993). Nisin in combination with lactate has been found to
reduce the numbers of Salmonella typhimurium attached to beef and nisin in
combination with EDTA has been reported to reduce the numbers of Escherichia coli
O157:H7 attached to beef (Cutter and Siragusa, 1995b).
5.2.1 Encapsulation of nisin
The immobilisation process can be used to confine molecules to a certain region in
space in such a way as to exhibit hydrodynamic characteristics which differ from those
of the surrounding environment (Willaert and Baron, 1996). This can be achieved by
attachment or adsorption onto a solid support or entrapment or encapsulation within a
matrix. Each of these methods has been reported for the immobilisation of nisin.
Encapsulation or immobilisation of the antimicrobial peptide nisin is a concept
which has only recently been reported (Daeschel et al., 1992; Lante et al., 1994; Bower
et al., 1995a, b; Fang and Lin, 1995; Cutter and Siragusa, 1996, 1997, 1998; Wan et al,
1997). There have been several reports on the immobilisation of nisin by adsorption
onto silica surfaces which have suggested that nisin may be adsorbed onto food contact
surfaces in order to prevent colonisation of food pathogens thus leading to a safer food
product (Daeschel et al., 1992; Bower et al., 1995a, b). Nisin has also been
immobilised on organic and inorganic matrices but will only display antimicrobial
activity when desorbed from these supports (Lante et al., 1994). Encapsulation of nisin
within a polymer gel has also been reported. Nisin immobilised within calcium alginate
resulted in a greater reduction in bacterial numbers and also nisin activity was sustained
for up to seven days under refrigeration conditions (Cutter and Siragusa, 1996, 1997).
The stability of nisin on cooked pork was also improved by immobilisation of nisin in
1-% calcium alginate (Fang and Lin, 1995). Incorporation of nisin within calcium
alginate micro-particles has been shown to protect the bacteriocin from degradation by
proteolytic enzymes (Wan et al, 1997). This suggests that encapsulation of nisin within
a polymer gel may be an effective delivery system for its protection and controlled
release to meat matrices.
Two methods of nisin delivery, using encapsulation in polymer gels, have been
investigated.
Immobilisation of the producer organism, Lactococcus lactis, in calcium
alginate beads
Encapsulation of nisin in capsules prepared using a combination of polymers.
5.2.1.1. Formulation of an optimal encapsulation system for nisin production by L.
lactis. Immobilisation of cells in calcium alginate beads allows incorporation of a
252
combination of nutrients and protective agents into the matrix, which enhances
inoculum survival (Heijnen et al., 1992). Formulation of a delivery system for a
microorganism by incorporating nutrient adjuncts into the bead microenvironment has
been previously reported. Including maize cob grits and wheat gluten in alginate beads
containing Aspergillus flavus spores enhanced the activity of the mould after field
release (Daigle and Cotty, 1995). The incorporation of a combination of skim milk and
bentonite resulted in the highest survival and colonisation rates of Pseudomonas
fluorescens inoculated into soil (van Elsas et al., 1992). Similarly with immobilised L.
lactis, co-entrapping a combination of nutrients and microenvironmental modifying
agents increased the activity of the immobilised cells. Thus, one of the obvious benefits
of immobilised cell technology is in the control and exploitation of the unique
microenvironment associated with gel entrapment and especially using this
microenvironment in the stabilisation of microbial cultures (Karel et al., 1985).
Figure 3: The effect of a combination of eo-immobilisation treatments on nisin activity by

cells immobilised within calcium alginate beads.
Treatment A; Cells only

Treatment B; Cells +1% glucose
Treatment C; Cells + 1% tricalcium
phosphate
Treatment D; Cells + 3% skim milk
Treatment E; Cells + 1% Tween 80
Treatment F; Cells + 1% tricalcium phosphate + 3% skim milk

Treatment G; Cells + 3% skim milk +1% glucose
Treatment H; Cells + 1% tricalcium phosphate +1% glucose
TreatmentI; Cells + 1% tricalcium phosphate + 3% skim milk
+1% Tween 80
Treatment J; Cells + 1% tricalcium phosphate + 3% skim milk
+ 1% glucose
253
Through manipulation of the bead microenvironmental conditions, nisin production

within a polymer gel can be optimised. The inclusion of nutrient and non-nutrient
adjuncts with L. lactis cells entrapped in calcium alginate beads has been observed to
have an advantageous effect on the activity of immobilised cells. An optimum delivery
system for L. lactis was formulated by co-entrapping a combination of adjuncts and
cells within the confines of the alginate bead (Figure 3). Locating both the nutrients and
the cells within the confines of the encapsulation matrix resulted in an increase in the
level of nisin produced by the entrapped cells.
The requirement to deliver microorganisms to dynamic environments such as meat and
the development of immobilisation technologies has resulted in the evolution of
inoculum delivery systems in a number of application areas which improve the
resistance of the culture to stress and regulate the release of cells from a protective
microniche (McLoughlin, 1994). The application of immobilisation in enhancing the
survival of starter cultures during freezing, lyophilisation and subsequent storage has
been mentioned previously and is well documented (Kim et al., 1988; Kearney et al.,
1990a; Sheu et al., 1993; Sheu and Marshall, 1993; Champagne et al., 1994). However
this is only one type of stress from which immobilisation has been shown to offer
protection
While the limited availability of nutrients has the ability to increase bacterial
resistance to stress (Gilbert and Brown, 1995), the inclusion of excess nutrients within
the bead microenvironment also increased the survival of cells exposed to acidic and
osmotic stresses. The activity of these cells was also enhanced. This means that the
incorporation of adjuncts within the bead can be used as a means of manipulating the
bead microenvironment to control bacteriocin activity (Figure 4). This is of particular
interest in light of the fact that lactic acid bacteria may react to physiological inducers
and so manipulation of the cellular environment may stimulate bacteriocin production
(De Vuyst et al., 1996). This stimulation of bacteriocin production may be of
significant importance when bacteriocin producing lactic acid bacteria are added to
foods as starter or protective cultures (De Vuyst et al., 1996). However, the specific
environment of the food may limit the capacity of the lactic acid bacteria to produce
these antimicrobials (De Vuyst et al., 1996). This was observed to be the case in the
presence of an acid and a salt stress (Figure 4). Therefore, designing the bead
microenvironment so that it provided a source of nutrients and a means of acid control,
as well as providing a physical barrier between the entrapped cells and the
macroenvironment, allowed enhanced bacteriocin production under adverse conditions
(Figure 4). This was significant in that it demonstrated the potential benefits of
entrapment in a polymer gel in the development of inocula for in situ bacteriocin
production.
254
Figure 4: The effect of o-immobilisation treatment on a) biomass development and b) nisin

production by immobilised cells grown in broth at pH 5 and 1% NaCl. and c) biomass
development and d) nisin production by immobilised cells grown in broth at pH 7 and 4%
NaCl.
Treatment A; Cells only
Treatment B; + 3% skim milk
Treatment C; + 1 % tri-calcium phosphate
Treatment D; + 3% skim milk + 1% tri-calciumphosphate
Treatment E; + 3% skim milk +I% glucose
Treatment F; + 1% tricalciumphosphate + 3% skim milk + 1% glucose
5.2.1.2 The application ofpolymer gels in the controlled release of nisin. One of the
disadvantages of nisin production in an immobilised cell system is retention of the
bacteriocin within the bead. Microenvironmental manipulation was shown to increase
nisin yield, however, this was also accompanied by an increase in nisin retention within
the encapsulation matrix. Therefore, the release of nisin from polymer gels and how the
release could be controlled and manipulated were investigated. There are numerous
advantages to be gained by the controlled release of nisin to the macroenvironment such
as enhanced stability and protection from proteinase degradation thus, prolonging
activity.
255
Figure 5: The release of nisin from polymer gel capsules. The release of nisin into liquid
medium DMI was followed over a one week period at 4C and 200 rpm.
For many bioactive agents it is recognised that there is a need to prolong the duration of
activity and develop a means of more efficient utilisation of the bioactive agent
(Schacht et al., 1993).
Within the area of pharmaceuticals, sustained release
formulations are now a relatively common method of delivering low molecular weight
drugs (Langer, 1990). The encapsulation of proteins and the development of slow
release formulations are viewed as enhancing the stability and thus the applications of
proteins in therapeutics (Putney and Burke, 1998). Natural polymers can serve as depot
systems in which the agent can be incorporated and from which it is subsequently
released over an extended and controllable period of time (Schacht et al., 1993).
Although the application of immobilisation or encapsulation technology in the
delivery of bacteriocins is a relatively new development (Degnan and Luchansky, 1992;
Kirby, 1993; Fang and Lin, 1995; Cutter and Siragusa, 1996, 1997, 1998; Wan et al.,
256
1997), the encapsulation of nisin in calcium alginate has been achieved with some
success. It has been observed to prolong the activity of the nisin and protect it from
degradation by proteolytic enzymes (Fang and Lin, 1995; Cutter and Siragusa, 1996,
1997; Wan et al., 1997). However, manipulation of the gel matrix in order to control
the release of nisin to the macroenvironment has not been reported. This, however, is
an important factor in the development of delivery systems for other bioactive agents,
such as therapeutic agents (Aydin and Akbuga, 1996; Berthold et al., 1996).
The influence of polymers on the release of nisin from the immobilisation matrix
has been examined with a view to developing a delivery system, which offered a
controlled mode of nisin release. Through exploitation of the polymer composition and
charge, immobilisation matrices were designed with the aim of producing encapsulates
which exhibited controlled release properties for the bacteriocin nisin. The preparation
of encapsulates from alginate and chitosan has been demonstrated as a means of
controlling the rate of nisin release, at least into a liquid macroenvironment (Figure 5).
The structure of these encapsulates (Figure 6) as well as the chemical properties of the
encapsulation matrix and the core material have been identified as factors with a role in
the mechanism of controlled release.
Figure 6: The internal structures of capsules preparedfrom a combination of chitosan and

alginate gels. (a + b) ACC capsules (alginate core chitosan coated capsules stabilised
with calcium chloride)
257
Figure 6: The internal structures of capsules prepared from a combination of chitosan

and alginate gels.
c) CCA capsules (chitosan core alginate coated capsules stabilised with calcium
chloride)
d) CAT capsules (chitosan core a lginate coated capsules stabilised with
tripolyphosphate)
Figure 6: The internal structures of capsules prepared from a combination of chitosan

and alginate gels. (e) ATCC capsules (alginate core chitosan coated capsules stabilised
with tripolyphosphate)
258
The modification of polymer beads by coating with a polymer of the opposite charge
has been previously reported (Huguet et al., 1996; Quong and Neufeld, 1998). It has
also been reported that better slow release properties can be achieved when chitosan or
polylysine are used to change the permeability of the alginate membrane (Nuissinovitch
et al., 1996). Chitosan is also acceptable for oral administration as well as being a good
matrix for sustained release properties (Chandy and Sharma, 1992, 1993). Alginate chitosan capsules can be produced as a result of complexing between two oppositely
charged polymers, alginate being polyanionic and chitosan being a polycationic
polymer (Huguet et al., 1996). Capsules have been prepared with an alginate core and a
chitosan coat and vice versa, and the effect of each polymer on the release properties of
the other evaluated. Low molecular weight counterions were included in the
preparation of these capsules (Knorr and Daly, 1988; Pandya and Knorr, 1991 ; Polk et
al., 1994; Chang et al., 1996; Hari et al., 1996; Nussinovitch et al., 1996) as this
stabilised the capsule structure. Although capsules can be produced solely from
alginate and chitosan (Vorlop and Klein, 1987) these were observed to be weak due to
narrow width of the capsule membrane compared to the overall diameter of the beads.
This may be overcome by producing capsules of very small diameter.
The release pattern from capsules was dependent on a number of factors. According
to Huguet and Dellacherie (1996), the release of materials encapsulated in alginate
beads coated with chitosan depends on 1) the molecular weight of the material, 2) the
chemical composition and 3) the conformation of the molecules. These factors can vary
depending on the composition of their environment.
Coating alginate beads with a polycation has been reported to drastically reduce
diffusion from the beads. Nussinovitch et al. (1996) reported that after 6 days only 50%
of the entrapped bovine albumin and 10% of entrapped gamma globulin was released
from alginate polylysine capsules. At the surface of the membrane a chemical reaction
occurs between the polycation and the polyanion and the strength of this membrane can
be controlled by the polycation used thus making it possible to tailor liquid-core beads
to specific aims (Nussinovitch et al., 1996). Chitosan is insoluble at a pH above 5.4 and
so, while it is unsuitable for biomolecules unstable at low pH values (Huguet at al.,
1996), this makes it a suitable gel for nisin encapsulation due to the high stability of
nisin at acidic pH values.
The molecular weight of nisin is 3,353 Da (Jung, 1991a, b), although, it often occurs
as stable dimers with a molecular mass of about 7,000 or tetramers of 14,000 Da
(Cheeseman and Berridge, 1957, 1959; Jarvis et al, 1968). While the rate of diffusion
decreases with increasing molecular weight (Martinsen et al., 1989), this should not
affect the diffusion of nisin due to its low molecular weight. Proteins with a molecular
weight of between 2,500 and 66,000 Da have been reported to diffuse readily from a
matrix (Nussinovitch et al., 1996).
The chemical nature of the environment, such as pH or ionic charge, is an important
factor in relation to nisin release as this determines how the bacteriocin reacts under
particular conditions. For example, the effect of pH on the solubility of nisin is well
known (Liu and Hansen, 1990). The pH is also an important factor in relation to the
physical structure of the encapsulating matrix. At high pH values chitosan forms an
insoluble precipitate while at acidic pH values an ionotrophic gel is formed (Vorlop and
Klein, 1987). The pH at which these capsules are prepared is an important factor in
259
relation to entrapment and release. For example, if alginate - chitosan encapsulates are
prepared at a pH of about 5.5, the surface of the beads formed is highly negative and the
positively charged chitosan can establish a strong ionic interaction with the alginate
(Huguet et al., 1996). On the other hand, if the capsules are prepared at a very low pH
(~ 2) the alginate will have a very low content of negative charges and so cannot
interact strongly with the chitosan (Huguet et al., 1996). In the preparation of alginate
core capsules, the pH of the alginate was 5.48, which would optimise ionic interactions
with the chitosan. In the preparation of chitosan core capsules, the pH of the alginate gel
was 7.17 and so it was highly negatively charged and so there was an ionic interaction
between the two gels. While normally such a high pH would cause precipitation of the
chitosan (Vorlop and Klein, 1987), the high molecular weight of the alginate would
prevent its diffusion through the chitosan. Therefore, the chitosan would not be
completely exposed to the high pH. The membrane produced as a result of the ionic
interaction between the alginate and the chitosan appeared to be the important factor in
relation to nisin release. The absence of any visible interaction between these two
polymers in chitosan-alginate-tripolyphosphate (CAT) and alginate-tripolyphosphatechitosan-calcium chloride (ATCC) capsules and the observed differences in release
from these capsules compared to chitosan-calcium chloride-alginate (CCA) and
alginate-chitosan-calcium chloride (ACC) capsules highlights this fact. The inclusion
of tripolyphosphate in the alginate increased its pH to approximately 8.5. Exposing
chitosan to such an alkaline pH causes it to precipitate (Vorlop and Klein, 1987) and in
the case of ATCC and CAT capsules this is likely to have prevented any interaction
between the chitosan and the alginate. CCA capsules exhibited a very low level of nisin
release and structural differences were observed between the chitosan core in these
capsules and the chitosan coat in ACC capsules. This was likely to be due to the pH
factor, The chitosan core of CCA capsules contained calcium chloride, which diffused
outwards to stabilise the alginate coating. There is no influx of ions into the bead and
so the pH of the core remained low (< 5). This is a stable environment for nisin (Liu
and Hansen, 1990) and as the alginate coat will be of a higher pH than the nisin
containing core the nisin was probably retained in this part of the capsule resulting in a
low level of release.
The solubility, stability and biological activity of nisin are highly dependent on pH.
They drop sharply and continuously as the pH is increased and nisin is almost insoluble
at neutral and alkaline conditions (Liu and Hansen, 1990). How the pH affects the
charge of a protein will affect its release (Huguet and Dellacherie, 1996). Proteins
which are stored under pH conditions lower than their isoelectric point present a
positive charge and their release remains low (Huguet and Dellacherie, 1996). In this
study, the low level of nisin release from CCA capsules, which have a low internal pH,
reflected this.
Investigating the application of polymer gels in the development of a delivery
system for nisin has shown that by entrapment of the nisin-producing organism, L.
lactis, in a polymer matrix, conditions for cell activity can be optimised. This permitted
nisin production to be enhanced under both stress and non-stress macroenvironmental
conditions. Manipulating the composition of the entrapment matrix and modifying the
entrapment/encapsulation process, enabled the production of nisin encapsulates, from
which the release of the bacteriocin to the external environment could be controlled.
260
Thus, it can be concluded that naturally occurring polymer gels can be successfully
applied to the development of a delivery system for nisin. The encapsulates which have
been developed for the delivery of L. lactis and nisin would now need to be evaluated in
a meat product. This would determine the full extent of the success of encapsulation in
polymer gels to nisin delivery.
6. Conclusions and future work
The production of safe food is a difficult task and while there will always be the risk of
foodborne disease there is an ongoing endeavour to control or minimise the risks. Risk
managers are continually looking for new options to minimise the risks associated with
foods and this in turn encourages researchers to focus on developing these options.
Encapsulation of food ingredients is one of these options and the technology offers great
potential in the area of food preservation. The pharmaceutical/medical industry has
extensively developed and used this technology for the purposes of time-release, flavour
masking and improved stability of pharmaceutical formulations (DeZarn, 1995) and
continues to do so to great benefit, such as limiting the side effects of drugs to the
development of techniques for in situ insulin production for diabetics (Badwan et al.,
1985; Langer, 1990; Duncan, 1993; Willaert and Baron, 1996).
The encapsulation of food ingredients was once regarded as too expensive and
customised for use in the food industry. However, the development of the technology,
although relatively unsophisticated when compared to other fields, means that the use of
encapsulated products has increased. Encapsulated ingredients are being designed to
meet the needs of a specific application. In order to do this a variety of factors must be
considered such as the functional properties of the finished product, the type of
encapsulation material, the processing conditions and the desired release properties
(Pszczola, 1998).
It is widely recognised that much remains to be done in the area of encapsulation of
food ingredients or additives. The necessity to use safe and edible materials and the
associated cost continue to impede the evolution of the technology. However the
potential for using naturally occurring polymer gels such as alginate, carrageenan and
chitosan as encapsulation matrices has been demonstrated here and they may provide a
means of overcoming such impediments.
Within the area of meat preservation, emerging and re-emerging pathogens are
challenging the existing methods of preservation. This means that it is necessary to
seek out new solutions to these new problems. Encapsulation may provide us with a
means of finding solutions and adapting and enhancing existing methods of
preservation. The amount of basic laboratory research being carried out in this area is
quite extensive and perhaps the greatest challenge, which faces the application of
encapsulation technology to particular areas in the food sector such as meat
preservation, is transposing the techniques from the laboratory to the industry.
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266
INDEX
Acetate ............................................................................. 24, 35, 83, 91, 92
Acidification.................. .239, 240, 241, 243, 246, 247, 248, 249, 251, 265
Activated sludge ............................................................................. 178, 181
Active dry yeast........................................ 33, 34, 36, 37, 38, 42, 43, 44, 47
Aerobic propagation ......................................................................... 90, 98
Aeromonas ............................................................................ 194, 196, 229
Aeromonas hydrophila ........................................................................... 196
AFLP ................. 193, 211, 212, 218, 220, 221, 225, 226, 229, 232, 233
Alcaligenes sp ................................................................................ 147, 150
Alcoholic fermentation ... 31, 32, 33, 34, 36, 37, 38, 39, 40, 42, 43, 44, 45,
46
Alginate.................................................................................. 244, 259, 264
Amplified ribosomal DNA restriction analysis ............................. 215, 232
Antagonistic activity ......................................................................... 19, 20
Antibacterial activity ........................................................ 14, 20, 252, 263
Antibiotics .............................................................................................. 18
Antimicrobial ..................................................................................... 1, 263
AP-PCR .................................................................. 211, 212, 222, 223, 228
ARDRA .......................................................................................... 215, 232
Aristolochene synthase ............................................................................ 27
Arming yeasts ..................................................................................... 59, 70
Arthrobacter nicotiana .......................................................................... 189
Aspergillus aculeatus .................................................................. 59, 61, 72
Aspergillus niger ......................................................................... 20, 28, 72
Aspergillus oryzae ................................................................................... 13
Aureobasidium pullulans ....................................................... 177, I78, 183
Azotobacter chroococcum .............................................................. 135, 139
Bacilli ..................................................................................................... 156
Bacillus cereus ................................................................. 20, 196, 233, 234
Bacillus stearothermophilus ........................................................ 59, 66, 73
Bacillus subtilis............................................................. 156, 162, 195, 265
Bacteria.............................................................................. 38, 52, 103, 263
261
Bacterial biomechanics .......................................................................... 157

Bacterial pathogens 1, 193, 194, 195, 196, 198, 201, 203, 205, 206, 208,
210, 212, 229, 235, 23 7
Bacterial typing ..................................................................... 193, 208, 210
Bacteriocins............................................. 20, 239, 250, 251, 252, 256, 262
Biogenic amines ....................................................................................... 42
Biomarker .............................................................................................. 145
Biomaterials ........................................................................................... 262
Biomechanics ................................................................................. 161, 162
Biosorption ............................................................................................ 183
Blue cheese .................................................................................. 13, 14, 29
Botryodiploidin ........................................................................................ 25
Brevibacterium flavum ................................................................ 51, 55, 57
Brucella.................................................................. 199, 200, 203, 230, 235
Campylobacter 193, 194, 195, 196, 199, 201, 209, 210, 226, 227, 231,
232, 233, 234, 235, 236, 237, 238
Campylobacter jejuni 193, 196, 226, 231, 232, 233, 234, 235, 236, 237,
238
Carbon sharing ...................................................................................... 147
Carrageenan .......................................................................................... 245
Cell growth rates ..................................................................................... 92
Cell wall ........................................................................................... 72, 156
Cellulase ................................................................................................ 138
Chitosan ......................................................................... 245, 259, 262, 263
Citric .................................................................................................. 41, 42
Classical typing ..................................................................................... 210
Cloning .............................................................. 44, 73, 131, 132, 190, 247
Cloning of genes ...................................................................................... 21
Clostridium ............................................ 20, 61, 72, 73, 195, 196, 201, 215
Clostridium perfringens .................................................................. 20, 196
Community physiology........................................................................... 150
Competition......................................... 14, 40, 43, 203, 239, 243, 246, 263
Contamination ........................... 65, 89, 193, 203, 204, 205, 206, 219, 243
Cyclopiazonic acid., .......................................................................... .23, 28
Detection1, 46, 144, 148, 149, 152, 153, 193, 194, 198, 199, 201, 202,
203, 204, 205, 206, 207, 218, 223, 224, 229, 230, 231, 232, 233, 234,
235, 236, 237, 238
DGGE ............................................................................................ 220, 234
Diacetyl............................................................................................. 35, 41
268
DNA amplification ................................................................................. 207

DNA chips. ............................................................................................. 220
DNA fingerprints ................................................................................... 219
DPH....................................................................................... 185, 186, 187
Effect of aeration ........................................................................... 137, 139
Effector protein ...................................................................................... 125
Effector proteins .................................................................................... 125
Electrophoretic karyotyping .................................................................... 17
Empedobacter sp.................................................................... 14 7, 149, 150
Encapsulation239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,
251, 252, 254, 255, 256, 257, 259, 260, 261, 262, 263, 265
Encapsulation matrices ................................................................ 244, 248
Enterococcus................................................................................. 195, 197
Escherichia coli 0157 ................................... 196, 229, 235, 237, 252, 263
Ethylcarbamate ........................................................................................ 45
Fattyacids 14, 33, 34, 38, 40, 46, 146, 148, 149, 150, 151, 152, 185, 187,
188
FCC............................................................................ 76, 77, 79, 80, 82, 83
Feather ................................................................................................... 165
Fermentation 2, 13, 14, 18, 20, 21, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
42, 43, 44, 46, 47, 52, 57, 60, 61, 65, 67, 71, 72, 79, 80, 89, 90, 96, 98,
99, 102, 107, 108, 109, 159, 160, 165, 166, 168, 171, 173, 175, 239,
240, 241, 242, 243, 244, 247, 249, 250, 252, 262, 263, 264, 265
Fermented feather meal ......................................................................... 171
Filamentous growth ...................................................................... 131, 132
flaA typing ...................................................................................... 193, 226
Flavor ............................................................ 14, 21, 28, 47, 242, 248, 261
Flocculation .......................................................... 92, 95, 96, 98, 129, 130
Fluorescence measurement., ................................................. 185, 186, 187
Food 27, 28, 29, 45, 47, 85, 227, 230, 231, 232, 233, 234, 235, 236, 237,
238, 249, 261, 262, 263, 264, 265, 266
Food components ........................................................................... 193, 203
Fumigaclavine ......................................................................................... 25
GAC ....................................................................... 177, 179, 181, 182, 183
Galactose .......................................................................................... 83, 85
Galactose metabolism., ............................................................................ 85
Gene disruption ........................................................................... 18, 21, 23
Genetically modified yeasts ..................................................................... 44
Genomic instability ................................................................................ 227
269
Glucoamylase..................................................................................... 59, 60
Glucose oxidase .................................................................. 20, 28, 29, 136
GRAS status ..................................................................... 20, 250, 251, 262
GTP analogues ...................................................................................... 187
GTP-binding proteins ................................................................... 109, 188
HACCP ......................................................................................... 194, 241
Histamine ......................................................................................... 42, 194
Hydrocarbon utilisation ......................................................................... 185
Hydrolytic enzyme .................................................................................... 61
Hydrolytic enzymes .................................................................................. 61
Hydrophobicity ...................................................................... 90, 91, 93, 96
Identification 37, 38, 42, 46, 77, 81, 84, 145, 147, 153, 154, 166, 175,
193, 194, 198, 199, 200, 203, 209, 214, 216, 220, 221, 225, 226, 229,
231, 232, 233, 234, 236, 237, 241
Idiophase .................................................................................................. 18
Immunomagnetic separation ........................................................ 206, 235
IMS......................................................................................................... 206
Infection ......................................... 195, 209, 223, 226, 227, 229, 265, 266
Inhibitory activity............................................................................ 20, 252
Insertion sequences....................................................................... 216, 236
Ion exchange resin ................................................................................. 181
IS200 typing ................................................................... 222, 223, 226, 235
Isoprenoid ......................................................................................... 23, 24
Isotopic fractionation. ............................................................................ 146
ITS regions ............................................................................................... 25
Kinetic fermentation .............................................................................. 171
Kinetic studies .......................................................................................... 80
Kocuria rosea ........................................ 165, 166, 167, 168, 170, 172, 173
Lactic acid ......................................................................... 14, 38, 262, 263
Lactic acid bacteria 14, 31, 32, 34, 38, 39, 41, 42, 44, 45, 46, 108, 239,
247, 250, 251, 254, 263, 264
Lactobacillus helveticus ................................................. 101, 102, 107, 108
Lactococcus lactis .................................................... 45, 252, 262, 263, 264
Leloir pathway. ........................................................................... 81, 82, 85
Line probe assay .................................................................................... 199
LiPA ....................................................................................................... 199
Lipase ....................................................................................................... 70
Lipases ............................................................................................... 14, 21
270
Listeria monocytogenes 14, 20, 195, 196, 201, 205, 227, 230, 231, 232,
233, 234, 235, 236, 237, 238, 262, 263, 264, 266
LPB-3 .................................................................... 167, 168, 170, 172, 173
Luedeking and Piret ............................................................................... 101
Lysine ................................................................................................. 55, 57
Malic .................................................................... 31, 38, 39, 40, 41, 42, 44
Malolactic fermentation ............................................. 31, 38, 39, 42, 43, 45
Malolactic starters ............................................................................. 38, 43
MAP kinase cascade 109, 113, 115, 117, 119, 120, 124, 125, 127, 131,
133
Marcfortines ............................................................................................ 25
Meat....................................................................... 241, 243, 262, 264, 265
Meatpreservation.................................................................. 239, 240, 241
Mechanical properties ........................................................................... 162
Metabolic control analysis .................................................... 75, 76, 82, 85
Metabolic engineering. ............................................................................ 85
Metabolic flux .................................................................................... 77, 78
Metabolicflux analysis .................................................... 75, 77, 79, 82, 84
Metabolic pathway analysis .................................................. 75, 76, 80, 81
Methylketones .......................................................................................... 14
Microencapsulation ...................................................... 262, 263, 264, 265
Micromanipulation ....................................................................... 158, 162
Molecular breeding .................................................................................. 73
Molecular detection ........................................................................... 1, 194
Molecular identification ....................................... 193, 198, 200, 201, 209
Molecular typing 15, 193, 208, 210, 211, 212, 215, 216, 217, 219, 220,
221, 225, 226, 227, 229, 230, 231, 232, 233, 238
Molecular weight..... 17, 135, 137, 138, 217, 219, 245, 256, 259, 260, 262
Mould fermented foods ...................................................................... 14, 21
Mould fermented meat products .............................................................. 13
MUC1 ............................................................................ 124, 125, 126, 127
Mutation........................... 18, 23, 24, 27, 28, 120, 121, 125, 130, 131, 217
Mycophenolic acid. ........................................................................... 25, 28
Mycotoxin............................................................................... 14, 23, 25, 28
NADPH accumulation ...................................................................... 55, 57
n-Alkane ................................................................................................. 185
NASBA ............................................................... 193, 198, 199, 200, 204, 237
NASBA method ....................................................................................... 200
n-Hexadecane ........................................................................................ 189
271
Nisin............................................... 250, 251, 252, 262, 263, 264, 265, 266

Nucleic acid based identification methods ............................................ 198
0157
h7 ....................................................... 195, 201, 202, 205, 217, 228, 250
Oenococcus oeni ...................................................................................... 38
Oil bioremediation ................................................................................. 190
Outbreak ........ 21 7, 222, 223, 224, 225, 229, 231, 234, 235, 236, 237, 238
Outer membrane proteins ...................................................................... 145
PAC........................................................................ 177, 179, 181, 182, 183
Pathogenic E. Coli ................................................................................. 198
Patulin., .................................................................................................... 25
Pb2+ removal........................................ 177, 178, 179, 180, 181, 182, 183
PCBC gene ............................................................................................... 17
PCR15, 17, 26, 27, 37, 193, 198, 199, 200, 202, 203, 204, 205, 206, 207,
210, 211, 212, 213, 214, 215, 218, 220, 222, 225, 226, 229, 230, 231,
232, 233, 235, 236, 237, 238
PCR detection ................................................................ 203, 205, 208, 238
PCR ribotyping ...................................................................................... 233
Penicillin.................................................................... 15, 17, 18, 19, 27, 28
Penicillin biosynthetic genes ................................................................... 17
Penicillin production ......................................................................... 17, 27
Penicillium camemberti ......................................................... 13, 23, 28, 29
Penicillium chrysogenum.................................................................. 27, 28
Penicillium commune ............................................................................... 28
Penicillium nalgiovense........................................................ 13, 15, 27, 28
Penicillium roqueforti........................................................... 25, 27, 28, 29
Penitrem A ............................................................................................... 25
Peptidoglycan ........................................................................................ 157
PFGE193, 211, 212, 216, 217, 220, 222, 223, 224, 225, 226, 228, 229,
230, 231, 234, 235, 236
Phage typing., ........................................................................................ 233
PHB........................................................................ 135, 136, 137, 138, 139
Pigments ................................................................................................ 173
Plasmid.......................................................................... 219, 230, 234, 235
Plasmidanalysis .................... 193, 212, 219, 222, 223, 224, 225, 226, 234
Polar lipids ................................................................................... 144, 146
PR imine............................................................................................. 25, 29
PR toxin ....................................................................................... 25, 26, 27
272
Primers 15, 17, 29, 37, 193, 198, 199, 201, 202, 213, 214, 218, 222, 232,
236, 238
Probes 46, 136, 153, 158, 162, 193, 198, 199, 201, 203, 207, 208, 216,
229. 231. 233. 235. 236
Propagation ............................................................................................. 90
Propagation conditions ........................................................................... 90
Protease ................................................................................. 171, 172, 174
Proteases .................................................................................... 14, 21, 173
Pseudomonas sp ....................................................... 57, 145, 147, 148, 150
Pulsedfield gel electrophoresis .................................... .193, 210, 216, 238
Quantification...................................................... 75, 78, 84, 193, 207, 234
rAPD15, 16, 27, 193, 211, 212, 213, 214, 220, 222, 223, 225, 226, 227,
228, 229, 232, 234, 235, 236, 238
rAPD analysis .......................................................................................... 15
rDNA...................................................................... 144, 147, 209, 215, 232
Reactivation ................................................................. 33, 34, 39, 244, 246
Relative resistance ................................................................................. 155
Repetitive elements ................................................................................ 213
REP-PCR............................... 193, 211, 214, 220, 222, 223, 225, 228, 231
Respiratory activity .................................................................................. 52
Restriction analysis........................ 193, 212, 215, 216, 219, 222, 225, 226
Restriction fragment length polymorphism ... 193, 211, 215, 230, 234, 236
RFLP................................................................ 37, 211, 215, 216, 228, 236
Rhizopus oryzae ....................................................................................... 59
Ribosomal RNA ...................................................................................... 204
Ribotyping 193, 211, 216, 222, 223, 224, 225, 226, 227, 228, 229, 231,
232, 233, 234, 235, 236, 23 7
rRNA .............. 145, 153, 193, 199, 201, 204, 215, 216, 230, 234, 235, 237
rRNA genes., .......................................................................................... 199
RT-PCR.................................................................................. 193, 198, 204
Saccharomyces cerevisiae. 32, 47, 59, 71, 72, 73, 97, 98, 99, 130, 131,
132, 133, 183
Salmonella 193, 194, 195, 196, 199, 200, 201, 202, 205, 206, 207, 209,
210, 214, 219, 221, 222, 223, 224, 225, 229, 230, 231, 232, 233, 234,
235, 236, 237, 238, 252, 262, 266
Salmonella sp ......................................................................................... 236
Salmonella typhimurium ................ 209, 234, 235, 236, 237, 238, 252, 266
Secondary metabolite ..................................... 14, 15, 17, 18, 23, 24, 25, 26
Sensitivity ............................................................................................... 203
273
Serotyping ............................................................................................. 227

Serovars ........................................................ 221, 222, 223, 225, 227, 228
Shigella ........................................................................... 194, 195, 197, 201
Signal transduction .......................................................................... 110, 113
Signal transduction modules .............................................................. 113
Spacer region ......................................................... 199, 200, 214, 225, 232
Spirillum ............................................................................................... 157
SRFH ............................................................................... 211, 216, 227, 229
Staphylococcus aureus ................................ 14, 20, 197, 201, 231, 234
Staphylococcus epidermis ............................................................. 155, 159
Starch degrading enzymes ............................................................... 127
Starter ................................................................................................... 243
Starter culture 13, 14, 18, 19, 20, 23, 25, 239, 241, 242, 243, 244, 246,
247, 248, 250, 251, 252, 254, 264, 265
Starters..................................................................................................... 11
Steady-state continuous cultivation ......................................................... 79
Streptococcus pyogenes ........................................................................ 197
Streptomyces......................... 166, 171, 174, 185, 186, 187, 188, 189, 190
Streptomyces griseoflavus ...................................................................... 185
Streptomyces plicatus ............................................................................ 185
Stuckfermentation ............................................................................ 32, 34
Substrate competition ............................................................................ 149
Surface charge ........................................................................................
91
Surface properties ............................................................................... 99
Target for molecular identification .................................... 198, 199, 200
Target genes ................................................................................ 83, 122
Taxon specific biomarkers ............................................................. 144
Taxonomic relationships .................................................................... 15
Taxonomy ........................................................................ 123, 131, 132
TEC1 ...................................................................................... 123, 131, 132
Tensile strength.............................................. 135, 136, 138, 139, 157, 162
Texture ..................................................................... 14, 242, 243, 249, 250
TGGE............................................................................................. 220, 234
Thermal stubility .............................................................. 65, 136, 137, I39
TLC analysis ............................................................................................ 23
Toxin ........................................................................................................ 29
Transcriptional regulators ............................................................. 113, 122
Transformation., ............................................. 19, 20, 32, 40, 44, 111, 227
Transformation vector ............................................................................ 19
274
tRNA-PCR ........................................................................................ 211, 214

Tryptophane ...................................................................................... 23, 24
Two dimensional TLC .............................................................................. 24
Typing1, 37, 193, 194, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,
218, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236, 238
Typing techniques .................................................................. 215, 218, 219
U-13 C...................................................................................... 148, 150, 151
Validation .................................................................................... 108, 205, 206
Viability of cells. .................................................................................... 204
Vibrio............................................................................. 195, 197, 200, 233
Volatile compounds .......................................................................... 14, 35
Whey....................................................................................................... 102
White cheese ..................................................................................... 13, 28
Wine .............................................................................................. 46, 109
Yeast starters .................................................................................... 33, 45
Yersinia.......................................... 194, 197, 198, 201, 229, 230, 233, 235
Yield coefficient ....................................................................................... 92
Zeolite ............................................................................................ I 78, 181
Zeta potential ......................................................................... 91, 94, 95, 96
275

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APPLIED MICROBIOLOGY

ALAIN DURIEUX and JEAN-PAUL SIMON

KLUWER ACADEMIC PUBLISHERS

2002 Kluwer Academic Publishers

All rights reserved

Created in the United States of America

Visit Kluwer Online at:

Editors: A. Durieux and J-P Simon

EDITORS PREFACE ....................................................................

TABLE OF CONTENTS ...............................................................

PART 1 - STARTERS ................................................................. 11

STARTERS FOR THE WINE INDUSTRY .............................................................. 31

PART 2 - PHYSIOLOGY, BIOSYNTHESIS AND METABOLIC

4.1. Kinetic studies of the glycolysis ...............................................................

PART 3 - STATE PARAMETERS AND CULTURE CONDITIONS

EFFECT OF THE MAIN CULTURE PARAMETERS ON THE GROWTH AND

PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES

PART 4 - NOVEL APPROACHES TO THE STUDY OF

SHARING OF NUTRITIONAL RESOURCES IN BACTERIAL COMMUNITIES

PART 5 - NOVEL APPLICATIONS ..........................................

KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA ........... 165

HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERIA . 185

1.3 Incorporation of radioactivity from labelled n-Hexadecane into mycelia .

PART 6 - FOOD SECURITY AND FOOD PRESERVATION ... 191

BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION ........... 239

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED

ROLF GEISEN AND PAUL FARBER

It should not produce a mycotoxin

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

ROLF GEISEN AND PAUL FARBER

Figure 1: RAPD pattern from different strains of P. nalgiovense generated

Figure 2: Comparison of the RAPD patterns of P. nalgiovense and P.

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

2.2. PENICILLIN PRODUCTION IS A COMMON FEATURE OF P.NALGIOVENSE

Figure 3: PCR analysis of the three penicillin biosynthetic genes of P.

ROLF GEISEN AND PAUL FARBER

compared to the chromosomes of P. chrysogenum they are smaller in size resulting in a

Figure 4: Scheme of the "gene disruption" approach.

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

Figure 5: Comparison of the antagonistic activity against Micrococcus

The transformed strain of P. nalgiovense is no longer able to produce penicillin. As a

ROLF GEISEN AND PAUL FARBER

2.3 HETEROLOGOUS GENE EXPRESSION IN P. NALGIOVENSE

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

inhibition could be observed with S. aureus, L. monocytogenes und S. enterititis as

Figure 6: Inhibitory activity of the transformed P. nalgiovense strains (2, 5

ROLF GEISEN AND PAUL FARBER

Figure 7: Proteolytic activity of the wild type (left) compared to the

ROLF GEISEN AND PAUL FARBER

Figure 9: Thin layer chromatogram of the chloroform extractable

ROLF GEISEN AND PAUL FARBER

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

ROLF GEISEN AND PAUL FARBER

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

STARTERS FOR THE WINE INDUSTRY

STARTERS FOR THE WINE INDUSTRY

STARTERS FOR THE WINE INDUSTRY

Figure 1 : Influence of the yeast strain on the relative intensity of the

Sauvignon that appears during alcoholic fermentation. The non-odorous precursors

Figure 2 : Influence of the yeast strain on the vinylphenol concentration of

STARTERS FOR THE WINE INDUSTRY

2.3 EVALUATION OF THE SETTLEMENT OF ACTIVE DRY YEAST DURING

Isolated Vat 1 inoculated

Vat 2 inoculated Vat 3 inoculated