BICTEL/e - ULg

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ABSIL, Olivier

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Exoplanet imaging with mid-infrared vector vortex coronagraphs: design, manufacture, validation and first light of the annular groove phase mask

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Geometric statistical processing of brain diffusion tensor images

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CHARLIER, Carole

GEORGES, Michel

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ROLLIN, Fr\ue9d\ue9ric

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Amisi, Safari

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la limite Pal\ue9oc\ue8ne-Eoc\ue8ne dans le bassin de Douala au Cameroun. Biostratigraphy et essai de reconstitution des pal\ue9oenvironnements/The Paleocene-Eocene boundary in the Douala basin in Cameroon. Biostratigraphy and tentative of palaeoenvironment reconstruction

Shafiei, Rasoul

Mahillon, Jacques

Joris, Bernard

Guiro, A.T.

Destain, Jacqueline

Delvigne, Franck

Dommes, Jacques

Thonart, Philippe

i 
 
Supervision committee 
 
Thesis supervisor 
Professor Philippe Thonart 
Walloon Center of Industrial Biology 
University of Liège and faculty of agricultural sciences of Gembloux, Belgium. 
 
Thesis committee 
Examination Committee 
 
 
 
 
 
© University of Liège, 2013 
 
 
 
                       
 
 
 
 
 
 
 
 
 
 
Prof.  F. Delvigne                              University of Gembloux  
Prof.  J. Destain University of Gembloux  
Prof.  J. Dommes             University of Liège  
Prof.  B. Joris University of Liège 
 
 
 
Prof.  J. Dommes             University of Liège Chairman 
Prof.  F. Delvigne                          University of Gembloux Examiner 
Prof.  J. Destain University of Gembloux Examiner 
Prof.  A.T. Guiro            Université du Sine Saloum de Kaolack Examiner 
Prof.  B. Joris University of Liège Examiner 
Prof.  J. Mahillon                 Université catholique de Louvain Examiner 
ii 
 
 
 
 
  
  
  
      
 
 
 
                                   
Faculty of Sciences-University of Liège 
Walloon Center of Industrial Biology 
 
Fundamental and Applied Studies on Freeze-dried  
Vinegar Starter 
 
 
 
 
 
 
 
 
 
 
 
Academic year 2013-2014           Thesis submitted by   
           Rasoul SHAFIEI  
            for the degree of                                                
Doctor of Philosophy in Sciences 
 
 
 
 
iii 
 
 
Abstarct 
Acetic acid bacteria (AAB) are used industrially to produce different kinds of 
bioproducts. AAB encounter very aggressive conditions during acetous fermentation 
(AF) including high acid and ethanol concentrations, low pH and also abrupt increases in 
temperature. In subtropics such as central and southern parts of Iran, fruits and also the 
by-products of the fruit processing industries are used to produce different kinds of 
foods. However, because of high temperature and deficiency in water resource, 
fermentation industries face many restrictions during spring and summer.  One of the 
main problematic restrictions is the low productivity or the ceases of fermentation due 
to the use of non-thermotolerant microorganisms. In this case, initiation of a new 
fermentation run needs efficient starter. 
In previous studies in Walloon Center of Industrial Biology, Acetobacter senegalensis, a 
novel thermo-tolerant bacterium, was isolated and used to produce vinegar starter and 
acetic acid at high temperature. However, in those studies, the viability and vitality of 
the starter were not evaluated under stress conditions. In addition, since most kinds of 
industrial vinegars have low prices, the use of high-priced nutrients for the production 
of low quantity of starter is not commercially cost-effective.  
In the present study, with a deep look to the acetous fermentation requirements, we 
analyzed the fundamental and applied aspects of A. senegalenisis resistance to stress 
inducers. Proteomics-based techniques and flow cytometry methods in combination 
with different biomass production techniques were used to develop a fermentation 
process improving cell viability and vitality during freeze-drying process and 
revitalization procedure. In addition, the trend of cell senescence during storage of 
starter and its effect on some bio-molecules were studied.   
In the first part of the study, the quality of the produced biomass was improved in order 
to achieve an acetic acid tolerant biomass. Adaptive laboratory evolution technique 
(ALE) enabled cells to grow rapidly in higher concentration of acetic acid. The results of 
2D-DiGE on the produced biomass revealed that structural and regulatory proteins were 
expressed differently under various conditions (Chapter II and III). Use of acetic acid in 
combination with glucose in a fed-batch fermentation mode could induce a physiological 
condition in A. senegalensis which was close to the physiological state of cells oxidizing 
ethanol. In addition, the presence of acetic acid in fermentation media could cause a 
cross-adaptation and improved the tolerance of cells to stressors (ethanol, low pH and 
acetic acid). Interestingly, by using this method for production of biomass, the rate of 
growth on ethanol improved significantly. 
In parallel to the first part of the study, we exhibited the influence of different stress on 
the produced biomass. Cell envelope integrity and respiration (dehydrogenase activity) 
were the two important targets for adverse effects of the stress. Assessment of the cell 
envelope integrity and respiration system of produced biomass by Multiparametric Flow 
Cytometry (MFC) method (Chapter II) demonstrated that the detrimental effects of 
ethanol and acetic acid depended on the carbon sources and fermentation conditions 
used for pre-adaptation. Respiration system and cell envelope integrity of cross-adapted 
iv 
 
cells were not compromised after exposure to different concentrations of ethanol and 
acetic acid. Thus, according to the obtained results, by  
 
using a mixture of acetic acid and glucose as carbon sources, it is possible to enhance not 
only the viability of cells but also induce tolerance to physicochemical stress during 
downstream process. 
Our investigation about the freeze-drying process provided a better understanding of 
lethal and sub-lethal damage to cells (Chapter IV). The results showed that drying 
process had the greatest effect on the viability and vitality of A. senegalensis especially 
by affecting on the cell envelope.  In addition, entrance into viable but non-culturable 
state (VBNC) was initiated during the drying process and enhanced during storage 
period. 
Analysis of the stored cell proteome by 2D-DiGE and western blotting (Chapter V) 
revealed that high storage temperature could induce a kind of senescence in the cells by 
different modifications in cellular proteome such as insolubility, degradation and 
carbonylation of cellular proteins and shift of isoelectric point. Carbonylation of the 
proteins involved in transcriptional and translational process could cause cell death  
whereas VBNC formation at low storage temperature seemed to be due to other 
deteriorative reactions such as fatty acid peroxidation. 
At the end of this dissertation, the discussion (Chapter VI) provides a general overview 
of the results and compares our findings with earlier studies. Potential industrial 
applications are reviewed and suggestions for further research are made.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
v 
 
Contents 
 
Supervision committee 
Preface 
Abstarct 
Publications related to this Thesis 
Acknowledgments 
Contents 
List of Figures 
List of Tables 
List of Abbreviations 
Chapter I 
Introduction 
Part I- Stress during Acetous Fermentation 
1.  Acetous fermentation 
1.1. Vinegar and its applications 
1.2. Acetic acid bacteria: Past, present and future 
2. Physiology of acetic acid bacteria 
3. Stress and stress responses in microorganisms 
4. Stressors during acetous fermentation 
4.1. Ethanol and its effect on cell physiology 
4.2. Acetic acid and its effect on cell physiology 
4.3. pH and its effect on acetic acid bacteria physiology 
4.4. Oxygen and its influence on AAB viability and vitality 
Part II- Starter Culture Production: Anhydrobiotic Engineering 
1. Starter culture and its production technology 
1.1. Fermentation 
1.2. Preservation of biomass 
2. Starter culture in vinegar industry and its limitations 
2.1. Vinegar production is a mixed fermentation 
2.2. Starter production entails availability of efficient AAB strains 
3. Stress during production of starter culture 
3.1. Stress during freeze-drying process 
3.2. Stress during storage 
4. Factors influencing the viability of bacteria during freeze-drying process and storage 
4.1. Bacterial species 
4.2. Growth conditions 
4.3. Protective additives 
4.4. Rehydration and recovery culture media 
4.5. Survival measurement 
4.6. Other factors 
Thesis objectives 
Chapter II 
Evaluation of Growth and Viability of Acetobacter senegalensis under Stress 
Conditions 
vi 
 
Preface 
Abstract 
1. Introduction 
2. Materials and methods 
2.1. Bacterial strain and culture media 
2.2. Preparation of pre-culture media 
2.3. Stress media 
2.4. Growth of A. senegalensis in stress media 
2.5. Leakage of cellular compounds 
2.6. Standard plate count procedure 
2.7. Flow cytometric analysis 
2.8. Determination of glucose, gluconic acid, acetic acid and ethanol concentrations 
2.9. Statistical analysis 
3. Results 
3.1. The effect of pre-adaptation on growth of A. senegalensis in stress media 
3.2. Cellular leakage in the presence of ethanol and acetic acid 
3.3. The effect of pre-adaptation and stressors on culturability of A. senegalensis 
3.4. Factors influencing cell envelope integrity and cell respiration in stress media 
4. Discussion 
5. Conclusions 
6. Acknowledgments 
Chapter III 
How Can Pre-adaptation Affect the Tolerance of Acetobacter senegalensis to 
Stressors: A Proteomic Approach 
Preface 
Abstract 
1. Introduction 
2. Materials and methods 
2.1. Bacterial strain and pre-culture media 
2.2. Sequential adaptation of A. senegalensis to different growth conditions 
2.3. Growth in Bioreactor 
2.4. Determination of glucose, acetic acid and ethanol concentrations 
2.5. Sample preparation for proteomic analysis 
2.6. Two-dimensional difference gel electrophoresis (2D-DiGE) and mass spectrometry 
2.7. Freeze-drying of produced biomass and storage 
2.8. Phenotype assays of evolved strains 
3. Results and discussion 
3.1. An overview of the methods used for the growth and adaptation of A. senegalensis 
3.2. Adaptation to different carbon sources 
3.3. Differentially expressed proteins 
3.4. Evaluation of phenotypic features of adapted cells 
4. Conclusions 
5. Acknowledgments 
Supplementary materials 
Chapter IV 
vii 
 
Flow-cytometric Assessment of Damage to  Acetobacter senegalensis during 
Freeze-drying Process and Storage 
Preface 
Abstract 
1. Introduction 
2. Materials and methods 
2.1. Bacterial strain 
2.2. Culture media and growth conditions 
2.3. Biomass production 
2.4. Freeze-drying Process 
2.5 Packaging and storage of freeze-dried cells 
2.6. Determination of culturability and total number of cells 
2.7. Preparation of cell suspension for flow cytometry 
2.8. Flow cytometry analysis 
2.9. Cell envelope integrity 
2.10 Cell respiration capacity 
3. Results 
3.1. The effect of freezing on cellular features 
3.2. The effect of drying on the main cellular features 
3.3. The effect of storage conditions on cellular features 
4. Discussion 
5. Conclusions 
6. Acknowledgments 
Chapter V 
The Role of Protein Modifications in Natural Senescence of Freeze-dried 
Acetobacter senegalensis during Storage 
Preface 
Abstract 
1. Introduction 
2. Results and discussion 
2.1. Heterogeneous populations are formed during storage of freeze-dried cells 
2.2. Formation of fluorescent compounds is enhanced during storage at high temperature 
2.3. The proteome is markedly affected in freeze-dried cells stored at high temperature 
2.4. Carbonylation of proteins increases massively during storage 
2.5 Solubility of cellular proteins is reduced during storage 
2.6. Changes in fatty acid profile may enhance protein modification 
3. Conclusions 
4. Materials and Methods 
4.1 Microorganisms and production of biomass 
4.2. Production and storage of freeze-dried cells 
4.3. Determination of total cell number and culturable cells after freeze-drying 
4.4. Flow-cytometric analysis 
4.5. Sequential extraction of cellular proteins 
4.6. 2D-DiGE analysis of freeze-dried cells stored at different temperature 
4.7. Quantification of carbonylated proteins 
4.8. Determination of carbonylated proteins by western blotting 
4.9. Total fatty acid analysis 
viii 
 
4.10. Statistical analysis 
Authors' contributions 
Acknowledgements 
Chapter VI 
General Discussion 
1. General overview 
1.1. Fermentation technology and its new-emerging requirements in the developing 
countries 
1.2. Starter productions and its limitations 
2. Pre-adaptation as a method for improving starter production 
2.1. Effect of acetic acid on pre-adaptation of A. senegalensis 
2.2. Effect of ethanol on pre-adaptation of A. senegalensis 
3. Advantages and drawbacks of pre-adaptation on downstream processes 
4. Modification of cellular compounds during storage 
Chapter VII 
Conclusions and Perspective 
1. Conclusions 
2. Perspectives and recommendations 
Chapter VIII 
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Fundamental and Applied Studies on Freeze-dried Vinegar Starter

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