Characterization of acetic acid bacteria and study of the molecular strategies involved in the resistance to acetic acid during oxidative fermentation

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Thesis

Reference

Characterization of acetic acid bacteria and study of the molecular strategies involved in the resistance to acetic acid during oxidative

fermentation

ANDRES BARRAO, Cristina

Abstract

Les bactéries acétiques (BA) sont des microorganismes largement répandus dans la nature, possédant un grand potentiel écologique et biotechnologique. Certaines souches des genres Acetobacter et Gluconacetobacter se caractérisent par leur capacité unique de transformer l'éthanol en acide acétique avec une grande efficacité et aussi de résister à de hautes concentrations de ce produit toxique. L'oxydation de l'éthanol se réalise grâce à l'action successive de deux enzymes situées sur la face externe de la membrane cytoplasmique:

l'alcool déshydrogénase (ADH) et l'aldéhyde déshydrogénase (ALDH). Cette réaction, connue sous le nom de "fermentation acétique", est utilisée de façon industrielle dans la production de vinaigre. Alors que la plupart des microorganismes sont sensibles à des concentrations d'acide acétique de 0.5%, certaines espèces du genre Acetobacter sont capables de résister à une concentration de 8%; chez les Gluconacetobacters des espèces peuvent même résister à une concentration allant jusqu'a 20%

ANDRES BARRAO, Cristina. Characterization of acetic acid bacteria and study of the molecular strategies involved in the resistance to acetic acid during oxidative fermentation. Thèse de doctorat : Univ. Genève, 2012, no. Sc. 4452

URN : urn:nbn:ch:unige-264137

DOI : 10.13097/archive-ouverte/unige:26413

Available at:

http://archive-ouverte.unige.ch/unige:26413

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Département de botanique et biologie végétale Professeure Teresa Fitzpatrick Unité de microbiologie Docteur François Barja

Characterization of Acetic Acid Bacteria and Study of the Molecular Strategies Involved in the Resistance to Acetic Acid During

Oxidative Fermentation

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Cristina Andrés Barrao de

Saragosse (Espagne)

Thèse n° 4452 Genève

Atélier d'impression ReproMail 2012

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To#my#parents#

A#mes#parents#

A#mis#padres#

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Acknowledgements

I would like first of all to thank the members of the Jury: Professor Theresa Fitzpatrick, Dr. Mauro Tonolla, Dr. Laurent Falquet, Dr. José Manuel Guillamón and Dr. François Barja, for having accepted to read and evaluate this work.

The culmination of this thesis has been possible by the support of my supervisor, François Barja.

Despite the difficulties and hard times, he has known how to encourage me to exploit the better of my capacities. He welcomed me when I arrived to Switzerland, and offered me a position in his laboratory, where I met many beautiful people, who had congratulate me with all their humanity and enthusiasm, creating the best environment for developing this work. Among them, Malou Chappuis and Ariane Fehr, with their perpetual good mood and efficiency, but also former collaborators and students: Prof. Reto Strasser, Dr. Rubén Ortega Pérez, Dr. Mukti Ohja, Marie France Blanc, Madeleine Fontana, Dr. Abdallah Oukarroum, Dr. M. Georgina Ceppi, Dr. Gert Schansker, Dr. Szilvia Z. Tòth, Aurélia Weber, Sibylle Barruchel, Géraldine Martinelli, Cathérine Wilson, Marco Dias, Fabrizio Molino, Edurne Martinez Sanz, Dr. Idoia Alonso Fauste, Pilar Okenve Ramos, Marta Alonso Nocelo, Vanesa Miguélez De la Torre and Nicolas Calo. I would like to thank specially Dr. Marta Cotado Sampayo, my colleague and friend, for all good and bad times passed together, whose support and wise advice have been indispensable for the finalization of this project.

I don't want to forget the people from the BIVEG Department and elsewhere that have kindly helped me and, directly or indirectly, contributed to the obtaining of most part of the results presented in this manuscript: Dr. Maged M. Saad, Dr. Nadia Bakkou, Dr. Xavier Perret, Grégory Thieler, Dr. Cinzia Benagli, Dr. Mauro Tonolla, Dr. Patrick Descombes, Dr. Laurent Falquet, Mike Parkan, Mauro Boffa, Daniel Bravo, Dr. Pilar Junier, Dr. Carlos A. Vegas and Dr. Albert Mas.

Thanks also to Rakel, Txema, Manu, Pedro and all my "non-biologist" close friends: Clara, Azucena, Leonor, Pawel, Mardokeo, Carolyne, Olga, Jorge, Paola, Javiera, Carlos, Eduardo, Sona and Rafaelle, for sharing moments of complicity and big laughs, so necessary during the hard moments; and the members of the Rugby Club CMSG, specially the Wildcats female team, for allowing me to evacuate the daily stress and to acquire confidence, while having a lot of fun.

I would like to address the last acknowledgement, the most special one, to my family: Maria Teresa, Juan, Tomás, Gina and Eric, for all their sometimes-misunderstood encouragements and their infinite love.

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Andrés-Barrao C, Falquet L, Calderón-Copete SP, Descombes P, Ortega Pérez R., Barja F. (2011) Genome sequences of the high-acetic acid-resistant bacteria Gluconacetobacter europaeus LMG 18890T and G. europaeus LMG 18494 (reference strains), G. europaeus 5P3, and Gluconacetobacter oboediens 174Bp2 (isolated from vinegar). J. Bacteriol. 193(10), 2670-2671. ARTICLE 5. ... 223 Schindhelm S, Weber A, Andrés-Barrao C, Schelling C, Stchigel AM, Cano J, Veuthey JL, Bourgeois J, Barja F (2009) Biochemical and morphological characterization of a new fungal contaminant in balsamic and cider vinegars. Food Addit. Contam. 26, 1306-1313.

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LIST OF ABREVIATIONS

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2D = Two dimensional AAB = Acetic acid bacteria ABC = ATP-binding cassette AcH = Acetic acid

ADH = Alcohol dehydrogenase

AE = Acetic acid-ethanol medium (Entani medium) AFLP = Amplified Fragment Length Polymorphism ALDH = Aldehyde dehydrogenase

ANOVA = Analysis of variance

ARDRA = Amplified Ribosomal DNA Restriction Analysis ASL = Alkali-stable lipids

ATP = Adenosine triphosphate BC = Before Christ

BCCM = Belgian Coordinated Collection of Microorganisms BLAST = Basic Local Alignment Search Tool

BME = Basal ethanol medium BSA = Bovine serum albumin CaCO3 = Calcium carbonate CDS = Coding Sequence

CECT = Spanish Type Culture Collection (Colección Española de Cultivos Tipo) CHAPS = 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

CHCA = α-cyano-4-hydroxycinamic acid CoA = Coenzyme A

CPS = Capsular polysaccharide

CRAFT = Cooperative Research Project CTAB = Cetyl trimethylammonium bromide Da = Dalton

DEM = Direct Epifluorescence Microscopy DGGE = Denaturing Gradient Gel Electrophoresis DHB = 2,5-dihydroxybenzoic acid

DIGE = Differential In-Gel Electrophoresis DMSO = Dimethyl sulfoxide

DNA = Deoxyribonucleic acid

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dNTP = Deoxyribonucleotide

DSMZ = German Collection of Microorganisms (Deutsche Sammlung vor Mikroorganismes und Zellkulturen)

DTE = Dithioerythritol

EAW= Ethanol-acetic acid-water

EDTA = Ethylenediaminetetraacetic acid EPS = Extracellular polysaccharide

ERIC = Enterobacterial Repetitive Intergenic Consensus sequences EtOH = Ethanol

H2O2 = Hydrogen peroxide Hz = Hertz

IEF = Isoelectric Focusing IPG = Immobilized pH gradient IS element = Insertion element ISO = Inside-out

ITS = Intergenic Spacer Sequence kDa = Kilodalton

kV = Kilovolts (10³ V) LAB = Lactic acid bacteria

LC-ESI-MS = Liquid Chromatography-Electrospray Ionization-Mass Spectrometry LPS = Lipopolysaccharide

LSU = Large subunit

MALDI-TOF = Matrix Assisted Laser Desorption/Ionization-Time of Flight MLSA = Multilocus Sequence Alignment

MLST = Multicolus Sequence Typing MS = Mass spectrometer

NaCl = Sodium chloride NaOH = Sodium hydroxide

NCBI = National Center for Biotechnology Information NL = Non-linear

ns = nanoseconds (10⁻⁹ s) OD = Optical density

ORF = Open Reading Frame OsO4 = Osmium tetroxide

PAGE = Polyacrylamide Gel Electrophoresis

PATAg = Periodic acid-thiocarbohydrazide-silver proteinate

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PBS = Phosphate buffered saline PC = Phosphatidylcholine

PCR = Polymerase Chain Reaction PE = Phosphatidylethanolamine

PFGE = Pulsed Field Gel Electrophoresis PG = Phosphatidylglycerol

PQQ = Pyrroloquinoline quinone PS = Polysaccharide

RAE = Reinforced AE medium (Reinforced Entani medium) RAPD = Random Amplified Polymorphic DNA

RDP = Ribosomal Database Project

REP = Repetitive Extragenic Palindromic sequences RFLP = Restriction Fragment Length Polymorphism rLPS = Rough lipopolysaccharide

RNA = Ribonucleic acid

rRNA = Ribosomal ribonucleic acid RSO = Right-side-out

SA = Sinapinic acid

SARAMIS™ = Spectral Archive and Microbial Identification System SC = Static surface culture

SDS = Sodium dodecyl sulfate

SEM = Scanning Electron Microscopy SF = Submerged fermentation

sLPS = Smooth lipopolysaccharide SSF = Solid-state fermentation SSU = Small subunit

TBE = Tris-borate-EDTA

TBV = Traditional Balsamic vinegar TCA = Tricarboxylic acid

TCH = Thiocarbohydrazide TE = Tris-EDTA

TEM = Transmission Electron Microscopy

YGC = Yeast extract-glucose-calcium carbonate medium YPM = Yeast extract-peptone-mannitol medium

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RÉSUMÉ

Les bactéries acétiques (BA) sont des microorganismes largement répandus dans la nature, possédant un grand potentiel écologique et biotechnologique. Elles jouent également un rôle fondamental dans l'industrie alimentaire. Ce sont des bactéries Gram-négatif et aérobies stricte et une de leurs principales caractéristiques est leur capacité d'oxyder les sucres, alcools et polyols en acides organiques. Certaines souches des genres Acetobacter et Gluconacetobacter se caractérisent par leur capacité unique de transformer l'éthanol en acide acétique avec une grande efficacité et aussi de résister à de hautes concentrations de ce produit toxique. L'oxydation de l'éthanol se réalise grâce à l'action successive de deux enzymes situées sur la face externe de la membrane cytoplasmique: l'alcool déshydrogénase (ADH) et l'aldéhyde déshydrogénase (ALDH). Cette réaction, connue sous le nom de "fermentation acétique", est utilisée de façon industrielle dans la production de vinaigre. Malgré le fait que ce condiment ait été considéré comme un produit secondaire provenant de l'oxydation du vin, la production de vinaigres de haute qualité a actuellement une grande importance. Du point de vue industriel, l'étude de la dynamique des populations microbiennes pendant le processus d'acétification est capital pour mener à bien la sélection des souches les plus performantes. Pour ce faire, diverses techniques de biologie moléculaires sont utilisées. Mais si ces techniques permettent l'identification précise de la plupart des BA, elles ne peuvent pas différencier certaines espèces qui se trouvent phylogénétiquement très proches. Une alternative aux techniques de biologie moléculaire est l'analyse des profils protéiques par "matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry", technique qui est actuellement très répandue dans les laboratoires médicaux en raison de sa grande fiabilité, sa rapidité et son bas coût.

La résistance des BA à l'acide acétique et à l'éthanol, deux puissants microbicides, varie selon les espèces. Alors que la plupart des microorganismes sont sensibles à des concentrations d'acide acétique de 0.5%, certaines espèces du genre Acetobacter sont capables de résister à une concentration de 8%; chez les Gluconacetobacters des espèces peuvent même résister à une concentration allant jusqu'a 20%. Les connaissances actuelles sur les mécanismes impliqués dans la résistance des BA à de hautes concentrations d'acide acétique sont très limitées. Les bactéries du genre Gluconacetobacter ont dû acquérir des mécanismes spécialisés qui leur permettent de survivre, en étant métaboliquement active, dans des environnements extrêmes (pH bas, haute concentration en acide acétique, haute oxygénation).

Dans ce travail de thèse doctorale, nous avons utilisé des techniques de biologie moléculaire pour caractériser la population de BA participant aux différents processus d'acétification par la

méthode immergée. Nous avons ensuite isolé 3 souches parmi les espèces majoritaires:

A. pasteurianus 3P3, Ga. europaeus 5P3 et Ga. oboediens 174Bp2. Pour rendre l'identification des BA plus rapide, plus simple, et dans le but de constituer une base de données fiable et accessible aux industriels, nous avons mis au point les analyses par MALDI-TOF. Afin de mieux comprendre les mécanismes de résistance et le changement des voies métaboliques pendant la fermentation acétique, nous avons fait appel à la technique "two-dimensional differential in-gel electrophoresis (2D-DIGE)". Nous avons ainsi étudié les modifications du protéome chez la souche type A. pasteurianus LMG 1262^T lors de la fermentation acétique, en essayant de différencier les effets dus à l'éthanol et à l'acide acétique. Nos résultats ont montré que les protéines GrpE, DnaK et Trx, parmi d'autres, sont spécifiquement induites en présence d'acide acétique. Pour faciliter l'analyse du protéome, mais aussi la compréhension de la résistance globale à l'acide acétique en déchiffrant les différences innées dans la résistance à l'acide acétique entre les genres Acetobacter et Gluconacetobacter, nous avons obtenu les séquences

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génomiques complètes de la souche type d' A. pasteurianus LMG 1262^T, d'une deuxième souche d’A. pasteurianus (3P3) ainsi que de quatre autres souches de Gluconacetobacter, de trois Ga. europaeus (LMG 18890T, LMG 18494, 5P3) et d’une Ga. oboediens (174Bp2).

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ABSTRACT

Acetic acid bacteria (AAB) are widespread microorganisms that play an important role in many natural processes leading to high-valued food and beverage products. They are mainly characterized by their capability to incompletely oxidize sugars, alcohols and sugar-alcohols into their corresponding organic acids. Specialized strains from the genera Acetobacter and Gluconacetobacter stand out because they are able to oxidize ethanol into acetic acid with a high efficiency, and also to resist high concentrations of the toxic product. The oxidation of ethanol results from the sequential activity of two enzymes that are located in the outer side of the cytoplasmic membrane of these microorganisms: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). The ethanol is derived from different raw materials such as wine, cider, spirit, fruits or flowers, and the action of AAB on these substrates let to the production of different types of vinegar. Although traditionally considered as a waste product issued from wine spoilage, high-quality vinegars are nowadays of great importance for industry.

From an industrial point of view, the study of microbial population dynamics during the acetification process to select the most performing strains is of paramount importance. The current molecular techniques for the identification of AAB are based on the analysis of different gene targets, but fail to discriminate between very closely related species. The analysis of whole proteome profiles by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), is a very promising alternative technique, that is nowadays routinely used in clinical laboratories due to its accuracy, rapidity and low cost.

The resistance of AAB to acetic acid and ethanol, potent microbicidals, varies among species.

While most microorganisms are sensitive to acetic acid concentrations as low as 0.5%, several species from the genus Acetobacter resist up to 8%, and species from the genus Gluconacetobacter up to 20%. Although the molecular mechanisms that confer resistance (or allow adaptation) to acetic acid still remain poorly understood, the Gluconacetobacter strains must have developed special characteristics for surviving and remaining metabolically active at extreme growth conditions (low pH, high acetic acid, high oxygenation).

During this PhD thesis we characterized the AAB population taking part in submerged acetifications of wine and spirit vinegars, and selected three isolates of the major species:

A. pasteurianus 3P3, Ga. europaeus 5P3 and Ga. oboediens 174Bp2. With the aim of applying MALDI-TOF MS for the rapid identification of AAB, we constructed a reference database and we validated the identification method by analyzing a set of 47 vinegar isolates, previously identified by molecular methods. Using two-dimensional differential in-gel electrophoresis (2D-DIGE), we tried to deepen in the study of the mechanisms responsible for acetic acid

resistance in vinegar related AAB. We studied the proteome changes during acetification in the A. pasteurianus type strain, LMG 1262^T, differentiating the effect of both ethanol and acetic

acid. Our results showed GrpE, DnaK and Trx, among others, as the main proteins induced by effect of the acetic acid during the acetic fermentation. To facilitate the proteome analysis and also the understanding of the acetic acid resistance globally, by unraveling the innate differences

between Acetobacter and Gluconacetobacter species, we sequenced the whole genome of the A. pasteurianus type strain (1262^T), a second A. pasteurianus strain (3P3), and four additional

Gluconacetobacter strains, three Ga. europaeus (18890T, 18494, 5P3) and one Ga. oboediens (174Bp2).

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CHAPTER 1 GENERAL INTRODUCTION

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1.1 What is vinegar?

1.1.2 Naturally occurring vinegar

Vinegar is an acidic food product of paramount importance for the enrichment of our diet. It results from the desired or controlled oxidation of ethanol containing (liquid) substrates into acetic acid by action of aerobic microorganisms (Holzapfel, 2009).

Nowadays well considered as a functional food, the benefits of vinegar for human health are known since ancient times. It has been documented that the Egyptians, Sumerians and Babylonians had experience and technical knowledge in making vinegar from barley and any kind of fruit. Vinegar was very popular both in ancient Greece and Rome, where it was used in food preparations and as a remedy against a great number of diseases. In Asia, the first records about vinegar date back to the Zhou Dynasty and probably China's ancient rice wines may have originally been driven from fruit, for which malted rice was substituted later (Holzapfel, 2009).

Little is known about its origin, but it is believed that alcoholic and acetic fermentations, that are nowadays two distinct and separate processes, were not discriminated in ancient times, simply because the two kinds of fermentation often followed on from each other in an uncontrolled process. The current alcoholic and acetous beverages, issue of very controlled processes, cannot then be compared to their primeval ancestors, who became aware of the transformation of alcoholic drinks into vinegar only after a historical period of observation and product improvement (Mazza and Muooka, 2009).

1.1.3.Vinegar composition

The chemical composition of vinegar depends on the origin and nature of the raw material used, which can be many kinds of fruits, cereals and other vegetables, animal derived products or distilled alcohol. Not only the raw material, but also the fabrication method and, in some cases, the ageing process, strongly influence the organoleptic properties of the final product. These properties are due to the presence in vinegar of different phenolic (volatile) compounds and pigments that are responsible for flavor and color, respectively. But even if these compounds are of paramount importance to ensure the final quality, their concentration in vinegar is minimal, even traces (Table 1, Figure 1). The main components of this acidic condiment are water (90% (w/v)) and acetic acid (6% (w/v)) (Figure 1), so it can be considered as a diluted solution of this organic acid.

Acetic acid is responsible of the pungent and strong-tasting, as well as the most benefits that vinegar shows for human health. It has been used since Classical Time (4^th century BC) as the main remedy against a great number of diseases, including the common cold and cough (Flandrin et al., 2000). Diluted vinegar mixed with water was used in the ancient Rome (60-50 BC) as a highly refreshing drink that was safer than water alone. Scandinavians and other northern Europeans used vinegar in the Middle Ages as a pickling agent to preserve meat and other food (Larsen, 1931). The famous Italian physician Tommaso del Garbo also suggested to use vinegar for washing hands, face and mouth to prevent the spread of the bubonic plague, in 1348 (Mazza and Murooka, 2009).

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Table 1. Composition of different types of vinegar (Bourgeois, personal communication).

Sample number Type of vinegar

P7472 Cider

P7473 Wine

P7474 Wine

P7475 Wine

P7476 Fruit^a

P7477 Fruit^a

P7478 Wine

Appearance Clear without sediment

Cloudy with much sediment

Clear without sediment Specific weight at 15°C 1.0159 1.0106 1.0111 1.0102 1.0131 1.0148 1.0107

Alcohol (%vol) 0.1 n.d. 0.3 0.1 0.1 0.2 0.3

Dry Extract (g/l) 21.1 8.6 10.3 7.9 15.1 19.7 11.3

Sugar (g/l) 3.5 0.7 0.8 0.4 1.2 2.6 0.5

Sugar free extract (g/l) 17.6 7.9 9.5 7.5 13.9 17.1 10.8

Total acidity

- Acetic acid (g/100 ml) 4.8 4.6 4.7 4.6 4.5 4.5 4.2

Non volatile acidity - Tartaric acid (g/100 ml)

- Malic acid (g/100 ml) 0.43 0.17 0.13 0.19

0.09 0.11 0.15

Ashes (g/l) 2.26 1.66 1.70 1.25 2.14 2.36 2.12

Heavy metals

- Pb, Cd, Zn, Cu, As traces traces traces traces traces traces traces

Iron (mg/l) 3 2 4 3 6 2 9

Artificial color n.d. n.d. Reddish-

purple n.d. n.d. n.d. n.d.

aa mixture of apple and pear were used as raw material for this type of vinegar n.d. = non detected

Figure 1. Chemical composition of a general table wine vinegar. A) Most abundant components, B) Volatile compounds present as traces, responsible for the rich organoleptic properties of the final product (García-García, personal communication).

A) B)

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The important use of vinegar as a disinfectant and preventer of disease along history fall on the great microbicidal action of acetic acid. This acid is a small polar molecule that, undissociated, is able to rapidly equilibrate across the cell membrane and dissociate inside the cell at the lower internal pH (pHi) (Gibson, 1981; Russel and Diez-Gonzalez, 1998). The products, particularly the released proton (H⁺), but also the acetate anion (CH3COO^–), are toxic for the cell (Russell, 1992). The sensitivity that most microorganisms show against the toxic effect of acetic acid contributes to the relative absence of contaminants during the fermentation process (Schindhelm et al., 2009).

1.1.4. Vinegar production methods

The majority of vinegars, especially from sugary and acidic fruits are easy to make, and this explains the relatively slow development in their science and technology through time.

Although vinegar can be manufactured from almost any product capable of yielding alcohol through fermentation, the current commercial production of vinegar relies on 3 main technologies: a) The static or surface culture and b) the solid-state fermentation methods are mainly applied to traditional vinegar production with a long fermentation phase, and entail low energy consumption. c) The most recently developed submerged fermentation is a quicker process, in which the typical parameters of oxidative conversion (temperature, oxygen, alcohol content and acidity) are strictly controlled. This process requires a strong energy input, and is implemented for industrial vinegar production (Holzapfel, 2009).

1) Static or Surface Culture (SC):

One of the most ancient methods that is still in use today, was developed by the corporation of Vinaigriers established in Orléans, France (the oldest corporation in the world specializing in vinegar making) and is so-called "Orléans or French process" or "slow process" (Figure 2).

The vinegar is obtained by starting from wine through a slow fermentation in wooden casks.

AAB form a thin biofilm on the surface of the growth medium that becomes thicker and more gelatinous with time. A large number of heterogeneous bacteria are embedded into this layer known as the “mother of vinegar”, a fraction of which is used as a starter to inoculate fresh mash. This process usually yields vinegars of high quality, but it is a slow process (several months) and entails high costs (Mazza and Murooka, 2009).

Figure 2. A) Set of oak barrels stocked in the open air (www.levinaigre.com). B) Scheme of a wood barrel used for the production of vinegar by the surface culture method. Must charge (c), air (a), window (w), level indicator (l), discharge tap (d) (Divies, 1995).

A) B)

c a

l d l w

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2) Solid-State Fermentation (SSF):

This method, mostly used for the production of traditional Chinese vinegars, refers to the growth of the microorganisms on moist solid substrates without or in the near absence of free- flowing water (Liu et al., 2004). It is considered to be a suitable and useful technique for the production of food and industrial goods due to its low technology and energy requirements, its use of cheap unrefined agricultural products as substrates, its moderate capital investment and operating costs, high productivity in a low reactor volume, and less stringent aseptic processing methods (Liu et al., 2004).

3) Submerged Fermentation (SF)

The semi-continuous submerged process involves the rapid mixing of AAB starters with forced aeration in specialized fermentors (acetators) (Figure 3), with a maximum oxygen-cell contact that leads to a very quick oxidation of ethanol into acetic acid. It is the main method used for producing spirit and wine industrial vinegars in large-scale, with an acetic acid concentration of 8-14%. The major physico-chemical parameters that affect vinegar production via SF need to be strictly controlled: temperature, pH, aeration rate and dissolved oxygen concentration, cell biomass, acetic acid and ethanol concentrations, as well as fermentor filling volume. The automation of the process permits the continuous monitoring of all these parameters during the acetification process (Divies, 1995).

Figure 3. A) Pilot fermentor used in the laboratory. Charge (c), discharge (d), regulation valve for air supply (as), refrigeration (r), thermostat fixed at 30°C (t), air filter using active carbon (af), air injection (ai). B) Scheme of an industrial fermentor (Frings Acetator) (http://www.moutarde-de-meaux.com/en/vinaigre-procedes- immersion.php)

as c

t r

af d

ai

A) B)

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1.2. Acetic acid bacteria 1.2.1. Natural habitats

Historically, acetic acid bacteria (AAB) were recognized as "vinegar bacteria" because the first studies were done on vinegar, and later on wine and beer spoilage. In fact, vinegar AAB are a subset of a larger AAB group that includes widespread bacteria that interact with flowers, fruits, rhizosphere of plants, insects and human beings (Table 2). Gluconobacter strains prefer sugar-enriched environments in contrast to Acetobacter strains, which prefer alcohol-enriched environments (Raspor and Goranovic, 2008).

Several Acetobacter strains have an active role in the fermentation of cocoa beans, during the manufacture of chocolate (Camu et al., 2007; Cleenweck et al., 2008; Lefeber et al., 2011).

Other AAB have been found among the characteristic microbial population of kombucha (Dutta and Gachhui, 2006; Nguyen et al., 2008). Some symbiotic nitrogen-fixing AAB strains have been studied to improve non-legume coffee, rice and sugarcane production (Pedraza, 2008). Recent research in microbe-insect symbiosis has shown that AAB establish symbiotic relationships with several insects as the fruit flies Drosophila melanogaster (vinegar fly) and Bactrocera oleae (olive fruit fly), but also with mosquitoes, honey bee and insects of other orders (Kounatidis et al., 2009; Crotti et al., 2010). However, a group of these bacteria have recently been described as emerging human opportunistic pathogens that are resistant to several microbiocidal agents (Greenberg et al., 2006; Alauzet et al., 2010).

1.2.2. Taxonomy

The main phenotypic traits of AAB are: Gram-negative or Gram-variable, acidophilic, ellipsoidal to rod shape; and are mainly characterized by their capacity to incompletely oxidize alcohols, sugars and sugar-alcohols into their corresponding organic acid, such as acetic acid from ethanol or gluconic acid from glucose (Adachi et al., 2003).

After the discovery that acetification was due to the transformation of alcohol into acetic acid by the British chemist and physicist Humprey Davy (1778-1829), several theories opposed the cause of the process to be biological or strictly inorganic. Christian Persoon (1822), a Dutch scientist, first identified the main agent of acetification as a microorganism that he called Mycoderma aceti. Forty years later, Louis Pasteur (1822-1895) confirmed the effectiveness of Persoon's studies. He identified the "vinegar mother" as a large mass of living organisms needed for the transformation, and found that although air should be kept from fermenting wine, it was necessary for the production of vinegar (Mazza and Murooka, 2009).

Although the taxonomic grouping of AAB started when Kützing (1837) observed for the first time the organism in vinegar (Asai 1968; Gullo and Giudici, 2009) (Figure 4), Hansen (1879) was the first to discover that the biological agent of acetification was a mixture of bacterial species, and Beijerinck (1899) used for the first time the terminology of Acetobacter. Visser’t Hooft (1925) was the first to use morphological, physiological and biochemical characteristics of microorganisms for classification and taxonomical purposes. Using these phenotypic traits, Asai (1935) proposed the classification of AAB in two genera: Acetobacter and Gluconobacter, in the family of Acetobacteraceae. The classification proposed by Frateur (1950) was based on 5 physiological tests: catalase, gluconic acid production from glucose, acetic acid oxidation to CO2 and water, lactic acid oxidation to CO2 and water and glycerol oxidation to dihydroxyacetone; and classified Acetobacter strains in 4 groups: peroxydans, oxydans, mesoxydans and suboxydans.

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Table 2. Vinegars of the world. Non-exhaustive list, modified from Gullo and Giudici, 2009).

Category Raw material Intermediate Vinegar name Geographical distribution

Vegetable^a Rice Moromi Komesu, kurosu (japan)

Heicu (china) East and Southeast Asia

Bamboo sap Fermented bamboo sap Bamboo vinegar^b Japan, Korea

Malt Beer Malt vinegar Northern Europe, USA

Palm sap Pal wine (toddy, tari, tuack, tuba) Palm or toddy vinegar Southeast Asia, Africa

Barley Beer Beer vinegar Germany, Austria, Netherlands

Millet Koji Black vinegar China, East Asia

Wheat Koji Black vinegar China, East Asia

Sorghum Koji Black vinegar China. East Asia

Tea and sugar Kombucha Kombucha vinegar Rusia, Asia (China, Japan,

Indonesia)

Onion Onion alcohol Onion vinegar East and Southeast Asia

Tomato --- Tomato vinegar Japan, East Asia

Sugarcane Fermented sugar cane juice

Basi Cane vinegar

Sukang iloko Kibizu

France, USA Philippines Japan

Fruit Apple Cider Cider vinegar USA, Canada

Pear Fermented pear juice Pear vinegar Europe, France

Pineapple Fermented pineapple juice Pineapple vinegar Africa

Grape Raisin

Red or white wine Sherry wine Cooked must

Raisin (grape) vinegar Wine vinegar Sherry vinegar Balsamic vinegar

Turkey and Middle East Widespread

Spain (Jerez) Italy

Fig Fermented fig juice Fig vinegar Turkey

Coconut Fermented coconut water Coconut vinegar Philippines, Sri Lanka

Date Fermented date juice Date vinegar Middle East

Mango Fermented mango juice Mango vinegar East and Southern Asia

Red date Fermented jujube juice Jujube vinegar China

Raspberry Fermented raspberry juice Raspberry vinegar East and Southern Asia Blackcurrant Fermented blackcurrant juice Blackcurrant vinegar East and Southern Asia Blackberry Fermented blackberry juice Blackberry vinegar East and Southern Asia Mulberry Fermented mulberry juice Mulberry vinegar East and Southern Asia

Plum Umeboshi^c fermented plum juice Ume-su Japan

Cranberry Fermented cranberry juice Cranberry vinegar East and Southern Asia Kaki Fermented persimmon juice Persimmon vinegar

Kakisu

South Korea Japan

Animal Whey Fermented whey Whey vinegar Europe

Honey Diluted honey wine, tej Honey vinegar Europe, America, Africa

aVegetable is not a botanical term and is used to refer to an edible plant part; some botanical fruits, such as tomatoes, are also generally considered to be vegetable.

bObtained by bamboo sap fermentation

cUmeboshi are pickled ume fruits. Ume is a species of fruit-bearing tree of the genus Prunus, which is often called a plum but is actually more closely related to apricot.

Figure 4. Scanning electron micrograph of AAB bacteria grown on synthetic medium. Bar = 1 µm

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The 1984 edition of the “Bergey’s Manual of Systematic Bacteriology”, the reference book for bacteria taxonomy, included additional molecular tests for the classification of microorganims, as the fatty acid composition, the ubiquinone nature, the electrophoretic pattern of soluble proteins, the plasmid fingerprinting; and other genome-based tests, as the

%G+C and the DNA-DNA hybridization. Using a polyphasic approach, the genera Gluconobacter and Acetobacter were considered phylogenetically close enough to be classified in the family Acetobacteraceae (De Ley et al., 1984). At that time, the genus Acetobacter included 4 species: A. aceti, A. pasteurianus, A. liquefaciens and A. hansenii, and the genus Gluconobacter only one, G. oxydans. The main difference was the ability of Acetobacter to completely oxidize ethanol into CO2 and water, which was missing in Gluconobacter. Further research in bacterial taxonomy based on genome studies: DNA-DNA or DNA-RNA hybridization, partial and complete 16S rRNA gene analysis, MLSA/MLST using a set of up to 7 housekeeping genes, changed considerably the bacterial classification, and the Acetobacteraceae have been no exception. After those two genera, four more genera have been described and included in the Acetobacteraceae family: Acidomonas (Urakami et al., 1989), Gluconacetobacter (Yamada et al., 1997) Asaia (Yamada et al., 2000) and Kozakia (Lisdiyanti et al., 2002). Also new species have been included and the group of AAB consists nowadays of 14 genera and 68 species (Table 3), where Acetobacter and Gluconacetobacter, with 20 and 19 species, respectively, are the genera which show the highest biodiversity (Yamada, 2003). Despite the high diversity, only a few species have been described to be related with vinegar (see Table 3, highlighted species). The genus Gluconacetobacter is well separated in two groups, the so-called Ga. xylinus group, which is mostly formed by vinegar related species, and Ga. liquiefaciens group, which include nitrogen-fixing strains among their members. Some researchers have proposed to separate these two groups in two different genera, but at present there is not yet sufficient evidence to support the reclassification (Cleenwerck et al., 2010).

Table 3. Currently recognized AAB genera and species (modified from Gullo and Giudici, 2009).

Phylum Class Order Family

Proteobacteria Alphaproteobacteria Rhodospirillales Acetobacteraceae

Genus Species Source^a Reference

ACETOBACTER

Acetobacter aceti Vinegar (Pasteur, 1864)

Beijerinck, 1898

Acetobacter cerevisiae Beer Cleenwerck et al., 2002

Acetobacter cibinongensis Fruit Lisdiyanti et al., 2002

Acetobacter estunensis Cider (Carr, 1958)

Lisdiyanti et al., 2002

Acetobacter fabarum Cocoa bean Cleenwerck et al., 2008

Acetobacter farinalis^b Fermented rice flour (khao-

khab)

Tanasupawat et al., 2011

Acetobacter ghanensis Cocoa bean Cleenwerck et al., 2007

Acetobacter indonesiensis Fruit and flower Lisdiyanti et al., 2000

Acetobacter lovaniensis Soil (Frateur, 1950)

Acetobacter malorum Apple Cleenwerck et al., 2002

Acetobacter nitrogenifigens Kombucha tea Dutta and Gachhui, 2006

Acetobacter oeni Wine Silva et al., 2006

Acetobacter orientalis Canna flower Lisdiyanti et al., 2002

Acetobacter orleanensis Beer (Henneberg, 1906)

Acetobacter pasteurianus Beer (Hansen, 1879)

Beijerinck and Folpmers, 1916

Acetobacter peroxydans Ditch water Visser't Hooft, 1925

Acetobacter pomorum Industrial vinegar

fermentation Sokollek et al., 1998

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Table 3. Currently recognized AAB genera and species (modified from Gullo and Giudici, 2009). (Cont.)

ACETOBACTER

Acetobacter senegalensis Mango fruit Ndoye et al., 2007

Acetobacter syzygii Organic apple juice Lisdiyanti et al., 2002

Acetobacter tropicalis Coconut Lisdiyanti et al., 2000

ACIDOMONAS

Acidomonas methanolica Yeast fermentation

process

(Uligh et al., 1986) Urakami et al., 1989 emend.

Yamashita et al., 2004 AMEYAMAEA^b

Ameyamaea chiangmaiensis^b Flower of red ginger Yukphan et al., 2009

ASAIA

Asaia bogorensis Flower or orchid tree Yamada et al., 2000

Asaia krungthepensis Heliconia flower Yukphan et al., 2004

Asaia lannaensis^b Flowers of the spider lily Malimas et al., 2008

Asaia siamensis Flower of crown flower Katsura et al., 2001

Asaia spathodeae^b Flowers of the african tulip Kommanee et al., 2010

GLUCONACETOBACTER

Gluconacetobacter asukensis Stone Tazato et al., 2011

Gluconacetobacter azotocaptans Coffe plant Fuentes-Ramírez et al., 2001

Gluconacetobacter diazotrophicus Sugarcane (Guillis et al., 1989)

Yamada et al., 1997

Gluconacetobacter entanii High-acid industrial vinegar

fermentation Schüller et al., 2000

Gluconacetobacter europaeus High-acid vinegar

fermentation (Sievers et al., 1992) Yamada et al., 1997

Gluconacetobacter hansenii Vinegar (Goseelé et al., 1997)

Yamada et al., 1997 emend.

Gluconacetobacter intermedius Kombucha beverage (Boesch et al., 1998)

Yamada, 2000

Gluconacetobacter johannae Coffe plant Fuentes-Ramírez et al., 2001

Gluconacetobacter kakiaceti Persimmon vinegar Lino et al., 2011

Gluconacetobacter kombuchae Kombucha tea Dutta and Gachhui, 2006

Gluconacetobacter liquefaciens Dried fruit (Asai, 1935)

Yamada et al., 1997

Gluconacetobacter nataicola Nata de coco Lisdiyanti et al., 2006

Gluconacetobacter oboediens Industrial red wine

vinegar fermentation

(Sokollet et al., 1998) Yamada, 2000

Gluconacetobacter rhaeticus Organic apple juice Dellaglio et al., 2005

Gluconacetobacter sacchari Sugarcane Franke et al., 1999

Gluconacetobacter saccharivorans Beet juice Lisdiyanti et al., 2006

Gluconacetobacter swingsii Organic apple juice Dellaglio et al., 2005

Gluconacetobacter tumulicola Stone Tazato et al., 2001

Gluconacetobacter xylinus Mountain-ash berries (Brown, 1886)

Yamada et al., 1997 GLUCONOBACTER

Gluconobacter albidus Flower of dahlia (ex Kondo and Ameyama, 1958)

Yukphan et al., 2005

Gluconobacter cerinus Cherry (ex Asai, 1935)

Yamada and Akita, 1984 emend.

Katsura et al., 2001

Gluconobacter frateurii Strawberry Mason and Claus, 1989

Gluconobacter japonicus Bayberry fruit Malimas et al., 2009

Gluconobacter kanchanaburiensis^b Spoiled Jackfruit Malimas et al., 2009

Gluconobacter kondonii^b Strawberry Malimas et al., 2007

Gluconobacter nephelii Fruit Kommannee et al., 2010

Gluconobacter oxydans Beer (Henneberg, 1897)

De Ley, 1961 emend.

Gosselé et al., 1983 emend.

Mason and Claus, 1989

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Table 3. Currently recognized AAB genera and species (modified from Gullo and Giudici, 2009). (Cont.)

GLUCONOBACTER

Gluconobacter roseus^b Persimmon fruit (ex Asai 1935)

Malimas et al., 2008

Gluconobacter sphaericus^b Fresh grape (Ameyama 1975)

Malimas et al., 2008

Gluconobacter thailandicus Flower Tanasupawat et al., 2004

Gluconobacter uchimurae^b Rakam fruit Tanasupawat et al., 2011

Gluconobacter wancherniae^b Unknown eeed Yukphan et al., 2010

GRANULIBACTER

Granulibacter bethesdensis Lymph node culture from a

patient with chronic granulomatous disease

Greenberg et al., 2006

KOZAKIA

Kozakia baliensis Palm brown sugar Lisdiyanti et al., 2002

NEOASAIA

Neoasaia changmaiensis Flower of red ginger Yukphan et al., 2005

NEOKOMAGATAEA^b

Neokomagataea tanensis^b Flowers of candle bush Yukphan et al., 2011

Neokomagataea tahilandica^b Flowers of lantana Yukphan et al., 2011

SACCHARIBACTER

Saccharibacter floricola Flower Jojima et al., 2004

SWAMINATHANIA

Swaminathania salitolerans Mangrove-associated wild

rice Loganathan and Nair, 2004

TANTICHAROENIA^b

Tanticharoenia sakaeratensis^b Soil Yukphan et al., 2008

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae

FRATEURIA

Frateuria aurantia Flower and fruit (ex Kondo and Ameyama, 1958)

Swings et al., 1980 Highlighted species (orange color) have been described to be related with vinegar production.

aRefers to the type strain isolation source.

bGenera and species waiting for validation by the Int. J. Syst. Bacteriol.

1.2.3. Molecular identification

AAB are currently identified by using a polyphasic approach, which includes the study of some phenotypical traits (morphology, physiology, biochemistry), but is mostly based on genomic information. The main molecular techniques used in ecological studies to analyze wine and vinegar bacterial diversity include:

1.2.3.A. Culture-dependent methods

1.2.3.A.1. Restriction Fragment Length Polymorphism (RFLP)

RFLP is one of the most widespread DNA-profiling methods that has been applied for multiple taxonomic studies on AAB. The principle of the method consists in the amplificaion by PCR of a genomic region of interest and the digestion of the amplification product with a set of restriction enzymes, followed by the separation of the resulting DNA fragments by

Characterization of acetic acid bacteria and study of the molecular strategies involved in the resistance to acetic acid during oxidative fermentation

Thesis

Reference