Claude Universite

Bernard -Lyon

M Le Professeur

Frédéric Fleury

Hamda Ben

Hadid M Le Professeur Didier

Revel M Le Professeur

Philippe Chevalier

M Fabrice

Vallée Mme

Dominique Marchand

Composantes Sante

G Rode

La Mme

C Professeure

Burillon

D Bourgeois

Vinciguerra

M X Perrot

A-M Professeure

Schott

Mauricio González

Jennifer Gil

Sergio Croquer

Beatriz Caraballo

Edwin Rodriguez

Julie Martel

Shant Shahbikian

Marie Lupien-Durocher

Iulian Zoltan

Judit Hunyadkurti

Jean Baptiste

" Beigbeder

Pedro Durao

Milad Ferdowsi

Bahman Khabiri

Jamie Sharp

Rajesh Guruswamy

Amirjalal Jalali

Tim Spitters

Luc Treutens

Graciela Chavez

Isabelle Arsenault

Valérie Larouche Moreover

Louise Carbonneau

Louise Chapdelaine

Beatriz Delgado

Erika Ligero

Gloria Chamorro

Jose Manuel

Keywords: Escherichia coli, 2, 3-butanediol, glucose, lactose, whey, permeate whey

Whey is a dairy effluent generated during the cheese manufacturing. It contains lactose (the main part of the dry matter of whey), proteins, etc. Due to its high biological (BOD) and chemical (COD) oxygen demands, it is necessary to treat the whey before releasing it in the environment. The valorization of whey via biotechnology will have to allow for a) a decrease of the BOD and COD values, and b) of a generation of products like 2,3-butanediol (2,3-BD). Bacteria like Enterobacter cloacae and Klebsiella pneumoniae are able to hydrolyze and transform saccharides like lactose into 2,3-BD. Although these bacteria are pathogenic, they are considered as the best 2,3-BD producers. Therefore, the use of other non-pathogenic bacterial strains is being developed for the hydrolysis and the fermentation of saccharides. In this way, a non-pathogenic strain of Escherichia coli able to hydrolyze and ferment a wide range of saccharides would be a good option. However, E. coli cannot produce 2,3-BD. A genetic modification in the E. coli strain has been performed in order to transfer the metabolic pathway from a natural 2,3-BD producer: E. cloacae. In this way, the main objective of this research is to valorize the lactose (a disaccharide) contained in the whey via fermentation in the presence of a genetically modified strain of E. coli K12 MG1655 in order to obtain 2,3-BD. Permeate whey, was also used as a substrate. Firstly, a literature review about the potential valorization of whey into 2,3-BD was performed to show the different kinds of bacteria and substrates used to produce 2,3-BD under different operating conditions. Secondly, the E. coli strain (E. coli JFR12) was used varying the substrate nature and concentration in M9 culture medium (previously chosen as the most appropriate among 4 culture media). The highest 2,3-BD yield (around 0.36 g 2,3-BD/g saccharide) was obtained in the presence of 25 g/L of glucose and lactose. Whatever galactose concentration used, low 2,3-BD yields were obtained. In the presence of a mixture of glucose-galactose (1:1, w/w), the 2,3-BD yields were similar to those obtained using galactose as a sole carbon source. Afterwards, three mixtures of whey or permeate whey in the presence or absence of M9 culture medium were used. Fermentations of these mixtures in absence of M9 improved the 2,3-BD yield up to 0.43 and 0.42 g 2,3-BD/g lactose for 72 h using whey and permeate whey, respectively. Thirdly, the effect of three operating conditions (inoculum size, initial pH and agitation rate) was evaluated. The highest 2,3-BD yields were 0.47 and 0.44 g 2,3-BD/g lactose for 72 h using whey and permeate whey, respectively. Finally, an increase of the reaction volume using a 2 L bioreactor to ferment whey and permeate whey was tested under anaerobic conditions. The 2,3-BD yields under anaerobic conditions were lower compared to those obtained in flask of 0.5 L. On the other hand, the addition of air (2 L/min) in a 2 L bioreactor was tested in order to study its effect on 2,3-BD production fermenting whey. The 2,3-BD yield was 0.40 g 2,3-BD/g lactose at 24 h. Therefore, the fermentation was improved using 2 L/min of air in the bioreactor since a similar 2,3-BD yield was obtained in comparison to the one in 0.5 L flasks, reducing the fermentation time from 72 h to 24 h. The E. coli JFR12 strain is a suitable strain to valorize whey and permeate whey into 2,3-BD.

COMPOSANTES ET DEPARTEMENTS DE SCIENCES ET TECHNOLOGIE

RÉSUMÉ

Le lactosérum est un résidu liquide obtenu lors de la fermentation du fromage. Il est composé de lactose, de protéines, etc. Étant donné les valeurs élevées de ses demandes biologique (DBO) et chimique (DCO) en oxygène, il est nécessaire de traiter le lactosérum avant de le rejeter. La valorisation du lactosérum par voie biotechnologique devrait permettre a) de réduire la DBO et la DCO et b) de générer des produits tels que le 2,3-butanediol [START_REF]Abstract Whey (W) is generated during cheese manufacturing and permeate whey (PW) is obtained after W deproteinization. As both effluents contain lactose, a carbon source, and they can cause environmental issues if they are released in the environment without a treatment. The aim of the present study was to test the transformation of lactose via fermentation into 2,3butanediol (2,3-BD) in the presence of a genetically modified strain of Escherichia coli K12 MG1655 (E. coli JFR12) in order to evaluate the potential of W and PW as lactose sources. Lactose (a disaccharide), glucose and galactose (monosaccharides of lactose) at concentrations of 12.5, 25 and 50 g/L were fermented in a synthetic culture medium (medium 9, M9) inoculated at a ratio of 10% (v/v), at 37ºC, 1 atm, initial pH 7.4 and 100 rpm for 72 h in flasks of 0.5 L with a working volume of 0.2 L. Under these operating conditions, the highest 2,3-BD yield was around 0.38 g/g saccharide in the presence of 25 g/L of glucose or lactose at 48 and 72 h, respectively, and 0.19 g/g galactose at 48 h. Therefore, E. coli JFR12 could transform galactose into 2,3-BD but being its yield the half of one observed with glucose at 48 h. Whey (31 g/L lactose) or PW (34 g/L lactose) were fermented in dilution with M9 (50:50, 75:25 and 100:0, v/v) under the same operating conditions. The fermentation of W and PW in absence of M9 (100:0, v/v) produced the highest 2,3-BD yield (up to 0.43 g/g lactose) at 72 h. Whereas, the highest acetoin (A) yield of 0.12 g/g lactose was obtained with[END_REF]. Des bactéries comme Enterobacter cloacae et Klebsiella pneumoniae peuvent hydrolyser et transformer des saccharides (ex. lactose) en 2,3-BD. Ces bactéries quoique pathogènes sont considérées comme étant les plus performantes pour générer du 2,3-BD. Par conséquent, l'utilisation d'autres souches bactériennes non pathogènes permettant l'hydrolyse et la fermentation des saccharides est recherchée. Dans ce contexte, une souche bactérienne non pathogène d'Escherichia coli capable d'hydrolyser et de fermenter une large variété de saccharides pourrait être une bonne option. Cependant, la souche E. coli ne produit pas de 2,3-BD. Une modification génétique d'E. coli a été effectuée afin de transférer la voie métabolique d'un producteur naturel de 2,3 BD à savoir l'E. cloacae. Par conséquent, l'objectif principal de cette étude est de valoriser le lactose (un disaccharide) contenu dans le lactosérum par fermentation en présence d'une souche génétiquement modifiée d'E. coli, afin d'obtenir du 2,3-BD. Un perméat de lactosérum a également été utilisé comme substrat. Dans un premier temps, une étude portant sur la valorisation potentielle du lactosérum en 2,3-BD a été réalisée. Les différentes bactéries et substrats utilisés pour produire du 2,3-BD sous différentes conditions opératoires sont détaillés. Dans un deuxième temps, la souche d'E. coli (E. coli JFR12) a été utilisée en variant la nature et la concentration du substrat dans le milieu de culture M9 (préalablement choisi comme le plus approprié parmi 4 milieux de culture). Le rendement en 2,3-BD le plus élevé (environ 0.36 g 2,3-BD/g saccharide) a été obtenu en présence de 25 g/L de glucose ou de lactose. Quelle que soit la concentration de galactose, les rendements en 2,3-BD étaient faibles. En présence du mélange glucose-galactose, les rendements en 2,3-BD obtenus étaient similaires à ceux obtenus avec le galactose. Ensuite, des mélanges de lactosérum ou de perméat de lactosérum ont été réalisés. Les fermentations de ces mélanges en absence de M9 améliorent le rendement en 2,3-BD jusqu'à 0.43 et 0.42 g 2,3-BD/g lactose en utilisant du lactosérum ou du perméat de lactosérum respectivement pendant 72 h. Finalement, l'effet des trois paramètres suivants (quantité d'inoculum, pH initial et vitesse d'agitation) ont été évalués. Les rendements en 2,3-BD les plus élevés ont été respectivement de 0.47 et de 0.44 g 2,3-BD/g lactose pendant 72 h en utilisant du lactosérum ou du perméat de lactosérum respectivement. Enfin, l'augmentation du volume réactionnel en utilisant un bioréacteur de 2 L pour la fermentation du lactosérum et du perméat de lactosérum a été testée en condition anaérobie. Les rendements en 2,3-BD ont été plus faibles par comparaison avec ceux obtenus à partir des expériences réalisées dans des fioles de 0.5 L. L'ajout d'air (2 L/min) dans le bioréacteur a été testé afin d'étudier son impact. Le rendement en 2,3-BD a été de 0.40 g 2,3-BD/g de lactose en 24 h. Le temps de fermentation a également été réduit de 72 h à 24 h. La souche E. coli JFR12 est appropriée pour valoriser le lactosérum et le perméat de lactosérum en 2,3-BD.

Mots-clefs: Escherichia coli, 2,3-butanediol, glucose, lactose, lactosérum, perméat de lactosérum. (A) and ABD yield (B) in M9 culture medium supplemented with 12.5 g/L of glucose as a function of time in the presence of E. coli JFR1. Source of nitrogen: urea at 0.6 g N/L (▲), 1.2 g N/L (∆), 2.5 g N/L (♦), 5.0 g N/L (■) and 7.0 g N/L (•). Additionally, the highest (▬) and lowest (+) ethanol yields obtained for 0.6 g N/L and without an ANS are represented respectively. Results are means ± SD of 3 replications performed in flasks at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% v/v of inoculum in a working volume of 200 mL (batch fermentation). ............................................................... 51 Figure 3.4: Glucose conversion (A) and ABD yield (B) in M9 culture medium supplemented with 25 g/L of glucose as a function of time in the presence of E. coli JFR1. Source of nitrogen: urea at 2.5 g/L of nitrogen (♦), 5.0 g/L of nitrogen (■), 7.0 g/L of nitrogen (•) and 10.0 g N/L (×). Additionally, the highest (□) and lowest (▬) ethanol yields obtained for 10.0 g N/L and 2.5 g N/L are represented respectively. Results are means ± SD of 3 replications performed in flasks at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% v/v of inoculum in a working volume of 200 mL (batch fermentation). ........................................ 53 Figure 4.1: Metabolic pathway to produce 2,3-BD from lactose fermentation in the presence of a genetically modified strain of Escherichia coli. Relevant reactions to transform pyruvic acid into 2,3-BD are represented by the names of the corresponding enzymes from Enterobacter cloacae: α-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC) and 2,3butanediol dehydrogenase (BDH). Reduced NADH) and oxidized (NAD+) form of ix nicotinamide adenine dinucleotide. Sources: [START_REF] Lazar | Awakening the endogenous Leloir pathway for efficient galactose utilization by Yarrowia lipolytica[END_REF], [START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF], [START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF], [START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF], [START_REF] Xu | Metabolic engineering of Escherichia coli for efficient production of (3R)-acetoin[END_REF] Table 2.1: Composition of sweet and acid wheys. Source: Hernández-Rojas and Vélez-Ruiz (2014); [START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF]; [START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF]; [START_REF] Tsakali | A review on whey composition and the methods used for its utilization for food and pharmaceutical products[END_REF] 2.3: Characteristics and particularities of some bacteria producers of 2,3 butanediol.25 Table 3.1: Composition of the 4 culture media in 1L of distilled water -LB medium [START_REF] Berney | Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection[END_REF]); M9 minimal medium [START_REF] Ecocyc | Escherichia coli K-12 substr[END_REF][START_REF] Maniatis | Molecular cloning: a laboratory manual[END_REF]; M63 minimal medium [START_REF] Pardee | The genetic control and cytoplasmic expression of "inducibility" in the synthesis of β-galactosidase by E. coli[END_REF] 3.2: Maximum glucose conversion (%) as a function of glucose concentration (g/L) and time (h) in the 4 studied culture media at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% (v/v) of inoculum in a working volume of 200 mL (batch fermentation). ..... 45 Table 3.3: Glucose conversion (%) as a function of nitrogen concentration (g N/L) in the presence of 12.5 and 25 g/L of glucose in M9 culture medium using NaNO3 as an ANS at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% (v/v) of inoculum in a working volume of 200 mL (batch fermentation). . 3.4: Acetic acid yield (%, g acetic acid/g glucose) as a function of nitrogen concentration (g N/L) in the presence of 12.5 and 25 g/L of glucose in M9 culture medium using NaNO3 as an ANS at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% v/v of inoculum in a working volume of 200 mL (batch fermentation). .............................. 50 Table 4.1: Operating conditions used to ferment saccharides (glucose, galactose, lactose and the mixture glucose-galactose, G-Gal) in M9, whey (W1) and permeate whey (PW1) diluted with M9, and the study of inoculum size, initial pH and agitation in flask. New batch of W (W2) and PW (PW2) diluted and undiluted were tested in flask and in a 2 L bioreactor. 4.2: Maximum Et yield fermenting glucose, galactose, lactose and a mixture (1:1, w/w) of glucose-galactose at different concentrations at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 7.4 and 100 rpm…………………………………………………………………………66 Table 4.3: Acetoin, 2,3-BD and ABD yields as a function of time fermenting W1 and PW1 at 37ºC, 1 atm and initial pH 7.4 for 48 and 72 h in flasks varying the agitation rate. ............ 84

viii

LIST OF FIGURES

CHAPTER 1. Introduction

In a world more and more industrialized to cover all the human needs, the environmental issues are getting a special attention since the industrialization is causing negative environmental impacts, which is trying to be contained by, for instance, international environmental agreements. It is possible to underline among the human requirements, the basic consumer goods like milk and bread, and their production have to be increased because of the rise of the worldwide population. The industrialization causes the increase of greenhouse gases (GHG) like carbon dioxide (CO 2 ) [START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF]. However, the upgrades of the productive processes have reduced the GHG emissions. For example, in the dairy industry, 1.63 kg CO 2 equivalent per L of milk cow produced were generated in 1961; whereas this amount was reduced to 0.92 kg CO 2 equivalent per L of milk cow produced in 2014 (FAO-FAOSTAT, 2017a).

The dairy industry example is important since it is one of the most important industries in the agro-food sector. The main dairy product is the milk, which worldwide production was 791.8 million tons in 2014 (FAO-FAOSTAT, 2017b). However, all of amount of milk produced is not only allocated for human consumption but also for manufacturing other products derived from milk like cheese. Cheese production, an important dairy processed product in terms of volume, needs around 10 L of milk to make 1 kg of cheese [START_REF] Bcb | British Cheese Board -Did you know that[END_REF]. During the cheese manufacturing, a liquid effluent is generated: the whey (W). Whey is the liquid obtained after coagulating the milk and is generated at a rate of 9 L per kg of cheese manufactured. If it is directly released into any body of water, these important quantities of W can affect the aquatic life of rivers, lakes, etc. (De Jesús et al. 2015[START_REF] Guimarães | Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey[END_REF]. Lactose, a disaccharide formed of glucose and galactose (monosaccharides), is the main dried solid compound of W with a concentration ranged between 46 and 54 g/L of W depending on the kind of W: sweet or acid (Hernández-Rojas and Vélez-Ruíz 2014, [START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF][START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF][START_REF] Tsakali | A review on whey composition and the methods used for its utilization for food and pharmaceutical products[END_REF]. One of the treatments that can be applied on W is its deproteinization by ultrafiltration (membranes), obtaining another liquid effluent: permeate whey (PW) [START_REF] De Wit | Lecturer's handbook on whey and whey products, 1st edn[END_REF]. Permeate whey can present a lactose concentration higher than 80 g/L [START_REF] Smith | Evaluation of whey, milk, and delactosed permeates as salt substitutes[END_REF].

Lactose can be used in order to produce hydrogen (H 2 ), biogas, acetoin (A) and 2,3-butanediol [START_REF]Abstract Whey (W) is generated during cheese manufacturing and permeate whey (PW) is obtained after W deproteinization. As both effluents contain lactose, a carbon source, and they can cause environmental issues if they are released in the environment without a treatment. The aim of the present study was to test the transformation of lactose via fermentation into 2,3butanediol (2,3-BD) in the presence of a genetically modified strain of Escherichia coli K12 MG1655 (E. coli JFR12) in order to evaluate the potential of W and PW as lactose sources. Lactose (a disaccharide), glucose and galactose (monosaccharides of lactose) at concentrations of 12.5, 25 and 50 g/L were fermented in a synthetic culture medium (medium 9, M9) inoculated at a ratio of 10% (v/v), at 37ºC, 1 atm, initial pH 7.4 and 100 rpm for 72 h in flasks of 0.5 L with a working volume of 0.2 L. Under these operating conditions, the highest 2,3-BD yield was around 0.38 g/g saccharide in the presence of 25 g/L of glucose or lactose at 48 and 72 h, respectively, and 0.19 g/g galactose at 48 h. Therefore, E. coli JFR12 could transform galactose into 2,3-BD but being its yield the half of one observed with glucose at 48 h. Whey (31 g/L lactose) or PW (34 g/L lactose) were fermented in dilution with M9 (50:50, 75:25 and 100:0, v/v) under the same operating conditions. The fermentation of W and PW in absence of M9 (100:0, v/v) produced the highest 2,3-BD yield (up to 0.43 g/g lactose) at 72 h. Whereas, the highest acetoin (A) yield of 0.12 g/g lactose was obtained with[END_REF] by catalytic reactions performed by enzymes, commonly named "fermentation" [START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF]Roncal et al., 2010). The fermentation is performed in the presence of bacteria and under controlled operating conditions. Several parameters such as temperature, pH, agitation, kind of bacteria and concentration of substrate, can affect the yield of the fermentative products.

To the author's best knowledge, no study discussed the fermentation of the lactose content in W and PW by a modified strain of Escherichia coli in order to obtain 2,3-butanediol. In addition, no study discussed the transformation of galactose as a sole carbon source by fermentation in the presence of an E. coli strain.

The objective of this research was the transformation of lactose contained in the W and PW into 2,3-BD by a genetically modified strain of Escherichia coli (ECGM), which hosts the metabolic pathway of 2,3-BD from Enterobacter cloacae. During this research, the ECGM was improved in terms of the 2,3-BD production. In this way, two ECGM strains were used: E. coli JFR1 and E. coli JFR12. To the best of our knowledge, this is the first time that a genetically modified strain (ECGM) has been used in order to catalyze the W and PW fermentation.

This study was carried out under seven specific objectives: 1) The inoculum size of E. coli JFR1 before performing fermentations in order to obtain the highest bacterial population;

2) The effect of three glucose (used as a control) concentrations supplementing four culture media in the presence of E. coli JFR1 on the formation of A and 2,3-BD (ABD);

3) The influence of the type and concentration of an additional nitrogen source (ANS) in a defined culture medium (M9) on the ABD production using E. coli JFR1; 4) The 2,3-BD yield fermenting three concentrations of glucose, galactose and lactose in M9 culture medium with E. coli JFR12; 5) The effect of the lactose dilution contained in W and PW on the 2,3-BD yield in the presence of M9 (W:M9 or PW:M9), varying the ratio substrate/M9 (50:50, 75:25 and 100:0); 6) The influence of the inoculum size, the initial pH and the agitation rate fermenting W and PW in absence of M9 on the 2,3-BD yield; 7) The effect of air addition (2 L/min) on the 2,3-BD yield in a 2 L bioreactor in the presence of E. coli JFR12 and W. This thesis contains 5 chapters including one review article and two research articles. In addition, one short paper is included as Annex 1.

Chapter 1 presents a general statement about the theme of the thesis. In this chapter, the whey and its permeate are introduced besides the objectives of the project.

Chapter 2 presents the state of art about the potential use of W in order to produce 2,3-BD. In this chapter, the W generation and its effects on the environment are introduced. In addition, the diverse kinds of treatments that can be performed on W in order to valorize it are discussed. Among these treatments, the biological process and particularly the transformation of several saccharides into 2,3-BD by different bacteria and how the operating conditions such as type and concentration of substrate, temperature, pH, agitation, etc. affect the 2,3-BD yield is also discussed.

Chapter 3 presents the estimation of the inoculum size by two experimental techniques: optical density (OD) and colony-forming unit (CFU). The wild and a genetically modified strain of Escherichia coli were characterized. Once the inoculum size was estimated, four culture media (LB, M9, M63 and MOPS) were tested at three glucose concentrations (4, 12.5 and 25 g/L) in the presence of the genetically modified E. coli strain (E. coli JFR1) to produce ABD. The culture medium M9 was selected according to the yield of ABD and to the cost of the culture medium. The chosen culture medium, M9, was supplemented with two ANS (sodium nitrate: NaNO 3 , and urea: (NH 2 ) 2 CO) and tested in the presence of 12.5 and 25 g/L of glucose. After

Introduction in French

Afin de répondre aux besoins des populations, l'industrialisation s'accroît ce qui provoque une augmentation de la pollution. Les besoins humains et notamment la production de produits de consommation courante ne cesse d'augmenter en lien avec la croissance de la population mondiale. Par conséquent, des problématiques environnementales apparaissent suite à une industrialisation intensive qui se traduit par une augmentation des émissions de gaz à effet de serre (GES) comme le dioxyde de carbone (CO 2 ) [START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF]. Cependant, l'optimisation des procédés a permis de réduire les émissions de GES notamment dans le domaine de la production laitière. Par exemple, dans l'industrie laitière, un litre de lait produit, émettait 1.63 kg de CO 2 en 1961 alors que cette quantité était de 0.92 kg de CO 2 équivalent par litre de lait produit en 2014 (FAO-FAOSTAT, 2017a). L'exemple de l'industrie laitière est important car il représente un secteur fort de l'industrie agroalimentaire. Le lait est le principal produit de cette industrie avec une production mondiale atteignant 791.8 millions de tonnes en 2014 (FAO-FAOSTAT, 2017b). Cependant, cette production annuelle se répartit entre le lait de consommation courante et les produits dérivés comme les fromages. La production de fromage, un important produit dérivé en termes de volume, nécessite 10 litres de lait par kilogramme de fromage produit [START_REF] Bcb | British Cheese Board -Did you know that[END_REF]. Au cours de la fabrication du fromage, un effluent liquide est généré: le lactosérum (W). Le W est le liquide résiduel issu du processus de coagulation du lait; 9 litres de W sont générés par kg de fromage manufacturé. En cas de rejets directs dans les eaux naturelles, ces quantités importantes de lactosérum peuvent nuire à la vie aquatique des fleuves, des lacs, etc. (De Jesús et al. 2015, Guimaraes et al. 2010). Le lactose, un disaccharide formé de glucose et galactose (monosaccharides), est le principal composant de la fraction sèche du W avec une concentration variant entre 46 et 54 g/L de W, selon le type de W: sucré ou acide [START_REF] Hernández-Rojas | Suero de leche y su aplicación en la elaboración de alimentos funcionales[END_REF][START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF][START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF][START_REF] Tsakali | A review on whey composition and the methods used for its utilization for food and pharmaceutical products[END_REF]. L'un des traitements pouvant être appliqué sur le W est sa déprotéinisation par ultrafiltration (membranes) afin d'obtenir du perméat (PW). Le PW peut avoir une concentration de lactose supérieure à 80 g/L [START_REF] Smith | Evaluation of whey, milk, and delactosed permeates as salt substitutes[END_REF].

Le lactose peut être utilisé pour la production d'hydrogène (H 2 ), de biogaz, d'acétoïne (A) et de 2,3-butanediol (2,3-BD) lors de réactions de catalyse enzymatique, appelée couramment "fermentation" [START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF]Roncal et al., 2010). La fermentation est effectuée en présence de bactéries et sous conditions contrôlées. Plusieurs facteurs comme la température, le pH, l'agitation, le type de bactéries, la nature et la concentration du substrat, peuvent modifier le rendement des produits de la fermentation.

À la connaissance de l'auteur, aucune étude n'a discuté de la fermentation du lactose présent dans W et PW par une souche modifiée d'Escherichia coli afin d'obtenir du 2,3-butanediol. En outre, aucune étude n'a examiné la transformation du galactose comme l'unique source de carbone par fermentation en présence d'une souche d'E. coli.

L'objectif de ce travail de recherche a porté sur la transformation du lactose contenu dans le W et le PW en 2,3-BD par une souche d'Escherichia coli génétiquement modifiée (ECGM). Tout d'abord, l'ECGM a été évaluée puis améliorée afin d'optimiser la production du 2,3-BD. Pour cela, deux souches d'ECGM ont été sélectionnées: E. coli JFR1 et E. coli JFR12. À notre connaissance, c'est la première fois qu'une souche génétiquement modifiée (ECGM) est utilisée pour la fermentation du W ou du PW. Lors de cette étude, les sept objectifs spécifiques suivants ont été évalués: 1) la taille de l'inoculum d'E. coli JFR1 nécessaire pour réaliser des fermentations; 2) l'effet de trois concentrations de glucose et de quatre milieux de culture en présence d'E. coli JFR1 sur la formation d'A et de 2,3-BD (ABD); 3) l'influence du type et de la concentration d'une source additionnelle d'azote (SAA) sur le rendement en ABD dans un milieu de culture défini (M9) en présence d'E. coli JFR1; 4) le rendement en 2,3-BD lors de la fermentation de trois concentrations de glucose, de galactose et de lactose dans le milieu de culture M9 en présence d'E. coli JFR12; 5) l'influence de la dilution du lactose contenu dans W et PW sur le rendement en 2,3-BD en mélangeant W ou PW avec M9 (W:M9 ou PW:M9) et en variant le ratio substrat/M9 (50:50, 75:25 et 100:0 (v/v)); 6) l'influence du volume d'inoculum, du pH initial et de la vitesse d'agitation lors de la fermentation du W et du PW en absence de M9 sur le rendement en 2,3-BD; 7) l'effet de l'oxygène (2 L/min d'air) sur le rendement en 2,3-BD lors d'une fermentation dans un bioréacteur de 2 L en présence d'E. coli JFR12 et de W.

Ce manuscrit de thèse contient 5 chapitres incluant un article de revue et deux articles de recherche. Un article complémentaire est annexé à ce document.

Le chapitre 1 est consacré à l'introduction du travail de recherche avec notamment des données relatives au lactosérum et au perméat. Les objectifs de l'étude sont ensuite précisés.

Le chapitre 2 présente l'état de l'art dans le domaine concerné et porte principalement sur la production du 2,3-BD à partir de W. Dans cette partie, la production de W et de ses effets sur l'environnement sont présentés. En outre, les différentes valorisations du W sont développées. Parmi ces traitements, le procédé biologique et en particulier la transformation de plusieurs types de saccharides en 2,3-BD par diverses souches bactériennes et les conditions d'opération (type et concentration de substrat, température, pH, vitesse d'agitation, etc.) sont discutés en détail.

Le chapitre 3 est consacré à l'estimation de la taille de l'inoculum. Deux techniques expérimentales ont été comparées : la densité optique (DO) et la méthode des unités formatrices de colonies (UFC). Les souches sauvage et génétiquement modifiée d'E. coli ont été caractérisées. Une fois la taille initiale de l'inoculum estimée, 4 milieux de culture (LB, M9, M63 et MOPS) ont été utilisés avec trois concentrations de glucose (4, 12.5 et 25 g/L) en présence de la souche d'E. coli génétiquement modifiée (E. coli JFR1). Le milieu de culture M9 fut sélectionné puisque le rendement en ABD était le plus élevé sur ce milieu et le coût de M9 était le plus faible. Le milieu de culture M9, a été complémenté avec deux sources additionnelles d'azote (SAA) (nitrate de sodium: NaNO 3 , et urée : (NH 2 ) 2 CO) et a été évalué en présence de 12.5 et 25 g/L de glucose. Il a été constaté que l'urée avait un effet positif sur le rendement en ABD; la teneur en azote a ensuite été optimisée.

Le chapitre 4 présente les rendements en 2,3-BD lors de la fermentation de W et PW en présence d'une souche d'E. coli optimisée: E. coli JFR12. Dans un premier temps, la fermentation du glucose, du galactose et du lactose (concentrations de 12.5, 25 et 50 g/L) dans le milieu de culture M9 enrichi de 15 g/L d'urée (7.0 g azote/L) a été étudiée. Les rendements en 2,3-BD en fonction de la nature du saccharide sont discutés. En complément, un mélange de glucose et de galactose sous deux concentrations (12.5 et 25 g/L pour chaque monosaccharide) a été réalisé puis utilisé comme substrat dans la réaction de fermentation. Enfin, un mélange de W1 (lot un (1) de lactosérum avec une concentration en lactose de 31 g/L) et de PW1 (lot un (1) de perméat avec une concentration en lactose de 34 g/L) a été ajouté au milieu de culture M9 dans les proportions suivantes: 50:50, 75:25 et 100:0 (v/v). L'effet du volume d'inoculum (5, 7.5 et 10.0%, v/v), du pH initial (6.5, 7.0 et 7.4) et de la vitesse d'agitation (50, 100 et 200 rpm) sur le rendement en 2,3-BD a été étudié lors de la fermentation des mélanges. Finalement, un nouveau lot de lactosérum (W2) et de perméat de lactosérum (PW2) avec une concentration de lactose de 51 et 47 g/L, respectivement, ont été fermentés en erlenmeyers de 0.5 L et en bioréacteur de 2 L (conditions anaérobies). L'effect d'air (2 L/min) en bioréacteur de 2 L (conditions micro-aérobies) sur le rendement en 2,3-BD a été testé avec W2.

Le chapitre 5 est la conclusion du travail de thèse. Des perspectives prenant en compte les difficultés rencontrées et les premiers résultats obtenus sont présentées. Contribution to the document: This paper presents a literature review on the generation of whey issued from the cheese manufacturing. Whey can cause diverse environmental issues due to its composition, which depends on the kind of whey. Performing fermentation of whey in order to produce 2,3-butanediol is a solution to avoid the environmental problems caused this kind of biomass. A fermentation process depends on different factors like the kind and concentration of the carbon source, temperature, pH, agitation, etc. Among the operational parameters, one of the most important is the kind and strain of bacteria. In the review, these parameters are presented. Therefore, this review paper is helpful and relevant to the thesis topic.

Biovalorization of saccharides derived from industrial wastes such as whey -A review

Résumé

Le lactosérum est un résidu liquide résultant de la transformation du lait en fromage. Le lactosérum pose un problème environnemental majeur pour l'industrie laitière dû à sa charge organique élevée reliée à sa forte teneur en lactose.

La valorisation du lactosérum peut se faire par des procédés biologiques basés sur la fermentation du lactose en différents produits comme l'acide lactique (utilisés comme additif alimentaire), le 2,3-butanediol (matière première pour obtenir des produits comme la méthyléthyl-cétone utilisée dans les industries pharmaceutique et chimique), le biogaz (pour obtenir de l'énergie). La production de 2,3-butanediol à partir de saccharides tel que le glucose a été étudiée depuis plusieurs années en utilisant plusieurs sortes de micro-organismes comme Enterobacter aerogenes, Panibacillus polymyxa, Klebsiella sp., Serratia marcescens et Escherillia coli dont certains ont été génétiquement modifiés pour améliorer la production de 2,3-butanediol. Le potentiel de fermentation du lactosérum en 2,3-butanediol dépend de plusieurs paramètres opératoires comme la nature des micro-organismes, la composition du milieu de culture, la température, le pH et la teneur en oxygène.

Cet article de revue présente, tout d'abord, un bilan de la production du lait et du fromage au Canada et dans le monde. Puis, il décrit les différentes variétés de lactosérum et leurs techniques de traitement. Enfin, cet article présente la production par catalyse enzymatique du 2,3-butanediol à partir de divers saccharides en présence de microorganismes sous différentes conditions.

Introduction

Dairy industry is one of the most important industries inside the agri-food sector around the world. For instance, in Canada in 2015, the dairy industry generated almost 0.17 billion CAD representing 16% of the total profits generated by the agri-food sector (Canadian Dairy Information Center (CDIC) 2017a). Therefore, the dairy industry not only has an important economic impact but creates thousands of direct and indirect jobs, representing local and regional economic benefits [START_REF] Martínez | La concentración de agroindustrias rurales de producción de quesos en el noroeste del Estado de México: un estudio de caracterización[END_REF]. For example, in 2015 in Canada and Quebec (a province of Canada), the number of people employed in the dairy industry was close to 23000 and 8900, respectively (CDIC 2017b). However, the dairy industry also generates detrimental compounds for the environment such as: (1) greenhouses gases (GHG); mainly methane (CH 4 ) and carbon dioxide (CO 2 ) (FAO 2010); ( 2) liquids (wastewater and whey); around 11000 million m 3 of wastewater/year are generated in the world containing high organic loads (i.e. between 30 and 50 g of biochemical oxygen demand (BOD)/L water) [START_REF] Mukhopadhyay | Whey processing with chitosan and isolation of lactose[END_REF][START_REF] Tikariha | Study of characteristics and treatments of dairy industry waste water[END_REF], which could perturb the aquatic life; and ( 3) solids (manure, a cow produces an average of 62 kg of manure per day (feces and urine)) (Statistic-Canada 2006).

The dairy industry generates effluents when milk is processed. One of the most important products is cheese. One kg of manufactured cheese generates 9 L of whey, the main effluent of dairy industry (Guimaraes et al. 2008;[START_REF] Pintado | Review: technology, chemistry and microbiology of whey cheeses[END_REF]). This must be processed to avoid the deterioration of the ecosystem. For this purpose, whey can be treated by coagulationflocculation [START_REF] Prazeres | Cheese whey management: a review[END_REF]. It can also be upgraded by physicochemical treatments like membranes in order to get proteins (de Wit 2001; [START_REF] Madureira | Bovine whey proteins-overview on their main biological properties[END_REF] or by biological treatments into biogas (hydrogen (H 2 )), lactic acid, and alcohols such as 2,3-butanediol (2,3-BD) [START_REF] Antonopoulou | Biohydrogen and methane production from cheese whey in a two-stage anaerobic process[END_REF][START_REF] Ghaly | Enhanced lactic acid production from cheese whey with nutrient supplement addition[END_REF][START_REF] Guimarães | Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey[END_REF][START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF][START_REF] Huertas | Whey: importance in the food industry[END_REF][START_REF] Ukpai | Comparative study of biogas production from cow dung, cow pea and cassava peeling using 45 L biogas digester[END_REF].

Current status of the dairy industry

Milk for human consumption can be produced from cows, goats, etc. [START_REF] Claeys | Consumption of raw or heated milk from different species: an evaluation of the nutritional and potential health benefits[END_REF]FAO-FAOSTAT 2017c). The worldwide production of milk was around 802 million tons in 2014, around 81% of the global production was cow milk (FAO-FAOSTAT 2017c).

Milk transformation produces a great variety of foods such as butter, yogurt or cheese. Cheese is one of the most consumed products (Muehlhoff et al. 2013). For example, in Canada, more than 1000 kinds of cheese are produced which include cheddar, cottage cheese and mozzarella (CDIC 2017c).

Current status in the world

In 2014, the worldwide production of cow milk was around 656 million tons. Among continents and countries, Europe and United States were respectively the largest global producers of cow milk, 33.1 and 14.3%, respectively (CDIC 2017d;FAO-FAOSTAT 2017c).

In the case of cheese, its worldwide production was 22.6 million tons of cheese produced in 2014 (FAO-FAOSTAT 2017d). Once again, Europe and United States were the largest producers of cheese in the world (52.4 and 24.7% of global cheese production respectively).

Milk and cheese production is an important part of the agro-food industry. Both products present an important role from an economic point of view for every country. For instance, in case of cheese, France exported around 33% of its cheese production [START_REF] Vlahović | International cheese market-current state and perspective[END_REF]).

Hence, this fact in addition to the number of employees needed to process milk and manufacture cheese makes the dairy industry an important worldwide economic support [START_REF] Hirsch | Persistence of firm level profitability in the European dairy processing industry[END_REF][START_REF] Prakash | A framework for sustainable food supply chain: reflections from the indian dairy producers[END_REF].

Current status in Canada and Québec

In 2015, the Canadian milk production was near 1.3% of world milk production, being the 5 th biggest producer of cow milk in the American continent (CDIC 2017e; FAO-FAOSTAT 2017c). Quebec and Ontario were the main producers of milk (36.7 and 32.9% of the milk produced in Canada, respectively) (CDIC 2017e).

In the case of cheese, Canada produced near 1.9% of the world cheese production in 2015, this being the 3 rd biggest producer of cheese in the American continent (FAO-FAOSTAT 2017d). Quebec and Ontario were the biggest producers (50.5 and 27.8%, respectively) of Canadian cheese (CDIC 2017f).

Hence, the dairy industry has a huge economic impact in Canada. For instance, in the particular case of Quebec, the dairy industry represented 38.2% of the number of employees in Canada (23322 in total) in 2015 (CDIC 2017b).

Environmental impacts of the dairy industry

On the other hand, milk production and its transformation cause important environmental impacts such as high consumption of energy and water, as well as emissions of gases, wastewater and solid waste [START_REF] Capper | The environmental impact of dairy production: 1944 compared with 2007[END_REF][START_REF] Saggar | A review of emissions of methane, ammonia, and nitrous oxide from animal excreta deposition and farm effluent application in grazed pastures[END_REF]).

Gases

The main atmospheric pollutants emitted by the dairy industry are: ammonia (NH 3 ), nitrogen dioxide (NO 2 ), nitric oxide (NO) and GHG like CH 4 , CO 2 and nitrous oxide (N 2 O) [START_REF] Saggar | A review of emissions of methane, ammonia, and nitrous oxide from animal excreta deposition and farm effluent application in grazed pastures[END_REF]United-Nations 2006). The gases are mainly due to animals metabolism (e.g., CH 4 ), the energy consumption necessary to operate barns and transport of raw materials and products, in addition to the transformation and preservation processes such as pasteurization, sterilization and refrigeration [START_REF] Bertsch | Efficient production of energy[END_REF]Ramirez et al. 2006). The dairy sector worldwide emissions were near 1970 million tons of CO 2 equivalent in 2007 (4% of the total emissions of GHG) (FAO 2010). Milk production is responsible for 2.6% of global emissions of GHG in 2007 (FAO 2010). In the world, the average emission rates linked to the production of 1 kg of milk and 1 kg of cheese are 1 kg of CO 2 equivalent/kg milk and 8.8 kg CO 2 equivalent/kg cheese [START_REF] Guignard | Environmental/ecological impact of the dairy sector: literature review on dairy products for an inventory of key issues, list of environmental initiative and influences on the dairy sector[END_REF]. Therefore, the dairy industry is an important source of GHG.

Liquid effluents and consumption of water

The )) [START_REF] Cristian | Characteristics of the untreated wastewater produced by food industry[END_REF][START_REF] Demirel | Anaerobic treatment of dairy wastewaters: a review[END_REF][START_REF] Omil | Anaerobic filter reactor performance for the treatment of complex dairy wastewater at industrial scale[END_REF]. In terms of the COD, its value can be higher than 60 g COD/L in streams from dairy products production (e.g., liquid stream from the cheese manufacturing, i.e. the whey) [START_REF] Demirel | Anaerobic treatment of dairy wastewaters: a review[END_REF][START_REF] Omil | Anaerobic filter reactor performance for the treatment of complex dairy wastewater at industrial scale[END_REF].

Another point of consideration in understanding the environmental impact of the dairy industry is the rate of water consumption expressed as water footprint. For example, to produce 1 L of milk and 1 kg of cheese, 1 m 3 and 5 m 3 of fresh water are needed, respectively [START_REF] Guignard | Environmental/ecological impact of the dairy sector: literature review on dairy products for an inventory of key issues, list of environmental initiative and influences on the dairy sector[END_REF][START_REF] Mekonnen | The green, blue and grey water footprint of farm animals and animal products[END_REF]Ridoutt and Pfister 2010).

Solid waste

Manure is the most important solid waste in the dairy industry. Solid cattle manure can contain up to 5 kg of N (NH 4 + , NH 3 ), 2.2 kg of P (phosphorus pentoxide (P 2 O 5 )), and 5 kg of K (potassium oxide (K 2 O)) per metric ton of solid manure [START_REF] Pennington | Nutrient and fertilizer value of dairy manure[END_REF]USEPA, 2012). Therefore, dry manure must be well managed because it can increase the salinity, pH and the concentrations of NH 4 + and NO 3 -in soils and emissions of NH 3 gas by decomposition of manure. Ammonia can cause respiratory illness (CDC 2017c; [START_REF] Lehtomäki | Laboratory investigations on co-digestion of energy crops and crop residues with cow manure for methane production: effect of crop to manure ratio[END_REF][START_REF] Moral | Characterisation of the organic matter pool in manures[END_REF]).

Whey

Whey is the liquid portion of milk which is obtained during the milk coagulation to produce cheese. Whey presents a high organic load (COD around 68 g/L) and thus it should be treated before being released to the environment as wastewater [START_REF] Saddoud | Anaerobic membrane reactor with phase separation for the treatment of cheese whey[END_REF]. Whey can also be valorized in high value added products such as nutritional food (e.g. as an additive for milk intended for children) and used in drugs manufacturing [START_REF] Gunasekaran | Use of whey proteins for encapsulation and controlled delivery applications[END_REF]). In addition, whey can be used to obtain fermentative products such as alcohols (e.g., ethanol or 2,3-BD) or biogas [START_REF] Chatzipaschali | Biotechnological utilization with a focus on anaerobic treatment of cheese whey: current status and prospects[END_REF][START_REF] De Wit | Lecturer's handbook on whey and whey products, 1st edn[END_REF][START_REF] Dragone | Characterisation of volatile compounds in an alcoholic beverage produced by whey fermentation[END_REF][START_REF] Huertas | Whey: importance in the food industry[END_REF][START_REF] Perego | 2,3-Butanediol production by Enterobacter aerogenes: selection of the optimal conditions and application to food industry residues[END_REF]. The valorization of whey depends on its composition which is a result of different factors including (1) the animal source of milk, (2) the kind of animal feed, (3) the stage of lactation, (4) the season of the year, (5) the acidity of whey and, (6) how the cheese is processed [START_REF] Madureira | Bovine whey proteins-overview on their main biological properties[END_REF][START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF][START_REF] Pintado | Review: technology, chemistry and microbiology of whey cheeses[END_REF]. Figure 2.1 shows an overall scheme of the different processes used to valorize the whey produced by the dairy industry.

Whey generation

Figure 2.2 shows a simplified schematic representation of cheese and whey production from milk transformation. Raw milk is treated using a pasteurization process (a thermal process to reduce the population of pathogens). After the pasteurization stage, a culture of microorganisms (e.g., a strain of Lactobacillus or Streptococcus) is inoculated to the pasteurized milk to transform lactose into lactic acid [START_REF] Miranda | Características físicoquímicas de sueros de queso dulce y ácido producidos en el combinado de quesos de Bayamo[END_REF]. Then, the coagulation process is performed. If the coagulation is carried out by rennet (complex of enzymes composed primarily of chymosin and pepsin), a sweet whey is generated [START_REF] Miranda | Hydrolysis of α-lactalbumin by chymosin and pepsin. Effect of conformation and pH[END_REF][START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF]; while if the coagulation process is performed by organic acids (e.g., acetic acid), an acid whey is obtained [START_REF] Pintado | Review: technology, chemistry and microbiology of whey cheeses[END_REF]. During the coagulation process, curd and whey are produced and thus have to be separated. The curd is then washed, cut, and pressed generating additional whey [START_REF] Spalatelu | Biotechnological valorisation of whey[END_REF][START_REF] Türkmenoglu | Organic acids production from cheese-whey[END_REF].

The cheese production has increased worldwide from 1.9x10 7 in 2005, to 2.2x10 7 tons in 2013. Hence, the whey generation around the world is estimated between 1.7x10 11 L in 2005, to near 1.9x10 11 L in 2013 (FAO-FAOSTAT 2017d;[START_REF] Smithers | Whey-ing up the options-Yesterday, today and tomorrow[END_REF]. According to the CDIC (2017f), the production of cheese was 4.3x10 8 kg in Canada in 2013 (i.e. 3.9x10 9 L of whey).

Composition of whey

Whey is mainly composed of water which represents 93-95% (w/w) and contains around 50% of milk nutrients [START_REF] Lievore | Chemical characterisation and application of acid whey in fermented milk[END_REF]. In 1 L of whey, there are between 60 and 70 g of solids (de Wit 2001; [START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF]). The dry matter fraction is composed as follows: 66-77% (w/w) of lactose, 8-15% (w/w) of proteins and 7-15% (w/w) of minerals; and a lower portion of non-protein nitrogen (e.g., amino acids), vitamins (e.g., vitamins A, D and B5), trace elements (e.g., zinc) and proteins (e.g., lactoperoxidase) (Casper et al. 1998;[START_REF] Chegini | Whey powder: process technology and physical properties. A review[END_REF][START_REF] De Wit | Lecturer's handbook on whey and whey products, 1st edn[END_REF][START_REF] Ghaly | Enhanced lactic acid production from cheese whey with nutrient supplement addition[END_REF][START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF]). The proteins of whey are albumins (up to 90% w/w), lactoferrin and immunoglobulins, which represent approximately 20% w/w of milk proteins [START_REF] Marshall | Therapeutic applications of whey protein[END_REF][START_REF] Patel | Emerging trends in nutraceutical applications of whey protein and its derivatives[END_REF]. These proteins have antiviral, antimicrobial and antioxidative properties and thus whey proteins have become a very valued product in the food industry [START_REF] Madureira | Bovine whey proteins-overview on their main biological properties[END_REF][START_REF] Patel | Emerging trends in nutraceutical applications of whey protein and its derivatives[END_REF].

Acidity of whey

Milk coagulation is performed to generate curd and whey. The coagulation process determines the acidity and the composition of whey. Table 2.1 shows the differences between both types of whey depending on the generation process.

1) Sweet whey has a pH value around 6.5 and contains near 70 g/L of dry matter. The composition of dry matter is mainly formed by up to 77% (w/w) of lactose, up to 15% (w/w) of proteins, 15% (w/w) of minerals, up to 3% (w/w) of lactic acid besides trace elements, vitamins and minor proteins (Hernández-Rojas and Vélez-Ruiz 2014; [START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF][START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF][START_REF] Tsakali | A review on whey composition and the methods used for its utilization for food and pharmaceutical products[END_REF]).

2) Acid whey has a pH value, between 4.5 and 4.7 and contains nearly 70 g/L of dry matter mainly formed by up to 72% (w/w) of lactose, up to 15% (w/w) of proteins, up to 15% (w/w) of minerals and up to 13% (w/w) of lactic acid. When comparing sweet and acid wheys, it is possible to observe a smaller amount of lactose present in acid whey. This is due to a higher formation of lactic acid in the acid whey (Hernández-Rojas and Vélez-Ruiz 2014; [START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF][START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF][START_REF] Tsakali | A review on whey composition and the methods used for its utilization for food and pharmaceutical products[END_REF]).

Whey as an environmental problem

Whey is the most polluting substance issued from the manufacture of cheese [START_REF] Prazeres | Cheese whey management: a review[END_REF]. It has been estimated that only 50% of the whey is valorized into different products used in the food and chemical industries [START_REF] Baldasso | Concentration and purification of whey proteins by ultrafiltration[END_REF][START_REF] Koutinas | Development of a hybrid fermentationenzymatic bioprocess for the production of ethyl lactate from dairy waste[END_REF][START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF]). When whey is released as a wastewater, it can cause environmental problems. In order to be able to discharge the effluent in the environment, whey must be treated, for example, by an ozonation treatment with hydrogen peroxide [START_REF] Martins | Final remediation of post-biological treated milk whey wastewater by ozone[END_REF]. This allows lactose and the rest of the organic loads of the whey to be degraded into CO 2 and water (H 2 O) thus decreasing its negative environmental impact.

Processes applied to treat whey

Whey can directly be used as an animal food supplement since it is a source of proteins and minerals, or as a fertilizer without treatment [START_REF] Audic | Non-food applications of milk components and dairy co-products: a review[END_REF][START_REF] Baldasso | Concentration and purification of whey proteins by ultrafiltration[END_REF][START_REF] Schaafsma | Lactose and lactose derivatives as bioactive ingredients in human nutrition[END_REF]. It is also possible to treat whey as a raw material in order to obtain addedvalue products (de Wit 2001; [START_REF] Karadag | A review on anaerobic biofilm reactors for the treatment of dairy industry wastewater[END_REF].

Physical and physicochemical processes

Whey can be treated by coagulation-flocculation using chemical compounds, such as iron (III) chloride (FeCl 3 ) or iron (II) sulfate (FeSO 4 ), to decrease the COD present in the whey. However, it is also possible to use membranes to obtain products (e.g., proteins) from whey [START_REF] Canli | Physico-chemical treatment of cheese whey effluent[END_REF]. Whey protein concentrates (WPC) are used in food such as emulsifier [START_REF] Huertas | Whey: importance in the food industry[END_REF]Rebouillat and Ortega-Requena 2015;[START_REF] Walzem | Whey components: millennia of evolution create functionalities for mammalian nutrition: what we know and what we may be overlooking[END_REF]. Whey possesses fat and casein particles in suspension which can be removed using a microfiltration process to prevent the obstruction of the ultrafiltration membranes [START_REF] Das | Recovery of whey proteins and lactose from dairy waste: a step towards green waste management[END_REF]. The streams after recovering WPC by ultrafiltration is named permeate whey and contains a lactose concentration higher than 80 g/L of permeate [START_REF] Becerra | Biobutanol from cheese whey[END_REF][START_REF] De Wit | Lecturer's handbook on whey and whey products, 1st edn[END_REF][START_REF] Esmaeili | PROKARYO: an illustrative and interactive computational model of the lactose operon in the bacterium Escherichia coli[END_REF]INTI 2017;USDairy 2017). Nanofiltration is also used to remove salts from whey. The non-salts whey can be used as follows: (1) in the food industry because it has a low salt content and thus prevents illnesses, such as hypertension; (2) in the pharmaceutical industry; and (3) in the chemical industry to make sugar-cellulose fibers (de Wit 2001; [START_REF] Minhalma | Optimization of "Serpa" cheese whey nanofiltration for effluent minimization and by-products recovery[END_REF]Rebouillat and Ortega-Requena 2015). The demineralization process can also be used to manufacture additives for milk intended for children [START_REF] Huertas | Whey: importance in the food industry[END_REF]. The removal of lactose from whey, by crystallization or by using hollow fiber membranes, can be used in the pharmaceutical industry to make tablets for people unable to digest lactose; or lactose can be hydrolyzed to produce glucose and galactose which could be valorized by chemical or biological processes [START_REF] Audic | Non-food applications of milk components and dairy co-products: a review[END_REF][START_REF] Das | Recovery of whey proteins and lactose from dairy waste: a step towards green waste management[END_REF][START_REF] De Wit | Lecturer's handbook on whey and whey products, 1st edn[END_REF].

Biological processes

Whey can be fermented to produce biogas, H 2 , lactic acid, and different types of alcohols, such as ethanol, butanol and 2,3-BD (Roncal et al. 2010;[START_REF] Roncal | Biotecnología-La biotecnología en el sector químico (I)-Producción industrial de productos químicos básicos[END_REF].

Biogas

Biogas can be produced by anaerobic digestion of organic compounds [START_REF] Gdjg | Biogás: una alternativa ecológica para la producción de energía[END_REF]. In this regard, whey can be used as feedstock because it contains lactose which could be fermented by microorganisms (e.g., Methanobacterium) under strict anaerobic conditions to produce a biogas containing 50-70% (v/v) of CH 4 , 30-40% (v/v) of CO 2 and 1-10% (v/v) of H 2 [START_REF] Coitavc | Valorización Energética: Producción de biogás mediante codigestión anaerobia de residuos/subproductos orgánicos agroindustriales[END_REF][START_REF] Ukpai | Comparative study of biogas production from cow dung, cow pea and cassava peeling using 45 L biogas digester[END_REF]. The biogas yield depends mainly on the temperature and pH. If bacteria are mesophilic, the optimal temperature is ranged between 30 and 40ºC, while if bacteria are thermophilic, the temperature is ranged between 50 and 60ºC [START_REF] Gerardi | The microbiology of anaerobic digesters, 1st edn[END_REF][START_REF] Khanal | Anaerobic biotechnology for bioenergy production. Chapter 1 -overview of anaerobic biotechnology[END_REF]). In the case of the pH, methanogenic bacteria produce biogas when the pH value is close to neutral [START_REF] Ertem | Improving biogas production by anaerobic digestion of different substrates: calculation of potential energy outcomes[END_REF]. Whey as a culture medium (50 mL in serum bottles) produced around 23 L CH 4 /L whey at 35ºC in 68 days [START_REF] Ergüder | Anaerobic biotransformation and methane generation potential of cheese whey in batch and UASB reactors[END_REF]. In Quebec (Canada), around 200000 m 3 of biogas/year are produced using more than 5 million of liters of whey [START_REF] Chatzipaschali | Biotechnological utilization with a focus on anaerobic treatment of cheese whey: current status and prospects[END_REF].

Hydrogen

Traditionally, H 2 is derived from fuel processing (e.g., hydrocarbon reforming or pyrolysis); or from water (e.g., hydrolysis) but H 2 can also be obtained by anaerobic fermentation. Its production has been widely studied as an energy source in order to substitute fossil fuels due its higher heating value (HHV) of 142 kJ/g (Blanco Londoño and Rodríguez Chaparro 2012; [START_REF] Holladay | An overview of hydrogen production technologies[END_REF][START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF][START_REF] Sinha | An evaluative report and challenges for fermentative biohydrogen production[END_REF]USDE 2017). Nowadays, only 1% of worldwide production of H 2 is produced by anaerobic fermentation using microorganisms such as Clostridia [START_REF] Ferchichi | Influence of initial pH on hydrogen production from cheese whey[END_REF][START_REF] Venetsaneas | Using cheese whey for hydrogen and methane generation in a two-stage continuous process with alternative pH controlling approaches[END_REF][START_REF] Wu | Simultaneous production of 2, 3-butanediol, ethanol and hydrogen with a Klebsiella sp. strain isolated from sewage sludge[END_REF]. For a day of production, 25 L of H 2 per L of whey were produced by means of fermentation in a 2-stage process (1 bar and 22ºC). A yield of 2.8 moles of H 2 /mol of lactose was attained using granular sludge (an aggregate of microorganisms) (Cota-Navarro et al. 2011).

Lactic acid

Lactic acid has several applications in food, pharmaceutical, and chemical industries to give flavor (to increase the sweetness) and texture of products, and as a precursor of biodegradable polymers [START_REF] Narayanan | Lactic acid fermentation and its product polymerization[END_REF][START_REF] Pescuma | Whey fermentation by thermophilic lactic acid bacteria: evolution of carbohydrates and protein content[END_REF][START_REF] Vijayakumar | Recent trends in the production, purification and application of lactic acid[END_REF]. Currently, near 90% of lactic acid produced around the world comes from the fermentation of saccharides by lactic acid bacteria (LAB) [START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF]. These bacteria can use whey and thereafter hydrolyze the lactose in order to produce glucose and galactose [START_REF] Black | Microbiology: principles and explorations[END_REF][START_REF] Pescuma | Whey-derived valuable products obtained by microbial fermentation[END_REF]. Glucose can be transformed into pyruvic acid by means of glycolysis, and the pyruvic acid can be fermented by lactic acid bacteria (e.g., Lactobacillus) into lactic acid [START_REF] Hugenholtz | Metabolic engineering of lactic acid bacteria: overview of the approaches and results of pathway rerouting involved in food fermentations[END_REF]. Therefore, whey is a potential feedstock for fermentation processes due to its high content of lactose. For instance, up to 95% w/v of lactose from the whey can be transformed into lactic acid by Lactobacillus casei at 37ºC, pH 6.5 and 100 rpm, producing 0.84 g lactic acid/g of lactose for a reaction time of 36h [START_REF] Panesar | Production of L (+) lactic acid using Lactobacillus casei from whey[END_REF]. The yield and concentration of lactic acid can be higher using a different species of Lactobacillus. A yield of 0.98 g lactic acid/g lactose was obtained for a reaction time of around 24h at 45ºC and pH 5.0 with L. bulgaricus NRRL B-548 [START_REF] Venkatesh | Kinetic model of growth and lactic acid production from lactose by Lactobacillus bulgaricus[END_REF]), whereas, Burgos-Rubio et al. (2000) found a lactic acid yield near 1.2 g lactic acid/g lactose for a reaction time of around 10h at 42ºC and pH 5.6 using the same bacteria (Burgos [START_REF] Rubio | Kinetic study of the conversion of different substrates to lactic acid using Lactobacillus bulgaricus[END_REF].

Alcohols

Whey can be transformed into alcohols such as ethanol, butanol, 1,3-propanediol, and 2,3-BD [START_REF] Becerra | Biobutanol from cheese whey[END_REF][START_REF] Petrov | High production of 2,3-butanediol from glycerol by Klebsiella pneumoniae G31[END_REF][START_REF] Wu | Simultaneous production of 2, 3-butanediol, ethanol and hydrogen with a Klebsiella sp. strain isolated from sewage sludge[END_REF].

The production of alcohols from whey is based on glucose fermentation; glucose is obtained from enzymatic hydrolysis (performed by enzymes, essentially β-galactosidases, at a temperature between 35 and 50ºC and a pH close to 7) or by chemical hydrolysis of lactose (using an acid, such as sulfuric acid, at a pH lower than 1.5 and a temperature higher than 150ºC) [START_REF] Carrascal | Desarrollo de un estudio que conlleve a la obtención de etanol a partir del lactosuero generado por la industria láctea. II Simposio Internacional y III Nacional Agroalimentario Universidad De Córdoba (Argentina) Facultad De Ciencias[END_REF][START_REF] Chatzipaschali | Biotechnological utilization with a focus on anaerobic treatment of cheese whey: current status and prospects[END_REF][START_REF] Jurado | A new kinetic model proposed for enzymatic hydrolysis of lactose by a β-galactosidase from Kluyveromyces fragilis[END_REF][START_REF] Khajavi | Hydrolysis kinetics of trisaccharides consisting of glucose, galactose, and fructose residues in subcritical water[END_REF][START_REF] Neri | Immobilization of β-galactosidase from Kluyveromyces lactis onto a polysiloxane-polyvinyl alcohol magnetic (mPOS-PVA) composite for lactose hydrolysis[END_REF][START_REF] Oomori | Hydrolysis of disaccharides containing glucose residue in subcritical water[END_REF][START_REF] Pereira-Rodríguez | Structural basis of specificity in tetrameric Kluyveromyces lactis βgalactosidase[END_REF][START_REF] Vieira | Hydrolysis of lactose in whole milk catalyzed by β-galactosidase from Kluyveromyces fragilis immobilized on chitosan-based matrix[END_REF]. The alcoholic fermentation of whey can be economically competitive if concentrated whey is used (higher than 50 kg of lactose/m 3 of whey) [START_REF] Guimarães | Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey[END_REF][START_REF] Prazeres | Cheese whey management: a review[END_REF]. Alcohols are interesting for petrochemical and pharmaceutical industries because they can be transformed into other intermediate or final products such as ethylene, butylamine, poly(trimethylene terephthalate), or methyl ethyl ketone (Roncal et al. 2010;[START_REF] Roncal | Biotecnología-La biotecnología en el sector químico (I)-Producción industrial de productos químicos básicos[END_REF].

A. Ethanol

Lactose can be directly fermented by microorganisms and therefore whey is an excellent option as feedstock to produce ethanol (Padín [START_REF] González | Fermentación alcohólica del lactosuero por Kluyveromyces marxianus y solventes orgánicos como extractantes[END_REF]. The transformation of lactose into ethanol using microorganisms like Kluyveromyces marxianus UFV-3 can produce up to 0.535 g ethanol/g lactose (the theoretical value is 0.538 g ethanol/g lactose), i.e. a yield of 99% (g/g) under hypoxic conditions (injecting N 2 ) at 30ºC (pH and reaction time not specified) [START_REF] Silveira | Ethanol production from cheese whey permeate by Kluyveromyces marxianus UFV-3: a flux analysis of oxido-reductive metabolism as a function of lactose concentration and oxygen levels[END_REF].

B. Biobutanol

Biobutanol encompasses 4 different isomers; iso-and n-butanol are dominantly produced. The other isomers are sec-and tert-butanol [START_REF] Becerra | Biobutanol from cheese whey[END_REF]. Biobutanol has been traditionally produced by acetone-butanol-ethanol (ABE) fermentation [START_REF] Green | Fermentative production of butanol-the industrial perspective[END_REF]. The mass ratio of ABE being 3:6:1 where butanol is the main product [START_REF] Khanal | Anaerobic biotechnology for bioenergy production. Chapter 1 -overview of anaerobic biotechnology[END_REF][START_REF] Lee | Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanolbutanol-ethanol fermentation[END_REF]). This process is carried out in 2 steps: (1) acidogenesis, which produces acetic and butyric acids and (2) solventogenesis, which produces acetone, butanol and ethanol. Diverse types of saccharides (e.g., lactose) have been fermented using a Clostridium strain [START_REF] Nk | Pre-optimization of medium for biobutanol production by a new isolate of solvent-producing Clostridium[END_REF][START_REF] Becerra | Biobutanol from cheese whey[END_REF]. In this case, using Clostridium acetobutylicum, the fermentation process yields 0.44 g ABE/g lactose after 470 h of fermentation at 35ºC, pH 5, and anaerobic conditions (Qureshi and Maddox 2005). However, a yield of 0.26 g butanol/g lactose using whey at 37ºC, pH 4.7 with a mean residence time of around 2h was obtained in a continuous fermentor using Clostridium acetobutylicum DSM792 (Raganati et al. 2013). Butanol possesses a HHV of 36.1 kJ/g, which is higher than the HHV of ethanol (29.8 kJ/g) and can be used as a biofuel [START_REF] Cleveland | Handbook of Energy: diagrams, charts, and tables -section 2 -biomass[END_REF]USDE 2017). Biobutanol is also the precursor of paints and products, such as butyl acrylate [START_REF] Green | Fermentative production of butanol-the industrial perspective[END_REF].

C. 1,3-propanediol

The diol, 1,3-propanediol (1,3-PD), is generally produced by microorganisms (e.g., Clostridia) which use glycerol as a carbon source, because no microorganism is able to ferment glucose into 1,3-PD in a natural way [START_REF] Biebl | Microbial production of 1, 3propanediol[END_REF][START_REF] Patel | The BREW project: medium and long-term opportunities and risks of the biotechnological production of bulk chemicals from renewable resources-the potential of white biotechnology[END_REF]. Therefore, 1,3-PD production from glucose is performed by genetically modified microorganisms e.g. Escherichia coli [START_REF] Roncal | Biotecnología-La biotecnología en el sector químico (I)-Producción industrial de productos químicos básicos[END_REF]). Here, glucose is transformed into glycerol [START_REF] Saxena | Microbial production of 1, 3-propanediol: recent developments and emerging opportunities[END_REF][START_REF] Zeng | Bulk chemicals from biotechnology: the case of 1, 3-propanediol production and the new trends[END_REF]. Using glycerol as a substrate, the yield of 1,3-PD can reach 0.90 g 1,3-PD/g glycerol by a modified strain of E. coli at pH 7 and with a combination of aerobic (0 to 10h) and anaerobic (10 to 40h) conditions at 30 and 42ºC, respectively [START_REF] Tang | Microbial conversion of glycerol to 1,3propanediol by an engineered strain of Escherichia coli[END_REF]. Using glucose as a carbon source, the yield of 1,3-PD reached 0.40 g 1,3-PD/g glucose with an engineered strain of E. coli at 37°C after 60h (pH not defined) [START_REF] Liang | Construction of stress-induced metabolic pathway from glucose to 1, 3-propanediol in Escherichia coli[END_REF].

Therefore, lactose contained in the whey may be used as feedstock to produce 1,3-PD if lactose is previously hydrolyzed into its 2 monosaccharides, glucose and galactose. 1,3-PD is employed to obtain polymers, such as polyesters, polyethers and polyurethanes [START_REF] Xiu | Present state and perspective of downstream processing of biologically produced 1, 3-propanediol and 2, 3-butanediol[END_REF].

D. 2,3-butanediol

The diol, 2,3-BD, can be obtained from petroleum by chemical processes, which present "hard" operating conditions (high temperature, superior to 150ºC, low pH, inferior to 1.5 and expensive catalyzers) [START_REF] Guimarães | Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey[END_REF][START_REF] Jiang | Microbial production of short chain diols[END_REF]. For this reason, the 2,3-BD could be produced by fermentation, a green technology with "softer" conditions of temperature and pH. Saccharides like arabinose, glucose, inulin, lactose, maltodextrin, sucrose could be used as substrates [START_REF] Celińska | Biotechnological production of 2, 3-butanediol-current state and prospects[END_REF][START_REF] Garg | A new method for industrial production of 2, 3-butanediol[END_REF][START_REF] Xiao | Thermophilic fermentation of acetoin and 2, 3-butanediol by a novel Geobacillus strain[END_REF]. Whey can also be used in order to produce 2,3-BD. But, whey has to be previously hydrolyzed into glucose and galactose by microorganisms like Klebsiella [START_REF] Afschar | Microbial production and downstream processing of 2, 3-butanediol[END_REF]Casper et al. 1998;[START_REF] Ghaly | Enhanced lactic acid production from cheese whey with nutrient supplement addition[END_REF][START_REF] Shin | High-yield production of meso-2,3-butanediol from cellodextrin by engineered E. coli biocatalysts[END_REF][START_REF] Wong | Effects of pH and fermentation strategies on 2, 3-butanediol production with an isolated Klebsiella sp. Zmd30 strain[END_REF][START_REF] Xiu | Present state and perspective of downstream processing of biologically produced 1, 3-propanediol and 2, 3-butanediol[END_REF].

The diol, 2,3-BD, can be used as additive for fuels due to its HHV (27.2 kJ/g), which is superior to the HHV of alcohol like methanol (22.7 kJ/g) (Ge et al. 2011; USDE 2017). 2,3-BD is also the precursor of (1) MEK, which is used as a fuel additive, ( 2) 1,3-butanediol, which is used to manufacture polyesters and plasticizers, (3) 2-butene, which is used in butadiene production (Roncal et al. 2010). The biological process to transform 2,3-BD into diverse products has recently attracted the interest of industries since bioprocesses reduce the dependence on petroleum and decrease the CO 2 emissions [START_REF] Savakis | Synthesis of 2, 3-butanediol by Synechocystis sp. PCC6803 via heterologous expression of a catabolic pathway from lactic acid-and enterobacteria[END_REF]).

Metabolic pathway from lactose to 2,3-BD

.3 presents a simplified diagram of the metabolic pathway from lactose to 2,3-BD which starts with the enzymatic hydrolysis of lactose [START_REF] Spalatelu | Biotechnological valorisation of whey[END_REF]. Lactose is hydrolyzed using the β-galactosidase enzyme encoded in the lacZ gene of the lactose operon, which is formed by the genes lacZ, lacY and lacA [START_REF] Alpers | Sequential transcription of the genes of the lactose operon and its regulation by protein synthesis[END_REF]. LacY encodes the enzyme galactoside permease that facilitates the transport of lactose into the cell, while lacA encodes the enzyme thiogalactoside transacetylase where its role is still not fully understood [START_REF] Esmaeili | PROKARYO: an illustrative and interactive computational model of the lactose operon in the bacterium Escherichia coli[END_REF].

Thus, the metabolism of lactose to 2,3-BD starts when the galactoside permease enzyme transports the lactose into the bacteria and the β-galactosidase enzyme breaks down 1 molecule of lactose and produces 2 molecules of monosaccharides (1 molecule of glucose and 1 molecule of galactose) [START_REF] Alpers | Sequential transcription of the genes of the lactose operon and its regulation by protein synthesis[END_REF][START_REF] Black | Microbiology: principles and explorations[END_REF]. Once glucose has been generated, glycolysis begins. In this way, glucose is transformed into pyruvic acid which is transformed into 2,3-BD [START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]. The enzyme α-acetolactate synthase (ALS) transforms pyruvic acid into α-acetolactate. This step is improved at approximately pH 6.0 [START_REF] Ji | Microbial 2, 3-butanediol production: a state-of-the-art review[END_REF]. Then, α-acetolactate is fermented to acetoin by the enzyme α-acetolactate decarboxylase (ALDC). Finally, acetoin is transformed into 2,3-BD by means of the enzyme 2,3-BD dehydrogenase (BDH) [START_REF] Ji | Microbial 2, 3-butanediol production: a state-of-the-art review[END_REF][START_REF] Magee | The microbial production of 2, 3-butanediol[END_REF].

Effect of microorganisms and operating conditions on 2,3-BD production

Fermentation of saccharides to produce 2,3-BD is influenced by different operating conditions (Table 2.2), such as the type of microorganisms, substrate concentration, macro and micronutrients, temperature, pH, residence time, agitation, aeration, etc. [START_REF] Celińska | Biotechnological production of 2, 3-butanediol-current state and prospects[END_REF][START_REF] Hespell | Fermentation of xylan, corn fiber, or sugars to acetoin and butanediol by Bacillus polymyxa strains[END_REF][START_REF] Nakashimada | Enhanced 2, 3butanediol production by addition of acetic acid in Paenibacillus polymyxa[END_REF].

Microorganism

The production of 2,3-BD from different carbon sources like glucose, lactose, sucrose, starch, etc. can be performed by fermentation in the presence of bacteria, such as Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Paenibacillus polymyxa, Serratia marcescens, etc. [START_REF] Fu | Metabolic engineering of Bacillus subtilis for chiral pure meso-2, 3-butanediol production[END_REF][START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF][START_REF] Nakashimada | Optimization of dilution rate, pH and oxygen supply on optical purity of 2, 3-butanediol produced by Paenibacillus polymyxa in chemostat culture[END_REF][START_REF] Perego | 2,3-Butanediol production by Enterobacter aerogenes: selection of the optimal conditions and application to food industry residues[END_REF][START_REF] Perego | Effects of temperature, inoculum size and starch hydrolyzate concentration on butanediol production by Bacillus licheniformis[END_REF][START_REF] Petrov | High production of 2,3-butanediol from glycerol by Klebsiella pneumoniae G31[END_REF][START_REF] Priya | Microbial production of 2, 3butanediol through a two-stage pH and agitation strategy in 150 L bioreactor[END_REF][START_REF] Shi | Microbial production of 2, 3-butanediol by a newlyisolated strain of Serratia marcescens[END_REF][START_REF] Xin | Production of 2,3-butanediol from sucrose by a Klebsiella species[END_REF][START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF]). Table 2.3 presents several characteristics and particularities of these bacteria. [START_REF] Fu | Metabolic engineering of Bacillus subtilis for chiral pure meso-2, 3-butanediol production[END_REF][START_REF] Wulff | Biochemical and molecular characterization of Bacillus amyloliquefaciens, B. subtilis and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv. campestris[END_REF][START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF] Natural producers of 2,3-BD like E. cloacae, K. oxytoca or S. marcescens possess the genes needed to produce the enzymatic machinery to be employed in the 2,3-BD metabolic pathway [START_REF] Ji | Microbial 2, 3-butanediol production: a state-of-the-art review[END_REF]). These natural producers of 2,3-BD gave 2,3-BD yields close to 0.50 g 2,3-BD/g saccharide such as glucose (monosaccharide), sucrose (disaccharide) or inulin (polysaccharide) [START_REF] Gao | Optimization of medium for one-step fermentation of inulin extract from Jerusalem artichoke tubers using Paenibacillus polymyxa ZJ-9 to produce R, R-2, 3-butanediol[END_REF][START_REF] Shi | Microbial production of 2, 3-butanediol by a newlyisolated strain of Serratia marcescens[END_REF][START_REF] Silveira | Production of 2,3-butanediol from sucrose by Klebsiella pneumoniae NRRL B199 in batch and fed-batch reactors[END_REF]. For example, a 2,3-BD yield of 0.49 g 2,3-BD/g glucose (calculated using the data provided by the authors) at 35ºC, pH 6.0 and 400 rpm was obtained in the presence of S. marcescens G12 (5% (v/v) of inoculum) after 68 h of fermentation. The aeration rate varied between 1 and 2.5 vvm using a working volume of 5 L (fed-batch fermentor and substrate concentration undefined) [START_REF] Shi | Microbial production of 2, 3-butanediol by a newlyisolated strain of Serratia marcescens[END_REF]. A 2,3-BD yield of 0.49 g 2,3-BD/g sucrose was obtained with K. pneumoniae NRRL B199 (10% (v/v) of inoculum) after 34.5 h of batch fermentation using 95 g/L of sucrose at 37ºC, pH 5.5, 0.8 vvm and 230 rpm in a fermentor of 2 L [START_REF] Silveira | Production of 2,3-butanediol from sucrose by Klebsiella pneumoniae NRRL B199 in batch and fed-batch reactors[END_REF]. Using a response surface methodology, [START_REF] Gao | Optimization of medium for one-step fermentation of inulin extract from Jerusalem artichoke tubers using Paenibacillus polymyxa ZJ-9 to produce R, R-2, 3-butanediol[END_REF] found that P. polymyxa ZJ-9 (8% (v/v) of inoculum) gave a similar yield (0.48 g 2,3-BD/g inulin) in a batch reactor (3 L of working volume) after 48 h fermenting 77 g/L of inulin at 30ºC, pH 6.0 and 240 rpm (0-24 h) and 120 rpm (24-48 h). Interestingly, E. aerogenes and E. cloacae can also ferment agro industrial wastes such as lactose derived from whey or a mixture of glucose and xylose. For example, E. aerogenes NCIMB 10102 (0.25 g/L of inoculum) fermented 20 g/L of pre-hydrolyzed whey in a working volume of 1.5 L, which resulted to a yield of 0.43 g 2,3-BD/g pre-hydrolyzed whey at 39ºC, pH 6.0, 150 rpm and 199 h of batch fermentation [START_REF] Perego | 2,3-Butanediol production by Enterobacter aerogenes: selection of the optimal conditions and application to food industry residues[END_REF]. On the other hand, E. cloacae SDM09 (5% (v/v) of inoculum) provided a yield of 0.50 g 2,3-BD/g glucose:xylose (3:1 (w/w), mixture concentration undefined) (calculated using the data provided by the authors) in a 1 L of fed-batch fermentor at 30ºC, pH 7.0, 1 vvm and 500 rpm for 50 h [START_REF] Li | Metabolic engineering of Enterobacter cloacae for high-yield production of enantiopure (2R, 3R)-2, 3-butanediol from lignocellulose-derived sugars[END_REF].

Klebsiella strains like K. pneumoniae and K. oxytoca are two of the most used microorganisms in the literature to produce 2,3-BD because they can generate high concentrations of 2,3-BD (up to 150 g 2,3-BD/L representing, for example, a 2,3-BD yield of 0.48 g 2,3-BD/g glucose) [START_REF] Ma | Enhanced 2, 3-butanediol production by Klebsiella pneumoniae SDM[END_REF]. They are also easy to cultivate and they grow relatively quickly (cell duplication time shorter than 1h) [START_REF] Białkowska | Strategies for efficient and economical 2,3-butanediol production: new trends in this field[END_REF][START_REF] Black | Microbiology: principles and explorations[END_REF]). However, the main disadvantage of Klebsiella is its biosafety level 2, i.e., it is a human pathogen microorganism [START_REF] Lu | Identification of factors regulating Escherichia coli 2, 3-butanediol production by continuous culture and metabolic flux analysis[END_REF]. Other promising 2,3-BD producers like E. aerogenes, E. cloacae and S. marcescens also possess biosafety level 2 (CDC 2017b; [START_REF] Tong | Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production[END_REF]. Therefore, the use of these bacteria for producing 2,3-BD at industrial scale is limited.

To overcome the biosecurity issue of Enterobacter, Klebsiella and Serratia strains, the 2,3-BD can be obtained by using genetically engineered E. coli. A wide number of E. coli strains, like E. coli W3110, K-12 or BL21, are harmless for humans with a biosafety level 1 (ABSA 2017;ATCC 2016;CDC 2017a;[START_REF] Fukiya | Extensive genomic diversity in pathogenic Escherichia coli and Shigella strains revealed by comparative genomic hybridization microarray[END_REF]iGEM 2017;[START_REF] Jin | Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157[END_REF][START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]).

Nevertheless, E. coli does not have the genes budB, budA and budC which encode the enzymes α-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC), and 2,3butanediol dehydrogenase (BDH) respectively required to produce 2,3-BD [START_REF] Shin | High-yield production of meso-2,3-butanediol from cellodextrin by engineered E. coli biocatalysts[END_REF][START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]. It is therefore necessary to add the 2,3-BD metabolic pathway into E. coli genome from other microorganisms like P. polymyxa or E. cloacae [START_REF] Lara | Producción de proteínas recombinantes en Escherichia coli[END_REF][START_REF] Portnoy | Aerobic fermentation of D-glucose by an evolved cytochrome oxidase-deficient Escherichia coli strain[END_REF][START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]). E. coli presents several advantages: (1) a wide knowledge of its genome, (2) an easy genetic manipulation to add metabolic pathways from other bacteria; and

(3) a wide and available number of substrates for fermentation (like glucose, lactose, starch, etc.) to produce 2,3-BD [START_REF] Tong | Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production[END_REF][START_REF] Wendisch | The flexible feedstock concept in Industrial Biotechnology: metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources[END_REF]. Moreover, it is also possible to block some metabolic pathways which produce end-products, such as acetate, ethanol, lactate and succinate, during the fermentation. The removal of these metabolic routes can affect the 2,3-BD yield produced by genetically modified E. coli in comparison to natural producer bacteria (e.g. E. cloacae, K. pneumoniae or S. marcescens) [START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF].

A 2,3-BD yield of 0.44 g 2,3-BD/g glucose was obtained after 17 h at a pH of 7.0, 1 vvm and 400 rpm in a batch reactor (0.8 L of working volume; glucose concentration undefined) in the presence of E. coli BL21 (10% (v/v) of inoculum) [START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]). E. coli YJ3 (10% (v/v) of inoculum) gave a 2,3-BD yield of 0.38 g 2,3-BD/g glucose after 18 h, fermenting 80 g/L of glucose at 37ºC, pH 6.5, 1.5 vvm and 400 rpm in a batch reactor (2 L of working volume) [START_REF] Tong | Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production[END_REF]. The modified strains of E. coli are potential 2,3-BD producers.

Furthermore, the production of specific 2,3-BD isomers ((S, S)-, meso-and (R, R)-2,3-BD) can also direct the selection of the bacteria. These isomers have different applications. For instance, R, R-2,3-BD is used in the production of antifreezes [START_REF] Qi | Deletion of meso-2,3butanediol dehydrogenase gene bud C for enhanced D-2,3-butanediol production in Bacillus licheniformis[END_REF], while meso-2,3-BD is dehydrated to obtain 1,3-butadiene, involved in the polyesters synthesis [START_REF] Qiu | Engineering Bacillus licheniformis for the production of meso-2,3-butanediol[END_REF]) and S, S-2,3-BD is dehydrogenated to synthesize butanedione [START_REF] Białkowska | Strategies for efficient and economical 2,3-butanediol production: new trends in this field[END_REF]. For example, a specific 2,3-BD isomer like R, R-2,3-BD can be obtained with E. coli by cloning the gene encoding the R, R-2,3-BDH enzyme obtained from B. licheniformis [START_REF] Jiang | Microbial production of short chain diols[END_REF]). In the presence of Klebsiella strains and S. marcescens, a mixture of 2,3-BD isomers can be produced, whereas the isomers (R, R)-and meso-2,3-BD can be formed at a purity of 98 and 99% (w/w) with P. polymyxa and B. subtilis respectively [START_REF] Fu | Metabolic engineering of Bacillus subtilis for chiral pure meso-2, 3-butanediol production[END_REF][START_REF] Ji | Microbial 2, 3-butanediol production: a state-of-the-art review[END_REF][START_REF] Ji | Constructing a synthetic metabolic pathway in Escherichia coli to produce the enantiomerically pure (R, R) 2, 3 butanediol[END_REF][START_REF] Jiang | Microbial production of short chain diols[END_REF].

The operating conditions influencing the 2,3-BD production like substrate concentration, macro and micronutrients, temperature, pH, residence time, agitation and aeration will be presented in the following subsections.

Influence of the substrate

The substrate can be a monosaccharide, a disaccharide or a polysaccharide like glucose, lactose and inulin, respectively. These saccharides can be transformed into 2,3-BD by bacteria and the price of 2,3-BD is strongly linked to the raw material (carbon substrate) cost [START_REF] Celińska | Biotechnological production of 2, 3-butanediol-current state and prospects[END_REF]. The food industry residues like corncob molasses and whey are good sources of saccharides since they are cheap, available and renewable [START_REF] Ji | Microbial 2, 3-butanediol production: a state-of-the-art review[END_REF]). Although, it could be necessary to perform a pretreatment at high temperature and in presence of acid in order to break the saccharide structure [START_REF] Białkowska | Strategies for efficient and economical 2,3-butanediol production: new trends in this field[END_REF]. On the other hand, the substrate concentration is also an important factor on the 2,3-BD production. The substrate could cause osmotic stress in bacterial cells if its concentration is higher than the one inside the cell [START_REF] Krämer | Bacterial stimulus perception and signal transduction: response to osmotic stress[END_REF].

Each microorganism possesses a substrate concentration threshold and beyond this value, an inhibition of metabolism or growth could occur [START_REF] Priya | Microbial production of 2, 3butanediol through a two-stage pH and agitation strategy in 150 L bioreactor[END_REF]. For example, a range of sucrose concentration varying between 20 and 204 g/L was fermented in the presence of K. pneumoniae NRRL B199 (10% (v/v) of inoculum) at 37ºC, pH 5.5, 230 rpm and 0.8 vvm in a fermentor of 2 L [START_REF] Silveira | Production of 2,3-butanediol from sucrose by Klebsiella pneumoniae NRRL B199 in batch and fed-batch reactors[END_REF]). In the range from 20 to 95 g/L of sucrose, the 2,3-BD yield increased by 48% (from 0.33 to 0.49 g 2,3-BD/g sucrose); whereas the fermentation time increased by 3 times (from 11.4 and 34.5 h fermenting 20 and 95 g/L, respectively). Nevertheless, when the sucrose concentration reached 204 g/L, the 2,3-BD yield and the fermentation time decreased by 16% and increased by 132% respectively (0.41 g 2,3-BD/g sucrose and 80 h) [START_REF] Silveira | Production of 2,3-butanediol from sucrose by Klebsiella pneumoniae NRRL B199 in batch and fed-batch reactors[END_REF]. [START_REF] Xin | Production of 2,3-butanediol from sucrose by a Klebsiella species[END_REF] also fermented sucrose in a range between 50 and 300 g/L in the presence of Klebsiella sp. XMR21 (2% (v/v) of inoculum) at 35ºC, pH 6.0 and 150 rpm in 0.05 L of culture medium during 48 h. The maximum 2,3-BD yield (0.43 g 2,3BD/g sucrose) was obtained using 50 and 100 g/L of sucrose. In the presence of 150 or 200 g/L of sucrose, the 2,3-BD yield dropped by 42% (0.25 g 2,3BD/g sucrose for both concentrations). Finally, the 2,3-BD yield continued falling down to 0.14 g 2,3BD/g sucrose at 300 g/L of sucrose.

The substrate threshold concentration might depend also on the substrates. For example, a range of maltodextrin (a polysaccharide) concentrations varying between 50 and 250 g/L, was fermented for 48 and 72 h respectively in the presence of K. oxytoca KMS005 (0.033 g/L of inoculum) at 37ºC, pH 6.0, 1 vvm and 400 rpm [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF]. For an increase of the maltodextrin concentration ranging from 50 to 200 g/L, the 2,3-BD yield increased up to 0.37 g 2,3-BD/g maltodextrin at 200 g/L of maltodextrin and decreased by 1.3 fold in the presence of 250 g/L of maltodextrin (calculated from the data provided by the authors).

On the other hand, certain bacterial strain can maximize the 2,3-BD yield at a low substrate concentration like 20 or 30 g/L. At a 30 g/L of glucose in the presence of E. cloacae SG1 (2% (v/v) of inoculum) at 30ºC (an initial pH of 6.5 and 200 rpm during 48 h), the 2,3-BD yield (0.48 g 2,3-BD/g glucose) increased by 27% compared to 60 g/L of glucose [START_REF] Hazeena | Evaluation of oil palm front hydrolysate as a novel substrate for 2, 3-butanediol production using a novel isolate Enterobacter cloacae SG1[END_REF]. In the presence of E. aerogenes NCIMB 10102 (0.25 g/L) a maximum 2,3-BD yield of 0.40 g 2,3-BD/g glucose was obtained by fermenting 20 g/L of glucose which increased by 18% compared to 100 g/L of glucose. The operating conditions were 39ºC, pH 6.0, 150 rpm and 1.5 L of fermentation medium [START_REF] Perego | 2,3-Butanediol production by Enterobacter aerogenes: selection of the optimal conditions and application to food industry residues[END_REF].

Influence of the compounds stimulating of the process

The fermentation of saccharides into 2,3-BD can be enhanced by adding nutrients like a nitrogen source (for instance yeast extract (YE)), phosphate sources like potassium monohydrogen phosphate (K 2 HPO 4 ) or ammonium hydrogen phosphate ((NH 4 ) 2 HPO 4 ), metal ions like magnesium (Mg 2+ ), manganese (Mn 2+ ) and cobalt (Co 2+ ), etc. [START_REF] Gao | Optimization of medium for one-step fermentation of inulin extract from Jerusalem artichoke tubers using Paenibacillus polymyxa ZJ-9 to produce R, R-2, 3-butanediol[END_REF][START_REF] Häßler | Enhanced fed-batch fermentation of 2, 3-butanediol by Paenibacillus polymyxa DSM 365[END_REF][START_REF] Nakashimada | Enhanced 2, 3butanediol production by addition of acetic acid in Paenibacillus polymyxa[END_REF][START_REF] Wong | Producing 2, 3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain[END_REF]).

a) Nitrogen

Yeast extract is a nitrogen source and it also provides essential substances like vitamins and amino acids needed for the microorganism development [START_REF] Garg | A new method for industrial production of 2, 3-butanediol[END_REF]. Yeast extract contains methionine, which is not synthesized by bacteria like P. polymyxa and could increase the 2,3-BD yield [START_REF] Li | Medium optimization and proteome analysis of (R, R)-2, 3-butanediol production by Paenibacillus polymyxa ATCC 12321[END_REF]). In addition, methionine is an essential amino acid for microorganisms and the precursor of proteins [START_REF] Garg | A new method for industrial production of 2, 3-butanediol[END_REF][START_REF] Levine | Oxidation of methionine in proteins: roles in antioxidant defense and cellular regulation[END_REF][START_REF] Sm | Science Museum -How are proteins made[END_REF]. Two concentrations of YE (5 and 60 g/L) were used in a fed-batch fermentor of 2 L (sucrose concentration undefined) in the presence of P. polymyxa DSM 365 (10% (v/v)) at 37ºC, pH 6.0, 500 rpm and 0.2 vvm for 54 h. The addition of 60 g/L of YE doubled the 2,3-BD productivity (up to 2.06 g 2,3-BD/(L•h)) in comparison with 5 g/L of YE (0.98 g 2,3-BD/(L•h)) [START_REF] Häßler | Enhanced fed-batch fermentation of 2, 3-butanediol by Paenibacillus polymyxa DSM 365[END_REF].

The price of YE being high, other nitrogen sources like urea were tested [START_REF] Ji | Microbial 2, 3-butanediol production: a state-of-the-art review[END_REF][START_REF] Wong | Producing 2, 3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain[END_REF]. For example, [START_REF] Wong | Producing 2, 3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain[END_REF] tested Klebsiella sp. Zmd30 (2% (v/v) of inoculum) with urea concentrations varying between 0 and 10 g/L at 30ºC and 200 rpm in 0.1 L of fermentation medium with 160 g/L of glucose. In the absence of urea, the 2,3-BD yield was practically null (0.02 g 2,3-BD/g glucose), while by adding 2 g/L of urea, the 2,3-BD yield reached 0.35 g 2,3-BD/g glucose. Furthermore, the 2,3-BD yield remained constant at 0.30 g 2,3-BD/g glucose with the urea concentrations varying from 4 to 10 g/L [START_REF] Wong | Producing 2, 3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain[END_REF]. However, the urea concentration needed to maximize the 2,3-BD yield could be higher than 2 g/L. For example, Ji et al. (2009b) reported a 2,3-BD yield of 0.39 g 2,3-BD/g glucosexylose using K. oxytoca ME-303 (5% (v/v) of inoculum) in the presence of 26.4 g/L of urea (ranged from 10 to 30 g/L) at 37ºC, pH 6.5 and 200 rpm in 2 L of basal medium supplemented with 60 g/L of a mixture of glucose-xylose (ratio of 2:1 (w/w)) (Ji et al. 2009b). Hence, the urea can be considered as an alternative nitrogen source to YE in order to produce 2,3-BD.

b) Phosphate

Phosphates like (NH 4 ) 2 HPO 4 and K 2 HPO 4 improve the 2,3-BD yield because: (1) they affect the performance of enzymes (e.g., ALS) involved in 2,3-BD production and ( 2) stimulate the metabolism of bacteria [START_REF] Garg | A new method for industrial production of 2, 3-butanediol[END_REF]Ji et al. 2009b;[START_REF] Jiayang | Production of 2, 3butanediol by Klebsiella pneumoniae using glucose and ammonium phosphate[END_REF][START_REF] Ma | Enhanced 2, 3-butanediol production by Klebsiella pneumoniae SDM[END_REF][START_REF] Sikdar | Expression of a gene encoding acetolactate synthase from rice complements two ilvH mutants in Escherichia coli[END_REF]. For example, using an experimental design approach, [START_REF] Jiayang | Production of 2, 3butanediol by Klebsiella pneumoniae using glucose and ammonium phosphate[END_REF] tested the influence of (NH 4 ) 2 HPO 4 on the yield of 2,3-BD. Increasing the (NH 4 ) 2 HPO 4 concentration 4 times (up to 24 g/L) in the presence of K. pneumoniae CICC 10011 (5% (v/v) of inoculum), the 2,3-BD yield enhanced (from 0.02 to 0.39 g 2,3-BD/g glucose). The fermentation conditions were: 37ºC and pH 6.0 in a 0.1 L of fermentation medium supplemented with 135 g/L of glucose during 55 h.

On the other hand, using an experimental design approach with response surface analysis, [START_REF] Gao | Optimization of medium for one-step fermentation of inulin extract from Jerusalem artichoke tubers using Paenibacillus polymyxa ZJ-9 to produce R, R-2, 3-butanediol[END_REF] studied the influence of K 2 HPO 4 and inulin concentrations on the 2,3-BD yield in the presence of P. polymyxa ZJ-9 (8% (v/v) of inoculum) in a working volume of 3 L. A variation of the K 2 HPO 4 and inulin concentrations (from 1 to 3.1 g/L and from 60 to 77 g/L respectively), improved the 2,3-BD yield by around 30% (from 0.37 to 0.48 g 2,3-BD/g inulin) at 30ºC and pH 6.0; the agitation rate was 240 rpm from 0 to 24 h and 120 rpm from 24 to 48 h [START_REF] Gao | Optimization of medium for one-step fermentation of inulin extract from Jerusalem artichoke tubers using Paenibacillus polymyxa ZJ-9 to produce R, R-2, 3-butanediol[END_REF].

c) Metal ions

As phosphate, metal ions affect the activity of enzymes like ALS, ALDH and BDH and stimulate the metabolism of bacteria enhancing the 2,3-BD yield [START_REF] Garg | A new method for industrial production of 2, 3-butanediol[END_REF]Ji et al. 2009b;[START_REF] Jiayang | Production of 2, 3butanediol by Klebsiella pneumoniae using glucose and ammonium phosphate[END_REF][START_REF] Ma | Enhanced 2, 3-butanediol production by Klebsiella pneumoniae SDM[END_REF][START_REF] Sikdar | Expression of a gene encoding acetolactate synthase from rice complements two ilvH mutants in Escherichia coli[END_REF]. For example, [START_REF] Wong | Producing 2, 3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain[END_REF] tested the metal ions concentration (Mn 2+ and Co 2+ ) influence on the 2,3-BD productivity in the presence of Klebsiella sp. Zmd30 (2% (v/v) of inoculum) at 30ºC and 200 rpm in a 0.1 L of fermentation medium supplemented with 160 g/L of glucose (the 2,3-BD yield was not mentioned). The 2,3-BD productivity was almost tripled (from 0.41 to 1.15 g 2,3-BD/(L•h)) when the concentration of MnCl 2 .4H 2 O was decreased from 2.2 to 0.7 mg/L and the CoCl 2 .6H 2 O concentration was increased from 5.3 to 5.7 mg/L. The effect of Mg 2+ on the 2,3-BD yield was studied using a surface response experiment design in the presence of K. pneumoniae CICC 10011 (5% (v/v) of inoculum) at 37ºC and pH 6.0 in a 0.1 L of fermentation medium supplemented with 135 g/L of glucose for 55 h. A MgSO 4 .7H 2 O concentration of around 0.9 g/L provided a 2,3-BD yield of 0.39 g 2,3-BD/g glucose; without Mg 2+ , the 2,3-BD yield was 0.16 g 2,3-BD/g glucose [START_REF] Jiayang | Production of 2, 3butanediol by Klebsiella pneumoniae using glucose and ammonium phosphate[END_REF]).

Nevertheless, not all the strains are affected by metal ions. For example, [START_REF] Gao | Optimization of medium for one-step fermentation of inulin extract from Jerusalem artichoke tubers using Paenibacillus polymyxa ZJ-9 to produce R, R-2, 3-butanediol[END_REF] reported that the concentration of Mn 2+ had no effect on 2,3-BD yield. A similar conclusion was observed by Ji et al. (2009b). Therefore, metal ions could increase the 2,3-BD yield, but their influence seems to depend on the strain.

d) Acetic acid

Acetic acid increases the concentration of protons which participate in the transformation of acetoin (2,3-BD precursor) to 2,3-BD [START_REF] Li | Medium optimization and proteome analysis of (R, R)-2, 3-butanediol production by Paenibacillus polymyxa ATCC 12321[END_REF][START_REF] Nakashimada | Enhanced 2, 3butanediol production by addition of acetic acid in Paenibacillus polymyxa[END_REF]. For example, [START_REF] Wang | The implementation of high fermentative 2, 3-butanediol production from xylose by simultaneous additions of yeast extract, Na 2 EDTA, and acetic acid[END_REF] studied the influence of acetic acid on 2,3-BD yield. The fermentation of xylose (90 g/L) was performed with an acetic acid concentration varying between 0 and 6.5 g/L in the presence of K. pneumoniae HR521 LDH (2% (v/v) of inoculum) at 37ºC, pH 6.5 and 200 rpm in a 0.1 L of culture medium for 48 h of fermentation. In the range from 0 to 4.5 g/L of acetic acid, the 2,3-BD yield increased by 22% (from 0.32 to 0.39 g 2,3-BD/g xylose).

From 4.5 to 6.5 g/L of acetic acid, the 2,3-BD yield was constant. In addition, a mixture of acetic acid (2.5 g/L) and YE (35.2 g/L) in the presence of K. pneumoniae HR521 LDH (5% (v/v) of inoculum) for 36 h at 37ºC, pH 6.5, 300 rpm and 2.5 vvm in a working volume of 4 L (90 g/L of xylose), raised the 2,3-BD yield up to 0.47 g 2,3-BD/g xylose [START_REF] Wang | The implementation of high fermentative 2, 3-butanediol production from xylose by simultaneous additions of yeast extract, Na 2 EDTA, and acetic acid[END_REF]).

The same conclusion was observed by [START_REF] Anvari | Enhancement of 2, 3-butanediol production by Klebsiella oxytoca PTCC 1402[END_REF] using an experimental design in the presence of K. oxytoca PTCC 1402 (8 g/L of inoculum) at 37ºC, pH 6.0 and 150 rpm in a 0.1 L of fermentation medium containing 6 g/L of glucose. The use of acetic acid at a concentration of 0.5 g/L enhanced the 2,3-BD yield up to 0.47 g 2,3-BD/g glucose [START_REF] Anvari | Enhancement of 2, 3-butanediol production by Klebsiella oxytoca PTCC 1402[END_REF]. However, the acetic acid can be inhibitory for bacteria like E. coli [START_REF] García | Estrategias de obtención de proteínas recombinantes en Escherichia coli[END_REF][START_REF] Wlaschin | Fedbatch culture and dynamic nutrient feeding[END_REF].

Influence of the temperature

The temperature of the fermentation medium affects the enzymatic activity and therefore the growth of microorganisms [START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF]. Metabolic activity of microorganisms decreases at a low temperature increasing the fermentation time. On the other hand, an enzymatic denaturation or a cellular breakdown can occur at a high temperature (e.g., > 50ºC in the presence of mesophilic microorganisms) thus preventing the 2,3-BD synthesis [START_REF] Garg | A new method for industrial production of 2, 3-butanediol[END_REF].

In order to produce 2,3-BD, most of studies tested mesophilic microorganisms able to grow in a temperature range between 20 and 50ºC; the optimal temperature range was usually between 30 and 40ºC (Boundless 2016a; [START_REF] Xiao | Thermophilic fermentation of acetoin and 2, 3-butanediol by a novel Geobacillus strain[END_REF]. For example, E. coli, a mesophilic microorganism, grows between 21 and 49ºC, having its optimal growth temperature in the vicinities of 37ºC [START_REF] Ferrer | Chaperonins govern growth of Escherichia coli at low temperatures[END_REF].

The growth temperature may be similar under different conditions for a bacterial genus like Bacillus. For example, 150 g/L of glucose (0.05 L of culture medium) was fermented between 25 and 40ºC in the presence of B. amyloliquefaciens B10-127 (4% (v/v) of inoculum) at an initial pH of 6.5 and 160 rpm. At 25ºC, the 2,3-BD yield was 0.18 g 2,3-BD/g glucose after 172 h of fermentation. However, a maximum 2,3-BD yield of 0.39 g 2,3-BD/g glucose (117% higher than at 25ºC) was obtained at 37ºC after 82 h of fermentation [START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF]. A similar temperature (37ºC) was reported by [START_REF] Perego | Effects of temperature, inoculum size and starch hydrolyzate concentration on butanediol production by Bacillus licheniformis[END_REF]. A maximum 2,3-BD yield of 0.11 g 2,3-BD/g glucose (calculated from data provided by the authors) was obtained in the presence of B. licheniformis NCIMB 8059 (0.5 g/L of inoculum) in a working volume of 1.5 L containing 20 g/L of glucose at a pH of 6.0 and 150 rpm (undefined time). On the other hand, a temperature superior to 40ºC could also be appropriate for the bacterial growth of some mesophilic bacteria. B. subtilis 168 (2% (v/v) of inoculum), was grown from 37 to 50ºC at an initial pH of 6.5, 100 rpm and 10 g/L of glucose in 0.1 L (time undefined). Between 37 and 46ºC, a 2,3-BD yield of 0.35 g 2,3-BD/g glucose was obtained (calculated from data provided by the authors); the yield decreased to 0.25 g 2,3-BD/g glucose at 50°C [START_REF] Fu | Metabolic engineering of Bacillus subtilis for chiral pure meso-2, 3-butanediol production[END_REF].

Influence of the pH

During fermentation, the production of alcohols like 2,3-BD increases under acid conditions, whereas the production of organic acids is induced under alkaline conditions [START_REF] Garg | A new method for industrial production of 2, 3-butanediol[END_REF]. In this way, if the pH of the fermentation medium is not controlled, the carbon source is transformed into organic acids like acetic acid and succinic acid and, consequently, the medium becomes acid [START_REF] Lu | Identification of factors regulating Escherichia coli 2, 3-butanediol production by continuous culture and metabolic flux analysis[END_REF][START_REF] Van Houdt | Quorum-sensing-dependent switch to butanediol fermentation prevents lethal medium acidification in Aeromonas hydrophila AH-1N[END_REF][START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]. In order to prevent the intracellular bacterial acidification, the formation of alcohols like 2,3-BD increases [START_REF] Hazeena | Evaluation of oil palm front hydrolysate as a novel substrate for 2, 3-butanediol production using a novel isolate Enterobacter cloacae SG1[END_REF]). Hence, the pH of the fermentation medium activates or deactivates the enzymes involved in the different metabolic pathways (e.g. ALS, ALDH and BDH enzymes involved in the 2,3-BD formation) [START_REF] Celińska | Biotechnological production of 2, 3-butanediol-current state and prospects[END_REF][START_REF] Garg | A new method for industrial production of 2, 3-butanediol[END_REF][START_REF] Hazeena | Evaluation of oil palm front hydrolysate as a novel substrate for 2, 3-butanediol production using a novel isolate Enterobacter cloacae SG1[END_REF][START_REF] Priya | Microbial production of 2, 3butanediol through a two-stage pH and agitation strategy in 150 L bioreactor[END_REF]Voloch et al. 1985;[START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF][START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF].

A pH close to 7.0 is usually required by bacteria like Enterobacter or Escherichia to generate a maximum 2,3-BD yield. For example, E. cloacae SG1 (2% (v/v) of inoculum) grew in a starting pH ranged between 5.0 and 8.0 at 30ºC, 200 rpm and 30 g/L of glucose for 48 h. In the range of pH from 5.0 to 6.5, the 2,3-BD yield improved from 0.02 to 0.47g 2,3-BD/g glucose. However, in the range of pH from 6.5 to 8.0, the 2,3-BD yield declined by 53% (from 0.47 to 0.22 g 2,3-BD/g glucose) [START_REF] Hazeena | Evaluation of oil palm front hydrolysate as a novel substrate for 2, 3-butanediol production using a novel isolate Enterobacter cloacae SG1[END_REF]. A pH close to 7.0 was also observed for E. coli YJ3 (10% (v/v) of inoculum). A maximum 2,3-BD yield of 0.38 g 2,3-BD/g glucose was obtained at a pH of 6.5, 37ºC, 1.5 vvm and 400 rpm with 80 g/L of glucose for 18 h [START_REF] Tong | Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production[END_REF]).

However, some authors obtained the maximum yield of 2,3-BD under acid or alkaline pH. A maximum 2,3-BD yield of 0.40 g 2,3-BD/g arabinose was obtained at a pH of 5.1, 30ºC and 200 rpm for 32 h with E. cloacae NRRL B-23289 (4% (v/v) of inoculum) in the presence of 50 g/L of arabinose [START_REF] Saha | Production of 2, 3-butanediol by newly isolated Enterobacter cloacae[END_REF]. On the other hand, the maximum 2,3-BD yield (0.43 g 2,3-BD/g glucose) was obtained at a pH of 8.0 for 18 h in the presence of E. coli BL21 (10% (v/v) of inoculum) at 1 vvm and 400 rpm (glucose concentration undefined) [START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]).

Influence of the fermentation time

The diol 2,3-BD, a secondary metabolite produced by bacteria and not involved in the bacterial development, is generated during the stationary phase, i.e., when the substrate begins to be depleted (Boundless 2016b; [START_REF] Köpke | 2, 3-Butanediol production by acetogenic bacteria, an alternative route to chemical synthesis, using industrial waste gas[END_REF]UNAC 2017).

The fermentation time depends on the beginning of the stationary phase inherent to each bacteria. In addition, the fermentation time depends also on: (1) the concentration of the carbon source in the culture medium, ( 2) the pH, (3) the temperature, etc. For example, the growths of K. oxytoca ME-UD-3 and K. oxytoca ME-XJ-8 were compared in terms of 2,3-BD yield at 37ºC, pH 6.5, 1 vvm and 200 rpm and 5% (v/v) of inoculum for 60 h in a culture medium of 2 L containing 200 g/L of glucose. K. oxytoca ME-XJ-8 grew faster and achieved the stationary phase 8 h in advance (40 h) compared to K. oxytoca ME-UD-3 (48 h). But the yields of 2,3-BD were quite similar (around 0.45 g 2,3-BD/g glucose) (calculated from data provided by the authors) [START_REF] Ji | Engineering Klebsiella oxytoca for efficient 2, 3-butanediol production through insertional inactivation of acetaldehyde dehydrogenase gene[END_REF].

The stationary phase could be delayed increasing the fermentation time and even reducing the yield of 2,3-BD at a high substrate concentration compared to its threshold. For example, [START_REF] Ui | Molecular generation of an Escherichia coli strain producing only the meso-isomer of 2, 3-butanediol[END_REF] studied the influence of different concentrations of glucose between 5 and 100 g/L on the maximum 2,3-BD yield in the presence of E. coli JM109 (2% (v/v) of inoculum) at 30ºC, pH 7.5 and 160 spm. The 2,3-BD yield increased from 0.20 to 0.27 g 2,3-BD/g glucose between 5 and 40 g/L of glucose (fermentation times of 17 and 24 h respectively). Nevertheless, for a glucose concentration ranging from 70 to 100 g/L, the 2,3-BD yield slightly dropped to 0.20 and 0.17 g 2,3-BD/g glucose respectively, for fermentation times varying between 48 and 58 h. The maximum 2,3-BD yield at 40 g/L of glucose was 37% higher than the maximum 2,3-BD yield using 100 g/L of glucose; whereas the fermentation time increased by 2.4 fold in the presence of 100 g/L of glucose [START_REF] Ui | Molecular generation of an Escherichia coli strain producing only the meso-isomer of 2, 3-butanediol[END_REF]. Fermenting a high glucose concentration not only could decrease the 2,3-BD yield but also increase the fermentation time.

Parameters like pH and temperature could also influence the fermentation time. For example, [START_REF] Petrov | High production of 2,3-butanediol from glycerol by Klebsiella pneumoniae G31[END_REF] tested the pH influence on the fermentation time. In the presence of K. pneumoniae G31 (10% (v/v) of inoculum) at an initial pH of 8.0, 37ºC, 0.44 vvm, 200 rpm in a fed-batch fermentor (volume undefined) using glycerol as substrate, a 0.36 g 2,3BD/g glycerol yield was obtained after 280 h of fermentation. In comparison, the fermentation time decreased by 16% (280 to 234 h) at an initial pH of 5.2, for a 2,3-BD yield of 0.38 g 2,3-BD/g glycerol.

Influence of agitation

Facultative anaerobic microorganisms, such as E. coli, can take energy either by aerobic respiration or by fermentation in low aeration conditions [START_REF] Sinha | Role of formate hydrogen lyase complex in hydrogen production in facultative anaerobes[END_REF][START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF].

A low agitation rate leads to a fermentative process since agitation is linked to the dissolved O 2 (DO) concentration in the culture medium [START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF][START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF]. The diol 2,3-BD is a fermentative product; a high agitation rate would decrease the production of 2,3-BD because higher amount of O 2 would be transferred to the culture medium causing the bacterial respiration (Voloch et al. 1985;[START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF]. Although, a low agitation decreases DO in the culture medium and enhances the fermentative process, moderate agitation generally improve the formation of 2,3-BD.

An agitation rate between 200 and 500 rpm in 1 L of culture medium was studied in the presence of Klebsiella oxytoca KMS005 (0.033 g/L of inoculum) for 48 h at 37ºC, pH 6.0, 1 vvm and 100 g/L of maltodextrin. At 200 rpm, the 2,3-BD yield was 0.32 g 2,3-BD/g maltodextrin; whereas at 300 rpm, the 2,3-BD yield increased by 16% (0.37 g 2,3-BD/g maltodextrin). Nevertheless, at 500 rpm, the 2,3-BD yield decreased by 35% compared to 300 rpm [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF]). On the other hand, the influence of agitation rate from 100 to 400 rpm in 0.05 L of culture medium containing 50 g/L of arabinose was studied in the presence of Enterobacter cloacae NRRL B-23289 (4% (v/v) of inoculum) at 30ºC and pH 5.0. The highest 2,3-BD yield was of 0.39 g 2,3-BD/g arabinose at 100 rpm and 144 h of cultivation; whereas at 300 rpm, the 2,3-BD yield decreased by 8% (0.36 g 2,3-BD/g arabinose) after 24 h of cultivation [START_REF] Saha | Production of 2, 3-butanediol by newly isolated Enterobacter cloacae[END_REF].

In the presence of E. coli BL21 (10% (v/v) of inoculum) in 0.8 L of culture medium (glucose concentration undefined); a 2,3-BD yield of 0.44 g 2,3-BD/g glucose was obtained. The 2,3-BD yield was 19% higher at 400 rpm than at 200 rpm for a cultivation of 17 h at a pH of 7.0 and 1 vvm [START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]. Using an experimental design approach, [START_REF] Tong | Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production[END_REF] found that the fermentation of 80 g/L of glucose in the presence of E. coli YJ3 (10% (v/v) of inoculum) for 18 h (2 L of culture medium) gave a 2,3-BD yield of 0.38 g 2,3-BD/g glucose at 400 rpm, 200% higher than at 200 rpm (aeration at 2 vvm). The operating conditions were 37ºC, pH 6.5 and 1.5 vvm.

Some authors varied the agitation rate in order to increase the formation of 2,3-BD. This variation consists in 2 steps: (1) a high agitation rate at the beginning of the culture favoring the bacterial growth and (2) a reduction of the agitation forcing the microaerobic conditions (a decrease of DO occurs) to improve the formation of 2,3-BD (Ji et al. 2009a;[START_REF] Priya | Microbial production of 2, 3butanediol through a two-stage pH and agitation strategy in 150 L bioreactor[END_REF]. For example, varying the agitation rate from 300 rpm (0-15 h) to 200 rpm (15-68 h) in the presence of Klebsiella oxytoca ME-UD-3 (5% (v/v) of inoculum) at 37ºC, pH 6.5, 1 vvm during the cultivation of 200 g/L of glucose in 2 L of culture medium increased slightly the 2,3-BD yield by 6% (0.48 g 2,3-BD/g glucose for 56 h of cultivation) in comparison to a constant agitation of 200 rpm for 64 h (Ji et al. 2009a). The 2,3-BD yield enhancement was also observed by varying the agitation (200 rpm up to 10 h and 150 rpm from 10 to 80 h) in the presence of Enterobacter cloacae TERI BD18 (2% (v/v) of inoculum) at 37ºC and pH 7.5 in 100 L of culture medium (substrate concentration undefined). [START_REF] Priya | Microbial production of 2, 3butanediol through a two-stage pH and agitation strategy in 150 L bioreactor[END_REF] found a 2,3-BD yield 17% higher (0.48 g 2,3-BD/g glucose for 50 h of cultivation) compared to a constant agitation rate of 200 rpm for 60 h [START_REF] Priya | Microbial production of 2, 3butanediol through a two-stage pH and agitation strategy in 150 L bioreactor[END_REF].

Agitation provides DO in the culture medium leading to microaerobic or aerobic conditions. It seems to be that keeping a moderate agitation (200-400 rpm for bioreactor volumes varying between 1 L and 150 L) is appropriate to maximize the 2,3-BD yield. However, a variation of the agitation rate could improve the 2,3-BD yield.

Influence of the aeration

The enzymes involved in the formation of 2,3-BD depend on the oxygen (O 2 ) because it inactivates ALS and BDH enzymes, reducing the 2,3-BD yield [START_REF] Celińska | Biotechnological production of 2, 3-butanediol-current state and prospects[END_REF][START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF]Voloch et al. 1985;[START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]. The aeration injects air in the culture medium and thus the DO increases [START_REF] Betts | Impact of aeration strategies on fed-batch cell culture kinetics in a single-use 24-well miniature bioreactor[END_REF][START_REF] Somerville | Cultivation conditions and the diffusion of oxygen into culture media: the rationale for the flask-to-medium ratio in microbiology[END_REF]. If the DO in the culture medium is high, the bacterial respiration is favored [START_REF] Alexeeva | Molecular physiology of responses to oxygen in Escherichia coli. Chapter 2 -concept of microaerobic cultivation[END_REF][START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF][START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF]. Under anaerobic conditions, the fermentation favors the production of 2,3-BD. However, under microaerobic conditions respiration and fermentation are activated in the presence of facultative anaerobic microorganisms like E. coli [START_REF] Alexeeva | Quantitative assessment of oxygen availability: perceived aerobiosis and its effect on flux distribution in the respiratory chain of Escherichia coli[END_REF][START_REF] Converti | Effect of specific oxygen uptake rate on Enterobacter aerogenes energetics: carbon and reduction degree balances in batch cultivations[END_REF][START_REF] Hanly | Dynamic metabolic modeling of a microaerobic yeast coculture: predicting and optimizing ethanol production from glucose/xylose mixtures[END_REF][START_REF] Nakashimada | Enhanced 2, 3butanediol production by addition of acetic acid in Paenibacillus polymyxa[END_REF][START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]. The microaerobic conditions activate the enzymes involved in 2,3-BD production because those conditions limit the bacterial respiration [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF]Voloch et al. 1985).

Different aeration rates were studied in the presence of K. oxytoca KMS005 (0.033 g/L of inoculum) at 37ºC, pH 6.0 and 200 rpm fermenting 100 g/L of maltodextrin in 1 L of culture medium for 48 h [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF]. Under anaerobic conditions, a 2,3-BD yield of 0.03 g 2,3-BD/g maltodextrin was reached, which doubled under an aeration rate of 0.1 vvm. The maximum 2,3-BD yield (0.35 g 2,3-BD/g maltodextrin) was obtained at 1.2 vvm [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF].

A maximum 2,3-BD yield can also be reached at a low aeration rate (close to full anaerobic conditions). An aeration range between 1.0 and 0.02 vvm was tested in the presence of B. subtilis 168 (2% (v/v) of inoculum) in 2.3 L of working volume containing 105 g/L of glucose at 37ºC, initial pH of 6.5 and 300 rpm [START_REF] Fu | Metabolic engineering of Bacillus subtilis for chiral pure meso-2, 3-butanediol production[END_REF]. From 1 to 0.02 vvm, the yield of 2,3-BD and the fermentation time tripled (0.16 to 0.49 g) and doubled (40 to 86 h), respectively.

The fermentation time increased because B. subtilis being an aerobic organism its metabolism is slower under anaerobic conditions [START_REF] Fu | Metabolic engineering of Bacillus subtilis for chiral pure meso-2, 3-butanediol production[END_REF].

It is also possible to perform a variation of the aeration rate during the fermentation in order to improve the 2,3-BD yield. This variation consists in 2 steps: (1) using an aeration rate to increase the bacterial population; and (2) decreasing the aeration rate forcing the microaerobic conditions in order to increase the formation of 2,3-BD [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF][START_REF] Fu | Metabolic engineering of Bacillus subtilis for chiral pure meso-2, 3-butanediol production[END_REF][START_REF] Shi | Microbial production of 2, 3-butanediol by a newlyisolated strain of Serratia marcescens[END_REF]).

Conclusion

During the last decade, the production and consumption of dairy products increased and the weight of the dairy industry in the worldwide economy is becoming more relevant. If the production of dairy products increases, the different environmental impacts of the dairy industry are higher. The main effluent of this industry, the whey, has to be treated in order to decrease its environmental impact. In this regard, whey can be treated to reduce its BOD content before being released in the environment, for example by coagulation and flocculation.

But the whey can also be valorized, on one hand extracting its main components (e.g., lactose or proteins) by a separation process like membranes and, on the other hand, transformed the lactose into value-added products (e.g., biogas or chemical products like 2,3-BD) using different types of bacteria.

Many types of bacteria (e.g., Klebsiella pneumoniae or Serratia marcencens) have been used to ferment saccharides, mainly glucose, to produce 2,3-BD. In this regard, lactose (a disaccharide) contained in the whey is a potential source of carbon, which can be transformed into glucose and galactose (monosaccharides) by means of hydrolysis and, therefore, can be used to produce 2,3-BD. This process depends on the kind of bacteria. Recently, researchers have been working with 2,3-BD wild strain producers and 2,3-BD genetically modified strains producers (e.g., E. cloacae TERI BD18, K. oxytoca ME-XJ-8, etc.) in order to obtain a high yield of 2,3-BD, i.e., doing a more efficient fermentation. Even microorganisms which do not possess the 2,3-BD pathway have been used, such as E. coli, because it is easy to perform genetic modifications in this bacteria, which could improve the transformation of saccharides into 2,3-BD.

During a fermentation, parameters like substrate concentration, macro and micro nutrients, temperature, pH, residence time, agitation and aeration influenced the 2,3-BD formation. Therefore, the success of the fermentation process is related to the optimization of these parameters.

Thus, many authors have reported a high 2,3-BD yield fermenting saccharides like glucose or sucrose in the presence of bacteria like P. polymyxa. However, the main obstacles for the use of a biological process have been the safety level of bacteria like E. cloacae or K. pneumoniae. The use of genetically modified strains may be an avenue to overcome this problem and promote the valorization of whey into 2,3-BD. Contribution to the document: This paper is relevant to the project since: i) the bacterial population was estimated by the colony-forming unit method for a genetically modified strain of Escherichia coli (E. coli JFR1) and compared with the optical density method, ii) the importance of the culture medium related to its composition in order to produce ABD (acetoin (A) and 2,3-butanediol (2,3-BD)) was shown in the presence of 3 glucose concentrations, and iii) the influence of nitrogen source, type and concentration, on ABD yield was studied.

Acknowledgement

Biovalorization of glucose in four culture media and effect of the nitrogen source on fermentative alcohols production by Escherichia coli

Introduction

The agro-food wastes can cause environmental problems due to their high organic load; for instance the lignocellulosic biomass presents values of biological oxygen demand (BOD) from 131 to 1237 expressed in g O 2 /kg of volatile solid at 30ºC for 28 days of incubation [START_REF] Bayard | Can biomethane potential (BMP) be predicted from other variables such as biochemical composition in lignocellulosic biomass and related organic residues?[END_REF]. However, the agro-food residues, such as: i) plant fibers, for example lignocellulosic fibers from corn, sugarcane and sugar-beet; or ii) wastewaters, for example from cheese manufacturing, can be valorized since they are composed of saccharides (monosaccharides, oligosaccharides, polysaccharides) [START_REF] Hazeena | Evaluation of oil palm front hydrolysate as a novel substrate for 2, 3-butanediol production using a novel isolate Enterobacter cloacae SG1[END_REF]. This turns agro-food wastes to plentiful, cheap and accessible sources of carbon to be fermented and transformed into added value products like ethanol, 2,3-butanediol (2,3-BD) and 3-hydroxy-2-butanone (acetoin (A)) [START_REF] Gao | Production of (3S)-acetoin from diacetyl by using stereoselective NADPH-dependent carbonyl reductase and glucose dehydrogenase[END_REF][START_REF] Wu | Simultaneous production of 2, 3-butanediol, ethanol and hydrogen with a Klebsiella sp. strain isolated from sewage sludge[END_REF]). In addition, biovalorization is a green technique, which respects the environmental policy and does not need high temperatures or high pressures like pyrolysis [START_REF] Noumi | Combined effect of pyrolysis pressure and temperature on the yield and CO 2 gasification Reactivity of acacia wood in macro-TG[END_REF].

Among the fermentative products, 2,3-BD and A (ABD) have attracted more attention because of their industrial applications [START_REF] Wong | Effects of pH and fermentation strategies on 2, 3-butanediol production with an isolated Klebsiella sp. Zmd30 strain[END_REF][START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF]. The diol, 2,3-BD, is widely used by chemical industries as: i) a precursor of methyl ethyl ketone (or butanone, an organic solvent) and 1,3-butadiene (for manufacturing synthetic rubber) obtained by 2,3-BD dehydration; ii) a biofuel additive due to its high heating value (27.2 kJ/g of 2,3-BD); iii) a component in perfume manufacturing; iv) an antifreeze due to its relatively low normal melting point, -60°C; and v) a raw material for the manufacturing of softening agents among other uses [START_REF] Białkowska | Strategies for efficient and economical 2,3-butanediol production: new trends in this field[END_REF][START_REF] Celińska | Biotechnological production of 2, 3-butanediol-current state and prospects[END_REF][START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF]Wang 2013).

Acetoin is widely used as: i) a flavoring agent, for instance, in dairy products due to its butter flavor; ii) a compound of perfumes due to its sweet fragrant smell; iii) a component of electronic cigarette; iv) a cleaning agent; and v) a precursor of compounds, such as 2,3,5,6tetramethylpyrazine [START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF].

Both, 2,3-BD and A can be produced by a chemical transformation. The diol 2,3-BD can be obtained performing the hydrolysis of butene oxide at a pressure of 50 bar and a temperature varying from 160 to 220°C, with a retention time unspecified [START_REF] Gräfje | Butanediols, butenediol, and butynediol[END_REF]; while 2butanone can be transformed into A using, for instance, a palladium based catalyst in a concentration up to 3.3 mmol/L in water-THF (50:50, v/v) at 25ºC and 1 atm, time unspecified (El-Qisairi and Qaseer 2002). However, the biological processes are usually selected because of environmental concerns about the reduction of fossil fuels consumption, improvements of green production technologies like fermentation, and soft operating conditions of those process (generally operating in a temperature range between 25 and 45°C, at atmospheric pressure and around neutral pH) [START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF][START_REF] Xu | Development of a mutant strain of Bacillus subtilis showing enhanced production of acetoin[END_REF]. ABD can be produced by a biological transformation of saccharides like glucose by yeast (i.e. Saccharomyces cerevisiae) or bacteria (i.e. Enterobacter cloacae, Klebsiella oxytoca or Serratia marcescens) [START_REF] Białkowska | Strategies for efficient and economical 2,3-butanediol production: new trends in this field[END_REF][START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF]. The mentioned bacteria naturally produce ABD. However, they possess a biosafety level 2, which means they are human pathogenic (PHAC 2017). Other bacteria (e.g., Escherichia coli) can be genetically modified to host the genes of metabolic pathways to produce ABD [START_REF] Nielsen | Metabolic engineering of acetoin and meso-2, 3-butanediol biosynthesis in E. coli[END_REF][START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF]. It is interesting to create genetically modified bacteria able to produce ABD because the genetic modification of bacteria can help: i) to eliminate the metabolic pathway of other sub products like acetic acid in order to improve the production of ABD; and ii) to use bacteria like E. coli K12 which possess a biological safety level 1 (non-human pathogenic) but do not have the metabolic pathway to produce ABD (iGEM 2017; [START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF]). In the metabolic pathways involved in ABD synthesis, glucose is first transformed into pyruvic acid (PA) (glycolysis).

Then, the transformation of PA into 2,3-BD is performed by 3 enzymes: α-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC) and 2,3-butanediol dehydrogenase (BDH) [START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF]. The enzyme ALS catalyzes the formation of α-acetolactate from PA. Following this step, ALDC transforms α-acetolactate into A, a 2,3-BD precursor [START_REF] Xu | Metabolic engineering of Escherichia coli for efficient production of (3R)-acetoin[END_REF]. Finally, the enzyme BDH converts A into 2,3-BD [START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF].

The biological production of ABD by bacteria or yeasts is performed using a culture medium, which is composed by at least a carbon source and nutrients like nitrogen, phosphorus, sulfur, etc. [START_REF] Tian | Optimization of fermentation medium for acetoin production by Bacillus subtilis SF4-3 using statistical methods[END_REF]). These nutrients might positively affect the ABD production depending on the bacteria used. For example, nitrogen sources like urea, yeast extract, tryptone or sodium nitrate, affect differently the production of ABD which may increase or decrease [START_REF] Wong | Producing 2, 3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain[END_REF]. On the other hand, the rise of nitrogen concentration might improve or hamper the ABD production fermenting diverse saccharides like sucrose, glucose or a mixture glucose:xylose (2:1, w/w) as reported in the presence of bacteria like Klebsiella sp. or Paenibacillus polymyxa (Häβler et al. 2012;Ji et al. 2009b;[START_REF] Wong | Producing 2, 3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain[END_REF].

The aim of this study was therefore to ferment glucose into ABD using a genetically modified strain of E. coli. Firstly, the bacterial population by the colony-forming unit (CFU) and the optical density (OD) methods of the genetically modified strain was estimated in a rich culture medium, lysogeny broth (LB) [START_REF] Berney | Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection[END_REF]. Secondly, since the formation of ABD varies depending on the type and concentration of nutrients, 4 different culture media (3 minimal culture media: medium 9 (M9) [START_REF] Ecocyc | Escherichia coli K-12 substr[END_REF][START_REF] Maniatis | Molecular cloning: a laboratory manual[END_REF], medium 63 (M63) [START_REF] Pardee | The genetic control and cytoplasmic expression of "inducibility" in the synthesis of β-galactosidase by E. coli[END_REF]) and potassium morpholinopropane sulfonate medium (MOPS) (Wisconsin-University 2017) and 1 rich medium (LB) [START_REF] Berney | Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection[END_REF] were supplemented with 3 glucose concentrations (4, 12.5 and 25 g/L). Finally, in order to observe the influence of an additional nitrogen source (ANS) and its concentration on ABD formation, the effect of 2 nitrogen sources (sodium nitrate (NaNO 3 ) and urea (NH 2 ) 2 CO)) and their concentrations (with an equivalent concentration of nitrogen for both sources: 2.5, 5.0 and 7.0 g N/L) were tested.

Materials and methods

Microorganisms

Two strains of E. coli MG1655, wild (ECW) and genetically modified (ECGM), were grown in the rich culture medium LB. The ECGM strain hosts the metabolic pathway to produce ABD from Enterobacter cloacae (E. coli MG1655/ALDH+budABC, named E. coli JFR1). Furthermore, the production of lactate was blocked by inactivation of the enzyme lactate dehydrogenase in E. coli JFR1 strain. Both strains were conserved into a mixture of glycerol and lysogeny broth (LB) culture medium (50:50, v/v) at -81ºC.

Inoculum preparation and population estimation

The seed culture medium was prepared as follows: 0.1 mL of E. coli (ECW or ECGM) from the conservation blend was mixed with 9.9 mL of fresh LB culture medium into a test tube (15 mL). The test tube was incubated overnight at 37ºC to obtain the pre-culture medium. The population estimation of the inoculum was performed using 2 mL of pre-culture medium transferred to a 500 mL Erlenmeyer flask containing 200 mL of fresh LB culture medium. The flask was incubated for 9 h in a rotary shaker incubator at 37ºC and 100 rpm. After this time (9 h), the seed culture medium obtained was used to inoculate the culture media.

Wild (ECW) and genetically modified (E. coli JFR1) strains were grown in LB culture medium in order to estimate and compare their bacterial population, using 9 replicates, to determine the CFU/mL of broth. In order to determine the CFU, 6 consecutive dilutions were carried out mixing 0.9 mL of fresh LB culture medium with 0.1 mL from the previous dilution to the following one. For example, in order to obtain the dilution 10 -3 , 0.1 mL from the dilution 10 -2 were taken and mixed with 0.9 mL of fresh LB culture medium. Then, samples of 0.1 mL were taken from the last dilution (10 -6 ), spread on LB agar plates and incubated for 9 h at 37ºC. In this way, colonies appeared on the agar surface and were directly counted.

Culture media and conditions

The fermentation of glucose using E. coli JFR1 was tested using 4 culture media (LB, M9, M63 and MOPS) and the ABD yields were compared. The composition of each culture medium is described in Table 3.1. The culture media were sterilized by autoclaving them at 121 º C and 1.2 atm for 0.25 h. All experiments were performed in triplicate using Erlenmeyer flasks with a working volume of 200 mL (10% (v/v) of inoculum) at 37ºC, 100 rpm, initial pH 6.5 and atmospheric pressure unless otherwise indicated. Flasks were placed on a rotary shaker incubator (Fermentation Design inc, Allentown, PA) for 96 h. Beyond this time, the ABD yield decreased. After selecting a culture medium (M9), 2 nitrogen sources (NaNO 3 and (NH 2 ) 2 CO) were tested for 72 h as a comparison purpose. The additional nitrogen concentration in M9 supplemented with glucose (12.5 and 25 g/L) was the same for both sources: 2.5, 5.0 and 7.0 g N/L.

Analytical methods and reagents

The concentration of glucose and fermentative products (A, 2,3-BD, ethanol and acetic acid) were determined by high performance liquid chromatography (HPLC). Raw broth withdraw from each flask was pre-filtered in order to remove the solids using 0.2 μm pore size filters. Ten (10) μL of sample were injected into an Agilent series 1100 chromatograph (Agilent Technologies, Palo Alto, CA) equipped with a column Aminex HPX-87H (300 x 7.8 mm) in order to separate glucose and products (2,3-BD, A, ethanol and acetic acid) at 25ºC using sulfuric acid 0.005M as a mobile phase (0.4 mL/min). Refractive index (RI) and ultraviolet (UV) detectors were used for the quantification of the products (glucose, 2,3-BD, ethanol and acetic acid concentration were quantified by RI detector; whereas acetoin concentration was estimated by UV detector).

The reagents used for the analytical method were HPLC grade. Glucose (≥ 99.5%), A and 2,3-BD (98%) were purchased from Sigma-Aldrich (Canada). Acetic acid (ACS pure) and ethanol (≥ 99%) were purchased from Fisher Scientific Inc. (Canada).

Statistical analysis

The significant parameters (i.e., culture medium, glucose concentration and nitrogen source and concentration) on ABD yield were determined by means of an analysis of variance (ANOVA) with a P-value lower than 0.05. Dixon's Q test was used to estimate and reject outlier values of ABD yield using a confidence level of 95% [START_REF] Rorabacher | Statistical treatment for rejection of deviant values: critical values of Dixon's" Q" parameter and related subrange ratios at the 95% confidence level[END_REF]).

Results and discussion

Optical density and colony-forming units for ECW and E. coli JFR1 strains

The bacterial growth can be defined by 3 phases: the lag phase, the exponential phase and the stationary phase, where the maximum bacterial population is reached. Figure 3.1 presents the results of OD at 600 nm (OD 600 ) and CFU/mL as a function of the fermentation time obtained in the presence of ECW (Figure 3.1A) and E. coli JFR1 (Figure 3.1B) in LB culture medium.

In the case of ECW, according to OD 600 , the exponential growth phase started after 2 h (OD 600 : 0.10) of fermentation until 4 h (OD 600 : 0.51). For a fermentation time higher than 4 h, the OD 600 increased slowly to reach 0.77 at 9 h. A similar tendency was observed in Figure 3.1B with the E. coli JFR1 strain. The exponential growth phase began after 2 h of fermentation (OD 600 : 0.05) and increased up to 4 h (OD 600 : 0.47). Finally, the E. coli JFR1 growth increased slowly to reach an OD 600 value close to 0.82 at 9 h. Taking into account the OD 600 values for both E. coli strains, both bacterial growths were similar. According to [START_REF] Sezonov | Escherichia coli physiology in Luria-Bertani broth[END_REF], the exponential phase ends when the OD 600 value is ranged between 0.6 and 1.0 in LB culture medium in the presence of E. coli K12 MG1655 at 37ºC and 180 rpm. This fact was also reported by Quigley (2008) using another E. coli strain (E. coli DH5α), where a typical OD 600 value at 37°C in LB culture medium was 0.8 at 6 h of fermentation, the end point of the exponential growth phase. In the present study, the growth tendency for ECW and E. coli JFR1 could be considered similar to a typical growth curve for E. coli bacteria in LB culture medium with an OD 600 close to 0.8 after 9 h of fermentation for both strains (ECW and E. coli JFR1).

In order to confirm the bacterial growth of each E. coli strain, the CFU/mL value was determined. In fact, both strains presented a relatively fast growth on LB agar plates (based on CFU/mL), where colonies appeared approximately after 9 h of incubation. In the presence of ECW (Figure 3.1A), the CFU/mL values were practically stable at 1.1x10 9 CFU/mL for fermentation times ranging from 6 to 9 h. This fact clearly confirms that the stationary growth phase is reached after 6 h. The CFU/mL value was similar to the one obtained for E. coli JM109 at 37ºC and 250 rpm using LB culture medium [START_REF] Matlock | Analyzing differences in bacterial optical density measurements between spectrophotometers[END_REF]). However, the CFU/mL obtained in the present study was 39% lower (1.8x10 9 CFU/mL) than the value reported by [START_REF] Yu | Effects of cell surface loading and phase of growth in cold atmospheric gas plasma inactivation of Escherichia coli K12[END_REF]. The authors used a tryptone soy broth as a culture medium, which presents a richer composition compared to LB culture medium [START_REF] Ecocyc | Escherichia coli K-12 substr[END_REF], in the presence of E. coli K12 for 24 h at 37°C and 140 rpm.

On the other hand, in the present study, E. coli JFR1 strain reached the stationary growth phase faster than ECW, since it was achieved after 5 h of fermentation. The CFU/mL value at 5 h was 6.5x10 8 CFU/mL, 41% lower compared to the CFU of ECW probably linked to the genetic modification performed in E. coli JFR1, since in recombinant strains a new gene might be toxic for the bacteria and thus their growth might be lower [START_REF] Rosano | Recombinant protein expression in Escherichia coli: advances and challenges[END_REF].

The difference between OD 600 and CFU techniques could be explained by the fact that OD 600 is a direct technique to measure the turbidity of the broth [START_REF] Sutton | Measurement of microbial cells by optical density[END_REF]). In the broth, there is a mixture of viable bacteria and dead bacteria. If many dead cells are in suspension, the measurement of the bacterial growth using OD 600 might have been overestimated, whereas the quantified bacterial population using the CFU method only takes into account the viable bacteria. Therefore, the CFU method seems to be more suitable than OD 600 to estimate the bacterial population in the inoculum.

Modeling of the bacterial growth

The experimental bacterial growth data of ECW and E. coli JFR1 strains were modeled using the modified Gompertz model [START_REF] Zwietering | Modeling of the bacterial growth curve[END_REF]):

where N is the CFU value (CFU/mL) for a given time and N 0 the CFU value (CFU/mL) at the initial time; K is the asymptote (ln [N ∞ /N 0 ]) in the stationary phase; μ m is the maximum specific growth rate (h -1 , which comes expressed as (ln(CFU 1 ) -ln(CFU 2 )) / (t 1 -t 0 )); λ is the lag time (h) and t is the time (h).

The modified Gompertz model was fitted with the experimental bacterial growth data by a non-linear regression (Figure 3.1). Determination coefficients (R 2 ) values of 0.981 and 0.999 were respectively obtained for the ECW and the E. coli JFR1 strains. The values of λ were 2.2 and 1.8 h for the ECW and E. coli JFR1, respectively. This fact points out that the E. coli JFR1 strain was able to adapt to the LB culture medium faster than ECW. However, ECW reached a higher μ m (2.8 h -1 ) compared to E. coli JFR1 (2.4 h -1 ), achieving a higher bacterial population.

Compared to the best μ m values obtained in the presence of 2 mutants of E. coli MG1655 (soluble transhydrogenase deficient and Entner-Doudoroff pathway deficient) and the wild strain (37°C and 200 rpm in M9 culture medium supplemented with 5 g/L of glucose), the E. coli JFR1 μ m obtained in the present study was more than 3 fold higher [START_REF] Sauer | The soluble and membranebound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli[END_REF]).

However, the value of E. coli JFR1 μ m was similar to the one obtained by [START_REF] Berney | Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection[END_REF] using LB without glucose as culture medium fermented at 37°C and 200 rpm in the presence of E. coli K12 MG1655 (wild strain).

Influence of glucose concentration supplementing 4 culture media on ABD yield in the presence of E. coli JFR1

The production of ABD by E. coli JFR1 was studied using 4 culture media (LB, M9, M63 and MOPS) for 96 h supplemented with 4, 12.5 and 25 g/L of glucose. In most of the experimental conditions, glucose was exhausted (conversion 100%) after a maximum of 72 h. However, a maximum glucose conversion of 92% after 96 h was observed using M9 culture medium supplemented with 25 g/L of glucose. In the same way, by fermenting 12.5 and 25 g/L of glucose in MOPS culture medium, a maximum glucose conversion of 93% and 66% respectively were observed after 96 h (Table 3.2).

Table 3.2: Maximum glucose conversion (%) as a function of glucose concentration (g/L) and time (h) in the 4 studied culture media at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% (v/v) of inoculum in a working volume of 200 mL (batch fermentation). 2A presents the ABD yield as a function of fermentation time using LB, a rich culture medium formed by tryptone, yeast extract and sodium chloride, supplemented with 3 glucose concentrations (4, 12.5 and 25 g/L). The maximum ABD yield was around 0.25 g ABD/g glucose independently of the glucose concentration. Therefore, the increase of glucose concentration had no influence on the maximum ABD yield using LB culture medium (p < 0.05), but the fermentation time to obtain the maximum ABD yield increased according to the glucose concentration, ranging from 24 h (4 g/L of glucose) to 72 h (25 g/L of glucose). On the other hand, after 24 h of fermentation, the ABD concentration decreased when LB culture medium was supplemented with 4 g/L of glucose (Figure 3.2A). As suggested by [START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF], once the carbon source (glucose) is depleted, bacteria could consume another carbon source like ABD during the stationary growth phase, causing a drop of its yield. This fact is observed in Figure 3.2A and thus it seems to confirm that E. coli JFR1 consumes ABD as a carbon source in order to maintain its metabolic activity. Lysogeny broth (LB) is a rich culture medium, which provides all nutrients (carbon, nitrogen, phosphorus, etc.) required by bacteria [START_REF] Ecocyc | Escherichia coli K-12 substr[END_REF]. In this way, the components present in LB culture medium like amino acids (e.g. leucine, methionine and tryptophan) could positively influence the ALS and ALDC enzymes [START_REF] Cho | Enhanced 2,3-butanediol production by optimizing fermentation conditions and engineering Klebsiella oxytoca M1 through overexpression of acetoin reductase[END_REF]. This fact may explain the similar ABD yields obtained whatever the glucose concentration, since the LB culture medium nutrients might efficiently stimulate the formation of ABD even at a relatively low glucose concentration.

Figures 3.2B and 3.2C present the ABD yield for E. coli JFR1 as a function of the fermentation time using M9 and M63 as culture media, respectively. When both culture media were supplemented with 4 g/L of glucose, the maximum ABD yield was obtained after 8 h of fermentation. For a higher fermentation time, the ABD yield constantly decreased down to zero (under the detection limit of the HPLC) since it was probably used as a carbon source. For both culture media, similar maximum ABD yield was obtained at 72 h using M9 (0.22 g ABD/g glucose) and M63 (0.25 g ABD/g glucose) in the presence of 12.5 and 25 g/L of glucose. The similar ABD yields obtained at 72 h for glucose concentrations of 12.5 and 25 g/L may be due to the inhibitory effects occasioned on E. coli JFR1 by a relatively high concentration of glucose [START_REF] Tian | Optimization of fermentation medium for acetoin production by Bacillus subtilis SF4-3 using statistical methods[END_REF]), which might be close to 25 g/L for E. coli JFR1.

The inhibitory effect of glucose concentration was also observed, for instance, in the presence of E. coli JM109 (2% (v/v) of inoculum) in LB culture medium at 30°C, pH 7.5 and 160 rpm when glucose concentration higher than 40 g/L was used [START_REF] Ui | Molecular generation of an Escherichia coli strain producing only the meso-isomer of 2, 3-butanediol[END_REF]). The maximum ABD yield (close to 0.25 g ABD/g glucose, at 72 h fermenting 12.5 and 25 g/L of glucose) obtained using LB, M9 and M63 as culture media was similar to the one reported in the presence of different E. coli strains. For instance, [START_REF] Ui | Molecular generation of an Escherichia coli strain producing only the meso-isomer of 2, 3-butanediol[END_REF] and [START_REF] Yan | Enantioselective synthesis of pure (R,R)-2,3-butanediol in Escherichia coli with stereospecific secondary alcohol dehydrogenases Organic[END_REF] reported ABD yields of 0.25 g ABD/g glucose (yield calculated using the experimental data provided by the authors) in the presence of E. coli JM109 (2% (v/v) of inoculum and 20 g/L of glucose) and E. coli JCL260 (1% (v/v) of inoculum and 40 g/L of glucose), respectively. The fermentations were performed at 30°C, pH 7.5 and 160 rpm for 20 h using E. coli JM109; and at 37°C (from 0 h to 3 h) and 30°C (from 3 h to 48 h), pH 7.0 and 250 rpm using E. coli JCL260.

Figure 3.2D presents the ABD yield variation as a function of time using MOPS as culture medium in the presence of E. coli JFR1. The maximum ABD yields were similar fermenting 4 and 12.5 g/L of glucose (around 0.18 g ABD/g glucose) after 24 and 48 h, respectively. However, the maximum ABD yield was 22% smaller (0.14 g ABD/g glucose at 48h) with MOPS supplemented with 25 g/L of glucose compared to the other glucose concentrations. A 25 g/L of glucose in MOPS may cause the bacterial inhibition and explain the lower ABD yield, associated with a 66% of glucose conversion (Table 3.2). The low glucose conversion and the low ABD formation was also observed by [START_REF] Nielsen | Metabolic engineering of acetoin and meso-2, 3-butanediol biosynthesis in E. coli[END_REF] with E. coli YYC202 strain (17% (v/v) of inoculum, estimated by the data provided by the authors) in LB culture medium. A maximum glucose conversion of 38% was obtained at 30°C for 120 h (pH and agitation non-defined) and the ABD yield was similar to the one of the present study (0.14 g ABD/g glucose, the yield was calculated using the experimental data provided by the authors). Moreover, MOPS contains several divalent metal ions like iron (Fe 2+ ), cobalt (Co 2+ ) and manganese (Mn 2+ ), which could influence the ABD formation [START_REF] Garg | A new method for industrial production of 2, 3-butanediol[END_REF]Jain 1995, Wong et al. 2012). The divalent metal ions present in MOPS culture medium might inhibit the enzymes involved in the formation of ABD, as reported by several authors [START_REF] Tian | Optimization of fermentation medium for acetoin production by Bacillus subtilis SF4-3 using statistical methods[END_REF][START_REF] Wong | Producing 2, 3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain[END_REF][START_REF] Yu | Characterization of a (2R,3R)-2,3-butanediol dehydrogenase from Rhodococcus erythropolis WZ010[END_REF].

The selection of a culture medium was based on the ABD yield and the respective cost of the medium. The cost of each culture medium is as follows: MOPS around 12 ($/L), M63 and LB around 8 ($/L) and M9 near 6 ($/L) (SigmaAldrich 2017). MOPS culture medium was not chosen because the lowest ABD yield among the 4 culture media tested in the presence of 12.5 and 25 g/L of glucose was obtained. LB and M63 culture media were rejected because they are more expensive and presented similar ABD yields (p < 0.05) than M9. In the subsequent experiments, M9 culture medium was supplemented with 12.5 and 25 g/L of glucose in order to test the effect of additional nitrogen sources on ABD yield.

Influence of the source and concentration of an additional nitrogen source on ABD yield in the presence of E. coli JFR1

Nitrogen is an important macronutrient since it is involved in the bacterial growth (constitution of coenzymes and amino acids, Todar 2017) and thus may improve the formation of ABD as shown in a study performed by [START_REF] Wong | Producing 2, 3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain[END_REF]. The authors reported that depending on the source and concentration of nitrogen, the ABD yield improved. In the present study, 2 additional inorganic nitrogen sources (urea and NaNO 3 ) were supplemented to the M9 culture medium in order to test the effect of the source and the concentration of nitrogen source on ABD formation at 2 glucose concentrations (12.5 and 25 g/L). Both urea and NaNO 3 concentration were kept at the same nitrogen (N) concentration (2.5, 5.0 and 7.0 g N/L) in order to allow a comparison. In the case of urea, additional concentrations of nitrogen (0.6 and 1.2 g N/L in the presence of 12.5 g/L of glucose; and 10 g N/L in the presence of 25 g/L of glucose) were tested.

The use of NaNO 3 hampered the formation of ABD and the consumption of glucose never exceeded 6 and 8 g/L at 48 h whatever nitrogen concentration used at 12.5 or 25 g/L of glucose respectively (Table 3.3) in all fermentations. This fact may point out i) that NaNO 3 inhibited the bacterial growth of E. coli JFR1 since E. coli may reduce nitrate (NO 3 -) into nitrite (NO 2 -), which is toxic for the cells [START_REF] Tiso | Nitrate reduction to nitrite, nitric oxide and ammonia by gut bacteria under physiological conditions[END_REF]; and ii) may explain the low glucose conversion (around 50% and 30% fermenting 12.5 and 25 g/L of glucose respectively). Table 3.3: Glucose conversion (%) as a function of nitrogen concentration (g N/L) in the presence of 12.5 and 25 g/L of glucose in M9 culture medium using NaNO 3 as an ANS at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% (v/v) of inoculum in a working volume of 200 mL (batch fermentation).

Nitrogen concentration (g N/L)

Glucose concentration (g/L) Maximum glucose conversion (%) On the other hand, E. coli JFR1 produced acetic acid (yield ranged from 0.06 to 0.09 g acetic acid/g glucose between 48 and 96 h, in the presence of 12.5 g/L of glucose; and was around 0.04 g acetic acid/g glucose at 48 h, fermenting 25 g/L of glucose for all nitrogen concentrations tested) in the presence of NaNO 3 (Table 3.4).

Acetic acid is a fermentative product formed by E. coli which inhibits its growth [START_REF] García | Estrategias de obtención de proteínas recombinantes en Escherichia coli[END_REF]. Hence, the production of acetic acid besides the reduction of NO 3 -prevents the formation of ABD and the consumption of glucose. The null ABD formation using NaNO 3 as a nitrogen source was also observed by [START_REF] Xiao | Thermophilic fermentation of acetoin and 2, 3-butanediol by a novel Geobacillus strain[END_REF] using Geobacillus sp. XT15 (5% (v/v) of inoculum) to ferment 20 g/L of glucose at 55°C, initial pH of 8 and 170 rpm (fermentation time unspecified). However, the authors reported an ABD yield of 0.40 g ABD/g glucose under the same operating conditions using a mixture of 20 and 10 g/L of peptone and yeast extract respectively. Table 3.4: Acetic acid yield (g/g glucose) as a function of nitrogen concentration (g N/L) in the presence of 12.5 and 25 g/L of glucose in M9 culture medium using NaNO 3 as an ANS at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% v/v of inoculum in a working volume of 200 mL (batch fermentation). and3.3B present the glucose conversion and ABD yield, respectively, as a function of time in the presence of 12.5 g/L of glucose. Urea was used as an ANS. Two additional nitrogen concentrations (0.6 and 1.2 g N/L) were also tested.

Glucose was exhausted after 24 h of fermentation when urea was added to M9 culture medium at nitrogen concentrations ranged from 2.5 to 7.0 g N/L, whereas glucose was totally consumed in the presence of 0.6 and 1.2 g N/L after 48 h (Figure 3.3A). After 24 h, the ABD yield increased by 21% and 17% in the presence respectively of 2.5 and 5.0 g N/L, in comparison of the ABD yield obtained without ANS (0.17 g ABD/g glucose, Figure 3.2B). However, adding 0.6, 1.2 and 7.0 g N/L did not improve the ABD yields, (0.14, 0.16 and 0.15 g ABD/g glucose) respectively (Figure 3.3B). The highest ABD yield, 0.26 g ABD/g glucose, was reached in the presence of 1.2 g N/L after 48 h, increasing by 29% compared to the one achieved without ANS. In all cases, the ABD yield remained practically constant once the highest yield was reached for each nitrogen concentration.

The formation of acetic acid, an inhibitory compound for E. coli, was not detected in the presence of urea fermenting 12.5 g/L of glucose (data not shown). Figure 3.3B presents the highest and lowest ethanol yields which were obtained with 0.6 g N/L and without ANS respectively. Ethanol was produced in a range varying between 0.07 and 0.11 g ethanol/g glucose for all the nitrogen concentrations. Additionally, the highest (▬) and lowest (+) ethanol yields obtained for 0.6 g N/L and without an ANS are represented respectively. Results are means ± SD of 3 replications performed in flasks at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% v/v of inoculum in a working volume of 200 mL (batch fermentation). Figures 3.4A and 3.4B show the glucose conversion and ABD yield, respectively, as a function of time when glucose (25 g/L) was fermented in the presence of E. coli JFR1 and urea as an ANS. The preliminary fermentations showed that increasing the nitrogen concentration provided a higher ABD yield. For this reason, a higher urea concentration (10.0 g N/L) was also tested. A 100% glucose conversion was reached at 48 h when 5.0 7.0 and 10.0 g N/L were used; whereas the glucose conversion was 75% in the presence of 2.5 g N/L. The 2.5 g N/L had no significant effect on the ABD yield (0.15 g ABD/g glucose) compared to the experiments performed without ANS (0.15 g ABD/g glucose) at a fermentation time of 48 h. However, the ABD yields compared to the one obtained in absence of urea increased by 66%, 47% and 41% using 5.0, 7.0 and 10.0 g N/L, respectively at 48 h. The highest ABD yield (0.27 g ABD/g glucose) was achieved at 7.0 g N/L after 96 h of fermentation. No acetic acid production was detected fermenting 25 g/L of glucose in the presence of 7.0 and 10.0 g N/L; whereas in the presence of lower nitrogen concentration like 2.5 and 5.0 g N/L acetic acid was produced (up to 0.06 and 0.02 g acetic acid/g glucose) at 96 h (data not shown). Therefore, the formation of acetic acid was avoided increasing the urea concentration (7.0 and 10.0 g N/L) in the presence of 25 g/L of glucose. Figure 3.4B presents the highest and lowest ethanol yields obtained for 10.0 and 2.5 g N/L respectively, where the ethanol yield was nearly constant, ranging from 0.04 to 0.07 g ethanol/g glucose when a concentration of 25 g/L of glucose was fermented. The rise of ABD yield in the presence of urea when fermenting carbon sources was also reported in the presence of other bacteria. For example, the ABD yield increased by adding a nitrogen concentration of 12.3 g N/L in the presence of K. oxytoca ME-303 (5% (v/v) of inoculum) for 44 h at 37°C, initial pH of 6.5 and 200 rpm fermenting 60 g/L of a mixture of glucose-xylose (2:1, w/w) (Ji et al. 2009b). [START_REF] Wong | Producing 2, 3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain[END_REF] showed the improvement of ABD yield using 0.9 g N/L in the presence of Klebsiella sp. Zmd30 (2% (v/v) of inoculum) at 30°C and 200 rpm (initial pH and fermentation time non specified) fermenting 160 g/L of glucose; whereas a concentration of 1.8 g N/L was reported as the optimal one fermenting 160 g/L of glucose in the presence of B. subtilis SF4-3 (5% (v/v) of inoculum) at 37ºC and 180 rpm (pH non specified) for 96 h [START_REF] Tian | Optimization of fermentation medium for acetoin production by Bacillus subtilis SF4-3 using statistical methods[END_REF].

The concentration and the type of nitrogen source are linked to the formation of ABD. The use of urea improved the ABD yield compared to the experiments performed without an ANS. On the other hand, the use of NaNO 3 did not generate ABD.

Conclusion

This study was based on the glucose fermentation using a genetically modified strain of Escherichia coli MG1655/ALDH+budABC (E. coli JFR1). In a first step, the growth of ECW and E. coli JFR1 was followed by 2 techniques: OD 600 and CFU. While the bacterial growth for both ECW and E. coli JFR1 in LB culture medium was the same according to the OD 600 method (near 0.8), the CFU method gave more realistic results about the bacterial growth. The population of ECW and E. coli JFR1 was 1.1x10 9 and 6.5x10 8 CFU/mL respectively, showing that the genetic modification of bacteria influences their growth decreasing the bacterial population, maybe due to the introduced gene toxicity.

The cheapest tested culture medium, M9, was selected to produce ABD (acetoin and 2,3butanediol). The fermentation of 12.5 and 25 g/L of glucose gave the lowest ABD yields (0.18 and 0.14 g ABD/g glucose, respectively) using MOPS, the most expensive culture medium.

The addition of urea to M9 culture medium improved the ABD yield. The highest ABD yield was 0.27 g ABD/g glucose using 7.0 g N/L at 96 h in the presence of 25 g/L of glucose. The use of sodium nitrate prevented the ABD formation.

Depending on the source, concentration and even the combination of different compounds present in the culture medium, the production of ABD could be affected, maybe in a critical way such as with NaNO 3 . In addition, it would be interesting and highly recommended to carry out an estimation of the bacterial population before performing fermentations since, as shown the results of ECW (1.1x10 9 CFU/mL) and E. coli JFR1 (6.5x10 8 CFU/mL), the number of bacteria (the inoculum) changes from a strain to another.

3.7

Chapter 4. Fermentation of whey and its permeate using a genetically modified strain of Escherichia coli to produce 2,3-butanediol Avant-propos:

L'article "Fermentation of whey and its permeate using a genetically modified strain of Escherichia coli to produce Contribution to the document: This paper is the relevant for the project since the main objective is to ferment the lactose contained in the whey (W) and permeate whey (PW) by an improved genetically modified strain of Escherichia coli (E. coli JFR12) in order to produce 2,3-butanediol [START_REF]Abstract Whey (W) is generated during cheese manufacturing and permeate whey (PW) is obtained after W deproteinization. As both effluents contain lactose, a carbon source, and they can cause environmental issues if they are released in the environment without a treatment. The aim of the present study was to test the transformation of lactose via fermentation into 2,3butanediol (2,3-BD) in the presence of a genetically modified strain of Escherichia coli K12 MG1655 (E. coli JFR12) in order to evaluate the potential of W and PW as lactose sources. Lactose (a disaccharide), glucose and galactose (monosaccharides of lactose) at concentrations of 12.5, 25 and 50 g/L were fermented in a synthetic culture medium (medium 9, M9) inoculated at a ratio of 10% (v/v), at 37ºC, 1 atm, initial pH 7.4 and 100 rpm for 72 h in flasks of 0.5 L with a working volume of 0.2 L. Under these operating conditions, the highest 2,3-BD yield was around 0.38 g/g saccharide in the presence of 25 g/L of glucose or lactose at 48 and 72 h, respectively, and 0.19 g/g galactose at 48 h. Therefore, E. coli JFR12 could transform galactose into 2,3-BD but being its yield the half of one observed with glucose at 48 h. Whey (31 g/L lactose) or PW (34 g/L lactose) were fermented in dilution with M9 (50:50, 75:25 and 100:0, v/v) under the same operating conditions. The fermentation of W and PW in absence of M9 (100:0, v/v) produced the highest 2,3-BD yield (up to 0.43 g/g lactose) at 72 h. Whereas, the highest acetoin (A) yield of 0.12 g/g lactose was obtained with[END_REF]. In addition, different operational parameters (dilution ratio with M9 culture medium, inoculum size, initial pH and agitation rate) were tested in order to study their influence on 2,3-BD yield. Moreover, a scaling up of the fermentation volume (4 times) was performed in a 2 L bioreactor besides the injection of air (2 L/min) in order to improve the formation of 2,3-BD. The potential of E. coli JFR12 to valorize W and PW was shown.

Fermentation of whey and its permeate using a genetically modified strain of Escherichia coli to produce 2,3-butanediol

Résumé

Le

Introduction

Nowadays, the worldwide manufacturing of the dairy products (e.g. cheese) has increased. For instance, the worldwide production of cheese increased by 25% between 2004 and 2014 to reach 22.6 million tons (FAO-FAOSTAT, 2017d). Cheese is produced by coagulating the casein present in milk, by the action of enzymes like chymosin or acids like citric acid [START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF]). Nine (9) L of whey (W) per kg of cheese are generated during the cheese making process (Guimaraes et al. 2008). Taking into account the amount of cheese produced worldwide in 2014, the volume of W generated can be estimated around 203 million m 3 . Whey can be valorized by ultrafiltration (membranes) in order to obtain whey proteins, which are used in food as emulsifier or as gelling and water binding agent (Rebouillat and Ortega-Requena 2015), generating another effluent: the permeate whey (PW). Whey contains up to 54 g/L of lactose (a disaccharide composed of glucose and galactose), whereas PW has a lactose concentration higher than 80 g/L [START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF][START_REF] Smith | Evaluation of whey, milk, and delactosed permeates as salt substitutes[END_REF]. Such lactose content represents high biological and chemical oxygen demands (BOD and COD, respectively). The BOD and COD contents are similar for W and PW at around 47 and 70 g/L, respectively [START_REF] Belhamidi | Recovery and purification of whey with the concentration, reincorporation and reuse protein and lactose[END_REF]. When these effluents are released in rivers or lakes without treatment, they cause an oxygen reduction perturbing the aquatic life [START_REF] Jesus | Biotechnological alternatives for the utilization of dairy industry waste products[END_REF]. Both W and PW can be valorized by means of lactose fermentation to produce, for example biogas, hydrogen (H 2 ), acetoin (A) and alcohols such as 2,3-butanediol (2,3-BD) [START_REF] Antonopoulou | Biohydrogen and methane production from cheese whey in a two-stage anaerobic process[END_REF][START_REF] Becerra | Biobutanol from cheese whey[END_REF][START_REF] Huertas | Whey: importance in the food industry[END_REF].

The diol, 2,3-butanediol [START_REF]Abstract Whey (W) is generated during cheese manufacturing and permeate whey (PW) is obtained after W deproteinization. As both effluents contain lactose, a carbon source, and they can cause environmental issues if they are released in the environment without a treatment. The aim of the present study was to test the transformation of lactose via fermentation into 2,3butanediol (2,3-BD) in the presence of a genetically modified strain of Escherichia coli K12 MG1655 (E. coli JFR12) in order to evaluate the potential of W and PW as lactose sources. Lactose (a disaccharide), glucose and galactose (monosaccharides of lactose) at concentrations of 12.5, 25 and 50 g/L were fermented in a synthetic culture medium (medium 9, M9) inoculated at a ratio of 10% (v/v), at 37ºC, 1 atm, initial pH 7.4 and 100 rpm for 72 h in flasks of 0.5 L with a working volume of 0.2 L. Under these operating conditions, the highest 2,3-BD yield was around 0.38 g/g saccharide in the presence of 25 g/L of glucose or lactose at 48 and 72 h, respectively, and 0.19 g/g galactose at 48 h. Therefore, E. coli JFR12 could transform galactose into 2,3-BD but being its yield the half of one observed with glucose at 48 h. Whey (31 g/L lactose) or PW (34 g/L lactose) were fermented in dilution with M9 (50:50, 75:25 and 100:0, v/v) under the same operating conditions. The fermentation of W and PW in absence of M9 (100:0, v/v) produced the highest 2,3-BD yield (up to 0.43 g/g lactose) at 72 h. Whereas, the highest acetoin (A) yield of 0.12 g/g lactose was obtained with[END_REF], is a valuable compound highly used in cosmetic, pharmaceutical and petrochemical industries (Ji et al. 2009b). For example, 2,3-BD is involved in the manufacture of perfumes, and can be used as an antifreeze agent (due to its normal melting point of -60ºC) [START_REF] Celińska | Biotechnological production of 2, 3-butanediol-current state and prospects[END_REF][START_REF] Garg | A new method for industrial production of 2, 3-butanediol[END_REF], Ji et al. 2009b[START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF]). 2,3-butanediol is a chemical platform to produce other chemicals, such 1,3-butadiene, used for synthetic rubber manufacturing, and methyl ethyl ketone, a solvent and fuel additive and polyurethane methacrylate, used in cardiovascular applications [START_REF] Cherng | Polyurethane-based drug delivery systems[END_REF][START_REF] Wong | Effects of pH and fermentation strategies on 2, 3-butanediol production with an isolated Klebsiella sp. Zmd30 strain[END_REF]. Acetoin, the 2,3-BD precursor via a biological process, is an interesting compound since it is widely used in cosmetics as perfume and lotion additive due to its sweet fragrance, as well as in food industry as an additive due to its buttery flavor [START_REF] Dai | High acetoin production by a newly isolated marine Bacillus subtilis strain with low requirement of oxygen supply[END_REF], Ji et al. 2009b[START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF]).

2,3-butanediol [START_REF]Abstract Whey (W) is generated during cheese manufacturing and permeate whey (PW) is obtained after W deproteinization. As both effluents contain lactose, a carbon source, and they can cause environmental issues if they are released in the environment without a treatment. The aim of the present study was to test the transformation of lactose via fermentation into 2,3butanediol (2,3-BD) in the presence of a genetically modified strain of Escherichia coli K12 MG1655 (E. coli JFR12) in order to evaluate the potential of W and PW as lactose sources. Lactose (a disaccharide), glucose and galactose (monosaccharides of lactose) at concentrations of 12.5, 25 and 50 g/L were fermented in a synthetic culture medium (medium 9, M9) inoculated at a ratio of 10% (v/v), at 37ºC, 1 atm, initial pH 7.4 and 100 rpm for 72 h in flasks of 0.5 L with a working volume of 0.2 L. Under these operating conditions, the highest 2,3-BD yield was around 0.38 g/g saccharide in the presence of 25 g/L of glucose or lactose at 48 and 72 h, respectively, and 0.19 g/g galactose at 48 h. Therefore, E. coli JFR12 could transform galactose into 2,3-BD but being its yield the half of one observed with glucose at 48 h. Whey (31 g/L lactose) or PW (34 g/L lactose) were fermented in dilution with M9 (50:50, 75:25 and 100:0, v/v) under the same operating conditions. The fermentation of W and PW in absence of M9 (100:0, v/v) produced the highest 2,3-BD yield (up to 0.43 g/g lactose) at 72 h. Whereas, the highest acetoin (A) yield of 0.12 g/g lactose was obtained with[END_REF] can also be obtained from A by its hydrogenation using a heterogeneous catalyst based on Ru at temperature and hydrogen partial pressures up to 175ºC and almost 20 atm, respectively, for 16 h [START_REF] Ochoa Gómez | Method for manufacturing 2,3-butanediol[END_REF]. Bacteria species like Bacillus, Enterobacter and Klebsiella are natural producers of 2,3-BD at high yields using lactose contained in W and PW [START_REF] Guo | Efficient production of 2,3-butanediol from cheese whey powder (CWP) solution by Klebsiella pneumoniae through integrating pulsed fed-batch fermentation with a two-stage pH control strategy[END_REF]. For example, [START_REF] Guo | Efficient production of 2,3-butanediol from cheese whey powder (CWP) solution by Klebsiella pneumoniae through integrating pulsed fed-batch fermentation with a two-stage pH control strategy[END_REF] reported a 2,3-BD yield of 0.43 g/g lactose using K. pneumoniae CICC 10781 fermenting 74 g/L of lactose with an inoculum of 10% (v/v) at 35ºC, pH 6, 300 rpm and 1 vvm after 32 h. However, some of these bacteria such as Enterobacter and Klebsiella species are pathogen, limiting their use in the industry [START_REF] Cho | Enhanced 2,3-butanediol production by optimizing fermentation conditions and engineering Klebsiella oxytoca M1 through overexpression of acetoin reductase[END_REF], PHAC 2017). Thus, it is necessary to modify genetically non-pathogen strains species such as Escherichia coli K12 MG1655 to produce 2,3-BD since they do not possess its metabolic pathway (Bialkowska 2016[START_REF] Fernández-Gutierrez | Biovalorization of saccharides derived from industrial wastes such as whey: a review[END_REF]. Hence, in the current study, E. coli K12 MG1655, an innocuous strain to human being (iGEM 2017), was considered to host the 2,3-BD metabolic pathway from Enterobacter cloacae (named as E. coli JFR12) in order to ferment both W and PW and produce 2,3-BD.

The main objective of this study was to ferment W and PW to produce 2,3-BD using E. coli JFR12. In addition, to the best of our knowledge, neither W or PW have been valorized into 2,3-BD using an E. coli strain.

Previously, M9 supplemented with 15 g/L of urea was determined as the most suitable culture medium to produce ABD (A + 2,3-BD) from glucose using the modified strain E. coli JFR1 [START_REF] Fernández-Gutierrez | Biovalorization of glucose in four culture media and effect of the nitrogen source on fermentative alcohols production by Escherichia coli[END_REF]). In the present study, the effect of glucose, galactose and lactose concentrations in M9 fermented with E. coli JFR12 was studied. In addition, two mixtures of glucose-galactose (1:1, w/w) in M9 for a total concentration of 25 and 50 g/L were fermented. Afterwards, two natural industrial effluents, W1 and PW1, were fermented in order to test the ability of E. coli JFR12 to grow and transform the lactose in W1 and PW1 into 2,3-BD at different dilution ratios with M9. The effects of initial pH, inoculum size and agitation on 2,3-BD yield were also studied in absence of M9. Moreover, 'second' batches of W (W2) (51 g lactose/L) and PW (PW2) (47 g lactose/L) were fermented in flasks in order to test the influence of a higher lactose concentration in both effluents. Finally, W2 and PW2 were studied in a 2 L bioreactor (0.8 L of working volume) to compare the 2,3-BD yields with those obtained in 0.5 L flask (0.2 L of working volume). The effect of the aeration on 2,3-BD and A yields was also performed fermenting only W2.

Materials and methods

Microorganism

The microorganism used in the present study was E. coli K12 MG1655, hosting the 2,3-BD metabolic pathway from Enterobacter cloacae (E. coli JFR12). The biosynthetic pathways of phosphate acetyltranferase (pta), fumarate reductase (frd) and fermentative D-lactate dehydrogenase (ldhA) were blocked to avoid the formation of acetic acid, succinic acid and lactic acid, respectively. The modified strain was E. coli K12 MG1655 pir + Dpta, DldhA, Dfrd + budABC (integrated in the chromosome) + plasmid pOSIP-TT + budABC (not integrated in the chromosome). The conservation of E. coli JFR12 was performed at -81ºC in a blend (50:50, v/v) of glycerol and lysogeny broth (LB) culture medium.

Culture media

Lysogeny broth (LB) culture medium was used to grow E. coli JFR12 (seed culture). LB culture medium has proved to be suitable for recombinant E. coli strains (Maniatis et al. 2001).

The composition of LB culture medium was prepared as follows: 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium chloride (NaCl) and distilled water [START_REF] Berney | Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection[END_REF] Tetracycline was added at a concentration of 15 μg/mL in order to favor the growth of the modified strain which contained the resistance plasmid, avoiding the biological contamination from other bacteria (SLH 2017).

The seed culture medium was prepared as follows: a sample of E. coli JFR12 from the conservation blend was taken with a tip and mixed with 10 mL of fresh LB medium in a test tube (15 mL). The test tube was incubated overnight at 37ºC (pre-culture medium). Then, 2 mL of the pre-culture medium were transferred into a 0.5 L Erlenmeyer flask containing 0.2 L of fresh LB medium and incubated in a rotary shaker incubator (Fermentation Design inc, Allentown, PA) at 37ºC, 1 atm, initial pH 6.5 and 100 rpm to reach a bacterial population of 4.8x10 7 colony-forming unit (CFU)/mL in 9 h. The seed culture medium was used to inoculate the culture media M9 and all industrial samples (diluted and undiluted with M9 or distilled water) at a ratio of 10% (v/v).

Fermentation parameters

The fermentations in 0.5 L flasks were performed using a working volume 0.2 L: glucose, galactose and lactose at initial concentration of 12.5, 25 and 50 g/L, mixture of glucosegalactose (1:1, w/w) at initial concentrations of 25 and 50 g/L, W1 and PW1 diluted with M9 (50:50, 75:25 and 100:0, v/v), and W2 and PW2 diluted with distilled water (dilution at 30 and 18%, v/v, respectively). The flasks were inoculated with a seed culture at a ratio of 10% (v/v) and incubated at 37ºC, 1 atm, initial pH 7.4 and 100 rpm for 72 h. In order to analyze the effect of fermentation operating conditions, W1 and PW1 were operated at: i) initial pHs of 6.5, 7.0 and 7.4, 10% (v/v) of inoculum, 37ºC, 1 atm and 100 rpm; ii) inoculum sizes of 5, 7.5 and 10% (v/v), 37ºC, 1 atm, pH 7.4 and 100 rpm; and iii) agitation rates of 50, 100 and 200 rpm, 10% (v/v) of inoculum, 37ºC, 1 atm and an initial pH of 7.4.

Whey (W1 and W2) and permeate whey (PW1 and PW2) were fermented using a 2 L bioreactor (0.8 L of working volume) at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 7.4 and 100 rpm for 72 h. The effect of air addition was tested using the 2 L bioreactor at 2.5 vvm fermenting diluted W2. All the operating conditions are summarized in Table 1.

Analytical methods

The concentration of saccharides (glucose, galactose and lactose), 2,3-BD, A and ethanol (Et) was determined by high performance liquid chromatography (HPLC) using an Agilent series 1100 chromatograph (Agilent Technologies, Palo Alto, CA) equipped with a column Aminex HPX-87H (300 x 7.8 mm) at a temperature of 25ºC; sulfuric acid 0.005M was used as a mobile phase (0.4 mL/min). Saccharides, 2,3-BD and Et were quantified by a Refractive index (RID) detector, and A by an ultraviolet (UVD) detector.

The reagents used for the analytical method were HPLC grade: Glucose (≥ 99.5%), 2,3-BD (98%) and A were purchased at Sigma-Aldrich (Canada), galactose (> 99%) was bought at Acros Organics (Belgium), lactose (≥ 99%) and Et (≥ 99%) were purchased at Fisher Scientific Inc. (Canada).

Statistical analysis

The influence of operating parameters (i.e., saccharide concentration, mixture ratios with M9 (W1 and PW1) and with distilled water (W2 and PW2), inoculum size, initial pH, agitation rate and anaerobic conditions) on 2,3-BD yield were determined by means of an analysis of variance (ANOVA) with a P-value lower than 0.05. Dixon's Q test was used to estimate and reject outlier values of fermentative products yield (2,3-BD, A and Et) using a confidence level of 95% [START_REF] Rorabacher | Statistical treatment for rejection of deviant values: critical values of Dixon's" Q" parameter and related subrange ratios at the 95% confidence level[END_REF].

Table 4.1: Operating conditions used to ferment saccharides (glucose, galactose, lactose and the mixture glucose-galactose, G-Gal) in M9, whey (W1) and permeate whey (PW1) diluted with M9, and the study of inoculum size, initial pH and agitation in flask. New batch of W (W2) and PW (PW2) diluted and undiluted were tested in flask and in a 2 L bioreactor. Fermentations performed at 37ºC, 1 atm and 72 h.

Results and discussion

Effect of kind and concentration saccharides on 2,3-butanediol yield

The modified E. coli JFR12 was used to produce 2,3-BD by fermentation of saccharides (lactose, glucose and galactose) and industrial wastes (W1 and PW1) as carbon sources. Figure 4.1 shows the metabolic pathway to produce 2,3-BD from lactose using a genetically modified strain of E. coli K12 MG1655 (E. coli JFR12). The disaccharide has to be first hydrolyzed into glucose and galactose (monosaccharides) by the enzyme β-galactosidase, breaking the glycosidic bond. Glucose is enzymatically fermented and transformed into pyruvic acid (PA) by glycolysis and galactose is transformed by the Leloir pathway into glucose-6-phosphate, which is transformed into PA by glycolysis [START_REF] Lazar | Awakening the endogenous Leloir pathway for efficient galactose utilization by Yarrowia lipolytica[END_REF]. Then, 3 successive enzymatic reactions are performed: i) from PA to α-acetolactate by the enzyme αacetolactate synthase (ALS), ii) from α-acetolactate to A by the enzyme α-acetolactate decarboxylase (ALDC), and iii) from A to 2,3-BD by the enzyme 2,3-butanediol dehydrogenase (BDH) [START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF], Xu et al. 2014, Xu et al. 2015). Under aerobic conditions, which may inhibit the ALS enzyme, α-acetolactate may be transformed into diacetyl; and then into A, being A transformed into 2,3-BD [START_REF] Shi | Microbial production of 2, 3-butanediol by a newlyisolated strain of Serratia marcescens[END_REF]. In these reactions, the coenzyme nicotinamide adenine dinucleotide (NAD), which has a reduced and an oxidized form (NADH + H + and NAD + , respectively), is involved in the transformation of diacetyl into A and of A into 2,3-BD where NADH is oxidized to NAD + [START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]. The fermentation conditions to perform this series of experiments are shown in Table 4.1. After the first 8 h, the conversion of glucose was around 60% for the [Glu] 0 of 12.5 g/L and 40% for the [Glu] 0 of 25 and 50 g/L. Glucose conversion was 100% at 24 h for a [Glu] 0 of 12.5 g/L, and at 48 h for a [Glu] 0 of 25 g/L. However, for a [Glu] 0 of 50 g/L, the conversions were close to 60 and 80% at 24 and 48 h, respectively; whereas the maximum conversion was slightly lower compared to 12.5 and 25 g/L of glucose and reached 94% after 72 h ( [Glu] 0 , at 8 h of fermentation, glucose conversions varied between 40 and 60%, which might be due to the proliferation of bacterial cells [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF]. The bacterial population might be affected by a high substrate concentration, leading to a slower bacterial growth [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF]. This may explain the glucose conversions observed for [Glu] 0 of 25 and 50 g/L, where longer times (48 and 72 h, respectively) were required in order to obtain the maximum conversion.

Glucose

For the [Glu] 0 of 12.5 g/L and 25 g/L, the 2,3-BD reached a maximum yield and then decreased. For 12.5 g/L, the 2,3 BD yield was 0.21 g/g glucose at 24 h and then decreased to 0.07 g/g glucose at 72 h; for 25 g/L it increased up to 0.38 g/g glucose at 48 h, and then decreased to 0.22 g/g glucose at 72 h (p < 0.05). In the case of [Glu] 0 of 50 g/L, the 2,3-BD yield increased up to 0.27 g/g glucose (p < 0.05) up to 72 h (Figure 4.2B). In this way, the maximum 2,3-BD yield increased from a [Glu] 0 of 12.5 to 25 g/L and decreased in the range from 25 to 50 g/L of glucose, which points out that a high substrate concentration not only may have an inhibitory effect on the bacterial growth, but also may affect the 2,3-BD formation, as suggested by other authors [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF][START_REF] Priya | Microbial production of 2, 3butanediol through a two-stage pH and agitation strategy in 150 L bioreactor[END_REF][START_REF] Xin | Production of 2,3-butanediol from sucrose by a Klebsiella species[END_REF][START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF].

The accumulation of A decreased when [Glu] 0 increased (p < 0.05). At 72 h for 12.5 g/L of glucose, the A yield increased up to 0.18 g/g glucose; whereas for 25 g/L, the maximum yield was 0.08 g/g glucose, and for 50 g/L, A was not detected (Figure 4.2C). During the conversion of glucose to PA, 2 molecules of NAD + are transformed into NADH.

On the other hand, when A is transformed into 2,3-BD by the enzyme BDH, a molecule of NADH is oxidized to NAD + . Therefore, BDH enzyme is regulated to produce A or 2,3-BD by the coenzymes NADH and NAD + (Figure 1). These transformations occur when glucose is available in the culture medium. However, when glucose is exhausted, the pool of free NAD + must be higher than NADH since NAD + cannot be reduced to NADH in the PA metabolic pathway; while NADH is oxidized to NAD + during the reaction from A to 2,3-BD. This causes the reversible reaction and transformation of 2,3-BD into A [START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF], being 2,3-BD considered as an energy-storing metabolite. This phenomenon was clearly observed in the present study for the lowest [Glu] 0 of 12.5 g/L between 24 and 72 h and for [Glu] 0 of 25 g/L between 48 and 72 h. It has also been reported by Li et al. (2010) using E. coli JM109 when glucose conversion was 100% for a [Glu] 0 of 60 g/L at 10% (v/v) of inoculum, 37ºC, pH 6.8 and 500 rpm for 48 h.

The Et maximum yields decreased from 0.09, to 0.05 and to 0.02 g Et/g glucose when glucose conversions were 100% for [Glu] 0 of 12.5, 25 and 50 g/L, respectively (Table 2). Ethanol formation, as 2,3-BD, depends on the use of NADH to be generated. The increase of the [Glu] 0 might have an effect on the enzymes involved in Et formation reducing its yield and thus increasing the 2,3-BD yield [START_REF] Ji | Engineering Klebsiella oxytoca for efficient 2, 3-butanediol production through insertional inactivation of acetaldehyde dehydrogenase gene[END_REF], as observed in the [Glu] 0 range between 12.5 and 25 g/L.

The highest 2,3-BD yield obtained with glucose at a concentration of 25 g/L was nearly 80% of the theoretical yield, i.e. 0.38 g/g glucose at 48 h. The 2,3-BD yield is close to the one reported for other E. coli strains using glucose concentrations higher than 25 g/L (from 50 to 80 g/L of glucose) ranging between 0.39 and 0.43 g/g glucose [START_REF] Tong | Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production[END_REF], Xu et al. 2014). For example, a [Glu] 0 of 50 g/L in M9 supplemented with 5 g/L of yeast extract fermented in the presence of E. coli BL21 gave a 2,3-BD yield of 0.43 g/g glucose at 1% (v/v) of inoculum, 37ºC and 180 rpm (pH not mentioned) for 24 h [START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF]. At 8 h, the galactose conversion was around 40% whatever the [Gal] 0 used. However, at 24 h of fermentation, the galactose conversion depended on the [Gal] 0 reaching 100, 92 and 59% for 12.5, 25 and 50 g/L respectively. At 72 h, for the highest [Gal] 0 (50 g/L), the galactose conversion reached 90% (Figure 4.3A). The trend for galactose conversion was similar to the one of glucose (Figure 2A), being the lowest conversion obtained fermenting 50 g/L of galactose.

For the [Gal] 0 of 12.5 g/L, the maximum 2,3-BD yield increased up to 0.12 g/g galactose at 24 h and then decreased by 33% (0.08 g/g galactose) at 72 h; whereas with the [Gal] 0 of 25 g/L, the maximum 2,3-BD yield reached 0.17 g/g galactose at 24 h, remained nearly constant (0.19 g/g galactose) at 48 h and decreased to 0.11 g/g galactose at 72 h (p < 0.05). The highest 2,3-BD yield (0.23 g/g galactose) was obtained with 50 g/L of galactose at 72 h, which was around 2.9 and 2.1 fold higher (p < 0.05) than the yields obtained at 72 h with 12.5 g/L (0.08 g/g galactose) and 25 g/L (0.11 g/g galactose), respectively (Figure 4.3B).

For the [Gal] 0 of 12.5 g/L, A was detected at 24 h. The A yield reached a plateau at 0.08 g/g galactose at 48 h. For the [Gal] 0 of 25 g/L, A formation occurred at 48 h and reached a similar A yield (0.07 g/g galactose) at 72 h compared to the one obtained for a [Gal] 0 of 12.5 g/L. On the other hand, A was not detected for a [Gal] 0 50 g/L (Figure 4.3C). The maximum Et yields for a [Gal] 0 of 12.5, 25 and 50 g/L were 0.08, 0.05 and 0.02 g/g galactose, respectively (Table 4.2). According to the best of our knowledge, E. coli has not been previously used for galactose fermentation in order to produce 2,3-BD. However, other bacteria like Raoultella were used for that purpose [START_REF] Kim | High production of 2,3butanediol (2,3-BD) by Raoultella ornithinolytica B6 via optimizing fermentation conditions and overexpressing 2,3-BD synthesis genes[END_REF]. For example, using R. ornithinolytica B6, the 2,3-BD yield was 0.24 g/g galactose for a [Gal] 0 of 82 g/L (concentration calculated using the data provided by the authors) at 10% (v/v) of inoculum, 25°C, pH 7, 200 rpm and 0.5 vvm for 36 h (Kim et al. 2016). The 2,3-BD yield was similar to the highest yield (0.23 g/g galactose) obtained in the present study with a [Gal] 0 of 50 g/L. As observed, E. coli JFR12 was able to transform galactose by fermentation into 2,3-BD but with lower 2,3-BD yields compared to those obtained with glucose. As an example, the 2,3-BD yield was twice lower at 48 h using 25 g/L of galactose and glucose: 0.19 g/g galactose and 0.38 g/g glucose, respectively. . Lactose conversion decreased with an increase of [Lac] 0 . After 8 h of fermentation, conversion of 75, 58 and 25% were obtained for [Lac] 0 of 12.5, 25 and 50 g/L, respectively. A lactose conversion of 100% was achieved at 24 h for 12.5 g/L, whereas it was 90 and 65% for 25 and 50 g/L, respectively.

Lactose

For the [Lac] 0 of 25 g/L, the lactose conversion was 100% at 48 h and nearly 100% for 50 g/L at 72 h (Figure 4.4A).

Using lactose, the 2,3-BD yields were similar to glucose for the same operating conditions. For the smallest [Lac] 0 of 12.5 g/L, the 2,3-BD yield increased from 0.13 g/g lactose at 8 h to 0.27 g/g lactose at 24 h. Then, the 2,3-BD yield decreased by 44% (0.15 g/g lactose) at 72 h (p < 0.05). For the 2 other glucose concentrations, this phenomenon was not observed and the 2,3-BD yield increased with the time reaching a plateau of 0.36 g/g lactose for [Lac] 0 of 25 g/L at 48 h and 0.28 g/g lactose for [Lac] 0 of 50 g/L at 72 h (Figure 4.4B).

For [Lac] 0 of 12.5 g/L, A was detected at 24 h, and then its yield increased 3 fold up to 72 h: from 0.05 g/g lactose at 24 h to 0.15 g/g lactose at 72 h (p < 0.05). For a [Lac] 0 of 25 g/L, the A yield was 0.04 g/g lactose at 48 h and 0.10 g/g lactose at 72 h; whereas A was not detected for the [Lac] 0 of 50 g/L (Figure 4.4C). The maximum Et yields for [Lac] 0 of 12.5, 25 and 50 g/L were 0.10, 0.05 and 0.02 g/g lactose, respectively (Table 4.2). The Et yields were similar to the yields obtained with glucose and galactose; thus, the Et formation seems not to depend on the carbon source, but on its concentration. The highest 2,3-BD yield (0.36 g/g lactose) obtained in the present study from [Lac] 0 of 25 g/L at 72 h was similar to those obtained using other wild bacteria like K. pneumoniae and K. oxytoca [START_REF] Guo | Efficient production of 2,3-butanediol from cheese whey powder (CWP) solution by Klebsiella pneumoniae through integrating pulsed fed-batch fermentation with a two-stage pH control strategy[END_REF]. For example, a maximum 2,3-BD yield was reached at 24 h in the presence of K. pneumoniae CICC 10781 (0.38 g/g lactose) and K. oxytoca CICC 21518 (0.36 g/g lactose) when both strains fermented 60 g/L of lactose at 10% (v/v) of inoculum, 37ºC, initial pH 6 and 200 rpm [START_REF] Guo | Efficient production of 2,3-butanediol from cheese whey powder (CWP) solution by Klebsiella pneumoniae through integrating pulsed fed-batch fermentation with a two-stage pH control strategy[END_REF]. Therefore, taking into account the results obtained in the present study, E. coli JFR12 was able to produce 2,3-BD from lactose and, in addition, is harmless for human health.

Results show that the use of galactose as sole carbon source gave 2,3-BD yields up to 2 fold lower compared to glucose as shown in Figures 4.2B and 4.3B (p < 0.05). However, galactose effect on the 2,3-BD yield was not observed using lactose as a carbon source after its hydrolysis by β-galactosidase enzyme (Figure 4.4B). In this way, it can be hypothesized that β-galactosidase enzyme plays an important role on the 2,3-BD formation. In order to break down saccharides, energy (e.g. that obtained by oxidizing ATP, adenosine triphosphate) is required at the first step of the fermentation [START_REF] Berg | Biochemistry[END_REF]. According to Ishikawa et al. (2015), the β-galactosidase enzyme provides both energy and carbon when lactose is hydrolyzed, and its activity is induced in the presence of lactose [START_REF] Chu | The effect of fructose, galactose, and glucose on the induction of β-galactosidase in Escherichia coli[END_REF]. This may explain why lactose and glucose gave similar maximum 2,3-BD yields at 12.5, 25 and 50 g/L and why galactose alone provided lower 2,3-BD yields.

Glucose-galactose

A mixture of glucose-galactose (G-Ga) was fermented to study the effect of the mixture on 2,3-BD formation and to understand the similar 2,3-BD yields obtained for lactose and glucose. The glucose (G) and galactose (Ga) concentrations for this assay were (1:1, w/w), i.e. 12.5 and 25 g/L of each monosaccharide for a total concentration of 25 and 50 g/L.

Figure 4.5 (A and B) shows the conversion, 2,3-BD and A yields for a mixture of G-Ga (25 and 50 g/L) as a function of time. For 25 g/L of G-Ga mixture (Figure 4.5A), conversions of glucose and galactose were 70 and 27%, respectively, at 8 h, and increased to 100% for both monosaccharides after 24 h of fermentation. The 2,3-BD yield increased and reached a maximum at 24 h (0.19 g/g mixture of G-Ga); then, the 2,3-BD yield decreased by 47 % down to 0.10 g/g mixture of G-Ga at 72 h (p < 0.05). Acetoin was not detected until 48 h and reached a yield of 0.07 g/g mixture of G-Ga at 72 h.

For the [G-Ga] 0 of 50 g/L (Figure 4.5B), the conversion of glucose and galactose was 60 and 30%, respectively, at 8 h. At 24 h, the glucose conversion reached 100%, whereas the galactose conversion was 76%. The galactose conversion reached 100% at 72 h. The maximum 2,3-BD yield increased with the time and reached 0.24 g/g mixture of G-Ga at 72 h. Acetoin was not detected during the mixture of monosaccharides fermentation at 50 g/L. The maximum Et yields were 0.05 and 0.02 g/g mixture of G-Ga for the [G-Ga] 0 of 25 and 50 g/L, respectively, which were similar to those using glucose, galactose and lactose alone at those saccharides concentration (Table 4.2). On one hand, glucose was preferred over galactose as a carbon source by E. coli JFR12 for both [G-Ga] 0 as shown in Figure 4.5 (A and B). As reported by [START_REF] Luo | The transport and mediation mechanisms of the common sugars in Escherichia coli[END_REF], glucose is at the top of the saccharide hierarchy to be metabolized by E. coli causing the phenomenon called glucose carbon catabolite repression setting galactose use aside, which may explain the higher glucose conversion for [G-Ga] 0 at 25 and 50 g/L. On the other hand, the 2,3-BD yields obtained for [G-Ga] 0 at 25 and 50 g/L were similar to those obtained with galactose (Figure 4.3B); thus, this might confirm that galactose hampers the formation of 2,3-BD by acting on the enzymes involved in its production. Acetoin and Et yields were similar in presence of glucose, galactose, lactose and mixture G-Ga at 25 and 50 g/L, which suggests that their formation depends on the saccharide concentration and not on the kind of carbon source. (A and B) presents the 2,3-BD yield obtained for the highest saccharide conversion at 25 and 50 g/L of saccharides (glucose, galactose, lactose and G-Ga). Figure 4.6A shows the 2,3-BD yield when saccharide conversion was 100%: glucose (48 h), galactose (48 h), lactose (48 h) and the mixture G-Ga (24 h) at 25 g/L. The 2,3-BD yields for glucose and lactose were around 0.35 g/g saccharide, whereas for galactose and the mixture G-Ga, it was 46% lower (0.19 g/g saccharide) (p < 0.05). Figure 4.6B shows that the 2,3-BD yields for glucose and lactose at a concentration of 50 g/L at 72 h were around 0.28 g/g saccharide and it was slightly smaller for galactose and the mixture G-Ga of around 0.25 g /g saccharide. 4.6A: 2,3-BD yield using glucose (48 h), galactose (48 h), lactose (48 h) and mixture G-Ga (24 h) when conversion was 100% in all cases. Figure 4.6B: 2,3-BD yield using glucose (conversion 94%), galactose (conversion 90%), lactose (conversion 96%) and mixture G-Ga at 72 h (conversion 100% for both monosaccharides). Results are means ± SD of 2 experiments performed in duplicate.

On one hand, the maximum conversion of saccharides (between 90 and 100% at 72 h) was similar at 50 g/L (Figures 4.2A,4.3A,4.4A and 4.5B). The explanation might be linked to the high saccharide concentration (50 g/L), which might cause the metabolism inhibition of E. coli JFR12. All bacteria have a substrate concentration limit where the bacterial metabolism may be inhibited if it is exceeded, affecting the maximum 2,3-BD yield (Chan et al. 2016, Priya et al. 2016). This phenomenon was also observed in the present study comparing the 2,3-BD yields at 50 g saccharides/L (glucose, galactose, lactose and mixture G-Ga) since the maximum 2,3-BD was similar. This might confirm that the substrate inhibitory effect on the bacterial metabolism is caused by a high saccharide concentration whose range might be between 25 and 50 g/L for E. coli JFR12, explaining similar 2,3-BD yield (around 0.25 g/g saccharide) at 50 g/L obtained whatever the saccharide used. Lactose can be transformed into 2,3-BD as efficiently as glucose since galactose obtained from lactose hydrolysis does not impede the formation of 2,3-BD, which was clearly observed at 25 g/L of saccharides.

Whey

The genetically modified E. coli JFR12 was also tested to ferment lactose contained in W1 and PW1. In order to determine the effect of substrate concentration on 2,3-BD yield, W1 and PW1 were fermented using three [Lac] 0 . The lactose concentration was adjusted by diluting W1 and PW1 with M9 medium. For W1, the [Lac] 0 tested were 15.5, 23 and 31 g/L while for PW1 17, 25.5 and 34 g/L were used. The fermentations were performed under the operating conditions shown in Table 1. (A, B and C) shows the lactose conversion, 2,3-BD and A yields using whey as a function of time and [Lac] 0 . For a [Lac] 0 of 15.5 g/L, the lactose conversion was 52% at 8 h and reached 100% at 24 h. On the other hand, using a [Lac] 0 of 23 g/L, the conversion increased with the time and reached 100% at 48 h; whereas with a [Lac] 0 of 31 g/L, the lactose conversion increased and reached 100% at 72 h (Figure 4.7A).

The 2,3-BD yield increased from 0.08 to 0.24 g/g lactose at 8 and 24 h, respectively in the presence of 15.5 g/L of lactose, and then decreased by 38% (0.15 g/g lactose) at 72 h (p < 0.05). Fermenting 23 g/L of lactose, the 2,3-BD yield increased from 0.04 g/g lactose at 8 h to 0.40 g/g lactose at 48 h remaining constant up to 72 h (p < 0.05). On the other hand, the 2,3-BD yield increased from 0.04 g/g lactose at 8 h to reach a maximum 2,3-BD yield of 0.43 g/g lactose at 72 h (p < 0.05) with 31 g/L of lactose (Figure 4.7B). Figure 4.7C shows that A yield increased from 0.03 g/g lactose at 24 h to 0.12 g/g lactose at 72 h for a [Lac] 0 of 15.5 g/L. Acetoin was not detected up to 72 h with 23 and 31 g/L of lactose, being the A yields 0.07 and 0.04 g/g lactose, respectively. These results were close to those obtained using lactose alone at 12.5 and 25 g/L in M9 (Figure 4.4B and C).

The formation of A was observed when the lactose conversion was 100% in presence of E. coli JFR12, i.e. at 24 h for the [Lac] 0 of 15.5 g/L and at 72 h for [Lac] 0 of 23 and 31 g/L. As mentioned before, the BDH enzyme catalyzes reversibly the reaction A 2,3-BD (NAD + /NADH + H + ). The reaction is directed to A when lactose conversion is 100% and the pool of free NAD + increases, which decreases the amount of 2,3-BD. This phenomena was clearly observed in the present study for the lowest [Lac] 0 of 15.5 g/L. (A, B and C) presents the lactose conversion, 2,3-BD and A yields as a function of time using PW1 diluted with M9. Fermenting 17 g/L of lactose the conversion was 59% at 8 h and then reached 100% at 24 h. In the presence the [Lac] 0 of 25.5 g/L, the lactose conversion increased from 54% at 8 h to 100% at 72 h. Similarly, the lactose conversion increased from 39% at 8 h to 100% at 72 h with 34 g/L (Figure 4.8A).

Similarly to W1, the 2,3-BD yield increased from 0.06 g/g lactose at 8 h to 0.26 g/g lactose at 24 h for the lowest [Lac] 0 of 17 g/L. Then, the 2,3-BD yield decreased by 38% down to 0.16 g/g lactose at 72 h (p < 0.05). For the 2 other [Lac] 0 , the 2,3-BD yield increased from 0.04 g/g lactose at 8 h to achieve the maximum 2,3-BD yield of 0.42 g/g lactose at 72 h (p < 0.05), as shown Figure 4.8B.

Acetoin was detected at 24 h fermenting 17 g/L of lactose, being its yield 0.03 g/g lactose increasing up to 0.12 g/g lactose at 72 h. On the other hand, A reached a yield of 0.07 g/g lactose for 25.5 g/L of lactose, whereas it was not detected at 34 g/L at 72 h (Figure 4.8C). As observed in Figures 4.7 and 4.8, the yields of 2,3-BD and A were quite similar for W1 and PW1 at the 3 dilution ratios, showing that the 2,3-BD and A yields are mainly determined by the lactose concentration. For W1 and PW1, the highest 2,3-BD yields were obtained at the dilution ratios of 75:25 and 100:0 (v/v). Therefore, the best results were obtained in absence of M9 because minerals and nutrients contained in W1 and PW1 [START_REF] Panesar | Bioutilisation of whey for lactic acid production[END_REF], USDEC 2017) could support the growth of E. coli JFR12 and the formation of 2,3-BD without adding other macro or micronutrients present in the synthetic culture medium. For this reason, neither W1 nor PW1 were diluted with M9 for the subsequent experiments. In addition, the use of PW1 will be preferred because it is an end effluent in dairy industry which should be valorized.

To our best knowledge, the fermentation of W and PW has not been performed using E. coli strains. These effluents have been fermented with other bacteria, e.g. E. aerogenes, K. oxytoca, K. pneumoniae or Lactococcus lactis to produce 2,3-BD obtaining yields in the range from 0.20 to 0.43 g/g lactose [START_REF] Kandasamy | Synthesis of (3R)-acetoin and 2,3-butanediol isomers by metabolically engineered Lactococcus lactis[END_REF][START_REF] Lee | Continuous production of 2,3-butanediol from whey permeate using Klebsiella pneumoniae immobilized in calcium alginate[END_REF][START_REF] Perego | 2,3-Butanediol production by Enterobacter aerogenes: selection of the optimal conditions and application to food industry residues[END_REF][START_REF] Vishwakarma | Bioconversion of whey to 2, 3-butanediol using Klebsiella oxytoca NRRL-13-199[END_REF]). For example, [START_REF] Perego | 2,3-Butanediol production by Enterobacter aerogenes: selection of the optimal conditions and application to food industry residues[END_REF] fermented W at a [Lac] 0 of 20 g/L using E. aerogenes NCIMB 10102 obtaining a 2,3-BD yield of 0.43 g/g lactose with an inoculum of 0.25 g/L and at 39ºC, pH 6 and 150 rpm for 250 h. Whereas, Vishwakarma (2014) reported a 2,3-BD yield of 0.20 g/g lactose (2,3-BD yield calculated from the data provided by the author) in the presence of K. oxytoca NRRl-13-199 using PW (30-32.5 g/L of lactose) and 1% (v/v) of inoculum at 30°C, pH 6.5 and 60 rpm for 96 h.

The use of E. coli JFR12 in the present study has several advantages in comparison to the previous mentioned bacteria: i) the strain E. coli JFR12 is harmless for human, whereas E. aerogenes, K. oxytoca and K. pneumoniae are known pathogens (iGEM 2017, PHAC 2017); ii) the highest 2,3-BD yield (0.43 g/g lactose) obtained with E. coli JFR12 is similar to the best yields obtained with pathogen strains; and iii) the fermentation time of 72 h to obtain the highest 2,3-BD yield in the present study was shorter than the times required for fermentations using E. aerogenes, K. oxytoca and K. pneumoniae, which vary from 96 to 250 h.

Effect of fermentation parameters

The effect of 3 operating conditions (initial pH, inoculum size and agitation rate) on 2,3-BD yield was studied. Both W and PW were fermented during 72 h using E. coli JFR12 under the operating conditions mentioned before (Table 4.1).

Initial pH

Figure 4.9 (A and B) present the evolution of 2,3-BD yield as a function of time and initial pH (pH 0 = 6.5, 7.0 and 7.4) for W1 and PW1 fermentation, respectively. Lactose conversions were 100% after 72 h of fermentation whatever the pH 0 (data not shown).

Between 8 and 72 h, the 2,3-BD yield varied from 0.05 to 0.47 g/g lactose for W1 and from 0.04 to 0.44 g/g lactose using PW1 for the 3 initial pH 0 s tested. Therefore, for the range of pH 0 tested, the 2,3-BD yield did not present any difference for W1 or PW1 (p < 0.05) in presence of E. coli JFR12.

The pH was uncontrolled during W1 and PW1 fermentation; for this reason, the pH decreased to values around 5.8 for all pH 0 . The acidification of the fermentation medium could be caused by the organic acids formation like PA. The pH stayed at 5.8 because metabolic pathways adjusted to avoid a higher medium acidification, favoring the formation of 2,3-BD. This mechanism to control the internal pool of PA was suggested by [START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF]. The lactose conversion with a 5% (v/v) of inoculum was close to 78%, whereas the lactose conversion was 100% for 7.5 and 10% (v/v) of inoculum after 72 h (Figure 4.10A). The 2,3-BD yield increased from 8 to 72 h for W1 and PW1. At 72 h, the highest 2,3-BD yield was 0.38 g/g lactose for 5% (v/v) of inoculum and 0.45 g/g lactose for 7.5 and 10% (v/v) of inoculum size fermenting W1 (Figure 4.10B). Lactose conversions were similar for PW1 and W1, being 79, 89 and 100% for 5, 7.5 and 10% (v/v), respectively at 72 h (Figure 4.11A). The maximum 2,3-BD yield was 0.33 g/g lactose for 5% (v/v) of inoculum and near 0.40 g/g lactose in the presence of 7.5 and 0.46 g/g lactose for 10% (v/v) of inoculum at 72 h (Figure 4.11B). The 2,3-BD yields (0.38 and 0.33 g/g lactose using W1 and PW1, respectively) obtained at 5% (v/v) of inoculum were around 20% lower (p < 0.05) compared to the other 2 inoculum size tested. This could be due to there was lactose in the culture medium without consuming at 72 h. In this way, the use of 5% (v/v) of inoculum might be a critical cellular density to reach a high 2,3-BD yield for a fermentation time of 72 h. Therefore, the use of a small inoculum size (5%, v/v) decreased the 2,3-BD yield, which in accordance with other studies [START_REF] Okonkwo | Process development for enhanced 2,3butanediol production by Paenibacillus polymyxa DSM 365[END_REF]. For example, [START_REF] Perego | Effects of temperature, inoculum size and starch hydrolyzate concentration on butanediol production by Bacillus licheniformis[END_REF] tested an inoculum of B. licheniformis of 0.5 and 10 g/L of dry weight (number of viable bacteria not defined) at a concentration of 20 g/L of glucose, 37ºC, pH 6 and 150 rpm (fermentation time not defined); the 2,3-BD yield increased with inoculum size from 0.1 to 0.35 g/g glucose.

Inoculum size

Agitation

Figures 4.12 and 4.13 (A and B) show the effect of agitation rate on lactose conversion and 2,3-BD yield as a function of time for W1 and PW1, respectively. Table 3 shows the 2,3-BD, A and ABD (A + 2,3-BD) yield at 48 and 72 h for W1 and PW1 as a function of agitation rate.

In the case of W1, at 50 rpm, lactose conversion increased with time from 14% at 8 h to 64% at 72 h, whereas for 100 and 200 rpm, it was close to 20% at 8 h and 100% at 72 h and 48 h, respectively (Figure 4.12A). The 2,3-BD yield increased with time, up to 0.26 g/g lactose at 72 h and 50 rpm. Using an agitation rate of 100 rpm, the 2,3-BD yield reached 0.43 g/g lactose at 72 h; whereas at 200 rpm, the maximum 2,3-BD yield (0.23 g/g lactose) was reached at 24 h and then decreased down to 0.14 g/g lactose (p < 0.05) at 72 h (Figure 4.12B). Acetoin was not detected at 50 rpm during the fermentation and A yield was 0.04 g/g lactose at 72 h and 100 rpm. However, A yield increased up to 0.23 g/g lactose (p < 0.05) at 72 h and 200 rpm (Table 4.3). As shown in Table 4.3, at 200 rpm, the reversible reaction [START_REF]Abstract Whey (W) is generated during cheese manufacturing and permeate whey (PW) is obtained after W deproteinization. As both effluents contain lactose, a carbon source, and they can cause environmental issues if they are released in the environment without a treatment. The aim of the present study was to test the transformation of lactose via fermentation into 2,3butanediol (2,3-BD) in the presence of a genetically modified strain of Escherichia coli K12 MG1655 (E. coli JFR12) in order to evaluate the potential of W and PW as lactose sources. Lactose (a disaccharide), glucose and galactose (monosaccharides of lactose) at concentrations of 12.5, 25 and 50 g/L were fermented in a synthetic culture medium (medium 9, M9) inoculated at a ratio of 10% (v/v), at 37ºC, 1 atm, initial pH 7.4 and 100 rpm for 72 h in flasks of 0.5 L with a working volume of 0.2 L. Under these operating conditions, the highest 2,3-BD yield was around 0.38 g/g saccharide in the presence of 25 g/L of glucose or lactose at 48 and 72 h, respectively, and 0.19 g/g galactose at 48 h. Therefore, E. coli JFR12 could transform galactose into 2,3-BD but being its yield the half of one observed with glucose at 48 h. Whey (31 g/L lactose) or PW (34 g/L lactose) were fermented in dilution with M9 (50:50, 75:25 and 100:0, v/v) under the same operating conditions. The fermentation of W and PW in absence of M9 (100:0, v/v) produced the highest 2,3-BD yield (up to 0.43 g/g lactose) at 72 h. Whereas, the highest acetoin (A) yield of 0.12 g/g lactose was obtained with[END_REF] was clearly observed at 48 and 72 h, when lactose conversion was 100% using W1. In this case, the ABD yield was practically constant (variation of 13%) showing that 2,3-BD might be used by E. coli JFR12 as a carbon source.

When PW1 was fermented, the lactose conversion varied with the time quite similarly to what has been observed for W1 at 50 rpm; 14% at 8 h of fermentation and reaching a maximum of 60% at 72 h. At 100 rpm, lactose conversion increased from 39% at 8 h up to 100% after 72 h. However, at 200 rpm, lactose conversion was 20% at 8 h and the maximum lactose conversion was 90% at 72 h (Figure 4.13A). At 50 rpm, 2,3-BD was not detected for the first 8 h of fermentation, increasing its yield up to 0.26 g/g lactose at 72 h. The highest 2,3-BD yield was reached at 100 rpm for 72 h, achieving 0.42 g/g lactose. However, at 200 rpm, the maximum 2,3-BD yield was 0.26 g/g lactose at 72 h (Figure 4.13B). On the other hand, A was not detected either at 50 or 100 rpm during fermentation. However, A yield at 48 and 72 h was 0.05 g/g lactose at 200 rpm even whether lactose was present in the medium (Table 4.3). Comparing the fermentation of W1 and PW1, the conversion of lactose was higher for W1 compared to PW1 at 200 rpm. This can be due to the presence of more nutrients in W1, because the PW1 is the by-product issued from protein removal of W1 [START_REF] Smith | Evaluation of whey, milk, and delactosed permeates as salt substitutes[END_REF]. This causes that PW1 has less nitrogen and amino acids than W1. The ABD yield was slightly higher for W1, confirming the possible effect of nutrients on ALDC enzyme as suggested by [START_REF] Cho | Enhanced 2,3-butanediol production by optimizing fermentation conditions and engineering Klebsiella oxytoca M1 through overexpression of acetoin reductase[END_REF]. Figures 4.12B and 4.13B show that the agitation rate affected lactose conversions and 2,3-BD yields. On one hand, at a low agitation rate (50 rpm), the lactose conversion was lower for both W1 and PW1. At a relatively high agitation rate (200 rpm) the formation of 2,3-BD and A are linked together and the maximum 2,3-BD yield is lower compared to the one obtained at 100 rpm using W1 and PW1. A high agitation would provide higher oxygen transfer to the culture medium and could cause the inactivation of enzyme ALDC [START_REF] Shi | Microbial production of 2, 3-butanediol by a newlyisolated strain of Serratia marcescens[END_REF]. In this way, a secondary reaction spontaneously may occur transforming α-acetolactate into diacetyl, which is transformed into A generating free NAD + . When A is transformed into 2,3-BD, free NAD + is also obtained and thus the amount of free NAD + increases in the broth (Figure 4.1). In this way, when lactose conversion was 100%, the reversible reaction A 2,3-BD was activated, decreasing the amount of 2,3-BD in the broth and increasing the A yield, as observed for 200 rpm using W1. In addition, according to [START_REF] Priya | Microbial production of 2, 3butanediol through a two-stage pH and agitation strategy in 150 L bioreactor[END_REF], A can accumulate as agitation rate increases because the oxygen dissolved in the broth and the oxidation potential of system also increases, causing the rise of NAD + in the broth even if the carbon source conversion is not 100%. This is in accordance with the assay of PW1 fermentation at 200 rpm of the present study.

4.5.3

Fermentations in bioreactor

Anaerobic fermentation

Whey and PW fermentations were also performed in a 2 L bioreactor with a working volume of 0.8 L. The operating conditions are shown in Table 4.1. Air was not supplied and thus anaerobic conditions were rapidly achieved, decreasing the dissolved oxygen (DO) near 30% (w/v) in the first 10 minutes and no DO was detected (DO close to 0%) in the following 10 minutes. For these experiments, W2 and PW2 were used. Fermentations of W2 and PW2 with or without dilution in distilled water were also performed in flasks; W1 and PW1 were used as controls.

Figure 4.14 (A and B) shows the 2,3-BD yield as a function of time for W1 and W2 fermentation in flasks (Figure 4.14A) and in bioreactor (Figure 4.14B). The 2,3-BD yields obtained in flasks using W1 ([Lac] 0 of 31 g/L) and diluted W2 ([Lac] 0 of 31 g/L) increased with time and were quite similar (nearly 0.40 g/g lactose) at 72 h. In contrast, the 2,3-BD yield obtained at 72 h using W2 ([Lac] 0 of 51 g/L) was around 30% lower than the one reached with W1 and diluted W2 ([Lac] 0 31 g/L) (p < 0.05). For fermentation in bioreactor, at 72 h, 2,3 BD yields of 0.26 and 0.23 g/g lactose were obtained using W1 and diluted W2 ([Lac] 0 31 g/L), respectively. These 2,3 BD yields were 41% lower than those observed in flasks. On the other hand, the 2,3-BD yield using W2 ([Lac] 0 of 51 g/L) in bioreactor was 0.18 g/g lactose (22% lower than the 2,3-BD yield obtained with 31 g/L of lactose). Acetoin was not detected either in flasks or bioreactor using W2 at [Lac] 0 of 31 and 51 g/L (Figure 4.14B). ). The 2,3-BD yield increased with the fermentation time and was similar at 72 h using PW1 ([Lac] 0 of 34 g/L) and diluted PW2 ([Lac] 0 of 34 g/L) with around 0.42 g/g lactose, while it was decreased by nearly the half (p < 0.05) with PW2 ([Lac] 0 of 47 g/L). The 2,3-BD yield using diluted PW2 ([Lac] 0 of 34 g/L) in bioreactor was also smaller (close to 60%) compared to fermentations in flasks (p < 0.05). Acetoin was not detected for any fermentation. Results showed that the 2,3-BD yields obtained in flasks were similar using W1 and diluted W2 ([Lac] 0 of 31 g/L). In addition, the 2,3-BD yields were higher using low lactose concentrations (W1 and diluted W2) than using higher lactose concentrations (W2). Similar results were obtained with PW. On the other hand, the 2,3-BD yield obtained using W2 and PW2 ([Lac] 0 of around 50 g/L) in flasks fermentation was similar to the one obtained with lactose (50 g/L) in M9 in flasks (Figure 4B). This confirms that the 2,3-BD accumulation in the broth is mainly a function of [Lac] 0 regardless of culture medium (M9, W or PW). In addition, these results can confirm that the threshold of lactose concentration may be around 35 g/L of lactose since the 2,3-BD yield increased compared to the results obtained at 25 g/L of lactose and decreased using around 50 g/L of lactose.

The amount of oxygen (O 2 ) in the broth is linked to the bacterial growth and thus to the formation of 2,3-BD (Ji et al. 2009a). However, microaerobic conditions may be used to improve the 2,3-BD yield [START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF]. Under microaerobic conditions, the amount of O 2 (less than 6.7 mg O 2 /L at 37ºC) is limited leading to a suitable bacterial population and avoiding the inhibition of ALS and BDH enzymes, increasing the 2,3-BD formation (Missouri 2017, [START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF]. In this way, the assays performed in the 2 L bioreactor (strict anaerobic conditions) with W2 and PW2 led to lower 2,3-BD yields (up to 43% in the case of W2 and 59% for PW2) compared to those carried out in flasks, where the agitation could permit to dissolve some atmospheric O 2 .

Aerated bioreactor

The effect of aeration to ferment diluted W2 ([Lac] 0 of 31 g/L) in the 2 L bioreactor was tested. The air flow rate was 2 L/min in a working volume of 0.8 L, equivalent to 2.5 vvm of aeration rate. The DO slightly decreased from the beginning of the fermentation of diluted W2 to 1% (w/v) after 12 h of fermentation. Figure 4.16 shows the lactose conversion, 2,3-BD, A and ABD yields as a function of time.

The lactose conversion reached 100% after 24 h of fermentation. The 2,3-BD yield increased from 0.08 g/g lactose at 8 h to 0.40 g/g lactose at 24 h and decreased to reach around 0.26 g/g lactose at 72 h. Acetoin was not detected within 24 h, while its yield reached 0.21 g/g lactose at 48 h and remained nearly constant until 72 h. The ABD yield was practically constant (around 0.40 g/g lactose) from 24 to 72 h. In fact, when the main carbon source (i.e. lactose) is not present in the culture medium (lactose conversion of 100%), the pool of NAD + increases, causing the use of 2,3-BD as a carbon source, decreasing its yield (observed after 24 h) and transforming 2,3-BD into A.

The maximum 2,3-BD yield (0.40 g/g lactose) was slightly lower at 24 h to that obtained in flask (0.43 g/g lactose) at 72 h under the same operating conditions. The air supply favored the lactose conversion (100%) and the 2,3-BD formation, reducing the time to reach the maximum 2,3-BD yield compared to the fermentations performed in flasks. However, 2,3-BD was transformed into A after 24 h. After 24 h, the ABD yield was nearly constant up to 72 h, which points out that 2,3-BD was used as a carbon source by means of BDH enzyme. Hence, on one hand, in an aerated bioreactor, it is possible to obtain the maximum 2,3-BD yield within 24 h; whereas on the other hand, after 24 h, the formation of A occurs in the presence of E. coli JFR12, being possible maximizing the production of either 2,3-BD or A. The 2,3-BD yield at 24 h in the aerated bioreactor was more than 2 fold higher than that in flask under the same operating conditions. According to [START_REF] Shi | Microbial production of 2, 3-butanediol by a newlyisolated strain of Serratia marcescens[END_REF] and [START_REF] Yang | Production of 2, 3 butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens[END_REF], a suitable amount of O 2 may improve the activity of ALS and BDH enzymes. Therefore, the results obtained in the present study show that the use of 2 L/min was appropriate to avoid the inactivation of ALS and BDH enzyme.

Conclusion

Fermentation of different saccharides derived from whey to produce 2,3-butanediol and acetoin using a genetically modified strain of Escherichia coli K12 MG1655 (E. coli JFR12) was performed. Different concentrations (12.5, 25 and 50 g/L) of glucose, galactose, lactose and a mixture of glucose-galactose were tested in a supplemented M9 culture medium at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 7.4 and 100 rpm in 0.5 L flask (0.2 L of working volume). Similar 2,3-BD yields (around 0.38 g/g saccharide) for 25 g/L of glucose or lactose were obtained at 48 h. Galactose gave lower 2,3-BD yields (50% less at 25 g/L and 48 h). However, galactose released by lactose hydrolysis seems not to interfere with the formation of 2,3-BD.

Mixing whey (W1, 31 g/L of lactose) or permeate whey (PW1, 34 g/L of lactose) with and without M9 (50:50, 75:25 and 100:0, v/v) showed that highest 2,3-BD yields were obtained in absence of M9 in the presence of E. coli JFR12, reaching 2,3-BD yields of 0.43 and 0.42 g/g lactose using W1 and PW1, respectively.

The influence of the initial pH (6.5, 7.0 and 7.4), inoculum size (5.0, 7.5 and 10.0%, v/v) and agitation rate (50, 100 and 200 rpm) were tested at 37ºC and 1 atm in flask of 0.5 L (0.2 L of working volume). The initial pH did not have a significant influence, being the 2,3-BD yields 0.47 and 0.44 g/g lactose for W1 and PW1, respectively. On the other hand, the use of 5% (v/v) of inoculum size gave a 2,3-BD yield slightly lower (14% and 18% using W1 and PW1, respectively) compared to 7.5 and 10% (v/v). The highest agitation rate (200 rpm) favored the acetoin formation; whereas the 2,3-BD yield was not improved. The 2,3-BD yield was 23% and 67% lower at 200 rpm compared to the one obtained at 100 rpm at 48 and 72 h, respectively using W1. On the other hand, fermenting PW1 at 200 rpm, the 2,3-BD yield was 61% and 38% lower at 48 h and 72 h, respectively, compared to the results at 100 rpm.

Another batch of W (W2, 51 g/L of lactose) and PW (PW2, 47 g/L of lactose) were tested in flasks. Higher lactose concentrations conducted to lower 2,3-BD yields: 30% and 46% using W2 and PW2, respectively.

Whey 2 and PW2 fermentation were also performed in a 2 L bioreactor under anaerobic conditions, which reduced the 2,3-BD formation for all the fermentation times, whereas the supply of air (2.5 vvm) reduced the time by 3 fold to obtain the highest 2,3-BD yield of 0.40 g/g lactose at 24 h, in comparison to the fermentations performed in flasks.

This study demonstrated that lactose from whey and permeate whey can be fermented in the presence of E. coli JFR12 in order to obtain 2,3-BD or acetoin according the experimental conditions.

CHAPTER 5. Conclusion

The valorization of byproducts of the agro-food industry like polysaccharides via enzymatic catalysis is currently a great challenge, which has to overcome in the following years. Diverse bacterial strains, such as Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae or Serratia marcescens, allow to transform polysaccharides into 2,3-butanediol (2,3-BD), a platform molecule. These microorganisms are divided into two group: i) natural producers of 2,3-BD like E. cloacae and K. pneumoniae, and ii) genetically modified strains like E. coli.

The biovalorization of saccharides via fermentation allows unlike the chemical process (160 and 220ºC, and 50 bar) working at softer operating conditions (temperatures between 25 and 40ºC, atmospheric pressure and pH close to 7.0) to produce of 2,3-BD without using petroleum sources. Each bacterial strain is differently affected by the operating conditions like kind and concentration of nutrients in the culture medium, temperature, pH, agitation, aeration, etc. In the present study, the main objective has been to study the ability of a genetically modified strain of Escherichia coli (E. coli JFR12) K12 MG1655 to transform lactose contained in whey (W) and permeate whey (PW) into 2,3-BD.

The first specific objective was to measure and quantify the amount of bacteria in the inoculum before performing the fermentation. In addition, a comparison between the wild (ECW) and the genetically modified (E. coli JFR1) strains of E. coli was carried out using the colony-forming unit (CFU) method. The E. coli JFR1 strain presented a CFU value of 6.5x10 8 CFU/mL, which was 41% lower compared to the value obtained in the presence of ECW. Therefore, the genetic modifications performed in ECW to obtain ECGM seem to affect the bacterial growth of E. coli.

The second specific objective was to evaluate the effect of the glucose concentration on the ABD (acetoin + 2,3-BD) production using 4 culture media (LB, M9, M63 and MOPS) in the presence of E. coli JFR1. The glucose concentrations in the 4 culture media were 4, 12.5 and 25 g/L. The highest ABD yield was close to 0.25 g ABD/g glucose at 48 and 72 h using 12.5 and 25 g/L of glucose, respectively, in LB, M9 and M63 at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 6.5 and 100 rpm. On the other hand, the use of MOPS culture medium decreased the ABD yields for those glucose concentrations (0.18 and 0.14 g ABD/g glucose, respectively, at 48 h). The selected culture medium was M9 since it presented an optimized relationship between the ABD yield and culture medium price.

The third specific objective was to study the effect of urea ((NH 2 ) 2 CO) and sodium nitrate (NaNO 3 ) as an additional nitrogen source (ANS) and their concentration in M9 culture medium on ABD yield in the presence of E. coli JFR1. The use of urea as an ANS improved the ABD yield up to 0.27 g ABD/g glucose at 96 h in the presence of 15 g/L of urea fermenting 25 g/L of glucose at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 6.5 and 100 rpm. The ABD formation was hampered when the ANS was NaNO 3 . Therefore, the ABD formation depends on the chemical nature of the nitrogen source.

The fourth specific objective of the study was to test the effect of glucose, galactose and lactose concentration in M9 culture medium to produce 2,3-BD in the presence of another mutant strain of E. coli: E. coli JFR12. This strain was obtained to improve the 2,3-BD production. The fermentation of glucose and lactose (25 g/L) provided similar 2,3-BD yields (close to 0.38 g 2,3-BD/g saccharide) at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 6.5 and 100 rpm for 48 and 72 h, respectively. Nevertheless, with galactose, lower 2,3-BD yields (0.19 g 2,3-BD/g galactose) at 48 h compared to those obtained with glucose and lactose. In order to understand these results, a mixture of glucose and galactose (12.5 and 25 g/L of each monosaccharide) were fermented. In the presence of galactose as a sole carbon source, the 2,3-BD concentration was lower compared to that one obtained when galactose was generated from lactose hydrolysis.

The fifth specific aim was to evaluate the 2,3-BD yield using mixtures of W (31 g/L of lactose, W1) and PW (34 g/L of lactose, PW1) in the presence of E. coli JFR12. In this case, W1 and PW1 were mixed with M9; three dilutions (W1 or PW1/W9) (50:50, 75:25 and 100:0, v/v) were tested. The maximum 2,3-BD yields (0.43 and 0.42 g 2,3-BD/g lactose with W1 and PW1, respectively) were reached at 72 h in absence of M9 culture medium (100:0, v/v) at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 7.4 and 100 rpm. Hence, E. coli JFR12 was able to transform efficiently the lactose contained in both effluents to 2,3-BD, with a value near the maximum 2,3-BD theoretical yield (0.52 g 2,3-BD/g lactose).

The sixth specific goal was focused on the influence of three parameters (initial pH, inoculum size and agitation rate) on 2,3-BD yield fermenting W1 and PW1 in absence of M9 culture medium and in the presence of E. coli JFR12. The inoculum size of 7.5% (v/v) gave a similar 2,3-BD yield to that one obtained using 10% (v/v) of inoculum, close to 0.45 and 0.42 g 2,3-BD/g lactose for W1 and PW1, respectively at 37ºC, 1 atm, initial pH 7.4 and 100 rpm.

Concerning the effect of the initial pH, no influence on the 2,3-BD yield was observed. In this particular case, in the presence of W1 and PW1, the 2,3-BD yields were 0.47 and 0.44 g 2,3-BD/g lactose, respectively at 10% (v/v) of inoculum, 37ºC, 1 atm and 100 rpm. As far as the W1 fermentation at 10% (v/v) of inoculum, 37ºC, 1 atm and initial pH 7.4 is concerned, an agitation rate of 200 rpm gave a maximum 2,3-BD yield of 0.23 g/g lactose at 24 h. This yield was two folds lower than the maximum yield obtained at 100 rpm at 72 h. For the fermentation of PW1 under similar operating conditions, an agitation rate of 200 rpm provided the same effects on the 2,3-BD yield.

A new batch of W (W2) and PW (PW2) were fermented in 0.5 L flasks and in a 2 L bioreactor (without aeration) at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 7.4 and 100 rpm. The W2 and PW2 contained, respectively, a lactose concentration of 51 and 47 g/L. Both effluents were directly fermented and diluted with distilled water to obtain a lactose concentration of 31 and 34 g/L of lactose, respectively, in 0.5 L flasks and the 2 L bioreactor. In flasks, the 2,3-BD yields obtained with diluted W2 and PW2 were comparable to those obtained with W1 and PW1. In both cases (W1 and W2 or PW1 and PW2) under anaerobic conditions, the 2,3-BD yields were lower.

Afterwards, in a 2 L bioreactor, the effect of the air at 2 L/min on 2,3-BD formation was tested in the presence of diluted W2. The addition of air increased the 2,3-BD yield by 135% (0.40 g 2,3-BD/g lactose) compared to the yield obtained in flask (0.5 L). By comparison, the 2,3-BD yields obtained in flasks for 24 h at 10%, v/v, of inoculum, 37ºC, 1 atm, initial pH 7.4 and 100 rpm were lower. Hence, the use of a bioreactor adding air allowed rapidly obtaining a high yield (reduction of the fermentation time from 72 h to 24 h).

Therefore, the main objective of this study, the optimization of W and PW fermentation by a mutant strain of E. coli, was achieved. The fermentation of both effluents gave high 2,3-BD yields, up to 89 and 84% of the theoretical yield (W and PW, respectively). In this way, the strain E. coli JFR12 can be considered a good candidate to valorize W and PW in order to produce 2,3-BD.

On the other hand, it has been demonstrated that both whey and permeate whey are two appropriate culture media for bacteria like E. coli JFR12. In this way, both industrial effluents can become innocuous for the environment since they can be valorized by a green biotechnology. It would be good to consider an in-depth study of the operating parameters of fermentation in order to determine the optimal ones, above all the aeration rate in a fermentor as this study has shown. Afterwards, the study at fed-batch and continuous operating modes in a pilot scale would be strongly interesting in order to implement the fermentation of whey and permeate whey at an industrial scale.

Conclusion in French

La valorisation des sous-produits de l'industrie agro-alimentaire tels les polysaccharides par catalyse enzymatique reste un défi considérable pour les années à venir. Diverses souches bactériennes, telles qu'Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae ou Serratia marcescens, permettent de transformer les polysaccharides en 2,3-butanediol ( Rebouillat S, Ortega-Requena S (2015) Potential applications of milk fractions and valorization of dairy by-products: a review of the state-of-the-art available data, outlining the innovation potential from a bigger data standpoint. J Biomater Nanobiotechnol 6: 176-203. doi: 10.4236/jbnb.2015.63018 Ridoutt BG, Pfister S (2010) A revised approach to water footprinting to make transparent the impacts of consumption and production on global freshwater scarcity. Glob Environ Change 20:113-120. doi: 10.1016/j.gloenvcha.2009.08.003 Roncal T, Cadiermo U, Torrecilla J, García Jaca J, Garmendia I, Azpeitia M, Ochoa-Gómez J (2010) La biotecnología en el sector químico (II). Nuevas tendencias en la producción industrial de productos químicos básicos. Ing Quím 478:152-160

A.3 Introduction

Nowadays there is a consciousness-raising about global warming and sustainability and, thus, the search of new green technologies taking a special interest to obtain chemical products coming from natural sources instead of petroleum [START_REF] Cho | Enhanced 2,3-butanediol production by optimizing fermentation conditions and engineering Klebsiella oxytoca M1 through overexpression of acetoin reductase[END_REF]. Among these green techniques, fermentation allows to use natural sources like mono-, oligo-and poly-saccharides which are wastes from the agro-food industry [START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF]. These saccharides can be transformed into fermentative products like acetoin (A) and 2,3-butanediol (BD) by bacteria like Enterobacter cloacae, Klebsiella oxytoca or Serratia marcescens [START_REF] Fernández-Gutierrez | Biovalorization of saccharides derived from industrial wastes such as whey: a review[END_REF]). However, other bacteria like Escherichia coli K12 MG1655 can host the metabolic pathway of BD from natural producers [START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF].

Escherichia coli K12 MG1655 presents several advantages compared to the previous mentioned strains: i) has a biosafety level 1 (non-pathogenic), ii) can consume a wide range of saccharides like glucose and iii) is easily modified to host metabolic pathways from other microorganisms (iGEM 2016; [START_REF] Tong | Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production[END_REF]). Among the different substrates which E. coli is able to break down are glucose and galactose (monosaccharides). In order to produce A and BD (ABD), E. coli has to transform both monosaccharides into pyruvic acid (PA), which occurs by different pathways: glycolysis for glucose and Leloir pathway + glycolysis for galactose [START_REF] Lazar | Awakening the endogenous Leloir pathway for efficient galactose utilization by Yarrowia lipolytica[END_REF], Xu et al. 2014). The enzymatic transformation from glucose and galactose into PA is shown in Figure 1. Afterwards, the PA is transformed into α-acetolactate by α-acetolactate synthase (ALS). Then, α-acetolactate is transformed into A by means of αacetolactate decarboxylase (ALDC) enzyme. Finally, A is converts into BD by 2,3-butanediol dehydrogenase (BDH) enzyme [START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF]. Figure 2 shows the metabolic pathway to obtain A and 2,3-BD from glucose and galactose.

In the present study, a genetically modified strain of E. coli K12 MG1655 (E. coli JFR1) was used to host the metabolic pathway of BD from E. cloacae in order to test the ability of E. coli JFR1 to produce ABD from glucose and galactose. Three monosaccharide concentrations (12.5, 25 and 50 g/L) were fermented at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 7.4 and 100 rpm and the ABD yields were compared. The culture medium, M9, was used since in preliminary studies and was selected as the best culture medium to obtain ABD from glucose [START_REF] Fernández-Gutierrez | Biovalorization of glucose in four culture media and effect of the nitrogen source on fermentative alcohols production by Escherichia coli[END_REF].

Glucose Galactose

Glucose-6-phosphate

Phosphoenol pyruvate

Fructose-6-phosphate Fructose-1,6-bisphosphate Dihydroxyacetone Glyceraldehyde-3-phosphate 1,3-bisphosphoglycerate 3-phosphoglycerate 2-phosphoglycerate

Pyruvic acid

Glucose-1-phosphate Galactose-1-phosphate Phosphoglucose isomerase

Hexokinase

Galactose

A.4 Materials and methods

A.4.1 Microorganism

The genetically modified strain of E. coli MG1655 hosts the metabolic pathway of 2,3butanediol to produce ABD from Enterobacter cloacae. The biosynthetic pathway of fermentative D-lactate dehydrogenase (ldhA) was blocked to avoid the formation of lactic acid. The modified strain was E. coli MG1655/DldhA + budABC, named as E. coli JFR1. The conservation of E. coli JFR1 was performed at -81ºC in a blend (50:50, v/v) of glycerol and lysogeny broth (LB) culture medium.

A.4.2 Culture media and conditions

Escherichia coli JFR1 was grown in LB culture medium since it is suitable for recombinant E. coli strains (Maniatis et al. 2001). The composition of LB culture medium was as follows: 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium chloride (NaCl) and α-acetolactate In the case of galactose, for a [Gal] 0 of 12.5 g/L, the ABD yield reached a plateau at 0.13 g/g galactose at 24 h (Figure A.4B). Using a [Gal] 0 of 25 g/L, the ABD yield increased up to 0.15 g/g galactose (p < 0.05) at 72 h and remained constant until the end of the experiment (120 h); whereas in the presence of [Gal] 0 of 50 g/L, the ABD yield increased to 0.08 g/g galactose at 72 h and remained nearly constant up to 120 h (p < 0.05). Comparing the 3 glucose concentrations, the maximum ABD yield was 0.25 g/g glucose at 72 h in the presence of 25 g/L of glucose, which was 28% and 39% higher compared to the maximum obtained using 12.5 and 50 g/L of glucose, respectively. In the case of galactose, the highest ABD yield (0.15 g/g galactose) was reached in the presence of a [Gal] 0 of 25 g/L at 72 h. However, this ABD yield (0.15 g/g galactose) was only 13% higher compared to the one obtained with 12.5 g/L of galactose (0.13 g/g galactose), obtained at 24 h, a fermentation time 3 fold shorter. Therefore, the galactose concentration that gave an ABD yield of 0.13 g/g galactose at a short fermentation time (24 h) was 12.5 g/L.

The difference between both monosaccharides in terms of ABD yield is that the use of glucose was more efficient than galactose since the ABD yields were 28%, 40% and 47% higher using glucose at 12.5, 25 and 50 g/L compared to galactose. As mentioned previously, the use of a high monosaccharide concentration (50 g/L) might lead to the inhibition of the bacterial growth, which affects the formation of ABD as suggested by [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF], [START_REF] Krämer | Bacterial stimulus perception and signal transduction: response to osmotic stress[END_REF] and [START_REF] Priya | Microbial production of 2, 3butanediol through a two-stage pH and agitation strategy in 150 L bioreactor[END_REF]. This phenomenon is clearly observed in the present study at 50 g/L of monosaccharide in the presence of E. coli JFR1 since the ABD yield at a [Glu] 0 of 25 g/L was 40% higher than the one obtained at a [Glu] 0 of 50 g/L; the ABD yield was 50% higher at a [Gal] 0 of 25 g/L than at 50 g/L of galactose.

As observed in Figure A.4 (A and B), the ABD yield reached a maximum and was nearly constant whatever the monosaccharide and concentration used. As shown in Figure 2, the diol (BD) is an end product which is in equilibrium with A, its precursor. Acetoin is transformed into BD by the BDH enzyme. [START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF] suggested that BD can be used by E. coli and, thus, can be transformed into A. This might explain why using E. coli JFR1, the ABD yield was nearly constant in the present study.

To the best of our knowledge, E. coli has not been used to ferment glucose and galactose in order to obtain ABD. However, other studies reported a comparison of glucose and galactose in the presence of bacteria like Enterobacter aerogenes, Geobacillus, Paenibacillus polymyxa and Saccharomyces cerevisiae [START_REF] Choi | Enhanced production of 2,3-butanediol in pyruvate decarboxylase-deficient Saccharomyces cerevisiae through optimizing ratio of glucose/galactose[END_REF][START_REF] Jiang | 2,3-butanediol and acetoin production from enzymatic hydrolysate of ionic liquid-pretreated cellulose by Paenibacillus polymyxa[END_REF][START_REF] Jung | Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production[END_REF][START_REF] Xiao | Thermophilic fermentation of acetoin and 2, 3-butanediol by a novel Geobacillus strain[END_REF]. For example, [START_REF] Xiao | Thermophilic fermentation of acetoin and 2, 3-butanediol by a novel Geobacillus strain[END_REF] reported an ABD yield of 0.34 and 0.21 g/g monosaccharide (ABD yield calculated from the data provided by the authors) fermenting 20 g/L of glucose and galactose respectively at 55ºC and 170 rpm (pH and time non-defined) in the presence of Geobacillus sp. XT15 (5% (v/v) of inoculum). Although the yields were superior to those obtained in the present study, the difference of ABD yield between both monosaccharides was 1.6 fold higher using glucose than galactose; a similar difference in terms of ABD yield in the presence of E. coli JFR1 using 25 g/L of monosaccharide at 96 h was observed. Therefore, the use of glucose seems to be more suitable than galactose in order to produce ABD in the presence of E. coli JFR1.

A.6 Conclusion

The present study was based on the fermentation of 2 monosaccharides in order to produce acetoin and 2,3-butanediol (ABD) in the presence of a genetically modified strain of Escherichia coli: E. coli JFR1. Different concentrations (12.5, 25 and 50 g/L) of glucose and galactose in M9 culture medium at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 7.4 and 100 rpm in 0.5 L flask (0.2 L of working volume) were fermented.

The use of whatever glucose concentration provided better results in terms of ABD yield than the fermentation of galactose. The ABD yields were between 28% and 47% higher in the presence of glucose for the range of monosaccharide concentrations tested. The highest ABD yield was 0.25 g/g glucose at 72 h and 25 g/L of glucose; whereas the best ABD yield was 0.13 g/g galactose at 24 h and 12.5 g/L of galactose.

Faculté

  Professorat et de l'Education Institut de Science Financière et d'Assurances Directeur : M. F. DE MARCHI Directeur : M. le Professeur F. THEVENARD Directeur : Mme C. FELIX Directeur : M. Hassan HAMMOURI Directeur : M. le Professeur S. AKKOUCHE Directeur : M. le Professeur G. TOMANOV Directeur : M. le Professeur H. BEN HADID Directeur : M. le Professeur J-C PLENET Directeur : M. Y.VANPOULLE Directeur : M. B. GUIDERDONI Directeur : M. le Professeur E.PERRIN Directeur : M. G. PIGNAULT Directeur : M. le Professeur C. VITON Directeur : M. le Professeur A. MOUGNIOTTE Directeur : M. N. LEBOISNE Essentially to Isa, who left everything back to support me i

Figure 2 . 1 :Figure 2 . 2 :Figure 2 . 3 :Figure 3 . 3 :

 21222333 Figure 2.1: An overall scheme of the whey valorization processes ..................................... 12

Figure 4 . 2 :Figure 4 . 3 :

 4243 Figure 4.2: Glucose conversion (Figure 4.2A), 2,3-BD (Figure 4.2B) and A (Figure 4.2C) yields as a function of time in the presence of E. coli JFR12 at 3 [Glu] 0 . Glucose concentration: 12.5 (○); 25 (•) and 50 (•) g/L. Results are means ± SD of 2 experiments performed in duplicate. ......................................................................................................... 63

  Figure 4.5: Glucose and galactose conversion and 2,3-BD and A yields as a function of time in the presence of E. coli JFR12. Two mixture [G-Ga] 0 were tested: 25 g/L (Figure 4.5A) and 50 g/L (Figure 4.5B).Figure 4.5A: Glucose conversion (□), galactose conversion (◊), 2,3-BD (○) and A (∆).Figure 4.5B: Glucose conversion (■), galactose conversion (♦), 2,3-BD (•) and A (▲). Results are means ± SD of 2 experiments performed in duplicate. ............................. 70

  Figure 4.5: Glucose and galactose conversion and 2,3-BD and A yields as a function of time in the presence of E. coli JFR12. Two mixture [G-Ga] 0 were tested: 25 g/L (Figure 4.5A) and 50 g/L (Figure 4.5B).Figure 4.5A: Glucose conversion (□), galactose conversion (◊), 2,3-BD (○) and A (∆).Figure 4.5B: Glucose conversion (■), galactose conversion (♦), 2,3-BD (•) and A (▲). Results are means ± SD of 2 experiments performed in duplicate. ............................. 70

  Figure 4.6: 2,3-BD yield at maximum saccharide conversion at 25 g/L (Figure 4.6A) and 50 g/L (Figure 4.6B) of saccharide concentration with E. coli JFR12. Figure 4.6A: 2,3-BD yield using glucose (48 h), galactose (48 h), lactose (48 h) and mixture G-Ga (24 h) when conversion was 100% in all cases. Figure 4.6B: 2,3-BD yield using glucose (conversion 94%), galactose (conversion 90%), lactose (conversion 96%) and mixture G-Ga at 72 h (conversion 100% for both monosaccharides). Results are means ± SD of 2 experiments performed in duplicate. .............................................................................................................................. 72

  Figure 4.14: 2,3-BD yield as a function of time fermenting W1 and W2 in the presence of E. coli JFR12 in flask (Figure 4.14A) and bioreactor (Figure 4.14B).Figure 4.14A: W1 (•), W2 undiluted (•) and W2 diluted (○).Figure 4.14B: W1 (•), W2 undiluted (•) and W2 diluted (○). Results are means ± SD of an experiment performed in duplicate. .............................. 85

  Figure 4.14: 2,3-BD yield as a function of time fermenting W1 and W2 in the presence of E. coli JFR12 in flask (Figure 4.14A) and bioreactor (Figure 4.14B).Figure 4.14A: W1 (•), W2 undiluted (•) and W2 diluted (○).Figure 4.14B: W1 (•), W2 undiluted (•) and W2 diluted (○). Results are means ± SD of an experiment performed in duplicate. .............................. 85

  Figure 4.15: 2,3-BD yields as a function of time fermenting PW1 and PW2 in the presence of E. coli JFR12 in flask (Figure 4.15A) and bioreactor (Figure 4.15B).Figure 4.15A: PW1 (•), PW2 undiluted (•) and PW2 diluted (○).Figure 4.15B: PW2 undiluted (•) and PW2 diluted (○). Results are means ± SD of an experiment performed in duplicate. .............................. 86

  Figure 4.15: 2,3-BD yields as a function of time fermenting PW1 and PW2 in the presence of E. coli JFR12 in flask (Figure 4.15A) and bioreactor (Figure 4.15B).Figure 4.15A: PW1 (•), PW2 undiluted (•) and PW2 diluted (○).Figure 4.15B: PW2 undiluted (•) and PW2 diluted (○). Results are means ± SD of an experiment performed in duplicate. .............................. 86

  . ........................ 14 Table 2.2: Operating conditions using different types of microorganisms and their influence on the maximum 2,3-butandediol yield. .................................................................................... 21 Table

  ); and MOPS (3-morpholinopropane-1-sulfonic acid) minimal medium (Wisconsin-University 2017). .............................................................................................. 40 Table

  .............................................................. 49 Table

  Fermentations performed at 37ºC, 1 atm and 72 h. .............................................................. 61 Table

  "Biovalorization of saccharides derived from industrial wastes such as whey -A review" a été publié dans le Journal "Reviews in Environmental Science and Bio/Technology" 16(1) (2017) 147-174. TITRE: Biovalorisation des saccharides dérivés des déchets industriels tels que le petit lait -Un revue de la littérature. Title: Biovalorization of saccharides derived from industrial wastes such as whey -A review. David Fernández-Gutiérrez a,b , Marc Veillette a , Anne Giroir-Fendler b , Antonio Avalos Ramirez c , Nathalie Faucheux a and Michèle Heitz a, d a Université de Sherbrooke, Department of Chemical Engineering and Biotechnological Engineering. Faculty of engineering, 2500 boul. de l'Université, Sherbrooke, (Québec), Canada, J1K 2R1 b Université Lyon 1, CNRS, UMR 5256, Institut de Recherche sur la Catalyse et l'Environnement de Lyon (IRCELYON), 2 avenue Albert Einstein, 69626 Villeurbanne, France c Centre National en Électrochimie et en Technologies Environnementales, Shawinigan, (Québec), Canada, G9N 6V8 d Corresponding author: E-mail address: Michele.Heitz@USherbrooke.ca

Figure 2

 2 Figure 2.1: An overall scheme of the whey valorization processes

Figure 2

 2 Figure 2.2: Whey production during cheese manufacturing

Figure 2 . 3 : 2 : 2 : 2 : 2 :

 232222 Figure 2.3: Representation of the metabolic pathway from lactose to 2,3-butanediol. Source:[START_REF] Alpers | Sequential transcription of the genes of the lactose operon and its regulation by protein synthesis[END_REF] and[START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF].

Figure 3 . 1 :

 31 Figure 3.1: Optical density at 600nm (OD600, ■), colony-forming units per mL of broth (CFU/mL, ♦) and modeling of the growth curve by the modified Gompertz model (-) as a function of time in the presence of ECW (A) and E. coli JFR1 (B) strain. Results are means ± SD of 9 replications performed in flasks at 37ºC, atmospheric pressure, initial pH of 6.5 and 100 rpm a working volume of 200 mL (batch fermentation).

  Figure 3.2A presents the ABD yield as a function of fermentation time using LB, a rich culture medium formed by tryptone, yeast extract and sodium chloride, supplemented with 3 glucose concentrations (4, 12.5 and 25 g/L). The maximum ABD yield was around 0.25 g ABD/g glucose independently of the glucose concentration. Therefore, the increase of glucose concentration had no influence on the maximum ABD yield using LB culture medium (p < 0.05), but the fermentation time to obtain the maximum ABD yield increased according to the glucose concentration, ranging from 24 h (4 g/L of glucose) to 72 h (25 g/L of glucose). On the other hand, after 24 h of fermentation, the ABD concentration decreased when LB culture medium was supplemented with 4 g/L of glucose (Figure3.2A). As suggested by[START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF], once the carbon source (glucose) is depleted, bacteria could consume another carbon source like ABD during the stationary growth phase, causing a drop of its yield. This fact is observed in Figure3.2A and thus it seems to confirm that E. coli JFR1 consumes ABD as a carbon source in order to maintain its metabolic activity. Lysogeny broth (LB) is a rich culture medium, which provides all nutrients (carbon, nitrogen, phosphorus, etc.) required by bacteria[START_REF] Ecocyc | Escherichia coli K-12 substr[END_REF]. In this way, the components present in LB culture medium like amino acids (e.g. leucine, methionine and tryptophan) could positively influence the ALS and ALDC enzymes[START_REF] Cho | Enhanced 2,3-butanediol production by optimizing fermentation conditions and engineering Klebsiella oxytoca M1 through overexpression of acetoin reductase[END_REF]. This fact may explain the similar ABD yields obtained whatever the glucose concentration, since the LB culture medium nutrients might efficiently stimulate the formation of ABD even at a relatively low glucose concentration.

Figure 3 . 2 :

 32 Figure 3.2: ABD yield as a function of time in the presence of E. coli JFR1 in LB (A), M9 (B), M63 (C) and MOPS (D) culture media at 4 g/L glucose (◊) , 12.5 g/L of glucose (□) and 25 g/L of glucose (○). Results are means ± SD of 3 replications performed in flasks at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% v/v of inoculum in a working volume of 200 mL (batch fermentation).

  Figures 3.3A and 3.3B present the glucose conversion and ABD yield, respectively, as a function of time in the presence of 12.5 g/L of glucose. Urea was used as an ANS. Two additional nitrogen concentrations (0.6 and 1.2 g N/L) were also tested.

Figure 3 . 3 :

 33 Figure 3.3: Glucose conversion(A) and ABD yield (B) in M9 culture medium supplemented with 12.5 g/L of glucose as a function of time in the presence of E. coli JFR1. Source of nitrogen: urea at 0.6 g N/L (▲), 1.2 g N/L (∆), 2.5 g N/L (♦), 5.0 g N/L (■) and 7.0 g N/L (•). Additionally, the highest (▬) and lowest (+) ethanol yields obtained for 0.6 g N/L and without an ANS are represented respectively. Results are means ± SD of 3 replications performed in flasks at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% v/v of inoculum in a working volume of 200 mL (batch fermentation).

Figure 3 . 4 :

 34 Figure 3.4: Glucose conversion(A) and ABD yield (B) in M9 culture medium supplemented with 25 g/L of glucose as a function of time in the presence of E. coli JFR1. Source of nitrogen: urea at 2.5 g/L of nitrogen (♦), 5.0 g/L of nitrogen (■), 7.0 g/L of nitrogen (•) and 10.0 g N/L (×). Additionally, the highest (□) and lowest (▬) ethanol yields obtained for 10.0 g N/L and 2.5 g N/L are represented respectively. Results are means ± SD of 3 replications performed in flasks at 37ºC, atmospheric pressure, initial pH of 6.5, 100 rpm and 10% v/v of inoculum in a working volume of 200 mL (batch fermentation).

Figure 4 . 1 :

 41 Figure 4.1: Metabolic pathway to produce 2,3-BD from lactose fermentation in the presence of a genetically modified strain of Escherichia coli. α-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC) and 2,3-butanediol dehydrogenase (BDH). Reduced NADH) and oxidized (NAD+) form of nicotinamide adenine dinucleotide. Sources:[START_REF] Lazar | Awakening the endogenous Leloir pathway for efficient galactose utilization by Yarrowia lipolytica[END_REF],[START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF],[START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF],[START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF],[START_REF] Xu | Metabolic engineering of Escherichia coli for efficient production of (3R)-acetoin[END_REF].

Figure 4 . 2 (

 42 Figure 4.2(A, B and C) shows the glucose conversion, 2,3-BD and A yields for 3 initial glucose concentrations ([Glu] 0 = 12.5, 25 and 50 g/L) as a function of time. After the first 8 h, the conversion of glucose was around 60% for the [Glu] 0 of 12.5 g/L and 40% for the [Glu] 0 of 25 and 50 g/L. Glucose conversion was 100% at 24 h for a [Glu] 0 of 12.5 g/L, and at 48 h for a [Glu] 0 of 25 g/L. However, for a [Glu] 0 of 50 g/L, the conversions were close to 60 and 80% at 24 and 48 h, respectively; whereas the maximum conversion was slightly lower compared to 12.5 and 25 g/L of glucose and reached 94% after 72 h (Figure4.2A). For the 3

Figure

  Figure 4.2(A, B and C) shows the glucose conversion, 2,3-BD and A yields for 3 initial glucose concentrations ([Glu] 0 = 12.5, 25 and 50 g/L) as a function of time. After the first 8 h, the conversion of glucose was around 60% for the [Glu] 0 of 12.5 g/L and 40% for the [Glu] 0 of 25 and 50 g/L. Glucose conversion was 100% at 24 h for a [Glu] 0 of 12.5 g/L, and at 48 h for a [Glu] 0 of 25 g/L. However, for a [Glu] 0 of 50 g/L, the conversions were close to 60 and 80% at 24 and 48 h, respectively; whereas the maximum conversion was slightly lower compared to 12.5 and 25 g/L of glucose and reached 94% after 72 h (Figure4.2A). For the 3

Figure 4 . 2 :

 42 Figure 4.2: Glucose conversion (Figure 4.2A), 2,3-BD (Figure 4.2B) and A (Figure 4.2C) yields as a function of time in the presence of E. coli JFR12 at 3 [Glu] 0 . Glucose concentration: 12.5 (○); 25 (•) and 50 (•) g/L. Results are means ± SD of 2 experiments performed in duplicate.

Figure 4 . 3 (

 43 Figure 4.3(A, B and C) presents the galactose conversion, 2,3-BD and A yields as a function of time and initial galactose concentrations of 12.5, 25 and 50 g/L. At 8 h, the galactose conversion was around 40% whatever the [Gal] 0 used. However, at 24 h of fermentation, the galactose conversion depended on the [Gal] 0 reaching 100, 92 and 59% for 12.5, 25 and 50 g/L respectively. At 72 h, for the highest [Gal] 0 (50 g/L), the galactose conversion reached 90% (Figure4.3A). The trend for galactose conversion was similar to the one of glucose (Figure2A), being the lowest conversion obtained fermenting 50 g/L of galactose.

Figure 4 . 3 :

 43 Figure 4.3: Galactose conversion (Figure 4.3A), 2,3-BD (Figure 4.3B) and A (Figure 4.3C) yields as a function of time in the presence of E. coli JFR12 at 3 [Gal] 0. Galactose concentration: 12.5 (◊); 25 (♦) and 50 (♦) g/L. Results are means ± SD of 2 experiments performed in duplicate.

Figure 4

 4 Figure 4.4(A, B and C) presents the lactose conversion, 2,3-BD and A yields as a function of time and initial lactose concentrations ([Lac] 0 = 12.5, 25 and 50 g/L). Lactose conversion decreased with an increase of [Lac] 0 . After 8 h of fermentation, conversion of 75, 58 and 25% were obtained for [Lac] 0 of 12.5, 25 and 50 g/L, respectively. A lactose conversion of 100% was achieved at 24 h for 12.5 g/L, whereas it was 90 and 65% for 25 and 50 g/L, respectively. For the [Lac] 0 of 25 g/L, the lactose conversion was 100% at 48 h and nearly 100% for 50 g/L at 72 h (Figure4.4A).

Figure 4

 4 Figure 4.4: Lactose conversion (Figure 4.4A), 2,3-BD (Figure 4.4B) and A (Figure 4.4C) yields as a function of time in the presence of E. coli JFR12 at 3 [Lac] 0 . Lactose concentration: 12.5 (□); 25 (■) and 50 (■) g/L. Results are means ± SD of 2 experiments performed in duplicate.

  Figure 4.5: Glucose and galactose conversion and 2,3-BD and A yields as a function of time in the presence of E. coli JFR12. Two mixture [G-Ga] 0 were tested: 25 g/L (Figure 4.5A) and 50 g/L (Figure 4.5B).Figure 4.5A: Glucose conversion (□), galactose conversion (◊), 2,3-BD (○) and A (∆).Figure 4.5B: Glucose conversion (■), galactose conversion (♦), 2,3-BD (•) and A (▲). Results are means ± SD of 2 experiments performed in duplicate.

  Figure 4.5: Glucose and galactose conversion and 2,3-BD and A yields as a function of time in the presence of E. coli JFR12. Two mixture [G-Ga] 0 were tested: 25 g/L (Figure 4.5A) and 50 g/L (Figure 4.5B).Figure 4.5A: Glucose conversion (□), galactose conversion (◊), 2,3-BD (○) and A (∆).Figure 4.5B: Glucose conversion (■), galactose conversion (♦), 2,3-BD (•) and A (▲). Results are means ± SD of 2 experiments performed in duplicate.

  Figure 4.5: Glucose and galactose conversion and 2,3-BD and A yields as a function of time in the presence of E. coli JFR12. Two mixture [G-Ga] 0 were tested: 25 g/L (Figure 4.5A) and 50 g/L (Figure 4.5B).Figure 4.5A: Glucose conversion (□), galactose conversion (◊), 2,3-BD (○) and A (∆).Figure 4.5B: Glucose conversion (■), galactose conversion (♦), 2,3-BD (•) and A (▲). Results are means ± SD of 2 experiments performed in duplicate.

Figure

  Figure 4.6(A and B) presents the 2,3-BD yield obtained for the highest saccharide conversion at 25 and 50 g/L of saccharides (glucose, galactose, lactose and G-Ga). Figure4.6A shows the 2,3-BD yield when saccharide conversion was 100%: glucose (48 h), galactose (48 h), lactose (48 h) and the mixture G-Ga (24 h) at 25 g/L. The 2,3-BD yields for glucose and lactose were around 0.35 g/g saccharide, whereas for galactose and the mixture G-Ga, it was 46% lower (0.19 g/g saccharide) (p < 0.05). Figure4.6B shows that the 2,3-BD yields for glucose and lactose at a concentration of 50 g/L at 72 h were around 0.28 g/g saccharide and it was slightly smaller for galactose and the mixture G-Ga of around 0.25 g /g saccharide.

  Figure 4.6: 2,3-BD yield at maximum saccharide conversion at 25 g/L (Figure 4.6A) and 50 g/L (Figure 4.6B) of saccharide concentration with E. coli JFR12.Figure4.6A: 2,3-BD yield using glucose (48 h), galactose (48 h), lactose (48 h) and mixture G-Ga (24 h) when conversion was 100% in all cases. Figure4.6B: 2,3-BD yield using glucose (conversion 94%), galactose (conversion 90%), lactose (conversion 96%) and mixture G-Ga at 72 h (conversion 100% for both monosaccharides). Results are means ± SD of 2 experiments performed in duplicate.

  Figure 4.6: 2,3-BD yield at maximum saccharide conversion at 25 g/L (Figure 4.6A) and 50 g/L (Figure 4.6B) of saccharide concentration with E. coli JFR12.Figure4.6A: 2,3-BD yield using glucose (48 h), galactose (48 h), lactose (48 h) and mixture G-Ga (24 h) when conversion was 100% in all cases. Figure4.6B: 2,3-BD yield using glucose (conversion 94%), galactose (conversion 90%), lactose (conversion 96%) and mixture G-Ga at 72 h (conversion 100% for both monosaccharides). Results are means ± SD of 2 experiments performed in duplicate.

Figure

  Figure 4.7(A, B and C) shows the lactose conversion, 2,3-BD and A yields using whey as a function of time and [Lac] 0 . For a [Lac] 0 of 15.5 g/L, the lactose conversion was 52% at 8 h and reached 100% at 24 h. On the other hand, using a [Lac] 0 of 23 g/L, the conversion increased with the time and reached 100% at 48 h; whereas with a [Lac] 0 of 31 g/L, the lactose conversion increased and reached 100% at 72 h (Figure4.7A).

Figure 4 . 7 :

 47 Figure 4.7: Lactose conversion (Figure 4.7A), 2,3-BD yield (Figure 4.7B) and A yield (Figure 4.7C) fermenting W1 diluted with M9 as a function of time in the presence of E. coli JFR12. Lactose concentration: 15.5 (○), 23 (•) and 31 (•) g/L of lactose. Results are means ± SD of 2 experiments performed in duplicate.

Figure 4 . 8 :

 48 Figure 4.8: Lactose conversion (Figure 4.8A), 2,3-BD yield (Figure 4.8B) and A yield (Figure 4.8C) fermenting PW1 diluted with M9 as a function of time in the presence of E. coli JFR12. Lactose concentration: 17 (○), 25.5 (•) and 34 (•) g/L. Results are means ± SD of 2 experiments performed in duplicate.

  Figure 4.9: 2,3-BD yield fermenting W1 (Figure 4.9A) and PW1 (Figure 4.9B) as a function of time in the presence of E. coli JFR12 at different initial pH: 6.5 (○), 7.0 (•) and 7.4 (•). Results are means ± SD of 2 experiments performed in duplicate.

Figures 4 .

 4 Figures 4.10 and 4.11 show the effect of the inoculum size (5, 7.5 and 10%, v/v) on the lactose conversion and the 2,3-BD yield as a function of time for the fermentation of W1 and PW1.

Figure 4 Figure 4

 44 Figure 4.10: Lactose conversion (Figure 4.10A) and 2,3-BD yield (Figure 4.10B) fermenting W1 as a function of time in the presence of E. coli JFR12 at different inoculum sizes (%, v/v). Inoculum size: 5% (□), 7.5% (■) and 10% (■). Results are means ± SD of 2 experiments performed in duplicate.

  Figure 4.12: Lactose conversion (Figure 4.12A) and 2,3-BD yield (Figure 4.12B) fermenting W1 as a function of time in the presence of E. coli JFR12 at different agitation rates. Agitation rate: 50 (◊), 100 (♦) and 200 rpm (♦). Results are means ± SD of 2 experiments performed in duplicate.

Figure 4 . 13 :

 413 Figure 4.13: Lactose conversion (Figure 4.13A) and 2,3-BD yield (Figure 4.13B) fermenting PW1 as a function of time in the presence of E. coli JFR12 at different agitation rates. Agitation rate: 50 (□), 100 (■) and 200 rpm (■). Results are means ± SD of 2 experiments performed in duplicate.

  Figure 4.14: 2,3-BD yield as a function of time fermenting W1 and W2 in the presence of E. coli JFR12 in flask (Figure 4.14A) and bioreactor (Figure 4.14B).Figure 4.14A: W1 (•), W2 undiluted (•) and W2 diluted (○).Figure 4.14B: W1 (•), W2 undiluted (•) and W2 diluted (○). Results are means ± SD of an experiment performed in duplicate.

  Figure 4.14: 2,3-BD yield as a function of time fermenting W1 and W2 in the presence of E. coli JFR12 in flask (Figure 4.14A) and bioreactor (Figure 4.14B).Figure 4.14A: W1 (•), W2 undiluted (•) and W2 diluted (○).Figure 4.14B: W1 (•), W2 undiluted (•) and W2 diluted (○). Results are means ± SD of an experiment performed in duplicate.

  Figure 4.14: 2,3-BD yield as a function of time fermenting W1 and W2 in the presence of E. coli JFR12 in flask (Figure 4.14A) and bioreactor (Figure 4.14B).Figure 4.14A: W1 (•), W2 undiluted (•) and W2 diluted (○).Figure 4.14B: W1 (•), W2 undiluted (•) and W2 diluted (○). Results are means ± SD of an experiment performed in duplicate.

Figure 4 .

 4 Figure 4.15(A and B) shows the 2,3-BD yield as a function of time for PW1 and PW2 fermentation in flasks (Figure4.15A) and in bioreactor (Figure4.15B). The 2,3-BD yield increased with the fermentation time and was similar at 72 h using PW1 ([Lac] 0 of 34 g/L) and diluted PW2 ([Lac] 0 of 34 g/L) with around 0.42 g/g lactose, while it was decreased by nearly the half (p < 0.05) with PW2 ([Lac] 0 of 47 g/L). The 2,3-BD yield using diluted PW2 ([Lac] 0 of 34 g/L) in bioreactor was also smaller (close to 60%) compared to fermentations in flasks (p < 0.05). Acetoin was not detected for any fermentation.

  Figure 4.15: 2,3-BD yields as a function of time fermenting PW1 and PW2 in the presence of E. coli JFR12 in flask (Figure 4.15A) and bioreactor (Figure 4.15B).Figure 4.15A: PW1 (•), PW2 undiluted (•) and PW2 diluted (○).Figure 4.15B: PW2 undiluted (•) and PW2 diluted (○). Results are means ± SD of an experiment performed in duplicate.

  Figure 4.15: 2,3-BD yields as a function of time fermenting PW1 and PW2 in the presence of E. coli JFR12 in flask (Figure 4.15A) and bioreactor (Figure 4.15B).Figure 4.15A: PW1 (•), PW2 undiluted (•) and PW2 diluted (○).Figure 4.15B: PW2 undiluted (•) and PW2 diluted (○). Results are means ± SD of an experiment performed in duplicate.

  Figure 4.15: 2,3-BD yields as a function of time fermenting PW1 and PW2 in the presence of E. coli JFR12 in flask (Figure 4.15A) and bioreactor (Figure 4.15B).Figure 4.15A: PW1 (•), PW2 undiluted (•) and PW2 diluted (○).Figure 4.15B: PW2 undiluted (•) and PW2 diluted (○). Results are means ± SD of an experiment performed in duplicate.

Figure 4 .

 4 Figure 4.16: Lactose conversion (■), 2,3-BD (○), A (∆) and ABD ( ) yields fermenting diluted W2, [Lac] 0 31 g/L, as a function of time in the presence of E. coli JFR12 in a bioreactor of 2 L. Results are means ± SD of an experiment performed in duplicate.

  123

Figure A. 1 :

 1 Figure A.1: Scheme of enzymatic reactions involved in the transformation of glucose and galactose into pyruvic acid.

Figure A. 2 :

 2 Figure A.2: Metabolic pathway to produce 2,3-BD from glucose and galactose fermentation in the presence of a genetically modified strain of Escherichia coli. Relevant reactions to transform pyruvic acid into 2,3-BD are represented by the names of the corresponding enzymes from Enterobacter cloacae: α-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC) and 2,3-butanediol dehydrogenase (BDH). Sources:[START_REF] Lazar | Awakening the endogenous Leloir pathway for efficient galactose utilization by Yarrowia lipolytica[END_REF],[START_REF] Mazumdar | Microbial production of 2, 3 butanediol from seaweed hydrolysate using metabolically engineered Escherichia coli[END_REF],[START_REF] Xiao | Strategies for enhancing fermentative production of acetoin: a review[END_REF],[START_REF] Xu | Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2, 3-butanediol[END_REF],[START_REF] Xu | Metabolic engineering of Escherichia coli for efficient production of (3R)-acetoin[END_REF].

3 :

 3 Figure A.3: Glucose (Figure A.3A) and galactose (Figure A.3B) conversion as a function of time. Results are means ± SD of 3 replications performed in flasks at 37ºC, 1 atm, initial pH 7.4, 100 rpm and 10% (v/v) of inoculum in the presence of E. coli JFR1 in M9 culture medium (200 mL of working volume) with 12.5 (•), 25 (•) and 50 (○) g/L of monosaccharide.

Figure A .

 A Figure A.4 (A and B) shows the ABD yield for 3 initial concentrations (12.5, 25 and 50 g/L) of glucose and galactose ([Glu] 0 and [Gal] 0 , respectively) as a function of time. For the [Glu] 0 of 12.5 g/L, the maximum ABD yield (0.18 g/g glucose) was obtained at 24 h and remained nearly constant up to 120 h. For a [Glu] 0 of 25 g/L, the ABD yield increased reaching a plateau at 0.25 g/g glucose at 72 h; whereas with the [Glu] 0 of 50 g/L, the ABD yield increased up to 0.15 g/g glucose at 120 h (p < 0.05) as shown inFigure A.4A.

  Figure A.4: ABD yield from glucose (Figure A.4A) and galactose (Figure A.4B) as a function of time. Results are means ± SD of 3 replications performed in flasks at 37ºC, 1 atm, initial pH 7.4, 100 rpm and 10% (v/v) of inoculum in the presence of E. coli JFR1 in M9 culture medium (200 mL of working volume) at 12.5 (▲), 25 (▲) and 50 (∆) g/L of monosaccharide.

Table 2

 2

	.1: Composition of sweet and acid wheys. Source: Hernández-Rojas and Vélez-Ruiz
	(2014); Panesar et al. (2007); Pescuma et al. (2015); Tsakali et al. (2010).

Components Sweet whey (% (w/w)) Acid whey (% (w/w))

		Enzymatic production	Acid production
	Water	93 -94	93 -95
	Dry matter	6.0 -7.0	5.0 -7.0
	Lactose *	70 -77	66 -72
	Proteins *	8 -15	8 -15
	Minerals *	7 -15	7 -15
	Lactic acid *	Up to 3	10 -13

Table 2 .

 2 3: Characteristics and particularities of some bacteria producers of 2,3 butanediol

	Genus	Strain	Characteristics	Particularities	Reference
			Gram-negative bacteria
				Human pathogen
	Enterobacter	E. cloacae E. aerogenes	Facultative anaerobic bacteria Rod-shaped bacteria	High ability to keep the damage and invasion of	Davin-Regli 2015; Santo Pereira et al. 2016
			Non-spore-forming	tissue host
			bacteria
				Human pathogen
			Gram-negative bacteria
				High ability and speed to
			Facultative anaerobic	grow
	Klebsiella	K. pneumoniae * K. oxytoca	bacteria Rod-shaped bacteria Non-motile bacteria	High resistance against penicillin *	PHAC 2017; Trivedi et al. 2015a, b
				High resistance against host
			Non-spore-forming	defenses due to its
			bacteria	prominent polysaccharide
				capsule
			Gram-positive bacteria		Bhandari et al. 2013; de
	Bacillus	B. licheniformis B. amyloliquefaciens	Spore-forming bacteria Facultative anaerobic/aerobic bacteria	Resistance to heat, chemical Generally regarded as safe (GRAS)	Clerck et al. 2004; Erem et al. 2009;
		B. subtilis	Rod-shaped bacteria	products and irradiation
			Motile bacteria

Biovalorization of glucose in four culture media and effect of the nitrogen source on fermentative alcohols production by Escherichia coli Avant-propos:

  Michèle Heitz is grateful to le Fond Québécois de la Recherche sur la Nature et les Technologies (FRQNT) in partnership with MAPAQ-NOVALAIT (Innovation en Production et en Transformation Laitières) for the grant attributed to the research program. The authors would like to thank Professor Ryszard Brzezinski of the Department of Biology, Université de Sherbrooke for his helpful discussion and advices. L'article "Biovalorization of glucose in four culture media and effect of the nitrogen source on fermentative alcohols production by Escherichia coli" a été publié dans le Journal "Environmental Technology". doi : https://doi.org/10.1080/09593330.2018.1494751. La version de l'article présenté dans le document diffère de ce qui a été publié. Biovalorization of glucose in four culture media and effect of the nitrogen source on fermentative alcohols production by Escherichia coli.David Fernández-Gutiérrez a,c , Marc Veillette a , Antonio Avalos Ramirez b , Anne Giroir-Fendler c , Nathalie Faucheux a and Michèle Heitz a, d

	Chapter 3.

TITRE: Biovalorisation du glucose dans quatre milieux de culture et effet de la source d'azote sur la production d'alcools par fermentation en présence d'Escherichia coli. Title: a Université de Sherbrooke, Department of Chemical Engineering and Biotechnological Engineering. Faculty of engineering, 2500 boul. de l'Université, Sherbrooke, (Québec), Canada, J1K 2R1 b Centre National en Électrochimie et en Technologies Environnementales, Shawinigan, (Québec), Canada, G9N 6V8 c Université Lyon 1, CNRS, UMR 5256, Institut de Recherche sur la Catalyse et l'Environnement de Lyon (IRCELYON), 2 avenue Albert Einstein, 69626 Villeurbanne, France d Corresponding author: E-mail address: Michele.Heitz@USherbrooke.ca

  Le glucose est l'un des monosaccharides les plus abondants. Il est la source de carbone la plus facile à 'consommer' par des bactéries. Dans cette étude, quatre milieux de culture (LB, M9, M63 et MOPS) ont été sélectionnés ainsi que trois solutions de glucose de concentration de 4, 12.5 et 25 g/L en présence d'une souche génétiquement modifiée d'Escherichia coli. Les quatre milieux de culture ont été testés pour obtenir de l'ABD (acétoïne (A) et le 2,3butanediol (2,3-BD)). Le milieu de culture sélectionné a été le M9, le plus économique, et permettant d'atteindre des rendements en ABD de 0.22 g/g glucose, lors de la fermentation de 12.5 et 25 g/L de glucose dans le milieu de culture M9 à 37ºC, pression atmosphérique, pH initial 6.5, 100 rpm et 10% (v/v) d'inoculum. L'influence de l'azote sur le rendement en ABD a été évalué en ajoutant du nitrate de sodium (NaNO 3 ) ou de l'urée ((NH 2 ) 2 CO) dans le milieu de culture M9 sous 3 concentrations d'azote(2.5, 5.0 et 7.0 g azote/L). Le rendement en ABD fut amélioré de 23% à 96 h en ajoutant de l'urée (7.0 g azote/L) dans le milieu M9 en présence de 25 g/L de glucose. Par contre, l'utilisation de NaNO 3 a eu un effet négligeable sur la formation d'ABD.

	3.1	Résumé

Table 3 .

 3

	Culture medium	Nutrient	Concentration (g/L)
		Tryptone	10.00
	LB	Yeast extract	5.00
		NaCl	10.00
		Na 2 HPO 4 • 7H 2 O	12.80
		KH 2 PO 4	3.00
	M9	NH 4 Cl	1.00
	(supplemented with MgSO 4 •7H 2 O and CaCl 2 )	NaCl	0.50
		MgSO 4 •7H 2 O	0.49
		CaCl 2	0.01
		KH 2 PO 4	13.50
	M63	(NH 4 ) 2 SO 4 MgSO 4 • 7 H 2 O	2.00 0.20
		FeSO 4	5.0x10 -4
		MOPS	8.30

1: Composition of the 4 culture media in 1L of distilled water -LB medium

[START_REF] Berney | Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection[END_REF]

); M9 minimal medium

[START_REF] Ecocyc | Escherichia coli K-12 substr[END_REF][START_REF] Maniatis | Molecular cloning: a laboratory manual[END_REF]

; M63 minimal medium

[START_REF] Pardee | The genetic control and cytoplasmic expression of "inducibility" in the synthesis of β-galactosidase by E. coli[END_REF]

; and MOPS (3-morpholinopropane-1-sulfonic acid) minimal medium (Wisconsin-University 2017).

  2,3-butanediol" a été soumis dans un journal scientifique international (Environmental Science and Pollution Research) le 10 juillet 2018. Un autre article relatif au chapitre 4 (partie 2) devrait être soumis ultérieurement dans un Journal scientifique international. Une autorisation pour publication de la 2 e partie du chapitre 4 doit être demandée au préalable à Novalait. La version de l'article présenté dans le document diffère de ce qui a été soumis.

	a Université de Sherbrooke, Department of Chemical Engineering and Biotechnological
	Engineering. Faculty of engineering, 2500 boul. de l'Université, Sherbrooke, (Québec),
	Canada, J1K 2R1
	b Centre National en Électrochimie et en Technologies Environnementales, Shawinigan,
	(Québec), Canada, G9N 6V8

TITRE:

Fermentation du lactosérum et de son perméat en présence d'une souche génétiquement modifiée d'Escherichia coli afin de produire du 2,3-butanediol Title: Fermentation of whey and its permeate using a genetically modified strain of Escherichia coli to produce 2,3-butanediol.

David Fernández-Gutiérrez a,c , Marc Veillette a , Antonio Avalos Ramirez b , Anne Giroir-Fendler c , Nathalie Faucheux a and Michèle Heitz a, d c Université Lyon 1, CNRS, UMR 5256, Institut de Recherche sur la Catalyse et l'Environnement de Lyon (IRCELYON), 2 avenue Albert Einstein, 69626 Villeurbanne, France d Corresponding author: E-mail address: Michele.Heitz@USherbrooke.ca

  lactosérum (W) est obtenu lors de la fabrication du fromage et le perméat de lactosérum (PW) est obtenu après la séparation des protéines du W. Les deux effluents contiennent du lactose, source de carbone, et ils peuvent affecter l'environnement s'ils sont rejetés dans la nature sans traitement préalable. L'objectif de ce travail de recherche a été d'étudier la transformation du lactose par fermentation en 2,3-butanediol (2,3-BD) en présence d'une souche d'Escherichia coli K12 MG1655 (E. coli JFR12) génétiquement modifiée afin d'évaluer le potentiel du W et PW comme sources de lactose. Le lactose (un disaccharide), le glucose et le galactose (monosaccharides présents dans le lactose) sous des concentrations de 12.5, 25 et 50 g/L ont été fermentés dans le milieu de culture synthétique (milieu M9) inoculé à 10% (v/v) d'inoculum, 37ºC, 1 atm, initial pH 7.4 et 100 rpm pendant 72 h dans des fioles de 0.5 L (volume de travail de 0.2 L). Dans ces conditions opératoires, le meilleur rendement en 2,3-BD a été de 0.38 g/g de saccharide en présence de 25 g/L de glucose ou de lactose à 48 et 72 h, respectivement, et de 0.19 g/g de galactose à 48 h. Donc, E. coli JFR12 peut transformer le galactose en 2,3-BD mais le rendement en 2,3-BD est deux fois plus faible si on le compare

	50:50
	(v/v) du W ou du PW avec M9 à 72 h. L'effet de trois paramètres (pH initial (de 6.5 à 7.4),
	quantité d'inoculum (de 5 à 10%, v/v) et vitesse d'agitation (de 50 à 200 rpm) a été testé pour
	étudier l'influence de ces paramètres sur le rendement en 2,3-BD en absence de M9. Le pH
	initial n'affecte pas le rendement en 2,3-BD (0.45 et 0.42 g/g lactose pour le W et le PW,
	respectivement). L'utilisation de 5% (v/v) d'inoculum a permis d'obtenir un rendement en 2,3-
	BD légèrement inférieur par comparaison avec les expériences réalisées à 10% (v/v)
	d'inoculum. Les résultats obtenus en faisant varier la vitesse d'agitation ont montré que le
	rendement en 2,3-BD a diminué et que celui de l'acétoïne a augmenté avec une vitesse
	d'agitation de 200 rpm. Ce phénomène peut s'expliquer par une augmentation de l'oxygène
	transféré au milieu de culture sous une agitation de 200 rpm en comparaison avec une
	agitation de 100 rpm. De plus, des fermentations du W et du PW dans un bioréacteur de 2 L
	ont été effectuées en conditions anaérobies et micro-aérobies pour évaluer l'augmentation du
	volume réactionnel sur le rendement en 2,3-BD. Les conditions anaérobies ont conduit à des
	rendements en 2,3-BD plus faibles, environ 0.20 g/g de lactose lors de la fermentation du W à
	72 h, tandis que l'ajout d'air (2 L/min) a permis l'obtention d'un rendement en 2,3-BD
	similaire à celui obtenu dans des fioles de 0.5 L, 0.40 g/g de lactose après 24 h en utilisant du
	W à 10% (v/v) d'inoculum, 37ºC, 1 atm, pH initial de 7.4 et vitesse d'agitation de 100 rpm.
	Les résultats ont montré que E. coli JFR12 est aussi efficace que les producteurs naturels de
	2,3-BD pour fermenter le lactose et obtenir du 2,3-BD.
	Mots-clefs: Lactose, fabrication de fromage, glucose, galactose, fermentation, 2,3-butanediol,
	acétoïne, Escherichia coli.

avec celui issu du glucose à 48 h. La fermentation du W (31 g/L) ou du PW (34 g/L) dilués avec le milieu M9 (50:50, 75:25 et 100:0, v/v) a été réalisée sous les mêmes conditions opératoires. La fermentation du W et du PW en absence de M9 (100:0, v/v) a fourni les rendements les plus élevés en 2,3-BD (jusqu'à 0.43 g/g lactose) à 72 h. Tandis que le rendement le plus élevé en acétoïne (0.12 g/g lactose) a été obtenu avec une dilution de

Table 4 .

 4 2: Maximum Et yield fermenting glucose, galactose, lactose and a mixture (1:1, w/w) of glucose-galactose at different concentrations at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 7.4 and 100 rpm.

	Saccharide	Saccharide concentration (g/L)	Et yield (g/g saccharide)
		12.5	0.09
	Glucose	25.0	0.05
		50.0	0.02
		12.5	0.08
	Galactose	25.0	0.05
		50.0	0.02
		12.5	0.10
	Lactose	25.0	0.05
		50.0	0.02
	Glucose-galactose	25.0 50.0	0.05 0.02
	4.5.1.2 Galactose

Table 4 .

 4 3: Acetoin, 2,3-BD and ABD yields as a function of time fermenting W1 and PW1 at 37ºC, 1 atm and initial pH 7.4 for 48 and 72 h in flasks varying the agitation rate.

	W1

  [START_REF]Abstract Whey (W) is generated during cheese manufacturing and permeate whey (PW) is obtained after W deproteinization. As both effluents contain lactose, a carbon source, and they can cause environmental issues if they are released in the environment without a treatment. The aim of the present study was to test the transformation of lactose via fermentation into 2,3butanediol (2,3-BD) in the presence of a genetically modified strain of Escherichia coli K12 MG1655 (E. coli JFR12) in order to evaluate the potential of W and PW as lactose sources. Lactose (a disaccharide), glucose and galactose (monosaccharides of lactose) at concentrations of 12.5, 25 and 50 g/L were fermented in a synthetic culture medium (medium 9, M9) inoculated at a ratio of 10% (v/v), at 37ºC, 1 atm, initial pH 7.4 and 100 rpm for 72 h in flasks of 0.5 L with a working volume of 0.2 L. Under these operating conditions, the highest 2,3-BD yield was around 0.38 g/g saccharide in the presence of 25 g/L of glucose or lactose at 48 and 72 h, respectively, and 0.19 g/g galactose at 48 h. Therefore, E. coli JFR12 could transform galactose into 2,3-BD but being its yield the half of one observed with glucose at 48 h. Whey (31 g/L lactose) or PW (34 g/L lactose) were fermented in dilution with M9 (50:50, 75:25 and 100:0, v/v) under the same operating conditions. The fermentation of W and PW in absence of M9 (100:0, v/v) produced the highest 2,3-BD yield (up to 0.43 g/g lactose) at 72 h. Whereas, the highest acetoin (A) yield of 0.12 g/g lactose was obtained with[END_REF], une molécule plateforme. Ces microorganismes se divisent en deux catégories: i) ceux produisant naturellement du 2,3-BD i.e. E. cloacae et K. pneumoniae, et ii) ceux génétiquement modifiés tels Escherichia coli.La biovalorisation des saccharides par fermentation permet contrairement au procédé chimique (160 à 220ºC, 50 bar) de travailler sous des conditions opératoires douces (température variant entre 25 et 40ºC, pression atmosphérique et pH 7.0) et de produire du 2,3-BD non issu de ressources pétrolières. Chaque souche bactérienne est affectée différemment en fonction des conditions opératoires telles le type et la concentration de nutriments dans le milieu de culture, la température, le pH, la vitesse d'agitation, l'aération, etc. Lors de cette étude, l'objectif principal a été d'étudier l'activité d'une souche génétiquement modifiée d'E. coli (E. coli JFR12) K12 MG1655 afin de transformer le lactose contenu dans le lactosérum (W) et le perméat de lactosérum (PW) en 2,3-BD.Le premier objectif spécifique a été de mesurer et de quantifier la quantité de bactéries présentes dans l'inoculum avant de réaliser des fermentations. De plus, une comparaison entre les souches sauvage (ECW) et génétiquement modifiée (E. coli JFR1) d'E. coli a été accomplie en utilisant la méthode des unités formatrices de colonies (CFU). Pour la souche E. coli JFR1, la valeur de CFU est de 6.5x10 8 CFU/mL, et est inférieure de 41% à celle obtenue Le sixième objectif spécifique a porté sur l'influence de trois paramètres (pH initial, volume d'inoculum et vitesse d'agitation) sur le rendement en 2,3-BD obtenu lors de la fermentation de W1 ou PW1 en absence du milieu de culture M9 et en présence d'E. coli JFR12. Le pourcentage d'inoculum à 7.5% (v/v) a donné un rendement similaire en 2,3-BD à celui obtenu avec le pourcentage d'inoculum à 10% (v/v), proches de 0.45 et 0.42 g 2,3-BD/g lactose pour W1 et PW1 respectivement à 37ºC, sous 1 atm, un pH initial 7.4 et une vitesse d'agitation de 100 rpm. En ce qui concerne l'influence du pH initial, il n'y a eu aucun changement constaté sur le rendement en 2,3-BD. Dans ce cas particulier, en présence de W1 et PW1, les rendements en 2,3-BD ont été de 0.47 et 0.44 g 2,3-BD/g lactose, respectivement à 10% (v/v) d'inoculum, à 37ºC, sous 1 atm et une vitesse d'agitation de 100 rpm. En ce qui concerne la fermentation du lactosérum à 10% (v/v) d'inoculum, à 37ºC, sous 1 atm et un pH initial de 7.4, une vitesse d'agitation de 200 rpm, un rendement maximum en 2,3-BD de 0.23 g/g lactose après 24 h a été obtenu. Ce rendement est deux fois plus faible que celui obtenu avec une vitesse d'agitation de 100 rpm après 72 h. Pour la fermentation du PW1 dans des conditions opératoires similaires, une vitesse d'agitation de 200 rpm a conduit aux mêmes effets sur le rendement en 2,3-BD.Un nouveau lot de W (W2) et de PW (PW2) a été fermenté dans des fioles de 0.5 L puis dans un bioréacteur de 2 L (sans aération) à 10% (v/v) d'inoculum, à 37ºC, sous 1 atm, avec un pH initial de 7.4 et une vitesse d'agitation de 100 rpm. Le W2 et le PW2 ont respectivement une concentration de lactose de 51 et 47 g/L. Les deux effluents ont été fermentés directement et dilués avec de l'eau distillée pour obtenir respectivement une concentration en lactose de 31 et 34 g/L que ce soit pour les expériences menées en fiole ou en bioréacteur de 2 L. En fiole, les rendements en 2,3-BD obtenus avec les substrats W2 et PW2 dilués sont comparables à ceux issus des expériences effectuées avec les substrats W1 et PW1. En condition anaérobie, il a été constaté dans les deux cas (W1 et W2 ou PW1 ou PW2) que les rendements en 2,3-BD sont plus faibles.Ensuite, en bioréacteur de 2 L, l'effet de l'apport d'air a été testé à 2 L/min pour étudier son impact sur la formation de 2,3-BD en présence de W2 dilué. L'addition d'air a permis d'augmenter le rendement en 2,3-BD de 135% (0.40 g, 2,3-BD/g lactose) par rapport à celui issu des expériences en bioréacteur de type fiole (0.5 L). Par comparaison, les rendements en 2,3-BD obtenus dans les fioles après 24 h à 10% (v/v) d'inoculum, à 37ºC, sous 1 atm, un pH initial de 7.4 et une vitesse d'agitation de 100 rpm sont plus faibles. Ainsi, l'utilisation d'un bioréacteur sous flux d'air, permet d'obtenir un meilleur rendement plus rapidement (diminution du temps de fermentation de 72 h à 24 h).Par conséquent, le principal objectif de cette étude, qui était d'optimiser la fermentation de W et PW en présence d'une souche génétiquement modifiée d'E. coli, a été atteint. La fermentation des deux effluents a donné des rendements élevés en 2,3-BD, représentant 89 et 84% des rendements théoriques(W et PW, respectivement). De cette façon, la souche E. coli JFR12 peut être considérée comme une bonne candidate pour la valorisation de W et PW afin de produire du 2,3-BD. Quigley T (2008) Monitoring the growth of E. coli with light scattering using the synergy™ 4 multi-mode microplate reader with hybrid technology. http://mktg.biotek.com/TekTalk/2008/0208/tek_corner.htm. Accessed 19 December 2017 Qureshi N, Maddox I (2005) Reduction in butanol inhibition by perstraction: utilization of concentrated lactose/whey permeate by Clostridium acetobutylicum to enhance butanol fermentation economics. Food Bioprod Process 83:43-52. doi: 10.1205/fbp.04163 Raganati F, Olivieri G, Procentese A, Russo M, Salatino P, Marzocchella A (2013) Butanol production by bioconversion of cheese whey in a continuous packed bed reactor. Bioresour Technol 138:259-265. doi: 10.1016/j.biortech.2013.03.180 Ramirez C, Patel M, Blok K (2006) From fluid milk to milk powder: energy use and energy

	efficiency	in	the	European	dairy	industry.	Energy	31:1984-2004.	doi:
	10.1016/j.energy.2005.10.014

Acknowledgement

Michèle Heitz is grateful to Fond Québécois de la Recherche sur la Nature et les Technologies (FRQNT) in partnership with MAPAQ-NOVALAIT (Innovation en Production et en Transformation Laitières) for the grant attributed to the research program "Développement d'une biotechnologie verte innovatrice pour valoriser des résidus fromagers". The authors are grateful to the laboratory technicians (Department of chemical and biotechnological engineering of Université de Sherbrooke) Isabelle Arsenault and Valérie Larouche for their support and help. The authors would like to particularly thank Pr. Sébastien Rodrigue and the master student Jean-François Rousseau (Department of biology of Université de Sherbrooke) for providing the genetically modified strain E. coli JFR1.

Acknowledgement

Michèle Heitz is grateful to Fond Québécois de la Recherche sur la Nature et les Technologies (FRQNT) in partnership with MAPAQ-NOVALAIT (Innovation en Production et en Transformation Laitières) for the grant attributed to the research program "Développement d'une biotechnologie verte innovatrice pour valoriser des résidus fromagers". The authors are grateful to the laboratory technicians (Department of chemical and biotechnological engineering of Université de Sherbrooke) Isabelle Arsenault and Valérie Larouche for their support and help. The authors would like to particularly thank Pr. Sébastien Rodrigue and the master student Jean-François Rousseau (Department of biology of Université de Sherbrooke) for providing the genetically modified strain E. coli JFR12. The authors would like to thank Pr. Denis Groleau (Department of chemical and biotechnological engineering of Université de Sherbrooke) for lending his bioreactor to perform the fermentations shown in the present chapter. Michèle Heitz would also like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for a financially contribution at the end of the study. Acknowledgement Michèle Heitz is grateful to Fond Québécois de la Recherche sur la Nature et les Technologies (FRQNT) in partnership with MAPAQ-NOVALAIT (Innovation en Production et en Transformation Laitières) for the grant attributed to the research program "Développement d'une biotechnologie verte innovatrice pour valoriser des résidus fromagers". The authors are grateful to the laboratory technicians (Department of chemical and biotechnological engineering of Université de Sherbrooke) Isabelle Arsenault and Valérie Larouche for their support and help. The authors would like to particularly thank Pr. Sébastien Rodrigue and the master student Jean-François Rousseau (Department of biology of Université de Sherbrooke) for providing the genetically modified strain E. coli JFR1. Michèle Heitz would also like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for a financially contribution at the end of the study.

Annex 1. Fermentation of glucose and galactose by a genetically modified strain of Escherichia coli into acetoin and 2,3-butanediol Avant-propos: L'article "Fermentation of glucose and galactose by a genetically modified strain of Escherichia coli into acetoin and 2,3-butanediol" sera soumis en tant que compte-rendu de conférence au cours de la prochaine année TITRE: Fermentation du glucose et du galactose par une souche génétiquement modifiée d'Escherichia coli afin de produire de l'acétoïne et de 2,3-butanediol Title: Fermentation of glucose and galactose by a genetically modified strain of Escherichia coli into acetoin and 2,3-butanediol Contribution to the document: This paper shows the potential of a genetically modified strain of Escherichia coli (E. coli JFR1) to transform glucose and galactose into ABD (acetoin (A) and 2,3-butanediol [START_REF]Abstract Whey (W) is generated during cheese manufacturing and permeate whey (PW) is obtained after W deproteinization. As both effluents contain lactose, a carbon source, and they can cause environmental issues if they are released in the environment without a treatment. The aim of the present study was to test the transformation of lactose via fermentation into 2,3butanediol (2,3-BD) in the presence of a genetically modified strain of Escherichia coli K12 MG1655 (E. coli JFR12) in order to evaluate the potential of W and PW as lactose sources. Lactose (a disaccharide), glucose and galactose (monosaccharides of lactose) at concentrations of 12.5, 25 and 50 g/L were fermented in a synthetic culture medium (medium 9, M9) inoculated at a ratio of 10% (v/v), at 37ºC, 1 atm, initial pH 7.4 and 100 rpm for 72 h in flasks of 0.5 L with a working volume of 0.2 L. Under these operating conditions, the highest 2,3-BD yield was around 0.38 g/g saccharide in the presence of 25 g/L of glucose or lactose at 48 and 72 h, respectively, and 0.19 g/g galactose at 48 h. Therefore, E. coli JFR12 could transform galactose into 2,3-BD but being its yield the half of one observed with glucose at 48 h. Whey (31 g/L lactose) or PW (34 g/L lactose) were fermented in dilution with M9 (50:50, 75:25 and 100:0, v/v) under the same operating conditions. The fermentation of W and PW in absence of M9 (100:0, v/v) produced the highest 2,3-BD yield (up to 0.43 g/g lactose) at 72 h. Whereas, the highest acetoin (A) yield of 0.12 g/g lactose was obtained with[END_REF]). In this study, the differences between glucose and galactose as a carbon source for the ABD production are shown, presenting the ability of E. coli to consume the monosaccharides of lactose and the potential of the mutant strain to valorize the disaccharide.

Fermentation of glucose and galactose by a genetically modified strain of Escherichia coli into acetoin and 2,3butanediol ). Toutes les expériences réalisées ont montré que la fermentation du galactose était moins efficace que celle du glucose (les rendements en ABD ont été de 28, 37 et 43% (concentration de galactose de 12.5, 25 et 50 g/L) inférieurs à ceux obtenus lors de la fermentation du glucose. Le rendement le plus élevé en ABD a été 0.25 g/g de glucose, lequel a été obtenu à 72 h pour une concentration de glucose de 25 g/L.

Mots-clefs: Monosaccharides, Escherichia coli, milieu de culture M9, biovalorisation.

A.2 Abstract

Acetoin (A) and 2,3-butanediol (BD) are two important chemical since they can be transformed into other chemical products like 1,3-propanediol. Their production can be performed by biological way in the presence of microorganisms, natural producers of A and 2,3-BD. However, the main issue of these bacteria to produce ABD (A + 2,3-BD), such as Klebsiella pneumoniae or K. oxytoca, is their biosafety level since they are pathogen. In this way, non-pathogenic bacteria can be genetically modified to produce ABD. In the present study, a genetically modified strain of Escherichia coli K12 MG1655 (non-pathogenic strain) was used in order to produce ABD. Two monosaccharides (glucose and galactose) at three concentrations (12.5, 25 and 50 g/L) were fermented for 120 h at 37ºC, 1 atm, initial pH 7.4, 100 rpm and 10% (v/v) of inoculum in a synthetic culture medium (M9). All experiments showed that the fermentation of galactose was less efficient than glucose (the ABD yields were 28%, 37% and 43% at 12.5, 25 and 50 g/L of galactose compared to those obtained fermenting glucose). The highest ABD yield was 0.25 (g ABD/g glucose), obtained at 96 h in the presence of 25 g/L of glucose at 72 h.

Keywords: Monosaccharide, glucose, galactose, M9 culture medium, urea, biovalorisation distilled water [START_REF] Berney | Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection[END_REF]). The pH of LB for growing E. coli JFR1 was adjusted at 6.5. The fermentation of glucose and galactose by E. coli JFR1 was tested using medium 9 (M9), which was made as follows: 12.8 g/L of sodium hydrogen phosphate heptahydrate (Na 2 HPO 4 •7H 2 O), 3 g/L of potassium dihydrogen phosphate (KH 2 PO 4 ), 1 g/L of ammonium chloride (NH 4 Cl), 0.5 g/L NaCl, 15 g/L urea ((NH 2 ) 2 CO), 0.49 g/L of magnesium sulfate heptahydrate (MgSO 4 •7H 2 O) and 0.01 g/L of calcium chloride (CaCl 2 ) and distilled water [START_REF] Fernández-Gutierrez | Biovalorization of glucose in four culture media and effect of the nitrogen source on fermentative alcohols production by Escherichia coli[END_REF]).

The seed culture medium was prepared as follows: a sample of E. coli JFR1 from the conservation blend was taken with a tip and mixed with 10 mL of fresh LB medium in a test tube (15 mL). The test tube was incubated overnight at 37ºC (pre-culture medium).

Then, 2 mL of the pre-culture medium were transferred into a 0.5 L Erlenmeyer flask containing 0.2 L of fresh LB medium and incubated in a rotary shaker incubator (Fermentation Design inc, Allentown, PA) at 37ºC, 1 atm, initial pH 6.5 and 100 rpm to reach a bacterial population of 6.5x10 8 colony-forming unit (CFU)/mL in 9 h. The seed culture medium was used to inoculate M9.

All fermentations were performed in triplicate at 10% (v/v) of inoculum, 37ºC, 1 atm, initial pH 7.4 and 100 rpm in 0.5 L flask (0.2 L of working volume).

A.4.3 Analytical methods and reagents

The analysis of glucose, galactose, BD and A was determined by high performance liquid chromatography (HPLC) as shown in previous studies [START_REF] Fernández-Gutierrez | Biovalorization of glucose in four culture media and effect of the nitrogen source on fermentative alcohols production by Escherichia coli[END_REF].

A.4.4 Statistical analysis

The ABD formation may be influenced by the type and concentration of the substrate. In this way, the effect of glucose and galactose concentration on ABD yield was statistically determined by means of an analysis of variance (ANOVA) at p < 0.05. In addition, Dixon's Q test was carried out to estimate and rule out the outlier values of ABD yield at a confidence level of 95% [START_REF] Rorabacher | Statistical treatment for rejection of deviant values: critical values of Dixon's" Q" parameter and related subrange ratios at the 95% confidence level[END_REF].

A.5 Results and discussion

The fermentation of glucose and galactose was performed in M9 culture medium in order to produce ABD by E. coli JFR1. The effect of 3 concentrations (12.5, 25 and 50 g/L) of glucose and galactose were tested.

Figure A.3 (A and B) shows the glucose and galactose conversion for 3 initial concentrations (12.5, 25 and 50 g/L) of glucose and galactose ([Glu] 0 and [Gal] 0 , respectively) as a function of time. At 24 h, for a [Glu] 0 of 12.5 g/L, the glucose conversion was 100%, whereas for a [Glu] 0 of 25 and 50 g/L conversions were 77% and 56%, respectively. Glucose conversion was 100% for a [Glu] 0 of 25 g/L at 72 h, while, the conversion was 70% fermenting 50 g/L. The maximum conversion for a [Glu] 0 of 50 g/L was 77%, obtained at 120 h (Figure A.3A).

Similarly, the galactose conversion was 100% for a [Gal] 0 of 12.5 g/L at 24 h; whereas it was 75 and 55% in the presence of 25 and 50 g/L of galactose, respectively. At 72 h, the galactose conversion was 100% using a [Gal] 0 of 25 g/L and it was 72% for a [Gal] 0 of 50 g/L. The maximum galactose conversion for a [Gal] 0 of 50 g/L was 76%, which was reached at 120 h (Figure A.3B).

As observed in Figure A.3A and B, the trend of glucose and galactose conversions was similar for both monosaccharides whatever the concentration used. On the other hand, the use of a high concentration (50 g/L) led to a low conversion (around 75%) even after 120 h of fermentation. Bacteria present a threshold concentration to which the bacterial population might be affected if it is exceeded, causing a slower bacterial growth as suggested by [START_REF] Chan | Process optimization on microaeration supply for high production yield of 2, 3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca[END_REF]. In the case of E. coli JFR1, this threshold concentration seems to be between 25 and 50 g/L since the 100% conversion was reached using 25 g/L for both monosaccharides at 72 h; however, it was not achieved for 50 g/L at 120 h.

Glucose conversion (%) Time (h)

A)

This study demonstrated that galactose can be used by E. coli JFR1 in order to produce ABD. Hence, an in depth study using this monosaccharide should be considered in the presence of E. coli JFR1 with the aim of improving the ABD yield.

A.7