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Table of contents
1 Chapter 1. Bibliographic study
1.1 Biobased 3-hydroxypropionic acid: a building block molecule for versatile industrial applications
1.1.1 Definition and physic-chemical properties of 3-HP
1.1.2 The increasing interest of biobased 3-HP and its applications
1.1.3 Summary of information
1.2 Bioproduction of 3-HP by using microorganisms
1.2.1 Biotechnological production of 3-HP: a challenge in the context of bioeconomy
1.2.2 Bioproduction of 3-HP from glucose
1.2.3 Bioproduction of 3-HP from glycerol
1.2.3.1 Transport of glycerol into the cells
1.2.3.2 Utilization of glycerol for growth
1.2.3.3 Bioproduction of 3-HP from glycerol via the Dha pathway
1.2.3.4 Bioproduction of 3-HP from glycerol via the Pdu pathway
1.2.4 Bioproduction of 3-HP from other carbon sources
1.2.5 The bacterium Lactobacillus reuteri: a promising strain for glycerol bioconversion
1.2.5.1 General characteristics of L. reuteri
1.2.5.2 General metabolic pathways for growth of L. reuteri
1.2.5.3 Bioproduction of bacteriocins by L. reuteri
1.2.5.4 Bioproduction of 3-HP by L. reuteri
1.2.6 Metabolic engineering approaches for 3-HP bioproduction
1.2.7 Summary of information
1.3 Effect of nutritional and environmental conditions on the growth of L. reuteri
1.3.1 Effects of nutritional conditions on L. reuteri growth
1.3.1.1 Carbon sources
1.3.1.2 Nitrogen sources
1.3.1.3 C/N ratio
1.3.1.4 Salts
1.3.1.5 Vitamins
1.3.1.6 Tween 80
1.3.1.7 Cysteine
1.3.1.8 Betaine
1.3.2 Effects of environmental conditions on the growth of L. reuteri in bioreactors
1.3.2.1 Agitation speed
1.3.2.2 Culture temperature
1.3.2.3 pH and base used for pH control
1.3.2.4 Osmolarity
1.3.2.5 Gaseous atmosphere
1.3.3 Summary of information
1.4 Glycerol bioconversion into 3-HP by L. reuteri
1.4.1 Major challenges of 3-HP bioproduction from glycerol by L. reuteri
1.4.1.1 Separation of growth and bioconversion steps
1.4.1.2 Limited conversion yield
1.4.1.3 Vitamin B12 supplementation
1.4.1.4 Inhibition of bioconversion by 3-HPA
1.4.1.5 Inhibition of bioconversion by 3-HP
1.4.2 Production modes used for glycerol bioconversion into 3-HP by L. reuteri
1.4.2.1 Bioconversion in batch process
1.4.2.2 Bioconversion in fed-batch process
1.4.2.3 Bioconversion using immobilized cells
1.4.2.4 Bioconversion associating a complexation of 3-HPA
1.4.3 Effects of environmental conditions during bioconversion on the performances of 3-HP production from glycerol
1.4.3.1 Composition of bioconversion medium
1.4.3.2 Temperature
1.4.3.3 pH
1.4.3.4 Gaseous atmosphere
1.4.3.5 Glycerol feeding rate and specific glycerol feeding rate
1.4.4 Effects of growth conditions on glycerol bioconversion into 3-HP
1.4.4.1 Harvesting time during growth
1.4.4.2 Addition of vitamin B12 in growth medium
1.4.4.3 Addition of 1,2-propanediol in the growth medium
1.4.5 Summary of information
1.5 Knowledge synthesis
2 Chapter 2. Materials and methods
2.1 Overall methodology
2.2 Materials
2.2.1 Bacterial strain
2.2.2 Chemicals used
2.2.3 Elemental analysis of L. reuteri DSM 17938 and complex media
2.2.3.1 Elemental analysis of complex media
2.2.3.2 Elemental analysis of L. reuteri DSM 17938
2.3 Two-step bioprocess for 3-HP bioproduction
2.3.1 Bacterial growth in batch mode
2.3.2 Cell harvesting and concentration
2.3.3 Glycerol bioconversion in fed-batch mode
2.4 Experimental designs
2.4.1 Plackett and Burman experimental design and statistical analysis
2.4.1.1 Experimental factors and their levels
2.4.1.2 Responses variables and statistical analyses
2.4.2 Central composite design
2.4.2.1 Experimental factors and their levels
2.4.2.2 Response variables and statistical analyses
2.5 Analytical methods
2.5.1 Characterization of cell concentration and cell physiological state
2.5.1.1 Quantification of cell dry weight
2.5.1.2 Measurement of optical density
2.5.1.3 Quantification of cell concentration and cell physiological state by flow cytometry
2.5.1.4 Correlations between optical density, cell dry weight and cell concentration obtained by flow cytometry
2.5.2 Assessment of intracellular energy
2.5.3 Measurement of intracellular pH
2.5.4 Quantification of substrates and metabolites by HPLC
2.5.5 Assessment of molecular balance and carbon mass balance
3 Chapter 3. Effect of culture conditions on L. reuteri DSM 17938 growth and ability to perform glycerol bioconversion 64
3.1 Introduction
3.2 Experimental strategy
3.3 Results and Discussion
3.3.1 Preliminary tests
3.3.1.1 Preliminary tests about glucose concentration
3.3.1.2 Preliminary tests about temperature
3.3.1.3 Preliminary tests about controlled pH
3.3.2 Kinetics of cell growth and glycerol bioconversion by L. reuteri DSM 17938 in the reference condition of the first experimental design
3.3.2.1 Kinetics of L. reuteri DSM 17938 growth in the reference condition 76
3.3.2.2 Kinetics of glycerol bioconversion by L. reuteri DSM 17938 in the reference condition
3.3.2.3 Summary of information
3.3.3 Effect of growth culture conditions on growth performance of L. reuteri DSM 17938
3.3.3.1 Effect of growth conditions on the final cell concentration
3.3.3.2 Effect of growth conditions on the lactic acid production yield
3.3.3.3 Effect of growth conditions on the ratio between [acetic acid + ethanol] and lactic acid
3.3.4 Effect of culture conditions on bioconversion performance of L. reuteri DSM 17938
3.3.4.1 Effect of glucose supplementation on 3-HP bioproduction
3.3.4.2 Effect of yeast extract and phytone peptone addition on 3-HP bioproduction
3.3.4.3 Effect of Tween 80 addition on 3-HP bioproduction
3.3.4.4 Effect of vitamin B12 addition on 3-HP bioproduction
3.3.4.5 Effect of 1,2-PDO addition on 3-HP bioproduction
3.3.4.6 Effect of cysteine addition on 3-HP bioproduction
3.3.4.7 Effect of the addition of betaine and KCl on 3-HP bioproduction
3.3.4.8 Effect of growth temperature on 3-HP bioproduction
3.3.4.9 Effect of growth pH and base used for pH control on 3-HP bioproduction
3.3.5 Selected growth conditions that improve L. reuteri DSM 17938 ability to convert glycerol into 3-HP
3.3.6 Effect of harvest time on the ability of L. reuteri DSM 17938 to produce 3- HP from glycerol
3.4 Conclusion of Chapter 3
4 Chapter 4. Effect of environmental conditions during bioconversion on 3-HP bioproduction by L. reuteri DSM 17938
4.1 Introduction
4.2 Experimental strategy
4.3 Preliminary tests to define the conditions to be used before and during the bioconversion step
4.3.1 Effect of freezing frozen storage on glycerol bioconversion by L. reuteri DSM 17938
4.3.2 Effect of two centrifugation steps on the ability of L. reuteri DSM 17938 to produce 3-HP from glycerol F
4.3.3 Effect of cell concentration and glycerol feeding rate on 3-HP bioproduction by L. reuteri DSM 17938
4.3.4 Summary of information
4.4 Preliminary tests to define the factors to be retained in the second experimental design
4.4.1 Effect of the bioconversion temperature on the ability of L. reuteri DSM 17938 to produce 3-HP from glycerol
4.4.2 Effect of the bioconversion pH on the ability of L. reuteri DSM 17938 to produce 3-HP from glycerol
4.4.3 Summary of information
4.5 Optimization of 3-HP bioproduction from glycerol by L. reuteri DSM 17938
4.5.1 Experimental approach
4.5.2 Results of the two-factors central composite rotatable design
4.5.3 Effect of pH and specific glycerol feeding rate on specific glycerol consumption rate
4.5.4 Effect of pH and specific glycerol feeding rate on 3-HP titer, 3-HP final quantity and 3-HP production yield by L. reuteri DSM 17938
4.5.5 Effect of pH and specific glycerol feeding rate on 3-HP production rate and specific 3-HP production rate of 3-HP by L. reuteri DSM 17938
4.5.6 Effect of pH and specific glycerol feeding rate on 3-HP volumetric productivity of L. reuteri DSM 17938
4.5.7 Summary of the information
4.6 Validation experiments for the optimization of 3-HP bioproduction by L. reuteri DSM 17938
4.6.1 Experimental approach
4.6.2 Kinetics of glycerol bioconversion into 3-HP by L. reuteri DSM 17938 at pH
6.0 and specific glycerol feeding rate of 60 mgglycerol·gCDW -1·h-1
4.6.3 Validation of the glycerol into 3-HP bioconversion kinetics by L. reuteri DSM 17938 at pH 6.0 and specific glycerol feeding rate of 80 mgglycerol·gCDW -1·h-1
4.6.4 Summary of the information
4.7 Assessment of intracellular pH and intracellular energy level during glycerol bioconversion into 3-HP by L. reuteri DSM 17938
4.7.1 Experimental approach
4.7.2 Results and discussion
4.7.3 Summary of information
4.8 Conclusion of chapter 4
5 Conclusion and prospects
6 References
