Effect of pH and specific glycerol feeding rate on specific glycerol consumption rate

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Effects of nutritional conditions on L. reuteri growth

During a bioprocess, nutritional conditions have to be defined in order to maximize the bacterial concentration and the specific growth rate, and to optimize the physiological state of the microorganism in order to produce efficiently the desired biomolecule. L. reuteri species has complex nutritional requirements. In addition to the common carbon and nitrogen sources, the growth of the bacteria needs to be supplied with specific amino acids, peptides, salts, fatty acids or fatty acid esters, nucleic acid derivatives and vitamins.

Carbon sources

As one of the first requirements for bacterial growth, the carbon source is necessary for biomass growth, product formation and maintenance of cell physiological state. When the cells enter the stationary phase as a consequence of substrate depletion, they are progressively exposed to carbohydrate starvation. The exhaustion of essential nutrients induces a decrease in the specific growth rate until cessation of growth. This is explained by a downregulation of nucleic acid and protein synthesis and a degradation of proteins as an adaptive response (De Angelis and Gobbetti, 2011; Wang et al., 2011). Besides, an increase in membrane fluidity, ascribed to a higher content of branched, unsaturated, and cyclic fatty acids was found in starved cells (Wang et al., 2011). Different carbon sources have been used to trigger L. reuteri growth, such as glucose alone (Årsköld et al., 2008; Burgé et al., 2015b), glucose plus fructose, saccharose (Årsköld et al., 2008), galactose, lactose, melibiose, raffinose, saccharose (Alazzeh et al., 2009) and industrial wheat or sugar beet syrup by-products that contained glucose and fructose (Couvreur et al., 2017). Among them, glucose is most widely used in research studies. As L. reuteri displays a heterofermentative metabolism, glucose is catabolized through the PK and EMP pathways that operate simultaneously and lead to the production of lactic acid, acetic acid, ethanol, CO2 and energy (Årsköld et al., 2008).
From (Bengtsson, 2020), the presence of glucose into the culture medium led to the highest biomass production of L. reuteri DSM 17938 (OD620 nm of 6.4) in comparison to the other carbon sources tested (maltose, maltose plus fructose, glucose plus fructose, sucrose alone and sucrose plus fructose. This was confirmed later by (Polak-Berecka et al., 2010) with Lactobacillus rhamnosus and studied as a component of an industrial byproduct by (Couvreur et al., 2017) with L. reuteri DSM 17938. One of advantage of using this sugar is its wide availability on the market. The suggested concentrations of glucose were comprised between 20 g·L-1 (Bengtsson, 2020) and 30 g·L-1 (Couvreur et al., 2017) in total.

Effects of environmental conditions on the growth of L. reuteri in bioreactors

Environmental conditions have to be controlled during any bioprocess, in order to potentiate the metabolic activity of the microorganisms. Agitation rate, temperature, pH, osmolarity and atmosphere are the most important factors that have to be taken into account (Hammes and Hertel, 2006).

Agitation speed

Agitation is compulsory in a bioreactor, to permit homogenization and avoid cell agglomeration and decantation, to facilitate temperature control and to reduce gradients (substrates and oxygen concentrations, pH). However, it can generate some shear stress, thus affecting membrane integrity and modifying cell functions. In addition, increasing agitation speed leads to the increase of dissolved oxygen concentration, which can engender an oxidative stress during anaerobic bioprocesses.
In the case of anaerobic bacteria like L. reuteri, the agitation speed only tends to ensure medium homogenization and microaerobic conditions. In addition, as L. reuteri does not form filaments unlike filamentous bacteria or fungi, the cells are not sensitive to shear stress. According to (Ju et al., 2020), low stirring rates (50 or 100 rpm) are more adapted to the production of biomass of L. reuteri CH53 than higher agitation rates (300 rpm). Consequently, a moderate value of stirring rate is commonly used during L. reuteri cultures, such as 100 rpm (Burgé et al., 2015c; Couvreur et al., 2017; Ju et al., 2020; Ortiz et al., 2015) or 200 rpm (Chen and Hatti-Kaul, 2017; Dishisha et al., 2014).

Culture temperature

Temperature is one of the most important environmental conditions that affect bacterial growth. Basically, increasing the temperature until the optimum value enhances the specific growth rate by improving the enzyme activities, modifying the membrane fluidity and altering the saturation level of the fatty acids of the phospholipids, both at intracellular and membrane levels. It also modifies the affinity between the microorganism and the substrates (Nedwell, 1999). However, at too high temperatures, denaturation of macromolecules occurs, thus leading to cell death.
Most lactobacilli species are mesophilic, with an optimum temperature ranging between 30 and 45 °C. They can also grow at lower temperatures (Van De Guchte et al., 2002) and until an upper limit of 53 °C, depending on the species (Ahmed et al., 2006).
Previous studies have investigated the optimal temperature of L. reuteri. By considering the DSM 17938 strain, it was demonstrated that 37 °C is the optimal temperature for cell growth. The specific growth rate was two-fold increased at 37 °C as compared to 32 °C, irrespective of culture pH (Hernández et al., 2019). A slight different value was found for L. reuteri CG001 that exhibited an optimal temperature of 38.6 °C (Chen et al., 2010).
It should be noticed that the optimum temperature for growth may differ from that maximizing a given metabolic activity. For example, the optimal temperature for lactic acid production by Lactobacillus acidophilus RD758 is 3 °C lower than the optimal growth temperature (Wang, 2005). In addition, L. reuteri I5007 cultured at 47 °C showed a better survival after freeze-drying, as compared to the optimum growth temperature of 37 °C (Liu et al., 2014). Consequently, this environmental factor has to be well defined for each strain and each purpose of bioprocess.

pH and base used for pH control

To ensure a high metabolic activity, the cells have to maintain their intracellular pH at a suitable value, even though the external pH is more acid or alkaline, since both disturb bacterial growth and survival (Wall et al., 2007). This necessity is explained by the mechanisms linked to the proton motive force (Chen et al., 2019). As a lactic acid bacterium, L. reuteri produces lactic acid as the main product of the glycolysis pathway. Lactic acid is excreted into the extracellular medium to help the cells to maintain their intracellular pH, thus leading to an acidification of the extracellular medium. Although L. reuteri is relatively acid tolerant (Teixeira et al., 2014), the accumulation of lactic acid and other organic acids associated, or not, with a pH decrease considerably affects the physiological state of the cells. Cytoplasmic acidification causes the inhibition of enzyme activities, the decrease in intracellular energy production, together with the increase in energy expenditure to cope with cytoplasmic acidification (Even et al., 2002). In these conditions, energetic limitations may occur, thus diminishing the specific growth rate and in some cases, the final biomass concentration (Schepers et al., 2002). The inhibition is more important at low pH, i.e. when the proportion of undissociated lactic acid is high as compared to the lactate form (Schepers et al., 2002). To enhance the bacterial growth, it is recommended to maintain the pH at a constant value corresponding to the optimum pH of bacterial cells. In that condition, the growth is improved (Bai et al., 2004) as a consequence of better enzymatic activities (Ampatzoglou et al., 2010).
By considering L. reuteri, the growth can occur in a large range of pH, between pH 5.0 and pH 7.5, depending on the strain. From (Kandler et al., 1980), the optimum pH is generally comprised between pH 6.0 and pH 6.8 for L. reuteri sp. It was identified at pH 5.5 for L. reuteri DSM 12246 (El-Ziney, 2018) and L. reuteri DSM 17938 (Hernández et al., 2019) and at pH 5.7 for L. reuteri I5007 (Liu et al., 2014). In addition, the maximum pH at which growth stopped has been reported at pH 8.1 for the L. reuteri JCM1112T strain (Sawatari and Yokota, 2007).
As for the temperature, the optimal pH for a given functionality may differ from the one for growth. For example, L. reuteri ATCC 55730 was more resistant to freeze-drying when the cells were cultured at pH 5.0 as compared to pH 6.0 (Palmfeldt and Hahn-Hägerdal, 2000). Three kinds of base have been reported to control the pH during L. reuteri growth: KOH (Burgé et al., 2015c), NaOH (Doleyres et al., 2005; Hernández et al., 2019; Kristjansdottir et al., 2019) and NH4OH (Dishisha et al., 2014; Ricci et al., 2015; Sardari et al., 2013a). However, the issue of whether the kind of supplied base affects or not the bacterial growth has not yet been studied.

Major challenges of 3-HP bioproduction from glycerol by L. reuteri

The bioconversion of glycerol into 3-HP by L. reuteri, according to the Pdu pathway shown in Section 1.2.3.4, shall take into account five main barriers that have to be controlled in order to achieve high performances during the bioprocess. These barriers are presented below, as well as the answers to avoid them.

Separation of growth and bioconversion steps

Due to the characteristics of the metabolic pathway that transforms glycerol into 3-HP (Figure 1.7), the growth stage of L. reuteri must be separated from the subsequent bioconversion phase, during which the cells will be used as whole-cell biocatalysts. Indeed, when glucose and glycerol are simultaneously brought in the medium, the cofactor NADH generated from the glycolytic pathway leads to a redox imbalance between NAD+ and NADH in the Pdu pathway (Dishisha et al., 2014), thus resulting in 1,3-PDO production only (Lüthi-Peng et al., 2002; Sriramulu et al., 2008). Besides, the biosynthesis of coproducts during the growth, such as lactic acid, ethanol and acetic acid will be a hindrance to the further 3-HP extraction processes (Burgé, 2015). Consequently, to separate the growth from the 3-HP bioproduction, a specific step of biomass harvesting coupled with a washing of cell pellets is necessary to eliminate the growth medium that contains metabolites and residual glucose. The solution used for washing and re-suspending cells is required to be not or less deleterious to cell physiological state. This solution can be a saline buffer, physiological water, or osmosis water that was recently used to re-suspend the resting cells L. reuteri DSM 17938 (Burgé, 2015). A recent study in our lab reported that cell pellets of L. reuteri DSM 17938 re-suspended directly in osmosis water without washing showed a better physiological state than cells suspended and washed by potassium phosphate buffer or by physiological water (Görge, 2016).
As a direct consequence of the separation of growth and bioconversion steps and of the dilution of the cells in osmosis water, a lack of intracellular energy may occur in the resting cells during the bioconversion, as there is no more carbon source for maintenance. Depletion of intracellular energy in resting cells may reduce the bioreaction kinetics. However, during the last step of the Pdu pathway that refers to the transformation of 3-hydroxypropionyl-phosphate into 3-HP, some ATP regeneration occurs (Figure 1.7) that may limit this energy depletion (Matsakas et al., 2018).

Limited conversion yield

The second challenge that 3-HP bioproduction shall take up concerns the limitation of the conversion yield of glycerol into 3-HP, that cannot be higher than a maximum value of 50 % in mole (Dishisha et al., 2014). This limitation is the result of the redox balance requirement that directs the catabolism of glycerol into equimolar proportions of 3-HP and 1,3-PDO (Figure 1.7). One solution to overcome this barrier is to couple the bioconversion process with another bioprocess to convert 1,3-PDO into 3-HP. This was proposed by (Dishisha et al., 2015) and (Spinnler et al., 2020) who demonstrated that 1,3-PDO produced by L. reuteri can subsequently be converted into 3-HP using an acetic acid bacterium such as G. oxydans (Dishisha et al., 2015) or Acetobacter sp. CIP 58.66 (Spinnler et al., 2020).

Vitamin B12 supplementation

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The mandatory presence of vitamin B12 constitutes the third challenge of the glycerol bioconversion process into 3-HP. Vitamin B12 is as a required cofactor of glycerol dehydratase, the enzyme involved in the dehydration of glycerol into 3-HPA (Toraya, 2002). Although L. reuteri owns the advantage of natural ability to synthesize this component (Matsakas et al., 2018), a supplementation of the bioconversion medium with an exogenous source of vitamin B12 may boost the glycerol metabolism (Couvreur et al., 2017). However, this supplementation is costly, which could be a serious impediment in the context of industrial production of 3-HP.

Bioconversion associating a complexation of 3-HPA

In order to alleviate the inhibitory effect of 3-HPA, a complexation of the molecule with scavengers can be considered. This approach was achieved by complexing 3-HPA with sodium bisulfite (Sardari et al., 2013b), carbohydrazide (Dishisha et al., 2014; Krauter et al., 2012) or semicarbazide (Talarico et al., 1988), with the aim to increase 3-HPA bioproduction or to protect cells from 3-HPA detrimental effects. When the complexation was performed with sodium bisulfite (43.4 g·L-1) during a fed-batch bioconversion with L. reuteri DSM 20016, the specific 3-HPA production and the overall molar ratio of 3-HPA to 1,3-PDO and 3-HP were increased by 2.2 times as compared to the fed-batch process without in situ complex formation (Sardari et al., 2013b). The protective effect of carbohydrazide has been studied during 3-HPA production by L. reuteri SD2112 (Krauter et al., 2012). The maximal titer of 150 g·L-1 3-HPA was achieved in batch biotransformation with an initial glycerol concentration of 184 g·L-1 and an initial carbohydrazide concentration of 180 g·L-1. These conditions led to the concomitant achievement of 3-HP 5.9 g·L-1 and 1,3-PDO 6.4 g·L-1. These authors showed that when 3-HPA was entrapped, its production was improved, without benefiting to 3-HP bioproduction. The component semicarbazide was utilized during reuterin production by L. reuteri 1063 (Talarico et al., 1988). Nevertheless, this complexation displayed an inhibition of the production of reuterin (i.e., the dimer form of 3-HPA) due to the covalent combination of semicarbazide with 3-hydroxypropionaldehyde. From this information, the addition of 3-HPA scavengers did not lead to an increase in 3-HP bioproduction.

Effects of environmental conditions during bioconversion on the performances of 3-HP production from glycerol

During the bioconversion step, L. reuteri cells are sensitive to environmental conditions such as temperature, pH and atmosphere composition. In addition, the substrate feeding conditions of the fed-batch process and the complementation with some specific components are also influent.

Composition of bioconversion medium

Before entering the bioconversion phase, cells resulting from the growth phase have to be  harvested and washed to eliminate growth products and remaining glucose. It is also possible to concentrate the cells to maximize the biomass concentration before starting the bioconversion. Cells are then re-suspended in a specific bioconversion medium that is supplied with glycerol in fed-batch. Different bioconversion media have been proposed to perform 3-HP bioproduction. As previously mentioned, a non-growing medium has to be used for 3-HP bioproduction, in order to prevent the synthesis of undesirable fermentation products (i.e. lactic acid, ethanol, acetic acid) (Burgé, 2015). In fact, the absence of glucose was shown to reduce the production of 1,3-PDO and to permit that of 3-HP, because of the balance between redox cofactors (Chen and Hatti-Kaul, 2017; Dishisha et al., 2014). Consequently, only glycerol shall be present as a carbon source in the bioconversion broth.
Moreover, from the study of (Görge, 2016), the re-suspension of washed cells of L. reuteri DSM 17938 in osmosis water gave better results compared to re-suspension in potassium phosphate buffer or in physiological water. Cells were able to better maintain their enzymatic activity and to ensure the maintenance of cell energy.
The addition of Na+ has also been documented. It leads to a decrease in the activity of the enzyme 1,3-PDO oxidoreductase (Malaoui et al., 2000) that explained the decrease of 3-HPA production by L. reuteri ATCC 53608 (Lüthi-Peng et al., 2002). However, as less 3-HPA was synthesized, it was less available for oxidation into 3-HP.
The presence of some free amino groups of peptides, that are included in complex media such as sodium caseinates, casein hydrolysates, peptones and Amicase (i.e., casein acid hydrolysate) was considered by (Lüthi-Peng et al., 2002). These components may react with 3-HPA as soon as it is produced, thus leading to a decrease in its concentration in the bioconversion medium.

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

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