Jean-Louis Uribelarrea

Transcriptomic Analyses during the Transition from Biomass Production to Lipid Accumulation in the Oleaginous Yeast Yarrowia lipolytica

We previously developed a fermentation protocol for lipid accumulation in the oleaginous yeast Y. lipolytica. This process was used to perform transcriptomic time-course analyses to explore gene expression in Y. lipolytica during the transition from biomass production to lipid accumulation. In this experiment, a biomass concentration of 54.6 gCDW/l, with 0.18 g/gCDW lipid was obtained in ca. 32 h, with low citric acid production. A transcriptomic profiling was performed on 11 samples throughout the fermentation. Through statistical analyses, 569 genes were highlighted as differentially expressed at one point during the time course of the experiment. These genes were classified into 9 clusters, according to their expression profiles. The combination of macroscopic and transcriptomic profiles highlighted 4 major steps in the culture: (i) a growth phase, (ii) a transition phase, (iii) an early lipid accumulation phase, characterized by an increase in nitrogen metabolism, together with strong repression of protein production and activity; (iv) a late lipid accumulation phase, characterized by the rerouting of carbon fluxes within cells. This study explores the potential of Y. lipolytica as an alternative oil producer, by identifying, at the transcriptomic level, the genes potentially involved in the metabolism of oleaginous species.

Impact of sustaining a controlled residual growth on polyhydroxybutyrate yield and production kinetics in Cupriavidus necator

In this study a complementary modeling and experimental approach was used to explore how growth controls the NADPH generation and availability, and the resulting impact on PHB (polyhydroxybutyrate) yields and kinetics. The results show that the anabolic demand allowed the NADPH production through the Entner-Doudoroff (ED) pathway, leading to a high maximal theoretical PHB production yield of 0.89 C mole C mole(-1); whereas without biomass production, NADPH regeneration is only possible via the isocitrate dehydrogenase leading to a theoretical yield of 0.67 C mole C mole(-1). Furthermore, the maximum specific rate of NADPH produced at maximal growth rate (to fulfil biomass requirement) was found to be the maximum set in every conditions, which by consequence determines the maximal PHB production rate. These results imply that sustaining a controlled residual growth improves the PHB specific production rate without altering production yield.

Polyhydroxybutyrate production by direct use of waste activated sludge in phosphorus-limited fed-batch culture.

Polyhydroxybutyrate (PHB) production directly by waste activated sludge (WAS) was investigated in aerobic fed-batch conditions using acetic acid as substrate. PHB production was induced by phosphorus limitation. WAS of different origin were tested with various degrees of phosphorus limitation and PHB contents of up to 70% (gCOD PHB/gCOD particulate) were obtained. This strategy showed the importance of maintaining cell growth for PHB production in order to increase PHB concentration and that the degree of phosphorus limitation has a direct impact on the quantity of PHB produced. Pyrosequencing of 16S rRNA transcripts showed changes in the active bacteria of the WAS microbial community as well as the acclimation of populations depending on sludge origin. The monitoring of the process appeared as the key factor for optimal PHB production by WAS. Different strategies are discussed and compared in terms of carbon yield and PHB content with the feast and famine selection process.

Dynamic model of temperature impact on cell viability and major product formation during fed-batch and continuous ethanolic fermentation in Saccharomyces cerevisiae.

The impact of the temperature on an industrial yeast strain was investigated in very high ethanol performance fermentation fed-batch process within the range of 30-47 °C. As previously observed with a lab strain, decoupling between growth and glycerol formation occurred at temperature of 36 °C and higher. A dynamic model was proposed to describe the impact of the temperature on the total and viable biomass, ethanol and glycerol production. The model validation was implemented with experimental data sets from independent cultures under different temperatures, temperature variation profiles and cultivation modes. The proposed model fitted accurately the dynamic evolutions for products and biomass concentrations over a wide range of temperature profiles. R2 values were above 0.96 for ethanol and glycerol in most experiments. The best results were obtained at 37 °C in fed-batch and chemostat cultures. This dynamic model could be further used for optimizing and monitoring the ethanol fermentation at larger scale.

Phosphorus limitation strategy to increase propionic acid flux towards 3-hydroxyvaleric acid monomers in Cupriavidus necator

Properties of polyhydroxybutyrate-co-hydroxyvalerate (P(3HB-co-3HV)) depend on their 3HV content. 3HV can be produced by Cupriavidus necator from propionic acid. Few studies explored carbon distribution and dynamics of 3HV and 3HB monomers production, and none of them have been done with phosphorus as limiting nutrient. In this study, fed-batch cultures of C. necator with propionic acid, as sole carbon source or mixed with butyric acid, were performed. Phosphorus deficiency allowed sustaining 3HV production rate and decreasing 3HB production rate, leading to an instant production of up to 100% of 3HV. When a residual growth is sustained by a phosphorus feeding, the maximum 3HV percentage produced from propionic acid is limited to 33% (Mole.Mole(-1)). The association of a second carbon source like butyric acid lead to higher conversion of propionic acid into 3HV. This study showed the importance of the limiting nutrient and of the culture strategy to get the appropriate product.

Real-time monitoring of metabolic shift and transcriptional induction of yciG::luxCDABE E. coli reporter strain to a glucose pulse of different concentrations

Ineffective mixing entailing heterogeneity issue within industrial bioreactors has been reported to affect microbial physiology and consequently bioprocess performances. Alteration of these performances results from microorganism ability to modulate their physiology at metabolic and/or transcriptional levels in order to survive in a given environment. Until now, dynamics of both metabolic and transcriptional microbial response to external stimuli have been investigated using mainly ex situ measurements with sampling and/or quenching constraints. This work showed an in situ bioluminescence approach for real-time monitoring of characteristic stress responses of Escherichia coli containing yciG::luxCDABE reporter to glucose pulses in well-controlled steady-state chemostat cultures. Reproducibility of in situ bioluminescence profiles was assessed. A dramatic transient increase in the bioluminescence intensity (sharp peak) was observed for a complete depletion of sugars and for a sudden decrease in the dilution rate. This response was connected to a sudden change of the metabolic activity. On the contrary a bell curve of bioluminescence intensity, dose-dependent, was related to an induction of transcriptional activity. Real-time monitoring of the bioluminescence signal with time-span less than a second gave access to the characteristic times of the metabolic shift and transcriptional induction of the stress response.

Understanding of polyhydroxybutyrate production under carbon and phosphorus-limited growth conditions in non-axenic continuous culture.

In a waste into resource strategy, a selection of polyhydroxybutyrate (PHB)-accumulating organisms from activated sludge was achieved in an open continuous culture under acetic acid and phosphorus limitation. Once the microbial population was selected at a dilution rate (D), an increase in phosphorus limitation degree was applied in order to study the intracellular phosphorus plasticity of selected bacteria and the resulting capacity to produce PHB. Whatever D, all selected populations were able to produce PHB. At a D, the phosphorus availability determined the phosphorus-cell content which in turn fixed the amount of cell. All the remaining carbon was thus directed toward PHB. By decreasing D, microorganisms adapted more easily to higher phosphorus limitation leading to higher PHB content. A one-stage continuous reactor operated at D=0.023h(-)(1) gave reliable high PHB productivity with PHB content up to 80%. A two-stage reactor could ensure better productivity while allowing tuning product quality.

Kinetic and stoichiometric characterization of organoautotrophic growth of Ralstonia eutropha on formic acid in fed-batch and continuous cultures.

Formic acid, acting as both carbon and energy source, is a safe alternative to a carbon dioxide, hydrogen and dioxygen mix for studying the conversion of carbon through the Calvin–Benson–Bassham (CBB) cycle into value‐added chemical compounds by non‐photosynthetic microorganisms. In this work, organoautotrophic growth of Ralstonia eutropha on formic acid was studied using an approach combining stoichiometric modeling and controlled cultures in bioreactors. A strain deleted of its polyhydroxyalkanoate production pathway was used in order to carry out a physiological characterization. The maximal growth yield was determined at 0.16 Cmole Cmole−1 in a formate‐limited continuous culture. The measured yield corresponded to 76% to 85% of the theoretical yield (later confirmed in pH‐controlled fed‐batch cultures). The stoichiometric study highlighted the imbalance between carbon and energy provided by formic acid and explained the low growth yields measured. Fed‐batch cultures were also used to determine the maximum specific growth rate (μmax = 0.18 h−1) and to study the impact of increasing formic acid concentrations on growth yields. High formic acid sensitivity was found in R eutropha since a linear decrease in the biomass yield with increasing residual formic acid concentrations was observed between 0 and 1.5 g l−1.

Dynamic metabolic modeling of lipid accumulation and citric acid production by Yarrowia lipolytica

Yarrowia lipolytica has the capacity to accumulate large amounts of lipids triggered by a depletion of nitrogen in excess of carbon source. However, under similar conditions this yeast also produces citric acid decreasing the lipid conversion yield. Three dynamic metabolic models are presented to describe lipid accumulation and citric acid production by Yarrowia lipolytica. First and second models were respectively based on the Hybrid Cybernetic Modeling (HCM) and the Macroscopic Bioreaction Modeling (MBM) approaches. The third model was a new approach based on the coupling between MBM and fuzzy sets. Simulation results of the three models fitted acceptably the experimental data sets for calibration and validation. However, MBM is time-dependent to consider metabolic shifts, and thus impractical for further applications. HCM and Fuzzy MBM adequately managed and described metabolic shifts presenting highlighting features for control and optimization. HCM and Fuzzy MBM were statistically compared reflecting similar results.

Improving carbon and energy distribution by coupling growth and medium chain length polyhydroxyalkanoate production from fatty acids by Pseudomonas putida KT2440

The production of medium chain length polyhydroxyalkanoates by Pseudomonas putida KT2440 from fatty acids leads to the loss of a large proportion of carbon. We studied the possibility of a shift of potentially available energy and carbon towards monitored residual growth during the production phase. A Fed-Batch culture achieving 125.6 g/L of total biomass containing 54.4% (g/g) of medium chain length polyhydroxyalkanoates was carried out leading to an overall experimental carbon yield of 0.7 Cmole/Cmole. The analysis of modeling fluxes deduced from experimental data indicated how carbon and reduced cofactors (NADH and FADH2) were managed to conclude that part of the carbon and reduced cofactors made available by polymer production were used in anabolic pathways. The strategy which consisted in coupled growth and medium chain length polyhydroxyalkanoate production enhanced the global yields compared to growth followed by a production phase. The understanding of carbon and energy fluxes distribution allowed deducing optimized culture strategy to perform the highest reported in the literature.