Corynebacterium glutamicum, a natural overproducer of succinic acid?

Corynebacterium glutamicum is well known as an important industrial amino acid producer. For a few years, its ability to produce organic acids, under micro-aerobic or anaerobic conditions was demonstrated. This study is focused on the identification of the culture parameters influencing the organic acids production and, in particular, the succinate production, by this bacterium. Corynebacterium glutamicum 2262, used throughout this study, was a wild-type strain, which was not genetically designed for the production of succinate. The oxygenation level and the residual glucose concentration appeared as two critical parameters for the organic acids production. The maximal succinate concentration (4.9 g L-1) corresponded to the lower kLa value of 5 h-1. Above 5 h-1, a transient accumulation of the succinate was observed. Interestingly, the stop in the succinate production was concomitant with a lower threshold glucose concentration of 9 g L-1. Taking into account this threshold, a fed-batch culture was performed to optimize the succinate production with C. glutamicum 2262. The results showed that this wild-type strain was able to produce 93.6 g L-1 of succinate, which is one of the highest concentration reported in the literature.

Pilot-scale biomethanation of cattle manure using dense membranes.

This study aimed at studying the biomethanation process using a 100 L pilot-scale digester equipped with a dense membrane for hydrogen injection. Hydrogen mass transfer was characterized and the impact of hydrogen flowrate, agitation rate and of the co-injection of CO2, on biogas production and composition, was precisely studied. A linear relationship between H2 flowrate and the CO2 and CH4 rates in biogas was found but no impact on biogas flowrate was shown. It was also noticed that, without exogenous CO2 injection, and for high H2 injection flowrates, residual H2 could be found at the digester outlet due to local CO2 limitation. Thus, this study suggested that biogas production in biomethanation process at the pilot scale was probably rather limited by the dissolved CO2 transport within the liquid phase than by the hydrogen mass transfer itself.

Control of IgG glycosylation by in situ and real-time estimation of specific growth rate of CHO cells cultured in bioreactor.

The cell-specific growth rate (µ) is a critical process parameter for antibody production processes performed by animal cell cultures, as it describes the cell growth and reflects the cell physiological state. When there are changes in these parameters, which are indicated by variations of µ, the synthesis and the quality of antibodies are often affected. Therefore, it is essential to monitor and control the variations of µto assure the antibody production and achieve high product quality. In this study, a novel approach for on-line estimation of µ was developed based on the process analytical technology initiative by using an in situ dielectric spectroscopy. Critical moments, such as significant µ decreases, were successfully detected by this method, in association with changes in cell physiology as well as with an accumulation of nonglycosylated antibodies. Thus, this method was used to perform medium renewals at the appropriate time points, maintaining the values of µ close to its maximum. Using this method, we demonstrated that the physiological state of cells remained stable, the quantity and the glycosylation quality of antibodies were assured at the same time, leading to better process performances compared with the reference feed-harvest cell cultures carried out by using off-line nutrient measurements.

Impact of shear stress and impeller design on the production of biogas in anaerobic digesters.

Today, intensification of anaerobic digestion is still a scientific and technical challenge. The present study proposed combined experimental and computational fluid dynamics simulations to characterize the impact of shear stress and impeller design on the biogas production after sequential additions of substrate. Liquid phase (cattle manure digestate) rheological law was experimentally determined and input in numerical simulations. The results showed that the original use of a double helical ribbon in digester allowed a significantly faster dispersion of fresh substrate than the use of a classical Rushton turbine, leading to a 50% higher methane production rate. However, with both impellers, too high agitation rates entailed a clear slow-down of production rate and a decrease in CH4 content. To avoid this loss of productivity, it was shown that the maximal value of shear stress, determined by numerical simulations, was a consistent parameter to set the upper agitation conditions in digesters.

Response surface methodology applied to Supercritical Fluid Extraction (SFE) of carotenoids from Persimmon (Diospyros kaki L.).

Supercritical carbon dioxide with ethanol as co-solvent was used to extract carotenoids from persimmon fruits (Diospyros kaki L.). Based on a response surface methodology (RSM), a predicting model describing the effects of CO2 temperature, pressure, flow rate, ethanol percentage and extraction time was set up for each of the four carotenoids of interest. The best extraction yields in our experimental domain were found at 300 bars, 60°C, 25% (w/w) ethanol, 3mL/min flow rate and 30min for xanthophylls (all-trans-lutein, all-trans-zeaxanthin and all-trans-ß-cryptoxanthin). The yields were 15.46±0.56, 16.81±1.74 and 33.23±2.91µg/g of persimmon powder for all-trans-lutein, all-trans-zeaxanthin and all-trans-ß-cryptoxanthin, respectively. As a non-oxygenated carotenoid, all-trans-ß-carotene was better extracted using 100 bars, 40°C, 25% (w/w) ethanol, 1mL/min flow rate and 30min extraction time, with an extraction yield of 11.19±0.47µg/g of persimmon powder.

Combination of yeast hydrolysates to improve CHO cell growth and IgG production.

Many studies underlined the great benefits of hydrolysates used as additives in animal free media on cell culture performances. However, to precisely define hydrolysate supplementation strategies, a deeper understanding of their effect on cell growth and protein production is required. In the present study, the effect of addition of one yeast extract (YE) and two yeast peptones (named YP.A and YP.B) in a chemically defined medium was first assessed on cell culture performances. Interestingly, specific effects were found depending on the degree of degradation of yeast hydrolysates. The YE at 1 g L(-1) increased the maximal cell density by 70 %, while a mixture of YE (1 g L(-1)) and YP.A (4 g L(-1)) increased IgG production by 180 %. These conditions were then evaluated on the CHO cell kinetics all over cultures. Hydrolysates extended the cell growth phase in Erlenmeyer flask and increased the maximal growth rate in bioreactor up to 20 %. Cell growth stimulation induced by hydrolysates addition was linked with energetic metabolism improvement suggesting that they promote oxidative pathway. Furthermore, hydrolysates provided an additional source of substrate that supported cell growth despite glutamine limitation.

Kinetic characterization of vero cell metabolism in a serum-free batch culture process.

A global kinetic study of the central metabolism of Vero cells cultivated in a serum-free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum-free medium which is a valuable help for the implementation of this cell line in serum-free industrial production processes.