Ivan Marc

Kinetic profile of the cellular lipid composition in an oleaginous Yarrowia lipolytica capable of producing a cocoa-butter substitute from industrial fats

Cell growth, lipid accumulation and cellular lipid composition of Yarrowia lipolytica growing on mixtures of industrial fats containing stearic, oleic, linoleic and palmitic acid have been studied. During growth, the strain incorporated oleic and linoleic acids more rapidly than the saturated fatty acids. Relatively high lipid accumulation (up to 0.44 g of lipids per g of dry matter) was observed when stearic acid was included in the culture medium. In contrast, substrates rich in oleic acid did not favor cellular lipid accumulation. The accumulated lipids, mainly composed of triacylglycerols (45-55% w/w), demonstrated a different total fatty acid composition compared with that of the substrate; in all cases, the microorganism showed the unusual capacity to increase its cellular stearic acid level, even if this fatty acid was not found in high concentrations in the substrate. This permitted the synthesis of interesting lipid profiles with high percentages of stearic acid and non-negligible percentages of palmitic and oleic acid, with a composition resembling that of cocoa-butter.

Accumulation of a cocoa-butter-like lipid by Yarrowia lipolytica cultivated on agro-industrial residues.

Yarrowia lipolytica was cultivated on mixtures of saturated free fatty acids (an industrial derivative of animal fat called stearin), technical glycerol (the main by-product of bio-diesel production facilities), and glucose. The utilization of technical glycerol and stearin as co-substrates resulted in higher lipid synthesis and increased citric acid production than the combination of glucose and stearin. The lipids produced contained significant amounts of stearic acid (50–70%, wt/wt) and lower ones of palmitic (15–20%, wt/wt), oleic (7–20%, wt/wt), and linoleic (2–7%, wt/wt) acid. Single-cell oil having a composition similar to cocoa-butter up to 3.4 g/L was produced, whereas in some cases relatively increased citric acid quantities (up to 14 g/L) were excreted into the growth medium. The microorganism presented a high specificity for lauric, myristic, and palmitic acid, while a discrimination for the stearic acid was observed. As a conclusion, microbial metabolism could be directed by using mixtures of inexpensive saturated fats, glycerol, and glucose as co-substrates, in order to accumulate lipids with predetermined composition, e.g., cocoa-butter equivalents.

Influence of Glucose and Saturated Free-Fatty Acid Mixtures on Citric Acid and Lipid Production by Yarrowia lipolytica

In the present report, the effect of glucose and stearin (substrate composed by saturated free-fatty acids) on the production of biomass, reserve lipid, and citric acid by Yarrowia lipolytica ACA-DC 50109 was investigated in nitrogen-limited cultures. Numerical models that were used in order to quantify the kinetic behavior of the above Yarrowia lipolytica strain showed successful simulation, while the optimized parameter values were similar to those experimentally measured and the predictive ability of the models was satisfactory. In nitrogen-limited cultures in which glucose was used as the sole substrate, satisfactory growth and no glucose inhibition occurred, although in some cases the initial concentration of glucose was significantly high (150 g/l). Citric acid production was observed in all trials, which was in some cases notable (final concentration 42.9 g/l, yield 0.56 g per g of sugar consumed). The concentration of unsaturated cellular fatty acids was slightly lower when the quantity of sugar in the medium was elevated.In the cases in which stearin and glucose were used as co-substrates, in spite of the fact that the quantity of cellular lipid inside the yeast cells varied remarkably (from 0.3 to 2.0 g/l – 4 to 20% wt/wt), de novo fatty acid biosynthesis was observed. This activity increased when the yeast cells assimilated higher sugar quantities. The citric acid produced was mainly derived from the catabolism of sugar. Nevertheless, citric acid yield on sugar consumed and citrate specific production rate, as evaluated by the numerical model, presented substantially higher values in the fermentation in which no fat was used as glucose co-substrate compared with the cultures with stearin used as co-substrate.

Multicriteria optimization of a single-cell oil production

Abstract The search of an experimental design based on D-optimality criterion to improve a bioprocess is proposed. The chosen model is a second-degree polynomial one selected for its satisfying precision leading to an accurate representation of non-linear systems with a low number of parameters. The optimization is carried out thanks to a real encoding diploid genetic algorithm developed within our laboratory. This study shows the setting of a multiobjective optimization procedure for bioprocesses having, in all cases, several criteria to optimize. A new approach by Pareto’s domain determination and ranking by Decision Engineering tool is presented.

CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form

The cetyltrimethylammonium bromide turbidimetric method (CTM) has been developed to quantify the hyaluronic acid (HA) in complex media to overcome the lack of selectivity and specificity of the standard carbazole method. The objective of this work is to assess the potential application of CTM to determine HA concentration. Factors such as duration of incubation, linearity range, HA size and form (natural linear HA or cross linked HA), pH and ionic environment impact were investigated. The incubation time was set to 10 min and the calibration curve was linear up to 0.6 g L(-1). The quantitative method was relevant whatever the HA size and form, and also for a wide range of conditions. The robustness of the CTM added to its high specificity and simplicity demonstrated that the CTM is a valuable method that would be an interesting substitute to the carbazole assay for HA quantification.

Selective extraction, structural characterisation and antifungal activity assessment of napins from an industrial rapeseed meal

This article reports an extraction-purification of napins from an industrial rapeseed meal and the assessment of their antimicrobial activity against Fusarium langsethiae. The best extraction conditions are observed at pH 2, 12% (w/w) of rapeseed meal after 15 min of extraction in water at room temperature. Under these conditions the extraction is highly selective, allowing a simple purification process (ammonium sulfate precipitation followed by desalting size exclusion chromatography) to get purified napins. These napins possessed significant anti-Fusarium activity (IC(50)=70 μM) and a compact secondary structure rich in α-helix, which may explain this bioactivity.

Kinetic study of the chemical reactivity of α-acetolactate as a function of pH in water, and in fresh and fermented culture media used for Lactococcus lactis spp. Lactis bv. diacetylactis cultivation

SummarySpontaneous oxidative decarboxylation of α-acetolactic acid (ALA) to diacetyl has been assessed under anaerobiosis as a function of pH in water, and in fresh and filtered Lactococcus lactis spp. lactis bv. diacetylactis SD 933 fermented culture media. Whatever the reaction medium, ALA was shown to be potentially reactive, depending on the pH of medium. Diacetyl production mechanism by this strain is discussed on the basis of these kinetic data.

Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement

Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates.

Quantification of extracellular α-acetolactate oxidative decarboxylation in diacetyl production by an α-acetolactate overproducing strain of Lactococcus lactis sp. lactis bv. diacetylactis

The quantification of α-acetolactate (AAL) extracellular oxidative decarboxylation during an AAL overproducing strain culture shows that this reaction is at the origin of about 90% of the diacetyl production and that only a small proportion of extracellular AAL is readily transformed to diacetyl. These results, compared with previous ones obtained with a non AAL accumulating strain, allow research options to be put forward for the improvement of microbiological diacetyl production.

Representation of changes in the metabolic pattern of baker's yeast from measurements of extracellular pyruvate, acetate, acetaldehyde and ethanol

SummaryExtracellular levels of pyruvate and acetate (HPLC method), acetaldehyde and ethanol (PTFE gas sensor method) are determined. Accurate measurements of these substances allow to definite more precisely the changes during aerobic growth of bakers yeast on a glucose medium (23 g/l). Unexpected variations in the level of extracellular acetaldehyde are noticed. In the oxydative mode, a successsive uptake of pyruvate, acetate, acetaldehyde and ethanol is pointed out. A model based onintracellular architecture is proposed to represent these experimental results.