Fermentation process for producing CFAs using Yarrowia lipolytica.

Past research has sought to improve the production of cyclopropane fatty acids by the oleaginous yeast Yarrowia lipolytica by heterologously expressing the E. coli fatty acid synthase gene and improving cultivation processes. Cyclopropane fatty acids display properties that hold promise for biofuel applications. The E. coli fatty acid synthase gene was introduced into several genetic backgrounds of the yeast Y. lipolytica to optimize lipid synthesis; the mean cyclopropane fatty acid productivity was 43 mg L-1 h-1 on glucose, and the production rate reached its maximum (3.06 g L-1) after 72 h of cultivation in a bioreactor. The best strain (JMY6851) overexpressed simultaneously the E. coli cyclopropane fatty acid synthase gene under a hybrid promoter (hp8d) and Y. lipolytica LRO1 gene. In fed-batch process using crude glycerol as carbon source, JMY6851 strain displayed high lipid accumulation (78% of dry cell weight) and high biomass production (56 g L-1). After 165 h of cultivation, cyclopropane fatty acids represented 22% of the lipids produced; cyclopropane fatty acid productivity (103.3 mg L-1 h-1) was maximal at 72.5 h of cultivation.

Optimization of cyclopropane fatty acids production in Yarrowia lipolytica.

Cyclopropane fatty acids, which can be simply converted to methylated fatty acids, are good unusual fatty acid candidates for long-term resistance to oxidization and low-temperature fluidity useful for oleochemistry and biofuels. Cyclopropane fatty acids are present in low amounts in plants or bacteria. In order to develop a process for large-scale biolipid production, we expressed 10 cyclopropane fatty acid synthases from various organisms in the oleaginous yeast Yarrowia lipolytica, a model yeast for lipid metabolism and naturally capable of producing large amounts of lipids. The Escherichia coli cyclopropane fatty acid synthase expression in Y. lipolytica allows the production of two classes of cyclopropane fatty acids, a C17:0 cyclopropanated form and a C19:0 cyclopropanated form, whereas others produce only the C17:0 form. Expression optimization and fed-batch fermentation set-up enable us to reach a specific productivity of 0.032 g·L-1 ·hr-1 with a genetically modified strain containing cyclopropane fatty acid up to 45% of the total lipid content corresponding to a titre of 2.3 ± 0.2 g/L and a yield of 56.2 ± 4.4 mg/g.

A metabolic engineering strategy for producing conjugated linoleic acids using the oleaginous yeast Yarrowia lipolytica.

Conjugated linoleic acids (CLAs) have been found to have beneficial effects on human health when used as dietary supplements. However, their availability is limited because pure, chemistry-based production is expensive, and biology-based fermentation methods can only create small quantities. In an effort to enhance microbial production of CLAs, four genetically modified strains of the oleaginous yeast Yarrowia lipolytica were generated. These mutants presented various genetic modifications, including the elimination of ß-oxidation (pox1-6∆), the inability to store lipids as triglycerides (dga1∆ dga2∆ are1∆ lro1∆), and the overexpression of the Y. lipolytica ∆12-desaturase gene (YlFAD2) under the control of the constitutive pTEF promoter. All strains received two copies of the pTEF-oPAI or pPOX-oPAI expression cassettes; PAI encodes linoleic acid isomerase in Propionibacterium acnes. The strains were cultured in neosynthesis or bioconversion medium in flasks or a bioreactor. The strain combining the three modifications mentioned above showed the best results: when it was grown in neosynthesis medium in a flask, CLAs represented 6.5% of total fatty acids and in bioconversion medium in a bioreactor, and CLA content reached 302 mg/L. In a previous study, a CLA degradation rate of 117 mg/L/h was observed in bioconversion medium. Here, by eliminating ß-oxidation, we achieved a much lower rate of 1.8 mg/L/h.

S. cerevisiae fermentation activity after moderate pulsed electric field pre-treatments.

The batch fermentation process, inoculated by Pulsed Electric Field (PEF) treated wine yeasts (Saccharomyces cerevisiae Actiflore F33), was studied. PEF treatment was applied to the aqueous yeast suspensions ([Y] = 0.012 g/L) at the electric field strengths of E = 100 and 6000 V/cm using the same treatment protocol (number of pulses n = 1000, pulse duration ti = 100 µs, and pulse repetition time Δt = 100 ms). Electrical conductivity was increasing during and after the PEF treatment, which reflected cell electroporation. Then, fermentation was run for 150 h in an incubator (30 °C) with synchronic agitation. Electro-stimulation was revealing itself by the improvement of fermentation characteristics, and thus increased yeast metabolism. At the end of the lag phase (t = 40 h), fructose consumption in samples with electrically activated inoculum exceeded that of the control samples by ≈ 2.33 times for E = 100 V/cm and by ≈ 3.98 for E = 6000 V/cm. At the end of the log phase (120 h of fermentation), ≈ 30% mass reduction was reached in samples with PEF-treated inocula (E = 6000 V/cm), whereas the same mass reduction of the control sample required approximately 20 extra hours of fermentation.

A simple and rapid one-time method to evaluate the non-acidic gas content from bioprocesses.

This paper presents a rapid less than 2 min and low-cost method involving the use of alkali solution to capture the acidic gasses from a biogas, thereby providing an estimate of the percentage of non-acidic gasses. Such a method was mentioned in the literature but never fully described or optimized. After sampling an aliquot of gas from bioprocess, gas was injected in a sealed flask with a 3 M NaOH solution, and after equilibrium was obtained, the non-acidic gas volume was measured. The method was first calibrated with certified gasses with an accuracy observed between 98 and 105%. Regarding the validation step, certified standard gas mixtures and nine biogas-laboratory batch reactors were used, the overall accuracy reported was 103 + 3%. This rapid and low-cost method may either be used in laboratory conditions as a quick and low cost alternative to standard analysis equipment or in addition as a routine field control method used on full-scale plants.