Anita Rywińska

Simultaneous production of citric acid and erythritol from crude glycerol by Yarrowia lipolytica Wratislavia K1

This study shows a possible microbial process for utilization of crude glycerol generated by the biodiesel industry for citric acid and erythritol production. Simultaneous production of citric acid and erythritol under nitrogen-limited conditions with glycerol as the carbon source was achieved with an acetate negative mutant of Y. lipolytica Wratislavia K1 in fed-batch cultivations. The effect of the initial glycerol concentration (from 30–180 g dm−3) on the citrate and erythritol production was investigated. As a result of the experiments, maximum citric acid production (110 g dm−3) and a very high amount of erythritol (81 g dm−3) were determined after 168 h of fed-batch cultivation with the initial glycerol concentration of 150 g dm−3 and the total glycerol concentration of 250 g dm−3. In addition, the citric acid to isocitric acid ratio of the products from this strain was 35.5:1.

EFFECT OF AGITATION AND AERATION ON THE CITRIC ACID PRODUCTION BY Yarrowia lipolytica GROWN ON GLYCEROL

The effects of agitation rates from 400 to 900 rpm and aeration rates ranging from 0.18 to 0.6 vvm on biomass and citric acid production on glycerol media by acetate-negative mutants of Yarrowia lipolytica, Wratislavia 1.31 and Wratislavia AWG7, in batch culture were studied. The agitation rates of 800 and 900 rpm (at a constant aeration rate of 0.36 vvm) and aeration rates within the range of 0.24–0.48 vvm (at a constant agitation rate of 800 rpm), which generated dissolved oxygen concentration (DO) higher than 40%, were found the best for citric acid biosynthesis from glycerol. An increase in agitation rate (higher than 800 rpm) and aeration rate (higher than 0.36 vvm) had no impact on DO and citric acid production. The highest citric acid concentration (92.8 g/L) and yield (0.63 g/g) were obtained with Wratislavia 1.31 strain at 0.24 vvm. The highest volumetric citric acid production rate (1.15 g/Lh) and specific citric acid production rate (0.071 g/gh) were reached at 0.48 vvm.

Optimization of Medium Composition for Erythritol Production from Glycerol by Yarrowia lipolytica Using Response Surface Methodology

Several factors affecting erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 strain were examined in batch fermentations. Ammonium sulfate, monopotassium phosphate, and sodium chloride were identified as critical medium components that determine the ratio of polyols produced. The central composite rotatable experimental design was used to optimize medium composition for erythritol production. The concentrations of ammonium sulfate, monopotassium phosphate, and sodium chloride in the optimized medium were 2.25, 0.22, and 26.4 g L−1, respectively. The C:N ratio was found as 81:1. In the optimized medium with 100 g L−1 of glycerol the Wratislavia K1 strain produced 46.9 g L−1 of erythritol, which corresponded to a 0.47 g g−1 yield and a productivity of 0.85 g L−1 hr−1. In the fed-batch mode and medium with the total concentration of glycerol at 300 g L−1 and C:N ratio at 81:1, 132 g L−1 of erythritol was produced with 0.44 g g−1 yield and a productivity of 1.01 g L−1 hr−1.

Enhanced production of erythritol and mannitol by Yarrowia lipolytica in media containing surfactants

Various chemical compounds, including surfactants, when introduced to culture media may increase the permeability of cellular membranes and thereby affect the quantity of metabolites excreted by cells. The aim of the present study was to evaluate the impact of detergents including Triton X-100, Span 20 and Tween 80 on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 in a shake-flask experiment, batch and fed-batch cultures. When Span 20 was added to a fed-batch culture with glycerol as a carbon source (300 g L−1), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142 g L−1 after 5 days, which corresponded to 0.47 g g−1 yield and productivity of 1.1 g L−1 h−1. Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol.

Efficient utilization of inulin and glycerol as fermentation substrates in erythritol and citric acid production using Yarrowia lipolytica expressing inulinase

Inulin and glycerol were used as substrates for efficient erythritol and citric acid production by newly engineered Yarrowia lipolytica strains. Hydrolysis of inulin by the Y. lipolytica Wratislavia K1 strain was established by expressing the Kluyveromyces marxianus INU1 gene. Erythritol was produced in two stages: inulin was used for biomass formation, followed by erythritol biosynthesis initiated by glycerol addition. The highest titer of erythritol obtained, 120.9 g L−1 with the yield of 0.6 g g−1, was produced by the K1 INU 6 strain. Moreover, the K1 INU 6 strain in fed-batch culture produced a high amount of citric acid: 105.2 g L−1 after 235 h from 200 g L−1 of inulin. Maximum activity of inulinase during this culture was 14000 U g−1 of cell dry mass. The presented study proves the potential of new Y. lipolytica transformants for efficient erythritol and citric acid production from inexpensive raw materials such as inulin and glycerol.

Continuous production of citric acid from raw glycerol by Yarrowia lipolytica in cell recycle cultivation

The kinetics of continuous citric acid (CA) fermentation, using two acetate-negative mutants of Yarrowia lipolytica (Wratislavia AWG7 and Wratislavia 1.31) with long-term cell recycle, were investigated at a dilution rate (D) of 0.011 h−1. The capacity of the mutants to produce CA from raw glycerol under steady state conditions was compared. The process involving the Wratislavia AWG7 strain showed that this strain was a better producer of CA than the Wratislavia 1.31 strain in this regime. In steady state, the concentration of CA in the effluent was constant and amounted to 116 g dm−3. This corresponded to a volumetric CA production rate of 1.3 g dm−3 h−1. A stable high-level production of CA was maintained for over 400 h of operation. The Wratislavia AWG7 strain employed remained highly stable throughout the experiment and demonstrated good viability, high genetic and phenotypic stability over the long-term continuous fermentation process.

An Effective Method of Continuous Production of Erythritol from Glycerol by Yarrowia lipolytica MK1

This study demonstrates the potential applicability of the UV mutant Yarrowia lipolytica MK1 for the valorisation of glycerol and erythritol production in a chemostat culture. The aim of this research is to investigate the optimal C:N ratio in the feeding medium in order to enhance erythritol production. The highest erythritol concentration, at 113.1 g/L with a volumetric erythritol production rate of 1.1 g/(L·h) and a yield of 0.57 g/g, was obtained in the feeding medium with a C:N ratio of 80:1. Moreover, no residual glycerol was observed in the culture broth during cultivation. The chemical composition of the biomass was analysed. The contents of lysine and threonine in the biomass protein amino acid profile were higher than those required by the FAO/WHO for fodder yeast.