Biosynthesis of citric and isocitric acids from ethanol by mutant Yarrowia lipolytica N 1 under continuous cultivation

The effect of ethanol, zinc, and iron (Fe²⁺ and Fe³⁺) concentration and of oxygen supply on cell growth and the production of citric acid (CA) and isocitric acid (ICA) from ethanol by mutant Yarrowia lipolytica N 1 was studied under continuous cultivation. The following peculiarities of Y. lipolytica metabolism were found: (1) intensive CA production occurred under yeast growth limitation by nitrogen; (2) inhibition of yeast growth by ethanol was accompanied by significant alterations in fatty acid composition of lipids; (3) the production of CA and ICA from ethanol required high concentrations of zinc and iron ions; (4) the intracellular iron concentration determined whether CA or ICA was predominantly formed; (5) the cell's requirement for oxygen depended on the intracellular iron concentration. The events taking place in the production of CA and ICA were evaluated through the activities of enzyme systems involved in the metabolism of ethanol and CA in this strain. Electronic Publication     

Characteristics of the growth on rapeseed oil and synthesis of citric and isocitric acids by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> yeasts

The native strain Yarrowia lipolytica VKMY-2373 grown in a complete medium exhibited the maximum lipase activity at the concentration of rapesseed oil of at least 5.0 g/l. In the course of yeast growth, no considerable changes were observed in the glycerol concentration, the proportions of the major free fatty acids formed via oil hydrolysis, or the fatty acid composition of oil. Under nitrogen limitation of cell growth, the accumulation of citric acids reached 77.1 g/l with predominance of isocitric acid at pH 6.0, whereas at pH 4.5, almost equal amounts of citric and isocitric acids were produced. Cultivation of the mutant strain Y. lipolytica N 1 at pH 4.5 resulted in the predominant accumulation of citric acid (66.6 g/l) with an insignificant amount of isocitric acid. In the period of intense acid synthesis, high production of lipase was observed.     

Synthesis and localization of L-lactate oxidase in yeasts Yarrowia lipolytica

Conditions for L-lactate oxidase synthesis by the yeast Yarrowia lipolytica were investigated. The enzyme was found to be synthesized during growth on L-lactate in the exponential growth phase. L-lactate oxidase synthesis was also observed on glucose after adaptation to stress conditions (oxidative or thermal stress) during the stationary growth phase after glucose consumption. The cells grown on L-lactate exhibited high levels of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase), which exceeded those of glucose-grown cells. Ultrastructurally, L-lactate-grown cells and the cells grown on glucose and adapted to various stress conditions were also found to be similar, with increased mitochondria, elevated number and size of peroxisomes, and formation of lipid and polyphosphate inclusions. In order to determine the intracellular localization of L-lactate oxidase, the cells were disintegrated by the lytic enzyme complex from Helix pomatia. Centrifugation of the homogenate in Percoll gradient resulted in the isolation of purified fractions of the native mitochondria and peroxisomes. L-lactate oxidase was shown to be localized in peroxisomes.     

Biosynthesis of citric acid by Yarrowia lipolytica repeat-batch culture on ethanol

After analysis of batch culture and identification of the ways for prolongation of citric acid active synthesis by yeast, repeat-batch (RB) cultivation was suggested. Yarrowia lipolytica strain RB cultivation was studied and optimal conditions for cultivation selected. It was shown that when applying RB cultivation, better results were obtained than for batch cultivation. The activity of the culture remained stable after cultivation for more than 700 h. Comparative analysis of enzyme activities confirmed the regularity of the effect described, as the activity of practically of all the enzymes participating in ethanol oxidation and citric acid biosynthesis remained stable over time during RB cultivation. Advantages of RB cultivation for the production of citric acid by yeast are discussed. Received: 1 March 1999 / Received revision: 28 June 1999 / Accepted: 5 July 1999     

Isocitric acid production from rapeseed oil by Yarrowia lipolytica yeast

Production of d _S-threo-isocitric acid (ICA) by yeast meets serious difficulties since it is accompanied by a simultaneous production of citric acid (CA) in significant amounts that reduces the yield of desired product. In order to develop an effective process of ICA production, 60 yeast strains of different genera (Candida, Pichia, Saccharomyces, Torulopsis, and Yarrowia) were tested for their ability to produce ICA from rapeseed oil; as a result, wild-type strain Yarrowia lipolytica VKM Y-2373 and its mutant Y. lipolytica 704-UV4-A/NG50 were selected as promising ICA producers. The effects of temperature, pH, aeration, and concentrations of rapeseed oil, iron, and itaconic acid on ICA production by selected strains were studied. Under optimal conditions (pH 6.0; aeration 50–55 %; rapeseed oil concentration of 20–60 gl^?1, iron ion concentration of 1.2 mg l^?1, and itaconic acid amount of 30 mM), selected strains of Y. lipolytica produced predominantly ICA with a low amount of a by-product, CA.     

Chemostat study of citric acid production from glycerol by Yarrowia lipolytica

The aim of the study was to examine how the dilution rate and the chemical composition of the production medium impacts on the synthesis of citric acid by the Yarrowia lipolytica strain Wratislavia AWG7 from glycerol in a chemostat culture. The yeast Y. lipolytica Wratislavia AWG7, an acetate (acet(-)) and morphological (fil(-)) mutant, was cultured in a nitrogen- and phosphorus-limited medium at the dilution rate of 0.009-0.031h(-1) in the chemostat. Under steady-state conditions, the increase in the dilution rate was paralleled by the decrease in citric acid concentration (from 86.5 to 51.2gL(-1)), as well as by the increase in the volumetric rate (from 0.78 to 1.59gL(-1)h(-1)) and specific rate (from 0.05 to 0.18gg(-1)h(-1)) of citric acid production. The yield of the production process varied from 0.59 to 0.67gg(-1). In a 550-h continuous culture of the yeast test, at a dilution rate of 0.01h(-1), in a medium with enhanced concentrations of carbon, nitrogen and phosphorus sources, the concentration of citric acid, the concentration of biomass and the volumetric rate of citric acid production were 97.8gL(-1), 22.2gL(-1) and 0.98gL(-1)h(-1), respectively. The yield of the process decreased to 0.49gg(-1). The number of dead cells did not exceed 1% while that of the budding cells accounted for about 20%. Owing to the low content of isocitric acid and polyols, the fermentation process was characterized by a high purity. This study has produced the following finding: the double mutant Y. lipolytica AWG7 is an effective citric acid producer, with the ability to preserve its properties unchanged during the long run of the continuous chemostat process. This is a valued technological feature of such mutants.     

High-yield production of citric acid by Yarrowia lipolytica on glycerol in repeated-batch bioreactors

An acetate negative mutant of Yarrowia lipolytica Wratislavia AWG7 was found to be suitable for the production of high amounts of citric acid in long-term repeated-batch cultures. When 40% of fresh replaced medium was fed, this strain produced 154 g l⁻¹, on average, which corresponded to a 0.78 g g⁻¹ yield and a productivity of 1.05 g l⁻¹ h⁻¹. The activity of the culture remained stable for more than 1,650 h, i.e., 16 cycles of the repeated-batch bioreactors.     

Acetate enhances citric acid production by Yarrowia lipolytica when grown on sunflower oil

It was discovered that the addition of 10 g/l acetate to a medium containing 30 g/l sunflower oil caused a drastic increase in citric acid production by Yarrowia lipolytica UOFS Y-1701 i.e. from 0.5 g/l in the absence of acetate to 18.7 g/l in the presence of acetate. Similarly, the ratio of citric acid:isocitric acid increased significantly from 1.7:1 in the absence of acetate to 3.7:1 in the presence of acetate after 240 h of growth.     

Simultaneous production of citric acid and invertase by Yarrowia lipolytica SUC+ transformants

Simultaneous production of citric acid (CA) and invertase by Yarrowia lipolytica A-101-B56-5 (SUC⁺ clone) growing from sucrose, mixture of glucose and fructose, glucose or glycerol was investigated. Among the tested substrates the highest concentration of CA was reached from glycerol (57.15 g/L) with high yield (Y_CA/S = 0.6 g/g). When sucrose was used, comparable amount of CA was secreted (45 g/L) with slightly higher yield (Y_CA/S = 0.643 g/g). In all cultures amount of isocitrate (ICA) was below 2% of total citrates. Considering invertase production, the best carbon source appeared to be sucrose (72 380 U/L). The highest yield of CA and invertase biosynthesis calculated for 1 g of biomass was obtained for cells growing from glycerol (9.9 g/g and 4325 U/g, respectively). Concentrates of extra- and intracellular invertase of the highest activity were obtained from sucrose as substrate (0.5 and 1.8 × 10⁶ U/L, respectively).     

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.     

The citric acid production from raw glycerol by Yarrowia lipolytica yeast and its regulation

The optimal cultivation conditions ensuring the maximal rate of citric acid (CA) biosynthesis by glycerol-grown mutant Yarrowia lipolytica NG40/UV7 were found to be as follows: growth limitation by inorganic nutrients (nitrogen, phosphorus, or sulfur), 28 °C, pH 5.0, dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and pulsed addition of glycerol from 20 to 80 g L^?1 depending on the rate of medium titration. Under optimal conditions of fed-batch cultivation, in the medium with pure glycerol, strain Y. lipolytica NG40/UV7 produced 115 g L^?1 of CA with the mass yield coefficient of 0.64 g g^?1 and isocitric acid (ICA) amounted to 4.6 g L^?1; in the medium with raw glycerol, CA production was 112 g L^?1 with the mass yield coefficient of 0.90 g g^?1 and ICA amounted to 5.3 g L^?1. Based on the activities of enzymes involved in the initial stages of raw glycerol assimilation, the tricarboxylic acid cycle and the glyoxylate cycle, the mechanism of increased CA yield from glycerol-containing substrates in Y. lipolytica yeast was explained.     

The peculiarities of succinic acid production from rapeseed oil by Yarrowia lipolytica yeast

The process of succinic acid (SA) production represents the combination of microbial synthesis of α-ketoglutaric acid from rapeseed oil by yeast Yarrowia lipolytica VKM Y-2412 and subsequent decarboxylation of α-ketoglutaric acid by hydrogen peroxide to SA that leads to the production of 69.0 g l^?1 of SA and 1.36 g l^?1 of acetic acid. SA was isolated from the culture broth filtrate in a crystalline form. The SA recovery from the culture filtrate has certain difficulties due to the presence of residual triglycerides of rapeseed oil. The effect of different methods of the culture filtrate treatment and various sorption materials on the coagulation of triglycerides was studied, and as a result, the precipitation of residual triglycerides by acetone was chosen. The subsequent isolation procedures involved the decomposition of H₂O₂ in the filtrate followed by filtrate bleaching and acidification with a mineral acid, evaporation of filtrate, and SA extraction with ethanol from the residue. The purity of crystalline SA isolated from the culture broth filtrate achieved 97.6–100 %. The product yield varied from 62.6 to 71.6 % depending on the acidity of the supernatant.     

Copper extrusion after accumulation during growth of copper-tolerant yeast Yarrowia lipolytica

The Cu2+-tolerant yeast Yarrowia lipolytica accumulated Cu2+ until the late logarithmic phase. Thereafter, Cu2+ was temperature-dependently extruded into phosphate-limited culture medium containing high concentrations of heavy metal ions but not into 10 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer (pH 6.0). Peptone in the culture medium played an important role in the extrusion, which proceeded even when peptone was substituted with cysteine or histidine, but not with any other amino acid tested.     

Repeated fed-batch fermentation using biosensor online control for citric acid production by Yarrowia lipolytica

Biosensor-controlled substrate feeding was used in a citric acid production process with the yeast strain Yarrowia lipolytica H222 with glucose as the carbon source. The application of an online glucose biosensor measurement facilitated the performance of long-time repeated fed-batch process with automated bioprocess control. Ten cycles of repeated fed-batch fermentation were carried out in order to validate both the stability of the microorganism for citric acid production and the robustness of the glucose biosensor in a long-time experiment. In the course of this fermentation with a duration of 553 h, a slight loss of productivity from 1.4 g/(L×h) to 1.1 g/(L×h) and of selectivity for citric acid from 91% to 88% was observed. The glucose biosensor provided 6,227 measurements without any loss of activity.     

Studies on citric acid production with immobilized Yarrowia lipolytica in repeated batch and continuous air-lift bioreactors

Citric acid was produced from glucose in repeated-batch shake-flask and continuous air-lift cultivations by calcium-alginate-immobilized Yarrowia lipolytica A-101 yeast. The medium composition was systematically studied in a batch system by using experimental design and empiric modelling. The highest citric acid product concentration of 39 g/l was reached with a medium containing 150 g/l of glucose, 0.105 g/l of potassium dihydrogen phosphate, 0.84 g/l of magnesium sulphate and 21 mg/l of copper sulphate (5.2 mg/l of copper). The results were further improved by hardening the alginate carrier beads with glutaraldehyde, and by activation of the immobilized biocatalyst in a nutrient solution. In continuous air-lift bioreactors with varying height-to-diameter ratio the highest productivity of 350 mg/l per hour with a dilution rate of 0.023 l/h and a citric acid product concentration of 12 g/l was reached with a ratio of 3. Correspondence to: H. Kautola     

Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 × His tag was found to be 22.6 U mg^?1 of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 °C, respectively and the inulinase was stable in the pH range of 3–8 and in the temperature range of 0–50 °C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g^?1 of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10 h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L^?1 citric acid and 5.3 g L^?1 iso-citric acid from inulin while 68.9 g L^?1 of citric acid and 4.1 g L^?1 iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.     

High specific rates of glucose utilisation under conditions of restricted growth are required for citric acid accumulation by Yarrowia lipolytica IMK 2

Yarrowia lipolytica strain IMK 2, a yeast capable of producing and excreting citric acid, only accumulated citric acid when cell growth was restricted by specific nutrient limitations. Effective citrate accumulation was linked with the ability of cells to maintain a high rate of glucose utilisation when growth was limited. Cells limited by nitrogen (N), sulphur (S), magnesium (Mg) or phosphorus (P) accumulated between 50 and 220 mm citric acid after 168 h, with N and S limitation resulting in the highest specific rates of production. In contrast, potassium (K)-limited cells accumulated 6 mm citric acid in the same time period. Cells limited by K or lower levels of Mg or P had rates of glucose utilisation that were less than 50% of those measured in cells limited by N or S. Although limitation of strain IMK 2 by Mg or P led to citrate production, significant accumulation occured only when the threshold concentration of the limiting nutrient was exceeded. There was no large accumulation of other tricarboxylic acid (TCA) cycle acids, acetate, pyruvate, lactate or ethanol, although K-limited cells produced quantities of intracellular glycogen. Mannitol was accumulated under N, S and P limitation (up to 40 mm), as were small quantities of 2-oxoglutarate, which preceded the accumulation of citrate and all of the other TCA cycle acids measured. A clear difference was evident between the rates of glucose utilisation under N limitation between two citrate-accumulating strains (IMK 2 and Candida guilliermondii IMK 1) and a citrate non-accumulating strain, Y. lipolytica YB 423. Although these strains had similar rates of glucose utilisation during exponential growth, both strains IMK 1 and IMK 2 had specific rates of glucose utilisation under N limitation more than double that of strain YB 423.Correspondence to: J. D. Brooks 2     

Uptake of fatty acids by the yeasts, Saccharomyces uvarum and Saccharomycopsis lipolytica

Yeast cells take up exogenous fatty acids with subsequent rapid incorporation into glycerolipids. beta-Oxidation does not occur in Saccharomyces uvarum and is observed in Saccharomycopsis lipolytica only 2-5 min after addition of radioactively labeled fatty acid. Rates of fatty acid uptake are linear up to 30 s with S. lipolytica and up to 2 min with S. uvarum. The uptake kinetics are consistent with a dual mode of transport, comprising a saturable component with KT values in the range 10(-5)-10(-6) M, and apparently simple diffusion that predominates at high substrate concentrations. Kinetics of fatty acid permeation are independent of metabolic energy and membrane potential. At least two fatty acid carrier systems exist in both S. lipolytica and S. uvarum, one being specific for fatty acids with 12 and 14 C atoms, respectively, the other for C16 and C18 saturated or unsaturated fatty acids. Octanoic acid and decanoic acid are not taken up by S. lipolytica. Internalization of lauric acid and oleic acid by S. lipolytica cells is preceded by a rapid (less than 5 s) initial uptake which most likely represents irreversible adsorption. This phenomenon was not observed with heat-inactivated S. lipolytica cells or with viable S. uvarum. In azide-poisoned cells of S. lipolytica an up to 20-fold accumulation of unesterified fatty acid was observed within 30 s after the addition of substrate.     

Substrate utilization by recombinant Yarrowia lipolytica growing on sucrose

We report the study of the dynamics of substrate utilization by the genetic modified strain Yarrowia lipolytica H222-S4(p67ICL1) T5. In contrast to its wild-type equivalent, this recombinant strain is able to excrete the sucrose cleaving enzyme invertase. Both the sucrose degradation rate and the glucose and fructose consumption rate have been investigated. In all experiments, satisfied amounts of invertase were produced so that all sucrose was cleaved into its monomers. While glucose and fructose as sole carbon sources were consumed with the same uptake rate, a clear preference for glucose uptake was detected in cultivations with sucrose as sole carbon source or mixed substrates when compared with fructose. Nevertheless, no real diauxie could be observed because of partly simultaneous consumption of both monosaccharides. Fructose being present in the cultivation medium at the beginning of the fermentation led to the retardation of glucose uptake. This effect was observed for various fructose starting concentrations in the range of 5–85 g/l.