Fed-batch production of citric acid by Candida lipolytica grown on n-paraffins

This study reports on the effects of fermentor agitation and fed-batch mode of operation on citric acid production from Candida lipolytica using n-paraffin as the carbon source. An optimum range of agitation speeds in the 800-1000 rpm range corresponding to Reynolds numbers of 50000-63000 (based on initial batch conditions) seemed to give the best balance between substrate utilization for biomass growth and citric acid production. Application of multiple fed-batch feedings can be used to extend the batch fermentation and increase final citric acid concentrations and product yield. The three-cycle fed-batch system increased overall citric acid yields to 0.8-1.0 g citricacid/g n-paraffin, approximately a 100% improvement in product yield from those observed in the single cycle fed-batch system and a 200% improvement over normal batch operation. The three-cycle fed-batch mode of operation also increased the final citric acid concentration to 42 g/l from about 12 and 6g/l for single fed-batch cycle and normal batch modes of operation, respectively. Increased citric acid concentrations in three-cycle fed-batch mode was achieved at longer fermentation times.     

Production of citric acid with immobilized Yarrowia lipolytica

Summary Citric acid was produced with immobilized Yarrowia lipolytica yeast in repeated batch-shake-flask and air-lift fermentations. In active and passive immobilization methods calcium alginate, -carrageenan, polyurethane gel, nylon web and polyurethane foams were tested as carriers in repeated-batch fermentations. The highest citric acid productivity of 155 mg l^–1 h^–1 was reached with alginate-bead-immobilized cells in the first batch. A decrease in bead diameter from 5–6 mm to 2–3 mm increased the volumetric citric acid productivity threefold. In an air-lift bioreactor the highest citric acid productivity of 120 mg l^–1 h^–1 with a product concentration of 16.4 g l^–1 was obtained with cells immobilized in -carrageenan beads. Offprint requests to: H. Kautola     

Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.     

Production of citric acid from sugars present in wood hemicellulose usingAspergillus niger andSaccharomycopsis lipolytica

Summary One strain each of the fungus,Aspergillus niger, and the yeast,Saccharomycopsis lipolytica, were investigated for their ability to produce citric acid from the sugars present in hemicellulose hydrolysates.S. lipolytica produced citric acid as efficiently from mannose as from glucose, but failed to assimilate xylose, arabinose or galactose.A. niger readily assimilated mannose, xylose and arabinose, and produced citric acid from these sugars although the yields were lower than from glucose. A possible inhibitory effect of arabinose on citric acid production from other sugars was observed usingA. niger.     

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.     

Obtaining of the mutant Yarrowia lipolytica strains producing citric acid from glucose

The possibility of obtaining mutant yeasts Yarrowia lipolytica VKM Y-2373 with increased ability to synthesize citric acid from glucose by using UV irradiation and N-methyl-NT-nitro-N-nitrosoguanidine was studied. Of 1500 colonies of the Y. lipolytica treated with either UV or N-methyl-NT-nitro-N-nitrosoguanidine, three mutants were selected that displayed higher (by 23%) biosynthetic ability as compared with the initial strain. Additionally, three mutants were selected from 1000 colonies of the Y. lipolytica exposed to a combined action of UV and N-methyl-NT-nitro-N-nitrosoguanidine; their biosynthetic activity exceeded that of the initial strain by 43.9%. The selective media with citrate and acetate were developed for a rapid selection of mutants as well as the express methods for the detection of active citric acid producers on the solid media with chalk and bromocresol containing a limiting concentration of amine nitrogen and an excess of glucose.     

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.     

Yarrowia lipolytica as a potential producer of citric acid from raw glycerol

AIMS: To study the biochemical response of Yarrowia lipolytica LGAM S(7)1 during growth on raw glycerol (the main by-product of bio-diesel production units) in order to produce metabolic products of industrial significance. METHODS AND RESULTS:Yarrowia lipolytica was cultivated on raw glycerol or glucose in flasks. Although nitrogen-limited media were employed, growth was not followed by production of reserve lipid. Nitrogen limitation led to citric acid excretion. Growth and citric acid production parameters on glycerol were similar to those obtained on glucose. When high initial glycerol media were used, citric acid up to 35 g l(-1) (yield 0.42-0.44 g acid g(-1) glycerol consumed) was produced. CONCLUSIONS: Raw glycerol was an adequate substrate for Y. lipolytica. Growth was not followed by reserve lipid accumulation, but amounts of citric acid were produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Raw glycerol is an industrial feedstock appearing in increasing quantities as the main by-product of bio-diesel production facilities. The present study describes an alternative way of glycerol valorization, with the production of remarkable amounts of citric acid, in addition to its main valorization way (production of 1,3-propanediol by bacteria).     

Optimization of citric acid production from Candida lipolytica Y-1095 using n-paraffin

Currently, the majority of worldwide microbial production of citric acid utilizes Aspergillus niger in a carbohydrate based submerged fermentation. Due to their high carbon content, hydrocarbons also have the potential of producing high concentrations of citric acid. Initial lab experiments conducted using 1875 ml batch fermentations with n-paraffin found that Candida lipolytica NRRL-Y-1095 assimilated the feedstock and had a citric acid productivity of 47 mg l(-1) h(-1). To determine the optimum level of initial biomass concentration, n-paraffin concentration, iron concentration and temperature for the production of citric acid, a central composite design was developed using 200 ml batch fermentations. The design involved conducting 31 batch fermentations under various combinations of high and low values of these four parameters. From this investigation empirical models were developed describing the interactions between the experimental parameters and citric acid production. It was found that the maximum concentration of citric acid produced was 9.8 g l(-1) and the optimum levels of each parameter for citric acid production were, 10--12% volume for initial biomass concentration, 10--15% volume for n-paraffin concentration, 10 mg l(-1) for ferric nitrate concentration, and 26--30 degrees C for temperature.     

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.     

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.     

Oxygen requirements for growth and citric acid production of Yarrowia lipolytica

During continuous cultivation of Yarrowia lipolytica N 1, oxygen requirements for growth and citric acid synthesis were found to depend on the iron concentration in the medium. A coupled effect of oxygen and iron concentrations on the functioning of the mitochondrial electron transport chain in Y. lipolytica N 1 was established. Based on the results obtained in continuous culture, conditions for citric acid production in a batch culture of Y. lipolytica N 1 were proposed. At relatively low pO(2) value and a high iron concentration, citric acid accumulation was as high as 120 g l(-1); the specific rate of citric acid synthesis reached 120 mg citric acid (g cells h)(-1). The mass yield coefficient was 0.87 and the energy yield coefficient was 0.31.     

Oxygen uptake and citric acid production by Candida lipolytica Y 1095

The rates of oxygen uptake and oxygen transfer during cell growth and citric acid production by Candida lipolytica Y 1095 were determined. The maximum cell growth rate, 1.43 g cell/L . h, and volumetric oxygen uptake rate, 343 mg O(2)/L . h, occurred approximately 21 to 22 h after inoculation. At the time of maximum oxygen uptake, the biomass concentration was 1.3% w/v and the specific oxygen uptake rate was slightly greater than 26 mg O(2)/g cell . h. The specific oxygen uptake rate decreased to approximately 3 mg O(2)/g cell . h by the end of the growth phase.During citric acid production, as the concentration of dissolved oxygen was increased from 20% to 80% saturation, the specific oxygen uptake and specific citric acid productivity (mg citric acid/g cell . h) increased by 160% and 71%, respectively, at a biomass concentration of 3% w/v. At a biomass concentration of 5% w/v, the specific oxygen uptake and specific citric acid productivity increased by 230% and 82%, respectively, over the same range of dissolved oxygen concentrations.The effect of dissolved oxygen on citric acid yields and productivities was also determined. Citric acid yields appeared to be independent of dissolved oxygen concentration during the initial production phase; however, volumetric productivity (g citric acid/L . h) increased sharply with an increase in dissolved oxygen. During the second or subsequent production phase, citric acid yields increased by approximately 50%, but productivities decreased by roughly the same percentage due to a loss of cell viability under prolonged nitrogen-deficient conditions. (c) 1994 John Wiley & Sons, Inc.     

Production of citric acid from starch-hydrolysate by Aspergillus niger

The present investigation explored the possible use of a rarely used agro-industrial by-product, maize starch-hydrolysate, for economic production of citric acid. To achieve this, seventeen strains of Aspergillus niger were screened for their capacity to produce citric acid using starch-hydrolysate as a substrate. The most efficient strain, ITCC-605 was selected for further improvement in citric acid content by mutation. Mutants developed by treatment with EMS and UV, singly and in combination, produced citric acid in the range of 0.51-64.7 g kg(-1) of glucose consumed. The mutant UE-1 produced the maximum citric acid which was about 130 times more than that produced by the parent strain, ITCC-605. For further increase in citric acid production from this substrate, the cultural conditions were optimized: concentration of starch-hydrolysate, 15% (glucose equivalent); ammonium nitrate, 0.25%; KH2PO4, 0.15%; nicotinic acid, 0.0001% and initial pH of 2.0. Under these conditions, the mutant strain UE-1 yielded 490 g citric acid kg(-1) of glucose consumed in 8 days of incubation at 30 degrees C. The productivity of 341 mgl(-1)h(-1) corresponded to 49% substrate conversion to citric acid.     

Aconitase and citric acid fermentation by Aspergillus niger

In view of the often-cited theory that citric acid accumulation is caused by an inhibition of aconitase activity, the equilibrium of the reaction of aconitase was investigated by comparing in vivo steady-state concentrations of citrate and isocitrate in Aspergillus niger grown under various citric acid-producing conditions. With the equilibrium catalyzed by the A. niger enzyme in vitro, similar values were obtained. The validity of our in vivo measurements was verified by the addition of the aconitase inhibitor fluorocitrate, which appreciably elevated the citrate:isocitrate ratio. The results strongly argue against an inhibition of aconitase during citric acid fermentation.     

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     

190 The peculiarities of regulation of Yarrowia lipolytica yeast and citric acid overproduction

The growth of Yarrowia lipolytica yeast as well the biosynthesis of citric acid on rapeseed oil were studied. It was indicated that the initial step of assimilation of rapeseed oil in the yeast Y. lipolytica is their hydrolysis by extracellular lipases with the formation of glycerol and fatty acids, which appear in the medium in the phase of active growth. The concentrations of these metabolites change insignificantly upon further cultivation. Lipase and the key enzymes of glycerol metabolism (glycerol kinase) and the glyoxylate cycle responsible for the metabolism of fatty acids (isocitrate lyase and malate synthase) are induced just at the beginning of the growth phase and remain active in the course of further cultivation. These results, taken together, suggest that glycerol and fatty acids according in the medium do not suppress the metabolism of each other. The fact that glycerol and fatty acids can be consumed simultaneously is of special importance for the development of the efficient regime of oil feeding, Y. lipolytica produced citric acid (175?g/L) with a yield of 150%. It should be noted that the simultaneous utilization of two different substrates is not typical of micro-organisms, which first assimilate one of the two available substrates (commonly, a carbohydrate), whereas the assimilation of the other substrate starts only after the first substrate is fully consumed from the medium. Indeed, upon the cultivation of Y. lipolytica on the mixture of glucose and oleic acid, the latter substrate began to be utilized only when the concentration of glucose decreased. The glycolytic enzyme pyruvate dehydrogenase was induced from the first hours of cultivation and remained at high levels until the exhaustion of glucose in the medium. At the same time, the activities of isocitrate lyase and malate synthase were very low during the metabolism of glucose, but were rapidly induced (approximately in 10 times) after the exhaustion of glucose in the medium. When Y. lipolytica was grown on the mixture of glucose and hexadecane, the dynamics of growth and substrate consumption was typical of the diauxie phenomenon: the utilization of hexadecane began only in several hours after the time when glucose was completely exhausted in the cultivation medium. In this case, the exhaustion of glucose arrested growth and the culture resumed growth only after a lag period. The assay of enzymes showed that the glycolytic enzyme pyruvate dehydrogenase was active during the phase of growth on glucose, whereas the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase were active during the phase of growth on hexadecane. In recent years in the literature, there are data that the different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins (Cho et al. 2009), but there are different circuits of repression for different groups of genes (Gancedo 1990). We will discuss the possible metabolic regulation in the case of Y. lipolytica.     

Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation

The genetically modified yeast strain Yarrowia lipolytica H222‐S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild‐type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose‐peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose‐peptone. Furthermore, two process strategies (fed‐batch and repeated fed‐batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed‐batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.