Continuous citric acid secretion by a high specific pH dependent active transport system in yeast Candida oleophila ATCC 20177

Abstract

The pH influence on continuous citric acid secretion was investigated in Candida oleophila ATCC 20177 (var.) under NH 4 + limiting state steady conditions, using glucose. Highest citric acid concentration of 57.8 g/l, citrate/isocitrate ratio of 15.6, space-time yield of 0.96 g/(l x hr) and biomass specific productivity of 0.041 g/(g x hr) were obtained at pH 5 and 60 hrs residence time. Only 22.8 g/l (39.4%) and a ratio of 9.9 were achieved at pH 6 pH and 12.4 g/l (21.5%) and a ratio of 3.7 at pH 3. Under non producing conditions, in excess of nitrogen, biomass concentration increased at raising pH. An iron concentration of 200 ppm was determined in biomass of C. oleophila at pH 5, compared with only 26 ppm found at pH 3 (factor 7.7). Intra-and extracellular concentrations of citrates and glucose confirmed the existence of a high specific, pH dependent active transport system for citrate secretion, while isocitrate isn't a high-affine substrate, displaying a strong correlation with ATP/ADP ratio. Differences between extra-and intracellular concentration of citrate higher than 1 and up to about 60 were determined. The active transport systemfor citrate excretion appears to be the main speed-determining factor in citrate overproduction by yeasts.Although citric acid production using mutant strains of A. niger or yeast strains has almost been extensively optimised, there is still no comprehensive explanation for citrate overproduction, and many aspects related to citrate accumulation and secretion remain unclear. There are many *Corresponding author similarities between A. niger and yeast strains in mechanism of citric acid synthesis, however, differences still exist in terms of triggering out and regulation of citrate overproduction. Many models have been developed describing the biochemistry of citrate synthesis, using glucose and other carbon sources, however a complete picture of formation pathway, regulation and secretion has not been described.Overproduction of citric acid in moulds and yeast has been reported to be triggered out by limitations of certain elements, like N, P, Mn, Fe or Zn, essential for citrate accumulation in A. niger (Shu and Johnson, 1948a;Shu and Johnson, 1948b;Noguchi and Johnson, 1961; Kisser et al. 1980; Kapoor et al. 1982;Kristiansen et al. 1982;Crueger and Crueger, 1989;Dawson and Maddox, 1989;Grewal and Kalra, 1995), as well as N, P, S and Mg in yeasts Yarrowia lipolytica and Candida oleophila (Lozinov et al. 1974;Behrens et al. 1987; Stottmeister and Hoppe, 1991;Anastassiadis, 1994;Anastassiadis et al. 2001;Anastassiadis et al. 2002; Anastassiadis et al. 2004). The yeasts can use various carbon sources for the formation of citric acid (Ikeno et al. 1975; Stottmeister and Hoppe, 1991;Grewal and Kalra, 1995; Mansfeld et al. 1995;Crolla and Kennedy, 2001;Crolla and Kennedy, 2004;Venter et al. 2004) or lipid production (Papanikolaou and Aggelis, 2002). Intracellular nitrogen limitation and low intracellular nitrogen content (Briffaud and Engasser, 1979;Moresi, 1994;Anastassiadis et...

Citric acid, biomass and cellular lipid production by Yarrowia lipolytica strains cultivated on olive mill wastewater‐based media

“…C. oleophila or Y. lipolytica strains have been shown capable of producing noticeable citric acid quantities after nitrogen exhaustion during growth on glucose or similarly metabolized substrates (e.g. glycerol) as secondary anabolic activity in several fermentation configurations 11, 23, 36, 41–44. The culture conditions (e.g.…”

Biosynthesis of lipids and organic acids by Yarrowia lipolytica strains cultivated on glucose

“…Furthermore, in all fermentations, a remarkable drop of the culture medium pH was observed, suggesting accumulation of organic acids into the culture medium. The pH was always maintained within the range 5.2-6.2, since in previous investigations it had been demonstrated that the maintenance of pH in this range was as a very important factor for the success of the fermentation [28,33]. Cit was the major organic acid accumulated into the culture medium, while other organic acids (e. g., a-ketoglutaric, acetic, pyruvic acid) were produced in negligible quantities (sum of organic acids produced was around 10% of Cit).…”

“…The bioprocess has also been successfully simulated and optimized with the aid of various models [6,11,12,[25][26][27]. Production of extracellular citric acid or intracellular lipid when sugars or similarly metabolized compounds are used as the sole substrate are, in their first steps, biochemically identical, and occur after intracellular nitrogen exhaustion [21][22][23]28]. The only difference in these processes is in the level of the enzyme ATP-citrate lyase, which catalyzes the cleavage of intracellular citric acid into oxaloacetate and acetyl-CoA, the latter being used as base-unit for the synthesis of intracellular fatty acids, by a quasi-inverted b-oxidation process [21,22,29].…”

“…The only difference in these processes is in the level of the enzyme ATP-citrate lyase, which catalyzes the cleavage of intracellular citric acid into oxaloacetate and acetyl-CoA, the latter being used as base-unit for the synthesis of intracellular fatty acids, by a quasi-inverted b-oxidation process [21,22,29]. In the absence of this enzyme, the carbon flow is directed towards the synthesis of citric acid that is secreted to the growth medium; yeast strains, hence, are classified into citric acid-pro-ducing or lipid-producing strains [23,28]. In the case of Y. lipolytica yeast, various strains of this species have the capability of producing large quantities of lipids that are located into lipid bodies when growth is conducted on hydrophobic substrates.…”

An overview of citric acid production

“…It is reported that below pH 5, citric acid production decreases and accumulation of other products like erythritol, arabitol and mannitol is favoured. Transport of citrate across cell membrane is also affected (Anastasisdis & Rehm, 2005;Karasu, Yalcin, Bozdemir & Ozhas, 2010).…”