Introduction and methods

Although citric acid production using mutant strains of A. niger has almost been extensively optimised (Röhr et al. 1983), there is still no comprehensive explanation for citrate overproduction and many aspects related to citrate accumulation and secretion remain unclear. A little information is found in literature regarding the mechanism of citric acid secretion in yeasts and fungi. Intracellular nitrogen limitation and the low intracellular nitrogen content (Briffaud and Engasser 1979; Anastassiadis 1994; Moresi 1994; Anastassiadis et al. 2002; Anastassiadis et al. 2005), occurring after the exhaustion of extracellular nitrogen and entering of the transition phase, and the increase in intracellular NH₄⁺ concentration are the most important factors influencing and triggering out citric acid formation and secretion in yeasts (Anastassiadis 1994; Anastassiadis et al. 1993; Anastassiadis et al. 1994; Anastassiadis et al. 2001; Anastassiadis et al. 2002; Anastassiadis et al. 2005). Intracellular accumulation of NH₄⁺ found in cytoplasm of A. niger (Röhr and Kubicek 1981; Habison et al. 1983) and in Candida oleophila (Anastassiadis 1994; Anastassiadis et al. 2002), possibly caused by a disturbances in protein or nucleic acid turn over and proteolysis, uncouples citrate feed back inhibitory effect on phosphofructokinase, enabling an unlimited flow through glycolysis. The central aspect of present work was to investigate the influence of pH on continuous citric acid secretion by a specific active transport system and on elemental biomass composition in free growing chemostat cultures of Candida oleophila ATCC 20177 (var.) (obtained from Dr. Siebert, Jungbunzlauer Co. and later H and R, Bayer, Germany). The investigations were carried out in magnetically stirred 1 litter double glass bioreactor (ResearchCenterJülich, RCJ, Germany) at a working volume of 460 ml, 30ºC, 1300 rpm and a constant aeration rate of 0.145 vvm pure oxygen. In generally, between five and 10 generations (residence times) are necessary for getting steady state conditions, depending on process and strain stability.

A basic fermentation medium of following composition was used for the investigation of pH influence on iron uptake and citrate formation (BM): 3 g/l NH₄Cl, 120 g/l glucose, 0.7 g/l KH₂PO₄, 0.35 g/l MgSO₄ x 7H₂O, 0.11 g/l (0.5 mM) MnSO₄ x 4H₂O, 5 ìM FeSO₄ x 7H₂O, 0.001 g/l CuSO₄ x 5H₂O, 0.021 g/l ZnSO₄ x 7H₂O, 0.004 g/l CoSO₄ x 7H₂O, 0.04 g/l H₃BO₃, 0.1 g/l CaCl₂, 0.1 g/l NaCl, 0.1 mg/l potassium iodide (KJ), 2.5 g/l citric acid, 0.2 mg/l Na₂MoO₄ x 2H₂O, 2 mg/l Thiamine-HCl, 0.25 mg/l Biotin, 0.625 mg/l Pyridoxine-HCl, 0.625 mg/l Ca-D-Pantothenate, 0.5 mg/l Nicotinic acid.

In a second series of experiments a production medium with 4.5 g/l NH₄C, analogously increased concentrations of residual compounds (factor 1.5), 250 g/l glucose, however with 1.125 mM manganese was used for the investigation of pH influence. No iron was added on intention to the medium. Silicon oil or polypropylene glycol was used as antifoaming agent. The 20 l medium was sterilized in autoclave for 30-60 min at 121ºC, however vitamins and NH₄Cl were added separately into media by sterile filtration through a 0.2 mm membrane filter (Sartorius, Göttingen, Germany). The analysis of ammonium nitrogen, biomass, intra and extracellular concentration of citric acid, isocitric acid and glucose were analysed as has been described in previous works (Anastassiadis, 1993; Anastassiadis et al. 1993; Anastassiadis, 1994; Anastassiadis, 2001; Anastassiadis, 2002). ATP and ADP ratio was determined based on the luciferin/luciferase method for ATP measurement (Lundin et al. 1976, modified; Schimz et al. 1981).

Results and Discussion

Kinetic data obtained in chemostat cultures give essential information for sophisticated process design, process development and scale up. However, this type of information for continuous citric acid fermentation is rather rare in literature. For instance, looking in the internet about 15,200 results was found as compared with 89 for chemostat. The influence of pH on the growth and elemental biomass composition of C. oleophila and citric acid secretion was investigated in different series of chemostat experiments, because no information was found about the influence of pH on continuous citric acid secretion and a little is known regarding the real pH effect on citrate formation.The pH influenced citric acid secretion and intracellular elemental cell composition. With 4.5 g/l NH₄Cl and 250 g/l glucose, 57.8 g/l citric acid was continuously produced with a citrate/isocitrate ratio of 15.6 at optimum pH of 5 and 60 hrs residence time (time that is required to replace the fermenter volume once). The highest formation rate for the generic product of 0.96 g/(l*h), specific citric acid productivity of 0.041 g/(g*h), selectivity of 44.3%, yield of 33.6%, conversion of 75.9% and ratio between ATP and ADP of 2.65 were determined at optimum pH 5 as well. Decreasing of pH resulted in continuous increase of biomass, whereas 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). Shavloskii et al. (1988) identified an active iron uptake system in Pichia quilliermondii with an optimum at pH 5.3 and 37ºC, whereas small pH alterations caused a dramatic lost of its activity (90%). The addition of iron resulted to enhanced biomass formation and affected continuous citric acid production significantly (Anastassiadis, 1994).

Citric acid production by yeasts seems to be a paradox, because citric acid accumulation occurs under a high ratio between ATP and ADP, although the process is considered to be non-growth related and citrate excretion is triggered out by nitrogen limitation. The active transport systemfor citrate excretion appears to be the main speed-determining factor in citrate overproduction by yeasts, reaching concentration gradients between extra- and intracellular concentration of citrate higher than 1 and up to about 60. It showed a very high specificity for citrate over isocitrate (specificity factor of about 33), indicating that isocitrate isn't a high-affine substrate. The highest intracellular concentrations of citrate, isocitrate and glucose and simultaneously the lowest extracellular citric acid concentrations were determined under none or low producing conditions. In contrary, maximum extracellular citric acid concentration was reached under conditions, where the lowest intracellular concentrations of citrate and isocitrate appeared. Intracellular isocitric acid concentration exceeded citric acid concentration significantly. Under high producing conditions citrate secretion resulted in a higher glycolysis rate and thus lowering of intracellular concentration of glucose and isocitric acid. Consequently, isocitric acid was drawn out from aconitase equilibrium (Anastassiadis, 1994; Anastassiadis et al. 1993; Anastassiadis et al. 1994; Anastassiadis et al. 2001).

Intracellular accumulation of citric acid and citrate secretion are obviously two different phenomena influencing however each other, every time in a different way based on varying environmental conditions. Present results are a very strong confirmation that not the intracellular citrate accumulation alone, however the active secretion of citrate over the plasma membrane is the speed determining factor for citric acid excretion, displaying an optimum at pH 5. The active transport system seems to be induced by other factors rather than by the intracellular accumulation of citric acid. However, a certain critical intracellular level of citrate (~20 mM), determined at optimum air saturation of 20%, is necessary for functioning of active transport system (Anastassiadis et al. 1993; Anastassiadis, 1994; Anastassiadis et al. 1994; Anastassiadis et al. 2001). A very high gradient between extra- and intracellular citric acid concentration of about 60 has been determined at citric acid concentration of 250 g/l that has been achieved using C. oleophila and Y. lipolytica. In contrary to reports of Marchal et al.(1980) and McKayet al.(cited in Gutierrez and Maddox, 1993), assuming a passive diffusion of citrate and isocitrate over the cell membrane, a specific active transport system for citric acid secretion was identified for the first time in Candida oleophila, preferring citrate over isocitrate. It is acting as the speed determining factor well explaining the overproduction of citric acid against a very high concentration gradient (Anastassiadis, 1994; Anastassiadis et al. 1993; Anastassiadis et al. 1994; Anastassiadis et al. 2001; Anastassiadis et al. 2002). Netik et al. (1997) reported later about a ÄpH-driven H⁺-symport dependent system for citric acid export in manganese-deficient cells of A. niger, claiming that only a passive diffusion through the plasma membrane had been reported before for citrate excretion in yeasts.

Citric acid production is obviously a very complicated process. Numerous events such as growth limitations, enzyme activities, energy gain and energy state, intracellular acid accumulation, as well as uptake and transport systems display different optima and regulation mechanisms, which are somehow interconnected and interrelated in a synergistic mode. The pH dependent specific active transport is providing the explanation for citrate overproduction in yeasts. The active transport seems to be a way for the regeneration of reduction equivalents and the conversion of excessive ATP that is gained by the intensive glycolysis under growth limiting conditions, indicating the presence of a kind of a "Crabtree effect". Thus under the aspect of regulation, fatty acid synthesis, citric acid secretion by active transport system as well as polyol formation can be considered as a means of cutting down energy overload and surplus amount of NAD(P)H₂. The existence of active transport system for citrate secretion and the strong correlation between ATP/ADP ratio and citrate overproduction found in C. oleophila goes very well together with the legendary reports of Lozinov and Finogenova (1982) about a non phosphorylating alternative oxidase, which had been identified in yeasts and completes electron flow without ATP regeneration, competing with the production of citric acid. Meyrath (1967) discussed on the other side the energy demand for growth and citrate excretion in relation to citrate overproduction in stationary cells of A. niger. A significant amount of energy is required, since the acid is excreted against a concentration gradient. Kristiansen and Sinclair (1979) proposed an other option explaining citrate secretion in A. niger under the consideration of cytoplasm streaming.

Acknowledgements

We thank Professor Dr. U. Stottmeister (UFZ Ctr. Envtl. Res. Leipzig-Halle, Germany), Prof. Dr. R. Krämer (Research Centre Jülich, RCJ; formerly known as Nuclear ResearchCenter, KFA, Germany) and Prof. Dr. Christian Wandrey (Research Centre Jülich RCJ, Germany) for their helpful advices and support.

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Declaration

The experiments of the present manuscript comply with the currant laws of the country Germany (Institute of Biotechnology 2 of Research Centre Jülich 2, RCJ; formerly known as Nuclear Research Centre Jülich, KFA) and Greece (Research in Biotechnology Co., Avgi/Sohos, 57002 Thessaloniki), where the experiments were performed.