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Electronic Journal of Biotechnology ISSN: 0717-3458  
© 2006 by Pontificia Universidad Católica de Valparaíso -- Chile  
BIP RESEARCH ARTICLE

Oxygen and temperature effect on continuous citric acid secretion in Candida oleophila

Savas Anastassiadis*
Pythia Institute of Biotechnology
Environmental Engineering Department
Democritus University of Thrace
Vat. #: 108851559
Avgi/Sohos, 57002 Thessaloniki, Greece
Tel: 302395051324
Tel/Fax: 302395051470
E-mail: sanasta@env.duth.gr
sanastassiadis@netscape.net

Hans-Jürgen Rehm
Institute of Molecular Microbiology and Biotechnology
University of Münster
Corrensstr. 3, 48149 Münster, Germany
(retired Professor)

Website: http://www.greekbiotechnologycenter.gr

*Corresponding author


Financial support: Part of the work that has been carried out at the Institute of Biotechnology 1 of Research Centre Jülich (Germany) was financed by Haarmann and Reimer, a daughter company of the company Bayer, Leverkusen, Germany.

Keywords: Candida oleophila , citric acid, citric acid secretion.

Abbreviations:

RT: Residence time, hrs
mp:Specific productivity of the generic product, g product/(g biomass x h)
Rj: Volumetric productivity of the generic product, g product/(l x h)
Rs: Volumetric glucose consumption rate, g/(l x h)

Reprint (BIP) Reprint (PDF)

The influence of air saturation and temperature on the continuous citric acid secretion was studied in chemostat cultures of Candida oleophila ATCC 20177 (var.). Simultaneous measurements of intra- and extracellular concentration of glucose, citric and isocitric acid confirmed the involvement of a specific active transport system in citrate secretion and overproduction, favoring citric acid over isocitrate. An optimum air saturation of 20% and temperature of 30-31ºC were determined, reaching at a residence time of 54 hrs 98 g/l of citric acid with a ratio between citrate and isocitrate of 33.3, a biomass specific citric acid productivity of 0.1 g/(g x hr) and a concentration ratio between extra- and intracellular concentration of citrate of 9.

Article

Citric acid is a tricarboxylic organic acid of industrial importance, which is produced in discontinuous aerobic bioreactors using almost exclusively selected mutant strains of Aspergillus niger, however an efficient continuous production of citric acid still remained an unachievable dream.The main actual problems in today's citric acid industries are still the low productivities, requiring long operational times, high investments and production costs (Anastassiadis and Rehm, 2005). Continuous operations for the production of citric acid by yeasts have increasingly received research interest in last years. The use of yeasts instead of moulds represents an innovative approach, which is also of human health; because A. niger possesses the formation potential of toxic compounds (mycotoxins) (Anastasssiadis et al. 2005). A little information exists in international bibliography regarding the mechanism of citric acid secretion in yeasts and fungi and the excretion mechanism of citrate from cytoplasm into the medium in A. niger still remains unclear (Grewal and Kalra, 1995; Netik et al. 1997; Anastassiadis and Rehm, 2005). A high specific active transport system for citric acid secretion has been detected for the first time in yeasts, acting as the speed determining factor and well explaining overproduction of citric acid against a concentration gradient (Anastassiadis, 1994; Anastassiadis et al. 1993; Anastassiadis et al. 1994; Anastassiadis et al. 2001; Anastassiadis and Rehm, 2005). Netik et al. (1997) reported later about a ΔpH-driven H+-symport dependent system for citric acid export in manganese-deficient cells of A. niger. The central aspect of present work was to investigate and optimize the effect of important fermentation parameters, such as the air saturation and temperature, on the continuous secretion of citric acid by a specific active transport system in C. oleophila ATCC 20177 (var.), in order to develop new continuous fermentation processes that would stand today's strong competition in citric acid industry.

Materials and Methods

Candida oleophila, strain ATCC 20177 (var.) was selected in previous studies under many yeast strains (Anastassiadis, 1994). The influence of temperature was investigated in 1 litter glass bioreactor (Research Center Jülich, Germany) at 1000 rpm and an aeration rate of 4 l/min pure oxygen for a working volume of 460 ml (corresponds to 0.145 vvm). The effect of oxygen was investigated in a 2 litter bioreactor (Biostat E, Braun-Diessel) at a working volume of 1.9 l, applying optimized fermentation medium and chemostat operation conditions (600 rpm, pH 4.5 and 30ºC) under nitrogen limited conditions (Anastassiadis and Rehm, 2005). A basal medium (BM) was used for the investigation of temperature effect containing: 3 g/l NH4Cl, 240 g/l glucose, 0.7 g/l KH2PO4, 0.35 g/l MgSO4 x 7H2O, 0.11 g/l MnSO4 x 4H2O, 0.002 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, 0.0001 g/l KJ, 2.5 g/l citric acid, 0.0002 g/l Na2MoO4 x 2H2O, 0.002 g/l Thiamine-HCl, 0.00025 g/l Biotin, 0.000625 g/l Pyridoxine-HCl, 0.000625 g/l Ca-D-Pantothenate, 0.0005 g/l Nicotinic acid. An optimized medium was used for the investigation of oxygen influence containing: 250 g/l glucose, 4.5 g/l NH4Cl, 1.05 g/l KH2PO4, 0.525 g/l MgSO4 x 7H2O, 0.2507 g/l MnSO4 x 4H2O, 0.00015 g/l CuSO4 5H2O, 0.0315 g/l ZnSO4 x 7H2O, 0.006 g/l CoSO4 x 7H2O, 0.06 g/l H3BO3, 0.15 g/l CaCl2, 0.15 g/l NaCl, 0.00015 g/l KJ, 2.5 g/l citric acid, 0.0003 g/l Na2MoO4 x 2H2O, 0.003 g/l Thiamine-HCl, 0.000375 g/l Biotin, 0.0009375 g/l Pyridoxine-HCl, 0.0009375 g/l Ca-D-Pantothenate, 0.00075 g/l Nicotinic acid. Vitamins and NH4Cl were added separately into autoclaved medium (30-60 min at 121ºC) by sterile filtration through 0.2 μm filters (Sartorius filter, Göttingen Germany). No Fe+2 salt has been intentionally added in to the media. Silicon oil or polypropylene glycol was used as antifoaming agent. The culture flow rate D was 0.0185 h-1. Optical density (OD660 nm), dry biomass (filter method), organic acids, glucose, ammonia nitrogen and intracellular concentrations were measured as has been described previously (Anastassiadis, 1993; Anastassiadis, 1994; Anastassiadis et al. 1993; Anastassiadis et al. 1994; Anastassiadis et al. 2001; Anastassiadis et al. 2002; Anastassiadis.

Results

Temperature effect

At 80 hrs RT and pH 4.5, the highest citrate concentration of 63.5 g/l, citrate/isocitrate ratio of 28.8, formation rate of the generic product (Rj) of 0.8 g/(l x hr) and specific citric acid productivity of 0.046 g/(g x hr) were achieved at 30ºC, compared with only 12 g/l (18.9%), 17 and 0.15 g/(l x hr) found at 24ºC. Only 80 g/l of citric acid was achieved at more than 100 hrs under still sub-optimal fermentation conditions. A greater intracellular ratio between citrate and isocitrate and decreasing intracellular isocitrate concentration is to record at higher temperatures, which is still lower than 1, in contrary to an almost identical remaining extracellular ratio and intracellular citrate concentration, indicating in accordance to previous studies that a minimum citrate concentration is required for functioning of active transport system (Anastassiadis and Rehm, 2005). An intracellular glucose concentration of 2.6 mg/g was identified at 27ºC and of 45.8 mg/g at 29ºC.

Oxygen effect

At 54 hrs RT (D = 0.0185 h-1), maximum values of 98 g/l citric acid, 70% molar selectivity (Mol citrate/Mol glucose), 1.81 g/(l x hr) formation rate of the generic product (Rj), 0.1 g/(g x hr) biomass specific citric acid productivity (mp) and a ratio between citrate and isocitrate of 33.3 (3% isocitrate) were reached using C. oleophila at the optimum air saturation of 20% by the experiment's lowest biomass of 18 g/l. Citrate concentrations of up to 150 g/l have been achieved at longer RT (Anastassiadis, 1994; Anastassiadis et al. 1993; Anastassiadis et al. 1994; Anastassiadis et al. 2001). Only 71.4 g/l citric acid (72.6%) was produced at 133% and 77.2 g/l (78.6%) at 5%. The maximum Rj of 55.6 mg/(l x h) for isocitric acid was identified at 50% and mp of 2.9 mg/(g x hr) at 20%. The lowest intracellular isocitrate concentration of 24.4 mg/g dry biomass, citrate + isocitrate of 53.1 mg/g (125.6 mM), glucose of 17.6 mg/g (8 g/l or 44.4 mM) or total acid plus glucose concentration of 70.72 mg/g and the highest intracellular ratio between citrate and isocitrate of 1.18 were determined at optimum air saturation of 20%, indicating a specificity and affinity maximum of active transport system towards citric over isocitric acid. A concentration ratio of 7.5 was calculated at 20% between the extra- (98 g/l) and intracellular citric acid (13.1 g/l or 67.96 mM). Isocitrate is obviously driven out from aconitase equilibrium towards citrate as a consequence of active secretion.

Discussion

Kinetic chemostat data for growth and citrate production are rare, they offer however important information for process development, optimization and scale up. Present work describes for the first time in international literature the very significant effect of oxygen and temperature on the continuous production and secretion of citric acid by free growing yeast cells. Enzyme activities as well as regulation and transport systems of microbial systems are in generally enormously affected by the temperature. In good agreement with batch fermentations (Rane and Sims, 1993; Crolla and Kennedy, 2001) C. oleophila grows and produces at temperatures between 24 and 31ºC, displaying an optimum at 30-31ºC. Citric acid production by yeasts and fungi is an obligatory aerobic process, strongly depending on oxygen supply in bioreactor. In contrary to chemostat cultures (20% saturation) a higher productivity and selectivity has been found in batch and repeated batch cultures at 80% or higher air saturations (Anastassiadis and Rehm 2006; Stottmeister et al. 1981; Stottmeister et al. 1986; Okoshi et al. 1987). Even short time interruptions of oxygen supply can cause irreversible changes or a complete production lost in A. niger (Anastassiadis, 1994; Grewal and Kalra, 1995).

Dissolved oxygen concentration appears to play a very important role in terms of influencing the activity of glycolysis and respiration chain. The higher glycolysis rate is possibly resulting in very high ATP levels (higher energy charge), thus intensifying citrate secretion by active transport system (Anastassiadis and Rehm, 2005). The lowest intracellular concentration of glucose, isocitrate, citrate plus isocitrate, total acid plus glucose, maximum intracellular citrate/isocitrate ratio and the highest extracellular citric acid concentration were found at optimum air saturation of 20% as a result of most intensive secretion and consequentially glycolysis rate. A certain minimum concentration of about 20 mM of citric acid is necessary for the proper functioning of active transport system. A similar effect of temperature on the intracellular accumulation and secretion of glutamic acid in Corynebacterium glutamicum has been reported by Lapujade et al. (1999). A kind of a Crabtree effect appears to occur in this case, simulating an anaerobic glycolytic pathway running under aerobic conditions. Energy consuming fatty acid synthesis and citric acid secretion can be considered as a means of cutting down energy overload and surplus amount of NAD(P)H2. Isocitric acid could somehow interact with ATP and AMP and regulate both, aconitase as well as transport system activity, thus also well explaining its very low affinity to the transport system. Whether the energy charge is the driving force for citrate excretion in A. niger is still unclear.

Concluding Remarks

Present results clearly show the feasibility of a continuous citric acid production by yeasts. 150 g/l of citric acid were continuously produced by C. oleophila and 200 g/l using a Yarrowia lipolytica strain (unpublished data). 170 g/l were reached in further continuously operating repeated batch experiments by C. oleophila (Anastassiadis, 1994; Anastassiadis et al. 1993; Anastassiadis et al. 1994; Anastassiadis et al. 2001; Anastassiadis and Rehm, 2006) and 250 g/l by a Y. lipolytica strain or 250 g/l in continuously operating fed batch experiments using C. oleophila. Significant citric acid concentrations have also been reached by Kamzolova et al. (2003). Citric acid fermentation is a very complex process. Numerous events including 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 active transport systemis the main speed-determining factor in citrate overproduction by yeasts, regulating in contrary to previous thoughts intracellular accumulation of citric acid and its secretion rather than aconitase activity, since isocitrate doesn't seem to be a high-affine substrate. Present results for the continuous production of citric acid by free growing cells are the best that have been published in international bibliography. The effect of air saturation was significant, which would also influence the costs of an industrial fermentation process enormously.

Acknowledgements

We thank Professor Dr. U. Stottmeister, Mrs. E. Weissbrodt (UFZ Ctr. Envtl. Res. Leipzig-Halle, Germany) and Prof. Dr. Christian Wandrey (Institute of Biotechnology 2 of Research Center Jülich, RCJ, Germany) for their helpful advices and support.

References

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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), where the experiments were performed.

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