Citric acid production from glucose by yeast Candida oleophila ATCC 20177 under batch, continuous and repeated batch cultivation

Savas Anastassiadis*^#
Research in Biotechnology, Co.
Vat. #: 108851559. Avgi/Sohos
57002 Thessaloniki, Greece
Tel: 30 2395 051324
Fax: 30 2395 051470
E-mail: sanastassiadis@netscape.net

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

http://www.greekbiotechnologycenter.gr

*Corresponding author

Present manuscript presents continuous processes using yeasts as alternatives for an industrial production of citric acid. The effect of air saturation on citric acid production by Candida oleophila in batch, repeated batch and chemostat cultures has been studied. In contrary to continuous fermentation (chemostat mode) displaying an optimum of 98 g/l of citric acid at 20%, 80% air saturation yielded higher values in repeated batch fermentation process. 167 g/l citric acid were produced continuously at 80% air saturation with the fill and drain technique after 4.85 days, compared with 157.6 g/l achieved within 5.4 days at 20%.

Introduction and Methods

Citric acid is an important multifunctional organic acid with a broad range of versatile uses in household and industrial applications that has been produced industrially since the beginning of 20^th century. Several hundreds of thousands metric tons of citric acid are produced worldwide every year almost exclusively by fermentation. Industry still employs selected strains of Aspergillus niger, however yeast processes that have been the focus of investigations in different aspects for the past 35 years using various C-sources are also sporadically used today. A little information can be found in literature regarding continuous citric acid production. For instance, looking in the internet about 15,200 results was found as compared with 89 for chemostat (Anastassiadis et al. 2005; Anastassiadis and Rehm, 2005). Previous continuous operations for the production of citric acid by yeasts didn’t yield efficient concentrations for a competitive industrial operation (Enzminger and Asenjo, 1986; Kim et al. 1987; Kim and Roberts, 1991; Klasson et al. 1989; Bubbico et al. 1996; Arzumanov et al. 2000; Kamzolova et al. 2003; Anastassiadis et al. 2005; Anastassiadis and Rehm, 2005). A new developed fermentation process using Candida oleophila ATCC 20177 (var.) (obtained from Dr. Siebert, Jungbunzlauer Co. and later H & R, Bayer, Germany) is presented as an alternative innovative approach for the continuous industrial production of citric acid.

The strain was selected under many yeast strains during an extensive screening program (Anastassiadis et al. 1993; Anastassiadis, 1994; Anastassiadis et al. 1994; Anastassiadis et al. 2001; Anastassiadis et al. 2002; Anastassiadis et al. 2005). Yeast malt extract agar plates inoculated with C. oleophila were incubated for 2-3 days, stored at 4ºC and refreshed every 2-3 months. The inoculums (10%) were prepared by transferring of cells from agar plates into 500 ml shake flasks with baffles on a medium containing production medium however with 30 g/l glucose. The shake flasks were incubated for 1 day at 30ºC and 200 rpm. Chemostat experiments were carried out in a 2 litter fermenter (Biostat E, Braun-Diessel) with the working volume of 1.9 litter at 30ºC, pH 4.5, air saturations of 5-133% and a dilution rate of D = 0.0185 h^-1 on a basal medium 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, 0.15 mg/l CuSO₄ x 5H₂O, 0.0315 g/l ZnSO₄ x 7H₂O, 6 mg/l CoSO₄ x 7H₂O, 0.06 g/l H₃BO₃, 0.15 g/l CaCl₂, 0.15 g/l NaCl, 0.15 mg/l KJ, 2.5 g/l citric acid, 0.3 mg/l Na₂MoO₄ x 2H₂O, 3 mg/l Thiamine-HCl, 0.375 mg/l Biotin, 0.9375 mg/l Pyridoxine-HCl, 0.9375 mg/l Ca-D-Pantothenate, 0.75 mg/l Nicotinic acid. When the flow rate had achieved a stationary level, the cultivation in each mode was continued until the culture medium in fermenter was replaced five times.

Batch and repeated batch experiments (RB) were carried out at 20% or 80% air saturation, 30ºC, pH 4.5 or 5 (optimum pH) and an agitation of 600 rpm on a basal production medium containing: 6 g/l NH₄Cl, 400 g/l glucose, 1.05 g/l KH₂PO₄, 1.4788 g/l MgSO₄ x 7H₂O, 0.33435 g/l MnSO₄ x 4H₂O, 4 mg/l CuSO₄ x 5H₂O, 0.0839 g/l ZnSO₄ x 7H₂O, 8 mg/l CoSO₄ x 7H₂O, 0.08 g/l H₃BO₃, 0.2 g/l CaCl₂, 0.2 g/l NaCl, 0.2 mg/l KJ, 0.4 mg/l Na₂MoO₄ x 2H₂O, 2.5 g/l citric acid, 4 mg/l Thiamine-HCl, 0.5 mg/l Biotin, 1.25 mg/l Pyridoxine-HCl, 1.25 mg/l Ca-D-Pantothenate, 1 mg/l Nicotinic acid. No iron was added on intention to the medium. Silicon oil or polypropylene glycol was used as antifoaming agent. At the end of each batch fermentation about the 1/7 of fermentation solution was kept in fermenter as starting inoculum and the fermenter was filled to 2 litters (Biostat E fermenter, Braun-Diessel) with a new sterile fermentation medium for the next repeated batch fermentation. The experiments were repeated as long as it was required for the repeated batch system to be stabilized and after this point the results were reproducible (a kind of steady state situation). 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). In all cases, nitrogen was a limiting factor of yeast growth. The analysis of ammonium nitrogen, biomass, and 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 et al. 1994; Anastassiadis et al. 2001; Anastassiadis et al. 2005).

Results, Discussion and Concluding Remarks

Air saturation has been found to have a very strong influence on continuous, batch and repeated batch fermentation of citric acid. New continuous processes for the production of citric acid have been developed and presented here. 98 g/l of citric acid were continuously produced at a residence time of 54 hrs (D = 0.0185 h^-1) with a ratio between citrate and isocitrate of 33.3 by the experiment’s lowest steady state biomass concentration of about 18 g/l and at lowest intracellular concentration of citrates at 20% air saturation under nitrogen limiting conditions. Up to 150 g/l were produced continuously at longer residence times using C. oleophila (Anastassiadis, 1994; Anastassiadis et al. 1993; Anastassiadis et al. 1994; Anastassiadis et al. 2001). The very high glycolytic flow rate determined at 20% air saturation means that a kind of a Crabtree effect was attained in this case, simulating an anaerobic glycolytic pathway under aerobic conditions.

In batch process, a biomass of 25.5 g/l, 134.3 g/l citric acid and 4.4 g/l glucose were obtained at fermentation end after 7.4 days with a ratio between citrate and isocitrate of 15.8 and a selectivity of above 40%. The initiation of citric acid secretion started a few hours after the complete depletion of nitrogen in the medium and was caused by the intracellular nitrogen limitation (following a transition phase) and the reduction of cellular nitrogen content that resulted in intracellular NH₄⁺ accumulation as has been described in Anastassiadis et al. 2002 and Anastassiadis et al. 2005. Acceleration in biomass formation and citrate production was observed in all of repeated batch experiments, compared with the initial batch fermentations. 20% air saturation resulted to lower productivity and concentration of citric acid, compared with 80%. Despite the very initial high glucose concentration of 336 g/l biomass increased at 80% air saturation after a very short lag phase showing the very high osmotolerance of yeast strains. Fermentation started with an initial citric acid concentration of 25.5 g/l and biomass concentration of 8.7 g/l from previous repeated batch experiment and reached 166.5 g/l and 34.3 g/l after 4.58 d with a citrate/isocitrate ratio of 20 at a residual glucose concentration of 7.6 g/l, whilst 93.7 g/l citric acid were already measured after 2.6 days of fermentation. Glucose was finally completely consumed at last measured point of 4.85 d, reducing total fermentation time by about 2 days. The increase of KH₂PO₄ to 2.1 g/l resulted in increasing of biomass by 20.5% and decreasing of specific productivity by about 28.5%. A biomass of 43.1 g/l, 147.9 g/l citric acid and 14.5 g/l residual glucose were determined after 4.29 d. In repeated batch experiment at 20% air saturation, 157.6 g/l citric acid and a residual glucose of 1.9 g/l were reached after 5.36 d starting with an initial concentration of 33 g/l and an initial glucose concentration of 336 g/l. In a further series of repeated batch experiments very high citric acid concentrations of 200-250 g/l were repeatedly achieved for about 20 times confirming the process stability (Research in Biotechnology, Greece). Oscillations (phased production and glucose consumption) were observed during the entire batch and repeated batch fermentation.

In view of economical aspects, continuous chemostat and repeated batch production of citric acid by yeasts seems to have many advantages compared with the traditional discontinuous industrial processes of the last 100 years utilizing Aspergillus niger. The presented results on citric acid production from glucose by yeast are the best reported in the international bibliography. The very high citrate/isocitrate ratio that was achieved by C. oleophila through a sophisticated process and medium optimisation is comparable with those that have been reached by high ratio mutant yeast strains (Akiyama et al. 1972; Akiyama et al. 1973a; Akiyama et al. 1973b). Summarizing present and previous findings, important events found in yeast process would include nitrogen depletion in fermentation medium, intracellular decrease of nitrogen content (~4%), elevation of NH₄⁺ concentration in the cell (~37.4 mM), enhanced energy charge in cells displaying a maximum at optimum fermentation conditions (e.g. pH, air saturation and temperature), induction of specific transport system for citrate secretion, citrate secretion by active transport system, fine tuning regulation of citrate secretion by ATP, CO₂, residence time etc.

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.

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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