Citric acid production from glucose by yeast Candida oleophila ATCC 20177 under batch, continuous and repeated batch cultivation Savas Anastassiadis*# Hans-Jürgen
Rehm http://www.greekbiotechnologycenter.gr *Corresponding author
Keywords: air saturation influence, chemostat, citric acid, citric acid fermentation, repeated batch, oxygen influence. Present address:
#Research
in Biotechnology, Co., Vat. 108851559. Avgi/Sohos, 57002
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%.
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 20th
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 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 Batch and repeated
batch experiments (RB) were carried out at 20% or 80% air saturation,
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
We thank Professor
Dr. U. Stottmeister, Mrs. E. Weissbrodt (UFZ Ctr. Envtl. Res.
The experiments of the
present manuscript comply with the currant laws of the country
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