Continuous gluconic acid production by Aureobasidium pullulans with and without biomass retention

New alternative processes for the continuous production of gluconic acid by Aureobasidium pullulans, using biomass retention by cell immobilization or cross over filtration, are described in the present work. 315 g/l gluconic acid was continuously produced in chemostat cultures at 21 hrs residence time without any biomass retention. 260 g/l gluconic acid was produced in fluidized bed reactor at 21 hrs residence time. The support carrier was overgrown resulting in limitations of oxygen transfer towards the inner layers of immobilized biomass. 375 g/l gluconic acid was produced under continuous cultivation at 22 hrs of residence time with a formation rate for the generic product of 17 g/(l x h) and a specific gluconic acid productivity of only 0.74 g/(g x h), using biomass retention by cross over filtration. 370 g/l were obtained at 19 hrs RT and 100 percent conversion with 25 g/l biomass and a formation rate of 19 g/(l x h). At 100 percent conversion, a selectivity of only 78 percent was determined at 22 hrs and of 77 percent at 19 hrs RT, because of the very high biomass concentration. Biomass retention makes it possible to break the existing link between growth and residence time.

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

The influence of air saturation and temperature on 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, favouring citric acid over isocitrate. An optimum air oxygen saturation of 20 percent and temperature of 30-31ºC were determined for the continuous citric acid secretion. The highest values of citric acid concentration (98 g/L), citrate to isocitrate ratio (33.3:1), volumetric citric productivity (1.8 g/(L x h)), and specific citric acid productivity (0.1 g/(g x h)), were reached at 20 percent air saturation at a residence time of 54 hrs by the experiment's lowest biomass of 18 g/L. The highest isocitic acid volumetric productivity (55.6 mg/(L x h)) and specific productivity (0.99 mg/(g x h)) were identified at 50 percent, instead. The fastest citrate excretion rate of the generic product of 0.046 g/(g*h) was found at 30-31ºC. A concentration ratio between extra- and intracellular concentration of citrate of up to 9 was identified. The highest extra-/intracellular ratio of citrate and lowest intracellular concentrations of glucose, citric and isocitric acid were determined at optimum air saturation as a consequence of active citrate export.

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

The effect of air saturation on citric acid production in batch, repeated batch and chemostat cultures has been studied. It was shown that, under continuous fermentation (chemostat mode), the highest concentration of citric acid equal of 98 g/l was produced at 20% of air saturation. In contrary to continuous fermentation, displaying an optimum at 20%, 80% air saturation yielded higher values in repeated batch fermentation process. 167 g/l citric acid were produced continuously with the fill and drain technique at 4.85 days, at 80% air saturation, compared with 157.6 g/l achieved within 5.4 days at 20%. Under repeated batch fermentation, the formation rate of the generic product (Rj) as well as the specific citric acid productivity (mp) reached a maximum of 1.283 g/(l x hr) at 4.01 days and of 0.0375 g/(g x hr) at 4.58 days, respectively. The glucose consumption rate (Rx) reached a maximum value of 3.33 g/(l x hr) entering stationary phase after 2.56 days at a glucose concentration of 131.2 g/l.

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

The pH influence on continuous citric acid secretion was investigated in Candida oleophila ATCC 20177 (var.) under NH4+ 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.