Continuous gluconic acid production by Aureobasidium pullulans with and without biomass retention
support: The work has been carried out at the Institute of Biotechnology 2 of Research Center Jülich (formerly known as Nuclear Research Center Jülich,
Keywords: biomass immobilization, continuous fermentation, cross over filtration, gluconic acid fermentation, reaction technique, residence time.
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% conversion with 25 g/l biomass and a formation rate of 19 g/(l x h). At 100% conversion, a selectivity of only 78% was determined at 22 hrs and of 77% 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.
As a multifunctional carbonic acid, belonging to the bulk chemicals and due to its physiological and chemical characteristics, gluconic acid itself, the gluconolatone form and its salts (e.g. alkali metal salts, in especially sodium gluconate) have found extensively versatile uses in the chemical, pharmaceutical (e.g. iron and calcium deficiency), food, beverage, textile and other industries (Hustede et al. 1989; Anastassiadis et al. 2003; Znad et al. 2004). Additionally, it can be exploited for cleaning purposes (e.g. diary industry) as well as for the extraction of trace elements like calcium, copper and iron. Gluconic acid can have further applications for the solubilization of phosphate (Fenice et al. 2000; Vassilev et al. 2001; Rodríguez et al. 2004) and as a cement additive in the construction industry, because it enhances the cement's resistance and stability under extreme climatic conditions, e.g. frost and water (Singh, 1976; Hustede et al. 1989).
methods have been extensively described for the production of gluconic
acid, including chemical and electrochemical catalysis, enzymatic
biocatalysis in enzyme bioreactor, microbial production using free
growing or immobilized cells of either Gluconobacter oxydans
Biomass retention by immobilization on porous sinter glass or by cross over filtration was applied in present work in order to investigate the feasibility of a further acceleration of continuous gluconic acid production and of the maximization of product concentration by an isolated Aureobasidium pullulans strain, as a comparison with the continuous cultivation of free growing cells.
Aureobasidium pullulans (de Bary) Arnaud isolate Nr. 70 (DSM 7085), which was isolated from wild flowers (
For the investigation of continuous gluconic acid fermentation with or without biomass retention, cells were grown in a
The fermentations were carried out in agitation
The fermentation medium was continuously added into the fermenter using a precision gravimetric dosing system (Sartorius,
The experiments for the continuous production of gluconic acid by biomass retention by means of cross over filtration were carried out in a
The experiments with biomass immobilization were carried out in a fluidized bed reactor at a working volume of about
Optical density (OD660 nm), dry biomass (filter method) and the concentration of glucose and gluconic acid were determined as has described in previous works (Anastassiadis, 1993; Anastassiadis et al. 1999; Anastassiadis et al. 2003; Anastassiadis et al. 2005).
Ammonium nitrogen was analyzed as has been described in Anastassiadis et al. (2002).
Novel superior processes were developed and optimized for the continuous and discontinuous production of gluconic acid by isolated strains of Aureobasidium pullulans during an extensive process development program (Anastassiadis et al. 1999; Anastassiadis et al. 2003; Anastassiadis et al. 2005). A defined fermentation medium was composed and optimized, which makes it easy to reproduce those data. Completing the process optimization program, reaction-technical investigations were accomplished, in order to examine the transferability of obtained results and to test different fermentation process variants (e.g. batches, fed batches, continuous fermentation without and with biomass retention) for the production of gluconic acid by Aureobasidium pullulans isolate 70 (DSM 7085) (Anastassiadis et al. 1999).
The continuous gluconic fermentation by free growing cells of A. pullulans and without any biomass retention was carried out in a
The continuous production of gluconic acid by cell immobilization on porous sinter glass (
A good alternative approach for the acceleration of product concentration maximization is the partial retention of biomass by means of microfiltration (cross over filtration). Biomass retention enables the breaking of the link between growth (biomass formation) and residence time and between growth and production. It makes feasible the performance of the process at very high oxygen concentrations, which are toxic for cell growth, resulting in higher biomass specific productivities. It should also be examined, whether very high gluconic acid concentrations can be reached at shortened retention times, using very high oxygen concentrations. In order to receive comparable data, the experiments were accomplished under the same conditions as the preceding continuous fermentations without biomass retention. About 80% of filtrate (filtered fermentation solution) and 20% bleed (unfiltered fermentation solution) were adjusted and maintained constant during the entire duration of the experiment. Applying a glucose concentration of 450 g/l, 375 g/l gluconic acid was continuously produced at a residence time of about 22 hrs, 100% conversion of glucose,
In previous studies, it has been extensively studied the influence of residence time on continuous production without biomass retention (Anastassiadis et al. 2003; Anastassiadis et al. 2005). In the present work, the maximization of gluconic acid concentration has been achieved by applying biomass retention in bioreactor by means of cross over filtration. According to present results, it seems very possible to still convert glucose completely at even very short residence times i.e. less than 7 hrs. The investigation of the performance of continuous gluconic acid production by biomass retention at very low residence times was not possible because of technical inadequacies.
Microbial production of metabolites of primary and intermediary metabolism usually takes place under stress conditions. The medium composition plays a crucial role for a successful metabolite production. Fermentation parameters such as pH, oxygen, temperature and medium composition, influencing continuous and discontinuous gluconic acid production by isolated strains of A. pullulans, have been identified and optimized in previous chemostat studies (Anastassiadis et al. 1999; Anastassiadis et al. 2003; Anastassiadis et al. 2005). In a conventional chemostat culture without any biomass retention, where growth and production occur simultaneously, the wash out effect of biomass is the limiting factor in terms of achieving very high product concentrations at very low residence times (high dilution rates). The highest formation rate and specific productivity for the generic product gluconic acid have been achieved at lower residence times. They decrease continuously at increasing residence time and very high gluconic acid concentrations of more than 230 g/l (Anastassiadis et al. 1999; Anastassiadis et al. 2003). The formation rate of the generic product (Rj) is a compensation effect between biomass concentration and biomass specific production rate. In continuous process, growth and production run parallel, influencing each other. For example, different optimum dissolved oxygen concentrations have been determined for growth, gluconic acid production and for various specific fermentation factors such as conversion, formation rate for the generic product, specific gluconic acid productivity, selectivity and yield. Hence, the optimum fermentation parameters that had been found for example for the pH, temperature and oxygen saturation are the compensation result between growth and production.
A gluconic acid concentration higher than 230 g/l was continuously produced at residence times of 12 hrs without any biomass retention (Anastassiadis et al. 2005). New alternative processes for the continuous production of very high gluconic acid concentrations using free growing cells or biomass retention of A. pullulans are described in the present work. Biomass retention by immobilization or cross over filtration enables the application of production optima in a continuous fermentation process even at very short times that would normally wash out the biomass from the fermenter. Thus, the maximum specific productivity of generic product can be maintained, uncoupling product formation from growth rate, although it differs from the optimum growth conditions. Biomass retention by means of cross over filtration makes it possible to break the existing link between growth and the residence time. A. pullulans was easily immobilized on porous sintered glass and suitable for a continuous gluconic acid production by immobilized cells. A good alternative for the acceleration of product concentration maximization offers the partial biomass retention by means of microfiltration (cross over filtration). Continuous fermentation can thus perform at optimum production parameters, which would not necessarily be optimal for the formation of biomass, thus breaking partially the interaction between growth and production. A very high formation rate and concentration of generic product can be obtained at simultaneously keeping very high specific productivities. Although continuous fermentation performing in a fluidized bed reactor with immobilized biomass on a support carrier may be more convenient from a process point of view, fermentation with non-supported microorganisms performing without or with biomass retention by cross over filtration has been found to be advantageously. In both cases, very high dissolved oxygen concentrations higher than 200% air saturation are applied successfully.
Early fungal fermentation processes for gluconic acid production employed species of Penicillium (Herrick and May, 1928). Improved strains of Aspergillus
To our knowledge, no comparable results reaching those high product concentrations (Anastassiadis et al. 1999; Anastassiadis et al. 2003; Anastassiadis et al. 2005; present work) can be found in the international literature, referring to the continuous gluconic acid production by free growing or immobilized cells of Aspergillus and Penicillium strains or other microorganisms, without any biomass retention. 260 g/l of gluconic acid were continuously produced without any biomass retention under optimized conditions by A. pullulans at 15.3 hrs RT and 305-315 g/l at about 21 hrs. 370 g/l of gluconic acid were also continuously produced at complete glucose conversion at 19 hrs residence time and up to 504 g/l were achieved in fed batch experiments (Anastassiadis et al. 1999; Anastassiadis et al. 2003; present work). Sankpal et al. (1999) reported about continuous gluconic acid production by immobilized mycelia of A.
The use of genetically engineered microorganisms and the immobilization of isolated glucose oxidizing enzymes or whole cells in specialized reactors appear in literature reports as the possible future developments regarding the advanced continuous production of gluconic acid (Hartmeier and Döppner, 1983; Milson, 1987; Szajani et al. 1987). Park et al. (2000) cloned and expressed for example glucose oxidase from A.
The process operation is very stable, under the condition that filter modules are cleaned (periodical reverse flow of filtrate). A precondition for an unproblematic long time operation is also keeping the system safe from contaminations, because some contaminants can overgrow the production strain. No strain instabilities have been observed so far during the extensive process optimization and development program, showing the genetic stability of the new yeast-like mold system.
Such data of reaching very high gluconic acid concentrations, which are stably obtained for a very long time under continuous cultivation using free growing chemostat cells without any biomass retention, have not been published in the international literature before. The very high product molar and mass selectivities, reaching more than 100% (g/g), show that the new process would be a favourable alternative for the industrial production of gluconic acid compared with the discontinuous fungi processes of the last 100 years. Even 504 g/l of gluconic acid were achieved in fed-batch fermentations using A. pullulans isolate 70 (Anastassiadis et al. 1999; Anastassiadis et al. 2003; Anastassiadis et al. 2005). Interesting future research works would include the investigation of continuous gluconic acid production, using biomass retention by cross over filtration, at different residence times and at varying the proportion ratio between filtrate and bleed stream. Mathematical models would enable the prediction of experimental results at various stream ratios and facilitate future research designs and experimental plans.
We thank Prof. Dr. Christian Wandrey (Institute of Biotechnology 2 of Research Center Jülich, RCJ,
The experiments of the present manuscript comply with the currant laws of the country
ANASTASSIADIS, Savas. Determination of organic acids, especially citricacid and isocitric acid, in fermentation solutions and fruit juices. In: HPLCApplications, MACHEREY-NAGEL GmbH & Co. KG (
ANASTASSIADIS, Savas, AIVASIDIS, Alexander and WANDREY, Christian. Process for the production of gluconic acid with a strain of Aureobasidium pullulans (De Bary) Arnaud. US Patent No. 5,962,286, October 5, 1999.
ANASTASSIADIS, Savas; AIVASIDIS, Alexander and WANDREY, Christian. Citric acid production by Candida strains under intracellular nitrogen limitation. Applied Microbiology Biotechnology, October 2002, vol. 60, no. 1-2, p. 81-87. [CrossRef]
ANASTASSIADIS, Savas; AIVASIDIS, Alexander; WANDREY, Christian and REHM, Hans-Jürgen. Process optimization of continuous gluconic acid fermentation by isolated yeast-like strains of Aureobasidium pullulans. Biotechnology and Bioengineering, August 2005, vol. 91, no. 4, p. 494-501. [CrossRef]
BANG, W.; LU, X.; DUQUENNE, A.M.; NIKOV,
BAO, Jie; FURUMOTO, Keiji; FUKUNAGA, Kimitoshi and NAKAO, Katsumi. A kinetic study on air oxidation of glucose catalyzed by immobilized glucose oxidase for production of calcium gluconate. Biochemical Engineering Journal, September 2001, vol. 8, no. 2, p. 91-102. [CrossRef]
BASSEGUY, R.; DELECOULS-SERVAT, K. and BERGEL, A. Glucose oxidase catalysed oxidation of glucose in a dialysis membrane electrochemical reactor (D-MER). Bioprocess and Biosystems Engineering, April 2004, vol. 26, no. 3, p. 165-168. [CrossRef]
BLANDINO, A.; MACIAS, M. and CANTERO, D. Immobilization of glucose oxidase within calcium alginate gel capsules. Process Biochemistry, February 2001, vol. 36, no. 7, p. 601-606. [CrossRef]
CHEEMA, Jitender Jit Singh; SANKPAL, Narendra V.; TAMBE, Sanjeev S. and KULKARNI, Bhaskar D. Genetic programming assisted stochastic optimization strategies for optimization of glucose to gluconic acid fermentation. Biotechnology Progress, December 2002, vol. 18, no. 6, p. 1356-1365. [CrossRef]
ELFARI, Mustafa; HA, Seung-Wook; BREMUS, Christoph; MERFORT, Marcel; KHODAVERDI, Viola; HERRMANN, Ute; SAHM, Hermann; GORISCH, Helmut. A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-D-gluconic acid. Applied Microbiology and Biotechnology, March 2005, vol. 66, no. 6, p. 668-674. [CrossRef]
FENICE, Massimiliano; SELBMAN, Laura; FEDERICI, Federico and VASSILEV, Nikolay. Application of encapsulated Penicillium variabile P16 in solubilization of rock phosphate. Bioresource Technology, June 2000, vol. 73, no. 2, p. 157-162. [CrossRef]
FERRAZ, Helen C.; BORGES, Cristiano P. and ALVES, Tito Lívio M. Sorbitol and gluconic acid production using permeabilized zymomonas mobilis cells confined by hollow-fiber membranes. Applied Biochemistry and Biotechnology, 2000, vol. 89, no. 1, p. 43-54.
GASTROCK, E.A.; FORGES, N.; WELLS, P.A. and MOYER, A.J. Gluconic acid production on pilot-plant scale, effect of variables on production by submerged mold growths. Industrial & Engineering Chemistry, 1938, vol. 30, p. 782-789.
GODJEVARGOVA, Tzonka; DAYAL, Rajeshwar and TURMANOVA, Sevdalina. Gluconic acid production in bioreactor with immobilized glucose oxidase plus catalase on polymer membrane adjacent to anion-exchange membrane. Macromolecular Bioscience, October 2004, vol. 4, no. 10, p. 950-956. [CrossRef]
GOLLHOFER, Dorothee; NIDETZKY, Bernd; FUERLINGER, Monika and KULBE, Klaus D. Efficient protection of glucose-fructose oxidoreductase from Zymomonas mobilis against irreversible inactivation during its catalytic action. Enzyme and Microbial Technology, March 1995, vol. 17, no. 3, p. 235-240. [CrossRef]
HALSALL-WHITNEY, H.; TAYLOR, D. and THIBAULT, J. Multicriteria optimization of gluconic acid production using net flow. Bioprocess and Biosystems Engineering, March 2003, vol. 25, no. 5, p. 299-307. [CrossRef]
HARTMEIER, W. and DÖPPNER, Theresia. Preparation and properties of mycelium bound glucose oxidase co-immobilized with excess catalase. Biotechnology Letters, November 1983, vol. 5, no. 11, p. 743-748. [CrossRef]
HERRICK, Horace T. and MAY, Orville E. The production of gluconic acid by the Penicillium luteum-purpurogenum Group II. Some optimal conditions for acid formation. The Journal of Biological Chemistry, April 1928, vol. 77, no. 1, p. 185-195.
KARA, A. and BOZDEMIR, T.O. Optimization of the growth parameters of Aspergillus foetidus. Acta Biotechnologica, 1998, vol. 18, no. 4, p. 327-338. [CrossRef]
KLEIN, Jaroslav; ROSENBERG, Michael; MARKO, Jozef; DOLGO, Ondrej; KROSLAK, Marek and KRISTOFIKOVA, L'udmila. Biotransformation of glucose to gluconic acid by Aspergillus niger-study of mass transfer in an airlift bioreactor. Biochemical Engineering Journal, April 2002, vol. 10, no. 3, p. 197-205. [CrossRef]
H.W.; SATO, S.; MUKATAKA, S. and TAKAHASHI, J. Studies on production
of gluconic acid by Aspergillus
MUKHOPADHYAY, R.; CHATTERJEE, S.; CHATTERJEE, B.P.; BANERJEE, P.C. and GUHA, A.K. Production of gluconic acid from whey by free and immobilized Aspergillus
NAKAO, Katsumi; KIEFNER, Andreas; FURUMOTO, Keiji and HARADA, Tsuyoshi. Production of gluconic acid with immobilized glucose oxidase in airlift reactors. Chemical Engineering Science, November 1997, vol. 52, no. 21-22, p. 4127-4133. [CrossRef]
PARK, Eun-Ha; SHIN, Young-Mi; LIM, Young-Yi; KWON, Tae-Ho; KIM, Dae-Hyuk and YANG, Moon-Sik. Expression of glucose oxidase by using recombinant yeast. Journal of Biotechnology, July 2000, vol. 81, no. 1, p. 35-44. [CrossRef]
PRONK, J.T.; LEVERING, P.R.; OLIJVE, W. and VAN DIJKEN, J.P. Role of NADP-dependent and quinoprotein glucose dehydrogenases in gluconic acid production by Gluconobacter oxydans. Enzyme and Microbial Technology, March 1989, vol. 11, no. 3, p. 160-164. [CrossRef]
REHR, Bert; WILHELM, Cornelia and SAHM, Hermann. Production of sorbitol and gluconic acid by permeabilized cells of Zymomonas mobilis. Applied Microbiology and Biotechnology, May 1991, vol. 35, no. 2, p. 144-148. [CrossRef]
RODRIGUEZ, Hilda; GONZALEZ, Tania; GOIRE, Isabel and BASHAN, Yoav. Gluconic acid production and phosphate solubilization by the plant growth-promoting bacterium Azospirillum spp. Naturwissenschaften, November 2004, vol. 91, no. 11, p. 552-555. [CrossRef]
ROUKAS, T. and HARVEY, L. The effect of pH on production of citric and gluconic acid from beet molasses using continuous culture. Biotechnology Letters, April 1988, vol. 10, no. 4, p. 289-294. [CrossRef]
SAKURAI, Hiroshi; LEE, Hang Woo; SATO, Seigo; MUKATAKA, Sukekuni and TAKAHASHI, Joji. Gluconic acid production at high concentrations by Aspergillus
SANKPAL, N.V.; JOSHI, A.P.; SUTAR, I.I. and KULKARNI, B.D. Continuous production of gluconic acid by Aspergillus
SANKPAL, N.V. and KULKARNI, B.D. Optimization of fermentation conditions for gluconic acid production using Aspergillus
SINGH, N.B. Effect of gluconates on the hydration of cement. Cement and Concrete Research, July 1976, vol. 6, no. 4, p. 455-460. [CrossRef]
SZAJANI, B.; MOLNAR, A.; KLAMAR, B. and KALMAN, M. Preparation, characterization, and potential application of an immobilized glucose oxidase. Applied Biochemistry and Biotechnology, 1987, vol. 14, p. 37-47.
TAKAO, S. and SASAKI, Y. Gluconic acid fermentation by Pullularia pullulans Part I. Screening of gluconic acid-producing strains and some conditions for its production. Agricultural and Biological Chemistry, 1964, vol. 28, no. 11, p. 752-756
VASSILEV, Nikolay; VASSILEVA, Maria; FENICE, Massimiliano and FEDERICI, Federico. Immobilized cell technology applied in solubilization of insoluble inorganic (rock) phosphates and P plant acquisition. Bioresource Technology, September 2001, vol. 79, no. 3, p. 263-271. [CrossRef]
VELIZAROV, S. and BESCHKOV, V. Biotransformation of glucose to free gluconic acid by Gluconobacter oxydans: substrate and product inhibition situations. Process Biochemistry, June 1998, vol. 33, no. 5, p. 527-534. [CrossRef]
ZNAD, H.; MARKOS, J. and BALES, V. Production of gluconic acid from glucose by Aspergillus
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