Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.9 No.5, Issue of October 15, 2006 © 2006 by Pontificia Universidad Católica de Valparaíso -- Chile Received June 6, 2005 / Accepted November 21, 2005 DOI: 10.2225/vol9-issue5-fulltext-18 RESEARCH ARTICLE

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

Savas Anastassiadis* Pythia Institute of Biotechnology of Research in Biotechnology Co., Vat. #: 108851559 Avgi/Sohos, 57002 Thessaloniki, Greece Tel. 30 2395 051324 Fax. 30 2395 051470 E-mail: [email protected]

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

Website: http://www.greekbiotechnologycenter.gr

Financial support: The work has been carried out at the Institute of Biotechnology 2 of Research Center Jülich (formerly known as Nuclear Research Center Jülich, Germany) and was financed by Haarmann and Reimer, a daughter company of the company Bayer, Leverkusen, Germany.

Keywords: biomass immobilization, continuous fermentation, cross over filtration, gluconic acid fermentation, reaction technique, residence time.

Abbreviations: Conversion (%): [(g consumed glucose/g feeding glucose) x 100], dilution of medium glucose by NaOH feeding was considered in the calculations mp: Specific gluconic acid productivity, [g gluconic acid/(g biomass x h)], Rj [g/(l x h)]/biomass concentration (g/l), g/(g x h) Rj: Formation rate of the generic product (volumetric productivity), g gluconic acid/(l x h), gluconic acid concentration (g/l)/RT (h), g/(l x h) Rs: Glucose consumption rate, g/(l x h), [(g feeding glucose-g consumed glucose)/RT (h)], g/(l x h) RT: Residence time - hours, [Bioreactor volume (ml)/(medium feeding rate (ml/h) + NaOH feeding rate (ml/h))], h Selectivity (%): [(g gluconic acid/g consumed glucose) x 100] Yield (%): [(g gluconic acid/g feeding glucose) x 100]

New alternative processes for the continuous production 22 hrs and of 77% at 19 hrs RT, because of the very of gluconic acid by Aureobasidium pullulans, using high biomass concentration. Biomass retention makes it biomass retention by cell immobilization or cross over possible to break the existing link between growth and filtration, are described in the present work. 315 g/l residence time. gluconic acid was continuously produced in chemostat cultures at 21 hrs residence time without any biomass As a multifunctional carbonic acid, belonging to the bulk retention. 260 g/l gluconic acid was produced in chemicals and due to its physiological and chemical fluidized bed reactor at 21 hrs residence time. The characteristics, gluconic acid itself, the gluconolatone support carrier was overgrown resulting in limitations form and its salts (e.g. alkali metal salts, in especially of oxygen transfer towards the inner layers of sodium gluconate) have found extensively versatile uses in immobilized biomass. 375 g/l gluconic acid was the chemical, pharmaceutical (e.g. iron and calcium produced under continuous cultivation at 22 hrs of deficiency), food, beverage, textile and other industries residence time with a formation rate for the generic (Hustede et al. 1989; Anastassiadis et al. 2003; Znad et al. product of 17 g/(l x h) and a specific gluconic acid 2004). Additionally, it can be exploited for cleaning productivity of only 0.74 g/(g x h), using biomass purposes (e.g. diary industry) as well as for the extraction retention by cross over filtration. 370 g/l were obtained of trace elements like calcium, copper and iron. Gluconic at 19 hrs RT and 100% conversion with 25 g/l biomass acid can have further applications for the solubilization of and a formation rate of 19 g/(l x h). At 100% phosphate (Fenice et al. 2000; Vassilev et al. 2001; conversion, a selectivity of only 78% was determined at Rodríguez et al. 2004) and as a cement additive in the

*Corresponding author

This paper is available on line at http://www.ejbiotechnology.info/content/vol9/issue5/full/18/ Anastassiadis, S. et al.

immobilized cells of A. niger on a highly porous cellulose Table 1. Results of the continuous gluconic acid support. fermentation with residence times of 21 and 25 hrs (3 g/l NH4Cl, 450 g/l glucose, 1 mM iron, 5 mM manganese, pH 6.5, Biomass retention by immobilization on porous sinter glass 30ºC and 155% oxygen saturation). 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 Residence time hr 21 25 maximization of product concentration by an isolated Aureobasidium pullulans strain, as a comparison with the Biomass g/l 6.8 6.8 continuous cultivation of free growing cells.

Optical density 660 nm 37 37 MATERIALS AND METHODS

Glucose g/l 80 70 Microorganism

Gluconic acid g/l 315 330 Aureobasidium pullulans (de Bary) Arnaud isolate Nr. 70 (DSM 7085), which was isolated from wild flowers (Jülich, Rj g/(l x h) 15 13.3 Germany) was used during the entire work (Anastassiadis et al. 1999; Anastassiadis et al. 2003; Anastassiadis et al. mp g/(g x h) 2.2 1.9 2005). Yeast malt extract agar plates (YME), inoculated with Aureobasidium pullulans, were incubated for 2-3 days Conversion % 81 82.5 and stored at 4ºC. The inoculum (10%) was prepared by Yield % 71 76 transferring of cells from agar plates into 500 ml shake flasks with buffels on a medium containing (g/l): Glucose Selectivity % 92 92.1 30 g/l, NH4Cl 3 g/l, KH2PO4 1.4 g/l, MgSO4 x 7 H2O 0.35 g/l, MnSO4 x 4 H2O 5 mM, FeSO4 x 7 H2O 1 mM, CuSO4 x 5 H2O 4 µM (1 mg/l), ZnSO4 x 7 H2O 0.01 g/l, CoSO4 x 7 H2O 4 mg/l, H3BO3 0.04 g/l, CaCl2 0.1 g/l, NaCl 0.1 g/l, construction industry, because it enhances the cement's citric acid 2.5 g/l, Na2MoO4 x 2 H2O 0.2 mg/l, thiamine- resistance and stability under extreme climatic conditions, HCl 2 mg/l, biotin 0.25 g/l, pyridoxine-HCl 0.625 mg/l, Ca- e.g. frost and water (Singh, 1976; Hustede et al. 1989). D-pantothenate 0.625 mg/l, nicotinic acid 0.5 mg/l.

Numerous methods have been extensively described for the Culture conditions production of gluconic acid, including chemical and electrochemical catalysis, enzymatic biocatalysis in enzyme For the investigation of continuous gluconic acid bioreactor, microbial production using free growing or fermentation with or without biomass retention, cells were immobilized cells of either Gluconobacter oxydans or grown in a 5 litre fermenter (Biostat E, Braun-Diessel) at a Aspergillus niger. The immobilization of whole cells or working volume of 3 l, 1000 rpm, pH 6.5 and 30ºC in glucose oxidase enzyme by various techniques has often chemostat mode on a basal defined medium containing been reported to be a useful approach to the production of (g/l): varying glucose concentration (s. results), NH4Cl 3 gluconic acid or other microbial metabolites (Hartmeier and g/l, KH2PO4 1.4 g/l, MgSO4 x 7 H2O 0.35 g/l, MnSO4 x 4 Döppner, 1983; Pronk et al. 1989; Sakurai et al. 1989; H2O 5 mM, FeSO4 x 7 H2O 1 mM, CuSO4 x 5 H2O 4 µM (1 Gromada and Fiedurek, 1997; Nakao et al. 1997; Kara and mg/l), ZnSO4 x 7 H2O 0.01 g/l, CoSO4 x 7 H2O 4 mg/l, Bozdemir, 1998; Velizarov and Beschkov, 1998; H3BO3 0.04 g/l, CaCl2 0.1 g/l, NaCl 0.1 g/l, citric acid 2.5 Anastassiadis et al. 1999; Sankpal et al. 1999; Bang et al. g/l, Na2MoO4 x 2H2O 0.2 mg/l, thiamine-HCl 2 mg/l, biotin 1999; Ferraz et al. 2000; Bao et al. 2001; Blandino et al. 0.25 g/l, pyridoxine-HCl 0.625 mg/l, Ca-D-pantothenate 2001; Klein et al. 2002; Sankpal and Kulkarni, 2002; 0.625 mg/l, nicotinic acid 0.5 mg/l [24,25,2]. Vitamins and Anastassiadis et al. 2003; Godjevargova et al. 2004; NH4Cl were added separately to autoclaved medium (30-60 Anastassiadis et al. 2005; Mukhopadhyay et al. 2005). min at 121ºC) by sterile filtration (Sartorius filter, Fluidized bed reactors have been often used for several Göttingen, Germany). The fermentations were carried out fermentation applications, in especially for anaerobic at 30ºC and pH 6.5 automatically adding a 45% NaOH processes. The separation of the biocatalyst is practically solution. connected with high expenses, substantially influencing the economics of a fermentation process. Cell immobilization would therefore be an economically efficient alternative option for the production of gluconic acid under either Fermentation equipment batch or continuous cultivation. Sankpal and Kulkarni (2002) described gluconic acid fermentation by The fermentations were carried out in agitation 5 litre fermenters (Figure 1) (Biostat M, Diessel-Braun,

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

Figure 1. Flow scheme of a stirrer fermenter with intergraded cross over filtration.

Germany). From the concept, the fermenters were laid out Biomass retention by cross over filtration for the continuous mode of operation. The fermenters worked according to the principle of chemostat, i.e. by The experiments for the continuous production of gluconic delimitation of a growth factor and at a constant supply of acid by biomass retention by means of cross over filtration nutritive solution into fermenter a stationary condition were carried out in a 5 litre stirrer fermenter. For the cross (steady state) can be adjusted. The temperature was over filtration, two parallel connected autoclavable controlled using laboratory thermostats Lauda M3 microfiltration tubing modules (cross over filtration) were (Meßgerätewerk Lauda, D6970-Lauda-Königshofen). The attached to the vessel (Figure 1). Fermenter, filters, medium air saturation (%) was measured as has been reported in and NaOH solution were externally sterilized in a large Anastassiadis et al. (2005). A sufficient oxygen supply was autoclave. In case of clogging appearances, the modules ensured adding pure oxygen over two parallel attached were exchanged or were cleaned by turning periodically the sterile filters (T-fitting) into the supply line, in order to pumping direction backwards. The goal was implemented maintain air saturations higher than 100% in fermenter by taking off a part of the fermentation broth as filtrate and (Anastassiadis et al. 2005). The oxygen was distributed by thus it is increased the stationary biomass concentration in means of a sinter glass frit in the culture liquid. The exhaust bioreactor. At the same time, the discharge remainder gas stream left likewise over two parallel sterile filters the (Bleed), containing fermentation broth and biomass, was

fermenter. A condenser prevented the discharge of water taken out of the fermenter by means of a second peristaltic pump. Both pumps were simultaneously controlled by a over the exhaust upstream of air. The experimental setup of level control system. By this way, it became feasible to such a fermenter is schematically represented in Figure 1. adjust a constant relationship between both types of The fermentation medium was continuously added into the discharges. The level control probe (Weathston's bridge) was calibrated, enabling the proportional adjustment of fermenter using a precision gravimetric dosing system desired level of filling under constant fermentation (Sartorius, Göttingen, Germany) and a peristalticpump conditions. For safety reasons, the substrate pump was also (Watson Marlow). Periodically, an antifoaming agent was interconnected to this control system and at reaching a pumped to the fermenter using a time switch clock (15 sec certain (adjustable) level of bioreactor broth the pump was performance every 1.5 hrs). The residence time was shut down automatically. The fermentation was performed determined based on the fermenter working volume via the at 30ºC, pH 6.5 and 290% air saturation. total medium and NaOH flux. More than five residence times were required for achieving steady state conditions Biomass retention by biomass immobilization and the cultivation in each mode was continued until the culture medium in fermenter was replaced at least five The experiments with biomass immobilization were carried times. out in a fluidized bed reactor at a working volume of about 0.9 litre (Figure 2). For the immobilization of biomass, about 350 g of porous sinter glass beads (SIRANR) (Schott AG, Mainz, Germany) was added in fluidized bed reactor,

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resulting in the ideal case to a total working volume of 0.9 et al. 1999; Anastassiadis et al. 2003; Anastassiadis et al. litre. Sinter glass balls with a three-dimensional cavity 2005). A defined fermentation medium was composed and structure and the optimized, which makes it easy to reproduce those data. Completing the process optimization program, reaction- following characteristics were used: ball form with 1-2 mm technical investigations were accomplished, in order to diameter, 60% porosity, distribution of pore sizes of 60-300 examine the transferability of obtained results and to test µm, from borosilicate. The carrier drawn away from different fermentation process variants (e.g. batches, fed fermenter was collected in a cyclone (precipitation vessel) batches, continuous fermentation without and with biomass in order to avoid clogging of tubing lines. The oxygen retention) for the production of gluconic acid by electrode was placed in an external glass vessel and the pH Aureobasidium pullulans isolate 70 (DSM 7085) electrode was placed in a conical glass vessel, which was (Anastassiadis et al. 1999). located between the large circulation pump and the fluidized bed fermenter, in order to avoid fouling by Continuous production of gluconic acid by free biofilm formation. Furthermore, the pH was periodically growing cells in stirrer fermenter checked using an external pH meter and readjusted. The reactor was securely placed and stabilized on a stand. An The continuous gluconic fermentation by free growing cells automatic dosing system (Sartorius, Germany) was used for of A. pullulans and without any biomass retention was the constant feeding rate of fermentation medium (Figure carried out in a 5 litre fermenter under optimized conditions 2). Because of the very high relationship between substrate (pH 6.5, 30ºC, air saturation 120-180%), using an flow rate and recirculation flow rate, the fluidized bed extensively optimized defined medium (Anastassiadis et al. reactor can be considered as a regular stirrer fermenter. 1999). More than 220 g/l were produced under continuous steady state cultivation in previous chemostat studies Analysis (Anastassiadis et al. 2005). With 450 g/l glucose in feeding medium and at 155% air saturation, 315 g/l gluconic acid Optical density (OD660 nm), dry biomass (filter method) and were continuously produced under steady state conditions the concentration of glucose and gluconic acid were at 21 hrs residence time with 6.8 g/l biomass and a determined as has described in previous works formation rate for the generic product of 15 g/(l x h). Figure (Anastassiadis, 1993; Anastassiadis et al. 1999; 3 shows the continuous production of gluconic acid at 25 Anastassiadis et al. 2003; Anastassiadis et al. 2005). hrs residence time, reaching steady state conditions about 170 hrs after the inoculation of fermenter. Table 1 Ammonium analysis illustrates the results that have been achieved at residence times of 21 and 25 hrs. 330 g/l were continuously produced Ammonium nitrogen was analyzed as has been described in at a conversion of 82.5% and 92% selectivity with a Anastassiadis et al. (2002). formation rate for the generic product of 13.2 g/(l x h) and a biomass of 6.8 g/l, corresponding to a specific productivity RESULTS of 1.94 g/(g x h). For comparison, a maximum specific Novel superior processes were developed and optimized for productivity of about 9.3 g/(g x h) and formation rate of the continuous and discontinuous production of gluconic about 18.6 g/(l x h) were reached in a chemostat under acid by isolated strains of Aureobasidium pullulans during optimized conditions without any biomass retention at very an extensive process development program (Anastassiadis short times of about 11.8 hrs (data not shown here). The fermentation continued to stably run without any technical Table 2. Continuous gluconic acid fermentation with and microbial stability problems for a very long period of biomass retention by means of cross over filtration. time. A continuous gluconic acid fermentation using a new strain of A. pullulans, which was isolated at the Research in Biotechnology Co. (Greece) was running for about 1 year Residence time hr 22 19 without any microbial stability problems, showing again the stability of Aureobasidium process. Biomass (g/l) 23 25 Continuous gluconic acid production by Gluconic acid (g/l) 375 370 immobilized cells

Rj (g/l x h) 17 19.5 The continuous production of gluconic acid by cell immobilization on porous sinter glass (Schott, Germany) mp (g/g x h) 0.74 0.78 was studied in a fluidized bed reactor under optimum fermentation condition, using an optimized defined medium Conversion (%) 100 100 with 450 g/l glucose. The fermenter was initially inoculated Selectivity (%) 78 77 with a two days culture of Aureobasidium pullulans isolate 70 (DSM 7085). The fermenter performed under batch conditions at the beginning of fermentation and the

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

Figure 2. Flow scheme of a magnetic stirrer fermenter with intergraded fluidized bed reactor.

continuous feeding of medium started after the biomass constant during the entire duration of the experiment. reached a certain concentration of more than 3 g/l, in order Applying a glucose concentration of 450 g/l, 375 g/l to increase the load of biomass on the porous glass beads. gluconic acid was continuously produced at a residence The glass beads were overgrown from cells of A. Pullulans. time of about 22 hrs, 100% conversion of glucose, 30ºC, 260 g/l gluconic acid could be continuously obtained in pH 6.5 and 290% air saturation by 23 g/l of accumulated fluidized bed reactor under steady state conditions at a biomass. 370 g/l of gluconic acid were also produced at 19 retention time of 21 hrs with a formation rate for the hrs and complete glucose conversion by a biomass of 25 g/l generic product gluconic acid of 12.4 g/(l x h). The (Table 1, Figure 4). adhesive behaviour of A. pullulans resulted in diffusion problems for the oxygen transfer and thus in oxygen In previous studies, it has been extensively studied the limitation of the immobilized biomass. influence of residence time on continuous production without biomass retention (Anastassiadis et al. 2003; Continuous gluconic acid fermentation with Anastassiadis et al. 2005). In the present work, the biomass retention by means of microfiltration maximization of gluconic acid concentration has been achieved by applying biomass retention in bioreactor by A good alternative approach for the acceleration of product means of cross over filtration. According to present results, concentration maximization is the partial retention of it seems very possible to still convert glucose completely at biomass by means of microfiltration (cross over filtration). even very short residence times i.e. less than 7 hrs. The Biomass retention enables the breaking of the link between investigation of the performance of continuous gluconic growth (biomass formation) and residence time and acid production by biomass retention at very low residence between growth and production. It makes feasible the times was not possible because of technical inadequacies. performance of the process at very high oxygen concentrations, which are toxic for cell growth, resulting in DISCUSSION higher biomass specific productivities. It should also be examined, whether very high gluconic acid concentrations Microbial production of metabolites of primary and can be reached at shortened retention times, using very high intermediary metabolism usually takes place under stress oxygen concentrations. In order to receive comparable data, conditions. The medium composition plays a crucial role the experiments were accomplished under the same for a successful metabolite production. Fermentation conditions as the preceding continuous fermentations parameters such as pH, oxygen, temperature and medium without biomass retention. About 80% of filtrate (filtered composition, influencing continuous and discontinuous fermentation solution) and 20% bleed (unfiltered gluconic acid production by isolated strains of A. pullulans, fermentation solution) were adjusted and maintained have been identified and optimized in previous chemostat studies (Anastassiadis et al. 1999; Anastassiadis et al. 2003;

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Anastassiadis et al. 2005). In a conventional chemostat over filtration enables the application of production optima culture without any biomass retention, where growth and in a continuous fermentation process even at very short production occur simultaneously, the wash out effect of times that would normally wash out the biomass from the biomass is the limiting factor in terms of achieving very fermenter. Thus, the maximum specific productivity of high product concentrations at very low residence times generic product can be maintained, uncoupling product (high dilution rates). The highest formation rate and formation from growth rate, although it differs from the specific productivity for the generic product gluconic acid optimum growth conditions. Biomass retention by means of have been achieved at lower residence times. They decrease cross over filtration makes it possible to break the existing continuously at increasing residence time and very high link between growth and the residence time. A. pullulans gluconic acid concentrations of more than 230 g/l was easily immobilized on porous sintered glass and (Anastassiadis et al. 1999; Anastassiadis et al. 2003). The suitable for a continuous gluconic acid production by formation rate of the generic product (Rj) is a compensation immobilized cells. A good alternative for the acceleration effect between biomass concentration and biomass specific of product concentration maximization offers the partial production rate. In continuous process, growth and biomass retention by means of microfiltration (cross over production run parallel, influencing each other. For filtration). Continuous fermentation can thus perform at example, different optimum dissolved oxygen optimum production parameters, which would not concentrations have been determined for growth, gluconic necessarily be optimal for the formation of biomass, thus acid production and for various specific fermentation breaking partially the interaction between growth and factors such as conversion, formation rate for the generic production. A very high formation rate and concentration of product, specific gluconic acid productivity, selectivity and generic product can be obtained at simultaneously keeping yield. Hence, the optimum fermentation parameters that very high specific productivities. Although continuous had been found for example for the pH, temperature and fermentation performing in a fluidized bed reactor with oxygen saturation are the compensation result between immobilized biomass on a support carrier may be more growth and production. convenient from a process point of view, fermentation with non-supported microorganisms performing without or with A gluconic acid concentration higher than 230 g/l was biomass retention by cross over filtration has been found to continuously produced at residence times of 12 hrs without be advantageously. In both cases, very high dissolved any biomass retention (Anastassiadis et al. 2005). New oxygen concentrations higher than 200% air saturation are alternative processes for the continuous production of very applied successfully. high gluconic acid concentrations using free growing cells or biomass retention of A. pullulans are described in the Early fungal fermentation processes for gluconic acid present work. Biomass retention by immobilization or cross production employed species of Penicillium (Herrick and

Figure 3. Continuous gluconic acid fermentation by free growing cells of A. pullulans at a residence time of 25 hrs (3 g/l NH4Cl, 450 g/l glucose, 1 mM iron, 5 mM manganese, pH 6.5, 30ºC and 155% oxygen saturation).

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

Figure 4. Continuous production of gluconic acid by cross over filtration.

May, 1928). Improved strains of Aspergillus niger continuous gluconic acid production by immobilized (predominantly) with recycled mycelia or Gluconobacter mycelia of A. niger achieving 120-140 g/l gluconic acid. suboxidans are used in discontinuous submerged The repeated reuse of cell mycelia has been reported as an fermentations in gluconic acid industry (Röhr et al. 1983). option of semi-continuous production of gluconic acid by Gluconic acid formation carried out in shake flasks by the A. niger (Hatcher, 1972). Alternatively, high gluconate yeast-like mold Aureobasidium (former Dematium or concentrations and very high productivities were achieved Pullularia) pullulans, using various carbon sources, has by the fed-batch-wise addition of large amounts of glucose been known since very long time (Pervozvansky, 1939; into fermenter using A. niger. However, the productivity Takao and Sasaki, 1964; Sasaki and Takao, 1970). became unattainable after a few repetitions, whereas 220 g/l However, gluconic acid production by A. pullulans on an gluconate were repeatedly produced 14 times, using industrial scale, which is well characterized for the immobilized mycelia on a non-woven fabric within a period production of pullulan, stayed out of question and of about 1,000 hrs (Sakurai et al. 1989). A product investigation. inhibition has been observed in A. pullulans process at gluconic acid concentrations above 250 g/l in good To our knowledge, no comparable results reaching those agreement to reports of Gastrock et al. (1938), Lee et al. high product concentrations (Anastassiadis et al. 1999; (1987) and Sakurai et al. (1989) for A. niger (300 g/l). Anastassiadis et al. 2003; Anastassiadis et al. 2005; present work) can be found in the international literature, referring Knowingly, A. niger is difficult to handle because of to the continuous gluconic acid production by free growing causing clogging problems during the fermentation and or immobilized cells of Aspergillus and Penicillium strains unsuitable for a continuous operation using free growing or other microorganisms, without any biomass retention. cells. Gluconobacter has been reported to produce a 260 g/l of gluconic acid were continuously produced relative large quantity of keto-acids during the production without any biomass retention under optimized conditions of gluconic acid (Elfari et al. 2005), thus complicating by A. pullulans at 15.3 hrs RT and 305-315 g/l at about 21 down streaming processing and the isolation of gluconic hrs. 370 g/l of gluconic acid were also continuously acid. Velizarov and Beschkov (1998) reported about a rapid produced at complete glucose conversion at 19 hrs decrease of conversion in Gluconobacter oxydans at residence time and up to 504 g/l were achieved in fed batch glucose concentrations higher than 0.56 M. Increasing experiments (Anastassiadis et al. 1999; Anastassiadis et al. additions of gluconic acid to the medium resulted in 2003; present work). Sankpal et al. (1999) reported about extended lag phases of growth and the maximum possible

500 Anastassiadis, S. et al.

gluconic acid concentration has been approximated at 0.7 competitiveness in industry, claiming numerous advantages M (Velizarov and Beschkov, 1998). For comparison, the over the traditional fungi or bacteria processes of the last continuous operations using A. pullulans show many 100 years. A continuous industrial production of the advantages compared with the traditional discontinuous responsible glucose oxidizing enzyme from A. pullulans fungi and bacterial processes. Under optimized appears feasible as well. fermentation conditions, it proved to be a superior gluconic acid producer, which can be used in continuous culture for The process operation is very stable, under the condition a very long period without any stability problems. The that filter modules are cleaned (periodical reverse flow of yeast like fungus Aureobasidium pullulans integrates the filtrate). A precondition for an unproblematic long time advantages of fungal and bacterial systems at once. It is operation is also keeping the system safe from enabled to utilize astonishingly very high glucose contaminations, because some contaminants can overgrow concentrations in continuous single or two stage mode the production strain. No strain instabilities have been reaching very high gluconic acid concentrations of 200-433 observed so far during the extensive process optimization at residence times between 10 and 38 hrs and a maximum and development program, showing the genetic stability of biomass specific productivity of about 10 g/(g x h) the new yeast-like mold system. (Anastassiadis et al. 1999; Anastassiadis et al. 2003; Anastassiadis et al. 2005). In comparison, Roukas and CONCLUDING REMARKS Harvey (1988) reached 16 g/l gluconic acid by free growing cells of A. niger at 83 hrs residence time. Babel and Müller Such data of reaching very high gluconic acid (1987) obtained 208 g/l of gluconic acid at 40 residence concentrations, which are stably obtained for a very long time using Acetobacter methanolicus, whereas up to 295 g/l time under continuous cultivation using free growing have been continuously produced by permeabilized cells of chemostat cells without any biomass retention, have not Zymomonas mobilis in about 75 hrs (Rehr et al. 1991). In a been published in the international literature before. The continuous process for the simultaneous production of very high product molar and mass selectivities, reaching sorbitol and gluconic acid, the use of cell-free glucose- more than 100% (g/g), show that the new process would be fructose oxidoreductase from Z. mobilis requires the a favourable alternative for the industrial production of efficient stabilization of the enzyme (Gollhofer et al. 1995). gluconic acid compared with the discontinuous fungi processes of the last 100 years. Even 504 g/l of gluconic The use of genetically engineered microorganisms and the acid were achieved in fed-batch fermentations using A. immobilization of isolated glucose oxidizing enzymes or pullulans isolate 70 (Anastassiadis et al. 1999; whole cells in specialized reactors appear in literature Anastassiadis et al. 2003; Anastassiadis et al. 2005). reports as the possible future developments regarding the Interesting future research works would include the advanced continuous production of gluconic acid investigation of continuous gluconic acid production, using (Hartmeier and Döppner, 1983; Milson, 1987; Szajani et al. biomass retention by cross over filtration, at different 1987). Park et al. (2000) cloned and expressed for example residence times and at varying the proportion ratio between glucose oxidase from A. niger in Saccharomyces cerevisiae filtrate and bleed stream. Mathematical models would using a yeast shuttle vector. Specialized reactors (Basseguy enable the prediction of experimental results at various et al. 2004; Godjevargova et al. 2004) and genetic stream ratios and facilitate future research designs and programming, as well as multicriteria optimization experimental plans. strategies (Cheema et al. 2002; Halsall-Whitney et al. 2003) have been applied for the production of gluconic acid as ACKNOWLEDGMENTS well. The fact that the present superior results were reached We thank Prof. Dr. Christian Wandrey (Institute of using a wild strain as it has been isolated from the nature Biotechnology 2 of Research Center Jülich, RCJ, Germany; and without the involvement of any classical or genetic formerly known as Nuclear Research Center Jülich, KFA) engineering mutagenesis approaches clearly emphasize the for his helpful advices and support. nature's great latent and still unknown potential for further future biotechnological achievements. The development of DECLARATION the present superior fermentation processes has accomplished the highest expectations of an expertise The experiments of the present manuscript comply with the researcher in the field of industrial microbiology and currant laws of the country Germany (Institute of biotechnology, extending the frontiers of known microbial Biotechnology 2 of Research Center Jülich 2, RCJ) where capabilities and emphasizing the importance of the deep the experiments were performed. understanding of classical and industrial microbiology in the field of biotechnology science. The novel continuous REFERENCES and discontinuous fermentation processes of Aureobasidium pullulans, applying or not biomass ANASTASSIADIS, Savas. Determination of organic acids, retention, offer new opportunities and are very promising especially citricacid and isocitric acid, in fermentation for future applications for the commercial production of solutions and fruit juices. In: HPLCApplications, gluconic acid. The new processes can last in today's high

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

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