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6, 2005 / Accepted November 21, 2005

This paper is available on line at http://www.ejbiotechnology.info/content/vol9/issue5/full/18/

DOI: 10.2225/vol9-issue5-fulltext-18

RESEARCH ARTICLE

Continuous gluconic acid production by

Aureobasidium pullulans

withand without biomass retention

Savas Anastassiadis*

Pythia Institute of Biotechnology of Research in BiotechnologyCo., Vat. #: 108851559Avgi/Sohos, 57002Thessaloniki, GreeceTel. 30 2395 051324Fax. 30 2395 051470E-mail: sanasta@env.duth.gr

Hans-Jürgen Rehm

Institute of Molecular Microbiology and BiotechnologyUniversity 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 ResearchCenter 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 thecalculationsm

: Specific gluconic acid productivity, [g gluconic acid/(g biomass x h)], Rj [g/(l x h)]/biomass concentration (g/l), g/(g x h)R

Formation rate of the generic product (volumetric productivity), g gluconic acid/(l x h), gluconic acid concentration (g/l)/RT (h), g/(l xh)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))], hSelectivity (%): [(g gluconic acid/g consumed glucose) x 100]Yield (%): [(g gluconic acid/g feeding glucose) x 100]

New alternative processes for the continuous productionof gluconic acid by

Aureobasidium pullulans

, usingbiomass retention by cell immobilization or cross overfiltration, are described in the present work. 315 g/lgluconic acid was continuously produced in chemostatcultures at 21 hrs residence time without any biomassretention. 260 g/l gluconic acid was produced influidized bed reactor at 21 hrs residence time. Thesupport carrier was overgrown resulting in limitationsof oxygen transfer towards the inner layers of immobilized biomass. 375 g/l gluconic acid wasproduced under continuous cultivation at 22 hrs of residence time with a formation rate for the genericproduct of 17 g/(l x h) and a specific gluconic acidproductivity of only 0.74 g/(g x h), using biomassretention by cross over filtration. 370 g/l were obtainedat 19 hrs RT and 100% conversion with 25 g/l biomassand a formation rate of 19 g/(l x h). At 100%conversion, a selectivity of only 78% was determined at

*Corresponding author

22 hrs and of 77% at 19 hrs RT, because of the veryhigh biomass concentration. Biomass retention makes itpossible to break the existing link between growth andresidence time.

As a multifunctional carbonic acid, belonging to the bulk chemicals and due to its physiological and chemicalcharacteristics, gluconic acid itself, the gluconolatoneform and its salts (

e.g.

alkali metal salts, in especiallysodium gluconate) have found extensively versatile uses inthe chemical, pharmaceutical (

e.g.

iron and calciumdeficiency), 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 extractionof trace elements like calcium, copper and iron. Gluconicacid 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

Anastassiadis, S. et al.

495

construction industry, because it enhances the cement'sresistance and stability under extreme climatic conditions,

e.g.

frost and water (Singh, 1976; Hustede et al. 1989). Numerous methods have been extensively described for the production of gluconic acid, including chemical andelectrochemical catalysis, enzymatic biocatalysis in enzyme bioreactor, microbial production using free growing or immobilized cells of either

Gluconobacter

oxydans

Aspergillus niger

. The immobilization of whole cells or glucose oxidase enzyme by various techniques has often been reported to be a useful approach to the production of gluconic acid or other microbial metabolites (Hartmeier andDöppner, 1983; Pronk et al. 1989; Sakurai et al. 1989;Gromada and Fiedurek, 1997; Nakao et al. 1997; Kara andBozdemir, 1998; Velizarov and Beschkov, 1998;Anastassiadis et al. 1999; Sankpal et al. 1999; Bang et al.1999; Ferraz et al. 2000; Bao et al. 2001; Blandino et al.2001; Klein et al. 2002; Sankpal and Kulkarni, 2002;Anastassiadis et al. 2003; Godjevargova et al. 2004;Anastassiadis et al. 2005; Mukhopadhyay et al. 2005).Fluidized bed reactors have been often used for severalfermentation applications, in especially for anaerobic processes. The separation of the biocatalyst is practicallyconnected with high expenses, substantially influencing theeconomics of a fermentation process. Cell immobilizationwould therefore be an economically efficient alternativeoption for the production of gluconic acid under either batch or continuous cultivation. Sankpal and Kulkarni(2002) described gluconic acid fermentation byimmobilized cells of

A. niger

on a highly porous cellulosesupport.Biomass retention by immobilization on porous sinter glassor by cross over filtration was applied in present work inorder to investigate the feasibility of a further accelerationof continuous gluconic acid production and of themaximization of product concentration by an isolated

Aureobasidium pullulans

strain, as a comparison with thecontinuous cultivation of free growing cells.

MATERIALS AND METHODS

Microorganism

Aureobasidium pullulans

(de Bary) Arnaud isolate Nr. 70(DSM 7085), which was isolated from wild flowers (Jülich,Germany) was used during the entire work (Anastassiadiset al. 1999; Anastassiadis et al. 2003; Anastassiadis et al.2005). Yeast malt extract agar plates (YME), inoculatedwith

Aureobasidium pullulans,

were incubated for 2-3 daysand stored at 4ºC. The inoculum (10%) was prepared bytransferring of cells from agar plates into 500 ml shakeflasks with buffels on a medium containing (g/l): Glucose30 g/l, NH

Cl 3 g/l, KH

1.4 g/l, MgSO

x 7 H

O 0.35g/l, MnSO

x 4 H

O 5 mM, FeSO

x 7 H

O 1 mM, CuSO

x5 H

O 4 µM (1 mg/l), ZnSO

x 7 H

O 0.01 g/l, CoSO

x 7H

O 4 mg/l, H

0.04 g/l, CaCl

0.1 g/l, NaCl 0.1 g/l,citric acid 2.5 g/l, Na

MoO

x 2 H

O 0.2 mg/l, thiamine-HCl 2 mg/l, biotin 0.25 g/l, pyridoxine-HCl 0.625 mg/l, Ca-D-pantothenate 0.625 mg/l, nicotinic acid 0.5 mg/l.

Culture conditions

For the investigation of continuous gluconic acidfermentation with or without biomass retention, cells weregrown in a 5 litre fermenter (Biostat E, Braun-Diessel) at aworking volume of 3 l, 1000 rpm, pH 6.5 and 30ºC inchemostat mode on a basal defined medium containing(g/l): varying glucose concentration (s. results), NH

Cl 3g/l, KH

1.4 g/l, MgSO

x 7 H

O 0.35 g/l, MnSO

x 4H

O 5 mM, FeSO

x 7 H

O 1 mM, CuSO

x 5 H

O 4 µM (1mg/l), ZnSO

x 7 H

O 0.01 g/l, CoSO

x 7 H

O 4 mg/l,H

0.04 g/l, CaCl

0.1 g/l, NaCl 0.1 g/l, citric acid 2.5g/l, Na

MoO

x 2H

O 0.2 mg/l, thiamine-HCl 2 mg/l, biotin0.25 g/l, pyridoxine-HCl 0.625 mg/l, Ca-D-pantothenate0.625 mg/l, nicotinic acid 0.5 mg/l [24,25,2]. Vitamins and NH

Cl were added separately to autoclaved medium (30-60min at 121ºC) by sterile filtration (Sartorius filter,Göttingen, Germany). The fermentations were carried outat 30ºC and pH 6.5 automatically adding a 45% NaOHsolution.

Fermentation equipment

The fermentations were carried out in agitation 5 litrefermenters (Figure 1) (Biostat M, Diessel-Braun,

Table 1. Results of the continuous gluconic acidfermentation with residence times of 21 and 25 hrs (3 g/lNH

Cl, 450 g/l glucose, 1 mM iron, 5 mM manganese, pH 6.5,30ºC and 155% oxygen saturation).

Residence time

Biomass

g/l

6.8

Optical density 660 nm

Glucose

g/l

Gluconic acid

g/l

315

330

g/(l x h)

13.3

g/(g x h)

2.2

1.9

Conversion

82.5

Yield

Selectivity

92.1

Continuous gluconic acid production by

Aureobasidium pullulans

with and without biomass retention

496

Germany). From the concept, the fermenters were laid outfor the continuous mode of operation. The fermentersworked according to the principle of chemostat,

i.e.

bydelimitation of a growth factor and at a constant supply of nutritive solution into fermenter a stationary condition(steady state) can be adjusted. The temperature wascontrolled using laboratory thermostats Lauda M3(Meßgerätewerk Lauda, D6970-Lauda-Königshofen). Theair saturation (%) was measured as has been reported inAnastassiadis et al. (2005). A sufficient oxygen supply wasensured adding pure oxygen over two parallel attachedsterile filters (T-fitting) into the supply line, in order tomaintain air saturations higher than 100% in fermenter (Anastassiadis et al. 2005). The oxygen was distributed bymeans of a sinter glass frit in the culture liquid. The exhaustgas stream left likewise over two parallel sterile filters thefermenter. A condenser prevented the discharge of water over the exhaust upstream of air. The experimental setup of such a fermenter is schematically represented in Figure 1.

The fermentation medium was continuously added into thefermenter using a precision gravimetric dosing system(Sartorius, Göttingen, Germany) and a peristalticpump(Watson Marlow). Periodically, an antifoaming agent was pumped to the fermenter using a time switch clock (15 sec performance every 1.5 hrs). The residence time wasdetermined based on the fermenter working volume via thetotal medium and NaOH flux. More than five residencetimes were required for achieving steady state conditionsand the cultivation in each mode was continued until theculture medium in fermenter was replaced at least fivetimes.

Biomass retention by cross over filtration

The experiments for the continuous production of gluconicacid by biomass retention by means of cross over filtrationwere carried out in a 5 litre stirrer fermenter. For the crossover filtration, two parallel connected autoclavablemicrofiltration tubing modules (cross over filtration) wereattached to the vessel (Figure 1). Fermenter, filters, mediumand NaOH solution were externally sterilized in a largeautoclave. In case of clogging appearances, the moduleswere exchanged or were cleaned by turning periodically the pumping direction backwards. The goal was implemented by taking off a part of the fermentation broth as filtrate andthus it is increased the stationary biomass concentration in bioreactor. At the same time, the discharge remainder (Bleed), containing fermentation broth and biomass, wastaken out of the fermenter by means of a second peristaltic pump. Both pumps were simultaneously controlled by alevel control system. By this way, it became feasible toadjust a constant relationship between both types of discharges. The level control probe (Weathston's bridge)was calibrated, enabling the proportional adjustment of desired level of filling under constant fermentationconditions. For safety reasons, the substrate pump was alsointerconnected to this control system and at reaching acertain (adjustable) level of bioreactor broth the pump wasshut down automatically. The fermentation was performedat 30ºC, pH 6.5 and 290% air saturation.

Biomass retention by biomass immobilization

The experiments with biomass immobilization were carriedout in a fluidized bed reactor at a working volume of about0.9 litre (Figure 2). For the immobilization of biomass,about 350 g of porous sinter glass beads (SIRAN

) (SchottAG, Mainz, Germany) was added in fluidized bed reactor,

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