Optimization of lactic acid production from whey by L casei NRRL B‐441 immobilized in chitosan stabilized Ca‐alginate beads

The production of lactic acid from whey by Lactobacillus casei NRRL B‐441 immobilized in chitosan‐stabilized Ca‐alginate beads was investigated. Higher lactic acid production and lower cell leakage were observed with alginate–chitosan beads compared with Ca‐alginate beads. The highest lactic acid concentration (131.2 g dm^?3) was obtained with cells entrapped in 1.3–1.7 mm alginate–chitosan beads prepared from 2% (w/v) Na‐alginate. The gel beads produced lactic acid for five consecutive batch fermentations without marked activity loss and deformation. Response surface methodology was used to investigate the effects of three fermentation parameters (initial sugar, yeast extract and calcium carbonate concentrations) on the concentration of lactic acid. Results of the statistical analysis showed that the fit of the model was good in all cases. Initial sugar, yeast extract and calcium carbonate concentrations had a strong linear effect on lactic acid production. The maximum lactic acid concentration of 136.3 g dm^?3 was obtained at the optimum concentrations of process variables (initial sugar 147.35 g dm^?3, yeast extract 28.81 g dm^?3, CaCO₃ 97.55 g dm^?3). These values were obtained by fitting of the experimental data to the model equation. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in lactic acid production using alginate–chitosan‐immobilized cells. Copyright © 2005 Society of Chemical Industry     

Immobilization of penicillin G acylase on a composite carrier with a biocompatible microenvironment of chitosan

BACKGROUND: Penicillin G acylase (PGA) has been used extensively in the β‐lactam antibiotics industry. As a biocatalyst, it is better to use immobilized enzymes than free enzymes, therefore, the immobilization of PGA on a composite carrier consisting of an adsorbent resin and biocompatible chitosan were investigated. RESULTS: First, FT‐IR, BET and SEM analysis confirmed the structure of the composite carrier. Then, the immobilization process was optimized. The activity of the immobilized PGA on the chitosan–resin (IP‐CsR) was about 1300 U (g dry carrier)^?1 with a protein loading of about 27 mg (g dry carrier)^?1. Compared with the immobilized PGA on unmodified resin (IP‐R), the specific activity of IP‐CsR was enhanced about 2‐fold. The operational, thermal and pH stability were investigated. IP‐CsR maintained more than 75% initial activity after 35 cycles, while IP‐R was active for only 10 cycles. The half‐life at 50 °C increased from 75 to 300 min and the most stable pH was changed from 8.0 to 5.5. CONCLUSION: A novel composite carrier containing a biocompatible chitosan was very effective for PGA immobilization. Copyright © 2008 Society of Chemical Industry     

Optimization of the production of chitosan from beet molasses by response surface methodology

Chitosan was produced by Rhizopus oryzae 00.4367 in shake flask culture and a stirred tank fermenter. Synthetic medium, treated and untreated beet molasses were used as cultivation media in shake flask cultures. In the stirred tank fermenter, the cultivation media were synthetic medium and untreated beet molasses. Shake flask culture containing untreated molasses with a sugar concentration of 40 g dm^?3 produced the maximum chitosan yield (961 mg dm^?3). Chitosan concentration reached its maximum value at the late exponential growth phase of R oryzae. In all experiments almost 8–10% of biomass and 32–38% of alkali‐insoluble material was extracted as chitosan. A central composite design was employed to determine the optimum values of process variables (aeration rate, agitation speed and initial sugar concentration) leading to maximum chitosan concentration in the stirred tank fermenter. In all cases, the fit of the model was found to be good. Aeration rate, agitation speed and initial sugar concentration had a strong linear effect on chitosan concentration. Moreover, the concentration of chitosan was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions. A maximum chitosan concentration of 1109.32 mg dm^?3 was obtained in untreated molasses medium containing an initial sugar concentration of 45.37 g dm^?3 with an aeration rate and agitation speed of 2.10 vvm and 338.93 rpm, respectively. Copyright © 2004 Society of Chemical Industry     

Immobilization of a nonspecific chitosan hydrolytic enzyme for application in preparation of water‐soluble low‐molecular‐weight chitosan

A nonspecific chitosan hydrolytic enzyme, cellulase, was immobilized onto magnetic chitosan microspheres, which was prepared in a well spherical shape by the suspension crosslinking technique. The morphology characterization of the microspheres was carried out with scanning electron microscope and the homogeneity of the magnetic materials (Fe₃O₄) in the microspheres was determined from optical micrograph. Factors affecting the immobilization, and the properties and stabilities of the immobilized enzyme were studied. The optimum concentration of the crosslinker and cellulase solution for the immobilization was 4% (v/v) and 6 mg/mL, respectively. The immobilized enzyme had a broader pH range of high activity and the loss of the activity of immobilized cellulase was lower than that of the free cellulase at high temperatures. This immobilized cellulase has higher apparent Michaelis–Menten constant K_m (1.28 mg/mL) than that of free cellulase (0.78 mg/mL), and the maximum apparent initial catalytic rate V_max of immobilized cellulase (0.39 mg mL^?1 h^?1) was lower than free enzyme (0.48 mg mL^?1 h^?1). Storage stability was enhanced after immobilization. The residual activity of the immobilized enzyme was 78% of original after 10 batch hydrolytic cycles, and the morphology of carrier was not changed. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 1334–1339, 2006     

Integration of flocculation and adsorptive immobilization of Pseudomonas delafieldii R‐8 for diesel oil biodesulfurization

BACKGROUND: Deep desulfurization of hydrocarbon fuels is receiving increasing worldwide attention because of the increasingly stringent regulations to meet the requirement of environmental protection. Biodesulfurization (BDS) is being explored as either an alternative or complementary process to the conventional oil refining technologies. The whole cell immobilization technique is of great importance for accelerating the industrialization of BDS. An effective technique for a BDS process employing flocculation and integration with immobilization was developed. RESULTS: Pseudomonas delafieldii R‐8 cells were successfully flocculated and immobilized by directly adding chitosan and celite into the culture broth. The one‐step immobilized R‐8 cells exhibited good catalytic activity and retained at least 85% activity after six cycles of repeated‐batch desulfurization. Extensive biodesulfurization of diesel oil resulted in 82% reduction of total sulfur from 123 to 22 µg g^?1 in 24 h. CONCLUSIONS: A novel and simple technique was developed using chitosan flocculation and integration with cell immobilization onto celite for dibenzothiophene BDS. The present report indicates that integration of flocculation and immobilization may provide a continuous and efficient method of BDS. Copyright © 2010 Society of Chemical Industry     

Continuous ethanol production from carob pod extract by immobilized Saccharomyces cerevisiae in a packed-bed reactor

The continuous production of ethanol from carob pod extract by immobilized Saccharomyces cerevisiae in a packed-bed reactor has been investigated. At a substrate concentration of 150 g dm^?3, maximum ethanol productivity of 16 g dm^?3 h^?1 was obtained at D = 0·4 h^?1 with 62·3% of theoretical yield and 83·6% sugars′ utilization. At a dilution rate of 0·1 h^?1, optimal ethanol productivity was achieved in the pH range 3·5–5·5, temperature range 30–35·C and initial sugar concentration of 200 g dm^?3. Maximum ethanol productivity of 24·5 g dm^?3 h^?1 was obtained at D = 0·5 h^?1 with 58·8% of theoretical yield and 85% sugars′ utilization when non-sterilized carob pod extract containing 200 g dm^?3 total sugars was used as feed material. The bioreactor system was operated at a constant dilution rate of 0·5 h^?1 for 30 days without loss of the original immobilized yeast activity. In this case, the average ethanol productivity, ethanol yield (% of theoretical) and sugars′ utilization were 25 g dm^?3 h^?1, 58·8% and 85·5%, respectively.     

Cinnamic ester of D‐sorbitol for immobilization of mushroom tyrosinase

Mushroom tyrosinase was immobilized by adsorption onto the totally cinnamoylated derivative of D ‐sorbitol. The polymerization and cross‐linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of tyrosinase on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. The pH value, enzyme concentration and immobilization time were all important parameters affecting immobilization efficiency; also, enzyme immobilization efficiency correlated well with the tyrosinase isoelectric point. The immobilized enzyme showed an optimum measuring pH of 3.5 and greater activity at acid and neutral pH values than the soluble enzyme. The optimal reaction temperature was 35 °C and the temperature profile was broader than that of the free enzyme or of the enzyme immobilized on other supports. The apparent Michaelis constant of mushroom tyrosinase immobilized on the SOTCN derivative acting on 4‐tert‐butylcatechol (TBC) was 0.40 ± 0.02 mmol dm^?3, which was lower than for the soluble enzyme, suggesting that the affinity of this enzyme for this substrate was greater when immobilized than when in solution. Immobilization stabilized the enzyme and made it less susceptible to activity loss during storage at pH values in the range 4–5.5, and the suicide inactivation of the immobilized tyrosinase was null or negligible in a reaction medium with 4‐tert‐butylcatechol at a concentration of 0.4 mmol dm^?3. The results show that cinnamic carbohydrate esters of D ‐sorbitol are an appropriate support for tyrosinase immobilization and could be of use for several tyrosinase applications. Copyright © 2005 Society of Chemical Industry     

L(+)‐Lactic acid production using poly(vinyl alcohol)‐cryogel‐entrapped Rhizopus oryzae fungal cells

A new immobilized biocatalyst based on Rhizopus oryzae fungal cells entrapped in poly(vinyl alcohol)‐cryogel was evaluated in both the batch and semi‐batch processes of L (+)‐lactic acid (LA) production, when glucose, acid hydrolysates of starch or gelatinized potato starch were used as the main substrates. Under the batch conditions, the immobilized biocatalyst developed produced LA with yields of 94% and 78% from glucose and acid starch hydrolysates, respectively. Semi‐batch conditions enabled product yields of 52% and 45% to be obtained with the corresponding substrates. The highest process productivity (up to 173 g L^?1) was reached under semi‐batch conditions. Potato starch (5–70 g L^?1) was also transformed into lactic acid by immobilized R. oryzae. It was shown that long‐term operation of the immobilized biocatalyst (for at least 480 h) produced a low decrease in metabolic activity. Copyright © 2006 Society of Chemical Industry     

Immobilization of invertase onto dimer acid‐co‐alkyl polyamine

Invertase was immobilized onto the dimer acid‐co‐alkyl polyamine after activation with 1,2‐diamine ethane and 1,3‐diamine propane. The effects of pH, temperature, substrate concentration, and storage stability on free and immobilized invertase were investigated. Kinetic parameters were calculated as 18.2 mM for K_m and 6.43 × 10^?5 mol dm^?3 min^?1 for V_max of free enzyme and in the range of 23.8–35.3 mM for K_m and 7.97–11.71 × 10^?5 mol dm^?3 min^?1 for V_max of immobilized enzyme. After storage at 4°C for 1 month, the enzyme activities were 21.0 and 60.0–70.0% of the initial activity for free and immobilized enzyme, respectively. The optimum pH values for free and immobilized enzymes were determined as 4.5. The optimum temperatures for free and immobilized enzymes were 45 and 50°C, respectively. After using immobilized enzyme in 3 days for 43 times, it showed 76–80% of its original activity. As a result of immobilization, thermal and storage stabilities were increased. The aim of this study was to increase the storage stability and reuse number of the immobilized enzyme and also to compare this immobilization method with others with respect to storage stability and reuse number. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1526–1530, 2004     

BTEX removal from contaminated groundwater by a co‐culture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a continuous fibrous‐bed bioreactor

A fibrous‐bed bioreactor with immobilized cells of Pseudomonas putida and Pseudomonas fluorescens was used to treat groundwater contaminated with benzene, toluene, ethylbenzene, and xylenes (collectively know as BTEX). The kinetics of BTEX biodegradation in the fibrous‐bed bioreactor operated under continuous well‐mixed conditions was studied at room temperature. Aeration was not used in the process fed with groundwater samples with an average total BTEX concentration of 2.75 mg dm^?3. All BTEX compounds present in the groundwater feed were concurrently and completely biodegraded even under oxygen‐limited or hypoxic conditions. Nearly 100% removal efficiency was obtained when the retention time was greater than 1 h. BTEX removal efficiency decreased with decreasing the retention time, with p‐ and o‐xylenes showed up first in the treated groundwater, followed by benzene and then other BTEX compounds. Biodegradation rates of BTEX generally increased with increasing BTEX concentration and loading rate. The maximum BTEX biodegradation rate was 5.76 mg h^?1 dm^?3 at the loading rate of 6.54 mg dm^?3 h^?1. The bioreactor had a stable performance, maintaining its ability for efficient BTEX degradation without requiring additional nutrients for more than 1 month. The good performance of the fibrous‐bed bioreactor was attributed to the high cell density (～15 g dm^?3 reactor volume) in the fibrous matrix. © 2002 Society of Chemical Industry     

Double‐functional characteristics of a surface molecular imprinted adsorbent with immobilization of nano‐TiO2

A new chitosan molecular imprinted adsorbent obtained by immobilization of nano‐TiO₂ on the adsorbent surface (surface‐imprinted adsorbent with nano‐TiO₂) was prepared. Based on photocatalytic reaction and the surface molecular imprinting technology, this new kind of surface‐imprinted adsorbent with immobilization of nano‐TiO₂ can not only adsorb template metal ions but can also degrade organic pollutants. The results showed that, after the nano‐TiO₂ was immobilized on the adsorbent surface, the adsorption ability for the imprinted ion (Ni²⁺) of this new imprinted adsorbent immobilized with nano‐TiO₂ was not affected, but the degradation ability for p‐nitrophenol (PNP) of the surface‐imprinted adsorbent with nano‐TiO₂ increased three‐fold compared with that of the surface‐imprinted adsorbent without nano‐TiO₂, from 23.8 to 76.1% (at an initial PNP concentration of 20 mg·dm^?3). The optimal TiO₂ concentration in the adsorbent preparation was 0.025 g·TiO₂ g^?1 adsorbent. The removal capacity for PNP reached 60.25 mg·g^?1 (at 400 mg·dm^?3 initial PNP concentration) under UV irradiation. The surface‐imprinted adsorbent with nano‐TiO₂ can be reused for at least five cycles without decreasing the removal ability for PNP and the imprinted ion (Ni²⁺). Copyright © 2006 Society of Chemical Industry     

Towards the design of non-amyloidogenic proteins

Glucose oxidase was immobilized onto poly(2-hydroxyethyl methacrylate) (pHEMA) membranes by two methods: by covalent bonding through epichlorohydrin and by entrapment between pHEMA membranes. The highest immobilization efficiency was found to be 17.4% and 93.7% for the covalent bonding and entrapment, respectively. The K_m values were 5.9 mmol dm^?3, 8.8 mmol dm^?3 and 12.4 mmol dm^?3 for free, bound and entrapped enzyme, respectively. The V_max values were 0.071 mmol dm^?3 min^?1, 0.067 mmol dm^?3 min^?1 and 0.056 mmol dm^?3 min^?1 for free, bound and entrapped enzyme. When the medium was saturated with oxygen, K_m was not significantly altered but V_max was. The optimum pH values for the free, covalently-bound and entrapped enzyme were determined to be 5, 6, and 7, respectively. The optimum temperature was 30°C for free or covalently-bound enzyme but 35°C for entrapped enzyme. The deactivation constant for bound enzyme was determined as 1.7 × 10^?4 min^?1 and 6.9 × 10^?4 min^?1 for the entrapped enzyme.     

Surface modification of atmospheric plasma activated BOPP by immobilizing chitosan

Summary Chitosan was immobilized onto plasma activated biaxially oriented polypropylene (BOPP) films aimed at producing antimicrobially active barrier film for food packaging applications. 1% chitosan dissolved into 0.1 M acetic acid was mixed with 0.1% glutaraldehyde (cross-linking agent) and applied onto N₂-plasma + NH₃ activated BOPP film. Amount of immobilized chitosan was 1.8 g/m². Films had strong antimicrobial activity against both Bacillus subtilis and Escherichia coli and they reduced the oxygen transmission rate (OTR) measured in dry conditions from 1500 down to 27 cm³/(m²·24 h). Migration tests for determining the total amounts of substances migrating into food simulants (3% acetic acid, 95% ethanol and iso-octane) indicated, that chitosan coating was permanently immobilized onto BOPP without any leaching (total migration < 2 mg/dm²), thus it met the requirements stipulated in Directive 2002/72/EC relating to plastic materials and articles intended to come into contact with foodstuffs. The results suggest that chitosan treated BOPP films may be exploited in various food packaging applications requiring high oxygen barrier and/or antimicrobially active packaging materials.     

Influence of chitosan derivatization on its physicochemical characteristics and its use as enzyme support

Chitosan was derivatized by two methodologies for analyzing their effect on chitosan physicochemical characteristics and its applicability as carrier for Bacillus circulans β‐galactosidase immobilization. Glutaraldehyde (GA) and epichlorohydrin (EPI) were used for crosslinking and activation of chitosan, producing the corresponding supports (C‐GA and C‐EPI‐EPI) after a one‐step and a two‐step process, respectively. The spherical shape and mean diameter of chitosan particles was not significantly affected by polymer derivatization, while Fourier transform infrared analysis showed that in both cases, chitosan polymer was chemically modified. TGA analysis indicated that C‐EPI‐EPI was the most thermally stable. The high degree of activation of C‐EPI‐EPI (586 μmol of aldehydes/g) resulted in the highest loss of activity during immobilization; hence a support with 100 μmol of aldehydes/g was produced (C‐EPI‐EPI₁₀₀). The highest expressed activity (89.3 IU/g) was obtained with the enzyme immobilized in C‐GA, while the biocatalyst with highest thermal stability at 60°C was obtained with C‐EPI‐EPI₁₀₀ (half‐life was 84‐fold higher than the one of the soluble enzyme). The best compromise between biocatalyst expressed activity and thermal stability corresponded to β‐galactosidase immobilized in C‐EPI‐EPI₁₀₀. According to this study, chitosan derivatized with EPI is a thermally stable carrier appropriate for producing highly stable immobilized B. circulans β‐galactosidase. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40171.     

Modulated gluconic acid production from immobilized cells of Aspergillus niger ORS‐4.410 utilizing grape must

BACKGROUND: Gluconic acid (GA) production by immobilized cells of mutant Aspergillus niger ORS‐4.410 on polyurethane sponge (PUS) and calcium‐alginate (Ca‐alginate) was evaluated in repeated batches of solid state surface fermentation (SSF) and submerged fermentation (SmF) conditions, respectively, utilizing rectified grape must as carbon source. RESULTS: The passive immobilization of cells in fermentation medium solid support of having 0.4 cm³ cube size, 4% spore suspension, 0.6 g inoculum of PUS immobilized cells at 32 °C and 2.0 L min^?1 resulted in the maximum GA production (88.16 g L^?1) with a 92.8% yield, while the Ca‐alginate matrix with a 0.5 cm diameter bead size, 2–3% spore suspension, 15 g inoculum at 34 °C and 150 rpm agitation speed revealed 67.19 g L^?1 GA with a 85.2% yield. Repeated use of PUS showed higher levels of GA (110.94 g L^?1) in the third–fourth fermentation cycles with 95–98% yield and 22.50 g L^?1 d^?1 productivity under SSF that was 2.5‐fold higher than the productivity obtained from a typical fermentation cycle, and 54% greater than the productivity obtained with repetitive use of Ca‐alginate immobilized cells of A. niger under SmF. CONCLUSION: Using immobilized cells of A. niger in PUS, the rectified form of grape must can be utilized for GA production as an alternative source of carbohydrate by replacing the conventional fermentation conditions. Copyright © 2008 Society of Chemical Industry     

Production of L‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor

Production of L ‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60 °C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (K_m and V_m) of immobilized pellets were found to be 7.99 mmol dm^?3 and 1.38 mmol dm^?3 h^?1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2 mol dm^?3. The L ‐methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4 mol dm^?3, respectively, at a flow rate of 7.5 cm³ h^?1 using the small‐scale packed bed reactor developed. The half‐life of the L ‐aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L ‐methionine. © 2002 Society of Chemical Industry     

High density culture of Coptis japonica cells increases berberine production

A high density culture method was devised to improve the yield of berberine from highly productive cells of Coptis japonica. By adjusting aeration and stirring, Coptis cells were cultured at densities of up to 75 g dm^?3 (dry weight) in a culture tank fitted with a hollow-paddle type stirrer. Whereas a maximum density of 30 g dm^?3 of C. japonica cells could be used in ordinary batch culture, 48 g dm^?3 could be used in a fed-batch culture in which the amounts of the nutrients in the medium were made proportional to the density of the inoculum. Moreover, in fed-batch culture done with modified medium, the composition of which had been determined from the amounts of components incorporated in cells grown at the usual density for ordinary batch culture, the cell yield was improved to 55 g dm^?3 and the berberine yield to 3.5 g dm^?3.     

Continuous ethanol fermentation using immobilized yeast in a fluidized bed reactor

Continuous ethanol fermentation of glucose using fluidized bed technology was studied. Saccharomyces cerevisiae were immobilized and retained on porous microcarriers. Over two-thirds of the total reactor yeast cell mass was immobilized. Ethanol productivity was examined as dilution rate was varied, keeping all other experimental parameters constant. Ethanol yield remained high at an average of 0.36 g ethanol g^?1 glucose (71% of theoretical yield) as the dilution rate was increased stepwise from 0.04 h^?1 to 0.14 h^?1. At a dilution rate of 0.15 h^?1, the ethanol yield steeply declined to 0.22 g ethanol g^?1 glucose (44% of theoretical yield). The low maximum percentage of theoretical yield is primarily due to an extended mean cell residence time, and possibly due to the inhibitory effect of a high dissolved carbon dioxide concentration, enhanced by the probable intermittent levels of low pH in the reactor. Constant ethanol production was possible at a high glucose loading rate of 840 g dm^?3 day^?1 (attained at a dilution rate of 0.14 h^?1). Although the highest average ethanol concentration (97.14 g dm^?3) occurred at the initial dilution rate of 0.04 h^?1, the peak average ethanol production rate (2.87 g (g yeast)^?1 day^?1) was reached at a greater dilution rate of 0.11 ^h-1. Thus, the optimal dilution rate was determined to be between 0.11 h^?1 and 0.14 h^?1. Ethanol inhibition on yeast cells was absent in the reactor at average bulk-liquid ethanol concentrations as high as 97.14 g dm^?3. In addition, zero-order kinetics on ethanol production and glucose utilization was evident.     

Maximizing the production of acetone—butanol in an alginate bead fluidized bed reactor using clostridium acetobutylicum

Low volumetric solvent productivities are one of the characteristics of an acetone-butanol fermentation by C. acetobutylicum. A calcium alginate immobilized continuous culture was used in a novel gas-sparged reactor to strip the solvents from the aqueous phase and reduce their toxicity. A dilution rate of 0.07 h^?1 was found to give maximum solvent productivity at 0.58 g dm^?3 h^?1, although at 0.12 h^?1 the productivity was slightly lower. In order to increase glucose uptake by the culture, feed glucose concentrations were increased over time to attempt to acclimatize the culture. This resulted in a productivity as high as 0.72 g dm^?3 h^?1 although this production rate was found to be unstable.     

Optimization of pullulan production using Ca‐alginate‐immobilized Aureobasidium pullulans by response surface methodology

BACKGROUND: The production of pullulan from synthetic medium by Aureobasidium pullulans P56 immobilized in Ca‐alginate beads was investigated using batch and repeated batch fermentation systems. RESULTS: The highest pullulan concentration (19.52 ± 0.37 g dm^?3) was obtained with 2.0‐2.4 mm beads prepared from 2% sodium alginate solution. Pullulan production was mainly accomplished by immobilized fungal cells since leaked cells in the fermentation medium comprised 17.4% of the total fungal population at the end of fermentation. The pullulan proportion was 84.5% of the total polysaccharide in the fermentation medium. Response surface methodology was used to investigate the effects of three fermentation parameters (initial pH, agitation speed and incubation time) on the concentration of pullulan. Results of the statistical analysis showed that the fit of the model was good in all cases. The maximum pullulan concentration of 21.07 ± 0.48 g dm^?3 was obtained at the optimum concentrations of process variables (pH 7.31, agitation speed 191.5 rpm, incubation time 101.2 h). The gel beads produced pullulan under the optimized conditions for six consecutive batch fermentations without marked activity loss and deformation. CONCLUSION: The results of this study suggest that the immobilization of A. pullulans cells in Ca‐alginate gel beads is suitable for batch and repeated batch production of pullulan. Copyright © 2007 Society of Chemical Industry