Polyvinyl alcohol-immobilized whole-cell preparations for the biotransformation of nitriles

Summary Resting cells of Rhodococcus sp. ATCC 39484 were immobilized in polyvinyl alcohol (PVA). The PVA-beads showed nitrile-hydrolysing activity with propionitrile as a model substrate. Drying of the beads resulted in a loss of weight of 85 %. Re-swollen beads showed no loss in activity. In a continuously-operated fixed-bed bioreactor, beads were able to convert substrate concentrations up to 100 mM.     

Enhancement and stabilization of the production of glucoamylase by immobilized cells of Aureobasidium pullulans in a fluidized-bed reactor

Summary Glucoamylase production by Aureobasidium pollulans A-124 was compared in free-living cells, cells immobilized in calcium alginate gel beads aerated on a rotary shaker (agitation rate 150 rpm), and immobilized cells aerated in an air bubble column reactor. Fermentation conditions in the bioreactor were established for bead concentration, substrate (starch) concentration, calcium chloride addition to the fermentation medium, and rate of aeration. Production of glucoamylase was optimized at approximately 1.5 units of enzyme activity/ml medium in the bioreactor under the following conditions: aeration rate, 2.0 vol air per working volume of the bioreactor (280 ml) per minute; gel bead concentration, 30% of the working volume; substrate (starch) concentration, at 0.3% (w/v); addition of calcium chloride to the medium at a final concentration of 0.01 M. Productivity levels were stabilized through the equivalent of ten batches of medium with the original inoculum of immobilized beads. Offprint requests to: M. Petruccioli     

Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

Biosynthesis of γ-aminobutyric acid (GABA) using immobilized whole cells of <Emphasis Type="Italic">Lactobacillus brevis</Emphasis>

On an industrial scale, the production of γ-aminobutyric acid (GABA) from the cheaper sodium L-glutamate (L-MSG) is a valuable process. By entrapping Lactobacillus brevis cells with higher glutamate decarboxylase (GAD) activity into Ca-alginate gel beads, the biotransformation conditions of L-MSG to GABA were optimized with the immobilized cells. The cells obtained from a 60-h culture broth showed the highest biotransformation efficiency from L-MSG to GABA. The optimal cell density in gel beads, reaction pH and temperature were 11.2 g dry cell weight (DCW) l⁻¹, 4.4 and 40°C respectively. The thermal stability of immobilized cells was significantly higher than free cells. Under the optimized reaction conditions, the yield of GABA reached above 90% during the initial five batches and the yield still remained 56% in the tenth batch. Continuous production of GABA was realized with a higher yield by incorporating cell re-cultivation using the packed bed reactor.     

Chainia Penicillin V Acylase: Strain Characteristics, Enzyme Immobilization, and Kinetic Studies

Aerobic cultures of an actinomycete were found to produce penicillin V acylase (PVA) (PA, EC-3.5.1.11) extracellularly. The presence of L-2-3 diamino-propionic acid in cell wall and formation of sclerotia on culture media led to its identification as Chainia, a sclerotial Streptomyces. Partially purified acylase was adsorbed on kieselguhr and entrapped in polyacrylamide gel. The immobilized preparation proved effective with respect to retention of enzyme and enzyme activity even after 15 successful cycles. The pH optimum for crude enzyme was in the range of pH 7.5–8.0, and for the (NH4)₂ SO₄ fraction it was pH 8.5. The immobilized enzyme showed maximal activity at pH 9.5. The optimum temperature for acylase activity was at 55°C. The crude enzyme, ammonium sulfate fraction, and immobilized enzyme showed K _m value for penicillin V of 6.13 mM, 14.3 mM, and 17.1 mM, respectively. Received: 11 December 1997 / Accepted: 9 April 1998     

Adsorptive Immobilization of a Pseudomonas Strain on Solid Carriers for Augmented Decolourization in a Chemostat Bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml^?1 carrier) in comparison with PVA particles (4.8 mg VS ml^?1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factor in the thicker biofilms (effectiveness factor η = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml^?1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l^?1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80 - 81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to ～80% by the augmented system.     

Ribonuclease production by free and immobilizedAspergillus clavatus cells

Several fungal strains ofAspergillus andPenicillium were immobilized by cryopolymerization in polyvinyl alcohol cryogel beads.Aspergillus clavatus was the best producer of extracellular ribonuclease. Enzyme productivity and growth of free and immobilized cells in shake flasks and agitated bioreactor were studied. Ribonuclease production and growth behaviour depended on concentrations of glucose, peptone and soybean in the culture medium. Enzyme production was influenced by agitation and aeration intensity. In repeated batch, shake-flask cultures, the immobilized cells showed 2 to 3.5 times higher enzyme activity than free cells. The optimal conditions in a bioreactor were at 150 rev/min agitation speed and 0.5 vol/vol.min aeration. Enzyme productivity of immobilized cells (237 units/g dry mycelium.h) was 2.1 times higher than the productivity of free cells in a bioreactor, and 2.3 times higher than that of a shake-flask culture.R.J. Manolov is with the Institute of Microbiology, Department of Enzymes, Bulgarian Academy of Sciences, Georgy Bonchev Street 26, 1113 Sofia, Bulgaria.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

Enhanced production of laccase activity by <Emphasis Type="Italic">Trametes versicolor </Emphasis>immobilized into alginate beads by the addition of different inducers

In the present paper the effect of adding veratryl alcohol and copper sulphate on laccase activity production by Trametes versicolor immobilized into alginate beads has been investigated. Employing copper sulphate as laccase inducer or supplementing the culture medium with veratryl alcohol, led to maximum values of laccase activity. However, the highest laccase activity (around 4,000 U l⁻¹) was obtained in cultures simultaneously supplemented with copper sulphate (3 mM) and veratryl alcohol (20 mM). These values implied a considerable enhancement in relation to␣control cultures without any inducer (around 200 U l⁻¹). The production of laccase by immobilized T. versicolor in a 2-l airlift bioreactor with the optimized inducer has been evaluated. Laccase activities around 1,500 U l⁻¹ were attained. The bioreactor operated for 44 days without operational problems and the bioparticles (fungus grows in alginate beads) maintained their shape throughout the fermentation. Moreover, the extracellular liquid obtained was studied in terms of pH and temperature activity and stability. On the other hand, anthracene was added in two-repeated batches in order to determine the efficiency of this process to degrade pollutants. Near complete degradation was reached in both batches. Moreover, in vitro degradation of several polycyclic aromatic hydrocarbons by crude laccase was also performed.     

Production of extracellular native and foreign proteins by immobilized growing cells of Myxococcus xanthus

Summary Several strains of the protein-secreting, Gram negative bacterium Myxococcus xanthus were immobilized in carrageenan beads and the production of extracellular proteins was followed.The extracellular proteolytic activity was enhanced and concentrated in the beads. In contrast, the amount of total protein secreted by the cells was not modified by immobilization, but it was also retained and concentrated in the beads, the more, the harder the gel. The amount of slime produced by the cells did not seem to influence protein retention.Foreign proteins expressed from genes cloned in Myxococcus xanthus chromosome can be secreted into the medium by immobilized recombinant strains. A polygalacturonate lyase, expressed from the pelC gene from Erwinia chrysanthemi was only detectable outside of the beads. The pH 2.5 acid phosphatase expressed from the appA gene from Escherichia coli was secreted by immobilized cells at the same rate than did the free cells. It was predominantly found in the medium outside of the beads which represented a first purification and facilitated the continuous production of this protein by immobilized recombinant cells packed in a reactor.     

Phytate degradation by immobilized<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phytase in soybean-curd whey

Saccharomyces cerevisiae CY phytase-producing cells were immobilized in calcium alginate beads and used for the degradation of phylate. The maximum activity and immobilization yield of the immobilized phytase reached 280 mU/g-bead and 43%, respectively. The optimal pH of the immobilized cell phytase was not different from that of the free cells. However, the optimum temperature for the immobilized phytase was 50°C, which was 10°C higher than that of the free cells; pH and thermal stability were enhanced as a consequence of immobilization. Using the immobilized phytase, phytate was degraded in a stirred tank bioreactor. Phytate degradation, both in a buffer solution and in soybean-curd whey mixture, showed very similar trends. At an enzyme dosage of 93.9 mU/g-phytate, half of the phytate was degraded after 1 h of hydrolysis. The operational stability of the immobilized beads was examined with repeated batchwise operations. Based on 50% conversion of the phytate and five times of reuse of the immobilized beads, the specific degradation (g phytate/g dry cell weight) for the immobilized phytase increased 170% compared to that of the free phytase.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Fructose production by immobilizedArthrobacter cells

Summary ImmobilizedArthrobacter cells (NRRL-B-3728) were used for continuous isomerization of glucose to fructose in a bioreactor system. The system utilized stationary phase (55h) cells (2.2×10⁹ CFU/ml saline) immobilized onto K-carrageenan (3% w/v) beads [cells were heated at 65°C for 10 min to inactivate endogenous proteolytic enzymes]. Immobilized-cell preparations were hardened using three different glutaraldehyde systems. Glutaraldehyde (0.2 M) treated-immobilized cells (pH 7.0, 5°C for 30 min) exhibited good gel strength and high glucose isomerase activities. Maximal bioreactor isomerization of 44% was achieved when a buffered feedstock containing 40% glucose was fed into the column (60°C) at a flow rate of 0.2 ml/min. The biological half-life of glucose isomerase activities in this system was 400 h. Scanning electron microscopy revealed large numbers of cells distributed within the beads. A thin layer surrounding the beads following glutaraldehyde treatment was mainly due to cross-linking reactions between cell proteins and glutaraldehyde. This layer prevented leaking of cells during continuous isomerization reaction.     

Removal and recovery of Cu(II) from industrial effluent by immobilized cells of Pseudomonas putida II-11

A copper [Cu(II)]-accumulating strain, Pseudomonas putida II-11, isolated from electroplating effluent removed a significantly high amount of Cu(II) from growth medium and buffer. A laboratory-scale fixed bed reactor with cells of P. putida II-11 immobilized in polyacrylamide gel was constructed. The adsorption of Cu(II) by the immobilized cells was pH-dependent. Maximum removal of Cu(II) by the immobilized cells was at pH 8.0. The presence of Cr(IV), Ni(II) and Zn(II) did not significantly inhibit Cu(II) uptake whereas the presence of Pb(II) reduced Cu(II) uptake by fivefold. The presence of borate, carbonate, chloride and sulphate did not significantly inhibit Cu(II) uptake. The Cu(II) removal capacity of the bioreactor with immobilized cells did not change significantly when operated at retention times greater than 3 min. More than 90% of Cu(II) adsorbed on immobilized cells could be recovered by eluting with 0.1 m HCl. The bioreactor could be used for at least five loading-elution cycles without loss of Cu(II) removal capacity. The feasibility of using this bioreactor to remove and recover Cu(II) from electroplating effluent is discussed. Correspondence to: P. K. Wong     

Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized <Emphasis Type="Italic">Aspergillus niger</Emphasis> cultures

The effects of cell immobilization on the secretion of extracellular proteases and glucoamylase production by Aspergillus niger were investigated under a variety of immobilization techniques and culture conditions. Immobilization was achieved by means of cell attachment on metal surfaces or spore entrapment and subsequent growth on porous Celite beads. Free-suspension cultures were compared with immobilized mycelium under culture conditions that included growth in shake flasks and an airlift bioreactor. Cell attachment on metal surfaces minimized the secretion of proteases while enhancing glucoamylase production by the fungus. Growth on Celite beads in shake-flask cultures reduced the specific activity of the secreted proteases from 128 to 61 U g⁻¹, while glucoamylase specific activity increased from 205 to 350 U g⁻¹. The effect was more pronounced in bioreactor cultures. A reduction of six orders of magnitude in protease specific activities was observed when the fungus grew immobilized on a rolled metal screen, which served as the draft tube of an airlift bioreactor. Received 29 October 2001/ Accepted in revised form 14 June 2002     

Protease production by immobilized growing cells of Serratia marcescens and Myxococcus xanthus in calcium alginate gel beads

Summary Serratia marcescens and Myxococcus xanthus cells were immobilized in calcium alginate gel beads. Immobilization under various conditions had no effect on the extracellular proteolytic activity of S. marcescens cells. Protease production seemed rather to depend on the free cells in the medium. However, the stability over time of enzyme production was enhanced, as immobilization increased protease production half-life from 5 to 12 days. On the other hand, Myxococcus xanthus produced proteases inside the gel beads which could diffuse into the medium. The proteolytic activity increased as a function of the initial cell content of the beads and of the bead inoculum. Compared to free cells, immobilized cells of Myxococcus xanthus could produce 8 times more proteolytic activity, with a very low free-cell concentration in the medium.     

Hydrodynamic characteristics of immobilized cell beads in a liquid-solid fluidized-bed bioreactor

This study examined the hydrodynamic characteristics of a liquid-solid fluidized-bed bioreactor using elastic particles (PVA gel beads) of various diameters as carriers. The drag coefficient-Reynolds number, velocity-voidage, and expansion index-Reynolds number relationships observed during fluidization of PVA gel beads in a fluidized bed in our experiments were compared with the published results. Predictions made from previous correlations were examined with our new experimental findings, revealing the inadequacy of most of these correlations. Thus, new correlations describing the above-mentioned relationships are suggested. The drag coefficient of immobilized cell beads is larger than that of free cell ones at the same Reynolds number because the surface of the immobilized cell beads is rougher. For multiparticle systems, the correction factor, f(epsilon), is a function of the falling gel bead properties (Reynolds number) as well as the fluidized gel bead properties (Archimedes number), and depend strongly on the bed voidage (epsilon). A new simple relation was developed to predict easily the epsilon value from 0.5-0.9 at 4,986 < A(r) < 40,745 or 34 < Re(t) < 186. For all the immobilized cell beads used in this study, the prediction error of the bed voidage was less than 5% at epsilon > 0.5. The prediction equations in this study can be further applied to analyzing the hydrodynamic characteristics of a fluidized-bed reactor using similar entrapped elastic particles as carriers.     

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized <Emphasis Type="Italic">Coprinus cinereus</Emphasis> in fluidized bed bioreactor

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g⁻¹ h⁻¹ with a 50% conversion time (t _1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.     

Physical Factors and Reuse Affect the Production of Cholesterol Oxidase in Reactors Containing Calcium Alginate-Immobilized Rhodococcus equi No. 23

Summary Some physical factors including initial pH of medium, cultivation temperature and shaking speed as well as reuse affecting the production of cholesterol oxidase (CholOx) in reactors containing calcium alginate-immobilized cells of Rhodococcus equi No. 23 were investigated. Results revealed that the free cells showed the maximum CholOx in the culture with an initial pH of 5.0, while culture inoculated with immobilized cells exhibited a broad pH range, 6.0–9.0, for maximum CholOx production. The immobilized and free cells produced the maximum CholOx in the culture incubated at 30 and 25°C, respectively. The CholOx production decreased upon increasing the cultivation temperature. Increasing CholOx activity was also noted for both immobilized and free cells of R. equi No. 23 in the culture with increasing shaking speed. Under the optimal culture conditions, that were established, a higher maximum CholOx production of 0.94 unit/ml was found for immobilized R. equi No. 23 compared to that of 0.84 unit/ml for free cells after 48 h of cultivation. Furthermore, no gel leakage was noted after re-use of the calcium alginate-immobilized R. equi No. 23 for seven consecutive 48 h batch culture. The CholOx production in the seventh cycle was about 60.4% of that obtained in the first cycle.     

Biotransformation of penicillin V to 6-aminopenicillanic acid using immobilized whole cells of E. coli expressing a highly active penicillin V acylase

The production of 6-aminopenicillanic acid (6-APA) is a key step in the manufacture of semisynthetic antibiotics in the pharmaceutical industry. The penicillin G acylase from Escherichia coli has long been utilized for this purpose. However, the use of penicillin V acylases (PVA) presents some advantages including better stability and higher conversion rates. The industrial application of PVAs has so far been limited due to the nonavailability of suitable bacterial strains and cost issues. In this study, whole-cell immobilization of a recombinant PVA enzyme from Pectobacterium atrosepticum expressed in E. coli was performed. Membrane permeabilization with detergent was used to enhance the cell-bound PVA activity, and the cells were encapsulated in calcium alginate beads and cross-linked with glutaraldehyde. Optimization of parameters for the biotransformation by immobilized cells showed that full conversion of pen V to 6-APA could be achieved within 1?hr at pH 5.0 and 35°C, till 4% (w/v) concentration of the substrate. The beads could be stored for 28 days at 4°C with minimal loss in activity and were reusable up to 10?cycles with 1-hr hardening in CaCl₂ between each cycle. The high enzyme productivity of the PVA enzyme system makes a promising case for its application for 6-APA production in the industry.