Production of acid proteinases byHumicola lutea immobilized in polyacrylamide gel

Summary Fungal spores ofHumicola lutea 120–5 were entrapped in 5% polyacrylamide gel and were cultivated for 44–48 h to form a mycelial network inside the beads. A dense mycelial growth also occurred on the surface of the beads. It was possible to reuse the immobilized mycelium for production of acid proteinases in 12 different batches without loss of mechanical stability. The inoculum size should be controlled prior to its transfer into fresh production medium. Maximal enzyme production exceeding the level of free cell fermentation was registered in the fourth to seventh cycles. According to the size of the inoculum, half of the initial production rate was reached after 7–14 batches.     

Production of casein hydrolysates by extracellular acid proteinases of immobilized Humicola lutea cells

Casein hydrolysis was studied during the cultivation of immobilized Humicola lutea cells producing acid proteinases. By monitoring the cultivation with time, various casein hydrolysates could be obtained, from partially modified proteins (yield 80%) with improved emulsion properties to peptones (yield > 50%) with a degree of hydrolysis >40%. The casein from the fermentation medium appeared to be simultaneously a nitrogen source, an inducer of proteinase biosynthesis, and a substrate for the production of casein hydrolysates. Casein (4%) and glucose (2%) ensured optimal cultivation conditions. The fungal cells, immobilized in calcium alginate beads, required a short cultivation time and demonstrated comparable hydrolysis of casein during five to seven reuses in batch mode. Correspondence to: B. Tchorbanov     

Production of acid proteinases by Humicola lutea mycelium immobilized in polyurethane sponge.

Humicola lutea 120-5 spores were entrapped in polyurethane sponge cubes and were cultivated inside the carrier to form an immobilized mycelium further used for production of acid proteinases in batch mode. A carrier—spore suspension ratio of 10:0.5 (wt) should be used to obtain optimal results. The polyurethane sponge-immobilized mycelium could be applied repeatedly, the enzyme activity secreted during the first 10 cycles being about the same as that produced by free cells. The advantages of immobilizing fungal cells by germinating conidia entrapped inside the supporting material are discussed.     

l-Glutamate production by protoplasts immobilized in carrageenan gel

Protoplastization of Brevibacterium flavum cultured in a medium containing 50 μg l⁻¹ and 5 units penicillin per ml was performed by lysozyme treatment. The protoplasts were immobilized in various polymer matrices, such as agar, polyacrylamide, calcium alginate, and κ-carrageenan and then used for l-glutamate production from glucose and urea in a batch system. The protoplasts immobilized in κ-carrageenan gels showed the highest productivity of l-glutamate being twice that of immobilized whole cells under optimum conditions. The maximum productivity reached 2.3 mg ml⁻¹ initially. The immobilized B. flavum protoplasts could be used 8 times (192 h) for l-glutamate production retaining about 22% of the initial productivity during the last reaction.     

Palatinose production by free and Ca-alginate gel immobilized cells of Erwinia sp.

Palatinose is a non-cariogenic disaccharide obtained from the enzymatic conversion of sucrose, used in food industries as a sugar substitute. Free and Ca-alginate immobilized cells of Erwinia sp. D12 were used to produce palatinose from sucrose. Palatinose production was studied in a repeated-batch process using different immobilized biocatalysts: whole cells, disrupted cells and glucosyltransferase. Successive batches were treated with the immobilized biocatalyst, but a decrease in palatinose production was observed. A continuous process using a packed-bed reactor was investigated, and found to produce 55–66% of palatinose during 17 days using immobilized cells treated with glutaraldehyde and a substrate flow speed of 0.56 ml min⁻¹. However, immobilized cells in a packed-bed reactor failed to maintain the palatinose production for a prolonged period. The free cells showed a high conversion rate using batch fermentation, obtaining a palatinose yield of 77%. The cells remained viable for 16 cycles with high palatinose yields (65–77%). Free Erwinia sp. D12 cells supported high production levels in repeated-batch operations, and the results showed the potential for repeated reuse.     

Scanning electron microscopy of immobilizedHumicola lutea during semicontinuous production of acid proteinases

The morphology of the fungusHumicola lutea (strain 120–5), immobilized in polyacrylamide and polyhydroxyethylmethacrylate and used for the semicontinuous production of acid proteinases, was examined by scanning electron microscopy. The fungus developed a dense mycelium below the bead surface as well as in the bead interior after precultivation of entrapped spores. During maximal semicontinuous enzyme biosynthesis, formation of numerous large bulbous cells with a different shape was observed. Lysis of the cells was observed mainly in the centre of the gel beads after 13 successive fermentations with polyacrylamide-immobilized cells or after 21 re-uses of polyhydroxyethylmethacrylate-immobilized mycelia, respectively. Growth and changes in the cellular morphology of immobilizedH. lutea, accompanying biosynthesis of acid proteinases, were comparable in both gel matrices but mycelia immobilized in polyhydroxyethylmethacrylate maintained their productivity twice as long.     

Benzene degradation by bacterial cells immobilized in polyacrylamide gel

Summary Whole cells ofPseudomonas putida were immobilized in polyacrylamide gel and their ability to utilise benzene was examined. On initial immobilization cells were found to lose 40–70% of their activity. This activity could be restored by incubation in a medium containing benzene and succinate. It was also found that partial activation could be achieved by incubation with iron salts, in the absence of a carbon source. Electron microscopy showed this activation to be accompanied by an increase in cell numbers, with the formation of cell conglomerates within gel interstices. However, under some conditions, prolonged elution with substrate resulted in cell disruption and loss of activity.     

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.     

Lipase production by immobilized Candida rugosa cells

Summary Immobilization of Candida rugosa cells on a solid support for extracellular lipase production has been explored. The use of Ca-alginate beads and of mixed matrix of polyurethane foam/Ca-alginate beads enabled us to operate a batch and a continuous four-phase fluidized bed bioreactor. Cells co-entrapped together with polyurethane into Ca-alginate did not show higher lipase production levels than the cells entrapped in Ca-alginate gels. The addition of gum arabic to the medium greatly enhanced lipase production without affecting the hydrodynamic operating conditions significantly. This fact demonstrates that the reactor system is limited in terms of organic substrate dispersion and direct contact with cells. Correspondence to: C. Solà     

Acetic acid production by immobilized acetobacter cells

Summary Using a pulsed gas and liquid flow and with cells directly adsorbed on to a suitably-formed support, aerobic transformations can be carried out in a fixed-cell reactor with significant gain in efficiency. Immobilized cells of Acetobacter on cordierite can produce acetic acid at a high rate which, at different dilution rates, may be limited either by product inhibition or by oxygen transfer requirements.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Glycerol production by immobilized cells of Saccharomyces cerevisiae

Summary Cells of Saccharomyces cerevisiae were immobilized in K-Carrageenan. Addition of sodium sulfite to the fermentation medium up to four percent led to glycerol yields of 25 to 27 g/l at temperatures below 31°C.These results demonstrate that it is possible to direct the metabolism of immobilized cells from ethanol fermentation to glycerol fermentation by sulfite.     

Galactose oxidase production by immobilized cells ofDactylium dendroides

Summary Dactylium dendroides cells were immobilized with calcium alginate, calcium pectate and k-carrageenan. Alginate immobilized cells produced relatively small amounts of (D-galactose: O₂ oxidoreductase, EC 1.1.3.9, GOase). Pectate immobilized cells gave the best yield of GOase, which was comparable with that obtained with free cells, and productivity could be extended up to 28 days (7 cycles). Controlled dosage of phosphate to the medium markedly improved GOase production with higher yields per cycle than with free cells.     

Continuous production of L-malic acid by immobilized cells

l-Malic acid is used extensively in the pharmaceutical industry and as a food additive. It is now produced on an industrial scale by the enzymatic conversion of fumaric acid using immobilized cells of Brevibacterium flavum. Recent improvements to this system, especially the use of x-carrageenan supports, have resulted in a continuous process capable of yielding 30 tonnes of l-malic acid per month.     

Ethanol production by immobilized cells of Zymomonas mobilis

Summary Columnar reactors containing immobilized cells of Zymomonas mobilis were utilized for the continuous production of ethanol from glucose. Two different immobilization strategies were investigated. In one case, cells were entrapped in borosilicate glass fiber pads, while in the other, cells were immobilized via flocculation. The reactors were operated in both the fixed-bed and expanded-bed manner. Ethanol productivities as high as 132 g/l·h were achieved. Data obtained from studies employing 5.0 and 10.0% glucose concentrations are presented. Problems encountered during the operation of the continuous, immobilized cell reactors are discussed.Operated by Union Carbide Corporation under contract W-7405-eng-26 with the U.S. Department of Energy.     

Lactic acid fermentation by cells of Lactobacillus rhamnosus immobilized in polyacrylamide gel

Summary The process of lactic acid fermentation of lactose to lactic acid by Lactobacillus rhamnosus ATCC 7469 has been studied. The following processes have been explored: growth kinetics, as well as lactose utilization, production of lactic acid and further degradation of lactic acid. The immobilization experiments were conducted with microbial cells entrapped in polyacrylamide gels. Gels with different ratios of the monomer (acrylamide) and the cross-linking agent (N,N′-methylene-bis-acrylamide) have been tested. These were used in a repeat-batch process. The current processes inside and outside the gel particles were subjects of examination. The evolution of the activity of immobilized cells with repeated use showed that the particles served mainly as a donor of cells for the free culture. In all experiments a very high degree of conversion, 85–90% was observed. After several runs however, the particles were exhausted for microbial cells. A kinetic model of the process of lactic acid production was developed. This model allowed the evaluation of the effect of microbial growth and diffusion limitations inside the gel particles on the process rate and the separate contribution of the free and immobilized cells to the overall fermentation process upon multiple use.     

Synthesis of l-dopa by escherichia intermedia cells immobilized in a polyacrylamide gel

Summary Escherichia intermedia cells were immobilized by entrapment in a polyacrylamide gel and used for l-dopa synthesis from pyrocatechol, pyruvate and ammonia. An immobilized cell preparation containing 75 mg cells/g gel retained 45%–50% of the activity of free cells. The effect of temperature, pH and substrate concentration of the initial rate of l-dopa synthesis was very similar for free and immobilized cells. Substrate inhibition was observed for pyrocatechol, pyruvate and ammonia. In a batch reactor, 5.4 g·l^-1 l-dopa was obtained, with 100% conversion yield of pyrocatechol and l-dopa productivity of 0.18 g·l^-1·h^-1. The use of a pyrocatechol-borate complex decreased by-product formation and catalyst inactivation.     

Isomaltulose production using immobilized cells

Three strains of Erwinia rhapontici especially suitable for use in the form of nongrowing immobilized cells were selected by screening strains of cells for high activity and operational stability in an immobilized form. Immobilization in calcium alginate gel pellets was easily the best method of immobilizing E. rhapontici. Much greater operational stabilities were obtained than when other immobilization methods were used. Conditions of operation which optimize the activity, stability, and yield and the ease of operation of the immobilized cell columns working in a steady state are described. These include the effects of substrate concentration, diffusional restrictions and water activity, the concentration of cells immobilized, and the type of reactor used. Thus, the immobilized cells produce about 1500 times their own weight of isomaltulose during one half-life of use (ca. 1 year). Loss of activity was most closely correlated with the volume of substrate processed and so presumably is due to the presence of low concentrations of a cummulative inhibitor in the substrate. Methods for regenerating the activity of the immobilized cells by the periodic administration of nutrients, of forming isomaltulose by continuously supplying nutrients to growing immobilized cells, and of crystallizing isomaltulose from the column eluate are also described.     

Hydrogen production by immobilized cells in the nozzle loop bioreactor

Summary The continuous production of hydrogen in a Nozzle Loop Bioreactor was investigated using immobilized Rhodospirillum rubum KS-301 with glucose as the growth-limiting substrate. The maximum hydrogen production rate in the experimental range was 91mL/h at dilution rate 0.4h^-1, initial glucose concentration 5.4g/L, and circulation rate 70h^-1 .     

Inactive proteinases in silkmoth moulting gel

The moulting gel of silkmoths lacks proteolytic activity but contains an inactive form of the proteinases which are later found in the moulting fluid. These inactive enzymes are activatable in vitro by dilution, activation proceeding most rapidly at low ionic strength. Activation proceeds as a first-order process and is not autocatalytic. Approximately the full amount of proteinases ultimately found in moulting fluid are already present in the gel. Moulting gel does not inhibit the active proteinases of moulting fluid; moreover, the proteolytic activity elicited by dilution of the moulting gel does not disappear upon reconcentration. These observations suggest that the proteinases in moulting gel are not inhibited by a stable, dissociable inhibitor; they may be present either as compartmentalized active enzymes or as proenzymes. Several possible mechanisms for the in vivo activation at the time of gel to fluid transformation are discussed.