Comparative study of d-xylose conversion to ethanol by immobilized growing or non-growing cells of the yeast Pachysolen tannophilus

Summary The direct conversion of d-xylose to ethanol was investigated using immobilized growing and non-growing cells of the yeast Pachysolen tannophilus. Both preparations produced ethanol from d-xylose, however the d-xylose conversion to ethanol was much better with immobilized growing cells. Ethanol concentration up to 22.9 g/l and ethanol yield of 0.351 g/g of d-xylose were obtained in batch fermentation by immobilized growing cells whereas only 17.0 g/l and 0.308 g/g of d-xylose were obtained by immobilized non-growing cells. With continuous systems, immobilized growing cells were necessary for the long-term operation, since a steady state ethanol concentration of 17.7 g/l was maintained for only one week by immobilized non-growing cell reactor. With simultaneous control of aeration rate and concentrations of nitrogen sources in feed medium, immobilized growing cells of P. tannophilus showed excellent performance. At a residence time of 25 h, the immobilized cell reactor produced 26.9 g/l of ethanol from 65 g/l of d-xylose in feed medium.     

Immobilization studies of whole microbial cells on transition metal activated inorganic supports

Summary Whole cells of Saccharomyces bayanus, Saccharomyces cerevisiae and Zymomonas mobilis were immobilized by chelation/metal-link processes onto porous inorganic carriers. The immobilized yeast cells displayed much higher sucrose hydrolyzing activities (90–517 U/g) than the bacterial, Z. mobilis, cells (0.76–1.65 U/g). The yeast cells chelated on hydrous metal oxide derivative of pumice stone presented higher initial -d-fructofuranosidase (invertase, EC 3.2.1.26) activity (161–517 U/g) than on other derivatives (90–201 U/g). The introduction of an organic bridge between the cells and the metal activator led to a decrease of the initial activity of the immobilized cells, however S. cerevisiae cells immobilized on the carbonyl derivative of titanium (IV) activated pumice stone, by covalent linkage, displayed a very stable behaviour, which in continuous operation at 30° C show only a slightly decrease on invertase activity for a two month period (half-life=470 days). The continuous hydrolysis of a 2% w/v sucrose solution at 30° C in an immobilized S. cerevisiae packed bed reactor was described by a simple kinetic model developed by the authors (Cabral et al., 1984a), which can also be used to predict the enzyme activity of the immobilized cells from conversion degree data.     

Conversion of D-sorbit to L-sorbose by cells of Acetobacter suboxydans immobilized in sintered glass

A simple method for immobilizing cells of Acetobacter suboxydans by adsorption on inorganic sintered glass carriers is described. The immobilized cell preparations exhibited 100% of the initial activity when converting d-sorbit to l-sorbose. This sintered glass was used in a fixed-bed loop reactor with a working volume of 0.2 l for semicontinuous and continuous experiments. A prolonged working life span was achieved, which could possibly satisfy requirements for scaled-up operations.     

Production of l-glutamate by immobilized protoplasts

Summary Protoplasts of Brevibacterium flavum cultured in a medium containing 50 g·l^-1 of biotin were prepared with lysozyme and immobilized in matrices of agar-acetylcellulose filters. The immobilized protoplasts were applied to l-glutamate production from glucose and urea in a batch system. The productivity of l-glutamate by the immobilized protoplasts was 2.5 times higher than that by immobilized whole cells under optimal conditions. Maximal productivity initially reached 1.5 mg·ml^-1. The immobilized protoplasts of B. flavum could be used six times for l-glutamate production with retention of about 70% of the initial productivity.     

Continuous production of L-arginine using immobilized growing Serratia marcescens cells: Effectiveness of supply of oxygen gas

Summary The immobilized growing cell system using Serratia marcescens was applied to continuous L-arginine production. From the determination of oxygen uptake rate, it was shown that the cells entrapped in carrageenan gel were in an oxygen-limited state due to the diffusion barrier to oxygen transport created by the gel layer. This limited state in gel was relieved by supply of oxygen-enriched gas instead of air into the medium. The maximum population of immobilized cells increased to five times that of free cells with the supply of pure oxygen gas. The L-arginine-producing activity of the immobilized growing cells was proportional to the concentration of oxygen gas supplied and was 6 mg/h per millilitre in gel supplied with pure oxyges gas. The continuous L-arginine containing production was constantly maintained by controlling the medium penicillin G at pH 6.5 and more than 10 mg/ml of L-arginine were obtained at 10h of residence time for at least 12 days.     

Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana

Escherichia coli cells expressing l-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t _1/2 = 43 days at 70°C) in a continuous recycling mode at 70°C produced 49 and 38 g d-tagatose/l from 180 and 90 g d-galactose/l, respectively, within 12 h.     

Recovery of uranium by immobilized microorganisms

Summary Some attempts were made to recover uranium from sea and fresh water using immobilized Streptomyces viridochromogenes and Chlorella regularis cells. The cells immobilized in polyacrylamide gel have the most favorable features for uranium recovery; high adsorption ability, good mechanical properties, and applicability in a column system. The adsorption of uranium by the immobilized cells is not affected by the pH values between 4 and 9. These results show that uranium adsorption becomes independent of pH after immobilization. The amounts of uranium adsorbed by the immobilized cells increased linearly with temperature, suggesting that the adsorption of uranium by the immobilized cells is an endothermic reaction. The immobilized cells can recover uranium almost quantitatively from both fresh and sea water containing uranium, and almost all uranium adsorbed is desorbed with a solution of Na₂CO₃. Thus the immobilized cells of Streptomyces and Chlorella can be used repeatedly in adsorption-desorption process.Studies on the Accumulation of Heavy Metal Elements in Biological Systems. XXI     

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     

The effects of immobilization on the biochemical,physiological and morphological features of Anabaena azollae

Anabaena azollae, a presumptive isolate from Azolla filiculoides, was immobilized in polyurethane foam, hydrophilic polyvinyl foam and alginate. When viewed by low-temperature scanning electron microscopy a thick mucilage layer covered the surface of both cells and matrix; this closely resembles the mode of attachment of the symbiont Anabaena in the Azolla leaf cavity. The heterocyst frequency of the immobilized A. azollae doubled relative to free-living cells and reached a level of 14–17%. Immobilization induced increases in both hydrogen production via nitrogenase or hydrogenase and in the rates and stabilization of acetylene reduction (N₂-fixation). Ammonia production by immobilized cells with L-methionine-D,L-sulfoximine (MSX) is greater than that of freeliving cells. Immobilized cells without MSX were, however, able to excrete ammonium at lower rates thus emulating the characteristic of the symbiotic cyanobacteria (A. azollae) in the leaf cavity of Azolla.Abbreviations Chl chlorophyll - GS glutamine synthetase - MSX L-methionine-D,L-sulfoximine - SEM scanning electron microscopy - PU polyurethane - PV polyvinyl     

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.     

Production of l (+) lactic acid by Lactobacillus delbrueckii immobilized in functionalized alginate matrices

The role of functionalized alginate gels as immobilized matrices in production of l (+) lactic acid by Lactobacillus delbrueckii was studied. L. delbrueckii cells immobilized in functionalized alginate beads showed enhanced bead stability and selectivity towards production of optically pure l (+) lactic acid in higher yields (1.74Yp/s) compared to natural alginate. Palmitoylated alginate beads revealed 99% enantiomeric selectivity (ee) in production of l (+) lactic acid. Metabolite analysis during fermentation indicated low by-product (acetic acid, propionic acid and ethanol) formation on repeated batch fermentation with functionalized immobilized microbial cells. The scanning electron microscopic studies showed dense entrapped microbial cell biomass in modified immobilized beads compared to native alginate. Thus the methodology has great importance in large-scale production of optically pure lactic acid.     

Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

Production of l-alanine from ammonium fumarate using two immobilized microorganisms

Summary For the efficient production of l-alanine from ammonium fumarate using the aspartase activity of immobilized Escherichia coli cells and l-aspartate -decarboxylase activity of immobilized Pseudomonas dacunhae cells, alanine racemase and fumarase activities should be eliminated. We investigated various procedures to eliminate these side reactions, and found that both activities of intact E. coli cells could be eliminated by treating the culture broth at pH 5.0 and 45° C for 1 h, and those of intact P. dacunhae cells could be eliminated by treating the culture broth at pH 4.75 and 30° C for 1 h. Further, it was confirmed that l-alanine was efficiently produced using these two immobilized pH-treated microorganisms.     

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E_(0.3)), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3 U/mg protein, which is 0.46 times less than that of the free lipase (35.6 U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 °C respectively. Kinetic parameters V_max and K_m were also determined for the immobilized lipase. It was observed that there is an increase of the K_m value (7.54 mM) and a decrease of the V_max value (145.0 U/mg-protein) comparing with that of the free lipase.     

Preparation of optically active β-hydroxy-α-amino acid by immobilized <Emphasis Type="Italic">Escherichia coli</Emphasis> cells with serine hydroxymethyl transferase activity

In this research, an improved method for preparation of optically pure β-hydroxy-α-amino acids, catalyzed by serine hydroxymethyl transferase with threonine aldolase activity, is reported. Using recombinant serine hydroxymethyl transferase (SHMT), an enzymatic resolution process was established. A series of new substrates, β-phenylserine, β-(nitrophenyl) serine and β-(methylsulfonylphenyl) serine were used in the resolution process catalyzed by immobilized Escherichia coli cells with SHMT activity. It was observed that the K _m for l-threonine was 28-fold higher than that for l-allo-threonine, suggesting that this enzyme can be classified as a low-specificity l-allo-threonine aldolase. The results also shows that SHMT activity with β-phenylserine as substrate was about 1.48-fold and 1.25-fold higher than that with β-(methylsulfonylphenyl) serine and β-(nitrophenyl) serine as substrate, respectively. Reaction conditions were optimized by using 200 mmol/l β-hydroxy-α-amino acid, and 0.1 g/ml of immobilized SHMT cells at pH 7.5 and 45°C. Under these conditions, the immobilized cells were continuously used 10 times, yielding an average conversion rate of 60.4%. Bead activity did not change significantly the first five times they were used, and the average conversion rate during the first five instances was 84.1%. The immobilized cells exhibited favourable operational stability.     

Improvement of production of l-aspartic acid using immobilized microbial cells

Summary In our laboratory, EAPc-7 a strain having higher aspartase activity was derived from Escherichia coli ATCC 11303. For the improvement of l-aspartic acid productivity using EAPc-7 cells immobilized in -carrageenan, it was necessary to eliminate the fumarase activity which converts fumaric acid to l-malic acid. Several treatments for specifically eliminating fumarase activity from EAPc-7 cells were tested and it was found that when EAPc-7 cells were treated in a culture broth (pH 4.9) containing 50 mM l-aspartic acid at 45° C for 1 h, fumarase activity was almost completely eliminated without inactivation of the aspartase.The treated cells, immobilized in -carrageenan, were used for continuous production of l-aspartic acid from ammonium fumarate. The formation of l-malic acid was negligible and the half-life of the immobilized preparation was 126 days.Productivity of immobilized preparation of treated EAPc-7 cells in l-aspartic acid production was six times of that of the parent cell preparation.     

Continuous production of l-serine by immobilized growing Corynebacterium glycinophilum cells

Summary Auxotrophic mutant cells of Corynebacterium glycinophilum with high l-serine production activity were immobilized by entrapment with various gel materials, such as synthetic prepolymers and natural polysaccharides. The entrapped cells were used for estimation of l-serine productivity in a medium supplemented with glycine as a precursor. Based on the above criteria, including cell growth in gels and cell leakage from gels, calcium alginate was the most suitable gel material. Continuous l-serine fermentation with calcium alginate-entrapped growing cells was successfully achieved in an air-bubbled reactor for at least 13 days.     

Physiological adsorption of Sulfolobus acidocaldarius on coal surfaces

Summary The adsorption of Sulfolobus acidocaldarius on bituminous coal surfaces and the respiration rate during adsorption at 70° C were enhanced at pH 1.0–2.0, in comparison with those at pH 3.0–5.0. The maximum number of bacterial cells adsorbed per unit area of coal attained a maximum (1.4 × 10¹¹ cells/m²) at pH 2.0. The rate of desulphurization at pH 2.2–2.5 was higher than at other pHs tested. Micrographs of S. acidocaldarius obtained by TEM and SEM indicated that the cells were adsorbed to the coal surfaces by extracellular slime. Specific inhibitors of membrane-bound ATPase (NaF, 20 mm) and respiration (NaN₃, 1 mm; KCN, 1 mm) had pronounced effects on suppressing adsorption. The amount of S. acidocaldarius adsorbed decreased when the coal particles were leached in advance with 2.0 m HNO₃. These facts lead to the conclusion that the adsorption of S. acidocaldarius on coal surfaces requires physiological activity relatd to respiration or energy conversion. Offprint requests to: V. B. Vitaya     

Immobilization of <Emphasis Type="Italic">Escherichia coli</Emphasis> cells with <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase activity for <Emphasis Type="SmallCaps">d</Emphasis>(−)-tartaric acid production

Immobilization of cis-epoxysuccinate hydrolase-containing E. coli for d(−)-tartaric acid production was screened by various methods. The highest recovery of activity was obtained by entrapment in κ-carrageenan gel. 23.6 g biomass/l and 43.4 g κ-carrageenan/l were the best immobilization conditions optimized by response surface methodology with 83% yield (114 U/g). Cell autolysis was observed after immobilization. Immobilized cells showed high pH (5–10) stability, thermal (up to 65°C) stability, conversion rate (>99.5%), enantioselectivity (ee > 99.6%), and were less affected by metal ions and surfactants compared with free cells. Conversion rate for immobilized cells preserved 93% after 10 repeated batches (5% for free cells).     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.