Properties of Trichoderma reesei cells immobilized by the irradiation technique

Aerobic cells of Trichoderma reesei have been immobilized by the radiation polymerization technique using fibrous substances and hydroxyethyl methacrylate. The enzyme [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] productivity and growth of the cells in the immobilized growing cells have been studied. The enzyme (filter paper) activity in the immobilized cells was comparable to that of the intact cells, showing that the cells immobilized with fibrous materials grow and become adhered to the surface of the fibrils. The filter paper activity of the immobilized cells was affected mainly by monomer concentration and the content of the fibrous materials, as well as the irradiation dose. It was demonstrated that in repeated batch culture of the immobilized cells the filter paper activity gave a constant value, and leakage of the cells was not observed.     

l-serine production from methanol and glycine with an immobilized methylotroph

l-Serine production from methanol and glycine was attempted using immobilized resting cells of a methylotroph, Protomonas extorquens NR 1, under automatically controlled conditions. A Ca-alginate system was selected. The conditions for l-serine formation were optimized at 30°C. A concentration of glycine 100 g·l⁻¹ which was the optimum concentration for l-serine production by free resting cells was used in the reaction mixture. The optimum concentrations of methanol and dissolved oxygen were 20 g·l⁻¹ and 5 ppm, respectively. Under the optimum conditions, 11.3 g·l⁻¹ of l-serine was produced within 36 h. The selectivities (mole of l-serine/mole of substrate consumed) of l-serine from methanol and glycine were 4.5% and 95.1%, respectively. The size of gel beads affected the l-serine formation rate. The initial rate of l-serine formation decreased with an increase in the size of beads. However, the l-serine formation rate increased at elevated concentrations of dissolved oxygen, even with large sized beads. This result implies that the oxygen diffusion inside the gel beads limited the l-serine formation rate. The observed effectiveness factor of the immobilized cells could be estimated by the theoretical effectiveness factor of the zero-order reaction with respect to the dissolved oxygen.Repeated use was not feasible without reactivation of the immobilized cells. Reusability was examined by reactivation of the immobilized resting cells in appropriate media for 12 h. The reactivated immobilized resting cells were used again in the next cycle. By this procedure, several cycles of l-serine formation were made possible.     

Preparation of immobilized whole cell biocatalyst and biodiesel production using a packed-bed bioreactor

Rhizopus oryzae NBRC 4697 was selected from among promising candidates as a biocatalyst for biodiesel production. This microorganism was immobilized on to polyurethane foam coated with activated carbon for reuse, and, for biodiesel production. Vacuum drying of the immobilized cells was found to be more efficient than natural or freeze-drying processes. Although the immobilized cells were severely inhibited by a molar ratio of methanol to soybean oil in excess of 2.0, stepwise methanol addition (3 aliquots at 24-h feeding intervals) significantly prevented methanol inhibition. A packed-bed bioreactor (PBB) containing the immobilized whole cell biocatalyst was then operated under circulating batch mode. Stepwise methanol feeding was used to mitigate methanol inhibition of the immobilized cells in the PBB. An increase in the feeding rate (circulating rate) of the reaction mixture barely affected biodiesel production, while an increase in the packing volume of the immobilized cells enhanced biodiesel production noticeably. Finally, repeated circulating batch operation of the PBB was carried out for five consecutive rounds without a noticeable decrease in the performance of the PBB for the three rounds.     

Dehydrogenation of cortisol by Arthrobacter simplex immobilized as supported monolayer

Arthrobacter simplex has been successfully immobilized by adhesion on glass, either by coating the support with colloidal particles of hydrous alumina or by pretreating the cells with aluminium ions. The use of glass slides as a model support has shown that a single, dense and regular layer of immobilized cells is achieved. The quantity of immobilized cells is about 7 × 10⁷ cells cm^?2. Immobilization on glass beads or glass wool packed as a bed in a column has also been successful. Transformation of cortisol to prednisolone has been tested under no-growth conditions in the absence of nutrients. The specific activity of immobilized cells is not significantly different from that of free cells. The use of a microreactor with the immobilized bacteria as biocatalyst demonstrated the feasibility of repeated use of the microorganisms.     

Biodegradation of benzene,toluene, ethylbenzene,and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (>1000 mg l⁻¹) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l⁻¹. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Characterization of Nitrosomonas europaea immobilized in calcium alginate

Nitrosomonas europaea cells have been immobilized in calcium alginate and the resulting preparation was used as a biocatalyst for the oxidation of NH⁺₄ to NO^?₂. Characterization of this immobilized biocatalyst was done according to the guidelines recommended by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The most important indications obtained from the results are: (a) at low concentrations of substrate, either ammonium ions or oxygen, diffusion limitation will play a role; (b) inhibition by nitrite ions accumulating in the support is not rapidly controlling the efficiency of the immobilized cells; (c) accumulation of hydrogen ions is a rate-limiting factor, especially in unbuffered solutions; (d) the activity of immobilized N. europaea can increase as a result of growth in the support under conditions which would cause washout of free cells. This last result shows the potential of immobilized N. europaea for nitrification of wastewater. The development of a system applying a cheaper and more stable support is, however, a prerequisite for this application.     

Enhancing the Viability of Lactobacillus plantarum Inoculum by Immobilizing the Cells in Calcium-Alginate Beads Incorporating Cryoprotectants

Many literature reports have cited the importance of the rehydration conditions of lyophilized cultures in determining viability. The rate of rehydration and the volume of fluid used have been identified as two important factors. One possible means of controlling these is by immobilizing the cells before lyophilization within a gel matrix in which the subsequent rehydration rate and fluid volume would be controlled by the properties of the gel. In this study Lactobacillus plantarum was immobilized and lyophilized in Ca-alginate beads in which 1 M glycerol or 0.75 M adonitol with skim milk were incorporated as a cryoprotectant. The properties of these Ca-alginate beads were examined before and after lyophilization and rehydration. The beads incorporating glycerol were smaller and stronger than those with adonitol. After lyophilization, size decreased and strength increased but to a greater extent in the beads with glycerol, indicating that the microenvironment within the two bead types was probably different. The protective effect of the bead microenvironment on immobilized L. plantarum was also examined. Lyophilization and rehydration within the alginate beads with either polyol yielded higher survival rates than that attained with free cell cultures during rehydration in optimal or suboptimal conditions. During rehydration under suboptimal conditions, the immobilized cell survival was greatest when 0.75 M adonitol was the incorporated cryoprotectant.     

Catalytic properties of a nitrile hydratase immobilized on activated chitosan

The catalytic properties of a nitrile hydratase, isolated from a strain of Rhodococcus ruber gt1 and immobilized by covalent cross-linking with chitosan activated with 0.1% benzoquinone solution, have been investigated. The kinetic parameters of acrylonitrile hydration catalyzed by immobilized nitrile hydratase and the enzyme in a solution have been determined. It is found that the immobilization does not lead to a decrease in the maximum reaction rate (V _max), whereas the Michaelis constant (K _M) is reduced by a factor of 2.4. The possibility of reusing an immobilized enzyme for 50 consecutive cycles of acrylonitrile transformation was shown, and the nitrile hydratase activity in the 50th cycle exceeded that in the first cycle by 3.5 times. It is shown that the effect of temperature on activity depended on the concentration of the enzyme, which confirms the dissociative nature of nitrile hydratase inactivation. It was found that immobilized nitrile hydratases remain active at pH 3.0–4.0, whereas the enzyme is inactivated in a solution under these conditions. The resulting biocatalyst can be effectively used to receive acrylamide from acrylonitrile.     

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 1. Preparation and enzymatic properties

Kluyveromyces marxianus cells with inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) activity have been immobilized in open pore gelatin pellets with retention of > 90% of the original activity. The open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of calcium alginate from the composite matrix, followed by crosslinking with glutaraldehyde. Enzymatic properties of the gelatin-entrapped cells were studied and compared with those of the free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation; the optimum for both free and immobilized cells was pH 6.0. The optimum temperature of inulin hydrolysis was 10°C higher for immobilized cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 6.35 and 2.26 kcal mol^?1, respectively. K_m values were 8 mM inulin for the free cells and 9.52 mM for the immobilized cells. The thermal stability of the enzyme was improved by immobilization. Free and immobilized cells showed fairly stable activities between pH 4 and 7, but free cell inulinase was more labile at pH values below 4 and above 7 compared to the immobilized form. There was no loss of enzyme activity of the immobilized cells on storage at 4°C for 30 days. Over the same period at room temperature only 6% of the original activity was lost.     

Diesel oil removal by immobilized Pseudoxanthomonas sp. RN402

Pseudoxanthomonas sp. RN402 was capable of degrading diesel, crude oil, n-tetradecane and n-hexadecane. The RN402 cells were immobilized on the surface of high-density polyethylene plastic pellets at a maximum cell density of 10⁸ most probable number (MPN) g^?1 of plastic pellets. The immobilized cells not only showed a higher efficacy of diesel oil removal than free cells but could also degrade higher concentrations of diesel oil. The rate of diesel oil removal by immobilized RN402 cells in liquid culture was 1,050 mg l^?1 day^?1. Moreover, the immobilized cells could maintain high efficacy and viability throughout 70 cycles of bioremedial treatment of diesel-contaminated water. The stability of diesel oil degradation in the immobilized cells resulted from the ability of living RN402 cells to attach to material surfaces by biofilm formation, as was shown by CLSM imaging. These characteristics of the immobilized RN402 cells, including high degradative efficacy, stability and flotation, make them suitable for the purpose of continuous wastewater bioremediation.     

Biological control of Stephanodiscus hantzschii (Bacillariophyceae) blooms in a field mesocosm by the immobilized algicidal bacterium Pseudomonas fluorescens HYK0210-SK09

The aim of the study was to determine the activity of an immobilized algicidal bacterium, Pseudomonas fluorescens HYK0210-SK09 (SK09), against blooms of the diatom Stephanodiscus hantzschii and to characterize its effect in an ecosystem. To this end, SK09 cells that had been immobilized using an activated carbon polyvinyl alcohol sponge (APVAS) were tested in a field mesocosm in the Nakdong River, South Korea. Immobilized SK09 cells showed species-specific activity toward S. hantzschii throughout the study, whereby up to 72 % of the population of this species was killed. The APVAS carrier absorbed nutrients in the mesocosms effectively, which reduced the free concentrations of nutrients. Other phytoplankton species, such as Cryptomonas ovata, did not show any fluctuation in abundance. The abundance of heterotrophic protists, such as heterotrophic flagellates and ciliates, increased significantly owing to the utilization of SK09 as a food source. The high level of algicidal activity of SK09 against S. hantzschii was sustained by the release of these algicidal cells from the carrier. Thus, the immobilization of algicidal bacteria has various advantages, which include high packing ability, enhancement of bacterial growth, protection from bacterial predators, effective nutrient removal, and improved algicidal activity. The present study demonstrates that immobilized SK09 is an effective biocontrol agent for natural S. hantzschii blooms. Taken together, the findings of this study clearly demonstrate that SK09 cells immobilized in APVAS can improve the water quality in mesocosm ecosystems without producing any ecological disturbance.     

Immobilization ofArthrobacter simplex cells in thermally reversible hydrogels: Comparative effects of organic solvent and polymeric surfactant on steroid conversion

Summary Organic solvents have sometimes been used to increase the solubility of water insoluble substrates for steroid transformation using immobilized whole cells, even though the cell viability is often damaged. Polymeric surfactants which form micelles in aqueous solutions could be used instead of organic solvents to solubilize the steroid. We have successfully utilized this approach by employing a poly(dimethyl siloxane)-poly(ethyleneoxide) (PDMS-PEO) block copolymer surfactant to enhance conversion of hydrocortisone to prednisolone by immobilizedArthrobacter simplex cells, without deactivation of the immobilized cells.     

Denitrification of PVA-immobilized denitrifying photosynthetic bacterium,Rhodobacter sphaeroides

A newly isolated denitrifying strain, Rhodobacter sphaeroides NII2 was immobilized in polyvinyl alcohol (PVA) gel, and the properties of the cells in the gel were examined. The immobilized cells had low or almost no denitrification activity, but the cells were activated by incubation in light with culture medium for denitrification containing 0.5% nitrate and no other nitrogen source. Cells grown in the dark were activated by incubation at an earlier stage and to a higher rate than the light-grown cells. The activation was markedly enhanced in the PVA gel with a low cell concentration. The immobilized cells consumed nitrate with a temporary accumulation of NO₂ and evolved nitrogen gas. The immobilized cells could use various organic compounds as electron donors for denitrification. Thus, the immobilized cells were applied to a continuous treatment of synthetic wastewater using an aparatus devised by this laboratory. The results showed an efficient removal of NO₃-N from the test water.     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.     

Methane production from organic acid-rich wastewaters by immobilized thermophilic methane-producing bacteria

Thermophilic methane-producing bacteria isolated from a wastewater treatment facility have been immobilized in acetylcellulose filter with agar. The immobilized cells produced methane from wastewaters in rich organic acid (acetic, propionic and butyric acids) at the rate of 1.4 μmol mg protein⁻¹ h⁻¹. The optimum conditions for methane production by immobilized whole cells were 52–55°C and pH 7.0–8.0. The immobilized cells retained 80% of the initial activity after exposure to air. The immobilized thermophilic bacteria produced methane continuously over 10 days at 52°C.     

An immobilized cell system in polyurethane foam for the lipophilic micro-alga Prototheca zopfii

Cells of the achlorophyllous micro-alga Prototheca zopfii were immobilized in 8-mm-cube polyurethane foam pieces. A 2-fold increase in the volumetric biodegradation rate of the immobilized cells for n-alkanes (mixture of C₁₄, C₁₅ and C₁₆) was observed compared with that of the immobilized system using calcium alginate gel in batch experiments using flasks agitated on a reciprocal shaker at 25°C. The apparent biodegradation rates were influenced significantly by the affinities between algal cells and matrix and/or between hydrocarbons to be degraded and matrix. Such affinities resulted in the improvement of the interaction between the substrates and algal cells. The stability of the immobilized cells was examined in repeated-batch culture and activity was stable over three successive cycles of cultivation. P. zopfii immobilized in polyurethane foam was incorporated into a bubble-column type bioreactor for degrading hydrocarbons and the potential effectiveness of the immobilized cell system was confirmed.     

Copper uptake by free and immobilized cyanobacterium

Abstract Copper uptake in free and immobilized cells of the cyanobacterium Nostoc calcicola has been examined. The immobilized cells invariably maintained a higher profile of Cu intake rate (12.7 nmol mg⁻¹ protein min⁻¹) over the free cells (6.0 nmol mg⁻¹ protein min⁻¹). The total Cu uptake in immobilized cells was almost two and a half-times more than their free cell counterpart under identical experimental conditions. Also, the immobilized cells showed a stronger positive correlation between Cu adsorption and uptake. The results have been discussed in terms of improved metabolic efficiency of immobilized cells.     

Peptide antibiotic production through immobilized biocatalyst technology

The use of immobilized biocatalysts for producing known or new antibiotics is presented. An evaluation of the applicability of this concept in the fascinating field of peptide antibiotic bioconversions and fermentations is also given.The use of immobilized enzymes, organelles and cells to synthesize antibiotics as an alternative method to conventional fermentation is discussed. In vitro total enzymatic antibiotic synthesis is illustrated with the ‘multienzyme thiotemplate mechanism’ of Bacillus brevis, the producer of gramicidin S. Total synthesis of peptide antibiotics, based on immobilized living cells, has recently been demonstrated with penicillin, bacitracin, nisin and a few other antibiotics.As an industrial example of the use of enzymes or cells to convert peptide antibiotics into therapeutically useful derivatives, free and immobilized penicillin acylases, producing the penicillin nucleus 6-aminopenicillanic acid (6-APA), are reviewed as well as their potential to synthesize semisynthetic β-lactams (penicillins, cephalosporins).Acylases, acetylesterases and α-amino acid ester hydrolases acting on cephalosporin-compounds and yielding valuable intermediary or end products have also gained wide interest. Stereospecific enzymic side-chain preparations for semisynthetic penicillin and cephalosporin production have recently reached the industrial stage. Bioconversion possibilities with the novel β-lactam compounds are suggested.These examples of simple single-step, as well as complex multi-step, enzyme reactions point to the vast potential of immobilized biocatalyst technology in fermentation science, in organic synthesis and in biotechnological processes in general.     

Immobilization of Glucose Isomerase-Containing Streptomyces phaeochromogenes Cells in Fine-Particle Form

A new preparation method for immobilizing Streptomyces phaeochromogenes cells in fine-particle form was investigated using radiation-induced polymerization at low temperatures with previously salted out hydrophilic monomers. Using this method, it was found that the glucose isomerase activity of the immobilized cell particles was markedly higher than that of immobilized cells in block form obtained without salting out of the monomer. The diameter of the particles was varied by changing the irradiation temperature or the concentrations of monomer and salt. The magnitude of the enzymatic activity increased with decreasing particle diameter. K_m values of the immobilized cell particles were close to that of the intact cell. These facts suggested that the cells were trapped on the surface of the particle.     

Hydrogen evolution by co-immobilized Chlorella vulgaris and Clostridium butyricum cells

Whole cells of Chlorella vulgaris and Clostridium butyricum were co-immobilized in 2% agar gel. NADP was suitable as an electron carrier. The rate of hydrogen evolution increased with increasing NADP concentration. The optimum conditions for hydrogen evolution were pH 7.0 and 37°C. The immobilized C. vulgaris-NADP-immobilized Cl. butyricum system continuously evolved hydrogen at a rate of 0.29–1.34 μmol/h per mg Chl for 6 days. On the other hand, the system without NADP evolved only a trace amount of hydrogen.