Immobilization of rice (Oryza sativa L.) callus in polyurethane foam using a turbine blade reactor

A turbine blade reactor (TBR) was employed to cultivate rice calli immobilized in polyurethane foam as a support. In the bioreactor, rice callus could be immobilized quickly in a 3 mm cube of the support, and then attached to the stainless mesh cylinder set at the center of the bioreactor. For improving the immobilization ratio of rice callus in the bioreactor, the optimum support volume and bioreactor operation and modification were investigated. The support volume had a pronounced effect on the immobilization ratio of rice callus, and the maximum volume was found to be 60 ml. By repeating a periodic operation three times (agitating at 300 rpm for 5 min and then 50 rpm for 2 min, and then 200 rpm of constant agitation speed during the remaining time), rice calli were uniformly entrapped in almost all supports and the immobilization ratio was improved as compared with that using a constant bioreactor operation at 200 rpm. When the inoculum concentration of rice callus was increased, the callus concentration after 7-day culture increased, but the immobilization ratio decreased. To improve the immobilization efficiency further at high cell concentration, the TBR was modified by setting an air sparger inside the stainless mesh cylinder. In the modified TBR, floating of the support by attached air bubbles was avoided, and the immobilization ratio increased further and reached 86.3% when we increased the support volume to 90 ml under the periodic bioreactor operation on a daily basis. The regeneration frequency of immobilized callus was increased by periodic operation and modification of the bioreactor.     

Characteristics of immobilized Rhizopus oryzae in polyurethane foam cubes

Summary Rhizopus oryzae was immobilized in polyurethane foam cubes. The effects of the cube size on cell immobilization, cell growth and L(+)-lactic acid production were studied. By the natural attachment method, R. oryzae could be easily immobilized in the polyurethane foam cubes larger than 2.5 × 5 × 5 mm³. The use of small cubes for R. oryzae immobilization was very effective to increase the productivity of L(+)-lactic acid by the immobilized cells. Although it was difficult for smaller cubes to be completely full of the mycelia, increasing the inoculum size in immobilizations was effective to increase the immobilization ratio (a ratio of the number of the cubes containing cells to the total number of cubes).     

Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods

Calcium-alginate immobilization method for the production of alkaline protease by Bacillus licheniformis NCIM-2042 was optimized statistically. Four variables, such as sodium-alginate concentration, calcium chloride concentration, inoculum size and agitation speed were optimized by 2(4) full factorial central composite design and subsequent analysis and model validation by a second-order regression equation. Eleven carbon, 11 organic nitrogen and seven inorganic nitrogen sources were screened by two-level Plackett-Burman design for maximum alkaline protease production by using optimized immobilized conditions. The levels of four variables, such as Na-alginate 2.78%; CaCl(2), 2.15%; inoculum size, 8.10% and agitation, 139 rpm were found to be optimum for maximal production of protease. Glucose, soybean meal and ammonium sulfate were resulted in maximum protease production at 644 U/ml, 720 U/ml, and 806 U/ml when screened for carbon, organic nitrogen and inorganic nitrogen sources, respectively, using optimized immobilization conditions. Repeated fed batch mode of operation, using optimized immobilized conditions, resulted in continuous operation for 12 cycles without disintegration of beads. Cross-sectional scanning electron microscope images have shown the growth pattern of B. licheniformis in Ca-alginate immobilized beads.     

Kinetic studies on urea hydrolysis by immobilized urease in a batch squeezer and flow reactor

The immobilization of urease on the reticulated polyurethane foam, and the kinetic phenomenon of urea hydrolysis by the resulting immobilized urease in both batch squeezer and circulated flow reactors were studied. Urease was immobilized with bovine serum albumin and glutaraldehyde on polyurethane foam support of 7 to 15 mum thickness. The residual apparent activity of urease after immobilization was about 50%. The good hydrodynamic property and flexibility of polyurethane foam were retained in solution after immobilization. A modified biofilm reactor model was used to describe the kinetic phenomenon of urea hydrolysis in both batch squeezer and circulated flow reactors. The characteristic parameters of the reactor model for both bioreactors were obtained by combining the Rosenbrock optimization method, the Rungs-Kutta method, and the Newton-Raphson method. The best-fit results were in good agreement with the experimental data. This study suggests another application of polyurethane foam in enzyme immobilization and immobilized enzyme reactors, which offers potential for practical applications in various bioreactors. (c) 1992 John Wiley & Sons, Inc.     

Ferrous iron oxidation by foam immobilized Acidithiobacillus ferrooxidans: Experiments and modeling

Ferrous iron bio‐oxidation by Acidithiobacillus ferrooxidans immobilized on polyurethane foam was investigated. Cells were immobilized on foams by placing them in a growth environment and fully bacterially activated polyurethane foams (BAPUFs) were prepared by serial subculturing in batches with partially bacterially activated foam (pBAPUFs). The dependence of foam density on cell immobilization process, the effect of pH and BAPUF loading on ferrous oxidation were studied to choose operating parameters for continuous operations. With an objective to have high cell densities both in foam and the liquid phase, pretreated foams of density 50 kg/m³ as cell support and ferrous oxidation at pH 1.5 to moderate the ferric precipitation were preferred. A novel basket‐type bioreactor for continuous ferrous iron oxidation, which features a multiple effect of stirred tank in combination with recirculation, was designed and operated. The results were compared with that of a free cell and a sheet‐type foam immobilized reactors. A fivefold increase in ferric iron productivity at 33.02 g/h/L of free volume in foam was achieved using basket‐type bioreactor when compared to a free cell continuous system. A mathematical model for ferrous iron oxidation by Acidithiobacillus ferrooxidans cells immobilized on polyurethane foam was developed with cell growth in foam accounted by an effectiveness factor. The basic parameters of simulation were estimated using the experimental data on free cell growth as well as from cell attachment to foam under nongrowing conditions. The model predicted the phase of both oxidation of ferrous in shake flasks by pBAPUFs as well as by fully activated BAPUFs for different cell loadings in foam. Model for stirred tank basket bioreactor predicted within 5% both transient and steady state of the experiments closely for the simulated dilution rates. Bio‐oxidation at high Fe²⁺ concentrations were simulated with experiments when substrate and product inhibition coefficients were factored into cell growth kinetics. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Production of gluconic acid by Aspergillus niger immobilized on polyurethane foam

Production of gluconic acid by cells of Aspergillus niger immobilized on polyurethane foam was studied in repeated-batch shake-flask and bubble-column fermentations. For passive immobilization, various amounts of polyurethane foam and spore suspension were tested in order to obtain a suitable combination for optimal concentration of immobilized biomass. Immobilized cells were sucessfully reused with higher levels of product formation being maintained for longer period (65–70h) than free cells. The highest gluconic acid concentration of about 143 g l^–1 was reached on hydrol-based production medium with 0.3-cm³ foam cubes in the bubble column, where the effect of more suitable aeration and particle volume: medium volume ratio scheme was also investigated.     

聚氨酯固定化热带假丝酵母发酵木糖醇

固定在多孔聚氨酯载体中的热带假丝酵母(Candida tropicalis), 可有效地利用玉米芯半纤维素水解液生产木糖醇。在摇瓶条件下, 采用分批发酵方式, 确立了适宜的发酵工艺参数为: 接种量7%, 聚氨酯加入量1.0 g/100 mL, 温度30°C, 初始pH值6.0, 分段改变摇床转速进行溶氧调节, 其中0~24 h 为200 r/min; 24 h~46 h为140 r/min。聚氨酯固定化提高了菌体对发酵抑制物的耐受力, 固定化细胞密度高, 发酵性能稳定, 发酵产率和体积生产速率都有所提高。水解液未经脱色与离子交换便可转化成木糖醇, 大幅降低了成本, 显示了良好的应用前景。固定化细胞连续重复进行12批次21 d的发酵, 木糖醇得率平均为67.6%, 体积生产速率平均为1.92 g/(L·h)。     

Immobilization and culture of coffee (Coffea arabica L.) cells on novel culture systems using a basket-shaped unit, “EGSTAR”

Summary A simple and new basket-shaped unit for agitation made of stainless steel (EGSTAR), in which immobilized coffee cells in Ca-alginate gel beads were packed, was placed in a jar fermentor (System-1). This system allowed the plant cells to grow submersed in the unit even at high agitation speed (650 rpm). Only a small amount of cells existed out of the EGSTAR. Most of the purine alkaloids produced were released into the medium. Suspended coffee cells in the jar fermentor were also possibly immobilized onto a polyurethane foam sheet fixed inside the net of the EGSTAR (System-2). The total cells in System-2 biotransformed theobromine to caffeine (77.9%). Other plant cell suspensions were also immobilized as efficiently as were the coffee cells in this system. Thus, System-2 is a simple and convenient system for immobilization of plant cells to produce secondary metabolites. This paper is Part 79 in the series of “Studies on Plant Tissue Cultures”. For Part 78, see Orihara, Y., Furuya, T., Hashimoto, N., Deguchi, Y., Tokoro, K., and Kanisawa, T., (1992)Phytochemistry 31: 827–831.     

Citric acid production by Aspergillus niger immobilized on polyurethane foam

Summary Citric acid was produced using Aspergillus niger immobilized on polyurethane foam in a bubble column reactor. Most of the adsorbed cells remained on the support and, as a result, high oxygen tension was maintained during the reactor operation. However, uncontrolled growth of the pellets made continuous reactor operation difficult. The citric acid productivity obtained from 15 vol.% foam particles containing immobilized cells was 0.135 g/l per hour. This productivity of immobilized cells was almost the same as that of free cells. The oxygen level dropped to half saturation in 5 days in the immobilized cell culture in contrast to 2 days in the free cell culture.     

聚氨酯泡沫固定产脂肪酶粗状假丝酵母（Candidav alida T2）细胞的研究

对聚氨酯泡沫固定产脂肪酶粗状假丝酵母（Candida validaT2）细胞的固定化条件进行了研究。实验结果表明,聚氨酯泡沫颗粒经密度和粒径筛选,酸碱处理,以及固液比优化,载体固定细胞干质量比达到157lmg／g,细胞脂肪酶的酶活为每克干细胞1415U。电镜图片显示粗状假丝酵母菌（Candida validaT2）在载体孔隙内和脊壁上缠绕充盈,生长良好,固定结构稳定。固定化细胞连续催化水解桐籽油5批次,细胞相对水解酶活保持率达73％,固定细胞的损失率为12．5％。固定化细胞颗粒显示出良好的操作稳定性和酶活保持率,为进一步的应用研究提供了实验基础。     

Haloalkane hydrolysis with an immobilized haloalkane dehalogenase.

Haloalkane dehalogenase from Rhodococcus rhodochrous was covalently immobilized onto a polyethyleneimine impregnated gamma-alumina support. The dehalogenating enzyme was found to retain greater than 40% of its original activity after immobilization, displaying an optimal loading (max. activity/supported protein) of 70 to 75 mg/g with an apparent maximum (max. protein/support) of 156 mg/g. The substrate, 1,2,3-trichloropropane, was found to favorably partition (adsorb) onto the inorganic alumina carrier (10 to 20 mg/g), thereby increasing the local reactant concentration with respect to the catalyst's environment, whereas the product, 2,3-dichloropropan-1-ol, demonstrated no affinity. Additionally, the inorganic alumina support exhibited no adverse effects because of solvent/component incompatibilities or deterioration due to pH variance (pH 7.0 to 10.5). As a result of the large surface area to volume ratio of the support matrix and the accessibility of the bound protein, the immobilized biocatalyst was not subject to internal mass transfer limitations. External diffusional restrictions could be eliminated with simple agitation (mixing speed: 50 rpm; flux: 4.22 cm/min). The pH-dependence of the immobilized dehalogenase was essentially the same as that for the native enzyme. Finally, both the thermostability and resistance toward inactivation by organic solvent were improved by more than an order of magnitude after immobilization.     

Aspergillus niger inulinase immobilized in polyurethane foam and treated in pressurized LPG: A potential catalyst for enzymatic synthesis of fructooligosaccharides

Inulinase from Aspergillus niger was immobilized in polyurethane foam (PU). Immobilized catalyst was treated in pressurized liquefied petroleum gas (LPG) system. This biocatalyst was used in the fructooligosaccharide production using sucrose as substrate in aqueous system. The main objective of this study was to evaluate the reaction yield and productivity by using polyurethane foam as a low-cost support for enzyme immobilization in an alternative processes for fructooligosaccharide production in pressurized LPG system with potential for industrial application. The total FOS concentration obtained were 31% as a result of sucrose concentration reduction, and formation of FOS long chain (GF3 and GF4) from kestose (GF2). FOS concentrations of 5%, 22%, and 3% were obtained for GF2, GF3, and GF4, respectively. The methodology suggested in this research work, enzyme immobilization in a low-cost support, and treatment in LPG, showed potential technology for fructooligosaccharide synthesis.     

Improving reuse cycles of Thermomyces lanuginosus lipase (NS-40116) by immobilization in flexible polyurethane

Enzyme immobilization using a low-cost support that allows increasing operational stability and reutilization arise as a great economic advantage for the industry. In this work, it was explored different methods of Thermomyces lanuginosus lipase (NS-40116) immobilization in flexible polyurethane foam (PU). PU polymer was synthesized using polyether and toluene diisocyanate as monomers. PU-NS-40116 immobilized was evaluated in terms of stability in a range of pH (7.0 and 9.0), temperature (24, 50 and 60?°C) for 24?h, and storage stability (room temperature and 4?°C) for 30?days. The results showed that after 30?days of storage immobilized enzyme kept 80% of initial enzyme activity. PU support before and after immobilization process was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Free and immobilized enzymes were compared in terms of hydrolysis of soybean oil. Immobilized enzyme by entrapment was evaluated in successive cycles of reuse showing catalytic activity above 50% even after 5 successive cycles of reuse, confirming the efficiency of immobilization process.     

Biosynthesis of pullulan using immobilized Aureobasidium pullulans cells

Immobilization of Aureobasidium pullulans by adsorption on solid supports and entrapment in open pore polyurethane foam were attempted. By adsorption, the highest cell loading of 0.012-0.018 g dry wt/cm(2) support was obtained in pH 2.0 medium. Under this acidic condition, the net surface charges (zeta potentials) of both the cells and supports were close to zero and no pullulan was synthesized. Cationic coatings of Cytodex and polyethylenimine were not efficient in enhancing the binding strength between the cells and the supports. Surface immobilized cells and polyurethane foam entrapped cells exhibited a similar fermentation characteristics resulting in ca. 18 g/L pullulan and ca. 5 g/L leaked cells. However, cells entrapped in the polyurethane foam were more shear resistant. The immobilized cells thus could be repeatedly used for pullulan biosynthesis.     

Immobilized cyanobacteria as a biofertilizer for rice crops; Intl. Conference on Applied Algology, Knysna, South Africa, April 1996.

N₂-fixing cyanobacteria (Anabaena azollae, symbiont strains) were immobilized in polyurethane foam and ammonia production by the cyanobacteria was investigated in the laboratory and rice field. The cyanobacterial symbiont, A. azollae - MPK-SK-AM-24 showed the highest growth rate and biomass production amongst the 5 isolates examined while A. azollae-AS-DS showed the highest nitrogenase activity followed by A. variabilis - SA₀ (wild type, non-symbiotic). Treatment of the foam-immobilized cyanobacteria with the systemic fungicide Bavistin stimulated nitrogenase activity while inhibiting glutamine synthetase (GS) activity. Free-living A. azollae-MPK-SK-AF-38, A. azollae - MPK-SK-AM-24 and A. azollae-MPK-SK-AM-27 excreted the highest amounts of ammonia into the growth medium; under foam - immobilized conditions the ammonia production increased further. Treatment of the foam - immobilized cyanobacteria with the fungicides Bavistin and Vitavax resulted in ammonia production at significantly higher rates. Rice seedlings (var. ADT 36) grown in the laboratory in conjunction with foam - immobilized A. azollae showed increased growth. A field experiment with paddy rice and foam - immobilized A. azollae strains indicated that the cyanobacteria excreted significant amounts of ammonia into the flood water in the rice fields resulting in increased chlorophyll content of the plants and increased the rice grain and straw yields. A combination of fertilizer nitrogen and inoculation with foam - immobilized cyanobacteria also significantly increased the rice grain and straw yield. Additionally, both A. azollae and A. variabilis were immobilized in sugarcane waste (bagasse), added to rice paddy and resulted in increased rice grain yield. This revised version was published online in September 2006 with corrections to the Cover Date.     

Optimization of immobilization conditions for vinegar production. Siran,wood chips and polyurethane foam as carriers for Acetobacter aceti

A complete experimental design has been developed to study the properties of three different solid carriers (Siran, wood chips and polyurethane foam) in the immobilization of acetic acid bacteria. Temperature-controlled reactors of 450-ml volume were employed to compare a standard immobilization procedure consisting of consecutive discontinuous acetic acid fermentations in the presence of the carrier. On reaching the final saturation condition, the different immobilized carriers were removed and introduced into identical sterilized reactors. These were then submitted to several semi-continuous fermentation cycles with the aim of characterising and comparing their acetification properties. Immobilization and acetification data obtained in this study have been evaluated in order to determine the best carrier material on the basis of several technical criteria. Polyurethane foam was the most successful because it allows a huge number of immobilized cells in the shortest time and leads to the highest acetification rate of the three assayed carriers.     

Effect of pore size and shape on the immobilization of coffee (Coffea arabica L.) cells in porous matrices

Summary The pore size and shape of porous matrices were evaluated as to their effect on the immobilization efficiency in cultured coffee (Coffea arabica L.)/cells. A hydrophilic porous matrix (13–20 pores/25 mm) and reticulate polyurethane foam (30 pores/25 mm) indicated more efficient immobilization than the others, in small cubes (1 cm³ × 9) and a strip (1 × 1 × 9 cm³) at the end of the fourth subculture. Among the large cubes (9 cm³), the reticulate one with the largest pore size (13 pores/25 mm) was the most advantageous for immobilization. In the strip-shaped matrices (1 × 1 × 9 cm³), immobilization was the most efficient in spite of its lower surface area as compared to the small cubes, except for those with the largest pore size. The strip-shaped foams, which were fixed on the inside of the flask against shaking, were effective for immobilization. Finally, strips (30 pores/25 mm) with slits to increase the surface area of the foam immobilized the largest amount of cells at the end of the fourth subculture. Caffeine production was not changed by diffenences in pore size.This paper is Part 76 in the series of Studies on Plant Tissue Cultures. For Part 75, see Furuya T., Orihara Y., Koge K. (1991) Plant Cell Rep 9:659–662 Offprint requests to: T. Furuya     

Highly efficient immobilization of glycosylated enzymes into polyurethane foams

Glycosylated enzymes, including aminoacylase from Aspergillus melleus, chloroperoxidase from Caldariomyces fumago, and phytase from Aspergillus ficuum, were covalently immobilized into polyurethane foams with very high enzyme loadings of up to 0.2 g protein per gram dry foam. The immobilization efficiency (retained activity) ranged from 100% at a low loading to 60% at high loadings. In contrast to many other immobilization methods no leaching of the enzyme from the support took place under the reaction conditions. In short, a universal method for the immobilization of enzymes from fungal sources was developed, affording a highly active, stable, and reusable biocatalyst.     

The effect of immobilization on the course of biotransformation reactions by plant cells

Summary The influence of different immobilization methods on the biotransformation of verbenol bySolanum aviculare plant cells has been studied. The biotransformation course was compared using free and immobilized cells. Immobilization techniques used included entrapment in alginate, pectate and carrageenane gels, in polyurethane foam, and on the surface of polyphenylenoxide.     

Effect of impeller type and stirring frequency on the behavior of an AnSBBR in the treatment of low-strength wastewater

The influence of impeller type and stirring frequency on the performance of a mechanically stirred anaerobic sequencing batch reactor containing immobilized biomass on an inert support (AnSBBR - Anaerobic Sequencing Batch Biofilm Reactor) was evaluated. The biomass was immobilized on polyurethane foam cubes placed in a stainless-steel basket inside a glass cylinder. Each 8-h batch run consisted of three stages: feed (10 min), reaction (460 min) and discharge (10 min) at 30 °C. Experiments were performed with four impeller types, i.e., helical, flat-blade, inclined-blade and curved-blade turbines, at stirring frequencies ranging from 100 to 1100 rpm. Synthetic wastewater was used in all experiments with an organic-matter concentration of 530 ± 37 mg/L measured as chemical oxygen demand (COD). The reactor achieved an organic-matter removal efficiency of around 87% under all investigated conditions. Analysis of the four impeller types and the investigated stirring frequencies showed that mass transfer in the liquid phase was affected not only by the applied stirring frequency but also by the agitation mode imposed by each impeller type. The best reactor performance at all stirring frequencies was obtained when agitation was provided by the flat-blade turbine impeller.