Production of anthraquinones by immobilized Frangula alnus Mill. plant cells in a four-phase air-lift bioreactor

 The production of anthraquinones by Frangula alnus Mill. plant cells was used as a model system to evaluate the performance of a liquid-liquid extractive product-recovery process. The shake flask experiments have shown higher production of anthraquinones in cell suspension and flask cultures of calcium-alginate-immobilized cells when silicone oil was incorporated into the medium, compared to a control without silicone oil. An external-loop air-lift bioreactor, developed and designed for the production and simultaneous extraction of extracellular plant cell products, was regarded as a four-phase system, with dispersed gas, non-aqueous solvent and calcium-alginate-immobilized plant cells in Murashige and Skoog medium. Continuous extraction of anthraquinones by silicone oil and n-hexadecane inside the bioreactor resulted in 10–30 times higher cell productivity, compared to that of immobilized cells in a flask. Based on the mixing pattern, immobilized biocatalyst extraparticle and intraparticle diffusional constraints and the kinetics of growth, substrate consumption and product formation, a mathematical model was developed to describe the time course of a batch plant cell culture. The model showed satisfactory agreement with four sets of shake flask experiments and three bioreactor production cycles. Received: 18 March 1994/Received revision: 20 September 1994/Accepted: 28 September 1994     

Immobilization of Acetobacter sp. CCTCC M209061 for efficient asymmetric reduction of ketones and biocatalyst recycling

ABSTRACT: BACKGROUND: The bacterium Acetobacter sp. CCTCC M209061 is a promising whole-cell biocatalyst with exclusive anti-Prelog stereoselectivity for the reduction of prochiral ketones that can be used to make valuable chiral alcohols such as (R)-4-(trimethylsilyl)-3-butyn-2-ol. Although it has promising catalytic properties, its stability and reusability are relatively poor compared to other biocatalysts. Hence, we explored various materials for immobilizing the active cells, in order to improve the operational stability of biocatalyst. RESULTS: It was found that Ca-alginate give the best immobilized biocatalyst, which was then coated with chitosan to further improve its mechanical strength and swelling-resistance properties. Conditions were optimized for formation of reusable immobilized beads which can be used for repeated batch asymmetric reduction of 4[prime]-chloroacetophenone. The optimized immobilized biocatalyst was very promising, with a specific activity of 85% that of the free-cell biocatalyst (34.66 mumol/min/g dw of cells for immobilized catalyst vs 40.54 mumol/min/g for free cells in the asymmetric reduction of 4[prime]-chloroacetophenone). The immobilized cells showed better thermal stability, pH stability, solvent tolerance and storability compared with free cells. After 25 cycles reaction, the immobilized beads still retained >50% catalytic activity, which was 3.5 times higher than degree of retention of activity by free cells reused in a similar way. The cells could be recultured in the beads to regain full activity and perform a further 25 cycles of the reduction reaction. The external mass transfer resistances were negligible as deduced from Damkohler modulus Da < <1, and internal mass transfer restriction affected the reduction action but was not the principal rate-controlling step according to effectiveness factors eta < 1 and Thiele modulus 0.3<[empty set] <1. CONCLUSIONS: Ca-alginate coated with chitosan is a highly effective material for immobilization of Acetobacter sp. CCTCC M209061 cells for repeated use in the asymmetric reduction of ketones. Only a small cost in terms of the slightly lower catalytic activity compared to free cells could give highly practicable immobilized biocatalyst.     

Immobilization of hydrocarbon-oxidizing bacteria in poly(vinyl alcohol) cryogels hydrophobized using a biosurfactant

A simple biosurfactant-based hydrophobization procedure for poly(vinyl alcohol) (PVA) cryogels was developed allowing effective immobilization of hydrocarbon-oxidizing bacteria. The resulting partially hydrophobized PVA cryogel granules (granule volume 5 microl) contained sufficient number (6.5 x 10(3)) of viable bacterial cells per granule, possessed high mechanical strength and spontaneously located at the interface in water-hydrocarbon system. Such interfacial location of PVA granules allowed high contact of immobilized biocatalyst with hydrophobic substrate and water phase, thus providing bacterial cells with mineral and organic nutrients. As a result, n-hexadecane oxidation efficiency of 51% after 10-day incubation was achieved using immobilized biocatalyst. PVA cryogels with increased hydrophobicity can be used for immobilization of bacterial cultures performing oxidative transformations of water-immiscible organic compounds. Immobilization of in situ biosurfactant producing Rhodococcus bacteria into PVA cryogel is discussed. PVA cryogel granules with entrapped alkanotrophic rhodococcal cells were stable after 10-month storage at room temperature.     

固定化细胞分布对动力学影响的模型化研究

通过对固定化Gluconobacter oxydans和Bacillus cereus的活细胞系统的研究,提出了固定化细胞不均匀分布模型,将此类分布与均匀分布(最劣分布模型)进行比较,分析了它们对固定化细胞内部的基质浓度分布、有效速率因子和选择率的影响,指出不均匀分布和均匀分布对于该固定化活细胞系统的动力学行为无显著影响,这一模型分析结果在实验中得以验证。通过无因次化分析,建立了适用于固定化生物催化剂动力学研究的模型方法。     

Use of immobilized Bacillus subtilis for the stereospecific hydrolysis of d,l-menthyl acetate

Bacillus subtilis possessing a stereospecific menthyl esterase has been immobilized in a polyurethane foam and used for the hydrolysis of menthyl ester. The specific activity of cells immobilized in polyurethane foam decreased as the cell loading was increased. The aqueous content of the immobilized biocatalyst particles influenced the activity. The activity half-life of about 400 h observed for the immobilized biocatalyst is 66 × greater than that observed for free bacteria.     

Degradation of thiodiglycol, the hydrolysis product of sulfur mustard, with bacteria immobilized within poly(vinyl) alcohol cryogels

Alcaligenes xylosoxidans subsp. xylosoxidans (SH91) capable of biodegradation of thiodiglycol (TDG) were immobilized in poly(vinyl) alcohol (PVA) cryogels. Cryoimmobilized biocatalyst was formed as spherical granules with a diameter of 0.5 mm; the biomass concentration inside the gel matrix was as high as 10% (w/w). The immobilized cells were capable of rapid degradation of TDG in tap water or potassium phosphate buffer (100 mM, pH 8.0) containing only (NH4)2 SO4. The immobilized biocatalyst did not show any substrate inhibition up to 200 mM TDG, and retained 100% activity during three months of continuous use in a repeated-batch bioreactor.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers, pH 3.5–7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2–4.6 and 7.5–8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 °C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 °C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 × 10³kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors. Received: 8 November 1996 / Received revision: 31 January 1997 / Accepted: 31 January 1997     

Comparative study of spent grains and delignified spent grains as yeast supports for alcohol production from molasses

Fresh, defrosted and delignified brewer's spent grains (BSG) were used as yeast supports for alcoholic fermentation of molasses. Glucose solution (12%) with and without nutrients was used for cell immobilization on fresh BSG, without nutrients for cell immobilization on defrosted and with nutrients for cell immobilization on delignified BSG. Repeated fermentation batches were performed by the immobilized biocatalysts in molasses of 7, 10 and 12 initial Baume density without additional nutrients at 30 and 20 degrees C. Defrosted BSG immobilized biocatalyst was used only for repeated fermentation batches of 7 initial Baume density of molasses without nutrients at 30 and 20 degrees C. After immobilization, the immobilized microorganism population was at 10(9) cells/g support for all immobilized biocatalysts. Fresh BSG immobilized biocatalyst without additional nutrients for yeast immobilization resulted in higher fermentation rates, lower final Baume densities and higher ethanol productivities in molasses fermentation at 7, 10 and 12 initial degrees Be densities than the other above biocatalysts. Adaptation of defrosted BSG immobilized biocatalyst in the molasses fermentation system was observed from batch to batch approaching kinetic parameters reported in fresh BSG immobilized biocatalyst. The results of this study concerning the use of fresh or defrosted BSG as yeast supports could be promising for scale-up operation.     

Biocatalytic scrubbing of gaseous acrylonitrile using Rhodococcus ruber immobilized in synthetic silicone polymer (ImmobaSil) rings

Microbial detoxification of acrylonitrile vapor was performed using Rhodococcus ruber immobilized into rings of a synthetic silicone polymer (ImmobaSil ) sponge. The immobilization matrix was used as both the cell support material and also as the trickle-bed column packing. The elimination capacity of the biocatalytic scrubber was determined for different influent concentrations of gaseous acrylonitrile. The working life of the nongrowing biocatalyst was also examined under test conditions and was found to be at least 70 days before biocatalyst replacement was required. An elimination capacity of 4.0 kg m(-3) h(-1) was achieved with a 95% removal efficiency, and an elimination capacity of over 7.2 kg m(-3) h(-1) acrylonitrile was achieved with a removal efficiency of 90%. This elimination capacity is 10 times that previously published in the literature.     

An efficient biocatalytic system for floxuridine biosynthesis based on Lactobacillus animalis ATCC 35046 immobilized in Sr-alginate

An efficient and green immobilized biocatalyst is herein reported to obtain 5-fluorouracil-2′-deoxyriboside (5FUradRib), an antimetabolite known as Floxuridine, used in gastrointestinal cancer treatment.Alginate is a natural polysaccharide used in the pharmaceutical industry due to its physicochemical properties, biocompatibility and non-toxicity. Multivalent cations, exposure time and cross-linking solution concentration were optimized, being Sr²⁺, 2 h and 0.2 M the best immobilization conditions. Furthermore, compression strength, swelling ratio and fracture frequency were evaluated, improving the mechanical stability of the biocatalyst favoring a future scale-up.On the other hand, the reaction parameters for 5FUradRib biosynthesis were optimized in order to obtain an immobilized biocatalyst with enhanced activity. Thus, Lactobacillus animalis ATCC 35046 immobilized in Sr-alginate showed yields of 96% at short reaction times.The obtained biocatalyst was stable for more than 25 days in storage conditions (4 °C) and could be reused at least 10 times without loss of its activity.Additionally, Sr-alginate biocatalyst stability was evaluated in different organic solvents to obtain hydrophobic compounds such as 5-bromouracil-2′-deoxyriboside (5BrUradRib), an effective radiosensitizing agent used in anti-cancer therapy, being hexane the best co-solvent.Finally, a smooth, cheap and environmentally friendly method to obtain anti-cancer drugs was developed in this study.     

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.     

Enzymatic transesterification in a solvent-free system: synthesis of sn-2 docosahexaenoyl monoacylglycerol

Abstract

The enzymatic transesterification of docosahexaenoic acid (DHA) ethyl ester with glycerol was carried out by using several immobilized lipases in a solvent-free system. This reaction involves the initial formation of sn-2 docosahexaenyl monoacylglycerol. This DHA derivative is highly relevant for improving the bioavailability of DHA and it has received increasing interest in the field of nutrition. Three commercial lipases, from Rhizomucor miehei (RML), Alcaligenes sp. (AQ) and Candida antarctica-fraction B (CALB) were immobilized by interfacial adsorption on a commercial hydrophobic support (a methacrylate resin containing octadecyl groups, Sepabeads C-18) and tested for glycerolysis of DHA ethyl ester. In certain cases (e.g. immobilized CALB), the transesterification reaction continues to the formation of triacylglycerol (80%) by using a very high excess of DHA ethyl ester ((115 mmols versus 1.24 mmols of glycerol and high temperatures (50?°C). However, the same biocatalyst working at lower temperatures, 37?°C, synthetizes a 90% of sn-2 monoacylglycerol even in the presence of that a high excess of DHA ethyl ester. Interestingly, immobilized RML derivative synthesizes a 98% of sn-2 monoacylglyceride (2-MG) in 15?min at 37?°C with a 4% of immobilized biocatalyst. These high activity and regioselectivity under very mild reaction conditions are very interesting for the thermal oxidative stability of the omega-3 fatty acid as well as for the thermal stability of the biocatalyst. Using Normal Phase HPLC-ELSD and accurate commercial markers, the formation of the 2-MG was confirmed.     

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis

(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (ϕ). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.     

Application of immobilized cells ofThermoanaerobium brockii for stereoselective reductions of oxo-acid esters

Summary Cells ofThermoanaerobium brockii were immobilized by entrapment methods as easy-to-handle biocatalyst for stereoselective reductions of oxo-acid esters. Different matrix materials were tested: agarose, k-carrageenan, alginate, polyacrylamide and polyurethanes. The two latter matrices allowed useful lifetimes of the immobilized biocatalysts of more than 2 months at thermophilic operation temperatures (around 65°C). Permeabilization of cells did not improve the catalytic activity. Immobilization of the cells did not enhance the thermostability. Only after a considerable period of operation could the immobilized biocatalysts be fed with medium lacking the complex substrates yeast extract and tryptone. Compared with freely suspended cells, reaction rates were lower. The immobilized system proved to be a relatively stable easy-to-handle biocatalyst, however, the freely suspended cells were superior with respect to flexibility of application and reaction velocity.     

Synthesis of organic acids by immobilized propionic bacteria in the flow system and stabilization of the process

The capacity of immobilized cells of propionic bacteria to synthesize organic acids was examined. Propionibacterium shermanii cells incorporated into polyacrylamide gel were capable to synthesize propionic, acetic and pyruvic acids in the flow system. As a carbon source glucose, lactate-Na or whey lactose was used. The greatest amount of the acids was synthesized with the use of lactate-Na. The life-time of the biocatalyst (immobilized cells) can be increased by its reactivation with a nutrient medium required for optimal cell proliferation.     

Preparation of new lipases derivatives with high activity–stability in anhydrous media: adsorption on hydrophobic supports plus hydrophilization with polyethylenimine

A novel method to prepare immobilized lipases derivatives is hereby proposed. Lipases are firstly adsorbed on supports having large internal surfaces covered by hydrophobic groups (e.g. polyacrylic resins covered by C18 moieties). Then, immobilized lipases are incubated in the presence of polyethyleneimine (PEI) at a pH value over the isoelectric point of the enzyme in order to cover the lipase surface with this polymer. In this way, we try to minimize all possible direct interactions between immobilized lipase and organic solvents when using these derivatives in anhydrous media.

Lipases from Rhizomucor miehie (RML) and Candida rugosa (CRL) were immobilized according to the proposed protocol. These derivatives were very active and very stable when catalyzing esterifications and transesterifications in anhydrous media. For example, RML derivatives exhibited a very high synthetic activity (more than 1000 Units/g immobilized biocatalyst) even when catalyzing the esterification of lauric acid with octanol at water activity values very close to zero. On the contrary, covalently immobilized derivatives exhibited a much lower synthetic activity under similar conditions (less than 10 Units/g of immobilized biocatalyst). Moreover, these new RML derivatives preserve 100% activity after incubation for 3 days in anhydrous butanone in the presence of molecular sieves. Under the same conditions, commercial immobilized RML lost more than 90% of activity in less than 10 min.     

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.     

Continuous production of L-phenylalanine by transamination

L-Phenylalanine was produced continuously from L-as-partate and phenylpyruvate by transaminase from a newly screened Pseudomonas putida strain. The process was carried out with an isolated enzyme in homogeneous phase in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively. Due to the difference in transport resistance, the productivity of the free enzyme in homogeneous phase (72 mmol/L h) was about 3 times higher than the productivity achieved using immobilized cells. However, a better stability of the biocatalyst was observed with immobilized cells.     

Oxidative biotransformation of thioanisole by Rhodococcus rhodochrous IEGM 66 cells

Comparative study of sulfoxidation activity of free and immobilized Rhodococcus rhodochrous IEGM 66 cells was performed. Free Rhodococcus cells (in the presence of 0.1 vol % n-hexadecane) displayed maximal oxidative activity towards thioanisole (0.5 g/l), a prochiral organic sulfide, added after 48-h cultivation of bacterial cells. Higher sulfide concentrations inhibited sulfoxidation activity of Rhodococcus. Use of immobilized cells allowed the 2-day preparatory stage to be omitted and a complete thioanisole bioconversion to be achieved in 24 h in the case that biocatalyst and 0.5 g/l thioanisole were added simultaneously. The biocatalyst immobilized on gel provides for complete thioanisole transformation into (S)-thioanisole sulfoxide (optical purity of 82.1%) at high (1.0-1.5 g/l) concentrations of sulfide substrate.     

Effect of high-cell-density fermentation of Candida utilis on kinetic parameters and the shift to respiro-fermentative metabolism

In this study, biodesulfurization (BDS) was carried out using immobilized Rhodococcus erythropolis KA2-5-1 in n-tetradecane containing dibenzothiophene (DBT) as a model oil (n-tetradecane/immobilized cell biphasic system). The cells were immobilized by entrapping them with calcium alginate, agar, photo-crosslinkable resin prepolymers (ENT-4000 and ENTP-4000), and urethane prepolymers (PU-3 and PU-6); and it was found that ENT-4000-immobilized cells had the highest DBT desulfurization activity in the model oil system without leakage of cells from the support. Furthermore, ENT4000-immobilized cells could catalyze BDS repeatedly in this system for more than 900 h with reactivation; and recovery of both the biocatalyst and the desulfurized model oil was easy. This study would give a solution to the problems in BDS, such as the troublesome process of recovering desulfurized oil and the short life of BDS biocatalysts.