Modelling of a fluidized bed bioreactor for immobilized enzymes (Part 2)

In the first part of this contribution, a mathematical model was presented for a liquid fluidized bed using immobilized enzymes, with reversible Michaelis-Menten kinetics. This part is focused on the experimental results. The reaction kinetics of native and immobilized enzymes was determined in continuous stirred tank reactors under comparable conditions. The influence of external mass transfer was investigated in a fixed bed reactor column. The extend of pore diffusional resistance was examined in a continuous stirred tank reactor and with a numerical simulation. Hydrodynamics was measured in different reactor columns (diameter d_t = 0.052 ? 0.225 m; length L: 1.0–2.0m) and with a static mixer. Further, the concentration profile was determined in a fluidized bed reactor with side stream analysis for different biocatalyst samples, fluid velocities and bed heights. The simulation of experimental results indicates that they are well described by the developed model. Furthermore, the model is well suited to predicting the influence of specific parameters on the effective kinetics of the biocatalyst and the expansion of the fluidized bed.     

Mass Spectrometry of Peptides,Proteins and Glycoproteins

Continuous alcoholic fermentation of untreated crude sugar beet molasses has been studied. The process was carried out in a vertical fluidized-bed reactor with beads of calcium-alginate containing immobilized Saccharomyces cerevisiae. The influence of hydraulic residence time, concentration of substrate and other variables have been studied.     

Catalytic Behaviour of Lipase Free and Immobilized in Biphasic Membrane Reactor with Different Low Water-Soluble Substrates

The catalytic activity and reaction rate of lipase have been studied using the biocatalyst free in an organic/aqueous emulsion and immobilized in a biphasic organic/aqueous membrane reactor. The first reaction system was realized in a stirred tank reactor. The other was obtained by immobilizing the enzyme in the sponge layer of an asymmetric capillary membrane and recirculating the two phases along the two separate circuits of the membrane module. The performance of the reactors has been studied using two different low water-soluble substrates: triglycerides present in commercial olive oil and (R,S)-cyanomethyl-[2-(4-isobutylphenyl)propionate] (CNE). The effects of substrate viscosity and flow dynamics conditions, such as organic phase flow rate, on the biphasic membrane reactor performance have been evaluated on the basis of observed reaction rate and catalytic activity of free and immobilized lipase for both substrates. It has been observed that free lipase showed higher catalytic activity with olive oil, while immobilized lipase showed higher catalytic activity with CNE which has a lower viscosity than olive oil. The increase of organic phase flow rate negatively affected the reactor performance, with a minor effect when using CNE rather than olive oil. The influence of temperature on the biocatalyst performance with the two substrates has also been investigated. The optimal temperature value of lipase was different for the two substrates: 28°C with CNE and 40°C with olive oil. © 1997 SCI.     

Synthesis of Oleic Acid Esters with Immobilized Lipase

Esterification of oleic acid and alcohols with immobilized lipase from fungus Mucor miehei in a stirred batch reactor is presented in this article. The degree of conversion of oleic acid to esters was determined by measurement of acid values. The dependence of the synthesis of n-butyl oleate on the amount of enzyme, the influence of temperature on the equilibrium conversion for this reaction and the influence of temperature on initial velocities is presented. The influence of water added to reaction mixture at various reaction conditions on the activity of the enzyme preparation is also presented.     

Determination of process parameters and modelling of lipase-catalyzed transesterification in a fixed bed reactor

Chemical transesterification is of major importance to the edible oil industry. While alkali catalysts randomize all the fatty acids in a triglyceride mixture, the use of a 1,3 specific lipase causes a more selective exchange of fatty acid residues. Basic process parameters for the development of a continuous solvent-free process in a fixed bed reactor have been determined. The kinetics of the transesterfication reaction and the influence of particle diameter, substrate and water concentration on the effective reaction rate were examined in batchwise experiments. Residence time distribution and parameters of inter- and intraparticle mass transfer were determined by modelling of experiments carried out in a fixed bed reactor under transient conditions. Fixed bed reactors with side stream analysis were used for continuous transesterification. A kinetic model was developed for the enzyme catalyzed reaction, thereby showing the analogy between heterogeneous catalytic and enzyme catalyzed reactions. A one-dimensional heterogeneous reactor model was formulated on the basis of the kinetic equation and different process parameters. For numerical calculations, an exponential enzyme distribution inside the carrier was assumed. The simulation of experimental results indicates that they are well described by the developed model. Water concentration and presence of other substances strongly influence the stability of the immobilized enzyme.     

Photocatalytic decolorization of rhodamine B by immobilized TiO2/UV in a fluidized-bed reactor

The photocatalytic oxidation of Rhodamine B (RhB) was studied by using a newly developed immobilized photocatalyst (TiO₂ immobilized by support consisting of a perlite and silicone sealant) and a fluidized-bed reactor. Three 8W germicidal lamps were used as the light source and the reactor volume was 2.8l. When this photocatalyst was employed in a batch process, a total decolorization of the RhB in reaction times lower than 60 min was observed. The optimum dosage of photocatalyst was 33.8 g/l. The initial RhB decolorization rate of the immobilized TiO₂ was higher than that of the suspended TiO₂ and this did not agree with pseudo first-order kinetics because of the adsorption onto the surface of the immobilized TiO₂. This result indicated that the adsorption capacity of the immobilized photocatalyst is very important in photocatalysis.     

Sol–gel-immobilized recombinant E. coli for biosorption of Cd

Recombinant Escherichia coli engineered with a metal-binding peptide was immobilized by entrapment in SiO₂ gel beads using the sol–gel method. Biosorption of Cd²⁺ ions by the immobilized cells was studied in both batch and continuous systems. Adsorption equilibrium could be established within 3 h and the kinetics was well described by the pseudo-second-order kinetic model. The equilibrium data were best described by the Langmuir isotherm with the maximum uptake capacity being 79.9 mg/g cell at 25 °C. More than 95% of the adsorbed Cd²⁺ could be removed with 0.1 M CaCl₂ during desorption. No loss in adsorption capacity was found up to five repeated adsorption/desorption cycles. From mass transfer analysis, only intraparticle diffusion effect was found to be important at low Cd²⁺ concentration (50 mg/dm³), while at high concentration (250 mg/dm³), both intraparticle and external mass transfer affected biosorption. Continuous removal and recovery of Cd²⁺ could be carried out by the immobilized cells in a packed-bed reactor.     

A new approach for describing and solving the reversible Briggs-Haldane mechanism using immobilized enzyme

Recently immobilized enzymes have been widely used in industrial processes due to their outstanding advantages, such as high stability and recyclability; however, their kinetic behaviour is generally controlled by mass diffusion effects. Thus, in order to improve these enzymatic processes, a clear discernment between the kinetic and diffusion mechanisms that control the production of the metabolite require investigation. In practice, it is typical to establish apparent kinetics for immobilized enzyme operations, and the validity of the apparent kinetics is restricted to the studied cases. In this work, a new approach for mathematically describing the kinetic and diffusion mechanics in an immobilized biocatalyst bead is established, in which the fraction of residual enzymatic activity is included, and is defined as a measure of the active and available enzymes in the bead porous network. In addition, the diffusion and kinetic mechanisms are described by the effective diffusion coefficient and the free enzyme kinetics, since the porous network of the bead is assumed as the bioreaction volume. Therefore, free enzyme kinetics were determined from glucose to fructose bioconversion using a stirred tank reactor with free glucose-isomerase, in which substrate and enzyme concentrations and temperature were varied. The fraction of residual enzymatic activity () and the effective diffusion coefficient () were obtained from the isomerization of glucose to fructose using a stirred tank reactor with immobilized glucose-isomerase in calcium alginate beads at different substrate and enzyme concentrations. Finally, simulations were carried out to establish the bioreaction solid-phase characteristics that most significantly influence productivity.     

Intensification of enzymatic conversion of glucose to lactic acid by reactive extraction

Lactic acid is an important commercial product and extracting this out of aqueous solution is a growing requirement in fermentation-based industries and recovery from waste streams. The design of an amine extraction process requires (i) equilibrium and (ii) kinetic data for the acid-amine (solvent) system used. Equilibria and kinetics for lactic acid extraction by Alamine 336 in octanol as a diluent have been determined and compared with other diluents studied earlier. An approach for extracting the lactic acid by a long-chain tertiary amine, which is in the dispersed phase as a liquid ion exchanger (LIX), is presented. A mathematical model for slurry phase reactor with glucose in the continuous aqueous phase, the amine with a diluent in the dispersed phase and the immobilized enzyme as the solid catalyst, has been developed using equilibrium and kinetic data for reactive extraction. Effects of various parameters affecting the conversion of glucose have been discussed. The model has been solved for batch and semi-batch modes. It has been shown that the semi-batch mode yields approximately five times higher productivity than batch mode.     

PRODUCTION OF ETHANOL WITH SACCHAROMYCES CEREVISIAE IN A CONTINUOUS REACTOR Note III: an experimental,kinetic study with suspended,recycled biomass

By way of comparison with the kinetic investigation of the continuous production of ethanol in a tubular reactor with immobilized yeasts, a study was carried out of continuous fermentation in a stirred reactor, with final separation and recycling of the microorganisms. Flocculation with potassium ferrocyanide and zinc sulphate was used to separate the biomass. The maintenance of the metabolic activity was assessed. Next, discontinuous fermentation at various substrate and biomass concentrations was performed to determine the kinetics of glucose-ethanol transformation by flocculated and reinoculated Saccharomyces Cerevisiae.

Once again, the kinetic expression was a Michaelis-Menten equation, with un-competitive inhibition of the substrate and linear inhibition of the product.

The values observed for the various parameters were not very different from those for a biomass immobilized on an inert support or for a free biomass. Finally, the productivities of several types of reactor are compared.     

Experimental determination of methane hydrate dissociation curve up to 55 MPa by using a small amount of surfactant as hydrate promoter

Methane hydrate equilibrium has been studied upon continuous heating of the water-hydrate-gas system within the temperature range of 275-300 K. This temperature range corresponds to equilibrium pressures of 3.15-55 MPa. The hydrate formation/dissociation experiments were carried out in a high-pressure reactor under isochoric conditions and with no agitation. A small amount of surfactant (0.02 wt% sodium dodecyl sulfate, SDS) was added to water to promote hydrate formation. It was demonstrated that SDS did not have any influence on the gas hydrate equilibrium, but increased drastically both the hydrate formation rate and the amount of water converted into hydrate, when compared with the experiments without surfactant. To understand and clarify the influence of SDS on hydrate formation, macroscopic observations of hydrate growth were carried out using gas propane as hydrate former in a fully transparent reactor. We observed that 10^-3 wt% SDS (230 times less than the Critical Micellar Concentration of SDS) were sufficient to prevent hydrate particles from agglomerating and forming a rigid hydrate film at the liquid-gas interface. In the presence of SDS, hydrates grew mainly on the reactor walls as a porous structure, which sucked the solution due to capillary forces. Hydrates grew with a high rate until about 97 wt% of the water present in the reactor was transformed into hydrate.Our data on methane hydrate equilibrium both confirm already published literature data and complement them within the pressure range of 20-55 MPa.     

Kinetic parameter determination in monoculture and monosubstrate biological reactors

The kinetic parameters of a biological reactor, operating under monoculture and monosubstrate conditions, have been determined. Pure cultures of Nocardia corallina and Pseudomonas fluorescens species have been used with phenol as the only organic carbon source. The experimental runs have been carried out both in a batch and in a continuous stirred reactor. The batch results have been interpreted by zero order kinetics in phenol and first order kinetics in biomass. The kinetic-constants have also been calculated. The activation energy has been determined only for the Nocardia species. Using this strain, the continuous reactor, working without biomass recycle, has confirmed the first order kinetics with respect to biomass.     

丝瓜瓤固定黄孢原毛平革菌对2,4-二氯酚的降解研究

利用植物载体丝瓜瓤对黄孢原毛平革菌进行固定,研究了固定化细胞对2,4-二氯酚的降解性能。利用固定化细胞流化床反应器连续处理质量浓度为20mg/L的2,4-二氯酚废水。考察了不同水力停留时间对处理效果的影响。结果表明,采用吸附生长法能有效实现黄孢原毛平革菌的固定,固定细胞量可达0.791g[细胞]/[丝瓜瓤]。固定化细胞最适降解温度和pH值分别为35℃、6.0。对于质量浓度在20mg/L以下的低浓度2,4-二氯酚,固定化细胞和游离细胞的降解速率相当;对于质量浓度在50～120mg/L的高浓度2,4-二氯酚,固定化细胞具有明显优势,不仅可以耐受更高浓度的2,4-二氯酚,其降解速率也高于游离细胞,最大降解速率是13.95mg/(L·d),是游离细胞的2倍。不同的水力停留时间对固定化细胞流化床反应器连续处理2,4-二氯酚的降解效率有很大影响,固定化细胞对2,4-二氯酚降解过程遵循Monod方程。     

Non-isothermal simulation of cyclohexane dehydrogenation in an inert membrane reactor with catalytic pellets in the feed-side chamber

A mathematical model is presented to simulate the performance of a non-isothermal inert membrane reactor with catalytic pellets in the feed-side chamber (IMRCF). The simulation takes into account the various heat exchanges that take place inside the reactor. The model consists of the full set of partial difference equations that describe the conservation of mass, momentum, energy and chemical species, coupled with chemical kinetics and appropriate boundary conditions for the physical problem. The set of equations is solved by finite difference method. The model is applied to investigate the endothermic dehydrogenation of cyclohexane in the IMRCF, where a permselective Vycor glass membrane is used. The simulation results show that the conversion of cyclohexane for non-isothermal IMRCF at the temperature of 550 K and below is higher than the equilibrium conversion. On the contrary, when the temperature is 570 K and above, the conversion will be lower than the equilibrium conversion. The heat effects have a greater influence on the IMRCF.     

THE EFFECT OF SUBSTRATE MASS TRANSFER LIMITATIONS IN AN IMMOBILIZED ENZYME MEMBRANE REACTOR

An unstirred ultrafiltration cell can be quite easily converted into an immobilized enzyme reactor. Indeed, if suitable amounts ofproteic solutions are fed and if the protein molecular weight exceeds the membrane cut off, gel precipitation occurs onto the active surface of the membrane because of concentration polarization phenomena. Two different gel formation procedures have been followed. Indeed, two different proteic solution have been injected, namely

1.a mixture of an inert protein and the enzyme

2.co-polymer obtained by co-cross-linking the enzyme and the inert protein.

Substrate mass transfer limitations which occur in the gel immobilized enzyme reactor have been considered. The relevance of mass transfer resistances upstream from the immobilized gel layer has been discussed together with the intrinsic enzyme kinetics.

A heterogeneous gel model has been proposed which adequately describes the experimental results. Effectiveness factor correlations of fairly general applicability have been also presented.     

Experimental investigation on mechanisms of fine particles generation for the Borealis Borstar multistage olefin polymerization process

Simultaneous production of large amount of undesired fine particles is a big trouble in the Borstar multistage olefin polymerization process. Aiming at reducing the fine particles, the formation mechanism and formation location of the fine particles were thus studied. First, the influence of catalyst nature was considered. Second, the ash content, bulk density, morphology, and molecular weight distribution of polyethylene with different particle sizes from the small‐scale loop prepolymerization reactor, supercritical loop reactor, and gas phase fluidized bed reactor were studied, respectively. In combination with particle growth model, scanning electron microscope, gel permeation chromatography, and laser particle size analyzer, the particle morphology and growth kinetics were investigated. The results showed that fine particles were mostly generated in the supercritical loop reactor, and were significantly affected by the particle size distribution, residence time distribution, and particle fragmentation of the catalyst. Furthermore, scanning electron microscope images showed the catalysts with low activity tended to generate more fine particles. Based on these results, several strategies for reducing the amount of fine particles were proposed, which could be applied in the industrial process. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46589.     

外循环喷雾式乙氧基化反应器的数学模型与模拟

对半间歇操作外循环喷雾式乙氧基化反应器的数学模型与模拟进行了研究,分析了反应器的特性,建立了同时考虑气液传质、动力学、汽液平衡、反应体积变化和惰性气氮气影响的反应器的数学模型和数值模拟算法。以合成聚乙二醇为例,模拟得到反应器中温度、压力、环氧乙烷浓度及乙氧基化产物随时间和空间位置的分布。模拟结果同工业操作数据进行了比较,结果表明了模型的可靠性。     

Glucoamylase immobilized on montmorillonite: influence of nature of binding on surface properties of clay-support and activity of enzyme

Glucoamylase was immobilized on acid activated montmorillonite clay via two different procedures namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N₂ adsorption measurements and the activity of immobilized glucoamylase for starch hydrolysis was determined in a batch reactor. XRD shows intercalation of enzyme into the clay matrix during both immobilization procedures. Intercalation occurs via the side chains of the amino acid residues, the entire polypeptide backbone being situated at the periphery of the clay matrix. ²⁷Al NMR studies revealed the different nature of interaction of enzyme with the support for both immobilization techniques. N₂ adsorption measurements indicated a sharp drop in surface area and pore volume for the covalently bound glucoamylase that suggested severe pore blockage. Activity studies were performed in a batch reactor. The adsorbed and covalently bound glucoamylase retained 49% and 66% activity of the free enzyme respectively. They showed enhanced pH and thermal stabilities. The immobilized enzymes also followed Michaelis–Menten kinetics. K _m was greater than the free enzyme that was attributed to an effect of immobilization. The immobilized preparations demonstrated increased reusability as well as storage stability.     

Production of structured lipids in a packed-bed reactor with <Emphasis Type="Italic">thermomyces lanuginosa</Emphasis> lipase

Lipase-catalyzed interesterification between fish oil and medium-chain TAG has been investigated in a packedbed reactor with a commercially immobilized enzyme. The enzyme, a Thermomyces lanuginosa lipase immobilized on silica by granulation (lipozyme TL IM; Novozymes A/S, Bagsvaerd, Denmark), has recently been developed for fat modification. This study focuses on the new characteristics of the lipase in a packed-bed reactor when applied to interesterification of TAG. The degree of reaction was strongly related to the flow rate (residence time) and temperature, whereas formation of hydrolysis by-products (DAG and FFA) were only slightly affected by reaction conditions. The degree of reaction reached equilibrium at 30–40 min residence time, and the most suitable temperature was 60°C or higher with respect to the maximal degree of reaction. The lipase was stable in a 2-wk continuous operation without adjustment of water content or activity of the column and the substrate mixture.