Enzymatic hydrolysis of sodium caseinate in a continuous ultrafiltration reactor using an inorganic membrane

Continuous hydrolysis of sodium caseinate by alcalase was investigated in a recycle bioreactor coupled to an inorganic M5 membrane module. The effects of various substrate concentrations and the role of an ultrafiltration membrane on conversion rate were reported. Although a high level of conversion was obtained in the retentate side at a steady state, only part of the products formed was transmitted through the inorganic membrane. Degree of hydrolysis and product concentration in the reactor seem to be the main factors limiting product output during the continuous hydrolysis.     

Study of performance of a tubular membrane reactor for an enzyme catalyzed reaction

A tubular membrane reactor offers many advantages over a solid wall reactor to carry out an enzyme catalyzed reaction. With proper membrane selectivity, the product, may be separated from the reacting stream and the enzyme recycled for continuous reuse. In most cases, enzyme reuse contributes to the economic feasibility of a continuous enzyme catalyzed process. Furthermore, the efficiency and performance of a membrane reactor is greater than that of a solid wall reactor. Continuous hydrolysis of starch by the enzyme β-amylase, carried out in a commercially available tubular membrane unit, is studied at different starch and enzyme concentrations for a given system pressure and inlet flow rate. Results show that the performance of the membrane reactor is in all cases greater than that of the solid wall reactor. A steady state in performance of permeation rate is, however, not reached by the membrane reactor, which shows a continuous decline within the periods examined in this study. This decline is caused in part by the aging of the starch solution, but mostly by the formation of a concentrated, or gel, layer at the membrane surface. This appears to be the main limiting factor for this process since the decline in reaction and permeation rate results in a severe decrease in the amount of maltose in the permeate.     

Hydrolysis of liquefied corn starch in a membrane reactor

The objective of this study was to develop a continuous hydrolysis process for the enzymatic saccharification of liquefied corn starch using a membrane reactor. A residence time distribution study confirmed that the membrane reactor could be modeled as a simple continuous stirred tank reactor (CSTR). Kinetic studies indicated that the continuous reactor operated in the first-order region with respect to substrate concentration at substrate concentrations greater than 200 g/L. At a residence time of 1 h and an enzyme concentration of 1 g/L, the maximum reaction velocity (V(m)) was 3.86 g glucose/L min and the apparent Michaelis constant (K(m) (')) was 562 g/L. The K(m) (') value for the continuous reactor was 2-7 times greater than that obtained in a batch reactor.Kinetic data were fit to a model based on the Michaelis-Menten rate expression and the design equation for a CSTR. Application of the model at low reactor space times was successful. At space times of 6 min or less, the model predicted the reactor's performance reasonably well. Additional work on the detection and quantitation of reversion products formed by glucoamylase is required. Isolation, detection, and quantitation of reversion products by HPLC was difficult. Detailed analysis on the formation of these reversion products could lead to better reactor designs in the future.     

Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: II. Quantification of inhibition and suitability of membrane reactors

Product inhibition of cellulolytic enzymes affects the efficiency of the biocatalytic conversion of lignocellulosic biomass to ethanol and other valuable products. New strategies that focus on reactor designs encompassing product removal, notably glucose removal, during enzymatic cellulose conversion are required for alleviation of glucose product inhibition. Supported by numerous calculations this review assesses the quantitative aspects of glucose product inhibition on enzyme-catalyzed cellulose degradation rates. The significance of glucose product inhibition on dimensioning of different ideal reactor types, i.e. batch, continuous stirred, and plug-flow, is illustrated quantitatively by modeling different extents of cellulose conversion at different reaction conditions. The main operational challenges of membrane reactors for lignocellulose conversion are highlighted. Key membrane reactor features, including system set-up, dilution rate, glucose output profile, and the problem of cellobiose are examined to illustrate the quantitative significance of the glucose product inhibition and the total glucose concentration on the cellulolytic conversion rate. Comprehensive overviews of the available literature data for glucose removal by membranes and for cellulose enzyme stability in membrane reactors are given. The treatise clearly shows that membrane reactors allowing continuous, complete, glucose removal during enzymatic cellulose hydrolysis, can provide for both higher cellulose hydrolysis rates and higher enzyme usage efficiency (kg_product/kg_enzyme). Current membrane reactor designs are however not feasible for large scale operations. The report emphasizes that the industrial realization of cellulosic ethanol requires more focus on the operational feasibility within the different hydrolysis reactor designs, notably for membrane reactors, to achieve efficient enzyme-catalyzed cellulose degradation.     

Increasing the efficiency of the enzymatic hydrolysis of potato starch in a membrane reactor

The aim of the work was to define the physicochemical parameters of a reaction system that alter the effectiveness of a continuous recycle membrane reactor during potato starch hydrolysis. The enzymatic hydrolysis of starch in an ultrafiltration reaction system proceeded with a continuous decrease in the permeate flux, accompanied by an increase in dry substance content in both the permeate and retentate fractions. The decrease in the permeate flux was caused by an increase in feed viscosity. If a prehydrolysis process was conducted, it was possible to enzymatically hydrolyse potato starch in solutions with concentrations up to 20%. A quasi-steady state of starch enzymatic hydrolysis was reached in the ultrafiltration reaction system by alternately supplementing it with starch solution and water.     

Mathematical modeling of maltose hydrolysis in different types of reactor

A commercial enzyme Dextrozyme was tested as catalyst for maltose hydrolysis at two different temperatures: 40 and 65 °C at pH 5.5. Its operational stability was studied in different reactor types: batch, repetitive batch, fed-batch and continuously operated enzyme membrane reactor. Dextrozyme was more active at 65 °C, but operational stability decay was observed during the prolonged use in the reactor at this temperature. The reactor efficiencies were compared according to the volumetric productivity, biocatalyst productivity and enzyme consumption. The best reactor type according to the volumetric productivity for maltose hydrolysis is batch and the best reactor type according to the biocatalyst productivity and enzyme consumption is continuously operated enzyme membrane reactor. The mathematical model developed for the maltose hydrolysis in the different reactors was validated by the experiments at both temperatures. The Michaelis–Menten kinetics describing maltose hydrolysis was used.     

A CSTR-hollow-fiber system for continuous hydrolysis of proteins. Factors affecting long-term stability of the reactor

Factors affecting the long-term operational stability of a CSTR-hollow-fiber reactor for continuous hydrolysis of proteins were studied. The activity declined in a stepwise manner during a run. Declining from 92% conversion to 60% conversion in about ten hours at a space time of four minutes. Initial decay appears to be due to leakage of small active fragments of the enzyme mixture (Pronase) through the membrane, and later decay due to thermal degradation and loss of activators such as calcium through the membrane. The rate of buildup of unconverted substrate in the reaction vessel was controlled by operational variables, but did not appear to affect the reactor output or the operation of the reactor. The decay of the reactor could be partially compensated for by appropriate manipulation of the space-time variables.     

A CSTR-hollow fiber system for continuous hydrolysis of proteins. Performance and kinetics

The effect of four operating variables (enzyme concentration, substrate concentration, flow rate, and reaction volume) on the performance of CSTR-hollow fiber membrane reactor was studied for the continuous hydrolysis of a soy protein isolate using Pronase. Based on a residence time distribution study, the reactor system was modeled as an ideal CSTR in combination with the Michaelis-Menten equation of enzyme kinetics. This kinetic model correlated conversion with a space-time parameter modified to include all four independent variables. An empirical model based on curvilinear regression analysis was also developed. Both models predicted conversion fairly well, although the kinetic model slightly underpredicts at high conversion.     

Selective and stable production of physiologically active chitosan oligosaccharides using an enzymatic membrane bioreactor

We investigated the production of chitosan oligosaccharides by continuous hydrolysis of chitosan in an enzyme membrane bioreactor, with the goal of improving the yield of physiologically active oligosaccharides (pentamers and hexamers) and achieving operational stability. The bioreactor was a continuous-flow stirred-tank reactor equipped with an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, and the hydrolysis was accomplished with chitosanase from Bacillus pumilus. After optimization of the reaction parameters, such as the amount of enzyme, the yield of the target oligosaccharides produced in the membrane bioreactor with free chitosanase reached 52% on the basis of the fed concentration of chitosan. An immobilized chitosanase prepared by the multipoint attachment method was used to improve the operational stability of the membrane bioreactor. Under the optimized conditions, pentameric and hexameric chitosan oligosaccharides were steadily produced at 2.3 g/L (46% yield) for a month. The half-life of the productivity of the reactor was estimated to be 50 d under the conditions examined.     

Enzymatic hydrolysis of sodium-hydroxide-pretreated sallow in an ultrafiltration membrane reactor

An ultrafiltration membrane reactor was used to investigate the recovery of biocatalysts during enzymatic hydrolysis of pretreated sallow. Product inhibition could be eliminated by continuous removal of products through the ultrafiltration membrane, thus retaining the macromolecular substrate and enzymes. In this way, the degree of conversion was improved from 40% in a batch hydrolysis to 95% (within 20 h), and the initial hydrolysis rate was increased up to seven times. The recovery studies were focused on mechanical deactivation and irreversible adsorption on to the nonconvertible fraction of the substrate. Cellulase deactivation during mechanical agitation was not significant, and the loss of activity was attributed mainly to strong adsorption of the enzymes onto undigested material. This process was studied in semicontinuous hydrolyses, where fresh substrate was added intermittently. The amount of reducing sugars produced in this experiment was 25.7 g/g enzyme, compared to 4.7 g/g enzyme in a batch hydrolysis.     

Covalent immobilization of lipase from Candida rugosa onto poly(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application

A simple way of fabricating enzymatic membrane reactor with high enzyme loading and activity retention from the conjugation between nanofibrous membrane and lipase was devised. Poly(acrylonitrile-co-2-hydroxyethyl methacrylate) (PANCHEMA) was electrospun into fibrous membrane and used as support for enzyme immobilization. The hydroxyl groups on the fibrous membrane surface were activated with epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively. Lipase from Candida rugosa was covalently immobilized on these fibrous membranes. The resulted bioactive fibrous membranes were examined in catalytic efficiency and activity for hydrolysis. The observed enzyme loading on the fibrous membrane with fiber diameter of 80–150 nm was up to 1.6% (wt/wt), which was as thrice as that on the fibrous membrane with fiber diameter of 800–1000 nm. Activity retention for the immobilized lipase varied between 32.5% and 40.6% with the activation methods of hydroxyl groups. Stabilities of the immobilized lipase were obviously improved. In addition, continuous hydrolysis was carried out with an enzyme-immobilized fibrous membrane bioreactor and a steady hydrolysis conversion (3.6%) was obtained at a 0.23 mL/min flow rate under optimum condition.     

Ultrafiltration membrane reactors for enzymatic resolution of amino acids: design model and optimization

In this paper the possibility of continuous resolution of DL-phenylalanine, catalyzed by L-aminoacylase in a ultrafiltration membrane reactor (UFMR) is presented. A simple design model, based on previous kinetic studies, has been demonstrated to be capable of describing the behavior of the experimental system. The model has been used to determine the optimal experimental conditions to carry out the asymmetrical hydrolysis of N-acetyl-DL-phenylalanine.     

Hydrolysis of triglyceride by the whole cell of Pseudomonas putida 3SK in two-phase batch and continuous reactors systems

Batch and continuous hydrolysis of olive oil in an organic-aqueous two-phase system using the live whole cell of Pseudomonas putida 3SK as a source of a lipase is investigated. The strain was not only fully viable and grown well, but also produced extracellular lipase simultaneously. The degree of hydrolysis, depending on olive oil concentration in the solvents, was maximal at 13.5% (w/v) and decreased with the increase of the substrate concentration. At the optimal condition, a degree of hydrolysis higher than 95% was achieved with 24 h at 30 degrees C when the reaction was carried out in a two-phase batch stirred reactor. For long-term operation a continuous stirred reactor was designed. When the reaction was carried out in a continuous stirred reactor, the degree was hydrolysis reached 86% at a dilution rate of 0.2 h(-1). Satisfactory performance of a two-phase bioreactor was obtained in a long-term continous operation, which lasted for at least 30 days by feeding organic solvent containing olive oil and aqueous media separately. (c) 1994 John Wiley & Sons, Inc.     

Column cellulose hydrolysis reactor: An efficient cellulose hydrolysis reactor with continuous cellulase recycling

Summary The use of a column cellulose hydrolysis reactor with continuous enzyme recycling was demonstrated by incorporating a continuous ultrafiltration apparatus at the effluent end of the column reactor. Using this setup, over 90% (w/v) cellulose hydrolysis was achieved, resulting in an average sugar concentration of 6.8% (w/v) in the effluent stream. The output of the system was 1.98 g of reducing sugar/l/h with a ratio of 87% (w/v) of the reducing sugars being monomeric sugars. Batch hydrolysis reactors were less effective, resulting in 57% (w/v) of the cellulose being hydrolyzed. The output of the batch reactor was 1.33 g of reducing sugar/l/h with similar product concentrations and percentage of monomeric sugars. The ratio of reducing sugar/filter paper unit of cellulase activity for the column method was 69.1 mg/U as compared to only 21.2 mg/U for the batch reactor.     

Mathematical modeling of diffusion and reaction in the hydrolysis of vegetable protein in an immobilized enzyme recycle reactor

Experimental investigation is by far the most effective approach for studying the behavior of physical systems. However, an enzymatic solubilization of vegetable protein is a complex combination of intrinsic problems, of which many are not easily adaptable to experimental investigation. Experimental designs to study enzyme vegetable protein reactions yield data which describe the extramembraneous activity of the immobilized enzyme. In a continuous recycle immobilized enzyme reactor, the microenvironment concentration of the substrate or product in the membrane phase, or the concentrations along the reactor axial length in the bulk phase are not discernible to the experimenter. However, the knowledge of such concentration profiles is important in weighing the significance of such factors as intermembrane diffusion, enzyme loading, wet membrane size, and the mode of operation of the reactor. The simulation of mathematical models, which describe the physical system within the constraints imposed, yields information which is vital to the understanding of the process occurring in the reactor. The kinetics and diffusion of an immobilized thermophilic Penicillium duponti enzyme at pH 3.4-3.7 and 50 degrees C was modeled mathematically. The kinetic parameters were evaluated by fitting a model to experimental data using nonlinear regression analysis. Simulation profiles of the effects of reactor geometry, substrate concentration, membrane thickness, and enzyme leading on the hydrolysis rate are presented. From the profiles generated by the mathematical model, the best operational reactor strategy is recommended.     

Continuous hydrolysis of carboxymethyl cellulose with cellulase aggregates trapped inside membranes

Enzymatic hydrolysis of cellulose is often conducted in batch processes in which hydrolytic products tend to inhibit enzyme activity. In this study, we report a method for continuous hydrolysis of carboxymethyl cellulose (CMC) by using cross-linked cellulase aggregate (XCA) trapped inside a membrane. XCA particles prepared by using a millifluidic reactor have a uniform size distribution around 350 nm. Because of their large size, XCA particles in solutions can be filtered through a polyethersulfone membrane to collect 87.1 ± 0.9% of XCA particles. The membrane with impregnated XCA can be used as a catalyst for hydrolysis of CMC in a continuous mode. When the CMC concentration is 1.0 g/l and the flow rate is 2 μl/min, 53.9% of CMC is hydrolyzed to reducing sugars. The membrane with XCA is very stable under continuously flowing solutions. After 72 h of reaction, 97.5% of XCA remains inside the membrane.     

Kinetics of continuous starch hydrolysis in a membrane reactor

Following a previous study on kinetics of enzymatic starch hydrolysis with Termamyl 120l (Novo Nordisk) in batch reactor, this paper deals with kinetics in a continuous recycled membrane reactor (CRMR). Starting from results obtained in various working conditions, an equation relating the production rates of small oligosaccharides (DP ranging from 1 to 5) to the sum of concentrations of oligosaccharides with a higher degree of polymerisation is proposed. This equation looks like the one already reported for a batch system, with the exception that in the CRMR the enzyme activity varies: an exponential decay of activity as a function of time must be introduced to smooth carefully data points.     

Lipase kinetics: hydrolysis of triacetin by lipase from Candida cylindracea in a hollow-fiber membrane reactor

The aptitude of a hollow-fiber membrane reactor to determine lipase kinetics was investigated using the hydrolysis of triacetin catalyzed by lipase from Canadida cylindracea as a model system. The binding of the lipase to the membrane appears not to be very specific (surface adsorption), and probably its conformation is hardly altered by immobilization, resulting in an activity comparable to that of the enzyme in its native form. The reaction kinetics defined on the membrane surface area were found to obey Michaelis-Menten kinetics. The specific activity of the lipase in the membrane reactor was found to be significantly higher than in an emulsion reactor. The activity and stability of the enzyme immobilized on a hydrophilic membrane surface seem not to be influenced significantly by the choice of the membrane material. The hollow-fiber membrane reactor is a suitable tool to assess lipase kinetics in a fast and convenient way.     

Application of immobilized invertase to continuous hydrolysis of concentrated sucrose solutions

Invertase immobilized onto corn grits was utilized in the hydrolysis of highly concentrated sucrose solutions producting liquid sugar solutions containing glucose and fructose. Comparisons of conversion efficiencies of this immobilized invertase in a continuous stirredtank reactor and a plug-flow reactor indicated that the plug-flow reactor has an higher efficiency. Continuous sucrose hydrolysis was then performed in 0.1- and 1-L tubular reactors. This tenforld scaling-up was achieved without any noticeable loss in efficiency. This process thus was scaled-up to a 17.6-L pilot reactor set in a cane sugar refinery. This reactor was fed with highly concentrated sucrose solutions [71% (w/w)] to produce invert sugar syrup with the desired inversion degree. It allows a productivity equal to 9.1 kg sucrose hydrolyzed/h in the case of a 69% (w/w) sucrose initial concentration with a 72% conversion rate.