Enzymatic Membrane Reactor System Research Articles

Integrated microsphere-packed bed enzymatic membrane reactor for enhanced bioconversion efficiency and stability: A proof-of-concept study

Fabricating high-performance enzyme reactors is requested for achieving efficient and stable bioconversions, but remains challenging, because few of them can possess high enzyme loading, sufficient mixing, and efficient mass transfer at the same time. Herein, we propose to develop a novel enzymatic packed bed membrane reactor (EPBMR) by integrating the advantages of both packed bed reactor (PBR) and enzymatic membrane reactor (EMR). A prototype study is conducted with the simplified enzyme-loaded microsphere-ultrafiltration EMR model (Mic-UF EMR). Invertase and dextranase are used in this work to produce glucose and oligodextran by hydrolysis of sucrose and dextran, respectively. Specifically, the use of microspheres can enlarge the contact area between enzymes and substrates and mitigate membrane fouling induced by free enzymes. Thus, Free&Mic-UF EMR (with both free and immobilized enzymes) exhibits a higher sucrose conversion rate (84%) than the EMR with free invertase (34%) and a negligible decline in sucrose conversion for 36 h continuous operation. Membrane fouling is ameliorated by alkaline cleaning and implementation of covalent bonding strategy. In addition, commercial resins with larger sizes are employed to replace konjac glucomannan microspheres (KGM) which reduce the pressure drop of EMR. Finally, by selecting the UF membrane with proper molecular weight cut-off (MWCO), the dextranase-based Mic-UF EMR system successfully produces oligodextran with desired molecular weight (Mw) and narrow Mw distribution. The outcome of this work not only offers a novel enzyme reactor construction strategy but also provides guidance for regulating the performance of EMR.

Mathematical modelling of reaction-separation in an enzymatic membrane reactor during oligodextran production

Producing oligodextran with an enzymatic membrane reactor (EMR) is a promising alternative to traditional fermentation processes due to its high efficiency and environmental compatibility. Process analysis and optimization of an EMR system used for producing oligodextran are critical to improving EMR operating stability and cost effectiveness. We herein developed a mathematical model to evaluate the effects of operating conditions and membrane properties on the reaction-separation behavior of an EMR. Our analysis shows that tailoring a membrane with large porosity and uniform pore size distribution can simultaneously improve the product quality and production efficiency. The optimal parameters of operating pressure and agitation speed for achieving high production efficiency depend on the selection of operating mode. EMR prefers higher operating pressure to increase the production efficiency under water feeding mode. However, since the membrane suffered severer fouling under substrate feeding mode, the extensive agitation is required to inhibit membrane fouling and enhance production yield. Our work paves a new avenue towards the fundamental understanding and designing of highly effective and low consumptive EMR for oligodextran production.

Modeling the design and operational mode of a continuous membrane reactor for enzymatic lignin modification

The modification of lignin-derived compounds such as technical lignins, which are highly aromatic and therefore valuable as renewable feedstocks for the biobased product industry, is still a challenging and multidisciplinary task. An enzyme membrane reactor system (EMRS) featuring a continuous stirred-tank reactor and an external ceramic crossflow ultrafiltration membrane is a promising configuration, particularly when combined with ligninolytic heme peroxidases (PODs) as biocatalysts, such as the new versatile peroxidases (VP) described herein. However, time-dependent irreversible enzyme inactivation caused by the co-substrate H2O2 and the fouling of the filtration membrane are limiting factors. To facilitate rational bioprocess development and reactor design, we present an overall modeling concept for a continuous operating mode that addresses both limitations. When including H2O2-related VP inactivation dynamic model analyses showed that two H2O2 molecules were required to convert one molecule of the reducing substrate (here, adlerol). In this context, an (initial) enzyme inactivation rate ki0 caused by factors other than H2O2 (e.g. the used buffer system) was introduced. Because oxygen utilization is characteristic of normal POD actions, the continuous online measurement of dissolved oxygen concentration was useful to monitor enzyme inactivation and therefore an excess of H2O2.

Efficient production of lactulose from whey powder by cellobiose 2-epimerase in an enzymatic membrane reactor

In this study, the gene encoding cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) was successfully expressed in Bacillus subtilis WB800. After the fermentation medium optimization, the activity of recombinant strain was 4.5-fold higher than the original medium in a 7.5L fermentor. The optimal catalytic pH and temperature of crude CsCE were 7.0 and 80°C, respectively. An enzymatic synthesis of lactulose was developed using cheese-whey lactose as its substrate. The maximum conversion rate of whey powder obtained was 58.5% using 7.5 U/mL CsCE. The enzymatic membrane reactor system exhibited a great operational stability, confirmed with the higher lactose conversion (42.4%) after 10 batches. To our best knowledge, this is the first report of lactulose synthesis in food grade strain, which improve the food safety, and we not only realize the biological production of lactulose, but also make good use of industrial waste, which have positive impact on environment.

Enzymatic production of fructooligosaccharides from inexpensive and abundant substrates using a membrane reactor system

ABSTRACTThe concept of zero waste in the sugar industry has encouraged the development of technologies that promote utilization of by-products, including the enzymatic conversion of waste feed stocks into nutraceutical products. Here we investigated a membrane reactor for the production of fructooligosaccharides (FOS) with immobilized enzyme (Pectinex Ultra SP-L) system using pretreated molasses as substrate with cleaning efficiency and less fouling as performance tools. The resulting spectra showed a high FOS yield of 63% with 89% conversion from available sucrose. So, the enzymatic membrane reactor system with innovative continuous separation of enzyme was proved to fit for the production of FOS.

Optimization of an enzymatic wheat gluten hydrolysis process in an enzyme membrane reactor using a design of experiment approach

The aim of this study was to determine the optimal conditions for operating an enzyme membrane reactor system to obtain a high yield of amino acids and peptides released from wheat gluten with Flavourzyme™. The optimal operating conditions were determined using multivariate analysis. A fractional factorial design made up of 27 runs amended by eight axial points was followed in a bench-scale enzyme membrane reactor system (V = 2 L), and responses were recorded over 8 h. Optimal conditions determined were a temperature of 50 ± 1 °C and a Flavourzyme™ activity of 116 ± 4 nkatLeu-pNA/mL. Modeling resulted in an optimal substrate feed rate of 19.2 g/L/h and a permeate flux of 57 L/m2/h. The enzymatic wheat gluten hydrolysis in an enzyme membrane reactor was conducted for 72 h with an average total product space–time yield of 12.6 ± 1.3 g/L/h (n = 4) and a free amino groups space–time yield of 4.4 ± 0.4 g/L/h (n = 4), and the substrate utilization was 0.82 ± 0.01 (g/g) (n = 4). The resulting product–enzyme ratios were 7.7 ± 0.6 mgTP/nkatLeu-pNA (n = 4) and 3.0 ± 0.2 mgFAA/nkatLeu-pNA (n = 4). The productivity of the enzyme membrane reactor improved compared to the reference batch process and doubled compared to previously proposed enzyme membrane reactor processes for the hydrolysis of wheat gluten.

Quantitative enzymatic production of sialylated galactooligosaccharides with an engineered sialidase from Trypanosoma rangeli

Sialylated galactooligosaccharides (GOS) represent a potential infant formula ingredient, which is believed to contribute with a combination of the beneficial properties of the prebiotic GOS as well as of sialylated human milk oligosaccharides. Sialylated GOS do not exist in natural milk, but can be produced from κ(kappa)-casein glycomacropeptide (CGMP), a sialylated side stream component from cheese-making, by sialidase-catalyzed transsialylation. Using a rationally designed mutant of the sialidase from Trypanosoma rangeli, Tr13, with enhanced transsialylation activity, six different GOS preparations with a varying degree of polymerization (DP) were effectively sialylated with molar yields of 20–30% on the CGMP sialyl in batch reactions. The rate of sialylation of the individual DPs was largely dependent on the DP distribution in each GOS preparation, and Tr13 catalysis did not discriminate against large GOS molecules, providing the novelty point that GOS molecules are sialylated independently of their size by Tr13. Using CGMP, GOS, and Tr13, the production of gram-scale quantities of sialyl-GOS was achieved in 20L volume reactions. Compared to the benchmark transsialidase from pathogenic Trypanosoma cruzi, the Tr13 was significantly more thermostable. By employing an enzymatic membrane reactor, Tr13 could be recycled and after seven consecutive 1-h reaction cycles, the biocatalytic productivity of the enzyme was increased 7-fold compared to the batch reaction. Assuming that the enzyme may be specific for α-2,3-bound sialyl moieties only, and that only 50% of sialyl linkages in CGMP are α-2,3-linked, the molar yield of sialyl-GOS on the available α-2,3-bound sialyl moieties in CGMP reached 80% in the enzymatic membrane reactor system.

Continuous Decolorization of Acid Blue 62 Solution in an Enzyme Membrane Reactor.

This paper focuses on using an enzyme membrane reactor (EMR) for the effective continuous decolorization of Acid Blue 62 (AB62). The following factors were considered for the effective use of Cerrena unicolor laccase immobilized in the EMR volume: the enzyme was stable in six successive runs in a batch reactor; no aeration was necessary; AB62 and the oxidized products were sorbed onto the membrane but were not rejected; and the enzyme was stable in the EMR system. It is obvious that any continuous process must be predictable, and thus, the objective was to verify the process model experimentally. For this reason, a proper isoenzyme kinetic equation was selected and the parameters were evaluated. The obtained kinetic parameters were used to plan processes and to verify their applicability to long-term AB62 decolorization, and a very good agreement between the calculated and the measured data was obtained. In the main designed continuous decolorization process, the conversion reached 98 % and was stable for 4 days. The membrane reactor with C. unicolor laccase appears to be very promising for AB62 decolorization.

Continuous removal of endocrine disruptors by versatile peroxidase using a two-stage system.

The oxidant Mn(3+) -malonate, generated by the ligninolytic enzyme versatile peroxidase in a two-stage system, was used for the continuous removal of endocrine disrupting compounds (EDCs) from synthetic and real wastewaters. One plasticizer (bisphenol-A), one bactericide (triclosan) and three estrogenic compounds (estrone, 17β-estradiol, and 17α-ethinylestradiol) were removed from wastewater at degradation rates in the range of 28-58 µg/L·min, with low enzyme inactivation. First, the optimization of three main parameters affecting the generation of Mn(3+) -malonate (hydraulic retention time as well as Na-malonate and H2 O2 feeding rates) was conducted following a response surface methodology (RSM). Under optimal conditions, the degradation of the EDCs was proven at high (1.3-8.8 mg/L) and environmental (1.2-6.1 µg/L) concentrations. Finally, when the two-stage system was compared with a conventional enzymatic membrane reactor (EMR) using the same enzyme, a 14-fold increase of the removal efficiency was observed. At the same time, operational problems found during EDCs removal in the EMR system (e.g., clogging of the membrane and enzyme inactivation) were avoided by physically separating the stages of complex formation and pollutant oxidation, allowing the system to be operated for a longer period (∼8 h). This study demonstrates the feasibility of the two-stage enzymatic system for removing EDCs both at high and environmental concentrations.

Influences of operating conditions on continuous lactulose synthesis in an enzymatic membrane reactor system: A basis prior to long-term operation

Lactulose synthesis was performed in a continuous stirred enzymatic membrane reactor. Each investigated operating condition (agitation, pH, feed molar ratio of lactose to fructose (mL/mF ratio), hydraulic residence time (HRT)) had an influence on reaction performances, in terms of lactulose concentration, productivity and selectivity. Lactulose concentration was maximum at an mL/mF ratio of 1/2. Higher than this ratio, synthesis of galactooligosaccharides was promoted rather than lactulose. At mL/mF ratios lower than 1/2, enzyme inhibition was pronounced to the detriment of lactulose production. At 7 or 9h HRT, higher lactulose concentrations were obtained than at shorter HRTs. Applying an mL/mF ratio of 1/2 and an HRT of 9h in a long-term operation, nearly constant lactulose concentration was reached after 23h and lasted up to 32h with a mean concentration of 14.51±0.07g/L and a reaction selectivity of 0.075–0.080mollactulose/molcons.lactose. After 7d, lactulose concentration reduced by 31%. A continuous synthesis of lactulose at lab-scale was shown to be amenable using a membrane reactor process. Moreover, for process evaluation, this study can bridge the gap between batch laboratory scale and continuous full-scale operation regarding lactulose synthesis.

Continuous long-term hydrolysis of wheat gluten using a principally food-grade enzyme membrane reactor system

The potential of the enzyme membrane reactor technology has been shown in several studies. In our study, we designed a principally food-grade continuous long-term hydrolysis process of wheat gluten in an enzyme membrane reactor with the proteolytic enzyme preparation Flavourzyme®. Among others, ethanol was the most suitable food-grade processing aid for avoiding contamination and showed high potential for application. The Flavourzyme®/wheat gluten process was comprehensively characterized in the enzyme membrane reactor. Critical factors for the enzyme stability were the temperature, pump stress and enzyme leakage through the membrane. The hydrolysis and process conditions were optimized to increase the space–time yield. Respective process parameters were chosen to obtain sufficient microbial, enzyme and process stability for the whole process time. The long-term hydrolysis was carried out in the presence of 8% (v/v) ethanol with a substrate concentration of 100gL−1 at 37°C and pH 7.5 for 96h. The continuous process resulted in stable product quality (degree of hydrolysis) and space–time yield (6.33gh−1L−1) over time. However, a discontinuous removal of accumulated dry matter was inevitable for the present enzyme membrane reactor process and was performed for 30min every 24h. Due to the fact that the enzymes were reused, the enzyme productivity could be increased by 450% compared to a reference batch process.

Performance of enzymatic wheat gluten hydrolysis in batch and continuous processes using Flavourzyme

The aim of this study was to investigate the process performance of wheat gluten hydrolysis with Flavourzyme in the conventional batch and in an enzyme membrane reactor system. The release of amino acids and peptides from wheat gluten using Flavourzyme in a batch process (T 50 °C, pH 5) is limited due to product inhibition. Investigation of Flavourzyme inhibition kinetics by l-amino acids revealed that the strongest inhibition was observed by l-cysteine. Thus, application of an enzyme membrane reactor with continuous product removal and substrate and enzyme retention was assessed. The highest productivity in the enzyme membrane reactor system was found using a 10 kDa molecular weight cut-off polyethersulfone membrane. This resulted in an increased product enzyme ratio from 2.1 mg product/nkat in the conventional batch process to 4.9 mg product/nkat with the enzyme membrane reactor after 20 h. The enzyme membrane reactor retained 80% of the initial space-time yield (STYinit 5 g/L/h) after nine days.

Effect of substrate and enzyme concentration on cyclodextrin production in a hollow fibre membrane reactor system

The batch and continuous production of cyclodextrins (CDs) was assessed by employing an enzymatic membrane reactor (EMR) system. The effects of tapioca starch substrate and cyclodextrin glycosyltransferase (CGTase) concentrations on the yield of CDs were studied. A similar effect on the behaviour of the ultrafiltration membrane in the EMR system (integration system) was also evaluated. The results for the batch process showed that incremental doses of CGTase caused gradual increments in CD yield; however, further addition of CGTase (above 1.0%) showed a 16% reduction in the total CD production. Further incremental in the tapioca starch concentration increased CD concentration (23g/L). However, addition above 8% w/v resulted in an insignificant yield of CDs. In the case of integration system, tapioca starch feeding rate that is higher than 4.41g/h caused adverse effects (lower CD yield and membrane flux). In particular at higher tapioca starch feeding rate (5.0g/h), the hydraulic resistance would reach as high as 1.31×1013m−1. Presumably this phenomenon was due to the unreacted substrates that were adsorbing onto the membrane surface and pores that subsequently led to greater fouling conditions and a severe flux decline. In addition, the weak adsorption (ra1) has been found to be the major fouling mechanism attributed to starch and its by products. Therefore, hydraulic cleaning is highly suggested as the procedure to be used for this EMR system.

Cyclodextrin production in hollow fiber membrane reactor system: Effect of substrate preparation

Production and separation of cyclodextrins (CDs) were assessed by employing an enzymatic membrane reactor (EMR) system. Comparison between gelatinized and ungelatinized tapioca starch was conducted to identify the pre-eminent type of tapioca starch via its reaction kinetic and the behavior of the ultrafiltration membrane in an EMR system. Results showed that the Michaelis constant value ( K m) for gelatinized tapioca starch was 47% lesser than the ungelatinized tapioca starch, which indicates higher affinity and strong substrate binding with the employed cyclodextrin glycosyltransferase (CGTase). However, gelatinized tapioca starch generated high membrane fouling, which demonstrated 60% higher hydraulic resistances and 7% lower percentage of flux recovery than ungelatinized tapioca starch, due to the cake deposition associated with large swollen tapioca starch molecules.

Continuous Recycle Enzymatic Membrane Reactor System for In-situ Production of Pure and Sterile Glucose Solution

In this study, an efficient Continuous Recycle Enzymatic Membrane Reactor (CREMR) system for production of in-situ glucose solution was developed and the Simultaneous Gelatinization, Liquefaction and Saccharification (SGLS) carried out at temperatures below 60°C, is proposed to replace the conventional starch hydrolysis. Using a 30 kD polysulfone hollow fibre membrane and 10% (w/w) tapioca starch concentration, it is found that during the steady state continuous operation, the SGLS process in the CREMR at temperatures of 55 and 60°C and trans-membrane pressures of 0.5 and 1 bar has produced a steady state glucose concentration in the permeate stream as high as 64 g L-1 over a period of eight hours operation. The glucose solution obtained is of high purity greater than 99.9% and sterile, hence can be utilised as intravenous dripping solution and other medical products without post-treatments. In addition, the CREMR system is also relatively easy to scale-up, has a smaller footprint c.f. conventional systems, thus allowing in-situ production. © 2007 Asian Network for Scientific Information.

Current technologies for the production of ( S)-ketoprofen: Process perspective

Ketoprofen (2-(3-benzoylphenyl)-propionic acid), belongs to the family of 2-arylpropionic acids (profens), is one of the nonstreroidal anti-inflammatory drugs. The production of single enantiomer of ketoprofen is expected to grow due to its enantiomer displaying better pharmacologic activities and benefits. Kinetic resolution is the most widely used method in the production of enantiomer pure ketoprofen. However, the process has a number of limitations, which are yet to be overcome. These limitations call for an innovative approach where two processes (enantioselective esterification and dynamic kinetic resolution) can be combined and operated under a single operating enzymatic membrane reactor system. The technology is more economical, more enantiomerically benign and has a potential maximum yield more than 50%. This paper will highlight the current technology in the production of biologically active ( S)-ketoprofen, the limitations associated with the production and solutions to overcome these limitations.

Chiral resolution of racemic ibuprofen ester in an enzymatic membrane reactor

In the present study, the production of ( S)-ibuprofen acid is reported using lipase-catalyzed hydrolysis reaction in a hollow fiber membrane reactor, as an efficient and cleaner route to obtain single-enantiomer compound. The performance of an enzymatic membrane reactor (EMR) was evaluated by varying operating temperature, organic phase flow rate, aqueous phase flow rate and enzyme loadings. The optimal operating conditions: temperature of 40 °C, pH 8 at EMR residence time ( τ org) of 10.87 h (organic inlet flow rate of 80 mL/min) with enzyme loading of 1.78 g/m 2 on the spongy layer of membrane were obtained. The kinetic resolution yielded an E value of 13, 31% ee S and 85% ee P. Both substrate and product purity were achieved by recovering the less reactive substrate in the organic stream while enhancing the optical purity of the desired product in the aqueous stream. The EMR system was modeled by incorporating Michaelis–Menten kinetics and treating the system as a continuous flow stirred tank reactor (CSTR). The simulated results agreed well with the experimental data.

Continuous conversion to optically pure l-methionine from d-enantiomer contaminated preparations by an immobilized enzyme membrane reactor

Continuous conversion to optically pure l-methionine from preparations contaminated by a small amount of the d-enantiomer (0.5–1.0%) has been developed with an enzyme membrane reactor system (slurry reactor) containing immobilized d-amino acid oxidase, catalase, and FAD. The optimal pH of the reaction system was from pH 7.0–8.0, and the optimum temperature was 25°C. The average conversion and the space-time yield were > 99% e.e. and nearly 90 g/ l·d, respectively with operation of the reactor for 7 d.

Forced‐flow bioreactor for sucrose inversion using ceramic membrane activated by silanization

A forced-flow enzyme membrane reactor system for sucrose inversion was investigated using three ceramic membranes having different pore sizes. Invertase was immobilized chemically to the inner surface of a ceramic membrane activated by a silane-glutaraldehyde technique. With the cross-flow filtration of sucrose solution, the reaction rate was a function of the permeate flux, easily controlled by pressure. Using 0.5 microm support pore size of membrane, the volumetric productivity obtained was 10 times higher than that in a reported immobilized enzyme column reactor, with a short residence time of 5 s and 100% conversion of the sucrose inversion.

Modelling of combined mass transfer‐kinetic effects in an enzyme membrane reactor system with a direct approach to the identification of intrinsic rate parameters

AbstractA methodology for identifying non‐linear parameters in a generalised Briggs‐Haldane rate expression for immobilised enzyme membrane flow reactors has been developed for the intermediate case where mutually rate‐controlling processes of mass transfer and enzymatic reaction prevail. The technological significance of the estimation procedure lies in the ability of the algorithm to handle parameter identification in non‐linear, two‐point boundary value problems which are prevalent in both immobilised enzyme and non‐enzymic heterogeneous catalysed reactor systems. The methodology was successfully implemented on an experimental level using quasi‐steady‐state conversion data for the model system of urea/collagen‐urease in a spiral‐wound, biocatalytic reactor module. By using a novel procedure of pepsin pretreatment of the collagen host membrane before enzyme impregnation, followed by glutaraldehyde crosslinking, it was possible to enhance the activity and stability of the immobilised enzyme complex to relatively high levels where interphase and intraphase diffusional limitations mutually controlled the rate of reaction. Information on the coefficient of mass transfer at the interface between solid and fluid phases was also obtained as part of the estimation procedure.