Biocatalytic Reactors Research Articles

Ontology-Based Laboratory Data Acquisition With EnzymeML for Process Simulation of Biocatalytic Reactors

The presented work explores the use of ontologies and standardized enzymatic data to set up enzymatic reactions in process simulators, such as DWSIM. Setting up an automated workflow to start a process simulation based on enzymatic data obtained from the laboratory can help save costs and time during the development phase. Standardized conditions are crucial for accurate comparison and analysis of enzymatic data, where ontologies provide a standardized vocabulary and semantic relations between relevant concepts. To ensure standardized data, an electronic lab notebook (ELN) is used based on EnzymeML, an open standard XML-based format for enzyme kinetics data. Furthermore, two ontologies are merged and the result is extended for the use in the Python-based workflow. The resulting data is stored in a knowledge graph for research data in a machine-accessible and human-readable format. Thus, the study demonstrates a workflow that allows for the direct translation of ELN data into a process simulation via ontologies.

Heterogeneous biocatalytic system for effective decolorization of textile dye effluent.

The current physicochemical methods for decolorizing toxic synthetic dyes are not sustainable to halt the environmental damage as they are expensive and often produce concentrated sludge, which may lead to secondary disposal problems. Biocatalysis (microbes and/or their enzymes) is a cost-effective, versatile, energy-saving and clean alternative. The most common enzymes involved in dye degradation are laccases, azoreductases and peroxidases. Toxic dyes could be converted into less harmful byproducts through the combined action of many enzymes or the utilization of whole cells. The action of whole cells to treat dye effluents is either by biosorption or degradation (aerobic or anaerobic). Using immobilized cells or enzymes will offer advantages such as superior stability, persistence against harsh environmental conditions, reusability and longer half-lives. This review envisages the recent strategies of immobilization and bioreactor considerations with the immobilized system as the effective treatment of textile dye effluents. Packed bed reactors are the most popular heterogeneous biocatalytic reactors for dye decolorization due to their efficiency and cost-effectiveness.

Carbonic Anhydrase Enhanced UV-Crosslinked PEG-DA/PEO Extruded Hydrogel Flexible Filaments and Durable Grids for CO2 Capture.

In this study, poly (ethylene glycol) diacrylate/poly (ethylene oxide) (PEG-DA/PEO) interpenetrating polymer network hydrogels (IPNH) were extruded into 1D filaments and 2D grids. The suitability of this system for enzyme immobilization and CO2 capture application was validated. IPNH chemical composition was verified spectroscopically using FTIR. The extruded filament had an average tensile strength of 6.5 MPa and elongation at break of 80%. IPNH filament can be twisted and bent and therefore is suitable for further processing using conventional textile fabrication methods. Initial activity recovery of the entrapped carbonic anhydrase (CA) calculated from esterase activity, showed a decrease with an increase in enzyme dose, while activity retention of high enzyme dose samples was over 87% after 150 days of repeated washing and testing. IPNH 2D grids that were assembled into spiral roll structured packings exhibited increased CO2 capture efficiency with increasing enzyme dose. Long-term CO2 capture performance of the CA immobilized IPNH structured packing was tested in a continuous solvent recirculation experiment for 1032 h, where 52% of the initial CO2 capture performance and 34% of the enzyme contribution were retained. These results demonstrate the feasibility of using rapid UV-crosslinking to form enzyme-immobilized hydrogels by a geometrically-controllable extrusion process that uses analogous linear polymers for both viscosity enhancement and chain entanglement purposes, and achieves high activity retention and performance stability of the immobilized CA. Potential uses for this system extend to 3D printing inks and enzyme immobilization matrices for such diverse applications as biocatalytic reactors and biosensor fabrication.

Systematic evaluation of agarose- and agar-based bioinks for extrusion-based bioprinting of enzymatically active hydrogels.

Extrusion-based 3D bioprinting enables the production of customized hydrogel structures that can be employed in flow reactors when printing with enzyme-containing inks. The present study compares inks based on either low-melt agarose or agar at different concentrations (3-6%) and loaded with the thermostable enzyme esterase 2 from the thermophilic organism Alicyclobacillus acidocaldarius (AaEst2) with regard to their suitability for the fabrication of such enzymatically active hydrogels. A customized printer setup including a heatable nozzle and a cooled substrate was established to allow for clean and reproducible prints. The inks and printed hydrogel samples were characterized using rheological measurements and compression tests. All inks were found to be sufficiently printable to create lattices without overhangs, but printing quality was strongly enhanced at 4.5% polymer or more. The produced hydrogels were characterized regarding mechanical strength and diffusibility. For both properties, a strong correlation with polymer concentration was observed with highly concentrated hydrogels being more stable and less diffusible. Agar hydrogels were found to be more stable and show higher diffusion rates than comparable agarose hydrogels. Enzyme leaching was identified as a major drawback of agar hydrogels, while hardly any leaching from agarose hydrogels was detected. The poor ability of agar hydrogels to permanently immobilize enzymes indicates their limited suitability for their employment in perfused biocatalytic reactors. Batch-based activity assays showed that the enzymatic activity of agar hydrogels was roughly twice as high as the activity of agarose hydrogels which was mostly attributed to the increased amount of enzyme leaching. Agarose bioinks with at least 4.5% polymer were identified as the most suitable of the investigated inks for the printing of biocatalytic reactors with AaEst2. Drawbacks of these inks are limited mechanical and thermal stability, not allowing the operation of a reactor at the optimum temperature of AaEst2 which is above the melting point of the employed low-melt agarose.

Biocatalytic Synthesis Using Self-Assembled Polymeric Nano- and Microreactors.

Biocatalysis is increasingly being explored for the sustainable development of green industry. Though enzymes show great industrial potential with their high efficiency, specificity, and selectivity, they suffer from poor usability and stability under abiological conditions. To solve these problems, researchers have fabricated nano- and micro-sized biocatalytic reactors based on the self-assembly of various polymers, leading to highly stable, functional, and reusable biocatalytic systems. This Review highlights recent progress in self-assembled polymeric nano- and microreactors for biocatalytic synthesis, including polymersomes, reverse micelles, polymer emulsions, Pickering emulsions, and static emulsions. We categorize these reactors into monophasic and biphasic systems and discuss their structural characteristics and latest successes with representative examples. We also consider the challenges and potential solutions associated with the future development of this field.

Biocatalytic Synthesis Using Self‐Assembled Polymeric Nano‐ and Microreactors

AbstractBiocatalysis is increasingly being explored for the sustainable development of green industry. Though enzymes show great industrial potential with their high efficiency, specificity, and selectivity, they suffer from poor usability and stability under abiological conditions. To solve these problems, researchers have fabricated nano‐ and micro‐sized biocatalytic reactors based on the self‐assembly of various polymers, leading to highly stable, functional, and reusable biocatalytic systems. This Review highlights recent progress in self‐assembled polymeric nano‐ and microreactors for biocatalytic synthesis, including polymersomes, reverse micelles, polymer emulsions, Pickering emulsions, and static emulsions. We categorize these reactors into monophasic and biphasic systems and discuss their structural characteristics and latest successes with representative examples. We also consider the challenges and potential solutions associated with the future development of this field.

Improved Productivity of NAD+ Reduction under Forced Convection in Aerated Solutions

AbstractThe regeneration of the NADH cofactor is crucial for bioelectrocatalytic reactors. This can be achieved by using an electrochemical mediator such as the rhodium complex [Cp*Rh(bpy)Cl]+, but the overall reduction process suffers from the interference of molecular oxygen. This interference can be avoided by using a second porous working electrode (acting as a real oxygen filter) positioned near the surface of the first working electrode. To fabricate the oxygen filter, platinum particles were deposited on the surface of a carbon paper. NADH could be produced in the presence of the oxygen filter with a faradaic yield at 63.4 %, very close to the yields obtained without the filter at 61.7 % in a fully N2‐degased medium, and only at 10.6 % in the presence of oxygen. Moreover, the productivity was also increased by nearly a factor of four. Therefore, the proposed concept of the oxygen filter offers a new avenue in the current strategies for NADH regeneration in biocatalytic reactors based on electrochemical methods.

Enzymatic Bioreactors: An Electrochemical Perspective

Biocatalysts provide a number of advantages such as high selectivity, the ability to operate under mild reaction conditions and availability from renewable resources that are of interest in the development of bioreactors for applications in the pharmaceutical and other sectors. The use of oxidoreductases in biocatalytic reactors is primarily focused on the use of NAD(P)-dependent enzymes, with the recycling of the cofactor occurring via an additional enzymatic system. The use of electrochemically based systems has been limited. This review focuses on the development of electrochemically based biocatalytic reactors. The mechanisms of mediated and direct electron transfer together with methods of immobilising enzymes are briefly reviewed. The use of electrochemically based batch and flow reactors is reviewed in detail with a focus on recent developments in the use of high surface area electrodes, enzyme engineering and enzyme cascades. A future perspective on electrochemically based bioreactors is presented.

3D-Printable and Enzymatically Active Composite Materials Based on Hydrogel-Filled High Internal Phase Emulsions.

The immobilization of enzymes in biocatalytic flow reactors is a common strategy to increase enzyme reusability and improve biocatalytic performance. Extrusion-based 3D bioprinting has recently emerged as a versatile tool for the fabrication of perfusable hydrogel grids containing entrapped enzymes for the use in such reactors. This study demonstrates the suitability of water-in-oil high internal phase emulsions (HIPEs) as 3D-printable bioinks for the fabrication of composite materials with a porous polymeric scaffold (polyHIPE) filled with enzyme-laden hydrogel. The prepared HIPEs exhibited excellent printability and are shown to be suitable for the printing of complex three-dimensional structures without the need for sacrificial support material. An automated activity assay method for the systematic screening of different material compositions in small-scale batch experiments is presented. The monomer mass fraction in the aqueous phase and the thickness of printed objects were found to be the most important parameters determining the apparent activity of the immobilized enzyme. Mass transfer limitations and enzyme inactivation were identified as probable factors reducing the apparent activity. The presented HIPE-based bioinks enable the fabrication of flow-optimized and more efficient biocatalytic reactors while the automated activity assay method allows the rapid screening of materials to optimize the biocatalytic efficiency further without time-consuming flow-through experiments involving whole printed reactors.

3D-Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4-Hydroxystilbene.

Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio‐ and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose‐based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor‐free phenacrylate decarboxylase enzymes suitable for the production of 4‐vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk‐shaped agarose‐based modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetate‐catalysed Heck reaction, 4‐hydroxystilbene was synthesized from p‐coumaric acid with a total yield of 14.7 % on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches.

Biotransporting Biocatalytic Reactors toward Therapeutic Nanofactories.

Drug‐delivery systems (DDSs), in which drug encapsulation in nanoparticles enables targeted delivery of therapeutic agents and their release at specific disease sites, are important because they improve drug efficacy and help to decrease side effects. Although significant progress has been made in the development of DDSs for the treatment of a wide range of diseases, new approaches that increase the scope and effectiveness of such systems are still needed. Concepts such as nanoreactors and nanofactories are therefore attracting much attention. Nanoreactors, which basically consist of vesicle‐encapsulated enzymes, provide prodrug conversion to therapeutic agents rather than simple drug delivery. Nanofactories are an extension of this concept and combine the features of nanoreactors and delivery carriers. Here, the required features of nanofactories are discussed and an overview of current strategies for the design and fabrication of different types of nanoreactors, i.e., systems based on lipid or polymer vesicles, capsules, mesoporous silica, viral capsids, and hydrogels, and their respective advantages and shortcomings, is provided. In vivo applications of biocatalytic reactors in the treatment of cancer, glaucoma, neuropathic pain, and alcohol intoxication are also discussed. Finally, the prospects for further progress in this important and promising field are outlined.

Synthesis of Geraniol Esters in a Continuous-Flow Packed-Bed Reactor of Immobilized Lipase: Optimization of Process Parameters and Kinetic Modeling.

With increasing demand for perfumes, flavors, beverages, and pharmaceuticals, the various associated industries are resorting to different approaches to enhance yields of desired compounds. The use of fixed-bed biocatalytic reactors in some of the processes for making fine chemicals will be of great value because the reaction times could be reduced substantially as well as high conversion and yields obtained. In the current study, a continuous-flow packed-bed reactor of immobilized Candida antarctica lipase B (Novozym 435) was employed for synthesis of various geraniol esters. Optimization of process parameters such as biocatalyst screening, effect of solvent, mole ratio, temperature and acyl donors was studied in a continuous-flow packed-bed reactor. Maximum conversion of ~87% of geranyl propionate was achieved in 15min residence time at 70°C using geraniol and propionic acid with a 1:1mol ratio. Novozym 435 was found to be the most active and stable biocatalyst among all tested. Ternary complex mechanism with propionic acid inhibition was found to fit the data.

Comparative analysis of stirred catalytic basket bio-reactor for the production of bio-ethanol using free and immobilized Saccharomyces cerevisiae cells

The successful industrial production of ethanol and fine chemicals requires the development of new biocatalytic reactors and support materials to achieve economically viable processes. In this work, a Stirred-Catalytic-Basket-BioReactor using various immobilizing foams as support material and compared to free cells were used, focusing mainly on; (i) effect of mass-transfer on cells physiology and (ii) ethanol productivity. The performance of the reactor was further evaluated by ethanol volumetric productivity, yield and time for process completion and it was found that the variation of ethanol production and diffusion of the substrate in fermentation process are co-related with the stirrer speed and initial glucose concentration. It was also observed that the time difference for glucose consumption between free and immobilized cells (alginate and sponges) tends to increase by increasing the glucose concentration in the medium. We found that at higher stirrer speed (500 rpm) when using higher glucose concentration (200 g/l), ethanol volumetric productivity increased significantly in the sponge (85 g/l) as compared to alginate beads (79 g/l) and free cells (60 g/l). From the data obtained, it can be concluded that sponges are the best support material for attaining higher ethanol productivity. A stirred catalytic basket bioreactor with yeast cells immobilized in polyethylene sponge gives higher ethanol production at a higher glucose consumption rate, and this productivity is due to higher mixing efficiency and reduced external as well as internal mass transfer limitations. The potentials of the reactor rank it as a remarkable ethanol/fine-chemical production approach that needs further investigations.

Biocatalytic reactors based on ribonuclease A immobilized on macroporous monolithic supports

Immobilized enzyme reactors (IMERs) produced by the covalent attachment of ribonuclease A to macroporous methacrylate-based monolithic supports using different experimental approaches are discussed and compared. Enzyme immobilization was carried out by direct covalent binding, as well as through attachment via a polymer spacer. The kinetic properties of an IMER operating in either recirculation mode or zonal elution mode were studied. Additionally, the effect of flow rate on the bioconversion efficiency of each IMER sample was examined.

Adsorption of Cr(VI) on ureolytic mixed culture from biocatalytic calcification reactor

Ca-rich water and wastewater have caused problems in water use, wastewater reuse and the operations of reactors treating Ca-rich wastewater. Nowadays, reuse of wastewaters is fast gaining importance as water sources have been polluted. Therefore, the concept of biocatalytic calcification reactors (BCR) based on urea hydrolysis, pH increase and calcite production has been studied to remove Ca from wastewaters. This biological process produces significant amounts of waste sludge. In the present study, Cr(VI) adsorption on the ureolytic mixed culture (UMC) waste by-product from BCR was investigated to evaluate its potential for metal removal. The biosorption process was investigated using equilibrium batch tests and the data were fit to the Langmuir, Freundlich and Temkin isotherm models. The Cr(VI) ion concentration dependence of sorption (1-100 mg/L) could be fit to the Langmuir isotherm model. Monolayer adsorption capacity, qm (mg/g), of the adsorbent was 8.67 and the Langmuir constant b (L/mg) was 0.881. Based on the obtained results, the waste UMC appears to be a potential biosorbent for the removal of Cr(VI) from wastewater, although its adsorptive capacity is lower than those of other biosorbents.

Fabrication and Activity of Silicate Nanoparticles and Nanosilicate-Entrapped Enzymes Using Polyethyleneimine As a Biomimetic Polymer

In nature, some peptides induce precipitation of silicic acid into silica nanoparticles such as is found in marine algae called diatoms. However, polybasic polymers can act as peptide mimics; one such polymer, polyethyleneimine (PEI), has the advantage that it is stable at room temperature and is inexpensive, in comparison with synthetic peptides. We describe the fabrication and characterization of biosilicate nanoparticles formed by mimicking the peptides using PEI. Brownian motion nanoparticle tracking analysis and field emission gun scanning electron microscopy have been used for the first time to characterize nanoparticles made with tetramethyl orthosilicate (TMOS) and PEI to investigate the fundamental factors that affect particle properties. These factors include the effect of phosphate concentration, PEI molecular weight, TMOS concentration, and species of alkoxy-silane used. The properties of the particles are compared with other particles made with polymers that induce silication. Our results show that using PEI gives differences in particle size compared with previous work using other polymers that induce silication. The entrapment of enzymes during the silication process, rationale for using nonphosphate and phosphate buffers during enzyme entrapment, and the analysis of enzyme activity are also presented. Because enzymes can be entrapped during fabrication, it means that there are many future possibilities for the use of silicate nanoparticles containing enzymes, such as biosensors and biocatalytic reactors.

Activity of immobilised rat hepatic microsomal CYP2E1 using alumina membrane as a support

Porous alumina membranes are attractive materials for the construction of biosensors and also have utility for the production of immobilised enzyme bioreactors. Microsomes from rat liver were adsorbed onto alumina membrane activated by silane. Microsomal membranes were pumped through the channels where they became immobilised by binding to amine groups on the surface of the alumina membrane. In an effort to gain a quantitative understanding of the effects of microsomal film growth on enzyme activity, we compared the para-nitrophenol (pNP) hydroxylase activity of the microsomes by varying the amount of microsomes fixed in alumina microchannels. The alumina membrane was placed in a fluidic device at a fast flow that afforded short residence time (seconds) to obtain transformation of pNP to 4-nitrocatechol (pNC), which was detected by LC-MS/MS. This enabled the use of this bioreactor where CYP2E1 activity is low and tissue sources are limiting. The microsomes, successfully immobilised on the alumina membranes, were used to produce stable biocatalytic reactors that can be used repeatedly over a period of 2 months.

Novel one-pot synthesis and characterization of bioactive thiol-silicate nanoparticles forbiocatalytic and biosensor applications

A novel one-pot neutral synthesis using bioinspired polymers to fabricate thiol-nanoparticles ispresented. The thiol-particles may be directly tethered to metal surfaces such asgold, allowing the production of self-assembled nanostructured biocatalytic orbiosensor surfaces. This one-pot method has also been used to entrap enzymeswithin the thiol-nanoparticles; it is apparent that once enzyme entrapment iscarried out a bimodal distribution of particles is formed, with particles of onemode being very similar in size to thiol-nanoparticles without enzyme entrapped,and particles of the other mode being much larger in size. To this end, effortshave been made to separate the two modes of particles for the sample containingenzyme and it has been observed that the larger mode thiol-nanoparticles do indeedcontain significant amounts of enzyme in comparison to the smaller mode ones.As the enzyme-containing thiol-nanoparticles can now be isolated, this meansthat there are many future possibilities for the use of thiol-particles containingenzyme, as they may be used in a wide range of processes and devices which requirecatalytic functionalized surfaces, such as biosensors and biocatalytic reactors.

Structure and homogeneity of pseudo-physiological phospholipid bilayers and their deposition characteristics on carboxylic acid terminated self-assembled monolayers

Supported phospholipid bilayers are frequently used to establish a pseudo-physiological environment required for the study of protein function or the design of enzyme-based biosensors and biocatalytic reactors. These membranes are deposited from bilayer vesicles (liposomes) that rupture and fuse into a planar membrane upon adhesion to a surface. However, the morphology and homogeneity of the resulting layer is affected by the characteristics of the precursor liposome suspension and the substrate. Here we show that two distinct liposome populations contribute to membrane formation – equilibrium liposomes and small unilamellar vesicles. Liposome deposition onto carboxylic acid terminated self-assembled monolayers resulted in planar mono- and multilayer, vesicular and composite membranes, as a function of liposome size and composition. Quartz crystal microbalance data provided estimates for layer thicknesses and sheer moduli and were used for classification of the final structure. Finally, atomic force microscopy data illustrated the inherently inhomogeneous and dynamic nature of these membranes.

Versatile bioelectronic interfaces on flexible non-conductive substrates

Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. These interfaces are commonly formed on gold films deposited using physical vapor deposition (PVD) or chemical vapor deposition (CVD). PVD and CVD require deposition of a primer layer, such as titanium or chromium, and require the use of expensive equipment and cannot be used on a wide range of substrates. This paper describes a versatile new bench-top method to form bioelectronic interfaces containing a gold film, electron mediator, cofactor, and dehydrogenase enzyme (secondary alcohol dehydrogenase, and sorbitol dehydrogenase) on nonconductive substrates such as polystyrene and glass. The method combines layer-by-layer deposition of polyelectrolytes, electroless metal deposition, and directed molecular self-assembly. Cyclic voltammetry, chronoamperometry, field emission X-ray dispersive spectroscopy, scanning electron microscopy, and atomic force microscopy were used to characterize the bioelectronic interfaces. Interfaces formed on flexible polystyrene slides were shown to retain their activity after bending to a radius of curvature of 18mm, confirming that the approach can be applied on cheap and flexible substrates for applications where traditional wafer-scale electronics is not suitable, such as personal or structural health monitors and rolled microtube biosensors.