Separation and purification of biomacromolecules based on microfluidics

Separation and purification of biomacromolecules either in biopharmaceuticals and fine chemicals manufacturing, or in diagnostics and biological characterization, can substantially benefit from application of microfluidic devices. Small volumes of equipment, very efficient mass and heat transfer together with high process control result in process intensification, high throughputs, low energy consumption and reduced waste production as compared to conventional processing. This review highlights microfluidics-based separation and purification of proteins and nucleic acids with the focus on liquid-liquid extractions, particularly with biocompatible aqueous two-phase systems, which represent a cost-effective and green alternative. A variety of microflow set-ups are shown to enable sustainable and efficient isolation of target biomolecules both for preparative, as well as for analytical purposes.publishe

Let the biocatalyst flow

Industrial biocatalysis has been identified as one of the key enabling technologies that, together with the transition to continuous processing, offers prospects for the development of cost-efficient manufacturing with high-quality products and low waste generation. This feature article highlights the role of miniaturized flow reactors with free enzymes and cells in the success of this endeavor with recent examples of their use in single or multiphase reactions. Microfluidics-based droplets enable ultrahigh-throughput screening and rapid biocatalytic process development. The use of unique microreactor configurations ensures highly efficient contacting of multiphase systems, resulting in process intensification and avoiding problems encountered in conventional batch processing. Further integration of downstream units offers the possibility of biocatalyst recycling, contributing to the cost-efficiency of the process. The use of environmentally friendly solvents supports effective reaction engineering, and thus paves the way for these highly selective catalysts to drive sustainable production

Biocatalytic process intensification via efficient biocatalyst immobilization, miniaturization, and process integration

Despite their sustainability, the potential of biocatalytic processes in industrial production is far from being realized. The main challenges in this field are the development of highly active, robust, and stable biocatalysts, the efficient regeneration of cofactors, and the prevention of biocatalyst deactivation under harsh industrial conditions. In addition to biocatalyst engineering, efficient enzyme and cell immobilization plays a crucial role in process feasibility. Reactor miniaturization, continuous operation, and integration with in situ product removal, process analytics, and cascade reactions that reduce the number of process steps enable process intensification. Mathematical model–based reactor and process design comprising time-scale analysis and efficient capacity increase can push the boundaries of biocatalytic processes toward industrial requirements. This review highlights the latest trends in efficient biocatalyst immobilization, miniaturization, and process integration to intensify biocatalytic processes

Lattice Boltzmann modeling-based design of a membrane-free liquid-liquid microseparator

The benefits of continuous processing and the challenges related to the integration with efficient downstream units for end-to-end manufacturing have spurred the development of efficient miniaturized continuously-operated separators. Membrane-free microseparators with specifically positioned internal structures subjecting fluids to a capillary pressure gradient have been previously shown to enable efficient gas-liquid separation. Here we present initial studies on the model-based design of a liquid-liquid microseparator with pillars of various diameters between two plates. For the optimization of in silico separator performance, mesoscopic lattice-Boltzmann modeling was used. Simulation results at various conditions revealed the possibility to improve the separation of two liquids by changing the geometrical characteristics of the microseparator

Copolymeric hydrogel-based immobilization of yeast cells for continuous biotransformation of fumaric acid in a microreactor

Although enzymatic microbioreactors have recently gained lots of attention, reports on the use of whole cells as biocatalysts in microreactors have been rather modest. In this work, an efficient microreactor with permeabilized Saccharomyces cerevisiae cells was developed and used for continuous biotransformation of fumaric into industrially relevant L-malic acid. The immobilization of yeast cells was achieved by entrapment in a porous structure of various hydrogels. Copolymers based on different ratios of sodium alginate (SA) and polyvinyl alcohol (PVA) were used for hydrogel formation, while calcium chloride and boric or phenylboronic acid were tested as crosslinking agents for SA and PVA, respectively. The influence of hydrogel composition on physico-chemical properties of hydrogels prepared in the form of thin films was evaluated. Immobilization of permeabilized S. cerevisiae cells in the selected copolymeric hydrogel resulted in up to 72% retained fumarase activity. The continuous biotransformation process using two layers of hydrogels integrated into a two-plate microreactor revealed high space time yield of 2.86 g/(L·h) while no activity loss was recorded during 7 days of continuous operation

Model-based design of continuous biotransformation in a microscale bioreactor with yeast cells immobilized in a hydrogel film

Miniaturized flow reactors with immobilized biocatalysts offer enormous potential for process intensification. They enable long-term use of biocatalysts, continuous operation that significantly outperforms batch processes, and efficient mass and heat transfer that results in highly controlled reaction conditions. Despite their increasing use in biocatalytic processes, optimization of reactor design and operating conditions based on mathematical description is very rare. This work aims to fill this gap by developing and validating a mathematical model for the continuous biotransformation process in a microreactor between two plates with immobilized whole cells in hydrogel layers on the bottom and top of the reactor. A biocatalytic production of L-malic acid by fumaric acid hydration using permeabilized Saccharomyces cerevisiae whole cells was used as a model reaction. The diffusivity of substrate and product in a liquid phase and in a copolymeric hydrogel layer and the reaction kinetic parameters considering the Michaelis-Menten kinetics of the reversible enzymatic reaction were estimated in initial batch experiments. The results obtained in a continuously operated microbioreactor with immobilized whole cells at different fumaric acid concentrations and flow rates were in excellent agreement with the predictions of the developed mathematical model comprising transport phenomena and reaction kinetics. Based on the validated model and using time-scale analysis with characteristic times, the optimal process and operating conditions for the developed microbioreactor system were determined. The model predicts an equilibrium conversion of fumaric acid at the highest inlet concentration tested when using a liquid height of 200 μm and a hydrogel thickness on both sides of the channel of 400 μm at a residence time of 30 min