Volume 2, Issue 6 - Special issue: 2nd International Conference Implementation of Microreactor Technology in Biotechnology (IMTB 2013) / Issue editors: Polona Žnidaršič-Plazl and Bruno Zelić

Enzymes are gaining increased importance as highly selective catalysts for green chemical synthesis. Multiphase microreaction systems are emerging tools for the development of enzyme-catalysed transformations involving two or more partly immiscible fluids in continuous flow. Mass transfer intensification due to miniaturisation of the flow dimensions and the associated enlargement of the interfacial area presents a powerful approach of effective reaction rate enhancement and thus reactor productivity increase for smart flow bioprocessing. Use of microstructured flow reactors for the study of multiphase (gas-liquid, liquid-liquid) biocatalytic conversions is reviewed. Multiphase flow characterisation based on dimensionless scaling parameters and flow-regime categorisation is presented with emphasis on the different flows applied to experimental studies of enzymatic reactions. Development of instrumented microsystems, flow instabilities, fast inactivation of biocatalysts and low conversion rates are problems often encountered with biotransformation under multiphase flow conditions. Key parameters controlling reaction performance are discussed along with some guidelines for design of scalable multiphase biocatalytic microreactors. Opportunities for biocatalytic process intensification are revealed in examples from fine chemical and materials synthesis.

Isoamyl acetate synthesis employing aqueous Candida antarctica lipase B (CaLB) solution was performed in a two-phase solvent system comprising hydrophilic ionic liquid 1-butyl-3-methylpyridinium dicyanamide and n -heptane. An X-junction glass microfluidic chip was used to obtain uniform microdroplets of n -heptane within a continuous phase of ionic liquid with dissolved enzyme and reactants, namely, isoamyl alcohol and acetic anhydride. A developed flow pattern resulted in a very large specific interfacial area for the reaction with amphiphilic CaLB and simultaneous extraction of isoamyl acetate in n -heptane. Biotransformation was performed within a continuously operated microfluidic system consisting of an X-junction chip and a silanized tube of a submillimeter diameter, which was further integrated with a microseparator based on a hydrophobic membrane. Efficient separation of n -heptane with product from ionic liquid phase containing enzyme and remaining substrates and products was achieved, enabling reuse of CaLB together with ionic liquid phase. More than 80% of productivity was preserved in each of the eight consecutive biotransformations within the integrated microfluidic system with reused lipase B in the ionic liquid phase. Subsequent ionic liquid regeneration accomplished by vacuum distillation enabled efficient reuse of this solvent in esterification with fresh enzyme.

A new concept of nicotinamide adenine dinucleotide hydrate (NADH) oxidation which combines advantages of the microreactor technology with the advantages of magnetic nanoparticles (MNPs) application is developed. Acetaldehyde was used as a substrate for the NADH regeneration process while the reaction was performed in a batch reactor and in a microreactor using alcohol dehydrogenase (ADH)-loaded MNPs. Three different microreactor systems with MNPs were studied, two with stationary MNPs trapped on the inner surface of microchannel by permanent magnetic field and one where the MNPs actively moved across the channel (movement inside microchannel allowed by an oscillating magnetic field). In a reactor system with an oscillating magnetic field and an actively moving ADH-loaded MNPs 100% NADH conversion was achieved for residence time of just 2 min.

Synthesis of ionic liquid 1 - heptyl-2,3-dimethylimidazolium bromide was accomplished with the assistance of ultrasound, microwave irradiation, and a continuously operated microreactor and was compared with a conventional laboratory scale process applying magnetic stirring and water-bath heating. Results were compared with respect to process productivity, energy consumption, and product colourisation as an indicator of its purity. By using nonconventional technologies, volumetric productivity was 10- to 30-fold superior, while energy consumption was reduced by 45%–65%. Among the alternatives tested, ultrasound-assisted synthesis was shown as the most efficient one in terms of volumetric productivity (4.40 mol l -1 h -1 ) and specific power consumption (909.1 W h mol -1 ), while microwave-assisted process was the least favourable. However, only a microreactor system enabled the synthesis of a noncoloured product resulting from very efficient mixing and temperature control. Due to significant process intensification along with high product quality and superior industrial perspectives, a continuous quaternisation within microchannels could be selected as the most promising green approach among the alternatives tested in this study. Integration of ultrasound and microreactor technology including miniaturised heat exchanger is foreseen for process intensification.

Droplet-based microfluidic technique is very suitable for a variety of screening processes. The cultivation within nanoliter segments and multidimensional microtoxicological screening of the Gram-negative bacterium Escherichia coli were studied under droplet-based microfluidic conditions. In order to evaluate the toxicity of the binary and ternary mixtures of antibiotic ampicillin and caffeine in the presence of the angiotensin-converting enzyme inhibitor captopril, a time-resolved optical double endpoint detection unit was applied. It included a microflow-through fluorimeter and photometer, which can simultaneously analyze changes in the endogenous autofluorescence signal and the cell density of E. coli cultivated inside 450-nl microfluid segments. As a result, strong nonlinear combination effects and a concentration-dependent antagonistic effect, as well as formation of activity summits on bolographic maps, were found. Our findings confirm the importance of multiparameter investigations for toxicological studies and could be taken into account in medical practice.

Magnetic nanoparticle (MNP)-supported catalysis is a new method to facilitate catalyst separation and reuse. This technique has recently been introduced for organocatalysis. MNP-supported organocatalysts have been evaluated for their activity, selectivity, and recyclability in a range of chemical transformations, especially for asymmetric synthesis. The synthesis and application of Fe 3 O 4 MNP-supported organocatalysis are highlighted in this short review article.

The role of dispersants on hydrocarbon phase behavior in seawater is an important problem that influences marine environment ecology. Offshore petroleum and natural gas catastrophes, such as the Deepwater Horizon spill of 2010, motive the need to understand how to minimize the introduction of potentially invasive compounds while maximizing their efficacy during emergency remediation. The microfluidic stabilities of mineral oil-seawater multiphase flows in the presence of model dispersants were studied for We <1. Introducing dispersants at varying dimensionless volumetric injection rates, ranging from 0.001 to 0.01, transitions from stable slug flow to the bubbly regime. Dimensionless mass ratios of three model dispersants to the mineral oil necessary to establish emulsions were estimated from 2.6×10 -3 to 7.7×10 -3 . Residence time distributions of seawater single-phase and mineral oil-seawater multiphase flows, laden with dispersants, were also investigated. Increasing the dimensionless dispersant injection rate from 0 to 0.01 was observed to increase convective dispersion, which was confirmed by estimations of the vessel dispersion number and the Bodenstein number. The observations undergird that microfluidics are useful laboratory techniques to identify the transition to bubbly flow where bacteria consumption rates could potentially be enhanced, while minimizing the dispersant mass introduced into calm-sea marine environments.