Polona Žnidaršič-Plazl

Steroid extraction in a microchannel system—mathematical modelling and experiments

The continuous ethyl acetate extraction of progesterone and 11alpha-hydroxyprogesterone, a reactant and the product of the biotransformation step involved in corticosteroid production, was studied in a microchannel at different flow velocities. In addition, non-steady state batch extraction without mixing was performed and modelled in order to verify the theoretically predicted parameters. In order to analyze experimental data and to forecast microreactor performance, a three-dimensional mathematical model with convection and diffusion terms was developed considering the velocity profile for laminar flow of two parallel phases in a microchannel at steady-state conditions. For the numerical solution of a complex equation system, non-equidistant finite differences were used. Very good agreement between model calculations and experimental data was achieved without any fitting procedure. Due to the efficient phase separation and high extraction yields obtained, the micro scale extraction units were found to be a promising tool for the development of an integrated system of 11alpha-hydroxylation of progesterone by Rhizopus nigricans in the form of pellets.

Microscale technology and biocatalytic processes: opportunities and challenges for synthesis

Despite the expanding presence of microscale technology in chemical synthesis and energy production as well as in biomedical devices and analytical and diagnostic tools, its potential in biocatalytic processes for pharmaceutical and fine chemicals, as well as related industries, has not yet been fully exploited. The aim of this review is to shed light on the strategic advantages of this promising technology for the development and realization of biocatalytic processes and subsequent product recovery steps, demonstrated with examples from the literature. Constraints, opportunities, and the future outlook for the implementation of these key green engineering methods and the role of supporting tools such as mathematical models to establish sustainable production processes are discussed.

The influence of β-cyclodextrin on the kinetics of progesterone transformation by Rhizopus nigricans

The process of progesterone 11α-hydroxylation by the pelleted growth form of the filamentous fungus Rhizopus nigricans has been described with a mathematical model, based on Michaelis-Menten enzyme kinetics and the rate of substrate dissolution. It was confirmed that the low water solubility of steroids is the limiting step of this process at high steroid concentrations. In order to overcome this problem, β-cyclodextrin, which is known to form inclusion complexes with these organic compounds, was added to the production medium. The phase solubility of the steroid-β-cyclodextrin system was investigated and the effect of β-cyclodextrin addition on progesterone biotransformation evaluated. Enhancement of steroid solubility was demonstrated and nearly two-fold increase in reaction rate was found in the presence of β-cyclodextrin.

Evaluation of Diffusion Coefficient Determination using a Microfluidic Device

A theoretical description of the convection-diffusion process in a homogeneous system enabling estimation of diffusion coefficients employing commercially available Y-junction microchannel is presented. A detailed numerical analysis based on finite volumes and finite differences, namely the explicit, implicit and Crank-Nicolson method, was performed and analyzed on the same domain in order to verify the proposed models. All numerical approaches provided stable solutions with certain numerical variations depending on the number of iterations defined by the mesh density. In addition, the method was validated with measurements of diffusion coefficients of some selected components in the short Y-junction microchannel. Benefits and possible pitfalls of this estimation method are discussed.

Batch and continuous transformation of progesterone by Rhizopus nigricans pellets in the presence of β-cyclodextrin

A pelleted morphological form of Rhizopus nigricans, which might be regarded as a naturally immobilized biomass, enabled development of a continuous process of progesterone 11α-hydroxylation in a laboratory-scale stirred tank bioreactor. β-Cyclodextrin was used to enhance steroid solubility in aqueous transformation media and consequently to increase the rate of steroid hydroxylation. Batch and continuous processes were performed with pellets of different average diameters, ranging from less than 1 mm to 7.5 mm. Mathematical model simulations of both operational modes confirmed the previously defined Michaelis–Menten kinetics for the biotransformation. A decrease in overall hydroxylation rate was noticed when pellets larger than 5 mm were used, which correlated with the lower area of outer surface of agglomerates where, presumably, most of the enzyme induction had taken place.

Continuous steroid biotransformations in microchannel reactors

The use of microchannel reactor based technologies within the scope of bioprocesses as process intensification and production platforms is gaining momentum. Such trend can be ascribed a particular set of characteristics of microchannel reactors, namely the enhanced mass and heat transfer, combined with easier handling and smaller volumes required, as compared to traditional reactors. In the present work, a continuous production process of 4-cholesten-3-one by the enzymatic oxidation of cholesterol without the formation of any by-product was assessed. The production was carried out within Y-shaped microchannel reactors in an aqueous-organic two-phase system. Substrate was delivered from the organic phase to aqueous phase containing cholesterol oxidase and the product formed partitions back to the organic phase. The aqueous phase was then forced through a plug-flow reactor, containing immobilized catalase. This step aimed at the reduction of hydrogen peroxide formed as a by-product during cholesterol oxidation, to avoid cholesterol oxidase deactivation due to said by-product. This setup was compared with traditional reactors and modes of operation. The results showed that microchannel reactor geometry outperformed traditional stirred tank and plug-flow reactors reaching similar conversion yields at reduced residence time. Coupling the plug-flow reactor containing catalase enabled aqueous phase reuse with maintenance of 30% catalytic activity of cholesterol oxidase while eliminating hydrogen peroxide. A final production of 36 m of cholestenone was reached after 300 hours of operation.

Ionic Liquids within Microfluidic Devices

In past decade, the use of ionic liquids as the replacement for conventional molecular solvents in organic reactions has tremendously increased. The present work aims to provide a literature review of the implementation of microreactor technology into the synthesis of ionic liquids, into (bio)catalyzed production of commercially important chemical compounds with ionic liquids as sustainable solvents, as well as in downstream processing with ionic liquids.

A comparative study of ultrasound, microwave and microreactor-assisted imidazolium-based ionic liquid synthesis

Abstract 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.

Continuous lipase B-catalyzed isoamyl acetate synthesis in a two-liquid phase system using corning® AFR™ module coupled with a membrane separator enabling biocatalyst recycle

The performance of the Corning AFR™ Low Flow (LF) fluidic module for Candida antarctica lipase B-catalyzed isoamyl acetate synthesis in an n-heptane–buffer two-liquid phase system was evaluated. Obtained flow regime of dispersed n-heptane droplets in a continuous buffer phase, which enables in situ extraction of the produced isoamyl acetate to the n-heptane phase, provides a very large interfacial area for the esterification catalyzed by an amphiphilic lipase B, which positions itself on the n-heptane–buffer interface. Productivities obtained were the highest reported so far for this reaction and indicate that Corning Advanced-Flow Reactor™ (AFR™) modules are also very efficient for carrying out biotransformations in two-phase systems. Additionally, for the separation of the n-heptane from the aqueous phase, a membrane separator consisting of a hydrophobic PTFE membrane was integrated, which enabled the reuse of biocatalyst in several consecutive biotransformations.

Development of microreactors with surface-immobilized biocatalysts for continuous transamination

The industrial importance of optically pure compounds has thrown a spotlight on ω-transaminases that have shown a high potential for the synthesis of bioactive compounds with a chiral amine moiety. The implementation of biocatalysts in industrial processes relies strongly on fast and cost effective process development, including selection of a biocatalyst form and the strategy for its immobilization. Here, microscale reactors with selected surface-immobilized amine-transaminase (ATA) in various forms are described as platforms for high-throughput process development. Wild type ATA (ATA-wt) from a crude cell extract, as well as Escherichia coli cells intracellularly overexpressing the enzyme, were immobilized on the surfaces of meander microchannels of disposable plastics by means of reactor surface silanization and glutaraldehyde bonding. In addition, a silicon/glass microchannel reactor was used for immobilization of an ATA-wt, genetically engineered to contain a silica-binding module (SBM) at the N-terminus (N-SBM-ATA-wt), leading to immobilization on the non-modified inner microchannel surface. Microreactors with surface-immobilized biocatalysts were coupled with a quenching system and at-line HPLC analytics and evaluated based on continuous biotransformation, yielding acetophenone and l-alanine. E. coli cells and N-SBM-ATA-wt were efficiently immobilized and yielded a volumetric productivity of up to 14.42 g L-1 h-1, while ATA-wt small load resulted in two orders of magnitude lower productivity. The miniaturized reactors further enabled in-operando characterization of biocatalyst stability, crucial for successful transfer to a production scale.