Micro scale reactor system development with integrated advanced sensor technology: A modular approach to the development of microfluidic screening platforms

Biotechnology is an increasingly relevant field, at a time when most industries strive for the development of greener processes by reducing and/or eliminating the environmental impact of industrial processes, often by limiting the use of certain compounds (e.g. harsh solvents, metal-based catalysts), but also by reducing the number of reaction steps and the quantity of generated waste.

The use of biological systems, such as biocatalysts and cells, enables operation at milder conditions, creating new synthetic routes, improving regio- and stereoselectivity, and avoiding (de)protection steps requiring harsh solvents or compounds, among other advantages. However, due to the complexity of biological systems, the development of fermentation or biocatalyst based processes is not straightforward.

Similar enzymes may act on similar substrates but operate at different temperatures. Combinations of enzymes in cascade systems may require the spatial separation of the involved enzymes due to incompatible side-products or inhibitions from the reaction components. Certain cells present a faster growth rate at high densities, or different production titres depending on the formation of aggregates or cell adherence.

The broad range of biological molecules and cells available for bioprocesses thus require the optimization of specific substrates or operation conditions, which as illustrated, can vary widely between them. Furthermore, the discovery and tailoring of new biocatalysts or cells involves environmental sampling and the generation of new variants, resulting in thousands of biological systems whose industrial or clinical potential needs to be evaluated, often in a relatively short timeframe.

High-throughput analytical systems are the main tool applied to biocatalyst screening. They enable the parallel operation of different reactions and/or fermentations at different conditions (e.g. substrate concentrations, different substrates, enzymes, medium, oxygen availability, etc.). Thus, high-throughput systems allow to cover the possible variations and narrow the feasible operation conditions, substrates and biocatalysts or cells for application at industrial scale.

The need for fast and comprehensive characterization of biocatalysts has also pushed the development of new screening platforms, based on microfluidics. Microfluidic systems involve the manipulation of small sample volumes (µL to nL) in miniaturized vessels and structures. Through miniaturization, mass and heat transfer becomes significantly faster, but surface and mass transfer limitations due to diffusion are also increased.

Furthermore, microfluidics allows the use of different strategies for each of the unit operations involved in such optimization and screening studies, as well as new sensing and monitoring approaches. Within microfluidics there are several approaches regarding the integration of the required unit operations, ranging from integration on a single chip to a fully modular approach, where the different units correspond to a single chip but are interconnected through fluidic devices.

The latter approach offers more flexibility at a lower cost in terms of the achievable studies with the same unit operations, since these can be placed in a different order depending on the purpose or sample being characterized. The main goal of this dissertation was to develop a biocatalyst screening platform based on modular microfluidics.

With this purpose in mind, three microfluidic modules are presented that can be integrated and used in such modular platform: a microreactor module with integrated oxygen sensors, a microfluidic dilution and quantification module compatible with electrochemical sensors and a module for continuous thermal inactivation of enzymes.

The last two modules were developed specifically for applications in online screening. The focus during development was on achieving user-friendly and simple to use platforms that were furthermore easy to connect with other existing platforms and compatible with a wide range of biocatalytic reactions.

The microreactor module enables the continuous monitoring of oxygen levels and was characterized with a biocatalytic oxidation reaction in order to highlight the operational limitations of the system in terms of oxygen depletion at certain enzyme and substrate concentrations. Strategies for in situ oxygen generation involving addition of catalase and hydrogen peroxide were applied as solutions to overcoming the identified oxygen depletion limitations.

Furthermore, the reactions carried out in the microfluidic system were modelled using computational fluid dynamics, with a good fit between the experimental and simulated data, and the results provided extra insight into the reaction dynamics. The same microreactor was applied to the screening of whole cell variants of a dioxygenase capable of converting alkene substrates.

It was used as a complement to the screening of genetically modified biocatalysts using end-point product quantification. The oxygen consumption rate of each variant in the presence of a standard substrate was used as the screening parameter to select the variant with the faster oxidation reaction rate as the best variant for a possible industrial application.

The second module was developed for integration of different types of sensors to achieve online quantification. The module presents a standardized fitting enabling the connection to either other microfluidic platforms or laboratory scale equipment. Screen-printed electrochemical sensors were integrated through pockets that allowed their easy replacement and thus the re-use of the microfluidics’ platform.

Also, the developed platform included a mixing/dilution channel enclosed by a two-sensor system, which allowed expanding the sensors’ detection range by controlling the sample dilution at which the measurements were performed. The dilution unit was optimized with computational fluid dynamic methods that enabled testing several geometries before fabrication, thus accelerating the platform development.

The third microfluidic module was developed to allow unspecific inactivation of biocatalysts (especially enzymes), and thus precisely control the reaction (residence) time at the point of product quantification in the second module. Such control is important when different modules – reactors and/ or sensing units – are used and frequently changed.

It can furthermore help to regulate the state of the biocatalyst, since it is depending on the temperature and exposure time. In this way, reversible or irreversible denaturation of the enzymes can be achieved. The different modules presented in the dissertation are useful additions to a modular microfluidic toolbox for biocatalyst screening.

They provide online monitoring of biocatalytic reactions or biotransformations, quantification of reaction products and controlled reaction end-points due to the potential to achieve precise temperature control. Furthermore, the developed computational fluid dynamic models allow for a better understanding of the reaction performed in the microsystem.

The model can be further improved to achieve online data acquisition of reaction kinetics by coupling with a mechanistic model. In the case of the developed mixing/dilution channel, the developed model enabled a fast optimization of the unit operation, thereby decreasing the cost and time spent on such endeavour.

The potential of modular microsystems in biotechnological applications was the main driver for the work performed and presented in this dissertation. The objective of this dissertation was to provide, beside three interesting microfluidic systems, a better understanding of the potential that microfluidics, especially in a modular approach and tightly connected to mathematical modelling, can offer to biotechnology and society.