Marco P.C. Marques

Microfluidic devices: useful tools for bioprocess intensification.

The dawn of the new millennium saw a trend towards the dedicated use of microfluidic devices for process intensification in biotechnology. As the last decade went by, it became evident that this pattern was not a short-lived fad, since the deliverables related to this field of research have been consistently piling-up. The application of process intensification in biotechnology is therefore seemingly catching up with the trend already observed in the chemical engineering area, where the use of microfluidic devices has already been upgraded to production scale. The goal of the present work is therefore to provide an updated overview of the developments centered on the use of microfluidic devices for process intensification in biotechnology. Within such scope, particular focus will be given to different designs, configurations and modes of operation of microreactors, but reference to similar features regarding microfluidic devices in downstream processing will not be overlooked. Engineering considerations and fluid dynamics issues, namely related to the characterization of flow in microchannels, promotion of micromixing and predictive tools, will also be addressed, as well as reflection on the analytics required to take full advantage of the possibilities provided by microfluidic devices in process intensification. Strategies developed to ease the implementation of experimental set-ups anchored in the use of microfluidic devices will be briefly tackled. Finally, realistic considerations on the current advantages and limitation on the use of microfluidic devices for process intensification, as well as prospective near future developments in the field, will be presented.

High Throughput in Biotechnology: From Shake-Flasks to Fully Instrumented Microfermentors

This review provides a broad overview of recent patents and developments in the field of small scale bioreactors. The scope of the work will focus on vessels from about 500 ml down to the submilliliter scale. This field of research is of acknowledged relevance, since the rational use of these devices is contributing to speeding up several areas of bioprocessing, in particular due to their intrinsic capability for high throughput. Within such framework, small scale bioreactors are currently used for a wide array of applications, from cell screening to process optimization. Small scale bioreactors are available in different working volumes, configurations and architectures, each of those presenting advantages and limitations. These issues will be tackled in the present work, which will thus address both shaken vessels, such as Erlenmeyer and microtiter plates, as well as miniature bioreactors, which are literally scaled down versions of bench/pilot scale reactors. Reference will be made to commercially available platforms that allow for parallel operation. Considerations will also be made on the necessary requirements for the effective use of the data gathered using these devices in bioprocess development.

Sitosterol bioconversion with resting cells in liquid polymer based systems.

The use of a biocompatible water-immiscible organic phase as a substrate and product pool has been acknowledged as an effective tool to overcome the low volumetric productivity of aqueous bioconversion systems involving hydrophobic compounds. The growing environmental and public health awareness is nevertheless leading to restrictions in the use of organic solvents in industrial processes, in order to render these more environmentally friendly. Different approaches are hence being assessed for the design of alternative bioconversion media, involving the use of supercritical fluids, ionic liquids and natural oils and liquid polymers, among others. In this work, the use of liquid polymers as key components in the bioconversion media for a multi-step microbial bioconversion was assessed. The model system used was the selective cleavage of the side-chain of beta-sitosterol by free resting cells of Mycobacterium sp. NRRL B-3805, a well established industrial multi-enzymatic process involving the use of nine catabolic enzymes in a fourteen-step metabolic pathway. High product yields were obtained when silicone B oil was used as substrate carrier/product pool, both in single oil and in oil:buffer two liquid phase system.

Miniaturization in biotechnology: speeding up the development of bioprocesses.

The use of miniaturized devices for fastening bioprocess development, even up to production scale, has expanded rapidly, a feature clearly noticeable in recent years. This matter was reviewed in a recent past, but several developments have occurred since. These will be addressed in the present work, which will provide some insight on the use of microfluidic /microstructured reactors and of micro-scale downstream processing as well, therefore broadening the scope of the review.

Recent Achievements on Siderophore Production and Application

Iron is the most abundant chemical element on Earth but its most common oxidation state is Fe(III) which presents a very low solubility under physiological conditions. During evolution, micro-organisms have developed sound strategies to acquire iron from both the environment and superior organisms, including direct uptake of iron ions from exogenous iron/heme sources and the synthesis of specialized Fe(III) chelators called siderophores. The present review paper aims at presenting and discussing the latest achievements in siderophore isolation and production, as well as novel applications of these molecules in therapies against iron-related diseases and in vaccines, and their application as antimicrobial agents and biosensors.

Conscious coupling: The challenges and opportunities of cascading enzymatic microreactors

The continuous production of high value or difficult to synthesize products is of increasing interest to the pharmaceutical industry. Cascading reaction systems have already been employed for chemical synthesis with great success, allowing a quick change in reaction conditions and addition of new reactants as well as removal of side products. A cascading system can remove the need for isolating unstable intermediates, increasing the yield of a synthetic pathway. Based on the success for chemical synthesis, the question arises how cascading systems could be beneficial to chemo-enzymatic or biocatalytic synthesis. Microreactors, with their rapid mass and heat transfer, small reaction volumes and short diffusion pathways, are promising tools for the development of such processes. In this mini-review, the authors provide an overview of recent examples of cascaded microreactors. Special attention will be paid to how microreactors are combined and the challenges as well as opportunities that arise from such combinations. Selected chemical reaction cascades will be used to illustrate this concept, before the discussion is widened to include chemo-enzymatic and multi-enzyme cascades. The authors also present the state of the art of online and at-line monitoring for enzymatic microreactor cascades. Finally, the authors review work-up and purification steps and their integration with microreactor cascades, highlighting the potential and the challenges of integrated cascades.

Characterization of 24-well microtiter plate reactors for a complex multistep bioconversion: from sitosterol to androstenedione.

Microtiter plates are commonly used for screening purposes and one-pot biotransformations. Nonetheless, there are scarce systematic studies focused on the application of these systems to complex whole cell multistep bioconversions, which typically require prolonged incubation periods, and on the evaluation of the reproducibility of data generated in such shaken miniature reactors. The present study aims to contribute to fill in this gap. The model system selected was the side-chain cleavage of beta-sitosterol performed by whole cells of Mycobacterium sp. NRRL B-3805 in 24-well microtiter plates. Reproducibility of the data was assessed within the 24 wells of the microtiter plate as well as matching different plates placed in different positions on the shaking platform. Results show the suitability of microtiter plates with proper oxygen and pH monitoring capabilities to carry out complex multistep bioconversion using relatively slow growing bacteria. Reproducibility levels are in the range of an interval of confidence of 92%.

Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

Abstract Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell−1 s−1, and 5 and 35 amol cell−1 s−1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies.

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.

Real‐time pH monitoring of industrially relevant enzymatic reactions in a microfluidic side‐entry reactor (μSER) shows potential for pH control

Monitoring and control of pH is essential for the control of reaction conditions and reaction progress for any biocatalytic or biotechnological process. Microfluidic enzymatic reactors are increasingly proposed for process development, however typically lack instrumentation, such as pH monitoring. We present a microfluidic side-entry reactor (μSER) and demonstrate for the first time real-time pH monitoring of the progression of an enzymatic reaction in a microfluidic reactor as a first step towards achieving pH control. Two different types of optical pH sensors were integrated at several positions in the reactor channel which enabled pH monitoring between pH 3.5 and pH 8.5, thus a broader range than typically reported. The sensors withstood the thermal bonding temperatures typical of microfluidic device fabrication. Additionally, fluidic inputs along the reaction channel were implemented to adjust the pH of the reaction. Time-course profiles of pH were recorded for a transketolase and a penicillin G acylase catalyzed reaction. Without pH adjustment, the former showed a pH increase of 1 pH unit and the latter a pH decrease of about 2.5 pH units. With pH adjustment, the pH drop of the penicillin G acylase catalyzed reaction was significantly attenuated, the reaction condition kept at a pH suitable for the operation of the enzyme, and the product yield increased. This contribution represents a further step towards fully instrumented and controlled microfluidic reactors for biocatalytic process development.