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  • 1 University of Ljubljana, SI-1000 Ljubljana, Slovenia
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Recent remarkable progress in understanding and engineering enzymes and whole cells as highly selective and environment-friendly catalysts enabling novel routes for the production of pharmaceuticals, fine and platform chemicals, and biofuels has spurred the quest for fast biocatalyst screening and development of efficient processes with long-term biocatalyst use. Besides this, current efforts towards more sustainable production systems and bio-based products have triggered an intense research on chemo-enzymatic cascades and establishment of continuous end-to-end processing. Microreaction technology, which has in the last two decades changed the paradigm in the laboratory and production scale organic synthesis, is recently gaining attention also in the field of applied biocatalysis. Based on the trends highlighted within this article, microfluidic systems linked with appropriate monitoring and feedback control can greatly contribute to successful implementation of biocatalysis in industrial production. Microflow-based droplets facilitate ultrahigh-throughput biocatalyst engineering, screening at various operational conditions, and very fast collection of data on reaction kinetics using minute amounts of time and reagents. Harnessing the benefits of microflow devices results in faster and cheaper selection of substrate(s) and media, and development of suitable immobilization methods for continuous biocatalyst use. Furthermore, the use of highly efficient reactor designs integrated with downstream processing enabling also faster and more reliable scale-up can bridge the gap between the academic research and industrial use of biocatalysts.

Abstract

Recent remarkable progress in understanding and engineering enzymes and whole cells as highly selective and environment-friendly catalysts enabling novel routes for the production of pharmaceuticals, fine and platform chemicals, and biofuels has spurred the quest for fast biocatalyst screening and development of efficient processes with long-term biocatalyst use. Besides this, current efforts towards more sustainable production systems and bio-based products have triggered an intense research on chemo-enzymatic cascades and establishment of continuous end-to-end processing. Microreaction technology, which has in the last two decades changed the paradigm in the laboratory and production scale organic synthesis, is recently gaining attention also in the field of applied biocatalysis. Based on the trends highlighted within this article, microfluidic systems linked with appropriate monitoring and feedback control can greatly contribute to successful implementation of biocatalysis in industrial production. Microflow-based droplets facilitate ultrahigh-throughput biocatalyst engineering, screening at various operational conditions, and very fast collection of data on reaction kinetics using minute amounts of time and reagents. Harnessing the benefits of microflow devices results in faster and cheaper selection of substrate(s) and media, and development of suitable immobilization methods for continuous biocatalyst use. Furthermore, the use of highly efficient reactor designs integrated with downstream processing enabling also faster and more reliable scale-up can bridge the gap between the academic research and industrial use of biocatalysts.

1. Introduction

Biological catalysts, either in the form of isolated enzyme preparations or whole cells, were in the last century recognized as extremely promising catalysts exhibiting several outperforming properties when compared to their chemical counterparts such as stereo- and regio-selectivity, application at mild conditions, biocompatibility, and biodegradability. Based on recent remarkable progress in enzymology, molecular biology, and metabolic engineering, enabling adjustment of enzyme activity, selectivity, and stability or cell metabolic pathways to industrial needs, biocatalysis has been in the last decade integrated into mainstream organic synthesis [1]. Moreover, biotransformations have been recognized as one of the key green engineering areas paving the way towards more sustainable technological platforms in pharma and fine chemicals production [2]. Implementations of biocatalytic steps in industrial productions of pharmaceuticals, e.g., pregabalin treating several central nervous system disorders [3], antidiabetic compound sitagliptin [4], and hepatitis C drug boceprevir [5], demonstrated significant improvement in the performance and sustainability of manufacturing processes, evident from E-factor reduction for 79.3% and 63.1% in the case of pregabalin [3] and boceprevir [5], respectively.

Within approximately the same time framework of biocatalytic expansion in organic synthesis applications, microreaction technology changed the paradigm of chemical engineering and proved its beneficial role in several processes [6]. Major motivation of industry to implement this new technology could be attributed to the improved safety, better temporal and spatial process control based on extremely efficient heat and mass transfer, and mostly laminar fluid flows in devices of typical volumes below 1 mL and with at least one characteristic dimension below 1 mm. Furthermore, the possibility to operate within novel process windows, integration of various processing steps and analytics, faster and easier transfer from laboratory to industrial scale by using numbering-up approach, and transfer from batch towards continuous operation are among advantages of microflow systems [79]. The latter was owing to several benefits compared to traditionally used batch mode of operation recognized as the crucial green engineering area in pharma and fine chemicals production, supported also by the FDA's first approval of switch from batch to continuous manufacturing process in 2016 for Janssen's HIV-1 treatment drug Prezista [2, 9].

Despite the growing importance of biocatalysis and microscale technology, merging of these two fields took much slower pace than implementation of microflow systems in organic synthesis. For the pioneers in the field of microreaction technology, the rising interest, as well as the resistance in application of microreactors in biotechnology, was like a “déjà vu” after almost two decades [10]. As evident from Figure 1a, there were only a few reports on biocatalytic reactions performed in microreactors before 2010, while lately a growing interest could be observed, especially in terms of number of citations these papers have achieved (Figure 1b). This trend is evident also from the topics of the conferences relevant for both fields: if at the 12th International Conference on Microreaction Technology (IMRET, 2012), gathering the community working on microreaction technology, only a few examples on biotransformations in microflow systems were present, the latest 14th IMRET edition in 2016 listed “Biotechnology in flow chemistry” among the conference topics. On the other side, on the last Biotrans 2017 conference, which serves as a major platform for discussions on novel trends regarding biotransformations, a session on “(Bio)process intensification by enzyme immobilization, compartmentalization and microreactors” was introduced, where a substantial amount of contributions showing (micro)flow-based biotransformations were presented. Besides this, in 2010, a series of conferences on Implementation of microreactor technology in biotechnology (IMTB) has emerged, having its 4th edition in 2017, aiming to promote the use of microscale devices in bioprocessing and to accelerate the knowledge transfer between the academia and industry [11]. The community created in the cross-section of microfluidics, life sciences, analytics, and bioprocess engineering focuses on enzymatic microreactors, cells within microdevices, and analytical microdevices, as well as bioprocess intensification and integration within microflow systems.

Figure 1.
Figure 1.

a) An estimation of the number of publications per year as determined by using “biocatal*” and “microfluid* OR microreact*” as a keyword search in Web of Science. b) The sum of citations that publications from a) have gained per year. Data were obtained on December 6, 2017

Citation: Journal of Flow Chemistry JFChem 7, 3-4; 10.1556/1846.2017.00021

This article will highlight some major applications and trends regarding biotransformations in micro- and meso-scale flow systems comprising biocatalyst screening, biocatalytic process development, biocatalyst immobilization, reactor design and optimization of process conditions, as well as biocatalytic process integration with downstream processing. Furthermore, the prospective of this emerging technology based on transdisciplinary research that crosses many disciplinary boundaries to create a holistic approach towards sustainable industrial production will be given.

2. Ultrahigh-Throughput Biocatalyst Screening

Implementation of biocatalytic steps in industrial organic synthesis mostly relies on the availability of commercially available and affordable enzymes or cells capable to achieve high productivities at non-natural conditions comprising high substrate concentrations, non-aqueous solvents, high temperatures, and/or high shear stress. Besides screening for promising novel enzyme activities among extremophiles and aquatic organisms, as well as from metagenomic libraries, metabolic and protein engineering play a crucial role in the expansion of industrial biotransformations [1, 12, 13]. Among others, the abovementioned industrial implementations of biocatalytic processes in sitagliptin and boceprevir production by ω-transaminase [3] and monoamine oxidase [4], respectively, resulted from successful combination of rational design and directed evolution. Thereby enzymes capable to stereoselectively transform non-natural substrates at high concentrations and in the presence of organic solvents [3], or with tolerance to substrate/product inhibition and improved thermostability [4], were developed.

Increasing demand for novel biocatalysts and millions of variants obtained by random mutagenesis, directed evolution, or in functional metagenomics have also spurred the development of novel techniques and tools for their screening end selection. Microtiter plates, which were brought to biotechnology from combinatorial chemistry in 1980s, were already a huge step forward from Petri dishes and Erlenmeyer flasks, traditionally used since late 19th century. Introduction of automated colony pickers and liquid handling robots significantly improved the performance of biocatalyst screening, but also increased the costs. At the end of the previous century, a breakthrough innovation in the field was the implementation of microfluidic-based droplets, where single cells are compartmentalized in water-in-oil droplets of a few picoliters volume, where they are typically incubated with a labelled substrate, incubated and then sorted based on the presence of a product, generally using fluorescence readouts [14]. Recently, the possibility to use microfluidic absorbance-activated cell sorting (Figure 2a) opened the opportunity for further expansion of this technique [15]. Furthermore, screening and selection of filamentous fungi, currently the most important producers of industrial enzymes, was recently demonstrated using nanoliter-volume droplets and microfluidic sorter (Figure 2b). Introduction of microfluidics reduced the time needed to sort the same amount of variants by the robotic microtiter plate-based screening platform from 16 days to less than 24 h and consumables costs from $8770 to $14 [16].

Figure 2.
Figure 2.

Implementation of microfluidic systems in ultrahigh-throughput screening and efficient and reliable gathering of enzyme kinetics data. a) Directed enzyme evolution by 180 pL droplets encapsulating single cells with expressed enzyme, released by lysis, incubated with substrate to produce dyed product, and selected based on absorbance-activated droplet sorting (AADS) [15]; b) creation and incubation of 10–20 nL droplets allowing growth of the branched mycelial network of mutants of filamentous fungus for the time needed to secret target enzyme and selected based on fluorescence-activated droplet sorting device adapted to sort nanoliter droplets (available from Beneyton et al. [16]); c) estimation of enzyme kinetics and inhibition using 33 pL droplets containing enzyme, various inhibitor concentrations barcoded by a fluorescent green dye, in which substrate was delivered using picoinjector module; orange fluorescent product and fluorescent green inhibitor were monitored along the channel at designated measurement points (MPs) (reproduced from [25] with permission from The Royal Society of Chemistry)

Citation: Journal of Flow Chemistry JFChem 7, 3-4; 10.1556/1846.2017.00021

A remarkable progress in in vitro protein engineering and directed evolution was gained by introduction of a droplet-based microfluidic screening based on the coupled transcription and translation of genes using cell-free systems comprising PCR reaction in droplets, which enabled reduction of the reagent costs as compared to microtiter plates by almost 105–fold [17]. Recently, monodisperse gel-shell beads containing a protein and its coding DNA from a single compartmentalized cell were used for performing ultrahigh-throughput evolution of enzyme catalysts within the picoliter-volume beads [18]. Microfluidic systems coupled directly to genetic-algorithm-controlled experimental systems are likely to make a major impact in the industrial selection of biocatalysts [19].

3. Fast Biocatalytic Process Development

Very short time-to-market requests in biopharmaceuticals production has led to high-throughput process development comprising model-aided experimental design and data mining, high-throughput analytics, transfer from batch to continuous operation, as well as upstream and downstream processing linking. Microfluidic bioreactors with integrated sensors for pH, dissolved oxygen, and biomass concentration were found as very efficient tools to test hundreds of process conditions, raising the need for highly automated systems enabling parallel experimentation, short time, and low material consumption [20]. Similarly, biocatalytic process development needs a holistic approach, where in addition to biocatalyst engineering, substrate and reaction medium engineering, biocatalyst immobilization strategy, and reactor design together with downstream processing should be considered [1, 21].

Microflow systems offer the possibility to shorten time and to reduce costs for obtaining basic data for process development. Examples on comparison of various types of catalysts comprise gluconic acid production, where nobel metal versus enzymatic oxidation and the oxidation under conventional process conditions versus under Novel Process Windows were explored [21]. Besides this, kinetic resolution of 2-methylene-substituted cycloalkanols using immobilized Pseudomonas fluorescens lipase and Candida antarctica lipase B was compared in a meso-flow reactor using various substrates and temperatures [22].

In order to determine the optimal reaction conditions and successfully design the reactor and process parameters, obtaining accurate kinetics is of utmost importance. By application of automated microreactor system that uses a sequential experimentation framework driven by model-based optimization feedback for online kinetic parameter determination, the speed and efficiency in gathering reaction rate model and parameters were substantially improved [23]. Besides this, a seminal work of Song and Ismagilov [24] using a droplet-based microfluidic system, where each droplet could be seen as a moving reactor, enabling monitoring of the rate of fluorogenic product development along the channel length (Figure 2c), has opened the possibility for extremely efficient estimation of enzyme kinetics [14]. The use the absorbance-based detection [15], as well as integration of picoinjector module for scanning of multiple inhibitor and substrate concentrations in a single high timely-resolved experiment (Figure 2c) [25], can even further expand the use of these devices in early stages of the biocatalytic process development. Further automatization of systems based on droplet-generating microfluidic chips enabling droplet manipulation and integration with corresponding detectors and software for data analysis might offer the possibility to obtain more reliable data and substantial time and cost advantage to conventional microplate-based methods, where inadequate mixing and heat transfer might interfere experimental data.

Multispecificity of enzymes, or so-called enzyme promiscuity, opens the ability to use them for biotransformations of substrates of higher economic and environmental interest or with better solubility, for the optimization of existing biotransformations, and to discover totally different reaction types [1]. The design of synthetic routes can be also oriented towards an abundant and inexpensive, preferably bio-based starting material [26]. A systematic substrate engineering relies on the knowledge on protein structure and the enzymatic transformation mechanism [1], which is not often available during the early stages of process development, so fast and low material-consuming testing on substrate specificity is a very useful tool in process design. Microreactors with immobilized biocatalysts were found very efficient for investigation of stereospecificity of thermophilic l-aminoacylase [27], as well as for testing of various 2-methylene-substituted cycloalkanols for lipase-catalyzed kinetic resolution [22] and for the highly enantioselective synthesis of various chiral amines from non-natural ketones using Escherichia coli cells overexpressing ω-transaminase [28]. Furthermore, the high sensitivity of microdroplet-based platforms for screening of unculturable bacterial communities revealed the possibility to isolate enzymes with promiscuous side activities that could not be predicted by bioinformatic harvesting of metagenomic sequencing [29].

Since many organic substrates or reaction products are only sparingly soluble in water, the use of organic solvents, ionic liquids, and supercritical fluids is getting considerable attention. Both free enzymes in two-liquid phase systems, as well as immobilized biocatalysts in non-conventional media, were frequently used within microfluidic devices, offering the possibility for intensified mass transfer among phases, better control of fluid flow, and the possibility for very efficient in situ product removal, which typically resulted in higher volumetric productivities as compared to conventional processing [3032]. Besides this, selection of the appropriate solvent could be facilitated and cheapened by using microreactors, as shown in the work on thermophilic l-aminoacylase, where the addition of various water-miscible organic solvents in aqueous media could be optimized with very small amounts of used material [27]. Current trends to use solvents that combine high solubilization capacity and cost-effectiveness with a low environmental footprint, such as deep eutectic solvents and protic ionic liquids that can even improve biocatalyst selectivity [1], might gather momentum by using high-throughput microflow system for their selection.

Among operational parameters affecting biotransformation outcome, pH and temperature are of significant importance. Again, several reports on their fast optimization using microflow devices were published, including various lipase- [22, 31], aminoacylase- [27], ω-transaminase- [28, 33], fumarase- [34], and invertase-catalyzed biotransformations [35], among others. Adjustment of pH based on an integrated pH sensor and a microfluidic side entry reactor enabled optimization of the transketolase and penicillin G acylase-catalyzed biotransformations [36]. Besides this, the decision on using free or immobilized enzymes or whole cells could be efficiently gained using microreactors, as demonstrated for alcohol dehydrogenase-catalyzed hexanal production with in situ coenzyme regeneration [37].

4. Cascade Reactions in Microreactors Employing Biocatalysts

The abovementioned trends towards establishment of chemo-enzymatic and multienzymatic cascades in continuously operated microflow reactors were recently reviewed by Gruber et al. [38]. Benefits of coupling the reaction steps in consecutively linked micro- or meso-reactors encompass the possibility for in situ generation of toxic reagents followed by their immediate use in the subsequent step, reduction of costs due to the removed need for intermediates isolation, and thereby an overall reduction of the number of synthesis steps. Furthermore, spatial control and fast heat transfer allowing the individual reactors to be run at optimal pH and temperature are among key advantages. As a consequence, larger synthetic yields and higher product qualities were typically obtained in microflow compartmentalized systems compared with their batch, one-pot equivalents. Furthermore, spatial separation empowers to monitor the progress of each reaction individually through the implementation of on-line or at-line monitoring tools [38, 39]. The use of highly controlled milliliter-scale tubular flow reactors also enables a rapid optimization of individual steps within the cascade and integration with in-line quenching and product separation, as recently shown in the study of a four-step chemo-enzymatic synthesis of enantiomerically pure captopril, a widely prescribed drug for the treatment of hypertension [40].

Among challenges that need to be addressed for a more widespread use of chemo-enzymatic systems is the engineering of the reaction media that would be compatible to subsequent reaction steps where enzymatic parts typically prefer aqueous solutions, while chemical steps are usually performed in organic solvents [38]. As highlighted above, the potential of non-conventional green solvents where biocatalysts might be stable for a long time could be explored. Furthermore, the use of thermophilic and engineered enzymes sustaining harsher conditions applicable in chemical steps could broaden the possibility for industrial implementation.

Similarly, in multi-enzyme cascade systems there are significant challenges such as matching of reaction conditions and overcoming the inhibition of the enzymes in the cascade, such as caused by reactants and products. Here, intermediate steps which remove inhibiting reactants or products might be necessary to achieve a fully functioning system. Recently, the first demonstration of free enzyme cascade within spatial confinement of reactions each having different pH optima resulted in full conversion in a short time [41]. Therefore, the ability to rapidly evaluate the effects of reaction conditions and different enzyme variants in microreactors provides a new paradigm for performing multi-step biosynthetic reactions with the aim to create de novo pathways for the production of new molecules [38].

5. Biocatalyst Immobilization in Microreactors

As successful implementation of biocatalysts in production systems vastly depends on their ability for a long-term use, biocatalyst immobilization is often preferred over the use of free cells or enzymes. Due to difficulties with recycling and possible contamination of usually expensive catalysts, as well as the possibility for the simultaneous reaction and separation, heterogeneous (bio)catalysis represents a trend of industrial application, especially for enantioselective syntheses.

Similarly as in their conventional counterparts, a vast variety of immobilization techniques might be applied in microstructured devices, depending on device design and material, as well as biocatalyst and media characteristics [42]. Harnessing extremely high surface-to-volume ratio of microflow systems was shown suitable for single-layer surface immobilization of various yeast and bacterial cells [34, 36, 43] and covalently linked enzymes [35, 44]. Furthermore, creation of bacterial biofilm used for styrene oxidation [45], as well as for oriented surface immobilization of d-amino acid oxidase [46], ω-transaminase [47], and sucrose phosphorylase [48] using positively charged protein tags benefited from this inherent feature of microflow devices, among others. Internal surfaces might be further increased by integration of nanostructures such as nanosprings, applied for the immobilization of β-galactosidase [49], threonine aldolase [50], and methanotrophic microorganisms [51], as well as by electrospun nanomats used for ω-transaminase-catalyzed biotransformation in a resealable flow cell, where His-tagged enzyme was complexed with metal ions in mats [52]. The latter approach presents also a challenging opportunity for simultaneous in-flow protein purification and surface tethering for continuous-flow biocatalysis in microflow systems, as it was recently demonstrated in a vortex fluidic device. There, a thin film of IMAC resins created at the reactor walls enabled purification of His-tagged enzymes from complex cell lysates, and the formation of distinct enzymatic zones for multi-step biocatalysis [53].

In order to get higher biocatalyst loads, meso- and microscale monolyths [27], integrated membranes [50], sol-gels [22], and packed bed reactors using porous beads [22, 28, 3133, 50] were also applied. Recently, the use of magnetic-field assisted microreactors using magnetic nanoparticles for retainment of enzymes [54, 55] or cells [56] were found as very promising approach for easily controlled biocatalyst immobilization.

One of the major challenges for better understanding and exploitation of heterogeneous biocatalytic conversions within flow reactors is to develop methods for characterization of immobilized biocatalyst. This comprises biocatalyst quantification and distribution within the reactor, as well as protein conformation enabling direct characterization of enzyme activity and stability. Some methods used for characterization of enzymes immobilized on solid supports in conventional reactors, reviewed by Bolivar et al. [57], could be implemented also in the micro- and meso-reactors with continuous operation mode, but there is still the need for more direct analytical methods. Quantification of enzymes immobilized on defined area of microreactor inner walls and their activity could be estimated from mathematical models comprising appropriate reaction-diffusion dynamics [44, 47].

Miniaturized reactors with immobilized cells or enzymes were shown to be very useful for process development studies, primarily due to the possibility for in-operando characterization of biocatalyst activity and stability, which is crucial for successful transfer to production scale [27, 28, 3133, 4446, 50, 57, 58].

6. Modular Flow Biocatalytic Reaction Platforms

Recent trends in pharma and fine chemicals production to shift the paradigm from batch towards continuous manufacturing, and to establish end-to-end production systems, shed the light towards development of efficient flow reactors and integrated unit operations. The inherent possibility of continuous operation and modularity of micro- and meso-reaction platforms that might be easily integrated with downstream processing units offer huge potential of these systems for industrial use, especially when considering scale-up/numbering-up concept for the capacity increase [6, 58, 59]. Furthermore, miniaturized reactors enable very efficient temperature control, fluid flow distribution, and mass transfer based on reduced paths between the molecules of the substrate and (bio)catalyst within the confined space, which lead to process intensification. Combining biotransformations and continuously operated miniaturized reactors therefore offers huge potential for high-added value chemicals manufacturing [50, 60] and for bio-based production of liquid fuels such as methanol [51] and biodiesel [61]. According to the recent market review reports, microreactors are expected to witness rapid growth with a CAGR of over 20% from 2016 to 2025, as it can be depicted from Figure 3, presenting U.S. flow chemistry market revenue predictions including continuous stirred tank reactors (CSTR), tubular plug flow reactors, microreactors and reactors comprising microwaves [62].

Figure 3.
Figure 3.

Current and expected US flow chemistry market revenue by reactor for the period from 2014 to 2025, in millions USD [62]

Citation: Journal of Flow Chemistry JFChem 7, 3-4; 10.1556/1846.2017.00021

As stated in recent reviews, continuously operated micro- and meso-reactors were found beneficial for several biocatalytic conversions, especially those performed in multiphase systems [26, 30, 45, 58]. Novel designs of miniaturized gas–liquid–solid flow reactors aim to harness very short diffusion times, high surface-to-volume area for high biocatalyst loads, and efficient introduction of gaseous substrates. This was demonstrated in the case of biocatalytic conversion of styrene to (S)-styrene oxide using biofilms of Pseudomonas sp., where oxygen availability and substrate inhibition were crucial for process efficiency [45]. A comparison of miniaturized glass tubular reactor with biofilm and a liquid phase containing dissolved substrates, a tubular membrane reactor enabling substrates supply through the permeable reactor walls, and the four-phase segmented flow reactor with oxygen bubbles revealed the benefit of the latter due to the increased oxygen availability [45]. Similarly, continuously operated bio-lamina bioreactor developed for methanol production from methane and oxygen encompassed nanomaterial- or porous polymer-supported biofilm of methanotrophic bacteria, where liquid phase and gas bubbles passing through the flow microchannels formed between two plates enabled high mass transfer rates for supplying nutrients and removing products and toxins [51]. Furthermore, a falling film microreactor with surface immobilized d-amino acid oxidase was used for efficient contacting of oxygen, heterogeneous biocatalyst, and the liquid phase containing substrate for biotransformation [46].

A unique microreactor with integrated dispersion membrane was developed for whole-cells catalyzed hydration of acrylonitrile to acrylamide, which is produced using a tons-scale industrial biotransformation. Microreactor design enabled formation of very small acrylonitrile droplets providing very high specific surface area for adsorption of free Rhodococcus ruber cells containing nitrile hydratase and faster substrate dissolution, resulting in significant acceleration of the apparent reaction rate. Together with reduced external diffusion resistance of acrylonitrile to the biocatalyst and short time of exposure of undissolved acrylonitrile to free cells, such reactor configuration led to very efficient acrylamide production [63].

Miniaturization was shown to be beneficial also for two-plate packed bed reactors, mostly due to efficient fluid flow distribution and accessibility of the biocatalyst [3133].

Although early studies envisioned simple multiplication of microreactors to scale-out to production levels, i.e., numbering-up, it became evident that such approach creates highly complex fluid flow distribution and control challenges. Based on the knowledge gained within recent years, scale-up is generally accomplished by a combined approach of increasing reactor dimensions with structures that preserve heat and mass transfer advantages followed by multiplying out [6]. Such scale-up from a microliter to the milliliter scale was used for a packed bed reactor with immobilized ω-transaminase used for production of optically active compounds [33].

Among commercially available microstructured reactors, Corning Advanced Flow Reactors™ from specialty glass ceramics offers scalable development and production systems considerably used in chemical industry. Recent study on lipase-catalyzed synthesis of isoamyl acetate, a flavor widely used in food industry, showed huge potential of these modules also for two-liquid phase reactions with dissolved biocatalyst. Productivities obtained within liquid flow (LF) reactor comprised of a chain of identical cells with variable cross sections, and internal obstacles enabling creation and maintenance of fine dispersions providing very large interfacial area for the reaction with amphiphilic enzyme and for in situ product extraction were the highest reported so far for this reaction. Additionally, a membrane separator integrated at the reactor outflow as shown in Figure 4 enabled phase separation and the reuse of the biocatalyst in several consecutive biotransformations [64].

Figure 4.
Figure 4.

Scheme of the integrated Corning LF fluidic module with a membrane separator. CaLB — Candida antarctica lipase B (with permission from Novak et al. [64])

Citation: Journal of Flow Chemistry JFChem 7, 3-4; 10.1556/1846.2017.00021

Such consecutive coupling of reactions conducted in micro- or meso-flow reactors with continuously operated miniaturized separation units, e.g., phase separation, concentration, and extraction, was shown to result in biocatalytic process intensification [32, 38, 60], analogous to their chemical counterparts [6, 59]. Moreover, in situ product removal could solve problems of reversible biocatalytic reactions, product inhibition or degradation, and unwanted side-reactions. This might be accomplished via either integrated solvent extraction or solid–liquid membranes, where microflow systems again allow for high interfacial areas and efficient mass transfer of product from the biocatalyst, as highlighted in recent reviews [38, 45, 60]. Furthermore, in situ substrate supply using side entry reactors or permeable microchannels can efficiently deal with substrate inhibition [38, 45].

For industrial implementation, process analytical and control technology is of crucial importance. Recent developments in sensors applications in microreactors allow for on-line monitoring of parameters such as pH and dissolved oxygen or carbon dioxide concentration [38, 39, 46], while most methods to detect substrate and product concentrations are performed in-line [23, 47]. Further development of integrated sensors will significantly contribute to the implementation of meso- and microflow systems in industrial environment, as well as to better evaluation of biocatalytic reaction and transport parameters used for model-based process design.

7. Conclusions and Perspectives

The quest for changing manufacturing paradigms in order to move towards a post-petroleum society has spurred the research of new bio-based value chains introducing biocatalytic steps as extremely efficient and sustainable routes to obtain vital products, ranging from food and feed to bio-based platform chemicals, drugs — especially optically pure compounds, and biofuels. Biotransformations therefore play a crucial role in the bioeconomy, which aims to separate economic growth from resource depletion and negative environmental impacts.

Microflow systems play an important role in biocatalyst screening and evolution, as well as in high-throughput biocatalytic process development enabling in-operando optimization of process parameters. Novel materials support innovative approaches for immobilization of engineered biocatalysts enabling long-term use in continuously operated reactors, gaining momentum in pharma and fine chemicals production. Development of integrated sensors enabling better control of process conditions and modularity of micro- and mesoflow reactors will contribute to further exploitation of the potential of cascade reaction systems employing heterogeneous biocatalysts. Furthermore, recent progress in microfabrication of extended nano-scale fluidics [65] opens the possibility to study mechanisms and kinetics of biocatalytic reactions at the molecular level.

Acknowledgment

Financial support from the EU FP7 Project EUROMBR — European Network for Innovative Microbioreactor Applications in Bioprocess Development (Grant No. 608104) and Grant P2-0191 provided by the Ministry of Higher Education, Science and Technology of the Republic of Slovenia as well as the support of the COST Action CM1303 “Systems Biocatalysis” are gratefully acknowledged.

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