Applications of Multiphasic Microreactors for Biocatalytic Reactions

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Applications of multiphasic microreactors for biocatalytic reactions Rohan Karande, Andreas Schmid, and Katja Buehler Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00352 • Publication Date (Web): 20 Jan 2016 Downloaded from http://pubs.acs.org on January 26, 2016

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Organic Process Research & Development

Applications of multiphasic microreactors for biocatalytic reactions Rohan Karande, Andreas Schmid and Katja Buehler * Helmholtz-Centre for Environmental Research − UFZ GmbH Department of Solar Materials Permoserstr. 15, 04318 Leipzig, Germany * [email protected]

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Table of content graphic


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Abstract: During the last decade, there was a rapid progress in integrating microfluidic reactors and biocatalytic reactions for various applications. The combination of miniaturized technologies and microfluidics allowed coupling of scale and time-dependent phenomena for bioprocess intensification. However, coupling microreactors and biocatalysis is highly complex, requiring an integrated approach addressing biocatalyst features, reaction kinetics, mass transfer and reactor engineering, as outlined in this review. Dimensionless numbers are discussed, which help identifying rate limiting steps and offer opportunities to enhance the overall reaction performance in solid-liquid biocatalytic reactions. This integrated concept is realized in a case study based on the biocatalytic conversion of styrene to (S)-styrene oxide using catalytic biofilms to demonstrate the pivotal role of this integrated approach for evolving the optimal (micro)reactor configuration and design.

Keywords: biocatalysis, microreactors, catalytic biofilms, multiphase flow, bioprocess intensification


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1) Introduction The progress of miniaturizing technologies is encountered in our everyday modern life. With the revolution of electronic miniaturization, the media device sizes of radios, computers and telephones changed substantially and are now fitting in our pockets. Subsequently, there is a strong trend towards integrating fluidics and miniaturized devices in academic and industrial research for the production of chemicals, polymers, fuel and energy.1 In microfluidics, the downsizing of scale makes viscous forces more and more important, and molecular diffusion might play a key role to provide efficient mixing at lower energy costs.2 Optimization of the microfluidics and reactor design improved mass and heat transfer rates and resulted in higher reactor performance compared to classical chemical reactors.1 Thus numerous examples of microreactors exists which have already been successfully established for chemical production on industrial scale.1 Also for biocatalytic reactions, the significance of microreactors has been evaluated with substantial progress.3 Typically, the application of microfluidic reactors for chemical synthesis is considered to be beneficial if reaction time scales are of similar order as mass transport time scales.4 The choice of microreactors for biocatalytic reactions depends on the overall goal of its application either as an analytical or production tool. In the recent past, several extensive reviews on the application of biocatalytic microreactors for analytical or synthesis purposes have been published.3, 5-9 This study reviews recent developments of multiphasic biocatalytic microreactors for chemical synthesis. Nature has provided a vast biocatalytic toolbox for reacting chemical compounds with significant industrial importance. A huge variety of biocatalytic reactions like reductions, oxidations, hydrolysis, hydrations, and others are already applied for synthesis at an industrial scale.10


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However, the use of biocatalytic microreactors on technical scales is not yet reported. A successful implementation of biocatalytic reactions in to microreactors for bioprocess intensification should rely on the engineering of three mutually interfering levels, such as biocatalyst, reaction and reactor (Figure 1). These design levels are mostly constrained by the physicochemical properties of the substrate and product such as water solubility, chirality, toxicity and volatility. A global study on kinetic parameters of enzymes clearly indicated that the physicochemical properties of the respective substrates determine the boundaries to kinetic parameters of enzymes.11 Thus according to the theoretical limitations, the apparent second order rate constant (kcat/Km) for a diffusion-limited enzymatic reaction is below 108-109 s-1 M-1, which means that catalytic turnover rates (kcat) for enzymatic transformation of low water soluble substrates cannot exceed 106-107 s-1.11 Physicochemical properties not only set constraints to the biocatalytic unit, but also have a severe influence on the selection of biocatalyst configuration, reaction setup and reactor design.12 The aim of this review is to set a framework for designing microreactors driven by biocatalysis using an integrated approach addressing biocatalyst, reaction, and (micro)-reactor engineering for bioprocess intensification. It is our belief that such a systematic approach to integrate different process levels will play a key role in future bioreactor development. The first section reviews biocatalyst and reaction features, and their implementation in microreactor modules. This is followed by a discussion of mass transfer and its impact on reactions in a solid-liquid biocatalytic microreactor using dimensionless numbers to identify rate limiting steps. In the final section, a case study based on the biocatalytic conversion of styrene to (S)-styrene oxide was accomplished to illustrate the development of multiphasic and multicomponent biofilm reactors using above described approaches.


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Low solubility and high toxicity

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high solubility and low toxicity

Impact of physicochemical properties of compounds on the four levels of bioprocess design boundaries defined by the kinetic parameters (kcat, Km, Ki) of enzymes

reaction environment defined by mass transfer, reaction and inhibition/toxicity

Biocatalyst level

Reactor level

substrate and product characteristics define up- and downstream process

Process level

multiple phases • reactor module • process design (membrane, and modelling substrate packed bed etc.) injection • • energy, ecology e.g. single-feed, • reactor and economic multiple-feed, viability operation • continuous-feed (continuous, semi-continuous, • flow dispersion batch) e.g. segmented flow and co-flow • reaction conditions Figure1. Graphical arrangement of substrates and products studied in the biocatalytic microreactor modules (upper panel). The physicochemical properties of substrates and products (water solubility, chirality, volatility and toxicity) impose constraints on the four level of bioprocess design such as the biocatalyst, the reaction, the reactor and the process engineering. An integrated approach is necessary to bridge the different physicochemical properties of reactants and the four levels of bioprocess design for bioprocess intensification. This work focused on coupling the first three process levels i.e. biocatalyst, reaction and reactor levels for the development and design of biocatalytic microreactors. The constraints imposed on the •

biocatalyst selection biocatalyst modification biocatalyst configuration e.g. soluble or immobilized biocatalyst

Reaction level

hardware module designed to match mixing time, mass transfer time and reaction

• •


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individual level of engineering need to be addressed in an integrated manner for successful bioprocess development. 2.1) Biocatalysts selection, modification and application in microreactors: Selection of the biocatalyst for catalytic transformation of a given chemical compound depends on its activity, specificity and stability. These parameters have a high impact on the feasibility of a process. While a high catalytic activity and stability are essential to minimize the necessary biocatalyst amount and the equipment size to achieve a given volumetric productivity, a high specificity is beneficial to lower byproduct formation and to improve the product yield. Biocatalyst selection for catalysis in microreactors: In order to perform biological reactions in microreactors, it is necessary to meet the time window for physical processes (heat and mass transfer) and the biocatalytic turnover time scale. As a thumb rule suggested by Renken et al., 2010, the time scale for the physical processes (mass and heat transfer) should be one order of magnitude smaller than the reaction time to overcome mass transfer limitations.13 The characteristic time range for physical processes is in between 10 to 10-2 s for conventional reactors and from 10-2 s to 10-5 s for microreactors.13 Therefore, very fast or instantaneous reactions having a time scale in the range of 10-3 to 10-5 s are influenced by physical parameters and do profit from the microreactor technology. The kinetic parameters of the biocatalysts need to be evaluated for describing the biological reaction time window and for evaluating whether it matches to the process time window in microreactors. Typically, an enzyme is considered to be highly efficient if it has a high second order rate constant (kcat/Km, M-1 s-1). In fact, several enzymes have been reported to perform catalysis at the second order rate constant of 108 to 109 M-1 s-1, which is considered to be the uppermost limit of bimolecular reactions.14,

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At these rate constants, the frequency of the

collision between substrate and enzyme and thus product formation rates are the same. Under


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such conditions, every collision of substrate molecules with a catalyst leads to the formation of a product molecule.14 Such bimolecular reactions are diffusion controlled. Nevertheless, most of the enzymes available in Nature have an average second order rate constant kcat/Km in the range of 104 to 106 M-1 s-1 and an average turnover number kcat in the range of 1 to 100 s-1.11 Assuming an organic compound to have low solubility (below 20 mM) in an aqueous environment, one may estimate that it would require 20 to 0.2 s for the conversion of this substrate, based on the presence of 1 mM enzyme and at the average turnover number kcat in the range of 1 to 100 s-1. The ratio between a given substrate concentration and a maximum reaction rate was used to estimate the reaction time with additional assumptions such as low Km and no inhibitory effects from substrate or product. Although this time range estimated for enzymes with moderate speed is higher than the time (10-2 s to 10-5 s) for physical processes in microreactors, such a basic estimation clearly indicates that not all enzymatic reactions will benefit if applied in microreactors. The statistical data presented in literature show that more than 98% of all enzymes are at least 1 order of magnitude slower than the diffusion rate (108 to 109 M-1 s-1).14 This reflects that the operating window of enzymes in single phase flow microreactors is strictly limited by the efficiency of enzymes used as catalysts. However, biological reactions vary greatly in their turnover rates and are often multi-substrate and/or multiphase dependent. This might involve interplay between the interfacial mass transfer and reaction rates, and open new opportunities to broaden the operating window of enzymes being applied in microreactors. Biocatalyst configuration and modification for its application in microreactors: Currently, different biocatalyst formats such as soluble enzymes, immobilized enzymes, and whole cells are explored in microreactors (Table 1A). The nature of the respective substrate is a very important criterion for the choice of the biocatalyst format. While hydrophobic substrates (logP>1) are well


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converted by whole cells, hydrophilic compounds (logP

Applications of Multiphasic Microreactors for Biocatalytic Reactions

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