Miniaturization in Biocatalysis

1. Introduction

2. Miniature Systems

2.1. Multi-well Plates as Bioreactors

Multi-well plates have been intensively used for high throughput screening of enzyme activity, in assays based in the production (depletion) of a chromogenic or fluorogenic product (substrate), allowing for the simultaneous evaluation of up to 10⁶ different variants [29,44]. Screening can be performed in liquid media or in solid media [45]. Within this scope, multi-well plate based screening has been mostly used for profiling hydrolases, and, to a minor extent, proteases [46]. Multi-well plates have also been used to evaluate the kinetics of biotransformation systems [24,47]. Dynamic pH monitoring as strategy for the determination of kinetic parameters has also been performed, using as model system the hydrolysis of 4-nitrophenol catalyzed by penicillin acylase, and based in the color change of a pH indicator [48]. A similar approach was developed recently for assessing fat quality in foods [49]. The test relied on the influence of fat composition in lipase activity and was monitored by absorbance shift. Recently, Rachinskiy and co-workers developed an experimental set-up based in multi-well plates that allows for the high throughput of enzyme performance and stability [50]. Enzymes are submitted to extreme conditions for short time periods in order to anticipate their long term stability under milder conditions. The methodology combines the high level of parallelization provided by multi-well plates, thorough temperature control, on line techniques to monitor shifts of given parameters (viz. pH) along the time course of the reaction and software for processing the raw data.

The commercialization of multi-well plates with pH (or dissolved oxygen) sensitive sensor spots embedded in each well, coupled to on-line data acquisition ( www.presens.de), favors the implementation of this approach for evaluation of kinetic data, although limited to bioconversion systems where pH (oxygen) changes are involved (Table 2).

Table 2. Characterization of bioconversion systems using multi-well plates.

To be representative such studies require an understanding of the impact in mass transfer phenomena and flow dynamics due to operation on a microliter scale. In the particular case of multi-well plates, mixing is typically promoted by shaking rather than by stirring. Some exceptions to this pattern are the use of micro stir bars in the screening of activity for the whole cell reduction of 6-bromo-β-tetralone to 6-bromo-β-tetralol within yeast and rhodococci libraries [53] and in the assessment of mixing conditions in the production of l-erythrulose promoted by a transketolase [47]; or gas sparging through porous membranes inserted into the base of each well, used for microbial cell growth [54]. The shape of the well (viz. round well vs square well), along with the static surface area to volume ratio and shaking intensity has been shown to affect biocatalyst activity in bioconversion systems requiring oxygen, hence gas-liquid mass transfer, such as the Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one [24]. Such behavior has been ascribed to the pattern of oxygen transfer. Given the relevance of gas-liquid transfer in oxygen dependent reactions, particular care has been given to the identification of operational condition that favor oxygen transfer into the liquid phase, hence preventing operating in biased conditions due to oxygen limitations. This is mostly due to: (a) enhanced shaking intensity within given boundaries, a lower boundary where oxygen transfer rate is distinguishable from static cultures, and an upper boundary, corresponding to a kinetic limited regime or to liquid spillage out of the vessel; (b) increased shaking amplitude; (c) a high static surface to volume ratio where it is assumed that oxygen transfer to the liquid phase mostly occurs at the surface of the liquid rather than by entrained bubbles; (d) improved mixing in square shaped wells, which emulate baffles [24,55–59]. Operation under suitable gas-liquid volumetric mass transfer coefficient, k_la, matching those observed in conventional laboratory (or higher)-scale bioreactor has been shown feasible in miniaturized systems, either based in given multi-well plates and in miniature bioreactors, and this parameter used as criterion for scale-up of fermentation systems [59,60–63]. When multi-well plates are used, a higher ratio of the gas–liquid exchange area to the volume of the bulk liquid can be made available. Miniaturization gives relevance to physical properties such as surface tension, which counters gravitational forces and bubbling, that are further enhanced alongside with size reduction. This pattern is therefore relatively unnoticed in 24-well plates, and increasingly noticeable in 48-, 96- and 384-well plates [55,64,65]. Given those two physical characteristics, a reasonable oxygen transfer rate can be obtained even under a poor degree of mixing of the bulk liquid in multiwall plates [65]. Optical methods for measuring surface tension of liquids contained in multiwall plates have hence been developed, in order to adequately cope with the high throughput nature of the process [66].

When gas-liquid transfer is not needed, the degree of mixing required is no longer related to a given demand for mass transfer between the two phases. Mixing takes place at different levels, macro-, meso- and micro-mixing. Macro-mixing relates to mixing at a macroscopic scale, hence at the scale of vessel, and leads to large-scale distribution patterns, such as residence-time distribution and concentration gradients. Meso-mixing relates to the blending of feed columns of fluid with the bulk fluid, and corresponds to the process where the reactant incoming into the reactor moves away from the plume and is reduced to turbulent eddies. Unlike macro-mixing, that relates to the whole lifetime of an element of fluid in the vessel, meso-mixing relates only to the early moments of an element of fluid that has entered the reactor. Micro-mixing promote contact of fluids at the microscopic or molecular scales. Micro-mixing comprehends a viscous-convective alteration of elements of fluid followed by diffusion [67,68]. In a relative timescale, micro-mixing and reaction times are comparable [69]. With miniaturization, micro-mixing tend to be predominant. Furthermore, and alongside with the decrease in linear dimensions, shorter mixing times are needed, hence diffusion limitation are minimized. Although this pattern is typical of microfluidic devices [20,85], the pattern is already observed at multi-well plate level. Thus, while assessing the relevance of mass transfer in miniaturized system, using as model system the production of l-erythrulose catalysed by transketolase, Matosevic and coworkers noticed that only when the 24-well reaction plate was vigorously mixed, the initial reaction rate statistically matched data obtained from non-shaking 96-well plate [47]. Multiwell plates are typically made of polypropylene or polystyrene, with the later being preferred due to the lack of leakage of toxic substances [56].

2.2. Microfluidic Systems

Microfluidic reactors provide another format for miniaturized bioconversion systems. Unlike micro-well plates, microfluidic reactors allow for processing small volumes (10⁻⁹ to 10⁻¹² m³) of fluid within channels, where at least one dimension is smaller than 1 mm (typically tens to hundreds of micrometers), and are compatible with continuous flow mode of operation. Microfluidic devices themselves are concomitantly of small dimensions, from some mm to micrometers [20,70,73,85]. Microfluidic reactors consist of a network of miniaturized channels, embedded in a flat surface, comonly called “chip” [20,71,72] (Figure 1). Along with this chip type of microreactors, simpler microcapillary devices are also extensively used, where the microchannel is the reaction space, and thus no control of microfluidics is required. On the other hand, they do not allow for the integration of different processes into one reaction device, unlike chip microreactors [73].

Figure 1. Scheme of a typical microfuidic device, with a microchannel and 2-ways inlet and outlet. Microreactors are commercially available from Chemtrix, CPC–Cellular Process Chemistry Systems GmbH Ehrfeld Mikrotechnik, Micronit/Future Chemistry, Microinnova, Mikroglas, Syrris, among others.

Scaling-up a bioconversion process for conventional bioreactors encompasses the use of a suitable engineering criterion, that is to be maintained constant throughout scales, such as the volumetric oxygen transfer coefficient (k_la), volumetric power consumption, mixing time or impeller tip speed, when microreactors are considered the operational conditions can be scaled by simply operating multiple systems in parallel, a process termed numbering-up or scaling-out. This feature is particularly attractive since it takes away the technical and financial risks associated with scaling a given process in traditional manner.

2.2.1. Materials

A wide array of materials has been used to build microfluidic reactors, among them polymers, glass, metals, and ceramics. Polymer-based devices typically present low cost and allow for the easy integration of flow control systems (viz. pneumatic valves, sensors), but are often sensitive to organic solvents [74]. Poly(dimethylsiloxane), or PDMS, a soft, optically transparent elastomer, is a typical choice for building microreactors [75]. This low-cost polymer is stable in aqueous environment and due to its low interfacial free energy there is a low probability of other polymers or fluids reacting with or attaching to its surface [75–77]. PDMS is however prone to swell or even dissolve in the presence of some organic solvents, and has a relatively limited temperature stability, although the later is compatible with common biological applications. Besides, its elastic properties lead to some restrictions in the design, since a high aspect ratio of the microchannels may result in pairing, where parallel structures attach to each other, whereas low aspect ratio can result in sagging, hence making the recommended aspect ratio for PDMS structures to be within 0.2 and 2 [76,78,79]. Other polymers commonly used are SU-8, an epoxy photosensitive epoxy resin that allows for adequate aspect ratio structures to be obtained [74,80], and silicone. The later polymer is actually also widely used for microreactor building. Not only fabrication methods that are well established for semi-conductor chip production are easily amenable, but also silicone has high thermal conductivity, allowing for excellent heat transfer capability. Furthermore, when oxidized, silicone becomes chemically inert towards most chemicals and solvents [71,74]. Glass is another widely used material for building microfluidic reactors, despite its rigid, brittle nature. Glass is however chemically inert to most reagents and solvents, and since it is transparent allows for visual inspection of events inside the reactor. Besides methods for fabrication, such as photolithography, are well established [70,71,78]. Stainless steel is the preferred metal for building microreactors. A well-established material, resistant to organic solvents and a wide array of chemicals, enables operation under pressure and high temperatures. Typically these reactors are larger than those made from polymers or glass, and are mostly used for pilot-plant or production scale, although they are also available for academic research purposes [70,71]. Methods for fabrication of microfluidic chip reactors include laser ablation, micro-injection molding, surface treatment and photolithography, perhaps the most popular. A detailed description of the different methodologies can be found in [70].

2.2.2. Flow in Microfluidic Devices

Continuous flow is by large the preferred mode of operation in large-scale processing in the chemical industry [81], so the use of microfluidic devices brings high-throughput, lab-scale process optimization closer to the large-scale processes [82–84]. Flow in microfluidic reactors can be single phase or multiphase, where in the later case two or more phases are separately added to the reactor, typically in co-current mode. Most common junctions for multiphase flow are T, cross, interdigitated multilamellar mixer, Ψ and Y junctions, the latter being widely used in two-phase bioconversion systems [70,85] (Figure 2).

Figure 2. Examples of junctions in microfluidic devices: (a) Y junction; (b) T junction (c) Ψ junction. Arrows suggest direction of flow.

The flow dynamics and mass transfer in a microfluidic environment, however, differs from larger systems. Given the small dimensions, the flow regime is typically laminar, interfacial forces are dominant over gravitational forces and since turbulence is not present due to the high viscous forces, mixing relies on molecular diffusion [85,86]. The laminar flow favors the control and modeling of the reaction, and provide high surface area to volume ratio (>200) and interfacial areas, a feature particularly advantageous in two-liquid extractive bioconversion systems [20,73,87]. In such environment Reynolds and Bond numbers are usually small and the Peclét number is high [88,89]. The Reynolds number, Re, represents the ratio inertial to viscous force (1):

$$\text{Re}=\frac{\text{u}\hspace{0.17em}\text{L}}{\nu }$$

(1)

where u is the linear velocity of the fluid, L is the hydraulic diameter of the microchannel and ν is the kinematic viscosity. The Bond number, Bo, expresses the ratio of gravitational to surface tension forces (2):

$$\text{Bo}=\frac{\Delta \rho \hspace{0.17em}\text{g}\hspace{0.17em}{\text{L}}^2}{\gamma }$$

(2)

where Δρ and γ are the density difference and the interfacial tension between two phases, respectively, and g is the gravitational acceleration. The Peclét number, Pe, expresses the ratio of forced convection to diffusion (3):

$$\text{Pe}=\frac{\text{u}\hspace{0.17em}\text{L}}{\text{D}}$$

(3)

where D is the diffusion coefficient. Also relevant in the characterization of multiphase flow is the Weber number, We, which provides the ratio between inertial and surface tension forces, (4), and is useful in the analysis of thin film flows and formation of droplets and bubbles. Despite of the small paths involved, We in the range of some hundreds can be obtained in microfluidic devices [70]

$$\text{We}=\frac{\rho \hspace{0.17em}\text{L}\hspace{0.17em}{\text{u}}^2}{\gamma }$$

(4)

Finally, the capillary number, Ca, (5), is also a relevant dimensionless parameter in the analysis of multiphase flow. It relates viscous (elongation) to surface tension forces acting across the interface:

$$\text{Ca}=\frac{\mu \hspace{0.17em}\text{u}}{\gamma }$$

(5)

where μ is the viscosity of the fluid. Tipically much lower than 1 [89], Ca allows to distinguish two-phase flow patterns and the mechanisms of break-up in T-junctions [90].

A common approach to improve diffusion-induced mixing is to use long and narrow microchannels (high aspect ratio channels) [86,91]. When channel dimensions are of tens of microns, the diffusion path is small enough for the mixture of different fluid streams to be complete in a few seconds, a time span that is considerably enlarged, to tens of seconds, when the channel dimensions are in the hundreds of microns [92]. Micro-mixers have hence been developed as to increase the interfacial surface and therefore decrease significantly the diffusion length [73,92]. Micro-mixers can be of passive or active nature. In the former case, mixing is promoted by diffusion or chaotic advection, whereas in the later, the flow is disturbed by the action of an external source. Passive mixing can be obtained by lamination, where the inlet streams are divided into several substreams, and then combined together in a single stream; or by chaotic advection, induced by special geometries in the mixing channel, such as zig-zag microchannel, and obstacles on the wall or in the microchannel [73,93] (Figure 3).

Figure 3. Examples of typical configurations to enhance mixing in microchannels: (a) obstacles on the wall of the microchannel; (b) obstacles in the microchannel (c) zig-zag microchannel. Arrows suggest direction of flow.

Typically fluids are delivered to the microreactor through the use of either syringe or HPLC pumps, within a broad range of flow rates, from μL min⁻¹ to L min⁻¹ [71]. The connection between microreactors and macroscopic fluid delivering device is performed using a large array of components, such as Luer-to-Luer or Luer-to-barb connectors, common HPLC fittings and high-pressure interconnect devices to interface with standard capillary tubing [94].

2.2.3. Applications of Microreactors in Biocatalysis

The use of microreactors for the analysis and development of bioconversion systems has mostly relied in the use of enzymes or at most cell lysates as biocatalysts [73]. Irrespective of the approach used, the microreactor approach has systematically been shown to outperform conventional batch-wise operation, namely when the rate of substrate processing is considered, which could be somehow anticipated, given the favorable mass and heat transfer characteristics of the former [73,95,96]. Different configurations have been used within the scope of microreactors. The simplest form is the operation in a single liquid phase, where substrate and enzyme aqueous solution are fed separately to the microreactor and the reaction takes place along the microchannel. This was used for the hydrolysis/transgalactosylation by glycosidase [97], for the bioluminescent reaction between ATP and luciferin, promoted by luciferase to ultimately yield oxyluciferin and AMP [98], and for the oxidation of 3,4-dihydroxy-l-phenylalanine (l-DOPA) with laccase [95]. In all cases the efficiency of the approach was demonstrated, which somehow paved the way for more complex approaches. One of these is a likely extension of the former, since organic-aqueous or ionic liquid-organic solvent two-liquid phase systems were used. This allowed for the conversion of sparingly water-soluble substrates or for the enzymatic resolution of chiral compounds. The experimental set-up for these occasionally featured Ψ-shaped microreactors, allowing for three independent feeding channels. Further details are given in Table 3.

Table 3. Examples of microfluidic systems for enzyme catalysis in liquid phase.

Enzymes can be advantageously used in immobilized form since this strategy allows for increased volumetric productivity and often improves stability. Continuous mode of operation is made possible, mostly using a packed-bed reactor (PBR) configuration, since this overcomes the continuous stirred tank reactor (CSTR) configuration, by allowing for higher conversion or space-time yields [2,104,105]. In order to retain the biocatalyst, particularly if in free form, membrane reactors operating in CSTR mode are commonly employed, where it is assumed that the contents of the reactor are perfectly mixed [2]. Other screening devices can be used if the biocatalyst is attached to a carrier or encapsulated. Ex-situ filtration or centrifugation, followed by recycling back into the reactor, is an alternative strategy for enabling continuous mode of operation [105]. Optional to PBR configuration, is the expanded or fluidized bed reactor, where the enzyme particles are retained by a hydrodynamic balance between gravity and drag forces promoted by the up-flow substrate stream [105,106]. The approaches commonly used for immobilization in conventional multiphase biocatalysis can also be used in microreactors, with particular focus on covalent methods, cross linked enzyme aggregates (CLEA) and adsorption (specific and non-specific) methods [107]. The experimental set-ups are preferably either chip-type reactors with activated channel walls where the enzyme binds, or enzyme immobilized monolith reactors, where a support is packed inside a capillary tube (Table 4).

Table 4. Examples of microfluidic systems with immobilized enzymes.

2.2.4. Visualization and Quantification of Fluid Flow

Gaining the in-depth in fluid mechanics, mixing and heat transfer required for the engineering characterization of miniature and microreactors has required both hardware and software tools, able to collect the required data, process them and generate models representative of the system dynamics. Contribution to the visualization of fluid flow has been made possible through the use of adequate high speed cameras, often combined with (micro) particle image velocimetry, (μ)PIV, and software for image analysis [17,121–127]. Generation of detailed predictive models, representative of the dynamics of flow, which can incorporate reaction data, has been made possible with computational fluid dynamics (CFD). CFD allows therefore for the design and analysis of a given system, as well as for the simulation of fluid flow, (bio)chemical reaction, mass and heat transfer [19,62,87,128,129]. The models developed within the scope of CFD methods are typically anchored in the Navier–Stokes equations and in the convection-diffusion equation [19,130].

3. Conclusions

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