Miniaturization in Biotechnology: Speeding up the

Recent Patents on Biotechnology 2011, 5, 000-000

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Miniaturization in Biotechnology: Speeding up the Development of Bioprocesses Pedro Fernandes1,2,*, Filipe Carvalho1,2* and Marco P.C. Marques1,2 1

Department of Bioengineering, Instituto Superior Técnico, Universidade Técnica de Lisboa, Portugal; 2IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

Received: June 27, 2011

Revised: July 25, 2011

Accepted: August 06, 2011

Abstract: 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.

Keywords: Automation, biocatalysis, downstream processing, fermentation, high throughput, microfluidic devices, microreactors, microsensors, microstructured reactors, microtiter plates, miniature bioreactors, miniaturization in biotechnology, monitoring and control of process variables, optodes, process intensification, shaken vessels. INTRODUCTION Process engineers are facing the need to improve or devise new bioprocesses in order to cope with the increasingly demanding challenges, namely involving lower costs in process development, reduced time from discovery to market, and use of renewable feedstocks [1,2]. These requirements have become particularly noticeable when the production of pharmaceuticals is addressed, given the increasingly relevant role played by biotechnology in this field. The requirements still stand for all other areas where biotechnology is an asset in the production of other goods or of energy [1,3,4]. High-throughput methods, with a wide level of parallelization and reduced volumes, are an obvious approach to comply with the need for fast and cost-effective process development. High throughput methods occasionally enable to move directly from laboratory to production scale, without the need for complex and eventually not fully successful scale-up, by simply numbering up small scale devices (scaling-out) [5,6]. These efforts strongly anchor on the use of miniaturized devices, that can be shaken vessels, either Erlenmeyer type or microtiter plates; miniature or microreactors; and on establishing methodologies relying on the use of such devices that are reproducible and scalable. The present work aims to update a previous review focused on highthroughput methods in biotechnology [7], by addressing developments made since within the scope of fermentation and biocatalysis, as well as matters that had been considered previously, such as the relevance of miniaturized devices in downstream processing. SHAKEN DEVICES – GENERAL Shaken vessels are possibly the most widely disseminated platforms for the early stages of bioprocess develop*Address correspondence to this author at the Department of Bioengineering, Instituto Superior Técnico, Universidade Técnica de Lisboa, Portugal; Tel: +351 21 8419065; Fax: +351 21 8419062; E-mail: [email protected]; 1872-2083/11 $100.00+.00

ment, an approach that has been long-established [5,7]. This can be ascribed to the simplicity of the vessels that were traditionally used, but also to the high parallelization that these devices allow, more so with the volume reduction of the vessels, viz. from 500 mL Erlenmeyer type flasks to as low as roughly 300 μL, the full volume of an individual well among standard 96-well microtiter plate [6,7]. The increased parallelization capacity came however with a cost, since with the minute filling volumes used, precise handling of fluids is a critical issue and evaporation can become a serious limitative issue [7,8]. Sampling, and concomitant analytical methodologies, is also a key aspect to be considered given the low volumes involved, which may require a “sacrificial well” approach or end point measurements, in order to prevent significant changes of the operational conditions, but resulting in low data density [6,8]. Minimization of evaporation can be achieved through the use of suitable sealing membranes, which preferably minimize the outflow of water vapor while having no relevant impact in gaseous exchanges. These microtiter plate covers are typically composed of polymeric materials, viz. copolymer of ethylene and vinyl acetate, polyethylene, silicone or rayon [9-12]. They are also fundamental in preventing crosscontamination, while assuring sterile conditions and preferably allow access to the sample wells without the need for removal of the sealing caps employed [7,12]. Among such covers are multilayered sealing plates composed of: a stainless steel lid, a microfiber filter, an expanded polytetrafluoroethylene filter, laminated between two polyester/ polyamide fabrics, a pinholed stainless steel foil (specifically for 96-lowwell microtiter plates) and a silicone layer [13]. Even with such sandwich covers, evaporation is observed, with rates that vary with operational conditions. some typical examples at 30ºC and 50% air humidity being of 50 Lwater day-1, per well, for 24-square deepwell plates, down to 6 Lwater day-1, for 96-round low-well plates [13]. Ireland had previously developed a flexible moulded closure composed of either a copolymer of ethylene and vinyl acetate or of low © 2011 Bentham Science Publishers

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either a copolymer of ethylene and vinyl acetate or of low density polyethylene, which include several downwardly depending cups adapted to be a force fit in the openings of microtiter plates. Each cup has a slot in the base that can be pierced by a metal probe [12]. Tyndorf and Cannataro developed a sealing tape using a thermoplastic elastomer membrane, viz. isoprene propylene or rubber, which is attached to the microtiter plate through an adhesive material. The membranes are disc-shaped and have a concave surface that face away from the well interior, so that they can be resealed as soon as the probe used for sampling is withdrawn from the well interior [14]. Treptow observed that in these approaches, a hermetic closure is not achieved, and suggested an alternative configuration. This consisted of a cover sheet composed of polyethylene or polystyrol, combined with an adhesive material on the lower side for attachment to the microtiter plate. The shape memory polymer that composes the cover sheet has so-called weakening zones, which are formed by at least one slit and a protuberance towards the downside into the well. Liquid can be taken out or filled up from the wells, using a suitable probe which is forced through the weakening zones. After withdrawal of the probe, the weakening zones close partly automatically, but once energy is introduced, viz. through heat or laser light, the initial shape is fully regained [15]. Handling lipophylic and volatile organics has been shown to be feasible in microtiter plates [16-18]. A particular limitation observed was the concentration decrease of organics due to sorption of such molecules into the glue of adhesive foils and into the polystyrene walls of the microtiter plates, a drawback that could be minimized by the use of plexiglass instead of polystyrene [18]. A reduction on evaporation from microtiter plates can also be achieved by placing the experimental setup under a hood, which is continuously flushed with humidified air [19,20]. Gas-tight boxes, that alongside from allowing operation at 100% humidity, fully preventing evaporative losses, also allow anaerobic cultivations and operation in the presence of CO2, required for mammalian cells [13]. Optimization of the design of the wells of microtiter plates has also been performed. Optimization aimed to allow high oxygen transfer rates, often a critical issue in fermentation processes, while minimizing the trend for drop formation or spilling of the liquid, furthermore contributing to reduce the risk of the bottom of the well running dry during rotation. This was obtained by introducing protrusions or indentations in the well wall, the most favorable resulting in a 6-petal flower shaped well, Fig. (1), commercially available from m2p Labs under the label Flowerplate® [21,22]. These microtiter plates allow for a maximum oxygen transfer rate (OTRmax) of 100 mM h-1 which doubles the best values reported so far in standartd microtiter plates. Besides, it matches the values reported in many standard batch fermentations, albeit in stirred tank reactors, OTRmax up to 500 mM h-1 can be reached [23]. Furthermore, the bottom is made of optically transparent materials, thus enabling measurements from the bottom, and provides several alternatives to fix the sealing cover on top of the microtiter plate. Microtiter plates with this design are at the center of the BioLector microreactor [22].

Fernandes et al.

Fig. (1). Examples of well formats in microtiter plates: from left to right, round-well bottom, square-well bottom and 6-petal shaped format (Flowerplate®).

Typically, microtiter plates with 4 to 96 wells are used in the development of bioprocesses, since these formats provide a suitable compromise between throughput and conservation of established operational conditions (viz. filling volume) and monitoring and control capabilities [7,10]. Recently, a 104-well format was introduced, the novel configuration corresponding to an extra column that allows for additional standards or controls without the need of using extra plates. The addition of the extra column required only a 4.5 mm shift to the left of the existing columns and negligible differences in mean absorbance readings (around 3x10-4 in optical density). The feasibility of using the novel plate in a 96 well environment was demonstrated by challenging the 104 well plate in an enzyme-linked immunosorbent assay on a standard liquid handler. No differences between formats (96 and 104 well) were observed [24]. Designs with further miniaturization of microtiter plates, comprising 1000 to 4000 wells, together with a lid to prevent evaporation, and allowing the use of confocal optics were presented. The novel plates are claimed to be useful in drug screening and to be able to carry biological molecules that bind to given substrates [25]. MONITORIZATION, CONTROL, OPERATION AND AUTOMATION OF SHAKEN DEVICES Significant developments have been made in microtiter and Erlenmeyer based platforms, leading to significant improvements from the original relatively crude devices. One of the most significant was the introduction of patch sensors for measurement of pH, dissolved oxygen tension, CO2 and optical density, with readings made with optical fiber sources. These patches, often referred to as sensor spots, are currently commercially available, and are bonded in Erlenmeyer flasks or in the base of each well in microtiter plates [5, 7, 26]. The on-line monitoring capability provided by these spots result form the combination of a suitable fluorescent dye, viz. a fluorescein amine, a ruthenium complex, or 8-hydroxy-pyrene-1,3,6-trisulfonate trisodium salt, and a bioconjugate, and suitable selection of materials can allow monitoring of further compounds, such as mono- and polysaccharides, alcohols, organic acids or steroids, as well as temperature [27-29]. These devices have been used for monitoring several fermentation processes, namely involving Bacillus cereus, Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae [19,20, 27,30,31] and mammalian cell culture, using Chinese ovary hamster (CHO) cells [32]. The latter was implemented using a 24-well microtiter plate platform commercially available from Pall Corporation, labeled Micro-24 [33]. Further examples are given in a recent review [5].

Miniaturized Tools and Methodologies to Fasten the Development of Bioprocesses

In order to increase the efficiency of densitometers used for measuring the optical density of fluids in microtiter plate wells, an apparatus was developed that allows for multiple reading both at single or different light frequencies. The apparatus comprehends two structures, one of them including several detectors, while the other incorporates several light emitting diodes, distributed in arrays that match each other, with similar number of columns and rows. The spacing between adjacent detectors and diodes is similar to spacing between the wells of a microtiter plate, so that detection can be performed without movement of the microtiter plate [34]. An approach for the quantification of biomass in microtiter plate wells using image analysis has also been suggested, which was shown to correlate nicely with off-line optical density measurements [35]. Recently, an apparatus was presented whereby a microtiter plate held in an orbital shaker is connected to a flatbed scanner, which can generate two-dimensional images of the bottoms of the wells present in the microtiter plate. The images are taken during an interrupt period of shaking, triggered by a dedicated processor, which can also adapt the shaking mode as a result of the analysis of the data scanned [36]. Care has to be taken to prevent any negative effects on oxygen supply due to stopping and hence on the performance of cells [37]. The dedicated image analysis software enables quantification of cell density, and generates growth curves. Up to twelve 96-well microtiter plates can be scanned, so the apparatus is commercially available from Enzyscreen as Growth Profiler 1152 [13, 36]. A limitation of traditional microtiter plate platforms was the lack of process control mechanisms. This clearly hampered pH control or fed-batch capability [7]. Two major approaches have been developed to counter this limitation. One relies in the use of feeding devices. Within such strategy, the microtiter plate can be integrated into a liquid handling workstation, which enables liquid dispensing for induction of microbial cell cultures, viz. IPTG (isopropyl--D-thiogalactopyranoside) stock solutions [31, 37]. Actually, dedicated work has been focused on the development of robotic hardware and corresponding control software, in order to allow suitable automatic displacement of microtiter plates [38,39]. Also within this scope, microfluidic devices can be integrated with microtiter plates, to deliver small volumes of fluid [5,7, 40-42]. Buchenauer and co-workers integrated a microfluidic device in a 48 well microtiter plate. The microfluidic device, made of polydimethylsiloxane (PDMS), allowed the delivery of acid or base and hence enabled pH control. The device is composed of three layers, one that contains the fluidic channels (100 m wide and 70 m deep); a second layer is composed of PDMS membrane 50 m thick that seals the fluidic channels; and a third, pneumatic, layer that controls the microvalves of the device, by guiding pressurized air to the microvalves. The pressurized air is controlled by external valves that are linked to compressed air connections placed on top of the microtiter plate. The apparatus was effectively tested for the pH-controlled fermentation of E. coli cultures [40]. Once established the feasibility of this concept, the apparatus was improved by incorporating a micropump in the microfluidic chip placed in the bottom of the 48-well microtiter plate. The set-up allows for pH-controlled fermentation, through adition of either

Recent Patents on Biotechnology 2011, Vol. 5, No. 3

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sodium hydroxyde or phosphoric acid solutions, or fed-batch fermentation, through feeding of a concentrated (500 gL-1 ) glucose solution, dosed at 2 L h-1, and using a measurement system for dissolved oxygen tension, pH and scattered light, slightly modified from the BioLector apparatus [19]. Puntambekar and co-workers integrated a microfluidic device with a 96-well microtiter plate for immunoassay processing [41]. Integration of microfluidic devices in microtiter plates seems thus a promising approach for feeding liquids in a controlled manner in the minute amounts required when operating in microtiter plates, and microtiter plates with integrated microfluidic structures are commercially available [43]. Van Leeuwen and co-workers used a Hagen-Poiseuille pump, or alternatively a syringe pump, for the delivery of liquid with flow rates up to 150 nL min-1, and integrated the micropump with a 96-well microtiter plate. The apparatus was effectively used for the fed-batch anaerobic cultivation of S. cerevisiae in the microtiter plate wells, where glucose solution was fed at different flow rates. CO 2 yield on glucose was determined and found to compare well with that determined on a 4 L stirred fermenter [44]. Another approach to provide controlled feeding of nutrients into shaken vessels, hence allowing for fed-batch cultures, relies on the use of materials that allow the controlled release of the desired molecule. This involves a two-phase system, a liquid phase corresponding to the cultivation medium, and a solid (or gel) phase, which is the course for controlled delivery of the desired nutrient, or of a precursor, that can be converted by an enzyme into the required substrate or pH controlling agent [45, 46, 47]. Early developments on this approach led to the development of a PDMS matrix from which glucose was released into the fermentation media. Fed-batch runs were performed in Erlenmeyer flask using as model system the production of green fluorescent protein by Hansenula polymorpha cells. Results clearly outmatched those obtained in shake flask operated in batch mode but were of the same magnitude as those from laboratory scale fermentors operated in fed-batch mode [45]. These PDMS/ glucose conjugates are commercially available under the label of FeedBeads®, and have also been effectively tested for the production of Yellow Fluorescent Protein (YFP) by E. coli mutants [48]. Disc-shaped silicone polymers were also developed to embed Na2CO3, which can be released in a controlled manner into cultivation media in order to control pH. Sodium carbonate is released form the polymer according to pre-determined kinetics, in such a manner that it compensated for the decrease in pH due to metabolic activity of E. coli cells grown with either glucose or glycerol as carbon source. In the former case, the use of this technology also allowed for a reduction of 50% in buffer concentration, whereas in the latter, no buffer was needed [49]. Further developments led to the use of an agar-based gel, containing starch, which diffused into the fermentation media to be hydrolysed by glucoamylase, thus providing the required glucose. This approach was effectively tested in shake flask and 96-well microtiter plates, using as model system the production of heterologous triosphosphate isomerase by E.coli cells [50]. This technology is currently commercially available under the label EnBase® [51], and also proved quite effective for fastening the development of a bioprocess aiming at the production of a RNase inhibitor by recombinant E. coli cells.


Data gathered in 96-, 24- well microtiter plate and shake flasks led to the selection of a system that was scaled up to a 10 L stirred bioreactor [52]. Krause and co-workers showed that EnBase® formulation can outmatch other currently used media, viz. Luria-Bertani, Terrific broth or mineral salt media, when a high yield of soluble recombinant proteins in shaken cultures (24-deepwell microtiter plates and 500 mL Erlenmeyer flasks) is aimed at, not only because high cell densities are obtained, but also because the formulation assures a controlled physiological state throughout the whole process leading to correctly folded proteins [53]. Continuous operation in cylinder-shaped type flasks has also been shown to be possible, by using the circulating motion of the bioreactor on a shaker, so that the fermentation broth reaches an outlet port and leaves the glass flask, whereas substrate is pumped in a controlled manner into the reactor [7, 54]. Still, the range of operational continuous conditions is rather limited and there is no pH and dissolved oxygen tension monitoring. SCALING-UP FROM MICROTITER PLATES The feasibility of scaling-up fermentation/bioconversion processes from microtiter plate has been established in several model systems [5, 7]. Recent examples include E. coli fermentations, under fed-batch operation using either linear or exponential feeding of a concentrated glucose solution. Scaling-up from microtiter plate to a fully controlled 2 L laboratory-scale inder constant volumetric mass transfer coefficient (kLa) 460 h-1 resulted in similar growth patterns of the E. coli cultures on both scales. This is suggestive of the reliability of this approach for process development and its efficient mimicking of large scale processes [20]. While addressing the microbial side chain cleavage of sitosterol to 4androstene-2,17-dione (AD), Marques and co-workers were also able to obtain similar AD production and dissolved oxygen profiles when scaling-up from 24-well microtiter plate to a 5 L bench-scale reactor under constant kLa [55]. The production of granulocyte macrophage colonystimulating factor (rhGM-CSF), through an open cell-free synthesis, anchored in cell extracts of an engineered K-12derived E. coli strain KGK10, was also shown to be linearly scalable over a change in fermentation volume from 250 μL (24 well microtiter plates) to 100 L (pilot scale bioreactor). Throughout different scales, the time course of rhGM-CSF formation was consistently observed, and concomitantly, a yield of 700 mg L-1 after 10 h of incubation was observed [56]. MINIATURE BIOREACTORS Miniature bioreactors are scaled-down versions of typical bench-scale bioreactors, which usually have volumes in excess of 1 L [5, 7]. Miniature bioreactors, which have volumes under 1 L, clearly mimic large scale bioreactors, including monitoring and feeding features, but has potential for automation and allows for parallelization, hence higher throughput, although this is below the throughput provided microtiter plates [8, 57, 58]. The application of miniature bioreactors for bioprocess development has been reviewed recently [5,7,8 59, 60]. A clearly distinctive feature of miniature bioreactors from microtiter plates is the approach used for promoting mixing, shaking in the later whereas stirring

Fernandes et al.

(or eventually liquid or gas impelled flow is used) [7]. A patent has however been disclosed where the rotor used to promote rotary or back and forth shaking motion can connected through a suitable drive to stirring rods placed in individual wells, so that fluids in the wells can be thus mixed by stirring [61]. Recently, a miniature (80 mL working volume), sixbladed turbine stirred miniature bioreactor was used in order to validate the hypothesis of transferring the production of anthrax vaccine from static culture to stirred tank operation, using as model system the production of anthrax vaccine by cultures of Bacillus anthracis Sterne 34F2. The approach proved successful and, when compared to the standard static cultures, the required culture time to achieve the maximum concentration of the two main components of the vaccine is halved. Furthermore, higher concentrations of said components were obtained in the stirred culture. However, nonaerated conditions were required, since aeration led to the degradation of one of the main components, the protective antigen [62]. Kloke and co-workers developed a miniature bioreactor with a volume of some milliliters (viz. 8 mL) that is put together by stacking individual polycarbonate elements and silicone gaskets in an alternate manner. The volume of the central reaction chamber can therefore be changed by the number of elements assembled. The authors claim that the design of the reactor is easily adaptable in size and functionality to experimental needs. Monitoring of dissolved oxygen tension and pH is performed through the use of either sensor spots or of optical sensors, and oxygen transfer can be controlled through the use of different flow rates and different nozzle configurations, ultimately allowing for a kLa of around 16 h-1, a relatively low performance, but for a flow rate of 15 mL min-1. The authors suggest that kLa can be enhanced by increasing the flow rate and improving nozzle design. Cultivation of E. coli with monitoring of pH and dissolved oxygen tension was performed. Furthermore, by connecting two reaction chambers as to generate a microbial fuel cell, the authors assessed the influence of outer membrane cytochromes on the performance of Shewanella oneidensis [63]. One of the relevant developments in miniature bioreactor technology relies in the apparatus developed by WeusterBotz and co-workers. Based in the use of magnetically driven gas-inducing impellers, allowing for high oxygen transfer capability, as demonstrated by the kLa values in excess of 1400 h-1, the apparatus enables parallel batch and fedbatch cultivations (up to 48 miniature reactors, with 10 mL of individual volume), with the possibility of monitoring pH, dissolved oxygen tension and stirring speed online. Furthermore, individual pH control of the bioreactors is possible, using sterile soda or sulfuric acid, fed through a liquid handling device. Proof of concept was provided using as model systems batch cultivations of E. coli and S. cerevisiae [7, 64]. This set-up was also used for evaluating the performance of different Bacillus subtilis strains that are producers of riboflavin. Using riboflavin yield as reference parameter, the authors established a correlation between the minimum number of parallel reactors and the difference in riboflavin yields after 48 h of incubation, which contributed to establish


criteria for statistically significant discrimination between the strains tested. Successful evaluation of the process performance under fed-batch operation at milliliter scale established the basis for further process optimization [65]. Since the impeller used in the original set-up was not adequate for the cultivation of mycelium forming microorganisms, a onesided paddle impeller was developed, to suit such role, otherwise using the same set-up. High volumetric oxygen transfer coefficients were obtained, as results from k La values in excess of 500 h-1. Using as scale-up parameter a constant mean power input of roughly 3 W L-1, the batch cultivation of Streptomyces tendae cells was reproduced when moving from 10 mL to 2 L scale, given the similarity of key parameters monitored, such as biomass concentration, mannitol consumption and production of the fungicide nikkomycin Z. The authors also reported a high reproducibility in 10 mL scale, with up to 48 bioreactors in operation, based on the low (under 8%) standard deviation [66]. It is expected that this set-up will become commercially available, alongside with set-ups referred to in Table 1. Apart from these, other groups assembled miniature stirred bioreactors in the 6 to 100 mL scale with monitoring capabilities (viz pH, dissolved oxygen tension) [67-71]. It was shown that scale-up to pilot scale could be performed, based on kLa similarity [69], or on matched power per unit volume, Pg/V [68, 71], although Gill and co-workers only found the later valid for values in excess of 1 kW m-3 [71]. It was also pointed out that the experimental condition used did not include the large biomass concentrations, typically over 200 gwet cell weight L-1 required in industrial processes [5]. Doig and co-workers also developed miniature (2 and 100 mL) bubble column reactors, which were used for E. coli fermentations. A kLa slightly in excess of 300 h-1 was reported and the effect of Pg/V on kLa was also correlated [72]. Aiming to gain further insight on the key aspects required to establish a reliable scale-up of bioprocesses from milliliter-scale to liter-scale reactors, Hortsch and Weuster-Botz determined the effect of operating conditions on the mean power consumption and maximum local energy dissipation, using a miniature (12 mL) bioreactor and a standard 1.2 L stirred tank bioreactor with Rushton turbines. These authors were able to establish that the maximum local energy dissipation in the miniature bioreactor was lower than that on the larger reactor at the same mean power input per unit mass. This result indicates that power is more uniformly distributed in the liquid medium in the case of the miniature bioreactor. Hence impeller speeds can be adjusted in order to achieve similar local energy dissipation at different scales [73]. MICROREACTORS In this section reactors with volumes under 1 mL, and other than microtiter plates, will be addressed. These reactors can provide the controllability and flexibility of large, standard reactors, with the advantage of the small size, potential for on line data and for automation and for operation in continuous mode. Furthermore the small dimensions clearly favor heat and mass transfer, although some care has to be taken, particularly when two-phase systems are involved and (micro)mixing is required [6]. The flow dynamics, heat and mass transfer in these environments, in the sub-micometer scale, differs from larger systems, most remarkably the flow


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regimes are typically laminar and interfacial forces are dominant over gravitational forces [6]. Microreactors also led to a paradigm shift in the development of bioprocesses, by allowing an easier transition from research to production stages, by simply numbering up of the reactors, preventing the need for time-consuming and expensive scale-up [6, 8, 84-87]. Different configurations of microreactors can be considered, such as [85]: a) submilliliter stirred reactors, which can integrate microfluidic devices (viz. micropumps, microvalves and mixers) if composed of PDMS [8, 84, 88, 89]; b) microchannels, typically with hydraulic diameters under 1 mm, often under 100 μm, and large surface area to volume ratios, within 1.0 x104 m2 m-3 and 5.0 x104 m2 m-3 [90,91]. Van Leeuwen and co-workers developed micro bioreactors of 100 μL working volume, based on the 96-well microtiter plate format, where each reactor was equipped with an electrochemical sensor array for the online measurement of the dissolved oxygen tension (using ultra-microelectrode arrays, through amperometric detection of oxygen reduction), pH (using a Ion Sensitive Field Effect Transistor, ISFET), temperature (platinum thermistor) and viable biomass concentration (impedance sensor). Electrochemical sensors have been shown a suitable option for optodes, particularly when relatively extreme pH values (viz. pH below 6) can occur, and where ISFET is superior to optodes [92-94]. The authors also developed a technology enabling the measurement of cumulative CO2 produced in micro bioreactor fermentations [95]. Using aerobic batch cultivations of Candida utilis, van Leeuwen and co-workers were able to show that data obtained both from the sensor array and from CO2 production measurements were quite reproducible, and roughly matched measurements performed during cultivations performed in a standard bench scale bioreactor, with a working volume of 4 L [94]. No definite criteria for scale-up was used, and furthermore the authors highlighted that, due to differences in kLa and aeration gas used, pure oxygen for the microreactor, rather than air in the bench scale reactor, oxygen profiles were only matched qualitatively. Microchannell(s) can be embedded in a flat surface made of polymer (viz PDMS, polymethylmethacrylate (PMMA), polycarbonate, Teflon), glass, ceramics or stainless steel [9698]. Simpler (micro)capillary devices are also used, where the microchannel is the reaction space. On the other hand, they do not allow for the integration of different processes into one reaction device, unlike chip microreactors [96]. An example of the potential provided by chip microreactors is a microfluidic chip developed by Jambovane and co-workers [99]. The microreactor integrates a microfluidic system with sample metering, mixing, and incubation functionalities, that allows for simultaneous parallel processing, so that eleven different conditions can be tested at the same time, by creating a gradient of reagent concentrations in eleven parallel processors. In order to validate the feasibility of their design, the authors determined the kinetic parameters of the hydrolysis of resorufin--D-galactopyranoside with -galactosidase, and also evaluated the effect of competitive inhibitors, phenylethyl -D-thiogalactoside and lactose. Other example is the microreactor developed by Jury and Angelino, where the platform includes mixing chambers, heating and dispersion units, reaction and separation units. The modular nature of


Table 1.

Fernandes et al.

Examples of Commercially Available Small Scale Bioreactors (Adapted From [5])

Bioreactor

Comments

Other References

MiniBio (Applikon)

250 mL up to 1 L volume. Microbial and cell culture. (Fed) batch and continuous operation. DOT, pH, temperature, agitation speed and foam level control

[74]

Dasgip AG

4 to 8 units, 60 to 200 mL. Microbial and mammalian cells, kLa up to 400 h-1, pH, agitation speed and DOT control, feed regime capability

[75]

Biostat® Qplus (Sartorius)

Up to 12 units, 0.5 to 1 L, Growth of microbial, mammalian, insect and plant cells. k La up to 400 h-1, pH, DOT and agitation speed control, feed regime capability

[76]

Medicel Cultivation Unit (Medicel Oy)

Up to 15 units, 100 to 200 mL or 200 to 500 mL. Various suspension culture, kLa up to 190 h-1, pH, DOT, temperature and agitation speed control, feed regime capability

[77]

CloneScreener® (Biospectra)

32 units, 400 mL, microbial and mammalian cultures, pH and DOT control

[78, 79]

Biopod f800 (Fogale biotech)

8 units, 80 mL, mixing by air sparging. Suspension cultures, pH, DOT, turbidity and temperature monitoring, temperature control

[80]

Cellsation ™ (Fluorometrix)

12 units, up to 35 mL, fermentation and cell culture, pH, DOT, turbidity and temperature monitoring, temperature and agitation control

[81]

AMBR™ (TAP Biosystems)

24 units, 10 mL, cell culture only, due to kLa lower than 30 h-1. Control of pH, DOT and CO2, liquid handling capability. Easily installed into laminar flow hood

[82]

Sixfors® (Infors HT)

Up to 6 units, 300–500 mL, with batch, fed-batch, and continuous operation capability. Control of pH, DOT, antifoam/level

[83]

DOT: dissolved oxygen tension

each device enables parallel or series operation, and oxygen, pH, pressure and temperature sensors can be embedded in the network. Separate feed streams empty into the mixing chamber under pressure, that allow for nutrient and gaseous feeding (including gaseous supply for pH control) and other feeding requirements (viz. antifoaming agents or solvents) [100]. Provided they have a defined internal structure, microchannel reactors are preferably termed microstructured reactors [85], as happens in: a) chip-type reactors [101, 102], where channel walls may be coated with enzymes or cells, using different methods [103-108]. A somehow alternate form of this approach relies on the adhesion of poly(ethylene glycol)-based hydrogel, enzyme containing microstructures, inside the microchannels by photolithography [109]; b) in monolith packed microreactors, where monolith is loaded with enzyme [110, 111]; c) fiber based microreactors [112]; folded-sheet mesoporous silicas [113]. Apart from these configurations, randomly packed bed microreactors have also been developed, as those using lipase immobilized onto acrylic resins (Novozym®435) for oxidation of 1-methylcyclohexene [114] or onto macroporous polymethyl methacrylate beads for the ring-opening polymerization of caprolactone to polycaprolactone [115]; alcohol dehydrogenase immobilized onto Sepabeads for asymmetric hydrogenation of ketones in a liquid-liquid segment flow microreactor [116]; or cross-linked lactamase, packed with controlled pore glass for the conversion of benzamide to benzoic acid [117]. Immobilization onto the walls of the bioreactor avoids the risk of back pressure, but there is a penalty on the load of biocatalyst inside the reactor. To overcome the latter, packing the reactor with the immobilized biocatalyst is the logical approach. The use of beads may however lead to pro-

hibitive back pressure in packed bed operation, a drawback that can be overcome by the use of monolith structures. The introduction of disk-shaped packed bed reactors has been suggested as another design alternative, since it displayed enhanced catalytic behavior, at a 6.5 times lower pressure drop, when compared to an equivalent packed bed reactor [118]. Since mixing by diffusion, the only natural mechanism available in these environments, is far from efficient, improving mixing is required, which can be performed either by increasing: a) the interfacial area between the two fluid phases, through the design of adequately structured microchannels. This can be achieved by lamination, where inlet streams are split into different substreams, and later combined; by introducing obstacles in the microchannel, by using a non-linear shaped channel, such as in zig-zag fomat or in the form of an hourglass, having a first outer end with a tangential inflow inlet and a tangential flow outlet, so that the mixing chamber narrows more or less slowly and then widens more or less rapidly, in the overall flow direction [119]. Such approach, that involves the generation of a contraction/expansion region, through a section which has a diameter smaller than the adjacent upstream and downstream channels was also used by Karp [120]; b) or by increasing the energy input, introduced into the flow from an external source, which can encompass ultrasounds, piezoelectric actuators, magnetic stirring using microsized mixers or through the generation of a polarized electric potential by applying an AC signal with high frequency, thus creating an electrical field [6, 100, 121-125]. Further detail on these matters can be found in recent reviews [125-128]. Micro-bioreactors and high throughput systems are commercially available from different sources, such as Seahorse Bioscience (SimCell Sys-


tem) [129, 130], Future Chemistry [131], Micronit Microfluidics [132,133], Chemtrix [134,135], Microinnova [136], Dolomite [137] and Syrris [138-140]. SimCell sytem has been recently used to perform multi-factorial experimentation for relating link between mammalian cell culture and glycoprotein productivity [141]. Microfluidic platforms, with integrated functionalities for fluid handling and on-line pH optical monitoring capabilities have been developed for application in three-dimensional cell culture [142, 143]. The microfluidic platform was shown to allow for the maintenance of homogenous and stable culture environments, along with the supply of multiple media and efficient cell/agarose (scaffold) loading functions. The platform allowed for a marked reduction in the need for human intervention, and enabled high throughput drug testing [144]. Recently a perfusion micro cell culture device was developed, aiming to evaluate tensile loading on cell physiology. The platform was able to generate the tensile strain range covering the physiological conditions that articular chondrocytes experience under human walking conditions [145]. SMALL SCALE REACTORS: SCALE-DOWN Large scale processes are performed under operational conditions that are prone to lead to broth heterogenities. The use of microreactors has been suggested within the scope of a scale-down approach, which consists on simulating in small-scale environment the environmental conditions experienced in large-scale vessels, and assess the physiological effects of the fluctuation [146]. The validity of this concept has been illustrated recently. Thus, the metabolic responses of E. coli cells to glucose gradients were monitored by de Mey and co-workers in short time periods of roughly 40 s [147]. The authors were furthermore able to establish that there was a good agreement between the dynamic behaviour of cells after glucose pulses in both microreactor and chemostat environment. Delvigne and co-workers studied the effect of glucose pulses in the synthesis of green fluorescent proteins (GFP) by recombinant E. coli cells [148]. These authors were able to conclude that under glucose intermittent feeding, GFP synthesis slowed down, leading to a binary repartition of GFP content among the microbial population. Lara and co-workers have recently used this approach to evaluate the effect of oxygen-poor and glucose-rich regions that can occur in large-scale bioreactors in cell response, using as model system E.coli cells [149]. The authors were able to establish that, after exposure to such gradients, production of metabolites, viz. formate, acetate, lactate, ethanol, and succinate, was observed. On the other hand, TaymazNikerel and co-workers showed that, under glucose-limited chemostat growth, and when exposed to a glucose pulse, the growth rate of E. coli cells cells increased roughly 4-fold and a new pseudo steay state could be reached in less than 2 minutes after fluctuation [150]. These examples are representative of the ability of the scale-down approach to cope with the dynamic phenomena that take place in large-scale bioreactors and thus contribute for the design of biological systems with improved behaviour under large-scale environment.


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MINIATURIZATION IN DOWNSTREAM PROCESSING The trend to miniaturization in downstream processing (dsp) is strongly related to scaling-down biochemical engineering procedures, designed for the recovery and purification of proteins, in order to reduce the risks of delays during bioprocess development and product launch [83, 151,152]. Research has thus focused on the development of methodologies enabling the prediction of the performance of largescale processes using laboratory-scale equipment, namely in the milliliter scale, within the scope of the so-called ultrascale down (USD) approach. Since the operations involved in dsp are potentially complex and given the interactions between operations, an easily interpretable representation of bioprocess analysis is desired. This can be achieved through a graphical user interface, which allows a straightforward display of the windows of operation tolerated [151, 153]. The USD methodology was used by Tustian and co-authors to predict the centrifugal clarification efficiency when high cell density fermentation broths (wet solids content within roughly 12 to 16% w/v) are addressed, where hindered settling and aggregation effects are noticeable. The suggested methodology relied on the dilution of the feed material to about 2% wet w/v prior to the clarification procedures, which was performed in a 2 mL scale. The dilution minimized cell-cell interactions effects, as well as the hindered settling in the feed suspensions. The resulting clarification curves had therefore to be corrected in order to properly mimic the original feed. The corrected USD curves were shown to accurately predict pilot-scale clarification performance of high cell density broths of E. coli and S. cerevisiae cells, which was performed in a disc stack centrifuge, with a Sigma factor of 1602 m2. These results validated the suggested methodology, whereas previous USD approaches typically led to a 5- to 10-fold over-prediction of clarification performance [154]. Within the scope of the USD approach, a 20mL rotating disc shear device was used to feed a 96-deep square well microtiter plate with mammalian cell cultures (CHO cells) with different levels of shearing, in order to establish the influence of cell growth and harvesting on following dsp, and subsequently developing a methodology for automated analysis of said impact. Also, the authors claim that their methodology can be applied to any centrifuge with known geometric features, using computational fluid dynamics modeling. In their work, the authors observed an increase in solids carry over with culture age, under no or marginal shear stress, whereas for high shear stress and poor settling nature, this pattern was inverted. With the validation of the suggested methodology, and given its high level of parallelization, the authors delivered a significant tool for high throughput process development [155]. Microfiltration characteristics of fermentation broths has been also assessed using miniaturized platforms, more specifically 96-well filter plates (0.3 cm2 per well) and custom designed 8–24-well filter plate (0.8 cm2 per well). Experimental filtration runs of an E. coli TOP10 fermentation broth runs were performed in parallel using an automated vacuum filtration manifold, and compared with data from a standard laboratory scale membrane cell (3.8 cm2). Using a 0.22 m Durapore polyvinylidene fluoride (PVDF) membrane in all formats, the authors were able to establish that both the measured mem-


brane resistance and the specific cake resistances were independent of the configuration of the filtration device. However, the custom designed microfiltration unit decreased the errors associated with the determination of specific cake resistance values, when compared with the multi-well unit, while enabling the simultaneous evaluation of membranes of different nature [156]. Miniaturization in chromatographic methods has been reviewed recently, encompassing systems where resin volumes are roughly under 500 μL [157]. Accordingly, Chhatre and Titchener-Hooker suggest differentiation of the devices used under three different formats, namely: microliter batch incubation, micropipette chromatography tips, and miniature packed columns [157]. The favored configuration for microliter batch incubation relies on the use of 96-well microtiter filter plates, where a given volume of resin is loaded into each well. Feed is added to each well, and once elapsed the intended loading time, the supernatant is recovered, either by centrifugation or under vacuum, using a manifold assembly, and collected in an underneath plate. Given the high level of parallelization, a wide array of operational conditions may be assessed simultaneously (viz. buffer nature and concentration, nature of the solid matrix). The methodology is easily integrated with robotic systems and prone to automation, thus allowing for high throughput [157-160]. This methodology was used to screen resins aiming to fasten the development of processes targeted for protein purification, using as models a monoclonal antibody and a Fc-fusion protein. Using 50 to 100 μL, dispensed in a 96-well plate format, and an incubation period of 20 minutes, the authors developed a high throughput method that compared favorably with a methodology relying on the use of hydrophobic interaction and anion-exchange lab-scale and production-scale chromatographic columns [161]. Optimization of either ionexchange or affinity chromatographic steps, using as model system a monoclonal antibody, was also performed, using this approach [162, 163]. A 96-well microtiter plate approach, fitted with monolithic ion-exchange resins was also used to define efficient separation conditions for the mobile phase. The gradient elution curves obtained were compared with those from a monolithic disk column and the peak salt concentration data for the microtiter plate system was shown to closely match those for the monolithic disk column, thus validating the scaled-down approach, which allows for amassing a large array of data with reduced amounts of reagents [164]. 96-well microtiter plates fitted with resins are available from several suppliers, such as Sigma-Aldrich (Discovery® SPE 96-Well Plates), GE Healthcare (PreDictor™ 96-well filter plates, MultiTrap™ 96-well filter plates), 3M Bioanalytical Technologies (Empore™ 96 Well C18 SPE Disk Plate), Millipore (96-well Multi-SPE Extraction Plates), Crawford Scientific (96 well SPE plates) or Phenomenex. Chromatography packing can also be inserted in pipette tips, thus forming microcolumns [157]. This methodology is also amenable to automation, and it was used by Wenger and co-authors for the recovery of virus like particles from lysates of yeast cells, using tips packed with either cation exchange resins or with ceramic hydroxypatite. Yeast lysates were obtained after adaptive focused acoustics treatment. The authors suggested that the combination of these two

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operations provides a promising methodology for the speedy process development of intracellular proteins [165]. The use of a tip packed with Protein G affinity matrix was nuclear in the development of a feasible and marketable methodology aimed at the quantification of antibody titers in bioprocess feedstocks [166]. A microscale approach based in the use of robotically operated pipette tips packed with 20 μL of Capto MMC was used to evaluate the effect of pH and loading buffer salt concentration on the recovery of polyclonal antibodies from ovine. The chosen operational conditions were tested in 10 mL packed bed column, and similar capacity, purity and yield were observed [167]. Pipette tips with chromatographic packings are commercially available from Milipore (ZipTip® Pipette Tips), Thermo Fisher Scientific Inc. (Thermo Scientific Aspire Protein A, Protein G and IMAC Cobalt Chromatography Tips Aspire™) or PhyNexus (PhyTip® columns). A third configuration used within the scope of microscale chromatography is the packed miniature column. This format is clearly reminiscent of standard columns, only smaller, and so is the way they are operated, but still miniature columns are amenable to automation [157]. Kee and co-workers used 1 mL Hi-trap Butyl-SepharoseVR columns for the capture of hepatitis B surface antigen (HBsAg) through hydrophobic interaction, and hence assess the effect of upstream processing on the yield in the chromatographic step. The upstream operations consisted of the centrifugation of the S. cerevisiae cell homogenate, followed by treatment with detergent under pressure (about 4x107 Pa). The authors established that the procedure suggested led to a cleaner stream fed to the chromatographic column, and concomitantly to an increase in the yield of the chromatography step [168]. Wierling and coauthors evaluated the feasibility of coupling high throughput screening of packed bed chromatography with mass spectrometric detection by SELDI-TOF MS. The model system used matched monoclonal antibodies to host cell protein from an industrial cell culture system. Chromatographic screening was performed in a robotic system, using miniature columns (200 L), packed with different resins, namely CM Hyper D, CM Sepharose FF, Toyopearl SP 650 and Fractogel SO3. The authors observed a lower sensitivity for protein A chromatography, when compared to SELDI-TOF MS analysis. Furthermore, a shift in the elution pattern was observed for one of the monoclonal antibodies irrespectively of the resin used, a feature that was ascribed to the glycosylation pattern of the antibody [169]. Brochier and co-workers screened different mixed-mode chromatography resins, more specifically (HEA, MEP and PPA HyperCelTM), using miniature columns of 2.5, 5 and 10 mL. Pre-packed MediaScout® MiniChrom columns, with volumes within 2.5 to 10mL were evaluated as testbeds for screening three mixedmode chromatography resins. The authors evaluated column performance based on dynamic binding capacity, separation of a protein mixture and partial purification of lactoglobulin from milk whey, and were able to establish that the resins outmatched typical hydrophobic interaction chromatography resins, and furthermore, that separations were scalable [170]. Miniature chromatography columns are available from several suppliers, such as Atoll GmbH [171], Spectrum Chromatography, Regis Technologies, Amsbio or Boca Scientific, whereas packings are available from manu-


facturers such as Applied Biosystems, BioRad, GE Healthcare, Millipore, Tosoh Biosciences, VWR or Whatman [157]. Further miniaturization of chromatographic columns has been achieved [172, 173]. One of these, with 1.5 L volume and 1 cm height, was developed by Shapiro and coworkers, in order to contribute for further enhancing the optimization of separation conditions for biopharmaceuticals. In order to establish the proof of concept, the column was tested packed with compressible, polydispersed porous agarose beads (70 m mean diameter), aimed at the binding of fluorescently labeled protein during isocratic loadin. Coupled to microscopic techniques, visualization of adsorption profiles within the bed was possible. Breakthrough curves were obtained for mobile phase velocities within 1.7x10-4 and 7.5x10-4 ms-1. The behavior of the microfluidic column proved to be suitable for performing studies at scales 20,000fold lower than those in current practice [174]. The microfluidic column was later packed with sieved Q Sepharose Fast Flow and used for establishing the quality of packing and for the generation of breakthrough and elution curves. Furthermore the performance (HETP), dynamic binding capacities and gradient elution separations of the microfluidic column were matched with those of laboratory scale columns. Tests were performed lysozyme, conalbumin and ovalbumin, all fluorescently labeled with fluorescein-5-ithocyanate. The authors reported a marked reproducibility of data throughout the different scales for both the maximum dynamic binding capacity and separation of binding and non-binding proteins. A significant decrease in protein requirements was also reported for the breakthrough curve and for elution (about 10,000- and 3,000-fold, respectively), when the microfluidic column was compared with a 30 mL laboratory column [175]. Two-liquid phase extraction has also been implemented at microfluidic scale, using either organic-aqueous or aqueous-aqueous two phase systems. The former has been used for the recovery of hydrophobic compounds, such as steroids [176], eventually integrating bioconversion and product recovery in a single step [177], typically using microfluidic devices with “Y” type inlet and outlet, thus allowing the feeding and separation of the two immiscible phases. Twoaqueous phase have on the other hand, been used for the recovery of large biomolecules, viz. proteins, and whole cells. The two-aqueous phase is composed of two immiscible polymers, viz. polyethylene glycol (PEG) and dextran, or of polymer and salt phases. Microfluidic devices with “Y” and “” shaped inlets/outlets are often used Fig. (2). The later configuration allows the separate feeding of the two-aqueous phases and a third stream with the material to be processed, whereas in the outlet enables the separate recovery of the interphase, in addition to the two remaining exit streams for the two immiscible aqueous phases. These subjects have been recently reviewed [178, 179]. CURRENT & FUTURE DEVELOPMENTS In recent years, significant progresses have taken place, when miniaturization in biotechnology is considered. With relation to a previous review [7], several aspects are worth highlighting. There is clearly a marked dissemination of the applications of small scale bioreactors – Erlenmeyer and microtiter plates, miniature reactors and microreactors - in


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different stages of bioprocess development, when fermentation and bioconversion systems are considered. Such trend reflects the increased recognition and acceptance of such miniaturized platforms, which offer unparallel highthroughput capability and easy automation, namely by handling through robotic systems. Still, and despite the advances in instrumentation, namely through the use of optodes and microsensors, and feedback control, for monitoring key variables namely pH and dissolved oxygen tension, Erlenmeyer type flasks and microtiter plates lack the full capabilities of conventional bioreactors (bench scale and higher). Shaken vessels, and particularly microtiter plates, are physically limited when the introduction of sensors for simultaneously monitoring further process variables (viz. a given substrate, or a product such as a green fluorescent protein) is envisaged. Most of the drawbacks related to operation of shaken devices relate to limitations in instrumentation needed for providing individual feeding capabilities. Thus pH, fed-batch or continuous operation is strongly limited, as it is also dissolved oxygen tension, despite some developments with the introduction of materials allowing for controlled delivery of given compounds or the use of micropumps for the same goal. Furthermore, the ability of existing platforms to cope with high cell densities is still questionable. These are therefore matters that will require further efforts. Nonetheless, the simple shaken vessels, if operated under well established condition, provide a reliable platform for the early stages of process development, although particular care as to be given to the control of evaporation, if microtiter plates are used in prolonged incubation runs. Also, when addressing microbial fermentations, oxygen transfer limitations may have to be taken into consideration, albeit novel designs for the wells of microtiter plates tend to overcome such drawback. Most of the constraints of the shaken vessels are overcome with the use of miniature bioreactors, namely the feeding capability, which enables, among others, operation in high cell density environment. Furthermore the working volume is enough for detailed characterization of product profile, a feature that may not be amenable in microtiter scale when the production of some metabolites (viz. siderophores) is considered. It is therefore understandable how several of such platforms are available commercially, albeit at some cost, with flexibility of operation burdened by upstream operations and cleaning procedures, which need further improvements. Tools to comply with those could include disposable items or “plug and play” devices. Also the burden of copying with large arrays of independently monitoring and control units for pH and dissolved oxygen tension (if none other) in multiple vessels is a complex task that requires additional efforts. Further miniaturization led to the introduction of microreactors, which, in adequate configuration, may enable integration of upstream and downstream process. Suitable sensors for monitoring and control have been developed, which include microfluidic pumps for delivery of nutrients or similar. In the particular case of microstructuured reactors, several application in biocatalysis have been implemented, highlighting the potential of such platforms for process development. These devices also bring along the possibility of scaling out rather than scaling-up, hence simplifying process development. Improvements in the design of microchannels or the use of other suitable devices have allowed an increased in mixing efficiency, which may prove critical when operation on


multi-phase environment is considered. Still more efforts are needed in order to further simplify operation with microfluidic devices, namely when the connection between macro and micro environment is considered; also the need for devising low-cost methods for the machining of chip devices required looking into. Efforts to introduce microfluidic chip analysis, alternatively to more conventional methods, such as chromatography, may contribute to significantly improve the role of small scale devices in the development of bioprocesses. Also, miniaturization has made its way into downstream processing, which has allowed to adequately handle small volumes and to adequately predict the behavior in large scale operation, with significant cuts in cost and reagents, although this approach can be expanded to a larger scale of applications. However, the range and the insight of unit operations addressed within the scope of miniaturization in a rational manner, is still relatively limited, thus more work is required. It is also considered that there is still further work required in order to operate a full bioprocess – upstream, transformation/fermentation and downstream - in miniaturized scale, including sample analysis, in such a manner that the performance in large, production scale can be adequately predicted with significant reduction in time and costs in process development, without hampering productivity or product quality; or otherwise, if adequate, further relying on simply numbering up micro bioreactors to move into production scale.

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DISCLOSURE The present work is an extended/updated version of a previous manuscript, High Throughput in Biotechnology: From Shake-Flasks to Fully Instrumented Microfermentors, published in Recent Patents in Biotechnology 2009; 3: 12440. The present manuscript thus focuses on developments that occurred since, as well as on matters related to the use of miniaturized tools and methodologies for the development of bioprocesses, that were not addressed previously. REFERENCES [1] [2] [3]

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Fig. (2). Examples of inlet and outlet configurations of microchip devices: from left to right, one inlet and one outlet, two inlets and two outlets (Y shaped), and three inlets and three outlets ( shaped). Drawings are not to scale.

CONFLICT OF INTEREST The authors have no conflict of interest to declare.

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ACKNOWLEDGEMENTS The authors would like to thank Fundação para a Ciência e a Tecnologia, Portugal, for financial support through contracts under the program Ciência 2007 awarded to P. Fernandes, for the post-doctoral grant SFRH/BPD/64160/2009 awarded to M.P.C. Marques and for the doctoral grant SFRH/BD/74818/2010 awarded to F. Carvalho.

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