Continuous aqueous two-phase systems devices for the recovery of biological products

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Food and Bioproducts Processing

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Review

Continuous aqueous two-phase systems devices for therecovery of biological products

Edith Espitia-Saloma1, Patricia Vázquez-Villegas1, Oscar Aguilar,Marco Rito-Palomares ∗

Centro de Biotecnología-FEMSA, Departamento de Biotecnología e Ingeniería de Alimentos, Tecnológico de Monterrey, Campus Monterrey,Avenue Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico

a b s t r a c t

Aqueous two-phase systems (ATPS) have proved to be a suitable technique for the recovery of biological products.

Although ATPS have been in the field of primary recovery and purification of products for several years, the majority of

the studies exploiting ATPS are usually based on batch mode operation. Reports on the potential of using continuous

ATPS are not common. This review attempts to present a practical analysis of selected devices employed for ATPS

continuous processing, from the conventional column contactors to novel designed mixer-settler units. A critical

analysis of operational and design parameters that impact the system performance is presented. Current trends on

the implementation of continuous ATPS approaches are discussed, together with the major challenges faced for the

generic adoption of the technique. Conclusions are drawn on the major contribution of previous studies in the field

to provide a better understanding of the technique for the newcomers.

© 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Aqueous two-phase system; Continuous extraction; Column contactors; Mixer-settler units

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012. Selected devices for continuous ATPS extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

2.1. Column contactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012.1.1. Spray column. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1032.1.2. Perforated rotating disk contactor (PRDC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1032.1.3. Pulsed cap columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1042.1.4. Other columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

2.2. Mixer-settler unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Abbreviations: AMC, axial mixing coefficient; Bot, bottom; BSA, bovine serum albumin; CS, citrate salts; CTG, cashew nut tree gum;Cy-b5, cytochrome b5; FE, fractionation efficiency; HD, hold up; H/D, height/diameter ratio; HPI, heavy phase inlet; HPO, heavy phaseoutlet; HRP, horseradish peroxidase; ID, internal diameter; KDa, mass transfer coefficient; Kp, partition coefficient; LLE, liquid–liquidextraction; LPI, light phase inlet; LPO, light phase outlet; LYZ, lysozyme; MW, molecular weight; NA, not available; PEG, polyethyleneglycol; PF, purification factor; PMMA, polymethylmethacrylate; PRDCs, perforated rotating disk contactors; PTFE, polytetrafluoroethylene;PVC, polyvinyl chloride; PS, phosphate salts; RE, recovery efficiency; SE, separation efficiency; SS, sulfate salts; TLL, tie line length; WPI,whey protein isolate.

∗ Corresponding author. Tel.: +52 81 83285060; fax: +52 81 83284136.E-mail address: [email protected] (M. Rito-Palomares).Received 10 September 2012; Received in revised form 3 May 2013; Accepted 28 May 2013

1 Both authors contributed equally to this work.0960-3085/$ – see front matter © 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.fbp.2013.05.006

Author's personal copy102 food and bioproducts processing 9 2 ( 2 0 1 4 ) 101–112

2.3. Other contactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1062.3.1. Raining bucket contactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

3. ATPS continuous operation potential problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1063.1. Flooding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1063.2. Backmixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1063.3. Emulsification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1083.4. Separation efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

4. Trends and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1085. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

1. Introduction

It is known that chromatography is usually preferred in mostpurification processes. However, some process disadvantages,such as low binding capacity and resin costs (Rosa et al.,2011), raise the need for alternative techniques to comple-ment the performance of chromatography-based downstreamstrategies. Among these alternatives, aqueous two-phase sys-tems (ATPS), a liquid–liquid extraction (LLE) strategy, is nowrecognized as a potential technique because of its multipleadvantages including: biologic compatibility, low interfacialtension, high load capacity and scale up easiness (Raghavaraoet al., 2003).

Since its discovery (Beijerinck, 1896) ATPS have beenexploited for the recovery of a wide variety of biomolecules– monoclonal antibodies (Azevedo et al., 2009; Rosa et al.,2010), proteins and enzymes (Liu et al., 2011; Platis and Labrou,2006; Xu et al., 2003)), antibiotics (Bora et al., 2005; Khederlouet al., 2009; Mokhtarani et al., 2008), dyes (Benavides andRito-Palomares, 2006; Mageste et al., 2009), low molecularweight compounds (Benavides et al., 2008; Willauer et al.,2002), DNA (Luechau et al., 2010; Mashayekhi et al., 2008),hormones (Haraguchi et al., 2004; Persson et al., 2005), cells(Edahiro et al., 2005; Yamada et al., 2004)) and membraneparticles (Cao et al., 2006; Everberg et al., 2006), from dif-ferent sources such as plant (Ibarra-Herrera et al., 2011;Aguilar and Rito-Palomares, 2010), animal (Boland, 2002),and insect (Benavides et al., 2006) cells as well as fermen-tation broths. Great part of this research has focused onfinding the ATPS physicochemical characteristics (composi-tion, viscosity, density, interfacial tension and pH) that betterfits the intrinsic properties of the molecule of interest andmatrix source (hydrophobicity, molecular weight (MW), iso-electric point) in order to obtain higher yields. Studies onphase separation (Cabezas, 1996) and partitioning phenom-ena (Huddleston et al., 1991), basic heuristic rules (Benavidesand Rito-Palomares, 2008) for ATPS with other recuperationtechniques (Aguilar et al., 2006) and pilot scale experiments(Kepka et al., 2003) have been performed. Most of them havebeen conducted in a batch mode and noteworthy knowledgehas been achieved. However, the suitable characteristics ofATPS as a technology for continuous process have been usuallyoverlooked.

In the biotechnology market, ATPS as a continuous orsemi-continuous operation would have clear competitiveadvantages: diminishing process time and costs and increas-ing process yields (Igarashi et al., 2004a). Many opportunityareas of research within continuous ATPS operation are nowpresent, such as bioaffinity partitioning (Ruiz-Ruiz et al., 2012),

fractionation of pegylated proteins (Mayolo-Deloisa et al.,2010), the use of alternative ATPS (alcohol-salt, micellar, andionic liquid based systems) to recover different biomolecules,the operational models for process optimization, phase recir-culation, large scale operation, etc. In order to achieve these, itis important to validate the equipment that outstands for itsversatility and thus can potentially be employed for all thesepurposes. There are few examples in literature of the use ofcontinuous ATPS performing a validation of different devicesfor the recovery of a model protein starting form a previouslyselected batch ATPS.

This review describes the main devices employed for ATPScontinuous processing, comparing their characteristics fromthe conventional column contactors to the novel designedmixer-settler units. A critical analysis of defined operationaland design parameters that have a significant impact onthe performance of equipment for continuous ATPS is pre-sented. Current trends and challenges are discussed in orderto present the areas of opportunity for the newcomers in thefield.

2. Selected devices for continuous ATPSextraction

For practical purposes, devices employed for continuous ATPSprocesses have been classified into three main groups: columncontactors, mixer-settler units and other contactors. Amongthe devices used to date, common column contactors are themost studied, probably due to their successful application inthe chemical industry.

2.1. Column contactors

In the following sections the most common designs in thisset of column contactors employed for continuous ATPS, arediscussed: spray columns, perforated rotating disk contac-tors (PRDCs), pulsed cap columns and other columns (packed,sieve plate and vanes agitated columns). All of them consistof a hollow pillar with two inlets for each one of the phasesinvolved and its corresponding outlets. The examples here dis-cussed (Table 1) share common building materials: glass andpolymethylmethacrylate (PMMA). The main variable amongthese columns is the mechanism by which the mass transferbetween the phases is promoted (pulsed caps, rotating discs,rotating vanes, spray mechanism, static packing, and staticmixer).

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Table 1 – Characteristics of selected column contactors and their performance parameters.

Device TrSSa, b (min) ATPS/product ofinterest

Disperse/loadedphase

Operational variables Ranges Performanceparameters

Ref.

PRDC

600PEG 8000-PS

Top/BotPhases flow rates 1–3 ml/min KDa:

0.003 min−1

Cavalcantiet al.(2008)�-Toxin Rotational speed of

discs35–140 rpm HD: 0.80

PF: 2.4

PRDC

55PEG 4000-CTG

Top/TopDisperse phase flowrate

2–5 ml/min SE: 96% Sarubboet al.(2005)BSA ATPS composition 2

No. of discs 3–4 discs

PRDC

NAPEG 550-PS

NA/TopDisperse phase flowrate

1–3 ml/min KDa:0.043–0.073 min−1 Figuereido et al. (2004)

Ascorbic oxidoreductase SE: 75–95%PF: 2–35

PRDCNA

PEG 550–1000/PSNA/Top

Disperse phase flowrate

1–3 ml/min HD: 0.01–0.45 Portoet al.(2000)BSA KDa:

0.03–0.125 min−1

PRDC60

PEG20000-CSTop/Bot

PEG MW 3350–20,000 kDa Kp: 3.3 Portoet al.(2010)

Ascorbate oxidase ATPS composition 3 PF: 1.46

Pulsed caps column70

PEG 6000-PSTop/Bot

Flow rates 2.8 and2.6 ml/min

PF: 33 BimandTeixeira(2000)

Xylanase Pulse frequencies 1/1 and 1/5(bottom/top)

Yield: 98%

Pulsed caps column30

PEG1000/1500-PSTop/Top

Flow rates 4.7–10.9 ml/min FE: 81% ± 8% RabeloandTambourgi(2003)

Cy-b5/ascorbicoxidoreductase

Pulse frequency 0.1–1.0 pulses/s

*Spray column

180PEG 6000-PS

Top/TopDispersed phaseflow rate

0.2–0.6 ml/min KDa:24 × 108 min−1

Srinivaset al.(2002)HRP Orifice diameter 0.50–1.33 mm

NaCl concentration 0.0–5.0% (w/w)

*Spray column packed withstatic mixer

180

PEG 4000-SS

Top/Top

Disperse phasesuperficial velocity

0.2–1.2 mm/s KDa: 396 min−1 RostamiandAlamshahi(2002)

�-Amylase ATPS composition 3 HD: 20Column diameter 47–65 mmStatic-mixer type 3

Packed column

40PEG 4000-PS

Top/BotPacking type Glass/polyestirene-

beads/ringsKDa: 0.1 min-1 Igarashi

et al.(2004a)Xylanase Void fraction 0.44–0.85 RE: 94%

Author's personal copy104 food and bioproducts processing 9 2 ( 2 0 1 4 ) 101–112

Tabl

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1

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Dev

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AT

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40PE

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2.1.1. Spray columnSpray columns consist of an empty shell filled by a liquid untila desired height, while a disperse liquid is distributed with theaid of an orifice or nozzle located at the bottom of the column(Fig. 1). The heavier phase is commonly the continuous liq-uid, while the lighter phase is the disperse flow distributed asspray.

Because of its operational advantages (construction sim-plicity, easy cleaning, and low operational and maintenancecosts) it seems to be one of the most popular choices byseveral researchers. Some characteristics of the column are:tendency to low values of height/diameter ratios (H/D), com-monly from 5 to 30 (Srinivas et al., 2002; Venancio and Teixeira,1995; Pawar et al., 1993), and distribution orifices (1 mm diam-eter) varying from 4 to 167 (Igarashi et al., 2004a; Venancioand Teixeira, 1995). The high degree of continuous back mix-ing observed in this equipment makes necessary the presenceof overflow-outlets located at different column heights. Addi-tionally, the time needed to reach a steady state (180 min) isone of the largest when compared with most continuous con-tactors (Rostami and Alamshahi, 2002; Srinivas et al., 2002).

Besides the column geometry, the operational parametersstudied in these devices include physicochemical character-istics governed by phase composition and flow rates. ATPScomposition and its related physical properties (viscosity, den-sity and interfacial tension) may limit the extraction and actas a resistance to mass transfer (Arsalani et al., 2005; Cuhnaand Aires-Barros, 2002; Igarashi et al., 2004a; Srinivas et al.,2002; Venancio and Teixeira, 1995; Pawar et al., 1993; Pawaret al., 1997).

Phases flow rate is another critical operational parame-ter in any contactor performance, since it plays a key role inmass transfer and in the column process related to operationalissues as back mixing and flooding. In spray columns, as wellas in most of continuous ATPS processes, an increase in thedispersed phase flow produces minor drop sizes that causehigher areas for mass transfer (Figuereido et al., 2004). How-ever flow rates cannot be increased unlimitedly, since they canhamper the separation efficiency.

Finally, the cost–benefit of the construction easiness of thisdevice should be considered before the selection for continu-ous ATPS operation. Although, high mass transfer coefficientscan be reached with spray columns, it lacks auxiliary elementsto contain back mixing, which is one of the biggest limita-tions of this equipment (Venancio and Teixeira, 1995). Theusefulness of this contactor can lie in being the first practi-cal approach for continuous ATPS, especially for systems withlow interfacial tension and biomolecules with high diffusivitycoefficients.

2.1.2. Perforated rotating disk contactor (PRDC)The PRDC consists of a cylindrical vessel containing perforateddisks mounted on a central rotating shaft (Cuhna and Aires-Barros, 2002) (Fig. 1). Among the advantages of these columnsare: the high throughput and efficiency, operational flexibil-ity and easiness of continuous mode of operation (Cavalcantiet al., 2008; Figuereido et al., 2004; Porto et al., 2010).

The main variables reported for these devices include:flow rates of disperse and continuous phase (1–5 ml/min)(Cavalcanti et al., 2008; Sarubbo et al., 2005), number of discs(3–6 with 6–20 holes each one) (Porto et al., 2000; Sarubbo et al.,2005), rotating discs speed (35–140 rpm) and ATPS composi-tion. The time to reach steady state ranges from 55 to 600 min(Cavalcanti et al., 2008; Porto et al., 2010).

Author's personal copyfood and bioproducts processing 9 2 ( 2 0 1 4 ) 101–112 105

Fig. 1 – Main column contactors comparison used for continuous ATPS.

PRDC have a better operational performance than spraycolumns, reducing back mixing and improving mass trans-fer coefficients (Figuereido et al., 2004). They have been usedto evaluate polymer-salt systems (PEG-PS), to recover modelproteins, such as BSA (bovine serum albumin); (Porto et al.,2000; Sarubbo et al., 2005), extracellular proteins, as cutinases(Cuhna et al., 2003); and proteins from complex mixtures,such as �-toxin from fermented broth of Clostridium perfringens(Cavalcanti et al., 2008) and ascorbate oxidase from Cucurbitamaxima (Porto et al., 2010). Processed feedstock loads rangefrom 0.06 to 2.0 mg/ml (Cavalcanti et al., 2008; Sarubbo et al.,2005).

PRDC seems to have a more robust design compared withthe rest of the column contactors. In contrast with otherequipment, PRDC performance is independent from the dis-persed phase velocity (Figuereido et al., 2004), which leadsthe mass transfer process optimization to other operationalparameters, such as number of discs and rotating discs speed.Separation efficiency seems to be inhibited by resistance ofthe flow toward the rotating discs at higher disperse flow rates(Figuereido et al., 2004). So, the number of rotating discs is a keydesign parameter, since this element promotes an adequatephases’ contact, but also interferes with the achievement ofan optimal separation of phases.

PRDC offers a more complex structure than spray columns,but at the same time improves mass transfer and decreasesback mixing. Flooding, as in the rest of the contactors,has shown to be a concern (see Section 3). Shearing phe-nomena and multistage redispersion makes of PRDC aninteresting option when having biomolecules with low diffu-sivity.

2.1.3. Pulsed cap columnsPulsed cap columns have a shaft in the center to which capsare welded (Fig. 1). These caps should be able to move upand down in order to agitate the inlet flow. The parametersstudied are flow rate (0.6–10.9 ml/min) and pulse frequency(0.1–2 pulses/s) (Bim and Teixeira, 2000; Rabelo and Tambourgi,2003). The time to reach steady (30–70 min) is usually lowerthan the one needed in PRDC probably due to a lowerturbulence generation (Bim and Teixeira, 2000; Rabelo andTambourgi, 2003).

The employment of this device has not shown an apprecia-ble advantage over batch operation. Bim and coworkers used aPEG-PS system to extract xylanase produced by Bacillus pumilusfrom the crude fermentation broth. The formation of a sta-ble emulsion was reported, avoiding further improvementsof mass transfer when compared to batch system (Bim andTeixeira, 2000).

Rabelo and collaborators (Rabelo and Tambourgi, 2003)extracted Cyt-b5 from Escherichia coli and ascorbic oxidoreduc-tase from Curcubita maxima using a PEG-PS ATPS. They showedthat high pulse frequencies and overall low flow rates werethe best conditions to generate an appropriate mass trans-fer. In contrast with the previous devices, in which a higherflow rate was needed, particularly for spray columns, in pulsedcap columns the agitation mechanism seems enough to pro-mote the desired mixing rate. However having low flow ratesas a condition for an optimal performance limits the devicethroughput, when compared with other column contactors.

Some conclusions can be drawn from the studies about thiscolumn. A remarkable gap that should be addressed is thestudy of the operational parameters, such as mass transfer,

Author's personal copy106 food and bioproducts processing 9 2 ( 2 0 1 4 ) 101–112

Fig. 2 – The main options for constituting the three stages of mixer-settlers units are shown; a mixer tank (a) or a staticmixer (b) for the mixing stage, a basic tank (c), a extended tubular settler (d) or a decanter (e) for the settling stage andfinally a decanter (f), a centrifuge (g) or a novel tubular separator (h) for the separating stage.

back mixing and hold-up rather than those most commonlyreported (yield and purification factor) as final process perfor-mance variables (Table 1). The pulsed-cap columns may resultcommercially unattractive if these gaps and emulsificationissues are not addressed.

2.1.4. Other columnsContinuous ATPS have also been operated in packed sieveplate and vanes agitated columns. The common operationalparameters studied are: vane agitation, flow rates, dispersedphase velocity, number of plates, void fraction, vane freearea and packing type. These devices share common phys-ical characteristics with an H/D ratio around 8, a commonvalue among several kinds of columns. Dispersed phasevelocity ranges (0.03–0.18 ml/s) are similar, as well as KDa(0.066–0.1 min−1) and recovery (70–94%) performance param-eters (Table 1).

Packed columns show certain advantages: they contributewith greater phase contact time, less space requirement,reduced back mixing and are easier to stabilize. On the otherhand the sieve plate column also reduces back mixing andprovides repeated coalescence and drops redispersion. Addi-tionally, mass transfer is enhanced due to the high disk holenumber (167) in comparison with the PRDC (6–20) (Igarashiet al., 2004a, 2004b). In the case of the vane-agitated col-umn, the geometry promotes a rapid coalescence mechanism,which increases with the rise of the vane free area and with adecrease of the vane rotation speed (Biazus et al., 2007).

These three devices (packed, sieve plate and vane agi-tated column) have been mainly employed with polymer-saltsystems, and seem to have an improved mass transfer, simul-taneously overcoming one of the major concerns of columnsperformance: back mixing. The time needed to reach steadystate is another noteworthy advantage of these devices. It isusually shorter (in average approx. 40 min) than the previ-ous devices. It should be noted that in exception of the vaneagitated column, sieve plate and packed column have staticmixing mechanisms that contribute to achieve these lowerstabilization times. What requires further evaluation is thecapacity to reach the same mass transfer coefficient valuesas dynamic mechanisms.

Diverse column contactors (Kühni, Karr-reciprocating,Scheibel and Oldshue-Rushton extractors) can be enumeratedunder this category. Although different static and dynamicmixing mechanisms can be observed, rapid coalescence is themost evident characteristic, which is not achieved in the pre-vious designs. These devices can be attractive when workingwith ATPS with low difference in phase density, and whenredispersion is needed to avoid emulsification.

2.2. Mixer-settler unit

Mixer-settler units were one of the first devices employed forcontinuous ATPS (Veide et al., 1984). The two different modesof operation (static and dynamic) basically consist of a mix-ing stage in tanks or columns, coupled to a series of settlingand separating units (Fig. 2). Ban and coworkers (Ban et al.,2001) employed a device consisting of an agitated tank andtwo inline columns (1 settler and 1 coalescer) using lipaseand lysozyme as model proteins with PEG 8000-PS ATPS. Allthe system was contained in a constant temperature bath.The total mixture coming from the mixer tank (agitated at150 rpm) is pumped into the coalescer, then to the settler andfinally again into the mixer, obtaining a close circuit whichneeds 180 min to reach steady state and process 1–2 g pro-tein/L. Steady state is reached when the partition coefficientobtained in the continuous mode is similar to the one in thebatch mode. A modification of the system with an impellerinduces larger throughputs by high suction pressures. Theconstruction and packing materials of the coalescer are impor-tant factors considered in this study, since the wettability ofthe surfaces, and chemical composition are related to prod-uct accumulation due to unspecific adsorption. The authorsstudied PTFE (polytetrafluoroethylene), ceramic and glass, toenhance phase separation, being PTFE the best coalescing pro-moter.

The use of centrifuges as a separator mechanism can beperceived as a disadvantage in comparison with column con-tactors in which phases separate easily and quickly by gravity(Porto et al., 2010). Therefore the introduction of an efficientnon-electric separator feasibility to be scaled up would beappreciated. In an attempt to improve continuous ATPS per-formance Rosa et al. (2012) have already assembled a fusionbetween a mixer-settler device and a packed column in a pilotscale, with optimal results of 85% yield for the recovery ofantibodies with PEG 3350-PS. In this context the use of a tubu-lar horizontal separator in line with an interphase harvestingport has been recently documented (Vázquez-Villegas et al.,2011). The continuous concurrent operated prototype devicehas been proved with dyes (Blue and Gentian violet), modelprotein (BSA) and whey protein isolate (WPI), with processingloads from 0.01 to 1.0 mg/ml. The use of flow rates of 50 ml/minallowed a mean time of 15 min to reach a steady state. Pro-tein samples can be individually injected and then mixed withboth phases into the static mixer. Separated outlet streamscan be used to collect phases. The tubular separator providesadditional time to increase interfacial contact between thephases, with minimal protein accumulation at the systeminterphase. The recoveries reported for the dyes, BSA and WPIwere up to 90%.

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Mathematical models and computational simulationsusing an impeller agitated reactor as the mixing stage andsettlers with different geometry inlets have been documented(Mistry et al., 1996; Salamanca et al., 1998). Although the sim-ulations provide data over separation kinetics as an aspectof continuous processing, the real application will usuallypresent implementation problems, especially with ATPS dif-ferent from PEG-PS. Typical process fluctuations, due toliquid infusing mechanism, hinder the equipment to oper-ate under steady state conditions. Process design has beenbased on empirical or pilot plant experiences (Saadat and AliMousavain, 2009).

In conclusion, mixer-settler units have an inherentassembling easiness compared with column contactors andsimultaneously they are suitable for individual stage (mix,coalescence, and separation) screening and surface mate-rial testing. Nevertheless there is a noteworthy gap in thehydrodynamics characterization, needed for a practical designplatform implementation.

2.3. Other contactors

2.3.1. Raining bucket contactorThe raining bucket or Graesser contactor consists of a hor-izontal cylinder containing an axial central shaft with anassembled rotor comprising fixed semicircular buckets (Cuhnaand Aires-Barros, 2002). Graesser contactors in union withmixer-settlers units and spray columns were one of the firstdevices to be used for ATPS continuous process. Interfacialrenewal and good mass transfer are achieved by the gentlemixing in the contactor, which promotes a natural break ofthe drops and helps to avoid the formation of stable emulsions(Coleby, 1983; Jarudilokkul et al., 2000). Consequently, the useof this equipment is specially recommended for ATPS withtendency to form emulsifications, such as reverse-micellarand aqueous-organic systems (Jarudilokkul et al., 2000; Dos-Reis et al., 1994).

As observed in Table 2, its mechanical characteristics allowa relevant operation flexibility employing high flow rates(20–80 ml/min) and a variety of phases flow ratios (0.025 upto 4) (Giraldo-Zuniga et al., 2006). It commonly needs 120 minto reach a steady state. Its separation efficiency (HD = 0.05756)is low, in comparison with vertical columns and it is availablein larger dimensions than the other common contactors (ID of100 mm, length of 1010 mm, 37 disks and 6 buckets per disk)(QVF Glastechnik, Germany).

These devices together with mixer-settler units have beenemployed for larger scale continuous ATPS studies. It has beenused for the purification of �-lactalbumin and �-lactoglobulinfrom whey, processing more than 600 g of whey proteins perday (Coleby, 1983). Lysozyme has been extracted from eggwhite with a reverse micellar extraction with a yield of 97–99%(Jarudilokkul et al., 2000). In summary, although it presentssome mechanical improvements for efficient phases mixingand separation, its mechanical complexity can be a charac-teristic that limits its adoption. Additionally, the absence ofa unique dispersed or continuous phase increases the timeneeded to achieve steady state.

3. ATPS continuous operation potentialproblems

As observed, in any continuous ATPS device the main control-lable variables are flow rates and mixing rates (rotor speed,

Fig. 3 – Key operational parameters relation with potentiallimiting phenomena within continuous ATPS.

number of discs, pulse frequency, and static mixer type).Drop size will be a consequence of both variables and thephysicochemical nature of the selected ATPS. A balance onthese parameters will have an impact on the mass transfercoefficients and consequently in the process efficiency. Havingdiscussed ATPS continuous devices, main operational prob-lems can be identified. Flooding, backmixing, emulsificationand poor phase separation degree will limit the selection ofoptimum operational parameters. The cause–effect relationamong these parameters should be carefully contemplated inorder to facilitate an appropriate device design (Fig. 3). In thissection, general guidelines are given indicating the key fac-tors that should be specially observed in order to have a moreefficient ATPS continuous extraction.

3.1. Flooding

In common column contactors due to a high continuous phasevelocity, the disperse phase droplets fall (or rise) more slowlyforming a dense region, which eventually coalesce. If smallerdroplets are produced by drastic agitation or with counter-current operation, and the droplets arrival rate exceeds thecoalescence rate at the main interphase, flooding related prob-lems may arise.

When using columns, high mass transfer between phasesis pursued. In the case of organic solvents, it is achieved bythe generation of enough exchange surfaces between phases(small drop size). Conversely in the case of ATPS the formationof very small droplets should be avoided to prevent flood-ing and decrease coalescence time (Cuhna and Aires-Barros,2002).

However, it should be kept in mind that in some kind ofcontactors optimal operation is near the flooding point, sincethe dispersion area is maximized and the mass transfer rate isthe highest (Wachs et al., 1997). Increasing cross section areascan be useful to overcome this phenomenon. An extra heightdesign is recommended contributing to a longer residencetime and enhancing mass transfer.

3.2. Backmixing

Most contactors have an agitation device that accelerates theinteraction of phases. Mixer design is one of the limiting fac-tors on mass transfer rate. While the physical presence ofthese devices decreases the contact of coalesced phases withthe unreacted fresh feedings (backmixing), simultaneouslythe agitation stimulated by these devices increases it.

Due to its relevancy backmixing (or axial mixing, dependingon the phases contact angle) has been considered in severalmodels, which can be used to minimize it. It has been high-lighted that computational models of residence time (Cuhnaand Aires-Barros, 2002), “tank in series”, “reactor network”,and “close dispersion” (Martin, 2000) are capable of predicting

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Table 2 – Characteristics of selected mixer-settler units and other continuous ATPS devices and their performance parameters.

Device TrSSa,b

(min)

ATPS/productof interest

Disperse/loadedphase

Operational variables Ranges Performanceparameters

Ref.

Raining bucketcontactor

120 PEG1500-PS/NA Top/NARotor speed 6.6–15.5 rpm HD: 0.5756 Giraldo-

Zunigaet al.(2006)

Phases flow ratio (flowrates)

0.025–4.0 (20–80 ml/min)

Raining bucketcontactor

NAPEG1550-PS/�-La and�-Lg

NA/Bot

Rotor speed 3–9 rpm HD: 0.58 Dos-Reiset al.(1994)

T-B phase flow rate ratio 0.5–2 AMC:1–4.2 × 10−6 m2/s�-La RE: 97%�-Lg: 99%

�Dynamicmixer-settler unit 180

PEG 8000-PS/lypaseand LYZ

NA/NACharacteristics of thepacked separator

Column volume (3) andpacking material (3), diameter(3), mode (3), shape (2), volume(2) and structure (3)

Lypase Kp: 0.071 Banet al.(2001)

Total flow rate 3.0–12.0 ml/min LYZ Kp: 0.54RE: 80%

�Static mixer-settlerunit 15

PEG1000-PS/dyes,BSA and WPI

NA/BotType of static mixer 4 RE >90% Vázquez-

Villegaset al.(2011)

Tubular separatorlength

3000 and 5000 mm BSA Kp: 3.6

Internal diameter 60 and 90 mm

aAll the shown devices have a continuous and countercurrent operation mode, except the ones marked with the symbol (�), which are concurrent.b Time to reach steady state. �-La: alpha-lactalbumin; �-Lg: beta-lactoglobulin.

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Fig. 4 – Continuous ATPS devices evolution. It arises with mixer-settler devices (static mixer/centrifuge) (A). Graessercontactors appeared afterwards (B). Later, different columns were employed, of which, spray columns and PRDCs can behighlighted (C and D).Nowadays mixer-settler units are being retrieved as: column allies (F) or novel settler/separators (E).

axial dispersion in a wide range of operative conditions(Lounes and Thibault, 1996). Basically the determination ofbackmixing can be accomplished by a pulse or step input ofsoluble tracer in one of the phases and no transferable intothe other. Stella and Clive (Stella and Clive, 2006) offered anextensive compilation of the main models originated in recip-rocate plate and pulsed-plate columns, with respect to axialmixing.

3.3. Emulsification

Emulsification is an issue that has not been extensively dis-cussed when working with continuous ATPS. The lack of studyof these phenomena may be due to the scarce analysis of otherkind of ATPS, other than PEG-PS systems, and larger scale trials(Leng, 2004; Selber et al., 2004)

A high emulsification degree, reduces mixing efficiency,and increases coalescing time. Aggressive agitation shouldbe avoided in order to restrain the problem; especially withlow interfacial tension systems larger specific surface (big-ger drop size) area also reduces the phenomena. The Graessercontactor has shown to have an enough gentle agitation mech-anism favorable for emulsification nature systems. However,the mass transfer rate is inversely proportional to drop size,and thus, an analysis of cost–benefit on these two parameters,should be performed before deciding the mixing rate.

3.4. Separation efficiency

A minimal value of this parameter is desired under the follow-ing definition: the volume of the continuous phase divided bythe total volume at the disperse phase outlet, and vice versa(Cavalcanti et al., 2008). It is directly proportional to the dis-perse phase flow rate and the drop inherent coalescence time,and inversely proportional to the purification factor and therecovery efficiency (RE) (Igarashi et al., 2004b; Rostami andAlamshahi, 2002). For any continuous ATPS process a phases’separation stage is fundamental. Even if the device promotesan excellent mixing; if the separation efficiency at the end ofthe process is reduced the extraction performance in generalis greatly diminished. Higher flow rates and agitation rates,if not having an adequate separation time will have perfor-mance parameters that will not be representative from thewhole process.

An efficient phases’ separation within column contactorsis achieved by large column heights (Igarashi et al., 2004b).This is a characteristic from most of the bench experimentspresented in Table 1. Yet, it is particularly critical in mixer-settler units in which one stage is focused on the demixing ofthe phases. This has led to the use of centrifuges in whichseparation is very efficient. However, less energy and costintensive alternatives are required. A separation device ableto handle with different range viscosities, interfacial tensionand density parameters will allow an easier generic continu-ous equipment design. In a first attempt empirical equationshave been proposed to study this essential kinetics prior to thedesign of large-scale gravity phase separators (Narayan et al.,2011).

4. Trends and challenges

Only a few companies in the world, such as Genentechhave reported to use batch mode ATPS for product recov-ery (Builder et al., 1993; Asenjo and Andrews, 2012). This cangive us a hint that continuous ATPS could be better adoptedby the industry given that some of the issues mentionedcould be addressed. Since the early 1980s, the first attemptsto operate ATPS continuously with mixer-settler units werereported. Afterwards column contactors became the mostattractive equipment. Spray columns were followed by differ-ent alternatives trying to enhance mass transfer and improvebiomolecules’ recovery. Nowadays mixer settler units seemto be recalled, in order to face the generic application chal-lenge of continuous ATPS operation (Fig. 4) (Saadat et al.,2009).

Although traditional polymer-salt systems are yet expectedto dominate applications, new kinds of ATPS, such as ionicliquids are the tracking trend. These systems are consid-ered a great option for the replacement of volatile organicsolvents in LLE (Novak et al., 2012) and are more eas-ily reusable and recycled. Among its advantages outstandthe lower viscosity, little or no formation of emulsionsand quick phase separation. Even though, toxicity is stilla concern for some of the compounds employed (Li et al.,2010).

The tendency toward the miniaturization of continuousATPS partition may highlight some characteristics of theATPS previously unknown, while studying the behavior of cer-tain high valuable pharmaceutical biomolecules (Rosa et al.,

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2013; Hardt and Hahn, 2012; Ingram et al., 2013). This wouldallow the shortening of data acquisition for the optimalpartition parameters and makes more efficient the use ofresources. This time reductions coupled with straightforwardscaling protocols should impulse ATPS industrial implemen-tation.

One of the great challenges of ATPS is to surpass theapparent unattractive economical image. Studies comparing,in detail, the costs involved in ATPS implementation withalternative technologies are not common (Aguilar et al., 2006;Huenupi et al., 1999). The use of commercial centrifuges doesnot contribute to the ATPS cost–benefit, due to the energy andcosts involved. A pre-evaluation of ATPS recuperation costsfor each biomolecule of interest should be made in order todetermine the viability of an ATPS process.

Another important challenge is the determination of anequipment design platform. Meanwhile column contactorshave more solid design guidelines, including its hydrody-namics characterization; mixer settler devices studies havemainly focused on the verification of its performance capac-ity. The development of novel separators for different ATPSphysicochemical characteristics would help for the imple-mentation of continuous ATPS (Huddleston et al., 1991). Phaserecycling is a missing gap that should be considered for con-tinuous operation in column contactors as well as in staticmixer units, given the environmental impact of phase form-ing compounds. New compounds such as carbohydrates andthermoseparating polymers are under study (De Brito Cardosoet al., 2013; Show et al., 2012; Li et al., 2002).

Finally, an integral view of the process before and afterATPS should be contemplated. ATPS and sample prepara-tion previous to the extraction procedure should be studied.This is particularly relevant when complex mixtures of differ-ent molecules of interest are processed. The recovery of theproduct of interest from the phase forming components isstill a matter of study that should be optimized. To date ionexchange chromatography, precipitation, ultrafiltration, dial-ysis, and supercritical CO2 extraction have been considered (Liet al., 2010). A device design, which reflects all of the treatedaspects in this review, will be very advantageous.

5. Conclusion

It is clear that ATPS offers valuable opportunity areas forprimary recovery processes. However, in the gradual transi-tion from batch to a continuous ATPS operation, the lack ofpractical rules for an effective implementation has limitedtheir generic application at commercial scale. Column con-tactors have been the common choice for continuous ATPSprocesses that use polymer-salt systems for proteins andenzymes extractions, but their lack of versatility and limitedmargin for mass transfer and separation performance havecaused the mixer-settler to re-appear in the stage. Some of themain areas of opportunity identified for the practical imple-mentation of continuous ATPS include: phases recycling, morerobust application, well-suited and feasible predictive perfor-mance models, practical guidelines for design and scale up,control and automation. Advances in these later aspects willovercome the greatest economic challenges for ATPS. It isanticipated that the development of generic suitable devicesfor continuous ATPS will represent an attractive, alternativeplatform in the establishment of commercial adoption of con-tinuous ATPS.

Acknowledgements

The authors wish to acknowledge the financial support ofTecnológico de Monterrey Bioprocess Research Chair (GrantCAT161) and CONACyT for the doctoral fellowships of E.Espitia-Saloma No. 246739 and P. Vázquez-Villegas No. 228073.

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