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chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738

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Chemical Engineering Research and Design

journa l homepage: www.e lsev ier .com/ locate /cherd

isualization of compound drops formation in multiphaserocesses for the identification of factors influencing bubblend water droplet inclusions in oil drops

. Corkidi ∗,1, A. Rojas1, A. Pimentel, E. Galindonstituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. 510-3, Cuernavaca 62250, Mor., Mexico

a b s t r a c t

An innovative methodology for visualizing and identifying some mechanisms by which complex structures such as

air-in-oil-in-water (A/O/W) and water-in-oil-in-water (W/O/W) may be formed inside mixing tanks dispersing various

phases is described. In the case of A/O/W inclusions, isolated inclusion events could be observed by the first time with

an experimental setup designed to produce sudden turbulence in a small confined space simulating a three-phase

fermentation system. It was observed that high-energy direct-collisions of the bodies are not required for inclusions

to occur; rather, a gentle contact between the phases was needed. Then, by maintaining an oil drop in a fixed position

while it was impacted by single air bubble, it was feasible to calculate the percentage of air-bubble inclusions into

oil drops for different compositions of the continuous phase. By adding biomass as a solid phase, the inclusion

occurrence reached 61%; likely this was caused by a mechanical effect of the added biomass (making the interface

breakable or unstable) with a minor contribution by the decreased surface tension. In the case of W/O/W, a basic

mechanism by which the inclusion of water droplets in oil drops may occur is described. This was derived from the

analysis of the hydrodynamic process of the formation of a water drop inside a volume of oil where the differential

pressures occurring along the water–oil interface were mapped. This is the first time that factors influencing water

and air inclusions in oil drops are identified, and possible mechanisms behind their occurrence are proposed, based

on visual evidence.

© 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Multiphase dispersion; Bubble; Drop; Particle formation; Compound drops; Mass transfer; High speed video

level of mechanical energy dissipated into the fluid by agita-

. Introduction

variety of chemical and biochemical processes involveultiphase dispersions. Particularly, in some fermentation

rocesses these dispersions consist of oil drops, air bubblesnd compound oil drops containing air bubbles and waterroplets. In multiphase processes, dispersion is a critical issueince it determines interfacial area, which in turn deter-ines mass transfer efficiency. It is generally assumed that in

as–liquid contactors (such as fermentors) the oxygen trans-er takes place from the air bubbles into the aqueous mediumy diffusion through the gas–liquid interface (Middleton andmith, 2004). However, our findings indicate that the diffu-

ion path is more complex, since there is a previous transfer

∗ Corresponding author. Tel.: +52 777 3291710; fax: +52 777 3172388.E-mail addresses: corkidi@ibt.unam.mx, corkidi@sgima.ceingebi.unReceived 27 April 2011; Received in revised form 28 March 2012; Accep

1 These authors contributed equally to the realization of this paper.263-8762/$ – see front matter © 2012 The Institution of Chemical Engittp://dx.doi.org/10.1016/j.cherd.2012.03.021

in the air-bubble-oil-drop interface (Galindo et al., 2000), fol-lowed by the transfer through the oil–water interface. This is amechanistic explanation to the fact that oils improve oxygentransfer and in some cases increase the secondary metabolitesproduction (Larralde-Corona et al., 2002; Dumont and Delmas,2003). Because of this, one of the most relevant phase inter-actions identified in fermentation processes involving liquidphases, is the multiphase (or compound) drops formation con-sisting in the presence of air bubbles and water droplets fromthe aqueous phase inside oil drops (Galindo et al., 2005). Theformation mechanism of these complex structures (Fig. 1) isnot known, but is expected to depend, for example, on the

am.mx (G. Corkidi).ted 29 March 2012

tion which may cause the deformation of the oil drops and

neers. Published by Elsevier B.V. All rights reserved.

1728 chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738

Fig. 1 – Compound drops (air and water-in-oil-in-water(A&W/O/W)).

experimental evidence documented in video of the inclusionof bubbles and water droplets inside oil drops is described.

air bubbles, such that their interactions could produce mul-tiphase drops. Although a number of papers have addressed,experimentally and theoretically, some aspects of the phasesinteraction, to the authors’ knowledge there is no litera-ture documenting the formation of these complex structuresoccurring in multiphase dispersions in mixing tanks. Further-more, no experimental visual evidence of the phenomenonhas been published.

Image analysis techniques are valuable tools that can pro-vide insights of interesting hydrodynamic events occurringin multiphase systems. Some recent and relevant examplesinclude the work of Guevara-López et al. (2008), Aarts et al.(2005), Borrell and Leal (2008), Lawson et al. (2004) and Rojasand Papadopoulos (2007).

Nevertheless, the inclusion of bubbles and water dropletsinside oil drops occurring in a mixing tank or fermentationprocess is extremely complex and so far technically impossi-ble to characterize in situ, due not only to the high speed atwhich the phenomena actually occurs (which can be solvedby the use of a high speed camera), but also due to the impos-sibility (with the technology currently available) to follow thephenomena in the relatively large windows of observation thatwould be required to fully characterize them. Therefore, theapproach followed in this work in order to overcome theseobstacles was to simulate (to the greatest extent possible) thecharacteristics of the actual phenomena likely occurring in amixing tank by means of simplified models. Visualization athigh speed rates and the characterization of the inclusion ofair bubbles and water droplets inside oil drops was carried outthrough experimental setups in which reproducible and con-trolled experiments of the dispersion processes occurring in amixing tank can be simulated to some extent.

In this way, qualitative and quantitative informationregarding the complex phase interactions and the oil-trappedbubbles and oil-trapped water droplets has been obtained. Byusing the proposed methodology it has been shown how thepresence of dispersed mycelium can increase considerably theinclusion of air bubbles in the oil drops. Furthermore, a mech-anism explaining how water droplets are included in oil dropsis proposed. This is a first step in the understanding of howcompound drops are produced in multiphase processes.

2. Materials and methods

2.1. Dispersion systems

2.1.1. Continuous phaseDistilled water and an aqueous salt solution simulating afermentation broth (Galindo et al., 2000) were used as thecontinuous phase. The surface tension of the solution was72 mN/m. In order to obtain different experimental condi-tions (see Sections 3.2 and 3.3), the surface tension for someof the experiments was decreased to 50 mN/m by addingeither bovine serum albumin (BSA) (Sigma Fraction V) 0.2 g/Lor a surfactant (Tween 40, Polyoxyethylene glycol sorbitanmonopalmitate, Sigma) 0.02 g/L.

2.1.2. Oil phaseFood-grade castor oil (Cosmopolita, Mexico) was used as theimmiscible liquid phase. Castor oil is a combination of fattyacids with about 90% of ricinoleic acid and the other significantcomponents being oleic and linoleic acids.

2.1.3. Solid phaseDispersed fungal biomass of Trichoderma harzianum IMI 206040was used as the solid phase (0.2 g/L). The biomass wasobtained following the procedure detailed by Lucatero et al.(2003) and had a mean diameter of 223 ± 1 �m and a circular-ity factor of 0.3 (these measures were determined using animage analysis system detailed also by Lucatero et al. (2003)).

2.1.4. Surface tension measurementsSurface tension of the solutions was measured according tothe Wilhelmy plate method (Holmberg, 2002) using a Tantec STTensiometer (USA) with a roughened platinum Plate 24.0 mmin length. After each measurement, the plate was rinsed withMilli-Q water and washed with an industrial degreaser (highalkaline degreaser) and acetone, and then flamed to removeany trace contaminants.

2.2. Image analysis equipment

Digital high-speed video was acquired by means of a high-speed video camera (MotionPro HS-4, Redlake, USA). Thecamera was coupled to a stereomicroscope with variablemagnification (SZ1145ESD, Olympus, USA) and illuminationprovided by an incandescent lamp that was placed behindthe experimental setups, facing the stereomicroscope andscreened by a translucent sheet of acrylic serving as a light dif-fuser. Image sequences of the experiments were acquired at300 fps and a magnification of 1.8× in all cases, except for theexperiment described in Fig. 4 (cf. Section 3.2(a)) in which theacquisition rate was 1000 fps. The image sequences were pro-cessed with the software package Image-Pro Plus V.7.0 (MediaCybernetics, USA) for measuring of the diameters of bubblesand drops. Meanwhile, the tracking of trajectories of small oildrops inside water droplets and their speeds were achievedby using the “manual-tracking” plug-in of the software ImageJV.1.34 (Fabrice Cordelieres, Institut Curie, Orsay, France).

2.3. Experimental setups for visualization andquantitative description of air and water in oil inclusions

In this section, the methodology followed in order to produce

chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738 1729

Table 1 – Comparison of designed setups.

Oil dropimpacting

water surfacefor observation

of W/O/Winclusions

Injection ofwater below oil

interface forobservation of

W/O/Winclusions

T-shaped tubefor observation

of A/O/Winclusions

Immobilizedoil drop for

observation ofA/O/W

inclusions

Purpose Qualitative isolatedobservation of apotentialmechanism forW/O/W inclusions.

Detailedobservation of theoil drop deforma-tion/recoveryprocess conducingto water capture.

Observation of anA/O/W inclusionproduced via asmall scalereproduction ofturbulentconditions similarto those inside amixing tank.

Repeatedobservation of airbubble inclusionsinto oil drops forstatistical analysesand identificationof its determiningfactors.

Observed phenomenon Water inclusionduringshape-recovery ofoil drop afterdeformation byimpact.

Water inclusionproduced after avolume of oildeformed via awater jet recoversits shape.

Inclusion of freelymoving air bubblesinto freely movingoil drops.

Air bubbleinclusions into oildrop under fourdifferentconditions(aqueous medium).

Pros Phenomenon takesplace with the oildrop moving freelyin the continuousphase.

Deformation of oilvolume can beobserved in detailand underreproducibleconditions. Thissetup enablescollectingquantitative data,such as the volumeof water injectedand flow patterns.

Within reasonableboundaries theconditions of theexperiment closelyresemble those ofthe real process ofinterest.

Several of theconditions in theexperiment can bechangedsystematicallyallowing theproduction ofquantitativeconclusions. Foreach conditiontested it is possibleto record severalinclusion eventswithout having torefit the setup.

Cons Lack of control overthe conditions thatmay promoteoccurrence of thephenomenon. Thissetup allows onlyfor a qualitativeanalysis of thephenomenon.

Confined oilvolume used inplace of a freelymoving oil drop is anecessaryapproximation tomore realconditions.

As particles areproduced andmove freely thereis no control overtheir interactionand the occurrenceof the inclusions.This setup onlyallows qualitativeobservations to beperformed.

The oil drop mustremain attached toa support in orderto keep thephenomenon inthe observationarea. The bubblegenerationmechanism can besusceptible toblockages andundesiredvariability due tothe small diameterof the tubes.

Ateftsii

2w(i

lso reported is a quantitative study that was carried out withhe purpose of determining which, of some selected param-ters, bear the strongest influence on the compound dropormation. The main characteristics, limitations and advan-ages of the different experimental rigs used to visualize and toome degree comprehend the phenomena occurring in a mix-ng tank/fermentor, are summarized in Table 1 and describedn the following section.

.3.1. Experiments regarding the formation ofater-in-oil-in-water (W/O/W) compound drops

a) Oil drop impacting a water surface: An experimental setup wasmplemented in order to observe and describe qualitatively

the deformation–recovery process of an oil drop and how thisprocess can induce the introduction of water droplets into theoil drop. For this purpose, an oil drop produced with a syringefilled with castor oil was dropped vertically from 1 m highinto a 10 × 10 × 10 cm3 glass container filled with water. Thehigh-speed video camera was installed just at the level of theair–water interface to capture the drop entering into the waterand touching the bottom of the glass container (see Fig. 2).

(b) Injection of water under an oil interface: The setup consistedof a glass tube containing water followed above by a columnof oil. Disregarding the actual geometry, it is assumed thatfor the purpose of the experiment, the interface between the

volumes of oil and water in the glass tube can be considered

1730 chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738

Fig. 2 – Scheme of experimental setup for observation of oildrop impacting a water surface (not to scale).

Fig. 4 – Experimental setup showing the assembledsolenoid, syringe and T-shaped tube containing water, oiland air (not to scale).

equivalent to that between an oil drop freely moving in waterand the medium surrounding it. In the first level (where thewater resides), a needle attached to a syringe containing wateritself was introduced (see Fig. 3). Using this syringe a water-flow of approximately 2 ml/s was produced that impactedthe oil column at the water–oil interface and promoted theformation of a water droplet inside the oil column. An obser-vation window was set at the water–oil interface in order toobserve and record the phenomena occurring.

2.3.2. Experiments regarding air-in-oil-in-water (A/O/W)compound drops(a) T-shaped tube for observation of air-in-oil-in-water (A/O/W)inclusions: An experimental setup was designed with the objec-tive of producing a turbulent flow which could mimic, to someextent, the conditions present inside a fermentation tank butin such a way that allowed the observation and recording ofthe interaction between the liquid-, oil- and air- phases of thedispersion system, as well as the possible formation of com-pound drops (by inclusion of air bubbles into oil drops). In orderto be able to record such interaction in a microscopic scale,the induced turbulence had to be contained and limited to a

small visualization area. The solution found was to employ a

Fig. 3 – Scheme of the experimental setup for injection ofwater under an oil interface (not to scale).

T-shaped glass tube (Ø12.5 mm) containing the components ofthe dispersion (water, oil and a small amount of air trappedin the joint of the T-shaped tube) and attached to the barrelof a plastic syringe (see Fig. 4). In turn, the syringe plungerwas attached to a solenoid in such a way that activating thesolenoid would rapidly pull the plunger a small distance andproduce turbulence inside the tube. The Reynolds numberof the produced turbulence was between 500 and 700 (cal-culated from the geometrical parameters of the system andthe velocity of the fluid generated by the suction). Thesedynamical conditions are comparable with those caused bya Rushton impeller operating at conventional stirring speedsused in fermentation tanks containing viscous fluids (Nienow,1993; Amanullah et al., 2004; Sánchez-Pérez et al., 2006). Theinduced turbulence would mix the components of the dis-persion in the volume inside the visualization area (1.0 mm2),allowing the recording of the interaction between the oil dropsand air bubbles thus produced.

(b) Immobilized oil drop and controlled air bubbling for obser-vation of air-in-oil-in-water (A/O/W) inclusions: An experimentalsetup was designed with the objective of observing and mea-suring the inclusion of air bubbles into one oil drop and testingthe effect of different physicochemical reproducible condi-tions. The basic idea was to keep the oil drop in a fixed position,while it was impacted by single air bubbles. The experimentalsetup consisted of an L-shaped glass capillary tube (Soda-lime Glass 25 �L microcapillary pipette (Kimble, USA), withan internal diameter of 650 �m) containing oil, with one endsubmerged into a tank filled with water or salt aqueous solu-tion (cf. Section 2.1). Single oil drops with a specific diameter(see Table 2) were produced at this end of the capillary tubeinside the water by means of a micropipette attached to theother end. Finally, air was injected below the oil drop througha single-bubble generator (similar to that reported by Seyyed-Najafi et al. (2008)), so that small bubbles (see Table 2) wouldimpact the oil drop one at a time (Fig. 5). The microcapillarytube was elaborated from a Soda-lime Glass 25 �L microcap-illary pipette (Kimble, USA) with an initial internal diameterof 650 �m that was reduced by heat-induced elongation of thepipette (the pipette was held vertically aligned from one endand heat was applied to its mid section so that its free end was

pulled by the effect of gravity) to a mean diameter of 70 �m.

chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738 1731

Table 2 – Oil and bubble diameters and percentage of air bubble inclusions within oil drops under different conditions.

Condition Surfacetension(mN/m)

Bubble meandiameter (�m)

Single dropdiameter (�m)

Number ofbubble impacts

Inclusions (%of impacts)

Aqueous solution medium (ASM) 72 468 ± 5 3016 262 1 ± 0.8ASM + Protein 0.2 g/L 50 484 ± 7 3110 276 6 ± 0.8ASM + Tween 40 0.02 g/L 50 470 ± 6 3000 309 6 ± 0.9ASM + Biomass 0.2 g/L 54 670 ± 8 4450 293 61 ± 2

Fig. 5 – Scheme of the experimental setup for studyingb

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w

Video 1

ubble impacts on a fixed oil drop (not to scale).

. Results and discussion

.1. Water-in-oil-in-water (W/O/W) compound dropormation

n compound drops, besides air bubbles, water droplets areresent (see Fig. 1). A proof of the nature of water dropletsas been reported previously (Córdova-Aguilar et al., 2008).owever, the mechanism by which the inclusions of waterroplets into oil drops occur has been only schematically pro-osed (Sajjadi et al., 2002; Brooks and Richmond, 1994). Thexperimental setups described in the previous section wereesigned to shed some light on this matter through directbservation of the phenomenon (Table 1 summarizes the mainharacteristics of these experimental setups).

(a) Oil drop impacting a water surface: Using the setupescribed in Fig. 2 (Section 2.3.1(a)), it was observed that theil drop is deformed by the impact against the water surface

and later against the bottom of the container). Impact withhe water interface produces drop deformation simulating thene likely occurring in high shear regions of the mixing tank.mpact with the bottom of the container simulates the likelyollision with the impeller blade. As the deformed body of oilecovers the spherical shape inside the water, it may incorpo-ate small volumes of water (see Fig. 6 and Video 1 ). This setupllowed us to observe inclusions of water into oil, and to con-ecture that the deformation of oil drops (and later recovery ofheir shape) may be acting as one of the mechanisms that pro-uce complex structures. Nevertheless, using this setup, theroduction of the water-inclusion event is random since there

s no control about the shape that the oil drop may adopt in itsynamic interaction with the water. For this reason, the setupescribed in Fig. 3 (Section 2.3.1(b)) was designed with thebjective of being able to control and reproduce the dynamiconditions that produce the water-inclusion events.

(b) Injection of water under an oil interface: In the experimentsith the setup described in Fig. 3 (Section 2.3.1(b)) a water

Video 2

jet directed toward a water–oil interface was produced. Thiswater flow displaces part of the oil creating a cavity-like sur-face that, swelled by the water, expands until a drop is formed(see Video 2 ). In what follows, a qualitative description ofthe hydrodynamics involved in the formation of this drop isoffered.

The focus of our analysis is the interaction between thecompressive forces (oil pressure and surface tension) and theexpansive forces (exerted by the water injected into the drop)acting over the drop surface. Since the dynamics of the flu-ids change over time, the drop formation process was dividedinto three distinctive stages, illustrated in Fig. 7a. Stage I cor-responds to the very initial formation of the drop; the instantsjust after the first contact of the water jet against the water–oil

interface that begins to produce a deformation of the latter(Fig. 7a(I)). Stage II describes the longest part of the process that

1732 chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738

Fig. 6 – Different stages of a single oil drop deformation and the inclusion of water droplets as it recovers the sphericalshape. (a) The drop enters the water and is deformed by impact; a cavern begins to form and a water droplet is “absorbed”by the oil drop. (b) The drop enters the water and is deformed by impact; the oil mass is further deformed against the

ape

bottom of the container and the oil returns to a spherical sh

in which the drop expands while receiving a nearly constantwater flow rate and in which the so-called drop neck appears(Fig. 7a(II)). Finally, Stage III corresponds to the final part of theprocess; the water flow decreases, the drop neck closes, andthe drop is about to complete its formation (Fig. 7a(III)).

The following discussion is limited to Stage II since it is thelongest and most complex part of the drop formation process.Consider the diagram in Fig. 7b where the forces exerted overan infinitesimal section of the oil–water interface are illus-trated. �F1 is the force related to the oil pressure, �F2 is theforce related to the pressure of the injected water, �T repre-sents the surface tension, and �Fr is the effective resultant forceover the section, defined as �Fr = �F1 + �F2 + �T. The direction of �Fr

determines whether the particular section of the drop surfaceexpands (

∣∣�Fr

∣∣ > 0),, is compressed (

∣∣�Fr

∣∣ < 0), or remains static

(∣∣�Fr

∣∣ = 0). To facilitate the analysis, three regions have been

identified in the drop structure playing different roles duringthe formation process due to the geometry of the interface andthe local relationship of the interacting forces (see Fig. 7c); thehydrodynamics are described accordingly.

In Region-1 the water flow pushes directly from below in itsvertical motion. The pressure of the water jet is larger than thepressure of the oil column above the drop (

∣∣�Fr

∣∣ > 0), and thus

the water displaces the interface upwards, expanding the dropand losing energy in this process. Since water is constantlyinjected, the excess water is deflected away from the centralaxis of the drop. The lateral walls in Region-1 are horizontallydisplaced by the deflected flow, so that the upper portion of thedrop expands horizontally too, but to a lesser degree. After thisprocess, the water stream has lost most of its momentum andmoves toward Region-2.

In Region-2 the deflected water-flow from Region-1 swirlsslowly (relatively to the speed of the incoming water jet) and

with water droplets inside the oil.

the stream lines of water are almost parallel to the drop sur-face. Therefore, the flow pushes weakly against the interface,but because the water is still coming into the drop, the con-tained water exerts a static pressure that prevents shrinkageof the interface under the oil pressure. The global balance ofpressures results in a moderate expansion of the interface(∣∣�Fr

∣∣ ≈ 0) but the drop shape in this region clearly indicates

that the dynamic water pressure becomes smaller as onemoves toward Region-3.

In Region-3 the incoming water flow has its highest speedand (according to Bernoulli’s principle) a low-pressure zone isproduced. Furthermore, due to the geometrical arrangementthe water flow is not opposing any dynamic force to the con-stant oil pressure pressing on the interface (

∣∣�Fr

∣∣ < 0) and as

a consequence a so-called drop neck is created. In turn, thegradual narrowing of the interface at the drop neck causes thelocal speed of the water jet to increase. The overall result is apositive-feedback process that eventually (as the water flowstarts to decrease) closes the drop neck. The drop then entersStage III where it detaches itself from the main body of waterand adopts a near-spherical shape.

In order to illustrate the process described above and as aproof of concept that the experimental setup used can be use-ful in obtaining quantitative data, the movement of severaltiny oil droplets present in the water flow while the drop wascreated inside the oil was tracked, so that these were usedas motion tracers. In doing so, it has been considered thatthe effect of the buoyancy force of the droplets was negligi-ble (due to a difference in densities between oil and water of∼4%) and only those droplets that approximately describedbidimensional trajectories (guided by whether these appeared

in- or out-of-focus in the experimental recordings) were used.After tracking, the corresponding velocity field of the droplets

chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738 1733

Fig. 7 – Formation of a water-droplet in oil and the forces involved in this process. (a) Representative stages of the dropformation: from the initial stage I, where the oil surface is displaced by a water jet, then to the stage II, where the incomingflow reaches a quasi-stationary state increasing the drop volume, to the final stage III, where the drop completes itsformation process. (b) Diagram of forces exerted over an infinitesimal section of the surface of the drop, �F1 is associated tothe oil pressure, �F2 represent the water flow pressure, �T is the surface tension and �Fr is the resultant force defined as�Fr = �F1 + �F2 + �T. The magnitude and sign of �Fr determines the direction of the section of the surface displaced. (c)Representative scheme of the magnitude and direction of �Fr across the surface of the drop: three regions are identified thatcorrespond to different portions of the droplet playing different roles in the drop formation process (see main text).

wtiemt(po

mbetadeeo

as derived; the result of this process is shown in Fig. 8. Noticehat, as expected, the main portion of the flow is concentratedn the vertical direction (Fig. 8a and b), but there also exist lat-rally curving branches that describe the re-circulating flowentioned before. Furthermore, the 10% highest velocity vec-

ors corresponding to the highest-energy regions were filteredFig. 8c), thus confirming that the highest-velocity (lowestressure) region is located at the drop neck and center portionf the drop.

A description of the hydrodynamics involved in the for-ation of a water droplet inside oil has been offered with

ase on the setup described previously. In spite of the nec-ssary controlled conditions of the experiment, some ofhe ideas regarding the interaction of local forces could bepplicable to more complex environments. There exist evi-ent differences between the phenomena occurring in ourxperimental setup and that occurring in a mixing tank, nev-

rtheless, the hydrodynamic conditions are comparable andur analysis provides the basis to demonstrate an important

result: if any local region into the system conjugates a gra-dient of pressures that deforms the oil drops in such away that they face a water flow specifically oriented rel-ative to said gradient, those compound structures may beformed.

3.2. Air-in-oil-in-water (A/O/W) compound dropformation

The volume of air bubbles trapped in the oil drops in a mixingtank seems to be determined mainly by oil drop deforma-tion and interfacial interactions (Moosai and Dawe, 2003).Moreover, the interaction of the phases themselves directlyinfluences their motion and their behavior (Massoudi, 2002).Several papers have reported the fluid mechanics of dropsand bubbles (Hagesaether et al., 1999); most of these studieshave been concerned with two-phase systems (liquid–liquid

or gas–liquid systems) under ideal controlled laboratory con-ditions (Kessler and York, 1970; Johnson and Sadhal, 1985;

1734 chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738

Fig. 8 – Steady water flow observed by tracking oil droplets moving inside a water drop. (a) Normalized water velocity fieldinside oil volume (red regions indicate highest velocity). (b) Velocity flow lines corresponding to several tracked tiny oildrops. (c) Velocity flow lines corresponding to the 10% highest-velocity streamlines measured. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of the article.)

Video 3

Sajjadi et al., 2002; Baldessari and Leal, 2006) or they havepredicted data based on the theoretical or numerical models(Khopkar et al., 2005). In this context, the experiments deal-ing with A/O/W inclusions described in the previous sectionwere designed to deliver important data regarding two specificquestions: first, how inclusions actually occur when oil dropsand air bubbles move freely in an aqueous medium under tur-bulent conditions. Second, what is the observable effect onthe frequency with which A/O/W inclusions occur, producedby (a) changing the surface tension of the continuous phase; (b)changing the surface tension by adding biomass to the system(i.e., converting it into a four phase system and introducingan agent that is expected to alter considerably some of themechanical aspects of the system). Table 1 summarizes themain characteristics of these experimental setups.

(a) T-shaped tube for observation of air-in-oil-in-water (A/O/W)inclusions: By means of the turbulence induced by the experi-mental setup shown in Fig. 4, oil drops and air bubbles wereproduced inside the T-tube and recorded as they freely inter-acted with each other. With this configuration, it was possibleto observe, in some of the experiments, the precise momentwhen air bubbles were trapped inside oil drops (Fig. 9 andVideo 3 ). To the authors’ knowledge, no previous evidencehad been obtained regarding the dynamics of inclusion andthis relatively simple setup allowed the visualization of suchevents.

From the many recordings made (Fig. 9 being an example),it was possible to document that air bubbles can enter an oildrop simply by gently touching it (that is, even if they do notfollow trajectories of direct collision against each other), andin a very small fraction of time (in the recordings, approxi-mately 35 ms from contact to complete engulfment of an airbubble by an oil drop was the time measured). Although thisobservation may seem contrary to some of the published the-ories (see Liao and Lucas, 2010) that attempt to explain thephenomenon of inclusion in turbulent flow mainly by the pres-ence of high energy impacts of the objects, it fits well into othermodels such as the “critical approach velocity” model, thatstates that small approach velocities favor coalescence (Liao

and Lucas, 2010). Furthermore, it seems reasonable that theincorporation of air bubbles into oil drops may also be favored

by the positive spreading of the oil employed, as proposed byMoosai and Dawe (2003); in other words, due to a strong affinitybetween these substances.

Even though this experimental setup produced conditionsthat are comparable to those occurring inside a mixing tank(in terms of the turbulent flow and the free-moving state of theobjects), it was not possible to reproduce an observation underexactly the same conditions. Therefore, another experimentalsetup based on a fixed oil drop and the generation of single air-bubbles was designed in order to enable the reproducibility ofthe observations.

(b) Immobilized oil drop and controlled air bubbling for obser-vation of air-in-oil-in-water (A/O/W) inclusions: Employing thesingle-bubble bubbler setup shown in Fig. 5, it was observedthat as the air bubble approaches and contacts the oil drop, itmay attach itself to the drop either instantly or after a vari-able period of time that they remain in contact with eachother (Fig. 10a, Videos 4 and 5 ). This occurs more often whenthe bubble makes contact with the drop at a point off the

vertical axis of the drop but it may even occur at an instant

chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738 1735

Fig. 9 – Sequence of images showing the inclusion phenomenon of an air bubble (dark small object indicated by the arrow)into an oil drop (brighter and larger object), occurring under turbulent flow conditions (by using the setup described inSection 2.3.2).

Fig. 10 – Sequence of images showing an air bubble getting trapped by an oil drop after contact. (a) Air bubble getting‘trapped’ by an oil drop after contact, (b) Air bubble bouncing before entering the oil drop.

Video 4

wfs“W

Video 5

without achieving to break the initial contact (Fig. 10b and

hen the bubble is already in the process of moving awayrom the drop after an initial off-axis contact (for a rigoroustudy of the phenomenon of coalescence after these so-calledglancing collisions”, conducted on drops, see Leal (2004)).

hen the bubble collides with the drop at a point along their

common vertical axis, the bubble may either bounce, or attachitself to the oil drop and attempt to bounce off the drop but

Video 6 ). During this process, deformation of the air bubble

1736 chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738

Video 7

Video 6

can be observed as it is pushed against, or pulled away from,the oil drop.

It has been observed that inclusions are not favored bydirect collision of the air bubbles against the oil drops, inso-far as the bubble bounces off the drop with more energyafter a head-on collision than after an off-axis collision,making it less probable that inclusion will occur at first con-tact (note that inclusion may still occur in a second contactwhen the bubble resumes its vertical trajectory, if it finds thedrop again). Furthermore, a reason why glancing collisionscan favor inclusions over head-on collisions is that they mayresult in prolonged contact time between the objects, allow-ing for what is known as film-drainage (see Chevaillier et al.(2006)) and thus leading to inclusion of the air into the oil, orotherwise called coalescence of the drop and the bubble(regarding film drainage and other aspects of the coalescenceprocess of drops, see also: Aarts and Lekkerkerker (2008),Yeung et al. (2003)).

3.3. Effect of biomass in air-in-oil-in-water (A/O/W)compound drop formation

Pulido-Mayoral and Galindo (2004) reported that a change inthe surface tension promotes the formation of oil drops witha high number of embedded bubbles. On the other hand,Giribabu and Ghosh (2007) observed that surfactants reducetension and this prevent drops and bubbles from coalescingwith one another. With the aim of observing the effect of thesurface tension on the number of inclusions of air bubblesinto oil drops, several experiments were performed using thesingle-bubble generator shown in Fig. 5 (cf. Section 2.3.2(b))and different aqueous salt solutions with different surfacetensions, as described in Section 2.1. Additionally to the solu-tions described in Section 2.1, another salt solution containing0.2 g/L of fungal biomass suspended in the liquid was alsoemployed, giving a total of four experimental conditions toconsider. These conditions simulate (at least partially) theconditions that can be found in the actual fermentation pro-cesses.

As bubbles moved upwards, they impacted the oil dropat a constant speed of 0.3 ± 0.02 m/s for all the experimen-

tal conditions (i.e., no significant differences in the velocitiesof the bubbles impacting the drop were observed, with a

confidence interval of 1%). Furthermore, in order to mini-mize the possible effect of the particular size of bubbles anddrops, the diameter ratio of air-bubbles to oil-drops was main-tained constant at 0.15 in all cases (experimental data do notemanate necessarily from Figs. 9 and 10, which were selectedto show illustrative cases). For each of the four experimentalconditions, at least six replica experiments were performed.Collisions were recorded and counted, as were the numberof bubbles that were captured within the oil drops (since notevery collision led to an inclusion).

The results of these experiments are reported in Table 2.The size of the drops and bubbles in each of the experiments isalso reported. For the experiments with the aqueous salt solu-tion, the proportion of collisions that led to inclusion was quitesmall, at 1 ± 0.8%, whereas in the presence of protein and ofthe surfactant Tween 40, the number of inclusions was higher,at 6 ± 0.9%. In contrast, when the fungal biomass was presentas the solid phase, the proportion of inclusions increased dra-matically to 61 ± 2%. To illustrate this last point, an oil dropin ASM + biomass media impacted by several consecutive airbubbles where most of them were included in the same dropis shown in Fig. 11 and Video 7 .

Contrary to the hypothesis that air-into-oil inclusionsmay be produced mainly by collisions of bubbles and drops(Larralde-Corona et al., 2002), it has been observed that, inmost of the cases, when freely moving bubbles led to aninclusion (Figs. 9 and 10), the air bubble approached the oildrop slowly (i.e., at a speed given by its own buoyancy) andmaintained contact with it during a certain period of timebefore the inclusion was produced. Also, in the case of theexperiments with immobilized oil drops, it was observed thatin many of the events that led to inclusion the air bubblesin their upward movement collided with the oil drop andeither initially bounced against it or moved quite closely andpresumably in contact around it before the inclusion was pro-duced (see Fig. 10). Results presented in Table 2 suggest that itwas not the decrease of surface tension the factor that mostlypromoted the bubbles inclusions as it has been proposed byPulido-Mayoral and Galindo (2004). The addition of biomass(which also decreased the surface tension because of thesoluble proteins that may contain, despite the extensive wash-

ing of the mycelia), increased dramatically the percentage ofinclusions. Surface tension seems to play a role in determining

chemical engineering research and design 9 0 ( 2 0 1 2 ) 1727–1738 1737

Fig. 11 – Oil drop in ASM + biomass media impacted by several consecutive air bubbles where most of them were includedi serve

iTmabbr

4

BstdasawtTttooTcbma

A

TSAdttba

R

A

A

nto the same drop (at the bottom of the images it can be ob

nclusions (indicated by the experiments with the surfactantween 40), although a minor one. The biomass may interactechanically over the contact surface between the oil drop

nd the air bubble, so the drop boundary or interface becamereakable or unstable, allowing that a high proportion of theubble impacts ended in inclusions as it was visualized in theecordings of the experiments with fungal biomass.

. Conclusions

y using simple mechanical devices, combined with highpeed video techniques, it was possible to observe, in real time,he formation of compound drops, i.e., A/O/W and W/O/W. Theeformation of oil drops (and later recovery of their shape) actss one of the mechanisms that produce W/O/W compoundtructures. Moreover, if any local region in the system displaysgradient of pressure that deforms the oil drops in such aay that they face a water flow specifically oriented relative

o said gradient, these compound structures may be formed.he presence of biomass (T. harzianum) as a solid phase led to

he inclusion into oil drops (A/O/W) of 61% of the air bubbleshat impacted them, while in other systems such as an aque-us medium containing protein or a surfactant, the inclusionf air into oil was only observed in about 6% of the impacts.his constitutes a first step to a deeper understanding of theomplex inclusion phenomenon of water droplets and air bub-les in the oil drops, which in turn will help to understand theechanisms of oxygen transfer in multiphase processes such

s those occurring in fermentations.

cknowledgements

his work was supported in part by CONACyT (grant 129676).upport was also provided to A. Rojas by the Nationalutonomous University of Mexico (UNAM), through its post-octoral scholarships program. The authors acknowledge withhanks M.S. Córdova and J. Iguiniz for their helpful collabora-ion in some experiments, D. Cuervo for providing the fungaliomass used in this work and A. Ocadiz for computationalssistance.

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