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Experimental Thermal and Fluid Science 32 (2007) 375–386

Effect of proteins on calcium phosphate deposition in turbulent flowas a function of surface properties

R. Rosmaninho *, L.F. Melo

LEPAE, Departamento de Engenharia Quımica, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal

Received 8 January 2007; received in revised form 26 April 2007; accepted 26 April 2007

Abstract

In order to reduce milk fouling during heat treatment, several approaches have been evaluated and one of them, the modification ofthe surface properties of the stainless steel used in heat exchangers, is analysed in this work. Fouling behaviour of stainless steel surfaces(2R) and modified stainless steel-based surfaces obtained by TiN sputtering was analysed. The combined effect of b-lactoglobulin and thesurface energy properties of the materials on the deposition behaviour of calcium phosphate was evaluated. The experiments were per-formed in turbulent flow regime in a heat flow cell where the fouling thermal resistance was continuously monitored. The presence ofprotein was responsible for changing the overall fouling curve independently of the surface nature, as well as for the appearance oftwo distinct growth periods. The second growth phase occurred after different delay times depending on the roughness, surface compo-sition and surface energy values of the deposition material. The resistance to cleaning of the different deposits was also evaluated and itwas proved to be dependent on the surface energy parameters, probably as a result of the formation of different deposit structures. Themain conclusion is that when protein is present, the non-modified 2R surfaces present the lowest amount of deposit, but the higher energysurfaces are the ones to retain less amount of deposit after the cleaning process.� 2007 Elsevier Inc. All rights reserved.

Keywords: Fouling; Calcium phosphate; Whey protein; Surface energy; Modified stainless steel surfaces

1. Introduction

The present work is focussed on the effect of whey pro-teins on the fouling behaviour of calcium phosphate underturbulent flow on different stainless steel modified surfaces.The importance of this study emerges from the fact that thedeposition of activated proteins and calcium phosphate arethe two major mechanisms of fouling during the heat treat-ment of milk, which results respectively from protein dena-turation and from the decrease of calcium phosphatesolubilization upon heating. Calcium ions, however, notonly contribute to the process through calcium salts depo-sition but they also influence the denaturation and aggrega-tion of the proteins prior to deposition, following a process

0894-1777/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.expthermflusci.2007.04.012

* Corresponding author. Tel.: +351 22 5081685; fax: +351 22 5081449.E-mail address: roxane@fe.up.pt (R. Rosmaninho).

not yet totally understood [1,2]. Considerable discussionhas been happening on whether fouling is primarily deter-mined by reactions in the bulk or at the heated surface. Infact, Belmar-Beiny et al. [3] outlined two simple modelsbased on wall and bulk reactions and showed that phenom-ena in the bulk are the ones to control the process. Morerecent work from Morison and Tie on milk simulatingsolutions [4] proved on the other hand that fouling ismainly caused by surface reactions of both calcium phos-phate and protein.

In the initial stages of milk processing, individual wheyprotein molecules are adsorbed onto the stainless steelheating surface covering most of the surface with a proteinmonolayer, after which the deposition of aggregatesformed in the bulk, both as calcium phosphate and as wheyprotein particles, occurs [5]. Consequently, it seems thatproteins are the ones to start the fouling process, asdefended by Morison and Tie [4] and by Belmar-Beiny

Thermocouplesconnection to computer

Flow Cell

OILOIL

econnection to computer

Flow Cell

OILOIL

Nomenclature

U overall heat transfer coefficient (W/m2 K)Rf fouling resistance (m2 K/W)cTOT total solid surface energy (mJ/m2)cLW Lifshitz–van der Waals component (mJ/m2)cAB acid–base component (mJ/m2)c� electron donor component (mJ/m2)

c+ electron acceptor component (mJ/m2)SMUF simulated milk ultrafiltrateMOD-SMUF modified simulated milk ultrafiltrateTiN titanium nitride

376 R. Rosmaninho, L.F. Melo / Experimental Thermal and Fluid Science 32 (2007) 375–386

and Fryer [6], who stated that the modification of the sur-face properties could be responsible for changing the num-ber and type of bonds between the protein and the surface.In the bulk, calcium ions promote b-lactoglobulin aggrega-tion and stabilization by binding between adjacent car-boxyl groups and, consequently, the presence of wheyprotein in solution will tend to retard the crystallizationprocess of calcium phosphate [7]. Once on the surface,the calcium ions form bridges between the first adsorbedproteins and the protein aggregates formed in the solution,thereby promoting the subsequent fouling stages. In somestudies however the addition of calcium seemed to decreasefouling caused by a whey protein concentrate which evenbecame negligible for some temperature ranges [8]. Theseapparently contradictory findings suggest that the interfer-ences between protein and mineral components dependalso on the stage of fouling considered.

The formation of the first protein deposition layer isknown to be affected by several factors, related to boththe nature of the heating surface and the bulk solutionproperties. The influence of surface characteristics on wheyprotein adsorption was studied by Santos et al. [9] and pre-viously by Adesso and Lund [10] but even if the surface wasconsidered an important parameter in every case, no directrelationship was found between surface energy parametersand the adsorption process. One way of obtaining a betterunderstanding of the overall fouling process in milk is tocontinuously monitor the formation of protein layers whencalcium phosphate is present in solution. An understandingon how the two components are affected by the surfaceproperties can be a significant step forward in the searchfor a surface able to reduce fouling in dairy processingequipment. The present paper focus on both the build-upand the cleaning of calcium phosphate–whey proteindeposits on modified stainless steel surfaces (in turbulentflow conditions), as well as on the interactions betweenthe two fouling agents and the surface properties.

Rotameter

SMUF/MOD SMUF

Rotameter

SMUF/MOD SMUF

Fig. 1. Schematic diagram of the heat flow cell.

2. Materials and methods

2.1. Fouling equipment – Heat flow cell

In the present work, flow equipment was used to evalu-ate the fouling process on the different surfaces in turbulentregime. The total setup is schematically presented in Fig. 1.

The fouling equipment was composed of: (a) a well stirredstainless steel tank of 120 l containing the foulant solutions(see Section 2.2) and kept at constant temperature by aheat resistance with an On–Off temperature controller(with derivative-proportional control); (b) a smaller tankwhich was used for heating and holding the oil used asheating medium; (c) a data acquisition board with a sensorinterface card (UPC 2100, Valiyne Engineering) connectedto a computer operating with a Labtech program (Mea-surement Computing Corporation) for temperature acqui-sition, processing and registration; and (d) the verticalfouling test section (flow cell) where the different surfaceswere placed and where the overall heat transfer coefficientwas measured (Fig. 2).

In the fouling experiments, the foulant solutions werepumped through the Flow Cell to generate fouling andreturned to the tank. For the cleaning experiments, a sim-ilar procedure was followed but using water instead of thesolutions. The 120 l tank was also used to hold the waterduring the cleaning experiments. The flow velocity wascontrolled and adjusted by a calibrated rotameter. Theoil, used as heating medium, was pumped through thebackside of the fouling test section and recycled to the oiltank.

Fig. 2. Detailed picture of the fouling test section (flow cell): (a) cross-section and (b) transversal view.

R. Rosmaninho, L.F. Melo / Experimental Thermal and Fluid Science 32 (2007) 375–386 377

The flow cell cross-section is semi-circular with a diam-eter of 0.03 m. The total length of the flow cell is 1.53 mand the fouling measuring section is placed 1.0 m afterthe entrance to allow complete boundary layer develop-ment for turbulent conditions [11]. The stainless steel rect-angular samples are imbedded in a stainless steel platefacing the fluid flow and sufficiently separated from eachother to avoid possible interferences. Three equal sampleswere tested each time in order to have the results in tripli-cate (Fig. 2b). For each sample, two pairs of thermocouples(Type E, Omega Engineering, Inc.) were inserted in thestainless steel plate in two different locations (two pairswere used in order to have the measurements in duplicate,the value considered being the average of the temperaturesat the same position in the heat transfer direction)(Fig. 2b), which allowed the determination of the tempera-ture along the plate (Fig. 2a). The fluid temperature in theflow cell was measured by two thermocouples (Type E,Omega Engineering, Inc.), one at the beginning of the mea-suring section and another one at the end; once more, thesolution temperature considered in the calculations wasthe average of these two values.

2.1.1. Overall heat transfer coefficient (U) and fouling

resistance (Rf)

Deposit formation on a surface can be monitored by fol-lowing the overall heat transfer coefficient (U) which is ameasure of the heat transferred between two fluids at differ-ent temperatures, through a wall. It is calculated as follows(1):

U ¼ kxðT 3 � T 2ÞðT 3 � T 1Þ

ð1Þ

k is the thermal conductivity of the wall, x the distance be-tween the two thermocouples embedded in the wall, whose

temperatures are T2 and T3, and T1 is the temperature ofthe foulant solution (see Fig. 2a).

As the fouling layer builds up, the fouling resistance (Rf)of the deposit increases and can be calculated as the differ-ence between the overall thermal resistance of the fouledsurface 1

U

� �and the overall thermal resistance of the ini-

tially clean surface 1U0

� �, according to Eq. (2):

Rf ¼1

U� 1

U 0

ð2Þ

The fouling process can then be represented by the evo-lution of Rf with time.

When the flow rate is kept constant along a channel, thepressure drop across the fouled section tends to increasedue to the deposit’s roughness. Both the fouling depositand the friction factor can be estimated by simultaneouslymonitoring the heat transfer coefficient and the pressuredrop across the measurement section [12]. In the presentcase, the increase in pressure drop measured between thebeginning and the end of the fouling test section was verysmall and consequently neglected. Therefore, only the heattransfer measurements were considered for fouling resis-tance calculations.

2.2. Calcium phosphate with and without milk proteins

Fouling experiments were made using SMUF (Simu-lated Milk Ultra Filtrate), which is an aqueous solutionthat has been used to study the deposition of calcium phos-phate under milk simulating conditions. It was preparedaccording to Jenness and Koops [13] and kept overnightat 5 �C for stabilization before use. The pH of the solutionwas adjusted, prior to use, to 6.80 using dilute NaOH. Tostudy the effect of whey protein on the deposition behav-iour of calcium phosphate, another aqueous solution (here

378 R. Rosmaninho, L.F. Melo / Experimental Thermal and Fluid Science 32 (2007) 375–386

called as Modified SMUF or ‘‘MOD-SMUF’’) was alsoprepared. To prepare MOD-SMUF, a commercial WPI(Whey Protein Isolate, Lacprodan DI-9224) provided byArla Foods (Arla Foods, Ingredients, Denmark) wasadded to the SMUF solution in order to obtain 3 g/l ofb-lactoglobulin (b-lg). The pH of this last solution was alsoadjusted to 6.80 before use.

2.3. Surface fouling materials

Stainless steel is the most used material on food treat-ment equipment and consequently stainless steel-based sur-faces will be tested as deposition supports for bothsolutions. Two types of surfaces were used: (1) non-modi-fied 316 stainless steel surfaces with bright annealing, coldrolled finish (2R) and (2) modified 316 stainless steel sur-faces obtained by reactive sputtering coating with TiN.Because this is a coating technique, all the surfaces becamecovered by an analogous layer of TiN which differed onlyon the surface energy from sample to sample. The surfaceswere named TiN 1, TiN 2 and TiN 3. All surfaces had15 · 60 · 1 mm as dimensions.

2.3.1. Surface’s cleaning proceduresBefore each deposition experiment, as well as before the

determination of the surface energy of each sample, allstainless steel samples were cleaned with a commercialdetergent (RBS35 from RBS Chemical Products) accordingto the following procedure: (1) samples were immersed in a2.0% w/v detergent (RBS35) solution in distilled water at65 �C for 5 min; (2) rinsed with distilled water at 65 �Cfor 5 min and (3) rinsed with distilled water at 20 �C. Boththe measurements and the experiments were performed24 h after the cleaning treatment.

2.4. Surface characterization

2.4.1. Surface energy characterization

Contact angle values were measured by the sessile dropmethod in a contact angle meter (DataPhysics OCA15Plus, Germany) using water, formamide and a-bromo-naphthalene as reference liquids. The contact angles weremeasured automatically using an image analyzing system.Contact angle measurements were performed on everystainless steel sample. At least 15 measurements with eachliquid were performed covering the whole sample surface.

The surface energy values for each surface was deter-mined according to van Oss [14] and the Lewis acid–basetheory. This theory evaluates the total surface energy of asolid (cTOT) as the sum of an apolar Lifshitz–van der Waalscomponent (cLW) and an acid–base polar component (cAB):

cTOT ¼ cLW þ cAB ð3Þ

The last one, cAB, composed by two non-additiveparameters, one for the electron donor component (c�)and one for the electron acceptor (c+) as follows [14]:

cAB ¼ 2ffiffiffiffiffiffiffiffiffifficþc�

pð4Þ

2.5. Deposition and cleaning experiments

The deposition experiments presented in this work canbe divided into two stages: (1) evaluation of the overallheat transfer coefficient with the clean surface. This stagewas performed with water at the deposition conditions(same temperature, heat flux and fluid velocity) and (2)monitoring the change in the overall heat transfer coeffi-cient during the fouling process. All experiments were car-ried out at a constant temperature of 50 �C correspondingto the beginning of bulk precipitation of calcium phos-phate/protein aggregates. The heating medium used inthe experiments was oil at a constant temperature of70 �C which created an average temperature differencebetween the deposition surface and the solution of 1.5 �C(measured during the experiments with clean water). Sincethe mass concentration of minerals in the solutions is assmall as 0.8%, their physical properties can be consideredidentical to the ones of water at the same temperature.The deposition experiments were performed at a Reynoldsnumber of 6256 (flow velocity of 0.20 m/s in the test sec-tion) while the cleaning experiments were performed atRe = 6256 (flow velocity 0.2 m/s) and Re = 11232 (flowvelocity 0.36 m/s). The ambient temperature in the labora-tory was controlled by air conditioning to a constant valueof 25 �C in all and throughout the experiments.

The solutions (SMUF and MOD-SMUF) were preparedin the tank and kept at a constant temperature and agita-tion throughout the whole experiment. The pH of bothsolutions was adjusted to 6.80 (WTW inolab pH level1) inthe beginning of the experiments and was allowed to varyalong the experiment as a consequence of the aggregatesformation and/or transformation. Along the experiment,bulk liquid samples were collected and vacuum filteredusing a 0.45 lm filter to analyse the particulate materialformed and determine its concentration. The pH value ofthe collected samples was measured to evaluate the progres-sion of the reactions in the bulk. The free calcium concen-tration in the bulk during the deposition tests was alsomeasured and registered (WTW-D82362 Weitheim).

The cleanability of the different deposits was alsoassessed. The cleaning process was accomplished with wateralso at 50 �C and was composed by two stages since two dif-ferent Reynolds numbers were successively applied to eachdeposit. At the first stage, the same Reynolds number usedduring deposition was used in order to assess the cleanabil-ity of the deposits due solely to the absence of foulant mate-rial in the fluid. The second one was accomplished by usinga Reynolds number of approximately double to evaluate theresistance of the deposits to detachment under higher stres-ses. For this process, the foulant solutions were removedfrom the container and replaced by water when a stablefouling layer was formed (detected by an approximatelyconstant fouling resistance value). This was performed

R. Rosmaninho, L.F. Melo / Experimental Thermal and Fluid Science 32 (2007) 375–386 379

without significant stopping periods since the flowing fluidwas immediately switched from the fouling solutions towater. The cleaning process proceeded with water at Rey-nolds of 6256 for approximately 24 h, after which the Rey-nolds was increased to 11232 and kept for another 24 happroximately.

2.6. Characterization e quantification of the deposit

After the cleaning steps, the flow cell was dismantledand the samples were removed. The amount of depositformed on each sample was determined by weighing thesamples using an analytical balance (AND GR-200,AND Engineering, California, USA), before and after amild drying process in air, in order to obtain respectively,the deposit’s wet and dry mass.

The distribution of the SMUF and MOD-SMUF depos-its throughout the surface and their structure were charac-terized by scanning electron microscopy observation(JEOL JSM6301F, Japan) and their chemical compositionwas assessed by X-ray microanalysis (Noran Voyager,Noran Instruments Inc., USA).

3. Results and discussion

3.1. Surface characterization by contact angle analysis

All the modified surfaces tested in this work were pro-duced by using the same technique under varying opera-tional parameters and consequently, the different TiNsputtered surfaces had a similar morphology and surfacecomposition (although with varying proportions of Tiand N) but different surface energy properties. These sur-faces could then be distinguished and characterized basedon their surface energy values, more precisely its electrondonor component (c�) as explained in previous work [15]and as shown in Table 1.

3.2. Deposition process of calcium phosphate with protein

on non-modified surfaces

To assess the repeatability of the fouling experimentsand the reliability of the measuring method, three trials(2R 1, 2R 2 and 2R 3) were performed on 2R surfaces(three samples were used in each trial). The three obtainedfouling curves (each one representing the average values forthe three surfaces tested in each experiment) are presentedin Fig. 3. The reproducibility of the measurements wasquite satisfactory, mainly for 2R 1 and 2R 3, and the over-all evolution of the fouling resistances (Rf) was similar for

Table 1Surface energy components for 2R non-modified surfaces and all TiNsputtered surfaces (standard deviation in brackets)

Surface TiN 1 TiN 25 TiN 3 2R

c� (mJ/m2) 55.3 (0.0) 23.0 (1.8) 46.2 (4.6) 39.0 (3.1)

the three experiments. Some differences in the Rf value werefound for the 2R 2 sample which might be related to pos-sible differences in the bulk liquid but the overall evolutionshape can be considered similar to the other two experi-ments.

The two different stages of the cleaning process can beeasily identified on the curves since the shift to a higherflow velocity caused a significant decrease in the Rf value.For the 2R 2 samples, a large decrease in the Rf valuewas immediately observed at the beginning of the cleaningprocess (first stage). That decrease was caused by an oper-ational problem when switching from the foulant solutionto water which provoked the removal of almost all theweakly attached upper layer of the deposit. This alsoexplains why no more deposit could be removed whenthe water velocity was increased in the second cleaningstage.

The dry mass and the fouling resistance of the depositsafter the cleaning process are presented in Fig. 4, showingthat Rf increases with the deposit mass.

3.3. Comparison between the fouling behaviour of calcium

phosphate with and without proteins on non-modifiedsurfaces

The deposition behaviour of the two different foulantsolutions on non-modified 2R surfaces was followed andtheir deposition curves are presented in Fig. 5. As can beseen, the deposit development in the presence of protein(MOD-SMUF) and in the absence of protein (SMUF) isclearly different.

When only minerals are present in solution, the foulingcurve increases smoothly during the whole time period con-sidered here. When protein is present the fouling starts tobe very similar to the one of SMUF but the fouling curveseems to stabilize after approximately 10 h of deposition,suffering a huge increase in the amount of fouling afteraround 20 h with a trend to a new stabilization period. Thisdifference in the fouling behaviour due to the presence ofprotein in solution was expected based on previous workfrom the authors under laminar regime [16]. It seemed,from the comparison between the two curves, that afteran initial deposition, the main calcium phosphate depositformation was delayed by the presence of protein.

This diversity in behaviour enhanced the interest inanalysing the fouling behaviour of the MOD-SMUF solu-tion on the different TiN surfaces in order to evaluate howthe surface energy of the deposition material could affectthe type of deposit build-up and its resistance to cleaning.

To better understand the differences in the depositsformed for SMUF and MOD-SMUF, both of them wereobserved under the scanning electron microscope (Figs. 6and 7). A calcium phosphate-based deposit with very sim-ilar individual structures were observed in the two cases(Figs. 6b and 7b), but the aggregates containing proteinwere less defined and presented a more continuous struc-ture than the ones without protein. In the MOD-SMUF

0.0E+00

1.0E-04

2.0E-04

3.0E-04

4.0E-04

5.0E-04

6.0E-04

7.0E-04

8.0E-04

0 10 20 30 40 50 60 70 80 90 100time (h)

Rf(m

2 K/W

)

2R1

2R2

2R3

fouling period cleaning period

velocityincrease

Fig. 3. Fouling curve for three trials on 2R surfaces, including the fouling and the cleaning period.

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

0.0E+00 1.0E-04 2.0E-04 3.0E-04 4.0E-04 5.0E-04 6.0E-04

Rf (m2K/W)

depo

sit d

ry m

ass

(mg)

Fig. 4. Relationship between the fouling resistance values for the three experiments on 2R surfaces (Rf) and the dry mass of the deposits.

0.0E+00

1.0E-04

2.0E-04

3.0E-04

4.0E-04

5.0E-04

6.0E-04

7.0E-04

8.0E-04

0 10 20 30 40 50time (h)

Rf (

m2 K

/W)

SMUF 2R MOD SMUF 2R

Fig. 5. Different fouling behaviour of SMUF and MOD-SMUF on 2R (non-modified) surfaces.

380 R. Rosmaninho, L.F. Melo / Experimental Thermal and Fluid Science 32 (2007) 375–386

Fig. 6. Deposition on 2R surfaces from SMUF solution: (a) aggregates distribution; (b) calcium phosphate structure.

Fig. 7. Deposition on 2R surfaces from MOD-SMUF solution: (a) aggregates distribution; (b) calcium phosphate/protein structure.

R. Rosmaninho, L.F. Melo / Experimental Thermal and Fluid Science 32 (2007) 375–386 381

case, it is possible to see some connecting structures withinthe calcium phosphate aggregates than can be responsiblefor a more consistently ‘‘glued’’ arrangement. In a macro-scopic observation, the differences between them are morevisible since the aggregates formed when protein was pres-ent (Fig. 7a) were much smaller and also displayed a morecontinuous spreading throughout the surface than whenonly minerals were present (Fig. 6a).

Nevertheless, X-ray microanalyses from both aggregatesshowed that their composition was mainly calcium phos-phate of similar type.

3.3.1. Evolution of bulk properties

The pH and the free calcium concentration in the bulksolution were monitored during the whole duration of theexperiments (Figs. 8 and 9). The evolutions of these twoparameters allowed to monitor the progressing of the reac-tions in the bulk as well as to compare the experiments inan easier way.

The curves are visibly different from each other indicat-ing differences in the bulk reactions occurring in each case.In the MOD-SMUF solution, the pH suffered two bigincreases, the first after around 5h of deposition and thesecond one after approximately 15 h (Fig. 8). In the sametime periods, a decrease in the free calcium concentrationin the solution was observed (Fig. 9). Such differences ledto the conclusion that for the MOD-SMUF solution thefouling process was composed by two distinct stages, eachone resulting in an increase of the pH and a decrease in the

free calcium concentration. These changes in both pH andcalcium concentration in the bulk solution were similar inall MOD-SMUF experiments, varying slightly in the timescale by no more than around one hour forward andbackward.

3.4. Fouling behaviour of MOD-SMUF on the different

surfaces

All the surfaces were subjected to fouling experimentswith MOD-SMUF solutions and the correspondentdeposit formation versus time is presented in Fig. 10.

The experiment on the surface TiN 3 is only presenteduntil around 18h of deposition since at that time, the heat-ing system had a failure and consequently the results afterthat moment could not be considered for analysis. How-ever, until that moment the surface was presenting a satis-factory behaviour and consequently is here considered forthe evaluation of the initial deposition behaviour.

The fouling behaviour of MOD-SMUF on the non-modified stainless steel surface (2R) can clearly be distin-guished from the one of the modified TiN surfaces. Onthe 2R surface, the first stabilization value was lower thanthe one for the TiN surfaces and the second increase infouling occured much later, with a second stabilizationvalue for the Rf also much lower, indicating the presenceof less deposit. One of the possible explanations for thisdistinction between the two types of surfaces can be relatedwith the fact that surfaces obtained by magnetic sputtering

6.6

6.8

7

7.2

7.4

7.6

7.8

8

8.2

0 10 15 20 25 30 35 40 45 50 55time (h)

pH

MOD SMUF

SMUF

5

Fig. 8. pH evolution of the SMUF and MOD-SMUF solutions along the experiments.

0

50

100

150

200

250

300

350

400

450

0 10 15 20 25 30 35 40 45 50 55time (h)

Cal

cium

in S

olut

ion

(mg/

L)

MOD SMUF

SMUF

5

Fig. 9. Concentration of calcium in solution along the experiments for SMUF and MOD-SMUF solutions.

0.0E+00

2.0E-04

4.0E-04

6.0E-04

8.0E-04

1.0E-03

1.2E-03

1.4E-03

1.6E-03

0 10 15 20 25 30time (h)

Rf (

m2 K

/W)

2R TiN 1 TiN 2 TiN 3

5

Fig. 10. Fouling behaviour of the MOD-SMUF on the different surfaces during the deposition period.

382 R. Rosmaninho, L.F. Melo / Experimental Thermal and Fluid Science 32 (2007) 375–386

Table 2Delay times and Rf plateau values for the first and second stages

Surface Delay time(h)

Rf first stage(m2 K/W)

Rf second stage(m2 K/W)

2R 21.5 1.4E�04 5.4E�04TiN 1 17.3 4.1E�04 1.2E�03TiN 2 10.2 3.1E�04 1.4E�03TiN 3 13.3 3.4E�04 –

R. Rosmaninho, L.F. Melo / Experimental Thermal and Fluid Science 32 (2007) 375–386 383

techniques tend to present higher surface roughness thanthe original stainless steel ones [17], and this parameterhas been shown to have great importance on fouling causedboth by proteins and calcium phosphate/proteins aggre-gates [2].

The first stabilization value was achieved after approxi-mately the same time for every surface. However, the sec-ond increase in deposit started at different momentsdepending on the surface. That second increase shouldmainly be caused by calcium phosphate deposit develop-ment which was delayed because of the presence of protein(to be explained later) and it will be called throughout thispaper as ‘‘delay time’’. For each surface, the ‘‘delay time’’was determined as being the time at which the secondgrowth period started, as presented for the 2R and TiN 2surfaces on Fig. 11. As a consequence of different ‘‘delaytimes’’, different new stabilization plateaus for the Rf valueswere observed for each surface.

The values for the ‘‘delay times’’ and the Rf values of thetwo stages for each surface are presented in Table 2.

The ‘‘delay time’’ for the lower surface energy TiN 2 (c�

of 23) was smaller than the one for the medium surfaceenergy TiN 3 (c� of 46) and even smaller than for thehigher surface energy TiN 1 (c� of 55) but any o themmuch smaller than for 2R surface (c� of 39), validatingthe statement that the fouling behaviour of calcium phos-phate/protein aggregates is greatly dependent on the sur-face properties (simultaneously roughness and surfaceenergy). The explanation for this different behaviour maylie on studies presented so far on the effect of proteins oncalcium phosphate deposition together with the effect oftitanium-based surfaces on the deposition process [18]although if most of those studies were done with bovineserum albumin (BSA). Other researchers showed that theadsorption of BSA has a modifying effect on the precipita-tion and crystallization of calcium phosphate. They alsoshowed that the structure and the properties of the

0.0E+00

2.0E-04

4.0E-04

6.0E-04

8.0E-04

1.0E-03

1.2E-03

1.4E-03

1.6E-03

0 10

Rf (

m2 K

/W)

5

Fig. 11. Fouling behaviour of the MOD-SMUF on the different surfaces duristage values (two illustrative cases).

adsorbed protein layers were influenced by the depositionsurface nature. Titanium (Ti) based surfaces affected theconformation of the BSA layers controlling the subsequentdeposition structure of calcium phosphate [18–20]. Adsorp-tion studies of BSA and calcium phosphate on TiO2 wereperformed in order to understand the role played by thedifferent conformations of the protein on the calcium phos-phate mineralization process. They showed that the proteinonly undergoes structural rearrangements until the surfaceis totally covered by one monolayer. After that, the adsorp-tion should be mainly controlled by electrostatic interac-tions with the surface and, consequently, affected by thesurface energy of the deposition material since the affinityof proteins is accepted to be higher to hydrophobic surfaces[19–21]. It is possible that depending on the surface, thechange in conformation of the proteins and its degree ofcoverage of the surface is different, which may explainthe difference in behaviour found in this work. However,independently of the surface considered the presence ofBSA retarded the transformation process of calcium phos-phate and it was suggested that the retardation on thetransformation rate is due to BSA shielding of the Ca2+

and PO�34 sites on the growing calcium phosphate [22]. Pro-

tein and calcium phosphate can coexist in the fouling layersince the protein was found to delay the growth of the min-eral part but not to inhibit its adhesion to the titanium sub-strata [23]. The explanation for the delay observed on thefouling curves obtained in this work can be based on the

15 20 25 30time (h)

2R TiN 2

ng the deposition period: determination of ‘‘delay time’’ and the different

Fig. 12. Deposition on 2R surfaces from MOD-SMUF solution: boundary space between aggregates after the first stage deposition.

384 R. Rosmaninho, L.F. Melo / Experimental Thermal and Fluid Science 32 (2007) 375–386

previously presented findings mainly since a similar behav-iour on the bulk retardation effect of the calcium phosphatecrystallization process was described by Tsuge et al. [7] inthe presence of whey protein.

The theory proposed to explain those curves can there-fore be the following: whey protein starts spreadingthroughout the surface, in a parallel process with calciumphosphate nucleation, forming the first detectable foulinglayer (corresponding to the first stage); subsequently, ontop of this layer calcium phosphate will continue to growas can be seen in Fig. 12 (image taken in the boundariesspaces between the aggregates presented in Fig. 7a).

The development of the first protein layer is dependenton the properties of the surfaces since the affinity of theproteins towards them is dependent on their surfaceenergy and consequently the available space left for thedevelopment of calcium phosphate-based deposits willalso be different from surface to surface. This will affectthe amount of deposit which will be formed. Similar dif-ferences on the deposits developed from protein and cal-

0.0E+00

2.0E-04

4.0E-04

6.0E-04

8.0E-04

1.0E-03

1.2E-03

1.4E-03

1.6E-03

30 35 40 45 50t

Rf (m

2 K/W

)

1st cleaning period

Fig. 13. Cleaning behaviour of the different surfaces when submitted t

cium phosphate were previously found by the authorson other modified surfaces but under laminar regime[16] in a rotating disk device. The different tendencies tobuild-up deposits can explain the different values obtainedfor Rf values for the first layer in each case. Being so andregarding the modified TiN surfaces, the lower surfaceenergy TiN 2 presented less deposit (smaller Rf) and thehighest surface energy TiN 1 presented more deposit(higher Rf). The average value obtained for the mediumsurface energy TiN 3 lied somewhere between the othertwo surfaces.

These layers will differ not only on the amount ofdeposit but also on the deposit structure and probably onthe arrangement of the proteins (even if the global aggre-gates distribution seemed similar macroscopically andunder small amplifications). That would explain the differ-ent effect on the retardation of the second growth periodand, consequently, the dependency of the ‘‘delay time’’on the surface energy. The different structures developedon each surface can therefore explain the fact that the final

55 60 65 70 75ime (h)

2R TiN 1 TiN 2

2nd cleaning period

o cleaning with water at two different values of Reynolds number.

R. Rosmaninho, L.F. Melo / Experimental Thermal and Fluid Science 32 (2007) 375–386 385

amount of deposit was also dependent on the surfaceenergy parameters of the supporting material.

3.5. Cleaning behaviour on the different surfaces

The cleaning behaviour of the deposits on the differentsurfaces is presented in Fig. 13.

Both the amount of deposit remaining on each surfaceand the behaviour during cleaning were different dependingon the surface, which indicates that deposits with differentstructure were in fact formed. Deposits formed on TiN 1surfaces were more sensitive to removal since the largestpart of the deposit was immediately removed at the firststage of the cleaning process. Opposed to that, the depositdeveloped on the lower surface energy TiN 2 showed differ-ent removal resistance: only a small part of the deposit wasremoved at the first stage of cleaning and only a little moremass was removed afterwards (in the second stage withhigher Reynolds number). As it happened during the depo-sition process, the 2R surface behaved differently also inthe cleaning process. In this case, hardly any deposit wasremoved during the whole cleaning period.

4. Conclusions

The effect of the presence of whey protein (b-lg) on thefouling behaviour of calcium phosphate from a milk simu-lating solution was evaluated in this work, mainly consid-ering its dependence on the surface energy of differentmodified surfaces used as deposition materials.

The deposition curves obtained in the presence andabsence of protein were considerably different and twogrowth periods were observed at different times when thewhey protein was present. The ‘‘delay time’’ after whichthe second growth period happened was proved to be depen-dent on the type of surface where fouling developed on, moreprecisely on its roughness, surface composition and surfaceenergy values. The most striking difference was found forthe non-modified 2R surface which presented the highest‘‘delay time’’ when compared to all the other TiN surfaces.Within the TiN modified surfaces, however, the ‘‘delay time’’was dependent on the energy of the surface: lower surfaceenergies showed smaller ‘‘delay times’’ and higher energysurfaces showed bigger ‘‘delay times’’. Also dependent onthe surfaces were the amounts of deposit formed in the firstand the second stage of deposition. As an outcome of thosedifferent dependences, different structures were formeddepending on the surface energy, resulting into differentresistance to removal (cleaning) of the different deposits.Consequently, after the cleaning process the higher energysurfaces were the ones to retain less amount of deposit.

Acknowledgement

The authors gratefully acknowledge the financial sup-port of the FCT (National Science Foundation) throughthe PhD Grant (SFRH/BD/13180/2003).

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