Continuous foam fractionation: Performance as a function of operating variables

Separation and Purification Technology 82 (2011) 10–18

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Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur

Continuous foam fractionation: Performance as a function of operating variables J. Merz a, B. Burghoff a, H. Zorn b, G. Schembecker a,⇑ a b

Technische Universität Dortmund, Emil-Figge-Str. 70, 44227 Dortmund, Germany Institut für Lebensmittelchemie und Lebensmittelbiotechnologie, Justus-Liebig-Universität Gießen, Heinrich-Buff-Ring 58, 35392 Gießen, Germany

a r t i c l e

i n f o

Article history: Received 14 June 2011 Received in revised form 13 July 2011 Accepted 16 July 2011 Available online 22 July 2011 Keywords: Continuous foam fractionation Cutinase Stripping mode Design of Experiments Factor interaction

a b s t r a c t In biotechnological processes, foaming often represents an undesired complication. Foam may induce cell death, and may limit the performance of down-stream processing steps. On the other hand, the differences in foamability of compounds can be used for separation of these substances. The process based on this principle is called foam fractionation, and has been subject of increased attention in recent years. Foam fractionation was employed for several separation tasks, for instance, the concentration of plant secondary metabolites. However, very few studies dealt with the systematic determination of significant operating parameters and their influence on the efficiency of continuous foam fractionation processes only. In this study, the influence of process parameters, like pH value or gas flow rate, were investigated on continuous foam fractionation of a fungal cutinase in stripping mode. Therefore, a Design of Experiments (DoE) was used to indicate significant parameters and their interactions. After the optimization of the foam fractionation process by means of the DoE, a maximal recovery of 98% active enzyme (enrichment: 5.6) or a maximal enrichment of 9.8 of cutinase (recovery: 79%) could be achieved. Ó 2011 Published by Elsevier B.V.

1. Introduction The foaming tendency caused by amphiphilic or surface-active substances of bioprocesses is normally undesired, and effort is put into the prevention of foam formation especially during downstream processing [1]. However, foaming does not necessarily need to be disadvantageous. Quite contrary to the current opinion, foaming can also be exploited for first recovery steps, rendering the use of anti-foaming agents is unnecessary. A downstream separation technique where foam is actually desired as a separation medium is foam fractionation. The principle of separation is the preferential adsorption of surface-active substances at a gas–liquid interface. Such an interface is generated by aerating the feed solution with a gas, like noble gas, nitrogen, oxygen, or air. The adsorption at the gas–liquid interface has two main effects. First, it lowers the surface tension and enhances bubble formation. Second, the molecules form an elastic film around the bubbles increasing foam stability [2]. The stabilized bubbles leave the liquid phase and stable foam emerges into the foam fractionation column. The enrichment of surface-active molecules in the foam phase is increased due to the backflow of entrained solution by gravitational and capillary forces (drainage). At the same time, more surface-active molecules can adsorb to the surface of rising bubbles and surface-inactive components drain back to the feed solution. The ⇑ Corresponding author. Address: Department of Biochemical and Chemical Engineering, Technische Universität Dortmund, Emil-Figge Str. 70, 44227 Dortmund, Germany. E-mail address: [email protected] (G. Schembecker). 1383-5866/$ - see front matter Ó 2011 Published by Elsevier B.V. doi:10.1016/j.seppur.2011.07.023

enriched foam leaves the column at the top, and is collapsed and collected in a receiving flask. The resulting liquid fraction is rich in the target molecule. The selective separation of different molecules is likely to be a result of differences in their adsorption/ desorption behavior at the gas–liquid interface. Thus, a chromatographic effect occurs inside the foam fractionation column [3] enhancing the separation performance especially of diluted solutions. For this reason foam fractionation is regarded as suitable for the separation and enrichment of dilute protein solutions [3– 5]. Thus, this technique is considered as an option for the early down-stream processing of microbial culture broths, where large volumes of crude starting material have to be handled. Continuous foam fractionation can be run in two different operation modes, namely stripping mode (Fig. 1a) and enriching or simple mode (Fig. 1b) [6]. In enriching mode, the feed enters the column in the liquid pool. Thus, drainage occurs in the foam over the whole foam column. Due to the drainage the excess liquid between the bubbles drains back to the liquid pool of the column resulting in a higher concentration of the target molecule [6]. The disadvantage of this method is that the formation of stable foams is a requirement. In stripping mode, the feed enters the column at the top of the foam column and trickles down through the rising foam (Fig. 1a) [6]. Due to this counter-current stream of foam and fresh feed, bulk surface-active molecules are present in the whole foam phase able to fill otherwise free adsorption places. Thus, stripping mode offers new alternatives for diluted and low surface-active systems which are not able to form stable foams for continuous foam fractionation in enriching mode (Fig. 1b). In literature, stripping mode has rarely

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mycelium of C. cinerea C29-2 was maintained on 2% agar plates with malt extract medium (20 g L1 malt extract). Pre-cultures of C. cinerea were prepared from a mycelium block (1 cm2) as inoculum, which was excised from the agar plate and placed in a 300 mL Erlenmeyer flask containing 100 mL malt extract medium. After treatment with an Ultra Turrax homogenizer (Ika Werke GmbH & CO. KG, Staufen, Germany), the cultures were incubated for 3 days at 150 rpm and 24 °C. For the production of lipolytic enzymes, a mineral salt medium with 0.4% (v/v) Tween 80 as carbon source was used as main culture medium [13]. The main cultures were prepared by inoculating 500 mL Erlenmeyer flasks containing 250 mL medium with 20 mL of the pre-culture. The cultures were incubated at 150 rpm and 24 °C for 3 days. For the foam fractionation experiments, the culture supernatant was separated from the mycelium by filtration under reduced pressure. Media and equipment were autoclaved prior to use, and sterile techniques were applied throughout the procedures. Fig. 1. Continuous foam fractionation column in (a) stripping mode and (b) enriching mode.

been discussed. Talmon and Rubin as well as Kinoshita et al. [7,8] studied the separation of dyes or metals using continuous foam fractionation in stripping mode. Oka et al. and Meikap et al. [9,10] studied the feasibility of counter-current foam fractionation in coiled columns or multistage reactors, respectively. However, the aim of these studies was to prove feasibility and not the systematic determination of important operating variables. To control foam fractionation in stripping mode, the influencing parameters, their interactions, and nonlinearities need to be investigated. To determine the influence of parameters like pH value or feed flow rate on the separation efficiency systematically, Design of Experiments (DoE) was used. Such studies of foam fractionation in stripping mode with the feed port at the top of the foam column involving DoE do not yet exist. The advantages compared to classical’’ one factor at a time experiments’’ are a reduced number of experimental runs and the possibility to determine impact factors as well as factor interactions and nonlinear behavior. As for previous studies [11,12], crude supernatants of submerged cultures of the basidiomycete Coprinopsis cinerea were used as model system for the foam fractionation experiments. 2. Materials and methods 2.1. Chemicals EDTA (P99%), gum Arabic (from acacia tree), magnesium sulfate anhydrous (99%), p-nitrophenyl palmitate (P98%), and sodium deoxycholate (P98%) were purchased from Sigma–Aldrich (Seelze, Germany). Agar–agar (Kobe), ammonium nitrate (P99%), hydrochloric acid (37%), calcium chloride (CaCl22H2O, P99%), copper sulfate (CuSO45H2O, P99%), ferric chloride (FeCl36H2O, P97%), isopropanol (HPLC grade), malt extract (96.5–98%), manganese sulfate (MnSO4H2O, P99%), potassium dihydrogen phosphate (P99%), di-potassium hydrogen phosphate (K2HPO43H2O, P99%), sodium chloride (P99.8%), sodium hydroxide (P99%), disodium hydrogen phosphate (P93%), Tween 80 (ph. Eur.), and zinc sulfate (ZnSO47H2O, P99%) were purchased from Roth (Karlsruhe, Germany). Substances were used as received. 2.2. Culture conditions and enzyme production The fungal strain C. cinerea C29-2 was obtained from the culture collection of the Friedrich Schiller University Jena [11]. The

2.3. Activity assay To quantify the esterolytic activity, a modified assay according to Winkler and Stuckmann with p-nitrophenylpalmitate (pNpp) as substrate was used [14]. The substrate solution was prepared by mixing 10 mL isopropanol and 30 mg pNpp with 90 mL of 0.05 M Soerensen phosphate buffer, pH 7.7, containing 207 mg sodium deoxycholate and 100 mg gum Arabic. The Soerensen phosphate buffer consisted of solution A (8.9 g L1 Na2HPO42H2O) and solution B (6.8 g L1 KH2PO4), which were mixed in a ratio of 7.4:1. A 1 mL aliquot of freshly prepared substrate solution was mixed with 42 lL culture supernatant or water (blank) and incubated for 15 min at 26 °C and 650 rpm. After incubation, the absorbance was measured at 410 nm. One enzyme unit U was defined as one lmol p-nitrophenol enzymatically released from the substrate per minute. Under the conditions described, the extinction coefficient of p-nitrophenol was determined to be e410nm = 0.028 L (lmol cm)1. All samples were analyzed at least as a duplicate. 2.4. Surface tension Surface tension of the culture supernatants was determined using the ring-tensiometer K10ST (Krüss GmbH, Hamburg, Germany). Approximately 20 mL of pH and temperature adjusted culture supernatant was filled into the test vessel, and the surface tension was measured according to the method of Lecomte Du Noüy [15]. Temperature and pH were varied according to the levels of the experimental plan. All samples were analyzed at least as a duplicate. 2.5. Configuration of equipment and experimental procedure for foam fractionation 2.5.1. Setup The foaming device consisted of a liquid column for the feed solution (length = 18 cm, diameter = 3 cm), with a porous glass frit at the bottom, a foam column (length = 56 cm, diameter = 1.6 cm), a horseshoe bend, and a receiving flask. A feed inlet (4.5 cm from top of the foam column) and one outlet for the depleted liquid were placed at the glass column (Fig. 2). To maintain a constant temperature during the experiments, the column was jacketed (temperature adjustment using thermostat; cooling/heating medium H2O). Additionally, the feed solution was preheated to the desired temperature. The experiments were started by pumping enzyme solution into the foam fractionation column till a height of 18 cm from

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Fig. 2. Schematic continuous foam fractionation column used for the experiments.

the bottom, and regulating the feed flow rate according to the experimental design. The liquid height was kept constant throughout the experiments by manually adjusting the speed of the peristaltic pump (Watson Marlow, Rommerskirchen, Germany) used to withdraw the depleted liquid from the bottom of the column. Air as foaming gas was passed through the porous frit. The emerging foam left the column through a horseshoe bend, was collapsed at reduced pressure, and collected in the receiving flask. This foam breaking method is conveniently used in laboratory scale setups and well documented in literature [1,2,12]. Samples were taken for activity assays from the feed solution, the depleted liquid (retentate), and the collapsed foam (foamate). The retentate and foamate samples were taken after the achievement of stationary operation. 2.5.2. Steady state operation Regarding the experimental design, the combinations of the lowest and highest feed flow rate (5 mL min1; 15 mL min1) with the two lowest gas flow rates (28 mL min1; 40 mL min1) were tested, while the other parameters were kept constant (pH 7.0, 25.0 °C, and a frit with a pore size of 40 lm (P3)). Each 5 to 10 min, a sample from the outflowing retentate stream was taken and analyzed over a period of up to 90 min. Steady state operation existed when the enzymatic activity and the flow of the retentate stream were constant. 2.6. Separation performance To describe the separation performance of the continuous foam fractionation the enrichment factor (EC) and the recovery of active enzyme (R) were defined as follows:

EC ¼ R¼

AF AI

V_ F AF  V_ F  100% ¼ EC   100% _ V_ I AI  V I

ð1Þ ð2Þ

In Eqs. (1), (2), AF and AI are the enzymatic activities [U L1] in the foamate and in the feed solution, respectively. V_ F and V_ I are the flow rates of the collapsed foam and the feed solution [mL min1]. For steady state operation where V_ R is constant over time, Eq. (2) can be expressed as follows:

R ¼ EC  ð1  XÞ

ð3Þ

In Eq. (3), X ¼ V_ R =V_ I as reflux ratio with an overall balance of V_ I ¼ V_ F þ V_ R , where V_ R is the flow rate of the retentate [mL min1]. Thus, the recovery rate is expressed with the control and actuating variable. Eq. (3) increases the accuracy of the calculation, because fluid loss due to wetting of the foam breaker and evaporation is taken into account (max. 5% fluid loss was observed). The results of both Eqs. (2) and (3) were in agreement. Thus, the simplification of Eq. (2) is regarded as valid for the experiments investigated.

2.7. Liquid holdup u The liquid holdup in the foam column was measured as a means to characterize the average liquid content of the foam phase. The liquid holdup u should not be used for the optimization of the foam fractionation process but should give explanations in differences of foam structure and drainage behavior within the experimental plan.

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In order to determine the liquid holdup, the whole foam fractionation column including the horseshoe bend was emptied and the foam was collapsed at the end of each experiment. The resulting liquid fraction was named Vtotal liquid. With this value, the liquid volume in the foam Vliquid in foam can be determined by the following Eq. (4):

V liquidinfoam ¼ V totalliquid  V liquidcolumn

ð4Þ

The foam and liquid column volume were constant during all experiments, as the same column was used and the liquid height was kept constant for each experiment. The liquid holdup u is defined according to Eq. (5) as the ratio between the liquid volume in the foam Vliquid in foam and the volume of the foam column Vfoam column:

u¼

V liquidinfoam V foamcolumn

ð5Þ

13

responses in the Design of Experiments. The relative liquid holdup was used to determine the influence of different process parameters on foam wetness only. To quantify the effects representing the relations between the factors and the responses, a multiple linear regression was used [16]. Previously, the orthogonality of the experimental plan was checked, because the factor E, i.e. pore diameter, was face-centered. The examination showed that the plan was orthogonal concerning all factors. An effect had a significant influence on the responses R, EC, or u if exceeding a confidence interval which was higher than the noise value. The confidence intervals were calculated via standard deviation of the center points and Student’s distribution [16]. The noise of the experiments was estimated by three factor interactions. 3. Results 3.1. Configuration of equipment

2.8. Design of Experiments (DoE) For the systematic investigation of continuous foam fractionation, a Design of Experiments was used. An experimental plan was developed in which the input variables (factors) were changed specifically to observe changes in the output variables (responses). Beside the determination of impact factors and interactions, nonlinear relations between the factors and responses were identified using a central composite design [16]. In this study, the parameters pH value, temperature, feed flow rate, gas flow rate, and frit pore size were investigated. These parameters were considered to have influence on adsorption strength and foam stability. Altogether, five factors were varied on five levels using the central composite design (Table 1). The design consisted of a 251 fractional factorial cube, a star and 15 center points, resulting in 41 experiments. The a value was calculated to ±2.19 [16]. In consideration of the adjustability of the used equipment, the values for the factor gas flow rate (D) slightly differed from the calculated data (flow meter: 5 mL steps). In case of the candle frits, the commercial sizes P4 (16 lm), P3 (40 lm), and P2 (100 lm) were used. Different sizes were not available. Thus, the calculated pore size for level 0 could not be realized. The fermentation of C. cinerea for cutinase production was limited to 7 L per week. The broth was stored at 4 °C (stable for 7 days), as freezing decreased the enzymatic activity of cutinase to 60%. To realize the experimental plan, 4 to 6 randomized experiments were arranged in a block. For each block, three center points were performed. Due to this experimental procedure, differences between the fermentation batches could be recognized and interpreted. Additionally, the standard deviation was calculated via the center points. Thus, the natural variation of the feed solution was taken into account. To describe the efficiency and quality of the foam fractionation process, the enrichment factor (EC), the recovery of active enzyme (R), and the liquid holdup (u) were used and represented the

Steady state operation is necessary for collecting reliable data. The variation of feed and gas flow rates resulted in each case in a stable retentate enzyme activity after approximately 40 min, as can be seen in Fig. 3. As a precaution, all foam fractionations were initially run for 60 min. This way, stationary operation was ensured. After 60 min, the receivers for foamate and retentate were changed and the experiments were performed for approximately ten minutes collecting foamate and retentate. 3.2. Design of Experiments The effects of single factors, factor interactions and nonlinearities were calculated and are depicted in Fig. 4a–c regarding EC, R, and u. A positive effect of a single factor X means, that an increase of factor X from level 1 to +1 increases the response. The increase of the pore diameter (E) from 16 lm to 100 lm enhanced EC about 1.2 in average (Fig. 4a). In case that the change from level 1 to +1 causes a negative effect, the response decreased. For instance, the same variation in pore diameter (E) decreased the recovery of active enzyme about 76% in average (Fig. 4). Significant quadratic effects XX indicate a nonlinear behavior of the response in the respective factor range. That means the change of the response value is not linear to the changes of the factor levels made. Thus, the value of the response could achieve a local

Table 1 Factors (A–E) and levels investigated. In brackets: Calculated factor values, which differ from the experimental used factor values.

A B C D E

Factor

Level a

1

0

1

+a

pH value [] Temperature [°C] Feed flow rate [mL min-1] Gas flow rate [mL min1] Pore diameter [lm]

4.8 22.0 5.0 30 (28) 16

6.0 25.0 7.74 40 16

7.0 27.5 10.0 50 40 (58)

8.0 30.0 12.29 60 100

9.2 33.0 15.0 70 (72) 100

Fig. 3. Activity in the retentate over time. (N gas flow rate 28 mL min1, feed flow rate 15 mL min1; j gas flow rate 28 mL min1, feed flow rate 5 mL min1; d gas flow rate 40 mL min1, feed flow rate 15 mL min1;  gas flow rate 40 mL min1, feed flow rate 5 mL min1).

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Fig. 4. Effects for the responses (a) EC, (b) R, and (c) u. Black columns show significant, gray columns insignificant effects. A: pH value, B: temperature, C: feed flow rate, D: gas flow rate, E: pore diameter.

maximum or minimum or be an asymptotic value. Quadratic effects can be significant, while the single effect X does not necessarily need to be significant. For instance, the quadratic effect of the factor gas flow rate (DD) for R was significant, while the single factor D was not (Fig. 4b). A factor interaction XY is the average difference in influence of factor X at levels 1 and +1 of factor Y. If a factor interaction XY is significant, both factors are interdependent. Thus, both factors have to be considered, although their single effects might not be significant. Factor interaction pH and temperature (AB) for the response EC, for instance, was significant, while both single effects were not (Fig. 4a). Significant interactions are important in establishing process variables for the optimization of the foam fractionation performance. As the factor E was face-centered (Section 2.8), the significance of the related quadratic effect had to be considered carefully. A nonlinear relation between factor pore diameter (E) and the response R was observed during the experiments of the statistical plan. Thus, the quadratic effect of E for response R was stated as significant. After identifying significant effects for each response, regression models were formed and depicted in Eqs. (6)–(8).

R ¼ 91:64  37:93  xE  7:81  xD  xD  32:98  xE  xE þ 6:40  xA  xE  4:08  xC  xD  6:58  xC  xE þ 5:28  xD  xE

ð6Þ

EC ¼ 2:15  0:63  xC þ 0:58  xE þ 0:21  xC  xC  0:20  xD  xD 0:17  xA  xB þ 0:22  xA  xC ¼ 0:17  xA  xE 0:25  xB  xD  0:22  xC  xD  0:46  xC  xE þ 0:18  xD  xE

ð7Þ

u ¼ 0:24 þ 0:064  xB þ 0:041  xC þ 0:039  xD  0:107  xE þ 0:078  xC  xC  0:09  xA  xC þ 0:042  xA  xD þ 0:061  xB  xD

ð8Þ

On the basis of the regression models, the performance of each experiment in the investigated range could be predicted. An overview of all possible level combinations for recovery and enrichment factor on basis of the models is displayed in a so called pareto plot (Fig. 5). The factor levels were chosen as discrete and not as continuous values, because not all factors were arbitrary. Fig. 5) shows that recoveries of almost 99% active enzyme (EC = 5.1) are possible. Also a maximal Enrichment factor of 9.4 is predicted, albeit at lower recovery of 82%. The level combinations predicted to aim maximal recovery of active enzyme or maximal enrichment are depicted in Table 2.

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Enrichment factor (EC)

10

15

surface tension is at a minimum at pH 6.0 or 7.0 depending on temperature. This is close to the isoelectric point of the protein cutinase investigated.

8 6

4. Discussion

4

4.1. Statistical analysis of single and quadratic effects

2 0 0

20

40

60

80

100

Recovery of activity (R) Fig. 5. Possible combinations for responses R and EC on basis of the regression models.

The corresponding experiments with factor levels depicted in table 2 yielded an activity recovery of 98% with an enrichment factor of 5.6, or a 9.8-fold concentration of active enzyme with a recovery of 79%. These experimental values show that an accurate prediction can be accomplished by the developed models. To visualize the dependencies between factor interactions, contour plots were generated using the regression models for each response. In the model equations, the factors of the interaction under evaluation were varied only, and all other factors were held constant at the mean level (0). The contour lines are curves of constant values for the respective responses. In Fig. 6, the contour plots for the responses R, EC, and u are shown. Not all factor interactions were significant for each response. Insignificant factor interactions were displayed as blank spaces in Fig. 6. In order to identify operational conditions which show both high enrichment and high recovery, the pareto plot can be used as well (Fig. 5). The highest enrichment (EC = 5.1) at maximum recovery (R = 99%) was found for the level combination depicted in Table 2. Secondary, the contour plots (Fig. 6) were used to predict high recovery (R = 107%) along with high enrichment (EC = 5.0) for the level combination: pH value: 1 (6.0), temperature: 0 (27.5 °C), feed flow rate: 2.19 (5 mL min1), gas flow rate: 0 (50 mL min1), pore diameter: 0 (40 lm). The corresponding experiment yielded 100% recovery with an enrichment factor of 4.8. Also, further level combinations yielded maximum recovery, but were not depicted in Fig. 5. Altogether, the experimental results were in good agreement with the regression models within the factor range investigated. Additionally, no loss of enzymatic activity was observed within the experimental plan. 3.3. Surface tension According to table 1, the surface tension of the culture supernatants of C. cinerea was measured for each combination of the factors pH (A) and temperature (B) (Fig. 7). Fig. 7 shows that the

Table 2 Calculated factor levels for the maximal responses R and EC. Variables

Level

Level value for maximal R

Level

Level value for maximal EC

xA xB xC xD xE

2.19 2.19 2.19 1 1

4.8 33.0 5.0 60.0 16

2.19 2.19 2.19 1 1

4.8 33.0 5.0 60.0 100

4.1.1. Recovery of activity (R) As shown in Fig. 4b), the recovery of cutinase was significantly influenced by the pore diameter of the frit (E). Additionally, the gas flow rate (D) and the pore diameter (E) showed significant quadratic effects, implying nonlinear dependencies. For high recovery, it was postulated that small pore diameters or rather small bubbles were necessary [17–21]. Small bubbles increase the interfacial area available for adsorption and cause high liquid hold-up promoting high enzyme removal from the feed solution and high recovery, respectively. The single effect was negative agreeing with the trends noted before. But also the quadratic effect was significant and various interactions involving the pore diameter existed. Thus, a generalization of a best pore diameter is difficult (Section 4.2). The gas flow rate (D) determines the residence time of the bubbles in the liquid as well as in the foam phase. The higher the gas flow rate is the shorter is the residence time and time for adsorption and drainage, respectively [22]. At the same time, high gas flow rates lead to a larger quantity of gas bubbles, because more pores of the frit were streamed with gas. The larger quantity of bubbles offer more hydrophobic surface for adsorption. Usually, high gas flow rates increase the recovery [17,22,23]. The single effect was not significant here. As well as for the frit pore size (E), the quadratic effect implied nonlinearities over the investigated factor range and several factor interactions existed. Thus, a general statement for a best value is difficult (Section 4.2). 4.1.2. Enrichment factor (EC) According to Fig. 4a), the enrichment factor was mainly affected by the feed flow rate (C) and the pore diameter (E) of the frit. Significant quadratic effects were observed for feed (CC) and gas flow rate (DD). The feed flow rate generally determines the residence time of the feed or rather the residence time of surface-active molecules in the foam fractionation column. In addition, the incoming mass of surface-active components depends on the feed flow. Thus, high feed flow rates lead to low enrichment factors, as the higher mass of surface-active molecules stabilizes the foam, thus increasing the volume of foamate collected. Despite the fact, that more surfaceactive molecules are taken into the foam (adsorbed at the gas– liquid interface and in the lamellae liquid) the enrichment decreases. Low feed flow rates increase the enrichment, because the incoming mass of surface-active molecules is lower, and the stability of the foam decreases, leading to lower liquid holdup. In this study, low levels of feed flow rate (C) increased the enrichment agreeing with the explanations given before and with observations from other authors [17,24]. However, the quadratic effect implied that a low feed flow level has not necessarily been the optimum. In case of the pore diameter (E), big pores were favorable for high enrichment factors. Large bubbles enhance the enrichment, because drainage is increased decreasing the liquid holdup in the foam [17,24]. The positive single effect of factor E confirmed this observation. As for the recovery of active enzyme, the gas flow rate (D) was only quadratic significant. In general, the enrichment factor decreases as the gas flow rate (D) increases due to the decreased entrainment of the foam [22,24].

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Fig. 6. Contour plots for the factor interactions of EC (a), R (b), and u (c). Blank contour plots resemble insignificant factor interactions.

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the experiments implying other surface-active substances stabilizing the foam. At high feed flow rates the opposite effect was observed. At pH values 67.0 the recovery decreased because of decreased residence time. For pH values >7.0 it can be assumed, that the incoming mass of other surface-active molecules stabilized the foam sufficiently. Cutinase, however, was transported not entirely as adsorbed species but in the interstitial liquid as well as on the bubble surface into the foamate.

Fig. 7. Surface tension of C. cinerea culture supernatants at varying pH values and temperatures ( 22.0 °C, j 25.0 °C,  27.5 °C, N 30.0 °C, d 32.5 °C).

4.1.3. Liquid holdup (u) The liquid holdup was affected by the temperature (B), the feed flow rate (C), the gas flow rate (D) and the pore diameter of the frit (E). Additionally, the feed flow rate (C) was quadratically significant (Fig. 4c). The single factors and their algebraic sign to minimize the liquid holdup were in line with the common opinion [19,21]. Long time for drainage (low D), less liquid loading of the foam and low mass of surface-active molecules (low C), big pores (high E) and high temperature (B) decreased the liquid holdup. 4.2. Statistical analysis of factor interactions 4.2.1. Recovery of activity (R) As mentioned before, various interactions with the frit pore size (E) existed. It is believed, that the nature and concentration of system components, temperature, gas flow rate, and pH value are primary variables, which determine secondary variables, like bubble size [25]. Thus, the bubble size is not affected by the frit pore size of the sparger only, but also by pH (AE), feed flow rate or rather incoming mass of surface-active components (CE), and the gas flow rate (DE) as seen in this study and observed by other scientists [19–21]. All in all, the interactions involving factor E could be explained with the theories given for the single factors. The quadratic influence of factor E and D on recovery can be seen for interaction CE and DE (Fig. 4b). Factor interaction CD was also consistent with the observations discussed in Section 4.1. The gas and feed flow rate determined the contact time between gas bubbles and surface-active molecules. Additionally, the feed flow fixed the mass of cutinase induced into the column. The quadratic effect of factor D indicated that a mean level of gas flow rate was necessary to recover cutinase efficiently. Another factor interaction between the pH value (A) and feed flow rate (C) existed. Generally, the pH value affects the enzyme’s surface-activity and the surface tension of the feed solution. It is expected that the adsorption strength at hydrophobic interfaces of the enzyme is enhanced at its isoelectric point (pI) as a result of both decreased repulsive forces and solubility [24,26,27]. The minimum surface tension was observed at pH 6.0–7.0, close to the isoelectric point of cutinase (pI = 6.7 [11]) (Fig. 7). At low feed flow rates and pH factor levels 60, i.e. pH values 67.0, high recoveries were obtained validating the statement that pH values close to the isoelectric point (low surface tension) were best. At pH values >7.0 the recovery decreased. This is a result of the increased surface tension, i.e. decreased adsorption strength of cutinase (Fig. 8). However, stable and wet foams resulted from

4.2.2. Enrichment factor (EC) For all factor interactions involving pore diameter (AE, CE, DE) a high factor level of the pore size (E) enhanced enrichment. Additionally, low factor values of feed flow favored high concentration of cutinase. These observations were consistent with literature [17,24,28]. For the mean level of the gas flow rate the best enrichment was observed, stating the significant quadratic effect as explained in Section 4.1. For factor interaction AC, high enrichment was achieved for low feed flow rates (long residence time and less liquid loading of the foam) and pH values corresponding to low surface tensions (Fig. 7). Higher pH values or feed flow rates decreased enrichment. This is in agreement with the previously described effects of these factors. Considering the factor interaction of temperature (B) and gas flow rate (D) the combination of either high or low levels was favorable for high enrichment. In general, high gas flow rates decreased the time for adsorption and drainage. Additionally, high temperature decreased the foam stability, but increased the foam formation. Increased drain rates could be explained by reduction of bulk and surface viscosity and increased evaporation of interstitial liquid. The increasing foam formation is likely to be a result of decreasing surface tension of the thin liquid lamellae improving foam bubble stability (Fig. 9) [29–31].

Fig. 8. Contour plot of factor interaction AC for response recovery (R) with surface tension measurements for pH values at a temperature of 27.5 °C.

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References

Fig. 9. Contour plot of factor interaction BD for response enrichment factor (EC) with surface tension measurements for temperatures consistent with level values at pH = 7.0.

For high gas flow rates and high temperatures the high enrichment was a result of combined destabilizing effects of these parameters. Bubbles collapsed partly in the upper part of the foam fractionation column and the foam was not leaving the column continuously. The cutinase rich liquid from the collapsed bubbles drained back and stabilized upcoming bubbles enabling them to leave the column. Hence, dry and cutinase rich foam left the column batch-wise increasing the enrichment factor. 5. Conclusion In this study, continuous foam fractionation in stripping mode was investigated. It could be shown, that the Design of Experiments was a valuable tool to get better insights into the foam fractionation process. Various factor interactions were determined and discussed. The regression models developed were in good agreement with the experimental data, and the optimization of the responses was successful. Experiments like surface tension measurements completed the investigation and supported the argumentation. With the knowledge gained, the foam fractionation process can be tailored fast and efficiently to different separation requirements for the system investigated. Hence, introducing foam as a separation medium can extend the possibilities in enzyme purification strategies. Acknowledgment Financial support from the Federal Ministry of Education and Research is gratefully acknowledged (‘‘Technology platform: Innovative Down-stream Processes’’, FKZ 0315520).

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