Separation and Purication Technology 123 (2014) 6468

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Separation and Purication Technology

journal homepage: www.elsevier.com/locate/seppur

Solidliquid extraction of phycocyanin from Spirulina platensis: Kinetic

modeling of inuential factors
Chia-Hung Su a,, Chang-Sung Liu a, Pei-Cheng Yang a, Kun-Siang Syu a, Chuang-Chun Chiuh b
a
b

Graduate School of Biochemical Engineering, Ming-Chi University of Technology, Taipei 24301, Taiwan
Far East Bio-Tec. Co., Ltd., Taipei 11503, Taiwan

a r t i c l e

i n f o

Article history:
Received 3 July 2013
Received in revised form 10 December 2013
Accepted 21 December 2013
Available online 3 January 2014
Keywords:
Phycocyanin
Spirulina platensis
Solidliquid extraction
Kinetics
Modeling

a b s t r a c t
To effectively extract value-added phycocyanin from Spirulina platensis, the effects of processing parameters (pH and temperature) on extraction performance and global kinetics were systematically studied.
The extraction kinetics was investigated by varying pH levels (58) and temperatures (3060 C). An
empirical kinetic model incorporating the aforementioned factors was developed. A good agreement
between the experimental and tted data was obtained, which indicated that the extraction process followed second-order kinetics. Furthermore, the model parameters (equilibrium concentration, extraction
rate constants, and initial rates of extraction) were calculated and formulated as a function of the operating factors. The activation energy of the extraction was 67.1 kJ mol1, indicating that the process was
endothermic. The predictions obtained from the developed model were compared with the experimental
data under the same operating conditions. The predicted and experimental data were consistent, indicating the reliability of the model.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Cultivation of Spirulina microalga is an effective process for
obtaining several valuable biochemicals, such as polysaccharides
[1], c-linolenic acid [2], b-carotene [3], chlorophyll a [4], and phycobiliproteins [5]. Phycobiliproteins, which are brightly colored pigments, function as a receiver of light for driving photosynthesis in
the Spirulina microalga [6]. Microalgal phycobiliproteins are classied into three major groups: phycoerythrin, allophycocyanin, and
phycocyanin [6]. The predominant pigment in the phycobiliprotein
family is phycocyanin [7]. Phycocyanin is commonly used as a natural colorant in food and cosmetic industries because it is inherently blue [6]. Moreover, it can be incorporated into health foods
because of its physiological properties, such as antioxidant, antiinammatory, and hepatoprotective activities [8,9]. Because of
these benets, numerous researchers have focused on developing
efcient processes for mass production of phycocyanin-producing
strains [10,11] and extraction of phycocyanin from microalgae
[5,12].
Isolating phycocyanin from microalgae typically begins with
solidliquid extraction using aqueous solvents [5]. In general,
solvent type, extraction temperature, and solidliquid ratio are
inuential factors in the extraction process [5,7]. The response
surface methodology has been used to optimize these operating fac-

Corresponding author. Tel.: +886 2 29089899x4665; fax: +886 2 29083072.

E-mail address: chsu@mail.mcut.edu.tw (C.-H. Su).
1383-5866/$ - see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.seppur.2013.12.026

tors for phycocyanin extraction [5]; however, this empirical

approach does not account for the mechanism governing the
process [13]. Developing a kinetic model coupling the operating factors on phycocyanin extraction is a solution that is crucial for designing an efcient phycocyanin extraction process. However, a relevant
kinetic model of phycocyanin extraction has not been developed.
In this study, Spirulina platensis was used as a source for phycocyanin. The effects of the operating factors (solvent pH and extraction temperature) on the aqueous solidliquid extraction of
phycocyanin from S. platensis were examined. Because a secondorder kinetic model effectively depicts solidliquid extraction
processes [1316], the kinetic model was used to determine corresponding kinetic parameters and predict the extraction process.
Finally, the predicted phycocyanin concentrations were veried
using actual experiments under the same conditions. This study is
required before developing and performing a systematic process
for phycocyanin extraction from S. platensis.

2. Materials and methods

2.1. Extraction procedure
The lyophilized S. platensis was provided by Far East Bio-Tec Co.,
Ltd. (Taipei, Taiwan). The dried microalgae were ground to reduce
the average particle size to less than 25 lm before examining the
extraction process. The extraction was conducted by mixing 2.5 g
of the ground biomass with 50 mL of sodium phosphate buffer

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C.-H. Su et al. / Separation and Purication Technology 123 (2014) 6468

solution (10 mM, pH 7.0) in a 125-mL stoppered conical ask

equipped with a magnetic stirrer. The pH level of the buffer solution was adjusted by mixing 10 mM KH2PO4 (pH 4.67) and
10 mM K2HPO4 (pH 8.99) stock solutions. The various pH levels
(58) and extraction temperatures (3060 C) were examined in
this experiment. The phycocyanin in the liquid extract was analyzed using the method described in Section 2.3.
2.2. Kinetic model
The second-order rate law provides a satisfactory representation of the solidliquid extraction process [1317]. Therefore, this
mathematic model was used to depict the kinetics of phycocyanin
extraction from S. platensis. The general second-order kinetic model can be expressed as

dC t
kC e  C t 2 ;
dt

t
where dC
represents the extraction rate (g L1 min1), k is the
dt
extraction rate constant (L g1 min1), Ce is the equilibrium concentration of phycocyanin (g L1) in the extract, and Ct is the concentration of phycocyanin (g L1) in the suspension at a specic extraction
time t. The initial extraction rate dened as h when t and Ct
approach 0 can be expressed as

h kC e ;

C 2e kt
;
1 C e kt

The linear form derived from Eq. (3) is shown as Eq. (4).

t
1
t

;
C t kC 2e C e

Thus, k and Ce can be determined experimentally from the slope and

intercept of a linear line by plotting t/Ct against t. After rearranging
Eqs. (2) and (3), Ct can be expressed as

Ct

t
;
1=h t=C e

The Arrhenius equation was used to evaluate the effect of extraction

temperature on the kinetic model, which is written as
Ea

k k0 e RT ;

2.4. Validity of model prediction

The consistency between the predicted and experimental data
was evaluated using the coefcient of determination (r2), which
is dened as

Pn
^i 2
yi  y
r2 1  Pi1
;
n
2
i1 yi  y

where n is the number of samples, yi is the actual experimental data

^i is the model-tting data of the ith sample, and y
of the ith sample, y
is the mean value of all experimental data. The coefcient r2 is normalized between 0 and 1, with a high r2 value indicating superior
consistency between the experimental data and model tting.
2.5. Statistical analysis
The data were analyzed by conducting a one-way analysis of
variance (ANOVA) or Fishers F-test using Microcal Origin 6.0
(Microcal Software, Inc., MA, USA) software. Statistical differences
were established according to a probability threshold (P) of 0.05.

To obtain the kinetic parameters (k and Ce), Eq. (1) was integrated
under the initial and boundary conditions, t = 0 to t and Ct = 0 to
Ct, as the following equation:

Ct

where PC is the phycocyanin concentration (g L1), and OD615 and

OD652 are the optical density of the sample at 615 and 652 nm,
respectively.

where k0 is the pre-exponential factor for extraction rate constant

(L g1 min1), Ea represents the activation energy of extraction
(J mol1), R is the ideal gas constant (J mol1 K1), and T is the
extraction temperature (K). The pre-exponential factor, k0, and the
activation energy, Ea, can be determined using the natural logarithm
of Eq. (6).

3. Results and discussion

3.1. Effect of pH
The effect of pH on solidliquid extraction is crucial because the
solubility of bio-compounds and apparent kinetic constants are
directly dependent on the pH variation [13,19]. The extraction
was performed with pH ranging from 5 to 8 at a temperature of
50 C. The pH range of 58 was selected because phycocyanin is
unstable below pH 5 and above pH 8 [7]. The results (Fig. 1) show
that a higher pH increased the extraction rate and equilibrium concentration. However, a substantial decrease in the equilibrium concentration occurred at pH 8. Protein denaturation may contribute
to this result [7]. Because the maximal equilibrium concentration
was obtained at pH 7, further experiments were performed at this
level to examine other operating conditions.
3.2. Effect of extraction temperature
The extraction temperature varied from 30 to 60 C to evaluate
its effect on the extraction efciency of phycocyanin at a pH of 7.

2.4
2.2
2.0
1.8

-1

The phycocyanin concentration during the extraction process

was determined according to the procedure used by Chen et al.
[4]. The sample withdrawn from the extraction broth was centrifuged and the optical density of the supernatant was determined
spectrophotometrically at 615 and 652 nm using an Ultrospec
3100 pro spectrophotometer (GE Healthcare Bio-Sciences Corp.,
USA). The phycocyanin concentration was estimated using Eq. (7)
[18].

PC

OD615  0:474  OD652

;
5:34

Ct (g L )

1.6

2.3. Analysis

1.4
1.2
1.0

pH 5
pH 5.5
pH 6
pH 6.5
pH 7
pH 8

0.8
0.6
0.4
0.2
0.0

50

100

150

200

250

Time (min)
Fig. 1. Effect of solvent pH on phycocyanin extraction. The data points and solid
lines represent the experimental results and model predictions, respectively.

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C.-H. Su et al. / Separation and Purication Technology 123 (2014) 6468

The evolution of the phycocyanin concentration for various temperatures is shown in Fig. 2. A faster rate was obtained with higher
temperatures. This may be attributed to the increased diffusion
coefcient of solutes at a higher temperature [17]. The gure also
shows that the equilibrium concentration of phycocyanin decreased slightly with increasing extraction temperature from 30
to 50 C. In general, charged proteins have aqueous solubilities that
increase substantially with temperature [20,21]. However, the
slight decrease in the equilibrium concentration indicates that
the phycocyanin was degraded by heat during the extraction process [22,23]. With further elevation of temperature to 60 C, the
equilibrium concentration of phycocyanin reached only 0.4 g L1
in 4 h. This result is similar to those of previous studies on the
thermal degradation kinetics of phycocyanin [22].

(A)

160
140

-1

t/Ct (min L g )

120
100
80
60

pH 5
pH 5.5
pH 6
pH 6.5
pH 7

40
20
0
0

50

100

150

200

250

Time (min)

3.3. Establishment of the kinetic model

120

kpH 1:37  103 X 2pH  1:32  102 X pH 4:51  102 ; r 2 0:99;

9
C e pH 1:46  101 X 2pH  1:49X pH 5:66; r 2 0:99;

10

hpH 1:98  102 X 2pH  2:05  101 X pH 5:78  101 ; r 2 0:99;

(B)
100

-1

t/Ct (min L g )

To establish the kinetic model, the model parameters (k, Ce, and
h) can be estimated using a linear equation, dened as Eq. (4). Fig. 3
shows the correlation between t/Ct and t under all experimental
conditions. A straight line is a clear indication that the proposed
model is valid. Hence, the equilibrium concentration of phycocyanin Ce and the extraction rate constant k can be calculated from the
slope and intercept of each straight line, respectively. Table 1
shows the calculated model parameters for various extraction
conditions.
The relationship between the model parameters and pH variation at 50 C is shown in Fig. 4. The ANOVA results indicated that
the pH variation signicantly (P < 0.05) affected the model parameters. The values of these parameters increased slightly with an increase in pH from 5 to 6. However, an obvious increase in those
quantities was observed with further elevation of the pH to 7. This
may be attributed to the fact that phycocyanin is negatively
charged at a pH above its isoelectric point; consequently, the
negatively charged protein is attracted by the aqueous solvent
[7]. Because k, Ce, and h were dependent on pH, the estimated
values were used to t the quadratic empirical equations (Eqs.
(9)(11)):

80

60
o

30 C
o
35 C
o
40 C
o
45 C
o
50 C

40
20

0
0

50

100

150

200

250

Time (min)
Fig. 3. Kinetic model to estimate the extraction rate constant and equilibrium
concentration for various (A) pH levels, and (B) extraction temperatures. In these
gures, the values of the abscissa and ordinate were obtained from the relationships t and t/Ct.

where XpH represents the variation of the pH level. The goodness-oft of the equations was evaluated using the coefcient of determination (r2). The high values of r2 indicated that the correlations
between the model parameters and pH variation are reliable.
The evolution of concentration Ct as a function of pH can be obtained by substituting Eqs. (10) and (11) into (5). The relationship
is described as
C t pH

t
1=1:98  102 X 2pH  2:05  101 X pH 5:78  101 t=1:46  101 X 2pH  1:49X pH 5:66

12

11

2.4
2.2
2.0
1.8

-1

Ct (g L )

1.6

30 C
35 C
40 C
45 C
50 C
60 C

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0

50

100

150

200

250

Time (min)
Fig. 2. Effect of temperature on phycocyanin extraction. The data points and solid
lines represent the experimental results and model predictions, respectively.

The model can be used to predict the phycocyanin extraction under

various pH levels at a specied time with an extraction temperature
of 50 C.
The effect of temperature on the model parameters at a pH of 7
is shown in Fig. 5. The ANOVA results indicated that the model
parameters were signicantly (P < 0.05) affected by varying the
temperatures from 30 C to 50 C. The temperature had an accelerative inuence on the extraction rate constant k and initial extraction rate h. However, a reverse trend was observed when the
equilibrium concentration Ce decreased with increasing temperature. The following empirical equations (Eqs. (13) and (14)) were
developed to correlate temperature with the equilibrium concentration of phycocyanin and the initial extraction rate. The reliability of these equations has been proved using r2.

C eT 2:6  103 T 2  0:25T 8:29; r2 0:98;

13

hT 1:14  104 T 2  5:55  103 T 1:03  101 ; r 2 0:99;

14

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C.-H. Su et al. / Separation and Purication Technology 123 (2014) 6468

Table 1
Model parameters (k and Ce) obtained for all tested conditions.
Model parameters

Solvent pH

ka
Ceb
r2
a
b

Temperature (C)

5.5

6.5

30

35

40

45

50

1.33  102
1.84
0.99

1.41  102
1.89
0.99

1.49  102
1.93
0.99

1.74  102
2.14
0.99

1.98  102
2.36
0.99

3.82  103
3.14
0.98

6.2  103
2.81
0.99

1.06  102
2.44
0.99

1.41  102
2.39
0.99

1.98  102
2.36
0.99

Unit of extraction rate constant is L g1 min1.

Unit of saturation extraction capacity is g L1.

12

2.0

-3.5

2.4

1.9

10

4
1.5

Ce

-1

-5.0

1.8
2

1.4
1.3

ln k

2.0

-4.5

-1

-1

1.6

h x10 (g L min )

2.2

Ce (g L )

-1

1.7

-1

k x10 (L g min )

-4.0
1.8

k
5.0

5.5

6.0

6.5

7.0

1.6

-5.5

-6.0
3.10

pH

3.15

3.20

3.25

3.30

Fig. 4. The effect of solvent pH on the extraction rate constant k, equilibrium

concentration Ce, and initial extraction rate h at 50 C. The solid lines represent
prediction results of the empirical equations (Eqs. (9)(11)).

Temperature reciprocal x10 (K-1)

Fig. 6. Arrhenius-Vant Hoff plot for phycocyanin extraction in the temperature
within a range of 3050 C.

12

2.5

3.4

3.0

2.6

6
4

0.8

2.4
2.2

0.4
30

35

40

45

50

2.0

-1

-1

-1

2.8

1.2

-1

Ce

Ce (g L )

1.6

-1

-1

k x10 (L g min )

3.2

Actual concentration (g L )

10

h x10 (g L min )

2.0

1.0

0.5

0.0
0.0

Fig. 5. The effect of extraction temperature on the extraction rate constant k,

equilibrium concentration Ce, and initial extraction rate h at a pH of 7. The solid
lines represent prediction results of the empirical equations (Eqs. (13)(15)).

The dependence of the rate constant k on the extraction temperature can be described using the Arrhenius equation (Eq. (6)). An
Arrhenius-Vant Hoff plot was used to determine the pre-exponential factor and the activation energy of extraction. As shown in
Fig. 6, the higher linear correlation coefcient (r2 = 0.98) indicated
that the Arrhenius model parameters, k0 and Ea, can be determined
from the slope and intercept of the straight line, respectively. Therefore, the relationship of k and T is written as


67100
; r 2 0:98;
8:314T 273:15

1.5

The predictions from Eq.12, r = 0.99

2
The predictions from Eq.16, r = 0.98

Temperature (C)

kT 1:47  109 exp

2.0

15

For the phycocyanin extraction process, the activation energy was

67.1 kJ mol1, indicating that the extraction is an endothermic
process [15].

0.5

1.0

1.5

2.0

2.5

-1

Predicted concentration (g L )
Fig. 7. Correlation between the actual and predicted concentration of phycocyanin.

Using a similar derivation as that in Eq. (12), substituting Eqs.

(13) and (14) into (5) yields an equation describing the evolution
of Ct versus time and temperature:
C tT

t
1=1:14  104 T 2  5:55  103 T 1:03  101 t=2:6  103 T 2  0:25T 8:29
16

This model can be used to predict the phycocyanin extraction under

various temperatures at a specied time with a pH level of 7.
3.4. Validity of the developed model
Because all kinetic parameters were determined, the developed
models (Eqs. (12) and (16)) were used for predicting phycocyanin
extraction from S. platensis under diverse operating conditions,
including various pH levels (57) and temperatures (3050 C).

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C.-H. Su et al. / Separation and Purication Technology 123 (2014) 6468

The phycocyanin concentration obtained using Eqs. (12) and (16)

was compared with the experimental results under the same operating conditions. As shown in Fig. 7, the predicted and experimental data were consistent with coefcients of determination (r2) of
0.98 and 0.99 for the extraction under diverse pH levels and temperatures, respectively. The results showed that the developed
models are valid for the extraction system. Thus, the kinetic
models can provide useful information on operation strategies
and economic descriptions of the extraction process.
4. Conclusions
This study examined the effects of solvent pH and extraction
temperature on the aqueous extraction of phycocyanin from
S. platensis. The results showed that a higher pH increased the
extraction rate and equilibrium concentration because the negatively charged phycocyanin is attracted by the aqueous solvent.
For the temperature tests, the extraction rate increased with
increased temperature, whereas the equilibrium concentration of
phycocyanin decreased with increased temperature. This indicated
that phycocyanin was degraded by heat during the extraction process. A second-order kinetic model for depicting the extraction
processes under diverse conditions was successfully developed.
The activation energy of phycocyanin extraction was 67.1 kJ mol1,
indicating that the extraction was an endothermic process. Finally,
the developed models (Eqs. (12) and (16)) were used for the prediction of phycocyanin concentration under various extraction
conditions. The predicted values were consistent with the actual
results, indicating their reliability.
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