Chromatographia (2011) 74:307–312
DOI 10.1007/s10337-011-2059-6
FULL SHORT COMMUNICATION
Recovery of C-Phycocyanin in the Presence of Cells
Using Expanded Bed IEC
Caroline Costa Moraes • Joana da Costa Ores •
Jorge Alberto Vieira Costa • Susana Juliano Kalil
Received: 19 January 2011 / Revised: 29 April 2011 / Accepted: 4 May 2011 / Published online: 21 May 2011
Ó Springer-Verlag 2011
Abstract C-Phycocyanin is a natural blue dye that can be
extracted from Spirulina platensis. In this paper, a crude
extract of C-phycocyanin in the presence of cells was
purified using ion exchange chromatography in an expan-
ded bed. The elution was studied aiming at its optimization
using a central composite rotatable design (CCRD), the
independent variables being the pH and elution volume,
and the responses purity, concentration, yield and recovery.
An evaluation of the contour curves showed that the best
conditions for purification were a pH value of 6.5 and
elution volume of about 150 mL. A run was carried out
under these conditions, combining a saline gradient
(0–1 M) with step elution using 0.1 M NaCl. Under these
conditions, it was possible to obtain C-phycocyanin with a
purity increment of 4.6-fold compared with the initial
extract working directly with unclarified extract.
Keywords Expanded bed  Ion exchange
chromatography  Purification  C-Phycocyanin
Introduction
Food colors create physiological and psychological
expectations and attitudes that are developed by experi-
ence, tradition, education and the environment [1], children
C. C. Moraes (&)
Engenharia de Alimentos, Universidade Federal do Pampa,
P.O. Box 07, Bagé, RS 96412-420, Brazil
e-mail: engcarolinemoraes@yahoo.com.br
J. da Costa Ores  J. A. V. Costa  S. J. Kalil
Escola de Quı́mica e Alimentos, Universidade Federal do Rio
Grande, P.O. Box 475, Rio Grande, RS 96201-900, Brazil
e-mail: susana.kalil@vetorial.net
being the main consumers of artificially colored food and
candies. Recently, a growing awareness of the importance
of natural colors has placed a great demand on the bio-
logical sources of natural colors [2].
Cyanobacteria and algae possess a wide range of colored
components including carotenoids, chlorophyll and phy-
cobiliproteins [2]. Species of the genera Spirulina are
inexpensive sources of phycobiliproteins [3], and have
been awarded a GRAS certificate (generally recognized as
safe) that permits their use in food.
C-phycocyanin (C-PC) is a natural blue dye extracted
from cyanobacteria such as Spirulina platensis, and has
raised interest because of its many commercial applications
in foods and cosmetics [4]. Recent studies have demon-
strated the hepatoprotective [5], anti-inflammatory [5, 6]
and antioxidant [6, 7] properties of C-PC. Due to their
limited distribution in nature [8] and high cost of obtaining,
resultant from the difficulty of purification process [9],
these pigments are rather expensive and thus obtaining
them as pure compounds is a potentially attractive endea-
vor [8].
Purity is defined as the ratio of the active substance
to the total quantity of material. For C-phycocyanin, the
A620/A280 ratio is considered to be a good indicator of the
purity of the preparation, especially when other forms of
protein contaminants are taken into account [10]. A purity
of 0.7 is considered as food grade, 3.9, as reactive grade,
and greater than 4.0, analytical grade [9, 11].
In the recovery and purification of protein products from
a variety of biological sources, the bio-separation of pro-
teins is an important unit operation in the food, pharma-
ceutical and biotechnology industries. Freitag and Horváth
[12] consider that ion exchange chromatography is the
most widely used technology in the preparation of proteins
on both laboratory and production scales, and the recovery
123
308
of biological activity is usually excellent. The reasons for
the success of ion exchange chromatography are its wide
applicability, high resolution power, high capacity and the
general simplicity and controllability of the method. Ion
exchange can be carried out in a fixed (generally for small-
scale operations) or expanded bed.
Adsorption in an expanded bed allows the elimination of
clarification step, and also produces concentrated and
partially purified product ready for the next purification
step [13], making the process attractive for large-scale use.
The use of controlled density adsorbents allows for the
production of a stable bed, each particle maintaining a
discreet position in the bed with a limited circular move-
ment [14].
Despite the great advantages offered for the use of
expanded bed, there are a few reports about the purification
of C-phycocyanin using an expanded bed, and the opti-
mization of this process still needs to be developed. To
date, there are no papers reporting the C-phycocyanin
purification using broth not clarified.
No papers were found reporting the use of EBA to purify
phycobiliproteins in the presence of cells. Thus, the aim of
this paper was to purify the crude extract, not clarified
C-phycocyanin, from S. platensis using ion exchange
chromatography in EBA, studying the elution step with an
aim to maximize the process yield and recovery without
diluting the product.
Experimental
Chromatography Media and Column
A Streamline 25 expanded bed column was used with a
column size of 100 cm 9 2.5 cm I.D., total bed volume
V t = 50 mL, and Streamline QXL adsorbent, all from GE
HealthcareTM.
Biomass and C-Phycocyanin Extraction
The cyanobacterium S. platensis LEB 52 culture [15] was
grown in outdoor 450 L capacity photo-bioreactors, using
Zarrouk’s synthetic medium diluted to 20%. The biomass
was dried at 40 °C and then frozen at -18 °C. Mechanical
rupture was carried out by grinding in a ball mill. The biomass
was added to the extractor solvent in the proportion of 24 g of
biomass: 100 mL of solvent, according to Moraes et al. [16].
C-Phycocyanin Purification
A StreamlineTM 25 column containing 50 mL of Streamline
QXL ion exchange resin (10 cm of bed height) was used in
all experiments. The column was equilibrated with 0.025 M
123
C. C. Moraes et al.
sodium phosphate buffer pH 7.5 with an upward flow rate
obtained using a peristaltic pump, so as to give an expansion
degree of twice its height (20 cm of bed height). This
equilibration phase was continued until the bed was stable.
A 145 mL portion of unclarified C-phycocyanin extract
diluted 10 times (cell concentration of 24 mg mL-1) at pH
7.5 was then loaded onto the column, maintaining the
expansion degree. The extract volume was based on the
adsorption capacity of adsorbent (data not shown) in order
to correspond to 10% of this capacity. After applying the
whole sample volume, the column was washed with 10 bed
volumes of starting buffer at the elution pH value to remove
the remaining cells and non-adsorbed proteins. After
washing in the expanded mode, the upward flow was
stopped and the bed was allowed to settle in the column.
The adaptor was then moved down to the surface of the
settled bed and the C-phycocyanin recovered using a linear
0–1 M NaCl gradient in 0.025 M sodium phosphate buffer
using the elution volume and pH values determined by the
central composite rotatable design (Table 1). The elution
volumes were calculated to obtain 3–17 bed volumes.
Fractions of 10 mL were collected during all steps of
purification process for the evaluation of their C-phycocy-
anin concentrations (Eq. 1) and extract purities (Eq. 2),
according to Bennet and Bogorad [17].
½A620  0:474 ðA652 Þ
C-PC (mg mL1 Þ ¼ ð1Þ
5:34
EP ¼ A620 =A280 ð2Þ
The eluted fractions showing purity greater than 0.7
were used to calculate the recovery (Eq. 3) and yield
(Eq. 4).
P
ðC  PC  volume collectedÞ
%Recovery ¼  100
Initial C-PC  initial volume
ð3Þ
P
C  PC  volume collected
Yield (mg g1 Þ ¼ ð4Þ
amount of biomass used
Central Composite Rotatable Design (CCRD)
The effects of pH and elution volume were studied using a
CCRD (22 plus axial and central points) with three replicates
at the central point giving a total of 11 trials, the responses
being the purity, C-phycocyanin concentration, recovery and
yield. Table 1 gives the values of the coded and real levels
used in the central composite rotatable design.
Results and Discussion
To have an optimized purification step is necessary to give
attention to some key parameters like the feeding of the
Recovery of C-Phycocyanin Using Expanded Bed IEC 309

deviation
extract (volume, concentration, flow rate), adsorption

Relative

-3.07

7.24

-4.62
9.03
-0.18
-3.32
0.42
2.66
-24.08

-23.76

25.02
conditions (pH and resin-type) and conditions of desorp-

(%)
tion/elution. The conditions of feeding and absorption were
studied and optimized previously (data not shown).
Table 1 Coded levels and real values (in parenthesis) for a CCRD, yield, recovery and C-phycocyanin concentration (C-PC), including the experimental and predictive results

The elution conditions were studied in this paper since

(mg mL-1)

this is a crucial step for more efficient purification. The

purity, concentration, recovery and purification factor were
C-PC

1.58
1.72
0.59
0.73
0.76
0.96
2.14
0.75
0.95
0.95
0.95
among the main responses that increased after optimizing
the elution [18]. The variable pH is directly linked to
purity, since the net negative charge of the protein varies
deviation
Relative

according to the pH, binding more or less strongly onto the

9.00
7.33
-1.08
-4.50
-4.70
-2.26
-4.47
2.35
-3.11
0.29
-1.51
(%)

resin, which could lead to better adsorption of the target

protein [18]. The elution volume promotes changes in the
concentration and purity, since when the elution volume
Recovery

increases, there is a tendency for protein separation to

66.14
64.69
78.12
52.70
70.47
51.53
69.88
69.88
69.88
69.88
69.88
improve, obtaining greater purity of the target molecule
(%)

[19] but a reduced concentration of the eluate and conse-

quently of the process recovery and yield. Thus, the elution
step was optimized using a CCRD to study the pH and
deviation
Relative

elution volume (Table 1).

3.65
3.68
-3.63
-4.87
0.34
0.24
-3.61
5.95
-4.32
-0.54
1.71
(%)

TM
The Streamline column is designed for use in expan-
ded bed adsorption, where the bed is expanded by the
upward liquid flow when feeding crude extract, without the
Predictive

(mg g-1)

need for prior clarification. When the crude extract of S.

37.49
38.29
47.81
35.23
39.57
31.27
41.48
46.61
44.05
44.05
44.05
Yield

platensis was applied to the column with an upward flow,

the phycobiliproteins were captured by the adsorbent while
the cell debris, particulates and contaminants in the extract
passed unhindered through the column.
(mg mL-1)

To construct a second order model that could predict the

C-PC

1.27
1.67
0.48
0.79
1.01
0.92
2.35
0.75
0.92
0.95
0.98

C-PC concentration, purity, recovery and yield as a func-

tion of the pH and elution volume, a statistical analysis was
carried out, evaluating the adequacy of fit. The analysis of
variance gave the following F values (Fcalculated/Ftable) at
Recovery

90% of confidence: 3.94 (R2 = 0.91) for yield, 4.59

72.68
69.80
77.29
50.44
67.31
50.39
66.89
71.56
67.77
70.08
68.83
(%)

(R2 = 0.86) for recovery and 3.01 (R2 = 0.91) for C-PC
concentration, indicating a significant and predictive
Experimental

model. The purity was not significant at 90% of confidence.

(mg g-1)

Equations 5, 6 and 7 present the codified models, and the

38.91
39.70
46.13
33.59
39.70
31.34
40.04
49.55
42.22
43.81
44.81
Yield

response surface and contour curves are presented in

Fig. 1.
Yield ðmg g1 Þ ¼ 44:03  2:92X1  4:35X12 þ 1:84X2
-1 (250)
-1 (250)
1 (750)
1 (750)
0 (500)
0 (500)
-1.41 (150)
1.41 (850)
0 (500)
0 (500)
0 (500)

 3:39X1 X2 ð5Þ
Recovery ð%Þ ¼ 69:84  6:64X1  4:44X12  6:08X1 X2
X2

X1 pH, X2 volume of elution

ð6Þ
1
C-PC ðmg mL Þ ¼ 0:95 þ 0:06X1  0:04X12  0:50X2
-1 (5.5)
1 (7.5)
-1 (5.5)
1 (7.5)
-1.41 (5.1)
1.41 (7.9)
0 (6.5)
0 (6.5)
0 (6.5)
0 (6.5)
0 (6.5)

þ 0:25X22 ð7Þ
X1

The values obtained for yield were between 31.34 and

49.55 mg g-1, and for recovery between 50.59 and
77.29%, the C-phycocyanin concentration varying
Run

10
11

between 0.48 and 2.35 mg mL-1 (Table 1), showing that

1
2
3
4
5
6
7
8
9

123
310 C. C. Moraes et al.

Fig. 1 Response surfaces and contour diagrams for the yield, recovery and C-PC concentration as a function of pH and elution volume

123
Recovery of C-Phycocyanin Using Expanded Bed IEC 311

Fig. 2 Chromatogram of A280 C-PC

C-phycocyanin purification in
[NaCl] EP
an expanded bed on Streamline 5.0 1.0
QXL using gradient elution 2.0
(0–1 M) in 150 mL, with a pH 4.5 0.9
6.5 buffer
4.0 0.8

1.5 3.5 0.7

C-PC (mg/mL) e EP
3.0 0.6

[NaCl]
2.5 0.5

A280
1.0
2.0 0.4

1.5 0.3
0.5
1.0 0.2

0.5 0.1

0.0 0.0 0.0

0 200 400 600 800 1000 1200 1400
Volume (mL)

the concentration was more affected by the variables
studied than the yield and recovery.
Table 1 and Fig. 1 show that the highest values for yield
and recovery were obtained at pH values around 5.1 and
the largest elution volume (850 mL). On the other hand,
the C-PC concentration was reduced to 80% when the
volume was increased, independent of the pH. However, it
is possible to avoid these losses in concentration if the
elution is performed at pH 6.5, because at this pH the losses
in yield and recovery, as compared to the optimum con-
ditions, were only about 13%. When the product to be
purified is added to foods as an additive, it is important to
get the concentration as high as possible to avoid the
addition of large amounts to the food product. In this case,
it was possible to work at pH 6.5 and an elution volume of
150 mL, or combine the process with an ultrafiltration step
and work under conditions that permitted the highest
recovery.
The models obtained agreed with the results observed in
the CCRD, since Table 1 presents values for relative
deviation of below 10% for the yield and recovery. For
concentration, the relative deviation was high (25%) at the
lower pH values, but at the other pH values the relative
deviation remained below 10%. Thus, it was considered
that the model did not fit the experimental data for the
C-PC concentration at pH values between 5.1 and 5.5, but
in the pH range considered as optimum, the experimental
data fitted the model well.
In the present study, C-phycocyanin was obtained with a
value for purity of 1.62 (an increase of 3.2 times compared
with the initial extract), for concentration of 2.35 mg
mL-1, yield of 40 mg g-1 and recovery of about 67%, in
the run 7. To confirm these results, the experimental data
were validated by carrying out three further assays of run 7
and comparing the results. The results obtained in these
three assays presented a percent deviation below 6% for all
the responses evaluated (purity, concentration, yield and
recovery). Figure 2 shows the chromatogram of one of
these assays, and it shows that 10 of the 15 fractions col-
lected during elution presented values for purity above 0.7,
corresponding to 67% of recovery, the value expected for
ion exchange.
Niu et al. [20] reported that with the use of an expanded
bed, many of the concentration and partial isolation steps
and procedures, such as precipitation in ammonium sulfate
and gel-filtration used in conventional chromatographic
purification protocols, are eliminated. A typical cycle of the
isolation process by EBA chromatography including
equilibration (30 min) ? loading (30 min) ? washing
(60 min) ? elution (30 min), only took 2.5–3.5 h.
Bermejo et al. [21] studied the purification of a crude
extract of C-phycocyanin without cells using expanded bed
ion exchange chromatography, and obtained a C-PC extract
three times purer than the initial extract, with a recovery of
68–80%. Niu et al. [20] used hydrophobic interaction in an
expanded bed to purify a crude extract of C-phycocyanin
without cells, and obtained a C-phycocyanin extract that
was four times purer with a recovery of 35% and yield of
31 mg g-1. In the present paper, if the fraction collected
were just more purified, a product with an increment of
four times in purity and a recovery of about 50% would be
obtained.
Although the results obtained were in agreement with
those found in the literature, Moraes and Kalil [18]

123
312
commented that it was possible to combine gradient elution
with step elution promoting greater resolution of the final
product with respect to purity. Thus, a new assay was
carried out, equal to run 7, but a pre-elution step was
carried out between the washing and elution steps using
100 mL of 0.1 M NaCl (two bed volumes). In this assay,
the pre-elution promoted the removal of protein contami-
nants that were weakly bound to the resin increasing
the purity of the C-phycocyanin eluted posteriori. The
C-phycocyanin obtained presented a concentration of
3 mg mL-1, purity of 2.9 (increment of 4.6 compared with
the initial extract), recovery of 60% and yield of
40 mg g-1.
In conclusion, the results similar or better to those pre-
viously reported were indeed obtained, but using crude
C-phycocyanin extract containing cells, justifying the use
of the expanded bed and eliminating the centrifugation step
which is too expensive industrially.
Acknowledgments The authors are grateful to CAPES and CNPq/
MCT for their financial support.
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