Appl Microbiol Biotechnol (2007) 74:244248

DOI 10.1007/s00253-006-0636-7

METHODS

A simple method for extracting C-phycocyanin

from Spirulina platensis using Klebsiella pneumoniae
Y. Zhu & X. B. Chen & K. B. Wang & Y. X. Li & K. Z. Bai &
T. Y. Kuang & H. B. Ji

Received: 26 December 2005 / Revised: 15 August 2006 / Accepted: 16 August 2006 / Published online: 30 September 2006
# Springer-Verlag 2006

Abstract C-phycocyanin (C-PC) was extracted from fresh

Spirulina platensis by deploying a species of non-pathogenic nitrogen-fixing bacteria, namely, Klebsiella pneumoniae. The algal slurry was neither washed nor centrifuged;
the bacterial culture was poured into the slurry, the vessel
sealed, and crude C-PC extracted after about 24 h. The
extraction was clean and efficient, and the purity and
concentration of C-PC proved to be of adequate quality.
Keywords C-phycocyanin . Klebsiella pneumoniae .
Extraction . Spirulina platensis

Introduction
C-phycocyanin (C-PC) is a water-soluble light-harvesting
protein that is used in the cosmetic and pharmaceutical
industries and in fluorescence labelling (Pulz and Gross

Y. Zhu : X. B. Chen : K. B. Wang : Y. X. Li : K. Z. Bai :

T. Y. Kuang (*)
Key Laboratory of Photosynthesis and Environmental Molecular
Physiology, Photosynthesis Research Center, Institute of Botany,
Chinese Academy of Sciences,
Beijing 100093, China
e-mail: kuang_tingyun@yahoo.com.cn
H. B. Ji (*)
Resource, Environment and GIS Key Laboratory of Beijing City,
Capital Normal University,
Beijing 100037, China
e-mail: hbji@mail.cnu.edu.cn
Y. Zhu
Graduate School of the Chinese Academy of Sciences,
Beijing 100039, China

2004; Cohen 1986; Glazer and Stryer 1984). C-PC is

extracted from cyanobacteria in a two-stage process. The
first stage involves the preparation of a cell-free extract to
dissolve C-PC in water to form a crude extract and the
second stage involves purifying the crude extract to obtain
C-PC in highly pure form. Extraction of phycobiliproteins
from cyanobacteria is difficult because the algae are small
and possess extremely resistant multilayered cell walls
(Stewart and Farmer 1984; Wyman 1992). Many conventional methods are used to obtain crude extracts of C-PC
including alternate freezing and thawing (Minkova et al.
2003), lysozyme digestion (Boussiba and Richmond 1979),
ultrasonication (Furuki et al. 2003), grinding with glass
beads (Scheer and Kufer 1977), grinding with silica-gel or
celite, and mechanical disruption using a pressure homogenizer (Abalde et al. 1998). Other methods include nitrogen
cavitation for algal-cell disruption (Gottlieb and Adachi
2003; Viskari and Colyer 2003), extraction using acids
(Sarada et al. 1999), treatment with linear alkylbenzene
sulfonate (Lin et al. 1998), and extraction by using high
pressure (Herrero et al. 2004). Each method has its
advantages and disadvantages. Methods for the second
stage include high-speed centrifugation, precipitation with
ammonium sulfate, and ion-exchange chromatography
(Boussiba and Richmond 1979; Hayashi et al. 1997;
Tchernov et al. 1999).
A comprehensive technique must include rapid and
efficient cell lysis and extraction and recovery of the
released C-PC in adequate quantities. The aim of the
present study was to develop a simple and easy method for
the extraction of C-PC, potentially suitable for industrial
production. We used a culture of Klebsiella pneumoniae
poured into fresh cyanobacteria (Spirulina platensis) to
achieve a high recovery of C-PC of adequate quality from
the crude extract.

Appl Microbiol Biotechnol (2007) 74:244248

Materials and methods

Reagents, buffers, and samples
Lysozyme was obtained from Sigma (St. Louis, MO, USA).
All other reagents were of analytical grade. Extraction media
for comparative studies and buffers for purification were
prepared by dissolving K-phosphate buffer stocks in distilled
deionized water (Millipore) so that the final concentration
was 0.001 M and the pH of the media was 7.0. All the
monitored samples of C-PC were diluted with this buffer,
which was stored at room temperature and sterilized by
autoclaving before use.
We used freshwater cyanobacterium S. platensis M2 (Lu
and Vonshak 1999) grown in a standard Zarrouks
medium (Zarrouk 1966) and cultivated as previously
reported (Sarada et al. 1999). Fresh cyanobacterial cells
were harvested when the optical density (OD) 560 was
approximately 0.9.
K. pneumoniae isolated from Mexico corn seeds was
identified with Biolog as K. pneumoniae spp. Pneumoniae.
The probability (PROB) is 95%, the similarity (SIM) is
0.56. The species was reported as non-pathogenic and was
one of the normal associate nitrogen-fixing bacteria. We
grew it in an improved HS medium (Harris 1989); the form
of trace elements were exchanged to 1 g yeast, and 20 g
sucrose per 1,000 ml was added to the medium. The culture
was inoculated from an agar slant of the same medium as
mentioned above, incubated for 1517 h and maintained at
about 30C until the OD 600 reached approximately 1.3;
the pH was down to about 4.5.
Enterobacter gergoviae and Klebsiella oxytoca were
isolated and preserved by the Institute of Botany, Chinese
Academy of Sciences; both are non-pathogenic nitrogenfixing species of bacteria and were grown in the same
medium as mentioned above as K. pneumoniae. In addition,
Bacillus subtilis 912, Fluorescence pseudomonas 78-101,
Escherichia coli DH52, and S. cerevisiae Y1980 were also
preserved by the Institute of Botany, Chinese Academy of
Sciences. The first three were grown in LB medium and S.
cerevisiae in YPD medium.
Extraction procedures
The rotary shaker was turned off for 2 h to allow S.
platensis to settle. As much of the medium was discarded
as possible and the algal layer saved; only a small amount
of the medium was left, but it did not affect the extraction
process. The algal layer was not rinsed; the layer was either
retained in Erlenmeyer flasks or transferred to the other
sterilized transparent containers. The pH of the algal culture
was 9.510.5. At this point, the prepared bacterial culture
was added to the algal layer; the ratio of the bacterial

245

culture to algae ranged from 1:1 to 2:1 (v/v). Then, the pH

of the mixed supernatant ranged from 6.0 to 7.0. The
containers were sealed and stored at room temperature,
protected from light, and the suspension allowed to settle.
Despite some vibrating, two layers were clearly visible: the
upper, yellowish, liquid layer, made up mostly of the
bacterial culture, and the bottom blue-green layer of algal
slurry. The algal slurry was immersed in the bacteria
culture. The contents were uncontaminated with other
living organisms. After about 12 h, the boundary of the
two layers could be seen as a thin light-blue layer from
the transparent containers; gradually, however, the whole
top layer (supernatant) took on a clean, bright blue hue
with a marked red fluorescence. The top layer grew and
held most of the crude extract containing C-PC whereas
the bottom layer was composed of broken algae and some
bacteria. The ratio of the supernatant and the bottom layer
increased to about 34:1. At this stage, the supernatant
was carefully removed and retained at room temperature
and the bottom layer discarded. The supernatant was
centrifuged at 10,000g for 30 min, the precipitate
discarded, and the supernatant containing the C-PC crude
extract cleaned by microfiltration (the pore size of the
membrane was 0.22 m). All subsequent procedures were
carried out at room temperature. The purification process
followed was a modification of an earlier method (Patel et
al. 2005).
Cell lysis efficiency, C-PC determination, and testing
the purity
The percentage of phycobiliprotein extracted, or the efficiency of extraction, was calculated using the equation
described by Kilpatrick (1985). This equation has been used
by other researchers as well (Viskari and Colyer 2003).
Extraction efficiency % A545supernatant =A545whole cell
 A750whole cell   100
C-PC concentration, purity, absorption spectra, and
fluorescence emission spectra were determined by the
methods described by Abalde et al. (1998) and Patel et al.
(2005), using an ultraviolet light-visible spectrophotometer
(SHIMADZU, Japan) and an F-4500 spectrofluorometer
(Hitachi, Japan). SDS-PAGE was conducted as reported
before (Bermejo et al. 1997). The protein marker was
obtained from Pharmacia (Uppsala, Sweden). The bacterial
samples were boiled for 5 min before incubation with the
sample solution.
The serial dilution method was used for measuring the
number of bacteria in the C-PC supernatant plated on the
same medium, namely, improved HS medium (Harris 1989)
with 1.5% agar. The plates were observed after 1 to 3 days.

246

Appl Microbiol Biotechnol (2007) 74:244248

The data are presented as the meansSD (n=10).

Statistical analysis was performed by one-way analysis of
variance or Duncans Multiple Range Test as appropriate
(SPSS 10.0; SPSS Inc., Chicago, IL, USA).

Results
The purity, extraction efficiency, and concentration were
higher in the products of the bacterial method of extraction
than in the products of methods based on sonication and
glass bead homogenization and the same as those in the
products of methods involving lysozymes or alternate
freezing and thawing (Table 1). However, repeated freezing
and thawing is time-consuming and, sometimes, energyintensive; four or more freezethaw cycles are usually
required. Lysozymes break the algal cell walls gently and
efficiently, and are potentially suitable for the large-scale
industrial extraction of C-PC. However, this process is not
only relatively expensive but also releases relatively unpleasant odours. Although the crude extract of K. pneumoniae also
gave off the characteristic odour of alcohol-fermentation, the
odour was mild and pleasant in comparison. The other four
methods require rinsing the algal slurry with K-phosphate
buffer three times. In the controls, formed by mixing the
algal slurry with either the algal-only or bacteria-only

medium or a mixture of the two, the slurry remained intact

even after several months.
Absorption and fluorescence emission spectra of the
crude extracts showed a major single peak at around
618 nm and 644 nm, respectively. The shoulder peak of
absorption at 652 nm is that of allophycocyanin (APC)
(Fig. 1). All were consistent with the C-PC characteristic
spectra. The profile of the bacterial extracts was similar to
that of the extracts obtained after lysozyme treatment or
after alternate freezing and thawing, which showed only
one major absorption peak of C-PC. While sonication and
glass-bead lysis were less efficient, the sample extracted
showed additional peaks at 680 and 450 nm because of
contamination with chlorophyll, which is indicative of the
disintegration of cells.
Figure 2 shows the SDS-PAGE of C-PC in each extract.
We did not find any obvious differences between lanes 4
and 7. Lane 4 was not contaminated with bacteria whereas
lane 6 was. Lanes 1 and 8 were not contaminated with other
proteins. The SDS-PAGE map suggests that there were no
differences in the levels of C-PC between the bacterial
extraction method and the freezethaw method. Two bands
(lanes 1 and 8) are visible, corresponding to the molecular
masses of 18,350 and 20,800 kDa, which represent the and
subunits of C-PC, respectively (Boussiba and Richmond
1979).

Table 1 Comparison of different C-PC extraction methods from Spirulina platensis

Extract methods

Treated
K. peneomieno
Control Medium of S. platensis
Medium of K. pneumoniae
Mix of the two
mediums above
Klebsiella oxytoca
Enterobacter gergoviae
Bacillus subtilis912
Fluorescence pseudomonas
78101
E. coli DH52
Yeast JRY4145
Lysozyme (2 mg/g wet weight)
Freezethaw (repeated four times from
20C to 25C)
Glass bead grinding (0.5 mm diameter)
Sonication (continued for 5 min,
frequency: repeated eight times)

Extraction
efficiency (%)

Phycocyanin concentration (mg/ml)

250 mg dry weight:10 ml

Purity ratio
(A615/280)

Time (h)

4.120.12a

1.090.08a

2030

pH
4.55
10.50
6.80
10.0

OD 600
1.36
0
0
0

91.002.95a
(+++)

3.92
4.85
7.98
7.88

1.37
1.32
1.30
1.33

++
++

5.72
4.67

1.35
1.35

89.343.17a
90.893.02a

4.080.08a
4.030.17a

1.070.06a
1.080.09a

2430
72

52.775.17b
42.654.34c

2.250.10b
2.130.09b

0.300.05b
0.250.04b

4
0.66

144

2430
144

The data was calculated from ten experiments, +++ refers to the best effect with K. pneumoniae, ++ refers to not perfect enough, is no
extract effect, refers to a little effect on the boundary. Mean values within a column with the same letters are not significantly different,
different letters indicate significantly different.

Appl Microbiol Biotechnol (2007) 74:244248

247

Discussion

3.0

2.0
1.5

bacteria
lysozyme
freeze-thaw
sonication
glass bead

4000

3000

2000

1.0
1000

Fluorescence intensity

Absorbance (a.u)

2.5

.5
0.0

0
300 400 500 600 700 650 750 800
Wavelength (nm)

Fig. 1 Comparison of the absorption and fluorescence spectra of CPC extracts from fresh S. platensis by different methods and recorded
from 600 to 800 nm (excitation and emission slits were 0.2 nm).
Fluorescence emission spectra were excited at 590 nm. Each curve is
the mean of three individual measurements; all data were recorded at
about 25C

Although K. pneumoniae population in the supernatant

was 105/ml when the crude C-PC was extracted, after
centrifuging at 10,000g for 30 min, the key procedure in
microfilteration, almost all bacteria were wiped off. We did
not find any K. pneumoniae growing on the plate.

Fig. 2 SDS-PAGE map. From left to right, lanes 1, 2, 3 and 4 are

from fresh S. platensis with K. pneumoniae: lane 1, pure C-PC; lanes
2 and 3, C-PC by the purification process; lane 4, crude extract; lane
5, the low-molecular-weight protein marker (-lactalbumin, 14,400;
trypsin inhibitor, 20,100; carbonic anhydrase, 30,000; ovalbumin,
43,000; albumin, 67,000; phosphorylase, 94,000); lane 6, bacterial
debris; lane 7, the crude extract after freezingthawing; lane 8, final
purified C-PC from the extract obtained after freezingthawing

When microorganisms do not naturally secrete products or

cannot be made to do so artificially, they must be
disintegrated to liberate their contents. When the intracellular components to be released are delicate, the disruption
process must be finely controlled to ensure that a balance is
maintained between efficient disruption and preservation of
the products (Follows et al. 1971). In this paper, we have
presented a new method for S. platensis cell lysis and
compared it with four popular methods for extracting C-PC
from fresh S. platensis (Table 1). Some of the difficulties
encountered during the conventional processes can be
overcome through the new method that deploys bacteria
for the lysis.
For further comparison, we chose some other familiar
bacteria, namely, E. gergoviae, K. oxytoca, B. subtilis 912, F.
pseudomonas 78-101, E. coli DH52, and S. cerevisiae
Y1980 (Table 1). When the OD 600 scale of these cultures
was approximately 1.3, they were mixed with fresh S.
platensis in a ratio of 2:1 (v/v) to observe the process of
extraction. Only E. gergoviae and K. oxytoca behaved
similarly to, although not as efficiently as, K. pneumoniae.
Occasionally, however, some samples showed no effect at
all. The other bacteria did not show any extraction at all after
24 h. S. platensis died out and changed the colour of the
mixture to yellow with the odour of decayed algae.
Unfortunately, crude C-PC did not show up in the extract.
After several days of cultivation, the yeast culture reached
low pH values, whereas a small amount of lysozyme could
be found in the B. subtilis culture after 72 h. A thin layer was
also seen at the boundary between bacteria and algae; this
boundary was dark blue, but the volumes of the two layers
separated by the boundary remained the same. The results
indicate that cell lysis may not be effective at acid pH values
or indeed the lysozyme itself, from the bacterial species in
question, may not be effective. It is also possible that
lysozyme is not the only enzyme responsible for algal cell
lysis: the bacteria may be synthesizing and secreting some
other enzymes that are responsible for the lysis.
We have also assessed other algae, including Synechococcus sp. PCC 7002, Anabaena sp. PCC 7120, Nostoc
flagelliforme, and Purple laver (data not shown), with
varying degree of success. We would like to emphasize that
it is important to control the vigour of the bacterial culture
because it affects the time taken for extracting C-PC.
Our method may be potentially useful in industrial
production. First, bacterial cultures are easy to obtain and
to maintain in culture, and the method is not limited by the
availability of machines. Second, bacterial cultures are
cheaper than specific enzymes for cell lysis. Third, this
method is relatively quick and labour-saving because it
does not involve repeated rinsing. Fourth, the whole

248

process occurs at about 25C with minimal demands on

temperature control. As for security, K. pneumoniae is a
non-pathogenic nitrogen-fixing species of bacteria; also,
after centrifugation, K. pneumoniae could be removed by
microfiltration. Thus, this method guarantees a clean supply
of CP-C. It should be noted that the phenomenon was
found incidentallyfurther experimentation and a wider,
more systematic search may turn up other bacteria that lyse
microalgal cells more efficiently. The new method opens a
fresh line of research.
Acknowledgements The authors are touched by the patience and
care shown by three unknown reviewers; the manuscript has benefited
from their precision and good advice. Thanks are also due to our
friends, Mr. Li B.X., Teng L.J. and Mrs. Qin X. Ch., for the
discussions on this study. This work was supported by the National
Natural Science Foundation of China (NSFC) grant (No. 40473051)
and Resource, Environment and GIS Key laboratory of Beijing City.

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