METHODS

A simple method for extracting C-phycocyaninfrom 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 freshSpirulina platensis by deploying a species of non-patho-genic nitrogen-fixing bacteria, namely, Klebsiella pneumo-niae. The algal slurry was neither washed nor centrifuged;the bacterial culture was poured into the slurry, the vesselsealed, and crude C-PC extracted after about 24 h. Theextraction was clean and efficient, and the purity andconcentration 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-harvestingprotein that is used in the cosmetic and pharmaceuticalindustries and in fluorescence labelling (Pulz and Gross

2004; Cohen 1986; Glazer and Stryer 1984). C-PC isextracted from cyanobacteria in a two-stage process. Thefirst stage involves the preparation of a cell-free extract todissolve C-PC in water to form a crude extract and thesecond stage involves purifying the crude extract to obtainC-PC in highly pure form. Extraction of phycobiliproteinsfrom cyanobacteria is difficult because the algae are smalland possess extremely resistant multilayered cell walls(Stewart and Farmer 1984; Wyman 1992). Many conven-tional methods are used to obtain crude extracts of C-PCincluding alternate freezing and thawing (Minkova et al.2003), lysozyme digestion (Boussiba and Richmond 1979),ultrasonication (Furuki et al. 2003), grinding with glassbeads (Scheer and Kufer 1977), grinding with silica-gel orcelite, and mechanical disruption using a pressure homog-enizer (Abalde et al. 1998). Other methods include nitrogencavitation for algal-cell disruption (Gottlieb and Adachi2003; Viskari and Colyer 2003), extraction using acids(Sarada et al. 1999), treatment with linear alkylbenzenesulfonate (Lin et al. 1998), and extraction by using highpressure (Herrero et al. 2004). Each method has itsadvantages and disadvantages. Methods for the secondstage include high-speed centrifugation, precipitation withammonium sulfate, and ion-exchange chromatography(Boussiba and Richmond 1979; Hayashi et al. 1997;Tchernov et al. 1999).

A comprehensive technique must include rapid andefficient cell lysis and extraction and recovery of thereleased C-PC in adequate quantities. The aim of thepresent study was to develop a simple and easy method forthe extraction of C-PC, potentially suitable for industrialproduction. We used a culture of Klebsiella pneumoniaepoured into fresh cyanobacteria (Spirulina platensis) toachieve a high recovery of C-PC of adequate quality fromthe crude extract.

Appl Microbiol Biotechnol (2007) 74:244–248DOI 10.1007/s00253-006-0636-7

Y. Zhu :X. B. Chen :K. B. Wang :Y. X. Li :K. Z. Bai :T. Y. Kuang (*)Key Laboratory of Photosynthesis and Environmental MolecularPhysiology, Photosynthesis Research Center, Institute of Botany,Chinese Academy of Sciences,Beijing 100093, Chinae-mail: kuang_tingyun@yahoo.com.cn

H. B. Ji (*)Resource, Environment and GIS Key Laboratory of Beijing City,Capital Normal University,Beijing 100037, Chinae-mail: hbji@mail.cnu.edu.cn

Y. ZhuGraduate School of the Chinese Academy of Sciences,Beijing 100039, China

Materials and methods

Reagents, buffers, and samples

Lysozyme was obtained from Sigma (St. Louis, MO, USA).All other reagents were of analytical grade. Extraction mediafor comparative studies and buffers for purification wereprepared by dissolving K-phosphate buffer stocks in distilleddeionized water (Millipore) so that the final concentrationwas 0.001 M and the pH of the media was 7.0. All themonitored samples of C-PC were diluted with this buffer,which was stored at room temperature and sterilized byautoclaving before use.

We used freshwater cyanobacterium S. platensis M2 (Luand Vonshak 1999) grown in a standard Zarrouks’medium (Zarrouk 1966) and cultivated as previouslyreported (Sarada et al. 1999). Fresh cyanobacterial cellswere harvested when the optical density (OD) 560 wasapproximately 0.9.

K. pneumoniae isolated from Mexico corn seeds wasidentified with Biolog as K. pneumoniae spp. Pneumoniae.The probability (PROB) is 95%, the similarity (SIM) is0.56. The species was reported as non-pathogenic and wasone of the normal associate nitrogen-fixing bacteria. Wegrew it in an improved HS medium (Harris 1989); the formof trace elements were exchanged to 1 g yeast, and 20 gsucrose per 1,000 ml was added to the medium. The culturewas inoculated from an agar slant of the same medium asmentioned above, incubated for 15–17 h and maintained atabout 30°C until the OD 600 reached approximately 1.3;the pH was down to about 4.5.

Enterobacter gergoviae and Klebsiella oxytoca wereisolated and preserved by the Institute of Botany, ChineseAcademy of Sciences; both are non-pathogenic nitrogen-fixing species of bacteria and were grown in the samemedium as mentioned above as K. pneumoniae. In addition,Bacillus subtilis 912, Fluorescence pseudomonas 78-101,Escherichia coli DH52, and S. cerevisiae Y1980 were alsopreserved by the Institute of Botany, Chinese Academy ofSciences. 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 discardedas possible and the algal layer saved; only a small amountof the medium was left, but it did not affect the extractionprocess. The algal layer was not rinsed; the layer was eitherretained in Erlenmeyer flasks or transferred to the othersterilized transparent containers. The pH of the algal culturewas 9.5–10.5. At this point, the prepared bacterial culturewas added to the algal layer; the ratio of the bacterial

culture to algae ranged from 1:1 to 2:1 (v/v). Then, the pHof the mixed supernatant ranged from 6.0 to 7.0. Thecontainers were sealed and stored at room temperature,protected from light, and the suspension allowed to settle.Despite some vibrating, two layers were clearly visible: theupper, yellowish, liquid layer, made up mostly of thebacterial culture, and the bottom blue-green layer of algalslurry. The algal slurry was immersed in the bacteriaculture. The contents were uncontaminated with otherliving organisms. After about 12 h, the boundary of thetwo layers could be seen as a thin light-blue layer fromthe transparent containers; gradually, however, the wholetop layer (supernatant) took on a clean, bright blue huewith a marked red fluorescence. The top layer grew andheld most of the crude extract containing C-PC whereasthe bottom layer was composed of broken algae and somebacteria. The ratio of the supernatant and the bottom layerincreased to about 3–4:1. At this stage, the supernatantwas carefully removed and retained at room temperatureand the bottom layer discarded. The supernatant wascentrifuged at 10,000×g for 30 min, the precipitatediscarded, and the supernatant containing the C-PC crudeextract cleaned by microfiltration (the pore size of themembrane was 0.22 μm). All subsequent procedures werecarried out at room temperature. The purification processfollowed was a modification of an earlier method (Patel etal. 2005).

Cell lysis efficiency, C-PC determination, and testingthe purity

The percentage of phycobiliprotein extracted, or the effi-ciency of extraction, was calculated using the equationdescribed by Kilpatrick (1985). This equation has been usedby other researchers as well (Viskari and Colyer 2003).

Extraction efficiency ð%Þ ¼ ½A545supernatant=ðA545whole cell

� A750whole cell Þ� � 100

C-PC concentration, purity, absorption spectra, andfluorescence emission spectra were determined by themethods 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 reportedbefore (Bermejo et al. 1997). The protein marker wasobtained from Pharmacia (Uppsala, Sweden). The bacterialsamples were boiled for 5 min before incubation with thesample solution.

The serial dilution method was used for measuring thenumber of bacteria in the C-PC supernatant plated on thesame medium, namely, improved HS medium (Harris 1989)with 1.5% agar. The plates were observed after 1 to 3 days.

Appl Microbiol Biotechnol (2007) 74:244–248 245

The data are presented as the means±SD (n=10).Statistical analysis was performed by one-way analysis ofvariance or Duncan’s Multiple Range Test as appropriate(SPSS 10.0; SPSS Inc., Chicago, IL, USA).

Results

The purity, extraction efficiency, and concentration werehigher in the products of the bacterial method of extractionthan in the products of methods based on sonication andglass bead homogenization and the same as those in theproducts of methods involving lysozymes or alternatefreezing and thawing (Table 1). However, repeated freezingand thawing is time-consuming and, sometimes, energy-intensive; four or more freeze–thaw cycles are usuallyrequired. Lysozymes break the algal cell walls gently andefficiently, and are potentially suitable for the large-scaleindustrial extraction of C-PC. However, this process is notonly relatively expensive but also releases relatively unpleas-ant odours. Although the crude extract of K. pneumoniae alsogave off the characteristic odour of alcohol-fermentation, theodour was mild and pleasant in comparison. The other fourmethods require rinsing the algal slurry with K-phosphatebuffer three times. In the controls, formed by mixing thealgal slurry with either the algal-only or bacteria-only

medium or a mixture of the two, the slurry remained intacteven after several months.

Absorption and fluorescence emission spectra of thecrude extracts showed a major single peak at around618 nm and 644 nm, respectively. The shoulder peak ofabsorption at 652 nm is that of allophycocyanin (APC)(Fig. 1). All were consistent with the C-PC characteristicspectra. The profile of the bacterial extracts was similar tothat of the extracts obtained after lysozyme treatment orafter alternate freezing and thawing, which showed onlyone major absorption peak of C-PC. While sonication andglass-bead lysis were less efficient, the sample extractedshowed additional peaks at 680 and 450 nm because ofcontamination with chlorophyll, which is indicative of thedisintegration of cells.

Figure 2 shows the SDS-PAGE of C-PC in each extract.We did not find any obvious differences between lanes 4and 7. Lane 4 was not contaminated with bacteria whereaslane 6 was. Lanes 1 and 8 were not contaminated with otherproteins. The SDS-PAGE map suggests that there were nodifferences in the levels of C-PC between the bacterialextraction method and the freeze–thaw method. Two bands(lanes 1 and 8) are visible, corresponding to the molecularmasses of 18,350 and 20,800 kDa, which represent the α andβ subunits of C-PC, respectively (Boussiba and Richmond1979).

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

Extract methods Extractionefficiency (%)

Phycocyanin concentration (mg/ml)250 mg dry weight:10 ml

Purity ratio(A615/280)

Time (h)

Treated pH OD 600 91.00±2.95a(+++)

4.12±0.12a 1.09±0.08a 20–30K. peneomieno 4.55 1.36

Control Medium of S. platensis 10.50 0 – 144Medium of K. pneumoniae 6.80 0 –Mix of the twomediums above

10.0 0 –

Klebsiella oxytoca 3.92 1.37 ++ 24–30Enterobacter gergoviae 4.85 1.32 ++Bacillus subtilis912 7.98 1.30 – 144Fluorescence pseudomonas78–101

7.88 1.33 –

E. coli DH52 5.72 1.35 –Yeast JRY4145 4.67 1.35 ±

Lysozyme (2 mg/g wet weight) 89.34±3.17a 4.08±0.08a 1.07±0.06a 24–30Freeze–thaw (repeated four times from−20°C to 25°C)

90.89±3.02a 4.03±0.17a 1.08±0.09a 72

Glass bead grinding (0.5 mm diameter) 52.77±5.17b 2.25±0.10b 0.30±0.05b 4Sonication (continued for 5 min,frequency: repeated eight times)

42.65±4.34c 2.13±0.09b 0.25±0.04b 0.66

The data was calculated from ten experiments, “+++” refers to the best effect with K. pneumoniae, “++” refers to not perfect enough, “−” is noextract 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.

246 Appl Microbiol Biotechnol (2007) 74:244–248

Although K. pneumoniae population in the supernatantwas 105/ml when the crude C-PC was extracted, aftercentrifuging at 10,000×g for 30 min, the key procedure inmicrofilteration, almost all bacteria were wiped off. We didnot find any K. pneumoniae growing on the plate.

Discussion

When microorganisms do not naturally secrete products orcannot be made to do so artificially, they must bedisintegrated to liberate their contents. When the intracel-lular components to be released are delicate, the disruptionprocess must be finely controlled to ensure that a balance ismaintained between efficient disruption and preservation ofthe products (Follows et al. 1971). In this paper, we havepresented a new method for S. platensis cell lysis andcompared it with four popular methods for extracting C-PCfrom fresh S. platensis (Table 1). Some of the difficultiesencountered during the conventional processes can beovercome through the new method that deploys bacteriafor the lysis.

For further comparison, we chose some other familiarbacteria, namely, E. gergoviae, K. oxytoca, B. subtilis 912, F.pseudomonas 78-101, E. coli DH52, and S. cerevisiaeY1980 (Table 1). When the OD 600 scale of these cultureswas approximately 1.3, they were mixed with fresh S.platensis in a ratio of 2:1 (v/v) to observe the process ofextraction. Only E. gergoviae and K. oxytoca behavedsimilarly to, although not as efficiently as, K. pneumoniae.Occasionally, however, some samples showed no effect atall. The other bacteria did not show any extraction at all after24 h. S. platensis died out and changed the colour of themixture 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 reachedlow pH values, whereas a small amount of lysozyme couldbe found in the B. subtilis culture after 72 h. A thin layer wasalso seen at the boundary between bacteria and algae; thisboundary was dark blue, but the volumes of the two layersseparated by the boundary remained the same. The resultsindicate that cell lysis may not be effective at acid pH valuesor indeed the lysozyme itself, from the bacterial species inquestion, may not be effective. It is also possible thatlysozyme is not the only enzyme responsible for algal celllysis: the bacteria may be synthesizing and secreting someother enzymes that are responsible for the lysis.

We have also assessed other algae, including Synecho-coccus sp. PCC 7002, Anabaena sp. PCC 7120, Nostocflagelliforme, and Purple laver (data not shown), withvarying degree of success. We would like to emphasize thatit is important to control the vigour of the bacterial culturebecause it affects the time taken for extracting C-PC.

Our method may be potentially useful in industrialproduction. First, bacterial cultures are easy to obtain andto maintain in culture, and the method is not limited by theavailability of machines. Second, bacterial cultures arecheaper than specific enzymes for cell lysis. Third, thismethod is relatively quick and labour-saving because itdoes not involve repeated rinsing. Fourth, the whole

Fig. 2 SDS-PAGE map. From left to right, lanes 1, 2, 3 and 4 arefrom fresh S. platensis with K. pneumoniae: lane 1, pure C-PC; lanes2 and 3, C-PC by the purification process; lane 4, crude extract; lane5, the low-molecular-weight protein marker (α-lactalbumin, 14,400;trypsin inhibitor, 20,100; carbonic anhydrase, 30,000; ovalbumin,43,000; albumin, 67,000; phosphorylase, 94,000); lane 6, bacterialdebris; lane 7, the crude extract after freezing–thawing; lane 8, finalpurified C-PC from the extract obtained after freezing–thawing

Wavelength (nm)

Flu

ores

cenc

e in

tens

ity

0

1000

2000

3000

4000

Abs

orba

nce

(a.u

)

0.0

.5

1.0

1.5

2.0

2.5

3.0

bacterialysozymefreeze-thawsonicationglass bead

300 400 500 600 750650 800700

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

Appl Microbiol Biotechnol (2007) 74:244–248 247

process occurs at about 25°C with minimal demands ontemperature control. As for security, K. pneumoniae is anon-pathogenic nitrogen-fixing species of bacteria; also,after centrifugation, K. pneumoniae could be removed bymicrofiltration. Thus, this method guarantees a clean supplyof CP-C. It should be noted that the phenomenon wasfound incidentally—further experimentation and a wider,more systematic search may turn up other bacteria that lysemicroalgal cells more efficiently. The new method opens afresh line of research.

Acknowledgements The authors are touched by the patience andcare shown by three unknown reviewers; the manuscript has benefitedfrom their precision and good advice. Thanks are also due to ourfriends, Mr. Li B.X., Teng L.J. and Mrs. Qin X. Ch., for thediscussions on this study. This work was supported by the NationalNatural Science Foundation of China (NSFC) grant (No. 40473051)and Resource, Environment and GIS Key laboratory of Beijing City.

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