Journal of Biotechnology 93 (2002) 73 85 www.elsevier.

com/locate/jbiotec

Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum

R. Bermejo Roma n a, J.M. Alva rez-Pez b, F.G. Acie n Ferna ndez c,*, E. Molina Grima c
a

Department of Physical and Analytical Chemistry, Jaen Uni6ersity, E.U.P. of Linares, Linares 23700, Spain b Department of Physical Chemistry, Granada Uni6ersity, Granada 18071, Spain c Department of Chemical Engineering, Almeria Uni6ersity, Almeria 04071, Spain Received 26 February 2001; received in revised form 3 July 2001; accepted 15 August 2001

Abstract Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria that is widely used as a uorescent probe and analytical reagent. In this paper, B-phycoerythrin and R -phycocyanin in native state, from the red alga Porphyridium cruentum were obtained by an inexpensive and simple process. The best results of this purication procedure were scaled up by a factor of 13 to a large preparative level using an anionic chromatographic column of DEAE cellulose. Gradient elution with acetic acid-sodium acetate buffer (pH 5.5) was used. In these conditions both 32% of B-phycoerythrin and 12% of R -phycocyanin contained in the biomass of the microalgae was recovered. B-phycoerythrin was homogeneous as determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE), yielding three migrating bands corresponding to its three subunits, consistent with the (ab)6g subunit composition characteristic of this biliprotein and the spectroscopic characterization of B-PE (UV visible absorption and emission spectroscopy; steady-state and polarization uorescence), is accompanied. Finally, a preliminary cost analysis of the recovery process is presented. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Porphyridium cruentum ; Phycobiliproteins; B-phycoerythrin; Scale-up

1. Introduction The phycobiliproteins are proteins with linear tetrapyrrole prosthetic groups (bilins) that, in their functional state, are covalently linked to specic cysteine residues of the proteins. These proteins are found in cyanobacteria (blue-green algae), in a class of biagellate unicellular eukaryotic algae (cryptomonads), and in Rhodophyta (red algae). In all of them the phycobiliproteins

Abbre6iations: APC, allophycocyanin; DEAE cellulose, diethylamino ethyl cellulose; PC, phycocyanin; PE, phycoerythrin; SDS-PAGE, sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. * Corresponding author. Tel.: + 34-950-015443; fax: + 34950-015484. E -mail address: facien@ual.es (F.G. Acie n Ferna ndez).

0168-1656/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 1 ) 0 0 3 8 5 - 6

74

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85

act as photosynthetic accessory pigments. Phycobiliproteins are used as colorants in food (chewing gums, dairy products, ice sherbaths, gellies, etc.) and cosmetics such as lipstick and eyeliners in Japan, Thailand and China (Dainippon Ink and Chemicals, 1985). It was also shown to have therapeutic value by immunomodulating activity and anticancer activity (Iijima and Shimamatsu, 1982). Owing to its uorescence properties it has gained importance in the development of phycouor probes for immunodiagnostics (Kronick and Grossman, 1983). In this sense, the red marine microalga Porphyridium cruentum is a Rhodophyta of increasing interest as a source of valuable phycobiliproteins, as well as sulphated exopolysaccharides, superoxide-dismutase, and polyunsaturated fatty acids (Vonshak, 1988) with applications in the food and pharmaceutical industries. The phycobiliproteins are assembled into an organized cellular structure, the phycobilisome. They absorb light, over a wide range of wavelengths in the visible part of the spectrum, and transfer the excitation energy by radiationless processes to the reaction centres in the photosynthetic membranes for conversion to chemical energy (Gantt, 1980; Glazer, 1976; Glazer and Wedemayer, 1995). The phycobiliproteins can be divided into three main classes depending on absorption properties: phycoerythrins (PE, umax 540 570 nm), phycocyanins (PC, umax 610 620 nm), and allophycocyanins (APC, umax 650 655 nm). Some cyanobacteria have a fourth type of biliprotein in place of PE, the phycoerythrocyanin (Gantt, 1981; Glazer, 1981). Visually, the phycoerythrins appear red, the phycocyanins range from purple (phycoerythrocyanin, R -phycocyanin) to deep blue (C-phycocyanin), and the allophycocyanins are blue with a hint of green. Phycoerythrins can be divided into three main classes, depending on their absorption spectrum, B-phycoerythrin (peaks at 545, 565 nm with a 499 shoulder), R -phycoerythrin (peaks at 499, 565 nm and a 545 shoulder) and C-phycoerythrin (peak at 565) (Glazer, 1984; Hilditch et al., 1991; GallandIrmouli et al., 2000). The introduction of phycobiliproteins as uorescent tags of cells and macromolecules was

followed by its widespread application in highly sensitive uorescence techniques such as uorescent activated cell sorting, ow cytometry, uorescence immunoassay and uorescence microscopy (Glazer and Stryer, 1984). B-PE has been shown to be particularly useful due to its large absorption coefcient and great uorescence properties just as the high quantum yield and high Stokes shift. B-PE uoresces in a spectral region that is distinct from the region of emission of the simple organic dyes commonly used as uorescent indicators. Therefore, B-PE is a valuable candidate in the design and characterization of light-sensing elements in biosensors (Ayyagari et al., 1995). Another interesting application of the phycobiliproteins is their use as natural dyes in foods and cosmetics replacing the synthetic dyes, since these are in general toxic, carcinogenic or otherwise unsafe. In Japan where algal cultivation is a well-developed industry, some natural pigments from phycobiliproteins have already been patented (Dainippon Ink and Chemicals Inc., 1979, 1987). Other colorants prepared from algae are also suitable for use in cosmetics (Arad and Yaron, 1992). Again, B-PE is the most valuable of the phycobiliproteins due to its intense and unique pink colour. On the other hand, its utilization as natural dyes in some kinds of heat-treated foods or in micelle solubilization makes it convenient to obtain smaller aggregates than hexameric B-PE (Bermejo et al., 2000). Pure phycobiliproteins from crude algal extracts are usually obtained by a combination of different and non-scaleable methods (Gray and Gantt, 1975; Grabowski and Gantt, 1978; Duerring et al., 1991; Ficner et al., 1992; Bermejo et al., 1997). Particularly, phycoerythrin is classically puried by a combination of several techniques such as ammonium sulphate precipitation, ion-exchange chromatography, gel ltration and chromatography on hydroxylapatite (Hilditch et al., 1991; DAgnolo et al., 1994; Schoelember et al., 1983; Ficner et al., 1992). Purication procedures are often long and complex. For this reason the use of this biliprotein has been somewhat limited by the tedious preparation of adequate amounts of the puried protein.

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85

75

In view of the multiple uses of phycoerythrin, in the present work a scaleable methodology for purication of large amounts of pure b-phycoerythrin from the red algae P. cruentum is proposed. The yield of the process at various stages is analysed. The high purity of B-PE obtained was conrmed by SDS-PAGE electrophoresis and the spectroscopic characterization of B-PE. Finally, the economic aspects of the process are discussed.

2. Materials and methods

2.1. Chemicals
Preswollen microgranular DEAE cellulose, molecular mass standards, dialysis tubing (avg. at. width 43 mm), dialysis tubing closures, ammonium sulphate and sodium azide were from Sigma diagnostics (St. Louis, MO, USA). The materials used for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were from Pharmacia (Uppsala, Sweden). All other chemicals were from Sigma and used without further purication.

chemical composition of the biomass is showed in Table 1. The biomass productivity was 1.2 g per l per day. The culture medium was Hemericks medium (1973), and the pH and temperature of the culture were controlled at 7.6 and 20 C, respectively. The photobioreactor consisted of an airlift pump that drove the culture uid through a horizontal tubular solar receiver (Camacho Rubio et al., 1999). The airlift section had a height of 3.5 m. The solar receiver was made of transparent Plexiglas tubes (0.05 m internal diameter, 0.005 m wall thickness) joined into a loop conguration by Plexiglas joints to obtain a total horizontal length of 98.8 m. The total culture volume in the bioreactor was 0.220 m3. Air was continuously supplied at a ow rate of 0.0124 mol s 1 whereas carbon was added as pure CO2 directly injected under pH requirement at a ow rate of 0.0014 mol s 1.

2.3. Phycobiliproteins purication 2.3.1. Initial stages (pre -treatments) All buffers mentioned in this paper contained 0.01% sodium azide unless otherwise specied. Initial stages followed the diagram shown in Fig. 1. Thus, frozen cells from P. cruentum were resuspended in 1 M acetic acid sodium acetate buffer (pH 5.5) at a ratio of 0.8 l per 1.0 kg of wet biomass. The slurry was homogenized and then sonicated for 10 min. After this, the slurry was centrifuged at 2500 g for 5 min in a Macrotronic Selecta centrifuge. The procedure was repeated again with the pellets, and the supernatants from both centrifugations were pooled. Extraction at high ionic strength leaves the phycobilisomes intact and would lead to the precipitation of all the phycobiliproteins present in the same centrifugation step (Glazer, 1976; Padgett and Krogmann, 1987). Solid (NH4)2SO4 was added to obtain 65% saturation, the resulting solution was allowed to stand for 12 h, and then centrifuged at 4500 g for 10 min. The pellets were resuspended in the same volume of 50 mM acetic acid sodium acetate buffer (pH 5.5) and dialyzed overnight against ten times volume of the same buffer (Fig. 1).

2.2. Microalgal biomass

The red microalga P. cruentum UTEX 161 was used. The biomass was obtained from chemostat cultures carried out in outdoor tubular photobioreactors at 0.049 h 1 dilution rate. The bioTable 1 Biochemical composition of the biomass produced in outdoor tubular reactors and utilised for purication of phycobiliproteins Content, %d.w. Main components Proteins Carbohydrates Lipids Ash Pigments Phycobiliproteins Chlorophylls Carotenoids

33.00 35.80 6.70 19.70 2.00 0.65 0.10

76

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85

maintained at 1300 ml h 1 (0.34 ml cm 2 min 1) until the eluate became coloured. The eluate was collected in 25 ml fractions. The coloured fractions were collected and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and UV visible absorption spectroscopy. The fractions were brought to 65% saturation with (NH4)2SO4 and left to stand overnight in the dark at 4 C prior to centrifugation at 16 000 g for 25 min in a J2-21 Beckman centrifuge. The pellets were resuspended in a small volume of 20 mM sodium phosphate buffer (pH 7.0) and dialyzed exhaustively overnight at 4 C against distilled water and freeze-dried for storage in a Edwards freezedryer, until use.

2.4. SDS -PAGE

Electrophoresis was carried out in a vertical slab gel apparatus (Miniprotean III, Bio-Rad) according to the tricine buffer system described by Shagger and Von (1987) using a 16.5% polyacrylamide slab gel containing 0.1% (w/v) SDS with a stacking gel of 4% polyacrylamide. Samples were pre-incubated with 4% (w/v) SDS, 12% (v/v) glycerol, 2% (v/v) b-mercaptoethanol, 0.025% (w/v) bromophenol blue, and 50 mM Tris, pH 6.8, for about 5 min at 95 C. Gels were run at room temperature, and visualized by staining for 30 min with 0.1% (w/v) Coomassie Brilliant Blue R-250, 40% methanol (v/v) with 7% (v/v) acetic acid and destained in dilute acetic acid. The following proteins were used as molecular mass markers: carbonic anhydrase (30 000), trypsin inhibitor (21 100), cytochrome (12 400) and aprotinin (6500).

Fig. 1. Initial stages on the B-PE and R-PC analytical purication process.

2.3.2. Chromatography For the small scale chromatography the dialyzed phycobiliproteins-containing solution was applied to a column (15.0 2.5 cm) of DEAE cellulose, pre-equilibrated with 50 mM acetate buffer (pH 5.5). After washing with 200 ml of starting buffer (50 mM), the column was developed with 200 ml of 0.25 M acetic acid sodium acetate buffer (pH 5.5). The column was then developed with 300 ml of 0.35 M acetic acidsodium acetate buffer (pH 5.5). The ow rate was maintained at 100 ml h 1 (0.34 ml cm 2 min 1) until the eluate became coloured. The eluate was collected in 2.5 ml fractions. For the large scale chromatography the dialyzed phycobiliproteins-containing solution was applied to a column (15.0 9.0 cm) of DEAE cellulose, pre-equilibrated with 50 mM acetate buffer (pH 5.5). After washing with 800 ml of starting buffer (50 mM), the column was developed with 800 ml of 0.25 M acetic acid sodium acetate buffer (pH 5.5). Then, the column was developed with 1000 ml of 0.35 M acetic acid sodium acetate buffer (pH 5.5). The ow rate was

2.5. Spectroscopic methods

Absorbance at 280, 545, 565, 620 and 650 nm and absorption spectra were recorded on a Perkin Elmer (Beaconseld, UK) Lambda-16 UV-Visible spectrophotometer with a 1 cm light path. Fluorescence emission spectra were recorded on a Shimadzu (Kyoto, Japan) RF5001 spectrouorometer. For steady state polarized uorescence measurements the spectrouorometer was

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85

77

equipped with polarizers in the excitation and emission paths. The anisotropy was calculated as A = (IVV GIVH)/(IVV + 2GIHH), where G = IHV/ IHH, a correction factor for the polarization due to the optics in the instrument. Protein concentrations were chosen so that re-absorption of the emission was negligible. All spectra were recorded at room temperature. The amounts of B-PE, R-PC and APC in the different extracts and biliprotein containing solutions were calculated from measurements of the absorbance at 565, 620 and 650 nm using the following equations. R-PC(mg ml 1) = (OD620 nm 0.7OD650 nm) 7.38 (OD650 nm 0.19OD620 nm) APC(mg ml 1) = 5.65 B-PE(mg ml 1) = (OD565 nm 2.8[R PC] 1.34[APC]) 12.7 (3) (1) (2)

These equations were established by using the simultaneous equations of Bennet and Bogorad (1973) and the extinction coefcients from Bryant et al. (1979).

3. Results The rst phycobiliproteins determination was carried out by precipitation of crude biomass extract with 1% (w/v) streptomycin sulphate for

Fig. 2. Absorption spectrum of crude extract obtained from the biomass, and pre-treated extract obtained after the initial stages.

30 min at 4 C and centrifugation at 2500 g for 10 min, in order to eliminate membrane fragments containing chlorophyll. The absorption spectrum of this crude extract is shown in Fig. 2. In this spectrum three peaks corresponded to the maxima of B-PE (565, 545 nm and 498 shoulder), the other peak corresponded to the maxima of R-PC (620 nm), and no peak was observed for A-PC (650 nm). This indicated that A-PC content is negligible or absent relative to R-PC and B-PE in P. cruentum. Analysis of the dry biomass indicated that the phycobiliproteins content was 1.66% d.wt. for B-PE and 0.34% d.wt. for R-PC. Thus, from 1.9 kg of biomass we can expect to obtain 31.3 g of B-PE and 6.4 g of R-PC. However, after the initial stages of processing a concentrated fraction with 13.8 g of B-PE and 2.0 g of R-PC was obtained, with concentrations of 0.55% d.wt. of B-PE and 0.08% d.wt. of R-PC (Table 2). The spectrum of this extract (Fig. 2) shows that the absorbance at 280 nm greatly decreases from the crude extract, whereas peaks of B-PE (545, 565 nm) are the main components. Peaks from R-PC also appear (620 nm) and no peaks corresponding to A-PC (650 nm) exist. This puried extract was utilized in the chromatography step, the content in phycobiliproteins being 5.52 g l 1 of B-PE and 0.80 g l 1 of R-PC, and with a composition of 87.2% of B-PE and 12.8% of R-PC. Before proceeding with the ion exchange chromatography, the pH and the initial ionic strength were selected by preliminary tests with continuous and simultaneous gradients of both pH and ionic strength (Bermejo et al., 2001). Results showed that an ionic strength discontinuous gradient was enough for separation of the phycoerythrin. The R-PC was eluted at 0.05 M acetic acid sodium acetate buffer (pH 5.5), whereas B-PE was eluted at 0.25 M acetic acid sodium acetate buffer (pH 5.5). The chromatography step was optimised on a small scale by using a column (2.5 15.0 cm) with different ow rates and different loadings of phycobiliproteins mixture. The best results were obtained with a ow rate of 100 ml h 1 (0.34 ml cm 2 min 1), whereas the optimum loading was 7.3 mg of phycobiliproteins. The recovery of pure fractions with A545/A280 \ 3 remained constant

78

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85

Table 2 Weight and composition of the materials obtained in each one of the steps (depicted in Fig. 1) of the initial stages Biomass Paste Step 1 Solid 1 Weight, g 1884 PC, %d.wt. 0.340 PE, %d.wt. 1.663 Phycobil., 2.003 %d.wt. PC, % of 16.97 TPB* PE, % of 83.03 TPB* Phycobil., 37.74 g PC, g 6.40 PE, g 31.34 18 902 0.033 0.161 0.193 16.81 83.19 36.56 6.15 30.42 10 000 0.021 0.113 0.134 15.86 84.14 13.43 2.13 11.30 Supern. 1 8860 0.042 0.213 0.256 16.55 83.45 22.65 3.75 18.90 Step 2 Filtered 8800 0.040 0.209 0.249 16.07 83.93 21.95 3.53 18.43 Step 3 Supern. 2 8000 0.010 0.039 0.049 21.03 78.97 3.93 0.83 3.10 Solid 2 2200 0.121 0.638 0.759 15.93 84.07 16.70 2.66 14.04 Step 4 Supern. 3 2500 0.081 0.552 0.633 12.82 87.18 15.83 2.03 13.80 Solid 3 505.8 0.064 0.403 0.468 13.77 86.23 2.37 0.33 2.04

TPB, Total phycobiliproteins content.

when the loading increases from 3.5 till 7.3 mg of phycobiliproteins then continuously decreasing, thus indicating the saturation of the stationary phase at this value (Fig. 3). By operating in this conditions, it was possible to obtain two fractions of phycobiliproteins: a blue coloured fraction I with absorption maxima at 280, 310, 375, 555 and 620 nm and with absorbance ratio A620/A280 \ 3 characteristic of R-PC and other colourless proteins, and a pink coloured fraction II with absorption maxima at 280, 310, 375, 545 and 565 nm a shoulder at 498 nm, and with absorbance ratio A565/A280 \ 4 characteristic of B-PE (Fig. 4). The chromatographic procedure was scaled to a large preparative level. This was done using the same supercial velocity in the column, but with a wider cross-sectional area to increase capacity. Flow characteristics in the packed bed were thus preserved and the concentration proles of different solutes were not greatly altered (Belter et al., 1988). The scale-up ratios of ow rate ( f ) and sample load (m ) were: f = m = D2 2 D2 1 L2D 2 2 L1D 2 1 (4) (5)

where D1 and D2 are the diameters of the smaller and the larger columns, respectively, and L1 and L2 are the respective lengths. We used L1 = L2 = 15 cm and then f and m were equal to 13. Thus, using a new column (15.0 9.0 cm) the mobile phase ow rate was 1300 ml h 1 (0.34 ml cm 2 min 1) and the loading of the phycobiliproteins mixture was 95 mg. Operating in these conditions (Fig. 5) the same two fractions were separated as at the small scale. Although the most utilized criteria to check biliprotein solutions purity is the absorbance ratio Amax visible/A280, the purity must be followed by

Fig. 3. Inuence of loading in the recovery of phycobiliproteins at small scale chromatography.

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85

79

Fig. 4. (A) Elution curve of different fractions from Porphyridium cruentum after small scale DEAE-cellulose chromatography. (B) Absorption spectrum of fraction I. (C) Absorption spectrum of fraction II.

electrophoresis experiments in order to conrm it. In this way, the spectroscopic and electrophoresis analysis of the fraction II (B-PE) obtained from the large-scale chromatography process was carried out (Fig. 6). Electrophoresis analysis shows that fraction II yields three bands corresponding to B-PE subunits: a, b, g (Fig. 6A). By comparison with standards, we obtained molecular mass of the a, b and g-subunits of about 16 500, 18 000 and 27 000 Da, respectively. Fig. 6B shows visible absorption, uorescence emission and anisotropy spectra of puried B-PE. Absorbance maximum is 545 nm, whereas the uorescence emission maximum is 574 nm and the anisotropy spectrum is similar to those earlier published for hexameric phycoerythrins from other red algae (MacColl, 1991; Guard-Friar et al., 1989).

4. Discussion The phycoerythrin productivity obtained from P. cruentum outdoor cultures was slightly lower than maximum values referenced indoor. Thus, in this work a productivity of 20 mg B-PE per l per day was obtained, much higher than 3 mg per l per day obtained from Calothrix spp. (OlveraRam rez et al., 2000), but similar to 25 mg per l per day referenced for P. cruentum (Lee and Tan, 1988), or 18 mg per l per day referenced for Gracilaria tenuistipatata (Carnicas et al., 1999), all of them at indoor conditions. Maximum productivity of 49 mg per l per day has been referenced in small-scale semicontinuous indoor cultures of P. cruentum (Fa bregas et al., 1998). Considering that synthesis of phycobiliproteins increases under

80

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85

light-limiting conditions, the high productivity reached outdoor under high irradiances indicates that this productivity could be yet enhanced, thus P. cruentum being a potential source of these products. In addition, P. cruentum had both types of phycobiliproteins; phycoerythrin and phycocyanin. However, there was only a compound for each type of phycobiliprotein, B-PE and R-PC, thus making easier the downstream process. Until now very different methodologies have been proposed for purifying phycobiliproteins from microalgae but only some of them are useful for scale-up. In addition, the yield of the proposed process is usually not calculated only focusing on the purity of the product. Most of the proposed methods could be divided into two steps: a rst step of extraction of the phycobiliproteins from the biomass, and a second step of purication of the obtained extract. In our work, evaluating the

yield of each step in the initial stages (Fig. 7) it was observed that mainly phycobiliproteins were released in the extraction (step 1) and precipitation (step 3). Thus, in the extraction process (step 1) only 60% of the phycobiliproteins were recovered, whereas in the precipitation process (step 3) the yield was 75%. The overall recovery at the end of the pre-treatments was only 43%. In addition, the yields were slightly different for the two phycobiliproteins, being higher for B-PE (45.4% recovery) and lower for R-PC (33.0% recovery). Thus, the yield of the initial stages needs to be enhanced. There are different possibilities, from optimisation of the cell disruption, extraction and precipitation conditions, to use of other methodologies such as expanded bed chromatography or ultraltration. Anywhere, the methodology used in this work provides better results than others referenced. Thus, Galland-Irmouli et al. (2000)

Fig. 5. (A) Elution curve of different fractions from Porphyridium cruentum after large scale DEAE-cellulose chromatography. (B) Absorption spectrum of fraction I. (C) Absorption spectrum of fraction II.

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85

81

Fig. 6. (A) Electrophoresis gel and spectral characteristics of B-PE puried in 20 mM sodium phosphate buffer pH 7.0. (B) Absorption spectra ( ), Fluorescence emission spectra (uex = 540 nm; Duex = Duem = 1.5 nm) (------). Absorption and uorescence spectra were normalized since these were only used on a comparative basis. Fluorescence excitation anisotropy spectra (uem = 595 nm, Duex = Duem = 3 nm and excitation wavelength range = 460 560 nm) (). The anisotropy values shown are the means of ve values and were calculated for each 5 nm. All spectra were recorded at room temperature.

Most of the works only indicate the overall yield. Thus, in the well documented processes the recovery yield is very low, lower than 5%. Thus, Jung and Dailey (1989) proposed a method based on LPLC to purify phycocyanin from Spirulina with a 1.2% yield. Galland-Irmouli et al. (2000) used electrophoresis to purify phycoerythrin from Palmaria palamata yielding 3.7%, whereas Hilditch et al. (1991) proposed a method based on three step chromatography from Corallina ofci nalis with a yield lower than 1%. Only the chromatographic methodology proposed by Tchernov et al. (1999) gave yields higher than 10%, being 24% for phycoerythrin from Nostoc spp. In this work we observed that the yield of the overall process was 32.7% of B-PE and 11.9% of R-PC at large scale. Similar results were obtained at small scale thus verifying that the scale up criteria used was valid in the range used. However, at large scale the purity of the fractions was higher than at small scale. The same behaviour has been previously referenced (Hilditch et al., 1991) although it had not been explained. Moreover, because most of the studies were carried out at small scale, in all of them a second purication step was needed (Hilditch et al., 1991; Tchernov et al., 1999; Galland-Irmouli et al., 2000; Bermejo et al., 1997, 2001). To analyse this phenomena the efciency at each scale was quantied by the number of theoretical plates (NTP) as (Christie, 1987), NTP = 5.54

 
tr wb

(6)

indicates 18% of recovery when the biomass is frozen with liquid nitrogen and extracted with phosphate solution, whereas Sarada et al. (1999) indicates that 50% of the proteins were lost when the biomass was dried. Only Tchernov et al. (1999) indicates a high yield of 60% using frozen biomass, phosphate buffer and rivanol to precipitate colourless proteins.

Fig. 7. Variation of recovery in each one of the steps depicted in Fig. 1.

82

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85

Table 3 Analysis of efciency of ion exchange chromatography at small and large scale for both phycobiliproteins: R-PC and B-PE Scale Small Small Large Large Phycobiliprotein R-PC B-PE R-PC B-PE t r, h 0.290 0.390 0.308 0.400 w b, h 0.149 0.473 0.092 0.287 NTP 21 4 62 11

where tr is the retention time and wb is the wide time of the peak. Results showed that the NTP at the large scale chromatography was higher than at the small scale chromatography for both proteins (Table 3). In both cases the same particles and conditions were used, the only difference is the column diameter to particle size ratio. This ratio was 250 for the small scale chromatography whereas it was 900 for the large scale. In both cases, the values were high enough to ensure high packing quality. The higher efciency of the large scale versus the small scale is, likely, due to the reduction of the extra-column band spreading at large scale. When a chromatographic process is scaled the ratio between extra-column to column band spreading decrease. This implies that if the effective performance of the column remained constant the total performance at larger scale is higher. Regarding the purity of the fractions, SDSPAGE shows that fraction II yields three bands corresponding to B-PE subunits (Fig. 6a). The a-subunit contains 164 amino-acid residues and a calculated molecular mass (including the two PEB chromophores) of 18 991 Da, therefore, its mobility is larger than that of the b-subunit that contains 177 residues and has a molecular mass of 20 315 Da including 3 PEB (Sidler et al., 1989), both are present in similar amounts. The third band corresponds to the g-subunit, a minor component of apparent molecular mass of about 27 000 Da (Ficner et al., 1992; Koller and Wehrmeyer, 1975; Redlinger and Gantt, 1981). By comparison with standards, we obtained molecular mass of the a, b and g-subunits of about 16 500, 18 000 and 27 000 Da, respectively. These values are in agreement with the previously reported values estimated by SDS-PAGE (Ficner et al., 1992; Koller and

Wehrmeyer, 1975; Redlinger and Gantt, 1981). The band intensities agree with a heteropolymer with the polypeptide composition (ab)6g. Fig. 6B shows visible absorption, uorescence emission and anisotropy spectra of puried B-PE and these agree well with those published for pure B-PE elsewhere (Ficner et al., 1992; Koller and Wehrmeyer, 1975; Kirk et al., 1993). In the pH range 5 7 and low ionic strength, phycoerythrins show an aggregation state type (ab)6g (MacColl et al., 1971). The uorescence emission maximum is 574 nm. In general, the uorescence spectra of phycobiliproteins are modulated by uorescence resonance energy transfer (FRET) between the several chromophores that they have in their different subunits (Dale and Teale, 1970), therefore, when the aggregation state of the phycobiliproteins changes from hexamers to monomers, energy transfer decreases and a dramatic increase in the uorescence excitation anisotropy spectra must be observed. The anisotropy spectrum shape of B-PE in 20 mM sodium phosphate buffer pH 7.0 is similar to those earlier published for hexameric phycoerythrins from other red algae (Grabowski and Gantt, 1978; GuardFriar et al., 1989; MacColl, 1991). Finally, the owchart in Fig. 8 shows the process by which highly concentrated B-PE and R-PC fractions are obtained from the biomass cultivated in an outdoor tubular photobioreactor, including all the unit operations of the downstream processing. The equipment has been sized considering a total capacity of the plant of 2.1 kg B-PE per day, and a yield of 33% of recovery thus being necessary to process 400 kg of biomass per day. Table 4 shows a preliminary estimation of the cost obtaining B-PE from wet P. cruentum biomass. The obtained unit production cost is around 55 000$ per kg B-PE, higher than 30 000$ per kg PC reported for phycocyanins by LPLC (Campanella et al., 2000), and lower than 60 000$ per kg PC for phycocyanins by HPLC (Campanella et al., 2000), anywhere this price being quite expensive for the food industry. The market price of the product for pharmaceutical or uorescent uses is 50$ mg 1 (Martek corporation, 1999; Haugland, 1996). The main factor inuencing this unit production cost is the xed capital per year (72%), whereas the raw material and others

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85

83

Fig. 8. Indicative owchart of B-PE and R-PC purication from wet Porphyridium cruentum biomass.

Table 4 Approximate estimation of the costs of obtaining B-PE from wet Porphyridium cruentum biomass Item Major Equipment List and Costs Fixed capital investment Fixed capital per year (US $) Raw materials Utilities Others Total direct production costs Total production costs Unit cost of producing PE ($/kg) Major Equipment List and Costs Item Slurry tank (m3, c.s., motor) Centrifuge (24 in. bowel solids discharge, s.s.), m3 h1 Homogenisers (m3 h1, c.s.) Filter (m2) Crystalliser (m3 per day) Dialysis unit (m3 per day) Chromatography system (s.s.) Total Size 4.00 2.00 2.00 80.00 4.00 0.40 0.40 Delivered cost 7888 98 486 53 183 6172 22 361 1691 1178 Number of units 3 1 1 1 1 1 1 Total cost 23 663 98 486 53 183 6172 22 361 1691 1178 Percentage 11.45 47.64 25.73 2.99 10.82 0.82 0.57 206 734 Cost, $ 206 734 791 857 86 061 16 425 4 16 977 33 406 119 467 56 566 Percentage (%)

72 14 0 14 28 100

Methodology used for cost analysis is detailed elsewhere (Kalk and Langlykke, 1986). Cost of the equipment calculated from Perry and Green (1999). Plant capacity, 2.1 kg B-PE per day. Biomass cost, 20$ per kg. Labor, 2 men per day. c.s., carbon steel; s.s, stainless steel.

84

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85 Bioseparation, Downstream Processing for Biotechnology. Wiley-Interscience Publication, Wiley, New York, pp. 209 213. Bennet, A., Bogorad, L., 1973. Complementary chromatic adaptation in a lamentous blue-green algae. J. Cell. Biol. 58, 419 422. Bermejo, R., Talavera, E.M., Alvarez-Pez, J.M., Orte, J.C., 1997. Chromatographic purication of phycobiliproteins from Spirulina platensis. High-performance liquid chromatographic separation of their alfa and beta subunits. J. Chromatogr. A 778, 441 450. Bermejo, R., Talavera, E.M., DelValle, C., Alvarez-Pez, J.M., 2000. C-phycocyanin incorporated into reverse micelles: a uorescence study. Coll. Surf. B Biointerf. 18, 51 59. Bermejo, R., Talavera, E.M., Alvarez-Pez, J.M., 2001. Chromatographic purication and characterization of B-phycoerythrin from Porphyridium cruentum. Semipreparative HPLC separation and characterization of its subunits. J. Chromatogr. A 917, 135 145. Bryant, D.A., Guglielmi, G., Tandeu de Marsac, N., Castets, A.M., Cohen-Bazire, G., 1979. The structure of cyanobacterial phycobilisomes: a model. Arch. Microbiol. 123, 113 127. Camacho Rubio, F., Acie n Ferna ndez, F.G., Sa nchez Pe rez, J.A., Garc a Camacho, F., Molina Grima, E., 1999. Prediction of dissolved oxygen and carbon dioxide concentration proles in tubular photobioreactors for microalgal culture. Biotechnol. Bioeng. 62 (1), 71 86. Campanella, L., Crescentini, G., Avino, P., Angiello, L., 2000. Simple and rapid procedure for analyzing two phycocyanins (C-PC and APC) from Spirulina platensis algae using LPLC and HPLC methods. Annali di Chimica 90, 153 161. Carnicas, E., Jimenez, C., Niell, F.X., 1999. Effects of changes of irradiance on the pigment composition of Gracilaria tenuistipitata var. liui Zhang et Xia. J. Photochem. Photobiol. B Biol. 50, 149 158. Christie, W.W., 1987. HPLC and Lipids: A Practical Guide, Chapter two: High Performance Liquid Chromatography: Theoretical Considerations and Equipment. Pergamon Press, Oxford, UK, pp. 9 41. DAgnolo, E., Rizzo, R., Paoleti, S., Murano, E., 1994. R -phycoerythrin from the red alga Gracilaria longa. Phytochemistry 35, 693 696. Dainippon Ink and Chemicals Inc., 1979, Japanese Patent 95: 770. Dainippon Ink and Chemicals Inc., 1987, Japanese Patent 06: 691. Dainippon Ink and Chemicals, 1985, Lina blue A (Natural blue colorant of Spirulina origin), Technical information. Tokyo, Japan: Dainippon Ink and Chemicals. Dale, R.E., Teale, F.W.J., 1970. Number and distribution of chromophore types in native phycobiliproteins. Photochem. Photobiol. 12, 99 117. Duerring, M., Schmidt, G.B., Huber, R., 1991. Isolation, crystallization, crystal structure analysis and renement of constitutive C-phycocyanin from the chromatically adapting

(mainly labour) only represent 28% of the unit production costs. The xed capital per year is directly a function of major equipment cost, the cost of centrifuges representing 50% of the total major equipment cost, whereas the ionic exchange chromatography step represent only 15% of this major equipment cost. Thus, the optimisation of these steps can strongly reduce this unit production cost.

5. Conclusions With these results we can conclude that the above described purication method is a one step scaleable chromatographic method that provides B-PE solutions in hexameric aggregation state, as well as pure fractions of R-PC. The feasibility of the method has been veried in a 13 fold scale up system, being the only method developed for phycoerythrin. In addition, the methodology described here demonstrates the feasibility of using the red algae P. cruentum for the preparation of large quantities of homogeneous B-PE and R-PC. However, the yield of the process must be enhanced mainly by optimising the initial stages of the downstream process.

Acknowledgements This research was supported by the Plan Propio de Investigacio n from University of Almer a and Plan Andaluz de Investigacio n II, Junta de Andaluc a.

References
Arad, S., Yaron, A., 1992. Natural pigments from red microalgae for use in foods and cosmetics. Trends Food Sci. Technol. 3, 92 97. Ayyagari, M., Pande, R., Kamtekar, S., Gao, H., Marx, K., Kumar, J., Tripathy, S., Akkara, J., Kaplan, D., 1995. Molecular assembly of proteins and conjugated polymers: toward development of biosensors. Biotechnol. Bioeng. 45, 116 121. Belter, P.A., Cussler, E.L., Hu, W.S., 1988. Elution chromatography. In: Belter, P.A., Cussler, E.L., Hu, W.S. (Eds.),

R. Bermejo Roma n et al. / Journal of Biotechnology 93 (2002) 73 85 cyanobacterium Fremyella diplosiphon at 166 D resolution. J. Mol. Biol. 217, 577 592. Fa bregas, J., Garc a, D., Morales, E., Dom nguez, A., Otero, A., 1998. Renewal rate of semicontinuous cultures of the microalga Porphyridium cruentum modies phycoerythrin, exopolysaccharides and fatty acid productivity. J. Ferment. Bioeng. 86 (5), 477 481. Ficner, R., Lobeck, K., Schmidt, G., Huber, R., 1992. Isolation, crystallization, crystal structure analysis and renement of B-phycoerythrin from the red alga Porphyridium sordidum at 2.2 A resolution. Mol. Biol. 228, 935 950. Galland-Irmouli, A.V., Pons, L., Lucon, M., Villaume, C., Mrabet, N.T., Gueant, J.L., Fleurence, J., 2000. One-step purication of R-phycoerythrin from the red macroalga Palmaria palmata using preparative polyacrylamide gel electrophoresis. J. Chromatogr. B 739 (1), 117 123. Gantt, E., 1980. Structure and function of phycobilisomes: light harvesting pigment complexes in red and blue-green algae. Int. Rev. Cytol. 66, 45 80. Gantt, E., 1981. Phycobilisomes. Ann. Rev. Plant Physiol. 32, 327 347. Glazer, A.N., 1976. Phycocyanins, structure and function. Photochem. Photobiol. Rev. 1, 71 115. Glazer, A.N., 1981. Photosynthetic Accesory Proteins with Bilin Prosthetic Groups. The Biochemistry of Plant, vol. 8. Academic Press, New York, p. 51. Glazer, A.N., 1984. Phycobilisome. A macromolecular complex optimized for energy tranfer. Biochim. Biophys. Acta 768, 29 51. Glazer, A.N., Stryer, L., 1984. Phycouor probes. Trends Biochem. Sci. 9, 423 427. Glazer, A.N., Wedemayer, G.J., 1995. Cryptomonad phycobiliproteins-an evolutionary perspective. Photosynthesis Res. 46, 93 105. Grabowski, J., Gantt, E., 1978. Photophysical properties of phycobiliproteins from phycobilisomes: uorescence lifetimes, quantum yields and polarization spectra. Photochem. Photobiol. 28, 39 45. Gray, B.H., Gantt, E., 1975. Spectral properties of phycobilisomes and phycobiliproteins from the blue-green alga Nostoc spp. Photochem. Photobiol. 21, 121 128. Guard-Friar, D., Hanlik, C., MacColl, R., 1989. Phycoerythrin 566. A uorescence study. Biochim. Biophys. Acta 973, 118 123. Haugland, R.P., 1996. Handbook of Fluorescent and Research Chemicals, Six ed. Molecular Probes, Eugene, OR ISBN:09652240-2-3. Hemerick, G., 1973. Culture Methods and Growth Measurements. In: Stein, J.R. (Ed.), Handbook of Physiological Methods. Cambridge University Press, Cambridge UK, pp. 259 260. Hilditch, C.M., Balding, P., Kakins, R., Smith, A.J., Rogers, L.J., 1991. C-Phycocyanin from the cyanobacterium Aph nothece halophytica. J. Appl. Phycol. 3, 345. Iijima, N., Shimamatsu, H., 1982. Antitumor agent and method of treatment there-with, US patent pending. Ref p1150-726A82679, Appl. 15. Jung, T.M., Dailey, M.O., 1989. A nobel and inexpensive source of allophycocyanin for multicolor ow cytometry. J. Immunol. Methods 121, 9 18.

85

Kalk, J.P., Langlykke, A.F., 1986. Cost estimated for biotechnology projects. In: Demain, A.L., Solomon, N.A., (Eds.), Manual of Industrial Microbiology and Biotechnology, Washington American Society of Microbiology. Kirk, E., Hoffman, N., Grossman, R., 1993. The gamma subunit of R -phycoerythrin ant its possible mode of transport into the plastid of red algae. J. Biol. Chem. 268, 16208 16215. Koller, K., Wehrmeyer, W., 1975. B-phycoerythrin from Rhodella 6iolacea. Arch. Microbiol. 104, 255 261. Kronick, M.N., Grossman, A.R., 1983. Immunoassay techniques with uorescent phycobiliprotein conjugates. Clin. Chem. 29, 1582 1586. Lee, Y.K., Tan, H.M., 1988. Effect of temperature, light intensity and dilution rate on the cellular composition of red alga in light-limited chemostat cultures. MIRCEN J. 4, 231 237. MacColl, R., Lee, L.J., Berns, D.S., 1971. Protein aggregation in C-phycocyanin. Studies at very low concentrations with the photoelectric scanner of the ultracentrifuge. Biochem. J. 122, 421 426. MacColl, R., 1991. Fluorescence Studies on R -phycoerythrin and C-phycoerythrin. J. Fluoresc. 1, 135 140. Martek corporation, 1999, web page: www.martekbio.com. Olvera-Ram rez, R., Coria-Cedillo, M., Can izares-Villanueva, R.O., Mart nez-Jero nimo, F., Ponce-Noyola, T., Rios-Leal, E., 2000. Growth evaluation and bioproducts characterization of Calothrix spp. Bioresource Technol. 72, 121 124. Padgett, M.P., Krogmann, D., 1987. Large scale preparation of pure phycobiliproteins. Photosynth. Res. 11, 225 235. Perry, R.H., Green, D.W., 1999. Perrys Chemical Engineers Handbook, seventh ed. Mc-Graw-Hill, New York, USA. Redlinger, T., Gantt, E., 1981. Phycobilisome structure of Porphyridium cruentum. Plant Physiol. 68, 1375 1379. Sarada, R., Pillai, M.G., Ravishankar, G.A., 1999. Phycocyanin from Spirulina spp: inuence of processing of biomass on phycocyanin yield, analysis of efcacy of extraction methods and stability studies on phycocyanin. Process Biochem. 34, 795 801. Schoelember, R.W., Leung, S., Lundell, D., Glazer, A.N., Rapoport, H., 1983. Chromopeptides from phycoerythrins. Structure and linkage of a phycoerythrobilin tryptic tripeptide derived from a B-phycoerythrin. J. Am. Chem. Soc. 105, 4072 4076. Shagger, H., Von, J., 1987. Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 KDa to 100 KDa. Anal. Biochem. 166, 368 379. Sidler, W., Kumpf, B.K., Suter, F., Klotz, A.V., Glazer, A.N., Zuber, H., 1989. The complete amino-acid sequence of the and subunits of B-phycoerythrin from the Rhodophytan alga Porphyridium cruentum. Biol. Chem. Hoppe Seyler 370, 115 124. Tchernov, A.A., Minkova, K.M., Houbavenska, N.B., Kovacheva, N.G., 1999. Purication of phycobiliproteins from Nostoc spp. by aminohexyl-Sepharose chromatography. J. Biotechnol. 69 (1), 69 73. Vonshak, A., 1988. Porphyridium. In: Borowitzka, M.A., Borowitzka, L.J. (Eds.), Microalgal Biotechnology. Cambridge University Press, Cambridge, pp. 122 134.