Journal of Applied Phycology 7: 17-23, 1995. 17© 1995 Kluwer Academic Publishers. Printed in Belgium.

Nitrogen-fixing cyanobacteria as source of phycobiliprotein pigments.Composition and growth performance of ten filamentousheterocystous strains

Jos6 Moreno, Herminia Rodriguez, M. Angeles Vargas, Joaquin Rivas & Miguel G. Guerrero*Instituto de Bioqu[mica Vegetal y Fotosntesis, Universidad de Sevilla - Consejo Superior de InvestigacionesCientificas, Apartado 1113, 41080 Sevilla, Spain(*Authorfor correspondence)

Received 1 June 1994; revised 1 August 1994; accepted 2 September 1994

Key words: blue-green algae, cyanobacteria, nitrogen fixation, outdoor culture, phycobiliproteins, phycoerythrin

Abstract

Ten strains of filamentous, heterocystous nitrogen-fixing blue-green algae (cyanobacteria) were screened for growthperformance and tolerance to temperature, pH, irradiance and salinity, together with their potential as producersof phycobiliprotein pigments. Phycobiliproteins typically accounted for about 50% total cell protein, the prevalenttype being C-phycocyanin, followed by allophycocyanin, with levels of 17 and 11% d.wt, respectively, in somestrains of Anabaena and Nostoc. C-phycoerythrin was the major pigment in several Nostoc strains, reaching 10%d.wt. Some strains represent, therefore, excellent sources of one or more phycobiliproteins. All strains toleratedan irradiance of ca 2000 mol photon m- 2 s- 1. Anabaena sp. ATCC 33047 and Nostoc sp. (Albufera) exhibitedthe widest optimum range of both temperature (30-45 and 25-40 C) and pH (6.5-9.5 and 6.0-9.0) for growth,the former also showing significant salt tolerance. In an outdoor open system, productivity of cultures of twophycoerythrin-rich strains of Nostoc was over 20 g (d.wt) m- 2 d-' during summer. The growth performance of theallophycocyanin-richAnabaena sp. ATCC 33047 in outdoor semi-continuous culture has been assessed throughoutthe year. Productivity values under optimized conditions ranged from 9 (winter) to 24 (summer) g (d.wt) m - 2 d- '1.

Introduction

Microalgae represent a potential source of commercial-ly important chemical and pharmaceutical products.Among them, phycobiliproteins stand out not only fortheir commercial value, but also because these pig-ments are exclusive to cyanobacteria and some eukary-otic algae. Cyanobacteria contain phycocyanin (blue)and allophycocyanin (blue-grey), whereas phycoery-thrin (red) is not always present. Phycobiliproteins canbe used as natural pigments for the food, drug and cos-metic industries to replace the currently used synthet-ical pigments (Cohen, 1986). They have found addi-tional applications in fluorescence immunoassays andfluorescence microscopy for diagnostics and biomed-ical research, exhibiting many advantages over tradi-tional fluorescence tracers (Glazer, 1994).

The nitrogen-fixing, heterocystous, filamentouscyanobacteria are particularly attractive for the pho-toproduction of phycobiliproteins and other chemi-cals (Borowitzka, 1988; Rodriguez et al., 1989, 1991,1992). They do not require the addition to the culturemedia of nitrogen fertilizer, since they can grow usingatmospheric nitrogen as the sole nitrogen source. Thelack of combined nitrogen in the culture media, asidefrom its economical implication, restricts the problemof contamination by other organisms. Moreover, thefilamentous nature of these organisms facilitates sepa-ration of biomass from the medium. Despite these obvi-ous advantages for mass production, available infor-mation regarding outdoor production of filamentousN2-fixing cyanobacteria is scarce (Fontes et al., 1987,Boussiba, 1993).

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This work deals with the screening of ten strainsof nitrogen-fixing cyanobacteria on the basis of specif-ic phycobiliprotein content, productivity and toleranceto salinity, temperature, pH and irradiance. To deriveoptimum conditions for outdoor production, the effectof nutritional and environmental factors on the pro-ductivity of selected strains, has also been studied inoutdoor cultures. Special emphasis has been given tothe allophycocyanin-rich strain Anabaena sp. ATCC33047.

Materials and methods

Biological material and culture conditions

The strains of nitrogen-fixing cyanobacteria used inthis work were:

Anabaena sp. ATCC 33047 and Anabaena vari-abilis ATCC 29413 from the American Type Cul-ture Collection, Rockville (USA); Nostoc communeB-1453-3 from the Culture Collection of GbttingenUniversity (Germany); Nostoc sp. PCC 73102 fromthe Pasteur Culture Collection, Paris (France); Nos-toc paludosum and Nostoc sp. (Albufera), isolatedby V. Vidal (E.T.S. Ingenieros Agr6nomos, Valencia,Spain) from the coastal lagoon Albufera de Valencia(Spain), the former being classified by M. Herndndez,and the latter by us; Anabaenopsis sp. (Albufera), iso-lated and classified by us from Albufera de Valencia(Spain); Nostoc sp. (Dofiana), isolated and classifiedby us from Santa Olalla lake in Dofiana National Park(Spain); Nostoc sp. (Chile), isolated from a lake inthe Andes (Chile) and classified by us; Nodularia sp.(Chucula) and Nostoc sp. (Chucula), isolated from SanPedro lake in the Andes (Chile) and classified by ourgroup.

Nostoc paludosum, Nostoc sp. (Albufera),Anabaenopsis sp. (Albufera), Nostoc sp. (Dofiana),Nostoc sp. (Chile) and Nostoc sp. (Chucula) have beenrecently deposited in the Pasteur Culture Collection,Paris (France), and designed as PCC 9206, PCC 9202,PCC 9215, PCC 9205, PCC 9201 and PCC 9203,respectively. Nodularia sp. (Chucula) is available atthe authors laboratory, and cultures will be providedupon request.

Anabaena sp. ATCC 33047 was grown on themedium described by Moreno et al. (1987), contain-ing 86 mM NaCI, 50 mM NaHCO 3, 8 mM KCI,1 mM K2 HPO4, 0.5 mM MgSO 4, 0.35 mM CaC12,as well as a supply of essential micronutrients and

Fe-EDTA (Arnon et al., 1974). The culture mediumof Arnon et al. (1974) was used without modifica-tion for Anabaena variabilis and modified to contain4 mM K2HPO 4 for the rest of the strains. In the case ofAnabaenopsis sp., the medium was also supplementedwith 30 mM NaCl. For the experiments aimed to deter-mine optimum pH, the pH of the media was adjustedby changing the proportions of KH2PO4 and K2HPO 4,keeping constant the total phosphate concentration. Forachieving pH values over 7.5, NaHCO 3 (10-100 mM)was also added.

At the laboratory, cells were grown photoau-totrophically by bubbling through the cell suspen-sion air supplemented with 0.4-1.5% (v/v) CO 2 as thesource of carbon and nitrogen at a flow rate of 50-130 L(L culture) - ' h - l . Cells were grown in 5 cm-deep 1 L-capacity Roux flasks, laterally illuminated with eitherfluorescent or mercury halide lamps at the indicatedirradiances, measured at the surface of the culture con-tainers. The cyanobacteria were grown, as indicated,either in semi-continuous culture subjected to 12 hlight/12 h dark cycles, in batch culture with continuousillumination or in continuous culture. Semi-continuouscultures were diluted with fresh medium to a cell den-sity of 0.4-0.6 g (d.wt) L- 1 at the beginning of thelight period, at which time samples were withdrawnfor analysis.

Outdoor cultures were performed in 1 m2 open con-tainers (miniponds) of 30 cm maximal depth (Fig. 1).Turbulence was provided by a rotating paddle-wheelmade up of three 28 x 32 cm paddles, operating, unlessotherwise indicated, at a rotating speed of about 18 rpm(culture flow rate, 0.35 m s-l). Pure CO2 (at the flowrequired to keep the pH at a given value) was supplied,from sunrise to sunset, through a PVC - porous tubeplaced on the bottom of the container. Cells were grownin semi-continuous culture, cultures being diluted withfresh medium early in the morning to the indicated celldensities. Minimum temperature of the cultures wasmaintained at 30 C, with the help of a 1.5 kW heatercontrolled by a thermostat. Growth rate and productiv-ity values were estimated on a dry weight basis.

Analytical methods

For dry weight determinations, 50-200 ml aliquotswere filtered through previously weighed GF/C What-man glass microfiber filters, washed twice with dis-tilled water and dried at 80 C for 24 h.

Chlorophyll a (Chl) was determined spectrophoto-metrically in methanol extracts employing the extinc-

19

Fig. 1. Containers (1 m2 surface) provided with a paddle-wheelsystem, utilized in this work for outdoor culture of filamentousnitrogen-fixing cyanobacteria.

tion coefficient given by Mackinney (1941). Proteinanalysis of sonicated cells (75 W, 60 s) was carried outaccording to Bradford (1976), following the techniqueproposed by Kochert (1978), using either lysozyme orbovine serum albumin as standards. Phycobiliproteinswere estimated spectrophotometrically, also using son-icated cells, employing the equations given by Siegel-man & Kycia (1978).

Results

Table 1 summarizes data regarding growth toler-ance to temperature and pH of several nitrogen-fixing cyanobacteria. The widest optimum temperatureranges found corresponded to 30-45 °C for Anabae-na sp. ATCC 33047 and 25-40 C for Nostoc sp.(Albufera). With respect to pH, the widest optimumranges were also exhibited by Anabaena sp. ATCC

33047 (6.5-9.5) and Nostoc sp. (Albufera) (6.0-9.0).The ample tolerance of Anabaena sp. ATCC 33047and Nostoc sp. (Albufera) with regard to both temper-ature and pH is advantageous for outdoor mass culture,where fluctuations in these factors are common.

The total net protein content of the ten strains ofnitrogen-fixing cyanobacteria assayed ranged between37 and 56% d.wt, being for most strains about 45-50%(Table 2). The high protein levels correlated with ahigh nitrogen content, that amounted to 8-12% d.wt,with C/N ratios of 4 to 5 (data not shown). Table 2also shows the phycobiliprotein composition of thesestrains. C-phycocyanin was the major phycobilipro-tein for most cases, with particularly high levels (13-18% d.wt)in Anabaena variabilis, Anabaenopsis sp.(Albufera) and Nostoc paludosum. Allophycocyaninranged from 2 to 11% d.wt, with Anabaena sp. ATCC33047, A. variabilis and Nostoc paludosum showingthe highest values. The level of C-phycoerythrin wasbelow 2% d.wt in most of the strains, although it isknown to amount to more than 6% d.wt in severalNostoc strains (Rodriguez et al., 1989, 1991, 1992).Remarkably high, 8-10% d.wt, was the phycoery-thrin content of Nostoc sp. (Albufera) and Nostoc sp.(Dofiana). Some of these nitrogen-fixing cyanobacteriarepresent, therefore, excellent sources of phycobilipro-teins, and should be viewed as a valid alternative tothose currently considered, namely the non nitrogen-fixing cyanobacterium Spirulina and the eukaryotic redalga Porphyridium (Richmond, 1988; Vonshak, 1988),in addition to some macrorhodophytes.

Tolerance to irradiance and salinity were also con-sidered as relevant features of the strains. With respectto salt tolerance, the case of the marine cyanobac-terium Anabaena sp. ATCC 33047 is especially rele-vant, since its growth at 0.5 M NaCl was as much as70% of the maximum value, attained at 0.085 M. Therest of the tested strains exhibited optimum growthat NaCI concentrations of up to 0.1 M (data notshown). The influence of irradiance on cell growthof Anabaena sp. ATCC 33047 in laboratory cultures isshown in Fig. 2. It can be observed that the doublingtime decreased (growth rate increased) in response toincreasing incident light energy. The light-responsecurve was hyperbolic, light saturation being reachedat about 1000 mol photon m- 2 s - 1, with an opti-mum doubling time of about 4 h. Light-responsecurves obtained for the different strains consideredwere rather similar, tolerating irradiance values of atleast 2000 pmol photon m - 2 s- .

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Table 1. Optimum temperature and pH for growth of several N2 -fixing cyanobacteria. Photonflux density was 180 mol photon m- 2 s- 1 (fluorescent lamps).

Strain Optimum Optimum Optimum Optimum

temperature temperature pH pH range*

(°C) range (0C)

Anabaena sp. ATCC 33047 40 30-45 8.5 6.5-9.5

Anabaenopsis sp. (Albufera) 35 25-35 8.5 7.0-9.0Nostoc commune 25 20-30 7.0 6.5-8.5

Nostoc paludosum 35 25-35 8.5 7.0-9.0

Nostoc sp. (Albufera) 35 25-40 6.5 6.0-9.0

Nostoc sp. (Doflana) 35 30-40 8.0 8.0-8.5

Nostoc sp. PCC 73102 30 25-30 8.0 6.5-8.0

* Growth at least 70% of maximum.

Table 2. Protein and phycobiliprotein content (% d.wt; mean ± SD; n>3) of several N2 -fixing cyanobacteria.Photon flux density was 180 mol photon m- 2 s -1 (fluorescent lamps). Underlined figures correspond toparticularly high contents in specific phycobiliproteins.

Strain Protein C-phycoerythrin C-phycocyanin Allophycocyanin

Anabaena sp. ATCC 33047 45.0 ± 1.8 1.3 0.1 9.2 ± 0.5 10.6 1.1

Anabaena variabilis 56.1 ± 2.2 0.8 ± 0.1 17.6 ±- 0.2 11.0 ± 0.1

Anabaenopsis sp. (Albufera) 52.2 ± 2.5 0.8 ± 0.1 13.0 ± 0.9 6.3 ± 0.2

Nodularia sp. (Chucula) 42.9 ± 2.5 1.6 ± 0.0 11.5 ± 0.1 7.7 ± 0.3

Nostoc commune 39.9 ± 1.0 1.7 ± 0.2 5.2 ± 0.2 2.2 ± 0.1

Nostoc paludosum 40.4 4.5 0.6 0.1 16.7 ± 0.5 10.1 ± 0.3

Nostoc sp. (Albufera) 47.0 ± 3.5 8.4 ± 0.8 7.3 ± 0.6 3.5 ± 0.4

Nostoc sp. (Chile) 47.3 ± 3.9 1.9 ± 0.1 9.7 ± 0.3 6.1 ± 0.3

Nostoc sp. (Chucula) 37.3 ± 0.5 6.6 ± 0.3 4.1 ± 0.1 3.7 ± 0.2

Nostoc sp. (Dofilana) 51.6 ± 4.6 10.1 ± 0.6 7.1 ± 0.5 5.9 ± 0.6

The productivity of some nitrogen-fixing cyanobac-teria under laboratory conditions at high irradiance hasbeen assessed. Nostoc sp. (Albufera) was the most pro-ductive strain among the cyanobacteria tested in semi-continuous culture, with a yield of about 37 g (d.wt)m- 2 d-1 when grown at 1400 jmol photon m2 s - l and12 h light/12 h dark cycles. Productivity values underthe same conditions for the other strains tested werebetween 18 g (d.wt) m- 2 d- l and 24 g (d.wt) m- 2

d-1 . When Anabaenopsis sp. (Albufera) and Nostocpaludosum were grown in continuous culture and withcontinuous illumination, productivities obtained wereabout 40 and 54 g (d.wt) m- 2 d- 1, respectively. Underthese conditions, Anabaena sp. ATCC 33047 was themost productive strain, with yields higher than 100 g(d.wt) m- 2 d- 1'.

According to the wide optima with regard to pH andtemperature, tolerance to high irradiances and NaClconcentrations, easy harvesting of the cells and highbiomass productivities achieved under fully controlledconditions, the allophycocyanin-rich strain Anabae-na sp. ATCC 33047, and the two phycoerythrin-richstrains Nostoc sp. (Albufera) and Nostoc sp. (Dofiana)were tested for outdoor growth performance. Theproductivity of these strains in 1 m2-surface openminiponds provided with paddle wheels was 21-23 g(d.wt) m - 2 d- l in summer. It is worth mentioning thatproductivity was not further enhanced by the addi-tion of combined nitrogen as 10 mM KNO 3 (datanot shown), indicating that these N2-fixing strains canderive all the nitrogen needed for growth from dinitro-gen present in the air.

Table 3. Effect of carbon supply and pH on produc-tivity (mean ± SD; n = 7) of Anabaena sp. ATCC33047 in outdoor semicontinuous culture. Culturedepth was 15 cm. Cell density was maintained ata minimal value of 0.18 g (d.wt) L- L. The experi-ment was carried out in autumn at an average dailyirradiance of 516 pmol photon m- 2 s- .

NaHCO 3 C0 2 pH Productivity(mM) (mol m- 2 (g (d.wt)

day- m-2 day-l)

0 6.5 6.5 7.2 + 2.3*10 4.9 7.4 11.3 - 2.5

50 2.8 8.6 12.1 - 1.7

50 0 9.3 11.7 2.1

0 1000 2000 3000

IRRADIANCE (mol photon m 2 s- 1)

Fig. 2. Effect of irradiance on doubling time of Anabaena sp. ATCC33047 in laboratory batch culture. Vertical bars represent standarddeviations; n = 3.

25

20

15

10

I

C3

.

DC

5

0

--- a-- summer· win

I I

* Under these conditions, growth ceased after threedays, the indicated productivity value correspond-ing to this short period.

Table 4. Effect of seasonal solar irradiance onproductivity (mean ± SD; n > 3) of Anabae-na sp. ATCC 33047 in outdoor semicontinu-ous culture. Cell density was maintained at theoptimum value for each season [spring, 0.20g (d.wt) L-l; summer, 0.23 g (d.wt) L-l;autumn, 0.21 g (d.wt) L-l; and winter, 0.11g (d.wt) L- 1].

Season Average daily Productivity

irradiance (g (d.wt)

(ftmol photon m-2 day- )m-2 S-1 )

Spring 996 18.0 1.6Summer 1177 24.2 ± 2.7Autumn 516 10.0 1.8Winter 458 9.2 1.0

0 0.1 0.2 0.3 0.4

CELL DENSITY (g (d.wt) LUl)

Fig. 3. Effect of cell density on productivity of Anabaena sp. ATCC33047 in outdoor semicontinuous culture. Culture depth was 10 cm.CO2 was provided at a flow rate of 1.0-1.5 mol m- 2 d-', as tokeep the pH of the cell suspension between 9.1 and 9.3. Averagesolar irradiance was about 426 H/mol photon m- 2 s- 1 in winterand about 1065 /,mol photon m- 2 s- 1 in summer. Vertical barsrepresent standard deviations; n = 4.

A more thorough study of the effect of some rel-evant factors on the productivity in outdoor semicon-tinuous culture throughout the year has been carriedout with the halotolerant allophycocyanin-rich strainAnabaena sp. ATCC 33047. Table 3 shows data on

yield of Anabaena sp. ATCC 33047 in semicontinuousculture under different conditions of carbon supply andpH in autumn, at a moderate average daily irradiance(516 mol photon m- 2 s-'). In the absence of addedbicarbonate, when inorganic carbon was supplied asCO 2 only, the pH became 6.5, growth was only mod-erate initially, and eventually ceased after the thirdday of culture. A stable and significant yield (about12 g (d.wt) m - 2 d - l) was obtained, however, in theabsence of added CO 2 if 50 mM NaHCO 3 was presentin the culture medium, the resulting pH being 9.3.Under the prevailing moderate average daily irradiance

18

30L9

5g

14

10

6

21

9

_

_

_

I

22

(516, mol photon m - 2 s- ) and when bicarbonate waspresent, an extra supply of CO2 resulted in decrease ofthe pH value, without any significant enhancement ofproductivity. These results thus indicate that mixing ofthe cell suspension by the paddle-wheel system provid-ed the culture with all the carbon (as CO2 present in air)required for a productivity of about 12 g (d.wt) m - 2

d- 1, optimum under conditions of moderate irradiance.Nevertheless, in summer, when irradiance was muchhigher, addition of CO2 in adequate amounts (1-2 molCO2 m- 2 d- 1) to the 50 mM NaHCO 3-supplementedsuspension was an absolute requirement for pH main-tenance and optimum productivity (over 20 g (d.wt)m- 2 d - l) (data not shown).

In microalgae mass culture, choice of appropri-ate depth, cell density and turbulence is of utmostimportance for achieving high productivity (Rich-mond, 1986). The effect of culture depth was stud-ied in containers filled at different heights (10-25 cm)with suspensions of the same cell density. The highestproductivity (23.4 g (d.wt) m- 2 d- ) was achieved for10-cm depth. Increasing the suspension depth resultedin decreased growth rate and productivity, the latterbecoming only 60% of maximum for a depth of 25 cm(data not shown).

The influence of the turbulence of the cell suspen-sion on the productivity of Anabaena sp. ATCC 33047has been assessed in two different seasons of the year.In summer, an increase in the flow speed from 0.17to 0.42 m s - 1 (obtained by increasing the rotatingspeed of the paddle wheel from 9 to 22 rpm) raisedthe biomass productivity by more of 50% (from 13 to20 g m- 2 d-l). On the contrary, in winter, productiv-ity was hardly influenced by increasing the flow rateover 0.17 m s- (data not shown). Thus, the beneficialeffect of enhanced turbulence is patent in summer, athigh solar irradiance values, but not in winter.

The population density represents a major parame-ter in the production of photoautotrophic mass, exert-ing far-reaching effects on the general performance andproductivity of the cultures. For a set of given condi-tions, there is an optimum value of population densitywhich yields the higher output rate and, therefore, thehighest photosynthetic efficiency (Richmond, 1986,1992). The effect of cell density throughout differentperiods of the year on the productivity of Anabaenasp. ATCC 33047 has also been assayed. Optimal celldensity varied along the year, according to seasonalirradiance. The response of productivity in outdoorsemicontinuous culture to varying cell density in sum-mer and winter is shown in Fig. 3. Under our exper-

imental conditions, optimum cell density was 0.23 g(d.wt) L- ' in summer, and about 0.11 g (d.wt) L- inwinter.

The results in Table 4 are a summary of productiv-ity values obtained throughout the year, and illustratethe influence of solar irradiance on the performanceof outdoor semicontinuous cultures of Anabaena sp.ATCC 33047 maintained at the optimum cell densi-ty for each period considered. Both productivity andgrowth rate of the cultures increased with solar irradi-ance, productivity values ranging from 9 g (d.wt) m- 2

d- 1 in winter, and 24 g (d.wt) m - 2 d-' in summer.The latter productivity values are among the highestreported for outdoor cultures of microalgae.

Discussion

The screening carried out among a variety of filamen-tous nitrogen-fixing blue-green algae has allowed theidentification of several strains, which exhibit unusu-ally high levels of specific phycobiliproteins of com-mercial interest. Some of these strains display besideswide tolerance to fluctuations in temperature and pH,also resistance to salinity and high irradiance.

The performance outdoors has been verified fortwo phycoerythrin-rich strains of Nostoc and theallophycocyanin-rich Anabaena sp. ATCC 33047.Under optimized growth conditions, peak productivi-ty values exceeding 20 g (d.wt) m - 2 d-' in summerwere obtained, with a mean annual productivity ofabout 15 g (d.wt) m- 2 d- 1. The productivity of phy-cobiliprotein achieved was higher than 1 g m- 2 d- 1

of allophycocyanin, phycocyanin, or phycoerythrin forthe respective producer. Outdoor mass culture of thesecyanobacteria thus represents an efficient way of pro-ducing these commercially interesting pigments, andconstitutes therefore an interesting and valid alterna-tive to the systems currently used.

The market price for phycobiliproteins is around50 US $ mg- ' (Gudin, 1988), which allows for highproduction costs. An evaluation of the latter for theN2-fixing cyanobacterial systems here reported is pre-mature, since the scale attempted is far from that ofa commercial facility. Although the results obtainedso far are encouraging, much work is still needed todevelop the mass cultivation of these organisms.

23

Acknowledgments

This study was supported by the Comisi6n Interminis-terial de Ciencia y Tecnologfa, Spain (grant no. BI09 1-0536) and Plan Andaluz de Investigaci6n, Spain.

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