Journal ofApplied Phycology 7: 17-23, 1995.

17
© 1995 Kluwer Academic Publishers. Printedin Belgium.

Nitrogen-fixing cyanobacteria as source of phycobiliprotein pigments.

Composition and growth performance of ten filamentous
heterocystous strains
Jos6 Moreno, Herminia Rodriguez, M. Angeles Vargas, Joaquin Rivas & Miguel G. Guerrero*
Instituto de Bioqu[mica Vegetal y Fotosntesis, Universidadde Sevilla - Consejo Superior de Investigaciones
Cientificas, 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 growth
performance and tolerance to temperature, pH, irradiance and salinity, together with their potential as producers
of phycobiliprotein pigments. Phycobiliproteins typically accounted for about 50% total cell protein, the prevalent
type being C-phycocyanin, followed by allophycocyanin, with levels of 17 and 11% d.wt, respectively, in some
strains 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 tolerated
an irradiance of ca 2000 mol photon m- 2 s- 1. Anabaena sp. ATCC 33047 and Nostoc sp. (Albufera) exhibited
the 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 two
phycoerythrin-rich strains of Nostoc was over 20 g (d.wt) m- 2 d-' during summer. The growth performance of the
allophycocyanin-richAnabaenasp. ATCC 33047 in outdoor semi-continuous culture has been assessed throughout
the year. Productivity values under optimized conditions ranged from 9 (winter) to 24 (summer) g (d.wt) m - 2 d - '1.

Introduction The nitrogen-fixing, heterocystous, filamentous

Microalgae represent a potential source of commercial-
ly important chemical and pharmaceutical products.
Among them, phycobiliproteins stand out not only for
their 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 can
be 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 and
fluorescence microscopy for diagnostics and biomed-
ical research, exhibiting many advantages over tradi-
tional fluorescence tracers (Glazer, 1994).
cyanobacteria 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 culture
media of nitrogen fertilizer, since they can grow using
atmospheric nitrogen as the sole nitrogen source. The
lack of combined nitrogen in the culture media, aside
from its economical implication, restricts the problem
of contamination by other organisms. Moreover, the
filamentous 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 filamentous
N2-fixing cyanobacteria is scarce (Fontes et al., 1987,
Boussiba, 1993).
 18
This work deals with the screening of ten strains
of nitrogen-fixing cyanobacteria on the basis of specif-
ic phycobiliprotein content, productivity and tolerance
to salinity, temperature, pH and irradiance. To derive
optimum conditions for outdoor production, the effect
of nutritional and environmental factors on the pro-
ductivity of selected strains, has also been studied in
outdoor cultures. Special emphasis has been given to
the allophycocyanin-rich strain Anabaena sp. ATCC
33047.
Materials and methods
Biologicalmaterialand culture conditions
The strains of nitrogen-fixing cyanobacteria used in
this work were:
Anabaena sp. ATCC 33047 and Anabaena vari-
abilis ATCC 29413 from the American Type Cul-
ture Collection, Rockville (USA); Nostoc commune
B-1453-3 from the Culture Collection of Gbttingen
University (Germany); Nostoc sp. PCC 73102 from
the Pasteur Culture Collection, Paris (France); Nos-
toc paludosum and Nostoc sp. (Albufera), isolated
by 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 classified
by us from Santa Olalla lake in Dofiana National Park
(Spain); Nostoc sp. (Chile), isolated from a lake in
the Andes (Chile) and classified by us; Nodularia sp.
(Chucula) and Nostoc sp. (Chucula), isolated from San
Pedro lake in the Andes (Chile) and classified by our
group.
Nostoc paludosum, Nostoc sp. (Albufera),
Anabaenopsis sp. (Albufera), Nostoc sp. (Dofiana),
Nostoc sp. (Chile) and Nostoc sp. (Chucula) have been
recently 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 at
the authors laboratory, and cultures will be provided
upon request.
Anabaena sp. ATCC 33047 was grown on the
medium described by Moreno et al. (1987), contain-
ing 86 mM NaCI, 50 mM NaHCO 3, 8 mM KCI,
1 mM K2 HPO 4, 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 medium
of Arnon et al. (1974) was used without modifica-
tion for Anabaena variabilis and modified to contain
4 mM K2HPO 4 for the rest of the strains. In the case of
Anabaenopsis sp., the medium was also supplemented
with 30 mM NaCl. For the experiments aimed to deter-
mine optimum pH, the pH of the media was adjusted
by changing the proportions of KH 2PO 4 and K2HPO 4,
keeping constant the total phosphate concentration. For
achieving 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 the
source 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 either
fluorescent or mercury halide lamps at the indicated
irradiances, measured at the surface of the culture con-
tainers. The cyanobacteria were grown, as indicated,
either in semi-continuous culture subjected to 12 h
light/12 h dark cycles, in batch culture with continuous
illumination or in continuous culture. Semi-continuous
cultures were diluted with fresh medium to a cell den-
sity of 0.4-0.6 g (d.wt) L- 1 at the beginning of the
light period, at which time samples were withdrawn
for analysis.
Outdoor cultures were performed in 1m2 open con-
tainers (miniponds) of 30 cm maximal depth (Fig. 1).
Turbulence was provided by a rotating paddle-wheel
made up of three 28 x 32 cm paddles, operating, unless
otherwise indicated, at a rotating speed of about 18 rpm
(culture flow rate, 0.35 m s-l). Pure CO 2 (at the flow
required to keep the pH at a given value) was supplied,
from sunrise to sunset, through a PVC - porous tube
placed on the bottom of the container. Cells were grown
in semi-continuous culture, cultures being diluted with
fresh medium early in the morning to the indicated cell
densities. Minimum temperature of the cultures was
maintained at 30 C, with the help of a 1.5 kW heater
controlled 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 aliquots
were 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-

2
Fig. 1. Containers (1 m surface) provided with a paddle-wheel
system, utilized in this work for outdoor culture of filamentous
nitrogen-fixing cyanobacteria.
tion coefficient given by Mackinney (1941). Protein
analysis of sonicated cells (75 W, 60 s) was carried out
according to Bradford (1976), following the technique
proposed by Kochert (1978), using either lysozyme or
bovine serum albumin as standards. Phycobiliproteins
were 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 temperature
ranges 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 optimum
ranges were also exhibited by Anabaena sp. ATCC
19
33047 (6.5-9.5) and Nostoc sp. (Albufera) (6.0-9.0).
The ample tolerance of Anabaena sp. ATCC 33047
and 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 of
nitrogen-fixing cyanobacteria assayed ranged between
37 and 56% d.wt, being for most strains about 45-50%
(Table 2). The high protein levels correlated with a
high nitrogen content, that amounted to 8-12% d.wt,
with C/N ratios of 4 to 5 (data not shown). Table 2
also shows the phycobiliprotein composition of these
strains. 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. Allophycocyanin
ranged from 2 to 11% d.wt, with Anabaena sp. ATCC
33047, A. variabilis and Nostoc paludosum showing
the highest values. The level of C-phycoerythrin was
below 2% d.wt in most of the strains, although it is
known to amount to more than 6% d.wt in several
Nostoc 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 cyanobacteria
represent, therefore, excellent sources of phycobilipro-
teins, and should be viewed as a valid alternative to
those currently considered, namely the non nitrogen-
fixing cyanobacterium Spirulinaand the eukaryotic red
algaPorphyridium(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 respect
to 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 as
70% of the maximum value, attained at 0.085 M. The
rest of the tested strains exhibited optimum growth
at NaCI concentrations of up to 0.1 M (data not
shown). The influence of irradiance on cell growth
of Anabaena sp. ATCC 33047 in laboratory cultures is
shown in Fig. 2. It can be observed that the doubling
time decreased (growth rate increased) in response to
increasing incident light energy. The light-response
curve was hyperbolic, light saturation being reached
at about 1000 mol photon m- 2 s - 1, with an opti-
mum doubling time of about 4 h. Light-response
curves obtained for the different strains considered
were rather similar, tolerating irradiance values of at
least 2000 pmol photon m - 2 s- .
20

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

Strain Optimum Optimum Optimum Optimum

temperature temperature pH pH range*
(°C) range (0C)

Anabaenasp. ATCC 33047 40 30-45 8.5 6.5-9.5

Anabaenopsis sp. (Albufera) 35 25-35 8.5 7.0-9.0
Nostoc 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.
-2 -1
Photon flux density was 180 mol photon m s (fluorescent lamps). Underlined figures correspond to
particularly 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 has
been 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 and
12 h light/12 h dark cycles. Productivity values under
the same conditions for the other strains tested were
between 18 g (d.wt) m- 2 d - l and 24 g (d.wt) m - 2
d -1 . When Anabaenopsis sp. (Albufera) and Nostoc
paludosum were grown in continuous culture and with
continuous illumination, productivities obtained were
about 40 and 54 g (d.wt) m- 2 d- 1, respectively. Under
these conditions, Anabaena sp. ATCC 33047 was the
most productive strain, with yields higher than 100 g
(d.wt) m- 2 d - '.1
According to the wide optima with regard to pH and
temperature, tolerance to high irradiances and NaCl
concentrations, easy harvesting of the cells and high
biomass productivities achieved under fully controlled
conditions, the allophycocyanin-rich strain Anabae-
na sp. ATCC 33047, and the two phycoerythrin-rich
strains Nostoc sp. (Albufera) and Nostoc sp. (Dofiana)
were tested for outdoor growth performance. The
productivity of these strains in 1 m2-surface open
miniponds provided with paddle wheels was 21-23 g
(d.wt) m - 2 d - l in summer. It is worth mentioning that
productivity was not further enhanced by the addi-
tion of combined nitrogen as 10 mM KNO 3 (data
not shown), indicating that these N 2-fixing strains can
derive all the nitrogen needed for growth from dinitro-
gen present in the air.
 21
18
Table 3. Effect of carbon supply and pH on produc-
tivity (mean ± SD; n = 7) of Anabaena sp. ATCC
33047 in outdoor semicontinuous culture. Culture
14 depth was 15 cm. Cell density was maintained at
a minimal value of 0.18 g (d.wt) L- L.The experi-
ment was carried out in autumn at an average daily
irradiance of 516 pmol photon m- 2 s- .
L9 10
0
3 NaHCO 3 C0 2 pH Productivity
5g (mM) (mol m- 2 (g (d.wt)
day- m-2 day-l)
6
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
9
50 0 9.3 11.7 2.1
0 1000 2000 3000

IRRADIANCE (mol photon m 2 s- 1) * Under these conditions, growth ceased after three
days, the indicated productivity value correspond-
Fig. 2. Effect of irradiance on doubling time of Anabaena sp. ATCC ing to this short period.
33047 in laboratory batch culture. Vertical bars represent standard
deviations; n = 3.
Table 4. Effect of seasonal solar irradiance on
25 productivity (mean ± SD; n > 3) of Anabae-
--- a-- summer na sp. ATCC 33047 in outdoor semicontinu-
· win ous culture. Cell density was maintained at the
20 _ optimum value for each season [spring, 0.20
g (d.wt) L-l; summer, 0.23 g (d.wt) L-l;
autumn, 0.21 g (d.wt) L-l; and winter, 0.11
I g (d.wt) L- 1].
15
Season Average daily Productivity
C3
irradiance (g (d.wt)
10 _
.
(ftmol photon m-2 day- )
DC m-2 S-1 )

5 _ Spring 996 18.0 1.6

Summer 1177 24.2 ± 2.7
I I
Autumn 516 10.0 1.8
I
0 Winter 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. ATCC

33047 in outdoor semicontinuous culture. Culture depth was 10 cm.
CO 2 was provided at a flow rate of 1.0-1.5 mol m- 2 d-', as to
keep the pH of the cell suspension between 9.1 and 9.3. Average
solar irradiance was about 426 H/mol photon m- 2 s- 1 in winter
and about 1065 /,mol photon m- 2 s- 1 in summer. Vertical bars
represent 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 carried
out with the halotolerant allophycocyanin-rich strain
Anabaena sp. ATCC 33047. Table 3 shows data on
yield of Anabaena sp. ATCC 33047 in semicontinuous
culture under different conditions of carbon supply and
pH in autumn, at a moderate average daily irradiance
(516 mol photon m- 2 s-'). In the absence of added
bicarbonate, when inorganic carbon was supplied as
CO 2 only, the pH became 6.5, growth was only mod-
erate initially, and eventually ceased after the third
day of culture. A stable and significant yield (about
12 g (d.wt) m - 2 d - l) was obtained, however, in the
absence of added CO 2 if 50 mM NaHCO 3 was present
in the culture medium, the resulting pH being 9.3.
Under the prevailing moderate average daily irradiance
22
(516, mol photon m - 2 s- ) and when bicarbonate was
present, an extra supply of CO2 resulted in decrease of
the pH value, without any significant enhancement of
productivity. These results thus indicate that mixing of
the 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 much
higher, addition of CO2 in adequate amounts (1-2 mol
CO 2 m- 2 d - 1) to the 50 mM NaHCO 3-supplemented
suspension 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 utmost
importance 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 highest
productivity (23.4 g (d.wt) m- 2 d- ) was achieved for
10-cm depth. Increasing the suspension depth resulted
in decreased growth rate and productivity, the latter
becoming 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 33047
has been assessed in two different seasons of the year.
In summer, an increase in the flow speed from 0.17
to 0.42 m s - 1 (obtained by increasing the rotating
speed of the paddle wheel from 9 to 22 rpm) raised
the biomass productivity by more of 50% (from 13 to
20 g m- 2 d-l). On the contrary, in winter, productiv-
ity was hardly influenced by increasing the flow rate
over 0.17 m s- (data not shown). Thus, the beneficial
effect of enhanced turbulence is patent in summer, at
high 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 and
productivity of the cultures. For a set of given condi-
tions, there is an optimum value of population density
which yields the higher output rate and, therefore, the
highest photosynthetic efficiency (Richmond, 1986,
1992). The effect of cell density throughout different
periods of the year on the productivity of Anabaena
sp. ATCC 33047 has also been assayed. Optimal cell
density varied along the year, according to seasonal
irradiance. The response of productivity in outdoor
semicontinuous 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- in
winter.
The results in Table 4 are a summary of productiv-
ity values obtained throughout the year, and illustrate
the influence of solar irradiance on the performance
of outdoor semicontinuous cultures of Anabaena sp.
ATCC 33047 maintained at the optimum cell densi-
ty for each period considered. Both productivity and
growth 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 highest
reported for outdoor cultures of microalgae.
Discussion
The screening carried out among a variety of filamen-
tous nitrogen-fixing blue-green algae has allowed the
identification of several strains, which exhibit unusu-
ally high levels of specific phycobiliproteins of com-
mercial interest. Some of these strains display besides
wide tolerance to fluctuations in temperature and pH,
also resistance to salinity and high irradiance.
The performance outdoors has been verified for
two phycoerythrin-rich strains of Nostoc and the
allophycocyanin-rich Anabaena sp. ATCC 33047.
Under optimized growth conditions, peak productivi-
ty values exceeding 20 g (d.wt) m - 2 d-' in summer
were obtained, with a mean annual productivity of
about 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 for
the respective producer. Outdoor mass culture of these
cyanobacteria thus represents an efficient way of pro-
ducing these commercially interesting pigments, and
constitutes therefore an interesting and valid alterna-
tive to the systems currently used.
The market price for phycobiliproteins is around
50 US $ mg- ' (Gudin, 1988), which allows for high
production costs. An evaluation of the latter for the
N2-fixing cyanobacterial systems here reported is pre-
mature, since the scale attempted is far from that of
a commercial facility. Although the results obtained
so far are encouraging, much work is still needed to
develop the mass cultivation of these organisms.

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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