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Journal of Biotechnology 103 (2003) 137 /152
Journal of Biotechnology 103 (2003) 137 /152
Journal of Biotechnology 103 (2003) 137 /152 www.else v ier.com/locate/jbiotec Outdoor production of Phaeodactylum

www.elsevier.com/locate/jbiotec

Outdoor production of Phaeodactylum tricornutum biomass in a helical reactor

F.G. Acie´n Ferna´ndez a , David O. Hall b,1 , E. Can˜ izares Guerrero a , K. Krishna Rao b , E. Molina Grima a, *

a Department of Chemical Engineering, University of Almerı´a, E-04071 Almerı´a, Spain b Division of Life Sciences, King’s College London, 150 Stamford Street, Waterloo, London SE1 8WA, UK

Received 19 November 2002; received in revised form 24 March 2003; accepted 3 April 2003

Abstract

The production of microalga Phaeodactylum tricornutum in an outdoor helical reactor was analysed. The influence of

temperature, solar irradiance and air flow rate on the yield of the culture was evaluated. Biomass productivities up to

1.5 g l 1 per day and photosynthetic efficiency up to 14% were obtained by maintaining the cultures below 30 8 C,

dissolved oxygen levels less than 400% Sat. (with respect to air saturated culture) and controlling the cell density in order to achieve an average irradiance within the culture below 250 mE m 2 s 1 . Under these conditions, the fluorescence parameter, Fv/Fm, which reflects the maximal efficiency of PSII photochemistry, remained roughly 0.6 /

0.7 and growth rates up to 0.050 h 1 were achieved. The average irradiance and the light/dark cycle frequency, were the

variables determining the behaviour of the cultures. A hyperbolic relationship between growth rate and biomass productivity with the average irradiance was observed, whereas both biomass productivity and photosynthetic efficiency linearly increased with the light/dark cycle frequencies. Optimum design and operational conditions which

maximise the production of P. tricornutum biomass in outdoor helical reactors were determined. # 2003 Elsevier Science B.V. All rights reserved.

Keywords: Microalgal; Photobioreactor; Stress; Light/dark frequency; Quantum efficiency

1. Introduction

Microalgae can be used for wastewater treat- ments (Travieso et al., 2002), as animal food (Knauer and Southgate, 1999), as human food (Villar et al., 1994) or to produce numerous high-

* Corresponding author. Tel.: /34-950-01-5032; fax: /34-

950-01-5484.

E-mail address: emolina@ual.es (E. Molina Grima). 1 Now deceased.

value bioactives (Borowitzka, 1986). Production of microalgal biomass can be carried out in fully contained photobioreactors or in open ponds and channels. Open-culture systems are almost always located outdoors and rely on natural light for illumination. Closed photobioreactors may be located indoors or outdoors, but outdoor location is more common because it can make use of free sunlight. Design and operation of the microalgal biomass production systems have been discussed extensively (Terry and Raymond, 1985; Boro-

0168-1656/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0168-1656(03)00101-9

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F.G. Acie´n Ferna´ndez et al. / Journal of Biotechnology 103 (2003) 137 /152

witzka, 1996; Pulz, 2001; Molina Grima et al., 1999; Tredici, 1999). One of the most promising closed photobio- reactors design is the helical reactor (Robinson et al., 1987). This helical tubular photobioreactor is advantageous because it allows: (1) a larger ratio of surface area to culture volume to receive illumination effectively, thereby increasing the incident light energy input per unit volume and reducing the self-shading phenomenon; (2) easy control of temperature and contaminants because it is a closed bioreactor; and (3) better CO 2 transfer from the gas stream to the liquid culture medium due to the extensive CO 2 absorbing pathway (Watanabe et al., 1995). For any photobioreactor design, the system productivity in continuous operating mode is obtained by multiplying the steady state biomass concentration by the imposed dilution rate. Both are related to the average irradiance inside the photobioreactor, which is a function of the irra- diance on the reactor surface, operational vari- ables (fluid-dynamic and dilution rate) and pigment content. In assessing the effect of light on reactor performance two main aspects have to be considered. Firstly, the light availability inside the culture is determined by the solar irradiance, the design of the reactor, the biomass concentra- tion and pigment content in the culture which leads to self-shading (Molina Grima et al., 1994). Secondly, the light regime, which is determined by the irradiance and duration of cell exposure inside the culture (Philipps and Myers, 1954; Terry, 1986; Grobbelaar, 1994; Schmidt-Staiger et al., 2000; Molina Grima et al., 2000). Thus, the effect of light can be modified by manipulating the light path (design of the reactor), the irradiance on the reactor surface (location of the reactor), fluid- dynamic and biomass concentration (operating conditions). Finally, the influence of culture con- ditions such as light availability, nutrient satura- tion, pH, culture temperature, and fluid-dynamic conditions on the biomass productivity must be determined in order to establish the optimal culture conditions. In the present paper, the influence of the following operational conditions: temperature, solar irradiance and air flow rate on the stress,

growth rate, biomass productivity, and photosyn- thetic efficiency of Phaeodactylum tricornutum cultures were analysed. Then, a previously devel- oped fluid-dynamic model of the reactor was utilised to determine the optimum design and operational conditions which maximise the pro- duction of P. tricornutum biomass in outdoor helical reactors.

2. Materials and methods

2.1. Organism and culture conditions

The microalga used, P. tricornutum UTEX 640, was obtained from the culture collection of the

University of Texas, Austin. P. tricornutum UTEX

640 is a freshwater strain but it tolerates a high

salinity (Yongmanitchai and Ward, 1991). The inoculum for the photobioreactor was grown indoors under artificial light (230 mE m 2 s 1 )

at controlled temperature (20 8 C) and using the Mann and Myers’ medium (1968). Outdoor cultures were operated in both discon- tinuous and continuous mode. In the continuous mode, fresh medium was supplied only during the 10-h daylight period and dilution stopped during the night. The cultures were maintained at pH 7.7 by injecting carbon dioxide automatically, as needed. Temperature was controlled by passing cold water through a heat exchanger system when temperature exceeded the set point. Nutrient limitation was prevented by using the Mann and Myers (1968) medium at three times the normal concentration.

2.2. Outdoor photobioreactor

A helical type tubular photobioreactor (Fig. 1) was used. The reactor was located outdoors in

Almerı´a (36 8 50?N, 2 8 27?W), Spain. The helical reactor had a volume of 75 l, and consisted of

106 m plastic tube of 0.03 m diameter, arranged

around a circular frame of 1.2 m diameter. The height of the helical section (the solar receiver, Fig. 1) was 0.8 m, the degasser head being 2 m above

the helical loop. Air was injected at the base of the air-lift riser for liquid circulation and oxygen

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al. / Journal of Biotechnology 103 (2003) 137 / 152 139 Fig. 1. Schematic drawing of

Fig. 1. Schematic drawing of the helical photobioreactor used.

desorption, whereas CO 2 was injected at the base of the helical section for pH control. A heat exchanger was installed before the helical section (coiled tube) for temperature control. Dissolved oxygen, pH and temperature probes were located at the end of the helical section. The probes were connected to a data acquisition board and com- puter for on-line registration and control.

2.3. Solar irradiance

The instantaneous photon-flux density of photosynthetically active radiation (PAR) was measured using a quantum scalar irradiance sensor (QSL 100, Biospherical Instruments Inc., San Diego, CA, USA). Solar irradiance (I) was measured periodically at different hours each day in an obstacles free reference surface. The instan- taneous values were numerically integrated and divided by the daylight time to obtain the mean daily irradiance (I md ). The light profiles within the culture were estimated using a solar irradiance model for tubular photobioreactors (Acie´n Fer- na´ndez et al., 1997). The model allows the determination of the light profiles inside the tube as a function of the irradiance on the culture surface, the reactor geometry, the biomass con- centration and the biomass absorption coefficient (K a ). The irradiance on the culture surface (I 0 ) was

calculated as 38% of solar irradiance. This rela- tionship was obtained from measuring the solar irradiance at different points on the reactor surface (16 points) at different hours of the day. The obtained ratio of 0.38 between the irradiance on the reactor surface and the solar irradiance is similar to the 0.30 value referenced for horizontal tubular reactors with no distance between the tubes (Acie´n Ferna´ndez et al., 2001). The absorp- tion coefficient depended on the biomass pigment content (X p ) (Molina Grima et al., 1996), in accordance with the equation:

(1)

K a 0:0105 0:0299X p

The average irradiance inside the culture (coiled tube) (I av ) was calculated as the volumetric integral of light profiles data (Acie´n Ferna´ndez et al., 1997). The irradiance profiles inside the reactor photostage were also used to determine the cross section of the solar receiver tubes in a ‘light’ zone with irradiance values up to saturation, or /200 mE /m 2 s 1 (Mann and Myers, 1968; Acie´n Fer- na´ndez et al., 1998); the rest of the cross section was considered as the ‘dark’ zone of the reactor.

2.4. Light/dark cycle frequencies

Light frequency was calculated as a function of illuminated proportion of culture ( f ), and the time expended in the dark zone (t d ) as (Molina Grima et al., 2000):

n 1 f

(2)

t d

where the time spent in the dark (t d ) was a function of the culture volume in the dark (V d ), the tube dark cross section (S d ) and the radial velocity of the liquid (U R ), this relationship being (Molina Grima et al., 2000):

t d

V d

U R S d

(3)

Both V d and S d were calculated from the light profiles inside the culture according to methodol- ogies reported elsewhere (Acie´n Ferna´ndez et al., 2000; Molina Grima et al., 2000). In Eq. (3), U R is the radial velocity of the liquid. Cells move

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radially within the fluid because of momentum transport between the turbulent core and the more quiescent boundary layer adjacent to walls. Thus, the radial movement of the fluid elements in the cross section of the tube was estimated as (Molina Grima et al., 2000):

7

U R 0:2 U d t r

L

m

1=8

(4)

which allows the calculation of the radial velocity (U R ) in the turbulent core as a function of the superficial liquid velocity (U L ), the tube diameter (d t ), and the density ( r ) and viscosity ( m ) of the culture broth.

2.5. Biomass concentration and daily growth rate

The biomass concentration was estimated from the measured optical density (OD) of the culture. The optical density was measured spectrophoto- metrically (Hitachi U-1000) at 625 nm wavelength in a cuvette with 1 cm light path. The relationship between the biomass concentration and the OD was:

(5)

where C b is the biomass concentration. The spectrophotometric determinations of biomass were periodically verified by dry weight measure- ments on samples that had been centrifuged (1800 /g ), washed with 0.5 M hydrochloric acid and distilled water to remove non-biological adhering materials such as mineral precipitates, and then freeze dried. The daily growth rate was calculated from the relation between the biomass concentration and time as:

(6)

ln C b ln C bo m t

C b (g l 1 ) 0:38 OD 625

where C b is the instantaneous biomass concentra- tion, C bo is the biomass concentration at the beginning of the solar period, and t is the solar hour. Thus, the daily growth rate was calculated as the slope of ln C b versus t; only values obtained from a minimum of four experimental data points being considered.

2.6. Photosynthetic efficiency

In microalgal cultures the photosynthetic effi- ciency C is defined as the amount of energy stored in the generated biomass per unit of radiation energy absorbed by the culture; it can be calcu- lated from the expression:

C

P b H biomass

F vol

(7)

where P b is the volumetric biomass productivity, H biomass is the combustion enthalpy of the bio- mass, and F vol is the photon flux absorbed in unit volume. Eq. (7) represent the efficiency in using solar irradiance by the biomass. The combustion enthalpy could be calculated from the biochemical composition, or directly measured. In this case, a value of 20.15 kJ g 1 has been considered (Thomas et al., 1984; Acie´n Ferna´ndez et al., 1998). Photon flux absorbed throughout the reactor volume may be obtained from I av , on a culture volume basis using the following equation (Molina Grima et al., 1997):

F vol I av K a C b

(8)

2.7. Analytical methods

The chlorophyll fluorescence of the cultures, as Fv/Fm ratio, was measured using a Hansatech portable plant efficiency analyser (PEA) (Hansa- tech Instruments Ltd., Norfolk, UK). For Fv/Fm determination, 0.5 ml samples were taken from the photobioreactor, incubated in the dark for 10 min, and then the fluorescence of chlorophyll was measured.

2.8. Statistical analysis

To determine the influence of each one of the variables studied, analysis of variance (ANOVA) were performed. The experimental data was grouped into different sets corresponding to simi- lar values of variables analysed. Statistical analysis of grouped series was performed using STAT-

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141

GRAPHICS v7.0 (Manugistics Inc. and Statistical Graphics Corporation, 1993).

3. Results

Influence of temperature, solar irradiance and superficial gas velocity in the riser on the yield of the system was analysed in both discontinuous and continuous culture experiments.

3.1. Batch cultures

The influence of the temperature was analysed in two experiments carried out at the same air flow

rate (4.0 l min 1 ) and mean daily irradiance (1100

the maximum

temperature at 35 8 C (Fig. 2) and 28 8 C (Fig. 3). In the two experiments the reactor was inoculated and operated in batch mode with an initial concentration of 0.2 g l 1 . By maintaining the temperature at 35 8 C (Fig. 2), the culture was photoinhibited on all days, where an Fv/Fm ratio of 0.6 in the early morning, decreases to 0.45 in the daylight period (Fig. 2A). The dissolved oxygen values were low although they were slightly higher than air saturation (100%) (Fig. 2B), thus indicat- ing cellular activity, although the growth rates were near to zero (Fig. 2C). The maximum daily growth rate achieved was 8 /10 3 h 1 , ranging

between 2 /10 3 and 8 /10 3 h 1 (Fig. 2C). The culture collapsed when the temperature ex- ceeded 35 8 C. By maintaining temperature at 28 8 C (Fig. 3), photoinhibition was measured in the first 2 days with Fv/Fm values of 0.4 at noon (Fig. 3A), in which growth was almost negligible (Fig. 3C). However, in the following days, the mean daily irradiance decreased to 800 mE m 2 s 1 and photoinhibition significantly reduced with Fv/Fm values of 0.7, and biomass concentration increased (Fig. 3A). From days 4 to 7, the mean daily irradiance again increased to 1100 mE m 2 s 1 but the biomass concentration had already reached approximately 0.5 g l 1 which prevented photo- inhibition. Growth rates based on daylight period of 0.038 h 1 were measured (Fig. 3C). Never- theless, the biomass productivity was low (0.25 g l 1 per day) because of the reduced value of the

mE m 2 s 1 ) but controlling

v alue of the m E m 2 s 1 ) but controlling Fig. 2. Variation
v alue of the m E m 2 s 1 ) but controlling Fig. 2. Variation
v alue of the m E m 2 s 1 ) but controlling Fig. 2. Variation

Fig. 2. Variation of culture parameters for batch culture carried out at 35 8C, 4.0 l min 1 and 1100 mE m 2 s 1 . (A) Biomass concentration and Fv/Fm ratio, (B) dissolved oxygen and temperature, (C) solar irradiance and daily growth rate.

biomass concentration in the culture. It was also observed that the growth rate decreased when dissolved oxygen values approached 400% Sat. (with respect to air saturated cultures; Fig. 3B). Once the dissolved oxygen exceeded this value, the culture collapsed. The influence of the irradiance was analysed at the same air flow rate (4.0 l min 1 ) and tempera- ture (28 8 C) but at mean daily irradiances of 1100

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et al. / Journal of Biotechnology 103 (2003) 137 / 152 Fig. 3. Variation of culture
et al. / Journal of Biotechnology 103 (2003) 137 / 152 Fig. 3. Variation of culture
et al. / Journal of Biotechnology 103 (2003) 137 / 152 Fig. 3. Variation of culture

Fig. 3. Variation of culture parameters for batch culture carried out at 28 8C, 4.0 l min 1 and 1100 mE m 2 s 1 . (A) Biomass concentration and Fv/Fm ratio, (B) dissolved oxygen and temperature, (C) solar irradiance and daily growth rate.

and temperature, (C) solar irradiance and daily growth rate. Fig. 4. Variation of culture parameters for
and temperature, (C) solar irradiance and daily growth rate. Fig. 4. Variation of culture parameters for
and temperature, (C) solar irradiance and daily growth rate. Fig. 4. Variation of culture parameters for

Fig. 4. Variation of culture parameters for batch culture carried out at 28 8C, 4.0 l min 1 and 1400 mE m 2 s 1 . (A) Biomass concentration and Fv/Fm ratio, (B) dissolved oxygen and temperature, (C) solar irradiance and daily growth rate.

m 2

(Fig. 3) and 1400 mE

cases, the reactor was inoculated and operated in batch mode. At the highest irradiance, 1400 mE m 2 s 1 , photoinhibition was observed on all days, being higher in the first few days during the midday hours of maximum irradiance, although when the biomass concentration increased this effect was reduced (Fig. 4A). However, the Fv/

s 1

(Fig. 4). In both

Fm values decreased at high dissolved oxygen concentrations and the culture finally collapsed at dissolved oxygen values higher than 400% Sat. (Fig. 4B). With respect to the growth and pro-

ductivity, the values reached were lower than those

The

obtained

maximum growth rate was 0.016 h 1 and the

at

1100

mE

m 2

s 1

(Fig.

3).

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143

al. / Journal of Biotechnology 103 (2003) 137 / 152 143 Fig. 5. Variation of culture
al. / Journal of Biotechnology 103 (2003) 137 / 152 143 Fig. 5. Variation of culture
al. / Journal of Biotechnology 103 (2003) 137 / 152 143 Fig. 5. Variation of culture

Fig. 5. Variation of culture parameters for batch culture carried out at 28 8C, 7.0 l min 1 and 1100 mE m 2 s 1 . (A) Biomass concentration and Fv/Fm ratio, (B) dissolved oxygen and temperature, (C) solar irradiance and daily growth rate.

maximum biomass productivity was 0.10 g l 1 per day (Fig. 4C). The influence of the air flow rate was analysed at the same temperature (28 8 C) and irradiance (1100 mE m 2 s 1 ) but at different riser air flow rates: 4.0 (Fig. 3), 7.0 (Fig. 5) and 11.0 l min 1 (Fig. 6). To reduce photoinhibition the batch cultures were inoculated at higher biomass con- centration (0.5 g l 1 ). At 7.0 l min 1 , although the

(0.5 g l 1 ). At 7.0 l min 1 , although the Fig. 6. Variation
(0.5 g l 1 ). At 7.0 l min 1 , although the Fig. 6. Variation
(0.5 g l 1 ). At 7.0 l min 1 , although the Fig. 6. Variation

Fig. 6. Variation of culture parameters for batch culture carried out at 28 8C, 11.0 l min 1 and 1100 mE m 2 s 1 . (A) Biomass concentration and Fv/Fm ratio, (B) dissolved oxygen and temperature, (C) solar irradiance and daily growth rate.

irradiance was high, it was observed that photo- inhibition was significantly reduced, including the values for the initial days in which the biomass concentrations were at their lowest (Fig. 5A). In addition, the dissolved oxygen concentration re- duced to values always below 350% (Fig. 5B). Under these conditions the biomass concentration reached values of 2.0 g l 1 in 7 days; whereas the biomass productivity was 0.9 g l 1 per day. The

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F.G. Acie´n Ferna´ndez et al. / Journal of Biotechnology 103 (2003) 137 /152

growth rate reached values of 0.052 h 1 , reflecting more favourable culture conditions (Fig. 5C). At 11.0 l min 1 (Fig. 6), the biomass concentration further increased up to 3.0 g l 1 in 7 days, whereas the biomass productivity reached values of 1.3 g l 1 per day. Photoinhibition was not observed with Fv/Fm values above 0.6 even at noon (Fig. 6A), whereas the growth rate reached values of

0.068 h 1

Both temperature and

dissolved oxygen were kept below 30 8 C and 350% Sat., respectively (Fig. 6B). These results confirmed the capability of the reactor to reach high biomass productivities when operated at high biomass concentrations and suitable fluid-dynamic conditions, due to an improved mass transfer capacity of the system, reducing the dissolved oxygen concentrations in the culture.

(Fig.

6C).

3.2. Continuous cultures

To verify the influence of fluid-dynamics condi- tions in the yield of the system, three chemostat cultures were carried out at the same dilution rate (0.038 h 1 ), temperature (28 8 C) and solar irra-

flow

rates of 12.0, 17.0 and 14 l min 1 (Fig. 7). The selected dilution rate had already shown to be the optimal in the culture of P. tricornutum in tubular reactors (Molina Grima et al., 1996; Acie´n Fer- na´ndez et al., 1998). The results confirm the significant impact of airflow rate on the yield of the system. Thus, the higher the airflow rate, the higher the biomass concentration, with biomass productivities up to 1.4 g l 1 per day being obtained. Photoinhibition was low, only being observed at noon, with minimum Fv/Fm values of 0.58 (Fig. 7A). Temperature and dissolved oxygen were well controlled, ranging from 25 to 30 8 C, and from 100 to 400% Sat., respectively (Fig. 7B). No correlation between air flow rate and dissolved oxygen level was noted. Thus, at the maximum air flow rate of 17.0 l min 1 the mean daily dissolved oxygen was 280% Sat., higher than mean daily dissolved oxygen of 250% Sat. mea- sured at 14.0 l min 1 . For an air flow rate of 12.0 l min 1 the mean daily dissolved oxygen was 300% Sat. The higher the airflow rate the higher the mass transfer capacity of the reactor, the biomass

diance (1100 mE m 2 s 1 ) but at different air

diance (1100 m E m 2 s 1 ) but at different air Fig. 7. Variation
diance (1100 m E m 2 s 1 ) but at different air Fig. 7. Variation
diance (1100 m E m 2 s 1 ) but at different air Fig. 7. Variation

Fig. 7. Variation of culture parameters with the air flow rate for chemostat cultures carried out at 0.038 h 1 , 28 8C and 1100 mE m 2 s 1 . (A) Biomass concentration and Fv/Fm ratio, (B) dissolved oxygen and temperature, (C) solar irradiance and daily growth rate.

productivity, the photosynthesis and the oxygen generation rate. The mean daily irradiance was 1100 mE m 2 s 1 , whereas the growth rate was quite similar throughout the culture period (Fig. 7C). No cellular damage was observed under the light microscope, even at the maximum airflow rate.

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4. Discussion

Analysis of the data showed that temperature and dissolved oxygen were the major factors influencing the collapse of the cultures. Cultures collapsed when temperature increased above 35 8 C or when dissolved oxygen values were higher than 400% Sat. In both cases, the value of Fv/Fm ratio was lower than 0.6 before the cells collapsed. To prevent these problems, a heat exchanger must be employed to control temperature and the mass transfer capacity must be maintained at levels higher than the maximum photosynthetic rate. Considering a maximum biomass productivity of 2 g l 1 per day with 50% of carbon content, and an O 2 /CO 2 photosynthesis ratio equal to 1 mol O 2 /

mol CO 2 , a mass transfer coefficient of 6.0 /10 3

s 1

would be needed to prevent dissolved oxygen

levels exceeding 400% Sat. In this work, the

maximum mass transfer coefficient through the

cultures was 3.8 /10 3 s 1 , and

therefore, a

maximum biomass productivity of approximately 1.5 g l 1 per day could be achieved for dissolved oxygen below 400% Sat. In addition to temperature and dissolved oxy- gen, excess of irradiance also reduces the yield of the system. Photoinhibition was observed at biomass concentrations below 0.5 g l 1 , the

growth rates being very low. To reduce this phenomenon the cultures should be inoculated at biomass concentrations above 0.5 g l 1 , or alter- natively the culture should be shaded at lower values of cell density. From this data, it may be inferred that the Fv/Fm ratio is also somewhat related not only with photoinhibition phenomena but also with cellular stress promoted by both dissolved oxygen and temperature. Experimental data showed as Fv/Fm values ranged between 0.65 and 0.70 for average irradiances lower than 280 mE m 2 s 1 , then linearly decreasing with average irradiance to values of 0.50 at 400 mE m 2 s 1 (Fig. 8A). This behaviour was observed for all the data except for those corresponding to experiment

l min 1 , 1100 mE m 2 s 1 and batch (28 8 C, 4.0 l min 1 , 1100 mE m 2 s 1

1 (35 8 C, 4.0 mode) and 2

and batch mode) for which Fv/Fm values were lower than expected for the same average irradi- ance. The lower value of Fv/Fm ratio for these two

experiments was related with the poor temperature control in 1 and an excess of dissolved oxygen in 2. Statistical analysis of the data showed as Fv/Fm was a function of dissolved oxygen (S.L. /0.0002), temperature (S.L. /0.0004) and average irradi- ance to which the cells were exposed to (S.L. / 0.0010). The Fv/Fm parameter decreased below 0.6 (strongly stressed cells) at dissolved oxygen higher than 350% (Fig. 9A), temperatures higher than 30 8 C (Fig. 9B) or average irradiances above

270 mE m 2 s 1

microalgal cultures at high irradiances has been reported in outdoor and indoor cultures (Rich- mond and Becker, 1986; Vonshak and Guy, 1988; Molina Grima et al., 1996; Torzillo et al., 1996; Acie´n Ferna´ndez et al., 1998) and it has been attributed to the reversible destruction of key components of PSII (Samuelsson et al., 1985; Jensen and Knutsen, 1993). Furthermore, photo- oxidation processes also take place in the cultures at high dissolved oxygen concentrations reducing the yield of the system. The effect of both photooxidation and photoinhibition being higher at non optimal temperatures (Richmond, 1990; Torzillo et al., 1998). Regarding the daily growth rate, experimental data showed that for all the experiments, a hyperbolic relationship between growth rate and average irradiance was observed, although the parameters of the model being significantly differ- ent (Fig. 8B). This behaviour was observed for average irradiance values below 250 mE m 2 s 1 . Above these values of irradiance, a linear decrease in the growth rate was observed. Fig. 8B shows data from experiments corresponding to stressed cultures with low values of Fv/Fm (experiments 1 / 3), which reached growth rates lower than ex- pected. For the rest of the experiments, the higher the air flow rate the higher the growth rate for the same average irradiance, or the lower the average irradiance for the same growth rate. By comparing experiment 4 and 5, corresponding to batch cultures at 7.0 and 11.0 l min 1 airflow rate, it was apparent that for the same average irradiance, higher growth rates were obtained at the higher airflow rates (Fig. 8B). Comparing the continuous cultures, experiments 6 /8, in which the growth rate was controlled by the imposed dilution rate

(Fig. 9C). The photoinhibition of

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et al. / Journal of Biotechnology 103 (2003) 137 / 152 Fig. 8. Variation of culture
et al. / Journal of Biotechnology 103 (2003) 137 / 152 Fig. 8. Variation of culture
et al. / Journal of Biotechnology 103 (2003) 137 / 152 Fig. 8. Variation of culture
et al. / Journal of Biotechnology 103 (2003) 137 / 152 Fig. 8. Variation of culture

Fig. 8. Variation of culture parameters with average irradiance inside the culture at different temperatures (T , 8C), air flow rate (Q , l min 1 ), mean daily irradiance (I md , mE m 2 s 1 ) and operation mode (D , batch D /0.00 h 1 or continuous mode D /0.04 h 1 ).

(D /0.038 h 1 ), the same growth rate was ob- tained at lower average irradiance when airflow rate increased from 12.0 to 17.0 l min 1 . However, for experiments 4 /8, the cultures were not stressed and the values of Fv/Fm were very similar, implying that another variable related to the air flow rate must be influencing the yield of the system. Statistical analysis of the data showed that the growth rate was a function of average irra- diance (S.L. /0.0100), Fv/Fm ratio (S.L. / 0.0066) and airflow (S.L. /0.0022) (Fig. 10). The growth rate hyperbolically increased with the average irradiance (Fig. 10A) and linearly in- creased with the Fv/Fm ratio (Fig. 10B). A linear increase of growth rate with airflow rate was also observed, although only for batch experiments, and not for chemostat cultures in which the growth rate was controlled (Fig. 10C). The mea- sured growth rates were similar to the maximum values reported for P. tricornutum in outdoor

reactors of 0.06 h 1 (Acie´n Ferna´ndez et al., 1998) but higher than reported at indoor condi- tions. Chrismadha and Borowitzka (1994) re- ported maximum values in an indoor 30 l helical reactor of 0.02 h 1 at irradiance on the reactor surface of 286 mE m 2 s 1 , whereas this growth

rate decreased

to 0.006 h 1 at 1700 mE m 2 s 1

due to photoinhibition. Although the growth rate is an important parameter of the culture, the yield of the system is quantified by the biomass productivity. Further- more, empirical data showed that the biomass productivity linearly decreased with the average irradiance for non stressed cultures (experiments 4 /8), whereas stressed cultures (experiments 1 /3) showed very low biomass productivities (Fig. 8C). It was also observed that for the same average irradiance, higher biomass productivities were reached at higher airflow rates, or the same biomass productivity was obtained at lower aver-

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al. / Journal of Biotechnology 103 (2003) 137 / 152 147 Fig. 9. Variation of F
al. / Journal of Biotechnology 103 (2003) 137 / 152 147 Fig. 9. Variation of F
al. / Journal of Biotechnology 103 (2003) 137 / 152 147 Fig. 9. Variation of F

Fig. 9. Variation of Fv/Fm ratio with (A) dissolved oxygen, (B) temperature and (C) average irradiance inside the culture. Data are the mean values and standard error obtained from variance analysis of experimental data.

age irradiances (Fig. 8C). This singular phenom- enon was also observed in horizontal tubular reactors (Acie´n Ferna´ndez et al., 2001). The maximum biomass productivity obtained in the present work with P. tricornutum (1.5 g l 1 per day) was much higher than the 0.14 g l 1 per day obtained in a indoor 30 l helical reactor with the same strain (Chrismadha and Borowitzka, 1994), or 0.51 g l 1 per day obtained in an indoor 15 l helical reactor with Spirulina platensis (Watanabe

15 l helical reactor with Spirulina platensis ( Watanabe Fig. 10. Variation of growth rate with

Fig. 10. Variation of growth rate with (A) average irradiance inside the culture, (B) Fv/Fm ratio and (C) air flow rate in the riser. Data are the mean values and standard error obtained from variance analysis of experimental data.

et al., 1995). Statistical analysis of the data showed that biomass productivity was a function of average irradiance (S.L. /0.0000), Fv/Fm ratio (S.L. /0.0000) and airflow rate (S.L. /0.0000) (Fig. 11). Biomass productivity is also a function of growth rate and biomass concentration, typi- cally showing a maximum value at growth rates half than the maximum specific growth rate of the micro-organism. Fig. 8C shows that the maximal

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et al. / Journal of Biotechnology 103 (2003) 137 / 152 Fig. 11. Variation of biomass
et al. / Journal of Biotechnology 103 (2003) 137 / 152 Fig. 11. Variation of biomass
et al. / Journal of Biotechnology 103 (2003) 137 / 152 Fig. 11. Variation of biomass

Fig. 11. Variation of biomass productivity with (A) average irradiance, (B) Fv/Fm ratio and (C) air flow rate in the riser. Data are the mean values and standard error obtained from variance analysis of experimental data.

biomass productivity was at average irradiances of 50 /150 mE m 2 s 1 , corresponding to growth rates of 0.035 /0.045 h 1 . Below 50 mE m 2 s 1 , the biomass concentration was too high and the culture was strongly photolimited, whereas at irradiances higher than 150 mE m 2 s 1 the yield of the culture was reduced by photoinhibition. This optimum range was previously reported for

this strain in horizontal tubular photobioreactors (Molina Grima et al., 1996; Acie´n Ferna´ndez et al., 1998). Another important parameter that quantified the yield of the system is the photosynthetic efficiency. As expected, the photosynthetic effi- ciency was very low for stressed cultures, slightly decreasing when average irradiance increased (Fig. 8D). In addition, an influence of air flow rate was

observ ed, where the photosynthetic efficiency in- creases with the air flow rate at the ved, where the photosynthetic efficiency in- creases with the air flow rate at the same average irradiance, and the photosynthetic efficiency re- maining the same for lower average irradiances at higher air flow rates (Fig. 8D). These variations were clearly observed from statistical analysis of the data. As the photosynthetic efficiency was also a function of average irradiance (S.L. /0.0000), Fv/Fm ratio (S.L. /0.0000) and airflow rate (S.L. /0.0000); photosynthetic efficiency linearly decreased with average irradiance (Fig. 12A), and increasing with the Fv/Fm ratio (Fig. 12B) and air flow rate in the riser (Fig. 12C). In the present work, a maximum photosynthetic efficiency of 15% was measured. This value correlates well with the 20% reported in outdoor horizontal reactors (Acie´n Ferna´ndez et al., 1998) or 13% reported in outdoor channels (Raymond, 1977) with P. tricor- nutum . Similar photosynthetic efficiency values were also obtained with other strains, including 20% (Tamiya, 1957) and 18% (Myers, 1980) reported for dense outdoor cultures of Chlorella . However, the measured photosynthetic efficiency was much higher than 8% reported for Spirulina

platensis in an indoor 15 l helical reactor (Wata- nabe et al., 1995) and 7% reported in a 8 l cone- shaped helical reactor also with Spirulina (Wata- nabe and Hall, 1996). Maximum value of 8% were reported for indoor chemostat cultures of Isochry- sis galbana in a 5 l photobioreactor, this value being obtained at the lowest irradiance (800 mE m 2 s 1 ) and decreasing at higher irradiances (Molina Grima et al., 1997). Air flow rate determines both the mass transfer and the fluid-dynamic conditions. Mass transfer influences the dissolved oxygen level in the culture; however, in continuous mode it was observed that there was not a linear relationship between dis- solved oxygen and airflow rate. On the other hand,

F.G. Acie´n Ferna´ndez et al. / Journal of Biotechnology 103 (2003) 137 /152

149

al. / Journal of Biotechnology 103 (2003) 137 / 152 149 Fig. 12. Variation of photosynthetic

Fig. 12. Variation of photosynthetic efficiency with (A) average irradiance, (B) Fv/Fm ratio and (C) air flow rate in the riser. Data are the mean values and standard error obtained from variance analysis of experimental data.

the fluid-dynamics determine the induced liquid velocity and, along with the light profiles within the reactor, the light/dark cycle frequency, n , at which the cells were exposed to light. Therefore, in only one variable, n incorporates both the influ- ence of the average irradiance and the air flow rate. Analysis of the data showed that biomass productivity and photosynthetic efficiency were a

biomass producti v ity and photosynthetic efficiency were a Fig. 13. Variation of (A) biomass producti

Fig. 13. Variation of (A) biomass productivity, (B) photosyn- thetic efficiency and (C) Fv/Fm ratio with the light/dark cycle frequency. Data are the mean values and standard error obtained from variance analysis of experimental data.

function of light frequency (S.L. /0.0000) and Fv/ Fm ratio (S.L. /0.0001), with biomass productiv- ity and photosynthetic efficiency linearly increas- ing with light frequency (Fig. 13). The linear relationship was not clearly observed at low frequency values because in these experiments the cultures were stressed (Fig. 13). Thus, a frequency near zero was obtained for diluted cultures devoid of dark zones (photoinhibited cells), whereas a

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frequency 0.38 Hz was obtained for cultures at low air flow rates in which dissolved oxygen also stressed the cultures. The n and biomass produc- tivities reached in the helical reactor shown in Fig. 13 agree well with those values reported by Molina Grima et al. (2000) for tubular reactors (Fig. 14). In this figure, data from the non-stressed experi- ments (experiments 4 /8) are plotted along with those obtained in horizontal placed tubular reac- tors of 0.03 and 0.06 m tube diameter. However, Molina Grima et al. (2000) determined a hyper- bolic relationship between biomass productivity and frequency, which was not observed in this present work. This could be due to the narrow range of n data obtained in the present work compared with that to reported in outdoor hor- izontal tubular photobioreactors (Fig. 14). To achieve higher biomass productivities n must be enhanced. For a specific biomass concentration the only way to increase n is to increase the liquid velocity. Thus, an optimum n value of 1 Hz has been proposed (Molina Grima et al., 2000; Schmidt-Staiger et al., 2000). Schmidt-Staiger et al. (2000) demonstrated that light/dark cycles of roughly 1 s enhanced productivity, in flat plate airlift reactors with static mixers to ensure the transportation of the algal cells in defined liquid loop, a 71% relative to flat plate airlift reactor

liquid loop, a 71% relati v e to flat plate airlift reactor Fig. 14. Variation of

Fig. 14. Variation of biomass productivity with light/dark cycle frequency. Only not stressed data has been indicated. Filled points corresponded to data reported by Molina Grima et al. (2000) for outdoor cultures of P. tricornutum in horizontal placed tubular reactors at similar level of irradiance. Line corresponded to model proposed by Molina Grima et al. (2000) for 1100 mE m 2 s 1 solar irradiances.

without static mixers. If one considers an average irradiance of 150 mE m 2 s 1 , it can be calculated (using equations 2, 3, 4 and light profiles) that a liquid flow rate of 0.5 m s 1 would be needed in the helical reactor to achieve a light/dark cycle frequency of 1 Hz.

5. Conclusions

The feasibility of a helical reactor for the out- door-continuous production of P. tricornutum biomass was demonstrated. It should be pointed out that the major issue to be considered is the mass transfer capacity of the reactor. Thus, the mass transfer must be improved in order to increase the productivity by reducing the stress caused by the high levels of dissolved oxygen. In addition, the liquid flow rate must be increased in order to approximate the light-dark cycle frequen- cies up to the optimum reported values (about 1 Hz). Thus, the liquid velocity through the tube must be increased up to 0.5 m s 1 . However, in practice it is quite difficult to reach this velocity by only increasing the height of the degasser of a single air-lift pump. An alternative approach would be to provide two airlift systems, where one pump creates a negative pressure to draw liquid from the outlet of photostage, while the second pump forces liquid into the inlet of the photostage as described herein. Alternatively, other types of pump should be considered, although cellular damage caused by excessive hydrodynamic shear forces within conventional pumps could be another issue to solve in these circumstances. Progressive cavity pumps (mono), peristaltic, lobe, gear and centrifugal pumps, all cause significant cell damage to microalgal bio- mass when coupled to tubular photobioreactors. An analysis of the influence of alternative pumps and the hydrodynamic stress inflicted on the biomass is the current aim of our work with this photobioreactor.

6. Notation

C b

biomass concentration (g l 1 )

F.G. Acie´n Ferna´ndez et al. / Journal of Biotechnology 103 (2003) 137 /152

151

D

dilution rate (h 1 )

g

gravitational acceleration

(m

s 2 )

I av

I md

I o

average irradiance inside the reactor (mE

m 2 s 1 )

mean daily irradiance (mE m 2 s 1 ) irradiance on photobioreactor surface (mE

m 2 s 1 )

k L a L volumetric gas /liquid mass transfer coeffi- cient (s 1 ) K a extinction coefficient for biomass (m 2 g 1 )

L straight tube length in loop (m)

P b R O 2

t

volumetric productivity of biomass (g l 1 ) volumetric rate of oxygen generation (mol

O 2 m 3

s 1 )

Time (s) Gas velocity in the riser zone (m s 1 )

U Gr

V Volume of culture (m 3 )

Acknowledgements

This research was supported by the European Union (contract BRPR CT97 0537), Ministerio de Ciencia y Tecnologı´a (PPQ2000-1220), and Junta de Andalucı´a, Plan Andaluz de Investigacio´n II (CVI 173). The authors thank Dr S. Skill for his comments on the draft of the manuscript.

References

Acie´n Ferna´ndez, F.G., Garcı´a Camacho, F., Sa´nchez Pe´rez, J.A., Ferna´ndez Sevilla, J.M., Molina Grima, E., 1997. A model for light distribution and average solar irradiance inside outdoor tubular photobioreactors for the microalgal mass culture. Biotechnol. Bioeng. 55 (5), 701 /714. Acie´n Ferna´ndez, F.G., Garcı´a Camacho, F., Sa´nchez Pe´rez, J.A., Ferna´ndez Sevilla, J.M., Molina Grima, E., 1998. Modelling of biomass productivity in tubular photobio- reactors for microalgal cultures: effects of dilution rate, tube diameter and solar irradiance. Biotechnol. Bioeng. 58 (6), 605 /616. Acie´n Ferna´ndez, F.G., Garcı´a Camacho, F., Sa´nchez Pe´rez, J.A., Ferna´ndez Sevilla, J.M., Molina Grima, E., 2000. Modeling of eicosapentaenoic acid (EPA) production from Phaeodactylum tricornutum cultures in tubular photobio- reactors. Effects of dilution rate, tube diameter and solar irradiance. Biotechnol. Bioeng. 68 (2), 173 /183. Acie´n Ferna´ndez, F.G., Ferna´ndez Sevilla, J.M., Sa´nchez Pe´rez, J.A., Molina Grima, E., Chisti, Y., 2001. Airlift-driven

external-loop tubular photobioreactors for outdoor produc- tion of microalgae: assessment of design and performance. Chem. Eng. Sci. 56, 2721 /2732. Borowitzka, M.A., 1986. Microalgae as sources of fine chemi-

cals. Microbiol. Sci. 3, 372 /375.

Borowitzka, M.A., 1996. Closed algal photobioreactors: design considerations for large-scale systems. J. Mar. Biotechnol. 70, 313 /321. Chrismadha, T., Borowitzka, M.A., 1994. Effect of cell density and irradiance on growth, proximate composition and

eicosapentaenoic acid production of Phaeodactylum tricor- nutum grown in a tubular photobioreactor. J. Appl. Phycol.

6, 67 /74. Grobbelaar, J.U., 1994. Turbulence in algal mass cultures and the role of light/dark fluctuations. J. Appl. Phycol. 6, 331 /

335.

Jensen, S., Knutsen, G., 1993. Influence of light and tempera- ture on photoinhibition of photosynthesis in Spirulina platensis . J. Appl. Phycol. 5, 495 /504. Knauer, J., Southgate, P.C., 1999. A review of the nutritional requirements of bivalves and the development of alternative

and artificial diets for bivalve aquaculture. Rev. Fisheries Sci. 7 (3 /4), 241 /280. Mann, J.E., Myers, J., 1968. On pigments, growth and

tricornutum . J. Phycol.

4, 349 /355. Molina Grima, E., Garcı´a Camacho, F., Sa´nchez Pe´rez, J.A., Acie´n Ferna´ndez, F.G., Ferna´ndez Sevilla, J.M., Valde´s

Sanz, F., 1994. Effect of dilution rate on eicosapentaenoic acid productivity of Phaeodactylum tricornutum UTEX 640 in outdoor chemostat culture. Biotechnol. Lett. 16 (10),

photosynthesis of Phaeodactylum

1035 /1040.

Molina Grima, E., Sa´nchez Pe´rez, J.A., Garcı´a Camacho, F., Ferna´ndez Sevilla, J.M., Acie´n Ferna´ndez, F.G., 1996. Productivity analysis of outdoor chemostat culture in tubular air-lift photobioreactors. J. Appl. Phycol. 8, 369 /380. Molina Grima, E., Garcı´a Camacho, F., Sa´nchez Pe´rez, J.A., Acie´n Ferna´ndez, F.G., Ferna´ndez Sevilla, J.M., 1997.

Growth yield determination in a chemostat culture of the marine microalga Isochrysis galbana . J. Appl. Phycol. 8,

529 /534.

Molina Grima, E., Acie´n, F.G., Garcı´a, F., Chisti, Y., 1999.

Photobioreactors: light regime, mass transfer, and scale-up.

J. Biotechnol. 70, 231 /247.

Molina Grima, E., Acie´n Ferna´ndez, F.G., Garcı´a Camacho,

F., Camacho Rubio, F., Chisti, Y., 2000. Scale-up of tubular photobioreactors. J. Appl. Phycol. 12, 355 /368. Myers, J., 1980. On the algae: thoughts about physiology and measurements of efficiency. In: Falkowsky, P.G. (Ed.), Primary Productivity in the Sea. Plenum Press, New York, pp. 1 /15. Philipps, J.N., Myers, J., 1954. Measurement of algal growth under controlled steady-state conditions. Plant Physiol. 29,

152 /161.

Pulz, O., 2001. Photobioreactors: production systems for

phototrophic microrganisms. Appl. Microbiol. Biotechnol. 57, 287 /293.

152

F.G. Acie´n Ferna´ndez et al. / Journal of Biotechnology 103 (2003) 137 /152

Raymond, L., 1977. Initial investigations of shallow-layer algal production system. Report to Hawaii Natural Energy Institute, University of Hawaii, and the Department of Planning and Economic Development, The State of Hawaii, Honolulu, 27. Richmond, A., 1990. Large scale microalgal culture and applications. In: Round, F.E., Chapman, D.J. (Eds.), Progress in Phycological Research, vol. 7. Biopress Ltd, pp. 269 /329. Richmond, A., Becker, E.W., 1986. Technological aspects of mass cultivation, a general outline. In: Richmond, A. (Ed.), CRC Handbook of Microalgal Mass Culture. CRC Press, Boca Raton, FL, pp. 245 /263. Robinson, L.F., Morrison, A.W., Bamforth M.R., 1987. European Pat. 0239272 (6 March 1987), to Biotechna Ltd. Samuelsson, G., Lonneborg, A., Rosenqvist, E., Gustafsson, P., Oquist, G., 1985. Photoinhibition and reactivation of photosynthesis in the cyanobacterium Anacystis nidulans . Plant Physiol. 79, 992 /995. Schmidt-Staiger, V., Degen, J., Retze, A., Krunn, A., Trosch, W., 2000. Light regime, the key to efficient mass culture of microalgae in photobioreactors. In: Proceedings of the Fourth International Congress on Biochemical Engineering, Frannhofer IRB Verlag, Stuttgart, Germany, pp. 139 /143. Tamiya, H., 1957. Mass culture of algae. Ann. Rev. Plant. Physiol. 8, 309 /333. Terry, K.L., 1986. Photosynthesis in modulated light: quanti- tative dependence of photosynthetic enhancement on flash- ing rate. Biotechnol. Bioengng. 28, 988 /995. Terry, K.L., Raymond, L.P., 1985. System design for the autotrophic production of microalgae. Enzyme Microb. Technol. 7, 474 /487. Thomas, W.H., Seibert, D.L.R., Alden, M., Neori, A., El- dridge, P., 1984. Yields, photosynthetic efficiencies and approximate composition of dense marine microalgal cul- tures. I. Introduction and Phaeodactylum tricornutum experiments. Biomass 5, 181 /209.

Torzillo, G., Bernardini, P., Masojidek, J., 1998. On-line monitoring of chlorophyll fluorescence to assess the extent of photoinhibition of photosynthesis induced by high oxygen concentration and low temperature and its effect on the productivity of outdoor cultures of Spirulina platensis (Cyanobacteria). J. Phycol. 34 (3), 504 /510. Torzillo, G., Accolla, P., Pinzani, E., Masojidek, J., 1996. In situ monitoring of chlorophyll fluorescence to assess the synergistic effect of low temperature and high irradiance stresses in Spirulina cultures grown outdoors in photobio- reactors. J. Appl. Phycol. 8, 283 /291. Travieso, L., Pellon, A., Benitez, F., Sanchez, E., Borja, R., O’Farrill, N., Weilland, P., 2002. BIOALGA reactor:

preliminary studies for heavy metals removal. Biochem. Eng. J. 12, 87 /91. Tredici, M.R., 1999. Bioreactors, photo. In: Flickinger, M.C., Drew, S.W. (Eds.), Encyclopedia of Bioprocess Technology:

Fermentation, Biocatalysis and Bioseparation, vol. 1. Wiley, New York, pp. 395 /419. Villar, R., Laguna, M.R., Cadavid, I., Calleja, J.M., 1994. Effects of aqueous extracts of six marine microalgae on smooth muscle contraction in rat duodenum and vas deferens. Planta Med. 60, 521 /526. Vonshak, A., Guy, R., 1988. Photoinhibition as a limiting factor in outdoor cultivation of Spirulina platensis . In:

Algal Biotechnology. Elsevier, Amsterdam, pp. 365 /370. Watanabe, Y., Hall, D.O., 1996. Photosynthetic production of the filamentous cyanobacterium Spirulina platensis in a cone-shaped helical tubular photobioreactor. Appl. Micro- biol. Biotechnol. 44, 693 /698. Watanabe, Y., de la Nou¨ e, J., Hall, D.O., 1995. Photosynthetic performance of a helical tubular photobioreactor incorpor- ating the cyanobacterium Spirulina platensis . Biotechnol. Bioeng. 47, 261 /269. Yongmanitchai, W., Ward, O.P., 1991. Growth of and Omega- 3 fatty acid production by Phaeodactylum tricornutum under different culture conditions. Appl. Environ. Micro- biol. 57 (2), 419 /425.