Acién-Fernández et al. 2003 Outdoor production of Phaeodactylum tricornutum biomass in a helical...

Outdoor production of Phaeodactylum tricornutum biomass ina helical reactor

F.G. Acien Fernandez a, David O. Hall b,1, E. Canizares Guerrero a,K. Krishna Rao b, E. Molina Grima a,*

a Department of Chemical Engineering, University of Almerıa, E-04071 Almerıa, Spainb 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 8C,

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-

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-

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

950-01-5484.

E-mail address: [email protected] (E. Molina Grima).1 Now deceased.

Journal of Biotechnology 103 (2003) 137�/152

www.elsevier.com/locate/jbiotec

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

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

mailto:[email protected]

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 andreducing the self-shading phenomenon; (2) easy

control of temperature and contaminants because

it is a closed bioreactor; and (3) better CO2

transfer from the gas stream to the liquid culture

medium due to the extensive CO2 absorbing

pathway (Watanabe et al., 1995).

For any photobioreactor design, the system

productivity in continuous operating mode isobtained 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 tobe 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 UTEX640 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 8C) and using the

Mann and Myers’ medium (1968).

Outdoor cultures were operated in both discon-

tinuous and continuous mode. In the continuousmode, 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 andMyers (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

Almerıa (36850?N, 2827?W), Spain. The helical

reactor had a volume of 75 l, and consisted of106 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

F.G. Acien Fernandez et al. / Journal of Biotechnology 103 (2003) 137�/152138

desorption, whereas CO2 was injected at the base

of the helical section for pH control. A heatexchanger 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 (Imd). The light profiles within the

culture were estimated using a solar irradiance

model for tubular photobioreactors (Acien Fer-nandez 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

(Ka). The irradiance on the culture surface (I0) 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 (Acien Fernandez et al., 2001). The absorp-tion coefficient depended on the biomass pigment

content (Xp) (Molina Grima et al., 1996), in

accordance with the equation:

Ka�0:0105�0:0299Xp (1)

The average irradiance inside the culture (coiled

tube) (Iav) was calculated as the volumetric integral

of light profiles data (Acien Fernandez 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�2s�1 (Mann and Myers, 1968; Acien Fer-nandez 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 timeexpended in the dark zone (td) as (Molina Grima

et al., 2000):

n�1 � f

td

(2)

where the time spent in the dark (td) was a functionof the culture volume in the dark (Vd), the tube

dark cross section (Sd) and the radial velocity of

the liquid (UR), this relationship being (Molina

Grima et al., 2000):

td�Vd

URSd

(3)

Both Vd and Sd were calculated from the light

profiles inside the culture according to methodol-

ogies reported elsewhere (Acien Fernandez et al.,

2000; Molina Grima et al., 2000). In Eq. (3), UR is

the radial velocity of the liquid. Cells move

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

F.G. Acien Fernandez et al. / Journal of Biotechnology 103 (2003) 137�/152 139

radially within the fluid because of momentumtransport 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):

UR�0:2

�U7

Lm

dtr

�1=8

(4)

which allows the calculation of the radial velocity

(UR) in the turbulent core as a function of the

superficial liquid velocity (UL), the tube diameter

(dt), and the density (r ) and viscosity (m) of the

culture broth.

2.5. Biomass concentration and daily growth rate

The biomass concentration was estimated fromthe 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:

Cb(g l�1)�0:38 � OD625 (5)

where Cb 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:

ln Cb� ln Cbo�mt (6)

where Cb is the instantaneous biomass concentra-tion, Cbo 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 Cb 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�PbHbiomass

Fvol

(7)

where Pb is the volumetric biomass productivity,

Hbiomass is the combustion enthalpy of the bio-

mass, and Fvol is the photon flux absorbed in unit

volume. Eq. (7) represent the efficiency in using

solar irradiance by the biomass. The combustionenthalpy 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; Acien Fernandez et al.,

1998). Photon flux absorbed throughout the

reactor volume may be obtained from Iav, on a

culture volume basis using the following equation

(Molina Grima et al., 1997):

Fvol�Iav � Ka � Cb (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-


GRAPHICS v7.0 (Manugistics Inc. and StatisticalGraphics 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 andcontinuous 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

mE m�2 s�1) but controlling the maximum

temperature at 35 8C (Fig. 2) and 28 8C (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 8C (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 higherthan 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 8C. By maintaining temperature at 28 8C(Fig. 3), photoinhibition was measured in the first2 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 butthe 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

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 8C) but at mean daily irradiances of 1100

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.


(Fig. 3) and 1400 mE m�2 s�1 (Fig. 4). In both

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/

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

obtained at 1100 mE m�2 s�1 (Fig. 3). The

maximum growth rate was 0.016 h�1 and the










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 8C) 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

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










growth rate reached values of 0.052 h�1, reflectingmore 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 (Fig. 6C). Both temperature anddissolved oxygen were kept below 30 8C 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.

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 8C) and solar irra-

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

rates of 12.0, 17.0 and 14 l min�1 (Fig. 7). Theselected dilution rate had already shown to be the

optimal in the culture of P. tricornutum in tubular

reactors (Molina Grima et al., 1996; Acien Fer-

nandez 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 beingobtained. 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 8C, 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 meandaily 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

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.

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.


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 8Cor 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. Toprevent 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

O2/CO2 photosynthesis ratio equal to 1 mol O2/

mol CO2, 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 beinferred 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

1 (35 8C, 4.0 l min�1, 1100 mE m�2 s�1 and batch

mode) and 2 (28 8C, 4.0 l min�1, 1100 mE m�2 s�1

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 temperaturecontrol 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 higherthan 30 8C (Fig. 9B) or average irradiances above

270 mE m�2 s�1 (Fig. 9C). The photoinhibition of

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;

Acien Fernandez et al., 1998) and it has been

attributed to the reversible destruction of keycomponents 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 decreasein 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 comparingexperiment 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


(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 (Acien Fernandez 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-

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 (Imd, mE m�2 s�1) and operation mode (D , batch D�/0.00 h�1 or continuous mode D�/0.04 h�1).


age irradiances (Fig. 8C). This singular phenom-

enon was also observed in horizontal tubular

reactors (Acien Fernandez 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

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

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.

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.


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; Acien Fernandez 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 wasobserved, 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(Acien Fernandez 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,

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.


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

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

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.

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.


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

without static mixers. If one considers an averageirradiance 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 stresscaused 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 asingle 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 excessivehydrodynamic 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 thebiomass is the current aim of our work with this

photobioreactor.

6. Notation

Cb biomass concentration (g l�1)

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.


D dilution rate (h�1)g gravitational acceleration (m s�2)

Iav average irradiance inside the reactor (mE

m�2 s�1)

Imd mean daily irradiance (mE m�2 s�1)

Io irradiance on photobioreactor surface (mE

m�2 s�1)

kLaL volumetric gas�/liquid mass transfer coeffi-

cient (s�1)Ka extinction coefficient for biomass (m2 g�1)

L straight tube length in loop (m)

Pb volumetric productivity of biomass (g l�1)

RO2volumetric rate of oxygen generation (mol

O2 m�3 s�1)

t Time (s)

UGr Gas velocity in the riser zone (m s�1)

V Volume of culture (m3)

Acknowledgements

This research was supported by the European

Union (contract BRPR CT97 0537), Ministerio de

Ciencia y Tecnologıa (PPQ2000-1220), and Junta

de Andalucıa, Plan Andaluz de Investigacion II

(CVI 173). The authors thank Dr S. Skill for his

comments on the draft of the manuscript.

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