fuentes - outdoor continuous culture of porphyridium cruentum in a

ELSEVIER Journal of Biotechnology 70 (1999) 271-288

~ Bi0

Outdoor continuous culture of Porphyridium cruentum in a tubular photobioreactor" quantitative analysis of the daily

cyclic variation of culture parameters

M.M. Rebolloso Fuentes, J.L. Garcia Sfinchez, J.M. Fernfindez Sevilla, F.G. Aci6n Fernfindez, J.A. Sfinchez P6rez, E. Molina Grima *

Departamento de Ingenieria Quimica, Universidad de Almeria, E-04071 Almeria, Spain

Received 27 October 1998; received in revised form 23 November 1998; accepted 22 December 1998

Abstract

The present work reports on the daily cyclic variation of oxygen generation rates, carbon consumption rates, photosynthetic activities, growth rates and biochemical composition of the biomass in a pilot plant continuous outdoor culture of the microalgae Porphyridium cruentum. A linear relationship between the external irradiance and the average irradiance inside the culture was found. In addition, the oxygen generation and carbon consumption rates were found to be a function of the average irradiance inside the culture. A reduction in photosynthetic activity of the cells at noon and recovery in the afternoon was also observed. Therefore, the cells showed a short-term response of parameters such as oxygen generation rate as well as carbon consumption rate with external and average irradiance; a model of photosynthesis rate considering photoinhibition is proposed. This model is a useful tool for the operation and scaleup of tubular photobioreactors, and can be used for determining CO2 requirements of the system. The higher the photosynthesis rates, the lower the carbon losses, ranging from 25% at noon to 100% during the night. The growth rate showed a linear relationship with the daily mean average irradiance inside the culture with a long-term response. Likewise, a linear relationship among the oxygen generation rate and the growth rate was obtained. With respect to the biochemical composition of the biomass, the cells showed a long-term response of metabolic routes to mean daily culture conditions. During the illuminated period, energy was stored as carbohydrates and synthesis of proteins was low. During the night, the stored carbohydrates were consumed. The fatty acid dry weight (DW) content decreased during the daylight period, whereas the fatty acid profile, as total fatty acids, was a function of growth rate. A short-term variation of exopolysaccharides synthesis with solar irradiance was also observed, i.e. the higher the external irradiance the higher the excretion of exopolysaccharides as a protection against adverse culture conditions. �9 1999 Elsevier Science B.V. All rights reserved.

Keywords: Porphyridium cruentum; Tubular photobioreactor: Oxygen generation rate: Carbon dioxide consumption rate; Continu- ous culture; Elemental analysis

* Corresponding author. Tel.: + 34-950-215032; fax: + 34-950-215484. E-mail address: [email protected] (E. Molina Grima)

0168-1656/99/$ - see front matter �9 1999 Elsevier Science B.V. All rights reserved. PII: S01 68- 1 656(99)00080-2

272 M.M. Rebolloso Fuentes et al. /'Journal of Biotechnology 70 (1999) 271-288

1. Introduction

Microalgae have traditionally been cultivated in open systems where the culture conditions are selected to favour algal growth while hindering the growth of potentially contaminating micro-or- ganisms. Open systems permit little operation control and a certain level of contamination always occurs. Consequently, it is not possible to use open systems to grow algae requiring conditions that would allow proliferation of other potentially contaminating microbes. The production of high-value algal products from strains that cannot be maintained in open ponds requires closed systems. One of these strains is the microalga Porphyridium cruentum which has been referenced as a potential source of several high- value products such as: arachidonic acid, an es- sential dietary constituent for man and precursor of a large group of C20 compounds (Nichols and Appleby, 1969; Ahem et al., 1983) polysaccharides containing about 10% half-sulphate esters (Ramus, 1972; Percival and Foyle, 1979; Arad et al., 1985), pigments such as phycocyanin and phycoerythrin (Arad, 1987), and antioxidants as superoxidedismutase (Gudin, 1989). Among the closed systems, the tubular photobioreactors, em- ploying an airlift device for low-shear propulsion of culture fluid through a horizontal tubular loop that constitutes the solar receiver, have been re- peatedly referenced to obtain the highest produc- tivities (Gudin and Chaumont, 1983; Gudin and Therpenier, 1986; Lee, 1986; Tredici and Mat- erassi, 1992; Richmond et al., 1993; Torzillo et al., 1993; Molina Grima et al., 1994a,b,c,d; Borowitzka, 1996).

The temperature, nutrients and light availability at which the cells are exposed (Little, 1953; Rich- mond et at., 1993) determine the productivity of microalgal cultures. The temperature can be easily controlled by several system such as water spray- ing (Torzillo et al., 1991a,b), termostation by a stainless steel tube placed inside the reactor (Chini Zitelli et at., 1996) or by immersion of the solar receiver in a termostated water pool (Molina Grima et al,, 1994a, b). Mineral nutrients can be easily supplied to the system although the optimal level has not been studied until now and they are

usually supplied in excess to attain nutrient-saturated conditions. In the same way, the addition of inorganic carbon, usually supplied as pure CO2 by an on-demand injection either mixed in the airflow (Chini Zitelli et al., 1996) or directly in the culture (Molina Grima et al., 1994a,b), has not been optimised either. The light availability is determined by the solar irradiance, a function of the geographic location and the local climatic parameters (Duffle and Beckman, 1980), the reactor design (Alfano et al., 1986; Lee and Low, 1992, 1993) and the attenuation produced by the cells, which is a function of the biomass concentration and the extinction coefficient of the biomass (Evers, 1991; Sukenik et al., 1986; M olina Grima et al., 1994c).

The influence of solar irradiance on the variation of culture conditions and the phenomena taking place in the culture has not been totally elucidated. Torzillo et al. (1991a,b) studied the influence of daily variations of temperature in the productivity of Spirulina platensis cultures devel- oped in open and closed reactors, concluding that the higher biomass productivity was attained with the best temperature control. Guterman et al. (1990) obtained a macromodel for open pond algal mass production of S. platensis in which the dissolved oxygen, growth rate and pH of the culture were functions of the solar irradiance, obtaining expressions for these relationships based on theoretical approximations. Camacho Rubio et al. (1998) studied the daily variation of pH, dissolved oxygen and carbon losses in outdoor cultures of Porphyridium cruentum during the day giving attention to the mass transfer phenomena involved, but disregarding the interre- lationship between the variables. Molina Grima et al. (1994b,d) described the daily variation of dissolved oxygen, biomass concentration and temperature of outdoor cultures of P. tricornutum and Isochrysis galbana, reporting a first approach to the phenomena taking place in such systems but not elucidating the relationships existing among the different variables. Qiang and Rich- mond (1996) studied the influence of biomass concentration on the daily growth rate of S. platensis cultures, showing that high biomass concentrations gave rise to low increases of growth

M.M. Rebolloso Fuentes et al. ,, Journal o f Biotechnology 70 (1999) 271-288 273

rate but also to low photoinhibition at noon. Photoinhibition phenomena, usually attributed to high irradiance levels, have been observed in S. platensis (Vonshak and Guy, 1992; Qiang and Richmond, 1996) and P. tricornutum (Aci6n Fer- n~mdez et al., 1998) outdoor cultures, resulting in a low yield of the system and even in inhibition of the nitrogen uptake in S. platensis cultures (Bocci et al., 1988).

Daily variations of the biochemical composition derived from the daily variation of culture parameters have also been referenced. Several studies have shown that microalgae and cyanobacteria when grown under high light intensity synthesise mostly carbohydrates during the light periods that are consumed in protein synthesis in the dark (Bocci et al., 1988; Collos and Slawyk, 1980; Molina Grima et al., 1994a,b, 1995). This behaviour presume different nutrients uptake rates for different times in the daily cycle, thus the supply of nutrients should be changed during the course of the day. Moreover, the accumulation of lipids (Bocci et al., 1988) and variation of fatty acids content and profiles (Molina Grima et al., 1995) has been referenced. The higher the adequacy of culture conditions to the requirements of the strain, the higher the synthesis of proteins and structural lipids and the lower the neutral lipids storage (Molina Grima et al., 1995). These variations in culture conditions determine different biochemical composition along the day, which could be taken into account in order to harvest a biomass with the most adequate composition.

The present work is aimed at studying the influence of the overall daily cyclic variations of several culture parameters and the phenomena taking place in P. cruentum outdoor cultures. The objective is to find the main variables determining the daily behaviour of the system, and the relationship between these variables in order to develop a model useful for the estimation of the culture parameters during the day. For this, the overall behaviour of a chemostat outdoor culture of P. cruentum in a tubular photobioreactor has been determined taking into account pH, temperature, dissolved oxygen, composition of the inlet and outlet gas, in addition to the elemental analy-

sis of the broth, supernatant and biomass, and the biochemical composition of the biomass.

2. Materials and methods

2.1. Micro-organism and culture conditions

The red microalga used was P. cruentum UTEX 161. The culture was carried out in continuous mode at a constant dilution rate of 0.049 h-~ for 10 h in the daylight period during March 1997. The culture medium was a modification of Hem- erick's medium (1973) prepared from sterilised seawater. The pH of the culture was controlled at 7.6 by an on-demand automatic injection of carbon dioxide. The temperature was controlled at 20~ by thermostating the water pond in which the solar receiver was submerged.

2.2. The photobioreactor

The photobioreactor (Fig. 1) consisted of an airlift pump that drove the culture fluid through a horizontal tubular solar receiver (Camacho Rubio et al., 1998). The airlift section (riser, downcomer and degasser) had a height of 3.5 m. The solar receiver was made of transparent Plexiglas tubes (0.05 m internal diameter, 0.005 m wall thickness) joined into a loop configuration by Plexiglas joints to obtain a total horizontal length of 98.8 m. The solar receiver was submerged (0.05 m) in a shallow pool of water thermostated by a heat pump (Calorex 4000, Andrews, Sykes Ltd., Mal- colm Essex, UK). The bottom and inside walls of the pool were painted in white to improve their reflectance. The surface area of the pool was 21.4 m 2. The total culture volume in the bioreactor was 0.220 m 3. A port located on the side of the degasser section was used to continuously harvest the culture. The temperature, dissolved oxygen and pH were measured by sensors located in the degassing zone. The data were read by a ML-4100 control unit (New Brunswick Scientific, New Brunswick, NY, USA) connected to a computer equipped with a data acquisition card for data logging. Air was continuously supplied at a flow rate of 0.0124 mol s-1 Carbon was added as

274 M.M. Rebolloso Fuentes et al./Journal of Biotechnology 70 (1999) 271-288

pure CO2 directly injected under pH requirement at a flow rate of 0.0014 mol s-1. The air supply point was located at the end of the loop (i.e. at the entrance of the riser) whereas the CO2 was injected at the entrance of the solar receiver. Both gas flows were continuously monitored.

2.3. Solar irradiance

The instantaneous photon-flux density of the photosynthetically active radiation (PAR) inside the thermostatic pond and therefore the irradiance on the reactor surface, Iw, was measured on-line using a quantum scalar irradiance meter (LI-190 SA, Licor Instruments, Lincoln, NE, USA) connected to a data acquisition card. The average irradiance inside the culture, Iav, was cal-

culated by using a solar irradiance model for tubular photobioreactors previously proposed by Aci6n Ferndndez et al. (1997).

2.4. Absorption coefficient of the biomass

The absorption coefficient of the biomass was determined using the method described by Aci6n Fern~.ndez et al. (1997). To ascertain the light attenuation at a given time of the day, culture samples were placed in a tubular device using the same type of tube as the external-loop with a sheath to house the quantum scalar irradiance meter sensor in the centre. Attenuation at the centre of the tube, A t , was calculated as,

At= L n ( ~ ) (1)

Air

(1) , 'B

"" ~-.i~#s ~s

Air

Mass flow

t (18) t O~ CO~

analyzer analyzer

Gasexhaust ~ i '

dium inlet . , !

.<:::!!!!!i::i : : ~ . . . . . . . . . . . . . . . . . . . . . . . . .

! . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . 2 " ; : : : : : : : : : : : : ' : ' " ~.

Unit ~ Data control acquisition

A

Mass now [ ~ - " C O 2

�9 ~ : ~ , 7 . , '; �9 :

(,.-~

S o l a r r e c e i v e r

Fig. 1. Schematic drawing of the photobioreactor.

M.M. Rebolloso Fuentes et al./Journal of Biotechnology 70 (1999) 271-288 275

with I0 and I being the irradiance measured at the tube centre when the biomass concentration is zero and not zero, respectively. The variation in attenuation with biomass concentration for each sample was obtained by consecutive dilution with seawater, obtaining a slope which is the product of absorption coefficient of the biomass, Ka, by the light path length, p. The light path length at a given time of the day was a function of sun position, and it was determined using the irradiance model proposed by Aci6n Fernfindez et al. (1997).

2.5. Oxygen generation rate, carbon consumption rate and C02 losses

The mass flow rates of both the inlet air and carbon dioxide were measured by two mass flow meters (Model 824-13-0V1-PV1-V4, SIERRA In- struments, Monterey, CA, USA). A gas analyser (Servomex Analyser Series 1400) was used to monitor the compositions of inlet and exhaust gases. The mass flow meters and the gas analyser were connected to a data acquisition card (Daq- Book/112 and DaqBoard/112, IOTECH Inc., Cleveland, OH, USA) and an IBM compatible 486 computer for monitoring. The photosynthesis rate in the entire volume of the culture in a given instant was calculated using the experimental measurement of the gas inlet and outlet compositions and an oxygen balance. The oxygen mass balance could be written as,

02 in + 02 generation

= 0 2 out + 02 accumulation (2)

Since the accumulation term resulted 100 times lower than the difference between the oxygen inlet and exhaust including at the first hours of light period when the slope of dissolved oxygen is the maximum, this term was rejected and the oxygen generation rate, Ro2 was calculated as,

R% = 02 out - 02 in (3)

In the same way, the carbon dioxide consumption, Rc% was determined from the difference between the amount injected into the loop and the amount present in the exhaust gas; the accumulation term was again rejected as being negligible. Hence,

R c o 2 - - C O 2 in - C O 2 out (4)

The losses of C O 2 w e r e expressed as the ratio of the net CO2 molar flow in the exhaust gas to the CO2 molar flow injected.

2. 6. Photosynthetic activity

The measurement of photosynthetic activity of the cells was carried out by a method similar to that described by Vonshak et al. (1985) based on the determination of the oxygen generation rate under constant laboratory conditions. For this, samples of culture were diluted to adjust the optical density to 0.1 at 625 nm, put into a termostated (22~ vessel (1 1 capacity), and irra- diated with 2000 laE m - 2 s-1 by a halogen lamp located 0.5 m from the vessel. Oxygen development was then measured every second during 30 min with a dissolved oxygen electrode (Lisle- Metrix 2200D/P91) connected to a data acquisition system. The photosynthetic activity of the cells was determined as the slope of the dissolved oxygen variation versus time.

2. 7. Analytical methods

The biomass concentration was determined by dry weight (DW). Duplicate culture samples were centrifuged (1800 x g), washed with 0.5 M NaC1 and distilled water to remove non-biological ma- terial such as mineral salt precipitates, lyophilised for 3 days, and weighed.

The elemental composition (C, H, N, S, O) of the biomass, broth and cell free culture medium was determined using an elemental analyser LECO CHNS-932 equipped with an additional furnace VT-900 for oxygen measurement. For the analysis of broth and cell free culture medium (supernatant), 5 lal samples were injected. For the analysis of lyophilised biomass, 2 mg were injected.

The protein content of the biomass was estimated as 6.25 times the elemental nitrogen content. These results were checked by the Kjehldahl method to confirm their accuracy (Rebolloso Fuentes et al., 1998). The chlorophyll content was measured according to the Hansmann method


w 5000

~.~. 4000

3000 e- ._m

I~ 2oo0

E 1000

o

o8~ 13 O o

& &o&& ~ A &

n ww i

0 4 8 12 Hour

16 20 24

I oDay 1 aDey 2 &Day 3]

Fig. 2. Daily variation of irradiance on the water pond.

140

r 120

N100 C ID ~ 80

o 60

0 = 2 0 r

0

, o lgo A

08 a A

l T ~ | r i

0 4 8 12 16 20 24 Hour

Fig. 3. Daily variation of dissolved oxygen within the culture.

(1973). Carotenoids determination was as described by Whyte (1987). The fatty acid profile of the biomass was determined by GC using a HP- 5938 gas chromatography analyser. Fresh centrifuged wet biomass was used for fatty acid analysis. Methylation was arried out by direct transesterification following a modification of the Lepage and Roy method (1984) proposed by Gar- cia S~.nchez et al. (1993). Nonadecanoic acid was used as an internal standard to quantify fatty acid content in biomass.

3. Results

The solar irradiance inside the water pond, Iw, presents a Gaussian-like variation during the day, from zero in the night to up 4000 gE m-2 s - 1 in the central hours of the day (Fig. 2). Day to day differences observed were due to weather variations. Thus, different daylight mean external irradiance on the reactor surface, Iwm, of 2000, 1800 and 1600 l, tE m-2 s-1 was measured for days 1 2 and 3, respectively. The relatively high solar irradiance values measured are due to the existence of albedo, e.g. the reflection of solar radiation in the walls and bottom of the pond. This effect has been referenced to increase the irradiance on the culture surface in tubular reactors compared to open systems (Aci6n Fern~indez et al., 1997).

The dissolved oxygen in the culture increases with irradiance up to 120% saturation with air in the daylight period due to photosynthesis (Fig. 3).

During the night, the dissolved oxygen decreases down to 60% saturation with air due to the consumption of oxygen by respiration (Fig. 3). On days 1 and 2, the dissolved oxygen reaches a maximum at 12:00 and then declines in spite of the high solar irradiance. On day 3, this decrease is not observed, the dissolved oxygen remaining high until the solar irradiance decreases, in this moment dissolved oxygen strongly decreasing (Fig. 3). This behaviour can be attributed to a photoinhibition effect on days 1 and 2 caused by the high external irradiance at noon (above 4000 ~E m - 2 S-1), being lower on day 3 (3000 laE m-2 s-1) due to shadowing by clouds. In order to verify the existence of photoinhibition in outdoor conditions, the variation of photosynthetic activity of the cells during the day was determined (Fig. 4). The results show that the photosynthetic activity of the cells is the highest at early morning, 0.0040 mol O~ m-3 s-1, whereas it is decreased to 0.0006 mol 02 m-3 s-X at noon, when the

0.0040 -

:~ 0.0030 -

.~ E "~ 0.0o20 - ~ _ o

c- 0 > ' E 0 ~ 0.oo10 - 13.

',,,,

0.0000 6 8 10 12 14 16 18 20

Hour

Fig. 4. Daily variation of photosynthetic activity of the cells.

M.M. Rebolloso Fuentes et al. j Journal o f Biotechnology 70 (1999) 271-288 277

21.6

.Co 21.2

20.8

~ 20.4

~ 20.0

19.6 . . . . . . 0.0 0.5 1.0 1.5 2.0 2.5 3.0

"13me, days

J �9 Oxygen .... ~ - - O ~ x ) n dioxide I t J

0.60 140

0.50 c 120

0.40 ~ 100 L

0.30 = -- ~" 80 O E o

0.20 (~ ,- o .~ 60

0.10 m r 40

0.00 3.5 20

Fig. 5. Daily variation of gas exhaust composition during the quasi steady state.

solar irradiance is the highest, and again an increase during the afternoon up to 0.0012 mol O2 m - 3 s - 1, when solar irradiance decreases (Fig. 4).

The gas exhaust composition varied according to the variations in dissolved oxygen. The oxygen molar fraction of the exhaust gas varied around the oxygen molar fraction in the air, ranging from 21.4% in the daylight period to 19.8% during the night (Fig. 5). The carbon dioxide molar fraction of the exhaust gas was always higher than in the air, ranging from 0.35% in the central hours of the day to ~ 0.50% during the night (Fig. 5). The high values of carbon dioxide molar fraction in the gas exhaust compared to air are due to the injection of pure CO2 bubbles for pH control. Therefore, when the CO2 injection is released inside the reactor, the broth becomes locally saturated with pure CO2. Then, when the culture is aerated in the airlift system, the carbon dioxide is stripped to air. This happened in both the daylight period and night period because the pH control system was not switched off at night. Moreover, the carbon dioxide generated during the night by respiration also increases the carbon dioxide molar fraction in the exhaust gas.

The daily variation of solar irradiance also influenced the pH of the culture and the carbon losses. Although the pH of the culture was controlled at 7.6_+0.3 by on-demand injection of pure CO2, a cyclic daily variation was observed (Fig. 6). The average pH of the culture in the daylight period was higher, 7.75 _+ 0.08, than during the night, 7.51 _+ 0.05. In the daylight period,

- 8 . 2

�9 , . t "

- 8.0

7.8 p.,

i] 7.6 �9

"6 7.4 :z:

7.2

. . . . . . 7.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Time, days

F i g .... Carbonlosses ,r PHI

Fig. 6. Daily variation of carbon losses and pH of the culture during the quasi steady state.

the pH was above the set point due to the carbon consumption by photosynthesis, whereas during the night, the pH was under the set point due to CO2 generation by respiration. Although the pH of the culture was controlled, the carbon losses were high, ~ 100% during the night, dawn and dusk, due to the low carbon consumption rate. In the central hours of the day, the carbon losses were lower, equal to 20%, due to the high carbon consumption rate (Fig. 6).

On the daylight period, the biomass concentration increased indicating that the growth rate was higher than the imposed dilution rate ( D - 0.049 h-1) and the biomass was accumulated in the reactor (Fig. 7). During the night, a fraction of the accumulated biomass was consumed, thus the

112 - 110 ~

1 08

o 106- r

r

..j 1 0 2 - 1 O0 0.98 - 0.96

Day 1, iJ=0.0107 h 1 BL=9% Day 2 IJ=0.0049 h "1 BL=20% ~ . ~

8 10 12 14 16 18 20 Hour

i O Day 1 IEI Day 2 A Day 3 I

Fig. 7. Daily variation of biomass concentration, additional growth rat in the daylight period, ~so~, and biomass losses, LB, during the quasi steady state.

278 M.M. Rebolloso Fuentes et al. Journal of Biotechnology 70 (1999) 271-288

biomass concentration at first hours in the morning being lower. The 3-day average quasi steady state biomass concentration was 3.50 g 1-1, this resulted in an average biomass productivity of 1.76 g 1- ld-~. In front, the biomass night losses were ~ 14% of the biomass concentration at the end of daylight time. The additional growth rate in the daylight period, ~tso~ (~t~o~ = actual growth rate imposed dilution rate) was different for each day, the maximum, 0.0107 h-1, being obtained on day 1, and the minimum, 0.0049 h - ~, on day 2 (Fig. 7). In addition, the biomass night losses also varied for each day. A maximum loss was observed on day 2 (20%) while a minimum was obtained on day 1 (9%) (Fig. 7).

Elemental analysis of the culture broth, cell free culture medium and liophylised biomass were carried out in order to determine the variation of metabolic routes of the cells during the day. The carbon content of the broth was in the range 0.14 + 0.02% , being higher in the morning and lower in the afternoon in spite of the higher biomass concentration (Fig. 8). This can only be due to either a carbon accumulation in the cell free culture medium (supernatant) during the morning that is eliminated in the afternoon, or to a decrease during the day of the carbon content in the biomass. The elemental analysis of the supernatant showed carbon accumulation during the morning in the liquid phase and elimination during the afternoon, as well as a reduction of nitrogen and sulphur content during the day (Fig. 8). Mean values ranges were 0.017 + 0.003, 0.025 + 0.005 and 0.024 + 0.004% DW of carbon, nitrogen and sulphur, respectively. With regard to elemental analysis of the biomass, the carbon and oxygen content increased during the daylight period (37.3 +_ 0.6 and 39.0 +_ 0.7% DW), whereas the nitrogen and sulphur content decreased (4.6 _+ 0.2 and 1.10 + 0.06%DW) and hydrogen content re- mained constant (5.5 _+ 0.3% DW) (Fig. 8).

Variations of the culture conditions also determined changes in the biochemical composition of the biomass. The pigment content, as chlorophylls and carotenoids, slightly decreased during the daylight period, being 0.36 + 0.02% at the beginning and decreasing to 0.32 _+ 0.02% at the end of daylight time (Table 1). This trend caused a de-

0.20 ~ �9

_ 0.15 j �9149 �9 e-

[_, 0.05 ~ ~176 ~J 0.00 ~ ~ - - ,

0.0 0.5 1.0

e

m o ( ~

1.5 2.0

-time, days

., , 1 ,

2.5 3.0 3.5

I �9 Carbon A Nitrogen m Sulphur I

0.040 T

0.03o

,.a 0.010 ~-

~ 0 . 0 0 0 ~

~, 0.0 ,,J ,,,,l

A

O O

A

�9 �9 �9 0 u

0.5 1.0 1.5 2.0 2.5 3.0 Time, days

�9 % Carbon ~ % Nitrogen o % Sulphur / J

3.5

"~ 42 +

g a s , -

~ ~" 32 ~

0 0.0

r r

7.0 6 . o

e e * 3.0~" 2.0

o o o o o o o o 1 0 ~ "

0.5 1.0 1.5 2.0 2.5 3.0 3.5 -time, days

. % o % Oxy n O/o Hyd ,,,,I =

A % Nitrogen o % Sulphur 1

Fig. 8. Variation of elemental composition of broth, cell free culture medium and liophylised biomass during the quasi steady state reached.

crease of the absorption coefficient of the biomass, K, the mean values varying from 0.041 + 0.001 m 2 g-1 at the beginning of the day to 0.038 + 0.001 m 2 g-1 at the end of daylight time (Table 1), because Ka is a function of pigment content (Molina Grima et al., 1994a,b,c). The protein content also decreased in the daylight period, from a mean value at the beginning of the daylight time of 30.3 + 0.6% to 27.0 ___ 0.6% at the end of the afternoon (Table 1). With regard to fatty acids, only the main ones were considered,

M . M . Rebo l loso Fuen te s et a l . / J o u r n a l o f B i o t e c h n o l o g y 70 (1999) 2 7 1 - 2 8 8 279

r~

.=_

e .

o

. . . . d ~

0

r c.q ~ q (.,q (.,q

~ ~ 1 r ~ ~ 1 r r162

e',l t '~ ,,O ~ , - - ,--. e 'q ~ ~

c5 o o o o o o o o

c5 c5 c5 c5 o o c5 c5 c5

~'q C'q C", l C"q C",l ~ l C'q r ~ l

280 M.M. Rebolloso Fuentes et al. /Journal of Biotechnology 70 (1999) 271-288

these being 16:0, 18:2, 20:4 and 20:5. The araquidonic acid (20:4) is the main fatty acid with 2%DW, the next being the palmitic acid (16:0) with 1.7%DW and eicosapentaenoic acid (20:5) with 1.1%DW (Table 1). All of the fatty acids content decrease in the daylight period, from 1.74 _+ 0.05, 0.67 _+ 0.0, 2.09 _+ 0.06 and 1.08 _+ 0.06% of biomass DW at the first hours in the morning to 1.63 _+ 0.05, 0.62 _+ 0.02, 1.93 _+ 0.06 and 0.99 +0.06% of biomass DW at the last hours in the afternoon, for 16:0, 18:2, 20:4 and 20:5, respectively. However, no variation of the fatty acids profile (as total fatty acids) during the day was observed (Table 1). The polyunsaturated fatty acids, araquidonic and eicosapentaenoic, represent more than 50% of total fatty acids (37.5% of araquidonic and 19.2% of eicosapentaenoic), the rest being mainly palmitic acid with 31.3% of total fatty acids (Table 1).

4. Analysis of the experimental results

Although in outdoor chemostat cultures of microalgae the pH, temperature and dilution rate are controlled, a daily cyclic variation of the culture parameters takes place mainly due to variations in the solar irradiance. In addition to solar irradiance, the average irradiance inside the culture, Iav, defined as the mean light intensity a cell randomly moving inside the culture intercepts, also varies during the day. The external irradiance, biomass concentration and pigment content of the biomass and thus the average irradiance inside the culture are modified during a day and between different days. A variance analysis indicates that the oxygen generation rate is influenced by the average irradiance inside the culture rather than by the irradiance on the culture surface. In this sense, a linear relationship between I~v and I,,. is observed (Fig. 9).

Iav = 0.089Iw + 4.05 r 2 = 0.9766 (5)

The linear dependence of light intensity inside fermentor and outdoor light intensity has been previously reported (Hirata et al., 1996). This linear variation of Iav with Iw brings about two important points. Firstly, a linear relationship

between Iw and lav implies the quotient Iav/Iw is a constant and therefore the product Ka 'P" C is also a constant during the quasi steady state. Thus, considering a mean light path, Pa,,erage, from Eq. (7) means that the product Ka" C must also be a constant.

I =/0 exp( - Ka'P " C) (6)

Ia__~ = exp( -- K a �9 Paverage " C) = m = cte (7) Iw

Secondly, the cells tend to adapt to the average irradiance inside the culture. Thus, the higher the external irradiance and biomass concentration, the lower the extinction coefficient of the biomass must be to keep the average irradiance constant. Eq. (5) makes it possible to determine the average irradiance inside the culture, Iav at any time during the quasi steady state directly from the external irradiance, Iw.

With regard to the oxygen generation rate, Ro, �9

it appears to be a function of the average lrrad~- ance inside the culture (Fig. 10). Vonshak et al. (1985) also observed this dependence as a hyperbolic variation of the photosynthesis rate with irradiance in the laboratory thin layer cultures of P. cruentum, while Jensen and Knutsen (1993) observed this same behaviour with S. platensis. However, in outdoor cultures, the existence of photoinhibition has been observed (Fig. 4). In the present study, the photosynthetic activity of the cells was very high at first hours in the morning, indicating a fast response of Porphyridium to light

400 7

i S ~ . w 200

100

l

O' 0 1000 2000 3000 4000

Iw, pE/m2s

Fig. 9. Correlation between the average irradiance inside the culture, Iav, and the external irradiance on the culture surface, Iw during the quasi steady state reached.


0.0020

o.oo15 -~ * # t .

e o o . o o l o . ~ -o 0.0005 �9

0.0000 - �9

-0.0005 ~ e

0 1 O0 200 300 400

lav, pE/m2s

Fig. 10. Variation of oxygen generation rate with the average irradiance inside the culture estimated by using Eq. (5). Line represents a smooth tendency curve.

availability. In the central hours of the day, when the solar irradiance is at the peak levels, the photosynthetic activity declined and then a slight increase again in the afternoon, when the solar radiation was reduced. This behaviour evidences the existence of photoinhibition that appears as a decrease in the efficiency of the culture to metabo- lize the available light when solar irradiance is increased, though the oxygen generation rate within the photobioreactor is not decreased because the loss in photosynthetic efficiency is com- pensated by a higher irradiance level. Different authors have referenced the existence of photoinhibition in outdoor cultures. Molina Grima et al. (1996a) observed the existence of photoinhibition in outdoor cultures in two ways. During the day, photoinhibition could be caused by an increase of the irradiance on the culture surface. With the position inside the culture, photoinhibition could arise caused by the strong irradiance gradient that exist in dense cultures. In this sense, Vonshak and Guy (1992) observed the existence of photoinhibition during the day in S. platensis cultures. Jensen and Knutsen (1993) demonstrated that photoinhibition is a reversible process in which degradation

and regeneration of key components of the photosynthetic apparatus coexist and the available amount of functional photosynthetic pigments is the result of an equilibrium between that two processes.

5. D i s c u s s i o n

The influence of photoinhibition in the behaviour of outdoor cultures must always be considered. In outdoor cultures of P. tricornutum (Aci6n Ferndndez et al., 1998) and L galbana indoor cultures (Molina Grima et al., 1996b), the growth rate has been observed to be a function of average irradiance rather than external irradiance. Molina Grima et al. (1996a,b) report that the growth rate varies hyperbolically with the average irradiance inside the culture although the external irradiance might influence the parameters of the growth model. Additionally, Aci6n Fern~indez et al. (1998) proposed a mathematical growth model that considers the existence of photolimitation and photoinhibition, taking into account a decrease in the efficiency of the cells when external irradiance increased. Bearing these references in mind, the variation of oxygen generation rate with the average irradiance inside the culture has been fitted to a hyperbolic expression (Eqn. 8) for each single day.

Ro2 R~ (8) = I~, + I~,, - R ~

Since the solar irradiance measured each day is different, the characteristic parameters of the model results are different (Table 2). The results show how the parameters of the model vary: n decreased while Ik increased linearly with the daily mean irradiance on the reactor surface, Iwm (Table

Table 2 Values of characteristics parameters of the model obtained by non-linear regression for each day during the quasi steady state

Day Iwm (laE m -2 s -1) Ro2ma x (mol 02 m -3 s -1) n Ik (laE m -2 s - l ) Ro2mi n (mol 02 m -3 s -1) r 2

1 2000 0.002968 0.837415 148.575 0.000482 2 1800 0.003238 0.963006 115.235 0.000886 3 1600 0.001663 1.325950 76.56 0.000082

0.99235 0.923979 0.959093


0.0020 1 Y = 0.91101x + 0.00005

"o 0.0015 t R2 = 0.93502 �9 �9

0.0005 . *

1 o . = 0.0000 1

-0.00__0.00050~40.00000.00050.00100.00150,0020 ROz experimental

Fig. 11. Correlat ion between oxygen generation rate estimated by the model Eq. (9) and experimental oxygen generation rate.

2). Equation 8 can therefore be enhanced to take these linear relationships of n and lk with Iw. Doing so, the following model of instantaneous oxygen generation rate with external irradiance and average irradiance inside the culture is obtained.

�9 I( a + b / w ) Ro2max -av

R~ = (c + d" Iw) (~ + b. Iw) + I(~v+ b. Zw) - R~ (9)

Ro2ma x - - 0.003979 mol 02 m - 3 s - ~, a = 0.635, b

= -0.000063 m2s ~tE-~, c

= 706.65, laE m-2 s-~, d

= - 0.025471, Ro2mi n

= 0.000317 mol 02 m-3 S-1, r2._. 0.9317.

The parameters of the model were obtained by non-linear regression, fitting to this equation all of the experimental data available (74 values). A representation of estimated oxygen generation rate Eq. (9) versus experimental oxygen generation rate show the adequacy of the model (Fig. 11). The maximum photosynthesis rate, 0.0040 mol O2 m - 3 s - 1 (600 ~tmol 0 2 h -~ mg Chlorophyl-~), is lower than the maximum value referenced for this strain, 1000 ~tmol 02 h-~ mg Chlorophyl-~ (Vonshak et al., 1985), this maximum being obtained under laboratory conditions. Ohta et al. (1992) observed a maximum CO2 consumption rate of 0.0044 mol m - 3 s - 1 in laboratory cultures Porphyridium under light- dark cycles. If an O2/CO2 ratio of one is accepted, the maximum photosynthesis rate obtained is the

same as predicted by the model. The minimum photosynthesis rate, 0.000317 mol O2 m-3 s-1, is the respiration rate during the night, and although it has been considered to be constant, a certain influence of the daily culture conditions can be expected. In this sense, Torzillo et al. (1991a,b) and Molina Grima et al. (1994b,d) observed that the respiration rate is a function of the temperature and the irradiance during the daylight time. Moreover, the experimental results show that the higher the growth rates in the daylight time the lower the biomass losses during the night (Fig. 7). This same behaviour was observed in laboratory light-dark cycle cultures of Porphyridium (Ohta et al., 1992). Thus, the lower the growth rate the higher the carbohydrates synthesis and the lower the protein synthesis, the carbohydrates being consumed during the night by respiration.

The obtained model Eq. (9) allows to determine the oxygen generation rate as a function of external and average irradiance inside the culture, thus being a useful tool in the design and scaleup of tubular photobioreactors. In this sense, one major problems in the scale up of tubular photobioreactors is to determine the acceptable maximum loop length, which will be limited by the maximum dissolved oxygen level that the cells can support without being affected by its toxicity. Therefore, for any given conditions of Iw and Iav, the model will estimate the oxygen generation rate. This, together with the liquid flow rate in the solar receiver, determines the maximum loop length admissible for a given maximum oxygen level. Alternatively, for a given length of the loop it is possible to calculate the liquid flow necessary to keep the dissolved oxygen under a given level and thus avoid its toxic effects. Moreover, by applying an oxygen mass balance to the overall reactor and by rejecting the accumulation term (Camacho Ru- bio et al., 1998), the model could be used to determine the mass transfer capacity necessary in the system in order to limit the dissolved oxygen level in the culture.

O2 in - 02 out = 02 generated + 02 accumulated (lo)


140

120

;~ 100

20

0 -0.0005 0 . ~ 0.0005 0.0010 0.111115

N(~, m~m~ I�9 Carbon losses,._ o Dissolv~ oxygen]

y = 41572x + 67.71 160 _ R 2 = 0 . 8 8 9 5 . 140 .'~

=

_ 100 i

tO***** �9 60 ! �9 ,= . q . � 9 40

" " ,W*~., . , 020 0.0020

Fig. 12. Variation of dissolved oxygen and carbon losses in the culture with the oxygen generation rate.

d[O2] Kla([02] - [O~ = ]) = Ro2 + d----~ (1 l)

Kla[02] - K/a[O~ ] = R%

1 [02] = ~a ~ + [0~']

(12)

(13)

A high photosynthesis rate causes a high dissolved oxygen level in the system. On the other hand, the higher the volumetric mass transfer, Kla, the lower the dissolved oxygen. In this sense, the dissolved oxygen linearly increases with the oxygen generation rate (Fig. 12), the slope of this line being the inverse of the volumetric mass transfer coefficient of the system. Thus, the Kla of the system can be determined from the value of this experimental slope, resulting as 0.0023 s - 1, which is very similar to that experimentally determined (Kla=O.O029 s -~) by Camacho Rubio et al.

0 .0020

0 .0015

E 0.0010

C~ 0.0005

0.0000

-0 .0005 | , , i

-0.0005 0 .0000 0 .0005 0 .0010 0 .0015

RO=, mol/mSs

0.0020

Fig. 13. Correlation between carbon consumption rate and oxygen generation rate during the quasi steady state.

(1998) for this same culture system. A linear relationship between oxygen genera-

tion and carbon consumption is also observed, the ratio being 1.004 mol CO2 per tool O2 (Fig. 13). This value is higher than others referenced for different strains like Chlorella pyrenoidosa (Myers, 1980) being 0.70 mol CO2 per mol O2. The difference can be attributed to the different metabolic routes of each strain. In particular, P. cruentum trends to accumulate carbohydrates and excrete polysaccharides, thus increasing the value of CO2 consumption to the 02 generation ratio. From the linearity between carbon consumption and oxygen generation, two conclusions can be drawn. First, the oxygen generation and carbon consumption are coupled, the ratio being constant and equal to one all the time and independent of cell metabolism or culture conditions. Second, the obtained model also allows to determine the carbon requirements of the system, thus becoming a tool useful to study how the carbon losses can be reduced by optimising the amount injected. In this sense, it is observed (Fig. 12) that the carbon losses are a function of oxygen generation rate. A high oxygen generation rate causes a high carbon consumption and low carbon losses. This fact points out how the injection of pure CO2 allows for reliable pH control but causes a high loss of CO2. In order to reduce these values, Camacho Rubio et al. (1998) proposed to modify the composition of the gas phase, reducing the COz molar fraction and thus reducing the carbon dioxide oversaturation in the culture. However, this mode of operation causes an increase of the pH in the culture, which lowers the carbon availability and could reduce the yield of the system (Molina Grima et al., 1996a).

With regard to the biomass productivity, the observed day to day variations of culture conditions determine different growth rates and values of maximum oxygen generation rate. However, these values are interrelated, the higher the growth rate in the daylight period, the higher the maximum oxygen generation rate (Fig. 14). This same behaviour was observed in Spirulina cultures (Guterman et al., 1990). The most significant vari- able determining both the growth rate in the daylight period and the maximum oxygen genera-

284 M.M. Rebolloso Fuentes et al. ~Journal of Biotechnology 70 (1999) 271-288

0.0022

o.ools 0 E

~ 0.0014

y = 0 . 1 2 1 6 x + 0 . 0 0 0 5

R 2 = 0.9627

C 0.0010 -- , , , ,

0.004 0.006 0.008 0.010 0.012

I~ol, h "1

Fig. 14. Variation of maximum oxygen generation rate with the additional growth rate in the daylight time, ~tso = for 3 days during the quasi steady state.

tion rate is the mean average irradiance inside the culture in the daylight period. Moreover, both the growth rate and the maximum oxygen generation rate linearly increase with the mean average irradiance inside the culture in the daylight period (Fig. 15). The higher the solar irradiance or the lower the biomass concentration, the higher the mean average irradiance and light availability, thus the growth rate and photosynthesis rate being highest. Aci~n Fernfindez et al. (1998) observed that the growth rate of P. tricornutum varies hyperbolically with the mean average irradiance inside the culture, this relationship being approximately linear until Iav values of 200 gE m -2 s-1, with the slope strongly decreasing for irradiances above this value. The linear relationship between growth rate and mean average irradiance observed agreed with the linear relationship observed for P. tricornutum at low

0.012

0.010

0.008

~.oo6 0.004

0.002

0.000

160

y = 0.00028x - 0.03985

R = = 0 . 9 ~ ,,,,,, ̀ ' ~

R 2 = 0.981510 I 1 ,

0.0030

O. 0025

0

0.0020 E

0.0015 n,,

0.0010

165 170 175 180 lawn, pE/rn=s

Fig. 15. Variation of additional growth rate in the daylight period, ~tso ~, and maximum oxygen generation rate with the mean average irradiance inside the culture, Iavm.

values of Iavm, thus indicating that the same behaviour could be accepted in this case although further research is necessary at different Iav values.

With regard to elemental analysis of culture, supernatant and biomass, the carbon content of the supernatant showed a variation parallel to the solar irradiance during the day, an increase during the morning and a decrease in the afternoon (Fig. 8). However, Camacho Rubio et al. (1998) previously demonstrated that cultures operated with pH control by on-demand CO2 injection had a lower carbon content in the central hours of the day, due to the consumption by the cells, than in the first and last hours of the daylight period, by a reduction in photosynthesis and respiration The carbon content of the supernatant changes in a opposing way. Thus, at noon the carbon content of the supernatant is 0.200 g 1-~ whereas Cama- cho Rubio et al. (1998) estimate that there must be 0.072 g 1-1 of total inorganic carbon in the culture. The difference corresponds to the organic carbon excreted to the medium by the cells, 0.128 g 1-1 consisting mainly of exopolysaccharides. Vonshak et al. (1985) observed exopolysaccharide concentrations of around 0.24 g 1-1, double than the values measured in the present study, however this could be due to the low dilution rate, 0.02 h-1, and low pH, 7.5, at which the cultures were operated. In the first and last hours of the day, the carbon content in the supernatant decreased, 0.13 g 1-1, while the total inorganic carbon estimated by Camacho Rubio et al. (1998) increased, 0.077 g 1-1. This difference indicates a lower exopolysaccharides concentration in these hours, 0.053 g 1-1, and reflects that exopolysaccharides synthesis modifies during the day increasing at noon due to the existence of adverse culture conditions by photoinhibition. Thus, Vonshak et al. (1985) found that Porphyridium excretes big amounts of exopolysaccharides under adverse conditions as nitrogen limitation, a low growth rate, etc. The cellular polysaccharides are origi- nated by the continuous excretion of polysaccharides, which form an envelope around the outer cell wall. The outer layer of this envelope is gradually sloughed off, enriching the growth medium and constituting the exocellular polysac- charide fraction (Vonshak et al., 1985).

M.M. Rebolloso Fuentes et al./"Journal of Biotechnology 70 (1999) 271-288 285

With respect to nitrogen, the second macronu- trient, the amount of nitrogen supplied daily to the system with the medium is 27.4 g d - 1. How- ever, the elemental analysis of the produced biomass indicated that only 16.03 g d-~ of nitrogen is taken up, thus the yield of this nutrient being 58%. In addition, the nitrogen content of supernatant decrease during the day, being a function of the daily variation of the cellular metabolism and not of the oxygen generation rate. Therefore, the results of elemental analysis of the biomass showed how during the illuminated period the metabolism is mainly directed to the synthesis of carbohydrates and lipids--carbon, oxygen and hydrogen content of the biomass increasing 1.2, 1.0 and 0.3%, respectively--while during the dark period, the accumulated carbohydrates were consumed and the metabolism was directed to the synthesis of proteins--nitrogen and sulphur content of the biomass increasing 3.7 and 0.2%, respectively--and structural lipids. The nitrogen is mainly taken during the afternoon and night when the protein synthesis is active. In this sense, the results confirm the idea of Professor Gudin (personal communication) that the nitrogen should be better supplied to the system during the night to avoid nitrogen limitation. However. in this research nitrogen limitation did not exist due to the excess of nitrogen supplied.

A daily variation of cellular metabolism was observed in outdoor cultures of P. tricornutum (Aci6n Fernfindez et al., 1998), S. platensis (Bocci et al., 1988; Torzillo et al., 1991 a,b) and Oscillato- ria agardhii (Van Liere et al., 1979) and it is attributed to a response of the cells to the daily solar cycle, because the cells store energy during the illuminated period which is metabolised in the night period. The mean elemental composition of the P. cruentum biomass was estimated as C1Hl.v6N0.1180.olO0.79, a value similar to that proposed by different authors for microalgal biomass (Borowitzka and Borowitzka, 1988).

The daily variation of the cellular metabolism also caused the variations on the fatty acids content of the biomass. The content of all the fatty acids decreases during the daylight time, the decrease being sharper for polyunsaturated fatty acids, 18:2, 20:4 and 20:5 (8-9%), than for satu-

rated and monounsaturated fatty acids, 16:0 (6%) (Fig. 14). In P. tricornutum (Aci6n Fernfindez, 1996) and I. galbana outdoor cultures (Molina Grima et al., 1995), an increase in the fatty acids content during the daylight period was observed, the increase being higher for the short-chain than for the long-chain fatty acids. This behaviour can be attributed to the different roles of the fatty acids in the cell metabolism. Short-chain saturated and monounsaturated fatty acids were found to be the main components of neutral lipids (storage lipids) (Molina Grima et al., 1994a,b, 1995), and the initial step in the path of fatty acid synthesis (Hodgoson et al., 1991). Thus, the amount of these fatty acids is subject to the daily cyclic variation of environmental conditions. On the other hand, polyunsaturated fatty acids are structural lipids, mainly found in glycolipids and phospholipids (Molina Grima et al., 1994a,b), and therefore, their contents are more related to state of growth than to short-term environmental variations. However, in this research a decrease in all the fatty acids content was observed. This decrease can be attributed to the accumulation of carbohydrates during the daylight period and highlights that the synthesis of lipids in P. cruentum takes place mainly in the darkness, when the carbohydrates accumulated are metabolised. Moreover, the lower decrease in the fatty acid content of 16:0 than 18:2, 20:4 and 20:5 indicates that the behaviour observed in P. tricornutum and I. galbana is also reproduced in P. cruentum, although variation of fatty acid profile during the day was not significant. With regard to the fatty acids profile (as total fatty acid), although the biomass was obtained in quasi steady state, the differences in the additional growth rate in each day determined a variation on the fatty acids profile. The higher the growth rate the higher the polyunsaturated fatty acid fraction and the lower the 16:0 and 18:2 fatty acid fraction (Fig. 16). This behaviour can be attributed to the reduction in the duplication time when growth rate increases, that increases the requirements of structural biomolecules while the storage of energy as lipids is reduced. This phenomenon causes a reduction in the fraction of saturated and monounsaturated fatty acids, and an increase of

286 M.M. Rebolloso Fuentes et al. / Journal of Biotechnology 70 (1999) 271-288

2.1

'~ 1.8 " o

o~ 1.5

1.2 u toO.9

m 0.6 u.

0.3

Z" T .T, ,,T.

..=_ l

�9 v , , , l

10 12 14 16 18 Hour

-~ 0.40

0.30 ~. " o

0.20 g c

O.lO .~ n

0.00

I --n-- 16:0 ~ 18:2 o 20:4 --e--20:5 --e--Pigmefcs I

4O

38 ai

,r ~ 5 3 4

d 32

"- 30

28

= I - a l .

0.004 0.006 0.008 0.010 psol, h "1

I n 16:0 e 20:4 ~ 18:2-~--20:51

24

2 0 . , r

w~ 12

4 ""

0 0.012

Fig. 16. Variation of biochemical composition of the biomass with the additional growth rate, gsol, during the quasi steady state reached.

polyunsaturated acids that are the main con- stituents of cellular membranes (Kates and Vol- cani, 1966).

6. C o n c l u s i o n s

In outdoor systems, daily variations of culture conditions take place due to the variation of solar irradiance. In order to respond to these variations, the cells adapt their behaviour. Some of these modifications are fast and are a direct function of culture conditions in a short time interval (seconds, minutes or a few hours). However, others are slower and are a function of culture conditions averaged over a long time interval (all the daylight time, all the night or all the day). Short term responses of oxygen generation rate, carbon consumption rate and exopolysaccharides production are observed whereas the growth rate and

metabolic routes show long-term responses. In this sense, in outdoor P. c r u e n t u m cultures, the oxygen generation rate is mainly determined by the average irradiance inside the culture, although the existence of photoinhibition, causing a reduction in the efficiency of the system to utilise the solar radiation, is observed. Considering this, a model for the photosynthesis rate as both oxygen generation rate and carbon dioxide consumption rate is obtained. This model allows to estimate the photosynthesis rate as a function of the solar irradiance--a function of system geometry and location of the factory--and average irradiance inside the cul ture--a function of the imposed dilution rate, biomass concentration and pigment content (Aci6n Fern~.ndez et al., 1998)gat which the cells are exposed to. Moreover, the linear relationship between the oxygen generation rate and carbon consumption observed allows the model to also estimate the carbon requirements of the system. The obtained model is therefore a useful tool in determining design parameters such as maximum solar receiver length of tubular external loops or the requirements of mass transfer in the system in order to avoid the deleterious effects of oxygen oversaturation.

The elemental analysis of the biomass revealed how the daily variation of the cellular metabolism differs in the variation of the photosynthesis rate, indicating that the storage of energy by photosynthesis and its use by the cellular metabolism are not simultaneous. During daylight time, carbohydrates were accumulated by the biomass as energy storage, to be metabolised later during the night to synthesise proteins and structural lipids. This behaviour indicates that a supply of nitrogen during the night could be necessary, while the carbon injection can be switched-off in order to reduce the carbon losses.

A c k n o w l e d g e m e n t s

This research was supported by the Comisi6n Interministerial de Ciencia y Tecnologia (CICYT) (BIO-95-0692) (Spain), and Plan Andaluz de In- vestigaci6n II, Junta de Andalucia.


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