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Production of Spirulina biomass in closed photobioreactors
Biomass l 1 ( 1986) 61-74
P r o d u c t i o n of Spirulina Biomass in Closed Photobioreactors G. Torzillo, B. Pushparaj, F. Bocci, W. Balloni, R. Materassi and G. Florenzano Centro di Studio dei Microrganismi Autotrofi del CNR e Istituto di Microbiologia Agraria e Tecnica dell'Universit~ di Firenze, Piazzale delle Cascine 27, 1-50144, Firenze, Italy (Received 20 June 1986; accepted 1 August 1986)
ABSTRACT The results of a six year investigation on the outdoor mass culture of Spirulina platensis and S. maxima in closed tubular photobioreactors are reported. On average, under the climatic conditions of central Italy, the annual yield of biomass obtained from the closed culture units was equivalent to 33 t dry weight ha -1 year-k In the same climatic conditions the yield of the same organisms grown in open ponds was about 18 t ha -I year -1. This considerable difference is due primarily to better temperature conditions in the closed culture system. The main problems encountered relate to the control of temperature and oxygen concentration in the culture suspension. This will require an appropriate design and management of the photobioreactor as well as the selection of strains specifically adapted to grow at high temperature and high oxygen concentration. Key words: Spirulina, outdoor mass culture, tubular reactor, photobioreactor.
INTRODUCTION In recent years several research groups have investigated the growth of different photosynthetic m i c r o o r g a n i s m s in tubular systems. Pirt et al. have developed the t h e o r y and design of a tubular photobioreactor. T h e y have c o n s i d e r e d s o m e i m p o r t a n t parameters, such as turbulent flow of the culture, e n e r g y r e q u i r e m e n t for culture circulation, 02 and c a r b o n dioxide d e m a n d and supply. 1 With a p h o t o b i o r e a c t o r m a d e of 61 Biomass 0144-4565/86/S03.50- © Elsevier Applied Science Publishers Ltd, England, 1986. Printed in Great Britain
62
G. Torzillo et al.
very narrow tubes (1 cm bore size) they attained very high photosynthetic efficiency of fight energy conversion into algal biomass. 2 Gudin and coworkers have investigated the growth of microalgae in closed culture devices of up to 100 m 2 with the aim of producing valuable extracellular products, such as polysaccharides from Porphyridium spp. and Chlamydomonas mexicana or hydrocarbons from Botryococcus spp. The problems investigated by Gudin include: thermal regulation of the culture, choice of material and culture management. 3,4 Tubular photobioreactors have several advantages over conventional open ponds: they can be erected over any open space, can operate at high biomass concentration and keep out atmosphere contaminants. Moreover, the process can be optimized by fermentation control principles and computer application, 5 so as to achieve the maximum utilization of solar energy at all times. Finally, loss of water by evaporation is eliminated. However, photobioreactors function like solar collectors in that they can reach high temperatures to adversely affect the growth of the majority of photosynthetic microorganisms. Culturing of typical mesophilic strains requires the installation of expensive cooling facilities; but the problem of temperature control becomes less critical if more thermotolerant organisms are grown. Spirulina platensis and Spirulina maxima can tolerate temperatures as high as 40°C for a few hours without appreciable adverse effects. For this reason the authors' Research Centre started an investigation several years ago on the use of tubular photobioreactors in the outdoor mass culture of Spirulina spp. The research was aimed at the optimization of biomass yield and its biochemical composition. This report illustrates the characteristics of the experimental culture equipment and the main results obtained. The performance of the tubular culture device is compared with that of open ponds. MATERIALS AND METHODS
General description of culture equipment The pilot plant for growing Spirufina and the equipment for circulating the culture suspension and harvesting the biomass are shown in Figs 1 and 2. The photobioreactors are made with transparent tubes. At the beginning flexible polyethylene tubes (14 cm diameter and 0-3 mm thickness) were used (Fig. 3). Owing to their inadequate mechanical strength, they were replaced by plexiglass tubes having 13 cm inner diameter and 4 mm thickness. These diameters were selected to achieve
Production of Spirulina biomass in closed photobioreactors
.... .....
63
: :i:!~:~ii;! iiiiiii!ii~'i!i~l'i li!iii i!
Fig. 1.
General view of the tubular pilot plant. Tubes made of polymethyl methacrylate (plexiglass).
Fig. 2.
Details of the ancillary equipments of the closed culture plant showing the harvesting and circulation systems.
a surface to volume ratio similar to that of open ponds (about 100 litres of culture suspension per m 2 of illuminated surface). Each photobioreactor is made up of several tubes laid side by side on a white polyethylene sheet and joined by PVC connections to form a loop. Each connection incorporates a narrow tube for oxygen degassing. At the exit
64
G. Torzillo et al.
Fig. 3. Viewof the photobioreactormadeof polyethylenetubes. of the tubular circuit the culture suspension falls into a receiving tank. A diaphragm pump (model MA 180, AGI POMPE, Milan) raises the culture to a feeding tank containing a siphon that allows an intermittent discharge into the photobioreactor. At intervals of 4 min about 350 litres of culture suspension are discharged into the photobioreactor, thus moving the culture in the tubes at a rate of 0.26 m s-1. This regime of circulation is obtained by adjusting the flow rate of the pump to 4000 litres h-1. The result was better than continuous circulation at the same flow rate of the pump. The maximal length of the circuit was 500 m, corresponding to a volume of 8000 litres and a surface of 80 m 2. The surface area of the photobioreactors was calculated on the basis of the surface area effectively occupied by the tubes plus the interspace between them (about 3 cm). The temperature of the culture suspension inside the tubular circuit is monitored with several thermistor probes (LSI model TT-3) appropriately positioned. The partial 02 pressure in the culture is determined polarographically with two Clark-type electrodes placed at the inlet of the culture in the photobioreactor and its outlet. The amount of solar radiation has been measured with a solarimeter MICROS equipped with a pyranometric sensor (Kipp and Zonen Type CM 5/6).
Organisms and culture conditions
Spirulina maxima strain 4MX and Spirulina platensis strain M2 of the culture collection of the authors' Research Centre were used. The
Production of Spirulina biomass in closed photobioreactors
65
culture medium has been described elsewhere. 6 It contains 24 g litre- ~of sodium bicarbonate + carbonate. The pH was maintained to 9.4-9.8 by CO2 addition. The temperature control of the culture suspension inside the photobioreactor was achieved in different ways (see results). The culture operated with a semicontinuous regimen. The amount of biomass harvested each day was adjusted in order to achieve a biomass concentration of 0.6 g dry weight litre- ~or 1.2 g dry weight litre-1, corresponding to an areal density of 60 and 120 g m -2 of illuminated area, respectively. During harvesting operations the culture suspension was withdrawn at a constant rate for a time corresponding to that required by the culture to cover the whole tubular circuit. The biomass was harvested by filtration on a vibroscreen with a net of 50 ktm pore size. The culture solution was recycled into the photobioreactor after the necessary additions of nutrients. The harvested biomass was washed twice with deionized water and sundried. Once a week a sample of biomass was analysed for nitrogen content. The concentration of nitrogen and phosphorus in the culture medium was checked once a week. The amino acid pattern and fatty acid composition were determined occasionally on representative samples of the biomass.
Analytical procedures Biomass concentration in the culture was determined daily. The cells from a representative sample were filtered, washed with deionized water and dried at 105°C for 3 h before weighing. Total nitrogen content of the biomass was determined with an automatic nitrogen analyser (ANA 1500, Carlo Erba Strumentazione, Milan). Fats were extracted with chloroform-methanol (2:1 v/v) for 24 h in a Soxlet extractor, dried at 75°C for 1 h and weighed. In order to determine the fatty acids composition, the lipid extract was dissolved in a methanol: H2SOa:benzene mixture (97.9:2:0-1% v/v), placed in a flame-closed vial and heated for 4 h at 75°C. The methyl esters of fatty acids were extracted with ether, washed with salt water and concentrated to a small volume for gas chromatographic analysis. GL analysis was performed with a Perkin-Elmer Sigma 2 gas chromatograph with a Supelco SP 2330 60 m capillary fused silica column and a FID detector. The temperature of the injector was 250°C and the oven was programmed from an isotherm of 130°C for 6°C rain-1 to a final temperature of 210°C with a rate of 3°C rain- 1. The carrier gas was He, at a pressure of 30 psi and the split was 80:1. The retention times of the peaks were compared with those of pure standards and the quantitative estimation was performed with the aid of a Perkin-Elmer LCI 100 integrator. The amino acid pattern was deter-
66
G. Torzilloet al.
mined with an automatic aminoacid analyser (Model 3A27 Carlo Erba Strumentazione, Milan). Phycocyanin content was estimated colorimetrically after extraction in phosphate buffer (pH 7), applying the extinction coefficient of O'hEocha. 7
RESULTS
Temperature control of the culture Since a large amount of heat is generated inside a photobioreactor exposed to natural sunlight, in the absence of an effective dissipation mechanism, a considerable increase in the temperature of the culture suspension can occur. This system is advantageous in temperate conditions but becomes problematic in warm climates. In Florence we observed that the temperature inside the photobioreactor on a sunny day reaches values 10-13°C higher than the air temperature for 2-3 h. Hence during summer months temperatures higher than 40°C were very common. Since our strains of Spirulina maxima fail to grow above 36°C and are killed by exposure to temperatures higher than 42°C for 2-3 h over 2-3 consecutive days, it was necessary to devise a cooling system. Three systems of cooling were tested: (i) Shading of the tubes with dark-coloured plastic sheets. For an effective control of the temperature it was necessary to cover about 80% of the surface for 5-6 h daily. This caused a strong reduction in the amount of solar radiation received by the culture and consequently in the yield of biomass. (ii) Overlapping two or three tubes. This system was difficult to instal and inadequate for effective control of the temperature inside the photobioreactor. (iii) Cooling the culture by spraying water on the surface. The system was operated when the temperature of the culture reached a critical value. For Spirufina maxima 4MX, whose maximum temperature for growth was 36°C, water spraying was started at 35°C and stopped at 33°C. In the climatic conditions of Florence the cooling device worked about 40 days year- 1. The amount of water lost by evaporation ranged from 1 to 2 litres m -2 day-1. This cooling system functioned very efficiently. However, in order to reduce the amount of water required for cooling, from 1983 a thermotolerant strain of Spirulina was utilized: Spirulina platensis M2. This organism was able to grow up to 42°C and could tolerate a daily exposure up to 46°C for at least 3 h without being killed. Cooling the circuit to avoid temperatures higher than 44°C
Production of Spirulina biomass in closed photobioreactors
67
proved adequate for attaining a high yield of biomass. In the last three years the cooling systems worked about 20 days year-1. The amount of water sprayed was less than 250 litres m-2 year- J. Only a fraction of this water was lost by evaporation. Influence of the circuit length on productivity and on protein content of the biomass
The length of the tubular circuit exerts a considerable influence on the oxygen concentration in the culture suspension. After the entry of the culture into the photobioreactor, the photosynthetic activity of the biomass causes an increase in the oxygen concentration in the liquid at a rate that depends on light intensity and temperature. For a given temperature an increase in light intensity causes an increase in the rate of oxygen evolution by the culture. Similarly, for a given light intensity an increase of temperature induces an increase in oxygen production, at least as long as the temperature remains below the optimum values for growth (Table 1). Obviously, the oxygen concentration in the culture suspension increases as the culture moves forward in the tubular circuit. On a summer day the oxygen concentration in the culture reaches 20-25 ppm one hour after entry of the culture into the photobioreactor. W h e n the culture comes out from the tubular circuit a certain amount of oxygen is lost in the atmosphere. The action of the circulation pump is very important in 02 degassing. In Fig. 4 the yields of two photobioreactors of 250 and 500 m length are shown. In the former the culture takes 2 h to cover the whole circuit length while in the latter it takes 4 h.
TABLE 1 Influence of Temperature and Light Intensity on the Increase in O2 Concentration (mg 02 litre 1) in a Culture of Spirulina' maxima 4MX After One Hour in the Tubular Photobioreactor
Light intensity (BE m -2 s-l)
200 200-400 400-?00 "/00-1000 1000-1400 1400-1800
Temperature C C) 16-20
21-25
26-30
0"9 2-9 3-0 5-0 ---
1'3 3"7 4-3 6-3 6-6 --
2"6 4'8 5'2 7-4 8"0 8"8
G. Torzillo et al.
68
2s E
m E o
";
5
>,
i
M
Fig. 4.
i
J
i
J months
I
A
i
S
Yield of Spirulina maxima 4MX grown in tubular photobioreactors of different length (as specified in the Figure) circulated at the same speed.
TABLE 2 Biochemical Composition of Spirufina maxima 4 M X Grown in Photobioreactors of Different Length (% of Dry Weight)
Length of photobioreactor
Crude protein Total lipids Carbohydrates Ash
250 m
500 m
62.0 13-3 18-0 6.7
55-5 13.2 22.6 8"7
The short photobioreactor gave higher yields. The biomass produced in the long photobioreactor had a lower protein content, counterbalanced by a higher carbohydrate content (Table 2). Taking into account that no significant differences in the temperature profile or in biomass concentrations occurred in the two photobioreactors, the influence of the length of the tubular circuit on the yield of biomass and on its biochemical composition must be attributed mostly to the adverse effect of high oxygen concentration on protein synthesis in Spirulina as described recently by the authors. 8 Influence of cellular concentration on the yield and composition of biomass The output of biomass of the photobioreactors was inversely related to the concentration of biomass in the culture. The yield of biomass
Production of S pirulina biomass in closed photobioreactors
69
obtained in various years from cultures maintained at 0.6 g and 1.2 g dry weight litre-~ are reshown in Fig 5. In general the cultures run at 0"6 g litre -1 were more productive. The differences in yield were more marked in July (about 35%) and declined in autumn, probably as a consequence of the limiting effect exerted by low temperature. Biomass concentrations below 0.6 g litre -1 are not practicable because the cyanobacterial cells are damaged by the combined effect of high light intensity and oxygen concentration. The biomass concentration of the cultures influences the phycocyanin level. In August the phycocyanin content in the culture at a concentration of 1"2 g litre-~ was 25% higher than in the culture at 0.6 g litre -l. This difference increased to 40% at the beginning of October, while at the end of October the phycocyanin content became equal. A decrease in total nitrogen content of the biomass occurred at the beginning of August (Fig. 6). The change was more marked in the culture run at low cellular concentration and coincided with a sudden lowering of cellular concentration due to a higher filtration rate of the cultures. After three days the normal cellular concentration was restored and the nitrogen content returned to the previous values. The protein content of the biomass (about 54%) was relatively stable, irrespective of the cellular concentration of the culture. In August the nitrogen content of biomass was higher in the culture grown at lower cellular concentration. Presumably in this culture the nucleic acid content was higher due to the higher growth rate reached by the cyanobacterial population. No significant differences in the fatty acids composition and in the amino acid pattern of the biomass grown at low and high cellular concentrations were found.
~
0,6 g t "+
I
1,2 g t -1
N ~.
months
Fig. 5.
Influence of cellular concentration on the yield of Spirulina platensis M2 grown in a tubular photobioreactor.
70
G. Torzillo et al. •
N*/,
1.2
gl1
o 0.6 gl -I
13
11 10 9 8
7 i
i
Ju(y
Fig. 6.
Nitrogen content of
i
August
September
Spirulina platensis M2
grown at different cellular concen-
trations.
photobioreacfor 25
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J
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20
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months
Fig. 7.
Comparison of the mean yield of Spirulina platensis M2 cultivated in photobioreactors and in open ponds.
Comparative yield of both open ponds and photobioreactors In Fig. 7 the course of productivity of Spirulina platensis M2 cultivated in photobioreactors and open ponds operating at the same areal density is shown. The daily yield of the photobioreactor was higher than that of the open ponds during the whole cultivation period. This was mainly due to better temperature profiles in the closed culture system. The culture in the photobioreactors reaches optimum temperature earlier in the
Production of
Spirulina biomass in closed photobioreactors
71
o
16
September photobioreacfor
~0
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i
1
1
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t 25 July. ~
E
I
i
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8.00
i
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I
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Fig. 8. Typicalcourseof the temperatureof Spirulina cultures in photobioreactorsand ponds in July and September.The arrow indicatesthe start of the cooling. morning and better temperatures for the growth are also reached during the months in which the cultivation in ponds is limited by low temperatures (Fig. 8). No significant differences in the protein content and amino acid composition between the biomass grown in photobioreactors and in open ponds were found (Table 3). However, some differences were found in the fatty acids composition. Biomass grown in open ponds showed a higher degree of unsaturation of fatty acids due mainly to a higher content in 7-linolenic acid (Table 4). It is reasonable to suppose that these differences are dependent on the higher temperatures reached in the photobioreactor.
DISCUSSION The results obtained from the investigations, extended for several years, on the growth of Spirulina platensis and S. maxima in a tubular photobioreactor at a pilot plant scale, allow a preliminary evaluation of the merits and drawbacks of closed culture systems, in comparison with open ponds, for mass culturing of oxygenic phototrophic microbes. Under the climatic conditions of central Italy, the average yearly output of biomass from the photobioreactor was nearly 90% higher than that obtained from open ponds. This difference is due primarily to the fact that closed photobioreactors allow a considerable extension of the cultivation period (215 days against 175 days with open ponds), because
G. Torzillo et al.
72
TABLE 3
Amino acid Composition of Spirulina platensis M2 Grown in Tubular Photobioreactor and in Open Pond (g amino acids 100 g protein- ~)
Amino a c i d s
Photobioreactor
Open pond
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Ammonia Arginine 19. a-e-Diamino pimelic acid
11.2 5.1 4.3 17"4 4.4 5"6 7'2 0'3 7.9 2.2 6.9 10.5 4.7 5'1 5"8 2"1 1"1 8"3 1.0
11.4 4.5 3.7 16.9 4.4 5.6 7.4 0"3 8.1 2"4 7.1 10"6 4.8 5.4 5"9 2.1 1.1 8'3 1.0
Protein %
53.6
51.0
TABLE 4
Main Differences in Fatty Acids Composition of Spirulina platensis M2 Grown in Tubular Photobioreactor and in Open Pond (% of total fatty acids)
Photobioreactor Open pond
CI6:0
CI6: l A 9
C18: I A 9
C18:2A 9,12
C18:3A 6, 9,12
52.3 48.0
4.2 6"5
3.8 3"0
23.0 21'5
13"8 18"1
inside the closed culture units the d a y t e m p e r a t u r e attains values considerably higher t h a n a m b i e n t temperature. Additionally, the tubular culture device gave slightly better yield also in the w a r m e s t p e r i o d of the year, w h e n air t e m p e r a t u r e s reach 3 0 - 3 5 ° C . Also in this p e r i o d a better t e m p e r a t u r e profile in the p h o t o b i o r e a c t o r , due to a m o r e rapid heating
Production of Spirulina biomass in closed photobioreactors
73
of the culture in the early morning, presumably plays a major role in determining the better yields observed. T h e main problems encountered in the operation of tubular photobioreactors are the control of oxygen concentration and of overheating in summer. T h e problem of temperature control has been greatly alleviated with the selection of a strain of Spirulina platensis able to grow up to 42°C and to withstand temperatures of 46°C for a few hours without adverse effect. With this strain cooling of the photobioreactor by spraying water on the culture tubes was required only for 20 days yearand the amount of water required was estimated to be 250 litres m-2 of cultivated area year-1. T h e control of oxygen concentration requires an appropriate design of the photobioreactors, so that the culture can be degassed before the oxygen tension in the culture suspension becomes harmful. The selection of a Spirulina strain less sensitive to the adverse effect of high O2 tension would contribute significantly to simplifying the construction of large photobioreactors as well as their management. Although the installation of large scale photobioreactors may be more costly than a production plant based on open ponds, the greater efficiency in solar energy conversion that can be achieved with closed systems makes them very attractive in photosynthetic biomass production through mass culture of microalgae and cyanobacteria.
REFERENCES 1. Pirt, S. J., Lee, Y. K., Walach, M. R., Pirt, M. W., Balyuzi, H. H. M. & Bazin, M. J. (1983). A tubular bioreactor for photosynthetic production of biomass from carbon dioxide: design and performance. J. Chem. Tech. Biotechnol., 33B, 35-58. 2. Pirt, S. J., Lee, Y. K., Richmond, A. & Watts Pirt, M. (1980). The photosynthetic efficiency of Chlorella biomass growth with reference to solar energy utilization. J. Chem. Tech. Biotechnol., 30, 25-34. 3. Gudin, C. & Chaumont, D. (1983). Solar biotechnology study and development of tubular solar receptors for controlled production of photosynthetic cellular biomass for methane production and specific exocellular biomass. In: Energy from biomass, Series E, Vol. 5, W. Palz and D. Pirrwitz (eds), D. Reidel, Dordrecht, pp. 184-93. 4. Gudin, C., Bernard, A. & Chaumond, D. (1983). Culture de microalgues en reacteur tubulaire clos. Actes du second Colloque de l'Association Franqaise d'Algologie Appliqu~e, Chfiteau de Fontager, 28 Octobre, AFAA Laboratoire de la Roquette Bauzille de Putois, France, pp. 105-54. 5. Walach, M. R., Balyuzi, H. H. M., Bazin, M. J., Lee, Y. K. & Pirt, S. J. (1983). Computer control of an algal bioreactor with simulated diurnal illumination. J. Chem. Tech. Biotechnol., 33B, 59-75.
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6. Paoletti, C., Pushparaj, B. & Tomaselli Feroci, L. (1975). Ricerche sulla nutrizione minerale di Spirulina platensis. In: Proc. XVII Nat. Congr. It. Soc. Microbiol., Padova 26-28 Ottobre, Vol. 2, Grafiche Erredici, Padova, pp. 845-853. 7. O'h Eocha, C. (1965). In: Chemistry and biochemistry of Plant Pigments, T. W. Goodwin (ed.), Academic Press, New York, pp. 175-6. 8. Torzillo, G., Giovannetti, L., Bocci, E & Materassi, R. (1984). Effect of oxygen concentration on the protein content of Spirulina biomass. Biotechnol. Bioeng., 26, 1134-5.
P r o d u c t i o n of Spirulina Biomass in Closed Photobioreactors G. Torzillo, B. Pushparaj, F. Bocci, W. Balloni, R. Materassi and G. Florenzano Centro di Studio dei Microrganismi Autotrofi del CNR e Istituto di Microbiologia Agraria e Tecnica dell'Universit~ di Firenze, Piazzale delle Cascine 27, 1-50144, Firenze, Italy (Received 20 June 1986; accepted 1 August 1986)
ABSTRACT The results of a six year investigation on the outdoor mass culture of Spirulina platensis and S. maxima in closed tubular photobioreactors are reported. On average, under the climatic conditions of central Italy, the annual yield of biomass obtained from the closed culture units was equivalent to 33 t dry weight ha -1 year-k In the same climatic conditions the yield of the same organisms grown in open ponds was about 18 t ha -I year -1. This considerable difference is due primarily to better temperature conditions in the closed culture system. The main problems encountered relate to the control of temperature and oxygen concentration in the culture suspension. This will require an appropriate design and management of the photobioreactor as well as the selection of strains specifically adapted to grow at high temperature and high oxygen concentration. Key words: Spirulina, outdoor mass culture, tubular reactor, photobioreactor.
INTRODUCTION In recent years several research groups have investigated the growth of different photosynthetic m i c r o o r g a n i s m s in tubular systems. Pirt et al. have developed the t h e o r y and design of a tubular photobioreactor. T h e y have c o n s i d e r e d s o m e i m p o r t a n t parameters, such as turbulent flow of the culture, e n e r g y r e q u i r e m e n t for culture circulation, 02 and c a r b o n dioxide d e m a n d and supply. 1 With a p h o t o b i o r e a c t o r m a d e of 61 Biomass 0144-4565/86/S03.50- © Elsevier Applied Science Publishers Ltd, England, 1986. Printed in Great Britain
62
G. Torzillo et al.
very narrow tubes (1 cm bore size) they attained very high photosynthetic efficiency of fight energy conversion into algal biomass. 2 Gudin and coworkers have investigated the growth of microalgae in closed culture devices of up to 100 m 2 with the aim of producing valuable extracellular products, such as polysaccharides from Porphyridium spp. and Chlamydomonas mexicana or hydrocarbons from Botryococcus spp. The problems investigated by Gudin include: thermal regulation of the culture, choice of material and culture management. 3,4 Tubular photobioreactors have several advantages over conventional open ponds: they can be erected over any open space, can operate at high biomass concentration and keep out atmosphere contaminants. Moreover, the process can be optimized by fermentation control principles and computer application, 5 so as to achieve the maximum utilization of solar energy at all times. Finally, loss of water by evaporation is eliminated. However, photobioreactors function like solar collectors in that they can reach high temperatures to adversely affect the growth of the majority of photosynthetic microorganisms. Culturing of typical mesophilic strains requires the installation of expensive cooling facilities; but the problem of temperature control becomes less critical if more thermotolerant organisms are grown. Spirulina platensis and Spirulina maxima can tolerate temperatures as high as 40°C for a few hours without appreciable adverse effects. For this reason the authors' Research Centre started an investigation several years ago on the use of tubular photobioreactors in the outdoor mass culture of Spirulina spp. The research was aimed at the optimization of biomass yield and its biochemical composition. This report illustrates the characteristics of the experimental culture equipment and the main results obtained. The performance of the tubular culture device is compared with that of open ponds. MATERIALS AND METHODS
General description of culture equipment The pilot plant for growing Spirufina and the equipment for circulating the culture suspension and harvesting the biomass are shown in Figs 1 and 2. The photobioreactors are made with transparent tubes. At the beginning flexible polyethylene tubes (14 cm diameter and 0-3 mm thickness) were used (Fig. 3). Owing to their inadequate mechanical strength, they were replaced by plexiglass tubes having 13 cm inner diameter and 4 mm thickness. These diameters were selected to achieve
Production of Spirulina biomass in closed photobioreactors
.... .....
63
: :i:!~:~ii;! iiiiiii!ii~'i!i~l'i li!iii i!
Fig. 1.
General view of the tubular pilot plant. Tubes made of polymethyl methacrylate (plexiglass).
Fig. 2.
Details of the ancillary equipments of the closed culture plant showing the harvesting and circulation systems.
a surface to volume ratio similar to that of open ponds (about 100 litres of culture suspension per m 2 of illuminated surface). Each photobioreactor is made up of several tubes laid side by side on a white polyethylene sheet and joined by PVC connections to form a loop. Each connection incorporates a narrow tube for oxygen degassing. At the exit
64
G. Torzillo et al.
Fig. 3. Viewof the photobioreactormadeof polyethylenetubes. of the tubular circuit the culture suspension falls into a receiving tank. A diaphragm pump (model MA 180, AGI POMPE, Milan) raises the culture to a feeding tank containing a siphon that allows an intermittent discharge into the photobioreactor. At intervals of 4 min about 350 litres of culture suspension are discharged into the photobioreactor, thus moving the culture in the tubes at a rate of 0.26 m s-1. This regime of circulation is obtained by adjusting the flow rate of the pump to 4000 litres h-1. The result was better than continuous circulation at the same flow rate of the pump. The maximal length of the circuit was 500 m, corresponding to a volume of 8000 litres and a surface of 80 m 2. The surface area of the photobioreactors was calculated on the basis of the surface area effectively occupied by the tubes plus the interspace between them (about 3 cm). The temperature of the culture suspension inside the tubular circuit is monitored with several thermistor probes (LSI model TT-3) appropriately positioned. The partial 02 pressure in the culture is determined polarographically with two Clark-type electrodes placed at the inlet of the culture in the photobioreactor and its outlet. The amount of solar radiation has been measured with a solarimeter MICROS equipped with a pyranometric sensor (Kipp and Zonen Type CM 5/6).
Organisms and culture conditions
Spirulina maxima strain 4MX and Spirulina platensis strain M2 of the culture collection of the authors' Research Centre were used. The
Production of Spirulina biomass in closed photobioreactors
65
culture medium has been described elsewhere. 6 It contains 24 g litre- ~of sodium bicarbonate + carbonate. The pH was maintained to 9.4-9.8 by CO2 addition. The temperature control of the culture suspension inside the photobioreactor was achieved in different ways (see results). The culture operated with a semicontinuous regimen. The amount of biomass harvested each day was adjusted in order to achieve a biomass concentration of 0.6 g dry weight litre- ~or 1.2 g dry weight litre-1, corresponding to an areal density of 60 and 120 g m -2 of illuminated area, respectively. During harvesting operations the culture suspension was withdrawn at a constant rate for a time corresponding to that required by the culture to cover the whole tubular circuit. The biomass was harvested by filtration on a vibroscreen with a net of 50 ktm pore size. The culture solution was recycled into the photobioreactor after the necessary additions of nutrients. The harvested biomass was washed twice with deionized water and sundried. Once a week a sample of biomass was analysed for nitrogen content. The concentration of nitrogen and phosphorus in the culture medium was checked once a week. The amino acid pattern and fatty acid composition were determined occasionally on representative samples of the biomass.
Analytical procedures Biomass concentration in the culture was determined daily. The cells from a representative sample were filtered, washed with deionized water and dried at 105°C for 3 h before weighing. Total nitrogen content of the biomass was determined with an automatic nitrogen analyser (ANA 1500, Carlo Erba Strumentazione, Milan). Fats were extracted with chloroform-methanol (2:1 v/v) for 24 h in a Soxlet extractor, dried at 75°C for 1 h and weighed. In order to determine the fatty acids composition, the lipid extract was dissolved in a methanol: H2SOa:benzene mixture (97.9:2:0-1% v/v), placed in a flame-closed vial and heated for 4 h at 75°C. The methyl esters of fatty acids were extracted with ether, washed with salt water and concentrated to a small volume for gas chromatographic analysis. GL analysis was performed with a Perkin-Elmer Sigma 2 gas chromatograph with a Supelco SP 2330 60 m capillary fused silica column and a FID detector. The temperature of the injector was 250°C and the oven was programmed from an isotherm of 130°C for 6°C rain-1 to a final temperature of 210°C with a rate of 3°C rain- 1. The carrier gas was He, at a pressure of 30 psi and the split was 80:1. The retention times of the peaks were compared with those of pure standards and the quantitative estimation was performed with the aid of a Perkin-Elmer LCI 100 integrator. The amino acid pattern was deter-
66
G. Torzilloet al.
mined with an automatic aminoacid analyser (Model 3A27 Carlo Erba Strumentazione, Milan). Phycocyanin content was estimated colorimetrically after extraction in phosphate buffer (pH 7), applying the extinction coefficient of O'hEocha. 7
RESULTS
Temperature control of the culture Since a large amount of heat is generated inside a photobioreactor exposed to natural sunlight, in the absence of an effective dissipation mechanism, a considerable increase in the temperature of the culture suspension can occur. This system is advantageous in temperate conditions but becomes problematic in warm climates. In Florence we observed that the temperature inside the photobioreactor on a sunny day reaches values 10-13°C higher than the air temperature for 2-3 h. Hence during summer months temperatures higher than 40°C were very common. Since our strains of Spirulina maxima fail to grow above 36°C and are killed by exposure to temperatures higher than 42°C for 2-3 h over 2-3 consecutive days, it was necessary to devise a cooling system. Three systems of cooling were tested: (i) Shading of the tubes with dark-coloured plastic sheets. For an effective control of the temperature it was necessary to cover about 80% of the surface for 5-6 h daily. This caused a strong reduction in the amount of solar radiation received by the culture and consequently in the yield of biomass. (ii) Overlapping two or three tubes. This system was difficult to instal and inadequate for effective control of the temperature inside the photobioreactor. (iii) Cooling the culture by spraying water on the surface. The system was operated when the temperature of the culture reached a critical value. For Spirufina maxima 4MX, whose maximum temperature for growth was 36°C, water spraying was started at 35°C and stopped at 33°C. In the climatic conditions of Florence the cooling device worked about 40 days year- 1. The amount of water lost by evaporation ranged from 1 to 2 litres m -2 day-1. This cooling system functioned very efficiently. However, in order to reduce the amount of water required for cooling, from 1983 a thermotolerant strain of Spirulina was utilized: Spirulina platensis M2. This organism was able to grow up to 42°C and could tolerate a daily exposure up to 46°C for at least 3 h without being killed. Cooling the circuit to avoid temperatures higher than 44°C
Production of Spirulina biomass in closed photobioreactors
67
proved adequate for attaining a high yield of biomass. In the last three years the cooling systems worked about 20 days year-1. The amount of water sprayed was less than 250 litres m-2 year- J. Only a fraction of this water was lost by evaporation. Influence of the circuit length on productivity and on protein content of the biomass
The length of the tubular circuit exerts a considerable influence on the oxygen concentration in the culture suspension. After the entry of the culture into the photobioreactor, the photosynthetic activity of the biomass causes an increase in the oxygen concentration in the liquid at a rate that depends on light intensity and temperature. For a given temperature an increase in light intensity causes an increase in the rate of oxygen evolution by the culture. Similarly, for a given light intensity an increase of temperature induces an increase in oxygen production, at least as long as the temperature remains below the optimum values for growth (Table 1). Obviously, the oxygen concentration in the culture suspension increases as the culture moves forward in the tubular circuit. On a summer day the oxygen concentration in the culture reaches 20-25 ppm one hour after entry of the culture into the photobioreactor. W h e n the culture comes out from the tubular circuit a certain amount of oxygen is lost in the atmosphere. The action of the circulation pump is very important in 02 degassing. In Fig. 4 the yields of two photobioreactors of 250 and 500 m length are shown. In the former the culture takes 2 h to cover the whole circuit length while in the latter it takes 4 h.
TABLE 1 Influence of Temperature and Light Intensity on the Increase in O2 Concentration (mg 02 litre 1) in a Culture of Spirulina' maxima 4MX After One Hour in the Tubular Photobioreactor
Light intensity (BE m -2 s-l)
200 200-400 400-?00 "/00-1000 1000-1400 1400-1800
Temperature C C) 16-20
21-25
26-30
0"9 2-9 3-0 5-0 ---
1'3 3"7 4-3 6-3 6-6 --
2"6 4'8 5'2 7-4 8"0 8"8
G. Torzillo et al.
68
2s E
m E o
";
5
>,
i
M
Fig. 4.
i
J
i
J months
I
A
i
S
Yield of Spirulina maxima 4MX grown in tubular photobioreactors of different length (as specified in the Figure) circulated at the same speed.
TABLE 2 Biochemical Composition of Spirufina maxima 4 M X Grown in Photobioreactors of Different Length (% of Dry Weight)
Length of photobioreactor
Crude protein Total lipids Carbohydrates Ash
250 m
500 m
62.0 13-3 18-0 6.7
55-5 13.2 22.6 8"7
The short photobioreactor gave higher yields. The biomass produced in the long photobioreactor had a lower protein content, counterbalanced by a higher carbohydrate content (Table 2). Taking into account that no significant differences in the temperature profile or in biomass concentrations occurred in the two photobioreactors, the influence of the length of the tubular circuit on the yield of biomass and on its biochemical composition must be attributed mostly to the adverse effect of high oxygen concentration on protein synthesis in Spirulina as described recently by the authors. 8 Influence of cellular concentration on the yield and composition of biomass The output of biomass of the photobioreactors was inversely related to the concentration of biomass in the culture. The yield of biomass
Production of S pirulina biomass in closed photobioreactors
69
obtained in various years from cultures maintained at 0.6 g and 1.2 g dry weight litre-~ are reshown in Fig 5. In general the cultures run at 0"6 g litre -1 were more productive. The differences in yield were more marked in July (about 35%) and declined in autumn, probably as a consequence of the limiting effect exerted by low temperature. Biomass concentrations below 0.6 g litre -1 are not practicable because the cyanobacterial cells are damaged by the combined effect of high light intensity and oxygen concentration. The biomass concentration of the cultures influences the phycocyanin level. In August the phycocyanin content in the culture at a concentration of 1"2 g litre-~ was 25% higher than in the culture at 0.6 g litre -l. This difference increased to 40% at the beginning of October, while at the end of October the phycocyanin content became equal. A decrease in total nitrogen content of the biomass occurred at the beginning of August (Fig. 6). The change was more marked in the culture run at low cellular concentration and coincided with a sudden lowering of cellular concentration due to a higher filtration rate of the cultures. After three days the normal cellular concentration was restored and the nitrogen content returned to the previous values. The protein content of the biomass (about 54%) was relatively stable, irrespective of the cellular concentration of the culture. In August the nitrogen content of biomass was higher in the culture grown at lower cellular concentration. Presumably in this culture the nucleic acid content was higher due to the higher growth rate reached by the cyanobacterial population. No significant differences in the fatty acids composition and in the amino acid pattern of the biomass grown at low and high cellular concentrations were found.
~
0,6 g t "+
I
1,2 g t -1
N ~.
months
Fig. 5.
Influence of cellular concentration on the yield of Spirulina platensis M2 grown in a tubular photobioreactor.
70
G. Torzillo et al. •
N*/,
1.2
gl1
o 0.6 gl -I
13
11 10 9 8
7 i
i
Ju(y
Fig. 6.
Nitrogen content of
i
August
September
Spirulina platensis M2
grown at different cellular concen-
trations.
photobioreacfor 25
:,..
5
A
M
J
J
A
5
0
20
4 ~g
10
2 .~_ *'<
o
r~
A
M
J
J
A
S
0
months
Fig. 7.
Comparison of the mean yield of Spirulina platensis M2 cultivated in photobioreactors and in open ponds.
Comparative yield of both open ponds and photobioreactors In Fig. 7 the course of productivity of Spirulina platensis M2 cultivated in photobioreactors and open ponds operating at the same areal density is shown. The daily yield of the photobioreactor was higher than that of the open ponds during the whole cultivation period. This was mainly due to better temperature profiles in the closed culture system. The culture in the photobioreactors reaches optimum temperature earlier in the
Production of
Spirulina biomass in closed photobioreactors
71
o
16
September photobioreacfor
~0
w 20 m
i
1
1
;Q. oC
t 25 July. ~
E
I
i
photobioreacfor
2o i
5.00
8.00
i
1100
I
lt, O0
17.00 hours
Fig. 8. Typicalcourseof the temperatureof Spirulina cultures in photobioreactorsand ponds in July and September.The arrow indicatesthe start of the cooling. morning and better temperatures for the growth are also reached during the months in which the cultivation in ponds is limited by low temperatures (Fig. 8). No significant differences in the protein content and amino acid composition between the biomass grown in photobioreactors and in open ponds were found (Table 3). However, some differences were found in the fatty acids composition. Biomass grown in open ponds showed a higher degree of unsaturation of fatty acids due mainly to a higher content in 7-linolenic acid (Table 4). It is reasonable to suppose that these differences are dependent on the higher temperatures reached in the photobioreactor.
DISCUSSION The results obtained from the investigations, extended for several years, on the growth of Spirulina platensis and S. maxima in a tubular photobioreactor at a pilot plant scale, allow a preliminary evaluation of the merits and drawbacks of closed culture systems, in comparison with open ponds, for mass culturing of oxygenic phototrophic microbes. Under the climatic conditions of central Italy, the average yearly output of biomass from the photobioreactor was nearly 90% higher than that obtained from open ponds. This difference is due primarily to the fact that closed photobioreactors allow a considerable extension of the cultivation period (215 days against 175 days with open ponds), because
G. Torzillo et al.
72
TABLE 3
Amino acid Composition of Spirulina platensis M2 Grown in Tubular Photobioreactor and in Open Pond (g amino acids 100 g protein- ~)
Amino a c i d s
Photobioreactor
Open pond
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Ammonia Arginine 19. a-e-Diamino pimelic acid
11.2 5.1 4.3 17"4 4.4 5"6 7'2 0'3 7.9 2.2 6.9 10.5 4.7 5'1 5"8 2"1 1"1 8"3 1.0
11.4 4.5 3.7 16.9 4.4 5.6 7.4 0"3 8.1 2"4 7.1 10"6 4.8 5.4 5"9 2.1 1.1 8'3 1.0
Protein %
53.6
51.0
TABLE 4
Main Differences in Fatty Acids Composition of Spirulina platensis M2 Grown in Tubular Photobioreactor and in Open Pond (% of total fatty acids)
Photobioreactor Open pond
CI6:0
CI6: l A 9
C18: I A 9
C18:2A 9,12
C18:3A 6, 9,12
52.3 48.0
4.2 6"5
3.8 3"0
23.0 21'5
13"8 18"1
inside the closed culture units the d a y t e m p e r a t u r e attains values considerably higher t h a n a m b i e n t temperature. Additionally, the tubular culture device gave slightly better yield also in the w a r m e s t p e r i o d of the year, w h e n air t e m p e r a t u r e s reach 3 0 - 3 5 ° C . Also in this p e r i o d a better t e m p e r a t u r e profile in the p h o t o b i o r e a c t o r , due to a m o r e rapid heating
Production of Spirulina biomass in closed photobioreactors
73
of the culture in the early morning, presumably plays a major role in determining the better yields observed. T h e main problems encountered in the operation of tubular photobioreactors are the control of oxygen concentration and of overheating in summer. T h e problem of temperature control has been greatly alleviated with the selection of a strain of Spirulina platensis able to grow up to 42°C and to withstand temperatures of 46°C for a few hours without adverse effect. With this strain cooling of the photobioreactor by spraying water on the culture tubes was required only for 20 days yearand the amount of water required was estimated to be 250 litres m-2 of cultivated area year-1. T h e control of oxygen concentration requires an appropriate design of the photobioreactors, so that the culture can be degassed before the oxygen tension in the culture suspension becomes harmful. The selection of a Spirulina strain less sensitive to the adverse effect of high O2 tension would contribute significantly to simplifying the construction of large photobioreactors as well as their management. Although the installation of large scale photobioreactors may be more costly than a production plant based on open ponds, the greater efficiency in solar energy conversion that can be achieved with closed systems makes them very attractive in photosynthetic biomass production through mass culture of microalgae and cyanobacteria.
REFERENCES 1. Pirt, S. J., Lee, Y. K., Walach, M. R., Pirt, M. W., Balyuzi, H. H. M. & Bazin, M. J. (1983). A tubular bioreactor for photosynthetic production of biomass from carbon dioxide: design and performance. J. Chem. Tech. Biotechnol., 33B, 35-58. 2. Pirt, S. J., Lee, Y. K., Richmond, A. & Watts Pirt, M. (1980). The photosynthetic efficiency of Chlorella biomass growth with reference to solar energy utilization. J. Chem. Tech. Biotechnol., 30, 25-34. 3. Gudin, C. & Chaumont, D. (1983). Solar biotechnology study and development of tubular solar receptors for controlled production of photosynthetic cellular biomass for methane production and specific exocellular biomass. In: Energy from biomass, Series E, Vol. 5, W. Palz and D. Pirrwitz (eds), D. Reidel, Dordrecht, pp. 184-93. 4. Gudin, C., Bernard, A. & Chaumond, D. (1983). Culture de microalgues en reacteur tubulaire clos. Actes du second Colloque de l'Association Franqaise d'Algologie Appliqu~e, Chfiteau de Fontager, 28 Octobre, AFAA Laboratoire de la Roquette Bauzille de Putois, France, pp. 105-54. 5. Walach, M. R., Balyuzi, H. H. M., Bazin, M. J., Lee, Y. K. & Pirt, S. J. (1983). Computer control of an algal bioreactor with simulated diurnal illumination. J. Chem. Tech. Biotechnol., 33B, 59-75.
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6. Paoletti, C., Pushparaj, B. & Tomaselli Feroci, L. (1975). Ricerche sulla nutrizione minerale di Spirulina platensis. In: Proc. XVII Nat. Congr. It. Soc. Microbiol., Padova 26-28 Ottobre, Vol. 2, Grafiche Erredici, Padova, pp. 845-853. 7. O'h Eocha, C. (1965). In: Chemistry and biochemistry of Plant Pigments, T. W. Goodwin (ed.), Academic Press, New York, pp. 175-6. 8. Torzillo, G., Giovannetti, L., Bocci, E & Materassi, R. (1984). Effect of oxygen concentration on the protein content of Spirulina biomass. Biotechnol. Bioeng., 26, 1134-5.