Biomass 2 (1982) 175-185

P R O D U C T I O N O F S P I R U L I N A B I O M A S S : E F F E C T S O F E N V I R O N M E N T A L F A C T O R S A N D P O P U L A T I O N D E N S I T Y

AVIGAD VONSHAK, AHARON ABELIOVICH, SAMY BOUSSIBA, SHOSHANA ARAD and AMOS RICHMOND

The Jacob Blaustein Institute for Desert Research at Sede Boqer and The Department of Biology in Beer Sheva, Ben-Gurion University of the Negev, Israel

(Received: 20 December, 1981)

ABSTRACT

The effects o f environmental conditions (solar irradiance and temperature) and population density on the production o f Spirulina biomass are reported for cultures grown in outdoor ponds. Both the specific rate o f photosynthesis, expressed on a chlorophyll basis, and the rate o f respiration, on a protein basis, decreased as algal concentration increased. Higher specific growth rates were observed at lower popula- tion densities. Lower growth rates were associated with the light limitation in dense cultures for optimum conditions in the summer. Seasonal variation was observed in productivity. In summer light was the limiting factor whereas in winter the low daytime temperature appeared to impose the ma/or limitation. It was found that the oxygen concentration in the culture can serve as a useful indicator o f limiting factors

and can also be used as a means o f estimating the extent o f such limitations.

Key words." Spirulina platensis, outdoor algal ponds, photosynthesis, population density.

INTRODUCTION

In recent years interest in the possibility of mass culturing microalgae, particularly for single-ceU protein production, has increased. 4,s'9,a4,lT'ls Many other applications for the large-scale commercial production of algae have also been suggested or investi- gated.2,3, m m ~6

We believe that algal biomass production in brackish or sea water could constitute an economically viable industry in many arid zones in the world ~s where scarcity o f

175 Biomass 0144-4565/82/0002-0175/$02.75© Applied Science Publishers Ltd, England, 1982 Printed in Great Britain

176 AVIGAD VONSHAK et al.

water suitable for conventional agriculture impairs the production of foodstuffs and natural products. The premise is that, through the cultivation of species and strains of algae which respond well to intense radiation and high temperature, saline water may be used to augment bioproduction in many arid regions of low bioproductivity.

The food potential of the genus Spirulina seems particularly promising. This is a cyanobacterium (family Oscillatoriaceae) with helical, multicellular filaments which may be 50-300 tam long and 10 #m in diameter. Several Spirulina species (e.g.S. platensis, S. maxima) are found as a single dominating organism in alkaline soda lakes with pH up to 11, and may grow well in waters containing up to 14 000 mg chloride litre -~ in arid or semi-arid regions in Africa and Central America. ~

Spirulina platensis has been collected from the salty lakes and ponds along the northern shores of Lake Tchad since time immemorial, sun-dried and eaten by the Kanembou people, s A similar alga was apparently collected and prepared by the Aztec Indians at the time of the arrival of Cortez in Mexico.TSpirulina filaments may be easily separated from their medium, have high digestibility and a mild flavour, and contain up to 70% protein of excellent quality.

Zarouk ~9 was the first to provide detailed experimental results on the basic nutritional and temperature requirements of Spirulina. Richmond and Vonshak ~3 and Richmond et al. is described various aspects that relate to the optimisation of outdoor systems for the production of Spirulina in Israel. Laboratory studies on the effects of population density and of the extent of stirring, as well as of various engineering aspects, on the production of Spirulina have been reported, 6 and the major conditions required for a semi-industrial facility suggested.

Our experience in outdoor production of algal biomass indicated that the output of algal biomass grown in pond cultures may be limited in practice by three aspects: (i) nutrient concentration; (ii) daily and seasonal variations in temperature and solar radiation; (iii) the degree to which a monoalgal culture is maintained. Whereas mineral and carbon limitations can be readily controlled, the other aspects require detailed research. In this work we studied the interrelations between solar irradiance and population density as well as the effects of temperature on the output rate of out- door cultures of Spirulina platensis. The maintenance of monoculture will be dealt with in another paper.

MATERIALS AND METHODS

The organism and growth conditions The blue-green alga Spirulina platensis was cultivated in either 1 m 2 fibreglass ponds

(miniponds) or in 100 m 2 ponds lined with 1.0 mm thick polyvinylchloride (PVC). The depth of the culture in the ponds was 13-15 cm, and stirring was provided by paddle wheels, producing a flow of about 50 cm s -1 in the 100 m 2 ponds and 20-30 cm s -1 in the miniponds.

PRODUCTION O F Spirulina BIOMASS 177

The growth medium Zarouk '9 medium was used, with modifications as follows: NaC1, 1.0 g 1-1; K2504,

1-0 g 1-1 ; KNO3, 3.0 g 1-1; EDTA, 0.08 g 1-1; FeSO4.7H20, 0.01 g F1; H3PO4, 0"25 ml 1-1 ; NaHCO3, 16-8 g 1-1 ; and As and B 6 microelement solutions, 1 ml of each per litre medium. As consisted of the following salts, in g l-l: H3BO3, 2.86; MnC12, 1.81; ZnSO4.7H20, 0.22; CuSO4.5H20, 0.079; MOO3, 0.015. B6 consisted of the following, in g 1-1 x 10-4: NH4VO3, 229.6; K2Ca2(SO4)4.24H20, 960; NiSO4.7H20, 478.5; Na2WO4.2H20, 179"4; Te2(SO4)3,400; CO(NO3)2.6H20, 439-8.

Dry weight measurements For dry weight determination, a 100 ml sample from the pond was filtered through

a cellulose nitrate filter (8.0 tam) and washed with an equal volume of acidified water (pH 4.0) to free the algal fdaments from insoluble minerals. Prior to filtration, the filter was dried in an oven for 24 h at 80°C and then placed in a desiccator. The weight of the filter was determined after it reached room temperature. The algal residue on the filter was dehydrated in an oven for 24 h at 80°C and, after cooling in a desiccator, was weighed.

Total chlorophyll concentration Five ml of algae suspension were taken from the pond and centrifuged for 10 min

at 2000g. The precipitate was resuspended in 5 ml methanol for 2 min in a water bath at 70°C. This suspension was then ground in a manual glass tissue homogeniser, and thereafter cleared of debris by centrifuging (10 min at 2000g). The optical density of the supernatant was determined at 665 nm.

Protein measurements The pellet remaining after chlorophyll extraction was used to determine the protein

content. The pellet was boiled for 15 min in 0-5 N NaOH, and the supernatant was collected after 10 min centrifuging (2000g). This procedure was repeated twice and the supernatants of the extraction were put together. Total protein was measured according to Lowry et al. '°

Measurement o f photosynthetic oxygen evolution and o f respiration All 02 kinetic measurements were performed in the laboratory in fresh Zarouk 19

Spirulina growth medium. Algae from the pond were washed in the medium, brought to a turbidity of 0-2A, and the 02 evolution rate was determined using a Yellow Spring 4004 02 electrode in a stirred and thermostatted glass cell at 30°C, illuminated by a tungsten lamp, l 0 s erg cm -1 s -1 at the surface of the cell. The values thus obtained were regarded as the photosynthetic potential of algal cells in the pond under optimal conditions.

178 AVIGAD VONSHAK et al.

Pond oxygen concentration In situ measurements of 02 concentrations were made with a WTW 02 meter. 02

concentration was determined at 08.00 h and 13.00 h daily.

Artificial brackish water Brackish water medium was produced by adding to tap water various salts, viz.

Zarouk 19 medium that contained, in addition: Na2SOa, 2-7 g F1; CaC12, 1.2 g l-l; MgC12, 1.6 g F 1. The aim was to simulate a prevalent source of such water in the hot Arava Valley in Israel.

RESULTS AND DISCUSSION

Population density was found to be a major factor in the production o f Spirul&a biomass, exerting far-reaching effects on the general performance of the culture. Plotting the potential photosynthetic activity, as indicated by 02 evolution, against the population density in the culture expressed in terms of chlorophyll concentra- tion revealed an inverse correlation between chlorophyll, or algal concentration, and the specific rate of photosynthesis per mg chlorophyll (assimilation number). Apparently, this reflects an adaptation on the part o f the algae to the low average light intensities available to the algal population when kept at high densities (Fig. 1). The effect of population density on the specific respiration rate (expressed on the basis o f protein) appeared to follow a similar inverse correlation (Fig. 2). This relationship, which existed at all population densities, was perhaps related to the decrease in pond 02 which was associated with increased population densities.

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P R O D U C T I O N O F Spirulina B I O M A S S 179

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The effect of population density on the specific rate of respiration in the summer of Spirulina grown in 100 m: ponds. • grown in tap water, o grown in brackish water.

The decreased photosynthetic potential observed in algal cells obtained from ponds maintained at medium to high density (i.e. 400-1000 mg 1-1) would appear to reflect a basic problem in biomass production. At these densities, most of the cells in the culture are at any given instant in complete darkness. Indeed, even at densities of 400- 500 mg (dry wt)1-1, which were found to be optimal for maximal photosynthetic efficiency, solar irradiation is almost completely absorbed in the upper 2-3 cm of the pond, leaving some 80% of the cells in complete darkness at any given instant) s Hence it would appear that an important factor which influences the growth rate and the photosynthetic efficiency is the 'light/dark' cycle. This cycle, which may be completed in a few seconds or extend to minutes, is a function of the speed of travel of the indi- vidual cells back and forth from the upper, illuminated layers of the pond to the lower, darkened ones. The dark/light regime which thus ensues for the average algal cell is influenced by the intensity of solar irradiance, and depends on the depth of the pond, the extent of turbulence, and the population density. The relationship between the population density and the specific growth rate throughout the seasons is shown in Fig. 3.

Clearly, the lower the population density, the higher the specific growth rate, as is to be expected in a system which is primarily light-limited. However, the relative effect of decreasing the population density, and thus of increasing the availability of light to each cell, is most pronounced in the summer and much less so in the winter. The obvious reason for this seems to be that in winter the major environmental factor

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Fig. 3. The specific growth rate of Spirulina as affected by population density and the seasons of the year. e--~-e summer (June to September), o--o--o autumn and spring (October to November and April to May), zx_~_A winter (December to February). The data represent averages obtained

from several experiments carried out throughout the year.

limiting the rates of growth and of output is essentially temperature, light exerting a relatively minor effect. In summer, on the other hand, light is the dominant limiting factor for growth, and thus the culture responds much more markedly to any change in this major limitation. Indeed, in light-limited growth, a close relationship must exist between the specific growth rate (/a) and cell density (X). Since/a is modified sub- stantially by temperature, this relationship varies greatly throughout the year, and the more severe the temperature limitation on/a, the smaller must be the dependence of/~ on X, which becomes hardly noticeable in winter.

A similar pattern was evident in relation to the limitation exerted by the population density. As the population density increased, the relative response of the culture to 'summer conditions' declined, until at very high densities, i.e. 0 . 8 0 D and over (1.5 g (dry wt)1-1), no response to and no seasonal effect on the specific growth rate was observed (Fig. 3). Clearly, in such dense cultures, light limitation due to mutual shading was so extreme that most of the growth rate potential of the algae was limited by light, thereby blocking any potential effect of temperature on growth.

Our extensive experimentation to elucidate the relationships between the specific growth and output rates and the optimal population dens i t y - defined as that cell concentration which yields the highest output rate under given environmental condi- tions - indicated that the optimal density varied according to the season. Plotting the pond's output rate against the population density during the course of the year (Fig. 4) revealed that the higher the temperature or the rate of available irradiance per cell, the more pronounced is the dependence of the output rate on population density. Thus, in the summer, the maximal output rate that was reached in a culture main-

PRODUCTION OF Spirulina BIOMASS 181

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The effect of population density on the output rate throughout the seasons of the year. e--o---e summer, o--o--o spring and autumn, ~ z x J , winter.

tained at optimal density was almost three times as high as that obtained in a culture maintained at the highest population density used in our experiments. In winter, when the total output was a fraction of that obtained in summer, the maximal output from the culture maintained at optimal density was only double that obtained from the culture with the highest density.

A decline in output rate was always associated with a high population density, i.e. over 500-600 mg 1-1. Since the output rate is a product of the specific growth rate and of the population density, this decline cannot be explained merely by the effect of increased mutual shading, since the increase in population density should compensate for the decrease in the specific growth rate. Thus, an additional factor involved in the phenomenon of decreased output at high densities seems to stem from the increase in maintenance energy relative to the photosynthetic activity.

A decrease in the population density below the optimal point also resulted in a significant decrease in the output rate. This apparently indicates that a high intensity of solar irradiance per unit area cannot be exploited at peak efficiency when the population density is relatively low. Thus, although the specific growth rate (/1) increased progressively in the culture as the population density (X) decreased (Fig. 3), this was not sufficient to compensate for the decline in output which was effected by the decline in the overall areal density. Indeed, the maximal output rate was obtained at a relatively high population density, exhibiting specific growth rates lower by some 50% than the maximal growth rates manifested in cultures maintained at as low a population density as was practically possible.

The decisive seasonal effect on output can be readily traced to variations in temperature, although seasonal fluctuations in radiation intensity must also have a

182 AVIGAD VONSHAK e t al.

significant effect. At Sede Boqer in winter, both day temperature and the rate of solar irradiance decline to about half their summer values. This effect of winter temperature on growth and output rate of Sp i ru l i na might be partially affected by the low night temperature (which, in our experimental area, may reach close to freezing point), or alternatively depend solely on daytime temperature. The latter, while being signifi- cantly higher than night temperatures, was in the winter 20-25°C below the optimal (37°C) for Sp i ru l ina . Experimentally it was shown that the decisive effect of winter temperature on decreased growth rate is largely due to lower daytime temperature (Table 1).

TABLE 1 The effect of modifying winter temperature a on the specific growth rate of Spirulina

Exper iment Conditions Specific growth no. rate, d -

1 Temperature not modified 0.03 2 Night temperature prevented from failing below 10°C; 0.03

day temperature not modified 3 Day and night temperature raised to 25°C 0-11 4 Day temperature raised to 25°C; night temperature 0.16

not modified

a Average maximum day temperatures and minimum night temperatures during the period of experimentation were 18°C and 5°C, respectively.

The 02 concentration in the culture may serve as a useful indicator for determining the limiting environmental factor and even for estimating the extent of such limitation. By following the daily course of temperature and radiation, and correlating them with the 02 concentration in the pond, it was possible to sort out the environmental limita- tion imposed on the culture. This is illustrated from measurements of pond 02 taken to estimate the relative rate of photosynthesis in the course of a day, in different seasons (Fig. 5). In winter, the peak of 02 concentration in the pond coincided with the peak in daily temperature, and during a typical day in winter (C in Fig. 5) the rate of increase in 02 evolution in the early morning, up to about 09.00 h, was clearly very low, until a rise in temperature became evident in the late morning. In contrast, peak 02 concentration coincided with the peak of illumination during the summer. More- over, when the temperature in the pond was maintained relatively high during the night (approximately 17°C; see A in Fig. 5), the rates of increase in 02 and in light intensity run very closely parallel. Clearly, 02 evolution under the temperatures prevailing in the pond in summer is controlled by the rate of light irradiance. The con- clusion was that during the winter, the rate of 02 evolution and thus the measured 02 concentration was not related to the irradiance as was clearly the case in summer.

As expected, the relationship between 02 evolution, temperature, and irradiance during early spring and autumn (Fig. 5, B) is such that the highest point in pond 02

PRODUCTION OF Spirulina BIOMASS 183

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Fig. 5. Rates of photosynthesis, as indicated by O~ evolution during the course of representative days in different seasons. A summer, B spring and autumn, C winter, o--~--D Temperature in °C;

o---o---o irradiance in klu×; ~---A---A 05, percentage of saturation.

does not coincide with the peaks of either temperature or irradiance, but occurs when temperature has not yet reached its maximum, whilst light intensity is beginning to decline. In these two seasons, the photosynthetic system is apparently limited by both light and temperature. Thus, in the mornings, no close parallel could be observed between the rise in 02 and the rise in temperature, as was evident in winter, and like- wise the daily increase in 02 concentration did not closely parallel the daily increase in the rate of irradiance, as was evident in summer.

A general response to light and temperature, similar to that observed for Spirulina, was recorded for Chlorella soroldniana, which is a high temperature strain. A marked response to temperature increase took place only at light intensities of >40 klux. As with Spirulina, the highest mark in pond oxygen was recorded at the highest points of light intensity and temperature. In contrast, in Chlorella vulgaris, the highest oxygen values in the pond were also recorded at the highest solar radiation (80-100 klux) but only at temperatures below 18°C. Higher temperatures resulted in lower oxygen concentrations in the pond. is Clearly, the oxygen concentration values reflected the fact that, for this strain, the optimal temperature was decisively lower than that for Spirulina platensis or for Chlorella sorokiniana.

In repeated experiments, the highest concentration of oxygen in the pond was associated with that cell density which yielded the highest output rate, and in general, our experience showed that pond oxygen may serve as a sensitive indicator for detecting divergence from steady state which, if left unattended, may quickly cause the deterioration of the culture.

184 AVIGAD VONSHAK et al.

Data collected daily over two years yielded the same pattern observed by following the daily course of pond oxygen concentration as follows: the higher the temperature and the intensity of solar irradiance, the higher the rise in pond oxygen. At any given light intensity, elevation of temperature resulted in increased concentration of pond oxygen and, similarly, for each temperature range, any increase in irradiance elevated pond oxygen (Fig. 6).

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Fig. 6. The interrelationships between maximum daily temperature, irradiance and photo- synthetic activity throughout the seasons. The data shown represent averages of a large number of observations, coUected during two years of experimentation with Spirulina platensis cultured in 1.0 m 2 ponds. The pH in the ponds was optimal for growth, ranging from 9.5 to 10.0, and no

nutrient limitation could be observed.

The fact that the highest oxygen values were recorded at the maximal light intensi- ties (800 W m -2) indicated that peak summer irradiance had no negative effects on Spirulina. Indeed, well-stirred cultures never reached light or temperature saturation even in summer days, when the highest irradiation and temperature values were recorded. This seemed to reflect the high degree o f adaptation ofSpirulina platensis to the climatic conditions prevalent in many arid and semi-arid zones.

ACKNOWLEDGEMENT

The authors wish to thank GSF, Manchen, West Germany, for their financial support of this research.

PRODUCTION OF Spirulina BIOMASS 18 5

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