Biomass ondBioenergy Vol. 1,No. 2,pp. 83-88, 1991 Printed in Great Britain. All rights reserved

0961-9534/91 $3.00+0.00 0 199 1 Pergamon Press plc

A CLOSED SYSTEM FOR OUTDOOR CULTIVATION OF MICROALGAE

EPHRAIM COHEN,* AVI KOREN~ and SHOSHANA (MALIS) ARAD* *The Institutes for Applied Research, Ben-Gurion University of the Negev, P.O. Box 1025, Beer-Sheva

84110, Israel tArava R & D Authority, Sapir Center, M.P. Arava 86825, Israel

(Received 26 September 1990; accepted 21 January 1991)

Abstract-The main problems in the large-scale cultivation of microalgae outdoors in open ponds are low productivity and contamination. To overcome these problems a closed system consisting of polyethylenes sleeves was developed. In a study conducted outdoors in the Negev area (Beer-Sheva, ‘En Yahav and Elat) the closed system was found to be superior to open ponds with respect to growth and production in a number of microalgae. In both closed and open systems, growth and production under continuous operation were higher than in batch cultivation. In continuous cultures the respective daily yields of dry matter and polysaccharides from the red microalga Porphyridium sp. were 17.7 and 7.4 g m-* in the sleeves compared with 7.6 and 2.4 g m-* in the ponds. With the aim of optimising growth and production the effect of sleeve diameters (10, 20 and 32cm) on these parameters was investigated. Growth and polysaccharide production were superior in the narrow sleeves than in the wider sleeves. To scale up the system a unit of connected sleeves was developed, and the peifotmance of various microalgae in the unit was investigated. The productivity in terms of biomass and polysaccharide production of the red microalga Rho&la reticulatu was higher in sleeve units than in open ponds. b-carotene production by the green microalga Dunuliellu bardawil and its isomer ratio (9-cis : all-tram) were better in the sleeve unit than in open ponds. The sleeve system seems to have a significant advantage over open ponds, and its development and optimisation are under way.

Keywords-DunalieNa, outdoor cultivation, microalgae, Porphyridium, Rhodella, Rhodophyta

1. INTRODUCTION

One of the main problems in outdoor algal cultivation is limited productivity, which ap- pears to be the result of the following factors: (i) limited light availability to the cells in the deeper layers of the ponds;’ (ii) photoinhibition in the summer caused by very high light intensities in the upper layer of the ponds;’ (iii) above optimal day temperatures in the summer and suboptimal temperatures in the winteq3 (iv) increased respiration and hence a loss of biomass caused by high night temperatures in the summer;4 (v) fluctuating salinity caused by intensive evapor- ation and (vi) inhibition of growth as a result of elevated oxygen concentrations.* The other major problem associated with outdoor cultiva- tion of algae is collapse of the cultures as a result of contamination by bacteria, fungi, and other algae as well as predation by zooplankton.’

As a result of the various problems there has been a tendency to change from open to closed systems, for example, different types of horizon- tally arranged glass or polyethylene tubes- or a covered convector.’ A closed system consisting of polyethylene sleeves, as proposed by Trotta” for rotifers, was adapted by us for outdoor

cultivation of red microalgae. Growth and production of biomass and polysaccharides were higher in the cultures grown in the sleeves than in those grown in the ponds.” Thus, we were encouraged to continue with the develop- ment and scaling up of this system.

In this study the productivity under desert conditions (in Elat) of the red microalgae Porphyridium sp. grown in batch and continu- ous culture in closed polyethylene sleeves was compared with that in open ponds. In addition, the effect of sleeve diameter on productivity was studied. The scaling up of the system into a unit of connected sleeves was undertaken for the red microalga Rhodella reticulata, which produces a sulphated polysaccharide, and for the green microalga Dunaliella bardawil, which produces the pigment p-carotene. The large-scale system was operated in Beer-Sheva and ‘En Yahav (Arava Valley) for R. reticulata and D. bardawil, respectively.

2. MATERIALS AND METHODS

2.1. Microalgae and growth conditions The red algae Porphyridium sp. (UTEX 637)

and Rhodella reticulata (UTEX LB3430) were

83

84 E. COHEN CI d.

obtained from the culture collection of the University of Texas. The green alga Dunaliellu hardawil (kindly supplied by A. Ben-Amotz and M. Avron) was a local isolated species deposited with the American type culture collection, Rockwill MD No. 30861.

Porphyridium sp. was grown in artificial sea water (ASW) prepared according to Jones et al.,” R. reticulata in a medium prepared according to Schlosser,‘3 and D. barduwil in a medium prepared according to Ben Amotz et aLI4 The cultures were started indoors as previously described.” When they reached the stationary phase of growth (maximum cell concentration) they were transferred to outdoor ponds or poly- ethylene sleeves at a density of 4 x 106, 2 x 106, and l-2 x 10’ cells ml-’ for Porphyridium sp., R. reticulata, and D. bardawil, respectively.

2.2. Outdoor cultivation 2.2.1. Single sleeves. Polyethylene sleeves,

each consisting of a transparent polyethylene tube (150 cm long, 0.2 mm thick, and 10, 20 or 32 cm in diameter), were sealed at one end and hung on the other end according to Cohen and Arad.” The lo-, 20-, and 32-cm diameter sleeves were filled with 10, 35, and 70 1 of medium, respectively. The cultures were mixed by means of an air stream containing 34% CO2 pumped into the sleeves at 4-5 1 min-’ via glass tubing. The pH of the cultures ranged between 7 and 8.

2.2.2. Unit of connected sleeves. This unit consisted of a long transparent polyethylene sleeve, 0.2 mm thick and 10 cm in diameter. The sleeve was looped around iron tubes connected to a suitable structure so as to form a row of vertical columns 2 m high. Each pair of columns was separated from the next at the point of

suspension on the iron frame. 25 1 of algal culture were pumped into each column via ;L plastic tube and mixed by means of an air stream containing 34% CO,. The unit was composed of 20 columns, with a total volume of 500 1 (Fig. 1).

2.2.3. Open ponds. Plastic containers of capacity 300 1 and raceway ponds of capacity 400 1, having surface areas of I and 2.5 m’, respectively, were used.” The containers were filled with a 100-150 1 of culture (l&-l5 cm deep) and the raceways with 375 1 of culture (15 cm deep). Mixing was achieved by means of paddle wheels, and CO, was supplied at a flow rate of 30 ml min-’ via porous tubing laid at the bottom of the containers or ponds.

2.2.4. Batch and continuous operation. Polyethylene sleeves and ponds were filled with medium and inoculated with algal cells, as described above. In the batch regime the Porphyridium sp. cells were harvested at the end of the growth and nutrients were not supplied during growth. In the continuous mode 25% and 20% of the total volume were harvested and diluted with fresh medium when reaching 20 x lo6 and 5 x lo5 cells.ml ’ for Porphyridium sp. and D. barduwil, respectively.

2.3. Measurements 2.3.1. Growth. For measurement of biomass

a cell sample was centrifuged, washed twice with water at pH 4.0, and filtered through a 0.45~pm filter. The precipitate was then oven dried (70°C) for 24 h and weighed. Determination of the cell wall polysaccharide was performed as previously described.15

2.3.2. Chlorophyll and b-carotene. The two pigments were extracted from a pellet of

Fig. 1. A unit of polyethylene sleeves

Outdoor cultivation of microalgae 85

D. bardawil with acetone, diluted with water to 80% acetone (v/v), and assayed as described previously.‘4 /?-carotene (9-cis and all-trans) was analyzed by HPLC according to Ben Amotz et a1.‘4

2.3.3. Light intensity. Light was measured with a Li-Cor instrument (Lincoln NB) with a spheric sensor (model SPN Quantum SR No. SPWA 0710) inserted vertically to a depth of 15 cm in the cultures grown in the sleeves.

3. RESULTS

3.1. Biomass and polysaccharide production in Porphyridium sp.

Biomass and total polysaccharide (bound + dissolved in the medium) produced by Por- phyridium sp. growing in batch and continuous cultures were compared in sleeves and mini- ponds (Table 1). For the same volume of culture (100 1) productivity was highest in the culture operated continuously in the sleeves in terms of both biomass and polysaccharide production- for biomass 160% (batch) and 130% (continu- ous) higher for sleeves vs. ponds and for polysaccharide 270% (batch) and 200% (continuous) higher for sleeves vs. ponds.

3.2. Influence of sleeve diameter on productivity

One of the main factors affecting productivity is light availability to the cells, which in the case of the sleeves is a function of the diameter. With the aim of optimising the sleeve diameter, growth and polysaccharide production in Porphyridium sp. were compared in sleeves of three different diameters-narrow 10 cm, medium 20 cm, and wide 32 cm. The highest values for cell number and biomass production were obtained for the narrow sleeves, being 1.5-2 and 3 times higher than those obtained for medium and wide sleeves, respectively (Fig. 2a and b). Similar results were also found for total

Table I. Biomass and polysaccharide produced by batch and continuous cultures of Porphyridium sp. in sleeves and

ponds

Biomass Polysaccharide (g day-‘) (g day-‘)

Batch Continuous Batch Continuous

Sleeves 10.2 17.7 5.6 7.4 Ponds 3.9 7.6 1.5 2.4

Results were calculated for a volume of 100 1 which was contained in 1 pond or 3 sleeves, each 2Ocm in diameter. The cultures were grown continuously for 40 days in the summer in Elat. On reaching a culture density of 20 x IO6 cells ml-’ in sleeves and ponds, 25% of the total volume was harvested. The sleeves were harvested 13 times and the ponds 9 times.

cm

cm

l-@eJw+ ,*,*&L~m- -0 6’ rn) 35 cm

0 10 20 1 3( 1

Days Fig. 2. Growth of Porphyridium sp. in sleeves of different diameters. (a) Cell number, (b) biomass, (c) total polysac- charide. The experiment was carried out in Beer-Sheva

during the winter.

polysaccharide production (Fig. 2~). Although the volume of the wide sleeve was three times that of the narrow one, calculated biomass production per l-m-long row of narrow sleeves was twice as high as that in a row of wide sleeves (Table 2). The experiment was carried out during the winter during which time wide fluctu- ations in light intensity are recorded. The differ- ences in light penetration into the sleeves with different diameters are shown in Table 3. Light intensity during the course of the experiment was higher in the two narrower sleeves, the differences between the sleeves being higher in the morning and at noon than in the afternoon. Towards the end of the experiment (day 23) light intensity decreased, reaching almost undetectable values in the wider sleeve.

3.3. Large-scale cultivation

The encouraging results obtained with the sleeves led us to proceed with the development

86 E. COHEN el al.

Table 2. Effect of diameter on biomass production of Porphyridium sp. cultivated in a I-m row of sleeves

Sleeve Sleeve Total vol in diam vol No. of sleeves 1 m of sleeves Biomass Biomass (cm) (1) in 1 my (1) (g 1-l) (g m-‘)

10 10 10 100 2.9 290 20 35 5 175 1.3 22-I 32 70 3 210 0.7 147

“Number of sleeves of different diameters that can be hung in a row 1 m long. Biomass was calculated at the end of 24 days of growth.

Table 3. Light intensity (pm01 quanta m-k’) under ambient conditions and in sleeves of different diameters during the day throughout the experiment

0800 1200 1600

Day 10 20 32 Amb 10 20 32 Amb 10 20 32 Amb

3 600 250 100 2400 500 200 200 2900 350 150 100 2300 6 300 35 20 1250 150 20 10 900 90

30 :.“o 5.0 400

10 115 8.0 2.0 1100 175 8.0 3.0 1650 1.5 180 23 90 0.9 0.1 1000 100 0.8 0.9 1000 9.0 0.15 0.1 150

10, 20 and 32 = sleeves of diameter 10, 20 and 32 cm, respectively; Amb. = ambient conditions.

of a system for large-scale cultivation. The large-scale unit consisted of a number of con- nected sleeves, as shown in Fig. 1. To investigate the effect of scaling up, R. reticulum was grown in a 2.5m-long sleeve unit composed of 20 sleeves (each with a diameter of 12 cm), contain- ing a total culture volume of 500 1. The algae were cultured in a semi-continuous mode, i.e., when a culture density of 5-6 x lo6 cells ml-’ was reached, 75% of the total volume was harvested. In this experiment six harvests, at intervals of 4-5 days, yielded 79 g day-’ of biomass and 67 g day-’ of polysaccharide. A comparison was not made with the results for open ponds, since we were unable to culture R. reticulata outdoors in open ponds.

The amount of p-carotene produced by D. bardawil growing continuously in sleeve units was compared with that in open mini-ponds (Table 4). In the fall/spring and winter B- carotene production in the sleeve unit was 100% higher than that in the ponds. In the summer B-carotene production in the sleeve unit was

Table 4. B-carotene production during different seasons by Dunaliella bardawil grown continuously for one month in

mini-ponds and sleeve units

b-carotene (g month-‘)

Miniponds Sleeve Unit

Summer 16.2 24.3 Fall/Spring 32.4 72.0 Winter 36.0 72.0

Results are given for two units of 20 sleeves (each 12 cm in diameter) for a total volume of 900 1 and for 3 ponds (each of 2.5 mm2 surface area and depth of 12 cm) for a total volume of 900 1. On reaching a culture density of 5 x lo5 cells ml-‘, 20% of the total volume was harvested.

only 50% higher. In addition, the ratio of isomers of b-carotene 94s : all-Puns was 1: 1 in the sleeves as compared with 0.8 : 1 .OO in ponds.

Summer and winter temperatures recorded at ‘En Yahav in the sleeve units and mini-ponds are presented in Fig. 3. In the winter the low morning temperatures in the sleeves increased rapidly to reach an optimal value of 25°C at noon. The maximum winter temperature in the mini-ponds reached only 10°C. In the summer the temperature in the ponds rose to 33°C but that in the sleeves was prevented from rising above 28°C by spraying the outer surface with water.

1 1 0 4 8 12 16 20 21

Time of day

Fig. 3. Daily temperature measured in the sleeve unit and mini-pond during algal growth. 0 = pond water, ??= sleeves winter, 0 = pond summer, 0 = sleeves summer. The experiment was conducted at ‘En Yahav during the summer and winter. The temperature in the sleeves was prevented from rising above 28°C by spraying

the sleeves with water from an automatic device.

Outdoor cultivation of microalgae

Table 5. Predicted productivity of Porphyridium sp. calculated for 100 m-* surface area of sleeve and nonds

87

Production in continuous growthb

Surface area of lOOm* Volume Biomass Polysaccharide provided by: (m’) (g m-* day-‘) (g m-* day-‘)

Sleeve unit with intervals of 1.5 m between rows 20” 35.4 14.8 Sleeve unit with intervals of 2 m between rows 15” 26.5 11.1 Pond 15 11.4 6.0

‘The volume was calculated for sleeves containing 60 1 (20 cm in diameter and 2 m high). Each row of sleeves (10m long) contained 50 sleeves.

bValues were calculated from results obtained in continuous cultivation of Porphyridium sp. in sleeves as described in Table 1.

The productivities predicted for Porphyridium sp. cultured continuously in sleeve units and ponds (based on results presented in Table 1) calculated and are presented in Table 5. For a surface area of 100 m2 the volume of culture in a sleeve unit with 1.5 m intervals between the rows was 33% higher than that in the ponds, whereas when 2 m intervals were allowed between the rows, the culture volume calculated for the sleeve unit was the same as that for the ponds. Biomass production in sleeve units with inter-row intervals of 1.5 and 2 m can be predicted to be 210 and 3 lo%, respectively, higher than that in the ponds. Similar results were obtained for calculations of polysaccharide production.

4. DISCUSSION

We have previously shown that algal cultiva- tion in sleeves is superior to that in mini- ponds.” The main factors contributing to the advantage of the sleeves over the ponds are the temperature fluctuations (high temperatures during the day and low temperatures during the night) and improved light availability. The differences in temperature between the sleeves and the ponds are already evident early in the morning. Thus, in the winter effective photosyn- thesis occurs over about 9 h of the day in the sleeves as compared with only 334 h in the ponds. During the summer spraying of the sleeves with water can prevent collapse of the cultures, which is a possibility in the desert where high temperatures prevail. The surface/volume ratio, which affects light availability, is better in the sleeves than in the ponds, By changing the sleeve diameter light intensity throughout the algal culture can be varied. By decreasing the sleeve diameter, productivity (biomass and polysaccharide) in Porphyridium sp. was increased. Although the productivities increased as the sleeve diameter

decreased, as was found by Pirt et al.,l6 we did not experiment with diameters of less than 10 cm, since such small diameters do not seem feasible from practical point of view for large- scale operation. Other factors also contribute to the superior productivity obtained in the sleeves: (1) the better turbulence improves light/dark cycles and reduces photo-oxidation, (2) evaporation is prevented by virtue of the system being closed, and (3) contamination is reduced.

The sleeve unit has certain advantages over both single sleeves and ponds. Since its installa- tion and operation are simple, operating costs which are high for single sleeves are reduced. The relative volume of culture in the sleeve unit is higher than or similar to that in the ponds and is dependent on the distance between the rows. Thus, a pond of surface area of 100 m* contains 15 m3 of culture volume, whereas a sleeve unit of the same surface area contains 15 or 20 m3 for inter-row distances of 2 or 1.5 m, respectively.

Calculation of predicted productivity based on results obtained in the Negev for various algal species indicates that productivities of 24 g m-* day-’ can be achieved. In certain cases the superiority of the sleeves, due to higher light availability to the cells, is evident not only in terms of biomass production but also in the quality of the product, as was shown for the isomer ratio of p-carotene.

Although the sleeve system appears to have advantages over ponds we do recognise the problems raised in such a comparison, i.e., the productivity of ponds, usually described in g m-? day-‘, cannot simply be transferred to the sleeve system. We make use of these productivity units since they are units commonly quoted in the literature (and are used by us only for purposes of comparison). Productivity per volume or per surface area can also be used. In addition, the comparison of the different factors affecting productivity in sleeves

88 E. COHEN et at.

and ponds, especially light availability, are not 6. D. Chaumont, C. Thepenier, C. Gudin and C. Junjas,

always simple, raising the problems of measure- Scaling up a tubular photoreactor for continuous culture of Porohvridium cruentum from laboratorv to

ment values, e.g., light per area or per cell or per day. All these and other problems have not yet been completely resolved. However, the sleeve unit represents in principle a totally new ap- proach to algal cultivation, i.e., vertical (sleeves) vs. horizontal (ponds). The horizontal concept also includes the tubular system suggested previously.6,8

way.

Acknowledgement-The authors wish to thank Dr. A. Ben- Amotz for kindly supplying the Dunalielta bardawil and for determination of b-carotene isomers.

At this stage, we do not feel that the system has been completely perfected. The develop- ment and optimisation of the sleeve unit towards large-scale production is now under

pilot plant ?I!&-1987). In Algal Biotechnology (T. Stadler, J. Mollion, M.-C. Verdus, Y. Karamanos, H. Morvan and D. Christiaen, Eds), pp. 199.-208. Elsevier Applied Science, London (1988).

7. A. Dicorato, Coltivazione sperimentals in sistema tubo- lare in CNR ed. Prospective della coltura di Spirulina in Italia, p. 261. Florenz, Italy (1980).

8. G. Torzillo, B. Pushparj, F. Bocci, W. Balloni, R. Materassi and G. Florenzano, Production of Spirulina biomass in closed photobioreactors. Biomass 11, 61--74 (1986).

Aquaculture 22, 383-387 (1980). 11. E. Cohen and S. (Malis) Arad, A closed system for

outdoor cultivation of Phorphyridium. Biomass 18, 59-67 (1989).

12. R. F. Jones, H. L. Speed and W. Kury, Studies on the growth of red alga Porphyridium cruentum. Physiol. Planf. 16, 636643 (1963).

9. D. B. Anderson and D. E. Eakin, A process for the production of polysaccharides from microalgae. Biotechnol. Bioeng. Symp. 5, 533-547 (1985).

10. P. A. Trotta, Simple and inexpensive system for con- tinuous monoxenic mass culture of marine microalgae.

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