Biomass 18 (1989) 59-67

A Closed System for Outdoor Cultivation of Porphyridium

E p h r a i m C o h e n & S h o s h a n a (Malis) A r a d

The Institutes for Applied Research, Ben-Gurion University of the Negev, PO Box 1025, Beer-Sheva 84110, Israel

(Received 9 June 1988; revised version received 17 November 1988: accepted 21 November 1988)

ABSTRA CT

Experience accumulated during the past few years indicates that the main problems in the large-scale cultivation of algae in open ponds are low productivity and contamination. Thus, the use of closed systems can be an alternative method of cultivation, b~ the present study, a closed system made of polyethylene sleeves was compared with open ponds with respect to growth and polysaccharide production of two species of Porphyridium: Porphyridium sp. and E aerugineum. For both species, cell number, biomass, and polysaccharide production were higher in the sleeves than in the ponds. It seems that polyethylene sleeves have the following advantages over open ponds: high light availability, high rate of heating and cooling, improved turbulence, relative lack of contamination, and prevention of evaporation and hence of fluctuation in salinity.

Key words: Porphyridium, outdoor cultivation, closed systems, poly- ethylene sleeves, open ponds, cell wall polysaccharide.

I N T R O D U C T I O N

Outdoor algal cultivation is conventionally carried out in open ponds in the form of raceways. Experience accumulated over a number of years has indicated two main problems with this technique: relatively low productivities and difficulties in maintaining clean cultures free of contamination by other micro-organisms.

Al though productivities of 46 g m -2 day-~ have been achieved in open ponds on a small scale for short times,~ such quantities could not be obtained on a large scale over long periods of time. The low produc-

59 Biomass 0144-4565/89/S03.50 -- © 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain

60 E. Cohen, X Arad

tivities achieved in outdoor open ponds seem to be the result of a variety of factors: limited availability of light to the cells 2 or very high light intensities cause photoinhibition; widely disparate day/night tempera- tures, e.g. low morning temperatures in the winter combined with high light intensities result in efficient photosynthesis beginning only late in the morning; accumulation of 02 , resulting from intensive photo- synthesis coupled with photo-oxidation, 3 intensive evaporation leads to fluctuating salinity. 4 Finally, contamination of the algal culture by predators including bacteria, fungi, zooplankton, or other algae, can take over the pond in a short time, causing collapse of the cultures. 5

In the past few years, as a result of the above-mentioned factors and other problems, there has been a tendency to change from open to closed systems. Various types of closed outdoor systems have been operated, including horizontally arranged glass and polyethylene tubes 6-8 and a covered convector. 9 A closed system composed of poly- ethylene sleeves has been used by Trotta "J for indoor cultivation of microalgae and rotifers as food for fish larvae.

In this study, the use of polyethylene sleeves outdoors for cultivation of two species of the unicellular red alga Porphyridium and for produc- tion of cell wall polysaccharides was investigated. The results were compared with those obtained with open ponds.

MATERIALS AND METHODS

Microaigae and growth conditions

The algae used in this experiment were Porphyridium sp. (UTEX 637) obtained from the culture collection of the University of Texas and P. aerugineum (111.79) obtained from the culture collection of the University of G6ttingen. Porphyridium sp. was grown in artificial sea- water (ASW) prepared according to Jones et al., ~ and P. aerugineum, in a medium prepared according to Ramus. ~2 When cultures grown indoors as previously described ~3 reached maximum density, they were transferred to outdoor ponds or to polyethylene sleeves at a density of 4 × 10 6 cells ml-l.

Outdoor cultivation

Closed system Each closed system consisted of a 150-cm-long transparent polyethylene sleeve, 0.2 mm thick and 32 cm wide. The bottom of the sleeve was

A closed system for outdoor cultivation of Porphyridium 61

sealed by heat-welding in a cone shape to prevent cell settling. The sleeve was then hung on an iron frame by means of an iron tube. Each sleeve was filled with 25 liters of medium and inoculated with 3-4 × 1 0 6 cells ml-1. The cultures were mixed by an air stream flow 4-5 liters min-J containing 3-4% CO 2 pumped into the sleeve via glass tubing. The pH of the culture ranged between 7 and 8.

Open ponds Fifty-litre plastic containers, (0.22 m 2 surface area) were used. The containers were filled with 25 liters of culture (12"5 cm deep). Mixing was achieved by means of a paddle wheel, and CO2 was supplied at a flow rate of 30 ml min-~ via porous tubing laid at the bottom of the containers.

Measurements

Growth Cells were counted with a hemocytometer using a Zeiss microscope. For the measurement of biomass, a cell sample was centrifuged, washed twice with water at pH 4.0, and filtered through a 0-45-/~m filter. The filtrate was then oven-dried (70°C) for 24 h and weighed.

Light intensity Light was measured with a Li-Cor instrument (Lincoln, NB) with a spheric sensor (model SPH Quantum SR no. SPWA 0710) inserted vertically to a depth of 15 cm in the cultures grown in the sleeves. In the ponds, the sensor was immersed horizontally at a depth of 5 cm.

Determination of the cell wall polysaccharide was performed as previ- ously described.l 3

TABLE 1 Temperature (°C) in the Sleeve, the Pond, and the Environment During the Daytime

Throughout the Experiment

Day 08.00 12.00 17.00

Sleeve Pond Ambient Sleeve Pond Ambient Sleeve Pond Ambient

0 18 10 16 29 23 23 33 28 26 4 11 9 13 23 18 18 23 18 17 8 19 13 23 33 27 23 35 30 25

12 17 12 20 33 26 23 30 27 20

62 E. Cohen, S. Arad

TABLE 2 Light Intensity (/~E m-2 s ~) in the Sleeve, the Pond and the Environment During the

Daytime Throughout the Experiment

Day 08.00 12.00 17.00

Sleeve Pond Ambient Sleeve Pond Ambient Sleeve Pond Ambient

0 420 145 2600 300 250 3800 250 90 2200 4 70 25 900 25 30 1350 40 30 850 8 30 15 2600 65 60 3400 35 10 1050

12 15 8 2400 7 10 2700 10 4 1600

Fig. 1.

1 0 ( 3

E

0 v

.o 10 E c

U

E ~2 E

E1 0

Ca) Sleeves

12 []

(b)

v v

I I I I I I I I t I 2 4 6 8 10 12 14 16 18, 20

Days

Growth of Porphyridium sp. in sleeves and ponds. (a) Cell number, (b) biomass.

RESULTS

The experiments were carried out during March. The temperatures measured in the morning (08.00), and at noon, and in the afternoon (17.00) were 5-8°C higher in the sleeves than in the ponds (Table 1 ). The light intensity in the sleeves and ponds was similar at noon but higher in the sleeves in the morning and afternoon (Table 2).

A closed system ~or outdoor cuhivation o/Porphyridium 63

E

0 v

~ 1 0 .Q E

E o~ E

E 0

rn

Ca)

(b) S l e e v e s

S l e e v e s

Ponds

P o n d s

Fig. 2.

1 I I I I I I I I I I 0 2 4 6 8 10 12 14 16 18 2 0

Days

Growth of Porphyridium aerugineurn in sleeves and ponds. (a) Cell number, (b) biomass.

Cell number and biomass of Porphyridium sp. and P. aerugineum grown in polyethylene sleeves and in ponds are presented in Figs 1 and 2. Porphyridium sp. grown in the ponds remained viable for only 13 days. After this time the culture became contaminated by dinoflagellates, and cell numbers started to decrease. No contamination by dinoflagel- lates was detected in the culture growing in polyethylene sleeves. For both algal species maximum cell number was higher in the sleeves than in the ponds, i.e. 130 and 50% higher for Porphyridium sp. and P. aerugineum, respectively (Figs 1 (a) and 2(a)). The difference between the sleeves and the ponds in terms of biomass was even higher, i.e. 300% higher in the sleeves for Porphyridium sp. growing for 13 days and 60% higher for P. aerugineum growing for 21 days (Figs l(b) and 2(b), respectively). Similar differences were found for total polysaccharide production (Fig. 3). The high concentration of total polysaccharide in the two cultures was the result of a significant increase in the bound and soluble polysaccharide (Figs 4 and 5). The high concentration of bound polysaccharide also contributed to the increase in the biomass. Since the ponds were contaminated on day 14, cell number and biomass could not

64 E. Cohen, S. Arad

be measured (Fig. l(a), (b); however, polysaccharide was measured until day 17 (Figs 3(a), 4(a) and 5(a)).

DISCUSSION

The results obtained in this study illustrate the potential of the poly- ethylene sleeve for outdoor algae cultivation. The experiments were carried out in March but similar results were obtained in repeated exper- iments carried out during the summer. Both growth and biomass produc- tion were higher in the sleeves than in open ponds. The increase in polysaccharide production was greater than can be accounted for by the rise in cell number. The conditions in the sleeves seem specifically favor- able for polysaccharide production.

Growth conditions in the sleeves, which are relatively small units, seem advantageous for photosynthetic micro-organisms for which light becomes limiting in a large body of water. The fact that the sleeves are

Fig. 3.

E

E

" 0

t - u

0 a .

I - .

2 - (a) /

(b)

m ~ ~

I I I I I I I I I I O 2 4 6 8 10 12 14 16 18 20

Days

Total polysaccharide production of (a) Porphyridium sp. and (b) Porphyridium aerugineum grown in sleeves and ponds.

A closed system for outdoor cultivation of Porphyridium 6 5

hung vertically, as opposed to horizontally, also results in efficient light harvesting. The integral sum of light absorbed throughout the day is currently under study. The subdivision of the culture into units of small volume has other advantages: the culture temperatures are more favor- able, and in the summer the temperature can be prevented from rising above 30°C by water spraying with an automatic device. The turbulence is more efficient than that effected by the paddle wheel of a pond, and large-scale collapse of the culture is obviously prevented. Although production in the sleeves is higher, we cannot yet pin-point the exact factors conferring these advantages.

In addition, it seems to us that the material used for constructing the sleeves - - polyethylene - - is inexpensive, and preliminary observation indicates that they can be continuously used for a number of runs. Since they are relatively inexpensive, their replacement a few times a year seems rather simple.

The results obtained in this study show the potential of such a system. However, in order to develop it for a large-scale system outdoors further

Fig. 4.

1-2

1.0

0-8

-~ 0.6 E

~ 0.4

0-2 "E tO

u

~ 2.e

°2 .4 "O

g 2.o 0

r n

1.6

(a)

(b)

1.2

0-8

0.2 0 2 4 6 8 10 12 14 16 18 20

Days Bound polysaccharide production of (a) Porphyridium sp. and (b) Porphyri-

dium aerugineum grown in sleeves and ponds.

66 E. Cohen, S. Arad

Fig. 5.

0.8

0.6,

E ~o.4 E

"Z 0.2 t -

o e~

"o 0.~ I J >

~ 0.4

0.3

(a)

(b}

0 . 2 ~ 0.1

[ l I I I i I l I I

0 2 4 6 8 10 12 14 16 18 20 Days

Dissolved polysaccharide production of (a) Porphyridium sp. and (b) Porphyri- dium aerugineum grown in sleeves and ponds.

study for longer periods and on a larger scale is required. Scaling up of the system for outdoor cultivation is now under way.

A C K N O W L E D G M E N T

The authors wish to thank Ms L. Uziel and Mr R. Gonen for their devoted technical assistance, and Ms I. Mureinik and Ms D. Imber for in-depth discussions and for styling of the manuscript.

R E F E R E N C E S

1. Laws, E. A., Taguchi, S., Hirrato, J. & Pang, L., High algal production rates achieved in a shallow outdoor flume. Biotech. Bioeng., 28 (1986) 191-7.

2. Goldman, J. C., Outdoor algal mass cultures. I. Applications. Water Res., 13 (1979) 1-19.

3. Powles, B. S., Photoinhibition of photosynthesis induced by visible light. Ann. Rev. Plant Physiol., 35 (1984) 15-44.

A closed system for outdoor cultivation of Porphyridium 67

4. Richmond, A. E., Microalgaculture. CRC Critical Reviews in BiotechnoL, 4 (1986) 369-438.

5. Canter, H. M. &Lund, J. W. G., The importance of protozoa in controlling the abundance of planctonic algae in lakes. Proc. Limn. Soc., Lond., 179 (1968) 203-7.

6. Chaumont, D., Thepenier, C., Gudin, C. & Junjas, C., Scaling up a tubular photoreactor for continuous culture of Porphyridiurn cruentum from laboratory to pilot plant (1981-1987). In Algal Biotechnology, ed. T. Stadler, J. Mollion, M.-C. Verdus, Y. Karamanos, H. Morvan & D. Christiaen. Elsevier Applied Science, London, 1988, pp. 199-208.

7. Dicorato, A., Coltivazione sperimentals in sistema tubolare in CNR ed. Prospective della coltura di Spirulina in Italia, Florenz, Italy, 1980, p. 261.

8. Torzillo, G., Pushparaj, B., Bocci, E, Balloni, W., Materassi, R. & Floren- zano, G., Production of Spirulina biomass in closed photobioreactors. Biomass, 11 (1986) 61-74. Anderson, D. B. & Eakin, D. E., A process for the production of poly- saccharides from microalgae. Biotechnol. Bioeng. Syrup., 5 (1985) 533-47. Trotta, E, A simple and inexpensive system for continuous monoxenic mass culture of marine microalgae. Aquaculture, 22 (1980) 383-7. Jones, R. E, Speer, H. L. & Kury, W., Studies on the growth of the red alga Porphyridium cruentum. Physiol. Plant., 16 (1963) 636-43. Ramus, J., The production of extracellular polysaccharide by the uni- cellular red algae. J. Phycol., 8 (1972) 97-111. Adda, M., Merchuk, J. G. & Arad (Malis) S., Effect of nitrate on growth and production of cell wall polysaccharides by the unicellular red alga Porphyri- dium. Biornass, 10 (1986) 131-40.

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