Large-Scale Algal Culture Systems: The Next Generation

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Michael A. Borowitzka, Algal Biotechnology Lab, School of Biological & Environmental Sciences, Murdoch University, Perth, W.A. 6150

The large scale commercial culture of microalgae for the production of health foods, for aquaculture feeds and for high value fine chemicals such as iS-carotene is a well established biotechnology. Other microalgal products close to commercialisation are astaxanthin (Borowitzka, M.A., 1992b) and essential long chain polyunsaturated fatty acids such as docosahexaenoic acid (Martek, USA - pers. com). The discovery of a range of unique biologically active substances with anti-neoplastic, anti-viral and anti-fungal activity produced by several microalgae, especially cyanobacteria, provides a further range of poten­tially valuable algal products (Knubel et al., 1990; Patterson et al., 1991, 1993) and studies to optimise production of these are already under way (e.g. Patterson & Bolis, 1993). Furthermore, progress is being made in genetic engineering of microalgae (Blankenship & Kindle, 1992; Davies et al., 1992; Gunson et al., 1993) and in the next few years it is likely that transgenic algae will also be used to produce selected chemicals.

In order to be able to commercialise these products new and more effective large-scale algal culture sys­tems are needed. The design of algal culture systems requires consideration of (a) light availability to the cells, (b) optimum turbulence (mixing), (c) C0

2-sup­

ply, (d) temperature control, and of course (e) capital and operating costs.

There are three types of large scale algal culture systems in use for comm:ercialmicroalgal culture at present. These are: (a) the very large (several hec­tares), shallow, unstirred ponds used in Australia by Western Biotechnology Ltd and Betatene Ltd for the culture of Dunaliella salina and in Mexico for the culture of Spirulina (Borowitzka, L.J. & Borowitzka, 1989b); (b) the circular stirred ponds (up to approx 500 m2) used in Taiwan for the culture of Chiarella; and (c) paddle-wheel driven raceway ponds (up to approx 1000 m2) used for the culture of Spirulina, Chiarella and D. salina in Thailand, Taiwan, China, the USA and Israel. Each of these systems has specific advantages, some of which only become apparent when the whole process is considered (Borowitzka, M.A., 1992a) and some of which are site-specific. However, since these are open-air systems their ap­plicability is generally limited to algae which live under very selective conditions such as high salinity, high pH or high nutrient conditions. There is also little scope for controlling the culture conditions so as to optimise cell and product yields. All of these systems also achieve only low cell densities of gener-

Australasian Biotechnology 4, 212-215 (1994)

ally < 1 g dry weight.I-1, which means that harvesting

costs are high.

Further significant development of new algal species and products, and also new geographical locations for algal culture requires new, cost effective closed culture systems. In the last few years a major effort has been undertaken to develop such systems and these include illuminated fermenters with either in­ternal or external light sources (Burgess et al., 1993), alveolar panels (Tredici & Materassi, 1992) and a variety of tubular photobioreactors (Borowitzka, L.J. & Borowitzka, 1989a). Although illuminated fermenters are very effective and high growth rates and culture densities can be achieved, the high capi­tal and operation costs limit their applicability for commercial culture to all but a very small number of high value products such as pharmaceuticals. The production of lower value products such as algae for health foodor animal feed, the production of carotenoids (i.e. astaxanthin), fatty acids (e.g. eicosapentaenoic acid & docosahexaenoic acid), polysaccharides etc. requires that the culture system be much cheaper.

Of the above systems, the tubular photobioreactors show the most potential. These reactors basically consist of long transparent tubes made of glass, polyacrilamide or PVC, some sort of pumping de­vice (e.g. centrifugal pump, diaphragm pump, lobe pump, air-lift), a means to remove 0

2 and add C02,

and a heat exchanger for temperature control. The tube diameter in these systems has varied from about 15 em to about 25 mm, and a number of large systems with volumes of up to 14,000 L have been operated very successfully in France, UK, Israel and Australia with a range of algae including Spirulina, Haematococcus, Porphyridium, Chiarella, Phaeodactylum, Tetraselmis, Anabaena and Isochrysis (Chaumont et al., 1988; Borowitzka, L.J. & Borowitzka, 1989a; Tredici &Materassi,1992;Richmondetai.,1993;Chrismadha & Borowitzka, 1994). Other species have been grown at laboratory scale and the Australian navy is experi­menting with such a system as a C02-scrubber for submarines. The tubes have been arranged horizon­tally on the ground, vertically in parallel arrays and wound helically around a frame. We have been working with the latter system (the BiocoiP system). The helical arrangement has several advantages; a very long total tube length can be arranged in a stable structure which occupies relatively little land while maximising the amount of light incident upon the tube and the helical arrangement generates a non­laminar flow pattern within the tube, giving better

'The design of the Biocoil system is patented by Biotechna Ltd, London (%%2758(Robinson et al., 1988)%%)

A U S T R A L A 6 I 4 N -

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Table 1. Comparison of the characteristics of different types of large-scale microalgal culture systems.

1 9 9 2 Chanawongse et al., 1994). Despite these limitations we have achieved excellent growth rates at high cell densities in this type of reactor both indoors and out­doors for periods of several months. Since this system is closed, it also has the advantage of good control of all culture variables including light and temperature, so that an algal prod­uct of uniform quality can be pro­duced.

Reactor Mixing Light Tempera- Gas Hydro- Species Scale-up Type utilisation ture Transfer dynamic Control

efiiciency control Stress

Unstirred Very Poor None Poor Very Low Difficult Very ShaUow poor difficult Ponds

ShaUow ExceUent ExceUent None High Low- Difficult Very Cascade High Difficult System

Paddle- Fair- Fair- None Poor Low Difficult Very Wheel .Good Good difficult Raceway Ponds

Stirred Largely Fair- ExceUent Low- lligh Easy Difficult Tank Uniform Good High I

Reactor I I

Air-Lift GeneraUy Good ExceUent High Low Easy Difficult !

Reactor Uniform '

Tubular Uniform ExceUent ExceUent Low- Low- Easy Easy Reactor High· High" :

(Biocoil-type)

Figure 1 illustrates the effects of cell

"Gas transfer efficiency depends on the design of the gas exchange system, and hydrodynamic stress is a function of the pumping system used to circulate the culture.

density and irradi­ance on the maximum biomass achieved in a semi­continuous culture of the marine diatom, Phaeodactylum tricornutum, grown in a 30 1 tubular photobioreactor in the laboratory. Increasing irradi­ance increases the maximum cell number achieved, and the addition of 5% C0

2 in air greatly increases

the biomass (ash free dry weight) due to an increase in cell size as well as the maximum cell number. However at high cell densities of about 15 x 106

cells.ml·l the cultures are no longer light limited and increasing the irradiance from 286 to 1712 J.!Einsteins.m·2 .sec·1 no longer results in an increase in biomass. Figure 2 shows the effects of these culture conditions on the productivity of eicosapentaenoic acid (EPA) in mg.l·1.day·1 in the same culture. Maxi­mum productivity of EPA occurs at a cell density of 8 x 106 cells.ml·1 and an irradiance of 286 J.!Einsteins. m·2.sec·1 with 5% C0

2 added. These results show how

the yield and productivity of a desires algal product can be optimised in a closed bioreactor by changing culture conditions. Such optimisation is not easily done in open large scale culture systems.

mixing than long straight tubes. Furthermore, the helical reactor has no suddenchanges in flow direc­tion which can give problems with algal accumula­tion in other arrangements. Through the use of small diameter tubing (about 25-60 mm i.d.) and appropri­ate flow rates the adhesion of the algae to the tube wall can be minimised or eliminated, thus ensuring that all cells receive the maximum amount of light available to the algae.

Table 1 compares some of the characteristics of tubu­lar photobioreactors with other algal culture sys­tems, and this table clearly shows the advantages of the Biocoil system over other kinds of algal culture systems.

The main limitations of tubular photobioreactors, such as the Biocoil system, relate to the pumps used to circulate the culture and to the efficiency of gas exchange. A wide range of pump types have been used in tubular reactors and no one type of pump has yet been shown . to be consistently better than an­other. The pump is the main source of shear in the system leading to cell damage and, ultimately, death (Gudin & Chaumont, 1991) . Cell growth in tubular reactors is also carbon limited and 0

2 build-up in the

long tubes may also lead to photorespiration and a reduction in growth. An efficient gas exchange sys­tem is therefore essential and some further design modifications may be necessary to minimise02 build­up . Alternatively, algal strains resistant to photorespiration could be used (Vonshak & Guy,

The microalgae are an extremely diverse group of organisms and this diversity is also expressed in the range of biomolecules produced by these algae. The potential of these organisms is still largely untapped and only a very small number of the available species has been screened so far for valuable compounds. The development of new culture systems means that the opportunity now exists to exploit the potential of these organisms.

VOLUME 4•NUMBER 4•AUGUST 1994 II

SPECIAL FEATURE • MARINE BIOTECHNOLOGY

Cell Number (x1QA6 Cells/ml)

18 lrradiance

(1JEifmA2fsec)

Borowitzka, M.A. (1992b) Compar­ing carotenogenesis in Dunaliella and Haematococcus: Implications for com­mercial production strategies. IN "Profiles on Biotechnology" edited by Villa, T.G., & Abalde, J. (Universidade de Santiago de Compostela: Santiago de Compostela) pages 301-310.

Burgess, J.G., Iwamoto, K., Miura, Y., Takano, H., & Matsunaga, T. (1993) An Optical fibre photobioreactor for enhanced pro­duction of the marine unicellular Alga Isochrysis aff. galbana T-Iso (UTEX-LB-2307) rich in docosahexaenoic acid. Appl. Microbial. Biotech., 39, 456-459

Figure 1. Changes in biomass (Ash Free Dry Weight in g.P) of Phaeodactylum tricornutum (strain MUR-136) grown in semi-continuous culture in a 30 l helical tubular photobioreactor at a range of cell densities (x106 cells.mP) and irradiances (Einsteins.m·2sec· 1). Culture conditions were: f/2 medium (Guillard & Ryther, 1962) with 1.764 mM nitrate

and 0.072 mM phosphate, pH 9-10, 14-18•C, with illumination provided by cool-white fluorescent lamps or a 1000-watt halogen lamp. The irradiance was measured at the surface of the reactor tubes. At the highest irradiances 5% C0

2 in air was added to the culture. The

analytical methods are described in (Chrismadha & Borowitzka, 1994).

Chanawongse, L., Tanticharoen, M., Bunnag, B., & Vonshak, A. (1994) Photoinhibition in Spirulina and its recovery. IN "Algal Biotechnology in the Asia-Pacific Region" edited by Phang, S.M., Lee, K., Borowitzka, M.A., & Whitton, B. (Institute of Ad­vanced Studies, University of Ma­laya: Kuala Lumpur) pages 118-121.

Chaumont, D., Thepenier, C., Gudin, C., & Junjas, C. (1988) Scaling up a tubular photoreactor for continuous Acknowlegements

The significant contribution of my collaborators, C. Chrismadha and T. Simpson, inthe development of tubular photobioreactors is gratefully acknowledged.

References Blankenship, J.E., & Kindle, K.L. (1992) Expression of chi­meric genes by the light-regulated cabll-1 promoter in Chlamydomonas reinhardtii- A cabll-1 I nitl gene functions as a dominant selectable marker in a nitl- nit2 strain. Molecular and Cellular Biology, 12, 5268-5279

Borowitzka, L.J., & Borowitzka, M.A. (1989a) Industrial production: meth­ods and economics. IN "Algal and Cyanobacterial Biotechnology" edited by Cresswell, R.C., Rees, T.A.V., & Shah, N. (Longman Scientific: Lon­don) pages 294-316.

Borowitzka, L.J., & Borowitzka, M.A. (1989b) -Carotene (Provitamin A) pro­duction with algae. IN "Biotechnology of Vitamins, Pigments and Growth Factors" edited by Vandamme, E.J. (Elsevier Applied Science: London) pages 15-26.

culture of Porphyridium cruentum from laboratory to pilot plant (1981 - 1987). IN "Algal Biotechnology" edited by Stadler, T.,Mollion,J., Verdus,M.C.,Karamanos, Y.,Morvan, H., & Christiaen, D. (Elsevier Applied Science: London) pages 199-208.

Chrismadha, T., & Borowitzka, M.A. (1994) Effect of cell density and irradiance on growth, proximate composition and eicosapentaenoic acid production of Phaeodactylum

10 11 12 Borowitzka, M.A. (1992a) Algal

biotechnology products and proc­esses: Matching science and econom­ics. f. Appl. Phycol., 4, 267-279

Cell Number 13 14 15 (X1QA6 Cells/ml) 16 17

18 lrradiance

(1JEi/mA2fsec)

Figure 2. Eicosapentaenoic acid (EPA) productivity in mg.l·1 .day·1 of the culture of Phaeodactylum tricornutum shown in Figure 1.

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tricornutum grown in a tubular photobioreactor. J. Appl. Phycol., 6, 67-74

Davies, J.P., Weeks, D.P., & Grossman, A.R. (1992) Expres­sion of the arylsulfatase gene from the beta2-tubulin pro- · rooter in Chlamydomonas reinhardtii. Nucl. Acids Res., 20, 2959-2965

Gudin, C., & Chaumont, D. (1991) Cell fragility- the key problem of microalgae mass production in closed photobioreactors. Bioresource Techno/., 38, 145-151

Guillard, R.R.L., & Ryther, J.H. (1962) Studies on marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can. J. Microbial., 8, 229-239

Gunson, C.S., Borowitzka, M.A., & Jones, M.G.K. (1993) Genetic transformation of the green alga Haematococcus pluvial is using a -glucuronidase reporter gene construct. IN "11th Australian Biotechnology Conference" edited by Sargeant, J., Washer, S., Jones, M., & Borowitzka, M. (Aus­tralian Biotechnology Association: Perth) pages 118-119.

Knubel, G., Larsen, L.K., Moore, R.E., Levine, LA., & Patterson, G.M.L. (1990) Cytotoxic, antiviralindolocarbazoles from a blue-green alga belonging to the Nostocaceae. J. Antibiot., 43, 1236-1239

Patterson, G.M.L., & Bolis, C.M. (1993) Regulation of scytophycin accumulation in cultures of Scytonema

oce/latum.1. Physical factors. Appl. Microbial. Biotech., 40, 375-381

Patterson, G.M.L., Baldwin, C.L., Bolis, C.M., Caplan, F.R., Karuso, H., Larsen, L.K., Levine, I.A., Moore, R.E., Nelson, C.S., Tschappat, K.D., Tuang, G. D., Furusawa, E., Furusawa, S., Norton, T.R., & Raybourne, R.B. (1991) Antineoplastic activity of cultured blue-green algae (Cyanophyta). J. Phycol., 27,530-536

Patterson, G.M.L., Baker, K.K., Baldwin, C.L., Bolis, C.M., Caplan, F.R., Larsen, L.K., Levine, I.A., Moore, R.E., Nelson, C.S., Tschappat, K.D., Tuang, G.D., Boyd, M.R., Cardellina, J.H., Collins, R.P., Gustafson, K.R., Snader, K.M., Weislow, O.S., & Lewin, R.A. (1993) Antiviral activity of cultured blue-green algae (Cyanophyta). J. Phycol., 29, 125-130

Richmond, A., Boussiba, S., Vonshak, A., & Kopel, R. (1993) A new tubular reactor for mass production of microalgae outdoors. J. Appl. Phycol., 5, 327-332

Tredici, M.R., & Materassi, R. (1992) From open ponds to vertical alveolar panels- The Italian experience in the devel­opment of reactors for the mass cultivation of phototrophic microorganisms. J. Appl. Phycol., 4, 221-231

Vonshak, A., & Guy, R. (1992) Photoadaptation, photoinhibition and productivity in the blue-green alga, Spirulina platensis grown outdoors. Pl. Cell Env., 15,613-616

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VOLUME 4•NUMBER 4•AUGUST 1994 II