Cultivation Systems

Many cultivation systems for microalgae have been designed and built. The choice of a suitable cultivation system and the adjustment of the cultivation regime must be worked out for each individual productive strain. In every cultivation system, several basic features must be considered: illumination, circulation, and gas exchange (supply of CO2 and O2 degassing).

Two basic approaches to microalgal mass production are used: the first applies to cultivation in open reservoirs large in area, while the second represents closed vessels - photobioreactors or fermentors. In this article, the term photobioreactor is used for closed or semiclosed systems using natural or artificial illumination. Generally, production from an open-pond culture is cheaper than from a culture in closed photobioreac-tors, but the use of the open pond is limited to a relatively small number of microalgal species. From a commercial point of view, the price of the final product is crucial.

Laboratory Cultivation

The simplest cultivation vessel is a flask containing the microalgal suspension placed on a shaker with illumination. In this way, stock cultures of microalgae are usually maintained. Glass cylinders or flat flasks kept in a temperature-controlled water bath and bubbled with a mixture of air with CO2 are commonly used to cultivate microalgae and cyanobacteria in small volumes up to 11 (Figure 1).

The outdoor culture is scaled up in stepwise fashion, starting with the laboratory culture in an approximately 1:5 dilution ratio. It is advisable not to expose diluted laboratory cultures outdoors to full sunlight during the first few days, in order to avoid the risk of photooxida-tion. However, a minimum biomass concentration corresponding to about 10 mg chlorophyll per liter is recommended.

Open Outdoor Systems

Natural or artificial ponds, raceways (ponds akin to racetracks), and cascades (i.e., inclined-surface systems) represent open cultivation systems for microalgae. All of them require a large area of anything from hundreds of square meters to hundreds of hectares. An overview of several open culture systems used for mass cultures of microalgae outdoors is presented in Figure 2.

Due to the limited control of cultivation conditions, open systems are suitable for microalgal strains that grow rapidly, or under very selective conditions. Numerous variations of open ponds are used: according

Figure 1 Laboratory cultivation using glass cylinders (i.d. 30 mm, volume 0.31) placed in a temperature-controlled waterbath with backside illumination from fluorescent lamps (Institute of Microbiology, Academy of Sciences, Trebon, Czech Republic). Mixing of the microalgal suspension is maintained by bubbling through a mixture of air + 2% CO2.

Figure 1 Laboratory cultivation using glass cylinders (i.d. 30 mm, volume 0.31) placed in a temperature-controlled waterbath with backside illumination from fluorescent lamps (Institute of Microbiology, Academy of Sciences, Trebon, Czech Republic). Mixing of the microalgal suspension is maintained by bubbling through a mixture of air + 2% CO2.

to local requirements, climate conditions, and materials available (concrete, polyvinyl chloride (PVC), fiberglass). Artificial shallow ponds of 50 ha are used for the cultivation of the halotolerant microalga Dunaliella in Western Australia to produce ^-carotene. Productivity in these ponds is very low (^1g dry weight m-2 day-1) due to the lack of stirring mechanisms and CO2 supply. Raceways, tanks, and circular ponds are mixed by impellers, rotating arms, paddle wheels, or by the stream of CO2-enriched air supplied into the culture (Figures 2a and 2b). The culture depth can vary between 10 and 30 cm. The cultures are usually operated at a biomass concentration ranging between 0.3 and 0.6 gl- depending on the culture depth. Outdoor cultures are considered a photo-limited system as they are operated at the optimal concentration rather than at the maximum growth rate. Open ponds are preferentially used for Spirulina and Chlorella production in Japan, Thailand, California, Hawaii, Taiwan, India, and China.

In inclined-surface systems, the microalgal suspension flows over sloping planes arranged in cascades, in such a way that the layer thickness below 1 cm and turbulent flow prevent self-shading (Figure 2c). Due to a very short optical path, high densities of biomass between 25 and 35 gl- can be operated in these units. High productivities over 40 g dry matter m- day- can be achieved in cascade cultivation units, even in moderate climate zones. Yet, up till now, the system has not been fully scaled up, due to its higher construction costs compared to traditional ponds.

Closed and Semiclosed Outdoor Photobioreactors

Closed photobioreactors are flexible systems that can be optimized according to the biological and physiological characteristics of the microalgal species involved. An overview of several closed or semiclosed culture systems used for mass cultures of microalgae outdoors is presented in Figure 3. Schematic diagram of a closed photobio-reactor is illustrated in Figure 4. Compared to open systems, photobioreactors have a number of advantages, namely reproducible cultivation conditions with regard to environmental influence; reduced risk of contamination; possibility of temperature regulation; low CO2 losses; and smaller area requirements. On the downside, closed systems are more difficult to clean, the tube material might partially decrease sunlight penetration, and the system must be effectively cooled and degassed since excessive oxygen produced by the growing cultures can reduce growth. Furthermore, the cost of construction is about 1 order of magnitude higher than that of open ponds (about 100 US$ m-2).

Figure 2 Examples of experimental, open outdoor systems for cultivation of microalgae, which can be scaled up to large production facilities (100001). (a) Raceway pond with a paddle-wheel mixer (25001) and (b) circular pond with a rotating arm (1001) at the Institute for Ecosystem Study of the CNR (Florence, Italy); (c) an inclined-surface system of sloping planes arranged in cascades (three modules of 22001 each) at the Institute of Microbiology, Academy of Sciences (Trebon, Czech Republic).

Figure 2 Examples of experimental, open outdoor systems for cultivation of microalgae, which can be scaled up to large production facilities (100001). (a) Raceway pond with a paddle-wheel mixer (25001) and (b) circular pond with a rotating arm (1001) at the Institute for Ecosystem Study of the CNR (Florence, Italy); (c) an inclined-surface system of sloping planes arranged in cascades (three modules of 22001 each) at the Institute of Microbiology, Academy of Sciences (Trebon, Czech Republic).

Plexiglas, acrylic, and glass tubes and flexible plastic coils can act as solar collectors - the microalgal suspension being circulated continuously through rows of connected transparent tubes or flexible coils. Here, a much greater biomass density can be maintained than in open ponds. Outdoor photobioreactors for commercial production are designed as modules.

Generally, the tubes are positioned horizontally or vertically, arranged as a serpentine loop or manifold rows. The culture suspension is circulated by a pump -or more preferably by air-lifting (injecting a stream of compressed air in upward-pointing tube). Peristaltic and membrane pumps are physically more 'friendly' to cells than centrifugal pumps, which cause high sheer stress. The cooling is maintained by submerging the tubes in a pool of water, by heat exchangers, or by spraying water on the tube surface. A further improvement is represented by the two-plane horizontal tubular photobioreactor built in Florence, Italy, which led to a high Spirulina productivity of 30 g dry weight m~2day_1 (Figure 3a). The biggest production plant to date has been built in a greenhouse in Klotze, Germany, with a volume of 700 m3 (20 modules of 35 m3). Horizontal glass tubes are arranged in a vertical fence-like system in order to utilize diffuse light (Figure 3b).

Helical tubular system, commonly called 'biocoils', seem to be another alternative for microalgae cultivation (see diagram in Figure 4). It consists of coiled, flexible, transparent tubes (3-6 cm in diameter) around an open cylindrical frame realized by the company Addavita Ltd. in UK. In Australia, 1001 laboratory photobioreactors have been scaled up to 10001 outdoor pilot plants.

Vertical-column photobioreactors are relatively simple systems in which mixing is achieved by air + CO2 bubbling from the bottom. A variation of this column system are annular photobioreactors, which consist of two glass or Plexiglas cylinders of different diameters

Figure 3 Examples of experimental, closed or semiclosed, photobioreactors for cultivation of microalgae, which can be scaled up to large production facilities. (a) A two-plane, horizontal tubular photobioreactor (Institute for Ecosystem Study of the CNR, Florence, Italy); (b) horizontal glass tubes arranged in a vertical fence-like photobioreactor mounted in a greenhouse (volume 700 m3) (Institut fur Getreideverarbeitung, Potsdam-Rehbrucke, Germany); (c) vertical-panel photobioreactors arranged in a series (Institut fur Getreideverarbeitung, Potsdam-Rehbrucke, Germany); (d) annular column photobioreactor (1001) consisting of two polyacrylate cylinders placed one inside the other to form the culture chamber (Institute of Physical Biology, University of South Bohemia, Nove Hrady, Czech Republic).

Figure 3 Examples of experimental, closed or semiclosed, photobioreactors for cultivation of microalgae, which can be scaled up to large production facilities. (a) A two-plane, horizontal tubular photobioreactor (Institute for Ecosystem Study of the CNR, Florence, Italy); (b) horizontal glass tubes arranged in a vertical fence-like photobioreactor mounted in a greenhouse (volume 700 m3) (Institut fur Getreideverarbeitung, Potsdam-Rehbrucke, Germany); (c) vertical-panel photobioreactors arranged in a series (Institut fur Getreideverarbeitung, Potsdam-Rehbrucke, Germany); (d) annular column photobioreactor (1001) consisting of two polyacrylate cylinders placed one inside the other to form the culture chamber (Institute of Physical Biology, University of South Bohemia, Nove Hrady, Czech Republic).

Heat exchanger

Heat exchanger

Pump

Figure 4 Schematic diagram of a photobioreactor. It consists of a photostage loop, heat exchanger, degasser, circulation pump, CO2 supply, and sensors (e.g., pH and oxygen electrodes, and thermometer). Created by PalSek.

Pump

Figure 4 Schematic diagram of a photobioreactor. It consists of a photostage loop, heat exchanger, degasser, circulation pump, CO2 supply, and sensors (e.g., pH and oxygen electrodes, and thermometer). Created by PalSek.

placed one inside the other to form a culture chamber, some 3-5 cm thick and 50-1501 in volume (Figure 3d). Illumination is provided by either natural or artificial light. In column photobioreactors, sensitive strains with fragile cells or filaments can be grown as the culture mixing is very gentle (e.g., Nostoc, Microcystis, Nannochloropsis).

Flat-panel photobioreactors with a short light path, with horizontal, inclined, or vertical orientation, have a high surface-to-volume ratio and give high volumetric productivities. These photobioreactors are made from plexiglass or polycarbonate alveolar sheets, 2-4 cm thick. In the system of vertical panels, where the flow is horizontal in alveolar channels using pumps, the panels are packed 20 cm apart in series (Figure 3c). Compared with tubular systems, flat-panel photobioreactors have several disadvantages: in temperature control, oxygen degassing, and fouling due to the lack of turbulent flow in the narrow, rectangular channels.

A similar principle of cultivation has been used in panel-type photobioreactors developed as flat-glass chambers connected in a series of plate-type photobio-reactors, vertical-arranged or inclined to the sun, and mixed by air-bubbling from the bottom.

Despite the higher yields attainable with photobior-eactors, its high construction and maintenance costs still make them uncompetitive for industrial production of microalgal biomass. Their use can be foreseen for the production of high-value bioactive substances, which require the adoption of sterile conditions.

Heterotrophic Fermentors

Nonilluminated heterotrophic fermentors for the cultivation of microalgae have gained in industrial importance since some microalgal strains can be grown heterotrophi-cally (e.g., Chlorella, Chlamydomonas, Phaeodactylum, and Haematococcus). Commercial fermentors come in a wide range of sizes: from 10 to 100 0001. The photobioreactors and fermentors have many features in common: pH and temperature control, harvesting, mixing, etc. The significant differences between fermentors and photobioreactors are the energy source, oxygen supply, and sterility. When grown heterotrophically, microalgal cultures utilize an organic compound (e.g., glucose or acetate) as both the carbon and energy source for growth. The crucial requirement is that the microalgal cultures must be axenic (i.e., free from other organisms) to avoid the growth of contaminants. It is absolutely essential that the fermentor and culture medium are sterilized before inoculation (e.g., by steam). In fermentors, adequate mixing is achieved with impellers and a stream of compressed air as a source of oxygen for the catabolic processes.

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