Biotechnol Lett (2008) 30:1525–1536 DOI 10.1007/s10529-008-9740-3

REVIEW

The technology of microalgal culturing Niels T. Eriksen

Received: 26 March 2008 / Revised: 23 April 2008 / Accepted: 28 April 2008 / Published online: 14 May 2008 Ó Springer Science+Business Media B.V. 2008

Abstract This review outlines the current status and recent developments in the technology of microalgal culturing in enclosed photobioreactors. Light distribution and mixing are the primary variables that affect productivities of photoautotrophic cultures and have strong impacts on photobioreactor designs. Process monitoring and control, physiological engineering, and heterotrophic microalgae are additional aspects of microalgal culturing, which have gained considerable attention in recent years. Keywords Heterotrophic microalgae
Light distribution
Mixing
Monitoring and control
Photobioreactors
Physiological engineering

Introduction Microalgal bioreactors are often designed differently from bioreactors used to grow other microorganisms. This is because most microalgae are photoautotrophs and depend on light as energy source. Supply, distribution and utilisation of light in microalgal cultures are therefore central aspects, which receive

N. T. Eriksen (&) Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, 9000 Aalborg, Denmark e-mail: [email protected]

particular attention in the design of photobioreactors. Mixing, process monitoring and control, and exploration of heterotrophic and recombinant microalgae are other aspects of microalgal culturing that have seen novel developments in recent years. Microalgae are presently used in foods and health foods, as aquaculture feeds, and for production of pigments, polyunsaturated fatty acids and other fine chemicals (Spolaore et al. 2006). Today microalgal biodiesel (Chisti 2007, 2008) and biohydrogen (Akkerman et al. 2002) production, and CO2 removal from flue gas (Vunjak-Novakovic et al. 2005; Doucha et al. 2005; de Morais and Costa 2007) are receiving particular attention. Molecular technologies have improved the performance of recombinant microalgae in photobioreactors (Mussgnug et al. 2007), recombinant products have been synthesised in microalgal cultures (Leo´n-Ban˜ares et al. 2004), and experimental phycology still create novel experimental tools and specialised photobioreactors. The purpose of this review is to outline the current status and recent developments in the technology of microalgal culturing, excluding open pond cultures. Culturing of cyanobacteria and other photoautotrophic prokaryotes is also included.

Design of photobioreactors The productivity of photoautotrophic cultures is primarily limited by the supply of light and suffers

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from low energy conversion efficiencies caused by inhomogeneous distribution of light inside the cultures (Grobbelaar 2000). At culture surfaces, light intensities are high but absorption and scattering result in decreasing light intensities and complex photosynthetic productivity profiles inside the cultures (Ogbonna and Tanaka 2000). High light intensities at culture surfaces may cause photoinhibition, and the efficiency of light energy conversion into biomass, the photosynthetic efficiency (PE) is low. The photosynthetic efficiency increase as light becomes limiting, but the productivity is negatively affected by central, light-deprived zones (Janssen et al. 2000a). Most of the recent research in microalgal culturing has been carried out in photobioreactors with external light supplies, designed as either tubular reactors, flat panel reactors, or column reactors with large surface areas, short internal light paths, and small dark zones (Janssen et al. 2002; Carvalho et al. 2006; Chisti 2006). Tubular photobioreactors The largest facilities for growing photoautotrophic cells in enclosed reactors, for example the 25 m3 reactors at Mera Pharmaceuticals, Hawaii (Olaizola 2003) and the 700 m3 plant in Klo¨tze, Germany (Pulz 2001; Janssen et al. 2002; Spolaore et al. 2006) are based on tubular reactors. In tubular photobioreactors, the cultures are pumped through long, transparent tubes. The tubes are organised horizontally (Molina et al. 2001; Carlozzi et al. 2006), vertically (Carlozzi 2000; Converti et al. 2006; Perner-Nochta et al. 2007), inclined (VunjakNovakovic et al. 2005), or as a helix (Hai et al. 2000; Travieso et al. 2001; Scragg et al. 2002; Fernande´z et al. 2003; Hall et al. 2003). Mechanical pumps or airlifts create the pumping force. The airlifts also allow CO2 and O2 to be exchanged between the liquid medium and the aeration gas (Molina et al. 2001; Travieso et al. 2001; Fernande´z et al. 2003; Hall et al. 2003; Converti et al. 2006), while almost no gas-exchange takes place in the tubes. Although tubular photobioreactors are often considered the most suitable for commercial largescale cultures of microalgae (Chisti 2006), the length of the tubes are limited by O2 accumulation, CO2 depletion, and pH variations. Tubular photobioreactors therefore cannot be scaled up indefinitely, and

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Biotechnol Lett (2008) 30:1525–1536

large-scale production plants partly rely on multiplication of reactor units (Janssen et al. 2002). Flat panel photobioreactors Flat panel (or flat plate) photobioreactors supports the highest densities of photoautotrophic cells, which can exceed 80 g l-1 (Hu et al. 1998). In these reactors, a thin layer of very dense culture is mixed or flown across a flat panel (Hu et al. 1998; Degen et al. 2001; Richmond et al. 2003), and incoming light is absorbed within the first few millimetres at the top of the culture. Also open, flat panel (or thin-layer) photobioreactors have recently been characterised with respect to growth and CO2 removal by Chlorella sp. (Doucha et al. 2005, Doucha and Lı´vansky´ 2006). Column photobioreactors Column photobioreactors are occasionally stirred tank reactors (Li et al. 2003; Sloth et al. 2006; Sobszuk et al. 2006; Eriksen et al. 2007), but more often bubble columns (Zitelli et al. 2006; Lee et al. 2006; Bosma et al. 2007; de Morais and Costa 2007) or airlifts (Merchuk et al. 2000; Suh and Lee 2001; Krichnavaruk et al. 2007). The columns are placed vertically, aerated from below, and illuminated through transparent walls. Light sources can also be installed internally (Cso¨go¨r et al. 2001; Suh and Lee 2001). Column bioreactors offer the most efficient mixing, the highest volumetric gas transfer rates, and the best controllable growth conditions. Experimental photobioreactors are often designed as columns. Miro´n et al. (1999) and Zitelli et al. (2006) have also argued that multiplication of vertical column bioreactors is a suitable strategy for scale-up of microalgal cultures. Photobioreactor productivity Table 1 summarises biomass productivities measured in various types photobioreactors in recent studies. Tubular reactors, flat panel reactors, and column reactors can all provide productivities of 20–40 g m-2 day-1 and PE as high as 5–9%. Highest productivities are obtained at high light intensities. The PE is maximal at low light intensities. In outdoor cultures, highest PE is seen in the morning and in the afternoon (Carlozzi et al. 2006). Alternative

1,126

2,690

Sun

Sun

–

Dome/sun

–

–

9.6

6.4

9.4

50.5

130

70

120

400

400

200

200

146

5.5

Volume l

Chlorococcum

Chlorococcum

Tetraselmis

Chlorella

Chlorella

Phaeodactylum

Phaeodactylum

Arthrospira

Spirulina

Algal strain

1–2

1–2

1.7

–

–

4.1

2.29

2.37

–

x g l-1

0.1

0.09

0.42

3.2

3.8

1.52

1.15

1.15

0.42

Pvolume g l-1 day-1

11.0

14.9

38.2

19.4

22.8

25.3

19.1

25.4

Parea g m-2 day-1

Open thin-layer culture

Calculated using a specific energy content of biomass of 25 kJ g-1 (Degen et al. 2001)

b

c

Calculated from PFFD using a value of 18.78 kJ lmol-1 photons (Converti et al. 2006)

a

References: A, Converti et al. (2006); B, Carlozzi (2000); C, Molina Grima et al. (2001); D, Doucha et al. (2005); E, Zitelli et al. (2006); F, Sato et al. (2006)

–

–

5

0.6

0.6

6

6

21.2a a

10

1.2

Light path cm

13.5

–

E MJ m-2 day-1

Values in italics are based on information in the references

–

Parabola/sun

Sun Alternative reactor designs

ca. 1,000

–

Sunc

Column photobioreactors

–

Sunc

Flat panel photobioreactors

80

–

Artificial

PFFD lmol m-2 s-1

Sun

Tubular photobioreactors

Reactor type light source

–

–

9.6

6.9

5.6

1.3b

2.3b

F

F

E

D

D

C

C

A B

4.7b

Ref.

8.1

PE %

Table 1 Biomass densities (x), volumetric and areal productivities (Pvolume and Parea), and photosynthetic efficiencies (PE) of selected photoautotrophic cultures grown in different types of enclosed photobioreactors and under various photosynthetic photon flux densities (PFFD) or light energy supplies (E)

Biotechnol Lett (2008) 30:1525–1536 1527

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photobioreactor configurations have also been tested (Sato et al. 2006) but not resulted in improved productivities.

Distribution of light inside photobioreactors Cells in photobioreactors are exposed to high light intensities close to reactor walls while central parts are dark. Currents in the liquid medium move the cells through the differently illuminated zones and individual cells experience fluctuating light regimens (see e.g. Richmond 2000). The light regimen itself is influenced by incident light intensity, reactor design and dimension, cell density, pigmentation of the cells, mixing pattern, and more. In outdoor photobioreactors the light regimen is also influenced by geographical location, time of the day, and weather conditions. Short light paths keep the dark zones at a minimum and are obtained in flat panel photobioreactors and tubular photobioreactors constructed of narrow, transparent pipes. In vertical column photobioreactors, it is more difficult to keep the dark zones small and at the same time maintain a large reactor surface for light collection. Zitelli et al. (2006) solved this dilemma by replacing an otherwise dark, unproductive central zone by an air-filled inner tube. Light distribution models Radial light distribution models assumes that light travels radially from the surface towards the centre of the reactor and that attenuation of the light can be attributed to absorption by pigments packed in the cells. Such models may describe the overall tendency of decreasing light gradients in microalgal cultures reasonably well (Sloth et al. 2006; Perner-Nochta et al. 2007), but they do not account for all processes affecting the light. Scattering by cells and other particles has a randomising effect on the direction of the light, and cells in microalgal cultures are therefore exposed to dispersed light coming from all directions (Katsuda et al. 2000). Also air bubbles affects light gradients (Miro´n et al. 1999), and can actually increase the light penetration depth (Berberoglu et al. 2007). Empirical relationships have been used to provide accurate descriptions of light gradients (Katsuda et al. 2000; Su et al. 2003), but the most complete

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Biotechnol Lett (2008) 30:1525–1536

descriptions of light distribution inside photobioreactors are obtained by diffused light distribution models. These take into account the absorption of light by pigments, scattering of light by cells and other particles, and the geometry of the reactor and the light source (Garcı´a Camacho et al. 1999; Katsuda et al. 2000; Pottier et al. 2005; Berberoglu et al. 2007). Bosma et al. (2007) demonstrated that a diffused light distribution model could actually be used to predict the productivity of Monodus subterraneus in an outdoor bubble column under variable weather conditions. Large amounts of experimental data that are needed to accurately describe light gradients by diffused light distribution and they are not employed routinely for example in cultures where cell density and pigmentation change over time.

Mixing Mixing of microbial cultures is important for homogeneous distribution of cells, metabolites, and heat, and for transfer of gasses across gas–liquid interfaces. In microalgal cultures, mixing also affects the light regimen (Richmond 2000; Grobbelaar 2000). Fluctuations in light intensity faster than 1 s-1 enhance specific growth rates and productivities of microalgal cultures (Nedbal et al. 1996; Ogbonna and Tanaka 2000; Janssen et al. 2001; Yoshimoto et al. 2005). In outdoor cultures exposed to photosynthetic photon flux densities above 1,000 lmol m-2 s-1 light exposure times should be as short as 10 ms to maintain high PE (Janssen et al. 2001). Fluctuating light intensities Only photosystems with oxidised quinone QA and which have not already been excited are able to process newly absorbed photons into electron transport. Excess photons absorbed by the same photosystem are dissipated as heat or fluorescence, and may work in formation of singlet oxygen that oxidise and damage photosystem II (Melis 1999). When surface light intensities are high, short light exposure times reduce saturation and inhibition of the photosynthetic systems. Nedbal et al. (1996) showed that photosynthetic inactivation proceeds at lower rates when light is supplied intermittently compared to continuously, and Camacho Rubio et al. (2003)

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was able to describe this observation by a model, in which photodamage was caused by reactive radicals formed in the photosystems. Camacho Rubio et al. (2003) also demonstrated that sufficiently short cycle times between dark and illuminated zones allow excitation energy to be carried into the dark zones and there do photosynthetic work at rates similar to what would have been found in continuous light. While light/dark cycles of 94/94 ms were sufficiently short to increase the PE in cultures of Dunaliella tertiolecta, light/dark cycles of 3/3 s were too long and the PE decreased in comparison to continuously illuminated cultures (Janssen et al. 2001). Light/dark cycles of 3/3 s–12/12 s also reduced the PE in Chlamydomonas reinhardtii cultures compared to continuous illumination (Janssen et al. 2000b). Flat panel photobioreactors are designed to take advantage of fluctuating light intensities. These reactors have short light path lengths and steep light gradients if operated at high cell densities. This enables rapid circulation of cells between illuminated and dark zones, and the cells are only exposed to high surface light intensities for fractions of a second. Richmond et al. (2003) found that the PE of Nanochloropsis sp. cultures was almost doubled when the light path length was shortened from 9 to 1 cm and the cell density increased from 3.9 to 43.5 g l-1. Biomass productivities in flat panel photobioreactors have been further improved by installation of stationary baffles that increase the rate of medium circulation through the light gradient (Degen et al. 2001). Also biomass productivities in tubular photobioreactors are dependent of turbulent flows (Richmond 2000), and improvements have been obtained by installation of stationary baffles (Ugwu et al. 2005a, b) or intermediary disks to induce swirling flow patterns (Muller-Feuga et al. 2003). In airlifts, the frequency of light exposure is largely determined by the circulation time through riser and down-comer, while it in bubble columns is the turbulent flow eddies alone that circulate cells between illuminated and dark zones. Airlifts are often regarded superior to bubble columns because of their well-defined flow patterns and circulation times, and some studies also report higher productivity in airlift compared to bubble column photobioreactors (Merchuk et al. 2000; Kaewpintong et al. 2007; Krichnavaruk et al. 2007). However, the circulation

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times in airlifts are in the order of several seconds (Janssen et al. 2002), which would be too slow to diminish light saturation and photo-inhibitory effects (Janssen et al. 2000b, 2001). Recent literature also do not unanimously support that airlifts are superior to bubble columns. Miro´n et al. (2002) and Barbosa et al. (2003b) found that bubble column photobioreactors were comparable or even superior to airlifts with regards to productivity. Growth of microalgal cultures in fluctuating light environments has been modelled by Merchuk and Wu (2003) and Wu and Merchuk (2004), who segregated photobioreactors into compartments of different light intensities, and structured the photosystems in the cells as open, closed (while processing an already absorbed photon), or inhibited (due to absorption of multiple photons by each antenna). Transfer of cells between the different reactor compartments and changes in status of the photosystems were described by kinetic rate constants. In reality the movement of algal cells through light gradients is very complex, but two recent approaches target this problem theoretically and experimentally. Perner-Nochta and Posten (2007) used computational fluid dynamic modelling to predict particle trajectories in a tubular photobioreactor equipped with a helical mixer, while Lou et al. (2003) and Lou and Al-Dahhan (2004) used computer-automated radioactive particle tracking to actually measure the trajectories of a small radioactive particle in bubble column and airlift bioreactors. Both approached visualised very complex movements of individual cells through light gradients. Shear sensitivity High liquid velocities and high degrees of turbulence in photobioreactors can damage microalgae due to shear stress, and shear damage is sometimes used as an argument against mechanical mixing in microalgal cultures. However, air bubbles may also damage microalgae (Barbosa et al. 2003a, Vega-Estrada et al. 2005). Often, shear stresses caused by eddies in the growth medium and air bubbles cannot readily be discriminated. Shear sensitive animal cell cultures are routinely supplemented with the non-ionic surfactant, Pluronic F-68, which prevents cell adhesion to gas bubbles and reduce their shear damage (see e.g. Ma et al. 2004). Sobszuk et al. (2006) has recently

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demonstrated, that Pluronic F-68 also makes microalgal cells less vulnerable to shear damage. Carboxymethyl cellulose may also reduce cell adherence to gas bubbles and protect microalgal cultures from shear damage (Garcı´a Camacho et al. 2001). This strongly suggests that air bubbles can be the major cause of shear damage also to microalgal cells, and shear stress can be as problematic in pneumatically mixed as in mechanically mixed microalgal bioreactors.

Biotechnol Lett (2008) 30:1525–1536

Photobioreactor monitoring and control Environmental conditions are important for the performance of all microbial cultures, and a range of environmental and physiological variables are often measured and possibly controlled. In cultures of photoautotrophic microorganisms, some methodologies not seen in cultures of other microorganisms have been developed for monitoring and/or control of light intensity and biomass density, the two most important process variables in microalgal culturing.

Gas exchange Lumostats Even though shear stress by mechanical mixing is probably over-estimated and shear stress by air bubbles over-looked, there may still be good reasons to design photobioreactors as bubble columns or airlifts rather than stirred tank reactors. Because of their simpler construction and absence of moving, mechanical parts, bubble column and airlift photobioreactors are less vulnerable to technical malfunctions, a very important feature for reactors used for long-term continuous cultivation of microalgae. Furthermore, photoautotrophic cultures are more than an order of magnitude less productive than many heterotrophic microbial cultures, and relatively low power inputs are needed in photoautotrophic compared to heterotrophic cultures in order to create sufficient gas transfer rates. In photoautotrophic cultures, particularly the magnitude of the CO2 transfer rate is of concern, but high CO2 transfer rates do not only depend on high power inputs. Eriksen et al. (1998) and Poulsen and Iversen (1999) described bubble column photobioreactors equipped with dual sparging devises. Large air bubbles (*4 mm in diameter) were supplied continuously through one sparger in order to mix the culture, while pure CO2 was added through a different, perforated rubber membrane sparger that created small bubbles (*1 mm in diameter) with only little mixing power but high surface to volume ratios. In a 1.7 l dual sparging bubble column photobioreactor, the CO2 transfer rate was increased 5 times compared to a similar reactor where the CO2 was blended into the aeration air (Eriksen et al. 1998), and in a 32 l dual sparging bubble column photobioreactor with a liquid height of 2 m, CO2 transfer efficiencies were 100% at certain conditions (Poulsen and Iversen 1999).

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It is neither possible to control or maintain constant light regimens in many microalgal cultures. In batch cultures, light gradients become steeper and the light availability per cell decrease as the cultures grow. Changes of light regimen have been minimised in a number of photobioreactors by feedback regulation of the incident light intensity. These photobioreactors, sometimes named Lumostats, have been used to grow photoautotrophic cultures under relatively constant light conditions, in order to optimise culture productivities and study microalgal growth kinetics. The average light intensity was used as control variable in the Lumostat photobioreactor described by Suh and Lee (2001). Off-line biomass measurements combined with a radial light distribution model were used to calculate average light intensities in the cultures, and the number of fluorescent tubes was successively turned on as the cell density increased. Batch cultures of Synechococcus sp. were grown to cell densities of approximately 1.5 g l-1 while the average light intensities were maintained within relatively narrow intervals between 30 and 90 lmol m-2 s-1. Choi et al. (2003) and Lee et al. (2006) used specific light uptake rate (number of photons supplied per time divided by biomass concentration) as control variable in their Lumostat photobioreactors. As the biomass concentration increased, the incident light intensity was also increased to maintain a constant, predetermined specific light uptake. This lumostatic operation more than doubled the maximal cell density of Haematococcus pluvialis cultures when compared to constant light cultures, and the specific growth rate was increased and sustained for more cell divisions.

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On-line determination of the photosynthetic rate was used to control incident light intensities in the Lumostat photobioreactors developed by Eriksen et al. (1996, 2007). CO2 was added in pulses to maintain constant pH. Since the photosynthetic CO2 uptake was the major cause of pH changes, CO2 addition rates were proportional to the rates of photosynthetic carbon fixation. At intervals the light intensity was automatically either increased or decreased. When this resulted in faster CO2 addition rate, the light intensity was after a new interval changed again, and in the same direction as the previous change. When a change of light intensity resulted in lower CO2 addition rate, the light intensity was next time changed in the opposite direction of the previous change. By this principle, incident light intensities were maintained at optimal levels with regards to productivity, and this independently of any predetermined knowledge of the actual light regimen or light demands of the cultures. Growth of Synechococcus sp. was faster than at any constant light intensity and exponential growth was maintained for more than 5 cell generations (Eriksen et al. 1996). In Chlamydomonas reinhardtii and Chlorella sp., constant specific growth rates were maintained until the nitrogen source was depleted, and effects of nitrogen limitation were studied independently of light limitation (Eriksen et al. 2007). Direct measurements of the physiological state of photosystem II (PSII) have also been used as control variable in a Lumostat-type photobioreactor, named Physiostat (Marxen et al. 2005). The quantum yield of PSII was estimated from in-line measurements of variable and maximal fluorescence and used to control the supply of UVB radiation to turbidostat cultures of Synechocystis sp. under constant UVBstress. Internal radiation photobioreactors Internal light photobioreactors with short light paths are technical solutions that minimise variations in light regimen in time and space (Suh and Lee 2001, 2003). Cso¨go¨r et al. (2001) described a stirred reactor in which an internal draught tube made of glass or acrylic glass was used as a light emitting tube. Fibre optics guided light from an external light source into the light emitting tube. This internal radiation photobioreactor was used to investigate growth kinetics

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of Phorphyridium purpureum under almost homogeneous light distribution (Flech-Schneider et al. 2007). Quantification of biomass and growth Off-line measurements of apparent absorbance, cell dry weight, or cell number are the most widely used methods to follow the biomass density in photoautotrophic cultures. On-line measurements of apparent absorbance have been implemented successfully in microalgal cultures (see e.g. Eriksen et al. 1996, Marxen et al. 2005). Photoautotrophic cultures seldom reach very high cell densities, and the limited dynamic range of apparent absorbance measurements is a lesser problem than in cultures of heterotrophic microorganisms. A number of indirect biomass measures have recently been developed specifically for cultures of photoautotrophic microorganisms. Biomass production was correlated to increasing headspace pressure from accumulation of O2 in enclosed photobioreactors by Cogne et al. (2001) or dissolved oxygen partial pressure by Li et al. (2003). Jung and Lee (2006) photographed their photobioreactor from the top, developed contour images of the light distribution profile in the reactor, and used image analysis to correlate light distribution profiles to biomass densities. Photosynthetic production has also been measured on-line in a photobioreactor designed as a calorimeter (Janssen et al. 2007). The difference between rates of light energy supplied and heat removed to maintain constant temperature was used as a measure of energy stored in the biomass. Biomass densities have also been estimated from the integrated number of CO2-additions to maintain constant pH in Lumostat cultures of cyanobacteria and green algae (Eriksen et al. 1996, 2007), and from H2-additions over a Pd-catalyst to remove photosynthetically produced O2 in an enclosed photobioreactor (Eriksen et al. 2007). The ratio between O2 removal and CO2 addition rates was further used to deduce the elementary composition of the produced biomass (Eriksen et al. 2007).

Engineering of microalgal physiology Genetic engineering of photoautotrophic microorganisms is a developing area, which can improve culture

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Biotechnol Lett (2008) 30:1525–1536

productivities and expand the number of microalgal products. Reduction of photosynthetic antenna sizes is a physiological way to increase photosynthetic efficiencies (Mussgnug et al. 2007). Reduced antenna sizes will reduce the rate by which photons are absorbed by individual antennas. Increased light intensities will be needed to saturate each reaction centre, and fewer photons will be dissipated as fluorescence or heat. Reduced antenna sizes will also result in lower absorption coefficients per unit of biomass. Light will penetrate deeper into the culture and dark, unproductive zones are reduced in volume (Berberoglu et al. 2007). Better use of absorbed photons in combination with smaller dark zones leads to an increase in overall culture productivity whether biomass or for example H2 is the primary product, although the specific growth rate may be reduced at low light intensities. Process optimisation can also be targeted via metabolic engineering. Zaslavskaia et al. (2001) inserted glucose transporter genes from Chlorella or humans into the obligate photoautotrophic diatom Phaeodactylum tricornutum, which became able to grow heterotrophically in darkness. Such transformants could be used in photo-heterotrophic microalgal processes, which may have higher energy conversion efficiencies than photoautotrophic cultures (Yang et al. 2000). Metabolic engineering has also been used to down-regulate the cyclic electron flow around PSI in H2 producing Chlamydomonas reinhardtii (Kruse et al. 2005). Thereby, the hydrogenases experienced less competition for excited electrons, and the yield of H2 was increased. Genetic engineering may also develop microalgae into

producers of recombinant products and thereby extend the range of products from these organisms (see e.g. Leo´n-Ban˜ares et al. 2004).

Heterotrophic microalgal cultures Heterotrophic microalgae are also receiving increased attention. They are grown in ordinary stirred tank bioreactors similar to the bioreactors used for most other microorganisms, and independently of light. Scale-up is much simpler with regards to reactor size, mixing, gas transfer, and productivity when high surface to volume ratios are not mandatory. Highly productive, high cell-density cultures of microalgae from various divisions, including the chlorophyte Chlorella spp. (Wu et al. 2007, Xiong et al. 2008), the euglenophyte Euglena gracilis (Ogbonna et al. 1998), the diatom Nitschia laevis (Wen and Chen 2001, 2003), the dinoflagellate Crypthecodinium cohnii (De Swaaf et al. 2003a, b), and the rhodophyte Galdieria sulphuraria (Schmidt et al. 2005, Graverholt and Eriksen 2007) have been described, in which cell densities and biomass productivities (Table 2) are much higher than in photoautotrophic cultures (Table 1).

Perspectives Despite the important roles played by microalgae as primary producers in aquatic environments of paramount importance to global CO2 fixation, fisheries, and human health, cultivation of these organisms has

Table 2 Maximal cell densities (xmax) and volumetric productivities (Pvolume) of high cell-density heterotrophic microalgal cultures Species

Product

Culture

xmax g l-1

Pvolume g l-1 day-1

Ref.

Euglena gracilis

a-tocopherol

Fed-batch

48

7.7a

A

Nitzchia laevis Crypthecodinium cohnii

Eicosapentaenoic acid Docosahexaenoic acid

Perfusion Fed-batch

ca. 30 83

6.75 10.0a

B C

Chlorella protothecoides

Biodiesel

Fed-batch

51.2

7.7a

D

Galdieria sulphuraria

C-phycocyanin

Continuous

83.3

50.0

E

Values in italics are based on information in the references References: A, Ogbonna et al. (1998); B, Wen and Chen (2001); C, de Swaaf et al. (2003a, b); D, Xiong et al. (2008); E, Graverholt and Eriksen (2007) a

Calculated based on data read from graphics

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Biotechnol Lett (2008) 30:1525–1536

never experienced the same growth as cultivation of heterotrophic microorganisms and mammalian cells. Microalgal cultures can synthesise a range of biological products, and potentially out-compete agricultural crops in terms of areal productivity. Microalgae should therefore be the ideal producers of bulk biological chemicals and first choices for the large and rapidly growing biofuel industry (Chisti 2007, 2008). However, their potential for synthesising bulk products has proven very difficult to realise in practise. Present days photobioreactors are at an advanced stage with high photosynthetic efficiencies of 5–10%, but these values decrease at high incident light intensities and to some extent with reactor size (Table 1). Bulk production of microalgal products are therefore still waiting for a break-through in the design of photobioreactors, in which high photosynthetic efficiencies are maintained at large scales and at high light intensities during long term operations. Separation of reactor and light collection system can allow photobioreactors to be optimised with respect to other variables than light harvesting. A novel internal light photobioreactor designed by Zijffers et al. (2008) uses Fresnel lenses to concentrate solar radiation onto light guides, which then distribute and re-emits the light into the photobioreactor at lower intensities. Thereby, high surface light intensities are avoided and all photosynthetically active radiation can potentially be utilised with high PE, but microalgal culture performance still needs to be demonstrated in this reactor. Novel reactor designs should also be accompanied by down stream processing procedures which handles the dilute algal cultures at reasonable costs (Molina Grima et al. 2003), and possibly engineered algal strains in which photosynthesis and product formation are optimised with respect to culture productivity rather than Nature’s choice of maximal productivity per individual cell. Microalgal cultures also provide fine chemicals of high value, but so far only a limited range of highvalued microalgal products cannot be obtained from alternative sources. The number of products therefore has to be increased, either by identification or engineering of novel microalgal products. Alternatively, heterotrophic microalgal cultures, that are not limited by external light, and much more productive than the photoautotrophic cultures (Table 2) can be used to broaden the range of economically viable

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microalgal products. The best example of such a product is probably the nutraceutical, docosahexaenoic acid (De Swaaf et al. 2003a, b), but even pigments are now being synthesised heterotrophically (Graverholt and Eriksen 2007, Wu et al. 2007). However, despite all the progress that has been made, microalgal culturing is still a small niche. Further developments still depend on continued research and developments in microalgal culturing technologies.

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