the technology of micro algal culturing- eriksen, 2008
TRANSCRIPT
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
� Springer Science+Business Media B.V. 2008
Abstract This review outlines the current status
and recent developments in the technology of mic-
roalgal 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 engi-
neering, 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
particular attention in the design of photobioreactors.
Mixing, process monitoring and control, and explo-
ration 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 (Leon-Banares et al. 2004),
and experimental phycology still create novel exper-
imental 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 photoautotro-
phic prokaryotes is also included.
Design of photobioreactors
The productivity of photoautotrophic cultures is
primarily limited by the supply of light and suffers
N. T. Eriksen (&)
Department of Biotechnology, Chemistry
and Environmental Engineering, Aalborg University,
Sohngaardsholmsvej 49, 9000 Aalborg, Denmark
e-mail: [email protected]
123
Biotechnol Lett (2008) 30:1525–1536
DOI 10.1007/s10529-008-9740-3
from low energy conversion efficiencies caused by
inhomogeneous distribution of light inside the cul-
tures (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 cul-
tures (Ogbonna and Tanaka 2000). High light
intensities at culture surfaces may cause photoinhi-
bition, 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 micro-
algal culturing has been carried out in photo-
bioreactors 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 Klotze, Germany (Pulz
2001; Janssen et al. 2002; Spolaore et al. 2006) are
based on tubular reactors. In tubular photobioreac-
tors, the cultures are pumped through long,
transparent tubes. The tubes are organised horizon-
tally (Molina et al. 2001; Carlozzi et al. 2006),
vertically (Carlozzi 2000; Converti et al. 2006;
Perner-Nochta et al. 2007), inclined (Vunjak-
Novakovic et al. 2005), or as a helix (Hai et al.
2000; Travieso et al. 2001; Scragg et al. 2002;
Fernandez 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; Fernandez
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 large-
scale cultures of microalgae (Chisti 2006), the length
of the tubes are limited by O2 accumulation, CO2
depletion, and pH variations. Tubular photobioreac-
tors therefore cannot be scaled up indefinitely, and
large-scale production plants partly rely on multipli-
cation 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 Lıvansky 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 (Csogor 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.
Miron 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
1526 Biotechnol Lett (2008) 30:1525–1536
123
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Biotechnol Lett (2008) 30:1525–1536 1527
123
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 photobioreac-
tors 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 photobiore-
actors and tubular photobioreactors constructed of
narrow, transparent pipes. In vertical column photo-
bioreactors, 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, unpro-
ductive 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
(Miron 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
descriptions of light distribution inside photobioreac-
tors 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 (Garcıa 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 subter-
raneus 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 homo-
geneous 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). Fluc-
tuations 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 expo-
sure 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 trans-
port. 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)
1528 Biotechnol Lett (2008) 30:1525–1536
123
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 Nano-
chloropsis 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 photobior-
eactors 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
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. Miron et al. (2002) and Barbosa
et al. (2003b) found that bubble column photobior-
eactors 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 theo-
retically 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 track-
ing to actually measure the trajectories of a small
radioactive particle in bubble column and airlift
bioreactors. Both approached visualised very com-
plex 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
Biotechnol Lett (2008) 30:1525–1536 1529
123
demonstrated, that Pluronic F-68 also makes
microalgal cells less vulnerable to shear damage.
Carboxymethyl cellulose may also reduce cell adher-
ence to gas bubbles and protect microalgal cultures
from shear damage (Garcıa 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 pneumat-
ically mixed as in mechanically mixed microalgal
bioreactors.
Gas exchange
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 photobi-
oreactors are less vulnerable to technical malfunc-
tions, a very important feature for reactors used for
long-term continuous cultivation of microalgae. Fur-
thermore, 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 com-
pared to heterotrophic cultures in order to create
sufficient gas transfer rates.
In photoautotrophic cultures, particularly the mag-
nitude 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 contin-
uously 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).
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 methodolo-
gies 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.
Lumostats
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 photobioreac-
tors, 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 measure-
ments 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 photobioreac-
tors. 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.
1530 Biotechnol Lett (2008) 30:1525–1536
123
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 Synecho-
coccus 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., con-
stant specific growth rates were maintained until the
nitrogen source was depleted, and effects of nitrogen
limitation were studied independently of light limi-
tation (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 UVB-
stress.
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). Csogor 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 pho-
tobioreactor was used to investigate growth kinetics
of Phorphyridium purpureum under almost homoge-
neous 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 photoauto-
trophic 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 sel-
dom 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 produc-
tion was correlated to increasing headspace pressure
from accumulation of O2 in enclosed photobioreac-
tors 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 distribu-
tion 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 photosyn-
thetically 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 microorgan-
isms is a developing area, which can improve culture
Biotechnol Lett (2008) 30:1525–1536 1531
123
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 transfor-
mants could be used in photo-heterotrophic micro-
algal processes, which may have higher energy
conversion efficiencies than photoautotrophic cul-
tures (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 hydrog-
enases 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. Leon-Banares 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 para-
mount 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 Pvolume Ref.
g l-1 g l-1 day-1
Euglena gracilis a-tocopherol Fed-batch 48 7.7a A
Nitzchia laevis Eicosapentaenoic acid Perfusion ca. 30 6.75 B
Crypthecodinium cohnii Docosahexaenoic acid Fed-batch 83 10.0a 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
1532 Biotechnol Lett (2008) 30:1525–1536
123
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 synthesis-
ing 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 photosyn-
thetic 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 pro-
cessing 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 individ-
ual cell.
Microalgal cultures also provide fine chemicals of
high value, but so far only a limited range of high-
valued 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. Alterna-
tively, 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
microalgal products. The best example of such a
product is probably the nutraceutical, docosahexae-
noic 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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