Microalgal Reactors: A Review of Enclosed System Designs and Performances
Ana P. Carvalho, Luı ´s A. Meireles, and F. Xavier Malcata*
Escola Superior de Biotecnologia, Universidade Cato ´lica Portuguesa, Rua Dr. Anto ´nio Bernardino de Almeida, P-4200-072
One major challenge to industrial microalgal culturing is to devise and develop technical apparata,
cultivation procedures and algal strains susceptible of undergoing substantial increases in
efficiency of use of solar energy and carbon dioxide. Despite several research efforts developed
to date, there is no such thing as “the best reactor system”- defined, in an absolute fashion, as
the one able to achieve maximum productivity with minimum operation costs, irrespective of
the biological and chemical system at stake. In fact, choice of the most suitable system is situation-
dependent, as both the species of alga available and the final purpose intended will play a role.
The need of accurate control impairs use of open-system configurations, so current investigation
has focused mostly on closed systems. In this review, several types of closed bioreactors described
in the technical literature as able to support production of microalgae are comprehensively
presented and duly discussed, using transport phenomenon and process engineering methodologi-
cal approaches. The text is subdivided into subsections on: reactor design, which includes tubular
reactors, flat plate reactors and fermenter-type reactors; and processing parameters, which include
gaseous transfer, medium mixing and light requirements.
2. Reactor design
2.1. Tubular reactors
2.1.1.Vertical tubular reactors
2.1.2.Horizontal tubular reactors
2.1.3.Helical tubular reactors
2.2. Flat plate reactors
2.3. Fermenter-type reactors
3. Processing parameters
3.1. Gaseous transfer
3.3. Light requirement
From an economic point of view, microalgae may be
described as microorganisms with the ability to “harvest the
sun” and hence transform its radiant energy into valuable
products, at the expense of (theoretically) inexpensive natural
resources (viz., CO2and H2O). The idea of producing microal-
gae at the technical scale first occurred to German scientists, in
concerted attempts to devise inexpensive sources of protein able
to replace those from animal sources, which were difficult to
obtain during World War II (1). In the U.S., research on mass
culture of microalgae began as a collateral development of
fundamental studies on photosynthesis; in attempts to translate
the biological requirements of microalgal culture into engineer-
ing specifications, a large-scale culture plant was made at
Stanford Research Institute, back in 1948. During 1951, Arthur
D. Little, Inc. (Cambridge, MA) made further advances through
construction and operation of a Chlorella pilot plant for the
Carnegie Institution (2). Other studies followed in Japan, under
the guidance of Tamiya (2). Although experimental results
showed that continuous culture was possible, many subjects still
needed improvement, as microalgal proteins could not compete
with such inexpensive plant sources as soybean meal.
Other developments were achieved by Oswald, who started
studying the role of microalgal photosynthesis in ponds and
accordingly developed a high-rate algal pond for photosyn-
thetic wastewater treatment. In the 1960s, Nichoporovich and
Semenenko intensively studied closed culture systems, for
extraterrestrial life support during prolonged missions in outer
space; this subject also received considerable attention by NASA
(1). The advent of the oil crisis in the 1970s led researchers to
investigate microalgae as potential sources of biomass, aiming
at methane production (3); more recently, advances have focused
on the production of fine chemicals and secondary metabolites,
which may reach high prices in the world market.
The evolution in goals throughout time has therefore been
driven by two major streamlines: (i) requirement for alternative
sources of several products, which were scarce as a result of
political or economic reasons; and (ii) perception that microal-
gal-mediated processes (as happens with most biotechnological
ones) are usually characterized by noncompetitive production
costs, the economic feasibility of which relies heavily on the
market value of the resulting compounds. As expected, the
* To whom correspondence should be addressed. Tel: 351 225 580 004.
Fax: 351 225 090 351. Email: email@example.com.
Biotechnol. Prog. 2006, 22, 1490−1506
10.1021/bp060065r CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical Engineers
Published on Web 11/15/2006
nature of these compounds has evolved with time, in response
to changing market demands.
A broad list of applications of microalga cultures has been
described and discussed in the literature. Those that have attained
commercial expression encompass the areas of healthy foods,
food additives, pigments, diets for aquaculture, growth-regulat-
ing agents, secondary metabolites and wastewater treatments
(4, 5) (see Table 1). The production of several bioactive
compounds such as hydrocarbons, isotopes, polysaccharides, and
antifungal, antitumor, antibacterial and antiviral substances is
currently under study; uses of microalgae for CO2 fixation,
removal of nitric oxide from flue gas, fuel production, recovery
of heavy metals from effluents and in outer space technologies
are also in order (3, 5-11). Nevertheless, despite such enormous
potential, the number of applications that has reached the
industrial scale is comparatively rather limited.
From the pioneer commercial large-scale cultures of microal-
gae in the 1960s in Japan using Chlorella (12), only a few more
species have been employed industrially ever since, which
include Spirulina sp. and Scenedesmus sp. for healthy food and
phycocyanin synthesis (a blue colorant in food and cosmetics),
Haematococcus pluVialis for production of astaxanthin (a food
colorant), and Dunaliella salina for the manufacture of ?-car-
otene (a vitamin A substitute and food colorant). In addition,
Crypthecodinium cohnii and Schizochytrium sp. are also com-
mercially used for production of polyunsaturated fatty acids
(e.g., docosahexaenoic acid), although following a fermentative
Open ponds were the ancient configurations proposed for
microalga production and are still the most widely applied in
industrial processes. They usually consist of either circular ponds
with a rotating arm to mix the culture, or long channels in a
single or multiple loop configuration stirred by paddle wheels
(3), although simpler configurations also exist. The main
constraints related to operation of these open systems are the
impossibility to control contamination, the difficulty to keep
the culture environment constant and the cost of the harvesting
stage. In order to avoid microbial contamination, highly selective
conditions are necessary, so as to guarantee dominance by the
selected strain (e.g., D. salina dominance requires highly salted
media, whereas Spirulina platensis demands high pH values);
unfortunately, both of these conditions are not suitable for most
microalgal species. The direct effect of weather conditions on
the characteristics of the open-pond cultivation media also makes
it very difficult to keep preset values for the environmental
parameters. Regarding the harvesting phase, the huge volume
of culture to be harvested (because of the low cell densities
attained) magnifies the cost of processing, thus substantially
increasing the final cost of the product. Because of the
aforementioned serious constraints, open systems have appar-
ently reached their upper limit, with little room for further
In view of these difficulties, another approach was envisaged,
which is based on the alternative use of closed systems. These
are more appropriate for sensitive strains (which grow in non-
extreme environments) or when the final product is highly
susceptible to microbial degradation (e.g., bacterial metaboliza-
tion of amino acids and polysaccharides). The closed config-
uration makes the control of contaminants easier, hence allowing
growth in photo-autotrophic, heterotrophic or mixotrophic
modes; because of the higher cell mass productivities attained
(up to 3-fold those obtained in open systems) (3), harvesting
costs per unit mass can also be significantly reduced. Neverthe-
less, the costs of closed systems are higher than their open-
system counterparts, in addition to several other disadvantages
(see Table 2). In fact, despite their higher volumetric productiv-
ity, closed systems were not a consensual industrial choice until
only recently; intensive capital investments and high production
costs account for this realization. In order to minimize produc-
tion costs, the major factors that play a role in the process ought
to be identified and their specific contributions comprehensively
studied, in order to maximize advantages and minimize disad-
vantages. The choice of which configuration is preferable
depends obviously on the objective function considered; e.g.,
wastewater treatment would preclude closed systems, owing to
the unacceptably high costs that arise from the large volumes
to be processed and the low added value of the feedstock
When improvement in efficacy of a closed photo-bioreactor
is the goal, light and CO2supply are key processing parameters,
especially owing to the difficulties associated with their control
(viz., assurance of stability throughout time and uniformity
throughout space); most of the so-called novel bioreactors do
in fact attempt to overcome the constraints related to control of
said parameters (14). However, the technology that supports
supply of adequate amounts of CO2to microalgal cells is still
poorly developed, which contrasts with the substantial research
efforts currently underway on the genetic improvement of native
freshwater and marine species for specific applications. On the
other hand, although the problem of light supply has sometimes
been circumvented by growing the microalgae heterotrophically,
not all microalgae (or microalgal products, for that matter) can
be produced in this way.
Therefore, issues such as contamination control, gaseous
exchanges, mixing patterns, suitability of light supply (which
comprises light quality and quantity), geometrical configuration
and building material are considered relevant, and will accord-
ingly be discussed to some extent in this review. The aspects
of nutrient supply, as well as pH and temperature control, will
not be considered here, because no major improvements are
Table 1. Generic Description of Commercial Microalgal Culture Systems Currently in Use
microorganismmetabolite commercial useculturing systemlocation
Chlorella spp.astaxanthinpigmenting agent circular ponds with
extensive open ponds
ferredoxin laboratory use
?-carotenepigmenting agent Australia, China, India,
Chile, U.S., Israel
functional food additive
functional food additive
Biotechnol. Prog., 2006, Vol. 22, No. 6
expected therefrom, as their control is rather simple. There has
been indeed substantial research on those topics, and the
solutions available to date already address conveniently the
intended purposes (15). Despite its relative importance, the
harvesting phase is also out of the scope of this review.
2. Reactor Design
The main parameter that affects reactor design is provision
for light penetration, which implies a high surface-to-volume
ratio; such penetration is crucial if one wants to improve the
photosynthetic efficiency, which is in turn a sine qua non
condition to reach high product and biomass productivities. In
order to achieve said high surface-to-volume ratio, several
shapes have been developed that met with success. These shapes
can be grouped in three basic types, viz., tubular, flat plate and
fermenter-type; the former two are specifically designed for
efficient harvest of sunlight, whereas the latter requires artificial
Tubular and flat plate reactors are undoubtedly the most
popular choices (12), considering that the light source required
is free and readily available. Those reactor types are based on
the same principle, viz., to guarantee the highest possible area-
to-volume ratio while ensuring reasonable working volume,
mixing pattern and carbon dioxide level. Both reactor configura-
tions may work with a separate unit for gas transfer, and several
layouts have been already tested with success (16-20). Such
systems comprise: a light-harvesting unit, which employs small
diameter tubing so as to provide a high area-to-volume ratio
that favors high photosynthetic activity, and a gas exchange unit,
in which CO2is supplied and biomass harvesting is processed.
The culture is circulated between those two units by a pump,
which needs to be carefully designed and operated in order to
prevent shear forces from disrupting cell integrity (17, 21-27).
Several reactor designs and corresponding productivities are
tabulated in Table 3.
2.1. Tubular Reactors. Most configurations of tubular
reactors (TR) are one of the following three types: (i) simple
airlift and bubble column, which is composed of vertical tubing
(in the form of a vertical tubular reactor) that is transparent so
as to allow for light penetration and where CO2is supplied via
bubbling; (ii) horizontal tubular reactor, which is composed of
horizontal transparent tubing, usually bearing gas transfer
systems attached to the connections; and (iii) helical tubular
reactor, which is composed of a flexible plastic tube coiled in
a circular framework. Another such reactor that deserves
particular attention is the R-shape tubular reactor, initially
conceived by Lee (28), because of its unique engineering design
that is characterized by a unidirectional, high liquid flow rate,
concomitant with a low air flow and an excellent angle relative
2.1.1. Vertical Tubular Reactors. The airlift and bubble
column reactors are examples of vertical tubular reactors (VTR),
regularly composed of polyethylene or glass tubes (Figure 1),
which are sufficiently transparent to allow good light penetration
but are manufactured with sufficiently common materials so as
to be nonexpensive. Air is bubbled at the bottom-a strategy
that provides good overall mixing, sufficient supply of CO2,
and efficient removal of O2. Polyethylene bags have frequently
been used, with advantage taken from their particularly low cost,
high transparency and good sterility at startup-due to the high
temperatures used during film extrusion (29, 30); 32 cm × 250
cm (ca. 25 L) bag reactors were employed by Cohen (31) for
cultivation of Porphyridium sp., which were able to reach cell
concentrations 3-fold those typically attained in open ponds.
Trotta (30) described a reactor composed of several 30 cm ×
180 cm (ca. 50 L) polyethylene bag reactors, with various
closing devices, and complemented with air and medium
supplies. Martine ´z-Jero ´nimo (32) also reported cultivation in
16.8 cm × 224 cm (ca. 40 L) bags to be practical, and to exhibit
an improved area-to-volume ratio. Tredici and Rodolfi (33)
improved this idea by using a culture chamber made of flexible
transparent plastic film contained in a rigid metal framework,
so as to form a vertical panel of reduced width. More recently,
Chae (34) reported a pilot-scale photo-bioreactor that uses
sunlight and flue gas, and consists of a vertical tubular part (kept
in the dark) and a horizontal tubular part (subject to sunlight).
Although cultivation of microalgae in the above systems is
simple and hence widely employed (including in hatcheries),
the corresponding technology is somehow primitive, with
obvious constraints derived from the high fragility and the low
versatility of the material in stake (21). Furthermore, scale-up
of these systems was initially thought to be easy, but ac-
cumulated experience (32) has indicated that increases in culture
volume decrease bag productivity.
Rigid VTR have also been frequently used. A 33.7 cm ×
250 cm (ca. 40 L) polyethylene reactor was discussed by Laing
(29), in which temperature was controlled by a refrigeration
fluid flown through its double wall and in which artificial light
was provided from the inside. Myamoto (35), James (36) and
Fukami (37) presented similar reactor configurations, but using
direct sunlight; their main advantages were low cost and ease
of operation. Unfortunately, scale-up is not straightforward;
furthermore, in order to provide enough culture volume, as well
as efficient gas transfer rate, the reactor diameter should be
relatively high when compared to flat plate or tubular loop
reactors, a requirement that in turn decreases the area-to-volume
ratio and consequently constrains photosynthetic efficiency.
Another major drawback is the large angle relative to the
direction of sunlight, which causes a high fraction of incident
Table 2. Main Design Features of Open and Closed Photobioreactors
featureopen systems closed systems
main criteria for species selection
water loss through evaporation
light utilization efficiency
most costly parameters
large (4-10 times higher than closed counterpart)
oxygen control, temperature control
aDependent on transparency of construction material.
Biotechnol. Prog., 2006, Vol. 22, No. 6
energy to be reflected back and thus lost in terms of biomass
growth purposes (38).
2.1.2. Horizontal Tubular Reactors. Horizontal tubular
reactors (HTR) have been on the rise; gas transfer takes place
in the tube connection or via a dedicated gas-exchange unit,
and the angle toward sunlight is particularly adequate for
efficient light harvesting. Such systems can handle large working
volumes, because they are not susceptible to contamination. On
the other hand, they may generate considerable amounts of heat,
which may attain temperature amplitudes of 20 °C within a
single day if (costly) temperature control systems are not
provided; thus, it will likely pose a problem for regular operation
A long tubular reactor, 14 cm in diameter, placed horizontally
and made of Plexiglas, was reported by Torzillo (40); said device
included a diaphragm pump designed to drive the culture to a
feeding tank, and gas transfer was allowed in the tube connec-
tions. The maximum working volume was 8000 L in a land
area of 80 m2. However, when the area-to-volume ratio was
set to that prevailing in open ponds, the photosynthetic efficiency
did not improve consistently:
attained was ca. 0.25 g L-1d-1when using S. platensis. The
major problem encountered was indeed the control of temper-
ature; three different methods were therefore tested, viz., shading
the tubes with dark plastic, overlapping the tubes and water-
spraying the surface of the tubes. The latter was shown to be
efficient, although assurance of low temperatures required a
large consumption of water. A thermo-tolerant strain of Spirulina
the maximum productivity
sp. (able to grow at 46 °C) was then used, and the maximum
temperature could be kept at 44 °C. A few companies have
adopted similar procedures for their industrial operation. For
example, the Aquasearch (currently MERA Pharmaceuticals)
growth module (42), which is essentially a long HTR for cultiv-
ation of H. pluVialis (a species tolerant to temperature changes),
was cooled down by water spraying whenever necessary.
Gudin and Chaumont (43) have been developing tubular
reactor configurations since 1984. They reported a reactor
system bearing a capacity of 7000 L in all, for which a
productivity of 36 g m-2d-1was claimed when using
Phorphyridium cruentum (44). This apparatus was made of
several 70-L glass VTR, all connected to a gas exchange unit,
and the culture was mechanically pumped between the light
harvesting and the gas exchange units-whereas the temperature
was controlled by submerging the culture in a water pool as
deemed necessary. A major limitation of this reactor was its
relatively high cost, coupled with the intrinsic fragility of its
Richmond (17) and Grima (45) reported similar layouts of
HTR, which led to productivities as high as 1.5 g L-1d-1with
S. platensis and 0.32 g L-1d-1with Isochrysis galbana. These
systems were based on an external unit designed for light
harvesting, connected to a gas exchange tower (placed on top)
via an airlift pump (see Figure 2A and B, respectively); the
working pressure was set so as to provide a low shear stress
and a good rate of homogenization, with typical linear velocities
of ca. 50 and 30 cm s-1, respectively. The main difference
between those two configurations pertains to the light harvesting
unit: Grima (45) presented a 2.6 cm × 80.8 m Plexiglas loop
submerged in a thermostatic pool, whereas Richmond (17)
described parallel sets of 3.0 cm × 20 m polycarbonate
transparent tubes connected by a manifold, with temperature
controlled by water spraying. Both of these systems were found
rather efficient and cost-effective. Richmond (17) claimed that
the best way to increase the working volume is to add more
tube sets, in order to maintain an adequate linear velocity and
avoid oxygen buildup. Therefore, scale-up in this case poses in
principle no problems, because the same tower exchange unit
can be duly connected to several extra sets of tubes. The major
drawback is the land area occupied; in fact, the tubes in the
light-harvesting unit are very narrow, so industrial volumes (i.e.,
5 000-10 000 L) can be reached only at the expense of a
paramount number of tubes, which are supposed to be placed
horizontally. Therefore, economic feasibility should be assessed
in advance; otherwise this type of apparatus may not be cost-
effective at all, as discussed elsewhere (46).
Another advance in microalgal cultivation systems is the near-
horizontal tubular reactor (NHTR) designed by Tredici (47) (see
Figure 3), which consists of sets of parallel tubes made of
flexible plastic (typically 6.4 m in length, 43 mm diameter and
0.15 mm thick), connected by PVC manifolds. The upper
manifolds are used as degassers, and a perforated pipe inserted
Table 3. Main Design Features of Closed Photobioreactors
requiredscale-up productivity (g L-1d-1); species ref
0.5; P. cruentum
0.25; S. platensis
0.7; Nannochloropsis sp.
0.4; S. platensis
46; 51 0.85; Nannochloropsis sp.
2.15; S. platensis
0.03-0.05; several fermenter type poor excellent excellent difficult48
Figure 1. Schematic representation of airlift (A) and bubble column
Biotechnol. Prog., 2006, Vol. 22, No. 6
in the lower manifolds is used to inject air in each individual
tube. The tubes are placed at an angle of 5-7° relative to the
horizontal plan. Temperature is controlled by activating water
spraying onto the reactor when the culture temperature exceeds
a preset value. The maximum volume tested, 4000 L, obtained
with a set of 8 parallel tubes 44 m in length, was associated
with a mean productivity of 0.7 g L-1d-1in the case of
Nannochloropsis sp. The main advantage of this system is the
high area-to-volume ratio and its easy scale-up, coupled with
the possibility for a higher degree of control (41). Surprisingly,
the temperature control was not efficient, and a day-night
temperature gap of ca. 14 °C during summer and of ca. 22 °C
during spring was actually recorded. Such a poor control led to
different biochemical compositions of the microalga throughout
the year. However, the major drawback of this system was
probably its low gas transfer rate, arising from a large length
and a small diameter (48).
2.1.3. Helical Tubular Reactors. Helical tubular reactors
(HeTR) are a suitable alternative to straight TR. The most
frequently used layout is the Biocoil, initially proposed by
Robinson (49) and currently traded by Biotechna (Melbourne,
Australia). This reactor is composed of a set of polyethylene
tubes (3.0 cm of inner diameter) coiled in an open circular
framework, coupled with a gas exchange tower and a heat
exchange system; a centrifugal pump drives the culture broth
through the long tube to the gas exchange tower (see Figure
4A). A few authors (12, 18, 20, 21, 50) experimented with and
eventually improved such a design, so it ranks at present among
the most effective ones as a result of its high area-to-volume
ratio on the one hand, and the requirement of only a small land
area for relatively large volumes on the other. Placing a light
source inside the coil and then providing a reasonable control
of light intensity may compensate for the large angle toward
sunlight. Scale-up of this type of system is easy; one has simply
to increase the number of parallel layers of tubes in the coil,
thus maintaining the hydraulic head essentially unchanged (21).
However, use of a centrifugal pump to drive the culture to the
upper side of the tubing might increase shear stress, which then
would become a limiting step for biomass productivity. There-
fore, not all algal species are suitable for culture in this system;
the recirculation pump may damage some, whereas others may
be engaged in fouling on the inside of the reactor tubing (12).
The system described by Travesio (20) operates with an airlift
column instead of a centrifugal pump, and may be useful toward
reduction of shear stress and consequent minimization of cell
Morita (51) proposed a conical helical reactor that encom-
passes a light-harvesting unit composed of PVC tubing (0.16
m of internal diameter) coiled in a conical framework (see Figure
4B), a degassing unit placed above and a heat exchanger. An
air pump is used to force the culture from the heat exchanger,
in an ascending trajectory, to the light-harvesting unit, which
is in turn connected to the degasser, placed at the same level;
the culture is then returned to the heat exchanger unit in a
descending trajectory. The same degasser and heat exchanger
can be connected to several light-harvesting units, as also tested
by the same author. The main advantage of this system its the
high efficiency in light harvesting; the conical shape distributes
the radiant energy input to a larger photo-receiving area, i.e., it
improves spatial distribution of light. The heat exchanger has
proven efficient in maintaining temperature within a narrow
interval (28 ( 3 °C). One major disadvantage is that, unlike
the Biocoil, this arrangement cannot be easily scaled-up. In fact,
although the conical light receptor is very efficient, the angle
and height are strictly defined, so the only way to maintain high
photosynthetic rates is to increase the number of light harvesting
units, which also leads to larger energy losses in the complicated
branches of the flow networks (52); hence the land area
productivity will be significantly reduced.
2.1.4. R-Shaped Reactors. Lee (28) described a 300-L
R-shaped tubular reactor, with sets of 2.5 cm × 25 m transparent
tubes made of PVC; it used an airlift pump to promote an
ascending/descending trajectory, with several CO2 injection
points along its path (see Figure 5). This configuration presents
several advantages from the engineering point of view. For
example, the fluid is pumped in a single direction (except in
the airlift tubes), so a high flow rate is possible at the expense
of relatively low air supply rates in the rising tubes. The
Figure 2. Schematic representation of horizontal tubular reactor with a degassing unit and a light harvesting unit, composed of parallel sets of
tubes (A) or a loop tube (B).
Figure 3. Schematic representation of near horizontal tubular reactor.
Biotechnol. Prog., 2006, Vol. 22, No. 6
ascending and descending trajectories are placed at a 45° angle
toward sunlight, so light harvesting is rather efficient.
2.2. Flat Plate Reactors. Flat plate reactors (FPR) are
conceptually designed to make efficient use of sunlight; hence,
narrow panels are usually built so as to attain high area-to-
volume ratios (see Figure 6A). In the early 1980s, FPR were
considered expensive and were even claimed to exhibit deficien-
cies in culture flow control (53). A 500-L FPR was developed
by Pulz (16), in which the culture was circulated from an open
gas exchange unit through several parallel panels placed hori-
zontally. The culture flew at a high linear speed (viz., 1.2 m
s-1), but hydrodynamic parameters usually lay in a safe oper-
ating range for the sake of cell integrity. The greatest advantage
of this system is its provision of an open gas transfer unit, which
has proven efficient in overcoming the problem of oxygen
buildup; however, such an open zone restricts effectiveness of
contamination control, as compared with completely closed
reactors. Richmond (54) presented a similar system, composed
of several 200-L units potted together, each unit being composed
of 200 cm × 100 cm × 10 cm glass plates. The main difference
was the absence of a gas transfer unit and instead bubbling of
compressed air at the bottom, through a perforated plastic tube.
A closed system of water spraying was employed to control
temperature; the sprayed water was then collected in troughs
and recirculated through a ventilated water column for refrigera-
tion. One such reactor, with an overall volume of 1000 L, was
tested with various light paths for cultivation of Nannochloropsis
sp.; the maximum volumetric productivity, 0.85 g L-1d-1, was
attained with the minimum light path, i.e., 1.3 cm.
A different FPR was reported by Iqbal (55), who described
a V-shaped apparatus characterized by unusually interesting
engineering features, viz., very high mixing rate and very low
shear stress (see Figure 6B); scale-up of its reduced capacity (2
L) is, however, still to be done.
Introduction of alveolar panels (see Figure 6C), made of PVC,
polycarbonate or polymethyl methacrylate, for microalga cul-
tivation has meanwhile emerged as a successful concept, because
of their high versatility and commercial availability. Several
systems using that type of panels have been built and duly tested.
Tredici (56-59) used double-row sets of alveolar plates placed
horizontally, where culture was circulated in the upper row and
thermostated water was circulated in the lower row. Tredici (59)
also described a bubble column FPR, in which alveolar plates
were mounted vertically, and the culture was mixed and
degassed simply by air bubbling at the bottom of each channel.
Its productivity was very high using S. platensis (2.15 g L-1
d-1), when compared with that obtained in open ponds under
similar conditions (0.15 g L-1d-1).
In general, the main advantages of FPR are their high
productivity and uniform distribution of light and, in the specific
case of bubbled column FPR, the absence of a driving pump.
Furthermore, these reactors can be oriented toward the sun,
hence permitting a better efficiency in terms of energy absorbed
from incident sunlight. Pusparaja (60) discussed a reactor
Figure 4. Schematic representation of helical tubular reactors: Biocoil (A) and conical framework (B).
Figure 5. Schematic representation of R-shaped reactor.
Biotechnol. Prog., 2006, Vol. 22, No. 6
encompassing an alveolar panel system oriented toward the sun,
coupled with an open raceway for gas transfer. The use of such
alveolar panels as solar receptors increased volumetric produc-
tivity from 0.18 g L-1d-1in open ponds to 0.31 g L-1d-1.
Although the volumetric productivity attained inside the panels
is higher, open raceways are the most often used cultivation
systems for microalgae, so said combination may be of great
practical significance. The main disadvantage of alveolar panels
is oxygen buildup, which arises from the high photosynthetic
activity reached, coupled with the small diameter of the reactors
used (57, 59).
2.3. Fermenter-Type Reactors. The least expanded systems
for microalga cultivation are conventional fermenter-type reac-
tors (FTR) (see Figure 7). These apparata present indeed an
intrinsic disadvantage: the area-to-volume ratio is quite low,
so sunlight harvesting efficiency is poor. To overcome this
nuclear drawback, sophisticated systems of internal illumination
were developed, which are able to provide a more homogeneous
distribution of light. When possible, microalgae may be het-
erotrophically cultivated in FTR, using appropriate organic
carbon sources. A 250-L FTR was built by Pohl (61) with
stainless steel and illuminated internally by fluorescent lamps
placed inside narrow glass (or Plexiglas) tubes; CO2-enriched
air was bubbled at the bottom, through a V-shaped (i.e., low
shear stress) stirrer. Such a system was operated batch-,
semicontinuous- and continuous-wise. The operation parameters
could be fully controlled, so axenic cultures were maintained
for long periods, as considered crucial for production of certain
high-value metabolites. Although biomass productivity was quite
low (typically 30-50 mg L-1d-1), there is still plenty of room
for enhancement of growth parameters with this reactor con-
Ogobona (62) has also developed an FTR using both sun and
artificial light, which may be a leap forward in terms of
reduction of operating costs.
Several laboratory-scale FTR have been developed (63-68),
but there is scarce information available on their large-scale
counterparts. The main advantage of these systems is of course
the accurate control of processing parameters, including light,
coupled with the vast experience accumulated in food and
pharmaceutical industries in terms of the scale-up thereof.
Therefore, should productivity be enhanced, FTR would cer-
tainly become a competitive alternative for industrial manufac-
ture of biochemical products brought about by microalgae.
3. Processing Parameters
Effective supply of light and carbon dioxide to the whole
cell culture, as well as other physical and nutritional require-
ments, demands uniform dispersion of microalgae in a non-
limiting nutrient culture medium. Since adequate mixing levels
in many reactor configurations are often obtained via injection
of gas into the system, mixing and gas transfer are thus closely
related to each other.
3.1. Gaseous Transfer
3.1.1. Carbon Source. Because nearly 50% of the whole
microalgal biomass is made up of carbon (69), this element is
a major nutrient for cell growth. When grown photo-litotro-
phycally, all microalgae use inorganic carbon sources to
synthesize organic compounds (70). In aqueous environments,
inorganic carbon may exist in several alternative chemical forms,
CO2(aq), H2CO3, HCO3-and CO32-(71), which are intercon-
vertible via reactions controlled by temperature and pH (69).
The possibility that microalgae use carbon in CO2form only
or that they also take up the HCO3-and CO32-forms is not a
critical issue, because reactions that interconvert CO2, H2CO3,
HCO3-and CO32-in soluble form are sufficiently fast not to
be limiting steps in CO2 demand by cells (14, 71). Besides,
e.g., for Chlorella Vulgaris, inorganic carbon affinity at the cell
surface was found to be very low (not above 1 µg carbon/L)
(71), and synthesis of carbonic anhydrase (which catalyzes the
aforementioned inorganic carbon interconversion) is enhanced
when cells are exposed to a low-CO2environment.
Detailed studies on the influence of the carbon source upon
microalga productivity (71) have indicated that, although HCO3-
is easily absorbed by cells, it is a poor source of carbon when
compared with CO2. In fact, it is possible to achieve a linear
response in microalgal carbon biomass with mass input of
Figure 6. Schematic representation of flat panel reactors: flat panel
bubbled in the bottom (A), V-shaped panel (B) and alveolar panel (C).
Figure 7. Schematic representation of fermenter-type bioreactor.
Biotechnol. Prog., 2006, Vol. 22, No. 6
carbon (which corresponds to an efficiency of virtually 100%)
only if limited inputs of inorganic carbon and narrow pH ranges
are permitted; e.g., Phaeodactylum tricornutum produced up to
25 mg d-1of algal carbon when pH of the culture was below
9.0 (71). Beyond a given threshold, pH control becomes difficult,
so chemical precipitation of salts containing CO32-, OH-and
PO43-will likely occur, hence leading to chemical deterioration
of the medium and possible cell injury. The upper threshold of
productivity is therefore considerably lower than that resulting
from light limitation, and about 1/10 that obtained by replacing
HCO3-by CO2. A general consensus exists about preference
of microalgae for CO2(as inorganic carbon source), as it is easily
controlled and produces minor pH changes. Note that CO2in
the open air accounts for only ca. 0.03% (v/v) (69), so fluxes
of carbon transfer to the culture are small, even in the presence
of extended interface areas or enhanced mixing. Consequently,
CO2-enriched air is the most commonly employed nutrient gas
mixture, so as to force light to become the sole limiting factor.
3.1.2. Transport Process. When CO2is injected at a given
point in a culture, a concentration gradient builds up as it is
consumed by cells and/or lost to the atmosphere. According to
the two-film theory, mass transfer of CO2from the gas phase
to the cell phase occurs through sequential stages: transport
from the bulk of the gas to the thin gaseous film at the immediate
vicinity of the interface; diffusion through this gas film; transport
across the gas/liquid interface; diffusion through the adjacent
liquid film; transport from the thin liquid film to the bulk of
the liquid; transport from the bulk of the liquid to the thin liquid
film at the immediate vicinity of the cell wall; diffusion through
the outer cell liquid film; and finally, metabolic uptake by the
cell. The overall resistance over the entire path distance can be
calculated by adding up the aforementioned single resistances,
as they occur in series; however, most of the resistance actually
lies on the liquid film. In an efficiently stirred bioreactor,
concentration gradients within the bulk liquid are marginal, so
(even in the presence of dense microalgal cultures) resistance
within the gas bubbles is larger than at the boundary layer of
cells (14). Consequently, resistance raised by the liquid film
adjacent to the interface essentially limits the rate of CO2
transfer. In fact, comparative studies (14) encompassing overall
mass-transfer resistances and gas/liquid mass-transfer resistance
revealed their similarity in order of magnitude, thus confirming
that CO2transport is mainly controlled by resistance offered
by the liquid film.
The rate of CO2uptake by cells eventually determines the
rate at which it will be transferred to the medium, if steady-
state conditions prevail. The rate of mass transfer is, in general,
proportional to the driving force for said transfer (expressed in
terms of a concentration difference involving one actual bulk
concentration of one phase and an equivalent bulk concentration
of the other phase) and the area available for transfer. The
proportionally coefficient is the sum of the reciprocals of all
resistances to transfer and is usually denoted as an overall mass-
transfer coefficient. In the present case, since liquid-phase mass
transfer resistance dominates overall resistance, the rate of mass
transfer of CO2(NCO2) is approximately given by
where kLis the liquid-phase mass transfer coefficient, a is the
specific area available for mass transfer, CCO2L* is the concen-
tration of CO2in the culture medium that would equilibrate its
actual partial pressure on the gas side, and CCO2L is the
concentration of CO2in the bulk of culture medium.
The lumped parameter kLa characterizes the CO2 mass
transfer capability of the reactor and thus determines whether a
certain reactor will be able to sustain a given rate of cell growth.
Regarding transfer of CO2, said parameter is of the utmost
importance in design, scale-up and operation steps for a biomass
culture system (72). Some properties of the bubbles affect
mainly kL, whereas others affect mainly a. Furthermore, the
amount of metabolites present in the medium depends on cell
concentration, which will also affect kLa since, in general,
metabolites modify the surface tension of the medium and thus
act as an extra barrier to mass transfer (73).
Studies (73, 74) have been undertaken to compare kLa values
obtained under bubbling (at different gas and liquid flow rates
and at distinct bubble sizes) and under diffusion through hollow-
fiber membranes (of different areas and distinct construction
materials); kLa typically increases with increasing liquid tan-
gential flow rate, because of thinning of the liquid boundary
layer. When comparing the rate of mass transfer obtained under
bubbling or by diffusion (with all other operating conditions
remaining similar) (75), it was observed that kLa values were
higher in the latter case: an overall volumetric coefficient for
CO2transfer of 1.48 × 10-2min-1was found for a hydrophobic
membrane, 1.33 × 10-2min-1for a hydrophilic membrane and
7.0 × 10-3min-1for plain bubbling (see Table 4). Such
increasingly lower values are a consequence of the substantially
shorter interfacial area available; in fact, the individual volu-
metric mass transfer coefficient values (kL) were higher for
bubbling (1.11 × 10-2m s-1) than for diffusion (1.45 × 10-7
and 1.59 × 10-6m s-1for hydrophobic and hydrophilic
membranes, respectively), because of the turbulent conditions
associated with the former. Therefore, use of microporous
hollow fibers instead of plain bubbling offers technological
enhancements in effectiveness of mass transfer at the expense
of larger a. In addition, this type of system offers opportunity
to recirculate gas and hence permits lower gas pressures to be
used, which in turn reduce operating costs. However, Carvalho
and Malcata (75) have demonstrated that overall metabolic
enhancement in microalgal growth is essentially nil, probably
because CO2- and light-rich periods did not coincide with each
other (both in space and time).
Other studies (71) pertaining to continuous cultures limited
in carbon afforded interesting relationships between bubble size,
gas flow rate and partial pressure of CO2. Optimum biomass
productivity was obtained by using either high bubbling rate
Table 4. Values for KLand KLa Using Several Gas Transfer Methods
1.48 × 10-2
1.33 × 10-2
(3.6-7.5) × 10-3
7.00 × 10-3
(7.59-21.7) × 10-2
(9-94) × 10-2
1.4 × 10-1
2.38 × 10-3
1.1 × 10-3
1.45 × 10-7
1.59 × 10-6
(1.26-2.64) × 10-5
1.11 × 10-4
(5.83-5.88) × 10-4
(3.51-10.6) × 10-5
aHIFO, hollow hydrophobic fiber membrane; HIFI, hollow hydrophilic fiber membrane.bNot available.
NCO2) kLa(CCO2L* - CCO2L) (1)
Biotechnol. Prog., 2006, Vol. 22, No. 6
(with small sized bubbles) with low inlet pressure of CO2or
low bubbling rate with high inlet pressure of CO2(irrespective
of bubble size). Although more efficient (47% vs 14%, in terms
of assimilation efficiency), the former option could bring about
problems of cell flotation and consequent washout. At 1% (v/
v) CO2, productivity was essentially independent of bubble size,
probably because partial pressure in the culture was already high
enough; it is indeed possible to achieve light limitation under
these conditions. In conclusion, total carbon input flux is
determined by both gas bubbling rate and partial pressure of
CO2. Selecting the proper combination of these two variables
is the key to avoid carbon limitation in intensive culturing.
In terms of biomass produced, the differences in value
between the several parameters affecting it make an overall
comparison difficult to establish. Concerning production costs,
the literature is scarce; hence, reliable economic data are to be
generated in the future, before a consistent comparison of
performances can be accurately done.
3.1.3. CO2Transfer Systems. In algal mass culture systems,
it is important to obtain a reliable prediction of CO2transfer
rates for accurate design, scale-up and operation. The occurrence
of chemical reactions between CO2and OH-, H2O and NH3in
the liquid phase may lead to enhanced rates of CO2absorption
by the culture medium (72); therefore, the CO2exchange flux
from the gaseous to liquid phase is governed not only by
diffusion kinetics, but also (depending on the relative magnitude
of the reaction rates observed) by kinetics of the reactions in
the liquid film at the vicinity of the interface.
The critical concentration of CO2 necessary for optimal
growth of a particular microalga cannot be stated in general, as
it strongly depends on the delivery system implemented in the
culture vessel. Since transfer of CO2occurs through the interface
between the gaseous mixture and the liquid medium culture,
two main processes to increase such an interface area can be
devised: (i) passive mode, where extensive gas/culture interface
areas are used and gas diffuses into the culture; and (ii) active
mode, where use of an extra apparatus for aeration either by
injecting the gas into the medium or spraying the medium into
the gas forces expansion of the contact area between gas and
culture (69) (see Table 5).
Regarding passive mode, large interface areas between the
gas and the culture medium can be obtained by: either (i) using
large open-air ponds, where gas exchanges occur by surface
driven aeration (see Figure 8A), e.g., cultivation of D. salina
in Australia, in natural lakes of 50-300 ha, or similar cultivation
of Chlorella and Spirulina spp. in paddle-wheel (or circular)
mixed ponds of ca. 1 ha; or (ii) via membranes, through which
gas diffuses into the culture. No examples have been reported
pertaining to surface aeration in closed systems, probably owing
to the huge areas that would be involved. Conversely, membrane-
mediated transfer has already been tested in closed systems
When membrane aeration is considered, gas diffuses through
a permeable membrane, which can be either microporous or
made of a material possessing high gas permeability (e.g.,
silicone). The membrane is often arranged as a coil or bank of
tubes, placed inside the reactor vessel (77). Alternatively, the
bank of tubes may be contained in an appropriate housing, which
will work as a gas exchanger, connected to the reactor vessel
by plastic tubing (75). One configuration tested with success
encompasses diffusion of pure CO2through permeable silicone
tubing, coiled up in order to maximize transfer area in a reduced
space (76). This kind of system theoretically offers several
advantages when compared with bubbling, viz., exclusion of
CO2losses to atmosphere, possibility to accurately control CO2
transfer rates, and no requirement for air/CO2mixing chamber
or even highly pure CO2(as it will not be in close contact with
the culture). Better overall efficiencies are expected for this
system when compared with bubbling (13-20%) or gas
exchange (25-65%) (76). However, such a system suffers from
severe drawbacks: (i) since the transfer rate is proportional to
the membrane area, long tubing membranes are normally
required, which raise the investment costs (21); (ii) especially
for highly salted media, high inner pressures are necessary in
order to balance transfer rates produced by bubbling, which force
the use of thick membrane walls (the only ones that can handle
such high pressures); and (iii) pressurization of the membrane
often promotes expansion of the material, thus originating
microspacing within the polymer network of the membrane, to
which bacteria may adhere and eventually grow, hence reducing
contact area between membrane and culture, and decreasing
transfer rate throughout time.
An alternative possibility relies on the potential of mi-
croporous hollow-fiber membranes, i.e., bundles of polymeric
porous fibers, potted to inlet/outlet ports in their ends and
contained in adequate housings (74, 75) (see Figure 8B), which
may also function as gas exchangers in the setup or as the whole
reactor itself. Commercial fiber modules usually contain tiny
hollow fibers, with typical inner diameters of ca. 250 µm; their
permeability obviously depends on the construction material.
As a result of the usually large number of fibers inside each
module, the ratio between membrane exposed area and external
Table 5. Generic Description of Methods for Gas Transfer in Photobioreactors
process type of reactoralgaref
namic stress scale-up
5 poor very poor very low difficult
Nannochloropsis sp.50 excellentuniforma
at bottom of
at given point
27good good (by
aDepends on mixing device.bLarge bags.
Biotechnol. Prog., 2006, Vol. 22, No. 6
volume of the module is typically high. Although such an
indirect way of supplying CO2by diffusion has similarities with
the process described above involving silicone tubing (76), this
system configuration makes it possible to use lower gas pres-
sures, as no need to counterbalance hydrostatic heads exists
According to Fick’s law, mass transfer flux increases with
transmembrane pressure and recirculation flow rate. Although
rises in either of these operating parameters would contribute
favorably, pressures are upperly constrained by mechanical
limits of the equipment (typically 40 psi). Hence, flux enhance-
ment usually takes advantage of increases in recirculation flow
rate, whereas inlet pressure is maintained close to the maximum
permitted. Use of hollow fiber devices has been reported for
culture of mammalian cells (78), in culture of the microalga
Chlamydomonas reinhardtii (79), in continuous culturing of the
microalga P. tricornutum in seawater (80), and in culture of
the cyanobacteria Anabaena Variabilis (81).
Figure 8. Schematic representation of major methods of gas exchange in microalgal reactors: surface driven aeration (A), microporous hollow-
fiber membranes (B), airlift loop (C), bubble column (D), stirrer blade bubbling (E), and gas exchanger system (F).
Biotechnol. Prog., 2006, Vol. 22, No. 6
In regard to active mode, two main transfer processes are
usually considered: (i) gas mixture is circulated together with
culture, either injected at a certain point of the system (riser
tube) (16, 17, 28, 41, 45, 53, 54, 82-90) or bubbled at the
bottom of the reactor vessel (29-32, 34-36, 46, 51, 52, 55,
56, 61-63, 91, 92); or (ii) gas is inserted into the culture when
it passes through a gas exchanger (44, 50) (see Table 5).
Bubbling of CO2-enriched air at the bottom of the reactor is
the most frequent mode of operation; it is usually performed
via sintered porous stones or pipes where tiny holes were
previously drilled, although gas can also be bubbled from the
perforated blades of a stirrer (see Figure 8C-E). For larger
reactor vessels, holes are usually drilled in the upper part of a
long, sealed pipe, which is placed longitudinally at the bottom
of the reactor. Such parameters as bubble size, gas flow rate
and CO2pressure can then be adjusted to meet the requirements
of each specific culture (72). In order to avoid microalgal
accumulation in certain (stagnant) areas of the bottom region,
the gas stream can be injected from the sides rather than on the
axis at the bottom, coupled with continuous stirring of the
bottom with a magnetic stirrer (69). Unfortunately, two major
drawbacks are often observed: (i) obstruction of the gas transfer
devices by biofouling, which requires frequent stops for
cleaning; and (ii) losses of CO2to the atmosphere, owing to
the low residence times of the gas in the culture (which increase
considerably operation costs, as CO2 is an expensive utility)
(21). Losses to the atmosphere can be reduced via installation
of a covered sparging-diffusion system, consisting of a transpar-
ent material tightened to a frame mounted below the waterline
(69); nevertheless, it is usually difficult to reach efficiencies
above 10% (69). By introducing the gas directly into the circuit
or via interconnected gas exchangers (see Figure 8F), it is
technically possible to saturate the culture suspension. In the
former situation, the gas is introduced into the circulation path
and is distributed over one or more parts of the reactor (usually
of tubular shape) through tiny holes. When using gas exchang-
ers, CO2 utilization is, in principle, rather efficient because
inflowing algal suspension is dispersed with a baffle plate and
flows down the walls of the cylinder as a thin film, through
which gas exchange can rapidly occur (69). However, this
configuration is effective, in terms of adequate gas supply, only
in small reactor systems. In fact, although levels of ca. 30 ppm
of CO2on the cell surface are considered sufficient to maintain
unlimited photosynthesis (69), experimental evidence has shown
that, after a 100-m path, microalgae have already used all gas
available, so further gas needs to be supplied (93).
Increasing awareness of the importance of CO2 led to
development of control systems, able to regulate the pH of the
culture and thus, indirectly, control the amount of CO2supplied.
The most common system employed for pH control is the on-
off type, in which CO2is injected into the culture when pH is
above a desired setpoint. However, the dynamics of the system
(e.g., mixing times) do not allow a fast response to disturbances.
An alternative is the use of model-based predictive control
(MPC), which relies on a model able to predict the process
output at future time points (horizon)-by calculating the control
sequence that minimizes a certain objective function and
implementing a receding strategy so that at each sampling instant
the horizon is shifted toward the future. When classical on-off
control was replaced by MPC (taking solar irradiance effect into
account), carbon losses in tubular reactors with P. tricornutum
were reduced from 19.7% to 5.5% (94). The main advantage
of using on-off MPC instead of a classical on-off controller is
the predictive capability of the former and the selection of an
adequate sampling time. The use of a model to predict future
behavior based on on-off signals helps to anticipate (and account
for) the delay associated with a cycle time, taking also into
account the on-off nature of the control signal (95). Other types
of mathematical models can predict the photosynthesis rate
(based on O2generation rate and CO2consumption rate) as a
function of solar irradiance (89). Such models also permit
accurate determination of the CO2requirements of the system,
thus allowing optimized use of this resource as well.
3.1.4. O2 RemoWal. As stressed above, light intensity is
regarded as an essential issue to be addressed in microalgal
systems; however, it is not the only potentially limiting factor
in photosynthesis, which is chemically described by
In fact, if oxygen build-up occurs (i.e., when the concentration
of dissolved oxygen in the culture is above its counterpart in
equilibrium with its partial pressure in the atmosphere), the
aforementioned reversible reaction is shifted to the left, thus
decreasing photosynthetic efficiency; in general, oxygen con-
centrations above air saturation inhibit photosynthesis in mi-
croalgae (46). Besides, accumulation of O2in the liquid culture
medium is one of the most difficult problems to overcome (58),
because it may become toxic above a certain threshold; dissolved
oxygen concentrations above 35 mg/L, which are toxic to most
microalgae, were reached in outdoor cultures of Spirulina (70).
This problem has not been reported in the literature pertaining
to most types of small-scale culture devices, probably because
the gas transfer systems employed were sufficiently powerful
to provide enough carbon dioxide and promptly remove the
oxygen concomitantly formed. However, when larger systems
are implemented, the magnitude of said problem increases
exponentially; it has even been claimed (46) to be responsible
for failures in a commercial device constituted by several-
Accumulation of photosynthetically generated oxygen be-
comes a particularly serious problem in high area-to-volume
ratio closed photobioreactors operated outdoors; among these,
HTR are described as particularly problematic. Most solutions
proposed to date rely on use of a degasser (or gas exchange
unit), where dissolved oxygen can be released (17, 20, 51, 53,
83, 87, 89, 90); however, to attain an effective separation
between gas and liquid, the distance between entrance and exit
of the degasser should be such that the smallest bubbles will
have a sufficient time to disengage from the liquid before it
leaves the unit. In closed tubular photobioreactors, in which
axial gradients in concentration of gases can develop, connec-
tions between the several tubes can also incorporate a narrow
tube for oxygen degassing (40). Other authors (41) tested the
performance of a NHTR, essentially consisting of a layer of
tubes arranged in parallel and connected by two manifolds: the
lower was used to inject air into the culture, whereas the higher
acted as a degasser; however, productivities were lower than
expected, probably due to build-up of oxygen tension during
periods of high light intensity. Especially when the exhausting
gas is recirculated, accumulation of oxygen may be avoided by
bubbling the exhaust gas through a sodium sulfite solution prior
to its return to the reactor (11).
It should be emphasized that the aforementioned devices for
oxygen degassing are only necessary when the reactor config-
uration does not provide an interface between culture and
surrounding atmosphere, e.g., reactors with tubular or flat panel
configuration. In stirred tanks and in the various bubbling-type
reactors available, oxygen leaves the culture when it reaches
energy + CO2+ H2O T sugar + O2
Biotechnol. Prog., 2006, Vol. 22, No. 6
the surface, hence allowing simultaneous degassing of the whole
culture. To overcome this incompatibility between a high area-
to-volume ratio (which characterizes tubular and flat panel
reactor configurations) and efficient gas transfer (of both oxygen
and carbon dioxide), a vertical alveolar panel was devised (56),
in which air with extra carbon dioxide is sparged at the bottom
plates and oxygen bubbles leave the liquid when they reach
Since the efficiencies of all techniques used to date to provide
oxygen removal from microalgal cultures are still not at a
satisfactory level, a competitive mode of operation (especially
in large culture systems) consists of using bubbling devices,
because they eliminate the need for (the still ineffective)
degassing devices. Another alternative relies on use of hollow-
fiber membrane apparata, as they seem to lead to lower dissolved
oxygen concentrations than bubbling systems do (74). The use
of several small-sized reactors instead of one larger unit may
also help alleviate this problem. Nevertheless, further research
should be conducted in order to accurately match the amount
of CO2 supplied to the actual requirement of the growing
microalga, and the amount of O2removed to the actual amount
In view of the above, one concludes that there is no
universally optimum gas transfer device; the choice of the most
suitable one depends on the microalga considered and the
objective function preselected.
3.2. Mixing. After fulfillment of nutritional growth require-
ments and assurance that environmental factors are non-limiting,
mixing becomes a critical parameter. In reactor configurations
that consist of more than one vessel (e.g., a carbonation tower
for gas exchange, coupled to one or several tubes for light
supply), adequate mixing is particularly required in order to
recirculate the culture between vessels. Even in the case of single
vessel reactors, efficient mixing should be provided in order to
produce a uniform dispersion of microalgae within the culture
medium, thus eliminating gradients of light, nutrient concentra-
tion (including CO2) and temperature; note that thermal
amplitudes of up to 8 °C have been recorded between the top
and bottom of unmixed cultures, leading to irreversible damage
(96). Finally, the effect of “mutual shading”, i.e., continuous
cell movement from and to dark/light zones, has been claimed
(69) to be essential to guarantee high biomass productivity. In
a dense culture, the region where microalgae receive enough
light for photosynthesis can be quite shallow (2-5 cm) (69),
so vigorous mixing is necessary to provide cells with a uniform
average exposure to light.
Current mixing techniques are often insufficient to attain the
aforementioned requirements, because the induced turbulence
is random, i.e., not all algal cells are submitted to a uniform
pattern of movement into and out of the illuminated area of the
reactor. Furthermore, mixing should be supplied to proper
extents, because very low values promote settling and hence
emergence of dead zones (especially toward the bottom of the
reactor) where anaerobic conditions prevail, thus leading to cell
deterioration and consequent decrease of productivity; in the
limit, toxic compounds will form that might compromise
viability of the whole culture (69, 70). Inadequate mixing also
permits clumping of cells into aggregates of varying sizes, hence
leading to development of a three-phase system (gas/liquid/solid)
inside the reactor, which is prone to decreasing mass transfer
rates (6). On the other hand, high mixing rates may lead to shear-
induced injury of cells (22, 23), which hamper their viability.
The most important mechanism of cell damage in sparged
cultures agitated at low intensities is bubble break-up at the
liquid-gas interface (i.e., microalgal cells are captured by the
rising gas bubbles and carried to the surface, where they die as
bubbles collapse). At higher agitation intensities, cell damage
caused by fluid-mechanical stress acquires a greater significance
as the specific aeration rate decreases (97).
The major mixing/recirculation systems commercially used
are described in Table 6. They can be tentatively divided in
pumping (used when more than one vessel is present), mechan-
ical stirring (usually present when only one vessel is used) and
gas mixing (which takes advantage of a gas, usually CO2-
enriched air, injected in the culture to promote turbulent mixing
and recirculation through the various vessels). Combinations
of these systems are also possible. Former recirculation systems
used exclusively pumps, either centrifugal, positive displace-
ment, peristaltic or diaphragm ones. As research progressed, it
was soon realized that cultures recirculated with distinct
pumping apparata attained different maximum specific growth
rates, and that the adverse effects caused by centrifugal and
rotary positive displacement pumps were essentially proportional
to their rotation speeds (38). The extent of damage observed in
various pumps was accordingly measured as a function of the
number of passes of algal suspension through the pumps in a
closed loop, and the concept of hydrodynamic stress was thus
introduced, which provided a framework to assess parameters
that determine magnitude of shear stress phenomena: geometry
of the reactor, type of pump involved, and morphological and
physiological conditions of the microalga (22, 24, 25).
Gas mixing systems, i.e., bubble column systems, cause less
extensive damage to fragile microalgal species than mechanical
pumping does. This is especially the case of air-lift units, in
which mixing is achieved by fluid flow obtained from sparging
air into a central draught tube (riser), where it decreases bulk
Table 6. Generic Description of Methods for Mixing in Photo-Bioreactors
process type of reactormicroalgaref
namic stress scale-up
rotary positive displacement
fair lowmedium easy
Mechanical Stirring Mode
Gas Mixing Mode
stirring with two or more bladescylindrical tank 48 uniformfair/highhigh medium
injection of gas
Biotechnol. Prog., 2006, Vol. 22, No. 6
liquid density hence causing the liquid to rise. The liquid then
flows downward through the outer tube, thus creating a natural
circulation. Although these systems appear to cause the least
extensive degree of cell damage (21), they are not completely
devoid of shear stress: a cell-damaging hydrodynamic effect
has been reported (46) in bubble columns and airlift reactors,
which was associated with so intense turbulence patterns that
the length scale of the fluid microeddies approached cellular
dimension. Barbosa (26) reported bubble formation at the
sparger as the main event leading to cell death. Furthermore,
increasing cell density leads to extra difficulties in producing
homogeneous cultures, so the need to provide mechanical
agitation arises (associated with lower speeds, so as to keep
shear stress to a minimum) (98). Subsequent studies on the
influence of increasing power supply on decreasing of mi-
croeddy length scale demonstrated that this scale was always
much higher than cell size, and therefore shear stress rather than
turbulence was responsible for the existence of stress phenomena
(27). Extra stirrers may provide such a mechanical agitation,
with one or more blades. Another alternative lies in the
introduction of baffles along the culture path, so as to create a
controlled turbulence pattern. Degen (99) used horizontal baffles
to subdivide the riser on a flat panel airlift photo-bioreactor,
thus creating a defined circulation path and exposing the cells
to intermittent light, hence creating a flashing-light effect.
When the reactor configuration corresponds to only one
vessel, the microalgal suspension can be agitated by means of
a stirrer, which usually consists of a central tube with two or
more blades. In order to enhance mixing without damaging cells,
a hybrid configuration has also been described (69) in which a
stream of CO2-enriched air is passed via the central tube into
the blades, from where it penetrates the medium through
capillary holes. This mixing device is the most usual in
As distinct microalgae have different sensitivities to shear,
there is no single optimum mixing system for all microalgal
species; the microorganism in use will again be the determinant
issue when evaluating mechanical and hydrodynamic shear
effects that are admissible during regular operation of the photo-
3.3. Light Requirement. Light is the basic energy source
for photo-autotrophic organisms, as microalgae are; hence, their
light harvesting efficiency is of crucial importance for bioreactor
engineering encompassing those microrganisms. The photosyn-
thetically active radiance (PAR) is normally assumed to be ca.
43-45% in the wavelength range 400-700 nm (100, 101). The
photosynthetic efficiency (PE) is defined as the fraction of
available light that is eventually stored as chemical energy in
biomass; however, there is no general consensus on how to
calculate its actual value. Note that PE is a theoretical upper
limit. In practice, especially in long-term cultures, PE is normally
below 6.5%, and under optimal conditions, only 40% of the
theoretical maximum yields can typically be reached (86).
Hence, increase in PE is likely the best approach in attempts to
enhance microalga productivity, which is a sine qua non
condition to guarantee commercial competitiveness of the
In order to estimate PE for a selected species in a given
reactor, one departs from calculation of the illuminated surface
area per unit volume of the culture, AV; in the case of a VTR
(probably the most popular reactor configuration), one has (53)
where r and ro are the inner and outer radius of the tubes,
respectively, provided that the tubes hold no gaps between them.
Applying the well-studied microbial-growth energy equation,
after the reasoning detailed by Pirt (83), one is led to
where q is the specific rate of light uptake, µ is the specific
rate of growth, YGis the maximum growth yield on light, and
m is the maintenance coefficient. The value of q may, in turn,
be calculated via
where φ is the fraction of photosynthetically available light, Io
is the total incident irradiance, and X is the biomass concentra-
Combining eqs 4 and 5 and assuming a light-limited culture
with a maintenance coefficient virtually nil, one obtains the
biomass output rate (µ‚X) as
In a reactor operated under a constant AV and a given Io, it is
possible to adjust µ and X (eq 6) so as to achieve the maximum
biomass yield (YG), which then also leads to the maximum PE
If a proper reactor design is sought, it is crucial to obtain the
maximum biomass yield at the expense of the light actually
available. The latter parameter depends on the reactor config-
uration and location. If one considers reactors illuminated by
direct sunlight, the maximum Iodepends on the latitude of the
facility, so it can be controlled only by changing the reactor
inclination toward sunlight (28). Several reactors have indeed
been developed or tuned accordingly (59). On the other hand,
in artificially illuminated reactors, the maximum Iodepends on
the nature, intensity and relative position of the light source,
all of which can be accurately manipulated and controlled.
Most reactors are normally designed so as to exhibit a high
AV; it is thus possible to work at higher biomass concentration
for a given yield (YG). Good examples of reactors designed to
harvest the maximum light are the alveolar panel reactor by
Tredici (59), and the tubular reactors by Grima (45), Richmond
(17) and Zittelli (41). Reactors that combine natural and arti-
ficial lights have also been described (3, 62). In terms of
artificially illuminated reactors, the need of small diameters to
increase AV can be circumvented via provision of internal
illumination. The higher biomass yield (YG) can be even more
important in the case of artificially illuminated reactors, as the
light provided represents a cost itself that adds to the overall
running costs of the process. Nevertheless, such cost may be
kept limited, as the latest few years have witnessed the
development of methods for in situ growth monitoring-from
flow injection analysis systems based on turbidimetric measure-
ments (68) to techniques based on real-time monitoring and
control of biomass by using light transmittance sensors (102).
The light energy available for each microalga cell (Iav)
depends on several factors, e.g., Ioand biomass concentration.
Several authors (103) have demonstrated that increasing light
intensity increases growth rate, but only until a saturation point
is reached. Above this threshold, increasing light produces no
positive effect on growth rate, hence generating a dramatic
decrease in YG, as a major fraction of light cannot be used by
AV ) 2
µ‚X ) φ‚Io‚YG‚AV
Biotechnol. Prog., 2006, Vol. 22, No. 6
the culture. Furthermore, the light will influence not only
biomass productivity, but also its biochemical profile (15, 19,
An interesting method to assess Iavon tubular reactors, based
on Ioand X, was developed by Evers (104) and later applied by
Grima (64). This model assumes that light attenuation caused
by mutual shading is dependent on Beer-Lambert law, which
calculates the light available at each single point at a given
distance from the surface, followed by integration on both the
cross sectional and the outer surface areas. Use of this model
allows one to fit Ioin order to work below the saturation point,
hence avoiding a decrease in YGand/or an adjustment of Iavfor
a more consistent biochemical profile.
Sunlight and artificial illumination data are compared in Table
7. The source of artificial illumination is of crucial importance,
because the wavelength spectrum should be specific in order
to take full advantage of the total Iomade available. As seen
before, PAR lies within the wavelength range 400-700 nm, so
a lamp with a vast spectrum and with its maximum radiation in
that range is normally chosen. However, different lamps will
generate distinct spectra; on the other hand, each microalga
species possesses its own absorption optimum, so each indi-
vidual case must be studied per se. You and Barnett (105) related
a dependence of the exponential growth rates on the energy of
radiation and spectrum of light, and concluded that blue light
(400-500 nm) increased cell growth and polysaccharide
production in P. cruentum.
The method to evaluate the efficiency of conversion, de-
scribed by Simmer (106), takes into account the photon flux
(P) emitted by the lamp in the PAR wavelength, the specific
absorption coefficient of each species and the quantum capacity,
hence allowing one to estimate the efficiency of conversion
where ERF is the emitted radiant flux, which is a function of
the wavelength, λ.
It is our belief that long-term trends of research in the field
of microalgae should encompass design and development of
reactor systems, as well as microalgal strains able to increase
specific metabolite productivity.
Whereas open raceway ponds are presently the primary
systems used in commercial production of microalgal biomass
outdoors, there is not a single recommended design in what
concerns closed systems. Closed systems are indeed more
complex than their open counterparts, thus requiring improved
technological skills to operate them and higher investment costs
to house them; otherwise, they possess the advantage of more
accurate control of various parameters during regular operation,
hence enhancing productivities.
The main challenge to the microalga research community now
appears to lie on the design of a closed system so effective in
biomass and metabolite productivity that it will eventually
overcome the extra investment cost required, and thus become
profitable and fully competitive.
To attain maximum productivity, several parameters are to
be controlled, viz., sufficient nutrient level and optimum
temperature and pH (topics not covered here, owing to limitation
of space), as well as adequate mixing and turbulence, coupled
with provision for oxygen degassing and light control (which
were the focus of this specific review). From the many reactor
configurations built to date (viz., HTR, VTR, HeTR, FPR and
FTR), none is able to effectively control all of those parameters
simultaneously. Some are indeed optimized for better sun-
harvesting capacity, but scale-up is not suitable; conversely,
others can be better controlled, but sun-harvesting efficiency
On the other hand, since continuous supply of CO2 to
microalgal cultures is expensive, it is necessary to provide
devices that supply it in a discontinuous fashion. In fact, during
the first few hours (or days, depending on the duration of the
lag phase) following inoculation of a batch culture, there is a
large amount of CO2 dispersed in the medium; during the
exponential growth phase, essentially all CO2supplied is used
up; and finally, in the stationary phase, the rate of utilization of
CO2declines again. The supply of CO2to the medium is in
general controlled via monitoring pH; nevertheless, such
underlying relationship may lead to erroneous values due to
the influence of other dissolved ions on pH. Consequently, it
would be more correct to measure CO2directly with a probe or
to choose another parameter closely related therewith. Maxi-
mization of CO2transfer may proceed via hollow-fiber mem-
branes, which enhances mass transfer and decreases costs, by
recirculating unused gas and using lower gas pressures.
In scale-up of culture devices, some of the conditions that
were easily controlled at a small scale may become difficult
problems. One such situation concerns degassing of oxygen,
which is believed to be responsible for failure of commercial
devices. There is not a single, straightforward solution, and a
feasible approach consists of using several small units for growth
rather than one larger unit. In this way, the various units may
function separately (depending on the production needs), and
the gaseous transfers will be more efficient, while minimizing
O2build-up. However, economic studies should be performed,
in order to adequately evaluate the feasibility of this solution.
When using hollow-fiber membrane devices to bring about
exchange of gases within the culture, the content of dissolved
oxygen was found to be lower than with the bubbling system,
so it may constitute a suitable alternative in terms of gaseous
changes. Another approach relies on use of bubbling devices,
as they eliminate the need for (the still ineffective) degassing
Concerning light intensity, operational issues are also not clear
cut. On the one hand, use of sunlight is cheaper but the light
cycle cannot be controlled, which often precludes higher biomass
productivities. On the other hand, artificially illuminated reactors
are typically expensive. The use of optical fibers (contaminated
with impurities that will lead to gradual dispersion of light) that
can introduce light inside reactors with large volumes and
effective monitoring of the light available for photosynthesis
Table 7. Generic Description of Methods for Light Supply in Photo-Bioreactors
modecost intensity control land area requiredreactor design productivity
sunlightsmall problematichigh must be designed for high
allows great flexibility
depends on weather
constant artificial lighthigh excellentsmall
Biotechnol. Prog., 2006, Vol. 22, No. 6
will probably constitute a breakthrough-that will eventually
turn closed reactors into fully competitive options.
In the past few years, studies on photo-bioreactor sensing
have also appeared, which presented innovative approaches to
assess photobioreactor states, hence allowing estimation of such
parameters as biomass density, specific growth rate, dissolved
oxygen concentration, photosynthetic efficiency and average
light intensity (107). Such type of control is expected to help
in decreasing operation costs. In addition, the continuous reuse
of biomass for harvesting of high-value compounds from
microalgae (“milking of microalgae”) has also been addressed
as a potential technology of production (108). Although such a
method has been employed for the production of ?-carotene
from D. salina, it is not applicable to all strains or products.
Studies on the physiological behavior of cells, particularly cell
membranes (among others), are still necessary before developing
a generic “milking” process.
Finally, the intrinsic characteristics of the microalgae selected
for use should also guarantee technological suitability. Selection
of strains should thus emphasize such features as specific growth
rate, tendency to clump and settle, ability to foam, adherence
to container walls, and shear and temperature sensitivities. In
addition, existing species should be carefully screened in order
to select the most performant strains, which hold the capacity
to overproduce specific end products with a market value. The
performance of those species could be even reinforced via
genetic engineering, using tools already available.
Mrs. Gisela Oliveira is hereby gratefully acknowledged for
her kind help in bibliographic search. Financial support for
A.P.C. (grant BD/2838/93-IF) and L.A.M. (grant BD/15890/
98-FCT) by PRAXIS XXI (Portugal) is also to be noted. Partial
funding was obtained from a project grant (MICROPESCA/
0072/04), issued by program MARE (Portugal).
References and Notes
(1) Soeder, C. J. A historical outline of applied algology. In Handbook
of Microalgal Mass Culture; Richmond, A., Ed.; CRC Press: Boca
Raton, FL, 1986; pp 25-41.
(2) Burlew, J. Current status of the large-scale culture of algae. In Algal
Culture: From Laboratory to Pilot Plant; Burlew, J. S., Ed.;
Carnegie Institution of Washington Publications: Washington, DC,
1976; pp 3-23.
(3) Chaumont, D. Biotechnology of algal biomass production: a review
of systems for outdoor mass culture. J. Appl. Phycol. 1993, 5, 593-
(4) Borowitzka, M. A. Microalgae for aquaculture: Opportunities and
constraints. J. Appl. Phycol. 1997, 9, 393-401.
(5) Yamaguchi, K. Recent advances in microalgal bioscience in Japan,
with special reference to utilization of biomass and metabolites: A
review. J. Appl. Phycol. 1996, 8, 487-502.
(6) Panda, A. K.; Mishra, S.; Bisaria, V. S.; Bhojwani, S. S. Plant cell
reactors-a perspective. Enzyme Microb. Technol. 1989, 11,
(7) Wang, G.; Chen, H.; Li, G.; Chen, L.; Li, D.; Hu, C.; Chen, K.;
Liu, Y. Population growth and physiological characteristics of
microalgae in a miniaturized bioreactor during space flight. Acta
Astronautica 2006, 58, 264-269.
(8) Travieso, L.; Pello ´n, A.; Benı ´tez, F.; Sa ´nchez, E.; Borja, R.; O’
Farrill, N.; Weiland, P. BIOALGA reactor: preliminary studies for
heavy metal removal. Biochem. Eng. J. 2002, 12, 87-91.
(9) Scragg, A. H.; Illman, A. M.; Carden, A.; Shales, S. W. Growth
of microalgae with increased calorific values in a tubular bioreactor.
Biomass Bioenergy 2002, 23, 67-73.
(10) Nagase, H.; Yoshihara, K.; Eguchi, K.; Okamoto, Y.; Murasaki,
S.; Yamashita, R.; Hirata, K.; Miyamoto, K. Uptake pathway and
continuous removal of nitric oxide from flue gas using microalgae.
Biochem. Eng. J. 2001, 7, 241-246.
(11) Acie ´n-Ferna ´ndez, F. G.; Fe ´rnandez-Sevilla, J. M.; Egorova-
Zachernyuk, T. A.; Molina-Grima, E. Cost-effective production of
13C,15N stable isotope-labelled biomass from phototrophic microal-
gae for various biotechnological applications. Biomol. Eng. 2005,
(12) Borowitzka, M. A. Commercial production of microalgae: ponds,
tanks, tubes and fermenters. J. Biotechnol. 1999, 70, 313-321.
(13) Becker, C. C.; Kyle, D. J. Developing functional foods containing
algal docosahexaenoic acid. Food Technol. 1998, 52, 68-71.
(14) Markl, H. CO2 transport and photosynthetic productivity of a
continuous culture of algae. Biotechnol. Bioeng. 1977, 19, 1851-
(15) Grima, E. M.; Ferna ´ndez, F. G. A.; Camacho, F. G.; Chisti, Y.
Photobioreactors: light regime, mass transfer, and scaleup. J.
Biotechnol. 1999, 70, 231-247.
(16) Pulz, O. Open-air and semi-closed cultivation systems for the mass
cultivation of microalgae. In First Asia-Pacific Conference on Algal
Biotechnology, Kuala Lumpur, Malaysia, 1992.
(17) Richmond, A.; Boussiba, S.; Vonshak, A.; Kopel, R. A new
tubular reactor for mass production of microalgae outdoors. J. Appl.
Phycol. 1993, 5, 327-332.
(18) Chrismada, T.; Borowitzka, M. A. Growth and lipid production
of Phaeodactylum tricornutum in a tubular photobioreactor. In Algal
Biotechnology in the Asia-Pacific Region, Kuala Lumpur, Malaysia,
(19) Grima, E. M.; Perez, J. A. S.; Camacho, F. G.; Fernandez, F. G.
A.; Sevilla, J. M. F.; Sanz, F. V. Effect of dilution rate on
eicosapentaenoic acid productivity of Phaeodactylum tricornutum
Utex-640 in outdoor chemostat culture. Biotechnol. Lett. 1994, 16,
(20) Travesio, L.; Hall, D. O.; Rao, K. K.; Benı ´tez, F.; Sa ´nchez, E.;
Borja, R. A helical tubular reactor producing Spirulina in a
semicontinuous mode. Int. Biodeterg. Biodegrad. 2001, 47, 151-
(21) Borowitzka, M. A. Closed algal photobioreactors: design con-
siderations for large-scale systems. J. Mar. Biotechnol. 1996, 4, 185-
(22) Gudin, C.; Chaumont, D. Cell fragility-the key problem of
microalgae mass production on closed photobioreactors. Bioresour.
Technol. 1991, 38, 145-151.
(23) Thomas, W. H.; Gibson, C. H. Effects of small-scale turbulence
on microalgae. J. Appl. Phycol. 1990, 2, 71-77.
(24) Jaouen, P.; Vandanjon, L.; Que ´me ´neur, F. The shear stress of
microalgal cell suspensions (Tetraselmis suecica) in tangential flow
filtration systems: the role of pumps. Bioresour. Technol. 1999, 68,
(25) Vandanjon, L.; Rossignol, N.; Jaouen, P.; Robert, J. M.; Que ´me ´-
neur, F. Effects of shear on two microalgae species. Contribution
of pumps and valves in tangential flow filtration systems. Biotechnol.
Bioeng. 1999, 63, 1-9.
(26) Barbosa, M. J.; Hadiyanto; Wijffels, R. H. Overcoming shear stress
of microalgae cultures in sparged photobioreactors. Biotechnol.
Bioeng. 2004, 85, 78-85.
(27) Alı ´as, C. B.; Lo ´pez, M. C. G. M.; Ferna ´ndez, F. G. A.; Sevilla,
J. M. F.; Sa ´nchez, J. L. G.; Grima, E. M. Influence of power supply
in the feasibility of Phaeodactylum tricornutum cultures. Biotechnol.
Bioeng. 2004, 87, 723-733.
(28) Lee, Y. K.; Ding, S. Y.; Low, C. S.; Chang, Y. C. Design and
performance of an R-type tubular photobioreactor for mass cultiva-
tion of microalgae. J. Appl. Phycol. 1995, 7, 47-51.
(29) Laing, I.; Jones, E. A turbidostat vessel for the continuous culture
of marine microalgae. Aquacult. Eng. 1988, 7, 89-96.
(30) Trotta, P. A simple and inexpensive system for continuous
monoxenic mass culture of marine microalgae. Aquaculture 1981,
(31) Cohen, E.; Arad, S. M. A closed system for outdoor cultivation
of Porphyridium. Biomass 1989, 18, 59-67.
(32) Martı ´nez-Jero ´nimo, F.; Espinosa-Chavez, F. A laboratory-scale
system for mass culture of freshwater microalgae in polyethylene
bags. J. Appl. Phycol. 1994, 6, 423-425.
(33) Tredici, M. R.; Rodolfi, L. University of Florence, Italy. Reactor
for industrial culture of photosynthetic micro-organisms. PCT WO
2004/074423 A2, 2004.
Biotechnol. Prog., 2006, Vol. 22, No. 6
(34) Chae, S. R.; Hwang, E. J.; Shin, H. S. Single cell protein
production of Euglena gracilis and carbon dioxide fixation in an
innovative photo-bioreactor. Bioresour. Technol. 2006, 97, 322-
(35) Miyamoto, K.; Wable, O.; Benemann, J. R. Vertical tubular reactor
for microalgae cultivation. Biotechnol. Lett. 1988, 10, 703-708.
(36) James, C. M.; al-Khars, A. M. An intensive continuous culture
system using tubular photobioreactors for producing microalgae.
Aquaculture 1990, 87, 381-393.
(37) Fukami, K.; Nishimura, S.; Ogusa, M.; Asada, M.; Nishijima, T.
Continuous culture with deep seawater of a benthic food diatom
Nitzchia sp. Hydrobiology 1997, 358, 245-249.
(38) Lee, Y. K. Enclosed bioreactors for the mass cultivation of
photosynthetic microorganisms: the future trend. TIBTECH 1986,
(39) Richmond, A. The challenge confronting industrial microagricul-
ture: high photosynthetic efficiency in large-scale reactors. Hydro-
biology 1987, 151/152, 117-121.
(40) Torzillo, G.; Puspararaj, B.; Bocci, F.; Balloni, W.; Materassi,
R.; Florenzano, G. Production of Spirulina biomass in closed
photobioreactors. Biomass 1986, 11, 61-74.
(41) Zittelli, G. C.; Lavista, F.; Bastianini, A.; Rodolfi, L.; Vincenzini,
M.; Tredici, M. R. Production of eicosapentaenoic acid by Nan-
nochloropsis sp. cultures in outdoor tubular photobioreactors. J.
Biotechnol. 1999, 70, 299-312.
(42) Olaizola, M. Commercial production of astaxanthin from Heama-
tococcus pluVialis using 25,000 liter outdoor photobioreactors. J.
Appl. Phycol. 2000, 12, 499-506.
(43) Gudin, C.; Chaumont, D. Solar biotechnology study and develop-
ment of tubular solar receptors for controlled production of
photosynthetic cellular biomass for methane production and specific
exocellular biomass. Sol. Energy R&D Eur. Community, Ser. E 1984,
(44) Chaumont, D.; Thepenier, C.; Gudin, C.; Junjas, C. Scaling up a
tubular photobioreactor for continuous culture of Porphyridium
cruentum from laboratory to pilot plant (1981-1987). In Algal
Biotechnology; Stadler, T., Mollion, J., Verdus, M.-C., Karamanos,
Y., Morvan, H., Christiaen, D., Eds.; Elsevier: New York, 1988;
(45) Grima, E. M.; Pe ´rez, J. A. S.; Camacho, F. G.; Sa ´nchez, J. L. G.;
Ferna ´ndez, F. G. A.; Alonso, D. L. Outdoor cultivation of Isochrysis
galbana ALII-4 in a closed tubular photobioreactor. J. Biotechnol.
1994, 37, 159-166.
(46) Miro ´n, A. S.; Go ´mez, A. C.; Camacho, F. G.; Grima, E. M.; Chisti,
Y. Comparative evaluation of compact photobioreactors for large-
scale monoculture of microalgae. J. Biotechnol. 1999, 70, 249-
(47) Tredici, M. R.; Zittelli, G. C. Efficiency of sunlight utilization:
tubular versus flat photobioreactors. Biotechnol. Bioeng. 1998, 57,
(48) Tredici, M. R. Closed photobioreactors: basic and applied aspects.
In Proceedings of Marine Biotechnology: Basics and Applications,
Matalascan ˜as, Spain, 2003; p 1.
(49) Robinson L. F.; Morrison A. W.; Bamforth M. R. Improvements
relating to biosynthesis. European Patent 261,872, 1988.
(50) Chrismada, T.; Borowitzka, M. A. Effect of cell density and
irradiance on growth, proximate composition and eicosapentaenoic
acid production of Phaeodactylum tricornutm grown in a tubular
photobioreactor. J. Appl. Phycol. 1994, 6, 67-74.
(51) Morita, M.; Watanable, Y.; Saiki, H. Investigation of photobiore-
actor design for enhancing the photosynthetic productivity of
microalgae. Biotechnol. Bioeng. 2000, 69, 693-698.
(52) Morita, M.; Watanable, Y.; Okawa, T.; Saiki, H. Photosynthetic
productivity of conical helical tubular photobioreactors incorporating
Chlorella sp. under various culture medium flow conditions.
Biotechnol. Bioeng. 2001, 74, 135-144.
(53) Pirt, S. J.; Lee, Y. K.; Walach, M. R.; Pirt, M. W.; Balyuzi, H.
H. M.; Bazin, M. J. A tubular bioreactor for photosynthetic
production of biomass from carbon-dioxide-design and performance.
J. Chem. Technol. Biotechnol. B 1983, 33, 35-58.
(54) Richmond, A.; Cheng-Wu, Z. Optimization of a flat plate glass
reactor for mass production of Nannochloropsis sp. outdoors. J.
Biotechnol. 2001, 85, 259-269.
(55) Iqbal, M.; Grey, D.; Stepan-Sarkissian, F.; Fowler, M. W. A flat-
sided photobioreactor for continuous culturing microalgae. Aquac-
ulture Eng. 1993, 12, 183-190.
(56) Tredici, M. R.; Carlozzi, P.; Zittelli, G. C.; Materassi, R. A vertical
alveolar panel (VAP) for outdoor mass cultivation of microalgae
and cyanobacteria. Bioresour. Technol. 1991, 38, 153-159.
(57) Tredici, M. R.; Materassi, R. 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.
1992, 4, 221-231.
(58) Tredici, M. R.; Zittelli, G. C.; Biagiolini, S.; Materassi, R. Novel
photobioreactor for the mass cultivation of Spirulina spp. Bull. Inst.
Oceanogr. 1993, 89-96.
(59) Tredici M. R. Bioreactors, photo. In Encyclopedia of Bioprocess
Technology: fermentation, biocatalysis and bioseparation; Flick-
inger, M.C., Drew, S.W., Eds.; Wiley: New York, 1999; Vol 1, pp
(60) Puspararaj, B.; Pelosi, E.; Tredici, M. R.; Pinzani, E.; Materassi,
R. An integrated culture system for outdoor production of microalgae
and cyanobacteria. J. Appl. Phycol. 1997, 9, 113-119.
(61) Pohl, P.; Kohlhase, M.; Martin, M. Photobioreactors for the axenic
mass cultivation of microalgae. In Algal Biotechnology; Stadler, T.,
Mollion, J., Verdus, M.-C., Karamanos, Y., Morvan, H., Christiaen,
D., Eds.; Elsevier: New York, 1988; pp 209-218.
(62) Ogbonna, J. C.; Soejima, T.; Tanaka, H. An integrated solar and
artificial light system for internal illumination of photobioreactors.
J. Biotechnol. 1999, 70, 289-297.
(63) Eriksen, N. T.; Geest, T.; Iversen, J. J. L. Phototrophic growth in
the lumostat: a photo-bioreactor with on-line optimization of light
intensity. J. Appl. Phycol. 1996, 8, 345-352.
(64) Grima, E. M.; Perez, J. A. S.; Camacho, F. G.; Sanchez, J. L. G.;
Alonso, D. L. n-3 PUFA productivity in chemostat cultures of
microalgae. Appl. Microbiol. Biotechnol. 1993, 38, 599-605.
(65) Grima, E. M.; Camacho, F. G.; Perez, J. A. S.; Sanchez, J. L. G.
Biochemical productivity and fatty acid profiles of Isochrysis galbana
Parke and Tetraselmis sp. as a function of incident light intensity.
Process Biochem. 1994, 29, 119-126.
(66) Grima, E. M.; Perez, J. A. S.; Camacho, F. G.; Sevilla, J. M. F.;
Fernandez, F. G. A. Effect of growth-rate on the eicosapentaenoic
acid and docosahexaenoic acid content of Isochrysis galbana in
chemostat culture. Appl. Microbiol. Biotechnol. 1994, 41, 23-27.
(67) Grima, E. M.; Camacho, E. G.; Perez, J. A. S.; Fernandez, E. G.
A.; Sevilla, J. M. F. Growth yield determination in a chemostat
culture of the marine microalga Isochrysis galbana. J. Appl. Phycol.
1996, 8, 529-534.
(68) Meireles, L. A.; Azevedo, J. L.; Cunha, J. P.; Malcata, F. X. On-
line determination of biomass in a microalga bioreactor using a novel
computerized flow injection analysis system. Biotechnol. Prog. 2002,
(69) Becker, E. W. Large-scale cultivation. In Microalgae: Biotech-
nology and Microbiology; Becker, E. W., Ed.; Cambridge University
Press: New York, 1994; pp 63-171.
(70) Richmond, A. Technological aspects of mass cultivation-a general
outline. In CRC Handbook of Microalgal Mass Culture; Richmond,
A., Ed.; CRC Press: Boca Raton, FL, 1986; pp 245-264.
(71) Goldman, J. C.; Dennett, M. R.; Riley, C. B. Inorganic carbon
sources and biomass regulation in intensive microalgal cultures.
Biotechnol. Bioeng. 1981, 23, 995-1014.
(72) Talbot, P.; Gortares, M. P.; Lencki, R. W.; de la Noue, J.
Absorption of CO2 in algal mass culture systems: a different
characterization approach. Biotechnol. Bioeng. 1991, 37, 834-842.
(73) Grima, E. M.; Sanchez-Perez, J. A.; Garcia-Camacho, F.; Robles-
Medina, A. Gas-liquid transfer of atmospheric CO2 in microalgal
cultures. Chem. Technol. Biotechnol. 1993, 56, 329-337.
(74) Ferreira, B. S.; Fernandes, H. L.; Reis, A.; Mateus, M. Mi-
croporous hollow fibres for carbon dioxide absorption: mass transfer
model fitting and the supplying of carbon dioxide to microalgal
cultures. Chem. Technol. Biotechnol. 1998, 71, 61-70.
(75) Carvalho, A. P.; Malcata, F. X. Transfer of carbon dioxide within
cultures of microalgae: plain bubbling versus hollow-fiber modules.
Biotechnol. Prog. 2001, 17, 265-272.
(76) Lee, Y.-K.; Hing, H.-K. Supplying CO2to photosynthetic algal
cultures by diffusion through gas-permeable membranes. Appl.
Microbiol. Biotechnol. 1989, 31, 298-301.
Biotechnol. Prog., 2006, Vol. 22, No. 6
(77) Aunins, J. G.; Henzler, H. Aeration in cell culture bioreactors. In
Biotechnology; Stephanopoulos, G., Ed.; Wiley-VCH: Weinheim,
1993; Vol. 3 (Bioprocessing), p 223.
(78) Gallagher, S. L.; Tharakan, J. T.; Chau, P. C. An intercalated-
spiral wound hollow fiber bioreactor for the culture of mammalian
cells. Biotechnol. Tech. 1987, 1, 91-96.
(79) Chen, F.; Johns, M. R. A strategy for high cell density culture of
heterotrophic microalgae with inhibitory substrates. J. Appl. Phycol.
1995, 7, 43-46.
(80) Marsot, P.; Cembella, A. D.; Mouhri, K. Croissance de la biomasse
azote ´e du Phaeodactylum tricornutum (Bacillariophyceae) en culture
discontinue dialysante et non-dialysante. Can. J. Microbiol. 1992,
(81) Markov, S. A.; Bazin, M. J.; Hall, D. O. Hydrogen photoproduc-
tion and carbon dioxide uptake by immobilized Anabaena Variabilis
in a hollow-fiber photobioreactor. Enzyme Microb. Technol. 1995,
(82) Pirt, S. J.; Panikov, N.; Lee, Y.-K. The miniloop: a small-scale
air-lift microbial culture vessel and photobiological reactor. J. Chem.
Technol. Biotechnol. 1979, 29, 437-441.
(83) Pirt, S. J. Microbial photosynthesis in the harnessing of solar-
energy. J. Chem. Technol. Biotechnol. 1982, 32, 198-202.
(84) Laws, E. A.; Terry, K. L.; Wickman, J.; Chalup, M. S. A simple
algal production system designed to utilize the flashing light effect.
Biotechnol. Bioeng. 1983, 25, 2319-2335.
(85) Laws, E. A.; Taguchi, S.; Harata, J.; Pang, L. High algal
production rates achieved in a shallow outdoor flume. Biotechnol.
Bioeng. 1986, 28, 191-197.
(86) Laws, E. A.; Taguchi, S.; Harata, J.; Pang, L. Optimization of
microalgal production in a shallow outdoor flume. Biotechnol.
Bioeng. 1988, 32, 140-147.
(87) Lee, C.-G.; Palsson, B. Ø. High-density algal photobioreactors
using light-emitting diodes. Biotechnol. Bioeng. 1994, 44, 1161-
(88) Muller-Feuga, A.; Gue ´des, R. L.; Herve ´, A.; Durand, P. Com-
parison of artificial light photobioreactors and other production
systems using Porphyridium cruentum. J. Appl. Phycol. 1998, 10,
(89) Fuentes, M. M. R.; Sa ´nchez, J. L. G.; Sevilla, J. M. F.; Ferna ´ndez,
F. G. A.; Pe ´rez, J. A. S.; Grima, E. M. Outdoor continuous culture
of Phorphyridium cruentum in a tubular photobioreactor: quantitative
analysis of the daily cyclic variation of culture parameters. J.
Biotechnol. 1999, 70, 271-288.
(90) Ferna ´ndez, A. F. G.; Sevilla, J. M. F.; Pe ´rez, J. A. S.; Molina-
Grima, E.; Chisti, Y. Airlift-driven external-loop tubular photobiore-
actors for outdoor production of microalgae: assessment of design
and performance. Chem. Eng. Sci. 2001, 56, 2721-2732.
(91) Marsot, P.; Fournier, R.; Blais, C. Culture a ` dialyse: emploi de
fibres creuses dialysantes pour la culture massive de phytoplankton.
Can. J. Fish. Aquat. Sci. 1981, 38, 905-911.
(92) Camacho, F. G.; Go ´mez, A. C.; Ferna ´ndez, F. G. A.; Sevilla, J.
F.; Grima, E. M. Use of concentric-tube airlift photobioreactors for
microalgal outdoor mass cultures. Enzyme Microb. Technol. 1999,
(93) Heussler, P.; Castillo, S. J.; Merino, M. F.; Vasquez, V. V.
Improvements in pond construction and CO2 supply for the mass
production of microalgae. Arch. Hydrobiol. Beih. Ergebn. Limnol.
1978, 11, 254-258.
(94) Sa ´nchez, J. L. G.; Berenguel, M.; Rodrı ´guez, F.; Sevilla, J. M.
F.; Alias, C. B.; Ferna ´ndez, F. G. A. Minimization of carbon losses
in pilot-scale outdoor photobioreactors by model-based predictive
control. Biotechnol. Bioeng. 2003, 84, 533-543.
(95) Berenguel, M.; Rodrı ´guez, F.; Acie ´n, F. G.; Garcı ´a, J. L. Model
predictive control of pH in tubular photobioreactors. J. Process
Control 2004, 14, 377-387.
(96) Oswald, W. J. Large-scale algal culture systems (engineering
aspects). In Micro-Algal Biotechnology; Borowitzka, M. A., Borow-
itzka, L. J., Eds.; Cambridge University Press: New York, 1988;
(97) Camacho, F. G.; Go ´mez, A. C.; Sobczuk, T. M.; Grima, E. M.
Effects of mechanical and hydrodynamic stress in agitated, sparged
cultures of Porphyridium cruentum. Process Biochem. 2000, 35,
(98) Singh, G. Reactor design for plant cell culture of food ingredients
and additives. Food Technol. 1997, 51, 62-66.
(99) Degen, J.; Uebele, A.; Retze, A.; Schmid-Staiger, U.; Tro ¨sch, W.
A novel airlift photobioreactor with baffles for improved light
utilization through the flashing light effect. J. Biotechnol. 2001, 92,
(100) Laws, E. A.; Taguchi, S.; Hirata, J.; Pang, L. Continued studies
of high algal productivities in a shallow flume. Biomass 1987, 11,
(101) Pirt, S. J.; Lee, Y. K.; Richmond, A.; Watts-Pirt, M. The
photosynthetic efficiency of Chlorella biomass growth with reference
to solar energy utilization. J. Chem. Technol. Biotechnol. 1980, 30,
(102) Sandnes, J. M.; Ringstad, T.; Wenner, D.; Heyerdahl, P. H.;
Ka ¨llqvist, T.; Gisler¢d, H. R. Real-time monitoring and automatic
density control of large-scale microalgal cultures using near infrared
(NIR) optical density sensors. J. Biotechnol. 2006, 122, 209-215.
(103) Dubinsky, Z.; Matsukawa, R.; Karube, I. Photobiological aspects
of algal mass culture. J. Mar. Biotechnol. 1985, 2, 61-65.
(104) Evers, E. G. A model for light-limited continuous cultures-
growth, shading, and maintenance. Biotechnol. Bioeng. 1991, 38,
(105) You, T.; Barnett, S. M. Effect of light quality on production of
extracellular polysaccharides and growth rate of Porphyridium
cruentum. Biochem. Eng. J. 2004, 19, 251-258.
(106) Simmer, J.; Tichy, V.; Doucha, J. What kind of lamp for the
cultivation of algae? J. Appl. Phycol. 1994, 6, 309-313.
(107) Li, J.; Xu, N. S.; Su, W. W. Online estimation of stirred-tank
microalgal photobioreactor cultures based on dissolved oxygen
measurement. Biochem. Eng. J. 2003, 14, 51-65.
(108) Hejazi, M. A.; Wijffels, R. H. Milking of microalgae. TIBTECH
2004, 22, 189-194.
Received March 7, 2006. Accepted August 3, 2006.
Biotechnol. Prog., 2006, Vol. 22, No. 6