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Third generation biofuels from microalgae
Giuliano Dragone, Bruno Fernandes, António A. Vicente, and José A. Teixeira
IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de
Gualtar, 4710-057 Braga, Portugal
Biofuel production from renewable sources is widely considered to be one of the most sustainable alternatives to
petroleum sourced fuels and a viable means for environmental and economic sustainability. Microalgae are currently being
promoted as an ideal third generation biofuel feedstock because of their rapid growth rate, CO
2
fixation ability and high
production capacity of lipids; they also do not compete with food or feed crops, and can be produced on non-arable land.
Microalgae have broad bioenergy potential as they can be used to produce liquid transportation and heating fuels, such as
biodiesel and bioethanol. In this review we present an overview about microalgae use for biodiesel and bioethanol
production, including their cultivation, harvesting, and processing. The most used microalgal species for these purposes as
well as the main microalgal cultivation systems (photobioreactors and open ponds) will also be discussed.
Keywords Microalgae; Biofuels; Biodiesel; Bioethanol; Global warming
1. Introduction
Concerns about shortage of fossil fuels, increasing crude oil price, energy security and accelerated global warming have
led to growing worldwide interests in renewable energy sources such as biofuels. An increasing number of developed
and rapidly developing nations see biofuels as a key to reducing reliance on foreign oil, lowering emissions of
greenhouse gases (GHG), mainly carbon dioxide (CO
2
) and methane (CH
4
), and meeting rural development goals [1].
Biofuels are referred to solid, liquid or gaseous fuels derived from organic matter. They are generally divided into
primary and secondary biofuels (Fig. 1). While primary biofuels such as fuelwood are used in an unprocessed form
primarily for heating, cooking or electricity production, secondary biofuels such as bioethanol and biodiesel are
produced by processing biomass and are able to be used in vehicles and various industrial processes. The secondary
biofuels can be categorized into three generations: first, second and third generation biofuels on the basis of different
parameters, such as the type of processing technology, type of feedstock or their level of development [2].
Fig. 1 Classification of biofuels (modified from [2]).
Biofuels
Primary
Secondary
Firewood, wood
chips, pellets,
animal waste,
forest and crop
residues, landfill
gas
1
st
generation
Bioethanol or butanol by
fermentation of starch
(from wheat, barley, corn,
potato) or sugars
(from sugarcane, sugar
beet, etc.)
Biodiesel by
transesterification of oil
crops (rapeseed, soybeans,
sunflower, palm, coconut,
used cooking oil, animal
fats, etc.)
2
nd
generation
Bioethanol and biodiesel
produced from
conventional technologies
but based on novel starch,
oil and sugar crops such as
Jatropha, cassava or
Miscanthus;
Bioethanol, biobutanol,
syndiesel produced from
lignocellulosic materials
(e.g. straw, wood, and
grass)
3
rd
generation
Biodiesel from microalgae
Bioethanol from
microalgae and seaweeds
Hydrogen from green
microalgae and microbes
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Although biofuel processes have a great potential to provide a carbon-neutral route to fuel production, first generation
production systems have considerable economic and environmental limitations. The most common concern related to
the current first generation biofuels is that as production capacities increase, so does their competition with agriculture
for arable land used for food production. The increased pressure on arable land currently used for food production can
lead to severe food shortages, in particular for the developing world where already more than 800 million people suffer
from hunger and malnutrition. In addition, the intensive use of land with high fertilizer and pesticide applications and
water use can cause significant environmental problems [3].
The advent of second generation biofuels is intended to produce fuels from lignocellulosic biomass, the woody part
of plants that do not compete with food production. Sources include agricultural residues, forest harvesting residues or
wood processing waste such as leaves, straw or wood chips as well as the non-edible components of corn or sugarcane.
However, converting the woody biomass into fermentable sugars requires costly technologies involving pre-treatment
with special enzymes, meaning that second generation biofuels cannot yet be produced economically on a large scale
[4].
Therefore, third generation biofuels derived from microalgae are considered to be a viable alternative energy
resource that is devoid of the major drawbacks associated with first and second generation biofuels [2, 5, 6]. Microalgae
are able to produce 15–300 times more oil for biodiesel production than traditional crops on an area basis. Furthermore
compared with conventional crop plants which are usually harvested once or twice a year, microalgae have a very short
harvesting cycle (≈1– 10 days depending on the process), allowing multiple or continuous harvests with significantly
increased yields [3].
2. Characteristics of microalgae
Microlgae, recognised as one of the oldest living organisms, are thallophytes (plants lacking roots, stems, and leaves)
that have chlorophyll a as their primary photosynthetic pigment and lack a sterile covering of cells around the
reproductive cells [4]. While the mechanism of photosynthesis in these microorganisms is similar to that of higher
plants, they are generally more efficient converters of solar energy because of their simple cellular structure. In
addition, because the cells grow in aqueous suspension, they have more efficient access to water, CO
2
, and other
nutrients [5].
Traditionally microalgae have been classified according to their colour and this characteristic continues to be of a
certain importance. The current systems of classification of microalgae are based on the following main criteria: kinds
of pigments, chemical nature of storage products and cell wall constituents. Additional criteria take into consideration
the following cytological and morphological characters: occurrence of flagellate cells, structure of the flagella, scheme
and path of nuclear and cell division, presence of an envelope of endoplasmic reticulum around the chloroplast, and
possible connection between the endoplasmic reticulum and the nuclear membrane [7]. There are two basic types of
cells in the algae, prokaryotic and eukaryotic. Prokaryotic cells lack membrane-bounded organelles (plastids,
mitochondria, nuclei, Golgi bodies, and flagella) and occur in the cyanobacteria. The remainder of the algae are
eukaryotic and have organelles [8].
Microalgae can be either autotrophic or heterotrophic. If they are autotrophic, they use inorganic compounds as a
source of carbon. Autotrophs can be photoautotrophic, using light as a source of energy, or chemoautotrophic, oxidizing
inorganic compounds for energy. If they are heterotrophic, microalgae use organic compounds for growth. Heterotrophs
can be photoheterotrophs, using light as a source of energy, or chemoheterotrophs, oxidizing organic compounds for
energy. Some photosynthetic microalgae are mixotrophic, combining heterotrophy and autotrophy by photosynthesis
[8]. For autotrophic algae, photosynthesis is a key component of their survival, whereby they convert solar radiation and
CO
2
absorbed by chloroplasts into adenosine triphosphate (ATP) and O
2
, the usable energy currency at cellular level,
which is then used in respiration to produce energy to support growth [4].
Microalgae are able to fix CO
2
efficiently from different sources, including the atmosphere, industrial exhaust gases,
and soluble carbonate salts. Fixation of CO
2
from atmosphere is probably the most basic method to sink carbon, and
relies on the mass transfer from the air to the microalgae in their aquatic growth environments during photosynthesis.
However, because of the relatively small percentage of CO
2
in the atmosphere (approximately 0.036 %), the use of
terrestrial plants is not an economically feasible option [4]. On the other hand, industrial exhaust gases such as flue gas
contains up to 15 % CO
2
, providing a CO
2
-rich source for microalgal cultivation and a potentially more efficient route
for CO
2
bio-fixation. Many microalgal species have also been able to utilize carbonates such as Na
2
CO
3
and NaHCO
3
for cell growth. Some of these species typically have high extracellular carboanhydrase activities, which is responsible
for the conversion of carbonate to free CO
2
to facilitate CO
2
assimilation. In addition, the direct uptake of bicarbonate
by an active transport system has also been found in several species [9].
Growth medium must provide the inorganic elements that constitute the algal cell. Essential elements include
nitrogen (N) and phosphorus (P). Minimal nutritional requirements can be estimated using the approximate molecular
formula of the microalgal biomass, which is CO
0.48
H
1.83
N
0.11
P
0.01
[5]. Nitrogen is mostly supplied as nitrate (NO
3-
), but
often ammonia (NH
4+
) and urea are also used. Urea is most favourable as the nitrogen source because, for an equivalent
nitrogen concentration, it gives higher yields and causes smaller pH fluctuations in the medium during algal growth
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[10]. On the other hand, nutrients such as P must be supplied in significant excess because the phosphates added
complex with metal ions, therefore, not all the added P is bio-available [5]. Furthermore, microalgae growth depends not
only on an adequate supply of essential macronutrient elements (carbon, nitrogen, phosphorus, silicon) and major ions
(Mg
2+
, Ca
2+
, Cl
-
, and SO
42-
) but also on a number of micronutrient metals such as iron, manganese, zinc, cobalt, copper,
and molybdenum [11].
3. Microalgae as a potential source of biofuel
There are several ways to convert microalgal biomass to energy sources, which can be classified into biochemical
conversion, chemical reaction, direct combustion, and thermochemical conversion (Fig. 2). Thus, microalgae can
provide feedstock for renewable liquid fuels such as biodiesel and bioethanol [12].
The idea of using microalgae as a source of biofuel is not new, but it is now being taken seriously because of the
rising price of petroleum and, more significantly, the emerging concern about global warming that is associated with
burning of fossil fuels [5]. The utilization of microalgae for biofuels production offers the following advantages over
higher plants: (1) microalgae synthesize and accumulate large quantities of neutral lipids (20–50 % dry weight of
biomass) and grow at high rates; (2) microalgae are capable of all year round production, therefore, oil yield per area of
microalgae cultures could greatly exceed the yield of best oilseed crops; (3) microalgae need less water than terrestrial
crops therefore reducing the load on freshwater sources; (4) microalgae cultivation does not require herbicides or
pesticides application; (5) microalgae sequester CO
2
from flue gases emitted from fossil fuel-fired power plants and
other sources, thereby reducing emissions of a major greenhouse gas (1 kg of dry algal biomass utilise about 1.83 kg of
CO2); (6) wastewater bioremediation by removal of NH
4+
, NO
3-
, PO
43-
from a variety of wastewater sources (e.g.
agricultural run-off, concentrated animal feed operations, and industrial and municipal wastewaters); (7) combined with
their ability to grow under harsher conditions and their reduced needs for nutrients, microalgae can be cultivated in
saline/brackish water/coastal seawater on non-arable land, and do not compete for resources with conventional
agriculture; (8) depending on the microalgae species other compounds may also be extracted, with valuable applications
in different industrial sectors, including a large range of fine chemicals and bulk products, such as polyunsaturated fatty
acids, natural dyes, polysaccharides, pigments, antioxidants, high-value bioactive compounds, and proteins [4, 12, 13].
Fig. 2 Conversion processes for biofuel production from microalgal biomass (modified from [9]).
Microalgal
Biomass
Chemical
Reaction
Thermochemical
Conversion
Biochemical
Conversion
Photobiological
Hydrogen Production Hydrogen
Fermentation
Anaerobic Digestion
Bioethanol, Acetone, Butanol
Methane, Hydrogen
Gasification
Pyrolysis
Liquefaction
Syngas
Bio-oil, Charcoal, Syngas
Bio-oil
Transesterification Biodiesel
Direct
Combustion
Power Generation Electricity
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4. Biodiesel and bioethanol production from microalgae
Recent studies have shown that microalgal biomass is one of the most promising sources of renewable biodiesel that is
capable of meeting the global demand for transport fuels. Biodiesel production by microalgae will not compromise
production of food, fodder and other products derived from crops [5].
Microalgal biomass contains three main components: proteins, carbohydrates, and lipids (oil) [13]. The biomass
composition of various microalgae in terms of those main components is shown in Table 1.
Table 1 Biomass composition of microalgae expressed on a dry matter basis ([13, 14]).
Strain Protein Carbohydrates Lipid
Anabaena cylindrica 43–56 25–30 4–7
Botryococcus braunii 40 2 33
Chlamydomonas rheinhardii 48 17 21
Chlorella pyrenoidosa 57 26 2
Chlorella vulgaris 41–58 12–17 10–22
Dunaliella bioculata 49 4 8
Dunaliella salina 57 32 6
Dunaliella tertiolecta 29 14 11
Euglena gracilis 39–61 14–18 14–20
Porphyridium cruentum 28–39 40–57 9–14
Prymnesium parvum 28–45 25–33 22–39
Scenedesmus dimorphus 8–18 21–52 16–40
Scenedesmus obliquus 50–56 10–17 12–14
Scenedesmus quadricauda 47 – 1.9
Spirogyra sp. 6–20 33–64 11–21
Spirulina maxima 60–71 13–16 6–7
Spirulina platensis 42–63 8–14 4–11
Synechoccus sp. 63 15 11
Tetraselmis maculata 52 15 3
Much of the on-going research work is focused on a small number of fast-growing microalgal species which have
been found to accumulate substantial quantities of lipids, though under specific conditions. Within the green algae,
typical species include Chlamydomonas reinhardtii, Dunaliella salina, and various Chlorella species, as well as
Botryococcus braunii, which although slow growing can accumulate large quantities of lipids [15]. While many
microalgae strains naturally have high lipid content, it is possible to increase that concentration by optimising growth-
determining factors such as the control of nitrogen level, light intensity, temperature, salinity, CO
2
concentration and
harvesting procedure.
However, increasing lipid accumulation will not result in increased lipid productivity as biomass productivity and
lipid accumulation are not necessarily correlated. Lipid accumulation refers to increased concentration of lipids within
the microalgae cells without consideration of the overall biomass production. Lipid productivity takes into account both
the lipid concentration within cells and the biomass produced by these cells and is therefore a more useful indicator of
the potential costs of liquid biofuel production [4].
An integrated production of biofuels from microalgae (Fig. 3) includes a microalgal cultivation step, followed by the
separation of the cells from the growth medium and subsequent lipid extraction for biodiesel production through
transesterification.
Fig. 3 Integrated process for biodiesel and bioethanol production from microalgae.
Microalgae
cultivation Harvesting Drying Cell disruption
and oil extraction Transesterification
Biodiesel
Starch
hydrolysis Fermentation Distillation
Light
Nutrients
Water CO
2
Culture
recycle
Lipids and
free fatty acids
Starch and
proteins
Bioethanol
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Following oil extraction, amylolytic enzymes are used to promote starch hydrolysis and formation of fermentable
sugars. These sugars are fermented and distilled into bioethanol using conventional ethanol distillation technology.
4.1 Cultivation systems
After selecting the microalgae strain to obtain the product of interest, it becomes necessary to develop a whole range of
bioprocesses that make viable its commercialization. Thus, the design and optimization of adequate bioreactors to
cultivate these microorganisms is a major step in the strategy that aims at transforming scientific findings into a
marketable product. Despite of many possible applications, only a few species of algae are cultured commercially
because of poorly developed microalgal bioreactor technology.
From a commercial point of view, a microalgae culture system must have as many of the following characteristics as
possible: high area productivity; high volumetric productivity; inexpensiveness (both in terms of investment and
maintenance costs); easiness of control of the culture parameters (temperature, pH, O
2
, turbulence); and reliability [16].
Cultivation systems of different designs attempt to achieve these characteristics differently. Although the term
“photobioreactor” (PBR) has been applied to open ponds and channels, applied phycologists have generally
distinguished between open-air systems and PBRs (devices that allow monoseptic culture). Thus in this chapter the term
PBR is used only for closed systems.
4.1.1 Open-air systems
Open-air systems were extensively studied in the past few years [17-19], but these algae cultivation systems have been
used since the 1950s. The classical open-air cultivation systems comprise lakes and natural ponds, circular ponds,
raceway ponds and inclined systems. Open-air systems are the most widespread growth systems and all very large
commercial systems used today are of this type. The reasons for this relate to economic and operational issues, since
these systems are easier and less expensive to build, operate more durably and have a larger production capacity than
most closed systems; further, they can utilize sunlight and the nutrients can be provided through runoff water from
nearby land areas or by channeling the water from sewage/water treatment plants [20] making it the cheapest method of
large-scale algal biomass production.
Although these systems are the most widely used at industrial level, open-air systems still present significant
technical challenges. Generally ponds are susceptive to weather conditions, not allowing control of water temperature,
evaporation and lighting, which make these systems dependent on the prevailing regional climate conditions (daily and
annual temperature range, annual rainfall and rainfall pattern, number of sunny days, and degree of cloud cover).
Furthermore, contamination by predators and other fast growing heterotrophs have restricted the commercial production
of algae in open culture systems to fast growing, naturally occurring or extremophilic species. Consequently, this
strictly limits the species of algae that can be grown in such systems. As a result, only Dunaliella (adaptable to very
high salinity), Spirulina (adaptable to high alkalinity) and Chlorella (adaptable to nutrient-rich media) have been
successfully grown in commercial open pond systems [20].
Natural and artificial ponds are only viable when a series of conditions are met. The existence of favorable climatic
conditions and sufficient nutrients in order to the microalgae grow is profusely unavoidable and it also requires that the
water presents selective characteristics (e.g. high salinity, high pH, high nutrients concentration) to ensure the existence
of a monoculture. Successful examples of this type of cultivation are the Arthrospira production in Lake Kossorom
(soda lake at the irregular northeast fringe of Lake Chad) where the Kanembu people harvest about 40 t/year of
Arthrospira (Spirulina), to use it as food [21] and in Myanmar, where four old volcanic craters, full of alkaline water
are used as cultivation system for the production of around 30 t/year of Arthrospira that are sold on the local market
[22]. The Australian producer of D. salina (extremely halophilic and highly light-tolerant green alga) Betatene Ltd, uses
very large ponds (up to 250 ha with an average depth of 0.2 to 0.3 m) at the extremely halophilic waters of Hutt-
Lagoon, Western Australia which are unmixed other than by wind and convection [24].
The inclined system (cascade system) is the only open-air system which achieves high sustainable cell densities (up
to 10 g l
-1
). This system is very well suited for algae such as Chlorella and Scenedesmus, which can tolerate repeated
pumping [23]. In inclined systems turbulence is created by gravity, the culture suspension flowing from the top to the
bottom of a sloping surface, thus achieving highly turbulent flow and allowing the adoption of very thin culture layers
(< 2 cm), facilitating higher cell concentrations and a higher surface-to-volume ratio (s/v) compared to raceway ponds.
Circular ponds with a centrally pivoted rotating agitator are widely used in Indonesia, Japan and Taiwan for the
production of Chlorella. Depth is about 0.3 m. The design of these systems, however, limits pond size to about
10,000 m
2
, because mixing by the rotating arm is no longer possible in larger ponds. Circular ponds are not favored in
commercial plants since they require expensive concrete construction and high energy input for mixing [24].
Raceway ponds are the most commonly used artificial system. They are typically made of a closed loop, oval shaped
recirculation channels, generally between 0.2 and 0.5 m deep, with mixing and circulation required to stabilize algae
growth and productivity (Table 2). In a continuous production cycle, algae broth and nutrients are introduced in front of
the paddlewheel and circulated through the loop to the harvest extraction point. The paddlewheel is in continuous
operation to prevent sedimentation. At water depths of 0.15-0.20 m, biomass concentrations of up to 1 g l
-1
and
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productivities of 10-25 g m
-2
d
-1
, are possible [25]. The largest raceway-based biomass production facility located in
Calipatria, CA (USA) occupies an area of 440,000 m
2
to grow Spirulina [26].
4.1.2 Photobioreactors
Photobioreactors (PBRs) are characterized by the regulation and control of nearly all the biotechnologically important
parameters as well as by a reduced contamination risk, no CO
2
losses, reproducible cultivation conditions, controllable
hydrodynamics and temperature, and flexible technical design [25]. These systems receive sunlight either directly
through the transparent container walls or via light fibres or tubes that channel it from sunlight collectors.
Despite the relative success of open systems, recent advances in microalgal mass culture require closed systems, as
many of the new algae and algal high-value products for use in the pharmaceutical and cosmetics industry must be
grown free of pollution and potential contaminants such as heavy metals and microorganisms.
Many different designs have been developed, but the main categories include: (1) tubular (e.g. helical, manifold,
serpentine, and α-shaped); (2) flat (e.g. alveolar panels and glass plates); and (3) column (e.g. bubble columns and
airlift). A great amount of developmental work has been carried out in order to optimize different PBR systems for
microalgae cultivation [17, 19, 27, 28].
4.1.2.1 Tubular photobioreactors
Tubular PBRs can be horizontal/serpentine- [29], near horizontal- [30], vertical- [31], inclined- [32] and conical-shaped
[33]. Microalgae are circulated through the tubes by a pump, or preferably with airlift technology. Generally these PBR
systems are relatively cheap, have a large illumination surface area and have fairly good biomass productivities.
Disadvantages include fouling, some degree of wall growth, dissolved oxygen and CO
2
along the tubes, and the pH
gradients that lead to frequent re-carbonation of the cultures, which would consequently increase the cost of algal
production (Table 2). The largest closed PBRs are tubular, e.g. the 25 m
3
plant at Mera Pharmaceuticals, Hawaii, and
the 700 m
3
plant in Klötze, Germany. A maximum productivity of 25 g m
-2
d
-1
(Spirulina) has been achieved in a 10 m
3
serpentine bioreactor with intermitted culture circulation [34]. Further improvements were obtained by constructing a
two-plane tubular photobioreactor with mean daylight productivities of about 30 g m
-2
d
-1
[35]. Helical tubular PBRs
are a suitable alternative to straight tubular PBRs. The most frequently used layout is the Biocoil, 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 [28]. A 300 l α-shaped tubular PBR has
been used for the cultivation of Chlorella pyrenoidosa [36]. That system comprises of an airlift pump to promote an
ascending/descending trajectory, with several CO
2
injection points along its path.
4.1.2.2 Flat photobioreactors
Some of the earliest forms of closed systems are flat PBRs which have received much research attention due to the large
surface area exposed to illumination and high densities (>80 g l
-1
) of photoautotrophic cells observed [4].
In these PBR a thin layer of very dense culture is mixed or flown across a flat transparent panel, which allows
radiation absorbance in the first few millimetres thickness. Flat PBRs are suitable for mass cultures of microalgae due
to the low accumulation of dissolved oxygen and the high photosynthetic efficiency achieved when compared to tubular
designs [4]. Usually, the panels are illuminated mainly on one side by direct sunlight and have the added advantage that
they can be positioned vertically or inclined at an optimum angle facing the sun permitting a better efficiency in terms
of energy absorbed from incident sunlight. Packed flat panels mixed by air bubbling can potentially achieve very high
overall ground-areal productivities through lamination of solar light. Limitations include difficulty in controlling culture
temperature, some degree of wall growth, scale-up requires many compartments and support materials, and possibility
of hydrodynamic stress to some algal strains [12] (Table 2).
4.1.2.3 Column photobioreactors
Column PBRs are occasionally stirred tank reactors [37], but more often bubble columns [38] or airlifts [39]. The
columns are placed vertically, aerated from the bottom, and illuminated through transparent walls or internally. Column
bioreactors offer the most efficient mixing, the highest volumetric gas transfer rates, and the best controllable growth
conditions. They are low-cost, compact and easy to operate. Their performance (i.e. final biomass concentration and
specific growth rate) compares favorably with the values typically reported for tubular PBRs.
Vertical bubble columns and airlift cylinders can attain substantially increased radial movement of fluid that is
necessary for improved light–dark cycling. These reactor designs have a low surface/volume, but substantially greater
gas hold-ups than horizontal reactors and a much more chaotic gas–liquid flow. Consequently, cultures suffer less from
photo-inhibition and photo-oxidation, and experience a more adequate light–dark cycle [12].
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4.1.3 Photobioreactor design and scale-up considerations
Despite various configurations, several essential issues need addressing when building a PBR: effective and efficient
provision of light; supply of CO
2
while minimizing desorption; efficient mixing and circulation of the culture; scalable
PBR technology and the material used in the construction of the PBR.
Light as the energy source for photoautotrophic life is the principal limiting factor in photobiotechnology. The light
regimen inside the PBR is influenced by incident light intensity, reactor design and dimension, cell density,
pigmentation of the cells, mixing pattern, etc. In outdoor PBRs the light regimen is also influenced by geographical
location, time of the day, and weather conditions. Due to the light gradient inside the reactor and depending on the
mixing properties, microalgae are subjected to light-dark cycles where the light period is characterized by a light
gradient. These light-dark cycles will determine productivity and biomass yield on light energy [40]. Information about
quantitative (photosynthetic photon flux density) and qualitative (spectral intensity distribution) aspects of light patterns
in different points of a PBR can be obtained by using optical fiber technology [40].
The supply of CO
2
to microalgal mass culture systems is one of the principal difficulties that must be solved [41].
The principal point of all considerations relating to the CO
2
budget is that, on the one hand, CO
2
must not reach the
upper concentration that produces inhibition and, on the other hand, must never fall below the minimum concentration
that limits growth. These maximum (inhibition) and minimum (limitation) concentrations varies from one species to
another and are not yet adequately known, ranging from 2.3 x 10
−2
M to 2.3 x 10
−4
M. Gas injection as minute bubbles
into a column of a downcoming culture in which the culture velocity is adjusted to that of the rising CO
2
bubbles may
increase the efficiency of absorption of CO
2
and thus the utilisation efficiency can be increased up to 70 % [42]. In a
dual sparging bubble column PBR, the CO
2
transfer rate was increased 5 times compared to a similar reactor where the
CO
2
was blended into the aeration air [43], while another study showed that, in the same PBR configuration, CO
2
transfer efficiencies were 100 % at certain conditions [44].
The level of mixing in a PBR strongly contributes to the growth of microalgae. Mixing is necessary to prevent cells
from settling, to avoid thermal stratification, to distribute nutrients and break down diffusion gradients at the cell
surface, to remove photosynthetically generated oxygen and to ensure that cells experience alternating periods of light
and darkness of adequate length [19]. The fluid dynamics of the culture medium and the type of mixing influence
average irradiance and the light regimen to which the cells are exposed, which in turns determine productivity.
Fluctuations in light intensity faster than 1 s
-1
enhance specific growth rates and productivities of microalgal cultures. In
outdoor cultures exposed to photosynthetic photon flux densities above 1 000 µmol m
-2
s light exposure times should be
as short as 10 ms to maintain high photosynthetic efficiency [45]. The choice of the mixing device and the intensity of
mixing should be dictated by the characteristics of the organism to be cultivated.
Tubular PBRs and raceway ponds are suitable for large-scale production [5]. The scalability of vertical air-lift PBR
and bubble columns was considered an advantage of these systems [46]. Scale-up of closed systems is only possible by
increasing the number of units in a production scheme. This method becomes extremely expensive, since each unit
requires a variety of devices that control the wide range of growth factors (e.g. pH, temperature, aeration, CO
2
supply,
nutrients supply). In addition, maintaining a monoculture in all of the units becomes challenging as the number of units
to monitor grows [45]. Other than scale-up by multiplication of identical modules, the only way to increase volume is
by increasing length or/and diameter or/and the light path of the PBR; however, this strategy is limited by the existence
of changes in the performance of the PBR. Commercial-scale closed PBR have not been widely reported in scientific
literature.
The type of material used is of fundamental importance for a suitable PBR construction. Materials such as plastic or
glass sheets, collapsible or rigid tubes should have high transparency, high mechanical strength, high durability,
chemical stability, low cost, must lack toxicity and be ease to clean [19].
Advantages and drawbacks of the most common materials used for building PBR have been reported in the literature
[47].
4.1.4 Photobioreactors versus open-air systems
Table 2 shows a comparison between PBR (tubular, flat and column) and open systems for several culture conditions
and growth parameters.
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Table 2 Advantages and limitations of various microalgae culture systems
Selection of a suitable production system clearly depends on the purpose of the production facility, microalgae strain
and product of interest. In conclusion, PBR and open ponds should not be viewed as competing technologies.
4.2 Harvesting methods
Given the relatively low biomass concentration obtainable in microalgal cultivation systems due to the limit of light
penetration (typically in the range of 1-5 g l
-1
) and the small size of microalgal cells (typically in the range of 2-20 µm
in diameter), costs and energy consumption for biomass harvesting are a significant concern that needs to be addressed
Culture Systems Advantages Limitations
Open systems
Relatively economical
Easy to clean up
Easy maintenance
Utilization of non-agricultural
land
Low energy inputs
Little control of culture conditions
Poor mixing, light and CO
2
utilization
Difficult to grow algal cultures for long
periods
Poor productivity
Limited to few strains
Cultures are easily contaminated
Tubular PBR
Relatively cheap
Large illumination surface area
Suitable for outdoor cultures
Good biomass productivities
Gradients of pH, dissolved oxygen and
CO
2
along the tubes
Fouling
Some degree of wall growth
Requires large land space
Photoinhibition
Flat PBR
Relatively cheap
Easy to clean up
Large illumination surface area
Suitable for outdoor cultures
Low power consumption
Good biomass productivities
Good light path
Readily tempered
Low oxygen build-up
Shortest oxygen path
Difficult scale-up
Difficult temperature control
Some degree of wall growth
Hydrodynamic stress to some algal
strains
Low photosynthetic efficiency
Column PBR
Low energy consumption
Readily tempered
High mass transfer
Good mixing
Best exposure to light-dark
cycles
Low shear stress
Easy to sterilize
Reduced photoinhibition
Reduced photo-oxidation
High photosynthetic efficiency
Small illumination surface area
Sophisticated construction materials
Shear stress to algal cultures
Decrease of illumination surface area
upon scale-up
Expensive compared to open ponds
Support costs
Modest scalability
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properly [6]. In this sense, harvesting of microalgal cultures has been considered as a major bottleneck towards the
industrial-scale processing of microalgae for biofuel production. The cost of biomass recovery from the broth can make
up to 20–30% of the total cost of producing the biomass [48]. Microalgal biomass harvesting can be achieved in several
physical, chemical or biological ways: flocculation, centrifugation, filtration, ultrafiltration, air-flotation, autoflotation,
etc. Generally, microalgae harvesting is a two stage process, involving: (1) Bulk harvesting: aimed at separation of
biomass from the bulk suspension. The concentration factors for this operation are generally 100–800 times to reach 2–
7 % total solid matter. This will depend on the initial biomass concentration and technologies employed, including
flocculation, flotation or gravity sedimentation; (2) Thickening: the aim is to concentrate the slurry through techniques
such as centrifugation, filtration and ultrasonic aggregation, hence, it is generally a more energy intensive step than bulk
harvesting .
4.2.1 Flocculation
Flocculation can be used as an initial dewatering step in the bulk harvesting process that will significantly enhance the
ease of further processing. This stage is intended to aggregate microalgal cells from the broth in order to increase the
effective ‘‘particle’’ size [49]. Since microalgae cells carry a negative charge that prevents them from self-aggregation
in suspension, addition of chemicals known as flocculants neutralises or reduces the negative surface charge. These
chemicals coagulate the algae without affecting the composition and toxicity of the product [48]. Multivalent metal salts
like ferric chloride (FeCl
3
), aluminium sulphate (Al
2
(SO
4
)
3
) and ferric sulphate (Fe
2
(SO
4
)
3
) are commonly used [4].
4.2.2 Flotation
Some strains naturally float at the surface of the water as the microalgal lipid content increase. Although flotation has
been mentioned as a potential harvesting method, there is very limited evidence of its technical or economic viability
[4].
4.2.3 Centrifugation
Centrifugation involves the application of centrifugal forces to separate microalgal biomass from growth medium. Once
separated, microalgae can be removed from the culture by simply draining the excess medium [49]. Centrifugal
recovery is a rapid method of recovering algal cells, especially for producing extended shelf-life concentrates for
aquaculture hatcheries and nurseries [48]. However, high gravitational and shear forces during the centrifugation
process can damage cell structure. Additionally, it is not cost effective due to high power consumption especially when
considering large volumes [49].
4.2.4 Filtration
Filtration is the method of harvesting that has proved to be the most competitive compared to other harvesting options.
There are many different forms of filtration, such as dead end filtration, microfiltration, ultra filtration, pressure
filtration, vacuum filtration and tangential flow filtration (TFF). Generally, filtration involves running the broth with
algae through filters on which the algae accumulate and allow the medium to pass through the filter. The broth
continually run through the microfilters until the filter contains a thick algae paste. Although filtration methods appear
to be an attractive dewatering option, they are associated with extensive running costs and hidden pre-concentration
requirements [49].
4.3 Extraction of microalgal lipids
4.3.1 Drying processes
Biomass drying before further lipid extraction and/or thermochemical processing is another step that needs to be taken
into consideration. Sun drying is probably the cheapest drying method that has been employed for the processing of
microalgal biomass. However, this method takes long drying time, requires large drying surface, and risks the loss of
some bioreactive products [6]. More efficient but more costly drying technologies having been investigated for drying
microalgae include drum drying, spray drying, fluidized bed drying, freeze drying and refractance window dehydration
technology [4].
4.3.2 Cell disruption
The majority of biodiesel today is produced from animal or plant oils through a transesterification process following oil
extraction with or without cell disruption [3]. Most cell disruption methods applicable to microalgae have been adapted
from applications on intracellular non-photosynthetic bioproducts [4]. Cell disruption methods that have been used
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successfully include high-pressure homogenisers, autoclaving, and addition of hydrochloric acid, sodium hydroxide, or
alkaline lysis [50].
4.3.3 Methods for extraction of lipids
Numerous methods for extraction of lipids from microalgae have been applied; but most common methods are
expeller/oil press, liquid–liquid extraction (solvent extraction), supercritical fluid extraction (SFE) and ultrasound
techniques [49].
Expeller/oil pressing is a mechanical method for extracting oil from raw materials such as nuts and seeds. Press uses
high pressure to squeeze and break cells. In order for this process to be effective, algae must first need to be dried.
Although this method can recover 75% of oil and no special skills is required, it was reported less effective due to
comparatively longer extraction time [49].
Solvent extraction proved to be successful in order to extract lipids from microalgae. In this approach, organic
solvents, such as benzene, cyclo-hexane, hexane, acetone, chloroform are added to algae paste. Solvent destroy algal
cell wall, and extract oil from aqueous medium because of their higher solubility in organic solvents than water. Solvent
extract can then be subjected to distillation process to separate oil from solvent. Latter can be reclaimed for further use.
Hexane is reported to be the most efficient solvent in extraction based on its highest extraction capability and low cost
[49].
Supercritical extraction makes use of high pressures and temperatures to rupture the cells. This particular method of
extraction has proved to be extremely time-efficient and is commonly employed [49].
Another promising method to be used in extraction of microalgae is the application of ultrasounds. This method
exposes algae to a high intensity ultrasonic wave, which creates tiny cavitation bubbles around cells. Collapse of
bubbles emits shockwaves, shattering the cell wall and releasing the desired compounds into solution. Although
extraction of oil from microalgae using ultrasound is already in extensive use at laboratory scale, sufficient information
on feasibility or cost for a commercial-scale operation is unavailable. This approach seems to have a high potential, but
more research is needed [49].
4.4 Biodiesel production
After the extraction processes, the resulting microalgal oil can be converted into biodiesel through a process called
transesterification. The transesterification reaction consists of transforming triglycerides into fatty acid alkyl esters, in
the presence of an alcohol, such as methanol or ethanol, and a catalyst, such as an alkali or acid, with glycerol as a by-
product [51].
For user acceptance, microalgal biodiesel needs to comply with existing standards, such as ASTM Biodiesel
Standard D 6751 (United States) or Standard EN 14214 (European Union). Microalgal oil contains a high degree of
polyunsaturated fatty acids (with four or more double bonds) when compared to vegetable oils, which makes it
susceptible to oxidation in storage and therefore reduces its acceptability for use in biodiesel. However, the extent of
unsaturation of microalgal oil and its content of fatty acids with more than four double bonds can be reduced easily by
partial catalytic hydrogenation of the oil, the same technology that is commonly used in making margarine from
vegetable oils [5]. Nevertheless, microalgal biodiesel has similar physical and chemical properties to petroleum diesel,
first generation biodiesel from oil crops and compares favourably with the international standard EN14214 [4].
4.5 Bioethanol production
The current interests in producing bioethanol are focusing on microalgae as a feedstock for fermentation process.
Microalgae provide carbohydrates (in the form of glucose, starch and other polysaccharides
)
and proteins that can be
used as carbon sources for fermentation by bacteria, yeast or fungi [49]. For instance, Chlorella vulgaris has been
considered as a potential raw material for bioethanol production because it can accumulate high levels of starch [52].
Chlorococum sp. was also used as a substrate for bioethanol production under different fermentation conditions. Results
showed a maximum bioethanol concentration of 3.83 g l
-1
obtained from 10 g l
-1
of lipid-extracted microalgae debris
[53].
Production of bioethanol by using microalgae can also be performed via self-fermentation. Previous studies reported
that dark fermentation in the marine green algae Chlorococcum littorale was able to produce 450 µmol ethanol g
-1
at
30 ºC [54].
Even though limited reports on microalgal fermentation were observed, a number of advantages were observed in
order to produce bioethanol from microalgae. Fermentation process requires less consumption of energy and simplified
process compared to biodiesel production system. Besides, CO
2
produced as by-product from fermentation process can
be recycled as carbon sources to microalgae in cultivation process thus reduce the greenhouse gases emissions.
However, the production of bioethanol from microalgae is still under investigation and this technology has not yet been
commercialized [49].
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5. Concluding remarks
Microalgae offer great potential as a sustainable feedstock for the production of third generation biofuels, such as
biodiesel and bioethanol. However, several important scientific and technical barriers remain to be overcome before the
large-scale production of microalgae derived biofuels can become a commercial reality. Technological developments,
including advances in photobioreactor design, microalgal biomass harvesting, drying, and processing are important
areas that may lead to enhanced cost-effectiveness and therefore, effective commercial implementation of the biofuel
from microalgae strategy.
Acknowledgements The authors acknowledge financial support from FCT (Fundação para a Ciência e a Tecnologia (Portugal))
through Giuliano Dragone grant (SFRH/BPD/44935/2008), Bruno Fernandes grant (SFRH/BD/44724/2008) and projects
INNOVALGAE (PTDC/AAC-AMB/108511/2008) and ALGANOL.
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