Comprehensive Scientific Report: Algae as a Biofuel

May 11, 2025

4. Algae Cultivation Techniques

Algae cultivation is the cornerstone of biofuel production, as it determines the biomass
yield, lipid content, and overall feasibility of the process. Algae, being photosynthetic
microorganisms, convert sunlight, carbon dioxide, and nutrients into energy-rich biomass
through photosynthesis. This biomass, particularly the lipid fraction, can be processed
into biodiesel, a renewable alternative to fossil fuels. The cultivation method chosen
impacts not only the productivity but also the scalability, environmental footprint, and
economic viability of the biofuel production process. Various techniques have been de-
veloped, each with distinct advantages, challenges, and applications.

Open Pond Systems

Open pond systems are the simplest and most traditional method for cultivating algae
on a large scale. These systems consist of shallow, open basinsoften called raceway
pondstypically 15 to 30 cm deep, with a surface area ranging from 1,000 to 5,000 m2 . The
ponds are designed with a looped configuration, where paddlewheels or pumps circulate
the algal culture to ensure even distribution of nutrients, sunlight, and CO2 . Open ponds
rely on natural sunlight as the primary energy source for photosynthesis, supplemented
by ambient CO2 from the atmosphere or industrial flue gases.
The primary advantage of open pond systems is their cost-effectiveness. Construction
costs are relatively low, averaging around $10,000 to $20,000 per hectare, and operational
costs are minimal, mainly involving water, nutrients, and energy for circulation (approx-
imately 0.10.5 kWh/m3 of culture). This makes open ponds economically attractive for
large-scale biofuel production, especially in regions with abundant sunlight and flat land,
such as arid or semi-arid areas. For example, commercial facilities in Australia and the
southwestern United States have successfully used open ponds to cultivate species like
Dunaliella salina and Chlorella vulgaris [3].
However, open pond systems face significant challenges. One major issue is con-
tamination by unwanted microorganisms, such as bacteria, fungi, or other algae species,
which compete with the desired algae for nutrients and space. For instance, predatory
zooplankton can reduce algal biomass by 50% within days if not controlled. Environmen-
tal fluctuations also pose a challenge: temperature variations (e.g., diurnal swings from
15°C to 35°C) and rainfall can dilute the culture or alter nutrient levels, while evaporation
in hot climates increases salinity, stressing the algae. These factors lead to inconsistent
biomass productivity, typically ranging from 10 to 25 g/m2 /day, which is lower than other
systems [4].

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 To quantify algal growth in open ponds, we can use the Monod growth model, which
describes the relationship between growth rate and substrate availability [20]:
S
µ = µmax ·
Ks + S
Here, µ is the specific growth rate (day-1 ), µmax is the maximum growth rate (e.g.,
0.5 day-1 for Chlorella), S is the substrate concentration (e.g., nitrogen or CO2 ), and
Ks is the half-saturation constant (e.g., 0.1 g/L for nitrogen). In open ponds, S can
vary widely due to environmental factors, leading to fluctuations in µ. To mitigate these
issues, operators often select robust algal species, use biocides to control contaminants,
and monitor water quality regularly.

Closed Photobioreactors
Closed photobioreactors (PBRs) are engineered systems designed to provide a controlled
environment for algal growth, minimizing external influences. PBRs come in various de-
signs, including tubular, flat-panel, and bag reactors, each constructed from transparent
materials like glass, acrylic, or polyethylene to allow light penetration. Tubular PBRs,
for example, consist of long, narrow tubes (1020 cm in diameter) arranged in horizontal
or vertical loops, with pumps circulating the culture to prevent sedimentation and ensure
uniform exposure to light and nutrients. Flat-panel PBRs use thin, rectangular cham-
bers (15 cm thick) to maximize light exposure, while bag reactors suspend plastic bags
vertically, often used for smaller-scale operations [23].
The key advantage of PBRs is their ability to control growth conditions precisely.
Parameters such as light intensity, temperature, pH, and CO2 concentration can be op-
timized to maximize algal productivity. For instance, temperature can be maintained
at 25°C (optimal for many species), pH at 7.5, and CO2 levels at 25% v/v, leading to
biomass productivities of 4050 g/m2 /daydouble that of open ponds. PBRs also signif-
icantly reduce contamination risks, as the enclosed system prevents entry of unwanted
microorganisms. This allows cultivation of high-value or sensitive species, such as Nan-
nochloropsis, which are prone to contamination in open systems. Additionally, PBRs
produce biomass with higher purity and lipid content, often reaching 3040% lipids by dry
weight, compared to 2025% in open ponds [6].
Despite these benefits, PBRs have notable drawbacks. The capital cost is high, often
exceeding $100,000 per hectare, due to the need for specialized materials and infrastruc-
ture. Operational costs are also significant, driven by energy requirements for mixing,
cooling, and CO2 delivery, totaling 13 kWh/m3 of culture. Overheating is a common
issue in tubular PBRs, as solar radiation can raise temperatures above 40°C, inhibiting
growth. Cooling systems, such as water sprays or heat exchangers, are often required,
further increasing costs. Another challenge is light limitation: as biomass concentra-
tion increases, self-shading reduces light penetration, which can be modeled using the
Beer-Lambert Law [1]:

Iz = I0 · e−α·C·z
Here, Iz is the light intensity at depth z, I0 is the incident light intensity (e.g., 1,000
ţmol photons/m2 /s), α is the absorption coefficient (e.g., 0.2 m2 /g), C is the biomass
concentration (e.g., 2 g/L), and z is the depth (e.g., 0.1 m). For a biomass concentration

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of 2 g/L, light intensity may drop by 50% within 5 cm, necessitating thin reactor designs
or artificial lighting, which adds to the cost.

Hybrid and Emerging Systems

Hybrid systems combine the strengths of open ponds and closed PBRs to achieve a
balance between cost and productivity. A common hybrid approach is a two-stage process:
in the first stage, algae are grown in a PBR to establish a pure, high-density culture,
minimizing contamination risks; in the second stage, the culture is transferred to an open
pond for mass production, reducing operational costs. For example, a 1,000 L PBR can
be used to inoculate a 100,000 L open pond, achieving productivities of 3040 g/m2 /day
while keeping costs at $50,000 per hectarelower than a fully closed system but higher
than a standalone open pond [7].
Emerging systems are also being developed to address the limitations of traditional
methods. Vertical bioreactors, for instance, stack algal cultures in vertically arranged
tubes or panels, maximizing land use efficiency. A vertical system occupying 100 m2
of land can have a photosynthetic surface area of 1,000 m2 , achieving biomass densities
of 6080 g/m2 . These systems are particularly suited for urban or space-constrained
areas, such as industrial rooftops. Another emerging technology is the biofilm reactor,
where algae grow as a thin layer on a solid surface, such as a rotating drum or a porous
membrane, while being exposed to air and a thin film of nutrient-rich water. Biofilm
reactors reduce water usage by 90% compared to suspended cultures, as only a minimal
amount of water is needed to supply nutrients. They can achieve biomass densities of
100150 g/m2 , significantly higher than traditional systems, due to the high surface area
and reduced light limitation. However, challenges include scaling up biofilm reactors, as
maintaining uniform nutrient delivery and preventing drying of the biofilm are technically
complex [21].
Hybrid and emerging systems are still evolving, but they offer promising solutions for
sustainable algae cultivation. Research is ongoing to optimize designs, such as integrating
renewable energy sources (e.g., solar-powered pumps) to reduce operational costs, and
developing genetically engineered algae strains that thrive in these systems.

Factors Affecting Algal Growth

Algal growth and lipid accumulation are influenced by several environmental and bio-
logical factors, each of which must be carefully managed to optimize biofuel production.
These factors interact in complex ways, affecting photosynthesis, metabolism, and cell
division [11].
- Light: Light is the primary energy source for photosynthesis, the process by which
algae convert CO2 and water into biomass. The optimal light intensity varies by species
but typically ranges from 100 to 500 ţmol photons/m2 /s. For example, Chlorella vulgaris
achieves maximum growth at 300 ţmol photons/m2 /s, while Nannochloropsis prefers 400
ţmol photons/m2 /s. The photosynthetic rate can be described using a light response
curve:
( )
α·I
P = Pmax · tanh
Pmax
Here, P is the photosynthetic rate (mg O2 /g/h), Pmax is the maximum rate (e.g.,
10 mg O2 /g/h), α is the initial slope (e.g., 0.05 mg O2 /g/h per ţmol/m2 /s), and I is

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the light intensity. At low light levels, growth is light-limited, while excessive light (e.g.,
1,000 ţmol photons/m2 /s) causes photoinhibition, where reactive oxygen species damage
photosynthetic machinery, reducing growth by 2030% [15].
- Temperature: Temperature affects enzymatic activity and metabolic rates. Most
algae grow optimally between 20°C and 30°C. For example, Spirulina platensis has an
optimal temperature of 28°C, with growth rates dropping by 50% at 35°C due to protein
denaturation. Low temperatures (e.g., 10°C) slow metabolism, reducing growth by 70%.
Temperature control is critical in PBRs, where cooling systems maintain stability, while
open ponds rely on ambient conditions, making site selection (e.g., tropical or subtropical
regions) crucial.
- Nutrients: Algae require macronutrients like nitrogen (for proteins and DNA) and
phosphorus (for ATP and phospholipids), as well as micronutrients like iron and mag-
nesium. Nitrogen concentrations of 0.51 g/L support exponential growth, but limitation
(e.g., 0.05 g/L) induces lipid accumulation, increasing lipid content from 20% to 50% in
species like Nannochloropsis. Phosphorus limitation has a similar effect but also slows
growth. Nutrient uptake can be modeled using Michaelis-Menten kinetics [18]:

[N ]
V = Vmax ·
Km + [N ]
Here, V is the uptake rate, Vmax is the maximum uptake rate, [N ] is the nutrient
concentration, and Km is the half-saturation constant.
- pH: Algae thrive at pH 79, where nutrient availability and enzyme activity are
optimal. For example, at pH 6, CO2 availability decreases (as it forms bicarbonate),
slowing photosynthesis, while pH 10 can denature proteins. pH is often controlled by
buffering systems or CO2 injection.
- CO2 Concentration: Algae use CO2 as a carbon source for photosynthesis. Am-
bient levels (0.04% v/v) are often limiting, but elevating CO2 to 25% v/v (e.g., using
industrial flue gas) can increase growth rates by 200300%. However, excessive CO2 (e.g.,
10% v/v) lowers pH to 56, causing toxicity. CO2 fixation can be quantified as:

Biomass (g) = YCO2 · CO2 absorbed (g)

where YCO2 is the biomass yield per gram of CO2 (typically 1.8 g biomass/g CO2 , as
1 g of biomass requires 1.83 g CO2 based on stoichiometry) [6].

5. Harvesting and Processing Algae Biomass

Harvesting and processing algal biomass are critical steps in biofuel production, as they
prepare the biomass for lipid extraction. These steps are energy-intensive, accounting
for 2030% of total production costs, and require efficient methods to ensure economic
viability. The process involves separating algae from the growth medium, reducing water
content, and disrupting cells to access lipids [4].

Harvesting Methods
Harvesting separates algal biomass from the culture medium, which typically contains
0.11 g/L of biomass, meaning 1,000 L of culture yields only 1001,000 g of dry biomass.
Several methods are used, each tailored to specific algal species, culture conditions, and
scale of operation.

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 - Centrifugation: Centrifugation uses high-speed rotation (5,00010,000 rpm) to sep-
arate algae based on density differences. A disc-stack centrifuge, for example, can process
10,000 L/h, recovering 9095% of biomass with a final concentration of 100150 g/L. The
process is highly efficient but energy-intensive, consuming 13 kWh/m3 of culture. For a
10,000 L batch, this translates to 1030 kWh, costing $13 at $0.1/kWh. Centrifugation
is ideal for high-value applications but less economical for large-scale biofuel production
[19].
- Flocculation: Flocculation aggregates algae cells into larger clumps, making them
easier to separate by sedimentation or filtration. Chemical flocculants, such as alum
(aluminum sulfate) or cationic polymers (e.g., chitosan), are added to neutralize the
negative surface charge of algae cells, promoting aggregation. For example, adding 100
mg/L of alum can flocculate 90% of Chlorella cells within 30 minutes. The process can
be modeled as:

Ceff = C0 · (1 − e−k·D )
Here, Ceff is the effective concentration of settled algae, C0 is the initial concentration
(e.g., 0.5 g/L), k is the flocculation rate constant (e.g., 0.01 L/mg), and D is the floccu-
lant dose (mg/L). Flocculation is cost-effective, with chemical costs of $0.10.5/kg of dry
biomass, but residues may contaminate the biomass, affecting downstream processes [24].
- Filtration: Filtration uses membranes or screens to retain algae while allowing
water to pass through. Microfiltration membranes (pore size 0.110 ţm) are effective for
larger species like Spirulina (510 ţm in size), achieving 8090% recovery. However, smaller
species like Chlorella (25 ţm) can cause membrane fouling, reducing flux rates from 100
L/m2 /h to 20 L/m2 /h within hours. Filtration systems cost $5,00010,000 per unit and
are best suited for small-scale operations or as a secondary step after flocculation [22].
- Flotation: Dissolved air flotation (DAF) introduces microbubbles (20100 ţm) into
the culture, which attach to algae cells and lift them to the surface for skimming. DAF
can recover 8590% of biomass, with energy consumption of 0.10.5 kWh/m3 , making it
more energy-efficient than centrifugation. However, the process is slower, requiring 3060
minutes per batch, and may require flocculants to enhance bubble attachment. DAF is
often used in wastewater treatment facilities growing algae, where air systems are already
available [25].
The choice of harvesting method depends on factors like algal size, culture density,
and energy budget. For large-scale biofuel production, a combination of flocculation and
centrifugation is often used to balance cost and efficiency.

Dewatering and Drying

After harvesting, the algal biomass contains 8090% water, which must be reduced to
1020% to facilitate lipid extraction. This process occurs in two stages: dewatering, which
produces a concentrated slurry, and drying, which removes residual moisture [8].
Dewatering uses techniques like sedimentation or mechanical pressing. Sedimentation
relies on gravity to settle algae cells, with settling velocity determined by Stokes Law:

2r2 (ρalgae − ρwater )g

v=
9η
Here, v is the settling velocity, r is the particle radius (e.g., 5 ţm for Chlorella),
ρalgae and ρwater are the densities of algae (1,050 kg/m3 ) and water (1,000 kg/m3 ), g is

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gravitational acceleration (9.81 m/s2 ), and η is the viscosity of water (0.001 Paůs). For
Chlorella, v is approximately 0.01 mm/s, meaning a 1 m deep tank takes 2448 hours
to settle, making sedimentation slow for large volumes. Mechanical pressing uses screw
presses or belt presses to squeeze out water, achieving 1520% solids content in minutes,
but requires energy (0.51 kWh/m3 ).
Drying removes the remaining moisture to prepare the biomass for extraction. Several
methods are used:
- Sun Drying: The biomass slurry is spread in thin layers (12 cm) on a flat surface
and dried using solar heat. This method is cost-free, requiring only labor and land, but
takes 23 days and depends on weather conditions. In humid or cloudy regions, drying
may take longer, and exposure to air can lead to oxidative degradation, reducing lipid
quality by 1020%. Sun drying is common in small-scale operations in tropical regions.
- Spray Drying: Spray drying atomizes the slurry into a hot chamber (150200°C),
where water evaporates rapidly, producing a dry powder (510% moisture) within seconds.
A typical spray dryer processes 100 kg/h of slurry, consuming 12 kWh/kg of dry biomass.
The high temperature preserves biomass quality by minimizing exposure time, but the
energy cost is significant, at $0.10.2/kg of dry biomass. Spray drying is widely used in
commercial facilities for its speed and consistency [13].
- Freeze-Drying: Freeze-drying (lyophilization) freezes the biomass at -50°C, then
removes water by sublimation under vacuum (0.01 mbar). This method produces high-
quality biomass with minimal lipid degradation, as low temperatures prevent oxidation.
However, it is energy-intensive, requiring 510 kWh/kg of dry biomass, and equipment
costs are high, at $50,000100,000 per unit. Freeze-drying is typically used for high-value
applications, such as pharmaceutical-grade algae products, rather than biofuel produc-
tion.
The choice of drying method depends on the scale of operation, energy availability,
and downstream requirements. For biofuel production, spray drying is often preferred for
its balance of speed and quality, despite higher costs.

Cell Disruption Techniques

Cell disruption breaks the algal cell walls to release intracellular lipids, which are often
stored in lipid bodies within the cytoplasm. Algal cell walls, composed of cellulose,
glycoproteins, and sometimes silica (in diatoms), are robust, with thicknesses of 0.10.5
ţm, requiring effective disruption methods. The choice of technique impacts lipid yield,
energy consumption, and scalability [14].
- Mechanical Methods: Mechanical disruption physically breaks cell walls using
shear forces. Bead milling, for example, uses small beads (0.11 mm diameter, typically
glass or ceramic) in a rotating chamber to grind the biomass. The process disrupts 8090%
of cells, releasing lipids within 510 minutes per batch. Energy consumption is high, at
0.52 kWh/kg of dry biomass, due to the mechanical work required. High-pressure ho-
mogenization (HPH) is another mechanical method, where the biomass is forced through
a narrow valve at 5001,000 bar, creating shear forces that disrupt cells. HPH achieves
8595% disruption but consumes 13 kWh/kg. Mechanical methods are scalable and ef-
fective for tough species like Chlorella, but their energy intensity increases production
costs.
- Chemical Methods: Chemical disruption uses solvents, acids, or alkalis to weaken
cell walls. For example, sulfuric acid (pH 2) or sodium hydroxide (pH 12) can be added to

6
the biomass slurry, breaking down cell wall components like cellulose and hemicellulose.
This increases lipid accessibility by 5060%, as the chemicals permeabilize the cell wall,
allowing lipids to be released. The process is energy-efficient, requiring only mixing
(0.10.2 kWh/kg), but chemical costs are $0.51/kg, and residues must be neutralized to
avoid affecting downstream processes like transesterification. Chemical methods are often
used in combination with mechanical methods to enhance efficiency [12].
- Enzymatic Methods: Enzymatic disruption uses enzymes like cellulase, lysozyme,
or pectinase to degrade specific cell wall components. For example, cellulase breaks down
cellulose into glucose, weakening the cell wall and releasing 7080% of lipids. The process
is conducted at mild conditions (40°C, pH 5), requiring low energy (0.10.5 kWh/kg), and
is highly specific, minimizing damage to lipids. However, enzymes are expensive, costing
$15/kg of dry biomass, and the process is slower, taking 24 hours per batch. Enzymatic
methods are typically used for high-value products or in research settings, but their cost
limits adoption in biofuel production [26].
The effectiveness of cell disruption depends on the algal species and cell wall composi-
tion. For example, Nannochloropsis has a thick, lipid-rich cell wall, requiring mechanical
methods, while Spirulina has a thinner wall, making chemical or enzymatic methods vi-
able. A combination of methods (e.g., enzymatic pretreatment followed by bead milling)
is often used to maximize lipid yield while minimizing energy use.

6. Lipid Extraction from Algae

Lipid extraction isolates the lipid fraction from algal biomass, primarily triglycerides,
which can be converted into biodiesel via transesterification. Algal lipids typically con-
stitute 2050% of dry weight, with neutral lipids (triglycerides) being the primary target
for biofuel, alongside polar lipids (phospholipids) and free fatty acids. The extraction
method must be efficient, scalable, and environmentally safe to ensure the viability of
algal biofuels [9].

Mechanical Extraction
Mechanical extraction uses physical force to disrupt algal cells and release lipids, avoiding
the use of chemicals. Two common methods are pressing and bead milling (also used in
cell disruption, as discussed earlier).
Pressing applies high pressure (1050 MPa) to the biomass using a screw press or hy-
draulic press, squeezing out lipids in a manner similar to oilseed processing (e.g., soybean
oil extraction). The biomass must be dried to 1015% moisture to prevent slippage during
pressing. For Chlorella, pressing can yield 1015% of total lipids, with the remaining lipids
requiring secondary extraction. The energy requirement for pressing can be estimated
as:

E =P ·V ·t
Here, E is the energy (kWh), P is the pressure (Pa), V is the volume of biomass (m3 ),
and t is the processing time (s). For a 1 kg batch at 20 MPa, processed over 60 s, the
energy is approximately 0.51 kWh/kg. Pressing is environmentally friendly, as it avoids
solvents, but its low yield makes it less efficient for algae compared to oilseeds, which
have higher lipid content (4060%) [16].

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 Bead milling, as a mechanical extraction method, not only disrupts cells but also fa-
cilitates lipid release by breaking lipid bodies. After milling, the biomass is centrifuged to
separate the lipid-rich supernatant, yielding 1520% of total lipids. The process is energy-
intensive, as noted earlier, but effective for small-scale operations or as a pretreatment
for solvent extraction.

Chemical and Solvent Extraction

Chemical and solvent extraction uses organic solvents to dissolve and extract lipids from
algal biomass. The most widely used method is the Bligh and Dyer protocol, which mixes
the biomass with chloroform, methanol, and water in a 1:2:0.8 ratio [2]. The mixture
forms a biphasic system: the organic phase (chloroform) contains the lipids, while the
aqueous phase (methanol-water) retains proteins and carbohydrates. After mixing for 12
hours, the phases are separated by centrifugation, and the chloroform is evaporated to
recover the lipids. This method can extract 9095% of total lipids, including neutral and
polar fractions, making it highly efficient.
Hexane is another common solvent, particularly for neutral lipids like triglycerides.
The biomass is mixed with hexane (1:10 w/v ratio) and agitated for 24 hours at 40°C,
after which the hexane-lipid mixture is separated and evaporated. Hexane extraction
yields 8090% of neutral lipids, with the extraction efficiency described by:
( )
Y = Ymax · 1 − e−k·t
Here, Y is the lipid yield, Ymax is the maximum extractable lipid (e.g., 40% of dry
weight), k is the extraction rate constant (e.g., 0.5 h-1 ), and t is the extraction time (h).
For a 4-hour extraction, the yield approaches 86% of Ymax .
Solvent extraction is effective but has drawbacks. Chloroform and hexane are toxic
and flammable, requiring safety measures like fume hoods and explosion-proof equipment,
which increase costs. Solvent disposal is also a concern, with costs of $0.10.5/kg of
solvent, and environmental regulations often require solvent recovery systems, adding
$5,00010,000 to setup costs. Despite these challenges, solvent extraction remains the
standard for commercial algal lipid extraction due to its high yield and scalability [17].

Supercritical Fluid Extraction

Supercritical fluid extraction (SFE) uses supercritical CO2 as a solvent to extract lipids.
At supercritical conditionsabove its critical point of 31.1°C and 73.8 barCO2 has a density
like a liquid (0.61 g/cm3 ) and viscosity like a gas (0.02 mPaůs), allowing it to penetrate
cells and dissolve lipids effectively. The process involves drying the biomass to 510%
moisture, then placing it in a high-pressure vessel. Supercritical CO2 is pumped through
the vessel at 4060°C and 200300 bar, extracting lipids over 12 hours. The CO2 -lipid
mixture is then depressurized, causing the CO2 to evaporate and leaving behind the
lipids.
SFE achieves yields of 8595%, comparable to solvent extraction, but with several
advantages. CO2 is non-toxic, non-flammable, and environmentally safe, eliminating the
need for hazardous waste disposal. The CO2 can be recycled, reducing operational costs
to $0.050.1/kg of biomass. SFE also allows selective extraction: by adjusting pressure
and temperature, neutral lipids can be targeted over polar lipids, improving biodiesel

8
quality. The solubility of lipids in supercritical CO2 can be modeled using the Chrastil
equation [5]:
a
S = k · ρc · e( T +b)
Here, S is the solubility (g/L), ρ is the density of CO2 (e.g., 0.8 g/cm3 at 300 bar), T is
the temperature (K), and k, c, a, and b are empirical constants (e.g., c = 3, a = −4000,
b = −10). At 50°C and 300 bar, lipid solubility is approximately 1020 g/L, allowing
efficient extraction.
The main drawback of SFE is its high capital cost, with equipment costing $12 million
for a commercial unit due to the need for high-pressure vessels and pumps. Energy
consumption is also significant, at 25 kWh/kg of dry biomass, due to the compression
and heating of CO2 . SFE is often used for high-value products or in facilities where
environmental regulations prohibit solvent use, but its adoption in biofuel production is
growing as equipment costs decrease [10].

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