Clean Techn Environ Policy (2017) 19:637–668

DOI 10.1007/s10098-016-1309-6

REVIEW

A review of biodiesel production from microalgae

Selena Dickinson1 • Miranda Mientus1 • Daniel Frey1 • Arsalon Amini-Hajibashi1 •
Serdar Ozturk1 • Faisal Shaikh1 • Debalina Sengupta2 • Mahmoud M. El-Halwagi2,3

Received: 18 July 2016 / Accepted: 5 November 2016 / Published online: 18 November 2016
Ó Springer-Verlag Berlin Heidelberg 2016

Abstract As the search for alternatives to fossil fuels drawn from industry and the literature, this review provides
continues, microalgae have emerged as a promising a practical approach for creating a microalgae biodiesel
renewable feedstock for biodiesel. Many species contain facility.
high lipid concentrations and require simple cultivation—
including reduced freshwater and land area needs—com- Keywords Biodiesel
Biofuel
Energy
Life cycle
pared to traditional crops used for biofuels. Recently, analysis
Microalgae
Sustainability
technological advancements have brought microalgae
biodiesel closer to becoming economically feasible through Abbreviations
increased efficiency of the cultivation, harvesting, pre- EER Energy efficiency ratio
treatment, lipid extraction, and transesterification subsys- FAEE Fatty acid ethyl ester
tems. The metabolism of microalgae can be favorably FAME Fatty acid methyl ester
manipulated to increase lipid productivity through envi- FFA Free fatty acids
ronmental stressors, and ‘‘green’’ techniques such as using HPH High-pressure homogenization
flue gas as a carbon source and wastewater as a media LCA Life cycle analysis
replacement can lower the environmental impact of bio- PEF Pulsed electric field
diesel production. Through life cycle assessment and the TAG Triacylglycerol
creation of process models, valuable insights have been % wt Weight percentage
made into the energy and material sinks of the manufac- v/v Volume ratio
turing process, helping to identify methods to successfully w/w Weight ratio
scale up microalgae biodiesel production. Several compa-
nies are already exploring the microalgae industry, offset- Units
ting operating costs through isolation of co-products and nm Nanometer
careful unit operation selection. With numerous examples lm Micrometer
ml Mililiter
mg Milligram
g Gram
& Serdar Ozturk kg Kilogram
ozturk@msoe.edu h Hour
1
Biomolecular Engineering Program, Physics and Chemistry
min Minute
Department, Milwaukee School of Engineering, 1025 N d Day
Broadway, Milwaukee, WI 53202, USA yr Year
2
Gas and Fuels Research Center, Texas A&M Engineering L Liter
Experiment Station, College Station, TX 77843, USA gal Gallon
3
Chemical Engineering Department, Texas A&M University, mol Mole number
College Station, TX 77843, USA MPa Megapascal

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638 S. Dickinson et al.

MJ Megajoule is used for cultivation of the microalgae. Choices in har-

GHz Gigahertz vesting and pretreatment affect the total amount of
microalgae captured and used for fuel, as well as the effi-
ciency of the lipid extraction process downstream. During
Introduction the extraction step, neutral lipids are separated from the
biomass and converted into biodiesel through a transes-
The increasing need for sustainable energy calls for the terification reaction. Extraction fluids vary in expense,
development of renewable and cost-effective alternative selectivity, and efficacy and thus affect the end product’s
energy sources to reduce the use of fossil fuels. Most cost, quantity, and purity. It is also during the extraction
biofuels manufactured in the USA come from food crops, step that valuable co-products are removed for further
which are not an ideal energy source due to inherent processing.
problems. These problems, listed in Table 1, include
inefficient use of land, low lipid content, high water con-
sumption for irrigation, and competition with food pro-
duction. The use of microalgae to produce oil that can be
converted to biodiesel provides a potential solution with
the promise of carbon sequestration and high oil yield at a
fraction of the land requirement for food crop-based bio-
diesel sources. Microalgae further eliminate ethical con-
cerns regarding changing food into fuel, or using valuable
cropland for cultivation. There exists a need to design and
optimize a sustainable, high-throughput production facility
in which biodiesel is made from microalgae at a compet-
itive market price. Microalgae biofuel has the potential to
shift national consumption of a nonrenewable resource to a
renewable and partly photosynthetic process, reduce net
carbon emissions, buffer fluctuating US oil prices, and be
easily implemented in current automobile models (Chisti
2007).
Microalgae biomass generation and processing options
that play an important role in the creation of a feasible
biodiesel production plant will be investigated in detail.
Many of the strategies being employed by researchers and
companies to produce microalgal biodiesel are given in
Fig. 1. Key considerations that must be addressed in the
design of a microalgae production facility include strain
selection, cultivation method, harvesting and pretreatment
processes, extraction choices, and transesterification sys-
tems. Strain selection plays an important role because
microalgae biodiversity is large and species differ in oil
productivity, oil content, and co-product generation. Land
and nutrient requirements differ greatly depending upon Fig. 1 Overview of the microalgae to biodiesel production process
whether open raceway ponds or a closed bioreactor system with the various potential reactor types and unit operations

Table 1 Characteristics of
microalgae biofuel versus
traditional crop-based biodiesel
and petro-diesel
Desirable characteristics of fuel sources Petroleum Crop Microalgae
Low sulfur content 4 4
Sequesters CO2 4 4
Higher lipid productivity 4
Lower freshwater needs 4
No competition with food sources 4 4
Can be obtained from arid land 4 4

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A review of biodiesel production from microalgae 639

Table 2 Differing strategies used with microalgal biodiversity in the production of biofuel
Name Carbon/ Advantages Disadvantages
energy
source

Phototrophic Sunlight No substrate energy or carbon source required; sequesters CO2; Low cell density in scale-up; light source
and CO2 less expensive than heterotrophic and mixotrophic cultivation required; sensitive to photoinhibition (Chen
(Wang et al. 2014) et al. 2011; Chisti 2007)
Heterotrophic Sugars or High oil productivity; growth is not light dependent (Brennan Substrate cost (Chen et al. 2011); vulnerable to
glycerol and Owende 2010) contamination (Wang et al. 2014); lower
concentration of high-value pigments;
production of CO2 (Lowrey et al. 2014)
Mixotrophic Sunlight, Sequesters CO2; substrate cost reduced compared to that of Lower energy conversion efficiency than
CO2, and heterotrophic microalgae (Brennan and Owende 2010; Chen heterotrophs; net CO2 production (Lowrey
sugars et al. 2011) et al. 2014)

Each step in the production process must be carefully
considered when developing a model regarding its capital
cost, operating cost, product yield, and environmental safety.
The environmental impact of each step also must be consid-
ered when producing a microalgal biofuel production facility.
Strain selection criteria
Because the cellular contents and growth behavior of dif-
ferent microalgae strains differ significantly, it is very
important to select species that best fit the process needs.
Specie characteristics such as concentration of high-value
side products in the cell, oil productivity, growth rate, opti-
mal growth conditions, and scale-up potential should be
taken into consideration (Leu and Boussiba 2014). After a
strain is selected, its growth parameters are optimized for the
production of the desired product such as biofuel or fine
chemicals. Upstream processing factors used in selection of
microalgae strains include resistivity to shear stress caused
by mixing, accessibility of genomic sequences, and tolerance
to harsh operating conditions caused by suboptimal pH,
temperature, or oxygen saturation (Chisti 2007). The strain
type is also crucial for downstream processing because the
ability for certain cell types to float might enable flotation-
type harvesting. Although strain selection is important for
assessment of environmental impact, process sustainability,
biofuel manufacturability, and process economics, there is
no perfect microalgae strain existing now that maximizes
each parameter.
Microalgae energy requirements
Microalgae are an ancient group of eukaryotic organisms
that constitute a range of species. Most microalgae are
known for their phototrophic nature, but microalgae strains
exhibit a high amount of variation. Microalgae can be
divided into three main categories based on whether energy
is obtained from sunlight and carbon dioxide (CO2), pre-
existing biomass, or both. Phototrophic microalgae use
sunlight to power glucose production and inorganic carbon,
typically CO2, as their carbon source. This inorganic car-
bon can also be supplied as soluble carbonates such as
Na2CO3 or NaHCO3 (Brennan and Owende 2010). Pho-
totrophic microalgae are the most widely used microalgae
for production-scale processes, in part due to their low
nutrient requirements and environmental benefits (Rawat
et al. 2012). Phototrophic microalgae typically have lower
lipid productivity levels than their heterotrophic counter-
parts (Brennan and Owende 2010). In contrast to pho-
totrophs, heterotrophic microalgae consume glucose from
biomass rather than synthesizing it from inorganic mole-
cules found in the environment. Mixotrophic microalgae
resemble both phototrophs and heterotrophs because they
are able to photosynthesize energy molecules as well as
feed off of biomass in their environment.
Table 2 shows the major microalgae metabolism classes
and characteristics associated with cultivation methods. Het-
erotrophic growth has the highest oil productivity and can be
grown in a conventional fermenter; however, high substrate
costs can limit the economic feasibility of this approach. Pho-
totrophic microalgae cultivation—especially in open ponds
rather than photobioreactors—are limited in achieving the high
cell densities compared to heterotrophic cultures. Mixotrophic
microalgae have the advantage of being able to use multiple
food sources, but may require more complex cultivation
equipment (Chen et al. 2011). Methods for reducing economic
costs associated with each microalgae type will be discussed
throughout the cultivation and harvesting sections.
Regardless of whether they are phototrophic, hetero-
trophic, or mixotrophic, microalgae contain the beta-oxi-
dation metabolic pathway, which allows for synthesis of
the class of lipids called acylglycerols. Acylglycerols,
which can be used as a secondary energy source or electron
sink during microalgal metabolism, can be converted via a
transesterification reaction into biodiesel.

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Microalgae biology
The basic biology of microalgae affects the amount and
quality of lipids isolated. Not only do the cells vary in
nutrition requirements to ensure optimum growth rates, but
stresses are required to shift cellular metabolism from
normal cellular functions to the generation of fuel or other
valuable co-products such as carotenoids. Once cultivation
has occurred, the cells must be harvested and are then often
lysed. Aspects of microalgae affect downstream process-
ing; for example, microalgae size can affect the ability to
float, flocculate, or be filtered efficiently, and thick cell
walls can make lysis difficult. Microalgae size can vary
greatly; for example, the diameter of Nannochloropsis
salina is 2.5 lm, but Haematococcus pluvialis is 5–25 lm
in diameter (Coustets et al. 2015). Some strains produce a
large amount of starch to store energy, while others—such
as Nannochloropsis or diatoms—may produce mostly
lipids (Vitova et al. 2015). Chlorella vulgaris has an
optimal growing temperature of 10 °C, while Nan-
nochloropsis flourishes at 25 °C (Georgianna and Mayfield
2012). With this in mind, generalizations of lipid produc-
tion, cell wall structure, cellular division, and microalgae
industrial applications are discussed below.
Lipid and starch production
Biodiesel is typically made up of fatty acid methyl ester
(FAME) and sometimes fatty acid ethyl ester (FAEE)
molecules, which have an approximate molar mass of
290 g/mol. Lipid precursors of these molecules are stored
Fig. 2 Accumulation of energy reserves in microalgae. Drawing adapted from Vitova et al. (2015). Figure courtesy of Wesley Zloza
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S. Dickinson et al.
in lipid bodies in the cytoplasm. Lipid bodies are round
organelles that not only store lipids, but are also respon-
sible for lipid transport, protein storage and degradation,
and other roles. If biodiesel is desired, then a strain with
high lipid production is preferred. If the desired product is
ethanol, a strain with a high starch concentration is ideal.
Starch functions as the primary energy source for
microalgae and is produced during light exposure in
chloroplasts. Triacylglycerol (TAG) and other fatty acid
derivatives function as secondary energy sources and are
often located in the microalgal cytoplasm. These biomo-
lecules act as both electron sinks and energy reserves for
microalgae metabolism. Figure 2 shows light entering the
cell and being absorbed by the chloroplast photosystem,
which causes an oxidation–reduction reaction that gener-
ates high-energy electrons that are then shuttled by
NADPH into the Calvin cycle. Products of this cycle are
used to create a variety of precursors to molecules
including starch and TAG. TAG can be synthesized in both
the chloroplast and endoplasmic reticulum (Vitova et al.
2015).
Starch and lipid molecules buffer microalgal cells
against light/nutrient irregularity and allow microalgae to
maintain cellular processes such as DNA replication,
nuclear division, cytokinesis, and cell survival in the face
of environmental stress. These cellular processes deplete
starch and lipid reserves that could otherwise be used to
produce fuel. Cellular lipid reserves can be controlled
through manipulation of the cultivation environment.
When processes such as DNA replication and proteosyn-
thesis are inhibited, either through chemical inhibitors or
A review of biodiesel production from microalgae
through environmental stress, rapid starch accumulation is
observed. In strains that use lipids as their main form of
energy storage, lipid synthesis follows hours (h) or days
(d) after the initial overproduction of starch, which is
gradually degraded. During phosphorous and nitrogen
deprivation, microalgae overproduce lipids as metabolism
shifts from primary metabolic pathways, such as protein
and DNA synthesis, to secondary metabolism used to
produce molecules such as those used for energy storage or
protection against oxidative conditions (Vitova et al. 2015).
Diatoms, another type of microalgae that use silica in their
cell walls, can be stressed by limiting silica concentration
in culture media. Additional variations in temperature and
pH have been shown to alter the type and amount of lipids
produced during microalgae cultivation. The effects of
these environmental variations are discussed in Sect. 3.
Cell wall
Like plants, microalgae have cell walls that become indus-
trially relevant during harvesting and pretreatment, specifi-
cally in cell lysis (Fasahati et al. 2015). Microalgae cell walls
are typically thicker than those of other plant cells that are
commonly used for biofuel, which make lipid extraction
more difficult. Microalgae cell walls contain polysaccha-
rides such as cellulose, as well as a variety of other
polysaccharides. Sulfated polysaccharides are present in
most microalgae cell walls including red microalgae. Dia-
toms, a type of phototrophic microalgae, have cell walls that
are synthesized from silicic acid, polymerized, and extruded
via intracellular reactions (Graham et al. 2012). One chal-
lenge and possible solution of enhancing lipid extraction is to
develop enzymes that break down the cell wall polysaccha-
rides during a pretreatment digest (Fasahati et al. 2015).
Enzyme-assisted pretreatment and other alternative methods
such as high-pressure homogenization, microwave-assisted
extraction are tactics for accessing lipids within the cell wall
and will be discussed later in this review.
Cell division
Microalgae divide via sexual and asexual reproduction
with both reproduction strategies having effects on the
level of starch and lipid accumulation within the cells.
Asexual reproduction is faster than sexual reproduction,
and less TAG is produced because of a heavier reliance on
primary metabolism. During sexual reproduction,
microalgae spend more time synthesizing metabolic
resources including lipids for secondary energy sources.
During cell division, starch is degraded for nuclear and
cellular division energy requirements. Endogenous respi-
ratory rate is accelerated during onset of nuclear division,
and photosynthesis is slowed due to chloroplast division
641
(Vitova et al. 2015). In phototrophically grown microalgae,
starch accumulates during the cell cycle prior to nuclear
and cellular division. An understanding of the timing and
mechanics of microalgae cell division allows for optimiz-
ing product yields by inhibiting certain cell cycle pro-
gressions and timing nutrient depletion properly.
Lipid productivity
The most important aspect of strain selection is the ability
for the microalgae to produce lipids. Three parameters are
considered when comparing microalgae in terms of lipid
production: biomass productivity, lipid content, and lipid
productivity. Many microalgae strains can be used in bio-
fuel production including phytoplankton, red microalgae,
green microalgae, and cyanobacteria (Wang et al. 2013).
Table 3 shows the comparison between the lipid produc-
tion rate, lipid content, and biomass productivity of
example strains that demonstrate high productivity levels.
Although Chlorella protothecoides has been shown to
have one of the highest biomass productivity levels at
2.2–7.4 grams per liter per day (g/L/d) in conjunction with
high lipid productivity of 1.2–3.7 g/L/d, heterotrophic
growth poses fermentation challenges and high substrate
costs. In comparison with heterotrophic biomass and lipid
productivity, phototrophic biomass productivity is mark-
edly lower at 0.10 g/L/d for Dunaliella tertiolecta and
0.07 g/L/d for Chaetoceros muellen. This lower pho-
totrophic biomass density has industrial ramifications due
in part to the importance of biomass productivity in lipid
and oil content. C. muellen produces 0.0218 g/L/d of lipids
and has roughly 4% of the biomass productivity rate of C.
protothecoides. Mixotrophic Chlorella vulgaris was
observed to have a lipid productivity of 22.0–54.0 mil-
ligrams per liter per day (mg/L/d) when grown under
sunlight with feed stream mixtures of glucose and glycerol
as carbon sources (Chen et al. 2011).
The biorefinery approach
A biorefinery uses as much of the microalgae cell as pos-
sible—not just the acylglycerols but also pigments, car-
bohydrates, enzymes, or other useful molecules found in
the cell. The extraction of multiple products from a single
microalgal slurry could offset the costs of biodiesel pro-
duction to improve the overall sustainability of the system.
Fuels and co-products
There are three main types of fuel that can be produced by
microalgae: biodiesel, bioethanol, and biomethane. The
simultaneous production of fuel products could reduce the
cost of a single fuel (Ubando et al. 2014). For example,
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Table 3 Popular algae strains and their metabolisms, biomass productivities, lipid contents, and lipid productivities (Chen et al. 2011)
Name Strain type Primary metabolism
Chlorella protothecoides Green microalgae Heterotrophic
Dunaliella tertiolecta Green microalgae Phototrophic
Chlorella vulgaris Green microalgae Mixotrophic
Chaetoceros muellen Green microalgae Phototrophic
integrated energy systems can be designed to produce bio-
diesel in addition to generating heat and electricity. This can
be done by feeding the residual biomass to an anaerobic
digestion plant for methane production and to a biochar plant
for production of a charcoal soil supplement. The methane can
then be partially used for heat and energy, but it can also be
converted into methanol (Ubando et al. 2014). Additionally,
carbohydrates and proteins not extracted during the biodiesel
production process could be used for manufacturing bioe-
thanol. Not only do microalgae provide products for the
energy market, but they also offer many products to the
nutritional, pharmaceutical, nutraceutical, and cosmetic
sectors.
Microalgae are rich in fatty acids with eighteen or more
carbons containing three or more double bonds, known as
long-chain polyunsaturated fatty acids. These include
omega-3 and omega-6 fatty acids, which can be found in
many species such as Nannochloropsis sp., Phaeodactylum
tricornutum, Thalassiosira, and Parietochloris incise.
They can be sold as components for important health
supplements (Leu and Boussiba 2014). Specific high-value
fatty acids such as y-linolenic, arachidonic, eicosapen-
taenoic (EPA), and docosahexaenoic acids (DHA) may be
extracted prior to transesterification and sold in their pure
forms as omega-3 nutritional supplements (Brennan and
Owende 2010; Cuellar-Bermudez et al. 2015; Yen et al.
2013).
In addition to ethanol production, microalgae carbohy-
drates can be applied to the food, cosmetic, and textile
industries as stabilizers, emulsifiers, and thickening agents.
Porphyridium cruentum accumulates large extracellular
layers of carbohydrates in commercially attractive amounts
and concentrations for the cosmetics and skin care industry
(Leu and Boussiba 2014). Sulfated polysaccharides such as
fucoidans, carrageenans, and agarans can also be particu-
larly useful as antioxidant, antitumor, anticoagulant, anti-
inflammatory, antiviral, and immunomodulating agents
(Yen et al. 2013). P. cruentum has been found to have
highly specific antiviral carbohydrates that can be devel-
oped into creams effective in herpes virus treatments (Leu
and Boussiba 2014). b-1,3-glucan, also known as
paramylon, may have a role in fighting tumors, lowering
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S. Dickinson et al.
Biomass Lipid content Lipid productivity
productivity (g/L/d) (% dry weight) (mg/L/d)
2.2–7.4 50.3–57.8 1209.6–3701.1
0.10 60.6–67.8 60.6–69.8
0.09–0.25 21.0–34.0 22.0–54.0
0.07 33.6 21.8
cholesterol levels, regulating the immune system, and
acting as an antioxidant (Grimm et al. 2015; Rodrı́guez-
Zavala et al. 2010).
Microalgal pigments including chlorophyll, carotenoids,
and phycobiliproteins are already finding themselves in
niche biotechnological and medical markets. Chlorophyll
has been used as a chelating agent in ointments and liver
and ulcer treatment. It has also been linked to higher levels
of hemoglobin in blood and cell growth (Harun et al.
2010). Pheophorbide A, a chlorophyll derivative, is being
investigated in photodynamic therapy to treat human
uterine sarcoma and other cancers (Yen et al. 2013).
Astaxanthin, a type of red carotenoid produced by the
microalgae species Haematococcus pluvialis (Fig. 3), is
currently marketed as a nutraceutical for its powerful
antioxidant properties and has a $200 million market
potential (Koller et al. 2014). Phycobiliproteins’ high flu-
orescence and nonspecific binding make them attractive
tools in the biotechnology industry (Cuellar-Bermudez
et al. 2015; Yen et al. 2013). For example, R-phycoery-
thrin is a stable, soluble, negatively charged, and highly
fluorescent red protein which is used in fluorescence-based
assays and currently sold by Thermo Fisher Scientific Inc.
for $105.50/mg (Cuellar-Bermudez et al. 2015; Thermo
Fisher Scientific Inc. 2009, 2015).
Other uses of microalgae include the production of
recombinant proteins and feed additives. Industrial
enzymes have also been produced in Dunaliella tertiolecta,
and a variety of high-value recombinant proteins have been
expressed in Phaeodactylum tricornutum including func-
tional antibodies, human growth hormone, and erythro-
poietin. Feed additives and pharmaceutical agents—such as
sporopollenin, a compound that protects against ultraviolet
radiation (Priyadarshani and Rath 2012)—produced by
Scenedesmus quadricauda grown in raceway ponds have a
$140 million market (Leu and Boussiba 2014). Microalgae
are useful for livestock and aquaculture because they
enrich food stocks with proteins; healthy fats; vitamins A,
C, D, and E; thiamin; riboflavin; niacin; pyridoxine; folic
acid; cobalamin; and biotin (Bishop and Zubeck 2012).
Table 4 discusses a selection of products that can be
refined from microalgae, along with their estimated global
A review of biodiesel production from microalgae 643

market value and some species that produce each
compound.
As Table 4 shows, the market for many microalgae
compounds is in the millions to billions of US dollars.
Although production of microalgal biodiesel is still eco-
nomically unfavorable, costs can be offset through refine-
ment of high-value co-products. Biorefinery approaches
have been explored by many pilot-scale companies and
commercial companies.
Fig. 3 Haematococcus pluvialis cells turn red from high astaxanthin
concentrations under nutrient-deficient conditions. Figure courtesy of
Kyra Gudgel, Brock Shilling, Caitlin Liddiard, Tori St. Martin, and
Alex Fuerst
Industrial large-scale production
As of the year 2015, only a few commercial-scale
microalgae biofuel facilities were in operation. Major
roadblocks that these businesses address are high operating
costs and energy requirements, low cell density in the
growing media, land needs, and others. Many of the
greatest opportunities for optimization lie within the cul-
tivation, co-product generation, harvesting, and pretreat-
ment subsystems. The cultivation cost was reported to be
65% of the total cost of biofuel production system, showing
that improvement in cultivation systems can reduce the
price of microalgal biodiesel dramatically (Iancu et al.
2012).
There are many businesses that manufacture products
using microalgae, though most are in the research or pilot
plant stages of production. Some of the facilities concerned
with the production of biofuel include Sapphire Energy
Inc., Cellana Inc., TerraVia Inc. (formerly Solazyme),
Algenol Biotech LLC, Joule Unlimited Inc., and Synthetic
Genomics Inc. (Table 5). Both Sapphire Energy Inc. and
Cellana Inc. are creating a commercial plant and already
have a pilot plant. Both companies make biofuel, but also
delve into co-products including animal and aquaculture
feed and omega-3 supplements (Cellana Inc. 2015; Sap-
phire Energy Inc 2016). TerraVia Inc. now uses its het-
erotrophic strains to produce an omega-9 supplement,
culinary oil, cosmetic ingredients, aquaculture feed, and
other products (AlgaWise 2016; TerraVia Inc. 2016).
Algenol Biotech LLC, a company in Florida that makes a

Table 4 Selected side-stream products of interest

Product Global market (US$ billion) Example species

b-Carotene 0.2 (Koller et al. 2014) Dunaliella salina (Li et al. 2008), Dunaliella bardawil
(Koller et al. 2014)
Astaxanthin 0.2 (Koller et al. 2014) Haematococcus pluvialis (Li et al. 2008)
Lutein 0.23 (Yaakob et al. 2014) Muriellopsis sp., Chlorella zofingensis, Chlorella
protothecoides (Ho et al. 2014)
Phycobiliproteins 0.05 (Koller et al. 2014) Spirulina platensis (Li et al. 2008)
b-1,3-Glucan 0.1 (estimate for USA) (Koller et al. 2014) Chlorella sp. (Yaakob et al. 2014)
Docosahexaenoic acid 7.2 (estimate for omega-3 packaged products) Schizochytrium sp., Crypthecodinium cohnii (Koller
(Yaakob et al. 2014) et al. 2014)
Eicosapentaenoic acid Chlorella minutissima (Li et al. 2008), Nannochloropsis
sp. (Koller et al. 2014)
UV-screening compounds 9 (Cosmetics Business 2015) Scenedesmus sp., Characium terrestre (Priyadarshani
and Rath 2012)
Antibodies, hormones 40 (estimate for therapeutic antibodies (Elvin Phaeodactylum tricornutum (Leu and Boussiba 2014)
et al. 2013)
Chlorophyll 1 (natural food colors market) (Lakshmi 2014) Chlorella sp. (Harun et al. 2010)
Aquaculture feed 106 (Yaakob et al. 2014) Spirulina sp., Hypnea cervicornis (Harun et al. 2010)
Vitamins, minerals, and nutritional/ 82 (Teichner and Lesko 2013) Nostoc sp., Anabaena sp. (Bishop and Zubeck 2012)
herbal supplements

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Table 5 Biorefinery production strategies followed by different companies

Company Type of bioreactor Products Approximate scale

Sapphire Energy Inc. Open ponds Animal/aquaculture feed; Phase 1 of commercial site is occurring; will be able to
(Sapphire Energy Inc. biofuel; omega-3 for human produce 1600 tons of biomass/yr
2016) nutrition
Cellana Inc. (Cellana Closed-culture Animal/aquaculture feed; 20 metric tons (dry weight) of whole microalgae since
Inc. 2015) bioreactors with biofuel; omega-3 for human 2009; planning to commercialize from current pilot plant
open ponds nutrition stage
TerraVia Inc. (Hitchings Closed fermenter Cosmetics; lubricant; food oil; 500,000 L of oil/yr
and Ward 2010) biofuel
Algenol Biotech LLC Closed vertical flat Ethanol; biodiesel; gasoline; jet 8000 gallons ethanol/acre/yr
(Lane 2015a) plate fuel
photobioreactors

variety of fuels from microalgae, is able to produce ethanol
for approximately $1.30/gal (Algenol Biotech LLC 2011;
Lane 2015a). Joule Unlimited Inc. plans to use genetically
modified cyanobacteria to produce bioethanol and biodie-
sel. The company has conducted pilot plant testing and
plans to build a commercial facility in 2017 to market
biodiesel by utilizing wastewater or brackish water for
nutrients (Lane 2015b, c). The production cost (which
appears not to include capital costs) is reported by Joule
Unlimited Inc. to be for $1.19/gal of biodiesel, which is
much lower than most costs reported in the literature (Lane
2015c). Synthetic Genomics Inc. is another company that
emphasizes genetic engineering to improve lipid secretion
(Schmidt 2010).
Commercial microalgal biofuel has not been able to
compete with the lower prices of petroleum-based fuels
yet, though many companies have delved into other pro-
duct lines to help offset the cost of biofuel production. In
the future, advancements in genetic engineering may have
a major impact on cellular metabolism and industry’s
approach to lipid generation to lower the price of biodiesel.
Genetic engineering
The goal of genetically engineering microalgae for biofuel
production is to manipulate the genome for greater oil or
starch production, easier processing, or higher concentra-
tions of valuable co-products to make microalgae feedstock
more profitable. Genetic engineering can be used to
increase lipid productivity, decrease sensitivity to oxygen
saturation, increase growth rates, introduce foreign genes
for additional side product production, and improve
microalgae tolerance to harsh operating conditions such as
pH, temperature, shear stress, and mixing. For example,
Chlamydomonas reinhardtii has been transformed with the
Haematococcus pluvialis enzyme coding for beta-carotene
ketolase in order to produce a new ketocarotenoid.
Attempts to enhance fatty acid synthase enzymes such as
acetyl-coenzyme A carboxylase—an enzyme that pushes
acetyl-coenzyme A down the fatty acid synthesis path-
way—have also been made with limited success (Ghosh
et al. 2016). In addition to direct metabolic pathway
modification, microalgae metabolic engineering can target
the manipulation of individual enzyme kinetics, molecular
transport, product secretion, cell wall structure, and regu-
latory functions within photosynthetic systems (Jinkerson
et al. 2011; Lu et al. 2011).
Efforts to influence the photosynthetic pathways have
been explored in diatoms using metabolic engineering
designs by targeting chloroplast protein expression
(Yoshihiko et al. 2015). In another study, molecular pumps
have been inserted into specific microalgae genome nodes
to control efflux pump expression and export biofuel out of
the cell. These pumps can be used to reduce the effects of
toxic buildup of by-products in strains with high lipid
productivity (Dunlop et al. 2010) and also may reduce the
costs of harvesting the oil from the microalgae cells.
Transformation methods typically used in microalgae
include electroporation and biolistic transformation. RNA
silencing to knock out genes and homologous recombina-
tion to provide more stable genes are also common tech-
niques to control gene expression. The goal is to produce a
stable transformation that will continue to express the gene
for several generations; transient transformations in which
the host system rejects the introduced DNA are one of the
challenges of genetic engineering. Thus, efficient trans-
formation systems must be used (Ghosh et al. 2016). Slow
progress in genetic engineering is due to the limited
number of available sequenced genomes in relation to the
sheer number of microalgae species. Ostreococcus tauri, C.
reinhardtii, and Dunaliella salina are among the few spe-
cies whose genomes have been sequenced (Ghosh et al.
2016). Researchers have begun sequencing and under-
standing the genome architecture, gene organization, and

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gene clusters associated with metabolic nodes in the fatty
acid biosynthesis of Nannochloropsis gaditana (Jinkerson
et al. 2013).
Cultivation parameters and requirements
To ensure the highest and most consistent productivity of a
microalgae culture, the cultivation parameters must be
optimized and validated. Nutritional requirements, amount
of light exposure, degree of agitation, and other environ-
mental effects impact the growth rate and metabolism of
the microalgae and will be discussed in this section.
Nutritional needs and potential sources
All microalgae strains require certain inorganic nutrients
such as carbon, nitrogen, and phosphorus in order to grow.
Each type of microalgal strain also thrives at a certain salt
concentration range, which is usually determined by the
microalgae’s original habitat, i.e., freshwater or seawater.
Wastewater and flue gases are two alternatives to tradi-
tional microalgae nutrient sources which will be discussed
further.
Wastewater
Inorganic nutrients that are tailored for each type of
microalgae are typically sold as media mixes. These
commercial medias are feasible for use at a laboratory or
even pilot scale. However, relying on purchased, premade
media becomes expensive when scaling up the microalgal
growth to industrial production levels. Wastewater has
been one suggested solution to this economic concern.
Wastewater is a cheap and readily available alternative
media that can allow for sustainable growth. Wastewater
contains nutrients, such as carbon, nitrates, phosphates,
ammonium, urea, essential trace elements of vitamins, and
certain trace metals, such as iron, cadmium, and zinc.
Using wastewater to cultivate microalgae would reduce
costs tremendously as well as significantly reduce the
amount of freshwater that need to be introduced to the
system (Rawat et al. 2012).
AlgaXperts LLC investigated the biofuel production
using diatom genera Cyclotella, Aulacoseira, Fragilaria,
and Synedra. The researchers studied the potential of
coupling diatom cultivation with wastewater treatment and
biofuel production. It was found that Cyclotella menegh-
iniana and Aulacoseira granulata had the highest lipid
content with C. meneghiniana having lipid values compa-
rable to genetically engineered cyanobacteria for lipid
production in biofuel. At optimal production levels, C.
meneghiniana grown on wastewater were found to have a
645
lipid concentration of 100 mg/L with a growth rate of
0.864 d-1 and time to stationary phase of 17 d. Using these
values, it is estimated that 116 gallons per day (gal/d) of
lipid can be produced per million gallons (gal) of
wastewater. Considering that average wastewater treatment
plants have a 3.2 million gal/d potential of wastewater
production, it is estimated that 371 gal/d or 135,500 gallons
per year (gal/yr) of lipids could be produced from C.
meneghiniana cultivation. It is estimated that there are
15,000 wastewater treatment facilities in the USA and that
coupling these with biofuel production through usage of C.
meneghiniana could produce an estimated 2 billion gal/yr
of biofuel (Graham et al. 2012). Therefore, if all wastew-
ater being produced in the USA was being treated by
microalgae and converted into biofuel, 2 billion gal, or 3%
of the annual US distillate fuel national consumption, could
be produced (US Department of Energy 2015).
There are potential problems associated with using
wastewater as a growth media. There may be viral or
bacterial contamination that affects the biomass production
or downstream processing. To prevent this, the system
design may require pretreatment of the wastewater, along
with frequent cleaning of the culturing system to limit the
potential for any contamination effects (Pittman et al.
2011). Potential pretreatment methods could include heat
treatments, filtration, and UV irradiation of the wastewater
prior to using it as media. The composition of the
wastewater could also become problematic. There are
many different sources of wastewater: industrial, munici-
pal, agricultural, etc. Each of these sources will contain
different types of nutrients. They could also contain toxic
chemicals that inhibit the microbial growth. High levels of
ammonia, for example, can be detrimental to microalgal
growth at high concentrations (Pittman et al. 2011). The
composition of wastewater from a single source may also
vary, meaning that the nutrients supplied to the microalgae
and their subsequent growth rates may not be consistent
(Singh et al. 2015).
Photosynthesis may also be significantly hindered if the
wastewater used is colored, suggesting that heterotrophic
or mixotrophic growth is much more feasible. Moreover,
the organic matter in wastewater could promote contami-
nation, leading to culture instability. Suggestions for
overcoming these challenges include the utilization of
multiple native microalgae species, as well as tighter
control on the cultivation process (Chen et al. 2015).
Flue gases
Flue gas has also been suggested as a potential nutrient
source for microalgae. Industrial companies frequently
look for methods to limit CO2 emissions. Some industrial
emissions can contain up to 15% CO2 (Mata et al. 2010),
123
646
which is much higher than the average 0.04% CO2 in the
atmosphere (Schneising et al. 2011). Since phototrophic
microalgae require CO2 for growth, the concentrated levels
of CO2 in the flue gases would help promote the microalgal
growth. Algenol Biotech LLC studied microalgae growth
and showed that the microalgae grown on CO2 from flue
gases grew 3–5% better than microalgae grown on com-
pressed CO2. They concluded that this improvement in
growth had been due to the fact that flue gases also had
much lower concentrations of oxygen, which is known to
inhibit microalgal photosynthesis at high levels, therefore
causing the growth to be much faster and more efficient
(Jessen 2015). The microalgal strain would need to be
resistant to any toxic chemicals that the flue gas contains,
such as NOx and SOx components, and the high tempera-
tures of flue gas, if it were directly used (Singh et al. 2015).
It has been reported that some microalgae strains are able
to utilize NO components in addition to CO2 in flue gases
(Chen et al. 2015). In order to identify a strain of
microalgae which is not majorly effected by the additional
chemicals in flue gases, further testing and investigation
needs to be done as it is a promising technology for aiding
in the reduction of power plant flue gases.
Environmental requirements
When growing microalgae at a production-scale level, it is
important to optimize the microalgae’s environment.
Minute changes in temperature, pH, or light intensity could
make a large impact on the overall amount of products
produced.
Temperature
Microalgae grow at a temperature between 15 and 40 °C
depending on the strain (Juneja et al. 2013). Studies have
shown that growth rate of the microalgae will increase with
temperature up to a species-dependent optimum tempera-
ture. For example, Nannochloropsis limnetica can be cul-
tured over the range of 15–27 °C, but its highest growth
rate occurs at 22 °C (Freire et al. 2016). After reaching this
optimum temperature, growth rate decreases. Additionally,
it is known that microalgal growth can be inhibited by
excess light intensities. Suboptimal temperature conditions
could encourage the photoinhibitory effects of less extreme
light intensities (Torzillo and Vonshak 2013).
pH
One of the most important environmental factors to con-
sider when growing microalgae is pH. This is because pH
can determine the solubility, and therefore availability, of
123
S. Dickinson et al.
CO2 for microalgal growth. Most microalgae species grow
maximally around a neutral pH range of 7.0–7.6 (Juneja
et al. 2013). The pH of the environment changes as nutri-
ents are metabolized by the microalgae. Because CO2
becomes carbonic acid when dissolved in water, CO2/car-
bonic acid consumption increases the pH and leads to a
more basic media. Consumption of nitrogen and phos-
phorus can also affect pH. When ammonium is consumed,
hydrogen is released in its consumption, causing the pH of
the medium to lower. In contrast, consumption of nitric and
phosphoric acids causes the pH to increase (Provust 2011).
The careful monitoring of pH is, therefore, vital when
cultivating the microalgae.
Light intensity
The amount of light exposed to microalgae affects biomass
productivity. Microalgal growth increases with light
intensity and/or exposure until it reaches a maximum
value, which is associated with the microalgae’s light sat-
uration. Light exposure past the microalgae’s maximum
light saturation may lead to photoinhibition and thus inhibit
growth, leading to less efficient CO2 fixation and other
nutrient intake rates (Judd et al. 2015).
Studies on Phaeodactylium tricornutum and Por-
phyridium cruentum have found light saturation to be
approximately ten times lower than the midday light
intensity in equatorial regions (Chisti 2007). Because
microalgae light saturation levels tend to be lower than the
amount available from the sun during midday, natural light
may not be preferred to artificial light due to photoinhibi-
tion. In addition, light sources utilizing technologies such
as light emitting diodes (LEDs), optical fibers, solar panels,
and wind power could greatly reduce the electrical
requirements of conventional light sources. Optical fibers
in tandem with wind- and solar-powered LEDs were able
to ensure continuous, sufficient, and stable light supply to
an indoor photobioreactor. This type of light system has the
potential for use in commercial systems (Chen et al. 2011),
because limiting the effects of photoinhibition through
controlled light intensity could greatly increase the
microalgae’s typical average daily growth.
Optimization of growth and production
Microalgae demonstrate the highest productivity levels in
well-mixed, nutrient-rich environments. By controlling the
nutrients available, the metabolism of microalgae can be
tailored to fit the needs of a biorefinery facility. Nanopar-
ticle applications also show promise in improving growth
and production.
A review of biodiesel production from microalgae
Mixing
Maintaining a well-mixed environment is necessary in
order to promote mass transfer processes in the cell. Pho-
tobioreactor technologies to improve CO2 mass transfer,
including membrane-sparged or air-lift systems, have been
investigated. Well-mixed conditions also ensure that heat is
transferred efficiently throughout the cell suspension, as
uneven temperature distribution may cause uneven growth.
Additionally, maintaining a well-mixed environment pre-
vents sedimentation and accumulation of biofilms within
the system. While mixing at speeds that are too low may
not cause even temperature, gas, and media distribution,
mixing at very high rates has the potential to cause
hydrodynamic shear stress to the cells. Rapid mixing also
has higher energy requirements. Further research must still
be done to better understand the effects of mixing within a
system (Provust 2011).
Stresses
Several techniques have been discovered in order to max-
imize the amount of lipids produced by microalgae. Many
different types of stresses such as nutrient concentration,
osmotic pressure changes, radiation, pH, temperature,
heavy metals, and other chemical stresses can be used to
increase the productivity of lipids. Dry microalgae biomass
typically contains about 5–20% lipids, including membrane
lipids. Out of the variety of lipid types found within the
cell, certain environmental stresses can target neutral
lipids, mainly acylglycerol, to increase their concentration
to 20–50% of the dry microalgae weight (Sharma et al.
2012).
Nutrient starvation is one stressor that increases lipid
concentration. Depriving a microalgae culture of certain
nutrients, such as nitrogen or phosphorus, decreases cell
division and growth. With enough sunlight and CO2
available, microalgae begin producing more fatty acids
to be converted into acylglycerols rather than undesirable
membrane lipids and compounds (Sharma et al. 2012).
Changes in temperature can also affect the composition
of the microalgal cells, since many chemical and bio-
chemical reaction rates are dependent on temperature.
Temperature fluctuation affects the amount and profile of
fatty acids produced within the microalgae cells (Juneja
et al. 2013). As a general trend, when temperature is
increased microalgae will produce more saturated fatty
acids; when temperature is decreased, the microalgae will
produce more unsaturated fatty acids (Sharma et al. 2012).
For example, one showed that Dunaliella salina had a
considerable increase in the amount of fatty acid unsatu-
ration when the temperature was changed from 30 to 12 °C
(Juneja et al. 2013). Since oxidation is known to decrease
647
the long-term storage of biodiesel, ensuring that the fatty
acids are saturated would increase its value (Pullen and
Saeed 2012).
Two additional stresses that can affect lipid content of
microalgae are pH changes and the presence of certain
heavy metals. Microalgae grow in a wide range of pH
levels, most falling between 7 and 9 (Lebeau and Robert
2003). Certain pH ranges encourage specific growth. For
example, multiple studies show that alkaline pH conditions
stress Chlorella species, which resulted in the promotion of
TAG and a decrease in membrane lipids; overall, cell
growth was also slowed. A study on growth at a pH of 1 on
a Chlamydomonas species has shown to increase the rela-
tive percentage of TAG out of the total lipids in the cells. In
addition to pH changes, the presence of heavy metals can
change lipid production. Heavy metals that can affect the
lipid content of microalgae include cadmium, iron, copper,
and zinc. However, the changes caused by both of these
stresses vary greatly depending on the strain (Sharma et al.
2012).
A specific class of microalgae called diatoms has a
siliceous cell wall, high lipid content, and a urea acid cycle
within their metabolism. This phytoplankton also grows
concurrently with filamentous green microalgae whose
cellulose could be separated out and used as feedstock for
heterogeneous fermentation (Graham et al. 2012). The urea
pathway in diatoms makes them able to reduce nitrogen
and phosphorous efficiently from wastewater which could
have large environmental benefits on the global water
supply. Silica is a nutrient uniquely needed for diatoms; if
depleted during stationary phase, silica can also be used as
a stressing agent to increase lipid productivity in the
microalgae cells.
Nanoparticle applications
Nanoparticles are nanosized particles typically ranging
from 1 to 100 nm in size that exhibit enhanced physico-
chemical properties (surface charge, composition, mag-
netism, etc.) characteristic of nanoscale quantum influence.
These physicochemical properties and emergent nanoef-
fects can be exploited for microalgae cultivation and har-
vesting (Lee et al. 2015).
There has been significant progress in cell growth,
pigment/lipid contents, efficiency and processing time of
microalgae separation and understanding of nanoparticle–
cell interactions. Indirect use of metal nanoparticles
placed outside of closed photobioreactors (PBRs) shows
about a [30% increase in cell growth for Chlamydomonas
reinhardtii by strong backscattering of blue light in
comparison with the control. Light flux and wavelength
can be controlled by nanoparticle size and can stimulate
specific microalgae species with different pigments.
123
648 S. Dickinson et al.

Table 6 Comparison of nutrient alternatives and cultivation optimization techniques

Alternative Description Advantages Disadvantages
or technique

Wastewater Water discarded after use
(sources include
agriculture, industry,
municipality)
Flue gases Industrial emissions which
contain high amounts of
CO2
Mixing Agitation of microalgae
culture to maintain
uniformity within system
Stresses Purposeful changes in
microalgae growth
conditions to induce a
desired response
Nanoparticle Application of nanoparticles
applications to utilize their unique
characteristics
Reduces or eliminates reliance on media;
cheap; contains readily available nutrients;
reduces freshwater usage
An inexpensive form of concentrated CO2;
could promote microalgae growth; improve
environmental impact of production
Promotes mass (e.g., nutrient) and heat
transfer, uniform growth of culture;
prevents sedimentation and accumulation
of biofilms
Increase the amount of desired lipids
produced; may help reduce costs,
depending on stress used
Allows for light flux and wavelength control;
antimicrobial effects can reduce culture
contaminations
Raises risk of contamination or culture
instability; may contain toxic chemicals; may
hinder phototrophic growth, if colored
High temperatures, if used directly after
produced, could harm microalgae; contains
toxic chemicals (e.g., NOx or SOx); requires
further research to understand affects
Requires additional energy; can cause shear stress
on cells at high rates; requires further research
to understand optimal rates
Dependent on the microalgae strain; slows
growth/reproduction rate if affecting essential
growth components
Recycling for reuse is not yet cost-effective;
requires further research (e.g., stability,
environmental effects)

Nanoscale MgSO4 particles were found to enhance pho-
tosynthesis and reduce glycerol consumption during
mixotrophic cultivation, resulting in increased oil yield
per glycerol consumed in C. vulgaris cultivated with
glycerol feedstock. Nanoparticle technology has the
potential to increase production of high-value carotenoid
pigments such as astaxanthin and lutein in closed photo-
bioreactor systems, while the incorporation of trace ele-
ments such as cobalt, manganese, boron, and
molybdenum in the nanoparticle form could improve
microalgae biomass production by 21–38%. Antimicrobial
or bacteriostatic effects of aminoclay-nanoparticle-coated
microalgae cells are also a promising way to reduce
culture contamination. Many critical issues such as cost-
effective nanoparticle recycling, rational nanoparticle
design, and environmental effects still remain. Analysis of
the safety and stability of nanoparticles is also needed in
order to assess the nanoparticle toxicity to microalgae
(Lee et al. 2015).
Cultivation is typically one of the most expensive and
lengthy steps of microalgae biodiesel production. By
selecting the most advantageous nutrient sources and
optimizing growth conditions, increases in cell densities
and oil productivity will help counteract this burden.
Table 6 summarizes the advantages and disadvantages of
nutrient alternatives and optimization techniques previ-
ously discussed.
Once the nutrient input sources and growth parameters
are established for the microalgae strain(s), the cultiva-
tion technique may be selected. The next section will
discuss potential unit operations suitable for large-scale
production.
Cultivation techniques
The cultivation of microalgae for biodiesel production
produces a much more efficient oil yield per acre than
current biodiesel sources from crops, waste cooking oil, or
animal fat. Table 7 shows a comparison of the oil effi-
ciency yield, required land areas, and percentage of the
required existing US cropping areas which would be nec-
essary to meet half of the current transport fuel needs of the
USA (Chisti 2007). As one can see, microalgae provide a
much higher oil yield for a fraction of the required land
area.
The type of conditions required to grow the microalgae
will determine the cultivation system used for scale-up.
There are three main types of culturing systems: open
ponds, closed PBRs, and conventional fermenters. An
additional type—hybrid systems—combines concepts of
open ponds and closed PBRs in an effort to maximize the
benefits associated with each.
Open systems: raceway ponds
Open ponds have been used for microalgae production
since the 1950s. This type of system consists of an open
lake, pond, or lagoon in which the microalgae are grown.
The most common type of open, outdoor cultivation system

123
A review of biodiesel production from microalgae
Table 7 Comparison of biodiesel production sources and the potential requirements in order to meet 50% of the US fuel transportation needs in
2007 (total needs = 0.53 billion m3)
Source Oil yield (L/ha)
Corn 172
Soybean 446
Canola 1190
Jatropha 1892
Coconut 2689
Oil palm 5950
Microalgae (30% oil by weight in biomass) 58,700
Microalgae (70% oil by weight in biomass) 136,900
Information was adapted from a review (Chisti 2007)
Fig. 4 Schematic drawing of a raceway pond, showing the major
parts of the system. Variations may occur with the number of
paddlewheels or the shape of the closed loop in which the microalgae
solution is directed. Adapted from a review by Chisti (2007) and a
case study by Jorquera et al. (2010). Figure courtesy of Wesley Zloza
is a raceway pond. This system, shown in Fig. 4, is typi-
cally oval-shaped with closed-loop recirculation channels
lined with materials such as plastic or concrete. Generally,
CO2 gas is bubbled up from the bottom of the pond to
supply carbon and agitation (Zitelli et al. 2013), and pad-
dlewheels mix the culture to prevent sedimentation. These
culturing systems are less expensive, require a lower
energy input, and need less regular maintenance than other
cultivation systems (Brennan and Owende 2010).
The growth conditions of raceways ponds are difficult to
control. Temperature, pH, and salinity variations create a
harsh environment for most microalgal strains. Such
growth inconsistency limits the ability to extract high-value
products and lipids (Sierra et al. 2008). Contamination is
likely, and many strains struggle to compete with predators
and microbial competition for sunlight and nutrients (Sierra
et al. 2008). Because raceway ponds are open to the
environment, their location is important. Threats of con-
tamination, evaporation losses, amount of sunlight supplied
throughout the day, and temperature fluctuations must all
be considered in order to maximize the microalgal growth
(Brennan and Owende 2010).
649
Required land area Percent of existing
(million hectares) US crop area
1540 846
594 326
223 122
140 77
99 54
45 24
4.5 2.5
2 1.1
Sapphire Energy Inc. is currently utilizing raceway ponds
for biodiesel production. It has over 70 working ponds and is
building a commercial demonstration facility that dedicates
300 acres of ponds to microalgae growth. Upon completion of
the facility, it is estimated that the microalgae in these ponds
will sequester approximately 56 metric tons of CO2 each day
(Sapphire Energy Inc. 2016). To minimize contamination,
Sapphire Energy Inc. has bred a strain of microalgae to
withstand high pH and saline conditions (Lane 2015d).
Closed systems: photobioreactors
PBRs are closed, mechanical systems that can be used
indoors or outdoors and provide controlled growth condi-
tions. With the ability to regulate culture parameters, PBRs
promote consistent, high cell densities and provide a con-
trollable environment to produce high-value products
(Sierra et al. 2008). However, these reactors require a large
amount of power and upfront capital costs, slowing their
implementation in commercial-scale microalgal production
until recently (Rawat et al. 2012).
There are elevated risks associated with closed systems
including biofilm formation, oxygen accumulation, and
overheating. A biofilm is formed when microalgae begin to
accumulate along the surfaces of the reactor. The biofilm
inhibits the microalgae from efficiently absorbing nutrients
and decreases light penetration to the rest of the culture.
Accumulation of oxygen, a by-product of photosynthesis,
threatens closed systems because high concentrations
inhibit microalgal growth. Similarly, heat accumulation
poses a threat. Overheating within the reactor could
potentially cause the temperature within the reactor to harm
the microalgae. Bioreactors can be redesigned to minimize
aforementioned risks. For example, agitation, heat dissi-
pation, and oxygen release mechanisms can be engineered
to help reduce biofilm formation, and heat and oxygen
accumulation. Optimized geometry of the clear tubes can
123
650
maximize the amount of available light, but minimize risks.
However, with more complex designs comes an increase in
cost of the reactor (Provust 2011), potentially jeopardizing
the feasibility of its use on a large scale. The two popular
types of PBRs—tubular bioreactors and flat plate reac-
tors—are discussed below.
Tubular photobioreactors
Tubular reactors consist of a holding tank to allow gas
exchange and long glass tubes orientated to maximize cell
exposure to light (Fig. 5). Microalgae and medium are
pumped mechanically or with an air-lift system through the
tubing.
The turbidity caused by the movement of media through
the tubes creates the agitation required to prevent sedimen-
tation of cells. These reactors are suitable for outdoor mass
cultures because they expose a large surface area to light.
The design of these reactors is limited by the length of the
tubes within the reactor. As the length of the tubes increases,
so do the potential oxygen accumulation, CO2 depletion, and
pH variation in the system (Brennan and Owende 2010).
High levels of dissolved oxygen, specifically, is a problem
because it can inhibit photosynthesis (Sierra et al. 2008).
Flat plate photobioreactors
A flat plate reactor, depicted in Fig. 6, does not accumulate
as much dissolved oxygen within their systems as tubular
reactors, since the microalgae do not travel long distances
within the plate. The reactors also have a higher photo-
synthetic efficiency due to their shape (Brennan and
Owende 2010). They can be situated outdoors and angled
to receive the most efficient amount of light. It has been
reported that aeration within these flat plate reactors can
Fig. 5 Schematic of a tubular photobioreactor showing the important
parts of the system adapted from a review by Chisti (2007) and a case
study by Jorquera et al. (2010). Figure courtesy of Wesley Zloza
123
S. Dickinson et al.
Fig. 6 Schematic drawing of a flat plate photobioreactor adapted
from a case study by Jorquera et al. (2010). Figure courtesy of Wesley
Zloza
sometimes cause shear stress on the microalgae, whereas
this has not been reported as a concern in tubular reactors
(Sierra et al. 2008).
Flat plate PBRs are currently being used by some compa-
nies to produce biofuels. Algenol Biotech LLC, for example,
produces ethanol, biodiesel, gasoline, and jet fuel using
polyethylene ‘‘bags’’ as flat plate reactors. Its pilot plant
facility currently spans 36 acres, in which 8000 gal of ethanol/
acre/yr is produced (Algenol Biotech LLC 2011). Joule
Unlimited Inc. also uses flat plate PBRs to produce 15,000 gal/
acre/yr of biodiesel and 25,000 gal/acre/yr of ethanol in a pilot
plant facility (Lane 2015c). Table 8 lists the advantages and
disadvantages of raceway ponds, flat plate PBRs, and tubular
PBRs.
When evaluating the performance of a production sys-
tem, it is important to analyze its effect on the overall
system, not just the cultivation. For example, while race-
way ponds appear to lower costs and energy requirements,
a study which produced biodiesel, methanol, glycerol,
biochar, electricity, and heat in a multiobjective model
found that a flat plate PBR proved to maximize economic
performance as well as minimize environmental impact
(Ubando et al. 2014).
Hybrid systems
Hybrid systems utilize both PBRs and open ponds to
combine the two systems in order to harness the advantages
of each. There are two types of hybrid systems: One type
uses both systems in two different steps, while the other
attempts to blend concepts of open and closed PBRs into
one reactor step.
A review of biodiesel production from microalgae 651

Table 8 Comparison of advantages and disadvantages of raceway ponds, flat plate PBRs, and tubular PBRs for microalgae cultivation
Cultivation Description Advantages Disadvantages
method

Raceway Open pond systems with
ponds continuous agitation and
directional current flow
Flat plate Closed mechanical systems in
PBRs which microalgae are
continuously pumped through
clear flat plates
Tubular Closed mechanical systems in
PBRs which microalgae are
continuously pumped through
long clear tubes
Low capital costs, energy requirements, and
dissolved oxygen levels; heat dissipated
easily via evaporation
High surface area-to-volume ratio; can select
growth conditions to maximize microalgae
growth density; artificial light can be
supplied for continuous growth; harvesting
costs can be reduced/lower than open
systems
High surface area-to-volume ratio; can select
growth conditions to maximize microalgae
growth density; artificial light can be
supplied for continuous growth; harvesting
costs can be reduced/lower than open
systems
Risk of contamination; growth conditions
depend on the environment/location of pond;
limited light supply during day time; long-
term growth may require resistant species to
contamination; requires large land space,
high harvesting costs
High energy requirements and capital costs;
shear stress due to mixing/turbulence within
plate
High energy requirements and capital costs;
risk of oxygen accumulation, CO2 depletion,
or pH variation with longer tubes

In hybrid two-step systems, PBRs are used to grow the
initial culture under controlled conditions to minimize any
contamination and maximize growth rates. Next, the cul-
ture is transferred to open ponds for less expensive oper-
ating conditions and easier exposure to nutrient stresses
that facilitate lipid accumulation within the cells. The two-
step cultivation method has been studied extensively
(Brennan and Owende 2010; Zitelli et al. 2013) and is
currently used by companies; for example, HR BioPe-
troleum operates such a system to produce astaxanthin
(Zitelli et al. 2013). A variation of the two-step hybrid
system first grows the microalgae photosynthetically to
sequester CO2, then heterotrophically to increase lipid
yields. In a study done by Xiong et al. (2010), Chlorella
protothecoides was grown phototrophically and then
heterotrophically to significantly increase the carbon con-
version efficiency of sugar to oil. Because of this, a 69%
higher lipid yield was observed in this model. Cellana
Inc.—which produces biofuel, omega-3 supplements, and
feed additives—claims that microalgae grow faster in open
ponds but at risk of contamination, so it first cultures
microalgae under the controlled environment of PBRs
(Cellana Inc. 2015).
The second type of hybrid system combines the setup of
raceway ponds and the closed characteristic of PBRs in a
one-step cultivation system by creating a covered raceway
pond. The cover allows sunlight penetration, but separates
the air directly above the pond from the atmosphere to
minimize contamination risks. This type of covered race-
way pond is currently used by Atacama Bio Natural
Products Inc. in the first stage of microalgae growth for
astaxanthin production, while the second stage of growth is
transferred to a standard, uncovered pond (Zitelli et al.
2013).
After the microalgae have been cultivated, downstream
processing must occur. There are several methods to select
the order of unit operations involved in a production
facility. While sequences of operations may be handpicked
based on research and experience, computer and mathe-
matical models can also be employed to generate the most
efficient process. For example, the forward–backward
approach involves matching forward synthesis of
microalgae biomass and reverse synthesis of a desired
product to intermediate compounds. With a start and end in
mind, intermediate procedures and equipment can be
identified and optimized until the correct configuration is
found (González-Delgado et al. 2015). Sections five
through eight will discuss the advantages and disadvan-
tages of the downstream processing steps.
Harvesting
After the microalgae have been cultivated, the cells need to
be collected from a very dilute solution at 0.3–1 g/L cell
density and concentrated for further processing in the
harvesting stage (Bhave et al. 2012). Concentration can be
completed using filtration, centrifugation, flocculation, or
flotation harvesting steps, which are summarized in
Table 9.
Harvesting and its subsequent unit operation, pretreat-
ment, account for 20–30% of the total production costs, so

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Table 9 Types of harvesting methods

Harvesting Description Advantages Disadvantages
method

Centrifugation Use of centrifugal force to
sediment microalgae
Flotation Use of microbubbles to carry
microalgae to the surface
Filtration Biomass filtered through pores
Flocculation Substance is added to the
culture to sediment the cells
Very high concentration efficiency, no
contamination
Can use very low energy, moderate
concentration efficiency
High concentration efficiency, moderate
energy usage, no contamination
Low energy, moderate concentration
efficiency, low expense
High energy, cost
Can be costly if flocculant is needed, depends
on the characteristics of microalgae
Fouling of the filter needs constant maintenance
Can contaminate microalgae, need to remove
flocculant

optimizing these steps have significant effects on process
economics (Milledge and Heaven 2013). Presented here are
the major characteristics of each option of the harvesting
unit operation.
Centrifugation
Centrifugation is a commonly used separation technique in
bioprocessing. An advantage of this process is that there is
no contamination of the microalgae (Mata et al. 2010). The
disadvantages associated with centrifugation include the
large energy requirements and operation costs of the cen-
trifugation process (Kim et al. 2013). Although improve-
ments are being made to reduce operation costs,
centrifugation still is too energy intensive and expensive to
make it a feasible option for the separation of the
microalgal biomass from water (Kim et al. 2013).
Flotation
Flotation is a process in which air microbubbles carry
microalgae to the surface of the culture where the biomass
can be collected. This process is dependent not only on the
size and surface characteristics of the microalgae being
used, but also on the size of the bubbles (Milledge and
Heaven 2013). The bubbles need to be small in order to
have a higher surface-to-volume ratio, which increases the
stability of the bubble. An ideal bubble has a diameter of
10–30 lm (Kim et al. 2013). The charge and hydropho-
bicity of the microalgal cell also play a major role in the
interaction between the cell and the bubbles, which impacts
the efficiency of the bubbles to be able to carry the cells
(Kim et al. 2013).
Microbubbles are produced via dissolved air flotation
(DAF), electrolytic flotation, or dispersed air flotation
(DiAF). DAF produces microbubbles by dissolving air in
water at a high pressure, leading to the nucleation of
bubbles when the pressure is reduced in the nozzle (Barros
et al. 2015). DiAF produces microbubbles by passing air
through a porous material. DiAF is less energy intensive
than DAF, but the equipment is more expensive (Barros
et al. 2015). Generally, flotation is less energy intensive
and less expensive than centrifugation. However, these
flotation methods can be more expensive than centrifuga-
tion if a flocculant is added to enhance aggregation.
Advancements in microbubble production need to be made
in order for flotation to be feasible as a harvesting method
for the production of biofuel from microalgae.
Filtration
There are several filtration methods used to filter microal-
gae. They can be classified by pore size of the filter or by
the process of filtration. For example, membrane filtration
is classified by the size of the pores. Ultrafiltration refers to
filters with pores from 0.02 to 0.2 lm, and microfiltration
refers to filters with pores from 0.1 to 10 lm (Milledge and
Heaven 2013). For most microalgae strains, it has been
shown that a pore size of 0.1–0.5 lm is preferable (Bhave
et al. 2012). The main disadvantage of membrane filtration
is the fouling of the filter, which causes flux reduction, and
consequently an increase in processing costs. Cross-flow
filtration has shown to be able to reduce fouling by using
tangential flow with respect to the membrane (Kim et al.
2013). One novel method that has been proposed involves
the use of hollow fiber membranes with diameters of about
1 mm. These hollow fibers are bundled together to form a
tube composed of 50–140 hollow fibers (Bhave et al.
2012). This method has been shown to concentrate the
biomass up to 75 times of the original concentration, and it
also recycles the permeate for use in the cultivation step
(Bhave et al. 2012).
Flocculation
Flocculation aggregates microalgae cells to form flocs,
which cause them to settle more quickly. This is the least
energy-intensive harvesting method because the addition of

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A review of biodiesel production from microalgae
a flocculant is all that is needed. There are many types of
flocculation methods available that should be evaluated
further. There are chemical flocculation methods in which
metal salts, polymers, or biopolymers are used to form flocs.
The metal salts and polymers may cause harmful contami-
nation to the microalgae for further processing, so biopoly-
mers are a safer option. Two biopolymers with potential are
chitosan and cationic starch. Cationic starch has proven to
work better than chitosan for microalgae flocculation
because it is pH independent (Vandamme et al. 2013).
There are also physical flocculation methods that can be
used such as electrocoagulation-flocculation and the use of
magnetic nanoparticles. Electrocoagulation-flocculation
releases metal ions from an anode to induce flocculation.
Magnetic nanoparticles adsorb onto the microalgal surface,
and a magnet can be used to gather the microalgae (Van-
damme et al. 2013). After this, the nanoparticles need to be
separated from the microalgae so they can be reused.
Bioflocculation occurs when bacteria or microalgae with
the capability of autoflocculation are grown with the
microalgae culture to induce flocculation. If wastewater is
used as the culture medium, the flocculation bacteria can
use the carbon source from the wastewater to grow. The
disadvantage of this method is that the microalgal biomass
would contain bacteria which could hinder other down-
stream processes. Another form of bioflocculation is the
use of bacteria- or microalgae-derived substances to floc-
culate the microalgal biomass. A study by Wan et al.
(2013) showed that a substance produced by a certain
bacteria was able to flocculate the microalgae Nan-
nochloropsis oceanica at an efficiency of about 85%. This
type of bioflocculation can be very inexpensive and envi-
ronmentally friendly, especially if the bioflocculant can be
reused. While harvesting methods are able to reduce the
downstream processing volume, another step, pretreatment,
is usually needed to aid in lipid extraction.
Magnetic nanoparticle flocculants have gained attention
recently due to their high efficiency shown in the literature.
The electrostatically positive magnetic nanoparticles inter-
act with the negatively charged microalgae cells, and then
they are separated from the stream magnetically. The main
concern regarding magnetic nanoparticles for flocculation is
their potential effect on the downstream processes (Lee et al.
2015). Fortunately, magnetic nanoparticles can be recovered
and recycled on the large scale if cost-effective recycling
processes are developed (Lee et al. 2015).
Pretreatment
After the cells have been collected and concentrated in the
harvesting step, there is an optional step of pretreatment in
order to increase extraction efficiency. One method is to
653
disrupt the microalgal cell in order to release its contents
into the extracellular fluid for better access for extraction.
For certain types of extraction, a drying step is needed.
Cell disruption
The cell disruption pretreatment method is aimed at
removing the contents from inside the cell to the extra-
cellular fluid. Bringing desired biomolecules outside the
cell enables the extraction fluid to more easily interact with
them, thereby increasing product yield. Table 10 shows the
comparison of the advantages and disadvantages of the
applicable cell disruption techniques.
High-pressure homogenization, bead milling, enzymatic
lysis, and electroporation can aid in cell disruption. All of
these disruption techniques will be discussed in the fol-
lowing subsections.
High-pressure homogenization
High-pressure homogenization (HPH) is a process in which
concentrated biomass is forced through a narrow tube at a
very high pressure, which causes shear forces to disrupt the
cell walls of the microalgae. The heat produced during the
process makes the cell wall softer so it is easier to lyse.
This method of cell disruption is very efficient, can be used
on wet biomass, and is very scalable (Samarasinghe et al.
2012). HPH has been shown to be able to process 50,000
L/h (Samarasinghe et al. 2012). The disadvantages of this
process can be listed as the long processing times, the large
amount of cell debris that can hinder extraction, and the
consumption of a large amount of energy.
Bead milling
In bead milling, the microalgae culture is mixed with beads
made of quartz or metal and the container is shaken. The
cells lyse due to the friction and shear forces caused by the
collisions with the beads. Some studies have shown that
bead milling is the best cell disruption technique for
microalgae compared to HPH and enzymatic degradation
(Geciova et al. 2002; Ramachandra et al. 2013) due to short
processing time and simple setup. On the other hand, it is
difficult to scale up and there is a possibility of thermal
degradation of the products (Kim et al. 2013).
Enzymatic lysis
Enzymatic lysis is the process of using enzymes to break
down the cell wall and membrane of the microalgae. This
method has the advantage of being able to use enzymes
specific for the type of bonds that need to be broken,
instead of destroying numerous kinds of bonds
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Table 10 Advantages and disadvantages of various cell disruption methods
Pretreatment method Description
High-pressure High pressure causes shear force to lyse
homogenization cells
Bead milling Beads crush microalgae to lyse them
Enzymatic lysis Enzymes degrade parts of the cell wall
Electroporation An electric field disrupts the cell
membrane
unspecifically like in mechanical methods (Kim et al.
2013). Enzymes such as lipase, trypsin, cellulase, neutrase,
papain, and pectinase have demonstrated this capability to
degrade cell walls and membranes (Kim et al. 2013); out of
these enzymes, it has been shown that neutrase is the best
single enzyme to use, and the best mix of enzymes has
been shown to be a combination of pectinase and papain
(Young et al. 2011). Enzymatic lysis has shown to be
comparable to that of mechanical cell disruption techniques
and can achieve lipid extraction efficiencies of up to 84%
(Samarasinghe et al. 2012). The disadvantage of this
approach is the high cost of the enzymes and that they can
only be reused a few times (Vanthoor-Koopmans et al.
2013). One solution to reduce the cost of enzymatic lysis is
to couple this process with a mechanical one.
Electroporation
Electroporation is the use of electric current to rupture the
microalgae cells in order for the contents to spill out. It is
already a prevalent biotechnology technique used to
transfect DNA into a cell. Similarly, but with the intention
to release cellular contents rather than to insert additional
products into the cell, electroporation is an emerging
technology in microalgae processing known as pulsed
electric field (PEF). This method sends rapid pulses of
electricity to disrupt the cell (Grimi et al. 2014), and it has
been proven to be feasible at the industrial scale with a
processing rate as high as 2000 L/h (Vanthoor-Koopmans
et al. 2013). The energy requirements are also favorable
compared to HPH and bead milling. PEF has been used
industrially to pasteurize food at low temperatures. Now, it
is being recognized as a viable treatment for aiding oil
extraction through cell membrane rupture in microalgae
and has already been put into action by the microalgae
company OriginOil Inc. (Vanthoor-Koopmans et al. 2013).
Drying
Drying the microalgal biomass before extraction is neces-
sary depending on the method of extraction. The three
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S. Dickinson et al.
Advantages Disadvantages
High extraction yield, easily scalable High energy
High extraction yield, simple process Not scalable, moderate
energy
Moderate extraction yield, breaks specific High cost
bonds
Scalable, low energy Moderate extraction yield
main types of drying are freeze-drying, oven-drying, and
spray-drying. All three types of drying are not very feasible
for industrial-scale operation due to the high energy con-
sumption and operation costs (Milledge and Heaven 2013).
Freeze-drying consists of freezing the microalgae at a
temperature and pressure below the triple point of water,
placing the microalgae in a vacuum, then heating the
microalgae enough to induce sublimation, which removes
the water molecules from the cells (Nireesha et al. 2013).
For oven-drying, the microalgal biomass is subjected to
heat in a large oven at 60 °C for about 3 h, depending on
the amount of moisture in the sample (Balasubramanian
et al. 2013). Spray-drying comprises the algal biomass
being sprayed through a nozzle that makes the biomass
droplets very small, so hot air can easily remove the liquid
(Kumar and Awasthi 2009).
Lipid extraction
While microalgae contain polar lipids (e.g., phospholipids
and glycolipids), neutral lipids (e.g., tri-, di- and monoa-
cylglycerols) are generally used for biodiesel production.
These neutral lipids must be extracted to make FAMEs.
Several types of lipid extraction processes exist, but there
is not yet one ideal method, and the effectiveness of each
process varies based on the lipid content of the microalgae
cell. Microalgae fats, which can be saturated or unsatu-
rated, make up 25% to over 50% of the cell’s dry biomass.
The most common chemical extraction methods are
organic solvent and supercritical fluid extraction, while
mechanical approaches typically rely on oil expellers or the
assistance of ultrasound or microwave radiation. Finally,
the merits of the new techniques of wet microalgae, ionic
fluid, and osmotic shock extraction are also being investi-
gated. The characteristics of these extraction systems are
discussed in Table 11.
Each type of extraction processes differs in their energy
consumption, efficiency, safety, and other parameters that
need to be considered when selecting the best extraction
method for a system. Many oil extraction techniques such
A review of biodiesel production from microalgae 655

Table 11 Various extraction fluids used to remove lipids from the microalgae biomass
Type Description Requirements Characteristics

Organic Uses principles of liquid–liquid Energy intensive due to distillation needed Moderate efficiency, organic solvents have
solvent extraction and polarity to extract for reuse of solvents; most efficient potential fire, health, and environmental
extraction nonpolar TAG lipids from rest of solvents are chloroform:methanol and risks; slow
cell hexane
Accelerated Solvent extraction at high Requires pressurized nonreactive gas (i.e., High efficiency, more rapid than traditional
solvent temperatures and pressures nitrogen), high temperatures, high energy solvent extraction, safety risks from
extraction organic solvents and high
temperatures/pressures
Supercritical Supercritical CO2 acts as the Dewatering; high energy because needs High efficiency, fast; crude lipids are devoid
CO2 ‘‘solvent’’ for lipids high-pressure gas of organic solvents; high mass transfer;
tunable
Expeller Mechanically crushes cells to Dewatering; must use solvent extraction Low efficiency; possibility of heat damage
press squeeze out oil afterward to remove pigments; high to products
energy
Ultrasound Ultrasound pulses cause shear Used in conjunction with another Low efficiency; energy intensive; difficult to
assisted stress on cells through waves of extraction method; improves yield upscale; temperatures are not as high as for
extraction high and low pressure especially if there is no pretreatment microwave-assisted extraction
Microwave- Heating causes water vapor pockets Used in conjunction with another High efficiency; high temperatures may
assisted to form in cell, inducing extraction method; improves yield damage thermosensitive products; faster
extraction membrane permeability especially if there is no pretreatment than ultrasonification
Ionic Extracts with salt solution rather Tailored synthetic salts with very specific Moderate efficiency; needs more research;
extraction than organic solvent solubility characteristics low safety risks
Osmotic Hypertonic/hypotonic conditions Lipids must be extracted from water Low efficiency; produces high volumes of
pressure damage cell membrane afterward wastewater; low-cost materials
extraction

as pulsed electric field (electroporation), enzymatic
degradation of cell membranes, and bead milling are
sometimes discussed in the context of oil extraction rather
than pretreatment, as it is discussed in this article.
Organic solvent extraction
The most common technique, organic solvent extraction, is
inexpensive but time-consuming and inherently hazardous
for human and environmental health, especially when less
polar solvents (e.g., hexane or chloroform) are used. Ideal
solvents are specific to acylglycerols and will not extract
phospholipids, which cannot be converted into diesel
through traditional transesterification reactions. When
selecting a solvent, characteristics such as volatility and
polarity must be considered. More volatile solvents require
less energy to remove through distillation, and more polar
solvents have better cell penetration but are less specific to
only neutral acylglycerols. Solvents can be used alone or in
solvent systems; examples include n-hexane, ethanol,
chloroform/methanol, ethyl acetate/methanol, and many
others. Degree of polarity is important to the yield and
types of lipids extracted. Polar solvents interrupt hydrogen
bonding between polar lipids, while nonpolar solvents
interfere with hydrophobic interactions of nonpolar lipids.
Cheng et al. (2011) conducted a study that demonstrated
that increasing polarity corresponded to increasing lipid
yield in three solvent systems—toluene:methanol, hex-
ane:methanol, and ethyl acetate:methanol—because polar
lipids found in the cell membrane and other regions were
extracted along with the acylglycerols (Table 12). How-
ever, higher crude lipid yields do not mean higher FAME
production, because only acylglycerols can be converted
into FAMEs through traditional transesterification.
The well-known types of organic solvent extraction
methods are the Folch, Soxhlet, and Bligh and Dyer. One
of the oldest extraction techniques, the Folch method, uses
a chloroform:methanol (2:1 v/v) mixture mixed with saline
solution and biomass; after the phases settle, the lipids are
concentrated in the upper organic phase (Halim et al. 2012;
Kumar et al. 2015). While the Folch method is fast and
simple, other techniques demonstrate increased sensitivity
(Kumar et al. 2015).
The Soxhlet method is one of the most popular and uses
hexane as the extraction solvent, though the Bligh and
Dyer’s method found chloroform:methanol (1:2 v/v) to
produce the higher lipid yields (Kumar et al. 2015). Rı́os
et al. (2013) noted that n-hexane has higher specificity for
neutral lipids than the chloroform and Bligh and Deyer
method, which may also extract unwanted phospholipids

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Table 12 Results of Cheng et al. (2011) show how increasing
polarity correlates with increasing lipid yield but decreasing speci-
ficity for acylglycerols
Crude lipid yield FAME yield Polarity
(% wt.) (% wt.) index
Hexane:methanol 13.8 10.6 0.1
Toluene:methanol 17.6 4.4 2.4
Ethyl acetate:methanol 44.7 15.6 4.4
and glycolipids. The use of acids (e.g., sodium chloride,
phosphoric acid, or hydrochloric acid) in the water phase
has been shown to improve yields (Kumar et al. 2015).
This method does not require drying of the microalgae cells
prior to extraction, though improved solvent penetration
occurs with dry biomass (Kim et al. 2013).
One limitation to the solvent extraction method is that
the concentration of lipids exists at equilibrium between
the liquid and solid phases. This problem can be circum-
vented by using a continuous flow process, though it is
more expensive and requires large solvent volumes.
Another problem faced by solvent extraction is that it can
take 2–24 h to complete. However, when high tempera-
tures are used, then lipid solubility and the overall process
are accelerated to take minutes. Accelerated solvent
extraction increases overall lipid yield and may provide
additional solvent recovery, but the high temperatures and
pressures used to prevent solvent vaporization increase the
cost of this method (Kim et al. 2013). Another type of
extraction that is less toxic than organic solvent extraction
but requires higher capital investment is supercritical fluid
extraction, discussed in the next section.
Supercritical CO2 (SCCO2)
Supercritical CO2 is a faster, more environmentally safe,
and often more efficient alternative to solvent extraction
(Demirbas and Demirbas 2011). SCCO2 extraction can
take 30–60 min, whereas solvent extraction can take up to
24 h (Taher et al. 2014). Though some researchers use
ethanol as a co-solvent, SCCO2 extraction has the potential
to produce completely solvent-free lipid, which makes it
much less toxic than traditional solvent extraction. SCCO2
also facilitates fast mass transfer due to its high diffusivity
and low surface tension (Halim et al. 2012). Because the
solubility of products in CO2 is pressure dependent, and
pressure (under constant volume and number of molecules)
is dictated by temperature, the extraction process can be
easily controlled. SCCO2 requires temperatures higher than
31.1 °C and pressures exceeding 7.4 MPa (Halim et al.
2012). Both the low critical temperature and the nonoxi-
dizing nature of CO2 prevent the breakdown of lipids; CO2
can then easily be separated from the extract at normal
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S. Dickinson et al.
room conditions. Li et al. (2014) compared the conven-
tional chloroform:methanol:water solvent system with
alternative dichloromethane:methanol and cyclohex-
ane:water solvent mixtures, direct saponification with
potassium hydroxide and ethanol, and SCCO2 extraction.
They concluded that SCCO2 was the most promising
technique; SCCO2 extraction yield from wet microalgae
paste was significantly higher than the yield from dry
microalgal biomass, which allows for more energy savings.
However, high water content may impede flowage of
SCCO2 through the biomass (Halim et al. 2012). It is also
important to note that the success of various extraction
techniques is species dependent (Li et al. 2014; Rashid
et al. 2014). For example, direct saponification with
potassium hydroxide:ethanol was poor with Tetraselmis sp.
but worked well for Thraustochytrium sp., Isochyrsis gal-
bana, and Phaeodactylum tricornutum (Li et al. 2014).
Through manipulation of both reaction temperature and
co-solvents, the solubility of different compounds in SCCO2
can be tuned. Because its specificity can be adjusted for
neutral lipids, polar lipids, or other compounds such as pig-
ments, the equipment and expertise needed for SCCO2 has
the potential to be reused for several different processes in
the biorefinery approach. A common way for nonpolar
SCCO2 to extract polar molecules is through the use of a
polar co-solvent. For example, SCCO2 alone can extract
nonpolar compounds such as TAG, but ethanol is needed as a
co-solvent to SCCO2 for medium-polarity molecules. Rela-
ted techniques, including pressurized solvents or gas-ex-
panded liquids, blend the excellent diffusive properties of
gases with the good solvent behavior of liquids to also
improve highly polar molecule extraction (Halim et al. 2012;
Herrero and Ibáñez 2015; Taher et al. 2014).
Though the energy cost of achieving critical temperature
is low, energy is required to condense CO2 from the air and
recollect it after the extraction process is finished. The
initial equipment costs are also complicated and expensive
to install (Halim et al. 2012). Research needs to be invested
into the life cycle analysis of SCCO2 usage before the
economic feasibility of this method can be determined. In
addition to chemical extraction methods, there are several
mechanical extraction techniques such as the expeller
press, ultrasound-assisted, and microwave-assisted extrac-
tion types.
Expeller press
Expeller presses, the oldest form of oil extraction, use
mechanical force to squeeze oil out of cells. Press bits can
be adjusted to fit the variety of physical characteristics of
different microalgae strains, with options including screw,
expeller, piston, or other heads. Often, mechanical pressing
is used in conjunction with solvent or other chemical
A review of biodiesel production from microalgae
extraction processes to improve yields to approximately
70–75%. A weakness of the expeller press is that it requires
microalgae drying pretreatment, which alone can require
30% of the production costs (Kumar et al. 2015). Expeller
presses are better suited for vegetables with thinner cell
walls than microalgae, which have thick cell walls that
impede extraction. Presses also extract molecules such as
pigments along with lipids, so additional steps must be
added to the process in order to remove the pigments from
the lipid fraction. In general, expeller presses have low
efficiency and high energy requirements (Rashid et al.
2014).
Ultrasound-assisted extraction
Ultrasound can be used to assist extraction and is often used
as a pretreatment tool to lyse cells prior to lipid extraction.
High-intensity sound waves ([20 kHz) pass through the
liquid medium and create rapidly moving waves of low and
high pressures (Rashid et al. 2014). Bubbles that form in low-
pressure areas implode vigorously when they come under
high pressure. Shear stress, known as cavitation, causes cells
to break open, often increasing oil yield by 50–500% in one-
tenth of the usual extraction time (Kim et al. 2013; Kumar
et al. 2015). Ultrasound does not induce as high temperatures
as other methods, such as microwave radiation, which helps
prevent thermal damage of the biomolecules (Kumar et al.
2015). Though effective at disrupting cells, disadvantages
include high energy input and difficulty reproducing this
process on a large scale (Rashid et al. 2014). Extended
sonication may harm the product through formation of free
radicals (Kim et al. 2013). Ultrasonification does not
improve lipid yields when using a chloroform:methanol
system because this solvent system already penetrates the
cell to extract lipids efficiently (Rı́os et al. 2013).
Three microalgae species—Botryococcus sp., Chlorella
vulgaris, and Scenedesmus sp.—were treated with micro-
wave, sonication, and osmotic shock extraction methods in
addition to autoclaving and bead beating (Lee et al. 2010).
A chloroform:methanol extraction method was used after
cell disruption in this study. The results showed that
microwave-assisted extraction was the most efficient at
extracting lipid, while sonication performed the poorest
with observed yields of 28 and 8.8% wt. lipid content for
microwave and ultrasound extraction, respectively, in
Botryococcus sp. However, the efficiency gap between
these two methods was much smaller in the other species
tested (Lee et al. 2010).
Microwave-assisted extraction
Microwave radiation can also be used in conjunction with
organic solvent or SCCO2 extraction to release lipids from
657
the microalgae cell. Unlike ultrasound, which used sound
waves to cause shear stress, microwave-assisted techniques
use electromagnetic radiation with frequencies between 0.3
gigahertz (GHz) and 300 GHz to heat samples (Kim et al.
2013). Used on polar, nonvolatile solutions, water and
other molecules with similar absorption spectrums absorb
microwave energy, which in turn heats the samples (Halim
et al. 2012; Kim et al. 2013). Intracellular water vapor
pockets form, and this pressure buildup causes cell mem-
brane permeability (Kumar et al. 2015; Ma et al. 2014). In
the process of extracting lipids from microalgae, micro-
wave radiation was in general faster and more efficient than
ultrasonification. Microwave radiation reached the maxi-
mum extraction yield in 75 s, whereas ultrasonification
reached maximum extraction at 1200 s (Ma et al. 2014).
Microwave radiation was also used to heat samples to 80,
100, and 120 °C within 5 min, followed by 15 min at
sustained temperature and a cooldown period. A maximum
acylglycerol yield of 56.6% dry weight was experienced at
120 °C (Iqbal and Theegala 2013). OriginOil Inc. employs
short electromagnetic and ultrasonic pulses to crack the
microalgae cell wall and achieve low energy extraction
(Brennan and Owende 2010). However, thermally sensitive
products may be damaged by this method.
Table 13 presents the work of different research groups
performing lipid extraction from microalgae and using a
variety of solvent systems, mechanical assistance, and
supercritical technology. While the lipid yield is presented
here, it is important to note that often lipid yield is not
indicative of FAME yield. For example, entry 26 used an
ethyl acetate:methanol:water system for a 44% (wt.) lipid
yield, but had a lower conversion to FAMEs than a bead
beating/SCCO2 system by the same author that had a 17.9%
lipid yield. This indicates the acetate:methanol:water system
extracted out many impurities—such as phospholipids—
that could not be converted through transesterification into
biodiesel.
In addition to conventional extraction methods, there are
emerging methods—such as ionic fluid and osmotic pres-
sure extraction—being currently investigated by
researchers.
Additional extraction methods
Novel methods of lipid extraction are beginning to appear,
showcasing techniques such as ionic fluid and osmotic
pressure extraction. These methods are less hazardous than
those that use organic solvents or high temperatures, but
more research is required to realize their full potential.
Ionic fluids are nonaqueous solutions with custom-made
complex salts. The cation of these salts is generally a
complex, asymmetric organic ion, which is paired with an
anion that is not necessarily organic and can be as small as
123
 Table 13 Crude lipid yields by % weight of dry microalgae biomass for selected techniques
658

Extraction method Parameters Biomass feedstock Lipid yield (% weight) Time (min) Source

123
1 Supercritical CO2 Lysozyme treatment ? 50 °C, Wet biomass (93.2% water content) 12.5 30 Taher et al. (2014)
500 bar, 13 ml/min
2 60 °C, 306 bar Dry microalgae powder 10.4 360 Cheng et al. (2011)
3 Bead beating ? 60 °C, 306 bar Dry microalgae powder 17.9a 360 Cheng et al. (2011)
4 65 °C, 30 MPa, 5% ethanol co- Dry microalgae powder 18.15 90 Solana et al. (2014)
solvent, 0.4 ± 05 kg/h
5 Soxhlet n-hexane Room temperature; ultrasonically Dry microalgae powder 13.5 900 Cheng et al. (2011)
shaken for 3 h
6 Bead beating ? room Dry microalgae powder 15.3 900 Cheng et al. (2011)
temperature; ultrasonically
shaken for 3 h
7 50 °C Wet biomass (93.2% water) 4b Overnight Taher et al. (2014)
8 Lysozyme treatment ? 50 °C Wet biomass (93.2% water) 16.6 Overnight Taher et al. (2014)
9 Temperature not specified Freeze-dried biomass 6.5 1440 Rı́os et al. (2013)c
10 Unspecified Oven-dried microalgae 4.99 180 Ramluckan et al. (2014)
11 Hexane 25 °C Oven-dried microalgae 4 180 Shin et al. (2014)
12 Hot compressed hexane 235 °C, 31 bar Oven-dried microalgae 16.3 5 Shin et al. (2014)
13 Hexane:isopropanol 3:1 25 °C Microalgae concentrate 6.8 450 Halim et al. (2012)
(v/v)
14 Chloroform:methanol 1:2 10-min ultrasound pretreatment, Freeze-dried microalgae 19.6 10 Rı́os et al. (2013)
(v/v) 25 °C
15 25 °C Freeze-dried microalgae 24.5 10 Rı́os et al. (2013)
16 Soxhlet chloroform Unspecified Oven-dried microalgae 3.49 180 Ramluckan et al. (2014)
17 Soxhlet methanol:chloroform 2:1 105 °C Dry microalgae powder 14.84 1080 Solana et al. (2014)
(v/v)
18 Chloroform:methanol:water 25 °C Oven-dried microalgae 14.5 120 Shin et al. (2014)
1:1:0.9 (v/v/v)
19 Chloroform:methanol:water 25 °C Dry microalgae powder 8.9 60 Halim et al. (2012)
1:2:0.8 (v/v/v)
20 Chloroform:methanol 2:1 (v/v) Bead beating pretreatment; Microalgae concentrate 28.6 50 Halim et al. (2012)
temperature not specified
21 Soxhlet chloroform:ethanol 1:1 (v/ Unspecified Oven-dried microalgae 11.76 Not specified Ramluckan et al. (2014)
v)
22 Soxhlet methylene Temperature not specified Dry microalgae powder 11.9 180 Halim et al. (2012)
chloride:methanol 2:1 (v/v)
23 Ethanol 25 °C Dry microalgae powder 6.3 1440 Halim et al. (2012)
24 Soxhlet ethanol Unspecified Oven-dried microalgae 5.71 180 Ramluckan et al. (2014)
S. Dickinson et al.
A review of biodiesel production from microalgae 659

This publication discussed values for three species of microalgae, each subjected to two different harvesting methods; however, only the values for Nannochloropsis gaditana harvested via
Conversion of lipids to FAMEs was observed to be the highest for SCCO2 with bead beating than for all other experiments reported in this publication (entries 3, 6, 7, and 26 in the table). This
shows that the 3-h ethyl acetate:methanol extraction that yielded 44 wt% crude lipid contained a larger quantity of impurities, which could not be converted to FAMEs during transesterification
a chloride ion. Each salt can be carefully constructed to
demonstrate the best qualities of polarity, conductivity,
Cheng et al. (2011)

Rı́os et al. (2013)

etc., on a case-by-case basis for a variety of microalgae
strains. The anion structure largely determines the lipid

Though yield appears high, the reference noted that the Soxhlet solvent extraction method was less selective for lipids and extracted more impurities than the SCCO2 method
solubility in the ionic liquid; hydrophilic ionic liquids that
Source

mix with water have been found to encourage higher

extraction percentages than hydrophobic, water-immiscible
liquids (Pragya et al. 2013). However, the presence of
hydrophobic side groups on the anion may help lipids
Time (min)

diffuse into the liquid. These ‘‘designer solvents’’ are less

toxic, less volatile, nonflammable, and more thermally
360

90

stable than organic solvents, making them a ‘‘greener’’

option for solvent extraction (Choi et al. 2014).
Lipid yield (% weight)

Another solvent-free technique, osmotic pressure

extraction, takes advantage of the cellular stress caused
when the extracellular concentration of solutes is greatly
different than the concentration within the cell. Cells
placed in hypertonic solution shrink as water diffuses
44.7d

6.2

through the cytoplasm in an attempt to reestablish equi-

librium with their environment, which injures the cell
envelope. In hypotonic environments, water enters the cell
causing it to swell and potentially burst. During lipid
extraction, alternating hypo-/hyper-osmotic conditions or
hypo-osmotic conditions alone are induced to stress the
Freeze-dried microalgae
Dry microalgae powder

cells. Osmotic stress is a simple technique with low-cost

Biomass feedstock

materials; however, it has low efficiency and produces

large volumes of waste water, though this water has the
potential to be recycled (Kim et al. 2013).
In conclusion, there are many types of lipid extraction
methods. Two of the most popular are organic solvent
extraction and SCCO2 technology. While hexane and
chloroform:methanol systems are the most popular organic
Room temperature; ultrasonically

solvents, many alternative solvent systems are being

cross-flow filtration and dewatered using a centrifuge are reported here

investigated for improved safety and performance. SCCO2,

on the other hand, is already environmentally friendly, and
Low yield believed to be due to water film forming over cells

the equipment can be reused to extract co-products.

shaken for 3 h

Techniques such as ultrasound- and microwave-assisted

80 °C, 250 bar

extraction are available to help improve extraction yields,

Parameters

and novel methods such as ionic fluid extraction and

osmotic shock are emerging novel techniques. Following
lipid extraction, the acylglycerols need to be changed into
biodiesel through a transesterification step.
acid:chloroform 10:1:1 (v/v/v)
Ethyl acetate:methanol:water

Transesterification
Direct transesterification
methanol:hydrochloric
Extraction method

After cell debris and solvent removal, the lipids are

2:1:0.4 (v/v/v)
Table 13 continued

converted into biodiesel, either through transesterification,

direct use and blending, microemulsions, or pyrolysis.
The most common technique is via transesterification
because the product from transesterification can be used
directly in engines pure or in blends without further
25

26

modification (Miao and Wu 2006). Transesterification

b

d
a

123
660
Fig. 7 Important reactions in
transesterification process,
reproduced from Halim et al.
(2012), showing a the
transesterification reaction of
triacylglycerol into biodiesel
and b the undesired soap
formation reaction
reduces the viscosity of TAG. The process usually
requires methanol, acid, or alkali catalysts for the reaction
to occur, producing glycerol, excess acid/alkali/methanol,
and contamination from lipids that did not undergo
transesterification as end products (Halim et al. 2012;
Rawat et al. 2012). The transesterification reactions are
shown in Fig. 7.
The most popular method currently used in industry is
homogenous alkali-catalyzed transesterification using
KOH or NaOH. Reaction conditions have low temperature
and pressure, and the reaction time is fast, but free fatty
acids (FFA) in the lipid fraction react with the base to form
soap in a saponification reaction, which causes gelling in
the reaction mixture and lowers overall conversion (see
Fig. 7). An acceptable concentration of FFA is between 0.5
and 2% (w/w) prior to transesterification (Kim et al. 2013).
To circumvent saponification, chemists have proposed
acid-catalyzed transesterification, using acids such as sul-
furic or hydrochloric acid (Ehimen et al. 2010). However,
this technique is generally avoided because the acid is
corrosive to equipment and it requires a high volume of
methanol and high temperatures and is about 4000 times
slower than alkali catalysis (Halim et al. 2012; Kim et al.
2013; Yen et al. 2013). Without a catalysis, transesterifi-
cation can occur under supercritical conditions of metha-
nol. High temperature and volume requirements of
methanol make this option also economically unfavorable
(Ehimen et al. 2010; Kim et al. 2013).
Currently, enzyme catalysis is being investigated as a
route to perform the transesterification reaction with high
specificity and at low temperatures. For example, Huang
et al. (2015) demonstrated that lipase GH2 could produce
both FAME and FAEE with over a 90% conversion effi-
ciency after 24 h. Table 14 discusses the major methods of
transesterification.
123
S. Dickinson et al.
Direct transesterification, which combines lipid extrac-
tion and transesterification in one step, is also possible. A
common approach to direct transesterification is through
supercritical fluids, often supercritical methanol. Though
traditional transesterification can often be negatively
impacted by water, supercritical methanol will form a
single phase with both lipid and water, thus decreasing
water’s inhibitory effect. Furthermore, this transesterifica-
tion can be coupled with co-solvents or compressed CO2 to
further improve yields (Najafabadia et al. 2015). Reddy
et al. (2014) used supercritical ethanol to produce biodiesel
from microalgae containing 60% water. Diesel yield
peaked at 67% of total lipids to produce FAEEs under
operating parameters of 265 °C, 20 min, and a dry
microalgae/ethanol ratio of 1:9 (w/v). It is important to
note the FAEEs have similar properties to FAMEs, but they
have a higher oxidative stability and calorific value.
Another study by Patil et al. (2012) compared direct
transesterification of wet microalgae via supercritical
methanol and dry microalgae via microwave irradiation.
They found that each produced a comparable amount of
FAMEs, but that supercritical methanol required higher
temperatures and longer reaction times (250 °C, 25 min),
while microwave-assisted transesterification needed only
4–5 min at about 60 °C. Though transesterification is more
energy intensive through the supercritical methanol
method, it does not require dry microalgae and produces a
purer product, which saves on downstream purification
costs. Supercritical fluid transesterification technology has
the advantages of being fast, safe, easily controlled, and
environmentally benign. However, its high capital and
operating costs make it less favorable.
Several research groups have investigated modeling the
various unit operations involved in producing biodiesel
from microalgae biomass to create optimized models.
A review of biodiesel production from microalgae 661

Table 14 Conventional techniques used for transesterification

Type Description Requirements Characteristics

Homogenous base Base catalyst is used to Best performed on FFA-free oil due to Fast; low cost; moderate temperatures;
convert TAG to FAME saponification of FFA by hydroxide saponification of FFA reduces yields; difficult to
base purify FAME from glycerol; chemical waste
from neutralization step
Homogenous acid Acid, usually sulfuric High temperatures; 4000 9 slower One-step esterification and transesterification;
acid, catalyzes reaction than base catalysis corrosive; high temperatures; slower than base
transesterification of catalysis; can process oils with FFA; additional
TAG to FAME processing costs; chemical waste from
neutralization step
Heterogeneous Heterogeneous CaO and High temperatures; requires low FFA One-step esterification and transesterification; less
catalyst MgO catalyze and water concentrations waste; sensitive to both water and FFA; high cost
transesterification
Direct Combines lipid extraction Typically uses supercritical fluids to Can be water sensitive; does not require catalyst;
transesterification and transesterification catalyze reaction most common system is supercritical MeOH;
into a single reaction pressurized CO2 or co-solvents used in
conjunction with supercritical fluid may improve
yields
Homogeneous Enzymes catalyze TAG High lipase enzyme cost; must maintain Less waste; low temperatures; can process oils
enzymes conversion into FAME minimal alcohol concentration and with FFA
neutral pH to prevent denaturation

Depending on each model’s parameters, researchers have
found microalgae biodiesel production costs to range from
almost $5/gal to over $30/gal. In the next section, seven
such systems are presented.
Techno-economic studies
Several techno-economic studies have been undertaken for
the performance and cost estimations of producing biofuel
from microalgae. Each study incorporates different
assumptions and production steps that affect its economic
feasibility and the environmental impact. Seven models
will be discussed to show the effects of the choices that
researchers make when designing their process models. A
summary of these studies is shown in Table 15.
The first model to be discussed was developed by Rı́os
et al. (2013) at Universitat Rovira i Virgili in Spain
(Fig. 8). This model was developed by proposing six
different computer-simulated production processes and
then selecting the most economical path. The best pro-
duction path used an open pond to cultivate, flocculation
and filtration to harvest, spray-drying to pretreat, and
direct transesterification to convert the lipids into biodie-
sel. This model had minimal energy recycling and did not
isolate by-products to mitigate the cost of production.
Therefore, the production cost for this model was high at
$15.96/gal.
Davis et al. (2011) from the National Renewable Energy
Laboratory modeled a microalgal biofuel production
process, comparing the use of PBRs and open ponds.
Dissolved air flotation and centrifugation were utilized
after cultivation, followed by high-pressure homogeniza-
tion and solvent extraction, which is shown in Fig. 9. The
raw oil production cost using an open pond was $8.52/gal,
contrasted by $18.10 when utilizing PBRs. This shows that
open ponds are currently less expensive than PBRs.
Members of the French Atomic Energy and Alternative
Energies Commission developed a model that focused on
using the least costly unit operations for each step. The
process that was selected used a raceway pond, followed by
flocculation, centrifugation, drying, and hexane extraction.
Although the model focused on economics, the multiple
harvesting procedures caused the production cost of this
process to be $8.12/gal (Delrue et al. 2012).
Another model was developed by researchers at North-
western University. This model was focused on choosing
the most environmentally friendly process. The selected
process used flue gas as a CO2 source, a tubular photo-
bioreactor as the cultivation method, filtration as the har-
vesting method, and hydroprocessing, which is a method to
remove impurities from the diesel to produce ‘‘green die-
sel.’’ Although this process focused mostly on the envi-
ronmental impact of the process, it also had a relatively low
production cost of $6.33/gal because it had only one har-
vesting unit (Gebreslassie et al. 2013).
Lundquist et al. (2010) at California Polytechnic State
University developed a low production cost method which
used an open pond, flocculation, and hexane extraction,
shown in Fig. 10. The researchers considered the revenue

123
662 S. Dickinson et al.

Table 15 Summary of various techno-economic studies of the production of biofuel from microalgae
Research group conducting study Technique Production cost
($/gal)

Universitat Rovira i Virgili (Rı́os et al. 2013) Little energy recycling/no co-products 15.96
National Renewable Energy Laboratory (Davis et al. Showed open pond is more economical than PBR 8.52
2011)
French Atomic Energy and Alternative Energies Algorithm focused on economics/many harvesting steps 8.12
Commission (Delrue et al. 2012)
Northwestern University (Gebreslassie et al. 2013) Algorithm focused on environmental impact/one harvesting step 6.33
California Polytechnic State University (Lundquist et al. Considered revenue from treating wastewater 5.71
2010)
Iowa State University (Thilakaratne et al. 2014) Showed mechanical drying is more economical than thermal 5.64
drying, does not consider cultivation
Texas A&M University (Pokoo-Aikins et al. 2010) Flue gas used in drying 4.20

Fig. 8 Best production path determined by Rı́os et al. (2013)

Fig. 9 Process flow diagram of production of biofuel from microalgae developed by Davis et al. (2011)

they would receive from treating wastewater, which low- Thilakaratne et al. (2014) from Iowa State University
ered the production cost to $5.71/gal. The addition of other studied the microalgal oil production process as well and
forms of revenue-generating processes may be the best way compared the usage of thermal drying with mechanical
of making microalgal biofuels affordable in the near future. dewatering. In their model, the biomass was dried by the two

123
A review of biodiesel production from microalgae
Fig. 10 Process flow diagram
of the least expensive
production of microalgal oil by
Lundquist et al. (2010)
methods being compared, then the oil was extracted by using
an organic solvent, and finally the mixture was fractionated
into gasoline and diesel. By using the mechanical dewater-
ing method, the minimum selling price of the fuel was
$5.64/gal, compared to a minimum selling price of $6.82
when using the thermal dewatering. This shows that
mechanical dewatering may be a more economical method
to dry microalgae. However, this model did not include the
costs for cultivating the microalgae.
Pokoo-Aikins et al. (2010) from Texas A&M University
developed a design and cost analysis of a biodiesel pro-
duction process grown through carbon sequestration. For
cultivation, an open pond and a reactor were tested sepa-
rately. The model used one harvesting technique—cen-
trifugation—which was assumed to generate a stream with
30% microalgae. A drying step was used to further dewater
the stream, and flue gas was used to reduce energy costs. An
expeller press was used to extract the microalgal oil. Finally,
base-catalyzed transesterification was used to convert the
oils to biodiesel. In addition, heat integration (heat recy-
cling) was used to help reduce the utility costs. This design
was shown to be profitable at a biodiesel selling price of
$4.20, as long as the conditions are favorable.
No matter which system is used, production facilities
create waste and use resources. Especially in fuel production
facilities, the net energy gain and CO2 emissions are of key
interest. Also of importance is the creation or use of toxic
chemicals and the consumption of water, which will be
discussed in the following environmental analysis section.
Environmental analysis
The production of biofuel from microalgae is of great
interest because of its superior environmental impact when
compared to that of petro-based fuel. On the other hand,
663
there are still concerns about the energy efficiency of the
production process. To identify possible areas of
improvement to tackle these issues, life cycle assessments
(LCAs) are done. LCA compares the inputs with the out-
puts of the process. The main inputs of concern for
microalgal biofuel production are CO2, water, nutrients,
and energy. The outputs of concern are oxygen, energy in
the form of biodiesel, and any co-product that is produced.
The main goal of the LCA is to determine how much the
proposed system impacts the environment. To do this, there
needs to be a quantification of each system component’s
impact. LCA components can be separated into 11 different
categories: carcinogenic substances, respiratory effects
(organic), respiratory effects (inorganic), climate change,
ionizing radiation, ozone layer depletion, ecotoxicity,
acidification, land use, depletion of fossil fuels, and
depletion of minerals. These ‘‘impact categories’’ are
placed into one of three ‘‘damage categories’’: damage to
human health, ecosystem quality, or resources depletion.
Within these damage categories, there are certain formulas
that quantify the environmental impact of each component,
giving researchers a tool to analyze the environmental
impact of systems (Gutiérrez-Arriaga et al. 2014).
Most of the consumption of water happens in the cultiva-
tion and harvesting steps. Depending on the assumed pro-
ductivities of the microalgae and the geographical location,
the amount of water used can vary greatly (Quinn and Davis
2015). Usually, the water can be recycled after harvesting to
reuse for cultivation. Yang et al. (2011) reported that the water
footprint of a process with no water recycling was 3700 kg-
water/kg-biodiesel, while the water footprint of a process with
all the water being recycled after harvesting was 600 kg-
water/kg-biodiesel. Some water loss is inevitable due to
evaporation during the cultivation stage. In addition to recy-
cling, the use of sea water or wastewater is able to reduce the
freshwater footprint even further (Yang et al. 2011).
123
664
Linked closely with water usage is the nutrient con-
sumption by the microalgae. The main nutrients needed are
nitrogen, phosphorous, potassium, magnesium, and sulfur.
When recycling water, nutrient usage decreases by 55%
compared to the case when there is no water recycling
(Yang et al. 2011). This is because the nutrients that were
not consumed are able to be fed back to the cultivation
step, reducing the amount of nutrients that need to be
added. The use of sea water and wastewater also reduces
the amount of nutrients needed initially because there are
already nutrients in those sources. For example, the use of
wastewater can reduce the additional nitrogen required by
94% (Yang et al. 2011). However, nutrient resources have
been shown to be a limiting factor when scaling microalgal
processes up to an industrial scale (Quinn and Davis 2015).
One of the most influential aspects of environmental
impact is the consumption and production of CO2. If bio-
diesel is to be more environmentally friendly than petro-
based diesel, then it is imperative that the CO2 footprint
should be smaller. In the production process, CO2 is con-
sumed during microalgae growth in cultivation but then
emitted during processes that require electricity, such as
cultivation and extraction. The CO2 that is emitted by
burning fuel can be reduced in many ways. Flue gas created
from burning fuel at nearby industrial site could be used for
cultivation and extraction processes, which would reduce
the amount of CO2 released by that company as well the
energy needed to isolate pure CO2. Renewable electricity
such as solar or wind power could also be used to give energy
to the production process, which would decrease the amount
of CO2 consumed by conventional electricity (Passell et al.
2013). In addition, the microalgae strains that are used in the
production process could be genetically engineered to
sequester more CO2 than usual, which could increase the
amount of CO2 converted into energy. According to a study
of production models by Quinn and Davis (2015), the net
CO2 emissions can vary greatly from net negative emissions
to over 500 grams of CO2 per megajoule (g CO2/MJ),
showing the lack of uniformity in the calculation of CO2
emissions in different studies.
Energy consumption is the greatest environmental con-
cern for the production process. The most energy-intensive
steps are pretreatment and extraction, due to high pressure
and/or a temperature increase. Lam and Lee (2012) com-
pared the energy efficiency ratio (EER) of the production
processes of crop and microalgae-based biofuel. A higher
value is better as the EER is defined as the energy output
divided by energy input. The range of EER for crop-based
biofuel was 1.44–5, while the range of EERs for microal-
gae-based biofuel was 0.35–4.34. Overall, the microalgae-
based biofuel production had a lower EER, but as
improvements to the process continue to be made, this ratio
may increase.
123
S. Dickinson et al.
Conclusions
Many companies and researchers have developed
microalgae to biofuel systems, but as of yet there have not
been any breakthroughs into commercial success. Many of
the limitations are due to technological advancements in
cultivation and harvesting, as well as in determining the
optimal growth conditions for the microalgae. It is
important that lipid productivity, energy sources, and co-
products are taken into account when determining how a
certain strain will affect the downstream processes. As
cultivation and harvesting technology advances, the cost
associated with those areas will shrink, providing better
opportunities for commercial success.
Several techno-economic case studies using computer-
aided simulation have been undertaken to assess the via-
bility of commercial processes for the production of bio-
diesel from microalgae. The results tend to indicate
relatively high production cost. Future research should
focus on several ways to offset costs of microalgae bio-
diesel production. First, flue gas and wastewater are
promising inexpensive nutrient sources for phototrophic
microalgae. Second, manipulating the environmental and
nutrient conditions throughout the cultivation process can
cause beneficial stresses, which can increase the amount of
neutral lipids in the microalgae cells. Finally, a biorefinery
concept should be implemented, in which additional co-
products are isolated from the biomass for additional rev-
enue to make the process economically feasible.
Given the merits of growing microalgae in reducing
CO2 concentration in the atmosphere, it is important to
conduct LCAs on a process to determine the environmental
effect of the system. One area of research is the reduction
of energy intensity in the pretreatment and extraction steps,
since they have been shown to be the most energy inten-
sive. Other areas of interest include clean and efficient
water recycling, in order to reduce water consumption and
to recycle unused nutrients. Energy integration strategies
should also be further researched in order to recycle as
much energy as possible during the process.
Microalgae-based biodiesel can potentially solve con-
straints imposed upon current petroleum-based transporta-
tion fuels. However, the operating costs of the process must
be reduced to make microalgae biodiesel competitive in the
market. Using biorefinery approaches, process integration,
and optimization strategies, microalgae-produced biodiesel
has the potential to be an environmentally friendly, eco-
nomically feasible alternative to crop-based biodiesel and
traditional petro-diesel.
Acknowledgements The authors would like to thank Wesley Zloza,
Kyra Gudgel, Caitlin Liddiard, Brock Shilling, Victoria St. Martin,
and Alex Fuerst for their assistance in image production.
A review of biodiesel production from microalgae
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
Algawise (2016) AlgaWise Ultra Omega-9. http://algawise.com/
ingredients/ultra-omega-9-algae-oil/. Accessed 17 April 2016
Algenol Biotech LLC (2011) About Algenol. http://www.algenol.
com/. Accessed 10 Oct 2015
Balasubramanian RK, Doan TT, Obbard JP (2013) Factors affecting
cellular lipid extraction from marine microalgae. Chem Eng J
215:929–936
Barros AI, Gonçalves AL, Simões M, Pires JC (2015) Harvesting
techniques applied to microalgae: a review. Renew Sustain
Energy Rev 41:1489–1500
Bhave R, Kuritz T, Powell L, Adcock D (2012) Membrane-based
energy efficient dewatering of microalgae in biofuels production
and recovery of value added co-products. Environ Sci Technol
46:5599–5606
Bishop WM, Zubeck HM (2012) Evaluation of microalgae for use as
nutraceuticals and nutritional supplements. J Ntr Food Sci 2:147.
doi:10.4172/2155-9600.1000147
Brennan L, Owende P (2010) Biofuels from microalgae—a review of
technologies for production, processing, and extractions of
biofuels and co-products. Renew Sustain Energy Rev
14:557–577
Cellana Inc (2015) Alduo technology. http://cellana.com/technology/
core-technology/. Accessed 8 Oct 2015
Chen CY, Yeh KL, Aisyah R, Lee DJ, Chang JS (2011) Cultivation,
photobioreactor design and harvesting of microalgae for
biodiesel production: a critical review. Bioresource Technol
102:71–81
Chen G, Zhao L, Qi Y (2015) Enhancing the productivity of
microalgae cultivated in wastewater toward biofuel production: a
critical revew. Appl Energ 137:282–291
Cheng CH, Du TB, Pi HC, Jang SM, Lin YH, Lee HT (2011)
Comparative study of lipid extraction from microalgae by
organic solvent and supercritical CO2. Bioresource Technol
102:10151–10153
Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv
25:294–306
Choi SA, Oh YK, Jeong MJ, Kim SW, Lee JS, Park JY (2014) Effects
of ionic liquid mixtures on lipid extraction from Chlorella
vulgaris. Renew Energ 65:169–174
Cosmetics Business (2015) Global sun care market to rise 6.4% by
2018. http://www.cosmeticsbusiness.com/news/article_page/Glo
bal_sun_care_market_to_rise_64_by_2018/105908. Accessed 14
Sep 2016
Coustets M, Joubert-Durigneux V, Hérault J, Schoefs B, Blanckaert
V, Garnier JP, Teissié J (2015) Optimization of protein
electroextraction from microalgae by a flow process. Bioelec-
trochemistry 103:74–81
Cuellar-Bermudez SP, Aguilar-Hernandez I, Cardenas-Chavez DL,
Ornelas-Soto N, Romero-Ogawa MA, Parra-Saldivar R (2015)
Extraction and purification of high-value metabolites from
microalgae: essential lipids, astaxanthin and phycobiliproteins.
Microb Biotechnol 8:190–209
Davis R, Aden A, Pienkos PT (2011) Techno-economic analysis of
autotrophic microalgae for fuel production. Appl Energ
88:3524–3531. doi:10.1016/j.apenergy.2011.04.018
Delrue F, Setier PA, Sahut C, Cournac L, Roubaud A, Peltier G,
Froment AK (2012) An economic, sustainability, and energetic
665
model of biodiesel production from microalgae. Bioresource
Technol 111:191–200
Demirbas A, Demirbas MF (2011) Importance of algae oil as a source
of biodiesel. Energ Convers Manage 52:163–170
Dunlop MJ, Keasling JD, Mukhopadhyay A (2010) A model for
improving microbial biofuel production using a synthetic
biology feedback loop. Syst Synth Biol 4:95–104
Ehimen EA, Sun ZF, Carrington CG (2010) Variables affecting the
in situ transesterification of microalgae lipids. Fuel 89:677–684
Elvin JG, Couston RG, van der Walle CF (2013) Therapeutic
antibodies: market considerations, disease targets and biopro-
cessing. Int J Pharm 440:83–98
Fasahati P, Woo HC, Liu JJ (2015) Industrial-scale bioethanol
production from brown algae: effects of pretreatment. Appl
Energ 139:175–187
Freire I, Cortina-Burgueño A, Grille P, Arizcun MA, Abellán E,
Segura M, Sousa FW, Otero A (2016) Nannochloropsis limnet-
ica: a freshwater microalga for marine aquaculture. Aquaculture
459:124–130
Gebreslassie BH, Waymire R, You F (2013) Sustainable design and
synthesis of algae-based biorefinery for simultaneous hydrocar-
bon biofuel production and carbon sequestration. AIChE J
59:1599–1621
Geciova J, Bury D, Jelen P (2002) Methods for disruption of
microbial cells for potential use in the diary industry—a review.
Int Diary J 12:541–553
Georgianna R, Mayfield SP (2012) Exploiting diversity and synthetic
biology for the production of algal biofuels. Nature 488:329–335
Ghosh A, Khanra S, Mondal M, Halder G, Tiwari ON, Saini S,
Bhowmick TK, Gayen K (2016) Progress toward isolation of
strains and genetically engineered strains of microalgae for
production of biofuel and other value added chemicals: a review.
Energ Convers Manage 113:104–118
González-Delgado ÁD, Kafarov V, El-Halwagi M (2015) Develop-
ment of a topology of microalgae-based biorefinery: process
synthesis and optimization using a combined forward–backward
screening and superstructure approach. Clean Technol Envir
17:2213–2228
Graham JM, Graham LE, Zulkifly SB, Pfleger BF, Hoover SW,
Yoshitani J (2012) Freshwater diatoms as a source of lipids for
biofuels. J Ind Microbiol Biot 39:419–428
Grimi N, Dubois A, Marchal L, Jubeau S, Lebovka NI, Vorobiev
(2014) Selective extraction from microalgae Nannochloropsis
sp. using different methods of cell disruption. Bioresource
Technol 153:254–259
Grimm P, Risse JM, Cholewa D, Müller JM, Beshay U, Friehs K,
Flaschel E (2015) Applicability of Euglena gracilis for biore-
fineries demonstrated by the production of a-tocopherol and
paramylon followed by anaerobic digestion. J Biotechnol
215:72–79
Gutiérrez-Arriaga CG, Serna-González M, Ponce-Ortega JM, El-
Halwagi MM (2014) Sustainable integration of algal biodiesel
production with steam electric power plants for greenhouse gas
mitigation. ACS Sustain Chem Eng 2:1388–1403
Halim R, Danquah MK, Webley PA (2012) Extraction of oil from
microalgae for biodiesel production: a review. Biotechnol Adv
30:709–732
Harun R, Singh M, Forde GM, Danquah MK (2010) Bioprocess
engineering of microalgae to produce a variety of consumer
products. Renew Sust Energ Rev 14:1037–1047
Herrero M, Ibáñez E (2015) Green processes and sustainability: an
overview on the extraction of high added-value products from
seaweeds and microalgae. J Supercrit Fluid 96:211–216
Hitchings MA, Ward T (2010) Solazyme wins first prize for
sustainable biofuels technology. Global Refining Fuels Today
2 (53). http://www.downstreambusiness.com/solazyme-wins-
123
666
first-prize-sustainable-biofuels-technology-361971. Accessed 11
Oct 2015
Ho SH, Chan MC, Liu CC, Chen CY, Lee WL, Lee DJ, Chang JS
(2014) Enhancing lutein productivity of an indigenous microalga
Scenedesmus obliquus FSP-3 using light-related strategies.
Bioresour Technol 152:275–282
Huang J, Xia J, Jiang W, Li Y, Li J (2015) Biodiesel production from
microalgae oil catalyzed by a recombinant lipase. Bioresource
Technol 180:47–53
Iancu P, Pleşu V, Velea S (2012) Flue gas CO2 capture by microalgae
in photobioreactor: a sustainable technology. Chem Eng Trans
29:799–804
Iqbal J, Theegala C (2013) Microwave assisted lipid extraction from
microalgae using biodiesel as co-solvent. Algal Res 2:34–42
Jessen H (2015) I have green expectations for algae. Ethanol Producer
Magazine. http://news.algaeworld.org/2015/03/holly-jessen-i-
have-green-expectations-for-algae/. Accessed 9 Dec 2015
Jinkerson RE, Subramanian V, Posewitz MC (2011) Improving
biofuel production in phototrophic microorganisms with systems
biology. Biofuels 2:125–144
Jinkerson RE, Radakovits R, Posewitz MC (2013) Genomic insights
from the oleaginous model alga Nannochloropsis gaditana.
Bioengineered 4:37–43
Jorquera O, Kiperstok A, Sales EA, Embiruçu M, Ghirardi ML (2010)
Comparative energy life-cycle analyses of microalgal biomass
production in open ponds and photobioreactors. Bioresource
Technol 101:1406–1413
Judd S, van den Broeke LJ, Shurair M, Kuti Y, Znad H (2015) Algal
remediation of CO2 and nutrient discharges: a review. Water Res
87:356–366
Juneja A, Ceballos RM, Murthy GS (2013) Effects of environmental
factors and nutrient availability on the biochemical composition
of algae for biofuels production: a review. Energies 6:4607–4638
Kim J, Yoo G, Lee H, Lim J, Kim K, Kim CW, Park MS, Yang JW
(2013) Methods of downstream processing for the production of
biodiesel from microalgae. Biotechnol Adv 31:862–876
Koller M, Muhr A, Braunegg G (2014) Microalgae as versatile
cellular factories for valued products. Algal Res 6:52–63
Kumar A, Awasthi A (2009) Bioseparation engineering. I.K. Inter-
national Publishing House Pvt Ltd, New Delhi
Kumar RR, Rao PH, Muthu A (2015) Lipid extraction methods from
microalgae: a comprehensive review. Front Energy Res 2:61
Lakshmi GC (2014) Food coloring: the natural way. Res J Chem Sci
4:87–96
Lam MK, Lee KT (2012) Microalgae biofuels: a critical review of
issues, problems and the way forward. Biotechnol Adv
30:673–690
Lane J (2015a) Algenol Algae fuels: coming soon to a Florida pump
near you. Biofuels Digest. http://www.biofuelsdigest.com/bdi
gest/2015/09/14/algenol-algae-fuels-coming-soon-to-a-florida-
pump-near-you/. Accessed 10 Oct 2015
Lane J (2015b) Joule raises $40 M, as ‘fuel from thin air’ preps for
commercial scale in 2017. Biofuels Digest. http://www.biofuels
digest.com/bdigest/2015/05/11/joule-raises-40m-as-fuel-from-
thin-air-preps-for-commercial-scale-in-2017. Accessed Oct 2015
Lane J (2015c) Joule unlimited: Biofuels Digest’s 2015 5-minute
guide. Biofuels Digest. http://www.biofuelsdigest.com/bdigest/
2015/02/03/joule-unlimited-biofuels-digests-2015-5-minute-
guide/. Accessed 10 Oct 2015
Lane J (2015d) Sapphire energy: Biofuels Digest’s 2015 5-minute
guide. Biofuels Digest. http://www.biofuelsdigest.com/bdigest/
2015/02/11/sapphire-energy-biofuels-digests-2015-5-minute-
guide/. Accessed 12 Dec 2015
Lebeau T, Robert JM (2003) Diatom cultivation and biotechnolog-
ically relevant products. Part I: cultivation at various scales. Eur
J Appl Microbiol 60:612–623
123
S. Dickinson et al.
Lee JY, Yoo C, Jun SY, Ahn CY, Oh HM (2010) Comparison of
several methods for effective lipid extraction from microalgae.
Bioresource Technol 101:S75–S77
Lee YC, Lee K, Oh YK (2015) Recent nanoparticle engineering
advances in microalgal cultivation and harvesting processes of
biodiesel production: a review. Bioresource Technol 184:63–72
Leu S, Boussiba S (2014) Advances in the production of high-value
products by microalgae. Industrial Biotechnology 10:169–183
Li Y, Horsman M, Wu N, Lan CQ, Dubois-Calero N (2008) Biofuels
from microalgae. Biotechnol Progr 24:815–820
Li Y, Nahdi FG, Garg S, Adarme-Vega TC, Thurecht KJ, Ghafor
WA, Tannock S, Schenk PM (2014) A comparative study: the
impact of different lipid extraction methods on current microal-
gal lipid research. Microb Cell Fact 13. doi:10.1186/1475-2859-
13-14
Lowrey J, Brooks MS, McGinn PJ (2014) Heterotrophic and
mixotrophic cultivation of microalgae for biodiesel production
in agricultural wastewaters and associated challenges—a critical
review. J Appl Phycol 27:1485–1498
Lu J, Sheahan C, Fu P (2011) Metabolic engineering of algae for
fourth generation biofuels production. Energ Environ Sci
4:2451–2466
Lundquist TJ, Woertz IC, Quinn NWT, Benemann JR (2010) A
realistic technology and engineering assessment of algae biofuel
production. Energy Biosciences Institute, University of Califor-
nia, Berkeley
Ma YA, Cheng YM, Huang JW, Jen JF, Huang YS, Yu CC (2014)
Effects of ultrasonic and microwave pretreatments on lipid
extraction of microalgae. Bioproc Biosyst Eng 37:1543–1549
Mata TM, Martins AA, Caetano NA (2010) Microalgae for biodiesel
production and other applications: a review. Renew Sust Energ
Rev 14:217–232
Miao X, Wu Q (2006) Biodiesel production from heterotrophic
microalgal oil. Bioresource Technol 97:841–846
Milledge JJ, Heaven S (2013) A review of the harvesting of micro-
algae for biofuel production. Rev Environ Sci Biotechnol
12:165–178
Najafabadia HA, Vossoughia M, Pazukic G (2015) The role of co-
solvents in improving the direct transesterification of wet
microalgal biomass under supercritical condition. Bioresource
Technol 193:90–96
Nireesha GR, Divya L, Sowmya C, Venkateshan N, Babu MN,
Lavakumar V (2013) Lyophilization/freeze drying—an review.
Inter J Novel Trends Pharm Sci 3:87–98
Passell H, Dhaliwal H, Reno M, Wu B, Amotz AB, Ivry E, Gay M,
Czartoski T, Laurin L, Ayer N (2013) Algae biodiesel life cycle
assessment using current commercial data. J Environ Manage
129:103–111
Patil PD, Gude VG, Mannarswamy A, Cooke P, Nirmalakhandan N,
Lammers P, Deng S (2012) Comparison of direct transesterifi-
cation of algal biomass under supercritical methanol and
microwave irradiation conditions. Fuel 97:822–831
Pittman JK, Dean AP, Osundeko O (2011) The potential of
sustainable algal biofuel production using wastewater resources.
Bioresource Technol 102:17–25
Pokoo-Aikins G, Nadim A, El-Halwagi MM, Mahalec V (2010)
Design and analysis of biodiesel production from algae grown
through carbon sequestration. Clean Technol Envir 12:239–254
Pragya N, Pandey KK, Sahoo PK (2013) A review on harvesting, oil
extraction and biofuels production technologies from microal-
gae. Renew Sust Energ Rev 24:159–171
Priyadarshani I, Rath B (2012) Commercial and industrial applica-
tions of micro algae—a review. J Algal Biomass Utln 3:89–
100
Provust J (2011) Cultivation of algae in photobioreactors for biodiesel
production. In: Pandey A, Larroche C, Ricke SC, Claude-Gilles
A review of biodiesel production from microalgae
Dussap CG, Gnansounou E (eds) Biofuels: alternative feedstocks
and conversion processes. Academic Press, London, pp 439–461
Pullen J, Saeed K (2012) An overview of biodiesel oxidation stability.
Renew Sust Energ Rev 16:5924–5950
Quinn JC, Davis R (2015) The potentials and challenges of algae based
biofuels: a review of the techno-economic, life cycle, and resource
assessment modeling. Bioresource Technol 184:444–452
Ramachandra TV, Madhab MD, Shilpi S, Joshi NV (2013) Algal
biofuel from urban wastewater in India: scope and challenges.
Renew Sust Energ Rev 21:767–777
Ramluckan K, Moodley KG, Bux F (2014) An evaluation of the
efficacy of using selected solvents for the extraction of lipids
from algal biomass by the soxhlet extraction method. Fuel
116:103–108
Rashid N, Rehman MSU, Sadiq M, Mahmood T, Han JI (2014)
Current status, issues and developments in microalgae derived
biodiesel production. Renew Sust Energ Rev 40:760–778
Rawat I, Kumar RR, Mutanda T, Bux F (2012) Biodiesel from
microalgae: a critical evaluation from laboratory to large scale
production. Appl Energ 103:444–467
Reddy HK, Muppaneni T, Patil PD, Ponnusamy S, Cooke P, Schaub
T, Deng S (2014) Direct conversion of wet algae to crude
biodiesel under supercritical ethanol conditions. Fuel
115:720–726
Rı́os SD, Torres CM, Torras C, Salvado J, Mateo-Sanz JM, Jimenez L
(2013) Microalgae-based biodiesel: economic analysis of down-
stream process realistic scenaRı́os. Bioresource Technol
136:617–625. doi:10.1016/j.biortech.2013.03.046
Rodrı́guez-Zavala JS, Ortiz-Cruz MA, Mendoza-Hernández G,
Moreno-Sánchez R (2010) Increased synthesis of a-tocopherol,
paramylon and tyrosine by Euglena gracilis under conditions of
high biomass production. J Appl Microbiol 109:2160–2172
Samarasinghe N, Fernando S, Lacey R, Faulkner WB (2012) Algal
cell rupture using high pressure homogenization as a prelude to
oil extraction. Renew Energ 48:300–308
Sapphire Energy Inc (2016) Sapphire energy. http://www.sapphiree
nergy.com/. Accessed 1 June 2016
Schmidt CW (2010) Synthetic biology: environmental health implica-
tions of a new field. Environ Health Perspect 118: A118–A123.
http://ehp.niehs.nih.gov/118-a118/?utm_source=rss&utm_
medium=rss&utm_campaign=118-a118. Accessed 9 June 2016
Schneising O, Buchwitz M, Reuter M, Heymann J, Bovensmann H,
Burrows JP (2011) Long-term analysis of carbon dioxide and
methane column-averaged mole fractions retrieved from SCIA-
MACHY. Atmos Chem Phys 11:2863–2880
Sharma KK, Schuhmann H, Schenk PM (2012) High lipid induction
in microalgae for biodiesel production. Energies 5:1532–1553
Shin HY, Ryu JH, Bae SY, Crofcheck C, Crocker M (2014) Lipid
extraction from Scenedesmus sp. microalgae for biodiesel
production using hot compressed hexane. Fuel 130:66–69
Sierra E, Acien FG, Fernandez JM, Garcia JL, Gonzalez C, Molina E
(2008) Characterization of a flat plate photobioreactor for the
production of microalgae. Chem Eng J 138:136–147
Singh B, Guldhe A, Singh P, Singh A, Rawat I, Bux F (2015)
Sustainable production of biofuels from microalgae using a
biorefinery approach. In: Kaushik G (ed) Applied environmental
biotechnology: present scenario and future trends. Springer,
India, pp 115–127
Solana M, Rizza CS, Bertucco A (2014) Exploiting microalgae as a
source of essential fatty acids by supercritical fluid extraction of
lipids: comparison between Scenedesmus obliquus, Chlorella
protothecoides and Nannochloropsis salina. J Supercrit Fluid
92:311–318
Taher H, Al-Zuhair S, Al-Marzouqi AH, Haik Y, Farid M (2014)
Effective extraction of microalgae lipids from wet biomass for
biodiesel production. Biomass Bioenerg 66:159–167
667
Teichner W, Lesko M (2013) Cashing in on the booming market for
dietary supplements. Prod. McKinsey & Company. https://www.
mckinseyonmarketingandsales.com/sites/default/files/pdf/CSI_
VMHS_FNL_0.pdf Accessed 15 June 2016
TerraVia Inc (2016) Reimagine what’s possible. http://terravia.com/.
Accessed 24 June 2016
Thermo Fisher Scientific Inc (2009) Phycobiliproteins. Invitrogen.
https://tools.thermofisher.com/content/sfs/manuals/mp00800.
pdf. Accessed 14 Dec 2015
Thermo Fisher Scientific Inc (2015) R-phycoerythrin (R-PE). https://
www.thermofisher.com/us/en/home/life-science/cell-analysis/
fluorophores/r-phycoerythrin.html. Accessed 14 Dec 2015
Thilakaratne R, Wright MM, Brown RC (2014) A techno-economic
analysis of microalgae remnant catalytic pyrolysis and upgrading
to fuels. Fuel 128:104–112
Torzillo G, Vonshak A (2013) Environmental stress physiology with
reference to mass cultures. In: Hu Q, Richmond A (ed)
Handbook of microalgal culture: applied phycology and biotech-
nology. Blackwell Publishing Ltd., Somerset, pp 90–113
Ubando AT, Culaba AB, Cuello JL, El-Halwagi MM, Tan RR (2014)
Multi-regional multi-objective optimization of an algal biofuel
polygeneration supply chain with fuzzy mathematical program-
ming. In: ASME 2014 8th international conference on energy
sustainability collocated with the ASME 2014 12th international
conference on fuel cell science, engineering and technology
US Department of Energy (2015) Petroleum & other liquids: sales of
distillate fuel oil by end use. US Energy Information Adminis-
tration. Washington, DC: US Department of Energy, December
22. https://www.eia.gov/dnav/pet/pet_cons_821dst_dcu_nus_a.
htm. Accessed 31 Jan 2016
Vandamme D, Foubert I, Muylaert K (2013) Flocculation as a low-
cost method for harvesting microalgae for bulk biomass
production. Trends Biotechnol 31:233–239
Vanthoor-Koopmans M, Wijffels RH, Barbosa MJ, Eppink MH
(2013) Biorefinery of microalgae for food and fuel. Bioresource
Technol 135:142–149
Vitova M, Bisova K, Kawano S, Zachleder V (2015) Accumulation of
energy reserves in algae: from cell cycles to biotechnological
applications. Biotechnol Adv 33:1205–1218
Wan C, Zhao XQ, Guo SL, Alam MA, Bai FW (2013) Bioflocculant
production from Solibacillus silvestris W01 and its application in
cost-effective harvest of marine microalga Nannochloropsis
oceanica by flocculation. Bioresource Technol 135:207–212
Wang WC, Allen E, Campos AA, Cade RK, Cade Killens, Dean L,
Dvora M, Immer JG, Mixson S, Srirangan S, Sauer ML, Schreck
S (2013) ASI: dunaliella marine microalgae to drop-in replace-
ment liquid transportation fuel. Environ Prog Sustain Energy
32:916–925
Wang J, Yang H, Wang F (2014) Mixotrophic cultivation of
microalgae for biodiesel production: status and prospects. Appl
Biochem Biotechnol 172:3307–3329
Xiong W, Gao C, Yan D, Wu C, Wu Q (2010) Double CO2 fixation in
photosynthesis-fermentation model enhances algal lipid synthesis
for biodiesel production. Bioresource Technol 101:2287–2293
Yaakob Z, Ali E, Zainal A, Mohamad M, Takriff MS (2014) An
overview: biomolecules from microalgae for animal feed and
aquaculture. J Biol Res 21
Yang J, Xu M, Zhang X, Hu Q, Sommerfeld M, Chen Y (2011) Life-
cycle analysis on biodiesel production from microalgae: water
footprint and nutrients balance. Bioresource Technol
102:159–165
Yen HW, Hu IC, Chen CY, Ho SH, Lee DJ, Chang JS (2013)
Microalgae-based biorefinery—from biofuels to natural prod-
ucts. Bioresource Technol 135:166–174
Yoshihiko S, Maeda Y, Yabuuchi T, Muto M, Yoshino T, Tanaka T
(2015) Chloroplast-targeting protein expression in the
123
668
oleaginous diatom fistulifera solaris JPCC DA0580 toward
metabolic engineering. J Biosci Bioeng 19:28–34
Young G, Nippen F, Titterbrandt S, Cooney MJ (2011) Direct
transesterification of biomass using an ionic liquid co-solvent
system. Adv Biochem Eng Biot 2:261–266
123
S. Dickinson et al.
Zitelli GC, Biondi N, Rodolfi L, Tredict MR (2013) Photobioreactors
for mass production of microalgae. In: Hu Q, Richmond A (eds)
Handbook of microalgal cultures: applied phycology and
biotechnology. Blackwell Publishing Ltd., Somerset,
pp 225–266