Biofuel production from microalgae: a review

Abstract

The shortage of fossil fuels is actually a major economic issue in the context of increasing energy demand. Renewable energies are thus gaining in importance. For instance, microalgae-based fuels are viewed as an alternative. Microalgae are microscopic unicellular plants, which typically grow in marine and freshwater environments. They are fast growing, have high photosynthetic efficiency, and have relatively small land requirement and water consumption in comparison with conventional land crops biofuels. Nonetheless, selling biofuels is still limited by high cost. Here, we review biofuel production from microalgae, including cultivation, harvesting, drying, extraction and conversion of microalgal lipids. Cost issues may be solved by upstream and downstream measures: (1) upstream measures, in which highly productive strains are obtained by strain selection, genetic engineering and metabolic engineering, and (2) downstream measures, in which high biofuels yields are obtained by enhancing the cellular lipid content and by advanced conversion of microalgal biomass to biofuels. Maximum biomass and high biofuels production can be achieved by two-stage culture strategies, which is a win–win approach because it solves the conflicts between cell growth and biomass accumulation.

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Environmental Chemistry Letters
https://doi.org/10.1007/s10311-019-00939-0
REVIEW
Biofuel production frommicroalgae: areview
LichengPeng1 · DongdongFu1· HuaqiangChu2· ZezhengWang1· HuaiyuanQi1
Received: 21 August 2019 / Accepted: 9 October 2019
© Springer Nature Switzerland AG 2019
Abstract
The shortage of fossil fuels is actually a major economic issue in the context of increasing energy demand. Renewable
energies are thus gaining in importance. For instance, microalgae-based fuels are viewed as an alternative. Microalgae are
microscopic unicellular plants, which typically grow in marine and freshwater environments. They are fast growing, have
high photosynthetic efficiency, and haverelatively small land requirement and water consumption in comparison with con-
ventional land crops biofuels. Nonetheless, selling biofuels is still limited by high cost. Here, we review biofuel production
from microalgae, including cultivation, harvesting, drying, extraction and conversion of microalgal lipids. Cost issues may
be solved by upstream and downstream measures: (1) upstream measures, in which highly productive strains are obtained
by strain selection, genetic engineering and metabolic engineering, and (2) downstream measures, in which high biofuels
yields are obtained by enhancing the cellular lipid content and by advanced conversion of microalgal biomass to biofuels.
Maximum biomass and high biofuels production can be achieved by two-stage culture strategies, which is a win–win approach
because it solves the conflicts between cell growth and biomass accumulation.
Keywords Microalgae· Biofuel· Commercialization· Challenges· Upstream and downstream measures
Introduction
Fossil fuel has played an important role in the industrial
era; however, they are non-renewable and less environment-
friendly. The statistics from Energy Information Adminis-
tration (EIA) estimated that the reserves of worldwide fos-
sil fuels will be exhausted in less than 50years (Plant and
Floorspace 2010). By comparison, biofuel is renewable
and sustainable alternative energy, which is regarded as
the replacement of fossil fuels. It is predicted by mobility
model results for the 2°C scenario (2DS) that the biofuels’
market share will account for 37% of total transportation
fuel consumption by 2060, and this number indicates a great
space for the production and market requirement as com-
pared with the current share of 4% published in 2016 (Oh
etal. 2018). In general, biofuels can be classified as three
generations such as the first generation, second generation,
and the third generation, corresponding to the feedstocks
of food sources sugarcane, wheat, corn, soybean, potato, or
sugar beet (Bringezu etal. 2007), lignocellulosic biomass
and agricultural wastes (Eisentraut 2010), and algae (i.e.,
macroalgae and microalgae) (Demirbas 2011), respectively.
Among the three types of feedstocks, microalgae-
based biofuel is considered as the promising one and has
attracted more and more attention in the past years, owing
to the characteristics such as fast growing rate, high-effi-
ciency photosynthesis, and high lipid content for some
species (Peng etal. 2016a). Generally speaking, microal-
gae are capable of converting nutrients either in medium
or wastewater into biomass and high-value cellular con-
stituents (Bouabidi etal. 2018; Wang etal. 2010). For
example, lipid, protein, carbohydrate, pigments, antioxi-
dant biomolecules derived from microalgae can be applied
in other sections of CO2 mitigation, wastewater treatment,
cosmetics, dyes, pharmaceuticals, functional food, food
additives, feeds for animals and aquaculture, fertilizers,
and others (Peng etal. 2013; Ramasamy etal. 2015; Umm-
alyma etal. 2017; Yun etal. 2014). Under such circum-
stance, microalgae-based biofuels have received more and
* Licheng Peng
lcpeng@hainanu.edu.cn
* Huaqiang Chu
hqchust@163.com
1 College ofEcology andEnvironment, Hainan University,
Haikou570228, HainanProvince, China
2 School ofEnergy andEnvironment, Anhui University
ofTechnology, Ma’anshan243002, China

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more concerns; however, the commercialization remains
to be expanded because of barriers such as the shortage
of feedstock, the blending wall of conventional biofuels,
and the most fundamental cause like high production cost.
This is derived from various challenges in the processes
from the selection of strain, mass-cultivation, harvesting,
drying, extraction, conversion, and others (Ho etal. 2014;
Rastogi etal. 2018; Bwapwa etal. 2017; Seo etal. 2018).
Although some major challenges for the production
of microalgae-based biofuels were presented in previous
studies, most are limited to partial sections of the produc-
tion process. This paper presents a brief review on the pro-
gress and challenges of microalgae-based biofuels in the
development of upstream and downstream technologies.
The main objective of this study was to introduce the pro-
gress of technologies involved in upstream such as strain
selection, genetic engineering, metabolic engineering for
obtaining high concentration of microalgae, and the pro-
gress of downstream measures in large-scale cultivation,
process control, harvesting, dewatering, extraction, and
conversion to biofuels products.
Microalgal species forproducing biofuels
Autotrophic microalgae consume carbon dioxide and pro-
duce carbohydrates or hydrogen, protein, and lipids, which
can be further utilized as feedstock of biofuels, including
biodiesel, biogas, biohydrogen, bioethanol, butanol, bio-
oil, char, and even power (Cheng etal. 2015; Francavilla
etal. 2015; Im etal. 2015; Khandelwal etal. 2018; Kim
etal. 2017; Lee etal. 2015; Song etal. 2017; Yun etal.
2016; Zhao etal. 2014) (Fig.1). Some typical microalgal
species can reach high concentrations of targeted biofu-
els; for example, Chlorella protothecoides is considered
as a perfect feedstock of biodiesel since they can accumu-
late 55% of lipid when cultivated heterotrophically under
nitrogen limitation (Xu etal. 2006) (Table1). Microalgae-
based biodiesel is generally obtained through two steps of
Fig. 1 Applications of micro-
algae
Table 1 Typical microalgal species for biofuels production
Microalgal species Targeted biofuels Microalgal growth and/or biofuels
production
Cultivation/reaction conditions References
C. protothecoides Biodiesel 55% of lipid Heterotrophy; nitrogen limitation Xu etal. (2006)
S. obliques Biohydrogen 300μmol H2/(mgChl*h) Indirect process; light biophotoly-
sis; (Fe–Fe) enzymes
Appel and Schulz (1998)
Nannochloropsis salina Biogas 0.70L biogas/g Photobioreactor, large scale, 35°C Quinn etal. (2014)
Chlorococum sp. Bioethanol 38wt% Fermentation Singh and Gu (2010)
Spirulina sp. Biomethanol Gasification/anaerobic fermenta-
tion
Rodionova etal. (2017)
Microalgal consortium Biochar 45.0 ± 5.9% dw solid biochar (with
energy density 8–10MJ/kg)
Hydrothermal liquefaction Roberts etal. (2013)

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firstly extract lipids from microalgal cells, and followed
by transesterification of lipid fraction using alcohol in the
presence of catalysts (Rodionova etal. 2017). A rate up
to 300μmolH2/(mgChlh) was reported in Scenedesmus
obliques, and this enables S. obliques to be suitable for
producing biohydrogen (Appel and Schulz 1998). The
general routes include indirect process with firstly pro-
duce biomass through photosynthesis and then covert the
biomass (particularly carbohydrates) to biohydrogen via
fermentation and/or photofermentation (Benemann 1996,
2000), while the other approach is splitting water to hydro-
gen and oxygen via processes of direct or indirect water
biophotolysis (Rodionova etal. 2017).
As also shown in Table1, some microalgal species
are also reported to produce biogas with productivity of
0.70L biogas/gVS (0.43L CH4/gVS), and the results
were obtained in the cultivation of Nannochloropsis salina
in photobioreactor at large scale at a temperature of 35°C
(Quinn etal. 2014). Moreover, microalgal biomass can
be also fermented into biogas, which can be illustrated
by that 3.83g/L obtained from 10g/L of lipid-extracted
microalgae debris of Chlorococum sp. (corresponds to
38wt%) when the microalgal biomass as a substrate via
yeast fermentation (Singh and Gu, 2010). In practice, the
addition of hydrogen is demonstrated to modify the com-
bustion characteristics of natural gas (Ren etal. 2019).
Biomethanol can be produced by some species such as
Spirulina sp., through gasification or anaerobic fermenta-
tion (Rodionova etal. 2017). It was also found that micro-
algal consortium has capability to take wastewater effluent
as growth medium in open ponds, and the biomass can be
harvested as the feedstock of biochar, reaching 45.0 ± 5.9%
dw solid biochar (with energy density 8–10MJ/kg) via
hydrothermal liquefaction (Roberts etal. 2013).
Development ofupstream measures
In general, the upstream technologies applicable to microal-
gae mainly include three aspects (Table2). Firstly, it is better
to select some proper strains characterized with robustness,
fast growing rate and rich in lipid from wild environment.
Secondly, advanced genetic engineering can be adopted to
modify and obtain the strains with fast growth rate and high
lipid productivity. Thirdly, other measures such as metabolic
engineering can be used to enhance the accumulation of
lipid and other fuel products. Genetic and metabolic engi-
neering of microalgae could be used to, for example, elimi-
nate photosaturation and photoinhibition, which is expected
to significantly increase productivity of outdoor cultures and
greatly improve the economics of microalgae oil production.
However, it will require long-term research and funding, to
overcome current strictures against the release of genetically
modified organisms. Thus, for the foreseeable future it would
be prudent to limit projections to what can be achieved with
wild-type strains (Rodolfi etal. 2010).
Strain selection
As estimated, there are one to ten million microalgal species
on the earth and more than 40,000 species have been identi-
fied (Wang etal. 2010). Microalgae are featured as having
fast growing rate and high lipid content; however, not all of
them are regarded as the best lipid producers but depend on
particular strain (Mata etal. 2010). In this regard, the fun-
damental requirement of microalgae-based biofuel is select-
ing suitable strains with the best combination of microalgal
biomass productivity and lipid content in outdoor culture.
For example, it would be promising when the strains can
substantially accumulate lipids even to nutrient deficiency.
Table 2 Upstream and downstream measures to enhance microalgal growth and biomass production
Type Approach Representative techniques/criteria References
Upstream measures Strain selection Robust strain; fast growing rate, high lipid
or other fuels’ yield
Arroussi etal. (2017) and Seo etal. (2018)
Genetic engineering Trans-conjugation; transformation; elec-
troporation; microinjection
Qin etal. (2012) and Ghosh etal. (2016)
Metabolic engineering Degradation of nutrients; biosynthesis;
preventing lipid catabolism
Dunahay etal. (1996) and Trentacoste etal.
(2013)
Downstream measures Large-scale cultivation Appropriate strains Singh and Gu (2010)
Process control Optimal cultivation system and conditions Quinn etal. (2012) and Peng etal. (2016a)
Harvesting and dewatering Primary harvesting; secondary dewatering Mata etal. (2010), Uduman etal. (2010) and
Buckwalter etal. (2013)
Extraction and conversion Transesterification; fermentation; hydro-
treatment; pyrolysis
Skorupskaite etal. (2016), Asada etal.
(2012), Plant and Floorspace (2010),
Sharma and Singh (2017) and Lee and Lee
(2016)

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The strains should be robust enough to withstand shear stress
generated by mixing or the interference by wild strains or
other microorganisms. Moreover, they are flexible to adapt
to the changes in physicochemical parameters of the grow-
ing environment (Arroussi etal. 2017; Rodolfi etal. 2010;
Seo etal. 2018).
The strain of marine or freshwater microalgae can be
generally isolated using a micropipette for isolation under a
microscope, cell dilution, and cultivation in liquid medium
or agar plate (Arroussi etal. 2017). Winkler test screening
protocol entailed a new and fast method for mutant strain
selection and analysis of algal hydrogen metabolism with-
out applying nutritional stress, and this method was recom-
mended to isolate the hydrogen-producing Chlamydomonas
reinhardtii strains (Rühle etal. 2008). Four strains, in which
two marine and two freshwater strains were screened among
thirty microalgal strains, were found with high biomass pro-
ductivity and lipid content. For example, up to 60% lipid
content was achieved in eustigmatophyte Nannochlorop-
sis sp. F&M-M24 under nitrogen starvation (Rodolfi etal.
2010). Besides, some locally isolated strains are more likely
accustomed in highly variable environment of outdoor cul-
tures, though they may have difficulties in dominating year-
round in the fluctuated cultivation conditions (Rodolfi etal.
2010).
Genetic engineering
Genetic engineering is defined as the direct manipulation of
organism’s genes using biotechnology. It has been applied
in the production of microalgae to satisfy the growing needs
and increasing quality of human life (Peng etal. 2016a;
Rodolfi etal. 2010).
In the past, some researchers adopted a crucial systemic
technology to obtain high microalgal biomass concentra-
tion for sustainable industrial applications, and to modify
the metabolic pathway for producing more anticipated high-
value products (Qin etal. 2012). Generally speaking, there
are several methods for transformation in marine algae,
including trans-conjugation, natural transformation and
induced transformation, electroporation (or electropermea-
bilization), biolistic transformation, glass beads, silicon car-
bon whiskers method, microinjection, artificial transposon
method, recombinant eukaryotic algal viruses, and agrobac-
terium tumefaciens-mediated genetic transformation (Qin
etal. 2012).
Among the methods, gene transfer by electroporation
is characterized by simplicity of the procedure and high
efficiency with a small amount of DNA and is a common
method for various cells and bacteria for over 30 years
(Neumann etal. 1982; Zimmermann et al. 1975). In
detail, an electrical field (e.g., 1–1.5kV, 250–750V/cm)
can be imposed on cells to increase the permeability of
cell membrane, and then chemicals, drugs, or DNA can
be introduced into the cells (Neumann etal. 1982; Sugar
and Neumann 1984). The method is applicable to both
prokaryotic cells and eukaryotic algae including red algae,
green algae, and diatoms. For example, marine alga Nan-
nochloropsis sp., suitable for potential biofuel production,
has been successfully genetically transformed with several
knockout genes involved in the nitrogen metabolism (Kil-
ian etal. 2011). However, this method is constrained in
brown algae because of undeveloped protoplast prepara-
tion and immature regeneration technologies (Qin etal.
2012).
Direct gene transfer by biolistic transformation (i.e.,
micro-particle bombardment) has been demonstrated to be
the most efficient method for many diatom strains (Qin etal.
2012; Cheney 1992), and it has been widely employed in
the transformation of the nuclear and chloroplast expression
systems (Qin etal. 2012). The method is characterized by
some advantages; for instance, it is the only effective method
that can repeatedly transform chloroplasts, mitochondria and
other organelles. It can introduce exogenous DNA into broad
cells and tissues of plants, animals, microbes, pollen, and
other peculiar acceptors. Furthermore, diversified endog-
enous vectors can be also used in biolistic transformation
through a controllable and mature manipulation procedure
(Qin etal. 2012). However, this method is highly reproduc-
ible and works under specialized and high-cost equipment
(Qin etal. 2012). Particularly, DNA is generally coated with
gold particles to target within the cell via pressurized helium
gas (Apt etal. 1996; Dunahay etal. 2010; Ghosh etal. 2016).
To date, more than twenty marine microalgal strains have
been successfully transformed with the aforementioned
transformation methods. For marine cyanobacteria, the
genetic transformation was successfully demonstrated in 5
strains of Synechococcus (i.e., Synechococystis, Pseudana-
baena) using the method of trans-conjugation (Sode etal.
1992), or natural transformation (Jiang etal. 2003).
For brown algae, five types of acceptor cells such as
juvenile sporophytes, male and female gametophytes, tissue
pieces from sporophytes, and parthenogenetic sporophytes
can all be transformed by particle bombardment (Jiang etal.
2003; Qin etal. 1999). For diatoms, biolistic transformation
was successfully demonstrated in centric diatom Thalassio-
sira weissflogii (Falciatore etal. 1999), Thalassiosira pseu-
donana (Poulsen etal. 2010), Chaetoceros sp. (Miyagawa-
Yamaguchi etal. 2011), Cyclotella cryptica (Dunahay etal.
2010), and the pinnate diatoms Navicula saprophila (Duna-
hay etal. 2010), Cylindrotheca fusiformis (Fischer etal.
2010), and Phaeodactylum tricornutum (Miyagawa etal.
2010). For green algae, foreign DNA can be also introduced
in marine microalga Dunaliella salina and freshwater micro-
alga C. reinhardtii using the agitation of glass beads (Feng
etal. 2009; Kindle 1998).

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Metabolic engineering ofmicroalgal pathways
Metabolic engineering is the practice of optimizing genetic
processes (i.e., remove and add genes) and regulatory pro-
cesses on an organism, to alter its metabolic functions in
a predetermined manner such as increasing the organisms’
production of a certain substance (Shuler and Kargi 2001).
The control of metabolic pathways is generally completed
by nutritional and environmental regulation in bioprocess
engineering (Park etal. 2019). In general, metabolic reac-
tions can be classified into three major types, including the
degradation of nutrients, biosynthesis of small molecules
(e.g., amino acids, nucleotides), and biosynthesis of large
molecules (Dunahay etal. 1996).
It was reported that fatty acid of diatom C. cryptica can
be increased by the overexpression of the acetyl-CoA car-
boxylase gene (ACCase) under stressed condition (Dunahay
etal. 1996), because ACCase controls the biosynthesis of
fatty acid (Ghosh etal. 2016). The oil content of diatoms
and green algae was also increased by adding heterologous
plant fatty acid synthetase enzymes gene (Blatti etal. 2012;
Radakovits etal. 2010). An increase up to 82% total neutral
lipid was achieved by knocking down the pyruvate carboxy-
lase kinase expression of diatom P. tricornutum using an
antisense cDNA construct (Ma etal. 2014).
In addition to pyruvate, the overexpression of endogenous
malic enzyme can result in a 2.5-fold increased lipid accu-
mulation of P. tricornutum under nutrient-replete condi-
tions (Xue etal. 2014). Some transgenic strategies are also
applied to increase the lipid accumulation of green alga and
diatom under nitrogen-starved conditions (Hamilton etal.
2014; Wang etal. 2009; Yongmanitchai and Ward 1991).
Furthermore, lipid accumulation can be enhanced by pre-
venting lipid catabolism, for example, as compared with the
wild-type, 3.3-fold higher total lipid content of the diatomT.
pseudonana was achieved by transforming antisense con-
structs (Trentacoste etal. 2013).
Development ofdownstream measures
The downstream technologies applicable to microalgae
mainly include the following aspects, such as large-scale
cultivation of suitable microalgal strains, the design of effi-
cient cultivation system and modified cultivation modes to
deal with the conflict between biomass and lipid production,
energy-saving approaches for harvesting and dewatering,
and the efficient technologies of extraction and conversion.
Large‑scale cultivation
Large-scale cultivation is the fundamental of commercial-
izing microalgae-derived biofuels, due to sufficient biomass
makes biofuels production possible. In general, it is limited
by those factors as appropriate strain, farming, process con-
trol, and other measures.
Appropriate strain guaranteeing high biomass production
As aforementioned, it is the priority to select promising
microalgae species with high oil content and can quickly
grow in the culture, and this is one of the essential keys to
produce biocrude, biodiesel, and drop-in fuels and further
develop economically viable project (Singh and Gu 2010;
Arroussi etal. 2017). As reported in Algae 2020 (Will 2009),
five key strategies such as “fatter (i.e., algae species with
high oil content), faster (i.e., grow more quickly), cheaper
(i.e., capital and operating costs), easier (i.e., manipulation
at each sub-sets of systems) and fractionation marketing
approaches (e.g., biomass co-product marketing strategies)”
have been identified as the key factors of driving a successful
commercialization of microalgae-based biofuels.
The strains with high lipid contents are promising spe-
cies feedstock of biodiesel, and this would be the base of
successful industrial farming of microalgae (Sadvakasova
etal. 2019). Some studies screened a series of local strains
(57 strains) from Moroccan coasts and found that diatoms
are generally rich in triglycerides (TAG), while the lipid con-
tent of marine microalgae Tetraselmis sp. and Dunaliella sp.
reached up to 56% and 50% of dry cell weight, respectively
(Arroussi etal. 2017).
Scale‑up cultivation
Commercialization of microalgae-based biofuels is depend-
ent on the high biomass concentration and lipid contents
through large-scale cultivation of microalgae (Quinn etal.
2012). However, large-scale cultivation faces formidable
challenges and the major barrier is the high production cost
(Borowitzka 2013), owing to the intensive energy demands
required for the complex processes of algal cultivation such
as sterilization, mixing, aeration, illumination, gas exchange,
and others (Peng etal. 2015, 2016b). For example, the pro-
cesses such as the sterilization of large volume of cultures
and maintenance of sterility for the whole cultivation sys-
tem, efficient illumination, and deoxygenation approach, and
other steps for better process control are difficult and costly.
Thus, it is important to cut down the cost of microalgal culti-
vation that is potentially caused by the above processes, and
meanwhile to obtain high biomass concentration and lipid
productivity. The routes of offsetting overall costs mainly
include co-producing value-added products, optimization
of algal cultivation processes, and lowering cultivation cost
via the utilization of wastewater or flue gas as nutrients and
carbon source (Peng etal.2013, 2015).

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Under the persistent efforts on the improvement and
development of technologies in the past years, some suc-
cessful examples of large-scale microalgae farming have
been implemented in companies. For instance, Sapphire
Energy was found in 2007 and has built up some multi-year
cooperation or agreement on algae-based research projects
with other companies, aiming to co-develop algae-to-fuel
cultivation systems at commercial-scale in the past few years
(SapphireEnergy 2016). A commercial demonstration algae-
to-energy facility, the Green Crude Farm, was announced
by the company in 2012, and the farm consists of 100 acres
of two ponds (at sizes of 1.1 acres and 2.2 acres), and the
planned 300 acre facility for the mechanical and processing
equipment that are applied in the processes such as harvest-
ing, extraction of algae, and recycle of water (SapphireEn-
ergy 2016).
Algenol is a notable world-class team assorted with state-
of-the-art facility and proprietary algae growing systems
(Algenol 2018). The company adopts photobioreactors with
production yields 2–3 times that of open ponds, and VIPER
manufacturing (i.e., Algenol’s proprietary photobioreac-
tors) with 40,000 square-foot facility. In addition to pro-
duce some high-value products including natural colorants,
protein, Spirulina, personal care ingredients, biofertilizers,
and biostimulants, Algenol also dedicates to biofuels such as
bioethanol and green crude oil with proprietary vapor com-
pression steam stripping unit (VCSS) for the purification of
the ethanol and hydrothermal liquefaction (HTL) technology
to make crude oil, respectively.
Process control duringthecultivation ofmicroalgae
Suitable cultivation system formicroalgal growth
Microalgae are capable of growing in natural habitats of
oceans, rivers, lakes, and ponds or artificial systems of open
ponds and photobioreactors (Wang etal. 2010). Open ponds
can be natural waters (e.g., lakes, shallow lagoons, ponds)
or artificial ponds such as circular ponds, raceway ponds,
shallow big ponds and tanks, and container-based systems
(e.g., hanging plastic bags) (Pulz 2001; Singh and Gu 2010;
Tredici and Materassi 1992). In general, open ponds are
characterized by some advantages of low cost, simplicity,
and easier to operate. However, they also have some major
limitations; for example, they require large areas of land,
more water evaporation, low biomass productivity, low uti-
lization of CO2 and light, poor mixing, and easier to be con-
taminated (Peng etal. 2013, 2015).
By comparison, closed systems like photobioreactors can
be sorted as vertical column photobioreactors, flat panel
photobioreactor, tubular photobioreactor, internally illumi-
nated photobioreactors, spectral shifting, membrane photo-
bioreactors, and plastic bag photobioreactors (Wang etal.
2012). They are capable of offering good control over key
operational parameters of microalgal cultivation, including
temperature, length of light path, pH, species control, and
others (Peng etal. 2016b). Thus, photobioreactors provide
a higher possibility of achieving much higher growth rate,
microalgal cell density, and volumetric biomass productiv-
ity compared to open ponds (Wang etal. 2012). The high
capital and operational costs result from complex configu-
ration, illumination, cooling, mixing, deoxygenation, and
other operational requirements. These limit the application
of photobioreactors at large-scale microalgal farming (Peng
etal. 2013, 2016b).
In the present scenario, it is necessary to design a suitable
cultivation system for microalgal growth in particular for
large-scale microalgal farming. Based on the advantages and
disadvantages of open ponds and photobioreactors, some
companies prefer to adopt photobioreactors than open ponds
systems or natural formations, but it is an attractive option
of cultivating microalgae in open ponds in the regions where
has sufficient lands and has no competition with arable lands
(Singh and Gu 2010). It was reported that most microalgae
systems today can produce a range of 2500–5000 gallon of
oil per surface acre in raceway ponds with 30% oil content
(Singh and Gu 2010).
Cultivation modes tosolve conicts betweenbiomass
andlipid production
Lipid synthesis is usually induced by environmental stresses
such as salinity, temperature, nutrients, and pH (Peng etal.
2015). Among these factors, nitrogen limitation is regarded
as the most effective approach to enhance lipid accumulation
of microalgae, whereas at the expense of cell growth (Park
etal. 2019). Some studies have demonstrated the feasibil-
ity of enhancing strains from low or medium initial lipid
content to super high level; for example, the lipid content of
Dunaliella tertiolecta was dramatically improved from the
initial level of 21 to up to 70% under saline stress (Arroussi
etal. 2015).
To solve the conflict between cell growth and biomass
accumulation, some efforts are made to select genetically
strains or screen local strains with fast growth rate and
high lipid content (Arroussi etal. 2017; Sadvakasova etal.
2019). High biomass concentration of microalgal cultures
can be achieved through simplified cultivation, including
the optimization of medium composition (Liu etal. 2007),
process optimization and control on light utilization, oxygen
accumulation mitigation, and contamination prevention (Liu
etal. 2007; Pulz 2001; Li etal. 2008), and improvement of
cultivation systems (Wang etal. 2012). Currently, two-stage
process has been demonstrated to be a win–win strategy for
obtaining both high cell density and biomass concentration
(i.e., carbohydrates, lipid or hydrogen) at lower operative

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and investment costs (Ra etal. 2015; Nagappan etal. 2019).
In the process, microalgal cells are cultivated in photoau-
totrophic conditions in the first stage, and then transfer the
biomass to a heterotrophic reactor where cells use organic
carbon to synthesize starch and lipids (Caprio etal. 2016). A
hetero-photoautotrophic microalgal growth model was also
studied for improved organic-rich wastewater treatment and
microalgal lipid yields, and therefore offering a sustainable
way to produce microalgae-based bioenergy and byproducts
(Zhou etal. 2012).
Energy‑saving approaches forharvesting
anddewatering
Harvesting and dewatering of microalgal biomass are
regarded as a major bottleneck to microalgae-based bio-
fuels due to their energy-intensive feature, and the pro-
cesses may account for 20–30% of the total production
costs (Mata etal. 2010; Uduman etal. 2010). Thus, finding
energy-saving approaches is very important to offset the
production cost of microalgae-based biofuels. In general,
microalgae can be harvested by two steps of bulk harvest-
ing and followed by thickening (or dewatering) (Chen etal.
2011). As shown in Table3, technologies of primary har-
vesting generally consist of coagulation and flocculation,
flotation, filtration, screening, ion exchange, gravity sedi-
mentation, precipitation, centrifugation, and other tech-
niques (Cheng etal. 2011; Heasman etal. 2000; Hwang
etal. 2013; Laamanen etal. 2016; Singh and Patidar 2018;
Uduman etal. 2010; Buckwalter etal. 2013). The sec-
ondary dewatering methods include filtration, drying, and
others such as microwave and fluid bed (Buckwalter etal.
2013; Laamanen etal. 2016; Shelef etal. 1984). Advanced
approaches such as a combined method of pulsed electric
field (PEF) and hydrothermal liquefaction (HTL) has been
proposed as a promising and suitable pretreatment for wet
extraction of microalgal residual biomass. It was reported
that PEF could accelerate the formation and extraction
efficiency of amino acids up to 150% in 60min, and this
promised a higher biocrude yield by 6wt% (Vlaskin etal.
2018). Besides, other methods are also applied in specially
cell wall disruption using high energy ultrasonic or micro-
wave-assisted extraction (Ranjan etal. 2010; Balasubra-
manian etal. 2011), or using supercritical fluid extraction
at high temperature and pressure (Crampon etal. 2013),
ionic liquids with very low melting point usually below
100°C (Kim etal. 2012).
Selection of harvesting technology is directly relevant
of the efficiency and cost, while the above technologies
have their own advantages and disadvantages. For exam-
ple, coagulation and flocculation are feasible for small size
of microalgae (e.g., < 5μm) and enable them to be larger
sizes flocks (1–5mm) (Park etal. 2011). Other technolo-
gies such as centrifugation have drawbacks such as being
energy intensive, having high capital and operating costs,
and resulting in shear stress to algal cells (Grima etal.
2003; Harun etal. 2010), while filtration is expensive
and easily to be fouling (Vonshak and Richmond 1988).
As reported, the combination of flocculation with flota-
tion was demonstrated to be capable of achieving a high
rate of solid–liquid separation (Rubio etal. 2007). In the
commercialization utilization, Global Algae Innovations
developed “ZOBI Harvester” which is renowned for an
automated membrane filtration system with 100% harvest
efficiency and no need for secondary dewatering in the
combined processes of harvest and dewatering (Equipment
2018). This system has been commercialized at 20,000L/h
with an energy use of 0.04kWh/m3, and it is capable of
reaching a 30 times reduction in harvest and dewatering
energy and 4 times reduction in water flow (EnergyGovOf-
fices 2016). The automated harvesting system is scalable
(with the size range of 5–200,000 gpm) and easy to oper-
ate. Particularly, it harvests microalgae at ambient pressure
without exposure to high shear stress or centrifugal forces
(Equipment 2018).
Table 3 Approaches for harvesting and dewatering
Types Approaches Examples References
Primary harvesting Sedimentation Gravity/centrifugal sedimentation Uduman etal. (2010)
Flocculation Chemical/biochemical/electro-flocculation Mubarak etal. (2019)
Centrifugation Decanter; centrifuge; hydrocyclones Knuckey etal. (2006)
Flotation Electro-/dissolved air/suspended air flotation Pragya etal. (2013) and Chen etal. (2011)
Others Screening; ion exchange; ultrasonic separa-
tion; electrophoresis techniques; magnetic
separation
Cerff etal. (2012), Cheng etal. (2011), Heasman etal.
(2000), Hwang etal. (2013), Laamanen etal. (2016),
Singh and Patidar (2018) and Uduman etal. (2010)
Drying Steam/spray/drum/freeze/oven/sun drying Shelef etal. (1984)
Secondary dewatering Filtration Forward osmosis; Belt/micro-/ultra-/rotary/
pressure/cross-flow/vacuum drum filtration
Buckwalter etal. (2013)
Others Microwave; fluid bed Buckwalter etal. (2013) and Shelef etal. (1984)

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Ecient technologies ofextraction andconversion
Improving theextraction eciency ofmicroalgal biomass
The dried microalgae biomass will be further used to extract
microalgal lipids through mechanical or non-mechanical
methods, such as cell homogenizers, autoclave, ultrasounds,
bead mills, and spray drying, and freezing, the utilization
of polar and non-polar solvents, osmotic shock, acid, base,
and enzymatic reactions, and supercritical carbon dioxide
(SCCO2) (Halim etal. 2011; Mata etal. 2010). However,
most of the extraction methods work at the laboratory scale
but to be challenging at large-scale extraction because of
the volume and complex (Lam and Lee 2012). For instance,
some previous studies (Jesus etal. 2019) found that large
quantities of solvent consumption are the largest expense
when extracting lipid from wet microalgae using green
solvents such as 2-methyltetrahydrofuran and cyclopentyl
methyl ether. They also compared various methods of lipid
extraction and demonstrated that the Hara and Radin method
was the most effective for extracting 1kg of fatty acids from
Chlorella pyrenoidosa (at 65.71% moisture) using hexane/
isopropanol (3:2v/v). However, the green solvents prices
are not competitive as compared with fossil-based solvents.
Moreover, a solvent-free osmotic shock pretreatment method
was used to extract lipid and subsequently produce meth-
ane from microalgae D. salina and Chaetoceros muelleri,
a lipid recovery efficiency of 21% and 72% was obtained,
respectively (González-González etal. 2019). Furthermore,
some recent methods including microwave-assisted extrac-
tion, supercritical fluid extraction, use of ionic liquids, and
switchable hydrophilicity solvents are also recommended for
economical lipid extraction (Deshmukh etal. 2019).
Enhancing theconversion ofmicroalgae tobiofuels
Microalgal biomass can be converted to renewable fuels
(e.g., power, heat, and fuels) and energy source through vari-
ous technologies including (1) transesterification (Skorup-
skaite etal. 2016), (2) thermochemical such as combustion,
pyrolysis, gasification, thermochemical liquefaction, and
(3) biochemical/biological conversion in terms of anaero-
bic digestion, fermentation, and photobiological hydrogen
production (Asada etal. 2012; Plant and Floorspace 2010;
Sharma and Singh 2017) (Fig.2).
Direct combustion is used to convert microalgal biomass
into hot gasses for energy production, which can power a
turbine and turn a generator to produce electricity, under
the condition of oxygen, furnace, boiler, or steam turbine at
around 1000°C (Lee and Lee 2016; Suganya etal. 2016).
For example, biomass for power (e.g., electricity) and heat
can be achieved by combustion direct-firing in a boiler,
where high-pressure steam is produced and introduced into
a steam turbine, and flows over a series of turbine blades
to make the turbine and electric generator rotate and there-
fore the electricity is produced (ClimateTechWiki 2006). A
Fig. 2 Conversion technologies of microalgal biomass to renewable
fuel. Note: The black solid frames indicate the major technologies
and techniques applied for the conversion of microalgal biomass to
the renewable fuels, respectively. The green dotted frames are the
targeted microalgal-based fuels or end products. The number repre-
sents the procedures or conditions in which, (1) direct combustion:
energy can be obtained under conditions of oxygen, furnace, boiler/
steam turbine, at 1000°C; (2) transesterification: direct/conventional
transesterification; (3) fermentation: dewatering → milling → liq-
uefaction → sacchar ification → fermentation → distillation → H2,
CH4; and the other route (3)′ anaerobic/dark environment → ethanol,
CO2 → pur ification → bioethanol; (4) hydrotreatment/gasification:
dehydration → pyrolysis → combustion → gasification → water–gas
shift reaction → jet fuel; (5) pyrolysis: conventional/fast/flash pyroly-
sis → bio-oil, char, syngas; (6) anaerobic digestion: hydrolysis → fer -
mentation → acetogenesis → methanogenesis → biogas CH4, CO2: and
the other route is (6)′ digested at pH 6–9, and methane is produced for
generating electricity

Environmental Chemistry Letters
1 3
limited amount of oxygen or air is required for the burning
of organic material to produce carbon dioxide and energy,
which drives a second reaction that is the conversion of fur-
ther organic material to hydrogen (H2) and additional CO2.
Some CH4 and CO2 would be produced when the third reac-
tion occurs in the way of carbon monoxide (CO) and residual
water (Rao etal. 2017).
Methyl esters (FAME) can be produced by direct (single-
stage) and conventional (two-stage) transesterification, and
the chemical equation is that triglyceride and methanol react
under the role of catalyst, to produce glycerol and methylest-
ers (Adeniyi etal. 2018; Lee and Lee 2016). By comparison,
hydrotreatment or gasification process of microalgal oil into
jet fuel is completed by hydrotreating fatty acid and esters
(Kuepker 2015) with the steps of dehydration, pyrolysis,
combustion, gasification, and/or water–gas shift reaction
(Rao etal. 2017). Pyrolysis is the thermal decomposition of
biomass in the absence of oxygen, and to produce liquid fuel
(bio-oil), solid fuel (biochar), and gaseous fuel products (H2,
CH4), the process can be classified as conventional pyrolysis,
fast pyrolysis and flash pyrolysis (Lee and Lee 2016; Sira-
junnisa and Surendhiran 2016).
Anaerobic digestion is a process of obtaining methane
from the delipidized algal biomass with carbon and nitrogen
content via the consecutive stages of hydrolysis, fermenta-
tion, acetogenesis, and methanogenesis (Lee and Lee 2016;
Sirajunnisa and Surendhiran 2016). This method converts
organic biomass into biogas (~ 60% CH4 and ~ 40% CO2),
and small-scale biogas digesters have been applied through-
out many developing countries such as China, India, Nepal,
Thailand, South Korea, and Brazil (ESMAP 2005). Fer-
mentation aims to convert the cellulose sugar or starch of
microalgal biomass into bioethanol, with consecutive stages
of dewatering, milling, liquefaction, saccharification, fer-
mentation, distillation and eventually the fuel products of
bioethanol is obtained (Lee and Lee 2016).
Conclusion
With the rapid process of economic development and energy
consumption, as well as the crisis of limited fossil fuel
resource, and the increasing requirement of environmental
protection, more and more concerns have been paid on the
development of environmentally friendly fuels such as bio-
fuels to solve the conflict. Microalgae-based biofuels have
been regarded as one of the promising feedstocks for the
new generation of biofuels. However, its commercialization
faces the biggest challenge of high production cost, which
results from the high capital and operational costs in terms
of complex configuration, illumination, cooling, mixing,
deoxygenation, and other operational requirements. Many
efforts have paid on exploring enormous advances in the
development of upstream and downstream technologies, to
offset the cost of obtaining high biomass concentration and
high content of anticipated fuels. Some appropriate microal-
gal strains have been screened or modified to guarantee high
microalgal biomass production, and win–win strategies such
as two-stage of cultivation have been demonstrated the pos-
sibility of obtaining both high biomass production and lipid
content or other fuels. Furthermore, advanced technologies
applied in harvesting and biomass-to-fuels conversion make
microalgae-based fuels more promising. However, some
significant challenges remain in the scale-up of microalgal
farming systems, and the constraints should be tackled in
the future.
Acknowledgements The authors are grateful for the financial sup-
ports provided by Natural Science Foundation of Hainan Province,
China (Grant No. 518QN212), National Natural Science Foundation
of China (41766003), and the Youth Fund Project of Hainan University
(hdkyxj201706).
References
Adeniyi OM, Azimov U, Burluka A (2018) Algae biofuel: current sta-
tus and future applications. Renew Sust Energ Rev 90:316–335.
https ://doi.org/10.1016/j.rser.2018.03.067
Algenol (2018) Sustainable products. https ://www.algen ol.com/susta
inabl e-produ cts/
Appel J, Schulz R (1998) Hydrogen metabolism in organisms with
oxygenic photosynthesis: hydrogenases as important regulatory
devices for a proper redox poising? J Photochem Photobiol B
47(1):1–11. https ://doi.org/10.1016/S1011 -1344(98)00179 -1
Apt KE, Kroth-Pancic PG, Grossman AR (1996) Stable nuclear trans-
formation of the diatom Phaeodactylum tricornutum. Mol Gene
Genet Mgg 252(5):572–579. https ://doi.org/10.1007/BF021
72403
Arroussi HE, Benhima R, Bennis I, Mernissi NE, Wahby I (2015)
Improvement of the potential of Dunaliella tertiolecta as a source
of biodiesel by auxin treatment coupled to salt stress. Renew
Energy 77:15–19. https ://doi.org/10.1016/j.renen e.2014.12.010
Arroussi HE, Benhima R, Mernissi NE, Bouhfid R, Tilsaghani C, Ben-
nis I, Wahby I (2017) Screening of marine microalgae strains
from Moroccan coasts for biodiesel production. Renew Energ
113:1515–1522. https ://doi.org/10.1016/j.renen e.2017.07.035
Asada C, Doi K, Sasaki C, Nakamura Y (2012) Efficient extraction
of starch from microalgae using ultrasonic homogenizer and its
conversion into ethanol by simultaneous saccharification and
fermentation. Nat Res 03(04):175–179. https ://doi.org/10.4236/
nr.2012.34023
Balasubramanian S, Allen JD, Kanitkar A, Boldor D (2011) Oil extrac-
tion from Scenedesmus obliquus using a continuous microwave
system-design, optimization, and quality characterization. Biore-
sour Technol 102:3396–3403. https ://doi.org/10.1016/j.biort
ech.2010.09.119
Benemann J (1996) Hydrogen biotechnology: progress and prospects.
Nat Biotechnol 14(9):1101–1103. https ://doi.org/10.1038/nbt09
96-1101
Benemann JR (2000) Hydrogen production by microalgae. J Appl Phy-
col 12(3):291–300. https ://doi.org/10.1023/A:10081 75112 704
Blatti JL, Beld J, Behnke C, Mendez M, Mayfield SP, Burkart MD
(2012) Manipulating fatty acid biosynthesis in microalgae

Environmental Chemistry Letters
1 3
for biofuel through protein–protein interactions. PLoS ONE
7(9):e42949. https ://doi.org/10.1371/journ al.pone.00429 49
Borowitzka MA (2013) High-value products from microalgae-their
development and commercialization. J Appl Phycol 25(3):743–
756. https ://doi.org/10.1007/s1081 1-013-9983-9
Bouabidi ZB, EI-Naas M, Zhang Z (2018) Immobilization of micro-
bial cells for the biotreatment of wastewater: a review. Environ
Chem Lett. https ://doi.org/10.1007/s1031 1-018-0795-7
Bringezu S, Ramesohl S, Arnold K, Fischedick M, Von Geibler J
(2007) Towards a sustainable biomass strategy: what we know
and what we should know. Wuppertal papers. https ://www.
resea rchga te.net/publi catio n/23752 2564_Towar ds_a_susta
inabl e_bioma ss_strat egy
Buckwalter P, Embaye T, Gormly S, Trent JD (2013) Dewatering
microalgae by forward osmosis. Desalination 312:19–22. https
://doi.org/10.1016/j.desal .2012.12.015
Bwapwa JK, Anandraj A, Trois C (2017) Possibilities for conver-
sion of microalgae oil into aviation fuel: a review. Renew
Sust Energ Rev 80:1345–1354. https ://doi.org/10.1016/j.
rser.2017.05.224
Caprio FD, Visca A, Altimari P, Toro L, Masciocchi B, Iaquaniello G,
Pagnaelli F (2016) Two stage process of microalgae cultivation
for starch and carotenoid production. Chem Eng Tran 49:415–
420. https ://doi.org/10.3303/CET16 49070
Cerff M, Morweiser M, Dillschneider R, Michel A, Menzel K, Posten
C (2012) Harvesting fresh water and marine algae by magnetic
separation: screening of separation parameters and high gradient
magnetic filtration. Bioresour Technol 118:289–295. https ://doi.
org/10.1016/j.biort ech.2012.05.020
Chen CY, Yeh KL, Aisyah R, Lee DJ, Chang JS (2011) Cultivation,
photobioreactor design and harvesting of microalgae for biodiesel
production: a critical review. Bioresour Technol 102(1):71–81.
https ://doi.org/10.1016/j.biort ech.2010.06.159
Cheney DPK (1992) Progress in protoplast fusion and gene transfer
in red algae. In: Proceedings of the XIV international seaweed
symposium, Brittany, p 68
Cheng YL, Juang YC, Liao GY, Tsai PW, Ho SH, Yeh KL, Chen CY,
Chang JS, Liu JC, Chen WM, Lee DJ (2011) Harvesting of
Scenedesmus obliquus FSP-3 using dispersed ozone flotation.
Bioresour Technol 102:82–87. https ://doi.org/10.1016/j.biort
ech.2010.04.083
Cheng HH, Whang LM, Chan KC, Chung MC, Wu SH, Liu CP, Tien
SY, Chen SY, Lee WJ (2015) Biological butanol production from
microalgae-based biodiesel residues by Clostridium acetobutyli-
cum. Bioresour Technol 184:379–385. https ://doi.org/10.1016/j.
biort ech.2014.11.017
ClimateTechWiki (2006) Biomass combustion and co-firing for elec-
tricty and heat. http://www.clima tetec hwiki .org/techn ology /
bioma ss
Crampon C, Mouahid A, Toudji SAA, Lépine O, Badens E (2013)
Influence of pretreatment on supercritical CO2 extraction from
Nannochloropsis oculata. J Supercrit Fluids 79:337–344. https
://doi.org/10.1016/j.supfl u.2012.12.022
Demirbas MF (2011) Biofuels from algae for sustainable development.
Appl Energ 88(10):3473–3480. https ://doi.org/10.1016/j.apene
rgy.2011.01.059
Deshmukh S, Kumar R, Bala K (2019) Microalgae biodiesel: a review
on oil extraction, fatty acid composition, properties and effect
on engine performance and emissions. Fuel Process Technol
191:232–247. https ://doi.org/10.1016/j.fupro c.2019.03.013
Dunahay TG, Jarvis EE, Dais SS, Roessler PG (1996) Manipulation
of microalgal lipid production using genetic engineering. Appl
Biochem Biotechnol 57–58(1):223–231. https ://doi.org/10.1007/
bf029 41703
Dunahay TG, Jarvis EE, Roessler PG (2010) Genetic transfor-
mation of the diatoms Cyclotella cryptica and Navicula
saprophila. J Phycol 31(6):1004–1012. https ://doi.org/10.111
1/j.0022-3646.1995.01004 .x
Eisentraut A (2010) Sustainable production of second-generation bio-
fuels, potential and perspectives in major economies and devel-
oping countries. International Energy Agency. https ://www.ouren
ergyp olicy .or g/resou r ces/susta inabl e-produ ction -of-secon d-gener
ation -biofu els-2/
EnergyGovOffices (2016) National algal biofuels technology review.
https ://www.energ y.gov/eere/bioen ergy/downl oads/2016-natio
nal-algal -biofu els-techn ology -revie w
Equipment (2018) Zobi harvester. Global Algae Innovations. http://
www.globa lgae.com/equip ment/
ESMAP (2005) Biomass conversion technologies. http://www.globa
lprob lems-globa lsolu tions files .org/gpgs_files /pdf/UNF_Bioen
ergy/UNF_Bioen ergy_5.pdf
Falciatore A, Casotti R, Leblanc C, Abrescia C, Bowler C (1999)
Transformation of nonselectable reporter genes in marine dia-
toms. Mar Biotechnol 1(3):239–251. https ://doi.org/10.1007/
pl000 11773
Feng S, Xue L, Liu H, Lu P (2009) Improvement of efficiency of
genetic transformation for Dunaliella salina by glass beads
method. Mol Biol Rep 36(6):1433–1439. https ://doi.org/10.1007/
s1103 3-008-9333-1
Fischer H, Robl I, Sumper M, Kröger N (2010) Targeting and cova-
lent modification of cell wall and membrane proteins heterolo-
gously expressed in the diatom Cylindrotheca fusiformis (Bacil-
lariophyceae). J Phycol 35(1):113–120. https ://doi.org/10.104
6/j.1529-8817.1999.35101 13.x
Francavilla M, Kamaterou P, Intini S, Monteleone M, Zabaniotou
A (2015) Cascading microalgae biorefinery: fast pyrolysis of
Dunaliella tertiolecta lipid extracted-residue. Algal Res 11:184–
193. https ://doi.org/10.1016/j.algal .2015.06.017
Ghosh A, Khanra S, Mondal M, Halder G, Tiwari ON, Saini S, Bhow-
mick TK, Gayen K (2016) Progress toward isolation of strains
and genetically engineered strains of microalgae for produc-
tion of biofuel and other value added chemicals: a review. Ene
Conver Manag 113:104–118. https ://doi.org/10.1016/j.encon
man.2016.01.050
González-González LM, Astal S, Pratt S, Jensen PD, Schenk PM
(2019) Impact of osmotic shock pre-treatment on microalgae
lipid extraction and subsequent methane production. Biore-
sour Technol Rep 7:100214. https ://doi.org/10.1016/j.biteb
.2019.10021 4
Grima EM, Belarbi EH, Fernández FGA, Medina AR, Chisti Y (2003)
Recovery of microalgal biomass and metabolites: process options
and economics. Biotechnol Adv 20(7–8):491–515. https ://doi.
org/10.1016/S0734 -9750(02)00050 -2
Halim R, Gladman B, Danquah MK, Webley PA (2011) Oil extraction
from microalgae for biodiesel production. Bioresour Technol
102(1):178–185. https ://doi.org/10.1016/j.biort ech.2010.06.136
Hamilton ML, Haslam RP, Napier JA, Sayanova O (2014) Metabolic
engineering of Phaeodactylum tricornutum for the enhanced
accumulation of omega-3 long chain polyunsaturated fatty acids.
Metab Eng 22:3–9. https ://doi.org/10.1016/j.ymben .2013.12.003
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(3):1037–1047. https ://doi.
org/10.1016/j.rser.2009.11.004
Heasman M, Diemar J, O’Connor W, Sushames T, Foulkes L, Nell JA
(2000) Development of extended shelf-life microalgae concen-
trate diets harvested by centrifugation for bivalve molluscs—a
summary. Aquac Res 31(8–9):637–659. https ://doi.org/10.104
6/j.1365-2109.2000.31849 2.x
Ho DP, Ngo HH, Guo W (2014) A mini review on renewable sources
for biofuel. Bioresour Technol 169:742–749. https ://doi.
org/10.1016/j.biort ech.2014.07.022

Environmental Chemistry Letters
1 3
Hwang T, Park SJ, Oh YK, Rashid N, Han JI (2013) Harvesting of
Chlorella sp. KR-1 using a cross-flow membrane filtration sys-
tem equipped with an anti-fouling membrane. Bioresour Technol
139:379–382. https ://doi.org/10.1016/j.biort ech.2013.03.149
Im H, Kim B, Lee JW (2015) Concurrent production of biodiesel and
chemicals through wet insitu transesterification of microalgae.
Bioresour Technol 193:386–392. https ://doi.org/10.1016/j.biort
ech.2015.06.122
Jesus SS, Ferreira GF, Moreira LS, Maciel MRW, Filho RM (2019)
Comparison of several methods for effective lipid extraction from
wet microalgae using green solvents. Renew Energy 143:130–
141. https ://doi.org/10.1016/j.renen e.2019.04.168
Jiang P, Qin S, Tseng CK (2003) Expression of the lacZ reporter gene
in sporophytes of the seaweed Laminaria japonica (Phaeophy-
ceae) by gametophyte-targeted transformation. Plant Cell Rep
21(12):1211–1216. https ://doi.org/10.1007/s0029 9-003-0645-2
Khandelwal A, Vijay A, Dixit A, Chhabra M (2018) Microbial fuel cell
powered by lipid extracted algae: a promising system for algal
lipids and power generation. Bioresour Technol 247:520–527.
https ://doi.org/10.1016/j.biort ech.2017.09.119
Kilian O, Benemann CSE, Niyogi KK, Vick B (2011) High-efficiency
homologous recombination in the oil-producing alga Nannochlo-
ropsis sp. Proc Natl Acad Sci 108(52):21265–21269. https ://doi.
org/10.1073/pnas.11058 61108
Kim YH, Choi YK, Park J, Lee S, Yang YH, Kim HJ, Kim YH, Lee SH
(2012) Ionic liquid-mediated extraction of lipids from algal bio-
mass. Bioresour Technol 109:312–315. https ://doi.org/10.1016/j.
biort ech.2011.04.064
Kim TH, Oh YK, Lee JW, Chang YK (2017) Levulinate production
from algal cell hydrolysis using insitu transesterification. Algal
Res 26:431–435. https ://doi.org/10.1016/j.algal .2017.06.024
Kindle KL (1998) High-frequency nuclear transformation of Chla-
mydomonas reinhardtii. Methods Enzymol 297:27–38. https ://
doi.org/10.1073/pnas.87.3.1228
Knuckey RM, Brown MR, Robert DR, Frampton DMF (2006) Produc-
tion of microalgal concentrates by flocculation and their assess-
ment as aquaculture feeds. Aquac Eng 35(3):300–313. https ://
doi.org/10.1016/j.aquae ng.2006.04.001
Kuepker BCE (2015) European renewable energy policy. European
Commission, Burussels, pp 1–10. https ://www.resea rchga te.net/
publi catio n/26420 5107_Europ ean_renew able_energ y_polic y
Laamanen CA, Ross GM, Scott JA (2016) Flotation harvesting of
microalgae. Renew Sust Energ Rev 58:75–86. https ://doi.
org/10.1016/j.rser.2015.12.293
Lam MK, Lee KT (2012) Microalgae biofuels: a critical review
of issues, problems and the way forward. Biotechnol Adv
30(3):673–690. https ://doi.org/10.1016/j.biote chadv .2011.11.008
Lee OK, Lee EY (2016) Sustainable production of bioethanol from
renewable brown algae biomass. Biomass Bioenerg 92:70–75.
https ://doi.org/10.1016/j.biomb ioe.2016.03.038
Lee OK, Oh YK, Lee EY (2015) Bioethanol production from carbo-
hydrate-enriched residual biomass obtained after lipid extraction
of Chlorella sp. KR-1. Bioresour Technol 196:22–27. https ://doi.
org/10.1016/j.biort ech.2015.07.040
Li Y, Horsman M, Wang B, Wu N, Lan CQ (2008) Effects of nitrogen
sources on cell growth and lipid accumulation of green alga Neo-
chloris oleoabundans. Appl Microbiol Biotechnol 81(4):629–
636. https ://doi.org/10.1007/s0025 3-008-1681-1
Liu W, Au DWT, Anderson DM, Lam PKS, Wu RSS (2007) Effects
of nutrients, salinity, pH and light: dark cycle on the production
of reactive oxygen species in the alga Chattonella marina. J Exp
Mar Biol Ecol 346(1–2):76–86. https ://doi.org/10.1016/j.jembe
.2007.03.007
Ma Y, Wang X, Niu Y, Yang Z, Zhang M, Wang Z, Yang W, Liu J,
Li H (2014) Antisense knockdown of pyruvate dehydrogenase
kinase promotes the neutral lipid accumulation in the diatom
Phaeodactylum tricornutum. Microb Cell Fact 13(1):100. https
://doi.org/10.1186/s1293 4-014-0100-9
Mata TM, Martins AA, Caetano NS (2010) Microalgae for biodiesel
production and other applications: a review. Renew Sust Energ
Rev 14(1):217–232. https ://doi.org/10.1016/j.rser.2009.07.020
Miyagawa A, Okami T, Kira N, Yamaguchi H, Ohnishi K, Adachi
M (2010) Research note: high efficiency transformation of the
diatom Phaeodactylum tricornutum with a promoter from the
diatom Cylindrotheca fusiformis. Phycol Res 57(2):142–146.
https ://doi.org/10.1111/j.1440-1835.2009.00531 .x
Miyagawa-Yamaguchi A, Okami T, Kira N, Yamaguchi H, Ohnishi
K, Adachi M (2011) Stable nuclear transformation of the dia-
tom Chaetoceros sp. Phycol Res 59(2):113–119. https ://doi.
org/10.1111/j.1440-1835.2011.00607 .x
Mubarak M, Shaija A, Suchithra TV (2019) Flocculation: an effec-
tive way to harvest microalgae for biodiesel production. J
Environ Chem Eng. https ://doi.org/10.1016/j.jece.2019.10322 1
Nagappan S, Devendran S, Tsai PC, Dahms HU, Ponnusamy VK
(2019) Potential of two-stage cultivation in microalgae bio-
fuel production. Fuel 252:339–349. https ://doi.org/10.1016/j.
fuel.2019.04.138
Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982)
Gene transfer into mouse lyoma cells by electroporation
in high electric fields. EMBO J 1(7):841–845. https ://doi.
org/10.1002/j.1460-2075.1982.tb012 57.x
Oh YK, Hwang KR, Kim C, Kim JR, Lee JS (2018) Recent develop-
ments and key barriers to advanced biofuels: a short review.
Bioresour Technol 257:320–333. https ://doi.org/10.1016/j.
biort ech.2018.02.089
Park JBK, Craggs RJ, Shilton AN (2011) Wastewater treatment high
rate algal ponds for biofuel production. Bioresour Technol
102:35–42. https ://doi.org/10.1016/j.biort ech.2010.06.158
Park S, Nguyen THT, Jin ES (2019) Improving lipid production
by strain development in microalgae: strategies, challenges
and perspectives. Bioresour Technol 292:121953. https ://doi.
org/10.1016/j.biort ech.2019.12195 3
Peng L, Lan CQ, Zhang Z (2013) Evolution, detrimental effects, and
removal of oxygen in microalga cultures: a review. Environ
Prog Sustain 32(4):982–988. https ://doi.org/10.1002/ep.11841
Peng L, Lan CQ, Zhang Z, Sarch C, Laporte M (2015) Control of
protozoa contamination and lipid accumulation in Neochlo-
ris oleoabundans culture: effects of pH and dissolved inor-
ganic carbon. Bioresour Technol 197:143–151. https ://doi.
org/10.1016/j.biort ech.2015.07.101
Peng L, Zhang Z, Cheng P, Wang Z, Lan CQ (2016a) Cultivation of
Neochloris oleoabundans in bubble column photobioreactor
with or without localized deoxygenation. Bioresour Technol
206:255–263. https ://doi.org/10.1016/j.biort ech.2016.01.081
Peng L, Zhang Z, Lan CQ, Basak A, Bond N, Ding X, Du J (2016b)
Alleviation of oxygen stress on Neochloris oleoabundans:
effects of bicarbonate and pH. J Appl Phycol 25(3):1–10. https
://doi.org/10.1007/s1081 1-016-0931-3
Plant P, Floorspace B (2010) Energy information administration.
Alphascript Publishing, New York. https ://www.eia.gov/
Poulsen N, Chesley PM, Kröger N (2010) Molecular genetic manip-
ulation of the diatom Thalassiosira pseudonana (Bacillari-
ophyceae). J Phycol 42(5):1059–1065. https ://doi.org/10.111
1/j.1529-8817.2006.00269 .x
Pragya N, Pandey KK, Sahoo PK (2013) A review on harvesting, oil
extraction and biofuels production technologies from micro-
algae. Renew Sustain Energy Rev 24:159–171. https ://doi.
org/10.1016/j.rser.2013.03.034
Pulz O (2001) Photobioreactors: production systems for phototrophic
microorganisms. Appl Microbiol Biotechnol 57(3):287–293.
https ://doi.org/10.1007/s0025 30100 7

Environmental Chemistry Letters
1 3
Qin S, Sun G, Jiang P, Zou L, Wu Y, Tseng CK (1999) Review of
genetic engineering of Laminaria japonica (Laminariales, Phae-
ophyta) in China. Hydrobiologia 398–399:469–472. https ://doi.
org/10.1023/a:10170 91629 539
Qin S, Lin H, Jiang P (2012) Advances in genetic engineering of marine
algae. Biotechnol Adv 30:1602–1613. https ://doi.org/10.1016/j.
biote chadv .2012.05.004
Quinn JC, Catton K, Wagner N, Bradley TH (2012) Current large-scale
US biofuel potential from microalgae cultivated in photobiore-
actors. Bioenerg Res 5(1):49–60. https ://doi.org/10.1007/s1215
5-011-9165-z
Quinn JC, Hanif A, Sharvelle S, Bradley TH (2014) Microalgae to bio-
fuels: life cycle impacts of methane production of anaerobically
digested lipid extracted algae. Bioresour Technol 171:37–43.
https ://doi.org/10.1016/j.biort ech.2014.08.037
Ra CH, Kang CH, Kim NK, Lee CG, Kim SK (2015) Cultivation of
four microalgae for biomass and oil production using a two-stage
culture strategy with salt stress. Renew Energy 80:117–122. https
://doi.org/10.1016/j.renen e.2015.02.002
Radakovits R, Jinkerson RE, Darzins A, Posewitz MC (2010) Genetic
engineering of algae for enhanced biofuel production. Eukaryot
Cell 9(4):486–501. https ://doi.org/10.1128/EC.00364 -09
Ramasamy P, Lee K, Lee J, Yk Oh (2015) Breaking dormancy: an
energy-efficient means of recovering astaxanthin from microal-
gae. Green Chem 17:1226–1234. https ://doi.org/10.1039/c4gc0
1413h
Ranjan A, Patil C, Moholkar VS (2010) Mechanistic assessment of
microalgal lipid extraction. Ind Eng Chem Res 49:2979–2985.
https ://doi.org/10.1021/ie901 6557
Rao H, Schmidt LC, Bonin J, Robert M (2017) Visible-light-driven
methane formation from CO2 with a molecular iron catalyst.
Nature 548(7665):74–77. https ://doi.org/10.1038/natur e2301 6
Rastogi RP, Pandey A, Larroche C, Madamwar D (2018) Algal green
energy—R&D and technological perspectives for biodiesel
production. Renew Sust Energ Rev 82:2946–2969. https ://doi.
org/10.1016/j.rser.2017.10.038
Ren F, Chu H, Xiang L, Han W, Gu M (2019) Effect of hydrogen addi-
tion on the laminar premixed combustion characteristics the main
components of natural gas. J Energy Inst 92(4):1178–1190. https
://doi.org/10.1016/j.joei.2018.05.011
Roberts GW, Fortier MP, Sturm BSM, Stagg-Williams SM (2013)
Promising pathway for algal biofuels through wastewater culti-
vation and hydrothermal conversion. Energ Fuel 27(2):857–867.
https ://doi.org/10.1021/ef302 0603
Rodionova MV, Poudyal RS, Tiwari I, Voloshin RA, Zharmukhamedov
SK, Nam HG, Zayadan BK, Bruce BD, Hou HJM, Allakhverdiev
SI (2017) Biofuel production: challenges and opportunities. Int
J Hydrog Energy 42(12):8450–8461. https ://doi.org/10.1016/j.
ijhyd ene.2016.11.125
Rodolfi L, Zittelli GC, Bassi N, Padovani G, Biondi N, Bonini G,
Tredici MR (2010) Microalgae for oil: strain selection, induc-
tion of lipid synthesis and outdoor mass cultivation in a low-cost
photobioreactor. Biotechnol Bioeng 102(1):100–112. https ://doi.
org/10.1002/bit.22033
Rubio J, Carissimi E, Rosa JJ, Rubio J, Rosa JJ (2007) Flotation in
water and wastewater treatment and reuse: recent trends in Bra-
zil. Int J Environ Pollut 30(3):197–212. https ://doi.org/10.1504/
IJEP.2007.01470 0
Rühle T, Hemschemeier A, Melis A, Happe T (2008) A novel screening
protocol for the isolation of hydrogen producing Chlamydomonas
reinhardtii strains. BMC Plant Biol 8(107):107. https ://doi.
org/10.1186/1471-2229-8-107
Sadvakasova AK, Akmukhanova NR, Bolatkhan KZ, Bolatkhan KU
(2019) Search for new strains of microalgae-producers of lipids
from natural sources for biodiesel production. Int J Hydrog
Energy. https ://doi.org/10.1016/j.ijhyd ene.2019.01.093
SapphireEnergy (2016) The sapphire story. Sapphire Energy, Inc.
http://www.sapph ireen ergy.com/
Seo JY, Jeon HJ, Kim JW, Lee J, Oh YK, Chi WA, Lee JW (2018)
Simulated-sunlight-driven cell lysis of magnetophoretically sepa-
rated microalgae using ZnFe2O4 octahedrons. Ind Eng Chem Res.
https ://doi.org/10.1021/acs.iecr.7b044 45
Sharma YC, Singh V (2017) Microalgal biodiesel: a possible solution
for India’s energy security. Renew Sust Energ Rev 67:72–78.
https ://doi.org/10.1016/j.rser.2016.08.031
Shelef GS, Sukenik A, Green M (1984) Microalgae harvesting and
processing: a literature review. Algae 8(3):237–244. https ://doi.
org/10.2172/62046 77
Shuler ML, Kargi F (2001) Bioprocess engineering basic concepts, 2nd
edn. Prentice Hall PTR, Upper Saddle River
Singh J, Gu S (2010) Commercialization potential of microalgae for
biofuels production. Renew Sust Energ Rev 14(9):2596–2610.
https ://doi.org/10.1016/j.rser.2010.06.014
Singh G, Patidar SK (2018) Microalgae harvesting techniques: a
review. J Environ Manag 217:499–508. https ://doi.org/10.1016/j.
jenvm an.2018.04.010
Sirajunnisa AR, Surendhiran D (2016) Algae—a quintessential and
positive resource of bioethanol production: a comprehen-
sive review. Renew Sust Energ Rev 66:248–267. https ://doi.
org/10.1016/j.rser.2016.07.024
Skorupskaite V, Makareviciene V, Gumbyte M (2016) Opportunities
for simultaneous oil extraction and transesterification during bio-
diesel fuel production from microalgae: a review. Fuel Process
Technol 150:78–87. https ://doi.org/10.1016/j.fupro c.2016.05.002
Sode K, Tatara M, Takeyama H, Burgess JG, Matsunaga T (1992) Con-
jugative gene transfer in marine cyanobacteria: Synechococcus
sp., Synechocystis sp. and Pseudanabaena sp. Appl Microbiol
Biotechnol 37(3):369–373. https ://doi.org/10.1007/bf002 10994
Song D, Park J, Kim K, Lee LS, Seo JY, Oh YK, Kim YJ, Ryou MH,
Lee YM, Lee K (2017) Recycling oil-extracted microalgal
biomass residues into nano/micro hierarchical Sn/C compos-
ite anode materials for lithium-ion batteries. Electrochim Acta
250:59–67. https ://doi.org/10.1016/j.elect acta.2017.08.045
Suganya T, Varman M, Masjuki HH, Renganathan S (2016) Macroal-
gae and microalgae as a potential source for commercial appli-
cations along with biofuels production: a biorefinery approach.
Renew Sust Energ Rev 55:909–941. https ://doi.org/10.1016/j.
rser.2015.11.026
Sugar IP, Neumann E (1984) Stochastic model for electric field-induced
membrane pores electroporation. Biophys Chem 19(3):211–225.
https ://doi.org/10.1016/0301-4622(84)87003 -9
Tredici M, Materassi R (1992) From open ponds to vertical alveolar
panels: the Italian experience in the development of reactors for
the mass cultivation of phototrophic microorganisms. J Appl
Phycol 4(3):221–231. https ://doi.org/10.1007/BF021 61208
Trentacoste EM, Shrestha RP, Smith SR, Glé C, Hartmann AC, Hilde-
brand M, Gerwick WH (2013) Metabolic engineering of lipid
catabolism increases microalgal lipid accumulation without
compromising growth. Pro Natl Acad Sci USA 110(49):19748–
19753. https ://doi.org/10.1073/pnas.13092 99110
Uduman N, Qi Y, Danquah MK, Forde GM, Hoadley A (2010)
Dewatering of microalgal cultures: a major bottleneck to algae-
based fuels. J Renew Sust Energ 2(1):23–571. https ://doi.
org/10.1063/1.32944 80
Ummalyma SB, Gnansounou E, Sukumaran RK, Sindhu R, Pandey
A, Sahoo D (2017) Bioflocculation: an alternative strategy for
harvesting of microalgae—an overview. Bioresour Technol
242:227–235. https ://doi.org/10.1016/j.biort ech.2017.02.097
Vlaskin M, Grigorenko AV, Chernova NI, Kiseleva SV (2018) Hydro-
thermal liquefaction of microalgae after different pre-treatments.
Energy Explor Exploit 36(6):014459871877710. https ://doi.
org/10.1177/01445 98718 77710 7

Environmental Chemistry Letters
1 3
Vonshak A, Richmond A (1988) Mass production of the blue-green
alga Spirulina: an overview. Biomass 15(4):233–247. https ://doi.
org/10.1016/0144-4565(88)90059 -5
Wang Z, Ullrich N, Joo S, Waffenschmidt S, Goodenough U (2009)
Algal lipid bodies: stress induction, purification, and biochemi-
cal characterization in wild-type and starchless Chlamydomonas
reinhardtii. Eukaryot Cell 8(12):1856–1868. https ://doi.
org/10.1128/ec.00272 -09
Wang B, Lan CQ, Courchesne N, Mu Y (2010) Microalgae for biofuel
production and CO2 sequestration. Nova Science Publishers Inc.,
New York
Wang B, Lan CQ, Horsman M (2012) Closed photobioreactors for pro-
duction of microalgal biomasses. Biotechnol Adv 30(4):904–912.
https ://doi.org/10.1016/j.biote chadv .2012.01.019
Will T (2009) Algae 2020: advanced biofuel markets and commer-
cialization outlook,Available via DIALOG. https ://www.emerg
ing-marke ts.com. Accessed June 2009
Xu H, Miao X, Wu Q (2006) High quality biodiesel production
from a microalga Chlorella protothecoides by heterotrophic
growth in fermenters. J Biotechnol 126(4):499–507. https ://doi.
org/10.1016/j.jbiot ec.2006.05.002
Xue J, Niu Y, Huang T, Yang W, Liu J, Li H (2014) Genetic improve-
ment of the microalga Phaeodactylum tricornutum for boost-
ing neutral lipid accumulation. Metab Eng 27:1–9. https ://doi.
org/10.1016/j.ymben .2014.10.002
Yongmanitchai W, Ward OP (1991) Growth of and omega-3 fatty acid
production by Phaeodactylum tricornutum under different culture
conditions. Appl Environ Microb 57(2):419–425
Yun YM, Kim DH, Oh YK, Shin HS, Jung KW (2014) Applica-
tion of a novel enzymatic pretreatment using crude hydrolytic
extracellular enzyme solution to microalgal biomass for dark fer-
mentative hydrogen production. Bioresour Technol 159:365–372.
https ://doi.org/10.1016/j.biort ech.2014.02.129
Yun YM, Shin HS, Lee CK, Oh YK, Kim HW (2016) Inhibition
of residual n-hexane in anaerobic digestion of lipid-extracted
microalgal wastes and microbial community shift. Environ Sci
Pollut Res 23(8):7138–7145. https ://doi.org/10.1007/s1135
6-015-4643-z
Zhao B, Ma J, Zhao Q, Laurens L, Jarvis E, Chen S, Frear C (2014)
Efficient anaerobic digestion of whole microalgae and lipid-
extracted microalgae residues for methane energy production.
Bioresour Technol 161:423–430. https ://doi.org/10.1016/j.biort
ech.2014.03.079
Zhou W, Min M, Li Y, Hu B, Ma X, Cheng Y, Liu Y, Chen P, Ruan
R (2012) A hetero-photoautotrophic two-stage cultivation pro-
cess to improve wastewater nutrient removal and enhance algal
lipid accumulation. Bioresour Technol 110:448–455. https ://doi.
org/10.1016/j.biort ech.2012.01.063
Zimmermann U, Pilwat G, Riemann F (1975) Preparation of eryth-
rocyte ghosts by dielectric breakdown of the cell membrane.
BBA Biomembr 375(2):209–219. https ://doi.org/10.1016/0005-
2736(75)90189 -3
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