Available online at www.sciencedirect.
com
ScienceDirect
Marine microalgae for production of biofuels and
chemicals
Yoshiaki Maeda1, Tomoko Yoshino1, Tadashi Matsunaga1,
Mitsufumi Matsumoto2 and Tsuyoshi Tanaka1
Marine microalgae are recognized as promising feedstocks for
biofuels and chemicals owing to their higher growth rates than
those of terrestrial crop plants. We aimed to summarize the
production of biofuels and chemicals by marine microalgae and
to discuss their advantages and potential from the aspect
of bioprocess. The present circumstances of the microalgae
industry were briefly described and large-scale industrial plants
for microalgae production, where some marine microalgae are
cultivated, were introduced. The advantages of marine
microalgae in terms of water and land usage were also
discussed. Finally, novel genome editing tools that could
further exploit the potential of marine microalgae were
reviewed. The present study provided comprehensive
information regarding current biotechnology using marine
microalgae.
Addresses
1 does not exist. Therefore, in the present study, several
Division of Biotechnology and Life Science, Institute of Engineering,
Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho,
Koganei, Tokyo 184-8588, Japan
2
Biotechnology Laboratory, Electric Power Development Co., Ltd., 1,
Yanagisaki-machi, Wakamatsu-ku, Kitakyusyu 808-0111, Japan
Corresponding author: Tanaka, Tsuyoshi (tsuyo@cc.tuat.ac.jp)
Current Opinion in Biotechnology 2018, 50:111–120
This review comes from a themed issue on Energy biotechnology
Edited by Akihiko Kondo and Hal Alper
For a complete overview see the Issue and the Editorial
Available online 10th December 2017
https://doi.org/10.1016/j.copbio.2017.11.018
0958-1669/ã 2017 Elsevier Ltd. All rights reserved.
Introduction
Vast marine environments support extremely diverse
microorganisms. Scientists have extensively studied a
number of marine microorganisms that synthesize useful
materials for various purposes including human food,
animal feed, and functional biochemical production, as
well as biofuel and bioenergy generation. Among such
microorganisms, marine microalgae have been recognized
as key subjects for biotechnology research owing to their
diversity generated via a unique evolutionary history,
CO2 fixation capacity by photosynthesis, and high
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biomass and lipid productivities. A variety of prokaryotic
(cyanobacteria) and eukaryotic microalgae have been
identified as promising candidate hosts for the production
of useful materials, in particular for biofuel and chemical
production, some of which are commercially available. In
the present review article, the current production of
biofuels and chemicals by marine microalgae is summa-
rized. The related synthetic pathways, in particular lipid
metabolism in microalgae [1–4] and enhancement by
conventional genetic engineering [5–8], have already
been heavily reviewed. Therefore, the present review
article does not contain these topics. Instead, the focus is
on the advantages of marine microalgae (including brack-
ish and saline water microalgae) for biotechnological
applications. Although numerous types of marine micro-
algae have been extensively studied, a comprehensive
review of their advantages over freshwater microalgae
modern examples of commercial applications of micro-
algae are listed. A number of companies currently operate
commercial-scale mass cultivation of microalgae, with
several of them utilizing marine strains. The cultivation
plants that are located at geographically suitable sites for
microalgae production are also introduced. Subsequently,
the water footprint and land usage required for marine
microalgae production is discussed because these two
factors are critical in dictating the scalability of microalgae
production. Recent progress in offshore cultivation sys-
tems is also described. Finally, the potential of genome
editing technology (e.g. transcription activator-like effec-
tor nuclease (TALEN) and clustered regularly inter-
spaced short palindromic repeats/CRISPR-associated
protein 9 (CRISPR/Cas9)) as novel tools for enhanced
biofuel and biochemical production by marine microalgae
are discussed.
Current commercial production of microalgae
Outdoor mass cultivation of microalgae (in particular
Chlorella) has been previously studied during the 1950s
[9]. Tamiya and colleagues studied mass cultivation of
Chlorella sp. in Japan to produce microalgal biomass for
human food or animal feed [10,11]. The commercial
application of microalgae started in the early 1960s [12]
and the subsequent growth of the microalgal industry has
been described previously [13,14]. Until a decade or so
ago, commercially produced microalgae has been mostly
consumed in the field of human and animal nutrition. To
date, Cyanotech Co. [15] and DIC Co. [16] (formerly
Current Opinion in Biotechnology 2018, 50:111–120
112 Energy biotechnology
Dainippon Ink and Chemicals Inc.) including the group
companies Earthrise Nutritionals, LLC (California, USA)
and Hainan-DIC Microalgae CO., Ltd. (Hainan, China)
have produced Arthrospira platensis for human nutrition.
In addition, microalgae have been utilized as producers of
high-value molecules such as polyunsaturated fatty acids
(PUFA, e.g. g-linolenic acid, arachidonic acid, eicosapen-
taenoic acid, and docosahexaenoic acid) and pigments (e.
g. b-carotene, astaxanthin, and phycoerythrin). b-Caro-
tene production by Dunaliella salina has been operated at
Hutt Lagoon (Western Australia, Australia), with this
cultivation site currently owned by BASF. Algatechnol-
ogies Ltd. (Ketura, Israel) produces astaxanthin from
Haematococcus pluvialis [17]. This company established
tubular photobioreactors (PBRs) for astaxanthin produc-
tion and similar systems are widely employed in other
companies.
During the recent decade, circumstances surrounding
industrial microalgal production have drastically changed.
As we face the global climate change, microalgae has been
attracting greater attention as feedstocks for biofuels
owing to high growth rates and lipid accumulation prop-
erties [18]. In the 2000s, a number of start-ups that
focused on biofuel production were established [19].
Representative examples include TerraVia Holdings,
Inc. (formerly Solazyme, Inc., California, USA) [20,21],
Cellana, Inc. (Hawaii, USA), Sapphire Energy, Inc. (Cali-
fornia, USA) [22], Solix Algredients, Inc. (formerly Solix
Biofuels, Inc., Colorado, USA) and Aurora Biofuels, Inc.
(afterwards Aurora Algae, Inc., California, USA). Several
of these companies no longer operate microalgal biofuel
production and others have diversified their business so
that microalgal production is not their primary target.
Table 1 is a summary of several companies that currently
operate large-scale cultivation of microalgae, which shows
the diversification of business strategies to high-value
molecule production and so on. This is because biofuel
production from microalgae requires the largest produc-
tion scale to make it economically feasible compared to
other high-value molecules (e.g. PUFAs and pigments).
Open-pond outdoor mass cultivation systems are widely
employed by the majority of companies for industrial
microalgal cultivation (Figure 1) [23–26], except for Ter-
raVia Holdings, Inc. that operates non-photosynthetic
fermentation in closed bioreactors (Table 1). Open-pond
outdoor cultivation is the most extensive system and,
thus, it can take advantage of low inputs of energy and
operation costs. Nonetheless, its performance can be
greatly affected by climatic conditions, as well as con-
tamination issues. In addition, geographic conditions are
important, that is, the open-pond systems generally
require a vast contiguous field with a slope no greater
than 2% or so to avoid an enormous earthmoving cost for
plant construction [27]. Therefore, selection of produc-
tion sites is crucial for stable microalgal cultivation.
Current Opinion in Biotechnology 2018, 50:111–120
A geographic information system (GIS) is a powerful tool
that can be utilized to assess the potential of candidate
cultivation sites. GIS considers numerous factors, for
example, temperature, rainfall, groundwater salinity, land
availability, and proximity to inputs such as a power plant
(as a source of CO2) and a wastewater treatment plant (as a
source of nutrients). GIS is also useful to generate global
[28] and local [27] maps (Figure 2(a) and (b), respec-
tively) of the potential microalgal production. By super-
imposing the locations of the commercial production sites
(Table 1) on the maps generated by GIS, we can reason-
ably assume that these production sites were carefully
selected in each nation to maximize productivity
(Figure 2).
Table 1 also demonstrates that many companies utilize
(including, but not limited to) marine, brackish, or saline
water microalgae. Selective cultivation of target micro-
algae under high salinity conditions is one of the methods
to decrease the contamination issue in open-pond culti-
vation [29,30]. Furthermore, marine microalgae have
advantages in terms of water footprint (WF) and land
usage as described in the following section.
Water footprint and land usage for the
production of marine microalgae
Water is one of the most fundamental natural resources
and is potentially required for any kind of human activity
including industry, agriculture, and energy production.
Among the approximately 1.4 billion km3 (1.4  1018 m3)
of water on Earth, available freshwater (including surface
water and groundwater) is believed to be approximately
200 thousands km3, which is less than 1% [31]. Besides
our potable water, water demand from the sectors men-
tioned above also needs to be supported by such limited
water resources. Therefore, it should be carefully consid-
ered whether water is utilized in the most efficient way by
all sectors.
To determine the water efficiency quantitatively, the
WF is a good indicator. The concept of the WF was
introduced by Hoekstra and Hung [32]. Gerbens-Leenes
et al. described that ‘the WF of a product (commodity,
good or service) is defined as the volume of freshwater
used for the production of that product at the place where
it was actually produced’ [33]. The WF for a product
consists of three components: blue WF (referring to the
amount of surface water and groundwater utilized during
the production process), green WF (referring to the
amount of rainwater stored in the soil strata during the
production process), and gray WF (referring to the
amount of water that is required to dilute the pollutants
emitted to the natural water system during the produc-
tion process) [32–34]. Gerbens-Leenes et al. reported
that the WF of bioenergy is larger than other forms of
energy (e.g. nuclear energy, natural gas, coal, and crude
oil) [33,35], indicating that increasing bioenergy
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 Marine microalgae for production of biofuels and chemicals Maeda et al. 113

Table 1

Industrial production of microalgae for biofuels and chemicals. Natural habitats of microalgae in bold letters are estimated to be marine,
brackish, or saline water microalgae

Company Microalgae (including, Technology Major products Major production Cultivation

but not limited to) site area
TerraVia Holdings, Inc. Prototheca spp. Non-photosynthetic Food oil Solazyme Bunge –
(Formerly Chlorella spp. fermentation in Omega3 PUFA Renewable Oils facility
Solazyme, Inc.) closed bioreactors Oil for cosmetics (in Bunge’s Moema
Whole algae sugarcane mill) Moema,
São Paulo, Brazil
Sapphire Energy, Inc. Scenedesmus dimorphus Open pond Green crude oil Green Crude Farm 55 ha
Nannochloropsis spp. (aka, Integrated Algal
Arthrospira platensis Bio-Refinery) Luna County,
(Formerly Spirulina New Mexico, USA
platensis)
Earthrise Nutritionals, Arthrospira platensis Open pond Whole algae Calipatria, California, USA 33 ha
LLC (DIC group) (Licensing agreement
with Sapphire
Energy, Inc.)
Hainan-DIC Arthrospira platensis Open pond Whole algae Haikou, Hainan, China 15 ha
Microalgae Co.,
Ltd. (DIC group)
Cellana, Inc. Nannochloropsis spp. Open pond Animal feed Kailua-Kona, Hawaii, USA 0.7 ha
Closed tube Biofuel
Omega3 PUFA
Whole algae
Cyanotech Co. Arthrospira platensis Open pond Whole algae Kailua-Kona, Hawaii, USA 25 ha
Haematococcus pluvialis Ultra-high pressure Astaxanthin
extraction of astaxanthin
from Haematococcus
BASF (Nutrition & Dunalliella salina Open pond Astaxanthin Hutt Lagoon, 740 ha
Health division) Oil for cosmetics Western Australia,
Betaine surfactant Australia
for cosmetics
(with TerraVia)
J-Power Fistulifera solairs Open pond Biofuel Kita-kyushu, Fukuoka, 0.2 ha
JPCC DA0580 Japan
Mayamaea
sp. JPCC CTDA0820
Euglena Co., Ltd. Euglena gracilis Open pond Whole algae Ishigaki Island, –
Biofuel Okinawa, Japan
IHI Co. Botryococcus braunii Open pond Whole algae Kagoshima, Japan 0.15 ha
Biofuel

Data were retrieved from the web sites for each company and patent files as of July 2017.

production will lead to increasing water consumption,
which could cause water scarcity in other sectors. None-
theless, these authors only calculated the WF of bioe-
nergy generated from terrestrial crop plants and excluded
that of microalgae.
The WF of bioenergy derived from microalgae was
reported by Yang et al. [36]. They calculated the total
of blue and green WFs (including water usage or
exchange during cultivation, harvest, drying, extraction,
and esterification) for the production of biodiesel by
Chlorella vulgaris, which is a model freshwater microalga
species with high growth rate and lipid content, cultured
in an open-pond system under conditions similar to the
summer in California, USA. The WF was estimated to be
3726 kg-water/kg-biodiesel, which is consistent with
other studies [37], and is lower than those for terrestrial
corps; soybean, rapeseed, and jatropha (Table 2). This
WF could be reduced to 591 kg-water/kg-biodiesel if the
water discharged during harvesting was fully recycled.
Benefits of water recycle were also emphasized in other
recent studies [37,38]. Utilization of seawater or waste-
water could significantly decrease the WF to 399 kg-
water/kg-biodiesel, and marine microalgae are more suit-
able for seawater-based culture media than freshwater
microalgae such as C. vulgaris. However, even the culti-
vation of marine microalgae in seawater-based media
requires a certain amount of freshwater usage during
cultivation because water evaporates from open ponds,
and freshwater needs to be added to prevent too great an
increase in salinity. Some marine microalgae such as
Chaetoceros gracilis, Cyclotella cryptica, and Nannochloropsis
sp., which show high growth rates and lipid contents,
have been estimated to show comparative WF to

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114 Energy biotechnology

Figure 1

(a) (b)

(c) (d) (e)

Current Opinion in Biotechnology

Cultivation sites of industrial microalgal production for biofuels and chemicals operated by (a) BASF (Hutt Lagoon, Western Australia, Australia), (b)
Earthrise Nutritionals, LLC (California, USA), (c) Sapphire Energy, Inc. (New Mexico, USA), (d) Cyanotech Co. (Hawaii, USA), and (e) Cellana, Inc.
(Hawaii, USA).
The figures are reproduced from previous studies [23–26], with some modifications.

C. vulgaris. Given the large WF for crop plants (Table 2),
cultivation of marine microalgae in seawater-based cul-
ture media with the aid of water recycling systems is a
promising way to decrease freshwater usage.
In line with the theoretical analyses described above, there
are several practical studies of outdoor cultivation of
marine microalgae using natural seawater-based culture
media. Tredici’s group has developed a closed-type biore-
actor (referred to as a Green Wall Panel [GWP]) that are
composed of low density polyethylene film and a rectan-
gular metal frame [39]. They first performed outdoor
cultivation of the marine eustigmatophyte Nannochloropsis
sp. F&M-M24 in the GWP containing 110 L of a natural
seawater-based medium [40], followed by application with
other marine or halo-tolerant microalgae [41,42]. The
seawater was treated with UV light and subsequently
filtered using 10-mm and 1.5-mm polypropylene filters
for sanitization, and enclosed in the GWP with sterile
nutrient solutions [40]. A series of GWPs (GWP-I [39],
GWP-II [41,43], and GWP-IIII [44]) have been devel-
oped and, in a recently constructed 1-ha GWP plant,
natural seawater was utilized for cultivation as well as
temperature control [45,46]. In contrast, Matsumoto and
Tanaka’s group utilized open-pond bioreactors for cultiva-
tion of marine oleaginous diatoms, Fistulifera solaris JPCC
DA0580 and Mayamaea sp. JPCC CTDA0820, in natural
seawater-based culture medium (up to 640 000 L) [47].
The high growth rates of these diatoms contributed to the
suppression of contamination, and allowed for the opera-
tion of open-pond cultivation with natural seawater over
the long-term. Natural seawater usage for cultivation has
also been employed, if geologically accessible, in the Algae
Tested Public–Private Partnership (ATP3). This is a
multi-region, long-term microalgae biomass cultivation
trial operated by the US Department of Energy (DOE)

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 Marine microalgae for production of biofuels and chemicals Maeda et al. 115

Figure 2

Current Opinion in Biotechnology

Global and local (Hawaii, USA) maps representing the suitable sites for microalgae production on which industrial microalgal production sites are
overlaid. (a) A global evaluation map of microalgal lipid yield. The thermal and biological growth models representing Nannochloropsis were
employed for the evaluation. (b) A local map of the suitable cultivation sites in Hawaii Island where geographic conditions (solar radiation, rainfall,
slope, and contiguous area) are considered. Contiguous areas wider than 4 km2 are highlighted in blue. The figures are reproduced from previous
studies [27,28], with some modifications.

Table 2

Water footprint for microalgae and terrestrial crops providing biodiesel

Source Water footprint Description Reference

(kg-water/kg-biodiesel)
Microalgae 3726 [36]
Microalgae 399 Sea water or wastewater use [36]
Soybean 15 541 Biodiesel density 0.88 kg/l [35]
Rapeseed 16 138 Biodiesel density 0.88 kg/l [35]
Jatropha 22 641 Biodiesel density 0.88 kg/l [35]

and private sectors, where the unified processes and sys-
tems (open, race-way type bioreactors containing 1000 L of
culture media, and N. oceanica and C. vulgaris as model
microalgae) are operated at 6 test sites (Arizona, Hawaii,
California, Ohio, Georgia, and Florida) in the USA [48].
These studies contribute to the accumulation of knowl-
edge in terms of stable and sustainable mass cultivation of
microalgae while utilizing natural seawater resources.
Vast land area is another essential requirement for the
mass cultivation of microalgae. Approximately a decade
ago, the estimation of how large an area was required to
supply sufficient amounts of biofuels was optimistic. In
2007, it was stated that the land area required to meet 30%
of all transport fuel needs of the USA was 1.2–2.7 Mha (i.
e. 2–4.5 Mha for 50%) [18]. A recent assessment by
Moody et al. in 2014 [28] revised this estimation to
34.4 Mha (i.e. 57.4 Mha for 50%). Their assessment,
however, was based on cultivation data obtained in a
closed-culture system and, thus, microalgae production
in an open-pond system could require additional land use.
Moody et al. emphasized that the productivity of micro-
algae biofuels is not a constant value, but can vary
temporally and geographically [28]. Their assessment
indicates that the potential of biofuel production using
microalgae has considerable promise for supplying abun-
dant energy and decreasing fossil fuel usage in several
countries (e.g. Canada, Brazil, China, and the USA) while
utilizing only a small portion of their non-arable land. In
contrast, in other countries (e.g. Japan) it is difficult to
ensure the allocation of enough land area to replace fossil
fuel with microalgae biofuel. For such countries, it would
be worthwhile to consider different approaches.
Offshore cultivation is an emerging technology for micro-
algal production in which microalgal culture is enclosed in
PBRs made of transparent polymer films (e.g. polyethyl-
ene and polyurethane) and the PBRs are floated on the
ocean surface [49–52,53]. As offshore cultivation uses a
vast amount of ocean area, terrestrial land is not required

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116 Energy biotechnology

Figure 3

(a) OMEGA System

(b)

(c) Gas out

Air/CO2 Air/CO2

Gas headspace
CO2 O2

Algae CO2 Microbial

Harvested
Wastewater O2 community
biomass
inflow
Nutrients BOD
Current Opinion in Biotechnology

Offshore microalgae cultivation systems. (a) Offshore Membrane Enclosures for Growing Algae (OMEGA) system (California, USA), (b) Floating
mass culture system for microalgae (Incheon, Korea), (c) Simultaneous process of microalgae cultivation and wastewater treatment offshore
(Alabama, USA). The figures are reproduced from previous studies [50–52,53], with some modifications.

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 Marine microalgae for production of biofuels and chemicals Maeda et al. 117
for microalgal cultivation. Several types of floating PBRs
have been developed (Figure 3), and the cultivation
performance was evaluated. Nonetheless, there are sev-
eral drawbacks to be overcome, such as environmental
pollution issues and difficulty in stable biomass produc-
tion. Further engineering efforts are required to ensure
that offshore cultivation systems are technologically and
economically feasible.
The potential of genome editing technology
for marine microalgae
Improvements of the biomass and/or chemicals produc-
tivities (e.g. lipids for biofuel) in the marine microalgae of
interest are the simplest ways to decrease production
costs, WF, and land use. Effective methodology, termed
genome editing, is now available that enables the
enhancement of these productivities in microalgae.
Genome editing is a genetic engineering technique capa-
ble of modifying the genomic DNA in a site-specific
manner. Several types of genome editing systems have
been developed based on ZFN (zinc-finger nuclease),
TALEN, and CRISPR/Cas9, while CRISPR/Cas9 and
TALEN are widely employed nowadays. Their mecha-
nisms have been reviewed elsewhere [54,55]. Genome
editing in microalgae was firstly demonstrated in the
model freshwater green microalgae Chlamydomonas rein-
hardtii with ZFN [56], followed by CRISPR/Cas9 [57–
60]. Model marine microalgae have also been engineered
with genome editing techniques. Genome editing of
marine diatoms Phaeodactylum tricornutum (with TALEN
[61–63] and CRISPR/Cas9 [64]) and Thalassiosira pseudo-
nana (with CRISPR/Cas9 [65]), and marine eustigmato-
phyte Nannochloropsis spp. with CRISPR/Cas9 [66,67]
has been successfully demonstrated.
Table 3 is a brief summary of the genome editing studies
for microalgae described above. One of the key steps in
the genome editing of microalgae is transformation
whereby the essential components for genome editing
need to be efficiently introduced into the microalgal cells.
Electroporation has been employed for C. reinhardtii and
Nannochloropsis spp. In contrast, particle bombardment
has been utilized for diatoms because this technique is
harsh enough to break the stiff silica cell walls, while it
could impair the viability of target microalgae. Electro-
poration is now available for diatoms [68,69] and it might
assist with the efficiency of genome editing. For knocking
out a gene of interest completely in diploid (2n) organ-
isms, it is necessary to screen the biallelic mutant clones,
since monoallelic mutation could simply result in the
attenuation of the target gene expression. This issue is
not problematic in the model green microalga C. rein-
hardtii, which is usually haploid (1n) [57]. To address this
issue, high throughput screening methods such as restric-
tion enzyme assay, where restriction site losses resulting
from mutations have been detected, and high-resolution
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melting analysis, where the melting point change of the
double strand DNA surrounding the target sites caused
by mutations have been detected, have been employed to
detect mutations. Considerable efforts need to be
devoted to optimize genome editing processes including
transformation and mutant screening of individual spe-
cies, particularly for non-model species.
Improvement of lipid productivity in marine microalga N.
gaditana by genome editing was recently demonstrated
[67]. In this study, a single transcription regulator gene
(a homolog of fungal Zn(ii)2Cys6-encoding genes) was
knocked out. The mutant clone showed double the lipid
productivity at 5.0 g/(m2 day) than the wild type did,
which suggests that genome editing can be a powerful
tool to enhance biofuel and chemical production.
The reality of outdoor cultivation of engineered micro-
algae, particularly in extensive open-pond or offshore
cultivation systems, is still a matter of debate owing to
the risk of diffusion into the environment. Recently, trials
approved by the US Environmental Protection Agency
were performed for the cultivation of the genetically
engineered freshwater microalga Acutodesmus dimorphus
in outdoor open-pond bioreactors, which demonstrated
that the genetically modified microalga did not outcom-
pete the native algal population [70]. Although further
assessments are required, this could be a first step to
utilize genetically engineered microalgae for efficient and
stable production of microalgal biomass and bioproducts.
In addition, DNA-free genome editing has already been
demonstrated in C. reinhardtii [59], and the resulting
microalga that is free from foreign DNA could be exempt
from genetically modified organism regulations. Based on
these studies, genetically engineered microalgae with
improved productivities of biofuels and chemicals will
keep attracting greater attention towards future develop-
ments of microalgal biotechnology.
Conclusions
Marine microalgae are promising feedstocks for the pro-
duction of biofuels and chemicals. A number of compa-
nies operate outdoor mass cultivation of marine micro-
algae at suitable cultivation sites in a variety of nations.
Since marine microalgae can be cultivated using seawater,
they have the advantage of a low WF. Furthermore, they
are well suited to offshore cultivation systems where the
vast ocean environment can be utilized as a cultivation
site, which could be an attractive option for nations that
only have narrow available land mass. To enhance the
productivities of biomass and bioproducts of marine
microalgae, genome editing techniques are powerful tools
to engineer the required metabolisms. As we face intrac-
table global problems such as exhaustion of fossil fuels,
global warming, and nutrition insufficiency caused by
population increase, marine microalgae could be the
key organisms to solve these problems.
Current Opinion in Biotechnology 2018, 50:111–120
118 Energy biotechnology

Table 3

Genome editing techniques applied for microalgae

Species Genome editing Transformation Introduce Cas9 Target Mutation detection Reference
technique method content codon methods
optimization
C. reinhardtii ZFN Glass beads Plasmid COP3 PCR, Western blotting, [56]
Cel1 Assay, Sequence
analysis
CRISPR/Cas9 Electroporation Plasmid + Hygro, mGFP, Gluc, RE assay, Sequence [57]
FKB12 analysis
CRISPR/Cas9 Electroporation Protein MAA7, CpSRP43, ChlM RT-PCR, Colony color [58]
change, Western blotting,
Southern blotting,
Sequence analysis *1
CRISPR/Cas9 Electroporation Protein ZEP, CpFTSY Colony color change or [59]
Chl a/b ratio change,
sequence analysis, Deep
sequence analysis
CRISPR/Cas9 Electroporation Plasmid + FKB12, Ku70, ALS, RE assay, Sequence [60]
ARG analysis
P. tricornutum TALEN Particle Plasmid UDP-GP, G3PD, EAR, PCR, T7EI assay, [61]
bombardment LACE, putative PPT, sequence analysis, deep
v3FAD, D12FAD sequence analysis
TALEN Particle Plasmid Urease PCR, Southern blotting, [62]
bombardment Western blotting
TALEN Particle Plasmid PtAureo1a PCR, Southern blotting, [63]
bombardment Western blotting,
Sequence analysis
CRISPR/Cas9 Particle Plasmid + CpSRP54 and other HRM analysis, sequence [64]
bombardment 2 genes analysis, qRT-PCR
T. pseudonana CRISPR/Cas9 Particle Plasmid Urease RE assay, PCR, [65]
bombardment sequence analysis
N. oceanica CRISPR/Cas9 Electroporation Plasmid + NR RE assay, RT-PCR, [66]
sequence analysis, deep
sequence analysis
N. gaditana CRISPR/Cas9 Electroporation Plasmid + Fungal Zn (II)2Cys6- Flow cytometry, Western [67]
encoding gene and blotting, PCR
other 17 genes

Abbreviations: ALS (acetolactate synthase), ARG (argininosuccinate lyase), ChlM (Mg-protoporphyrin IX S-adenosylmethionine O-methyl transfer-
ase), COP3 (light-activated ion channel rhodopsin-1), CpFTSY (chloroplast signal recognition particle receptor), CpSRP43 (chloroplast signal
recognition particle 43), CpSRP54 (chloroplast signal recognition particle 54), CRISPR/Cas9 (clustered regularly interspaced short palindromic
repeat/CRISPR associated protein 9), EAR (enoyl-acyl carrier protein reductase), FKB12 (peptidyl prolyl cis-trans isomerase), G3PD (glycerol-3-
phosphate dehydrogenase), Gluc (Gaussia luciferase), HRM analysis (high-resolution melting analysis), Hygro (hygromycin resistance gene), Ku70
(DNA-repairing protein), LACE (long chain acyl-CoA elongase), MAA7 (beta subunit of tryptophan synthase), mGFP (mutant green fluorescence
protein), NR (nitrate reductase), PCR (polymerase chain reaction), PtAureo1a (P. tricornutum aureochrome 1a), putative PPT (putative palmitoyl-
protein thioesterase), qRT-PCR (quantitative real time PCR), RE assay (restrict enzyme assay), RNPs (ribonucleoproteins), RT-PCR (reverse
transcription PCR), T7EI assay (T7 endonuclease I assay), TALEN (transcription activator-like effector nuclease), UDP-GP (UDP-glucose pyropho-
sphorylase), ZEP (zeaxanthin epoxidase), ZNF (zinc finger nuclease), D12FAD (delta 12-fatty acid desaturase), v3FAD (omega-3 fatty acid
desaturase).

Conflict of interest statement References and recommended reading

The authors declare that they have no conflict of interest Papers of particular interest, published within the period of review,
have been highlighted as:
regarding the publication of this article.
 of special interest
 of outstanding interest

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